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.

Patient-specific lean body mass can be estimated from limited-coverage computed tomography images.

PubMed

Devriese, Joke; Beels, Laurence; Maes, Alex; van de Wiele, Christophe; Pottel, Hans

2018-06-01

In PET/CT, quantitative evaluation of tumour metabolic activity is possible through standardized uptake values, usually normalized for body weight (BW) or lean body mass (LBM). Patient-specific LBM can be estimated from whole-body (WB) CT images. As most clinical indications only warrant PET/CT examinations covering head to midthigh, the aim of this study was to develop a simple and reliable method to estimate LBM from limited-coverage (LC) CT images and test its validity. Head-to-toe PET/CT examinations were retrospectively retrieved and semiautomatically segmented into tissue types based on thresholding of CT Hounsfield units. LC was obtained by omitting image slices. Image segmentation was validated on the WB CT examinations by comparing CT-estimated BW with actual BW, and LBM estimated from LC images were compared with LBM estimated from WB images. A direct method and an indirect method were developed and validated on an independent data set. Comparing LBM estimated from LC examinations with estimates from WB examinations (LBMWB) showed a significant but limited bias of 1.2 kg (direct method) and nonsignificant bias of 0.05 kg (indirect method). This study demonstrates that LBM can be estimated from LC CT images with no significant difference from LBMWB.

Determination of linuron and related compounds in soil by microwave-assisted solvent extraction and reversed-phase liquid chromatography with UV detection.

PubMed

Molins, C; Hogendoorn, E A; Dijkman, E; Heusinkveld, H A; Baumann, R A

2000-02-11

The combination of microwave-assisted solvent extraction (MASE) and reversed-phase liquid chromatography (RPLC) with UV detection has been investigated for the efficient determination of phenylurea herbicides in soils involving the single-residue method (SRM) approach (linuron) and the multi-residue method (MRM) approach (monuron, monolinuron, isoproturon, metobromuron, diuron and linuron). Critical parameters of MASE, viz, extraction temperature, water content and extraction solvent were varied in order to optimise recoveries of the analytes while simultaneously minimising co-extraction of soil interferences. The optimised extraction procedure was applied to different types of soil with an organic carbon content of 0.4-16.7%. Besides freshly spiked soil samples, method validation included the analysis of samples with aged residues. A comparative study between the applicability of RPLC-UV without and with the use of column switching for the processing of uncleaned extracts, was carried out. For some of the tested analyte/matrix combinations the one-column approach (LC mode) is feasible. In comparison to LC, coupled-column LC (LC-LC mode) provides high selectivity in single-residue analysis (linuron) and, although less pronounced in multi-residue analysis (all six phenylurea herbicides), the clean-up performance of LC-LC improves both time of analysis and sample throughput. In the MRM approach the developed procedure involving MASE and LC-LC-UV provided acceptable recoveries (range, 80-120%) and RSDs (<12%) at levels of 10 microg/kg (n=9) and 50 microg/kg (n=7), respectively, for most analyte/matrix combinations. Recoveries from aged residue samples spiked at a level of 100 microg/kg (n=7) ranged, depending of the analyte/soil type combination, from 41-113% with RSDs ranging from 1-35%. In the SRM approach the developed LC-LC procedure was applied for the determination of linuron in 28 sandy soil samples collected in a field study. Linuron could be determined in soil with a limit of quantitation of 10 microg/kg.

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.

Simultaneous determination of residues of emamectin and its metabolites, and milbemectin, ivermectin, and abamectin in crops by liquid chromatography with fluorescence detection.

PubMed

Yoshii, K; Kaihara, A; Tsumura, Y; Ishimitsu, S; Tonogai, Y

2001-01-01

A liquid chromatographic (LC) method was developed for the determination of emamectin and its metabolites (8,9-Z-isomer, N-demethylated, N-formylated, and N-methylformylated emamectin) in various crops. The analytes were extracted with acetone, cleaned up on cartridge columns (C18 and NH2), derivatized with trifluoroacetic anhydride and 1-methylimidazole, and determined by LC with fluorescence detection. Because radish inhibited the formation of the fluorescent derivatives, an additional Bond Elut PRS cartridge was used in the cleanup of Japanese radish samples. During sample preparation, N-formylated emamectin partially degraded to emamectin B1b and emamectin B1a, and the 8,9-Z-isomer partially degraded to N-demethylated emamectin. Therefore, emamectin and its metabolites were determined as total emamectin, i.e., their sum was estimated as emamectin benzoate. Their recoveries from most crops were approximately 80-110% with the developed method. The detection limits for the analytes in vegetables were 0.1-0.3 parts per trillion (ppt). The results for these compounds were confirmed by LC/mass spectrometry (LC/MS; electrospray ionization mode). Because the fluorescent derivative of emamectin was undetectable by LC/MS, the results for the analyte were confirmed by using a sample solution without derivatization. Limits of detection by LC/MS were similar to the fluorescence detection limits, 0.1-0.3 ppt in vegetables. In addition to the emamectins, milbemectin, ivermectin, and abamectin were also determined by the developed method.

Multi-residue analysis method for analysis of pharmaceuticals using liquid chromatography-time of flight/mass spectrometry (LC-TOF/MS) in water sample

NASA Astrophysics Data System (ADS)

Al-Qaim, Fouad Fadhil; Abdullah, Md Pauzi; Othman, Mohamed Rozali

2013-11-01

In this work, a developed method using solid - phase extraction (SPE) followed by liquid chromatography - time of flight mass spectrometry (LC-ESI-TOF/MS) was developed and validated for quantification and confirmation of eleven pharmaceuticals with different therapeutic classes in water samples, Malaysia. These compounds are caffeine (CAF), prazosin (PRZ), enalapril (ENL), carbamazepine (CBZ), nifedipine (NFD), levonorgestrel (LNG), simvastatin (SMV), hydrochlorothiazide (HYD), gliclazide (GLIC), diclofenac-Na (DIC-Na) and mefenamic acid (MEF). LC was performed on a Dionex Ultimate 3000/LC 09115047 (USA) system. Chromatography was performed on a Thermo Scientific C18 (250 mm × 2.1 mm, i.d.: 5μm) column. Several parameters were optimised such as; mobile phase, gradient elution, collision energy and solvent elution for extraction of compounds from water. The recoveries obtained ranged from 30-148 % in river water. Five pharmaceutical compounds were detected in the surface water samples: caffeine, prazosin, enalpril, diclofenac-Na and mefenamic acid. The developed method is precise and accepted recoveries were got. In addition, this method is suitable to identify and quantify trace concentrations of pharmaceuticals in surface water.

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.

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.

NEW SCX PEPTIDE ELUTION SCORE FOR PH/SALT-GRADIENT SCX CHROMATOGRAPHY IN 2D-NANO-LC/MSMS ANALYSIS OF PROTEIN DIGESTS

EPA Science Inventory

A new automated 2D-(SCX/RP)-nano-LC/MSMS method was developed. Separation of the peptides in the first LC dimension was the main focus of this work, and it was optimized using human serum albumin (HSA) and human lung cell lysate tryptic digests. Samples were reduced and alkylated...

Self-assembled lecithin/chitosan nanoparticles for oral insulin delivery: preparation and functional evaluation

PubMed Central

Liu, Liyao; Zhou, Cuiping; Xia, Xuejun; Liu, Yuling

2016-01-01

Purpose Here, we investigated the formation and functional properties of self-assembled lecithin/chitosan nanoparticles (L/C NPs) loaded with insulin following insulin–phospholipid complex preparation, with the aim of developing a method for oral insulin delivery. Methods Using a modified solvent-injection method, insulin-loaded L/C NPs were obtained by combining insulin–phospholipid complexes with L/C NPs. The nanoparticle size distribution was determined by dynamic light scattering, and morphologies were analyzed by cryogenic transmission electron microscopy. Fourier transform infrared spectroscopy analysis was used to disclose the molecular mechanism of prepared insulin-loaded L/C NPs. Fast ultrafiltration and a reversed-phase high-performance liquid chromatography assay were used to separate free insulin from insulin entrapped in the L/C NPs, as well as to measure the insulin-entrapment and drug-loading efficiencies. The in vitro release profile was obtained, and in vivo hypoglycemic effects were evaluated in streptozotocin-induced diabetic rats. Results Our results indicated that insulin-containing L/C NPs had a mean size of 180 nm, an insulin-entrapment efficiency of 94%, and an insulin-loading efficiency of 4.5%. Cryogenic transmission electron microscopy observations of insulin-loaded L/C NPs revealed multilamellar structures with a hollow core, encircled by several bilayers. In vitro analysis revealed that insulin release from L/C NPs depended on the L/C ratio. Insulin-loaded L/C NPs orally administered to streptozotocin-induced diabetic rats exerted a significant hypoglycemic effect. The relative pharmacological bioavailability following oral administration of L/C NPs was 6.01%. Conclusion With the aid of phospholipid-complexation techniques, some hydrophilic peptides, such as insulin, can be successfully entrapped into L/C NPs, which could improve oral bioavailability, time-dependent release, and therapeutic activity. PMID:26966360

Life cycle tools combined with multi-criteria and participatory methods for agricultural sustainability: Insights from a systematic and critical review.

PubMed

De Luca, Anna Irene; Iofrida, Nathalie; Leskinen, Pekka; Stillitano, Teodora; Falcone, Giacomo; Strano, Alfio; Gulisano, Giovanni

2017-10-01

Life cycle (LC) methodologies have attracted a great interest in agricultural sustainability assessments, even if, at the same time, they have sometimes been criticized for making unrealistic assumptions and subjective choices. To cope with these weaknesses, Multi-Criteria Decision Analysis (MCDA) and/or participatory methods can be used to balance and integrate different sustainability dimensions. The purpose of this study is to highlight how life cycle approaches were combined with MCDA and participatory methods to address agricultural sustainability in the published scientific literature. A systematic and critical review was developed, highlighting the following features: which multi-criterial and/or participatory methods have been associated with LC tools; how they have been integrated or complemented (methodological relationships); the intensity of the involvement of stakeholders (degree of participation); and which synergies have been achieved by combining the methods. The main typology of integration was represented by multi-criterial frameworks integrating LC evaluations. LC tools can provide MCDA studies with local and global information on how to reduce negative impacts and avoid burden shifts, while MCDA methods can help LC practitioners deal with subjective assumptions in an objective way, to take into consideration actors' values and to overcome trade-offs among the different dimensions of sustainability. Considerations concerning the further development of Life Cycle Sustainability Assessment (LCSA) have been identified as well. Copyright © 2017 Elsevier B.V. All rights reserved.

Determination of Gentamicin Sulphate Composition and Related Substances in Pharmaceutical Preparations by LC with Charged Aerosol Detection

PubMed Central

Stypulkowska, Karolina; Fijalek, Zbigniew; Sarna, Katarzyna

2010-01-01

A new, simple and repeatable liquid chromatography method with charged aerosol detection (LC-CAD) for the determination of gentamicin sulphate composition and related substances has been developed. Gentamicin lacks of chromophores, therefore its determination is quite problematic. Using a universal CAD enables to achieve good separation without sample derivatization. Mass spectrometry was employed to confirm the LC-CAD peak profile. The proposed method was validated and applied for the determination of gentamicin sulphate composition and related substances in pharmaceutical preparations. PMID:21212825

[Sublethal effects of spinetoram and azadirachtin on development and reproduction of Frankliniella occidentalis (Pergande).

PubMed

Yang, Guang Ming; Zhi, Jun Rui; Li, Shun Xin; Liu, Li

2016-11-18

To evaluate the sublethal effects of spinetoram and azadirachtin on western flower thrips, Frankliniella occidentalis, leaf dipping method was used to determine their sublethal concentrations (LC 25 ) on the 2 nd instar nymph, and their influences on development and reproduction of F. occidentalis were studied. The results showed exposure of sublethal concentrations of spinetoram and azadirachtin to F. accidentalis had different degrees of effects on this insect pest. Under bisexual reproduction, the LC 25 spinetoram had no significant influences on pre-oviposition period, female adult longevity and fecundity, but male adult longevity was significantly shorter than the control. The LC 25 azadirachtin significantly reduced fecundity and prolonged pre-oviposition period. Under parthenogenesis, the LC 25 spinetoram and azadirachtin extended the pre-oviposition duration, whereas the LC 25 azadirachtin shortened the female adult longevity and significantly decreased fecundity. The LC 25 spinetoram and azadirachtin had different influences on developmental duration of each stage of next generation. The immature stage in treatment group of the LC 25 spinetoram was shorter than that in treatment group of the LC 25 azadirachtin, under bisexual reproduction or parthenogenesis. Intrinsic rate of increase (r m ) and finite rate of increase (λ) of population treated by the LC 25 spinetoram were higher than those of the control, whereas the r m , R 0 , and λ of population treated by the LC 25 azadirachtin were lower than those of the control. The findings indicated that the effects of the LC 25 spinetoram and azadirachtin on the development and reproduction of F. accidentalis were different. The LC 25 spinetoram had certain stimulating effect, whereas the LC 25 azadirach-tinon had significant inhibitory effect. Two biopesticides' influences were related with the reproductive patterns of F. accidentalis.

A simple method to determine IgG light chain to heavy chain polypeptide ratios expressed by CHO cells.

PubMed

Gerster, Anja; Wodarczyk, Claas; Reichenbächer, Britta; Köhler, Janet; Schulze, Andreas; Krause, Felix; Müller, Dethardt

2016-12-01

To establish a high-throughput method for determination of antibodies intra- and extracellular light chain (LC) to heavy chain (HC) polypeptide ratio as screening parameter during cell line development. Chinese Hamster Ovary (CHO) TurboCell pools containing different designed vectors supposed to result in different LC:HC polypeptide ratios were generated by targeted integration. Cell culture supernatants and cell lysates of a fed batch experiment were purified by combined Protein A and anti-kappa affinity batch purification in 96-well format. Capture of all antibodies and their fragments allowed the determination of the intra- and extracellular LC:HC peptide ratios by reduced SDS capillary electrophoresis. Results demonstrate that the method is suitable to show the significant impact of the vector design on the intra- and extracellular LC:HC polypeptide ratios. Determination of LC:HC polypeptide ratios can give important information in vector design optimization leading to CHO cell lines with optimized antibody assembly and preferred product quality.

An assessment of two-step linear regression and a multifactor probit analysis as alternatives to acute to chronic ratios in the estimation of chronic response from acute toxicity data to derive water quality guidelines.

PubMed

Slaughter, Andrew R; Palmer, Carolyn G; Muller, Wilhelmine J

2007-04-01

In aquatic ecotoxicology, acute to chronic ratios (ACRs) are often used to predict chronic responses from available acute data to derive water quality guidelines, despite many problems associated with this method. This paper explores the comparative protectiveness and accuracy of predicted guideline values derived from the ACR, linear regression analysis (LRA), and multifactor probit analysis (MPA) extrapolation methods applied to acute toxicity data for aquatic macroinvertebrates. Although the authors of the LRA and MPA methods advocate the use of extrapolated lethal effects in the 0.01% to 10% lethal concentration (LC0.01-LC10) range to predict safe chronic exposure levels to toxicants, the use of an extrapolated LC50 value divided by a safety factor of 5 was in addition explored here because of higher statistical confidence surrounding the LC50 value. The LRA LC50/5 method was found to compare most favorably with available experimental chronic toxicity data and was therefore most likely to be sufficiently protective, although further validation with the use of additional species is needed. Values derived by the ACR method were the least protective. It is suggested that there is an argument for the replacement of ACRs in developing water quality guidelines by the LRA LC50/5 method.

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.

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.

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.

Review of life-cycle approaches coupled with data envelopment analysis: launching the CFP + DEA method for energy policy making.

PubMed

Vázquez-Rowe, Ian; Iribarren, Diego

2015-01-01

Life-cycle (LC) approaches play a significant role in energy policy making to determine the environmental impacts associated with the choice of energy source. Data envelopment analysis (DEA) can be combined with LC approaches to provide quantitative benchmarks that orientate the performance of energy systems towards environmental sustainability, with different implications depending on the selected LC + DEA method. The present paper examines currently available LC + DEA methods and develops a novel method combining carbon footprinting (CFP) and DEA. Thus, the CFP + DEA method is proposed, a five-step structure including data collection for multiple homogenous entities, calculation of target operating points, evaluation of current and target carbon footprints, and result interpretation. As the current context for energy policy implies an anthropocentric perspective with focus on the global warming impact of energy systems, the CFP + DEA method is foreseen to be the most consistent LC + DEA approach to provide benchmarks for energy policy making. The fact that this method relies on the definition of operating points with optimised resource intensity helps to moderate the concerns about the omission of other environmental impacts. Moreover, the CFP + DEA method benefits from CFP specifications in terms of flexibility, understanding, and reporting.

Review of Life-Cycle Approaches Coupled with Data Envelopment Analysis: Launching the CFP + DEA Method for Energy Policy Making

PubMed Central

Vázquez-Rowe, Ian

2015-01-01

Life-cycle (LC) approaches play a significant role in energy policy making to determine the environmental impacts associated with the choice of energy source. Data envelopment analysis (DEA) can be combined with LC approaches to provide quantitative benchmarks that orientate the performance of energy systems towards environmental sustainability, with different implications depending on the selected LC + DEA method. The present paper examines currently available LC + DEA methods and develops a novel method combining carbon footprinting (CFP) and DEA. Thus, the CFP + DEA method is proposed, a five-step structure including data collection for multiple homogenous entities, calculation of target operating points, evaluation of current and target carbon footprints, and result interpretation. As the current context for energy policy implies an anthropocentric perspective with focus on the global warming impact of energy systems, the CFP + DEA method is foreseen to be the most consistent LC + DEA approach to provide benchmarks for energy policy making. The fact that this method relies on the definition of operating points with optimised resource intensity helps to moderate the concerns about the omission of other environmental impacts. Moreover, the CFP + DEA method benefits from CFP specifications in terms of flexibility, understanding, and reporting. PMID:25654136

Stability-indicating RP-HPLC method for the simultaneous determination of escitalopram oxalate and clonazepam.

PubMed

Kakde, Rajendra B; Satone, Dinesh D; Gadapayale, Kamalesh K; Kakde, Megha G

2013-07-01

The objective of the current study was to develop a validated, specific stability-indicating reversed-phase liquid chromatographic (LC) method for the quantitative determination of escitalopram oxalate and clonazepam and their related substances in bulk drugs and pharmaceutical dosage forms in the presence of degradation products. Forced degradation studies were performed on the pure drugs of escitalopram oxalate and clonazepam, as per the stress conditions prescribed by the International Conference on Harmonization (ICH) using acid, base, oxidation, thermal stress and photolytic degradation to show the stability-indicating power of the method. Significant degradation was observed during acid and alkaline hydrolysis and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from forced degradation studies. Good resolution between the peaks corresponded to the active pharmaceutical ingredients, escitalopram oxalate and clonazepam, and degradation products from the analyte were achieved on an ODS Hypersil C18 column (250 × 4.6 mm) using a mobile phase consisting of a mixture of acetonitrile-50 mM phosphate buffer + 10 mM triethylamine (70:30, v/v). The detection was conducted at 268 nm. The limit of detection and the limit of quantitation for escitalopram oxalate and clonazepam were established. The stress test solutions were assayed against the qualified working standards of escitalopram oxalate and clonazepam, which indicated that the developed LC method was stability-indicating. Validation of the developed LC method was conducted as per ICH requirements. The developed LC method was found to be suitable to check the quality of bulk samples of escitalopram oxalate and clonazepam.

Development of an Analytical Method for Explosive Residues in Soil,

DTIC Science & Technology

1987-06-01

confirm peak identities. The eluent for both columns should be 50:50 methanol-water. The elution time for all the analytes of interest on the LC -18 column...nitrate at 1.77 min for LC -8, 1.73 min for LC -DP, and 1.80 for LC -1. 23 Table A2. Instrument calibration results for HMX. Concentration Solution Soil* Peak ...LCT 12 AUG 2 0 W 1M 2j TNT Owl ""r’ L ,,,O MRYX TN L DNS 2 HMX 0 12 LC -CN 110 KMX S 8 TETRYL 6 RDXW 4 DNB and TNB 0 Approved for public release

Simultaneous determination of centchroman and tamoxifen along with their metabolites in rat plasma using LC-MS/MS.

PubMed

Rama Raju, Kanumuri Siva; Taneja, Isha; Singh, Sheelendra Pratap; Tripathi, Amit; Mishra, Durga Prasad; Hussain, K Mahaboob; Gayen, Jiaur Rahman; Singh, Shio Kumar; Wahajuddin, Muhammad

2015-01-01

Tamoxifen and centchroman are two non-steroidal, selective estrogen receptors modulators, intended for long term therapy in the woman. Because of their wide spread use, there is a possibility of co-prescription of these agents. We studied the probable pharmacokinetic interaction between these agents in breast cancer model rats. A simple, sensitive and rapid LC-ESI-MS/MS method was developed and validated for the simultaneous determination of tamoxifen, centchroman and their active metabolites. The method was linear over a range of 0.2-200 ng/ml. All validation parameters met the acceptance criteria according to regulatory guidelines. LC-MS/MS method for determination of tamoxifen, centchroman and their metabolites was developed and validated. Results show the potential of drug-drug interaction upon co-administration these two marketed drugs.

LC-UV assay method and UPLC/Q-TOF-MS characterisation of saponins from Ilex paraguariensis A. St. Hil. (mate) unripe fruits.

PubMed

Peixoto, Maria Paula Garofo; Kaiser, Samuel; Verza, Simone Gasparin; de Resende, Pedro Ernesto; Treter, Janine; Pavei, Cabral; Borré, Gustavo Luís; Ortega, George González

2012-01-01

Ilex paraguariensis A. St. Hil. (mate) is known in several South American countries because of the use of its leaves in stimulant herbal beverages. High saponin contents were reported in mate leaves and unripe fruits that possess a dissimilar composition. Two LC-UV methods previously reported for mate saponins assay focused on mate leaves and the quantification of the less polar saponin fraction in mate fruits. To develop and validate a LC-UV method to assay the total content of saponins in unripe mate fruits and characterise the chemical structure of triterpenic saponins by UPLC/Q-TOF-MS. From unripe fruits of mate a crude ethanolic extract was prepared (EX40) and the mate saponin fraction (MSF) purified by solid phase extraction. The LC-UV method was validated using ilexoside II as external standard. UPLC/Q-TOF-MS was adjusted from the LC-UV method to obtain the fragmentation patterns of the main saponins present in unripe fruits. Both LC-UV and UPLC/Q-TOF-MS methods indicate a wide range of Ilex saponins polarity. The ilexoside II and total saponin content of EX40 were 8.20% (w/w) and 47.60% (w/w), respectively. The total saponin content in unripe fruits was 7.28% (w/w). The saponins present in MSF characterised by UPLC/Q-TOF-MS are derived mainly from ursolic/oleanolic, acetyl ursolic or pomolic acid. The validated LC-UV method was shown to be linear, precise, accurate and to cover several saponins previously isolated from Ilex species and could be applied for the quality control of unripe fruit saponins. Copyright © 2011 John Wiley & Sons, Ltd.

Quantifying MMA by SLE LC-MS/MS: Unexpected challenges in assay development.

PubMed

Lo, Sheng-Ying; Gordon, Cindy; Sadilkova, Katerina; Jack, Rhona M; Dickerson, Jane A

2016-09-01

Analysis of serum/plasma methylmalonic acid (MMA) is important for the diagnosis and management of methylmalonic acidemia in pediatric populations. This work focuses on developing and validating a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to monitor methylmalonic acidemia using a simple method preparation. MMA and stable isotope labeled d3-MMA was extracted using supported liquid extraction (SLE). Assay imprecision, bias, linearity, recovery and carryover were determined. The relationship between MMA and propionyl acylcarnitine (C3-acylcarnitine) was also evaluated using historical paired results from 51 unique individuals. Baseline separation between MMA and succinic acid was completed in 7min. The assay was linear from 0.1 to 500μM. The intra-day and inter-day imprecision CV ranged from 4.1 to 13.2% (0.3 to 526μM) and 5.0 to 15.7% (0.3 to 233μM), respectively. Recovery ranged from 93 to 125%. The correlation with a national reference laboratory LC-MS/MS assay showed a Deming regression of 1.026 and intercept of -1.335. Carryover was determined to be <0.04%. Patient-specific correlation was observed between MMA and C3-acylcarnitine. This report describes the first LC-MS/MS method using SLE for MMA extraction. In addition, we illustrate the challenges encountered during this method development that should be assessed and resolved by any laboratory implementing a SLE LC-MS/MS assay designed to quantify analytes across several orders of magnitude. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Simultaneous determination of five coumarins in Angelicae dahuricae Radix by HPLC/UV and LC-ESI-MS/MS.

PubMed

Park, Ah Yeon; Park, So-Young; Lee, Jaehyun; Jung, Mihye; Kim, Jinwoong; Kang, Sam Sik; Youm, Jeong-Rok; Han, Sang Beom

2009-10-01

Rapid, simple and reliable HPLC/UV and LC-ESI-MS/MS methods for the simultaneous determination of five active coumarins of Angelicae dahuricae Radix, byakangelicol (1), oxypeucedanin (2), imperatorin (3), phellopterin (4) and isoimperatorin (5) were developed and validated. The separation condition for HPLC/UV was optimized using a Develosil RPAQUEOUS C(30) column using 70% acetonitrile in water as the mobile phase. This HPLC/UV method was successful for providing the baseline separation of the five coumarins with no interfering peaks detected in the 70% ethanol extract of Angelicae dahuricae Radix. The specific determination of the five coumarins was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source (LC-ESI-MS/MS). Multiple reaction monitoring (MRM) in the positive mode was used to enhance the selectivity of detection. The LC-ESI-MS/MS methods were successfully applied for the determination of the five major coumarins in Angelicae dahuricae Radix. These HPLC/UV and LC-ESI-MS/MS methods were validated in terms of recovery, linearity, accuracy and precision (intra- and inter-day validation). Taken together, the shorter analysis time involved makes these HPLC/UV and LC-ESI-MS/MS methods valuable for the commercial quality control of Angelicae dahuricae Radix extracts and its pharmaceutical preparations. Copyright (c) 2009 John Wiley & Sons, Ltd.

Nanomaterials as stationary phases and supports in liquid chromatography.

PubMed

Beeram, Sandya R; Rodriguez, Elliott; Doddavenkatanna, Suresh; Li, Zhao; Pekarek, Allegra; Peev, Darin; Goerl, Kathryn; Trovato, Gianfranco; Hofmann, Tino; Hage, David S

2017-10-01

The development of various nanomaterials over the last few decades has led to many applications for these materials in liquid chromatography (LC). This review will look at the types of nanomaterials that have been incorporated into LC systems and the applications that have been explored for such systems. A number of carbon-based nanomaterials and inorganic nanomaterials have been considered for use in LC, ranging from carbon nanotubes, fullerenes and nanodiamonds to metal nanoparticles and nanostructures based on silica, alumina, zirconia and titanium dioxide. Many ways have been described for incorporating these nanomaterials into LC systems. These methods have included covalent immobilization, adsorption, entrapment, and the synthesis or direct development of nanomaterials as part of a chromatographic support. Nanomaterials have been used in many types of LC. These applications have included the reversed-phase, normal-phase, ion-exchange, and affinity modes of LC, as well as related methods such as chiral separations, ion-pair chromatography and hydrophilic interaction liquid chromatography. Both small and large analytes (e.g., dyes, drugs, amino acids, peptides and proteins) have been used to evaluate possible applications for these nanomaterial-based methods. The use of nanomaterials in columns, capillaries and planar chromatography has been considered as part of these efforts. Potential advantages of nanomaterials in these applications have included their good chemical and physical stabilities, the variety of interactions many nanomaterials can have with analytes, and their unique retention properties in some separation formats. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The future of liquid chromatography-mass spectrometry (LC-MS) in metabolic profiling and metabolomic studies for biomarker discovery

PubMed Central

Metz, Thomas O.; Zhang, Qibin; Page, Jason S.; Shen, Yufeng; Callister, Stephen J.; Jacobs, Jon M.; Smith, Richard D.

2008-01-01

SUMMARY The future utility of liquid chromatography-mass spectrometry (LC-MS) in metabolic profiling and metabolomic studies for biomarker discover will be discussed, beginning with a brief description of the evolution of metabolomics and the utilization of the three most popular analytical platforms in such studies: NMR, GC-MS, and LC-MS. Emphasis is placed on recent developments in high-efficiency LC separations, sensitive electrospray ionization approaches, and the benefits to incorporating both in LC-MS-based approaches. The advantages and disadvantages of various quantitative approaches are reviewed, followed by the current LC-MS-based tools available for candidate biomarker characterization and identification. Finally, a brief prediction on the future path of LC-MS-based methods in metabolic profiling and metabolomic studies is given. PMID:19177179

Development and application of an LC-MS/MS method for the detection of the vinyl chloride-induced DNA adduct N2,3-ethenoguanine in tissues of adult and weanling rats following the exposure to [13C2]-VC

EPA Science Inventory

In the 1970s exposure to vinyl chloride (VC) was shown to cause liver angiosarcoma in VC workers. We have developed a new LC-MS/MS method for analyzing the promutagenic DNA adduct N2,3-ethenoguanine and have applied this to DNA from tissues of both adult and weanling rats expose...

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.

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.

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.

Identification of hydroxycinnamoylquinic acids of arnica flowers and burdock roots using a standardized LC-DAD-ESI/MS profiling method.

PubMed

Lin, Long-Ze; Harnly, James M

2008-11-12

A screening method using LC-DAD-ESI/MS was developed for the identification of common hydroxycinnamoylquinic acids based on direct comparison with standards. A complete standard set for mono-, di-, and tricaffeoylquinic isomers was assembled from commercially available standards, positively identified compounds in common plants (artichokes, asparagus, coffee bean, honeysuckle flowers, sweet potato, and Vernonia amygdalina leaves) and chemically modified standards. Four C18 reversed phase columns were tested using the standardized profiling method (based on LC-DAD-ESI/MS) for 30 phenolic compounds, and their elution order and retention times were evaluated. Using only two columns under standardized LC condition and the collected phenolic compound database, it was possible to separate all of the hydroxycinnamoylquinic acid conjugates and to identify 28 and 18 hydroxycinnamoylquinic acids in arnica flowers (Arnica montana L.) and burdock roots (Arctium lappa L.), respectively. Of these, 22 are reported for the first time.

Development of Chiral LC-MS Methods for small Molecules and Their Applications in the Analysis of Enantiomeric Composition and Pharmacokinetic Studies

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

Desai, Meera Jay

The purpose of this research was to develop sensitive LC-MS methods for enantiomeric separation and detection, and then apply these methods for determination of enantiomeric composition and for the study of pharmacokinetic and pharmacodynamic properties of a chiral nutraceutical. Our first study, evaluated the use of reverse phase and polar organic mode for chiral LC-API/MS method development. Reverse phase methods containing high water were found to decrease ionization efficiency in electrospray, while polar organic methods offered good compatibility and low limits of detection with ESI. The use of lower flow rates dramatically increased the sensitivity by an order of magnitude.more » Additionally, for rapid chiral screening, the coupled Chirobiotic column afforded great applicability for LC-MS method development. Our second study, continued with chiral LC-MS method development in this case for the normal phase mode. Ethoxynonafluorobutane, a fluorocarbon with low flammability and no flashpoint, was used as a substitute solvent for hexane/heptane mobile phases for LC-APCI/MS. Comparable chromatographic resolutions and selectivities were found using ENFB substituted mobile phase systems, although, peak efficiencies were significantly diminished. Limits of detection were either comparable or better for ENFB-MS over heptane-PDA detection. The miscibility of ENFB with a variety of commonly used organic modifiers provided for flexibility in method development. For APCI, lower flow rates did not increase sensitivity as significantly as was previously found for ESI-MS detection. The chiral analysis of native amino acids was evaluated using both APCI and ESI sources. For free amino acids and small peptides, APCI was found to have better sensitivities over ESI at high flow rates. For larger peptides, however, sensitivity was greatly improved with the use of electrospray. Additionally, sensitivity was enhanced with the use of non-volatile additives, This optimized method was then used to simultaneously separate all 19 native amino acids enantiomerically in less than 20 minutes, making it suitable for complex biological analysis. The previously developed amino acid method was then used to enantiomerically separate theanine, a free amino acid found in tea leaves. Native theanine was found to have lower limits of detection and better sensitivity over derivatized theanine samples. The native theanine method was then used to determine the enantiomeric composition of six commercially available L-theanine products. Five out of the six samples were found to be a racemic mixture of both D- and L-theanine. Concern over the efficacy of these theanine products led to our final study evaluating the pharmacokinetics and pharmacodynamics of theanine in rats using LC-ESI/MS. Rats were administered D-, L, and QL-theanine both orally and intra-peritoneally. Oral administration data demonstrated that intestinal absorption of L-theanine was greater than that of D-theanine, while i.p. data showed equal plasma uptake of both isomers. This suggested a possible competitive binding effect with respect to gut absorption. Additionally, it was found that regardless of administration method, the presence of the other enantiomer always decreased overall theanine plasma concentration. This indicated that D- and L- theanine exhibit competitive binding with respect to urinary reabsorption as well. The large quantities of D-theanine detected in the urine suggested that D-themine was eliminated with minimal metabolism, while L-theanine was preferentially reabsorbed and metabolized to ethylamine. Clearly, the metabolic fate of racemic theanine and its individual enantiomers was quite different, placing into doubt the utility of the commercial theanine products.« less

Simultaneous monitoring of oxidation, deamidation, isomerization, and glycosylation of monoclonal antibodies by liquid chromatography-mass spectrometry method with ultrafast tryptic digestion.

PubMed

Wang, Yi; Li, Xiaojuan; Liu, Yan-Hui; Richardson, Daisy; Li, Huijuan; Shameem, Mohammed; Yang, Xiaoyu

Monoclonal antibodies are subjected to a wide variety of post-translational modifications (PTMs) that cause structural heterogeneity. Characterization and control of these modifications or quality attributes are critical to ensure antibody quality and to define any potential effects on the ultimate safety and potency of antibody therapeutics. The biopharmaceutical industry currently uses numerous tools to analyze these quality attributes individually, which requires substantial time and resources. Here, we report a simple and ultrafast bottom-up liquid chromatography-mass spectrometry (uLC-MS) method with 5 min tryptic digestion to simultaneously analyze multiple modifications, including oxidation, deamidation, isomerization, glycation, glycosylation, and N-terminal pyro-glutamate formation, which can occur during antibody production in mammalian cell culture, during purification and/or on storage. Compared to commonly used preparation procedures, this uLC-MS method eliminates assay artifacts of falsely-increased Met oxidation, Asp isomerization, and Asn deamidation, a problem associated with long digestion times in conventional LC-MS methods. This simple, low artifact multi-attribute uLC-MS method can be used to quickly and accurately analyze samples at any stage of antibody drug development, in particular for clone and media selection during cell culture development.

Volumetric adsorptive microsampling-liquid chromatography tandem mass spectrometry assay for the simultaneous quantification of four antibiotics in human blood: Method development, validation and comparison with dried blood spot.

PubMed

Barco, Sebastiano; Castagnola, Elio; Moscatelli, Andrea; Rudge, James; Tripodi, Gino; Cangemi, Giuliana

2017-10-25

In this paper we show the development and validation of a volumetric absorptive microsampling (VAMS™)-LC-MS/MS method for the simultaneous quantification of four antibiotics: piperacillin-tazobactam, meropenem, linezolid and ceftazidime in 10μL human blood. The novel VAMS-LC-MS/MS method has been compared with a dried blood spot (DBS)-based method in terms of impact of hematocrit (HCT) on accuracy, reproducibility, recovery and matrix effect. Antibiotics were extracted from VAMS and DBS by protein precipitation with methanol after a re-hydration step at 37°C for 10min. LC-MS/MS was carried out on a Thermo Scientific™ TSQ Quantum™ Access MAX triple quadrupole coupled to an Accela ™UHPLC system. The VAMS-LC-MS/MS method is selective, precise and reproducible. In contrast to DBS, it allows an accurate quantification without any HCT influence. It has been applied to samples derived from pediatric patients under therapy. VAMS is a valid alternative sampling strategy for the quantification of antibiotics and is valuable in support of clinical PK/PD studies and consequently therapeutic drug monitoring (TDM) in pediatrics. Copyright © 2017 Elsevier B.V. All rights reserved.

High-Throughput Quantification of GFP-LC3+ Dots by Automated Fluorescence Microscopy.

PubMed

Bravo-San Pedro, J M; Pietrocola, F; Sica, V; Izzo, V; Sauvat, A; Kepp, O; Maiuri, M C; Kroemer, G; Galluzzi, L

2017-01-01

Macroautophagy is a specific variant of autophagy that involves a dedicated double-membraned organelle commonly known as autophagosome. Various methods have been developed to quantify the size of the autophagosomal compartment, which is an indirect indicator of macroautophagic responses, based on the peculiar ability of microtubule-associated protein 1 light chain 3 beta (MAP1LC3B; best known as LC3) to accumulate in forming autophagosomes upon maturation. One particularly convenient method to monitor the accumulation of mature LC3 within autophagosomes relies on a green fluorescent protein (GFP)-tagged variant of this protein and fluorescence microscopy. In physiological conditions, cells transfected temporarily or stably with a GFP-LC3-encoding construct exhibit a diffuse green fluorescence over the cytoplasm and nucleus. Conversely, in response to macroautophagy-promoting stimuli, the GFP-LC3 signal becomes punctate and often (but not always) predominantly cytoplasmic. The accumulation of GFP-LC3 in cytoplasmic dots, however, also ensues the blockage of any of the steps that ensure the degradation of mature autophagosomes, calling for the implementation of strategies that accurately discriminate between an increase in autophagic flux and an arrest in autophagic degradation. Various cell lines have been engineered to stably express GFP-LC3, which-combined with the appropriate controls of flux, high-throughput imaging stations, and automated image analysis-offer a relatively straightforward tool to screen large chemical or biological libraries for inducers or inhibitors of autophagy. Here, we describe a simple and robust method for the high-throughput quantification of GFP-LC3 + dots by automated fluorescence microscopy. © 2017 Elsevier Inc. All rights reserved.

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

Determination of 17alpha-ethynylestradiol, carbamazepine, diazepam, simvastatin, and oxybenzone in fish livers.

PubMed

Kwon, Jeong-Wook; Armbrust, Kevin L; Vidal-Dorsch, Doris; Bay, Steven M

2009-01-01

A method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed for the determination of 17alpha-ethynylestradiol in fish liver; a second method using LC/MS was developed for the determination of carbamazepine, diazepam, simvastatin, and oxybenzone in fish liver. The fish liver samples were extracted and cleaned up by using liquid-liquid extraction and solid-phase extraction before the extracts were analyzed by LC/MS or LC/MS/MS with electrospray negative and positive ionization. Recoveries of the 5 target compounds from spiked catfish liver ranged from 72 +/- 2 to 100 +/- 3%. Limits of quantification for the 5 compounds were between 4.2 and 12.3 ng/g (wet weight). Ten turbot (Pleuronichthys verticalis) liver samples were analyzed; levels of 17alpha-ethynylestradiol, carbamazepine, simvastatin, and oxybenzone were below the detection limits. Diazepam was detected in all 10 fish liver samples at concentrations ranging from 23 to 110 ng/g (wet weight).

Assessment of Intrathecal Free Light Chain Synthesis: Comparison of Different Quantitative Methods with the Detection of Oligoclonal Free Light Chains by Isoelectric Focusing and Affinity-Mediated Immunoblotting.

PubMed

Zeman, David; Kušnierová, Pavlína; Švagera, Zdeněk; Všianský, František; Byrtusová, Monika; Hradílek, Pavel; Kurková, Barbora; Zapletalová, Olga; Bartoš, Vladimír

2016-01-01

We aimed to compare various methods for free light chain (fLC) quantitation in cerebrospinal fluid (CSF) and serum and to determine whether quantitative CSF measurements could reliably predict intrathecal fLC synthesis. In addition, we wished to determine the relationship between free kappa and free lambda light chain concentrations in CSF and serum in various disease groups. We analysed 166 paired CSF and serum samples by at least one of the following methods: turbidimetry (Freelite™, SPAPLUS), nephelometry (N Latex FLC™, BN ProSpec), and two different (commercially available and in-house developed) sandwich ELISAs. The results were compared with oligoclonal fLC detected by affinity-mediated immunoblotting after isoelectric focusing. Although the correlations between quantitative methods were good, both proportional and systematic differences were discerned. However, no major differences were observed in the prediction of positive oligoclonal fLC test. Surprisingly, CSF free kappa/free lambda light chain ratios were lower than those in serum in about 75% of samples with negative oligoclonal fLC test. In about a half of patients with multiple sclerosis and clinically isolated syndrome, profoundly increased free kappa/free lambda light chain ratios were found in the CSF. Our results show that using appropriate method-specific cut-offs, different methods of CSF fLC quantitation can be used for the prediction of intrathecal fLC synthesis. The reason for unexpectedly low free kappa/free lambda light chain ratios in normal CSFs remains to be elucidated. Whereas CSF free kappa light chain concentration is increased in most patients with multiple sclerosis and clinically isolated syndrome, CSF free lambda light chain values show large interindividual variability in these patients and should be investigated further for possible immunopathological and prognostic significance.

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.

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.

Advantages of a validated UPLC-MS/MS standard addition method for the quantification of A-type dimeric and trimeric proanthocyanidins in cranberry extracts in comparison with well-known quantification methods.

PubMed

van Dooren, Ines; Foubert, Kenn; Theunis, Mart; Naessens, Tania; Pieters, Luc; Apers, Sandra

2018-01-30

The berries of Vaccinium macrocarpon, cranberry, are widely used for the prevention of urinary tract infections. This species contains A-type proanthocyanidins (PACs), which intervene in the initial phase of the development of urinary tract infections by preventing the adherence of Escherichia coli by their P-type fimbriae to uroepithelial cells. Unfortunately, the existing clinical studies used different cranberry preparations, which were poorly standardized. Because of this, the results were hard to compare, which led sometimes to conflicting results. Currently, PACs are quantified using the rather non-specific spectrophotometric 4-dimethylaminocinnamaldehyde (DMAC) method. In addition, a normal phase HPTLC-densitometric method, a HPLC-UV method and three LC-MS/MS methods for quantification of procyanidin A2 were recently published. All these methods contain some shortcomings and errors. Hence, the development and validation of a fast and sensitive standard addition LC-MS/MS method for the simultaneous quantification of A-type dimers and trimers in a cranberry dry extract was carried out. A linear calibration model could be adopted for dimers and, after logaritmic transformation, for trimers. The maximal interday and interconcentration precision was found to be 4.86% and 4.28% for procyanidin A2, and 5.61% and 7.65% for trimeric PACs, which are all acceptable values for an analytical method using LC-MS/MS. In addition, twelve different cranberry extracts were analyzed by means of the newly validated method and other widely used methods. There appeared to be an enormous variation in dimeric and trimeric PAC content. Comparison of these results with LC-MS/MS analysis without standard addition showed the presence of matrix effects for some of the extracts and proved the necessity of standard addition. A comparison of the well-known and widely used DMAC method, the butanol-HCl assay and this newly developed LC-MS/MS method clearly indicated the need for a reliable method able to quantify A-type PACs, which are considered to be the pharmacologically active constituents of cranberry, since neither the DMAC or butanol-HCl assays are capable of distinguishing between A and B-type PACs and therefore cannot detect adulterations with, for example, extracts with a high B-type PAC content. Hence, the combination of the DMAC method or butanol-HCl assay with this more specific LC-MS/MS assay could overcome these shortcomings. Copyright © 2017 Elsevier B.V. All rights reserved.

High-Performance Liquid Chromatography with Diode Array Detector and Electrospray Ionization Ion Trap Time-of-Flight Tandem Mass Spectrometry to Evaluate Ginseng Roots and Rhizomes from Different Regions.

PubMed

Wang, Hong-Ping; Zhang, You-Bo; Yang, Xiu-Wei; Yang, Xin-Bao; Xu, Wei; Xu, Feng; Cai, Shao-Qing; Wang, Ying-Ping; Xu, Yong-Hua; Zhang, Lian-Xue

2016-05-09

Ginseng, Panax ginseng C. A. Meyer, is an industrial crop in China and Korea. The functional components in ginseng roots and rhizomes are characteristic ginsenosides. This work developed a new high-performance liquid chromatography coupled with electrospray ionization ion trap time-of-flight multistage mass spectrometry (LC-ESI-IT-TOF-MS(n)) method to identify the triterpenoids. Sixty compounds (1-60) including 58 triterpenoids were identified from the ginseng cultivated in China. Substances 1, 2, 7, 15-20, 35, 39, 45-47, 49, 55-57, 59, and 60 were identified for the first time. To evaluate the quality of ginseng cultivated in Northeast China, this paper developed a practical liquid chromatography-diode array detection (LC-DAD) method to simultaneously quantify 14 interesting ginsenosides in ginseng collected from 66 different producing areas for the first time. The results showed the quality of ginseng roots and rhizomes from different sources was different due to growing environment, cultivation technology, and so on. The developed LC-ESI-IT-TOF-MS(n) method can be used to identify many more ginsenosides and the LC-DAD method can be used not only to assess the quality of ginseng, but also to optimize the cultivation conditions for the production of ginsenosides.

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.

Investigation of Soman Adducts of Human Hemoglobin by Liquid Chromatography

DTIC Science & Technology

2004-04-01

acid standard, with fifteen primary amino acids , was used to evaluate and refine the chromatographic methods . An LC/MS/MS was used to analyze the non...several chromatographic conditions and stationary phases were used to create an LC/MS/MS method to directly analyze the amino acids , these studies...terminated because of a lack of resolution of the amino acid peaks. Also, initial attempts to develop an HPLC method to separate individual amino acids

Differential recruitment efficacy of patient-derived amyloidogenic and myeloma light chain proteins by synthetic fibrils-A metric for predicting amyloid propensity.

PubMed

Martin, Emily B; Williams, Angela; Wooliver, Craig; Heidel, R Eric; Adams, Sarah; Dunlap, John; Ramirez-Alvarado, Marina; Blancas-Mejia, Luis M; Lands, Ronald H; Kennel, Stephen J; Wall, Jonathan S

2017-01-01

Monoclonal free light chain (LC) proteins are present in the circulation of patients with immunoproliferative disorders such as light chain (AL) amyloidosis and multiple myeloma (MM). Light chain-associated amyloid is a complex pathology composed of proteinaceous fibrils and extracellular matrix proteins found in all patients with AL and in ~10-30% of patients who presented with MM. Amyloid deposits systemically in multiple organs and tissues leading to dysfunction and ultimately death. The overall survival of patients with amyloidosis is worse than for those with early stage MM. We have developed a sensitive binding assay quantifying the recruitment of full length, patient-derived LC proteins by synthetic amyloid fibrils, as a method for studying their amyloidogenic potential. In a survey of eight urinary LC, both AL and MM-associated proteins were recruited by synthetic amyloid fibrils; however, AL-associated LC bound significantly more efficiently (p < 0.05) than did MM LCs. The LC proteins used in this study were isolated from urine and presumed to represent a surrogate of serum free light chains. The binding of LC to synthetic fibrils in this assay accurately differentiated LC with amyloidogenic propensity from MM LC that were not associated with clinical amyloid disease. Notably, the LC from a MM patient who subsequently developed amyloid behaved as an AL-associated protein in the assay, indicating the possibility for identifying MM patients at risk for developing amyloidosis based on the light chain recruitment efficacy. With this information, at risk patients can be monitored more closely for the development of amyloidosis, allowing timely administration of novel, amyloid-directed immunotherapies-this approach may improve the prognosis for these patients.

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

Self-assembled lecithin/chitosan nanoparticles for oral insulin delivery: preparation and functional evaluation.

PubMed

Liu, Liyao; Zhou, Cuiping; Xia, Xuejun; Liu, Yuling

2016-01-01

Here, we investigated the formation and functional properties of self-assembled lecithin/chitosan nanoparticles (L/C NPs) loaded with insulin following insulin-phospholipid complex preparation, with the aim of developing a method for oral insulin delivery. Using a modified solvent-injection method, insulin-loaded L/C NPs were obtained by combining insulin-phospholipid complexes with L/C NPs. The nanoparticle size distribution was determined by dynamic light scattering, and morphologies were analyzed by cryogenic transmission electron microscopy. Fourier transform infrared spectroscopy analysis was used to disclose the molecular mechanism of prepared insulin-loaded L/C NPs. Fast ultrafiltration and a reversed-phase high-performance liquid chromatography assay were used to separate free insulin from insulin entrapped in the L/C NPs, as well as to measure the insulin-entrapment and drug-loading efficiencies. The in vitro release profile was obtained, and in vivo hypoglycemic effects were evaluated in streptozotocin-induced diabetic rats. Our results indicated that insulin-containing L/C NPs had a mean size of 180 nm, an insulin-entrapment efficiency of 94%, and an insulin-loading efficiency of 4.5%. Cryogenic transmission electron microscopy observations of insulin-loaded L/C NPs revealed multilamellar structures with a hollow core, encircled by several bilayers. In vitro analysis revealed that insulin release from L/C NPs depended on the L/C ratio. Insulin-loaded L/C NPs orally administered to streptozotocin-induced diabetic rats exerted a significant hypoglycemic effect. The relative pharmacological bioavailability following oral administration of L/C NPs was 6.01%. With the aid of phospholipid-complexation techniques, some hydrophilic peptides, such as insulin, can be successfully entrapped into L/C NPs, which could improve oral bioavailability, time-dependent release, and therapeutic activity.

Mixed-mode chromatography/isotope ratio mass spectrometry.

PubMed

McCullagh, James S O

2010-03-15

Liquid chromatography coupled to molecular mass spectrometry (LC/MS) has been a standard technique since the early 1970s but liquid chromatography coupled to high-precision isotope ratio mass spectrometry (LC/IRMS) has only been available commercially since 2004. This development has, for the first time, enabled natural abundance and low enrichment delta(13)C measurements to be applied to individual analytes in aqueous mixtures creating new opportunities for IRMS applications, particularly for the isotopic study of biological molecules. A growing number of applications have been published in a range of areas including amino acid metabolism, carbohydrates studies, quantification of cellular and plasma metabolites, dietary tracer and nucleic acid studies. There is strong potential to extend these to new compounds and complex matrices but several challenges face the development of LC/IRMS methods. To achieve accurate isotopic measurements, HPLC separations must provide baseline-resolution between analyte peaks; however, the design of current liquid interfaces places severe restrictions on compatible flow rates and in particular mobile phase compositions. These create a significant challenge on which reports associated with LC/IRMS have not previously focused. Accordingly, this paper will address aspects of chromatography in the context of LC/IRMS, in particular focusing on mixed-mode separations and their benefits in light of these restrictions. It aims to provide an overview of mixed-mode stationary phases and of ways to improve high aqueous separations through manipulation of parameters such as column length, temperature and mobile phase pH. The results of several practical experiments are given using proteogenic amino acids and nucleosides both of which are of noted importance in the LC/IRMS literature. This communication aims to demonstrate that mixed-mode stationary phases provide a flexible approach given the constraints of LC/IRMS interface design and acts as a practical guide for the development of new chromatographic methods compatible with LC/IRMS applications. Copyright 2010 John Wiley & Sons, Ltd.

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...

APPLICATION OF DNPH DERIVATIZATION WITH LC/MS TO THE IDENTIFICATION OF POLAR CARBONYL DRINKING WATER DISINFECTION BY-PRODUCTS IN DRINKING WATER

EPA Science Inventory

A qualitative method using 2,4-dinitrophenylhydrazine (DNPH) derivatization followed by analysis with liquid chromatography (LC)/negative ion-electrospray mass spectrometry (MS) was developed for analyzing and identifying highly polar aldehydes and ketones in ozonated drinking wa...

Determination of 7alpha-OH cholesterol by LC-MS/MS: Application in assessing the activity of CYP7A1 in cholestatic minipigs

USDA-ARS?s Scientific Manuscript database

An LC-MS/MS method was developed and validated to determine 7alpha-OH cholesterol in liver microsome. This method was convenient and fast with high specificity and sensitivity. Briefly, a gradient elution was performed on a Synergi polar-C18 column (50 x 4.6mm i.d., 3microm). The mobile phase (cons...

Development of a new screening method for the detection of antibiotic residues in muscle tissues using liquid chromatography and high resolution mass spectrometry with a LC-LTQ-Orbitrap instrument.

PubMed

Hurtaud-Pessel, D; Jagadeshwar-Reddy, T; Verdon, E

2011-10-01

A liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was developed for screening meat for a wide range of antibiotics used in veterinary medicine. Full-scan mode under high resolution mass spectral conditions using an LTQ-Orbitrap mass spectrometer with resolving power 60,000 full width at half maximum (FWHM) was applied for analysis of the samples. Samples were prepared using two extraction protocols prior to LC-HRMS analysis. The scope of the method focuses on screening the following main families of antibacterial veterinary drugs: penicillins, cephalosporins, sulfonamides, macrolides, tetracyclines, aminoglucosides and quinolones. Compounds were successfully identified in spiked samples from their accurate mass and LC retention times from the acquired full-scan chromatogram. Automated data processing using ToxId software allowed rapid treatment of the data. Analyses of muscle tissues from real samples collected from antibiotic-treated animals was carried out using the above methodology and antibiotic residues were identified unambiguously. Further analysis of the data for real samples allowed the identification of the targeted antibiotic residues but also non-targeted compounds, such as some of their metabolites.

Method Development and Monitoring of Cyanotoxins in Water

EPA Science Inventory

This presentation describes method development of two ambient water LC/MS/MS methods for microcystins, cylindrospermopsin and anatoxin-a. Ruggedness of the methods will be demonstrated by evaluation of quality control samples derived from various water bodies across the country.

Development of high-throughput multi-residue method for non-steroidal anti-inflammatory drugs monitoring in swine muscle by LC-MS/MS.

PubMed

Castilhos, Tamara S; Barreto, Fabiano; Meneghini, Leonardo; Bergold, Ana Maria

2016-07-01

A reliable and simple method for the detection and quantification of residues of 14 non-steroidal anti-inflammatory drugs and a metamizole metabolite in swine muscle was developed using liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS). The samples were extracted with acetonitrile (ACN) in solid-liquid extraction followed by a low-temperature partitioning (LLE-LTP) process at -20 ± 2°C. After evaporation to dryness, the residue was reconstituted with hexane and a mixture of water:acetonitrile (1:1). LC separation was achieved on a reversed-phase (RP18) column with gradient elution using water (phase A) and ACN (phase B) both containing 1 mmol l(-)(1) ammonium acetate (NH4COO) with 0.025% acetic acid. Analysis was carried out on a triple-quadrupole tandem mass spectrometer (LC-MS/MS) in multiple reaction monitoring mode using an electrospray interface in negative and positive mode in a single run. Method validation was performed according to the criteria of Commission Decision No. 2002/657/EC. The matrix effect and linearity were evaluated. Decision limit (CCα), detection capability (CCβ), accuracy and repeatability of the method are also reported. The proposed method proved to be simple, easy and adequate for high-throughput analysis and was applied to routine analysis by the Brazilian Ministry of Agriculture, Livestock and Food Supply.

Current progress and technical challenges of flexible liquid crystal displays

NASA Astrophysics Data System (ADS)

Fujikake, Hideo; Sato, Hiroto

2009-02-01

We focused on several technical approaches to flexible liquid crystal (LC) display in this report. We have been developing flexible displays using plastic film substrates based on polymer-dispersed LC technology with molecular alignment control. In our representative devices, molecular-aligned polymer walls keep plastic-substrate gap constant without LC alignment disorder, and aligned polymer networks create monostable switching of fast-response ferroelectric LC (FLC) for grayscale capability. In the fabrication process, a high-viscosity FLC/monomer solution was printed, sandwiched and pressed between plastic substrates. Then the polymer walls and networks were sequentially formed based on photo-polymerization-induced phase separation in the nematic phase by two exposure processes of patterned and uniform ultraviolet light. The two flexible backlight films of direct illumination and light-guide methods using small three-primary-color light-emitting diodes were fabricated to obtain high-visibility display images. The fabricated flexible FLC panels were driven by external transistor arrays, internal organic thin film transistor (TFT) arrays, and poly-Si TFT arrays. We achieved full-color moving-image displays using the flexible FLC panel and the flexible backlight film based on field-sequential-color driving technique. Otherwise, for backlight-free flexible LC displays, flexible reflective devices of twisted guest-host nematic LC and cholesteric LC were discussed with molecular-aligned polymer walls. Singlesubstrate device structure and fabrication method using self-standing polymer-stabilized nematic LC film and polymer ceiling layer were also proposed for obtaining LC devices with excellent flexibility.

An Improved LC-MS/MS Method for Simultaneous Determination of the Eleven Bioactive Constituents for Quality Control of Radix Angelicae Pubescentis and Its Related Preparations

PubMed Central

Li, Jin; Zhang, Qiu-Hong; He, Jun; Liu, Er-wei; Gao, Xiu-mei; Chang, Yan-xu

2015-01-01

An improved LC-MS/MS method was developed for simultaneous determination of eleven bioactive constituents of Radix Angelicae Pubescentis and its related preparations. It was the first report on the quantification of bioactive constituents in different preparations of Radix Angelicae Pubescentis by LC-MS/MS analytical method. These samples were separated with an Agilent Zorbax Extend reversed-phase C18 column (1.8 μm, 4.6 × 100 mm) by linear gradient elution using aqueous ammonium acetate and acetonitrile as mobile phase. The flow rate was 0.3 mL min−1. The eleven bioactive constituents showed good regression (R > 0.990) within test ranges and the recoveries were in the range of 87.1–110%. The limit of detections and quantifications for most of the major constituents were less than 0.5 and 1.0 ng mL−1, respectively. All results indicated that the developed method could be readily utilized as a suitable quality control method for Radix Angelicae Pubescentis and related preparations. PMID:26078992

Assessment of Intrathecal Free Light Chain Synthesis: Comparison of Different Quantitative Methods with the Detection of Oligoclonal Free Light Chains by Isoelectric Focusing and Affinity-Mediated Immunoblotting

PubMed Central

Kušnierová, Pavlína; Švagera, Zdeněk; Všianský, František; Byrtusová, Monika; Hradílek, Pavel; Kurková, Barbora; Zapletalová, Olga; Bartoš, Vladimír

2016-01-01

Objectives We aimed to compare various methods for free light chain (fLC) quantitation in cerebrospinal fluid (CSF) and serum and to determine whether quantitative CSF measurements could reliably predict intrathecal fLC synthesis. In addition, we wished to determine the relationship between free kappa and free lambda light chain concentrations in CSF and serum in various disease groups. Methods We analysed 166 paired CSF and serum samples by at least one of the following methods: turbidimetry (Freelite™, SPAPLUS), nephelometry (N Latex FLC™, BN ProSpec), and two different (commercially available and in-house developed) sandwich ELISAs. The results were compared with oligoclonal fLC detected by affinity-mediated immunoblotting after isoelectric focusing. Results Although the correlations between quantitative methods were good, both proportional and systematic differences were discerned. However, no major differences were observed in the prediction of positive oligoclonal fLC test. Surprisingly, CSF free kappa/free lambda light chain ratios were lower than those in serum in about 75% of samples with negative oligoclonal fLC test. In about a half of patients with multiple sclerosis and clinically isolated syndrome, profoundly increased free kappa/free lambda light chain ratios were found in the CSF. Conclusions Our results show that using appropriate method-specific cut-offs, different methods of CSF fLC quantitation can be used for the prediction of intrathecal fLC synthesis. The reason for unexpectedly low free kappa/free lambda light chain ratios in normal CSFs remains to be elucidated. Whereas CSF free kappa light chain concentration is increased in most patients with multiple sclerosis and clinically isolated syndrome, CSF free lambda light chain values show large interindividual variability in these patients and should be investigated further for possible immunopathological and prognostic significance. PMID:27846293

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.

Toward an operational framework for fine-scale urban land-cover mapping in Wallonia using submeter remote sensing and ancillary vector data

NASA Astrophysics Data System (ADS)

Beaumont, Benjamin; Grippa, Tais; Lennert, Moritz; Vanhuysse, Sabine; Stephenne, Nathalie; Wolff, Eléonore

2017-07-01

Encouraged by the EU INSPIRE directive requirements and recommendations, the Walloon authorities, similar to other EU regional or national authorities, want to develop operational land-cover (LC) and land-use (LU) mapping methods using existing geodata. Urban planners and environmental monitoring stakeholders of Wallonia have to rely on outdated, mixed, and incomplete LC and LU information. The current reference map is 10-years old. The two object-based classification methods, i.e., a rule- and a classifier-based method, for detailed regional urban LC mapping are compared. The added value of using the different existing geospatial datasets in the process is assessed. This includes the comparison between satellite and aerial optical data in terms of mapping accuracies, visual quality of the map, costs, processing, data availability, and property rights. The combination of spectral, tridimensional, and vector data provides accuracy values close to 0.90 for mapping the LC into nine categories with a minimum mapping unit of 15 m2. Such a detailed LC map offers opportunities for fine-scale environmental and spatial planning activities. Still, the regional application poses challenges regarding automation, big data handling, and processing time, which are discussed.

Identification and characterization of stress degradants of lacosamide by LC-MS and ESI-Q-TOF-MS/MS: development and validation of a stability indicating RP-HPLC method.

PubMed

Ramisetti, Nageswara Rao; Kuntamukkala, Ramakrishna; Lakshetti, Sridhar; Sripadi, Prabhakar

2014-07-01

The current study dealt with the degradation behavior of lacosamide (LAC) under ICH prescribed stress conditions. LAC was found to be labile under acid and base hydrolytic stress conditions, while it was stable to neutral hydrolytic, oxidative, photolytic and thermal stress. In total, seven degradation products (DPs) were formed, which were separated on a C18 column using a stability-indicating method. LC-MS analyses indicated that one of the DPs had the same molecular mass as that of the drug. Structural characterization of DPs was carried out using ESI-Q-TOF-MS/MS technique. The degradation pathways and mechanisms of degradation of the drug were delineated by carrying out the degradation in different co-solvents viz. methanol, deuterated methanol, ethanol, 1-propanol and acetonitrile. The developed LC method was validated for the determination of related substances and assay of LAC as per ICH guidelines. This study demonstrates a comprehensive approach of LAC degradation studies during its development phase. Copyright © 2014. Published by Elsevier B.V.

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.

Multivalency regulates activity in an intrinsically disordered transcription factor

PubMed Central

Clark, Sarah; Myers, Janette B; King, Ashleigh; Fiala, Radovan; Novacek, Jiri; Pearce, Grant; Heierhorst, Jörg; Reichow, Steve L

2018-01-01

The transcription factor ASCIZ (ATMIN, ZNF822) has an unusually high number of recognition motifs for the product of its main target gene, the hub protein LC8 (DYNLL1). Using a combination of biophysical methods, structural analysis by NMR and electron microscopy, and cellular transcription assays, we developed a model that proposes a concerted role of intrinsic disorder and multiple LC8 binding events in regulating LC8 transcription. We demonstrate that the long intrinsically disordered C-terminal domain of ASCIZ binds LC8 to form a dynamic ensemble of complexes with a gradient of transcriptional activity that is inversely proportional to LC8 occupancy. The preference for low occupancy complexes at saturating LC8 concentrations with both human and Drosophila ASCIZ indicates that negative cooperativity is an important feature of ASCIZ-LC8 interactions. The prevalence of intrinsic disorder and multivalency among transcription factors suggests that formation of heterogeneous, dynamic complexes is a widespread mechanism for tuning transcriptional regulation. PMID:29714690

Performance of plasma free metanephrines measured by liquid chromatography-tandem mass spectrometry in the diagnosis of pheochromocytoma.

PubMed

Peaston, Robert T; Graham, Kendon S; Chambers, Erin; van der Molen, Jan C; Ball, Stephen

2010-04-02

Plasma free metanephrines have proved a highly sensitive biochemical test for the diagnosis of pheochromocytoma. We have developed and validated a simple, LC-MS/MS method to determine plasma metanephrines and compared the diagnostic efficacy of the method with an enzyme immunoassay procedure in 151 patients, 38 with histologically confirmed pheochromocytoma. Off-line solid phase extraction in a 96-well plate format was used to isolate metanephrines from 100-microL of plasma, followed by rapid separation with hydrophilic interaction chromatography. Mass spectrometry detection was performed in multiple-reaction monitoring mode using a tandem quadrupole mass spectrometer with positive electrospray ionization. Detection limits were <0.1nmol/l with method linearity up to 23.0nmol/L for normetanephrine (NMN), metanephrine (MN) and 3-methoxytyramine (3-MT). Method comparison with an automated LC-MS/MS yielded Deming regression slopes of r=0.94 for NMN, r=0.98 for MN and r=0.94 for 3-MT. Method comparison with enzyme immunoassay revealed regression slope of r=1.28 (NMN) and 1.25 (MN) with values approximately 25% lower than LC-MS/MS. Plasma metanephrines by LC-MS/MS identified all 38 patients with phaeochromocytoma compared with 36 cases by immunoassay. Plasma metanephrines measured by LC-MS/MS are a reliable and sensitive test for the biochemical detection of pheochromocytoma. 2010 Elsevier B.V. All rights reserved.

Measurement of citrate in urine using liquid chromatography tandem mass spectrometry: comparison with an enzymatic method.

PubMed

Keevil, B G; Owen, L; Thornton, S; Kavanagh, J

2005-09-01

Measurement of urine citrate is used to assess the risk of further urinary stone formation and to assess the benefit of treatment in affected individuals. We wanted to develop a simple and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the analysis of urinary citrate and to compare it with our current enzymatic assay. For the LC-MS/MS assay, samples were prepared in a deep-well block by adding 10 microL of urine and 20 microL of internal standard to 400 microL of water. After mixing, 3 microL of the diluted sample was injected into the LC-MS/MS system. An LC system was used to isocratically elute a C18 column (50 x 2.1 mm) with 0.4 mL/min water containing 2 mmol/L ammonium acetate and 0.1% (v/v) formic acid. A step gradient of 100% methanol containing 2 mmol/L ammonium acetate and 0.1% (v/v) formic acid was used to wash the column. The retention times were 1.4 min for citrate and 1.4 min for d4-citrate. Cycle time was 4.0 min, injection to injection. The analytes were monitored using a tandem mass spectrometer operated in multiple reaction monitoring mode using the following transitions, citrate m/z 191.0>111.0 and d4-citrate m/z 195.0>113.0. Within and between-batch coefficients of variation were <3% over the range 480-3800 micromol/L. The lower limit of quantification was 24.0 micromol/L. Regression analysis showed LC-MS/MS = 0.8781 (enzymatic assay) + 102.5, r = 0.964, n = 73. We have developed a simple LC-MS/MS method for urinary citrate measurement that shows acceptable performance.

Rapid LC-MS Determination of Acromelic Acids A and B, Toxic Constituents of the Mushroom Paralepistopsis acromelalga.

PubMed

Yoshioka, Naoki; Ouchi, Hitoshi; Kan, Toshiyuki; Yoshida, Masashi; Nomura, Motoyuki

2017-01-01

A rapid LC-MS method was developed for determination of acromelic acids A and B, which are toxic constituents of Paralepistopsis acromelalga (=Clitocybe acromelalga), in mushroom samples. Acromelic acids were extracted twice with 50% methanol and the extract was passed through a syringe filter, and then analyzed by LC-MS. The LC separation was performed on a multi-mode ODS column. The recoveries of acromelic acids A and B spiked into blank mushroom samples at 2.5 μg/g were 93 and 74%, respectively. This method was applied to the remaining mushroom sample from a food poisoning case. Acromelic acids A and B were detected at 2.0 and 1.4 μg/g, respectively, in the remaining sample. Another toxic constituent, which appeared to be clitidine, was also detected in the sample.

An Autophagic Flux Probe that Releases an Internal Control.

PubMed

Kaizuka, Takeshi; Morishita, Hideaki; Hama, Yutaro; Tsukamoto, Satoshi; Matsui, Takahide; Toyota, Yuichiro; Kodama, Akihiko; Ishihara, Tomoaki; Mizushima, Tohru; Mizushima, Noboru

2016-11-17

Macroautophagy is an intracellular degradation system that utilizes the autophagosome to deliver cytoplasmic components to the lysosome. Measuring autophagic activity is critically important but remains complicated and challenging. Here, we have developed GFP-LC3-RFP-LC3ΔG, a fluorescent probe to evaluate autophagic flux. This probe is cleaved by endogenous ATG4 proteases into equimolar amounts of GFP-LC3 and RFP-LC3ΔG. GFP-LC3 is degraded by autophagy, while RFP-LC3ΔG remains in the cytosol, serving as an internal control. Thus, autophagic flux can be estimated by calculating the GFP/RFP signal ratio. Using this probe, we re-evaluated previously reported autophagy-modulating compounds, performed a high-throughput screen of an approved drug library, and identified autophagy modulators. Furthermore, we succeeded in measuring both induced and basal autophagic flux in embryos and tissues of zebrafish and mice. The GFP-LC3-RFP-LC3ΔG probe is a simple and quantitative method to evaluate autophagic flux in cultured cells and whole organisms. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

Research and Development Services: Methods Development. Appendices

DTIC Science & Technology

1982-07-23

0.010-inch stainless-steel tubing. The order of the detectors is as follows: 1. Perkin-Elmer LC-75 variable-wavelength spectrometer with autocontrol . 2...reset it prior to the next injection. At 60 minutes, the micro- processor signals the Perkin-Elmer LC-75 with autocontrol to switch detection...variable-wavelength spectrometer with autocontrol set at 230 am for first 60 minutes of chromatogram then switched to 280 m. b. Altex Model 153 fixed

Method Development and Monitoring of Cyanotoxins in Water ...

EPA Pesticide Factsheets

This presentation describes method development of two ambient water LC/MS/MS methods for microcystins, cylindrospermopsin and anatoxin-a. Ruggedness of the methods will be demonstrated by evaluation of quality control samples derived from various water bodies across the country. Presentation at the Central Regional meeting of the American Chemical Society ambient water methods development for cyanotoxins

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.

Direct analysis of prostaglandin-E2 and -D2 produced in an inflammatory cell reaction and its application for activity screening and potency evaluation using turbulent flow chromatography liquid chromatography-high resolution mass spectrometry.

PubMed

Shin, Jeong-Sook; Peng, Lei; Kang, Kyungsu; Choi, Yongsoo

2016-09-09

Direct analysis of prostaglandin-E2 (PGE2) and -D2 (PGD2) produced from a RAW264.7 cell-based reaction was performed by liquid chromatography high-resolution mass spectrometry (LC-HRMS), which was online coupled with turbulent flow chromatography (TFC). The capability of this method to accurately measure PG levels in cell reaction medium containing cytokines or proteins as a reaction byproduct was cross-validated by two conventional methods. Two methods, including an LC-HRMS method after liquid-liquid extraction (LLE) of the sample and a commercial PGE2 enzyme-linked immunosorbent assay (ELISA), showed PGE2 and/or PGD2 levels almost similar to those obtained by TFC LC-HRMS over the reaction time after LPS stimulation. After the cross-validation, significant analytical throughputs, allowing simultaneous screening and potency evaluation of 80 natural products including 60 phytochemicals and 20 natural product extracts for the inhibition of the PGD2 produced in the cell-based inflammatory reaction, were achieved using the TFC LC-HRMS method developed. Among the 60 phytochemicals screened, licochalcone A and formononetin inhibited PGD2 production the most with IC50 values of 126 and 151nM, respectively. For a reference activity, indomethacin and diclofenac were used, measuring IC50 values of 0.64 and 0.21nM, respectively. This method also found a butanol extract of Akebia quinata Decne (AQ) stem as a promising natural product for PGD2 inhibition. Direct and accurate analysis of PGs in the inflammatory cell reaction using the TFC LC-HRMS method developed enables the high-throughput screening and potency evaluation of as many as 320 samples in less than 48h without changing a TFC column. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of Two Analytical Methods Based on Reverse Phase Chromatographic and SDS-PAGE Gel for Assessment of Deglycosylation Yield in N-Glycan Mapping.

PubMed

Eckard, Anahita D; Dupont, David R; Young, Johnie K

2018-01-01

N -lined glycosylation is one of the critical quality attributes (CQA) for biotherapeutics impacting the safety and activity of drug product. Changes in pattern and level of glycosylation can significantly alter the intrinsic properties of the product and, therefore, have to be monitored throughout its lifecycle. Therefore fast, precise, and unbiased N -glycan mapping assay is desired. To ensure these qualities, using analytical methods that evaluate completeness of deglycosylation is necessary. For quantification of deglycosylation yield, methods such as reduced liquid chromatography-mass spectrometry (LC-MS) and reduced capillary gel electrophoresis (CGE) have been commonly used. Here we present development of two additional methods to evaluate deglycosylation yield: one based on LC using reverse phase (RP) column and one based on reduced sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE gel) with offline software (GelAnalyzer). With the advent of rapid deglycosylation workflows in the market for N -glycan profiling replacing overnight incubation, we have aimed to quantify the level of deglycosylation in a selected rapid deglycosylation workflow. Our results have shown well resolved peaks of glycosylated and deglycosylated protein species with RP-LC method allowing simple quantification of deglycosylation yield of protein with high confidence. Additionally a good correlation, ≥0.94, was found between deglycosylation yields estimated by RP-LC method and that of reduced SDS-PAGE gel method with offline software. Evaluation of rapid deglycosylation protocol from GlycanAssure™ HyPerformance assay kit performed on fetuin and RNase B has shown complete deglycosylation within the recommended protocol time when evaluated with these techniques. Using this kit, N -glycans from NIST mAb were prepared in 1.4 hr and analyzed by hydrophilic interaction chromatography (HILIC) ultrahigh performance LC (UHPLC) equipped with a fluorescence detector (FLD). 37 peaks were resolved with good resolution. Excellent sample preparation repeatability was found with relative standard deviation (RSD) of <5% for peaks with >0.5% relative area.

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.

Processing methods for differential analysis of LC/MS profile data

PubMed Central

Katajamaa, Mikko; Orešič, Matej

2005-01-01

Background Liquid chromatography coupled to mass spectrometry (LC/MS) has been widely used in proteomics and metabolomics research. In this context, the technology has been increasingly used for differential profiling, i.e. broad screening of biomolecular components across multiple samples in order to elucidate the observed phenotypes and discover biomarkers. One of the major challenges in this domain remains development of better solutions for processing of LC/MS data. Results We present a software package MZmine that enables differential LC/MS analysis of metabolomics data. This software is a toolbox containing methods for all data processing stages preceding differential analysis: spectral filtering, peak detection, alignment and normalization. Specifically, we developed and implemented a new recursive peak search algorithm and a secondary peak picking method for improving already aligned results, as well as a normalization tool that uses multiple internal standards. Visualization tools enable comparative viewing of data across multiple samples. Peak lists can be exported into other data analysis programs. The toolbox has already been utilized in a wide range of applications. We demonstrate its utility on an example of metabolic profiling of Catharanthus roseus cell cultures. Conclusion The software is freely available under the GNU General Public License and it can be obtained from the project web page at: . PMID:16026613

Processing methods for differential analysis of LC/MS profile data.

PubMed

Katajamaa, Mikko; Oresic, Matej

2005-07-18

Liquid chromatography coupled to mass spectrometry (LC/MS) has been widely used in proteomics and metabolomics research. In this context, the technology has been increasingly used for differential profiling, i.e. broad screening of biomolecular components across multiple samples in order to elucidate the observed phenotypes and discover biomarkers. One of the major challenges in this domain remains development of better solutions for processing of LC/MS data. We present a software package MZmine that enables differential LC/MS analysis of metabolomics data. This software is a toolbox containing methods for all data processing stages preceding differential analysis: spectral filtering, peak detection, alignment and normalization. Specifically, we developed and implemented a new recursive peak search algorithm and a secondary peak picking method for improving already aligned results, as well as a normalization tool that uses multiple internal standards. Visualization tools enable comparative viewing of data across multiple samples. Peak lists can be exported into other data analysis programs. The toolbox has already been utilized in a wide range of applications. We demonstrate its utility on an example of metabolic profiling of Catharanthus roseus cell cultures. The software is freely available under the GNU General Public License and it can be obtained from the project web page at: http://mzmine.sourceforge.net/.

Identification of Forced Degradation Products of Itopride by LC-PDA and LC-MS.

PubMed

Joshi, Payal; Bhoir, Suvarna; Bhagwat, A M; Vishwanath, K; Jadhav, R K

2011-05-01

Degradation products of itopride formed under different forced conditions have been identified using LC-PDA and LC-MS techniques. Itopride was subjected to forced degradation under the conditions of hydrolysis, photolysis, oxidation, dry and wet heat, in accordance with the International Conference on Harmonization. The stress solutions were chromatographed on reversed phase C18 (250×4.6 mm, 5 μm) column with a mobile phase methanol:water (55:45, v/v) at a detection wavelength of 215 nm. Itopride degraded in acid, alkali and oxidative stress conditions. The stability indicating method was developed and validated. The degradation pathway of the drug to products II-VIII is proposed.

Identification of Forced Degradation Products of Itopride by LC-PDA and LC-MS

PubMed Central

Joshi, Payal; Bhoir, Suvarna; Bhagwat, A. M.; Vishwanath, K.; Jadhav, R. K.

2011-01-01

Degradation products of itopride formed under different forced conditions have been identified using LC-PDA and LC-MS techniques. Itopride was subjected to forced degradation under the conditions of hydrolysis, photolysis, oxidation, dry and wet heat, in accordance with the International Conference on Harmonization. The stress solutions were chromatographed on reversed phase C18 (250×4.6 mm, 5 μm) column with a mobile phase methanol:water (55:45, v/v) at a detection wavelength of 215 nm. Itopride degraded in acid, alkali and oxidative stress conditions. The stability indicating method was developed and validated. The degradation pathway of the drug to products II-VIII is proposed. PMID:22457552

Cognitive training transfer using a personal computer-based game: A close quarters battle case study

NASA Astrophysics Data System (ADS)

Woodman, Michael D.

In this dissertation, liquid crystal (LC) materials and devices are investigated in order to meet the challenges for photonics and displays applications. We have studied three kinds of liquid crystal materials: positive dielectric anisotropic LCs, negative dielectric anisotropic LCs, and dual-frequency LCs. For the positive dielectric anisotropic LCs, we have developed some high birefringence isothiocyanato tolane LC compounds with birefringence ˜0.4, and super high birefringence isothiocyanato biphenyl-bistolane LC compounds with birefringence as high as ˜0.7. Moreover, we have studied the photostability of several high birefringence LC compounds, mixtures, and LC alignment layers in order to determine the failure mechanism concerning the lifetime of LC devices. Although cyano and isothiocyanato LC compounds have similar absorption peaks, the isothiocyanato compounds are more stable than their cyano counterparts under the same illumination conditions. This ultraviolet-durable performance of isothiocyanato compounds originates from its molecular structure and the delocalized electron distribution. We have investigated the alignment performance of negative dielectric anisotropic LCs in homeotropic (vertical aligned, VA) LC cell. Some (2, 3) laterally difluorinated biphenyls, terphenyls and tolanes are selected for this study. Due to the strong repulsive force between LCs and alignment layer, (2,3) laterally difluorinated terphenyls and tolanes do not align well in a VA cell resulting in a poor contrast ratio for the LC panel. We have developed a novel method to suppress the light leakage at dark state. By doping positive Deltaepsilon or non-polar LC compounds or mixtures into the host negative LC mixtures, the repulsive force is reduced and the cell exhibits an excellent dark state. In addition, these dopants increase the birefringence and reduce the viscosity of the host LCs which leads to a faster response time. In this dissertation, we investigate the dielectric heating effect of dual-frequency LCs. Because the absorption peak of imaginary dielectric constant occurs at high frequency region (˜ MHz), there is a heat generated when the LC cell is operated at a high frequency voltage. We have formulated a new dual-frequency LC mixture which greatly reduces the dielectric heating effect while maintaining good physical properties. Another achievement in this thesis is that we have developed a polarization independent phase modulator by using a negative dielectric anisotropic LC gel. (Abstract shortened by UMI.)

Simultaneous detection of diagnostic biomarkers of alkaptonuria, ornithine carbamoyltransferase deficiency, and neuroblastoma disease by high-performance liquid chromatography/tandem mass spectrometry.

PubMed

Hsu, Wei-Yi; Chen, Ching-Ming; Tsai, Fuu-Jen; Lai, Chien-Chen

2013-05-01

Urinary homovanillic acid (HVA)/vanillylmandelic acid (VMA), orotic acid (OA), and homogentisic acid (HGA) are diagnostic biomarkers of neuroblastoma, ornithine carbamoyl transferase deficiency (OCTD), and alkaptonuria (AKU), respectively. In this study, a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous quantification of HVA, VMA, OA, and HGA in urine. After sample preparation, which involved only the dilution procedure, samples were quantified by LC-MS/MS. Full-scan MS/MS mode enabled the urinary markers to be quantified with a high degree of specificity and sensitivity. Rather than using a separate enzymatic method to normalize the concentration of creatinine in urine, we quantified the level of creatinine in urine in one LC-MS run. The limits of detection were 10 μg/l for HGA, 25 μg/l for HVA/VMA, and 50 μg/l for OA with a single-to-noise ratio of 3; the limits of quantification were 50 μg/l for HVA and HGA, 100 μg/l for VMA, and 250 μg/l for OA. The linear dynamic range for quantification of the analytes covered 2 to 3 orders of magnitude, depending on the analyte. The relative standard deviation of the developed LC-MS/MS method was less than 4% for the intra-day validation and 10% for the inter-day validation. The results show that our LC-MS/MS technique is a highly sensitive and rapid method for screening for biomarkers that are diagnostic of three metabolic diseases. Copyright © 2012 Elsevier B.V. All rights reserved.

Dried blood spot testing for seven steroids using liquid chromatography-tandem mass spectrometry with reference interval determination in the Korean population.

PubMed

Kim, Borahm; Lee, Mi Na; Park, Hyung Doo; Kim, Jong Won; Chang, Yun Sil; Park, Won Soon; Lee, Soo Youn

2015-11-01

Conventional screening for congenital adrenal hyperplasia (CAH) using immunoassays generates a large number of false-positive results. A more specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been introduced to minimize unnecessary follow-ups. However, because of limited data on its use in the Korean population, LC-MS/MS has not yet been incorporated into newborn screening programs in this region. The present study aims to develop and validate an LC-MS/MS method for the simultaneous determination of seven steroids in dried blood spots (DBS) for CAH screening, and to define age-specific reference intervals in the Korean population. We developed and validated an LC-MS/MS method to determine the reference intervals of cortisol, 17-hydroxyprogesterone, 11-deoxycortisol, 21-deoxycortisol, androstenedione, corticosterone, and 11-deoxycorticosterone simultaneously in 453 DBS samples. The samples were from Korean subjects stratified by age group (78 full-term neonates, 76 premature neonates, 89 children, and 100 adults). The accuracy, precision, matrix effects, and extraction recovery were satisfactory for all the steroids at three concentrations; values of intra- and inter-day precision coefficients of variance, bias, and recovery were 0.7-7.7%, -1.5-9.8%, and 49.3-97.5%, respectively. The linearity range was 1-100 ng/mL for cortisol and 0.5-50 ng/mL for other steroids (R²>0.99). The reference intervals were in agreement with the previous reports. This LC-MS/MS method and the reference intervals validated in the Korean population can be successfully applied to analyze seven steroids in DBS for the diagnosis of CAH.

METHOD 544. DETERMINATION OF MICROCYSTINS AND ...

EPA Pesticide Factsheets

Method 544 is an accurate and precise analytical method to determine six microcystins (including MC-LR) and nodularin in drinking water using solid phase extraction and liquid chromatography tandem mass spectrometry (SPE-LC/MS/MS). The advantage of this SPE-LC/MS/MS is its sensitivity and ability to speciate the microcystins. This method development task establishes sample preservation techniques, sample concentration and analytical procedures, aqueous and extract holding time criteria and quality control procedures. Draft Method 544 undergone a multi-laboratory verification to ensure other laboratories can implement the method and achieve the quality control measures specified in the method. It is anticipated that Method 544 may be used in UCMR 4 to collect nationwide occurrence data for selected microcystins in drinking water. The purpose of this research project is to develop an accurate and precise analytical method to concentrate and determine selected MCs and nodularin in drinking water.

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.

A simple, rapid atmospheric pressure chemical ionization liquid chromatography tandem mass spectrometry method for the determination of 25-hydroxyvitamin D2 and D3.

PubMed

Garg, Uttam; Munar, Ada; Frazee, Clinton; Scott, David

2012-09-01

Vitamin D plays a vital role not only in bone health but also in pathophysiology of many other body functions. In recent years, there has been significant increase in testing of 25-hydroxyvitamin D (25-OH vitamin D), a marker of vitamin D deficiency. The most commonly used methods for the measurement of 25-OH vitamin D are immunoassays and liquid chromatography tandem mass spectrometry (LC-MS-MS). Since immunoassays suffer from inaccuracies and interferences, LC-MS-MS is a preferred method. In LC-MS-MS methods, 25-OH vitamin D is extracted from serum or plasma by solid-phase or liquid-phase extraction. Because these extraction methods are time consuming, we developed an easy method that uses simple protein precipitation followed by injection of the supernatant to LC-MS-MS. Several mass-to-charge (m/z) ratio transitions, including commonly used transitions based on water loss, were evaluated and several tube types were tested. The optimal transitions for 25-OH vitamin D2 and D3 were 395.5 > 269.5 and 383.4 > 257.3, respectively. The reportable range of the method was 1-100 ng/mL, and repeatability (within-run) and within-laboratory imprecision were <4% and <6%, respectively. The method agreed well with the solid-phase extraction methods. © 2012 Wiley Periodicals, Inc.

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.

Detection of levamisole exposure in cocaine users by liquid chromatography-tandem mass spectrometry.

PubMed

Lynch, Kara L; Dominy, Stephen S; Graf, Jonathan; Kral, Alexander H

2011-04-01

Levamisole, a veterinary antihelminthic, was recently recognized as an adulterant in cocaine and is known to cause severe adverse reactions in some cocaine users. Because of the health concerns involving levamisole-adulterated cocaine, we developed a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the detection of levamisole in urine. This method was used to determine the prevalence of levamisole in cocaine-positive patient samples. All cocaine-positive urine samples that were sent to the San Francisco General Hospital Clinical Laboratory were tested for levamisole for one month. For LC, an Agilent 1200 series was used with a C(18) column and a gradient of mobile phase A (0.05% formic acid) and B (acetonitrile/methanol). Detection was carried out with an Applied Biosystems QTRAP(®) LC-MS-MS. The levamisole LC-MS-MS method was linear over the range of 5-2500 ng/mL (r > 0.996). Interassay and intraassay CVs were < 6%. The lower limit of detection for levamisole was 0.5 ng/mL. Out of 949 total urine drug screens, 20% were positive for benzoylecgonine, and of those, 88% were positive for levamisole. The high prevalence of levamisole-adulterated cocaine and potential toxicity in cocaine users is a serious public health concern. These findings validate the utility of an LC-MS-MS method for the detection of levamisole.

Dopant-assisted atmospheric pressure photoionization of patulin in apple juice and apple-based food with liquid chromatography-tandem mass spectrometry.

PubMed

Zhang, Kai; Wong, Jon W; Mai, Huy; Trucksess, Mary W

2014-05-07

A dopant-assisted atmospheric pressure photoionization (APPI) with liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed to determine patulin in apple juice and apple-based food. Different dopants, dopant flow rates, and LC separation conditions were evaluated. Using toluene as the dopant, the LC-APPI-MS/MS method achieved a linear calibration from 12.5 to 2000 μg/L (r(2) > 0.99). Matrix-dependent limits of quantitation (LOQs) were from 8 μg/L (solvent) to 12 μg/L (apple juice). [(13)C]-Patulin-fortified apple juice samples were directly analyzed by the LC-APPI-MS/MS method. Other apple-based food was fortified with [(13)C]-patulin, diluted using water (1% formic acid), centrifuged, and filtered, followed by LC-APPI-MS/MS analysis. In clear apple juice, unfiltered apple cider, applesauce, and apple-based baby food, average recoveries were 101 ± 6% (50 μg/kg), 103 ± 5% (250 μg/kg), and 102 ± 5% (1000 μg/kg) (av ± SD, n = 16). Using the suggested method, patulin was detected in 3 of 30 collected market samples with concentrations ranging from

[Simultaneous determination of pesticide residues in agricultural products by LC-MS/MS].

PubMed

Watanabe, Minae; Ueno, Eiji; Inoue, Tomomi; Ohno, Haruka; Ikai, Yoshitomo; Morishita, Toshio; Oshima, Harumi; Hayashi, Rumiko

2013-01-01

A method for the simultaneous determination of multiple pesticide residues in agricultural products was developed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The sample was extracted with acetonitrile. Co-extractives were removed by GPC/graphitized carbon column SPE, and silica gel/PSA cartridge column SPE. Pesticides in the test solution were determined by LC-MS/MS using scheduled MRM. Recoveries of 124 pesticides from spinach, brown rice, soybean, orange and tomato were tested at the level of 0.1 µg/g, and those of 121 pesticides ranged from 70 to 120% (RSD≤15%). Pesticide residues in 239 agricultural products were investigated by this method, and residues of 49 pesticides were detected in 98 agricultural products.

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

Simultaneous Quantification of Apolipoprotein A-I and Apolipoprotein B by Liquid-Chromatography–Multiple-Reaction–Monitoring Mass Spectrometry

PubMed Central

Agger, Sean A.; Marney, Luke C.; Hoofnagle, Andrew N.

2011-01-01

BACKGROUND If liquid-chromatography–multiple-reaction–monitoring mass spectrometry (LC-MRM/MS) could be used in the large-scale preclinical verification of putative biomarkers, it would obviate the need for the development of expensive immunoassays. In addition, the translation of novel biomarkers to clinical use would be accelerated if the assays used in preclinical studies were the same as those used in the clinical laboratory. To validate this approach, we developed a multiplexed assay for the quantification of 2 clinically well-known biomarkers in human plasma, apolipoprotein A-I and apolipoprotein B (apoA-I and apoB). METHODS We used PeptideAtlas to identify candidate peptides. Human samples were denatured with urea or trifluoroethanol, reduced and alkylated, and digested with trypsin. We compared reversed-phase chromatographic separation of peptides with normal flow and microflow, and we normalized endogenous peptide peak areas to internal standard peptides. We evaluated different methods of calibration and compared the final method with a nephelometric immunoassay. RESULTS We developed a final method using trifluoroethanol denaturation, 21-h digestion, normal flow chromatography-electrospray ionization, and calibration with a single normal human plasma sample. For samples injected in duplicate, the method had intraassay CVs <6% and interassay CVs <12% for both proteins, and compared well with immunoassay (n = 47; Deming regression, LC-MRM/MS = 1.17 × immunoassay – 36.6; Sx|y = 10.3 for apoA-I and LC-MRM/MS = 1.21 × immunoassay + 7.0; Sx|y = 7.9 for apoB). CONCLUSIONS Multiplexed quantification of proteins in human plasma/serum by LC-MRM/MS is possible and compares well with clinically useful immunoassays. The potential application of single-point calibration to large clinical studies could simplify efforts to reduce day-to-day digestion variability. PMID:20923952

Comparison of various liquid chromatographic methods involving UV and atmospheric pressure chemical ionization mass spectrometric detection for the efficient trace analysis of phenylurea herbicides in various types of water samples.

PubMed

van der Heeft, E; Dijkman, E; Baumann, R A; Hogendoorn, E A

2000-05-19

The performance of mass spectrometric (MS) detection and UV detection in combination with reversed-phase liquid chromatography without and with the use of coupled column RPLC (LC-LC) has been compared for the trace analysis of phenylurea herbicides in environmental waters. The selected samples of this comparative study originated from an inter-laboratory study. For both detection modes, a 50 mm x 4.6 mm I.D. column and a 100 mm x 4.6 mm I.D. column packed with 3 microm C18 were used as the first (C-1) and second (C-2) column, respectively. Atmospheric pressure chemical ionization mass spectrometry was performed on a magnetic sector instrument. The LC-LC-MS analysis was carried out on-line by means of direct large volume (11.7 ml) injection (LVI). The performance of both on-line (LVI, 4 ml of sample) and off-line LC-LC-UV (244 nm) analysis was investigated. The latter procedure consisted of a solid-phase extraction (SPE) of 250 ml of water sample on a 500 mg C18 cartridge. The comparative study showed that LC-LC-MS is more selective then LC-LC-UV and, in most cases, more sensitive. The LVI-LC-LC-MS approach combines direct quantification and confirmation of most of the analytes down to a level of 0.01 microg/l in water samples in less then 30 min. As regards LC-LC-UV, the off-line method appeared to be a more viable approach in comparison with the on-line procedure. This method allows the screening of phenylurea's in various types of water samples down to a level of at least 0.05 microg/l. On-line analysis with LVI provided marginal sensitivity (limits of detection of about 0.1 microg/l) and selectivity was sometimes less in case of surface water samples. Both the on-line LVI-LC-LC-MS method and the LC-LC-UV method using off-line SPE were validated by analysing a series of real-life reference samples. These samples were part of an inter-laboratory test and contained residues of herbicides ranging from 0.02 to 0.8 microg/l. Beside good correlation between the methods the data agreed very well with the true values of the samples.

A harmonized immunoassay with liquid chromatography-mass spectrometry analysis in egg allergen determination.

PubMed

Nimata, Masaomi; Okada, Hideki; Kurihara, Kei; Sugimoto, Tsukasa; Honjoh, Tsutomu; Kuroda, Kazuhiko; Yano, Takeo; Tachibana, Hirofumi; Shoji, Masahiro

2018-01-01

Food allergy is a serious health issue worldwide. Implementing allergen labeling regulations is extremely challenging for regulators, food manufacturers, and analytical kit manufacturers. Here we have developed an "amino acid sequence immunoassay" approach to ELISA. The new ELISA comprises of a monoclonal antibody generated via an analyte specific peptide antigen and sodium lauryl sulfate/sulfite solution. This combination enables the antibody to access the epitope site in unfolded analyte protein. The newly developed ELISA recovered 87.1%-106.4% ovalbumin from ovalbumin-incurred model processed foods, thereby demonstrating its applicability as practical egg allergen determination. Furthermore, the comparison of LC-MS/MS and the new ELISA, which targets the amino acid sequence conforming to the LC-MS/MS detection peptide, showed a good agreement. Consequently the harmonization of two methods was demonstrated. The complementary use of the new ELISA and LC-MS analysis can offer a wide range of practical benefits in terms of easiness, cost, accuracy, and efficiency in food allergen analysis. In addition, the new assay is attractive in respect to its easy antigen preparation and predetermined specificity. Graphical abstract The ELISA composing of the monoclonal antibody targeting the amino acid sequence conformed to LC-MS detection peptide, and the protein conformation unfolding reagent was developed. In ovalbumin determination, the developed ELISA showed a good agreement with LC-MS analysis. Consequently the harmonization of immunoassay with LC-MS analysis by using common target amino acid sequence was demonstrated.

Development and Bioanalytical Validation of a Liquid Chromatographic-Tandem Mass Spectrometric (LC-MS/MS) Method for the Quantification of the CCR5 Antagonist Maraviroc in Human Plasma

PubMed Central

Emory, Joshua F.; Seserko, Lauren A.; Marzinke, Mark A.

2014-01-01

Background Maraviroc is a CCR5 antagonist that has been utilized as a viral entry inhibitor in the management of HIV-1. Current clinical trials are pursuing maraviroc drug efficacy in both oral and topical formulations. Therefore, in order to fully understand drug pharmacokinetics, a sensitive method is required to quantify plasma drug concentrations. Methods Maraviroc-spiked plasma was combined with acetonitrile containing an isotopically-labeled internal standard, and following protein precipitation, samples were evaporated to dryness and reconstituted for liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. Chromatographic separation was achieved on a Waters BEH C8, 50 × 2.1 mm UPLC column, with a 1.7 μm particle size and the eluent was analyzed using an API 4000 mass analyzer in selected reaction monitoring mode. The method was validated as per FDA Bioanalytical Method Validation guidelines. Results The analytical measuring range of the LC-MS/MS method is 0.5-1000 ng/ml. Calibration curves were generated using weighted 1/x2 quadratic regression. Inter-and intra-assay precision was ≤ 5.38% and ≤ 5.98%, respectively; inter-and intra-assay accuracy (%DEV) was ≤ 10.2% and ≤ 8.44%, respectively. Additional studies illustrated similar matrix effects between maraviroc and its internal standard, and that maraviroc is stable under a variety of conditions. Method comparison studies with a reference LC-MS/MS method show a slope of 0.948 with a Spearman coefficient of 0.98. Conclusions Based on the validation metrics, we have generated a sensitive and automated LC-MS/MS method for maraviroc quantification in human plasma. PMID:24561264

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.

Principles and clinical applications of liquid chromatography - tandem mass spectrometry for the determination of adrenal and gonadal steroid hormones.

PubMed

Kulle, A E; Welzel, M; Holterhus, P-M; Riepe, F G

2011-10-01

Liquid-chromatography - tandem mass spectrometry (LC-MS/MS) is becoming the method of choice for clinical steroid analysis. In most instances, it has the advantage of higher sensitivity, better reproducibility and greater specificity than commercial immunoassay techniques. The method requires only minimal sample preparation and a small sample volume. Furthermore, it has the potential to analyze multiple steroids simultaneously. Modern instruments guarantee high throughput, allowing an affordable price for the individual assay. All this makes LC-MS/MS an attractive method for use in a clinical setting. Reliable reference ranges for the detected analytes are the pre-requisite for their clinical use. If these are available, LC-MS/MS can find application in congenital disorders of steroid metabolism, such as congenital adrenal hyperplasia, disorders of sex development and disorders of salt homeostasis, as well as in acquired disorders of steroid metabolism, such as primary aldosteronism, Cushing's disease, Addison's disease, and hyperandrogenemia, as well as in psychiatric disease states such as depression or anxiety disorders. The principles of LC-MS/MS for steroid measurement, the pros and cons of LC-MS/MS compared with conventional immunoassays and the possible applications in clinical routine, with a special focus on pediatric endocrinology needs, are discussed here.

A Non-Biological Method for Screening Active Components against Influenza Virus from Traditional Chinese Medicine by Coupling a LC Column with Oseltamivir Molecularly Imprinted Polymers

PubMed Central

Yang, Ya-Jun; Li, Jian-Yong; Liu, Xi-Wang; Zhang, Ji-Yu; Liu, Yu-Rong; Li, Bing

2013-01-01

To develop a non-biological method for screening active components against influenza virus from traditional Chinese medicine (TCM) extraction, a liquid chromatography (LC) column prepared with oseltamivir molecularly imprinted polymer (OSMIP) was employed with LC-mass spectrometry (LC-MS). From chloroform extracts of compound TCM liquid preparation, we observed an affinitive component m/z 249, which was identified to be matrine following analysis of phytochemical literatures, OSMIP-LC column on-line of control compounds and MS/MS off-line. The results showed that matrine had similar bioactivities with OS against avian influenza virus H9N2 in vitro for both alleviating cytopathic effect and hemagglutination inhibition and that the stereostructures of these two compounds are similar while their two-dimensional structures were different. In addition, our results suggested that the bioactivities of those affinitive compounds were correlated with their chromatographic behaviors, in which less difference of the chromatographic behaviors might have more similar bioactivities. This indicates that matrine is a potential candidate drug to prevent or cure influenza for human or animal. In conclusion, the present study showed that molecularly imprinted polymers can be used as a non-biological method for screening active components against influenza virus from TCM. PMID:24386385

On-line solid-phase microextraction of triclosan, bisphenol A, chlorophenols, and selected pharmaceuticals in environmental water samples by high-performance liquid chromatography-ultraviolet detection.

PubMed

Kim, Dalho; Han, Jungho; Choi, Yongwook

2013-01-01

A method using on-line solid-phase microextraction (SPME) on a carbowax-templated fiber followed by liquid chromatography (LC) with ultraviolet (UV) detection was developed for the determination of triclosan in environmental water samples. Along with triclosan, other selected phenolic compounds, bisphenol A, and acidic pharmaceuticals were studied. Previous SPME/LC or stir-bar sorptive extraction/LC-UV for polar analytes showed lack of sensitivity. In this study, the calculated octanol-water distribution coefficient (log D) values of the target analytes at different pH values were used to estimate polarity of the analytes. The lack of sensitivity observed in earlier studies is identified as a lack of desorption by strong polar-polar interactions between analyte and solid-phase. Calculated log D values were useful to understand or predict the interaction between analyte and solid phase. Under the optimized conditions, the method detection limit of selected analytes by using on-line SPME-LC-UV method ranged from 5 to 33 ng L(-1), except for very polar 3-chlorophenol and 2,4-dichlorophenol which was obscured in wastewater samples by an interfering substance. This level of detection represented a remarkable improvement over the conventional existing methods. The on-line SPME-LC-UV method, which did not require derivatization of analytes, was applied to the determination of TCS including phenolic compounds and acidic pharmaceuticals in tap water and river water and municipal wastewater samples.

Simultaneous determination of bifonazole and tinctures of calendula flower in pharmaceutical creams by reversed-phase liquid chromatography.

PubMed

Ferreyra, Carola F; Ortiz, Cristina S

2005-01-01

The aim of this research was to develop and validate a sensitive, rapid, easy, and precise reversed-phase liquid chromatography (LC) method for stability studies of bifonazole (I) formulated with tinctures of calendula flower (II). The method was especially developed for the analysis and quantitative determination of I and II in pure and combined forms in cream pharmaceutical formulations without using gradient elution and at room temperature. The influence on the stability of compound I of temperature, artificial radiation, and drug II used for the new pharmaceutical design was evaluated. The LC separation was carried out using a Supelcosil LC-18 column (25 cm x 4.6 mm id, 5 microm particle size); the mobile phase was composed of methanol-0.1 M ammonium acetate buffer (85 + 15, v/v) pumped isocratically at a flow rate of 1 mL/min; and ultraviolet detection was at 254 nm. The analysis time was less than 10 min. Calibration graphs were found to be linear in the 0.125-0.375 mg/mL (rI = 0.9991) and 0.639-1.916 mg/mL (rII = 0.9995) ranges for I and II, respectively. The linearity, precision, recovery, and limits of detection and quantification were satisfactory for I and II. The results obtained suggested that the developed LC method is selective and specific for the analysis of I and II in pharmaceutical products, and that it can be applied to stability studies.

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.

Rapid and precise measurement of serum branched-chain and aromatic amino acids by isotope dilution liquid chromatography tandem mass spectrometry.

PubMed

Yang, Ruiyue; Dong, Jun; Guo, Hanbang; Li, Hongxia; Wang, Shu; Zhao, Haijian; Zhou, Weiyan; Yu, Songlin; Wang, Mo; Chen, Wenxiang

2013-01-01

Serum branched-chain and aromatic amino acids (BCAAs and AAAs) have emerged as predictors for the future development of diabetes and may aid in diabetes risk assessment. However, the current methods for the analysis of such amino acids in biological samples are time consuming. An isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) method for serum BCAAs and AAAs was developed. The serum was mixed with isotope-labeled BCAA and AAA internal standards and the amino acids were extracted with acetonitrile, followed by analysis using LC/MS/MS. The LC separation was performed on a reversed-phase C18 column, and the MS/MS detection was performed via the positive electronic spray ionization in multiple reaction monitoring mode. Specific analysis of the amino acids was achieved within 2 min. Intra-run and total CVs for the amino acids were less than 2% and 4%, respectively, and the analytical recoveries ranged from 99.6 to 103.6%. A rapid and precise method for the measurement of serum BCAAs and AAAs was developed and may serve as a quick tool for screening serum BCAAs and AAAs in studies assessing diabetes risk.

Development of LC-MS determination method and back-propagation ANN pharmacokinetic model of corynoxeine in rat.

PubMed

Ma, Jianshe; Cai, Jinzhang; Lin, Guanyang; Chen, Huilin; Wang, Xianqin; Wang, Xianchuan; Hu, Lufeng

2014-05-15

Corynoxeine(CX), isolated from the extract of Uncaria rhynchophylla, is a useful and prospective compound in the prevention and treatment for vascular diseases. A simple and selective liquid chromatography mass spectrometry (LC-MS) method was developed to determine the concentration of CX in rat plasma. The chromatographic separation was achieved on a Zorbax SB-C18 (2.1 mm × 150 mm, 5 μm) column with acetonitrile-0.1% formic acid in water as mobile phase. Selective ion monitoring (SIM) mode was used for quantification using target ions m/z 383 for CX and m/z 237 for the carbamazepine (IS). After the LC-MS method was validated, it was applied to a back-propagation artificial neural network (BP-ANN) pharmacokinetic model study of CX in rats. The results showed that after intravenous administration of CX, it was mainly distributed in blood and eliminated quickly, t1/2 was less than 1h. The predicted concentrations generated by BP-ANN model had a high correlation coefficient (R>0.99) with experimental values. The developed BP-ANN pharmacokinetic model can be used to predict the concentration of CX in rats. Copyright © 2014 Elsevier B.V. All rights reserved.

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...

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.

Evaluation of analytical techniques to determine AQUI-S(R) 20E (eugenol) concentrations in water

USGS Publications Warehouse

Meinertz, Jeffery R.; Hess, Karina R.

2013-01-01

There is a critical need in U.S. public aquaculture and fishery management programs for an immediate-release sedative, i.e. a compound that can be safely and effectively used to sedate fish and subsequently, allow for their immediate release. AQUI-S® 20E (10% active ingredient, eugenol; any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government) is being pursued for U.S. approval as an immediate-release sedative. As part of the approval process, data describing animal safety and efficacy are needed. Essential to conducting studies that generate those data, is a method to accurately and precisely determine AQUI-S® 20E concentrations in exposure baths. Spectrophotometric and solid phase extraction (SPE)–high pressure liquid chromatography (LC) methods were developed and evaluated as methods to determine AQUI-S® 20E (eugenol) concentrations in water, methods that could be applied to any situation where eugenol was being evaluated as a fish sedative. The spectrophotometric method was accurate and precise (accuracy, > 87%; precision, < 0.70 %CV) when determining eugenol concentrations in solutions of 50 to 1000 mg/L AQUI-S® 20E made with LC grade water and water with varying pH and hardness. The spectrophotometric method's accuracy was negatively affected when analyzing water containing fish feed. The SPE–LC method was also accurate and precise (accuracy > 86%; precision < 8.9 %CV) when determining eugenol concentrations in solutions of 50 to 1000 mg/L AQUI-S® 20E made with LC grade water and water with varying pH and hardness. The SPE–LC method was influenced to a lesser degree by the presence of fish feed indicating greater specificity for eugenol.

Impact of glucuronide interferences on therapeutic drug monitoring of posaconazole by tandem mass spectrometry.

PubMed

Krüger, Ralf; Vogeser, Michael; Burghardt, Stephan; Vogelsberger, Rita; Lackner, Karl J

2010-12-01

Posaconazole is a novel antifungal drug for oral application intended especially for therapy of invasive mycoses. Due to variable gastrointestinal absorption, adverse side effects, and suspected drug-drug interactions, therapeutic drug monitoring (TDM) of posaconazole is recommended. A fast ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of posaconazole with a run-time <3 min was developed and compared to a LC-MS/MS method and HPLC method with fluorescence detection. During evaluation of UPLC-MS/MS, two earlier eluting peaks were observed in the MRM trace of posaconazole. This was only seen in patient samples, but not in spiked calibrator samples. Comparison with LC-MS/MS disclosed a significant bias with higher concentrations measured by LC-MS/MS, while UPLC-MS/MS showed excellent agreement with the commercially available HPLC method. In the LC-MS/MS procedure, comparably wide and left side shifted peaks were noticed. This could be ascribed to in-source fragmentation of conjugate metabolites during electrospray ionisation. Precursor and product ion scans confirmed the assumption that the additional compounds are posaconazole glucuronides. Reducing the cone voltage led to disappearance of the glucuronide peaks. Slight modification of the LC-MS/MS method enabled separation of the main interference, leading to significantly reduced deviation. These results highlight the necessity to reliably eliminate interference from labile drug metabolites for correct TDM results, either by sufficient separation or selective MS conditions. The presented UPLC-MS/MS method provides a reliable and fast assay for TDM of posaconazole.

A new LC-MS/MS bioanalytical method for atenolol in human plasma and milk.

PubMed

Phyo Lwin, Ei Mon; Gerber, Cobus; Song, Yunmei; Leggett, Catherine; Ritchie, Usha; Turner, Sean; Garg, Sanjay

2017-04-01

A new sensitive LC-MS/MS method for the quantification of atenolol in human plasma and milk has been developed for clinical lactation studies. Atenolol and the internal standard, phenazone, were extracted from biological matrices by protein precipitation. A Phenomenex ® C-18 column and gradient chromatographic conditions were used for separation of the analyte, followed by detection with MS. Stability of samples was confirmed for atenolol in human plasma and milk for up to 3 months. Linearity range of 1-800 ng/ml (r 2 = 0.9995), the precision within 15% CV and the recovery of the analyte (80-100% range) were achieved. A new validated analytical method for atenolol in plasma and milk was developed.

Determination of coumarin, vanillin, and ethyl vanillin in vanilla extract products: liquid chromatography mass spectrometry method development and validation studies.

PubMed

de Jager, Lowri S; Perfetti, Gracia A; Diachenko, Gregory W

2007-03-23

A LC-MS method was developed for the determination of coumarin, vanillin, and ethyl vanillin in vanilla products. Samples were analyzed using LC-electrospray ionization (ESI)-MS in the positive ionization mode. Limits of detection for the method ranged from 0.051 to 0.073 microg mL(-1). Using the optimized method, 24 vanilla products were analyzed. All samples tested negative for coumarin. Concentrations ranged from 0.38 to 8.59 mg mL(-1) (x =3.73) for vanillin and 0.33 to 2.27 mg mL(-1) (x =1.03) for ethyl vanillin. The measured concentrations are compared to values calculated using UV monitoring and to results reported in a similar survey in 1988. Analytical results, method precision, and accuracy data are presented.

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.

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.

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.

Radical scavenging activity and LC-MS metabolic profiling of petals, stamens, and flowers of Crocus sativus L.

PubMed

Montoro, Paola; Maldini, Mariateresa; Luciani, Leonilda; Tuberoso, Carlo I G; Congiu, Francesca; Pizza, Cosimo

2012-08-01

Radical scavenging activities of Crocus sativus petals, stamens and entire flowers, which are waste products in the production of the spice saffron, by employing ABTS radical scavenging method, were determined. At the same time, the metabolic profiles of different extract (obtained by petals, stamens and flowers) were obtained by LC-ESI-IT MS (liquid chromatography coupled with electrospray mass spectrometry equipped with Ion Trap analyser). LC-ESI-MS is a techniques largely used nowadays for qualitative fingerprint of herbal extracts and particularly for phenolic compounds. To compare the different extracts under an analytical point of view a specific method for qualitative LC-MS analysis was developed. The high variety of glycosylated flavonoids found in the metabolic profiles could give value to C. sativus petals, stamens and entire flowers. Waste products obtained during saffron production, could represent an interesting source of phenolic compounds, with respect to the high variety of compounds and their free radical scavenging activity. © 2012 Institute of Food Technologists®

Round robin test on quantification of amyloid-β 1-42 in cerebrospinal fluid by mass spectrometry.

PubMed

Pannee, Josef; Gobom, Johan; Shaw, Leslie M; Korecka, Magdalena; Chambers, Erin E; Lame, Mary; Jenkins, Rand; Mylott, William; Carrillo, Maria C; Zegers, Ingrid; Zetterberg, Henrik; Blennow, Kaj; Portelius, Erik

2016-01-01

Cerebrospinal fluid (CSF) amyloid-β 1-42 (Aβ42) is an important biomarker for Alzheimer's disease, both in diagnostics and to monitor disease-modifying therapies. However, there is a great need for standardization of methods used for quantification. To overcome problems associated with immunoassays, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a critical orthogonal alternative. We compared results for CSF Aβ42 quantification in a round robin study performed in four laboratories using similar sample preparation methods and LC-MS instrumentation. The LC-MS results showed excellent correlation between laboratories (r(2) >0.98), high analytical precision, and good correlation with enzyme-linked immunosorbent assay (r(2) >0.85). The use of a common reference sample further decreased interlaboratory variation. Our results indicate that LC-MS is suitable for absolute quantification of Aβ42 in CSF and highlight the importance of developing a certified reference material. Copyright © 2016 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.

A new approach based on off-line coupling of high-performance liquid chromatography with gas chromatography-mass spectrometry to determine acrylamide in coffee brew.

PubMed

Blanch, Gracia Patricia; Morales, Francisco José; Moreno, Fernando de la Peña; del Castillo, María Luisa Ruiz

2013-01-01

A new method based on off-line coupling of LC with GC in replacement of conventional sample preparation techniques is proposed to analyze acrylamide in coffee brews. The method involves the preseparation of the sample by LC, the collection of the selected fraction, its concentration under nitrogen, and subsequent analysis by GC coupled with MS. The composition of the LC mobile phase and the flow rate were studied to select those conditions that allowed separation of acrylamide without coeluting compounds. Under the conditions selected recoveries close to 100% were achieved while LODs and LOQs equal to 5 and 10 μg/L for acrylamide in brewed coffee were obtained. The method developed enabled the reliable detection of acrylamide in spiked coffee beverage samples without further clean-up steps or sample manipulation. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of Isomeric Glycans by Reversed Phase Liquid Chromatography-Electronic Excitation Dissociation Tandem Mass Spectrometry

NASA Astrophysics Data System (ADS)

Tang, Yang; Wei, Juan; Costello, Catherine E.; Lin, Cheng

2018-04-01

The occurrence of numerous structural isomers in glycans from biological sources presents a severe challenge for structural glycomics. The subtle differences among isomeric structures demand analytical methods that can provide structural details while working efficiently with on-line glycan separation methods. Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful tool for mixture analysis, the commonly utilized collision-induced dissociation (CID) method often does not generate a sufficient number of fragments at the MS2 level for comprehensive structural characterization. Here, we studied the electronic excitation dissociation (EED) behaviors of metal-adducted, permethylated glycans, and identified key spectral features that could facilitate both topology and linkage determinations. We developed an EED-based, nanoscale, reversed phase (RP)LC-MS/MS platform, and demonstrated its ability to achieve complete structural elucidation of up to five structural isomers in a single LC-MS/MS analysis. [Figure not available: see fulltext.

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.

Quantification of residual EDU (N-ethyl-N'-(dimethylaminopropyl) carbodiimide (EDC) hydrolyzed urea derivative) and other residual by LC-MS/MS.

PubMed

Lei, Q Paula; Lamb, David H; Shannon, Anthony G; Cai, Xinxing; Heller, Ronald K; Huang, Michael; Zablackis, Earl; Ryall, Robert; Cash, Patricia

2004-12-25

An LC-MS/MS method for determination of the break down product of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) urea derivative, EDU, has been developed and validated for monitoring the residual coupling reagents. Results indicate that the method exhibits suitable specificity, sensitivity, precision, linearity and accuracy for quantification of residual EDU in the presence of meningococcal polysaccharide-diphtheria toxoid conjugate vaccine and other vaccine matrix compounds. The assay has been validated for a detection range of 10-100 ng/mL and then successfully transferred to quality control (QC) lab. This same method has also been applied to the determination of residual diaminohexane (DAH) in the presence of EDU. LC-MS/MS has proven to be useful as a quick and sensitive approach for simultaneous determination of multiple residual compounds in glycoconjugate vaccine samples.

Characterization of rhamnolipids by liquid chromatography/mass spectrometry after solid-phase extraction.

PubMed

Behrens, Beate; Engelen, Jeannine; Tiso, Till; Blank, Lars Mathias; Hayen, Heiko

2016-04-01

Rhamnolipids are surface-active agents with a broad application potential that are produced in complex mixtures by bacteria of the genus Pseudomonas. Analysis from fermentation broth is often characterized by laborious sample preparation and requires hyphenated analytical techniques like liquid chromatography coupled to mass spectrometry (LC-MS) to obtain detailed information about sample composition. In this study, an analytical procedure based on chromatographic method development and characterization of rhamnolipid sample material by LC-MS as well as a comparison of two sample preparation methods, i.e., liquid-liquid extraction and solid-phase extraction, is presented. Efficient separation was achieved under reversed-phase conditions using a mixed propylphenyl and octadecylsilyl-modified silica gel stationary phase. LC-MS/MS analysis of a supernatant from Pseudomonas putida strain KT2440 pVLT33_rhlABC grown on glucose as sole carbon source and purified by solid-phase extraction revealed a total of 20 congeners of di-rhamnolipids, mono-rhamnolipids, and their biosynthetic precursors 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) with different carbon chain lengths from C8 to C14, including three rhamnolipids with uncommon C9 and C11 fatty acid residues. LC-MS and the orcinol assay were used to evaluate the developed solid-phase extraction method in comparison with the established liquid-liquid extraction. Solid-phase extraction exhibited higher yields and reproducibility as well as lower experimental effort.

Quality evaluation of LC-MS/MS-based E. coli H antigen typing (MS-H) through label-free quantitative data analysis in a clinical sample setup.

PubMed

Cheng, Keding; Sloan, Angela; McCorrister, Stuart; Peterson, Lorea; Chui, Huixia; Drebot, Mike; Nadon, Celine; Knox, J David; Wang, Gehua

2014-12-01

The need for rapid and accurate H typing is evident during Escherichia coli outbreak situations. This study explores the transition of MS-H, a method originally developed for rapid H antigen typing of E. coli using LC-MS/MS of flagella digest of reference strains and some clinical strains, to E. coli isolates in clinical scenario through quantitative analysis and method validation. Motile and nonmotile strains were examined in batches to simulate clinical sample scenario. Various LC-MS/MS batch run procedures and MS-H typing rules were compared and summarized through quantitative analysis of MS-H data output for a standard method development. Label-free quantitative data analysis of MS-H typing was proven very useful for examining the quality of MS-H result and the effects of some sample carryovers from motile E. coli isolates. Based on this, a refined procedure and protein identification rule specific for clinical MS-H typing was established and validated. With LC-MS/MS batch run procedure and database search parameter unique for E. coli MS-H typing, the standard procedure maintained high accuracy and specificity in clinical situations, and its potential to be used in a clinical setting was clearly established. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Antioxidant activity of leaf extracts from different Hibiscus sabdariffa accessions and simultaneous determination five major antioxidant compounds by LC-Q-TOF-MS.

PubMed

Wang, Jin; Cao, Xianshuang; Jiang, Hao; Qi, Yadong; Chin, Kit L; Yue, Yongde

2014-12-17

Hibiscus sabdariffa has gained attention for its antioxidant activity. There are many accessions of H. sabdariffa in the world. However, information on the quantification of antioxidant compounds in different accessions is rather limited. In this paper, a liquid chromatography/quadrupole-time-of-flight mass spectrometry (LC-Q-TOF-MS) method for simultaneous determination of five antioxidant compounds (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, and isoquercitrin) in H. sabdariffa leaves was developed. The method was validated for linearity, sensitivity, precision, repeatability and accuracy. The validated method has been successfully applied for determination of the five analytes in eight accessions of H. sabdariffa. The eight accessions of H. sabdariffa were evaluated for their antioxidant activities by DPPH free radical scavenging assay. The investigated accessions of H. sabdariffa were rich in rutin and exhibited strong antioxidant activity. The two accessions showing the highest antioxidant activities were from Cuba (No. 2) and Taiwan (No. 5). The results indicated that H. sabdariffa leaves could be considered as a potential antioxidant source for the food industry. The developed LC-Q-TOF-MS method is helpful for quality control of H. sabdariffa.

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 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.

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.

Enhancing analysis throughput, sensitivity and specificity in LC/ESI-MS/MS assay of plasma 25-hydroxyvitamin D3 by derivatization with triplex 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) isotopologues.

PubMed

Ogawa, Shoujiro; Kittaka, Hiroki; Nakata, Akiho; Komatsu, Kenji; Sugiura, Takahiro; Satoh, Mamoru; Nomura, Fumio; Higashi, Tatsuya

2017-03-20

The plasma/serum concentration of 25-hydroxyvitamin D 3 [25(OH)D 3 ] is a diagnostic index for vitamin D deficiency/insufficiency, which is associated with a wide range of diseases, such as rickets, cancer and diabetes. We have reported that the derivatization with 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) works well in the liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) assay of the serum/plasma 25(OH)D 3 for enhancing the sensitivity and the separation from a potent interfering metabolite, 3-epi-25-hydroxyvitamin D 3 [3-epi-25(OH)D 3 ]. However, enhancing the analysis throughput remains an issue in the LC/ESI-MS/MS assay of 25(OH)D 3 . The most obvious restriction of the LC/MS/MS throughput is the chromatographic run time. In this study, we developed an enhanced throughput method for the determination of the plasma 25(OH)D 3 by LC/ESI-MS/MS combined with the derivatization using the triplex ( 2 H 0 -, 2 H 3 - and 2 H 6 -) DAPTAD isotopologues. After separate derivatization with 1 of 3 different isotopologues, the 3 samples were combined and injected together into LC/ESI-MS/MS. Based on the mass differences between the isotopologues, the derivatized 25(OH)D 3 in the 3 different samples were quantified within a single run. The developed method tripled the hourly analysis throughput without sacrificing assay performance, i.e., ease of pretreatment of plasma sample (only deproteinization), limit of quantification (1.0ng/mL when a 5μL-plasma was used), precision (intra-assay RSD≤5.9% and inter-assay RSD≤5.5%), accuracy (98.7-102.2%), matrix effects, and capability of separating from an interfering metabolite, 3-epi-25(OH)D 3 . The multiplexing of samples by the isotopologue derivatization was applied to the analysis of plasma samples of healthy subjects and the developed method was proven to have a satisfactory applicability. Copyright © 2016 Elsevier B.V. All rights reserved.

Measuring geographical accessibility to palliative and end of life (PEoLC) related facilities: a comparative study in an area with well-developed specialist palliative care (SPC) provision.

PubMed

Pearson, Clare; Verne, Julia; Wells, Claudia; Polato, Giovanna M; Higginson, Irene J; Gao, Wei

2017-01-26

Geographical accessibility is important in accessing healthcare services. Measuring it has evolved alongside technological and data analysis advances. High correlations between different methods have been detected, but no comparisons exist in the context of palliative and end of life care (PEoLC) studies. To assess how geographical accessibility can affect PEoLC, selection of an appropriate method to capture it is crucial. We therefore aimed to compare methods of measuring geographical accessibility of decedents to PEoLC-related facilities in South London, an area with well-developed SPC provision. Individual-level death registration data in 2012 (n = 18,165), from the Office for National Statistics (ONS) were linked to area-level PEoLC-related facilities from various sources. Simple and more complex measures of geographical accessibility were calculated using the residential postcodes of the decedents and postcodes of the nearest hospital, care home and hospice. Distance measures (straight-line, travel network) and travel times along the road network were compared using geographic information system (GIS) mapping and correlation analysis (Spearman rho). Borough-level maps demonstrate similarities in geographical accessibility measures. Strong positive correlation exist between straight-line and travel distances to the nearest hospital (rho = 0.97), care home (rho = 0.94) and hospice (rho = 0.99). Travel times were also highly correlated with distance measures to the nearest hospital (rho range = 0.84-0.88), care home (rho = 0.88-0.95) and hospice (rho = 0.93-0.95). All correlations were significant at p < 0.001 level. Distance-based and travel-time measures of geographical accessibility to PEoLC-related facilities in South London are similar, suggesting the choice of measure can be based on the ease of calculation.

Development of a solid-phase extraction method for determination of pheophorbide a and pyropheophorbide a in health foods by liquid chromatography.

PubMed

Oshima, Harumi; Ueno, Eiji; Saito, Isao; Matsumoto, Hiroshi

2004-01-01

A simple solid-phase extraction (SPE) method was developed for the liquid chromatography (LC) determination of pheophorbide (Phor) a and pyropheophorbide (Pyro) a in health foods such as chlorella, spirulina, etc. The food sample was extracted with 85% (v/v) acetone. The extract was acidified with hydrochloric acid and loaded on a C18 cartridge. After washing with water, Phor a and Pyro a were eluted with the LC mobile phase. Phor a and Pyro a were separated by isocratic reversed-phase LC and quantitated by fluorescence detection. The recoveries for spiked samples of chlorella and the extract were 87.1-102.0%. Commercial health foods (chlorella, spirulina, aloe, kale, Jews mallow, and green tea leaves) were analyzed using the SPE method. The values found for Phor a and Pyro a ranged from 2 to 788 microg/g and from <1 to 24 microg/g, respectively. There was no significant difference between the SPE method and the official method in Japan (spectrophotometry after liquid-liquid extraction). The advantages of the SPE method are the short extraction times, lack of emulsions, and reduced consumption of organic solvents compared with the official method in Japan. The SPE method is considered to be useful for the screening of Phor a and Pyro a in health foods.

Quantitative Method for Simultaneous Analysis of Acetaminophen and 6 Metabolites.

PubMed

Lammers, Laureen A; Achterbergh, Roos; Pistorius, Marcel C M; Romijn, Johannes A; Mathôt, Ron A A

2017-04-01

Hepatotoxicity after ingestion of high-dose acetaminophen [N-acetyl-para-aminophenol (APAP)] is caused by the metabolites of the drug. To gain more insight into factors influencing susceptibility to APAP hepatotoxicity, quantification of APAP and metabolites is important. A few methods have been developed to simultaneously quantify APAP and its most important metabolites. However, these methods require a comprehensive sample preparation and long run times. The aim of this study was to develop and validate a simplified, but sensitive method for the simultaneous quantification of acetaminophen, the main metabolites acetaminophen glucuronide and acetaminophen sulfate, and 4 Cytochrome P450-mediated metabolites by using liquid chromatography with mass spectrometric (LC-MS) detection. The method was developed and validated for the human plasma, and it entailed a single method for sample preparation, enabling quick processing of the samples followed by an LC-MS method with a chromatographic run time of 9 minutes. The method was validated for selectivity, linearity, accuracy, imprecision, dilution integrity, recovery, process efficiency, ionization efficiency, and carryover effect. The method showed good selectivity without matrix interferences. For all analytes, the mean process efficiency was >86%, and the mean ionization efficiency was >94%. Furthermore, the accuracy was between 90.3% and 112% for all analytes, and the within- and between-run imprecision were <20% for the lower limit of quantification and <14.3% for the middle level and upper limit of quantification. The method presented here enables the simultaneous quantification of APAP and 6 of its metabolites. It is less time consuming than previously reported methods because it requires only a single and simple method for the sample preparation followed by an LC-MS method with a short run time. Therefore, this analytical method provides a useful method for both clinical and research purposes.

Evaluation of tamoxifen and metabolites by LC-MS/MS and HPLC methods.

PubMed

Heath, D D; Flat, S W; Wu, A H B; Pruitt, M A; Rock, C L

2014-01-01

Epidemiological and laboratory evidence suggests that quantification of serum or plasma levels of tamoxifen and its metabolites, 4-hydroxy-N-desmethyl-tamoxifen (endoxifen), Z-4-hydroxytamoxifen (4HT), N-desmethyl-tamoxifen (ND-tam), is a clinically useful tool in the assessment and monitoring of breast cancer status in patients taking adjuvant tamoxifen. A liquid chromatographic mass spectrometric method (LC-MS/MS) was used to measure the blood levels of tamoxifen and its metabolites. This fully automated analytical method is specific, accurate and sensitive. The LC-MS/MS automated technique has now become a widely accepted reference method. This study analysed a randomly selected batch of blood samples from participants enrolled in a breast cancer study to compare results from this reference method in 40 samples with those obtained from a recently developed high-performance liquid chromatography (HPLC) method with fluorescence detection. The mean (SD) concentrations for the LC-MS/MS method (endoxifen 12.6 [7.5] ng/mL, tamoxifen 105 [44] ng/mL, 4-HT 1.9 [1.0] ng/mL, ND-tam 181 [69] ng/mL) and the HPLC method (endoxifen 13.1 [7.8] ng/mL, tamoxifen 108 [55] ng/mL, 4-HT 1.8 [0.8] ng/mL, ND-tam 184 [81] ng/mL) did not show any significant differences. The results confirm that the HPLC method offers an accurate and comparable alternative for the quantification of tamoxifen and tamoxifen metabolites.

Automated Online Solid-Phase Derivatization for Sensitive Quantification of Endogenous S-Nitrosoglutathione and Rapid Capture of Other Low-Molecular-Mass S-Nitrosothiols.

PubMed

Wang, Xin; Garcia, Carlos T; Gong, Guanyu; Wishnok, John S; Tannenbaum, Steven R

2018-02-06

S-Nitrosothiols (RSNOs) constitute a circulating endogenous reservoir of nitric oxide and have important biological activities. In this study, an online coupling of solid-phase derivatization (SPD) with liquid chromatography-mass spectrometry (LC-MS) was developed and applied in the analysis of low-molecular-mass RSNOs. A derivatizing-reagent-modified polymer monolithic column was prepared and adapted for online SPD-LC-MS. Analytes from the LC autosampler flowed through the monolithic column for derivatization and then directly into the LC-MS for analysis. This integration of the online derivatization, LC separation, and MS detection facilitated system automation, allowing rapid, laborsaving, and sensitive detection of RSNOs. S-Nitrosoglutathione (GSNO) was quantified using this automated online method with good linearity (R 2 = 0.9994); the limit of detection was 0.015 nM. The online SPD-LC-MS method has been used to determine GSNO levels in mouse samples, 138 ± 13.2 nM of endogenous GSNO was detected in mouse plasma. Besides, the GSNO concentrations in liver (64.8 ± 11.3 pmol/mg protein), kidney (47.2 ± 6.1 pmol/mg protein), heart (8.9 ± 1.8 pmol/mg protein), muscle (1.9 ± 0.3 pmol/mg protein), hippocampus (5.3 ± 0.9 pmol/mg protein), striatum (6.7 ± 0.6 pmol/mg protein), cerebellum (31.4 ± 6.5 pmol/mg protein), and cortex (47.9 ± 4.6 pmol/mg protein) were also successfully quantified. When the derivatization was performed within 8 min, followed by LC-MS detection, samples could be rapidly analyzed compared with the offline manual method. Other low-molecular-mass RSNOs, such as S-nitrosocysteine and S-nitrosocysteinylglycine, were captured by rapid precursor-ion scanning, showing that the proposed method is a potentially powerful tool for capture, identification, and quantification of RSNOs in biological samples.

The Association Between Lung Carcinoma and Tuberculosis

PubMed Central

Cukic, Vesna

2017-01-01

Introduction: The association between lung tuberculosis and lung carcinoma is still controversial. Objective: to describe the characteristics of patients with associated lung tuberculosis (TB) and lung carcinoma (LC) in patients treated in Clinic for pulmonary diseases and TB “Podhrastovi”. Material and Methods: This is the retrospective study of patients with LC associated with TB treated in Clinic for pulmonary diseases and TB “Podhrastovi” in five-year period -from 2012 to 2016. We analyzed sex and age of patients, whether TB preceded LC or LC preceded TB, a time period between the developments of these two diseases, activity of TB, the histopathological type of LC, localization of LC in lungs (bronchial, peripheral, cavern) according to histopathological type. Results: In this period there were 2608 patients treated for LC. Among them there were 34 patients with diagnosed TB or 1.3%. All of them were smokers. No one had active TB. TB was the first diagnosis in all these patients. Each patient was previously treated for TB in hospital and had regular anti TB treatment. TB preceded LC in median time of 5 years (interquartile range 2 to 25 years). In 21 cases it was carcinoma of the drainage bronchus, in 11 cases it was peripheral lung carcinoma and 2 cases it was cavern carcinoma. Conlusion: patients with cured pulmonary tuberculosis represent a group at risk for developing lung carcinoma. Changes in the bronchial and alveolar mucosa which tuberculosis leaves behind in the lungs must be taken as a possible place of later malignant alteration. Patients with any form of pulmonary tuberculosis have to be controlled continuously. PMID:28974836

Seeding Experiment of Liquid Carbon Dioxide for Enhancing Winter-time Precipitation in Saga Prefecture,Northern Kyushu,Japan

NASA Astrophysics Data System (ADS)

Wakimizu, K.; Nishiyama, K.; Tomine, K.; Maki, T.; Suzuki, Y.; Morita, O.

2012-12-01

Many droughts (shortage of water) have broken out by extreme small amount rainfall in recent Japan. So far,in order to prevent these droughts,artificial rainfall methods with 'AgI' or 'dry ice' have been widely used in Japan. However,these methods have many problems,which a large amount of overcooling liquid in the cumulus cloud was not able to be converted into precipitation efficiently. So as to solve these problems,new artificial rainfall method using liquid carbon dioxide (LC) was proposed by Fukuta (1996). This new method consists of the generation of ice particles by homogeneous nucleation using LC and the subsequent more effective growth for ice particles without competition process. And, this method is called 'Low-Level Penetration Seeding of Homogeneous Ice Nucleant (LOLEPSHIN)' ; this induces a 'Roll-up Expansion of Twin Horizontal Ice-crystal Thermals (RETHIT)' and a subsequent 'Falling growth-Induced Lateral Air Spreading (FILAS)'. This LC method was applied to thin super-cooled cumulus clouds in Saga prefecture, Northern Kyushu, Japan on February 4,2006. The seeding airplane took off the Atugi Airport in Kanagawa Prefecture toward the Iki Island around 0830JST. Many cloud bands were cofirmed in the flight going to the experimental area and the cloud base temperature was approximately -9C (1200m). Scince some young developing thin cumuli were found over the Iki Island, LC seeding to these clouds was carried out two times from 0841JST until 0919JST penetrating the -9C (1200m) altitude. The first precipitation seeding ebded in failure. The second penetration seeding was done for 115 seconds around 0917JST. This penetration led to success of developing one artificial echo (Echo I) in the leeward side of the Iki Island. Eco I moved from NNW to SSW. The maximum area of Echo I were 48km2 (at 1033JST) and first comfirmed by the Kyushu University radar (KU radar) at 1006JST (46 min. after LC seeding) around Mt.Sefuri in Saga Prefecture. It can be inferred that ice perticles formed by LC seeding grew to the precipitable size and resultant snowfall was detected by radar in approximately 120 min. after seeding operation. In this study, based on these observed facts, optimum design for enhancing winter-time water resources by LC seeding method was suggested. Successive low-level horizontal penetrations of operational aircraft with seeding LC into many moving super-cooled cumuli towards the Japan Islands will lead to the spreading of cloud volume and subsequent coversion of large amount of iv active cloud volume into newly exploited artificial precipitation. As a result, these experiments succeeded, and the total amount of estimated radar precipitation of the be able to secure a large amount of water resource from these experiment results.

Evaluation of various QuEChERS based methods for the analysis of herbicides and other commonly used pesticides in polished rice by LC-MS/MS.

PubMed

Pareja, Lucía; Cesio, Verónica; Heinzen, Horacio; Fernández-Alba, Amadeo R

2011-02-15

Four different extraction and clean-up protocols based on the QuEChERS method were compared for the development of an optimized sample preparation procedure for the multiresidue analysis of 16 commonly applied herbicides in rice crops using LC-QqQ/MS. Additionally the methods were evaluated for the analysis of 26 insecticides and fungicides currently used in rice crops. The methods comprise, in general, the hydratation of the sample with water followed by the extraction with acetonitrile, phase separation with the addition of different salts and finally a clean-up step with various sorbents. Matrix effects were evaluated for the 4 studied methods using LC-QqQ/MS. Additionally LC-TOF/MS was used to compare the co-extractants obtained with the four assayed methodologies. Thirty-six pesticides presented good performance with recoveries in the range 70-120% and relative standard deviations below 20% using 7.5 g of milled polished rice and the buffered acetate QuEChERS method without clean-up at both fortification levels: 10 and 300 μg kg(-1). The other six pesticides presented low recovery rates, nevertheless all these analytes could be analyzed with at least one of the other three studied procedures. Copyright © 2010. Published by Elsevier B.V.

Rapid method for the measurement of circulating thyroid hormones in low volumes of teleost fish plasma by LC-ESI/MS/MS

PubMed Central

Noyes, Pamela D.; Lema, Sean C.; Roberts, Simon C.; Cooper, Ellen M.

2014-01-01

Thyroid hormones are critical regulators of normal development and physiological functioning in all vertebrates. Radioimmunoassay (RIA) approaches have been the method of choice for measuring circulating levels of thyroid hormones in vertebrates. While sensitive, RIA-based approaches only allow for a single analyte measurement per assay, can lack concordance across platforms and laboratories, and can be prone to analytical interferences especially when used with fish plasma. Ongoing advances in liquid chromatography tandem mass spectrometry (LC/MS/MS) have led to substantial decreases in detection limits for thyroid hormones and other biomolecules in complex matrices, including human plasma. Despite these advances, current analytical approaches do not allow for the measurement of native thyroid hormone in teleost fish plasma by mass spectrometry and continue to rely on immunoassay. In this study, we developed a new method that allows for the rapid extraction and simultaneous measurement of total T4 (TT4) and total T3 (TT3) in low volumes (50 μL) of fish plasma by LC/MS/MS. Methods were optimized initially in plasma from rainbow trout (Oncorhynchus mykiss) and applied to plasma from other teleost fishes, including fathead minnows (Pimephales promelas), mummichogs (Fundulus heteroclitus), sockeye salmon (Oncorhynchus nerka), and coho salmon (Oncorhynchus kisutch). Validation of method performance with T4- and T3-spiked rainbow trout plasma at 2 and 4 ng/mL produced mean recoveries ranging from 82 to 95 % and 97 to 105 %, respectively. Recovery of 13C12-T4 internal standard in plasma extractions was: 99±1.8 % in rainbow trout, 85±11 % in fathead minnow, 73±5.0 % in mummichog, 73±1.7 % in sockeye salmon, and 80±8.4 % in coho salmon. While absolute levels of thyroid hormones measured in identical plasma samples by LC/MS/MS and RIA varied depending on the assay used, T4/T3 ratios were generally consistent across both techniques. Less variability was measured among samples subjected to LC/MS/MS suggesting a more precise estimate of thyroid hormone homeostasis in the species targeted. Overall, a sensitive and reproducible method was established that takes advantage of LC/MS/MS techniques to rapidly measure TT4 and TT3 with negligible interferences in low volumes of plasma across a variety of teleost fishes. PMID:24343452

Determination of nicotianamine in soy sauce and other plant-based foods by LC-MS/MS.

PubMed

Yamaguchi, Hitomi; Uchida, Riichiro

2012-10-10

Nicotianamine is a nonproteinogenic amino acid, known to be an important metal chelator in plants. Recently, the antihypertensive effect of nicotianamine was discovered. In this study, a simple method to determine nicotianamine was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a multimode ODS column. This method does not need derivatizing or ion-pairing reagents to retain nicotianamine, which is known for its poor retention on reversed-phase columns because of its high polarity. Moreover, this method showed a sufficient limit of detection (0.5 ng/mL), so it was found to be suitable for the analysis of nicotianamine in soy sauce and other foods, without cleanup. To subtract the matrix effect during LC-MS/MS analysis, a standard addition method was used. The levels of nicotianamine in soy sauce ranged from <0.25 to 71 μg/g. Nicotianamine was also determined in other foods, including soy milk, vegetable juice, fruit juice, and bottled tea.

Reduction of Solvent Effect in Reverse Phase Gradient Elution LC-ICP-MS

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

Sullivan, Patrick Allen

2005-12-17

Quantification in liquid chromatography (LC) is becoming very important as more researchers are using LC, not as an analytical tool itself, but as a sample introduction system for other analytical instruments. The ability of LC instrumentation to quickly separate a wide variety of compounds makes it ideal for analysis of complex mixtures. For elemental speciation, LC is joined with inductively coupled plasma mass spectrometry (ICP-MS) to separate and detect metal-containing, organic compounds in complex mixtures, such as biological samples. Often, the solvent gradients required to perform complex separations will cause matrix effects within the plasma. This limits the sensitivity ofmore » the ICP-MS and the quantification methods available for use in such analyses. Traditionally, isotope dilution has been the method of choice for LC-ICP-MS quantification. The use of naturally abundant isotopes of a single element in quantification corrects for most of the effects that LC solvent gradients produce within the plasma. However, not all elements of interest in speciation studies have multiple naturally occurring isotopes; and polyatomic interferences for a given isotope can develop within the plasma, depending on the solvent matrix. This is the case for reverse phase LC separations, where increasing amounts of organic solvent are required. For such separations, an alternative to isotope dilution for quantification would be is needed. To this end, a new method was developed using the Apex-Q desolvation system (ESI, Omaha, NE) to couple LC instrumentation with an ICP-MS device. The desolvation power of the system allowed greater concentrations of methanol to be introduced to the plasma prior to destabilization than with direct methanol injection into the plasma. Studies were performed, using simulated and actual linear methanol gradients, to find analyte-internal standard (AIS) pairs whose ratio remains consistent (deviations {+-} 10%) over methanol concentration ranges of 5%-35% (simulated) and 8%-32% (actual). Quadrupole (low resolution) and sector field (high resolution) ICP-MS instrumentation were utilized in these studies. Once an AIS pair is determined, quantification studies can be performed. First, an analysis is performed by adding both elements of the AIS pair post-column while performing the gradient elution without sample injection. A comparison of the ratio of the measured intensities to the atomic ratio of the two standards is used to determine a correction factor that can be used to account for the matrix effects caused by the mobile phase. Then, organic and/or biological molecules containing one of the two elements in the AIS pair are injected into the LC column. A gradient method is used to vary the methanol-water mixture in the mobile phase and to separate out the compounds in a given sample. A standard solution of the second ion in the AIS pair is added continuously post-column. By comparing the ratio of the measured intensities to the atomic ratio of the eluting compound and internal standard, the concentration of the injected compound can be determined.« less

A Liquid Chromatographic-Mass Spectrometric (LC-MS) Method for the Analysis of the Bis-pyridinium Oxime ICD-585 in Plasma: Application in a Guinea Pig Model

DTIC Science & Technology

2010-01-01

Chrom LC –MS...Literature 3. DATES COVERED (From - To) 4. TITLE AND SUBTITLE A liquid chromatographic–mass spectrometric ( LC –MS) method for the analysis of the 5a...Journal of Chromatography B journa l homepage: www.e lsev ier .com/ locate /chromb A liquid chromatographic–mass spectrometric ( LC –MS) method for

Development of LC-MS/MS method for the simultaneous analysis of the cardioprotective drug dexrazoxane and its metabolite ADR-925 in isolated cardiomyocytes and cell culture medium.

PubMed

Kovarikova, Petra; Pasakova-Vrbatova, Ivana; Vavrova, Anna; Stariat, Jan; Klimes, Jiri; Simunek, Tomas

2013-03-25

Dexrazoxane (DEX) is the only clinically used drug effective against anthracycline-induced cardiotoxicity and extravasation injury. However, the mechanism of its cardioprotective action still remains elusive. This paucity of comprehensive data is at least partially caused by the analytical difficulties associated with selective and sensitive simultaneous determination of the parent drug and its putative active metabolite ADR-925 in the relevant biological material. The aim of this study was to develop and validate the first LC-MS/MS method for simultaneous determination of DEX and ADR-925 in the isolated rat neonatal ventricular cardiomyocytes (NVCMs) and the cell culture medium. The analysis was performed on a Synergi Polar-RP column using the gradient profile of the mobile phase composed of 2mM ammonium formate and methanol. Electrospray ionization and ion trap mass analyzer were used as ionization and detection techniques, respectively. NVCMs were precipitated with methanol and the cell culture medium samples were diluted with the same solvent prior the LC-MS/MS analysis. The method was validated within the range of 4-80pmol/10(6) NVCMs and 7-70pmol/10(6) NVCMs for DEX and ADR-925, respectively, and at the concentrations of 8-100μM for both compounds in the culture cell medium. The practical applicability of this method was confirmed by the pilot analysis of NVCMs and the corresponding cell medium samples from relevant in vitro experiment. Hence, the LC-MS/MS method developed in this study represents a modern analytical tool suitable for investigation of DEX bioactivation inside the cardiomyocytes. In addition, the basic utility of the method for the analysis of DEX and ADR-925 in plasma samples was proved in a pilot experiment. Copyright © 2013 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.

Evaluation of Tamoxifen and metabolites by LC-MS/MS and HPLC Methods

PubMed Central

Heath, D.D.; Flatt, S.W.; Wu, A.H.B.; Pruitt, M.A.; Rock, C.L.

2015-01-01

Epidemiological and laboratory evidence suggests that quantification of serum or plasma levels of tamoxifen and the metabolites of tamoxifen, 4-hydroxy-N-desmethyl-tamoxifen (endoxifen), Z-4-hydroxy-tamoxifen (4HT), N-desmethyl-tamoxifen (ND-tam) is a clinically useful tool in the assessment and monitoring of breast cancer status in patients taking adjuvant tamoxifen. A liquid chromatographic mass spectrometric method (LC-MS/MS) was used to measure the blood levels of tamoxifen and the metabolites of tamoxifen. This fully automated analytical method is specific, accurate and sensitive. The LC-MS/MS automated technique has now become a widely accepted reference method. We analyzed a randomly selected batch of blood samples from participants enrolled in a breast cancer study to compare results from this reference method in 40 samples with those obtained from a recently developed high performance liquid chromatography (HPLC) method with fluorescence detection. The mean (SD) concentration for the LC-MS/MS (endoxifen 12.6 [7.5] ng/mL, tamoxifen 105 [44] ng/mL, 4-HT 1.9 [1.0] ng/mL, ND-tam 181 [69] ng/mL) and the HPLC (endoxifen 13.1 [7.8] ng/mL, tamoxifen 108[55]ng/mL, 4-HT 1.8 [0.8] ng/mL, ND-tam 184 [81] ng/mL), the methods did not show any significant differences. Our results confirm that the HPLC method offers an accurate and comparable alternative for the quantification of tamoxifen and tamoxifen metabolites. PMID:24693573

Localisation and semi-quantitative measurement of lipocortin 1 in rat anterior pituitary cells by fluorescence-activated cell analysis/sorting and electron microscopy.

PubMed

Christian, H C; Flower, R J; Morris, J F; Buckingham, J C

1999-09-01

Lipocortin 1 (LC1, also called annexin 1), a Ca2(+)- and phospholipid-binding protein, is an important mediator of glucocorticoid action in the anterior pituitary gland. Previous studies based on immunoprecipitation and Western blot analysis suggest that LC1 is found intracellularly both in the cytoplasm and in association with membranes and also on the cell surface where it attaches to the membrane by a Ca2(+)-dependent mechanism. However, as yet it is unclear which anterior pituitary cell types express the protein. Accordingly, we have developed a method based on a combination of fluorescence activated cell (FAC) analysis/sorting and electron microscopy to detect and quantify intracellular LC1 in rat anterior pituitary cells and to identify the cell types in which it is expressed. In addition, we have measured cell surface LC1 and examined the influence of glucocorticoids on the cellular disposition of the protein. Anterior pituitary cells were dispersed with collagenase. For experiments measuring intracellular LC1, three cell fixation/permeabilisation methods were examined initially, i.e. (1) Zamboni's fluid (30 min) and Triton-X-100 (0.12%, 1 or 12 h); (2) paraformaldehyde (2%, 1 h) and Triton-X-100 (0.2%, 10 min); and (3) paraformaldehyde (0.2%, 15 min) and saponin (0.1%, 5 min). The protocol using paraformaldehyde/Triton-X-100 provided optimal preservation of cell ultrastructure and of LC1 immunoreactivity (ir-LC1) while also effectively permeabilising the cells; it was therefore used in subsequent studies. Using an anti-LC1 monoclonal antibody as a probe, 82+/-5% of the secretory cells in the heterogeneous anterior pituitary cell preparation were shown by FAC analysis to display specific fluorescence for intracellular ir-LC1. Morphological analysis and immunogold-histochemistry of cells separated by FAC sorting identified corticotrophs, lactotrophs, somatotrophs and gonadotrophs in the population displaying LC1 immunofluorescence. LC1 was also detected on the surface of anterior pituitary cells by FACS analysis. Incubation of anterior pituitary cells with dexamethasone or corticosterone (0.1 and 1.0 microM) prior to fixation and analysis produced a significant, concentration-dependent decrease in intracellular ir-LC1 and a concomitant increase in the amount of ir-LC1 detected on the surface of the cells; the effects of the two steroids were indistinguishable quantitatively. In conclusion, we report a novel method which permits (1) the detection and semi-quantitative measurement of intracellular and surface LC1 in anterior pituitary cells; and (2) the identification of the cell types in which the protein is found.

Analysis of 70 Environmental Protection Agency priority pharmaceuticals in water by EPA Method 1694.

PubMed

Ferrer, Imma; Zweigenbaum, Jerry A; Thurman, E Michael

2010-09-03

The U.S. Environmental Protection Agency (EPA) Method 1694 for the determination of pharmaceuticals in water recently brought a new challenge for treatment utilities, where pharmaceuticals have been reported in the drinking water of 41-million Americans. This proposed methodology, designed to address this important issue, consists of solid-phase extraction (SPE) followed by liquid chromatography-mass spectrometry (LC/MS-MS) using triple quadrupole. Under the guidelines of Method 1694, a multi-residue method was developed, validated, and applied to wastewater, surface water and drinking water samples for the analysis of 70 pharmaceuticals. Four distinct chromatographic gradients and LC conditions were used according to the polarity and extraction of the different pharmaceuticals. Positive and negative ion electrospray were used with two MRM transitions (a quantifier and a qualifier ion for each compound), which adds extra confirmation not included in the original Method 1694. Finally, we verify, for the first time, EPA Method 1694 on water samples collected in several locations in Colorado, where positive identifications for several pharmaceuticals were found. This study is a valuable indicator of the potential of LC/MS-MS for routine quantitative multi-residue analysis of pharmaceuticals in drinking water and wastewater samples and will make monitoring studies much easier to develop for water utilities across the US, who are currently seeking guidance on analytical methods for pharmaceuticals in their water supplies. 2010 Elsevier B.V. All rights reserved.

Study of optical design of Blu-ray pickup head system with a liquid crystal element.

PubMed

Fang, Yi-Chin; Yen, Chih-Ta; Hsu, Jui-Hsin

2014-10-10

This paper proposes a newly developed optical design and an active compensation method for a Blu-ray pickup head system with a liquid crystal (LC) element. Different from traditional pickup lens design, this new optical design delivers performance as good as the conventional one but has more room for tolerance control, which plays a role in antishaking devices, such as portable Blu-ray players. A hole-pattern electrode and LC optics with external voltage input were employed to generate a symmetric nonuniform electrical field in the LC layer that directs LC molecules into the appropriate gradient refractive index distribution, resulting in the convergence or divergence of specific light beams. LC optics deliver fast and, most importantly, active compensation through optical design when errors occur. Simulations and tolerance analysis were conducted using Code V software, including various tolerance analyses, such as defocus, tilt, and decenter, and their related compensations. Two distinct Blu-ray pickup head system designs were examined in this study. In traditional Blu-ray pickup head system designs, the aperture stop is always set on objective lenses. In the study, the aperture stop is on the LC lens as a newly developed lens. The results revealed that an optical design with aperture stop set on the LC lens as an active compensation device successfully eliminated up to 57% of coma aberration compared with traditional optical designs so that this pickup head lens design will have more space for tolerance control.

The NISTmAb tryptic peptide spectral library for monoclonal antibody characterization.

PubMed

Dong, Qian; Liang, Yuxue; Yan, Xinjian; Markey, Sanford P; Mirokhin, Yuri A; Tchekhovskoi, Dmitrii V; Bukhari, Tallat H; Stein, Stephen E

2018-04-01

We describe the creation of a mass spectral library composed of all identifiable spectra derived from the tryptic digest of the NISTmAb IgG1κ. The library is a unique reference spectral collection developed from over six million peptide-spectrum matches acquired by liquid chromatography-mass spectrometry (LC-MS) over a wide range of collision energy. Conventional one-dimensional (1D) LC-MS was used for various digestion conditions and 20- and 24-fraction two-dimensional (2D) LC-MS studies permitted in-depth analyses of single digests. Computer methods were developed for automated analysis of LC-MS isotopic clusters to determine the attributes for all ions detected in the 1D and 2D studies. The library contains a selection of over 12,600 high-quality tandem spectra of more than 3,300 peptide ions identified and validated by accurate mass, differential elution pattern, and expected peptide classes in peptide map experiments. These include a variety of biologically modified peptide spectra involving glycosylated, oxidized, deamidated, glycated, and N/C-terminal modified peptides, as well as artifacts. A complete glycation profile was obtained for the NISTmAb with spectra for 58% and 100% of all possible glycation sites in the heavy and light chains, respectively. The site-specific quantification of methionine oxidation in the protein is described. The utility of this reference library is demonstrated by the analysis of a commercial monoclonal antibody (adalimumab, Humira®), where 691 peptide ion spectra are identifiable in the constant regions, accounting for 60% coverage for both heavy and light chains. The NIST reference library platform may be used as a tool for facile identification of the primary sequence and post-translational modifications, as well as the recognition of LC-MS method-induced artifacts for human and recombinant IgG antibodies. Its development also provides a general method for creating comprehensive peptide libraries of individual proteins.

The NISTmAb tryptic peptide spectral library for monoclonal antibody characterization

PubMed Central

Dong, Qian; Liang, Yuxue; Yan, Xinjian; Markey, Sanford P.; Mirokhin, Yuri A.; Tchekhovskoi, Dmitrii V.; Bukhari, Tallat H.; Stein, Stephen E.

2018-01-01

ABSTRACT We describe the creation of a mass spectral library composed of all identifiable spectra derived from the tryptic digest of the NISTmAb IgG1κ. The library is a unique reference spectral collection developed from over six million peptide-spectrum matches acquired by liquid chromatography-mass spectrometry (LC-MS) over a wide range of collision energy. Conventional one-dimensional (1D) LC-MS was used for various digestion conditions and 20- and 24-fraction two-dimensional (2D) LC-MS studies permitted in-depth analyses of single digests. Computer methods were developed for automated analysis of LC-MS isotopic clusters to determine the attributes for all ions detected in the 1D and 2D studies. The library contains a selection of over 12,600 high-quality tandem spectra of more than 3,300 peptide ions identified and validated by accurate mass, differential elution pattern, and expected peptide classes in peptide map experiments. These include a variety of biologically modified peptide spectra involving glycosylated, oxidized, deamidated, glycated, and N/C-terminal modified peptides, as well as artifacts. A complete glycation profile was obtained for the NISTmAb with spectra for 58% and 100% of all possible glycation sites in the heavy and light chains, respectively. The site-specific quantification of methionine oxidation in the protein is described. The utility of this reference library is demonstrated by the analysis of a commercial monoclonal antibody (adalimumab, Humira®), where 691 peptide ion spectra are identifiable in the constant regions, accounting for 60% coverage for both heavy and light chains. The NIST reference library platform may be used as a tool for facile identification of the primary sequence and post-translational modifications, as well as the recognition of LC-MS method-induced artifacts for human and recombinant IgG antibodies. Its development also provides a general method for creating comprehensive peptide libraries of individual proteins. PMID:29425077

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).

Multi-mycotoxin analysis of animal feed and animal-derived food using LC-MS/MS system with timed and highly selective reaction monitoring.

PubMed

Zhao, Zhiyong; Liu, Na; Yang, Lingchen; Deng, Yifeng; Wang, Jianhua; Song, Suquan; Lin, Shanhai; Wu, Aibo; Zhou, Zhenlei; Hou, Jiafa

2015-09-01

Mycotoxins have the potential to enter the human food chain through carry-over of contaminants from feed into animal-derived products. The objective of the study was to develop a reliable and sensitive method for the analysis of 30 mycotoxins in animal feed and animal-derived food (meat, edible animal tissues, and milk) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In the study, three extraction procedures, as well as various cleanup procedures, were evaluated to select the most suitable sample preparation procedure for different sample matrices. In addition, timed and highly selective reaction monitoring on LC-MS/MS was used to filter out isobaric matrix interferences. The performance characteristics (linearity, sensitivity, recovery, precision, and specificity) of the method were determined according to Commission Decision 2002/657/EC and 401/2006/EC. The established method was successfully applied to screening of mycotoxins in animal feed and animal-derived food. The results indicated that mycotoxin contamination in feed directly influenced the presence of mycotoxin in animal-derived food. Graphical abstract Multi-mycotoxin analysis of animal feed and animal-derived food using LC-MS/MS.

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.

High-speed homogenization coupled with microwave-assisted extraction followed by liquid chromatography-tandem mass spectrometry for the direct determination of alkaloids and flavonoids in fresh Isatis tinctoria L. hairy root cultures.

PubMed

Jiao, Jiao; Gai, Qing-Yan; Zhang, Lin; Wang, Wei; Luo, Meng; Zu, Yuan-Gang; Fu, Yu-Jie

2015-06-01

A new, simple and efficient analysis method for fresh plant in vitro cultures-namely, high-speed homogenization coupled with microwave-assisted extraction (HSH-MAE) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-was developed for simultaneous determination of six alkaloids and eight flavonoids in Isatis tinctoria hairy root cultures (ITHRCs). Compared with traditional methods, the proposed HSH-MAE offers the advantages of easy manipulation, higher efficiency, energy saving, and reduced waste. Cytohistological studies were conducted to clarify the mechanism of HSH-MAE at cellular/tissue levels. Moreover, the established LC-MS/MS method showed excellent linearity, precision, repeatability, and reproducibility. The HSH-MAE-LC-MS/MS method was also successfully applied for screening high-productivity ITHRCs. Overall, this study opened up a new avenue for the direct determination of secondary metabolic profiles from fresh plant in vitro cultures, which is valuable for improving quality control of plant cell/organ cultures and sheds light on the metabolomic analysis of biological samples. Graphical Abstract HSH-MAE-LC-MS/MS opened up a new avenue for the direct determination of alkaloids and flavonoids in fresh Isatis tinctoria hairy root cultures.

A liquid chromatography/tandem mass spectrometry assay for the analysis of atomoxetine in human plasma and in vitro cellular samples

PubMed Central

Appel, David I.; Brinda, Bryan; Markowitz, John S.; Newcorn, Jeffrey H.; Zhu, Hao-Jie

2012-01-01

A simple, rapid and sensitive method for quantification of atomoxetine by liquid chromatography- tandem mass spectrometry (LC-MS/MS) was developed. This assay represents the first LC-MS/MS quantification method for atomoxetine utilizing electrospray ionization. Deuterated atomoxetine (d3-atomoxetine) was adopted as the internal standard. Direct protein precipitation was utilized for sample preparation. This method was validated for both human plasma and in vitro cellular samples. The lower limit of quantification was 3 ng/ml and 10 nM for human plasma and cellular samples, respectively. The calibration curves were linear within the ranges of 3 ng/ml to 900 ng/ml and 10 nM to 10 μM for human plasma and cellular samples, respectively (r2 > 0.999). The intra- and inter-day assay accuracy and precision were evaluated using quality control samples at 3 different concentrations in both human plasma and cellular lysate. Sample run stability, assay selectivity, matrix effect, and recovery were also successfully demonstrated. The present assay is superior to previously published LC-MS and LC-MS/MS methods in terms of sensitivity or the simplicity of sample preparation. This assay is applicable to the analysis of atomoxetine in both human plasma and in vitro cellular samples. PMID:22275222

Development of an Analytic Method for Sulfur Compounds in Aged Garlic Extract with the Use of a Postcolumn High Performance Liquid Chromatography Method with Sulfur-Specific Detection.

PubMed

Matsutomo, Toshiaki; Kodera, Yukihiro

2016-02-01

Garlic and its processed preparations contain numerous sulfur compounds that are difficult to analyze in a single run using HPLC. The aim of this study was to develop a rapid and convenient sulfur-specific HPLC method to analyze sulfur compounds in aged garlic extract (AGE). We modified a conventional postcolumn HPLC method by employing a hexaiodoplatinate reagent. Identification and structural analysis of sulfur compounds were conducted by LC-mass spectrometry (LC-MS) and nuclear magnetic resonance. The production mechanisms of cis-S-1-propenylcysteine (cis-S1PC) and S-allylmercaptocysteine (SAMC) were examined by model reactions. Our method has the following advantages: less interference from nonsulfur compounds, high sensitivity, good correlation coefficients (r > 0.98), and high resolution that can separate >20 sulfur compounds, including several isomers, in garlic preparations in a single run. This method was adapted for LC-MS analysis. We identified cis-S1PC and γ-glutamyl-S-allyl-mercaptocysteine in AGE. The results of model reactions suggest that cis-S1PC is produced from trans-S1PC through an isomerization reaction and that SAMC is produced by a reaction involving S-allylcysteine/S1PC and diallyldisulfide during the aging period. We developed a rapid postcolumn HPLC method for both qualitative and quantitative analyses of sulfur compounds, and this method helped elucidate a potential mechanism of cis-S1PC and SAMC action in AGE. © 2016 American Society for Nutrition.

Dermal and inhalation acute toxic class methods: test procedures and biometric evaluations for the Globally Harmonized Classification System.

PubMed

Holzhütter, H G; Genschow, E; Diener, W; Schlede, E

2003-05-01

The acute toxic class (ATC) methods were developed for determining LD(50)/LC(50) estimates of chemical substances with significantly fewer animals than needed when applying conventional LD(50)/LC(50) tests. The ATC methods are sequential stepwise procedures with fixed starting doses/concentrations and a maximum of six animals used per dose/concentration. The numbers of dead/moribund animals determine whether further testing is necessary or whether the test is terminated. In recent years we have developed classification procedures for the oral, dermal and inhalation routes of administration by using biometric methods. The biometric approach assumes a probit model for the mortality probability of a single animal and assigns the chemical to that toxicity class for which the best concordance is achieved between the statistically expected and the observed numbers of dead/moribund animals at the various steps of the test procedure. In previous publications we have demonstrated the validity of the biometric ATC methods on the basis of data obtained for the oral ATC method in two-animal ring studies with 15 participants from six countries. Although the test procedures and biometric evaluations for the dermal and inhalation ATC methods have already been published, there was a need for an adaptation of the classification schemes to the starting doses/concentrations of the Globally Harmonized Classification System (GHS) recently adopted by the Organization for Economic Co-operation and Development (OECD). Here we present the biometric evaluation of the dermal and inhalation ATC methods for the starting doses/concentrations of the GHS and of some other international classification systems still in use. We have developed new test procedures and decision rules for the dermal and inhalation ATC methods, which require significantly fewer animals to provide predictions of toxicity classes, that are equally good or even better than those achieved by using the conventional LD(50)/LC(50) methods. In order to cope with rather narrow dose/concentration classes of the GHS we have, as in our previous publications, combined the outcome of all results that can be obtained during testing for the allocation to one of the defined toxicity classes of the GHS. Our results strongly recommend the deletion of the dermal LD(50) and the inhalation LC(50) test as regulatory tests and the adoption of the dermal and inhalation ATC methods as internationally accepted alternatives.

Development and validation of an LC-UV method for the quantification and purity determination of the novel anticancer agent C1311 and its pharmaceutical dosage form.

PubMed

den Brok, Monique W J; Nuijen, Bastiaan; Hillebrand, Michel J X; Grieshaber, Charles K; Harvey, Michael D; Beijnen, Jos H

2005-09-01

C1311 (5-[[2-(diethylamino)ethyl]amino]-8-hydroxyimidazo [4,5,1-de]-acridin-6-one-dihydrochloride trihydrate) is the lead compound from the group of imidazoacridinones, a novel group of rationally designed anticancer agents. The pharmaceutical development of C1311 necessitated the availability of an assay for the quantification and purity determination of C1311 active pharmaceutical ingredient (API) and its pharmaceutical dosage form. A reversed-phase liquid chromatographic method (RP-LC) with ultraviolet (UV) detection was developed, consisting of separation on a C18 column with phosphate buffer (60 mM; pH 3 with 1 M citric acid)-acetonitrile-triethylamine (83:17:0.05, v/v/v) as the mobile phase and UV-detection at 280 nm. The method was found to be linear over a concentration range of 2.50-100 microg/mL, precise and accurate. Accelerated stress testing showed degradation products, which were well separated from the parent compound, confirming its stability-indicating capacity. Moreover, the use of LC-MS and on-line photo diode array detection enabled us to propose structures for four degradation products. Two of these products were also found as impurities in the API and more abundantly in an impure lot of API.

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.

Simultaneous assay of multiple antibiotics in human plasma by LC-MS/MS: importance of optimizing formic acid concentration.

PubMed

Chen, Feng; Hu, Zhe-Yi; Laizure, S Casey; Hudson, Joanna Q

2017-03-01

Optimal dosing of antibiotics in critically ill patients is complicated by the development of resistant organisms requiring treatment with multiple antibiotics and alterations in systemic exposure due to diseases and extracorporeal drug removal. Developing guidelines for optimal antibiotic dosing is an important therapeutic goal requiring robust analytical methods to simultaneously measure multiple antibiotics. An LC-MS/MS assay using protein precipitation for cleanup followed by a 6-min gradient separation was developed to simultaneously determine five antibiotics in human plasma. The precision and accuracy were within the 15% acceptance range. The formic acid concentration was an important determinant of signal intensity, peak shape and matrix effects. The method was designed to be simple and successfully applied to a clinical pharmacokinetic study.

Analysis of penicillin G in milk by liquid chromatography.

PubMed

Boison, J O; Keng, L J; MacNeil, J D

1994-01-01

A liquid chromatographic (LC) method that was previously developed for penicillin G residues in animal tissues has been adapted to milk and milk products. After protein precipitation with sodium tungstate, samples are applied to a C18 solid-phase extraction cartridge, from which penicillin is eluted, derivatized with 1,2,4-triazole-mercuric chloride solution, and analyzed by isocratic liquid chromatography (LC) on a C18 column with UV detection at 325 nm. Quantitation is done with reference to penicillin V as an internal standard. Penicillin G recoveries were determined to be > 70% on standards fortified at 3-60 ppb. Accuracy approached 100% using the penicillin V internal standard. The detection limit for penicillin G residues was 3 ppb in fluid milk. Samples may be confirmed by thermospray/LC at concentrations approaching the detection limit of the UV method.

Direct high-performance liquid chromatographic determination of the enantiomeric purity of levodopa and methyldopa: comparison with pharmacopoeial polarimetric methods.

PubMed

Dolezalová, M; Tkaczyková, M

1999-03-01

Chiral high-performance liquid chromatography was employed for determination of the enantiomeric purity of levodopa and methyldopa. The determination of D-DOPA in levodopa was accomplished using a chiral ligand-exchange chromatograpy with an ordinary C18 column and a chiral mobile phase containing N,N-dimethyl-L-phenylalanine and Cu(II) acetate or by means of LC on a teicoplanin column in conjunction with ethanol-water (65:35, v/v). Both methods gave good performance, however, the latter was faster and more convenient and suitable for routine analyses. For the determination of D-methyldopa a LC method based on the use of a teicoplanin column in polar organic mode with methanol-acetic acid-triethylamine (1,000:0.05:0.05, v/v/v) mobile phase was developed. The precision, accuracy, linearity and selectivity were satisfactory. In comparison with pharmacopoeial polarimetric methods (according to the European Pharmacopoeia and the Pharmacopoea Bohemoslovaca), the LC methods proved to be much more sensitive giving detection limits 0.04% of D-DOPA and 0.3% of D-methyldopa.

An automated online turboflow cleanup LC/MS/MS method for the determination of 11 plasticizers in beverages and milk.

PubMed

Ates, Ebru; Mittendorf, Klaus; Senyuva, Hamide

2013-01-01

An automated sample preparation technique involving cleanup and analytical separation in a single operation using an online coupled TurboFlow (RP-LC system) is reported. This method eliminates time-consuming sample preparation steps that can be potential sources for cross-contamination in the analysis of plasticizers. Using TurboFlow chromatography, liquid samples were injected directly into the automated system without previous extraction or cleanup. Special cleanup columns enabled specific binding of target compounds; higher MW compounds, i.e., fats and proteins, and other matrix interferences with different chemical properties were removed to waste, prior to LC/MS/MS. Systematic stepwise method development using this new technology in the food safety area is described. Selection of optimum columns and mobile phases for loading onto the cleanup column followed by transfer onto the analytical column and MS detection are critical method parameters. The method was optimized for the assay of 10 phthalates (dimethyl, diethyl, dipropyl, butyl benzyl, diisobutyl, dicyclohexyl, dihexyl, diethylhexyl, diisononyl, and diisododecyl) and one adipate (diethylhexyl) in beverages and milk.

Discovery, identification and mitigation of isobaric sulfate metabolite interference to a phosphate prodrug in LC-MS/MS bioanalysis: Critical role of method development in ensuring assay quality.

PubMed

Yuan, Long; Ji, Qin C

2018-06-05

Metabolite interferences represent a major risk of inaccurate quantification when using LC-MS/MS bioanalytical assays. During LC-MS/MS bioanalysis of BMS-919194, a phosphate ester prodrug, in plasma samples from rat and monkey GLP toxicology studies, an unknown peak was detected in the MRM channel of the prodrug. This peak was not observed in previous discovery toxicology studies, in which a fast gradient LC-MS/MS method was used. We found out that this unknown peak would co-elute with the prodrug peak when the discovery method was used, therefore, causing significant overestimation of the exposure of the prodrug in the discovery toxicology studies. To understand the nature of this interfering peak and its impact to bioanalytical assay, we further investigated its formation and identification. The interfering compound and the prodrug were found to be isobaric and to have the same major product ions in electrospray ionization positive mode, thus, could not be differentiated using a triple quadrupole mass spectrometer. By using high-resolution mass spectrometry (HRMS), the interfering metabolite was successfully identified to be an isobaric sulfate metabolite of BMS-919194. To the best of our knowledge, this is the first report that a phosphate prodrug was metabolized in vivo to an isobaric sulfate metabolite, and this metabolite caused significant interference to the analysis of the prodrug. This work demonstrated the presence of the interference risk from isobaric sulfate metabolites to the bioanalysis of phosphate prodrugs in real samples. It is critical to evaluate and mitigate potential metabolite interferences during method development, therefore, minimize the related bioanalytical risks and ensure assay quality. Our work also showed the unique advantages of HRMS in identifying potential metabolite interference during LC-MS/MS bioanalysis. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

High throughput LC-MS/MS method for the simultaneous analysis of multiple vitamin D analytes in serum.

PubMed

Jenkinson, Carl; Taylor, Angela E; Hassan-Smith, Zaki K; Adams, John S; Stewart, Paul M; Hewison, Martin; Keevil, Brian G

2016-03-01

Recent studies suggest that vitamin D-deficiency is linked to increased risk of common human health problems. To define vitamin D 'status' most routine analytical methods quantify one particular vitamin D metabolite, 25-hydroxyvitamin D3 (25OHD3). However, vitamin D is characterized by complex metabolic pathways, and simultaneous measurement of multiple vitamin D metabolites may provide a more accurate interpretation of vitamin D status. To address this we developed a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to analyse multiple vitamin D analytes, with particular emphasis on the separation of epimer metabolites. A supportive liquid-liquid extraction (SLE) and LC-MS/MS method was developed to quantify 10 vitamin D metabolites as well as separation of an interfering 7α-hydroxy-4-cholesten-3-one (7αC4) isobar (precursor of bile acid), and validated by analysis of human serum samples. In a cohort of 116 healthy subjects, circulating concentrations of 25-hydroxyvitamin D3 (25OHD3), 3-epi-25-hydroxyvitamin D3 (3-epi-25OHD3), 24,25-dihydroxyvitamin D3 (24R,25(OH)2D3), 1,25-dihydroxyvitamin D3 (1α,25(OH)2D3), and 25-hydroxyvitamin D2 (25OHD2) were quantifiable using 220μL of serum, with 25OHD3 and 24R,25(OH)2D3 showing significant seasonal variations. This high-throughput LC-MS/MS method provides a novel strategy for assessing the impact of vitamin D on human health and disease. Copyright © 2016 Elsevier B.V. All rights reserved.

Determination of thiol metabolites in human urine by stable isotope labeling in combination with pseudo-targeted mass spectrometry analysis

NASA Astrophysics Data System (ADS)

Liu, Ping; Qi, Chu-Bo; Zhu, Quan-Fei; Yuan, Bi-Feng; Feng, Yu-Qi

2016-02-01

Precursor ion scan and multiple reaction monitoring scan (MRM) are two typical scan modes in mass spectrometry analysis. Here, we developed a strategy by combining stable isotope labeling (IL) with liquid chromatography-mass spectrometry (LC-MS) under double precursor ion scan (DPI) and MRM for analysis of thiols in 5 types of human cancer urine. Firstly, the IL-LC-DPI-MS method was applied for non-targeted profiling of thiols from cancer samples. Compared to traditional full scan mode, the DPI method significantly improved identification selectivity and accuracy. 103 thiol candidates were discovered in all cancers and 6 thiols were identified by their standards. It is worth noting that pantetheine, for the first time, was identified in human urine. Secondly, the IL-LC-MRM-MS method was developed for relative quantification of thiols in cancers compared to healthy controls. All the MRM transitions of light and heavy labeled thiols were acquired from urines by using DPI method. Compared to DPI method, the sensitivity of MRM improved by 2.1-11.3 folds. In addition, the concentration of homocysteine, γ-glutamylcysteine and pantetheine enhanced more than two folds in cancer patients compared to healthy controls. Taken together, the method demonstrated to be a promising strategy for identification and comprehensive quantification of thiols in human urines.

Determination of thiol metabolites in human urine by stable isotope labeling in combination with pseudo-targeted mass spectrometry analysis

PubMed Central

Liu, Ping; Qi, Chu-Bo; Zhu, Quan-Fei; Yuan, Bi-Feng; Feng, Yu-Qi

2016-01-01

Precursor ion scan and multiple reaction monitoring scan (MRM) are two typical scan modes in mass spectrometry analysis. Here, we developed a strategy by combining stable isotope labeling (IL) with liquid chromatography-mass spectrometry (LC-MS) under double precursor ion scan (DPI) and MRM for analysis of thiols in 5 types of human cancer urine. Firstly, the IL-LC-DPI-MS method was applied for non-targeted profiling of thiols from cancer samples. Compared to traditional full scan mode, the DPI method significantly improved identification selectivity and accuracy. 103 thiol candidates were discovered in all cancers and 6 thiols were identified by their standards. It is worth noting that pantetheine, for the first time, was identified in human urine. Secondly, the IL-LC-MRM-MS method was developed for relative quantification of thiols in cancers compared to healthy controls. All the MRM transitions of light and heavy labeled thiols were acquired from urines by using DPI method. Compared to DPI method, the sensitivity of MRM improved by 2.1–11.3 folds. In addition, the concentration of homocysteine, γ-glutamylcysteine and pantetheine enhanced more than two folds in cancer patients compared to healthy controls. Taken together, the method demonstrated to be a promising strategy for identification and comprehensive quantification of thiols in human urines. PMID:26888486

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

Simultaneous determination of eight chemicals in Fufang Xueshuantong capsules by LC-MS-MS with periodic polarity switching.

PubMed

Zhou, Chunhua; Su, Linfei; Shang, Weiding; Rong, Zhihuan; Sun, Mengmeng; Zhang, Kai; Shi, Xiaowei; Wang, Qiao

2015-01-01

A rapid, sensitive, selective and periodic polarity-switching liquid chromatography-tandem mass spectrometry(LC-MS-MS) method was developed for the simultaneous determination of eight components in an important traditional Chinese medicinal capsule, Fufang Xueshuantong. LC analysis was carried out on a Kinetex 2.6 µm XB-C18 column by gradient elution. The detection was performed by multiple reaction monitoring (MRM) with a periodic polarity-switching electrospray ionization (ESI) source across five periods of switching between positive and negative mode. Periodic polarity-switching was used to acquire maximal responses and to simultaneously detect eight constituents in one analysis run lasting 8 min. All of the analytes showed good linearity (r > 0.9947) in the tested ranges. The average recoveries were in the range of 95.4-103.9% with relative standard deviations ≤6.3%, and the intra- and inter-day variations were ≤3.7%. The developed method was successfully employed to analyze three batches of Fufang Xueshuantong capsule samples. The results showed that the levels of notoginsenoside R1, ginsenoside Rb1 and salvianolic acid B were clearly different in different batches of Fufang Xueshuantong capsules. Furthermore, the new established periodic polarity-switching LC-MS-MS method was proven to be highly sensitive and effective in evaluating the quality of Fufang Xueshuantong capsules. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Analysis of catecholamines in urine by unique LC/MS suitable ion-pairing chromatography.

PubMed

Bergmann, Marianne L; Sadjadi, Seyed; Schmedes, Anne

2017-07-01

The catecholamines, epinephrine (E) and norepinephrine (NE) are small polar, hydrophilic molecules, posing significant challenges to liquid chromatography - tandem mass spectrometry (LC-MS/MS) method development. Specifically, these compounds show little retention on conventional reversed-phase liquid chromatography columns. This work presents development and validation of an LC-MS/MS method for determining catecholamines in urine, based on a new approach to ion-pairing chromatography (IPC), in which the ion-pairing reagent (IPR), 1-Heptane Sulfonic Acid (HSA), is added to the extracted samples instead of the mobile phases. A Hamilton STARlet workstation carried out the solid phase extraction of urine samples. The extracted samples were diluted with 60mmol/L HSA and injected on a Kinetex core-shell biphenyl column with conventional LC-MS/MS suitable mobile phases. Chromatographic separation of E and NE was achieved successfully with very stable retention times (RT). In 484 injections, the RTs were steady with a CV of less than ±4%. Furthermore, HSA was separated from E and NE, allowing HSA to be diverted to waste instead of entering the mass spectrometer ion chamber. The method was validated with good analytical performance, and even though the analysis for urinary catecholamines is increasingly being replaced by plasma free metanephrines in diagnosing pheochromocytomas, this work represents the application of a new analytical technique that can be transferred to other small polar molecules, that are difficult to chromatograph on traditional reversed phase columns. Copyright © 2017 Elsevier B.V. All rights reserved.

Developing analytical approaches to explore the connectionbetween endocrine-active pharmaceuticals in waterto effects in fish

EPA Science Inventory

The emphasis of this research project was to develop, and optimize, a solid-phase extraction (SPE) method and high performance liquid chromatography-electrospray ionization- mass spectrometry (LC-MS/MS) method, such that a linkage between the detection of endocrine active pharma...

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.

Development of molecularly imprinted column-on line-two dimensional liquid chromatography for rapidly and selectively monitoring estradiol in cosmetics.

PubMed

Guo, Pengqi; Xu, Xinya; Xian, Liang; Ge, Yanhui; Luo, Zhimin; Du, Wei; Jing, Wanghui; Zeng, Aiguo; Chang, Chun; Fu, Qiang

2016-12-01

Nowadays, the illegal use of estradiol in cosmetics has caused a series of events which endangering public health seriously. Therefore, it is imperative to establish a simple, fast and specific method for monitoring the illegal use of estradiol in cosmetics. In current study, we developed a molecular imprinted monolithic column two dimensional liquid chromatography method (MIMC-2D-LC) for rapid and selective determination of estradiol in various cosmetic samples. The best polymerization, morphology, structure property, surface groups, and the adsorption performance of the prepared material were investigated. The MIMC-2D-LC was validated and successfully used for detecting estradiol in cosmetic samples with good selectivity, sensitivity, efficiency and reproducibility. The linear range of the MIMC-2D-LC for estradiol was 0.5-50μgg -1 with the limit of detection of 0.08μgg -1 . Finally, six batches of cosmetic samples obtained from local markets were tested by the proposed method. The test results showed that the illegal use of estradiol still existed in the commercially available samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Analytical determination of virginiamycin drug residues in edible porcine tissues by LC-MS with confirmation by LC-MS/MS.

PubMed

Boison, Joe; Lee, Stephen; Gedir, Ron

2009-01-01

A liquid chromatographic-mass spectrometric (LC-MS) method was developed and validated for the determination and confirmation of virginiamycin (VMY) M1 residues in porcine liver, kidney, and muscle tissues at concentrations > or =2 ng/g. Porcine liver, kidney, or muscle tissue is homogenized with methanol-acetonitrile. After centrifugation, the supernatant is diluted with phosphate buffer and cleaned up on a C18 solid-phase extraction cartridge. VMY in the eluate is partitioned into chloroform and the aqueous upper layer is removed by aspiration. After evaporating the chloroform in the residual mixture to dryness, the dried extract is reconstituted in mobile phase and VMY is quantified by LC-MS. Any samples eliciting quantifiable levels of VMY M1 (i.e., at concentrations > or =2 ng/g) are subjected to confirmatory analysis by LC-MSIMS. VMY S1, a minor component of the VMY complex, is monitored but not quantified or confirmed.

Misleading measures in Vitamin D analysis: A novel LC-MS/MS assay to account for epimers and isobars

PubMed Central

2011-01-01

Background Recently, the accuracies of many commercially available immunoassays for Vitamin D have been questioned. Liquid chromatography tandem mass spectrometry (LC- MS/MS) has been shown to facilitate accurate separation and quantification of the major circulating metabolite 25-hydroxyvitamin-D3 (25OHD3) and 25-hydroxyvitamin-D2 (25OHD2) collectively termed as 25OHD. However, among other interferents, this method may be compromised by overlapping peaks and identical masses of epimers and isobars, resulting in inaccuracies in circulating 25OHD measurements. The aim of this study was to develop a novel LC-MS/MS method that can accurately identify and quantitate 25OHD3 and 25OHD2 through chromatographic separation of 25OHD from its epimers and isobars. Methods A positive ion electrospray ionisation (ESI) LC-MS/MS method was used in the Multiple Reaction Monitoring (MRM) mode for quantification. It involved i) liquid-liquid extraction, ii) tandem columns (a high resolution ZORBAX C18 coupled to an ULTRON chiral, with guard column and inlet filter), iii) Stanozolol-D3 as internal standard, and iv) identification via ESI and monitoring of three fragmentation transitions. To demonstrate the practical usefulness of our method, blood samples were collected from 5 healthy male Caucasian volunteers; age range 22 to 37 years and 25OHD2, 25OHD3 along with co-eluting epimers and analogues were quantified. Results The new method allowed chromatographic separation and quantification of 25OHD2, 25OHD3, along with 25OHD3 epimer 3-epi-25OHD3 and isobars 1-α-hydroxyvitamin-D3 (1αOHD3), and 7-α-hydroxy-4-cholesten-3-one (7αC4). The new assay was capable of detecting 0.25 ng/mL of all analytes in serum. Conclusions To our knowledge, this is the first specific, reliable, reproducible and robust LC-MS/MS method developed for the accurate detection of 25OHD (Vitamin D). The method is capable of detecting low levels of 25OHD3 and 25OHD2 together with chromatographic separation from the co-eluting epimers and isobars and circumvents other instrumental/analytical interferences. This analytical method does not require time-consuming derivatisation and complex extraction techniques and could prove very useful in clinical studies. PMID:21569549

Viral-genetic tracing of the input-output organization of a central noradrenaline circuit.

PubMed

Schwarz, Lindsay A; Miyamichi, Kazunari; Gao, Xiaojing J; Beier, Kevin T; Weissbourd, Brandon; DeLoach, Katherine E; Ren, Jing; Ibanes, Sandy; Malenka, Robert C; Kremer, Eric J; Luo, Liqun

2015-08-06

Deciphering how neural circuits are anatomically organized with regard to input and output is instrumental in understanding how the brain processes information. For example, locus coeruleus noradrenaline (also known as norepinephrine) (LC-NE) neurons receive input from and send output to broad regions of the brain and spinal cord, and regulate diverse functions including arousal, attention, mood and sensory gating. However, it is unclear how LC-NE neurons divide up their brain-wide projection patterns and whether different LC-NE neurons receive differential input. Here we developed a set of viral-genetic tools to quantitatively analyse the input-output relationship of neural circuits, and applied these tools to dissect the LC-NE circuit in mice. Rabies-virus-based input mapping indicated that LC-NE neurons receive convergent synaptic input from many regions previously identified as sending axons to the locus coeruleus, as well as from newly identified presynaptic partners, including cerebellar Purkinje cells. The 'tracing the relationship between input and output' method (or TRIO method) enables trans-synaptic input tracing from specific subsets of neurons based on their projection and cell type. We found that LC-NE neurons projecting to diverse output regions receive mostly similar input. Projection-based viral labelling revealed that LC-NE neurons projecting to one output region also project to all brain regions we examined. Thus, the LC-NE circuit overall integrates information from, and broadcasts to, many brain regions, consistent with its primary role in regulating brain states. At the same time, we uncovered several levels of specificity in certain LC-NE sub-circuits. These tools for mapping output architecture and input-output relationship are applicable to other neuronal circuits and organisms. More broadly, our viral-genetic approaches provide an efficient intersectional means to target neuronal populations based on cell type and projection pattern.

Scientific developments of liquid crystal-based optical memory: a review

NASA Astrophysics Data System (ADS)

Prakash, Jai; Chandran, Achu; Biradar, Ashok M.

2017-01-01

The memory behavior in liquid crystals (LCs), although rarely observed, has made very significant headway over the past three decades since their discovery in nematic type LCs. It has gone from a mere scientific curiosity to application in variety of commodities. The memory element formed by numerous LCs have been protected by patents, and some commercialized, and used as compensation to non-volatile memory devices, and as memory in personal computers and digital cameras. They also have the low cost, large area, high speed, and high density memory needed for advanced computers and digital electronics. Short and long duration memory behavior for industrial applications have been obtained from several LC materials, and an LC memory with interesting features and applications has been demonstrated using numerous LCs. However, considerable challenges still exist in searching for highly efficient, stable, and long-lifespan materials and methods so that the development of useful memory devices is possible. This review focuses on the scientific and technological approach of fascinating applications of LC-based memory. We address the introduction, development status, novel design and engineering principles, and parameters of LC memory. We also address how the amalgamation of LCs could bring significant change/improvement in memory effects in the emerging field of nanotechnology, and the application of LC memory as the active component for futuristic and interesting memory devices.

Scientific developments of liquid crystal-based optical memory: a review.

PubMed

Prakash, Jai; Chandran, Achu; Biradar, Ashok M

2017-01-01

The memory behavior in liquid crystals (LCs), although rarely observed, has made very significant headway over the past three decades since their discovery in nematic type LCs. It has gone from a mere scientific curiosity to application in variety of commodities. The memory element formed by numerous LCs have been protected by patents, and some commercialized, and used as compensation to non-volatile memory devices, and as memory in personal computers and digital cameras. They also have the low cost, large area, high speed, and high density memory needed for advanced computers and digital electronics. Short and long duration memory behavior for industrial applications have been obtained from several LC materials, and an LC memory with interesting features and applications has been demonstrated using numerous LCs. However, considerable challenges still exist in searching for highly efficient, stable, and long-lifespan materials and methods so that the development of useful memory devices is possible. This review focuses on the scientific and technological approach of fascinating applications of LC-based memory. We address the introduction, development status, novel design and engineering principles, and parameters of LC memory. We also address how the amalgamation of LCs could bring significant change/improvement in memory effects in the emerging field of nanotechnology, and the application of LC memory as the active component for futuristic and interesting memory devices.

Development of an on-line mixed-mode gel liquid chromatography×reversed phase liquid chromatography method for separation of water extract from Flos Carthami.

PubMed

Wang, Yu-Qing; Tang, Xu; Li, Jia-Fu; Wu, Yun-Long; Sun, Yu-Ying; Fang, Mei-Juan; Wu, Zhen; Wang, Xiu-Min; Qiu, Ying-Kun

2017-10-13

A novel on-line comprehensive two-dimensional liquid chromatography (2D-LC) method by coupling mixed-mode gel liquid chromatography (MMG-LC) with reversed phase liquid chromatography (RPLC) was developed. A mixture of 17 reference compounds was used to study the separation mechanism. A crude water extract of Flos Carthami was applied to evaluate the performance of the novel 2D-LC system. In the first dimension, the extract was eluted with a gradient of water/methanol over a cross-linked dextran gel Sephadex LH-20 column. Meanwhile, the advantages of size exclusion, reversed phase partition and adsorption separation mechanism were exploited before further on-line reversed phase purification on the second dimension. This novel on-line mixed-mode Sephadex LH-20×RPLC method provided higher peak resolution, sample processing ability (2.5mg) and better orthogonality (72.9%) versus RPLC×RPLC and hydrophilic interaction liquid chromatography (HILIC)×RPLC. To the best of our knowledge, this is the first report of a mixed-mode Sephadex LH-20×RPLC separation method with successful applications in on-line mode, which might be beneficial for harvesting targets from complicated medicinal plants. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Detection of Peptide-Based Nanoparticles in Blood Plasma by ELISA

PubMed Central

Bode, Gerard H.; Pickl, Karin E.; Sanchez-Purrà, Maria; Albaiges, Berta; Borrós, Salvador; Pötgens, Andy J. G.; Schmitz, Christoph; Sinner, Frank M.; Losen, Mario; Steinbusch, Harry W. M.; Frank, Hans-Georg; Martinez-Martinez, Pilar

2015-01-01

Aims The aim of the current study was to develop a method to detect peptide-linked nanoparticles in blood plasma. Materials & Methods A convenient enzyme linked immunosorbent assay (ELISA) was developed for the detection of peptides functionalized with biotin and fluorescein groups. As a proof of principle, polymerized pentafluorophenyl methacrylate nanoparticles linked to biotin-carboxyfluorescein labeled peptides were intravenously injected in Wistar rats. Serial blood plasma samples were analyzed by ELISA and by liquid chromatography mass spectrometry (LC/MS) technology. Results The ELISA based method for the detection of FITC labeled peptides had a detection limit of 1 ng/mL. We were able to accurately measure peptides bound to pentafluorophenyl methacrylate nanoparticles in blood plasma of rats, and similar results were obtained by LC/MS. Conclusions We detected FITC-labeled peptides on pentafluorophenyl methacrylate nanoparticles after injection in vivo. This method can be extended to detect nanoparticles with different chemical compositions. PMID:25996618

Multiplexed LC-MS/MS analysis of horse plasma proteins to study doping in sport.

PubMed

Barton, Chris; Beck, Paul; Kay, Richard; Teale, Phil; Roberts, Jane

2009-06-01

The development of protein biomarkers for the indirect detection of doping in horse is a potential solution to doping threats such as gene and protein doping. A method for biomarker candidate discovery in horse plasma is presented using targeted analysis of proteotypic peptides from horse proteins. These peptides were first identified in a novel list of the abundant proteins in horse plasma. To monitor these peptides, an LC-MS/MS method using multiple reaction monitoring was developed to study the quantity of 49 proteins in horse plasma in a single run. The method was optimised and validated, and then applied to a population of race-horses to study protein variance within a population. The method was finally applied to longitudinal time courses of horse plasma collected after administration of an anabolic steroid to demonstrate utility for hypothesis-driven discovery of doping biomarker candidates.

L-C Measurement Acquisition Method for Aerospace Systems

NASA Technical Reports Server (NTRS)

Woodard, Stanley E.; Taylor, B. Douglas; Shams, Qamar A.; Fox, Robert L.

2003-01-01

This paper describes a measurement acquisition method for aerospace systems that eliminates the need for sensors to have physical connection to a power source (i.e., no lead wires) or to data acquisition equipment. Furthermore, the method does not require the sensors to be in proximity to any form of acquisition hardware. Multiple sensors can be interrogated using this method. The sensors consist of a capacitor, C(p), whose capacitance changes with changes to a physical property, p, electrically connected to an inductor, L. The method uses an antenna to broadcast electromagnetic energy that electrically excites one or more inductive-capacitive sensors via Faraday induction. This method facilitates measurements that were not previously possible because there was no practical means of providing power and data acquisition electrical connections to a sensor. Unlike traditional sensors, which measure only a single physical property, the manner in which the sensing element is interrogated simultaneously allows measurement of at least two unrelated physical properties (e.g., displacement rate and fluid level) by using each constituent of the L-C element. The key to using the method for aerospace applications is to increase the distance between the L-C elements and interrogating antenna; develop all key components to be non-obtrusive and to develop sensing elements that can easily be implemented. Techniques that have resulted in increased distance between antenna and sensor will be presented. Fluid-level measurements and pressure measurements using the acquisition method are demonstrated in the paper.

Design of experiments as a tool for LC-MS/MS method development for the trace analysis of the potentially genotoxic 4-dimethylaminopyridine impurity in glucocorticoids.

PubMed

Székely, Gy; Henriques, B; Gil, M; Ramos, A; Alvarez, C

2012-11-01

The present study reports on a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method development strategy supported by design of experiments (DoE) for the trace analysis of 4-dimethylaminopyridine (DMAP). The conventional approaches for development of LC-MS/MS methods are usually via trial and error, varying intentionally the experimental factors which is time consuming and interactions between experimental factors are not considered. The LC factors chosen for the DoE study include flow (F), gradient (G) and injection volume (V(inj)) while cone voltage (E(con)) and collision energy (E(col)) were chosen as MS parameters. All of the five factors were studied simultaneously. The method was optimized with respect to four responses: separation of peaks (Sep), peak area (A(peak)), length of the analysis (T) and the signal to noise ratio (S/N). A quadratic model, namely central composite face (CCF) featuring 29 runs was used instead of a less powerful linear model since the increase in the number of injections was insignificant. In order to determine the robustness of the method a new set of DoE experiments was carried out applying robustness around the optimal conditions was evaluated applying a fractional factorial of resolution III with 11 runs, wherein additional factors - such as column temperature and quadrupole resolution - were considered. The method utilizes a Phenomenex Gemini NX C-18 HPLC analytical column with electrospray ionization and a triple quadrupole mass detector in multiple reaction monitoring (MRM) mode, resulting in short analyses with a 10min runtime. Drawbacks of derivatization, namely incomplete reaction and time consuming sample preparation, have been avoided and the change from SIM to MRM mode resulted in increased sensitivity and lower LOQ. The DoE method development strategy led to a method allowing the trace analysis of DMAP at 0.5 ng/ml absolute concentration which corresponds to a 0.1 ppm limit of quantification in 5mg/ml mometasone furoate glucocorticoid. The obtained method was validated in a linear range of 0.1-10 ppm and presented a %RSD of 0.02% for system precision. Regarding DMAP recovery in mometasone furoate, spiked samples produced %recoveries between 83 and 113% in the range of 0.1-2 ppm. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

A review of lipidomic technologies applicable to sphingolipidomics and their relevant applications

PubMed Central

Han, Xianlin; Jiang, Xuntian

2009-01-01

Sphingolipidomics, a branch of lipidomics, focuses on the large-scale study of the cellular sphingolipidomes. In the current review, two main approaches for the analysis of cellular sphingolipidomes (i.e. LC-MS- or LC-MS/MS-based approach and shotgun lipidomics-based approach) are briefly discussed. Their advantages, some considerations of these methods, and recent applications of these approaches are summarized. It is the authors’ sincere hope that this review article will add to the readers understanding of the advantages and limitations of each developed method for the analysis of a cellular sphingolipidome. PMID:19690629

A combined A431 cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening compounds from total alkaloid of Radix Caulophylli acting on the human EGFR.

PubMed

Sun, Meng; Ren, Jing; Du, Hui; Zhang, Yanmin; Zhang, Jie; Wang, Sicen; He, Langchong

2010-10-15

We have developed an online analytical method that combines A431 cell membrane chromatography (A431/CMC) with high performance liquid chromatography and mass spectrometry (LC/MS) for identifying active components from Radix Caulophylli acting on human EGFR. Retention fractions on A431/CMC model were captured onto an enrichment column and the components were directly analyzed by combining a 10-port column switcher with an LC/MS system for separation and preliminary identification. Using Sorafenib tosylate as a positive control, taspine and caulophine from Radix Caulophylli were identified as the active molecules which could act on the EGFR. This A431/CMC-online-LC/MS method can be applied for screening active components acting on EGFR from traditional Chinese medicines exemplified by Radix Caulophylli and will be of great utility in drug discovery using natural medicinal herbs as a source of novel compounds. Copyright © 2010 Elsevier B.V. All rights reserved.

Characterization of cellulose acetates according to DS and molar mass using two-dimensional chromatography.

PubMed

Ghareeb, Hewa Othman; Radke, Wolfgang

2013-11-06

A two-dimensional liquid chromatographic method (2D LC) was developed to analyze the heterogeneities of cellulose acetates (CA) in the DS-range DS=1.5-2.9 with respect to both, molar mass and degree of substitution (DS). The method uses gradient liquid chromatography (HPLC) as the first dimension in order to separate by DS followed by separation of the different fractions by size (SEC) in the second dimension. The 2D experiments revealed different correlations between gradient and SEC elution volume. These correlations might arise from differences in the synthetic conditions. The newly developed 2D LC separation therefore provides new insights into the heterogeneity of CAs. Copyright © 2013 Elsevier Ltd. All rights reserved.

Precision Nutrition and Omega-3 Polyunsaturated Fatty Acids: A Case for Personalized Supplementation Approaches for the Prevention and Management of Human Diseases

PubMed Central

Chilton, Floyd H.; Dutta, Rahul; Reynolds, Lindsay M.; Sergeant, Susan; Mathias, Rasika A.; Seeds, Michael C.

2017-01-01

Background: Dietary essential omega-6 (n-6) and omega-3 (n-3) 18 carbon (18C-) polyunsaturated fatty acids (PUFA), linoleic acid (LA) and α-linolenic acid (ALA), can be converted (utilizing desaturase and elongase enzymes encoded by FADS and ELOVL genes) to biologically-active long chain (LC; >20)-PUFAs by numerous cells and tissues. These n-6 and n-3 LC-PUFAs and their metabolites (ex, eicosanoids and endocannabinoids) play critical signaling and structural roles in almost all physiologic and pathophysiologic processes. Methods: This review summarizes: (1) the biosynthesis, metabolism and roles of LC-PUFAs; (2) the potential impact of rapidly altering the intake of dietary LA and ALA; (3) the genetics and evolution of LC-PUFA biosynthesis; (4) Gene–diet interactions that may lead to excess levels of n-6 LC-PUFAs and deficiencies of n-3 LC-PUFAs; and (5) opportunities for precision nutrition approaches to personalize n-3 LC-PUFA supplementation for individuals and populations. Conclusions: The rapid nature of transitions in 18C-PUFA exposure together with the genetic variation in the LC-PUFA biosynthetic pathway found in different populations make mal-adaptations a likely outcome of our current nutritional environment. Understanding this genetic variation in the context of 18C-PUFA dietary exposure should enable the development of individualized n-3 LC-PUFA supplementation regimens to prevent and manage human disease. PMID:29068398

Quasi-targeted analysis of hydroxylation-related metabolites of polycyclic aromatic hydrocarbons in human urine by liquid chromatography-mass spectrometry.

PubMed

Tang, Caiming; Tan, Jianhua; Fan, Ruifang; Zhao, Bo; Tang, Caixing; Ou, Weihui; Jin, Jiabin; Peng, Xianzhi

2016-08-26

Metabolite identification is crucial for revealing metabolic pathways and comprehensive potential toxicities of polycyclic aromatic hydrocarbons (PAHs) in human body. In this work, a quasi-targeted analysis strategy was proposed for metabolite identification of monohydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) in human urine using liquid chromatography triple quadruple mass spectrometry (LC-QqQ-MS/MS) combined with liquid chromatography high resolution mass spectrometry (LC-HRMS). Potential metabolites of OH-PAHs were preliminarily screened out by LC-QqQ-MS/MS in association with filtering in a self-constructed information list of possible metabolites, followed by further identification and confirmation with LC-HRMS. The developed method can provide more reliable and systematic results compared with traditional untargeted analysis using LC-HRMS. In addition, data processing for LC-HRMS analysis were greatly simplified. This quasi-targeted analysis method was successfully applied to identifying phase I and phase II metabolites of OH-PAHs in human urine. Five metabolites of hydroxynaphthalene, seven of hydroxyfluorene, four of hydroxyphenanthrene, and three of hydroxypyrene were tentatively identified. Metabolic pathways of PAHs in human body were putatively revealed based on the identified metabolites. The experimental results will be valuable for investigating the metabolic processes of PAHs in human body, and the quasi-targeted analysis strategy can be expanded to the metabolite identification and profiling of other compounds in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

Validated reversed phase LC method for quantitative analysis of polymethoxyflavones in citrus peel extracts.

PubMed

Wang, Zhenyu; Li, Shiming; Ferguson, Stephen; Goodnow, Robert; Ho, Chi-Tang

2008-01-01

Polymethoxyflavones (PMFs), which exist exclusively in the citrus genus, have biological activities including anti-inflammatory, anticarcinogenic, and antiatherogenic properties. A validated RPLC method was developed for quantitative analysis of six major PMFs, namely nobiletin, tangeretin, sinensetin, 5,6,7,4'-tetramethoxyflavone, 3,5,6,7,3',4'-hexamethoxyflavone, and 3,5,6,7,8,3',4'-heptamethoxyflavone. The polar embedded LC stationary phase was able to fully resolve the six analogues. The developed method was fully validated in terms of linearity, accuracy, precision, sensitivity, and system suitability. The LOD of the method was calculated as 0.15 microg/mL and the recovery rate was between 97.0 and 105.1%. This analytical method was successfully applied to quantify the individual PMFs in four commercially available citrus peel extracts (CPEs). Each extract shows significant difference in the PMF composition and concentration. This method may provide a simple, rapid, and reliable tool to help reveal the correlation between the bioactivity of the PMF extracts and the individual PMF content.

[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.

Misleading measures in Vitamin D analysis: a novel LC-MS/MS assay to account for epimers and isobars.

PubMed

Shah, Iltaf; James, Ricky; Barker, James; Petroczi, Andrea; Naughton, Declan P

2011-05-14

Recently, the accuracies of many commercially available immunoassays for Vitamin D have been questioned. Liquid chromatography tandem mass spectrometry (LC- MS/MS) has been shown to facilitate accurate separation and quantification of the major circulating metabolite 25-hydroxyvitamin-D3 (25OHD3) and 25-hydroxyvitamin-D2 (25OHD2) collectively termed as 25OHD. However, among other interferents, this method may be compromised by overlapping peaks and identical masses of epimers and isobars, resulting in inaccuracies in circulating 25OHD measurements. The aim of this study was to develop a novel LC-MS/MS method that can accurately identify and quantitate 25OHD3 and 25OHD2 through chromatographic separation of 25OHD from its epimers and isobars. A positive ion electrospray ionisation (ESI) LC-MS/MS method was used in the Multiple Reaction Monitoring (MRM) mode for quantification. It involved i) liquid-liquid extraction, ii) tandem columns (a high resolution ZORBAX C18 coupled to an ULTRON chiral, with guard column and inlet filter), iii) Stanozolol-D3 as internal standard, and iv) identification via ESI and monitoring of three fragmentation transitions. To demonstrate the practical usefulness of our method, blood samples were collected from 5 healthy male Caucasian volunteers; age range 22 to 37 years and 25OHD2, 25OHD3 along with co-eluting epimers and analogues were quantified. The new method allowed chromatographic separation and quantification of 25OHD2, 25OHD3, along with 25OHD3 epimer 3-epi-25OHD3 and isobars 1-α-hydroxyvitamin-D3 (1αOHD3), and 7-α-hydroxy-4-cholesten-3-one (7αC4). The new assay was capable of detecting 0.25 ng/mL of all analytes in serum. To our knowledge, this is the first specific, reliable, reproducible and robust LC-MS/MS method developed for the accurate detection of 25OHD (Vitamin D). The method is capable of detecting low levels of 25OHD3 and 25OHD2 together with chromatographic separation from the co-eluting epimers and isobars and circumvents other instrumental/analytical interferences. This analytical method does not require time-consuming derivatisation and complex extraction techniques and could prove very useful in clinical studies.

Simultaneous determination of water-soluble vitamins in SRM 1849 Infant/Adult Nutritional Formula powder by liquid chromatography-isotope dilution mass spectrometry.

PubMed

Goldschmidt, Robert J; Wolf, Wayne R

2010-05-01

Assessing dietary intake of vitamins from all sources, including foods, dietary supplements, and fortified foods, would be aided considerably by having analytical methodologies that are capable of simultaneous determination of several vitamins. Vitamins naturally present in foods may occur in different chemical forms, with levels ranging over several orders of magnitude. Vitamins in dietary supplements and fortified foods, however, are typically added in a single chemical form, and matrix issues are usually not as complex. These sources should thus be relatively amenable to approaches that aim for simultaneous determination of multiple vitamins. Our recent work has focused on development of liquid chromatography (LC)-UV/fluorescence and LC-tandem mass spectrometry methods for the simultaneous determination of water-soluble vitamins (thiamine, niacin, pyridoxine, pantothenic acid, folic acid, biotin, and riboflavin) in dietary supplement tablets and fortified foods, such as formula powders and breakfast cereals. As part of the validation of our methods and collaboration in characterization of a new NIST SRM 1849 Infant/Adult Nutritional Formula powder, we report data on SRM 1849 using isotope dilution mass spectrometric methods. Use of available NIST Standard Reference Materials(R) as test matrices in our method development and validation gives a benchmark for future application of these methods. We compare three chromatographic approaches and provide data on stability of vitamin standard solutions for LC-based multiple vitamin determinations.

Data representing two separate LC-MS methods for detection and quantification of water-soluble and fat-soluble vitamins in tears and blood serum.

PubMed

Khaksari, Maryam; Mazzoleni, Lynn R; Ruan, Chunhai; Kennedy, Robert T; Minerick, Adrienne R

2017-04-01

Two separate liquid chromatography (LC)-mass spectrometry (MS) methods were developed for determination and quantification of water-soluble and fat-soluble vitamins in human tear and blood serum samples. The water-soluble vitamin method was originally developed to detect vitamins B 1 , B 2 , B 3 (nicotinamide), B 5 , B 6 (pyridoxine), B 7 , B 9 and B 12 while the fat-soluble vitamin method detected vitamins A, D 3 , 25(OH)D 3, E and K 1 . These methods were then validated with tear and blood serum samples. In this data in brief article, we provide details on the two LC-MS methods development, methods sensitivity, as well as precision and accuracy for determination of vitamins in human tears and blood serum. These methods were then used to determine the vitamin concentrations in infant and parent samples under a clinical study which were reported in "Determination of Water-Soluble and Fat-Soluble Vitamins in Tears and Blood Serum of Infants and Parents by Liquid Chromatography/Mass Spectrometry DOI:10.1016/j.exer.2016.12.007 [1]". This article provides more details on comparison of vitamin concentrations in the samples with the ranges reported in the literature along with the medically accepted normal ranges. The details on concentrations below the limits of detection (LOD) and limits of quantification (LOQ) are also discussed. Vitamin concentrations were also compared and cross-correlated with clinical data and nutritional information. Significant differences and strongly correlated data were reported in [1]. This article provides comprehensive details on the data with slight differences or slight correlations.

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.

Analysis of beta-lactam antibiotics in incurred raw milk by rapid test methods and liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

PubMed

Riediker, S; Diserens, J M; Stadler, R H

2001-09-01

A recently developed confirmatory LC-MS method has been applied to the quantification of five major beta-lactam antibiotics in suspect raw bovine milk samples that gave a positive response with receptor-based (BetaStar) and rapid microbial inhibitory screen tests (Delvotest SP). In total, 18 presumptive positive raw milk samples were reanalyzed; 16 samples showed traces of antibiotic residues that could be identified and quantified by the LC-MS method, ranging from the limits of confirmation up to 38 microg/kg. Of the positive samples, only five (approximately 30%) were found to be violative of EU maximum residue limits. The most frequently detected antibiotic residues were cloxacillin and penicillin G, the former often in combination with amoxicillin or ampicillin. This study compares the results obtained by the three methods on identical samples and addresses how these relate to certain criteria such as sensitivity and selectivity. Furthermore, the limitations of the LC-MS method and the potential impact of the presence of frequently more than one residue in the same milk sample on the response of the rapid test methods are discussed.

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.

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.

General unknown screening procedure for the characterization of human drug metabolites in forensic toxicology: applications and constraints.

PubMed

Sauvage, François-Ludovic; Picard, Nicolas; Saint-Marcoux, Franck; Gaulier, Jean-Michel; Lachâtre, Gérard; Marquet, Pierre

2009-09-01

LC coupled to single (LC-MS) and tandem (LC-MS/MS) mass spectrometry is recognized as the most powerful analytical tools for metabolic studies in drug discovery. In this article, we describe five cases illustrating the utility of screening xenobiotic metabolites in routine analysis of forensic samples using LC-MS/MS. Analyses were performed using a previously published LC-MS/MS general unknown screening (GUS) procedure developed using a hybrid linear IT-tandem mass spectrometer. In each of the cases presented, the presence of metabolites of xenobiotics was suspected after analyzing urine samples. In two cases, the parent drug was also detected and the metabolites were merely useful to confirm drug intake, but in three other cases, metabolite detection was of actual forensic interest. The presented results indicate that: (i) the GUS procedure developed is useful to detect a large variety of drug metabolites, which would have been hardly detected using targeted methods in the context of clinical or forensic toxicology; (ii) metabolite structure can generally be inferred from their "enhanced" product ion scan spectra; and (iii) structure confirmation can be achieved through in vitro metabolic experiments or through the analysis of urine samples from individuals taking the parent drug.

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

Determination of benzoylurea insecticides in food by pressurized liquid extraction and LC-MS.

PubMed

Brutti, Monia; Blasco, Cristina; Picó, Yolanda

2010-01-01

A method based on pressurized liquid extraction and LC-MS/MS has been developed for determining nine benzoylureas (BUs) in fruit, vegetable, cereals, and animal products. Samples (5 g) were homogenized with diatomaceous earth and extracted in a 22 mL cell with 22 mL of ethyl acetate at 80 degrees C and 1500 psi. After solvent concentration and exchange to methanol, BUs were analyzed by LC-MS/MS using an IT mass analyzer, which achieved several transitions of precursor ions that increase selectivity providing identification. LOQs were between 0.002 and 0.01 mg/kg, which are equal or lower than maximum residue limits established by the Codex Alimentarius. Excellent linearity was achieved over a range of concentrations from 0.01 to 1 mg/kg with correlation coefficients 0.995-0.999 (n=7). Validation of the total method was performed by analyzing in quintuplicate seven different commodities (milk, eggs, meat, rice, lettuce, avocado, and lemon) at three concentration levels (0.01, 0.1, and 1 mg/kg). The recoveries ranged from 58 to 97% and the RSDs from 5 to 19% depending on the compound and the commodity. The combination of pressurized liquid extraction with LC-MS/MS provides a sensitive and selective method for the determination of BUs in food.

Validation of LC-TOF-MS screening for drugs, metabolites, and collateral compounds in forensic toxicology specimens.

PubMed

Guale, Fessessework; Shahreza, Shahriar; Walterscheid, Jeffrey P; Chen, Hsin-Hung; Arndt, Crystal; Kelly, Anna T; Mozayani, Ashraf

2013-01-01

Liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) analysis provides an expansive technique for identifying many known and unknown analytes. This study developed a screening method that utilizes automated solid-phase extraction to purify a wide array of analytes involving stimulants, benzodiazepines, opiates, muscle relaxants, hypnotics, antihistamines, antidepressants and newer synthetic "Spice/K2" cannabinoids and cathinone "bath salt" designer drugs. The extract was applied to LC-TOF-MS analysis, implementing a 13 min chromatography gradient with mobile phases of ammonium formate and methanol using positive mode electrospray. Several common drugs and metabolites can share the same mass and chemical formula among unrelated compounds, but they are structurally different. In this method, the LC-TOF-MS was able to resolve many isobaric compounds by accurate mass correlation within 15 ppm mass units and a narrow retention time interval of less than 10 s of separation. Drug recovery yields varied among spiked compounds, but resulted in overall robust area counts to deliver an average match score of 86 when compared to the retention time and mass of authentic standards. In summary, this method represents a rapid, enhanced screen for blood and urine specimens in postmortem, driving under the influence, and drug facilitated sexual assault forensic toxicology casework.

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

METHOD 544. DETERMINATION OF MICROCYSTINS AND NODULARIN IN DRINKING WATER BY SOLID PHASE EXTRACTION AND LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY (LC/MS/MS)

EPA Science Inventory

Method 544 is an accurate and precise analytical method to determine six microcystins (including MC-LR) and nodularin in drinking water using solid phase extraction and liquid chromatography tandem mass spectrometry (SPE-LC/MS/MS). The advantage of this SPE-LC/MS/MS is its sensi...

Primary care physicians' educational needs and learning preferences in end of life care: A focus group study in the UK.

PubMed

Selman, Lucy Ellen; Brighton, Lisa Jane; Robinson, Vicky; George, Rob; Khan, Shaheen A; Burman, Rachel; Koffman, Jonathan

2017-03-09

Primary care physicians (General Practitioners (GPs)) play a pivotal role in providing end of life care (EoLC). However, many lack confidence in this area, and the quality of EoLC by GPs can be problematic. Evidence regarding educational needs, learning preferences and the acceptability of evaluation methods is needed to inform the development and testing of EoLC education. This study therefore aimed to explore GPs' EoLC educational needs and preferences for learning and evaluation. A qualitative focus group study was conducted with qualified GPs and GP trainees in the UK. Audio recordings were transcribed and analysed thematically. Expert review of the coding frame and dual coding of transcripts maximised rigour. Twenty-eight GPs (10 fully qualified, 18 trainees) participated in five focus groups. Four major themes emerged: (1) why education is needed, (2) perceived educational needs, (3) learning preferences, and (4) evaluation preferences. EoLC was perceived as emotionally and clinically challenging. Educational needs included: identifying patients for palliative care; responsibilities and teamwork; out-of-hours care; having difficult conversations; symptom management; non-malignant conditions; and paediatric palliative care. Participants preferred learning through experience, working alongside specialist palliative care staff, and discussion of real cases, to didactic methods and e-learning. 360° appraisals and behavioural assessment using videoing or simulated interactions were considered problematic. Self-assessment questionnaires and patient and family outcome measures were acceptable, if used and interpreted correctly. GPs require education and support in EoLC, particularly the management of complex clinical care and counselling. GPs value mentoring, peer-support, and experiential learning alongside EoLC specialists over formal training.

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.

Online immunoaffinity LC/MS/MS. A general method to increase sensitivity and specificity: How do you do it and what do you need?

PubMed

Dufield, Dawn R; Radabaugh, Melissa R

2012-02-01

There is an increased emphasis on hyphenated techniques such as immunoaffinity LC/MS/MS (IA-LC/MS/MS) or IA-LC/MRM. These techniques offer competitive advantages with respect to sensitivity and selectivity over traditional LC/MS and are complementary to ligand binding assays (LBA) or ELISA's. However, these techniques are not entirely straightforward and there are several tips and tricks to routine sample analysis. We describe here our methods and procedures for how to perform online IA-LC/MS/MS including a detailed protocol for the preparation of antibody (Ab) enrichment columns. We have included sample trapping and Ab methods. Furthermore, we highlight tips, tricks, minimal and optimal approaches. This technology has been shown to be viable for several applications, species and fluids from small molecules to proteins and biomarkers to PK assays. Copyright © 2011 Elsevier Inc. All rights reserved.

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.

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.

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.

Determination of drugs in surface water and wastewater samples by liquid chromatography-mass spectrometry: Methods and preliminary results including toxicity studies with Vibrio fischeri

USGS Publications Warehouse

Farre, M.; Ferrer, I.; Ginebreda, A.; Figueras, M.; Olivella, L.; Tirapu, L.; Vilanova, M.; Barcelo, D.

2001-01-01

In the present work a combined analytical method involving toxicity and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) was developed for the determination of pharmaceutical compounds in water samples. The drugs investigated were the analgesics: ibuprofen, ketoprofen, naproxen, and diclofenac, the decomposition product of the acetyl salicylic acid: salicylic acid and one lipid lowering agent, gemfibrozil. The selected compounds are acidic substances, very polar and all of them are analgesic compounds that can be purchased without medical prescription. The developed protocol consisted, first of all, on the use Microtox?? and ToxAlert??100 toxicity tests with Vibrio fischeri for the different pharmaceutical drugs. The 50% effective concentration (EC50) values and the toxicity units (TU) were determined for every compound using both systems. Sample enrichment of water samples was achieved by solid-phase extraction procedure (SPE), using the Merck LiChrolut?? EN cartridges followed by LC-ESI-MS. Average recoveries loading 1 l of samples with pH=2 varied from 69 to 91% and the detection limits in the range of 15-56 ng/l. The developed method was applied to real samples from wastewater and surface-river waters of Catalonia (north-east of Spain). One batch of samples was analyzed in parallel also by High Resolution Gas Chromatography coupled with Mass Spectrometry (HRGC-MS) and the results have been compared with the LC-ESI-MS method developed in this work. ?? 2001 Elsevier Science B.V. All rights reserved.

Detecting a wide range of environmental contaminants in human blood samples--combining QuEChERS with LC-MS and GC-MS methods.

PubMed

Plassmann, Merle M; Schmidt, Magdalena; Brack, Werner; Krauss, Martin

2015-09-01

Exposure to environmental pollution and consumer products may result in an uptake of chemicals into human tissues. Several studies have reported the presence of diverse environmental contaminants in human blood samples. However, previously developed multi-target methods for the analysis of human blood include a fairly limited amount of compounds stemming from one or two related compound groups. Thus, the sample preparation method QuEChERS (quick easy cheap effective rugged and safe) was tested for the extraction of 64 analytes covering a broad compound domain followed by detection using liquid and gas chromatography coupled to mass spectrometry (LC- and GC-MS). Forty-seven analytes showed absolute recoveries above 70% in the first QuEChERS step, being a simple liquid-liquid extraction (LLE) using acetonitrile and salt. The second QuEChERS step, being a dispersive solid phase extraction, did not result in an overall improvement of recoveries or removal of background signals. Using solely the LLE step, eight analytes could subsequently be detected in human blood samples from the German Environmental Specimen Bank. Using a LC-multiple reaction monitoring (MRM) method with a triple quadrupole instrument, better recoveries were achieved than with an older LC-high-resolution (HR) MS full scan orbitrap instrument, which required a higher concentration factor of the extracts. However, the application of HRMS full scan methods could be used for the detection of additional compounds retrospectively.

The rapid and direct determination of ATP-ase activity by ion exchange chromatography and the application to the activity of heat shock protein-90

PubMed Central

Bartolini, Manuela; Wainer, Irving W.; Bertucci, Carlo; Andrisano, Vincenza

2012-01-01

Adenosine nucleotides are involved as substrates or co-factors in several biochemical reactions, catalyzed by enzymes, which modulate energy production, signal transduction and cell proliferation. We here report the development and optimization of an ion exchange liquid chromatography (LC) method for the determination of ATP, ADP and AMP. This method is specifically aimed at the determination of the ATP-ase activity of human heat shock protein 90 (Hsp90), a molecular chaperone that has emerged as target enzyme in cancer therapy. Separation of the three nucleotides was achieved in a 15-min run by using a disk shaped monolithic ethylene diamine stationary phase of small dimensions (2×6 mm i.d.), under a three-solvent gradient elution mode and UV detection at 256 nm. The described direct LC method resulted highly specific as a consequence of the baseline separation of the three adenosine nucleotides and could be applied to the determination of the enzymatic activity of ADP/ATP generating or consuming enzymes (such as kinases). Furthermore, comparison of the LOD and LOQ values of the LC method with those obtained with the malachite green assay, which is one of the most used indirect screening methodologies for ATP-ase activity, showed that the LC method has a similar range of application without presenting the drawbacks related to contamination by inorganic phosphate ions and glycerol, which are present in Hsp90 commercial samples. PMID:22497853

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.

Application of a new vitamin D blood test on the Emirati population.

PubMed

Shah, Iltaf; Al-Dabbagh, Bayan; Gariballa, Salah; Al-Menhali, Asma; Muhammad, Neak; Yasin, Javed; Ashraf, S Salman

2018-06-01

Research shows that immunoassay techniques are not the best choice for the estimation of vitamin D in human blood samples. The main reasons are that some immunoassays are not able to distinguish between 25-OHD3 and 25-OHD2 vitamin D metabolites. Furthermore, immunoassays cannot differentiate between 25OHD and inactive epimers of vitamin D. Vitamin D epimers and isobars have been known to overlap with the 25OHD signals and give false positives when tested. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) can differentiate between 25OHD3 and 25OHD2. Separating epimers and isobars (which have the same molecular weight) from vitamin D is achieved through chromatographic separation from actual 25OHD peaks, although this could also cause inaccuracies in vitamin D measurements. The main aim of this study was to develop and validate an improved LC-MS/MS method (using a Shimadzu 8060 system) that could accurately detect and quantitate up to 10 different metabolites of vitamin D, as well as differentiate the epimers and isobars. The secondary aim was to apply the developed LC-MS/MS method for the accurate measurement of blood vitamin D levels in the Emirati population. The Shimadzu 8060 system was run using positive ion electrospray ionization (ESI) in Dynamic Multiple Reaction Monitoring (DMRM) mode for quantification. The method involved blood sample collection from 80 Emirati volunteers, followed by serum extraction and liquid-liquid extraction. The chromatography column used for the analysis was an Ascentis Express F5. Precursor and product ions were detected using a Shimadzu 8060 LC-MS/MS system, and 10 metabolites of vitamin D were detected and quantified, including epimers and isobars. The method validation showed good sensitivity, recovery, linearity, precision, specificity, and accuracy. Furthermore, the data showed that vitamin D epimer 3-epi-25OHD and isobar 7-α-hydroxy-4-cholesten-3-one (7αC4) accounted for a significant portion of vitamin D results in the Emirati population. We report a more reliable, reproducible, and robust LC-MS/MS method for the accurate detection of 25OHD (vitamin D) in the Emirati population. The method has the capacity to detect and separate 10 metabolites of vitamin D as well as separate 25OHD from co-eluting epimers and isobars. The method has also been successfully implemented in gauging vitamin D deficiency in the Emirati population. Thus, this improved LC-MS/MS method could prove very useful in accurately estimating the levels of vitamin D in the Emirati population and in further clinical studies. Copyright © 2018 Elsevier Ltd. All rights reserved.

Improvement of a stability-indicating method by Quality-by-Design versus Quality-by-Testing: a case of a learning process.

PubMed

Hubert, C; Lebrun, P; Houari, S; Ziemons, E; Rozet, E; Hubert, Ph

2014-01-01

The understanding of the method is a major concern when developing a stability-indicating method and even more so when dealing with impurity assays from complex matrices. In the presented case study, a Quality-by-Design approach was applied in order to optimize a routinely used method. An analytical issue occurring at the last stage of a long-term stability study involving unexpected impurities perturbing the monitoring of characterized impurities needed to be resolved. A compliant Quality-by-Design (QbD) methodology based on a Design of Experiments (DoE) approach was evaluated within the framework of a Liquid Chromatography (LC) method. This approach allows the investigation of Critical Process Parameters (CPPs), which have an impact on Critical Quality Attributes (CQAs) and, consequently, on LC selectivity. Using polynomial regression response modeling as well as Monte Carlo simulations for error propagation, Design Space (DS) was computed in order to determine robust working conditions for the developed stability-indicating method. This QbD compliant development was conducted in two phases allowing the use of the Design Space knowledge acquired during the first phase to define the experimental domain of the second phase, which constitutes a learning process. The selected working condition was then fully validated using accuracy profiles based on statistical tolerance intervals in order to evaluate the reliability of the results generated by this LC/ESI-MS stability-indicating method. A comparison was made between the traditional Quality-by-Testing (QbT) approach and the QbD strategy, highlighting the benefit of this QbD strategy in the case of an unexpected impurities issue. On this basis, the advantages of a systematic use of the QbD methodology were discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

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

Comparison study of two procedures for the determination of emamectin benzoate in medicated fish feed.

PubMed

Farer, Leslie J; Hayes, John M

2005-01-01

A new method has been developed for the determination of emamectin benzoate in fish feed. The method uses a wet extraction, cleanup by solid-phase extraction, and quantitation and separation by liquid chromatography (LC). In this paper, we compare the performance of this method with that of a previously reported LC assay for the determination of emamectin benzoate in fish feed. Although similar to the previous method, the new procedure uses a different sample pretreatment, wet extraction, and quantitation method. The performance of the new method was compared with that of the previously reported method by analyses of 22 medicated feed samples from various commercial sources. A comparison of the results presented here reveals slightly lower assay values obtained with the new method. Although a paired sample t-test indicates the difference in results is significant, this difference is within the method precision of either procedure.

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.

Simple and rapid LC-MS/MS method for the absolute determination of cetuximab in human serum using an immobilized trypsin.

PubMed

Shibata, Kaito; Naito, Takafumi; Okamura, Jun; Hosokawa, Seiji; Mineta, Hiroyuki; Kawakami, Junichi

2017-11-30

Proteomic approaches using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) without an immunopurification technique have not been applied to the determination of serum cetuximab. This study developed a simple and rapid LC-MS/MS method for the absolute determination of cetuximab in human serum and applied it to clinical settings. Surrogate peptides derived from cetuximab digests were selected using a Fourier transform mass spectrometer. Reduced-alkylated serum cetuximab without immunopurification was digested for 20minutes using immobilized trypsin, and the digestion products were purified by solid-phase extraction. The LC-MS/MS was run in positive ion multiple reaction monitoring mode. This method was applied to the determination of serum samples in head and neck cancer patients treated with cetuximab. The chromatographic run time was 10minutes and no peaks interfering with surrogate peptides in serum digestion products were observed. The calibration curve of absolute cetuximab in serum was linear over the concentration range of 4-200μg/mL. The lower limit of quantification of cetuximab in human serum was 4μg/mL. The intra-assay and inter-assay precision and accuracy were less than 13.2% and 88.0-100.7%, respectively. The serum concentration range of cetuximab was 19-140μg/mL in patients. The serum cetuximab concentrations in LC-MS/MS were correlated with those in ELISA (r=0.899, P <0.01) and the mean bias was 1.5% in cancer patients. In conclusion, the present simple and rapid method with acceptable analytical performance can be helpful for evaluating the absolute concentration of serum cetuximab in clinical settings. Copyright © 2017 Elsevier B.V. All rights reserved.

Determination of a flame retardant hydrolysis product in human urine by SPE and LC-MS. Comparison of molecularly imprinted solid-phase extraction with a mixed-mode anion exchanger.

PubMed

Möller, Kristina; Crescenzi, Carlo; Nilsson, Ulrika

2004-01-01

Diphenyl phosphate is a hydrolysis product and possible metabolite of the flame retardant and plasticiser additive triphenyl phosphate. A molecularly imprinted polymer solid-phase extraction (MISPE) method for extracting diphenyl phosphate from aqueous solutions has been developed and compared with SPE using a commercially available mixed-mode anion exchanger. The imprinted polymer was prepared using 2-vinylpyridine (2-Vpy) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linker, and a structural analogue of the analyte as the template molecule. The imprinted polymer was evaluated for use as a SPE sorbent, in tests with both aqueous standards and spiked urine samples, by comparing recovery and breakthrough data obtained using the imprinted form of the polymer and a non-imprinted form (NIP). Extraction from aqueous solutions resulted in more than 80% recovery. Adsorption by the molecularly imprinted polymer (MIP) was non-selective, but selectivity was achieved by selective desorption in the wash steps. Diphenyl phosphate could also be selectively extracted from urine samples, although the urine matrix reduced the capacity of the MISPE cartridges. Recoveries from urine extraction were higher than 70%. It was important to control pH during sample loading. The MISPE method was found to yield a less complex LC-ESI-MS chromatogram of the urine extracts compared with the mixed-mode anion-exchanger method. An LC-ESI-MS method using a Hypercarb LC column with a graphitised carbon stationary phase was also evaluated for organophosphate diesters. LC-ESI-MS using negative-ion detection in selected ion monitoring (SIM) mode was shown to be linear for diphenyl phosphate in the range 0.08-20 ng microL(-1).

Simultaneous quantitation of five Panax notoginseng saponins by multi heart-cutting two-dimensional liquid chromatography: Method development and application to the quality control of eight Notoginseng containing Chinese patent medicines.

PubMed

Yao, Chang-liang; Yang, Wen-zhi; Wu, Wan-Yyng; Da, Juan; Hou, Jin-jun; Zhang, Jing-xian; Zhang, Yan-hai; Jin, Yan; Yang, Min; Jiang, Bao-hong; Liu, Xuan; Guo, De-an

2015-07-10

Current China Pharmacopoeia (ChP) standards employ diversified and case-dependent assay methods to evaluate the quality of different Chinese patent medicines (CPMs) that contain Panax notoginseng as the monarch drug. These conventional, HPLC-based approaches, utilizing a complex sample preparation procedure, can easily result in low analytical efficiency and possible component loss. Here, a "monomethod-heterotrait matrix" (MHM) strategy is proposed, that is, developing a universal multi heart-cutting two-dimensional liquid chromatography (MHC-2D-LC) approach that facilitates the simultaneous quantitation of five P. notoginseng saponins (noto-R1, Re, Rg1, Rb1, and Rd) in eight different CPMs. The MHC-2D-LC system was constructed on a dual-gradient liquid chromatography instrument equipped with a Poroshell SB C18 column and a Zorbax SB-Aq column for respective (1)D and (2)D separation. Method validation was performed in terms of specificity, linearity (r(2) and F-test), intra-/inter-day precision (0.4-7.9%), stability (1.2-3.9%), and recovery (90.2-108.7%), and the LODs and LOQs (loaded masses) of the five analytes varied between 4.0-11.0ng and 6.0-33.0ng, respectively. The validated MHC-2D-LC approach was subsequently applied to quantify the five saponins in thirty batches of different CPMs. The method demonstrated superiority over the current ChP assay methods in respect of specificity (avoiding co-elution), resolution (Rs>1.5), sample preparation (easy-to-implement ultrasonic extraction without repeated re-extraction), and transfer rate (minimum component loss). This is the first application of an MHC-2D-LC method for the quantitative assessment of the constituents of CPMs. The MHM approach represents a new, strategically significant methodology for the quality control of CPMs that involve complex chemical matrix. Copyright © 2015 Elsevier B.V. All rights reserved.

Semi-permeable surface analytical reversed-phase column for the improved trace analysis of acidic pesticides in water with coupled-column reversed-phase liquid chromatography with UV detection. Determination of bromoxynil and bentazone in surface water.

PubMed

Hogendoorn, E A; Westhuis, K; Dijkman, E; Heusinkveld, H A; den Boer, A C; Evers, E A; Baumann, R A

1999-10-08

The coupled-column (LC-LC) configuration consisting of a 3 microm C18 column (50 x 4.6 mm I.D.) as the first column and a 5 microm C18 semi-permeable-surface (SPS) column (150 x 4.6 mm I.D.) as the second column appeared to be successful for the screening of acidic pesticides in surface water samples. In comparison to LC-LC employing two C18 columns, the combination of C18/SPS-C18 significantly decreased the baseline deviation caused by the hump of the co-extracted humic substances when using UV detection (217 nm). The developed LC-LC procedure allowed the simultaneous determination of the target analytes bentazone and bromoxynil in uncleaned extracts of surface water samples to a level of 0.05 microg/l in less than 15 min. In combination with a simple solid-phase extraction step (200 ml of water on a 500 mg C18-bonded silica) the analytical procedure provides a high sample throughput. During a period of about five months more than 200 ditch-water samples originating from agricultural locations were analyzed with the developed procedure. Validation of the method was performed by randomly analyzing recoveries of water samples spiked at levels of 0.1 microg/l (n=10), 0.5 microg/l (n=7) and 2.5 microg/l (n=4). Weighted regression of the recovery data showed that the method provides overall recoveries of 95 and 100% for bentazone and bromoxynil, respectively, with corresponding intra-laboratory reproducibilities of 10 and 11%, respectively. Confirmation of the analytes in part of the samples extracts was carried out with GC-negative ion chemical ionization MS involving a derivatization step with bis(trifluoromethyl)benzyl bromide. No false negatives or positives were observed.

Measurement of "total" microcystins using the MMPB/LC/MS/MS method, and application to HAB impacted surface waters-presentation

EPA Science Inventory

The detection and quantification of microcystins, a family of toxins associated with harmful algal blooms, is complicated by their structural diversity and a lack of commercially available analytical standards for method development. As a result, most detection methods have focus...

Development of LC/MS/MS Methods for Implementation in US EPA’s Drinking Water Unregulated Contaminant Monitoring Regulations

EPA Science Inventory

Well-characterized and standardized methods are the foundation upon which monitoring of regulated and unregulated contaminants in drinking water are based. To obtain reliable, high quality data for trace analysis of contaminants, these methods must be rugged, selective and sensit...

A novel method for quantification of gemcitabine and its metabolites 2',2'-difluorodeoxyuridine and gemcitabine triphosphate in tumour tissue by LC-MS/MS: comparison with (19)F NMR spectroscopy.

PubMed

Bapiro, Tashinga E; Richards, Frances M; Goldgraben, Mae A; Olive, Kenneth P; Madhu, Basetti; Frese, Kristopher K; Cook, Natalie; Jacobetz, Michael A; Smith, Donna-Michelle; Tuveson, David A; Griffiths, John R; Jodrell, Duncan I

2011-11-01

To develop a sensitive analytical method to quantify gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and its metabolites 2',2'-difluorodeoxyuridine (dFdU) and 2',2'-difluorodeoxycytidine-5'-triphosphate (dFdCTP) simultaneously from tumour tissue. Pancreatic ductal adenocarcinoma tumour tissue from genetically engineered mouse models of pancreatic cancer (KP ( FL/FL ) C and KP ( R172H/+) C) was collected after dosing the mice with gemcitabine. (19)F NMR spectroscopy and LC-MS/MS protocols were optimised to detect gemcitabine and its metabolites in homogenates of the tumour tissue. A (19)F NMR protocol was developed, which was capable of distinguishing the three analytes in tumour homogenates. However, it required at least 100 mg of the tissue in question and a long acquisition time per sample, making it impractical for use in large PK/PD studies or clinical trials. The LC-MS/MS protocol was developed using porous graphitic carbon to separate the analytes, enabling simultaneous detection of all three analytes from as little as 10 mg of tissue, with a sensitivity for dFdCTP of 0.2 ng/mg tissue. Multiple pieces of tissue from single tumours were analysed, showing little intra-tumour variation in the concentrations of dFdC or dFdU (both intra- and extra-cellular). Intra-tumoural variation was observed in the concentration of dFdCTP, an intra-cellular metabolite, which may reflect regions of different cellularity within a tumour. We have developed a sensitive LC-MS/MS method capable of quantifying gemcitabine, dFdU and dFdCTP in pancreatic tumour tissue. The requirement for only 10 mg of tissue enables this protocol to be used to analyse multiple areas from a single tumour and to spare tissue for additional pharmacodynamic assays.

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.

Determination of chloramphenicol and zeranols in pig muscle by immunoaffinity column clean-up and LC-MS/MS analysis.

PubMed

Wang, Qing; Zhao, Hua; Xi, Cunxian; Wang, Guomin; Chen, Dongdong; Ding, Shijia

2014-01-01

An immunoaffinity column clean-up and LC-MS/MS method was successfully developed for simultaneous determination of chloramphenicol, zearalanone, α-zearalanol, β-zearalanol, zearalenone, α-zearalenol and β-zearalenol in pig muscle. The sample was extracted with diethyl ether after enzymatic digestion by β-glucuronidase/sulfatase. The extracted solution was evaporated to dryness and the residue was then dissolved in 1 ml of 50% acetonitrile solution. After filtration and dilution with phosphate buffer solution (PBS), the reconstituted solution was cleaned-up with an IAC-CZ immunoaffinity column and then analysed by HPLC-MS/MS. The established method were validated by linearity (r ≥ 0.9990), precision (RSD ≥ 2.9%), average recovery (74.5-105.0%) and limit of detection (0.04-0.10 μg kg(-1)). The developed method is rapid, reliable, sensitive, accurate and has good applicability for real samples.

Detection of Peptide-based nanoparticles in blood plasma by ELISA.

PubMed

Bode, Gerard H; Pickl, Karin E; Sanchez-Purrà, Maria; Albaiges, Berta; Borrós, Salvador; Pötgens, Andy J G; Schmitz, Christoph; Sinner, Frank M; Losen, Mario; Steinbusch, Harry W M; Frank, Hans-Georg; Martinez-Martinez, Pilar

2015-01-01

The aim of the current study was to develop a method to detect peptide-linked nanoparticles in blood plasma. A convenient enzyme linked immunosorbent assay (ELISA) was developed for the detection of peptides functionalized with biotin and fluorescein groups. As a proof of principle, polymerized pentafluorophenyl methacrylate nanoparticles linked to biotin-carboxyfluorescein labeled peptides were intravenously injected in Wistar rats. Serial blood plasma samples were analyzed by ELISA and by liquid chromatography mass spectrometry (LC/MS) technology. The ELISA based method for the detection of FITC labeled peptides had a detection limit of 1 ng/mL. We were able to accurately measure peptides bound to pentafluorophenyl methacrylate nanoparticles in blood plasma of rats, and similar results were obtained by LC/MS. We detected FITC-labeled peptides on pentafluorophenyl methacrylate nanoparticles after injection in vivo. This method can be extended to detect nanoparticles with different chemical compositions.

Determination of pesticides in coconut (Cocos nucifera Linn.) water and pulp using modified QuEChERS and LC-MS/MS.

PubMed

Ferreira, Jordana Alves; Ferreira, Joana Maria Santos; Talamini, Viviane; Facco, Janice de Fátima; Rizzetti, Tiele Medianeira; Prestes, Osmar Damian; Adaime, Martha Bohrer; Zanella, Renato; Bottoli, Carla Beatriz Grespan

2016-12-15

The use of pesticides is directly linked to improvements in productivity and to the preservation of coconut palms. However pesticide analysis is necessary to determine whether pesticide residues in the food products containing coconut are within the maximum residue limits (MRLs), ensuring the quality of these products. This work aimed to develop a method for multiresidue determination of ten pesticides in coconut water and pulp using QuEChERS and LC-MS/MS. The method was effective in terms of selectivity, linearity, matrix effect, accuracy and precision, providing LOD of 3μgkg(-1), LOQ of 10μgkg(-1) and recoveries between 70 and 120% with RSD lower than 20%. The developed method was applied to 36 samples in which residues of carbendazim, carbofuran, cyproconazole and thiabendazole were found below the LOQ in coconut water and pulp. Copyright © 2016 Elsevier Ltd. All rights reserved.

Analysis of formononetin from black cohosh (Actaea racemosa).

PubMed

Jiang, B; Kronenberg, F; Balick, M J; Kennelly, E J

2006-07-01

Black cohosh has been widely used as an herbal medicine for the treatment of symptoms related to menopause in America and Europe during the past several decades, but the bioactive constituents are still unknown. Formononetin is an isoflavone with known estrogen-like activity. This compound was first reported to be isolated from black cohosh in 1985, but subsequent research in 2002 using HPLC-PDA and LC-MS revealed no evidence to show the presence of formononetin in 13 populations of American black cohosh. A more recent report published in 2004 claimed to detect formononetin in an extract of black cohosh rhizomes using a TLC-fluorescent densitometry method. To further resolve these conflicting reports, we analyzed black cohosh roots and rhizomes for the presence of formononetin, using a combined TLC, HPLC-PDA and LC-MS method. We examined both methanolic and aqueous methanolic black cohosh extracts by HPLC-PDA and LC-MS methods, and did not detect formononetin in any extracts. We further determined the limits of detection of formononetin by HPLC-PDA and LC-MS. Our experimental results indicated that the sensitivity and accuracy of the HPLC-PDA and LC-MS methods for the analysis of formononetin were slightly higher than those of the reported fluorescent method, suggesting that the HPLC-PDA and LC-MS methods were reliable for the analysis of formononetin from black cohosh. We also repeated the reported TLC method to concentrate two fractions from a modern black cohosh sample and an 86-year-old black cohosh sample, respectively, and then analyzed these two fractions for formononetin using the HPLC-PDA and LC-MS method instead of the fluorescent method. Formononetin was not detected by HPLC-PDA or LC-MS. From the results of the present study it is not reasonable to attribute the estrogen-like activity of black cohosh extracts to formononetin.

Timing of The First Zygotic Cleavage Affects Post-Vitrification Viability of Murine Embryos Produced In Vivo

PubMed Central

Jusof, Wan-Hafizah Wan; Khan, Nor-Ashikin Mohamed Noor; Rajikin, Mohd Hamim; Satar, Nuraliza Abdul; Mustafa, Mohd-Fazirul; Jusoh, Norhazlin; Dasiman, Razif

2015-01-01

Background Timing of the first zygotic cleavage is an accurate predictor of embryo quality. Embryos that cleaved early (EC) have been shown to exhibit higher develop- mental viability compared to those that cleaved at a later period (LC). However, the vi- ability of EC embryos in comparison to LC embryos after vitrification is unknown. The present study aims to investigate the post-vitrification developmental viability of murine EC versus LC embryos. Materials and Methods In this experimental study, female ICR mice (6-8 weeks old) were superovulated and cohabited with fertile males for 24 hours. Afterwards, their ovi- ducts were excised and embryos harvested. Embryos at the 2-cell stage were catego- rized as EC embryos, while zygotes with two pronuclei were categorized as LC embryos. Embryos were cultured in M16 medium supplemented with 3% bovine serum albumin (BSA) in a humidified 5% CO2atmosphere. Control embryos were cultured until the blastocyst stage without vitrification. Experimental embryos at the 2-cell stage were vitri- fied for one hour using 40% v/v ethylene glycol, 18% w/v Ficoll-70 and 0.5 M sucrose as the cryoprotectant. We recorded the numbers of surviving embryos from the control and experimental groups and their development until the blastocyst stage. Results were analyzed using the chi-square test. Results A significantly higher proportion of EC embryos (96.7%) from the control group developed to the blastocyst stage compared with LC embryos (57.5%, P<0.0001). Similarly, in the experimental group, a significantly higher percentage of vitrified EC embryos (69.4%) reached the blastocyst stage compared to vitrified LC embryos (27.1%, P<0.0001). Conclusion Vitrified EC embryos are more vitrification tolerant than LC embryos. Prese- lection of EC embryos may be used as a tool for selection of embryos that exhibit higher developmental competence after vitrification. PMID:26246881

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.

Use of Imipenem To Detect KPC, NDM, OXA, IMP, and VIM Carbapenemase Activity from Gram-Negative Rods in 75 Minutes Using Liquid Chromatography-Tandem Mass Spectrometry

PubMed Central

Kulkarni, M. V.; Zurita, A. N.; Pyka, J. S.; Murray, T. S.; Hodsdon, M. E.

2014-01-01

Resistance to extended-spectrum β-lactam antibiotics has led to a greater reliance upon carbapenems, but the expression of carbapenemases threatens to limit the utility of these drugs. Current methods to detect carbapenemase activity are suboptimal, requiring prolonged incubations during which ineffective therapy may be prescribed. We previously described a sensitive and specific assay for the detection of carbapenemase activity using ertapenem and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, we assessed 402 Gram-negative rods, including both Enterobacteriaceae and non-Enterobacteriaceae expressing IMP, VIM, KPC, NDM, and/or OXA carbapenemases, by using imipenem, meropenem, and ertapenem with LC-MS/MS assays. LC-MS/MS methods for the detection of intact and hydrolyzed carbapenems from an enrichment broth were developed. No ion suppression was observed, and the limits of detection for all three drugs were below 0.04 μg/ml. The sensitivity and specificity of meropenem and ertapenem for carbapenemase activity among non-Enterobacteriaceae were low, but imipenem demonstrated a sensitivity and specificity of 96% and 95%, respectively, among all Gram-negative rods (GNR) tested, including both Enterobacteriaceae and non-Enterobacteriaceae. LC-MS/MS allows for the analysis of more complex matrices, and this LC-MS/MS assay could easily be adapted for use with primary specimens requiring growth enrichment. PMID:24789180

Recent Advance in Liquid Chromatography/Mass Spectrometry Techniques for Environmental Analysis in Japan

PubMed Central

Suzuki, Shigeru

2014-01-01

The techniques and measurement methods developed in the Environmental Survey and Monitoring of Chemicals by Japan’s Ministry of the Environment, as well as a large amount of knowledge archived in the survey, have led to the advancement of environmental analysis. Recently, technologies such as non-target liquid chromatography/high resolution mass spectrometry and liquid chromatography with micro bore column have further developed the field. Here, the general strategy of a method developed for the liquid chromatography/mass spectrometry (LC/MS) analysis of environmental chemicals with a brief description is presented. Also, a non-target analysis for the identification of environmental pollutants using a provisional fragment database and “MsMsFilter,” an elemental composition elucidation tool, is presented. This analytical method is shown to be highly effective in the identification of a model chemical, the pesticide Bendiocarb. Our improved micro-liquid chromatography injection system showed substantially enhanced sensitivity to perfluoroalkyl substances, with peak areas 32–71 times larger than those observed in conventional LC/MS. PMID:26819891

IDENTIFICATION OF POLAR DRINKING WATER DISINFECTION BY-PRODUCTS USING LIQUID CHROMATOGRAPHY - MASS SPECTROMETRY

EPA Science Inventory

A qualitative method using 2,4-dinitrophenylhydrazine (DNPH) derivatization followed by analysis with liquid chromatography (LC)/negative ion-electrospray mass spectrometry (MS) was developed for identifying polar aldehydes and ketones in ozonated drinking water. This method offe...

Development and validation of LC methods for the separation of misoprostol related substances and diastereoisomers.

PubMed

Kahsay, Getu; Song, Huiying; Eerdekens, Fran; Tie, Yaxin; Hendriks, Danny; Van Schepdael, Ann; Cabooter, Deirdre; Adams, Erwin

2015-01-01

Misoprostol is a synthetic prostaglandin E1 analogue which is mainly used for prevention and treatment of gastric ulcers, but also for abortion due to its labour inducing effect. Misoprostol exists as a mixture of diastereoisomers (1:1) and has several related impurities owing to its instability at higher temperatures and moisture. A simple and robust reversed phase liquid chromatographic (RPLC) method is described for the separation of the related substances and a normal phase (NP) LC method for the separation of misoprostol diastereoisomers. The RPLC method was performed using an Ascentis Express C18 (150 mm × 4.6 mm, 5 μm) column kept at 35 °C. The mobile phase was a gradient mixture of mobile phase A (ACN-H2O-MeOH, 28:69:3 v/v/v) and mobile phase B (ACN-H2O-MeOH, 47:50:3 v/v/v) eluted at a flow rate of 1.5 mL/min. UV detection was performed at 200 nm. The NPLC method was undertaken by using an XBridge bare silica (150 mm × 2.1 mm, 3.5 μm) column at 35 °C. The mobile phase contained 1-propanol-heptane-TFA (4:96:0.1%, v/v/v), pumped at a flow rate of 0.5 mL/min. UV detection was performed at 205 nm. This LC method can properly separate the two diastereoisomers (Rs > 2) within an analysis time of less than 20 min. Both methods were validated according to the ICH guidelines. Furthermore, these new LC methods have been successfully applied for purity control and diastereoisomers ratio determination of misoprostol bulk drug, tablets and dispersion. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification of alkyl dimethylbenzylammonium surfactants in water samples by solid-phase extraction followed by ion trap LC/MS and LC/MS/MS

USGS Publications Warehouse

Ferrer, I.; Furlong, E.T.

2001-01-01

A novel methodology was developed for the determination of alkyl (C12, C14, and C16) dimethylbenzylammonium chloride (benzalkonium chloride or BAC, Chemical Abstract Service number: 8001-54-5) in water samples. This method is based on solid-phase extraction (SPE) using polymeric cartridges, followed by high-performance liquid chromatography/ion trap mass spectrometry (LC/MS) and tandem mass spectrometry(MS/MS) detection, equipped with an electrospray interface in positive ion mode. Chromatographic separation was achieved for three BAC homologues by using a C18 column and a gradient of acetonitrile/10 millimolar aqueous ammonium formate. Total method recoveries were higher than 71% in different water matrices. The main ions observed by LC/MS were at mass-to-charge ratios (m/z) of 304, 332, and 360, which correspond to the molecular ions of the C12, C14, and C16 alkyl BAC, respectively. The unequivocal structural identification of these compounds in water samples was performed by LC/MS/MS after isolation and subsequent fragmentation of each molecular ion. The main fragmentation observed for the three different homologues corresponded to the loss of the toluyl group in the chemical structure, which leads to the fragment ions at m/z 212, 240, and 268 and a tropylium ion, characteristic of all homologues, at m/z 91. Detection limits for the methodology developed in this work were in the low nanogram-per-liter range. Concentration levels of BAC - ranging from 1.2 to 36.6 micrograms per liter - were found in surface-water samples collected downstream from different wastewater-treatment discharges, thus indicating its input and persistence through the wastewater-treatment process.

Simultaneous LC-MS/MS determination of phenylbutyrate, phenylacetate benzoate and their corresponding metabolites phenylacetylglutamine and hippurate in blood and urine.

PubMed

Laryea, Maurice D; Herebian, Diran; Meissner, Thomas; Mayatepek, Ertan

2010-12-01

Inborn errors of urea metabolism result in hyperammonemia. Treatment of urea cycle disorders can effectively lower plasma ammonium levels and results in survival in the majority of patients. Available medications for treating urea cycle disorders include sodium benzoate (BA), sodium phenylacetate (PAA), and sodium phenylbutyrate (PBA) and are given to provide alternate routes for disposition of waste nitrogen excretion. In this study, we develop and validate a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of benzoic acid, phenylacetic acid, phenylbutyric acid, phenylacetylglutamine, and hippuric acid in plasma and urine from children with inborn errors of urea synthesis. Plasma extracts and diluted urine samples were injected on a reverse-phase column and identified and quantified by selected reaction monitoring (SRM) in negative ion mode. Deuterated analogues served as internal standards. Analysis time was 7 min. Assay precision, accuracy, and linearity and sample stability were determined using enriched samples. Quantification limits of the method were 100 ng/ml (0.3-0.8 μmol/L) for all analytes, and recoveries were >90%. Inter- and intraday relative standard deviations were <10%. Our newly developed LC-MS/MS represents a robust, sensitive, and rapid method that allows simultaneous determination of the five compounds in plasma and urine.

Development of an LC/MS/MS method in order to determine arctigenin in rat plasma: its application to a pharmacokinetic study.

PubMed

Zou, Quanfei; Gu, Yuan; Lu, Rong; Zhang, Tiejun; Zhao, Guang-Rong; Liu, Changxiao; Si, Duanyun

2013-09-01

In this study, a simple and sensitive LC/MS/MS method was developed and validated for the determination of arctigenin in rat plasma. The MS detection was performed using multiple reaction monitoring at the transitions of m/z 373.2 → 137.3 for arctigenin and m/z 187.1 → 131.0 for psoralen (internal standard) with a Turbo IonSpray electrospray in positive mode. The calibration curves fitted a good linear relationship over the concentration range of 0.2-500 ng/mL. It was found that arctigenin is not stable enough at both room temperature and -80 °C unless mixed with methanol before storage. The validated LC/MS/MS method was successfully applied for the pharmacokinetic study of arctigenin in rats. After intravenous injection of 0.3 mg/kg arctigenin injection to rats, the maximum concentration, half-life and area under the concentration-time curve were 323 ± 65.2 ng/mL, 0.830 ± 0.166 and 81.0 ± 22.1 h ng/mL, respectively. Copyright © 2013 John Wiley & Sons, Ltd.

Screening Natural Content of Water-Soluble B Vitamins in Fish: Enzymatic Extraction, HILIC Separation, and Tandem Mass Spectrometric Determination.

PubMed

Chatterjee, Niladri Sekhar; Kumar, K Ashok; Ajeeshkumar, K K; Kumari, K R Remya; Vishnu, K V; Anandan, Rangasamy; Mathew, Suseela; Ravishankar, C N

2017-05-01

Despite the potential of LC with tandem MS (MS/MS) in improving sensitivity and selectivity, analytical methods are scarce for the determination of protein-bound and phosphorylated forms of B vitamins in food. This prompted us to develop a method for LC-MS/MS determination of naturally occurring nicotinamide, nicotinic acid, thiamine, pyridoxine, riboflavin, pantothenic acid, biotin, folic acid, and cyanocobalamin in fish. Baseline separation of the vitamins was achieved in a hydrophilic interaction LC condition. An ultrasonication-assisted enzymatic extraction protocol for sample preparation was optimized and validated. The time required for extraction was significantly reduced (to 4 h), while maintaining good extraction efficiency. Acetonitrile content (80%, v/v) in the prepared sample was found to be optimum for excellent peak shape and sensitivity. The dynamic linear range of the vitamins ranged from 2.5 to 500 ng/g, and the regression coefficient values were greater than 0.99. LOQ values ranged from 0.4 to 50 ng/g for the different vitamins. The spike recovery values at 50 and 100 ng/g ranged from 87.5 to 97.5%. The intra- and interday precision values were satisfactory. Accuracy of the developed method was determined by analysis of a Certified Reference Material. The method could also be used for unambiguous determination of the natural content of the target vitamins in fish.

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.

Identification and characterization of chlorogenic acids, chlorogenic acid glycosides and flavonoids from Lonicera henryi L. (Caprifoliaceae) leaves by LC-MSn.

PubMed

Jaiswal, Rakesh; Müller, Heiko; Müller, Anja; Karar, Mohamed Gamaleldin Elsadig; Kuhnert, Nikolai

2014-12-01

The chlorogenic acids, chlorogenic acid glycosides and flavonoids of the leaves of Lonicera henryi L. (Caprifoliaceae) were investigated qualitatively by liquid chromatography tandem mass spectrometry. Thirty-one chlorogenic acids and their glycosides were detected and characterized to their regioisomeric level on the basis of their unique fragmentation pattern in the negative ion mode tandem MS spectra. All of them were extracted for the first time from this source and thirteen of them were not reported previously in nature. For the positive identification of chlorogenic acid glycosides by LC-MS(n), multiple reaction monitoring and targeted MS(n) experiments were performed. We have developed an LC-MS(n) method for the systematic identification of chlorogenic acid glycosides and were also able to discriminate between chlorogenic acids and their isobaric glycosides. It was also possible to discriminate between 5-O-(3'-O-caffeoyl glucosyl)quinic acid and 5-O-(4'-O-caffeoyl glucosyl)quinic acid by LC-MS(n). This method can be applied for the rapid and positive identification of chlorogenic acids and their glycosides in plant materials, food and beverages. Copyright © 2014 Elsevier Ltd. All rights reserved.

A novel four-dimensional analytical approach for analysis of complex samples.

PubMed

Stephan, Susanne; Jakob, Cornelia; Hippler, Jörg; Schmitz, Oliver J

2016-05-01

A two-dimensional LC (2D-LC) method, based on the work of Erni and Frei in 1978, was developed and coupled to an ion mobility-high-resolution mass spectrometer (IM-MS), which enabled the separation of complex samples in four dimensions (2D-LC, ion mobility spectrometry (IMS), and mass spectrometry (MS)). This approach works as a continuous multiheart-cutting LC system, using a long modulation time of 4 min, which allows the complete transfer of most of the first - dimension peaks to the second - dimension column without fractionation, in comparison to comprehensive two-dimensional liquid chromatography. Hence, each compound delivers only one peak in the second dimension, which simplifies the data handling even when ion mobility spectrometry as a third and mass spectrometry as a fourth dimension are introduced. The analysis of a plant extract from Ginkgo biloba shows the separation power of this four-dimensional separation method with a calculated total peak capacity of more than 8700. Furthermore, the advantage of ion mobility for characterizing unknown compounds by their collision cross section (CCS) and accurate mass in a non-target approach is shown for different matrices like plant extracts and coffee. Graphical abstract Principle of the four-dimensional separation.

Larvicidal Activity of Citrus Limonoids against Aedes albopictus Larvae

PubMed Central

Bilal, Hazrat; Akram, Waseem; Ali-Hassan, Soaib

2012-01-01

Background: Development of insecticide resistance occurred due to the continuous and misuse of synthetic insecticides therefore, the recent study was conducted to explore eco-friendly plant extracts that have some potential to suppress mosquito larval population. Methods: WHO recommended mosquito larval bioassay method for insecticide was used while for the analysis of citrus oils for limonin and nomilin content HPLC was used. Results: Among the two citrus cultivars tested as larvicide against Aedes albopictus, valencia late (Citrus sinensis) was the best in terms of LC50 (297 ppm), % mortality (97%) and LT50 (18.49 hours) then freutrall early (Citrus reticulate) with LC50 (377.4 ppm), % mortality (88%) and LT50 (31 hours), While nomilin gave lowest LC50 (121.04 ppm) than limonin (382.22 ppm) after 72 hours of exposure. Valencia late also had more limonin and nomilin (377 μg/ml and 21.19 μg/ml) than freutrall early (5.29 μg/ml and 3.89 μg/ml) respectively. Conclusion: Valencia late showed best results in term of LC50, LT50 and percentage mortality against Aedes albopictus as it has more amount of nomilin then freutrall early, however further evaluation in the field conditions is required. PMID:23378967

Separation and identification of twelve catechins in tea using liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry.

PubMed

Zeeb, D J; Nelson, B C; Albert, K; Dalluge, J J

2000-10-15

A method has been developed for the direct microscale determination of 12 catechins in green and black tea infusions. The method is based on liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS). Standard catechin mixtures and tea infusions were analyzed by LC/APCI-MS with detection of protonated molecular ions and characteristic fragment ions for each compound. The identities of eight major catechins and caffeine in tea were established based on LC retention times and simultaneously recorded mass spectra. In addition, monitoring of the catechin-specific retro Diels-Alder fragment ion at m/z 139 throughout the chromatogram provided a unique fingerprint for catechin content in the samples that led to the identification of four minor chemically modified catechin derivatives in the infusions. This report is the first to describe the comprehensive determination of all 12 reported catechins in a single analysis. The utility of LC/APCI-MS for providing routine separation and identification of catechins at femtomole to low-picomole levels without extraction or sample pretreatment, and its potential as a standard analytical tool for the determination of polyphenols in natural products and biological fluids, are discussed.

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.

Determination of acrylamide in various food matrices: evaluation of LC and GC mass spectrometric methods.

PubMed

Becalski, Adam; Lau, Benjamin P Y; Lewis, David; Seaman, Stephen W; Sun, Wing F

2005-01-01

Recent concerns surrounding the presence of acrylamide in many types of thermally processed food have brought about the need for the development of analytical methods suitable for determination of acrylamide in diverse matrices with the goals of improving overall confidence in analytical results and better understanding of method capabilities. Consequently, the results are presented of acrylamide testing in commercially available food products--potato fries, potato chips, crispbread, instant coffee, coffee beans, cocoa, chocolate and peanut butter, obtained by using the same sample extract. The results obtained by using LC-MS/MS, GC/MS (El), GC/HRMS (El)--with or without derivatization--and the use of different analytical columns, are discussed and compared with respect to matrix borne interferences, detection limits and method complexities.

Accumulation of three important bioactive compounds in different plant parts of Withania somnifera and its determination by the LC-ESI-MS-MS (MRM) method.

PubMed

Gajbhiye, Narendra A; Makasana, Jayanti; Kumar, Satyanshu

2015-01-01

A comprehensive experiment was conducted to study the accumulation pattern and determination of three important bioactive compounds namely withaferin-A (WA), 12-deoxywithastramonolide (WO) and withanolide-A (WD) and its determination by the liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) method in root, stem, fruits and leaves of Withania somnifera. A rapid and sensitive LC-ESI-MS-MS method was developed and validated for the determination of these three important bioactive compounds, having same molecular weight. The multiple reaction monitoring method was established by two transitions for each analyte and intense transition used for quantification. Separation of the three analytes was achieved within a run time of 5 min on an RP-18 column using a mobile phase consisting of acetonitrile and 0.1% acetic acid in water in an isocratic condition. The developed method was validated as per the ICH guidelines. The developed method was found to be suitable for identification and quantification of WA, WO and WD in different plant parts such as roots, stems, fruits and leaves of W. somnifera. The accumulation of WA was highest in leaves samples (8.84 ± 0.37 mg/g) and it was 2.23, 5.85 and 27.26 times higher than its concentration in fruits, stems and roots, respectively. WO and WD contents were highest (0.44 ± 0.016 and 0.72 ± 0.016 mg/g, respectively) in root. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

[Comparison of surface light scattering of acrylic intraocular lenses made by lathe-cutting and cast-molding methods--long-term observation and experimental study].

PubMed

Nishihara, Hitoshi; Ayaki, Masahiko; Watanabe, Tomiko; Ohnishi, Takeo; Kageyama, Toshiyuki; Yaguchi, Shigeo

2004-03-01

To compare the long-term clinical and experimental results of soft acrylic intraocular lenses(IOLs) manufactured by the lathe-cut(LC) method and by the cast-molding(CM) method. This was a retrospective study of 20 patients(22 eyes) who were examined in a 5- and 7-year follow-up study. Sixteen eyes were implanted with polyacrylic IOLs manufactured by the LC method and 6 eyes were implanted with polyacrylic IOLs manufactured by the CM method. Postoperative measurements included best corrected visual acuity, contrast sensitivity, biomicroscopic examination, and Scheimpflug slit-lamp images to evaluate surface light scattering. Scanning electron microscopy and three-dimensional surface analysis were conducted. At 7 years, the mean visual acuity was 1.08 +/- 0.24 (mean +/- standard deviation) in the LC group and 1.22 +/- 0.27 in the CM group. Surface light-seatter was 12.0 +/- 4.0 computer compatible tapes(CCT) in the LC group and 37.4 +/- 5.4 CCT in the CM group. Mean surface roughness was 0.70 +/- 0.07 nm in the LC group and 6.16 +/- 0.97 nm in the CM group. Acrylic IOLs manufactured by the LC method are more stable in long-termuse.

Increasing Flexibility in Two-Dimensional Liquid Chromatography by Pulsed Elution of the First Dimension: A Proof of Concept.

PubMed

Jakobsen, Simon S; Christensen, Jan H; Verdier, Sylvain; Mallet, Claude R; Nielsen, Nikoline J

2017-09-05

This work demonstrates the development of an online two-dimensional liquid chromatography (2D-LC) method where the first dimension column is eluted by a sequence of pulses of increasing eluotropic strength generated by the LC pumps (pulsed-elution 2D-LC). Between the pulses, the first dimension is kept in a no-elution state using low eluent strength. The eluate from the first dimension is actively modulated using trap columns and subsequently analyzed in the second dimension. We demonstrate that by tuning the length and eluotropic strength of the pulses, peaks with retention factors in water, k w , above 150 can be manipulated to elute in 3-4 pulses. The no-elution state can be kept for 1-10 min with only minor changes as to which and how many pulses the peaks elute in. Pulsed-elution 2D-LC combined with active modulation tackles three of the main challenges encountered in 2D-LC and specifically online comprehensive 2D-LC: undersampling, difficulties in refocusing, and lack of flexibility in the selection of column dimensions and flow rates because the two dimensions constrain each other. The pulsed-elution 2D-LC was applied for the analysis of a basic fraction of vacuum gas oil. Peak capacity was 4018 for a 540 min analysis and 4610 for a 1040 min analysis.

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

E2F8 as a Novel Therapeutic Target for Lung Cancer

PubMed Central

Park, Sin-Aye; Platt, James; Lee, Jong Woo; López-Giráldez, Francesc; Herbst, Roy S.

2015-01-01

Background: The E2F members have been divided into transcription activators (E2F1-E2F3) and repressors (E2F4-E2F8). E2F8 with E2F7 has been known to play an important physiologic role in embryonic development and cell cycle regulation by repressing E2F1. However, the function of E2F8 in cancer cells is unknown. Methods: E2F8 expression was assessed by immunoblotting or immunofluorescence staining in human lung cancer (LC) cells and tissues from LC patients (n = 45). Cell proliferation, colony formation, and invasion analysis were performed to evaluate the role of E2F8 in LC. Microarray analysis was used to determine the target genes of E2F8. The regulation of E2F8 on the expression of ubiquitin-like PHD and RING domain-containing 1 (UHRF1), one of E2F8 target genes, was determined using chromatin immunoprecipitation and promoter activity assays. Human LC xenograft models were used to determine the effects of inhibiting E2F8 by siRNAs (n = 7 per group) or antisense morpholino (n = 8 per group) on tumor growth. Survival was analyzed using the Kaplan-Meier method and group differences by the Student’s t test. All statistical tests were two-sided. Results: LC tumors overexpressed E2F8 compared with normal lung tissues. Depletion of E2F8 inhibited cell proliferation and tumor growth. E2F8 knockdown statistically significantly reduced the expression of UHRF1 (~60%-70%, P < .001), and the direct binding of E2F8 on the promoter of UHRF1 was identified. Kaplan-Meier analysis with a public database showed prognostic significance of aberrant E2F8 expression in LC (HR = 1.91 95% CI = 1.21 to 3.01 in chemo-naïve patients, P = .0047). Conclusions: We demonstrated that E2F8 is overexpressed in LC and is required for the growth of LC cells. These findings implicate E2F8 as a novel therapeutic target for LC treatment. PMID:26089541

Determination of acrylamide in processed foods by LC/MS using column switching.

PubMed

Takatsuki, Satoshi; Nemoto, Satoru; Sasaki, Kumiko; Maitani, Tamio

2003-04-01

An LC/MS method was developed for the determination of acrylamide (AA) in processed or cooked foods. AA was extracted with a mixture of water and acetone from homogenized food samples after the addition of 13C-labeled acrylamide (AA-1-(13)C) as an internal standard. The extract was concentrated, washed with dichloromethane for defatting, and cleaned up on Bond Elut C18, PSA and ACCUCAT cartridge-columns, and then AA was determined by LC/MS in the selected ion recording (SIR) mode. For the LC/MS analysis, four LC columns were connected in-line and the flow of the mobile phase was switched according to a time-program. Monitoring ions for AA were m/z 72 and 55, and those for AA-1-(13)C were m/z 73 and 56. AA and AA-1-(13)C were determined without interference from the matrices in all samples. The recoveries of AA from potato chips, corn snack, pretzel and roasted tea spiked at the level of 500 ng/g of AA were 99.5-101.0% with standard deviations (SD) in the range from 0.3 to 1.6%. The limits of detection and quantification of the developed method were 9 and 30 ng/g for AA in samples, respectively. The method was applied to the analysis of AA in various processed or cooked food samples purchased from retail markets. High levels of AA were found in potato chips and French-fried potato (467-3,544 ng/g). Fried and sugar-coated dough cakes (karinto) contained 374 and 1,895 ng/g. Corn snacks contained 117-535 ng/g of AA. Roasted foods (such as roasted sesame seed, roasted barley (mugi-cha), roasted tea (hoji-cha), coffee beans and curry powder) contained 116-567 ng/g of AA. Foods made from fish, egg and meat contained lower levels of AA than the plant-based foods. Foods containing much water showed a tendency to have low levels of AA compared with dry foods. The proposed method was applicable to the analysis of AA in variety of processed foods.

Multi-mycotoxin stable isotope dilution LC-MS/MS method for Fusarium toxins in beer.

PubMed

Habler, Katharina; Gotthardt, Marina; Schüler, Jan; Rychlik, Michael

2017-03-01

A stable isotope dilution LC-MS/MS multi-mycotoxin method was developed for 12 different Fusarium toxins including modified mycotoxins in beer (deoxynivalenol-3-glucoside, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyl-deoxynivalenol, HT2-toxin, T2-toxin, enniatin B, B1, A1, A, beauvericin and zearalenone). As sample preparation and purification of beer a combined solid phase extraction for trichothecenes, enniatins, beauvericin and zearalenone was firstly developed. The validation of the new method gave satisfying results: intra-day and inter-day precision and recoveries were 1-5%, 2-8% and 72-117%, respectively. In total, 61 different organic and conventional beer samples from Germany and all over the world were analyzed by using the newly developed multi-mycotoxin method. In summary, deoxynivalenol, deoxynivalenol-3-glucoside, 3-acetyldeoxynivaleneol and enniatin B were quantified in rather low contents in the investigated beer samples. None of the other monitored Fusarium toxins like 15-acetyldeoxynivalenol, HT2- and T2-toxin, zearalenone, enniatin B1, A1, A or beauvericin were detectable. Copyright © 2016 Elsevier Ltd. All rights reserved.

Lung cancer development in patients with connective tissue disease-related interstitial lung disease: A retrospective observational study.

PubMed

Enomoto, Yasunori; Inui, Naoki; Yoshimura, Katsuhiro; Nishimoto, Koji; Mori, Kazutaka; Kono, Masato; Fujisawa, Tomoyuki; Enomoto, Noriyuki; Nakamura, Yutaro; Iwashita, Toshihide; Suda, Takafumi

2016-12-01

Previous studies have reported that patients with idiopathic pulmonary fibrosis occasionally develop lung cancer (LC). However, in connective tissue disease (CTD)-related interstitial lung disease (ILD), there are few data regarding the LC development. The aim of the present study was to evaluate the clinical significance of LC development in patients with CTD-ILD. A retrospective review of our database of 562 patients with ILD between 2000 and 2014 identified 127 patients diagnosed with CTD-ILD. The overall and cumulative incidences of LC were calculated. In addition, the risk factors and prognostic impact of LC development were evaluated. The median age at the ILD diagnosis was 63 years (range 37-84 years), and 73 patients (57.5%) were female. The median follow-up period from the ILD diagnosis was 67.4 months (range 10.4-322.1 months). During the period, 7 out of the 127 patients developed LC (overall incidence 5.5%). The cumulative incidences at 1, 3, and 5 years were 0.0%, 1.8%, and 2.9%, respectively. The risk of LC development was significantly higher in patients with higher smoking pack-year (odds ratio [OR] 1.028; 95% confidence interval [CI] 1.008-1.049; P = 0.007) and emphysema on chest high-resolution computed tomography (OR 14.667; 95% CI 2.871-74.926; P = 0.001). The median overall survival time after developing LC was 7.0 months (95% CI 4.9-9.1 months), and the most common cause of death was LC, not ILD. According to the Cox proportional hazard model analysis with time-dependent covariates, patients who developed LC showed significantly poorer prognosis than those who did not (hazard ratio 87.86; 95% CI 19.56-394.67; P < 0.001). In CTD-ILD, clinicians should be careful with the risk of LC development in patients with a heavy smoking history and subsequent emphysema. Although not so frequent, the complication could be a poor prognostic determinant.

Development and Validation of RP-LC Method for the Determination of Cinnarizine/Piracetam and Cinnarizine/Heptaminol Acefyllinate in Presence of Cinnarizine Reported Degradation Products

PubMed Central

EL-Houssini, Ola M.; Zawilla, Nagwan H.; Mohammad, Mohammad A.

2013-01-01

Specific stability indicating reverse-phase liquid chromatography (RP-LC) assay method (SIAM) was developed for the determination of cinnarizine (Cinn)/piracetam (Pira) and cinnarizine (Cinn)/heptaminol acefyllinate (Hept) in the presence of the reported degradation products of Cinn. A C18 column and gradient mobile phase was applied for good resolution of all peaks. The detection was achieved at 210 nm and 254 nm for Cinn/Pira and Cinn/Hept, respectively. The responses were linear over concentration ranges of 20–200, 20–1000 and 25–1000 μgmL−1 for Cinn, Pira, and Hept respectively. The proposed method was validated for linearity, accuracy, repeatability, intermediate precision, and robustness via statistical analysis of the data. The method was shown to be precise, accurate, reproducible, sensitive, and selective for the analysis of Cinn/Pira and Cinn/Hept in laboratory prepared mixtures and in pharmaceutical formulations. PMID:24137049

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

Determination of chlormequat in pig serum and sow milk by LC-MS/MS.

PubMed

Poulsen, M E; Christensen, H B; Sørensen, M T; Leffers, H; Andersen, J H

2007-11-01

Chlormequat is a plant growth regulator widely used on cereals, and there is general concern that it may impair human fertility. A LC-MS/MS method for the analysis of chlormequat in milk and serum was developed and validated in connection with an investigation on the effect of chlormequat on pig reproduction. Validation of the method was based on recovery tests at three spiking levels, determined as double determinations and repeated at least four times. Samples were extracted with methanol-water-acetic acid, centrifuged, filtrated and determined by LC-MS/MS. The mean recoveries were in the range 80-110%, and the LOD was 0.2 ng/g for serum and 0.3 ng/g for milk. The values for repeatability and reproducibility were within 2/3 of the limits given by the Horwitz equation. Samples of pig serum (59) and sow milk (27) were analyzed using the method. Chlormequat was determined in four milk samples in the range of 0.4 ng/g to 1.2 ng/g and in all serum samples in the range of 0.2 ng/g-4.0 ng/g.

Determination of alkylphenol and alkylphenolethoxylates in biota by liquid chromatography with detection by tandem mass spectrometry and fluorescence spectroscopy

USGS Publications Warehouse

Schmitz-Afonso, I.; Loyo-Rosales, J.E.; de la Paz Aviles, M.; Rattner, B.A.; Rice, C.P.

2003-01-01

A quantitative method for the simultaneous determination of octylphenol, nonylphenol and the corresponding ethoxylates (1 to 5) in biota is presented. Extraction methods were developed for egg and fish matrices based on accelerated solvent extraction followed by a solid-phase extraction cleanup, using octadecylsilica or aminopropyl cartridges. Identification and quantitation were accomplished by liquid chromatography-electrospray tandem mass spectrometry (LC-MS-MS) and compared to the traditional liquid chromatography with fluorescence spectroscopy detection. LC-MS-MS provides high sensitivity and specificity required for these complex matrices and an accurate quantitation with the use of 13C-labeled internal standards. Quantitation limits by LC-MS-MS ranged from 4 to 12 ng/g in eggs, and from 6 to 22 ng/g in fish samples. These methods were successfully applied to osprey eggs from the Chesapeake Bay and fish from the Great Lakes area. Total levels found in osprey egg samples were up to 18 ng/g wet mass and as high as 8.2 ug/g wet mass in the fish samples.

Application of handheld and portable spectrometers for screening acrylamide content in commercial potato chips.

PubMed

Ayvaz, Huseyin; Rodriguez-Saona, Luis E

2015-05-01

The most common methods for acrylamide analysis in foods require the use of LC-MS/MS and GC-MS. Although these methods have great analytical performance, they need intensive sample preparation, highly specialised instrumentation, and are time consuming. In this study, portable and handheld infrared spectrometers were evaluated as rapid methods for screening acrylamide in potato chips and their performances were compared to those of benchtop infrared systems. The acrylamide content of 64 commercial potato chips (169-2453 μg/kg) was determined by LC-MS/MS. Spectral data were collected using mid-infrared (MIR) and near-infrared (NIR) spectrometers. Partial least squares regression (PLSR) calibration models were developed to predict acrylamide levels. Overall, good linear correlation was found between the predicted acrylamide levels and actual measured acrylamide concentrations by LC-MS/MS (rPred > 0.90 and SEP < 100 μg/kg). Our results indicate that portable and handheld spectrometers can be used as simple and rapid alternatives for acrylamide analysis in potato chips. Copyright © 2014 Elsevier Ltd. All rights reserved.

Acute toxicity of ochratoxin-A in marine water-reared sea bass (Dicentrarchus labrax L.).

PubMed

El-Sayed, Yasser Said; Khalil, Riad Hassan; Saad, Talaat Talaat

2009-05-01

The toxic effects of ochratoxin-A (OTA) on sea bass, Dicentrarchus labrax L., have not been previously documented. A flow-through bioassay test system was conducted in two series and a total 180 of adult marine-reared sea bass was used to estimate the acute oral 96 h median lethal concentration (LC(50)) value and behavioral changes of OTA. The data obtained were statistically evaluated using Finney's Probit Analysis Method developed by EPA. The 96 h LC(50) value for adult D. labrax was found to be 277 microg kg(-1)bwt with 95% confidence limits of 244-311 microg kg(-1)bwt. This value was calculated to be 285 microg kg(-1) bwt with Behrens-Karber's method. The two methods were relatively comparable. The acute dietary 96 h LC(50) of OTA is 9.23 mg kg(-1) diet. Additionally, the behavioral changes of sea bass were primarily observed as nervous and respiratory manifestations. We concluded that sea bass is a species highly sensitive to OTA making them a useful experimental model for aquatic mycotoxigenic problems.

A Simple Method to Measure the Twist Elastic Constant of a Nematic Liquid Crystal

DTIC Science & Technology

2015-01-01

for measuring the twist elastic constant (K22) of a nematic liquid crystal (LC). By adding some chiral dopant to an LC host, the LC directors rotate......of Optics and Photonics , University of Central Florida, Orlando, FL, USA (Received 14 June 2015; accepted 6 July 2015) We demonstrate a simple method

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.

Analysis of anabolic steroids in human hair using LC-MS/MS.

PubMed

Deshmukh, Nawed; Hussain, Iltaf; Barker, James; Petroczi, Andrea; Naughton, Declan P

2010-10-01

New highly sensitive, specific, reliable, reproducible and robust LC-MS/MS methods were developed to detect the anabolic steroids, nandrolone and stanozolol, in human hair for the first time. Hair samples from 180 participants (108 males, 72 females, 62% athletes) were screened using ELISA which revealed 16 athletes as positive for stanozolol and 3 for nandrolone. Positive samples were confirmed on LC-MS/MS in selective reaction monitoring (SRM) mode. The assays for stanozolol and nandrolone showed good linearity in the range 1-400pg/mg and 5-400pg/mg, respectively. The methods were validated for LLOD, interday precision, intraday precision, specificity, extraction recovery and accuracy. The assays were capable of detecting 0.5pg stanozolol and 3.0pg nandrolone per mg of hair, when approximately 20mg of hair were processed. Analysis using LC-MS/MS confirmed 11 athletes' positive for stanozolol (5.0pg/mg to 86.3pg/mg) and 1 for nandrolone (14.0pg/mg) thus avoiding false results from ELISA screening. The results obtained demonstrate the application of these hair analysis methods to detect both steroids at low concentrations, hence reducing the amount of hair required significantly. The new methods complement urinalysis or blood testing and facilitate improved doping testing regimes. Hair analysis benefits from non-invasiveness, negligible risk of infection and facile sample storage and collection, whilst reducing risks of tampering and cross-contamination. Owing to the wide detection window, this approach may also offer an alternative approach for out-of-competition testing.

First report on the pharmacokinetic profile of nimbolide, a novel anticancer agent in oral and intravenous administrated rats by LC/MS method.

PubMed

Baira, Shandilya Mahamuni; Khurana, Amit; Somagoni, Jaganmohan; Srinivas, R; Godugu, Chandraiah; Talluri, M V N Kumar

2018-06-02

Nimbolide is a novel, natural compound with promising potential as a drug candidate for anticancer activity. It is isolated from the Indian traditional medicinal plant Azadirachta indica popularly known as neem. The present study was undertaken to explore the oral bioavailability and pharmacokinetic characteristics of nimbolide in rats using the LC/QTOF/MS method. A simple protein precipitation method using acetonitrile was employed for extracting nimbolide from rat plasma. The chromatographic separation of nimbolide and the internal standard (regorafenib) was attained on an Aquity BEH C18 column (100 × 2.1 mm, 2.7 μm), using ACN and 0.1% of formic acid in water as mobile phase components in a gradient elution mode at a flow rate of 0.45 mL/min over a short run time of 4 min. A mass detection was carried out using target ions of [M + H] + at m/z 467.2074 for nimbolide and m/z 483.0847 for the internal standard. The LC/MS method was validated and all the parameters were found well within the specified limits. The calibration curve was constructed in the range of 1-1000 ng/mL. The method shows good accuracy (91.66-97.12%) and precision (intra 2.21-6.92% CV and inter-day 2.56-4.62% CV). This developed LC/MS method was effectively applied to the pharmacokinetic study of nimbolide upon oral and intravenous administration in rats. In concordance with its physicochemical properties and high lipophilicity, we found that it shows poor oral absorption at different doses (10, 30 and 50 mg/kg). As expected, higher plasma levels were observed upon intravenous (10 mg/kg) administration. This method can be extended for evaluation of drug interaction and drug metabolism in rats as well as in humans. Moreover, our rapid and sensitive method may cater the need to accelerate the preclinical formulation development and lead optimization for future drug development of this potent anticancer agent. Further, our oral bioavailability studies demonstrated that nimbolide possesses poor oral absorption, which could be the probable reason for the delay in therapeutic translation of this promising agent for clinical use. Copyright © 2018 Elsevier B.V. All rights reserved.

A validated specific stability-indicating RP-HPLC assay method for Ambrisentan and its related substances.

PubMed

Narayana, M B V; Chandrasekhar, K B; Rao, B M

2014-09-01

A validated specific stability-indicating reverse-phase liquid chromatographic method was developed for the quantitative determination of Ambrisentan as well as its related substances in bulk samples, pharmaceutical dosage forms in the presence of degradation products and its related impurities. Forced degradation studies were performed on bulk samples of Ambrisentan as per the ICH-prescribed stress conditions using acid, base, oxidative, thermal stress and photolytic degradation to show the stability-indicating power of the LC method. Significant degradation in acidic, basic stress conditions was observed and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from the forced degradation studies and the impurity-spiked solution. Good resolution between the peaks corresponds to Ambrisentan-related impurities and degradation products from the analyte were achieved on a SunFire C18 column using a mobile phase consisting of a mixture of potassium dihydrogen orthophosphate at a pH adjusted to 2.5 with ortho-phosphoric acid in water and a mixture of acetonitrile:methanol using a simple linear gradient. The detection was carried out at 225 nm. The limit of detection and the limit of quantification for the Ambrisentan and its related impurities were established. The stressed test solutions were assayed against the qualified working standard of Ambrisentan and the mass balance in each case was between 98.9 and 100.3%, indicating that the developed LC method was stability indicating. Validation of the developed LC method was carried out as per the ICH requirements. The developed method was found to be suitable to check the quality of bulk samples of Ambrisentan at the time of batch release and also during its storage (long-term and accelerated stability). © The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A validated stability-indicating RP-HPLC method for levofloxacin in the presence of degradation products, its process related impurities and identification of oxidative degradant.

PubMed

Lalitha Devi, M; Chandrasekhar, K B

2009-12-05

The objective of current study was to develop a validated specific stability indicating reversed-phase liquid chromatographic method for the quantitative determination of levofloxacin as well as its related substances determination in bulk samples, pharmaceutical dosage forms in the presence of degradation products and its process related impurities. Forced degradation studies were performed on bulk sample of levofloxacin as per ICH prescribed stress conditions using acid, base, oxidative, water hydrolysis, thermal stress and photolytic degradation to show the stability indicating power of the method. Significant degradation was observed during oxidative stress and the degradation product formed was identified by LCMS/MS, slight degradation in acidic stress and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from forced degradation studies and the impurity spiked solution. Good resolution between the peaks corresponds to process related impurities and degradation products from the analyte were achieved on ACE C18 column using the mobile phase consists a mixture of 0.5% (v/v) triethyl amine in sodium dihydrogen orthophosphate dihydrate (25 mM; pH 6.0) and methanol using a simple linear gradient. The detection was carried out at 294 nm. The limit of detection and the limit of quantitation for the levofloxacin and its process related impurities were established. The stressed test solutions were assayed against the qualified working standard of levofloxacin and the mass balance in each case was in between 99.4 and 99.8% indicating that the developed LC method was stability indicating. Validation of the developed LC method was carried out as per ICH requirements. The developed LC method was found to be suitable to check the quality of bulk samples of levofloxacin at the time of batch release and also during its stability studies (long term and accelerated stability).

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.

Development of a quantitative LC-MS/MS analytical method coupled with turbulent flow chromatography for digoxin for the in vitro P-gp inhibition assay.

PubMed

Smalley, James; Marino, Anthony M; Xin, Baomin; Olah, Timothy; Balimane, Praveen V

2007-07-01

Caco-2 cells, the human colon carcinoma cells, are typically used for screening compounds for their permeability characteristics and P-glycoprotein (P-gp) interaction potential during discovery and development. The P-gp inhibition of test compounds is assessed by performing bi-directional permeability studies with digoxin, a well established P-gp substrate probe. Studies performed with digoxin alone as well as digoxin in presence of test compounds as putative inhibitors constitute the P-gp inhibition assay used to assess the potential liability of discovery compounds. Radiolabeled (3)H-digoxin is commonly used in such studies followed by liquid scintillation counting. This manuscript describes the development of a sensitive, accurate, and reproducible LC-MS/MS method for analysis of digoxin and its internal standard digitoxin using an on-line extraction turbulent flow chromatography coupled to tandem mass spectrometric detection that is amendable to high throughput with use of 96-well plates. The standard curve for digoxin was linear between 10 nM and 5000 nM with regression coefficient (R(2)) of 0.99. The applicability and reliability of the analysis method was evaluated by successful demonstration of efflux ratio (permeability B to A over permeability A to B) greater than 10 for digoxin in Caco-2 cells. Additional evaluations were performed on 13 marketed compounds by conducting inhibition studies in Caco-2 cells using classical P-gp inhibitors (ketoconazole, cyclosporin, verapamil, quinidine, saquinavir etc.) and comparing the results to historical data with (3)H-digoxin studies. Similarly, P-gp inhibition studies with LC-MS/MS analytical method for digoxin were also performed for 21 additional test compounds classified as negative, moderate, and potent P-gp inhibitors spanning multiple chemo types and results compared with the historical P-gp inhibition data from the (3)H-digoxin studies. A very good correlation coefficient (R(2)) of 0.89 between the results from the two analytical methods affords an attractive LC-MS/MS analytical option for labs that need to conduct the P-gp inhibition assay without using radiolabeled compounds.

The rapid and direct determination of ATPase activity by ion exchange chromatography and the application to the activity of heat shock protein-90.

PubMed

Bartolini, Manuela; Wainer, Irving W; Bertucci, Carlo; Andrisano, Vincenza

2013-01-25

Adenosine nucleotides are involved as substrates or co-factors in several biochemical reactions, catalyzed by enzymes, which modulate energy production, signal transduction and cell proliferation. We here report the development and optimization of an ion exchange liquid chromatography (LC) method for the determination of ATP, ADP and AMP. This method is specifically aimed at the determination of the ATP-ase activity of human heat shock protein 90 (Hsp90), a molecular chaperone that has emerged as target enzyme in cancer therapy. Separation of the three nucleotides was achieved in a 15-min run by using a disk shaped monolithic ethylene diamine stationary phase of small dimensions (2mm×6mm i.d.), under a three-solvent gradient elution mode and UV detection at 256nm. The described direct LC method resulted highly specific as a consequence of the baseline separation of the three adenosine nucleotides and could be applied to the determination of the enzymatic activity of ADP/ATP generating or consuming enzymes (such as kinases). Furthermore, comparison of the LOD and LOQ values of the LC method with those obtained with the malachite green assay, which is one of the most used indirect screening methodologies for ATP-ase activity, showed that the LC method has a similar range of application without presenting the drawbacks related to contamination by inorganic phosphate ions and glycerol, which are present in Hsp90 commercial samples. Copyright © 2012 Elsevier B.V. All rights reserved.

A simple, fast and sensitive screening LC-ESI-MS/MS method for antibiotics in fish.

PubMed

Guidi, Letícia Rocha; Santos, Flávio Alves; Ribeiro, Ana Cláudia S R; Fernandes, Christian; Silva, Luiza H M; Gloria, Maria Beatriz A

2017-01-15

The objective of this study was to develop and validate a fast, sensitive and simple liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the screening of six classes of antibiotics (aminoglycosides, beta-lactams, macrolides, quinolones, sulfonamides and tetracyclines) in fish. Samples were extracted with trichloroacetic acid. LC separation was achieved on a Zorbax Eclipse XDB C18 column and gradient elution using 0.1% heptafluorobutyric acid in water and acetonitrile as mobile phase. Analysis was carried out in multiple reaction monitoring mode via electrospray interface operated in the positive ionization mode, with sulfaphenazole as internal standard. The method was suitable for routine screening purposes of 40 antibiotics, according to EC Guidelines for the Validation of Screening Methods for Residues of Veterinary Medicines, taking into consideration threshold value, cut-off factor, detection capability, limit of detection, sensitivity and specificity. Real fish samples (n=193) from aquaculture were analyzed and 15% were positive for enrofloxacin (quinolone), one of them at a higher concentration than the level of interest (50µgkg -1 ), suggesting possible contamination or illegal use of that antibiotic. Copyright © 2016 Elsevier B.V. All rights reserved.

High-throughput determination of vancomycin in human plasma by a cost-effective system of two-dimensional liquid chromatography.

PubMed

Sheng, Yanghao; Zhou, Boting

2017-05-26

Therapeutic drug monitoring (TDM) is one of the most important services of clinical laboratories. Two main techniques are commonly used: the immunoassay and chromatography method. We have developed a cost-effective system of two-dimensional liquid chromatography with ultraviolet detection (2D-LC-UV) for high-throughput determination of vancomycin in human plasma that combines the automation and low start-up costs of the immunoassay with the high selectivity and sensitivity of the liquid chromatography coupled with mass spectrometric detection without incurring their disadvantages, achieving high cost-effectiveness. This 2D-LC system offers a large volume injection to provide sufficient sensitivity and uses simulated gradient peak compression technology to control peak broadening and to improve peak shape. A middle column was added to reduce the analysis cycle time and make it suitable for high-throughput routine clinical assays. The analysis cycle time was 4min and the peak width was 0.8min. Compared with other chromatographic methods that have been developed, the analysis cycle time and peak width for vancomycin was reduced significantly. The lower limit of quantification was 0.20μg/mL for vancomycin, which is the same as certain LC-MS/MS methods that have been recently developed and validated. The method is rapid, automated, and low-cost and has high selectivity and sensitivity for the quantification of vancomycin in human plasma, thus making it well-suited for use in hospital clinical laboratories. Copyright © 2017 Elsevier B.V. All rights reserved.

Measurement of "total" microcystins using the MMPB/LC/MS ...

EPA Pesticide Factsheets

The detection and quantification of microcystins, a family of toxins associated with harmful algal blooms, is complicated by their structural diversity and a lack of commercially available analytical standards for method development. As a result, most detection methods have focused on either a subset of microcystin congeners, as in US EPA Method 544, or on techniques which are sensitive to structural features common to most microcystins, as in the anti-ADDA ELISA method. A recent development has been the use of 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB), which is produced by chemical oxidation the ADDA moiety in most microcystin congeners, as a proxy for the sum of congeners present. Conditions for the MMPB derivatization were evaluated and applied to water samples obtained from various HAB impacted surface waters, and results were compared with congener-based LC/MS/MS and ELISA methods. The detection and quantification of microcystins, a family of toxins associated with harmful algal blooms, is complicated by their structural diversity and a lack of commercially available analytical standards for method development. As a result, most detection methods have focused on either a subset of microcystin congeners, as in US EPA Method 544, or on techniques which are sensitive to structural features common to most microcystins, as in the anti-ADDA ELISA method. A recent development has been the use of 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB), which is produce

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

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 .

Constructing Understandings of End-of-Life Care in Europe: A Qualitative Study Involving Cognitive Interviewing with Implications for Cross-National Surveys

PubMed Central

Bechinger-English, Dorothee; Bausewein, Claudia; Simon, Steffan T.; Harding, Richard; Higginson, Irene J.; Gomes, Barbara

2011-01-01

Abstract Background Although national findings regarding people's end-of-life care (EoLC) preferences and priorities are available within Europe, a lack of research coordination between countries has meant that cross-national understandings of EoLC remain unknown. Purpose To (1) identify English and German understandings of EoLC within the context of an EoLC survey, and (2) to synthesise these understandings to aid interpretation of results from a cross-national survey. Methods An inductive and interpretive two-phased sequential design involving (1) qualitative analysis of cognitive interview data from 15 English and 15 German respondents to develop country-related categories, and (2) qualitative synthesis to identify a conceptually coherent understanding of EoLC. Results Open and axial coding resulted in six English and six German categories. Commonalities included (a) the importance of social and relational dimensions, (b) dynamic decision making comprising uncertainty, (c) a valuing of life's quality and quantity, and (d) expectations for holistic care involving autonomy, choice, and timely information from trusted professionals. Differences involved attention to practical matters, and thoughts about prolongation of life, preferred place of death, and the role of media and context. Synthesis resulted in four concepts with underlying coherence: expectations of a high standard of EoLC involving autonomy, choice, and context; evolving decision making amid anticipated change; thoughts about living and existing; and worldviews shaping EoLC preferences in real and hypothetical scenarios. Conclusion Individual and country-related diversity must be remembered when quantifying EoLC understandings. Inductive-interpretive analysis of cognitive interview data aids interpretation of survey findings. Cross-national research coordination and qualitative synthesis assists EoLC in Europe. PMID:21306232

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.

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.

[Simultaneous determination of ochratoxin A, B and citrinin in foods by HPLC-FL and LC/MS/MS].

PubMed

Tabata, Setsuko; Iida, Kenji; Kimura, Keisuke; Iwasaki, Yumiko; Nakazato, Mitsuo; Kamata, Kunihiro; Hirokado, Masako

2008-04-01

Methods using high-performance liquid chromatography with fluorescence detection (HPLC-FL) and using liquid chromatography with tandem mass spectrometry (LC/MS/MS) were developed for simultaneous determination of ochratoxin A (OTA), ochratoxin B (OTB) and citrinin (CIT) in cereal, fruit, and coffee products. The samples were extracted with ethyl acetate under an acidic condition, and then cleaned up with liquid-liquid separation. The test solutions were analyzed by reverse-phase HPLC-FL and LC/MS/MS. Mass spectral acquisition was performed in positive ion mode by applying multiple reaction monitoring. The performances of both detectors were almost equivalent. The recoveries of OTA and OTB were 87-111%, and that of CIT were 70-88%. The limits of quantification (S/N> or =10) of OTA, OTB and CIT was 0.1 mug/kg or less. These methods were considered to be useful for the determination of the three mycotoxins at low levels (0.1 microg/kg).

LC-MS-sMRM Method Development and Validation of Different Classes of Pain Panel Drugs and Analysis of Clinical Urine Samples.

PubMed

Athar Masood, M; Veenstra, Timothy D

2017-08-26

Urine Drug Testing (UDT) is an important analytical/bio-analytical technique that has inevitably become an integral and vital part of a testing program for diagnostic purposes. This manuscript presents a tailor-made LC-MS/MS quantitative assay method development and validation for a custom group of 33 pain panel drugs and their metabolites belonging to different classes (opiates, opioids, benzodiazepines, illicit, amphetamines, etc.) that are prescribed in pain management and depressant therapies. The LC-MS/MS method incorporates two experiments to enhance the sensitivity of the assay and has a run time of about 7 min. with no prior purification of the samples required and a flow rate of 0.7 mL/min. The method also includes the second stage metabolites for some drugs that belong to different classes but have first stage similar metabolic pathways that will enable to correctly identify the right drug or to flag the drug that might be due to specimen tampering. Some real case examples and difficulties in peak picking were provided with some of the analytes in subject samples. Finally, the method was deliberated with some randomly selected de-identified clinical subject samples, and the data evaluated from "direct dilute and shoot analysis" and after "glucuronide hydrolysis" were compared. This method is now used to run routinely more than 100 clinical subjects samples on a daily basis. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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.

Label-free assay based on immobilized capillary enzyme reactor of Leishmania infantum nucleoside triphosphate diphosphohydrolase (LicNTPDase-2-ICER-LC/UV).

PubMed

Magalhães, Luana; de Oliveira, Arthur Henrique Cavalcante; de Souza Vasconcellos, Raphael; Mariotini-Moura, Christiane; de Cássia Firmino, Rafaela; Fietto, Juliana Lopes Rangel; Cardoso, Carmen Lúcia

2016-01-01

Nucleoside triphosphate diphosphohydrolase (NTPDase) is an enzyme belonging to the apyrase family that participates in the hydrolysis of the nucleosides di- and triphosphate to the corresponding nucleoside monophosphate. This enzyme underlies the virulence of parasites such as Leishmania. Recently, an NTPDase from Leishmania infantum (LicNTPDase-2) was cloned and expressed and has been considered as a new drug target for the treatment of leishmaniasis. With the intent of developing label-free online screening methodologies, LicNTPDase-2 was covalently immobilized onto a fused silica capillary tube in the present study to create an immobilized capillary enzyme reactor (ICER) based on LicNTPDase-2 (LicNTPDase-2-ICER). To perform the activity assays, a multidimensional chromatographic method was developed employing the LicNTPDase-2-ICER in the first dimension, and an analytical Ascentis C8 column was used in the second dimension to provide analytical separation of the substrates and products. The validated LicNTPDase-2-ICER method provided the following kinetic parameters of the immobilized enzyme: KM of 2.2 and 1.8mmolL(-1) for the ADP and ATP substrates, respectively. Suramin (1mmolL(-1)) was also shown to inhibit 32.9% of the enzymatic activity. The developed method is applicable to kinetic studies and enables the recognition of the ligands. Furthermore, a comparison of the values of LicNTPDase-2-ICER with those obtained with an LC method using free enzyme in solution showed that LicNTPDase-2-ICER-LC/UV was an accurate and reproducible method that enabled automated measurements for the rapid screening of ligands. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

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.

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.

A high-throughput urinalysis of abused drugs based on a SPE-LC-MS/MS method coupled with an in-house developed post-analysis data treatment system.

PubMed

Cheng, Wing-Chi; Yau, Tsan-Sang; Wong, Ming-Kei; Chan, Lai-Ping; Mok, Vincent King-Kuen

2006-10-16

A rapid urinalysis system based on SPE-LC-MS/MS with an in-house post-analysis data management system has been developed for the simultaneous identification and semi-quantitation of opiates (morphine, codeine), methadone, amphetamines (amphetamine, methylamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA)), 11-benzodiazepines or their metabolites and ketamine. The urine samples are subjected to automated solid phase extraction prior to analysis by LC-MS (Finnigan Surveyor LC connected to a Finnigan LCQ Advantage) fitted with an Alltech Rocket Platinum EPS C-18 column. With a single point calibration at the cut-off concentration for each analyte, simultaneous identification and semi-quantitation for the above mentioned drugs can be achieved in a 10 min run per urine sample. A computer macro-program package was developed to automatically retrieve appropriate data from the analytical data files, compare results with preset values (such as cut-off concentrations, MS matching scores) of each drug being analyzed and generate user-defined Excel reports to indicate all positive and negative results in batch-wise manner for ease of checking. The final analytical results are automatically copied into an Access database for report generation purposes. Through the use of automation in sample preparation, simultaneous identification and semi-quantitation by LC-MS/MS and a tailored made post-analysis data management system, this new urinalysis system significantly improves the quality of results, reduces the post-data treatment time, error due to data transfer and is suitable for high-throughput laboratory in batch-wise operation.

Screening for toxic phorbol esters in jerky pet treat products using LC-MS.

PubMed

Nishshanka, Upul; Jayasuriya, Hiranthi; Chattopadhaya, Chaitali; Kijak, Philip J; Chu, Pak-Sin; Reimschuessel, Renate; Tkachenko, Andriy; Ceric, Olgica; De Alwis, Hemakanthi G

2016-05-01

Since 2007, the U.S. FDA's Center for Veterinary Medicine (CVM) has been investigating reports of pets becoming ill after consuming jerky pet treats. Jerky used in pet treats contains glycerin, which can be made from vegetable oil or as a byproduct of biodiesel production. Because some biodiesel is produced using oil from Jatropha curcas, a plant that contains toxic compounds including phorbol esters, CVM developed a liquid chromatography-mass spectrometry (LC-MS) screening method to evaluate investigational jerky samples for the presence of these toxins. Results indicated that the samples analyzed with the new method did not contain Jatropha toxins at or above the lowest concentration tested. Published by Elsevier B.V.

Measurement of trace levels of antibiotics in river water using on-line enrichment and triple-quadrupole LC-MS/MS.

PubMed

Dinh, Quoc Tuc; Alliot, Fabrice; Moreau-Guigon, Elodie; Eurin, Joëlle; Chevreuil, Marc; Labadie, Pierre

2011-09-15

This study presents the development of an automated on-line solid phase extraction (SPE)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of 23 antibiotics in environmental water samples. After optimisation of LC-MS/MS conditions, SPE parameters such as sorbent type, sample pH or sample volume were optimised. Antibiotic recoveries ranged from 64% to 98% and compared favourably with those achieved using off-line SPE. Limits of detection were in the range 0.5-13.7 ng L(-1). This on-line SPE-LC-MS/MS procedure was applied to the analysis of water samples taken in three rivers within the Seine River basin, near Paris (France). The obtained results revealed the occurrence of 12 antibiotics, including tylosin, erythromycin, tetracycline, amoxicillin, trimethoprim, sulfamethoxazole, oxolinic acid, flumequine, norfloxacin, ciprofloxacin, ofloxacin, and vancomycin (2-1435 ng L(-1)). Copyright © 2011 Elsevier B.V. All rights reserved.

Bioanalytical method for in vitro metabolism study of repaglinide using 96-blade thin-film solid-phase microextraction and LC-MS/MS.

PubMed

Simões, Rodrigo Almeida; Bonato, Pierina Sueli; Mirnaghi, Fatemeh S; Bojko, Barbara; Pawliszyn, Janusz

2015-01-01

A high-throughput bioanalytical method using 96-blade thin film microextraction (TFME) and LC-MS/MS for the analysis of repaglinide (RPG) and two of its main metabolites was developed and used for an in vitro metabolism study. The target analytes were extracted from human microsomal medium by a 96-blade-TFME system employing the low-cost prototype 'SPME multi-sampler' using C18 coating. Method validation showed recoveries around 90% for all analytes and was linear over the concentration range of 2-1000 ng ml(-1) for RPG and of 2-500 ng ml(-1) for each RPG metabolite. The method was applied to an in vitro metabolism study of RPG employing human liver microsomes and proved to be very useful for this purpose.

Principles and Applications of Liquid Chromatography-Mass Spectrometry in Clinical Biochemistry

PubMed Central

Pitt, James J

2009-01-01

Liquid chromatography-mass spectrometry (LC-MS) is now a routine technique with the development of electrospray ionisation (ESI) providing a simple and robust interface. It can be applied to a wide range of biological molecules and the use of tandem MS and stable isotope internal standards allows highly sensitive and accurate assays to be developed although some method optimisation is required to minimise ion suppression effects. Fast scanning speeds allow a high degree of multiplexing and many compounds can be measured in a single analytical run. With the development of more affordable and reliable instruments, LC-MS is starting to play an important role in several areas of clinical biochemistry and compete with conventional liquid chromatography and other techniques such as immunoassay. PMID:19224008

Determination of carbon isotope ratios for honey samples by means of a liquid chromatography/isotope ratio mass spectrometry system coupled with a post-column pump.

PubMed

Kawashima, Hiroto; Suto, Momoka; Suto, Nana

2018-05-20

Liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) has been used to authenticate and trace products such as honey, wine, and lemon juice, and compounds such as caffeine and pesticides. However, LC/IRMS has several disadvantages, including the high cost of the CO 2 membrane and blocking by solidified sodium persulfate. Here, we developed an improved system for determining carbon isotope ratios by LC/IRMS. The main improvement was the use of a post-column pump. Using the improved system, we determined δ 13 C values for glucose with high accuracy and precision (0.1‰ and 0.1‰, respectively; n = 3). The glucose, fructose, disaccharide, trisaccharide, and organic acid constituents of the honey samples were analyzed by LC/IRMS. The δ 13 C values for glucose, fructose, disaccharides, trisaccharides, and organic acids ranged from -27.0 to -24.2‰, -26.8 to -24.0‰, -28.8 to -24.0‰, -27.8 to -22.8‰, and -30.6 to -27.4‰, respectively. The analysis time was 1/3-1/2 the times required for analysis by previously reported methods. The column flow rate could be arbitrarily adjusted with the post-column pump. We applied the improved method to 26 commercial honey samples. Our results can be expected to be useful for other researchers who use LC/IRMS. This article is protected by copyright. All rights reserved.

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.

An improved method for fast and selective separation of carotenoids by LC-MS.

PubMed

Abate-Pella, Daniel; Freund, Dana M; Slovin, Janet P; Hegeman, Adrian D; Cohen, Jerry D

2017-11-01

Carotenoids are a large class of compounds that are biosynthesized by condensation of isoprene units in plants, fungi, bacteria, and some animals. They are characteristically highly conjugated through double bonds, which lead to many isomers as well susceptibility to oxidation and other chemical modifications. Carotenoids are important because of their potent antioxidant activity and are the pigments responsible for color in a wide variety of foods. Human consumption is correlated to many health benefits including prevention of cancer, cardiovascular disease, and age-related disease. Extreme hydrophobicity, poor stability, and low concentration in biological samples make these compounds difficult to analyze and difficult to develop analytical methods for aimed towards identification and quantification. Examples in the literature frequently report the use of exotic stationary phases, solvents, and additives, such as ethyl acetate, dichloromethane, and methyl tert-butyl ether that are incompatible with liquid chromatography mass spectrometry (LC-MS). In order to address these issues, we implemented the use of LC-MS friendly conditions using a low-hydrophobicity cyano-propyl column (Agilent Zorbax SB-CN). We successfully differentiated between isomeric carotenoids by optimizing two gradient methods and using a mixture of 11 standards and LC-MS in positive ionization mode. Three complex biological samples from strawberry leaf, chicken feed supplement, and the photosynthetic bacterium Chloroflexus aurantiacus were analyzed and several carotenoids were resolved in these diverse backgrounds. Our results show this methodology is a significant improvement over other alternatives for analyzing carotenoids because of its ease of use, rapid analysis time, high selectivity, and, most importantly, its compatibility with typical LC-MS conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

New liquid chromatography-tandem mass spectrometry method for routine TDM of vancomycin in patients with both normal and impaired renal functions and comparison with results of polarization fluoroimmunoassay in light of varying creatinine concentrations.

PubMed

Brozmanová, Hana; Kacířová, Ivana; Uřinovská, Romana; Šištík, Pavel; Grundmann, Milan

2017-06-01

A new LC-MS/MS method with simple sample extraction and a relatively short period of vancomycin analysis for routine therapeutic drug monitoring was developed and validated. 50μL serum was precipitated using 20μL 33% trichloroacetic acid and 0.5mol/L NH 4 OH was added to increase pH before analysis. A RP BEH C18, 1.7μm, 2.1×50mm column maintained at 30°C and tobramycin as internal standard were used. Mass detection was performed in positive electrospray mode. The results obtained with LC-MS/MS method were correlated with an FPIA assay (Abbott AxSYM) using mouse monoclonal antibody. Subjects were divided into three groups according to creatinine levels (53.5±19.1, 150.2±48.4, 471.7±124.7μmol/L) and Passing-Bablok regression analysis and Bland-Altman analysis were used to compare vancomycin concentrations. The results of subjects with both normal and higher creatinine levels correlated very well and the linear regression model equations were near ideal (LC-MS VAN =0.947×Abbott VAN +0.192 and LC-MS VAN =0.973×Abbott VAN -0.411 respectively). Dialyzed patients with the highest creatinine levels showed about 14% greater vancomycin concentration with the FPIA assay (LC-MS VAN =0.866×Abbott VAN +2.127). This overestimation probably due to the presence of the metabolite CDP ought not to be of clinical relevance owing to the wide range of recommended vancomycin concentration. Copyright © 2017 Elsevier B.V. All rights reserved.

Analysis of street drugs in seized material without primary reference standards.

PubMed

Laks, Suvi; Pelander, Anna; Vuori, Erkki; Ali-Tolppa, Elisa; Sippola, Erkki; Ojanperä, Ilkka

2004-12-15

A novel approach was used to analyze street drugs in seized material without primary reference standards. Identification was performed by liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS), essentially based on accurate mass determination using a target library of 735 exact monoisotopic masses. Quantification was carried out by liquid chromatography/chemiluminescence nitrogen detection (LC/CLND) with a single secondary standard (caffeine), utilizing the detector's equimolar response to nitrogen. Sample preparation comprised dilution, first with methanol and further with the LC mobile phase. Altogether 21 seized drug samples were analyzed blind by the present method, and results were compared to accredited reference methods utilizing identification by gas chromatography/mass spectrometry and quantification by gas chromatography or liquid chromatography. The 31 drug findings by LC/TOFMS comprised 19 different drugs-of-abuse, byproducts, and adulterants, including amphetamine and tryptamine designer drugs, with one unresolved pair of compounds having an identical mass. By the reference methods, 27 findings could be confirmed, and among the four unconfirmed findings, only 1 apparent false positive was found. In the quantitative analysis of 11 amphetamine, heroin, and cocaine findings, mean relative difference between the results of LC/CLND and the reference methods was 11% (range 4.2-21%), without any observable bias. Mean relative standard deviation for three parallel LC/CLND results was 6%. Results suggest that the present combination of LC/TOFMS and LC/CLND offers a simple solution for the analysis of scheduled and designer drugs in seized material, independent of the availability of primary reference standards.

Sensitive and simultaneous determination of HIV protease inhibitors in rat biological samples by liquid chromatography-mass spectrometry.

PubMed

Gao, Weihua; Kishida, Tomoyuki; Kimura, Keisuke; Kageyama, Michiharu; Sumi, Masaki; Yoshikawa, Yukako; Shibata, Nobuhito; Takada, Kanji

2002-06-01

A sensitive and simultaneous liquid chromatographic-mass spectrometric (LC/MS) method for the determination of current four HIV protease inhibitors (PIs), indinavir (IDV), saquinavir (SQV), nelfinavir (NFV) and amprenavir (APV) in rat plasma and liver dialysate by a microdialysis method was described. An isocratic LC/MS method in combination with atmospheric pressure chemical ionization was developed for the determination of these four PIs in biological samples in the same run. The analytes including an internal standard were extracted from 100 microL of plasma or 150 microL of liver dialysate samples by salting-out with 100 microL of ice-cold 2 M K(3)PO(4) followed by ether extraction. The separation of analytes was carried out on a reversed-phase semi-micro column using 50% of acetonitrile containing 1% acetic acid as mobile phase at a flow rate of 0.2mL/min(-1). The separation was completed within 5 min. Precision, recovery and limits of detection indicated that the method was suitable for the quantitative determination of these PIs in rat plasma or liver dialysate. This simple, sensitive and highly specific LC/MS method is suitable for pharmacokinetic studies and therapeutic drug monitoring in AIDS patients who receive double protease therapy. Copyright 2002 John Wiley & Sons, Ltd.

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%.

Quantitative determination of atenolol in dried blood spot samples by LC-HRMS: a potential method for assessing medication adherence.

PubMed

Lawson, Graham; Cocks, Elizabeth; Tanna, Sangeeta

2012-05-15

The use of blood spot collection cards was investigated as a means of obtaining small volume samples for the quantification of therapeutic drugs for assessing medication adherence. A liquid chromatography-high resolution TOF mass spectrometry (LC-HRMS) method, based on the measurement at the accurate mass to charge ratio of the target analyte, was used to ensure specificity for atenolol in the dried blood spot (DBS) samples. A working method was developed and validated. For the preparation of DBS samples whole blood spiked with analyte was used to produce 30 μl blood spots on specimen collection cards. A 5mm disc was cut from the dried blood spot and extracted using methanol:water (60:40, v/v) containing the internal standard, atenolol-d(7). Extracts were vortexed, sonicated and then centrifuged. Gradient chromatographic elution was achieved using an Ascentis Express C18 100mm×2.1mm column and a mobile phase flow rate of 0.2 ml/min and the column oven temperature at 30 °C. MS detection was carried out in electrospray positive ion mode for target ions at accurate mass m/z 267.1703 for atenolol and 274.2143 for the IS. Drug extraction efficiency from spiked blood spots was demonstrated to be 96±5% and the drug was stable in DBS for at least 10 weeks. The developed LC-HRMS method was linear within the tested calibration range of 25-1500 ng/ml and validation showed the accuracy (relative error) and precision (coefficient of variation) values were within the pre-defined limits of ≤ 5% at all concentrations with a limit of quantification of 25 ng/ml. Factors with potential to affect drug quantification measurements such as the matrix effects, volume of blood applied onto the collection card and effect of different sampling cards were investigated. The developed LC-HRMS method was applied to blood spots on sampling card taken from adult healthy volunteers previously administered a 50mg atenolol tablet and a DBS concentration-time profile was obtained for atenolol. Requiring only a micro volume (30 μl) blood sample for analysis, the developed DBS based assay has the potential to assess patient adherence to atenolol. Copyright © 2012 Elsevier B.V. All rights reserved.

Relationships between chronic obstructive pulmonary disease and lung cancer: biological insights

PubMed Central

Bustamante, Víctor; Curull, Víctor; Gea, Joaquim; López-Campos, José Luis; Muñoz, Xavier

2016-01-01

Lung cancer (LC) has become one of the leading causes of preventable death in the last few decades. Cigarette smoking (CS) stays as the main etiologic factor of LC despite that many other causes such as occupational exposures, air pollution, asbestos, or radiation have also been implicated. Patients with chronic obstructive pulmonary disease (COPD), which also represents a major cause of morbidity and mortality in developed countries, exhibit a significantly greater risk of LC. The study of the underlying biological mechanisms that may predispose patients with chronic respiratory diseases to a higher incidence of LC has also gained much attention in the last few years. The present review has been divided into three major sections in which different aspects have been addressed: (I) relevant etiologic agents of LC; (II) studies confirming the hypothesis that COPD patients are exposed to a greater risk of developing LC; and (III) evidence on the most relevant underlying biological mechanisms that support the links between COPD and LC. Several carcinogenic agents have been described in the last decades but CS remains to be the leading etiologic agent in most geographical regions in which the incidence of LC is very high. Growing evidence has put the line forward the implications of COPD and especially of emphysema in LC development. Hence, COPD represents a major risk factor of LC in patients. Different avenues of research have demonstrated the presence of relevant biological mechanisms that may predispose COPD patients to develop LC. Importantly, the so far identified biological mechanisms offer targets for the design of specific therapeutic strategies that will further the current treatment options for patients with LC. Prospective screening studies, in which patients with COPD should be followed up for several years will help identify biomarkers that may predict the risk of LC among these patients. PMID:27867578

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.

CHARACTERIZATION OF DANSYLATED CYSTEINE, GLUTATHIONE DISULFIDE, CYSTEINE AND CYSTINE BY NARROW BORE LIQUID CHROMATOGRAPHY/ELECTROSPRAY IONIZATION MASS SPECTROMETRY

EPA Science Inventory

A method using reversed phase high performance liquid chromatography/electrospray ionization-mass spectrometric (RP-LC/ESI-MS) method has been developed to confirm the identity of dansylated derivatives of cysteine and glutathione, and their respective dimers. Cysteine, GSH, CSSC...

GC/FT-IR ANALYSIS OF THE THERMALLY LABILE COMPOUND TRIS (2,3-DIBROMOPROPYL) PHOSPHATE

EPA Science Inventory

A fast and convenient GC method has been developed for a compound [tris(2,3-dibromopropyl)phosphate] that poses a difficult analytical problem for both GC (thermal instability/low volatility) and LC (not amenable to commonly available, sensitive detectors) analysis. his method em...

Kaempferol loaded lecithin/chitosan nanoparticles: preparation, characterization, and their potential applications as a sustainable antifungal agent.

PubMed

Ilk, Sedef; Saglam, Necdet; Özgen, Mustafa

2017-08-01

Flavonoid compounds are strong antioxidant and antifungal agents but their applications are limited due to their poor dissolution and bioavailability. The use of nanotechnology in agriculture has received increasing attention, with the development of new formulations containing active compounds. In this study, kaempferol (KAE) was loaded into lecithin/chitosan nanoparticles (LC NPs) to determine antifungal activity compared to pure KAE against the phytopathogenic fungus Fusarium oxysporium to resolve the bioavailability problem. The influence of formulation parameters on the physicochemical properties of KAE loaded lecithin chitosan nanoparticles (KAE-LC NPs) were studied by using the electrostatic self-assembly technique. KAE-LC NPs were characterized in terms of physicochemical properties. KAE has been successfully encapsulated in LC NPs with an efficiency of 93.8 ± 4.28% and KAE-LC NPs showed good physicochemical stability. Moreover, in vitro evaluation of the KAE-LC NP system was made by the release kinetics, antioxidant and antifungal activity in a time-dependent manner against free KAE. Encapsulated KAE exhibited a significantly inhibition efficacy (67%) against Fusarium oxysporium at the end of the 60 day storage period. The results indicated that KAE-LC NP formulation could solve the problems related to the solubility and loss of KAE during use and storage. The new nanoparticle system enables the use of smaller quantities of fungicide and therefore, offers a more environmentally friendly method of controlling fungal pathogens in agriculture.

Comparison of multiple methods for the determination of sulphite in Allium and Brassica vegetables

PubMed Central

Robbins Carlos, Katherine S.; de Jager, Lowri S.

2018-01-01

Sulphites are a family of additives regulated for use worldwide in food products. They must be declared on the label if they are present in concentrations greater than 10 mg kg−1, determined as sulphur dioxide (SO2). The current US regulatory method for sulphites, the optimised Monier–Williams method (OMW), produces false-positive results with vegetables from the Allium (garlic) and Brassica (cabbage) genera due to extraction conditions that are thought to cause endogenous sulphur compounds to release SO2. Recently, modifications to the OMW method (2× MW) were published that reportedly reduced this false-positive in garlic. However, no other vegetables from these genera have been investigated. In addition, an LC-MS/MS method was developed for sulphite analysis, but it has not yet been tested with these problematic matrices. Ten vegetable species were analysed using these sulphite methods (OMW titration, OMW gravimetric, 2× MW and LC-MS/MS) to determine the false-positive rate. Sulphite concentrations > 10 mg kg−1 SO2 were observed with the OMW analyses. The 2× MW method reduced the measured concentration in unsulphited samples to ≤ 10 mg kg−1 SO2 for all matrices analysed. The LC-MS/MS method showed concentrations < 10 mg kg−1 for the Brassica samples, but only displayed a slight reduction in the Allium matrices. Spiked recovery studies were conducted to determine if these methods can detect added sulphite. The 2× MW had recoveries of 17% and 42% for water and fresh garlic, respectively, and the LC-MS/MS had recoveries of 108%, 125%, 116% and 107% for water, fresh garlic, roasted garlic, and hummus, respectively. The low recoveries of the 2× MW may indicate that sulphur compounds cannot be properly quantified with this method. The ability to eliminate false-positives will enable accurate determination of added sulphite to ensure compliance with sulphite labelling requirements. PMID:27592824

One-pot preparation of a mixed-mode organic-silica hybrid monolithic capillary column and its application in determination of endogenous gibberellins in plant tissues.

PubMed

Zhang, Zheng; Hao, Yan-Hong; Ding, Jun; Xu, Sheng-Nan; Yuan, Bi-Feng; Feng, Yu-Qi

2015-10-16

A newly improved one-pot method, based on "thiol-ene" click chemistry and sol-gel approach in microemulsion system, was developed for the preparation of C8/PO(OH)2-silica hybrid monolithic capillary column. The prepared monolith possesses large specific surface area, narrow mesopore size distribution and high column efficiency. The monolithic column was demonstrated to have cation exchange/reversed-phase (CX/RP) mixed-mode retention for analytes on nano-liquid chromatography (nano-LC). On the basis of the developed nano-LC system with MS detector coupled to pipette tip solid phase extraction (PT-SPE) and derivatization process, we then realized simultaneous determination of 10 gibberellins (GAs) with low limits of detection (LODs, 0.003-0.025 ng/mL). Furthermore, 6 endogenous GAs in only 5mg rice leaves (fresh weight) were successfully detected and quantified. The developed PT-SPE-nano-LC-MS strategy may offer promising applications in the determination of low abundant bioactive molecules from complex matrix. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

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

Chiral HPLC for a study of the optical purity of new liquid crystalline materials derived from lactic acid

NASA Astrophysics Data System (ADS)

Vojtylová, T.; Kašpar, M.; Hamplová, V.; Novotná, V.; Sýkora, D.

2014-08-01

New liquid crystalline (LC) materials were prepared by derivatization of lactic acid. First compound possesses the lactic acid unit as the only chiral center and the second group of LC materials contains two chiral centers. Mesomorphic properties of both the newly synthesized LC materials were studied and the presence of the SmA*-SmC* or exhibit the twist grain boundary (TGB) phases, namely TGBA and TGBC, in a wide range of temperatures down to the room temperature was established. The potential of high-performance liquid chromatography (HPLC) applying chiral stationary phases to separate enantiomers or diastereoisomers of the synthesized LC compounds was evaluated. Two different brands of commercial chiral sorbents, Lux Amylose-2 and Chiralpak AD-3, both based on modified silica with derivatized polysaccharide, were employed in the development of separation procedures. The optimized chiral HPLC method provided a baseline separation of the individual enantiomers for the LC material containing one chiral center. In the case of the more complex compound with two asymmetric carbon atoms, where four isomers exist, partial separation was reached only using the current chiral HPLC.

Development and Multi-laboratory Verification of U.S. EPA Method 540 for the Analysis of Drinking Water Contaminants by Solid Phase Extraction-LC/MS/MS

EPA Science Inventory

A drinking water method for 12 chemicals, predominately pesticides, is presented that addresses the occurrence monitoring needs of the U.S. Environmental Protection Agency (EPA) for a future Unregulated Contaminant Monitoring Regulation (UCMR). The method employs solid phase ext...

Quantitative determination of triterpenes from Amphiptherygium adstringens by liquid chromatography and thin-layer chromatography and morphological analysis of cuachalalate preparations.

PubMed

Navarrete, Andres; Avula, Bharathi; Joshi, Vaishali C; Ji, Xiuhong; Hersh, Paul; Khan, Ikhlas A

2006-01-01

Amphiptherygium adstringens (Anacardiaceae/Julianaceae), local name "cuachalalate," is used in folk medicine for the treatment of cholelithiasis, fevers, fresh wounds, hypercholesterolemia, gastritis, gastric ulcers, and cancer of the gastrointestinal tract. The development of column high-performance liquid chromatography-photodiode array detector (LC-PDA) and high-performance thin-layer chromatography (HPTLC)-densitometry methods for the determination of masticadienonic acid and 3-hydroxymasticadienonic acid in cuachalalate preparations is described in this paper. Good separation of the compounds could be achieved by both methods. Either might be preparable depending on the requirements. The LC separation was performed on a Phenomenex Synergi MAX-RP 80A reversed-phase column operated at 40 degrees C with detection at 215 nm. The plant materials were extracted with methanol by sonication. The triterpenes present in the plant material and commercial extracts were separated with an acetonitrile-water reagent alcohol isocratic system. The limit of detection was 0.1-0.2 microg/mL. The relative standard deviation values for the determination of triterpenes in plant extracts were less than 1.00%. This is the first report of an analytical method developed for the quantitative analysis of triterpenes from Amphiptherygium adstringens by LC-PDA and HPTLC. The stem bark showed higher amounts of triterpenes, and low amounts in root and stem root. The microscopic description of the crude drug of cuachalalate was also provided.

Direct-injection screening for acidic drugs in plasma and neutral drugs in equine urine by differential-gradient LC-LC coupled MS/MS.

PubMed

Stanley, Shawn M R; Wee, Wei Khee; Lim, Boon Huat; Foo, Hsiao Ching

2007-04-01

Direct-injection LC-LC hybrid tandem MS methods have been developed for undertaking broad-based screening for acidic drugs in protein-precipitated plasma and neutral doping agents in equine urine. In both analyses, analytes present in the matrix were trapped using a HLB extraction column before being refocused and separated on a Chromolith RP-18e monolithic analytical column using a controlled differential gradient generated by proportional dilution of the first column's eluent with water. Each method has been optimised by the adoption of a mobile phase and gradient that was tailored to enhance ionisation in the MS source while maintaining good chromatographic behaviour for the majority of the target drugs. The analytical column eluent was fed into the heated nebulizer (HN) part of the Duospray interface attached to a 4000 QTRAP mass spectrometer. Information dependent acquisition (IDA) with dynamic background subtraction (DBS) was configured to trigger a sensitive enhanced product ion (EPI) scan when a multiple reaction monitoring (MRM) survey scan signal exceeded the defined criteria. Ninety-one percent of acidic drugs in protein-precipitated plasma and 80% of the neutral compounds in equine urine were detected when spiked at 10 ng/ml.

Pentacyclic triterpene in Olea europaea L: A simultaneous determination by high-performance liquid chromatography coupled to mass spectrometry.

PubMed

Giménez, Estela; Juan, M Emília; Calvo-Melià, Sara; Barbosa, José; Sanz-Nebot, Victoria; Planas, Joana M

2015-09-04

Pentacyclic triterpenes are gaining interest due to their beneficial health effects, as anti-inflammatory, anti-diabetic and anti-tumoral, among others. In this study, an analytical LC-MS method was developed for the simultaneous determination of maslinic, oleanolic and ursolic acids along with erythrodiol and uvaol, which are the main triterpenic compounds present in the fruits and leaves of Olea europaea L. A Zorbax Eclipse PAH column at 30°C with mobile phase of water (17%) and methanol (83%) at 0.8mL/min conformed the optimal chromatographic conditions that allowed the separation of the compounds of interest, two pairs of which are isomers differing only in the position of one methyl group (oleanolic-ursolic and erythrodiol-uvaol). The ionization was performed in an APCI source at 450°C programmed in negative mode for the triterpenic acids, and in positive for the alcohols. An ion trap (LC-IT-MS) and a triple quadrupole (LC-QqQ-MS) were assessed for maximal sensitivity that was achieved with LC-QqQ-MS. The LODs of triterpenic acids were lower than 1nM, whereas for erythrodiol and uvaol were 4.5 and 7.5nM, respectively. The method was linear for the five analytes in the range of concentrations from 0.005 to 15μM with correlation coefficients exceeding 0.99. The precision and accuracy were ≤9.90% and ≤9.57%, respectively. The applicability of the validated method was assessed in the analysis of the pentacyclic triterpenes in Marfil table olives, after the optimization of the extraction procedure. The developed method constitutes the first step for future studies of triterpenic compounds present in foods that would allow establishing their effects on human health. Copyright © 2015 Elsevier B.V. All rights reserved.

MS/MS library facilitated MRM quantification of native peptides prepared by denaturing ultrafiltration

PubMed Central

2012-01-01

Naturally occurring native peptides provide important information about physiological states of an organism and its changes in disease conditions but protocols and methods for assessing their abundance are not well-developed. In this paper, we describe a simple procedure for the quantification of non-tryptic peptides in body fluids. The workflow includes an enrichment step followed by two-dimensional fractionation of native peptides and MS/MS data management facilitating the design and validation of LC- MRM MS assays. The added value of the workflow is demonstrated in the development of a triplex LC-MRM MS assay used for quantification of peptides potentially associated with the progression of liver disease to hepatocellular carcinoma. PMID:22304756

Characterization of matrix effects in developing rugged high-throughput LC-MS/MS methods for bioanalysis.

PubMed

Li, Fumin; Wang, Jun; Jenkins, Rand

2016-05-01

There is an ever-increasing demand for high-throughput LC-MS/MS bioanalytical assays to support drug discovery and development. Matrix effects of sofosbuvir (protonated) and paclitaxel (sodiated) were thoroughly evaluated using high-throughput chromatography (defined as having a run time ≤1 min) under 14 elution conditions with extracts from protein precipitation, liquid-liquid extraction and solid-phase extraction. A slight separation, in terms of retention time, between underlying matrix components and sofosbuvir/paclitaxel can greatly alleviate matrix effects. High-throughput chromatography, with proper optimization, can provide rapid and effective chromatographic separation under 1 min to alleviate matrix effects and enhance assay ruggedness for regulated bioanalysis.

High throughput screening of active pharmaceutical ingredients by UPLC.

PubMed

Al-Sayah, Mohammad A; Rizos, Panagiota; Antonucci, Vincent; Wu, Naijun

2008-07-01

Ultra performance LC (UPLC) was evaluated as an efficient screening approach to facilitate method development for drug candidates. Three stationary phases were screened: C-18, phenyl, and Shield RP 18 with column dimensions of 150 mm x 2.1 mm, 1.7 microm, which should theoretically generate 35,000 plates or 175% of the typical column plate count of a conventional 250 mm x 4.6 mm, 5 microm particle column. Thirteen different active pharmaceutical ingredients (APIs) were screened using this column set with a standardized mobile-phase gradient. The UPLC method selectivity results were compared to those obtained for these compounds via methods developed through laborious trial and error screening experiments using numerous conventional HPLC mobile and stationary phases. Peak capacity was compared for columns packed with 5 microm particles and columns packed with 1.7 microm particles. The impurities screened by UPLC were confirmed by LC/MS. The results demonstrate that simple, high efficiency UPLC gradients are a feasible and productive alternative to more conventional multiparametric chromatographic screening approaches for many compounds in the early stages of drug development.

Second-tier test for quantification of underivatized amino acids in dry blood spot for metabolic diseases in newborn screening.

PubMed

Wang, Chunyan; Zhu, Hongbin; Zhang, Wenyan; Song, Fengrui; Liu, Zhiqiang; Liu, Shuying

2013-02-01

The quantitative analysis of amino acids (AAs) in single dry blood spot (DBS) samples is an important issue for metabolic diseases as a second-tier test in newborn screening. An analytical method for quantifying underivatized AAs in DBS was developed by using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The sample preparation in this method is simple and ion-pairing agent is not used in the mobile phase that could avoid ion suppression, which happens in mass spectrometry and avoids damage to the column. Through chromatographic separation, some isomeric compounds could be identified and quantified, which cannot be solved through only appropriate multiple reactions monitoring transitions by MS/MS. The concentrations of the different AAs were determined using non-deuterated internal standard. All calibration curves showed excellent linearity within test ranges. For most of the amino acids the accuracy of extraction recovery was between 85.3 and 115 %, and the precision of relative standard deviation was <7.0 %. The 35 AAs could be identified in DBS specimens by the developed LC-MS/MS method in 17-19 min, and eventually 24 AAs in DBS were quantified. The results of the present study prove that this method as a second-tier test in newborn screening for metabolic diseases could be performed by the quantification of free AAs in DBS using the LC-MS/MS method. The assay has advantages of high sensitive, specific, and inexpensive merits because non-deuterated internal standard and acetic acid instead of ion-pairing agent in mobile phase are used in this protocol.

Automated solid-phase extraction-liquid chromatography-tandem mass spectrometry analysis of 6-acetylmorphine in human urine specimens: application for a high-throughput urine analysis laboratory.

PubMed

Robandt, P V; Bui, H M; Scancella, J M; Klette, K L

2010-10-01

An automated solid-phase extraction-liquid chromatography- tandem mass spectrometry (SPE-LC-MS-MS) method using the Spark Holland Symbiosis Pharma SPE-LC coupled to a Waters Quattro Micro MS-MS was developed for the analysis of 6-acetylmorphine (6-AM) in human urine specimens. The method was linear (R² = 0.9983) to 100 ng/mL, with no carryover at 200 ng/mL. Limits of quantification and detection were found to be 2 ng/mL. Interrun precision calculated as percent coefficient of variation (%CV) and evaluated by analyzing five specimens at 10 ng/mL over nine batches (n = 45) was 3.6%. Intrarun precision evaluated from 0 to 100 ng/mL ranged from 1.0 to 4.4%CV. Other opioids (codeine, morphine, oxycodone, oxymorphone, hydromorphone, hydrocodone, and norcodeine) did not interfere in the detection, quantification, or chromatography of 6-AM or the deuterated internal standard. The quantified values for 41 authentic human urine specimens previously found to contain 6-AM by a validated gas chromatography (GC)-MS method were compared to those obtained by the SPE-LC-MS-MS method. The SPE-LC-MS-MS procedure eliminates the human factors of specimen handling, extraction, and derivatization, thereby reducing labor costs and rework resulting from human error or technique issues. The time required for extraction and analysis was reduced by approximately 50% when compared to a validated 6-AM procedure using manual SPE and GC-MS analysis.

A sensitive LC-MS/MS method for measurement of organophosphorus pesticides and their oxygen analogs in air sampling matrices

PubMed Central

ARMSTRONG, JENNA L.; DILLS, RUSSELL L.; YU, JIANBO; YOST, MICHAEL G.; FENSKE, RICHARD A.

2018-01-01

A rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for determination of levels of the organophosphorus (OP) pesticides chlorpyrifos (CPF), azinphos methyl (AZM), and their oxygen analogs chlorpyrifos-oxon (CPF-O) and azinphos methyl-oxon (AZM-O) on common active air sampling matrices. XAD-2 resin and polyurethane foam (PUF) matrices were extracted with acetonitrile containing stable-isotope labeled internal standards (ISTD). Analysis was accomplished in Multiple Reaction Monitoring (MRM) mode, and analytes in unknown samples were identified by retention time (±0.1 min) and qualifier ratio (±30% absolute) as compared to the mean of calibrants. For all compounds, calibration linearity correlation coefficients were ≥0.996. Limits of detection (LOD) ranged from 0.15–1.1 ng/sample for CPF, CPF-O, AZM, and AZM-O on active sampling matrices. Spiked fortification recoveries were 78–113% from XAD-2 active air sampling tubes and 71–108% from PUF active air sampling tubes. Storage stability tests also yielded recoveries ranging from 74–94% after time periods ranging from 2–10 months. The results demonstrate that LC-MS/MS is a sensitive method for determining these compounds from two different matrices at the low concentrations that can result from spray drift and long range transport in non-target areas following agricultural applications. In an inter-laboratory comparison, the limit of quantification (LOQ) for LC-MS/MS was 100 times lower than a typical gas chromatography-mass spectrometry (GC-MS) method. PMID:24328542

A sensitive LC-MS/MS method for measurement of organophosphorus pesticides and their oxygen analogs in air sampling matrices.

PubMed

Armstrong, Jenna L; Dills, Russell L; Yu, Jianbo; Yost, Michael G; Fenske, Richard A

2014-01-01

A rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for determination of levels of the organophosphorus (OP) pesticides chlorpyrifos (CPF), azinphos methyl (AZM), and their oxygen analogs chlorpyrifos-oxon (CPF-O) and azinphos methyl-oxon (AZM-O) on common active air sampling matrices. XAD-2 resin and polyurethane foam (PUF) matrices were extracted with acetonitrile containing stable-isotope labeled internal standards (ISTD). Analysis was accomplished in Multiple Reaction Monitoring (MRM) mode, and analytes in unknown samples were identified by retention time (±0.1 min) and qualifier ratio (±30% absolute) as compared to the mean of calibrants. For all compounds, calibration linearity correlation coefficients were ≥0.996. Limits of detection (LOD) ranged from 0.15-1.1 ng/sample for CPF, CPF-O, AZM, and AZM-O on active sampling matrices. Spiked fortification recoveries were 78-113% from XAD-2 active air sampling tubes and 71-108% from PUF active air sampling tubes. Storage stability tests also yielded recoveries ranging from 74-94% after time periods ranging from 2-10 months. The results demonstrate that LC-MS/MS is a sensitive method for determining these compounds from two different matrices at the low concentrations that can result from spray drift and long range transport in non-target areas following agricultural applications. In an inter-laboratory comparison, the limit of quantification (LOQ) for LC-MS/MS was 100 times lower than a typical gas chromatography-mass spectrometry (GC-MS) method.

Determination of Aristolochic Acid in Botanicals and Dietary Supplements by Liquid Chromatography with Ultraviolet Detection and by Liquid Chromatography/Mass Spectrometry: Single Laboratory Validation Confirmation

PubMed Central

Trujillo, William A.; Sorenson, Wendy R.; La Luzerne, Paul; Austad, John W.; Sullivan, Darryl

2008-01-01

The presence of aristolochic acid in some dietary supplements is a concern to regulators and consumers. A method has been developed, by initially using a reference method as a guide, during single laboratory validation (SLV) for the determination of aristolochic acid I, also known as aristolochic acid A, in botanical species and dietary supplements at concentrations of approximately 2 to 32 μg/g. Higher levels were determined by dilution to fit the standard curve. Through the SLV, the method was optimized for quantification by liquid Chromatography with ultraviolet detection (LC-UV) and LC/mass Spectrometry (MS) confirmation. The test samples were extracted with organic solvent and water, then injected on a reverse phase LC column. Quantification was achieved with linear regression using a laboratory automation system. The SLV study included systematically optimizing the LC-UV method with regard to test sample size, fine grinding of solids, and solvent extraction efficiency. These parameters were varied in increments (and in separate optimization studies), in order to ensure that each parameter was individually studied; the test results include corresponding tables of parameter variations. In addition, the chromatographic conditions were optimized with respect to injection volume and detection wavelength. Precision studies produced overall relative standard deviation values from 2.44 up to 8.26% for aristolochic acid I. Mean recoveries were between 100 and 103% at the 2 μg/g level, between 102 and 103% at the 10 μg/g level, and 104% at the 30 μg/g level. PMID:16915829

Determination of aristolochic acid in botanicals and dietary supplements by liquid chromatography with ultraviolet detection and by liquid chromatography/mass spectrometry: single laboratory validation confirmation.

PubMed

Trujillo, William A; Sorenson, Wendy R; La Luzerne, Paul; Austad, John W; Sullivan, Darryl

2006-01-01

The presence of aristolochic acid in some dietary supplements is a concern to regulators and consumers. A method has been developed, by initially using a reference method as a guide, during single laboratory validation (SLV) for the determination of aristolochic acid I, also known as aristolochic acid A, in botanical species and dietary supplements at concentrations of approximately 2 to 32 microg/g. Higher levels were determined by dilution to fit the standard curve. Through the SLV, the method was optimized for quantification by liquid chromatography with ultraviolet detection (LC-UV) and LC/mass spectrometry (MS) confirmation. The test samples were extracted with organic solvent and water, then injected on a reverse phase LC column. Quantification was achieved with linear regression using a laboratory automation system. The SLV study included systematically optimizing the LC-UV method with regard to test sample size, fine grinding of solids, and solvent extraction efficiency. These parameters were varied in increments (and in separate optimization studies), in order to ensure that each parameter was individually studied; the test results include corresponding tables of parameter variations. In addition, the chromatographic conditions were optimized with respect to injection volume and detection wavelength. Precision studies produced overall relative standard deviation values from 2.44 up to 8.26% for aristolochic acid I. Mean recoveries were between 100 and 103% at the 2 microg/g level, between 102 and 103% at the 10 microg/g level, and 104% at the 30 microg/g level.

Development and Validation of a Rapid and Sensitive LC-MS/MS Method for the Pharmacokinetic Study of Osimertinib in Rats.

PubMed

Xiong, Shan; Deng, Zhipeng; Sun, Peilu; Mu, Yanling; Xue, Mingxing

2017-11-01

Osimertinib is a new-generation epidermal growth factor inhibitor for the treatment of non-small cell lung cancer. In the present study, a rapid and sensitive LC with tandem MS method was developed and validated for the determination of osimertinib in rat plasma. Chromatographic separation was carried out on a C18 column using acetonitrile and water containing 0.1% formic acid. The assay was validated over a concentration range of 1.0-1000 ng/mL for osimertinib, with a lower LOQ of 1.0 ng/mL. The intra- and interday accuracy values for osimertinib ranged from 92.66 to 101.50% and from 97.08 to 99.15%, respectively, and the intra- and interday precision values for osimertinib ranged from 6.25 to 10.34% and from 3.43 to 10.44%, respectively. The method was successfully applied in a pharmacokinetic study of osimertinib after oral administration of osimertinib (4.5 mg/kg) to rats.

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.

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.

LC-MSMS assays of urinary cortisol, a comparison between four in-house assays.

PubMed

Brossaud, Julie; Leban, Monique; Corcuff, Jean-Benoit; Boux de Casson, Florence; Leloupp, Anne-Gaëlle; Masson, Damien; Moal, Valérie; Bach-Ngohou, Kalyane

2018-06-27

Twenty-four hour urinary free cortisol (UFC) determination can be used for screening and follow-up of Cushing syndrome (CS). As immunoassay methods lack specificity for UFC measurement, the use of high-performance liquid chromatography coupled to mass spectrometer (LC-MSMS) is recommended. The aim of our study was to compare UFC results using four LC-MSMS methods performed in four independent laboratories in order to evaluate interlaboratory agreement. Frozen aliquots of 24-h urine samples (78 healthy volunteers and 20 patients with CS) were sent to four different laboratories for analysis. Following liquid-liquid or solid-liquid extraction, UFC were determined using four different LC-MSMS assay. UFC intra- and interassays variation coefficients were lower than 10% for each centre. External quality control results were not significantly different. UFC normal ranges (established from healthy volunteers) were 17-126, 15-134, 12-118 and 27-157 nmol/day, respectively. Classification of UFC from healthy volunteers and patients with CS using a 95th percentile threshold was similar. However, for extreme UFC values (<50 or >270 nmol/day), negative or positive bias was noted. Even for highly specific methods such as LC-MSMS, variations of results can be found depending on analytical process. Validation of LC-MSMS methods including determination of the reference range is essential.

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

Unclosed fascial defect: is it the risk to develop port-site hernia after laparoscopic cholecystectomy?

PubMed

Tangjaroen, Somard; Watanapa, Prasit

2014-02-01

Port-site hernia (PSH) is one of the complications after laparoscopic cholecystectomy (LC). Closure of the fascial defect has been mentioned to prevent such complication. However, the results are still controversial. The present study was done to clarify whether unclosed fascial defect was actually the risk factor for the development of PSH MATERIAL AND METHOD: Two hundred ninety four patients underwent LC by a single surgeon at Kalasin Hospital between 2007 and 2010. The procedure was done by using a four-port technique without closure of any fascial defects. The male:female ratio was 85:209, and the mean body mass index was 24.38 +/- 3.33 (SD). The mean operative time was 18.71 +/- 3.76 minutes and there was no postoperative wound infection. Patients were regularly followed-up and underwent both supine and upright physical examination. The mean duration of follow-up period was 4.94 +/- 1.31 years with the shortest follow-up period of two years. None of the patients in the present study developed PSH in any port sites during the follow-up period. Unclosed fascial defect may not have the significant risk factor of developing PSH after LC.

Amnesic shellfish poisoning toxins in shellfish: estimation of uncertainty of measurement for a liquid chromatography/tandem mass spectrometry method.

PubMed

Holland, Patrick T; McNabb, Paul; Selwood, Andrew I; Neil, Tracey

2003-01-01

A liquid chromatography/mass spectrometry (LC/MS) method for amnesic shellfish poisoning toxins in shellfish was developed and validated. Tissue homogenate (4 g) was extracted with 16 mL methanol-water (1 + 1, v/v). Dilution into acetonitrile-water (1 + 9, v/v) was followed by C18 solid-phase extraction cleanup. Domoic acid (DA) and epi-domoic acid were determined by LC/MS/MS with electrospray ionization and multiple reaction monitoring. External calibration was performed with dilutions of a certified reference standard. Advantages of this method include speed, lower detection limits, and a very high degree of specificity. The LC/MS response was highly linear, and there were no significant interferences to the determination of DA. Formal method validation was performed on 4 shellfish species. Fortification studies gave recoveries (mean +/- SD; n = 24) of 93 +/- 14% at 1 mg/kg, and 93.3 +/- 7.6% at 20 mg/kg over all the species. Analysis of a mussel certified reference material showed the bias as < 5%. The limits of detection and quantitation were 0.15 and 0.5 mg/kg, respectively. Routine application of the method over 4 months gave a recovery for the QC sample (1 mg/kg fortified blank mussel homogenate) run with each batch of 88.9 +/- 5.5% (mean +/- SD; n = 37). The total uncertainty of measurement results were estimated as 0.12 (12%) at 0.25-5 mg/kg and 0.079 (7.9%) at 5-50 mg/kg. The major contribution to the uncertainty was the repeatability of the LC/MS determination, probably arising from subtle matrix effects.

Analysis of benzodiazepines and their metabolites using DBS cards and LC-MS/MS.

PubMed

Lee, Heesang; Park, Yujin; Jo, Jiyeong; In, Sangwhan; Park, Yonghoon; Kim, Eunmi; Pyo, Jaesung; Choe, Sanggil

2015-10-01

Dried Blood Spot (DBS) has been used a blood extraction method for inherited metabolic disorder screening since 1960s. With introduction of LC-MS/MS, not only DBS could be used to analysis drugs in small blood volume, but in various fields, such as toxicology, drug therapeutic monitoring, drug diagnostic screening, and illicit drugs. In toxicology field, many drugs (e.g. benzodiazepines, acetaminophen, small molecule drugs) have been tested with DBS. Compared with earlier blood extraction methods (SPE and LLE), DBS has lots of advantages; lower blood volume (less than 50μL), shorter analysis time caused by a more concise analysis procedure and lower cost. We optimized the DBS procedure and LC-MS/MS conditions for 18 benzodiazepines, seven benzodiazepine metabolites, and one z-drug (zolpidem) analysis in blood. 30μL of whole blood was spotted on FTA DMPK card C and dried for 2h in a desiccator. A 6-mm disk was punched and vortexed for 1min in a centrifuge tube with 300μL methanol/acetonitrile mixture (1:1, v/v). After evaporation, redissolved in 100μL mobile phase of LC-MS/MS and 5μL was injected. In the analysis for 26 target compounds in blood, all of the method validation parameters - LLOD, LLOQ, accuracy (intra- and inter-assay), and precision (intra- and inter-assay) - were satisfied with method validation criteria, within 15%. The results of matrix effect, recovery, and process efficiency were good. We developed a fast and reliable sample preparation method using DBS for 26 benzodiazepines, benzodiazepine metabolites, and z-drug (zolpidem). Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

Evaluation of analytical techniques to determine AQUI-S® 20E (eugenol) concentrations in water

USGS Publications Warehouse

Meinertz, Jeffery R.; Hess, Karina R.

2014-01-01

There is a critical need in U.S. public aquaculture and fishery management programs for an immediate-release sedative, i.e. a compound that can be safely and effectively used to sedate fish and subsequently, allow for their immediate release. AQUI-S® 20E (10% active ingredient, eugenol; any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government) is being pursued for U.S. approval as an immediate-release sedative. As part of the approval process, data describing animal safety and efficacy are needed. Essential to conducting studies that generate those data, is a method to accurately and precisely determine AQUI-S® 20E concentrations in exposure baths. Spectrophotometric and solid phase extraction (SPE)–high pressure liquid chromatography (LC) methods were developed and evaluated as methods to determine AQUI-S® 20E (eugenol) concentrations in water, methods that could be applied to any situation where eugenol was being evaluated as a fish sedative. The spectrophotometric method was accurate and precise (accuracy, > 87%; precision, 86%; precision < 8.9 %CV) when determining eugenol concentrations in solutions of 50 to 1000 mg/L AQUI-S® 20E made with LC grade water and water with varying pH and hardness. The SPE–LC method was influenced to a lesser degree by the presence of fish feed indicating greater specificity for eugenol.

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.

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.

Validated enantiospecific LC method for determination of (R)-enantiomer impurity in (S)-efavirenz.

PubMed

Seshachalam, U; Narasimha Rao, D V L; Chandrasekhar, K B

2008-02-01

A high-performance liquid chromatographic method was developed for separation of the enantiomers of efavirenz. The developed method was applied for the determination of (R)-enantiomer in (S)-efavirenz and satisfactory results were achieved. The base line separation with a resolution of more than 4.0 was achieved on Chiralcel OD (250 mm x 4.6 mm, 10 microm) column containing tris-(3,5-dimethylphenylcarbomate) as stationary phase. The mobile phase consists of n-hexane: isopropyl alcohol (80:20 v/v) with 0.1% (v/v) of formic acid as additive. The flow rate was kept at 1.0 ml/min and the UV detection was monitored at 254 nm. The (R)-enantiomer was found linear over the range of 0.1 microg/ml--6 microg/ml. The limit of detection (LOD) was 0.03 microg/ml and the limit of quantification (LOQ) was 0.1 microg/ml (n=3. The precision of (R)-enantiomer at LOQ level was evaluated through six replicate injections and the RSD of the peak response was achieved as 1.34%. The results demonstrated that the developed LC method was simple, precise, robust and applicable for the purity determination of efavirenz.

Highly sensitive LC-MS/MS-ESI method for determination of phenelzine in human plasma and its application to a human pharmacokinetic study.

PubMed

Kallem, Raja Reddy; Jillela, Bhupathi; Ravula, Arun Reddy; Samala, Ramakrishna; Andy, Adinarayana; Ramesh, Mullangi; Rao, Jvln Seshagiri

2016-06-01

A selective, sensitive and rapid LC-MS/MS method has been developed and validated for quantification of the phenelzine (PZ) in 200μL of human plasma using hydroxyzine (HZ) as an internal standard (IS) as per regulatory guidelines. The sample preparation involved the derivatization of PZ using pentaflurobenzaldehyde followed by solid phase extraction process to extract PZ and HZ from human plasma. LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electro spray ionization technique in positive ion mode and the transitions of m/z 305.1→105.1 and m/z 375.3→201.1 were used to measure the derivative of PZ and IS, respectively. The total run time was 3.5min and the elution of PZ and HZ occurred at 2.53, and 1.92min, respectively; this was achieved with a mobile phase consisting of 10mM ammonium acetate: acetonitrile (20:80, v/v) at a flow rate of 1.0mL/min on an Ace C18 column with a split ratio of 70:30. The developed method was validated in human plasma with a lower limit of quantitation 0.51ng/mL. A linear response function was established for the range of concentrations 0.51-25.2ng/mL (r>0.995) for PZ. The intra- and inter-day precision values met the acceptance criteria. PZ was stable in the battery of stability studies viz., stock solution, bench-top, auto-sampler, long-term and freeze/thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans. Copyright © 2016 Elsevier B.V. All rights reserved.

Simultaneous targeted analysis of trimethylamine-N-oxide, choline, betaine, and carnitine by high performance liquid chromatography tandem mass spectrometry.

PubMed

Liu, Jia; Zhao, Mingming; Zhou, Juntuo; Liu, Changjie; Zheng, Lemin; Yin, Yuxin

2016-11-01

Trimethylamine-N-oxide (TMAO) is a metabolite generated from choline, betaine and carnitine in a gut microbiota-dependent way. This molecule is associated with development of atherosclerosis and cardiovascular events. A sensitive liquid chromatographic electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) has been developed and validated for the simultaneous determination of TMAO related molecules including TMAO, betaine, choline, and carnitine in mouse plasma. Analytes are extracted after protein precipitation by methanol and subjected to LC-ESI-MS/MS without preliminary derivatization. Separation of analytes was achieved on an amide column with acetonitrile-water as the mobile phase. This method has been fully validated in this study in terms of selectivity, linearity, sensitivity, precision, accuracy, and carryover effect, and the stability of the analyte under various conditions has been confirmed. This developed method has successfully been applied to plasma samples of our mouse model. Copyright © 2016 Elsevier B.V. All rights reserved.

Porous Graphitic Carbon Liquid Chromatography-Mass Spectrometry Analysis of Drought Stress-Responsive Raffinose Family Oligosaccharides in Plant Tissues.

PubMed

Jorge, Tiago F; Florêncio, Maria H; António, Carla

2017-01-01

Drought is a major limiting factor in agriculture and responsible for dramatic crop yield losses worldwide. The adjustment of the metabolic status via accumulation of drought stress-responsive osmolytes is one of the many strategies that some plants have developed to cope with water deficit conditions. Osmolytes are highly polar compounds, analysis of whcih is difficult with typical reversed-phase chromatography. Porous graphitic carbon (PGC) has shown to be a suitable alternative to reversed-phase stationary phases for the analysis of highly polar compounds typically found in the plant metabolome. In this chapter, we describe the development and validation of a PGC-based liquid chromatography tandem mass spectrometry (LC-MS n ) method suitable for the target analysis of water-soluble carbohydrates, such as raffinose family oligosaccharides (RFOs). We present detailed information regarding PGC column equilibration, LC-MS n system operation, data analysis, and important notes to be considered during the steps of method development and validation.

A dilute-and-shoot flow-injection tandem mass spectrometry method for quantification of phenobarbital in urine.

PubMed

Alagandula, Ravali; Zhou, Xiang; Guo, Baochuan

2017-01-15

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) is the gold standard of urine drug testing. However, current LC-based methods are time consuming, limiting the throughput of MS-based testing and increasing the cost. This is particularly problematic for quantification of drugs such as phenobarbital, which is often analyzed in a separate run because they must be negatively ionized. This study examined the feasibility of using a dilute-and-shoot flow-injection method without LC separation to quantify drugs with phenobarbital as a model system. Briefly, a urine sample containing phenobarbital was first diluted by 10 times, followed by flow injection of the diluted sample to mass spectrometer. Quantification and detection of phenobarbital were achieved by an electrospray negative ionization MS/MS system operated in the multiple reaction monitoring (MRM) mode with the stable-isotope-labeled drug as internal standard. The dilute-and-shoot flow-injection method developed was linear with a dynamic range of 50-2000 ng/mL of phenobarbital and correlation coefficient > 0.9996. The coefficients of variation and relative errors for intra- and inter-assays at four quality control (QC) levels (50, 125, 445 and 1600 ng/mL) were 3.0% and 5.0%, respectively. The total run time to quantify one sample was 2 min, and the sensitivity and specificity of the method did not deteriorate even after 1200 consecutive injections. Our method can accurately and robustly quantify phenobarbital in urine without LC separation. Because of its 2 min run time, the method can process 720 samples per day. This feasibility study shows that the dilute-and-shoot flow-injection method can be a general way for fast analysis of drugs in urine. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Determination of naltrexone and 6beta-naltrexol in human blood: comparison of high-performance liquid chromatography with spectrophotometric and tandem-mass-spectrometric detection.

PubMed

Brünen, Sonja; Krüger, Ralf; Finger, Susann; Korf, Felix; Kiefer, Falk; Wiedemann, Klaus; Lackner, Karl J; Hiemke, Christoph

2010-02-01

We present data for a comparison of a liquid-chromatographic method coupled with tandem mass spectrometry (LC-MS/MS) and a high-performance liquid-chromatographic method with column switching and UV spectrophotometric detection. The two methods were developed for determination of naltrexone and 6beta-naltrexol in blood serum or plasma aiming to be used for therapeutic drug monitoring to guide the treatment of patients with naltrexone. For the high-performance liquid chromatography (HPLC)/UV detection, online sample cleanup was conducted on Perfect Bond C(18) material with 2% (vol/vol) acetonitrile in deionized water. Drugs were separated on a C(18) column using 11.5% (vol/vol) acetonitrile and 0.4% (vol/vol) N,N,N,N-tetramethylethylenediamine within 20 min. LC-MS/MS used naltrexone-d (3) and 6beta-naltrexol-d (4) as internal standards. After protein precipitation, the chromatographic separation was performed on a C(18) column by applying a methanol gradient (5-100%, vol/vol) with 0.1% formic acid over 9.5 min. The HPLC/UV method was found to be linear for concentrations ranging from 2 to 100 ng/ml, with a regression correlation coefficient of r (2) > 0.998 for naltrexone and 6beta-naltrexol. The limit of quantification was 2 ng/ml for naltrexone and 6beta-naltrexol. For the LC-MS/MS method the calibration curves were linear (r(2) > 0.999) from 0.5 to 200 ng/ml for both substances, and the limit of quantification was 0.5 ng/ml. The concentrations measured by the two methods correlated significantly for both substances (r(2) > 0.967; p < 0.001). Both methods could be used for therapeutic drug monitoring. The HPLC/UV method was advantageous regarding automatization and costs, whereas LC-MS/MS was superior with regard to sensitivity.

THE DEVELOPMENT AND INTER-LABORATORY VERIFICATION OF LC-MS LIBRARIES FOR ORGANIC CHEMICALS OF ENVIRONMENTAL CONCERN

EPA Science Inventory

The development, verification, and comparison study between LC-MS libraries for two manufacturers’ instruments and a verified protocol are discussed. The LC-MS library protocol was verified through an inter-laboratory study that involved Federal, State, and private laboratories. ...

Forensic determination of ricin and the alkaloid marker ricinine from castor bean extracts.

PubMed

Darby, S M; Miller, M L; Allen, R O

2001-09-01

Liquid chromatography/mass spectrometry (LC/ MS) and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS methods were developed for the presumptive identification of ricin toxin and the alkaloid marker ricinine from crude plant materials. Ricin is an extremely potent poison, which is of forensic interest due to its appearance in terrorism literature and its potential for use as a homicide agent. Difficulties arise in attempting to analyze ricin because it is a large heterogeneous protein with glycosylation. The general protein identification scheme developed uses LC/MS or MALDI-TOF for size classification followed by the use of the same instrumentation for the analysis of the tryptic digest. Fragments of the digest can be searched in an online database for tentative identification of the unknown protein and then followed by comparison to authentic reference materials. LC fractionation or molecular weight cutoff filtration was used for preparation of the intact toxin before analysis. Extracts from two types of castor beans were prepared using a terrorist handbook procedure and determined to contain 1% ricin. Additionally, a forensic sample suspected to contain ricin was analyzed using the presented identification scheme (data not shown). The identification of the alkaloid ricinine by GC/MS and LC/MS was shown to be a complementary technique for the determination of castor bean extracts.

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.

A microbiological method for determining serum levels of broad spectrum β-lactam antibiotics in critically ill patients.

PubMed

Fridlund, Jimmy; Woksepp, Hanna; Schön, Thomas

2016-10-01

Recent studies show that suboptimal blood levels of β-lactam antibiotics are present in intensive care unit (ICU) patients. A common reference method for assessing drug concentrations is liquid chromatography coupled with mass-spectrometry (LC-MS) which is highly accurate but rarely available outside reference centres. Thus, our aim was to develop a microbiological method for monitoring β-lactam antibiotic serum levels which could be used at any hospital with a microbiological laboratory. The method was developed as a 96-well broth microdilution format to assess the concentrations of cefotaxime (CTX), meropenem (MER), and piperacillin (PIP). Patient serum containing antibiotics were diluted in suspensions of bacteria with known minimal inhibitory concentrations (MICs). Serum antibiotic concentrations were calculated by dividing the MIC with the dilution factor at which the serum inhibited growth of the bacterial suspension. Serum (n=88) from ICU patients at four hospitals in south-east Sweden were analysed and compared to LC-MS analysis. The overall accuracy and precision for spiked samples and patient samples was within the pre-set target of ±20.0% for all drugs. There was a significant correlation between the microbiological assay and LC-MS for the patient samples (CTX: r=0.86, n=31; MER: r=0.96, n=11; PIP: r=0.88, n=39) and the agreement around the clinical cut-off for CTX (4.0mg/l), MER (2.0mg/l) and PIP (16.0mg/l) was 90%, 100% and 87%, respectively. The microbiological method has a performance for determination of serum levels of meropenem, piperacillin and cefotaxime suitable for clinical use. It is an inexpensive method applicable in any microbiology laboratory. Copyright © 2016 Elsevier B.V. All rights reserved.

UNE-EN ISO/IEC 17025:2005-accredited method for the determination of pesticide residues in fruit and vegetable samples by LC-MS/MS.

PubMed

Camino-Sánchez, F J; Zafra-Gómez, A; Oliver-Rodríguez, B; Ballesteros, O; Navalón, A; Crovetto, G; Vílchez, J L

2010-11-01

A rapid, simple and sensitive multi-residue method was developed and validated for the simultaneous quantification and confirmation of 69 pesticides in fruit and vegetables using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The samples were extracted following the quick, easy, cheap, effective, rugged and safe method known as QuEChERS. Mass spectrometric conditions were individually optimised for each analyte in order to achieve maximum sensitivity in multiple reaction monitoring (MRM) mode. Using the developed chromatographic conditions, 69 pesticides can be separated in less than 17 min. Two selected reaction monitoring (SRM) assays were used for each pesticide to obtain simultaneous quantification and identification in one run. With this method in SRM mode, more than 150 pesticides can be analysed and quantified, but their confirmation is not possible in all cases according to the European regulations on pesticide residues. Nine common representative matrices (zucchini, melon, cucumber, watermelon, tomato, garlic, eggplant, lettuce and pepper) were selected to investigate the effect of different matrices on recovery and precision. Mean recoveries ranged from 70% to 120%, with relative standard deviations (RSDs) lower than 20% for all the pesticides. The proposed method was applied to the analysis of more than 2000 vegetable samples from the extensive greenhouse cultivation in the province of Almeria, Spain, during one year. The methodology combines the advantages of both QuEChERS and LC-MS/MS producing a very rapid, sensitive, accurate and reliable procedure that can be applied in routine analytical laboratories. The method was validated and accredited according to UNE-EN-ISO/IEC 17025:2005 international standard (accreditation number 278/LE1027).

Measurement of Intracellular Ribavirin Mono-, Di- and Triphosphate Using Solid Phase Extraction and LC-MS/MS Quantification

PubMed Central

Jimmerson, Leah C.; Ray, Michelle L.; Bushman, Lane R.; Anderson, Peter L.; Klein, Brandon; Rower, Joseph E.; Zheng, Jia-Hua; Kiser, Jennifer J.

2014-01-01

Ribavirin (RBV) is a nucleoside analog used to treat a variety of DNA and RNA viruses. RBV undergoes intracellular phosphorylation to a mono- (MP), di- (DP), and triphosphate (TP). The phosphorylated forms have been associated with the mechanisms of antiviral effect observed in vitro, but the intracellular pharmacology of the drug has not been well characterized in vivo. A highly sensitive LC-MS/MS method was developed and validated for the determination of intracellular RBV MP, DP, and TP in multiple cell matrix types. For this method, the individual MP, DP, and TP fractions were isolated from lysed intracellular matrix using strong anion exchange solid phase extraction, dephosphorylated to parent RBV, desalted and concentrated and quantified using LC-MS/MS. The method utilized a stable labeled internal standard (RBV-13C5) which facilitated accuracy (% deviation within ±15%) and precision (coefficient of variation of ≤15%). The quantifiable linear range for the assay was 0.50 to 200 pmol/sample. The method was applied to the measurement of RBV MP, DP, and TP in human peripheral blood mononuclear cells (PBMC), red blood cells (RBC), and dried blood spot (DBS) samples obtained from patients taking RBV for the treatment of chronic Hepatitis C virus infection. PMID:25555148

[Simultaneous determination of pyraclostrobin and thiophanate-methyl and its metabolite carbendazim residues in soil and citrus by QuEChERS-liquid chromatography- tandem mass spectrometry].

PubMed

Li, Fuqin; Shi, Lihong; Wang, Fei; Sun, Caiyuan; Kang, Di; Zhang, Yuping; Chen, Lingzhu; Hu, Deyu

2017-06-08

A QuEChERS-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of pyraclostrobin, thiophanate-methyl and its metabolite carbendazim in soil and citrus. The samples were extracted with methanol or acetonitrile, purified by primary secondary amine (PSA), then separated by LC, detected in multiple reaction monitoring (MRM) mass spectrometry mode via positive electrospray ionization. The analytes were quantified by matrix-matched standard solutions with external standard method. The limits of quantification (LOQs) of pyraclostrobin, thiophanate-methyl and carbendazim in different matrices were 5.8-7.0 μg/kg, 9.3-14.1 μg/kg and 2.1-2.6 μg/kg, respectively. For all the samples, the spiked recoveries ranged from 75.48% to 109.18%, and the relative standard deviations (RSDs) were 0.60%-5.11% ( n =5). The method is quick, easy, effective, sensitive and accurate. The matrix-matched calibration solutions can efficiently compensate matrix effects of the pyraclostrobin, thiophanate-methyl and carbendazim in LC-MS/MS analysis. The established method can be applied to the residue analysis of the real samples of soil, citrus peel, citrus pulp and citrus fruits.

A method for the direct injection and analysis of small volume human blood spots and plasma extracts containing high concentrations of organic solvents using revered-phase 2D UPLC/MS.

PubMed

Rainville, Paul D; Simeone, Jennifer L; Root, Dan S; Mallet, Claude R; Wilson, Ian D; Plumb, Robert S

2015-03-21

The emergence of micro sampling techniques holds great potential to improve pharmacokinetic data quality, reduce animal usage, and save costs in safety assessment studies. The analysis of these samples presents new challenges for bioanalytical scientists, both in terms of sample processing and analytical sensitivity. The use of two dimensional LC/MS with, at-column-dilution for the direct analysis of highly organic extracts prepared from biological fluids such as dried blood spots and plasma is demonstrated. This technique negated the need to dry down and reconstitute, or dilute samples with water/aqueous buffer solutions, prior to injection onto a reversed-phase LC system. A mixture of model drugs, including bromhexine, triprolidine, enrofloxacin, and procaine were used to test the feasibility of the method. Finally an LC/MS assay for the probe pharmaceutical rosuvastatin was developed from dried blood spots and protein-precipitated plasma. The assays showed acceptable recovery, accuracy and precision according to US FDA guidelines. The resulting analytical method showed an increase in assay sensitivity of up to forty fold as compared to conventional methods by maximizing the amount loaded onto the system and the MS response for the probe pharmaceutical rosuvastatin from small volume samples.

Chromatographic methods for determination of macrolide antibiotic residues in tissues and milk of food-producing animals.

PubMed

Moats, W A

1985-01-01

Tylosin, an antibiotic developed specifically for agricultural use, and erythromycin are the main macrolide antibiotics used in animal production. Two-dimensional thin layer chromatography has been used for detection of tylosin in poultry meat, eggs, and milk and for erythromycin in poultry meat. Detection limits reported are, for tylosin, 0.1 ppm in poultry meat, 0.05 ppm in egg, and 0.01 ppm in milk, and for erythromycin, 0.25 ppm in poultry meat. Liquid chromatography (LC) has also been used for determination of tylosin in milk, blood, and tissues of animals. Samples (milk, blood serum, or tissue homogenates in water or pH 2.2 buffer) were deproteinized with acetonitrile, tylosin was partitioned into methylene chloride, and the extracts were concentrated and dissolved in acetonitrile. Chromatography was done on a reverse phase end-capped C18 column using 0.002-0.005 M ammonium dihydrogen phosphate-acetonitrile-methanol (10 + 60 + 30-5 + 80 + 15). Solvent composition was varied with the type of sample analyzed. The method will detect 0.1 ppm tylosin in tissues and less in milk and blood serum. The LC method was more sensitive than microbiological assays for detection of tylosin in tissues of treated swine; recoveries of tylosin by the LC method were frequently several-fold higher.

PREDICTION OF THE ACUTE TOXICITY OF ORGANIC COMPOUNDS TO THE FATHEAD MINNOW (PIMEPHALES PROMALAS) USING A GROUP CONTRIBUTION METHOD

EPA Science Inventory

A group contribution method has been developed to correlate the acute toxicity (96 h LC50) to the fathead minnow (Pimephales promelas) for 379 organic chemicals. Multilinear regression and computational neural networks (CNNs) were used for model building. The multilinear linear m...

Determination of tafenoquine in dried blood spots and plasma using LC and fluorescence detection.

PubMed

Römsing, Susanne; Lindegardh, Niklas; Bergqvist, Yngve

2011-08-01

The growing problem of parasites developing resistance to the traditional antimalarial drugs makes the development of new effective and safe drugs crucial. Tafenoquine is a new promising antimalarial drug for prophylaxis and treatment. A bioanalytical method for the determination of tafenoquine in 100 µl of capillary blood applied onto sampling paper and in 100 µl of plasma has been developed and validated. The Whatman 31 ET Chr paper was treated with 0.6 mol/l tartaric acid to improve the extraction recovery and solid-phase extraction was used for cleanup procedure of the blood samples. Plasma samples were precipitated with methanol. Tafenoquine and internal standard were separated on a Zorbax SB-CN column by reversed-phase LC and detected with fluorescence detection at 262 and 470 nm. The within- and between-day variations were below 10 and 14%, respectively, over the range 50-200 nmol/l for capillary blood on sampling paper and below 6 and 10% for plasma samples. The LLOQ of the method was 50 nmol/l. The developed method has adequate sensitivity and is highly suitable for clinical studies in dried blood spots and plasma.

Determination of a steroid profile in heel prick blood using LC-MS/MS.

PubMed

Boelen, Anita; Ruiter, An F C; Claahsen-van der Grinten, Hedi L; Endert, Erik; Ackermans, Mariette T

2016-01-01

The aim of this study was to improve the sensitivity of the congenital adrenal hyperplasia (CAH) neonatal screening by including second-tier steroid profiling on a DBS using LC-MS. We developed a method to measure the steroid profile in DBS and established gestational age-specific reference ranges of cortisol, cortisone, 11-deoxycortisol, 21-deoxycortisol, 17-hydroxyprogesterone, testosterone, Δ4-androstenedione, corticosterone and 11-deoxycorticosterone using 450 heel prick samples of neonates, participating in the Dutch Screening Program. Analyzing 92 cards with a positive CAH screening showed that only 21-deoxycortisol was 100% specific for diagnosed CAH patients. Steroid precursors can be measured in DBS and we suggest to implement the method as a second tier testing for CAH in The Netherlands.

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.

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.

LC-MS/MS-based quantification of kynurenine metabolites, tryptophan, monoamines and neopterin in plasma, cerebrospinal fluid and brain.

PubMed

Fuertig, René; Ceci, Angelo; Camus, Sandrine M; Bezard, Erwan; Luippold, Andreas H; Hengerer, Bastian

2016-09-01

The kynurenine (KYN) pathway is implicated in diseases such as cancer, psychiatric, neurodegenerative and autoimmune disorders. Measurement of KYN metabolite levels will help elucidating the involvement of the KYN pathway in the disease pathology and inform drug development. Samples of plasma, cerebrospinal fluid or brain tissue were spiked with deuterated internal standards, processed and analyzed by LC-MS/MS; analytes were chromatographically separated by gradient elution on a C18 reversed phase analytical column without derivatization. We established an LC-MS/MS method to measure 11 molecules, namely tryptophan, KYN, 3-OH-KYN, 3-OH-anthranilic acid, quinolinic acid, picolinic acid, kynurenic acid, xanthurenic acid, serotonin, dopamine and neopterin within 5.5 min, with sufficient sensitivity to quantify these molecules in small sample volumes of plasma, cerebrospinal fluid and brain tissue.

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.

Liquid crystal based optical platform for the detection of Pb2+ ions using NiFe2O4 nanoparticles

NASA Astrophysics Data System (ADS)

Zehra, Saman; Gul, Iftikhar Hussain; Hussain, Zakir

2018-06-01

A simple, sensitive, selective and real time detection protocol was developed for Pb2+ ions in water using liquid crystals (LCs). In this method, NiFe2O4 nanoparticles were synthesized using chemical co-precipitation method. Crystallite size, morphological, functional groups and magnetization studies were confirmed using X-ray diffraction, Scanning Electron Microscopy, and Fourier transform infrared spectroscopy techniques, respectively. The nanoparticles were mono dispersed with average particle size of 20 ± 2 nm. The surfactant stabilized magnetic nanoparticles were incubated in liquid crystal based sensor system for the detection of Pb+2 ions. The bright to dark transition of LC was observed through optical microscope. When this system was further immersed with a solution containing Pb2+ ions, it caused homeotropic to planar orientation of LC. This interaction is attributed to the presence of abundant hydroxyl groups in such as M-OH, Fe-OH on the surface of spinel ferrites nanoparticles. These groups interact with metal ions at aqueous interface, causing disruption in LCs orientation giving bright texture. This sensor showed higher selectivity towards Pb2+ ions. The detection limit was estimated to be 100 ppb. The cheap and effective protocol reported here should make promising development of LC based sensor for lead ion detection.

Feasibility Research on Alternative Approaches for Sampling and Extraction Methods in the TO-4A Method for Pesticides in Ambient Air with Analysis by GC/MS and LC/MS/MS

EPA Science Inventory

This compilation of methods is the result of a Regional Methods project between the U.S. Environmental Protection Agency Region 4 and the EPA’s Office of Research and Development. The research leading to these methods was conducted in response to an observed need to update an EPA...

Development of a Suitable Dissolution Method for the Combined Tablet Formulation of Atorvastatin and Ezetimibe by RP-LC Method.

PubMed

Ozkan Cansel, Kose; Ozgur, Esim; Sevinc, Kurbanoglu; Ayhan, Savaser; Ozkan, Sibel A; Yalcin, Ozkan

2016-01-01

Pharmaceutical preparations of ezetimibe and atorvastatin are generally used to regulate the lipid level in blood. It decreases the secondary events for patients with high cholesterol and clinical cardiovascular disease such as non-fatal or fatal heart attack. There is no any pharmacopoeia method available for the dissolution testing recommended by the FDA. Development of dissolution tests method is very critical parameter especially for the pharmaceutical preparations that contain Class II drugs (slightly soluble, good permeable). In the proposed method, the effects of pH and surfactant on the dissolution of poorly water soluble combined drug therapy with a different pKa values in an in vitro environment is investigated. The content of our study was designed to answer these open-ended questions. The optimized test conditions achieved under sink conditions with USP apparatus 2 at a paddle rotation speed of 75 rpm and 900 ml in 0.01 M Acetate buffer (pH= 6.8) containing 0.45% SDS as a dissolution medium. Quantification of dissolution samples were analyzed with a new fully validated RP-LC method with UV detection at 242 nm.

Rapid determination of fumonisins in corn-based products by liquid chromatography/tandem mass spectrometry.

PubMed

Li, Wei; Herrman, Timothy J; Dai, Susie Y

2010-01-01

A simple, fast, and robust method was developed for the determination of fumonisin B1 (FB1), fumonisin B2 (FB2), and fumonisin B3 (FB3) in corn-based human food and animal feed (cornmeal). The method involves a single extraction step followed by centrifugation and filtration before analysis by ultra-performance liquid chromatographylelectrospray ionization (UPLC/ESI)-MS/MS. The LC/MS/MS method developed here represents the fastest and simplest procedure (<30 min) among both conventional HPLC methods and other LC/MS methods using SPE cleanup. The potential for high throughput analysis makes the method particularly beneficial for regulatory agencies and analytical laboratories with a high sample volume. A single-laboratory validation was conducted by testing three different spiking levels (200, 500, and 1000 ng/g for FB1 and FB2; 100, 250, and 500 ng/g for FB3) for accuracy and precision. Recoveries of FB1 ranged from 93 to 98% with RSD values of 3-8%. Recoveries of FB2 ranged from 104 to 108%, with RSD values of 2-6%. Recoveries of FB3 ranged from 94 to 108%, with RSD values of 2-5%.

LC-MS determination of steroidal glycosides from Dioscorea deltoidea Wall cell suspension culture: Optimization of pre-LC-MS procedure parameters by Latin Square design.

PubMed

Sarvin, Boris; Fedorova, Elizaveta; Shpigun, Oleg; Titova, Maria; Nikitin, Mikhail; Kochkin, Dmitry; Rodin, Igor; Stavrianidi, Andrey

2018-03-30

In this paper, the ultrasound assisted extraction method for isolation of steroidal glycosides from D. deltoidea plant cell suspension culture with a subsequent HPLC-MS determination was developed. After the organic solvent was selected via a two-factor experiment the optimization via Latin Square 4 × 4 experimental design was carried out for the following parameters: extraction time, organic solvent concentration in extraction solution and the ratio of solvent to sample. It was also shown that the ultrasound assisted extraction method is not suitable for isolation of steroidal glycosides from the D. deltoidea plant material. The results were double-checked using the multiple successive extraction method and refluxing extraction. Optimal conditions for the extraction of steroidal glycosides by the ultrasound assisted extraction method were: extraction time, 60 min; acetonitrile (water) concentration in extraction solution, 50%; the ratio of solvent to sample, 400 mL/g. Also, the developed method was tested on D. deltoidea cell suspension cultures of different terms and conditions of cultivation. The completeness of the extraction was confirmed using the multiple successive extraction method. Copyright © 2018 Elsevier B.V. All rights reserved.

LC-MS/MS determination of alectinib and its major human metabolite M4 in human urine: prevention of nonspecific binding.

PubMed

Heinig, Katja; Herzog, Denis; Ferrari, Luca; Fraier, Daniela; Miya, Kazuhiro; Morcos, Peter N

2017-03-01

Alectinib (Alecensa ® ) is an anaplastic lymphoma kinase inhibitor for the treatment of anaplastic lymphoma kinase positive non-small-cell lung cancer, and M4 is its major pharmacologically active metabolite. To characterize the pharmacokinetics and excretion of alectinib and M4 in human urine, a bioanalytical method was required. An LC-MS/MS method using supported liquid extraction was developed for the determination of alectinib and M4 in human urine over the concentration range 0.5-500 ng/ml. Accuracy ranged from 92.0 to 112.2% and precision (CV) was below 9.6%. The method was successfully employed to determine alectinib and M4 concentrations in urine samples from a clinical mass balance study. Addition of the surfactant Tween-20 to urine prevented nonspecific binding of the analytes.

Determination of capsaicinoids in topical cream by liquid-liquid extraction and liquid chromatography.

PubMed

Kaale, Eliangiringa; Van Schepdael, Ann; Roets, Eugène; Hoogmartens, Jos

2002-11-07

A reversed-phase liquid chromatography (LC) method has been developed, optimised and validated for the separation and quantitation of capsaicin (CP) and dihydrocapsaicin (DHCP) in a topical cream formulation. Sample preparation involves liquid-liquid extraction prior to LC analysis. The method uses a Hypersil C(18) BDS, 5 micrometer, 250x4.6 mm I.D. column maintained at 35 degrees C. The mobile phase comprises methanol, water, acetonitrile (ACN) and acetic acid (47:42:10:1, v/v/v/v) at a flow rate of 1.0 ml/min. Robustness was evaluated by performing a central composite face-centred design (CCF) experiment. The method shows good selectivity, linearity, sensitivity and repeatability. The conditions allow the separation and quantitation of CP and DHCP without interference from the other substances contained in the cream.

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.

Development of a quantitative multi-compound method for the detection of 14 nitrogen-rich adulterants by LC-MS/MS in food materials.

PubMed

Frank, Nancy; Bessaire, Thomas; Tarres, Adrienne; Goyon, Alexandre; Delatour, Thierry

2017-11-01

The increasing number of food frauds using exogenous nitrogen-rich adulterants to artificially raise the protein content for economically motivated adulteration has demonstrated the need for a robust analytical methodology. This method should be applicable for quality control in operations covering a wide range of analyte concentrations to be able to analyse high levels as usually found in adulteration, as well as low levels due to contamination. The paper describes a LC-MS/MS method covering 14 nitrogen-rich adulterants using a simple and fast sample preparation based on dilution and clean-up by dispersive SPE. Quantification is carried out by isotopic dilution reaching LOQs of 0.05-0.20 mg/kg in a broad range of food matrices (infant formula, liquid milk, dairy ingredient, high protein meal, cereal, infant cereal, and meat/fish powder). Validation of seven commodity groups was performed according to SANCO 12571/2013, giving satisfactory results demonstrating the method's fitness for purpose at the validated range at contamination level. Method ruggedness was further assessed by transferring the developed method into another laboratory devoted to routine testing for quality control. Next to the method description, emphasis is placed on challenges and problems appearing during method development as well as validation. They are discussed in detail and solutions are provided.

LANDSCAPE CHARACTERIZATION AND CHANGE DETECTION METHODS DEVELOPMENT RESEARCH (2005-2007)

EPA Science Inventory

The characterization of land-cover (LC) type, extent, and distribution represent important landscape characterization element required for monitoring ecosystem conditions and for primary data input to biogenic emission and atmospheric deposition models. Current spectral-based ch...

Sensitivity enhancement by chromatographic peak concentration with ultra-high performance liquid chromatography-nuclear magnetic resonance spectroscopy for minor impurity analysis.

PubMed

Tokunaga, Takashi; Akagi, Ken-Ichi; Okamoto, Masahiko

2017-07-28

High performance liquid chromatography can be coupled with nuclear magnetic resonance (NMR) spectroscopy to give a powerful analytical method known as liquid chromatography-nuclear magnetic resonance (LC-NMR) spectroscopy, which can be used to determine the chemical structures of the components of complex mixtures. However, intrinsic limitations in the sensitivity of NMR spectroscopy have restricted the scope of this procedure, and resolving these limitations remains a critical problem for analysis. In this study, we coupled ultra-high performance liquid chromatography (UHPLC) with NMR to give a simple and versatile analytical method with higher sensitivity than conventional LC-NMR. UHPLC separation enabled the concentration of individual peaks to give a volume similar to that of the NMR flow cell, thereby maximizing the sensitivity to the theoretical upper limit. The UHPLC concentration of compound peaks present at typical impurity levels (5.0-13.1 nmol) in a mixture led to at most three-fold increase in the signal-to-noise ratio compared with LC-NMR. Furthermore, we demonstrated the use of UHPLC-NMR for obtaining structural information of a minor impurity in a reaction mixture in actual laboratory-scale development of a synthetic process. Using UHPLC-NMR, the experimental run times for chromatography and NMR were greatly reduced compared with LC-NMR. UHPLC-NMR successfully overcomes the difficulties associated with analyses of minor components in a complex mixture by LC-NMR, which are problematic even when an ultra-high field magnet and cryogenic probe are used. Copyright © 2017 Elsevier B.V. All rights reserved.

Two-Dimensional Liquid Chromatography Analysis of Polystyrene/Polybutadiene Block Copolymers.

PubMed

Lee, Sanghoon; Choi, Heejae; Chang, Taihyun; Staal, Bastiaan

2018-05-15

A detailed characterization of a commercial polystyrene/polybutadiene block copolymer material (Styrolux) was carried out using two-dimensional liquid chromatography (2D-LC). The Styrolux is prepared by statistical linking reaction of two different polystyrene- block-polybutadienyl anion precursors with a multivalent linking agent. Therefore, it is a mixture of a number of branched block copolymers different in molecular weight, composition, and chain architecture. While individual LC analysis, including size exclusion chromatography, interaction chromatography, or liquid chromatography at critical condition, is not good enough to resolve all the polymer species, 2D-LC separations coupling two chromatography methods were able to resolve all polymer species present in the sample; at least 13 block copolymer species and a homopolystyrene blended. Four different 2D-LC analyses combining a different pair of two LC methods provide their characteristic separation results. The separation characteristics of the 2D-LC separations are compared to elucidate the elution characteristics of the block copolymer species.

Quasi-optic millimeter-wave device application of liquid crystal material by using porous PMMA matrix

NASA Astrophysics Data System (ADS)

Nose, T.; Watanabe, Y.; Kon, A.; Ito, R.; Honma, M.

2018-02-01

Recently, millimeter-waves (MMWs) have become indispensable for application in next-generation high-speed wireless communication i.e., 5G, in addition to conventional applications such as in automobile collision avoidance radars and airport security inspection systems. Some manageable devices to control MMW propagation will be necessary with the development of this new technology field. We believe that liquid crystal (LC) devices are one of the major candidates for such applications because it is known that LC materials are excellent electro-optic materials. However, as the wavelength of MMWs is extremely longer than the optics region, extremely thick LC layers are necessary if we choose the quasioptic approach to attain LC MMW control devices. Therefore, we adopt a PDLC structure to attain the extremely thick LC layers by using porous (polymethyl methacrylate) PMMA materials, which can be easily obtained using a solvent consisting of a mixture of ethanol/water and a little heating. In this work, we focus on Fresnel lens, which is an important quasi-optic device for MMW application, to introduce a tunable property by using LC materials. Here, we adopt the thin film deposition method to obtain a porous PMMA matrix with the aim of obtaining final composite structure based on the Fresnel substrate. First, the fundamental material properties of porous PMMA are investigated to control the microscopic porous structure. Then, the LC-MMW Fresnel lens substrate is prepared using a 3D printer, and the fundamental MMW focusing properties of the prototype composite Fresnel structure are investigated.

Acute, sublethal and combination effects of azadirachtin and Bacillus thuringiensis toxins on Helicoverpa armigera (Lepidoptera: Noctuidae) larvae.

PubMed

Singh, G; Rup, P J; Koul, Opender

2007-08-01

The efficacy of neem (1500 ppm azadirachtin (AI)), Delfin WG, a biological insecticide based on selected strain of Bacillus thuringiensis Berliner (Bt) subspecies kurstaki, and Cry1Ac protein, either individually or in combination, were examined against first to fourth instar Helicoverpa armigera (Hübner) larvae. Using an oral administration method, various growth inhibitory concentrations (EC) and lethal concentrations (LC) were determined for each bioagent. Combinations of sublethal concentrations of Bt spray formulation with azadirachtin at EC50 or EC95 levels not only enhanced the toxicity, but also reduced the duration of action when used in a mixture. The LC20 and LC50 values for Cry1Ac toxin were 0.06 and 0.22 microg ml-1, respectively. Bt-azadirachtin combinations of LC50+EC20 and LC50+EC50 result in 100% mortality. The mortality also was significant in LC20+EC20 and LC20+EC50 mixtures. These studies imply that the combined action is not synergistic but complimentary, with azadirachtin particularly facilitating the action of Bt. The Bt spray-azadirachtin combination is more economical than combinations that involve isolating the toxic protein, as the Bt spray formulations can be combined in a spray mixture with neem. These combinations may be useful for controlling bollworm populations that have acquired resistance to Bt as they may not survive the effect of mixture. Azadirachtin may be useful as a means of reducing the endotoxin concentrations in a mixture, to promote increased economic savings and further reduce the probability of resistance development to either insect control agent.

Development of an analytical method for detecting nitrofurans in bee pollen by liquid chromatography-electrospray ionization tandem mass spectrometry.

PubMed

Park, Mi Sun; Kim, Kyung Tae; Kang, Jong Seong

2017-03-01

Bee pollen collected by honeybees, which is in powdered form, is a good nutritional supplement. Nitrofuran antibiotics are assumed not to be present in bee pollen, which is important as the level of antibiotics in bee pollen is strongly regulated in many countries. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect nitrofurans in honey has been developed, but this method is not suitable for bee pollen because of it being in powdered form. During preparation of bee pollen samples, the dispersal of powder particles in an aqueous solution often makes them susceptible to forming an emulsion with solvent components such as hexane and ethyl acetate. This may reduce the reproducibility and sensitivity of analyses of nitrofuran levels in bee pollen. Therefore, we attempted to optimize the sample preparation conditions to detect nitrofurans in bee pollen by determining three nitrofuran residues, namely, 3-amino-2-oxazolidinone (AOZ), 3-amino-5-methyl-morpholino-2-oxazolidinone (AMOZ), and 1-aminohydantoin (AHD), using LC-MS/MS. The optimized method prevented the formation of powder-induced emulsion. To verify the reproducibility and sensitivity of this method, it was validated using nitrofuran-free bee pollen spiked with analytes with different side chains at 1.0, 2.0, and 5.0μgkg -1 . The accuracy levels were 94.1%-104.0% and the coefficients of variation were less than 12%. The limits of detection for AOZ, AMOZ, and AHD were 0.18, 0.25, and 0.30μgkg -1 , respectively, while their limits of quantitation were 0.59, 0.83, and 1.00μgkg -1 . The LC-MS/MS method developed to analyze nitrofuran in bee pollen should contribute to the quality control of bee pollen and food safety. Copyright © 2016 Elsevier B.V. All rights reserved.

Glycidyl fatty acid esters in food by LC-MS/MS: method development.

PubMed

Becalski, A; Feng, S Y; Lau, B P-Y; Zhao, T

2012-07-01

An improved method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the analysis of glycidyl fatty acid esters in oils was developed. The method incorporates stable isotope dilution analysis (SIDA) for quantifying the five target analytes: glycidyl esters of palmitic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2) and linolenic acid (C18:3). For the analysis, 10 mg sample of edible oil or fat is dissolved in acetone, spiked with deuterium labelled analogs of glycidyl esters and purified by a two-step chromatography on C18 and normal silica solid phase extraction (SPE) cartridges using methanol and 5% ethyl acetate in hexane, respectively. If the concentration of analytes is expected to be below 0.5 mg/kg, 0.5 g sample of oil is pre-concentrated first using a silica column. The dried final extract is re-dissolved in 250 μL of a mixture of methanol/isopropanol (1:1, v/v), 15 μL is injected on the analytical C18 LC column and analytes are eluted with 100% methanol. Detection of target glycidyl fatty acid esters is accomplished by LC-MS/MS using positive ion atmospheric pressure chemical ionization operating in Multiple Reaction Monitoring mode monitoring 2 ion transitions for each analyte. The method was tested on replicates of a virgin olive oil which was free of glycidyl esters. The method detection limit was calculated to be in the range of 70-150 μg/kg for each analyte using 10 mg sample and 1-3 μg/kg using 0.5 g sample of oil. Average recoveries of 5 glycidyl esters spiked at 10, 1 and 0.1 mg/kg were in the range 84% to 108%. The major advantage of our method is use of SIDA for all analytes using commercially available internal standards and detection limits that are lower by a factor of 5-10 from published methods when 0.5 g sample of oil is used. Additionally, MS/MS mass chromatograms offer greater specificity than liquid chromatography-mass spectrometry operated in selected ion monitoring mode. The method will be applied to the survey of glycidyl fatty acid esters in food products on the Canadian market.

[Multi-residue method for screening of pesticides in crops by liquid chromatography with tandem mass spectrometry].

PubMed

Tanizawa, Haruna; Shima, Mikie; Ikehara, Chieko; Kobata, Masakazu; Sato, Motoaki

2005-10-01

A simple and rapid method was developed for the screening of 82 pesticides/metabolites in a wide variety of crops, using solid-phase extraction and liquid chromatography with tandem mass spectrometry (LC/MS/MS). After extraction with methanol, the filtered extracts were made up to 100 mL and a 2 mL aliquot was subjected to solid-phase extraction. Co-extractives were removed with a C18 mini-column, while pesticides were retained on 3 kinds of mini-columns (HLB, SAX, activated carbon), and then eluted with acetonitrile. Analysis was performed by LC/MS/MS, and MS acquisition parameters were established in positive and negative ESI modes. The utility of the method was demonstrated by the analysis of 6 crops (carrot, cabbage, onion, spinach, lemon, brown rice) and one mixed vegetable juice. Of 82 compounds tested, 75 in carrot and 62 in lemon were obtained with recoveries ranging from 70-120%. For all samples tested, 75 compounds could be obtained with recoveries of over 50%, and the detection limits of most compounds were lower than 0.01 microg/g. This method provides acceptable performance for analysis of these 75 compounds. Further, by using aliquots of the extracts with small-scale mini-columns, purified samples could be obtained. This proposed method with small matrix effects, is effective and suitable for screening of multiple residual pesticides by using LC/MS/MS.

A statistical evaluation of the effects of gender differences in assessment of acute inhalation toxicity

PubMed Central

Price, Charlotte; Stallard, Nigel; Creton, Stuart; Indans, Ian; Guest, Robert; Griffiths, David; Edwards, Philippa

2010-01-01

Acute inhalation toxicity of chemicals has conventionally been assessed by the median lethal concentration (LC50) test (organisation for economic co-operation and development (OECD) TG 403). Two new methods, the recently adopted acute toxic class method (ATC; OECD TG 436) and a proposed fixed concentration procedure (FCP), have recently been considered, but statistical evaluations of these methods did not investigate the influence of differential sensitivity between male and female rats on the outcomes. This paper presents an analysis of data from the assessment of acute inhalation toxicity for 56 substances. Statistically significant differences between the LC50 for males and females were found for 16 substances, with greater than 10-fold differences in the LC50 for two substances. The paper also reports a statistical evaluation of the three test methods in the presence of unanticipated gender differences. With TG 403, a gender difference leads to a slightly greater chance of under-classification. This is also the case for the ATC method, but more pronounced than for TG 403, with misclassification of nearly all substances from Globally Harmonised System (GHS) class 3 into class 4. As the FCP uses females only, if females are more sensitive, the classification is unchanged. If males are more sensitive, the procedure may lead to under-classification. Additional research on modification of the FCP is thus proposed. PMID:20488841

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.

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.

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.

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.

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

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.

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.

A multiclass multiresidue LC-MS/MS method for analysis of veterinary drugs in bovine kidney

USDA-ARS?s Scientific Manuscript database

The increased efficiency permitted by multiclass, multiresidue methods has made such approaches very attractive to laboratories involved in monitoring veterinary drug residues in animal tissues. In this current work, evaluation of a multiclass multiresidue LC-MS/MS method in bovine kidney is describ...

Method of preparing a tunable-focus liquid-crystal (LC) lens

NASA Astrophysics Data System (ADS)

Li, Xiaolong; Zhou, Zuowei; Ren, Hongwen

2018-02-01

A liquid crystal (LC) lens is prepared by controlling the alignment of a LC using a homogeneous polyimide (PI) layer and a homeotropic PI layer. The rubbed homogeneous PI layer has a concave surface and the homeotropic PI layer is flat. The LC sandwiched between the two PI layers obtains a hybrid alignment which has the largest gradient of refractive index (GRIN) distribution. The LC layer exhibits a lens character because of its convex shape. Since the effective refractive index of the LC is larger than that of the homogeneous PI, the LC lens can focus a light with the shortest focal length in the voltage-off state. By applying an external voltage, the LC molecules can be reoriented along the electric field. As a result, the focal length of the LC lens is reduced. The focal length of the LC lens can be tuned from 30 to 120 μm when the voltage is changed from 0 to 7 Vrms. This LC lens has the advantages of no threshold, low operating voltage, and simple fabrication.

A simple LC method with UV detection for the analysis of creatine and creatinine and its application to several creatine formulations.

PubMed

Dash, Alekha K; Sawhney, Angeli

2002-07-31

The objective of this study was to develop a simple and sensitive LC method for the determination of creatine and creatinine in various creatine supplement formulations. The chromatographic system comprised of a LC-600 pump, SCL-6B system controller, and SPD-6AV detector (Shimadzu, Japan). The mobile phase consisted of 0.045 M ammonium sulfate in water. The chromatographic separation was achieved at ambient temperature on a Betabasic C-18 column (250 x 4.6 mm, Keystone Sci.). The flow rate was maintained at 0.75 ml/min and effluents are monitored at 205 nm. 4-(2-Aminoethyl)benzene sulfonamide was used as an internal standard (IS). This method required less than 7 min of chromatographic time. The standard curves were linear over the concentration range of 1-100 microg/ml for creatine and 2-100 microg/ml for creatinine, respectively. The relative standard deviations (RSD) for the within-day and day-to-day precision for creatine were within 1.0-4.6 and 2.2-4.7%, respectively. The RSD for the accuracy of creatine assay was in the range of 2.4-4.7%. The RSD values for the within-day precision, day-to-day precision and accuracy for creatinine validation were 1.7-4.4, 2.3-5.4 and 2.4-4.8%, respectively. This method was used to determine: (i) the creatine concentration in various marketed products; (ii) saturated solubility of various creatine salts; and (iii) stability of creatine in aqueous solution. In conclusion, a simple and sensitive LC method with UV detection was developed for the simultaneous determination of creatine and creatinine in formulations. Di-creatine citrate salt showed a higher aqueous solubility (at 25 degrees C) as compared to creatine and creatine monohydrate. Some of the over-the-counter (OTC) products tested contained a very low level of creatine in contrast to their label claim. Substantial conversion of creatine into creatinine was noticed in liquid formulation.

A simple LC-MS/MS method for determination of deferasirox in human plasma: Troubleshooting of interference from ferric ion in method development and its application.

PubMed

Li, Tengfei; Cui, Zhimin; Wang, Yan; Yang, Wen; Li, Duo; Song, QinXin; Sun, Luning; Ding, Li

2018-03-20

As an orally active iron chelator, deferasirox forms its ion complexes in the prepared plasma samples and LC-MS mobile phase where ferric ion exists, and then comparing with the nominal concentration level, a lower detected concentration level of deferasirox would be obtained after LC-MS analysis, if no proper treatment was adopted. Meanwhile, the phenomenon would be observed that multiple repeat injections of the same deferasirox plasma sample in the same tube would show the lower and lower detected concentration levels of deferasirox, which caused by more and more ferric ions from the injection needle dissolved in the sample solution as multiple repeated injections. The addition of a proper concentration of EDTA in the mobile phase and the sample will competitively inhibit deferasirox from complexing with ferric ion, and prevent the decrease of deferasirox concentration. In this paper, an LC-MS/MS method was developed and validated for the determination of deferasirox in human plasma. To achieve the protein precipitation, the analytes were extracted from aliquots of 200 μL human plasma with acetonitrile. Chromatographic separation was performed on an ODS-C18 column with the mobile phase consisted of methanol and 0.1% formic acid containing 0.04 mM ethylenediamine tetraacetate dihydrate (EDTA) (80:20, v/v) at a flow rate of 0.5 mL/min. Deferasirox and the internal standard (IS, mifepristone) were detected using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the precursor-to-product ion transitions m/z 374.2 → 108.1 for deferasirox and m/z 430.1 → 372.2 for the IS. The method exhibited good linearity over the concentration range of 0.04-40 μg/mL for deferasirox. The method was successfully applied to a pharmacokinetic study in 10 Chinese healthy volunteers after oral administration of deferasirox. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

Development and Characterization of Liquid Crystal-Gold Nanoparticle Hybrid Materials for Optical Applications

NASA Astrophysics Data System (ADS)

Quint, Makiko T.

Hybrid material, mixtures of two or more materials with new properties, represents an exciting class of new materials for a variety of potential applications such as displays, optoelectronics, and sensors due to their unique physical and optical properties. The scope of this dissertation is to produce two new plasmonic applications by combining liquid crystals with gold nanoparticles. The first application is gold nanoparticle coated liquid crystal thin film. Most liquid crystal (LC) thin films require external voltage to reorient LC molecules. Recent advances in optical controlling technology of LC molecule behavior, resulting in the reduction of energy consumption, have stimulated research and development of new LC thin films. In order to re-orient LC molecules by just using light, the common approach is to include either a photo-responsive LC host, one that require high power light and severely narrows the range of usable materials, or add photo-active dye or polymer layer, photodegradation over time. Our work designing an all-optical method for LC re-orientation that overcomes all the limitations mentioned above. We have successfully both in- and out-of-plane spatial orientation of nematic liquid crystal (LC) molecules by leveraging the highly localized electric fields produced in the near-field regime of a gold nanoparticle (AuNP) layer. This re-orientation of LC molecules in thin LC-AuNP film is all-optical, driven by a small resonance excitation power with the localized surface plasmon absorption of the AuNPs at room temperature. The second application is LC mediated nano-assembled gold microcapsules. This application has a potential in controlled-release cargo-style delivery system. Targeted delivery systems with controlled release mechanisms have been the subject of extensive research more than fifty years. One is to control the release process remotely by using optical excitation. Optical actuation of delivery capsules, which plasmonic nanoparticle such as gold, allows rapid release at specific locations and uses the photothermal effect to unload contents. Almost all gold-based delivery applications including Au coated nanocrystals or AuNPs with soft materials like gels and polymers are not suitable for control release applications in real life since these applications do not provide robust leakage-free containment lower than the American National Standards Institute (ANSI) maximum permissible light exposure limit. We have successfully managed the difficulties mentioned above and produced a new gold-based delivery application. The application is spherical capsules with a densely packed wall of AuNPs. The rigid capsule wall allows encapsulation of cargo that can be contained, virtually leakage-free, over several months. Further, by leveraging LSPR of AuNPs, we can rupture the microshells using optical excitation with ultralow power (< 2 mW), controllably and rapidly releasing the encapsulated contents in less than 5 seconds. Our results exhibiting the capture and optically regulated release of encapsulated substances are a novel platform that combines controlled-release cargo-style delivery and photothermal therapy in one versatile and multifunctional unit. Both our applications are overcoming current limitations and promising future research directions towards the next generation of LC-AuNPs hybrid material research and developments.

Establishing and performing targeted multi-residue analysis for lipid mediators and fatty acids in small clinical plasma samples.

USDA-ARS?s Scientific Manuscript database

LC-MS/MS and GC-MS based targeted metabolomics is typically conducted by analyzing and quantifying a cascade of metabolites with methods specifically developed for the metabolite class. Here we describe an approach for the development of multi-residue analytical profiles, calibration standards, and ...

Five Decades of Microforms at the Library of Congress.

ERIC Educational Resources Information Center

Sullivan, Robert C.

1988-01-01

Discusses the history of the microform collections at the Library of Congress (LC), highlighting the exchange of microfilmed newspapers between LC and the Biblioteca Nacional, Brazil. Recent developments in preservation microfilming, LC's acquisition of microforms, and the activities of the LC Preservation Microfilming Office are described. (3…

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.

Liquid chromatography/mass spectrometry-based plasma metabolic profiling study of escitalopram in subjects with major depressive disorder.

PubMed

Bandu, Raju; Lee, Hyun Jeong; Lee, Hyeong Min; Ha, Tae Hyon; Lee, Heon-Jeong; Kim, Se Joo; Ha, Kyooseob; Kim, Kwang Pyo

2018-05-01

Liquid chromatography-mass spectrometry (LC-MS) method revealed the plasma metabolite profiles in major depressive disorder patients treated with escitalopram (ECTP) (n = 7). Depression severity was assessed according to the 17-item Hamilton Depression Rating Scale. Metabolic profiles were derived from major depressive disorder subject blood samples collected after ECTP treatment. Blood plasma was separated and processed in order to effectively extract metabolites, which were then analyzed using LC-MS. We identified 19 metabolites and elucidated their structures using LC-tandem MS (LC-MS/MS) combined with elemental compositions derived from accurate mass measurements. We further used online H/D exchange experiments to verify the structural elucidations of each metabolite. Identifying molecular metabolites may provide critical insights into the pharmacological and clinical effects of ECTP treatment and may also provide useful information informing the development of new antidepressant treatments. These detailed plasma metabolite analyses may also be used to identify optimal dose concentrations in psychopharmacotherapeutic treatment through drug monitoring, as well as forming the basis for response predictions in depressed subjects. Copyright © 2018 John Wiley & Sons, Ltd.

Determination of Dextromethorphan in Oral Fluid by LC-MS-MS.

PubMed

Amaratunga, Piyadarsha; Clothier, Morgan; Lorenz Lemberg, Bridget; Lemberg, Dave

2016-06-01

Dextromethorphan (DXM) is an antitussive drug found in commonly used nonprescription cold and cough medications. At low doses, DXM is a safe drug that does not produce adverse reactions. However, abuse of DXM has been reported among adolescents and young adults using the drug at higher doses. DXM is not a scheduled drug in the USA, and the primary reason for its abuse is the ease of availability. DXM is available to purchase in the form of over-the-counter cough medications, such as Robitussin(®) and Coricidin(®), or it can be purchased over the Internet in the form of a powder. In this research work, we developed an LC-MS-MS method that can quantify DXM and dextrorphan (DXO) in oral fluid in a high-throughput toxicology laboratory setting. The developed method was validated according to the Scientific Working Group for Forensic Toxicology guidelines. The linear dynamic range was 5-100 ng/mL with a lowest limit of quantitation (LLOQ) of 5.0 ng/mL for DXM and DXO. Overall, the results of the accuracy and the precision values were within the acceptance criteria for both drugs. In addition, selectivity, matrix effect and recovery were calculated for the LC-MS-MS method. Authentic samples (n = 59) were tested to evaluate the applicability of the method. Thirty samples were found to be positive for DXM and DXO and two samples were found to be positive for DXM only. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Development and validation of a sensitive LC/MS/MS method for the simultaneous determination of naloxone and its metabolites in mouse plasma.

PubMed

Jiang, Hongliang; Wang, Yurong; Shet, Manjunath S; Zhang, Yang; Zenke, Duane; Fast, Douglas M

2011-09-01

A rapid, specific, and reliable LC-MS/MS based bioanalytical method was developed and validated for the simultaneous determination of naloxone (NLX) and its two metabolites, 6β-naloxol (NLL) and naloxone-3β-D-glucuronide (NLG) in mouse plasma. The optimal chromatographic behavior of these analytes was achieved on an Aquasil C18 column (50 mm × 2.1 mm, 5 μm) using reversed phase chromatography. The total LC analysis time per injection was 2.5 min with a flow rate of 1.0 mL/min with gradient elution. Sample preparation via protein precipitation with acetonitrile in a 96-well format was applied for analyses of these analytes. The analytes were monitored by electrospray ionization in positive ion multiple reaction monitoring (MRM) mode. Modification of collision energy besides chromatographic separation was applied to further eliminate interference peaks for NLL and NLG. The method validation was conducted over the curve range of 0.200/0.400/0.500 to 100/200/250 ng/mL for NLX/NLL/NLG, respectively, using 0.0250 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed ≤ 6.5% relative standard deviation (RSD) and -8.3 to -2.5% relative error (RE). The method was successfully applied to determine the concentrations of NLX, NLL, and NLG in incurred mouse plasma samples. Copyright © 2011 Elsevier B.V. All rights reserved.

[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.

A validated stability-indicating LC method for the separation of enantiomer and potential impurities of Linezolid using polar organic mode.

PubMed

Satyanarayana Raju, T; Vishweshwari Kutty, O; Ganesh, V; Yadagiri Swamy, P

2012-08-01

Although a number of methods are available for evaluating Linezolid and its possible impurities, a common method for separation if its potential impurities, degradants and enantiomer in a single method with good efficiency remain unavailable. With the objective of developing an advanced method with shorter runtimes, a simple, precise, accurate stability-indicating LC method was developed for the determination of purity of Linezolid drug substance and drug products in bulk samples and pharmaceutical dosage forms in the presence of its impurities and degradation products. This method is capable of separating all the related substances of Linezolid along with the chiral impurity. This method can also be used for the estimation of assay of Linezolid in drug substance as well as in drug product. The method was developed using Chiralpak IA (250 mm×4.6 mm, 5 μm) column. A mixture of acetonitrile, ethanol, n-butyl amine and trifluoro acetic acid in 96:4:0.10:0.16 (v/v/v/v) ratio was used as a mobile phase. The eluted compounds were monitored at 254 nm. Linezolid was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. The degradation products were well resolved from main peak and its impurities, proving the stability-indicating power of the method. The developed method was validated as per International Conference on Harmonization (ICH) guidelines with respect to specificity, limit of detection, limit of quantification, precision, linearity, accuracy, robustness and system suitability.

Lung cancer in patients with chronic obstructive pulmonary disease. Development and validation of the COPD Lung Cancer Screening Score.

PubMed

de-Torres, Juan P; Wilson, David O; Sanchez-Salcedo, Pablo; Weissfeld, Joel L; Berto, Juan; Campo, Arantzazu; Alcaide, Ana B; García-Granero, Marta; Celli, Bartolome R; Zulueta, Javier J

2015-02-01

Patients with chronic obstructive pulmonary disease (COPD) are at high risk for lung cancer (LC) and represent a potential target to improve the diagnostic yield of screening programs. To develop a predictive score for LC risk for patients with COPD. The Pamplona International Early Lung Cancer Detection Program (P-IELCAP) and the Pittsburgh Lung Screening Study (PLuSS) databases were analyzed. Only patients with COPD on spirometry were included. By logistic regression we determined which factors were independently associated with LC in PLuSS and developed a COPD LC screening score (COPD-LUCSS) to be validated in P-IELCAP. By regression analysis, age greater than 60, body mass index less than 25 kg/m(2), pack-years history greater than 60, and emphysema presence were independently associated with LC diagnosis and integrated into the COPD-LUCSS, which ranges from 0 to 10 points. Two COPD-LUCSS risk categories were proposed: low risk (scores 0-6) and high risk (scores 7-10). In comparison with low-risk patients, in both cohorts LC risk increased 3.5-fold in the high-risk category. The COPD-LUCSS is a good predictor of LC risk in patients with COPD participating in LC screening programs. Validation in two different populations adds strength to the findings.

A robust, sensitive assay for genomic uracil determination by LC/MS/MS reveals lower levels than previously reported.

PubMed

Galashevskaya, Anastasia; Sarno, Antonio; Vågbø, Cathrine B; Aas, Per A; Hagen, Lars; Slupphaug, Geir; Krokan, Hans E

2013-09-01

Considerable progress has been made in understanding the origins of genomic uracil and its role in genome stability and host defense; however, the main question concerning the basal level of uracil in DNA remains disputed. Results from assays designed to quantify genomic uracil vary by almost three orders of magnitude. To address the issues leading to this inconsistency, we explored possible shortcomings with existing methods and developed a sensitive LC/MS/MS-based method for the absolute quantification of genomic 2'-deoxyuridine (dUrd). To this end, DNA was enzymatically hydrolyzed to 2'-deoxyribonucleosides and dUrd was purified in a preparative HPLC step and analyzed by LC/MS/MS. The standard curve was linear over four orders of magnitude with a quantification limit of 5 fmol dUrd. Control samples demonstrated high inter-experimental accuracy (94.3%) and precision (CV 9.7%). An alternative method that employed UNG2 to excise uracil from DNA for LC/MS/MS analysis gave similar results, but the intra-assay variability was significantly greater. We quantified genomic dUrd in Ung(+/+) and Ung(-/-) mouse embryonic fibroblasts and human lymphoblastoid cell lines carrying UNG mutations. DNA-dUrd is 5-fold higher in Ung(-/-) than in Ung(+/+) fibroblasts and 11-fold higher in UNG2 dysfunctional than in UNG2 functional lymphoblastoid cells. We report approximately 400-600 dUrd per human or murine genome in repair-proficient cells, which is lower than results using other methods and suggests that genomic uracil levels may have previously been overestimated. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Improving peak detection in high-resolution LC/MS metabolomics data using preexisting knowledge and machine learning approach.

PubMed

Yu, Tianwei; Jones, Dean P

2014-10-15

Peak detection is a key step in the preprocessing of untargeted metabolomics data generated from high-resolution liquid chromatography-mass spectrometry (LC/MS). The common practice is to use filters with predetermined parameters to select peaks in the LC/MS profile. This rigid approach can cause suboptimal performance when the choice of peak model and parameters do not suit the data characteristics. Here we present a method that learns directly from various data features of the extracted ion chromatograms (EICs) to differentiate between true peak regions from noise regions in the LC/MS profile. It utilizes the knowledge of known metabolites, as well as robust machine learning approaches. Unlike currently available methods, this new approach does not assume a parametric peak shape model and allows maximum flexibility. We demonstrate the superiority of the new approach using real data. Because matching to known metabolites entails uncertainties and cannot be considered a gold standard, we also developed a probabilistic receiver-operating characteristic (pROC) approach that can incorporate uncertainties. The new peak detection approach is implemented as part of the apLCMS package available at http://web1.sph.emory.edu/apLCMS/ CONTACT: tyu8@emory.edu Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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.

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.

Multiresidue analysis of 24 Water Framework Directive priority substances by on-line solid phase extraction-liquid chromatography tandem mass spectrometry in environmental waters.

PubMed

Rubirola, Adrià; Boleda, Mª Rosa; Galceran, Mª Teresa

2017-04-14

This paper reports the development of a fully multiresidue and automated on-line solid phase extraction (SPE) - liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of 24 priority substances (PS) belonging to different classes (pesticides, hormones or pharmaceuticals) included in the Directive 2013/39/UE and the recent Watch List (Decision 2015/495) in water samples (drinking water, surface water, and effluent wastewaters). LC-MS/MS conditions and on-line SPE parameters such as sorbent type, sample and wash volumes were optimized. The developed method is highly sensitive (limits of detection between 0.1 and 1.4ngL -1 ) and precise (relative standard deviations lower than 8%). As part of the method validation studies, linearity, accuracy and matrix effects were assessed. The main advantage of this method over traditional off-line procedures is the minimization of tedious sample preparation increasing productivity and sample throughput. The optimized method was applied to the analysis of water samples and the results revealed the presence of 16 PS in river water and effluent water of wastewater treatment plants. Copyright © 2017 Elsevier B.V. All rights reserved.

AMP-regulated protein kinase activity in the hearts of mice treated with low- or high-fat diet measured using novel LC-MS method.

PubMed

Rybakowska, I M; Slominska, E M; Romaszko, P; Olkowicz, M; Kaletha, K; Smolenski, R T

2015-06-01

AMP-regulated protein kinase (AMPK) is involved in regulation of energy-generating pathways in response to the metabolic needs in different organs including the heart. The activity of AMPK is mainly controlled by AMP concentration that in turn could be affected by nucleotide metabolic pathways. This study aimed to develop a procedure for measurement of AMPK activity together with nucleotide metabolic enzymes and its application for studies of mice treated with high-fat diet. The method developed was based on analysis of conversion of AMARA peptide to pAMARA by partially purified heart homogenate by liquid chromatography/mass spectrometry (LC/MS). Activities of the enzymes of nucleotide metabolism were evaluated by analysis of conversion of substrates into products by HPLC. The method was applied for analysis of hearts of mice fed 12 weeks with low- (LFD) or high-fat diet (HFD). The optimized method for AMPK activity analysis (measured in presence of AMP) revealed change of activity from 0.089 ± 0.035 pmol/min/mg protein in LFD to 0.024 ± 0.002 in HFD. This coincided with increase of adenosine deaminase (ADA) activity from 0.11 ± 0.02 to 0.19 ± 0.06 nmol/mg tissue/min and decrease of AMP-deaminase (AMPD) activity from 1.26 ± 0.35 to 0.56 ± 0.15 nmol/mg tissue/min for LFD and HFD, respectively. We have proven quality of our LC/MS method for analysis of AMPK activity. We observed decrease in AMPK activity in the heart of mice treated with high-fat diet. However, physiological consequences of this change could be modulated by decrease in AMPD activity.

Fully automated method for the liquid chromatographic determination of cyproterone acetate in plasma using restricted access material for sample pre-treatment.

PubMed

Christiaens, B; Chiap, P; Rbeida, O; Cello, D; Crommen, J; Hubert, Ph

2003-09-25

A new fully automated method for the quantitative analysis of an antiandrogenic substance, cyproterone acetate (CPA), in plasma samples has been developed using on-line solid-phase extraction (SPE) prior to the determination by reversed-phase liquid chromatography (LC). The automated method was based on the use of a precolumn packed with an internal-surface reversed-phase packing material (LiChrospher RP-4 ADS) for sample clean-up coupled to LC analysis on an octadecyl stationary phase using a column-switching system. A 200-microL volume of plasma sample was injected directly on the precolumn packed with restricted access material using a mixture of water-acetonitrile (90:10, v/v) as washing liquid. The analyte was then eluted in the back-flush mode with the LC mobile phase which consisted of a mixture of phosphate buffer, pH 7.0-acetonitrile (54:46, v/v). The elution profiles of CPA and blank plasma samples on the precolumn and the time needed for analyte transfer from the precolumn to the analytical column were determined. Different compositions of washing liquid and mobile phase were tested to reduce the interference of plasma endogenous components. UV detection was achieved at 280 nm. Finally, the developed method was validated using a new approach, namely the application of the accuracy profile based on the interval confidence at 90% of the total measurement error (bias+standard deviation). The limit of quantification of cyproterone acetate in plasma was determined at 15 ng mL(-1). The validated method should be applicable to the determination of CPA in patients treated by at least 50 mg day(-1).

On-Line Ion Exchange Liquid Chromatography as a Process Analytical Technology for Monoclonal Antibody Characterization in Continuous Bioprocessing.

PubMed

Patel, Bhumit A; Pinto, Nuno D S; Gospodarek, Adrian; Kilgore, Bruce; Goswami, Kudrat; Napoli, William N; Desai, Jayesh; Heo, Jun H; Panzera, Dominick; Pollard, David; Richardson, Daisy; Brower, Mark; Richardson, Douglas D

2017-11-07

Combining process analytical technology (PAT) with continuous production provides a powerful tool to observe and control monoclonal antibody (mAb) fermentation and purification processes. This work demonstrates on-line liquid chromatography (on-line LC) as a PAT tool for monitoring a continuous biologics process and forced degradation studies. Specifically, this work focused on ion exchange chromatography (IEX), which is a critical separation technique to detect charge variants. Product-related impurities, including charge variants, that impact function are classified as critical quality attributes (CQAs). First, we confirmed no significant differences were observed in the charge heterogeneity profile of a mAb through both at-line and on-line sampling and that the on-line method has the ability to rapidly detect changes in protein quality over time. The robustness and versatility of the PAT methods were tested by sampling from two purification locations in a continuous mAb process. The PAT IEX methods used with on-line LC were a weak cation exchange (WCX) separation and a newly developed shorter strong cation exchange (SCX) assay. Both methods provided similar results with the distribution of percent acidic, main, and basic species remaining unchanged over a 2 week period. Second, a forced degradation study showed an increase in acidic species and a decrease in basic species when sampled on-line over 7 days. These applications further strengthen the use of on-line LC to monitor CQAs of a mAb continuously with various PAT IEX analytical methods. Implementation of on-line IEX will enable faster decision making during process development and could potentially be applied to control in biomanufacturing.

Chlorotoxin-mediated disinhibition of noradrenergic locus coeruleus neurons using a conditional transgenic approach.

PubMed

Salbaum, J Michael; Cirelli, Chiara; Walcott, Elisabeth; Krushel, Les A; Edelman, Gerald M; Tononi, Giulio

2004-07-30

The noradrenergic locus coeruleus (LC) has been implicated in the promotion of arousal, in focused attention and learning, and in the regulation of the sleep/waking cycle. The complex biological functions of the central noradrenergic system have been investigated largely through electrophysiological recordings and neurotoxic lesions of LC neurons. Activation of LC neurons through electrical or chemical stimulation has also led to important insights, although these techniques have limited cellular specificity and short-term effects. Here, we describe a novel method aimed at stimulating the central noradrenergic system in a highly selective manner for prolonged periods of time. This was achieved through the conditional expression of a transgene for chlorotoxin (Cltx) in the LC of adult mice. Chlorotoxin is a component of scorpion venom that partially blocks small conductance chloride channels. In this manner, the influence of GABAergic and glycinergic inhibitory inputs on LC cells is greatly reduced, while their ability to respond to excitatory inputs is unaffected. We demonstrate that the unilateral induction of Cltx expression in the LC is associated with a concomitant ipsilateral increase in the expression of markers of noradrenergic activity in LC neurons. Moreover, LC disinhibition is associated with the ipsilateral induction of the immediate early gene NGFI-A in cortical and subcortical target areas. Unlike previous gain of function approaches, transgenic disinhibition of LC cells is highly selective and persists for at least several weeks. This method represents a powerful new tool to assess the long-term effects of LC activation and is potentially applicable to other neuronal systems.

A LC/UV/Vis method for determination of cyanocobalamin in multivitamin dietary supplements with on-line sample clean-up

USDA-ARS?s Scientific Manuscript database

A HPLC-UV method using a two-column strategy with a switching valve for on-line sample clean-up was developed for the determination of cyanocobalamin (CN-CBL-vitamin B12, in dietary supplements. The method uses two columns, an Agilent Zorbax C8 (150 mm x 4.6 mm, 5 um particle) reversed-phase column...

Evaluation of Larvicidal Activity of Essential Oil from Leaves of Coccinia grandis against Three Mosquito Species

PubMed Central

Mohammed, Shahid Iqbal; Vishwakarma, Kishor Sukhlal; Maheshwari, Vijay Laxminarayan

2017-01-01

Background: To study the chemical constituents and larvicidal activity of essential oil extracted from the leaves of Coccinia grandis against three mosquito species. Methods: Essential oil was extracted by hydro distillation using clevenger apparatus and was analyzed for chemical constituents by gas chromatography-mass spectrophotometry (GC-MS). Larvicidal activity was recorded after 12 and 24h of post-exposure against three mosquito species, Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus. Dead larvae were identified when they failed to move after probing with a needle in the siphon or cervical region. The LC50 and LC90 values for three mosquito larvae were calculated by Probit analysis. Results: The GC-MS analysis revealed that essential oil contains 23 different constituents. Out of these 23 constituents, major constituents identified were n-tetracosane (39.18%), n-eicosane (30.04%), tetratriacotane (2.97%), 7-octadecanal (2.81%), and tricosane (2.31%). Essential oil from leaves of Coccinia grandis exhibited significant larvicidal activity against An. stephensi with LC50 and LC90 values 39.41ppm and 123.24ppm, respectively. This was followed by Ae. aegypti and Cx. quinquefasciatus with LC50 and LC90 values of 48.20ppm, 131.84ppm and 52.80ppm, 135.48ppm, respectively after 24h of exposure. Conclusion: The results could be useful in developing a cost effective, ecofriendly, region specific and practical strategy for the control of mosquito vectors. PMID:29062847

Photoswitchable gas permeation membranes based on azobenzene-doped liquid crystals II. Permeation-switching characterization under variable volume and variable pressure conditions

NASA Astrophysics Data System (ADS)

Glowacki, E.; Hunt, K.; Abud, D.; Marshall, K. L.

2010-08-01

Stimuli-responsive gas permeation membranes hold substantial potential for industrial processes as well as in analytical and screening applications. Such "smart" membrane systems, although prevalent in liquid mass-transfer manipulations, have yet to be realized for gas applications. We report our progress in developing gas permeation membranes in which liquid crystalline (LC) phases afford the active region of permeation. To achieve rapid and reversible switching between LC and isotropic permeation states, we harnessed the photomechanical action of mesogenic azobenzene dyes that can produce isothermal nematic-isotropic transitions. Both polymeric and low-molecular-weight LC materials were tested. Three different dye-doped LC mixtures with mesogenic azo dyes were infused into commercially available track-etched porous membranes with regular cylindrical pores (0.4 to 10.0 μm). Photoinduced isothermal phase changes in the imbibed material produced large and fully reversible changes in the permeability of the membrane to nitrogen with 5 s of irradiation at 2 mW/cm2. Using two measurement tools constructed in-house, the permeability of the photoswitched membranes was determined by both variable-pressure and variable-volume methods. Both the LC and photogenerated isotropic states demonstrate a linear permeability/pressure (ideal sorption) relationship, with up to a 16-fold difference in their permeability coefficients. Liquid crystal compositions can be chosen such that the LC phase is more permeable than the isotropic-or vice versa. This approach is the first system offering reversible tunable gas permeation membranes.

RapidRIP quantifies the intracellular metabolome of 7 industrial strains of E. coli.

PubMed

McCloskey, Douglas; Xu, Julia; Schrübbers, Lars; Christensen, Hanne B; Herrgård, Markus J

2018-04-25

Fast metabolite quantification methods are required for high throughput screening of microbial strains obtained by combinatorial or evolutionary engineering approaches. In this study, a rapid RIP-LC-MS/MS (RapidRIP) method for high-throughput quantitative metabolomics was developed and validated that was capable of quantifying 102 metabolites from central, amino acid, energy, nucleotide, and cofactor metabolism in less than 5 minutes. The method was shown to have comparable sensitivity and resolving capability as compared to a full length RIP-LC-MS/MS method (FullRIP). The RapidRIP method was used to quantify the metabolome of seven industrial strains of E. coli revealing significant differences in glycolytic, pentose phosphate, TCA cycle, amino acid, and energy and cofactor metabolites were found. These differences translated to statistically and biologically significant differences in thermodynamics of biochemical reactions between strains that could have implications when choosing a host for bioprocessing. Copyright © 2018. Published by Elsevier Inc.

The versatility of heart-cutting and comprehensive two-dimensional liquid chromatography in monoclonal antibody clone selection.

PubMed

Sandra, Koen; Steenbeke, Mieke; Vandenheede, Isabel; Vanhoenacker, Gerd; Sandra, Pat

2017-11-10

In recent years, two-dimensional liquid chromatography (2D-LC) has seen an enormous evolution and one of the fields where it is being widely adopted is in the analysis of therapeutic monoclonal antibodies (mAbs). We here further add to the many flavours of this powerful technology. Workflows based on heart-cutting (LC-LC) and comprehensive (LC×LC) 2D-LC are described that allow to guide the clone selection process in mAb and biosimilar development. Combining Protein A affinity chromatography in the first dimension with size exclusion (SEC), cation exchange (CEX) or reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) in the second dimension simultaneously allows to assess mAb titer and critical structural aspects such as aggregation, fragmentation, charge heterogeneity, molecular weight (MW), amino acid sequence and glycosylation. Complementing the LC-LC measurements at intact protein level with LC×LC based peptide mapping provides the necessary information to make clear decisions on which clones to take further into development. Copyright © 2017 Elsevier B.V. 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

Analytical Stability-Indicating Methods for Alogliptin in Tablets by LC-CAD and LC-UV.

PubMed

Bertol, Charise Dallazem; Friedrich, Maria Tereza; Carlos, Graciela; Froehlich, Pedro Eduardo

2017-03-01

Stability-indicating LC methods using a UV detector and a charged aerosol detector (CAD) simultaneously were validated for the assessment of alogliptin (ALG) in tablets. The analysis was performed on a C8 column (250 × 4.6 mm, 5 μm) at a flow of 0.8 mL/min, using acetonitrile-10 mM ammonium acetate buffer (pH 3.5; 90 + 10, v/v) as mobile phase and UV detection at 275 nm. Validation followed the International Conference on Harmonization guidelines. The method was linear over the range of 25-200 μg/mL. Normality of the residuals showed a normal distribution, no autocorrelation, and homoscedasticity. LODs were 6.25 and 2.65 µg/mL and LOQs were 20.85 and 8.84 µg/mL for the CAD and the UV detector, respectively. The methods were precise and accurate. Excipients and degradation products did not interfere in the methods in studies of specificity. None of the factors studied in the analysis of robustness had a significant effect on the quantification of the ALG by the Pareto chart. The results of the assay obtained with LC-CAD and LC-UV were similar. The methods could be considered interchangeable and stability-indicating, and can be applied as an appropriate QC tool for analysis of ALG in tablets.

Quantitative Multiclass Pesticide Residue Analysis in Apple, Pear, and Grape by Modified QuEChERS and Liquid Chromatography Coupled to High-Resolution Mass Spectrometry.

PubMed

Munaretto, Juliana S; Viera, Mariela de S; Martins, Manoel L; Adaime, Martha B; Zanella, Renato

2016-11-01

Most of the analytical methods currently applied in food control laboratories are focused on the determination of target compounds using LC coupled to tandem MS, which is an effective technique, but low-resolution MS is limited. Thus, a method for determination of pesticide multiresidues in fruits (pear, apple, and grape) using a modified quick, easy, cheap, effective, rugged, and safe method and LC coupled to quadrupole time-of-flight (Q-TOF) MS was developed and validated. The proposed method showed good linearity (r2 > 0.99) from 1 to 100 μg/L. Recoveries for blank samples spiked at 0.01, 0.04, and 0.10 mg/kg were between 66 and 122%, with RSDs <28%. Respective LOQs for apple, pear, and grape matrixes were 0.01 mg/kg for 112, 120, and 118 compounds, and 0.04 mg/kg for 22, 12, and 17 compounds, and average mass accuracy error was 3.2 ppm. LC with Q-TOF MS detection using protonated molecular ion and/or adducts and mass accuracy provided reliability for the method. The proposed method is effective for pesticide residue determination in apple, pear, and grape samples, proving that high-resolution MS using full scan mode can be a powerful and reliable technique for quantification purposes, being adequate for application in the surveillance of maximum residue limits set by different legislations.

Determination of illegal adulteration of dietary supplements with synthetic hair-growth compounds by UPLC and LC-Q-TOF/MS.

PubMed

Lee, Ji Hyun; Kang, Gihaeng; Park, Han Na; Kim, Jihee; Kim, Nam Sook; Park, Seongsoo; Park, Sung-Kwan; Baek, Sun Young; Kang, Hoil

2018-02-01

In this study, we developed a UPLC-PDA and LC-Q-TOF/MS method to identify and measure the following prohibited substances that may be found in dietary supplements:triaminodil, minoxidil, bimatoprost, alimemazine, diphenylcyclopropenone, α-tradiol, finasteride, methyltestosterone, spironolatone, flutamide, cyproterone, dutasteride, and testosterone 17-propionate.The method was validated according to International Conference on Harmonization guidelines in terms of specificity, linearity, accuracy, precision, LOD, LOQ, recovery, and stability. The method was completely validated showing satisfactory data for all method validation parameters. The linearity was good (R 2  > 0.999) with intra- and inter-day precision values of 0.2-3.4% and 0.3-2.9%, respectively. Moreover, the intra- and inter-day accuracies were 87-102% and 86-103%, respectively, and the precision was better than 9.4% (relative standard deviation).Hence, the proposed method is precise and has high quality,and can be utilised to comprehensively and continually monitor illegal drug adulteration in various forms of dietary supplements. Furthermore, to evaluate the applicability of the proposed method, we analysed 13 hair-growth compounds in 78 samples including food and dietary supplements. Minoxidil and triaminodil were detected in capsules at concentrations of 4.69 mg/g and 6.54 mg/g. In addition, finasteride was detected in a tablet at 13.45 mg/g. In addition, the major characteristic fragment ions were confirmed once again using LC-Q-TOF/MS for higher accuracy.

Development and validation of a stability-indicating size-indicating size-exclusion LC method for the determination of rhIFN-alpha2a in pharmaceutical formulations.

PubMed

Zimmermann, Estevan Sonego; da Silva, Lucélia Magalhães; Calegari, Guilherme Zanini; Stamm, Fernanda Pavani; Souto, Ricardo Bizogne; Dalmora, Sérgio Luiz

2013-01-01

A size-exclusion LC method was validated for the determination of interferon-a2a (rhlFN-alpha2a) in pharmaceutical formulations without interference from human serum albumin. Chromatographic separation was performed on a BioSep-SEC-S 2000 column (300 x 7.8 mm id). The mobile phase consisted of 0.001 M monobasic potassium phosphate, 0.008 M sodium phosphate dibasic; 0.2 M sodium chloride buffer, pH 7.4, run at a gradient flow rate and using photodiode array detection at 214 nm, was used. Chromatographic separation was achieved with a retention time of 17.2 min, and the analysis was linear over the concentration range of 1.98 to 198 microg/mL (r2 = 0.9996). The accuracy was 101.39%, with bias lower than 1.67%. The LOD and LOQ were 0.87 and 1.98 microg/mL, respectively. Moreover, method validation demonstrated acceptable results for precision and robustness. The method was applied to the assessment of rhlFN-alpha2a and related proteins in biopharmaceutical dosage forms, and the content/potencies were correlated to those given by a validated RP-LC method and an in vitro bioassay. It was concluded that use of the methods in conjunction allows a great improvement in monitoring stability and QC, thereby ensuring the therapeutic efficacy of the biotechnology-derived medicine.

Development of isotope labeling LC-MS for human salivary metabolomics and application to profiling metabolome changes associated with mild cognitive impairment.

PubMed

Zheng, Jiamin; Dixon, Roger A; Li, Liang

2012-12-18

Saliva is a readily available biofluid that may contain metabolites of interest for diagnosis and prognosis of diseases. In this work, a differential (13)C/(12)C isotope dansylation labeling method, combined with liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS), is described for quantitative profiling of the human salivary metabolome. New strategies are presented to optimize the sample preparation and LC-MS detection processes. The strategies allow the use of as little of 5 μL of saliva sample as a starting material to determine the concentration changes of an average of 1058 ion pairs or putative metabolites in comparative saliva samples. The overall workflow consists of several steps including acetone-induced protein precipitation, (12)C-dansylation labeling of the metabolites, and LC-UV measurement of the total concentration of the labeled metabolites in individual saliva samples. A pooled sample was prepared from all the individual samples and labeled with (13)C-dansylation to serve as a reference. Using this metabolome profiling method, it was found that compatible metabolome results could be obtained after saliva samples were stored in tubes normally used for genetic material collection at room temperature, -20 °C freezer, and -80 °C freezer over a period of 1 month, suggesting that many saliva samples already collected in genomic studies could become a valuable resource for metabolomics studies, although the effect of much longer term of storage remains to be determined. Finally, the developed method was applied for analyzing the metabolome changes of two different groups: normal healthy older adults and comparable older adults with mild cognitive impairment (MCI). Top-ranked 18 metabolites successfully distinguished the two groups, among which seven metabolites were putatively identified while one metabolite, taurine, was definitively identified.

Development of a Highly Automated and Multiplexed Targeted Proteome Pipeline and Assay for 112 Rat Brain Synaptic Proteins

PubMed Central

Colangelo, Christopher M.; Ivosev, Gordana; Chung, Lisa; Abbott, Thomas; Shifman, Mark; Sakaue, Fumika; Cox, David; Kitchen, Rob R.; Burton, Lyle; Tate, Stephen A; Gulcicek, Erol; Bonner, Ron; Rinehart, Jesse; Nairn, Angus C.; Williams, Kenneth R.

2015-01-01

We present a comprehensive workflow for large scale (>1000 transitions/run) label-free LC-MRM proteome assays. Innovations include automated MRM transition selection, intelligent retention time scheduling (xMRM) that improves Signal/Noise by >2-fold, and automatic peak modeling. Improvements to data analysis include a novel Q/C metric, Normalized Group Area Ratio (NGAR), MLR normalization, weighted regression analysis, and data dissemination through the Yale Protein Expression Database. As a proof of principle we developed a robust 90 minute LC-MRM assay for Mouse/Rat Post-Synaptic Density (PSD) fractions which resulted in the routine quantification of 337 peptides from 112 proteins based on 15 observations per protein. Parallel analyses with stable isotope dilution peptide standards (SIS), demonstrate very high correlation in retention time (1.0) and protein fold change (0.94) between the label-free and SIS analyses. Overall, our first method achieved a technical CV of 11.4% with >97.5% of the 1697 transitions being quantified without user intervention, resulting in a highly efficient, robust, and single injection LC-MRM assay. PMID:25476245

Dispersion-correcting potentials can significantly improve the bond dissociation enthalpies and noncovalent binding energies predicted by density-functional theory

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

DiLabio, Gino A., E-mail: Gino.DiLabio@nrc.ca; Department of Chemistry, University of British Columbia, Okanagan, 3333 University Way, Kelowna, British Columbia V1V 1V7; Koleini, Mohammad

2014-05-14

Dispersion-correcting potentials (DCPs) are atom-centered Gaussian functions that are applied in a manner that is similar to effective core potentials. Previous work on DCPs has focussed on their use as a simple means of improving the ability of conventional density-functional theory methods to predict the binding energies of noncovalently bonded molecular dimers. We show in this work that DCPs developed for use with the LC-ωPBE functional along with 6-31+G(2d,2p) basis sets are capable of simultaneously improving predicted noncovalent binding energies of van der Waals dimer complexes and covalent bond dissociation enthalpies in molecules. Specifically, the DCPs developed herein for themore » C, H, N, and O atoms provide binding energies for a set of 66 noncovalently bonded molecular dimers (the “S66” set) with a mean absolute error (MAE) of 0.21 kcal/mol, which represents an improvement of more than a factor of 10 over unadorned LC-ωPBE/6-31+G(2d,2p) and almost a factor of two improvement over LC-ωPBE/6-31+G(2d,2p) used in conjunction with the “D3” pairwise dispersion energy corrections. In addition, the DCPs reduce the MAE of calculated X-H and X-Y (X,Y = C, H, N, O) bond dissociation enthalpies for a set of 40 species from 3.2 kcal/mol obtained with unadorned LC-ωPBE/6-31+G(2d,2p) to 1.6 kcal/mol. Our findings demonstrate that broad improvements to the performance of DFT methods may be achievable through the use of DCPs.« less

Immunoextraction-Tandem Mass Spectrometry Method for Measuring Intact Human Chorionic Gonadotropin, Free β-Subunit, and β-Subunit Core Fragment in Urine

PubMed Central

Woldemariam, Getachew A.; Butch, Anthony W.

2015-01-01

BACKGROUND Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles. Because of the potential for abuse, hCG is banned (males only) in most sports and has been placed on the World Anti-Doping Agency list of prohibited substances. Intact hCG, free β-subunit (hCGβ), and β-subunit core fragment (hCGβcf) are the major variants or isoforms in urine. Immunoassays are used by antidoping laboratories to measure urinary hCG. Cross-reactivity with isoforms differs among immunoassays, resulting in widely varying results. We developed a sequential im-munoextraction method with LC-MS/MS detection for quantification of intact hCG, hCGβ, and hCGβcf in urine. METHODS hCG isoforms were immunoextracted with antibody-conjugated magnetic beads and digested with trypsin, and hCGβ and hCGβcf unique peptides were quantified by LC-MS/MS with the corresponding heavy peptides as internal standard. hCG isoform concentrations were determined in urine after administration of hCG, and the intact hCG results were compared to immunoassay results. RESULTS The method was linear to 20 IU/L. Total imprecision was 6.6%-13.7% (CV), recovery ranged from 91% to 109%, and the limit of quantification was 0.2 IU/L. Intact hCG predominated in the urine after administration of 2 hCG formulations. The window of detection ranged from 6 to 9 days. Mean immunoassay results were 12.4-15.5 IU/L higher than LC-MS/MS results. CONCLUSIONS The performance characteristics of the method are acceptable for measuring hCG isoforms, and the method can quantify intact hCG and hCGβ separately. The limit of quantification will allow LC-MS/MS hCG reference intervals to be established in nondoping male athletes for improved doping control. PMID:24899693

Saving time and money: a validation of the self ratings on the prospective NIMH Life-Chart Method (NIMH-LCM).

PubMed

Born, Christoph; Amann, Benedikt L; Grunze, Heinz; Post, Robert M; Schärer, Lars

2014-05-07

Careful observation of the longitudinal course of bipolar disorders is pivotal to finding optimal treatments and improving outcome. A useful tool is the daily prospective Life-Chart Method, developed by the National Institute of Mental Health. However, it remains unclear whether the patient version is as valid as the clinician version. We compared the patient-rated version of the Lifechart (LC-self) with the Young-Mania-Rating Scale (YMRS), Inventory of Depressive Symptoms-Clinician version (IDS-C), and Clinical Global Impression-Bipolar version (CGI-BP) in 108 bipolar I and II patients who participated in the Naturalistic Follow-up Study (NFS) of the German centres of the Bipolar Collaborative Network (BCN; formerly Stanley Foundation Bipolar Network). For statistical evaluation, levels of severity of mood states on the Lifechart were transformed numerically and comparison with affective scales was performed using chi-square and t tests. For testing correlations Pearson´s coefficient was calculated. Ratings for depression of LC-self and total scores of IDS-C were found to be highly correlated (Pearson coefficient r = -.718; p < .001), whilst the correlation of ratings for mania with YMRS compared to LC-self were slightly less robust (Pearson coefficient r = .491; p = .001). These results were confirmed by good correlations between the CGI-BP IA (mania), IB (depression) and IC (overall mood state) and the LC-self ratings (Pearson coefficient r = .488, r = .721 and r = .65, respectively; all p < .001). The LC-self shows a significant correlation and good concordance with standard cross sectional affective rating scales, suggesting that the LC-self is a valid and time and money saving alternative to the clinician-rated version which should be incorporated in future clinical research in bipolar disorder. Generalizability of the results is limited by the selection of highly motivated patients in specialized bipolar centres and by the open design of the study.

Determination of Antimycin-A in water by liquid chromatographic/mass spectrometry: single-laboratory validation

USGS Publications Warehouse

Bernardy, Jeffry A.; Hubert, Terrance D.; Ogorek, Jacob M.; Schmidt, Larry J.

2013-01-01

An LC/MS method was developed and validated for the quantitative determination and confirmation of antimycin-A (ANT-A) in water from lakes or streams. Three different water sample volumes (25, 50, and 250 mL) were evaluated. ANT-A was stabilized in the field by immediately extracting it from water into anhydrous acetone using SPE. The stabilized concentrated samples were then transported to a laboratory and analyzed by LC/MS using negative electrospray ionization. The method was determined to have adequate accuracy (78 to 113% recovery), precision (0.77 to 7.5% RSD with samples ≥500 ng/L and 4.8 to 17% RSD with samples ≤100 ng/L), linearity, and robustness over an LOQ range from 8 to 51 600 ng/L.

Determination of antimycin-A in water by liquid chromatographic/mass spectrometry: single-laboratory validation.

PubMed

Bernardy, Jeffry A; Hubert, Terrance D; Ogorek, Jacob M; Schmidt, Larry J

2013-01-01

An LC/MS method was developed and validated for the quantitative determination and confirmation of antimycin-A (ANT-A) in water from lakes or streams. Three different water sample volumes (25, 50, and 250 mL) were evaluated. ANT-A was stabilized in the field by immediately extracting it from water into anhydrous acetone using SPE. The stabilized concentrated samples were then transported to a laboratory and analyzed by LC/MS using negative electrospray ionization. The method was determined to have adequate accuracy (78 to 113% recovery), precision (0.77 to 7.5% RSD with samples > or = 500 ng/L and 4.8 to 17% RSD with samples < or = 100 ng/L), linearity, and robustness over an LOQ range from 8 to 51 600 ng/L.

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.

Time resolved analysis of quetiapine and 7-OH-quetiapine in hair using LC/MS-MS.

PubMed

Binz, Tina M; Yegles, Michel; Schneider, Serge; Neels, Hugo; Crunelle, Cleo L

2014-09-01

Hair analysis is a powerful tool for retrospective drug analysis and has a wide application window. This article describes the simultaneous determination and quantification of the short-acting atypical antipsychotic drug quetiapine and its main metabolite 7-OH quetiapine in hair. A sensitive and accurate method for the determination of these two compounds was developed using high-performance liquid chromatography coupled to tandem mass spectrometry detection (LC-MS/MS). The method was applied to 10 real case samples. For five patients, a time resolved hair analysis was done. Results varied from 0.35 ng/mg to 10.21 ng/mg hair for quetiapine and from 0.02 ng/mg to 3.19 ng/mg hair for 7-OH-quetiapine. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Clinical Implications of In Vivo Lamina Cribrosa Imaging in Glaucoma.

PubMed

Kim, Yong Woo; Jeoung, Jin Wook; Kim, Young Kook; Park, Ki Ho

2017-09-01

The lamina cribrosa (LC) is a multilayered, collagenous, sieve-like structure at the deep optic nerve head, and is presumed to be the primary site of axonal injury. According to biomechanical theory, intraocular pressure-induced posterior deformation of the LC causes blockage of axonal transport and alters the ocular blood flow, so that the axons of the retinal ganglion cells lead to apoptosis, which results in glaucomatous optic disc change. Although most of the research on the LC to date has been limited to experimental animal or histologic studies, the recent advances in optical coherence tomography devices and image processing techniques have made possible the visualization of the LC structure in vivo. LC deformation in glaucoma typically has been evaluated in terms of its position from a structural reference plane (LC depth), entire curvature or shape, thickness, or localized structural change (focal LC defects or LC pore change). In this review, we highlight the methods of assessing LC deformation from in vivo optical coherence tomography scans, and we discuss the clinical implications of the recent investigations of the in vivo structure of LC in glaucoma.

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.

LC-MS/MS method for the simultaneous determination of Lys-MCC-DM1, MCC-DM1 and DM1 as potential intracellular catabolites of the antibody-drug conjugate trastuzumab emtansine (T-DM1).

PubMed

Liu, Yazhong; Zhou, Fang; Sang, Hua; Ye, Hui; Chen, Qianying; Yao, Lan; Ni, Ping; Wang, Guangji; Zhang, Jingwei

2017-04-15

Lysine-MCC-DM1, MCC-DM1 and DM1 are potential catabolites of trastuzumab emtansine (T-DM1). A convenient liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to detect these catabolites simultaneously in in vitro investigations for the first time. Protein precipitation was utilized to prepare the samples. Chromatographic separation was achieved on a Phenomenex Kinetex C18 column (100×2.1mm, 2.6μm) with mobile-phase gradient elution. The calibration curves of each analyte ranging from 1 to 100nM showed good linearity (r 2 >0.995). The method was validated successfully and applied to the intracellular catabolism and regulation of T-DM1. Copyright © 2017 Elsevier B.V. All rights reserved.

Screening of 23 β-lactams in foodstuffs by LC-MS/MS using an alkaline QuEChERS-like extraction.

PubMed

Bessaire, Thomas; Mujahid, Claudia; Beck, Andrea; Tarres, Adrienne; Savoy, Marie-Claude; Woo, Pei-Mun; Mottier, Pascal; Desmarchelier, Aurélien

2018-04-01

A fast and robust high performance LC-MS/MS screening method was developed for the analysis of β-lactam antibiotics in foods of animal origin: eggs, raw milk, processed dairy ingredients, infant formula, and meat- and fish-based products including baby foods. QuEChERS extraction with some adaptations enabled 23 drugs to be simultaneously monitored. Screening target concentrations were set at levels adequate to ensure compliance with current European, Chinese, US and Canadian regulations. The method was fully validated according to the European Community Reference Laboratories Residues Guidelines using 93 food samples of different composition. False-negative and false-positive rates were below 5% for all analytes. The method is adequate for use in high-routine laboratories. A 1-year study was additionally conducted to assess the stability of the 23 analytes in the working standard solution.

Determination of dimethyltryptamine and β-carbolines (ayahuasca alkaloids) in plasma samples by LC-MS/MS.

PubMed

Oliveira, Carolina Dizioli Rodrigues; Okai, Guilherme Gonçalves; da Costa, José Luiz; de Almeida, Rafael Menck; Oliveira-Silva, Diogo; Yonamine, Mauricio

2012-07-01

Ayahuasca is a psychoactive plant beverage originally used by indigenous people throughout the Amazon Basin, long before its modern use by syncretic religious groups established in Brazil, the USA and European countries. The objective of this study was to develop a method for quantification of dimethyltryptamine and β-carbolines in human plasma samples. The analytes were extracted by means of C18 cartridges and injected into LC-MS/MS, operated in positive ion mode and multiple reaction monitoring. The LOQs obtained for all analytes were below 0.5 ng/ml. By using the weighted least squares linear regression, the accuracy of the analytical method was improved at the lower end of the calibration curve (from 0.5 to 100 ng/ml; r(2)> 0.98). The method proved to be simple, rapid and useful to estimate administered doses for further pharmacological and toxicological investigations of ayahuasca exposure.

LC-MS/MS Identification of Species-Specific Muscle Peptides in Processed Animal Proteins.

PubMed

Marchis, Daniela; Altomare, Alessandra; Gili, Marilena; Ostorero, Federica; Khadjavi, Amina; Corona, Cristiano; Ru, Giuseppe; Cappelletti, Benedetta; Gianelli, Silvia; Amadeo, Francesca; Rumio, Cristiano; Carini, Marina; Aldini, Giancarlo; Casalone, Cristina

2017-12-06

An innovative analytical strategy has been applied to identify signature peptides able to distinguish among processed animal proteins (PAPs) derived from bovine, pig, fish, and milk products. Proteomics was first used to elucidate the proteome of each source. Starting from the identified proteins and using a funnel based approach, a set of abundant and well characterized peptides with suitable physical-chemical properties (signature peptides) and specific for each source was selected. An on-target LC-ESI-MS/MS method (MRM mode) was set up using standard peptides and was then applied to selectively identify the PAP source and also to distinguish proteins from bovine carcass and milk proteins. We believe that the method described meets the request of the European Commission which has developed a strategy for gradually lifting the "total ban" toward "species to species ban", therefore requiring official methods for species-specific discrimination in feed.

Review of software tools for design and analysis of large scale MRM proteomic datasets.

PubMed

Colangelo, Christopher M; Chung, Lisa; Bruce, Can; Cheung, Kei-Hoi

2013-06-15

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. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

DETERMINATION OF AROMATIC AMINES IN SOILS

EPA Science Inventory

A rapid liquid chromatographic(LC)method with ultraviolet(UV)or fluorescence detection was developed for parts-per-billion levels of aromatic amines in soils. 2,4-Diaminotoluene, pyridine,aniline,2-picoline,2-toluidine,5-nitro-2-toluidine,2-methyl-6-ethylaniline,4-aminobiphenyl,4...

Recent development in liquid chromatography stationary phases for separation of Traditional Chinese Medicine components.

PubMed

Jin, Hongli; Liu, Yanfang; Guo, Zhimou; Wang, Jixia; Zhang, Xiuli; Wang, Chaoran; Liang, Xinmiao

2016-10-25

Traditional Chinese Medicine (TCM) is an ancient medical practice which has been used to prevent and cure diseases for thousands of years. TCMs are frequently multi-component systems with mainly unidentified constituents. The study of the chemical compositions of TCMs remains a hotspot of research. Different strategies have been developed to manage the significant complexity of TCMs, in an attempt to determine their constituents. Reversed-phase liquid chromatography (RPLC) is still the method of choice for the separation of TCMs, but has many problems related to limited selectivity. Recently, enormous efforts have been concentrated on the development of efficient liquid chromatography (LC) methods for TCMs, based on selective stationary phases. This can improve the resolution and peak capacity considerably. In addition, high-efficiency stationary phases have been applied in the analysis of TCMs since the invention of ultra high-performance liquid chromatography (UHPLC). This review describes the advances in LC methods in TCM research from 2010 to date, and focuses on novel stationary phases. Their potential in the separation of TCMs using relevant applications is also demonstrated. Copyright © 2016 Elsevier B.V. All rights reserved.

LC-UV/MS methods for the analysis of prochelator - boronyl salicylaldehyde isonicotinoyl hydrazone (BSIH) and its active chelator salicylaldehyde isonicotinoyl hydrazone (SIH)

PubMed Central

Bureš, Jan; Jansová, Hana; Stariat, Ján; Filipský, Tomáš; Mladěnka, Přemysl; Šimůnek, Tomáš; Kučera, Radim; Klimeš, Jiří; Wang, Qin; Franz, Katherine J.; Kovaříková, Petra

2015-01-01

Salicylaldehyde isonicotinoyl hydrazone (SIH) is an intracellular iron chelator with well documented potential to protect against oxidative injury both in vitro and in vivo. However, it suffers from short biological half-life caused by fast hydrolysis of the hydrazone bond. Recently, a concept of boronate prochelators has been introduced as a strategy that might overcome these limitations. This study presents two complementary analytical methods for detecting the prochelator BSIH (boronyl salicylaldehyde isonicotinoyl hydrazone) along with its active metal-binding chelator SIH in different solution matrices and concentration ranges. An LC-UV method for determination of BSIH and SIH in buffer and cell culture medium was validated over concentrations of 7 – 115 and 4 – 115 μM, respectively, and applied to BSIH activation experiments in vitro. An LC-MS assay was validated for quantification of BSIH and SIH in plasma over the concentration range of 0.06 – 23 and 0.24 – 23 μM, respectively, and applied to stability studies in plasma in vitro as well as analysis of plasma taken after i.v. administration of BSIH to rats. A Zorbax-RP bonus column and mobile phases containing either phosphate buffer with EDTA or ammonium formate and methanol/acetonitrile mixture provided suitable conditions for the LC-UV and LC-MS analysis, respectively. Samples were diluted or precipitated with methanol prior to analysis. These separative analytical techniques establish the first validated protocols to investigate BSIH activation by hydrogen peroxide in multiple matrices, directly compare the stabilities of the prochelator and chelator in plasma, and provide the first basic pharmacokinetic data of this prochelator. Experiments reveal that BSIH is stable in all media tested and is partially converted to SIH by H2O2. The observed integrity of BSIH in plasma samples from the in vivo study suggest that the concept of prochelation might be a promising strategy for further development of aroylhydrazone cytoprotective agents. PMID:25527982

Post-column mobile phase adjustment: a strategy to eliminate the contradiction between liquid chromatography and mass spectrometry in the determination of flavonoids in rat plasma.

PubMed

Zheng, Shirui; Ma, Zhiyuan; Han, Haixia; Ye, Jianfeng; Wang, Ruwei; Cai, Sheng; Zhou, Hui; Yu, Lushan; Zeng, Su; Jiang, Huidi

2014-07-01

Flavonoids are a group of important naturally occurring polyphenolic compounds with a wide range of biological effects. In this study, a sensitive liquid chromatography tandem mass spectrometry method was developed to simultaneously determine multiple active flavonoids, including quercetin (Que), kaempferol (Kae), apigenin (Api), isorhamnetin (Iso), luteolin (Lut), and naringenin (Nar), in rat plasma. To achieve a satisfied peak shape and LC separation, formic acid with the concentration between 0.05 and 0.2%, or in some case 5%, was generally used to acidify the LC mobile phase in reported studies. Here we found that even 0.05% formic acid could lead to strong mass signal suppression, and the absence of formic acid could reverse the signal suppression but cause serious peak tailing. There is an irreconcilable contradiction between liquid chromatography (LC) and mass spectrometry (MS). In order to simultaneously satisfy LC and MS, LC mobile phase with 0.00075% formic acid and post column mobile phase adjustment with 0.0677% ammonium solution in isopropanol were applied. Compared with the conventional method with mobile phase containing 0.05% formic acid, the mass signal response of Que, Kae, Api, Iso, Lut, Nar, and Oka increased 26.2, 18.6, 13.6, 23.5, 17.5, 15.6 and 15.4 fold, respectively. In addition, the post column mobile phase addition exhibited the better peak shape for the reduction of analytes longitudinal diffusion. The method has been fully validated according to FDA guidelines within the linear range between 0.328 ng mL⁻¹ and 168 ng mL⁻¹, and successfully applied to a pilot pharmacokinetic study of rats after administering 5.43 g kg⁻¹ Pollen of Brassica campestris. Copyright © 2014 Elsevier B.V. All rights reserved.

Quantitative Methods Based on Twisted Nematic Liquid Crystals for Mapping Surfaces Patterned with Bio/Chemical Functionality Relevant to Bioanalytical Assays

PubMed Central

Lowe, Aaron M.; Bertics, Paul J.; Abbott, Nicholas L.

2009-01-01

We report methods for the acquisition and analysis of optical images formed by thin films of twisted nematic liquid crystals (LCs) placed into contact with surfaces patterned with bio/chemical functionality relevant to surface-based assays. The methods are simple to implement and are shown to provide easily interpreted maps of chemical transformations on surfaces that are widely exploited in the preparation of analytic devices. The methods involve acquisition of multiple images of the LC as a function of the orientation of a polarizer; data analysis condenses the information present in the stack of images into a spatial map of the twist angle of the LC on the analytic surface. The potential utility of the methods is illustrated by mapping (i) the displacement of a monolayer formed from one alkanethiol on a gold film by a second thiol in solution, (ii) coadsorption of mixtures of amine-terminated and ethyleneglycol-terminated alkanethiols on gold films, which leads to a type of mixed monolayer that is widely exploited for immobilization of proteins on analytic surfaces, and (iii) patterns of antibodies printed onto surfaces. These results show that maps of the twist angle of the LC constructed from families of optical images can be used to reveal surface features that are not apparent in a single image of the LC film. Furthermore, the twist angles of the LC can be used to quantify the energy of interaction of the LC with the surface with a spatial resolution of <10 µm. When combined, the results described in this paper suggest non-destructive methods to monitor and validate chemical transformations on surfaces of the type that are routinely employed in the preparation of surface-based analytic technologies. PMID:18355089

Methods for Studying Interactions Between Atg8/LC3/GABARAP and LIR-Containing Proteins.

PubMed

Johansen, T; Birgisdottir, Å B; Huber, J; Kniss, A; Dötsch, V; Kirkin, V; Rogov, V V

2017-01-01

LC3/GABARAP proteins (LC3/GABARAPs) are mammalian orthologues of yeast Atg8, small ubiquitin (Ub)-like proteins (UBLs) whose covalent attachment to lipid membranes is crucial for the growth and closure of the double membrane vesicle called the autophagosome. In the past decade, it was demonstrated that Atg8/LC3/GABARAPs are also required for autophagic degradation of cargos in a selective fashion. Cargo selectivity is ensured by receptor proteins, such as p62/SQSTM1, NBR1, Cue5, Atg19, NIX, Atg32, NCOA4, and FAM134B, which simultaneously bind Atg8/LC3/GABARAPs and the cargo together, thereby linking the core autophagic machinery to the target structure: a protein, an organelle, or a pathogen. LC3-interacting regions (LIRs) are short linear motifs within selective autophagy receptors and some other structural and signaling proteins (e.g., ULK1, ATG13, FIP200, and Dvl2), which mediate binding to Atg8/LC3/GABARAPs. Identification and characterization of LIR-containing proteins have provided important insights into the biology of the autophagy pathway, and studying their interactions with the core autophagy machinery represents a growing area of autophagy research. Here, we present protocols for the identification of LIR-containing proteins, i.e., by yeast-two-hybrid screening, glutathione S-transferase (GST) pulldown experiments, and peptide arrays. The use of two-dimensional peptide arrays also represents a powerful method to identify the residues of the LIR motif that are critical for binding. We also describe a biophysical method for studying interactions between Atg8/LC3/GABARAP and LIR-containing proteins and a protocol for preparation and purification of LIR peptides. © 2017 Elsevier Inc. All rights reserved.

Comparison of methods, storage conditions, and time to analysis of serum and urine creatinine measured from microsamples by liquid chromatography mass spectrometery (LC/MS) vs. Jaffe.

PubMed

Askenazi, David J; Moore, John F; Fineberg, Naomi; Koralkar, Rajesh; Clevenger, Stephanie; Sharer, Jon Daniel

2014-09-01

Measurement of serum creatinine (SCr) and urine creatinine (UCr) is regularly used in clinical and research settings. For small animal experiments and for studies in which sample collection is spare (i.e. neonatal cohorts), measuring SCr and UCr using tiny amounts of sample (as low as 10 mcl) would maximize exploration and minimize iatrogenic blood loss. We performed an evaluation in six healthy adults to determine differences between SCr and UCr values in different methodologies and storage environments and time. Study was conducted using 20 mcl of sample. Analyses were done using two-way repeated measures of ANOVA. Scr values showed no significant differences between LC/MS vs. Jaffe. However, the SCr using LC/MS method was lowest when measured immediately compared to other time points (F = 7.2; P< 0.001). Similarly, Jaffe measurements showed changes in the mean differences over time; however, these were not significant. UCr values were consistently higher using LC/MS than Jaffe (F = 19; P< 0.01), and UCr changed over time (F = 8.7; P < 0.02). In addition, the interaction term for method and time was also significant (F = 5.8; P < 0.04) which reflects the stability of the Jaffe measurements over time whereas the LC/MS measurements declined; especially after being frozen for 1 year (P < 0.001). UCr measured by Jaffe is lower than samples measured by LC/MS. UCr measurements by LC/MS vary more over time, mostly due to the sample measured after 1 year; therefore, storage of urine for more than 90 days measured by LC/MS may provide altered results. © 2014 Wiley Periodicals, Inc.

[Latest development in mass spectrometry for clinical application].

PubMed

Takino, Masahiko

2013-09-01

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has seen enormous growth in special clinical chemistry laboratories. It significantly increases the analytic potential in clinical chemistry, especially in the field of low molecular weight biomarker analysis. This review summarizes the state of the art in mass spectrometry and related techniques for clinical application with a main focus on recent developments in LC-MS. Current trends in ionization techniques, automated online sample preparation techniques coupled with LC-MS, and ion mobility spectrometry are discussed. Emerging mass spectrometric approaches complementary to LC-MS are discussed as well.

Larvicidal activity of essential oil and methanol extract of Nepeta menthoides against malaria vector Anopheles stephensi.

PubMed

Mahnaz, Khanavi; Alireza, Fallah; Hassan, Vatandoost; Mahdi, Sedaghat; Reza, Abai Mohammad; Abbas, Hadjiakhoondi

2012-12-01

To investigate the larvicidal activity of essential oil and methanol extract of the Nepeta menthoides (N. menthoides) against main malaria vector, Anopheles stephensi (An. stephensi). The essential oil of plant was obtained by Clevenger type apparatus and the methanol extract was supplied with Percolation method. Larvicidal activity was tested by WHO method. Twenty five fourth-instar larvae of An. stephensi were used in the larvicidal assay and four replicates were tested for each concentration. Five different concentrations of the oil and extract were tested for calculation of LC(50) and LC(90) values. The LC(50) and LC(90) values were determined by probit analysis. LC(50) was 69.5 and 234.3 ppm and LC(90) was 175.5 and 419.9 ppm for the extract and essential oil respectively. According to the results of this study methanolic extract of plant exhibited more larvicidal activity than essential oil. This could be useful for investigation of new natural larvicidal compounds. Copyright © 2012 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

The toxicity evaluation of prepared Lantana camara nano extract against Spodoptera litura (Lepidoptera: Noctuidae)

NASA Astrophysics Data System (ADS)

Kasmara, Hikmat; Melanie, Nurfajri, Dea Audia; Hermawan, Wawan; Panatarani, Camellia

2018-02-01

Nanotechnology plays an important role in providing opportunities and possibilities for the development of a plant extracts, particularly applied for agriculture using the organic nanoparticles extract as bioinsecticide. This paper reports the extraction of L. camaraas an insecticidal compound applied to control the S. litura larvae which is the main pest for agricultural commodities in Indonesia. The objective of this research is to evaluate the toxicity performance of nano extract to S. litura larvae. The extract was prepared by maceration and evaporation and followed by particles stabilization. The received suspension was characterized using a Particles Size Analyzer (PSA). The performance evaluation was conducted using dye feed method by observing at 24 h and 48 h. The toxicity performance of L. camara nano extract on S. litura larvae was evaluated based on the lethal concentration which was obtained at 50% of 3rd instar larvae S. litura (LC50) and the toxicity level. The larval mortality data was analyzed by probit analysis to obtain LC50 values. The results showed that the LC50 value of L. camara nano extract after 24 h was 16,347 ppm and after 48 h the LC50 value was 3,548 ppm. The smaller LC50 values indicate a stronger toxicity level, in contrast the best toxicity was LC50 after 48 h with the concentration of 3,548 ppm. Referring to toxicity category standard with LC50 pesticide value, the range 0.5-5 g/L is considered as medium toxic category. Toxicity level of L. camara nano extract to S. litura 3,548 ppm or 3,548 mg/L is considered as medium toxic category.

Development and validation of an LC-UV method for the determination of sulfonamides in animal feeds.

PubMed

Kumar, P; Companyó, R

2012-05-01

A simple LC-UV method was developed for the determination of residues of eight sulfonamides (sulfachloropyridazine, sulfadiazine, sulfadimidine, sulfadoxine, sulfamethoxypyridazine, sulfaquinoxaline, sulfamethoxazole, and sulfadimethoxine) in six types of animal feed. C18, Oasis HLB, Plexa and Plexa PCX stationary phases were assessed for the clean-up step and the latter was chosen as it showed greater efficiency in the clean-up of interferences. Feed samples spiked with sulfonamides at 2 mg/kg were used to assess the trueness (recovery %) and precision of the method. Mean recovery values ranged from 47% to 66%, intra-day precision (RSD %) from 4% to 15% and inter-day precision (RSD %) from 7% to 18% in pig feed. Recoveries and intra-day precisions were also evaluated in rabbit, hen, cow, chicken and piglet feed matrices. Calibration curves with standards prepared in mobile phase and matrix-matched calibration curves were compared and the matrix effects were ascertained. The limits of detection and quantification in the feeds ranged from 74 to 265 µg/kg and from 265 to 868 µg/kg, respectively. Copyright © 2011 John Wiley & Sons, Ltd.

WE-AB-207B-02: A Bayesian Network Approach for Joint Prediction of Tumor Control and Radiation Pneumonitis (RP) in Non-Small-Cell Lung Cancer (NSCLC)

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

Luo, Y; McShan, D; Matuszak, M

Purpose: NSCLC radiotherapy treatment is a trade-off between controlling the tumor while limiting radiation-induced toxicities. Here we identify hierarchical biophysical relationships that could simultaneously influence both local control (LC) and RP by using an integrated Bayesian Networks (BN) approach. Methods: We studied 79 NSCLC patients treated on prospective protocol with 56 cases of LC and 21 events of RP. Beyond dosimetric information, each patient had 193 features including 12 clinical factors, 60 circulating blood cytokines before and during radiotherapy, 62 microRNAs, and 59 single-nucleotide polymorphisms (SNPs). The most relevant biophysical predictors for both LC and RP were identified using amore » Markov blanket local discovery algorithm and the corresponding BN was constructed using a score-learning algorithm. The area under the free-response receiver operating characteristics (AU-FROC) was used for performance evaluation. Cross-validation was employed to guard against overfitting pitfalls. Results: A BN revealing the biophysical interrelationships jointly in terms of LC and RP was developed and evaluated. The integrated BN included two SNPs, one microRNA, one clinical factor, three pre-treatment cytokines, relative changes of two cytokines between pre and during-treatment, and gEUDs of the GTV (a=-20) and lung (a=1). On cross-validation, the AUC prediction of independent LC was 0.85 (95% CI: 0.75–0.95) and RP was 0.83 (0.73–0.92). The AU-FROC of the integrated BN to predict both LC/RP was 0.81 (0.71–0.90) based on 2000 stratified bootstrap, indicating minimal loss in joint prediction power. Conclusions: We developed a new approach for multiple outcome utility application in radiotherapy based on integrated BN techniques. The BN developed from large-scale retrospective data is able to simultaneously predict LC and RP in NSCLC treatments based on individual patient characteristics. The joint prediction is only slightly compromised compared to independent predictions. Our approach shows promise for use in clinical decision support system for personalized radiotherapy subject to multiple endpoints. These studies were supported by a grant from the NCI/NIH P01-CA59827.« less

Survivability Versus Time

NASA Technical Reports Server (NTRS)

Joyner, James J., Sr.

2014-01-01

Develop Survivability vs Time Model as a decision-evaluation tool to assess various emergency egress methods used at Launch Complex 39B (LC 39B) and in the Vehicle Assembly Building (VAB) on NASAs Kennedy Space Center. For each hazard scenario, develop probability distributions to address statistical uncertainty resulting in survivability plots over time and composite survivability plots encompassing multiple hazard scenarios.

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

Direct chiral determination of free amino acid enantiomers by two-dimensional liquid chromatography: application to control transformations in E-beam irradiated foodstuffs.

PubMed

Guillén-Casla, Vanesa; León-González, María Eugenia; Pérez-Arribas, Luis Vicente; Polo-Díez, Luis María

2010-05-01

Changes in free amino acids content and its potential racemization in ready-to-eat foods treated with E-beam irradiation between 1 and 8 kGy for sanitation purposes were studied. A simple heart cut two-dimensional high performance liquid chromatographic method (LC-LC) for the simultaneous enantiomeric determination of three pairs of amino acids used as markers (tyrosine, phenylalanine, and tryptophan) is presented. The proposed method involves the use of two chromatographs in an LC-LC achiral-chiral coupling. Amino acids and their decomposition products were firstly separated in a primary column (C(18)) using a mixture of ammonium acetate buffer (20 mM, pH 6) (94%) and methanol (6%) as the mobile phase. Then, a portion of each peak was transferred by heart cutting through a switching valve to a teicoplanin-chiral column. Methanol (90%)/water (10%) was used as the mobile phase. Ultraviolet detection was at 260 nm. Detection limits were between 0.16 and 3 mg L(-1) for each enantiomer. Recoveries were in the range 79-98%. The LC-LC method combined with the proposed sample extraction procedure is suitable for complex samples; it involves an online cleanup, and it prevents degradation of protein, racemization of L-enantiomers, and degradation of tryptophan. Under these conditions, D-amino acids were not found in any of the analyzed samples at detection levels of the proposed method.

Development of Fluorescent Substrates and Assays for the Key Autophagy-Related Cysteine Protease Enzyme, ATG4B

PubMed Central

Nguyen, Thanh G.; Honson, Nicolette S.; Arns, Steven; Davis, Tara L.; Dhe-Paganon, Sirano; Kovacic, Suzana; Kumar, Nag S.; Pfeifer, Tom A.

2014-01-01

Abstract The cysteine protease ATG4B plays a role in key steps of the autophagy process and is of interest as a potential therapeutic target. At an early step, ATG4B cleaves proLC3 isoforms to form LC3-I for subsequent lipidation to form LC3-II and autophagosome membrane insertion. ATG4B also cleaves phosphatidylethanolamine (PE) from LC3-II to regenerate LC3-I, enabling its recycling for further membrane biogenesis. Here, we report several novel assays for monitoring the enzymatic activity of ATG4B. An assay based on mass spectrometric analysis and quantification of cleavage of the substrate protein LC3-B was developed and, while useful for mechanistic studies, was not suitable for high throughput screening (HTS). A doubly fluorescent fluorescence resonance energy transfer (FRET) ligand YFP-LC3B-EmGFP (FRET-LC3) was constructed and shown to be an excellent substrate for ATG4B with rates of cleavage similar to that for LC3B itself. A HTS assay to identify candidate inhibitors of ATG4B utilizing FRET-LC3 as a substrate was developed and validated with a satisfactory Z′ factor and high signal-to-noise ratio suitable for screening small molecule libraries. Pilot screens of the 1,280-member library of pharmacologically active compounds (LOPAC™) and a 3,481-member library of known drugs (KD2) gave hit rates of 0.6% and 0.5% respectively, and subsequent titrations confirmed ATG4B inhibitory activity for three compounds, both in the FRET and mass spectrometry assays. The FRET- and mass spectrometry–based assays we have developed will allow for both HTS for inhibitors of ATG4B and mechanistic approaches to study inhibition of a major component of the autophagy pathway. PMID:24735444

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.

Determination of antibacterial agent tilmicosin in pig plasma by LC/MS/MS and its application to pharmacokinetics.

PubMed

Li, Bing; Gong, Shi-Yue; Zhou, Xu-Zheng; Yang, Ya-Jun; Li, Jian-Yong; Wei, Xiao-Juan; Cheng, Fu-Sheng; Niu, Jian-Rong; Liu, Xi-Wang; Zhang, Ji-Yu

2017-03-01

A rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to quantify tilmicosin in pig plasma. Plasma samples were prepared by liquid-liquid extraction. Chromatographic separation was achieved on a C 18 column (2.1 × 30 mm, 3.5 μm) using acetonitrile-water (90:10, v/v; water included 0.1% formic acid) as the mobile phase. Mass detection was carried out using positive electrospray ionization in multiple reaction monitoring mode. The calibration curve was linear from 0.5 to 2000 ng/mL (r 2  = 0.9998). The intra- and inter-day accuracy and precision were within the acceptable limits of ±10% for all tilmicosin concentrations. The recoveries ranged from 95 to 99% for the three tested concentrations. The LC-MS/MS method described herein was simple, fast and less laborious than other methods, achieved high sensitivity using a small sample volume, and was successfully applied to pharmacokinetic studies of tilmicosin enteric granules after oral delivery to pigs. In comparison with tilmicosin premix, tilmicosin enteric granules slowed the elimination rate of tilmicosin, prolonged its period of action and significantly improved its bioavailability. Copyright © 2016 John Wiley & Sons, Ltd.

Rapid determination of polycyclic aromatic hydrocarbons in rainwater by liquid-liquid microextraction and LC with core-shell particles column and fluorescence detection.

PubMed

Vinci, Giuliana; Antonelli, Marta L; Preti, Raffaella

2013-02-01

Liquid-liquid microextraction coupled to LC with fluorescence detection for the determination of Environmental Protection Agency's 16 priority pollutant polycyclic aromatic hydrocarbons in rainwater has been developed. The optimization of the extraction method has involved several parameters, including the comparison between an ultrasonic bath and a magnetic stirrer as extractant apparatus, the choice of the extractant solvent, and the optimization of the extraction time. Liquid-liquid microextraction gave good results in terms of recoveries (from 73.6 to 102.8% in rainwater) and repeatability, with a very simple procedure and low solvent consumption. The reported chromatographic method uses a Core-Shell technology column, with particle size <3 μm instead of classical 5-μm particles column. The resulting backpressure was below 300 bar, allowing the use of a conventional HPLC system rather than the more expensive ultrahigh performance LC (UHPLC). An average decrease of 59% in run time and 75% in eluent consumption has been obtained, compared to classical HPLC methods, keeping good separation, sensitivity, and repeatability. The proposed conditions were successfully applied to the determinations of polycyclic aromatic hydrocarbons in genuine rainwater samples. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ultra-sensitive LC-MS/MS method for the quantification of gemcitabine and its metabolite 2',2'-difluorodeoxyuridine in human plasma for a microdose clinical trial.

PubMed

van Nuland, M; Hillebrand, M J X; Rosing, H; Burgers, J A; Schellens, J H M; Beijnen, J H

2018-03-20

In microdose clinical trials a maximum of 100 μg of drug substance is administered to participants, in order to determine the pharmacokinetic properties of the agents. Measuring low plasma concentrations after administration of a microdose is challenging and requires the use of ulta-sensitive equipment. Novel liquid chromatography-mass spectrometry (LC-MS/MS) platforms can be used for quantification of low drug plasma levels. Here we describe the development and validation of an LC-MS/MS method for quantification of gemcitabine and its metabolite 2',2'-difluorodeoxyuridine (dFdU) in the low picogram per milliliter range to support a microdose trial. The validated assay ranges from 2.5-500 pg/mL for gemcitabine and 250-50,000 pg/mL for dFdU were linear, with a correlation coefficient (r 2 ) of 0.996 or better. Sample preparation with solid phase extraction provided a good and reproducible recovery. All results were within the acceptance criteria of the latest US FDA guidance and EMA guidelines. In addition, the method was successfully applied to measure plasma concentrations of gemcitabine in a patient after administration of a microdose of gemcitabine. Copyright © 2017 Elsevier B.V. All rights reserved.

Screening and monitoring microbial xenobiotics' biodegradation by rapid, inexpensive and easy to perform microplate UV-absorbance measurements.

PubMed

Herzog, Bastian; Lemmer, Hilde; Horn, Harald; Müller, Elisabeth

2014-02-22

Evaluation of xenobiotics biodegradation potential, shown here for benzotriazoles (corrosion inhibitors) and sulfamethoxazole (sulfonamide antibiotic) by microbial communities and/or pure cultures normally requires time intensive and money consuming LC/GC methods that are, in case of laboratory setups, not always needed. The usage of high concentrations to apply a high selective pressure on the microbial communities/pure cultures in laboratory setups, a simple UV-absorbance measurement (UV-AM) was developed and validated for screening a large number of setups, requiring almost no preparation and significantly less time and money compared to LC/GC methods. This rapid and easy to use method was evaluated by comparing its measured values to LC-UV and GC-MS/MS results. Furthermore, its application for monitoring and screening unknown activated sludge communities (ASC) and mixed pure cultures has been tested and approved to detect biodegradation of benzotriazole (BTri), 4- and 5-tolyltriazole (4-TTri, 5-TTri) as well as SMX. In laboratory setups, xenobiotics concentrations above 1.0 mg L(-1) without any enrichment or preparation could be detected after optimization of the method. As UV-AM does not require much preparatory work and can be conducted in 96 or even 384 well plate formats, the number of possible parallel setups and screening efficiency was significantly increased while analytic and laboratory costs were reduced to a minimum.

On-line high-performance liquid chromatography-ultraviolet-nuclear magnetic resonance method of the markers of nerve agents for verification of the Chemical Weapons Convention.

PubMed

Mazumder, Avik; Gupta, Hemendra K; Garg, Prabhat; Jain, Rajeev; Dubey, Devendra K

2009-07-03

This paper details an on-flow liquid chromatography-ultraviolet-nuclear magnetic resonance (LC-UV-NMR) method for the retrospective detection and identification of alkyl alkylphosphonic acids (AAPAs) and alkylphosphonic acids (APAs), the markers of the toxic nerve agents for verification of the Chemical Weapons Convention (CWC). Initially, the LC-UV-NMR parameters were optimized for benzyl derivatives of the APAs and AAPAs. The optimized parameters include stationary phase C(18), mobile phase methanol:water 78:22 (v/v), UV detection at 268nm and (1)H NMR acquisition conditions. The protocol described herein allowed the detection of analytes through acquisition of high quality NMR spectra from the aqueous solution of the APAs and AAPAs with high concentrations of interfering background chemicals which have been removed by preceding sample preparation. The reported standard deviation for the quantification is related to the UV detector which showed relative standard deviations (RSDs) for quantification within +/-1.1%, while lower limit of detection upto 16mug (in mug absolute) for the NMR detector. Finally the developed LC-UV-NMR method was applied to identify the APAs and AAPAs in real water samples, consequent to solid phase extraction and derivatization. The method is fast (total experiment time approximately 2h), sensitive, rugged and efficient.

Validation of highly sensitive simultaneous targeted and untargeted analysis of keto-steroids by Girard P derivatization and stable isotope dilution-liquid chromatography-high resolution mass spectrometry.

PubMed

Frey, Alexander J; Wang, Qingqing; Busch, Christine; Feldman, Daniel; Bottalico, Lisa; Mesaros, Clementina A; Blair, Ian A; Vachani, Anil; Snyder, Nathaniel W

2016-12-01

A multiplexed quantitative method for the analysis of three major unconjugated steroids in human serum by stable isotope dilution liquid chromatography-high resolution mass spectrometry (LC-HRMS) was developed and validated on a Q Exactive Plus hybrid quadrupole/Orbitrap mass spectrometer. This quantification utilized isotope dilution and Girard P derivatization on the keto-groups of testosterone (T), androstenedione (AD) and dehydroepiandrosterone (DHEA) to improve ionization efficiency using electrospray ionization. Major isomeric compounds to T and DHEA; the inactive epimer of testosterone (epiT), and the metabolite of AD, 5α-androstanedione (5α-AD) were completely resolved on a biphenyl column within an 18min method. Inter- and intra-day method validation using LC-HRMS with qualifying product ions was performed and acceptable analytical performance was achieved. The method was further validated by comparing steroid levels from 100μL of serum from young vs older subjects. Since this approach provides high-dimensional HRMS data, untargeted analysis by age group was performed. DHEA and T were detected among the top analytes most significantly different across the two groups after untargeted LC-HRMS analysis, as well as a number of other still unknown metabolites, indicating the potential for combined targeted/untargeted analysis in steroid analysis. Copyright © 2016 Elsevier Inc. All rights reserved.

Screening and monitoring microbial xenobiotics’ biodegradation by rapid, inexpensive and easy to perform microplate UV-absorbance measurements

PubMed Central

2014-01-01

Background Evaluation of xenobiotics biodegradation potential, shown here for benzotriazoles (corrosion inhibitors) and sulfamethoxazole (sulfonamide antibiotic) by microbial communities and/or pure cultures normally requires time intensive and money consuming LC/GC methods that are, in case of laboratory setups, not always needed. Results The usage of high concentrations to apply a high selective pressure on the microbial communities/pure cultures in laboratory setups, a simple UV-absorbance measurement (UV-AM) was developed and validated for screening a large number of setups, requiring almost no preparation and significantly less time and money compared to LC/GC methods. This rapid and easy to use method was evaluated by comparing its measured values to LC-UV and GC-MS/MS results. Furthermore, its application for monitoring and screening unknown activated sludge communities (ASC) and mixed pure cultures has been tested and approved to detect biodegradation of benzotriazole (BTri), 4- and 5-tolyltriazole (4-TTri, 5-TTri) as well as SMX. Conclusions In laboratory setups, xenobiotics concentrations above 1.0 mg L-1 without any enrichment or preparation could be detected after optimization of the method. As UV-AM does not require much preparatory work and can be conducted in 96 or even 384 well plate formats, the number of possible parallel setups and screening efficiency was significantly increased while analytic and laboratory costs were reduced to a minimum. PMID:24558966

Pharmacokinetic Evaluation of Empagliflozin in Healthy Egyptian Volunteers Using LC-MS/MS and Comparison with Other Ethnic Populations.

PubMed

Ayoub, Bassam M; Mowaka, Shereen; Elzanfaly, Eman S; Ashoush, Nermeen; Elmazar, Mohamed M; Mousa, Shaker A

2017-05-31

The present study considered the pharmacokinetic evaluation of empagliflozin after administration to Egyptian volunteers, and the results were compared with other ethnic populations. The FDA recognizes that standard methods of defining racial subgroups are necessary to compare results across pharmacokinetic studies and to assess potential subgroup differences. The design of the study was as an open labeled, randomized, one treatment, one period, single dose pharmacokinetic study. The main pharmacokinetic parameters estimated were C max , T max , t 1/2 , elimination rate constant, AUC 0-t and AUC 0-inf . The insignificant difference in pharmacokinetic parameters between Egyptians and white German subjects suggests that no dose adjustment should be considered with administration of 25 mg empagliflozin to Egyptian population. A new LC-MS/MS method was developed and validated, allowing sensitive estimation of empagliflozin (25-600 ng mL -1 ) in human plasma using dapagliflozin as an internal standard (IS). The method was applied successfully on the underlying pharmacokinetic study with enhanced sample preparation that involved liquid-liquid extraction. Multiple Reaction Monitoring (MRM) of the transition pairs of m/z 449.01 to 371.21 for empagliflozin and m/z 407.00 to 328.81 for dapagliflozin (IS) was employed utilizing negative mode Electro Spray Ionization (ESI). The validated LC-MS/MS method is suitable for further toxicodynamic and bioequivalence studies.

The role of orexin type-1 receptors in the development of morphine tolerance in locus coeruleus neurons: An electrophysiological perspective.

PubMed

Abdollahi, Hakime; Ghaemi-Jandabi, Masoumeh; Azizi, Hossein; Semnanian, Saeed

2016-09-01

Long-term exposure to opioid agonists results in tolerance to their analgesic effects, so the effectiveness of opioid agonists in the management of pain becomes limited. The locus coeruleus (LC) nucleus has been involved in the development of tolerance to opiates. Orexin type-1 receptors (OX1Rs) are highly expressed in LC nucleus. Orexin plays a noteworthy role in the occurrence of morphine tolerance. The purpose of the present study is to investigate the role of orexin type-1 receptors in the development of morphine tolerance in LC neurons. In this study, adult male Wistar rats weighing 250-300g were utilized. Induction of morphine tolerance was obtained by single injection of morphine per day for 6 successive days. An orexin type-1 receptor antagonist (SB-334867) was injected into the lateral ventricle instantly prior to morphine injection. On day 7, the effect of morphine on the electrical activity of LC neurons was studied using in vivo extracellular single unit recording. The results demonstrate that morphine injection for 6 consecutive days led to the development of morphine-induced tolerance in LC neurons. In other words, there was a significant decrease in LC neuronal responsiveness to morphine injection. Inhibitory responses of LC neurons to intraperitoneally applied morphine can be observed with the treatment of the SB-334867 prior to morphine injection. This study showed that OX1R blockade by SB-334867 prevents the development of morphine tolerance in LC neurons. We hope that further studies will lead to considerable progress in understanding the molecular adaptations that contribute to morphine tolerance. Copyright © 2016 Elsevier B.V. All rights reserved.

Correlation between Serum Levels of 3,3',5'-Triiodothyronine and Thyroid Hormones Measured by Liquid Chromatography-Tandem Mass Spectrometry and Immunoassay.

PubMed

Sakai, Hiroyuki; Nagao, Hidenori; Sakurai, Mamoru; Okumura, Takako; Nagai, Yoshiyuki; Shikuma, Junpei; Ito, Rokuro; Imazu, Tetsuya; Miwa, Takashi; Odawara, Masato

2015-01-01

For measuring serum 3,3',5'-triiodothyronine (rT3) levels, radioimmunoassay (RIA) has traditionally been used owing to the lack of other reliable methods; however, it has recently become difficult to perform. Meanwhile, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has recently been attracting attention as a novel alternative method in clinical chemistry. To the best of our knowledge, there are no studies to date comparing results of the quantification of human serum rT3 between LC-MS/MS and RIA. We therefore examined the feasibility of LC-MS/MS as a novel alternative method for measuring serum rT3, thyroxine (T4), and 3,5,3'-triiodothyronine (T3) levels. Assay validation was performed by LC-MS/MS using quality control samples of rT3, T4, and T3 at 4 various concentrations which were prepared from reference compounds. Serum samples of 50 outpatients in our department were quantified both by LC-MS/MS and conventional immunoassay for rT3, T4, and T3. Correlation coefficients between the 2 measurement methods were statistically analyzed respectively. Matrix effects were not observed with our method. Intra-day and inter-day precisions were less than 10.8% and 9.6% for each analyte at each quality control level, respectively. Intra-day and inter-day accuracies were between 96.2% and 110%, and between 98.3% and 108.6%, respectively. The lower limit of quantification was 0.05 ng/mL. Strong correlations were observed between the 2 measurement methods (correlation coefficient, T4: 0.976, p < 0.001; T3: 0.912, p < 0.001; rT3: 0.928, p < 0.001). Our LC-MS/MS system requires no manual cleanup operation, and the process after application of a sample is fully automated; furthermore, it was found to be highly sensitive, and superior in both precision and accuracy. The correlation between the 2 methods over a wide range of concentrations was strong. LC-MS/MS is therefore expected to become a useful tool for clinical diagnosis and research.

Comprehensive Optimization of LC-MS Metabolomics Methods Using Design of Experiments (COLMeD)

PubMed Central

Rhoades, Seth D.

2017-01-01

Introduction Both reverse-phase and HILIC chemistries are deployed for liquid-chromatography mass spectrometry (LC-MS) metabolomics analyses, however HILIC methods lag behind reverse-phase methods in reproducibility and versatility. Comprehensive metabolomics analysis is additionally complicated by the physiochemical diversity of metabolites and array of tunable analytical parameters. Objective Our aim was to rationally and efficiently design complementary HILIC-based polar metabolomics methods on multiple instruments using Design of Experiments (DoE). Methods We iteratively tuned LC and MS conditions on ion-switching triple quadrupole (QqQ) and quadrupole-time-of-flight (qTOF) mass spectrometers through multiple rounds of a workflow we term COLMeD (Comprehensive optimization of LC-MS metabolomics methods using design of experiments). Multivariate statistical analysis guided our decision process in the method optimizations. Results LC-MS/MS tuning for the QqQ method on serum metabolites yielded a median response increase of 161.5% (p<0.0001) over initial conditions with a 13.3% increase in metabolite coverage. The COLMeD output was benchmarked against two widely used polar metabolomics methods, demonstrating total ion current increases of 105.8% and 57.3%, with median metabolite response increases of 106.1% and 10.3% (p<0.0001 and p<0.05 respectively). For our optimized qTOF method, 22 solvent systems were compared on a standard mix of physiochemically diverse metabolites, followed by COLMeD optimization, yielding a median 29.8% response increase (p<0.0001) over initial conditions. Conclusions The COLMeD process elucidated response tradeoffs, facilitating improved chromatography and MS response without compromising separation of isobars. COLMeD is efficient, requiring no more than 20 injections in a given DoE round, and flexible, capable of class-specific optimization as demonstrated through acylcarnitine optimization within the QqQ method. PMID:28348510

Simple and robust diagnosis of early, small and AFP-negative primary hepatic carcinomas: an integrative approach of serum fluorescence and conventional blood tests.

PubMed

Wang, Ting; Zhang, Kun-He; Hu, Piao-Ping; Huang, Zeng-Yong; Zhang, Pan; Wan, Qin-Si; Huang, De-Qiang; Lv, Nong-Hua

2016-09-27

The diagnosis of early, small and alpha-fetoprotein (AFP)-negative primary hepatic carcinomas (PHCs) remains a significant challenge. We developed a simple and robust approach to noninvasively detect these PHCs. A rapid, high-throughput and single-tube method was firstly developed to measure serum autofluorescence and cell-free DNA (cfDNA)-related fluorescence using a real-time PCR system, and both types of serum fluorescence were measured and routine laboratory data were collected in 1229 subjects, including 353 PHC patients, 331 liver cirrhosis (LC) patients, 213 chronic hepatitis (CH) patients and 332 normal controls (NC). The results showed that fluorescence indicators of PHC differed from those of NC, CH and LC to various extents, and all of them were not associated with age, gender, or AFP level. The logistic regression models established with the fluorescence indicators alone and combined with AFP, hepatic function tests and blood cell analyses were valuable for distinguishing early, small, AFP-negative and all PHC from LC, CH, NC and all non-PHC, with areas under the receiver operating characteristic curves 0.857-0.993 and diagnostic accuracies 80.2-97.7%. Conclusively, serum autofluorescence and cfDNA-related fluorescence are able to be rapidly and simultaneously measured by our simple method and valuable for diagnosing early, small and AFP-negative PHCs, especially integrating with AFP and conventional blood tests.

Quantification of the IgG2/4 kappa Monoclonal Therapeutic Eculizumab from Serum Using Isotype Specific Affinity Purification and Microflow LC-ESI-Q-TOF Mass Spectrometry.

PubMed

Ladwig, Paula M; Barnidge, David R; Willrich, Maria A V

2017-05-01

As therapeutic monoclonal antibodies (mAbs) become more humanized, traditional tryptic peptide approaches used to measure biologics in serum become more challenging since unique clonotypic peptides used for quantifying the mAb may also be found in the normal serum polyclonal background. An alternative approach is to monitor the unique molecular mass of the intact light chain portion of the mAbs using liquid chromatography-mass spectrometry (LC-MS). Distinguishing a therapeutic mAb from a patient's normal polyclonal immunoglobulin (Ig) repertoire is the primary limiting factor when determining the limit of quantitation (LOQ) in serum. The ability to selectively extract subclass specific Igs from serum reduces the polyclonal background in a sample. We present here the development of an LC-MS method to quantify eculizumab in serum. Eculizumab is a complement component 5 (C5) binding mAb that is fully humanized and contains portions of both IgG2 and IgG4 subclasses. Our group developed a method that uses Life Technologies CaptureSelect IgG4 (Hu) affinity matrix. We show here the ability to quantitate eculizumab with a LOQ of 5 mcg/mL by removing the higher abundance IgG1, IgG2, and IgG3 from the polyclonal background, making this approach a simple and efficient procedure. Graphical Abstract ᅟ.

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.

Quantification of the IgG2/4 kappa Monoclonal Therapeutic Eculizumab from Serum Using Isotype Specific Affinity Purification and Microflow LC-ESI-Q-TOF Mass Spectrometry

NASA Astrophysics Data System (ADS)

Ladwig, Paula M.; Barnidge, David R.; Willrich, Maria A. V.

2017-05-01

As therapeutic monoclonal antibodies (mAbs) become more humanized, traditional tryptic peptide approaches used to measure biologics in serum become more challenging since unique clonotypic peptides used for quantifying the mAb may also be found in the normal serum polyclonal background. An alternative approach is to monitor the unique molecular mass of the intact light chain portion of the mAbs using liquid chromatography-mass spectrometry (LC-MS). Distinguishing a therapeutic mAb from a patient's normal polyclonal immunoglobulin (Ig) repertoire is the primary limiting factor when determining the limit of quantitation (LOQ) in serum. The ability to selectively extract subclass specific Igs from serum reduces the polyclonal background in a sample. We present here the development of an LC-MS method to quantify eculizumab in serum. Eculizumab is a complement component 5 (C5) binding mAb that is fully humanized and contains portions of both IgG2 and IgG4 subclasses. Our group developed a method that uses Life Technologies CaptureSelect IgG4 (Hu) affinity matrix. We show here the ability to quantitate eculizumab with a LOQ of 5 mcg/mL by removing the higher abundance IgG1, IgG2, and IgG3 from the polyclonal background, making this approach a simple and efficient procedure.

Evaluation of bilirubin interference and accuracy of six creatinine assays compared with isotope dilution-liquid chromatography mass spectrometry.

PubMed

Nah, Hyunjin; Lee, Sang-Guk; Lee, Kyeong-Seob; Won, Jae-Hee; Kim, Hyun Ok; Kim, Jeong-Ho

2016-02-01

The aim of this study was to estimate bilirubin interference and accuracy of six routine methods for measuring creatinine compared with isotope dilution-liquid chromatography mass spectrometry (ID-LC/MS). A total of 40 clinical serum samples from 31 patients with serum total bilirubin concentration >68.4μmol/L were collected. Serum creatinine was measured using two enzymatic reagents and four Jaffe reagents as well as ID-LC/MS. Correlations between bilirubin concentration and percent difference in creatinine compared with ID-LC/MS were analyzed to investigate bilirubin interference. Bias estimations between the six reagents and ID-LC/MS were performed. Recovery tests using National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 967a were also performed. Both the enzymatic methods showed no bilirubin interference. However, three of the four Jaffe methods demonstrated significant bilirubin concentration-dependent interference in samples with creatinine levels <53μmol/L, and two of them showed significant bilirubin interference in samples with creatinine levels ranging from 53.0 to 97.2μmol/L. Comparison of these methods with ID-LC/MS using patients' samples with elevated bilirubin revealed that the tested methods failed to achieve the bias goal at especially low levels of creatinine. In addition, recovery test using NIST SRM 967a showed that bias in one Jaffe method and two enzymatic methods did not achieve the bias goal at either low or high level of creatinine, indicating they had calibration bias. One enzymatic method failed to achieve all the bias goals in both comparison experiment and recovery test. It is important to understand that both bilirubin interference and calibration traceability to ID-LC/MS should be considered to improve the accuracy of creatinine measurement. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Microwave assisted solvent extraction and coupled-column reversed-phase liquid chromatography with UV detection use of an analytical restricted-access-medium column for the efficient multi-residue analysis of acidic pesticides in soils.

PubMed

Hogendoom, E A; Huls, R; Dijkman, E; Hoogerbrugge, R

2001-12-14

A screening method has been developed for the determination of acidic pesticides in various types of soils. Methodology is based on the use of microwave assisted solvent extraction (MASE) for fast and efficient extraction of the analytes from the soils and coupled-column reversed-phase liquid chromatography (LC-LC) with UV detection at 228 nm for the instrumental analysis of uncleaned extracts. Four types of soils, including sand, clay and peat, with a range in organic matter content of 0.3-13% and ten acidic pesticides of different chemical families (bentazone, bromoxynil, metsulfuron-methyl, 2,4-D, MCPA, MCPP, 2,4-DP, 2,4,5-T, 2,4-DB and MCPB) were selected as matrices and analytes, respectively. The method developed included the selection of suitable MASE and LC-LC conditions. The latter consisted of the selection of a 5-microm GFF-II internal surface reversed-phase (ISRP, Pinkerton) analytical column (50 x 4.6 mm, I.D.) as the first column in the RAM-C18 configuration in combination with an optimised linear gradient elution including on-line cleanup of sample extracts and reconditioning of the columns. The method was validated with the analysis of freshly spiked samples and samples with aged residues (120 days). The four types of soils were spiked with the ten acidic pesticides at levels between 20 and 200 microg/kg. Weighted regression of the recovery data showed for most analyte-matrix combinations, including freshly spiked samples and aged residues, that the method provides overall recoveries between 60 and 90% with relative standard deviations of the intra-laboratory reproducibility's between 5 and 25%; LODs were obtained between 5 and 50 microg/kg. Evaluation of the data set with principal component analysis revealed that the parameters (i) increase of organic matter content of the soil samples and (ii) aged residues negatively effect the recovery of the analytes.

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.

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/...

Single Laboratory Validated Method for Determination of Cylindrospermopsin and Anatoxin-a in Ambient Freshwaters by Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS)

EPA Pesticide Factsheets

This document is a standardized single laboratory validated liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the detection and quantification of cyanotoxins (combined intracellular and extracellular) in ambient freshwaters.

Amplification of interference color by using liquid crystal for protein detection

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

Zhu, Qingdi; Yang, Kun-Lin, E-mail: cheyk@nus.edu.sg

Micrometer-sized, periodic protein lines printed on a solid surface cause interference color which is invisible to the naked eye. However, the interference color can be amplified by using a thin layer of liquid crystal (LC) covered on the surface to form a phase diffraction grating. Strong interference color can thus be observed under ambient light. By using the LC-amplified interference color, we demonstrate naked-eye detection of a model protein—immunoglobulin G (IgG). Limit of detection can reach 20 μg/ml of IgG without using any instrumentation. This detection method is potentially useful for the development of low-cost and portable biosensors.

Single Lab Validation of a LC/UV/FLD/MS Method for Simultaneous Determination of Water-soluble Vitamins in Multi-Vitamin Dietary Supplements

USDA-ARS?s Scientific Manuscript database

The purpose of this study was to develop a Single-Lab Validated Method using high-performance liquid chromatography (HPLC) with different detectors (diode array detector - DAD, fluorescence detector - FLD, and mass spectrometer - MS) for determination of seven B-complex vitamins (B1 - thiamin, B2 – ...

Advantages of tandem LC-MS for the rapid assessment of tissue-specific metabolic complexity using a pentafluorophenylpropyl stationary phase.

PubMed

Lv, Haitao; Palacios, Gustavo; Hartil, Kirsten; Kurland, Irwin J

2011-04-01

In this study, a tandem LC-MS (Waters Xevo TQ) MRM-based MS method was developed for rapid, broad profiling of hydrophilic metabolites from biological samples, in either positive or negative ion modes without the need for an ion pairing reagent, using a reversed-phase pentafluorophenylpropyl (PFPP) column. The developed method was successfully applied to analyze various biological samples from C57BL/6 mice, including urine, duodenum, liver, plasma, kidney, heart, and skeletal muscle. As result, a total 112 of hydrophilic metabolites were detected within 8 min of running time to obtain a metabolite profile of the biological samples. The analysis of this number of hydrophilic metabolites is significantly faster than previous studies. Classification separation for metabolites from different tissues was globally analyzed by PCA, PLS-DA and HCA biostatistical methods. Overall, most of the hydrophilic metabolites were found to have a "fingerprint" characteristic of tissue dependency. In general, a higher level of most metabolites was found in urine, duodenum, and kidney. Altogether, these results suggest that this method has potential application for targeted metabolomic analyzes of hydrophilic metabolites in a wide ranges of biological samples.

Qualitative identification of permitted and non-permitted colour additives in food products.

PubMed

Harp, Bhakti Petigara; Miranda-Bermudez, Enio; Baron, Carolina I; Richard, Gerald I

2012-01-01

Colour additives are dyes, pigments or other substances that can impart colour when added or applied to food, drugs, cosmetics, medical devices, or the human body. The substances must be pre-approved by the US Food and Drug Administration (USFDA) and listed in Title 21 of the US Code of Federal Regulations before they may be used in products marketed in the United States. Some also are required to be batch certified by the USFDA prior to their use. Both domestic and imported products sold in interstate commerce fall under USFDA jurisdiction, and the USFDA's district laboratories use a combination of analytical methods for identifying or confirming the presence of potentially violative colour additives. We have developed a qualitative method for identifying 17 certifiable, certification exempt, and non-permitted colour additives in various food products. The method involves extracting the colour additives from a product and isolating them from non-coloured components with a C(18) Sep-Pak cartridge. The colour additives are then separated and identified by liquid chromatography (LC) with photodiode array detection, using an Xterra RP18 column and gradient elution with aqueous ammonium acetate and methanol. Limits of detection (LODs) ranged from 0.02 to 1.49 mg/l. This qualititative LC method supplements the visible spectrophotometric and thin-layer chromatography methods currently used by the USFDA's district laboratories and is less time-consuming and requires less solvent compared to the other methods. The extraction step in the new LC method is a simple and an efficient process that can be used for most food types.

Performance Evaluation and Online Realization of Data-driven Normalization Methods Used in LC/MS based Untargeted Metabolomics Analysis.

PubMed

Li, Bo; Tang, Jing; Yang, Qingxia; Cui, Xuejiao; Li, Shuang; Chen, Sijie; Cao, Quanxing; Xue, Weiwei; Chen, Na; Zhu, Feng

2016-12-13

In untargeted metabolomics analysis, several factors (e.g., unwanted experimental &biological variations and technical errors) may hamper the identification of differential metabolic features, which requires the data-driven normalization approaches before feature selection. So far, ≥16 normalization methods have been widely applied for processing the LC/MS based metabolomics data. However, the performance and the sample size dependence of those methods have not yet been exhaustively compared and no online tool for comparatively and comprehensively evaluating the performance of all 16 normalization methods has been provided. In this study, a comprehensive comparison on these methods was conducted. As a result, 16 methods were categorized into three groups based on their normalization performances across various sample sizes. The VSN, the Log Transformation and the PQN were identified as methods of the best normalization performance, while the Contrast consistently underperformed across all sub-datasets of different benchmark data. Moreover, an interactive web tool comprehensively evaluating the performance of 16 methods specifically for normalizing LC/MS based metabolomics data was constructed and hosted at http://server.idrb.cqu.edu.cn/MetaPre/. In summary, this study could serve as a useful guidance to the selection of suitable normalization methods in analyzing the LC/MS based metabolomics data.

Performance Evaluation and Online Realization of Data-driven Normalization Methods Used in LC/MS based Untargeted Metabolomics Analysis

PubMed Central

Li, Bo; Tang, Jing; Yang, Qingxia; Cui, Xuejiao; Li, Shuang; Chen, Sijie; Cao, Quanxing; Xue, Weiwei; Chen, Na; Zhu, Feng

2016-01-01

In untargeted metabolomics analysis, several factors (e.g., unwanted experimental & biological variations and technical errors) may hamper the identification of differential metabolic features, which requires the data-driven normalization approaches before feature selection. So far, ≥16 normalization methods have been widely applied for processing the LC/MS based metabolomics data. However, the performance and the sample size dependence of those methods have not yet been exhaustively compared and no online tool for comparatively and comprehensively evaluating the performance of all 16 normalization methods has been provided. In this study, a comprehensive comparison on these methods was conducted. As a result, 16 methods were categorized into three groups based on their normalization performances across various sample sizes. The VSN, the Log Transformation and the PQN were identified as methods of the best normalization performance, while the Contrast consistently underperformed across all sub-datasets of different benchmark data. Moreover, an interactive web tool comprehensively evaluating the performance of 16 methods specifically for normalizing LC/MS based metabolomics data was constructed and hosted at http://server.idrb.cqu.edu.cn/MetaPre/. In summary, this study could serve as a useful guidance to the selection of suitable normalization methods in analyzing the LC/MS based metabolomics data. PMID:27958387

A method for the determination of acrylamide in bakery products using ion trap LC-ESI-MS/MS.

PubMed

Claus, Achim; Weisz, Georg M; Kammerer, Dietmar R; Carle, Reinhold; Schieber, Andreas

2005-10-01

Acrylamide levels of bakery products, e. g., bread and bread rolls, are usually below 100 microg/kg , often even below 50 microg/kg. Therefore, usual analytical methods which have an LOQ greater, not dbl equals 25 microg/kg are not sensitive enough for detailed investigations on acrylamide formation within these commodities. An improved method for trace level determination of acrylamide in bakery products was developed using ion trap LC-ESI-MS/MS. Samples were divided into crumbs and crusts to achieve an initial concentration by removing the crumbs since these are devoid of acrylamide. After sample extraction and clean-up using multimode SPE cartridges, further analyte enrichment was accomplished by solid-phase-supported liquid-liquid extraction with ethyl acetate prior to LC-MS/MS analysis. The method was evaluated using bread, bread rolls, alkali-baked bread rolls, and toast. LOQ was calculated from the confidence interval of the calibration curve and found to be 1.7 ng/mL, corresponding to 17 microg/kg of product. When crumbs and crusts were separated, an LOQ of 10.2 microg/kg of bakery product could be obtained. As demonstrated in preliminary comparative analyses, accuracy of the method met the requirements for determination of trace level acrylamide formation in bakery products. Mean recovery was 102.4% (CV 4.5%), intermediate reproducibility revealed a CV of 2.1%, and a repeatability of CV 6.0%.

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.

Tissue proteomics of the low-molecular weight proteome using an integrated cLC-ESI-QTOFMS approach.

PubMed

Alvarez, MeiHwa Tanielle Bench; Shah, Dipti Jigar; Thulin, Craig D; Graves, Steven W

2013-05-01

Analysis of the protein/peptide composition of tissue has provided meaningful insights into tissue biology and even disease mechanisms. However, little has been published regarding top down methods to investigate lower molecular weight (MW) (500-5000 Da) species in tissue. Here, we evaluate a tissue proteomics approach involving tissue homogenization followed by depletion of large proteins and then cLC-MS (where c stands for capillary) analysis to interrogate the low MW/low abundance tissue proteome. In the development of this method, sheep heart, lung, liver, kidney, and spleen were surveyed to test our ability to observe tissue differences. After categorical tissue differences were demonstrated, a detailed study of this method's reproducibility was undertaken to determine whether or not it is suitable for analyzing more subtle differences in the abundance of small proteins and peptides. Our results suggest that this method should be useful in exploring the low MW proteome of tissues. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Comprehensive Optimization of LC-MS Metabolomics Methods Using Design of Experiments (COLMeD).

PubMed

Rhoades, Seth D; Weljie, Aalim M

2016-12-01

Both reverse-phase and HILIC chemistries are deployed for liquid-chromatography mass spectrometry (LC-MS) metabolomics analyses, however HILIC methods lag behind reverse-phase methods in reproducibility and versatility. Comprehensive metabolomics analysis is additionally complicated by the physiochemical diversity of metabolites and array of tunable analytical parameters. Our aim was to rationally and efficiently design complementary HILIC-based polar metabolomics methods on multiple instruments using Design of Experiments (DoE). We iteratively tuned LC and MS conditions on ion-switching triple quadrupole (QqQ) and quadrupole-time-of-flight (qTOF) mass spectrometers through multiple rounds of a workflow we term COLMeD (Comprehensive optimization of LC-MS metabolomics methods using design of experiments). Multivariate statistical analysis guided our decision process in the method optimizations. LC-MS/MS tuning for the QqQ method on serum metabolites yielded a median response increase of 161.5% (p<0.0001) over initial conditions with a 13.3% increase in metabolite coverage. The COLMeD output was benchmarked against two widely used polar metabolomics methods, demonstrating total ion current increases of 105.8% and 57.3%, with median metabolite response increases of 106.1% and 10.3% (p<0.0001 and p<0.05 respectively). For our optimized qTOF method, 22 solvent systems were compared on a standard mix of physiochemically diverse metabolites, followed by COLMeD optimization, yielding a median 29.8% response increase (p<0.0001) over initial conditions. The COLMeD process elucidated response tradeoffs, facilitating improved chromatography and MS response without compromising separation of isobars. COLMeD is efficient, requiring no more than 20 injections in a given DoE round, and flexible, capable of class-specific optimization as demonstrated through acylcarnitine optimization within the QqQ method.

Improved sensitivity of ochratoxin A analysis in coffee using high-performance liquid chromatography with hybrid triple quadrupole-linear ion trap mass spectrometry (LC-QqQLIT-MS/MS).

PubMed

Kokina, Aija; Pugajeva, Iveta; Bartkevics, Vadims

2016-01-01

A novel and sensitive method utilising high-performance liquid chromatography coupled to triple quadrupole-linear ion trap mass spectrometry (LC-QqQLIT-MS/MS) was developed in order to analyse the content of ochratoxin A (OTA) in coffee samples. The introduction of the triple-stage MS scanning mode (MS(3)) has been shown to increase greatly sensitivity and selectivity by eliminating the high chromatographic baseline caused by interference of complex coffee matrices. The analysis included the sample preparation procedure involving extraction of OTA using a methanol-water mixture and clean-up by immunoaffinity columns and detection using the MS(3) scanning mode of LC-QqQLIT-MS/MS. The proposed method offered a good linear correlation (r(2) > 0.998), excellent precision (RSD < 2.9%) and recovery (94%). The limit of quantification (LOQ) for coffee beans and espresso beverages was 0.010 and 0.003 µg kg(-1), respectively. The developed procedure was compared with traditional methods employing liquid chromatography coupled to fluorescent and tandem quadrupole detectors in conjunction with QuEChERS and solid-phase extraction. The proposed method was successfully applied to the determination of OTA in 15 samples of coffee beans and in 15 samples of espresso coffee beverages obtained from the Latvian market. OTA was found in 10 samples of coffee beans and in two samples of espresso in the ranges of 0.018-1.80 µg kg(-1) and 0.020-0.440 µg l(-1), respectively. No samples exceeded the maximum permitted level of OTA in the European Union (5.0 µg kg(-1)).

Separation of Cis-Trans Phospholipid Isomers using Reversed Phase LC with High Resolution MS Detection

PubMed Central

Bird, Susan S.; Marur, Vasant R.; Stavrovskaya, Irina G.; Kristal, Bruce S.

2012-01-01

The increased presence of synthetic trans fatty acids into western diets has been shown to have deleterious effects on physiology and raising an individual’s risk of developing metabolic disease, cardiovascular disease, and stroke. The importance of these fatty acids for health and the diversity of their (patho) physiological effects suggest that not only should the free trans fatty acids be studied, but that monitoring the presence of these fats into the side-chains of biological lipids, such as glycerophospholipids, is also essential. We developed a high resolution LC-MS method that quantitatively monitors the major lipid classes found in biospecimens in an efficient, sensitive and robust manner while also characterizing individual lipid side-chains through the use of HCD fragmentation and chromatographic alignment. We herein show how this previously described reversed phase method can baseline separate the cis-trans isomers of phosphatidylglycerol and phosphatidylcholine (PC) with two 18:1 side chains, in both positive and negative mode, as neat solutions and when spiked into a biological matrix. Endogenous PC (18:1/18:1) cis and PC (18:1/18:1) trans isomers were examined in mitochondrial and serum profiling studies, where rats were fed diets enriched in either trans 18:1 fatty acids, or cis 18:1 fatty acids. In this study, we determined the cis:trans isomer ratios of PC (18:1/18:1) and related this ratio to dietary composition. This generalized LC-MS method enables the monitoring of trans fats in biological lipids in the context of a non-targeted method, allowing for relative quantitation and enhanced identification of unknown lipids in complex matrices. PMID:22656324

Simultaneous determination of five plant growth regulators in fruits by modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction and liquid chromatography-tandem mass spectrometry.

PubMed

Shi, Xiaomei; Jin, Fen; Huang, Yuting; Du, Xinwei; Li, Chunmei; Wang, Miao; Shao, Hua; Jin, Maojun; Wang, Jing

2012-01-11

An effective method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and optimized to obtain a complete separation of five representative plant growth regulators (PGRs) [gibberellic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), thidiazuron, forchlorfenuron, and paclobutrazol] in fruits. Extraction was performed with acetonitrile containing 0.1% (v/v) acetic acid, applying modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) methodology. LC-MS/MS conditions including composition of mobile phases and mass spectrometry (MS) conditions were evaluated to achieve the highest sensitivity in MS detection. All of the data acquisition was employed in the segmented multiple-reaction monitoring mode for the selected negative and positive transition ions. The octadecylsilyl (C18) dispersive solid-phase extraction (SPE) sorbent was found to provide the more satisfied recoveries than primary secondary amine (PSA) and graphitized carbon black (GCB) for five target PGRs. The optimized method allowed for recoveries of 76-112% for the five PGRs from fruit samples with relative standard deviation (RSD) values less than 10%. Limits of quantification (0.5-16.5 μg/kg) were lower than the maximum limit of residues established for PGRs. The results demonstrated that the developed LC-MS/MS and QuEChERS extraction method is highly effective for analyzing trace amounts of target PGRs in fruit samples. Finally, the method was successfully used to detect residual PGRs in Beijing, China, in 2010. The concentrations of 2,4-D (5.1-1503 μg/kg) and paclobutrazol (1-1381 μg/kg) found in orange and peach, respectively, suggesting that the use of these PGRs in these fruits should be regulated in China in the future.