Comparison of Quantitative Mass Spectrometry Platforms for Monitoring Kinase ATP Probe Uptake in Lung Cancer.

PubMed

Hoffman, Melissa A; Fang, Bin; Haura, Eric B; Rix, Uwe; Koomen, John M

2018-01-05

Recent developments in instrumentation and bioinformatics have led to new quantitative mass spectrometry platforms including LC-MS/MS with data-independent acquisition (DIA) and targeted analysis using parallel reaction monitoring mass spectrometry (LC-PRM), which provide alternatives to well-established methods, such as LC-MS/MS with data-dependent acquisition (DDA) and targeted analysis using multiple reaction monitoring mass spectrometry (LC-MRM). These tools have been used to identify signaling perturbations in lung cancers and other malignancies, supporting the development of effective kinase inhibitors and, more recently, providing insights into therapeutic resistance mechanisms and drug repurposing opportunities. However, detection of kinases in biological matrices can be challenging; therefore, activity-based protein profiling enrichment of ATP-utilizing proteins was selected as a test case for exploring the limits of detection of low-abundance analytes in complex biological samples. To examine the impact of different MS acquisition platforms, quantification of kinase ATP uptake following kinase inhibitor treatment was analyzed by four different methods: LC-MS/MS with DDA and DIA, LC-MRM, and LC-PRM. For discovery data sets, DIA increased the number of identified kinases by 21% and reduced missingness when compared with DDA. In this context, MRM and PRM were most effective at identifying global kinome responses to inhibitor treatment, highlighting the value of a priori target identification and manual evaluation of quantitative proteomics data sets. We compare results for a selected set of desthiobiotinylated peptides from PRM, MRM, and DIA and identify considerations for selecting a quantification method and postprocessing steps that should be used for each data acquisition strategy.

Determination of selected non-authorized insecticides in peppers by liquid chromatography time-of-flight mass spectrometry and tandem mass spectrometry.

PubMed

Mezcua, Milagros; Ferrer, Carmen; García-Reyes, Juan F; Martínez-Bueno, María Jesús; Albarracín, Micaela; Claret, María; Fernández-Alba, Amadeo R

2008-05-01

In this work, two analytical methods based on liquid chromatography coupled to electrospray time-of-flight mass spectrometry (LC/ESI-TOFMS) and tandem mass spectrometry (LC/ESI-MS/MS) are described for the identification, confirmation and quantitation of three insecticides non-authorized in the European Union (nitenpyram, isocarbophos and isofenphos-methyl) but detected in recent monitoring programmes in pepper samples. The proposed methodologies involved a sample extraction procedure using liquid-liquid partition with acetonitrile followed by a cleanup step based on dispersive solid-phase extraction. Recovery studies performed on peppers spiked at different fortification levels (10 and 50 microg kg(-1)) yielded average recoveries in the range 76-100% with relative standard deviation (RSD) (%) values below 10%. Identification, confirmation and quantitation were carried out by LC/TOFMS and LC/MS/MS using a hybrid triple quadrupole linear ion trap (QqLIT) instrument in multiple-reaction monitoring (MRM) mode. The obtained limits of quantitation (LOQs) were in the range 0.1-5 microg kg(-1), depending on each individual technique. Finally, the proposed methods were successfully applied to the analysis of suspected pepper samples. Copyright (c) 2008 John Wiley & Sons, Ltd.

LFQuant: a label-free fast quantitative analysis tool for high-resolution LC-MS/MS proteomics data.

PubMed

Zhang, Wei; Zhang, Jiyang; Xu, Changming; Li, Ning; Liu, Hui; Ma, Jie; Zhu, Yunping; Xie, Hongwei

2012-12-01

Database searching based methods for label-free quantification aim to reconstruct the peptide extracted ion chromatogram based on the identification information, which can limit the search space and thus make the data processing much faster. The random effect of the MS/MS sampling can be remedied by cross-assignment among different runs. Here, we present a new label-free fast quantitative analysis tool, LFQuant, for high-resolution LC-MS/MS proteomics data based on database searching. It is designed to accept raw data in two common formats (mzXML and Thermo RAW), and database search results from mainstream tools (MASCOT, SEQUEST, and X!Tandem), as input data. LFQuant can handle large-scale label-free data with fractionation such as SDS-PAGE and 2D LC. It is easy to use and provides handy user interfaces for data loading, parameter setting, quantitative analysis, and quantitative data visualization. LFQuant was compared with two common quantification software packages, MaxQuant and IDEAL-Q, on the replication data set and the UPS1 standard data set. The results show that LFQuant performs better than them in terms of both precision and accuracy, and consumes significantly less processing time. LFQuant is freely available under the GNU General Public License v3.0 at http://sourceforge.net/projects/lfquant/. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

LC-MS Data Processing with MAVEN: A Metabolomic Analysis and Visualization Engine

PubMed Central

Clasquin, Michelle F.; Melamud, Eugene; Rabinowitz, Joshua D.

2014-01-01

MAVEN is an open-source software program for interactive processing of LC-MS-based metabolomics data. MAVEN enables rapid and reliable metabolite quantitation from multiple reaction monitoring data or high-resolution full-scan mass spectrometry data. It automatically detects and reports peak intensities for isotope-labeled metabolites. Menu-driven, click-based navigation allows visualization of raw and analyzed data. Here we provide a User Guide for MAVEN. Step-by-step instructions are provided for data import, peak alignment across samples, identification of metabolites that differ strongly between biological conditions, quantitation and visualization of isotope-labeling patterns, and export of tables of metabolite-specific peak intensities. Together, these instructions describe a workflow that allows efficient processing of raw LC-MS data into a form ready for biological analysis. PMID:22389014

LC-MS data processing with MAVEN: a metabolomic analysis and visualization engine.

PubMed

Clasquin, Michelle F; Melamud, Eugene; Rabinowitz, Joshua D

2012-03-01

MAVEN is an open-source software program for interactive processing of LC-MS-based metabolomics data. MAVEN enables rapid and reliable metabolite quantitation from multiple reaction monitoring data or high-resolution full-scan mass spectrometry data. It automatically detects and reports peak intensities for isotope-labeled metabolites. Menu-driven, click-based navigation allows visualization of raw and analyzed data. Here we provide a User Guide for MAVEN. Step-by-step instructions are provided for data import, peak alignment across samples, identification of metabolites that differ strongly between biological conditions, quantitation and visualization of isotope-labeling patterns, and export of tables of metabolite-specific peak intensities. Together, these instructions describe a workflow that allows efficient processing of raw LC-MS data into a form ready for biological analysis.

Method and platform standardization in MRM-based quantitative plasma proteomics.

PubMed

Percy, Andrew J; Chambers, Andrew G; Yang, Juncong; Jackson, Angela M; Domanski, Dominik; Burkhart, Julia; Sickmann, Albert; Borchers, Christoph H

2013-12-16

There exists a growing demand in the proteomics community to standardize experimental methods and liquid chromatography-mass spectrometry (LC/MS) platforms in order to enable the acquisition of more precise and accurate quantitative data. This necessity is heightened by the evolving trend of verifying and validating candidate disease biomarkers in complex biofluids, such as blood plasma, through targeted multiple reaction monitoring (MRM)-based approaches with stable isotope-labeled standards (SIS). Considering the lack of performance standards for quantitative plasma proteomics, we previously developed two reference kits to evaluate the MRM with SIS peptide approach using undepleted and non-enriched human plasma. The first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). Here, these kits have been refined for practical use and then evaluated through intra- and inter-laboratory testing on 6 common LC/MS platforms. For an identical panel of 22 plasma proteins, similar concentrations were determined, regardless of the kit, instrument platform, and laboratory of analysis. These results demonstrate the value of the kit and reinforce the utility of standardized methods and protocols. The proteomics community needs standardized experimental protocols and quality control methods in order to improve the reproducibility of MS-based quantitative data. This need is heightened by the evolving trend for MRM-based validation of proposed disease biomarkers in complex biofluids such as blood plasma. We have developed two kits to assist in the inter- and intra-laboratory quality control of MRM experiments: the first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). In this paper, we report the use of these kits in intra- and inter-laboratory testing on 6 common LC/MS platforms. This article is part of a Special Issue entitled: Standardization and Quality Control in Proteomics. © 2013.

In vivo D2O labeling to quantify static and dynamic changes in cholesterol and cholesterol esters by high resolution LC/MS[S

PubMed Central

Castro-Perez, Jose; Previs, Stephen F.; McLaren, David G.; Shah, Vinit; Herath, Kithsiri; Bhat, Gowri; Johns, Douglas G.; Wang, Sheng-Ping; Mitnaul, Lyndon; Jensen, Kristian; Vreeken, Robert; Hankemeier, Thomas; Roddy, Thomas P.; Hubbard, Brian K.

2011-01-01

High resolution LC/MS-MS and LC/APPI-MS methods have been established for the quantitation of flux in the turnover of cholesterol and cholesterol ester. Attention was directed toward quantifying the monoisotopic mass (M0) and that of the singly deuterated labeled (M+1) isotope. A good degree of isotopic dynamic range has been achieved by LC/MS-MS ranging from 3-4 orders of magnitude. Correlation between the linearity of GC/MS and LC atmospheric pressure photoionization (APPI)-MS are complimentary (r2 = 0.9409). To prove the viability of this particular approach, male C57Bl/6 mice on either a high carbohydrate (HC) or a high fat (HF) diet were treated with 2H2O for 96 h. Gene expression analysis showed an increase in the activity of stearoyl-CoA desaturase (Scd1) in the HC diet up to 69-fold (P < 0.0008) compared with the HF diet. This result was supported by the quantitative flux measurement of the isotopic incorporation of 2H into the respective cholesterol and cholesterol ester (CE) pools. We concluded that it is possible to readily obtain static and dynamic measurement of cholesterol and CEs in vivo by coupling novel LC/MS methods with stable isotope-based protocols. PMID:20884843

Development of high-performance chemical isotope labeling LC-MS for profiling the human fecal metabolome.

PubMed

Xu, Wei; Chen, Deying; Wang, Nan; Zhang, Ting; Zhou, Ruokun; Huan, Tao; Lu, Yingfeng; Su, Xiaoling; Xie, Qing; Li, Liang; Li, Lanjuan

2015-01-20

Human fecal samples contain endogenous human metabolites, gut microbiota metabolites, and other compounds. Profiling the fecal metabolome can produce metabolic information that may be used not only for disease biomarker discovery, but also for providing an insight about the relationship of the gut microbiome and human health. In this work, we report a chemical isotope labeling liquid chromatography-mass spectrometry (LC-MS) method for comprehensive and quantitative analysis of the amine- and phenol-containing metabolites in fecal samples. Differential (13)C2/(12)C2-dansyl labeling of the amines and phenols was used to improve LC separation efficiency and MS detection sensitivity. Water, methanol, and acetonitrile were examined as an extraction solvent, and a sequential water-acetonitrile extraction method was found to be optimal. A step-gradient LC-UV setup and a fast LC-MS method were evaluated for measuring the total concentration of dansyl labeled metabolites that could be used for normalizing the sample amounts of individual samples for quantitative metabolomics. Knowing the total concentration was also useful for optimizing the sample injection amount into LC-MS to maximize the number of metabolites detectable while avoiding sample overloading. For the first time, dansylation isotope labeling LC-MS was performed in a simple time-of-flight mass spectrometer, instead of high-end equipment, demonstrating the feasibility of using a low-cost instrument for chemical isotope labeling metabolomics. The developed method was applied for profiling the amine/phenol submetabolome of fecal samples collected from three families. An average of 1785 peak pairs or putative metabolites were found from a 30 min LC-MS run. From 243 LC-MS runs of all the fecal samples, a total of 6200 peak pairs were detected. Among them, 67 could be positively identified based on the mass and retention time match to a dansyl standard library, while 581 and 3197 peak pairs could be putatively identified based on mass match using MyCompoundID against a Human Metabolome Database and an Evidence-based Metabolome Library, respectively. This represents the most comprehensive profile of the amine/phenol submetabolome ever detected in human fecal samples. The quantitative metabolome profiles of individual samples were shown to be useful to separate different groups of samples, illustrating the possibility of using this method for fecal metabolomics studies.

Large-Scale and Deep Quantitative Proteome Profiling Using Isobaric Labeling Coupled with Two-Dimensional LC-MS/MS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gritsenko, Marina A.; Xu, Zhe; Liu, Tao

Comprehensive, quantitative information on abundances of proteins and their post-translational modifications (PTMs) can potentially provide novel biological insights into diseases pathogenesis and therapeutic intervention. Herein, we introduce a quantitative strategy utilizing isobaric stable isotope-labelling techniques combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) for large-scale, deep quantitative proteome profiling of biological samples or clinical specimens such as tumor tissues. The workflow includes isobaric labeling of tryptic peptides for multiplexed and accurate quantitative analysis, basic reversed-phase LC fractionation and concatenation for reduced sample complexity, and nano-LC coupled to high resolution and high mass accuracy MS analysis for high confidence identification andmore » quantification of proteins. This proteomic analysis strategy has been successfully applied for in-depth quantitative proteomic analysis of tumor samples, and can also be used for integrated proteome and PTM characterization, as well as comprehensive quantitative proteomic analysis across samples from large clinical cohorts.« less

Large-Scale and Deep Quantitative Proteome Profiling Using Isobaric Labeling Coupled with Two-Dimensional LC-MS/MS.

PubMed

Gritsenko, Marina A; Xu, Zhe; Liu, Tao; Smith, Richard D

2016-01-01

Comprehensive, quantitative information on abundances of proteins and their posttranslational modifications (PTMs) can potentially provide novel biological insights into diseases pathogenesis and therapeutic intervention. Herein, we introduce a quantitative strategy utilizing isobaric stable isotope-labeling techniques combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) for large-scale, deep quantitative proteome profiling of biological samples or clinical specimens such as tumor tissues. The workflow includes isobaric labeling of tryptic peptides for multiplexed and accurate quantitative analysis, basic reversed-phase LC fractionation and concatenation for reduced sample complexity, and nano-LC coupled to high resolution and high mass accuracy MS analysis for high confidence identification and quantification of proteins. This proteomic analysis strategy has been successfully applied for in-depth quantitative proteomic analysis of tumor samples and can also be used for integrated proteome and PTM characterization, as well as comprehensive quantitative proteomic analysis across samples from large clinical cohorts.

A software suite for the generation and comparison of peptide arrays from sets of data collected by liquid chromatography-mass spectrometry.

PubMed

Li, Xiao-jun; Yi, Eugene C; Kemp, Christopher J; Zhang, Hui; Aebersold, Ruedi

2005-09-01

There is an increasing interest in the quantitative proteomic measurement of the protein contents of substantially similar biological samples, e.g. for the analysis of cellular response to perturbations over time or for the discovery of protein biomarkers from clinical samples. Technical limitations of current proteomic platforms such as limited reproducibility and low throughput make this a challenging task. A new LC-MS-based platform is able to generate complex peptide patterns from the analysis of proteolyzed protein samples at high throughput and represents a promising approach for quantitative proteomics. A crucial component of the LC-MS approach is the accurate evaluation of the abundance of detected peptides over many samples and the identification of peptide features that can stratify samples with respect to their genetic, physiological, or environmental origins. We present here a new software suite, SpecArray, that generates a peptide versus sample array from a set of LC-MS data. A peptide array stores the relative abundance of thousands of peptide features in many samples and is in a format identical to that of a gene expression microarray. A peptide array can be subjected to an unsupervised clustering analysis to stratify samples or to a discriminant analysis to identify discriminatory peptide features. We applied the SpecArray to analyze two sets of LC-MS data: one was from four repeat LC-MS analyses of the same glycopeptide sample, and another was from LC-MS analysis of serum samples of five male and five female mice. We demonstrate through these two study cases that the SpecArray software suite can serve as an effective software platform in the LC-MS approach for quantitative proteomics.

Enhanced sensitivity and multiplexing with 2D LC/MRM-MS and labeled standards for deeper and more comprehensive protein quantitation.

PubMed

Percy, Andrew J; Simon, Romain; Chambers, Andrew G; Borchers, Christoph H

2014-06-25

Mass spectrometry (MS)-based protein quantitation is increasingly being employed to verify candidate protein biomarkers. Multiple or selected reaction monitoring-mass spectrometry (MRM-MS or SRM-MS) with isotopically labeled internal standards has proven to be a successful approach in that regard, but has yet to reach its full potential in terms of multiplexing and sensitivity. Here, we report the development of a new MRM method for the quantitation of 253 disease-associated proteins (represented by 625 interference-free peptides) in 13 LC fractions. This 2D RPLC/MRM-MS approach extends the depth and breadth of the assay by 2 orders of magnitude over pre-fractionation-free assays, with 31 proteins below 10 ng/mL and 41 proteins above 10 ng/mL now quantifiable. Standard flow rates are used in both chromatographic dimensions, and up-front depletion or antibody-based enrichment is not required. The LC separations utilize high and low pH conditions, with the former employing an ammonium hydroxide-based eluent, instead of the conventional ammonium formate, resulting in improved LC column lifetime and performance. The high sensitivity (determined concentration range: 15 mg/mL to 452 pg/mL) and robustness afforded by this method makes the full MRM panel, or subsets thereof, useful for the verification of disease-associated plasma protein biomarkers in patient samples. The described research extends the breadth and depth of protein quantitation in undepleted and non-enriched human plasma by employing standard-flow 2D RPLC/MRM-MS in conjunction with a complex mixture of isotopically labeled peptide standards. The proteins quantified are mainly putative biomarkers of non-communicable (i.e., non-infectious) disease (e.g., cardiovascular or cancer), which require pre-clinical verification and validation before clinical implementation. Based on the enhanced sensitivity and multiplexing, this quantitative plasma proteomic method should prove useful in future candidate biomarker verification studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Quantitative proteomics analysis using 2D-PAGE to investigate the effects of cigarette smoke and aerosol of a prototypic modified risk tobacco product on the lung proteome in C57BL/6 mice.

PubMed

Elamin, Ashraf; Titz, Bjoern; Dijon, Sophie; Merg, Celine; Geertz, Marcel; Schneider, Thomas; Martin, Florian; Schlage, Walter K; Frentzel, Stefan; Talamo, Fabio; Phillips, Blaine; Veljkovic, Emilija; Ivanov, Nikolai V; Vanscheeuwijck, Patrick; Peitsch, Manuel C; Hoeng, Julia

2016-08-11

Smoking is associated with several serious diseases, such as lung cancer and chronic obstructive pulmonary disease (COPD). Within our systems toxicology framework, we are assessing whether potential modified risk tobacco products (MRTP) can reduce smoking-related health risks compared to conventional cigarettes. In this article, we evaluated to what extent 2D-PAGE/MALDI MS/MS (2D-PAGE) can complement the iTRAQ LC-MS/MS results from a previously reported mouse inhalation study, in which we assessed a prototypic MRTP (pMRTP). Selected differentially expressed proteins identified by both LC-MS/MS and 2D-PAGE approaches were further verified using reverse-phase protein microarrays. LC-MS/MS captured the effects of cigarette smoke (CS) on the lung proteome more comprehensively than 2D-PAGE. However, an integrated analysis of both proteomics data sets showed that 2D-PAGE data complement the LC-MS/MS results by supporting the overall trend of lower effects of pMRTP aerosol than CS on the lung proteome. Biological effects of CS exposure supported by both methods included increases in immune-related, surfactant metabolism, proteasome, and actin cytoskeleton protein clusters. Overall, while 2D-PAGE has its value, especially as a complementary method for the analysis of effects on intact proteins, LC-MS/MS approaches will likely be the method of choice for proteome analysis in systems toxicology investigations. Quantitative proteomics is anticipated to play a growing role within systems toxicology assessment frameworks in the future. To further understand how different proteomics technologies can contribute to toxicity assessment, we conducted a quantitative proteomics analysis using 2D-PAGE and isobaric tag-based LC-MS/MS approaches and compared the results produced from the 2 approaches. Using a prototypic modified risk tobacco product (pMRTP) as our test item, we show compared with cigarette smoke, how 2D-PAGE results can complement and support LC-MS/MS data, demonstrating the much lower effects of pMRTP aerosol than cigarette smoke on the mouse lung proteome. The combined analysis of 2D-PAGE and LC-MS/MS data identified an effect of cigarette smoke on the proteasome and actin cytoskeleton in the lung. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Differentiation of Panax quinquefolius grown in United States and China using LC/MS-based chromatographic fingerprinting and metabolomic approaches

USDA-ARS?s Scientific Manuscript database

American ginseng (Panax quinquefolius) is one of the most commonly used herbal medicines in the world. Discriminating between P. quinquefolius grown in different countries is difficult using the traditional quantitation methods. In this study, a liquid chromatographic mass spectrometry (LC-MS) fing...

Application of survival analysis methodology to the quantitative analysis of LC-MS proteomics data.

PubMed

Tekwe, Carmen D; Carroll, Raymond J; Dabney, Alan R

2012-08-01

Protein abundance in quantitative proteomics is often based on observed spectral features derived from liquid chromatography mass spectrometry (LC-MS) or LC-MS/MS experiments. Peak intensities are largely non-normal in distribution. Furthermore, LC-MS-based proteomics data frequently have large proportions of missing peak intensities due to censoring mechanisms on low-abundance spectral features. Recognizing that the observed peak intensities detected with the LC-MS method are all positive, skewed and often left-censored, we propose using survival methodology to carry out differential expression analysis of proteins. Various standard statistical techniques including non-parametric tests such as the Kolmogorov-Smirnov and Wilcoxon-Mann-Whitney rank sum tests, and the parametric survival model and accelerated failure time-model with log-normal, log-logistic and Weibull distributions were used to detect any differentially expressed proteins. The statistical operating characteristics of each method are explored using both real and simulated datasets. Survival methods generally have greater statistical power than standard differential expression methods when the proportion of missing protein level data is 5% or more. In particular, the AFT models we consider consistently achieve greater statistical power than standard testing procedures, with the discrepancy widening with increasing missingness in the proportions. The testing procedures discussed in this article can all be performed using readily available software such as R. The R codes are provided as supplemental materials. ctekwe@stat.tamu.edu.

Matrix effects break the LC behavior rule for analytes in LC-MS/MS analysis of biological samples

USDA-ARS?s Scientific Manuscript database

High-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are generally accepted as the preferred techniques for detecting and quantitating analytes of interest in biological matrices on the basis of the rule that one chemical compound yields one LC-...

CPTAC Evaluates Long-Term Reproducibility of Quantitative Proteomics Using Breast Cancer Xenografts | Office of Cancer Clinical Proteomics Research

Cancer.gov

Liquid chromatography tandem-mass spectrometry (LC-MS/MS)- based methods such as isobaric tags for relative and absolute quantification (iTRAQ) and tandem mass tags (TMT) have been shown to provide overall better quantification accuracy and reproducibility over other LC-MS/MS techniques. However, large scale projects like the Clinical Proteomic Tumor Analysis Consortium (CPTAC) require comparisons across many genomically characterized clinical specimens in a single study and often exceed the capability of traditional iTRAQ-based quantification.

Determination of Grayanotoxins from Rhododendron brachycarpum in Dietary Supplements and Homemade Wine by Liquid Chromatography-Quadrupole Time-of-Flight-Mass Spectrometry and Liquid Chromatography-Tandem Mass Spectrometry.

PubMed

Hwang, Taeik; Noh, Eunyoung; Jeong, Ji Hye; Park, Sung-Kwan; Shin, Dongwoo; Kang, Hoil

2018-02-28

A sensitive and specific high-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry (LC-QTOF-MS) method combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the determination of grayanotoxins I and III in dietary supplements and homemade wine. Grayanotoxins I and III were successfully extracted using solid-phase extraction cartridges, characterized by LC-QTOF-MS, and quantitated by LC-MS/MS. The LC-MS/MS calibration curves were linear over concentrations of 10-100 ng/mL (grayanotoxin I) and 20-400 ng/mL (grayanotoxin III). Grayanotoxins I and III were found in 51 foodstuffs, with quantitative determinations revealing total toxin concentrations of 18.4-101 000 ng/mL (grayanotoxin I) and 15.3-56 000 ng/mL (grayanotoxin III). The potential of the validated method was demonstrated by successful quantitative analysis of grayanotoxins I and III in dietary supplements and homemade wine; the method appears suitable for the routine detection of grayanotoxins I and III from Rhododendron brachycarpum.

Analysis of Ergot Alkaloids

PubMed Central

Crews, Colin

2015-01-01

The principles and application of established and newer methods for the quantitative and semi-quantitative determination of ergot alkaloids in food, feed, plant materials and animal tissues are reviewed. The techniques of sampling, extraction, clean-up, detection, quantification and validation are described. The major procedures for ergot alkaloid analysis comprise liquid chromatography with tandem mass spectrometry (LC-MS/MS) and liquid chromatography with fluorescence detection (LC-FLD). Other methods based on immunoassays are under development and variations of these and minor techniques are available for specific purposes. PMID:26046699

Reliable LC-MS quantitative glycomics using iGlycoMab stable isotope labeled glycans as internal standards.

PubMed

Zhou, Shiyue; Tello, Nadia; Harvey, Alex; Boyes, Barry; Orlando, Ron; Mechref, Yehia

2016-06-01

Glycans have numerous functions in various biological processes and participate in the progress of diseases. Reliable quantitative glycomic profiling techniques could contribute to the understanding of the biological functions of glycans, and lead to the discovery of potential glycan biomarkers for diseases. Although LC-MS is a powerful analytical tool for quantitative glycomics, the variation of ionization efficiency and MS intensity bias are influencing quantitation reliability. Internal standards can be utilized for glycomic quantitation by MS-based methods to reduce variability. In this study, we used stable isotope labeled IgG2b monoclonal antibody, iGlycoMab, as an internal standard to reduce potential for errors and to reduce variabililty due to sample digestion, derivatization, and fluctuation of nanoESI efficiency in the LC-MS analysis of permethylated N-glycans released from model glycoproteins, human blood serum, and breast cancer cell line. We observed an unanticipated degradation of isotope labeled glycans, tracked a source of such degradation, and optimized a sample preparation protocol to minimize degradation of the internal standard glycans. All results indicated the effectiveness of using iGlycoMab to minimize errors originating from sample handling and instruments. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Orthogonal analytical methods for botanical standardization: Determination of green tea catechins by qNMR and LC-MS/MS

PubMed Central

Napolitano, José G.; Gödecke, Tanja; Lankin, David C.; Jaki, Birgit U.; McAlpine, James B.; Chen, Shao-Nong; Pauli, Guido F.

2013-01-01

The development of analytical methods for parallel characterization of multiple phytoconstituents is essential to advance the quality control of herbal products. While chemical standardization is commonly carried out by targeted analysis using gas or liquid chromatography-based methods, more universal approaches based on quantitative 1H NMR (qHNMR) measurements are being used increasingly in the multi-targeted assessment of these complex mixtures. The present study describes the development of a 1D qHNMR-based method for simultaneous identification and quantification of green tea constituents. This approach utilizes computer-assisted 1H iterative Full Spin Analysis (HiFSA) and enables rapid profiling of seven catechins in commercial green tea extracts. The qHNMR results were cross-validated against quantitative profiles obtained with an orthogonal LC-MS/MS method. The relative strengths and weaknesses of both approaches are discussed, with special emphasis on the role of identical reference standards in qualitative and quantitative analyses. PMID:23870106

Screening Antioxidants Using LC-MS: A Case Study with Cocoa

PubMed Central

Calderón, Angela I.; Wright, Brian J.; Hurst, W. Jeffrey; van Breemen, Richard B.

2009-01-01

Oxidative stress enhances pathological processes contributing to cancer, cardiovascular disease and neurodegenerative diseases, and dietary antioxidants may counteract these deleterious processes. Since rapid methods to evaluate and compare food products for antioxidant benefits are needed, a new assay based on liquid chromatography-mass spectrometry (LC-MS) was developed for the identification and quantitative analysis of antioxidants in complex natural product samples such as food extracts. This assay is based on the comparison of electrospray LC-MS profiles of sample extracts before and after treatment with reactive oxygen species such as hydrogen peroxide or DPPH (2,2-diphenyl-1-picrylhydrazyl radical). Using this assay, methanolic extracts of cocoa powder were analyzed, and procyanidins were found to be the most potent antioxidant species. These species were identified using LC-MS, LC-MS-MS, accurate mass measurement, and comparison with reference standards. Furthermore, LC-MS was used to determine the levels of these species in cocoa samples. Catechin and epicatechin were the most abundant antioxidants followed by their dimers and trimers. The most potent antioxidants in cocoa were trimers and dimers of catechin and epicatechin, such as procyanidin B2, followed by catechin and epicatechin. This new LC-MS assay facilitates the rapid identification and then the determination of the relative antioxidant activities of individual antioxidant species in complex natural product samples and food products such as cocoa. PMID:19489609

Quantification of peptides from immunoglobulin constant and variable regions by LC-MRM MS for assessment of multiple myeloma patients.

PubMed

Remily-Wood, Elizabeth R; Benson, Kaaron; Baz, Rachid C; Chen, Y Ann; Hussein, Mohamad; Hartley-Brown, Monique A; Sprung, Robert W; Perez, Brianna; Liu, Richard Z; Yoder, Sean J; Teer, Jamie K; Eschrich, Steven A; Koomen, John M

2014-10-01

Quantitative MS assays for Igs are compared with existing clinical methods in samples from patients with plasma cell dyscrasias, for example, multiple myeloma (MM). Using LC-MS/MS data, Ig constant region peptides, and transitions were selected for LC-MRM MS. Quantitative assays were used to assess Igs in serum from 83 patients. RNA sequencing and peptide-based LC-MRM are used to define peptides for quantification of the disease-specific Ig. LC-MRM assays quantify serum levels of Igs and their isoforms (IgG1-4, IgA1-2, IgM, IgD, and IgE, as well as kappa (κ) and lambda (λ) light chains). LC-MRM quantification has been applied to single samples from a patient cohort and a longitudinal study of an IgE patient undergoing treatment, to enable comparison with existing clinical methods. Proof-of-concept data for defining and monitoring variable region peptides are provided using the H929 MM cell line and two MM patients. LC-MRM assays targeting constant region peptides determine the type and isoform of the involved Ig and quantify its expression; the LC-MRM approach has improved sensitivity compared with the current clinical method, but slightly higher inter-assay variability. Detection of variable region peptides is a promising way to improve Ig quantification, which could produce a dramatic increase in sensitivity over existing methods, and could further complement current clinical techniques. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A mass spectrometry-based method for comprehensive quantitative determination of post-transcriptional RNA modifications: the complete chemical structure of Schizosaccharomyces pombe ribosomal RNAs

PubMed Central

Taoka, Masato; Nobe, Yuko; Hori, Masayuki; Takeuchi, Aiko; Masaki, Shunpei; Yamauchi, Yoshio; Nakayama, Hiroshi; Takahashi, Nobuhiro; Isobe, Toshiaki

2015-01-01

We present a liquid chromatography–mass spectrometry (LC-MS)-based method for comprehensive quantitative identification of post-transcriptional modifications (PTMs) of RNA. We incorporated an in vitro-transcribed, heavy isotope-labeled reference RNA into a sample RNA solution, digested the mixture with a number of RNases and detected the post-transcriptionally modified oligonucleotides quantitatively based on shifts in retention time and the MS signal in subsequent LC-MS. This allowed the determination and quantitation of all PTMs in Schizosaccharomyces pombe ribosomal (r)RNAs and generated the first complete PTM maps of eukaryotic rRNAs at single-nucleotide resolution. There were 122 modified sites, most of which appear to locate at the interface of ribosomal subunits where translation takes place. We also identified PTMs at specific locations in rRNAs that were altered in response to growth conditions of yeast cells, suggesting that the cells coordinately regulate the modification levels of RNA. PMID:26013808

Analysis of Mammalian Sphingolipids by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) and Tissue Imaging Mass Spectrometry (TIMS)

PubMed Central

Sullards, M. Cameron; Liu, Ying; Chen, Yanfeng; Merrill, Alfred H.

2011-01-01

Sphingolipids are a highly diverse category of molecules that serve not only as components of biological structures but also as regulators of numerous cell functions. Because so many of the structural features of sphingolipids give rise to their biological activity, there is a need for comprehensive or “sphingolipidomic” methods for identification and quantitation of as many individual subspecies as possible. This review defines sphingolipids as a class, briefly discusses classical methods for their analysis, and focuses primarily on liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS). Recently, a set of evolving and expanding methods have been developed and rigorously validated for the extraction, identification, separation, and quantitation of sphingolipids by LC-MS/MS. Quantitation of these biomolecules is made possible via the use of an internal standard cocktail. The compounds that can be readily analyzed are free long-chain (sphingoid) bases, sphingoid base 1-phosphates, and more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides sulfatides, and novel compounds such as the 1-deoxy- and 1-(deoxymethyl)-sphingoid bases and their N-acyl-derivatives. These methods can be altered slightly to separate and quantitate isomeric species such as glucosyl/galactosylceramide. Because these techniques require the extraction of sphingolipids from their native environment, any information regarding their localization in histological slices is lost. Therefore, this review also describes methods for TIMS. This technique has been shown to be a powerful tool to determine the localization of individual molecular species of sphingolipids directly from tissue slices. PMID:21749933

HPLC and LC-MS/MS methods for determination of sodium benzoate and potassium sorbate in food and beverages: performances of local accredited laboratories via proficiency tests in Turkey.

PubMed

Gören, Ahmet C; Bilsel, Gökhan; Şimşek, Adnan; Bilsel, Mine; Akçadağ, Fatma; Topal, Kevser; Ozgen, Hasan

2015-05-15

High Performance Liquid Chromatography LC-UV and LC-MS/MS methods were developed and validated for quantitative analyses of sodium benzoate and potassium sorbate in foods and beverages. HPLC-UV and LC-MS/MS methods were compared for quantitative analyses of sodium benzoate and potassium sorbate in a representative ketchup sample. Optimisation of the methods enabled the chromatographic separation of the analytes in less than 4 min. A correlation coefficient of 0.999 was achieved over the measured calibration range for both compounds and methods (HPLC and LC-MS/MS). The uncertainty values of sodium benzoate and potassium sorbate were found as 0.199 and 0.150 mg/L by HPLC and 0.072 and 0.044 mg/L by LC-MS/MS, respectively. Proficiency testing performance of Turkish accredited laboratories between the years 2005 and 2013 was evaluated and reported herein. The aim of the proficiency testing scheme was to evaluate the performance of the laboratories, analysing benzoate and sorbate in tomato ketchup. Copyright © 2014 Elsevier Ltd. All rights reserved.

Confirmatory and quantitative analysis of beta-lactam antibiotics in bovine kidney tissue by dispersive solid-phase extraction and liquid chromatography-tandem mass spectrometry.

PubMed

Fagerquist, Clifton K; Lightfield, Alan R; Lehotay, Steven J

2005-03-01

A simple, rapid, rugged, sensitive, and specific method for the confirmation and quantitation of 10 beta-lactam antibiotics in fortified and incurred bovine kidney tissue has been developed. The method uses a simple solvent extraction, dispersive solid-phase extraction (dispersive-SPE) cleanup, and liquid chromatography-tandem mass spectrometry (LC/MS/MS) for confirmation and quantitation. Dispersive-SPE greatly simplifies and accelerates sample cleanup and improves overall recoveries compared with conventional SPE cleanup. The beta-lactam antibiotics tested were as follows: deacetylcephapirin (an antimicrobial metabolite of cephapirin), amoxicillin, desfuroylceftiofur cysteine disulfide (DCCD, an antimicrobial metabolite of ceftiofur), ampicillin, cefazolin, penicillin G, oxacillin, cloxacillin, naficillin, and dicloxacillin. Average recoveries of fortified samples were 70% or better for all beta-lactams except DCCD, which had an average recovery of 58%. The LC/MS/MS method was able to demonstrate quantitative recoveries at established tolerance levels and provide confirmatory data for unambiguous analyte identification. The method was also tested on 30 incurred bovine kidney samples obtained from the USDA Food Safety and Inspection Service, which had previously tested the samples using the approved semiquantitative microbial assay. The results from the quantitative LC/MS/MS analysis were in general agreement with the microbial assay for 23 samples although the LC/MS/MS method was superior in that it could specifically identify which beta-lactam was present and quantitate its concentration, whereas the microbial assay could only identify the type of beta-lactam present and report a concentration with respect to the microbial inhibition of a penicillin G standard. In addition, for 6 of the 23 samples, LC/MS/MS analysis detected a penicillin and a cephalosporin beta-lactam, whereas the microbial assay detected only a penicillin beta-lactam. For samples that do not fall into the "general agreement" category, the most serious discrepancy involves two samples where the LC/MS/MS method detected a violative level of a cephalosporin beta-lactam (deacetylcephapirin) in the first sample and a possibly violative level of desfuroylceftiofur in the second, whereas the microbial assay identified the two samples as having only violative levels of a penicillin beta-lactam.

Analysis of phytochemical variations in dioecious Tinospora cordifolia stems using HPLC/QTOF MS/MS and UPLC/QqQLIT -MS/MS.

PubMed

Bajpai, Vikas; Singh, Awantika; Chandra, Preeti; Negi, M P S; Kumar, Nikhil; Kumar, Brijesh

2016-01-01

The stem of dioecious Tinospora cordifolia (Menispermaceae) is a commonly used traditional Ayurvedic medicine in India having several therapeutic properties. To develop and validate LC-MS methods for the identification and simultaneous quantitation of various secondary metabolites and to study metabolomic variations in the stem of male and female plants. Ethanolic extract of stems were analysed by HPLC/ESI-QTOF-MS/MS for rapid screening of bioactive phytochemicals. High resolution MS and MS/MS in positive ESI mode were used for structural investigation of secondary metabolites. An UPLC/ESI-QqQ(LIT) -MS/MS method in MRM mode was developed and validated for the simultaneous quantitation of five bioactive alkaloids. Identification and characterisation of 36 metabolites including alkaloids, sesquiterpenes and phytoecdysteroids were performed using LC-MS and MS/MS techniques. The bioactive alkaloids such as jatrorrhizine, magnoflorine, isocorydine, palmatine and tetrahydropalmatine were successfully quantified in male and female plants. The mean abundances of magnoflorine jatrorrhizine, and oblongine were significantly (P < 0.05) higher in male plants while mean abundances of tetrahydropalmatine, norcoclaurine, and reticuline were significantly (P < 0.05) higher in female plants. Phytochemicals in the stem of male and female Tinospora cordifolia showed significant qualitative and quantitative variations. LC-MS and MS/MS methods can be used to differentiate between male and female plants based on their chemical profiles and quantities of the marker bioactive alkaloids. This chemical composition difference was also evident during vegetative stage when there were no male and female flowers. Copyright © 2015 John Wiley & Sons, Ltd.

Integrated identification, qualification and quantification strategy for pharmacokinetic profile study of Guizhi Fuling capsule in healthy volunteers

PubMed Central

Zhong, Yun-Xi; Jin, Xiao-Liang; Gu, Shi-Yin; Peng, Ying; Zhang, Ke-Rong; Ou-Yang, Bing-Chen; Wang, Yu; Xiao, Wei; Wang, Zhen-Zhong; Aa, Ji-Ye; Wang, Guang-Ji; Sun, Jian-Guo

2016-01-01

Guizhi Fuling capsule (GZFL), a traditional Chinese medicine formulation, is widely used in China to relieve pain from dysmenorrhea and is now in a Phase II clinical trial in the USA. Due to the low exposure of the five main medicative ingredients (amygdalin, cinnamic acid, gallic acid, paeoniflorin and paeonol) of GZFL in human, a strategy was built to qualitatively and quantitatively identify the possible metabolites of GZFL and to describe the pharmacokinetic profiles of GZFL in human. In this strategy, LC-Q-TOF/MS was used to identify and structurally elucidate the possible metabolites of GZFL in vivo; and a time-based metabolite-confirming step (TBMCs) was used to confirm uncertain metabolites. The simultaneously quantitation results by LC-MS/MS showed low exposure of the five medicative ingredients. According to the strategy we built, a total of 36 metabolites were found and structurally elucidated. The simultaneously semi-quantitative analysis by LC-MS/MS showed that obvious time-concentration curves could be established for 12 of the metabolites, and most of them showed a relatively higher exposure. This study provides a better understanding of the metabolic processes of GZFL in human. PMID:27527657

Integrated identification, qualification and quantification strategy for pharmacokinetic profile study of Guizhi Fuling capsule in healthy volunteers.

PubMed

Zhong, Yun-Xi; Jin, Xiao-Liang; Gu, Shi-Yin; Peng, Ying; Zhang, Ke-Rong; Ou-Yang, Bing-Chen; Wang, Yu; Xiao, Wei; Wang, Zhen-Zhong; Aa, Ji-Ye; Wang, Guang-Ji; Sun, Jian-Guo

2016-08-16

Guizhi Fuling capsule (GZFL), a traditional Chinese medicine formulation, is widely used in China to relieve pain from dysmenorrhea and is now in a Phase II clinical trial in the USA. Due to the low exposure of the five main medicative ingredients (amygdalin, cinnamic acid, gallic acid, paeoniflorin and paeonol) of GZFL in human, a strategy was built to qualitatively and quantitatively identify the possible metabolites of GZFL and to describe the pharmacokinetic profiles of GZFL in human. In this strategy, LC-Q-TOF/MS was used to identify and structurally elucidate the possible metabolites of GZFL in vivo; and a time-based metabolite-confirming step (TBMCs) was used to confirm uncertain metabolites. The simultaneously quantitation results by LC-MS/MS showed low exposure of the five medicative ingredients. According to the strategy we built, a total of 36 metabolites were found and structurally elucidated. The simultaneously semi-quantitative analysis by LC-MS/MS showed that obvious time-concentration curves could be established for 12 of the metabolites, and most of them showed a relatively higher exposure. This study provides a better understanding of the metabolic processes of GZFL in human.

Qualitative and quantitative analyses of flavonoids in Spirodela polyrrhiza by high-performance liquid chromatography coupled with mass spectrometry.

PubMed

Qiao, Xue; He, Wen-ni; Xiang, Cheng; Han, Jian; Wu, Li-jun; Guo, De-an; Ye, Min

2011-01-01

Spirodela polyrrhiza (L.) Schleid. is a traditional Chinese herbal medicine for the treatment of influenza. Despite its wide use in Chinese medicine, no report on quality control of this herb is available so far. To establish qualitative and quantitative analytical methods by high-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) for the quality control of S. polyrrhiza. The methanol extract of S. polyrrhiza was analysed by HPLC/ESI-MS(n). Flavonoids were identified by comparing with reference standards or according to their MS(n) (n = 2-4) fragmentation behaviours. Based on LC/MS data, a standardised HPLC fingerprint was established by analysing 15 batches of commercial herbal samples. Furthermore, quantitative analysis was conducted by determining five major flavonoids, namely luteolin 8-C-glucoside, apigenin 8-C-glucoside, luteolin 7-O-glucoside, apigenin 7-O-glucoside and luteolin. A total of 18 flavonoids were identified by LC/MS, and 14 of them were reported from this herb for the first time. The HPLC fingerprints contained 10 common peaks, and could differentiate good quality batches from counterfeits. The total contents of five major flavonoids in S. polyrrhiza varied significantly from 4.28 to 19.87 mg/g. Qualitative LC/MS and quantitative HPLC analytical methods were established for the comprehensive quality control of S. polyrrhiza. Copyright © 2011 John Wiley & Sons, Ltd.

Quantitation of Melatonin and N-acetylserotonin in Human Plasma by Nanoflow LC-MS/MS and Electrospray LC-MS/MS

PubMed Central

Carter, Melissa D.; Calcutt, M. Wade; Malow, Beth A.; Rose, Kristie L.; Hachey, David L.

2012-01-01

Melatonin (MEL) and its chemical precursor N-acetylserotonin (NAS) are believed to be potential biomarkers for sleep-related disorders. Measurement of these compounds, however, has proven to be difficult due to their low circulating levels, especially that of NAS. Few methods offer the sensitivity, specificity and dynamic range needed to monitor MEL and its precursors and metabolites in small blood samples, such as those obtained from pediatric patients. In support of our ongoing study to determine the safety, tolerability, and PK dosing strategies for MEL in treating insomnia in children with autism spectrum disorder, two highly sensitive LC-MS/MS assays were developed for the quantitation of MEL and precursor NAS at pg/mL levels in small volumes of human plasma. A validated electrospray ionization (ESI) method was used to quantitate high levels of MEL in PK studies and a validated nanospray (nESI) method was developed for quantitation of MEL and NAS at endogenous levels. In both assays plasma samples were processed by centrifugal membrane dialysis after addition of stable isotopic internal standards, and the components were separated by either conventional LC using a Waters SymmetryShield RP18 column (2.1×100 mm, 3.5 μm) or on a polyimide-coated, fused-silica capillary self-packed with 17 cm AquaC18 (3 μm, 125 Å). Quantitation was done using the SRM transitions m/z 233→174 and m/z 219→160 for MEL and NAS, respectively. The analytical response ratio vs. concentration curves were linear for MEL (nanoflow LC: 11.7–1165 pg/mL, LC: 1165–116500 pg/mL) and for NAS (nanoflow LC: 11.0–1095 pg/mL). PMID:22431453

Rapid and interference-free analysis of nine B-group vitamins in energy drinks using trilinear component modeling of liquid chromatography-mass spectrometry data.

PubMed

Hu, Yong; Wu, Hai-Long; Yin, Xiao-Li; Gu, Hui-Wen; Xiao, Rong; Xie, Li-Xia; Liu, Zhi; Fang, Huan; Wang, Li; Yu, Ru-Qin

2018-04-01

The aim of the present work was to develop a rapid and interference-free method based on liquid chromatography-mass spectrometry (LC-MS) for the simultaneous determination of nine B-group vitamins in various energy drinks. A smart and green strategy that modeled the three-way data array of LC-MS with second-order calibration methods based on alternating trilinear decomposition (ATLD) and alternating penalty trilinear decomposition (APTLD) algorithms was developed. By virtue of "mathematical separation" and "second-order advantage", the proposed strategy successfully solved the co-eluted peaks and unknown interferents in LC-MS analysis with the elution time less than 4.5min and simple sample preparation. Satisfactory quantitative results were obtained by the ATLD-LC-MS and APTLD-LC-MS methods for the spiked recovery assays, with the average spiked recoveries ranging from 87.2-113.9% to 92.0-111.7%, respectively. These results acquired from the proposed methods were confirmed by the LC-MS/MS method, which shows a quite good consistency with each other. All these results demonstrated that the developed chemometrics-assisted LC-MS strategy had advantages of being rapid, green, accurate and low-cost, and it could be an attractive alternative for the determination of multiple vitamins in complex food matrices, which required no laborious sample preparation, tedious condition optimization or more sophisticated instrumentations. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantitative ionspray liquid chromatographic/tandem mass spectrometric determination of reserpine in equine plasma.

PubMed

Anderson, M A; Wachs, T; Henion, J D

1997-02-01

A method based on ionspray liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed for the determination of reserpine in equine plasma. A comparison was made of the isolation of reserpine from plasma by liquid-liquid extraction and by solid-phase extraction. A structural analog, rescinnamine, was used as the internal standard. The reconstituted extracts were analyzed by ionspray LC/MS/MS in the selected reaction monitoring (SRM) mode. The calibration graph for reserpine extracted from equine plasma obtained using liquid-liquid extraction was linear from 10 to 5000 pg ml-1 and that using solid-phase extraction from 100 to 5000 pg ml-1. The lower level of quantitation (LLQ) using liquid-liquid and solid-phase extraction was 50 and 200 pg ml-1, respectively. The lower level of detection for reserpine by LC/MS/MS was 10 pg ml-1. The intra-assay accuracy did not exceed 13% for liquid-liquid and 12% for solid-phase extraction. The recoveries for the LLQ were 68% for liquid-liquid and 58% for solid-phase extraction.

A Proof of Concept to Bridge the Gap between Mass Spectrometry Imaging, Protein Identification and Relative Quantitation: MSI~LC-MS/MS-LF.

PubMed

Théron, Laëtitia; Centeno, Delphine; Coudy-Gandilhon, Cécile; Pujos-Guillot, Estelle; Astruc, Thierry; Rémond, Didier; Barthelemy, Jean-Claude; Roche, Frédéric; Feasson, Léonard; Hébraud, Michel; Béchet, Daniel; Chambon, Christophe

2016-10-26

Mass spectrometry imaging (MSI) is a powerful tool to visualize the spatial distribution of molecules on a tissue section. The main limitation of MALDI-MSI of proteins is the lack of direct identification. Therefore, this study focuses on a MSI~LC-MS/MS-LF workflow to link the results from MALDI-MSI with potential peak identification and label-free quantitation, using only one tissue section. At first, we studied the impact of matrix deposition and laser ablation on protein extraction from the tissue section. Then, we did a back-correlation of the m / z of the proteins detected by MALDI-MSI to those identified by label-free quantitation. This allowed us to compare the label-free quantitation of proteins obtained in LC-MS/MS with the peak intensities observed in MALDI-MSI. We managed to link identification to nine peaks observed by MALDI-MSI. The results showed that the MSI~LC-MS/MS-LF workflow (i) allowed us to study a representative muscle proteome compared to a classical bottom-up workflow; and (ii) was sparsely impacted by matrix deposition and laser ablation. This workflow, performed as a proof-of-concept, suggests that a single tissue section can be used to perform MALDI-MSI and protein extraction, identification, and relative quantitation.

A Proof of Concept to Bridge the Gap between Mass Spectrometry Imaging, Protein Identification and Relative Quantitation: MSI~LC-MS/MS-LF

PubMed Central

Théron, Laëtitia; Centeno, Delphine; Coudy-Gandilhon, Cécile; Pujos-Guillot, Estelle; Astruc, Thierry; Rémond, Didier; Barthelemy, Jean-Claude; Roche, Frédéric; Feasson, Léonard; Hébraud, Michel; Béchet, Daniel; Chambon, Christophe

2016-01-01

Mass spectrometry imaging (MSI) is a powerful tool to visualize the spatial distribution of molecules on a tissue section. The main limitation of MALDI-MSI of proteins is the lack of direct identification. Therefore, this study focuses on a MSI~LC-MS/MS-LF workflow to link the results from MALDI-MSI with potential peak identification and label-free quantitation, using only one tissue section. At first, we studied the impact of matrix deposition and laser ablation on protein extraction from the tissue section. Then, we did a back-correlation of the m/z of the proteins detected by MALDI-MSI to those identified by label-free quantitation. This allowed us to compare the label-free quantitation of proteins obtained in LC-MS/MS with the peak intensities observed in MALDI-MSI. We managed to link identification to nine peaks observed by MALDI-MSI. The results showed that the MSI~LC-MS/MS-LF workflow (i) allowed us to study a representative muscle proteome compared to a classical bottom-up workflow; and (ii) was sparsely impacted by matrix deposition and laser ablation. This workflow, performed as a proof-of-concept, suggests that a single tissue section can be used to perform MALDI-MSI and protein extraction, identification, and relative quantitation. PMID:28248242

Quantitative Determination of Perfluorochemicals and Fluorotelomer Alcohols in Plants from Biosolid-Amended Fields using LC/MS/MS and GC/MS

EPA Science Inventory

Analytical methods for determining perfluorochemicals (PFCs) and fluorotelomer alcohols (FTOHs) in plants using liquid chromatography/tandem mass spectrometry (LC/MS/MS) and gas chromatography/mass spectrometry (GC/MS) were developed, and applied to quantify a suite of analytes i...

A quantitative method for residues of macrolide antibiotics in porcine kidney by liquid chromatography/tandem mass spectrometry.

PubMed

Dickson, Leslie C; O'Byrne, Collin; Chan, Wayne

2012-01-01

An LC/MS/MS-based multiresidue quantitative method was developed for the macrolides erythromycin A, neospiramycin I, oleandomycin, spiramycin I, tilmicosin, and tylosin A in porcine kidney tissues. The Canadian Food Inspection Agency (CFIA) had as part of its analytical scope an LC/UV method for quantification of residues of two macrolide antibiotics, tilmicosin and tylosin A, in the kidney, liver, and muscle of cattle, swine, and poultry. The method could not reliably detect concentrations below 10 microg/kg. To increase the scope of the CFIA's analytical capabilities, a sensitive multiresidue quantitative method for macrolide residues in food animal tissues was required. Porcine kidney samples were extracted with acetonitrile and alkaline buffer and cleaned-up using silica-based C18 SPE cartridges. Sample extracts were analyzed using LC/MS/MS with positive electrospray ionization. Fitness for purpose was verified in a single-laboratory validation study using a second analyst. The working analytical range was 5 to 50 microg/kg. LOD and LOQ were 0.5 to 0.6 microg/kg and 1.5 to 3.0 microg/kg, respectively. Limits of identification were 0.5 to 2.0 microg/kg. Relative intermediate precisions were 8 to 17%. Average absolute recoveries were 68 to 76%.

Application of HRAM screening and LC-MS/MS confirmation of active pharmaceutical ingredient in "natural" herbal supplements.

PubMed

Pascali, Jennifer P; Fais, Paolo; Vaiano, Fabio; Bertol, Elisabetta

2018-05-01

The growing market of herbal remedies worldwide could pose severe problems to consumers' health due to the possible presence of potentially harmful, undeclared synthetic substances or analogues of prescription drugs. The present work shows a simple but effective approach to unequivocally identify synthetic anorectic compounds in allegedly 'natural' herbal extracts, by exploiting liquid chromatography/time of flight (Q-TOF LC/MS) technology coupled to liquid chromatography/triple quadrupole (LC-MS/MS) confirmation and quantitation. The procedure was applied to five tea herbal extracts and pills sold as coadjutant for weigh loss. The method exploited liquid-liquid sample extraction (LLE) and separation in a C18 (2.1mm×150mm, 1.8μm) column. QTOF acquisitions were carried out both in scan mode and all ion MS/MS mode and results were obtained after search against ad hoc prepared library. Sibutramine, 4-hydroxyamphetamine, caffeine and theophylline were preliminary identified samples. Confirmation and quantitation of the preliminary identified compounds were obtained in LC-MS/MS after preparation of appropriated standards. Sibutramine, caffeine and theophylline were finally confirmed and quantitate. Copyright © 2018 Elsevier B.V. All rights reserved.

Development and validation of a LC-MS method for quantitation of ergot alkaloids in lateral saphenous vein tissue

USDA-ARS?s Scientific Manuscript database

A liquid chromatography-mass spectrometry (LC/MS) method for simultaneous quantitation of seven ergot alkaloids (lysergic acid, ergonovine, ergovaline, ergocornine, ergotamine, ergocryptine and ergocrystine) in vascular tissue was developed and validated. Reverse-phase chromatography, coupled to an...

Rapid Quantification of 25-Hydroxyvitamin D3 in Human Serum by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

NASA Astrophysics Data System (ADS)

Qi, Yulin; Müller, Miriam; Stokes, Caroline S.; Volmer, Dietrich A.

2018-04-01

LC-MS/MS is widely utilized today for quantification of vitamin D in biological fluids. Mass spectrometric assays for vitamin D require very careful method optimization for precise and interference-free, accurate analyses however. Here, we explore chemical derivatization and matrix-assisted laser desorption/ionization (MALDI) as a rapid alternative for quantitative measurement of 25-hydroxyvitamin D3 in human serum, and compare it to results from LC-MS/MS. The method implemented an automated imaging step of each MALDI spot, to locate areas of high intensity, avoid sweet spot phenomena, and thus improve precision. There was no statistically significant difference in vitamin D quantification between the MALDI-MS/MS and LC-MS/MS: mean ± standard deviation for MALDI-MS—29.4 ± 10.3 ng/mL—versus LC-MS/MS—30.3 ± 11.2 ng/mL (P = 0.128)—for the sum of the 25-hydroxyvitamin D epimers. The MALDI-based assay avoided time-consuming chromatographic separation steps and was thus much faster than the LC-MS/MS assay. It also consumed less sample, required no organic solvents, and was readily automated. In this proof-of-concept study, MALDI-MS readily demonstrated its potential for mass spectrometric quantification of vitamin D compounds in biological fluids.

Design, implementation and multisite evaluation of a system suitability protocol for the quantitative assessment of instrument performance in liquid chromatography-multiple reaction monitoring-MS (LC-MRM-MS).

PubMed

Abbatiello, Susan E; Mani, D R; Schilling, Birgit; Maclean, Brendan; Zimmerman, Lisa J; Feng, Xingdong; Cusack, Michael P; Sedransk, Nell; Hall, Steven C; Addona, Terri; Allen, Simon; Dodder, Nathan G; Ghosh, Mousumi; Held, Jason M; Hedrick, Victoria; Inerowicz, H Dorota; Jackson, Angela; Keshishian, Hasmik; Kim, Jong Won; Lyssand, John S; Riley, C Paige; Rudnick, Paul; Sadowski, Pawel; Shaddox, Kent; Smith, Derek; Tomazela, Daniela; Wahlander, Asa; Waldemarson, Sofia; Whitwell, Corbin A; You, Jinsam; Zhang, Shucha; Kinsinger, Christopher R; Mesri, Mehdi; Rodriguez, Henry; Borchers, Christoph H; Buck, Charles; Fisher, Susan J; Gibson, Bradford W; Liebler, Daniel; Maccoss, Michael; Neubert, Thomas A; Paulovich, Amanda; Regnier, Fred; Skates, Steven J; Tempst, Paul; Wang, Mu; Carr, Steven A

2013-09-01

Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation <0.15, peak width coefficient of variation <0.15, standard deviation of RT <0.15 min (9 s), and the RT drift <0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the use and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to ensure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread use of MRM-MS technology by the basic biomedical and clinical laboratory research communities.

Design, Implementation and Multisite Evaluation of a System Suitability Protocol for the Quantitative Assessment of Instrument Performance in Liquid Chromatography-Multiple Reaction Monitoring-MS (LC-MRM-MS)*

PubMed Central

Abbatiello, Susan E.; Mani, D. R.; Schilling, Birgit; MacLean, Brendan; Zimmerman, Lisa J.; Feng, Xingdong; Cusack, Michael P.; Sedransk, Nell; Hall, Steven C.; Addona, Terri; Allen, Simon; Dodder, Nathan G.; Ghosh, Mousumi; Held, Jason M.; Hedrick, Victoria; Inerowicz, H. Dorota; Jackson, Angela; Keshishian, Hasmik; Kim, Jong Won; Lyssand, John S.; Riley, C. Paige; Rudnick, Paul; Sadowski, Pawel; Shaddox, Kent; Smith, Derek; Tomazela, Daniela; Wahlander, Asa; Waldemarson, Sofia; Whitwell, Corbin A.; You, Jinsam; Zhang, Shucha; Kinsinger, Christopher R.; Mesri, Mehdi; Rodriguez, Henry; Borchers, Christoph H.; Buck, Charles; Fisher, Susan J.; Gibson, Bradford W.; Liebler, Daniel; MacCoss, Michael; Neubert, Thomas A.; Paulovich, Amanda; Regnier, Fred; Skates, Steven J.; Tempst, Paul; Wang, Mu; Carr, Steven A.

2013-01-01

Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation <0.15, peak width coefficient of variation <0.15, standard deviation of RT <0.15 min (9 s), and the RT drift <0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the use and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to ensure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread use of MRM-MS technology by the basic biomedical and clinical laboratory research communities. PMID:23689285

How much separation for LC-MS/MS quantitative bioanalysis of drugs and metabolites?

PubMed

Tan, Aimin; Fanaras, John C

2018-05-01

LC-MS/MS has been the dominant analytical technology for quantitative bioanalysis of drugs and metabolites for more than two decades. Despite this, a very fundamental question like how much separation is required for LC-MS/MS quantitative bioanalysis of drugs and metabolites has not been adequately addressed. Some think that no or only very limited separation is necessary thanks to the unparalleled selectivity offered by tandem mass spectrometry. Others think that the more separation, the better, because of the potential detrimental impact of matrix effect (ion suppression or enhancement). Still others just use a rule-of-thumb approach by keeping the adjusted retention/capacity factor always between 2 and 5. The purpose of this article is to address this fundamental question through rational thinking together with various real case examples drawn from regulated bioanalytical laboratories. Copyright © 2018 Elsevier B.V. All rights reserved.

Automated on-line SPE LC-MS/MS method to quantitate 6beta-hydroxycortisol and cortisol in human urine: use of the 6beta-hydroxycortisol to cortisol ratio as an indicator of CYP3A4 activity.

PubMed

Barrett, Yu Chen; Akinsanya, Billy; Chang, Shu-Ying; Vesterqvist, Ole

2005-07-25

A sensitive method for quantitation of urinary 6beta-hydroxycortisol (6beta-HC) and cortisol using on-line SPE and LC-MS/MS was developed and validated. Human urine samples were injected directly onto an on-line solid phase extraction apparatus, Prospekt-2, followed by HPLC separation and electrospray triple quadrupole LC-MS/MS detection. The inter-day precision for the 6beta-HC:cortisol ratio was 7-9%. The lower limit of quantitation was 1 and 0.2 ng/mL for 6beta-HC and cortisol, respectively. Using the method we observed a diurnal variation on the 6beta-HC:cortisol ratio in healthy volunteers with the maximal ratio observed in the 2-10 pm urine collection period.

Non-gel Based Proteomics to Study Steroid Receptor Agonists in the Fathead Minnow

EPA Science Inventory

Toxicoproteomics is an emerging field that is greatly enabled by non-gel based methods using LC MS/MS for biomarker discovery and characterization for endocrine disrupting chemicals. Using iTRAQ (isobaric tagging for relative and absolute quantitation), we quantified a diverse r...

Development of liquid chromatography/mass spectrometry based screening assay for PfTrxR inhibitors using relative quantitation of intact thioredoxin.

PubMed

Munigunti, Ranjith; Calderón, Angela I

2012-09-15

Plasmodium falciparum (Pf) thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin disulfide (Trx-S(2)) to thioredoxin dithiol (Trx-(SH)(2)) that is essential for antioxidant defense mechanism and DNA synthesis in the parasite and is a validated drug target for new antimalarial agents. In this study, we have developed a liquid chromatography/mass spectrometry (LC/MS)-based functional assay to identify inhibitors of PfTrxR by quantifying the product formed (Trx-(SH)(2)) in the enzymatic reaction. Relative quantitation of the reaction product (intact Trx-(SH)(2)) was carried out using an Agilent 6520 QTOF mass spectrometer equipped with a positive mode electrospray ionization (ESI) source. The calibration curve prepared for Trx-(SH)(2) at concentrations ranging from 1.8 to 116.5 µg/mL was linear (R(2) >0.998). The limit of detection (LOD) and limit of quantification (LOQ) of Trx-(SH)(2) were at 0.45 and 1.8 µg/mL respectively. To validate the developed functional assay we have screened reference compounds 1, 2 and 3 for their PfTrxR inhibitory activity and ten natural compounds (at 10 μM) which were earlier identified as ligands of PfTrxR by a UF-LC/MS based binding assay. The developed LC/MS-based functional assay for identification of inhibitors of PfTrxR is a sensitive and reliable method that is also amendable for high-throughput format. This is the first representation of a relative quantitation of intact Trx-(SH)(2) using LC/MS. Copyright © 2012 John Wiley & Sons, Ltd.

Determination of formetanate hydrochloride in fruit samples using liquid chromatography-mass selective detection or -tandem mass spectrometry.

PubMed

Podhorniak, Lynda V; Kamel, Alaa; Rains, Diane M

2010-05-26

A rapid multiresidue method that captures residues of the insecticide formetanate hydrochloride (FHCl) in selected fruits is described. The method was used to provide residue data for dietary exposure determinations of FHCl. Using an acetonitrile extraction with a dispersive cleanup based on AOAC International method 2007.01, also known as QuEChERS, which was further modified and streamlined, thousands of samples were successfully analyzed for FHCl residues. FHCl levels were determined both by liquid chromatography-single-stage mass spectrometry (LC-MS) and ultraperformance liquid chromatography (UPLC)-tandem mass spectrometry (LC-MS/MS). The target limit of detection (LOD) and the limit of quantitation (LOQ) achieved for FHCl were 3.33 and 10 ng/g, respectively, with LC-MS and 0.1 and 0.3 ng/g, respectively, with LC-MS/MS. Recoveries at these previously unpublished levels ranged from 95 to 109%. A set of 20-40 samples can be prepared in one working day by two chemists.

On-line capillary electrophoresis/laser-induced fluorescence/mass spectrometry analysis of glycans labeled with Teal™ fluorescent dye using an electrokinetic sheath liquid pump-based nanospray ion source.

PubMed

Khan, Shaheer; Liu, Jenkuei; Szabo, Zoltan; Kunnummal, Baburaj; Han, Xiaorui; Ouyang, Yilan; Linhardt, Robert J; Xia, Qiangwei

2018-06-15

N-linked glycan analysis of recombinant therapeutic proteins, such as monoclonal antibodies, Fc-fusion proteins, and antibody-drug conjugates, provides valuable information regarding protein therapeutics glycosylation profile. Both qualitative identification and quantitative analysis of N-linked glycans on recombinant therapeutic proteins are critical analytical tasks in the biopharma industry during the development of a biotherapeutic. Currently, such analyses are mainly carried out using capillary electrophoresis/laser-induced fluorescence (CE/LIF), liquid chromatography/fluorescence (LC/FLR), and liquid chromatography/fluorescence/mass spectrometry (LC/FLR/MS) technologies. N-linked glycans are first released from glycoproteins by enzymatic digestion, then labeled with fluorescence dyes for subsequent CE or LC separation, and LIF or MS detection. Here we present an on-line CE/LIF/MS N-glycan analysis workflow that incorporates the fluorescent Teal™ dye and an electrokinetic pump-based nanospray sheath liquid capillary electrophoresis/mass spectrometry (CE/MS) ion source. Electrophoresis running buffer systems using ammonium acetate and ammonium hydroxide were developed for the negative ion mode CE/MS analysis of fluorescence-labeled N-linked glycans. Results show that on-line CE/LIF/MS analysis can be readily achieved using this versatile CE/MS ion source on common CE/MS instrument platforms. This on-line CE/LIF/MS method using Teal™ fluorescent dye and electrokinetic pump-based nanospray sheath liquid CE/MS coupling technology holds promise for on-line quantitation and identification of N-linked glycans on recombinant therapeutic proteins. Copyright © 2018 John Wiley & Sons, Ltd.

Identification and Quantification of Gingerols and Related Compounds in Ginger Dietary Supplements Using High Performance Liquid Chromatography-Tandem Mass Spectrometry

PubMed Central

TAO, YI; LI, WENKUI; LIANG, WENZHONG; VAN BREEMEN, RICHARD B.

2009-01-01

Dietary supplements containing preparations of ginger roots/rhizomes (Zingiber officinale Roscoe) are being used by consumers, and clinical trials using ginger dietary supplements have been carried out to evaluate their anti-inflammatory or anti-emetic properties with inconsistent results. Chemical standardization of these products is needed for quality control and to facilitate the design of clinical trials and the evaluation of data from these studies. To address this issue, methods based on liquid chromatography-tandem mass spectrometry (LC-MS-MS) were developed for the detection, characterization and quantitative analysis of gingerol-related compounds in botanical dietary supplements containing ginger roots/rhizomes. During negative ion electrospray with collision induced-dissociation, the cleavage of the C4-C5 bond with a neutral loss of 194 u and benzylic cleavage leading to the neutral loss of 136 u were found to be class characteristic fragmentation patterns of the pharmacologically active gingerols or shogaols, respectively. Based on these results, an assay using LC-MS-MS with neutral loss scanning (loss of 194 u or 136 u) was developed that is suitable for the fingerprinting of ginger dietary supplements based on the selective detection of gingerols, shogaols, paradols, and gingerdiones. In addition, a quantitative assay based on LC-MS-MS with selected reaction monitoring was developed for the quantitative analysis of 6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8-shogaol, and 10-shogaol in ginger dietary supplements. After method validation, the quantities of these compounds in three commercially available ginger dietary supplements were determined. This assay showed excellent sensitivity, accuracy and precision and may be used to address the need for quality control and standardization of ginger dietary supplements. PMID:19817455

Tracing the metabolism of HT-2 toxin and T-2 toxin in barley by isotope-assisted untargeted screening and quantitative LC-HRMS analysis

USDA-ARS?s Scientific Manuscript database

An extensive study of the metabolism of the type-A trichothecene mycotoxins HT-2 toxin and T-2 toxin in barley using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) is reported. A recently developed untargeted approach based on stable isotopic labelling, LC-Orbitrap-MS a...

Evaluation of peptide adsorption-controlled liquid chromatography-tandem mass spectrometric (PAC-LC-MS/MS) method for simple and simultaneous quantitation of amyloid β 1-38, 1-40, 1-42 and 1-43 peptides in dog cerebrospinal fluid.

PubMed

Goda, Ryoya; Kobayashi, Nobuhiro

2012-05-01

To evaluate the usefulness of the peptide adsorption-controlled liquid chromatography-tandem mass spectrometry (PAC-LC-MS/MS) for reproducible measurement of peptides in biological fluids, simultaneous quantitation of amyloid β 1-38, 1-40, 1-42 and 1-43 peptides (Aβ38, Aβ40, Aβ42 and Aβ43) in dog cerebrospinal fluid (CSF) was tried. Each stable isotope labeled Aβ was used as the internal standard to minimize the influence of CSF matrix on the reproducible Aβ quantitation. To reduce a loss of Aβ during the pretreatment procedures, the dog CSF diluted by water-acetic acid-methanol (2:6:1, v/v/v) was loaded on PAC-LC-MS/MS directly. Quantification of the Aβ in the diluted dog CSF was carried out using multiple reaction monitoring (MRM) mode. The [M+5H(5+)] and b(5+) ion fragment of each peptide were chosen as the precursor and product ions for MRM transitions of each peptide. The calibration curves were drawn from Aβ standard calibration solutions using PAC-LC-MS/MS. Analysis of dog CSF samples suggests that the basal concentration of Aβ38, Aβ40, Aβ42 and Aβ43 in dog CSF is approximately 300, 900, 200 and 30 pM, respectively. This is the first time Aβ concentrations in dog CSF have been reported. Additionally, the evaluation of intra- and inter-day reproducibility of analysis of Aβ standard solution, the freeze-thaw stability and the room temperature stability of Aβ standard solution suggest that the PAC-LC-MS/MS method enables reproducible Aβ quantitation. Copyright © 2012 Elsevier B.V. All rights reserved.

Predicting ESI/MS Signal Change for Anions in Different Solvents.

PubMed

Kruve, Anneli; Kaupmees, Karl

2017-05-02

LC/ESI/MS is a technique widely used for qualitative and quantitative analysis in various fields. However, quantification is currently possible only for compounds for which the standard substances are available, as the ionization efficiency of different compounds in ESI source differs by orders of magnitude. In this paper we present an approach for quantitative LC/ESI/MS analysis without standard substances. This approach relies on accurately predicting the ionization efficiencies in ESI source based on a model, which uses physicochemical parameters of analytes. Furthermore, the model has been made transferable between different mobile phases and instrument setups by using a suitable set of calibration compounds. This approach has been validated both in flow injection and chromatographic mode with gradient elution.

Development and validation of an LC-MS/MS method for quantification of cyclic guanosine 3',5'-monophosphate (cGMP) in clinical applications: a comparison with a EIA method.

PubMed

Zhang, Yanhua; Dufield, Dawn; Klover, Jon; Li, Wenlin; Szekely-Klepser, Gabriella; Lepsy, Christopher; Sadagopan, Nalini

2009-02-15

An LC-MS/MS method was developed and validated to quantify endogenous cyclic guanosine 3',5'-monophosphate (cGMP) in human plasma. The LC-MS/MS and competitive enzyme immunoassay (EIA) assays were compared. cGMP concentrations of 20 human plasma samples were measured by both methods. For the MS-based assay, plasma samples were subjected to a simple protein precipitation procedure by acetonitrile prior to analysis by electrospray ionization LC-MS/MS. De-protonated analytes generated in negative ionization mode were monitored through multiple reaction monitoring (MRM). A stable isotope-labeled internal standard, (13)C(10),(15)N(5)-cGMP, which was biosynthesized in-house, was used in the LC-MS/MS method. The competitive EIA was validated using a commercially available cGMP fluorescence assay kit. The intra-assay accuracy and precision for MS-based assay for cGMP were 6-10.1% CV and -3.6% to 7.3% relative error (RE), respectively, while inter-assay precision and accuracy were 5.6-8.1% CV and -2.1% to 6.3% RE, respectively. The intra-assay accuracy and precision for EIA were 17.9-27.1% CV and -4.9% to 24.5% RE, respectively, while inter-assay precision and accuracy were 15.1-39.5% CV and -30.8% to 4.37% RE, respectively. Near the lower limits of detection, there was little correlation between the cGMP concentration values in human plasma generated by these two methods (R(2)=0.197, P=0.05). Overall, the MS-based assay offered better selectivity, recovery, precision and accuracy over a linear range of 0.5-20ng/mL. The LC-MS/MS method provides an effective tool for the quantitation of cGMP to support clinical mechanistic studies of curative pharmaceuticals.

Metabolic profiling of Vitex agnus castus leaves, fruits and sprouts: analysis by LC/ESI/(QqQ)MS and (HR) LC/ESI/(Orbitrap)/MS n.

PubMed

Mari, Angela; Montoro, Paola; D'Urso, Gilda; Macchia, Mario; Pizza, Cosimo; Piacente, Sonia

2015-01-01

Food supplements based on Vitex agnus castus L. (Verbenaceae) fruits, also known as chasteberry, are routinely used by women against somatic and psychic premenstrual symptoms such as depression, sadness or irritability. With the aim of highlighting the differences in the chemical profiles of cultivated fruits and different parts of wild plants (fruits, leaves and sprouts) of V. agnus castus, a method concerning with the quali-quantitative study of the derived hydroalcoholic extracts was carried out by using high-performance liquid chromatography coupled to electrospray negative ionization Orbitrap multicollisional high resolution mass spectrometry (LC/ESI/(Orbitrap)MS(n)) and high-performance liquid chromatography coupled to electrospray negative ionization triple quadrupole tandem mass spectrometry (LC/ESI/(QqQ)MS) in multiple reaction monitoring (MRM) mode. Copyright © 2014 Elsevier B.V. All rights reserved.

Determination of Perfluorocarboxylic Acids in Sludge

EPA Science Inventory

Methods were developed for the extraction from wastewater-treatment sludge and quantitation by LC/MS/MS of perfluorocarboxylic acids (PFCAs, C6 to C12), 7-3 fluorotelomer carboxylic acid (7-3 FTCA) and 8-2 fluorotelomer 2-unsaturated carboxylic acid (8-2 FTUCA) using LC/MS/MS.

Matrix effect and correction by standard addition in quantitative liquid chromatographic-mass spectrometric analysis of diarrhetic shellfish poisoning toxins.

PubMed

Ito, Shinya; Tsukada, Katsuo

2002-01-11

An evaluation of the feasibility of liquid chromatography-mass spectrometry (LC-MS) with atmospheric pressure ionization was made for quantitation of four diarrhetic shellfish poisoning toxins, okadaic acid, dinophysistoxin-1, pectenotoxin-6 and yessotoxin in scallops. When LC-MS was applied to the analysis of scallop extracts, large signal suppressions were observed due to coeluting substances from the column. To compensate for these matrix signal suppressions, the standard addition method was applied. First, the sample was analyzed and then the sample involving the addition of calibration standards is analyzed. Although this method requires two LC-MS runs per analysis, effective correction of quantitative errors was found.

Lipid Informed Quantitation and Identification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kevin Crowell, PNNL

2014-07-21

LIQUID (Lipid Informed Quantitation and Identification) is a software program that has been developed to enable users to conduct both informed and high-throughput global liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based lipidomics analysis. This newly designed desktop application can quickly identify and quantify lipids from LC-MS/MS datasets while providing a friendly graphical user interface for users to fully explore the data. Informed data analysis simply involves the user specifying an electrospray ionization mode, lipid common name (i.e. PE(16:0/18:2)), and associated charge carrier. A stemplot of the isotopic profile and a line plot of the extracted ion chromatogram are also provided to showmore » the MS-level evidence of the identified lipid. In addition to plots, other information such as intensity, mass measurement error, and elution time are also provided. Typically, a global analysis for 15,000 lipid targets« less

Quantitation of melatonin and n-acetylserotonin in human plasma by nanoflow LC-MS/MS and electrospray LC-MS/MS.

PubMed

Carter, Melissa D; Calcutt, M Wade; Malow, Beth A; Rose, Kristie L; Hachey, David L

2012-03-01

Melatonin (MEL) and its chemical precursor N-acetylserotonin (NAS) are believed to be potential biomarkers for sleep-related disorders. Measurement of these compounds, however, has proven to be difficult due to their low circulating levels, especially that of NAS. Few methods offer the sensitivity, specificity and dynamic range needed to monitor MEL and its precursors and metabolites in small blood samples, such as those obtained from pediatric patients. In support of our ongoing study to determine the safety, tolerability and PK dosing strategies for MEL in treating insomnia in children with autism spectrum disorder, two highly sensitive LC-MS/MS assays were developed for the quantitation of MEL and precursor NAS at pg/mL levels in small volumes of human plasma. A validated electrospray ionization (ESI) method was used to quantitate high levels of MEL in PK studies, and a validated nanospray (nESI) method was developed for quantitation of MEL and NAS at endogenous levels. In both assays, plasma samples were processed by centrifugal membrane dialysis after addition of stable isotopic internal standards, and the components were separated by either conventional LC using a Waters SymmetryShield RP18 column (2.1 × 100  mm, 3.5 µm) or on a polyimide-coated, fused-silica capillary self-packed with 17 cm AquaC18 (3 µm, 125 Å). Quantitation was done using the SRM transitions m/z 233 → 174 and m/z 219 → 160 for MEL and NAS, respectively. The analytical response ratio versus concentration curves were linear for MEL (nanoflow LC: 11.7-1165  pg/mL, LC: 1165-116,500  pg/mL) and for NAS (nanoflow LC: 11.0-1095  pg/mL). Copyright © 2012 John Wiley & Sons, Ltd.

An FD-LC-MS/MS Proteomic Strategy for Revealing Cellular Protein Networks: A Conditional Superoxide Dismutase 1 Knockout Cells

PubMed Central

Ichibangase, Tomoko; Sugawara, Yasuhiro; Yamabe, Akio; Koshiyama, Akiyo; Yoshimura, Akari; Enomoto, Takemi; Imai, Kazuhiro

2012-01-01

Systems biology aims to understand biological phenomena in terms of complex biological and molecular interactions, and thus proteomics plays an important role in elucidating protein networks. However, many proteomic methods have suffered from their high variability, resulting in only showing altered protein names. Here, we propose a strategy for elucidating cellular protein networks based on an FD-LC-MS/MS proteomic method. The strategy permits reproducible relative quantitation of differences in protein levels between different cell populations and allows for integration of the data with those obtained through other methods. We demonstrate the validity of the approach through a comparison of differential protein expression in normal and conditional superoxide dismutase 1 gene knockout cells and believe that beginning with an FD-LC-MS/MS proteomic approach will enable researchers to elucidate protein networks more easily and comprehensively. PMID:23029042

Quinone-based stable isotope probing for assessment of 13C substrate-utilizing bacteria

NASA Astrophysics Data System (ADS)

Kunihiro, Tadao; Katayama, Arata; Demachi, Toyoko; Veuger, Bart; Boschker, Henricus T. S.; van Oevelen, Dick

2015-04-01

In this study, we attempted to establish quinone-stable-isotope probing (SIP) technique to link substrate-utilizing bacterial group to chemotaxonomic group in bacterial community. To identify metabolically active bacterial group in various environments, SIP techniques combined with biomarkers have been widely utilized as an attractive method for environmental study. Quantitative approaches of the SIP technique have unique advantage to assess substrate-incorporation into bacteria. As a most major quantitative approach, SIP technique based on phospholipid-derived fatty acids (PLFA) have been applied to simultaneously assess substrate-incorporation rate into bacteria and microbial community structure. This approach is powerful to estimate the incorporation rate because of the high sensitivity due to the detection by a gas chromatograph-combustion interface-isotope ratio mass spectrometer (GC-c-IRMS). However, its phylogenetic resolution is limited by specificity of a compound-specific marker. We focused on respiratory quinone as a biomarker. Our previous study found a good correlation between concentrations of bacteria-specific PLFAs and quinones over several orders of magnitude in various marine sediments, and the quinone method has a higher resolution (bacterial phylum level) for resolving differences in bacterial community composition more than that of bacterial PLFA. Therefore, respiratory quinones are potentially good biomarkers for quantitative approaches of the SIP technique. The LC-APCI-MS method as molecular-mass based detection method for quinone was developed and provides useful structural information for identifying quinone molecular species in environmental samples. LC-MS/MS on hybrid triple quadrupole/linear ion trap, which enables to simultaneously identify and quantify compounds in a single analysis, can detect high molecular compounds with their isotope ions. Use of LC-MS/MS allows us to develop quinone-SIP based on molecular mass differences due to 13C abundance in the quinone. In this study, we verified carbon stable isotope of quinone compared with bulk carbon stable isotope of bacterial culture. Results indicated a good correlation between carbon stable isotope of quinone compared with bulk carbon stable isotope. However, our measurement conditions for detection of quinone isotope-ions incurred underestimation of 13C abundance in the quinone. The quinone-SIP technique needs further optimization for measurement conditions of LC-MS/MS.

A systematic strategy for the identification and determination of pharmaceuticals in environment using advanced LC-MS tools: application to ground water samples.

PubMed

Jindal, Kriti; Narayanam, Mallikarjun; Singh, Saranjit

2015-04-10

In the present study, a novel analytical strategy was employed to study the occurrence of 40 drug residues belonging to different medicinal classes, e.g., antibiotics, β blockers, NSAIDs, antidiabetics, proton pump inhibitors, H2 receptor antagonists, antihypertensives, antihyperlipidemics, etc. in ground water samples collected from villages adjoining to S.A.S. Nagar, Punjab, India. The drug residues were extracted from the samples using solid-phase extraction, and LC-ESI-HRMS and LC-ESI-MS/MS were used for identification and quantitation of the analytes. Initially, qualifier and quantifier MRM transitions were classified for 40 targeted drugs, followed by development of LC-MS methods for the separation of all the drugs, which were divided into three categories to curtail overlapping of peaks. Overall identification was done through matching of retention times and MRM transitions; matching of intensity ratio of qualifier to quantifier transitions; comparison of base peak MS/MS profiles; and evaluation of isotopic abundances (wherever applicable). Final confirmation was carried out through comparison of accurate masses obtained from HRMS studies for both standard and targeted analytes in the samples. The application of the strategy allowed removal of false positives and helped in identification and quantitation of diclofenac in the ground water samples of four villages, and pitavastatin in a sample of one village. Copyright © 2015 Elsevier B.V. All rights reserved.

Advanced hyphenated chromatographic-mass spectrometry in mycotoxin determination: current status and prospects.

PubMed

Li, Peiwu; Zhang, Zhaowei; Hu, Xiaofeng; Zhang, Qi

2013-01-01

Mass spectrometric techniques are essential for advanced research in food safety and environmental monitoring. These fields are important for securing the health of humans and animals, and for ensuring environmental security. Mycotoxins, toxic secondary metabolites of filamentous fungi, are major contaminants of agricultural products, food and feed, biological samples, and the environment as a whole. Mycotoxins can cause cancers, nephritic and hepatic diseases, various hemorrhagic syndromes, and immune and neurological disorders. Mycotoxin-contaminated food and feed can provoke trade conflicts, resulting in massive economic losses. Risk assessment of mycotoxin contamination for humans and animals generally depends on clear identification and reliable quantitation in diversified matrices. Pioneering work on mycotoxin quantitation using mass spectrometry (MS) was performed in the early 1970s. Now, unambiguous confirmation and quantitation of mycotoxins can be readily achieved with a variety hyphenated techniques that combine chromatographic separation with MS, including liquid chromatography (LC) or gas chromatography (GC). With the advent of atmospheric pressure ionization, LC-MS has become a routine technique. Recently, the co-occurrence of multiple mycotoxins in the same sample has drawn an increasing amount of attention. Thus, modern analyses must be able to detect and quantitate multiple mycotoxins in a single run. Improvements in tandem MS techniques have been made to achieve this purpose. This review describes the advanced research that has been done regarding mycotoxin determination using hyphenated chromatographic-MS techniques, but is not a full-circle survey of all the literature published on this topic. The present work provides an overview of the various hyphenated chromatographic-MS-based strategies that have been applied to mycotoxin analysis, with a focus on recent developments. The use of chromatographic-MS to measure levels of mycotoxins, including aflatoxins, ochratoxins, patulin, trichothecenes, zearalenone, and fumonisins, is discussed in detail. Both free and masked mycotoxins are included in this review due to different methods of sample preparation. Techniques are described in terms of sample preparation, internal standards, LC/ultra performance LC (UPLC) optimization, and applications and survey. Several future hyphenated MS techniques are discussed as well, including multidimensional chromatography-MS, capillary electrophoresis-MS, and surface plasmon resonance array-MS. © 2013 Wiley Periodicals, Inc.

Development of an LC-MS based enzyme activity assay for MurC: application to evaluation of inhibitors and kinetic analysis.

PubMed

Deng, Gejing; Gu, Rong-Fang; Marmor, Stephen; Fisher, Stewart L; Jahic, Haris; Sanyal, Gautam

2004-06-29

An enzyme activity assay, based on mass spectrometric (MS) detection of specific reaction product following HPLC separation, has been developed to evaluate pharmaceutical hits identified from primary high throughput screening (HTS) against target enzyme Escherichia coli UDP-N-acetyl-muramyl-L-alanine ligase (MurC), an essential enzyme in the bacterial peptidoglycan biosynthetic pathway, and to study the kinetics of the enzyme. A comparative analysis of this new liquid chromatographic-MS (LC-MS) based assay with a conventional spectrophotometric Malachite Green (MG) assay, which detects phosphate produced in the reaction, was performed. The results demonstrated that the LC-MS assay, which determines specific ligase activity of MurC, offers several advantages including a lower background (0.2% versus 26%), higher sensitivity (> or = 10 fold), lower limit of quantitation (LOQ) (0.02 microM versus 1 microM) and wider linear dynamic range (> or = 4 fold) than the MG assay. Good precision for the LC-MS assay was demonstrated by the low intraday and interday coefficient of variation (CV) values (3 and 6%, respectively). The LC-MS assay, free of the artifacts often seen in the Malachite Green assay, offers a valuable secondary assay for hit evaluation in which the false positives from the primary high throughput screening can be eliminated. In addition, the applicability of this assay to the study of enzyme kinetics has also been demonstrated. Copyright 2004 Elsevier B.V.

Recent developments in qualitative and quantitative analysis of phytochemical constituents and their metabolites using liquid chromatography-mass spectrometry.

PubMed

Wu, Haifeng; Guo, Jian; Chen, Shilin; Liu, Xin; Zhou, Yan; Zhang, Xiaopo; Xu, Xudong

2013-01-01

Over the past few years, the applications of liquid chromatography coupled with mass spectrometry (LC-MS) in natural product analysis have been dramatically growing because of the increasingly improved separation and detection capabilities of LC-MS instruments. In particular, novel high-resolution hybrid instruments linked to ultra-high-performance LC and the hyphenations of LC-MS with other separation or analytical techniques greatly aid unequivocal identification and highly sensitive quantification of natural products at trace concentrations in complex matrices. With the aim of providing an up-to-date overview of LC-MS applications on the analysis of plant-derived compounds, papers published within the latest years (2007-2012) involving qualitative and quantitative analysis of phytochemical constituents and their metabolites are summarized in the present review. After briefly describing the general characteristics of natural products analysis, the most remarkable features of LC-MS and sample preparation techniques, the present paper mainly focuses on screening and characterization of phenols (including flavonoids), alkaloids, terpenoids, steroids, coumarins, lignans, and miscellaneous compounds in respective herbs and biological samples, as well as traditional Chinese medicine (TCM) prescriptions using tandem mass spectrometer. Chemical fingerprinting analysis using LC-MS is also described. Meanwhile, instrumental peculiarities and methodological details are accentuated. Copyright © 2012 Elsevier B.V. All rights reserved.

Systematic assessment of survey scan and MS2-based abundance strategies for label-free quantitative proteomics using high-resolution MS data.

PubMed

Tu, Chengjian; Li, Jun; Sheng, Quanhu; Zhang, Ming; Qu, Jun

2014-04-04

Survey-scan-based label-free method have shown no compelling benefit over fragment ion (MS2)-based approaches when low-resolution mass spectrometry (MS) was used, the growing prevalence of high-resolution analyzers may have changed the game. This necessitates an updated, comparative investigation of these approaches for data acquired by high-resolution MS. Here, we compared survey scan-based (ion current, IC) and MS2-based abundance features including spectral-count (SpC) and MS2 total-ion-current (MS2-TIC), for quantitative analysis using various high-resolution LC/MS data sets. Key discoveries include: (i) study with seven different biological data sets revealed only IC achieved high reproducibility for lower-abundance proteins; (ii) evaluation with 5-replicate analyses of a yeast sample showed IC provided much higher quantitative precision and lower missing data; (iii) IC, SpC, and MS2-TIC all showed good quantitative linearity (R(2) > 0.99) over a >1000-fold concentration range; (iv) both MS2-TIC and IC showed good linear response to various protein loading amounts but not SpC; (v) quantification using a well-characterized CPTAC data set showed that IC exhibited markedly higher quantitative accuracy, higher sensitivity, and lower false-positives/false-negatives than both SpC and MS2-TIC. Therefore, IC achieved an overall superior performance than the MS2-based strategies in terms of reproducibility, missing data, quantitative dynamic range, quantitative accuracy, and biomarker discovery.

Systematic Assessment of Survey Scan and MS2-Based Abundance Strategies for Label-Free Quantitative Proteomics Using High-Resolution MS Data

PubMed Central

2015-01-01

Survey-scan-based label-free method have shown no compelling benefit over fragment ion (MS2)-based approaches when low-resolution mass spectrometry (MS) was used, the growing prevalence of high-resolution analyzers may have changed the game. This necessitates an updated, comparative investigation of these approaches for data acquired by high-resolution MS. Here, we compared survey scan-based (ion current, IC) and MS2-based abundance features including spectral-count (SpC) and MS2 total-ion-current (MS2-TIC), for quantitative analysis using various high-resolution LC/MS data sets. Key discoveries include: (i) study with seven different biological data sets revealed only IC achieved high reproducibility for lower-abundance proteins; (ii) evaluation with 5-replicate analyses of a yeast sample showed IC provided much higher quantitative precision and lower missing data; (iii) IC, SpC, and MS2-TIC all showed good quantitative linearity (R2 > 0.99) over a >1000-fold concentration range; (iv) both MS2-TIC and IC showed good linear response to various protein loading amounts but not SpC; (v) quantification using a well-characterized CPTAC data set showed that IC exhibited markedly higher quantitative accuracy, higher sensitivity, and lower false-positives/false-negatives than both SpC and MS2-TIC. Therefore, IC achieved an overall superior performance than the MS2-based strategies in terms of reproducibility, missing data, quantitative dynamic range, quantitative accuracy, and biomarker discovery. PMID:24635752

Quantitative evaluation of protein conformation in pharmaceuticals using cross-linking reactions coupled with LC-MS/MS analysis.

PubMed

Yamaguchi, Hideto; Hirakura, Yutaka; Shirai, Hiroki; Mimura, Hisashi; Toyo'oka, Toshimasa

2011-06-01

The need for a simple and high-throughput method for identifying the tertiary structure of protein pharmaceuticals has increased. In this study, a simple method for mapping the protein fold is proposed for use as a complementary quality test. This method is based on cross-linking a protein using a [bis(sulfosuccinimidyl)suberate (BS(3))], followed by peptide mapping by LC-MS. Consensus interferon (CIFN) was used as the model protein. The tryptic map obtained via liquid chromatography tandem mass spectroscopy (LC-MS/MS) and the mass mapping obtained via matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy were used to identify cross-linked peptides. While LC-MS/MS analyses found that BS(3) formed cross-links in the loop region of the protein, which was regarded as the biologically active site, sodium dodecyl-sulfate polyacrylamide gel electrophoresis demonstrated that cross-linking occurred within a protein molecule, but not between protein molecules. The occurrence of cross-links at the active site depends greatly on the conformation of the protein, which is determined by the denaturing conditions. Quantitative evaluation of the tertiary structure of CIFN was thus possible by monitoring the amounts of cross-linked peptides generated. Assuming that background information is available at the development stage, this method may be applicable to process development as a complementary test for quality control. Copyright © 2011 Elsevier B.V. All rights reserved.

Simultaneous determination of rhamnose, xylitol, arabitol, fructose, glucose, inositol, sucrose, maltose in jujube (Zizyphus jujube Mill.) extract: comparison of HPLC-ELSD, LC-ESI-MS/MS and GC-MS.

PubMed

Sun, Shihao; Wang, Hui; Xie, Jianping; Su, Yue

2016-01-01

Jujube extract is commonly used as a food additive and flavoring. The sensory properties of the extract, especially sweetness, are a critical factor determining the product quality and therefore affecting consumer acceptability. Small molecular carbohydrates make major contribution to the sweetness of the jujube extract, and their types and contents in the extract have direct influence on quality of the product. So, an appropriate qualitative and quantitative method for determination of the carbohydrates is vitally important for quality control of the product. High performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD), liquid chromatography-electronic spay ionization tandem mass spectrometry (LC-ESI-MS/MS), and gas chromatography-mass spectrometry (GC-MS) methods have been developed and applied to determining small molecular carbohydrates in jujube extract, respectively. Eight sugars and alditols were identified from the extract, including rhamnose, xylitol, arabitol, fructose, glucose, inositol, sucrose, and maltose. Comparisons were carried out to investigate the performance of the methods. Although the methods have been found to perform satisfactorily, only three sugars (fructose, glucose and inositol) could be detected by all these methods. Meanwhile, a similar quantitative result for the three sugars can be obtained by the methods. Eight sugars and alditols in the jujube extract were determined by HPLC-ELSD, LC-ESI-MS/MS and GC-MS, respectively. The LC-ELSD method and the LC-ESI-MS/MS method with good precision and accuracy were suitable for quantitative analysis of carbohydrates in jujube extract; although the performance of the GC-MS method for quantitative analysis was inferior to the other methods, it has a wider scope in qualitative analysis. A multi-analysis technique should be adopted in order to obtain complete constituents of about the carbohydrates in jujube extract, and the methods should be employed according to the purpose of analysis.

Metabolic profiling of roots of liquorice (Glycyrrhiza glabra) from different geographical areas by ESI/MS/MS and determination of major metabolites by LC-ESI/MS and LC-ESI/MS/MS.

PubMed

Montoro, Paola; Maldini, Mariateresa; Russo, Mariateresa; Postorino, Santo; Piacente, Sonia; Pizza, Cosimo

2011-02-20

Liquid chromatography electrospray mass spectrometry (LC-ESI/MS) has been applied to the full characterization of saponins and phenolics in hydroalcoholic extracts of roots of liquorice (Glycyrrhiza glabra). Relative quantitative analyses of the samples with respect to the phenolic constituents and to a group of saponins related to glycyrrhizic acid were performed using LC-ESI/MS. For the saponin constituents, full scan LC-MS/MS fragmentation of the protonated (positive ion mode) or deprotonated (negative ion mode) molecular species generated diagnostic fragment ions that provided information concerning the triterpene skeleton and the number and nature of the substituents. On the basis of the specific fragmentation of glycyrrhizic acid, an LC-MS/MS method was developed in order to quantify the analyte in the liquorice root samples. Chinese G. glabra roots contained the highest levels of glycyrrhizic acid, followed by those from Italy (Calabria). Copyright © 2010 Elsevier B.V. All rights reserved.

Rugged LC-MS/MS survey analysis for acrylamide in foods.

PubMed

Roach, John A G; Andrzejewski, Denis; Gay, Martha L; Nortrup, David; Musser, Steven M

2003-12-17

The described liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection of acrylamide in food entails aqueous room temperature extraction, SPE cleanup, and analysis by LC-MS/MS. The method is applicable to a wide variety of foods. [(13)C(3)]acrylamide is the internal standard. The limit of quantitation is 10 ppb (microg/kg). Data were obtained in duplicate from >450 products representing >35 different food types. The variability in analyte levels in certain food types suggests that it may be possible to reduce acrylamide levels in those foods.

Label-Free, LC-MS-Based Assays to Quantitate Small-Molecule Antagonist Binding to the Mammalian BLT1 Receptor.

PubMed

Chen, Xun; Stout, Steven; Mueller, Uwe; Boykow, George; Visconti, Richard; Siliphaivanh, Phieng; Spencer, Kerrie; Presland, Jeremy; Kavana, Michael; Basso, Andrea D; McLaren, David G; Myers, Robert W

2017-08-01

We have developed and validated label-free, liquid chromatography-mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds 1 and 2, were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands 1 and 2. Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.

Nonesterified fatty acid determination for functional lipidomics: comprehensive ultrahigh performance liquid chromatography-tandem mass spectrometry quantitation, qualification, and parameter prediction.

PubMed

Hellmuth, Christian; Weber, Martina; Koletzko, Berthold; Peissner, Wolfgang

2012-02-07

Despite their central importance for lipid metabolism, straightforward quantitative methods for determination of nonesterified fatty acid (NEFA) species are still missing. The protocol presented here provides unbiased quantitation of plasma NEFA species by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Simple deproteination of plasma in organic solvent solution yields high accuracy, including both the unbound and initially protein-bound fractions, while avoiding interferences from hydrolysis of esterified fatty acids from other lipid classes. Sample preparation is fast and nonexpensive, hence well suited for automation and high-throughput applications. Separation of isotopologic NEFA is achieved using ultrahigh-performance liquid chromatography (UPLC) coupled to triple quadrupole LC-MS/MS detection. In combination with automated liquid handling, total assay time per sample is less than 15 min. The analytical spectrum extends beyond readily available NEFA standard compounds by a regression model predicting all the relevant analytical parameters (retention time, ion path settings, and response factor) of NEFA species based on chain length and number of double bonds. Detection of 50 NEFA species and accurate quantification of 36 NEFA species in human plasma is described, the highest numbers ever reported for a LC-MS application. Accuracy and precision are within widely accepted limits. The use of qualifier ions supports unequivocal analyte verification. © 2012 American Chemical Society

Differential Mobility Spectrometry for Improved Selectivity in Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry Analysis of Paralytic Shellfish Toxins

NASA Astrophysics Data System (ADS)

Beach, Daniel G.

2017-08-01

Paralytic shellfish toxins (PSTs) are neurotoxins produced by dinoflagellates and cyanobacteria that cause paralytic shellfish poisoning in humans. PST quantitation by LC-MS is challenging because of their high polarity, lability as gas-phase ions, and large number of potentially interfering analogues. Differential mobility spectrometry (DMS) has the potential to improve the performance of LC-MS methods for PSTs in terms of selectivity and limits of detection. This work describes a comprehensive investigation of the separation of 16 regulated PSTs by DMS and the development of highly selective LC-DMS-MS methods for PST quantitation. The effects of all DMS parameters on the separation of PSTs from one another were first investigated in detail. The labile nature of 11α-gonyautoxin epimers gave unique insight into fragmentation of labile analytes before, during, and after the DMS analyzer. Two sets of DMS parameters were identified that either optimized the resolution of PSTs from one another or transmitted them at a limited number of compensation voltage (CV) values corresponding to structural subclasses. These were used to develop multidimensional LC-DMS-MS/MS methods using existing HILIC-MS/MS parameters. In both cases, improved selectivity was observed when using DMS, and the quantitative capabilities of a rapid UPLC-DMS-MS/MS method were evaluated. Limits of detection of the developed method were similar to those without DMS, and differences were highly analyte-dependant. Analysis of shellfish matrix reference materials showed good agreement with established methods. The developed methods will be useful in cases where specific matrix interferences are encountered in the LC-MS/MS analysis of PSTs in complex biological samples.

Quantitative Metabolome Analysis Based on Chromatographic Peak Reconstruction in Chemical Isotope Labeling Liquid Chromatography Mass Spectrometry.

PubMed

Huan, Tao; Li, Liang

2015-07-21

Generating precise and accurate quantitative information on metabolomic changes in comparative samples is important for metabolomics research where technical variations in the metabolomic data should be minimized in order to reveal biological changes. We report a method and software program, IsoMS-Quant, for extracting quantitative information from a metabolomic data set generated by chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS). Unlike previous work of relying on mass spectral peak ratio of the highest intensity peak pair to measure relative quantity difference of a differentially labeled metabolite, this new program reconstructs the chromatographic peaks of the light- and heavy-labeled metabolite pair and then calculates the ratio of their peak areas to represent the relative concentration difference in two comparative samples. Using chromatographic peaks to perform relative quantification is shown to be more precise and accurate. IsoMS-Quant is integrated with IsoMS for picking peak pairs and Zero-fill for retrieving missing peak pairs in the initial peak pairs table generated by IsoMS to form a complete tool for processing CIL LC-MS data. This program can be freely downloaded from the www.MyCompoundID.org web site for noncommercial use.

Tissue Localization of Glycosphingolipid Accumulation in a Gaucher Disease Mouse Brain by LC-ESI-MS/MS and High-Resolution MALDI Imaging Mass Spectrometry.

PubMed

Jones, E Ellen; Zhang, Wujuan; Zhao, Xueheng; Quiason, Cristine; Dale, Stephanie; Shahidi-Latham, Sheerin; Grabowski, Gregory A; Setchell, Kenneth D R; Drake, Richard R; Sun, Ying

2017-12-01

To better understand regional brain glycosphingolipid (GSL) accumulation in Gaucher disease (GD) and its relationship to neuropathology, a feasibility study using mass spectrometry and immunohistochemistry was conducted using brains derived from a GD mouse model (4L/PS/NA) homozygous for a mutant GCase (V394L [4L]) and expressing a prosaposin hypomorphic (PS-NA) transgene. Whole brains from GD and control animals were collected using one hemisphere for MALDI FTICR IMS analysis and the other for quantitation by LC-ESI-MS/MS. MALDI IMS detected several HexCers across the brains. Comparison with the brain hematoxylin and eosin (H&E) revealed differential signal distributions in the midbrain, brain stem, and CB of the GD brain versus the control. Quantitation of serial brain sections with LC-ESI-MS/MS supported the imaging results, finding the overall HexCer levels in the 4L/PS-NA brains to be four times higher than the control. LC-ESI-MS/MS also confirmed that the elevated hexosyl isomers were glucosylceramides rather than galactosylceramides. MALDI imaging also detected differential analyte distributions of lactosylceramide species and gangliosides in the 4L/PS-NA brain, which was validated by LC-ESI-MS/MS. Immunohistochemistry revealed regional inflammation, altered autophagy, and defective protein degradation correlating with regions of GSL accumulation, suggesting that specific GSLs may have distinct neuropathological effects.

Use of specific peptide biomarkers for quantitative confirmation of hidden allergenic peanut proteins Ara h 2 and Ara h 3/4 for food control by liquid chromatography-tandem mass spectrometry.

PubMed

Careri, M; Costa, A; Elviri, L; Lagos, J-B; Mangia, A; Terenghi, M; Cereti, A; Garoffo, L Perono

2007-11-01

A liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS-MS) method based on the detection of biomarker peptides from allergenic proteins was devised for confirming and quantifying peanut allergens in foods. Peptides obtained from tryptic digestion of Ara h 2 and Ara h 3/4 proteins were identified and characterized by LC-MS and LC-MS-MS with a quadrupole-time of flight mass analyzer. Four peptides were chosen and investigated as biomarkers taking into account their selectivity, the absence of missed cleavages, the uniform distribution in the Ara h 2 and Ara h 3/4 protein isoforms together with their spectral features under ESI-MS-MS conditions, and good repeatability of LC retention time. Because of the different expression levels, the selection of two different allergenic proteins was proved to be useful in the identification and univocal confirmation of the presence of peanuts in foodstuffs. Using rice crisp and chocolate-based snacks as model food matrix, an LC-MS-MS method with triple quadrupole mass analyzer allowed good detection limits to be obtained for Ara h 2 (5 microg protein g(-1) matrix) and Ara h 3/4 (1 microg protein g(-1) matrix). Linearity of the method was established in the 10-200 microg g(-1) range of peanut proteins in the food matrix investigated. Method selectivity was demonstrated by analyzing tree nuts (almonds, pecan nuts, hazelnuts, walnuts) and food ingredients such as milk, soy beans, chocolate, cornflakes, and rice crisp.

Qualitative and quantitative analysis of poly(amidoamine) dendrimers in an aqueous matrix by liquid chromatography-electrospray ionization-hybrid quadrupole/time-of-flight mass spectrometry (LC-ESI-QTOF-MS).

PubMed

Uclés, A; Ulaszewska, M M; Hernando, M D; Ramos, M J; Herrera, S; García, E; Fernández-Alba, A R

2013-07-01

This work introduces a liquid chromatography-electrospray ionization-hybrid quadrupole/time-of-flight mass spectrometry (LC-ESI-QTOF-MS)-based method for qualitative and quantitative analysis of poly(amidoamine) (PAMAM) dendrimers of generations 0 to 3 in an aqueous matrix. The multiple charging of PAMAM dendrimers generated by means of ESI has provided key advantages in dendrimer identification by assignation of charge state through high resolution of isotopic clusters. Isotopic distribution in function of abundance of isotopes (12)C and (13)C yielded valuable and complementarity data for confident characterization. A mass accuracy below 3.8 ppm for the most abundant isotopes (diagnostic ions) provided unambiguous identification of PAMAM dendrimers. Validation of the LC-ESI-QTOF-MS method and matrix effect evaluation enabled reliable and reproducible quantification. The validation parameters, limits of quantification in the range of 0.012 to 1.73 μM, depending on the generation, good linear range (R > 0.996), repeatability (RSD < 13.4%), and reproducibility (RSD < 10.9%) demonstrated the suitability of the method for the quantification of dendrimers in aqueous matrices (water and wastewater). The added selectivity, achieved by multicharge phenomena, represents a clear advantage in screening aqueous mixtures due to the fact that the matrix had no significant effect on ionization, with what is evidenced by an absence of sensitivity loss in most generations of PAMAM dendrimers. Fig Liquid chromatography-electrospray ionization-hybrid quadrupole/time of flight mass spectrometry (LC-ESI-QTOF-MS) based method for qualitative and quantitative analysis of PAMAM dendrimers in aqueous matrix.

Development and application of a quantitative method based on LC-QqQ MS/MS for determination of steviol glycosides in Stevia leaves.

PubMed

Molina-Calle, M; Sánchez de Medina, V; Delgado de la Torre, M P; Priego-Capote, F; Luque de Castro, M D

2016-07-01

Stevia is a currently well-known plant thanks to the presence of steviol glycosides, which are considered as sweeteners obtained from a natural source. In this research, a method based on LC-MS/MS by using a triple quadrupole detector was developed for quantitation of 8 steviol glycosides in extracts from Stevia leaves. The ionization and fragmentation parameters for selected reaction monitoring were optimized. Detection and quantitation limits ranging from 0.1 to 0.5ng/mL and from 0.5 to 1ng/mL, respectively, were achieved: the lowest attained so far. The steviol glycosides were quantified in extracts from leaves of seven varieties of Stevia cultivated in laboratory, greenhouse and field. Plants cultivated in field presented higher concentration of steviol glycosides than those cultivated in greenhouse. Thus, the way of cultivation clearly influences the concentration of these compounds. The inclusion of branches together with leaves as raw material was also evaluated, showing that this inclusion modifies, either positively or negatively, the concentration of steviol glycosides. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of liquid chromatography-tandem mass spectrometry methods for the quantitation of Anisakis simplex proteins in fish.

PubMed

Fæste, Christiane Kruse; Moen, Anders; Schniedewind, Björn; Haug Anonsen, Jan; Klawitter, Jelena; Christians, Uwe

2016-02-05

The parasite Anisakis simplex is present in many marine fish species that are directly used as food or in processed products. The anisakid larvae infect mostly the gut and inner organs of fish but have also been shown to penetrate into the fillet. Thus, human health can be at risk, either by contracting anisakiasis through the consumption of raw or under-cooked fish, or by sensitisation to anisakid proteins in processed food. A number of different methods for the detection of A. simplex in fish and products thereof have been developed, including visual techniques and PCR for larvae tracing, and immunological assays for the determination of proteins. The recent identification of a number of anisakid proteins by mass spectrometry-based proteomics has laid the groundwork for the development of two quantitative liquid chromatography-tandem mass spectrometry methods for the detection of A. simplex in fish that are described in the present study. Both, the label-free semi-quantitative nLC-nESI-Orbitrap-MS/MS (MS1) and the heavy peptide-applying absolute-quantitative (AQUA) LC-TripleQ-MS/MS (MS2) use unique reporter peptides derived from anisakid hemoglobin and SXP/RAL-2 protein as analytes. Standard curves in buffer and in salmon matrix showed limits of detection at 1μg/mL and 10μg/mL for MS1 and 0.1μg/mL and 2μg/mL for MS2. Preliminary method validation included the assessment of sensitivity, repeatability, reproducibility, and applicability to incurred and naturally-contaminated samples for both assays. By further optimization and full validation in accordance with current recommendations the LC-MS/MS methods could be standardized and used generally as confirmative techniques for the detection of A. simplex protein in fish. Copyright © 2016 Elsevier B.V. All rights reserved.

Isotope-coded ESI-enhancing derivatization reagents for differential analysis, quantification and profiling of metabolites in biological samples by LC/MS: A review.

PubMed

Higashi, Tatsuya; Ogawa, Shoujiro

2016-10-25

The analysis of the qualitative and quantitative changes of metabolites in body fluids and tissues yields valuable information for the diagnosis, pathological analysis and treatment of many diseases. Recently, liquid chromatography/electrospray ionization-(tandem) mass spectrometry [LC/ESI-MS(/MS)] has been widely used for these purposes due to the high separation capability of LC, broad coverage of ESI for various compounds and high specificity of MS(/MS). However, there are still two major problems to be solved regarding the biological sample analysis; lack of sensitivity and limited availability of stable isotope-labeled analogues (internal standards, ISs) for most metabolites. Stable isotope-coded derivatization (ICD) can be the answer for these problems. By the ICD, different isotope-coded moieties are introduced to the metabolites and one of the resulting derivatives can serve as the IS, which minimize the matrix effects. Furthermore, the derivatization can improve the ESI efficiency, fragmentation property in the MS/MS and chromatographic behavior of the metabolites, which lead to a high sensitivity and specificity in the various detection modes. Based on this background, this article reviews the recently-reported isotope-coded ESI-enhancing derivatization (ICEED) reagents, which are key components for the ICD-based LC/MS(/MS) studies, and their applications to the detection, identification, quantification and profiling of metabolites in human and animal samples. The LC/MS(/MS) using the ICEED reagents is the powerful method especially for the differential analysis (relative quantification) of metabolites in two comparative samples, simultaneous quantification of multiple metabolites whose stable isotope-labeled ISs are not available, and submetabolome profiling. Copyright © 2016 Elsevier B.V. All rights reserved.

The current preference for the immuno-analytical ELISA method for quantitation of steroid hormones (endocrine disruptor compounds) in wastewater in South Africa.

PubMed

Manickum, Thavrin; John, Wilson

2015-07-01

The availability of national test centers to offer a routine service for analysis and quantitation of some selected steroid hormones [natural estrogens (17-β-estradiol, E2; estrone, E1; estriol, E3), synthetic estrogen (17-α-ethinylestradiol, EE2), androgen (testosterone), and progestogen (progesterone)] in wastewater matrix was investigated; corresponding internationally used chemical- and immuno-analytical test methods were reviewed. The enzyme-linked immunosorbent assay (ELISA) (immuno-analytical technique) was also assessed for its suitability as a routine test method to quantitate the levels of these hormones at a sewage/wastewater treatment plant (WTP) (Darvill, Pietermaritzburg, South Africa), over a 2-year period. The method performance and other relevant characteristics of the immuno-analytical ELISA method were compared to the conventional chemical-analytical methodology, like gas/liquid chromatography-mass spectrometry (GC/LC-MS), and GC-LC/tandem mass spectrometry (MSMS), for quantitation of the steroid hormones in wastewater and environmental waters. The national immuno-analytical ELISA technique was found to be sensitive (LOQ 5 ng/L, LOD 0.2-5 ng/L), accurate (mean recovery 96%), precise (RSD 7-10%), and cost-effective for screening and quantitation of these steroid hormones in wastewater and environmental water matrix. A survey of the most current international literature indicates a fairly equal use of the LC-MS/MS, GC-MS/MS (chemical-analytical), and ELISA (immuno-analytical) test methods for screening and quantitation of the target steroid hormones in both water and wastewater matrix. Internationally, the observed sensitivity, based on LOQ (ng/L), for the steroid estrogens E1, E2, EE2, is, in decreasing order: LC-MSMS (0.08-9.54) > GC-MS (1) > ELISA (5) (chemical-analytical > immuno-analytical). At the national level, the routine, unoptimized chemical-analytical LC-MSMS method was found to lack the required sensitivity for meeting environmental requirements for steroid hormone quantitation. Further optimization of the sensitivity of the chemical-analytical LC-tandem mass spectrometry methods, especially for wastewater screening, in South Africa is required. Risk assessment studies showed that it was not practical to propose standards or allowable limits for the steroid estrogens E1, E2, EE2, and E3; the use of predicted-no-effect concentration values of the steroid estrogens appears to be appropriate for use in their risk assessment in relation to aquatic organisms. For raw water sources, drinking water, raw and treated wastewater, the use of bioassays, with trigger values, is a useful screening tool option to decide whether further examination of specific endocrine activity may be warranted, or whether concentrations of such activity are of low priority, with respect to health concerns in the human population. The achievement of improved quantitation limits for immuno-analytical methods, like ELISA, used for compound quantitation, and standardization of the method for measuring E2 equivalents (EEQs) used for biological activity (endocrine: e.g., estrogenic) are some areas for future EDC research.

LC/MS/MS Bioanalysis of Protein-Drug Conjugates-The Importance of Incorporating Succinimide Hydrolysis Products.

PubMed

Shi, Chuan; Goldberg, Shalom; Lin, Tricia; Dudkin, Vadim; Widdison, Wayne; Harris, Luke; Wilhelm, Sharon; Jmeian, Yazen; Davis, Darryl; O'Neil, Karyn; Weng, Naidong; Jian, Wenying

2018-04-17

Bioanalysis of antibody-drug conjugates (ADCs) is challenging due to the complex, heterogeneous nature of their structures and their complicated catabolism. To fully describe the pharmacokinetics (PK) of an ADC, several analytes are commonly quantified, including total antibody, conjugate, and payload. Among them, conjugate is the most challenging to measure, because it requires detection of both small and large molecules as one entity. Existing approaches to quantify the conjugated species of ADCs involve a ligand binding assay (LBA) for conjugated antibody or hybrid LBA/liquid chromatography/tandem mass spectrometry (LC/MS/MS) for quantitation of conjugated drug. In our current work for a protein-drug conjugate (PDC) using the Centyrin scaffold, a similar concept to ADCs but with smaller protein size, an alternative method to quantify the conjugate by using a surrogate peptide approach, was utilized. The His-tagged proteins were isolated from biological samples using immobilized metal affinity chromatography (IMAC), followed by trypsin digestion. The tryptic peptide containing the linker attached to the payload was used as a surrogate of the conjugate and monitored by LC/MS/MS analysis. During method development and its application, we found that hydrolysis of the succinimide ring of the linker was ubiquitous, taking place at many stages during the lifetime of the PDC including in the initial drug product, in vivo in circulation in the animals, and ex vivo during the trypsin digestion step of the sample preparation. We have shown that hydrolysis during trypsin digestion is concentration-independent and consistent during the work flow-therefore, having no impact on assay performance. However, for samples that have undergone extensive hydrolysis prior to trypsin digestion, significant bias could be introduced if only the non-hydrolyzed form is considered in the quantitation. Therefore, it is important to incorporate succinimide hydrolysis products in the quantitation method in order to provide an accurate estimation of the total conjugate level. More importantly, the LC/MS/MS-based method described here provides a useful tool to quantitatively evaluate succinimide hydrolysis of ADCs in vivo, which has been previously reported to have significant impact on their stability, exposure, and efficacy.

Analytical method for the accurate determination of tricothecenes in grains using LC-MS/MS: a comparison between MRM transition and MS3 quantitation.

PubMed

Lim, Chee Wei; Tai, Siew Hoon; Lee, Lin Min; Chan, Sheot Harn

2012-07-01

The current food crisis demands unambiguous determination of mycotoxin contamination in staple foods to achieve safer food for consumption. This paper describes the first accurate LC-MS/MS method developed to analyze tricothecenes in grains by applying multiple reaction monitoring (MRM) transition and MS(3) quantitation strategies in tandem. The tricothecenes are nivalenol, deoxynivalenol, deoxynivalenol-3-glucoside, fusarenon X, 3-acetyl-deoxynivalenol, 15-acetyldeoxynivalenol, diacetoxyscirpenol, and HT-2 and T-2 toxins. Acetic acid and ammonium acetate were used to convert the analytes into their respective acetate adducts and ammonium adducts under negative and positive MS polarity conditions, respectively. The mycotoxins were separated by reversed-phase LC in a 13.5-min run, ionized using electrospray ionization, and detected by tandem mass spectrometry. Analyte-specific mass-to-charge (m/z) ratios were used to perform quantitation under MRM transition and MS(3) (linear ion trap) modes. Three experiments were made for each quantitation mode and matrix in batches over 6 days for recovery studies. The matrix effect was investigated at concentration levels of 20, 40, 80, 120, 160, and 200 μg kg(-1) (n = 3) in 5 g corn flour and rice flour. Extraction with acetonitrile provided a good overall recovery range of 90-108% (n = 3) at three levels of spiking concentration of 40, 80, and 120 μg kg(-1). A quantitation limit of 2-6 μg kg(-1) was achieved by applying an MRM transition quantitation strategy. Under MS(3) mode, a quantitation limit of 4-10 μg kg(-1) was achieved. Relative standard deviations of 2-10% and 2-11% were reported for MRM transition and MS(3) quantitation, respectively. The successful utilization of MS(3) enabled accurate analyte fragmentation pattern matching and its quantitation, leading to the development of analytical methods in fields that demand both analyte specificity and fragmentation fingerprint-matching capabilities that are unavailable under MRM transition.

The great importance of normalization of LC-MS data for highly-accurate non-targeted metabolomics.

PubMed

Mizuno, Hajime; Ueda, Kazuki; Kobayashi, Yuta; Tsuyama, Naohiro; Todoroki, Kenichiro; Min, Jun Zhe; Toyo'oka, Toshimasa

2017-01-01

The non-targeted metabolomics analysis of biological samples is very important to understand biological functions and diseases. LC combined with electrospray ionization-based MS has been a powerful tool and widely used for metabolomic analyses. However, the ionization efficiency of electrospray ionization fluctuates for various unexpected reasons such as matrix effects and intraday variations of the instrument performances. To remove these fluctuations, normalization methods have been developed. Such techniques include increasing the sensitivity, separating co-eluting components and normalizing the ionization efficiencies. Normalization techniques allow simultaneously correcting of the ionization efficiencies of the detected metabolite peaks and achieving quantitative non-targeted metabolomics. In this review paper, we focused on these normalization methods for non-targeted metabolomics by LC-MS. Copyright © 2016 John Wiley & Sons, Ltd.

Microdose clinical trial: quantitative determination of nicardipine and prediction of metabolites in human plasma.

PubMed

Yamane, Naoe; Takami, Tomonori; Tozuka, Zenzaburo; Sugiyama, Yuichi; Yamazaki, Akira; Kumagai, Yuji

2009-01-01

A sample treatment procedure and high-sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for quantitative determination of nicardipine in human plasma were developed for a microdose clinical trial with nicardipine, a non-radioisotope labeled drug. The calibration curve was linear in the range of 1-500 pg/mL using 1 mL of plasma. Analytical method validation for the clinical dose, for which the calibration curve was linear in the range of 0.2-100 ng/mL using 20 microL of plasma, was also conducted. Each method was successfully applied to making determinations in plasma using LC/MS/MS after administration of a microdose (100 microg) and clinical dose (20 mg) to each of six healthy volunteers. We tested new approaches in the search for metabolites in plasma after microdosing. In vitro metabolites of nicardipine were characterized using linear ion trap-fourier transform ion cyclotron resonance mass spectrometry (LIT-FTICRMS) and the nine metabolites predicted to be in plasma were analyzed using LC/MS/MS. There is a strong possibility that analysis of metabolites by LC/MS/MS may advance to utilization in microdose clinical trials with non-radioisotope labeled drugs.

A new LC-MS based method to quantitate exogenous recombinant transferrin in cerebrospinal fluid: a potential approach for pharmacokinetic studies of transferrin-based therapeutics in the central nervous system

PubMed Central

Wang, Shunhai; Bobst, Cedric E.; Kaltashov, Igor A.

2018-01-01

Transferrin (Tf) is an 80 kDa iron-binding protein which is viewed as a promising drug carrier to target the central nervous system due to its ability to penetrate the blood-brain barrier (BBB). Among the many challenges during the development of Tf-based therapeutics, sensitive and accurate quantitation of the administered Tf in cerebrospinal fluid (CSF) remains particularly difficult due to the presence of abundant endogenous Tf. Herein, we describe the development of a new LC-MS based method for sensitive and accurate quantitation of exogenous recombinant human Tf in rat CSF. By taking advantage of a His-tag present in recombinant Tf and applying Ni affinity purification, the exogenous hTf can be greatly enriched from rat CSF, despite the presence of the abundant endogenous protein. Additionally, we applied a newly developed O18-labeling technique that can generate internal standards at the protein level, which greatly improved the accuracy and robustness of quantitation. The developed method was investigated for linearity, accuracy, precision and lower limit of quantitation, all of which met the commonly accepted criteria for bioanalytical method validation. PMID:26307718

Absolute quantitation of disease protein biomarkers in a single LC-MS acquisition using apolipoprotein F as an example.

PubMed

Kumar, Abhinav; Gangadharan, Bevin; Cobbold, Jeremy; Thursz, Mark; Zitzmann, Nicole

2017-09-21

LC-MS and immunoassay can detect protein biomarkers. Immunoassays are more commonly used but can potentially be outperformed by LC-MS. These techniques have limitations including the necessity to generate separate calibration curves for each biomarker. We present a rapid mass spectrometry-based assay utilising a universal calibration curve. For the first time we analyse clinical samples using the HeavyPeptide IGNIS kit which establishes a 6-point calibration curve and determines the biomarker concentration in a single LC-MS acquisition. IGNIS was tested using apolipoprotein F (APO-F), a potential biomarker for non-alcoholic fatty liver disease (NAFLD). Human serum and IGNIS prime peptides were digested and the IGNIS assay was used to quantify APO-F in clinical samples. Digestion of IGNIS prime peptides was optimised using trypsin and SMART Digest™. IGNIS was 9 times faster than the conventional LC-MS method for determining the concentration of APO-F in serum. APO-F decreased across NAFLD stages. Inter/intra-day variation and stability post sample preparation for one of the peptides was ≤13% coefficient of variation (CV). SMART Digest™ enabled complete digestion in 30 minutes compared to 24 hours using in-solution trypsin digestion. We have optimised the IGNIS kit to quantify APO-F as a NAFLD biomarker in serum using a single LC-MS acquisition.

Multiplicative effects model with internal standard in mobile phase for quantitative liquid chromatography-mass spectrometry.

PubMed

Song, Mi; Chen, Zeng-Ping; Chen, Yao; Jin, Jing-Wen

2014-07-01

Liquid chromatography-mass spectrometry assays suffer from signal instability caused by the gradual fouling of the ion source, vacuum instability, aging of the ion multiplier, etc. To address this issue, in this contribution, an internal standard was added into the mobile phase. The internal standard was therefore ionized and detected together with the analytes of interest by the mass spectrometer to ensure that variations in measurement conditions and/or instrument have similar effects on the signal contributions of both the analytes of interest and the internal standard. Subsequently, based on the unique strategy of adding internal standard in mobile phase, a multiplicative effects model was developed for quantitative LC-MS assays and tested on a proof of concept model system: the determination of amino acids in water by LC-MS. The experimental results demonstrated that the proposed method could efficiently mitigate the detrimental effects of continuous signal variation, and achieved quantitative results with average relative predictive error values in the range of 8.0-15.0%, which were much more accurate than the corresponding results of conventional internal standard method based on the peak height ratio and partial least squares method (their average relative predictive error values were as high as 66.3% and 64.8%, respectively). Therefore, it is expected that the proposed method can be developed and extended in quantitative LC-MS analysis of more complex systems. Copyright © 2014 Elsevier B.V. All rights reserved.

Twoplex 12/13 C6 aniline stable isotope and linkage-specific sialic acid labeling 2D-LC-MS workflow for quantitative N-glycomics.

PubMed

Albrecht, Simone; Mittermayr, Stefan; Smith, Josh; Martín, Silvia Millán; Doherty, Margaret; Bones, Jonathan

2017-01-01

Quantitative glycomics represents an actively expanding research field ranging from the discovery of disease-associated glycan alterations to the quantitative characterization of N-glycans on therapeutic proteins. Commonly used analytical platforms for comparative relative quantitation of complex glycan samples include MALDI-TOF-MS or chromatographic glycan profiling with subsequent data alignment and statistical evaluation. Limitations of such approaches include run-to-run technical variation and the potential introduction of subjectivity during data processing. Here, we introduce an offline 2D LC-MS E workflow for the fractionation and relative quantitation of twoplex isotopically labeled N-linked oligosaccharides using neutral 12 C 6 and 13 C 6 aniline (Δmass = 6 Da). Additional linkage-specific derivatization of sialic acids using 4-(4,6-dimethoxy-1,3,5-trizain-2-yl)-4-methylmorpholinium chloride offered simultaneous and advanced in-depth structural characterization. The potential of the method was demonstrated for the differential analysis of structurally defined N-glycans released from serum proteins of patients diagnosed with various stages of colorectal cancer. The described twoplex 12 C 6 / 13 C 6 aniline 2D LC-MS platform is ideally suited for differential glycomic analysis of structurally complex N-glycan pools due to combination and analysis of samples in a single LC-MS injection and the associated minimization in technical variation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A proteomics performance standard to support measurement quality in proteomics.

PubMed

Beasley-Green, Ashley; Bunk, David; Rudnick, Paul; Kilpatrick, Lisa; Phinney, Karen

2012-04-01

The emergence of MS-based proteomic platforms as a prominent technology utilized in biochemical and biomedical research has increased the need for high-quality MS measurements. To address this need, National Institute of Standards and Technology (NIST) reference material (RM) 8323 yeast protein extract is introduced as a proteomics quality control material for benchmarking the preanalytical and analytical performance of proteomics-based experimental workflows. RM 8323 yeast protein extract is based upon the well-characterized eukaryote Saccharomyces cerevisiae and can be utilized in the design and optimization of proteomics-based methodologies from sample preparation to data analysis. To demonstrate its utility as a proteomics quality control material, we coupled LC-MS/MS measurements of RM 8323 with the NIST MS Quality Control (MSQC) performance metrics to quantitatively assess the LC-MS/MS instrumentation parameters that influence measurement accuracy, repeatability, and reproducibility. Due to the complexity of the yeast proteome, we also demonstrate how NIST RM 8323, along with the NIST MSQC performance metrics, can be used in the evaluation and optimization of proteomics-based sample preparation methods. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Increasing the throughput and productivity of Caco-2 cell permeability assays using liquid chromatography-mass spectrometry: application to resveratrol absorption and metabolism.

PubMed

Li, Yongmei; Shin, Young Geun; Yu, Chongwoo; Kosmeder, Jerome W; Hirschelman, Wendy H; Pezzuto, John M; van Breemen, Richard B

2003-12-01

The Caco-2 cell monolayer permeability assay has become a standard model of human intestinal absorption and transport. This paper reviews recent progress in increasing the throughput of Caco-2 cell monolayer assays and in expanding the scope of this assay to include modeling intestinal drug metabolism. The state-of-the-art in Caco-2 cell monolayer permeability assays combines multi-well plates fitted with semi-permeable inserts on which Caco-2 cells have been cultured with liquid chromatography-mass spectrometry (LC-MS) or LC-tandem mass spectrometry (LC-MS-MS) for the quantitative analysis of test compounds and the identification of their intestinal metabolites. After reviewing the progress in increasing the throughput of Caco-2 cell monolayer assays for both modeling human intestinal permeability or transport and the metabolism of xenobiotic compounds, we demonstrate the application of LC-MS and LC-MS-MS to the measurement of resveratrol permeability and metabolism in the Caco-2 model. trans-Resveratrol (trans-3,5,4'-trihydroxystilbene) is a polyphenolic compound occurring in grapes, peanuts and other food sources, that is under investigation as a cancer chemoprevention agent. The apparent permeability coefficient for apical (AP) to basolateral (BL) movement of resveratrol was 2.0 x 10(-5)cm/sec. Resveratrol was not a substrate for P-glycoprotein or the multi-drug resistance associated proteins (MRP). No phase I metabolites were observed, but the phase II conjugates resveratrol-3-glucuronide and resveratrol-3-sulfate was identified based on LC-MS and LC-MS-MS analysis and comparison with synthetic standards. Although these data indicate that resveratrol diffuses rapidly across the intestinal epithelium, extensive phase II metabolism during absorption might reduce resveratrol bioavailability.

A Collaborative Study: Determination of Mycotoxins in Corn, Peanut Butter, and Wheat Flour Using Stable Isotope Dilution Assay (SIDA) and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

PubMed

Zhang, Kai; Schaab, Matthew R; Southwood, Gavin; Tor, Elizabeth R; Aston, Linda S; Song, Wenlu; Eitzer, Brian; Majumdar, Sanghamitra; Lapainis, Theodore; Mai, Huy; Tran, Kevin; El-Demerdash, Aref; Vega, Victor; Cai, Yanxuan; Wong, Jon W; Krynitsky, Alexandra J; Begley, Timothy H

2017-08-23

A collaborative study was conducted to evaluate stable isotope dilution assay (SIDA) and LC-MS/MS for the simultaneous determination of aflatoxins B 1 , B 2 , G 1 , and G 2 ; deoxynivalenol; fumonisins B 1 , B 2 , and B 3 ; ochratoxin A; HT-2 toxin; T-2 toxin; and zearalenone in foods. Samples were fortified with 12 13 C uniformly labeled mycotoxins ( 13 C-IS) corresponding to the native mycotoxins and extracted with acetonitrile/water (50:50 v/v), followed by centrifugation, filtration, and LC-MS/MS analysis. In addition to certified reference materials, the six participating laboratories analyzed corn, peanut butter, and wheat flour fortified with the 12 mycotoxins at concentrations ranging from 1.0 to 1000 ng/g. Using their available LC-MS/MS platform, each laboratory developed in-house instrumental conditions for analysis. The majority of recoveries ranged from 80 to 120% with relative standard derivations (RSDs) <20%. Greater than 90% of the average recoveries of the participating laboratories were in the range of 90-110%, with repeatability RSD r (within laboratory) < 10% and reproducibility RSD R (among laboratory) < 15%. All Z scores of the results of certified reference materials were between -2 and 2. Using 13 C-IS eliminated the need for matrix-matched calibration standards for quantitation, simplified sample preparation, and achieved simultaneous identification and quantitation of multiple mycotoxins in a simple LC-MS/MS procedure.

Utility of high-resolution accurate MS to eliminate interferences in the bioanalysis of ribavirin and its phosphate metabolites.

PubMed

Wei, Cong; Grace, James E; Zvyaga, Tatyana A; Drexler, Dieter M

2012-08-01

The polar nucleoside drug ribavirin (RBV) combined with IFN-α is a front-line treatment for chronic hepatitis C virus infection. RBV acts as a prodrug and exerts its broad antiviral activity primarily through its active phosphorylated metabolite ribavirin 5´-triphosphate (RTP), and also possibly through ribavirin 5´-monophosphate (RMP). To study RBV transport, diffusion, metabolic clearance and its impact on drug-metabolizing enzymes, a LC-MS method is needed to simultaneously quantify RBV and its phosphorylated metabolites (RTP, ribavirin 5´-diphosphate and RMP). In a recombinant human UGT1A1 assay, the assay buffer components uridine and its phosphorylated derivatives are isobaric with RBV and its phosphorylated metabolites, leading to significant interference when analyzed by LC-MS with the nominal mass resolution mode. Presented here is a LC-MS method employing LC coupled with full-scan high-resolution accurate MS analysis for the simultaneous quantitative determination of RBV, RMP, ribavirin 5´-diphosphate and RTP by differentiating RBV and its phosphorylated metabolites from uridine and its phosphorylated derivatives by accurate mass, thus avoiding interference. The developed LC-high-resolution accurate MS method allows for quantitation of RBV and its phosphorylated metabolites, eliminating the interferences from uridine and its phosphorylated derivatives in recombinant human UGT1A1 assays.

Rapid and reliable QuEChERS-based LC-MS/MS method for determination of acrylamide in potato chips and roasted coffee

NASA Astrophysics Data System (ADS)

Stefanović, S.; Đorđevic, V.; Jelušić, V.

2017-09-01

The aim of this paper is to verify the performance characteristics and fitness for purpose of rapid and simple QuEChERS-based LC-MS/MS method for determination of acrylamide in potato chips and coffee. LC-MS/MS is by far the most suitable analytical technique for acrylamide measurements given its inherent sensitivity and selectivity, as well as capability of analyzing underivatized molecule. Acrylamide in roasted coffee and potato chips wasextracted with water:acetonitrile mixture using NaCl and MgSO4. Cleanup was carried out with MgSO4 and PSA. Obtained results were satisfactory. Recoveries were in the range of 85-112%, interlaboratory reproducibility (Cv) was 5.8-7.6% and linearity (R2) was in the range of 0.995-0.999. LoQ was 35 μg kg-1 for coffee and 20 μg kg-1 for potato chips. Performance characteristic of the method are compliant with criteria for analytical methods validation. Presented method for quantitative determination of acrylamide in roasted coffee and potato chips is fit for purposes of self-control in food industry as well as regulatory controls carried out by the governmental agencies.

Development of Immunocapture-LC/MS Assay for Simultaneous ADA Isotyping and Semiquantitation

PubMed Central

2016-01-01

Therapeutic proteins and peptides have potential to elicit immune responses resulting in anti-drug antibodies that can pose problems for both patient safety and product efficacy. During drug development immunogenicity is usually examined by risk-based approach along with specific strategies for developing “fit-for-purpose” bioanalytical approaches. Enzyme-linked immunosorbent assays and electrochemiluminescence immunoassays are the most widely used platform for ADA detection due to their high sensitivity and throughput. During the past decade, LC/MS has emerged as a promising technology for quantitation of biotherapeutics and protein biomarkers in biological matrices, mainly owing to its high specificity, selectivity, multiplexing, and wide dynamic range. In fully taking these advantages, we describe here an immunocapture-LC/MS methodology for simultaneous isotyping and semiquantitation of ADA in human plasma. Briefly, ADA and/or drug-ADA complex is captured by biotinylated drug or anti-drug Ab, immobilized on streptavidin magnetic beads, and separated from human plasma by a magnet. ADA is then released from the beads and subjected to trypsin digestion followed by LC/MS detection of specific universal peptides for each ADA isotype. The LC/MS data are analyzed using cut-point and calibration curve. The proof-of-concept of this methodology is demonstrated by detecting preexisting ADA in human plasma. PMID:27034966

Development of Immunocapture-LC/MS Assay for Simultaneous ADA Isotyping and Semiquantitation.

PubMed

Chen, Lin-Zhi; Roos, David; Philip, Elsy

2016-01-01

Therapeutic proteins and peptides have potential to elicit immune responses resulting in anti-drug antibodies that can pose problems for both patient safety and product efficacy. During drug development immunogenicity is usually examined by risk-based approach along with specific strategies for developing "fit-for-purpose" bioanalytical approaches. Enzyme-linked immunosorbent assays and electrochemiluminescence immunoassays are the most widely used platform for ADA detection due to their high sensitivity and throughput. During the past decade, LC/MS has emerged as a promising technology for quantitation of biotherapeutics and protein biomarkers in biological matrices, mainly owing to its high specificity, selectivity, multiplexing, and wide dynamic range. In fully taking these advantages, we describe here an immunocapture-LC/MS methodology for simultaneous isotyping and semiquantitation of ADA in human plasma. Briefly, ADA and/or drug-ADA complex is captured by biotinylated drug or anti-drug Ab, immobilized on streptavidin magnetic beads, and separated from human plasma by a magnet. ADA is then released from the beads and subjected to trypsin digestion followed by LC/MS detection of specific universal peptides for each ADA isotype. The LC/MS data are analyzed using cut-point and calibration curve. The proof-of-concept of this methodology is demonstrated by detecting preexisting ADA in human plasma.

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) based bioavailability determination of the major classes of phytochemicals.

PubMed

Stylos, Evgenios; Chatziathanasiadou, Maria V; Syriopoulou, Aggeliki; Tzakos, Andreas G

2017-03-15

Natural products derived from plants have served as an inexhausted source for drug discovery and drug development. They have been evolutionary amplified with drug-like properties and have already illustrated immense therapeutic potential over an array of different diseases. However, their incorporation in the drug discovery pipeline has been diminished the last two decades. This was probably due to barriers related to their inherent difficulties to be integrated in high-throughput screening assays as also their largely unexplored bioavailability. Analytical procedures have come into the spotlight, a result of the continuous development of the instrumentation's capabilities as far as detection and separation is concerned. Integral part of this technological evolution is LC-MS instrumentation and its extended use for the determination of various compounds. The fact that it provides extra sensitivity, specificity and good separation in complex samples, makes LC-MS/MS the ultimate tool in the determination of many types of chemical compounds, such as phytochemicals. Herein, we focus on the achievements of the last five years in quantitative analysis of the major classes of phytochemicals (flavonoids, alkaloids, terpenes, glycosides and saponins) in plasma, through LC-MS/MS, as also their bioavailability. Copyright © 2016 Elsevier B.V. All rights reserved.

Structure Annotation and Quantification of Wheat Seed Oxidized Lipids by High-Resolution LC-MS/MS.

PubMed

Riewe, David; Wiebach, Janine; Altmann, Thomas

2017-10-01

Lipid oxidation is a process ubiquitous in life, but the direct and comprehensive analysis of oxidized lipids has been limited by available analytical methods. We applied high-resolution liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS) to quantify oxidized lipids (glycerides, fatty acids, phospholipids, lysophospholipids, and galactolipids) and implemented a platform-independent high-throughput-amenable analysis pipeline for the high-confidence annotation and acyl composition analysis of oxidized lipids. Lipid contents of 90 different naturally aged wheat ( Triticum aestivum ) seed stocks were quantified in an untargeted high-resolution LC-MS experiment, resulting in 18,556 quantitative mass-to-charge ratio features. In a posthoc liquid chromatography-tandem mass spectrometry experiment, high-resolution MS/MS spectra (5 mD accuracy) were recorded for 8,957 out of 12,080 putatively monoisotopic features of the LC-MS data set. A total of 353 nonoxidized and 559 oxidized lipids with up to four additional oxygen atoms were annotated based on the accurate mass recordings (1.5 ppm tolerance) of the LC-MS data set and filtering procedures. MS/MS spectra available for 828 of these annotations were analyzed by translating experimentally known fragmentation rules of lipids into the fragmentation of oxidized lipids. This led to the identification of 259 nonoxidized and 365 oxidized lipids by both accurate mass and MS/MS spectra and to the determination of acyl compositions for 221 nonoxidized and 295 oxidized lipids. Analysis of 15-year aged wheat seeds revealed increased lipid oxidation and hydrolysis in seeds stored in ambient versus cold conditions. © 2017 The author(s). All Rights Reserved.

An Artifact in LC-MS/MS Measurement of Glutamine and Glutamic Acid: In-Source Cyclization to Pyroglutamic Acid

PubMed Central

2015-01-01

Advances in metabolomics, particularly for research on cancer, have increased the demand for accurate, highly sensitive methods for measuring glutamine (Gln) and glutamic acid (Glu) in cell cultures and other biological samples. N-terminal Gln and Glu residues in proteins or peptides have been reported to cyclize to pyroglutamic acid (pGlu) during liquid chromatography (LC)-mass spectrometry (MS) analysis, but cyclization of free Gln and Glu to free pGlu during LC-MS analysis has not been well-characterized. Using an LC-MS/MS protocol that we developed to separate Gln, Glu, and pGlu, we found that free Gln and Glu cyclize to pGlu in the electrospray ionization source, revealing a previously uncharacterized artifact in metabolomic studies. Analysis of Gln standards over a concentration range from 0.39 to 200 μM indicated that a minimum of 33% and maximum of almost 100% of Gln was converted to pGlu in the ionization source, with the extent of conversion dependent on fragmentor voltage. We conclude that the sensitivity and accuracy of Gln, Glu, and pGlu quantitation by electrospray ionization-based mass spectrometry can be improved dramatically by using (i) chromatographic conditions that adequately separate the three metabolites, (ii) isotopic internal standards to correct for in-source pGlu formation, and (iii) user-optimized fragmentor voltage for acquisition of the MS spectra. These findings have immediate impact on metabolomics and metabolism research using LC-MS technologies. PMID:24892977

An artifact in LC-MS/MS measurement of glutamine and glutamic acid: in-source cyclization to pyroglutamic acid.

PubMed

Purwaha, Preeti; Silva, Leslie P; Hawke, David H; Weinstein, John N; Lorenzi, Philip L

2014-06-17

Advances in metabolomics, particularly for research on cancer, have increased the demand for accurate, highly sensitive methods for measuring glutamine (Gln) and glutamic acid (Glu) in cell cultures and other biological samples. N-terminal Gln and Glu residues in proteins or peptides have been reported to cyclize to pyroglutamic acid (pGlu) during liquid chromatography (LC)-mass spectrometry (MS) analysis, but cyclization of free Gln and Glu to free pGlu during LC-MS analysis has not been well-characterized. Using an LC-MS/MS protocol that we developed to separate Gln, Glu, and pGlu, we found that free Gln and Glu cyclize to pGlu in the electrospray ionization source, revealing a previously uncharacterized artifact in metabolomic studies. Analysis of Gln standards over a concentration range from 0.39 to 200 μM indicated that a minimum of 33% and maximum of almost 100% of Gln was converted to pGlu in the ionization source, with the extent of conversion dependent on fragmentor voltage. We conclude that the sensitivity and accuracy of Gln, Glu, and pGlu quantitation by electrospray ionization-based mass spectrometry can be improved dramatically by using (i) chromatographic conditions that adequately separate the three metabolites, (ii) isotopic internal standards to correct for in-source pGlu formation, and (iii) user-optimized fragmentor voltage for acquisition of the MS spectra. These findings have immediate impact on metabolomics and metabolism research using LC-MS technologies.

Analysis of the differentially expressed low molecular weight peptides in human serum via an N-terminal isotope labeling technique combining nano-liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry.

PubMed

Leng, Jiapeng; Zhu, Dong; Wu, Duojiao; Zhu, Tongyu; Zhao, Ningwei; Guo, Yinlong

2012-11-15

Peptidomics analysis of human serum is challenging due to the low abundance of serum peptides and interference from the complex matrix. This study analyzed the differentially expressed (DE) low molecular weight peptides in human serum integrating a DMPITC-based N-terminal isotope labeling technique with nano-liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry (nano-LC/MALDI-MS). The workflow introduced a [d(6)]-4,6-dimethoxypyrimidine-2-isothiocyanate (DMPITC)-labeled mixture of aliquots from test samples as the internal standard. The spiked [d(0)]-DMPITC-labeled samples were separated by nano-LC then spotted on the MALDI target. Both quantitative and qualitative studies for serum peptides were achieved based on the isotope-labeled peaks. The DMPITC labeling technique combined with nano-LC/MALDI-MS not only minimized the errors in peptide quantitation, but also allowed convenient recognition of the labeled peptides due to the 6 Da mass difference. The data showed that the entire research procedure as well as the subsequent data analysis method were effective, reproducible, and sensitive for the analysis of DE serum peptides. This study successfully established a research model for DE serum peptides using DMPITC-based N-terminal isotope labeling and nano-LC/MALDI-MS. Application of the DMPITC-based N-terminal labeling technique is expected to provide a promising tool for the investigation of peptides in vivo, especially for the analysis of DE peptides under different biological conditions. Copyright © 2012 John Wiley & Sons, Ltd.

Development of a pressurized liquid extraction-solid-phase extraction followed by liquid chromatography-electrospray ionization tandem mass spectrometry method for the quantitative determination of benzoxazolinones and their degradation products in agricultural soil.

PubMed

Villagrasa, M; Guillamón, M; Navarro, A; Eljarrat, E; Barceló, D

2008-02-01

A new analytical method for the quantitative determination of benzoxazolinones and their degradation products in agricultural soils based on the use of pressurized liquid extraction (PLE) followed by solid-phase extraction (SPE) and then instrumental determination using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) is described. Using this method, the characterization, separation and quantitative detection of a mixture of two benzoxazolinones, benzoxazolin-2-one (BOA) and 6-methoxybenzoxazolin-2-one (MBOA) and their degradation products, 2-aminophenol (APH), N-(2-hydroxyphenyl)malonamic acid (HMPMA), 2-amino-3-H-phenoxazin-3-one (APO), 9-methoxy-2-amino-3-H-phenoxazin-3-one (AMPO), 2-acetylamino-3-H-phenoxazin-3-one (AAPO) and 2-acetylamino-9-methoxy-2-amino-3-H-phenoxazin-3-one (AAMPO) was achieved. The complete LC-ESI-MS-MS precursor-product ion fragmentation pathways for the degradation products of benzoxazolinones are described for the first time. Quantitative analysis was done in the multiple reaction mode using two specific combinations of precursor-product ion transitions for each compound. The optimized method was quality assessed by the measure of parameter as recovery, linearity, sensitivity, repeatability and reproducibility. Recoveries of the analytes ranged from 53 to 123%. The developed method offered improvements to the sensitivity as compared with our previously LC-MS method, with detection limits down to 2.4-21 ng/g of dry weight. This achievement allows us to identify and quantify for the first time degradation products of benzoxazolinones in real agricultural soil samples. Analytes were found in the range of 20.6-149 ng/g dry weight.

Hematocrit-Independent Quantitation of Stimulants in Dried Blood Spots: Pipet versus Microfluidic-Based Volumetric Sampling Coupled with Automated Flow-Through Desorption and Online Solid Phase Extraction-LC-MS/MS Bioanalysis.

PubMed

Verplaetse, Ruth; Henion, Jack

2016-07-05

A workflow overcoming microsample collection issues and hematocrit (HCT)-related bias would facilitate more widespread use of dried blood spots (DBS). This report describes comparative results between the use of a pipet and a microfluidic-based sampling device for the creation of volumetric DBS. Both approaches were successfully coupled to HCT-independent, fully automated sample preparation and online liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis allowing detection of five stimulants in finger prick blood. Reproducible, selective, accurate, and precise responses meeting generally accepted regulated bioanalysis guidelines were observed over the range of 5-1000 ng/mL whole blood. The applied heated flow-through solvent desorption of the entire spot and online solid phase extraction (SPE) procedure were unaffected by the blood's HCT value within the tested range of 28.0-61.5% HCT. Enhanced stability for mephedrone on DBS compared to liquid whole blood was observed. Finger prick blood samples were collected using both volumetric sampling approaches over a time course of 25 h after intake of a single oral dose of phentermine. A pharmacokinetic curve for the incurred phentermine was successfully produced using the described validated method. These results suggest that either volumetric sample collection method may be amenable to field-use followed by fully automated, HCT-independent DBS-SPE-LC-MS/MS bioanalysis for the quantitation of these representative controlled substances. Analytical data from DBS prepared with a pipet and microfluidic-based sampling devices were comparable, but the latter is easier to operate, making this approach more suitable for sample collection by unskilled persons.

A comparative analysis of human plasma and serum proteins by combining native PAGE, whole-gel slicing and quantitative LC-MS/MS: Utilizing native MS-electropherograms in proteomic analysis for discovering structure and interaction-correlated differences.

PubMed

Wen, Meiling; Jin, Ya; Manabe, Takashi; Chen, Shumin; Tan, Wen

2017-12-01

MS identification has long been used for PAGE-separated protein bands, but global and systematic quantitation utilizing MS after PAGE has remained rare and not been reported for native PAGE. Here we reported on a new method combining native PAGE, whole-gel slicing and quantitative LC-MS/MS, aiming at comparative analysis on not only abundance, but also structures and interactions of proteins. A pair of human plasma and serum samples were used as test samples and separated on a native PAGE gel. Six lanes of each sample were cut, each lane was further sliced into thirty-five 1.1 mm × 1.1 mm squares and all the squares were subjected to standardized procedures of in-gel digestion and quantitative LC-MS/MS. The results comprised 958 data rows that each contained abundance values of a protein detected in one square in eleven gel lanes (one plasma lane excluded). The data were evaluated to have satisfactory reproducibility of assignment and quantitation. Totally 315 proteins were assigned, with each protein assigned in 1-28 squares. The abundance distributions in the plasma and serum gel lanes were reconstructed for each protein, named as "native MS-electropherograms". Comparison of the electropherograms revealed significant plasma-versus-serum differences on 33 proteins in 87 squares (fold difference > 2 or < 0.5, p < 0.05). Many of the differences matched with accumulated knowledge on protein interactions and proteolysis involved in blood coagulation, complement and wound healing processes. We expect this method would be useful to provide more comprehensive information in comparative proteomic analysis, on both quantities and structures/interactions. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Liquid chromatography with isotope-dilution mass spectrometry for determination of water-soluble vitamins in foods.

PubMed

Phillips, Melissa M

2015-04-01

Vitamins are essential for improving and maintaining human health, and the main source of vitamins is the diet. Measurement of the quantities of water-soluble vitamins in common food materials is important to understand the impact of vitamin intake on human health, and also to provide necessary information for regulators to determine adequate intakes. Liquid chromatography (LC) and mass spectrometry (MS) based methods for water-soluble vitamin analysis are abundant in the literature, but most focus on only fortified foods or dietary supplements or allow determination of only a single vitamin. In this work, a method based on LC/MS and LC/MS/MS has been developed to allow simultaneous quantitation of eight water-soluble vitamins, including multiple forms of vitamins B3 and B6, in a variety of fortified and unfortified food-matrix Standard Reference Materials (SRMs). Optimization of extraction of unbound vitamin forms and confirmation using data from external laboratories ensured accuracy in the assigned values, and addition of stable isotope labeled internal standards for each of the vitamins allowed for increased precision.

Mass spectrometric analysis of sulfur mustard-induced biomolecular adducts: Are DNA adducts suitable biomarkers of exposure?

PubMed

Zubel, Tabea; Bürkle, Alexander; Mangerich, Aswin

2018-09-01

The bi-functional chemical warfare agent sulfur mustard (SM), whose release in asymmetric conflicts or terrorist attacks represents a realistic threat, induces several kinds of biomolecular adducts, including highly toxic DNA adducts. Isotope dilution liquid chromatographic tandem mass spectrometry (ID-LC-MS/MS) is considered the gold standard for highly accurate, precise, specific and sensitive quantification of DNA adducts in general. Recently, a number of LC-MS/MS approaches have been established to analyze SM-induced protein and DNA adducts in cell culture and rodent animal models. As DNA adducts are mechanism-based biomarkers for SM exposure, results from such studies provide a deeper understanding of the etiology of SM-induced pathologies, especially of long-term effects such as cancer formation. As a result, medical treatment of SM-exposed individuals might be improved. Yet, despite the progress that has been made during the last years, there is still a need for advanced methods of ID-LC-MS/MS for the detection and quantitation of SM adducts. Copyright © 2017 Elsevier B.V. All rights reserved.

Development and validation of a serum total testosterone liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay calibrated to NIST SRM 971.

PubMed

French, Deborah

2013-01-16

At our institution, serum testosterone in adult males is measured by immunoassay while female and pediatric specimens are sent to a reference laboratory for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis due to low concentrations. As this is of significant cost, a testosterone LC-MS/MS assay was developed in-house. A 5500 QTRAP® using electrospray ionization and a Shimadzu Prominence with a C18 column were used. Gradient elution with formic acid, water and methanol:acetonitrile at 0.5 ml/min had a 7-min run-time. A liquid-liquid extraction with hexane:ethyl acetate was carried out on 200 μl of serum. Multiple reaction monitoring was employed. Sample preparation took ~80 min for 21 samples. Six calibrators were used (0-1263 ng/dl; concentration assigned by NIST SRM 971) with 3 quality controls (9, 168 and 532 ng/dl). The limits of detection and quantitation were 1 and 2 ng/dl respectively. Extraction recovery was ~90% and ion suppression ~5%. Within-run and total precision studies yielded <15% CV at the limit of quantitation and <7% CV through the rest of the linear range. Isobaric interferences were baseline separated from testosterone. Method comparisons between this assay, an immunoassay, and another LC-MS/MS assay were completed. An accurate and sensitive LC-MS/MS assay for total testosterone was developed. Bringing this assay in-house reduces turnaround time for clinicians and patients and saves our institution funds. Copyright © 2012 Elsevier B.V. All rights reserved.

Development and validation of sensitive LC-MS/MS assays for quantification of HP-β-CD in human plasma and CSF

PubMed Central

Jiang, Hui; Sidhu, Rohini; Fujiwara, Hideji; De Meulder, Marc; de Vries, Ronald; Gong, Yong; Kao, Mark; Porter, Forbes D.; Yanjanin, Nicole M.; Carillo-Carasco, Nuria; Xu, Xin; Ottinger, Elizabeth; Woolery, Myra; Ory, Daniel S.; Jiang, Xuntian

2014-01-01

2-Hydroxypropyl-β-cyclodextrin (HP-β-CD), a widely used excipient for drug formulation, has emerged as an investigational new drug for the treatment of Niemann-Pick type C1 (NPC1) disease, a neurodegenerative cholesterol storage disorder. Development of a sensitive quantitative LC-MS/MS assay to monitor the pharmacokinetics (PKs) of HP-β-CD required for clinical trials has been challenging owing to the dispersity of the HP-β-CD. To support a phase 1 clinical trial for ICV delivery of HP-β-CD in NPC1 patients, novel methods for quantification of HP-β-CD in human plasma and cerebrospinal fluid (CSF) using LC-MS/MS were developed and validated: a 2D-LC-in-source fragmentation-MS/MS (2D-LC-IF-MS/MS) assay and a reversed phase ultra performance LC-MS/MS (RP-UPLC-MS/MS) assay. In both assays, protein precipitation and “dilute and shoot” procedures were used to process plasma and CSF, respectively. The assays were fully validated and in close agreement, and allowed determination of PK parameters for HP-β-CD. The LC-MS/MS methods are ∼100-fold more sensitive than the current HPLC assay, and were successfully employed to analyze HP-β-CD in human plasma and CSF samples to support the phase 1 clinical trial of HP-β-CD in NPC1 patients. PMID:24868096

Development and validation of sensitive LC-MS/MS assays for quantification of HP-β-CD in human plasma and CSF.

PubMed

Jiang, Hui; Sidhu, Rohini; Fujiwara, Hideji; De Meulder, Marc; de Vries, Ronald; Gong, Yong; Kao, Mark; Porter, Forbes D; Yanjanin, Nicole M; Carillo-Carasco, Nuria; Xu, Xin; Ottinger, Elizabeth; Woolery, Myra; Ory, Daniel S; Jiang, Xuntian

2014-07-01

2-Hydroxypropyl-β-cyclodextrin (HP-β-CD), a widely used excipient for drug formulation, has emerged as an investigational new drug for the treatment of Niemann-Pick type C1 (NPC1) disease, a neurodegenerative cholesterol storage disorder. Development of a sensitive quantitative LC-MS/MS assay to monitor the pharmacokinetics (PKs) of HP-β-CD required for clinical trials has been challenging owing to the dispersity of the HP-β-CD. To support a phase 1 clinical trial for ICV delivery of HP-β-CD in NPC1 patients, novel methods for quantification of HP-β-CD in human plasma and cerebrospinal fluid (CSF) using LC-MS/MS were developed and validated: a 2D-LC-in-source fragmentation-MS/MS (2D-LC-IF-MS/MS) assay and a reversed phase ultra performance LC-MS/MS (RP-UPLC-MS/MS) assay. In both assays, protein precipitation and "dilute and shoot" procedures were used to process plasma and CSF, respectively. The assays were fully validated and in close agreement, and allowed determination of PK parameters for HP-β-CD. The LC-MS/MS methods are ∼100-fold more sensitive than the current HPLC assay, and were successfully employed to analyze HP-β-CD in human plasma and CSF samples to support the phase 1 clinical trial of HP-β-CD in NPC1 patients. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

Interlaboratory Validation of a Stable Isotope Dilution and Liquid Chromatography Tandem Mass Spectrometry Method for the Determination of Aflatoxins in Milk, Milk-Based Infant Formula, and Feed.

PubMed

Zhang, Kai; Liao, Chia-Ding; Prakash, Shristi; Conway, Michael; Cheng, Hwei-Fang

2018-05-01

An interlaboratory study was conducted to evaluate stable isotope dilution and LC tandem MS (MS/MS) for the determination of aflatoxins B1, B2, G1, G2, and M1 (AFB1, AFB2, AFG1, AFG2, and AFM1) in milk, milk-based infant formula (formula), and feed. Samples were first fortified with five 13C uniformly labeled aflatoxins {[13C]-internal standard (IS)} corresponding to the five native aflatoxins, which were subsequently extracted with acetonitrile-water (50 + 50, v/v), followed by centrifugation, filtration, and LC-MS/MS analysis. In addition to certified milk powder and animal feed, the three participating laboratories also analyzed milk, formula, and feed fortified with the five aflatoxins at concentrations ranging from 0.5 to 50 ng/g. The majority of recoveries ranged from 80 to 120%, with RSDs < 20%. Method LOQs were determined by the three laboratories using the three sample matrixes in replicates (n = 8), and the determined LOQs of AFB1, AFB2, AFG1, AFG2, and AFM1 ranged from 0.1 to 0.91, 0.24 to 0.64, 0.28 to 1.52, 0.19 to 3.80, and 0.12 to 0.45 ng/g, respectively. For detected aflatoxins in the certified materials, all measured concentrations were within ±25% of the certified values. Using [13C]-IS eliminated the need for matrix-matched calibration standards for quantitation, simplified sample preparation, and achieved simultaneous identification and quantitation of the aflatoxins in a simple LC-MS/MS procedure.

Single hair analysis of small molecules using MALDI-triple quadrupole MS imaging and LC-MS/MS: investigations on opportunities and pitfalls.

PubMed

Poetzsch, Michael; Steuer, Andrea E; Roemmelt, Andreas T; Baumgartner, Markus R; Kraemer, Thomas

2014-12-02

Single hair analysis normally requires extensive sample preparation microscale protocols including time-consuming steps like segmentation and extraction. Matrix assisted laser desorption and ionization mass spectrometric imaging (MALDI-MSI) was shown to be an alternative tool in single hair analysis, but still, questions remain. Therefore, an investigation of MALDI-MSI in single hair analysis concerning the extraction process, usage of internal standard (IS), and influences on the ionization processes were systematically investigated to enable the reliable application to hair analysis. Furthermore, single dose detection, quantitative correlation to a single hair, and hair strand LC-MS/MS results were performed, and the performance was compared to LC-MS/MS single hair monitoring. The MALDI process was shown to be independent from natural hair color and not influenced by the presence of melanin. Ionization was shown to be reproducible along and in between different hair samples. MALDI image intensities in single hair and hair snippets showed good semiquantitative correlation to zolpidem hair concentrations obtained from validated routine LC-MS/MS methods. MALDI-MSI is superior to LC-MS/MS analysis when a fast, easy, and cheap sample preparation is necessary, whereas LC-MS/MS showed higher sensitivity with the ability of single dose detection for zolpidem. MALDI-MSI and LC-MS/MS segmental single hair analysis showed good correlation, and both are suitable for consumption monitoring of drugs of abuse with a high time resolution.

Accurate inclusion mass screening: a bridge from unbiased discovery to targeted assay development for biomarker verification.

PubMed

Jaffe, Jacob D; Keshishian, Hasmik; Chang, Betty; Addona, Theresa A; Gillette, Michael A; Carr, Steven A

2008-10-01

Verification of candidate biomarker proteins in blood is typically done using multiple reaction monitoring (MRM) of peptides by LC-MS/MS on triple quadrupole MS systems. MRM assay development for each protein requires significant time and cost, much of which is likely to be of little value if the candidate biomarker is below the detection limit in blood or a false positive in the original discovery data. Here we present a new technology, accurate inclusion mass screening (AIMS), designed to provide a bridge from unbiased discovery to MS-based targeted assay development. Masses on the software inclusion list are monitored in each scan on the Orbitrap MS system, and MS/MS spectra for sequence confirmation are acquired only when a peptide from the list is detected with both the correct accurate mass and charge state. The AIMS experiment confirms that a given peptide (and thus the protein from which it is derived) is present in the plasma. Throughput of the method is sufficient to qualify up to a hundred proteins/week. The sensitivity of AIMS is similar to MRM on a triple quadrupole MS system using optimized sample preparation methods (low tens of ng/ml in plasma), and MS/MS data from the AIMS experiments on the Orbitrap can be directly used to configure MRM assays. The method was shown to be at least 4-fold more efficient at detecting peptides of interest than undirected LC-MS/MS experiments using the same instrumentation, and relative quantitation information can be obtained by AIMS in case versus control experiments. Detection by AIMS ensures that a quantitative MRM-based assay can be configured for that protein. The method has the potential to qualify large number of biomarker candidates based on their detection in plasma prior to committing to the time- and resource-intensive steps of establishing a quantitative assay.

Fast targeted analysis of 132 acidic and neutral drugs and poisons in whole blood using LC-MS/MS.

PubMed

Di Rago, Matthew; Saar, Eva; Rodda, Luke N; Turfus, Sophie; Kotsos, Alex; Gerostamoulos, Dimitri; Drummer, Olaf H

2014-10-01

The aim of this study was to develop an LC-MS/MS based screening technique that covers a broad range of acidic and neutral drugs and poisons by combining a small sample volume and efficient extraction technique with simple automated data processing. After protein precipitation of 100μL of whole blood, 132 common acidic and neutral drugs and poisons including non-steroidal anti-inflammatory drugs, barbiturates, anticonvulsants, antidiabetics, muscle relaxants, diuretics and superwarfarin rodenticides (47 quantitated, 85 reported as detected) were separated using a Shimadzu Prominence HPLC system with a C18 separation column (Kinetex XB-C18, 4.6mm×150mm, 5μm), using gradient elution with a mobile phase of 25mM ammonium acetate buffer (pH 7.5)/acetonitrile. The drugs were detected using an ABSciex(®) API 2000 LC-MS/MS system (ESI+ and -, MRM mode, two transitions per analyte). The method was fully validated in accordance with international guidelines. Quantification data obtained using one-point calibration compared favorably to that using multiple calibrants. The presented LC-MS/MS assay has proven to be applicable for determination of the analytes in blood. The fast and reliable extraction method combined with automated processing gives the opportunity for high throughput and fast turnaround times for forensic and clinical toxicology. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Targeted liquid chromatography tandem mass spectrometry to quantitate wheat gluten using well-defined reference proteins.

PubMed

Schalk, Kathrin; Koehler, Peter; Scherf, Katharina Anne

2018-01-01

Celiac disease (CD) is an inflammatory disorder of the upper small intestine caused by the ingestion of storage proteins (prolamins and glutelins) from wheat, barley, rye, and, in rare cases, oats. CD patients need to follow a gluten-free diet by consuming gluten-free products with gluten contents of less than 20 mg/kg. Currently, the recommended method for the quantitative determination of gluten is an enzyme-linked immunosorbent assay (ELISA) based on the R5 monoclonal antibody. Because the R5 ELISA mostly detects the prolamin fraction of gluten, a new independent method is required to detect prolamins as well as glutelins. This paper presents the development of a method to quantitate 16 wheat marker peptides derived from all wheat gluten protein types by liquid chromatography tandem mass spectrometry (LC-MS/MS) in the multiple reaction monitoring mode. The quantitation of each marker peptide in the chymotryptic digest of a defined amount of the respective reference wheat protein type resulted in peptide-specific yields. This enabled the conversion of peptide into protein type concentrations. Gluten contents were expressed as sum of all determined protein type concentrations. This new method was applied to quantitate gluten in wheat starches and compared to R5 ELISA and gel-permeation high-performance liquid chromatography with fluorescence detection (GP-HPLC-FLD), which resulted in a strong correlation between LC-MS/MS and the other two methods.

Improvement of Nicotinic Acid and Nicotinamide Analysis in Meats and Meat Products by HPLC and LC-MS/MS with Solid-Phase Extraction.

PubMed

Hiki, Asako; Yamajima, Yukiko; Uematsu, Yoko

2016-01-01

A method for nicotinic acid (NA) and nicotinamide (NAA) analysis in meats was developed. NA and NAA were extracted from meats or meat products with metaphosphate aqueous solution. The extract was cleaned up with an Oasis MCX cartridge. The cartridge was washed with 2% acetic acid (v/v) and acetic acid-methanol solution. NA and NAA were eluted with ammonia-methanol solution. NA and NAA in the eluate were chromatographed on a Scherzo SM-C18 (3.0×150 mm, 3.0 μm) column with 20 mmol/L ammonium acetate containing 0.1% acetic acid-acetonitrile (97 : 3) as a mobile phase and were monitored at 261 nm. Quantification was performed by LC and LC-MS/MS. Calibration curves showed high linearity (correlation coefficient>0.998) between 1-25 μg/mL for LC and LC-MS/MS. Recoveries were 84-108% (CV≦5.8%) by HPLC and 79-105% (CV≦9.0%) by LC-MS/MS. The limit of quantitation for NA was 0.005-0.01 g/kg and that for NAA was 0.01-0.02 g/kg.

Preprocessing and Analysis of LC-MS-Based Proteomic Data

PubMed Central

Tsai, Tsung-Heng; Wang, Minkun; Ressom, Habtom W.

2016-01-01

Liquid chromatography coupled with mass spectrometry (LC-MS) has been widely used for profiling protein expression levels. This chapter is focused on LC-MS data preprocessing, which is a crucial step in the analysis of LC-MS based proteomics. We provide a high-level overview, highlight associated challenges, and present a step-by-step example for analysis of data from LC-MS based untargeted proteomic study. Furthermore, key procedures and relevant issues with the subsequent analysis by multiple reaction monitoring (MRM) are discussed. PMID:26519169

Improvement of Quantitative Measurements in Multiplex Proteomics Using High-Field Asymmetric Waveform Spectrometry.

PubMed

Pfammatter, Sibylle; Bonneil, Eric; Thibault, Pierre

2016-12-02

Quantitative proteomics using isobaric reagent tandem mass tags (TMT) or isobaric tags for relative and absolute quantitation (iTRAQ) provides a convenient approach to compare changes in protein abundance across multiple samples. However, the analysis of complex protein digests by isobaric labeling can be undermined by the relative large proportion of co-selected peptide ions that lead to distorted reporter ion ratios and affect the accuracy and precision of quantitative measurements. Here, we investigated the use of high-field asymmetric waveform ion mobility spectrometry (FAIMS) in proteomic experiments to reduce sample complexity and improve protein quantification using TMT isobaric labeling. LC-FAIMS-MS/MS analyses of human and yeast protein digests led to significant reductions in interfering ions, which increased the number of quantifiable peptides by up to 68% while significantly improving the accuracy of abundance measurements compared to that with conventional LC-MS/MS. The improvement in quantitative measurements using FAIMS is further demonstrated for the temporal profiling of protein abundance of HEK293 cells following heat shock treatment.

Quantitation of acrylamide in foods by high-resolution mass spectrometry.

PubMed

Troise, Antonio Dario; Fiore, Alberto; Fogliano, Vincenzo

2014-01-08

Acrylamide detection still represents one of the hottest topics in food chemistry. Solid phase cleanup coupled to liquid chromatography separation and tandem mass spectrometry detection along with GC-MS detection are nowadays the gold standard procedure for acrylamide quantitation thanks to high reproducibility, good recovery, and low relative standard deviation. High-resolution mass spectrometry (HRMS) is particularly suitable for the detection of low molecular weight amides, and it can provide some analytical advantages over other MS techniques. In this paper a liquid chromatography (LC) method for acrylamide determination using HRMS detection was developed and compared to LC coupled to tandem mass spectrometry. The procedure applied a simplified extraction, no cleanup steps, and a 4 min chromatography. It proved to be solid and robust with an acrylamide mass accuracy of 0.7 ppm, a limit of detection of 2.65 ppb, and a limit of quantitation of 5 ppb. The method was tested on four acrylamide-containing foods: cookies, French fries, ground coffee, and brewed coffee. Results were perfectly in line with those obtained by LC-MS/MS.

The future of targeted peptidomics.

PubMed

Findeisen, Peter

2013-12-01

Targeted MS is becoming increasingly important for sensitive and specific quantitative detection of proteins and respective PTMs. In this article, Ceglarek et al. [Proteomics Clin. Appl. 2013, 7, 794-801] present an LC-MS-based method for simultaneous quantitation of seven apolipoproteins in serum specimens. The assay fulfills many necessities of routine diagnostic applications, namely, low cost, high throughput, and good reproducibility. We anticipate that validation of new biomarkers will speed up with this technology and the palette of laboratory-based diagnostic tools will hopefully be augmented significantly in the near future. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Intact stable isotope labeled plasma proteins from the SILAC-labeled HepG2 secretome.

PubMed

Mangrum, John B; Martin, Erika J; Brophy, Donald F; Hawkridge, Adam M

2015-09-01

The plasma proteome remains an attractive biospecimen for MS-based biomarker discovery studies. The success of these efforts relies on the continued development of quantitative MS-based proteomics approaches. Herein we report the use of the SILAC-labeled HepG2 secretome as a source for stable isotope labeled plasma proteins for quantitative LC-MS/MS measurements. The HepG2 liver cancer cell line secretes the major plasma proteins including serum albumin, apolipoproteins, protease inhibitors, coagulation factors, and transporters that represent some of the most abundant proteins in plasma. The SILAC-labeled HepG2 secretome was collected, spiked into human plasma (1:1 total protein), and then processed for LC-MS/MS analysis. A total of 62 and 56 plasma proteins were quantified (heavy:light (H/L) peptide pairs) from undepleted and depleted (serum albumin and IgG), respectively, with log2 H/L = ± 6. Major plasma proteins quantified included albumin, apolipoproteins (e.g., APOA1, APOA2, APOA4, APOB, APOC3, APOE, APOH, and APOM), protease inhibitors (e.g., A2M and SERPINs), coagulation factors (e.g., Factor V, Factor X, fibrinogen), and transport proteins (e.g., TTR). The average log2 H/L values for shared plasma proteins in both undepleted and depleted plasma samples were 0.43 and 0.44, respectively. This work further expands the SILAC strategy into MS-based biomarker discovery of clinical biospecimens. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A serially coupled stationary phase method for the determination of urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine by liquid chromatography ion trap tandem mass spectrometry☆

PubMed Central

Rota, Cristina; Cristoni, Simone; Trenti, Tommaso; Cariani, Elisabetta

2013-01-01

Oxidative attack to DNA is of particular interest since DNA modifications can lead to heritable mutations. The most studied product of DNA oxidation is 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG). While 8-oxodG determination in blood and tissue cells is prone to artifacts, its measurement in urine employing liquid chromatography tandem mass spectrometry (LC-MS/MS) has gained more and more interest for increased reliability. LC-MS/MS can be affected by matrix effects and this is particularly true when ion trap is used as MS analyzer, due to ion accumulation in the trap and related space charge effect. In the present work, we have developed a LC-MS/MS method where the combination of cation exchange and reverse phase solid phases resulted in LC separation optimization. This together with the employment of an isotopically labeled internal standard, allowed the usage of ion trap LC-MS/MS, typically not employed for quantitative measurement in biological samples, for the measurement of 8-oxodG in urine samples from control populations. Four different urine matrices were employed for method validation. Limit of quantitation was set at least at 0.5 ng/ml. While analyzing urine samples from healthy volunteers, 8-oxodG levels reported as ng/ml were statistically different comparing males with females (p<0.05, Mann Whitney test); while comparing results normalized for creatinine no statistical significant difference was found. Mean urinary 8-oxodG level found in healthy volunteers was 1.16±0.46 nmol/mmol creatinine. The present method by enhancing at best the chromatographic performances allows the usage of ion trap LC-MS/MS for the measurement of 8-oxodG in urine samples from control populations. PMID:24251117

Towards a proteome signature for invasive ductal breast carcinoma derived from label-free nanoscale LC-MS protein expression profiling of tumorous and glandular tissue.

PubMed

Röwer, Claudia; Vissers, Johannes P C; Koy, Cornelia; Kipping, Marc; Hecker, Michael; Reimer, Toralf; Gerber, Bernd; Thiesen, Hans-Jürgen; Glocker, Michael O

2009-12-01

As more and more alternative treatments become available for breast carcinoma, there is a need to stratify patients and individual molecular information seems to be suitable for this purpose. In this study, we applied label-free protein quantitation by nanoscale LC-MS and investigated whether this approach could be used for defining a proteome signature for invasive ductal breast carcinoma. Tissue samples from healthy breast and tumor were collected from three patients. Protein identifications were based on LC-MS peptide fragmentation data which were obtained simultaneously to the quantitative information. Hereby, an invasive ductal breast carcinoma proteome signature was generated which contains 60 protein entries. The on-column concentrations for osteoinductive factor, vimentin, GAP-DH, and NDKA are provided as examples. These proteins represent distinctive gene ontology groups of differentially expressed proteins and are discussed as risk markers for primary tumor pathogenesis. The developed methodology has been found well applicable in a clinical environment in which standard operating procedures can be kept; a prerequisite for the definition of molecular parameter sets that shall be capable for stratification of patients.

Effective determination of a pharmaceutical, sulpiride, in river water by online SPE-LC-MS using a molecularly imprinted polymer as a preconcentration medium.

PubMed

Kubo, Takuya; Kuroda, Kenta; Tominaga, Yuichi; Naito, Toyohiro; Sueyoshi, Kenji; Hosoya, Ken; Otsuka, Koji

2014-02-01

We report an effective and a quantitative analysis method for one of pharmaceuticals, sulpiride, in river water by online solid phase extraction (SPE) connected with liquid chromatography-mass spectrometry (LC-MS) using a molecularly imprinted polymer as a preconcentration medium. The polymer prepared with a pseudo template molecule showed the selective retention ability based on the interval recognition of functional groups in sulpiride. Also, the imprinted polymer provided an effective concentration of a trace level of sulpiride in offline SPE with dual washing processes using water and acetonitrile, although another imprinted polymer prepared by an authentic method using sulpiride and methacrylic acid as a template and a functional monomer, respectively, showed the selective adsorption only in organic solvents. Furthermore, we employed the imprinted polymer as the preconcentration column of online SPE-LC-MS and the results supposed that the proposed system allowed the quantitative analysis of sulpiride with high sensitivity and recovery (10ng/L at 96%). Additionally, the determination of sulpiride in real river water without an additional spiking was effectively achieved by the system. Copyright © 2013 Elsevier B.V. All rights reserved.

Quantitative LC–MS for water-soluble heterocyclic amines in fine aerosols (PM2.5) at Duke Forest, USA

EPA Science Inventory

In this study, a quantitative liquid chromatography-mass spectrometry (LC-MS) technique capable of measuring the concentrations of heterocyclic nitrogen compounds in ambient fine aerosols (PM2.5) has been developed. Quadrupole time-of-flight (Q-TOF) MS technology is used to provi...

Endogenous glucocorticoid analysis by liquid chromatography-tandem mass spectrometry in routine clinical laboratories.

PubMed

Hawley, James M; Keevil, Brian G

2016-09-01

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful analytical technique that offers exceptional selectivity and sensitivity. Used optimally, LC-MS/MS provides accurate and precise results for a wide range of analytes at concentrations that are difficult to quantitate with other methodologies. Its implementation into routine clinical biochemistry laboratories has revolutionised our ability to analyse small molecules such as glucocorticoids. Whereas immunoassays can suffer from matrix effects and cross-reactivity due to interactions with structural analogues, the selectivity offered by LC-MS/MS has largely overcome these limitations. As many clinical guidelines are now beginning to acknowledge the importance of the methodology used to provide results, the advantages associated with LC-MS/MS are gaining wider recognition. With their integral role in both the diagnosis and management of hypo- and hyperadrenal disorders, coupled with their widespread pharmacological use, the accurate measurement of glucocorticoids is fundamental to effective patient care. Here, we provide an up-to-date review of the LC-MS/MS techniques used to successfully measure endogenous glucocorticoids, particular reference is made to serum, urine and salivary cortisol. Copyright © 2016 Elsevier Ltd. All rights reserved.

Online extraction LC-MS/MS method for the simultaneous quantitative confirmation of urine drugs of abuse and metabolites: amphetamines, opiates, cocaine, cannabis, benzodiazepines and methadone.

PubMed

de Jager, Andrew D; Bailey, Neville L

2011-09-01

A rapid LC-MS/MS method for confirmatory testing of five major categories of drugs of abuse (amphetamine-type substances, opiates, cocaine, cannabis metabolites and benzodiazepines) in urine has been developed. All drugs of abuse mandated by the Australian/New Zealand Standard AS/NZS 4308:2008 are quantified in a single chromatographic run. Urine samples are diluted with a mixture of isotope labelled internal standards. An on-line trap-and-flush approach, followed by LC-ESI-MS/MS has been successfully used to process samples in a functioning drugs of abuse laboratory. Following injection of diluted urine samples, compounds retained on the trap cartridge are flushed onto a reverse-phase C18 HPLC column (5-μm particle size) with embedded hydrophylic functionality. A total chromatographic run-time of 15 min is required for adequate resolution. Automated quantitation software algorithms have been developed in-house using XML scripting to partially automate the identification of positive samples, taking into account ion ratio (IR) and retention times (Rt). The sensitivity of the assay was found to be adequate for the quantitation of drugs in urine at and below the confirmation cut-off concentrations prescribed by AS/NZS 4308:2008. Copyright © 2011 Elsevier B.V. All rights reserved.

Rapid quantitative analysis of 8-iso-prostaglandin-F(2alpha) using liquid chromatography-tandem mass spectrometry and comparison with an enzyme immunoassay method.

PubMed

Dahl, Jeffrey H; van Breemen, Richard B

2010-09-15

A rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed for the measurement of urinary 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)), a biomarker of lipid peroxidation. Because urine contains numerous F(2) prostaglandin isomers, each with identical mass and similar mass spectrometric fragmentation patterns, chromatographic separation of 8-iso-PGF(2alpha) from its isomers is necessary for its quantitative analysis using MS/MS. We were able to achieve this separation using an isocratic LC method with a run time of less than 9min, which is at least threefold faster than previous methods, while maintaining sensitivity, accuracy, precision, and reliability. The limits of detection and quantitation were 53 and 178pg/ml urine, respectively. We compared our method with a commercially available affinity purification and enzyme immunoassay kit and found both assays to be in agreement. Despite the high sensitivity of the enzyme immunoassay method, it is more expensive and has a narrower dynamic range than LC-MS/MS. Our method was optimized for rapid measurement of 8-iso-PGF(2alpha) in urine, and it is ideally suited for clinical sample analysis. 2010 Elsevier Inc. All rights reserved.

Comparison of the docetaxel concentration in human plasma measured with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and a nanoparticle immunoassay and clinical applications of that assay.

PubMed

Geng, Chunmei; Li, Pingli; Chen, Xuwang; Yuan, Guiyan; Guo, Nan; Liu, Huanjun; Zhang, Rui; Guo, Ruichen

2017-05-23

To determine the feasibility of using a nanoparticle immunoassay for clinical therapeutic drug monitoring (TDM) of docetaxel concentrations, a sensitive and simple method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established to measure the docetaxel concentration in human plasma and the results of LC-MS/MS and the immunoassay were compared. Docetaxel and paclitaxel (the internal standard, or IS) in human plasma were extracted through protein precipitation, separated on a Diamonsil C18 column (150 mm × 4.6 mm, 5 μm), ionized with positive ions, and detected with LC-MS/MS in multi-reaction monitoring (MRM) mode. Plasma samples from 248 cancer patients were assayed with LC-MS/MS and a nanoparticle immunoassay. Data from the samples were analyzed with the statistical software SPSS and the software MedCalc. Results indicated that the calibration curve of the validated method of LC-MS/MS was linear over the range of 10-2,000 ng/mL, with an lowest limit of quantitation (LLOQ) of 10 ng/mL, and the intra- and inter- day precision and accuracy were both < ± 15%. Comparison of the two methods indicated that results of the LC-MS/MS were closely related to those of the nanoparticle immunoassay, with a correlation coefficient (R) of 0.965 and acceptable 95% confidence intervals (CI) of ‒ 231.7-331.1 ng/mL. Overall, the established method of LC-MC/MS and the nanoparticle immunoassay were both suitable for measurement of the docetaxel concentration in human plasma, and the immunoassay was far more cost-effective and better at clinical TDM of docetaxel in clinical practice.

Application of the accurate mass and time tag approach in studies of the human blood lipidome

PubMed Central

Ding, Jie; Sorensen, Christina M.; Jaitly, Navdeep; Jiang, Hongliang; Orton, Daniel J.; Monroe, Matthew E.; Moore, Ronald J.; Smith, Richard D.; Metz, Thomas O.

2008-01-01

We report a preliminary demonstration of the accurate mass and time (AMT) tag approach for lipidomics. Initial data-dependent LC-MS/MS analyses of human plasma, erythrocyte, and lymphocyte lipids were performed in order to identify lipid molecular species in conjunction with complementary accurate mass and isotopic distribution information. Identified lipids were used to populate initial lipid AMT tag databases containing 250 and 45 entries for those species detected in positive and negative electrospray ionization (ESI) modes, respectively. The positive ESI database was then utilized to identify human plasma, erythrocyte, and lymphocyte lipids in high-throughput LC-MS analyses based on the AMT tag approach. We were able to define the lipid profiles of human plasma, erythrocytes, and lymphocytes based on qualitative and quantitative differences in lipid abundance. PMID:18502191

An evaluation of the DRI-ETG EIA method for the determination of ethyl glucuronide concentrations in clinical and post-mortem urine.

PubMed

Turfus, Sophie C; Vo, Tu; Niehaus, Nadia; Gerostamoulos, Dimitri; Beyer, Jochen

2013-06-01

A commercial enzyme immunoassay for the qualitative and semi-quantitative measurement of ethyl glucuronide (EtG) in urine was evaluated. Post-mortem (n=800), and clinical urine (n=200) samples were assayed using a Hitachi 902 analyzer. The determined concentrations were compared with those obtained using a previously published liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of EtG and ethyl sulfate. Using a cut-off of 0.5 µg/ml and LC-MS/MS limit of reporting of 0.1 µg/ml, there was a sensitivity of 60.8% and a specificity of 100% for clinical samples. For post-mortem samples, sensitivity and specificity were 82.4% and 97.1%, respectively. When reducing the cut-off to 0.1 µg/ml, the sensitivity and specificity were 83.3% and 100% for clinical samples whereas for post-mortem samples the sensitivity and specificity were 90.3 % and 88.3 %, respectively. The best trade-offs between sensitivity and specificity for LC-MS/MS limits of reporting of 0.5 and 0.1 µg/ml were achieved when using immunoassay cut-offs of 0.3 and 0.092 µg/ml, respectively. There was good correlation between quantitative results obtained by both methods but analysis of samples by LC-MS/MS gave higher concentrations than by enzyme immunoassay (EIA), with a statistically significant proportional bias (P<0.0001, Deming regression) for both sample types. The immunoassay is reliable for the qualitative and semi-quantitative presumptive detection of ethyl glucuronide in urine. Copyright © 2012 John Wiley & Sons, Ltd.

Development of a QuEChERS-Based Stable-Isotope Dilution LC-MS/MS Method To Quantitate Ferulic Acid and Its Main Microbial and Hepatic Metabolites in Milk.

PubMed

Waterstraat, Martin; Hildebrand, Andreas; Rosler, Margit; Bunzel, Mirko

2016-11-16

Forage plants of the Poaceae family are grown as pasturage or used for the production of hay, straw, corn stover, etc. Although ferulic acid contents of grasses are generally high, the amount of ingested ferulic acid differs depending on the type of forage, resulting in varying contents of ferulic acid and its microbial and hepatic metabolites in milk. Concentrations and patterns of these metabolites may be used as markers to track different forages in livestock feeding. Therefore, we developed a stable isotope dilution assay to quantitate ferulic acid, 12 ferulic acid-based metabolites, p-coumaric acid, and cinnamic acid in milk. Because most analytes were not commercially available as stable isotope labeled standard compounds, they were synthesized as 13 C- or deuterium-labeled standard compounds. A modification of the QuEChERS method, a Quick, Easy, Cheap, Effective, Rugged, and Safe approach usually applied to analyze pesticides in plant-based products, was used to extract the phenolic acids from milk. Determination was carried out by LC-ESI-MS/MS in scheduled multiple reaction monitoring modus. By using three different milk samples, the applicability of the validated approach was demonstrated.

Bayesian Normalization Model for Label-Free Quantitative Analysis by LC-MS

PubMed Central

Nezami Ranjbar, Mohammad R.; Tadesse, Mahlet G.; Wang, Yue; Ressom, Habtom W.

2016-01-01

We introduce a new method for normalization of data acquired by liquid chromatography coupled with mass spectrometry (LC-MS) in label-free differential expression analysis. Normalization of LC-MS data is desired prior to subsequent statistical analysis to adjust variabilities in ion intensities that are not caused by biological differences but experimental bias. There are different sources of bias including variabilities during sample collection and sample storage, poor experimental design, noise, etc. In addition, instrument variability in experiments involving a large number of LC-MS runs leads to a significant drift in intensity measurements. Although various methods have been proposed for normalization of LC-MS data, there is no universally applicable approach. In this paper, we propose a Bayesian normalization model (BNM) that utilizes scan-level information from LC-MS data. Specifically, the proposed method uses peak shapes to model the scan-level data acquired from extracted ion chromatograms (EIC) with parameters considered as a linear mixed effects model. We extended the model into BNM with drift (BNMD) to compensate for the variability in intensity measurements due to long LC-MS runs. We evaluated the performance of our method using synthetic and experimental data. In comparison with several existing methods, the proposed BNM and BNMD yielded significant improvement. PMID:26357332

TRIC: an automated alignment strategy for reproducible protein quantification in targeted proteomics

PubMed Central

Röst, Hannes L.; Liu, Yansheng; D’Agostino, Giuseppe; Zanella, Matteo; Navarro, Pedro; Rosenberger, George; Collins, Ben C.; Gillet, Ludovic; Testa, Giuseppe; Malmström, Lars; Aebersold, Ruedi

2016-01-01

Large scale, quantitative proteomic studies have become essential for the analysis of clinical cohorts, large perturbation experiments and systems biology studies. While next-generation mass spectrometric techniques such as SWATH-MS have substantially increased throughput and reproducibility, ensuring consistent quantification of thousands of peptide analytes across multiple LC-MS/MS runs remains a challenging and laborious manual process. To produce highly consistent and quantitatively accurate proteomics data matrices in an automated fashion, we have developed the TRIC software which utilizes fragment ion data to perform cross-run alignment, consistent peak-picking and quantification for high throughput targeted proteomics. TRIC uses a graph-based alignment strategy based on non-linear retention time correction to integrate peak elution information from all LC-MS/MS runs acquired in a study. When compared to state-of-the-art SWATH-MS data analysis, the algorithm was able to reduce the identification error by more than 3-fold at constant recall, while correcting for highly non-linear chromatographic effects. On a pulsed-SILAC experiment performed on human induced pluripotent stem (iPS) cells, TRIC was able to automatically align and quantify thousands of light and heavy isotopic peak groups and substantially increased the quantitative completeness and biological information in the data, providing insights into protein dynamics of iPS cells. Overall, this study demonstrates the importance of consistent quantification in highly challenging experimental setups, and proposes an algorithm to automate this task, constituting the last missing piece in a pipeline for automated analysis of massively parallel targeted proteomics datasets. PMID:27479329

The EIPeptiDi tool: enhancing peptide discovery in ICAT-based LC MS/MS experiments.

PubMed

Cannataro, Mario; Cuda, Giovanni; Gaspari, Marco; Greco, Sergio; Tradigo, Giuseppe; Veltri, Pierangelo

2007-07-15

Isotope-coded affinity tags (ICAT) is a method for quantitative proteomics based on differential isotopic labeling, sample digestion and mass spectrometry (MS). The method allows the identification and relative quantification of proteins present in two samples and consists of the following phases. First, cysteine residues are either labeled using the ICAT Light or ICAT Heavy reagent (having identical chemical properties but different masses). Then, after whole sample digestion, the labeled peptides are captured selectively using the biotin tag contained in both ICAT reagents. Finally, the simplified peptide mixture is analyzed by nanoscale liquid chromatography-tandem mass spectrometry (LC-MS/MS). Nevertheless, the ICAT LC-MS/MS method still suffers from insufficient sample-to-sample reproducibility on peptide identification. In particular, the number and the type of peptides identified in different experiments can vary considerably and, thus, the statistical (comparative) analysis of sample sets is very challenging. Low information overlap at the peptide and, consequently, at the protein level, is very detrimental in situations where the number of samples to be analyzed is high. We designed a method for improving the data processing and peptide identification in sample sets subjected to ICAT labeling and LC-MS/MS analysis, based on cross validating MS/MS results. Such a method has been implemented in a tool, called EIPeptiDi, which boosts the ICAT data analysis software improving peptide identification throughout the input data set. Heavy/Light (H/L) pairs quantified but not identified by the MS/MS routine, are assigned to peptide sequences identified in other samples, by using similarity criteria based on chromatographic retention time and Heavy/Light mass attributes. EIPeptiDi significantly improves the number of identified peptides per sample, proving that the proposed method has a considerable impact on the protein identification process and, consequently, on the amount of potentially critical information in clinical studies. The EIPeptiDi tool is available at http://bioingegneria.unicz.it/~veltri/projects/eipeptidi/ with a demo data set. EIPeptiDi significantly increases the number of peptides identified and quantified in analyzed samples, thus reducing the number of unassigned H/L pairs and allowing a better comparative analysis of sample data sets.

Matrix Effect Evaluation and Method Validation of Azoxystrobin and Difenoconazole Residues in Red Flesh Dragon Fruit (Hylocereus polyrhizus) Matrices Using QuEChERS Sample Preparation Methods Followed by LC-MS/MS Determination.

PubMed

Noegrohati, Sri; Hernadi, Elan; Asviastuti, Syanti

2018-06-01

Production of red flesh dragon fruit (Hylocereus polyrhizus) was hampered by Colletotrichum sp. Pre-harvest application of azoxystrobin and difenoconazole mixture is recommended, therefore, a selective and sensitive multi residues analytical method is required in monitoring and evaluating the commodity's safety. LC-MS/MS is a well-established analytical technique for qualitative and quantitative determination in complex matrices. However, this method is hurdled by co-eluted coextractives interferences. This work evaluated the pH effect of acetate buffered and citrate buffered QuEChERS sample preparation in their effectiveness of matrix effect reduction. Citrate buffered QuEChERS proved to produce clean final extract with relative matrix effect 0.4%-0.7%. Method validation of the selected sample preparation followed by LC-MS/MS for whole dragon fruit, flesh and peel matrices fortified at 0.005, 0.01, 0.1 and 1 g/g showed recoveries 75%-119%, intermediate repeatability 2%-14%. The expanded uncertainties were 7%-48%. Based on the international acceptance criteria, this method is valid.

Quantitative Profiling of DNA Damage and Apoptotic Pathways in UV Damaged Cells Using PTMScan Direct

PubMed Central

Stokes, Matthew P.; Silva, Jeffrey C.; Jia, Xiaoying; Lee, Kimberly A.; Polakiewicz, Roberto D.; Comb, Michael J.

2013-01-01

Traditional methods for analysis of peptides using liquid chromatography and tandem mass spectrometry (LC-MS/MS) lack the specificity to comprehensively monitor specific biological processes due to the inherent duty cycle limitations of the MS instrument and the stochastic nature of the analytical platform. PTMScan Direct is a novel, antibody-based method that allows quantitative LC-MS/MS profiling of specific peptides from proteins that reside in the same signaling pathway. New PTMScan Direct reagents have been produced that target peptides from proteins involved in DNA Damage/Cell Cycle and Apoptosis/Autophagy pathways. Together, the reagents provide access to 438 sites on 237 proteins in these signaling cascades. These reagents have been used to profile the response to UV damage of DNA in human cell lines. UV damage was shown to activate canonical DNA damage response pathways through ATM/ATR-dependent signaling, stress response pathways and induce the initiation of apoptosis, as assessed by an increase in the abundance of peptides corresponding to cleaved, activated caspases. These data demonstrate the utility of PTMScan Direct as a multiplexed assay for profiling specific cellular responses to various stimuli, such as UV damage of DNA. PMID:23344034

Adhesive blood microsampling systems for steroid measurement via LC-MS/MS in the rat.

PubMed

Heussner, Kirsten; Rauh, Manfred; Cordasic, Nada; Menendez-Castro, Carlos; Huebner, Hanna; Ruebner, Matthias; Schmidt, Marius; Hartner, Andrea; Rascher, Wolfgang; Fahlbusch, Fabian B

2017-04-01

Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) allows for the direct analysis of multiple hormones in a single probe with minimal sample volume. Rodent-based animal studies strongly rely on microsampling, such as the dry blood spot (DBS) method. However, DBS suffers the drawback of hematocrit-dependence (non-volumetric). Hence, novel volumetric microsampling techniques were introduced recently, allowing sampling of fixed accurate volumes. We compared these methods for steroid analysis in the rat to improve inter-system comparability. We analyzed steroid levels in blood using the absorptive microsampling devices Whatman® 903 Protein Saver Cards, Noviplex™ Plasma Prep Cards and the Mitra™ Microsampling device and compared the obtained results to the respective EDTA plasma levels. Quantitative steroid analysis was performed via LC-MS/MS. For the determination of the plasma volume factor for each steroid, their levels in pooled blood samples from each human adults and rats (18weeks) were compared and the transferability of these factors was evaluated in a new set of juvenile (21days) and adult (18weeks) rats. Hematocrit was determined concomitantly. Using these approaches, we were unable to apply one single volume factor for each steroid. Instead, plasma volume factors had to be adjusted for the recovery rate of each steroid and device individually. The tested microsampling systems did not allow the use of one single volume factor for adult and juvenile rats based on an unexpectedly strong hematocrit-dependency and other steroid specific (pre-analytic) factors. Our study provides correction factors for LC-MS/MS steroid analysis of volumetric and non-volumetric microsampling systems in comparison to plasma. It argues for thorough analysis of chromatographic effects before the use of novel volumetric systems for steroid analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

Quantitative twoplex glycan analysis using 12C6 and 13C6 stable isotope 2-aminobenzoic acid labelling and capillary electrophoresis mass spectrometry.

PubMed

Váradi, Csaba; Mittermayr, Stefan; Millán-Martín, Silvia; Bones, Jonathan

2016-12-01

Capillary electrophoresis (CE) offers excellent efficiency and orthogonality to liquid chromatographic (LC) separations for oligosaccharide structural analysis. Combination of CE with high resolution mass spectrometry (MS) for glycan analysis remains a challenging task due to the MS incompatibility of background electrolyte buffers and additives commonly used in offline CE separations. Here, a novel method is presented for the analysis of 2-aminobenzoic acid (2-AA) labelled glycans by capillary electrophoresis coupled to mass spectrometry (CE-MS). To ensure maximum resolution and excellent precision without the requirement for excessive analysis times, CE separation conditions including the concentration and pH of the background electrolyte, the effect of applied pressure on the capillary inlet and the capillary length were evaluated. Using readily available 12/13 C 6 stable isotopologues of 2-AA, the developed method can be applied for quantitative glycan profiling in a twoplex manner based on the generation of extracted ion electropherograms (EIE) for 12 C 6 'light' and 13 C 6 'heavy' 2-AA labelled glycan isotope clusters. The twoplex quantitative CE-MS glycan analysis platform is ideally suited for comparability assessment of biopharmaceuticals, such as monoclonal antibodies, for differential glycomic analysis of clinical material for potential biomarker discovery or for quantitative microheterogeneity analysis of different glycosylation sites within a glycoprotein. Additionally, due to the low injection volume requirements of CE, subsequent LC-MS analysis of the same sample can be performed facilitating the use of orthogonal separation techniques for structural elucidation or verification of quantitative performance.

Quantitative determination of four nitrofuran metabolites in meat by isotope dilution liquid chromatography-electrospray ionisation-tandem mass spectrometry.

PubMed

Mottier, Pascal; Khong, Seu-Ping; Gremaud, Eric; Richoz, Janique; Delatour, Thierry; Goldmann, Till; Guy, Philippe A

2005-03-04

A confirmatory method based on isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the low-level determination of residues of four nitrofuran veterinary drugs in meat, e.g., furazolidone, furaltadone, nitrofurantoin, and nitrofurazone. The procedure entails an acid-catalysed release of protein-bound metabolites, followed by their in situ conversion into the 2-nitrobenzaldehyde (NBA) imine-type derivatives. Liquid-liquid extraction and clean-up on a polymeric solid phase extraction cartridge are then performed before LC-MS/MS analysis by positive electrospray ionisation (ESI) applying multiple reaction monitoring of three transition reactions for each compound. Reliable quantitation is obtained by using one deuterated analogue per analyte (d4-NBA derivative) as internal standard (IS). Validation of the method in chicken meat was conducted following the European Union (EU) criteria for the analysis of veterinary drug residues in foods. The decision limits (CCalpha) were 0.11-0.21 microg/kg, and the detection capabilities (CCbeta) 0.19-0.36 microg/kg, thus below the minimum required performance limit (MRPL) set at 1 microg/kg by the EU. The method is robust and suitable for routine quality control operations, and more than 200 sample injections were performed without excessive pollution of the mass spectrometer or loss of LC column performance.

Toward best practices in data processing and analysis for intact biotherapeutics by MS in quantitative bioanalysis.

PubMed

Kellie, John F; Kehler, Jonathan R; Karlinsey, Molly Z; Summerfield, Scott G

2017-12-01

Typically, quantitation of biotherapeutics from biological matrices by LC-MS is based on a surrogate peptide approach to determine molecule concentration. Recent efforts have focused on quantitation of the intact protein molecules or larger mass subunits of monoclonal antibodies. To date, there has been limited guidance for large or intact protein mass quantitation for quantitative bioanalysis. Intact- and subunit-level analyses of biotherapeutics from biological matrices are performed at 12-25 kDa mass range with quantitation data presented. Linearity, bias and other metrics are presented along with recommendations made on the viability of existing quantitation approaches. This communication is intended to start a discussion around intact protein data analysis and processing, recognizing that other published contributions will be required.

Targeted and non-targeted detection of lemon juice adulteration by LC-MS and chemometrics.

PubMed

Wang, Zhengfang; Jablonski, Joseph E

2016-01-01

Economically motivated adulteration (EMA) of lemon juice was detected by LC-MS and principal component analysis (PCA). Twenty-two batches of freshly squeezed lemon juice were adulterated by adding an aqueous solution containing 5% citric acid and 6% sucrose to pure lemon juice to obtain 30%, 60% and 100% lemon juice samples. Their total titratable acidities, °Brix and pH values were measured, and then all the lemon juice samples were subject to LC-MS analysis. Concentrations of hesperidin and eriocitrin, major phenolic components of lemon juice, were quantified. The PCA score plots for LC-MS datasets were used to preview the classification of pure and adulterated lemon juice samples. Results showed a large inherent variability in the chemical properties among 22 batches of 100% lemon juice samples. Measurement or quantitation of one or several chemical properties (targeted detection) was not effective in detecting lemon juice adulteration. However, by using the LC-MS datasets, including both chromatographic and mass spectrometric information, 100% lemon juice samples were successfully differentiated from adulterated samples containing 30% lemon juice in the PCA score plot. LC-MS coupled with chemometric analysis can be a complement to existing methods for detecting juice adulteration.

Targeted liquid chromatography tandem mass spectrometry to quantitate wheat gluten using well-defined reference proteins

PubMed Central

Schalk, Kathrin; Koehler, Peter

2018-01-01

Celiac disease (CD) is an inflammatory disorder of the upper small intestine caused by the ingestion of storage proteins (prolamins and glutelins) from wheat, barley, rye, and, in rare cases, oats. CD patients need to follow a gluten-free diet by consuming gluten-free products with gluten contents of less than 20 mg/kg. Currently, the recommended method for the quantitative determination of gluten is an enzyme-linked immunosorbent assay (ELISA) based on the R5 monoclonal antibody. Because the R5 ELISA mostly detects the prolamin fraction of gluten, a new independent method is required to detect prolamins as well as glutelins. This paper presents the development of a method to quantitate 16 wheat marker peptides derived from all wheat gluten protein types by liquid chromatography tandem mass spectrometry (LC-MS/MS) in the multiple reaction monitoring mode. The quantitation of each marker peptide in the chymotryptic digest of a defined amount of the respective reference wheat protein type resulted in peptide-specific yields. This enabled the conversion of peptide into protein type concentrations. Gluten contents were expressed as sum of all determined protein type concentrations. This new method was applied to quantitate gluten in wheat starches and compared to R5 ELISA and gel-permeation high-performance liquid chromatography with fluorescence detection (GP-HPLC-FLD), which resulted in a strong correlation between LC-MS/MS and the other two methods. PMID:29425234

Mycotoxin analysis: an update.

PubMed

Krska, Rudolf; Schubert-Ullrich, Patricia; Molinelli, Alexandra; Sulyok, Michael; MacDonald, Susan; Crews, Colin

2008-02-01

Mycotoxin contamination of cereals and related products used for feed can cause intoxication, especially in farm animals. Therefore, efficient analytical tools for the qualitative and quantitative analysis of toxic fungal metabolites in feed are required. Current methods usually include an extraction step, a clean-up step to reduce or eliminate unwanted co-extracted matrix components and a separation step with suitably specific detection ability. Quantitative methods of analysis for most mycotoxins use immunoaffinity clean-up with high-performance liquid chromatography (HPLC) separation in combination with UV and/or fluorescence detection. Screening of samples contaminated with mycotoxins is frequently performed by thin layer chromatography (TLC), which yields qualitative or semi-quantitative results. Nowadays, enzyme-linked immunosorbent assays (ELISA) are often used for rapid screening. A number of promising methods, such as fluorescence polarization immunoassays, dipsticks, and even newer methods such as biosensors and non-invasive techniques based on infrared spectroscopy, have shown great potential for mycotoxin analysis. Currently, there is a strong trend towards the use of multi-mycotoxin methods for the simultaneous analysis of several of the important Fusarium mycotoxins, which is best achieved by LC-MS/MS (liquid chromatography with tandem mass spectrometry). This review focuses on recent developments in the determination of mycotoxins with a special emphasis on LC-MS/MS and emerging rapid methods.

Certification of NIST standard reference material 2389a, amino acids in 0.1 mol/L HCl--quantification by ID LC-MS/MS.

PubMed

Lowenthal, Mark S; Yen, James; Bunk, David M; Phinney, Karen W

2010-05-01

An isotope-dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) measurement procedure was developed to accurately quantify amino acid concentrations in National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 2389a-amino acids in 0.1 mol/L hydrochloric acid. Seventeen amino acids were quantified using selected reaction monitoring on a triple quadrupole mass spectrometer. LC-MS/MS results were compared to gravimetric measurements from the preparation of SRM 2389a-a reference material developed at NIST and intended for use in intra-laboratory calibrations and quality control. Quantitative mass spectrometry results and gravimetric values were statistically combined into NIST-certified mass fraction values with associated uncertainty estimates. Coefficients of variation (CV) for the repeatability of the LC-MS/MS measurements among amino acids ranged from 0.33% to 2.7% with an average CV of 1.2%. Average relative expanded uncertainty of the certified values including Types A and B uncertainties was 3.5%. Mean accuracy of the LC-MS/MS measurements with gravimetric preparation values agreed to within |1.1|% for all amino acids. NIST SRM 2389a will be available for characterization of routine methods for amino acid analysis and serves as a standard for higher-order measurement traceability. This is the first time an ID LC-MS/MS methodology has been applied for quantifying amino acids in a NIST SRM material.

ANALYSIS OF PERFLUORINATED CARBOXYLIC ACIDS IN SOILS: DETECTION AND QUANTITATION ISSUES AT LOW CONCENTRATIONS

EPA Science Inventory

Methods were developed for the extraction from soil, identification, confirmation and quantitation by LC/MS/MS of trace levels of perfluorinated octanoic acid (PFOA), perfluorinated nonanoic acid (PFNA) and perfluorinated decanoic acid (PFDA). Whereas PFOA, PFNA and PFDA all can...

Standardized protocols for quality control of MRM-based plasma proteomic workflows.

PubMed

Percy, Andrew J; Chambers, Andrew G; Smith, Derek S; Borchers, Christoph H

2013-01-04

Mass spectrometry (MS)-based proteomics is rapidly emerging as a viable technology for the identification and quantitation of biological samples, such as human plasma--the most complex yet commonly employed biofluid in clinical analyses. The transition from a qualitative to quantitative science is required if proteomics is going to successfully make the transition to a clinically useful technique. MS, however, has been criticized for a lack of reproducibility and interlaboratory transferability. Currently, the MS and plasma proteomics communities lack standardized protocols and reagents to ensure that high-quality quantitative data can be accurately and precisely reproduced by laboratories across the world using different MS technologies. Toward addressing this issue, we have developed standard protocols for multiple reaction monitoring (MRM)-based assays with customized isotopically labeled internal standards for quality control of the sample preparation workflow and the MS platform in quantitative plasma proteomic analyses. The development of reference standards and their application to a single MS platform is discussed herein, along with the results from intralaboratory tests. The tests highlighted the importance of the reference standards in assessing the efficiency and reproducibility of the entire bottom-up proteomic workflow and revealed errors related to the sample preparation and performance quality and deficits of the MS and LC systems. Such evaluations are necessary if MRM-based quantitative plasma proteomics is to be used in verifying and validating putative disease biomarkers across different research laboratories and eventually in clinical laboratories.

Determination of Acid Herbicides Using Modified QuEChERS with Fast Switching ESI(+)/ESI(-) LC-MS/MS.

PubMed

Sack, Chris; Vonderbrink, John; Smoker, Michael; Smith, Robert E

2015-11-04

A method for the determination of 35 acid herbicides in food matrices was developed, validated, and implemented. It utilizes a modified QuEChERS extraction procedure coupled with quantitation by liquid chromatography tandem mass spectrometry (LC-MS/MS). The acid herbicides analyzed are all organic carboxylic acids, including the older chlorophenoxy acid herbicides such as 2,4-dichlorophenoxyacetic acid (2,4-D), dicamba, 4-chlorophenoxyacetic acid (4-CPA), quinclorac, and many of the newer imidazolinone herbicides such as imazethapyr and imazaquin. In the procedure, 10 mL of water is added to 5 g of sample and then extracted with 1% formic acid in acetonitrile for 1 min. The acetonitrile phase is salted out of the extract by adding sodium chloride and magnesium sulfate, followed by centrifugation. The acetonitrile is diluted 1:1 with water to enable quantitation by LC-MS/MS using fast switching between positive and negative electrospray ionization modes. The average recoveries for all the compounds except aminocyclopyrachlor were 95% with a precision of 8%. The method detection limits for all residues were less than 10 ng/g, and the correlation coefficients for the calibration curves was greater than 0.99 for all but two compounds tested. The method was used successfully for the quantitation of acid herbicides in the FDA's total diet study. The procedure proved to be accurate, precise, linear, sensitive, and rugged.

Protocol for an electrospray ionization tandem mass spectral product ion library: development and application for identification of 240 pesticides in foods.

PubMed

Zhang, Kai; Wong, Jon W; Yang, Paul; Hayward, Douglas G; Sakuma, Takeo; Zou, Yunyun; Schreiber, André; Borton, Christopher; Nguyen, Tung-Vi; Kaushik, Banerjee; Oulkar, Dasharath

2012-07-03

Modern determination techniques for pesticides must yield identification quickly with high confidence for timely enforcement of tolerances. A protocol for the collection of liquid chromatography (LC) electrospray ionization (ESI)-quadruple linear ion trap (Q-LIT) mass spectrometry (MS) library spectra was developed. Following the protocol, an enhanced product ion (EPI) library of 240 pesticides was developed by use of spectra collected from two laboratories. A LC-Q-LIT-MS workflow using scheduled multiple reaction monitoring (sMRM) survey scan, information-dependent acquisition (IDA) triggered collection of EPI spectra, and library search was developed and tested to identify the 240 target pesticides in one single LC-Q-LIT MS analysis. By use of LC retention time, one sMRM survey scan transition, and a library search, 75-87% of the 240 pesticides were identified in a single LC/MS analysis at fortified concentrations of 10 ng/g in 18 different foods. A conventional approach with LC-MS/MS using two MRM transitions produced the same identifications and comparable quantitative results with the same incurred foods as the LC-Q-LIT using EPI library search, finding 1.2-49 ng/g of either carbaryl, carbendazim, fenbuconazole, propiconazole, or pyridaben in peaches; carbendazim, imazalil, terbutryn, and thiabendazole in oranges; terbutryn in salmon; and azoxystrobin in ginseng. Incurred broccoli, cabbage, and kale were screened with the same EPI library using three LC-Q-LIT and a LC-quadruple time-of-flight (Q-TOF) instruments. The library search identified azoxystrobin, cyprodinil, fludioxinil, imidacloprid, metalaxyl, spinosyn A, D, and J, amd spirotetramat with each instrument. The approach has a broad application in LC-MS/MS type targeted screening in food analysis.

Absolute Quantification of Middle- to High-Abundant Plasma Proteins via Targeted Proteomics.

PubMed

Dittrich, Julia; Ceglarek, Uta

2017-01-01

The increasing number of peptide and protein biomarker candidates requires expeditious and reliable quantification strategies. The utilization of liquid chromatography coupled to quadrupole tandem mass spectrometry (LC-MS/MS) for the absolute quantitation of plasma proteins and peptides facilitates the multiplexed verification of tens to hundreds of biomarkers from smallest sample quantities. Targeted proteomics assays derived from bottom-up proteomics principles rely on the identification and analysis of proteotypic peptides formed in an enzymatic digestion of the target protein. This protocol proposes a procedure for the establishment of a targeted absolute quantitation method for middle- to high-abundant plasma proteins waiving depletion or enrichment steps. Essential topics as proteotypic peptide identification and LC-MS/MS method development as well as sample preparation and calibration strategies are described in detail.

Development of carbon plasma-coated multiwell plates for high-throughput mass spectrometric analysis of highly lipophilic fermentation products.

PubMed

Heinig, Uwe; Scholz, Susanne; Dahm, Pia; Grabowy, Udo; Jennewein, Stefan

2010-08-01

Classical approaches to strain improvement and metabolic engineering rely on rapid qualitative and quantitative analyses of the metabolites of interest. As an analytical tool, mass spectrometry (MS) has proven to be efficient and nearly universally applicable for timely screening of metabolites. Furthermore, gas chromatography (GC)/MS- and liquid chromatography (LC)/MS-based metabolite screens can often be adapted to high-throughput formats. We recently engineered a Saccharomyces cerevisiae strain to produce taxa-4(5),11(12)-diene, the first pathway-committing biosynthetic intermediate for the anticancer drug Taxol, through the heterologous and homologous expression of several genes related to isoprenoid biosynthesis. To date, GC/MS- and LC/MS-based high-throughput methods have been inherently difficult to adapt to the screening of isoprenoid-producing microbial strains due to the need for extensive sample preparation of these often highly lipophilic compounds. In the current work, we examined different approaches to the high-throughput analysis of taxa-4(5),11(12)-diene biosynthesizing yeast strains in a 96-deep-well format. Carbon plasma coating of standard 96-deep-well polypropylene plates allowed us to circumvent the inherent solvent instability of commonly used deep-well plates. In addition, efficient adsorption of the target isoprenoid product by the coated plates allowed rapid and simple qualitative and quantitative analyses of the individual cultures. Copyright 2010 Elsevier Inc. All rights reserved.

MASS SPECTROMETRY OF FATTY ALDEHYDES

PubMed Central

Berdyshev, Evgeny V.

2011-01-01

Fatty aldehydes are important components of the cellular lipidome. Significant interest has been developed towards the analysis of the short chain α,β-unsaturated and hydroxylated aldehydes formed as a result of oxidation of polyunsaturated fatty acids. Multiple gas chromatography-mass spectrometry (GC/MS) and subsequently liquid chromatography-mass spectrometry (LC/MS) approaches have been developed to identify and quantify short-chain as well as long-chain fatty aldehydes. Due to the ability to non-enzymaticaly form Schiff bases with amino groups of proteins, lipids, and with DNA guanidine, free aldehydes are viewed as a marker or metric of fatty acid oxidation and not the part of intracellular signaling pathways which has significantly limited the overall attention this group of molecules have received. This review provides an overview of current GC/MS and LC/MS approaches of fatty aldehyde analysis as well as discusses technical challenges standing in the way of free fatty aldehyde quantitation. PMID:21930240

Large-Scale Interlaboratory Study to Develop, Analytically Validate and Apply Highly Multiplexed, Quantitative Peptide Assays to Measure Cancer-Relevant Proteins in Plasma*

PubMed Central

Abbatiello, Susan E.; Schilling, Birgit; Mani, D. R.; Zimmerman, Lisa J.; Hall, Steven C.; MacLean, Brendan; Albertolle, Matthew; Allen, Simon; Burgess, Michael; Cusack, Michael P.; Gosh, Mousumi; Hedrick, Victoria; Held, Jason M.; Inerowicz, H. Dorota; Jackson, Angela; Keshishian, Hasmik; Kinsinger, Christopher R.; Lyssand, John; Makowski, Lee; Mesri, Mehdi; Rodriguez, Henry; Rudnick, Paul; Sadowski, Pawel; Sedransk, Nell; Shaddox, Kent; Skates, Stephen J.; Kuhn, Eric; Smith, Derek; Whiteaker, Jeffery R.; Whitwell, Corbin; Zhang, Shucha; Borchers, Christoph H.; Fisher, Susan J.; Gibson, Bradford W.; Liebler, Daniel C.; MacCoss, Michael J.; Neubert, Thomas A.; Paulovich, Amanda G.; Regnier, Fred E.; Tempst, Paul; Carr, Steven A.

2015-01-01

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens to hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility, and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here, we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data, we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy-isotope-labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an interlaboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality control measures, enables sensitive, specific, reproducible, and quantitative measurements of proteins and peptides in complex biological matrices such as plasma. PMID:25693799

Determination of n-hexane metabolites by liquid chromatography/mass spectrometry. 2. Glucuronide-conjugated metabolites in untreated urine samples by electrospray ionization.

PubMed

Manini, P; Andreoli, R; Mutti, A; Bergamaschi, E; Niessen, W M

1998-01-01

A liquid chromatography atmospheric pressure electrospray mass spectrometry (ESI-LC/MS) system was evaluated for the identification and characterization of n-hexane conjugated metabolites (glucuronides) in untreated urine samples. Chromatography of glucuronides was obtained under ion-suppressed reversed-phase conditions, by using high-speed (3 cm, 3 microns) columns and formic acid (2 mM) as modifier in the mobile phase. The mass spectrometer was operated in negative ion (NI) mode. For the first time, four glucuronides were identified by ESI-LC/MS in untreated urine samples of rats exposed to n-hexane: 2-hexanol-glucuronide, 5-hydroxy-2-hexanone-glucuronide, 2,5-hexanediol-glucuronide and 4,5-dihydroxy-2-hexanone-glucuronide. Confirmation of the conjugated metabolites was obtained by LC/MS/MS experiments. Gas chromatography/mass spectrometry (GC/MS) and atmospheric pressure chemical ionization (APCI) LC/MS analyses were performed on the same samples. An integrated approach GC/MS-LC/MS for the semi-quantitative analysis of n-hexane glucuronides, whose standards are not commercially available, is discussed and proposed here. In order to understand the fate of the metabolites during sample pre-treatment, a study about the effects of enzymatic and acid hydrolysis on urine samples was conducted on glucuronides isolated by solid-phase extraction. Combined analyses by GC/MS and LC/MS enabled us to distinguish 'true' n-hexane metabolites from compounds resulting from sample treatment and handling (i.e. enzymatic and acid hydrolysis, extraction and GC injection).

Advancing a High Throughput Glycotope-centric Glycomics Workflow Based on nanoLC-MS2-product Dependent-MS3 Analysis of Permethylated Glycans.

PubMed

Hsiao, Cheng-Te; Wang, Po-Wei; Chang, Hua-Chien; Chen, Yen-Ying; Wang, Shui-Hua; Chern, Yijuang; Khoo, Kay-Hooi

2017-12-01

The intrinsic nature of glycosylation, namely nontemplate encoded, stepwise elongation and termination with a diverse range of isomeric glyco-epitopes (glycotopes), translates into ambiguity in most cases of mass spectrometry (MS)-based glycomic mapping. It is arguable that whether one needs to delineate every single glycomic entity, which may be counterproductive. Instead, one should focus on identifying as many structural features as possible that would collectively define the glycomic characteristics of a cell or tissue, and how these may change in response to self-programmed development, immuno-activation, and malignant transformation. We have been pursuing this line of analytical strategy that homes in on identifying the terminal sulfo-, sialyl, and/or fucosylated glycotopes by comprehensive nanoLC-MS 2 -product dependent MS 3 analysis of permethylated glycans, in conjunction with development of a data mining computational tool, GlyPick, to enable an automated, high throughput, semi-quantitative glycotope-centric glycomic mapping amenable to even nonexperts. We demonstrate in this work that diagnostic MS 2 ions can be relied on to inform the presence of specific glycotopes, whereas their possible isomeric identities can be resolved at MS 3 level. Both MS 2 and associated MS 3 data can be acquired exhaustively and processed automatically by GlyPick. The high acquisition speed, resolution, and mass accuracy afforded by top-notch Orbitrap Fusion MS system now allow a sensible spectral count and/or summed ion intensity-based glycome-wide glycotope quantification. We report here the technical aspects, reproducibility and optimization of such an analytical approach that uses the same acidic reverse phase C18 nanoLC conditions fully compatible with proteomic analysis to allow rapid hassle-free switching. We further show how this workflow is particularly effective when applied to larger, multiply sialylated and fucosylated N-glycans derived from mouse brain. The complexity of their terminal glycotopes including variants of fucosylated and disialylated type 1 and 2 chains would otherwise not be adequately delineated by any conventional LC-MS/MS analysis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A new strategy for faster urinary biomarkers identification by Nano-LC-MALDI-TOF/TOF mass spectrometry

PubMed Central

Benkali, K; Marquet, P; Rérolle, JP; Le Meur, Y; Gastinel, LN

2008-01-01

Background LC-MALDI-TOF/TOF analysis is a potent tool in biomarkers discovery characterized by its high sensitivity and high throughput capacity. However, methods based on MALDI-TOF/TOF for biomarkers discovery still need optimization, in particular to reduce analysis time and to evaluate their reproducibility for peak intensities measurement. The aims of this methodological study were: (i) to optimize and critically evaluate each step of urine biomarker discovery method based on Nano-LC coupled off-line to MALDI-TOF/TOF, taking full advantage of the dual decoupling between Nano-LC, MS and MS/MS to reduce the overall analysis time; (ii) to evaluate the quantitative performance and reproducibility of nano-LC-MALDI analysis in biomarker discovery; and (iii) to evaluate the robustness of biomarkers selection. Results A pool of urine sample spiked at increasing concentrations with a mixture of standard peptides was used as a specimen for biological samples with or without biomarkers. Extraction and nano-LC-MS variabilities were estimated by analyzing in triplicates and hexaplicates, respectively. The stability of chromatographic fractions immobilised with MALDI matrix on MALDI plates was evaluated by successive MS acquisitions after different storage times at different temperatures. Low coefficient of variation (CV%: 10–22%) and high correlation (R2 > 0.96) values were obtained for the quantification of the spiked peptides, allowing quantification of these peptides in the low fentomole range, correct group discrimination and selection of "specific" markers using principal component analysis. Excellent peptide integrity and stable signal intensity were found when MALDI plates were stored for periods of up to 2 months at +4°C. This allowed storage of MALDI plates between LC separation and MS acquisition (first decoupling), and between MS and MSMS acquisitions while the selection of inter-group discriminative ions is done (second decoupling). Finally the recording of MSMS spectra to obtain structural information was focused only on discriminative ions in order to minimize analysis time. Conclusion Contrary to other classical approaches with direct online coupling of chromatographic separation and on the flight MS and/or MSMS data acquisition for all detected analytes, our dual decoupling strategy allowed us to focus on the most discriminative analytes, giving us more time to acquire more replicates of the same urine samples thus increasing detection sensitivity and mass precision. PMID:19014585

Investigation of the "true" extraction recovery of analytes from multiple types of tissues and its impact on tissue bioanalysis using two model compounds.

PubMed

Yuan, Long; Ma, Li; Dillon, Lisa; Fancher, R Marcus; Sun, Huadong; Zhu, Mingshe; Lehman-McKeeman, Lois; Aubry, Anne-Françoise; Ji, Qin C

2016-11-16

LC-MS/MS has been widely applied to the quantitative analysis of tissue samples. However, one key remaining issue is that the extraction recovery of analyte from spiked tissue calibration standard and quality control samples (QCs) may not accurately represent the "true" recovery of analyte from incurred tissue samples. This may affect the accuracy of LC-MS/MS tissue bioanalysis. Here, we investigated whether the recovery determined using tissue QCs by LC-MS/MS can accurately represent the "true" recovery from incurred tissue samples using two model compounds: BMS-986104, a S1P 1 receptor modulator drug candidate, and its phosphate metabolite, BMS-986104-P. We first developed a novel acid and surfactant assisted protein precipitation method for the extraction of BMS-986104 and BMS-986104-P from rat tissues, and determined their recoveries using tissue QCs by LC-MS/MS. We then used radioactive incurred samples from rats dosed with 3 H-labeled BMS-986104 to determine the absolute total radioactivity recovery in six different tissues. The recoveries determined using tissue QCs and incurred samples matched with each other very well. The results demonstrated that, in this assay, tissue QCs accurately represented the incurred tissue samples to determine the "true" recovery, and LC-MS/MS assay was accurate for tissue bioanalysis. Another aspect we investigated is how the tissue QCs should be prepared to better represent the incurred tissue samples. We compared two different QC preparation methods (analyte spiked in tissue homogenates or in intact tissues) and demonstrated that the two methods had no significant difference when a good sample preparation was in place. The developed assay showed excellent accuracy and precision, and was successfully applied to the quantitative determination of BMS-986104 and BMS-986104-P in tissues in a rat toxicology study. Copyright © 2016 Elsevier B.V. All rights reserved.

An integrated platform for directly widely-targeted quantitative analysis of feces part I: Platform configuration and method validation.

PubMed

Song, Yuelin; Song, Qingqing; Li, Jun; Zheng, Jiao; Li, Chun; Zhang, Yuan; Zhang, Lingling; Jiang, Yong; Tu, Pengfei

2016-07-08

Direct analysis is of great importance to understand the real chemical profile of a given sample, notably biological materials, because either chemical degradation or diverse errors and uncertainties might be resulted from sophisticated protocols. In comparison with biofluids, it is still challenging for direct analysis of solid biological samples using high performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Herein, a new analytical platform was configured by online hyphenating pressurized liquid extraction (PLE), turbulent flow chromatography (TFC), and LC-MS/MS. A facile, but robust PLE module was constructed based on the phenomenon that noticeable back-pressure can be generated during rapid fluid passing through a narrow tube. TFC column that is advantageous at extracting low molecular analytes from rushing fluid was employed to link at the outlet of the PLE module to capture constituents-of-interest. An electronic 6-port/2-position valve was introduced between TFC column and LC-MS/MS to fragment each measurement into extraction and elution phases, whereas LC-MS/MS took the charge of analyte separation and monitoring. As a proof of concept, simultaneous determination of 24 endogenous substances including eighteen steroids, five eicosanoids, and one porphyrin in feces was carried out in this paper. Method validation assays demonstrated the analytical platform to be qualified for directly simultaneous measurement of diverse endogenous analytes in fecal matrices. Application of this integrated platform on homolog-focused profiling of feces is discussed in a companion paper. Copyright © 2016 Elsevier B.V. All rights reserved.

Mycotoxin Analysis of Human Urine by LC-MS/MS: A Comparative Extraction Study

PubMed Central

Escrivá, Laura; Font, Guillermina

2017-01-01

The lower mycotoxin levels detected in urine make the development of sensitive and accurate analytical methods essential. Three extraction methods, namely salting-out liquid–liquid extraction (SALLE), miniQuEChERS (quick, easy, cheap, effective, rugged, and safe), and dispersive liquid–liquid microextraction (DLLME), were evaluated and compared based on analytical parameters for the quantitative LC-MS/MS measurement of 11 mycotoxins (AFB1, AFB2, AFG1, AFG2, OTA, ZEA, BEA, EN A, EN B, EN A1 and EN B1) in human urine. DLLME was selected as the most appropriate methodology, as it produced better validation results for recovery (79–113%), reproducibility (RSDs < 12%), and repeatability (RSDs < 15%) than miniQuEChERS (71–109%, RSDs <14% and <24%, respectively) and SALLE (70–108%, RSDs < 14% and < 24%, respectively). Moreover, the lowest detection (LODS) and quantitation limits (LOQS) were achieved with DLLME (LODs: 0.005–2 μg L−1, LOQs: 0.1–4 μg L−1). DLLME methodology was used for the analysis of 10 real urine samples from healthy volunteers showing the presence of ENs B, B1 and A1 at low concentrations. PMID:29048356

High-resolution mass spectrometry method for the detection, characterization and quantitation of pharmaceuticals in water.

PubMed

Pinhancos, Rebeca; Maass, Sara; Ramanathan, Dil M

2011-11-01

The presence of pharmaceuticals in drinking water is an emerging environmental concern. In most environmental testing laboratories, LC-MS/MS assays based on selected reaction monitoring are used as part of a battery of tests used to assure water quality. Although LC-MS/MS continues to be the best tool for detecting pharmaceuticals in water, the combined use of hybrid high-resolution mass spectrometry (HRMS) and ultrahigh pressure liquid chromatography (UHPLC) is starting to become a practical tool to study emerging environmental contaminants. The hybrid LTQ-orbitrap mass spectrometer is suitable for integrated quantitative and qualitative bioanalysis because of the following reasons: (1) the ability to collect full-scan HRMS spectra with scan speeds suitable for UHPLC separations, (2) routine measurement of mass with less than 5 ppm mass accuracy, (3) high mass resolving power, and (4) ability to perform on-the-fly polarity switching in the linear ion trap (LTQ). In the present work, we provide data demonstrating the application of UHPLC-LTQ-orbitrap for the detection, characterization and quantification of pharmaceuticals and their metabolites in drinking water. Copyright © 2011 John Wiley & Sons, Ltd.

Epitope mapping and targeted quantitation of the cardiac biomarker troponin by SID-MRM mass spectrometry.

PubMed

Zhao, Cheng; Trudeau, Beth; Xie, Helen; Prostko, John; Fishpaugh, Jeffrey; Ramsay, Carol

2014-06-01

The absolute quantitation of the targeted protein using MS provides a promising method to evaluate/verify biomarkers used in clinical diagnostics. In this study, a cardiac biomarker, troponin I (TnI), was used as a model protein for method development. The epitope peptide of TnI was characterized by epitope excision followed with LC/MS/MS method and acted as the surrogate peptide for the targeted protein quantitation. The MRM-based MS assay using a stable internal standard that improved the selectivity, specificity, and sensitivity of the protein quantitation. Also, plasma albumin depletion and affinity enrichment of TnI by anti-TnI mAb-coated microparticles reduced the sample complexity, enhanced the dynamic range, and further improved the detecting sensitivity of the targeted protein in the biological matrix. Therefore, quantitation of TnI, a low abundant protein in human plasma, has demonstrated the applicability of the targeted protein quantitation strategy through its epitope peptide determined by epitope mapping method. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Methods for the analysis of organophosphorus flame retardants-Comparison of GC-EI-MS, GC-NCI-MS, LC-ESI-MS/MS, and LC-APCI-MS/MS.

PubMed

Tokumura, Masahiro; Miyake, Yuichi; Wang, Qi; Nakayama, Hayato; Amagai, Takashi; Ogo, Sayaka; Kume, Kazunari; Kobayashi, Takeshi; Takasu, Shinji; Ogawa, Kumiko

2018-04-16

Organophosphorus flame retardants (PFRs) are extensively used as alternatives to banned polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCD). In this study, we analyzed 14 PFRs by means of four mass-spectrometry-based methods: gas chromatography combined with electron-impact mass spectrometry (GC-EI-MS) or negative-chemical-ionization mass spectrometry (GC-NCI-MS) and liquid chromatography combined with tandem mass spectrometry using electrospray ionization (LC-ESI-MS/MS) or atmospheric pressure chemical ionization (LC-APCI-MS/MS). The limits of quantification (LOQs) for LC-ESI-MS/MS and LC-APCI-MS/MS (0.81-970 pg) were 1-2 orders of magnitude lower than the LOQs for GC-EI-MS and GC-NCI-MS (2.3-3900 pg). LC-APCI-MS/MS showed the lowest LOQs (mean = 41 pg; median = 3.4 pg) for all but two of the PFRs targeted in this study. For LC-APCI-MS/MS, the lowest LOQ was observed for tributyl phosphate (TBP) (0.81 pg), and the highest was observed for tris(butoxyethyl) phosphate (TBOEP) (36 pg). The results of this study indicate that LC-APCI-MS/MS is the optimum analytical method for the target PFRs, at least in terms of LOQ.

Phenolic composition of pomegranate peel extracts using an LC-MS approach with silica hydride columns

USDA-ARS?s Scientific Manuscript database

The peels of different pomegranate cultivars (Molla Nepes, Parfianka, Purple Heart, Wonderful and Vkunsyi) were compared in terms of phenolic composition and total phenolics. Analyses were performed on two silica hydride-based stationary phases: phenyl and undecenoic acid columns. Quantitation was ...

Multiplexed, quantitative, and targeted metabolite profiling by LC-MS/MRM.

PubMed

Wei, Ru; Li, Guodong; Seymour, Albert B

2014-01-01

Targeted metabolomics, which focuses on a subset of known metabolites representative of biologically relevant metabolic pathways, is a valuable tool to discover biomarkers and link disease phenotypes to underlying mechanisms or therapeutic modes of action. A key advantage of targeted metabolomics, compared to discovery metabolomics, is its immediate readiness for extracting biological information derived from known metabolites and quantitative measurements. However, simultaneously analyzing hundreds of endogenous metabolites presents a challenge due to their diverse chemical structures and properties. Here we report a method which combines different chromatographic separation conditions, optimal ionization polarities, and the most sensitive triple-quadrupole MS-based data acquisition mode, multiple reaction monitoring (MRM), to quantitatively profile 205 endogenous metabolites in 10 min.

Quantitation of intracellular purine intermediates in different Corynebacteria using electrospray LC-MS/MS.

PubMed

Peifer, Susanne; Schneider, Konstantin; Nürenberg, Gudrun; Volmer, Dietrich A; Heinzle, Elmar

2012-11-01

Intermediates of the purine biosynthesis pathway play key roles in cellular metabolism including nucleic acid synthesis and signal mediation. In addition, they are also of major interest to the biotechnological industry as several intermediates either possess flavor-enhancing characteristics or are applied in medical therapy. In this study, we have developed an analytical method for quantitation of 12 intermediates from the purine biosynthesis pathway including important nucleotides and their corresponding nucleosides and nucleobases. The approach comprised a single-step acidic extraction/quenching procedure, followed by quantitative electrospray LC-MS/MS analysis. The assay was validated in terms of accuracy, precision, reproducibility, and applicability for complex biological matrices. The method was subsequently applied for determination of free intracellular pool sizes of purine biosynthetic pathway intermediates in the two Gram-positive bacteria Corynebacterium glutamicum and Corynebacterium ammoniagenes. Importantly, no ion pair reagents were applied in this approach as usually required for liquid chromatography analysis of large classes of diverse metabolites.

Development of a liquid chromatography-mass spectrometry based enzyme activity assay for phosphatidylcholine-specific phospholipase C.

PubMed

Murakami, Chiaki; Mizuno, Satoru; Kado, Sayaka; Sakane, Fumio

2017-06-01

Phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) hydrolyzes PC to generate the second messenger 1,2-diacylglycerol (DG) and phosphocholine. PC-PLC plays pivotal roles in inflammation, carcinogenesis, tumor progression, atherogenesis, and subarachnoid hemorrhage. Although the activity of PC-PLC in mammalian tissues was discovered approximately 40 years ago, neither the protein nor its gene has been identified. In the present study, we developed a non-radioactive enzyme activity assay for PC-PLC based on mass spectrometric detection of DG following HPLC separation. This new liquid chromatography-mass spectrometry (LC-MS) assay directly determines a specific reaction product, 1-palmitoyl-2-oleoyl-DG, that is generated from 1-palmitoyl-2-oleoyl-PC by purified Bacillus cereus PC-PLC. The LC-MS assay offers several advantages including a lower background (0.02% versus 91%), higher signal background ratio (4242 versus 1.06)/signal noise ratio (7494 versus 4.4), higher sensitivity (≥32-fold), and lower limit of quantitation (0.04 pmol versus 0.69 pmol of PC-PLC), than a conventional fluorometric assay, which indirectly detects phosphocholine produced in the reaction. In addition to Bacillus cereus PC-PLC, the LC-MS assay was applicable to the measurement of mammalian PC-PLC prepared from the mouse brain. The radioisotope-free, highly sensitive and precise LC-MS assay for PC-PLC would be useful for the purification and identification of PC-PLC protein. Copyright © 2017 Elsevier Inc. All rights reserved.

Pushing quantitation limits in micro UHPLC-MS/MS analysis of steroid hormones by sample dilution using high volume injection.

PubMed

Márta, Zoltán; Bobály, Balázs; Fekete, Jenő; Magda, Balázs; Imre, Tímea; Mészáros, Katalin Viola; Szabó, Pál Tamás

2016-09-10

Ultratrace analysis of sample components requires excellent analytical performance in terms of limits of quantitation (LoQ). Micro UHPLC coupling with sensitive tandem mass spectrometry provides state of the art solutions for such analytical problems. Decreased column volume in micro LC limits the injectable sample volume. However, if analyte concentration is extremely low, it might be necessary to inject high sample volumes. This is particularly critical for strong sample solvents and weakly retained analytes, which are often the case when preparing biological samples (protein precipitation, sample extraction, etc.). In that case, high injection volumes may cause band broadening, peak distortion or even elution in dead volume. In this study, we evaluated possibilities of high volume injection onto microbore RP-LC columns, when sample solvent is diluted. The presented micro RP-LC-MS/MS method was optimized for the analysis of steroid hormones from human plasma after protein precipitation with organic solvents. A proper sample dilution procedure helps to increase the injection volume without compromising peak shapes. Finally, due to increased injection volume, the limit of quantitation can be decreased by a factor of 2-5, depending on the analytes and the experimental conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

Strategy to improve the quantitative LC-MS analysis of molecular ions resistant to gas-phase collision induced dissociation: application to disulfide-rich cyclic peptides.

PubMed

Ciccimaro, Eugene; Ranasinghe, Asoka; D'Arienzo, Celia; Xu, Carrie; Onorato, Joelle; Drexler, Dieter M; Josephs, Jonathan L; Poss, Michael; Olah, Timothy

2014-12-02

Due to observed collision induced dissociation (CID) fragmentation inefficiency, developing sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assays for CID resistant compounds is especially challenging. As an alternative to traditional LC-MS/MS, we present here a methodology that preserves the intact analyte ion for quantification by selectively filtering ions while reducing chemical noise. Utilizing a quadrupole-Orbitrap MS, the target ion is selectively isolated while interfering matrix components undergo MS/MS fragmentation by CID, allowing noise-free detection of the analyte's surviving molecular ion. In this manner, CID affords additional selectivity during high resolution accurate mass analysis by elimination of isobaric interferences, a fundamentally different concept than the traditional approach of monitoring a target analyte's unique fragment following CID. This survivor-selected ion monitoring (survivor-SIM) approach has allowed sensitive and specific detection of disulfide-rich cyclic peptides extracted from plasma.

Semi-automated 96-well liquid-liquid extraction for quantitation of drugs in biological fluids.

PubMed

Zhang, N; Hoffman, K L; Li, W; Rossi, D T

2000-02-01

A semi-automated liquid-liquid extraction (LLE) technique for biological fluid sample preparation was introduced for the quantitation of four drugs in rat plasma. All liquid transferring during the sample preparation was automated using a Tomtec Quadra 96 Model 320 liquid handling robot, which processed up to 96 samples in parallel. The samples were either in 96-deep-well plate or tube-rack format. One plate of samples can be prepared in approximately 1.5 h, and the 96-well plate is directly compatible with the autosampler of an LC/MS system. Selection of organic solvents and recoveries are discussed. Also, precision, relative error, linearity and quantitation of the semi automated LLE method are estimated for four example drugs using LC/MS/MS with a multiple reaction monitoring (MRM) approach. The applicability of this method and future directions are evaluated.

Analysis of coffee for the presence of acrylamide by LC-MS/MS.

PubMed

Andrzejewski, Denis; Roach, John A G; Gay, Martha L; Musser, Steven M

2004-04-07

A variety of popular instant, ground, and brewed coffees were analyzed using a modified liquid chromatography-tandem mass spectrometry (LC-MS/MS) method specifically developed for the determination of acrylamide in foods. Coffee test portions were spiked with 13C3-labeled acrylamide as an internal standard prior to their extraction and cleanup. Ground coffees (1 g) and instant coffees (0.5 g) were extracted by shaking with 9 mL of water for 20 min. Brewed coffee test portions (9 mL) were taken through the cleanup procedure without further dilution with extraction solvent. Coffee test portions were cleaned up by passing 1.5 mL first through an Oasis HLB (hydrophilic/lipophilic copolymer sorbent) solid phase extraction (SPE) cartridge and then a Bond Elut-Accucat (cation and anion exchange sorbent) SPE cartridge. The cleaned up extracts were analyzed by positive ion electrospray LC-MS/MS. The MS/MS data was used to detect, confirm, and quantitate acrylamide. The limit of quantitation of the method was 10 ng/g for ground and instant coffees and 1.0 ng/mL for brewed coffee. The levels of acrylamide ranged from 45 to 374 ng/g in unbrewed coffee grounds, from 172 to 539 ng/g in instant coffee crystals, and from 6 to 16 ng/mL in brewed coffee.

Determination of type B fumonisin mycotoxins in maize and maize-based products by liquid chromatography/tandem mass spectrometry using a QqQlinear ion trap mass spectrometer.

PubMed

Faberi, Angelo; Foglia, Patrizia; Pastorini, Elisabetta; Samperi, Roberto; Laganà, Aldo

2005-01-01

A sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for determining the type B fumonisin mycotoxins in corn-based foodstuffs is described. Fumonisins FB1 and FB2 were extracted from a 1 g sample by homogenization with acetonitrile/water (75:25, v/v, 50 mmol/L formic acid, 25 mL final volume) and the extract was defatted on C18 phase. Volumes of 5 mL of crude extracts were cleaned up on Carbograph-4 cartridges. The final solution was analyzed by HPLC with electrospray ionization mass spectrometry in positive ion mode using multiple reaction monitoring with a QqQ linear ion trap mass spectrometer. Recoveries for spiked corn-based foodstuffs ranged from 91-105% (RSD% < or =8%), and method detection limits were < or =2 ng/g for FB1 and < or =1 ng/g for FB2. Two different spiking levels were tested (5000 and 100 ng/g for FB1, 1000 and 20 ng/g for FB2). Quantitation was achieved by an external calibration procedure using matrix-matched standards, with diclofenac added post-cleanup as internal standard for the LC/MS/MS analyses. Calibration curves showed linearity in the concentration range 0.005-5 ng/microL of final extract (0.992 < or = R2< or =0.995). Two other fumonisins, FB3 and FB4, were identified in naturally contaminated samples of corn meal using an information-dependent acquisition protocol that looped three experiments, including neutral loss scan, enhanced resolution scan, and enhanced product ion scan. FB3 and FB4 quantitation was estimated as peak area ratios relative to the FB2 response in view of the lack of both standards. This work also includes an application of the present LC/MS/MS method to some maize and maize-based product samples (corn meal, cornflakes and popcorn) collected from Italian stores. FB1 and FB2 contamination levels exceeding the European Union recommendation were found in 8 out of 15 corn meal samples.

Quantitative interference by cysteine and N-acetylcysteine metabolites during the LC-MS/MS bioanalysis of a small molecule.

PubMed

Barricklow, Jason; Ryder, Tim F; Furlong, Michael T

2009-08-01

During LC-MS/MS quantification of a small molecule in human urine samples from a clinical study, an unexpected peak was observed to nearly co-elute with the analyte of interest in many study samples. Improved chromatographic resolution revealed the presence of at least 3 non-analyte peaks, which were identified as cysteine metabolites and N-acetyl (mercapturic acid) derivatives thereof. These metabolites produced artifact responses in the parent compound MRM channel due to decomposition in the ionization source of the mass spectrometer. Quantitative comparison of the analyte concentrations in study samples using the original chromatographic method and the improved chromatographic separation method demonstrated that the original method substantially over-estimated the analyte concentration in many cases. The substitution of electrospray ionization (ESI) for atmospheric pressure chemical ionization (APCI) nearly eliminated the source instability of these metabolites, which would have mitigated their interference in the quantification of the analyte, even without chromatographic separation. These results 1) demonstrate the potential for thiol metabolite interferences during the quantification of small molecules in pharmacokinetic samples, and 2) underscore the need to carefully evaluate LC-MS/MS methods for molecules that can undergo metabolism to thiol adducts to ensure that they are not susceptible to such interferences during quantification.

Analytical techniques and method validation for the measurement of selected semivolatile and nonvolatile organofluorochemicals in air.

PubMed

Reagen, William K; Lindstrom, Kent R; Thompson, Kathy L; Flaherty, John M

2004-09-01

The widespread use of semi- and nonvolatile organofluorochemicals in industrial facilities, concern about their persistence, and relatively recent advancements in liquid chromatography/mass spectrometry (LC/MS) technology have led to the development of new analytical methods to assess potential worker exposure to airborne organofluorochemicals. Techniques were evaluated for the determination of 19 organofluorochemicals and for total fluorine in ambient air samples. Due to the potential biphasic nature of most of these fluorochemicals when airborne, Occupational Safety and Health Administration (OSHA) versatile sampler (OVS) tubes were used to simultaneously trap fluorochemical particulates and vapors from workplace air. Analytical methods were developed for OVS air samples to quantitatively analyze for total fluorine using oxygen bomb combustion/ion selective electrode and for 17 organofluorochemicals using LC/MS and gas chromatography/mass spectrometry (GC/MS). The experimental design for this validation was based on the National Institute of Occupational Safety and Health (NIOSH) Guidelines for Air Sampling and Analytical Method Development and Evaluation, with some revisions of the experimental design. The study design incorporated experiments to determine analytical recovery and stability, sampler capacity, the effect of some environmental parameters on recoveries, storage stability, limits of detection, precision, and accuracy. Fluorochemical mixtures were spiked onto each OVS tube over a range of 0.06-6 microg for each of 12 compounds analyzed by LC/MS and 0.3-30 microg for 5 compounds analyzed by GC/MS. These ranges allowed reliable quantitation at 0.001-0.1 mg/m3 in general for LC/MS analytes and 0.005-0.5 mg/m3 for GC/MS analytes when 60 L of air are sampled. The organofluorochemical exposure guideline (EG) is currently 0.1 mg/m3 for many analytes, with one exception being ammonium perfluorooctanoate (EG is 0.01 mg/m3). Total fluorine results may be used to determine if the individual compounds quantified provide a suitable mass balance of total airborne organofluorochemicals based on known fluorine content. Improvements in precision and/or recovery as well as some additional testing would be needed to meet all NIOSH validation criteria. This study provided valuable information about the accuracy of this method for organofluorochemical exposure assessment.

The flexibility of a generic LC-MS/MS method for the quantitative analysis of therapeutic proteins based on human immunoglobulin G and related constructs in animal studies.

PubMed

Lanshoeft, Christian; Wolf, Thierry; Walles, Markus; Barteau, Samuel; Picard, Franck; Kretz, Olivier; Cianférani, Sarah; Heudi, Olivier

2016-11-30

An increasing demand of new analytical methods is associated with the growing number of biotherapeutic programs being prosecuted in the pharmaceutical industry. Whilst immunoassay has been the standard method for decades, a great interest in assays based on liquid chromatography tandem mass spectrometry (LC-MS/MS) is evolving. In this present work, the development of a generic method for the quantitative analysis of therapeutic proteins based on human immunoglobulin G (hIgG) in rat serum is reported. The method is based on four generic peptides GPSVFPLAPSSK (GPS), TTPPVLDSDGSFFLYSK (TTP), VVSVLTVLHQDWLNGK (VVS) and FNWYVDGVEVHNAK (FNW) originating from different parts of the fraction crystallizable (Fc) region of a reference hIgG1 (hIgG1A). A tryptic pellet digestion of rat serum spiked with hIgG1A and a stable isotope labeled protein (hIgG1B) used as internal standard (ISTD) was applied prior LC-MS/MS analysis. The upper limit of quantification was at 1000μg/mL. The lower limit of quantitation was for GPS, TTP and VVS at 1.00μg/mL whereas for FNW at 5.00μg/mL. Accuracy and precision data met acceptance over three days. The presented method was further successfully applied to the quantitative analysis of other hIgG1s (hIgG1C and hIgG1D) and hIgG4-based therapeutic proteins on spiked quality control (QC) samples in monkey and rat serum using calibration standards (Cs) prepared with hIgG1A in rat serum. In order to extend the applicability of our generic approach, a bispecific-bivalent hIgG1 (bb-hIgG1) and two lysine conjugated antibody-drug conjugates (ADC1 and ADC2) were incorporated as well. The observed values on spiked QC samples in monkey serum were satisfactory with GPS for the determination of bb-hIgG1 whereas the FNW and TTP peptides were suitable for the ADCs. Moreover, comparable mean concentration-time profiles were obtained from monkeys previously dosed intravenously with ADC2 measured against Cs samples prepared either with hIgG1A in rat serum (presented approach) or with the actual ADC2 in monkey serum (conventional approach). The results of this study highlight the great flexibility of our newly developed generic approach and that the choice of the surrogate peptide still remains critical when dealing with different matrix types or modalities. Copyright © 2016 Elsevier B.V. All rights reserved.

Potential of capillary-column-switching liquid chromatography-tandem mass spectrometry for the quantitative trace analysis of small molecules. Application to the on-line screening of drugs in water.

PubMed

Pitarch, Elena; Hernandez, Felix; ten Hove, Jan; Meiring, Hugo; Niesing, Willem; Dijkman, Ellen; Stolker, Linda; Hogendoorn, Elbert

2004-03-26

We have investigated the potential of capillary-column-switching liquid chromatography coupled to tandem mass spectrometry (cLC-MS-MS) for the quantitative on-line trace analysis of target compounds in aqueous solutions. The technical design of the nano-scale cLC system developed at our Institute for peptide and protein identification has been tested and evaluated for the direct trace analysis of drugs in water samples. Sulphametoxazole, bezafibrate, metoprolol, carbamazepine and bisoprolol occurring frequently in Dutch waters, were selected as test compounds. Adequate conditions for trapping, elution and MS-MS detection were investigated by employing laboratory made 200 microm i.d. capillary columns packed with 5 microm aqua C18 material. In the final cLC-MS-MS conditions, a 1 cm length trapping column and a 4 cm length analytical column were selected. Under these conditions, the target compounds could be directly determined in water down to a level of around 50 ng/l employing only 25 microl of water sample. Validation was done by recovery experiments in ground-, surface- and drinking-water matrices as well as by the analysis of water samples with incurred residues and previously analyzed with a conventional procedure involving off-line solid-phase extraction and narrow-bore LC with MS-MS detection. The new methodology provided recoveries (50-500 ng/l level) between 50 and 114% with RSDs (n = 3, each level) below 20% for most of the compounds. Despite the somewhat less analytical performance in comparison to the conventional procedure, the on-line approach of the new methodology is very suitable for screening of drugs in aqueous samples.

Identification and determination of the saikosaponins in Radix bupleuri by accelerated solvent extraction combined with rapid-resolution LC-MS.

PubMed

Yang, Yun-Yun; Tang, You-Zhi; Fan, Chun-Lin; Luo, Hui-Tai; Guo, Peng-Ran; Chen, Jian-Xin

2010-07-01

A method based on accelerated solvent extraction combined with rapid-resolution LC-MS for efficient extraction, rapid separation, online identification and accurate determination of the saikosaponins (SSs) in Radix bupleuri (RB) was developed. The RB samples were extracted by accelerated solvent extraction using 70% aqueous ethanol v/v as solvent, at a temperature of 120 degrees C and pressure of 100 bar, with 10 min of static extraction time and three extraction cycles. Rapid-resolution LC separation was performed by using a C(18) column at gradient elution of water (containing 0.5% formic acid) and acetonitrile, and the major constituents were well separated within 20 min. A TOF-MS and an IT-MS were used for online identification of the major constituents, and 27 SSs were identified or tentatively identified. Five major bioactive SSs (SSa, SSc, SSd, 6''-O-acetyl-SSa and 6''-O-acetyl-SSd) with obvious peak areas and good resolution were chosen as benchmark substances, and a triple quadrupole MS operating in multiple-reaction monitoring mode was used for their quantitative analysis. A total of 16 RB samples from different regions of China were analyzed. The results indicated that the method was rapid, efficient, accurate and suitable for use in the quality control of RB.

Automated solid-phase extraction workstations combined with quantitative bioanalytical LC/MS.

PubMed

Huang, N H; Kagel, J R; Rossi, D T

1999-03-01

An automated solid-phase extraction workstation was used to develop, characterize and validate an LC/MS/MS method for quantifying a novel lipid-regulating drug in dog plasma. Method development was facilitated by workstation functions that allowed wash solvents of varying organic composition to be mixed and tested automatically. Precision estimates for this approach were within 9.8% relative standard deviation (RSD) across the calibration range. Accuracy for replicate determinations of quality controls was between -7.2 and +6.2% relative error (RE) over 5-1,000 ng/ml(-1). Recoveries were evaluated for a wide variety of wash solvents, elution solvents and sorbents. Optimized recoveries were generally > 95%. A sample throughput benchmark for the method was approximately equal 8 min per sample. Because of parallel sample processing, 100 samples were extracted in less than 120 min. The approach has proven useful for use with LC/MS/MS, using a multiple reaction monitoring (MRM) approach.

An evaluation of an aptamer for use as an affinity reagent with MS: PCSK9 as an example protein.

PubMed

Gupta, Vinita; Lassman, Michael E; McAvoy, Thomas; Lee, Anita Yh; Chappell, Derek L; Laterza, Omar F

2016-08-01

For quantitative immunoaffinity IA-LC-MS, the utility of antibodies has been demonstrated many times but the utility of aptamers as affinity reagents is unproven. Immunoaffinity reagents including a monoclonal antibody and an aptamer were coupled to magnetic beads and used as part of an enrichment strategy for PCSK9 quantitation in plasma. With limited method development, we have established a comparison of an anti-PCSK9 aptamer with an anti-PCSK9 monoclonal antibody. The background that results from a tryptic digest of affinity enrichment in plasma was demonstrated for each reagent using high-resolution full scan MS. The assay recovery was demonstrated for multiple concentrations of aptamer in plasma with different concentrations of PCSK9 protein. The aptamer achieved comparable enrichment to the antibody, but with lower peptide background, thus demonstrating the potential use of aptamers for IA-LC-MS.

LC-MS/MS method development for quantitative analysis of acetaminophen uptake by the aquatic fungus Mucor hiemalis.

PubMed

Esterhuizen-Londt, Maranda; Schwartz, Katrin; Balsano, Evelyn; Kühn, Sandra; Pflugmacher, Stephan

2016-06-01

Acetaminophen is a pharmaceutical, frequently found in surface water as a contaminant. Bioremediation, in particular, mycoremediation of acetaminophen is a method to remove this compound from waters. Owing to the lack of quantitative analytical method for acetaminophen in aquatic organisms, the present study aimed to develop a method for the determination of acetaminophen using LC-MS/MS in the aquatic fungus Mucor hiemalis. The method was then applied to evaluate the uptake of acetaminophen by M. hiemalis, cultured in pellet morphology. The method was robust, sensitive and reproducible with a lower limit of quantification of 5 pg acetaminophen on column. It was found that M. hiemalis internalize the pharmaceutical, and bioaccumulate it with time. Therefore, M. hiemalis was deemed a suitable candidate for further studies to elucidate its pharmaceutical tolerance and the longevity in mycoremediation applications. Copyright © 2016 Elsevier Inc. All rights reserved.

The clinical impact of recent advances in LC-MS for cancer biomarker discovery and verification.

PubMed

Wang, Hui; Shi, Tujin; Qian, Wei-Jun; Liu, Tao; Kagan, Jacob; Srivastava, Sudhir; Smith, Richard D; Rodland, Karin D; Camp, David G

2016-01-01

Mass spectrometry (MS) -based proteomics has become an indispensable tool with broad applications in systems biology and biomedical research. With recent advances in liquid chromatography (LC) and MS instrumentation, LC-MS is making increasingly significant contributions to clinical applications, especially in the area of cancer biomarker discovery and verification. To overcome challenges associated with analyses of clinical samples (for example, a wide dynamic range of protein concentrations in bodily fluids and the need to perform high throughput and accurate quantification of candidate biomarker proteins), significant efforts have been devoted to improve the overall performance of LC-MS-based clinical proteomics platforms. Reviewed here are the recent advances in LC-MS and its applications in cancer biomarker discovery and quantification, along with the potentials, limitations and future perspectives.

Validation of the Mass-Extraction-Window for Quantitative Methods Using Liquid Chromatography High Resolution Mass Spectrometry.

PubMed

Glauser, Gaétan; Grund, Baptiste; Gassner, Anne-Laure; Menin, Laure; Henry, Hugues; Bromirski, Maciej; Schütz, Frédéric; McMullen, Justin; Rochat, Bertrand

2016-03-15

A paradigm shift is underway in the field of quantitative liquid chromatography-mass spectrometry (LC-MS) analysis thanks to the arrival of recent high-resolution mass spectrometers (HRMS). The capability of HRMS to perform sensitive and reliable quantifications of a large variety of analytes in HR-full scan mode is showing that it is now realistic to perform quantitative and qualitative analysis with the same instrument. Moreover, HR-full scan acquisition offers a global view of sample extracts and allows retrospective investigations as virtually all ionized compounds are detected with a high sensitivity. In time, the versatility of HRMS together with the increasing need for relative quantification of hundreds of endogenous metabolites should promote a shift from triple-quadrupole MS to HRMS. However, a current "pitfall" in quantitative LC-HRMS analysis is the lack of HRMS-specific guidance for validated quantitative analyses. Indeed, false positive and false negative HRMS detections are rare, albeit possible, if inadequate parameters are used. Here, we investigated two key parameters for the validation of LC-HRMS quantitative analyses: the mass accuracy (MA) and the mass-extraction-window (MEW) that is used to construct the extracted-ion-chromatograms. We propose MA-parameters, graphs, and equations to calculate rational MEW width for the validation of quantitative LC-HRMS methods. MA measurements were performed on four different LC-HRMS platforms. Experimentally determined MEW values ranged between 5.6 and 16.5 ppm and depended on the HRMS platform, its working environment, the calibration procedure, and the analyte considered. The proposed procedure provides a fit-for-purpose MEW determination and prevents false detections.

Retention time alignment of LC/MS data by a divide-and-conquer algorithm.

PubMed

Zhang, Zhongqi

2012-04-01

Liquid chromatography-mass spectrometry (LC/MS) has become the method of choice for characterizing complex mixtures. These analyses often involve quantitative comparison of components in multiple samples. To achieve automated sample comparison, the components of interest must be detected and identified, and their retention times aligned and peak areas calculated. This article describes a simple pairwise iterative retention time alignment algorithm, based on the divide-and-conquer approach, for alignment of ion features detected in LC/MS experiments. In this iterative algorithm, ion features in the sample run are first aligned with features in the reference run by applying a single constant shift of retention time. The sample chromatogram is then divided into two shorter chromatograms, which are aligned to the reference chromatogram the same way. Each shorter chromatogram is further divided into even shorter chromatograms. This process continues until each chromatogram is sufficiently narrow so that ion features within it have a similar retention time shift. In six pairwise LC/MS alignment examples containing a total of 6507 confirmed true corresponding feature pairs with retention time shifts up to five peak widths, the algorithm successfully aligned these features with an error rate of 0.2%. The alignment algorithm is demonstrated to be fast, robust, fully automatic, and superior to other algorithms. After alignment and gap-filling of detected ion features, their abundances can be tabulated for direct comparison between samples.

LC-MS/MS-based approach for obtaining exposure estimates of metabolites in early clinical trials using radioactive metabolites as reference standards.

PubMed

Zhang, Donglu; Raghavan, Nirmala; Chando, Theodore; Gambardella, Janice; Fu, Yunlin; Zhang, Duxi; Unger, Steve E; Humphreys, W Griffith

2007-12-01

An LC-MS/MS-based approach that employs authentic radioactive metabolites as reference standards was developed to estimate metabolite exposures in early drug development studies. This method is useful to estimate metabolite levels in studies done with non-radiolabeled compounds where metabolite standards are not available to allow standard LC-MS/MS assay development. A metabolite mixture obtained from an in vivo source treated with a radiolabeled compound was partially purified, quantified, and spiked into human plasma to provide metabolite standard curves. Metabolites were analyzed by LC-MS/MS using the specific mass transitions and an internal standard. The metabolite concentrations determined by this approach were found to be comparable to those determined by valid LC-MS/MS assays. This approach does not requires synthesis of authentic metabolites or the knowledge of exact structures of metabolites, and therefore should provide a useful method to obtain early estimates of circulating metabolites in early clinical or toxicological studies.

A statistical framework for protein quantitation in bottom-up MS-based proteomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karpievitch, Yuliya; Stanley, Jeffrey R.; Taverner, Thomas

2009-08-15

ABSTRACT Motivation: Quantitative mass spectrometry-based proteomics requires protein-level estimates and confidence measures. Challenges include the presence of low-quality or incorrectly identified peptides and widespread, informative, missing data. Furthermore, models are required for rolling peptide-level information up to the protein level. Results: We present a statistical model for protein abundance in terms of peptide peak intensities, applicable to both label-based and label-free quantitation experiments. The model allows for both random and censoring missingness mechanisms and provides naturally for protein-level estimates and confidence measures. The model is also used to derive automated filtering and imputation routines. Three LC-MS datasets are used tomore » illustrate the methods. Availability: The software has been made available in the open-source proteomics platform DAnTE (Polpitiya et al. (2008)) (http://omics.pnl.gov/software/). Contact: adabney@stat.tamu.edu« less

Benchmarking quantitative label-free LC-MS data processing workflows using a complex spiked proteomic standard dataset.

PubMed

Ramus, Claire; Hovasse, Agnès; Marcellin, Marlène; Hesse, Anne-Marie; Mouton-Barbosa, Emmanuelle; Bouyssié, David; Vaca, Sebastian; Carapito, Christine; Chaoui, Karima; Bruley, Christophe; Garin, Jérôme; Cianférani, Sarah; Ferro, Myriam; Van Dorssaeler, Alain; Burlet-Schiltz, Odile; Schaeffer, Christine; Couté, Yohann; Gonzalez de Peredo, Anne

2016-01-30

Proteomic workflows based on nanoLC-MS/MS data-dependent-acquisition analysis have progressed tremendously in recent years. High-resolution and fast sequencing instruments have enabled the use of label-free quantitative methods, based either on spectral counting or on MS signal analysis, which appear as an attractive way to analyze differential protein expression in complex biological samples. However, the computational processing of the data for label-free quantification still remains a challenge. Here, we used a proteomic standard composed of an equimolar mixture of 48 human proteins (Sigma UPS1) spiked at different concentrations into a background of yeast cell lysate to benchmark several label-free quantitative workflows, involving different software packages developed in recent years. This experimental design allowed to finely assess their performances in terms of sensitivity and false discovery rate, by measuring the number of true and false-positive (respectively UPS1 or yeast background proteins found as differential). The spiked standard dataset has been deposited to the ProteomeXchange repository with the identifier PXD001819 and can be used to benchmark other label-free workflows, adjust software parameter settings, improve algorithms for extraction of the quantitative metrics from raw MS data, or evaluate downstream statistical methods. Bioinformatic pipelines for label-free quantitative analysis must be objectively evaluated in their ability to detect variant proteins with good sensitivity and low false discovery rate in large-scale proteomic studies. This can be done through the use of complex spiked samples, for which the "ground truth" of variant proteins is known, allowing a statistical evaluation of the performances of the data processing workflow. We provide here such a controlled standard dataset and used it to evaluate the performances of several label-free bioinformatics tools (including MaxQuant, Skyline, MFPaQ, IRMa-hEIDI and Scaffold) in different workflows, for detection of variant proteins with different absolute expression levels and fold change values. The dataset presented here can be useful for tuning software tool parameters, and also testing new algorithms for label-free quantitative analysis, or for evaluation of downstream statistical methods. Copyright © 2015 Elsevier B.V. All rights reserved.

High Resolution Separations and Improved Ion Production and Transmission in Metabolomics

PubMed Central

Metz, Thomas O.; Page, Jason S.; Baker, Erin S.; Tang, Keqi; Ding, Jie; Shen, Yufeng; Smith, Richard D.

2008-01-01

The goal of metabolomics analyses is the detection and quantitation of as many sample components as reasonably possible in order to identify compounds or “features” that can be used to characterize the samples under study. When utilizing electrospray ionization to produce ions for analysis by mass spectrometry (MS), it is important that metabolome sample constituents be efficiently separated prior to ion production, in order to minimize ionization suppression and thereby extend the dynamic range of the measurement, as well as the coverage of the metabolome. Similarly, optimization of the MS inlet and interface can lead to increased measurement sensitivity. This perspective review will focus on the role of high resolution liquid chromatography (LC) separations in conjunction with improved ion production and transmission for LC-MS-based metabolomics. Additional emphasis will be placed on the compromise between metabolome coverage and sample analysis throughput. PMID:19255623

Comparison of clonazepam compliance by measurement of urinary concentration by immunoassay and LC-MS/MS in pain management population.

PubMed

West, Robert; Pesce, Amadeo; West, Cameron; Crews, Bridgit; Mikel, Charles; Almazan, Perla; Rosenthal, Murray; Latyshev, Sergey

2010-01-01

Physicians determine patient compliance with their medications by use of urine drug testing. It is known that measurement of benzodiazepines is limited by immunoassay specificity and cutoff limits and therefore does not offer physicians an accurate picture of their patients' compliance with these medications. A few studies have used lower cutoffs to demonstrate patient compliance. To define more appropriate cutoffs for compliance monitoring of patients prescribed clonazepam as determined using immunoassay and liquid chromatography-tandem mass spectrometry (LC-MS/MS). A diagnostic accuracy study of the urinary excretion of clonazepam. Millennium Laboratories performed measurements on the urinary excretion of pain patients prescribed clonazepam as the indicator test. This benzodiazepine was chosen because it forms one major metabolite, 7-aminoclonazepam which is specific for that drug. Patients whose only benzodiazepine medication was clonazepam were selected as the test population. The Millennium Laboratories test database was filtered first to select patients on clonazepam, then a second filter was used to eliminate patients with any other listed benzodiazepine medications. Samples were tested using the Microgenics DRI benzodiazepine assay with a 200 ng/mL cutoff. The same samples were quantitatively assessed for 7-aminoclonazepam by LC-MS/MS with a cutoff of 40 ng/mL. The results from the immunoassay were scored as positive or negative while the quantitative results from the LC-MS/MS were also scored as positive or negative depending upon their concentration. Samples from 180 patients met these medication criteria. The positivity rates were 21% (38 samples) by immunoassay. The positivity rate was 70% (126 samples) if the LC-MS/MS cutoff was set at 200 ng/mL. However, the positivity rate was 87% (157 samples) if the LC-MS/MS was set at 40 ng/mL. Concentration distributions revealed a significant fraction (7%) in the 40 - 100 ng/mL range. A limitation of the study was the inability to measure lower than 40 ng/mL. There may be another fraction of the population that was positive below the cutoff value. The difference in positivity rate between the immunoassay and the LC-MS/MS result showed that the nominal 200 ng/mL cutoff of the immunoassay did not apply to 7-aminoclonazepam. This low immunoassay positivity rate is inconsistent with the manufacturer's published cross reactivity data for clonazepam and 7-aminoclonazepam. These data illustrate the limitations of using a 200 ng/mL cutoff to monitor clonazepam compliance and suggest that a cutoff of 40 ng/mL or less is needed to reliably monitor use of this drug.

Sensitivity of GC-EI/MS, GC-EI/MS/MS, LC-ESI/MS/MS, LC-Ag(+) CIS/MS/MS, and GC-ESI/MS/MS for analysis of anabolic steroids in doping control.

PubMed

Cha, Eunju; Kim, Sohee; Kim, Ho Jun; Lee, Kang Mi; Kim, Ki Hun; Kwon, Oh-Seung; Lee, Jaeick

2015-01-01

This study compared the sensitivity of various separation and ionization methods, including gas chromatography with an electron ionization source (GC-EI), liquid chromatography with an electrospray ionization source (LC-ESI), and liquid chromatography with a silver ion coordination ion spray source (LC-Ag(+) CIS), coupled to a mass spectrometer (MS) for steroid analysis. Chromatographic conditions, mass spectrometric transitions, and ion source parameters were optimized. The majority of steroids in GC-EI/MS/MS and LC-Ag(+) CIS/MS/MS analysis showed higher sensitivities than those obtained with other analytical methods. The limits of detection (LODs) of 65 steroids by GC-EI/MS/MS, 68 steroids by LC-Ag(+) CIS/MS/MS, 56 steroids by GC-EI/MS, 54 steroids by LC-ESI/MS/MS, and 27 steroids by GC-ESI/MS/MS were below cut-off value of 2.0 ng/mL. LODs of steroids that formed protonated ions in LC-ESI/MS/MS analysis were all lower than the cut-off value. Several steroids such as unconjugated C3-hydroxyl with C17-hydroxyl structure showed higher sensitivities in GC-EI/MS/MS analysis relative to those obtained using the LC-based methods. The steroids containing 4, 9, 11-triene structures showed relatively poor sensitivities in GC-EI/MS and GC-ESI/MS/MS analysis. The results of this study provide information that may be useful for selecting suitable analytical methods for confirmatory analysis of steroids. Copyright © 2015 John Wiley & Sons, Ltd.

Quantitation of 25-hydroxyvitamin D in dried blood spots by 2D LC-MS/MS without derivatization and correlation with serum in adult and pediatric studies.

PubMed

Jensen, Berit P; Saraf, Rajneeta; Ma, Jing; Berry, Sarah; Grant, Cameron C; Camargo, Carlos A; Sies, Christiaan W

2018-06-01

Demand for measurement of 25-hydroxyvitamin D (25OHD) is growing and dried blood spot (DBS) sampling is attractive as samples are easier to collect, transport and store. A 2D LC-MS/MS assay without derivatization was developed. DBS punches (3.2 mm) were ultrasonicated with d 6 -25OHD 3 in 70% methanol followed by hexane extraction, dry-down and reconstitution. The assay was validated and applied to two studies comparing whole blood adult DBS with serum samples (n = 40) and neonatal whole blood DBS with cord serum samples (n = 80). The assay was validated in whole blood DBS over the range 13-106 nmol/L 25OHD 3 and 11-91 nmol/L 25OHD 2 with a limit of detection of 3 nmol/L. Intra- and inter-day imprecision was <13% CV and bias <12%. The assay had high recovery and minimal matrix effects. Triplicate DBS study samples had a mean CV of ≤13% for 25OHD 3. No 25OHD 2 was detected. DBS calculated serum 25OHD 3 concentrations correlated strongly with serum concentrations in the adult DBS/serum study (r = 0.94) and moderately in the neonatal DBS/cord serum study (r = 0.69). Direct quantitation of 25OHD in DBS by 2D LC-MS/MS without derivatization was found to be an alternative to serum quantitation applicable to clinical research studies on adult DBS samples. Copyright © 2018 Elsevier B.V. All rights reserved.

A comparative proteomics method for multiple samples based on a 18O-reference strategy and a quantitation and identification-decoupled strategy.

PubMed

Wang, Hongbin; Zhang, Yongqian; Gui, Shuqi; Zhang, Yong; Lu, Fuping; Deng, Yulin

2017-08-15

Comparisons across large numbers of samples are frequently necessary in quantitative proteomics. Many quantitative methods used in proteomics are based on stable isotope labeling, but most of these are only useful for comparing two samples. For up to eight samples, the iTRAQ labeling technique can be used. For greater numbers of samples, the label-free method has been used, but this method was criticized for low reproducibility and accuracy. An ingenious strategy has been introduced, comparing each sample against a 18 O-labeled reference sample that was created by pooling equal amounts of all samples. However, it is necessary to use proportion-known protein mixtures to investigate and evaluate this new strategy. Another problem for comparative proteomics of multiple samples is the poor coincidence and reproducibility in protein identification results across samples. In present study, a method combining 18 O-reference strategy and a quantitation and identification-decoupled strategy was investigated with proportion-known protein mixtures. The results obviously demonstrated that the 18 O-reference strategy had greater accuracy and reliability than other previously used comparison methods based on transferring comparison or label-free strategies. By the decoupling strategy, the quantification data acquired by LC-MS and the identification data acquired by LC-MS/MS are matched and correlated to identify differential expressed proteins, according to retention time and accurate mass. This strategy made protein identification possible for all samples using a single pooled sample, and therefore gave a good reproducibility in protein identification across multiple samples, and allowed for optimizing peptide identification separately so as to identify more proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of a SIDA-LC-MS/MS Method for the Determination of Phomopsin A in Legumes.

PubMed

Schloß, Svenja; Koch, Matthias; Rohn, Sascha; Maul, Ronald

2015-12-09

A novel method for the determination of phomopsin A (1) in lupin flour, pea flour, and bean flour as well as whole lupin plants was established based on stable isotope dilution assay (SIDA) LC-MS/MS using (15)N6-1 as an isotopically labeled internal standard. Artificially infected samples were used to develop an optimized extraction procedure and sample pretreatment. The limits of detection were 0.5-1 μg/kg for all matrices. The limits of quantitation were 2-4 μg/kg. The method was used to analyze flour samples generated from selected legume seeds and lupin plant samples that had been inoculated with Diaporthe toxica and two further fungal strains. Finally, growing lupin plants infected with D. toxica were investigated to simulate a naturally in-field mycotoxicosis. Toxin levels of up to 10.1 μg/kg of 1 were found in the pods and 7.2 μg/kg in the stems and leaves.

Analysis of phenolic choline esters from seeds of Arabidopsis thaliana and Brassica napus by capillary liquid chromatography/electrospray- tandem mass spectrometry.

PubMed

Böttcher, Christoph; von Roepenack-Lahaye, Edda; Schmidt, Jürgen; Clemens, Stephan; Scheel, Dierk

2009-04-01

Total phenolic choline ester fractions prepared from seeds of Arabidopsis thaliana and Brassica napus were analyzed by capillary LC/ESI-QTOF-MS and direct infusion ESI-FTICR-MS. In addition to the dominating sinapoylcholine, 30 phenolic choline esters could be identified based on accurate mass measurements, interpretation of collision-induced dissociation (CID) mass spectra, and synthesis of selected representatives. The compounds identified so far include substituted hydroxycinnamoyl- and hydroxybenzoylcholines, respective monohexosides as well as oxidative coupling products of phenolic choline esters and monolignols. Phenolic choline esters are well separable by reversed-phase liquid chromatography and sensitively detectable using electrospray ionization mass spectrometry in positive ion mode. CID mass spectra obtained from molecular ions facilitate the characterization of both the type and substitution pattern of such compounds. Therefore, LC/ESI-MS/MS represents a valuable tool for comprehensive qualitative and quantitative analysis of this compound class. Copyright (c) 2009 John Wiley & Sons, Ltd.

Rapid and reliable quantitation of amino acids and myo-inositol in mouse brain by high performance liquid chromatography and tandem mass spectrometry.

PubMed

Bathena, Sai P; Huang, Jiangeng; Epstein, Adrian A; Gendelman, Howard E; Boska, Michael D; Alnouti, Yazen

2012-04-15

Amino acids and myo-inositol have long been proposed as putative biomarkers for neurodegenerative diseases. Accurate measures and stability have precluded their selective use. To this end, a sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method based on multiple reaction monitoring was developed to simultaneously quantify glutamine, glutamate, γ-aminobutyric acid (GABA), aspartic acid, N-acetyl aspartic acid, taurine, choline, creatine, phosphocholine and myo-inositol in mouse brain by methanol extractions. Chromatography was performed using a hydrophilic interaction chromatography silica column within in a total run time of 15 min. The validated method is selective, sensitive, accurate, and precise. The method has a limit of quantification ranging from 2.5 to 20 ng/ml for a range of analytes and a dynamic range from 2.5-20 to 500-4000 ng/ml. This LC-MS/MS method was validated for biomarker discovery in models of human neurological disorders. Copyright © 2012 Elsevier B.V. All rights reserved.

Electronic tongue-based discrimination of Korean rice wines (makgeolli) including prediction of sensory evaluation and instrumental measurements.

PubMed

Kang, Bo-Sik; Lee, Jang-Eun; Park, Hyun-Jin

2014-05-15

A commercial electronic tongue was used to discriminate Korean rice wines (makgeolli) brewed from nine cultivars of rice with different amino acid and fatty acid compositions. The E-tongue was applied to establish prediction models with sensory evaluation or LC-MS/MS by partial least squares regression (PLSR). All makgeollis were classified into three groups by principal components analysis, and the separation pattern was affected by rice qualities and yeast fermentation. Makgeolli taste changed from the complicated comprising sweetness, saltiness, and umami to the uncomplicated, such as bitterness and then, sourness, with a decrease of amino acids and fatty acids in the rice. The quantitative correlation between E-tongue and sensory scores or LC-MS/MS by PLSR demonstrated that E-tongue could well predict most of the sensory attributes with relatively acceptable r(2), except for bitterness, but could not predict most of the chemical compounds responsible for taste attributes, except for ribose, lactate, succinate, and tryptophan. Copyright © 2013 Elsevier Ltd. All rights reserved.

Improved profiling of estrogen metabolites by orbitrap LC/MS

PubMed Central

Li, Xingnan; Franke, Adrian A.

2015-01-01

Estrogen metabolites are important biomarkers to evaluate cancer risks and metabolic diseases. Due to their low physiological levels, a sensitive and accurate method is required, especially for the quantitation of unconjugated forms of endogenous steroids and their metabolites in humans. Here, we evaluated various derivatives of estrogens for improved analysis by orbitrap LC/MS in human serum samples. A new chemical derivatization reagent was applied modifying phenolic steroids to form 1-methylimidazole-2-sulfonyl adducts. The method significantly improves the sensitivity 2–100 fold by full scan MS and targeted selected ion monitoring MS over other derivatization methods including, dansyl, picolinoyl, and pyridine-3-sulfonyl products. PMID:25543003

Mass spectrometric based approaches in urine metabolomics and biomarker discovery.

PubMed

Khamis, Mona M; Adamko, Darryl J; El-Aneed, Anas

2017-03-01

Urine metabolomics has recently emerged as a prominent field for the discovery of non-invasive biomarkers that can detect subtle metabolic discrepancies in response to a specific disease or therapeutic intervention. Urine, compared to other biofluids, is characterized by its ease of collection, richness in metabolites and its ability to reflect imbalances of all biochemical pathways within the body. Following urine collection for metabolomic analysis, samples must be immediately frozen to quench any biogenic and/or non-biogenic chemical reactions. According to the aim of the experiment; sample preparation can vary from simple procedures such as filtration to more specific extraction protocols such as liquid-liquid extraction. Due to the lack of comprehensive studies on urine metabolome stability, higher storage temperatures (i.e. 4°C) and repetitive freeze-thaw cycles should be avoided. To date, among all analytical techniques, mass spectrometry (MS) provides the best sensitivity, selectivity and identification capabilities to analyze the majority of the metabolite composition in the urine. Combined with the qualitative and quantitative capabilities of MS, and due to the continuous improvements in its related technologies (i.e. ultra high-performance liquid chromatography [UPLC] and hydrophilic interaction liquid chromatography [HILIC]), liquid chromatography (LC)-MS is unequivocally the most utilized and the most informative analytical tool employed in urine metabolomics. Furthermore, differential isotope tagging techniques has provided a solution to ion suppression from urine matrix thus allowing for quantitative analysis. In addition to LC-MS, other MS-based technologies have been utilized in urine metabolomics. These include direct injection (infusion)-MS, capillary electrophoresis-MS and gas chromatography-MS. In this article, the current progresses of different MS-based techniques in exploring the urine metabolome as well as the recent findings in providing potentially diagnostic urinary biomarkers are discussed. © 2015 Wiley Periodicals, Inc. Mass Spec Rev 36:115-134, 2017. © 2015 Wiley Periodicals, Inc.

LC-MS/MS imaging with thermal film-based laser microdissection.

PubMed

Oya, Michiko; Suzuki, Hiromi; Anas, Andrea Roxanne J; Oishi, Koichi; Ono, Kenji; Yamaguchi, Shun; Eguchi, Megumi; Sawada, Makoto

2018-01-01

Mass spectrometry (MS) imaging is a useful tool for direct and simultaneous visualization of specific molecules. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to evaluate the abundance of molecules in tissues using sample homogenates. To date, however, LC-MS/MS has not been utilized as an imaging tool because spatial information is lost during sample preparation. Here we report a new approach for LC-MS/MS imaging using a thermal film-based laser microdissection (LMD) technique. To isolate tissue spots, our LMD system uses a 808-nm near infrared laser, the diameter of which can be freely changed from 2.7 to 500 μm; for imaging purposes in this study, the diameter was fixed at 40 μm, allowing acquisition of LC-MS/MS images at a 40-μm resolution. The isolated spots are arranged on a thermal film at 4.5-mm intervals, corresponding to the well spacing on a 384-well plate. Each tissue spot is handled on the film in such a manner as to maintain its spatial information, allowing it to be extracted separately in its individual well. Using analytical LC-MS/MS in combination with the spatial information of each sample, we can reconstruct LC-MS/MS images. With this imaging technique, we successfully obtained the distributions of pilocarpine, glutamate, γ-aminobutyric acid, acetylcholine, and choline in a cross-section of mouse hippocampus. The protocol we established in this study is applicable to revealing the neurochemistry of pilocarpine model of epilepsy. Our system has a wide range of uses in fields such as biology, pharmacology, pathology, and neuroscience. Graphical abstract Schematic Indication of LMD-LC-MS/MS imaging.

Absolute quantification of histone PTM marks by MRM-based LC-MS/MS.

PubMed

Gao, Jun; Liao, Rijing; Yu, Yanyan; Zhai, Huili; Wang, Yingqi; Sack, Ragna; Peters, Antoine H F M; Chen, Jiajia; Wu, Haiping; Huang, Zheng; Hu, Min; Qi, Wei; Lu, Chris; Atadja, Peter; Oyang, Counde; Li, En; Yi, Wei; Zhou, Shaolian

2014-10-07

The N-terminal tails of core histones harbor the sites of numerous post-translational modifications (PTMs) with important roles in the regulation of chromatin structure and function. Profiling histone PTM marks provides data that help understand the epigenetics events in cells and their connections with cancer and other diseases. Our previous study demonstrated that specific derivatization of histone peptides by NHS propionate significantly improved their chromatographic performance on reversed phase columns for LC/MS analysis. As a step forward, we recently developed a multiple reaction monitoring (MRM) based LC-MS/MS method to analyze 42 targeted histone peptides. By using stable isotopic labeled peptides as internal standards that are spiked into the reconstituted solutions, this method allows to measure absolute concentration of the tryptic peptides of H3 histone proteins extracted from cancer cell lines. The method was thoroughly validated for the accuracy and reproducibility through analyzing recombinant histone proteins and cellular samples. The linear dynamic range of the MRM assays was achieved in 3 orders of magnitude from 1 nM to 1 μM for all targeted peptides. Excellent intrabatch and interbatch reproducibility (<15% CV) was obtained. This method has been used to study translocated NSD2 (a histone lysine methyltransferase that catalyzes the histone lysine 36 methylation) function with its overexpression in KMS11 multiple myeloma cells. From the results we have successfully quantitated both individual and combinatorial histone marks in parental and NSD2 selective knockout KMS11 cells.

The application of multiple analyte adduct formation in the LC-MS3 analysis of valproic acid in human serum.

PubMed

Dziadosz, Marek

2017-01-01

LC-MS using electrospray ionisation (negative ion mode) and low-energy collision-induced dissociation tandem mass spectrometric (CID-MS/MS) analysis, together with the multiple analyte adduct formation with the components of the mobile phase, were applied to analyse valproic acid in human serum with LC-MS 3 . The CID-fragmentation of the precursor analyte adduct [M+2CH 3 COONa-H] - was applied in the method validation (307.1/225.1/143.0). Chromatographic separation was performed with a Luna 5μm C18 (2) 100A, 150mm×2mm column and the elution with a mobile phase consisting of A (H 2 O/methanol=95/5, v/v) and B (H 2 O/methanol=3/97, v/v), both with 10mM ammonium acetate and 0.1% acetic acid. A binary flow pumping mode with a total flow rate of 0.400mL/min was used. The calculated limit of detection/quantification of the method calibrated in the range of 10-200μg/mL was 0.31/1.0μg/mL. The sample preparation based on protein precipitation with 1mL of H 2 O/methanol solution (3/97, v/v) with 10mM sodium acetate and 100mM acetic acid. On the basis of the experiments performed could be demonstrated, that multiple analyte adduct formation can be applied to generate MS 3 quantitation of analytes with problematic fragmentation. The presented new strategy makes the analysis of small drugs, which do not produce any stable product ions at all, on the basis of LC-MS 3 possible. Copyright © 2016 Elsevier B.V. All rights reserved.

Heterogeneous distribution of alectinib in neuroblastoma xenografts revealed by matrix-assisted laser desorption ionization mass spectrometry imaging: a pilot study.

PubMed

Ryu, Shoraku; Hayashi, Mitsuhiro; Aikawa, Hiroaki; Okamoto, Isamu; Fujiwara, Yasuhiro; Hamada, Akinobu

2018-01-01

The penetration of the anaplastic lymphoma kinase (ALK) inhibitor alectinib in neuroblastomas and the relationship between alectinib and ALK expression are unknown. The aim of this study was to perform a quantitative investigation of the inter- and intra-tumoural distribution of alectinib in different neuroblastoma xenograft models using matrix-assisted laser desorption ionization MS imaging (MALDI-MSI). The distribution of alectinib in NB1 (ALK amplification) and SK-N-FI (ALK wild-type) xenograft tissues was analysed using MALDI-MSI. The abundance of alectinib in tumours and intra-tumoural areas was quantified using ion signal intensities from MALDI-MSI after normalization by correlation with LC-MS/MS. The distribution of alectinib was heterogeneous in neuroblastomas. The penetration of alectinib was not significantly different between ALK amplification and ALK wide-type tissues using both LC-MS/MS concentrations and MSI intensities. Normalization with an internal standard increased the quantitative property of MSI by adjusting for the ion suppression effect. The distribution of alectinib in different intra-tumoural areas can alternatively be quantified from MS images by correlation with LC-MS/MS. The penetration of alectinib into tumour tissues may not be homogenous or influenced by ALK expression in the early period after single-dose administration. MALDI-MSI may prove to be a valuable pharmaceutical method for elucidating the mechanism of action of drugs by clarifying their microscopic distribution in heterogeneous tissues. © 2017 The British Pharmacological Society.

Dynamic calibration approach for determining catechins and gallic acid in green tea using LC-ESI/MS.

PubMed

Bedner, Mary; Duewer, David L

2011-08-15

Catechins and gallic acid are antioxidant constituents of Camellia sinensis, or green tea. Liquid chromatography with both ultraviolet (UV) absorbance and electrospray ionization mass spectrometric (ESI/MS) detection was used to determine catechins and gallic acid in three green tea matrix materials that are commonly used as dietary supplements. The results from both detection modes were evaluated with 14 quantitation models, all of which were based on the analyte response relative to an internal standard. Half of the models were static, where quantitation was achieved with calibration factors that were constant over an analysis set. The other half were dynamic, with calibration factors calculated from interpolated response factor data at each time a sample was injected to correct for potential variations in analyte response over time. For all analytes, the relatively nonselective UV responses were found to be very stable over time and independent of the calibrant concentration; comparable results with low variability were obtained regardless of the quantitation model used. Conversely, the highly selective MS responses were found to vary both with time and as a function of the calibrant concentration. A dynamic quantitation model based on polynomial data-fitting was used to reduce the variability in the quantitative results using the MS data.

Reassessment of the Access Testosterone chemiluminescence assay and comparison with LC-MS method.

PubMed

Dittadi, Ruggero; Matteucci, Mara; Meneghetti, Elisa; Ndreu, Rudina

2018-03-01

To reassess the imprecision and Limit of Quantitation, to evaluate the cross-reaction with dehydroepiandrosterone-sulfate (DHEAS), the accuracy toward liquid chromatography-mass spectrometry (LC-MS) and the reference interval of the Access Testosterone method, performed by DxI immunoassay platform (Beckman Coulter). Imprecision was evaluated testing six pool samples assayed in 20 different run using two reagents lots. The cross-reaction with DHEAS was studied both by a displacement curve and by spiking DHEAS standard in two serum samples with known amount of testosterone. The comparison with LC-MS was evaluated by Passing-Bablock analysis in 21 routine serum samples and 19 control samples from an External Quality Assurance (EQA) scheme. The reference interval was verified by an indirect estimation on 2445 male and 2838 female outpatients. The imprecision study showed a coefficient of variation (CV) between 2.7% and 34.7% for serum pools from 16.3 and 0.27 nmol/L. The value of Limit of Quantitation at 20% CV was 0.53 nmol/L. The DHEAS showed a cross-reaction of 0.0074%. A comparison with LC-MS showed a trend toward a slight underestimation of immunoassay vs LC-MS (Passing-Bablock equations: DxI=-0.24+0.906 LCMS in serum samples and DxI=-0.299+0.981 LCMS in EQA samples). The verification of reference interval showed a 2.5th-97.5th percentile distribution of 6.6-24.3 nmol/L for male over 14 years and <0.5-2.78 nmol/L for female subjects, in accord with the reference intervals reported by the manufacturer. The Access Testosterone method could be considered an adequately reliable tool for the testosterone measurement. © 2017 Wiley Periodicals, Inc.

Importance of highly selective LC-MS/MS analysis for the accurate quantification of tamoxifen and its metabolites: focus on endoxifen and 4-hydroxytamoxifen.

PubMed

Jager, N G L; Rosing, H; Linn, S C; Schellens, J H M; Beijnen, J H

2012-06-01

The antiestrogenic effect of tamoxifen is mainly attributable to the active metabolites endoxifen and 4-hydroxytamoxifen. This effect is assumed to be concentration-dependent and therefore quantitative analysis of tamoxifen and metabolites for clinical studies and therapeutic drug monitoring is increasing. We investigated the large discrepancies in reported mean endoxifen and 4-hydroxytamoxifen concentrations. Two published LC-MS/MS methods are used to analyse a set of 75 serum samples from patients treated with tamoxifen. The method from Teunissen et al. (J Chrom B, 879:1677-1685, 2011) separates endoxifen and 4-hydroxytamoxifen from other tamoxifen metabolites with similar masses and fragmentation patterns. The second method, published by Gjerde et al. (J Chrom A, 1082:6-14, 2005) however lacks selectivity, resulting in a factor 2-3 overestimation of the endoxifen and 4-hydroxytamoxifen levels, respectively. We emphasize the use of highly selective LC-MS/MS methods for the quantification of tamoxifen and its metabolites in biological samples.

Integrated work-flow for quantitative metabolome profiling of plants, Peucedani Radix as a case.

PubMed

Song, Yuelin; Song, Qingqing; Liu, Yao; Li, Jun; Wan, Jian-Bo; Wang, Yitao; Jiang, Yong; Tu, Pengfei

2017-02-08

Universal acquisition of reliable information regarding the qualitative and quantitative properties of complicated matrices is the premise for the success of metabolomics study. Liquid chromatography-mass spectrometry (LC-MS) is now serving as a workhorse for metabolomics; however, LC-MS-based non-targeted metabolomics is suffering from some shortcomings, even some cutting-edge techniques have been introduced. Aiming to tackle, to some extent, the drawbacks of the conventional approaches, such as redundant information, detector saturation, low sensitivity, and inconstant signal number among different runs, herein, a novel and flexible work-flow consisting of three progressive steps was proposed to profile in depth the quantitative metabolome of plants. The roots of Peucedanum praeruptorum Dunn (Peucedani Radix, PR) that are rich in various coumarin isomers, were employed as a case study to verify the applicability. First, offline two dimensional LC-MS was utilized for in-depth detection of metabolites in a pooled PR extract namely universal metabolome standard (UMS). Second, mass fragmentation rules, notably concerning angular-type pyranocoumarins that are the primary chemical homologues in PR, and available databases were integrated for signal assignment and structural annotation. Third, optimum collision energy (OCE) as well as ion transition for multiple monitoring reaction measurement was online optimized with a reference compound-free strategy for each annotated component and large-scale relative quantification of all annotated components was accomplished by plotting calibration curves via serially diluting UMS. It is worthwhile to highlight that the potential of OCE for isomer discrimination was described and the linearity ranges of those primary ingredients were extended by suppressing their responses. The integrated workflow is expected to be qualified as a promising pipeline to clarify the quantitative metabolome of plants because it could not only holistically provide qualitative information, but also straightforwardly generate accurate quantitative dataset. Copyright © 2016 Elsevier B.V. All rights reserved.

Simultaneous Proteomic Discovery and Targeted Monitoring using Liquid Chromatography, Ion Mobility Spectrometry, and Mass Spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burnum-Johnson, Kristin E.; Nie, Song; Casey, Cameron P.

Current proteomics approaches are comprised of both broad discovery measurements as well as more quantitative targeted measurements. These two different measurement types are used to initially identify potentially important proteins (e.g., candidate biomarkers) and then enable improved quantification for a limited number of selected proteins. However, both approaches suffer from limitations, particularly the lower sensitivity, accuracy, and quantitation precision for discovery approaches compared to targeted approaches, and the limited proteome coverage provided by targeted approaches. Herein, we describe a new proteomics approach that allows both discovery and targeted monitoring (DTM) in a single analysis using liquid chromatography, ion mobility spectrometrymore » and mass spectrometry (LC-IMS-MS). In DTM, heavy labeled peptides for target ions are spiked into tryptic digests and both the labeled and unlabeled peptides are broadly detected using LC-IMS-MS instrumentation, allowing the benefits of discovery and targeted approaches. To understand the possible improvement of the DTM approach, it was compared to LC-MS broad measurements using an accurate mass and time tag database and selected reaction monitoring (SRM) targeted measurements. The DTM results yielded greater peptide/protein coverage and a significant improvement in the detection of lower abundance species compared to LC-MS discovery measurements. DTM was also observed to have similar detection limits as SRM for the targeted measurements indicating its potential for combining the discovery and targeted approaches.« less

Rapid LC-MS method for the detection of common fragrances in personal care products without sample preparation.

PubMed

Famiglini, Giorgio; Termopoli, Veronica; Palma, Pierangela; Capriotti, Fabiana; Cappiello, Achille

2014-05-01

An LC-MS method for the analysis of personal care and household products without sample preparation is presented. The method takes advantage of the Direct-electron ionization (EI) LC-MS interface for the quantitation of principal components, as well as for the identification of unknown or undeclared ingredients. The technique has proven its inertness toward matrix effects and the electron ionization allows quantitation and library identification. Commercially available products (shower gel, perfume, and hand cream) were diluted with methanol and injected directly into a nano-LC column. Limonene, linalool, and citral were selected as target compounds because of their use as fragrances in toiletry and detergent products. These and all other fragrances are commonly determined with GC-MS analysis, prior to sample cleanup, a procedure that can lead to analytes loss. The selected compounds are not detected with ESI because of their poor or very low response. Figures of merit and validation studies were executed and special attention was devoted to matrix-effects evaluation, because a sample preparation procedure is not involved. No matrix effects were observed, and the repeatability was excellent even after several weeks of operation. Products composition was investigated in full scan mode to determine the presence of unknown or not listed ingredients. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rapid LC-MS/MS quantification of the major benzodiazepines and their metabolites on dried blood spots using a simple and cost-effective sample pretreatment.

PubMed

Déglon, Julien; Versace, François; Lauer, Estelle; Widmer, Christèle; Mangin, Patrice; Thomas, Aurélien; Staub, Christian

2012-06-01

Dried blood spots (DBS) sampling has gained popularity in the bioanalytical community as an alternative to conventional plasma sampling, as it provides numerous benefits in terms of sample collection and logistics. The aim of this work was to show that these advantages can be coupled with a simple and cost-effective sample pretreatment, with subsequent rapid LC-MS/MS analysis for quantitation of 15 benzodiazepines, six metabolites and three Z-drugs. For this purpose, a simplified offline procedure was developed that consisted of letting a 5-µl DBS infuse directly into 100 µl of MeOH, in a conventional LC vial. The parameters related to the DBS pretreatment, such as extraction time or internal standard addition, were investigated and optimized, demonstrating that passive infusion in a regular LC vial was sufficient to quantitatively extract the analytes of interest. The method was validated according to international criteria in the therapeutic concentration ranges of the selected compounds. The presented strategy proved to be efficient for the rapid analysis of the selected drugs. Indeed, the offline sample preparation was reduced to a minimum, using a small amount of organic solvent and consumables, without affecting the accuracy of the method. Thus, this approach enables simple and rapid DBS analysis, even when using a non-DBS-dedicated autosampler, while lowering the costs and environmental impact.

Automated extraction of lysergic acid diethylamide (LSD) and N-demethyl-LSD from blood, serum, plasma, and urine samples using the Zymark RapidTrace with LC/MS/MS confirmation.

PubMed

de Kanel, J; Vickery, W E; Waldner, B; Monahan, R M; Diamond, F X

1998-05-01

A forensic procedure for the quantitative confirmation of lysergic acid diethylamide (LSD) and the qualitative confirmation of its metabolite, N-demethyl-LSD, in blood, serum, plasma, and urine samples is presented. The Zymark RapidTrace was used to perform fully automated solid-phase extractions of all specimen types. After extract evaporation, confirmations were performed using liquid chromatography (LC) followed by positive electrospray ionization (ESI+) mass spectrometry/mass spectrometry (MS/MS) without derivatization. Quantitation of LSD was accomplished using LSD-d3 as an internal standard. The limit of quantitation (LOQ) for LSD was 0.05 ng/mL. The limit of detection (LOD) for both LSD and N-demethyl-LSD was 0.025 ng/mL. The recovery of LSD was greater than 95% at levels of 0.1 ng/mL and 2.0 ng/mL. For LSD at 1.0 ng/mL, the within-run and between-run (different day) relative standard deviation (RSD) was 2.2% and 4.4%, respectively.

LC-MS metabolic profiling of Arabidopsis thaliana plant leaves and cell cultures: optimization of pre-LC-MS procedure parameters.

PubMed

t'Kindt, Ruben; De Veylder, Lieven; Storme, Michael; Deforce, Dieter; Van Bocxlaer, Jan

2008-08-01

This study treats the optimization of methods for homogenizing Arabidopsis thaliana plant leaves as well as cell cultures, and extracting their metabolites for metabolomics analysis by conventional liquid chromatography electrospray ionization mass spectrometry (LC-ESI/MS). Absolute recovery, process efficiency and procedure repeatability have been compared between different pre-LC-MS homogenization/extraction procedures through the use of samples fortified before extraction with a range of representative metabolites. Hereby, the magnitude of the matrix effect observed in the ensuing LC-MS based metabolomics analysis was evaluated. Based on relative recovery and repeatability of key metabolites, comprehensiveness of extraction (number of m/z-retention time pairs) and clean-up potential of the approach (minimum matrix effects), the most appropriate sample pre-treatment was adopted. It combines liquid nitrogen homogenization for plant leaves with thermomixer based extraction using MeOH/H(2)O 80/20. As such, an efficient and highly reproducible LC-MS plant metabolomics set-up is achieved, as illustrated by the obtained results for both LC-MS (8.88%+/-5.16 versus 7.05%+/-4.45) and technical variability (12.53%+/-11.21 versus 9.31%+/-6.65) data in a comparative investigation of A. thaliana plant leaves and cell cultures, respectively.

Chip-LC-MS for label-free profiling of human serum.

PubMed

Horvatovich, Peter; Govorukhina, Natalia I; Reijmers, Theo H; van der Zee, Ate G J; Suits, Frank; Bischoff, Rainer

2007-12-01

The discovery of biomarkers in easily accessible body fluids such as serum is one of the most challenging topics in proteomics requiring highly efficient separation and detection methodologies. Here, we present the application of a microfluidics-based LC-MS system (chip-LC-MS) to the label-free profiling of immunodepleted, trypsin-digested serum in comparison to conventional capillary LC-MS (cap-LC-MS). Both systems proved to have a repeatability of approximately 20% RSD for peak area, all sample preparation steps included, while repeatability of the LC-MS part by itself was less than 10% RSD for the chip-LC-MS system. Importantly, the chip-LC-MS system had a two times higher resolution in the LC dimension and resulted in a lower average charge state of the tryptic peptide ions generated in the ESI interface when compared to cap-LC-MS while requiring approximately 30 times less (~5 pmol) sample. In order to characterize both systems for their capability to find discriminating peptides in trypsin-digested serum samples, five out of ten individually prepared, identical sera were spiked with horse heart cytochrome c. A comprehensive data processing methodology was applied including 2-D smoothing, resolution reduction, peak picking, time alignment, and matching of the individual peak lists to create an aligned peak matrix amenable for statistical analysis. Statistical analysis by supervised classification and variable selection showed that both LC-MS systems could discriminate the two sample groups. However, the chip-LC-MS system allowed to assign 55% of the overall signal to selected peaks against 32% for the cap-LC-MS system.

Capillary liquid chromatography-microchip atmospheric pressure chemical ionization-mass spectrometry.

PubMed

Ostman, Pekka; Jäntti, Sirkku; Grigoras, Kestas; Saarela, Ville; Ketola, Raimo A; Franssila, Sami; Kotiaho, Tapio; Kostiainen, Risto

2006-07-01

A miniaturized nebulizer chip for capillary liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (capillary LC-microchip APCI-MS) is presented. The APCI chip consists of two wafers, a silicon wafer and a Pyrex glass wafer. The silicon wafer has a DRIE etched through-wafer nebulizer gas inlet, an edge capillary insertion channel, a stopper, a vaporizer channel and a nozzle. The platinum heater electrode and pads for electrical connection were patterned on to the Pyrex glass wafer. The two wafers were joined by anodic bonding, creating a microchip version of an APCI-source. The sample inlet capillary from an LC column is directly connected to the vaporizer channel of the APCI chip. The etched nozzle in the microchip forms a narrow sample plume, which is ionized by an external corona needle, and the formed ions are analyzed by a mass spectrometer. The nebulizer chip enables for the first time the use of low flow rate separation techniques with APCI-MS. The performance of capillary LC-microchip APCI-MS was tested with selected neurosteroids. The capillary LC-microchip APCI-MS provides quantitative repeatability and good linearity. The limits of detection (LOD) with a signal-to-noise ratio (S/N) of 3 in MS/MS mode for the selected neurosteroids were 20-1000 fmol (10-500 nmol l(-1)). LODs (S/N = 3) with commercial macro APCI with the same compounds using the same MS were about 10 times higher. Fast heat transfer allows the use of the optimized temperature for each compound during an LC run. The microchip APCI-source provides a convenient and easy method to combine capillary LC to any API-MS equipped with an APCI source. The advantages and potentials of the microchip APCI also make it a very attractive interface in microfluidic APCI-MS.

Analysis of Perfluorinated Chemicals in Sludge: Method Development and Initial Results

EPA Science Inventory

A fast, rigorous method was developed to maximize the extraction efficacy for ten perfluorocarboxylic acids and perfluorooctanesulfonate from wastewater-treatment sludge and to quantitate using liquid chromatography, tandem-mass spectrometry (LC/MS/MS). First, organic solvents w...

Optimized liquid chromatography tandem mass spectrometry approach for the determination of diquat and paraquat herbicides.

PubMed

Hao, Chunyan; Zhao, Xiaoming; Morse, David; Yang, Paul; Taguchi, Vince; Morra, Franca

2013-08-23

Liquid chromatography tandem mass spectrometry (LC-MS/MS) determination of quaternary ammonium herbicides diquat (DQ) and paraquat (PQ) can be very challenging due to their complicated chromatographic and mass spectrometric behaviors. Various multiple reaction monitoring (MRM) transitions from radical cations M(+) and singly charged cations [M-H](+), have been reported for LC-MS/MS quantitation under different chromatographic and mass spectrometric conditions. However, interference peaks were observed for certain previously reported MRM transitions in our study. Using a Dionex Acclaim(®) reversed-phase and HILIC mixed-mode LC column, we evaluated the most sensitive MRM transitions from three types of quasi-molecular ions of DQ and PQ, elucidated the cross-interference phenomena, and demonstrated that the rarely mentioned MRM transitions from dications M(2+) offered the best selectivity for LC-MS/MS analysis. Experimental parameters, such as IonSpray (IS) voltage, source temperature, declustering potential (DP), column oven temperature, collision energy (CE), acid and salt concentrations in the mobile phases were also optimized and an uncommon electrospray ionization (ESI) capillary voltage of 1000V achieved the highest sensitivity. Employing the proposed dication transitions 92/84.5 for DQ and 93/171 for PQ, the direct aqueous injection LC-MS/MS method developed was able to provide a method detection limit (MDL) of 0.1μg/L for the determination of these two herbicides in drinking water. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Investigation of epigallocatechin-3-O-caffeoate and epigallocatechin-3-O-p-coumaroate in tea leaves by LC/MS-MS analysis.

PubMed

Umehara, Masahiro; Yanae, Koji; Maruki-Uchida, Hiroko; Sai, Masahiko

2017-12-01

(-)-Epigallocatechin-3-O-gallate (EGCG), the major catechin present in green tea, exhibits potent antioxidant activity. We thereby investigated the presence of unknown components bearing the (-)-epigallocatechin (EGC) moiety in fresh tea leaf samples. Initially, liquid chromatography tandem mass spectrometry (LC-MS/MS) was employed to examine fresh tea leaves of the Yabukita, the most popular tea cultivar in Japan, which suggested the presence of the EGC phenylpropanoid derivatives, (-)-epigallocatechin-3-O-p-coumaroate (EGCpCA) and (-)-epigallocatechin-3-O-caffeoate (EGCCA). The structures of the detected EGCpCA and EGCCA were then confirmed by LC-MS/MS using synthesized EGCpCA and EGCCA as standards. In addition, EGCpCA and EGCCA were evaluated for their antioxidant activity in the ORAC (oxygen radical antioxidant capacity) and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assays, where EGCCA (8.60μmolTE/μmol, TE=Trolox equivalents) exhibited a stronger antioxidant activity than EGCG (5.52μmolTE/μmol) in the ORAC assay. Finally, EGCpCA and EGCCA were quantitated in several tea leaf samples using LC-MS/MS, and it was found that these compounds were present in lower quantities (EGCpCA, 16.8-345.8μg/g, EGCCA, 4.3-75.1μg/g in the dry tea leaves) than the major catechins. In this study, we found the potent antioxidant EGCCA using LC-MS/MS and revealed its wide existence in various tea leaves. Copyright © 2017 Elsevier Ltd. All rights reserved.

IsoMS: automated processing of LC-MS data generated by a chemical isotope labeling metabolomics platform.

PubMed

Zhou, Ruokun; Tseng, Chiao-Li; Huan, Tao; Li, Liang

2014-05-20

A chemical isotope labeling or isotope coded derivatization (ICD) metabolomics platform uses a chemical derivatization method to introduce a mass tag to all of the metabolites having a common functional group (e.g., amine), followed by LC-MS analysis of the labeled metabolites. To apply this platform to metabolomics studies involving quantitative analysis of different groups of samples, automated data processing is required. Herein, we report a data processing method based on the use of a mass spectral feature unique to the chemical labeling approach, i.e., any differential-isotope-labeled metabolites are detected as peak pairs with a fixed mass difference in a mass spectrum. A software tool, IsoMS, has been developed to process the raw data generated from one or multiple LC-MS runs by peak picking, peak pairing, peak-pair filtering, and peak-pair intensity ratio calculation. The same peak pairs detected from multiple samples are then aligned to produce a CSV file that contains the metabolite information and peak ratios relative to a control (e.g., a pooled sample). This file can be readily exported for further data and statistical analysis, which is illustrated in an example of comparing the metabolomes of human urine samples collected before and after drinking coffee. To demonstrate that this method is reliable for data processing, five (13)C2-/(12)C2-dansyl labeled metabolite standards were analyzed by LC-MS. IsoMS was able to detect these metabolites correctly. In addition, in the analysis of a (13)C2-/(12)C2-dansyl labeled human urine, IsoMS detected 2044 peak pairs, and manual inspection of these peak pairs found 90 false peak pairs, representing a false positive rate of 4.4%. IsoMS for Windows running R is freely available for noncommercial use from www.mycompoundid.org/IsoMS.

Ultra-performance liquid chromatography/tandem mass spectrometric quantification of structurally diverse drug mixtures using an ESI-APCI multimode ionization source.

PubMed

Yu, Kate; Di, Li; Kerns, Edward; Li, Susan Q; Alden, Peter; Plumb, Robert S

2007-01-01

We report in this paper an ultra-performance liquid chromatography/tandem mass spectrometric (UPLC(R)/MS/MS) method utilizing an ESI-APCI multimode ionization source to quantify structurally diverse analytes. Eight commercial drugs were used as test compounds. Each LC injection was completed in 1 min using a UPLC system coupled with MS/MS multiple reaction monitoring (MRM) detection. Results from three separate sets of experiments are reported. In the first set of experiments, the eight test compounds were analyzed as a single mixture. The mass spectrometer was switching rapidly among four ionization modes (ESI+, ESI-, APCI-, and APCI+) during an LC run. Approximately 8-10 data points were collected across each LC peak. This was insufficient for a quantitative analysis. In the second set of experiments, four compounds were analyzed as a single mixture. The mass spectrometer was switching rapidly among four ionization modes during an LC run. Approximately 15 data points were obtained for each LC peak. Quantification results were obtained with a limit of detection (LOD) as low as 0.01 ng/mL. For the third set of experiments, the eight test compounds were analyzed as a batch. During each LC injection, a single compound was analyzed. The mass spectrometer was detecting at a particular ionization mode during each LC injection. More than 20 data points were obtained for each LC peak. Quantification results were also obtained. This single-compound analytical method was applied to a microsomal stability test. Compared with a typical HPLC method currently used for the microsomal stability test, the injection-to-injection cycle time was reduced to 1.5 min (UPLC method) from 3.5 min (HPLC method). The microsome stability results were comparable with those obtained by traditional HPLC/MS/MS.

A Statistical Framework for Protein Quantitation in Bottom-Up MS-Based Proteomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karpievitch, Yuliya; Stanley, Jeffrey R.; Taverner, Thomas

2009-08-15

Motivation: Quantitative mass spectrometry-based proteomics requires protein-level estimates and associated confidence measures. Challenges include the presence of low quality or incorrectly identified peptides and informative missingness. Furthermore, models are required for rolling peptide-level information up to the protein level. Results: We present a statistical model that carefully accounts for informative missingness in peak intensities and allows unbiased, model-based, protein-level estimation and inference. The model is applicable to both label-based and label-free quantitation experiments. We also provide automated, model-based, algorithms for filtering of proteins and peptides as well as imputation of missing values. Two LC/MS datasets are used to illustrate themore » methods. In simulation studies, our methods are shown to achieve substantially more discoveries than standard alternatives. Availability: The software has been made available in the opensource proteomics platform DAnTE (http://omics.pnl.gov/software/). Contact: adabney@stat.tamu.edu Supplementary information: Supplementary data are available at Bioinformatics online.« less

Development of a Liquid Chromatography High Resolution Mass Spectrometry (LC-HRMS) Method for the Quantitation of Viral Envelope Glycoprotein in Ebola Virus-Like Particle Vaccine Preparations

DTIC Science & Technology

2016-09-05

46 was performed on an LTQ-Orbitrap Elite MS and the final quantitation was derived by 47 comparing the relative response of the 200 fmol AQUA...shown in Figure 3B, the final quantitation is derived by comparing the 527 relative response of the 200 fmol AQUA standards (SEE and IRSEE: Set 1) to...measure of eVLP quality, the western blot 553 and LC-HRMS quantitation results were compared to survival data in mice for each of these 554 eVLP vaccine

Quantitative measurement of a candidate serum biomarker peptide derived from α2-HS-glycoprotein, and a preliminary trial of multidimensional peptide analysis in females with pregnancy-induced hypertension.

PubMed

Hamamura, Kensuke; Yanagida, Mitsuaki; Ishikawa, Hitoshi; Banzai, Michio; Yoshitake, Hiroshi; Nonaka, Daisuke; Tanaka, Kenji; Sakuraba, Mayumi; Miyakuni, Yasuka; Takamori, Kenji; Nojima, Michio; Yoshida, Koyo; Fujiwara, Hiroshi; Takeda, Satoru; Araki, Yoshihiko

2018-03-01

Purpose We previously attempted to develop quantitative enzyme-linked immunosorbent assay (ELISA) systems for the PDA039/044/071 peptides, potential serum disease biomarkers (DBMs) of pregnancy-induced hypertension (PIH), primarily identified by a peptidomic approach (BLOTCHIP®-mass spectrometry (MS)). However, our methodology did not extend to PDA071 (cysteinyl α2-HS-glycoprotein 341-367 ), due to difficulty to produce a specific antibody against the peptide. The aim of the present study was to establish an alternative PDA071 quantitation system using liquid chromatography-multiple reaction monitoring (LC-MRM)/MS, to explore the potential utility of PDA071 as a DBM for PIH. Methods We tested heat/acid denaturation methods in efforts to purify serum PDA071 and developed an LC-MRM/MS method allowing for specific quantitation thereof. We measured serum PDA071 concentrations, and these results were validated including by three-dimensional (3D) plotting against PDA039 (kininogen-1 439-456 )/044 (kininogen-1 438-456 ) concentrations, followed by discriminant analysis. Results PDA071 was successfully extracted from serum using a heat denaturation method. Optimum conditions for quantitation via LC-MRM/MS were developed; the assayed serum PDA071 correlated well with the BLOTCHIP® assay values. Although the PDA071 alone did not significantly differ between patients and controls, 3D plotting of PDA039/044/071 peptide concentrations and construction of a Jackknife classification matrix were satisfactory in terms of PIH diagnostic precision. Conclusions Combination analysis using both PDA071 and PDA039/044 concentrations allowed PIH diagnostic accuracy to be attained, and our method will be valuable in future pathophysiological studies of hypertensive disorders of pregnancy.

Quantitative and comparative liquid chromatography-electrospray ionization-mass spectrometry analyses of hydrogen sulfide and thiol metabolites derivaitized with 2-iodoacetanilide isotopologues.

PubMed

Lee, Der-Yen; Huang, Wei-Chieh; Gu, Ting-Jia; Chang, Geen-Dong

2018-06-01

Hydrogen sulfide (H 2 S), previously known as a toxic gas, is now recognized as a gasotransmitter along with nitric oxide and carbon monoxide. However, only few methods are available for quantitative determination of H 2 S in biological samples. 2-Iodoacetanilide (2-IAN), a thiol-reacting agent, has been used to tag the reduced cysteine residues of proteins for quantitative proteomics and for detection of cysteine oxidation modification. In this article, we proposed a new method for quantitative analyses of H 2 S and thiol metabolites using the procedure of pre-column 2-IAN derivatization coupled with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). 13 C 6 -Labeled and label-free 2-IAN efficiently react with H 2 S and thiol compounds at pH 9.5 and 65 °C. The derivatives exhibit excellent stability at alkaline conditions, high resolution on reverse phase liquid chromatography and great sensitivity for ESI-MS detection. The measurement of H 2 S, l-cysteine, glutathione, and DL-homocysteine derivatives was validated using 13 C 6 -labeled standard in LC-ESI-MS analyses and exhibited 10 nM-1 μM linear ranges for DL-homocysteine and glutathione and 1 nM-1 μM linear ranges for l-cysteine and H 2 S. In addition, the sequence of derivatization and extraction of metabolites is important in the quantification of thiol metabolites suggesting the presence of matrix effects. Most importantly, labeling with 2-IAN and 13 C 6 -2-IAN isotopologues could achieve quantitative and matched sample comparative analyses with minimal bias using our extraction and labeling procedures before LC-MS analysis. Copyright © 2018 Elsevier B.V. All rights reserved.

High-coverage quantitative proteomics using amine-specific isotopic labeling.

PubMed

Melanson, Jeremy E; Avery, Steven L; Pinto, Devanand M

2006-08-01

Peptide dimethylation with isotopically coded formaldehydes was evaluated as a potential alternative to techniques such as the iTRAQ method for comparative proteomics. The isotopic labeling strategy and custom-designed protein quantitation software were tested using protein standards and then applied to measure proteins levels associated with Alzheimer's disease (AD). The method provided high accuracy (10% error), precision (14% RSD) and coverage (70%) when applied to the analysis of a standard solution of BSA by LC-MS/MS. The technique was then applied to measure protein abundance levels in brain tissue afflicted with AD relative to normal brain tissue. 2-D LC-MS analysis identified 548 unique proteins (p<0.05). Of these, 349 were quantified with two or more peptides that met the statistical criteria used in this study. Several classes of proteins exhibited significant changes in abundance. For example, elevated levels of antioxidant proteins and decreased levels of mitochondrial electron transport proteins were observed. The results demonstrate the utility of the labeling method for high-throughput quantitative analysis.

LC-MS/MS quantification of next-generation biotherapeutics: a case study for an IgE binding Nanobody in cynomolgus monkey plasma.

PubMed

Sandra, Koen; Mortier, Kjell; Jorge, Lucie; Perez, Luis C; Sandra, Pat; Priem, Sofie; Poelmans, Sofie; Bouche, Marie-Paule

2014-05-01

Nanobodies(®) are therapeutic proteins derived from the smallest functional fragments of heavy chain-only antibodies. The development and validation of an LC-MS/MS-based method for the quantification of an IgE binding Nanobody in cynomolgus monkey plasma is presented. Nanobody quantification was performed making use of a proteotypic tryptic peptide chromatographically enriched prior to LC-MS/MS analysis. The validated LLOQ at 36 ng/ml was measured with an intra- and inter-assay precision and accuracy <20%. The required sensitivity could be obtained based on the selectivity of 2D LC combined with MS/MS. No analyte specific tools for affinity purification were used. Plasma samples originating from a PK/PD study were analyzed and compared with the results obtained with a traditional ligand-binding assay. Excellent correlations between the two techniques were obtained, and similar PK parameters were estimated. A 2D LC-MS/MS method was successfully developed and validated for the quantification of a next generation biotherapeutic.

Multi-allergen Quantitation and the Impact of Thermal Treatment in Industry-Processed Baked Goods by ELISA and Liquid Chromatography-Tandem Mass Spectrometry.

PubMed

Parker, Christine H; Khuda, Sefat E; Pereira, Marion; Ross, Mark M; Fu, Tong-Jen; Fan, Xuebin; Wu, Yan; Williams, Kristina M; DeVries, Jonathan; Pulvermacher, Brian; Bedford, Binaifer; Zhang, Xi; Jackson, Lauren S

2015-12-16

Undeclared food allergens account for 30-40% of food recalls in the United States. Compliance with ingredient labeling regulations and the implementation of effective manufacturing allergen control plans require the use of reliable methods for allergen detection and quantitation in complex food products. The objectives of this work were to (1) produce industry-processed model foods incurred with egg, milk, and peanut allergens, (2) compare analytical method performance for allergen quantitation in thermally processed bakery products, and (3) determine the effects of thermal treatment on allergen detection. Control and allergen-incurred cereal bars and muffins were formulated in a pilot-scale industry processing facility. Quantitation of egg, milk, and peanut in incurred baked goods was compared at various processing stages using commercial enzyme-linked immunosorbent assay (ELISA) kits and a novel multi-allergen liquid chromatography (LC)-tandem mass spectrometry (MS/MS) multiple-reaction monitoring (MRM) method. Thermal processing was determined to negatively affect the recovery and quantitation of egg, milk, and peanut to different extents depending on the allergen, matrix, and analytical test method. The Morinaga ELISA and LC-MS/MS quantitative methods reported the highest recovery across all monitored allergens, whereas the ELISA Systems, Neogen BioKits, Neogen Veratox, and R-Biopharm ELISA Kits underperformed in the determination of allergen content of industry-processed bakery products.

Qualification of a Quantitative Method for Monitoring Aspartate Isomerization of a Monoclonal Antibody by Focused Peptide Mapping.

PubMed

Cao, Mingyan; Mo, Wenjun David; Shannon, Anthony; Wei, Ziping; Washabaugh, Michael; Cash, Patricia

Aspartate (Asp) isomerization is a common post-translational modification of recombinant therapeutic proteins that can occur during manufacturing, storage, or administration. Asp isomerization in the complementarity-determining regions of a monoclonal antibody may affect the target binding and thus a sufficiently robust quality control method for routine monitoring is desirable. In this work, we utilized a liquid chromatography-mass spectrometry (LC/MS)-based approach to identify the Asp isomerization in the complementarity-determining regions of a therapeutic monoclonal antibody. To quantitate the site-specific Asp isomerization of the monoclonal antibody, a UV detection-based quantitation assay utilizing the same LC platform was developed. The assay was qualified and implemented for routine monitoring of this product-specific modification. Compared with existing methods, this analytical paradigm is applicable to identify Asp isomerization (or other modifications) and subsequently develop a rapid, sufficiently robust quality control method for routine site-specific monitoring and quantitation to ensure product quality. This approach first identifies and locates a product-related impurity (a critical quality attribute) caused by isomerization, deamidation, oxidation, or other post-translational modifications, and then utilizes synthetic peptides and MS to assist the development of a LC-UV-based chromatographic method that separates and quantifies the product-related impurities by UV peaks. The established LC-UV method has acceptable peak specificity, precision, linearity, and accuracy; it can be validated and used in a good manufacturing practice environment for lot release and stability testing. Aspartate isomerization is a common post-translational modification of recombinant proteins during manufacture process and storage. Isomerization in the complementarity-determining regions (CDRs) of a monoclonal antibody A (mAb-A) has been detected and has been shown to have impact on the binding affinity to the antigen. In this work, we utilized a mass spectrometry-based peptide mapping approach to detect and quantitate the Asp isomerization in the CDRs of mAb-A. To routinely monitor the CDR isomerization of mAb-A, a focused peptide mapping method utilizing reversed phase chromatographic separation and UV detection has been developed and qualified. This approach is generally applicable to monitor isomerization and other post-translational modifications of proteins in a specific and high-throughput mode to ensure product quality. © PDA, Inc. 2016.

Counting missing values in a metabolite-intensity data set for measuring the analytical performance of a metabolomics platform.

PubMed

Huan, Tao; Li, Liang

2015-01-20

Metabolomics requires quantitative comparison of individual metabolites present in an entire sample set. Unfortunately, missing intensity values in one or more samples are very common. Because missing values can have a profound influence on metabolomic results, the extent of missing values found in a metabolomic data set should be treated as an important parameter for measuring the analytical performance of a technique. In this work, we report a study on the scope of missing values and a robust method of filling the missing values in a chemical isotope labeling (CIL) LC-MS metabolomics platform. Unlike conventional LC-MS, CIL LC-MS quantifies the concentration differences of individual metabolites in two comparative samples based on the mass spectral peak intensity ratio of a peak pair from a mixture of differentially labeled samples. We show that this peak-pair feature can be explored as a unique means of extracting metabolite intensity information from raw mass spectra. In our approach, a peak-pair peaking algorithm, IsoMS, is initially used to process the LC-MS data set to generate a CSV file or table that contains metabolite ID and peak ratio information (i.e., metabolite-intensity table). A zero-fill program, freely available from MyCompoundID.org , is developed to automatically find a missing value in the CSV file and go back to the raw LC-MS data to find the peak pair and, then, calculate the intensity ratio and enter the ratio value into the table. Most of the missing values are found to be low abundance peak pairs. We demonstrate the performance of this method in analyzing an experimental and technical replicate data set of human urine metabolome. Furthermore, we propose a standardized approach of counting missing values in a replicate data set as a way of gauging the extent of missing values in a metabolomics platform. Finally, we illustrate that applying the zero-fill program, in conjunction with dansylation CIL LC-MS, can lead to a marked improvement in finding significant metabolites that differentiate bladder cancer patients and their controls in a metabolomics study of 109 subjects.

Factors That Contribute to Assay Variation in Quantitative Analysis of Sex Steroid Hormones Using Liquid and Gas Chromatography-Mass Spectrometry

ERIC Educational Resources Information Center

Xu, Xia; Veenstra, Timothy D.

2012-01-01

The list of physiological events in which sex steroids play a role continues to increase. To decipher the roles that sex steroids play in any condition requires high quality cohorts of samples and assays that provide highly accurate quantitative measures. Liquid and gas chromatography coupled with mass spectrometry (LC-MS and GC-MS) have…

ComplexQuant: high-throughput computational pipeline for the global quantitative analysis of endogenous soluble protein complexes using high resolution protein HPLC and precision label-free LC/MS/MS.

PubMed

Wan, Cuihong; Liu, Jian; Fong, Vincent; Lugowski, Andrew; Stoilova, Snejana; Bethune-Waddell, Dylan; Borgeson, Blake; Havugimana, Pierre C; Marcotte, Edward M; Emili, Andrew

2013-04-09

The experimental isolation and characterization of stable multi-protein complexes are essential to understanding the molecular systems biology of a cell. To this end, we have developed a high-throughput proteomic platform for the systematic identification of native protein complexes based on extensive fractionation of soluble protein extracts by multi-bed ion exchange high performance liquid chromatography (IEX-HPLC) combined with exhaustive label-free LC/MS/MS shotgun profiling. To support these studies, we have built a companion data analysis software pipeline, termed ComplexQuant. Proteins present in the hundreds of fractions typically collected per experiment are first identified by exhaustively interrogating MS/MS spectra using multiple database search engines within an integrative probabilistic framework, while accounting for possible post-translation modifications. Protein abundance is then measured across the fractions based on normalized total spectral counts and precursor ion intensities using a dedicated tool, PepQuant. This analysis allows co-complex membership to be inferred based on the similarity of extracted protein co-elution profiles. Each computational step has been optimized for processing large-scale biochemical fractionation datasets, and the reliability of the integrated pipeline has been benchmarked extensively. This article is part of a Special Issue entitled: From protein structures to clinical applications. Copyright © 2012 Elsevier B.V. All rights reserved.

Simultaneous quantitative analysis of nine vitamin D compounds in human blood using LC-MS/MS.

PubMed

Abu Kassim, Nur Sofiah; Gomes, Fabio P; Shaw, Paul Nicholas; Hewavitharana, Amitha K

2016-01-01

It has been suggested that each member of the family of vitamin D compounds may have different function(s). Therefore, selective quantification of each compound is important in clinical research. Development and validation attempts of a simultaneous determination method of 12 vitamin D compounds in human blood using precolumn derivatization followed by LC-MS/MS is described. Internal standard calibration with 12 stable isotope labeled analogs was used to correct for matrix effects in MS detector. Nine vitamin D compounds were quantifiable in blood samples with detection limits within femtomole levels. Serum (compared with plasma) was found to be a more suitable sample type, and protein precipitation (compared with saponification) a more effective extraction method for vitamin D assay.

Multicomponent Calibration and Quantitation Methods.

DTIC Science & Technology

1984-11-01

liquid chromatographic peaks (45). Applying rank annihilation to LC /UV data requires that the elution profiles of each individual component in the...measurements, e.g. LC /UV, CC/MS, or GC/FTIR analyses. The response of a single component can be described as M = a x y’ (45) 0 where M contains the...in the second dimension. For example, a GC/MS peak consisting - 3 of 50 mass spectra each composed of 20 distinct m/e ratios would result in a matrix

Development and Application of an MSALL-Based Approach for the Quantitative Analysis of Linear Polyethylene Glycols in Rat Plasma by Liquid Chromatography Triple-Quadrupole/Time-of-Flight Mass Spectrometry.

PubMed

Zhou, Xiaotong; Meng, Xiangjun; Cheng, Longmei; Su, Chong; Sun, Yantong; Sun, Lingxia; Tang, Zhaohui; Fawcett, John Paul; Yang, Yan; Gu, Jingkai

2017-05-16

Polyethylene glycols (PEGs) are synthetic polymers composed of repeating ethylene oxide subunits. They display excellent biocompatibility and are widely used as pharmaceutical excipients. To fully understand the biological fate of PEGs requires accurate and sensitive analytical methods for their quantitation. Application of conventional liquid chromatography-tandem mass spectrometry (LC-MS/MS) is difficult because PEGs have polydisperse molecular weights (MWs) and tend to produce multicharged ions in-source resulting in innumerable precursor ions. As a result, multiple reaction monitoring (MRM) fails to scan all ion pairs so that information on the fate of unselected ions is missed. This Article addresses this problem by application of liquid chromatography-triple-quadrupole/time-of-flight mass spectrometry (LC-Q-TOF MS) based on the MS ALL technique. This technique performs information-independent acquisition by allowing all PEG precursor ions to enter the collision cell (Q2). In-quadrupole collision-induced dissociation (CID) in Q2 then effectively generates several fragments from all PEGs due to the high collision energy (CE). A particular PEG product ion (m/z 133.08592) was found to be common to all linear PEGs and allowed their total quantitation in rat plasma with high sensitivity, excellent linearity and reproducibility. Assay validation showed the method was linear for all linear PEGs over the concentration range 0.05-5.0 μg/mL. The assay was successfully applied to the pharmacokinetic study in rat involving intravenous administration of linear PEG 600, PEG 4000, and PEG 20000. It is anticipated the method will have wide ranging applications and stimulate the development of assays for other pharmaceutical polymers in the future.

Stereoselective quantitation of mecoprop and dichlorprop in natural waters by supramolecular solvent-based microextraction, chiral liquid chromatography and tandem mass spectrometry.

PubMed

Caballo, C; Sicilia, M D; Rubio, S

2013-01-25

Liquid chromatography (LC)/tandem mass spectrometry (MS/MS) after supramolecular solvent-based microextraction (SUSME) was firstly used in this work for the enantioselective determination of chiral pesticides in natural waters. The method developed for the quantitation of the R- and S-enantiomers of mecoprop (MCPP) and dichlorprop (DCPP) involved the extraction of the herbicides in a supramolecular solvent (SUPRAS) made up of reverse aggregates of dodecanoic acid (DoA), analyte re-extraction in acetate buffer (pH = 5.0), separation of the target enantiomers on a chiral column of permethylated α-cyclodextrin under isocratic conditions, and detection of the daughter ions (m/z = 140.9 and 160.6 for MCPP and DCPP, respectively) using a hybrid triple quadrupole mass spectrometer equipped with an electrospray source operating in the negative ion mode. Similar recoveries (ca. 75%) and actual concentration factors (ca. 94) were obtained for both phenoxypropanoic acids (PPAs). The quantitation limits were 1 ng L(-1) for R- and S-MCPP, and 4 ng L(-1) for R- and S-DCPP, and the precision, expressed as relative standard deviation (n = 6) was in the ranges 2.4-2.7% ([R-MCPP] = [S-MCPP] = 5 ng L(-1) and [R-DCPP] = [S-DCPP] = 15 ng L(-1)) and 1.6-1.8% (100 ng L(-1) of each enantiomer). The SUSME-LC-MS/MS method was successfully applied to the determination of the enantiomers of MCPP and DCPP in river and underground waters, fortified at concentrations between 15 and 180 ng L(-1) at variable enantiomeric ratios (ER = 1-9). Copyright © 2012 Elsevier B.V. All rights reserved.

Challenges in the indirect quantitation of acyl-glucuronide metabolites of a cardiovascular drug from complex biological mixtures in the absence of reference standards.

PubMed

Srinivasan, Karthik; Nouri, Parya; Kavetskaia, Olga

2010-07-01

This paper describes the quantitation of acyl-glucuronide metabolites (M26 and M5) of a cardiovascular-drug (torcetrapib) from monkey urine, in the absence of their reference standards. LC/MS/MS assays for M1 and M4 (aglycones of M26 and M5, respectively) were characterized from normal and base-treated urine, as their respective reference standards were available. The in vivo study samples containing M26 and M5 were treated with 1 n sodium hydroxide to hydrolyze them to their respective aglycones. The study samples were assayed for M1 and M4 before and after alkaline hydrolysis and the difference in the concentrations provided an estimate of the urinary levels of M26 and M5. Prior to the main sample analysis, conditions for alkaline hydrolysis of the glucuronides were optimized by incubating pooled study samples. During incubations, a prolonged increase in M4 levels over time was observed, which is inconsistent with the base-hydrolysis of an acyl-glucuronide (expected to hydrolyze rapidly). Possible interference of the metabolite M9 (an ether-glucuronide metabolite isobaric to M4) was investigated to explain this observation using chromatographic and wet-chemistry approaches. The strategies adopted herein established that the LC/MS/MS assay and our approach were reliable. The metabolite exposure was then correlated to toxicological observations to gain initial insights into the physiological role of these metabolites. Copyright (c) 2009 John Wiley & Sons, Ltd.

Simultaneous LC/MS/MS determination of thiols and disulfides in urine samples based on differential labeling with ferrocene-based maleimides.

PubMed

Seiwert, Bettina; Karst, Uwe

2007-09-15

A method for the simultaneous determination of a series of thiols and disulfides in urine samples has been developed based on the sequential labeling of free and bound thiol functionalities with two ferrocene-based maleimide reagents. The sample is first exposed to N-(2-ferroceneethyl)maleimide, thus leading to the derivatization of free thiol groups in the sample. After quantitative reaction and subsequent reduction of the disulfide-bound thiols by tris(2-carboxyethyl)phosphine, the newly formed thiol functionalities are reacted with ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide. The reaction products are determined by LC/MS/MS in the multiple reaction mode, and precursor ion scan as well as neutral loss scan is applied to detect unknown further thiols. The method was successfully applied to the analysis of free and disulfide-bound thiols in urine samples. Limits of detection are 30 to 110 nM, and the linear range comprises two decades of concentration, thus covering the relevant concentration range of thiols in urine samples. The thiol and disulfide concentrations were referred to the creatinine content to compensate for different sample volumes. As some calibration standards for the disulfides are not commercially available, they were synthesized in an electrochemical flow-through cell. This allowed the synthesis of hetero- and homodimeric disulfides.

Chemical Isotope Labeling LC-MS for High Coverage and Quantitative Profiling of the Hydroxyl Submetabolome in Metabolomics.

PubMed

Zhao, Shuang; Luo, Xian; Li, Liang

2016-11-01

A key step in metabolomics is to perform accurate relative quantification of the metabolomes in comparative samples with high coverage. Hydroxyl-containing metabolites are an important class of the metabolome with diverse structures and physical/chemical properties; however, many of them are difficult to detect with high sensitivity. We present a high-performance chemical isotope labeling liquid chromatography mass spectrometry (LC-MS) technique for in-depth profiling of the hydroxyl submetabolome, which involves the use of acidic liquid-liquid extraction to enrich hydroxyl metabolites into ethyl acetate from an aqueous sample. After drying and then redissolving in acetonitrile, the metabolite extract is labeled using a base-activated 12 C- or 13 C-dansylation reaction. A fast step-gradient LC-UV method is used to determine the total concentration of labeled metabolites. On the basis of the concentration information, a 12 C-labeled individual sample is mixed with an equal mole amount of a 13 C-labeled pool or control for relative metabolite quantification. The 12 C-/ 13 C-labeled mixtures are individually analyzed by LC-MS, and the resultant peak pairs of labeled metabolites in MS are measured for relative quantification and metabolite identification. A standard library of 85 hydroxyl compounds containing MS, retention time, and MS/MS information was constructed for positive metabolite identification based on matches of two or all three of these parameters with those of an unknown. Using human urine as an example, we analyzed samples of 1:1 12 C-/ 13 C-labeled urine in triplicate with triplicate runs per sample and detected an average of 3759 ± 45 peak pairs or metabolites per run and 3538 ± 71 pairs per sample with 3093 pairs in common (n = 9). Out of the 3093 peak pairs, 2304 pairs (75%) could be positively or putatively identified based on metabolome database searches, including 20 pairs positively identified using the dansylated hydroxyl standards library. The majority of detected metabolites were those containing hydroxyl groups. This technique opens a new avenue for the detailed characterization of the hydroxyl submetabolome in metabolomics research.

LC-DAD-MS (ESI+) analysis and antioxidant capacity of crocus sativus petal extracts.

PubMed

Termentzi, Aikaterini; Kokkalou, Eugene

2008-04-01

In this study, various fractions isolated from the petals of Crocus sativus were assessed at first for their phenolic content both qualitatively and quantitatively and secondly for their antioxidant activity. The phytochemical analysis was carried out by LC-DAD-MS (ESI (+)) whereas the antioxidant potential was evaluated by applying two methodologies, the DPPH. radical scavenging activity test and the Co(II)-induced luminol chemiluminescence procedure. According to data obtained from these antioxidant tests, the diethyl ether, ethyl acetate and aqueous fractions demonstrated the strongest antioxidant capacity. Interestingly, the major constituents identified in these fractions correspond to kaempferol, quercetin, naringenin and some flavanone and flavanol derivatives glycosylated and esterified with phenylpropanoic acids. In addition, the presence of some nitrogen-containing substances, as well as other phenolics and phenylpropanoic derivatives was also traced. The identification and structural elucidation of all substances isolated in this study was achieved by both comparing available literature data and by proposed fragmentation mechanisms based on evaluating the LC-DAD-MS (ESI (+)) experimental data. The quantitative analysis data obtained thus far have shown that Crocus sativus petals are a rich source of flavonoids. Such a fact suggests that the good antioxidant capacity detected in the various fractions of Crocus sativus petals could be attributed to the presence of flavonoids, since it is already known that these molecules exert antioxidant capability. The latter, along with the use of Crocus sativus in food and pharmaceutical industry is discussed.

Positive and negative electrospray LC-MS-MS methods for quantitation of the antiparasitic endectocide drugs, abamectin, doramectin, emamectin, eprinomectin, ivermectin, moxidectin and selamectin in milk.

PubMed

Durden, David A

2007-05-01

Avermectin endectocides are used for the treatment of cattle against a variety of nematode and arthropod parasites, and consequently may appear in milk after normal or off-label use. The compounds abamectin, doramectin, and ivermectin, contain only C, H and O and may be expected to be detected by LC-MS in negative ion mode. The others contain nitrogen in addition and would be expected to be preferentially ionized in positive mode. The use of positive ion and negative ion methods with electrospray LC-MS-MS were compared. Using negative ion the compounds abamectin, doramectin, ivermectin, emamectin, eprinomectin, and moxidectin gave a curvilinear response and were quantified in raw milk by LC-MS-MS with a triethylamine-acetonitrile buffer over the concentration range 1-60 ppb (microg/kg) using selamectin as the internal standard. The limits of detection (LOD) were between 0.19 ppb (doramectin) and 0.38 ppb (emamectin). The compounds gave maximum sensitivity with positive ionisation from a formic acid-ammonium formate-acetonitrile buffer and were detected in milk (LC-MS-MS) also with a curvilinear response over the range 0.5-60 ppb. Although the positive ion signals were larger, with somewhat lower limits of detection (LOD between 0.06 ppb (doramectin) and 0.32 ppb (moxidectin) the negative ion procedure gave a more linear response and more consistent results. Comparison of spiked samples in the range 2-50 ppb showed a high degree of correlation between the two methods.

Simultaneous quantitation of hydroxychloroquine and its metabolites in mouse blood and tissues using LC-ESI-MS/MS: An application for pharmacokinetic studies.

PubMed

Chhonker, Yashpal S; Sleightholm, Richard L; Li, Jing; Oupický, David; Murry, Daryl J

2018-01-01

Hydroxychloroquine (HCQ) has been shown to disrupt autophagy and sensitize cancer cells to radiation and chemotherapeutic agents. However, the optimal delivery method, dose, and tumor concentrations required for these effects are not known. This is in part due to a lack of sensitive and reproducible analytical methods for HCQ quantitation in small animals. As such, we developed and validated a selective and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for simultaneous quantitation of hydroxychloroquine and its metabolites in mouse blood and tissues. The chromatographic separation and detection of analytes were achieved on a reversed phase Thermo Aquasil C 18 (50×4.6mm, 3μ) column, with gradient elution using 0.2% formic acid and 0.1% formic acid in methanol as mobile phase at a flow rate of 0.5mL/min. Simple protein precipitation was utilized for extraction of analytes from the desired matrix. Analytes were separated and quantitated using MS/MS with an electrospray ionization source in positive multiple reaction monitoring (MRM) mode. The MS/MS response was linear over the concentration range from 1 to 2000ng/mL for all analytes with a correlation coefficient (R 2 ) of 0.998 or better. The within- and between-day precision (relative standard deviation, % RSD) and accuracy were within the acceptable limits per FDA guidelines. The validated method was successfully applied to a preclinical pharmacokinetic mouse study involving low volume blood and tissue samples for hydroxychloroquine and metabolites. Copyright © 2017 Elsevier B.V. All rights reserved.

Aptamer based peptide enrichment for quantitative analysis of gonadotropin-releasing hormone by LC-MS/MS.

PubMed

Richards, S L; Cawley, A T; Cavicchioli, R; Suann, C J; Pickford, R; Raftery, M J

2016-04-01

Over recent years threats to racing have expanded to include naturally occurring biological molecules, such as peptides and proteins, and their synthetic analogues. Traditionally, antibodies have been used to enable detection of these compounds as they allow purification and concentration of the analyte of interest. The rapid expansion of peptide-based therapeutics necessitates a similarly rapid development of suitable antibodies or other means of enrichment. Potential alternative enrichment strategies include the use of aptamers, which offer the significant advantage of chemical synthesis once the nucleic acid sequence is known. A method was developed for the enrichment, detection and quantitation of gonadotropin-releasing hormone (GnRH) in equine urine using aptamer-based enrichment and LC-MS/MS. The method achieved comparable limits of detection (1 pg/mL) and quantification (2.5 pg/mL) to previously published antibody-based enrichment methods. The intra- and inter-assay precision achieved was less than 10% at both 5 and 20 pg/mL, and displayed a working dynamic range of 2.5-100 pg/mL. Significant matrix enhancement (170 ± 8%) and low analytical recovery (29 ± 15%) was observed, although the use of an isotopically heavy labelled GnRH peptide, GnRH (Pro(13)C5,(15)N), as the internal standard provides compensation for these parameters. Within the current limits of detection GnRH was detectable up to 1h post administration in urine and identification of a urinary catabolite extended this detection window to 4h. Based on the results of this preliminary investigation we propose the use of aptamers as a viable alternative to antibodies in the enrichment of peptide targets from equine urine. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantitative determination of opioids in whole blood using fully automated dried blood spot desorption coupled to on-line SPE-LC-MS/MS.

PubMed

Verplaetse, Ruth; Henion, Jack

2016-01-01

Opioids are well known, widely used painkillers. Increased stability of opioids in the dried blood spot (DBS) matrix compared to blood/plasma has been described. Other benefits provided by DBS techniques include point-of-care collection, less invasive micro sampling, more economical shipment, and convenient storage. Current methodology for analysis of micro whole blood samples for opioids is limited to the classical DBS workflow, including tedious manual punching of the DBS cards followed by extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalysis. The goal of this study was to develop and validate a fully automated on-line sample preparation procedure for the analysis of DBS micro samples relevant to the detection of opioids in finger prick blood. To this end, automated flow-through elution of DBS cards was followed by on-line solid-phase extraction (SPE) and analysis by LC-MS/MS. Selective, sensitive, accurate, and reproducible quantitation of five representative opioids in human blood at sub-therapeutic, therapeutic, and toxic levels was achieved. The range of reliable response (R(2)  ≥0.997) was 1 to 500 ng/mL whole blood for morphine, codeine, oxycodone, hydrocodone; and 0.1 to 50 ng/mL for fentanyl. Inter-day, intra-day, and matrix inter-lot accuracy and precision was less than 15% (even at lower limits of quantitation (LLOQ) level). The method was successfully used to measure hydrocodone and its major metabolite norhydrocodone in incurred human samples. Our data support the enormous potential of DBS sampling and automated analysis for monitoring opioids as well as other pharmaceuticals in both anti-doping and pain management regimens. Copyright © 2015 John Wiley & Sons, Ltd.

Simultaneous quantitation of multiple contraceptive hormones in human serum by LC-MS/MS.

PubMed

Blue, Steven W; Winchell, Andrea J; Kaucher, Amy V; Lieberman, Rachel A; Gilles, Christopher T; Pyra, Maria N; Heffron, Renee; Hou, Xuanlin; Coombs, Robert W; Nanda, Kavita; Davis, Nicole L; Kourtis, Athena P; Herbeck, Joshua T; Baeten, Jared M; Lingappa, Jairam R; Erikson, David W

2018-04-01

The objective was to develop a method to simultaneously quantify five commonly used hormonal contraceptives (HCs) and two endogenous sex steroids by liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS/MS) and apply this method to human serum samples. We developed a method to simultaneously analyze ethinyl estradiol (EE2), etonogestrel (ENG), levonorgestrel (LNG), medroxyprogesterone acetate (MPA) and norethisterone (NET), along with estradiol (E2) and progesterone (P4), in human serum for a Shimadzu Nexera-LCMS-8050 LC-MS/MS platform. We analyzed serum collected from women self-reporting use of oral contraceptives, contraceptive implants or injectable contraceptives (n=14) and normally cycling women using no HC (n=15) as well as pooled samples from women administered various HCs (ENG, n=6; LNG, n=14; MPA, n=7; NET, n=5). Limits of quantitation were 0.010ng/mL for E2, EE2 and P4; 0.020ng/mL for ENG, LNG and MPA; and 0.040ng/mL for NET. Precisions for all assays, as indicated by coefficient of variation, were less than or equal to 12.1%. Accuracies for all assays were in the range of 95%-108%. Endogenous hormone values obtained from analysis of human serum samples are in agreement with levels previously reported in the literature for normally cycling women as well as for women taking the appropriate HC. We have developed a robust, accurate and sensitive method for simultaneously analyzing commonly used contraceptive steroids and endogenous sex steroids in human serum. This analytical method can be used for quantitating contraceptive steroid levels in women for monitoring systemic exposure to determine drug interactions, nonadherence, misreporting and proper dosing. Copyright © 2018 Elsevier Inc. All rights reserved.

Evaluation of Normalization Methods to Pave the Way Towards Large-Scale LC-MS-Based Metabolomics Profiling Experiments

PubMed Central

Valkenborg, Dirk; Baggerman, Geert; Vanaerschot, Manu; Witters, Erwin; Dujardin, Jean-Claude; Burzykowski, Tomasz; Berg, Maya

2013-01-01

Abstract Combining liquid chromatography-mass spectrometry (LC-MS)-based metabolomics experiments that were collected over a long period of time remains problematic due to systematic variability between LC-MS measurements. Until now, most normalization methods for LC-MS data are model-driven, based on internal standards or intermediate quality control runs, where an external model is extrapolated to the dataset of interest. In the first part of this article, we evaluate several existing data-driven normalization approaches on LC-MS metabolomics experiments, which do not require the use of internal standards. According to variability measures, each normalization method performs relatively well, showing that the use of any normalization method will greatly improve data-analysis originating from multiple experimental runs. In the second part, we apply cyclic-Loess normalization to a Leishmania sample. This normalization method allows the removal of systematic variability between two measurement blocks over time and maintains the differential metabolites. In conclusion, normalization allows for pooling datasets from different measurement blocks over time and increases the statistical power of the analysis, hence paving the way to increase the scale of LC-MS metabolomics experiments. From our investigation, we recommend data-driven normalization methods over model-driven normalization methods, if only a few internal standards were used. Moreover, data-driven normalization methods are the best option to normalize datasets from untargeted LC-MS experiments. PMID:23808607

HPLC-MS/MS method optimisation for matrix metalloproteinase 3 and matrix metalloproteinase 9 determination in human blood serum using target analysis.

PubMed

Kotnik, Petra; Krajnc, Metka Koren; Pahor, Artur; Finšgar, Matjaž; Knez, Željko

2018-02-20

A quantitative analysis of zinc endopeptidases matrix metalloproteinase 9 (MMP9) and matrix metalloproteinase 3 (MMP3) from human blood serum are presented. Both matrix metalloproteinases (MMP) are present in human blood serum and can be used as biomarkers for different diseases. The analysis was performed using LC-MS/MS with a triple quadrupole mass spectrometer, based on two specific peptides of each MMP in comparison with an enzyme-linked immunosorbent assay (ELISA). While the conditions for the LC-MS/MS analysis of MMP9 peptides were previously reported for bronchoalveolar lavage fluid, the analysis of MMP3 peptides was newly quantified for human blood serum herein for the first time. For MMP3, the linear behaviour was determined in the concentration range from 1.0-200.0ng/mL (R 2 =0.997) with an LLOD of 0.5ng/mL. For MMP9, linearity was determined in the concentration range from 6.5-65.0ng/mL (R 2 =0.995) with an LLOD of 2.0ng/mL. Copyright © 2017 Elsevier B.V. All rights reserved.

Differential Plasma Glycoproteome of p19 Skin Cancer Mouse Model Using the Corra Label-Free LC-MS Proteomics Platform.

PubMed

Letarte, Simon; Brusniak, Mi-Youn; Campbell, David; Eddes, James; Kemp, Christopher J; Lau, Hollis; Mueller, Lukas; Schmidt, Alexander; Shannon, Paul; Kelly-Spratt, Karen S; Vitek, Olga; Zhang, Hui; Aebersold, Ruedi; Watts, Julian D

2008-12-01

A proof-of-concept demonstration of the use of label-free quantitative glycoproteomics for biomarker discovery workflow is presented here, using a mouse model for skin cancer as an example. Blood plasma was collected from 10 control mice, and 10 mice having a mutation in the p19(ARF) gene, conferring them high propensity to develop skin cancer after carcinogen exposure. We enriched for N-glycosylated plasma proteins, ultimately generating deglycosylated forms of the modified tryptic peptides for liquid chromatography mass spectrometry (LC-MS) analyses. LC-MS runs for each sample were then performed with a view to identifying proteins that were differentially abundant between the two mouse populations. We then used a recently developed computational framework, Corra, to perform peak picking and alignment, and to compute the statistical significance of any observed changes in individual peptide abundances. Once determined, the most discriminating peptide features were then fragmented and identified by tandem mass spectrometry with the use of inclusion lists. We next assessed the identified proteins to see if there were sets of proteins indicative of specific biological processes that correlate with the presence of disease, and specifically cancer, according to their functional annotations. As expected for such sick animals, many of the proteins identified were related to host immune response. However, a significant number of proteins also directly associated with processes linked to cancer development, including proteins related to the cell cycle, localisation, trasport, and cell death. Additional analysis of the same samples in profiling mode, and in triplicate, confirmed that replicate MS analysis of the same plasma sample generated less variation than that observed between plasma samples from different individuals, demonstrating that the reproducibility of the LC-MS platform was sufficient for this application. These results thus show that an LC-MS-based workflow can be a useful tool for the generation of candidate proteins of interest as part of a disease biomarker discovery effort.

Differential Plasma Glycoproteome of p19ARF Skin Cancer Mouse Model Using the Corra Label-Free LC-MS Proteomics Platform

PubMed Central

Letarte, Simon; Brusniak, Mi-Youn; Campbell, David; Eddes, James; Kemp, Christopher J.; Lau, Hollis; Mueller, Lukas; Schmidt, Alexander; Shannon, Paul; Kelly-Spratt, Karen S.; Vitek, Olga; Zhang, Hui; Aebersold, Ruedi; Watts, Julian D.

2010-01-01

A proof-of-concept demonstration of the use of label-free quantitative glycoproteomics for biomarker discovery workflow is presented here, using a mouse model for skin cancer as an example. Blood plasma was collected from 10 control mice, and 10 mice having a mutation in the p19ARF gene, conferring them high propensity to develop skin cancer after carcinogen exposure. We enriched for N-glycosylated plasma proteins, ultimately generating deglycosylated forms of the modified tryptic peptides for liquid chromatography mass spectrometry (LC-MS) analyses. LC-MS runs for each sample were then performed with a view to identifying proteins that were differentially abundant between the two mouse populations. We then used a recently developed computational framework, Corra, to perform peak picking and alignment, and to compute the statistical significance of any observed changes in individual peptide abundances. Once determined, the most discriminating peptide features were then fragmented and identified by tandem mass spectrometry with the use of inclusion lists. We next assessed the identified proteins to see if there were sets of proteins indicative of specific biological processes that correlate with the presence of disease, and specifically cancer, according to their functional annotations. As expected for such sick animals, many of the proteins identified were related to host immune response. However, a significant number of proteins also directly associated with processes linked to cancer development, including proteins related to the cell cycle, localisation, trasport, and cell death. Additional analysis of the same samples in profiling mode, and in triplicate, confirmed that replicate MS analysis of the same plasma sample generated less variation than that observed between plasma samples from different individuals, demonstrating that the reproducibility of the LC-MS platform was sufficient for this application. These results thus show that an LC-MS-based workflow can be a useful tool for the generation of candidate proteins of interest as part of a disease biomarker discovery effort. PMID:20157627

Development and validation of a LC-MS/MS method for quantitation of fosfomycin - Application to in vitro antimicrobial resistance study using hollow-fiber infection model.

PubMed

Gandhi, Adarsh; Matta, Murali; Garimella, Narayana; Zere, Tesfalem; Weaver, James

2018-06-01

Extensive use and misuse of antibiotics over the past 50 years has contributed to the emergence and spread of antibiotic-resistant bacterial strains, rendering them as a global health concern. To address this issue, a dynamic in vitro hollow-fiber system, which mimics the in vivo environment more closely than the static model, was used to study the emergence of bacterial resistance of Escherichia coli against fosfomycin (FOS). To aid in this endeavor we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for quantitative analysis of FOS in lysogeny broth. FOS was resolved on a Kinetex HILIC (2.1 × 50 mm, 2.6 μm) column with 2 mm ammonium acetate (pH 4.76) and acetonitrile as mobile phase within 3 min. Multiple reaction monitoring was used to acquire data on a triple quadrupole mass spectrometer. The assay was linear from 1 to 1000 μg/mL. Inter- and intra-assay precision and accuracy were <15% and between ±85 and 115% respectively. No significant matrix effect was observed when corrected with the internal standard. FOS was stable for up to 24 h at room temperature, up to three freeze-thaw cycles and up to 24 h when stored at 4°C in the autosampler. In vitro experimental data were similar to the simulated plasma pharmacokinetic data, further confirming the appropriateness of the experimental design to quantitate antibiotics and study occurrence of antimicrobial resistance in real time. The validated LC-MS/MS assays for quantitative determination of FOS in lysogeny broth will help antimicrobial drug resistance studies. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

Characterization of inhibitory constituents of NO production from Catalpa ovata using LC-MS coupled with a cell-based assay.

PubMed

Park, Sangmin; Shin, Hyeji; Park, Yeeun; Choi, Ilgyu; Park, Byoungduck; Lee, Ki Yong

2018-05-25

An effective screening method for inhibitors of NO production in natural products using LC-QTOF MS/MS coupled with a cell-based assay was proposed. The ethyl acetate fraction of Catalpa ovata exhibited a strong inhibitory effect on NO production in lipopolysaccharide-induced BV2 microglia cells. We attempted to identify the active constituents of C. ovata by using LC-QTOF MS/MS coupled with a cell-based assay. Peaks at approximately 14-15 min on the MS chromatogram were estimated to be the bioactive constituents. A new iridoid compound, 6-O-trans-feruloyl-3β-hydroxy-7-deoxyrehamaglutin A (4), and nine known compounds (1-3, 5-10) were isolated from the ethyl acetate fraction of C. ovata by repeated column chromatography. Compounds 3, 4, 5, 7, and 8 significantly attenuated lipopolysaccharide-stimulated NO production in BV2 cells. Our results indicate that LC-QTOF MS/MS coupled with a cell-based NO production inhibitory assay successfully predicted active compounds without a time-consuming isolation process. Copyright © 2018 Elsevier Inc. All rights reserved.

Quantification of low molecular weight selenium metabolites in human plasma after treatment with selenite in pharmacological doses by LC-ICP-MS.

PubMed

Flouda, Konstantina; Dersch, Julie Maria; Gabel-Jensen, Charlotte; Stürup, Stefan; Misra, Sougat; Björnstedt, Mikael; Gammelgaard, Bente

2016-03-01

The paper presents an analytical method for quantification of low molecular weight (LMW) selenium compounds in human plasma based on liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS) and post column isotope dilution-based quantification. Prior to analysis, samples were ultrafiltrated using a cut-off value of 3000 Da. The method was validated in aqueous solution as well as plasma using standards of selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), selenite, and the selenosugar Se-methylseleno-N-acetylgalactosamine (SeGal) for linearity, precision, recoveries, and limits of detection and quantitation with satisfactory results. The method was applied for analysis of a set of plasma samples from cancer patients receiving selenite treatment in a clinical trial. Three LMW selenium compounds were observed. The main compounds, SeGal and selenite were tentatively identified by retention time matching with standards in different chromatographic systems, while the third minor compound was not identified. The identity of the selenosugar was verified by ESI-MS-MS product ion scanning, while selenite was identified indirectly as the glutathione (GSH) reaction product, GS-Se-SG.

Surrogate analyte approach for quantitation of endogenous NAD(+) in human acidified blood samples using liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

PubMed

Liu, Liling; Cui, Zhiyi; Deng, Yuzhong; Dean, Brian; Hop, Cornelis E C A; Liang, Xiaorong

2016-02-01

A high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the quantitative determination of NAD(+) in human whole blood using a surrogate analyte approach was developed and validated. Human whole blood was acidified using 0.5N perchloric acid at a ratio of 1:3 (v:v, blood:perchloric acid) during sample collection. 25μL of acidified blood was extracted using a protein precipitation method and the resulting extracts were analyzed using reverse-phase chromatography and positive electrospray ionization mass spectrometry. (13)C5-NAD(+) was used as the surrogate analyte for authentic analyte, NAD(+). The standard curve ranging from 0.250 to 25.0μg/mL in acidified human blood for (13)C5-NAD(+) was fitted to a 1/x(2) weighted linear regression model. The LC-MS/MS response between surrogate analyte and authentic analyte at the same concentration was obtained before and after the batch run. This response factor was not applied when determining the NAD(+) concentration from the (13)C5-NAD(+) standard curve since the percent difference was less than 5%. The precision and accuracy of the LC-MS/MS assay based on the five analytical QC levels were well within the acceptance criteria from both FDA and EMA guidance for bioanalytical method validation. Average extraction recovery of (13)C5-NAD(+) was 94.6% across the curve range. Matrix factor was 0.99 for both high and low QC indicating minimal ion suppression or enhancement. The validated assay was used to measure the baseline level of NAD(+) in 29 male and 21 female human subjects. This assay was also used to study the circadian effect of endogenous level of NAD(+) in 10 human subjects. Copyright © 2015 Elsevier B.V. All rights reserved.

Streaming visualisation of quantitative mass spectrometry data based on a novel raw signal decomposition method

PubMed Central

Zhang, Yan; Bhamber, Ranjeet; Riba-Garcia, Isabel; Liao, Hanqing; Unwin, Richard D; Dowsey, Andrew W

2015-01-01

As data rates rise, there is a danger that informatics for high-throughput LC-MS becomes more opaque and inaccessible to practitioners. It is therefore critical that efficient visualisation tools are available to facilitate quality control, verification, validation, interpretation, and sharing of raw MS data and the results of MS analyses. Currently, MS data is stored as contiguous spectra. Recall of individual spectra is quick but panoramas, zooming and panning across whole datasets necessitates processing/memory overheads impractical for interactive use. Moreover, visualisation is challenging if significant quantification data is missing due to data-dependent acquisition of MS/MS spectra. In order to tackle these issues, we leverage our seaMass technique for novel signal decomposition. LC-MS data is modelled as a 2D surface through selection of a sparse set of weighted B-spline basis functions from an over-complete dictionary. By ordering and spatially partitioning the weights with an R-tree data model, efficient streaming visualisations are achieved. In this paper, we describe the core MS1 visualisation engine and overlay of MS/MS annotations. This enables the mass spectrometrist to quickly inspect whole runs for ionisation/chromatographic issues, MS/MS precursors for coverage problems, or putative biomarkers for interferences, for example. The open-source software is available from http://seamass.net/viz/. PMID:25663356

A surrogate analyte-based LC-MS/MS method for the determination of γ-hydroxybutyrate (GHB) in human urine and variation of endogenous urinary concentrations of GHB.

PubMed

Kang, Soyoung; Oh, Seung Min; Chung, Kyu Hyuck; Lee, Sooyeun

2014-09-01

γ-Hydroxybutyrate (GHB) is a drug of abuse with a strong anesthetic effect; however, proving its ingestion through the quantification of GHB in biological specimens is not straightforward due to the endogenous presence of GHB in human blood, urine, saliva, etc. In the present study, a surrogate analyte approach was applied to accurate quantitative determination of GHB in human urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in order to overcome this issue. For this, (2)H6-GHB and (13)C2-dl-3-hydroxybutyrate were used as a surrogate standard and as an internal standard, respectively, and parallelism between the surrogate analyte approach and standard addition was investigated at the initial step. The validation results proved the method to be selective, accurate, and precise, with acceptable linearity within calibration ranges (0.1-1μg/ml). The limit of detection and the limit of quantification of (2)H6-GHB were 0.05 and 0.1μg/ml, respectively. No significant variations were observed among urine matrices from different sources. The stability of (2)H6-GHB was satisfactory under sample storage and in-process conditions. However, in vitro production of endogenous GHB was observed when the urine sample was kept under the in-process condition for 4h and under the storage conditions of 4 and -20°C. In order to facilitate the practical interpretation of urinary GHB, endogenous GHB was accurately measured in urine samples from 79 healthy volunteers using the surrogate analyte-based LC-MS/MS method developed in the present study. The unadjusted and creatinine-adjusted GHB concentrations in 74 urine samples with quantitative results ranged from 0.09 to 1.8μg/ml and from 4.5 to 530μg/mmol creatinine, respectively. No significant correlation was observed between the unadjusted and creatinine-adjusted GHB concentrations. The urinary endogenous GHB concentrations were affected by gender and age while they were not significantly influenced by habitual smoking, alcohol drinking, or caffeine-containing beverage drinking. Copyright © 2014 Elsevier B.V. All rights reserved.

[Improvement of 2-mercaptoimidazoline analysis in rubber products containing chlorine].

PubMed

Kaneko, Reiko; Haneishi, Nahoko; Kawamura, Yoko

2012-01-01

An improved analysis method for 2-mercaptoimidazoline in rubber products containing chlorine was developed. 2-Mercaptoimidazoline (20 µg/mL) is detected by means of TLC with two developing solvents in the official method. But, this method is not quantitative. Instead, we employed HPLC using water-methanol (9 : 1) as the mobile phase. This procedure decreased interfering peaks, and the quantitation limit was 2 µg/mL of standard solution. 2-Mercaptoimidazoline was confirmed by GC-MS (5 µg/mL) and LC/MS (1 µg/mL) in the scan mode. For preparation of test solution, a soaking extraction method, in which 20 mL of methanol was added to the sample and allowed to stand overnight at about 40°C, was used. This gave similar values to the Soxhlet extraction method (official method) and was more convenient. The results indicate that our procedure is suitable for analysis of 2-mercaptoimidazoline. When 2-mercaptoimidazoline is detected, it is confirmed by either GC/MS or LC/MS.

Quantitative characterization of solid epoxy resins using comprehensive two dimensional liquid chromatography coupled with electrospray ionization-time of flight mass spectrometry.

PubMed

Julka, Samir; Cortes, Hernan; Harfmann, Robert; Bell, Bruce; Schweizer-Theobaldt, Andreas; Pursch, Matthias; Mondello, Luigi; Maynard, Shawn; West, David

2009-06-01

A comprehensive multidimensional liquid chromatography system coupled to Electrospray Ionization-Mass Spectrometry (LCxLC-ESI-MS) was developed for detailed characterization and quantitation of solid epoxy resin components. The two orthogonal modes of separation selected were size exclusion chromatography (SEC) in the first dimension and liquid chromatography at critical conditions (LCCC) in the second dimension. Different components present in the solid epoxy resins were separated and quantitated for the first time based on the functional groups and molecular weight heterogeneity. Coupling LCxLC separations with mass spectrometry enabled the identification of components resolved in the two-dimensional space. Several different functional group families of compounds were separated and identified, including epoxy-epoxy and epoxy-alpha-glycol functional oligomers, and their individual molecular weight ranges were determined. Repeatability obtained ranged from 0.5% for the main product to 21% for oligomers at the 0.4% concentration level.

Recent trends in analytical procedures in forensic toxicology.

PubMed

Van Bocxlaer, Jan F

2005-12-01

Forensic toxicology is a very demanding discipline,heavily dependent on good analytical techniques. That is why new trends appear continuously. In the past years. LC-MS has revolutionized target compound analysis and has become the trend, also in toxicology. In LC-MS screening analysis, things are less straightforward and several approaches exist. One promising approach based on accurate LC-MSTOF mass measurements and elemental formula based library searches is discussed. This way of screening has already proven its applicability but at the same time it became obvious that a single accurate mass measurement lacks some specificity when using large compound libraries. CE too is a reemerging approach. The increasingly polar and ionic molecules encountered make it a worthwhile addition to e.g. LC, as illustrated for the analysis of GHB. A third recent trend is the use of MALDI mass spectrometry for small molecules. It is promising for its ease-of-use and high throughput. Unfortunately, re-ports of disappointment but also accomplishment, e.g. the quantitative analysis of LSD as discussed here, alternate, and it remains to be seen whether MALDI really will establish itself. Indeed, not all new trends will prove themselves but the mere fact that many appear in the world of analytical toxicology nowadays is, in itself, encouraging for the future of (forensic) toxicology.

Quantitative determination of metformin, glyburide and its metabolites in plasma and urine of pregnant patients by LC-MS/MS.

PubMed

Zhang, Xing; Wang, Xiaoming; Vernikovskaya, Daria I; Fokina, Valentina M; Nanovskaya, Tatiana N; Hankins, Gary D V; Ahmed, Mahmoud S

2015-04-01

This report describes the development and validation of an LC-MS/MS method for the quantitative determination of glyburide (GLB), its five metabolites (M1, M2a, M2b, M3 and M4) and metformin (MET) in plasma and urine of pregnant patients under treatment with a combination of the two medications. The extraction recovery of the analytes from plasma samples was 87-99%, and that from urine samples was 85-95%. The differences in retention times among the analytes and the wide range of the concentrations of the medications and their metabolites in plasma and urine patient samples required the development of three LC methods. The lower limit of quantitation (LLOQ) of the analytes in plasma samples was as follows: GLB, 1.02 ng/mL; its five metabolites, 0.100-0.113 ng/mL; and MET, 4.95 ng/mL. The LLOQ in urine samples was 0.0594 ng/mL for GLB, 0.984-1.02 ng/mL for its five metabolites and 30.0 µg/mL for MET. The relative deviation of this method was <14% for intra-day and inter-day assays in plasma and urine samples, and the accuracy was 86-114% in plasma, and 94-105% in urine. The method described in this report was successfully utilized for determining the concentrations of the two medications in patient plasma and urine. Copyright © 2014 John Wiley & Sons, Ltd.

Quantitative determination of metformin, glyburide and its metabolites in plasma and urine of pregnant patients by LC-MS/MS

PubMed Central

Zhang, Xing; Wang, Xiaoming; Vernikovskaya, Daria I.; Fokina, Valentina M.; Nanovskaya, Tatiana N.; Hankins, Gary D.V.; Ahmed, Mahmoud S.

2014-01-01

This report describes the development and validation of an LC-MS/MS method for the quantitative determination of glyburide (GLB), its five metabolites (M1, M2a, M2b, M3, and M4) and metformin (MET) in plasma and urine of pregnant patients under treatment with a combination of the two medications. The extraction recovery of the analytes from plasma samples ranged between 87% and 99%, and 85%–95% for urine samples. The differences in retention times among the analytes, and the wide range of the concentrations of the medications and their metabolites in plasma and urine patient samples required the development of three LC methods. The lower limit of quantitation (LLOQ) of the analytes in plasma samples was as follows: GLB, 1.02 ng/mL; its five metabolites, 0.100–0.113 ng/mL and 4.95 ng/mL for MET. LLOQ in urine samples was 0.0594 ng/mL for GLB, 0.984–1.02 ng/mL for its five metabolites and 30.0 μg/mL for MET. The relative deviation of this method was < 14% for intra-day and inter-day assays in plasma and urine samples, and the accuracy ranged between 86% and 114% in plasma, and 94% to 105% in urine. The method described in this report was successfully utilized for determining the concentrations of the two medications in patient plasma and urine. PMID:25164921

Validation of an LC-MS/MS salivary assay for glucocorticoid status assessment: Evaluation of the diurnal fluctuation of cortisol and cortisone and of their association within and between serum and saliva.

PubMed

Mezzullo, Marco; Fanelli, Flaminia; Fazzini, Alessia; Gambineri, Alessandra; Vicennati, Valentina; Di Dalmazi, Guido; Pelusi, Carlotta; Mazza, Roberta; Pagotto, Uberto; Pasquali, Renato

2016-10-01

Salivary steroid testing represents a valuable source of biological information; however, the proper measurement of low salivary levels is challenging for direct immunoassays, lacking adequate sensitivity and specificity and causing poor inter-laboratory reproducibility. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has overcome previous analytical limits, often providing results deviating from previous knowledge. Nowadays, LC-MS/MS is being introduced in clinical laboratories for salivary cortisol testing; however, so far only a few studies have reported thorough biological validation based on LC-MS/MS data. In this study, we provide a thorough analytical, pre-analytical and biological validation of an LC-MS/MS method for the measurement of salivary cortisol (F) and of its inactive metabolite cortisone (E). Analytes were extracted from 50μl of saliva, were then separated in 7.5min LC-gradient and detected by negative electrospray ionization-multiple reaction monitoring. The reliability of a widely diffused collection device, Salivette(®), was assessed and the overall procedure was validated. The diurnal cortisol and cortisone fluctuation in saliva and serum was described by a four paired collection protocol (8 am, 12 am, 4 pm and 8 pm) in 19 healthy subjects. The assay allowed the quantitation of F and E down to 39.1 and 78.1pg/ml, with an imprecision range of 5.5-9.5%, 3.9-14.1% and 2.6-14.4%, and an accuracy range of 105.5-113.1%, 88.5-98.7% and 90.7-96.7% for both analytes at low, medium and high levels, respectively. Salivette(®) provided comparable results and better precision (CV<1.0%) as referred to direct spitting (CV<13.0%). A parallel diurnal rhythm in saliva and serum was observed for cortisol and cortisone, with values lowering from the morning to the evening time points (P<0.0001). While salivary E linearly correlated to total serum F (R(2)=0.854, P<0.001), salivary F showed an exponential relationship (R(2)=0.903, P<0.001) with serum F reflecting the free circulating fraction. A non linear association between E and F was observed in saliva (R(2)=0.941, p<0.001) consistent with the type II 11β-HSD activity. We concluded that our LC-MS/MS method allowed a sensitive evaluation of salivary levels of cortisol and cortisone. The simultaneous determination of both hormones in saliva allowed the differential estimation of the active and of the total glucocorticoid exposure over the daytime. The assay could provide further insight into the comprehension of normal and dysfunctional glucocorticoid circadian rhythm. Copyright © 2016 Elsevier Ltd. All rights reserved.

Study of different HILIC, mixed-mode, and other aqueous normal-phase approaches for the liquid chromatography/mass spectrometry-based determination of challenging polar pesticides.

PubMed

Vass, Andrea; Robles-Molina, José; Pérez-Ortega, Patricia; Gilbert-López, Bienvenida; Dernovics, Mihaly; Molina-Díaz, Antonio; García-Reyes, Juan F

2016-07-01

The aim of the study was to evaluate the performance of different chromatographic approaches for the liquid chromatography/mass spectrometry (LC-MS(/MS)) determination of 24 highly polar pesticides. The studied compounds, which are in most cases unsuitable for conventional LC-MS(/MS) multiresidue methods were tested with nine different chromatographic conditions, including two different hydrophilic interaction liquid chromatography (HILIC) columns, two zwitterionic-type mixed-mode columns, three normal-phase columns operated in HILIC-mode (bare silica and two silica-based chemically bonded columns (cyano and amino)), and two standard reversed-phase C18 columns. Different sets of chromatographic parameters in positive (for 17 analytes) and negative ionization modes (for nine analytes) were examined. In order to compare the different approaches, a semi-quantitative classification was proposed, calculated as the percentage of an empirical performance value, which consisted of three main features: (i) capacity factor (k) to characterize analyte separation from the void, (ii) relative response factor, and (iii) peak shape based on analytes' peak width. While no single method was able to provide appropriate detection of all the 24 studied species in a single run, the best suited approach for the compounds ionized in positive mode was based on a UHPLC HILIC column with 1.8 μm particle size, providing appropriate results for 22 out of the 24 species tested. In contrast, the detection of glyphosate and aminomethylphosphonic acid could only be achieved with a zwitterionic-type mixed-mode column, which proved to be suitable only for the pesticides detected in negative ion mode. Finally, the selected approach (UHPLC HILIC) was found to be useful for the determination of multiple pesticides in oranges using HILIC-ESI-MS/MS, with limits of quantitation in the low microgram per kilogram in most cases. Graphical Abstract HILIC improves separation of multiclass polar pesticides.

Comparing identified and statistically significant lipids and polar metabolites in 15-year old serum and dried blood spot samples for longitudinal studies: Comparing lipids and metabolites in serum and DBS samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kyle, Jennifer E.; Casey, Cameron P.; Stratton, Kelly G.

The use of dried blood spots (DBS) has many advantages over traditional plasma and serum samples such as smaller blood volume required, storage at room temperature, and ability for sampling in remote locations. However, understanding the robustness of different analytes in DBS samples is essential, especially in older samples collected for longitudinal studies. Here we analyzed DBS samples collected in 2000-2001 and stored at room temperature and compared them to matched serum samples stored at -80°C to determine if they could be effectively used as specific time points in a longitudinal study following metabolic disease. Four hundred small molecules weremore » identified in both the serum and DBS samples using gas chromatograph-mass spectrometry (GC-MS), liquid chromatography-MS (LC-MS) and LC-ion mobility spectrometry-MS (LC-IMS-MS). The identified polar metabolites overlapped well between the sample types, though only one statistically significant polar metabolite in a case-control study was conserved, indicating degradation occurs in the DBS samples affecting quantitation. Differences in the lipid identifications indicated that some oxidation occurs in the DBS samples. However, thirty-six statistically significant lipids correlated in both sample types indicating that lipid quantitation was more stable across the sample types.« less

Determination of hydroxylated polychlorinated biphenyls (HO-PCBs) in blood plasma by high-performance liquid chromatography-electrospray ionization-tandem quadrupole mass spectrometry.

PubMed

Letcher, R J; Li, H X; Chu, S G

2005-01-01

Hydroxylated metabolites of polychlorinated biphenyls (HO-PCBs) and pentachlorophenol (PCP) are halogenated phenolic compounds, and they are increasingly common as environmental contaminants mainly in the blood of wildlife and humans. A methodology based on high-performance liquid chromatography (reversed-phase)-electrospray (negative) ionization-tandem quadrupole mass spectrometry (LC-ESI(-)-MS-MS) in the select ion monitoring or multiple reaction monitoring modes was developed for HO-PCB and PCP determination in blood plasma and serum. Among 11 environmentally relevant HO-PCB congeners and PCP spiked to fetal calf serum, quantitative assessments, including matrix effects on ESI(-) suppression/ enhancement, showed process (recovery) efficiencies of 73% to 89% without internal standard (IS) correction, and 88% to 103% with IS correction, and method limits of quantification ranging from 1 to 50 pg/g (wet weight). Using the developed LC-ESI(-)-MS methodology in comparison with GC-MS and GC-ECD based approaches, similar results were found for HO-PCB identification and quantification in the plasma of polar bear (Ursus maritimus) from the Canadian arctic. LC-ESI(-)-MS identified four HO-PCB congeners [4'-HO-2,2',4,6,6'-pentachlorobiphenyl (4'-HO-CB104), 4-HO-2,3,3',4',5-pentachlorobiphenyl (4-HO-CB107), 4-HO-2,3,3',5,5',6-hexachlorobiphenyl (4-HO-CB165) and 3'-HO-2,2',3,4,4',5,5'-heptachlorobiphenyl (3'-HO-CB180)], and 14 additional tetra- to hepta-chlorinated HO-PCBs isomers in the polar bear plasma.

Complementarity of SOMAscan to LC-MS/MS and RNA-seq for quantitative profiling of human embryonic and mesenchymal stem cells.

PubMed

Billing, Anja M; Ben Hamidane, Hisham; Bhagwat, Aditya M; Cotton, Richard J; Dib, Shaima S; Kumar, Pankaj; Hayat, Shahina; Goswami, Neha; Suhre, Karsten; Rafii, Arash; Graumann, Johannes

2017-01-06

Dynamic range limitations are challenging to proteomics, particularly in clinical samples. Affinity proteomics partially overcomes this, yet suffers from dependence on reagent quality. SOMAscan, an aptamer-based platform for over 1000 proteins, avoids that issue using nucleic acid binders. Targets include low expressed proteins not easily accessible by other approaches. Here we report on the potential of SOMAscan for the study of differently sourced mesenchymal stem cells (MSC) in comparison to LC-MS/MS and RNA sequencing. While targeting fewer analytes, SOMAscan displays high precision and dynamic range coverage, allowing quantification of proteins not measured by the other platforms. Expression between cell types (ESC and MSC) was compared across techniques and uncovered the expected large differences. Sourcing was investigated by comparing subtypes: bone marrow-derived, standard in clinical studies, and ESC-derived MSC, thought to hold similar potential but devoid of inter-donor variability and proliferating faster in vitro. We confirmed subtype-equivalency, as well as vesicle and extracellular matrix related processes in MSC. In contrast, the proliferative nature of ESC was captured less by SOMAscan, where nuclear proteins are underrepresented. The complementary of SOMAscan allowed the comprehensive exploration of CD markers and signaling molecules, not readily accessible otherwise and offering unprecedented potential in subtype characterization. Mesenchymal stem cells (MSC) represent promising stem cell-derived therapeutics as indicated by their application in >500 clinical trials currently registered with the NIH. Tissue-derived MSC require invasive harvesting and imply donor-to-donor differences, to which embryonic stem cell (ESC)-derived MSC may provide an alternative and thus warrant thorough characterization. In continuation of our previous study where we compared in depth embryonic stem cells (ESC) and MSC from two sources (bone marrow and ESC-derived), we included the aptamer-based SOMAscan assay, complementing LC-MS/MS and RNA-seq data. Furthermore, SOMAscan, a targeted proteomics platform developed for analyzing clinical samples, has been benchmarked against established analytical platforms (LC-MS/MS and RNA-seq) using stem cell comparisons as a model. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Isotope Inversion Experiment evaluating the suitability of calibration in surrogate matrix for quantification via LC-MS/MS-Exemplary application for a steroid multi-method.

PubMed

Suhr, Anna Catharina; Vogeser, Michael; Grimm, Stefanie H

2016-05-30

For quotable quantitative analysis of endogenous analytes in complex biological samples by isotope dilution LC-MS/MS, the creation of appropriate calibrators is a challenge, since analyte-free authentic material is in general not available. Thus, surrogate matrices are often used to prepare calibrators and controls. However, currently employed validation protocols do not include specific experiments to verify the suitability of a surrogate matrix calibration for quantification of authentic matrix samples. The aim of the study was the development of a novel validation experiment to test whether surrogate matrix based calibrators enable correct quantification of authentic matrix samples. The key element of the novel validation experiment is the inversion of nonlabelled analytes and their stable isotope labelled (SIL) counterparts in respect to their functions, i.e. SIL compound is the analyte and nonlabelled substance is employed as internal standard. As a consequence, both surrogate and authentic matrix are analyte-free regarding SIL analytes, which allows a comparison of both matrices. We called this approach Isotope Inversion Experiment. As figure of merit we defined the accuracy of inverse quality controls in authentic matrix quantified by means of a surrogate matrix calibration curve. As a proof-of-concept application a LC-MS/MS assay addressing six corticosteroids (cortisol, cortisone, corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, and 17-OH-progesterone) was chosen. The integration of the Isotope Inversion Experiment in the validation protocol for the steroid assay was successfully realized. The accuracy results of the inverse quality controls were all in all very satisfying. As a consequence the suitability of a surrogate matrix calibration for quantification of the targeted steroids in human serum as authentic matrix could be successfully demonstrated. The Isotope Inversion Experiment fills a gap in the validation process for LC-MS/MS assays quantifying endogenous analytes. We consider it a valuable and convenient tool to evaluate the correct quantification of authentic matrix samples based on a calibration curve in surrogate matrix. Copyright © 2016 Elsevier B.V. All rights reserved.

To elute or not to elute in immunocapture bottom-up LC-MS.

PubMed

Levernæs, Maren Christin Stillesby; Broughton, Marianne Nordlund; Reubsaet, Léon; Halvorsen, Trine Grønhaug

2017-06-15

Immunocapture-based bottom-up LC-MS is a promising technique for the quantification of low abundant proteins. Magnetic immunocapture beads provide efficient enrichment from complex samples through the highly specific interaction between the target protein and its antibody. In this article, we have performed the first thorough comparison between digestion of proteins while bound to antibody coated beads versus after elution from the beads. Two previously validated immunocapture based MS methods for the quantification of pro-gastrin releasing peptide (ProGRP) and human chorionic gonadotropin (hCG) were used as model systems. The tryptic peptide generation was shown to be protein dependent and influenced by protein folding and accessibility towards trypsin both on-beads and in the eluate. The elution of proteins bound to the beads was also shown to be incomplete. In addition, the on-beads digestion suffered from non-specific binding of the trypsin generated peptides. A combination of on-beads digestion and elution may be applied to improve both the quantitative (peak area of the signature peptides) and qualitative yield (number of missed cleavages, total number of identified peptides, coverage, signal intensity and number of zero missed cleavage peptides) of the target proteins. The quantitative yield of signature peptides was shown to be reproducible in all procedures tested. Copyright © 2017 Elsevier B.V. All rights reserved.

Simultaneous serum nicotine, cotinine, and trans-3'-hydroxycotinine quantitation with minimal sample volume for tobacco exposure status of solid organ transplant patients.

PubMed

Shu, Irene; Wang, Ping

2013-06-01

Concentrations of nicotine and its metabolites in blood are indicative of patients' current tobacco exposure, and their quantifications have been clinically applied to multiple assessments including demonstration of abstinence prior to heart-lung transplantation. For the purpose of transplant evaluation, the laboratory work up is extensive; thereby an assay with minimal sample volume is preferred. We developed and validated a rapid LC-MS/MS assay to simultaneously quantitate nicotine and its major metabolites, Cotinine and trans-3'-OH-cotinine (3-OH-Cot), in serum. 100μL of serum was spiked with deuterated internal standards and extracted by Oasis HLB solid phase extraction cartridge. Nicotine and metabolites in the reconstituted serum extract were separated by Agilent Eclipse XDB-C8 3.5μm 2.1mm×50mm HPLC column within 4.7min, and quantified by MS/MS with positive mode electrospray ionization and multiple reaction monitoring. Ion suppression was insignificant, and extraction efficiency was 79-110% at 50ng/mL for all compounds. Limit of detection was 1.0ng/mL for nicotine and 3-OH-Cot, and <0.5ng/mL for Cotinine. Linearity ranges for nicotine, cotinine and 3-OH-Cot were 2-100, 2-1000, and 5-1000ng/mL with recoveries of 86-115%. Within-day and twenty-day imprecision at nicotine/cotinine/3-OH-Cot levels of 22/150/90, 37/250/150, and 50/800/500ng/mL were all 1.1-6.5%. The reconstituted serum extracts were stable for at least 7 days stored in the HPLC autosampler at 5°C. Our method correlates well with alternative LC-MS/MS methods. We successfully developed and validated an LC-MS/MS assay to quantitate concentrations of nicotine and its metabolites in serum with minimal sample volume to assess tobacco exposure of heart-lung transplant patients. Copyright © 2013 Elsevier B.V. All rights reserved.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for analysis of endogenous steroids in the luteal phase and early pregnancy in dogs: a pilot study.

PubMed

Holst, Bodil S; Kushnir, Mark M; Bergquist, Jonas

2015-12-01

Blood samples from dogs are often limited in volume, only allowing few steroids to be quantified with immunoassays. In addition, immunoassays may be compromised by interferences such as anti-reagent antibodies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can be used for the simultaneous quantitation of several steroids. This has not been described in dogs before. The aims were to use LC-MS/MS to study steroid profiles in early pregnancy and luteal phase in dogs, and to determine if differences exist between pregnant (P) and nonpregnant (NP) dogs. Nine female dogs were included, 4 during a NP luteal phase, 4 during a P luteal phase, and one during one NP and one P luteal phase. Blood samples were collected around the time of the LH surge (Day 0) and on Day 26. Serum was analyzed for 5 classes of steroids, including glucocorticoids, androgens, estrogens, pregnanes, and progestins, using LC-MS/MS methods. The concentration of progesterone was significantly higher on Day 26 in P than in NP bitches. Distribution of concentrations of glucocorticoids, androgens, estrogens, or pregnanes in P and NP dogs were not statistically different. The predominating glucocorticoid was cortisol, and dihydroepiandrosterone (DHEA) was the predominating androgen. Concentration of estrone was comparable to oestradiol, whereas concentrations of pregnenolone were higher than those of 17-OH pregnenolone. Only concentration of progesterone differed between P and NP bitches, being significantly higher on Day 26 in P than in NP bitches. LC-MS/MS offers interesting possibilities for studies of canine reproductive endocrinology. © 2015 American Society for Veterinary Clinical Pathology.

Direct total and free testosterone measurement by liquid chromatography tandem mass spectrometry across two different platforms.

PubMed

Rhea, Jeanne M; French, Deborah; Molinaro, Ross J

2013-05-01

To develop and validate liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for the direct measurement of total and free testosterone in patient samples on two different analytical systems. An API 4000 and 5000 triple quadropoles were used and compared; the former is reported to be 3-5 times less sensitive, as was used to set the quantitation limits. Free testosterone was separated from the protein-bound fraction by equilibrium dialysis followed by derivatization. Either free or total testosterone, and a deuterated internal standard (d3-testosterone) were extracted by liquid-liquid extraction. The validation results were compared to two different clinical laboratories. The use of d2-testosterone was found to be unacceptable for our method. The total testosterone LC-MS/MS methods on both systems were linear over a wide concentration range of 1.5-2000ng/dL. Free testosterone was measured directly using equilibrium dialysis coupled LC-MS/MS and linear over the concentration range of 2.5-2500pg/mL. Good correlation (total testosterone, R(2)=0.96; free testosterone, R(2)=0.98) was observed between our LC-MS/MS systems and comparator laboratory. However, differences in absolute values for both free and total testosterone measurements were observed while a comparison to a second published LC-MS/MS method showed excellent correlation. Free and total testosterone measurements correlated well with clinical observations. To our knowledge, this is the first published validation of free and total testosterone methods across two analytical systems of different analytical sensitivities. A less sensitive system does not sacrifice analytical or clinical sensitivity to directly measure free and total testosterone in patient samples. Copyright © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

BatMass: a Java Software Platform for LC-MS Data Visualization in Proteomics and Metabolomics.

PubMed

Avtonomov, Dmitry M; Raskind, Alexander; Nesvizhskii, Alexey I

2016-08-05

Mass spectrometry (MS) coupled to liquid chromatography (LC) is a commonly used technique in metabolomic and proteomic research. As the size and complexity of LC-MS-based experiments grow, it becomes increasingly more difficult to perform quality control of both raw data and processing results. In a practical setting, quality control steps for raw LC-MS data are often overlooked, and assessment of an experiment's success is based on some derived metrics such as "the number of identified compounds". The human brain interprets visual data much better than plain text, hence the saying "a picture is worth a thousand words". Here, we present the BatMass software package, which allows for performing quick quality control of raw LC-MS data through its fast visualization capabilities. It also serves as a testbed for developers of LC-MS data processing algorithms by providing a data access library for open mass spectrometry file formats and a means of visually mapping processing results back to the original data. We illustrate the utility of BatMass with several use cases of quality control and data exploration.

BatMass: a Java software platform for LC/MS data visualization in proteomics and metabolomics

PubMed Central

Avtonomov, Dmitry; Raskind, Alexander; Nesvizhskii, Alexey I.

2017-01-01

Mass spectrometry (MS) coupled to liquid chromatography (LC) is a commonly used technique in metabolomic and proteomic research. As the size and complexity of LC/MS based experiments grow, it becomes increasingly more difficult to perform quality control of both raw data and processing results. In a practical setting, quality control steps for raw LC/MS data are often overlooked and assessment of an experiment's success is based on some derived metrics such as “the number of identified compounds”. Human brain interprets visual data much better than plain text, hence the saying “a picture is worth a thousand words”. Here we present BatMass software package which allows to perform quick quality control of raw LC/MS data through its fast visualization capabilities. It also serves as a testbed for developers of LC/MS data processing algorithms by providing a data access library for open mass spectrometry file formats and a means of visually mapping processing results back to the original data. We illustrate the utility of BatMass with several use cases of quality control and data exploration. PMID:27306858

LC-MS based analysis of endogenous steroid hormones in human hair.

PubMed

Gao, Wei; Kirschbaum, Clemens; Grass, Juliane; Stalder, Tobias

2016-09-01

The quantification of endogenous steroid hormone concentrations in hair is increasingly used as a method for obtaining retrospective information on long-term integrated hormone exposure. Several different analytical procedures have been employed for hair steroid analysis, with liquid chromatography-mass spectrometry (LC-MS) being recognized as a particularly powerful analytical tool. Several methodological aspects affect the performance of LC-MS systems for hair steroid analysis, including sample preparation and pretreatment, steroid extraction, post-incubation purification, LC methodology, ionization techniques and MS specifications. Here, we critically review the differential value of such protocol variants for hair steroid hormones analysis, focusing on both analytical quality and practical feasibility issues. Our results show that, when methodological challenges are adequately addressed, LC-MS protocols can not only yield excellent sensitivity and specificity but are also characterized by relatively simple sample processing and short run times. This makes LC-MS based hair steroid protocols particularly suitable as a high-quality option for routine application in research contexts requiring the processing of larger numbers of samples. Copyright © 2016 Elsevier Ltd. All rights reserved.

Multiplexed Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry Quantification of Cancer Signaling Proteins

PubMed Central

Chen, Yi; Fisher, Kate J.; Lloyd, Mark; Wood, Elizabeth R.; Coppola, Domenico; Siegel, Erin; Shibata, David; Chen, Yian A.; Koomen, John M.

2017-01-01

Quantitative evaluation of protein expression across multiple cancer-related signaling pathways (e.g. Wnt/β-catenin, TGF-β, receptor tyrosine kinases (RTK), MAP kinases, NF-κB, and apoptosis) in tumor tissues may enable the development of a molecular profile for each individual tumor that can aid in the selection of appropriate targeted cancer therapies. Here, we describe the development of a broadly applicable protocol to develop and implement quantitative mass spectrometry assays using cell line models and frozen tissue specimens from colon cancer patients. Cell lines are used to develop peptide-based assays for protein quantification, which are incorporated into a method based on SDS-PAGE protein fractionation, in-gel digestion, and liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM/MS). This analytical platform is then applied to frozen tumor tissues. This protocol can be broadly applied to the study of human disease using multiplexed LC-MRM assays. PMID:28808993

Lipidomic analysis of glycerolipid and cholesteryl ester autooxidation products.

PubMed

Kuksis, Arnis; Suomela, Jukka-Pekka; Tarvainen, Marko; Kallio, Heikki

2009-06-01

Thin-layer chromatography (TLC), gas chromatography (GC), and liquid chromatography (LC) in combination with mass spectrometry (MS) have been adopted for the isolation and identification of oxolipids and for determining their functionality. TLC provides a rapid separation and access to most oxolipids as intact molecules and has recently been effectively interfaced with time-of-flight (TOF) MS (TOF-MS). GC with flame ionization (FI) (GC/FI) and electron impact (EI) MS (GC/EI-MS) has been extensively utilized in the analysis of isoprostanes and other low-molecular-weight oxolipids, although these methods require derivatization of the analytes. In contrast, LC with ultraviolet (UV) absorption (LC/UV) or evaporate light scattering detection (ELSD) (LC/ELSD) as well as electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) MS (LC/ESI-MS) or LC/APCI-MS has proven to be well suited for the analysis of intact oxolipids and their conjugates without or with minimal derivatization. Nevertheless, kit-based colorimetric and fluorescent procedures continue to serve as sensitive indicators of the presence of hydroperoxides and aldehydes.

Optimization of Sample Preparation for the Identification and Quantification of Saxitoxin in Proficiency Test Mussel Sample using Liquid Chromatography-Tandem Mass Spectrometry

PubMed Central

Harju, Kirsi; Rapinoja, Marja-Leena; Avondet, Marc-André; Arnold, Werner; Schär, Martin; Burrell, Stephen; Luginbühl, Werner; Vanninen, Paula

2015-01-01

Saxitoxin (STX) and some selected paralytic shellfish poisoning (PSP) analogues in mussel samples were identified and quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample extraction and purification methods of mussel sample were optimized for LC-MS/MS analysis. The developed method was applied to the analysis of the homogenized mussel samples in the proficiency test (PT) within the EQuATox project (Establishment of Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk). Ten laboratories from eight countries participated in the STX PT. Identification of PSP toxins in naturally contaminated mussel samples was performed by comparison of product ion spectra and retention times with those of reference standards. The quantitative results were obtained with LC-MS/MS by spiking reference standards in toxic mussel extracts. The results were within the z-score of ±1 when compared to the results measured with the official AOAC (Association of Official Analytical Chemists) method 2005.06, pre-column oxidation high-performance liquid chromatography with fluorescence detection (HPLC-FLD). PMID:26610567

Multiclass determination and confirmation of antibiotic residues in honey using LC-MS/MS.

PubMed

Lopez, Mayda I; Pettis, Jeffery S; Smith, I Barton; Chu, Pak-Sin

2008-03-12

A multiclass method has been developed for the determination and confirmation in honey of tetracyclines (chlortetracycline, doxycycline, oxytetracycline, and tetracycline), fluoroquinolones (ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, and sarafloxacin), macrolides (tylosin), lincosamides (lincomycin), aminoglycosides (streptomycin), sulfonamides (sulfathiazole), phenicols (chloramphenicol), and fumagillin residues using liquid chromatography tandem mass spectrometry (LC-MS/MS). Erythromycin (a macrolide) and monensin (an ionophore) can be detected and confirmed but not quantitated. Honey samples (approximately 2 g) are dissolved in 10 mL of water and centrifuged. An aliquot of the supernatant is used to determine streptomycin. The remaining supernatant is filtered through a fine-mesh nylon fabric and cleaned up by solid phase extraction. After solvent evaporation and sample reconstitution, 15 antibiotics are assayed by LC-MS/MS using electrospray ionization (ESI) in positive ion mode. Afterward, chloramphenicol is assayed using ESI in negative ion mode. The method has been validated at the low part per billion levels for most of the drugs with accuracies between 65 and 104% and coefficients of variation less than 17%. The evaluation of matrix effects caused by honey of different floral origin is presented.

Power of Orbitrap-based LC-high resolution-MS/MS for comprehensive drug testing in urine with or without conjugate cleavage or using dried urine spots after on-spot cleavage in comparison to established LC-MSn or GC-MS procedures.

PubMed

Michely, Julian A; Meyer, Markus R; Maurer, Hans H

2018-01-01

Reliable, sensitive, and comprehensive urine screening procedures by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS) with low or high resolution (HR) are of high importance for drug testing, adherence monitoring, or detection of toxic compounds. Besides conventional urine sampling, dried urine spots are of increasing interest. In the present study, the power of LC-HR-MS/MS was investigated for comprehensive drug testing in urine with or without conjugate cleavage or using dried urine spots after on-spot cleavage in comparison to established LC-MS n or GC-MS procedures. Authentic human urine samples (n = 103) were split in 4 parts. One aliquot was prepared by precipitation (UP), one by UP with conjugate cleavage (UglucP), one spot on filter paper cards and prepared by on-spot cleavage followed by liquid extraction (DUSglucE), and one worked-up by acid hydrolysis, liquid-liquid extraction, and acetylation for GC-MS analysis. The 3 series of LC-HR-MS/MS results were compared among themselves, to corresponding published LC-MS n data, and to screening results obtained by conventional GC-MS. The reference libraries used for the 3 techniques contained over 4500 spectra of parent compounds and their metabolites. The number of all detected hits (770 drug intakes) was set to 100%. The LC-HR-MS/MS approach detected 80% of the hits after UP, 89% after UglucP, and 77% after DUSglucE, which meant over one-third more hits in comparison to the corresponding published LC-MS n results with ≤49% detected hits. The GC-MS approach identified 56% of all detected hits. In conclusion, LC-HR-MS/MS provided the best screening results after conjugate cleavage and precipitation. Copyright © 2017 John Wiley & Sons, Ltd.

Development and validation of a highly sensitive LC-ESI-MS/MS method for estimation of IIIM-MCD-211, a novel nitrofuranyl methyl piperazine derivative with potential activity against tuberculosis: Application to drug development.

PubMed

Magotra, Asmita; Sharma, Anjna; Gupta, Ajai Prakash; Wazir, Priya; Sharma, Shweta; Singh, Parvinder Pal; Tikoo, Manoj Kumar; Vishwakarma, Ram A; Singh, Gurdarshan; Nandi, Utpal

2017-08-15

In the present study, a simple, sensitive, specific and rapid liquid chromatography (LC) tandem mass spectrometry (MS/MS) method was developed and validated according to the Food and Drug Administration (FDA) guidelines for estimation of IIIM-MCD-211 (a potent oral candidate with promising action against tuberculosis) in mice plasma using carbamazepine as internal standard (IS). Bioanalytical method consisted of one step protein precipitation for sample preparation followed by quantitation in LC-MS/MS using positive electrospray ionization technique (ESI) operating in multiple reaction monitoring (MRM) mode. Elution was achieved in gradient mode on High Resolution Chromolith RP-18e column with mobile phase comprised of acetonitrile and 0.1% (v/v) formic acid in water at the flow rate of 0.4mL/min. Precursor to product ion transitions (m/z 344.5/218.4 and m/z 237.3/194.2) were used to measure analyte and IS, respectively. All validation parameters were well within the limit of acceptance criteria. The method was successfully applied to assess the pharmacokinetics of the candidate in mice following oral (10mg/kg) and intravenous (IV; 2.5mg/kg) administration. It was also effectively used to quantitate metabolic stability of the compound in mouse liver microsomes (MLM) and human liver microsomes (HLM) followed by its in-vitro-in-vivo extrapolation. Copyright © 2017 Elsevier B.V. All rights reserved.

Native protein mapping and visualization of protein interactions in the area of human plasma high-density lipoprotein by combining nondenaturing micro 2DE and quantitative LC-MS/MS.

PubMed

Jin, Ya; Bu, Shujie; Zhang, Jun; Yuan, Qi; Manabe, Takashi; Tan, Wen

2014-07-01

A human plasma sample was subjected to nondenaturing micro 2DE and a gel area (5 mm × 18 mm) that includes high-density lipoprotein (HDL) was cut into 1 mm × 1 mm squares, then the proteins in the 90 gel pieces were analyzed by quantitative LC-MS/MS. Grid-cutting of the gel was employed to; (i) ensure the total analysis of the proteins in the area, (ii) standardize the conditions of analysis by LC-MS/MS, (iii) reconstruct the protein distribution patterns from the quantity data. Totally 154 proteins were assigned in the 90 gel pieces and the quantity distribution of each was reconstructed as a color density pattern (a native protein map). The map of apolipoprotein (Apo) A-I showed a wide apparent mass distribution characteristic to HDL and was compared with the maps of the other 153 proteins. Eleven proteins showed maps of wide distribution that overlapped with the map of Apo A-I, and all have been reported to be the components of HDL. Further, seven minor proteins associated with HDL were detected at the gel positions of high Apo A-I quantity. These results for the first time visualized the localization of HDL apolipoproteins on a nondenaturing 2DE gel and strongly suggested their interactions. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development and validation of sensitive LC/MS/MS method for quantitative bioanalysis of levonorgestrel in rat plasma and application to pharmacokinetics study.

PubMed

Ananthula, Suryatheja; Janagam, Dileep R; Jamalapuram, Seshulatha; Johnson, James R; Mandrell, Timothy D; Lowe, Tao L

2015-10-15

Rapid, sensitive, selective and accurate LC/MS/MS method was developed for quantitative determination of levonorgestrel (LNG) in rat plasma and further validated for specificity, linearity, accuracy, precision, sensitivity, matrix effect, recovery efficiency and stability. Liquid-liquid extraction procedure using hexane:ethyl acetate mixture at 80:20 v:v ratio was employed to efficiently extract LNG from rat plasma. Reversed phase Luna column C18(2) (50×2.0mm i.d., 3μM) installed on a AB SCIEX Triple Quad™ 4500 LC/MS/MS system was used to perform chromatographic separation. LNG was identified within 2min with high specificity. Linear calibration curve was drawn within 0.5-50ng·mL(-1) concentration range. The developed method was validated for intra-day and inter-day accuracy and precision whose values fell in the acceptable limits. Matrix effect was found to be minimal. Recovery efficiency at three quality control (QC) concentrations 0.5 (low), 5 (medium) and 50 (high) ng·mL(-1) was found to be >90%. Stability of LNG at various stages of experiment including storage, extraction and analysis was evaluated using QC samples, and the results showed that LNG was stable at all the conditions. This validated method was successfully used to study the pharmacokinetics of LNG in rats after SubQ injection, providing its applicability in relevant preclinical studies. Copyright © 2015 Elsevier B.V. All rights reserved.

Establishment and comparison of three novel methods for the determination of the photodynamic therapy agent 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH) in human serum.

PubMed

Chen, Lin; Xiao, Qingqing; Zhang, Xian; Yang, Jin

2016-03-20

2-[1-Hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH) is a second-generation photosensitizer that has been applied in clinical studies of photodynamic therapy for a variety of malignant lesions. Based on the differences in selectivity and labour intensity, three novel methods - fluorescence detection coupled with high performance liquid chromatography (LC-FLD), LC-tandem mass spectrometry (LC-MS/MS) and fluorescence-based microplate reader methods - were developed for the determination of HPPH in human serum, which allowed comparison of fluorescence and MS platform for HPPH quantification. All three methods have been validated and successfully applied to support the clinical pharmacokinetic study of HPPH. The concentrations measured by LC-FLD matched those by LC-MS/MS with a correlation coefficient (r=0.994) and coefficient of determination (r(2)=0.989). Data consistency was also found between the measurements of microplate reader and LC-MS/MS with a correlation coefficient (r=0.999) and coefficient of determination (r(2)=0.998), indicating that fluorescence assay, the low cost alternative with a relatively poorer selectivity, is clearly suitable for the quantification of HPPH. Calibration curves in the methods of LC-FLD and microplate reader were linear (r˃0.998) over the concentration range from 50 to 5000 ng/mL, and linearity was obtained over the concentration range from 5 to 1000 ng/mL in the LC-MS/MS method. Compared with the other two methods, the fluorescence-based microplate reader method with proven high selectivity should be strongly recommended because of obvious advantages such as the lowest labour intensity, the lowest instrument cost, a better sensitivity than LC-FLD and the very rapid determination of large number of samples (24 samples/40 s). Copyright © 2015 Elsevier B.V. All rights reserved.

ICPD-a new peak detection algorithm for LC/MS.

PubMed

Zhang, Jianqiu; Haskins, William

2010-12-01

The identification and quantification of proteins using label-free Liquid Chromatography/Mass Spectrometry (LC/MS) play crucial roles in biological and biomedical research. Increasing evidence has shown that biomarkers are often low abundance proteins. However, LC/MS systems are subject to considerable noise and sample variability, whose statistical characteristics are still elusive, making computational identification of low abundance proteins extremely challenging. As a result, the inability of identifying low abundance proteins in a proteomic study is the main bottleneck in protein biomarker discovery. In this paper, we propose a new peak detection method called Information Combining Peak Detection (ICPD ) for high resolution LC/MS. In LC/MS, peptides elute during a certain time period and as a result, peptide isotope patterns are registered in multiple MS scans. The key feature of the new algorithm is that the observed isotope patterns registered in multiple scans are combined together for estimating the likelihood of the peptide existence. An isotope pattern matching score based on the likelihood probability is provided and utilized for peak detection. The performance of the new algorithm is evaluated based on protein standards with 48 known proteins. The evaluation shows better peak detection accuracy for low abundance proteins than other LC/MS peak detection methods.

Characterisation and quantification of phenolic compounds of extra-virgin olive oils according to their geographical origin by a rapid and resolutive LC-ESI-TOF MS method.

PubMed

Ouni, Youssef; Taamalli, Ameni; Gómez-Caravaca, Ana Maria; Segura-Carretero, Antonio; Fernández-Gutiérrez, Alberto; Zarrouk, Mokhtar

2011-08-01

The phenolic compounds present in seven samples of olive fruits were analysed by a rapid and resolutive LC-ESI-TOF MS method. All samples were collected during the normal picking period for olive oil production, in central and south Tunisia, and were obtained from the Oueslati variety cultivated in different olive growing areas. In the Tunisian samples, 22 compounds have been characterised by LC-ESI-TOF MS analysis. Results showed no qualitative differences in the phenolic fractions between virgin olive oils from different geographical region. However, significant quantitative differences were observed in a wide number of phenolic compounds. These results permit to use the phenolic fractions as an indicator of each region. Copyright © 2011 Elsevier Ltd. All rights reserved.

SIMPATIQCO: a server-based software suite which facilitates monitoring the time course of LC-MS performance metrics on Orbitrap instruments.

PubMed

Pichler, Peter; Mazanek, Michael; Dusberger, Frederico; Weilnböck, Lisa; Huber, Christian G; Stingl, Christoph; Luider, Theo M; Straube, Werner L; Köcher, Thomas; Mechtler, Karl

2012-11-02

While the performance of liquid chromatography (LC) and mass spectrometry (MS) instrumentation continues to increase, applications such as analyses of complete or near-complete proteomes and quantitative studies require constant and optimal system performance. For this reason, research laboratories and core facilities alike are recommended to implement quality control (QC) measures as part of their routine workflows. Many laboratories perform sporadic quality control checks. However, successive and systematic longitudinal monitoring of system performance would be facilitated by dedicated automatic or semiautomatic software solutions that aid an effortless analysis and display of QC metrics over time. We present the software package SIMPATIQCO (SIMPle AuTomatIc Quality COntrol) designed for evaluation of data from LTQ Orbitrap, Q-Exactive, LTQ FT, and LTQ instruments. A centralized SIMPATIQCO server can process QC data from multiple instruments. The software calculates QC metrics supervising every step of data acquisition from LC and electrospray to MS. For each QC metric the software learns the range indicating adequate system performance from the uploaded data using robust statistics. Results are stored in a database and can be displayed in a comfortable manner from any computer in the laboratory via a web browser. QC data can be monitored for individual LC runs as well as plotted over time. SIMPATIQCO thus assists the longitudinal monitoring of important QC metrics such as peptide elution times, peak widths, intensities, total ion current (TIC) as well as sensitivity, and overall LC-MS system performance; in this way the software also helps identify potential problems. The SIMPATIQCO software package is available free of charge.

EasyLCMS: an asynchronous web application for the automated quantification of LC-MS data

PubMed Central

2012-01-01

Background Downstream applications in metabolomics, as well as mathematical modelling, require data in a quantitative format, which may also necessitate the automated and simultaneous quantification of numerous metabolites. Although numerous applications have been previously developed for metabolomics data handling, automated calibration and calculation of the concentrations in terms of μmol have not been carried out. Moreover, most of the metabolomics applications are designed for GC-MS, and would not be suitable for LC-MS, since in LC, the deviation in the retention time is not linear, which is not taken into account in these applications. Moreover, only a few are web-based applications, which could improve stand-alone software in terms of compatibility, sharing capabilities and hardware requirements, even though a strong bandwidth is required. Furthermore, none of these incorporate asynchronous communication to allow real-time interaction with pre-processed results. Findings Here, we present EasyLCMS (http://www.easylcms.es/), a new application for automated quantification which was validated using more than 1000 concentration comparisons in real samples with manual operation. The results showed that only 1% of the quantifications presented a relative error higher than 15%. Using clustering analysis, the metabolites with the highest relative error distributions were identified and studied to solve recurrent mistakes. Conclusions EasyLCMS is a new web application designed to quantify numerous metabolites, simultaneously integrating LC distortions and asynchronous web technology to present a visual interface with dynamic interaction which allows checking and correction of LC-MS raw data pre-processing results. Moreover, quantified data obtained with EasyLCMS are fully compatible with numerous downstream applications, as well as for mathematical modelling in the systems biology field. PMID:22884039

MicroSPE-nanoLC-ESI-MS/MS Using 10-μm-i.d. Silica-Based Monolithic Columns for Proteomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luo, Quanzhou; Page, Jason S.; Tang, Keqi

2007-01-01

Silica-based monolithic narrow bore capillary columns (25 cm x 10 µm i.d.) with an integrated nanoESI emitter has been developed to provide high quality and robust microSPE-nanoLC-ESI-MS analyses. The integrated nanoESI emitter adds no dead volume to the LC separation, allowing stable electrospray performance to be obtained at flow rates of ~10 nL/min. In an initial application we identified 5510 unique peptides covering 1443 distinct Shewanella oneidensis proteins from a 300 ng tryptic digest sample in a single 4-h LC-MS/MS analysis using a linear ion trap MS (LTQ). We found the use of an integrated monolithic ESI emitter provided enhancedmore » resistance to clogging and good run-to-run reproducibility.« less

Analysis of selected sugars and sugar phosphates in mouse heart tissue by reductive amination and liquid chromatography-electrospray ionization mass spectrometry.

PubMed

Han, Jun; Tschernutter, Vera; Yang, Juncong; Eckle, Tobias; Borchers, Christoph H

2013-06-18

Sensitive and reliable analysis of sugars and sugar phosphates in tissues and cells is essential for many biological and cell engineering studies. However, the successful analysis of these endogenous compounds in biological samples by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is often difficult because of their poor chromatographic retention properties in reversed-phase LC, the complex biological matrices, and the ionization suppression in ESI. This situation is further complicated by the existence of their multiple structural isomers in vivo. This work describes the combination of reductive amination using 3-amino-9-ethylcarbazole, with a new LC approach using a pentafluorophenyl core-shell ultrahigh performance (UP) LC column and methylphosphonic acid as an efficient tail-sweeping reagent for improved chromatographic separation. This new method was used for selected detection and accurate quantitation of the major free and phosphorylated reducing sugars in mouse heart tissue. Among the detected compounds, accurate quantitation of glyceraldehyde, ribose, glucose, glycerylaldehyde-3-phosphate, ribose-5-phosphate, glucose-6-phosphate, and mannose-6-phosphate was achieved by UPLC/multiple-reaction monitoring (MRM)-MS, with analytical accuracies ranging from 87.4% to 109.4% and CVs of ≤8.5% (n = 6). To demonstrate isotope-resolved metabolic profiling, we used UPLC/quadrupole time-of-flight (QTOF)-MS to analyze the isotope distribution patterns of C3 to C6 free and phosphorylated reducing sugars in heart tissues from (13)C-labeled wild type and knockout mice. In conclusion, the preanalytical derivatization-LC/ESI-MS method has resulted in selective determination of free and phosphorylated reducing sugars without the interferences from their nonreducing structural isomers in mouse heart tissue, with analytical sensitivities in the femtomole to low picomole range.

Simultaneous Quantitation of Advanced Glycation End Products in Soy Sauce and Beer by Liquid Chromatography-Tandem Mass Spectrometry without Ion-Pair Reagents and Derivatization.

PubMed

Nomi, Yuri; Annaka, Hironori; Sato, Shinji; Ueta, Etsuko; Ohkura, Tsuyoshi; Yamamoto, Kazuhiro; Homma, Seiichi; Suzuki, Emiko; Otsuka, Yuzuru

2016-11-09

The aim of this study was to develop a simple and sensitive method to analyze several advanced glycation end products (AGEs) simultaneously using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and to apply this method to the quantitation of AGEs in brown-colored foods. The developed method enabled to separate and quantitate simultaneously seven AGEs, and was applied to the determination of free AGEs contained in various kinds of soy sauce and beer. The major AGEs in soy sauce and beer were N ε -carboxymethyllysine (CML), N ε -carboxyethyllysine (CEL), and N δ -(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine (MG-H1). Using the developed LC-MS/MS method, recovery test on soy sauce and beer samples showed the recovery values of 85.3-103.9% for CML, 95.9-107.4% for CEL, and 69.5-123.2% for MG-H1. In particular, it is the first report that free CML, CEL, and MG-H1 were present in beer. Furthermore, long-term storage and heating process of soy sauce increased CML and MG-H1.

Extraction and Quantitation of Ketones and Aldehydes from Mammalian Cells Using Fluorous Tagging and Capillary LC-MS.

PubMed

Yuan, Wei; Li, Shuwei; Edwards, James L

2015-08-04

The extraction and quantitation of carbonyl metabolites from cell lysate was accomplished using a carbonyl-reactive fluorous tag and capillary liquid chromatography coupled to mass spectrometry (capLC-MS). Selective fluorous tagging for ketones and aldehydes provided a 30-fold increase in sensitivity using electrospray ionization MS. Separation of fluorous tagged carbonyl resulted in good separation of all components, and tandem MS was able to differentiate structural carbonyl isomers. The average limit of detection for carbonyl standards was 37 nM (range 1.5-250 nM), with linearity of R(2) > 0.99. Reproducibility for metabolites in cell lysate averaged 9% RSD. Human aortic endothelial cells (HAECs) were exposed to varying levels of glucose, and their carbonyl metabolite levels were quantified. Significant metabolite changes were seen in glycolysis and the propanoate pathway from a glucose challenge. Using an untargeted approach, 120 carbonyl metabolites were found to change in hyperglycemic HAECs. From this list of compounds, multiple metabolites from the pentose phosphate and tryptophan metabolic pathways were discovered. This system provides excellent sensitivity and quantitation of carbonyl metabolites without the need for isotope standards or labels.

Absolute quantitation of NAPQI-modified rat serum albumin by LC-MS/MS: monitoring acetaminophen covalent binding in vivo.

PubMed

LeBlanc, André; Shiao, Tze Chieh; Roy, René; Sleno, Lekha

2014-09-15

Acetaminophen is known to cause hepatoxicity via the formation of a reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), as a result of covalent binding to liver proteins. Serum albumin (SA) is known to be covalently modified by NAPQI and is present at high concentrations in the bloodstream and is therefore a potential biomarker to assess the levels of protein modification by NAPQI. A newly developed method for the absolute quantitation of serum albumin containing NAPQI covalently bound to its active site cysteine (Cys34) is described. This optimized assay represents the first absolute quantitation of a modified protein, with very low stoichiometric abundance, using a protein-level standard combined with isotope dilution. The LC-MS/MS assay is based on a protein standard modified with a custom-designed reagent, yielding a surrogate peptide (following digestion) that is a positional isomer to the target peptide modified by NAPQI. To illustrate the potential of this approach, the method was applied to quantify NAPQI-modified SA in plasma from rats dosed with acetaminophen. The resulting method is highly sensitive (capable of quantifying down to 0.0006% of total RSA in its NAPQI-modified form) and yields excellent precision and accuracy statistics. A time-course pharmacokinetic study was performed to test the usefulness of this method for following acetaminophen-induced covalent binding at four dosing levels (75-600 mg/kg IP), showing the viability of this approach to directly monitor in vivo samples. This approach can reliably quantify NAPQI-modified albumin, allowing direct monitoring of acetaminophen-related covalent binding.

Silica-based ionic liquid coating for 96-blade system for extraction of aminoacids from complex matrixes.

PubMed

Mousavi, Fatemeh; Pawliszyn, Janusz

2013-11-25

1-Vinyl-3-octadecylimidazolium bromide ionic liquid [C18VIm]Br was prepared and used for the modification of mercaptopropyl-functionalized silica (Si-MPS) through surface radical chain-transfer addition. The synthesized octadecylimidazolium-modified silica (SiImC18) was characterized by thermogravimetric analysis (TGA), infrared spectroscopy (IR), (13)C NMR and (29)Si NMR spectroscopy and used as an extraction phase for the automated 96-blade solid phase microextraction (SPME) system with thin-film geometry using polyacrylonitrile (PAN) glue. The new proposed extraction phase was applied for extraction of aminoacids from grape pulp, and LC-MS-MS method was developed for separation of model compounds. Extraction efficiency, reusability, linearity, limit of detection, limit of quantitation and matrix effect were evaluated. The whole process of sample preparation for the proposed method requires 270min for 96 samples simultaneously (60min preconditioning, 90min extraction, 60min desorption and 60min for carryover step) using 96-blade SPME system. Inter-blade and intra-blade reproducibility were in the respective ranges of 5-13 and 3-10% relative standard deviation (RSD) for all model compounds. Limits of detection and quantitation of the proposed SPME-LC-MS/MS system for analysis of analytes were found to range from 0.1 to 1.0 and 0.5 to 3.0μgL(-1), respectively. Standard addition calibration was applied for quantitative analysis of aminoacids from grape juice and the results were validated with solvent extraction (SE) technique. Copyright © 2013 Elsevier B.V. All rights reserved.

Multiple reaction monitoring and multiple reaction monitoring cubed based assays for the quantitation of apolipoprotein F.

PubMed

Kumar, Abhinav; Gangadharan, Bevin; Zitzmann, Nicole

2016-10-15

Apolipoprotein F (APO-F) is a novel low abundance liver fibrosis biomarker and its concentration decreases in human serum and plasma across liver fibrosis stages. Current antibody based assays for APO-F suffer from limitations such as unspecific binding, antibody availability and undetectable target if the protein is degraded; and so an antibody-free assay has the potential to be a valuable diagnostic tool. We report an antibody-free, rapid, sensitive, selective and robust LC-MS/MS (MRM and MRM(3)) method for the detection and quantitation of APO-F in healthy human plasma. With further analysis of clinical samples, this LC-MS based method could be established as the first ever antibody-free biomarker assay for liver fibrosis. We explain the use of Skyline software for peptide selection and the creation of a reference library to aid in true peak identification of endogenous APO-F peptides in digests of human plasma without protein or peptide enrichment. Detection of a glycopeptide using MRM-EPI mode and reduction of interferences using MRM3 are explained. The amount of APO-F in human plasma from a healthy volunteer was determined to be 445.2ng/mL, the coefficient of variation (CV) of precision for 20 injections was <12% and the percentage error of each point along the calibration curve was calculated to be <8%, which is in line with the assay requirements for clinical samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantification of proteins in urine samples using targeted mass spectrometry methods.

PubMed

Khristenko, Nina; Domon, Bruno

2015-01-01

Numerous clinical proteomics studies are focused on the development of biomarkers to improve either diagnostics for early disease detection or the monitoring of the response to the treatment. Although, a wealth of biomarker candidates are available, their evaluation and validation in a true clinical setup remains challenging. In biomarkers evaluation studies, a panel of proteins of interest are systematically analyzed in a large cohort of samples. However, in spite of the latest progresses in mass spectrometry, the consistent detection of pertinent proteins in high complex biological samples is still a challenging task. Thus, targeted LC-MS/MS methods are better suited for the systematic analysis of biomarkers rather than shotgun approaches. This chapter describes the workflow used to perform targeted quantitative analyses of proteins in urinary samples. The peptides, as surrogates of the protein of interest, are commonly measured using a triple quadrupole mass spectrometers operated in selected reaction monitoring (SRM) mode. More recently, the advances in targeted LC-MS/MS analysis based on parallel reaction monitoring (PRM) performed on a quadrupole-orbitrap instrument have allowed to increase the specificity and selectivity of the measurements.

Comparison of Gluten Extraction Protocols Assessed by LC-MS/MS Analysis.

PubMed

Fallahbaghery, Azadeh; Zou, Wei; Byrne, Keren; Howitt, Crispin A; Colgrave, Michelle L

2017-04-05

The efficiency of gluten extraction is of critical importance to the results derived from any analytical method for gluten detection and quantitation, whether it employs reagent-based technology (antibodies) or analytical instrumentation (mass spectrometry). If the target proteins are not efficiently extracted, the end result will be an under-estimation in the gluten content posing a health risk to people affected by conditions such as celiac disease (CD) and nonceliac gluten sensitivity (NCGS). Five different extraction protocols were investigated using LC-MRM-MS for their ability to efficiently and reproducibly extract gluten. The rapid and simple "IPA/DTT" protocol and related "two-step" protocol were enriched for gluten proteins, 55/86% (trypsin/chymotrypsin) and 41/68% of all protein identifications, respectively, with both methods showing high reproducibility (CV < 15%). When using multistep protocols, it was critical to examine all fractions, as coextraction of proteins occurred across fractions, with significant levels of proteins existing in unexpected fractions and not all proteins within a particular gluten class behaving the same.

Quantitative measurement of betaine and free choline in plasma, cereals and cereal products by isotope dilution LC-MS/MS.

PubMed

Bruce, Stephen J; Guy, Philippe A; Rezzi, Serge; Ross, Alastair B

2010-02-24

Betaine and choline are important components of the one-carbon metabolism cycle, linked with the amino acid homocysteine and lipid metabolism. Analyses of broad ranges of foods point to cereal based foods being important sources of betaine and choline, however to date there has been no detailed analysis of these compounds in cereal flours or cereal products. An analytical method based on optimization of an existing extraction followed by LC-MS/MS analysis was used to analyze 47 plasma samples, 32 cereal flours and cereal fractions, and 51 cereal products. For the method validation LLOQ, recovery, inter- and intraday repeatability were all performed. Whole-grain wheat and rye flours, and products based on these were the best whole cereal sources of betaine (747-1508 microg/g) and to a lesser extent choline (76-159 microg/g), while the bran fraction contained the highest concentrations of betaine and free-choline (2350-2899 microg/g and 366-384 microg/g respectively). Refined wheat flour and products contained lower concentrations, while rice and maize contained only very low and no detectable amounts of betaine respectively (0-10 microg/g), and low amounts of free-choline (<31 microg/g). These results were mirrored in cereal products analyzed, with whole-grain wheat or rye-based cereal products having the highest concentrations of the two metabolites. Plasma concentrations for betaine and free-choline in a group of 47 subjects ranged from 15.2-66.3 and 9.8-18.5 micromol/L respectively, within the range of previous reports. This LC-MS/MS method can be used to rapidly and sensitively quantify betaine and free-choline in plasma and cereal products. Whole-grain cereal products and products containing cereal bran appear to be excellent dietary sources of betaine and free-choline.

A Guideline to Univariate Statistical Analysis for LC/MS-Based Untargeted Metabolomics-Derived Data

PubMed Central

Vinaixa, Maria; Samino, Sara; Saez, Isabel; Duran, Jordi; Guinovart, Joan J.; Yanes, Oscar

2012-01-01

Several metabolomic software programs provide methods for peak picking, retention time alignment and quantification of metabolite features in LC/MS-based metabolomics. Statistical analysis, however, is needed in order to discover those features significantly altered between samples. By comparing the retention time and MS/MS data of a model compound to that from the altered feature of interest in the research sample, metabolites can be then unequivocally identified. This paper reports on a comprehensive overview of a workflow for statistical analysis to rank relevant metabolite features that will be selected for further MS/MS experiments. We focus on univariate data analysis applied in parallel on all detected features. Characteristics and challenges of this analysis are discussed and illustrated using four different real LC/MS untargeted metabolomic datasets. We demonstrate the influence of considering or violating mathematical assumptions on which univariate statistical test rely, using high-dimensional LC/MS datasets. Issues in data analysis such as determination of sample size, analytical variation, assumption of normality and homocedasticity, or correction for multiple testing are discussed and illustrated in the context of our four untargeted LC/MS working examples. PMID:24957762

A Guideline to Univariate Statistical Analysis for LC/MS-Based Untargeted Metabolomics-Derived Data.

PubMed

Vinaixa, Maria; Samino, Sara; Saez, Isabel; Duran, Jordi; Guinovart, Joan J; Yanes, Oscar

2012-10-18

Several metabolomic software programs provide methods for peak picking, retention time alignment and quantification of metabolite features in LC/MS-based metabolomics. Statistical analysis, however, is needed in order to discover those features significantly altered between samples. By comparing the retention time and MS/MS data of a model compound to that from the altered feature of interest in the research sample, metabolites can be then unequivocally identified. This paper reports on a comprehensive overview of a workflow for statistical analysis to rank relevant metabolite features that will be selected for further MS/MS experiments. We focus on univariate data analysis applied in parallel on all detected features. Characteristics and challenges of this analysis are discussed and illustrated using four different real LC/MS untargeted metabolomic datasets. We demonstrate the influence of considering or violating mathematical assumptions on which univariate statistical test rely, using high-dimensional LC/MS datasets. Issues in data analysis such as determination of sample size, analytical variation, assumption of normality and homocedasticity, or correction for multiple testing are discussed and illustrated in the context of our four untargeted LC/MS working examples.

A novel approach for quantitation of glucosylceramide in human dried blood spot using LC-MS/MS.

PubMed

Ji, Allena Ji; Wang, Haixing; Ziso-Qejvanaj, Enida; Zheng, Kefei; Chung, Lee Lee; Foley, Timothy; Chuang, Wei-Lien; Richards, Susan; Sung, Crystal

2015-01-01

Glucosylceramide, an efficacy biomarker for Gaucher Type 1 disease, exhibits poor solubility in polar solvents and whole blood which makes it difficult to prepare a homogenous blood standard. We developed a novel method using standard addition approach by spiking a small volume of analyte solution on the surface of prespotted dried blood spot. The whole spots were punched out for subsequent extraction and LC-MS/MS analysis. The assay performance met all validation acceptance criteria. Glucosylceramide concentrations in 50 paired plasma and dry blood spot samples obtained from Gaucher Type 1 patients were tested and the results demonstrated the feasibility of using the DBS method for clinical biomarker monitoring. The new approach greatly improves assay precision and accuracy.

Quantification of CSF cystatin C using liquid chromatography tandem mass spectrometry.

PubMed

Matsuda, Chikashi; Shiota, Yuri; Sheikh, Abdullah Md; Okazaki, Ryota; Yamada, Kazuo; Yano, Shozo; Minohata, Toshikazu; Matsumoto, Ken-Ichi; Yamaguchi, Shuhei; Nagai, Atsushi

2018-03-01

Cystatin C (CST3), a ubiquitously expressed cysteine protease inhibitor, is implicated in several neurological diseases. Here, we have developed an accurate CST3 measurement system based on liquid chromatography tandem mass spectrometry (LC-MS/MS). LC-MS/MS based measurement for CSF CST3 was validated by determination of assay precision, accuracy and recovery. The values were compared with those measured by immunoassay. Glycosylation of CST3 in CSF was analyzed by Western blotting and lectin blotting. Measuring standard CST3 by LC-MS/MS produced a linear standard curve that correlated with assigned values (r 2 =0.99). Both intra- and inter-assay variation was <10%. Although showed a correlation, the average CST3 concentration measured by LC-MS/MS was significantly higher than that of immunoassay. Western blotting showed the presence of a 25KDa species along with CST3 monomer (14KDa) in CSF. The volume of 25KDa species was decreased by deglycosylation. Lectin blotting revealed a 25KDa glycosylated protein in sialidase-treated CSF, which was decreased by deglycosylation. However, deglycosylation did not alter CST3 concentration measured by immunoassay. Our results suggest that LC-MS/MS-based CST3 measurement is a robust method with higher detection ability. Such method could be useful for the diagnosis and monitoring of neurological diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterizing fluorotelomer and polyfluoroalkyl substances in new and aged fluorotelomer-based polymers for degradation studies with GC/MS and LC/MS/MS.

PubMed

Washington, John W; Naile, Jonathan E; Jenkins, Thomas M; Lynch, David G

2014-05-20

Fluorotelomer-based polymers (FTPs), the dominant product of the fluorotelomer industry, are antistaining and antiwetting agents that permeate the products and surfaces of modern society. However, the degree to which these materials expose humans and the environment to fluorotelomer and perfluorinated compounds, including recalcitrant and toxic compounds such as perfluorooctanoic acid (PFOA), is ill-defined. The design intent of FTPs, to minimize interaction with other substances, including solvents, heretofore has stymied efforts to develop robust methods to characterize the content of monomers and associated compounds of new commercial FTPs, as well as commercial FTPs that have been aged in environmental media for degradation testing. Here we show that FTPs can be exhausted of these compounds and quantitated by (i) drying the FTP on a suitable substrate at elevated temperature to achieve low, constant monomer concentrations; (ii) serial extraction with MTBE for fluorotelomer-monomer analysis by GC/MS in PCI mode; followed by (iii) serial extraction with 90/10 ACN/H2O for polyfluorocompound analysis by LC/MS/MS in negative ESI mode. This approach yields exhaustive, internally consistent accounting of monomers and associated compounds for FTPs, either alone or in a soil matrix (representing an environmental medium), for both new and simulated-aged FTPs to allow degradation testing, and for fluorinated compounds at least as long as C12.

Quantitative profiling of oxylipins through comprehensive lc-ms/ms analysis: Application in cardiac surgery

USDA-ARS?s Scientific Manuscript database

Oxylipins, including eicosanoids, affect a broad range of biological processes, such as the initiation and resolution of inflammation. These compounds, also referred to as lipid mediators, are (non-) enzymatically generated by oxidation of polyunsaturated fatty acids such as arachidonic acid (AA). A...

78 FR 11126 - Receipt of Several Pesticide Petitions Filed for Residues of Pesticide Chemicals in or on Various...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-15

... using capillary gas liquid chromatography (GLC) with flame ionization detector (FID). Contact: Andrew...) purification. Detection and quantitation are conducted by gas chromatograph equipped with nitrogen phosphorus... pressure liquid chromatography/triple stage quadrupole mass spectrometry (LC/MS/MS) using the stable...

Simultaneous Proteomic Discovery and Targeted Monitoring using Liquid Chromatography, Ion Mobility Spectrometry, and Mass Spectrometry*

PubMed Central

Burnum-Johnson, Kristin E.; Nie, Song; Casey, Cameron P.; Monroe, Matthew E.; Orton, Daniel J.; Ibrahim, Yehia M.; Gritsenko, Marina A.; Clauss, Therese R. W.; Shukla, Anil K.; Moore, Ronald J.; Purvine, Samuel O.; Shi, Tujin; Qian, Weijun; Liu, Tao; Baker, Erin S.; Smith, Richard D.

2016-01-01

Current proteomic approaches include both broad discovery measurements and quantitative targeted analyses. In many cases, discovery measurements are initially used to identify potentially important proteins (e.g. candidate biomarkers) and then targeted studies are employed to quantify a limited number of selected proteins. Both approaches, however, suffer from limitations. Discovery measurements aim to sample the whole proteome but have lower sensitivity, accuracy, and quantitation precision than targeted approaches, whereas targeted measurements are significantly more sensitive but only sample a limited portion of the proteome. Herein, we describe a new approach that performs both discovery and targeted monitoring (DTM) in a single analysis by combining liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS). In DTM, heavy labeled target peptides are spiked into tryptic digests and both the labeled and unlabeled peptides are detected using LC-IMS-MS instrumentation. Compared with the broad LC-MS discovery measurements, DTM yields greater peptide/protein coverage and detects lower abundance species. DTM also achieved detection limits similar to selected reaction monitoring (SRM) indicating its potential for combined high quality discovery and targeted analyses, which is a significant step toward the convergence of discovery and targeted approaches. PMID:27670688

Validation of a LC/MS method for the determination of gemfibrozil in human plasma and its application to a pharmacokinetic study

PubMed Central

Rower, Joseph E.; Bushman, Lane R.; Hammond, Kyle P.; Kadam, Rajendra S.; Aquilante, Christina L.

2011-01-01

Gemfibrozil, a fibric acid hypolipidemic agent, is increasingly being used in clinical drug-drug interaction studies as an inhibitor of drug metabolizing enzymes and drug transporters. The validation of a fast, accurate, and precise LC/MS method is described for the quantitative determination of gemfibrozil in an EDTA-anticoagulated human plasma matrix. Briefly, gemfibrozil was extracted from human plasma by an acetonitrile protein precipitation method. The assay was reproducible with intra-assay precision between 1.6% and 10.7%, and inter-assay precision ranging from 4.4% to 7.8%. The assay also showed good accuracy, with intra-assay concentrations within 85.6% and 108.7% of the expected value, and inter-assay concentrations within 89.4 to 104.0% of the expected value. The linear concentration range was between 0.5 and 50 μg/mL with a lower limit of quantitation of 0.5 μg/mL when 125 μL of plasma were extracted. This LC/MS method yielded a quick, simple, and reliable protocol for determining gemfibrozil concentrations in plasma and is applicable to clinical pharmacokinetic studies. PMID:21077249

Validation of an LC/MS method for the determination of gemfibrozil in human plasma and its application to a pharmacokinetic study.

PubMed

Rower, Joseph E; Bushman, Lane R; Hammond, Kyle P; Kadam, Rajendra S; Aquilante, Christina L

2010-12-01

Gemfibrozil, a fibric acid hypolipidemic agent, is increasingly being used in clinical drug-drug interaction studies as an inhibitor of drug metabolizing enzymes and drug transporters. The validation of a fast, accurate and precise LC/MS method is described for the quantitative determination of gemfibrozil in an EDTA-anticoagulated human plasma matrix. Briefly, gemfibrozil was extracted from human plasma by an acetonitrile protein precipitation method. The assay was reproducible with intra-assay precision between 1.6 and 10.7%, and inter-assay precision ranging from 4.4 to 7.8%. The assay also showed good accuracy, with intra-assay concentrations within 85.6-108.7% of the expected value, and inter-assay concentrations within 89.4-104.0% of the expected value. The linear concentration range was between 0.5 and 50 µg/mL with a lower limit of quantitation of 0.5 µg/mL when 125 µL of plasma were extracted. This LC/MS method yielded a quick, simple and reliable protocol for determining gemfibrozil concentrations in plasma and is applicable to clinical pharmacokinetic studies. Copyright © 2010 John Wiley & Sons, Ltd.

Method development aspects for the quantitation of pharmaceutical compounds in human plasma with a matrix-assisted laser desorption/ionization source in the multiple reaction monitoring mode.

PubMed

Kovarik, Peter; Grivet, Chantal; Bourgogne, Emmanuel; Hopfgartner, Gérard

2007-01-01

The present work investigates various method development aspects for the quantitative analysis of pharmaceutical compounds in human plasma using matrix-assisted laser desorption/ionization and multiple reaction monitoring (MALDI-MRM). Talinolol was selected as a model analyte. Liquid-liquid extraction (LLE) and protein precipitation were evaluated regarding sensitivity and throughput for the MALDI-MRM technique and its applicability without and with chromatographic separation. Compared to classical electrospray liquid chromatography/mass spectrometry (LC/ESI-MS) method development, with MALDI-MRM the tuning of the analyte in single MS mode is more challenging due to interfering matrix background ions. An approach is proposed using background subtraction. With LLE and using a 200 microL human plasma aliquot acceptable precision and accuracy could be obtained in the range of 1 to 1000 ng/mL without any LC separation. Approximately 3 s were required for one analysis. A full calibration curve and its quality control samples (20 samples) can be analyzed within 1 min. Combining LC with the MALDI analysis allowed improving the linearity down to 50 pg/mL, while reducing the throughput potential only by two-fold. Matrix effects are still a significant issue with MALDI but can be monitored in a similar way to that used for LC/ESI-MS analysis.

On the Reproducibility of Label-Free Quantitative Cross-Linking/Mass Spectrometry

NASA Astrophysics Data System (ADS)

Müller, Fränze; Fischer, Lutz; Chen, Zhuo Angel; Auchynnikava, Tania; Rappsilber, Juri

2018-02-01

Quantitative cross-linking/mass spectrometry (QCLMS) is an emerging approach to study conformational changes of proteins and multi-subunit complexes. Distinguishing protein conformations requires reproducibly identifying and quantifying cross-linked peptides. Here we analyzed the variation between multiple cross-linking reactions using bis[sulfosuccinimidyl] suberate (BS3)-cross-linked human serum albumin (HSA) and evaluated how reproducible cross-linked peptides can be identified and quantified by LC-MS analysis. To make QCLMS accessible to a broader research community, we developed a workflow that integrates the established software tools MaxQuant for spectra preprocessing, Xi for cross-linked peptide identification, and finally Skyline for quantification (MS1 filtering). Out of the 221 unique residue pairs identified in our sample, 124 were subsequently quantified across 10 analyses with coefficient of variation (CV) values of 14% (injection replica) and 32% (reaction replica). Thus our results demonstrate that the reproducibility of QCLMS is in line with the reproducibility of general quantitative proteomics and we establish a robust workflow for MS1-based quantitation of cross-linked peptides.

Analysis of acrylamide by LC-MS/MS and GC-MS in processed Japanese foods.

PubMed

Ono, H; Chuda, Y; Ohnishi-Kameyama, M; Yada, H; Ishizaka, M; Kobayashi, H; Yoshida, M

2003-03-01

Acrylamide concentrations in processed foods (63 samples covering 31 product types) from Japan were analysed by LC-MS/MS and GC-MS methods. The limit of detection and limit of quantification of acrylamide were 0.2 ng x ml(-1) (6 fmol) and 0.8 ng x ml(-1) (22 fmol), respectively, by LC-MS/MS, and those of 2,3-dibromopropionamide derived from acrylamide were 12 ng x ml(-1) (52 fmol) and 40 ng x ml(-1) (170 fmol), respectively, by GC-MS. Repeatability given as RSD was <5 and <15% for the LC-MS/MS and GC-MS methods, respectively. High correlation (r(2) - 0.946) was observed between values obtained by the two methods. Most potato crisps and whole potato-based fried snacks showed acrylamide concentrations >1000 microg x kg(-1). The concentrations in non-whole potato-based snacks, rice crackers processed by grilling or frying, and candied sweet potatoes were lower compared with those in the potato crisps and the whole potato-based fried snacks. One of the whole potato-based fried snacks, however, showed low acrylamide concentration (<50 microg x kg(-1)) suggesting the formation of acrylamide is strongly influenced by processing conditions. Acrylamide concentrations in instant precooked noodles and won-tons were <100 microg x kg(-1) with only one exception. Roasted barley grains for 'Mugi-cha' tea contained 200-600 microg x kg(-1) acrylamide.

Targeted methods for quantitative analysis of protein glycosylation

PubMed Central

Goldman, Radoslav; Sanda, Miloslav

2018-01-01

Quantification of proteins by LC-MS/MS-MRM has become a standard method with broad projected clinical applicability. MRM quantification of protein modifications is, however, far less utilized, especially in the case of glycoproteins. This review summarizes current methods for quantitative analysis of protein glycosylation with a focus on MRM methods. We describe advantages of this quantitative approach, analytical parameters that need to be optimized to achieve reliable measurements, and point out the limitations. Differences between major classes of N- and O-glycopeptides are described and class-specific glycopeptide assays are demonstrated. PMID:25522218

Effect of chlorination by-products on the quantitation of microcystins in finished drinking water.

PubMed

Rosenblum, Laura; Zaffiro, Alan; Adams, William A; Wendelken, Steven C

2017-11-01

Microcystins are toxic peptides that can be produced by cyanobacteria in harmful algal blooms (HABs). Various analytical techniques have been developed to quantify microcystins in drinking water, including liquid chromatography tandem mass spectrometry (LC/MS/MS), enzyme linked immunosorbent assay (ELISA), and oxidative cleavage to produce 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) with detection by LC/MS/MS, the "MMPB method". Both the ELISA and MMPB methods quantify microcystins by detecting a portion of the molecule common to most microcystins. However, there is little research evaluating the effect of microcystin chlorination by-products potentially produced during drinking water treatment on analytical results. To evaluate this potential, chlorinated drinking water samples were fortified with various microcystin congeners in bench-scale studies. The samples were allowed to react, followed by a comparison of microcystin concentrations measured using the three methods. The congener-specific LC/MS/MS method selectively quantified microcystins and was not affected by the presence of chlorination by-products. The ELISA results were similar to those obtained by LC/MS/MS for most microcystin congeners, but results deviated for a particular microcystin containing a variable amino acid susceptible to oxidation. The concentrations measured by the MMPB method were at least five-fold higher than the concentrations of microcystin measured by the other methods and demonstrate that detection of MMPB does not necessarily correlate to intact microcystin toxins in finished drinking water. Published by Elsevier Ltd.

LC-MSsim – a simulation software for liquid chromatography mass spectrometry data

PubMed Central

Schulz-Trieglaff, Ole; Pfeifer, Nico; Gröpl, Clemens; Kohlbacher, Oliver; Reinert, Knut

2008-01-01

Background Mass Spectrometry coupled to Liquid Chromatography (LC-MS) is commonly used to analyze the protein content of biological samples in large scale studies. The data resulting from an LC-MS experiment is huge, highly complex and noisy. Accordingly, it has sparked new developments in Bioinformatics, especially in the fields of algorithm development, statistics and software engineering. In a quantitative label-free mass spectrometry experiment, crucial steps are the detection of peptide features in the mass spectra and the alignment of samples by correcting for shifts in retention time. At the moment, it is difficult to compare the plethora of algorithms for these tasks. So far, curated benchmark data exists only for peptide identification algorithms but no data that represents a ground truth for the evaluation of feature detection, alignment and filtering algorithms. Results We present LC-MSsim, a simulation software for LC-ESI-MS experiments. It simulates ESI spectra on the MS level. It reads a list of proteins from a FASTA file and digests the protein mixture using a user-defined enzyme. The software creates an LC-MS data set using a predictor for the retention time of the peptides and a model for peak shapes and elution profiles of the mass spectral peaks. Our software also offers the possibility to add contaminants, to change the background noise level and includes a model for the detectability of peptides in mass spectra. After the simulation, LC-MSsim writes the simulated data to mzData, a public XML format. The software also stores the positions (monoisotopic m/z and retention time) and ion counts of the simulated ions in separate files. Conclusion LC-MSsim generates simulated LC-MS data sets and incorporates models for peak shapes and contaminations. Algorithm developers can match the results of feature detection and alignment algorithms against the simulated ion lists and meaningful error rates can be computed. We anticipate that LC-MSsim will be useful to the wider community to perform benchmark studies and comparisons between computational tools. PMID:18842122

ICPD-A New Peak Detection Algorithm for LC/MS

PubMed Central

2010-01-01

Background The identification and quantification of proteins using label-free Liquid Chromatography/Mass Spectrometry (LC/MS) play crucial roles in biological and biomedical research. Increasing evidence has shown that biomarkers are often low abundance proteins. However, LC/MS systems are subject to considerable noise and sample variability, whose statistical characteristics are still elusive, making computational identification of low abundance proteins extremely challenging. As a result, the inability of identifying low abundance proteins in a proteomic study is the main bottleneck in protein biomarker discovery. Results In this paper, we propose a new peak detection method called Information Combining Peak Detection (ICPD ) for high resolution LC/MS. In LC/MS, peptides elute during a certain time period and as a result, peptide isotope patterns are registered in multiple MS scans. The key feature of the new algorithm is that the observed isotope patterns registered in multiple scans are combined together for estimating the likelihood of the peptide existence. An isotope pattern matching score based on the likelihood probability is provided and utilized for peak detection. Conclusions The performance of the new algorithm is evaluated based on protein standards with 48 known proteins. The evaluation shows better peak detection accuracy for low abundance proteins than other LC/MS peak detection methods. PMID:21143790

Spectroscopic characterization and quantitative determination of atorvastatin calcium impurities by novel HPLC method

NASA Astrophysics Data System (ADS)

Gupta, Lokesh Kumar

2012-11-01

Seven process related impurities were identified by LC-MS in the atorvastatin calcium drug substance. These impurities were identified by LC-MS. The structure of impurities was confirmed by modern spectroscopic techniques like 1H NMR and IR and physicochemical studies conducted by using synthesized authentic reference compounds. The synthesized reference samples of the impurity compounds were used for the quantitative HPLC determination. These impurities were detected by newly developed gradient, reverse phase high performance liquid chromatographic (HPLC) method. The system suitability of HPLC analysis established the validity of the separation. The analytical method was validated according to International Conference of Harmonization (ICH) with respect to specificity, precision, accuracy, linearity, robustness and stability of analytical solutions to demonstrate the power of newly developed HPLC method.

Quantitation of acrylamide in food products by liquid chromatography/mass spectrometry.

PubMed

Eberhart, B Loye; Ewald, Deborah K; Sanders, Robert A; Tallmadge, Daniel H; Zyzak, David V; Strothers, Melissa A

2005-01-01

A simple and inexpensive liquid chromatography/mass spectrometry (LC/MS) method was developed for the quantitation of acrylamide in various food products. The method involved spiking the isotope-substituted internal standard (1-C13 acrylamide) onto 6.00 g of the food product, adding 40 mL distilled/deionized water, and heating at 65 degrees C for 30 min. Afterwards, 10 mL ethylene dichloride was added and the mixture was homogenized for 30 s and centrifuged at 2700 x g for 30 min, and then 8 g supernatant was extracted with 10, 5, and 5 mL portions of ethyl acetate. The extracts were combined, dried with sodium sulfate, and concentrated to 100-200 microL. Acrylamide was determined by analysis of the final extract on a single quadrupole, bench-top mass spectrometer with electrospray ionization, using a 2 mm id C18 column and monitoring m/z = 72 (acrylamide) and m/z = 73 (internal standard). For difficult food matrixes, such as coffee and cocoa, a solid-phase extraction cleanup step was incorporated to improve both chromatography and column lifetime. The method had a limit of quantitation of 10 ppb, and coefficients of determination (r2) for calibration curves were typically better than 0.998. Acceptable spike recovery results were achieved in 11 different food matrixes. Precision in potato chip analyses was 5-8% (relative standard deviation). This method provides an LC/MS alternative to the current LC/MS/MS methods and derivatization gas chromatography/mass spectrometry methods, and is applicable to difficult food products such as coffee, cocoa, and high-salt foods.

High-Throughput Quantitation of Neonicotinoids in Lyophilized Surface Water by LC-APCI-MS/MS.

PubMed

Morrison, Lucas M; Renaud, Justin B; Sabourin, Lyne; Sumarah, Mark W; Yeung, Ken K C; Lapen, David R

2018-05-21

Background : Neonicotinoids are among the most widely used insecticides. Recently, there has been concern associated with unintended adverse effects on honeybees and aquatic invertebrates at low parts-per-trillion levels. Objective : There is a need for LC-MS/MS methods that are capable of high-throughput measurements of the most widely used neonicotinoids at environmentally relevant concentrations in surface water. Methods : This method allows for quantitation of acetamiprid, clothianidin, imidacloprid, dinotefuran, nitenpyram, thiacloprid, and thiamethoxam in surface water. Deuterated internal standards are added to 20 mL environmental samples, which are concentrated by lyophilisation and reconstituted with methanol followed by acetonitrile. Results : A large variation of mean recovery efficiencies across five different surface water sampling sites within this study was observed, ranging from 45 to 74%. This demonstrated the need for labelled internal standards to compensate for these differences. Atmospheric pressure chemical ionization (APCI) performed better than electrospray ionization (ESI) with limited matrix suppression, achieving 71-110% of the laboratory fortified blank signal. Neonicotinoids were resolved on a C18 column using a 5 min LC method, in which MQL ranged between 0.93 and 4.88 ng/L. Conclusions : This method enables cost effective, accurate, and reproducible monitoring of these pesticides in the aquatic environment. Highlights : Lyophilization is used for high throughput concentration of neonicotinoids in surface water. Variations in matrix effects between samples was greatly reduced by using APCI compared with ESI. Clothianidin and thiamethoxam were detected in all samples with levels ranging from below method quantitation limit to 65 ng/L.

Comparative chromatography-mass spectrometry studies on the antiretroviral drug nevirapine-analytical performance characteristics in human plasma determination.

PubMed

Sichilongo, Kwenga; Chinyama, Mompati; Massele, Amos; Vento, Sandro

2014-01-15

A contrast between the analytical performance characteristics using gas chromatography-mass spectrometry (GC-MS) liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-ultraviolet (LC-UV) detection for the determination of the antiretroviral drug (ARV) nevirapine (NVP) in fortified human plasma after QuEChERS extraction has been made. Analytical performance characteristics, i.e. linearities, instrument detection limits (IDLs), limits of quantitation (LOQs), method detection limits (MDLs), % mean recoveries and the corresponding relative standard deviations (%RSDs) were estimated using techniques above. Using GC-MS, the correlation coefficients (r(2)) were ≥0.990, which were deemed acceptable linearities. The MDLs ranged between 11.1-29.8μg/L and 13.7-36.0μg/L using helium and hydrogen carrier gases respectively. The LOQs ranged between 16.5-66.7μg/L and 28.4-98.7μg/L using helium and hydrogen carrier gases respectively with a % mean recovery of 83% and %RSD of 4.6%. Using LC-MS and LC-UV, the correlation coefficients (r(2)) were ≥0.990. The MDLs were ranged between 3.14 and 47.1μg/L. The LOQs ranged between 2.85 and 90.0μg/L respectively. The MDLs using GC-MS, LC-MS and LC-UV were below the therapeutic range for NVP in human plasma is considered to be between 2300μg/L (Cmin) and 8000μg/L (Cmax). This study also demonstrated that helium can be substituted with hydrogen which is relatively cheaper and easily obtainable even by use of a generator. Copyright © 2013 Elsevier B.V. All rights reserved.

Development of an ESI-LC-MS-based assay for kinetic evaluation of Mycobacterium tuberculosis shikimate kinase activity and inhibition.

PubMed

Simithy, Johayra; Gill, Gobind; Wang, Yu; Goodwin, Douglas C; Calderón, Angela I

2015-02-17

A simple and reliable liquid chromatography-mass spectrometry (LC-MS) assay has been developed and validated for the kinetic characterization and evaluation of inhibitors of shikimate kinase from Mycobacterium tuberculosis (MtSK), a potential target for the development of novel antitubercular drugs. This assay is based on the direct determination of the reaction product shikimate-3-phosphate (S3P) using electrospray ionization (ESI) and a quadrupole time-of-flight (Q-TOF) detector. A comparative analysis of the kinetic parameters of MtSK obtained by the LC-MS assay with those obtained by a conventional UV-assay was performed. Kinetic parameters determined by LC-MS were in excellent agreement with those obtained from the UV assay, demonstrating the accuracy, and reliability of this method. The validated assay was successfully applied to the kinetic characterization of a known inhibitor of shikimate kinase; inhibition constants and mode of inhibition were accurately delineated with LC-MS.

Ultrasensitive Hybridization-Based ELISA Method for the Determination of Phosphorodiamidate Morpholino Oligonucleotides in Biological samples.

PubMed

Burki, Umar; Straub, Volker

2017-01-01

Determining the concentration of oligonucleotide in biological samples such as tissue lysate and serum is essential for determining the biodistribution and pharmacokinetic profile, respectively. ELISA-based assays have shown far greater sensitivities compared to other methods such as HPLC and LC/MS. Here, we describe a novel ultrasensitive hybridization-based ELISA method for quantitating morpholino oligonucleotides in mouse tissue lysate and serum samples. The assay has a linear detection range of 5-250 pM (R2 > 0.99).

LC-MS/MS Detection of Karlotoxins Reveals New Variants in Strains of the Marine Dinoflagellate Karlodinium veneficum from the Ebro Delta (NW Mediterranean).

PubMed

Krock, Bernd; Busch, Julia A; Tillmann, Urban; García-Camacho, Francisco; Sánchez-Mirón, Asterio; Gallardo-Rodríguez, Juan J; López-Rosales, Lorenzo; Andree, Karl B; Fernández-Tejedor, Margarita; Witt, Matthias; Cembella, Allan D; Place, Allen R

2017-12-18

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the detection and quantitation of karlotoxins in the selected reaction monitoring (SRM) mode. This novel method was based upon the analysis of purified karlotoxins (KcTx-1, KmTx-2, 44-oxo-KmTx-2, KmTx-5), one amphidinol (AM-18), and unpurified extracts of bulk cultures of the marine dinoflagellate Karlodinium veneficum strain CCMP2936 from Delaware (Eastern USA), which produces KmTx-1 and KmTx-3. The limit of detection of the SRM method for KmTx-2 was determined as 2.5 ng on-column. Collision induced dissociation (CID) spectra of all putative karlotoxins were recorded to present fragmentation patterns of each compound for their unambiguous identification. Bulk cultures of K. veneficum strain K10 isolated from an embayment of the Ebro Delta, NW Mediterranean, yielded five previously unreported putative karlotoxins with molecular masses 1280, 1298, 1332, 1356, and 1400 Da, and similar fragments to KmTx-5. Analysis of several isolates of K. veneficum from the Ebro Delta revealed small-scale diversity in the karlotoxin spectrum in that one isolate from Fangar Bay produced KmTx-5, whereas the five putative novel karlotoxins were found among several isolates from nearby, but hydrographically distinct Alfacs Bay. Application of this LC-MS/MS method represents an incremental advance in the determination of putative karlotoxins, particularly in the absence of a complete spectrum of purified analytical standards of known specific potency.

LC-MS/MS Detection of Karlotoxins Reveals New Variants in Strains of the Marine Dinoflagellate Karlodinium veneficum from the Ebro Delta (NW Mediterranean)

PubMed Central

Krock, Bernd; Busch, Julia A.; García-Camacho, Francisco; Sánchez-Mirón, Asterio; Gallardo-Rodríguez, Juan J.; López-Rosales, Lorenzo; Andree, Karl B.; Fernández-Tejedor, Margarita; Witt, Matthias; Place, Allen R.

2017-01-01

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the detection and quantitation of karlotoxins in the selected reaction monitoring (SRM) mode. This novel method was based upon the analysis of purified karlotoxins (KcTx-1, KmTx-2, 44-oxo-KmTx-2, KmTx-5), one amphidinol (AM-18), and unpurified extracts of bulk cultures of the marine dinoflagellate Karlodinium veneficum strain CCMP2936 from Delaware (Eastern USA), which produces KmTx-1 and KmTx-3. The limit of detection of the SRM method for KmTx-2 was determined as 2.5 ng on-column. Collision induced dissociation (CID) spectra of all putative karlotoxins were recorded to present fragmentation patterns of each compound for their unambiguous identification. Bulk cultures of K. veneficum strain K10 isolated from an embayment of the Ebro Delta, NW Mediterranean, yielded five previously unreported putative karlotoxins with molecular masses 1280, 1298, 1332, 1356, and 1400 Da, and similar fragments to KmTx-5. Analysis of several isolates of K. veneficum from the Ebro Delta revealed small-scale diversity in the karlotoxin spectrum in that one isolate from Fangar Bay produced KmTx-5, whereas the five putative novel karlotoxins were found among several isolates from nearby, but hydrographically distinct Alfacs Bay. Application of this LC-MS/MS method represents an incremental advance in the determination of putative karlotoxins, particularly in the absence of a complete spectrum of purified analytical standards of known specific potency. PMID:29258236

Stable isotope labeling-solid phase extraction-mass spectrometry analysis for profiling of thiols and aldehydes in beer.

PubMed

Zheng, Shu-Jian; Wang, Ya-Lan; Liu, Ping; Zhang, Zheng; Yu, Lei; Yuan, Bi-Feng; Feng, Yu-Qi

2017-12-15

In this study, we developed a strategy for profiling of thiols and aldehydes in beer samples by stable isotope labeling-solid phase extraction-liquid chromatography-double precursor ion scan/double neutral loss scan-mass spectrometry analysis (SIL-SPE-LC-DPIS/DNLS-MS). A pair of isotope reagents (ω-bromoacetonylquinolinium bromide, BQB; ω-bromoacetonylquinolinium-d 7 bromide, BQB-d 7 ) were used to label thiols; while for the aldehydes, a pair of isotope reagents (4-(2-(trimethylammonio) ethoxy) benzenaminium halide, 4-APC; 4-(2-(trimethylammonio) ethoxy) benzenaminium halide-d 4 , 4-APC-d 4 ) were used. The labeled thiols and aldehydes were extracted and purified with solid-phase extraction, respectively, followed by LC-MS analysis. Using the proposed SIL-SPE-LC-DPIS/DNLS-MS methods, 76 thiol and 25 aldehyde candidates were found in beer. Furthermore, we established SIL-SPE-LC-MRM-MS methods for the relative quantitation of thiols and aldehydes in different beer samples. The results showed that the contents of thiols and aldehydes are closely related to the brands and origins of beers. Copyright © 2017 Elsevier Ltd. All rights reserved.

Simultaneous Determination of 11 Illicit Phenethylamines in Hair by LC-MS-MS: In Vivo Application.

PubMed

Nieddu, Maria; Burrai, Lucia; Demontis, Maria Piera; Varoni, Maria Vittoria; Baralla, Elena; Trignano, Claudia; Boatto, Gianpiero

2015-09-01

Existing phenethylamines are a class of synthetic compounds that differ from each other only in small changes to a largely conserved chemical structure. The recreational and illicit use of phenethylamines is a widespread problem. A simple procedure for the simultaneous quantitative determination in hair of 11 phenethylamines that are officially recognized as illicit by Italian legislation (p-methoxyamphetamine; p-methoxymethamphetamine; 3,4,5-trimethoxyamphetamine; 2,5-dimethoxyamphetamine; 2,5-dimethoxy-4-methylamphetamine; 2,5-dimethoxy-4-ethylamphetamine; 2,5-dimethoxy-4-bromoamphetamine; 2,5-dimethoxy-4-bromophenethylamine; 2,5-dimethoxy-4-iodophenethylamine; 2,5-dimethoxy-4-ethylthiophenethylamine and 2,5-dimethoxy-4-n-propylthiophenethylamine) has been developed and validated. Extraction from the matrix was performed after incubation in methanolic HCl and filtered reconstituted extracts were injected into a liquid chromatography/tandem mass spectrometry system (LC-MS-MS) without any further purification steps. This validated LC-MS-MS method has been used to determine the in vivo accumulation/retention of the above target analytes in hair after repeat oral administration to rats. This experiment further permitted investigation of the effect of pigmentation on the uptake of these phenethylamines by hair and the effect of hair pigmentation. The developed method could potentially be used for forensic and toxicological purposes, in the detection and quantitation of these illicit substances in human hair in workplace drug testing; drug-facilitated crime investigation; driver re-licensing; determining drug abuse history and postmortem toxicology. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Milrinone therapeutic drug monitoring in a pediatric population: Development and validation of a quantitative liquid chromatography-tandem mass spectrometry method.

PubMed

Raizman, Joshua E; Taylor, Katherine; Parshuram, Christopher; Colantonio, David A

2017-05-01

Milrinone is a potent selective phosphodiesterase type III inhibitor which stimulates myocardial function and improves myocardial relaxation. Although therapeutic monitoring is crucial to maintain therapeutic outcome, little data is available. A proof-of-principle study has been initiated in our institution to evaluate the clinical impact of optimizing milrinone dosing through therapeutic drug monitoring (TDM) in children following cardiac surgery. We developed a robust LC-MS/MS method to quantify milrinone in serum from pediatric patients in real-time. A liquid-liquid extraction procedure was used to prepare samples for analysis prior to measurement by LC-MS/MS. Performance characteristics, such as linearity, limit of quantitation (LOQ) and precision, were assessed. Patient samples were acquired post-surgery and analyzed to determine the concentration-time profile of the drug as well as to track turn-around-times. Within day precision was <8.3% across 3 levels of QC. Between-day precision was <12%. The method was linear from 50 to 800μg/l; the lower limit of quantification was 22μg/l. Comparison with another LC-MS/MS method showed good agreement. Using this simplified method, turnaround times within 3-6h were achievable, and patient drug profiles demonstrated that some milrinone levels were either sub-therapeutic or in the toxic range, highlighting the importance for milrinone TDM. This simplified and quick method proved to be analytically robust and able to provide therapeutic monitoring of milrinone in real-time in patients post-cardiac surgery. Copyright © 2017. Published by Elsevier B.V.

Quantitative analysis of polyethylene glycol (PEG) and PEGylated proteins in animal tissues by LC-MS/MS coupled with in-source CID.

PubMed

Gong, Jiachang; Gu, Xiaomei; Achanzar, William E; Chadwick, Kristina D; Gan, Jinping; Brock, Barry J; Kishnani, Narendra S; Humphreys, W Griff; Iyer, Ramaswamy A

2014-08-05

The covalent conjugation of polyethylene glycol (PEG, typical MW > 10k) to therapeutic peptides and proteins is a well-established approach to improve their pharmacokinetic properties and diminish the potential for immunogenicity. Even though PEG is generally considered biologically inert and safe in animals and humans, the slow clearance of large PEGs raises concerns about potential adverse effects resulting from PEG accumulation in tissues following chronic administration, particularly in the central nervous system. The key information relevant to the issue is the disposition and fate of the PEG moiety after repeated dosing with PEGylated proteins. Here, we report a novel quantitative method utilizing LC-MS/MS coupled with in-source CID that is highly selective and sensitive to PEG-related materials. Both (40K)PEG and a tool PEGylated protein (ATI-1072) underwent dissociation in the ionization source of mass spectrometer to generate a series of PEG-specific ions, which were subjected to further dissociation through conventional CID. To demonstrate the potential application of the method to assess PEG biodistribution following PEGylated protein administration, a single dose study of ATI-1072 was conducted in rats. Plasma and various tissues were collected, and the concentrations of both (40K)PEG and ATI-1072 were determined using the LC-MS/MS method. The presence of (40k)PEG in plasma and tissue homogenates suggests the degradation of PEGylated proteins after dose administration to rats, given that free PEG was absent in the dosing solution. The method enables further studies for a thorough characterization of disposition and fate of PEGylated proteins.

An automation-assisted generic approach for biological sample preparation and LC-MS/MS method validation.

PubMed

Zhang, Jie; Wei, Shimin; Ayres, David W; Smith, Harold T; Tse, Francis L S

2011-09-01

Although it is well known that automation can provide significant improvement in the efficiency of biological sample preparation in quantitative LC-MS/MS analysis, it has not been widely implemented in bioanalytical laboratories throughout the industry. This can be attributed to the lack of a sound strategy and practical procedures in working with robotic liquid-handling systems. Several comprehensive automation assisted procedures for biological sample preparation and method validation were developed and qualified using two types of Hamilton Microlab liquid-handling robots. The procedures developed were generic, user-friendly and covered the majority of steps involved in routine sample preparation and method validation. Generic automation procedures were established as a practical approach to widely implement automation into the routine bioanalysis of samples in support of drug-development programs.

Quantitative measurement of salivary testosterone in Korean adults by stable isotope-dilution liquid chromatographyelectrospray-tandem mass spectrometry.

PubMed

Lee, Sanghoo; Kwon, Soonho; Shin, Hye-Jin; Park, Jimyeong; Lim, Hwan-Sub; Lee, Kyoung-Ryul; Kim, Young-Jin

2010-11-01

Salivary testosterone levels in Korean adults were quantitatively measured for the first time by liquid chromatography-electrospray-tandem mass spectrometry (LC ESI MS/MS). Salivary testosterone was separated on a multiple reaction monitoring (MRM) chromatogram within 7 min. The LC ESI MS/MS assay was validated over the linearity range of 0.01-2.00 ng/ml (r=0.99987) using testosterone-d(3) as an internal standard. The lower limit of quantification (LOQ) was 0.01 ng/ml. The intra- and inter-assay precisions were 1.54% to 4.09% and 0.96% to 4.29%, respectively. The mean recovery was 93.32% (range 88.43-98.05%). The validated assay was then applied to measure the salivary testosterone levels of Korean adults. In men, the salivary testosterone level collected between 9:00-11:00 am was approximately 2.8 times higher than that in women (P < 0.0001). Salivary testosterone levels in both sexes negatively correlated with age. The present assay would also be useful in measuring salivary testosterone levels in clinical laboratories.

Evaluation of a multiresidue method for measuring fourteen chemical groups of pesticides in water by use of LC-MS-MS.

PubMed

Carvalho, J J; Jerónimo, P C A; Gonçalves, C; Alpendurada, M F

2008-11-01

European Council Directive 98/83/EC on the quality of water intended for human consumption brought a new challenge for water-quality control routine laboratories, mainly on pesticides analysis. Under the guidelines of ISO/IEC 17025:2005, a multiresidue method was developed, validated, implemented in routine, and studied with real samples during a one-year period. The proposed method enables routine laboratories to handle a large number of samples, since 28 pesticides of 14 different chemical groups can be quantitated in a single procedure. The method comprises a solid-phase extraction step and subsequent analysis by liquid chromatography-mass spectrometry (LC-MS-MS). The accuracy was established on the basis of participation in interlaboratory proficiency tests, with encouraging results (majority |z-score| <2), and the precision was consistently analysed over one year. The limits of quantitation (below 0.050 microg L(-1)) are in agreement with the enforced threshold value for pesticides of 0.10 microg L(-1). Overall method performance is suitable for routine use according to accreditation rules, taking into account the data collected over one year.

Quantitative Analysis of Global Proteome and Lysine Acetylome Reveal the Differential Impacts of VPA and SAHA on HL60 Cells.

PubMed

Zhu, Xiaoyu; Liu, Xin; Cheng, Zhongyi; Zhu, Jun; Xu, Lei; Wang, Fengsong; Qi, Wulin; Yan, Jiawei; Liu, Ning; Sun, Zimin; Liu, Huilan; Peng, Xiaojun; Hao, Yingchan; Zheng, Nan; Wu, Quan

2016-01-29

Valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) are both HDAC inhibitors (HDACi). Previous studies indicated that both inhibitors show therapeutic effects on acute myeloid leukaemia (AML), while the differential impacts of the two different HDACi on AML treatment still remains elusive. In this study, using 3-plex SILAC based quantitative proteomics technique, anti-acetyllysine antibody based affinity enrichment, high resolution LC-MS/MS and intensive bioinformatic analysis, the quantitative proteome and acetylome in SAHA and VPA treated AML HL60 cells were extensively studied. In total, 5,775 proteins and 1,124 lysine acetylation sites were successfully obtained in response to VAP and SAHA treatment. It is found that VPA and SAHA treatment differently induced proteome and acetylome profiling in AML HL60 cells. This study revealed the differential impacts of VPA and SAHA on proteome/acetylome in AML cells, deepening our understanding of HDAC inhibitor mediated AML therapeutics.

A sensitive LC-MS/MS method for the simultaneous determination of amoxicillin and ambroxol in human plasma with segmental monitoring.

PubMed

Dong, Xin; Ding, Li; Cao, Xiaomei; Jiang, Liyuan; Zhong, Shuisheng

2013-04-01

Amoxicillin (AMO) degrades in plasma at room temperature and readily undergoes hydrolysis by the plasma amidase. In this paper, a novel, rapid and sensitive LC-MS/MS method operated in segmental and multiple reaction monitoring has been developed for the simultaneous determination of amoxicillin and ambroxol in human plasma. The degradation of amoxicillin in plasma was well prevented by immediate addition of 20 μL glacial acetic acid to 200 μL aliquot of freshly collected plasma samples before storage at -80°C. The sensitivity of the method was improved with segmental monitoring of the analytes, and lower limits of quantitation of 0.5 ng/mL for ambroxol and 5 ng/mL for amoxicillin were obtained. The sensitivity of our method was five times better than those of the existing methods. Furthermore, the mass response saturation problem with amoxicillin was avoided by diluting the deproteinized plasma samples with water before injection into the LC-MS/MS system. The method was successfully employed in a pharmacokinetic study of the compound amoxicillin and ambroxol hydrochloride tablets. Copyright © 2012 John Wiley & Sons, Ltd.

Characterization of phenolic compounds and antinociceptive activity of Sempervivum tectorum L. leaf juice.

PubMed

Alberti, Ágnes; Béni, Szabolcs; Lackó, Erzsébet; Riba, Pál; Al-Khrasani, Mahmoud; Kéry, Ágnes

2012-11-01

Sempervivum tectorum L. (houseleek) leaf juice has been known as a traditional herbal remedy. The aim of the present study was the chemical characterization of its phenolic compounds and to develop quantitation methods for its main flavonol glycoside, as well as to evaluate its antinociceptive activity. Lyophilized houseleek leaf juice was studied by HPLC-DAD coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to identify flavonol glycosides, hydroxy-benzoic and hydroxy-cinnamic acids. Ten flavonol glycosides and sixteen phenolic acid compounds were identified or tentatively characterized. Structure of the main flavonol compound was identified by nuclear magnetic resonance spectroscopy. Three characteristic kaempferol glycosides were isolated and determined by LC-ESI-MS/MS with external calibration method, using the isolated compounds as standard. The main flavonol glycoside was also determined by HPLC-DAD. Validated HPLC-DAD and LC-ESI-MS/MS methods were developed to quantify kaempferol-3-O-rhamnosyl-glucoside-7-O-rhamnoside and two other kaempferol glycosides. Antinociceptive activity of houseleek leaf juice was investigated by writhing test of mice. Sempervivum extract significantly reduced pain in the mouse writhing test. Copyright © 2012 Elsevier B.V. All rights reserved.

Quantitation of Tenofovir and Emtricitabine in Dried Blood Spots (DBS) with LC-MS/MS

PubMed Central

Zheng, Jia-Hua; Guida, Louis A; Rower, Caitlin; Castillo-Mancilla, Jose; Meditz, Amie; Klein, Brandon; Kerr, Becky Jo; Langness, Jacob; Bushman, Lane; Kiser, Jennifer; Anderson, Peter L.

2013-01-01

A reversed-phase high performance liquid chromatographic (LC), tandem mass spectrometry (MS/MS) assay for the determination of tenofovir (TFV) and emtricitabine (FTC) in dried blood spots (DBS) from human whole blood was developed and validated. Whole blood samples were spotted, dried, and a 3mm punch was extracted with methanol for analysis by LC-MS/MS utilizing stable isotope labeled internal standards. The assay was validated over the range of 2.5ng/mL to 1,000ng/mL for TFV and 2.5ng/mL to 5,000ng/mL for FTC. The method was accurate (within ± 15% of control) and precise (coefficient of variation ≤ 15%) for hematocrit concentrations ranging from 25% to 76%; using edge punches versus center punches; and spot volumes of 10µL to 50µL. Analytes were stable for five freeze/thaw cycles and up to 6 days at room temperature, whereas long-term storage required −20°C or −80°C. Comparison of TFV and FTC in DBS versus plasma yielded r2 ≥ 0.96, indicating that DBS can be used as a plasma alternative for pharmacokinetic analyses in vivo. PMID:24055850

LC-MS/MS assay for the quantitation of the tyrosine kinase inhibitor neratinib in human plasma.

PubMed

Kiesel, Brian F; Parise, Robert A; Wong, Alvin; Keyvanjah, Kiana; Jacobs, Samuel; Beumer, Jan H

2017-02-05

Neratinib is an orally available tyrosine kinase inhibitor targeting HER2 (ERBB2) and EGFR (ERBB). It is being clinically evaluated for the treatment of breast and other solid tumors types as a single agent or in combination with other chemotherapies. In support of several phase I/II clinical trials investigating neratinib combinations, we developed and validated a novel LC-MS/MS assay for the quantification of neratinib in 100μL of human plasma with a stable isotopic internal standard. Analytes were extracted from plasma using protein precipitation and evaporation of the resulting supernatant followed by resuspension. Chromatographic separation was achieved using an Acquity UPLC BEH Shield RP18 column and a gradient methanol-water mobile phase containing 10% ammonium acetate. An ABI 4000 mass spectrometer and electrospray positive mode ionization were used for detection. The assay was linear from 2 to 1,000ng/mL and proved to be accurate (98.9-106.5%) and precise (<6.2%CV), and met the FDA guidance for bioanalytical method validation. This LC-MS/MS assay will be an essential tool to further define the pharmacokinetics of neratinib. Copyright © 2016 Elsevier B.V. All rights reserved.

Improved LC-MS/MS method for the quantification of hepcidin-25 in clinical samples.

PubMed

Abbas, Ioana M; Hoffmann, Holger; Montes-Bayón, María; Weller, Michael G

2018-06-01

Mass spectrometry-based methods play a crucial role in the quantification of the main iron metabolism regulator hepcidin by singling out the bioactive 25-residue peptide from the other naturally occurring N-truncated isoforms (hepcidin-20, -22, -24), which seem to be inactive in iron homeostasis. However, several difficulties arise in the MS analysis of hepcidin due to the "sticky" character of the peptide and the lack of suitable standards. Here, we propose the use of amino- and fluoro-silanized autosampler vials to reduce hepcidin interaction to laboratory glassware surfaces after testing several types of vials for the preparation of stock solutions and serum samples for isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Furthermore, we have investigated two sample preparation strategies and two chromatographic separation conditions with the aim of developing a LC-MS/MS method for the sensitive and reliable quantification of hepcidin-25 in serum samples. A chromatographic separation based on usual acidic mobile phases was compared with a novel approach involving the separation of hepcidin-25 with solvents at high pH containing 0.1% of ammonia. Both methods were applied to clinical samples in an intra-laboratory comparison of two LC-MS/MS methods using the same hepcidin-25 calibrators with good correlation of the results. Finally, we recommend a LC-MS/MS-based quantification method with a dynamic range of 0.5-40 μg/L for the assessment of hepcidin-25 in human serum that uses TFA-based mobile phases and silanized glass vials. Graphical abstract Structure of hepcidin-25 (Protein Data Bank, PDB ID 2KEF).

GeLC-MS-based proteomics of Chromobacterium violaceum: comparison of proteome changes elicited by hydrogen peroxide

PubMed Central

Lima, D. C.; Duarte, F. T.; Medeiros, V. K. S.; Carvalho, P. C.; Nogueira, F. C. S.; Araujo, G. D. T.; Domont, G. B.; Batistuzzo de Medeiros, S. R.

2016-01-01

Chromobacterium violaceum is a free-living bacillus with several genes that enables it survival under different harsh environments such as oxidative and temperature stresses. Here we performed a label-free quantitative proteomic study to unravel the molecular mechanisms that enable C. violaceum to survive oxidative stress. To achieve this, total proteins extracted from control and C. violaceum cultures exposed during two hours with 8 mM hydrogen peroxide were analyzed using GeLC-MS proteomics. Analysis revealed that under the stress condition, the bacterium expressed proteins that protected it from the damage caused by reactive oxygen condition and decreasing the abundance of proteins responsible for bacterial growth and catabolism. GeLC-MS proteomics analysis provided an overview of the metabolic pathways involved in the response of C. violaceum to oxidative stress ultimately aggregating knowledge of the response of this organism to environmental stress. This study identified approximately 1500 proteins, generating the largest proteomic coverage of C. violaceum so far. We also detected proteins with unknown function that we hypothesize to be part of new mechanisms related to oxidative stress defense. Finally, we identified the mechanism of clustered regularly interspaced short palindromic repeats (CRISPR), which has not yet been reported for this organism. PMID:27321545

GeLC-MS-based proteomics of Chromobacterium violaceum: comparison of proteome changes elicited by hydrogen peroxide.

PubMed

Lima, D C; Duarte, F T; Medeiros, V K S; Carvalho, P C; Nogueira, F C S; Araujo, G D T; Domont, G B; Batistuzzo de Medeiros, S R

2016-06-20

Chromobacterium violaceum is a free-living bacillus with several genes that enables it survival under different harsh environments such as oxidative and temperature stresses. Here we performed a label-free quantitative proteomic study to unravel the molecular mechanisms that enable C. violaceum to survive oxidative stress. To achieve this, total proteins extracted from control and C. violaceum cultures exposed during two hours with 8 mM hydrogen peroxide were analyzed using GeLC-MS proteomics. Analysis revealed that under the stress condition, the bacterium expressed proteins that protected it from the damage caused by reactive oxygen condition and decreasing the abundance of proteins responsible for bacterial growth and catabolism. GeLC-MS proteomics analysis provided an overview of the metabolic pathways involved in the response of C. violaceum to oxidative stress ultimately aggregating knowledge of the response of this organism to environmental stress. This study identified approximately 1500 proteins, generating the largest proteomic coverage of C. violaceum so far. We also detected proteins with unknown function that we hypothesize to be part of new mechanisms related to oxidative stress defense. Finally, we identified the mechanism of clustered regularly interspaced short palindromic repeats (CRISPR), which has not yet been reported for this organism.

Comparative analysis of soybean plasma membrane proteins under osmotic stress using gel-based and LC MS/MS-based proteomics approaches.

PubMed

Nouri, Mohammad-Zaman; Komatsu, Setsuko

2010-05-01

To study the soybean plasma membrane proteome under osmotic stress, two methods were used: a gel-based and a LC MS/MS-based proteomics method. Two-day-old seedlings were subjected to 10% PEG for 2 days. Plasma membranes were purified from seedlings using a two-phase partitioning method and their purity was verified by measuring ATPase activity. Using the gel-based proteomics, four and eight protein spots were identified as up- and downregulated, respectively, whereas in the nanoLC MS/MS approach, 11 and 75 proteins were identified as up- and downregulated, respectively, under PEG treatment. Out of osmotic stress responsive proteins, most of the transporter proteins and all proteins with high number of transmembrane helices as well as low-abundance proteins could be identified by the LC MS/MS-based method. Three homologues of plasma membrane H(+)-ATPase, which are transporter proteins involved in ion efflux, were upregulated under osmotic stress. Gene expression of this protein was increased after 12 h of stress exposure. Among the identified proteins, seven proteins were mutual in two proteomics techniques, in which calnexin was the highly upregulated protein. Accumulation of calnexin in plasma membrane was confirmed by immunoblot analysis. These results suggest that under hyperosmotic conditions, calnexin accumulates in the plasma membrane and ion efflux accelerates by upregulation of plasma membrane H(+)-ATPase protein.

Unraveling Molecular Differences of Gastric Cancer by Label-Free Quantitative Proteomics Analysis.

PubMed

Dai, Peng; Wang, Qin; Wang, Weihua; Jing, Ruirui; Wang, Wei; Wang, Fengqin; Azadzoi, Kazem M; Yang, Jing-Hua; Yan, Zhen

2016-01-21

Gastric cancer (GC) has significant morbidity and mortality worldwide and especially in China. Its molecular pathogenesis has not been thoroughly elaborated. The acknowledged biomarkers for diagnosis, prognosis, recurrence monitoring and treatment are lacking. Proteins from matched pairs of human GC and adjacent tissues were analyzed by a coupled label-free Mass Spectrometry (MS) approach, followed by functional annotation with software analysis. Nano-LC-MS/MS, quantitative real-time polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry were used to validate dysregulated proteins. One hundred forty-six dysregulated proteins with more than twofold expressions were quantified, 22 of which were first reported to be relevant with GC. Most of them were involved in cancers and gastrointestinal disease. The expression of a panel of four upregulated nucleic acid binding proteins, heterogeneous nuclear ribonucleoprotein hnRNPA2B1, hnRNPD, hnRNPL and Y-box binding protein 1 (YBX-1) were validated by Nano-LC-MS/MS, qRT-PCR, western blot and immunohistochemistry assays in ten GC patients' tissues. They were located in the keynotes of a predicted interaction network and might play important roles in abnormal cell growth. The label-free quantitative proteomic approach provides a deeper understanding and novel insight into GC-related molecular changes and possible mechanisms. It also provides some potential biomarkers for clinical diagnosis.

The mzqLibrary--An open source Java library supporting the HUPO-PSI quantitative proteomics standard.

PubMed

Qi, Da; Zhang, Huaizhong; Fan, Jun; Perkins, Simon; Pisconti, Addolorata; Simpson, Deborah M; Bessant, Conrad; Hubbard, Simon; Jones, Andrew R

2015-09-01

The mzQuantML standard has been developed by the Proteomics Standards Initiative for capturing, archiving and exchanging quantitative proteomic data, derived from mass spectrometry. It is a rich XML-based format, capable of representing data about two-dimensional features from LC-MS data, and peptides, proteins or groups of proteins that have been quantified from multiple samples. In this article we report the development of an open source Java-based library of routines for mzQuantML, called the mzqLibrary, and associated software for visualising data called the mzqViewer. The mzqLibrary contains routines for mapping (peptide) identifications on quantified features, inference of protein (group)-level quantification values from peptide-level values, normalisation and basic statistics for differential expression. These routines can be accessed via the command line, via a Java programming interface access or a basic graphical user interface. The mzqLibrary also contains several file format converters, including import converters (to mzQuantML) from OpenMS, Progenesis LC-MS and MaxQuant, and exporters (from mzQuantML) to other standards or useful formats (mzTab, HTML, csv). The mzqViewer contains in-built routines for viewing the tables of data (about features, peptides or proteins), and connects to the R statistical library for more advanced plotting options. The mzqLibrary and mzqViewer packages are available from https://code.google.com/p/mzq-lib/. © 2015 The Authors. PROTEOMICS Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Profiling of anthocyanins in transgenic purple-fleshed sweet potatoes by HPLC-MS/MS.

PubMed

Ge, Jingqiu; Hu, Yijie; Wang, Hongxia; Huang, Yuanshe; Zhang, Peng; Liao, Zhihua; Chen, Min

2017-11-01

Anthocyanins in purple-fleshed sweet potato (PSP) are beneficial to human health. The leaf color (Lc) gene is a transcription factor involved in regulating anthocyanin biosynthesis. The anthocyanin profiles of wild-type PSP of Ayamurasaki and its three Lc-transgenic lines were investigated by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). In vitro antioxidant activities of wild-type and Lc-transgenic lines, including reducing power activity, DPPH radical scavenging activity, hydroxyl radical scavenging activity, linoleic acid autoxidation inhibition activity, ABTS free radical scavenging activity and oxygen radical absorbance capacity activity, were measured. The results showed that the total anthocyanin contents increased 1.5-1.9 times in three transgenic lines compared with that in wild-type PSP. Seventeen anthocyanins were found in wild-type PSP, while 19 in Lc-transgenic lines including cyanidin-based, peonidin-based and pelargonidin-based anthocyanins. Three pelargonidin-based anthocyanins were detected in three Lc-transgenic lines. Among them, the relative contents of cyanidin-based and pelargonidin-based anthocyanins increased 1.9-2.0 and 3.4-4.5 times respectively, while peonidin-based anthocyanins decreased 1.8-1.9 times in Lc-transgenic lines, compared with wild-type PSP. PSP from wild-type Ayamurasaki and three Lc-transgenic lines exhibited potent antioxidant activities, whereas there was no distinct difference among them. The transgene Lc significantly increased the content of total anthocyanins and remarkably changed the anthocyanin profiles in Ayamurasaki. Such novel and high content of anthocyanins obtained in the Lc-transgenic lines with potent antioxidant activities may provide unique functional products with potential helpful for human health. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Multiple reaction monitoring targeted LC-MS analysis of potential cell death marker proteins for increased bioprocess control.

PubMed

Albrecht, Simone; Kaisermayer, Christian; Reinhart, David; Ambrose, Monica; Kunert, Renate; Lindeberg, Anna; Bones, Jonathan

2018-05-01

The monitoring of protein biomarkers for the early prediction of cell stress and death is a valuable tool for process characterization and efficient biomanufacturing control. A representative set of six proteins, namely GPDH, PRDX1, LGALS1, CFL1, TAGLN2 and MDH, which were identified in a previous CHO-K1 cell death model using discovery LC-MS E was translated into a targeted liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) platform and verified. The universality of the markers was confirmed in a cell growth model for which three Chinese hamster ovary host cell lines (CHO-K1, CHO-S, CHO-DG44) were grown in batch culture in two different types of basal media. LC-MRM-MS was also applied to spent media (n = 39) from four perfusion biomanufacturing series. Stable isotope-labelled peptide analogues and a stable isotope-labelled monoclonal antibody were used for improved protein quantitation and simultaneous monitoring of the workflow reproducibility. Significant increases in protein concentrations were observed for all viability marker proteins upon increased dead cell numbers and allowed for discrimination of spent media with dead cell densities below and above 1 × 10 6  dead cells/mL which highlights the potential of the selected viability marker proteins in bioprocess control. Graphical abstract Overview of the LC-MRM-MS workflow for the determination of proteomic markers in conditioned media from the bioreactor that correlate with CHO cell death.

The clinical impact of recent advances in LC-MS for cancer biomarker discovery and verification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Hui; Shi, Tujin; Qian, Wei-Jun

2015-12-04

Mass spectrometry-based proteomics has become an indispensable tool in biomedical research with broad applications ranging from fundamental biology, systems biology, and biomarker discovery. Recent advances in LC-MS have made it become a major technology in clinical applications, especially in cancer biomarker discovery and verification. To overcome the challenges associated with the analysis of clinical samples, such as extremely wide dynamic range of protein concentrations in biofluids and the need to perform high throughput and accurate quantification, significant efforts have been devoted to improve the overall performance of LC-MS bases clinical proteomics. In this review, we summarize the recent advances inmore » LC-MS in the aspect of cancer biomarker discovery and quantification, and discuss its potentials, limitations, and future perspectives.« less

Quantitative mass spectrometry imaging of emtricitabine in cervical tissue model using infrared matrix-assisted laser desorption electrospray ionization

PubMed Central

Bokhart, Mark T.; Rosen, Elias; Thompson, Corbin; Sykes, Craig; Kashuba, Angela D. M.; Muddiman, David C.

2015-01-01

A quantitative mass spectrometry imaging (QMSI) technique using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is demonstrated for the antiretroviral (ARV) drug emtricitabine in incubated human cervical tissue. Method development of the QMSI technique leads to a gain in sensitivity and removal of interferences for several ARV drugs. Analyte response was significantly improved by a detailed evaluation of several cationization agents. Increased sensitivity and removal of an isobaric interference was demonstrated with sodium chloride in the electrospray solvent. Voxel-to-voxel variability was improved for the MSI experiments by normalizing analyte abundance to a uniformly applied compound with similar characteristics to the drug of interest. Finally, emtricitabine was quantified in tissue with a calibration curve generated from the stable isotope-labeled analog of emtricitabine followed by cross-validation using liquid chromatography tandem mass spectrometry (LC-MS/MS). The quantitative IR-MALDESI analysis proved to be reproducible with an emtricitabine concentration of 17.2±1.8 μg/gtissue. This amount corresponds to the detection of 7 fmol/voxel in the IR-MALDESI QMSI experiment. Adjacent tissue slices were analyzed using LC-MS/MS which resulted in an emtricitabine concentration of 28.4±2.8 μg/gtissue. PMID:25318460

Visualizing spatial distribution of alectinib in murine brain using quantitative mass spectrometry imaging.

PubMed

Aikawa, Hiroaki; Hayashi, Mitsuhiro; Ryu, Shoraku; Yamashita, Makiko; Ohtsuka, Naoto; Nishidate, Masanobu; Fujiwara, Yasuhiro; Hamada, Akinobu

2016-03-30

In the development of anticancer drugs, drug concentration measurements in the target tissue have been thought to be crucial for predicting drug efficacy and safety. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is commonly used for determination of average drug concentrations; however, complete loss of spatial information in the target tissue occurs. Mass spectrometry imaging (MSI) has been recently applied as an innovative tool for detection of molecular distribution of pharmacological agents in heterogeneous targets. This study examined the intra-brain transitivity of alectinib, a novel anaplastic lymphoma kinase inhibitor, using a combination of matrix-assisted laser desorption ionization-MSI and LC-MS/MS techniques. We first analyzed the pharmacokinetic profiles in FVB mice and then examined the effect of the multidrug resistance protein-1 (MDR1) using Mdr1a/b knockout mice including quantitative distribution of alectinib in the brain. While no differences were observed between the mice for the plasma alectinib concentrations, diffuse alectinib distributions were found in the brain of the Mdr1a/b knockout versus FVB mice. These results indicate the potential for using quantitative MSI for clarifying drug distribution in the brain on a microscopic level, in addition to suggesting a possible use in designing studies for anticancer drug development and translational research.

Visualizing spatial distribution of alectinib in murine brain using quantitative mass spectrometry imaging

PubMed Central

Aikawa, Hiroaki; Hayashi, Mitsuhiro; Ryu, Shoraku; Yamashita, Makiko; Ohtsuka, Naoto; Nishidate, Masanobu; Fujiwara, Yasuhiro; Hamada, Akinobu

2016-01-01

In the development of anticancer drugs, drug concentration measurements in the target tissue have been thought to be crucial for predicting drug efficacy and safety. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is commonly used for determination of average drug concentrations; however, complete loss of spatial information in the target tissue occurs. Mass spectrometry imaging (MSI) has been recently applied as an innovative tool for detection of molecular distribution of pharmacological agents in heterogeneous targets. This study examined the intra-brain transitivity of alectinib, a novel anaplastic lymphoma kinase inhibitor, using a combination of matrix-assisted laser desorption ionization–MSI and LC-MS/MS techniques. We first analyzed the pharmacokinetic profiles in FVB mice and then examined the effect of the multidrug resistance protein-1 (MDR1) using Mdr1a/b knockout mice including quantitative distribution of alectinib in the brain. While no differences were observed between the mice for the plasma alectinib concentrations, diffuse alectinib distributions were found in the brain of the Mdr1a/b knockout versus FVB mice. These results indicate the potential for using quantitative MSI for clarifying drug distribution in the brain on a microscopic level, in addition to suggesting a possible use in designing studies for anticancer drug development and translational research. PMID:27026287

Isolation and determination of ivermectin in post-mortem and in vivo tissues of dung beetles using a continuous solid phase extraction method followed by LC-ESI+-MS/MS

PubMed Central

Ortiz, Antonio J.; Cortez, Vieyle; Azzouz, Abdelmonaim

2017-01-01

A new analytical method based on solvent extraction, followed by continuous solid-phase extraction (SPE) clean-up using a polymeric sorbent, was demonstrated to be applicable for the detection of ivermectin in complex biological matrices of dung beetles (hemolymph, excreta or dry tissues) using liquid chromatography combined with positive electrospray ionization tandem mass spectrometry (LC/ESI+–MS/MS). Using a signal-to-noise ratio of 3:1, the limit of detection (LOD) in the insect matrices at trace levels was 0.01 ng g–1 and the limit of quantification (LOQ) was 0.1 ng g–1. The proposed method was successfully used to quantitatively determine the levels of ivermectin in the analysis of small samples in in vivo and post mortem samples, demonstrating the usefulness for quantitative analyses that are focused on future pharmacokinetic and bioavailability studies in insects and the establishment of a new protocol to study the impact of ivermectin on non-target arthropods such as dung beetles and other insects that are related with the “dung community”. Because satisfactory precision and accuracy values were obtained in both in vivo matrices, we suggest that the method can be consistently used for quantitative determinations that are focused on future pharmacokinetic and bioavailability studies in insects. Furthermore, this new analytical method was successfully applied to biological samples of dead dung beetles from the field suggesting that the method can be used to establish a new routine analysis of ivermectin residues in insect carcasses that is applied to complement typical mortality tests. PMID:28207908

Quantitation of the enantiomers of tramadol and its three main metabolites in human whole blood using LC-MS/MS.

PubMed

Haage, Pernilla; Kronstrand, Robert; Carlsson, Björn; Kugelberg, Fredrik C; Josefsson, Martin

2016-02-05

The analgesic drug tramadol and its metabolites are chiral compounds, with the (+)- and (-)-enantiomers showing different pharmacological and toxicological effects. This novel enantioselective method, based on LC-MS/MS in reversed phase mode, enabled measurement of the parent compound and its three main metabolites O-desmethyltramadol, N-desmethyltramadol and N,O-didesmethyltramadol simultaneously. Whole blood samples of 0.5g were fortified with internal standards (tramadol-(13)C-D3 and O-desmethyl-cis-tramadol-D6) and extracted under basic conditions (pH 11) by liquid-liquid extraction. Chromatography was performed on a chiral alpha-1-acid glycoprotein (AGP) column preceded by an AGP guard column. The mobile phase consisted of 0.8% acetonitrile and 99.2% ammonium acetate (20mM, pH 7.2). A post-column infusion with 0.05% formic acid in acetonitrile was used to enhance sensitivity. Quantitation as well as enantiomeric ratio measurements were covered by quality controls. Validation parameters for all eight enantiomers included selectivity (high), matrix effects (no ion suppression/enhancement), calibration model (linear, weight 1/X(2), in the range of 0.25-250ng/g), limit of quantitation (0.125-0.50ng/g), repeatability (2-6%) and intermediate precision (2-7%), accuracy (83-114%), dilution integrity (98-115%), carry over (not exceeding 0.07%) and stability (stable in blood and extract). The method was applied to blood samples from a healthy volunteer administrated a single 100mg dose and to a case sample concerning an impaired driver, which confirmed its applicability in human pharmacokinetic studies as well as in toxicological and forensic investigations. Copyright © 2015 Elsevier B.V. All rights reserved.

Improving liquid chromatography-tandem mass spectrometry determinations by modifying noise frequency spectrum between two consecutive wavelet-based low-pass filtering procedures.

PubMed

Chen, Hsiao-Ping; Liao, Hui-Ju; Huang, Chih-Min; Wang, Shau-Chun; Yu, Sung-Nien

2010-04-23

This paper employs one chemometric technique to modify the noise spectrum of liquid chromatography-tandem mass spectrometry (LC-MS/MS) chromatogram between two consecutive wavelet-based low-pass filter procedures to improve the peak signal-to-noise (S/N) ratio enhancement. Although similar techniques of using other sets of low-pass procedures such as matched filters have been published, the procedures developed in this work are able to avoid peak broadening disadvantages inherent in matched filters. In addition, unlike Fourier transform-based low-pass filters, wavelet-based filters efficiently reject noises in the chromatograms directly in the time domain without distorting the original signals. In this work, the low-pass filtering procedures sequentially convolve the original chromatograms against each set of low pass filters to result in approximation coefficients, representing the low-frequency wavelets, of the first five resolution levels. The tedious trials of setting threshold values to properly shrink each wavelet are therefore no longer required. This noise modification technique is to multiply one wavelet-based low-pass filtered LC-MS/MS chromatogram with another artificial chromatogram added with thermal noises prior to the other wavelet-based low-pass filter. Because low-pass filter cannot eliminate frequency components below its cut-off frequency, more efficient peak S/N ratio improvement cannot be accomplished using consecutive low-pass filter procedures to process LC-MS/MS chromatograms. In contrast, when the low-pass filtered LC-MS/MS chromatogram is conditioned with the multiplication alteration prior to the other low-pass filter, much better ratio improvement is achieved. The noise frequency spectrum of low-pass filtered chromatogram, which originally contains frequency components below the filter cut-off frequency, is altered to span a broader range with multiplication operation. When the frequency range of this modified noise spectrum shifts toward the high frequency regimes, the other low-pass filter is able to provide better filtering efficiency to obtain higher peak S/N ratios. Real LC-MS/MS chromatograms, of which typically less than 6-fold peak S/N ratio improvement achieved with two consecutive wavelet-based low-pass filters remains the same S/N ratio improvement using one-step wavelet-based low-pass filter, are improved to accomplish much better ratio enhancement 25-folds to 40-folds typically when the noise frequency spectrum is modified between two low-pass filters. The linear standard curves using the filtered LC-MS/MS signals are validated. The filtered LC-MS/MS signals are also reproducible. The more accurate determinations of very low concentration samples (S/N ratio about 7-9) are obtained using the filtered signals than the determinations using the original signals. Copyright 2010 Elsevier B.V. All rights reserved.

Accidental intoxication with Veratrum album.

PubMed

Grobosch, T; Binscheck, T; Martens, F; Lampe, D

2008-01-01

A 49-year-old man consumed two glasses (approximately 2 x 20 mL) of a beverage containing yellow gentian (Gentiana lutea). Shortly after ingestion, he developed nausea, vomiting, and oral paraesthesia. On admission to the hospital he suffered from severe bradycardia (35 beats/min) and hypotension (50/30 mm Hg), and he was treated with activated charcoal, antiemetics (metoclopramide, ondansetron), atropine, and intravenous electrolytic solution. The initial suspicion of Veratrum poisoning could be confirmed by identifying protoveratrines A (ProA) and protoveratrine B (ProB) in a sample from the beverage as well as in the patients serum by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS). The yellow-colored beverage contained 25% ethanol (by headspace gas chromatography), 20.4 mg/L ProA, and 13.7 mg/L ProB. The serum concentration of ProA was 1162 ng/L and ProB was 402 ng/L. Veratridine, cevadine, and jervine were not detected, neither in the beverage nor in the serum sample. The lower limits of quantitation for all compounds is 10 microg/L (S/N > 10, beverage) and 100 ng/L (S/N > 10, serum). After treatment, the patient completely recovered from the symptoms within 24 h and was discharged from the hospital. The analytical method described was developed for the simultaneous identification and quantitation of five Veratrum alkaloids. The method is based on a liquid-liquid extraction followed by LC-MS-MS analysis. The time needed for analysis was 6 min.

Development and single-laboratory validation of a UHPLC-MS/MS method for quantitation of microcystins and nodularin in natural water, cyanobacteria, shellfish and algal supplement tablet powders.

PubMed

Turner, Andrew D; Waack, Julia; Lewis, Adam; Edwards, Christine; Lawton, Linda

2018-02-01

A simple, rapid UHPLC-MS/MS method has been developed and optimised for the quantitation of microcystins and nodularin in wide variety of sample matrices. Microcystin analogues targeted were MC-LR, MC-RR, MC-LA, MC-LY, MC-LF, LC-LW, MC-YR, MC-WR, [Asp3] MC-LR, [Dha7] MC-LR, MC-HilR and MC-HtyR. Optimisation studies were conducted to develop a simple, quick and efficient extraction protocol without the need for complex pre-analysis concentration procedures, together with a rapid sub 5min chromatographic separation of toxins in shellfish and algal supplement tablet powders, as well as water and cyanobacterial bloom samples. Validation studies were undertaken on each matrix-analyte combination to the full method performance characteristics following international guidelines. The method was found to be specific and linear over the full calibration range. Method sensitivity in terms of limits of detection, quantitation and reporting were found to be significantly improved in comparison to LC-UV methods and applicable to the analysis of each of the four matrices. Overall, acceptable recoveries were determined for each of the matrices studied, with associated precision and within-laboratory reproducibility well within expected guidance limits. Results from the formalised ruggedness analysis of all available cyanotoxins, showed that the method was robust for all parameters investigated. The results presented here show that the optimised LC-MS/MS method for cyanotoxins is fit for the purpose of detection and quantitation of a range of microcystins and nodularin in shellfish, algal supplement tablet powder, water and cyanobacteria. The method provides a valuable early warning tool for the rapid, routine extraction and analysis of natural waters, cyanobacterial blooms, algal powders, food supplements and shellfish tissues, enabling monitoring labs to supplement traditional microscopy techniques and report toxicity results within a short timeframe of sample receipt. The new method, now accredited to ISO17025 standard, is simple, quick, applicable to multiple matrices and is highly suitable for use as a routine, high-throughout, fast turnaround regulatory monitoring tool. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization and quantitation of yohimbine and its analogs in botanicals and dietary supplements using LC/QTOF-MS and LC/QQQ-MS for determination of the presence of bark extract and yohimbine adulteration.

PubMed

Lucas, Derick; Neal-Kababick, James; Zweigenbaum, Jerry

2015-01-01

The compound yohimbine HCl has been restricted in Australia and categorized as a scheduled prescription drug in other parts of the world, including the United States where it is monographed as a drug in the U. S. Pharmacopeia. However, the bark of the yohimbe plant and its extract is considered a botanical that can be used as a dietary supplement in some parts of the world. For these reasons, methods to characterize the indole alkaloids of the bark and quantify yohimbine and its analogs are presented using accurate mass LC/quadrupole time-of-flight (QTOF)-MS and triple quadrupole LC/MS, respectively. Samples were extracted with a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method to characterize and quantify the indole alkaloids. With the LC/QTOF-MS in auto MS/MS mode the indole alkaloids were identified, and the isomeric response of each could be used to determine whether the actual bark or extract was in samples of dietary supplements and not adulteration with yohimbine HCl. Analogs were identified and include yohimbic acid, methyl yohimbine, and hydroxyl yohimbine. Many isomers of each were also detected, but identified only by the number of chromatographic peaks. Quantification of yohimbine and ajmalicine spiked extracts showed recoveries of 99 to 103% with RSD of 3.6% or lower and LODs of less than 100 ppt. Calibration of the two standards gave r(2) = 0.9999 in a range from 0.1 to 100 ppb. Dietary supplements quantified for these two compounds showed a range from not detected to 3x the amounts found in the bark.

A Method for Comprehensive Glycosite-Mapping and Direct Quantitation of Serum Glycoproteins.

PubMed

Hong, Qiuting; Ruhaak, L Renee; Stroble, Carol; Parker, Evan; Huang, Jincui; Maverakis, Emanual; Lebrilla, Carlito B

2015-12-04

A comprehensive glycan map was constructed for the top eight abundant glycoproteins in plasma using both specific and nonspecific enzyme digestions followed by nano liquid chromatography (LC)-chip/quadrupole time-of-flight mass spectrometry (MS) analysis. Glycopeptides were identified using an in-house software tool, GPFinder. A sensitive and reproducible multiple reaction monitoring (MRM) technique on a triple quadrupole MS was developed and applied to quantify immunoglobulins G, A, M, and their site-specific glycans simultaneously and directly from human serum/plasma without protein enrichments. A total of 64 glycopeptides and 15 peptides were monitored for IgG, IgA, and IgM in a 20 min ultra high performance (UP)LC gradient. The absolute protein contents were quantified using peptide calibration curves. The glycopeptide ion abundances were normalized to the respective protein abundances to separate protein glycosylation from protein expression. This technique yields higher method reproducibility and less sample loss when compared with the quantitation method that involves protein enrichments. The absolute protein quantitation has a wide linear range (3-4 orders of magnitude) and low limit of quantitation (femtomole level). This rapid and robust quantitation technique, which provides quantitative information for both proteins and glycosylation, will further facilitate disease biomarker discoveries.

Development and validation of the simultaneous measurement of four vitamin D metabolites in serum by LC-MS/MS for clinical laboratory applications.

PubMed

Satoh, Mamoru; Ishige, Takayuki; Ogawa, Shoujiro; Nishimura, Motoi; Matsushita, Kazuyuki; Higashi, Tatsuya; Nomura, Fumio

2016-11-01

The quantification of serum 25-hydroxyvitamin D [25(OH)D] as an indicator of vitamin D status is currently primarily conducted by immunoassays, yet LC-MS/MS would allow more accurate determination. Furthermore, LC-MS/MS would allow simultaneous measurement of multiple analytes. The aim of this study was to develop and validate an LC-MS/MS method to simultaneously measure four vitamin D metabolites (25(OH)D 3 , 3-epi-25(OH)D 3 , 25(OH)D 2 , and 24,25(OH) 2 D 3 ) in serum for clinical laboratory applications. Serum samples were first prepared in a 96-well supported liquid extraction plate and the eluate was derivatized using the Cookson-type reagent 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD), which rapidly and quantitatively reacts with the s-cis-diene structure of vitamin D metabolites. The derivatized samples were subjected to LC-MS/MS, ionized by electrospray ionization (positive-ion mode), and detected by selected reaction monitoring. The lower limits of quantification for 25(OH)D 3 , 3-epi-25(OH)D 3 , 25(OH)D 2 , and 24,25(OH) 2 D 3 were 0.091, 0.020, 0.013, and 0.024 ng/mL, respectively. The accuracy values and the extraction recoveries for these four metabolites were satisfactory. Serum 25(OH)D levels determined by our LC-MS/MS were compared with those obtained by conventional radioimmunoassay (RIA) that cannot distinguish 25(OH)D 3 and 25(OH)D 2 . The values obtained by the RIA method exhibited a mean bias of about 8.35 ng/mL, most likely as a result of cross reaction of the antibody with low-abundance metabolites, including 24,25(OH) 2 D 3 . Various preanalytical factors, such as long sample sitting prior to serum separation, repeated freeze-thaw cycles, and the presence of anticoagulants, had no significant effects on these determinations. This high-throughput LC-MS/MS simultaneous assay of the four vitamin D metabolites 25(OH)D 3 , 3-epi-25(OH)D 3 , 25(OH)D 2 , and 24,25(OH) 2 D 3 required as little as 20 μL serum. This method will aid further understanding of low-abundance vitamin D metabolites, as well as the accurate determination of 25(OH)D 3 and 25(OH)D 2 .

Simultaneous analysis of 17 diuretics in dietary supplements by HPLC and LC-MS/MS.

PubMed

Woo, H; Kim, J W; Han, K M; Lee, J H; Hwang, I S; Lee, J H; Kim, J; Kweon, S J; Cho, S; Chae, K R; Han, S Y; Kim, J

2013-01-01

In order to test health foods for illegally added diuretics for weight loss, we developed simple, rapid, selective, and sensitive methods using HPLC and LC-MS/MS for the simultaneous analysis of 17 diuretics in dietary supplements. HPLC conditions were set with a Capcell-pak C18, using a mobile phase consisting of gradient conditions, UV detection at 254 nm and validated for linearity (r(2)> 0.999), precision (CV ≤ 3%), recoveries (90.4-102.8%) and reproducibility. Identification and quantification of 17 diuretics were accomplished by ion-spray LC-MS/MS using multiple reaction monitoring (MRM). The chromatographic separation was carried out under the reversed-phase mechanism on an HSS-T3 column. The LC-MS/MS method was validated for linearity (r(2)> 0.99) and precision (CV < 13%). Sixteen dietary supplements were tested with the developed methods. Diuretics were not detected in all samples. Extraction recovery was also investigated and the extraction recoveries in different formulations were from 88% to 110% and from 81% to 116% using HPLC and LC-MS/MS, respectively. There was no significant difference in recoveries in the type of dietary supplements. Based on this result, the developed methods to monitor illegal drug adulterations in dietary supplements using HPLC and LC-MS/MS are simple, fast and reliable. Therefore, it is applicable to routine drug-adulteration screening.

msCompare: A Framework for Quantitative Analysis of Label-free LC-MS Data for Comparative Candidate Biomarker Studies*

PubMed Central

Hoekman, Berend; Breitling, Rainer; Suits, Frank; Bischoff, Rainer; Horvatovich, Peter

2012-01-01

Data processing forms an integral part of biomarker discovery and contributes significantly to the ultimate result. To compare and evaluate various publicly available open source label-free data processing workflows, we developed msCompare, a modular framework that allows the arbitrary combination of different feature detection/quantification and alignment/matching algorithms in conjunction with a novel scoring method to evaluate their overall performance. We used msCompare to assess the performance of workflows built from modules of publicly available data processing packages such as SuperHirn, OpenMS, and MZmine and our in-house developed modules on peptide-spiked urine and trypsin-digested cerebrospinal fluid (CSF) samples. We found that the quality of results varied greatly among workflows, and interestingly, heterogeneous combinations of algorithms often performed better than the homogenous workflows. Our scoring method showed that the union of feature matrices of different workflows outperformed the original homogenous workflows in some cases. msCompare is open source software (https://trac.nbic.nl/mscompare), and we provide a web-based data processing service for our framework by integration into the Galaxy server of the Netherlands Bioinformatics Center (http://galaxy.nbic.nl/galaxy) to allow scientists to determine which combination of modules provides the most accurate processing for their particular LC-MS data sets. PMID:22318370

Triple Quadrupole Versus High Resolution Quadrupole-Time-of-Flight Mass Spectrometry for Quantitative LC-MS/MS Analysis of 25-Hydroxyvitamin D in Human Serum

NASA Astrophysics Data System (ADS)

Geib, Timon; Sleno, Lekha; Hall, Rabea A.; Stokes, Caroline S.; Volmer, Dietrich A.

2016-08-01

We describe a systematic comparison of high and low resolution LC-MS/MS assays for quantification of 25-hydroxyvitamin D3 in human serum. Identical sample preparation, chromatography separations, electrospray ionization sources, precursor ion selection, and ion activation were used; the two assays differed only in the implemented final mass analyzer stage; viz. high resolution quadrupole-quadrupole-time-of-flight (QqTOF) versus low resolution triple quadrupole instruments. The results were assessed against measured concentration levels from a routine clinical chemiluminescence immunoassay. Isobaric interferences prevented the simple use of TOF-MS spectra for extraction of accurate masses and necessitated the application of collision-induced dissociation on the QqTOF platform. The two mass spectrometry assays provided very similar analytical figures of merit, reflecting the lack of relevant isobaric interferences in the MS/MS domain, and were successfully applied to determine the levels of 25-hydroxyvitamin D for patients with chronic liver disease.

Use of phenyl/tetrazolyl-functionalized magnetic microspheres and stable isotope labeled internal standards for significant reduction of matrix effect in determination of nine fluoroquinolones by liquid chromatography-quadrupole linear ion trap mass spectrometry.

PubMed

Xu, Fei; Liu, Feng; Wang, Chaozhan; Wei, Yinmao

2018-02-01

In this study, the strategy of unique adsorbent combined with isotope labeled internal standards was used to significantly reduce the matrix effect for the enrichment and analysis of nine fluoroquinolones in a complex sample by liquid chromatography coupled to quadrupole linear ion trap mass spectrometry (LC-QqQ LIT -MS/MS). The adsorbent was prepared conveniently by functionalizing Fe 3 O 4 @SiO 2 microspheres with phenyl and tetrazolyl groups, which could adsorb fluoroquinolones selectively via hydrophobic, electrostatic, and π-π interactions. The established magnetic solid-phase extraction (MSPE) method as well as using stable isotope labeled internal standards in the next MS/MS detection was able to reduce the matrix effect significantly. In the process of LC-QqQ LIT -MS/MS analysis, the precursor and product ions of the analytes were monitored quantitatively and qualitatively on a QTrap system equipped simultaneously with the multiple reaction monitoring (MRM) and enhanced product ion (EPI) scan. Subsequently, the enrichment method combined with LC-QqQ LIT -MS/MS demonstrated good analytical features in terms of linearity (7.5-100.0 ng mL -1 , r > 0.9960), satisfactory recoveries (88.6%-118.3%) with RSDs < 12.0%, LODs = 0.5 μg kg -1 and LOQs = 1.5 μg kg -1 for all tested analytes. Finally, the developed MSPE-LC-QqQ LIT -MS/MS method had been successfully applied to real pork samples for food-safety risk monitoring in Ningxia Province, China. Graphical abstract Mechanism of reducing matrix effect through the as-prepared adsorbent.

Reagent for Evaluating Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Performance in Bottom-Up Proteomic Experiments.

PubMed

Beri, Joshua; Rosenblatt, Michael M; Strauss, Ethan; Urh, Marjeta; Bereman, Michael S

2015-12-01

We present a novel proteomic standard for assessing liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrument performance, in terms of chromatographic reproducibility and dynamic range within a single LC-MS/MS injection. The peptide mixture standard consists of six peptides that were specifically synthesized to cover a wide range of hydrophobicities (grand average hydropathy (GRAVY) scores of -0.6 to 1.9). A combination of stable isotope labeled amino acids ((13)C and (15)N) were inserted to create five isotopologues. By combining these isotopologues at different ratios, they span four orders of magnitude within each distinct peptide sequence. Each peptide, from lightest to heaviest, increases in abundance by a factor of 10. We evaluate several metrics on our quadrupole orbitrap instrument using the 6 × 5 LC-MS/MS reference mixture spiked into a complex lysate background as a function of dynamic range, including mass measurement accuracy (MMA) and the linear range of quantitation of MS1 and parallel reaction monitoring experiments. Detection and linearity of the instrument routinely spanned three orders of magnitude across the gradient (500 fmol to 0.5 fmol on column) and no systematic trend was observed for MMA of targeted peptides as a function of abundance by analysis of variance analysis (p = 0.17). Detection and linearity of the fifth isotopologue (i.e., 0.05 fmol on column) was dependent on the peptide and instrument scan type (MS1 vs PRM). We foresee that this standard will serve as a powerful method to conduct both intra-instrument performance monitoring/evaluation, technology development, and inter-instrument comparisons.

MassSieve: Panning MS/MS peptide data for proteins

PubMed Central

Slotta, Douglas J.; McFarland, Melinda A.; Markey, Sanford P.

2010-01-01

We present MassSieve, a Java-based platform for visualization and parsimony analysis of single and comparative LC-MS/MS database search engine results. The success of mass spectrometric peptide sequence assignment algorithms has led to the need for a tool to merge and evaluate the increasing data set sizes that result from LC-MS/MS-based shotgun proteomic experiments. MassSieve supports reports from multiple search engines with differing search characteristics, which can increase peptide sequence coverage and/or identify conflicting or ambiguous spectral assignments. PMID:20564260

Development and validation of LC-MS/MS method for the determination of Ochratoxin A and its metabolite Ochratoxin α in poultry tissues and eggs.

PubMed

Paoloni, Angela; Solfrizzo, Michele; Bibi, Rita; Pecorelli, Ivan

2018-05-04

The objective of this study was to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of Ochratoxin A (OTA) and Ochratoxin α (OTα) in poultry tissues and eggs. The two toxins were extracted by a mixture of acetonitrile/water, purified with a reversed phase C18 solid phase extraction column (SPE) and determined by LC-MS/MS. The LC-MS/MS method performances were evaluated in terms of linearity in solvent and in matrix (ranged from 0.5 to 15.10 µg L -1 for OTA and from 0.60 to 17.85 µg L -1 for OTα), limit of detection (LOD), limit of quantitation (LOQ), specificity, accuracy and precision in repeatability conditions. Recovery experiments were performed by spiking poultry liver, kidney, muscle and eggs around 1 µg kg -1 and 10 µg kg -1 . LODs were 0.27 and 0.26 µg kg -1 while LOQs were fixed at 1.0 and 1.2 µg kg -1 for OTA and OTα, respectively. Main recoveries for OTA ranged from 82 to 109% and for OTα ranged from 55 to 89%. The values of within-laboratory relative standard deviation (RSD r ) were equal to or below 20%. Considering the results obtained and that all analytical performance criteria were fulfilled, the new extraction and purification method developed for OTA and OTα determination in animal tissues and eggs was found appropriate for control laboratories and research activities designed to ensure food safety.

Optimized approaches for quantification of drug transporters in tissues and cells by MRM proteomics.

PubMed

Prasad, Bhagwat; Unadkat, Jashvant D

2014-07-01

Drug transporter expression in tissues (in vivo) usually differs from that in cell lines used to measure transporter activity (in vitro). Therefore, quantification of transporter expression in tissues and cell lines is important to develop scaling factor for in vitro to in vivo extrapolation (IVIVE) of transporter-mediated drug disposition. Since traditional immunoquantification methods are semiquantitative, targeted proteomics is now emerging as a superior method to quantify proteins, including membrane transporters. This superiority is derived from the selectivity, precision, accuracy, and speed of analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode. Moreover, LC-MS/MS proteomics has broader applicability because it does not require selective antibodies for individual proteins. There are a number of recent research and review papers that discuss the use of LC-MS/MS for transporter quantification. Here, we have compiled from the literature various elements of MRM proteomics to provide a comprehensive systematic strategy to quantify drug transporters. This review emphasizes practical aspects and challenges in surrogate peptide selection, peptide qualification, peptide synthesis and characterization, membrane protein isolation, protein digestion, sample preparation, LC-MS/MS parameter optimization, method validation, and sample analysis. In particular, bioinformatic tools used in method development and sample analysis are discussed in detail. Various pre-analytical and analytical sources of variability that should be considered during transporter quantification are highlighted. All these steps are illustrated using P-glycoprotein (P-gp) as a case example. Greater use of quantitative transporter proteomics will lead to a better understanding of the role of drug transporters in drug disposition.

Mass spectrometric-based stable isotopic 2-aminobenzoic acid glycan mapping for rapid glycan screening of biotherapeutics.

PubMed

Prien, Justin M; Prater, Bradley D; Qin, Qiang; Cockrill, Steven L

2010-02-15

Fast, sensitive, robust methods for "high-level" glycan screening are necessary during various stages of a biotherapeutic product's lifecycle, including clone selection, process changes, and quality control for lot release testing. Traditional glycan screening involves chromatographic or electrophoretic separation-based methods, and, although reproducible, these methods can be time-consuming. Even ultrahigh-performance chromatographic and microfluidic integrated LC/MS systems, which work on the tens of minute time scale, become lengthy when hundreds of samples are to be analyzed. Comparatively, a direct infusion mass spectrometry (MS)-based glycan screening method acquires data on a millisecond time scale, exhibits exquisite sensitivity and reproducibility, and is amenable to automated peak annotation. In addition, characterization of glycan species via sequential mass spectrometry can be performed simultaneously. Here, we demonstrate a quantitative high-throughput MS-based mapping approach using stable isotope 2-aminobenzoic acid (2-AA) for rapid "high-level" glycan screening.

Data from quantitative label free proteomics analysis of rat spleen.

PubMed

Dudekula, Khadar; Le Bihan, Thierry

2016-09-01

The dataset presented in this work has been obtained using a label-free quantitative proteomic analysis of rat spleen. A robust method for extraction of proteins from rat spleen tissue and LC-MS-MS analysis was developed using a urea and SDS-based buffer. Different fractionation methods were compared. A total of 3484 different proteins were identified from the pool of all experiments run in this study (a total of 2460 proteins with at least two peptides). A total of 1822 proteins were identified from nine non-fractionated pulse gels, 2288 proteins and 2864 proteins were identified by SDS-PAGE fractionation into three and five fractions respectively. The proteomics data are deposited in ProteomeXchange Consortium via PRIDE PXD003520, Progenesis and Maxquant output are presented in the supported information. The generated list of proteins under different regimes of fractionation allow assessing the nature of the identified proteins; variability in the quantitative analysis associated with the different sampling strategy and allow defining a proper number of replicates for future quantitative analysis.

Quantification of Peptides from Immunoglobulin Constant and Variable Regions by Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry for Assessment of Multiple Myeloma Patients

PubMed Central

Remily-Wood, Elizabeth R.; Benson, Kaaron; Baz, Rachid C.; Chen, Y. Ann; Hussein, Mohamad; Hartley-Brown, Monique A.; Sprung, Robert W.; Perez, Brianna; Liu, Richard Z.; Yoder, Sean; Teer, Jamie; Eschrich, Steven A.; Koomen, John M.

2014-01-01

Purpose Quantitative mass spectrometry assays for immunoglobulins (Igs) are compared with existing clinical methods in samples from patients with plasma cell dyscrasias, e.g. multiple myeloma. Experimental design Using LC-MS/MS data, Ig constant region peptides and transitions were selected for liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM). Quantitative assays were used to assess Igs in serum from 83 patients. Results LC-MRM assays quantify serum levels of Igs and their isoforms (IgG1–4, IgA1–2, IgM, IgD, and IgE, as well as kappa(κ) and lambda(λ) light chains). LC-MRM quantification has been applied to single samples from a patient cohort and a longitudinal study of an IgE patient undergoing treatment, to enable comparison with existing clinical methods. Proof-of-concept data for defining and monitoring variable region peptides are provided using the H929 multiple myeloma cell line and two MM patients. Conclusions and Clinical Relevance LC-MRM assays targeting constant region peptides determine the type and isoform of the involved immunoglobulin and quantify its expression; the LC-MRM approach has improved sensitivity compared with the current clinical method, but slightly higher interassay variability. Detection of variable region peptides is a promising way to improve Ig quantification, which could produce a dramatic increase in sensitivity over existing methods, and could further complement current clinical techniques. PMID:24723328

Simultaneous Proteomic Discovery and Targeted Monitoring using Liquid Chromatography, Ion Mobility Spectrometry, and Mass Spectrometry.

PubMed

Burnum-Johnson, Kristin E; Nie, Song; Casey, Cameron P; Monroe, Matthew E; Orton, Daniel J; Ibrahim, Yehia M; Gritsenko, Marina A; Clauss, Therese R W; Shukla, Anil K; Moore, Ronald J; Purvine, Samuel O; Shi, Tujin; Qian, Weijun; Liu, Tao; Baker, Erin S; Smith, Richard D

2016-12-01

Current proteomic approaches include both broad discovery measurements and quantitative targeted analyses. In many cases, discovery measurements are initially used to identify potentially important proteins (e.g. candidate biomarkers) and then targeted studies are employed to quantify a limited number of selected proteins. Both approaches, however, suffer from limitations. Discovery measurements aim to sample the whole proteome but have lower sensitivity, accuracy, and quantitation precision than targeted approaches, whereas targeted measurements are significantly more sensitive but only sample a limited portion of the proteome. Herein, we describe a new approach that performs both discovery and targeted monitoring (DTM) in a single analysis by combining liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS). In DTM, heavy labeled target peptides are spiked into tryptic digests and both the labeled and unlabeled peptides are detected using LC-IMS-MS instrumentation. Compared with the broad LC-MS discovery measurements, DTM yields greater peptide/protein coverage and detects lower abundance species. DTM also achieved detection limits similar to selected reaction monitoring (SRM) indicating its potential for combined high quality discovery and targeted analyses, which is a significant step toward the convergence of discovery and targeted approaches. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of RIP-II toxins by affinity enrichment, enzymatic digestion and LC-MS.

PubMed

Fredriksson, Sten-Åke; Artursson, Elisabet; Bergström, Tomas; Östin, Anders; Nilsson, Calle; Åstot, Crister

2015-01-20

Type 2 ribosome-inactivating protein toxins (RIP-II toxins) were enriched and purified prior to enzymatic digestion and LC-MS analysis. The enrichment of the RIP-II family of plant proteins, such as ricin, abrin, viscumin, and volkensin was based on their affinity for galactosyl moieties. A macroporous chromatographic material was modified with a galactose-terminated substituent and packed into miniaturized columns that were used in a chromatographic system to achieve up to 1000-fold toxin enrichment. The galactose affinity of the RIP-II proteins enabled their selective enrichment from water, beverages, and extracts of powder and wipe samples. The enriched fractions were digested with trypsin and RIP-II peptides were identified based on accurate mass LC-MS data. Their identities were unambiguously confirmed by LC-MS/MS product ion scans of peptides unique to each of the toxins. The LC-MS detection limit achieved for ricin target peptides was 10 amol and the corresponding detection limit for the full method was 10 fmol/mL (0.6 ng/mL). The affinity enrichment method was applied to samples from a forensic investigation into a case involving the illegal production of ricin and abrin toxins.

Quali-quantitative analysis of the phenolic fraction of the flowers of Corylus avellana, source of the Italian PGI product "Nocciola di Giffoni": Isolation of antioxidant diarylheptanoids.

PubMed

Masullo, Milena; Mari, Angela; Cerulli, Antonietta; Bottone, Alfredo; Kontek, Bogdan; Olas, Beata; Pizza, Cosimo; Piacente, Sonia

2016-10-01

There is only limited information available on the chemical composition of the non-edible parts of Corylus avellana, source of the Italian PGI product "Nocciola di Giffoni" (hazelnut). An initial LC-MS profile of the methanolic extract of the male flowers of C. avellana, cultivar 'Tonda di Giffoni' led to the isolation of 12 compounds, of which the structures were elucidated by NMR spectroscopy. These were identified as three previously undescribed diarylheptanoids, named giffonins Q-S, along with nine known compounds. Furthermore, the quantitative determination of the main compounds occurring in the methanolic extract of C. avellana flowers was carried out by an analytical approach based on LC-ESI(QqQ)MS, using the Multiple Reaction Monitoring (MRM) experiment. In order to explore the antioxidant ability of C. avellana flowers, the methanolic extract and the isolated compounds were evaluated for their inhibitory effects on human plasma lipid peroxidation induced by H2O2 and H2O2/Fe(2+), by measuring the concentration of TBARS. Copyright © 2016 Elsevier Ltd. All rights reserved.

Quantitative evaluation of the matrix effect in bioanalytical methods based on LC-MS: A comparison of two approaches.

PubMed

Rudzki, Piotr J; Gniazdowska, Elżbieta; Buś-Kwaśnik, Katarzyna

2018-06-05

Liquid chromatography coupled to mass spectrometry (LC-MS) is a powerful tool for studying pharmacokinetics and toxicokinetics. Reliable bioanalysis requires the characterization of the matrix effect, i.e. influence of the endogenous or exogenous compounds on the analyte signal intensity. We have compared two methods for the quantitation of matrix effect. The CVs(%) of internal standard normalized matrix factors recommended by the European Medicines Agency were evaluated against internal standard normalized relative matrix effects derived from Matuszewski et al. (2003). Both methods use post-extraction spiked samples, but matrix factors require also neat solutions. We have tested both approaches using analytes of diverse chemical structures. The study did not reveal relevant differences in the results obtained with both calculation methods. After normalization with the internal standard, the CV(%) of the matrix factor was on average 0.5% higher than the corresponding relative matrix effect. The method adopted by the European Medicines Agency seems to be slightly more conservative in the analyzed datasets. Nine analytes of different structures enabled a general overview of the problem, still, further studies are encouraged to confirm our observations. Copyright © 2018 Elsevier B.V. All rights reserved.

Monolithic Integration of Two-Dimensional Liquid Chromatography-Capillary Electrophoresis and Electrospray Ionization on a Microfluidic Device

PubMed Central

Chambers, Andrew G.; Mellors, J. Scott; Henley, W. Hampton; Ramsey, J. Michael

2011-01-01

A microfluidic device capable of two-dimensional reversed-phase liquid chromatography-capillary electrophoresis with integrated electrospray ionization (LC-CE-ESI) for mass spectrometry (MS)-based proteomic applications is described. Traditional instrumentation was used for the LC sample injection and delivery of the LC mobile phase. The glass microfabricated device incorporated a sample-trapping region and an LC channel packed with reversed-phase particles. Rapid electrokinetic injections of the LC effluent into the CE dimension were performed at a cross channel intersection. The CE separation channel terminated at a corner of the square device, which functioned as an integrated electrospray tip. In addition to LC-CE-ESI, this device was used for LC-ESI without any instrumental modifications. To evaluate the system, LC-MS and LC-CE-MS analysis of protein digests were performed and compared. PMID:21214194

Comparison of Two ELISA Methods and Mass Spectrometry for Measurement of Vitamin D-Binding Protein: Implications for the Assessment of Bioavailable Vitamin D Concentrations across Genotypes

PubMed Central

Denburg, Michelle R.; Hoofnagle, Andrew N.; Sayed, Samir; Gupta, Jayanta; de Boer, Ian H.; Appel, Lawrence J.; Durazo-Arvizu, Ramon; Whitehead, Krista; Feldman, Harold I.; Leonard, Mary B.

2016-01-01

Studies using vitamin D-binding protein (DBP) concentrations to estimate free and bioavailable vitamin D have increased dramatically in recent years. Combinations of two single-nucleotide polymorphisms produce three major DBP isoforms (Gc1f, Gc1s and Gc2). A recent study showed that DBP concentrations quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) did not differ by race, while a widely used monoclonal ELISA quantified DBP differentially by isoform, yielding significantly lower DBP concentrations in black versus white individuals. We compared measurements of serum DBP using a monoclonal ELISA, a polyclonal ELISA, and LC-MS/MS in 125 participants in the Chronic Renal Insufficiency Cohort. Serum free and bioavailable 25OHD were calculated based on DBP concentrations from these three assays in homozygous participants, and race differences were compared. We confirmed that the monoclonal ELISA quantifies DBP differentially by isoform and demonstrated that the polyclonal ELISA is not subject to this bias. While ≤9% of the variability in DBP concentrations quantified using either LC-MS/MS or the polyclonal ELISA was explained by genotype, 85% of the variability in the monoclonal ELISA-based measures was explained by genotypes. DBP concentrations measured by the monoclonal ELISA were disproportionately lower than LC-MS/MS-based results for Gc1f homozygotes [median difference −67%; interquartile range (IQR) −71%, −64%], 95% of whom were black. In contrast, the polyclonal ELISA yielded consistently and similarly higher measurements of DBP than LC-MS/MS, irrespective of genotype, with a median percent difference of +50% [IQR +33%, +65%]. Contrary to findings using the monoclonal ELISA, DBP concentrations did not differ by race, and free and bioavailable 25OHD were significantly lower in black versus white participants based on both the polyclonal ELISA and LC-MS/MS, consistent with their lower total 25OHD. Future studies of DBP and free or bioavailable vitamin D metabolites should employ DBP assays that are not biased by DBP genotype. PMID:27250744

Quantitative liquid chromatography-mass spectrometry-multiple reaction monitoring (LC-MS-MRM) analysis of site-specific glycoforms of haptoglobin in liver disease.

PubMed

Sanda, Miloslav; Pompach, Petr; Brnakova, Zuzana; Wu, Jing; Makambi, Kepher; Goldman, Radoslav

2013-05-01

Development of liver disease is associated with the appearance of multiply fucosylated glycoforms of haptoglobin. To analyze the disease-related haptoglobin glycoforms in liver cirrhosis and hepatocellular carcinoma, we have optimized an LC-MS-multiple reaction monitoring (MRM) workflow for glycopeptide quantification. The final quantitative analysis included 24 site-specific glycoforms generated by treatment of a tryptic digest of haptoglobin with α(2-3,6,8)-neuraminidase and β(1-4)-galactosidase. The combination of LC-MS-MRM with exoglycosidase digests allowed resolution of isobaric glycoforms of the haptoglobin-T3 glycopeptide for quantification of the multiply fucosylated Lewis Y-containing glycoforms we have identified in the context of liver disease. Fourteen multiply fucosylated glycoforms of the 20 examined increased significantly in the liver disease group compared with healthy controls with an average 5-fold increase in intensity (p < 0.05). At the same time, two tri-antennary glycoforms without fucoses did not increase in the liver disease group, and two tetra-antennary glycoforms without fucoses showed a marginal increase (at most 40%) in intensity. Our analysis of 30 individual patient samples (10 healthy controls, 10 cirrhosis patients, and 10 hepatocellular carcinoma patients) showed that these glycoforms were substantially increased in a small subgroup of liver disease patients but did not significantly differ between the groups of hepatocellular carcinoma and cirrhosis patients. The tri- and tetra-antennary singly fucosylated glycoforms are associated with a MELD score and low platelet counts (p < 0.05). The exoglycosidase-assisted LC-MS-MRM workflow, optimized for the quantification of fucosylated glycoforms of haptoglobin, can be used for quantification of these glycoforms on other glycopeptides with appropriate analytical behavior.

Quantitative Liquid Chromatography-Mass Spectrometry-Multiple Reaction Monitoring (LC-MS-MRM) Analysis of Site-specific Glycoforms of Haptoglobin in Liver Disease*

PubMed Central

Sanda, Miloslav; Pompach, Petr; Brnakova, Zuzana; Wu, Jing; Makambi, Kepher; Goldman, Radoslav

2013-01-01

Development of liver disease is associated with the appearance of multiply fucosylated glycoforms of haptoglobin. To analyze the disease-related haptoglobin glycoforms in liver cirrhosis and hepatocellular carcinoma, we have optimized an LC-MS-multiple reaction monitoring (MRM) workflow for glycopeptide quantification. The final quantitative analysis included 24 site-specific glycoforms generated by treatment of a tryptic digest of haptoglobin with α(2–3,6,8)-neuraminidase and β(1–4)-galactosidase. The combination of LC-MS-MRM with exoglycosidase digests allowed resolution of isobaric glycoforms of the haptoglobin-T3 glycopeptide for quantification of the multiply fucosylated Lewis Y-containing glycoforms we have identified in the context of liver disease. Fourteen multiply fucosylated glycoforms of the 20 examined increased significantly in the liver disease group compared with healthy controls with an average 5-fold increase in intensity (p < 0.05). At the same time, two tri-antennary glycoforms without fucoses did not increase in the liver disease group, and two tetra-antennary glycoforms without fucoses showed a marginal increase (at most 40%) in intensity. Our analysis of 30 individual patient samples (10 healthy controls, 10 cirrhosis patients, and 10 hepatocellular carcinoma patients) showed that these glycoforms were substantially increased in a small subgroup of liver disease patients but did not significantly differ between the groups of hepatocellular carcinoma and cirrhosis patients. The tri- and tetra-antennary singly fucosylated glycoforms are associated with a MELD score and low platelet counts (p < 0.05). The exoglycosidase-assisted LC-MS-MRM workflow, optimized for the quantification of fucosylated glycoforms of haptoglobin, can be used for quantification of these glycoforms on other glycopeptides with appropriate analytical behavior. PMID:23389048

Rapid screening and determination of 11 new psychoactive substances by direct analysis in real time mass spectrometry and liquid chromatography/quadrupole time-of-flight mass spectrometry.

PubMed

Nie, Honggang; Li, Xianjiang; Hua, Zhendong; Pan, Wei; Bai, Yanping; Fu, Xiaofang

2016-08-01

With the amounts and types of new psychoactive substances (NPSs) increasing rapidly in recent years, an excellent high-throughput method for the analysis of these compounds is urgently needed. In this article, a rapid screening method and a quantitative analysis method for 11 NPSs are described and compared, respectively. A simple direct analysis in real time mass spectrometry (DART-MS) method was developed for the analysis of 11 NPSs including three categories of these substances present on the global market such as four cathinones, one phenylethylamine, and six synthetic cannabinoids. In order to analyze these compounds quantitatively with better accuracy and sensitivity, another rapid analytical method with a low limit of detection (LOD) was also developed using liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (LC/QTOFMS). The 11 NPSs could be determined within 0.5 min by DART-MS. Furthermore, they could also be separated and determined within 5 min by the LC/QTOFMS method. The two methods both showed good linearity with correlation coefficients (r(2) ) higher than 0.99. The LODs for all these target NPSs by DART-MS and LC/QTOFMS ranged from 5 to 40 ng mL(-1) and 0.1 to 1 ng mL(-1) , respectively. Confiscated samples, named as "music vanilla" and "bath salt", and 11 spiked samples were firstly screened by DART-MS and then determined by LC/QTOFMS. The identification of NPSs in confiscated materials was successfully achieved, and the proposed analytical methodology could offer rapid screening and accurate analysis results. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

LC-MS/MS strategies for therapeutic antibodies and investigation into the quantitative impact of antidrug-antibodies.

PubMed

Ewles, Matthew; Mannu, Ranbir; Fox, Chris; Stanta, Johannes; Evans, Graeme; Goodwin, Lee; Duffy, James; Bell, Len; Estdale, Sian; Firth, David

2016-12-01

We aimed to establish novel, high-throughput LC-MS/MS strategies for quantification of monoclonal antibodies in human serum and examine the potential impact of antidrug antibodies. We present two strategies using a thermally stable immobilized trypsin. The first strategy uses whole serum digestion and the second introduces Protein G enrichment to improve the selectivity. The impact of anti-trastuzumab antibodies on the methods was tested. Whole serum digestion has been validated for trastuzumab (LLOQ 0.25 µg/ml). Protein G enrichment has been validated for trastuzumab (LLOQ 0.1 µg/ml), bevacizumab (LLOQ 0.1 µg/ml) and adalimumab (LLOQ 0.25 µg/ml). We have shown the potential for anti-drug antibodies to impact on the quantification and we have subsequently established a strategy to overcome this impact where total quantification is desired.

Quantitative analysis of factor P (Properdin) in monkey serum using immunoaffinity capturing in combination with LC-MS/MS.

PubMed

Gao, Xinliu; Lin, Hui; Krantz, Carsten; Garnier, Arlette; Flarakos, Jimmy; Tse, Francis L S; Li, Wenkui

2016-01-01

Factor P (Properdin), an endogenous glycoprotein, plays a key role in innate immune defense. Its quantification is important for understanding the pharmacodynamics (PD) of drug candidate(s). In the present work, an immunoaffinity capturing LC-MS/MS method has been developed and validated for the first time for the quantification of factor P in monkey serum with a dynamic range of 125 to 25,000 ng/ml using the calibration standards and QCs prepared in factor P depleted monkey serum. The intra- and inter-run precision was ≤7.2% (CV) and accuracy within ±16.8% (%Bias) across all QC levels evaluated. Results of other evaluations (e.g., stability) all met the acceptance criteria. The validated method was robust and implemented in support of a preclinical PK/PD study.

A porous graphitized carbon LC-ESI/MS method for the quantitation of metronidazole and fluconazole in breast milk and human plasma.

PubMed

Geballa-Koukoula, Ariadni; Panderi, Irene; Zervas, Konstantinos; Geballa-Koukoulas, Khalil; Kavvalou, Eirini; Panteri-Petratou, Eirini; Vourna, Panagiota; Gennimata, Dimitra

2018-05-01

Information on drug transfer into the breast milk is essential to protect the infant from undesirable adverse effects of maternal consumption of drugs and to allow effective pharmacological treatment of breastfeeding mothers. Metronidazole and fluconazole are two drugs frequently used in nursing women to treat various infections, thus questioning infant's safety due to drug exposure through breast milk. In this article a porous graphitized carbon LC/ESI-MS assay was developed for the quantitation of metronidazole and fluconazole in breast milk and human plasma. The assay was based on the use of 150 μL of biological samples, following acetonitrile precipitation of proteins and filtration that enabled injection into the LC/ESI-MS system. All analytes and the internal standard, ropinirole, were separated by using a porous graphitized carbon analytical column (150 × 2.1 mm i.d., particle size 5 μm) with isocratic elution. The mobile phase consists of 55% acetonitrile in water acidified with 0.1% concentrated formic acid and pumped at a flow rate of 0.25 mL min -1 . The assay was linear over a concentration range of 0.1 to 15 μg mL -1 for all analytes in both biological samples. Intermediate precision was found to be <8.4% over the tested concentration ranges. A run time of <5 min for each sample made it possible to analyze a large number of biological samples per day. The method is the first reported application for the analysis of metronidazole and fluconazole in both breast milk and human plasma and it can be used to support a wide range of clinical studies. Copyright © 2018 Elsevier B.V. All rights reserved.

Quantitative analysis of glycation and its impact on antigen binding

PubMed Central

Mo, Jingjie; Yan, Qingrong; Sokolowska, Izabela; Lewis, Michael J.; Hu, Ping

2018-01-01

ABSTRACT Glycation has been observed in antibody therapeutics manufactured by the fed-batch fermentation process. It not only increases the heterogeneity of antibodies, but also potentially affects product safety and efficacy. In this study, non-glycated and glycated fractions enriched from a monoclonal antibody (mAb1) as well as glucose-stressed mAb1 were characterized using a variety of biochemical, biophysical and biological assays to determine the effects of glycation on the structure and function of mAb1. Glycation was detected at multiple lysine residues and reduced the antigen binding activity of mAb1. Heavy chain Lys100, which is located in the complementary-determining region of mAb1, had the highest levels of glycation in both stressed and unstressed samples, and glycation of this residue was likely responsible for the loss of antigen binding based on hydrogen/deuterium exchange mass spectrometry analysis. Peptide mapping and intact liquid chromatography-mass spectrometry (LC-MS) can both be used to monitor the glycation levels. Peptide mapping provides site specific glycation results, while intact LC-MS is a quicker and simpler method to quantitate the total glycation levels and is more useful for routine testing. Capillary isoelectric focusing (cIEF) can also be used to monitor glycation because glycation induces an acidic shift in the cIEF profile. As expected, total glycation measured by intact LC-MS correlated very well with the percentage of total acidic peaks or main peak measured by cIEF. In summary, we demonstrated that glycation can affect the function of a representative IgG1 mAb. The analytical characterization, as described here, should be generally applicable for other therapeutic mAbs. PMID:29436927

Tissue distribution, metabolism and excretion of 3, 3′-dichloro-4′-sulfooxy-biphenyl in the rat

PubMed Central

Grimm, Fabian A.; He, Xianran; Teesch, Lynn M.; Lehmler, Hans-Joachim; Robertson, Larry W.; Duffel, Michael W.

2015-01-01

Polychlorinated biphenyls (PCBs) with lower numbers of chlorine atoms exhibit a greater susceptibility to metabolism than their higher-chlorinated counterparts. Following initial hydroxylation of these lower chlorinated PCBs, metabolic sulfation to form PCB sulfates is increasingly recognized as an important component of their toxicology. Since procedures for the quantitative analysis of PCB sulfates in tissue samples have not been previously available, we have now developed an efficient, LC-ESI-MS/MS based, protocol for the quantitative analysis of 4-PCB 11 sulfate in biological samples. This procedure was used to determine the distribution of 4-PCB 11 sulfate in liver, kidney, lung, and brain, as well as its excretion profile, following its intravenous administration to male Sprague-Dawley rats. Following initial uptake of 4-PCB 11 sulfate, its concentration in these tissues and serum declined within the first hour following injection. Although biliary secretion was detected, analysis of 24 hour collections of urine and feces revealed recovery of less than 4% of the administered 4-PCB 11 sulfate. High-resolution LC-MS analysis of bile, urine, and feces showed metabolic products derived from 4-PCB 11 sulfate. Thus, 4-PCB 11 sulfate at this dose was not directly excreted in the urine, but was, instead, re-distributed to tissues and/or subjected to further metabolism. PMID:26046945

Simultaneous determination of creatinine and creatine in human serum by double-spike isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS).

PubMed

Fernández-Fernández, Mario; Rodríguez-González, Pablo; Añón Álvarez, M Elena; Rodríguez, Felix; Menéndez, Francisco V Álvarez; García Alonso, J Ignacio

2015-04-07

This work describes the first multiple spiking isotope dilution procedure for organic compounds using (13)C labeling. A double-spiking isotope dilution method capable of correcting and quantifying the creatine-creatinine interconversion occurring during the analytical determination of both compounds in human serum is presented. The determination of serum creatinine may be affected by the interconversion between creatine and creatinine during sample preparation or by inefficient chemical separation of those compounds by solid phase extraction (SPE). The methodology is based on the use differently labeled (13)C analogues ((13)C1-creatinine and (13)C2-creatine), the measurement of the isotopic distribution of creatine and creatinine by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the application of multiple linear regression. Five different lyophilized serum-based controls and two certified human serum reference materials (ERM-DA252a and ERM-DA253a) were analyzed to evaluate the accuracy and precision of the proposed double-spike LC-MS/MS method. The methodology was applied to study the creatine-creatinine interconversion during LC-MS/MS and gas chromatography-mass spectrometry (GC-MS) analyses and the separation efficiency of the SPE step required in the traditional gas chromatography-isotope dilution mass spectrometry (GC-IDMS) reference methods employed for the determination of serum creatinine. The analysis of real serum samples by GC-MS showed that creatine-creatinine separation by SPE can be a nonquantitative step that may induce creatinine overestimations up to 28% in samples containing high amounts of creatine. Also, a detectable conversion of creatine into creatinine was observed during sample preparation for LC-MS/MS. The developed double-spike LC-MS/MS improves the current state of the art for the determination of creatinine in human serum by isotope dilution mass spectrometry (IDMS), because corrections are made for all the possible errors derived from the sample preparation step.

Liquid to liquid extraction and liquid chromatography-tandem mass spectrometry determination of hainanmycin in feed.

PubMed

Wang, Ze Ping; Shen, Jian Zhong; Linhardt, Robert J; Jiang, Hui; Cheng, Lin Li

2017-03-01

Hainanmycin is a new veterinary polyether antibiotic and has few sensitive analytical method in present days. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) relying on multiple reaction monitoring (MRM) detection was developed for analysis of hainanmycin in animal feed. Feed samples were extracted with ethyl acetate and purified by two steps of liquid-liquid extraction (LLE) to get rid of water solvable matrix and lipids one by one. The final simple was analyzed by LC-MS/MS. The LC mobile phase was composed of 0.1% aqueous formic acid and 0.1% formic acidified acetonitrile by gradient elution. Average recoveries ranged from 74.22% to 87.85%, as determined by spiking with 2.0 (LOQ) ∼2500μgkg -1 of hainanmycin. The inter-day and intra-day coefficient of variation was 9.21% to 11.77% and 7.67% to 13.49%, respectively. The limit of detection (LOD) and the limit of quantitation (LOQ) were 0.36μgkg -1 and 2.0μgkg -1 , respectively. Copyright © 2016. Published by Elsevier B.V.

High-throughput screening for multi-class veterinary drug residues in animal muscle using liquid chromatography/tandem mass spectrometry with on-line solid-phase extraction.

PubMed

Tang, Hubert Po-On; Ho, Clare; Lai, Shirley Sau-Ling

2006-01-01

A rapid qualitative method using on-line column-switching liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed and validated for screening 13 target veterinary drugs: four macrolides - erythromycin A, josamycin (leucomycin A3), kitasamycin (leucomycin A5), and tylosin A; six (fluoro)quinolones - ciprofloxacin, danofloxacin, enrofloxacin, flumequine, oxolinic acid, and sarafloxacin; and lincomycin, virginiamycin M1, and trimethoprim in different animal muscles. Clindamycin, norfloxacin, nalidixic acid, oleandomycin, ormetoprim, and roxithromycin were used as the internal standards. After simple deproteination and analyte extraction of muscle samples using acetonitrile, the supernatant was subjected to on-line cleanup and direct analysis by LC/MS/MS. On-line cleanup with an extraction cartridge packed with hydrophilic-hydrophobic polymer sorbent followed by fast LC using a short C18 column resulted in a total analysis cycle of 6 min for 19 drugs. This screening method considerably reduced the time and the cost for the quantitative and confirmatory analyses. The application of a control point approach was also introduced and explained. Copyright (c) 2006 John Wiley & Sons, Ltd.

The Short Cosyntropin Test Revisited: New Normal Reference Range Using LC-MS/MS.

PubMed

Ueland, Grethe Å; Methlie, Paal; Øksnes, Marianne; Thordarson, Hrafnkell B; Sagen, Jørn; Kellmann, Ralf; Mellgren, Gunnar; Ræder, Maria; Dahlqvist, Per; Dahl, Sandra R; Thorsby, Per M; Løvås, Kristian; Husebye, Eystein S

2018-04-01

The cosyntropin test is used to diagnose adrenal insufficiency (AI) and nonclassical congenital adrenal hyperplasia (NCCAH). Current cutoffs for cortisol and 17-hydroxyprogesterone (17-OHP) are derived from nonstandardized immunoassays. Liquid chromatography tandem mass spectrometry (LC-MS/MS) offers direct measurement of steroids, prompting the need to re-establish normal ranges. The goal of this study was to define cutoff values for cortisol and 17-OHP in serum by LC-MS/MS 30 and 60 minutes after intravenous administration of 250 µg tetracosactide acetate to healthy volunteers and to compare the results with LC-MS/MS with routine immunoassays. Cosyntropin testing was performed in healthy subjects (n = 138) and in patients referred for evaluation of adrenocortical function (n = 94). Steroids were assayed by LC-MS/MS and compared with two immunoassays used in routine diagnostics (Immulite and Roche platforms). The cutoff level for cortisol was defined as the 2.5% percentile in healthy subjects not using oral estrogens (n = 121) and for 17-OHP as the 97.5% percentile. Cortisol cutoff levels for LC-MS/MS were 412 and 485 nmol/L at 30 and 60 minutes, respectively. Applying the new cutoffs, 13 of 60 (22%) subjects who had AI according to conventional criteria now had a normal test result. For 17-OHP, the cutoff levels were 8.9 and 9.0 nmol/L at 30 and 60 minutes, respectively. LC-MS/MS provides cutoff levels for cortisol and 17-OHP after cosyntropin stimulation that are lower than those based on immunoassays, possibly because cross-reactivity between steroid intermediates and cortisol is eliminated. This reduces the number of false-positive tests for AI and false-negative tests for NCCAH.

Multi-profile Bayesian alignment model for LC-MS data analysis with integration of internal standards

PubMed Central

Tsai, Tsung-Heng; Tadesse, Mahlet G.; Di Poto, Cristina; Pannell, Lewis K.; Mechref, Yehia; Wang, Yue; Ressom, Habtom W.

2013-01-01

Motivation: Liquid chromatography-mass spectrometry (LC-MS) has been widely used for profiling expression levels of biomolecules in various ‘-omic’ studies including proteomics, metabolomics and glycomics. Appropriate LC-MS data preprocessing steps are needed to detect true differences between biological groups. Retention time (RT) alignment, which is required to ensure that ion intensity measurements among multiple LC-MS runs are comparable, is one of the most important yet challenging preprocessing steps. Current alignment approaches estimate RT variability using either single chromatograms or detected peaks, but do not simultaneously take into account the complementary information embedded in the entire LC-MS data. Results: We propose a Bayesian alignment model for LC-MS data analysis. The alignment model provides estimates of the RT variability along with uncertainty measures. The model enables integration of multiple sources of information including internal standards and clustered chromatograms in a mathematically rigorous framework. We apply the model to LC-MS metabolomic, proteomic and glycomic data. The performance of the model is evaluated based on ground-truth data, by measuring correlation of variation, RT difference across runs and peak-matching performance. We demonstrate that Bayesian alignment model improves significantly the RT alignment performance through appropriate integration of relevant information. Availability and implementation: MATLAB code, raw and preprocessed LC-MS data are available at http://omics.georgetown.edu/alignLCMS.html Contact: hwr@georgetown.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24013927

Analysis of antithyroid drugs in surface water by using liquid chromatography-tandem mass spectrometry.

PubMed

Pérez-Fernández, Virginia; Marchese, Stefano; Gentili, Alessandra; García, María Ángeles; Curini, Roberta; Caretti, Fulvia; Perret, Daniela

2014-11-07

This paper describes development and validation of a new method for the simultaneous determination of six antithyroid drugs (ATDs) in surface waters by using liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS). Target compounds include two ATD classes: thiouracil derivatives (thiouracil (TU), methyl-thiouracil (MTU), propyl-thiouracil (PTU), phenyl-thiouracil (PhTU)) and imidazole derivatives (tapazole (TAP), and mercaptobenzimidazole (MBI)). Sensitivity and selectivity of the LC-multiple reaction monitoring (MRM) analysis allowed applying a simple pre-concentration procedure and "shooting" the concentrated sample into the LC-MS/MS system without any other treatment. Recoveries were higher than 75% for all analytes. Intra-day precision and inter-day precision, calculated as relative standard deviation (RSD), were below 19 and 22%, respectively. Limits of detection (LODs) ranged from 0.05 to 0.25 μg/L; limits of quantitation (LOQs) varied between 0.15 and 0.75 μg/L. The validated method was successfully applied to the analysis of ATD residues in surface water samples collected from the Tiber River basin and three lakes of Lazio (central Italy). The analytes were quantified based on matrix-matched calibration curves with mercaptobenzimidazole-d4 (MBI-d4) as the internal standard (IS). The most widespread compound was TAP, one of the most common ATDs used in human medicine, but also TU and MBI were often detected in the analysed samples. Copyright © 2014 Elsevier B.V. All rights reserved.

LIQUID: an-open source software for identifying lipids in LC-MS/MS-based lipidomics data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kyle, Jennifer E.; Crowell, Kevin L.; Casey, Cameron P.

2017-01-31

We introduce an open-source software, LIQUID, for semi-automated processing and visualization of LC-MS/MS based lipidomics data. LIQUID provides users with the capability to process high throughput data and contains a customizable target library and scoring model per project needs. The graphical user interface provides visualization of multiple lines of spectral evidence for each lipid identification, allowing rapid examination of data for making confident identifications of lipid molecular species.

LC-MS/MS assay for the quantitation of the ATR kinase inhibitor VX-970 in human plasma.

PubMed

Kiesel, Brian F; Scemama, Jonas; Parise, Robert A; Villaruz, Liza; Iffland, Andre; Doyle, Austin; Ivy, Percy; Chu, Edward; Bakkenist, Christopher J; Beumer, Jan H

2017-11-30

DNA damaging chemotherapy and radiation are widely used standard-of-care modalities for the treatment of cancer. Nevertheless, the outcome for many patients remains poor and this may be attributed, at least in part, to highly effective DNA repair mechanisms. Ataxia-telangiectasia mutated and Rad3-related (ATR) is a key regulator of the DNA-damage response (DDR) that orchestrates the repair of damaged replication forks. ATR is a serine/threonine protein kinase and ATR kinase inhibitors potentiate chemotherapy and radiation. The ATR kinase inhibitor VX-970 (NSC 780162) is in clinical development in combination with primary cytotoxic agents and as a monotherapy for tumors harboring specific mutations. We have developed and validated an LC-MS/MS assay for the sensitive, accurate and precise quantitation of VX-970 in human plasma. A dilute-and-shoot method was used to precipitate proteins followed by chromatographic separation with a Phenomenex Polar-RP 80Å (4μm, 50×2mm) column and a gradient acetonitrile-water mobile phase containing 0.1% formic acid from a 50μL sample volume. Detection was achieved using an API 4000 mass spectrometer using electrospray positive ionization mode. The assay was linear from 3 to 5,000ng/mL, proved to be accurate (94.6-104.2%) and precise (<8.4% CV), and fulfilled criteria from the FDA guidance for bioanalytical method validation. This LC-MS/MS assay will be a crucial tool in defining the clinical pharmacokinetics and pharmacology of VX-970 as it progresses through clinical development. Copyright © 2017 Elsevier B.V. All rights reserved.

Salivary testosterone measurement by liquid chromatography tandem mass spectrometry in adult males and females.

PubMed

Keevil, B G; MacDonald, P; Macdowall, W; Lee, D M; Wu, F C W

2014-05-01

Salivary testosterone (Sal-T) may be a useful surrogate of serum free testosterone. The study aims were to use a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) assay to determine whether Sal-T concentrations accurately reflect Sal-T concentrations in both sexes and to investigate practical aspects of sample collection. Saliva and serum samples were collected in 104 male and 91 female subjects. A more sensitive LC-MS/MS assay was developed to enable Sal-T quantitation in the low concentrations found in females. Saliva (200 µL) was extracted with 1 mL of methyl-tert-butyl ether following the addition of D5-testosterone. Quantitation was performed using a Waters TQ-S mass spectrometer. The assay achieved a lower limit of quantification of 5 pmol/L, sufficiently sensitive to measure testosterone in female saliva. Sal-T showed a diurnal variation but samples taken at weekly and monthly intervals showed no significant differences. Sal-T was stable at ambient temperature for up to 5 days, after freeze-thawing and 3 years frozen storage. Reference intervals for Sal-T were 93-378 pmol/L in males and 5-46 pmol/L in females. Sal-T correlated significantly with serum calculated free-T in males (r = 0.71, P < 0.001) and in females (r = 0.39, P < 0.001). These results confirm that testosterone can be reliably and accurately measured by LC-MS/MS in both adult male and female saliva samples. These results lay the foundation for further exploration of the clinical application of Sal- T as a reliable alternative to serum testosterone in the diagnosis and management of androgen disorders and assessment of androgen status in clinical research.

Proteomic analysis of cellular soluble proteins from human bronchial smooth muscle cells by combining nondenaturing micro 2DE and quantitative LC-MS/MS. 1. Preparation of more than 4000 native protein maps.

PubMed

Jin, Ya; Zhang, Jun; Yuan, Qi; Manabe, Takashi; Tan, Wen

2015-08-01

Soluble proteins of human bronchial smooth muscle cells (HBSMC) were separated by nondenaturing micro 2DE and a 30 mm × 40 mm area of the CBB-stained slab gel (1.0 mm thick) was cut into 1.1 mm × 1.1 mm squares, then the proteins in the 972 gel pieces (squares) were applied to quantitative LC-MS/MS. Grid-cutting of the gel was employed to; (i) ensure the total analysis of the proteins in the area, (ii) standardize the conditions of analysis by LC-MS/MS, (iii) reconstruct the protein distribution patterns from the quantity data [1]. Totally 4323 proteins were identified in successfully analyzed 967 squares and the quantity distribution of each was reconstructed as a color density pattern (a native protein map). The quantity of the proteins distributed from 3.6% to 1 × 10(-5) % of the total protein quantity in the grid area. Each protein map was characterized by several features, including the position of quantity peak square, number of detected squares, and degree of concentration (focused or dispersed). About 4% of the proteins were detected in 100 or more squares, suggesting that they might be ubiquitous and interacting with other proteins. In contrast, many proteins showed more concentrated quantity distribution and the quantity peak positions of 565 proteins with a defined degree of concentration were summarized into a quantity peak map. These results for the first time visualized the distribution patterns of cellular proteins on a nondenaturing 2D gel. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Global mapping of rat plasma proteins with a native proteomic approach using nondenaturing micro 2DE and quantitative LC-MS/MS.

PubMed

Chen, Shumin; Wen, Meiling; Bu, Shujie; Wang, Ahui; Jin, Ya; Tan, Wen

2016-12-01

Plasma samples from adult male rats were separated by nondenaturing micro 2DE and a reference gel was selected, on which 136 CBB-stained spots were numbered and subjected to in-gel digestion and quantitative LC-MS/MS. The analysis provided the assignment of 1-25 (average eight) non-redundant proteins in each spot and totally 199 proteins were assigned in the 136 spots. About 40% of the proteins were detected in more than one spot and 15% in more than ten spots. We speculate this complexity arose from multiple causes, including protein heterogeneity, overlapping of protein locations and formation of protein complexes. Consequently, such results could not be appropriately presented as a conventional 2DE map, i.e. a list or a gel pattern with one or a few proteins annotated to each spot. Therefore, the LC-MS/MS quantity data was used to reconstruct the gel distribution of each protein and a library containing 199 native protein maps was established for rat plasma. Since proteins that formed a complex would migrate together during the nondenaturing 2DE and thus show similar gel distributions, correlation analysis was attempted for similarity comparison between the maps. The protein pairs showing high correlation coefficients included some well-known complexes, suggesting the promising application of native protein mapping for interaction analysis. With the importance of rat as the most commonly used laboratory animal in biomedical research, we expect this work would facilitate relevant studies by providing not only a reference library of rat plasma protein maps but a means for functional and interaction analysis. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A fit-for-purpose LC-MS/MS method for the simultaneous quantitation of ATP and 2,3-DPG in human K2EDTA whole blood.

PubMed

Kim, Hyeryun; Kosinski, Penelope; Kung, Charles; Dang, Lenny; Chen, Yue; Yang, Hua; Chen, Yuan-Shek; Kramer, Jordyn; Liu, Guowen

2017-09-01

Many hemolytic anemias results in major metabolic abnormalities: two common metabolite abnormalities include increased levels of 2,3-diphosphoglycerate (2,3-DPG) and decreased levels of adenosine triphosphate (ATP). To better monitor the concentration changes of these metabolites, the development of a reliable LC-MS/MS method to quantitatively profile the concentrations of 2, 3-DPG and ATP in whole blood is essential to understand the effects of investigational therapeutics. Accurate quantification of both compounds imposes great challenges to bioanalytical scientists due to their polar, ionic and endogenous nature. Here we present an LC-MS/MS method for the reliable quantification of 2,3-DPG and ATP from K 2 EDTA human whole blood (WB) simultaneously. Whole blood samples were spiked with stable isotope labeled internal standards, processed by protein precipitation extraction, and analyzed using zwitterionic ion chromatography-hydrophilic interaction chromatography (ZIC-HILIC) coupled with tandem mass spectrometry. The linear analytical range of the assay was 50-3000μg/mL. The fit-for-purpose method demonstrated excellent accuracy and precision. The overall accuracy was within ±10.5% (%RE) for both analytes and the intra- and inter-assay precision (%CV) were less than 6.7% and 6.2% for both analytes, respectively. ATP and 2,3-DPG were found to be stable in human K 2 EDTA blood for at least 8h at 4°C, 96days when stored at -70°C and after three freeze/thaw cycles. The assay has been successfully applied to K 2 EDTA human whole blood samples to support clinical studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Analysis of anthocyanins in commercial fruit juices by using nano-liquid chromatography-electrospray-mass spectrometry and high-performance liquid chromatography with UV-vis detector.

PubMed

Fanali, Chiara; Dugo, Laura; D'Orazio, Giovanni; Lirangi, Melania; Dachà, Marina; Dugo, Paola; Mondello, Luigi

2011-01-01

Nano-LC and conventional HPLC techniques were applied for the analysis of anthocyanins present in commercial fruit juices using a capillary column of 100 μm id and a 2.1 mm id narrow-bore C(18) column. Analytes were detected by UV-Vis at 518 nm and ESI-ion trap MS with HPLC and nano-LC, respectively. Commercial blueberry juice (14 anthocyanins detected) was used to optimize chromatographic separation of analytes and other analysis parameters. Qualitative identification of anthocyanins was performed by comparing the recorded mass spectral data with those of published papers. The use of the same mobile phase composition in both techniques revealed that the miniaturized method exhibited shorter analysis time and higher sensitivity than narrow-bore chromatography. Good intra-day and day-to-day precision of retention time was obtained in both methods with values of RSD less than 3.4 and 0.8% for nano-LC and HPLC, respectively. Quantitative analysis was performed by external standard curve calibration of cyanidin-3-O-glucoside standard. Calibration curves were linear in the concentration ranges studied, 0.1-50 and 6-50 μg/mL for HPLC-UV/Vis and nano-LC-MS, respectively. LOD and LOQ values were good for both methods. In addition to commercial blueberry juice, qualitative and quantitative analysis of other juices (e.g. raspberry, sweet cherry and pomegranate) was performed. The optimized nano-LC-MS method allowed an easy and selective identification and quantification of anthocyanins in commercial fruit juices; it offered good results, shorter analysis time and reduced mobile phase volume with respect to narrow-bore HPLC. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rapid and High-Throughput Detection and Quantitation of Radiation Biomarkers in Human and Nonhuman Primates by Differential Mobility Spectrometry-Mass Spectrometry

NASA Astrophysics Data System (ADS)

Chen, Zhidan; Coy, Stephen L.; Pannkuk, Evan L.; Laiakis, Evagelia C.; Hall, Adam B.; Fornace, Albert J.; Vouros, Paul

2016-10-01

Radiation exposure is an important public health issue due to a range of accidental and intentional threats. Prompt and effective large-scale screening and appropriate use of medical countermeasures (MCM) to mitigate radiation injury requires rapid methods for determining the radiation dose. In a number of studies, metabolomics has identified small-molecule biomarkers responding to the radiation dose. Differential mobility spectrometry-mass spectrometry (DMS-MS) has been used for similar compounds for high-throughput small-molecule detection and quantitation. In this study, we show that DMS-MS can detect and quantify two radiation biomarkers, trimethyl-L-lysine (TML) and hypoxanthine. Hypoxanthine is a human and nonhuman primate (NHP) radiation biomarker and metabolic intermediate, whereas TML is a radiation biomarker in humans but not in NHP, which is involved in carnitine synthesis. They have been analyzed by DMS-MS from urine samples after a simple strong cation exchange-solid phase extraction (SCX-SPE). The dramatic suppression of background and chemical noise provided by DMS-MS results in an approximately 10-fold reduction in time, including sample pretreatment time, compared with liquid chromatography-mass spectrometry (LC-MS). DMS-MS quantitation accuracy has been verified by validation testing for each biomarker. Human samples are not yet available, but for hypoxanthine, selected NHP urine samples (pre- and 7-d-post 10 Gy exposure) were analyzed, resulting in a mean change in concentration essentially identical to that obtained by LC-MS (fold-change 2.76 versus 2.59). These results confirm the potential of DMS-MS for field or clinical first-level rapid screening for radiation exposure.

iPhos: a toolkit to streamline the alkaline phosphatase-assisted comprehensive LC-MS phosphoproteome investigation

PubMed Central

2014-01-01

Background Comprehensive characterization of the phosphoproteome in living cells is critical in signal transduction research. But the low abundance of phosphopeptides among the total proteome in cells remains an obstacle in mass spectrometry-based proteomic analysis. To provide a solution, an alternative analytic strategy to confidently identify phosphorylated peptides by using the alkaline phosphatase (AP) treatment combined with high-resolution mass spectrometry was provided. While the process is applicable, the key integration along the pipeline was mostly done by tedious manual work. Results We developed a software toolkit, iPhos, to facilitate and streamline the work-flow of AP-assisted phosphoproteome characterization. The iPhos tookit includes one assister and three modules. The iPhos Peak Extraction Assister automates the batch mode peak extraction for multiple liquid chromatography mass spectrometry (LC-MS) runs. iPhos Module-1 can process the peak lists extracted from the LC-MS analyses derived from the original and dephosphorylated samples to mine out potential phosphorylated peptide signals based on mass shift caused by the loss of some multiples of phosphate groups. And iPhos Module-2 provides customized inclusion lists with peak retention time windows for subsequent targeted LC-MS/MS experiments. Finally, iPhos Module-3 facilitates to link the peptide identifications from protein search engines to the quantification results from pattern-based label-free quantification tools. We further demonstrated the utility of the iPhos toolkit on the data of human metastatic lung cancer cells (CL1-5). Conclusions In the comparison study of the control group of CL1-5 cell lysates and the treatment group of dasatinib-treated CL1-5 cell lysates, we demonstrated the applicability of the iPhos toolkit and reported the experimental results based on the iPhos-facilitated phosphoproteome investigation. And further, we also compared the strategy with pure DDA-based LC-MS/MS phosphoproteome investigation. The results of iPhos-facilitated targeted LC-MS/MS analysis convey more thorough and confident phosphopeptide identification than the results of pure DDA-based analysis. PMID:25521246

LC-MS/MS Peptide Mapping with Automated Data Processing for Routine Profiling of N-Glycans in Immunoglobulins

NASA Astrophysics Data System (ADS)

Shah, Bhavana; Jiang, Xinzhao Grace; Chen, Louise; Zhang, Zhongqi

2014-06-01

Protein N-Glycan analysis is traditionally performed by high pH anion exchange chromatography (HPAEC), reversed phase liquid chromatography (RPLC), or hydrophilic interaction liquid chromatography (HILIC) on fluorescence-labeled glycans enzymatically released from the glycoprotein. These methods require time-consuming sample preparations and do not provide site-specific glycosylation information. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping is frequently used for protein structural characterization and, as a bonus, can potentially provide glycan profile on each individual glycosylation site. In this work, a recently developed glycopeptide fragmentation model was used for automated identification, based on their MS/MS, of N-glycopeptides from proteolytic digestion of monoclonal antibodies (mAbs). Experimental conditions were optimized to achieve accurate profiling of glycoforms. Glycan profiles obtained from LC-MS/MS peptide mapping were compared with those obtained from HPAEC, RPLC, and HILIC analyses of released glycans for several mAb molecules. Accuracy, reproducibility, and linearity of the LC-MS/MS peptide mapping method for glycan profiling were evaluated. The LC-MS/MS peptide mapping method with fully automated data analysis requires less sample preparation, provides site-specific information, and may serve as an alternative method for routine profiling of N-glycans on immunoglobulins as well as other glycoproteins with simple N-glycans.

Simultaneous Detection of Human C-Terminal p53 Isoforms by Single Template Molecularly Imprinted Polymers (MIPs) Coupled with Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)-Based Targeted Proteomics.

PubMed

Jiang, Wenting; Liu, Liang; Chen, Yun

2018-03-06

Abnormal expression of C-terminal p53 isoforms α, β, and γ can cause the development of cancers including breast cancer. To date, much evidence has demonstrated that these isoforms can differentially regulate target genes and modulate their expression. Thus, quantification of individual isoforms may help to link clinical outcome to p53 status and to improve cancer patient treatment. However, there are few studies on accurate determination of p53 isoforms, probably due to sequence homology of these isoforms and also their low abundance. In this study, a targeted proteomics assay combining molecularly imprinted polymers (MIPs) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for simultaneous quantification of C-terminal p53 isoforms. Isoform-specific surrogate peptides (i.e., KPLDGEYFTLQIR (peptide-α) for isoform α, KPLDGEYFTLQDQTSFQK (peptide-β) for isoform β, and KPLDGEYFTLQMLLDLR (peptide-γ) for isoform γ) were first selected and used in both MIPs enrichment and mass spectrometric detection. The common sequence KPLDGEYFTLQ of these three surrogate peptides was used as single template in MIPs. In addition to optimization of imprinting conditions and characterization of the prepared MIPs, binding affinity and cross-reactivity of the MIPs for each surrogate peptide were also evaluated. As a result, a LOQ of 5 nM was achieved, which was >15-fold more sensitive than that without MIPs. Finally, the assay was validated and applied to simultaneous quantitative analysis of C-terminal p53 isoforms α, β, and γ in several human breast cell lines (i.e., MCF-10A normal cells, MCF-7 and MDA-MB-231 cancer cells, and drug-resistant MCF-7/ADR cancer cells). This study is among the first to employ single template MIPs and cross-reactivity phenomenon to select isoform-specific surrogate peptides and enable simultaneous quantification of protein isoforms in LC-MS/MS-based targeted proteomics.

Development and validation of an LC-ESI-MS/MS method for the quantification of D-84, reboxetine and citalopram for their use in MS Binding Assays addressing the monoamine transporters hDAT, hSERT and hNET.

PubMed

Neiens, Patrick; De Simone, Angela; Ramershoven, Anna; Höfner, Georg; Allmendinger, Lars; Wanner, Klaus T

2018-03-03

MS Binding Assays represent a label-free alternative to radioligand binding assays. In this study, we present an LC-ESI-MS/MS method for the quantification of (R,R)-4-(2-benzhydryloxyethyl)-1-(4-fluorobenzyl)piperidin-3-ol [(R,R)-D-84, (R,R)-1], (S,S)-reboxetine [(S,S)-2], and (S)-citalopram [(S)-3] employed as highly selective nonlabeled reporter ligands in MS Binding Assays addressing the dopamine [DAT, (R,R)-D-84], norepinephrine [NET, (S,S)-reboxetine] and serotonin transporter [SERT, (S)-citalopram], respectively. The developed LC-ESI-MS/MS method uses a pentafluorphenyl stationary phase in combination with a mobile phase composed of acetonitrile and ammonium formate buffer for chromatography and a triple quadrupole mass spectrometer in the multiple reaction monitoring mode for mass spectrometric detection. Quantification is based on deuterated derivatives of all three analytes serving as internal standards. The established LC-ESI-MS/MS method enables fast, robust, selective and highly sensitive quantification of all three reporter ligands in a single chromatographic run. The method was validated according to the Center for Drug Evaluation and Research (CDER) guideline for bioanalytical method validation regarding selectivity, accuracy, precision, calibration curve and sensitivity. Finally, filtration-based MS Binding Assays were performed for all three monoamine transporters based on this LC-ESI-MS/MS quantification method as read out. The affinities determined in saturation experiments for (R,R)-D-84 toward hDAT, for (S,S)-reboxetine toward hNET, and for (S)-citalopram toward hSERT, respectively, were in good accordance with results from literature, clearly demonstrating that the established MS Binding Assays have the potential to be an efficient alternative to radioligand binding assays widely used for this purpose so far. Copyright © 2018 John Wiley & Sons, Ltd.

Applying quantitative metabolomics based on chemical isotope labeling LC-MS for detecting potential milk adulterant in human milk.

PubMed

Mung, Dorothea; Li, Liang

2018-02-25

There is an increasing demand for donor human milk to feed infants for various reasons including that a mother may be unable to provide sufficient amounts of milk for their child or the milk is considered unsafe for the baby. Selling and buying human milk via the Internet has gained popularity. However, there is a risk of human milk sold containing other adulterants such as animal or plant milk. Analytical tools for rapid detection of adulterants in human milk are needed. We report a quantitative metabolomics method for detecting potential milk adulterants (soy, almond, cow, goat and infant formula milk) in human milk. It is based on the use of a high-performance chemical isotope labeling (CIL) LC-MS platform to profile the metabolome of an unknown milk sample, followed by multivariate or univariate comparison of the resultant metabolomic profile with that of human milk to determine the differences. Using dansylation LC-MS to profile the amine/phenol submetabolome, we could detect an average of 4129 ± 297 (n = 9) soy metabolites, 3080 ± 470 (n = 9) almond metabolites, 4256 ± 136 (n = 18) cow metabolites, 4318 ± 198 (n = 9) goat metabolites, 4444 ± 563 (n = 9) infant formula metabolites, and 4020 ± 375 (n = 30) human metabolites. This high level of coverage allowed us to readily differentiate the six different types of samples. From the analysis of binary mixtures of human milk containing 5, 10, 25, 50 and 75% other type of milk, we demonstrated that this method could be used to detect the presence of as low as 5% adulterant in human milk. We envisage that this method could be applied to detect contaminant or adulterant in other types of food or drinks. Copyright © 2017 Elsevier B.V. All rights reserved.

Recent advances of liquid chromatography-(tandem) mass spectrometry in clinical and forensic toxicology - An update.

PubMed

Remane, Daniela; Wissenbach, Dirk K; Peters, Frank T

2016-09-01

Liquid chromatography (LC) coupled to mass spectrometry (MS) or tandem mass spectrometry (MS/MS) is a well-established and widely used technique in clinical and forensic toxicology as well as doping control especially for quantitative analysis. In recent years, many applications for so-called multi-target screening and/or quantification of drugs, poisons, and or their metabolites in biological matrices have been developed. Such methods have proven particularly useful for analysis of so-called new psychoactive substances that have appeared on recreational drug markets throughout the world. Moreover, the evolvement of high resolution MS techniques and the development of data-independent detection modes have opened new possibilities for applications of LC-(MS/MS) in systematic toxicological screening analysis in the so called general unknown setting. The present paper will provide an overview and discuss these recent developments focusing on the literature published after 2010. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Systematic Optimization of Long Gradient Chromatography Mass Spectrometry for Deep Analysis of Brain Proteome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Hong; Yang, Yanling; Li, Yuxin

2015-02-06

Development of high resolution liquid chromatography (LC) is essential for improving the sensitivity and throughput of mass spectrometry (MS)-based proteomics. Here we present systematic optimization of a long gradient LC-MS/MS platform to enhance protein identification from a complex mixture. The platform employed an in-house fabricated, reverse phase column (100 μm x 150 cm) coupled with Q Exactive MS. The column was capable of achieving a peak capacity of approximately 700 in a 720 min gradient of 10-45% acetonitrile. The optimal loading level was about 6 micrograms of peptides, although the column allowed loading as many as 20 micrograms. Gas phasemore » fractionation of peptide ions further increased the number of peptide identification by ~10%. Moreover, the combination of basic pH LC pre-fractionation with the long gradient LC-MS/MS platform enabled the identification of 96,127 peptides and 10,544 proteins at 1% protein false discovery rate in a postmortem brain sample of Alzheimer’s disease. As deep RNA sequencing of the same specimen suggested that ~16,000 genes were expressed, current analysis covered more than 60% of the expressed proteome. Further improvement strategies of the LC/LC-MS/MS platform were also discussed.« less

Development of primary standards for mass spectrometry to increase accuracy in quantifying environmental contaminants.

PubMed

Oates, R P; Mcmanus, Michelle; Subbiah, Seenivasan; Klein, David M; Kobelski, Robert

2017-07-14

Internal standards are essential in electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS) to correct for systematic error associated with ionization suppression and/or enhancement. A wide array of instrument setups and interfaces has created difficulty in comparing the quantitation of absolute analyte response across laboratories. This communication demonstrates the use of primary standards as operational qualification standards for LC-MS instruments and their comparison with commonly accepted internal standards. In monitoring the performance of internal standards for perfluorinated compounds, potassium hydrogen phthalate (KHP) presented lower inter-day variability in instrument response than a commonly accepted deuterated perfluorinated internal standard (d3-PFOS), with percent relative standard deviations less than or equal to 6%. The inter-day precision of KHP was greater than d3-PFOS over a 28-day monitoring of perfluorooctanesulfonic acid (PFOS), across concentrations ranging from 0 to 100μg/L. The primary standard trometamol (Trizma) performed as well as known internal standards simeton and tris (2-chloroisopropyl) phosphate (TCPP), with intra-day precision of Trizma response as low as 7% RSD on day 28. The inter-day precision of Trizma response was found to be greater than simeton and TCPP, across concentrations of neonicotinoids ranging from 1 to 100μg/L. This study explores the potential of primary standards to be incorporated into LC-MS/MS methodology to improve the quantitative accuracy in environmental contaminant analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

Electrochemistry coupled online to liquid chromatography-mass spectrometry for fast simulation of biotransformation reactions of the insecticide chlorpyrifos.

PubMed

Mekonnen, Tessema F; Panne, Ulrich; Koch, Matthias

2017-05-01

An automated method is presented for fast simulation of (bio)transformation products (TPs) of the organophosphate insecticide chlorpyrifos (CPF) based on electrochemistry coupled online to liquid chromatography-mass spectrometry (EC-LC-MS). Oxidative TPs were produced by a boron doped diamond (BDD) electrode, separated by reversed phase HPLC and online detected by electrospray ionization-mass spectrometry (ESI-MS). Furthermore, EC oxidative TPs were investigated by HPLC-tandem mass spectrometry (LC-MS/MS) and FT-ICR high resolution mass spectrometry (HRMS) and compared to in vitro assay metabolites (rat and human liver microsomes). Main phase I metabolites of CPF: chlorpyrifos oxon (CPF oxon), trichloropyridinol (TCP), diethylthiophosphate (DETP), diethylphosphate (DEP), desethyl chlorpyrifos (De-CPF), and desethyl chlorpyrifos oxon (De-CPF oxon), were successfully identified by the developed EC-LC-MS method. The EC-LC-MS method showed similar metabolites compared to the in vitro assay with possibilities of determining reactive species. Our results reveal that online EC-(LC)-MS brings an advantage on time of analysis by eliminating sample preparation steps and matrix complexity compared to conventional in vivo or in vitro methods.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the direct detection of 2-monochloropropanediol (2-MCPD) esters in edible oils.

PubMed

MacMahon, Shaun; Ridge, Clark D; Begley, Timothy H

2014-12-03

A new analytical method has been developed and validated for the detection and quantification of 2-monochloropropanediol (2-MCPD) esters in edible oils. The target compounds are potentially carcinogenic contaminants formed during the processing of edible oils. As the 2-MCPD esters that occur most frequently in refined edible oils were not commercially available, standards were synthesized with identity and purity (95+%) confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and (1)H NMR. Target analytes are separated from edible oil matrices using a two-step solid-phase extraction (SPE) procedure. The extracts are then analyzed using LC-MS/MS with electrospray ionization (ESI). The method has been validated for 11 2-MCPD diesters and 3 2-MCPD monoesters in soybean oil, olive oil, and palm oil using an external calibration curve. The ranges of average recoveries and relative standard deviations (RSD) across the three oil matrices at three spiking concentrations are 79-106% (3-13% RSD) for the 2-MCPD diesters and 72-108% (4-17% RSD) for the 2-MCPD monoesters, with limits of quantitation at or below 30 ng/g for the diesters and 90 ng/g for the monoesters.

[LC-MS/MS analysis of determination of strychnine and brucine in formaldehyde fixed tissue].

PubMed

Zhan, Lan-fen; Liu, Ming-dong; Yan, You-yi; Ye, Yi; Wang, Wei; Wang, Zhi-hui; Zhao, Jun-hong; Liao, Lin-chuan

2012-10-01

To establish a method for determination of strychnine and brucine in formaldehyde fixed tissue by LC-MS/MS analysis. The samples were pretreated with solid phase extraction using SCX cartridges and separated on SB-C18 column with mobile phase 0.1% formic acid : 0.1% formic acid-acetonitrile (75:25). Electrospray ionization (ESI) source was utilized and operated in positive ion mode. Multiple reactions monitoring (MRM) mode was applied. External standard method was applied for quantitation. The chromatographic separation of strychnine and brucine in formaldehyde fixed nephritic and hepatic tissues resulted successfully. The standard curve was linear in the range of 0.002-2.0 microg/g for strychnine and brucine in formaldehyde fixed tissues, and the correlation coefficient was more than 0.996. The limits of detection (LOD) of strychnine and brucine in nephritic tissues were 0.06ng/g and 0.03 ng/g, respectively. The LOD of both chemicals were 0.3 ng/g in hepatic tissues. The extraction recovery rate was more than 74.5%. The precision of intra-day and inter-day were both less than 8.2%. Strychnine and brucine can be sensitive to be determined in formaldehyde fixed tissue by LC-MS/MS analysis. It can be applied in the forensic toxicological analysis.

GC-MS olfactometric and LC-DAD-ESI-MS/MS characterization of key odorants and phenolic compounds in black dry-salted olives.

PubMed

Selli, Serkan; Kelebek, Hasim; Kesen, Songul; Sonmezdag, Ahmet Salih

2018-02-01

Olives are processed in different ways depending on consumption habits, which vary between countries. Different de-bittering methods affect the aroma and aroma-active compounds of table olives. This study focused on analyzing the aroma and aroma-active compounds of black dry-salted olives using gas chromatography-mass spectrometry-olfactometry (GC-MS-O) techniques. Thirty-nine volatile compounds which they have a total concentration of 29 459 µg kg -1 , were determined. Aroma extract dilution analysis (AEDA) was used to determine key aroma compounds of table olives. Based on the flavor dilution (FD) factor, the most powerful aroma-active compounds in the sample were methyl-2-methyl butyrate (tropical, sweet; FD: 512) and (Z)-3-hexenol (green, flowery; FD: 256). Phenolic compounds in table olives were also analyzed by LC-DAD-ESI-MS/MS. A total of 20 main phenolic compounds were identified and the highest content of phenolic compound was luteolin-7-glucoside (306 mg kg -1 ), followed by verbascoside (271 mg kg -1 ), oleuropein (231 mg kg -1 ), and hydroxytyrosol (3,4-DHPEA) (221 mg kg -1 ). Alcohols, carboxylic acids, and lactones were qualitatively and quantitatively the dominant volatiles in black dry-salted olives. Results indicated that esters and alcohols were the major aroma-active compounds. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

Application of a trap-free two-dimensional liquid chromatography combined with ion trap/time-of-flight mass spectrometry for separation and characterization of impurities and isomers in cefpiramide.

PubMed

Wang, Jian; Xu, Yu; Wen, Chunmei; Wang, Zhijian

2017-11-01

High-resolution mass spectrometry had been routinely used for structure identification of impurity. However, all LC-MS methods were based on a volatile mobile phase, and a non-volatile system is used in the official analytical method of United States Pharmacopoeia for cefpiramide which limited the use of mass spectrometry for structure characterization of the impurities. Here we presented the utilization of a trap-free two-dimensional liquid chromatography coupled to high resolution ion trap/time-of-flight mass spectrometry (2D LC-IT-TOF MS) with positive and negative modes of electrospray ionization for characterization of eight impurities in cefpiramide. Trap-free two-dimensional liquid chromatography and online desalting technique made it possible to characterize the impurity in cefpiramide in the condition of official standard, and the TIC chromatogram of LC-MS was in conformity with the LC chromatogram of the official analytical method in the peak sequence of impurities, which could further improve the method of official monographs in pharmacopoeias. Each peak separated by the non-volatile mobile phase was trapped by a 20 μL quantitative loop then transferred into a system with a volatile mobile phase connected to a MS detector. In the first dimension, the column was Kromasil C 8 analytical column (250 mm × 4.6 mm, 5 μm) with a non-volatile salt mobile phase at the flow rate of 0.8 mL min -1 . In the second dimension, the column was Shimadzu Shim-pack GISS C 18 (50 mm × 2.1 mm, 1.9 μm) with a volatile salt mobile phase at the flow rate of 0.3 mL min -1 . Through the multiple heart-cutting 2D-LC approach and online desalting technique, the problem of incompatibility between non-volatile salt mobile phase and mass spectrometry was solved completely. The fragmentation behavior of cefpiramide and its eight impurities were studied. The structures of eight impurities in cefpiramide drug substance were deduced based on the HPLC-MS n data, in which seven impurities were novel impurities. The forming mechanisms of degradation products in cefpiramide were also studied. Copyright © 2017 Elsevier B.V. All rights reserved.

Rapid Verification of Candidate Serological Biomarkers Using Gel-based, Label-free Multiple Reaction Monitoring

PubMed Central

Tang, Hsin-Yao; Beer, Lynn A.; Barnhart, Kurt T.; Speicher, David W.

2011-01-01

Stable isotope dilution-multiple reaction monitoring-mass spectrometry (SID-MRM-MS) has emerged as a promising platform for verification of serological candidate biomarkers. However, cost and time needed to synthesize and evaluate stable isotope peptides, optimize spike-in assays, and generate standard curves, quickly becomes unattractive when testing many candidate biomarkers. In this study, we demonstrate that label-free multiplexed MRM-MS coupled with major protein depletion and 1-D gel separation is a time-efficient, cost-effective initial biomarker verification strategy requiring less than 100 μl serum. Furthermore, SDS gel fractionation can resolve different molecular weight forms of targeted proteins with potential diagnostic value. Because fractionation is at the protein level, consistency of peptide quantitation profiles across fractions permits rapid detection of quantitation problems for specific peptides from a given protein. Despite the lack of internal standards, the entire workflow can be highly reproducible, and long-term reproducibility of relative protein abundance can be obtained using different mass spectrometers and LC methods with external reference standards. Quantitation down to ~200 pg/mL could be achieved using this workflow. Hence, the label-free GeLC-MRM workflow enables rapid, sensitive, and economical initial screening of large numbers of candidate biomarkers prior to setting up SID-MRM assays or immunoassays for the most promising candidate biomarkers. PMID:21726088

Multiplexed MRM-based assays for the quantitation of proteins in mouse plasma and heart tissue.

PubMed

Percy, Andrew J; Michaud, Sarah A; Jardim, Armando; Sinclair, Nicholas J; Zhang, Suping; Mohammed, Yassene; Palmer, Andrea L; Hardie, Darryl B; Yang, Juncong; LeBlanc, Andre M; Borchers, Christoph H

2017-04-01

The mouse is the most commonly used laboratory animal, with more than 14 million mice being used for research each year in North America alone. The number and diversity of mouse models is increasing rapidly through genetic engineering strategies, but detailed characterization of these models is still challenging because most phenotypic information is derived from time-consuming histological and biochemical analyses. To expand the biochemists' toolkit, we generated a set of targeted proteomic assays for mouse plasma and heart tissue, utilizing bottom-up LC/MRM-MS with isotope-labeled peptides as internal standards. Protein quantitation was performed using reverse standard curves, with LC-MS platform and curve performance evaluated by quality control standards. The assays comprising the final panel (101 peptides for 81 proteins in plasma; 227 peptides for 159 proteins in heart tissue) have been rigorously developed under a fit-for-purpose approach and utilize stable-isotope labeled peptides for every analyte to provide high-quality, precise relative quantitation. In addition, the peptides have been tested to be interference-free and the assay is highly multiplexed, with reproducibly determined protein concentrations spanning >4 orders of magnitude. The developed assays have been used in a small pilot study to demonstrate their application to molecular phenotyping or biomarker discovery/verification studies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rapid verification of candidate serological biomarkers using gel-based, label-free multiple reaction monitoring.

PubMed

Tang, Hsin-Yao; Beer, Lynn A; Barnhart, Kurt T; Speicher, David W

2011-09-02

Stable isotope dilution-multiple reaction monitoring-mass spectrometry (SID-MRM-MS) has emerged as a promising platform for verification of serological candidate biomarkers. However, cost and time needed to synthesize and evaluate stable isotope peptides, optimize spike-in assays, and generate standard curves quickly becomes unattractive when testing many candidate biomarkers. In this study, we demonstrate that label-free multiplexed MRM-MS coupled with major protein depletion and 1D gel separation is a time-efficient, cost-effective initial biomarker verification strategy requiring less than 100 μL of serum. Furthermore, SDS gel fractionation can resolve different molecular weight forms of targeted proteins with potential diagnostic value. Because fractionation is at the protein level, consistency of peptide quantitation profiles across fractions permits rapid detection of quantitation problems for specific peptides from a given protein. Despite the lack of internal standards, the entire workflow can be highly reproducible, and long-term reproducibility of relative protein abundance can be obtained using different mass spectrometers and LC methods with external reference standards. Quantitation down to ~200 pg/mL could be achieved using this workflow. Hence, the label-free GeLC-MRM workflow enables rapid, sensitive, and economical initial screening of large numbers of candidate biomarkers prior to setting up SID-MRM assays or immunoassays for the most promising candidate biomarkers.

A novel mass spectrometry-based method for simultaneous determination of asymmetric and symmetric dimethylarginine, l-arginine and l-citrulline optimized for LC-MS-TOF and LC-MS/MS.

PubMed

Wiśniewski, Jerzy; Fleszar, Mariusz G; Piechowicz, Joanna; Krzystek-Korpacka, Małgorzata; Chachaj, Angelika; Szuba, Andrzej; Lorenc-Kukula, Katarzyna; Masłowski, Leszek; Witkiewicz, Wojciech; Gamian, Andrzej

2017-11-01

Nitric oxide (NO) is a regulatory molecule involved in many biological processes. NO is produced by nitric oxide synthase by conversion of l-arginine to l-citrulline. l-Arginine methylated derivatives, asymmetric and symmetric dimethylarginines (asymmetric dimethylarginine, ADMA, and symmetric dimethylarginine, SDMA), regulate l-arginine availability and the activity of nitric oxide synthase. As such, they have been frequently investigated as potential biomarkers in pathologies associated with dysfunctions in NO synthesis. Here, we present a new multistep analytical methodology based on liquid chromatography combined with mass spectrometry for the accurate identification of l-arginine, l-citrulline, ADMA and SDMA. Compounds are measured as stable 2,3,4,5,6-pentafluorobenzoyl chloride derivatives, which allows for simultaneous analysis of all compounds through chromatographic separation of ADMA and SDMA using a reverse-phase column. Serum aliquots (100 μL) were spiked with isotope-labeled internal standards and sodium carbonate buffer. The derivatization process was carried out at 25°C for 10 minu using pentafluorobenzoyl chloride as derivatization reagent. Calibration demonstrated good linearity (R 2  = 0.9966-0.9986) for all derivatized compounds. Good accuracy (94.67-99.91%) and precision (1.92-11.8%) were observed for the quality control samples. The applicability of the method was evaluated in a cohort of angiological patients and healthy volunteers. The method discerned significantly lower l-arginine and l-citrulline in angiologic patients. This robust and fast LC-ESI-MS method may be a useful tool in quantitative analysis of l-arginine, ADMA, SDMA and l-citrulline. Copyright © 2017 John Wiley & Sons, Ltd.

Data Dependent Peak Model Based Spectrum Deconvolution for Analysis of High Resolution LC-MS Data

PubMed Central

2015-01-01

A data dependent peak model (DDPM) based spectrum deconvolution method was developed for analysis of high resolution LC-MS data. To construct the selected ion chromatogram (XIC), a clustering method, the density based spatial clustering of applications with noise (DBSCAN), is applied to all m/z values of an LC-MS data set to group the m/z values into each XIC. The DBSCAN constructs XICs without the need for a user defined m/z variation window. After the XIC construction, the peaks of molecular ions in each XIC are detected using both the first and the second derivative tests, followed by an optimized chromatographic peak model selection method for peak deconvolution. A total of six chromatographic peak models are considered, including Gaussian, log-normal, Poisson, gamma, exponentially modified Gaussian, and hybrid of exponential and Gaussian models. The abundant nonoverlapping peaks are chosen to find the optimal peak models that are both data- and retention-time-dependent. Analysis of 18 spiked-in LC-MS data demonstrates that the proposed DDPM spectrum deconvolution method outperforms the traditional method. On average, the DDPM approach not only detected 58 more chromatographic peaks from each of the testing LC-MS data but also improved the retention time and peak area 3% and 6%, respectively. PMID:24533635

TOXICITY-BASED IDENTIFICATION OF DRINKING WATER DISINFECTION BY-PRODUCTS USING LC/MS AND LC/MS/MS

EPA Science Inventory

The goal of this research is to use a bio-assay directed approach to focus identification work on the most toxicologically important disinfection by-products. To this end, drinking water is being collected from full-scale treatment plants that use chlorine, ozone, chlorine dioxi...

LC/MS/MS quantitation assay for pharmacokinetics of naringenin and double peaks phenomenon in rats plasma.

PubMed

Ma, Yan; Li, Peibo; Chen, Dawei; Fang, Tiezheng; Li, Haitian; Su, Weiwei

2006-01-13

A highly sensitive and specific electrospray ionization (ESI) liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for quantitation of naringenin (NAR) and an explanation for the double peaks phenomenon was developed and validated. NAR was extracted from rat plasma and tissues along with the internal standard (IS), hesperidin, with ethyl acetate. The analytes were analyzed in the multiple-reaction-monitoring (MRM) mode as the precursor/product ion pair of m/z 273.4/151.3 for NAR and m/z 611.5/303.3 for the IS. The assay was linear over the concentration range of 5-2500 ng/mL. The lower limit quantification was 5 ng/mL, available for plasma pharmacokinetics of NAR in rats. Accuracy in within- and between-run precisions showed good reproducibility. When NAR was administered orally, only little and predominantly its glucuronidation were into circulation in the plasma. There existed double peaks phenomenon in plasma concentration-time curve leading to the relatively slow elimination of NAR in plasma. The results showed that there was a linear relationship between the AUC of total NAR and dosages. And the double peaks are mainly due to enterohepatic circulation.

Quantitative analysis of a novel HIV fusion inhibitor (sifuvirtide) in HIV infected human plasma using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.

PubMed

Che, Jinjing; Meng, Qingfang; Chen, Zhihang; Hou, Yunan; Shan, Chengqi; Cheng, Yuanguo

2010-03-11

A sensitive method for measuring sifuvirtide, a novel HIV fusion inhibitor peptide drug in HIV-1(+) human plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The plasma samples were treated by solvent/detergent (S/D) method to inactivate viral activity before analysis. After protein precipitation sifuvirtide was determined by LC-MS/MS. A structure analog was used as internal standard (IS). The mass spectrometer was operated in positive ion and multiple reaction monitoring mode with transitions m/z 946.3-->159.0 for sifuvirtide and 951.7-->159.2 for IS. The intra-day precision ranged from 2.74% to 7.57% with accuracy from 91.63% to 102.53%. The inter-day precision ranged from 2.65% to 3.58% and the accuracy from 95.53% to 105.28%. Stability studies showed that sifuvirtide was stable both during the assay procedure and long-term storage. The lower limit of quantitation (LLOQ) was 9.75ngml(-1). The method was used for analyzing samples from phase IIa clinical study of sifuvirtide in China. Copyright 2009 Elsevier B.V. All rights reserved.

Identification, quantitation, and method validation for flavan-3-ols in fermented ready-to-drink teas from the Italian market using HPLC-UV/DAD and LC-MS/MS.

PubMed

Cordero, Chiara; Canale, Francesca; Del Rio, Daniele; Bicchi, Carlo

2009-11-01

The present study is focused on flavan-3-ols characterizing the antioxidant properties of fermented tea (Camellia sinensis). These bioactive compounds, object of nutritional claims in commercial products, should be quantified with rigorous analytical procedures whose accuracy and precision have been stated with a certain level of confidence. An HPLC-UV/DAD method, able to detect and quantify flavan-3-ols in infusions and ready-to-drink teas, has been developed for routine analysis and validated by characterizing several performance parameters. The accuracy assessment has been run through a series of LC-MS/MS analyses. Epigallocatechin, (+)-catechin, (-)-epigallocatechingallate, (-)-epicatechin, (-)-gallocatechingallate, (-)-epicatechingallate, and (-)-catechingallate were chosen as markers of the polyphenolic fraction. Quantitative results showed that samples obtained from tea leaves infusion were richer in polyphenolic antioxidants than those obtained through other industrial processes. The influence of shelf-life and packaging material on the flavan-3-ols content was also considered; markers decreased, with an exponential trend, as a function of time within the shelf life while packaging materials demonstrated to influence differently the flavan-3-ol fraction composition over time. The method presented here provides quantitative results with a certain level of confidence and is suitable for a routine quality control of iced teas whose antioxidant properties are object of nutritional claim.

A novel liquid chromatography/tandem mass spectrometry method for the quantification of glycine as biomarker in brain microdialysis and cerebrospinal fluid samples within 5min.

PubMed

Voehringer, Patrizia; Fuertig, René; Ferger, Boris

2013-11-15

Glycine is an important amino acid neurotransmitter in the central nervous system (CNS) and a useful biomarker to indicate biological activity of drugs such as glycine reuptake inhibitors (GRI) in the brain. Here, we report how a liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the fast and reliable analysis of glycine in brain microdialysates and cerebrospinal fluid (CSF) samples has been established. Additionally, we compare this method with the conventional approach of high performance liquid chromatography (HPLC) coupled to fluorescence detection (FD). The present LC-MS/MS method did not require any derivatisation step. Fifteen microliters of sample were injected for analysis. Glycine was detected by a triple quadrupole mass spectrometer in the positive electrospray ionisation (ESI) mode. The total running time was 5min. The limit of quantitation (LOQ) was determined as 100nM, while linearity was given in the range from 100nM to 100μM. In order to demonstrate the feasibility of the LC-MS/MS method, we measured glycine levels in striatal in vivo microdialysates and CSF of rats after administration of the commercially available glycine transporter 1 (GlyT1) inhibitor LY 2365109 (10mg/kg, p.o.). LY 2365109 produced 2-fold and 3-fold elevated glycine concentrations from 1.52μM to 3.6μM in striatal microdialysates and from 10.38μM to 36μM in CSF, respectively. In conclusion, we established a fast and reliable LC-MS/MS method, which can be used for the quantification of glycine in brain microdialysis and CSF samples in biomarker studies. Copyright © 2013 Elsevier B.V. All rights reserved.

Periodic modulation-based stochastic resonance algorithm applied to quantitative analysis for weak liquid chromatography-mass spectrometry signal of granisetron in plasma

NASA Astrophysics Data System (ADS)

Xiang, Suyun; Wang, Wei; Xiang, Bingren; Deng, Haishan; Xie, Shaofei

2007-05-01

The periodic modulation-based stochastic resonance algorithm (PSRA) was used to amplify and detect the weak liquid chromatography-mass spectrometry (LC-MS) signal of granisetron in plasma. In the algorithm, the stochastic resonance (SR) was achieved by introducing an external periodic force to the nonlinear system. The optimization of parameters was carried out in two steps to give attention to both the signal-to-noise ratio (S/N) and the peak shape of output signal. By applying PSRA with the optimized parameters, the signal-to-noise ratio of LC-MS peak was enhanced significantly and distorted peak shape that often appeared in the traditional stochastic resonance algorithm was corrected by the added periodic force. Using the signals enhanced by PSRA, this method extended the limit of detection (LOD) and limit of quantification (LOQ) of granisetron in plasma from 0.05 and 0.2 ng/mL, respectively, to 0.01 and 0.02 ng/mL, and exhibited good linearity, accuracy and precision, which ensure accurate determination of the target analyte.

Identification and Characterization of Human Proteoforms by Top-Down LC-21 Tesla FT-ICR Mass Spectrometry.

PubMed

Anderson, Lissa C; DeHart, Caroline J; Kaiser, Nathan K; Fellers, Ryan T; Smith, Donald F; Greer, Joseph B; LeDuc, Richard D; Blakney, Greg T; Thomas, Paul M; Kelleher, Neil L; Hendrickson, Christopher L

2017-02-03

Successful high-throughput characterization of intact proteins from complex biological samples by mass spectrometry requires instrumentation capable of high mass resolving power, mass accuracy, sensitivity, and spectral acquisition rate. These limitations often necessitate the performance of hundreds of LC-MS/MS experiments to obtain reasonable coverage of the targeted proteome, which is still typically limited to molecular weights below 30 kDa. The National High Magnetic Field Laboratory (NHMFL) recently installed a 21 T FT-ICR mass spectrometer, which is part of the NHMFL FT-ICR User Facility and available to all qualified users. Here we demonstrate top-down LC-21 T FT-ICR MS/MS of intact proteins derived from human colorectal cancer cell lysate. We identified a combined total of 684 unique protein entries observed as 3238 unique proteoforms at a 1% false discovery rate, based on rapid, data-dependent acquisition of collision-induced and electron-transfer dissociation tandem mass spectra from just 40 LC-MS/MS experiments. Our identifications included 372 proteoforms with molecular weights over 30 kDa detected at isotopic resolution, which substantially extends the accessible mass range for high-throughput top-down LC-MS/MS.

Quantitative bioanalysis of antibody-conjugated payload in monkey plasma using a hybrid immuno-capture LC-MS/MS approach: Assay development, validation, and a case study.

PubMed

Liu, Ang; Kozhich, Alexander; Passmore, David; Gu, Huidong; Wong, Richard; Zambito, Frank; Rangan, Vangipuram S; Myler, Heather; Aubry, Anne-Françoise; Arnold, Mark E; Wang, Jian

2015-10-01

Antibody drug conjugates (ADCs) are complex molecules composed of two pharmacologically distinct components, the cytotoxic payload and the antibody. The measurement of the payload molecules that are attached to the antibody in vivo is important for the evaluation of the safety and efficacy of ADCs, and can also provide distinct information compared to the antibody-related analytes. However, analyzing the antibody-conjugated payload is challenging and in some cases may not be feasible. The in vivo change in drug antibody ratio (DAR), due to deconjugation, biotransformation or other clearance phenomena, generates unique and additional challenges for ADC analysis in biological samples. Here, we report a novel hybrid approach with immuno-capture of the ADC, payload cleavage by specific enzyme, and LC-MS/MS of the cleaved payload to quantitatively measure the concentration of payload molecules still attached to the antibody via linker in plasma. The ADC reference material used for the calibration curve is not likely to be identical to the ADC measured in study samples due to the change in DAR distribution over the PK time course. The assay clearly demonstrated that there was no bias in the measurement of antibody-conjugated payload for ADC with varying DAR, which thus allowed accurate quantification even when the DAR distribution dynamically changes in vivo. This hybrid assay was fully validated based on a combination of requirements for both chromatographic and ligand binding methods, and was successfully applied to support a GLP safety study in monkeys. Copyright © 2015 Elsevier B.V. All rights reserved.

Phenolic compounds from Byrsonima crassifolia L. bark: phytochemical investigation and quantitative analysis by LC-ESI MS/MS.

PubMed

Maldini, Mariateresa; Montoro, Paola; Pizza, Cosimo

2011-08-25

Phytochemical investigation of the methanolic extract of Byrsonima crassifolia's bark led to the isolation of 8 known phenolic compounds 5-O-galloylquinic acid, 3-O-galloylquinic acid, 3,4-di-O-galloylquinic acid, 3,5-di-O-galloylquinic acid, 3,4,5-tri-O-galloylquinic acid, (+)-epicatechin-3-gallate along with (+)-catechin and (+)-epicatechin. Due to their biological value, in the present study, a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, working in multiple reaction monitoring (MRM) mode, has been developed to quantify these compounds. B. crassifolia bark resulted in a rich source of phenolic compounds and particularly of galloyl derivates. The proposed analytical method is promising to be applied to other galloyl derivatives to quantify these bioactive compounds in raw material and final products. Copyright © 2011 Elsevier B.V. All rights reserved.

Nano-flow vs standard-flow: Which is the more suitable LC/MS method for quantifying hepcidin-25 in human serum in routine clinical settings?

PubMed

Vialaret, Jérôme; Picas, Alexia; Delaby, Constance; Bros, Pauline; Lehmann, Sylvain; Hirtz, Christophe

2018-06-01

Hepcidin-25 peptide is a biomarker which is known to have considerable clinical potential for diagnosing iron-related diseases. Developing analytical methods for the absolute quantification of hepcidin is still a real challenge, however, due to the sensitivity, specificity and reproducibility issues involved. In this study, we compare and discuss two MS-based assays for quantifying hepcidin, which differ only in terms of the type of liquid chromatography (nano LC/MS versus standard LC/MS) involved. The same sample preparation, the same internal standards and the same MS analyzer were used with both approaches. In the field of proteomics, nano LC chromatography is generally known to be more sensitive and less robust than standard LC methods. In this study, we established that the performances of the standard LC method are equivalent to those of our previously developed nano LC method. Although the analytical performances were very similar in both cases. The standard-flow platform therefore provides the more suitable alternative for accurately determining hepcidin in clinical settings. Copyright © 2018 Elsevier B.V. All rights reserved.

Non-enzymolytic adenosine barcode-mediated dual signal amplification strategy for ultrasensitive protein detection using LC-MS/MS.

PubMed

Yang, Wen; Li, Tengfei; Shu, Chang; Ji, Shunli; Wang, Lei; Wang, Yan; Li, Duo; Mtalimanja, Michael; Sun, Luning; Ding, Li

2018-05-10

A method is described for the determination of proteins with LC-MS/MS enabled by a small molecule (adenosine) barcode and based on a double-recognition sandwich structure. The coagulation protein thrombin was chosen as the model analyte. Magnetic nanoparticles were functionalized with aptamer29 (MNP/apt29) and used to capture thrombin from the samples. MNP/apt29 forms a sandwich with functionalized gold nanoparticles modified with (a) aptamer15 acting as thrombin-recognizing element and (b) a large number of adenosine as mass barcodes. The sandwich formed (MNP/apt29-thrombin-apt15/AuNP/adenosine) can ben magnetically separated from the sample. Mass barcodes are subsequently released from the sandwiched structure for further analysis by adding 11-mercaptoundecanoic acid. Adenosine is then detected by LC-MS/MS as it reflects the level of thrombin with impressively amplified signal. Numerous adenosines introduced into the sandwich proportional to the target concentration further amplify the signal. Under optimized conditions, the response is linearly proportional to the thrombin concentration in the range of 0.02 nM to 10 nM, with a detection limit of 9 fM. The application of this method to the determination of thrombin in spiked plasma samples gave recoveries that ranged from 92.3% to 104.7%. Graphical abstract Schematic representation of a method for the determination of thrombin with LC-MS/MS. The method is based on a double-recognition sandwiched structure. With LC-MS/MS, mass barcodes (adenosine) are detected to quantify thrombin, which amplifies the detection signal impressively.

Chemical derivatization for enhancing sensitivity during LC/ESI-MS/MS quantification of steroids in biological samples: a review.

PubMed

Higashi, Tatsuya; Ogawa, Shoujiro

2016-09-01

Sensitive and specific methods for the detection, characterization and quantification of endogenous steroids in body fluids or tissues are necessary for the diagnosis, pathological analysis and treatment of many diseases. Recently, liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has been widely used for these purposes due to its specificity and versatility. However, the ESI efficiency and fragmentation behavior of some steroids are poor, which lead to a low sensitivity. Chemical derivatization is one of the most effective methods to improve the detection characteristics of steroids in ESI-MS/MS. Based on this background, this article reviews the recent advances in chemical derivatization for the trace quantification of steroids in biological samples by LC/ESI-MS/MS. The derivatization in ESI-MS/MS is based on tagging a proton-affinitive or permanently charged moiety on the target steroid. Introduction/formation of a fragmentable moiety suitable for the selected reaction monitoring by the derivatization also enhances the sensitivity. The stable isotope-coded derivatization procedures for the steroid analysis are also described. Copyright © 2015 Elsevier Ltd. All rights reserved.

High-field asymmetric waveform ion mobility spectrometry for mass spectrometry-based proteomics.

PubMed

Swearingen, Kristian E; Moritz, Robert L

2012-10-01

High-field asymmetric waveform ion mobility spectrometry (FAIMS) is an atmospheric pressure ion mobility technique that separates gas-phase ions by their behavior in strong and weak electric fields. FAIMS is easily interfaced with electrospray ionization and has been implemented as an additional separation mode between liquid chromatography (LC) and mass spectrometry (MS) in proteomic studies. FAIMS separation is orthogonal to both LC and MS and is used as a means of on-line fractionation to improve the detection of peptides in complex samples. FAIMS improves dynamic range and concomitantly the detection limits of ions by filtering out chemical noise. FAIMS can also be used to remove interfering ion species and to select peptide charge states optimal for identification by tandem MS. Here, the authors review recent developments in LC-FAIMS-MS and its application to MS-based proteomics.

DBS-LC-MS/MS assay for caffeine: validation and neonatal application.

PubMed

Bruschettini, Matteo; Barco, Sebastiano; Romantsik, Olga; Risso, Francesco; Gennai, Iulian; Chinea, Benito; Ramenghi, Luca A; Tripodi, Gino; Cangemi, Giuliana

2016-09-01

DBS might be an appropriate microsampling technique for therapeutic drug monitoring of caffeine in infants. Nevertheless, its application presents several issues that still limit its use. This paper describes a validated DBS-LC-MS/MS method for caffeine. The results of the method validation showed an hematocrit dependence. In the analysis of 96 paired plasma and DBS clinical samples, caffeine levels measured in DBS were statistically significantly lower than in plasma but the observed differences were independent from hematocrit. These results clearly showed the need for extensive validation with real-life samples for DBS-based methods. DBS-LC-MS/MS can be considered to be a good alternative to traditional methods for therapeutic drug monitoring or PK studies in preterm infants.

Development and application of a sol-gel immunosorbent-based method for the determination of isoproturon in surface water.

PubMed

Zhang, Xiuli; Martens, Dieter; Krämer, Petra M; Kettrup, Antonius A; Liang, Xinmiao

2006-01-13

An immunosorbent was fabricated by encapsulation of monoclonal anti-isoproturon antibodies in sol-gel matrix. The immunosorbent-based loading, rinsing and eluting processes were optimized. Based on these optimizations, the sol-gel immunosorbent (SG-IS) selectively extracted isoproturon from an artificial mixture of 68 pesticides. In addition to this high selectivity, the SG-IS proved to be reusable. The SG-IS was combined with liquid chromatography-tandem mass spectrometry (LC-MS-MS) to determine isoproturon in surface water, and the linear range was up to 2.2 microg/l with correlation coefficient higher than 0.99 and relative standard deviation (RSD) lower than 5% (n=8). The limit of quantitation (LOQ) for 25-ml surface water sample was 5 ng/l.

Streamlined system for purifying and quantifying a diverse library of compounds and the effect of compound concentration measurements on the accurate interpretation of biological assay results.

PubMed

Popa-Burke, Ioana G; Issakova, Olga; Arroway, James D; Bernasconi, Paul; Chen, Min; Coudurier, Louis; Galasinski, Scott; Jadhav, Ajit P; Janzen, William P; Lagasca, Dennis; Liu, Darren; Lewis, Roderic S; Mohney, Robert P; Sepetov, Nikolai; Sparkman, Darren A; Hodge, C Nicholas

2004-12-15

As part of an overall systems approach to generating highly accurate screening data across large numbers of compounds and biological targets, we have developed and implemented streamlined methods for purifying and quantitating compounds at various stages of the screening process, coupled with automated "traditional" storage methods (DMSO, -20 degrees C). Specifically, all of the compounds in our druglike library are purified by LC/MS/UV and are then controlled for identity and concentration in their respective DMSO stock solutions by chemiluminescent nitrogen detection (CLND)/evaporative light scattering detection (ELSD) and MS/UV. In addition, the compound-buffer solutions used in the various biological assays are quantitated by LC/UV/CLND to determine the concentration of compound actually present during screening. Our results show that LC/UV/CLND/ELSD/MS is a widely applicable method that can be used to purify, quantitate, and identify most small organic molecules from compound libraries. The LC/UV/CLND technique is a simple and sensitive method that can be easily and cost-effectively employed to rapidly determine the concentrations of even small amounts of any N-containing compound in aqueous solution. We present data to establish error limits for concentration determination that are well within the overall variability of the screening process. This study demonstrates that there is a significant difference between the predicted amount of soluble compound from stock DMSO solutions following dilution into assay buffer and the actual amount present in assay buffer solutions, even at the low concentrations employed for the assays. We also demonstrate that knowledge of the concentrations of compounds to which the biological target is exposed is critical for accurate potency determinations. Accurate potency values are in turn particularly important for drug discovery, for understanding structure-activity relationships, and for building useful empirical models of protein-ligand interactions. Our new understanding of relative solubility demonstrates that most, if not all, decisions that are made in early discovery are based upon missing or inaccurate information. Finally, we demonstrate that careful control of compound handling and concentration, coupled with accurate assay methods, allows the use of both positive and negative data in analyzing screening data sets for structure-activity relationships that determine potency and selectivity.

Quantitation of Human Cytochrome P450 2D6 Protein with Immunoblot and Mass Spectrometry Analysis

PubMed Central

Yu, Ai-Ming; Qu, Jun; Felmlee, Melanie A.; Cao, Jin; Jiang, Xi-Ling

2009-01-01

Accurate quantification of cytochrome P450 (P450) protein contents is essential for reliable assessment of drug safety, including the prediction of in vivo clearance from in vitro metabolism data, which may be hampered by the use of uncharacterized standards and existence of unknown allelic isozymes. Therefore, this study aimed to delineate the variability in absolute quantification of polymorphic CYP2D6 drug-metabolizing enzyme and compare immunoblot and nano liquid chromatography coupled to mass spectrometry (nano-LC/MS) methods in identification and relative quantification of CYP2D6.1 and CYP2D6.2 allelic isozymes. Holoprotein content of in-house purified CYP2D6 isozymes was determined according to carbon monoxide difference spectrum, and total protein was quantified with bicinchoninic acid protein assay. Holoprotein/total CYP2D6 protein ratio was markedly higher for purified CYP2D6.1 (71.0%) than that calculated for CYP2D6.1 Supersomes (35.5%), resulting in distinct linear calibration range (0.05–0.50 versus 0.025–0.25 pmol) that was determined by densitometric analysis of immunoblot bands. Likewise, purified CYP2D6.2 and CYP2D6.10 and the CYP2D6.10 Supersomes all showed different holoprotein/total CYP2D6 protein ratios and distinct immunoblot linear calibration ranges. In contrast to immunoblot, nano-LC/MS readily distinguished CYP2D6.2 (R296C and S486T) from CYP2D6.1 by isoform-specific proteolytic peptides that contain the altered amino acid residues. In addition, relative quantitation of the two allelic isozymes was successfully achieved with label-free protein quantification, consistent with the nominated ratio. Because immunoblot and nano-LC/MS analyses measure total P450 protein (holoprotein and apoprotein) in a sample, complete understanding of holoprotein and apoprotein contents in P450 standards is desired toward reliable quantification. Our data also suggest that nano-LC/MS not only facilitates P450 quantitation but also provides genotypic information. PMID:18832475

[Rapid identification of two new isomers in bear bile powder by LC-Q-TOF-MS combined with PCC oxidation].

PubMed

Jian, Long-Hai; Hu, Chun; Yu, Hong; Wang, Ke; Ji, Shen

2013-07-01

A rapid method of Liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) combined with pyridinium chlorochromate (PCC) oxidation has been developed to determine chemical structures of two novel isomers in bear bile powder. Derivatives of ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA) were semi-synthesized by PCC oxidation, then were analyzed by LC-Q-TOF-MS. Separation was carried out on a reverse column with the mobile phase of acetonitrile-0.1% formic acid (45:55). The data of Q-TOF-MS was acquired by MS, MS/MS, positive and negative modes. Since UDCA and CDCA were stereochemical isomeric at an alcohol position, two oxidation products were same and have been confirmed by LC-Q-TOF-MS. Other two products were also determined based on the PCC oxidation theory. Samples of bear bile powder were dissolved by methanol and measured by LC-Q-TOF-MS. Two unknown peaks were found and identified by matching their retention times and accurate mass spectra ions with PCC oxidation productS. Finally, the structures of two new bile acids in bear bile powder were confirmed as 3alpha-hydroxy-7-oxo-5beta-cholanic acid, 7alpha-hydroxy-3-oxo-5beta-cholanic acid, respectively.

Optimization of hydrophilic interaction liquid chromatography/mass spectrometry and development of solid-phase extraction for the determination of paralytic shellfish poisoning toxins.

PubMed

Turrell, Elizabeth; Stobo, Lesley; Lacaze, Jean-Pierre; Piletsky, Sergey; Piletska, Elena

2008-01-01

The combination of hydrophilic interaction liquid chromatography (HILIC) and liquid chromatography/mass spectrometry (LC/MS) for the determination of paralytic shellfish poisoning (PSP) toxins has been proposed for use in routine monitoring of shellfish. In this study, methods for the detection of multiple PSP toxins [saxitoxin (STX), neosaxitoxin (NEO), decarbamoyl saxitoxin (dcSTX), decarbamoyl neosaxitoxin (dcNEO), gonyautoxins 1-5 (GTX1, GTX2, GTX3, GTX4, GTX5), decarbamoyl gonyautoxins (dcGTX2 and dcGTX3), and the N-sulfocarbamoyl C toxins (C1 and C2)] were optimized using single (MS) and triple quadrupole (MS/MS) instruments. Chromatographic separation of the toxins was achieved by using a TSK-gel Amide-80 analytical column, although superior chromatography was observed through application of a ZIC-HILIC column. Preparative procedures used to clean up shellfish extracts and concentrate PSP toxins prior to analysis were investigated. The capacity of computationally designed polymeric (CDP) materials and HILIC solid-phase extraction (SPE) cartridges to retain highly polar PSP toxins was explored. Three CDP materials and 2 HILIC cartridges were assessed for the extraction of PSP toxins from aqueous solution. Screening of the CDPs showed that all tested polymers adsorbed PSP toxins. A variety of elution procedures were examined, with dilute 0.01% acetic acid providing optimum recovery from a CDP based on 2-(trifluoromethyl)acrylic acid as the monomer. ZIC-HILIC SPE cartridges were superior to the PolyLC equivalent, with recoveries ranging from 70 to 112% (ZIC-HILIC) and 0 to 90% (PolyLC) depending on the PSP toxin. It is proposed that optimized SPE and HILIC-MS methods can be applied for the quantitative determination of PSP toxins in shellfish.

Development of a high performance liquid chromatography method and a liquid chromatography-tandem mass spectrometry method with the pressurized liquid extraction for the quantification and confirmation of sulfonamides in the foods of animal origin.

PubMed

Yu, Huan; Tao, Yanfei; Chen, Dongmei; Wang, Yulian; Huang, Lingli; Peng, Dapeng; Dai, Menghong; Liu, Zhenli; Wang, Xu; Yuan, Zonghui

2011-09-01

The residues of sulfonamides (SAs) in the foods of animal origin are of the major concern because they are harmful to the consumer's health and could induce pathogens to develop resistance. Rapid and efficient determination methods are urgently in need. A quantitative high performance liquid chromatography method (HPLC) and a confirmative liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of 18 sulfonamides such as sulfamidinum, sulfanilamide, sulfisomidine, sulfadiazine, sulfapyridine, sulfathiazole, sulfamerazine, sulfadimidine, sulfamethoxypyridazine, sulfamethoxydiazine, sulfisoxazole, sulfachloropyridazine, sulfamethoxazole, sulfamonomethoxine, sulfadoxine, sulfaclozine, sulfadimethoxine, sulfaquinoxaline in the muscles, livers and kidneys of swine, bovine and chicken were developed and validated. The sample preparation procedures included a pressurized liquid extraction (PLE) with acetonitrile conducted at elevated temperature (70°C) and pressure (1400 psi). After clean-up with hydrophilic-lipophilic balance cartridge, the extraction solution was concentrated and analyzed by HPLC and LC-MS/MS analysis. 18 SAs were separated by the HPLC with a Zorbax SB-Aq-C18 column and the mobile phase of methanol/acetonitrile/1% acetic acid with a gradient system. The wavelength of UV for the HPLC detection was set at 285 nm. The LC-MS/MS analysis was achieved with a Hypersil Golden column and the mobile phase of acetonitrile and 0.1% formic acid aqueous solution with two gradient systems. The Limits of detection (LOD) and the limits of quantitation (LOQ) were 3 μg/kg and 10 μg/kg, respectively, for both of the HPLC and LC-MS/MS. Linearity was obtained with an average coefficient of determination (R) higher than 0.9980 over a dynamic range from the LOQ value up to 5000 μg/kg. The recoveries of the methods range from 71.1% to 118.3% with the relative standard derivation less than 13%. The peaks of interest with no interferences were observed throughout the chromatographic run. The sample pretreatment provided efficient extraction and cleanup that enables a sensitive and rugged determination of 18 SAs, the obtained results revealed that PLE, in comparison with other sample preparation methods applied, has significantly higher efficacy for SAs isolation from animal tissues. Copyright © 2011 Elsevier B.V. All rights reserved.

Mapping Antiretroviral Drugs in Tissue by IR-MALDESI MSI Coupled to the Q Exactive and Comparison with LC-MS/MS SRM Assay

NASA Astrophysics Data System (ADS)

Barry, Jeremy A.; Robichaud, Guillaume; Bokhart, Mark T.; Thompson, Corbin; Sykes, Craig; Kashuba, Angela D. M.; Muddiman, David C.

2014-12-01

This work describes the coupling of the IR-MALDESI imaging source with the Q Exactive mass spectrometer. IR-MALDESI MSI was used to elucidate the spatial distribution of several HIV drugs in cervical tissues that had been incubated in either a low or high concentration. Serial sections of those analyzed by IR-MALDESI MSI were homogenized and analyzed by LC-MS/MS to quantify the amount of each drug present in the tissue. By comparing the two techniques, an agreement between the average intensities from the imaging experiment and the absolute quantities for each drug was observed. This correlation between these two techniques serves as a prerequisite to quantitative IR-MALDESI MSI. In addition, a targeted MS2 imaging experiment was also conducted to demonstrate the capabilities of the Q Exactive and to highlight the added selectivity that can be obtained with SRM or MRM imaging experiments.

Comparative Proteomics of Human Monkeypox and Vaccinia Intracellular Mature and Extracellular Enveloped Virions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Manes, Nathan P.; Estep, Ryan D.; Mottaz, Heather M.

2008-03-07

Orthopoxviruses are the largest and most complex of the animal viruses. In response to the recent emergence of monkeypox in Africa and the threat of smallpox bioterrorism, virulent (monkeypox virus) and benign (vaccinia virus) orthopoxviruses were proteomically compared with the goal of identifying proteins required for pathogenesis. Orthopoxviruses were grown in HeLa cells to two different viral forms (intracellular mature virus and extracellular enveloped virus), purified by sucrose gradient ultracentrifugation, denatured using RapiGest™ surfactant, and digested with trypsin. Unfractionated samples and strong cation exchange HPLC fractions were analyzed by reversed-phase LC-MS/MS, and analyses of the MS/MS spectra using SEQUEST® andmore » X! Tandem resulted in the identification of hundreds of monkeypox, vaccinia, and copurified host proteins. The unfractionated samples were additionally analyzed by LC-MS on an LTQ-Orbitrap™, and the accurate mass and elution time tag approach was used to perform quantitative comparisons. Possible pathophysiological roles of differentially expressed orthopoxvirus genes are discussed.« less

Verification of propofol sulfate as a further human propofol metabolite using LC-ESI-QQQ-MS and LC-ESI-QTOF-MS analysis.

PubMed

Maas, Alexandra; Maier, Christoph; Michel-Lauter, Beate; Broecker, Sebastian; Madea, Burkhard; Hess, Cornelius

2017-03-01

Propofol (2,6-diisopropylphenol) is a water-insoluble, intravenous anesthetic that is widely used for the induction and maintenance of anesthesia as well as for endoscopic and pediatric sedation. After admission, propofol undergoes extensive hepatic and extrahepatic metabolism, including direct conjugation to propofol glucuronide and hydroxylation to 2,6-diisopropyl-1,4-quinol. The latter substance subsequently undergoes phase II metabolism, resulting in the formation of further metabolites (1quinolglucuronide, 4quinolglucuronide and 4quinol-sulfate). Further minor phase I propofol metabolites (2-(ω-propanol)-6-isopropylphenol and 2-(ω-propanol)-6-isopropyl-1,4-quinol)) are also described. Due to its chemical structure with the phenolic hydroxyl group, propofol is also an appropriate substrate for sulfation by sulfotransferases. The existence of propofol sulfate was investigated by liquid chromatography electrospray ionization triple quadrupole mass spectrometry (LCESIQQQ-MS) and liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LCESI-QTOF-MS). A propofol sulfate reference standard was used for identification and method development, yielding a precursor at m/z 257 (deprotonated propofol sulfate) and product ions at m/z 177 (deprotonated propofol) and m/z 80 ([SO3]-). Propofol sulfate - a further phase II metabolite of propofol - was verified in urine samples by LC-ESI-QQQ-MS and LC-ESI-QTOF-MS. Analyses of urine samples from five volunteers collected before and after propofol-induced sedation verified the presence of propofol sulfate in urine following propofol administration, whereas ascertained concentrations of this metabolite were significantly lower compared with detected propofol glucuronide concentrations. The existence of propofol sulfate as a further phase II propofol metabolite in humans could be verified by two different detection techniques (LCESIQQQ-MS and LC-ESI-QTOFMS) on the basis of a propofol sulfate reference standard. Evaluation of the quantitative analyses of propofol sulfate imply that propofol sulfate represents a minor metabolite of propofol and is only slightly involved in human propofol clearance.

General Unknown Screening by Ion Trap LC/MS/MS

DTIC Science & Technology

2010-04-01

Subtitle 5 . Report Date April 2010 General Unknown Screening by Ion Trap LC/MS/MS 6 . Performing Organization Code 7. Author(s) 8... 5 Table 1: Analytical Data for Each of the...359 Compounds in the LC/MS/MS Library . . . . . . . . . . . 6 1 General Unknown ScreeninG by ion Trap lc/MS/MS INTrOduCTION The Federal Aviation

Methods for Quantitative Creatinine Determination.

PubMed

Moore, John F; Sharer, J Daniel

2017-04-06

Reliable measurement of creatinine is necessary to assess kidney function, and also to quantitate drug levels and diagnostic compounds in urine samples. The most commonly used methods are based on the Jaffe principal of alkaline creatinine-picric acid complex color formation. However, other compounds commonly found in serum and urine may interfere with Jaffe creatinine measurements. Therefore, many laboratories have made modifications to the basic method to remove or account for these interfering substances. This appendix will summarize the basic Jaffe method, as well as a modified, automated version. Also described is a high performance liquid chromatography (HPLC) method that separates creatinine from contaminants prior to direct quantification by UV absorption. Lastly, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described that uses stable isotope dilution to reliably quantify creatinine in any sample. This last approach has been recommended by experts in the field as a means to standardize all quantitative creatinine methods against an accepted reference. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Quantitative Determination of Fluorinated Alkyl Substances by Large-Volume-Injection LC/MS/MS—Characterization of Municipal Wastewaters

PubMed Central

Schultz, Melissa M.; Barofsky, Douglas F.; Field, Jennifer A.

2008-01-01

A quantitative method was developed for the determination of fluorinated alkyl substances in municipal wastewater influents and effluents. The method consisted of centrifugation followed by large-volume injection (500 μL) of the supernatant onto a liquid chromatograph with a reverse-phase column and detection by electrospray ionization, and tandem mass spectrometry (LC/MS/MS). The fluorinated analytes studied include perfluoroalkyl sulfonates, fluorotelomer sulfonates, perfluorocarboxylates, and select fluorinated alkyl sulfonamides. Recoveries of the fluorinated analytes from wastewater treatment plant (WWTP) raw influents and final effluent ranged from 77% – 96% and 80% – 99%, respectively. The lower limit of quantitation ranged from 0.5 to 3.0 ng/L depending on the analyte. The method was applied to flow-proportional composites of raw influent and final effluent collected over a 24 hr period from ten WWTPs nationwide. Fluorinated alkyl substances were observed in wastewater at all treatment plants and each plant exhibited unique distributions of fluorinated alkyl substances despite similarities in treatment processes. In nine out of the ten plants sampled, at least one class of fluorinated alkyl substances exhibited increased concentrations in the effluent as compared to the influent concentrations. In some instances, decreases in certain fluorinated analyte concentrations were observed and attributed to sorption to sludge. PMID:16433363

Mining the human urine proteome for monitoring renal transplant injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sigdel, Tara K.; Gao, Yuqian; He, Jintang

The human urinary proteome reflects systemic and inherent renal injury perturbations and can be analyzed to harness specific biomarkers for different kidney transplant injury states. 396 unique urine samples were collected contemporaneously with an allograft biopsy from 396 unique kidney transplant recipients. Centralized, blinded histology on the graft was used to classify matched urine samples into categories of acute rejection (AR), chronic allograft nephropathy (CAN), BK virus nephritis (BKVN), and stable graft (STA). Liquid chromatography–mass spectrometry (LC-MS) based proteomics using iTRAQ based discovery (n=108) and global label-free LC-MS analyses of individual samples (n=137) for quantitative proteome assessment were used inmore » the discovery step. Selected reaction monitoring (SRM) was applied to identify and validate minimal urine protein/peptide biomarkers to accurately segregate organ injury causation and pathology on unique urine samples (n=151). A total of 958 proteins were initially quantified by iTRAQ, 87% of which were also identified among 1574 urine proteins detected in LC-MS validation. 103 urine proteins were significantly (p<0.05) perturbed in injury and enriched for humoral immunity, complement activation, and lymphocyte trafficking. A set of 131 peptides corresponding to 78 proteins were assessed by SRM for their significance in an independent sample cohort. A minimal set of 35 peptides mapping to 33 proteins, were modeled to segregate different injury groups (AUC =93% for AR, 99% for CAN, 83% for BKVN). Urinary proteome discovery and targeted validation identified urine protein fingerprints for non-invasive differentiation of kidney transplant injuries, thus opening the door for personalized immune risk assessment and therapy.« less

Sample preparation and liquid chromatography-tandem mass spectrometry for multiple steroids in mammalian and avian circulation.

PubMed

Koren, Lee; Ng, Ella S M; Soma, Kiran K; Wynne-Edwards, Katherine E

2012-01-01

Blood samples from wild mammals and birds are often limited in volume, allowing researchers to quantify only one or two steroids from a single sample by immunoassays. In addition, wildlife serum or plasma samples are often lipemic, necessitating stringent sample preparation. Here, we validated sample preparation for simultaneous liquid chromatography--tandem mass spectrometry (LC-MS/MS) quantitation of cortisol, corticosterone, 11-deoxycortisol, dehydroepiandrosterone (DHEA), 17β-estradiol, progesterone, 17α-hydroxyprogesterone and testosterone from diverse mammalian (7 species) and avian (5 species) samples. Using 100 µL of serum or plasma, we quantified (signal-to-noise (S/N) ratio ≥ 10) 4-7 steroids depending on the species and sample, without derivatization. Steroids were extracted from serum or plasma using automated solid-phase extraction where samples were loaded onto C18 columns, washed with water and hexane, and then eluted with ethyl acetate. Quantitation by LC-MS/MS was done in positive ion, multiple reaction-monitoring (MRM) mode with an atmospheric pressure chemical ionization (APCI) source and heated nebulizer (500°C). Deuterated steroids served as internal standards and run time was 15 minutes. Extraction recoveries were 87-101% for the 8 analytes, and all intra- and inter-run CVs were ≤ 8.25%. This quantitation method yields good recoveries with variable lipid-content samples, avoids antibody cross-reactivity issues, and delivers results for multiple steroids. Thus, this method can enrich datasets by providing simultaneous quantitation of multiple steroids, and allow researchers to reimagine the hypotheses that could be tested with their volume-limited, lipemic, wildlife samples.

Definitive screening design enables optimization of LC-ESI-MS/MS parameters in proteomics.

PubMed

Aburaya, Shunsuke; Aoki, Wataru; Minakuchi, Hiroyoshi; Ueda, Mitsuyoshi

2017-12-01

In proteomics, more than 100,000 peptides are generated from the digestion of human cell lysates. Proteome samples have a broad dynamic range in protein abundance; therefore, it is critical to optimize various parameters of LC-ESI-MS/MS to comprehensively identify these peptides. However, there are many parameters for LC-ESI-MS/MS analysis. In this study, we applied definitive screening design to simultaneously optimize 14 parameters in the operation of monolithic capillary LC-ESI-MS/MS to increase the number of identified proteins and/or the average peak area of MS1. The simultaneous optimization enabled the determination of two-factor interactions between LC and MS. Finally, we found two parameter sets of monolithic capillary LC-ESI-MS/MS that increased the number of identified proteins by 8.1% or the average peak area of MS1 by 67%. The definitive screening design would be highly useful for high-throughput analysis of the best parameter set in LC-ESI-MS/MS systems.

Comparative examination of adsorption of serum proteins on HSA- and PLGA-based nanoparticles using SDS-PAGE and LC-MS.

PubMed

Gossmann, R; Fahrländer, E; Hummel, M; Mulac, D; Brockmeyer, J; Langer, K

2015-06-01

The behavior of nanosized drug carrier systems under cell culture conditions and therefore also the destiny in the body are highly influenced by the protein corona, which is formed upon entering a biological environment. Some of the adsorbed proteins, named opsonins, lead to a shortened plasma circulation half-life of the nanoparticles. Others are attributed to promote the transport of nanoparticles into other compartments of the body, just to mention two examples. Hence, detailed knowledge concerning the composition of the protein corona is of great importance. The aim of this work was to investigate the influence of the nanoparticle starting material and the surface modification on the composition of the adsorbed serum proteins in a cell culture environment. Therefore, positively charged nanoparticles based on the biodegradable polymer poly(dl-lactide-co-glycolide) (PLGA) stabilized with didodecyldimethylammonium bromide (DMAB) and negatively charged nanoparticles based on human serum albumin (HSA) were prepared and modified with hydrophilic polymers. By incubating the nanoparticles with fetal bovine serum (FBS) the adsorption of serum proteins on the colloidal system was investigated. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) a semi-quantitative analysis of the protein corona was performed and after enzymatic in-solution-digestion the adsorbed proteins were identified using high resolution LC-MS. Our study accentuates the influence of the core material, surface charge, and surface modification on the amount and nature of the adsorbed proteins. The combination of SDS-PAGE and LC-MS turns out to be a simple and reliable method to investigate the protein corona of nanoparticles. Copyright © 2015 Elsevier B.V. All rights reserved.

Review of Software Tools for Design and Analysis of Large scale MRM Proteomic Datasets

PubMed Central

Colangelo, Christopher M.; Chung, Lisa; Bruce, Can; Cheung, Kei-Hoi

2013-01-01

Selective or Multiple Reaction monitoring (SRM/MRM) is a liquid-chromatography (LC)/tandem-mass spectrometry (MS/MS) method that enables the quantitation of specific proteins in a sample by analyzing precursor ions and the fragment ions of their selected tryptic peptides. Instrumentation software has advanced to the point that thousands of transitions (pairs of primary and secondary m/z values) can be measured in a triple quadrupole instrument coupled to an LC, by a well-designed scheduling and selection of m/z windows. The design of a good MRM assay relies on the availability of peptide spectra from previous discovery-phase LC-MS/MS studies. The tedious aspect of manually developing and processing MRM assays involving thousands of transitions has spurred to development of software tools to automate this process. Software packages have been developed for project management, assay development, assay validation, data export, peak integration, quality assessment, and biostatistical analysis. No single tool provides a complete end-to-end solution, thus this article reviews the current state and discusses future directions of these software tools in order to enable researchers to combine these tools for a comprehensive targeted proteomics workflow. PMID:23702368

Ranking Fragment Ions Based on Outlier Detection for Improved Label-Free Quantification in Data-Independent Acquisition LC-MS/MS

PubMed Central

Bilbao, Aivett; Zhang, Ying; Varesio, Emmanuel; Luban, Jeremy; Strambio-De-Castillia, Caterina; Lisacek, Frédérique; Hopfgartner, Gérard

2016-01-01

Data-independent acquisition LC-MS/MS techniques complement supervised methods for peptide quantification. However, due to the wide precursor isolation windows, these techniques are prone to interference at the fragment ion level, which in turn is detrimental for accurate quantification. The “non-outlier fragment ion” (NOFI) ranking algorithm has been developed to assign low priority to fragment ions affected by interference. By using the optimal subset of high priority fragment ions these interfered fragment ions are effectively excluded from quantification. NOFI represents each fragment ion as a vector of four dimensions related to chromatographic and MS fragmentation attributes and applies multivariate outlier detection techniques. Benchmarking conducted on a well-defined quantitative dataset (i.e. the SWATH Gold Standard), indicates that NOFI on average is able to accurately quantify 11-25% more peptides than the commonly used Top-N library intensity ranking method. The sum of the area of the Top3-5 NOFIs produces similar coefficients of variation as compared to the library intensity method but with more accurate quantification results. On a biologically relevant human dendritic cell digest dataset, NOFI properly assigns low priority ranks to 85% of annotated interferences, resulting in sensitivity values between 0.92 and 0.80 against 0.76 for the Spectronaut interference detection algorithm. PMID:26412574

DMS-prefiltered mass spectrometry for the detection of biomarkers

NASA Astrophysics Data System (ADS)

Coy, Stephen L.; Krylov, Evgeny V.; Nazarov, Erkinjon G.

2008-04-01

Technologies based on Differential Mobility Spectrometry (DMS) are ideally matched to rapid, sensitive, and selective detection of chemicals like biomarkers. Biomarkers linked to exposure to radiation, exposure to CWA's, exposure to toxic materials (TICs and TIMs) and to specific diseases are being examined in a number of laboratories. Screening for these types of exposure can be improved in accuracy and greatly speeded up by using DMS-MS instead of slower techniques like LC-MS and GC-MS. We have performed an extensive series of tests with nanospray-DMS-mass spectroscopy and standalone nanospray-DMS obtaining extensive information on chemistry and detectivity. DMS-MS systems implemented with low-resolution, low-cost, portable mass-spectrometry systems are very promising. Lowresolution mass spectrometry alone would be inadequate for the task, but with DMS pre-filtration to suppress interferences, can be quite effective, even for quantitative measurement. Bio-fluids and digests are well suited to ionization by electrospray and detection by mass-spectrometry, but signals from critical markers are overwhelmed by chemical noise from unrelated species, making essential quantitative analysis impossible. Sionex and collaborators have presented data using DMS to suppress chemical noise, allowing detection of cancer biomarkers in 10,000-fold excess of normal products 1,2. In addition, a linear dynamic range of approximately 2,000 has been demonstrated with accurate quantitation 3. We will review the range of possible applications and present new data on DMS-MS biomarker detection.

Development of a general method for quantifying IgG-based therapeutic monoclonal antibodies in human plasma using protein G purification coupled with a two internal standard calibration strategy using LC-MS/MS.

PubMed

Chiu, Huai-Hsuan; Liao, Hsiao-Wei; Shao, Yu-Yun; Lu, Yen-Shen; Lin, Ching-Hung; Tsai, I-Lin; Kuo, Ching-Hua

2018-08-17

Monoclonal antibody (mAb) drugs have generated much interest in recent years for treating various diseases. Immunoglobulin G (IgG) represents a high percentage of mAb drugs that have been approved by the Food and Drug Administration (FDA). To facilitate therapeutic drug monitoring and pharmacokinetic/pharmacodynamic studies, we developed a general liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify the concentration of IgG-based mAbs in human plasma. Three IgG-based drugs (bevacizumab, nivolumab and pembrolizumab) were selected to demonstrate our method. Protein G beads were used for sample pretreatment due to their universal ability to trap IgG-based drugs. Surrogate peptides that were obtained after trypsin digestion were quantified by using LC-MS/MS. To calibrate sample preparation errors and matrix effects that occur during LC-MS/MS analysis, we used two internal standards (IS) method that include the IgG-based drug-IS tocilizumab and post-column infused IS. Using two internal standards was found to effectively improve quantification accuracy, which was within 15% for all mAb drugs that were tested at three different concentrations. This general method was validated in term of its precision, accuracy, linearity and sensitivity for 3 demonstration mAb drugs. The successful application of the method to clinical samples demonstrated its' applicability in clinical analysis. It is anticipated that this general method could be applied to other mAb-based drugs for use in precision medicine and clinical studies. Copyright © 2018 Elsevier B.V. All rights reserved.

Proteomics wants cRacker: automated standardized data analysis of LC-MS derived proteomic data.

PubMed

Zauber, Henrik; Schulze, Waltraud X

2012-11-02

The large-scale analysis of thousands of proteins under various experimental conditions or in mutant lines has gained more and more importance in hypothesis-driven scientific research and systems biology in the past years. Quantitative analysis by large scale proteomics using modern mass spectrometry usually results in long lists of peptide ion intensities. The main interest for most researchers, however, is to draw conclusions on the protein level. Postprocessing and combining peptide intensities of a proteomic data set requires expert knowledge, and the often repetitive and standardized manual calculations can be time-consuming. The analysis of complex samples can result in very large data sets (lists with several 1000s to 100,000 entries of different peptides) that cannot easily be analyzed using standard spreadsheet programs. To improve speed and consistency of the data analysis of LC-MS derived proteomic data, we developed cRacker. cRacker is an R-based program for automated downstream proteomic data analysis including data normalization strategies for metabolic labeling and label free quantitation. In addition, cRacker includes basic statistical analysis, such as clustering of data, or ANOVA and t tests for comparison between treatments. Results are presented in editable graphic formats and in list files.

Differential expression profiling of serum proteins and metabolites for biomarker discovery

NASA Astrophysics Data System (ADS)

Roy, Sushmita Mimi; Anderle, Markus; Lin, Hua; Becker, Christopher H.

2004-11-01

A liquid chromatography-mass spectrometry (LC-MS) proteomics and metabolomics platform is presented for quantitative differential expression analysis. Proteome profiles obtained from 1.5 [mu]L of human serum show ~5000 de-isotoped and quantifiable molecular ions. Approximately 1500 metabolites are observed from 100 [mu]L of serum. Quantification is based on reproducible sample preparation and linear signal intensity as a function of concentration. The platform is validated using human serum, but is generally applicable to all biological fluids and tissues. The median coefficient of variation (CV) for ~5000 proteomic and ~1500 metabolomic molecular ions is approximately 25%. For the case of C-reactive protein, results agree with quantification by immunoassay. The independent contributions of two sources of variance, namely sample preparation and LC-MS analysis, are respectively quantified as 20.4 and 15.1% for the proteome, and 19.5 and 13.5% for the metabolome, for median CV values. Furthermore, biological diversity for ~20 healthy individuals is estimated by measuring the variance of ~6500 proteomic and metabolomic molecular ions in sera for each sample; the median CV is 22.3% for the proteome and 16.7% for the metabolome. Finally, quantitative differential expression profiling is applied to a clinical study comparing healthy individuals and rheumatoid arthritis (RA) patients.

High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) for Mass Spectrometry-Based Proteomics

PubMed Central

Swearingen, Kristian E.; Moritz, Robert L.

2013-01-01

SUMMARY High field asymmetric waveform ion mobility spectrometry (FAIMS) is an atmospheric pressure ion mobility technique that separates gas-phase ions by their behavior in strong and weak electric fields. FAIMS is easily interfaced with electrospray ionization and has been implemented as an additional separation mode between liquid chromatography (LC) and mass spectrometry (MS) in proteomic studies. FAIMS separation is orthogonal to both LC and MS and is used as a means of on-line fractionation to improve detection of peptides in complex samples. FAIMS improves dynamic range and concomitantly the detection limits of ions by filtering out chemical noise. FAIMS can also be used to remove interfering ion species and to select peptide charge states optimal for identification by tandem MS. Here, we review recent developments in LC-FAIMS-MS and its application to MS-based proteomics. PMID:23194268

Effect of Genetics, Environment, and Phenotype on the Metabolome of Maize Hybrids Using GC/MS and LC/MS.

PubMed

Tang, Weijuan; Hazebroek, Jan; Zhong, Cathy; Harp, Teresa; Vlahakis, Chris; Baumhover, Brian; Asiago, Vincent

2017-06-28

We evaluated the variability of metabolites in various maize hybrids due to the effect of environment, genotype, phenotype as well as the interaction of the first two factors. We analyzed 480 forage and the same number of grain samples from 21 genetically diverse non-GM Pioneer brand maize hybrids, including some with drought tolerance and viral resistance phenotypes, grown at eight North American locations. As complementary platforms, both GC/MS and LC/MS were utilized to detect a wide diversity of metabolites. GC/MS revealed 166 and 137 metabolites in forage and grain samples, respectively, while LC/MS captured 1341 and 635 metabolites in forage and grain samples, respectively. Univariate and multivariate analyses were utilized to investigate the response of the maize metabolome to the environment, genotype, phenotype, and their interaction. Based on combined percentages from GC/MS and LC/MS datasets, the environment affected 36% to 84% of forage metabolites, while less than 7% were affected by genotype. The environment affected 12% to 90% of grain metabolites, whereas less than 27% were affected by genotype. Less than 10% and 11% of the metabolites were affected by phenotype in forage and grain, respectively. Unsupervised PCA and HCA analyses revealed similar trends, i.e., environmental effect was much stronger than genotype or phenotype effects. On the basis of comparisons of disease tolerant and disease susceptible hybrids, neither forage nor grain samples originating from different locations showed obvious phenotype effects. Our findings demonstrate that the combination of GC/MS and LC/MS based metabolite profiling followed by broad statistical analysis is an effective approach to identify the relative impact of environmental, genetic and phenotypic effects on the forage and grain composition of maize hybrids.

A Label-Free Porous Silicon Immunosensor for Broad Detection of Opiates in a Blind Clinical Study and Result Comparison to Commercial Analytical Chemistry Techniques

PubMed Central

Bonanno, Lisa M.; Kwong, Tai C.; DeLouise, Lisa A.

2010-01-01

In this work we evaluate for the first time the performance of a label-free porous silicon (PSi) immunosensor assay in a blind clinical study designed to screen authentic patient urine specimens for a broad range of opiates. The PSi opiate immunosensor achieved 96% concordance with liquid chromatography-mass spectrometry/tandem mass spectrometry (LC-MS/MS) results on samples that underwent standard opiate testing (n=50). In addition, successful detection of a commonly abused opiate, oxycodone, resulted in 100% qualitative agreement between the PSi opiate sensor and LC-MS/MS. In contrast, a commercial broad opiate immunoassay technique (CEDIA®) achieved 65% qualitative concordance with LC-MS/MS. Evaluation of important performance attributes including precision, accuracy, and recovery was completed on blank urine specimens spiked with test analytes. Variability of morphine detection as a model opiate target was < 9% both within-run and between-day at and above the cutoff limit of 300 ng ml−1. This study validates the analytical screening capability of label-free PSi opiate immunosensors in authentic patient samples and is the first semi-quantitative demonstration of the technology’s successful clinical use. These results motivate future development of PSi technology to reduce complexity and cost of diagnostic testing particularly in a point-of-care setting. PMID:21062030

Chemometrically assisted development and validation of LC-MS/MS method for the analysis of potential genotoxic impurities in meropenem active pharmaceutical ingredient.

PubMed

Grigori, Katerina; Loukas, Yannis L; Malenović, Anđelija; Samara, Vicky; Kalaskani, Anastasia; Dimovasili, Efi; Kalovidouri, Magda; Dotsikas, Yannis

2017-10-25

A sensitive Liquid Chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitative analysis of three potential genotoxic impurities (318BP, M9, S5) in meropenem Active Pharmaceutical Ingredient (API). Due to the requirement for LOD values in ppb range, a high concentration of meropenem API (30mg/mL) had to be injected. Therefore, efficient determination of meropenem from its impurities became a critical aim of this study, in order to divert meropenem to waste, via a switching valve. ‎ After the selection of the important factors affecting analytes' elution, a Box-Behnken design was utilized to set the plan of experiments conducted with UV detector. As responses, the separation factor s between the last eluting impurity and meropenem, as well as meropenem retention factor k were used. Grid point search methodology was implemented aiming to obtain the optimal conditions that simultaneously comply to the conflicted criteria. Optimal mobile phase consisted of ACN, methanol and 0.09% HCOOH at a ratio 71/3.5/15.5v/v. All impurities and internal standard omeprazole were eluted before 7.5min and at 8.0min the eluents were directed to waste. The protocol was transferred to LC-MS/MS and validated according to ICH guidelines. Copyright © 2017 Elsevier B.V. All rights reserved.

A high-throughput mass spectrometry assay to simultaneously measure intact insulin and C-peptide.

PubMed

Taylor, Steven W; Clarke, Nigel J; Chen, Zhaohui; McPhaul, Michael J

2016-04-01

Measurements of fasting levels of insulin and C-peptide are useful in documenting insulin resistance and may help predict development of diabetes mellitus. However, the specific insulin and C-peptide levels associated with specific degrees of insulin resistance have not been defined, owing to marked variability among immunoassays and lack of standardization. Herein, we describe a multiplexed liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for intact insulin and C-peptide. Insulin and C-peptide were enriched from patient sera using monoclonal antibodies immobilized on magnetic beads and processed on a robotic liquid handler. Eluted peptides were analyzed by LC-MS/MS. Bovine insulin and a stable isotopically-labeled (13C/15N) C-peptide were utilized as internal standards. The assay had an analytical measurement range of 3 to 320 μIU/ml (18 to 1920 pmol/l) for insulin and 0.11 to 27.2 ng/ml (36 to 9006 pmol/l) for C-peptide. Intra- and inter-day assay variation was less than 11% for both peptides. Of the 5 insulin analogs commonly prescribed to treat diabetes, only the recombinant drug insulin lispro caused significant interference for the determination of endogenous insulin. There were no observed interferences for C-peptide. We developed and validated a high-throughput, quantitative, multiplexed LC-MS/MS assay for intact insulin and C-peptide. Copyright © 2016 Elsevier B.V. All rights reserved.

Liquid chromatography-tandem mass spectrometry detection of the quaternary ammonium compound mebezonium as an active ingredient in t61.

PubMed

Kirschbaum, Katrin M; Grellner, Wolfgang; Rochholz, Gertrud; Musshoff, Frank; Madea, Burkhard

2011-03-01

Quaternary ammonium compounds pose an analytical challenge. Mebezonium, a muscle-relaxing agent contained in veterinary euthanasia solution T61, was analyzed in body fluids, organs, and injection sites of a veterinarian by liquid chromatography-tandem mass spectrometry (LC-MS-MS) method. Additionally, embutramide and tetracaine, which are two other active ingredients contained in T61, methadone, xylazine, and analgesics were detected by LC-MS-MS and high-performance liquid chromatography-ultraviolet detection methods. For detection of mebezonium a solid-phase extraction (SPE) combined with ionpairing reagent heptafluorobutyric acid was developed. Separation was achieved on Phenomenex Synergi Hydro RP C(18) column combined with ammonium formate buffer and acetonitrile (pH 3.5). To enrich other drugs, liquid-liquid extraction procedures were used. Most of these drugs were separated on a Restek Allure PFP Propyl column using the mentioned mobile phase. Mebezonium and embutramide were detected in femoral vein serum in concentrations of 10.9 and 2.0 mg/L, respectively. The concentration of xylazine and methadone in serum was 2.0 and 0.4 mg/L, respectively. The LC-MS-MS method with SPE combined with an ion-pairing reagent allowed the quantitation of mebezonium. Methadone was detected in toxic concentrations and was, in combination with xylazine and T61, considered to be the cause of death.

Variations in plasma and urinary lipids in response to enzyme replacement therapy for Fabry disease patients by nanoflow UPLC-ESI-MS/MS.

PubMed

Byeon, Seul Kee; Kim, Jin Yong; Lee, Jin-Sung; Moon, Myeong Hee

2016-03-01

A deficiency of α-galactosidase A causes Fabry disease (FD) by disrupting lipid metabolism, especially trihexosylceramide (THC). Enzyme replacement therapy (ERT) is clinically offered to FD patients in an attempt to lower the accumulated lipids. Studies on specific types of lipids that are directly or indirectly altered by FD are very scarce, even though they are crucial in understanding the biological process linked to the pathogenesis of FD. We performed a comprehensive lipid profiling of plasma and urinary lipids from FD patients with nanoflow liquid chromatography electrospray-ionization tandem mass spectrometry (nLC-ESI-MS/MS) and identified 129 plasma and 111 urinary lipids. Among these, lipids that exhibited alternations (>twofold) in patients were selected as targets for selected reaction monitoring (SRM)-based high-speed quantitation using nanoflow ultra-performance LC-ESI-MS/MS (nUPLC-ESI-MS/MS) and 31 plasma and 26 urinary lipids showed significant elevation among FD patients. Higher percentages of sphingolipids (SLs; 48% for plasma and 42% for urine) were highly elevated in patients; whereas, a smaller percentage of phospholipids (PLs; 15% for plasma and 13% for urine) were significantly affected. Even though α-galactosidase A is reported to affect THC only, the results show that other classes of lipids (especially SLs) are changed as well, indicating that FD not only alters metabolism of THC but various classes of lipids too. Most lipids showing significant increases in relative amounts before ERT decreased after ERT, but overall, ERT influenced plasma lipids more than urinary lipids.

Post-acquisition data mining techniques for LC-MS/MS-acquired data in drug metabolite identification.

PubMed

Dhurjad, Pooja Sukhdev; Marothu, Vamsi Krishna; Rathod, Rajeshwari

2017-08-01

Metabolite identification is a crucial part of the drug discovery process. LC-MS/MS-based metabolite identification has gained widespread use, but the data acquired by the LC-MS/MS instrument is complex, and thus the interpretation of data becomes troublesome. Fortunately, advancements in data mining techniques have simplified the process of data interpretation with improved mass accuracy and provide a potentially selective, sensitive, accurate and comprehensive way for metabolite identification. In this review, we have discussed the targeted (extracted ion chromatogram, mass defect filter, product ion filter, neutral loss filter and isotope pattern filter) and untargeted (control sample comparison, background subtraction and metabolomic approaches) post-acquisition data mining techniques, which facilitate the drug metabolite identification. We have also discussed the importance of integrated data mining strategy.

Mass spectrometry data from label-free quantitative proteomic analysis of harmless and pathogenic strains of infectious microalgae, Prototheca spp.

PubMed

Murugaiyan, Jayaseelan; Eravci, Murat; Weise, Christoph; Roesler, Uwe

2017-06-01

Here, we provide the dataset associated with our research article 'label-free quantitative proteomic analysis of harmless and pathogenic strains of infectious microalgae, Prototheca spp.' (Murugaiyan et al., 2017) [1]. This dataset describes liquid chromatography-mass spectrometry (LC-MS)-based protein identification and quantification of a non-infectious strain, Prototheca zopfii genotype 1 and two strains associated with severe and mild infections, respectively, P. zopfii genotype 2 and Prototheca blaschkeae . Protein identification and label-free quantification was carried out by analysing MS raw data using the MaxQuant-Andromeda software suit. The expressional level differences of the identified proteins among the strains were computed using Perseus software and the results were presented in [1]. This DiB provides the MaxQuant output file and raw data deposited in the PRIDE repository with the dataset identifier PXD005305.

Isolation of n-decyl-alpha(1-->6) isomaltoside from a technical APG mixture and its identification by the parallel use of LC-MS and NMR spectroscopy

PubMed

Billian; Hock; Doetzer; Stan; Dreher

2000-10-15

The identification of n-decyl alpha(1-->6)isomaltoside as a main component of technical alkyl polyglucoside (APG) mixtures by the parallel use of liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy is described. Following enrichment on a styrene-divinylbenzene-based solid-phase extraction material, unknown components were separated by reversed-phase liquid chromatography (LC). Chemical characterization was achieved by both mass spectrometry and NMR spectroscopy. It is demonstrated that the combination of LC-MS with various NMR techniques is very suitable for stereochemical assignment of unknown components in technical APG mixtures.

A simultaneous quantitative method for vitamins A, D and E in human serum using liquid chromatography-tandem mass spectrometry.

PubMed

Albahrani, Ali A; Rotarou, Victor; Roche, Peter J; Greaves, Ronda F

2016-05-01

Non-classical roles of fat-soluble vitamins (FSVs) in many pathologies including cancer have been identified. There is also evidence of hormonal interactions between two of these vitamins, A and D. As a result of this enhanced clinical association with disease, translational clinical research and laboratory requests for FSV measurement has significantly increased. However there are still gaps in the analytical methods available for the measurement of these vitamins. This study aimed to develop a method for simultaneous quantification of 25-hydroxyvitamin-D2 (25-OHD2), 25-hydroxyvitamin-D3 (25-OHD3) and its 3-epimer (epi-25-OHD3), retinol and α-tocopherol in human serum using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The procedure was developed and validated across two LC-MS/MS platforms, using commercial calibrators referenced to certified reference materials, controls, and deuterated internal standards. The samples were prepared by liquid-liquid extraction prior to injection and LC separation (using a Pursuit-PFP column) on two Agilent MS/MS systems (6410 and 6490) in electrospray ionisation positive mode with multiple reaction monitoring. Identification and quantification of 25-OHD3 from its 3-epimer as well as 25-OHD2, retinol and α-tocopherol were achieved. The dynamic ranges were 4-160 nmol/L for 25-OHD2 and epi-25-OHD3, 4-200 nmol/L for 25-OHD3, 0.1-4.0μmol/L for retinol and 4-70μmol/L for α-tocopherol with correlation (r(2)) of 0.997-0.998. Based on participation in an external quality assurance program, the overall performance of the simultaneous methods were: imprecision (CV%) and inaccuracy (average bias) 3.0% and 3.2 nmol/L, respectively, for 25-OHD3; 5.0% and 0.04μmol/L, respectively, for retinol; and 4.7% and 0.2μmol/L, respectively, for α-tocopherol. In summary, two simple LC-MS/MS methods were successfully developed and validated for the simultaneous quantification of the three vitamin D metabolites (25-OHD2, 25-OHD3 and 3-epimer of 25-OHD3), vitamin A (retinol) and vitamin E (α-tocopherol) in serum. Copyright © 2016 Elsevier Ltd. All rights reserved.

Attribution of the discrepancy between ELISA and LC-MS/MS assay results of a PEGylated scaffold protein in post-dose monkey plasma samples due to the presence of anti-drug antibodies.

PubMed

Wang, Shujie J; Wu, Steven T; Gokemeijer, Jochem; Fura, Aberra; Krishna, Murli; Morin, Paul; Chen, Guodong; Price, Karen; Wang-Iverson, David; Olah, Timothy; Weiner, Russell; Tymiak, Adrienne; Jemal, Mohammed

2012-01-01

High-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA) methods were developed for the quantification of a PEGylated scaffold protein drug in monkey plasma samples. The LC-MS/MS method was based on the extraction of the therapeutic protein with a water-miscible organic solvent and the subsequent trypsin digestion of the extract followed by the detection of a surrogate peptide. The assay was linear over a range of 10-3,000 ng/mL. The ELISA method utilized a therapeutic target-binding format in which the recombinant target antigen was used to capture the drug in the sample, followed by detection with an anti-PEG monoclonal antibody. The assay range was 30-2,000 ng/mL. A correlation study between the two methods was performed by measuring the drug concentrations in plasma samples from a single-dose pharmacokinetic (PK) study in cynomolgus monkeys following a 5-mg/kg subcutaneous administration (n = 4). In the early time points of the PK profile, the drug concentrations obtained by the LC-MS/MS method agreed very well with those obtained by the ELISA method. However, at later time points, the drug concentrations measured by the LC-MS/MS method were consistently higher than those measured by the ELISA method. The PK parameters calculated based on the concentration data showed that the two methods gave equivalent peak exposure (C(max)) at 24-48 h. However, the LC-MS/MS results exhibited about 1.53-fold higher total exposure (AUC(tot)) than the ELISA results. The discrepancy between the LC-MS/MS and ELISA results was investigated by conducting immunogenicity testing, anti-drug antibody (ADA) epitope mapping, and Western blot analysis of the drug concentrations coupled with Protein G separation. The results demonstrated the presence of ADA specific to the engineered antigen-binding region of the scaffold protein drug that interfered with the ability of the drug to bind to the target antigen used in the ELISA method. In the presence of the ADAs, the ELISA method measured only the active circulating drug (target-binding), while the LC-MS/MS method measured the total circulating drug. The work presented here indicates that the bioanalysis of protein drugs may be complicated owing to the presence of drug-binding endogenous components or ADAs in the post-dose (incurred) samples. The clear understanding of the behavior of different bioanalytical techniques vis-à-vis the potentially interfering components found in incurred samples is critical in selecting bioanalytical strategies for measuring protein drugs.

Liquid Chromatography Electrospray Ionization Mass Spectrometric (LC-ESI-MS) and Desorption Electrospray Ionization Mass Spectrometric (DESI-MS) Identification of Chemical Warfare Agents in Consumer Products

DTIC Science & Technology

2007-06-01

T ACanadaY Approved for PublicR Distribution Uln& Liquid Chromatography Electrospray Ionization Mass Spectrometric ( LC -ESI- MS) and Desorption...consumer products with chemical warfare agents or other toxic chemicals. Liquid chromatography electrospray ionization mass spectrometry ( LC -ESI-MS) and...house LC -ESI-MS and LC -ESI-MS/MS methods were evaluated for the determination of chemical warfare agents in spiked bottled water samples. The

Identification and Characterization of Reaction Products of 5-Hydroxytryptamine with Methylglyoxal and Glyoxal by LC/MS/MS.

PubMed

Sai Sachin, L; Nagarjuna Chary, R; Pavankumar, P; Prabhakar, S

2018-06-06

The methylglyoxal (MGO) and glyoxal (GO) are known to be at high levels in the diabetic humans. They react with amine containing proteins and amino acids to form advanced glycation end products, however, the reactivity with the other amine containing metabolites, such as neurotransmitters are not explored. In this study, we aimed at studying the reactivity of 5-hydroxytryptamine (5-HT) with MGO or GO, which may alter the metabolic function of 5-HT. The stock solutions of 5-HT, MGO and GO were made in PBS buffer at pH 7.4 and incubated 5-HT with MGO or GO at difference concentrations. The reactions were also performed at physiological concentrations. The reaction mixtures collected at different incubation times were analyzed by direct ESI-HRMS, LC/MS and LC/MS/MS conditions to detect/characterize the products. Agilent 6545 Q-TOF and Agilent 6420 triple quadrupole mass spectrometer were used for the study, and LC separations were performed on a C18 column. The direct ESI-HRMS data of the reaction mixtures showed formation of three and four reaction products when 5-HT reacted with MGO and GO, respectively. All the products showed dominant [M+H] + ions. The products were characterized by HRMS, LC/MS/MS and the literature reports on similar compounds. The products can easily be identified by LC/MS based on the accurate mass values together with retention time information. The MS/MS of the reaction products showed structure indicative fragment ions. 5-HT reacts with one or two MGO/GO to form a set of reaction products. The reaction between 5-HT and MGO or GO was faster at higher concentrations of MGO/GO (<10 min), and the same products were found even at physiological concentrations (<48 hrs). The LC-MS/MS (SRM) method can be used to screen the reaction products when present at low level. This article is protected by copyright. All rights reserved.

Development of a liquid chromatography-tandem mass spectrometry method for quantitative analysis of trace d-amino acids.

PubMed

Nakano, Yosuke; Konya, Yutaka; Taniguchi, Moyu; Fukusaki, Eiichiro

2017-01-01

d-Amino acids have recently attracted much attention in various research fields including medical, clinical and food industry due to their important biological functions that differ from l-amino acid. Most chiral amino acid separation techniques require complicated derivatization procedures in order to achieve the desirable chromatographic behavior and detectability. Thus, the aim of this research is to develop a highly sensitive analytical method for the enantioseparation of chiral amino acids without any derivatization process using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By optimizing MS/MS parameters, we established a quantification method that allowed the simultaneous analysis of 18 d-amino acids with high sensitivity and reproducibility. Additionally, we applied the method to food sample (vinegar) for the validation, and successfully quantified trace levels of d-amino acids in samples. These results demonstrated the applicability and feasibility of the LC-MS/MS method as a novel, effective tool for d-amino acid measurement in various biological samples. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

An On-Line Solid Phase Extraction-Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Perfluoroalkyl Acids in Drinking and Surface Waters

PubMed Central

Mazzoni, Michela; Rusconi, Marianna; Valsecchi, Sara; Martins, Claudia P. B.; Polesello, Stefano

2015-01-01

An UHPLC-MS/MS multiresidue method based on an on-line solid phase extraction (SPE) procedure was developed for the simultaneous determination of 9 perfluorinated carboxylates (from 4 to 12 carbon atoms) and 3 perfluorinated sulphonates (from 4 to 8 carbon atoms). This work proposes using an on-line solid phase extraction before chromatographic separation and analysis to replace traditional methods of off-line SPE before direct injection to LC-MS/MS. Manual sample preparation was reduced to sample centrifugation and acidification, thus eliminating several procedural errors and significantly reducing time-consuming and costs. Ionization suppression between target perfluorinated analytes and their coeluting SIL-IS were detected for homologues with a number of carbon atoms less than 9, but the quantitation was not affected. Total matrix effect corrected by SIL-IS, inclusive of extraction efficacy, and of ionization efficiency, ranged between −34 and +39%. The percentage of recoveries, between 76 and 134%, calculated in different matrices (tap water and rivers impacted by different pollutions) was generally satisfactory. LODs and LOQs of this on-line SPE method, which also incorporate recovery losses, ranged from 0.2 to 5.0 ng/L and from 1 to 20 ng/L, respectively. Validated on-line SPE-LC/MS/MS method has been applied in a wide survey for the determination of perfluoroalkyl acids in Italian surface and ground waters. PMID:25834752

Analysis of Natural Toxins by Liquid Chromatography-Chemiluminescence Nitrogen Detection and Application to the Preparation of Certified Reference Materials.

PubMed

Thomas, Krista; Wechsler, Dominik; Chen, Yi-Min; Crain, Sheila; Quilliam, Michael A

2016-09-01

The implementation of instrumental analytical methods such as LC-MS for routine monitoring of toxins requires the availability of accurate calibration standards. This is a challenge because many toxins are rare, expensive, dangerous to handle, and/or unstable, and simple gravimetric procedures are not reliable for establishing accurate concentrations in solution. NMR has served as one method of qualitative and quantitative characterization of toxin calibration solution Certified Reference Materials (CRMs). LC with chemiluminescence N detection (LC-CLND) was selected as a complementary method for comprehensive characterization of CRMs because it provides a molar response to N. Here we report on our investigation of LC-CLND as a method suitable for quantitative analysis of nitrogenous toxins. It was demonstrated that a wide range of toxins could be analyzed quantitatively by LC-CLND. Furthermore, equimolar responses among diverse structures were established and it was shown that a single high-purity standard such as caffeine could be used for instrument calibration. The limit of detection was approximately 0.6 ng N. Measurement of several of Canada's National Research Council toxin CRMs with caffeine as the calibrant showed precision averaging 2% RSD and accuracy ranging from 97 to 102%. Application of LC-CLND to the production of calibration solution CRMs and the establishment of traceability of measurement results are presented.

Isotope-Encoded Carboxyl Group Footprinting for Mass Spectrometry-Based Protein Conformational Studies

NASA Astrophysics Data System (ADS)

Zhang, Hao; Liu, Haijun; Blankenship, Robert E.; Gross, Michael L.

2016-01-01

We report an isotope-encoding method coupled with carboxyl-group footprinting to monitor protein conformational changes. The carboxyl groups of aspartic/glutamic acids and of the C-terminus of proteins can serve as reporters for protein conformational changes when labeled with glycine ethyl ester (GEE) mediated by carbodiimide. In the new development, isotope-encoded "heavy" and "light" GEE are used to label separately the two states of the orange carotenoid protein (OCP) from cyanobacteria. Two samples are mixed (1:1 ratio) and analyzed by a single LC-MS/MS experiment. The differences in labeling extent between the two states are represented by the ratio of the "heavy" and "light" peptides, providing information about protein conformational changes. Combining isotope-encoded MS quantitative analysis and carboxyl-group footprinting reduces the time of MS analysis and improves the sensitivity of GEE and other footprinting.

Isotope-encoded Carboxyl Group Footprinting for Mass Spectrometry-based Protein Conformational Studies

PubMed Central

Zhang, Hao; Liu, Haijun; Blankenship, Robert E.; Gross, Michael L.

2015-01-01

We report an isotope-encoding method coupled with carboxyl-group footprinting to monitor protein conformational changes. The carboxyl groups of aspartic/glutamic acids and of the C-terminus of proteins can serve as reporters for protein conformational changes when labeled with glycine ethyl ester (GEE) mediated by carbodiimide. In the new development, isotope-encoded “heavy” and “light” GEE are used to label separately the two states of the Orange Carotenoid Protein (OCP) from cyanobacteria. Two samples are mixed (1:1 ratio) and analyzed by a single LC-MS/MS experiment. The differences in labeling extent between the two states are represented by the ratio of the “heavy” and “light” peptides, providing information about protein conformational changes. Combining isotope-encoded MS quantitative analysis and carboxyl-group footprinting reduces the time of MS analysis and improves the sensitivity of GEE and other footprinting. PMID:26384685

Isotope-Encoded Carboxyl Group Footprinting for Mass Spectrometry-Based Protein Conformational Studies

DOE PAGES

Zhang, Hao; Liu, Haijun; Blankenship, Robert E.; ...

2015-09-18

Here, we report an isotope-encoding method coupled with carboxyl-group footprinting to monitor protein conformational changes. The carboxyl groups of aspartic/glutamic acids and of the C-terminus of proteins can serve as reporters for protein conformational changes when labeled with glycine ethyl ester (GEE) mediated by carbodiimide. In the new development, isotope-encoded “heavy” and “light” GEE are used to label separately the two states of the orange carotenoid protein (OCP) from cyanobacteria. Two samples are mixed (1:1 ratio) and analyzed by a single LC-MS/MS experiment. The differences in labeling extent between the two states are represented by the ratio of the “heavy”more » and “light” peptides, providing information about protein conformational changes. Combining isotope-encoded MS quantitative analysis and carboxyl-group footprinting reduces the time of MS analysis and improves the sensitivity of GEE and other footprinting.« less

A peptide-retrieval strategy enables significant improvement of quantitative performance without compromising confidence of identification.

PubMed

Tu, Chengjian; Shen, Shichen; Sheng, Quanhu; Shyr, Yu; Qu, Jun

2017-01-30

Reliable quantification of low-abundance proteins in complex proteomes is challenging largely owing to the limited number of spectra/peptides identified. In this study we developed a straightforward method to improve the quantitative accuracy and precision of proteins by strategically retrieving the less confident peptides that were previously filtered out using the standard target-decoy search strategy. The filtered-out MS/MS spectra matched to confidently-identified proteins were recovered, and the peptide-spectrum-match FDR were re-calculated and controlled at a confident level of FDR≤1%, while protein FDR maintained at ~1%. We evaluated the performance of this strategy in both spectral count- and ion current-based methods. >60% increase of total quantified spectra/peptides was respectively achieved for analyzing a spike-in sample set and a public dataset from CPTAC. Incorporating the peptide retrieval strategy significantly improved the quantitative accuracy and precision, especially for low-abundance proteins (e.g. one-hit proteins). Moreover, the capacity of confidently discovering significantly-altered proteins was also enhanced substantially, as demonstrated with two spike-in datasets. In summary, improved quantitative performance was achieved by this peptide recovery strategy without compromising confidence of protein identification, which can be readily implemented in a broad range of quantitative proteomics techniques including label-free or labeling approaches. We hypothesize that more quantifiable spectra and peptides in a protein, even including less confident peptides, could help reduce variations and improve protein quantification. Hence the peptide retrieval strategy was developed and evaluated in two spike-in sample sets with different LC-MS/MS variations using both MS1- and MS2-based quantitative approach. The list of confidently identified proteins using the standard target-decoy search strategy was fixed and more spectra/peptides with less confidence matched to confident proteins were retrieved. However, the total peptide-spectrum-match false discovery rate (PSM FDR) after retrieval analysis was still controlled at a confident level of FDR≤1%. As expected, the penalty for occasionally incorporating incorrect peptide identifications is negligible by comparison with the improvements in quantitative performance. More quantifiable peptides, lower missing value rate, better quantitative accuracy and precision were significantly achieved for the same protein identifications by this simple strategy. This strategy is theoretically applicable for any quantitative approaches in proteomics and thereby provides more quantitative information, especially on low-abundance proteins. Published by Elsevier B.V.

Microsomal metabolism of calycosin, formononetin and drug-drug interactions by dynamic microdialysis sampling and HPLC-DAD-MS analysis.

PubMed

Wen, Xiao-Dong; Qi, Lian-Wen; Li, Bin; Li, Ping; Yi, Ling; Wang, Ya-Qiong; Liu, E-Hu; Yang, Xiao-Lin

2009-08-15

A dynamic microdialysis sampling method with liquid chromatography-diode array detection and time-of-flight mass spectrometry (LC-DAD-TOF/MS) analysis was developed to investigate rat microsomal metabolisms of calycosin and formononetin, and their drug-drug interactions. Two hydroxylated metabolites from calycosin, and three hydroxylated or 4'-O-demethylated derivatives from formononetin were detected and identified after co-incubation with microsomes. Calibration curves offered linear ranges of two orders of magnitude with r(2)>0.999 for calycosin, formononetin and daidzein. The quantitative LC method provides a range of 0.028-0.034microg/mL for limits of detection, overall precision less than 5% and accuracy less than 3% by RSD. Besides, calycosin and formononetin were found to produce the depressive effect on the CYP450 enzyme reaction, and inhibit phase I enzyme reaction of each other when they are concurrent. Dynamic microdialysis sampling with LC-DAD-TOF/MS analysis developed in this work is a powerful tool for in vitro metabolism studies of drugs and metabolic interactions.

ANALYTICAL METHODS AND QUALITY ASSURANCE CRITERIA FOR LC/ES/MS DETERMINATION OF PFOS IN FISH

EPA Science Inventory

PFOS, perfluorooctanesulfonate, has recently received much attention from environmental researchers. Previous analytical methods were based upon complexing with a strong ion-pairing reagent and extraction into MTBE. Detection was done on a concentrate using negative ion LC/ES/MS/...

Characterization of cocoa liquors by GC-MS and LC-MS/MS: focus on alkylpyrazines and flavanols.

PubMed

Magi, Emanuele; Bono, Luca; Di Carro, Marina

2012-09-01

Flavor is one of the most important characteristics of chocolate products and is due to a complex volatile fraction, depending both on the cocoa bean genotype and the several processes occurring during chocolate production (fermentation, drying, roasting and conching). Alkylpyrazines are among the most studied volatiles, being one of the main classes of odorant compounds in cocoa products. In this work, a mass spectrometric approach was used for the comparison of cocoa liquors from different countries. A headspace solid-phase microextraction gas chromatography-mass spectrometry method was developed for the qualitative study of the volatile fraction; the standard addition method was then used for the quantitative determination of five pyrazines (2-methylpyrazine, 2,3-dimethylpyrazine, 2,5-dimethylpyrazine, 2,3,5-trimethylpyrazine and tetramethylpyrazine). Satisfactory figures of merit were obtained: Limits of quantitation were in the range 0.1-2.7 ng/g; repeatability and reproducibility varied between 3% and 7% and between 8% and 14%, respectively. The total content of the pyrazines was remarkably different in the considered samples, ranging from 99 to 708 ng/g. Tetramethylpyrazine showed the highest concentration in all samples, with a maximum value of 585 ng/g. A preliminary study was also performed on the nonvolatile fraction using LC-MS/MS, identifying some flavanols such as catechin, epicatechin and procyanidins. Copyright © 2012 John Wiley & Sons, Ltd.

Novel approach in LC-MS/MS using MRM to generate a full profile of acyl-CoAs: discovery of acyl-dephospho-CoAs.

PubMed

Li, Qingling; Zhang, Shenghui; Berthiaume, Jessica M; Simons, Brigitte; Zhang, Guo-Fang

2014-03-01

A metabolomic approach to selectively profile all acyl-CoAs was developed using a programmed multiple reaction monitoring (MRM) method in LC-MS/MS and was employed in the analysis of various rat organs. The programmed MRM method possessed 300 mass ion transitions with the mass difference of 507 between precursor ion (Q1) and product ion (Q3), and the precursor ion started from m/z 768 and progressively increased one mass unit at each step. Acyl-dephospho-CoAs resulting from the dephosphorylation of acyl-CoAs were identified by accurate MS and fragmentation. Acyl-dephospho-CoAs were also quantitatively scanned by the MRM method with the mass difference of 427 between Q1 and Q3 mass ions. Acyl-CoAs and dephospho-CoAs were assayed with limits of detection ranging from 2 to 133 nM. The accuracy of the method was demonstrated by assaying a range of concentrations of spiked acyl-CoAs with the results of 80-114%. The distribution of acyl-CoAs reflects the metabolic status of each organ. The physiological role of dephosphorylation of acyl-CoAs remains to be further characterized. The methodology described herein provides a novel strategy in metabolomic studies to quantitatively and qualitatively profile all potential acyl-CoAs and acyl-dephospho-CoAs.

Quantitation of mycotoxins in food and feed from Burkina Faso and Mozambique using a modern LC-MS/MS multitoxin method.

PubMed

Warth, Benedikt; Parich, Alexandra; Atehnkeng, Joseph; Bandyopadhyay, Ranajit; Schuhmacher, Rainer; Sulyok, Michael; Krska, Rudolf

2012-09-12

In this study an LC-MS/MS multitoxin method covering a total of 247 fungal and bacterial metabolites was applied to the analysis of different foods and feedstuffs from Burkina Faso and Mozambique. Overall, 63 metabolites were determined in 122 samples of mainly maize and groundnuts and a few samples of sorghum, millet, rice, wheat, soy, dried fruits, other processed foods and animal feeds. Aflatoxin B(1) was observed more frequently in maize (Burkina Faso, 50% incidence, median = 23.6 μg/kg; Mozambique, 46% incidence, median = 69.9 μg/kg) than in groundnuts (Burkina Faso, 22% incidence, median = 10.5 μg/kg; Mozambique, 14% incidence, median = 3.4 μg/kg). Fumonisin B(1) concentrations in maize were higher in Mozambique (92% incidence, median = 869 μg/kg) than in Burkina Faso (81% incidence, median = 269 μg/kg). In addition, ochratoxin A, zearalenone, deoxynivalenol, nivalenol, and other less reported mycotoxins such as citrinin, alternariol, cyclopiazonic acid, sterigmatocystin, moniliformin, beauvericin, and enniatins were detected. Up to 28 toxic fungal metabolites were quantitated in a single sample, emphasizing the great variety of mycotoxin coexposure. Most mycotoxins have not been reported before in either country.

Comparison of enzyme kinetics of warfarin analyzed by LC-MS/MS QTrap and differential mobility spectrometry.

PubMed

Shaik, Abdul Naveed; Grater, Richard; Lulla, Mukesh; Williams, David A; Gan, Lawrence L; Bohnert, Tonika; LeDuc, Barbara W

2016-01-01

Warfarin is an anticoagulant used in the treatment of thrombosis and thromboembolism. It is given as a racemic mixture of R and S enantiomers. These two enantiomers show differences in metabolism by CYPs: S-warfarin undergoes 7 hydroxylation by CYP2C9 and R-warfarin by CYP3A4 to form 10 hydroxy warfarin. In addition, warfarin is acted upon by different CYPs to form the minor metabolites 3'-hydroxy, 4'-hydroxy, 6-hydroxy, and 8-hydroxy warfarin. For analysis, separation of these metabolites is necessary since all have the same m/z ratio and similar fragmentation pattern. Enzyme kinetics for the formation of all of the six hydroxylated metabolites of warfarin from human liver microsomes were determined using an LC-MS/MS QTrap and LC-MS/MS with a differential mobility spectrometry (DMS) (SelexION™) interface to compare the kinetic parameters. These two methods were chosen to compare their selectivity and sensitivity. Substrate curves for 3'-OH, 4'-OH, 6-OH, 7-OH, 8-OH and 10-OH warfarin formation were generated to determine the kinetic parameters (Km and Vmax) in human liver microsomal preparations. The limit of quantitation (LOQ) for all the six hydroxylated metabolites of warfarin were in the range of 1-3nM using an LC-MS/MS QTrap method which had a run time of 22min. In contrast, the LOQ for all the six hydroxylated metabolites using DMS interface technology was 100nM with a run time of 2.8min. We compare these two MS methods and discuss the kinetics of metabolite formation for the metabolites generated from racemic warfarin. In addition, we show inhibition of major metabolic pathways of warfarin by sulfaphenazole and ketoconazole which are known specific inhibitors of CYP2C9 and CYP3A4 respectively. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantitative analysis of immunoglobulin subclasses and subclass specific glycosylation by LC-MS-MRM in liver disease.

PubMed

Yuan, Wei; Sanda, Miloslav; Wu, Jing; Koomen, John; Goldman, Radoslav

2015-02-26

Aberrant glycosylation of IgGs has been linked to human diseases, including liver disease. In this study, we have quantified plasma immunoglobulins in cirrhosis (CIR) and hepatocellular carcinoma (HCC) and employed a novel LC-MS-MRM assay to quantify glycoforms of IgG subclasses 1-4. Glycan oxonium ions and peptide-GlcNAc fragment ions were utilized to quantify the IgG glycoforms purified by affinity chromatography with normalization to the unique peptide for each IgG subclass. Our results indicate that HCC patients have increased circulating IgG1, IgG3, IgA1, and IgM compared to healthy controls; comparison of HCC and CIR patients shows that HCC patients have significantly higher concentration of IgG1 and IgM but lower concentration of IgG2. An increase in galactose-deficient core fucosylated glycoforms was consistently observed in CIR and HCC patients. The FA2G0 and FA2BG0 glycoforms increase approximately 2-fold in all IgG subclasses accompanied by a decrease in the FA2G2 glycoform. Fucosylation changes are less pronounced but we have detected increased degree of fucosylation in the IgG1 and IgG3 glycoforms. In conclusion, we have optimized a sensitive and selective LC-MS-MRM method for the quantification of immunoglobulin subclasses and their site specific glycoforms, demonstrating that both quantities and glycoforms of immunoglobulins change significantly in liver disease progression to HCC. We have demonstrated that both quantities and glycoforms of immunoglobulin subclasses change significantly in liver disease progression to HCC through quantitative study of immunoglobulin subclasses and their site specific glycoforms using a sensitive and selective LC-MS-MRM method. Redistribution of the glycoforms of specific immunoglobulin subclasses could have important implications for receptor mediated responses affecting the progression of liver disease. Copyright © 2015 Elsevier B.V. All rights reserved.

ALLocator: an interactive web platform for the analysis of metabolomic LC-ESI-MS datasets, enabling semi-automated, user-revised compound annotation and mass isotopomer ratio analysis.

PubMed

Kessler, Nikolas; Walter, Frederik; Persicke, Marcus; Albaum, Stefan P; Kalinowski, Jörn; Goesmann, Alexander; Niehaus, Karsten; Nattkemper, Tim W

2014-01-01

Adduct formation, fragmentation events and matrix effects impose special challenges to the identification and quantitation of metabolites in LC-ESI-MS datasets. An important step in compound identification is the deconvolution of mass signals. During this processing step, peaks representing adducts, fragments, and isotopologues of the same analyte are allocated to a distinct group, in order to separate peaks from coeluting compounds. From these peak groups, neutral masses and pseudo spectra are derived and used for metabolite identification via mass decomposition and database matching. Quantitation of metabolites is hampered by matrix effects and nonlinear responses in LC-ESI-MS measurements. A common approach to correct for these effects is the addition of a U-13C-labeled internal standard and the calculation of mass isotopomer ratios for each metabolite. Here we present a new web-platform for the analysis of LC-ESI-MS experiments. ALLocator covers the workflow from raw data processing to metabolite identification and mass isotopomer ratio analysis. The integrated processing pipeline for spectra deconvolution "ALLocatorSD" generates pseudo spectra and automatically identifies peaks emerging from the U-13C-labeled internal standard. Information from the latter improves mass decomposition and annotation of neutral losses. ALLocator provides an interactive and dynamic interface to explore and enhance the results in depth. Pseudo spectra of identified metabolites can be stored in user- and method-specific reference lists that can be applied on succeeding datasets. The potential of the software is exemplified in an experiment, in which abundance fold-changes of metabolites of the l-arginine biosynthesis in C. glutamicum type strain ATCC 13032 and l-arginine producing strain ATCC 21831 are compared. Furthermore, the capability for detection and annotation of uncommon large neutral losses is shown by the identification of (γ-)glutamyl dipeptides in the same strains. ALLocator is available online at: https://allocator.cebitec.uni-bielefeld.de. A login is required, but freely available.

Clinical laboratory verification of thyroglobulin concentrations in the presence of autoantibodies to thyroglobulin: comparison of EIA, radioimmunoassay and LC MS/MS measurements in an Urban Hospital.

PubMed

Wheeler, Sarah E; Liu, Li; Blair, Harry C; Sivak, Richard; Longo, Nancy; Tischler, Jeffery; Mulvey, Kathryn; Palmer, Octavia M Peck

2017-12-08

Thyroglobulin (Tg) measurements assess recurrence in post-thyroidectomy thyroid cancer patients. Tg measurements by enzyme immunoassays (EIA) can be falsely elevated by interference from Tg autoantibodies (TgAb). Radioimmunoassay (RIA) is less susceptible to TgAb interference and has been the standard-of-care test for TgAb positive patients. Recently developed liquid chromatography tandem mass spectrometry (LC-MS/MS) methods may eliminate TgAb interference. We assessed the performance of Tg measurements by EIA, RIA and LC-MS/MS to evaluate TgAb interference differences. We measured TgAb and Tg in 50 plasma samples from 40 patients in whom Tg measurement was part of their routine follow-up and 10 healthy volunteers. Discrepancy between EIA and both LC-MS/MS and RIA was observed at low Tg concentrations (≤ 7.55 ng/mL) in TgAb positive specimens (LC-MS/MS = 1.9 * EIA - 0.03, r = 0.68). RIA and LC-MS/MS Tg measurements in TgAb positive specimens with low Tg concentrations had improved correlation but demonstrated bias (LC MS/MS = 0.6 * RIA - 1.4, r = 0.90). Disagreement between methods may be attributed to LC-MS/MS reported Tg concentrations as undetectable compared to RIA. It seems likely that most discrepant cases are falsely elevated in RIA due to TgAb interference, however, some cases appear below the detection limit of LC-MS/MS; implementation of LC-MS/MS by clinicians will require lower detection limits.

Matrix Effect Compensation in Small-Molecule Profiling for an LC-TOF Platform Using Multicomponent Postcolumn Infusion.

PubMed

González, Oskar; van Vliet, Michael; Damen, Carola W N; van der Kloet, Frans M; Vreeken, Rob J; Hankemeier, Thomas

2015-06-16

The possible presence of matrix effect is one of the main concerns in liquid chromatography-mass spectrometry (LC-MS)-driven bioanalysis due to its impact on the reliability of the obtained quantitative results. Here we propose an approach to correct for the matrix effect in LC-MS with electrospray ionization using postcolumn infusion of eight internal standards (PCI-IS). We applied this approach to a generic ultraperformance liquid chromatography-time-of-flight (UHPLC-TOF) platform developed for small-molecule profiling with a main focus on drugs. Different urine samples were spiked with 19 drugs with different physicochemical properties and analyzed in order to study matrix effect (in absolute and relative terms). Furthermore, calibration curves for each analyte were constructed and quality control samples at different concentration levels were analyzed to check the applicability of this approach in quantitative analysis. The matrix effect profiles of the PCI-ISs were different: this confirms that the matrix effect is compound-dependent, and therefore the most suitable PCI-IS has to be chosen for each analyte. Chromatograms were reconstructed using analyte and PCI-IS responses, which were used to develop an optimized method which compensates for variation in ionization efficiency. The approach presented here improved the results in terms of matrix effect dramatically. Furthermore, calibration curves of higher quality are obtained, dynamic range is enhanced, and accuracy and precision of QC samples is increased. The use of PCI-ISs is a very promising step toward an analytical platform free of matrix effect, which can make LC-MS analysis even more successful, adding a higher reliability in quantification to its intrinsic high sensitivity and selectivity.

Quantitative analysis of drug distribution by ambient mass spectrometry imaging method with signal extinction normalization strategy and inkjet-printing technology.

PubMed

Luo, Zhigang; He, Jingjing; He, Jiuming; Huang, Lan; Song, Xiaowei; Li, Xin; Abliz, Zeper

2018-03-01

Quantitative mass spectrometry imaging (MSI) is a robust approach that provides both quantitative and spatial information for drug candidates' research. However, because of complicated signal suppression and interference, acquiring accurate quantitative information from MSI data remains a challenge, especially for whole-body tissue sample. Ambient MSI techniques using spray-based ionization appear to be ideal for pharmaceutical quantitative MSI analysis. However, it is more challenging, as it involves almost no sample preparation and is more susceptible to ion suppression/enhancement. Herein, based on our developed air flow-assisted desorption electrospray ionization (AFADESI)-MSI technology, an ambient quantitative MSI method was introduced by integrating inkjet-printing technology with normalization of the signal extinction coefficient (SEC) using the target compound itself. The method utilized a single calibration curve to quantify multiple tissue types. Basic blue 7 and an antitumor drug candidate (S-(+)-deoxytylophorinidine, CAT) were chosen to initially validate the feasibility and reliability of the quantitative MSI method. Rat tissue sections (heart, kidney, and brain) administered with CAT was then analyzed. The quantitative MSI analysis results were cross-validated by LC-MS/MS analysis data of the same tissues. The consistency suggests that the approach is able to fast obtain the quantitative MSI data without introducing interference into the in-situ environment of the tissue sample, and is potential to provide a high-throughput, economical and reliable approach for drug discovery and development. Copyright © 2017 Elsevier B.V. All rights reserved.

Determination of spinetoram and its metabolites in amaranth and parsley using QuEChERS-based extraction and liquid chromatography-tandem mass spectrometry.

PubMed

Park, Ki Hun; Choi, Jeong-Heui; Abd El-Aty, A M; Cho, Soon-Kil; Park, Jong-Hyouk; Kim, Bo Mi; Yang, Angel; Na, Tae Woong; Rahman, Musfiqur; Im, Geon-Jae; Shim, Jae-Han

2012-10-15

In this study, a simultaneous method was developed for the determination of spinetoram (XDE-175-J and XDE-175-L) and its demethyl metabolites (N-demethyl-175-J and N-demethyl-175-L) and formyl metabolites (N-formyl-175-J and N-formyl-175-L) in the minor crops; amaranth and parsley. The method uses quick, easy, cheap, effective, rugged, and safe (QuEChERS)-based extraction. Afterwards, the analytes were quantified and confirmed via liquid chromatography-electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) in the positive ion mode using multiple reaction monitoring (MRM). Calibration curves were linear over the calibration ranges for all the analytes tested with r(2)>0.993. Limits of detection and quantitation were 0.01 and 0.03 mg/kg for all the tested analytes in amaranth and parsley, respectively. Recovery values, at spiking levels 0.05 and 0.25 mg/kg, ranged from 71.0% to 115.2% with relative standard deviations <15%, except for N-formyl-175-J in both amaranth and parsley. This method was applied to field-incurred samples and was shown to provide an adequate sensitivity and performance for the simultaneous determination of spinetoram and metabolites. To the best of our knowledge, this is the first time spinetoram and its metabolites were quantified using LC-MS/MS in minor crops. Copyright © 2012 Elsevier Ltd. All rights reserved.

Shotgun proteomics of plant plasma membrane and microdomain proteins using nano-LC-MS/MS.

PubMed

Takahashi, Daisuke; Li, Bin; Nakayama, Takato; Kawamura, Yukio; Uemura, Matsuo

2014-01-01

Shotgun proteomics allows the comprehensive analysis of proteins extracted from plant cells, subcellular organelles, and membranes. Previously, two-dimensional gel electrophoresis-based proteomics was used for mass spectrometric analysis of plasma membrane proteins. In order to get comprehensive proteome profiles of the plasma membrane including highly hydrophobic proteins with a number of transmembrane domains, a mass spectrometry-based shotgun proteomics method using nano-LC-MS/MS for proteins from the plasma membrane proteins and plasma membrane microdomain fraction is described. The results obtained are easily applicable to label-free protein semiquantification.

Determination of phenolic acids and flavonoids in Taraxacum formosanum Kitam by liquid chromatography-tandem mass spectrometry coupled with a post-column derivatization technique.

PubMed

Chen, Hung-Ju; Inbaraj, Baskaran Stephen; Chen, Bing-Huei

2012-01-01

A liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed for the determination of phenolic acids and flavonoids in a medicinal Chinese herb Taraxacum formosanum Kitam. Initially, both phenolic acids and flavonoids were extracted with 50% ethanol in a water-bath at 60 °C for 3 h and eventually separated into acidic fraction and neutral fraction by using a C(18) cartridge. A total of 29 compounds were separated within 68 min by employing a Gemini C(18) column and a gradient solvent system of 0.1% formic acid and acetonitrile at a flow rate of 1.0 mL/min. Based on the retention behavior as well as absorption and mass spectra, 19 phenolic acids and 10 flavonoids were identified and quantified in T. formosanum, with the former ranging from 14.1 μg/g to 10,870.4 μg/g, and the latter from 9.9 μg/g to 325.8 μg/g. For further identification of flavonoids, a post-column derivatization method involving shift reagents such as sodium acetate or aluminum chloride was used and the absorption spectral characteristics without or with shift reagents were compared. An internal standard syringic acid was used for quantitation of phenolic acids, whereas (±) naringenin was found suitable for quantitation of flavonoids. The developed LC-MS/MS method showed high reproducibility, as evident from the relative standard deviation (RSD) values for intra-day and inter-day variability being 1.0-6.8% and 2.0-7.7% for phenolic acids and 3.7-7.4% and 1.5-8.1% for flavonoids, respectively, and thus may be applied for simultaneous determination of phenolic acids and flavonoids in Chinese herb and nutraceuticals.

Rapid determination of underivatized pyroglutamic acid, glutamic acid, glutamine and other relevant amino acids in fermentation media by LC-MS-MS.

PubMed

Qu, Jun; Chen, Wei; Luo, Guoan; Wang, Yiming; Xiao, Shengyuan; Ling, Zhihua; Chen, Guoqiang

2002-01-01

Determination of amino acids in a complex matrix without derivatization is advantageous, however, difficulties are found in both the detection and the separation of those compounds. In this study, a rapid and reliable LC-MS-MS method for the quantitation of underivatized amino acids in exocellular media was established. Injections were made directly after centrifugation of the samples, without further preparation. The separation of seven underivatized amino acids was achieved on a reversed-phase C18 column with pentadecafluorooctanoic acid as a volatile ion-pair reagent, and the specific detection of most amino acids was achieved by MS-MS of the specific transitions [M + H]+-->[M + H - 46]+. The calibration curves of all analytes were linear over the range of 1.0-1000 microg ml(-1) and the detection limits ranged from 0.1 to 5 ng ml(-1), with an injection volume of 20 microl. The inter-day and intra-day precisions ranged from 2.6 to 5.7% and 4.8 to 8.2%, respectively; the mean recoveries of the seven analytes were 81-104%, 91-107% and 93-101% respectively at the spiked level of 10, 40 and 200 microg ml(-1). A large number of fermentation samples were analysed using this method. The technique is simple, rapid, selective and sensitive, and shows potential for the high-throughput quantitation of amino acids from other biological matrices.

speaq 2.0: A complete workflow for high-throughput 1D NMR spectra processing and quantification.

PubMed

Beirnaert, Charlie; Meysman, Pieter; Vu, Trung Nghia; Hermans, Nina; Apers, Sandra; Pieters, Luc; Covaci, Adrian; Laukens, Kris

2018-03-01

Nuclear Magnetic Resonance (NMR) spectroscopy is, together with liquid chromatography-mass spectrometry (LC-MS), the most established platform to perform metabolomics. In contrast to LC-MS however, NMR data is predominantly being processed with commercial software. Meanwhile its data processing remains tedious and dependent on user interventions. As a follow-up to speaq, a previously released workflow for NMR spectral alignment and quantitation, we present speaq 2.0. This completely revised framework to automatically analyze 1D NMR spectra uses wavelets to efficiently summarize the raw spectra with minimal information loss or user interaction. The tool offers a fast and easy workflow that starts with the common approach of peak-picking, followed by grouping, thus avoiding the binning step. This yields a matrix consisting of features, samples and peak values that can be conveniently processed either by using included multivariate statistical functions or by using many other recently developed methods for NMR data analysis. speaq 2.0 facilitates robust and high-throughput metabolomics based on 1D NMR but is also compatible with other NMR frameworks or complementary LC-MS workflows. The methods are benchmarked using a simulated dataset and two publicly available datasets. speaq 2.0 is distributed through the existing speaq R package to provide a complete solution for NMR data processing. The package and the code for the presented case studies are freely available on CRAN (https://cran.r-project.org/package=speaq) and GitHub (https://github.com/beirnaert/speaq).

speaq 2.0: A complete workflow for high-throughput 1D NMR spectra processing and quantification

PubMed Central

Pieters, Luc; Covaci, Adrian

2018-01-01

Nuclear Magnetic Resonance (NMR) spectroscopy is, together with liquid chromatography-mass spectrometry (LC-MS), the most established platform to perform metabolomics. In contrast to LC-MS however, NMR data is predominantly being processed with commercial software. Meanwhile its data processing remains tedious and dependent on user interventions. As a follow-up to speaq, a previously released workflow for NMR spectral alignment and quantitation, we present speaq 2.0. This completely revised framework to automatically analyze 1D NMR spectra uses wavelets to efficiently summarize the raw spectra with minimal information loss or user interaction. The tool offers a fast and easy workflow that starts with the common approach of peak-picking, followed by grouping, thus avoiding the binning step. This yields a matrix consisting of features, samples and peak values that can be conveniently processed either by using included multivariate statistical functions or by using many other recently developed methods for NMR data analysis. speaq 2.0 facilitates robust and high-throughput metabolomics based on 1D NMR but is also compatible with other NMR frameworks or complementary LC-MS workflows. The methods are benchmarked using a simulated dataset and two publicly available datasets. speaq 2.0 is distributed through the existing speaq R package to provide a complete solution for NMR data processing. The package and the code for the presented case studies are freely available on CRAN (https://cran.r-project.org/package=speaq) and GitHub (https://github.com/beirnaert/speaq). PMID:29494588

Microwave assisted synthesis for A2E and development of LC-ESI-MS method for quantification of ocular bisretinoids in human retina.

PubMed

Kotnala, A; Senthilkumari, S; Halder, N; Kumar, A; Velpandian, T

2018-01-15

To develop a microwave assisted method for the rapid synthesis of A2E and also to develop a method to quantify N-retinylidene-N-retinylethanolamine(A2E), all-trans retinal dimer (ATRD), A2-glycerophospho ethanolamine (A2GPE), dihydropyridine phosphatidyl ethanolamine (A2DHPE) and monofuran A2E (MFA2E) in age matched retina. The development of microwave assisted synthesis of A2E, its purification and characterization for its utility in quantification in human retina. The semi-quantitative method development using LC-ESI-MS, LC-ESI-MS/MS and LC-APCI-MS/MS from pooled macula and peripheral retina for the bisretinoid analysis has been done. Maximum A2E conversion using microwave assisted process took place at 80°C for 45min with a yield of 55.01%. Highly sensitive and specific mass spectrometric method was developed using reverse phase C-18 separation with positive electrospray ionization and positive atmospheric phase chemical ionization of tandom mass spectrometry. A gradient mobile phase separation was achieved using water and methanol with 0.1% TFA. Multiple reaction monitoring acquisition for ESI and APCI was performed at ATRD m/z 551.2/522.2, A2GPE m/z 746.4/729.5, A2DHPEm/z 594.4/576.5, MFA2E m/z 608.2/591.2, A2E m/z 592.4/418.2. Method was validated using LC-ESI-SIM mode to determine selectivity, linearity, sensitivity, precision and accuracy. An attempt towards optimization of the synthetic procedure of A2E was made so as to reduce the lengthy reaction time without compromising the yield. Developed method was capable enough for the detection of low level of bisretinids in retina. Copyright © 2017 Elsevier B.V. All rights reserved.