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Sample records for quantitative mass spectrometry

  1. Quantitative mass spectrometry: an overview

    NASA Astrophysics Data System (ADS)

    Urban, Pawel L.

    2016-10-01

    Mass spectrometry (MS) is a mainstream chemical analysis technique in the twenty-first century. It has contributed to numerous discoveries in chemistry, physics and biochemistry. Hundreds of research laboratories scattered all over the world use MS every day to investigate fundamental phenomena on the molecular level. MS is also widely used by industry-especially in drug discovery, quality control and food safety protocols. In some cases, mass spectrometers are indispensable and irreplaceable by any other metrological tools. The uniqueness of MS is due to the fact that it enables direct identification of molecules based on the mass-to-charge ratios as well as fragmentation patterns. Thus, for several decades now, MS has been used in qualitative chemical analysis. To address the pressing need for quantitative molecular measurements, a number of laboratories focused on technological and methodological improvements that could render MS a fully quantitative metrological platform. In this theme issue, the experts working for some of those laboratories share their knowledge and enthusiasm about quantitative MS. I hope this theme issue will benefit readers, and foster fundamental and applied research based on quantitative MS measurements. This article is part of the themed issue 'Quantitative mass spectrometry'.

  2. Quantitative mass spectrometry: an overview

    PubMed Central

    2016-01-01

    Mass spectrometry (MS) is a mainstream chemical analysis technique in the twenty-first century. It has contributed to numerous discoveries in chemistry, physics and biochemistry. Hundreds of research laboratories scattered all over the world use MS every day to investigate fundamental phenomena on the molecular level. MS is also widely used by industry—especially in drug discovery, quality control and food safety protocols. In some cases, mass spectrometers are indispensable and irreplaceable by any other metrological tools. The uniqueness of MS is due to the fact that it enables direct identification of molecules based on the mass-to-charge ratios as well as fragmentation patterns. Thus, for several decades now, MS has been used in qualitative chemical analysis. To address the pressing need for quantitative molecular measurements, a number of laboratories focused on technological and methodological improvements that could render MS a fully quantitative metrological platform. In this theme issue, the experts working for some of those laboratories share their knowledge and enthusiasm about quantitative MS. I hope this theme issue will benefit readers, and foster fundamental and applied research based on quantitative MS measurements. This article is part of the themed issue ‘Quantitative mass spectrometry’. PMID:27644965

  3. Targeted Quantitation of Proteins by Mass Spectrometry

    PubMed Central

    2013-01-01

    Quantitative measurement of proteins is one of the most fundamental analytical tasks in a biochemistry laboratory, but widely used immunochemical methods often have limited specificity and high measurement variation. In this review, we discuss applications of multiple-reaction monitoring (MRM) mass spectrometry, which allows sensitive, precise quantitative analyses of peptides and the proteins from which they are derived. Systematic development of MRM assays is permitted by databases of peptide mass spectra and sequences, software tools for analysis design and data analysis, and rapid evolution of tandem mass spectrometer technology. Key advantages of MRM assays are the ability to target specific peptide sequences, including variants and modified forms, and the capacity for multiplexing that allows analysis of dozens to hundreds of peptides. Different quantitative standardization methods provide options that balance precision, sensitivity, and assay cost. Targeted protein quantitation by MRM and related mass spectrometry methods can advance biochemistry by transforming approaches to protein measurement. PMID:23517332

  4. Quantitative interaction proteomics using mass spectrometry.

    PubMed

    Wepf, Alexander; Glatter, Timo; Schmidt, Alexander; Aebersold, Ruedi; Gstaiger, Matthias

    2009-03-01

    We present a mass spectrometry-based strategy for the absolute quantification of protein complex components isolated through affinity purification. We quantified bait proteins via isotope-labeled reference peptides corresponding to an affinity tag sequence and prey proteins by label-free correlational quantification using the precursor ion signal intensities of proteotypic peptides generated in reciprocal purifications. We used this method to quantitatively analyze interaction stoichiometries in the human protein phosphatase 2A network.

  5. Protein Quantitation of the Developing Cochlea Using Mass Spectrometry.

    PubMed

    Darville, Lancia N F; Sokolowski, Bernd H A

    2016-01-01

    Mass spectrometry-based proteomics allows for the measurement of hundreds to thousands of proteins in a biological system. Additionally, mass spectrometry can also be used to quantify proteins and peptides. However, observing quantitative differences between biological systems using mass spectrometry-based proteomics can be challenging because it is critical to have a method that is fast, reproducible, and accurate. Therefore, to study differential protein expression in biological samples labeling or label-free quantitative methods can be used. Labeling methods have been widely used in quantitative proteomics, however label-free methods have become equally as popular and more preferred because they produce faster, cleaner, and simpler results. Here, we describe the methods by which proteins are isolated and identified from cochlear sensory epithelia tissues at different ages and quantitatively differentiated using label-free mass spectrometry.

  6. Liquid Chromatography-Mass Spectrometry-based Quantitative Proteomics

    SciTech Connect

    Xie, Fang; Liu, Tao; Qian, Weijun; Petyuk, Vladislav A.; Smith, Richard D.

    2011-07-22

    Liquid chromatography-mass spectrometry (LC-MS)-based quantitative proteomics has become increasingly applied for a broad range of biological applications due to growing capabilities for broad proteome coverage and good accuracy in quantification. Herein, we review the current LC-MS-based quantification methods with respect to their advantages and limitations, and highlight their potential applications.

  7. Quantitative aspects of inductively coupled plasma mass spectrometry

    NASA Astrophysics Data System (ADS)

    Bulska, Ewa; Wagner, Barbara

    2016-10-01

    Accurate determination of elements in various kinds of samples is essential for many areas, including environmental science, medicine, as well as industry. Inductively coupled plasma mass spectrometry (ICP-MS) is a powerful tool enabling multi-elemental analysis of numerous matrices with high sensitivity and good precision. Various calibration approaches can be used to perform accurate quantitative measurements by ICP-MS. They include the use of pure standards, matrix-matched standards, or relevant certified reference materials, assuring traceability of the reported results. This review critically evaluates the advantages and limitations of different calibration approaches, which are used in quantitative analyses by ICP-MS. Examples of such analyses are provided. This article is part of the themed issue 'Quantitative mass spectrometry'.

  8. Quantitative aspects of inductively coupled plasma mass spectrometry.

    PubMed

    Bulska, Ewa; Wagner, Barbara

    2016-10-28

    Accurate determination of elements in various kinds of samples is essential for many areas, including environmental science, medicine, as well as industry. Inductively coupled plasma mass spectrometry (ICP-MS) is a powerful tool enabling multi-elemental analysis of numerous matrices with high sensitivity and good precision. Various calibration approaches can be used to perform accurate quantitative measurements by ICP-MS. They include the use of pure standards, matrix-matched standards, or relevant certified reference materials, assuring traceability of the reported results. This review critically evaluates the advantages and limitations of different calibration approaches, which are used in quantitative analyses by ICP-MS. Examples of such analyses are provided.This article is part of the themed issue 'Quantitative mass spectrometry'.

  9. Statistical design of quantitative mass spectrometry-based proteomic experiments.

    PubMed

    Oberg, Ann L; Vitek, Olga

    2009-05-01

    We review the fundamental principles of statistical experimental design, and their application to quantitative mass spectrometry-based proteomics. We focus on class comparison using Analysis of Variance (ANOVA), and discuss how randomization, replication and blocking help avoid systematic biases due to the experimental procedure, and help optimize our ability to detect true quantitative changes between groups. We also discuss the issues of pooling multiple biological specimens for a single mass analysis, and calculation of the number of replicates in a future study. When applicable, we emphasize the parallels between designing quantitative proteomic experiments and experiments with gene expression microarrays, and give examples from that area of research. We illustrate the discussion using theoretical considerations, and using real-data examples of profiling of disease.

  10. Quantitative mass spectrometry of unconventional human biological matrices

    NASA Astrophysics Data System (ADS)

    Dutkiewicz, Ewelina P.; Urban, Pawel L.

    2016-10-01

    The development of sensitive and versatile mass spectrometric methodology has fuelled interest in the analysis of metabolites and drugs in unconventional biological specimens. Here, we discuss the analysis of eight human matrices-hair, nail, breath, saliva, tears, meibum, nasal mucus and skin excretions (including sweat)-by mass spectrometry (MS). The use of such specimens brings a number of advantages, the most important being non-invasive sampling, the limited risk of adulteration and the ability to obtain information that complements blood and urine tests. The most often studied matrices are hair, breath and saliva. This review primarily focuses on endogenous (e.g. potential biomarkers, hormones) and exogenous (e.g. drugs, environmental contaminants) small molecules. The majority of analytical methods used chromatographic separation prior to MS; however, such a hyphenated methodology greatly limits analytical throughput. On the other hand, the mass spectrometric methods that exclude chromatographic separation are fast but suffer from matrix interferences. To enable development of quantitative assays for unconventional matrices, it is desirable to standardize the protocols for the analysis of each specimen and create appropriate certified reference materials. Overcoming these challenges will make analysis of unconventional human biological matrices more common in a clinical setting. This article is part of the themed issue 'Quantitative mass spectrometry'.

  11. Attomole quantitation of protein separations with accelerator mass spectrometry

    SciTech Connect

    Vogel, J S; Grant, P G; Buccholz, B A; Dingley, K; Turteltaub, K W

    2000-12-15

    Quantification of specific proteins depends on separation by chromatography or electrophoresis followed by chemical detection schemes such as staining and fluorophore adhesion. Chemical exchange of short-lived isotopes, particularly sulfur, is also prevalent despite the inconveniences of counting radioactivity. Physical methods based on isotopic and elemental analyses offer highly sensitive protein quantitation that has linear response over wide dynamic ranges and is independent of protein conformation. Accelerator mass spectrometry quantifies long-lived isotopes such as 14C to sub-attomole sensitivity. We quantified protein interactions with small molecules such as toxins, vitamins, and natural biochemicals at precisions of 1-5% . Micro-proton-induced-xray-emission quantifies elemental abundances in separated metalloprotein samples to nanogram amounts and is capable of quantifying phosphorylated loci in gels. Accelerator-based quantitation is a possible tool for quantifying the genome translation into proteome.

  12. Neuropeptidomics: Mass Spectrometry-Based Identification and Quantitation of Neuropeptides

    PubMed Central

    2016-01-01

    Neuropeptides produced from prohormones by selective action of endopeptidases are vital signaling molecules, playing a critical role in a variety of physiological processes, such as addiction, depression, pain, and circadian rhythms. Neuropeptides bind to post-synaptic receptors and elicit cellular effects like classical neurotransmitters. While each neuropeptide could have its own biological function, mass spectrometry (MS) allows for the identification of the precise molecular forms of each peptide without a priori knowledge of the peptide identity and for the quantitation of neuropeptides in different conditions of the samples. MS-based neuropeptidomics approaches have been applied to various animal models and conditions to characterize and quantify novel neuropeptides, as well as known neuropeptides, advancing our understanding of nervous system function over the past decade. Here, we will present an overview of neuropeptides and MS-based neuropeptidomic strategies for the identification and quantitation of neuropeptides. PMID:27103886

  13. Issues and Applications in Label-Free Quantitative Mass Spectrometry

    PubMed Central

    Lai, Xianyin; Wang, Lianshui; Witzmann, Frank A.

    2013-01-01

    To address the challenges associated with differential expression proteomics, label-free mass spectrometric protein quantification methods have been developed as alternatives to array-based, gel-based, and stable isotope tag or label-based approaches. In this paper, we focus on the issues associated with label-free methods that rely on quantitation based on peptide ion peak area measurement. These issues include chromatographic alignment, peptide qualification for quantitation, and normalization. In addressing these issues, we present various approaches, assembled in a recently developed label-free quantitative mass spectrometry platform, that overcome these difficulties and enable comprehensive, accurate, and reproducible protein quantitation in highly complex protein mixtures from experiments with many sample groups. As examples of the utility of this approach, we present a variety of cases where the platform was applied successfully to assess differential protein expression or abundance in body fluids, in vitro nanotoxicology models, tissue proteomics in genetic knock-in mice, and cell membrane proteomics. PMID:23401775

  14. Quantitation of DNA adducts by stable isotope dilution mass spectrometry

    PubMed Central

    Tretyakova, Natalia; Goggin, Melissa; Janis, Gregory

    2012-01-01

    Exposure to endogenous and exogenous chemicals can lead to the formation of structurally modified DNA bases (DNA adducts). If not repaired, these nucleobase lesions can cause polymerase errors during DNA replication, leading to heritable mutations potentially contributing to the development of cancer. Due to their critical role in cancer initiation, DNA adducts represent mechanism-based biomarkers of carcinogen exposure, and their quantitation is particularly useful for cancer risk assessment. DNA adducts are also valuable in mechanistic studies linking tumorigenic effects of environmental and industrial carcinogens to specific electrophilic species generated from their metabolism. While multiple experimental methodologies have been developed for DNA adduct analysis in biological samples – including immunoassay, HPLC, and 32P-postlabeling – isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) generally has superior selectivity, sensitivity, accuracy, and reproducibility. As typical DNA adducts concentrations in biological samples are between 0.01 – 10 adducts per 108 normal nucleotides, ultrasensitive HPLC-ESI-MS/MS methodologies are required for their analysis. Recent developments in analytical separations and biological mass spectrometry – especially nanoflow HPLC, nanospray ionization MS, chip-MS, and high resolution MS – have pushed the limits of analytical HPLC-ESI-MS/MS methodologies for DNA adducts, allowing researchers to accurately measure their concentrations in biological samples from patients treated with DNA alkylating drugs and in populations exposed to carcinogens from urban air, drinking water, cooked food, alcohol, and cigarette smoke. PMID:22827593

  15. Accelerator mass spectrometry for quantitative in vivo tracing

    SciTech Connect

    Vogel, J S

    2005-04-19

    Accelerator mass spectrometry (AMS) counts individual rare, usually radio-, isotopes such as radiocarbon at high efficiency and specificity in milligram-sized samples. AMS traces very low chemical doses ({micro}g) and radiative doses (100 Bq) of isotope labeled compounds in animal models and directly in humans for pharmaceutical, nutritional, or toxicological research. Absorption, metabolism, distribution, binding, and elimination are all quantifiable with high precision after appropriate sample definition.

  16. [Identification and quantitative determination of baclofen in human blood by HPLC with mass spectrometry detection].

    PubMed

    Dukova, O A; Kotlovsky, M Yu; Pokrovsky, A A; Suvorova, E V; Shivrina, T G; Krasnov, E A; Efremov, A A

    2016-03-01

    A method of identification and quantitative determination of baclofen in blood by HPLC with mass spectrometry detection has been developed. It is characterized by high sensitivity, specificity, linearity, accuracy, reproducibility, and a low detection for quantitative determination. The method has been used for diagnostics of acute baclofen poisoning in patients.

  17. Assessing the Phagosome Proteome by Quantitative Mass Spectrometry.

    PubMed

    Peltier, Julien; Härtlova, Anetta; Trost, Matthias

    2017-01-01

    Phagocytosis is the process that engulfs particles in vesicles called phagosomes that are trafficked through a series of maturation steps, culminating in the destruction of the internalized cargo. Because phagosomes are in direct contact with the particle and undergo constant fusion and fission events with other organelles, characterization of the phagosomal proteome is a powerful tool to understand mechanisms controlling innate immunity as well as vesicle trafficking. The ability to isolate highly pure phagosomes through the use of latex beads led to an extensive use of proteomics to study phagosomes under different stimuli. Thousands of different proteins have been identified and quantified, revealing new properties and shedding new light on the dynamics and composition of maturing phagosomes and innate immunity mechanisms. In this chapter, we describe how quantitative-based proteomic methods such as label-free, dimethyl labeling or Tandem Mass Tag (TMT) labeling can be applied for the characterization of protein composition and translocation during maturation of phagosomes in macrophages.

  18. Mass spectrometry.

    NASA Technical Reports Server (NTRS)

    Burlingame, A. L.; Johanson, G. A.

    1972-01-01

    Review of the current state of mass spectrometry, indicating its unique importance for advanced scientific research. Mass spectrometry applications in computer techniques, gas chromatography, ion cyclotron resonance, molecular fragmentation and ionization, and isotope labeling are covered. Details are given on mass spectrometry applications in bio-organic chemistry and biomedical research. As the subjects of these applications are indicated alkaloids, carbohydrates, lipids, terpenes, quinones, nucleic acid components, peptides, antibiotics, and human and animal metabolisms. Particular attention is given to the mass spectra of organo-inorganic compounds, inorganic mass spectrometry, surface phenomena such as secondary ion and electron emission, and elemental and isotope analysis. Further topics include mass spectrometry in organic geochemistry, applications in geochronology and cosmochemistry, and organic mass spectrometry.

  19. Identification and Quantitative Measurements of Chemical Species by Mass Spectrometry

    NASA Technical Reports Server (NTRS)

    Zondlo, Mark A.; Bomse, David S.

    2005-01-01

    The development of a miniature gas chromatograph/mass spectrometer system for the measurement of chemical species of interest to combustion is described. The completed system is a fully-contained, automated instrument consisting of a sampling inlet, a small-scale gas chromatograph, a miniature, quadrupole mass spectrometer, vacuum pumps, and software. A pair of computer-driven valves controls the gas sampling and introduction to the chromatographic column. The column has a stainless steel exterior and a silica interior, and contains an adsorbent of that is used to separate organic species. The detection system is based on a quadrupole mass spectrometer consisting of a micropole array, electrometer, and a computer interface. The vacuum system has two miniature pumps to maintain the low pressure needed for the mass spectrometer. A laptop computer uses custom software to control the entire system and collect the data. In a laboratory demonstration, the system separated calibration mixtures containing 1000 ppm of alkanes and alkenes.

  20. Mass spectrometry

    SciTech Connect

    Burlingame, A.L.; Baillie, T.A.; Derrick, P.J.

    1986-04-01

    It is the intention of the review to bring together in one source the direction of major developments in mass spectrometry and to illustrate these by citing key contributions from both fundamental and applied research. The Review is intended to provide the reader with a sense of the main currents, their breadth and depth, and probable future directions. It is also intended to provide the reader with a glimpse of the diverse discoveries and results that underpin the eventual development of new methods and instruments - the keys to obtaining new insights in all the physical, chemical, and biological sciences which depend on mass spectrometry at various levels of sophistication. Focal points for future interdisciplinary synergism might be selective quantitative derivatization of large peptides, which would convey properties that direct fragmentation providing specific sequence information, or optimization of LCMS for biooligomer sequencing and mixture analysis, or the perfect way to control or enhance the internal energy of ions of any size, or many others. 1669 references.

  1. An assessment of software solutions for the analysis of mass spectrometry based quantitative proteomics data.

    PubMed

    Mueller, Lukas N; Brusniak, Mi-Youn; Mani, D R; Aebersold, Ruedi

    2008-01-01

    Over the past decade, a series of experimental strategies for mass spectrometry based quantitative proteomics and corresponding computational methodology for the processing of the resulting data have been generated. We provide here an overview of the main quantification principles and available software solutions for the analysis of data generated by liquid chromatography coupled to mass spectrometry (LC-MS). Three conceptually different methods to perform quantitative LC-MS experiments have been introduced. In the first, quantification is achieved by spectral counting, in the second via differential stable isotopic labeling, and in the third by using the ion current in label-free LC-MS measurements. We discuss here advantages and challenges of each quantification approach and assess available software solutions with respect to their instrument compatibility and processing functionality. This review therefore serves as a starting point for researchers to choose an appropriate software solution for quantitative proteomic experiments based on their experimental and analytical requirements.

  2. Method for quantitative proteomics research by using metal element chelated tags coupled with mass spectrometry.

    PubMed

    Liu, Huiling; Zhang, Yangjun; Wang, Jinglan; Wang, Dong; Zhou, Chunxi; Cai, Yun; Qian, Xiaohong

    2006-09-15

    The mass spectrometry-based methods with a stable isotope as the internal standard in quantitative proteomics have been developed quickly in recent years. But the use of some stable isotope reagents is limited by the relative high price and synthetic difficulties. We have developed a new method for quantitative proteomics research by using metal element chelated tags (MECT) coupled with mass spectrometry. The bicyclic anhydride diethylenetriamine-N,N,N',N' ',N' '-pentaacetic acid (DTPA) is covalently coupled to primary amines of peptides, and the ligand is then chelated to the rare earth metals Y and Tb. The tagged peptides are mixed and analyzed by LC-ESI-MS/MS. Peptides are quantified by measuring the relative signal intensities for the Y and Tb tag pairs in MS, which permits the quantitation of the original proteins generating the corresponding peptides. The protein is then identified by the corresponding peptide sequence from its MS/MS spectrum. The MECT method was evaluated by using standard proteins as model sample. The experimental results showed that metal chelate-tagged peptides chromatographically coeluted successfully during the reversed-phase LC analysis. The relative quantitation results were accurate for proteins using MECT. DTPA modification of the N-terminal of peptides promoted cleaner fragmentation (only y-series ions) in mass spectrometry and improved the confidence level of protein identification. The MECT strategy provides a simple, rapid, and economical alternative to current mass tagging technologies available.

  3. Quantitative analysis of multiple components based on liquid chromatography with mass spectrometry in full scan mode.

    PubMed

    Xu, Min Li; Li, Bao Qiong; Wang, Xue; Chen, Jing; Zhai, Hong Lin

    2016-08-01

    Although liquid chromatography with mass spectrometry in full scan mode can obtain all the signals simultaneously in a large range and low cost, it is rarely used in quantitative analysis due to several problems such as chromatographic drifts and peak overlap. In this paper, we propose a Tchebichef moment method for the simultaneous quantitative analysis of three active compounds in Qingrejiedu oral liquid based on three-dimensional spectra in full scan mode of liquid chromatography with mass spectrometry. After the Tchebichef moments were calculated directly from the spectra, the quantitative linear models for three active compounds were established by stepwise regression. All the correlation coefficients were more than 0.9978. The limits of detection and limits of quantitation were less than 0.11 and 0.49 μg/mL, respectively. The intra- and interday precisions were less than 6.54 and 9.47%, while the recovery ranged from 102.56 to 112.15%. Owing to the advantages of multi-resolution and inherent invariance properties, Tchebichef moments could provide favorable results even in the situation of peaks shifting and overlapping, unknown interferences and noise signals, so it could be applied to the analysis of three-dimensional spectra in full scan mode of liquid chromatography with mass spectrometry.

  4. Building the connectivity map of epigenetics: Chromatin profiling by quantitative targeted mass spectrometry

    PubMed Central

    Creech, Amanda L.; Taylor, Jordan E.; Maier, Verena K.; Wu, Xiaoyun; Feeney, Caitlin M.; Udeshi, Namrata D.; Peach, Sally E.; Boehm, Jesse S.; Lee, Jeannie T.; Carr, Steven A.; Jaffe, Jacob D.

    2014-01-01

    Epigenetic control of genome function is an important regulatory mechanism in diverse processes such as lineage commitment and environmental sensing, and in disease etiologies ranging from neuropsychiatric disorders to cancer. Here we report a robust, high-throughput targeted, quantitative mass spectrometry (MS) method to rapidly profile modifications of the core histones of chromatin that compose the epigenetic landscape, enabling comparisons among cells with differing genetic backgrounds, genomic perturbations, and drug treatments. PMID:25448295

  5. Analysis of Cell Surface Proteome Changes via Label-free, Quantitative Mass Spectrometry*S⃞

    PubMed Central

    Schiess, Ralph; Mueller, Lukas N.; Schmidt, Alexander; Mueller, Markus; Wollscheid, Bernd; Aebersold, Ruedi

    2009-01-01

    We present a mass spectrometry-based strategy for the specific detection and quantification of cell surface proteome changes. The method is based on the label-free quantification of peptide patterns acquired by high mass accuracy mass spectrometry using new software tools and the cell surface capturing technology that selectively enriches glycopeptides exposed to the cell exterior. The method was applied to monitor dynamic protein changes in the cell surface glycoproteome of Drosophila melanogaster cells. The results led to the construction of a cell surface glycoprotein atlas consisting of 202 cell surface glycoproteins of D. melanogaster Kc167 cells and indicated relative quantitative changes of cell surface glycoproteins in four different cellular states. Furthermore we specifically investigated cell surface proteome changes upon prolonged insulin stimulation. The data revealed insulin-dependent cell surface glycoprotein dynamics, including insulin receptor internalization, and linked these changes to intracellular signaling networks. PMID:19036722

  6. 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.

  7. Quantitative Caffeine Analysis Using a Surface Sampling Probe Electrospray Ionization Tandem Mass Spectrometry System

    SciTech Connect

    Ford, Michael J; Deibel, Michael A.; Tomkins, Bruce A; Van Berkel, Gary J

    2005-01-01

    Quantitative determination of caffeine on reversed-phase C8 thin-layer chromatography plates using a surface sampling electrospray ionization system with tandem mass spectrometry detection is reported. The thin-layer chromatography/electrospray tandem mass spectrometry method employed a deuterium-labeled caffeine internal standard and selected reaction monitoring detection. Up to nine parallel caffeine bands on a single plate were sampled in a single surface scanning experiment requiring 35 min at a surface scan rate of 44 {mu}m/s. A reversed-phase HPLC/UV caffeine assay was developed in parallel to assess the mass spectrometry method performance. Limits of detection for the HPLC/UV and thin-layer chromatography/electrospray tandem mass spectrometry methods determined from the calibration curve statistics were 0.20 ng injected (0.50 {mu}L) and 1.0 ng spotted on the plate, respectively. Spike recoveries with standards and real samples ranged between 97 and 106% for both methods. The caffeine content of three diet soft drinks (Diet Coke, Diet Cherry Coke, Diet Pepsi) and three diet sport drinks (Diet Turbo Tea, Speed Stack Grape, Speed Stack Fruit Punch) was measured. The HPLC/UV and mass spectrometry determinations were in general agreement, and these values were consistent with the quoted values for two of the three diet colas. In the case of Diet Cherry Coke and the diet sports drinks, the determined caffeine amounts using both methods were consistently higher (by 8% or more) than the literature values.

  8. Investigation of Elemental Mass Spectrometry in Pharmacology for Peptide Quantitation at Femtomolar Levels

    PubMed Central

    Cordeau, Emmanuelle; Arnaudguilhem, Carine; Bouyssiere, Brice; Hagège, Agnès; Martinez, Jean; Subra, Gilles; Cantel, Sonia

    2016-01-01

    In the search of new robust and environmental-friendly analytical methods able to answer quantitative issues in pharmacology, we explore liquid chromatography (LC) associated with elemental mass spectrometry (ICP-MS) to monitor peptides in such complex biological matrices. The novelty is to use mass spectrometry to replace radiolabelling and radioactivity measurements, which represent up-to now the gold standard to measure organic compound concentrations in life science. As a proof of concept, we choose the vasopressin (AVP)/V1A receptor system for model pharmacological assays. The capacity of ICP-MS to provide highly sensitive quantitation of metallic and hetero elements, whatever the sample medium, prompted us to investigate this technique in combination with appropriate labelling of the peptide of interest. Selenium, that is scarcely present in biological media, was selected as a good compromise between ICP-MS response, covalent tagging ability using conventional sulfur chemistry and peptide detection specificity. Applying selenium monitoring by elemental mass spectrometry in pharmacology is challenging due to the very high salt content and organic material complexity of the samples that produces polyatomic aggregates and thus potentially mass interferences with selenium detection. Hyphenation with a chromatographic separation was found compulsory. Noteworthy, we aimed to develop a straightforward quantitative protocol that can be performed in any laboratory equipped with a standard macrobore LC-ICP-MS system, in order to avoid time-consuming sample treatment or special implementation of instrumental set-up, while allowing efficient suppression of all mass interferences to reach the targeted sensitivity. Significantly, a quantification limit of 57 ng Se L-1 (72 femtomoles of injected Se) was achieved, the samples issued from the pharmacological assays being directly introduced into the LC-ICP-MS system. The established method was successfully validated and

  9. Quantitation of vitamin B6 in biological samples by isotope dilution mass spectrometry

    SciTech Connect

    Hachey, D.L.; Coburn, S.P.; Brown, L.T.; Erbelding, W.F.; DeMark, B.; Klein, P.D.

    1985-11-15

    Methods have been developed for the simultaneous quantitative analysis of vitamin B6 forms in biological samples by isotope dilution mass spectrometry using deuterated forms of pyridoxine, pyridoxal, pyridoxamine, and pyridoxic acid. The biological fluid or tissue sample was homogenized and then treated with a cocktail containing appropriate amounts of each deuterated vitamer, as well as the deuterated, phosphorylated vitamer forms. The individual vitamers were isolated from the homogenate by a complex high-performance liquid chromatographic procedure that provided separate fractions for each of the six vitamers found in biological samples. Aldehydic B6 vitamers were reduced to the alcohol form prior to acetylation and analysis by gas chromatography/mass spectrometry (GC/MS). The three resulting vitamers were analyzed by electron ionization GC/MS using a silicone capillary column. The methods have been applied to analysis of vitamin B6 in liver, milk, urine, and feces at levels as low as 0.02 nmol/ml.

  10. Quantitative analysis of phytosterols in edible oils using APCI liquid chromatography-tandem mass spectrometry.

    PubMed

    Mo, Shunyan; Dong, Linlin; Hurst, W Jeffrey; van Breemen, Richard B

    2013-09-01

    Previous methods for the quantitative analysis of phytosterols have usually used GC-MS and require elaborate sample preparation including chemical derivatization. Other common methods such as HPLC with absorbance detection do not provide information regarding the identity of the analytes. To address the need for an assay that utilizes mass selectivity while avoiding derivatization, a quantitative method based on LC-tandem mass spectrometry (LC-MS-MS) was developed and validated for the measurement of six abundant dietary phytosterols and structurally related triterpene alcohols including brassicasterol, campesterol, cycloartenol, β-sitosterol, stigmasterol, and lupeol in edible oils. Samples were saponified, extracted with hexane and then analyzed using reversed phase HPLC with positive ion atmospheric pressure chemical ionization tandem mass spectrometry and selected reaction monitoring. The utility of the LC-MS-MS method was demonstrated by analyzing 14 edible oils. All six compounds were present in at least some of the edible oils. The most abundant phytosterol in all samples was β-sitosterol, which was highest in corn oil at 4.35 ± 0.03 mg/g, followed by campesterol in canola oil at 1.84 ± 0.01 mg/g. The new LC-MS-MS method for the quantitative analysis of phytosterols provides a combination of speed, selectivity and sensitivity that exceed those of previous assays.

  11. Quantitative analysis of phytosterols in edible oils using APCI liquid chromatography-tandem mass spectrometry

    PubMed Central

    Mo, Shunyan; Dong, Linlin; Hurst, W. Jeffrey; van Breemen, Richard B.

    2014-01-01

    Previous methods for the quantitative analysis of phytosterols have usually used GC-MS and require elaborate sample preparation including chemical derivatization. Other common methods such as HPLC with absorbance detection do not provide information regarding the identity of the analytes. To address the need for an assay that utilizes mass selectivity while avoiding derivatization, a quantitative method based on LC-tandem mass spectrometry (LC-MS-MS) was developed and validated for the measurement of six abundant dietary phytosterols and structurally related triterpene alcohols including brassicasterol, campesterol, cycloartenol, β-sitosterol, stigmasterol, and lupeol in edible oils. Samples were saponified, extracted with hexane and then analyzed using reversed phase HPLC with positive ion atmospheric pressure chemical ionization tandem mass spectrometry and selected reaction monitoring. The utility of the LC-MS-MS method was demonstrated by analyzing 14 edible oils. All six compounds were present in at least some of the edible oils. The most abundant phytosterol in all samples was β-sitosterol, which was highest in corn oil at 4.35 ± 0.03 mg/g, followed by campesterol in canola oil at 1.84 ± 0.01 mg/g. The new LC-MS-MS method for the quantitative analysis of phytosterols provides a combination of speed, selectivity and sensitivity that exceed those of previous assays. PMID:23884629

  12. Hydroponic isotope labeling of entire plants and high-performance mass spectrometry for quantitative plant proteomics.

    PubMed

    Bindschedler, Laurence V; Mills, Davinia J S; Cramer, Rainer

    2012-01-01

    Hydroponic isotope labeling of entire plants (HILEP) combines hydroponic plant cultivation and metabolic labeling with stable isotopes using (15)N-containing inorganic salts to label whole and mature plants. Employing (15)N salts as the sole nitrogen source for HILEP leads to the production of healthy-looking plants which contain (15)N proteins labeled to nearly 100%. Therefore, HILEP is suitable for quantitative plant proteomic analysis, where plants are grown in either (14)N- or (15)N-hydroponic media and pooled when the biological samples are collected for relative proteome quantitation. The pooled (14)N-/(15)N-protein extracts can be fractionated in any suitable way and digested with a protease for shotgun proteomics, using typically reverse phase liquid chromatography nanoelectrospray ionization tandem mass spectrometry (RPLC-nESI-MS/MS). Best results were obtained with a hybrid ion trap/FT-MS mass spectrometer, combining high mass accuracy and sensitivity for the MS data acquisition with speed and high-throughput MS/MS data acquisition, increasing the number of proteins identified and quantified and improving protein quantitation. Peak processing and picking from raw MS data files, protein identification, and quantitation were performed in a highly automated way using integrated MS data analysis software with minimum manual intervention, thus easing the analytical workflow. In this methodology paper, we describe how to grow Arabidopsis plants hydroponically for isotope labeling using (15)N salts and how to quantitate the resulting proteomes using a convenient workflow that does not require extensive bioinformatics skills.

  13. Enabling Quantitative Analysis in Ambient Ionization Mass Spectrometry: Internal Standard Coated Capillary Samplers

    PubMed Central

    Liu, Jiangjiang; Cooks, R. Graham; Ouyang, Zheng

    2013-01-01

    We describe a sampling method using glass capillaries for quantitative analysis of trace analytes in small volumes of complex mixtures (~1 μL) using ambient ionization mass spectrometry. The internal surface of a sampling glass capillary was coated with internal standard then used to draw liquid sample and so transfer both the analyte and internal standard in a single fixed volume onto a substrate for analysis. The internal standard was automatically mixed into the sample during this process and the volumes of the internal standard solution and sample are both fixed by the capillary volume. Precision in quantitation is insensitive to variations in length of the capillary, making the preparation of the sampling capillary simple and providing a robust sampling protocol. Significant improvements in quantitation accuracy were obtained for analysis of 1 μL samples using various ambient ionization methods. PMID:23731380

  14. Quantitative imaging of subcellular metabolism with stable isotopes and multi-isotope imaging mass spectrometry.

    PubMed

    Steinhauser, Matthew L; Lechene, Claude P

    2013-01-01

    Multi-isotope imaging mass spectrometry (MIMS) is the quantitative imaging of stable isotope labels in cells with a new type of secondary ion mass spectrometer (NanoSIMS). The power of the methodology is attributable to (i) the immense advantage of using non-toxic stable isotope labels, (ii) high resolution imaging that approaches the resolution of usual transmission electron microscopy and (iii) the precise quantification of label down to 1 part-per-million and spanning several orders of magnitude. Here we review the basic elements of MIMS and describe new applications of MIMS to the quantitative study of metabolic processes including protein and nucleic acid synthesis in model organisms ranging from microbes to humans.

  15. New sample preparation for quantitative laser desorption mass spectrometry and optical spectroscopy

    NASA Astrophysics Data System (ADS)

    Haefliger, Olivier P.; Zenobi, Renato

    1998-04-01

    Several analytical mass spectrometric and optical spectroscopic methods require a step during which a nonvolatile substance is desorbed by a laser pulse. It is, however, very difficult to use these methods for quantitative measurements because an accurate control over the amount desorbed by the laser pulse is generally not possible, especially when mixtures of several substances are used. We report a new fast and convenient sample preparation procedure that solves these problems. A solution of the analytes is mixed with a solution of poly(vinyl chloride) to obtain a homogeneous and vacuum-stable thin polymer membrane after the solvent has evaporated. Laser ablation is then performed directly from this membrane, allowing an accurate control of the amount of ablated analytes and excellent reproducibility. Quantitative laser desorption mass spectrometry over three orders of magnitude as well as optical spectroscopic measurements using this sample preparation method are demonstrated for polycyclic aromatic hydrocarbons.

  16. Current challenges in software solutions for mass spectrometry-based quantitative proteomics.

    PubMed

    Cappadona, Salvatore; Baker, Peter R; Cutillas, Pedro R; Heck, Albert J R; van Breukelen, Bas

    2012-09-01

    Mass spectrometry-based proteomics has evolved as a high-throughput research field over the past decade. Significant advances in instrumentation, and the ability to produce huge volumes of data, have emphasized the need for adequate data analysis tools, which are nowadays often considered the main bottleneck for proteomics development. This review highlights important issues that directly impact the effectiveness of proteomic quantitation and educates software developers and end-users on available computational solutions to correct for the occurrence of these factors. Potential sources of errors specific for stable isotope-based methods or label-free approaches are explicitly outlined. The overall aim focuses on a generic proteomic workflow.

  17. Identifying Kinase Substrates via a Heavy ATP Kinase Assay and Quantitative Mass Spectrometry

    PubMed Central

    Müller, André C.; Giambruno, Roberto; Weißer, Juliane; Májek, Peter; Hofer, Alexandre; Bigenzahn, Johannes W.; Superti-Furga, Giulio; Jessen, Henning J.; Bennett, Keiryn L.

    2016-01-01

    Mass spectrometry-based in vitro kinase screens play an essential role in the discovery of kinase substrates, however, many suffer from biological and technical noise or necessitate genetically-altered enzyme-cofactor systems. We describe a method that combines stable γ-[18O2]-ATP with classical in vitro kinase assays within a contemporary quantitative proteomic workflow. Our approach improved detection of known substrates of the non-receptor tyrosine kinase ABL1; and identified potential, new in vitro substrates. PMID:27346722

  18. Beyond hairballs: the use of quantitative mass spectrometry data to understand protein-protein interactions

    PubMed Central

    Gingras, Anne-Claude; Raught, Brian

    2012-01-01

    The past 10 years have witnessed a dramatic proliferation in the availability of protein interaction data. However, for interaction mapping based on affinity purification coupled with mass spectrometry (AP-MS), there is a wealth of information present in the datasets that often goes unrecorded in public repositories, and as such remains largely unexplored. Further, how this type of data is represented and used by bioinformaticians has not been well established. Here, we point out some common mistakes in how AP-MS data are handled, and describe how protein complex organization and interaction dynamics can be inferred using quantitative AP-MS approaches. PMID:22710165

  19. Quantitation of Insulin-Like Growth Factor 1 in Serum by Liquid Chromatography High Resolution Accurate-Mass Mass Spectrometry.

    PubMed

    Ketha, Hemamalini; Singh, Ravinder J

    2016-01-01

    Insulin-like growth factor 1 (IGF-1) is a 70 amino acid peptide hormone which acts as the principal mediator of the effects of growth hormone (GH). Due to a wide variability in circulating concentration of GH, IGF-1 quantitation is the first step in the diagnosis of GH excess or deficiency. Majority (>95 %) of IGF-1 circulates as a ternary complex along with its principle binding protein insulin-like growth factor 1 binding protein 3 (IGFBP-3) and acid labile subunit. The assay design approach for IGF-1 quantitation has to include a step to dissociate IGF-1 from its ternary complex. Several commercial assays employ a buffer containing acidified ethanol to achieve this. Despite several modifications, commercially available immunoassays have been shown to have challenges with interference from IGFBP-3. Additionally, inter-method comparison between IGF-1 immunoassays has been shown to be suboptimal. Mass spectrometry has been utilized for quantitation of IGF-1. In this chapter a liquid chromatography high resolution accurate-mass mass spectrometry (LC-HRAMS) based method for IGF-1 quantitation has been described.

  20. A General Method for Targeted Quantitative Cross-Linking Mass Spectrometry

    PubMed Central

    Chavez, Juan D.; Eng, Jimmy K.; Schweppe, Devin K.; Cilia, Michelle; Rivera, Keith; Zhong, Xuefei; Wu, Xia; Allen, Terrence; Khurgel, Moshe; Kumar, Akhilesh; Lampropoulos, Athanasios; Larsson, Mårten; Maity, Shuvadeep; Morozov, Yaroslav; Pathmasiri, Wimal; Perez-Neut, Mathew; Pineyro-Ruiz, Coriness; Polina, Elizabeth; Post, Stephanie; Rider, Mark; Tokmina-Roszyk, Dorota; Tyson, Katherine; Vieira Parrine Sant'Ana, Debora; Bruce, James E.

    2016-01-01

    Chemical cross-linking mass spectrometry (XL-MS) provides protein structural information by identifying covalently linked proximal amino acid residues on protein surfaces. The information gained by this technique is complementary to other structural biology methods such as x-ray crystallography, NMR and cryo-electron microscopy[1]. The extension of traditional quantitative proteomics methods with chemical cross-linking can provide information on the structural dynamics of protein structures and protein complexes. The identification and quantitation of cross-linked peptides remains challenging for the general community, requiring specialized expertise ultimately limiting more widespread adoption of the technique. We describe a general method for targeted quantitative mass spectrometric analysis of cross-linked peptide pairs. We report the adaptation of the widely used, open source software package Skyline, for the analysis of quantitative XL-MS data as a means for data analysis and sharing of methods. We demonstrate the utility and robustness of the method with a cross-laboratory study and present data that is supported by and validates previously published data on quantified cross-linked peptide pairs. This advance provides an easy to use resource so that any lab with access to a LC-MS system capable of performing targeted quantitative analysis can quickly and accurately measure dynamic changes in protein structure and protein interactions. PMID:27997545

  1. Emerging flow injection mass spectrometry methods for high-throughput quantitative analysis.

    PubMed

    Nanita, Sergio C; Kaldon, Laura G

    2016-01-01

    Where does flow injection analysis mass spectrometry (FIA-MS) stand relative to ambient mass spectrometry (MS) and chromatography-MS? Improvements in FIA-MS methods have resulted in fast-expanding uses of this technique. Key advantages of FIA-MS over chromatography-MS are fast analysis (typical run time <60 s) and method simplicity, and FIA-MS offers high-throughput without compromising sensitivity, precision and accuracy as much as ambient MS techniques. Consequently, FIA-MS is increasingly becoming recognized as a suitable technique for applications where quantitative screening of chemicals needs to be performed rapidly and reliably. The FIA-MS methods discussed herein have demonstrated quantitation of diverse analytes, including pharmaceuticals, pesticides, environmental contaminants, and endogenous compounds, at levels ranging from parts-per-billion (ppb) to parts-per-million (ppm) in very complex matrices (such as blood, urine, and a variety of foods of plant and animal origin), allowing successful applications of the technique in clinical diagnostics, metabolomics, environmental sciences, toxicology, and detection of adulterated/counterfeited goods. The recent boom in applications of FIA-MS for high-throughput quantitative analysis has been driven in part by (1) the continuous improvements in sensitivity and selectivity of MS instrumentation, (2) the introduction of novel sample preparation procedures compatible with standalone mass spectrometric analysis such as salting out assisted liquid-liquid extraction (SALLE) with volatile solutes and NH4(+) QuEChERS, and (3) the need to improve efficiency of laboratories to satisfy increasing analytical demand while lowering operational cost. The advantages and drawbacks of quantitative analysis by FIA-MS are discussed in comparison to chromatography-MS and ambient MS (e.g., DESI, LAESI, DART). Generally, FIA-MS sits 'in the middle' between ambient MS and chromatography-MS, offering a balance between analytical

  2. Novel aspects of quantitation of immunogenic wheat gluten peptides by liquid chromatography-mass spectrometry/mass spectrometry.

    PubMed

    Sealey-Voyksner, Jennifer A; Khosla, Chaitan; Voyksner, Robert D; Jorgenson, James W

    2010-06-18

    A novel, specific and sensitive non-immunological liquid chromatography-mass spectrometry (LC-MS) based assay has been developed to detect and quantify trace levels of wheat gluten in food and consumer products. Detection and quantification of dietary gluten is important, because gluten is a principle trigger of a variety of immune diseases including food allergies and intolerances. One such disease, celiac sprue, can cause intestinal inflammation and enteropathy in patients who are exposed to dietary gluten. At present, immunochemistry is the leading analytical method for gluten detection in food. Consequently, enzyme-linked immunosorbent assays (ELISAs), such as the sandwich or competitive type assays, are the only commercially available methods to ensure that food and consumer products are accurately labeled as gluten-free. The availability of a comprehensive, fast and economic alternative to the immunological ELISA may also facilitate research towards the development of new drugs, therapies and food processing technologies to aid patients with gluten intolerances and for gluten-free labeling and certification purposes. LC-MS is an effective and efficient analytical technique for the study of cereal grain proteins and to quantify trace levels of targeted dietary gluten peptides in complex matrices. Initial efforts in this area afforded the unambiguous identification and structural characterization of six unique physiologically relevant wheat gluten peptides. This paper describes the development and optimization of an LC-MS/MS method that attempts to provide the best possible accuracy and sensitivity for the quantitative detection of trace levels of these six peptides in various food and consumer products. The overall performance of this method was evaluated using native cereal grains. Experimental results demonstrated that this method is capable of detecting and quantifying select target peptides in food over a range from 10pg/mg to 100ng/mg (corresponding to

  3. Identification of hypoxia-regulated proteins using MALDI-mass spectrometry imaging combined with quantitative proteomics.

    PubMed

    Djidja, Marie-Claude; Chang, Joan; Hadjiprocopis, Andreas; Schmich, Fabian; Sinclair, John; Mršnik, Martina; Schoof, Erwin M; Barker, Holly E; Linding, Rune; Jørgensen, Claus; Erler, Janine T

    2014-05-02

    Hypoxia is present in most solid tumors and is clinically correlated with increased metastasis and poor patient survival. While studies have demonstrated the role of hypoxia and hypoxia-regulated proteins in cancer progression, no attempts have been made to identify hypoxia-regulated proteins using quantitative proteomics combined with MALDI-mass spectrometry imaging (MALDI-MSI). Here we present a comprehensive hypoxic proteome study and are the first to investigate changes in situ using tumor samples. In vitro quantitative mass spectrometry analysis of the hypoxic proteome was performed on breast cancer cells using stable isotope labeling with amino acids in cell culture (SILAC). MS analyses were performed on laser-capture microdissected samples isolated from normoxic and hypoxic regions from tumors derived from the same cells used in vitro. MALDI-MSI was used in combination to investigate hypoxia-regulated protein localization within tumor sections. Here we identified more than 100 proteins, both novel and previously reported, that were associated with hypoxia. Several proteins were localized in hypoxic regions, as identified by MALDI-MSI. Visualization and data extrapolation methods for the in vitro SILAC data were also developed, and computational mapping of MALDI-MSI data to IHC results was applied for data validation. The results and limitations of the methodologies described are discussed.

  4. High Throughput Quantitative Analysis of Serum Proteins Using Glycopeptide Capture and Liquid Chromatography Mass Spectrometry

    SciTech Connect

    Zhang, Hui; Yi, Eugene C.; Li, Xiao-jun; Mallick, Parag; Kelly-Spratt, Karen S.; Masselon, Christophe D.; Camp, David G.; Smith, Richard D.; Kemp, Christopher J.; Aebersold, Reudi

    2005-02-01

    It is expected that the composition of the serum proteome can provide valuable information about the state of the human body in health and disease and that this information can be extracted via quantitative proteomic measurements. Suitable proteomic techniques need to be sensitive, reproducible, and robust to detect potential biomarkers below the level of highly expressed proteins, generate data sets that are comparable between experiments and laboratories, and have high throughput to support statistical studies. Here we report a method for high throughput quantitative analysis of serum proteins. It consists of the selective isolation of peptides that are N-linked glycosylated in the intact protein, the analysis of these now deglycosylated peptides by liquid chromatography electrospray ionization mass spectrometry, and the comparative analysis of the resulting patterns. By focusing selectively on a few formerly N-linked glycopeptides per serum protein, the complexity of the analyte sample is significantly reduced and the sensitivity and throughput of serum proteome analysis are increased compared with the analysis of total tryptic peptides from unfractionated samples. We provide data that document the performance of the method and show that sera from untreated normal mice and genetically identical mice with carcinogen-induced skin cancer can be unambiguously discriminated using unsupervised clustering of the resulting peptide patterns. We further identify, by tandem mass spectrometry, some of the peptides that were consistently elevated in cancer mice compared with their control littermates.

  5. Quantitative determination of 1-deoxynojirimycin in mulberry leaves using liquid chromatography-tandem mass spectrometry.

    PubMed

    Nuengchamnong, Nitra; Ingkaninan, Kornkanok; Kaewruang, Wiroje; Wongareonwanakij, Sathaporn; Hongthongdaeng, Bhinai

    2007-08-15

    A novel HPLC-MS/MS method was developed for the quantitative determination of 1-deoxynojirimycin (DNJ), a potent glucosidase inhibitor present in mulberry leaves (Morus alba L.). DNJ was isolated from the mulberry leave extract on a TSKgel Amide-80 column using a mixture of 0.1% formic acid and acetonitrile as a mobile phase at a flow rate of 0.6 ml/min. A triple quadrupole mass spectrometry using electrospray ionization source in a positive ion mode under multiple reaction monitoring with the [M+H]+ ions, m/z 164.4/109.9 was used. The detection limit (S/N=3) was 75 pg and quantitation limit (S/N=10) was 100 pg. The comparison of mulberry leaves of different ages showed that the DNJ level was higher in mulberry shoots than young and mature leaves.

  6. Advances in liquid chromatography-high-resolution mass spectrometry for quantitative and qualitative environmental analysis.

    PubMed

    Aceña, Jaume; Stampachiacchiere, Serena; Pérez, Sandra; Barceló, Damià

    2015-08-01

    This review summarizes the advances in environmental analysis by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) during the last decade and discusses different aspects of their application. LC-HRMS has become a powerful tool for simultaneous quantitative and qualitative analysis of organic pollutants, enabling their quantitation and the search for metabolites and transformation products or the detection of unknown compounds. LC-HRMS provides more information than low-resolution (LR) MS for each sample because it can accurately determine the mass of the molecular ion and its fragment ions if it can be used for MS-MS. Another advantage is that the data can be processed using either target analysis, suspect screening, retrospective analysis, or non-target screening. With the growing popularity and acceptance of HRMS analysis, current guidelines for compound confirmation need to be revised for quantitative and qualitative purposes. Furthermore, new commercial software and user-built libraries are required to mine data in an efficient and comprehensive way. The scope of this critical review is not to provide a comprehensive overview of the many studies performed with LC-HRMS in the field of environmental analysis, but to reveal its advantages and limitations using different workflows.

  7. Spatial Quantitation of Drugs in tissues using Liquid Extraction Surface Analysis Mass Spectrometry Imaging.

    PubMed

    Swales, John G; Strittmatter, Nicole; Tucker, James W; Clench, Malcolm R; Webborn, Peter J H; Goodwin, Richard J A

    2016-11-24

    Liquid extraction surface analysis mass spectrometry imaging (LESA-MSI) has been shown to be an effective tissue profiling and imaging technique, producing robust and reliable qualitative distribution images of an analyte or analytes in tissue sections. Here, we expand the use of LESA-MSI beyond qualitative analysis to a quantitative analytical technique by employing a mimetic tissue model previously shown to be applicable for MALDI-MSI quantitation. Liver homogenate was used to generate a viable and molecularly relevant control matrix for spiked drug standards which can be frozen, sectioned and subsequently analyzed for the generation of calibration curves to quantify unknown tissue section samples. The effects of extraction solvent composition, tissue thickness and solvent/tissue contact time were explored prior to any quantitative studies in order to optimize the LESA-MSI method across several different chemical entities. The use of a internal standard to normalize regional differences in ionization response across tissue sections was also investigated. Data are presented comparing quantitative results generated by LESA-MSI to LC-MS/MS. Subsequent analysis of adjacent tissue sections using DESI-MSI is also reported.

  8. Spatial Quantitation of Drugs in tissues using Liquid Extraction Surface Analysis Mass Spectrometry Imaging

    PubMed Central

    Swales, John G.; Strittmatter, Nicole; Tucker, James W.; Clench, Malcolm R.; Webborn, Peter J. H.; Goodwin, Richard J. A.

    2016-01-01

    Liquid extraction surface analysis mass spectrometry imaging (LESA-MSI) has been shown to be an effective tissue profiling and imaging technique, producing robust and reliable qualitative distribution images of an analyte or analytes in tissue sections. Here, we expand the use of LESA-MSI beyond qualitative analysis to a quantitative analytical technique by employing a mimetic tissue model previously shown to be applicable for MALDI-MSI quantitation. Liver homogenate was used to generate a viable and molecularly relevant control matrix for spiked drug standards which can be frozen, sectioned and subsequently analyzed for the generation of calibration curves to quantify unknown tissue section samples. The effects of extraction solvent composition, tissue thickness and solvent/tissue contact time were explored prior to any quantitative studies in order to optimize the LESA-MSI method across several different chemical entities. The use of a internal standard to normalize regional differences in ionization response across tissue sections was also investigated. Data are presented comparing quantitative results generated by LESA-MSI to LC-MS/MS. Subsequent analysis of adjacent tissue sections using DESI-MSI is also reported. PMID:27883030

  9. Qualitative and Quantitative Proteome Analysis of Oral Fluids in Health and Periodontal Disease by Mass Spectrometry.

    PubMed

    Salih, Erdjan

    2017-01-01

    The significance of protein identification and characterization by classical protein chemistry approaches is clearly highlighted by our detailed understanding of the biological systems assembled over a time period of almost a century. The advent of state-of-the-art mass spectrometry (MS) with sensitivity, speed, and global protein analysis capacity without individual protein purification has transformed the classical protein chemistry with premise to accelerate discovery. These combined with the ability of the oral fluids such as whole saliva (WS) and gingival crevicular fluid (GCF) to reflect both systemic and locally derived proteins have generated significant interest to characterize these fluids more extensively by MS technology. This chapter deals with the experimental details of preanalytical steps using multidimensional protein separation combined with MS analysis of WS and GCF to achieve detailed protein composition at qualitative and quantitative levels. These approaches are interfaced with gold standard "stable-isotope" labeling technologies for large-scale quantitative MS analysis which is a prerequisite to determine accurate alterations in protein levels as a function of disease progression. The latter incorporates two stable-isotope chemistries one specific for cysteine containing proteins and the other universal amine-specific reagent in conjunction with oral fluids in health and periodontal disease to perform quantitative MS analysis. In addition, specific preanalytical steps demanded by the oral fluids such as GCF and WS for sample preparations to overcome limitations and uncertainties are elaborated for reliable large-scale quantitative MS analysis.

  10. Spatial Quantitation of Drugs in tissues using Liquid Extraction Surface Analysis Mass Spectrometry Imaging

    NASA Astrophysics Data System (ADS)

    Swales, John G.; Strittmatter, Nicole; Tucker, James W.; Clench, Malcolm R.; Webborn, Peter J. H.; Goodwin, Richard J. A.

    2016-11-01

    Liquid extraction surface analysis mass spectrometry imaging (LESA-MSI) has been shown to be an effective tissue profiling and imaging technique, producing robust and reliable qualitative distribution images of an analyte or analytes in tissue sections. Here, we expand the use of LESA-MSI beyond qualitative analysis to a quantitative analytical technique by employing a mimetic tissue model previously shown to be applicable for MALDI-MSI quantitation. Liver homogenate was used to generate a viable and molecularly relevant control matrix for spiked drug standards which can be frozen, sectioned and subsequently analyzed for the generation of calibration curves to quantify unknown tissue section samples. The effects of extraction solvent composition, tissue thickness and solvent/tissue contact time were explored prior to any quantitative studies in order to optimize the LESA-MSI method across several different chemical entities. The use of a internal standard to normalize regional differences in ionization response across tissue sections was also investigated. Data are presented comparing quantitative results generated by LESA-MSI to LC-MS/MS. Subsequent analysis of adjacent tissue sections using DESI-MSI is also reported.

  11. Comparison between liquid chromatography-time of-flight mass spectrometry and selected reaction monitoring liquid chromatography-mass spectrometry for quantitative determination of idoxifene in human plasma.

    PubMed

    Zhang, H; Henion, J

    2001-06-05

    This study compares HPLC electrospray time-of-flight mass spectrometry and selected reaction monitoring (SRM) LC-MS for high throughput quantitative determination of a small molecule drug in biological samples. A high throughput LC-MS method was developed for quantitatative determination of idoxifene in human plasma and the evaluation was accomplished with the cross-validation of the developed LC-MS method between the time-of-flight mass spectrometer, and a triple quadrupole mass spectrometer operated in the SRM mode. A simple one-step semi-automated 96-well liquid-liquid extraction procedure was used to prepare 96 samples in approximately 30 min and a rapid gradient was used to shorten the LC run time. Time-of-flight mass spectrometry provides acquisition of full-scan mass spectra and extracted ion current chromatograms, which may be extracted from the total ion current chromatogram for peak area determination. The limit of quantitation for idoxifene in human plasma obtained with the time-of-flight mass spectrometer was 5 ng/ml based on 100-microl aliquots of human plasma, and the linear dynamic range was from 5 ng/ml to 2000 ng/ml. The quantitative LC-MS results from the time-of-flight mass spectrometer demonstrated that precision did not exceed 7.1% and accuracy did not exceed 1.7% with reference to quality control samples at three concentration levels in replicates of six. In contrast, the limit of quantitation for idoxifene in human plasma using a tandem triple quadrupole mass spectrometer was 0.5 ng/ml with a linear dynamic range to 1000 ng/ml. The results from the triple quadrupole instrument show that the precision did not exceed 2.2% and accuracy did not exceed 2.9%. The overall results suggest time-of-flight mass spectrometry may be a viable technique for high throughput bioanalytical work for the quantitative determination of a representative small molecule drug in the low ng/ml range in human plasma.

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

    PubMed

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

    2016-03-15

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

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

    PubMed

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

    2011-11-01

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

  14. Quantitation of Free Metanephrines in Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

    PubMed

    Heideloff, Courtney; Payto, Drew; Wang, Sihe

    2016-01-01

    Plasma metanephrines are measured to aid in the diagnosis of pheochromocytomas. In patients with pheochromocytomas there is excessive production of catecholamines and metanephrines. Measurement of plasma free metanephrines is one of the first-line clinical tests that are used for the diagnosis and follow-up of pheochromocytoma. We describe here a liquid chromatography-tandem mass spectrometry method to measure free metanephrines in plasma. Free metanephrine and normetanephrine are extracted via solid-phase extraction. After extraction and evaporation, the reconstituted supernatant is analyzed by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The MS/MS is set to selective reaction monitoring mode (180.1 → 148.1 m/z for metanephrine, 183.1 → 168.1 for d3-metanephrine, 166.1 → 134.1 m/z for normetanephrine, and 169.1 → 137.2 m/z for d3-normetanephrine) with positive electrospray ionization. Quantitation is based on peak area ratio of the analyte to its respective deuterated internal standard. The assay is linear from 5.9 to 4090.0 pg/mL for metanephrine and 22.0 to 4386.7 pg/mL for normetanephrine with precision of <6 % over the ranges.

  15. Quantitative Clinical Chemistry Proteomics (qCCP) using mass spectrometry: general characteristics and application.

    PubMed

    Lehmann, Sylvain; Hoofnagle, Andrew; Hochstrasser, Denis; Brede, Cato; Glueckmann, Matthias; Cocho, José A; Ceglarek, Uta; Lenz, Christof; Vialaret, Jérôme; Scherl, Alexander; Hirtz, Christophe

    2013-05-01

    Proteomics studies typically aim to exhaustively detect peptides/proteins in a given biological sample. Over the past decade, the number of publications using proteomics methodologies has exploded. This was made possible due to the availability of high-quality genomic data and many technological advances in the fields of microfluidics and mass spectrometry. Proteomics in biomedical research was initially used in 'functional' studies for the identification of proteins involved in pathophysiological processes, complexes and networks. Improved sensitivity of instrumentation facilitated the analysis of even more complex sample types, including human biological fluids. It is at that point the field of clinical proteomics was born, and its fundamental aim was the discovery and (ideally) validation of biomarkers for the diagnosis, prognosis, or therapeutic monitoring of disease. Eventually, it was recognized that the technologies used in clinical proteomics studies [particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS)] could represent an alternative to classical immunochemical assays. Prior to deploying MS in the measurement of peptides/proteins in the clinical laboratory, it seems likely that traditional proteomics workflows and data management systems will need to adapt to the clinical environment and meet in vitro diagnostic (IVD) regulatory constraints. This defines a new field, as reviewed in this article, that we have termed quantitative Clinical Chemistry Proteomics (qCCP).

  16. Quantitative thin-layer chromatography/mass spectrometry analysis of caffeine using a surface sampling probe electrospray ionization tandem mass spectrometry system.

    PubMed

    Ford, Michael J; Deibel, Michael A; Tomkins, Bruce A; Van Berkel, Gary J

    2005-07-15

    Quantitative determination of caffeine on reversed-phase C8 thin-layer chromatography plates using a surface sampling electrospray ionization system with tandem mass spectrometry detection is reported. The thin-layer chromatography/electrospray tandem mass spectrometry method employed a deuterium-labeled caffeine internal standard and selected reaction monitoring detection. Up to nine parallel caffeine bands on a single plate were sampled in a single surface scanning experiment requiring 35 min at a surface scan rate of 44 mum/s. A reversed-phase HPLC/UV caffeine assay was developed in parallel to assess the mass spectrometry method performance. Limits of detection for the HPLC/UV and thin-layer chromatography/electrospray tandem mass spectrometry methods determined from the calibration curve statistics were 0.20 ng injected (0.50 muL) and 1.0 ng spotted on the plate, respectively. Spike recoveries with standards and real samples ranged between 97 and 106% for both methods. The caffeine content of three diet soft drinks (Diet Coke, Diet Cherry Coke, Diet Pepsi) and three diet sport drinks (Diet Turbo Tea, Speed Stack Grape, Speed Stack Fruit Punch) was measured. The HPLC/UV and mass spectrometry determinations were in general agreement, and these values were consistent with the quoted values for two of the three diet colas. In the case of Diet Cherry Coke and the diet sports drinks, the determined caffeine amounts using both methods were consistently higher (by approximately 8% or more) than the literature values.

  17. Quantitative Thin-Layer Chromatography/Mass Spectrometry Analysis of Caffeine Using a Surface Sampling Probe Electrospray Ionization Tandem Mass Spectrometry System

    SciTech Connect

    Ford, Michael J; Deibel, Michael A.; Tomkins, Bruce A; Van Berkel, Gary J

    2005-01-01

    Quantitative determination of caffeine on reversed-phase C8 thin-layer chromatography plates using a surface sampling electrospray ionization system with tandem mass spectrometry detection is reported. The thin-layer chromatography/electrospray tandem mass spectrometry method employed a deuterium-labeled caffeine internal standard and selected reaction monitoring detection. Up to nine parallel caffeine bands on a single plate were sampled in a single surface scanning experiment requiring 35 min at a surface scan rate of 44 {mu}m/s. A reversed-phase HPLC/UV caffeine assay was developed in parallel to assess the mass spectrometry method performance. Limits of detection for the HPLC/UV and thin-layer chromatography/electrospray tandem mass spectrometry methods determined from the calibration curve statistics were 0.20 ng injected (0.50 {mu}L) and 1.0 ng spotted on the plate, respectively. Spike recoveries with standards and real samples ranged between 97 and 106% for both methods. The caffeine content of three diet soft drinks (Diet Coke, Diet Cherry Coke, Diet Pepsi) and three diet sport drinks (Diet Turbo Tea, Speed Stack Grape, Speed Stack Fruit Punch) was measured. The HPLC/UV and mass spectrometry determinations were in general agreement, and these values were consistent with the quoted values for two of the three diet colas. In the case of Diet Cherry Coke and the diet sports drinks, the determined caffeine amounts using both methods were consistently higher (by 8% or more) than the literature values.

  18. Shotgun Approach for Quantitative Imaging of Phospholipids Using Nanospray Desorption Electrospray Ionization Mass Spectrometry

    SciTech Connect

    Lanekoff, Ingela T.; Thomas, Mathew; Laskin, Julia

    2014-02-04

    Mass spectrometry imaging (MSI) has been extensively used for determining spatial distributions of molecules in biological samples, and there is increasing interest in using MSI for quantification. Nanospray desorption electrospray ionization, or nano-DESI, is an ambient MSI technique where a solvent is used for localized extraction of molecules followed by nanoelectrospray ionization. Doping the nano-DESI solvent with carefully selected standards enables online quantification during MSI experiments. In this proof-of-principle study, we demonstrate this quantification approach can be extended to provide shotgun-like quantification of phospholipids in thin brain tissue sections. Specifically, two phosphatidylcholine (PC) standards were added to the nano-DESI solvent for simultaneous imaging and quantification of 22 PC species observed in nano-DESI MSI. Furthermore, by combining the quantitative data obtained in the individual pixels, we demonstrate quantification of these PC species in seven different regions of a rat brain tissue section.

  19. Facilitated diffusion of acetonitrile revealed by quantitative breath analysis using extractive electrospray ionization mass spectrometry.

    PubMed

    Li, Ming; Ding, Jianhua; Gu, Haiwei; Zhang, Yan; Pan, Susu; Xu, Ning; Chen, Huanwen; Li, Hongmei

    2013-01-01

    By using silver cations (Ag⁺) as the ionic reagent in reactive extractive electrospray ionization mass spectrometry (EESI-MS), the concentrations of acetonitrile in exhaled breath samples from the volunteers including active smokers, passive smokers, and non-smokers were quantitatively measured in vivo, without any sample pretreatment. A limit of detection (LOD) and relative standard deviation (RSD) were 0.16 ng/L and 3.5% (n = 8), respectively, for the acetonitrile signals in MS/MS experiments. Interestingly, the concentrations of acetonitrile in human breath continuously increased for 1-4 hours after the smoker finished smoking and then slowly decreased to the background level in 7 days. The experimental data of a large number of (> 165) samples indicated that the inhaled acetonitrile is excreted most likely by facilitated diffusion, instead of simple diffusion reported previously for other volatile compounds.

  20. Serum Insulin-like Growth Factor I Quantitation by Mass Spectrometry: Insights for Protein Quantitation with this Technology.

    PubMed

    Kam, Richard Kin Ting; Ho, Chung Shun; Chan, Michael Ho Ming

    2016-12-01

    Liquid chromatography mass spectrometry (LC-MS) is a widely used technique in the clinical laboratory, especially for small molecule quantitation in biological specimens, for example, steroid hormones and therapeutic drugs. Analysis of circulating macromolecules, including proteins and peptides, is largely dominated by traditional enzymatic, spectrophotometric, or immunological assays in clinical laboratories. However, these methodologies are known to be subjected to interfering substances, for example heterophilic antibodies, as well as subjected to non-specificity issues. In recent years, there has been a growing interest in using LC-MS platforms for protein analysis in the clinical setting, due to the superior specificity compared to immunoassay, and the possibility of simultaneous quantitation of multiple proteins. Different analytical approaches are possible using LC-MS-based methodology, including accurate mass measurement of intact molecules, protein digestion followed by detection of proteolytic peptides, and in combination with immunoaffinity purification. Proteins with different complexity, isoforms, variants, or chemical alteration can be simultaneously analysed by LC-MS, either by targeted or non-targeted approaches. While the LC-MS platform offers a more specific determination of proteins, there remain issues of LC-MS assay harmonization, correlation with current existing platforms, and the potential impact in making clinical decision. In this review, the clinical utility, historical aspect, and challenges in using LC-MS for protein analysis in the clinical setting will be discussed, using insulin-like growth factor (IGF) as an example.

  1. Serum Insulin-like Growth Factor I Quantitation by Mass Spectrometry: Insights for Protein Quantitation with this Technology

    PubMed Central

    Ho, Chung Shun; Chan, Michael Ho Ming

    2016-01-01

    Liquid chromatography mass spectrometry (LC-MS) is a widely used technique in the clinical laboratory, especially for small molecule quantitation in biological specimens, for example, steroid hormones and therapeutic drugs. Analysis of circulating macromolecules, including proteins and peptides, is largely dominated by traditional enzymatic, spectrophotometric, or immunological assays in clinical laboratories. However, these methodologies are known to be subjected to interfering substances, for example heterophilic antibodies, as well as subjected to non-specificity issues. In recent years, there has been a growing interest in using LC-MS platforms for protein analysis in the clinical setting, due to the superior specificity compared to immunoassay, and the possibility of simultaneous quantitation of multiple proteins. Different analytical approaches are possible using LC-MS-based methodology, including accurate mass measurement of intact molecules, protein digestion followed by detection of proteolytic peptides, and in combination with immunoaffinity purification. Proteins with different complexity, isoforms, variants, or chemical alteration can be simultaneously analysed by LC-MS, either by targeted or non-targeted approaches. While the LC-MS platform offers a more specific determination of proteins, there remain issues of LC-MS assay harmonization, correlation with current existing platforms, and the potential impact in making clinical decision. In this review, the clinical utility, historical aspect, and challenges in using LC-MS for protein analysis in the clinical setting will be discussed, using insulin-like growth factor (IGF) as an example. PMID:28149264

  2. Relative quantitation of transfer RNAs using liquid chromatography mass spectrometry and signature digestion products.

    PubMed

    Castleberry, Colette M; Limbach, Patrick A

    2010-09-01

    Transfer ribonucleic acids (tRNAs) are challenging to identify and quantify from unseparated mixtures. Our lab previously developed the signature digestion approach for identifying tRNAs without specific separation. Here we describe the combination of relative quantification via enzyme-mediated isotope labeling with this signature digestion approach for the relative quantification of tRNAs. These quantitative signature digestion products were characterized using liquid chromatography mass spectrometry (LC-MS), and we find that up to 5-fold changes in tRNA abundance can be quantified from sub-microgram amounts of total tRNA. Quantitative tRNA signature digestion products must (i) incorporate an isotopic label during enzymatic digestion; (ii) have no m/z interferences from other signature digestion products in the sample and (iii) yield a linear response during LC-MS analysis. Under these experimental conditions, the RNase T1, A and U2 signature digestion products that potentially could be used for the relative quantification of Escherichia coli tRNAs were identified, and the linearity and sequence identify of RNase T1 signature digestion products were experimentally confirmed. These RNase T1 quantitative signature digestion products were then used in proof-of-principle experiments to quantify changes arising due to different culturing media to 17 tRNA families. This method enables new experiments where information regarding tRNA identity and changes in abundance are desired.

  3. Direct quantitative analysis of nicotine alkaloids from biofluid samples using paper spray mass spectrometry.

    PubMed

    Wang, He; Ren, Yue; McLuckey, Morgan N; Manicke, Nicholas E; Park, Jonghyuck; Zheng, Lingxing; Shi, Riyi; Cooks, R Graham; Ouyang, Zheng

    2013-12-03

    The determination of tobacco derived nicotine alkaloids in biofluid samples is of great importance to testing for tobacco use, tobacco cessation treatment, and studies on exposure to secondhand smoke. Paper spray mass spectrometry (MS) has been adapted for direct, quantitative analysis of tobacco alkaloids from biofluid samples, such as blood, urine, and saliva in liquid and dried form. Limits of quantitation as low as several nanograms per milliliter were obtained for nicotine, cotinine, trans-3'-hydroxycotinine, and anabasine. Direct analysis of fresh blood samples has also been achieved with improved sensitivity using print paper substrates of high density. Quantitation of the cotinine in the blood of a rat was performed with both direct analysis using paper spray and a traditional analysis protocol using liquid chromatography MS. Comparable results were obtained and the precision of the two methods was similar. The paper spray MS method is rapid and shows potential for significantly improved analytical efficiency in clinical laboratories as well as for point-of-care tobacco use assessment.

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

    PubMed

    Huan, Tao; Li, Liang

    2015-07-21

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

  5. Quantitative detection of nitric oxide in exhaled human breath by extractive electrospray ionization mass spectrometry

    NASA Astrophysics Data System (ADS)

    Pan, Susu; Tian, Yong; Li, Ming; Zhao, Jiuyan; Zhu, Lanlan; Zhang, Wei; Gu, Haiwei; Wang, Haidong; Shi, Jianbo; Fang, Xiang; Li, Penghui; Chen, Huanwen

    2015-03-01

    Exhaled nitric oxide (eNO) is a useful biomarker of various physiological conditions, including asthma and other pulmonary diseases. Herein a fast and sensitive analytical method has been developed for the quantitative detection of eNO based on extractive electrospray ionization mass spectrometry (EESI-MS). Exhaled NO molecules selectively reacted with 2-phenyl-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) reagent, and eNO concentration was derived based on the EESI-MS response of 1-oxyl-2-phenyl-4, 4, 5, 5-tetramethylimidazoline (PTI) product. The method allowed quantification of eNO below ppb level (~0.02 ppbv) with a relative standard deviation (RSD) of 11.6%. In addition, eNO levels of 20 volunteers were monitored by EESI-MS over the time period of 10 hrs. Long-term eNO response to smoking a cigarette was recorded, and the observed time-dependent profile was discussed. This work extends the application of EESI-MS to small molecules (<30 Da) with low proton affinity and collision-induced dissociation efficiency, which are usually poorly visible by conventional ion trap mass spectrometers. Long-term quantitative profiling of eNO by EESI-MS opens new possibilities for the research of human metabolism and clinical diagnosis.

  6. Field-assisted paper spray mass spectrometry for the quantitative evaluation of imatinib levels in plasma.

    PubMed

    D'Aronco, Sara; Calandra, Eleonora; Crotti, Sara; Toffoli, Giuseppe; Marangon, Elena; Posocco, Bianca; Traldi, Pietro; Agostini, Marco

    2016-01-01

    Drug levels in patients' bloodstreams vary among individuals and consequently therapeutic drug monitoring (TDM) is fundamental to controlling the effective therapeutic range. For TDM purposes, different analytical approaches have been used, mainly based on immunoassay, liquid chromatography- ultraviolet, liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. More recently a matrix-assisted laser desorption/ionisation method has been proposed for the determination of irinotecan levels in the plasma of subjects under therapy and this method has been cross- validated by comparison with data achieved by LC-MS/MS. However, to reach an effective point-of-care monitoring of plasma drug concentrations, a TDM platform technology for fast, accurate, low-cost assays is required. In this frame, recently the use of paper spray mass spectrometry, which is becoming a popular and widely employed MS method, has been proposed. In this paper we report the results obtained by the development of a paper spray-based method for quantitative analysis in plasma samples of imatinib, a new generation of anticancer drug. Preliminary experiments showed that poor sensitivity, reproducibility and linear response were obtained by the "classical" paper spray set-up. In order to achieve better results, it was thought of interest to operate in presence of a higher and more homogeneous electrical field. For this aim, a stainless steel needle connected with the high voltage power supply was mounted below the paper triangle. Furthermore, in order to obtain valid quantitative data, we analysed the role of the different equilibria participating to the phenomena occurring in paper spray experiments, depending either on instrumental parameters or on the chemical nature of analyte and solvents. A calibration curve was obtained by spiking plasma samples containing different amounts of imatinib (1) with known amounts of deuterated imatinib (1d3) as

  7. Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication

    PubMed Central

    Gunning, Yvonne; Watson, Andrew D.; Rigby, Neil M.; Philo, Mark; Peazer, Joshua K.; Kemsley, E. Kate

    2016-01-01

    We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. Central to the method is the identification of 'corresponding proteins' in the species of interest, in other words proteins that are nominally the same but possess species-specific sequence differences. When subject to proteolysis, corresponding proteins will give rise to some peptides which are likewise similar but with species-specific variants. These are 'corresponding peptides'. Species-specific peptides can be used as markers for species determination, while pairs of corresponding peptides permit relative quantitation of two species in a mixture. The peptides are detected using multiple reaction monitoring (MRM) mass spectrometry, a highly specific technique that enables peptide-based species determination even in complex systems. In addition, the ratio of MRM peak areas deriving from corresponding peptides supports relative quantitation. Since corresponding proteins and peptides will, in the main, behave similarly in both processing and in experimental extraction and sample preparation, the relative quantitation should remain comparatively robust. In addition, this approach does not need the standards and calibrations required by absolute quantitation methods. The protocol is described in the context of red meats, which have convenient corresponding proteins in the form of their respective myoglobins. This application is relevant to food fraud detection: the method can detect 1% weight for weight of horse meat in beef. The corresponding protein, corresponding peptide (CPCP) relative quantitation using MRM peak area ratios gives good estimates of the weight for weight composition of a horse plus beef mixture. PMID:27685654

  8. Quantitative Analysis of Tetramethylenedisulfotetramine ("Tetramine") Spiked into Beverages by Liquid Chromatography Tandem Mass Spectrometry with Validation by Gas Chromatography Mass Spectrometry

    SciTech Connect

    Owens, J; Hok, S; Alcaraz, A; Koester, C

    2008-11-13

    Tetramethylenedisulfotetramine, commonly known as tetramine, is a highly neurotoxic rodenticide (human oral LD{sub 50} = 0.1 mg/kg) used in hundreds of deliberate food poisoning events in China. Here we describe a method for quantitation of tetramine spiked into beverages, including milk, juice, tea, cola, and water and cleaned up by C8 solid phase extraction and liquid-liquid extraction. Quantitation by high performance liquid chromatography tandem mass spectrometry (LC/MS/MS) was based upon fragmentation of m/z 347 to m/z 268. The method was validated by gas chromatography mass spectrometry (GC/MS) operated in SIM mode for ions m/z 212, 240, and 360. The limit of quantitation was 0.10 {micro}g/mL by LC/MS/MS versus 0.15 {micro}g/mL for GC/MS. Fortifications of the beverages at 2.5 {micro}g/mL and 0.25 {micro}g/mL were recovered ranging from 73-128% by liquid-liquid extraction for GC/MS analysis, 13-96% by SPE and 10-101% by liquid-liquid extraction for LC/MS/MS analysis.

  9. MSQuant, an open source platform for mass spectrometry-based quantitative proteomics.

    PubMed

    Mortensen, Peter; Gouw, Joost W; Olsen, Jesper V; Ong, Shao-En; Rigbolt, Kristoffer T G; Bunkenborg, Jakob; Cox, Jürgen; Foster, Leonard J; Heck, Albert J R; Blagoev, Blagoy; Andersen, Jens S; Mann, Matthias

    2010-01-01

    Mass spectrometry-based proteomics critically depends on algorithms for data interpretation. A current bottleneck in the rapid advance of proteomics technology is the closed nature and slow development cycle of vendor-supplied software solutions. We have created an open source software environment, called MSQuant, which allows visualization and validation of peptide identification results directly on the raw mass spectrometric data. MSQuant iteratively recalibrates MS data thereby significantly increasing mass accuracy leading to fewer false positive peptide identifications. Algorithms to increase data quality include an MS(3) score for peptide identification and a post-translational modification (PTM) score that determines the probability that a modification such as phosphorylation is placed at a specific residue in an identified peptide. MSQuant supports relative protein quantitation based on precursor ion intensities, including element labels (e.g., (15)N), residue labels (e.g., SILAC and ICAT), termini labels (e.g., (18)O), functional group labels (e.g., mTRAQ), and label-free ion intensity approaches. MSQuant is available, including an installer and supporting scripts, at http://msquant.sourceforge.net .

  10. Quantitation of benzodiazepines in whole blood by electron impact-gas chromatography-mass spectrometry.

    PubMed

    Tiscione, Nicholas B; Shan, Xiaoqin; Alford, Ilene; Yeatman, Dustin Tate

    2008-10-01

    Benzodiazepines are frequently encountered in forensic toxicology. A literature search was conducted to find a simple method using electron impact-gas chromatography-mass spectrometry (EI-GC-MS) to examine whole blood specimens for the most commonly encountered benzodiazepines in the United States. A recently published method was identified in the literature search and used as a starting point for development of a new procedure to be used for routine analysis of forensic toxicology case samples. The procedure was then developed and validated as a rapid and efficient method for the screening and quantitation of benzodiazepines in blood using liquid-liquid extraction and EI-GC-MS in selective ion monitoring mode. Materials and instrumentation common to most forensic toxicology laboratories were utilized while obtaining LODs from 5 to 50 ng/mL and LOQs of 50 ng/mL or less using 1 mL of sample. Target compounds were chosen based on availability and common use in the United States and include diazepam, desalkylflurazepam, nordiazepam, midazolam, oxazepam, temazepam, lorazepam, clonazepam, and alprazolam (relative elution order). The linear range (r2 > 0.990) was validated from 50 to 1000 ng/mL for all analytes. The CV of replicate analyses at both 50 and 200 ng/mL was less than 4%. Quantitative accuracy was within +/- 16% at 50 ng/mL and within +/- 7% at 200 ng/mL. The validated method provides an efficient procedure for the quantitation of a broad range of the most common benzodiazepines in blood at meaningful limits of detection and quantitation using standard laboratory equipment and a small amount of sample.

  11. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Applications in Quantitative Proteomics.

    PubMed

    Chahrour, Osama; Malone, John

    2016-12-12

    Recent advances in inductively coupled plasma mass spectrometry (ICP-MS) hyphenated to different separation techniques have promoted it as a valuable tool in protein/peptide quantification. These emerging ICP-MS applications allow absolute quantification by measuring specific elemental responses. One approach quantifies elements already present in the structure of the target peptide (e.g. phosphorus and sulphur) as natural tags. Quantification of these natural tags allows the elucidation of the degree of protein phosphorylation in addition to absolute protein quantification. A separate approach is based on utilising bi-functional labelling substances (those containing ICP-MS detectable elements), that form a covalent chemical bond with the protein thus creating analogs which are detectable by ICP-MS. Based on the previously established stoichiometries of the labelling reagents, quantification can be achieved. This technique is very useful for the design of precise multiplexed quantitation schemes to address the challenges of biomarker screening and discovery. This review discusses the capabilities and different strategies to implement ICP-MS in the field of quantitative proteomics.

  12. Quantitative cross-linking/mass spectrometry reveals subtle protein conformational changes

    PubMed Central

    2016-01-01

    Quantitative cross-linking/mass spectrometry (QCLMS) probes protein structural dynamics in solution by quantitatively comparing the yields of cross-links between different conformational statuses. We have used QCLMS to understand the final maturation step of the proteasome lid and also to elucidate the structure of complement C3(H2O). Here we benchmark our workflow using a structurally well-described reference system, the human complement protein C3 and its activated cleavage product C3b. We found that small local conformational changes affect the yields of cross-linking residues that are near in space while larger conformational changes affect the detectability of cross-links. Distinguishing between minor and major changes required robust analysis based on replica analysis and a label-swapping procedure. By providing workflow, code of practice and a framework for semi-automated data processing, we lay the foundation for QCLMS as a tool to monitor the domain choreography that drives binary switching in many protein-protein interaction networks. PMID:27976756

  13. Quantitative analysis of chemical warfare agent degradation products in beverages by liquid chromatography tandem mass spectrometry.

    PubMed

    Owens, Janel; Koester, Carolyn

    2009-09-23

    Though chemical warfare agents (CWAs) have been banned by the Chemical Weapons Convention, the threat that such chemicals may be used, including their deliberate addition to food, remains. In such matrixes, CWAs may hydrolyze to phosphonic acids, which are good surrogate markers of CWA contamination. The method described here details the extraction of five CWA degradation products, including methylphosphonic acid (MPA), ethyl-MPA, isopropyl-MPA, cyclohexyl-MPA, and pinacolyl-MPA, from five different beverages by strata-X solid phase extraction cartridges. Samples were analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS) with multiple reaction monitoring. The limit of quantitation ranged from 0.05 to 0.5 ng on-column, and the limit of detection was >0.02 ng on-column. Beverages were fortified with the five phosphonic acids at 1 microg/mL and 0.25 microg/mL and quantitated using both an internally standardized method and matrix-matched standards. Reasonable recoveries (>50%) were achieved for ethyl, isopropyl, cyclohexyl, and pinacolyl-MPA for most matrixes.

  14. Quantitation of the Noncovalent Cellular Retinol-Binding Protein, Type 1 Complex Through Native Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Li, Wenjing; Yu, Jianshi; Kane, Maureen A.

    2017-01-01

    Native mass spectrometry (MS) has become a valuable tool in probing noncovalent protein-ligand interactions in a sample-efficient way, yet the quantitative application potential of native MS has not been fully explored. Cellular retinol binding protein, type I (CrbpI) chaperones retinol and retinal in the cell, protecting them from nonspecific oxidation and delivering them to biosynthesis enzymes where the bound (holo-) and unbound (apo-) forms of CrbpI exert distinct biological functions. Using nanoelectrospray, we developed a native MS assay for probing apo- and holo-CrbpI abundance to facilitate exploring their biological functions in retinoid metabolism and signaling. The methods were developed on two platforms, an Orbitrap-based Thermo Exactive and a Q-IMS-TOF-based Waters Synapt G2S, where similar ion behaviors under optimized conditions were observed. Overall, our results suggested that within the working range ( 1-10 μM), gas-phase ions in the native state linearly correspond to solution concentration and relative ion intensities of the apo- and holo-protein ions can linearly respond to the solution ratios, suggesting native MS is a viable tool for relative quantitation in this system.

  15. High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry

    PubMed Central

    Lechene, Claude; Hillion, Francois; McMahon, Greg; Benson, Douglas; Kleinfeld, Alan M; Kampf, J Patrick; Distel, Daniel; Luyten, Yvette; Bonventre, Joseph; Hentschel, Dirk; Park, Kwon Moo; Ito, Susumu; Schwartz, Martin; Benichou, Gilles; Slodzian, Georges

    2006-01-01

    Background Secondary-ion mass spectrometry (SIMS) is an important tool for investigating isotopic composition in the chemical and materials sciences, but its use in biology has been limited by technical considerations. Multi-isotope imaging mass spectrometry (MIMS), which combines a new generation of SIMS instrument with sophisticated ion optics, labeling with stable isotopes, and quantitative image-analysis software, was developed to study biological materials. Results The new instrument allows the production of mass images of high lateral resolution (down to 33 nm), as well as the counting or imaging of several isotopes simultaneously. As MIMS can distinguish between ions of very similar mass, such as 12C15N- and 13C14N-, it enables the precise and reproducible measurement of isotope ratios, and thus of the levels of enrichment in specific isotopic labels, within volumes of less than a cubic micrometer. The sensitivity of MIMS is at least 1,000 times that of 14C autoradiography. The depth resolution can be smaller than 1 nm because only a few atomic layers are needed to create an atomic mass image. We illustrate the use of MIMS to image unlabeled mammalian cultured cells and tissue sections; to analyze fatty-acid transport in adipocyte lipid droplets using 13C-oleic acid; to examine nitrogen fixation in bacteria using 15N gaseous nitrogen; to measure levels of protein renewal in the cochlea and in post-ischemic kidney cells using 15N-leucine; to study DNA and RNA co-distribution and uridine incorporation in the nucleolus using 15N-uridine and 81Br of bromodeoxyuridine or 14C-thymidine; to reveal domains in cultured endothelial cells using the native isotopes 12C, 16O, 14N and 31P; and to track a few 15N-labeled donor spleen cells in the lymph nodes of the host mouse. Conclusion MIMS makes it possible for the first time to both image and quantify molecules labeled with stable or radioactive isotopes within subcellular compartments. PMID:17010211

  16. Quantitation of Phenol Levels in Oil of Wintergreen Using Gas Chromatography-Mass Spectrometry with Selected Ion Monitoring

    ERIC Educational Resources Information Center

    Sobel, Robert M.; Ballantine, David S.; Ryzhov, Victor

    2005-01-01

    Industrial application of gas chromatography-mass spectrometry (GC-MS) analysis is a powerful technique that could be used to elucidate components of a complex mixture while offering the benefits of high-precision quantitative analysis. The natural wintergreen oil is examined for its phenol concentration to determine the level of refining…

  17. Quantitative Histone Mass Spectrometry Identifies Elevated Histone H3 Lysine 27 (Lys27) Trimethylation in Melanoma*

    PubMed Central

    Sengupta, Deepanwita; Byrum, Stephanie D.; Avaritt, Nathan L.; Davis, Lauren; Shields, Bradley; Mahmoud, Fade; Reynolds, Matthew; Orr, Lisa M.; Mackintosh, Samuel G.; Shalin, Sara C.; Tackett, Alan J.

    2016-01-01

    Normal cell growth is characterized by a regulated epigenetic program that drives cellular activities such as gene transcription, DNA replication, and DNA damage repair. Perturbation of this epigenetic program can lead to events such as mis-regulation of gene transcription and diseases such as cancer. To begin to understand the epigenetic program correlated to the development of melanoma, we performed a novel quantitative mass spectrometric analysis of histone post-translational modifications mis-regulated in melanoma cell culture as well as patient tumors. Aggressive melanoma cell lines as well as metastatic melanoma were found to have elevated histone H3 Lys27 trimethylation (H3K27me3) accompanied by overexpressed methyltransferase EZH2 that adds the specific modification. The altered epigenetic program that led to elevated H3K27me3 in melanoma cell culture was found to directly silence transcription of the tumor suppressor genes RUNX3 and E-cadherin. The EZH2-mediated silencing of RUNX3 and E-cadherin transcription was also validated in advanced stage human melanoma tissues. This is the first study focusing on the detailed epigenetic mechanisms leading to EZH2-mediated silencing of RUNX3 and E-cadherin tumor suppressors in melanoma. This study underscores the utility of using high resolution mass spectrometry to identify mis-regulated epigenetic programs in diseases such as cancer, which could ultimately lead to the identification of biological markers for diagnostic and prognostic applications. PMID:26621846

  18. Quantitation of opioids in whole blood by electron impact-gas chromatography-mass spectrometry.

    PubMed

    Tiscione, Nicholas B; Shan, Xiaoqin; Alford, Ilene; Yeatman, Dustin Tate

    2011-03-01

    Opioids are frequently encountered in Forensic Toxicology casework. A PubMed literature search was conducted to find a method using electron impact-gas chromatography-mass spectrometry to examine whole blood specimens. A previously published method was identified, and an updated version was provided by the State of North Carolina Office of the Chief Medical Examiner. This procedure was used as a starting point for development and validation of a refined procedure to be used in the Palm Beach County Sheriff's Office Forensic Toxicology laboratory for routine analysis of antemortem forensic toxicology case samples. Materials and instrumentation common to most forensic toxicology laboratories were utilized while obtaining detection limits from 1 to 10 ng/mL and quantitation limits of 2.5 to 10 ng/mL using 1 mL of whole blood. Target compounds were chosen based on applicability to the method as well as availability and common use in the United States and include dihydrocodeine, codeine, morphine, hydrocodone, 6-monoacetylmorphine, hydromorphone, oxycodone, and oxymorphone. Each analyte demonstrated two zero-order linear ranges (r(2) > 0.990) over the concentrations evaluated (from 2.5 to 500 ng/mL). The coefficient of variation of replicate analyses was less than 12%. Quantitative accuracy was within ± 27% at 2.5 ng/mL, ± 11% at 10 ng/mL, and ± 8% at 50 ng/mL. The validated method provides a more sensitive procedure for the quantitation of common opioids in blood using standard laboratory equipment and a small amount of sample.

  19. The role of quantitative mass spectrometry in the discovery of pancreatic cancer biomarkers for translational science

    PubMed Central

    2014-01-01

    In the post-genomic era, it has become evident that genetic changes alone are not sufficient to understand most disease processes including pancreatic cancer. Genome sequencing has revealed a complex set of genetic alterations in pancreatic cancer such as point mutations, chromosomal losses, gene amplifications and telomere shortening that drive cancerous growth through specific signaling pathways. Proteome-based approaches are important complements to genomic data and provide crucial information of the target driver molecules and their post-translational modifications. By applying quantitative mass spectrometry, this is an alternative way to identify biomarkers for early diagnosis and personalized medicine. We review the current quantitative mass spectrometric technologies and analyses that have been developed and applied in the last decade in the context of pancreatic cancer. Examples of candidate biomarkers that have been identified from these pancreas studies include among others, asporin, CD9, CXC chemokine ligand 7, fibronectin 1, galectin-1, gelsolin, intercellular adhesion molecule 1, insulin-like growth factor binding protein 2, metalloproteinase inhibitor 1, stromal cell derived factor 4, and transforming growth factor beta-induced protein. Many of these proteins are involved in various steps in pancreatic tumor progression including cell proliferation, adhesion, migration, invasion, metastasis, immune response and angiogenesis. These new protein candidates may provide essential information for the development of protein diagnostics and targeted therapies. We further argue that new strategies must be advanced and established for the integration of proteomic, transcriptomic and genomic data, in order to enhance biomarker translation. Large scale studies with meta data processing will pave the way for novel and unexpected correlations within pancreatic cancer, that will benefit the patient, with targeted treatment. PMID:24708694

  20. The role of quantitative mass spectrometry in the discovery of pancreatic cancer biomarkers for translational science.

    PubMed

    Ansari, Daniel; Aronsson, Linus; Sasor, Agata; Welinder, Charlotte; Rezeli, Melinda; Marko-Varga, György; Andersson, Roland

    2014-04-05

    In the post-genomic era, it has become evident that genetic changes alone are not sufficient to understand most disease processes including pancreatic cancer. Genome sequencing has revealed a complex set of genetic alterations in pancreatic cancer such as point mutations, chromosomal losses, gene amplifications and telomere shortening that drive cancerous growth through specific signaling pathways. Proteome-based approaches are important complements to genomic data and provide crucial information of the target driver molecules and their post-translational modifications. By applying quantitative mass spectrometry, this is an alternative way to identify biomarkers for early diagnosis and personalized medicine. We review the current quantitative mass spectrometric technologies and analyses that have been developed and applied in the last decade in the context of pancreatic cancer. Examples of candidate biomarkers that have been identified from these pancreas studies include among others, asporin, CD9, CXC chemokine ligand 7, fibronectin 1, galectin-1, gelsolin, intercellular adhesion molecule 1, insulin-like growth factor binding protein 2, metalloproteinase inhibitor 1, stromal cell derived factor 4, and transforming growth factor beta-induced protein. Many of these proteins are involved in various steps in pancreatic tumor progression including cell proliferation, adhesion, migration, invasion, metastasis, immune response and angiogenesis. These new protein candidates may provide essential information for the development of protein diagnostics and targeted therapies. We further argue that new strategies must be advanced and established for the integration of proteomic, transcriptomic and genomic data, in order to enhance biomarker translation. Large scale studies with meta data processing will pave the way for novel and unexpected correlations within pancreatic cancer, that will benefit the patient, with targeted treatment.

  1. Comparison of Different Time of Flight-Mass Spectrometry Modes for Small Molecule Quantitative Analysis.

    PubMed

    Chindarkar, Nandkishor S; Park, Hyung-Doo; Stone, Judith A; Fitzgerald, Robert L

    2015-01-01

    Currently, the use of time of flight (TOF)-mass spectrometry (MS) in quantitative analysis of small molecules is rare. Recently, the quantitative performance of TOF mass analyzers has improved due to the advancements in TOF technology. We evaluated a Q-TOF-MS in different modes, i.e., Q-TOF-full scan (Q-TOF-FS), Q-TOF-enhanced-full scan (Q-TOF-En-FS), MS(E), Q-TOF-targeted (Q-TOF-TGT), Q-TOF-enhanced-targeted (Q-TOF-En-TGT), and compared their quantitative performance against a unit resolution LC-MS-MS (tandem quadrupole) platform. The five modes were investigated for sensitivity, linearity, signal-to-noise ratio, recovery and precision using 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THC-COOH) as a model compound in electrospray ionization (ESI) with negative polarity. Preliminary studies indicated that Q-TOF-FS mode was the least linear and precise; hence, it was eliminated from further investigation. Total imprecision in remaining four modes was <10%. The Q-TOF-En-FS and Q-TOF-En-TGT showed better signal intensity than their respective modes without enhancement. Overall, peak signal intensity was the highest in MS(E) mode, whereas the signal-to-noise ratio was the best in the Q-TOF-En-TGT mode. Relatively, MS(E) and Q-TOF-En-TGT modes were the best overall performers compared with the other modes. Both MS(E) and Q-TOF-En-TGT modes showed excellent precision (coefficient of variation <6%), patient correlation (r > 0.99) and linearity (range, 5-455 ng/mL) for THC-COOH analysis when compared with LC-MS-MS. We also investigated the performance of the same four modes using methamphetamine in positive ESI. Quantitative data obtained by Q-TOF-En-TGT and MS(E), using both positive and negative ESI, suggest that these modes performed better than the other modes. While unit resolution LC-MS-MS remains the optimal technique for quantification, our data showed that Q-TOF-MS can also be used to quantify small molecules in complex biological specimens.

  2. A quantitative multiplexed mass spectrometry assay for studying the kinetic of residue-specific histone acetylation.

    PubMed

    Kuo, Yin-Ming; Henry, Ryan A; Andrews, Andrew J

    2014-12-01

    Histone acetylation is involved in gene regulation and, most importantly, aberrant regulation of histone acetylation is correlated with major human diseases. Although many lysine acetyltransferases (KATs) have been characterized as being capable of acetylating multiple lysine residues on histones, how different factors such as enzyme complexes or external stimuli (e.g. KAT activators or inhibitors) alter KAT specificity remains elusive. In order to comprehensively understand how the homeostasis of histone acetylation is maintained, a method that can quantitate acetylation levels of individual lysines on histones is needed. Here we demonstrate that our mass spectrometry (MS)-based method accomplishes this goal. In addition, the high throughput, high sensitivity, and high dynamic range of this method allows for effectively and accurately studying steady-state kinetics. Based on the kinetic parameters from in vitro enzymatic assays, we can determine the specificity and selectivity of a KAT and use this information to understand what factors influence histone acetylation. These approaches can be used to study the enzymatic mechanisms of histone acetylation as well as be adapted to other histone modifications. Understanding the post-translational modification of individual residues within the histones will provide a better picture of chromatin regulation in the cell.

  3. Quantitative analysis of denatured collagen by collagenase digestion and subsequent MALDI-TOF mass spectrometry.

    PubMed

    Nimptsch, Ariane; Schibur, Stephanie; Ihling, Christian; Sinz, Andrea; Riemer, Thomas; Huster, Daniel; Schiller, Jürgen

    2011-03-01

    Collagens are the most abundant proteins in vertebrate tissues and constitute significant moieties of the extracellular matrix (ECM). The determination of the collagen content is of relevance not only in the field of native tissue research, but also regarding the quality assessment of bioengineered tissues. Here, we describe a quantitative method to assess small amounts of collagen based on MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry (MS) subsequent to digestion of collagen with clostridial collagenase (clostridiopeptidase A) in order to obtain characteristic oligopeptides. Among the resulting peptides, Gly-Pro-Hyp, which is highly indicative of collagen, has been used to assess the amount of collagen by comparing the Gly-Pro-Hyp peak intensities with the intensities of a spiked tripeptide (Arg-Gly-Asp). The approach presented herein is both simple and convenient and allows the determination of collagen in microgram quantities. In tissue samples such as cartilage, the actual collagen content has additionally been determined for comparative purposes by nuclear magnetic resonance spectroscopy subsequent to acidic hydrolysis. Both methods give consistent data within an experimental error of ±10%. Although the differentiation of the different collagen types cannot be achieved by this approach, the overall collagen contents of tissues can be easily determined.

  4. Liquid chromatography/tandem mass spectrometry method for quantitation of cremophor el and its applications.

    PubMed

    Vijaya Bhaskar, V; Middha, Anil

    2013-01-01

    A rapid sensitive and selective MRM based method for the determination of Cremophor EL (CrEL) in rat plasma was developed using liquid chromatography/tandem mass spectrometry (LC-MS/MS). CrEL and polypropylene glycol (internal standard) were extracted from rat plasma with acetonitrile and analysed on C18 column (XBridge, 50 × 4.6 mm, 3.5  μ m). The most abundant molecular ions corresponding to PEG oligomers at m/z 828, 872, 916 and 960 with daughter ion at m/z 89 were selected for multiple reaction monitoring (MRM) in electrospray mode of ionisation. Plasma concentrations of CrEL were quantified after administration through oral and intravenous routes in male sprague dawley rats at a dose of 0.26 g/kg. The standard curve was linear (0.9972) over the concentration range of 1.00 to 200  μ g/mL. The lower limit of quantitation for CrEL was 1.00  μ g/mL using 50  μ L plasma. The coefficient of variation and relative error for inter and intra assay at three QC levels were 0.69 to 9.21 and -7.60 to 4.74 respectively. A novel proposal was conveyed to the scientific community, where formulation excipient can be analysed as qualifier in the analysis of NCEs to address the spiky plasma concentration profiles.

  5. Quantitative lipidomic analysis of plasma and plasma lipoproteins using MALDI-TOF mass spectrometry.

    PubMed

    Serna, Jorge; García-Seisdedos, David; Alcázar, Alberto; Lasunción, Miguel Ángel; Busto, Rebeca; Pastor, Óscar

    2015-07-01

    Knowledge of the plasma lipid composition is essential to clarify the specific roles of different lipid species in various pathophysiological processes. In this study, we developed an analytical strategy combining high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) and off-line coupling with matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF/MS) to determine the composition of plasma and major lipoproteins at two levels, lipid classes and lipid species. We confirmed the suitability of MALDI-TOF/MS as a quantitative measurement tool studying the linearity and repeatability for triglycerides (TG), phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Moreover, data obtained with this method were correlated with other lipid classes and species measurements using currently available technologies. To establish the potential utility of our approach, human plasma very low density- (VLDL), low density- (LDL) and high density- (HDL) lipoproteins from 10 healthy donors were separated using ultracentrifugation, and compositions of nine lipid classes, cholesteryl esters (CE), TG, free cholesterol (FC), PE, phosphatidylinositol (PI), sulfatides (S), PC, lysophosphatidylcholine (LPC) and sphingomyelin (SM), analyzed. In total, 157 lipid species in plasma, 182 in LDL, 171 in HDL, and 148 in VLDL were quantified. The lipidomic profile was consistent with known differences in lipid classes, but also revealed unexpected differences in lipid species distribution of lipoproteins, particularly for LPC and SM. In summary, the methodology developed in this study constitutes a valid approach to determine the lipidomic composition of plasma and lipoproteins.

  6. Quantitation of a recombinant monoclonal antibody in monkey serum by liquid chromatography-mass spectrometry.

    PubMed

    Liu, Hongcheng; Manuilov, Anton V; Chumsae, Chris; Babineau, Michelle L; Tarcsa, Edit

    2011-07-01

    A method including protein A purification, limited Lys-C digestion, and mass spectrometry analysis was used in the study to quantify a recombinant monoclonal antibody in cynomolgus monkey serum. The same antibody that was isotopically labeled was used as an internal standard. Interferences from serum proteins were first significantly reduced by protein A purification and then by limited Lys-C digestion of protein A bound IgG, including both monkey and the recombinant IgG. Fab fragment of the recombinant human IgG was analyzed directly by LC-MS, while monkey IgG and the Fc fragment of the recombinant human IgG remained bound to protein A resin. Quantitation was achieved by measuring the peak intensity of the Fab from the recombinant human IgG and comparing it to that of the Fab from the stable isotope-labeled internal standard. The results were in good agreement with the values from ELISA. LC-MS can therefore be used as a complementary approach to ELISA to quantify recombinant monoclonal antibodies in serum for pharmacokinetics studies and it can also be used where specific reagents such as antigens are not readily available for ELISA.

  7. Large-scale human skin lipidomics by quantitative, high-throughput shotgun mass spectrometry

    PubMed Central

    Sadowski, Tomasz; Klose, Christian; Gerl, Mathias J.; Wójcik-Maciejewicz, Anna; Herzog, Ronny; Simons, Kai; Reich, Adam; Surma, Michal A.

    2017-01-01

    The lipid composition of human skin is essential for its function; however the simultaneous quantification of a wide range of stratum corneum (SC) and sebaceous lipids is not trivial. We developed and validated a quantitative high-throughput shotgun mass spectrometry-based platform for lipid analysis of tape-stripped SC skin samples. It features coverage of 16 lipid classes; total quantification to the level of individual lipid molecules; high reproducibility and high-throughput capabilities. With this method we conducted a large lipidomic survey of 268 human SC samples, where we investigated the relationship between sampling depth and lipid composition, lipidome variability in samples from 14 different sampling sites on the human body and finally, we assessed the impact of age and sex on lipidome variability in 104 healthy subjects. We found sebaceous lipids to constitute an abundant component of the SC lipidome as they diffuse into the topmost SC layers forming a gradient. Lipidomic variability with respect to sampling depth, site and subject is considerable, and mainly accredited to sebaceous lipids, while stratum corneum lipids vary less. This stresses the importance of sampling design and the role of sebaceous lipids in skin studies. PMID:28266621

  8. Mass spectrometry-based quantitative metabolomics revealed a distinct lipid profile in breast cancer patients.

    PubMed

    Qiu, Yunping; Zhou, Bingsen; Su, Mingming; Baxter, Sarah; Zheng, Xiaojiao; Zhao, Xueqing; Yen, Yun; Jia, Wei

    2013-04-12

    Breast cancer accounts for the largest number of newly diagnosed cases in female cancer patients. Although mammography is a powerful screening tool, about 20% of breast cancer cases cannot be detected by this method. New diagnostic biomarkers for breast cancer are necessary. Here, we used a mass spectrometry-based quantitative metabolomics method to analyze plasma samples from 55 breast cancer patients and 25 healthy controls. A number of 30 patients and 20 age-matched healthy controls were used as a training dataset to establish a diagnostic model and to identify potential biomarkers. The remaining samples were used as a validation dataset to evaluate the predictive accuracy for the established model. Distinct separation was obtained from an orthogonal partial least squares-discriminant analysis (OPLS-DA) model with good prediction accuracy. Based on this analysis, 39 differentiating metabolites were identified, including significantly lower levels of lysophosphatidylcholines and higher levels of sphingomyelins in the plasma samples obtained from breast cancer patients compared with healthy controls. Using logical regression, a diagnostic equation based on three metabolites (lysoPC a C16:0, PC ae C42:5 and PC aa C34:2) successfully differentiated breast cancer patients from healthy controls, with a sensitivity of 98.1% and a specificity of 96.0%.

  9. Identification of CRM1-dependent Nuclear Export Cargos Using Quantitative Mass Spectrometry.

    PubMed

    Thakar, Ketan; Karaca, Samir; Port, Sarah A; Urlaub, Henning; Kehlenbach, Ralph H

    2013-03-01

    Chromosome region maintenance 1/exportin1/Exp1/Xpo1 (CRM1) is the major transport receptor for the export of proteins from the nucleus. It binds to nuclear export signals (NESs) that are rich in leucines and other hydrophobic amino acids. The prediction of NESs is difficult because of the extreme recognition flexibility of CRM1. Furthermore, proteins can be exported upon binding to an NES-containing adaptor protein. Here we present an approach for identifying targets of the CRM1-export pathway via quantitative mass spectrometry using stable isotope labeling with amino acids in cell culture. With this approach, we identified >100 proteins from HeLa cells that were depleted from cytosolic fractions and/or enriched in nuclear fractions in the presence of the selective CRM1-inhibitor leptomycin B. Novel and validated substrates are the polyubiquitin-binding protein sequestosome 1, the cancerous inhibitor of protein phosphatase 2A (PP2A), the guanine nucleotide-binding protein-like 3-like protein, the programmed cell death protein 2-like protein, and the cytosolic carboxypeptidase 1 (CCP1). We identified a functional NES in CCP1 that mediates direct binding to the export receptor CRM1. The method will be applicable to other nucleocytoplasmic transport pathways, as well as to the analysis of nucleocytoplasmic shuttling proteins under different growth conditions.

  10. Quantitative Detection of Trace Malachite Green in Aquiculture Water Samples by Extractive Electrospray Ionization Mass Spectrometry

    PubMed Central

    Fang, Xiaowei; Yang, Shuiping; Chingin, Konstantin; Zhu, Liang; Zhang, Xinglei; Zhou, Zhiquan; Zhao, Zhanfeng

    2016-01-01

    Exposure to malachite green (MG) may pose great health risks to humans; thus, it is of prime importance to develop fast and robust methods to quantitatively screen the presence of malachite green in water. Herein the application of extractive electrospray ionization mass spectrometry (EESI-MS) has been extended to the trace detection of MG within lake water and aquiculture water, due to the intensive use of MG as a biocide in fisheries. This method has the advantage of obviating offline liquid-liquid extraction or tedious matrix separation prior to the measurement of malachite green in native aqueous medium. The experimental results indicate that the extrapolated detection limit for MG was ~3.8 μg·L−1 (S/N = 3) in lake water samples and ~0.5 μg·L−1 in ultrapure water under optimized experimental conditions. The signal intensity of MG showed good linearity over the concentration range of 10–1000 μg·L−1. Measurement of practical water samples fortified with MG at 0.01, 0.1 and 1.0 mg·L−1 gave a good validation of the established calibration curve. The average recoveries and relative standard deviation (RSD) of malachite green in lake water and Carassius carassius fish farm effluent water were 115% (6.64% RSD), 85.4% (9.17% RSD) and 96.0% (7.44% RSD), respectively. Overall, the established EESI-MS/MS method has been demonstrated suitable for sensitive and rapid (<2 min per sample) quantitative detection of malachite green in various aqueous media, indicating its potential for online real-time monitoring of real life samples. PMID:27529262

  11. Quantitative Detection of Trace Malachite Green in Aquiculture Water Samples by Extractive Electrospray Ionization Mass Spectrometry.

    PubMed

    Fang, Xiaowei; Yang, Shuiping; Chingin, Konstantin; Zhu, Liang; Zhang, Xinglei; Zhou, Zhiquan; Zhao, Zhanfeng

    2016-08-11

    Exposure to malachite green (MG) may pose great health risks to humans; thus, it is of prime importance to develop fast and robust methods to quantitatively screen the presence of malachite green in water. Herein the application of extractive electrospray ionization mass spectrometry (EESI-MS) has been extended to the trace detection of MG within lake water and aquiculture water, due to the intensive use of MG as a biocide in fisheries. This method has the advantage of obviating offline liquid-liquid extraction or tedious matrix separation prior to the measurement of malachite green in native aqueous medium. The experimental results indicate that the extrapolated detection limit for MG was ~3.8 μg·L(-1) (S/N = 3) in lake water samples and ~0.5 μg·L(-1) in ultrapure water under optimized experimental conditions. The signal intensity of MG showed good linearity over the concentration range of 10-1000 μg·L(-1). Measurement of practical water samples fortified with MG at 0.01, 0.1 and 1.0 mg·L(-1) gave a good validation of the established calibration curve. The average recoveries and relative standard deviation (RSD) of malachite green in lake water and Carassius carassius fish farm effluent water were 115% (6.64% RSD), 85.4% (9.17% RSD) and 96.0% (7.44% RSD), respectively. Overall, the established EESI-MS/MS method has been demonstrated suitable for sensitive and rapid (<2 min per sample) quantitative detection of malachite green in various aqueous media, indicating its potential for online real-time monitoring of real life samples.

  12. Quantitative mass spectrometry measurements reveal stoichiometry of principal postsynaptic density proteins.

    PubMed

    Lowenthal, Mark S; Markey, Sanford P; Dosemeci, Ayse

    2015-06-05

    Quantitative studies are presented of postsynaptic density (PSD) fractions from rat cerebral cortex with the ultimate goal of defining the average copy numbers of proteins in the PSD complex. Highly specific and selective isotope dilution mass spectrometry assays were developed using isotopically labeled polypeptide concatemer internal standards. Interpretation of PSD protein stoichiometry was achieved as a molar ratio with respect to PSD-95 (SAP-90, DLG4), and subsequently, copy numbers were estimated using a consensus literature value for PSD-95. Average copy numbers for several proteins at the PSD were estimated for the first time, including those for AIDA-1, BRAGs, and densin. Major findings include evidence for the high copy number of AIDA-1 in the PSD (144 ± 30)-equivalent to that of the total GKAP family of proteins (150 ± 27)-suggesting that AIDA-1 is an element of the PSD scaffold. The average copy numbers for NMDA receptor sub-units were estimated to be 66 ± 18, 27 ± 9, and 45 ± 15, respectively, for GluN1, GluN2A, and GluN2B, yielding a total of 34 ± 10 NMDA channels. Estimated average copy numbers for AMPA channels and their auxiliary sub-units TARPs were 68 ± 36 and 144 ± 38, respectively, with a stoichiometry of ∼1:2, supporting the assertion that most AMPA receptors anchor to the PSD via TARP sub-units. This robust, quantitative analysis of PSD proteins improves upon and extends the list of major PSD components with assigned average copy numbers in the ongoing effort to unravel the complex molecular architecture of the PSD.

  13. Quantitative Analysis of Human Salivary Gland-Derived Intact Proteome Using Top-Down Mass Spectrometry

    SciTech Connect

    Wu, Si; Brown, Joseph N.; Tolic, Nikola; Meng, Da; Liu, Xiaowen; Zhang, Haizhen; Zhao, Rui; Moore, Ronald J.; Pevzner, Pavel A.; Smith, Richard D.; Pasa-Tolic, Ljiljana

    2014-05-31

    There are several notable challenges inherent to fully characterizing the entirety of the human saliva proteome using bottom-up approaches, including polymorphic isoforms, post-translational modifications, unique splice variants, deletions, and truncations. To address these challenges, we have developed a top-down based liquid chromatography-mass spectrometry (LC-MS) approach, which cataloged 20 major human salivary proteins with a total of 83 proteoforms, containing a broad range of post-translational modifications. Among these proteins, several previously reported disease biomarker proteins were identified at the intact protein level, such as beta-2 microglobulin (B2M). In addition, intact glycosylated proteoforms of several saliva proteins were also characterized, including intact N-glycosylated protein prolactin inducible protein (PIP) and O-glycosylated acidic protein rich protein (aPRP). These characterized proteoforms constitute an intact saliva proteoform database, which was used for quantitative comparison of intact salivary proteoforms among six healthy individuals. Human parotid (PS) and submandibular/sublingual gland (SMSL) secretion samples (2 μg of protein each) from six healthy individuals were compared using RPLC coupled with the 12T FTICR mass spectrometer. Significantly different protein and PTM patterns were resolved with high reproducibility between PS and SMSL glands. The results from this study provide further insight into the potential mechanisms of PTM pathways in oral glandular secretion, expanding our knowledge of this complex yet easily accessible fluid. Intact protein LC-MS approach presented herein can potentially be applied for rapid and accurate identification of biomarkers from only a few microliters of human glandular saliva.

  14. Complex mixtures of antibodies generated from a single production qualitatively and quantitatively evaluated by native Orbitrap mass spectrometry.

    PubMed

    Thompson, Natalie J; Hendriks, Linda J A; de Kruif, John; Throsby, Mark; Heck, Albert J R

    2014-01-01

    Composite antibody mixtures designed to combat diseases present a new, rapidly emerging technology in the field of biopharmaceuticals. The combination of multiple antibodies can lead to increased effector response and limit the effect of escape variants that can propagate the disease. However, parallel development of analytical technologies is required to provide fast, thorough, accurate, and robust characterization of these mixtures. Here, we evaluate the utility of native mass spectrometry on an Orbitrap platform with high mass resolving power to characterize composite mixtures of up to 15 separate antibodies. With this technique, unambiguous identification of each antibody in the mixtures was achieved. Mass measurements of the intact antibodies varied 7 ppm on average, allowing highly reproducible identification and quantitation of each compound in these complex mixtures. We show that with the high mass-resolving power and robustness of this technology, high-resolution native mass spectrometry can be used efficiently even for batch-to batch characterization.

  15. Mass Spectrometry-Based Quantitative Strategies for Assessing the Biological Consequences and Repair of DNA Adducts.

    PubMed

    You, Changjun; Wang, Yinsheng

    2016-02-16

    The genetic integrity of living organisms is constantly threatened by environmental and endogenous sources of DNA damaging agents that can induce a plethora of chemically modified DNA lesions. Unrepaired DNA lesions may elicit cytotoxic and mutagenic effects and contribute to the development of human diseases including cancer and neurodegeneration. Understanding the deleterious outcomes of DNA damage necessitates the investigation about the effects of DNA adducts on the efficiency and fidelity of DNA replication and transcription. Conventional methods for measuring lesion-induced replicative or transcriptional alterations often require time-consuming colony screening and DNA sequencing procedures. Recently, a series of mass spectrometry (MS)-based strategies have been developed in our laboratory as an efficient platform for qualitative and quantitative analyses of the changes in genetic information induced by DNA adducts during DNA replication and transcription. During the past few years, we have successfully used these MS-based methods for assessing the replicative or transcriptional blocking and miscoding properties of more than 30 distinct DNA adducts. When combined with genetic manipulation, these methods have also been successfully employed for revealing the roles of various DNA repair proteins or translesion synthesis DNA polymerases (Pols) in modulating the adverse effects of DNA lesions on transcription or replication in mammalian and bacterial cells. For instance, we found that Escherichia coli Pol IV and its mammalian ortholog (i.e., Pol κ) are required for error-free bypass of N(2)-(1-carboxyethyl)-2'-deoxyguanosine (N(2)-CEdG) in cells. We also found that the N(2)-CEdG lesions strongly inhibit DNA transcription and they are repaired by transcription-coupled nucleotide excision repair in mammalian cells. In this Account, we focus on the development of MS-based approaches for determining the effects of DNA adducts on DNA replication and transcription

  16. Quantitative mass spectrometry reveals plasticity of metabolic networks in Mycobacterium smegmatis.

    PubMed

    Chopra, Tarun; Hamelin, Romain; Armand, Florence; Chiappe, Diego; Moniatte, Marc; McKinney, John D

    2014-11-01

    Mycobacterium tuberculosis has a remarkable ability to persist within the human host as a clinically inapparent or chronically active infection. Fatty acids are thought to be an important carbon source used by the bacteria during long term infection. Catabolism of fatty acids requires reprogramming of metabolic networks, and enzymes central to this reprogramming have been targeted for drug discovery. Mycobacterium smegmatis, a nonpathogenic relative of M. tuberculosis, is often used as a model system because of the similarity of basic cellular processes in these two species. Here, we take a quantitative proteomics-based approach to achieve a global view of how the M. smegmatis metabolic network adjusts to utilization of fatty acids as a carbon source. Two-dimensional liquid chromatography and mass spectrometry of isotopically labeled proteins identified a total of 3,067 proteins with high confidence. This number corresponds to 44% of the predicted M. smegmatis proteome and includes most of the predicted metabolic enzymes. Compared with glucose-grown cells, 162 proteins showed differential abundance in acetate- or propionate-grown cells. Among these, acetate-grown cells showed a higher abundance of proteins that could constitute a functional glycerate pathway. Gene inactivation experiments confirmed that both the glyoxylate shunt and the glycerate pathway are operational in M. smegmatis. In addition to proteins with annotated functions, we demonstrate carbon source-dependent differential abundance of proteins that have not been functionally characterized. These proteins might play as-yet-unidentified roles in mycobacterial carbon metabolism. This study reveals several novel features of carbon assimilation in M. smegmatis, which suggests significant functional plasticity of metabolic networks in this organism.

  17. Quantitation of Cotinine in Nonsmoker Saliva Using Chip Based Nanoelectrospray Tandem Mass Spectrometry

    SciTech Connect

    Tomkins, Bruce A; Van Berkel, Gary J; Jenkins, Roger A; Counts, Richard Wayne

    2006-01-01

    A new analytical procedure was developed for the quantitation of nonsmoker salivary cotinine. Small volumes of saliva were diluted with water, fortified with cotinine-d{sub 3} (internal standard), then passed through small extraction columns. The analyte and internal standard were eluted with 0.1% (v/v) acetic acid/acetonitrile. Aliquots of each extract were analyzed directly, without chromatographic separation, using chip-based (NanoMate{sup TM}) nanospray tandem mass spectrometry. The calculated detection limit was 0.49 ng cotinine/mL saliva. This method was used to quantify salivary cotinine collected from nonsmoking human subjects living in one of three environmental tobacco smoke (ETS) exposure categories or 'cells': 1. smoking home/smoking workplace; 2. smoking home/nonsmoking workplace; and 3. nonsmoking home/smoking workplace. Samples were collected during five sequential days, including Saturday, as part of a larger study to evaluate potential variability in exposure to ETS. Salivary cotinine measurements were made for the purpose of excluding misclassified smokers and for comparison with known levels of exposure to airborne nicotine in each exposure category. The concentrations observed were consistent with those reported from other large studies reported elsewhere. A non-parametric statistical test was applied to the data within each cell. No statistically significant differences were found between the mean cotinine concentrations collected on a weekday as compared to those collected on a weekend day. When the non-parametric test was applied to the three cells, a statistically significant difference was observed between cell 1 compared to cells 2 and 3. The salivary cotinine concentrations were thus statistically invariant over a five-day exposure period, and they were greatest under the conditions of smoking home and smoking workplace.

  18. Towards cracking the epigenetic code using a combination of high-throughput epigenomics and quantitative mass spectrometry-based proteomics.

    PubMed

    Stunnenberg, Hendrik G; Vermeulen, Michiel

    2011-07-01

    High-throughput genomic sequencing and quantitative mass spectrometry (MS)-based proteomics technology have recently emerged as powerful tools, increasing our understanding of chromatin structure and function. Both of these approaches require substantial investments and expertise in terms of instrumentation, experimental methodology, bioinformatics, and data interpretation and are, therefore, usually applied independently from each other by dedicated research groups. However, when applied reiteratively in the context of epigenetics research these approaches are strongly synergistic in nature.

  19. Quantitative bioanalysis of strontium in human serum by inductively coupled plasma-mass spectrometry

    PubMed Central

    Somarouthu, Srikanth; Ohh, Jayoung; Shaked, Jonathan; Cunico, Robert L; Yakatan, Gerald; Corritori, Suzana; Tami, Joe; Foehr, Erik D

    2015-01-01

    Aim: A bioanalytical method using inductively-coupled plasma-mass spectrometry to measure endogenous levels of strontium in human serum was developed and validated. Results & methodology: This article details the experimental procedures used for the method development and validation thus demonstrating the application of the inductively-coupled plasma-mass spectrometry method for quantification of strontium in human serum samples. The assay was validated for specificity, linearity, accuracy, precision, recovery and stability. Significant endogenous levels of strontium are present in human serum samples ranging from 19 to 96 ng/ml with a mean of 34.6 ± 15.2 ng/ml (SD). Discussion & conclusion: Calibration procedures and sample pretreatment were simplified for high throughput analysis. The validation demonstrates that the method was sensitive, selective for quantification of strontium (88Sr) and is suitable for routine clinical testing of strontium in human serum samples. PMID:28031925

  20. MASS SPECTROMETRY

    DOEpatents

    Nier, A.O.C.

    1959-08-25

    A voltage switching apparatus is described for use with a mass spectrometer in the concentratron analysis of several components of a gas mixture. The system automatically varies the voltage on the accelerating electrode of the mass spectrometer through a program of voltages which corresponds to the particular gas components under analysis. Automatic operation may be discontinued at any time to permit the operator to manually select any desired predetermined accelerating voltage. Further, the system may be manually adjusted to vary the accelerating voltage over a wide range.

  1. Quantitation of aflatoxins from corn and other food related materials by direct analysis in real time - mass spectrometry (DART-MS)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ambient ionization coupled to mass spectrometry continues to be applied to new analytical problems, facilitating the rapid and convenient analysis of a variety of analytes. Recently, demonstrations of ambient ionization mass spectrometry applied to quantitative analysis of mycotoxins have been shown...

  2. Analysis on the go: quantitation of drugs of abuse in dried urine with digital microfluidics and miniature mass spectrometry.

    PubMed

    Kirby, Andrea E; Lafrenière, Nelson M; Seale, Brendon; Hendricks, Paul I; Cooks, R Graham; Wheeler, Aaron R

    2014-06-17

    We report the development of a method coupling microfluidics and a miniature mass spectrometer, applied to quantitation of drugs of abuse in urine. A custom digital microfluidic system was designed to deliver droplets of solvent to dried urine samples and then transport extracted analytes to an array of nanoelectrospray emitters for analysis. Tandem mass spectrometry (MS/MS) detection was performed using a fully autonomous 25 kg instrument. Using the new method, cocaine, benzoylecgonine, and codeine can be quantified from four samples in less than 15 min from (dried) sample to analysis. The figures of merit for the new method suggest that it is suitable for on-site screening; for example, the limit of quantitation (LOQ) for cocaine is 40 ng/mL, which is compatible with the performance criteria for laboratory analyses established by the United Nations Office on Drugs and Crime. More importantly, the LOQ of the new method is superior to the 300 ng/mL cutoff values used by the only other portable analysis systems we are aware of (relying on immunoassays). This work serves as a proof-of-concept for integration of microfluidics with miniature mass spectrometry. The system is attractive for the quantitation of drugs of abuse from urine and, more generally, may be useful for a wide range of applications that would benefit from portable, quantitative, on-site analysis.

  3. MASS SPECTROMETRY

    DOEpatents

    Friedman, L.

    1962-01-01

    method is described for operating a mass spectrometer to improve its resolution qualities and to extend its period of use substantially between cleanings. In this method, a small amount of a beta emitting gas such as hydrogen titride or carbon-14 methane is added to the sample being supplied to the spectrometer for investigation. The additive establishes leakage paths on the surface of the non-conducting film accumulating within the vacuum chamber of the spectrometer, thereby reducing the effect of an accumulated static charge on the electrostatic and magnetic fields established within the instrument. (AEC)

  4. Fast quantitative detection of cocaine in beverages using nanoextractive electrospray ionization tandem mass spectrometry.

    PubMed

    Hu, Bin; Peng, Xuejiao; Yang, Shuiping; Gu, Haiwei; Chen, Huanwen; Huan, Yanfu; Zhang, Tingting; Qiao, Xiaolin

    2010-02-01

    Without any sample pretreatment, effervescent beverage fluids were manually sprayed into the primary ion plume created by using a nanoelectrospray ionization source for direct ionization, and the analyte ions of interest were guided into an ion trap mass spectrometer for tandem mass analysis. Functional ingredients (e.g., vitamins, taurine, and caffeine, etc.) and spiked impurity (e.g., cocaine) in various beverages, such as Red Bull energy drink, Coco-cola, and Pepsi samples were rapidly identified within 1.5 s. The limit of detection was found to be 7-15 fg (S/N = 3) for cocaine in different samples using the characteristic fragment (m/z 150) observed in the MS(3) experiments. Typical relative standard deviation and recovery of this method were 6.9%-8.6% and 104%-108% for direct analysis of three actual samples, showing that nanoextractive electrospray ionization tandem mass spectrometry is a useful technique for fast screening cocaine presence in beverages.

  5. Qualitative and quantitative metabolomic investigation of single neurons by capillary electrophoresis electrospray ionization mass spectrometry

    PubMed Central

    Nemes, Peter; Rubakhin, Stanislav S.; Aerts, Jordan T.; Sweedler, Jonathan V.

    2013-01-01

    Single-cell mass spectrometry (MS) empowers metabolomic investigations by decreasing analytical dimensions to the size of individual cells and subcellular structures. We describe a protocol for investigating and quantifying metabolites in individual isolated neurons using single-cell capillary electrophoresis hyphenated to electrospray ionization time-of-flight MS. The protocol requires ~2 h for sample preparation, neuron isolation, and metabolite extraction, and 1 h for metabolic measurement. The approach was used to detect more than 300 distinct compounds in the mass range of typical metabolites in various individual neurons (25–500-µm in diameter) isolated from the sea slug (Aplysia californica) central and rat (Rattus norvegicus) peripheral nervous systems. A subset of identified compounds was sufficient to reveal metabolic differences among freshly isolated neurons of different types and changes in the metabolite profiles of cultured neurons. The protocol can be applied to the characterization of the metabolome in a variety of smaller cells and/or subcellular domains. PMID:23538882

  6. Quantitative analysis of biomolecules by time-of-flight secondary-ion mass spectrometry: Fundamental considerations

    SciTech Connect

    Muddiman, D.C.; Nicola, A.J.; Proctor, A.

    1995-12-31

    Static Time-of-Flight Secondary-Ion Mass Spectrometry (TOF-SIMS) has been applied to investigate an extensive assortment of analytical systems; from semiconductors to DNA sequencing. Recently, the TOF-SIMS method has been successfully applied to real biological systems. This report focuses on some important aspects that must be taken into consideration when conducting measurements on biomaterials in order to observe the potential the TOF-SIMS method affords. The current data are presented using Cyclosporin A (CsA, 1202 Da) and cocaine (303 Da) as model compounds. CsA is observed in the TOF-SIMS mass spectrum predominately as a Ag-cationized species and cocaine as a protonated species; thus, they are complementary probe molecules.

  7. Biological Matrix Effects in Quantitative Tandem Mass Spectrometry-Based Analytical Methods: Advancing Biomonitoring

    PubMed Central

    Panuwet, Parinya; Hunter, Ronald E.; D’Souza, Priya E.; Chen, Xianyu; Radford, Samantha A.; Cohen, Jordan R.; Marder, M. Elizabeth; Kartavenka, Kostya; Ryan, P. Barry; Barr, Dana Boyd

    2015-01-01

    The ability to quantify levels of target analytes in biological samples accurately and precisely, in biomonitoring, involves the use of highly sensitive and selective instrumentation such as tandem mass spectrometers and a thorough understanding of highly variable matrix effects. Typically, matrix effects are caused by co-eluting matrix components that alter the ionization of target analytes as well as the chromatographic response of target analytes, leading to reduced or increased sensitivity of the analysis. Thus, before the desired accuracy and precision standards of laboratory data are achieved, these effects must be characterized and controlled. Here we present our review and observations of matrix effects encountered during the validation and implementation of tandem mass spectrometry-based analytical methods. We also provide systematic, comprehensive laboratory strategies needed to control challenges posed by matrix effects in order to ensure delivery of the most accurate data for biomonitoring studies assessing exposure to environmental toxicants. PMID:25562585

  8. Chemical modification of deoxyribonucleic acids: Quantitation of 3-methylthymidine and O4-methylthymidine by tandem mass spectrometry

    NASA Astrophysics Data System (ADS)

    Wood, Joe M.; Hoke, Steven H., II; Graham Cooks, R.; Chae, Whi-Gun; Chang, Ching-Jer

    1991-12-01

    Quantitation of 3-methylthymidine and O4-methylthymidine generated in the reaction of calf thymus DNA with methyl methanesulfonate (MeMS) and 1-methyl-1nitrosourea (MeNU) by mass spectrometry is reported. Quantitative precision of 7% or better is achieved on samples of 10-12 -10-13 mole in the HPLC and a final stage of separation before quantification by tandem mass spectrometry using desorption chemical ionization. Synthetic CD3-labeled nucleosides were used as internal standards for mass spectral quantification. A unique mass spectrometric scanning procedure, which allowed simultaneous MS--MS product ion analysis of both the analyte and the internal standard, was utilized to enchance precision and accuracy in these low level determinations. MeNU (a potent carcinogen) resulted in 18&%; 3-methylation and 0.17% O4-methylation of deoxythymidine whereas MeMS (a weak carcinogen) produced only 6.8% 3-methylation and 0.005% of deoxythymidine. These results demonstrate that the sensitivity and accuracy of this method should be adequate for the detection and quantification of methyl-nucleosides at the sub-picomole level at which mutation is induced in cell cultures.

  9. Quantitative analysis of phenibut in rat brain tissue extracts by liquid chromatography-tandem mass spectrometry.

    PubMed

    Grinberga, Solveiga; Zvejniece, Liga; Liepinsh, Edgars; Dambrova, Maija; Pugovics, Osvalds

    2008-12-01

    Phenibut (3-phenyl-4-aminobutyric acid) is a gamma-aminobutyric acid mimetic drug, which is used clinically as a mood elevator and tranquilizer. In the present work, a rapid, selective and sensitive liquid chromatography-tandem mass spectrometry method for quantification of phenibut in biological matrices has been developed. The method is based on protein precipitation with acidic acetonitrile followed by isocratic chromatographic separation using acetonitrile-formic acid (0.1% in water; 8:92, v/v) mobile phase on a reversed-phase column. Detection of the analyte was performed by electrospray ionization mass spectrometry in multiple reaction monitoring mode with the precursor-to-product ion transition m/z 180.3 --> m/z 117.2. The calibration curve was linear over the concentration range 50-2000 ng/mL. The lower limit of quantification for phenibut in rat brain extracts was 50 ng/mL. Acceptable precision and accuracy were obtained over the whole concentration range. The validated method was successfully applied in a pharmacological study to analyze phenibut concentration in rat brain tissue extract samples.

  10. Quantitative mass spectrometry in proteomics: critical review update from 2007 to the present.

    PubMed

    Bantscheff, Marcus; Lemeer, Simone; Savitski, Mikhail M; Kuster, Bernhard

    2012-09-01

    Mass-spectrometry-based proteomics is continuing to make major contributions to the discovery of fundamental biological processes and, more recently, has also developed into an assay platform capable of measuring hundreds to thousands of proteins in any biological system. The field has progressed at an amazing rate over the past five years in terms of technology as well as the breadth and depth of applications in all areas of the life sciences. Some of the technical approaches that were at an experimental stage back then are considered the gold standard today, and the community is learning to come to grips with the volume and complexity of the data generated. The revolution in DNA/RNA sequencing technology extends the reach of proteomic research to practically any species, and the notion that mass spectrometry has the potential to eventually retire the western blot is no longer in the realm of science fiction. In this review, we focus on the major technical and conceptual developments since 2007 and illustrate these by important recent applications.

  11. Liquid chromatography tandem mass spectrometry applied to quantitation of the organophosphorus nerve agent VX in microdialysates from blood probes.

    PubMed

    Stubbs, S J; Read, R W

    2010-05-15

    VX (O-ethyl-S-[2(di-isopropylamino)ethyl] methylphosphonothiolate) is a low volatility organophosphorus (OP) nerve agent and therefore the most likely route of exposure is via percutaneous absorption. Microdialysis has been used as a tool to study percutaneous poisoning by VX in the anesthetised guinea pig. A liquid chromatography tandem mass spectrometry (LC-MS-MS) method using positive electrospray ionisation (ESI) was used to quantitate VX in microdialysate samples collected from microdialysis probes, implanted into a blood vessel of anesthetised guinea pigs. The method resulted from modification of a LC-MS-MS method previously developed for the analysis of dermal microdialysates. Modification increased the sensitivity of the method, allowing quantitation of the trace levels of VX in blood microdialysates, over the range 0.002-1 ng/ml, with linear calibration. Quantitative results have been used to determine the time course of VX concentrations in the blood of guinea pigs following percutaneous poisoning.

  12. The quantitative surface analysis of an antioxidant additive in a lubricant oil matrix by desorption electrospray ionization mass spectrometry

    PubMed Central

    Da Costa, Caitlyn; Reynolds, James C; Whitmarsh, Samuel; Lynch, Tom; Creaser, Colin S

    2013-01-01

    RATIONALE Chemical additives are incorporated into commercial lubricant oils to modify the physical and chemical properties of the lubricant. The quantitative analysis of additives in oil-based lubricants deposited on a surface without extraction of the sample from the surface presents a challenge. The potential of desorption electrospray ionization mass spectrometry (DESI-MS) for the quantitative surface analysis of an oil additive in a complex oil lubricant matrix without sample extraction has been evaluated. METHODS The quantitative surface analysis of the antioxidant additive octyl (4-hydroxy-3,5-di-tert-butylphenyl)propionate in an oil lubricant matrix was carried out by DESI-MS in the presence of 2-(pentyloxy)ethyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate as an internal standard. A quadrupole/time-of-flight mass spectrometer fitted with an in-house modified ion source enabling non-proximal DESI-MS was used for the analyses. RESULTS An eight-point calibration curve ranging from 1 to 80 µg/spot of octyl (4-hydroxy-3,5-di-tert-butylphenyl)propionate in an oil lubricant matrix and in the presence of the internal standard was used to determine the quantitative response of the DESI-MS method. The sensitivity and repeatability of the technique were assessed by conducting replicate analyses at each concentration. The limit of detection was determined to be 11 ng/mm2 additive on spot with relative standard deviations in the range 3–14%. CONCLUSIONS The application of DESI-MS to the direct, quantitative surface analysis of a commercial lubricant additive in a native oil lubricant matrix is demonstrated. © 2013 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons, Ltd. PMID:24097398

  13. Quantitative Organic Acids in Urine by Two Dimensional Gas Chromatography-Time of Flight Mass Spectrometry (GCxGC-TOFMS).

    PubMed

    Sweetman, Lawrence; Ashcraft, Paula; Bennett-Firmin, Jeanna

    2016-01-01

    Seventy-six organic acids in urine specimens are determined with quantitative two dimensional Gas Chromatography-Time of Flight Mass Spectrometry (GCxGC-TOFMS). The specimen is treated with urease to remove urea then derivatized to form pentafluorobenzyl oximes (PFBO) of oxoacids. The sample is then treated with ethyl alcohol to precipitate proteins and centrifuged. After drying the supernatant, the organic acids are derivatized to form volatile trimethylsilyl (TMS) derivatives for separation by capillary two dimensional Gas Chromatography (GCxGC) with temperature programming and modulation. Detection is by Time of Flight Mass Spectrometry (TOFMS) with identification of the organic acids by their mass spectra. Organic acids are quantitated by peak areas of reconstructed ion chromatograms with internal standards and calibration curves. Organic acids are quantified to determine abnormal patterns for the diagnosis of more than 100 inherited disorders of organic acid metabolism. Characteristic abnormal metabolites are quantified to monitor dietary and other modes of treatment for patients who are diagnosed with specific organic acid disorders.

  14. Hybrid Quadrupole-Orbitrap mass spectrometry for quantitative measurement of quorum sensing inhibition.

    PubMed

    Todd, Daniel A; Zich, David B; Ettefagh, Keivan A; Kavanaugh, Jeffrey S; Horswill, Alexander R; Cech, Nadja B

    2016-08-01

    Drug resistant bacterial infections cause significant morbidity and mortality worldwide, and new strategies are needed for the treatment of these infections. The anti-virulence approach, which targets non-essential virulence factors in bacteria, has been proposed as one way to combat the problem of antibiotic resistance. Virulence in methicillin-resistant Staphylococcus aureus (MRSA) and many other Gram-positive bacterial pathogens is controlled by the quorum sensing system. Thus, there is excellent therapeutic potential for compounds that target this system. With this project, we have developed and validated a novel approach for measuring quorum sensing inhibition in vitro. Ultraperformance liquid chromatography coupled to mass spectrometry (UPLC-MS) was employed to directly measure one of the important outputs of the quorum sensing system in MRSA, auto-inducing peptide I (AIP I) in bacterial cultures. The method for AIP detection was validated and demonstrated limits of detection and quantification of range of 0.0035μM and 0.10μM, respectively. It was shown that the known quorum sensing inhibitor ambuic acid inhibited AIP I production by a clinically relevant strain of MRSA, with an IC50 value of 2.6±0.2μM. The new method performed similarly to previously published methods using GFP reporter assays, but has the advantage of being applicable without the need for engineering of a reporter strain. Additionally, the mass spectrometry-based method could be applicable in situations where interference by the inhibitor prevents the application of fluorescence-based methods.

  15. Quantitative Analysis and Fingerprint Profiles for Quality Control of Fructus Schisandrae by Gas Chromatography: Mass Spectrometry

    PubMed Central

    Xia, Yong-Gang; Yang, Bing-You; Liang, Jun; Yang, Qi; Wang, Di; Kuang, Hai-Xue

    2014-01-01

    This paper describes a simple, rapid, and effective quality assessment method for Fructus Schisandrae by gas chromatography-mass spectrum (GC-MS). The method was established by using specific lignan fingerprint profiles and quantitation of characteristic compounds in this herbal medicine. The GC-MS fingerprints of 15 batches of Schisandra samples from different regions of China showed similar lignan profiles. Five peaks were selected as characteristic peaks, and all of these were identified by using GC-MS techniques. The relative retention times of these characteristic peaks in the GC-MS fingerprint were established as an important parameter for identification of Schisandra samples. Meanwhile, relative peak areas may be a feasible approach to discriminate the S. chinensis and S. sphenanthera. Finally, these pharmacologically active constituents in the titled plant, schisandrins A–C and schizandrols A and B, were quantitatively determined using a validated GC-MS method. PMID:24574919

  16. A quantitative assay for reductive metabolism of a pesticide in fish using electrochemistry coupled with liquid chromatography tandem mass spectrometry.

    PubMed

    Bussy, Ugo; Chung-Davidson, Yu-Wen; Li, Ke; Li, Weiming

    2015-04-07

    This is the first study to use electrochemistry to generate a nitro reduction metabolite as a standard for a liquid chromatography-mass spectrometry-based quantitative assay. This approach is further used to quantify 3-trifluoromethyl-4-nitrophenol (TFM) reductive metabolism. TFM is a widely used pesticide for the population control of sea lamprey (Petromyzon marinus), an invasive species of the Laurentian Great Lakes. Three animal models, sea lamprey, lake sturgeon (Acipenser fulvescens), and rainbow trout (Oncorhynchus mykiss), were selected to evaluate TFM reductive metabolism because they have been known to show differential susceptibilities to TFM toxicity. Amino-TFM (aTFM; 3-trifluoromethyl-4-aminophenol) was the only reductive metabolite identified through liquid chromatography-high-resolution mass spectrometry screening of liver extracts incubated with TFM and was targeted for electrochemical synthesis. After synthesis and purification, aTFM was used to develop a quantitative assay of the reductive metabolism of TFM through liquid chromatography and tandem mass spectrometry. The concentrations of aTFM were measured from TFM-treated cellular fractions, including cytosolic, nuclear, membrane, and mitochondrial protein extracts. Sea lamprey extracts produced the highest concentrations (500 ng/mL) of aTFM. In addition, sea lamprey and sturgeon cytosolic extracts showed concentrations of aTFM substantially higher than those of rainbow trout. However, other fractions of lake sturgeon extracts tend to show aTFM concentrations similar to those of rainbow trout but not with sea lamprey. These data suggest that the level of reductive metabolism of TFM may be associated with the sensitivities of the animals to this particular pesticide.

  17. Quantitative analysis of synthetic polymers using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

    PubMed

    Chen, Hui; He, Meiyu; Pei, Jian; He, Haifeng

    2003-12-01

    Quantitative analyses of synthetic polymers were accomplished using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). Many factors have hindered the development of quantitative measurement of polymers via MALDI TOF MS, e.g., laser power, matrix, cation salt, and cocrystallization. By probing the optimal conditions, two sets of polymers were studied. Fair repeatability of the samples ensures acceptable results. In set 1, two poly(ethylene glycols) with different end groups showed equal desorption/ionization efficiencies. Two synthetic polymers in set 2 with different chemical properties resulted in different MALDI responses. Good linearity was achieved by plotting the relationship between the sample concentration ratio and the total signal intensity ratio in both sets.

  18. Quantitative analysis of modified proteins and their positional isomers by tandem mass spectrometry: human histone H4.

    PubMed

    Pesavento, James J; Mizzen, Craig A; Kelleher, Neil L

    2006-07-01

    Here we show that fragment ion abundances from dissociation of ions created from mixtures of multiply modified histone H4 (11 kDa) or of N-terminal synthetic peptides (2 kDa) correspond to their respective intact ion abundances measured by Fourier transform mass spectrometry. Isomeric mixtures of modified forms of the same protein are resolved and quantitated with a precision of quantitative information on the highly related and often isomeric protein forms created by combinatorial arrays of posttranslational modifications.

  19. Multiplexed Isobaric Tagging Protocols for Quantitative Mass Spectrometry Approaches to Auditory Research.

    PubMed

    Vetter, Douglas E; Basappa, Johnvesly

    2016-01-01

    Modern biologists have at their disposal a large array of techniques used to assess the existence and relative or absolute quantity of any molecule of interest in a sample. However, implementing most of these procedures can be a daunting task for the first time, even in a lab with experienced researchers. Just choosing a protocol to follow can take weeks while all of the nuances are examined and it is determined whether a protocol will (a) give the desired results, (b) result in interpretable and unbiased data, and (c) be amenable to the sample of interest. We detail here a robust procedure for labeling proteins in a complex lysate for the ultimate differential quantification of protein abundance following experimental manipulations. Following a successful outcome of the labeling procedure, the sample is submitted for mass spectrometric analysis, resulting in peptide quantification and protein identification. While we will concentrate on cells in culture, we will point out procedures that can be used for labeling lysates generated from tissues, along with any minor modifications required for such samples. We will also outline, but not fully document, other strategies used in our lab to label proteins prior to mass spectrometric analysis, and describe under which conditions each procedure may be desirable. What is not covered in this chapter is anything but the most brief introduction to mass spectrometry (instrumentation, theory, etc.), nor do we attempt to cover much in the way of software used for post hoc analysis. These two topics are dependent upon one's resources, and where applicable, one's collaborators. We strongly encourage the reader to seek out expert advice on topics not covered here.

  20. Simultaneous screening and quantitation of 18 antihistamine drugs in blood by liquid chromatography ionspray tandem mass spectrometry.

    PubMed

    Gergov, M; Robson, J N; Ojanperä, I; Heinonen, O P; Vuori, E

    2001-09-15

    A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is presented for the simultaneous screening and quantitation of 18 antihistamine drugs in blood samples. Sample pretreatment involved liquid-liquid extraction of the basic antihistamines followed by a second extraction of the acidic antihistamines. The recoveries were 43-113% for basic drugs and 23-66% for acidic drugs. The combined extracts were run by LC on C(18) reversed phase column using acetonitrile-ammonium acetate mobile phase at pH 3.2. The mass spectrometric analysis was performed with a triple stage quadrupole mass analyzer. Screening was performed using multiple reaction monitoring (MRM) and any compounds tentatively identified as antihistamine drugs were then automatedly verified by their Product Ion Spectra in a subsequent MS/MS run. Quantitation was based on the MRM data from the screening step. In validation tests, the method showed good linearity at the relevant concentrations. The attained limits of quantitation varied between 0.0005 and 0.01mg/l in blood and were lower than the therapeutic concentrations (C(max)). The limits for identification by Product Ion Spectra were also lower than C(max), except for clemastine, which has exceptionally low concentrations in blood. The intra-assay relative standard deviations were better than 10% and the inaccuracy varied between 39% for levocabastine and 5% for cyclizine, the majority of the values being <20%.

  1. Identification and quantitation of urinary dicarboxylic acids as their dicyclohexyl esters in disease states by gas chromatography mass spectrometry.

    PubMed

    Norman, E J; Berry, H K; Denton, M D

    1979-12-01

    Clinical studies were conducted by gas chromatography mass spectrometry selected ion monitoring of urinary dicarboxylic acids as dicyclohexyl esters. The dicyclohexyl esters of the dicarboxylic acids give characteristic electron impact mass spectra suitable for selected ion monitoring. The mass spectra exhibit a prominent acid + 1H ion and an (acid + 1H)-H2O ion for use as quantitating and confirming ions. The cyclohexyl esters are stable for days at room temperature and have excellent chromatographic properties. Dicarboxylic acid quantitation is performed within one hour using only 50 microliter of unpurified urine. A rapid method specifically for methylmalonic acid quantitation is described which has assisted physicians in the diagnosis of pernicious anemia and methylmalonic aciduria. This procedure is applicable for screening urinary organic acids for detection of inborn errors of metabolism. The detection of a child with elevated medium length dicarboxylic acids in the terminal urine specimen is reported. This condition, previously described as an inborn error, is attributed to a terminal event. Finally, an increase in urinary succinic acid paralleling putrescine levels is described during a response to cancer chemotherapy.

  2. Isotopologue Distributions of Peptide Product Ions by Tandem Mass Spectrometry: Quantitation of Low Levels of Deuterium Incorporation1

    PubMed Central

    Wang, Benlian; Sun, Gang; Anderson, David R.; Jia, Minghong; Previs, Stephen; Anderson, Vernon E.

    2007-01-01

    Protonated molecular peptide ions and their product ions generated by tandem mass spectrometry appear as isotopologue clusters due to the natural isotopic variations of carbon, hydrogen, nitrogen, oxygen and sulfur. Quantitation of the isotopic composition of peptides can be employed in experiments involving isotope effects, isotope exchange, isotopic labeling by chemical reactions, and studies of metabolism by stable isotope incorporation. Both ion trap and quadrupole-time of flight mass spectrometry are shown to be capable of determining the isotopic composition of peptide product ions obtained by tandem mass spectrometry with both precision and accuracy. Tandem mass spectra obtained in profile-mode of clusters of isotopologue ions are fit by non-linear least squares to a series of Gaussian peaks (described in the accompanying manuscript) which quantify the Mn/M0 values which define the isotopologue distribution (ID). To determine the isotopic composition of product ions from their ID, a new algorithm that predicts the Mn/M0 ratios is developed which obviates the need to determine the intensity of all of the ions of an ID. Consequently a precise and accurate determination of the isotopic composition a product ion may be obtained from only the initial values of the ID, however the entire isotopologue cluster must be isolated prior to fragmentation. Following optimization of the molecular ion isolation width, fragmentation energy and detector sensitivity, the presence of isotopic excess (2H, 13C, 15N, 18O) is readily determined within 1%. The ability to determine the isotopic composition of sequential product ions permits the isotopic composition of individual amino acid residues in the precursor ion to be determined. PMID:17559791

  3. Protocol: A simple protocol for quantitative analysis of bio-oils through gas- chromatography/mass spectrometry.

    PubMed

    Bartoli, Mattia; Rosi, Luca; Frediani, Marco; Frediani, Piero

    2016-01-01

    A new and simple protocol for quantitative analysis of bio-oils using gas-chromatography/mass spectrometry is suggested. Compounds were identified via their mass spectra, and then unavailable response factors were calculated with respect to diphenyl as the internal standard using a modified method previously suggested for gas chromatography with flame ionization detection. This new protocol was applied to the characterization of bio-oils obtained from the pyrolysis of woods of different sources or using different pyrolysis procedures. This protocol allowed evaluation of the yields of products from poplar pyrolysis (among 50% and 99%), while a reduced amounts of products were identified from the pyrolysis of cellulose (between 46% and 58%). The main product was always acetic acid, but it was formed in very large yields from poplar while lower yields were obtained from cellulose.

  4. Quantitative determination of trisiloxane surfactants in beehive environments based on liquid chromatography coupled to mass spectrometry.

    PubMed

    Chen, Jing; Mullin, Christopher A

    2013-08-20

    Organosilicone surfactants are increasingly being applied to agricultural agro-ecosystems as spray adjuvants, and were recently shown to impact the learning ability of honey bees. Here we developed a method for analyzing three trisiloxane surfactants (single polyethoxylate (EO) chain and end-capped with methyl, acetyl, or hydroxyl groups; TSS-CH3, TSS-COCH3, or TSS-H) in beehive matrices based on liquid chromatography coupled to mass spectrometry (LC-MS) and the QuEChERS (quick, easy, cheap, effective, rugged, and safe) approach from less than 2 g of honey, pollen, or beeswax. Recoveries for each oligomer (2-13 EO) were between 66 and 112% in all matrices. Average method detection limits (MDL) were 0.53, 0.60, 0.56 ng/g in honey, 0.63, 0.81, 0.78 ng/g in pollen, and 0.51, 0.69, 0.63 ng/g in beeswax. Five honey, 10 pollen, and 10 beeswax samples were analyzed. Trisiloxane surfactants were detected in every beeswax and 60% of the pollen samples. Total trisiloxane surfactant concentrations were up to 390 and 39 ng/g in wax and pollen. The described method is proved suitable for analyzing trisiloxane surfactants in beehive samples. The presence of trisiloxane surfactants in North American beehives calls for renewed effort to investigate the consequence of these adjuvants to bee health and the ongoing global bee decline.

  5. DetectTLC: Automated Reaction Mixture Screening Utilizing Quantitative Mass Spectrometry Image Features.

    PubMed

    Kaddi, Chanchala D; Bennett, Rachel V; Paine, Martin R L; Banks, Mitchel D; Weber, Arthur L; Fernández, Facundo M; Wang, May D

    2016-02-01

    Full characterization of complex reaction mixtures is necessary to understand mechanisms, optimize yields, and elucidate secondary reaction pathways. Molecular-level information for species in such mixtures can be readily obtained by coupling mass spectrometry imaging (MSI) with thin layer chromatography (TLC) separations. User-guided investigation of imaging data for mixture components with known m/z values is generally straightforward; however, spot detection for unknowns is highly tedious, and limits the applicability of MSI in conjunction with TLC. To accelerate imaging data mining, we developed DetectTLC, an approach that automatically identifies m/z values exhibiting TLC spot-like regions in MS molecular images. Furthermore, DetectTLC can also spatially match m/z values for spots acquired during alternating high and low collision-energy scans, pairing product ions with precursors to enhance structural identification. As an example, DetectTLC is applied to the identification and structural confirmation of unknown, yet significant, products of abiotic pyrazinone and aminopyrazine nucleoside analog synthesis. Graphical Abstract ᅟ.

  6. Commercially available antibodies can be applied in quantitative multiplexed peptide immunoaffinity enrichment targeted mass spectrometry assays

    PubMed Central

    Schoenherr, Regine M.; Zhao, Lei; Ivey, Richard G.; Voytovich, Uliana J.; Kennedy, Jacob; Yan, Ping; Lin, Chenwei; Whiteaker, Jeffrey R.; Paulovich, Amanda G.

    2016-01-01

    Immunoaffinity enrichment of peptides coupled to multiple reaction monitoring-mass spectrometry (immuno-MRM) enables highly specific, sensitive, and precise quantification of peptides and post-translational modifications. Major obstacles to developing a large number of immuno-MRM assays are the poor availability of monoclonal antibodies (mAbs) validated for immunoaffinity enrichment of peptides and the cost and lead time of developing the antibodies de novo. Although many thousands of mAbs are commercially offered, few have been tested for application to immunoaffinity enrichment of peptides. In this study we tested the success rate of using commercially available mAbs for peptide immuno-MRM assays. We selected 105 commercial mAbs (76 targeting non-modified “pan” epitopes, 29 targeting phosphorylation) to proteins associated with the DNA damage response network. We found that 8 of the 76 pan (11%) and 5 of the 29 phospho-specific mAbs (17%) captured tryptic peptides (detected by LC-MS/MS) of their protein targets from human cell lysates. Seven of these mAbs were successfully used to configure and analytically characterize immuno-MRM assays. By applying selection criteria upfront, the results indicate that a screening success rate of up to 24% is possible, establishing the feasibility of screening a large number of catalog antibodies to provide readily-available assay reagents. PMID:27094115

  7. Spatial Localization and Quantitation of Androgens in Mouse Testis by Mass Spectrometry Imaging

    PubMed Central

    2016-01-01

    Androgens are essential for male development and reproductive function. They are transported to their site of action as blood-borne endocrine hormones but can also be produced within tissues to act in intracrine and paracrine fashions. Because of this, circulating concentrations may not accurately reflect the androgenic influence within specific tissue microenvironments. Mass spectrometry imaging permits regional analysis of small molecular species directly from tissue surfaces. However, due to poor ionization and localized ion suppression, steroid hormones are difficult to detect. Here, derivatization with Girard T reagent was used to charge-tag testosterone and 5α-dihydrotestosterone allowing direct detection of these steroids in mouse testes, in both basal and maximally stimulated states, and in rat prostate. Limits of detection were ∼0.1 pg for testosterone. Exemplary detection of endogenous steroids was achieved by matrix-assisted laser desorption ionization and either Fourier transform ion cyclotron resonance detection (at 150 μm spatial resolution) or quadrupole-time-of-flight detection (at 50 μm spatial resolution). Structural confirmation was achieved by collision induced fragmentation following liquid extraction surface analysis and electrospray ionization. This application broadens the scope for derivatization strategies on tissue surfaces to elucidate local endocrine signaling in health and disease. PMID:27676129

  8. DetectTLC: Automated Reaction Mixture Screening Utilizing Quantitative Mass Spectrometry Image Features

    NASA Astrophysics Data System (ADS)

    Kaddi, Chanchala D.; Bennett, Rachel V.; Paine, Martin R. L.; Banks, Mitchel D.; Weber, Arthur L.; Fernández, Facundo M.; Wang, May D.

    2016-02-01

    Full characterization of complex reaction mixtures is necessary to understand mechanisms, optimize yields, and elucidate secondary reaction pathways. Molecular-level information for species in such mixtures can be readily obtained by coupling mass spectrometry imaging (MSI) with thin layer chromatography (TLC) separations. User-guided investigation of imaging data for mixture components with known m/z values is generally straightforward; however, spot detection for unknowns is highly tedious, and limits the applicability of MSI in conjunction with TLC. To accelerate imaging data mining, we developed DetectTLC, an approach that automatically identifies m/z values exhibiting TLC spot-like regions in MS molecular images. Furthermore, DetectTLC can also spatially match m/z values for spots acquired during alternating high and low collision-energy scans, pairing product ions with precursors to enhance structural identification. As an example, DetectTLC is applied to the identification and structural confirmation of unknown, yet significant, products of abiotic pyrazinone and aminopyrazine nucleoside analog synthesis.

  9. Quantitation of ibuprofen in blood using gas chromatography-mass spectrometry (GC-MS).

    PubMed

    Huber, Gerry; Garg, Uttam

    2010-01-01

    Ibuprofen is a non-narcotic, non-steroidal anti-inflammatory drug used for the treatment of pain, fever, and inflammatory diseases such as rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis. It is also used for induction of closure of patent ductus arteriosus (PDA) in neonates. Although the exact mechanism of action of ibuprofen is not known, it is believed to mediate its therapeutic effects through the inhibition of cyclooxygenase and subsequently by the inhibition of prostacyclin production. As the drug has a number of side effects, which correlate to its circulating concentration, monitoring of ibuprofen in plasma or serum is desired for patients receiving high-dose therapy. Chromatographic methods are frequently used for the assay of ibuprofen, as no immunoassays are currently available.In the method described, the drug is extracted from the serum or plasma using methylene chloride and phosphate buffer (pH 6). Meclofenamic acid is used as an internal standard. The organic phase containing the drug is separated and dried under stream of nitrogen. After trimethylsilyl derivatization, analysis is done using gas-chromatography/ mass spectrometry (GC-MS). Quantification of the drug in a sample is achieved by comparing responses of the unknown sample to the responses of the calibrators using selected ion monitoring.

  10. Laser Ablation/Ionisation Mass Spectrometry: Sensitive and Quantitative Chemical Depth Profiling of Solid Materials.

    PubMed

    Riedo, Andreas; Grimaudo, Valentine; Moreno-García, Pavel; Neuland, Maike B; Tulej, Marek; Broekmann, Peter; Wurz, Peter

    2016-01-01

    Direct quantitative and sensitive chemical analysis of solid materials with high spatial resolution, both in lateral and vertical direction is of high importance in various fields of analytical research, ranging from in situ space research to the semiconductor industry. Accurate knowledge of the chemical composition of solid materials allows a better understanding of physical and chemical processes that formed/altered the material and allows e.g. to further improve these processes. So far, state-of-the-art techniques such as SIMS, LA-ICP-MS or GD-MS have been applied for chemical analyses in these fields of research. In this report we review the current measurement capability and the applicability of our Laser Ablation/Ionisation Mass Spectrometer (instrument name LMS) for the chemical analysis of solids with high spatial resolution. The most recent chemical analyses conducted on various solid materials, including e.g. alloys, fossils and meteorites are discussed.

  11. Simultaneous quantitative analysis of isobars by tandem mass spectrometry from unresolved chromatographic peaks.

    PubMed

    Kushnir, Mark M; Rockwood, Alan L; Nelson, Gordon J

    2004-05-01

    A method was developed for the simultaneous quantitation of isobars from unresolved chromatographic peaks. The method is based on differences in branching ratios of ion abundances in their tandem mass spectra and an assumption that the product ion mass spectra of a mixture can be considered as a linear combination of the spectra of individual constituents. We present analytical equations and a matrix-based approach for deconvoluting the concentration of individual components from the total peak intensity for two and three isobars and also a matrix-based generalization to any number of compounds. The feasibility of the simultaneous analysis of mixtures containing two compounds was assessed. The approach was evaluated for the analysis of structural isomers of methylmalonic and succinic acids in human plasma and urine samples for a group of 270 samples. The linear regression equation, standard error and correlation coefficient for the agreement with a traditional method utilizing chromatographic separation of the isomers were y = 0.999x - 0.005, 0.024 micro mol l(-1), and 0.985, respectively. The utility of a spectral contrast angle as a predictor of analysis feasibility was evaluated.

  12. Quantitative analysis of aberrant protein glycosylation in liver cancer plasma by AAL-enrichment and MRM mass spectrometry.

    PubMed

    Ahn, Yeong Hee; Shin, Park Min; Kim, Yong-Sam; Oh, Na Ree; Ji, Eun Sun; Kim, Kwang Hoe; Lee, Yeon Jung; Kim, Sung Ho; Yoo, Jong Shin

    2013-11-07

    A lectin-coupled mass spectrometry (MS) approach was employed to quantitatively monitor aberrant protein glycosylation in liver cancer plasma. To do this, we compared the difference in the total protein abundance of a target glycoprotein between hepatocellular carcinoma (HCC) plasmas and hepatitis B virus (HBV) plasmas, as well as the difference in lectin-specific protein glycoform abundance of the target glycoprotein. Capturing the lectin-specific protein glycoforms from a plasma sample was accomplished by using a fucose-specific aleuria aurantia lectin (AAL) immobilized onto magnetic beads via a biotin-streptavidin conjugate. Following tryptic digestion of both the total plasma and its AAL-captured fraction of each HCC and HBV sample, targeted proteomic mass spectrometry was conducted quantitatively by a multiple reaction monitoring (MRM) technique. From the MRM-based analysis of the total plasmas and AAL-captured fractions, differences between HCC and HBV plasma groups in fucosylated glycoform levels of target glycoproteins were confirmed to arise from both the change in the total protein abundance of the target proteins and the change incurred by aberrant fucosylation on target glycoproteins in HCC plasma, even when no significant change occurs in the total protein abundance level. Combining the MRM-based analysis method with the lectin-capturing technique proved to be a successful means of quantitatively investigating aberrant protein glycosylation in cancer plasma samples. Additionally, it was elucidated that the differences between HCC and control groups in fucosylated biomarker candidates A1AT and FETUA mainly originated from an increase in fucosylation levels on these target glycoproteins, rather than an increase in the total protein abundance of the target glycoproteins.

  13. Rapid and High-Throughput Detection and Quantitation of Radiation Biomarkers in Human and Nonhuman Primates by Differential Mobility Spectrometry-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Chen, Zhidan; Coy, Stephen L.; Pannkuk, Evan L.; Laiakis, Evagelia C.; Hall, Adam B.; Fornace, Albert J.; Vouros, Paul

    2016-10-01

    Radiation exposure is an important public health issue due to a range of accidental and intentional threats. Prompt and effective large-scale screening and appropriate use of medical countermeasures (MCM) to mitigate radiation injury requires rapid methods for determining the radiation dose. In a number of studies, metabolomics has identified small-molecule biomarkers responding to the radiation dose. Differential mobility spectrometry-mass spectrometry (DMS-MS) has been used for similar compounds for high-throughput small-molecule detection and quantitation. In this study, we show that DMS-MS can detect and quantify two radiation biomarkers, trimethyl-L-lysine (TML) and hypoxanthine. Hypoxanthine is a human and nonhuman primate (NHP) radiation biomarker and metabolic intermediate, whereas TML is a radiation biomarker in humans but not in NHP, which is involved in carnitine synthesis. They have been analyzed by DMS-MS from urine samples after a simple strong cation exchange-solid phase extraction (SCX-SPE). The dramatic suppression of background and chemical noise provided by DMS-MS results in an approximately 10-fold reduction in time, including sample pretreatment time, compared with liquid chromatography-mass spectrometry (LC-MS). DMS-MS quantitation accuracy has been verified by validation testing for each biomarker. Human samples are not yet available, but for hypoxanthine, selected NHP urine samples (pre- and 7-d-post 10 Gy exposure) were analyzed, resulting in a mean change in concentration essentially identical to that obtained by LC-MS (fold-change 2.76 versus 2.59). These results confirm the potential of DMS-MS for field or clinical first-level rapid screening for radiation exposure.

  14. Quantitative profiling of perfluoroalkyl substances by ultrahigh-performance liquid chromatography and hybrid quadrupole time-of-flight mass spectrometry.

    PubMed

    Picó, Yolanda; Farré, Marinella; Barceló, Damià

    2015-06-01

    The accurate determination of perfluoroalkyl substances (PFSAs) in water, sediment, fish, meat, and human milk was achieved by ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QqTOF-MS) with an ABSciex Triple TOF®. A group of 21 PFSAs was selected as target to evaluate the quantitative possibilities. Full scan MS acquisition data allows quantification at relevant low levels (0.1-50 ng L(-1) in water, 0.05-2 ng g(-1) in sediments, 0.01-5 ng g(-1) in fish and meat, and 0.005-2 ng g(-1) in human milk depending on the compound). Automatic information dependent acquisition product ion mass spectrometry (IDA-MS/MS) confirms the identity even for those compounds that presented only one product ion. The preparation of a homemade database using the extracted ion chromatogram (XIC) Manager of the software based upon retention time, accurate mass, isotopic pattern, and MS/MS library searching achieves not only the successful identification of PFSAs but also of some pharmaceuticals, such as acetaminophen, ibuprofen, salicylic acid, and gemfibrozid. Mean recoveries and relative standard deviation (RSD) were 67-99% (9-16% RSD) for water, 62-103% (8-18% RSD) for sediment, 60-95% (8-17% RSD) for fish, 64-95% (8-15% RSD) for meat, and 63-95% (8-16%) for human milk. The quantitative data obtained for 60 samples by UHPLC-QqTOF-MS agree with those obtained by LC-MS/MS with a triple quadrupole (QqQ).

  15. Pathology Tissue-quantitative Mass Spectrometry Analysis to Profile Histone Post-translational Modification Patterns in Patient Samples.

    PubMed

    Noberini, Roberta; Uggetti, Andrea; Pruneri, Giancarlo; Minucci, Saverio; Bonaldi, Tiziana

    2016-03-01

    Histone post-translational modifications (hPTMs) generate a complex combinatorial code that has been implicated with various pathologies, including cancer. Dissecting such a code in physiological and diseased states may be exploited for epigenetic biomarker discovery, but hPTM analysis in clinical samples has been hindered by technical limitations. Here, we developed a method (PAThology tissue analysis of Histones by Mass Spectrometry - PAT-H-MS) that allows to perform a comprehensive, unbiased and quantitative MS-analysis of hPTM patterns on formalin-fixed paraffin-embedded (FFPE) samples. In pairwise comparisons, histone extracted from formalin-fixed paraffin-embedded tissues showed patterns similar to fresh frozen samples for 24 differentially modified peptides from histone H3. In addition, when coupled with a histone-focused version of the super-SILAC approach, this method allows the accurate quantification of modification changes among breast cancer patient samples. As an initial application of the PAThology tissue analysis of Histones by Mass Spectrometry method, we analyzed breast cancer samples, revealing significant changes in histone H3 methylation patterns among Luminal A-like and Triple Negative disease subtypes. These results pave the way for retrospective epigenetic studies that combine the power of MS-based hPTM analysis with the extensive clinical information associated with formalin-fixed paraffin-embedded archives.

  16. Calibration of laser ablation inductively coupled plasma mass spectrometry using dried solution aerosols for the quantitative analysis of solid samples

    SciTech Connect

    Leach, James

    1999-02-12

    Inductively coupled plasma mass spectrometry (ICP-MS) has become the method of choice for elemental and isotopic analysis. Several factors contribute to its success. Modern instruments are capable of routine analysis at part per trillion levels with relative detection limits in part per quadrillion levels. Sensitivities in these instruments can be as high as 200 million counts per second per part per million with linear dynamic ranges up to eight orders of magnitude. With standards for only a few elements, rapid semiquantitative analysis of over 70 elements in an individual sample can be performed. Less than 20 years after its inception ICP-MS has shown to be applicable to several areas of science. These include geochemistry, the nuclear industry, environmental chemistry, clinical chemistry, the semiconductor industry, and forensic chemistry. In this introduction, the general attributes of ICP-MS will be discussed in terms of instrumentation and sample introduction. The advantages and disadvantages of current systems are presented. A detailed description of one method of sample introduction, laser ablation, is given. The paper also gives conclusions and suggestions for future work. Chapter 2, Quantitative analysis of solids by laser ablation inductively coupled plasma mass spectrometry using dried solution aerosols for calibration, has been removed for separate processing.

  17. Simultaneous quantitation of paracetamol, caffeine, pseudoephedrine, chlorpheniramine and cloperastine in human plasma by liquid chromatography-tandem mass spectrometry.

    PubMed

    Li, Hao; Zhang, Chao; Wang, Jiang; Jiang, Yao; Fawcett, J Paul; Gu, Jingkai

    2010-02-05

    A rapid and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous quantitation of paracetamol, caffeine, pseudoephedrine, chlorpheniramine and cloperastine in human plasma has been developed and validated. After sample preparation by liquid-liquid extraction, the analytes and internal standard (diphenhydramine) were analyzed by reversed-phase HPLC on a Venusil Mp-C(18) column (50mmx4.6mm, 5microm) using formic acid:10mM ammonium acetate:methanol (1:40:60, v/v/v) as mobile phase in a run time of 2.6min. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring mode. The method was linear for all analytes over the following concentration (ng/ml) ranges: paracetamol 5.0-2000; caffeine 10-4000; pseudoephedrine 0.25-100; chlorpheniramine 0.05-20; cloperastine 0.10-40. Intra- and inter-day precisions (as relative standard deviation) were all < or =11.3% with accuracy (as relative error) of +/-5.0%. The method was successfully applied to a study of the pharmacokinetics of the five analytes after administration of a combination oral dose to healthy Chinese volunteers.

  18. Quantitation of midazolam in human plasma by automated chip-based infusion nanoelectrospray tandem mass spectrometry.

    PubMed

    Kapron, James T; Pace, Ellen; Van Pelt, Colleen K; Henion, Jack

    2003-01-01

    An automated chip-based infusion nanoelectrospray ionization (nanoESI) platform was used to demonstrate reproducible quantitation of drug molecules from biological matrices. Three sample preparation strategies were explored including protein precipitation of plasma with acetonitrile, de-salting of the plasma, and a combination of protein precipitation with subsequent de-salting of the dried and reconstituted extract. The best results were obtained when fortified human plasma samples containing midazolam were precipitated with acetonitrile containing alprazolam as the internal standard (IS). The supernatant was concentrated to dryness, reconstituted in aqueous acid, and de-salted by automated reversed-phase solid-phase extraction (SPE) prior to infusion nanoESI-MS/MS. Analyses employed a triple quadrupole mass spectrometer operated in selected reaction monitoring (SRM) mode. Each sample was infused for approximately 10 s and the resulting ion current profiles were integrated. Area ratios were used for regression analysis of standard samples (1.5-500 ng/mL). Quality control samples (3, 250, and 400 ng/mL) in five replicates from three different analysis days demonstrated intra-assay precision (< or =16%), inter-assay precision (< or =5%), and overall accuracy (+/-9% deviation). Infusion reproducibility of the assay was established by analyzing extracts after storage for 24 h at ambient temperature. Control plasma samples from six different sources probed the potential utility of this technique for the analysis of clinical samples. At the lower limit of quantitation (LLQ), variability and mean overall accuracy were < or =13% CV and +/-3% deviation, respectively, while at the upper limit of quantitation (ULQ) variability and mean overall accuracy were < or =9% CV and +/-9% deviation, respectively. Inter-chip variability was established by determining standard sample extracts across five different chips (< or =12% CV). Throughput for the assay was 55 s per sample

  19. Toxin screening in phytoplankton: detection and quantitation using MALDI triple quadrupole mass spectrometry.

    PubMed

    Sleno, Lekha; Volmer, Dietrich A

    2005-03-01

    The investigation of a MALDI triple quadrupole instrument for the analysis of spirolide toxins in phytoplankton samples is described in this study. A high-frequency (kHz) laser was employed for MALDI, generating a semicontinuous ion beam, thus taking advantage of the high duty cycle obtained in sensitive triple quadrupole MRM experiments. Initially, several experimental parameters such as type of organic matrix and concentration, solvent composition, and matrix-to-analyte ratio were optimized, and their impact on sensitivity and precision of the obtained ion currents for a reference spirolide, 13-desmethyl-C, was studied. In all quantitative experiments, excellent linearities in the concentration range between 0.01 and 1.75 microg/mL were obtained, with R2 values of 0.99 or higher. The average precision of the quantitative MALDI measurements was 7.4+/-2.4% RSD. No systematic errors were apparent with this method as shown by a direct comparison to an electrospray LC/MS/MS method. Most importantly, the MALDI technique was very fast; each sample spot was analyzed in less than 5 s as compared to several minutes with the electrospray assay. To demonstrate the potential of the MALDI triple quadrupole method, its application to quantitative analysis in several different phytoplankton samples was investigated, including crude extracts and samples from mass-triggered fractionation experiments. 13-Desmethyl spirolide C was successfully quantified in these complex samples at concentration levels from 0.05 to 90.4 microg/mL (prior to dilution to have samples fall within the dynamic range of the method) without extensive sample preparation steps. The versatility of the MALDI triple quadrupole method was also exhibited for the identification of unknown spirolide analogues. Through the use of dedicated linked scan functions such as precursor ion and neutral loss scans, several spirolide compounds were tentatively identified directly from the crude extract, without the usual time

  20. Quantitative analysis of human salivary gland-derived intact proteome using top-down mass spectrometry.

    PubMed

    Wu, Si; Brown, Joseph N; Tolić, Nikola; Meng, Da; Liu, Xiaowen; Zhang, Haizhen; Zhao, Rui; Moore, Ronald J; Pevzner, Pavel; Smith, Richard D; Paša-Tolić, Ljiljana

    2014-05-01

    There are several notable challenges inherent for fully characterizing the entirety of the human saliva proteome using bottom-up approaches, including polymorphic isoforms, PTMs, unique splice variants, deletions, and truncations. To address these challenges, we have developed a top-down based LC-MS/MS approach, which cataloged 20 major human salivary proteins with a total of 83 proteoforms, containing a broad range of PTMs. Among these proteins, several previously reported disease biomarker proteins were identified at the intact protein level, such as beta-2 microglobulin. In addition, intact glycosylated proteoforms of several saliva proteins were also characterized, including intact N-glycosylated protein prolactin inducible protein and O-glycosylated acidic protein rich protein. These characterized proteoforms constitute an intact saliva proteoform database, which was used for quantitative comparison of intact salivary proteoforms among six healthy individuals. Human parotid and submandibular/sublingual gland secretion samples (2 μg of protein each) from six healthy individuals were compared using RPLC coupled with the 12T FT-ICR mass spectrometer. Significantly different proteoform profiles were resolved with high reproducibility between parotid secretion and submandibular/sublingual glands. The results from this study provide further insight into the potential mechanisms of PTM pathways in oral glandular secretion, expanding our knowledge of this complex yet easily accessible fluid. Intact protein LC-MS approach presented herein can potentially be applied for rapid and accurate identification of biomarkers from only a few microliters of human glandular saliva.

  1. A perspective on relative quantitation of a polydisperse polymer using chromatography and mass spectrometry

    NASA Astrophysics Data System (ADS)

    Liu, X. Michael; Maziarz, E. Peter; Quinn, Edmond; Lai, Yu-Chin

    2004-11-01

    High throughput analysis of polymeric materials has become increasingly important in today's medical device industry. Direct matrix-assisted laser desorption ionisation (MALDI)-TOF MS analysis of polymers has been "the method of choice" for industrial analytical chemists due to its high speed, ease of use, and soft ionization. However, using this approach we experience difficulties for the analysis of poly(dimethyl siloxane) samples containing UV curable end groups. For example we observe a considerable amount of fragment products that act as chemical noise to the peaks of interest. This makes it difficult to obtain any meaningful quantitative information about the sample. In this study, we demonstrate that this dilemma can be remedied by coupling gel permeation chromatography (GPC) with MALDI-TOF MS analysis. With this approach a true impurity in the sample is clearly detected throughout the molecular weight distribution where direct analysis provided no information due to the chemical noise of the fragment peaks. This impurity is positively identified from exact mass measurements. The content of this impurity is calculated to be 33.0% by using a multiple data point approach from both GPC and MALDI-TOF analysis.

  2. Isotope dilution mass spectrometry for quantitative elemental analysis of powdered samples by radiofrequency pulsed glow discharge time of flight mass spectrometry.

    PubMed

    Alvarez-Toral, Aitor; Fernandez, Beatriz; Malherbe, Julien; Claverie, Fanny; Molloy, John L; Pereiro, Rosario; Sanz-Medel, Alfredo

    2013-10-15

    In recent years particular effort is being devoted to the development of pulsed glow discharges (PGDs) for mass spectrometry because this powering operation mode could offer important ionization analytical advantages. However, the capabilities of radiofrequency (RF) PGD coupled to a time of flight mass spectrometry (ToFMS) for accurate isotope ratio measurements have not been demonstrated yet. This work is focused on investigating different time positions along the pulse profile for the accurate measurement of isotope ratios. As a result, a method has been developed for the direct and simultaneous multielement determination of trace elements in powdered geological samples by RF-PGD-ToFMS in combination with isotope dilution mass spectrometry (IDMS) as an absolute measurement method directly traceable to the International System of Units. Optimized operating conditions were 70 W of applied radiofrequency power, 250 Pa of pressure, 2 ms of pulse width and 4 ms of pulse period, being argon the plasma gas used. To homogeneously distribute the added isotopically-enriched standards, lithium borate fusion of powdered solid samples was used as sample preparation approach. In this way, Cu, Zn, Ba and Pb were successfully determined by RF-PGD-ToF(IDMS) in two NIST Standard Reference Materials (SRM 2586 and SRM 2780) representing two different matrices of geological interest (soil and rock samples). Cu, Zn, Ba and Pb concentrations determined by RF-PGD-ToF(IDMS) were well in agreement with the certified values at 95% confidence interval and precisions below 12% relative standard deviation were observed for three independent analyses. Elemental concentrations investigated were in the range of 81-5770 mg/kg, demonstrating the potential of RF-PGD-ToF(IDMS) for a sensitive, accurate and robust analysis of powdered samples.

  3. Quantitative analysis of [Dmt(1)]DALDA in ovine plasma by capillary liquid chromatography-nanospray ion-trap mass spectrometry.

    PubMed

    Wan, Haibao; Umstot, Edward S; Szeto, Hazel H; Schiller, Peter W; Desiderio, Dominic M

    2004-04-15

    The synthetic opioid peptide analog Dmt-D-Arg-Phe-Lys-NH(2) ([Dmt(1)]DALDA; [Dmt= 2',6'-dimethyltyrosine) is a highly potent and selective mu opioid-receptor agonist. A very sensitive and robust capillary liquid chromatography/nanospray ion-trap (IT) mass spectrometry method has been developed to quantify [Dmt(1)]DALDA in ovine plasma, using deuterated [Dmt(1)]DALDA as the internal standard. The standard MS/MS spectra of d(0)- and d(5)-[Dmt(1)]DALDA were obtained, and the collision energy was experimentally optimized to 25%. The product ion [ M + 2H-NH(3)](2+) (m/z 312.2) was used to identify and to quantify the synthetic opioid peptide analog in ovine plasma samples. The MS/MS detection sensitivity for [Dmt(1)]DALDA was 625 amol. A calibration curve was constructed, and quantitative analysis was performed on a series of ovine plasma samples.

  4. Simultaneous quantitation of six major quassinoids in Tongkat Ali dietary supplements by liquid chromatography with tandem mass spectrometry.

    PubMed

    Han, Young Min; Jang, Moonhee; Kim, In Sook; Kim, Seung Hyun; Yoo, Hye Hyun

    2015-07-01

    Tongkat Ali (Eurycoma longifolia) is one of the most popular traditional herbs in Southeast Asia and generally consumed as forms of dietary supplements, tea, or drink additives for coffee or energy beverages. In this study, the liquid chromatography with tandem mass spectrometry method for the simultaneous quantitation of six major quassinoids of Tongkat Ali (eurycomanone, 13,21-dihydroeurycomanone, 13α(21)-epoxyeurycomanone, 14,15β-dihydroxyklaineanone, eurycomalactone, and longilactone) was developed and validated. Using the developed method, the content of the six quassinoids was measured in Tongkat Ali containing dietary supplement tablets or capsules, and the resulting data were used to confirm the presence of Tongkat Ali in those products. Among the six quassinoids, eurycomanone was the most abundant quassinoid in all samples tested. The developed method would be useful for the quality assessment of Tongkat Ali containing dietary supplements.

  5. Potato glycoalkaloids in soil-optimising liquid chromatography-time-of-flight mass spectrometry for quantitative studies.

    PubMed

    Jensen, Pia H; Juhler, René K; Nielsen, Nikoline J; Hansen, Thomas H; Strobel, Bjarne W; Jacobsen, Ole S; Nielsen, John; Hansen, Hans Christian B

    2008-02-22

    Potato glycoalkaloids are produced in high amounts in potato fields during the growth season and losses to soil potentially impact shallow groundwater and via tiles to fresh water ecosystems. A quantitative liquid chromatography-electrospray ionization time-of-flight mass spectrometry (LC-ESI-TOF-MS) method for determination and quantification of potato glycoalkaloids and their metabolites in aqueous soil extracts was developed. The LC-ESI-TOF-MS method had linearities up to 2000microg/L for alpha-solanine and alpha-chaconine and up to 760microg/L for solanidine. No matrix effect was observed, and the detection limits found were in the range 2.2-4.7microg/L. The method enabled quantification of the potato glycoalkaloids in environmental samples.

  6. Analysis and Quantitation of Glycated Hemoglobin by Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Hattan, Stephen J.; Parker, Kenneth C.; Vestal, Marvin L.; Yang, Jane Y.; Herold, David A.; Duncan, Mark W.

    2016-03-01

    Measurement of glycated hemoglobin is widely used for the diagnosis and monitoring of diabetes mellitus. Matrix assisted laser desorption/ionization (MALDI) time of flight (TOF) mass spectrometry (MS) analysis of patient samples is used to demonstrate a method for quantitation of total glycation on the β-subunit of hemoglobin. The approach is accurate and calibrated with commercially available reference materials. Measurements were linear (R2 > 0.99) across the clinically relevant range of 4% to 20% glycation with coefficients of variation of ≤ 2.5%. Additional and independent measurements of glycation of the α-subunit of hemoglobin are used to validate β-subunit glycation measurements and distinguish hemoglobin variants. Results obtained by MALDI-TOF MS were compared with those obtained in a clinical laboratory using validated HPLC methodology. MALDI-TOF MS sample preparation was minimal and analysis times were rapid making the method an attractive alternative to methodologies currently in practice.

  7. Quantitative and Selective Analysis of Feline Growth Related Proteins Using Parallel Reaction Monitoring High Resolution Mass Spectrometry

    PubMed Central

    Sundberg, Mårten; Strage, Emma M.; Bergquist, Jonas; Holst, Bodil S.

    2016-01-01

    Today immunoassays are widely used in veterinary medicine, but lack of species specific assays often necessitates the use of assays developed for human applications. Mass spectrometry (MS) is an attractive alternative due to high specificity and versatility, allowing for species-independent analysis. Targeted MS-based quantification methods are valuable complements to large scale shotgun analysis. A method referred to as parallel reaction monitoring (PRM), implemented on Orbitrap MS, has lately been presented as an excellent alternative to more traditional selected reaction monitoring/multiple reaction monitoring (SRM/MRM) methods. The insulin-like growth factor (IGF)-system is not well described in the cat but there are indications of important differences between cats and humans. In feline medicine IGF–I is mainly analyzed for diagnosis of growth hormone disorders but also for research, while the other proteins in the IGF-system are not routinely analyzed within clinical practice. Here, a PRM method for quantification of IGF–I, IGF–II, IGF binding protein (BP) –3 and IGFBP–5 in feline serum is presented. Selective quantification was supported by the use of a newly launched internal standard named QPrEST™. Homology searches demonstrated the possibility to use this standard of human origin for quantification of the targeted feline proteins. Excellent quantitative sensitivity at the attomol/μL (pM) level and selectivity were obtained. As the presented approach is very generic we show that high resolution mass spectrometry in combination with PRM and QPrEST™ internal standards is a versatile tool for protein quantitation across multispecies. PMID:27907059

  8. Quantitative determination of cyclic polylactic acid oligomers in serum by direct injection liquid chromatography tandem mass spectrometry.

    PubMed

    Osaka, Issey; Yoshimoto, Arihumi; Watanabe, Mikio; Takama, Masashi; Murakami, Masahiro; Kawasaki, Hideya; Arakawa, Ryuichi

    2008-07-15

    Polylactic acid (PLA) is a biodegradable polymer, currently used in pharmaceutical and surgical devices. There is a concern that cyclic polylactic acid (CPLA), which is a by-product of PLA synthesis, may be introduced into the human body as an undesirable contaminant. We carried out a quantitation investigation of the CPLA heptamer (CPLA-7) by liquid chromatography mass spectrometry (LC-MS). We found that CPLA-7 binds strongly with serum proteins and that only 62% of CPLA-7 was recovered after routine deproteination; therefore, we directly injected serum into the LC-MS/MS system after passage through a bovine serum albumin (BSA)-coated chromatographic column and found the recovery of CPLA-7 was improved to 84%, and that the detection (S/N=3) and quantitation limit (S/N=10 and below 15% relative standard deviation) were 1.5 and 2.5 ng/mL, respectively. We conclude that direct injection LC-MS/MS, using a BSA column, is a simple and effective quantitative analysis method for CPLA in serum.

  9. Qualitative and quantitative analysis of Andrographis paniculata by rapid resolution liquid chromatography/time-of-flight mass spectrometry.

    PubMed

    Song, Yong-Xi; Liu, Shi-Ping; Jin, Zhao; Qin, Jian-Fei; Jiang, Zhi-Yuan

    2013-09-30

    A rapid resolution liquid chromatography/time-of-flight tandem mass spectrometry (RRLC-TOF/MS) method was developed for qualitative and quantitative analysis of the major chemical constituents in Andrographis paniculata. Fifteen compounds, including flavonoids and diterpenoid lactones, were unambiguously or tentatively identified in 10 min by comparing their retention times and accurate masses with standards or literature data. The characteristic fragmentation patterns of flavonoids and diterpenoid lactones were summarized, and the structures of the unknown compounds were predicted. Andrographolide, dehydroandrographolide and neoandrographolide were further quantified as marker substances. It was found that the calibration curves for all analytes showed good linearity (R² > 0.9995) within the test ranges. The overall limits of detection (LODs) and limits of quantification (LOQs) were 0.02 μg/mL to 0.06 μg/mL and 0.06 μg/mL to 0.2 μg/mL, respectively. The relative standard deviations (RSDs) for intra- and inter-day precisions were below 3.3% and 4.2%, respectively. The mean recovery rates ranged from 96.7% to 104.5% with the relative standard deviations (RSDs) less than 2.72%. It is concluded that RRLC-TOF/MS is powerful and practical in qualitative and quantitative analysis of complex plant samples due to time savings, sensitivity, precision, accuracy and lowering solvent consumption.

  10. Selective quantitation of the neurotoxin BMAA by use of hydrophilic-interaction liquid chromatography-differential mobility spectrometry-tandem mass spectrometry (HILIC-DMS-MS/MS).

    PubMed

    Beach, Daniel G; Kerrin, Elliott S; Quilliam, Michael A

    2015-11-01

    The neurotoxin β-N-methylamino-L-alanine (BMAA) has been reported in cyanobacteria and shellfish, raising concerns about widespread human exposure. However, inconsistent results for BMAA analysis have led to controversy. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the most appropriate method for analysis of BMAA, but the risk of interference from isomers, other sample components, and the electrospray background is still present. We have investigated differential mobility spectrometry (DMS) as an ion filter to improve selectivity in the hydrophilic interaction liquid chromatographic (HILIC)-MS/MS determination of BMAA. We obtained standards for two BMAA isomers not previously analyzed by HILIC-MS, β-amino-N-methylalanine and 3,4-diaminobutanoic acid, and the typically used 2,4-diaminobutanoic acid and N-(2-aminoethyl)glycine. DMS separation of BMAA from these isomers was achieved and optimized conditions were used to develop a sensitive and highly selective multidimensional HILIC-DMS-MS/MS method. This work revealed current technical limitations of DMS for trace quantitation, and practical solutions were implemented. Accurate control of low levels of DMS carrier gas modifier was essential, but required external metering. The linearity of our optimized method was excellent from 0.01 to 6 μmol L(-1). The instrumental LOD was 0.4 pg BMAA injected on-column and the estimated method LOD was 20 ng g(-1) dry weight for BMAA in sample matrix. The method was used to analyze cycad plant tissue, a cyanobacterial reference material, and mussel tissues, by use of isotope-dilution quantitation with deuterated BMAA. This confirmed the presence of BMAA and several of its isomers in cycad and mussel tissues, including commercially available mussel tissue reference materials certified for other biotoxins. Graphical Abstract Differential Mobility Spectrometry is used to increases the selectivity of BMAA analysis by HILIC-MS/MS.

  11. Highly sensitive capillary electrophoresis-mass spectrometry for rapid screening and accurate quantitation of drugs of abuse in urine.

    PubMed

    Kohler, Isabelle; Schappler, Julie; Rudaz, Serge

    2013-05-30

    The combination of capillary electrophoresis (CE) and mass spectrometry (MS) is particularly well adapted to bioanalysis due to its high separation efficiency, selectivity, and sensitivity; its short analytical time; and its low solvent and sample consumption. For clinical and forensic toxicology, a two-step analysis is usually performed: first, a screening step for compound identification, and second, confirmation and/or accurate quantitation in cases of presumed positive results. In this study, a fast and sensitive CE-MS workflow was developed for the screening and quantitation of drugs of abuse in urine samples. A CE with a time-of-flight MS (CE-TOF/MS) screening method was developed using a simple urine dilution and on-line sample preconcentration with pH-mediated stacking. The sample stacking allowed for a high loading capacity (20.5% of the capillary length), leading to limits of detection as low as 2 ng mL(-1) for drugs of abuse. Compound quantitation of positive samples was performed by CE-MS/MS with a triple quadrupole MS equipped with an adapted triple-tube sprayer and an electrospray ionization (ESI) source. The CE-ESI-MS/MS method was validated for two model compounds, cocaine (COC) and methadone (MTD), according to the Guidance of the Food and Drug Administration. The quantitative performance was evaluated for selectivity, response function, the lower limit of quantitation, trueness, precision, and accuracy. COC and MTD detection in urine samples was determined to be accurate over the range of 10-1000 ng mL(-1) and 21-1000 ng mL(-1), respectively.

  12. Development and validation of the liquid chromatography-tandem mass spectrometry method for quantitative estimation of candesartan from human plasma

    PubMed Central

    Prajapati, Shailesh T.; Patel, Pratik K.; Patel, Marmik; Chauhan, Vijendra B.; Patel, Chhaganbhai N.

    2011-01-01

    Introduction: A simple and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for estimation of candesartan in human plasma using the protein precipitation technique. Materials and Methods: The chromatographic separation was performed on reverse phase using a Betasil C8 (100 × 2.1 mm) 5-μm column, mobile phase of methanol:ammonium tri-floro acetate buffer with formic acid (60:40 v/v) and flow rate of 0.45 ml/min. The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 441.2 → 263.2 and 260.2 → 116.1 were used to measure candesartan by using propranolol as an internal standard. Results: The linearity of the developed method was achieved in the range of 1.2–1030 ng/ml (r2 ≥ 0.9996) for candesartan. Conclusion: The developed method is simple, rapid, accurate, cost-effective and specific; hence, it can be applied for routine analysis in pharmaceutical industries. PMID:23781443

  13. Quantitative determination of phencyclidine in pigmented and nonpigmented hair by ion-trap mass spectrometry.

    PubMed

    Slawson, M H; Wilkins, D G; Foltz, R L; Rollins, D E

    1996-10-01

    A sensitive and specific method has been developed for the quantitative analysis of phencyclidine (PCP) in pigmented and nonpigmented rat hair. After the addition of PCP-d5 as the internal standard, hair samples (10 mg) were digested overnight in 1N NaOH at 30 degrees C. Digested solutions were then extracted using a solid-phase procedure with Bond Elut CertifyTM extraction columns. Reconstituted extracts were analyzed on a Finnigan ion trap (MagnumTM) mass spectrometer in the electron ionization mode using helium as the carrier gas, and a DB-5 MS (30 m x 0.25-mm i.d.; 25-microns film thickness) capillary column. The assay is linear from 0.1 to 50 ng/mg with a correlation coefficient of > 0.99 and is capable of detecting 25 pg of PCP on column. The accuracy of this assay was estimated using fortified hair standards at PCP concentrations of 0.5 and 10 ng/mg. Intra-assay coefficients of variation were determined to be less than 6% at 0.5, 2, and 10 ng/mg. Interassay coefficients of variation were determined to be less than 15% at 0.5, 2, and 10 ng/mg. The method has been used to evaluate PCP incorporation into Long-Evans rat hair but could also be used to evaluate the incorporation of PCP into human hair. Male rats were shaved prior to dosing such that both pigmented and nonpigmented hair was collected. Animals were administered 12 mg/kg PCP by intraperitoneal injection daily for five days. Fourteen days after the first dose, pigmented and nonpigmented hair were collected and analyzed for PCP. The mean plus or minus the standard error of the mean (n = 5) concentrations of PCP in pigmented and nonpigmented hair were 14.33 +/- 1.43 ng/mg of hair and 0.47 +/- 0.04 ng/mg of hair, respectively. This method is also being used to evaluate PCP as a model xenobiotic for studies of the incorporation of xenobiotics into hair.

  14. Semi-quantitative analysis of microstructures by secondary ion mass spectrometry

    SciTech Connect

    Phinney, D L

    2005-02-14

    The focus of this review is on trace-element quantitation of microstructures in solids. This review is aimed at the non-specialist who wants to know how SIMS quantitation is achieved. Despite 35 years of SIMS research and applications, SIMS quantitation remains a fundamentally empirical enterprise and is based on standards. The most used standards are ''bulk standards''--solids with a homogeneous distribution of a trace element--and ion-implanted solids. The SIMS systematics of bulk standards and ion-implanted solids are reviewed.

  15. Quantitation by Portable Gas Chromatography: Mass Spectrometry of VOCs Associated with Vapor Intrusion

    PubMed Central

    Fair, Justin D.; Bailey, William F.; Felty, Robert A.; Gifford, Amy E.; Shultes, Benjamin; Volles, Leslie H.

    2010-01-01

    Development of a robust reliable technique that permits for the rapid quantitation of volatile organic chemicals is an important first step to remediation associated with vapor intrusion. This paper describes the development of an analytical method that allows for the rapid and precise identification and quantitation of halogenated and nonhalogenated contaminants commonly found within the ppbv level at sites where vapor intrusion is a concern. PMID:20885969

  16. Quantitation by Portable Gas Chromatography: Mass Spectrometry of VOCs Associated with Vapor Intrusion.

    PubMed

    Fair, Justin D; Bailey, William F; Felty, Robert A; Gifford, Amy E; Shultes, Benjamin; Volles, Leslie H

    2010-01-01

    Development of a robust reliable technique that permits for the rapid quantitation of volatile organic chemicals is an important first step to remediation associated with vapor intrusion. This paper describes the development of an analytical method that allows for the rapid and precise identification and quantitation of halogenated and nonhalogenated contaminants commonly found within the ppbv level at sites where vapor intrusion is a concern.

  17. STRUCTURAL DETERMINATION AND QUANTITATIVE ANALYSIS OF BACTERIAL PHOSPHOLIPIDS USING LIQUID CHROMATOGRAPHY/ELECTROSPRAY IONIZATION/MASS SPECTROMETRY

    EPA Science Inventory

    This report presents a comprehensive spectral analysis of common bacterial phospholipids using electrospray/mass spectrometry (ESI/MS) under both negative and positive ionization conditions. Phospholipids under positive ionization yield sodium-adduct molecular ions which are mos...

  18. A sensitive liquid chromatography/mass spectrometry-based assay for quantitation of amino-containing moieties in lipid A

    PubMed Central

    Kalhorn, Thomas F.; Kiavand, Anahita; Cohen, Ilana E.; Nelson, Amanda K.; Ernst, Robert K.

    2009-01-01

    A novel sensitive liquid chromatography/mass spectrometry-based assay was developed for the quantitation of aminosugars, including 2-amino-2-deoxyglucose (glucosamine, GlcN), 2-amino-2-deoxygalactose (galactosamine, GalN), and 4-amino-4-deoxyarabinose (aminoarabinose, AraN), and for ethanolamine (EtN), present in lipid A. This assay enables the identification and quantitation of all amino-containing moieties present in lipopolysaccharide or lipid A from a single sample. The method was applied to the analysis of lipid A (endotoxin) isolated from a variety of biosynthetic and regulatory mutants of Salmonella enterica serovar Typhimurium and Francisella tularensis subspecies novicida. Lipid A is treated with trifluoroacetic acid to liberate and deacetylate individual aminosugars and mass tagged with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, which reacts with primary and secondary amines. The derivatives are separated using reversed-phase chromatography and analyzed using a single quadrupole mass spectrometer to detect quantities as small as 20 fmol. GalN was detected only in Francisella and AraN only in Salmonella, while GlcN was detected in lipid A samples from both species of bacteria. Additionally, we found an approximately 10-fold increase in the level of AraN in lipid A isolated from Salmonella grown in magnesium-limited versus magnesium-replete conditions. Salmonella with defined mutations in lipid A synthesis and regulatory genes were used to further validate the assay. Salmonella with null mutations in the phoP, pmrE, and prmF genes were unable to add AraN to their lipid A, while Salmonella with constitutively active phoP and pmrA exhibited AraN modification of lipid A even in the normally repressive magnesium-replete growth condition. The described assay produces excellent repeatability and reproducibility for the detection of amino-containing moieties in lipid A from a variety of bacterial sources. PMID:19130491

  19. Quantitative Profiling of Major Neutral Lipid Classes in Human Meibum by Direct Infusion Electrospray Ionization Mass Spectrometry

    PubMed Central

    Chen, Jianzhong; Green, Kari B.; Nichols, Kelly K.

    2013-01-01

    Purpose. The purpose of this investigation was to better understand lipid composition in human meibum. Methods. Intact lipids in meibum samples were detected by direct infusion electrospray ionization mass spectrometry (ESI-MS) analysis in positive detection mode using sodium iodide (NaI) as an additive. The peak intensities of all major types of lipid species, that is, wax esters (WEs), cholesteryl esters (CEs), and diesters (DEs) were corrected for peak overlapping and isotopic distribution; an additional ionization efficiency correction was performed for WEs and CEs, which was simplified by the observation that the corresponding ionization efficiency was primarily dependent on the specific lipid class and saturation degree of the lipids while independent of the carbon chain length. A set of WE and CE standards was spiked in meibum samples for ionization efficiency determination and absolute quantitation. Results. The absolute amount (μmol/mg) for each of 51 WEs and 31 CEs in meibum samples was determined. The summed masses for 51 WEs and 31 CEs accounted for 48 ± 4% and 40 ± 2%, respectively, of the total meibum lipids. The mass percentages of saturated and unsaturated species were determined to be 75 ± 2% and 25 ± 1% for CEs and 14 ± 1% and 86 ± 1% for WEs. The profiles for two types of DEs were also obtained, which include 42 α,ω Type II DEs, and 21 ω Type I-St DEs. Conclusions. Major neutral lipid classes in meibum samples were quantitatively profiled by ESI-MS analysis with NaI additive. PMID:23847307

  20. High-throughput quantitative analysis of domoic acid directly from mussel tissue using Laser Ablation Electrospray Ionization - tandem mass spectrometry.

    PubMed

    Beach, Daniel G; Walsh, Callee M; McCarron, Pearse

    2014-12-15

    Eliminating sample extraction or liquid chromatography steps from methods for analysis of the neurotoxin Domoic Acid (DA) in shellfish could greatly increase throughput in food safety testing laboratories worldwide. To this end, we have investigated the use of Laser Ablation Electrospray Ionization (LAESI) with tandem mass spectrometry (MS/MS) detection for DA analysis directly from mussel tissue homogenates without sample extraction, cleanup or separation. DA could be selectively detected directly from mussel tissue homogenates using MS/MS in selected reaction monitoring scan mode. The quantitative capabilities of LAESI-MS/MS for DA analysis from mussel tissue were evaluated by analysis of four mussel tissue reference materials using matrix-matched calibration. Linear response was observed from 1 mg/kg to 40 mg/kg and the method limit of detection was 1 mg/kg. Results for DA analysis in tissue within the linear range were in good agreement with two established methods, LC-UV and LC-MS/MS (recoveries from 103 to 125%). Beyond the linear range, extraction and clean-up were required to achieve good quantitation. Most notable is the extremely rapid analysis time of about 10 s per sample by LAESI-MS/MS, which corresponds to a significant increase in sample throughput compared with existing methodology for routine DA analysis.

  1. Coumarin and furanocoumarin quantitation in citrus peel via ultraperformance liquid chromatography coupled with mass spectrometry (UPLC-MS).

    PubMed

    Dugrand, Audray; Olry, Alexandre; Duval, Thibault; Hehn, Alain; Froelicher, Yann; Bourgaud, Frédéric

    2013-11-13

    Coumarins and furanocoumarins are secondary metabolites commonly found in citrus plants. These molecules are allelochemical compounds in plants that have controversial effects on humans, such as phototoxicity and the commonly described interactions with drugs, referred to as the "grapefruit juice effect". Thus, it is important to develop a reliable method to identify and quantitate the coumarins and furanocoumarins in citrus extracts. For this purpose, we herein describe an ultraperformance liquid chromatography coupled with mass spectrometry (UPLC-MS)-based method. We first developed a rapid UPLC method (20 min) to separate the isomers of each furanocoumarin. A subsequent single ion monitoring MS detection method was performed to distinguish between the molecules, which were possibly coeluting but had different molecular weights. The method was successfully used to separate and quantitate 6 coumarins and 21 furanocoumarins in variable amounts within peel extracts (flavedo and albedo) of 6 varieties of Citrus (sweet orange, lemon, grapefruit, bergamot, pummelo, and clementine). This method combines high selectivity and sensitivity in a rapid analysis and is useful for fingerprinting Citrus species via their coumarin and furanocoumarin contents.

  2. Quantitative determination of folic acid in multivitamin/multielement tablets using liquid chromatography/tandem mass spectrometry.

    PubMed

    Nelson, Bryant C; Sharpless, Katherine E; Sander, Lane C

    2006-12-01

    Two different isotope-dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods for the quantitative determination of folic acid (FA) in multivitamin/multielement tablets are reported. These methods represent distinct improvements in terms of speed and specificity over most existing microbiological and chromatographic methods for the determination of FA in dietary supplements. The first method utilizes an aqueous/organic-based extraction solvent combined with positive-ion mode LC/MS/MS detection of protonated [M + H]+ FA molecules and the second method utilizes a pure aqueous-based extraction solvent combined with negative-ion mode LC/MS/MS detection of deprotonated [M - H]- FA molecules. The LC/MS/MS methods exhibit comparable linear dynamic ranges (> or =3 orders of magnitude), limits of detection (0.02 ng on-column) and limits of quantification (0.06 ng on-column) for FA. Two methods employing different extraction and different MS detection modes were developed to allow method cross-validation. Successful validation of each measurement procedure supports the use of either method for the certification of FA levels in dietary supplements. The accuracy and precision of each measurement procedure were evaluated by applying each method to the quantitative determination of FA in a NIST standard reference material (NIST SRM 3280 multivitamin/multielement tablets). The FA measurement accuracy for both methods was > or =95% (based on the manufacturer's assessment of the FA level in SRM 3280) with corresponding measurement precision values (% RSD) of approximately 1%.

  3. Evaluation of liquid chromatography-ion trap-time of flight hybrid mass spectrometry on the quantitative analysis for ginsenosides.

    PubMed

    Xing, Lu; Xie, Lin; Liang, Yan; Xing, Rong; Rao, Tai; Zhou, Lijun; Wang, Qian; Fu, Hanxu; Ye, Wei; Wang, Guangji

    2014-07-01

    It is ideal and desirable for a single instrument to meet the requirement of both qualitative and quantitative analysis of complicated components in pharmacokinetic research for herbal medicine. Liquid chromatography combined with hybrid ion trap and time-of-flight mass spectrometry (LCMS-IT-TOF) was recently confirmed as a very powerful tool for the qualitative analysis of both target and nontarget components in herbal medicines. The present study was designed to investigate the feasibility of LCMS-IT-TOF on quantitative analysis of ginsenosides in biological matrices. A simple liquid-liquid extraction procedure was followed by injection of the extracts onto a C₁₈ column with gradient elution and detection based on LCMS-IT-TOF system in negative scan mode. The developed method was validated with respect to the limit of quantification, linear dynamic range, precision, accuracy, matrix effects and stabilities. All the results suggested that the presently developed method was sufficiently sensitive and robust enough to simultaneously monitor 15 ginsenosides with diverse properties and a large range of concentration differences. Therefore, this method would be expected to be highly useful for comprehensive studies of ginsenosides in complicated matrix.

  4. Quantitative Gingival Crevicular Fluid Proteome in Health and Periodontal Disease Using Stable-Isotope Chemistries and Mass Spectrometry

    PubMed Central

    Carneiro, Leandro G.; Nouh, Hesham; Salih, Erdjan

    2014-01-01

    Aim Application of quantitative stable-isotope-labeling chemistries and mass spectrometry (MS) to determine alterations in gingival crevicular fluid (GCF) proteome in periodontal disease. Materials and Methods Quantitative proteome of GCF from 40 healthy individuals versus 40 patients with periodontal disease was established using 320 GCF samples and stable-isotope-labeling reagents, ICAT and mTRAQ, with MS technology and validated by enzyme-linked immunosorbent methods. Results We have identified 238 distinct proteins of which 180 were quantified in GCF of both healthy and periodontal patients with additional 26 and 32 distinct proteins that were found only in GCF of healthy or periodontal patients. In addition, 42 pathogenic bacterial proteins and 11 yeast proteins were quantified. The data highlighted a series of proteins not quantified previously by large-scale MS approaches in GCF with relevance to periodontal disease, such as host derived Ig alpha-2 chain C, Kallikrein-4, S100-A9, transmembrane proteinase 13, peptidase S1 domain, several collagen types and pathogenic bacterial proteins e.g., formamidase, leucine amidopeptidase and virulence factor OMP85. Conclusions The innovative analytical approaches provided detailed novel changes in both host and microbial derived GCF proteomes of periodontal patients. The study defined 50 host and 16 pathogenic bacterial proteins significantly elevated in periodontal disease most of which were novel with significant potential for application in the clinical arena of periodontal disease. PMID:24738839

  5. Metabolomics relative quantitation with mass spectrometry using chemical derivatization and isotope labeling

    DOE PAGES

    O'Maille, Grace; Go, Eden P.; Hoang, Linh; ...

    2008-01-01

    Comprehensive detection and quantitation of metabolites from a biological source constitute the major challenges of current metabolomics research. Two chemical derivatization methodologies, butylation and amination, were applied to human serum for ionization enhancement of a broad spectrum of metabolite classes, including steroids and amino acids. LC-ESI-MS analysis of the derivatized serum samples provided a significant signal elevation across the total ion chromatogram to over a 100-fold increase in ionization efficiency. It was also demonstrated that derivatization combined with isotopically labeled reagents facilitated the relative quantitation of derivatized metabolites from individual as well as pooled samples.

  6. Fourier Transform Mass Spectrometry.

    ERIC Educational Resources Information Center

    Gross, Michael L.; Rempel, Don L.

    1984-01-01

    Discusses the nature of Fourier transform mass spectrometry and its unique combination of high mass resolution, high upper mass limit, and multichannel advantage. Examines its operation, capabilities and limitations, applications (ion storage, ion manipulation, ion chemistry), and future applications and developments. (JN)

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

    ERIC Educational Resources Information Center

    Xu, Xia; Veenstra, Timothy D.

    2012-01-01

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

  8. Rapid qualitative and quantitative analysis of proanthocyanidin oligomers and polymers by ultra-performance liquid chromatography – tandem mass spectrometry (UPLC-MS/MS)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We developed a rapid method with ultra-performance liquid chromatography – tandem mass spectrometry (UPLC-MS/MS) for the qualitative and quantitative analysis of plant proanthocyanidins (PAs) directly from crude plant extracts. The method utilizes a range of cone voltages to achieve the depolymeriza...

  9. Online nanoliquid chromatography-mass spectrometry and nanofluorescence detection for high-resolution quantitative N-glycan analysis.

    PubMed

    Kalay, Hakan; Ambrosini, Martino; van Berkel, Patrick H C; Parren, Paul W H I; van Kooyk, Yvette; García Vallejo, Juan J

    2012-04-01

    The characterization of the repertoire of glycans at the quantitative and qualitative levels on cells and glycoproteins is a necessary step to the understanding of glycan functions in biology. In addition, there is an increasing demand in the field of biotechnology for the monitoring of glycosylation of recombinant glycoproteins, an important issue with regard to their safety and biological activity. The enzymatic release followed by fluorescent derivatization of glycans and separation by normal phase high-performance liquid chromatography (HPLC) has proven for many years to be a powerful approach to the quantification of glycans. Characterization of glycans has classically been performed by mass spectrometry (MS) with external standardization. Here, we report a new method for the simultaneous quantification and characterization of the N-glycans on glycoproteins without the need for external standardization. This method, which we call glycan nanoprofiling, uses nanoLC-coupled electrospray ionization (ESI)-MS with an intercalated nanofluorescence reader and provides effective single glycan separation with subpicomolar sensitivity. The method relies on the isolation and coumaric derivatization of enzymatically released glycans collected by solid phase extraction with porous graphitized carbon and their separation over polyamide-based nanoHPLC prior to serial nanofluorescence and nanoelectrospray mass spectrometric analysis. Glycan nanoprofiling is a broadly applicable and powerful approach that is sufficient to identify and quantify many glycan oligomers in a single run. Glycan nanoprofiling was successfully applied to resolve the glycans of monoclonal antibodies, showing that this method is a fast and sensitive alternative to available methods.

  10. Characterization and quantitative analysis of surfactants in textile wastewater by liquid chromatography/quadrupole-time-of-flight mass spectrometry.

    PubMed

    González, Susana; Petrović, Mira; Radetic, Maja; Jovancic, Petar; Ilic, Vesna; Barceló, Damià

    2008-05-01

    A method based on the application of ultra-performance liquid chromatography (UPLC) coupled to hybrid quadrupole-time-of-flight mass spectrometry (QqTOF-MS) with an electrospray (ESI) interface has been developed for the screening and confirmation of several anionic and non-ionic surfactants: linear alkylbenzenesulfonates (LAS), alkylsulfate (AS), alkylethersulfate (AES), dihexyl sulfosuccinate (DHSS), alcohol ethoxylates (AEOs), coconut diethanolamide (CDEA), nonylphenol ethoxylates (NPEOs), and their degradation products (nonylphenol carboxylate (NPEC), octylphenol carboxylate (OPEC), 4-nonylphenol (NP), 4-octylphenol (OP) and NPEO sulfate (NPEO-SO4). The developed methodology permits reliable quantification combined with a high accuracy confirmation based on the accurate mass of the (de)protonated molecules in the TOFMS mode. For further confirmation of the identity of the detected compounds the QqTOF mode was used. Accurate masses of product ions obtained by performing collision-induced dissociation (CID) of the (de)protonated molecules of parent compounds were matched with the ions obtained for a standard solution. The method was applied for the quantitative analysis and high accuracy confirmation of surfactants in complex mixtures in effluents from the textile industry. Positive identification of the target compounds was based on accurate mass measurement of the base peak, at least one product ion and the LC retention time of the analyte compared with that of a standard. The most frequently surfactants found in these textile effluents were NPEO and NPEO-SO4 in concentrations ranging from 0.93 to 5.68 mg/L for NPEO and 0.06 to 4.30 mg/L for NPEO-SO4. AEOs were also identified.

  11. Quantitative Proteomics using Nano-LC with High Accuracy Mass Spectrometry

    SciTech Connect

    Pasa-Tolic, Liljiana; Jacobs, Jon M.; Qian, Weijun; Smith, Richard D.

    2008-01-29

    Despite significant advances in LC-MS based technologies, challenges remain in implementing a proteomics platform for routine clinical applications. These include the needed robustness as well as the sensitivity and dynamic range of detection to both effectively address extremely small tissue samples, for example microdissected or biopsy tissues, or high dynamic range samples, such as blood plasma. Other key components include providing the needed throughput to enable statistically meaningful number of analyses for clinical setting within a robust platform that utilizes effective quantitative approaches for high accuracy and reproducibility. This chapter describes the key components of a nanoLC- MS based technological approach that is designed to target these challenges by virtue of enhancing sensitivity, dynamic range coverage, and throughput, for the generation of robust quantitative measurements in support of clinical studies.

  12. High-throughput pesticide residue quantitative analysis achieved by tandem mass spectrometry with automated flow injection.

    PubMed

    Nanita, Sergio C; Pentz, Anne M; Bramble, Frederick Q

    2009-04-15

    The use of automated flow injection with MS/MS detection for fast quantitation of agrochemicals in food and water samples was demonstrated in this study. Active ingredients from the sulfonylurea herbicide and carbamate insecticide classes were selected as model systems. Samples were prepared using typical procedures from residue methods, placed in an autosampler, and injected directly into a triple quadrupole instrument without chromatographic separation. The technique allows data acquisition in 15 s per injection, with samples being injected every 65 s, representing a significant improvement from the 15-30 min needed in typical HPLC/MS/MS methods. The availability of HPLC systems is an advantage since they can be used in flow-injection mode (bypassing the column compartment). Adequate accuracy, linearity, and precision (R(2) > 0.99 and RSD < 20%) were obtained using external standards prepared in each control matrix. The limit of quantitation (LOQ) achieved for all analytes was 0.01 mg/kg in food samples and 0.1 ng/mL in water; while limits of detection (LOD) were estimated to be about 0.003 mg/kg and 0.03 ng/mL in food and water, respectively. The advantages and limitations of flow injection MS/MS for ultratrace-level quantitative analysis in complex matrixes are discussed.

  13. Simultaneous quantitation of urinary cotinine and acrylonitrile-derived mercapturic acids with ultraperformance liquid chromatography-tandem mass spectrometry.

    PubMed

    Wu, Chia-Fang; Uang, Shi-Nian; Chiang, Su-Yin; Shih, Wei-Chung; Huang, Yu-Fang; Wu, Kuen-Yuh

    2012-02-01

    Acrylonitrile (AN), a widely used industrial chemical also found in tobacco smoke, has been classified as a possible human carcinogen (group 2B) by the International Agency for Research on Cancer. AN can be detoxified by glutathione S-transferase (GST) to form glutathione (GSH) conjugates in vivo. It can be metabolically activated by cytochrome P450 2E1 to form 2-cyanoethylene oxide, which can also be detoxified by GST to generate GSH conjugates. The GSH conjugates can be further metabolized to mercapturic acids (MAs), namely, N-acetyl-S-(2-cyanoethyl)cysteine (CEMA), N-acetyl-S-(2-hydroxyethyl)cysteine (HEMA), and N-acetyl-S-(1-cyano-2-hydroxyethyl)cysteine (CHEMA). This study developed an ultraperformance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) method to quantitatively profile the major AN urinary metabolites (CEMA, HEMA, and CHEMA) to assess AN exposure, as well as analyze urinary cotinine (COT) as an indicator for tobacco smoke exposure. The limits of quantitation were 0.1, 0.1, 1.0, and 0.05 μg/L for HEMA, CEMA, CHEMA, and COT, respectively. This method was applied to analyze the three AN-derived MAs in 36 volunteers with no prior occupational AN exposure. Data analysis showed significant correlations between the level of COT and the levels of these MAs, suggesting them as biomarkers for exposure to low levels of AN. The results demonstrate that a highly specific and sensitive UPLC-MS/MS method has been successfully developed to quantitatively profile the major urinary metabolites of AN in humans to assess low AN exposure.

  14. Mass Spectrometry for the Masses

    ERIC Educational Resources Information Center

    Persinger, Jared D.; Hoops, Geoffrey, C.; Samide, Michael J.

    2004-01-01

    A simple, qualitative experiment is developed for implementation, where the gas chromatography-mass spectrometry (GC-MS) plays an important role, into the laboratory curriculum of a chemistry course designed for nonscience majors. This laboratory experiment is well suited for the students as it helps them to determine the validity of their…

  15. Forensic Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Hoffmann, William D.; Jackson, Glen P.

    2015-07-01

    Developments in forensic mass spectrometry tend to follow, rather than lead, the developments in other disciplines. Examples of techniques having forensic potential born independently of forensic applications include ambient ionization, imaging mass spectrometry, isotope ratio mass spectrometry, portable mass spectrometers, and hyphenated chromatography-mass spectrometry instruments, to name a few. Forensic science has the potential to benefit enormously from developments that are funded by other means, if only the infrastructure and personnel existed to adopt, validate, and implement the new technologies into casework. Perhaps one unique area in which forensic science is at the cutting edge is in the area of chemometrics and the determination of likelihood ratios for the evaluation of the weight of evidence. Such statistical techniques have been developed most extensively for ignitable-liquid residue analyses and isotope ratio analysis. This review attempts to capture the trends, motivating forces, and likely impact of developing areas of forensic mass spectrometry, with the caveat that none of this research is likely to have any real impact in the forensic community unless: (a) The instruments developed are turned into robust black boxes with red and green lights for positives and negatives, respectively, or (b) there are PhD graduates in the workforce who can help adopt these sophisticated techniques.

  16. Forensic Mass Spectrometry.

    PubMed

    Hoffmann, William D; Jackson, Glen P

    2015-01-01

    Developments in forensic mass spectrometry tend to follow, rather than lead, the developments in other disciplines. Examples of techniques having forensic potential born independently of forensic applications include ambient ionization, imaging mass spectrometry, isotope ratio mass spectrometry, portable mass spectrometers, and hyphenated chromatography-mass spectrometry instruments, to name a few. Forensic science has the potential to benefit enormously from developments that are funded by other means, if only the infrastructure and personnel existed to adopt, validate, and implement the new technologies into casework. Perhaps one unique area in which forensic science is at the cutting edge is in the area of chemometrics and the determination of likelihood ratios for the evaluation of the weight of evidence. Such statistical techniques have been developed most extensively for ignitable-liquid residue analyses and isotope ratio analysis. This review attempts to capture the trends, motivating forces, and likely impact of developing areas of forensic mass spectrometry, with the caveat that none of this research is likely to have any real impact in the forensic community unless: (a) The instruments developed are turned into robust black boxes with red and green lights for positives and negatives, respectively, or (b) there are PhD graduates in the workforce who can help adopt these sophisticated techniques.

  17. A Quantitative Proteomic Workflow for Characterization of Frozen Clinical Biopsies: Laser Capture Microdissection Coupled with Label-Free Mass Spectrometry

    PubMed Central

    Shapiro, John P.; Biswas, Sabyasachi; Merchant, Anand S.; Satoskar, Anjali; Taslim, Cenny; Lin, Shili; Rovin, Brad H.; Sen, Chandan K.; Roy, Sashwati; Freitas, Michael A.

    2013-01-01

    This paper describes a simple, highly efficient and robust proteomic workflow for routine liquid-chromatography tandem mass spectrometry analysis of Laser Microdissection Pressure Catapulting (LMPC) isolates. Highly efficient protein recovery was achieved by optimization of a “one-pot” protein extraction and digestion allowing for reproducible proteomic analysis on as few as 500 LMPC isolated cells. The method was combined with label-free spectral count quantitation to characterize proteomic differences from 3,000–10,000 LMPC isolated cells. Significance analysis of spectral count data was accomplished using the edgeR tag-count R package combined with hierarchical cluster analysis. To illustrate the capability of this robust workflow, two examples are presented: 1) analysis of keratinocytes from human punch biopsies of normal skin and a chronic diabetic wound and 2) comparison of glomeruli from needle biopsies of patients with kidney disease. Differentially expressed proteins were validated by use of immunohistochemistry. These examples illustrate that tissue proteomics carried out on limited clinical material can obtain informative proteomic signatures for disease pathogenesis and demonstrate the suitability of this approach for biomarker discovery. PMID:23022584

  18. A quantitative proteomic workflow for characterization of frozen clinical biopsies: laser capture microdissection coupled with label-free mass spectrometry.

    PubMed

    Shapiro, John P; Biswas, Sabyasachi; Merchant, Anand S; Satoskar, Anjali; Taslim, Cenny; Lin, Shili; Rovin, Brad H; Sen, Chandan K; Roy, Sashwati; Freitas, Michael A

    2012-12-21

    This paper describes a simple, highly efficient and robust proteomic workflow for routine liquid-chromatography tandem mass spectrometry analysis of Laser Microdissection Pressure Catapulting (LMPC) isolates. Highly efficient protein recovery was achieved by optimization of a "one-pot" protein extraction and digestion allowing for reproducible proteomic analysis on as few as 500 LMPC isolated cells. The method was combined with label-free spectral count quantitation to characterize proteomic differences from 3000-10,000 LMPC isolated cells. Significance analysis of spectral count data was accomplished using the edgeR tag-count R package combined with hierarchical cluster analysis. To illustrate the capability of this robust workflow, two examples are presented: 1) analysis of keratinocytes from human punch biopsies of normal skin and a chronic diabetic wound and 2) comparison of glomeruli from needle biopsies of patients with kidney disease. Differentially expressed proteins were validated by use of immunohistochemistry. These examples illustrate that tissue proteomics carried out on limited clinical material can obtain informative proteomic signatures for disease pathogenesis and demonstrate the suitability of this approach for biomarker discovery.

  19. Quantitative determination of methamphetamine in oral fluid by liquid-liquid extraction and gas chromatography/mass spectrometry.

    PubMed

    Bahmanabadi, L; Akhgari, M; Jokar, F; Sadeghi, H B

    2017-02-01

    Methamphetamine abuse is one of the most medical and social problems many countries face. In spite of the ban on the use of methamphetamine, it is widely available in Iran's drug black market. There are many analytical methods for the detection of methamphetamine in biological specimen. Oral fluid has become a popular specimen to test for the presence of methamphetamine. The purpose of the present study was to develop a method for the extraction and detection of methamphetamine in oral fluid samples using liquid-liquid extraction (LLE) and gas chromatography/mass spectrometry (GC/MS) methods. An analytical study was designed in that blank and 50 authentic oral fluid samples were collected to be first extracted by LLE and subsequently analysed by GC/MS. The method was fully validated and showed an excellent intra- and inter-assay precision (reflex sympathetic dystrophy ˂ 10%) for external quality control samples. Recovery with LLE methods was 96%. Limit of detection and limit of quantitation were 5 and 15 ng/mL, respectively. The method showed high selectivity, no additional peak due to interfering substances in samples was observed. The introduced method was sensitive, accurate and precise enough for the extraction of methamphetamine from oral fluid samples in forensic toxicology laboratories.

  20. Quantitation of the immunodominant 33-mer peptide from α-gliadin in wheat flours by liquid chromatography tandem mass spectrometry

    PubMed Central

    Schalk, Kathrin; Lang, Christina; Wieser, Herbert; Koehler, Peter; Scherf, Katharina Anne

    2017-01-01

    Coeliac disease (CD) is triggered by the ingestion of gluten proteins from wheat, rye, and barley. The 33-mer peptide from α2-gliadin has frequently been described as the most important CD-immunogenic sequence within gluten. However, from more than 890 published amino acid sequences of α-gliadins, only 19 sequences contain the 33-mer. In order to make a precise assessment of the importance of the 33-mer, it is necessary to elucidate which wheat species and cultivars contain the peptide and at which concentrations. This paper presents the development of a stable isotope dilution assay followed by liquid chromatography tandem mass spectrometry to quantitate the 33-mer in flours of 23 hexaploid modern and 15 old common (bread) wheat as well as two spelt cultivars. All flours contained the 33-mer peptide at levels ranging from 91–603 μg/g flour. In contrast, the 33-mer was absent (

  1. A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E. coli

    PubMed Central

    Sashital, Dipali G; Greeman, Candacia A; Lyumkis, Dmitry; Potter, Clinton S; Carragher, Bridget; Williamson, James R

    2014-01-01

    Ribosome assembly is a complex process involving the folding and processing of ribosomal RNAs (rRNAs), concomitant binding of ribosomal proteins (r-proteins), and participation of numerous accessory cofactors. Here, we use a quantitative mass spectrometry/electron microscopy hybrid approach to determine the r-protein composition and conformation of 30S ribosome assembly intermediates in Escherichia coli. The relative timing of assembly of the 3′ domain and the formation of the central pseudoknot (PK) structure depends on the presence of the assembly factor RimP. The central PK is unstable in the absence of RimP, resulting in the accumulation of intermediates in which the 3′-domain is unanchored and the 5′-domain is depleted for r-proteins S5 and S12 that contact the central PK. Our results reveal the importance of the cofactor RimP in central PK formation, and introduce a broadly applicable method for characterizing macromolecular assembly in cells. DOI: http://dx.doi.org/10.7554/eLife.04491.001 PMID:25313868

  2. Ultra Performance Liquid Chromatography with Tandem Mass Spectrometry for the Quantitation of Seventeen Sedative Hypnotics in Six Common Toxicological Matrices.

    PubMed

    Mata, Dani C; Davis, John F; Figueroa, Ariana K; Stanford, Mary June

    2016-01-01

    An ultra performance liquid chromatography triple quadrupole mass spectrometry (LC-MS-MS) method for the quantification of 14 benzodiazepines and three sedative hypnotics is presented. The fast and inexpensive assay was developed for California's Orange County Crime Lab for use in antemortem (AM) and postmortem casework. The drugs were rapidly cleaned up from AM blood, postmortem blood, urine, liver, brain and stomach contents using DPX(®) Weak Anion Exchange (DPX WAX) tips fitted on a pneumatic extractor, which can process up to 48 samples at one time. Assay performance was determined for validation based on recommendations by the Scientific Working Group for Forensic Toxicology for linearity, limit of quantitation, limit of detection, bias, precision (within run and between run), dilution integrity, carry-over, selectivity, recovery, ion suppression and extracted sample stability. Linearity was verified using the therapeutic and toxic ranges of all 17 analytes. Final verification of the method was confirmed by four analysts using 20 blind matrix matched samples. All results were within 20% of each other and the expected value.

  3. Colostrum protein uptake in neonatal lambs examined by descriptive and quantitative liquid chromatography-tandem mass spectrometry.

    PubMed

    Hernández-Castellano, Lorenzo E; Argüello, Anastasio; Almeida, André M; Castro, Noemí; Bendixen, Emøke

    2015-01-01

    Colostrum intake is a key factor for newborn ruminant survival because the placenta does not allow the transfer of immune components. Therefore, newborn ruminants depend entirely on passive immunity transfer from the mother to the neonate, through the suckling of colostrum. Understanding the importance of specific colostrum proteins has gained significant attention in recent years. However, proteomics studies of sheep colostrum and their uptake in neonate lambs has not yet been presented. The aim of this study was to describe the proteomes of sheep colostrum and lamb blood plasma, using sodium dodecyl sulfate-PAGE for protein separation and in-gel digestion, followed by liquid chromatography-tandem mass spectrometry of resulting tryptic peptides for protein identification. An isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomics approach was subsequently used to provide relative quantification of how neonatal plasma protein concentrations change as an effect of colostrum intake. The results of this study describe the presence of 70 proteins in the ovine colostrum proteome. Furthermore, colostrum intake resulted in an increase of 8 proteins with important immune functions in the blood plasma of lambs. Further proteomic studies will be necessary, particularly using the selected reaction monitoring approach, to describe in detail the role of specific colostrum proteins for immune transfer to the neonate.

  4. From "clinical proteomics" to "clinical chemistry proteomics": considerations using quantitative mass-spectrometry as a model approach.

    PubMed

    Lehmann, Sylvain; Poinot, Pauline; Tiers, Laurent; Junot, Christophe; Becher, François; Hirtz, Christophe

    2011-10-08

    Clinical Proteomics biomarker discovery programs lead to the selection of putative new biomarkers of human pathologies. Following an initial discovery phase, validation of these candidates in larger populations is a major task that recently started relying upon the use of mass spectrometry approaches, especially in cases where classical immune-detection methods were lacking. Thanks to highly sensitive spectrometers, adapted measurement methods like selective reaction monitoring (SRM) and various pre-fractionation methods, the quantitative detection of protein/peptide biomarkers in low concentrations is now feasible from complex biological fluids. This possibility leads to the use of similar methodologies in clinical biology laboratories, within a new proteomic field that we shall name "Clinical Chemistry Proteomics" (CCP). Such evolution of Clinical Proteomics adds important constraints with regards to the in vitro diagnostic (IVD) application. As measured values of analytes will be used to diagnose, follow-up and adapt patient treatment on a routine basis; medical utility, robustness, reference materials and clinical feasibility are among the new issues of CCP to consider.

  5. Quantitation of the immunodominant 33-mer peptide from α-gliadin in wheat flours by liquid chromatography tandem mass spectrometry.

    PubMed

    Schalk, Kathrin; Lang, Christina; Wieser, Herbert; Koehler, Peter; Scherf, Katharina Anne

    2017-03-22

    Coeliac disease (CD) is triggered by the ingestion of gluten proteins from wheat, rye, and barley. The 33-mer peptide from α2-gliadin has frequently been described as the most important CD-immunogenic sequence within gluten. However, from more than 890 published amino acid sequences of α-gliadins, only 19 sequences contain the 33-mer. In order to make a precise assessment of the importance of the 33-mer, it is necessary to elucidate which wheat species and cultivars contain the peptide and at which concentrations. This paper presents the development of a stable isotope dilution assay followed by liquid chromatography tandem mass spectrometry to quantitate the 33-mer in flours of 23 hexaploid modern and 15 old common (bread) wheat as well as two spelt cultivars. All flours contained the 33-mer peptide at levels ranging from 91-603 μg/g flour. In contrast, the 33-mer was absent (

  6. Quantitative assessment of chemical artefacts produced by propionylation of histones prior to mass spectrometry analysis.

    PubMed

    Soldi, Monica; Cuomo, Alessandro; Bonaldi, Tiziana

    2016-07-01

    Histone PTMs play a crucial role in regulating chromatin structure and function, with impact on gene expression. MS is nowadays widely applied to study histone PTMs systematically. Because histones are rich in arginine and lysine, classical shot-gun approaches based on trypsin digestion are typically not employed for histone modifications mapping. Instead, different protocols of chemical derivatization of lysines in combination with trypsin have been implemented to obtain "Arg-C like" digestion products that are more suitable for LC-MS/MS analysis. Although widespread, these strategies have been recently described to cause various side reactions that result in chemical modifications prone to be misinterpreted as native histone marks. These artefacts can also interfere with the quantification process, causing errors in histone PTMs profiling. The work of Paternoster V. et al. is a quantitative assessment of methyl-esterification and other side reactions occurring on histones after chemical derivatization of lysines with propionic anhydride [Proteomics 2016, 16, 2059-2063]. The authors estimate the effect of different solvents, incubation times, and pH on the extent of these side reactions. The results collected indicate that the replacement of methanol with isopropanol or ACN not only blocks methyl-esterification, but also significantly reduces other undesired unspecific reactions. Carefully titrating the pH after propionic anhydride addition is another way to keep methyl-esterification under control. Overall, the authors describe a set of experimental conditions that allow reducing the generation of various artefacts during histone propionylation.

  7. Discovery of mouse spleen signaling responses to anthrax using label-free quantitative phosphoproteomics via mass spectrometry.

    PubMed

    Manes, Nathan P; Dong, Li; Zhou, Weidong; Du, Xiuxia; Reghu, Nikitha; Kool, Arjan C; Choi, Dahan; Bailey, Charles L; Petricoin, Emanuel F; Liotta, Lance A; Popov, Serguei G

    2011-03-01

    Inhalational anthrax is caused by spores of the bacterium Bacillus anthracis (B. anthracis), and is an extremely dangerous disease that can kill unvaccinated victims within 2 weeks. Modern antibiotic-based therapy can increase the survival rate to ∼50%, but only if administered presymptomatically (within 24-48 h of exposure). To discover host signaling responses to presymptomatic anthrax, label-free quantitative phosphoproteomics via liquid chromatography coupled to mass spectrometry was used to compare spleens from uninfected and spore-challenged mice over a 72 h time-course. Spleen proteins were denatured using urea, reduced using dithiothreitol, alkylated using iodoacetamide, and digested into peptides using trypsin, and the resulting phosphopeptides were enriched using titanium dioxide solid-phase extraction and analyzed by nano-liquid chromatography-Linear Trap Quadrupole-Orbitrap-MS(/MS). The fragment ion spectra were processed using DeconMSn and searched using both Mascot and SEQUEST resulting in 252,626 confident identifications of 6248 phosphopeptides (corresponding to 5782 phosphorylation sites). The precursor ion spectra were deisotoped using Decon2LS and aligned using MultiAlign resulting in the confident quantitation of 3265 of the identified phosphopeptides. ANOVAs were used to produce a q-value ranked list of host signaling responses. Late-stage (48-72 h postchallenge) Sterne strain (lethal) infections resulted in global alterations to the spleen phosphoproteome. In contrast, ΔSterne strain (asymptomatic; missing the anthrax toxin) infections resulted in 188 (5.8%) significantly altered (q<0.05) phosphopeptides. Twenty-six highly tentative phosphorylation responses to early-stage (24 h postchallenge) anthrax were discovered (q<0.5), and ten of these originated from eight proteins that have known roles in the host immune response. These tentative early-anthrax host response signaling events within mouse spleens may translate into presymptomatic

  8. Discovery of Mouse Spleen Signaling Responses to Anthrax using Label-Free Quantitative Phosphoproteomics via Mass Spectrometry*

    PubMed Central

    Manes, Nathan P.; Dong, Li; Zhou, Weidong; Du, Xiuxia; Reghu, Nikitha; Kool, Arjan C.; Choi, Dahan; Bailey, Charles L.; Petricoin, Emanuel F.; Liotta, Lance A.; Popov, Serguei G.

    2011-01-01

    Inhalational anthrax is caused by spores of the bacterium Bacillus anthracis (B. anthracis), and is an extremely dangerous disease that can kill unvaccinated victims within 2 weeks. Modern antibiotic-based therapy can increase the survival rate to ∼50%, but only if administered presymptomatically (within 24–48 h of exposure). To discover host signaling responses to presymptomatic anthrax, label-free quantitative phosphoproteomics via liquid chromatography coupled to mass spectrometry was used to compare spleens from uninfected and spore-challenged mice over a 72 h time-course. Spleen proteins were denatured using urea, reduced using dithiothreitol, alkylated using iodoacetamide, and digested into peptides using trypsin, and the resulting phosphopeptides were enriched using titanium dioxide solid-phase extraction and analyzed by nano-liquid chromatography-Linear Trap Quadrupole-Orbitrap-MS(/MS). The fragment ion spectra were processed using DeconMSn and searched using both Mascot and SEQUEST resulting in 252,626 confident identifications of 6248 phosphopeptides (corresponding to 5782 phosphorylation sites). The precursor ion spectra were deisotoped using Decon2LS and aligned using MultiAlign resulting in the confident quantitation of 3265 of the identified phosphopeptides. ANOVAs were used to produce a q-value ranked list of host signaling responses. Late-stage (48–72 h postchallenge) Sterne strain (lethal) infections resulted in global alterations to the spleen phosphoproteome. In contrast, ΔSterne strain (asymptomatic; missing the anthrax toxin) infections resulted in 188 (5.8%) significantly altered (q<0.05) phosphopeptides. Twenty-six highly tentative phosphorylation responses to early-stage (24 h postchallenge) anthrax were discovered (q<0.5), and ten of these originated from eight proteins that have known roles in the host immune response. These tentative early-anthrax host response signaling events within mouse spleens may translate into presymptomatic

  9. Identification and quantitation of amphetamines, cocaine, opiates, and phencyclidine in oral fluid by liquid chromatography-tandem mass spectrometry.

    PubMed

    Fritch, Dean; Blum, Kristen; Nonnemacher, Sheena; Haggerty, Brenda J; Sullivan, Matthew P; Cone, Edward J

    2009-01-01

    Analytical methods for measuring multiple licit and illicit drugs and metabolites in oral fluid require high sensitivity, specificity, and accuracy. With the limited volume available for testing, comprehensive methodology is needed for simultaneous measurement of multiple analytes in a single aliquot. This report describes the validation of a semi-automated method for the simultaneous extraction, identification, and quantitation of 21 analytes in a single oral fluid aliquot. The target compounds included are amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxy-amphetamine, 3,4-methylenedioxyethylamphetamine, pseudoephedrine, cocaine, benzoylecgonine, codeine, norcodeine, 6-acetylcodeine, morphine, 6-acetylmorphine, hydrocodone, norhydrocodone, dihydrocodeine, hydromorphone, oxycodone, noroxycodone, oxymorphone, and phencyclidine. Oral fluid specimens were collected with the Intercept device and extracted by solid-phase extraction (SPE). Drug recovery from the Intercept device averaged 84.3%, and SPE extraction efficiency averaged 91.2% for the 21 analytes. Drug analysis was performed by liquid chromatography-tandem mass spectrometry in the positive electrospray mode using ratios of qualifying product ions within +/-25% of calibration standards. Matrix ion suppression ranged from -57 to 8%. The limit of quantitation ranged from 0.4 to 5 ng/mL using 0.2 mL of diluted oral fluid sample. Application of the method was demonstrated by testing oral fluid specimens from drug abuse treatment patients. Thirty-nine patients tested positive for various combinations of licit and illicit drugs and metabolites. In conclusion, this validated method is suitable for simultaneous measurement of 21 licit and illicit drugs and metabolites in oral fluid.

  10. Mass spectrometry-based, label-free quantitative proteomics of round spermatids in mice

    PubMed Central

    WANG, HAILONG; LI, YAN; YANG, LIJUAN; YU, BAOFENG; YAN, PING; PANG, MIN; LI, XIAOBING; YANG, HONG; ZHENG, GUOPING; XIE, JUN; GUO, RUI

    2014-01-01

    Round haploid spermatids are formed at the completion of meiosis. These spermatids then undergo morphological and cytological changes during spermiogenesis. Although sperm proteomes have been extensively studied, relatively few studies have specifically investigated the proteome of round spermatids. We developed a label-free quantitative method in combination with 2D-nano-LC-ESI-MS/MS to investigate the proteome of round spermatids in mice. Analysis of the proteomic data identified 2,331 proteins in the round spermatids. Functional classification of the proteins based on Gene Ontology terms and enrichment analysis further revealed the following: 504 of the identified proteins are predicted to be involved in the generation of precursor metabolites and energy; 343 proteins in translation and protein targeting; 298 proteins in nucleotide and nucleic acid metabolism; 275 and 289 proteins in transport and cellular component organization, respectively. A number of the identified proteins were associated with cytoskeleton organization (183), protein degradation (116) and response to stimulus (115). KEGG pathway analysis identified 68 proteins that are annotated as components of the ribosomal pathway and 17 proteins were related to aminoacyl-tRNA biosynthesis. The round spermatids also contained 28 proteins involved in the proteasome pathway and 40 proteins in the lysosome pathway. A total of 60 proteins were annotated as parts of the spliceosome pathway, in which heterogeneous nuclear RNA is converted to mRNA. Approximately 94 proteins were identified as actin-binding proteins, involved in the regulation of the actin cytoskeleton. In conclusion, using a label-free shotgun proteomic approach, we identified numerous proteins associated with spermiogenesis in round spermatids. PMID:25109358

  11. Precision and accuracy in the quantitative analysis of biological samples by accelerator mass spectrometry: application in microdose absolute bioavailability studies.

    PubMed

    Gao, Lan; Li, Jing; Kasserra, Claudia; Song, Qi; Arjomand, Ali; Hesk, David; Chowdhury, Swapan K

    2011-07-15

    Determination of the pharmacokinetics and absolute bioavailability of an experimental compound, SCH 900518, following a 89.7 nCi (100 μg) intravenous (iv) dose of (14)C-SCH 900518 2 h post 200 mg oral administration of nonradiolabeled SCH 900518 to six healthy male subjects has been described. The plasma concentration of SCH 900518 was measured using a validated LC-MS/MS system, and accelerator mass spectrometry (AMS) was used for quantitative plasma (14)C-SCH 900518 concentration determination. Calibration standards and quality controls were included for every batch of sample analysis by AMS to ensure acceptable quality of the assay. Plasma (14)C-SCH 900518 concentrations were derived from the regression function established from the calibration standards, rather than directly from isotopic ratios from AMS measurement. The precision and accuracy of quality controls and calibration standards met the requirements of bioanalytical guidance (U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Veterinary Medicine. Guidance for Industry: Bioanalytical Method Validation (ucm070107), May 2001. http://www.fda.gov/downloads/Drugs/GuidanceCompilanceRegulatoryInformation/Guidances/ucm070107.pdf ). The AMS measurement had a linear response range from 0.0159 to 9.07 dpm/mL for plasma (14)C-SCH 900158 concentrations. The CV and accuracy were 3.4-8.5% and 94-108% (82-119% for the lower limit of quantitation (LLOQ)), respectively, with a correlation coefficient of 0.9998. The absolute bioavailability was calculated from the dose-normalized area under the curve of iv and oral doses after the plasma concentrations were plotted vs the sampling time post oral dose. The mean absolute bioavailability of SCH 900518 was 40.8% (range 16.8-60.6%). The typical accuracy and standard deviation in AMS quantitative analysis of drugs from human plasma samples have been reported for the first time, and the impact of these

  12. Quantitation of sirolimus using liquid chromatography-tandem mass spectrometry (LC-MS-MS).

    PubMed

    Korecka, Magdalena; Shaw, Leslie M

    2010-01-01

    A multiple reaction monitoring positive ion HPLC method with tandem mass spectrometric detection (MS-MS) for determination of sirolimus in human blood samples is described. This method utilizes an online cleanup step that provides simple and rapid sample preparation with a switching valve technique. This procedure includes: instrumentation, API 3000 triple quadrupole with turbo-ion spray (Applied Biosystems, Foster City, CA); HPLC system (Agilent Technologies series 1100, Wilmington, DE); two position switching valve (Valco, Houston, TX); 10 mm guard cartridge (C(18)) used as an extraction column (Perkin Elmer, Norwalk, CT); analytical column (Nova-Pak C(18) column, 2.1 x 150 mm I.D., 4 microm, Waters Corp, Milford, MA) maintained at 65 degrees C; extraction solution, ammonium acetate (30 mM, pH 5.2), flow rate 1.0 mL/min; eluting solution, methanol:30 mM ammonium acetate buffer (pH 5.2, 97:3 v/v), flow rate 0.8 mL/min with 1/3 of the flow split post-column into the MS-MS; total run-time 3.5 min. Sample preparation is based on simple protein precipitation with a mixture of methanol and zinc sulfate (7:3, v/v) followed by online sample cleanup. This procedure provides a decreased sample preparation time by a factor of four compared to a method that uses an SPE column. The first and third quadrupoles were set to detect the ammonium adduct ion and a high mass fragment of sirolimus (m/z 931.8-->864.6), and of an internal standard (ascomycin) (m/z 809.5-->756.5). The lower limit of quantification of this method is 2.5 microg/L. The quantification of drug is made from standard curve using peak-area ratio of analyte vs. internal standard. Calibration curve is constructed using non-weighted linear through zero regression.

  13. Simultaneous quantitation of amphetamines and opiates in human hair by liquid chromatography-tandem mass spectrometry.

    PubMed

    Liu, Hsiu-Chuan; Liu, Ray H; Lin, Dong-Liang

    2015-04-01

    In this study, an incubation, solid-phase extraction (SPE) and LC-MS-MS procedure was developed, validated and used for simultaneous analysis of amphetamine (AP), methamphetamine (MA), morphine (MOR), codeine (COD), 6-acetylmorphine (6-AM) and 6-acetylcodeine (6-AC) in hair. Hair samples were initially cut into sections, washed with dichloromethane, then sonicated in a methanol-trifluoroacetic acid mixture. The resulting solutions were processed with a SPE procedure before undergoing LC-MS-MS analysis. Mass spectrometric analysis was performed in positive-ion, multiple reactions monitoring (MRM) mode, using appropriate collision energy for each selected precursor ion. The overall protocol, when applied to the analysis of hair (50 mg) samples fortified with 100-10,000 pg/mg of the analytes, was found to achieve 55.5-74.6% recovery of the six analytes with the following analytical parameters: (i) intra- and interday precision/accuracy data for the six analytes in the 1.6-7.6%/-6.0-12.8% and 1.3-6.6%/-6.9-9.3% ranges, respectively; (ii) r(2) > 0.998 for all six analytes and (iii) LOD 2 pg/mg for AP and MA, and 8 pg/mg for MOR, COD, 6-AM and 6-AC; LOQ 10 pg/mg for all six analytes. This method was then utilized to (i) analyze hair samples collected from 86 self-reported drug users and (ii) evaluate the deposition pattern of drugs in head hairs from four female MA and heroin users in a rehabilitation facility. This relatively simple protocol was found superior over the GC-MS methods we have previously developed and utilized in our laboratory for the analysis of these six analytes.

  14. Ambient ionization mass spectrometry

    NASA Astrophysics Data System (ADS)

    Lebedev, A. T.

    2015-07-01

    Ambient ionization mass spectrometry emerged as a new scientific discipline only about ten years ago. A considerable body of information has been reported since that time. Keeping the sensitivity, performance and informativity of classical mass spectrometry methods, the new approach made it possible to eliminate laborious sample preparation procedures and triggered the development of miniaturized instruments to work directly in the field. The review concerns the theoretical foundations and design of ambient ionization methods. Their advantages and drawbacks, as well as prospects for application in chemistry, biology, medicine, environmetal analysis, etc., are discussed. The bibliography includes 194 references.

  15. Determination of loperamide in mdr1a/1b knock-out mouse brain tissue using matrix-assisted laser desorption/ionization mass spectrometry and comparison with quantitative electrospray-triple quadrupole mass spectrometry analysis.

    PubMed

    Shin, Young G; Dong, Teresa; Chou, Bilin; Menghrajani, Kapil

    2011-11-01

    Recently matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) imaging has been used to analyze small molecule pharmaceutical compounds directly on tissue sections to determine spatial distribution within target tissue and organs. The data presented to date usually indicate relative amounts of drug within the tissue. The determination of absolute amounts is still done using tissue homogenization followed by traditional liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, the quantitative determination of loperamide, an antidiarrheal agent and a P-glycoprotein substrate, in mdr1a/1b (-/-) mouse brain tissue sections using MALDI MS on a quadrupole time-of-flight mass spectrometry is described. 5 mg/mL α-cyano-4-hydroxycinnamic acid in 50% acetonitrile with 0.1% trifluoroacetic acid and 0.5 μM reserpine was used as the MALDI matrix. The calibration curve constructed by the peak intensities of standard samples from MALDI MS was linear from 0.025 to 0.5 μM with r² = 0.9989. The accuracy of calibration curve standards was 78.3-105.9% and the percent deviation was less than 25%. Comparison between direct MALDI tissue analysis and conventional tissue analysis using homogenization followed by electrospray LC-MS/MS was also explored.

  16. A device for automated direct sampling and quantitation from solid-phase sorbent extraction cards by electrospray tandem mass spectrometry.

    PubMed

    Wachs, Timothy; Henion, Jack

    2003-04-01

    A new solid-phase extraction (SPE) device in the 96-well format (SPE Card) has been employed for automated off-line sample preparation of low-volume urine samples. On-line automated analyte elution via SPE and direct quantitation by micro ion spray mass spectrometry is reported. This sample preparation device has the format of a microtiter plate and is molded in a plastic frame which houses 96 separate sandwiched 3M Empore sorbents (0.5-mm-thickness, 8-microm particles) covered on both sides by a microfiber support material. Ninety-six discrete SPE zones, each 7 mm in diameter, are imbedded into the sheet in the conventional 9-mm pitch (spacing) of a 96-well microtiter plate. In this study one-quarter of an SPE Card (24 individual zones) was used merely as a convenience. After automated off-line interference elution of applied human urine from 24 samples, a section of SPE Card is mounted vertically on a computer-controlled X, Y, Z positioner in front of a micro ion spray direct sampling tube equipped with a beveled tip. The beveled tip of this needle robotically penetrates each SPE elution zone (sorbent disk) or stationary phase in a serial fashion. The eluted analytes are sequentially transferred directly to a microelectrosprayer to obtain tandem mass spectrometric (MS/MS) analysis. This strategy precludes any HPLC separation and the associated method development. The quantitative determination of Ritalin (methylphenidate) from fortified human urine samples is demonstrated. A trideuterated internal standard of methylphenidate was used to obtain ion current response ratios between the parent drug and the internal standard. Human control urine samples fortified from 6.6 to 3300 ng/mL (normal therapeutic levels have been determined in other studies to be between 50 and 100 ng/mL urine) were analyzed and a linear calibration curve was obtained with a correlation coefficient of 0.9999, where the precision of the quality control (QC) samples ranged from 9.6% at the 24

  17. Using ProtMAX to create high-mass-accuracy precursor alignments from label-free quantitative mass spectrometry data generated in shotgun proteomics experiments.

    PubMed

    Egelhofer, Volker; Hoehenwarter, Wolfgang; Lyon, David; Weckwerth, Wolfram; Wienkoop, Stefanie

    2013-03-01

    Recently, new software tools have been developed for improved protein quantification using mass spectrometry (MS) data. However, there are still limitations especially in high-sample-throughput quantification methods, and most of these relate to extensive computational calculations. The mass accuracy precursor alignment (MAPA) strategy has been shown to be a robust method for relative protein quantification. Its major advantages are high resolution, sensitivity and sample throughput. Its accuracy is data dependent and thus best suited for precursor mass-to-charge precision of ∼1 p.p.m. This protocol describes how to use a software tool (ProtMAX) that allows for the automated alignment of precursors from up to several hundred MS runs within minutes without computational restrictions. It comprises features for 'ion intensity count' and 'target search' of a distinct set of peptides. This procedure also includes the recommended MS settings for complex quantitative MAPA analysis using ProtMAX (http://www.univie.ac.at/mosys/software.html).

  18. MASS SPECTROMETRY-BASED METABOLOMICS

    PubMed Central

    Dettmer, Katja; Aronov, Pavel A.; Hammock, Bruce D.

    2007-01-01

    This review presents an overview of the dynamically developing field of mass spectrometry-based metabolomics. Metabolomics aims at the comprehensive and quantitative analysis of wide arrays of metabolites in biological samples. These numerous analytes have very diverse physico-chemical properties and occur at different abundance levels. Consequently, comprehensive metabolomics investigations are primarily a challenge for analytical chemistry and specifically mass spectrometry has vast potential as a tool for this type of investigation. Metabolomics require special approaches for sample preparation, separation, and mass spectrometric analysis. Current examples of those approaches are described in this review. It primarily focuses on metabolic fingerprinting, a technique that analyzes all detectable analytes in a given sample with subsequent classification of samples and identification of differentially expressed metabolites, which define the sample classes. To perform this complex task, data analysis tools, metabolite libraries, and databases are required. Therefore, recent advances in metabolomics bioinformatics are also discussed. PMID:16921475

  19. Analytical mass spectrometry

    SciTech Connect

    Not Available

    1990-01-01

    This 43rd Annual Summer Symposium on Analytical Chemistry was held July 24--27, 1990 at Oak Ridge, TN and contained sessions on the following topics: Fundamentals of Analytical Mass Spectrometry (MS), MS in the National Laboratories, Lasers and Fourier Transform Methods, Future of MS, New Ionization and LC/MS Methods, and an extra session. (WET)

  20. Analytical mass spectrometry. Abstracts

    SciTech Connect

    Not Available

    1990-12-31

    This 43rd Annual Summer Symposium on Analytical Chemistry was held July 24--27, 1990 at Oak Ridge, TN and contained sessions on the following topics: Fundamentals of Analytical Mass Spectrometry (MS), MS in the National Laboratories, Lasers and Fourier Transform Methods, Future of MS, New Ionization and LC/MS Methods, and an extra session. (WET)

  1. Simultaneous quantitative analysis of eight vitamin D analogues in milk using liquid chromatography-tandem mass spectrometry.

    PubMed

    Gomes, Fabio P; Shaw, P Nicholas; Whitfield, Karen; Hewavitharana, Amitha K

    2015-09-03

    Milk is an important source of nutrients for various risk populations, including infants. The accurate measurement of vitamin D in milk is necessary to provide adequate supplementation advice for risk groups and to monitor regulatory compliance. Currently used liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods are capable of measuring only four analogues of vitamin D in unfortified milk. We report here an accurate quantitative analytical method for eight analogues of vitamin D: Vitamin D2 and D3 (D2 and D3), 25-hydroxy D2 and D3, 24,25-dihydroxy D2 and D3, and 1,25-dihydroxyD2 and D3. In this study, we compared saponification and protein precipitation for the extraction of vitamin D from milk and found the latter to be more effective. We also optimised the pre-column derivatisation using 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD), to achieve the highest sensitivity and accuracy for all major vitamin D forms in milk. Chromatography was optimised to reduce matrix effects such as ion-suppression, and the matrix effects were eliminated using co-eluting stable isotope labelled internal standards for the calibration of each analogue. The analogues, 25-hydroxyD3 (25(OH)D3) and its epimer (3-epi-25(OH)D3) were chromatographically resolved, to prevent over-estimation of 25(OH)D3. The method was validated and subsequently applied for the measurement of total vitamin D levels in human, cow, mare, goat and sheep milk samples. The detection limits, repeatability standard deviations, and recovery ranges were from 0.2 to 0.4 femtomols, 6.30-13.5%, and 88.2-105%, respectively.

  2. Quantitation of ceramides in nude mouse skin by normal-phase liquid chromatography and atmospheric pressure chemical ionization mass spectrometry.

    PubMed

    Liou, Yi-Bo; Sheu, Ming-Thau; Liu, Der-Zen; Lin, Shan-Yang; Ho, Hsiu-O

    2010-06-01

    A sensitive and accurate normal-phase liquid chromatography and atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS) method for determining the standard ceramide [NS] (Cer[NS]) was developed and validated so as to improve the traditional thin-layer chromatography (TLC) technique and LC-electrospray ionization (ESI)-MS method to profile and quantify ceramides in nude mouse skin. Normal-phase LC-APCI-MS was optimized to separate the nine classes of ceramides presented in the stratum corneum (SC) of nude mouse skin. A normal-phase silica column eluted with the gradient system from heptane:acetone/butanol (90:10, v/v) of 75:25 to 100% acetone/butanol (90:10, v/v) (with each solvent containing 0.1% [v/v] triethylamine and 0.1% [v/v] formic acid) at a flow rate of 0.8 ml/min was found to be optimal for analyzing standard Cer[NS]. The analysis of Cer[NS] was validated and employed as the standard for constructing a calibration curve to quantitate all classes of ceramides. This method was applied to profile the classes and contents of ceramides in the SC of nude mouse skin and proved to be workable. It was concluded that this improved method can be used to directly detect and quantify all classes of ceramides in the SC of nude mouse skin and that it is more convenient and labor-saving than the traditional TLC method.

  3. Structure of Complement C3(H2O) Revealed By Quantitative Cross-Linking/Mass Spectrometry And Modeling*

    PubMed Central

    Pellarin, Riccardo; Sali, Andrej; Barlow, Paul N.

    2016-01-01

    The slow but spontaneous and ubiquitous formation of C3(H2O), the hydrolytic and conformationally rearranged product of C3, initiates antibody-independent activation of the complement system that is a key first line of antimicrobial defense. The structure of C3(H2O) has not been determined. Here we subjected C3(H2O) to quantitative cross-linking/mass spectrometry (QCLMS). This revealed details of the structural differences and similarities between C3(H2O) and C3, as well as between C3(H2O) and its pivotal proteolytic cleavage product, C3b, which shares functionally similarity with C3(H2O). Considered in combination with the crystal structures of C3 and C3b, the QCMLS data suggest that C3(H2O) generation is accompanied by the migration of the thioester-containing domain of C3 from one end of the molecule to the other. This creates a stable C3b-like platform able to bind the zymogen, factor B, or the regulator, factor H. Integration of available crystallographic and QCLMS data allowed the determination of a 3D model of the C3(H2O) domain architecture. The unique arrangement of domains thus observed in C3(H2O), which retains the anaphylatoxin domain (that is excised when C3 is enzymatically activated to C3b), can be used to rationalize observed differences between C3(H2O) and C3b in terms of complement activation and regulation. PMID:27250206

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

    PubMed

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

    2009-06-01

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

  5. Using quantitative mass spectrometry to better understand the influence of genetics and nutritional perturbations on the virulence potential of Staphylococcus aureus.

    PubMed

    Chapman, Jessica R; Balasubramanian, Divya; Tam, Kayan; Askenazi, Manor; Copin, Richard; Shopsin, Bo; Torres, Victor J; Ueberheide, Beatrix

    2017-02-14

    Staphylococcus aureus (Sa) is the leading cause of a variety of bacterial infections ranging from superficial skin infections to invasive and life threatening diseases such as septic bacteremia, necrotizing pneumonia, and endocarditis. The success of Sa as a human pathogen is due to its ability to adapt to the environment by changing expression, production, or secretion of virulence factors. Although Sa immune evasion is well-studied, the regulation of virulence factors under different nutrient and growth conditions is still not well understood. Here, we used label-free quantitative mass spectrometry to quantify and compare the secreted Sa proteins (i.e. exoproteomes) of master regulator mutants or established reference strains. Different environmental conditions were addressed by growing the bacteria in rich or minimal media at different phases of growth. We observed clear differences in the composition of the exoproteomes depending on the genetic background or growth conditions. The relative abundance of cytotoxins determined in our study correlated well with differences in cytotoxicity measured by lysis of human neutrophils. Our findings demonstrate that label-free quantitative mass spectrometry is a versatile tool for predicting the virulence of bacterial strains and highlights the importance of the experimental design for in vitro studies. Furthermore, the results indicate that label-free proteomics can be used to cluster isolates into groups with similar virulence properties and genetic lineages, highlighting the power of label-free quantitative mass spectrometry to distinguish Sa strains.

  6. Mass spectrometry with accelerators.

    PubMed

    Litherland, A E; Zhao, X-L; Kieser, W E

    2011-01-01

    As one in a series of articles on Canadian contributions to mass spectrometry, this review begins with an outline of the history of accelerator mass spectrometry (AMS), noting roles played by researchers at three Canadian AMS laboratories. After a description of the unique features of AMS, three examples, (14)C, (10)Be, and (129)I are given to illustrate the methods. The capabilities of mass spectrometry have been extended by the addition of atomic isobar selection, molecular isobar attenuation, further ion acceleration, followed by ion detection and ion identification at essentially zero dark current or ion flux. This has been accomplished by exploiting the techniques and accelerators of atomic and nuclear physics. In 1939, the first principles of AMS were established using a cyclotron. In 1977 the selection of isobars in the ion source was established when it was shown that the (14)N(-) ion was very unstable, or extremely difficult to create, making a tandem electrostatic accelerator highly suitable for assisting the mass spectrometric measurement of the rare long-lived radioactive isotope (14)C in the environment. This observation, together with the large attenuation of the molecular isobars (13)CH(-) and (12)CH 2(-) during tandem acceleration and the observed very low background contamination from the ion source, was found to facilitate the mass spectrometry of (14)C to at least a level of (14)C/C ~ 6 × 10(-16), the equivalent of a radiocarbon age of 60,000 years. Tandem Accelerator Mass Spectrometry, or AMS, has now made possible the accurate radiocarbon dating of milligram-sized carbon samples by ion counting as well as dating and tracing with many other long-lived radioactive isotopes such as (10)Be, (26)Al, (36)Cl, and (129)I. The difficulty of obtaining large anion currents with low electron affinities and the difficulties of isobar separation, especially for the heavier mass ions, has prompted the use of molecular anions and the search for alternative

  7. MASS SPECTROMETRY IN ENVIRONMENTAL SCIENCES

    EPA Science Inventory

    This review covers applications of mass spectrometry to the environmental sciences. From the early applications of mass spectrometry to environmental research in the 1960s and 1970s, mass spectrometry has played an important role in aiding our understanding of environmental poll...

  8. Desorption in Mass Spectrometry.

    PubMed

    Usmanov, Dilshadbek Tursunbayevich; Ninomiya, Satoshi; Chen, Lee Chuin; Saha, Subhrakanti; Mandal, Mridul Kanti; Sakai, Yuji; Takaishi, Rio; Habib, Ahsan; Hiraoka, Kenzo; Yoshimura, Kentaro; Takeda, Sen; Wada, Hiroshi; Nonami, Hiroshi

    2017-01-01

    In mass spectrometry, analytes must be released in the gas phase. There are two representative methods for the gasification of the condensed samples, i.e., ablation and desorption. While ablation is based on the explosion induced by the energy accumulated in the condensed matrix, desorption is a single molecular process taking place on the surface. In this paper, desorption methods for mass spectrometry developed in our laboratory: flash heating/rapid cooling, Leidenfrost phenomenon-assisted thermal desorption (LPTD), solid/solid friction, liquid/solid friction, electrospray droplet impact (EDI) ionization/desorption, and probe electrospray ionization (PESI), will be described. All the methods are concerned with the surface and interface phenomena. The concept of how to desorb less-volatility compounds from the surface will be discussed.

  9. Desorption in Mass Spectrometry

    PubMed Central

    Usmanov, Dilshadbek Tursunbayevich; Ninomiya, Satoshi; Chen, Lee Chuin; Saha, Subhrakanti; Mandal, Mridul Kanti; Sakai, Yuji; Takaishi, Rio; Habib, Ahsan; Hiraoka, Kenzo; Yoshimura, Kentaro; Takeda, Sen; Wada, Hiroshi; Nonami, Hiroshi

    2017-01-01

    In mass spectrometry, analytes must be released in the gas phase. There are two representative methods for the gasification of the condensed samples, i.e., ablation and desorption. While ablation is based on the explosion induced by the energy accumulated in the condensed matrix, desorption is a single molecular process taking place on the surface. In this paper, desorption methods for mass spectrometry developed in our laboratory: flash heating/rapid cooling, Leidenfrost phenomenon-assisted thermal desorption (LPTD), solid/solid friction, liquid/solid friction, electrospray droplet impact (EDI) ionization/desorption, and probe electrospray ionization (PESI), will be described. All the methods are concerned with the surface and interface phenomena. The concept of how to desorb less-volatility compounds from the surface will be discussed. PMID:28337398

  10. Hybrid instruments for mass spectrometry/mass spectrometry

    SciTech Connect

    Glish, G.L.; McLuckey, S.A.

    1986-01-01

    In order to refine further the technique of mass spectrometry/mass spectrometry efforts are being made to combine the desirable features of sector based tandem instruments with those of triple quadrupole mass spectrometers. This has resulted in the construction of tandem mass spectrometers which incorporate both sector type analyzers and quadrupole mass filters. These so-called hybrid instruments, designed specifically for mass spectrometry/mass spectrometry applications, are appearing in a variety of geometries each with unique features. This review describes the hybrid instruments reported to data and discusses general considerations for evaluating hybrid instruments with regard to application. 100 references.

  11. Quantitative analysis of polar lipids in the nanoliter level of rat serum by liquid chromatography/mass spectrometry/mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polar lipids in serum, including lysophospholipids (LPLs) and free fatty acids (FFAs), have a broad range of biological activities and require a suitable method for their quantitative analysis. Conventional methods use multistep procedures to simultaneously purify and analyze polar lipids and non-po...

  12. Characterization of microbial siderophores by mass spectrometry.

    PubMed

    Pluháček, Tomáš; Lemr, Karel; Ghosh, Dipankar; Milde, David; Novák, Jiří; Havlíček, Vladimír

    2016-01-01

    Siderophores play important roles in microbial iron piracy, and are applied as infectious disease biomarkers and novel pharmaceutical drugs. Inductively coupled plasma and molecular mass spectrometry (ICP-MS) combined with high resolution separations allow characterization of siderophores in complex samples taking advantages of mass defect data filtering, tandem mass spectrometry, and iron-containing compound quantitation. The enrichment approaches used in siderophore analysis and current ICP-MS technologies are reviewed. The recent tools for fast dereplication of secondary metabolites and their databases are reported. This review on siderophores is concluded with their recent medical, biochemical, geochemical, and agricultural applications in mass spectrometry context.

  13. A Temporal Examination of the Planktonic and Biofilm Proteome of Whole Cell Pseudomonas aeruginosa PAO1 Using Quantitative Mass Spectrometry*

    PubMed Central

    Park, Amber J.; Murphy, Kathleen; Krieger, Jonathan R.; Brewer, Dyanne; Taylor, Paul; Habash, Marc; Khursigara, Cezar M.

    2014-01-01

    Chronic polymicrobial lung infections are the chief complication in patients with cystic fibrosis. The dominant pathogen in late-stage disease is Pseudomonas aeruginosa, which forms recalcitrant, structured communities known as biofilms. Many aspects of biofilm biology are poorly understood; consequently, effective treatment of these infections is limited, and cystic fibrosis remains fatal. Here we combined in-solution protein digestion of triplicate growth-matched samples with a high-performance mass spectrometry platform to provide the most comprehensive proteomic dataset known to date for whole cell P. aeruginosa PAO1 grown in biofilm cultures. Our analysis included protein–protein interaction networks and PseudoCAP functional information for unique and significantly modulated proteins at three different time points. Secondary analysis of a subgroup of proteins using extracted ion currents validated the spectral counting data of 1884 high-confidence proteins. In this paper we demonstrate a greater representation of proteins related to metabolism, DNA stability, and molecular activity in planktonically grown P. aeruginosa PAO1. In addition, several virulence-related proteins were increased during planktonic growth, including multiple proteins encoded by the pyoverdine locus, uncharacterized proteins with sequence similarity to mammalian cell entry protein, and a member of the hemagglutinin family of adhesins, HecA. Conversely, biofilm samples contained an uncharacterized protein with sequence similarity to an adhesion protein with self-association characteristics (AidA). Increased levels of several phenazine biosynthetic proteins, an uncharacterized protein with sequence similarity to a metallo-beta-lactamase, and lower levels of the drug target gyrA support the putative characteristics of in situ P. aeruginosa infections, including competitive fitness and antibiotic resistance. This quantitative whole cell approach advances the existing P. aeruginosa

  14. Use of proton transfer reaction time-of-flight mass spectrometry for quantitative monitoring of toxic nitramines in the environment

    NASA Astrophysics Data System (ADS)

    Wisthaler, A.; Zhu, L.; Stenstrøm, Y.; Nielsen, C. J.

    2014-12-01

    Naturally occurring aliphatic amines and industrially emitted aromatic amines and alkanolamines produce toxic nitramines, (R1R2)-N-NO2, when photo-oxidized in the atmosphere in the presence of nitrogen oxides (NOx). Particular concerns arise from amine-based CO2 capture where the amine solution may get nitrated by NOx in the flue gas. An on-line analytical technique for measuring nitramines in industrial emissions and in ambient air is thus in high demand. Proton-Transfer-Reaction Mass Spectrometry (PTR-MS) is a state-of-the-art technique for on-line measurements of volatile organic compounds (VOCs) in air. Herein, we report on the use of high mass resolution proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS) for on-line analysis of nitramines. We generated a mass spectral library from a series of nitramines and investigated the analytical performance of PTR-ToF-MS in terms of sensitivity, precision, accuracy and detection limit. We will discuss limitations of the innovative technique and propose measurement strategies for future emission and ambient measurements.

  15. [Qualitative and quantitative analysis of amygdalin and its metabolite prunasin in plasma by ultra-high performance liquid chromatography-tandem quadrupole time of flight mass spectrometry and ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry].

    PubMed

    Gao, Meng; Wang, Yuesheng; Wei, Huizhen; Ouyang, Hui; He, Mingzhen; Zeng, Lianqing; Shen, Fengyun; Guo, Qiang; Rao, Yi

    2014-06-01

    A method was developed for the determination of amygdalin and its metabolite prunasin in rat plasma after intragastric administration of Maxing shigan decoction. The analytes were identified by ultra-high performance liquid chromatography-tandem quadrupole time of flight mass spectrometry and quantitatively determined by ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry. After purified by liquid-liquid extraction, the qualitative analysis of amygdalin and prunasin in the plasma sample was performed on a Shim-pack XR-ODS III HPLC column (75 mm x 2.0 mm, 1.6 microm), using acetonitrile-0.1% (v/v) formic acid aqueous solution. The detection was performed on a Triple TOF 5600 quadrupole time of flight mass spectrometer. The quantitative analysis of amygdalin and prunasin in the plasma sample was performed by separation on an Agilent C18 HPLC column (50 mm x 2.1 mm, 1.7 microm), using acetonitrile-0.1% (v/v) formic acid aqueous solution. The detection was performed on an AB Q-TRAP 4500 triple quadrupole mass spectrometer utilizing electrospray ionization (ESI) interface operated in negative ion mode and multiple-reaction monitoring (MRM) mode. The qualitative analysis results showed that amygdalin and its metabolite prunasin were detected in the plasma sample. The quantitative analysis results showed that the linear range of amygdalin was 1.05-4 200 ng/mL with the correlation coefficient of 0.999 0 and the linear range of prunasin was 1.25-2 490 ng/mL with the correlation coefficient of 0.997 0. The method had a good precision with the relative standard deviations (RSDs) lower than 9.20% and the overall recoveries varied from 82.33% to 95.25%. The limits of detection (LODs) of amygdalin and prunasin were 0.50 ng/mL. With good reproducibility, the method is simple, fast and effective for the qualitative and quantitative analysis of the amygdalin and prunasin in plasma sample of rats which were administered by Maxing shigan decoction.

  16. Real-Time Quantitative Analysis of H2, He, O2, and Ar by Quadrupole Ion Trap Mass Spectrometry

    NASA Technical Reports Server (NTRS)

    Ottens, Andrew K.; Harrison, W. W.; Griffin, Timothy P.; Helms, William R.; Voska, N. (Technical Monitor)

    2002-01-01

    The use of a quadrupole ion trap mass spectrometer for quantitative analysis of hydrogen and helium as well as other permanent gases is demonstrated. The customized instrument utilizes the mass selective instability mode of mass analysis as with commercial instruments; however, this instrument operates at a greater RF trapping frequency and without a buffer gas. With these differences, a useable mass range from 2 to over 50 Da is achieved, as required by NASA for monitoring the Space Shuttle during a launch countdown. The performance of the ion trap is evaluated using part-per-million concentrations of hydrogen, helium, oxygen and argon mixed into a nitrogen gas stream. Relative accuracy and precision when quantitating the four analytes were better than the NASA-required minimum of 10% error and 5% deviation, respectively. Limits of detection were below the NASA requirement of 25-ppm hydrogen and 100-ppm helium; those for oxygen and argon were slightly higher than the requirement. The instrument provided adequate performance at fast data recording rates, demonstrating the utility of an ion trap mass spectrometer as a real-time quantitative monitoring device for permanent gas analysis.

  17. Characterization and quantitative amino acids analysis of analgesic peptides in cinobufacini injection by size exclusion chromatography, matrix-assisted laser desorption/ionization time of flight mass spectrometry and gas chromatography mass spectrometry.

    PubMed

    Wu, Xu; Si, Nan; Bo, Gao; Hu, Hao; Yang, Jian; Bian, Baolin; Zhao, Hai Yu; Wang, Hongjie

    2015-01-01

    Cinobufacini injection that comes from the water extract of Bufo bufo gargarizans Cantor skin is widely used for cancer treatment in China. Peptide is one of its major types of constituents, however the biological effects and content of this injection are little reported. In present study, the analgesic effect of peptides was determined and evaluated by in-vivo models. To characterize and quantitatively analyze these peptides, a reliable and efficient method combining size exclusion chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with amino acid analysis was developed. The peptides presented as a series of analogs with similar molecular weights mostly ranging from 2 to 8 kDa. The amino acid analysis by gas chromatography mass spectrometry (GC-MS) was developed to determine both free and combined amino acids (FAA and CAA) in cinobufacini injection. This method achieved good linearity (R(2) , 0.9909-0.9999) and low limit of detection and quantification. FAA and CAA samples were efficiently analyzed by modified Phenomenex EZ: faast procedure. For the sample analysis, the method showed good repeatability (relative standard deviation, RSD ≤ 10%). For most FAA and CAA the mean recoveries were >80% with RSD <10%. The GC-MS based method is useful for quality assurance of both FAA and CAA in cinobufacini injection.

  18. "Magic" Ionization Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Trimpin, Sarah

    2016-01-01

    The systematic study of the temperature and pressure dependence of matrix-assisted ionization (MAI) led us to the discovery of the seemingly impossible, initially explained by some reviewers as either sleight of hand or the misinterpretation by an overzealous young scientist of results reported many years before and having little utility. The "magic" that we were attempting to report was that with matrix assistance, molecules, at least as large as bovine serum albumin (66 kDa), are lifted into the gas phase as multiply charged ions simply by exposure of the matrix:analyte sample to the vacuum of a mass spectrometer. Applied heat, a laser, or voltages are not necessary to achieve charge states and ion abundances only previously observed with electrospray ionization (ESI). The fundamentals of how solid phase volatile or nonvolatile compounds are converted to gas-phase ions without added energy currently involves speculation providing a great opportunity to rethink mechanistic understanding of ionization processes used in mass spectrometry. Improved understanding of the mechanism(s) of these processes and their connection to ESI and matrix-assisted laser desorption/ionization may provide opportunities to further develop new ionization strategies for traditional and yet unforeseen applications of mass spectrometry. This Critical Insights article covers developments leading to the discovery of a seemingly magic ionization process that is simple to use, fast, sensitive, robust, and can be directly applied to surface characterization using portable or high performance mass spectrometers.

  19. "Magic" Ionization Mass Spectrometry.

    PubMed

    Trimpin, Sarah

    2016-01-01

    The systematic study of the temperature and pressure dependence of matrix-assisted ionization (MAI) led us to the discovery of the seemingly impossible, initially explained by some reviewers as either sleight of hand or the misinterpretation by an overzealous young scientist of results reported many years before and having little utility. The “magic” that we were attempting to report was that with matrix assistance, molecules, at least as large as bovine serum albumin (66 kDa), are lifted into the gas phase as multiply charged ions simply by exposure of the matrix:analyte sample to the vacuum of a mass spectrometer. Applied heat, a laser, or voltages are not necessary to achieve charge states and ion abundances only previously observed with electrospray ionization (ESI). The fundamentals of how solid phase volatile or nonvolatile compounds are converted to gas-phase ions without added energy currently involves speculation providing a great opportunity to rethink mechanistic understanding of ionization processes used in mass spectrometry. Improved understanding of the mechanism(s) of these processes and their connection to ESI and matrix-assisted laser desorption/ionization may provide opportunities to further develop new ionization strategies for traditional and yet unforeseen applications of mass spectrometry. This Critical Insights article covers developments leading to the discovery of a seemingly magic ionization process that is simple to use, fast, sensitive, robust, and can be directly applied to surface characterization using portable or high performance mass spectrometers.

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

    PubMed

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

    2016-12-01

    Capillary electrophoresis (CE) offers excellent efficiency and orthogonality to liquid chromatographic (LC) separations for oligosaccharide structural analysis. Combination of CE with high resolution mass spectrometry (MS) for glycan analysis remains a challenging task due to the MS incompatibility of background electrolyte buffers and additives commonly used in offline CE separations. Here, a novel method is presented for the analysis of 2-aminobenzoic acid (2-AA) labelled glycans by capillary electrophoresis coupled to mass spectrometry (CE-MS). To ensure maximum resolution and excellent precision without the requirement for excessive analysis times, CE separation conditions including the concentration and pH of the background electrolyte, the effect of applied pressure on the capillary inlet and the capillary length were evaluated. Using readily available (12/13)C6 stable isotopologues of 2-AA, the developed method can be applied for quantitative glycan profiling in a twoplex manner based on the generation of extracted ion electropherograms (EIE) for (12)C6 'light' and (13)C6 'heavy' 2-AA labelled glycan isotope clusters. The twoplex quantitative CE-MS glycan analysis platform is ideally suited for comparability assessment of biopharmaceuticals, such as monoclonal antibodies, for differential glycomic analysis of clinical material for potential biomarker discovery or for quantitative microheterogeneity analysis of different glycosylation sites within a glycoprotein. Additionally, due to the low injection volume requirements of CE, subsequent LC-MS analysis of the same sample can be performed facilitating the use of orthogonal separation techniques for structural elucidation or verification of quantitative performance.

  1. Mass Spectrometry Applications for the Identification and Quantitation of Biomarkers Resulting from Human Exposure to Chemical Warfare Agents

    NASA Astrophysics Data System (ADS)

    Smith, J. Richard; Capacio, Benedict R.

    In recent years, a number of analytical methods using biomedical samples such as blood and urine have been developed for the verification of exposure to chemical warfare agents. The majority of methods utilize gas or liquid chromatography in conjunction with mass spectrometry. In a small number of cases of suspected human exposure to chemical warfare agents, biomedical specimens have been made available for testing. This chapter provides an overview of biomarkers that have been verified in human biomedical samples, details of the exposure incidents, the methods utilized for analysis, and the biomarker concentration levels determined in the blood and/or urine.

  2. Application of mass spectrometry in proteomics.

    PubMed

    Guerrera, Ida Chiara; Kleiner, Oliver

    2005-01-01

    Mass spectrometry has arguably become the core technology in proteomics. The application of mass spectrometry based techniques for the qualitative and quantitative analysis of global proteome samples derived from complex mixtures has had a big impact in the understanding of cellular function. Here, we give a brief introduction to principles of mass spectrometry and instrumentation currently used in proteomics experiments. In addition, recent developments in the application of mass spectrometry in proteomics are summarised. Strategies allowing high-throughput identification of proteins from highly complex mixtures include accurate mass measurement of peptides derived from total proteome digests and multidimensional peptide separations coupled with mass spectrometry. Mass spectrometric analysis of intact proteins permits the characterisation of protein isoforms. Recent developments in stable isotope labelling techniques and chemical tagging allow the mass spectrometry based differential display and quantitation of proteins, and newly established affinity procedures enable the targeted characterisation of post-translationally modified proteins. Finally, advances in mass spectrometric imaging allow the gathering of specific information on the local molecular composition, relative abundance and spatial distribution of peptides and proteins in thin tissue sections.

  3. Mass spectrometry data from a quantitative analysis of protein expression in gills of immuno-challenged blue mussels (Mytilus edulis).

    PubMed

    Hörnaeus, K; Guillemant, J; Mi, J; Hernroth, B; Bergquist, J; Lind, S Bergström

    2016-09-01

    Here, we provide the dataset associated with our research article on the potential effects of ocean acidification on antimicrobial peptide (AMP) activity in the gills of Mytilus edulis, "Impact of ocean acidification on antimicrobial activity in gills of the blue mussel (Mytilus edulis)" [1]. Blue mussels were stimulated with lipopolysaccharides and samples were collected at different time points post injection. Protein extracts were prepared from the gills, digested using trypsin and a full in-depth proteome investigation was performed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Protein identification and quantification was performed using the MaxQuant 1.5.1.2 software, "MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification" [2].

  4. Quantitative determination of bovine caseinoglycomacropeptide in infant formulas by ultra-high-performance liquid chromatography-electrospray-ionization mass spectrometry.

    PubMed

    Zhang, Jingshun; Ren, Yiping; Ma, Zhenyi; Huang, Baifen; Cai, Zengxuan; Li, Duo

    2011-10-01

    An ultra-high-performance liquid chromatography-electrospray ionization coupled to mass spectrometry method has been developed for determining caseinoglycomacropeptide (CGMP) in infant formulas by selected ion reaction and area monitoring modes. The present study focused on the optimization of sample pretreatment, chromatographic resolution and mass spectrometry parameters. After a simple sample pretreatment, the two genetic variants of caseinoglycomacropeptide, CGMP(A) and CGMP(B), were separated using a BEH300 C(18) column by gradient elution. The established method was extensively validated by determining the linearity (R(2)>0.999), average recovery (95.8-118.4%), inter-day precision (relative standard deviation ≤7.81%) and intra-day precision (relative standard deviation ≤6.99%) based on two scan modes. To further verify the applicability of the method, 21 brands of commercial available infant formulas were analyzed. The results showed that the present method is selective, sensitive and reliable for separating and quantifying two genetic variants (CGMP(A) and CGMP(B)) of caseinoglycomacropeptide in infant formulas with complex matrix.

  5. Quantitation of protein S-glutathionylation by liquid chromatography-tandem mass spectrometry: correction for contaminating glutathione and glutathione disulfide.

    PubMed

    Bukowski, Michael R; Bucklin, Christopher; Picklo, Matthew J

    2015-01-15

    Protein S-glutathionylation is a posttranslational modification that links oxidative stimuli to reversible changes in cellular function. Protein-glutathione mixed disulfide (PSSG) is commonly quantified by reduction of the disulfide and detection of the resultant glutathione species. This methodology is susceptible to contamination by free unreacted cellular glutathione (GSH) species, which are present in 1000-fold greater concentration. A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method was developed for quantification of glutathione and glutathione disulfide (GSSG), which was used for the determination of PSSG in biological samples. Analysis of rat liver samples demonstrated that GSH and GSSG coprecipitated with proteins similar to the range for PSSG in the sample. The use of [(13)C2,(5)N]GSH and [(13)C4,(5)N2]GSSG validated these results and demonstrated that the release of GSH from PSSG did not occur during sample preparation and analysis. These data demonstrate that GSH and GSSG contamination must be accounted for when determining PSSG content in cellular/tissue preparations. A protocol for rinsing samples to remove the adventitious glutathione species is demonstrated. The fragmentation patterns for glutathione were determined by high-resolution mass spectrometry, and candidate ions for detection of PSSG on protein and protein fragments were identified.

  6. Quantitative analysis of cytokinins in plants by high performance liquid chromatography: electronspray ionization ion trap mass spectrometry.

    PubMed

    Chen, Weiqi; Gai, Ying; Liu, Shichang; Wang, Renxiao; Jiang, Xiangning

    2010-10-01

    The present paper introduces a highly sensitive and selective method for simultaneous quantification of 12 cytokinins (free form and their conjugates). The method includes a protocol of extraction with methanol/water/formic acid (15/4/1, v/v/v) to the micro-scale samples, pre-purification with solid phase extraction (SPE) cartridges of the extracts, separation with a high performance liquid chromatography (HPLC) and detection by an electrospray ionization ion trap mass spectrometry (ESI-Ion trap-MS) system in a consecutive ion monitoring (CRM) mode at the three stage fragmentation of mass spectrometry (MS(3) ). The lowest detection level of the cytokinins of the method reaches 0.1-2.0 pg with a very wide range of linear regression from 1-512 pg, at the coefficient factors of 0.98-0.99. The feasibility of this method has been proven in the application of the method to the analysis of the trace-amount contents of cytokinins in the micro-scale samples of various types of plant materials, such as aerial parts of rice and poplar leaves etc. 12 endogenous cytokinins had been identified and quantified in the plant tissues, with an acceptable relatively higher recovery rate from 40% to 70%.

  7. Quantitative real-time monitoring of chemical reactions by autosampling flow injection analysis coupled with atmospheric pressure chemical ionization mass spectrometry.

    PubMed

    Zhu, Zhenqian; Bartmess, John E; McNally, Mary Ellen; Hoffman, Ron M; Cook, Kelsey D; Song, Liguo

    2012-09-04

    Although qualitative and/or semiquantitative real-time monitoring of chemical reactions have been reported with a few mass spectrometric approaches, to our knowledge, no quantitative mass spectrometric approach has been reported so far to have a calibration valid up to molar concentrations as required by process control. This is mostly due to the absence of a practical solution that could well address the sample overloading issue. In this study, a novel autosampling flow injection analysis coupled with an atmospheric pressure chemical ionization mass spectrometry (FIA/APCI-MS) system, consisting of a 1 μL automatic internal sample injector, a postinjection splitter with 1:10 splitting ratio, and a detached APCI source connected to the mass spectrometer using a 4.5 in. long, 0.042 in. inner diameter (ID) stainless-steel capillary, was thus introduced. Using this system together with an optional FIA solvent modifier, e.g., 0.05% (v/v) isopropylamine, a linear quantitative calibration up to molar concentration has been achieved with 3.4-7.2% relative standard deviations (RSDs) for 4 replicates. As a result, quantitative real-time monitoring of a model reaction was successfully performed at the 1.63 M level. It is expected that this novel autosampling FIA/APCI-MS system can be used in quantitative real-time monitoring of a wide range of reactions under diverse reaction conditions.

  8. Analytical aspects of hydrogen exchange mass spectrometry

    PubMed Central

    Engen, John R.; Wales, Thomas E.

    2016-01-01

    The analytical aspects of measuring hydrogen exchange by mass spectrometry are reviewed. The nature of analytical selectivity in hydrogen exchange is described followed by review of the analytical tools required to accomplish fragmentation, separation, and the mass spectrometry measurements under restrictive exchange quench conditions. In contrast to analytical quantitation that relies on measurements of peak intensity or area, quantitation in hydrogen exchange mass spectrometry depends on measuring a mass change with respect to an undeuterated or deuterated control, resulting in a value between zero and the maximum amount of deuterium that could be incorporated. Reliable quantitation is a function of experimental fidelity and to achieve high measurement reproducibility, a large number of experimental variables must be controlled during sample preparation and analysis. The method also reports on important qualitative aspects of the sample, including conformational heterogeneity and population dynamics. PMID:26048552

  9. Capillary isoelectric focusing-tandem mass spectrometry and reversed-phase liquid chromatography-tandem mass spectrometry for quantitative proteomic analysis of differentiating PC12 cells by eight-plex isobaric tags for relative and absolute quantification.

    PubMed

    Zhu, Guijie; Sun, Liangliang; Keithley, Richard B; Dovichi, Norman J

    2013-08-06

    We report the application of capillary isoelectric focusing for quantitative analysis of a complex proteome. Biological duplicates were generated from PC12 cells at days 0, 3, 7, and 12 following treatment with nerve growth factor. These biological duplicates were digested with trypsin, labeled using eight-plex isobaric tags for relative and absolute quantification (iTRAQ) chemistry, and pooled. The pooled peptides were separated into 25 fractions using reversed-phase liquid chromatography (RPLC). Technical duplicates of each fraction were separated by capillary isoelectric focusing (cIEF) using a set of amino acids as ampholytes. The cIEF column was interfaced to an Orbitrap Velos mass spectrometer with an electrokinetically pumped sheath-flow nanospray interface. This HPLC-cIEF-electrospray-tandem mass spectrometry (ESI-MS/MS) approach identified 835 protein groups and produced 2,329 unique peptides IDs. The biological duplicates were analyzed in parallel using conventional strong-cation exchange (SCX)-RPLC-ESI-MS/MS. The iTRAQ peptides were first separated into eight fractions using SCX. Each fraction was then analyzed by RPLC-ESI-MS/MS. The SCX-RPLC approach generated 1,369 protein groups and 3,494 unique peptide IDs. For protein quantitation, 96 and 198 differentially expressed proteins were obtained with RPLC-cIEF and SCX-RPLC, respectively. The combined set identified 231 proteins. Protein expression changes measured by RPLC-cEIF and SCX-RPLC were highly correlated.

  10. Quantitative determination of phenothiazine derivatives in human plasma using monolithic silica solid-phase extraction tips and gas chromatography-mass spectrometry.

    PubMed

    Kumazawa, Takeshi; Hasegawa, Chika; Uchigasaki, Seisaku; Lee, Xiao-Pen; Suzuki, Osamu; Sato, Keizo

    2011-05-06

    Solid-phase extraction (SPE) using micropipette tips is a useful technique to prepare samples prior to mass spectrometry. However, most commercial SPE tips have loading capacities that are insufficient for quantitative determination. In this paper, we describe a rapid method for quantitative microanalysis of five phenothiazine derivatives, chlorpromazine, levomepromazine, promazine, promethazine and trimeprazine, using a recently introduced C(18) monolithic silica SPE tip, the MonoTip C(18), for extraction from human plasma. The drugs could be extracted within 5 min from 0.1-mL plasma samples, eluted with methanol, and the eluate injected directly into a gas chromatograph prior to mass spectrometry analysis. Only 0.7 mL of solvent was required for each step of the extraction process. The recoveries of the five phenothiazines spiked into plasma were 91-95% and the limits of quantification for each drug were between 0.25 and 2.0 ng/0.1 mL. The maximum intra- and inter-day coefficient of variation was 11%. The validated method was successfully used to quantify the plasma concentration of levemepromazine in a human subject after oral administration of the drug. This new method is expected to have wide applications as a pretreatment for the rapid, quantitative determination of drug concentrations in plasma samples.

  11. Single event mass spectrometry

    DOEpatents

    Conzemius, Robert J.

    1990-01-16

    A means and method for single event time of flight mass spectrometry for analysis of specimen materials. The method of the invention includes pulsing an ion source imposing at least one pulsed ion onto the specimen to produce a corresponding emission of at least one electrically charged particle. The emitted particle is then dissociated into a charged ion component and an uncharged neutral component. The ion and neutral components are then detected. The time of flight of the components are recorded and can be used to analyze the predecessor of the components, and therefore the specimen material. When more than one ion particle is emitted from the specimen per single ion impact, the single event time of flight mass spectrometer described here furnis This invention was made with Government support under Contract No. W-7405-ENG82 awarded by the Department of Energy. The Government has certain rights in the invention.

  12. Liquid chromatography-high-resolution mass spectrometry for pesticide residue analysis in fruit and vegetables: screening and quantitative studies.

    PubMed

    Gómez-Ramos, M M; Ferrer, C; Malato, O; Agüera, A; Fernández-Alba, A R

    2013-04-26

    This work reviews the current state-of-the-art of liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) techniques applied to the analysis of pesticides in fruit-based and vegetable-based matrices. Nowadays, simultaneous trace analysis of hundreds of pesticides from different classes is required, preferably in just one run. The most commonly used QqQ-MS technology presents certain limitations in its application in a cost and effective way when analyzing a large number of pesticides. Thus, this review includes HRMS technology as a reliable complementary alternative allowing the analysis of a wide range of pesticides in food. Its capabilities and limitations in identifying, confirming and quantifying pesticides are discussed. HRMS instruments can adequately address such issues; however, the main drawbacks are as a result of insufficient prior optimization of the operational parameters during non-target analysis in full-scan mode and due to software shortcomings.

  13. Quantitative time-of-flight secondary ion mass spectrometry for the characterization of multicomponent adsorbed protein films

    NASA Astrophysics Data System (ADS)

    Wagner, M. S.; Shen, M.; Horbett, T. A.; Castner, David G.

    2003-01-01

    Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is ideal for the characterization of adsorbed proteins due to its chemical specificity and surface sensitivity. We have employed ToF-SIMS and multivariate analysis to determine the surface composition of adsorbed protein films from binary mixtures, blood serum, and blood plasma. Good correlation between ToF-SIMS data and independent radiolabeling studies was achieved for binary mixtures, though these results depended on the substrate. Qualitative insight into the composition of the serum and plasma protein films was obtained via comparison to standard single protein film spectra. ToF-SIMS and multivariate analysis were able to measure the surface composition of multicomponent adsorbed protein films.

  14. Benefits of prolonged gradient separation for high-performance liquid chromatography-tandem mass spectrometry quantitation of plasma total 15-series F-isoprostanes.

    PubMed

    Taylor, Alan W; Bruno, Richard S; Frei, Balz; Traber, Maret G

    2006-03-01

    The F(2)-isoprostanes are products of free-radical-induced oxidation of arachidonic acid (AA) that are stereoisomers of prostaglandin F(2alpha) (PGF(2alpha)). We describe a method for quantitation of several 15-series PGF isomers (15-PGFs) and AA by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS). Plasma samples were subjected to alkaline hydrolysis and acidified, and total (free + esterified) 15-PGFs and AA were extracted with organic solvents. The analytes were separated by gradient reverse-phase HPLC and detected by multiple reaction monitoring on a triple-quadrupole mass spectrometer, using deuterated internal standards for quantitation. The assay had a linear range of 1-40 pg of 8-iso-PGF(2alpha) on column and can quantify as little as 40 pg/mL (0.11 nM) in plasma. Outcomes significantly correlated (p < 0.0001) with data obtained by gas chromatography-mass spectrometry GC-MS or enzyme-linked immunosorbent assay. All plasma 15-PGF isomers increased over time with in vitro cigarette smoke exposure and correlated (p < 0.0001) with each other. The same strong inter-15-PGF correlations were observed in plasma from healthy young adult subjects. The coefficients of variation of HPLC-MS-MS measurements (24-32%) were smaller than those obtained by GC-MS (53%). Thus, HPLC-MS-MS potentially offers greater precision and allows quantitation of more compounds with simpler sample preparation than existing methods. Ours is the first validated quantitative assay using HPLC-tandem MS applied to plasma total 15-PGFs.

  15. Quantitation of organophosphorus nerve agent metabolites in human urine using isotope dilution gas chromatography-tandem mass spectrometry.

    PubMed

    Driskell, W Jack; Shih, Ming; Needham, Larry L; Barr, Dana B

    2002-01-01

    An isotope dilution gas chromatography-tandem mass spectrometric (GC-MS-MS) method was developed for quantitating the urinary metabolites of the organophosphorus nerve agents sarin, soman, tabun (GA), VX, and GF. Urine samples were concentrated by codistillation with acetonitrile, derivatized by methylation with diazomethane, and analyzed by GC-MS-MS. The limits of detection were less than 4 microg/L for all the analytes except for the GA metabolite, which had a limit of detection of less than 20 microg/L.

  16. A Quantitative Tool to Distinguish Isobaric Leucine and Isoleucine Residues for Mass Spectrometry-Based De Novo Monoclonal Antibody Sequencing

    NASA Astrophysics Data System (ADS)

    Poston, Chloe N.; Higgs, Richard E.; You, Jinsam; Gelfanova, Valentina; Hale, John E.; Knierman, Michael D.; Siegel, Robert; Gutierrez, Jesus A.

    2014-07-01

    De novo sequencing by mass spectrometry (MS) allows for the determination of the complete amino acid (AA) sequence of a given protein based on the mass difference of detected ions from MS/MS fragmentation spectra. The technique relies on obtaining specific masses that can be attributed to characteristic theoretical masses of AAs. A major limitation of de novo sequencing by MS is the inability to distinguish between the isobaric residues leucine (Leu) and isoleucine (Ile). Incorrect identification of Ile as Leu or vice versa often results in loss of activity in recombinant antibodies. This functional ambiguity is commonly resolved with costly and time-consuming AA mutation and peptide sequencing experiments. Here, we describe a set of orthogonal biochemical protocols, which experimentally determine the identity of Ile or Leu residues in monoclonal antibodies (mAb) based on the selectivity that leucine aminopeptidase shows for n-terminal Leu residues and the cleavage preference for Leu by chymotrypsin. The resulting observations are combined with germline frequencies and incorporated into a logistic regression model, called Predictor for Xle Sites (PXleS) to provide a statistical likelihood for the identity of Leu at an ambiguous site. We demonstrate that PXleS can generate a probability for an Xle site in mAbs with 96% accuracy. The implementation of PXleS precludes the expression of several possible sequences and, therefore, reduces the overall time and resources required to go from spectra generation to a biologically active sequence for a mAb when an Ile or Leu residue is in question.

  17. Evaluation of Flow-Injection Tandem Mass Spectrometry for Rapid and High-Throughput Quantitative Determination of B-Vitamins in Nutritional Supplements

    SciTech Connect

    Bhandari, Deepak; Van Berkel, Gary J

    2012-01-01

    The use of flow-injection electrospray ionization tandem mass spectrometry for rapid and high-throughput mass spectral analysis of selected B-vitamins, viz. B1, B2, B3, B5, and B6, in nutritional formulations was demonstrated. A simple and rapid (~5 min) in-tube sample preparation was performed by adding extraction solvent to a powdered sample aliquot followed by agitation, centrifugation, and filtration to recover an extract for analysis. Automated flow injection introduced 1 L of the extracts directly into the mass spectrometer ion source without chromatographic separation. Sample-to-sample analysis time was 60 s representing significant improvement over conventional liquid chromatography approaches which typically require 25-45 min, and often require more significant sample preparation procedures. Quantitative capabilities of the flow-injection analysis were tested using the method of standard additions and NIST standard reference material (SRM 3280) multivitamin/multielement tablets. The quantity determined for each B-vitamin in SRM 3280 was within the statistical range provided for the respective certified values. The same sample preparation and analysis approach was also applied to two different commercial vitamin supplement tablets and proved to be successful in the quantification of the selected B-vitamins as evidenced by an agreement with the labels values and the results obtained using isotope dilution liquid chromatography/mass spectrometry.

  18. Quantitative determination of oxytocin receptor antagonist atosiban in rat plasma by liquid chromatography-tandem mass spectrometry.

    PubMed

    Kannan, Vivekanandan; Gadamsetty, Deepak; Rose, Madhankumar; Maria, Stella; Mustafa, Imran; Khedkar, Anand; Dave, Nitesh; Arumugam, Muruganandam; Iyer, Harish

    2010-05-01

    A kinetic study of atosiban was conducted following repeated intravenous administration in Wistar rats. Sample analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) following full validation of an in-house method. Eptifibatide, a cyclic peptide, was used as an internal standard (IS). The analyte and internal standard were extracted using solid phase extraction (SPE) method. Chromatographic separation was carried out using an ACE C18 5 microm 50 mm x 4.6 mm column with gradient elution. Mass spectrometric detection was performed using TSQ Quantum ultra AM. The lower limit of quantification was 0.01 microg/ml when 100 microl rat plasma was used. Plasma concentrations of atosiban were measured at 0 (pre-dose), 2, 15, 30, 45, 60, 120 min at the dosage levels of 0.125 mg/kg (low dose), 0.250 mg/kg (mid dose), and 0.500 mg/kg (high dose), respectively. Atosiban plasma concentration measured at Day 1 showed mean peak atosiban concentration (C(max)) 0.40, 0.57, 1.95 microg/ml for low, mid and high dose treated animals and mean peak concentration on Day 28 was 0.41, 0.88, 1.31microg/ml on Day 28 for low, mid and high dose treated animals.

  19. A Simple and Sensitive Method for Quantitative Measurement of Methylmalonic Acid by Turbulent Flow Chromatography and Tandem Mass Spectrometry

    PubMed Central

    Tecleab, AG; Schofield, RC; Ramanathan, LV; Carlow, Dean C

    2016-01-01

    A simple and sensitive method for the detection of methylmalonic acid in serum without derivatization has been developed. This method implements protein precipitation using methanol followed by additional sample clean up by turbulent flow liquid chromatography (TFLC). The sample was directly injected into the turbulent flow liquid chromatography tandem mass spectrometry system (TFLC-MS/MS) for online extraction followed by HPLC separation. The eluent was transferred to the mass spectrometer and ionized by heated electrospray negative ionization (HESI) and the analyte was quantified using a six-point calibration curve. The validated analytical measurement range (AMR) is 30–1,000 nMol/L. Dilutions of 10 and 200-fold were validated giving a clinical reportable range (CRR) of 30–200,000 nMol/L. The between-day and within-day imprecision values at concentrations spanning the AMR were less than 15%. This method was compared to an established LC-MS/MS method at a CLIA certified national reference laboratory and shows an excellent correlation with our TFLC-MS/MS method. PMID:27833786

  20. Development of a quantitative mass spectrometry multi-attribute method for characterization, quality control testing and disposition of biologics

    PubMed Central

    Rogers, Richard S; Nightlinger, Nancy S; Livingston, Brittney; Campbell, Phil; Bailey, Robert; Balland, Alain

    2015-01-01

    Regulatory agencies have recently recommended a Quality by Design (QbD) approach for the manufacturing of therapeutic molecules. A QbD strategy requires deep understanding at the molecular level of the attributes that are crucial for safety and efficacy and for insuring that the desired quality of the purified protein drug product is met at the end of the manufacturing process. A mass spectrometry (MS)-based approach to simultaneously monitor the extensive array of product quality attributes (PQAs) present on therapeutic molecules has been developed. This multi-attribute method (MAM) uses a combination of high mass accuracy / high resolution MS data generated by Orbitrap technology and automated identification and relative quantification of PQAs with dedicated software (Pinpoint). The MAM has the potential to replace several conventional electrophoretic and chromatographic methods currently used in Quality Control to release therapeutic molecules. The MAM represents an optimized analytical solution to focus on the attributes of the therapeutic molecule essential for function and implement QbD principles across process development, manufacturing and drug disposition. PMID:26186204

  1. Quantitative analysis of intracellular coenzymes in Saccharomyces cerevisiae using ion pair reversed phase ultra high performance liquid chromatography tandem mass spectrometry.

    PubMed

    Seifar, Reza Maleki; Ras, Cor; Deshmukh, Amit T; Bekers, Katelijne M; Suarez-Mendez, Camilo A; da Cruz, Ana L B; van Gulik, Walter M; Heijnen, Joseph J

    2013-10-11

    A fast, sensitive and specific analytical method, based on ion pair reversed phase ultrahigh performance liquid chromatography tandem mass spectrometry, IP-RP-UHPLC-MS/MS, was developed for quantitative determination of intracellular coenzyme A (CoA), acetyl CoA, succinyl CoA, phenylacetyl CoA, flavin mononucleotide, (FMN), flavin adenine dinucleotide, (FAD), NAD, NADH, NADP, NADPH. Dibutylammonium acetate (DBAA) was used as volatile ion pair reagent in the mobile phase. Addition of DBAA to the sample solutions resulted in an enhanced sensitivity for the phosphorylated coenzymes. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP·HCl), was added to keep CoA in the reduced form. Isotope dilution mass spectrometry (IDMS) was applied for quantitative measurements for which culture derived global U-(13)C-labeled cell extract was used as internal standard. The analytical method was validated by determining the limit of detection, the limit of quantification, repeatability and intermediate precision. The method was successfully applied for quantification of coenzymes in the cell extracts of Saccharomyces cerevisiae.

  2. Quantitation of lysergic acid diethylamide in urine using atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry.

    PubMed

    Cui, Meng; McCooeye, Margaret A; Fraser, Catharine; Mester, Zoltán

    2004-12-01

    A quantitative method was developed for analysis of lysergic acid diethylamide (LSD) in urine using atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry (AP MALDI-ITMS). Following solid-phase extraction of LSD from urine samples, extracts were analyzed by AP MALDI-ITMS. The identity of LSD was confirmed by fragmentation of the [M + H](+) ion using tandem mass spectrometry. The quantification of LSD was achieved using stable-isotope-labeled LSD (LSD-d(3)) as the internal standard. The [M + H](+) ion fragmented to produce a dominant fragment ion, which was used for a selected reaction monitoring (SRM) method for quantitative analysis of LSD. SRM was compared with selected ion monitoring and produced a wider linear range and lower limit of quantification. For SRM analysis of samples of LSD spiked in urine, the calibration curve was linear in the range of 1-100 ng/mL with a coefficient of determination, r(2), of 0.9917. This assay was used to determine LSD in urine samples and the AP MALDI-MS results were comparable to the HPLC/ ESI-MS results.

  3. Integrating qualitative and quantitative characterization of traditional Chinese medicine injection by high-performance liquid chromatography with diode array detection and tandem mass spectrometry.

    PubMed

    Xie, Yuan-yuan; Xiao, Xue; Luo, Juan-min; Fu, Chan; Wang, Qiao-wei; Wang, Yi-ming; Liang, Qiong-lin; Luo, Guo-an

    2014-06-01

    The present study aims to describe and exemplify an integrated strategy of the combination of qualitative and quantitative characterization of a multicomponent mixture for the quality control of traditional Chinese medicine injections with the example of Danhong injection (DHI). The standardized chemical profile of DHI has been established based on liquid chromatography with diode array detection. High-performance liquid chromatography coupled with time-of-flight mass spectrometry and high-performance liquid chromatography with electrospray multistage tandem ion-trap mass spectrometry have been developed to identify the major constituents in DHI. The structures of 26 compounds including nucleotides, phenolic acids, and flavonoid glycosides were identified or tentatively characterized. Meanwhile, the simultaneous determination of seven marker constituents, including uridine, adenosine, danshensu, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid, and salvianolic acid B, in DHI was performed by multiwavelength detection based on high-performance liquid chromatography with diode array detection. The integrated qualitative and quantitative characterization strategy provided an effective and reliable pattern for the comprehensive and systematic characterization of the complex traditional Chinese medicine system.

  4. Qualitative and quantitative analysis of anthraquinones in rhubarbs by high performance liquid chromatography with diode array detector and mass spectrometry.

    PubMed

    Wei, Shao-yin; Yao, Wen-xin; Ji, Wen-yuan; Wei, Jia-qi; Peng, Shi-qi

    2013-12-01

    Rhubarb is well known in traditional Chinese medicines (TCMs) mainly due to its effective purgative activity. Anthraquinones, including anthraquinone derivatives and their glycosides, are thought to be the major active components in rhubarb. To improve the quality control method of rhubarb, we studied on the extraction method, and did qualitative and quantitative analysis of widely used rhubarbs, Rheum tanguticum Maxim. ex Balf. and Rheum palmatum L., by HPLC-photodiode array detection (HPLC-DAD) and HPLC-mass spectrum (HPLC-MS) on a Waters SymmetryShield RP18 column (250 mm × 4.6 mm i.d., 5 μm). Amount of five anthraquinones was viewed as the evaluating standard. A standardized characteristic fingerprint of rhubarb was provided. From the quantitative analysis, the rationality was demonstrated for ancestors to use these two species of rhubarb equally. Under modern extraction methods, the amount of five anthraquinones in Rheum tanguticum Maxim. ex Balf. is higher than that in Rheum palmatum L. Among various extraction methods, ultrasonication with 70% methanol for 30 min is a promising one. For HPLC analysis, mobile phase consisted of methanol and 0.1% phosphoric acid in water with a gradient program, the detection wavelength at 280nm for fingerprinting analysis and 254 nm for quantitative analysis are good choices.

  5. Qualitative and quantitative analysis of branches in dextran using high-performance anion exchange chromatography coupled to quadrupole time-of-flight mass spectrometry.

    PubMed

    Yi, Lin; Ouyang, Yilan; Sun, Xue; Xu, Naiyu; Linhardt, Robert J; Zhang, Zhenqing

    2015-12-04

    Dextran, a family of natural polysaccharides, consists of an α (1→6) linked-glucose main (backbone) chain having a number of branches. The determination of the types and the quantities of branches in dextran is important in understanding its various biological roles. In this study, a hyphenated method using high-performance anion exchange chromatography (HPAEC) in parallel with pulsed amperometric detection (PAD) and mass spectrometry (MS) was applied to qualitative and quantitative analysis of dextran branches. A rotary cation-exchange cartridge array desalter was used for removal of salt from the HPAEC eluent making it MS compatible. MS and MS/MS were used to provide structural information on the enzymatically prepared dextran oligosaccharides. PAD provides quantitative data on the ratio of enzyme-resistant, branched dextran oligosaccharides. Both the types and degree of branching found in a variety of dextrans could be simultaneously determined online using this method.

  6. Quantitative analysis of THC, 11-OH-THC, and THCCOOH in human hair by negative ion chemical ionization mass spectrometry.

    PubMed

    Wilkins, D; Haughey, H; Cone, E; Huestis, M; Foltz, R; Rollins, D

    1995-10-01

    A sensitive and specific method was developed for the quantitative analysis of delta9-tetrahydrocannabinol (THC), 11 -hydroxy-THC (11-OH-THC), and 11-nor-9-carboxy-THC (THCCOOH) in human hair. Deuterated internal standards were added to hair samples, and samples were digested overnight in 1 N NaOH at 37 degrees C. Digest solutions were extracted with a liquid-liquid extraction procedure, which was previously developed in our laboratory for the analysis of plasma and whole blood. Derivatized extracts were analyzed on a Finnigan 4500" mass spectrometer in negative ion chemical ionization mode using methane as the reagent gas, hydrogen as the carrier gas, and a Restek Rtx 200-15M-0.25-microm capillary column. The assay was linear up to 50 ng/mg hair (r, 0.99) for all three compounds and was capable of detecting 10 pg THC and THCCOOH and 100 pg 11-OH-THC on column. The intra-assay precision ranged from 2.1 to 11.2% for the three analytes; the interassay precision ranged from 4.4 to 13.0%. The method was used to detect and quantitate the presence of THC, 11-OH-THC, and THCCOOH in human hair obtained from eight regular users of cannabis. THC, but not 11-OH-THC or THCCOOH, was detectable in the hair shaft above the assay limit of quantitation. Four laboratory wash procedures were also evaluated for their effect on the measured concentration of THC in hair. in seven of eight subjects, a methylene chloride wash procedure substantially reduced the measured THC concentration by up to 50%. The gas chromatographic-mass spectrometric assay is currently being used to support pharmacokinetic studies of drug disposition into the hair of humans and animals.

  7. Quantitation of triacylglycerols in edible oils by off-line comprehensive two-dimensional liquid chromatography-atmospheric pressure chemical ionization mass spectrometry using a single column.

    PubMed

    Wei, Fang; Hu, Na; Lv, Xin; Dong, Xu-Yan; Chen, Hong

    2015-07-24

    In this investigation, off-line comprehensive two-dimensional liquid chromatography-atmospheric pressure chemical ionization mass spectrometry using a single column has been applied for the identification and quantification of triacylglycerols in edible oils. A novel mixed-mode phenyl-hexyl chromatographic column was employed in this off-line two-dimensional separation system. The phenyl-hexyl column combined the features of traditional C18 and silver-ion columns, which could provide hydrophobic interactions with triacylglycerols under acetonitrile conditions and can offer π-π interactions with triacylglycerols under methanol conditions. When compared with traditional off-line comprehensive two-dimensional liquid chromatography employing two different chromatographic columns (C18 and silver-ion column) and using elution solvents comprised of two phases (reversed-phase/normal-phase) for triacylglycerols separation, the novel off-line comprehensive two-dimensional liquid chromatography using a single column can be achieved by simply altering the mobile phase between acetonitrile and methanol, which exhibited a much higher selectivity for the separation of triacylglycerols with great efficiency and rapid speed. In addition, an approach based on the use of response factor with atmospheric pressure chemical ionization mass spectrometry has been developed for triacylglycerols quantification. Due to the differences between saturated and unsaturated acyl chains, the use of response factors significantly improves the quantitation of triacylglycerols. This two-dimensional liquid chromatography-mass spectrometry system was successfully applied for the profiling of triacylglycerols in soybean oils, peanut oils and lord oils. A total of 68 triacylglycerols including 40 triacylglycerols in soybean oils, 50 triacylglycerols in peanut oils and 44 triacylglycerols in lord oils have been identified and quantified. The liquid chromatography-mass spectrometry data were analyzed

  8. Quantitative fingerprinting by headspace--two-dimensional comprehensive gas chromatography-mass spectrometry of solid matrices: some challenging aspects of the exhaustive assessment of food volatiles.

    PubMed

    Nicolotti, Luca; Cordero, Chiara; Cagliero, Cecilia; Liberto, Erica; Sgorbini, Barbara; Rubiolo, Patrizia; Bicchi, Carlo

    2013-10-10

    The study proposes an investigation strategy that simultaneously provides detailed profiling and quantitative fingerprinting of food volatiles, through a "comprehensive" analytical platform that includes sample preparation by Headspace Solid Phase Microextraction (HS-SPME), separation by two-dimensional comprehensive gas chromatography coupled with mass spectrometry detection (GC×GC-MS) and data processing using advanced fingerprinting approaches. Experiments were carried out on roasted hazelnuts and on Gianduja pastes (sugar, vegetable oil, hazelnuts, cocoa, nonfat dried milk, vanilla flavorings) and demonstrated that the information potential of each analysis can better be exploited if suitable quantitation methods are applied. Quantitation approaches through Multiple Headspace Extraction and Standard Addition were compared in terms of performance parameters (linearity, precision, accuracy, Limit of Detection and Limit of Quantitation) under headspace linearity conditions. The results on 19 key analytes, potent odorants, and technological markers, and more than 300 fingerprint components, were used for further processing to obtain information concerning the effect of the matrix on volatile release, and to produce an informative chemical blueprint for use in sensomics and flavoromics. The importance of quantitation approaches in headspace analysis of solid matrices of complex composition, and the advantages of MHE, are also critically discussed.

  9. Biomedical accelerator mass spectrometry

    NASA Astrophysics Data System (ADS)

    Freeman, Stewart P. H. T.; Vogel, John S.

    1995-05-01

    Ultrasensitive SIMS with accelerator based spectrometers has recently begun to be applied to biomedical problems. Certain very long-lived radioisotopes of very low natural abundances can be used to trace metabolism at environmental dose levels ( [greater-or-equal, slanted] z mol in mg samples). 14C in particular can be employed to label a myriad of compounds. Competing technologies typically require super environmental doses that can perturb the system under investigation, followed by uncertain extrapolation to the low dose regime. 41Ca and 26Al are also used as elemental tracers. Given the sensitivity of the accelerator method, care must be taken to avoid contamination of the mass spectrometer and the apparatus employed in prior sample handling including chemical separation. This infant field comprises the efforts of a dozen accelerator laboratories. The Center for Accelerator Mass Spectrometry has been particularly active. In addition to collaborating with groups further afield, we are researching the kinematics and binding of genotoxins in-house, and we support innovative uses of our capability in the disciplines of chemistry, pharmacology, nutrition and physiology within the University of California. The field can be expected to grow further given the numerous potential applications and the efforts of several groups and companies to integrate more the accelerator technology into biomedical research programs; the development of miniaturized accelerator systems and ion sources capable of interfacing to conventional HPLC and GMC, etc. apparatus for complementary chemical analysis is anticipated for biomedical laboratories.

  10. Extractionless and sensitive method for high-throughput quantitation of cetirizine in human plasma samples by liquid chromatography-tandem mass spectrometry.

    PubMed

    de Jager, A D; Hundt, H K L; Swart, K J; Hundt, A F; Els, J

    2002-06-25

    Following a single 10-mg oral dose of cetirizine dihydrochloride to 24 healthy volunteers, the analyte was quantified in human plasma. Protein precipitation using acetonitrile (ACN) was followed by reversed-phase liquid chromatography and tandem mass spectrometry. The MS/MS method was optimised using a PE Sciex API 2000 triple quadrupole mass spectrometer in selected reaction monitoring (SRM) mode, using electrospray with positive ionisation. Oxybutynin was used as the internal standard. The assay method represents a robust, high-throughput, highly specific and sensitive quantitative assay procedure, with 0.5 ng/ml being the lowest plasma concentration that could be reliably quantified. The procedure involves minimal sample preparation, and is well suited to clinical studies of the drug involving large numbers of generated samples. Pre-dose as well as post-dose samples up to and including 48 h were quantified, and the data generated were used to determine the pharmacokinetic profile of the drug.

  11. Mass Spectrometry Applications for Toxicology

    PubMed Central

    Mbughuni, Michael M.; Jannetto, Paul J.

    2016-01-01

    Toxicology is a multidisciplinary study of poisons, aimed to correlate the quantitative and qualitative relationships between poisons and their physiological and behavioural effects in living systems. Other key aspects of toxicology focus on elucidation of the mechanisms of action of poisons and development of remedies and treatment plans for associated toxic effects. In these endeavours, Mass spectrometry (MS) has become a powerful analytical technique with a wide range of application used in the Toxicological analysis of drugs, poisons, and metabolites of both. To date, MS applications have permeated all fields of toxicology which include; environmental, clinical, and forensic toxicology. While many different analytical applications are used in these fields, MS and its hyphenated applications such as; gas chromatography MS (GC-MS), liquid chromatography MS (LC-MS), inductively coupled plasma ionization MS (ICP-MS), tandem mass spectrometry (MS/MS and MSn) have emerged as powerful tools used in toxicology laboratories. This review will focus on these hyphenated MS technologies and their applications for toxicology. PMID:28149262

  12. Mass Spectrometry Applications for Toxicology.

    PubMed

    Mbughuni, Michael M; Jannetto, Paul J; Langman, Loralie J

    2016-12-01

    Toxicology is a multidisciplinary study of poisons, aimed to correlate the quantitative and qualitative relationships between poisons and their physiological and behavioural effects in living systems. Other key aspects of toxicology focus on elucidation of the mechanisms of action of poisons and development of remedies and treatment plans for associated toxic effects. In these endeavours, Mass spectrometry (MS) has become a powerful analytical technique with a wide range of application used in the Toxicological analysis of drugs, poisons, and metabolites of both. To date, MS applications have permeated all fields of toxicology which include; environmental, clinical, and forensic toxicology. While many different analytical applications are used in these fields, MS and its hyphenated applications such as; gas chromatography MS (GC-MS), liquid chromatography MS (LC-MS), inductively coupled plasma ionization MS (ICP-MS), tandem mass spectrometry (MS/MS and MS(n)) have emerged as powerful tools used in toxicology laboratories. This review will focus on these hyphenated MS technologies and their applications for toxicology.

  13. Simultaneous quantitation of acetylsalicylic acid and clopidogrel along with their metabolites in human plasma using liquid chromatography tandem mass spectrometry.

    PubMed

    Chhonker, Yashpal S; Pandey, Chandra P; Chandasana, Hardik; Laxman, Tulsankar Sachin; Prasad, Yarra Durga; Narain, V S; Dikshit, Madhu; Bhatta, Rabi S

    2016-03-01

    The interest in therapeutic drug monitoring has increased over the last few years. Inter- and intra-patient variability in pharmacokinetics, plasma concentration related toxicity and success of therapy have stressed the need of frequent therapeutic drug monitoring of the drugs. A sensitive, selective and rapid liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of acetylsalicylic acid (aspirin), salicylic acid, clopidogrel and carboxylic acid metabolite of clopidogrel in human plasma. The chromatographic separations were achieved on Waters Symmetry Shield(TM) C18 column (150 × 4.6 mm, 5 µm) using 3.5 mm ammonium acetate (pH 3.5)-acetonitrile (10:90, v/v) as mobile phase at a flow rate of 0.75 mL/min. The present method was successfully applied for therapeutic drug monitoring of aspirin and clopidogrel in 67 patients with coronary artery disease.

  14. Quantitative measurement of dihydrouridine in RNA using isotope dilution liquid chromatography-mass spectrometry (LC/MS).

    PubMed Central

    Dalluge, J J; Hashizume, T; McCloskey, J A

    1996-01-01

    A method has been developed for the microscale determination of 5,6-dihydrouridine, the most common post-transcriptional modification in bacterial and eukaryotic tRNA. The method is based on stable isotope dilution liquid chromatography-mass spectrometry (LC/MS) using [1,3-15N2]dihydrouridine and [1,3-15N2]uridine as internal standards. RNA samples were enzymatically digested to nucleosides before addition of the internal standards and subsequently analyzed by LC/MS with selected ion monitoring of protonated molecular ions of the labeled and unlabeled nucleosides. Sample quantities of approximately 1 pmol tRNA and 5 pmol 23S rRNA were analyzed for mole% dihydrouridine. Dihydrouridine content of Escherichia coli tRNASer(VGA) and tRNAThr(GGU) as controls were measured as 2.03 and 2.84 residues/tRNA molecule, representing accuracies of 98 and 95%. Overall precision values for the analyses of E. coli tRNASer(VGA) and E. coli tRNAThr(GGU), unfractionated tRNA from E. coli and 23S rRNA from E. coli were within the range 0.43-2.4%. The mole% dihydrouridine in unfractionated tRNA and 23S rRNA from E. coli were determined as 1.79 and 0.0396%, corresponding to 1.4 and 1.1 residues/RNA molecule respectively. PMID:8774907

  15. Monolith immuno-affinity enrichment liquid chromatography tandem mass spectrometry for quantitative protein analysis of recombinant bovine somatotropin in serum.

    PubMed

    Smits, Nathalie G E; Blokland, Marco H; Wubs, Klaas L; Nessen, Merel A; van Ginkel, Leen A; Nielen, Michel W F

    2015-08-01

    The use of recombinant bovine somatotropin (rbST) to enhance milk production is approved in several countries, but it is prohibited in the European Union. According to EU legislation, it is necessary to confirm positive screening results prior to enforcement. Although adequate screening assays are available nowadays, development of liquid chromatography tandem mass spectrometry (LC-MS/MS) confirmatory methods to detect low levels of rbST is still a challenge. Here, we present a novel approach using immuno-affinity enrichment on monolithic micro-columns in combination with state-of-the-art ultra-high pressure LC-MS/MS (UHPLC-MS/MS) detection. The developed approach enables detection and confirmation of rbST in serum at a decision limit (CCα) concentration of 0.8 ng mL(-1). Furthermore, the method is easy to handle, robust and reproducible. We successfully applied the confirmatory method to serum samples from rbST treated cows that were found suspect after immunoassay-based screening. The use of rbST could be confirmed over 1 week after treatment, and the developed method demonstrated the sensitivity needed for effective control. Graphical Abstract Graphical summary of the workflow, for serum preparation, enrichment with monolith microcolumns and LC-MS/MS measurement of rbST.

  16. Quantitative ester analysis in cachaca and distilled spirits by gas chromatography-mass spectrometry (GC-MS).

    PubMed

    Nascimento, Eduardo S P; Cardoso, Daniel R; Franco, Douglas W

    2008-07-23

    An analytical procedure for the separation and quantification of ethyl acetate, ethyl butyrate, ethyl hexanoate, ethyl lactate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, isoamyl octanoate, and ethyl laurate in cachaca, rum, and whisky by direct injection gas chromatography-mass spectrometry was developed. The analytical method is simple, selective, and appropriated for the determination of esters in distilled spirits. The limit of detection ranged from 29 (ethyl hexanoate) to 530 (ethyl acetate) microg L(-1), whereas the standard deviation for repeatability was between 0.774% (ethyl hexanoate) and 5.05% (isoamyl octanoate). Relative standard deviation values for accuracy vary from 90.3 to 98.5% for ethyl butyrate and ethyl acetate, respectively. Ethyl acetate was shown to be the major ester in cachaca (median content of 22.6 mg 100 mL(-1) anhydrous alcohol), followed by ethyl lactate (median content of 8.32 mg 100 mL(-1) anhydrous alcohol). Cachaca produced in copper and hybrid alembic present a higher content of ethyl acetate and ethyl lactate than those produced in a stainless-steel column, whereas cachaca produced by distillation in a stainless-steel column present a higher content of ethyl octanoate, ethyl decanoate, and ethyl laurate. As expected, ethyl acetate is the major ester in whiskey and rum, followed by ethyl lactate for samples of rum. Nevertheless, whiskey samples exhibit ethyl lactate at contents lower or at the same order of magnitude of the fatty esters.

  17. Quantitation of five organophosphorus nerve agent metabolites in serum using hydrophilic interaction liquid chromatography and tandem mass spectrometry.

    PubMed

    Hamelin, Elizabeth I; Schulze, Nicholas D; Shaner, Rebecca L; Coleman, Rebecca M; Lawrence, Richard J; Crow, Brian S; Jakubowski, E M; Johnson, Rudolph C

    2014-08-01

    Although nerve agent use is prohibited, concerns remain for human exposure to nerve agents during decommissioning, research, and warfare. Exposure can be detected through the analysis of hydrolysis products in urine as well as blood. An analytical method to detect exposure to five nerve agents, including VX, VR (Russian VX), GB (sarin), GD (soman), and GF (cyclosarin), through the analysis of the hydrolysis products, which are the primary metabolites, in serum has been developed and characterized. This method uses solid-phase extraction coupled with high-performance liquid chromatography for separation and isotopic dilution tandem mass spectrometry for detection. An uncommon buffer of ammonium fluoride was used to enhance ionization and improve sensitivity when coupled with hydrophilic interaction liquid chromatography resulting in detection limits from 0.3 to 0.5 ng/mL. The assessment of two quality control samples demonstrated high accuracy (101-105%) and high precision (5-8%) for the detection of these five nerve agent hydrolysis products in serum.

  18. Reductive amination-assisted quantitation of tamoxifen and its metabolites by liquid phase chromatography tandem mass spectrometry.

    PubMed

    Liang, Shih-Shin; Wang, Tsu-Nai; Chiu, Chien-Chih; Kuo, Po-Lin; Huang, Mei-Fang; Liu, Meng-Chieh; Tsai, Eing-Mei

    2016-02-19

    Tamoxifen, a hormonal therapy drug against estrogen receptor-positive breast cancer, can be metabolized by cytochrome P450 enzymes such as CYP3A4 and CYP3A5, and converted to N-desmethyltamoxifen, which is subsequently, metabolized by CYP2D6 and inverted to form 4-hydroxy-N-desmethyltamoxifen (endoxifen). Conventional mass spectrometry (MS) analyses of tamoxifen and its metabolites require isotopic internal standards (ISs). In this study, endoxifen and N-desmethyltamoxifen amine groups were modified by reductive amination with formaldehyde-D2 to produce new metabolite molecules. Both endoxifen and N-desmethyltamoxifen generated their corresponding D2-methyl modified analogs. This method is expected to simplify MS detection and overcome the difficulty in selecting adequate ISs when tamoxifen metabolites are analyzed by absolute quantification. It identified tamoxifen, D2-methyl modified endoxifen, and D2-methyl modified N-desmethyltamoxifen with a linearity ranging from 2 to 5000 ng/mL with correlation coefficient (R(2)) values of 0.9868, 0.9849, and 0.9880, respectively. Furthermore, this reductive amination-based method may enhance the signal intensities of D2-methyl modified N-desmethyltamoxifen and endoxifen, thus facilitating the MS detection.

  19. Quantitation of five organophosphorus nerve agent metabolites in serum using hydrophilic interaction liquid chromatography and tandem mass spectrometry

    PubMed Central

    Hamelin, Elizabeth I.; Schulze, Nicholas D.; Shaner, Rebecca L.; Coleman, Rebecca M.; Lawrence, Richard J.; Crow, Brian S.; Jakubowski, E. M.; Johnson, Rudolph C.

    2015-01-01

    Although nerve agent use is prohibited, concerns remain for human exposure to nerve agents during decommissioning, research, and warfare. Exposure can be detected through the analysis of the hydrolysis products in urine as well as blood. An analytical method to detect exposure to five nerve agents, including VX, VR (Russian VX), GB (sarin), GD (soman) and GF (cyclosarin), through the analysis of the hydrolysis products, which are the primary metabolites, in serum has been developed and characterized. This method uses solid phase extraction coupled with high performance liquid chromatography for separation and isotopic dilution tandem mass spectrometry for detection. An uncommon buffer of ammonium fluoride was used to enhance ionization and improve sensitivity when coupled with hydrophilic interaction liquid chromatography resulting in detection limits from 0.3–0.5 ng/mL. The assessment of two quality control samples demonstrated high accuracy (101–105%) and high precision (5–8%) for the detection of these five nerve agent hydrolysis products in serum. PMID:24633507

  20. 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.

  1. Combination of ESI and MALDI mass spectrometry for qualitative, semi-quantitative and in situ analysis of gangliosides in brain

    PubMed Central

    Zhang, Yangyang; Wang, Jun; Liu, Jian’an; Han, Juanjuan; Xiong, Shaoxiang; Yong, Weidong; Zhao, Zhenwen

    2016-01-01

    Gangliosides are a family of complex lipids that are abundant in the brain. There is no doubt the investigations about the distribution of gangliosides in brian and the relationship between gangliosides and Alzheimer’s disease is profound. However, these investigations are full of challenges due to the structural complexity of gangliosides. In this work, the method for efficient extraction and enrichment of gangliosides from brain was established. Moreover, the distribution of gangliosides in brain was obtained by matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI). It was found that 3-aminoquinoline (3-AQ) as matrix was well-suited for MALDI MS analysis of gangliosides in negative ion mode. In addition, the pretreatment by ethanol (EtOH) cleaning brain section and the addition of ammonium formate greatly improved the MS signal of gangliosides in the brain section when MALDI MSI analysis was employed. The distribution of ganliosides in cerebral cortex, hippocampus and cerebellum was respectively acquired by electrospray ionization (ESI) MS and MALDI MSI, and the data were compared for reliability evaluation of MALDI MSI. Further, applying MALDI MSI technology, the distribution of gangliosides in amyloid precursor protein transgenic mouse brain was obtained, which may provide a new insight for bioresearch of Alzheimer’s disease (AD). PMID:27142336

  2. Quantitative analysis of menthol in human urine using solid phase microextraction and stable isotope dilution gas chromatography-mass spectrometry.

    PubMed

    Huang, Wenlin; Blount, Benjamin C; Watson, Clifford H; Watson, Christina; Chambers, David M

    2017-02-15

    To accurately measure menthol levels in human urine, we developed a method using gas chromatography/electron ionization mass spectrometry with menthol-d4 stable isotope internal standardization. We used solid phase microextraction (SPME) headspace sampling for collection, preconcentration and automation. Conjugated forms of menthol were released using β-glucuronidase/sulfatase to allow for measuring total menthol. Additionally, we processed the specimens without using β-glucuronidase/sulfatase to quantify the levels of unconjugated (free) menthol in urine. This method was developed to verify mentholated cigarette smoking status to study the influence of menthol on smoking behaviour and exposure. This objective was accomplished with this method, which has no carryover or memory from the SPME fiber assembly, a method detection limit of 0.0017μg/mL, a broad linear range of 0.002-0.5μg/mL for free menthol and 0.01-10μg/mL for total menthol, a 7.6% precision and 88.5% accuracy, and an analysis runtime of 17min. We applied this method in analysis of urine specimens collected from cigarette smokers who smoke either mentholated or non-mentholated cigarettes. Among these smokers, the average total urinary menthol levels was three-fold higher (p<0.001) among mentholated cigarette smokers compared with non-mentholated cigarette smokers.

  3. Stoichiometry of chromatin-associated protein complexes revealed by label-free quantitative mass spectrometry-based proteomics.

    PubMed

    Smits, Arne H; Jansen, Pascal W T C; Poser, Ina; Hyman, Anthony A; Vermeulen, Michiel

    2013-01-07

    Many cellular proteins assemble into macromolecular protein complexes. The identification of protein-protein interactions and quantification of their stoichiometry is therefore crucial to understand the molecular function of protein complexes. Determining the stoichiometry of protein complexes is usually achieved by mass spectrometry-based methods that rely on introducing stable isotope-labeled reference peptides into the sample of interest. However, these approaches are laborious and not suitable for high-throughput screenings. Here, we describe a robust and easy to implement label-free relative quantification approach that combines the detection of high-confidence protein-protein interactions with an accurate determination of the stoichiometry of the identified protein-protein interactions in a single experiment. We applied this method to two chromatin-associated protein complexes for which the stoichiometry thus far remained elusive: the MBD3/NuRD and PRC2 complex. For each of these complexes, we accurately determined the stoichiometry of the core subunits while at the same time identifying novel interactors and their stoichiometry.

  4. Quantitative determination of the diastereoisomers of hexabromocyclododecane in human plasma using liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

    PubMed

    Tang, Caiming

    2010-12-01

    A sensitive, simple and feasible method has been developed and validated for the simultaneous determination of three diastereoisomers of hexabromocyclododecane (HBCD) in human plasma using liquid chromatography tandem mass spectrometry (LC-MS/MS). The simple pretreatment generally involved protein precipitation with methanol (MeOH). The separation was performed with a C18 reverse phase column. The mobile phases were 5mM ammonium acetate (NH(4)AC) in water and acetonitrile (ACN). The mass spectrometer was operated using negative electrospray ionization (ESI) source and the data acquisition was carried out with multiple reaction monitoring (MRM) mode. The analyte quantifications were performed by external standard method with matrix-matched calibration curves. The method was partially validated with the evaluations of accuracy, precision, linearity, limit of quantification (LOQ), limit of detection (LOD), recovery, matrix effect and carryover effect. With the present method, the intra-batch accuracies were 94.7-104.3%, 91.9-109.3% and 89.8-105.0% for α-, β- and γ-HBCD, respectively. And the inter-batch accuracies were ranged from 94.2% to 109.7%. Both intra-batch and inter-batch precisions (relative standard deviation, RSD, %) of the analytes were no more than 11.2%. The recoveries were from 79.0% to 108.9% and the LOQ was 10pg/mL for each diastereoisomer. The linear range was 10-10,000pg/mL with the linear correlation coefficient R(2)>0.996. No significant matrix effect and carryover effect of the analytes were observed in this study. This method is in possession of sufficient resolution, high sensitivity as well as selectivity and convenient to be applied to the trace determination of HBCDs in human plasma.

  5. Separation, Sizing, and Quantitation of Engineered Nanoparticles in an Organism Model Using Inductively Coupled Plasma Mass Spectrometry and Image Analysis.

    PubMed

    Johnson, Monique E; Hanna, Shannon K; Montoro Bustos, Antonio R; Sims, Christopher M; Elliott, Lindsay C C; Lingayat, Akshay; Johnston, Adrian C; Nikoobakht, Babak; Elliott, John T; Holbrook, R David; Scott, Keana C K; Murphy, Karen E; Petersen, Elijah J; Yu, Lee L; Nelson, Bryant C

    2017-01-24

    For environmental studies assessing uptake of orally ingested engineered nanoparticles (ENPs), a key step in ensuring accurate quantification of ingested ENPs is efficient separation of the organism from ENPs that are either nonspecifically adsorbed to the organism and/or suspended in the dispersion following exposure. Here, we measure the uptake of 30 and 60 nm gold nanoparticles (AuNPs) by the nematode, Caenorhabditis elegans, using a sucrose density gradient centrifugation protocol to remove noningested AuNPs. Both conventional inductively coupled plasma mass spectrometry (ICP-MS) and single particle (sp)ICP-MS are utilized to measure the total mass and size distribution, respectively, of ingested AuNPs. Scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDS) imaging confirmed that traditional nematode washing procedures were ineffective at removing excess suspended and/or adsorbed AuNPs after exposure. Water rinsing procedures had AuNP removal efficiencies ranging from 57 to 97% and 22 to 83%, while the sucrose density gradient procedure had removal efficiencies of 100 and 93 to 98%, respectively, for the 30 and 60 nm AuNP exposure conditions. Quantification of total Au uptake was performed following acidic digestion of nonexposed and Au-exposed nematodes, whereas an alkaline digestion procedure was optimized for the liberation of ingested AuNPs for spICP-MS characterization. Size distributions and particle number concentrations were determined for AuNPs ingested by nematodes with corresponding confirmation of nematode uptake via high-pressure freezing/freeze substitution resin preparation and large-area SEM imaging. Methods for the separation and in vivo quantification of ENPs in multicellular organisms will facilitate robust studies of ENP uptake, biotransformation, and hazard assessment in the environment.

  6. A universal SI-traceable isotope dilution mass spectrometry method for protein quantitation in a matrix by tandem mass tag technology.

    PubMed

    Li, Jiale; Wu, Liqing; Jin, Youxun; Su, Ping; Yang, Bin; Yang, Yi

    2016-05-01

    Isotope dilution mass spectrometry (IDMS), an important metrological method, is widely used for absolute quantification of peptides and proteins. IDMS employs an isotope-labeled peptide or protein as an internal standard although the use of a protein provides improved accuracy. Generally, the isotope-labeled protein is obtained by stable isotope labeling by amino acids in cell culture (SILAC) technology. However, SILAC is expensive, laborious, and time-consuming. To overcome these drawbacks, a novel universal SI-traceable IDMS method for absolute quantification of proteins in a matrix is described with human transferrin (hTRF). The hTRF and a human serum sample were labeled with different tandem mass tags (TMTs). After mixing the TMT-labeled hTRF and serum sample together followed by digestion, the peptides were separated by nano-liquid chromatography and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Using the signature peptides, we calculated the ratios of reporter ions from the TMT-labeled peptides which, in turn, allowed determination of the mass fraction of hTRF. The recovery ranged from 97% to 105% with a CV of 3.9%. The LOD and LOQ were 1.71 × 10(-5) g/g and 5.69 × 10(-5) g/g of hTRF in human serum, respectively, and the relative expanded uncertainty was 4.7% with a mass fraction of 2.08 mg/g. For comparison, an enzyme-linked immunosorbent assay (ELISA) method for hTRF yielded a mass fraction of 2.03 mg/g. This method provides a starting point for establishing IDMS technology to accurately determine the mass fractions of protein biomarkers in a matrix with traceability to SI units. This technology should support the development of a metrological method useful for quantification of a wide variety of proteins.

  7. Morpheus Spectral Counter: A computational tool for label-free quantitative mass spectrometry using the Morpheus search engine.

    PubMed

    Gemperline, David C; Scalf, Mark; Smith, Lloyd M; Vierstra, Richard D

    2016-03-01

    Label-free quantitative MS based on the Normalized Spectral Abundance Factor (NSAF) has emerged as a straightforward and robust method to determine the relative abundance of individual proteins within complex mixtures. Here, we present Morpheus Spectral Counter (MSpC) as the first computational tool that directly calculates NSAF values from output obtained from Morpheus, a fast, open-source, peptide-MS/MS matching engine compatible with high-resolution accurate-mass instruments. NSAF has distinct advantages over other MS-based quantification methods, including a greater dynamic range as compared to isobaric tags, no requirement to align and re-extract MS1 peaks, and increased speed. MSpC features an easy-to-use graphic user interface that additionally calculates both distributed and unique NSAF values to permit analyses of both protein families and isoforms/proteoforms. MSpC determinations of protein concentration were linear over several orders of magnitude based on the analysis of several high-mass accuracy datasets either obtained from PRIDE or generated with total cell extracts spiked with purified Arabidopsis 20S proteasomes. The MSpC software was developed in C# and is open sourced under a permissive license with the code made available at http://dcgemperline.github.io/Morpheus_SpC/.

  8. Morpheus Spectral Counter: A Computational Tool for Label-Free Quantitative Mass Spectrometry using the Morpheus Search Engine

    PubMed Central

    Gemperline, David C.; Scalf, Mark; Smith, Lloyd M.; Vierstra, Richard D.

    2016-01-01

    Label-free quantitative MS based on the Normalized Spectral Abundance Factor (NSAF) has emerged as a straightforward and robust method to determine the relative abundance of individual proteins within complex mixtures. Here, we present Morpheus Spectral Counter (MSpC) as the first computational tool that directly calculates NSAF values from output obtained from Morpheus, a fast, open-source, peptide-MS/MS matching engine compatible with high-resolution accurate-mass instruments. NSAF has distinct advantages over other MS-based quantification methods, including a higher dynamic range as compared to isobaric tags, no requirement to align and re-extract MS1 peaks, and increased speed. MSpC features an easy to use graphic user interface that additionally calculates both distributed and unique NSAF values to permit analyses of both protein families and isoforms/proteoforms. MSpC determinations of protein concentration were linear over several orders of magnitude based on the analysis of several high-mass accuracy datasets either obtained from PRIDE or generated with total cell extracts spiked with purified Arabidopsis 20S proteasomes. The MSpC software was developed in C# and is open sourced under a permissive license with the code made available at http://dcgemperline.github.io/Morpheus_SpC/. PMID:26791624

  9. Accelerator mass spectrometry.

    PubMed

    Hellborg, Ragnar; Skog, Göran

    2008-01-01

    In this overview the technique of accelerator mass spectrometry (AMS) and its use are described. AMS is a highly sensitive method of counting atoms. It is used to detect very low concentrations of natural isotopic abundances (typically in the range between 10(-12) and 10(-16)) of both radionuclides and stable nuclides. The main advantages of AMS compared to conventional radiometric methods are the use of smaller samples (mg and even sub-mg size) and shorter measuring times (less than 1 hr). The equipment used for AMS is almost exclusively based on the electrostatic tandem accelerator, although some of the newest systems are based on a slightly different principle. Dedicated accelerators as well as older "nuclear physics machines" can be found in the 80 or so AMS laboratories in existence today. The most widely used isotope studied with AMS is 14C. Besides radiocarbon dating this isotope is used in climate studies, biomedicine applications and many other fields. More than 100,000 14C samples are measured per year. Other isotopes studied include 10Be, 26Al, 36Cl, 41Ca, 59Ni, 129I, U, and Pu. Although these measurements are important, the number of samples of these other isotopes measured each year is estimated to be less than 10% of the number of 14C samples.

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

    PubMed Central

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

    2016-01-01

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

  11. Quantitative analysis of costunolide and dehydrocostuslactone in rat plasma by ultraperformance liquid chromatography-electrospray ionization-mass spectrometry.

    PubMed

    Hu, Fangdi; Feng, Shilan; Wu, Yuqiong; Bi, Yingyan; Wang, Chunming; Li, Wen

    2011-05-01

    Costunolide and dehydrocostuslactone are well-known sesquiterpene lactones contained in many plants used as popular herbs, such as Saussurea lappa and Laurus novocanariensis, and have been considered as potential candidates for the treatment of various types of tumor. In the present work, a sensitive UPLC-MS/MS for the quantification of costunolide and dehydrocostuslactone in biological matrices has been developed. The method is based on protein precipitation with acetonitrile followed by isocratic ultraperformance liquid chromatographic separation using methanol-formic acid (0.1% in water; 70:30, v/v) mobile phase. Detection was performed by ESI mass spectrometry in MRM mode with the precursor-to-product ion transitions m/z 233-187 and m/z 231-185, respectively. The calibration curves of analytes showed good linearity within the established range 0.19-760  ng/mL for costunolide and 0.23-908  ng/mL for dehydrocostuslactone. The lower limits of quantification of costunolide and dehydrocostuslactone were found to be 0.19 and 0.23  ng/mL, respectively. The intra-day and inter-day presicions of this method for the entire validation were less than coefficient of variation of 7% and the accuracy was within ±8% (relative error). The mean extraction recoveries were 73.8 and 75.3%, respectively. The method was found to be precise, accurate and specific during the study, and was successfully used to analyze the pharmacokinetics of costunolide and dehydrocostuslactone.

  12. Quantitative analysis of arbutin and hydroquinone in strawberry tree (Arbutus unedo L., Ericaceae) leaves by gas chromatography-mass spectrometry.

    PubMed

    Jurica, Karlo; Karačonji, Irena Brčić; Šegan, Sandra; Opsenica, Dušanka Milojković; Kremer, Dario

    2015-09-01

    The phenolic glycoside arbutin and its metabolite with uroantiseptic activity hydroquinone occur naturally in the leaves of various medicinal plants and spices. In this study, an extraction procedure coupled with gas chromatography-mass spectrometry (GC-MS) was developed to determine arbutin and hydroquinone content in strawberry tree (Arbutus unedo L., Ericaceae) leaves. The method showed good linearity (R2>0.9987) in the tested concentration range (0.5-200 μg mL(-1)), as well as good precision (RSD<5%), analytical recovery (96.2-98.0%), and sensitivity (limit of detection=0.009 and 0.004 μg mL(-1) for arbutin and hydroquinone, respectively). The results obtained by the validated GC-MS method corresponded well to those obtained by high performance liquid chromatography (HPLC) method. The proposed method was then applied for determining arbutin and hydroquinone content in methanolic leaf extracts. The amount of arbutin in the leaves collected on the island of Koločep (6.82 mg g(-1) dry weight) was found to be higher (tpaired=43.57, tc=2.92) in comparison to the amount of arbutin in the leaves collected on the island of Mali Lošinj (2.75 mg g(-1) dry weight). Hydroquinone was not detected in any of the samples. The analytical features of the proposed GC-MS method demonstrated that arbutin and hydroquinone could be determined alternatively by gas chromatography. Due to its wide concentration range, the method could also be suitable for arbutin and hydroquinone analysis in leaves of other plant families (Rosaceae, Lamiaceae, etc.).

  13. Pyrolysis Mass Spectrometry of Complex Organic Materials.

    ERIC Educational Resources Information Center

    Meuzelaar, Henk L. C.; And Others

    1984-01-01

    Illustrates the state of the art in pyrolysis mass spectrometry techniques through applications in: (1) structural determination and quality control of synthetic polymers; (2) quantitative analysis of polymer mixtures; (3) classification and structural characterization of fossil organic matter; and (4) nonsupervised numerical extraction of…

  14. Quantitative Metabolite Profiling of an Amino Group Containing Pharmaceutical in Human Plasma via Precolumn Derivatization and High-Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry.

    PubMed

    Li, Sanwang; Klencsár, Balázs; Balcaen, Lieve; Cuyckens, Filip; Lynen, Frederic; Vanhaecke, Frank

    2017-02-07

    Quantitative determination of the candidate drug molecule and its metabolites in biofluids and tissues is an inevitable step in the development of new pharmaceuticals. Because of the time-consuming and expensive nature of the current standard technique for quantitative metabolite profiling, i.e., radiolabeling followed by high-performance liquid chromatography (HPLC) with radiodetection, the development of alternative methodologies is of great interest. In this work, a simple, fast, sensitive, and accurate method for the quantitative metabolite profiling of an amino group containing drug (levothyroxine) and its metabolites in human plasma, based on precolumn derivatization followed by HPLC-inductively coupled plasma mass spectrometry (ICPMS), was developed and validated. To introduce a suitable "heteroelement" (defined here as an element that is detectable with ICPMS), an inexpensive and commercially available reagent, tetrabromophthalic anhydride (TBPA) was used for the derivatization of free NH2-groups. The presence of a known number of I atoms in both the drug molecule and its metabolites enabled a cross-validation of the newly developed derivatization procedure and quantification based on monitoring of the introduced Br. The formation of the derivatives was quantitative, providing a 4:1 stoichiometric Br/NH2 ratio. The derivatives were separated via reversed-phase HPLC with gradient elution. Bromine was determined via ICPMS at a mass-to-charge ratio of 79 using H2 as a reaction gas to ensure interference-free detection, and iodine was determined at a mass-to-charge ratio of 127 for cross-validation purposes. The method developed shows a fit-for-purpose accuracy (recovery between 85% and 115%) and precision (repeatability <15% RSD). The limit of quantification (LoQ) for Br was approximately 100 μg/L.

  15. Quantitative fluorescence spectroscopy and flow cytometry analyses of cell-penetrating peptides internalization pathways: optimization, pitfalls, comparison with mass spectrometry quantification

    NASA Astrophysics Data System (ADS)

    Illien, Françoise; Rodriguez, Nicolas; Amoura, Mehdi; Joliot, Alain; Pallerla, Manjula; Cribier, Sophie; Burlina, Fabienne; Sagan, Sandrine

    2016-11-01

    The mechanism of cell-penetrating peptides entry into cells is unclear, preventing the development of more efficient vectors for biotechnological or therapeutic purposes. Here, we developed a protocol relying on fluorometry to distinguish endocytosis from direct membrane translocation, using Penetratin, TAT and R9. The quantities of internalized CPPs measured by fluorometry in cell lysates converge with those obtained by our previously reported mass spectrometry quantification method. By contrast, flow cytometry quantification faces several limitations due to fluorescence quenching processes that depend on the cell line and occur at peptide/cell ratio >6.108 for CF-Penetratin. The analysis of cellular internalization of a doubly labeled fluorescent and biotinylated Penetratin analogue by the two independent techniques, fluorometry and mass spectrometry, gave consistent results at the quantitative and qualitative levels. Both techniques revealed the use of two alternative translocation and endocytosis pathways, whose relative efficacy depends on cell-surface sugars and peptide concentration. We confirmed that Penetratin translocates at low concentration and uses endocytosis at high μM concentrations. We further demonstrate that the hydrophobic/hydrophilic nature of the N-terminal extremity impacts on the internalization efficiency of CPPs. We expect these results and the associated protocols to help unraveling the translocation pathway to the cytosol of cells.

  16. Quantitative fluorescence spectroscopy and flow cytometry analyses of cell-penetrating peptides internalization pathways: optimization, pitfalls, comparison with mass spectrometry quantification.

    PubMed

    Illien, Françoise; Rodriguez, Nicolas; Amoura, Mehdi; Joliot, Alain; Pallerla, Manjula; Cribier, Sophie; Burlina, Fabienne; Sagan, Sandrine

    2016-11-14

    The mechanism of cell-penetrating peptides entry into cells is unclear, preventing the development of more efficient vectors for biotechnological or therapeutic purposes. Here, we developed a protocol relying on fluorometry to distinguish endocytosis from direct membrane translocation, using Penetratin, TAT and R9. The quantities of internalized CPPs measured by fluorometry in cell lysates converge with those obtained by our previously reported mass spectrometry quantification method. By contrast, flow cytometry quantification faces several limitations due to fluorescence quenching processes that depend on the cell line and occur at peptide/cell ratio >6.10(8) for CF-Penetratin. The analysis of cellular internalization of a doubly labeled fluorescent and biotinylated Penetratin analogue by the two independent techniques, fluorometry and mass spectrometry, gave consistent results at the quantitative and qualitative levels. Both techniques revealed the use of two alternative translocation and endocytosis pathways, whose relative efficacy depends on cell-surface sugars and peptide concentration. We confirmed that Penetratin translocates at low concentration and uses endocytosis at high μM concentrations. We further demonstrate that the hydrophobic/hydrophilic nature of the N-terminal extremity impacts on the internalization efficiency of CPPs. We expect these results and the associated protocols to help unraveling the translocation pathway to the cytosol of cells.

  17. Enhanced Trace-Fiber Color Discrimination by Electrospray Ionization Mass Spectrometry: A Quantitative and Qualitative Tool for the Analysis of Dyes Extracted from Sub-millimeter Nylon Fibers

    SciTech Connect

    2002-09-26

    The application of electrospray-ionization mass spectrometry (ESI-MS) to trace-fiber color analysis is explored using acidic dyes commonly employed to color nylon-based fibers, as well as extracts from dyed nylon fibers. Qualitative information about constituent dyes and quantitative information about the relative amounts of those dyes present on a single fiber become readily available using this technique. Sample requirements for establishing the color-identity of different samples (i.e., comparative trace-fiber analysis) are shown to be sub-millimeter. Absolute verification of dye-mixture identity (beyond the comparison of molecular weights derived from ESI-MS) can be obtained by expanding the technique to include tandem mass spectrometry (ESI-MS/MS). For dyes of unknown origin, the ESI-MS/MS analyses may offer insights into the chemical structure of the compound--information not available from chromatographic techniques alone. This research demonstrates that ESI-MS is viable as a sensitive technique for distinguishing dye constituents extracted from a minute amount of trace fiber evidence. A protocol is suggested to establish/refute the proposition that two fibers--one of which is available in minute quantity only--are of the same origin.

  18. Quantitative analysis of trace-level benzene, toluene, ethylbenzene, and xylene in cellulose acetate tow using headspace heart-cutting multidimensional gas chromatography with mass spectrometry.

    PubMed

    Ji, Xiaorong; Zhang, Jing; Guo, Yinlong

    2016-06-01

    This study describes a method for the quantification of trace-level benzene, toluene, ethylbenzene, and xylene in cellulose acetate tow by heart-cutting multidimensional gas chromatography with mass spectrometry in selected ion monitoring mode. As the major volatile component in cellulose acetate tow samples, acetone would be overloaded when attempting to perform a high-resolution separation to analyze trace benzene, toluene, ethylbenzene, and xylene. With heart-cutting technology, a larger volume injection was achieved and acetone was easily cut off by employing a capillary column with inner diameter of 0.32 mm in the primary gas chromatography. Only benzene, toluene, ethylbenzene, and xylene were directed to the secondary column to result in an effective separation. The matrix interference was minimized and the peak shapes were greatly improved. Finally, quantitative analysis of benzene, toluene, ethylbenzene, and xylene was performed using an isotopically labeled internal standard. The headspace multidimensional gas chromatography mass spectrometry system was proved to be a powerful tool for analyzing trace volatile organic compounds in complex samples.

  19. Quantitative fluorescence spectroscopy and flow cytometry analyses of cell-penetrating peptides internalization pathways: optimization, pitfalls, comparison with mass spectrometry quantification

    PubMed Central

    Illien, Françoise; Rodriguez, Nicolas; Amoura, Mehdi; Joliot, Alain; Pallerla, Manjula; Cribier, Sophie; Burlina, Fabienne; Sagan, Sandrine

    2016-01-01

    The mechanism of cell-penetrating peptides entry into cells is unclear, preventing the development of more efficient vectors for biotechnological or therapeutic purposes. Here, we developed a protocol relying on fluorometry to distinguish endocytosis from direct membrane translocation, using Penetratin, TAT and R9. The quantities of internalized CPPs measured by fluorometry in cell lysates converge with those obtained by our previously reported mass spectrometry quantification method. By contrast, flow cytometry quantification faces several limitations due to fluorescence quenching processes that depend on the cell line and occur at peptide/cell ratio >6.108 for CF-Penetratin. The analysis of cellular internalization of a doubly labeled fluorescent and biotinylated Penetratin analogue by the two independent techniques, fluorometry and mass spectrometry, gave consistent results at the quantitative and qualitative levels. Both techniques revealed the use of two alternative translocation and endocytosis pathways, whose relative efficacy depends on cell-surface sugars and peptide concentration. We confirmed that Penetratin translocates at low concentration and uses endocytosis at high μM concentrations. We further demonstrate that the hydrophobic/hydrophilic nature of the N-terminal extremity impacts on the internalization efficiency of CPPs. We expect these results and the associated protocols to help unraveling the translocation pathway to the cytosol of cells. PMID:27841303

  20. Quantitative determination of the neurotoxin β-N-methylamino-L-alanine (BMAA) by capillary electrophoresis-tandem mass spectrometry.

    PubMed

    Kerrin, Elliott S; White, Robert L; Quilliam, Michael A

    2017-02-01

    Recent reports of the widespread occurrence of the neurotoxin β-N-methylamino-L-alanine (BMAA) in cyanobacteria and particularly seafood have raised concerns for public health. LC-MS/MS is currently the analytical method of choice for BMAA determinations but incomplete separation of isomeric and isobaric compounds, matrix suppression and conjugated forms are plausible limitations. In this study, capillary electrophoresis (CE) coupled with MS/MS has been developed as an alternative method for the quantitative determination of free BMAA. Using a bare fused silica capillary, a phosphate buffer (250 mM, pH 3.0) and UV detection, it was possible to separate BMAA from four isomers, but the limit of detection (LOD) of 0.25 μg mL(-1) proved insufficient for analysis of typical samples. Coupling the CE to a triple quadrupole MS was accomplished using a custom sheath-flow interface. The best separation was achieved with a 5 M formic acid in water/acetonitrile (9:1) background electrolyte. Strong acid hydrolysis of lyophilized samples was used to release BMAA from conjugated forms. Field-amplified stacking after injection was achieved by lowering sample ionic strength with a cation-exchange cleanup procedure. Quantitation was accomplished using isotope dilution with deuterium-labelled BMAA as internal standard. An LOD for BMAA in solution of 0.8 ng mL(-1) was attained, which was equivalent to 16 ng g(-1) dry mass in samples using the specified extraction procedure. This was comparable with LC-MS/MS methods. The method displayed excellent resolution of amino acid isomers and had no interference from matrix components. The presence of BMAA in cycad, mussel and lobster samples was confirmed by CE-MS/MS, but not in an in-house cyanobacterial reference material, with quantitative results agreeing with those from LC-MS/MS. Graphical Abstract CE-MS separation and detection of BMAA, its isomers and the internal standard BMAA-d3.

  1. Mass spectrometry in environmental toxicology.

    PubMed

    Groh, Ksenia J; Suter, Marc J-F

    2014-01-01

    In environmental toxicology, mass spectrometry can be applied to evaluate both exposure to chemicals as well as their effects in organisms. Various ultra-trace techniques are employed today to measure pollutants in different environmental compartments. Increasingly, effect-directed analysis is being applied to focus chemical monitoring on sites of ecotoxicological concern. Mass spectrometry is also very instrumental for studying the interactions of chemicals with organisms on the molecular and cellular level, providing new insights into mechanisms of toxicity. In the future, diverse mass spectrometry-based techniques are expected to become even more widely used in this field, contributing to the refinement of currently used environmental risk assessment strategies.

  2. Rapid Quantitation of Ascorbic and Folic Acids in SRM 3280 Multivitamin/Multielement Tablets using Flow-Injection Tandem Mass Spectrometry

    SciTech Connect

    Bhandari, Deepak; Kertesz, Vilmos; Van Berkel, Gary J

    2013-01-01

    RATIONALE: Ascorbic acid (AA) and folic acid (FA) are water-soluble vitamins and are usually fortified in food and dietary supplements. For the safety of human health, proper intake of these vitamins is recommended. Improvement in the analysis time required for the quantitative determination of these vitamins in food and nutritional formulations is desired. METHODS: A simple and fast (~5 min) in-tube sample preparation was performed, independently for FA and AA, by mixing extraction solvent with a powdered sample aliquot followed by agitation, centrifugation, and filtration to recover an extract for analysis. Quantitative detection was achieved by flow-injection (1 L injection volume) electrospray ionization tandem mass spectrometry (ESI-MS/MS) in negative ion mode using the method of standard addition. RESULTS: Method of standard addition was employed for the quantitative estimation of each vitamin in a sample extract. At least 2 spiked and 1 non-spiked sample extract were injected in triplicate for each quantitative analysis. Given an injection-to-injection interval of approximately 2 min, about 18 min was required to complete the quantitative estimation of each vitamin. The concentration values obtained for the respective vitamins in the standard reference material (SRM) 3280 using this approach were within the statistical range of the certified values provided in the NIST Certificate of Analysis. The estimated limit of detections of FA and AA were 13 and 5.9 ng/g, respectively. CONCLUSIONS: Flow-injection ESI-MS/MS was successfully applied for the rapid quantitation of FA and AA in SRM 3280 multivitamin/multielement tablets.

  3. Ion mobility-mass spectrometry.

    PubMed

    Kanu, Abu B; Dwivedi, Prabha; Tam, Maggie; Matz, Laura; Hill, Herbert H

    2008-01-01

    This review article compares and contrasts various types of ion mobility-mass spectrometers available today and describes their advantages for application to a wide range of analytes. Ion mobility spectrometry (IMS), when coupled with mass spectrometry, offers value-added data not possible from mass spectra alone. Separation of isomers, isobars, and conformers; reduction of chemical noise; and measurement of ion size are possible with the addition of ion mobility cells to mass spectrometers. In addition, structurally similar ions and ions of the same charge state can be separated into families of ions which appear along a unique mass-mobility correlation line. This review describes the four methods of ion mobility separation currently used with mass spectrometry. They are (1) drift-time ion mobility spectrometry (DTIMS), (2) aspiration ion mobility spectrometry (AIMS), (3) differential-mobility spectrometry (DMS) which is also called field-asymmetric waveform ion mobility spectrometry (FAIMS) and (4) traveling-wave ion mobility spectrometry (TWIMS). DTIMS provides the highest IMS resolving power and is the only IMS method which can directly measure collision cross-sections. AIMS is a low resolution mobility separation method but can monitor ions in a continuous manner. DMS and FAIMS offer continuous-ion monitoring capability as well as orthogonal ion mobility separation in which high-separation selectivity can be achieved. TWIMS is a novel method of IMS with a low resolving power but has good sensitivity and is well intergrated into a commercial mass spectrometer. One hundred and sixty references on ion mobility-mass spectrometry (IMMS) are provided.

  4. Label-free high-throughput screening via mass spectrometry: a single cystathionine quantitative method for multiple applications.

    PubMed

    Holt, Tom G; Choi, Bernard K; Geoghagen, Neil S; Jensen, Kristian K; Luo, Qi; LaMarr, William A; Makara, Gergely M; Malkowitz, Lorraine; Ozbal, Can C; Xiong, Yusheng; Dufresne, Claude; Luo, Ming-Juan

    2009-10-01

    Label-free mass spectrometric (MS) technologies are particularly useful for enzyme assay design for drug discovery screens. MS permits the selective detection of enzyme substrates or products in a wide range of biological matrices without need for derivatization, labeling, or capture technologies. As part of a cardiovascular drug discovery effort aimed at finding modulators of cystathionine beta-synthase (CBS), we used the RapidFire((R)) label-free high-throughput MS (HTMS) technology to develop a high-throughput screening (HTS) assay for CBS activity. The in vitro assay used HTMS to quantify the unlabeled product of the CBS reaction, cystathionine. Cystathionine HTMS analyses were carried out with a throughput of 7 s per sample and quantitation over a linear range of 80-10,000 nM. A compound library of 25,559 samples (or 80 384-well plates) was screened as singlets using the HTMS assay in a period of 8 days. With a hit rate of 0.32%, the actives showed a 90% confirmation rate. The in vitro assay was applied to secondary screens in more complex matrices with no additional analytical development. Our results show that the HTMS method was useful for screening samples containing serum, for cell-based assays, and for liver explants. The novel extension of the in vitro analytical method, without modification, to secondary assays resulted in a significant and advantageous economy of development time for the drug discovery project.

  5. High performance liquid chromatography coupled to mass spectrometry for profiling and quantitative analysis of folate monoglutamates in tomato.

    PubMed

    Tyagi, Kamal; Upadhyaya, Pallawi; Sarma, Supriya; Tamboli, Vajir; Sreelakshmi, Yellamaraju; Sharma, Rameshwar

    2015-07-15

    Folates are essential micronutrients for animals as they play a major role in one carbon metabolism. Animals are unable to synthesize folates and obtain them from plant derived food. In the present study, a high performance liquid chromatography coupled to mass spectrometric (HPLC-MS/MS) method was developed for the high throughput screening and quantitative analysis of folate monoglutamates in tomato fruits. For folate extraction, several parameters were optimized including extraction conditions, pH range, amount of tri-enzyme and boiling time. After processing the extract was purified using ultra-filtration with 10 kDa membrane filter. The ultra-filtered extract was chromatographed on a RP Luna C18 column using gradient elution program. The method was validated by determining linearity, sensitivity and recovery. This method was successfully applied to folate estimation in spinach, capsicum, and garden pea and demonstrated that this method offers a versatile approach for accurate and fast determination of different folate monoglutamates in vegetables.

  6. Quantitative Determination of Irinotecan and the Metabolite SN-38 by Nanoflow Liquid Chromatography-Tandem Mass Spectrometry in Different Regions of Multicellular Tumor Spheroids

    NASA Astrophysics Data System (ADS)

    Liu, Xin; Hummon, Amanda B.

    2015-04-01

    A new and simple method was developed to evaluate the distribution of therapeutics in three-dimensional multicellular tumor spheroids (MCTS) by combining serial trypsinization and nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS). This methodology was validated with quantitative measurements of irinotecan and its bioactive metabolite, SN-38, in distinct spatial regions of HCT 116 MCTS. Irinotecan showed a time-dependent permeability into MCTS with most of the drug accumulating in the core after 24 h of treatment. The amount of SN-38 detected was 30 times lower than that of the parent drug, and was more abundant in the outer rim and intermediate regions of MCTS where proliferating cells were present. This method can be used to investigate novel and established drugs. It enables investigation of drug penetration properties and identification of metabolites with spatial specificity in MCTS. The new approach has great value in facilitating the drug evaluation process.

  7. Quantitative Mass Spectrometry-Based Analysis of β-D-Glucosyl-5-Hydroxymethyluracil in Genomic DNA of Trypanosoma brucei

    NASA Astrophysics Data System (ADS)

    Liu, Shuo; Ji, Debin; Cliffe, Laura; Sabatini, Robert; Wang, Yinsheng

    2014-10-01

    β-D-glucosyl-5-hydroxymethyluracil (base J) is a hyper-modified nucleobase found in the nuclear DNA of kinetoplastid parasites. With replacement of a fraction of thymine in DNA, J is localized primarily in telomeric regions of all organisms carrying this modified base. The biosynthesis of J occurs in two putative steps: first, a specific thymine in DNA is recognized and converted into 5-hydroxymethyluracil (5-HmU) by J-binding proteins (JBP1 and JBP2); a glucosyl transferase (GT) subsequently glucosylates the 5-HmU to yield J. Although several recent studies revealed the roles of internal J in regulating transcription in kinetoplastids, functions of telomeric J and proteins involved in J synthesis remain elusive. Assessing the functions of base J and understanding fully its biosynthesis necessitate the measurement of its level in cells and organisms. In this study, we reported a reversed-phase HPLC coupled with tandem mass spectrometry (LC-MS/MS) method, together with the use of a surrogate internal standard (β-D-glucosyl-5-hydroxymethyl-2'-deoxycytidine, 5-gHmdC), for the accurate detection of β-D-glucosyl-5-hydroxymethyl-2'-deoxyuridine (dJ) in Trypanosoma brucei DNA. For comparison, we also measured the level of the precursor for dJ synthesis [i.e. 5-hydroxymethyl-2'-deoxyuridine (5-HmdU)]. We found that base J was not detectable in the JBP-null cells whereas it replaced approximately 0.5% thymine in wild-type cells, which was accompanied with a markedly decreased level of 5-HmdU in JBP1/JBP2-null strain relative to the wild-type strain. These results provided direct evidence supporting that JBP proteins play an important role in oxidizing thymidine to form 5-HmdU, which facilitated the generation of dJ. This is the first report about the application of LC-MS/MS for the quantification of base J. The analytical method built a solid foundation for dissecting the molecular mechanisms of J biosynthesis and assessing the biological functions of base J in the

  8. Quantitative determination of phosphatidylcholine hydroperoxides during copper oxidation of LDL and HDL by liquid chromatography/mass spectrometry.

    PubMed

    Hui, Shu-Ping; Taguchi, Yudai; Takeda, Seiji; Ohkawa, Futaba; Sakurai, Toshihiro; Yamaki, Shinobu; Jin, Shigeki; Fuda, Hirotoshi; Kurosawa, Takao; Chiba, Hitoshi

    2012-06-01

    1-Palmitoyl-2-linoleoylphosphatidylcholine monohydroperoxide (PC 16:0/18:2-OOH) and 1-stearoyl-2-linoleoylphosphatidylcholine monohydroperoxide (PC 18:0/18:2-OOH) were measured by liquid chromatography/mass spectrometry (LC/MS) using nonendogenous 1-palmitoyl-2-heptadecenoylphosphatidylcholine monohydroperoxide as an internal standard. The calibration curves for synthetic PC 16:0/18:2-OOH and PC 18:0/18:2-OOH, which were obtained by direct injection of the internal standard into the LC/MS system, were linear throughout the calibration range (0.8-12.8 pmol). Within-day and between-day coefficients of variation were less than 10%, and the recoveries were between 86% and 105%. The limit of detection (LOD) and the limit of quantification (LOQ) were determined using synthetic standards. The LOD (signal-to-noise ratio 3:1) was 0.01 pmol, and the LOQ (signal-to-noise ratio 6:1) was 0.08 pmol for both PC 16:0/18:2-OOH and PC 18:0/18:2-OOH. With use of this method, the concentrations of PC 16:0/18:2-OOH and PC 18:0/18:2-OOH in the lipoprotein fractions during copper-mediated oxidation were determined. We prepared oxLDL and oxHDL by incubating native LDL and native HDL from human plasma (n =  10) with CuSO(4) for up to 4 h. The time course of the PC 16:0/18:2-OOH and PC 18:0/18:2-OOH levels during oxidation consisted of three phases. For oxidized LDL, both compounds exhibited a slow lag phase and a subsequent rapidly increasing propagation phase, followed by a gradually decreasing degradation phase. In contrast, for oxidized HDL, both compounds initially exhibited a prompt propagation phase with a subsequent plateau phase, followed by a rapid degradation phase. The analytical LC/MS method for phosphatidylcholine hydroperoxides might be useful for the analysis of biological samples.

  9. 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

  10. Quantitative determination of pioglitazone in human serum by direct-injection high-performance liquid chromatography mass spectrometry and its application to a bioequivalence study.

    PubMed

    Xue, Y-J; Turner, Kenneth C; Meeker, Jeff B; Pursley, Janice; Arnold, Mark; Unger, Steve

    2003-10-05

    A simple, high throughput, direct-injection high-performance liquid chromatography tandem mass spectrometry method (LC/MS/MS) has been developed and validated for the quantitation of pioglitazone in human serum. After mixing the internal standard with a sample, a 10 microl portion of the mixture was directly injected into a high-flow LC/MS/MS system, which included an extraction column, an analytical column and a six-port switching valve. The on-line extraction was achieved on an Oasis HLB column (1 mm x 50 mm, 30 microm) with a 100% aqueous loading mobile phase containing 5 mM ammonium acetate (pH 4.0) at a flow rate of 4 ml/min. The extracted analyte was eluted by a mobile phase which contained 5 mM ammonium acetate and acetonitrile. The analytical column was a Luna C18 column (4.6 mm x 50 mm, 5 microm). Detection was achieved by positive ion electrospray tandem mass spectrometry. The lower limit of quantitation of the method was 9 ng/ml. The standard curve, which ranged from 9 to 1350 ng/ml, was fitted by a weighted (1/x2) quadratic regression model. The validation results demonstrated that this method had satisfactory precision and accuracy across the calibration range. There was no evidence of instability of the analyte in human serum following three freeze-thaw cycles, and samples could be stored for at least 2 weeks at -30 degrees C. This method was used to analyze pioglitazone concentrations in human serum samples from a bioequivalence study of a blinded Actos formulation (encapsulated 15 mg tablet) and an Actos 15 mg tablet. The blinded formulation was shown to be bioequivalent to an Actos 15 mg tablet.

  11. Qualitative and quantitative analysis on chemical constituents from Curculigo orchioides using ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

    PubMed

    He, Yongjing; Dong, Xin; Jia, Xiaoxuan; Li, Mei; Yuan, Tingting; Xu, Hongtao; Qin, Luping; Han, Ting; Zhang, Qiaoyan

    2015-01-01

    A rapid ultra-high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC-ESI-Q-TOF/MS) method was developed for qualitative and quantitative determination of constituents in the rhizome of Curculigo orchioides. Qualitative analysis was performed on a Waters ACQUITY UHPLC @ HSS T3 column (1.8 μm 100 × 2.1mm) using gradient elution with mobile phase of 0.1% formic acid and acetonitrile. Quantitative analysis was performed on an Agilent ZORBAX Eclipse plus C18 column (1.7 μm 100 × 2.1mm) using gradient elution with mobile phase of 0.1% acetic acid and acetonitrile for at least 20 min. Quadrupole TOF/MS in either full scan mode or extracted ion mode was used for qualitative and quantitative analysis of the constituents. According to the mass spectrometric fragmentation mechanism and UHPLC-ESI-Q-TOF-MS data, chemical structures of 45 constituents in the rhizome of Curculigo orchioides, including 19 phenols and phenolic glycosides, 16 lignans and lignan glycosides, 8 triterpenoid saponins, one flavone and one sesquiterpene, were identified tentatively on-line without the time-consuming process of isolation. In addition, 8 phenolic glycosides including 5-hydroxymethylfurfural (HMF), 2-hydroxy-5-(2-hydroxyethyl) phenyl-β-D-glucopyranoside (HPG), anacardoside (ACD), orcinol glucoside (OGD), orcinol-1-O-β-D-apiofuranosyl-(1 → 6)-β-D-glucopyranoside (OAG), 2,6-dimethoxybenzoic acid (DBA), curculigoside (CUR) and curculigine A (CCL) were quantitated in 11 collected samples and 10 commercial samples from different providers. The results show that UHPLC-ESI-Q-TOF-MS is a viable method for analysis and quality evaluation of the constituents from the rhizome of Curculigo orchioides.

  12. Sensitive, Efficient Quantitation of 13C-Enriched Nucleic Acids via Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry for Applications in Stable Isotope Probing.

    PubMed

    Wilhelm, Roland; Szeitz, András; Klassen, Tara L; Mohn, William W

    2014-12-01

    Stable isotope probing (SIP) of nucleic acids is a powerful tool for studying the functional traits of microbial populations within complex communities, but SIP involves a number of technical challenges. Many of the difficulties in DNA-SIP and RNA-SIP experiments can be effectively overcome with an efficient, sensitive method for quantitating the isotopic enrichment of nucleic acids. Here, we present a sensitive method for quantitating (13)C enrichment of nucleic acids, requiring a few nanograms of sample, and we demonstrate its utility in typical DNA-SIP and RNA-SIP experiments. All five nucleobases (adenine, guanine, cytosine, thymine, and uracil) were separated and detected by using ultrahigh-performance liquid chromatography-tandem mass spectrometry. We detected all isotopic species in samples with as low as 1.5 atom% (13)C above natural abundance, using 1-ng loadings. Quantitation was used to characterize the isotopic enrichment kinetics of cellulose- and lignin-based microcosm experiments and to optimize the recovery of enriched nucleic acids. Application of our method will minimize the quantity of expensive isotopically labeled substrates required and reduce the risk of failed experiments due to insufficient recovery of labeled nucleic acids for sequencing library preparation.

  13. Sensitive, Efficient Quantitation of 13C-Enriched Nucleic Acids via Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry for Applications in Stable Isotope Probing

    PubMed Central

    Wilhelm, Roland; Szeitz, András; Klassen, Tara L.

    2014-01-01

    Stable isotope probing (SIP) of nucleic acids is a powerful tool for studying the functional traits of microbial populations within complex communities, but SIP involves a number of technical challenges. Many of the difficulties in DNA-SIP and RNA-SIP experiments can be effectively overcome with an efficient, sensitive method for quantitating the isotopic enrichment of nucleic acids. Here, we present a sensitive method for quantitating 13C enrichment of nucleic acids, requiring a few nanograms of sample, and we demonstrate its utility in typical DNA-SIP and RNA-SIP experiments. All five nucleobases (adenine, guanine, cytosine, thymine, and uracil) were separated and detected by using ultrahigh-performance liquid chromatography–tandem mass spectrometry. We detected all isotopic species in samples with as low as 1.5 atom% 13C above natural abundance, using 1-ng loadings. Quantitation was used to characterize the isotopic enrichment kinetics of cellulose- and lignin-based microcosm experiments and to optimize the recovery of enriched nucleic acids. Application of our method will minimize the quantity of expensive isotopically labeled substrates required and reduce the risk of failed experiments due to insufficient recovery of labeled nucleic acids for sequencing library preparation. PMID:25217022

  14. Combination of quantitative analysis and chemometric analysis for the quality evaluation of three different frankincenses by ultra high performance liquid chromatography and quadrupole time of flight mass spectrometry.

    PubMed

    Zhang, Chao; Sun, Lei; Tian, Run-tao; Jin, Hong-yu; Ma, Shuang-Cheng; Gu, Bing-ren

    2015-10-01

    Frankincense has gained increasing attention in the pharmaceutical industry because of its pharmacologically active components such as boswellic acids. However, the identity and overall quality evaluation of three different frankincense species in different Pharmacopeias and the literature have less been reported. In this paper, quantitative analysis and chemometric evaluation were established and applied for the quality control of frankincense. Meanwhile, quantitative and chemometric analysis could be conducted under the same analytical conditions. In total 55 samples from four habitats (three species) of frankincense were collected and six boswellic acids were chosen for quantitative analysis. Chemometric analyses such as similarity analysis, hierarchical cluster analysis, and principal component analysis were used to identify frankincense of three species to reveal the correlation between its components and species. In addition, 12 chromatographic peaks have been tentatively identified explored by reference substances and quadrupole time-of-flight mass spectrometry. The results indicated that the total boswellic acid profiles of three species of frankincense are similar and their fingerprints can be used to differentiate between them.

  15. A rapid, sensitive method for quantitative analysis of underivatized amino acids by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

    PubMed

    Le, Anthony; Ng, Angelina; Kwan, Tony; Cusmano-Ozog, Kristina; Cowan, Tina M

    2014-01-01

    The quantitation of free amino acids from physiologic samples is essential for diagnosing and monitoring patients with inherited metabolic disorders. Current methods are hindered by long preparative and/or analysis times, expensive reagents, and often suboptimal performance characteristics. To overcome these challenges, a improved method for amino acid analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed and validated. Samples were deproteinized with sulfosalicylic acid and supernatants diluted with tridecafluoroheptanoic acid. Chromatographic separation of amino acids occurred using two columns, with conditions favoring resolution of isobaric compounds and minimizing ion suppression. Eluted compounds were detected by selective reaction monitoring, and quantitated by relating peak areas of amino acids to externally run standards. Validation studies evaluated linearity, within- and between-run imprecision, lower limits of detection and quantification for 33 amino acids, and correlation with the Biochrom 30 Amino Acid Analyzer. Total run time including re-equilibration was 15min per sample. Within-run precision averaged 2.8% for all compounds, with an average linear correlation coefficient of 0.995. The majority of compounds were reliably quantitated at ≤0.1μM. Between-run precision averaged 4.0%. Results showed excellent correlation with the Biochrom 30 amino acid analyzer with an average overall correlation of 0.908. We conclude that our method is extremely sensitive, specific and reproducible and represents an improvement over other currently available technologies.

  16. Quantitative and qualitative analysis of hemicellulose, cellulose and lignin bio-oils by comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry.

    PubMed

    Michailof, Chrysoula; Sfetsas, Themistoklis; Stefanidis, Stylianos; Kalogiannis, Konstantinos; Theodoridis, Georgios; Lappas, Angelos

    2014-11-21

    Thermal and catalytic pyrolysis are efficient processes for the transformation of biomass to bio-oil, a liquid energy carrier and a general source of chemicals. The elucidation of the bio-oil's composition is essential for a rational design of both its production and utilization process. However, the complex composition of bio-oils hinders their complete qualitative and quantitative analysis, and conventional chromatographic techniques lack the necessary separation power. Two-dimensional gas chromatography with time-of-flight mass spectrometry (GC×GC-ToFMS) is considered a suitable technique for bio-oil analysis due to its increased separation and resolution capacity. This work presents the tentative qualitative and quantitative analysis of bio-oils resulting from the thermal and catalytic pyrolysis of standard xylan, cellulose, lignin and their mixture by GC×GC-ToFMS. Emphasis is placed on the development of the quantitative method using phenol-d6 as internal standard. During the method development, a standard solution of 39 compounds was used for the determination of the respective Relative Response Factors (RRF) employing statistical methods, ANOVA and WLSLR, for verification of the data. The developed method was applied to the above mentioned bio-oils and their detailed analysis is presented. The different compounds produced and their diverse concentration allows for an elucidation of the pyrolysis mechanism and highlight the effect of the catalyst.

  17. Linear electric field mass spectrometry

    DOEpatents

    McComas, D.J.; Nordholt, J.E.

    1992-12-01

    A mass spectrometer and methods for mass spectrometry are described. The apparatus is compact and of low weight and has a low power requirement, making it suitable for use on a space satellite and as a portable detector for the presence of substances. High mass resolution measurements are made by timing ions moving through a gridless cylindrically symmetric linear electric field. 8 figs.

  18. Linear electric field mass spectrometry

    DOEpatents

    McComas, David J.; Nordholt, Jane E.

    1992-01-01

    A mass spectrometer and methods for mass spectrometry. The apparatus is compact and of low weight and has a low power requirement, making it suitable for use on a space satellite and as a portable detector for the presence of substances. High mass resolution measurements are made by timing ions moving through a gridless cylindrically symmetric linear electric field.

  19. Analysis of protein complexes using mass spectrometry.

    PubMed

    Gingras, Anne-Claude; Gstaiger, Matthias; Raught, Brian; Aebersold, Ruedi

    2007-08-01

    The versatile combination of affinity purification and mass spectrometry (AP-MS) has recently been applied to the detailed characterization of many protein complexes and large protein-interaction networks. The combination of AP-MS with other techniques, such as biochemical fractionation, intact mass measurement and chemical crosslinking, can help to decipher the supramolecular organization of protein complexes. AP-MS can also be combined with quantitative proteomics approaches to better understand the dynamics of protein-complex assembly.

  20. Qualitative and quantitative analysis of polycyclic polyprenylated acylphloroglucinols from Garcinia species using ultra performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

    PubMed

    Zhou, Yan; Lee, Stephanie; Choi, Franky Fung Kei; Xu, Gang; Liu, Xin; Song, Jing-Zheng; Li, Song-Lin; Qiao, Chun-Feng; Xu, Hong-Xi

    2010-09-23

    Polycyclic polyprenylated acylphloroglucinols (PPAPs) are a group of natural products isolated from different Garcinia species with a wide range of important biological activities. In this study, an ultra performance liquid chromatography (UPLC) coupled to photodiode-array detection and quadrupole time-of-flight mass spectrometry (Q-TOF) method was developed to characterize 16 PPAPs in 10 Garcinia species. In source dissociation techniques based on cone voltage fragmentation were used to fragment the deprotonated molecules and multiple mass spectrometry (MS/MS) using ramping collision energy were used to further break down the resulting product ions. The resulting characteristic fragment ions were generated by cleavage of C1-C5 bond and C7-C8 bond through concerted pericyclic reaction, which is especially valuable for differentiating three types of PPAPs isomers. As such, two new PPAPs isomers present in minor amount in the extracts of Garcinia oblongifolia were tentatively characterized by comparing their tandem mass spectra to the known ones. In addition, an UPLC-Q-TOF-MS method was validated for the quantitative determination of PPAPs. The method exhibited limits of detection from 2.7 to 21.4 ng mL(-1) and intra-day and inter-day variations were less than 3.7% and the recovery was in the range of 89-107% with RSD less than 9.0%. This UPLC-Q-TOF-MS method has successfully been applied to quantify 16 PPAPs in 32 samples of 10 Garcinia species, which were found to be a rich source of PPAPs.

  1. Quantitative analysis of autoinducing peptide I (AIP-I) from Staphylococcus aureus cultures using ultrahigh performance liquid chromatography-high resolving power mass spectrometry.

    PubMed

    Junio, Hiyas A; Todd, Daniel A; Ettefagh, Keivan A; Ehrmann, Brandie M; Kavanaugh, Jeffrey S; Horswill, Alexander R; Cech, Nadja B

    2013-07-01

    Staphylococcus aureus infections acquired in hospitals now cause more deaths per annum in the US than does HIV/AIDS. Perhaps even more alarming is the rise in community associated methicillin-resistant S. aureus (CA-MRSA) infections, which have spread out of hospital settings and are infecting otherwise healthy individuals. The mechanism of enhanced pathogenesis in CA-MRSA remains unclear, but it has been postulated that high activity in the agr quorum-sensing system could be a contributing factor. The purpose of this study was to develop a quantitative method for analysis of autoinducing peptide I (AIP-I), the activating signal for the agr system in S. aureus. An effective method was developed using ultrahigh performance liquid chromatography (UHPLC) coupled to electrospray ionization mass spectrometry with an LTQ Orbitrap mass spectrometer. Relying on the exceptional resolving power and mass accuracy of this instrument configuration, it was possible to quantify AIP-I directly from the complex growth media of S. aureus cultures with a limit of detection (LOD) of 0.25μM and a linear dynamic range of 2.6 to 63μM. The method was then employed to monitor time-dependent production of AIP-I by S. aureus cultures, and it was observed that AIP-I production reached a maximum and leveled off after approximately 16h. Finally, it was determined that virulence of S. aureus was correlated with AIP-I production in some (but not all) strains analyzed.

  2. Mass spectrometry. [in organic chemistry

    NASA Technical Reports Server (NTRS)

    Burlingame, A. L.; Shackleton, C. H. L.; Howe, I.; Chizhov, O. S.

    1978-01-01

    A review of mass spectrometry in organic chemistry is given, dealing with advances in instrumentation and computer techniques, selected topics in gas-phase ion chemistry, and applications in such fields as biomedicine, natural-product studies, and environmental pollution analysis. Innovative techniques and instrumentation are discussed, along with chromatographic-mass spectrometric on-line computer techniques, mass spectral interpretation and management techniques, and such topics in gas-phase ion chemistry as electron-impact ionization and decomposition, photoionization, field ionization and desorption, high-pressure mass spectrometry, ion cyclotron resonance, and isomerization reactions of organic ions. Applications of mass spectrometry are examined with respect to bio-oligomers and their constituents, biomedically important substances, microbiology, environmental organic analysis, and organic geochemistry.

  3. Streamlined pentafluorophenylpropyl column liquid chromatography-tandem quadrupole mass spectrometry and global (13)C-labeled internal standards improve performance for quantitative metabolomics in bacteria.

    PubMed

    Yang, Song; Sadilek, Martin; Lidstrom, Mary E

    2010-11-19

    Streamlined quantitative metabolomics in central metabolism of bacteria would be greatly facilitated by a high-efficiency liquid chromatography (LC) method in conjunction with accurate quantitation. To achieve this goal, a methodology for LC-tandem quadrupole mass spectrometry (LC-MS/MS) involving a pentafluorophenylpropyl (PFPP) column and culture-derived global (13)C-labeled internal standards (I.Ss.) has been developed and compared to hydrophilic interaction liquid chromatography (HILIC)-MS/MS and published combined two-dimensional gas chromatography and LC methods. All 50 tested metabolite standards from 5 classes (amino acids, carboxylic acids, nucleotides, acyl-CoAs and sugar phosphates) displayed good chromatographic separation and sensitivity on the PFPP column. In addition, many important critical pairs such as isomers/isobars (e.g. isoleucine/leucine, methylsuccinic acid/ethylmalonic acid and malonyl-CoA/3-hydroxybutyryl-CoA) and metabolites of similar structure (e.g. malate/fumarate) were resolved better on the PFPP than on the HILIC column. Compared to only one (13)C-labeled I.S., the addition of global (13)C-labeled I.Ss. improved quantitative linearity and accuracy. PFPP-MS/MS with global (13)C-labeled I.Ss. allowed the absolute quantitation of 42 metabolite pool sizes in Methylobacterium extorquens AM1. A comparison of metabolite level changes published previously for ethylamine (C2) versus succinate (C4) cultures of M. extorquens AM1 indicated a good consistency with the data obtained by PFPP-MS/MS, suggesting this single approach has the capability of providing comprehensive metabolite profiling similar to the combination of methods. The more accurate quantification obtained by this method forms a fundamental basis for flux measurements and can be used for metabolism modeling in bacteria in future studies.

  4. Instrumentation for mass spectrometry: 1997

    SciTech Connect

    McLuckey, S.A.

    1997-08-01

    All mass spectrometry experiments involve the manipulation of material, an interface with the mass spectrometer, ionization, ion manipulation/analysis, detection and data collection/reduction. Each of these elements involve instrumentation. The wide range of species now amenable to mass spectrometry and the diverse areas of physical science in which it plays a role have led to a seemingly unlimited array of instrumental combinations. However, only a limited number of mass analyzers, and their combinations, dominate. The dominant analyzers include time-of-flight, Fourier transform ion cyclotron resonance, the Paul trap, the mass filter, and the sector mass spectrometer. Why there are so few (or so many, depending upon one`s point of view) can be understood upon consideration of a set of mass analyzer figures of merit. These include mass resolution, mass accuracy, mass range, dynamic range, abundance sensitivity, precision, efficiency, speed, MS{sup n} capability, compatibility with the ionizer, cost, and size. The most appropriate form of mass spectrometry is determined by the priorities of the particular measurement placed on the various mass analyzer characteristics and the relative strengths of the analyzers in meeting the requirements. Each of the analyzer types has a unique set of figures of merit that makes it optimally suited for particular applications. This paper discusses these figures of merit, provides data illustrating recent developments for each analyzer type, and gives the figures of merit of each type of analyzer as they stand in 1997. 101 refs., 24 figs.

  5. The effect of co-occurring polychlorinated biphenyls on quantitation of toxaphene in fish tissue samples by gas chromatography negative ion mass spectrometry.

    PubMed

    Lao, Wenjian; Tsukada, David; Maruya, Keith A

    2012-12-28

    Determinative methods based on gas chromatography-negative chemical ionization mass spectrometry (GC-NCI/MS) provide improved sensitivity and specificity for toxaphene in environmental samples, but are subject to misidentification due to oxygen reaction in the presence of polychlorinated biphenyls (PCBs). The goal of this study was to quantify the impact of co-occurring PCBs in fish tissue samples when utilizing single quadrupole instruments to implement this method. Mixtures of PCB congeners and technical toxaphene, and extracts of fish tissue with varying concentrations of PCBs were analyzed for individual congener and total toxaphene concentrations by GC-NCI/MS. The contribution of co-injected PCB 204 ranged from 23% to 88% of the total peak area for the Cl-9 toxaphene homolog quantitation ion, a contribution that increased as the ratio of technical toxaphene to PCB 204 decreased. PCB interferences in fish tissue extracts, including a standard reference material, were subtracted using a three-step procedure featuring spectral analysis of isotopic patterns for target peaks. Total toxaphene concentrations without PCB subtraction in three fish tissue samples with low, intermediate and high co-occurring PCBs were overestimated by 33, 55 and 745%, respectively, underscoring the need for practical strategies to account for PCB interferences in GC-NCI/MS based protocols. In contrast, no appreciable interference or resulting positive bias in concentrations was observed for quantitation of eight common toxaphene residue congeners.

  6. High-resolution gas chromatography/mass spectrometry method for characterization and quantitative analysis of ginkgolic acids in Ginkgo biloba plants, extracts, and dietary supplements.

    PubMed

    Wang, Mei; Zhao, Jianping; Avula, Bharathi; Wang, Yan-Hong; Avonto, Cristina; Chittiboyina, Amar G; Wylie, Philip L; Parcher, Jon F; Khan, Ikhlas A

    2014-12-17

    A high-resolution gas chromatography/mass spectrometry (GC/MS) with selected ion monitor method focusing on the characterization and quantitative analysis of ginkgolic acids (GAs) in Ginkgo biloba L. plant materials, extracts, and commercial products was developed and validated. The method involved sample extraction with (1:1) methanol and 10% formic acid, liquid-liquid extraction with n-hexane, and derivatization with trimethylsulfonium hydroxide (TMSH). Separation of two saturated (C13:0 and C15:0) and six unsaturated ginkgolic acid methyl esters with different positional double bonds (C15:1 Δ8 and Δ10, C17:1 Δ8, Δ10, and Δ12, and C17:2) was achieved on a very polar (88% cyanopropyl) aryl-polysiloxane HP-88 capillary GC column. The double bond positions in the GAs were determined by ozonolysis. The developed GC/MS method was validated according to ICH guidelines, and the quantitation results were verified by comparison with a standard high-performance liquid chromatography method. Nineteen G. biloba authenticated and commercial plant samples and 21 dietary supplements purported to contain G. biloba leaf extracts were analyzed. Finally, the presence of the marker compounds, terpene trilactones and flavonol glycosides for Ginkgo biloba in the dietary supplements was determined by UHPLC/MS and used to confirm the presence of G. biloba leaf extracts in all of the botanical dietary supplements.

  7. Quantitative imaging of platinum based on laser ablation-inductively coupled plasma-mass spectrometry to investigate toxic side effects of cisplatin.

    PubMed

    Köppen, C; Reifschneider, O; Castanheira, I; Sperling, M; Karst, U; Ciarimboli, G

    2015-12-01

    This work presents a quantitative bioimaging method for platinum based on laser ablation-inductively coupled plasma-mass spectrometry and its application for a biomedical study concerning toxic side effects of cisplatin. To trace the histopathology back to cisplatin, platinum was localized and quantified in major functional units of testicle, cochlea, kidney, nerve and brain sections from cisplatin treated mice. The direct consideration of the histology enables precise interpretation of the Pt images and the novel quantitative evaluation approach allows significantly more precise investigations than the pure image. For the first time, platinum was detected and quantified in all major injured structures including organ of Corti of cochlea and seminiferous tubule of testicle. In this way, proximal tubule in kidney, Leydig cells in testicle, stria vascularis and organ of Corti in cochlea and nerve fibers in sciatic nerves are confirmed as targets of cisplatin in these organs. However, the accumulation of platinum in almost all investigated structures also raises questions about more complex pathogenesis including direct and indirect interruption of several biological processes.

  8. A selective and sensitive method for quantitation of lysergic acid diethylamide (LSD) in whole blood by gas chromatography-ion trap tandem mass spectrometry.

    PubMed

    Libong, Danielle; Bouchonnet, Stéphane; Ricordel, Ivan

    2003-01-01

    A gas chromatography-ion trap tandem mass spectrometry (GC-ion trap MS-MS) method for detection and quantitation of LSD in whole blood is presented. The sample preparation process, including a solid-phase extraction step with Bond Elut cartridges, was performed with 2 mL of whole blood. Eight microliters of the purified extract was injected with a cold on-column injection method. Positive chemical ionization was performed using acetonitrile as reagent gas; LSD was detected in the MS-MS mode. The chromatograms obtained from blood extracts showed the great selectivity of the method. GC-MS quantitation was performed using lysergic acid methylpropylamide as the internal standard. The response of the MS was linear for concentrations ranging from 0.02 ng/mL (detection threshold) to 10.0 ng/mL. Several parameters such as the choice of the capillary column, the choice of the internal standard and that of the ionization mode (positive CI vs. EI) were rationalized. Decomposition pathways under both ionization modes were studied. Within-day and between-day stability were evaluated.

  9. Simultaneous quantitative determination of 11 sesquiterpene lactones in Jerusalem artichoke (Helianthus tuberosus L.) leaves by ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry.

    PubMed

    Yuan, Xiaoyan; Yang, Qianxu

    2017-04-01

    A method of ultra high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry was developed for the simultaneous quantification of 11 sesquiterpene lactones in 11 Jerusalem artichoke leaf samples harvested in a number of areas at different periods. The optimal chromatographic conditions were achieved on a ZORBAX Eclipse Plus C18 column (3.0 × 150 mm, 1.8 μm) with linear gradient elution of methanol and water in 8 min. Quantitative analysis was carried out under selective ion monitoring mode. All of the sesquiterpene lactones showed good linearity (R(2) ≥ 0.9949), repeatability (relative standard deviations < 4.66%), and intra- and interday precisions (relative standard deviations < 4.52%) with an accuracy of 95.24-104.84%. The recoveries measured at three concentration levels varied from 95.07 to 104.87% with relative standard deviations less than 4.9%. The limit of detection and limit of quantitation for this method were 0.89-5.05 and 1.12-44.33 ng/mL, respectively. The results showed that the contents of sesquiterpene lactones varied significantly in the Jerusalem artichoke leaf samples from different areas. Among them, the content of sesquiterpene lactones in the sample collected from Dalian, Liaoning province was the highest and the early flowering period was considered to be the optimal harvest time.

  10. Gas chromatography-mass spectrometry method optimized using response surface modeling for the quantitation of fungal off-flavors in grapes and wine.

    PubMed

    Sadoughi, Navideh; Schmidtke, Leigh M; Antalick, Guillaume; Blackman, John W; Steel, Christopher C

    2015-03-25

    An optimized method for the quantitation of volatile compounds responsible for off-aromas, such as earthy odors, found in wine and grapes was developed. The method involved a fast and simple headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) for simultaneous determination of 2-isopropyl-3-methoxypyrazine, 2-isobutyl-3-methoxypyrazine, 3-octanone, fenchone, 1-octen-3-one, trans-2-octen-1-ol, fenchol, 1-octen-3-ol, 2-methylisoborneol, 2,4,6-trichloroanisole, geosmin, 2,4,6-tribromoanisole, and pentachloroanisole. The extraction of the temperature and time were optimized using response surface methodology in both wine base (WB) and grape base (GB). Low limits of detection (0.1-5 ng/L in WB and 0.05-1.6 in GB) and quantitation (0.3-17 in WB and 0.2-6.2 in GB) with good recoveries (83-131%) and repeatability [4.3-9.8% coefficient of variation (CV) in WB and 5.1-11.1% CV in GB] and reproducibility (3.6-10.2 in WB and 1.9-10.9 in GB) indicate that the method has excellent sensitivity and is suitable for the analysis of these off-flavor compounds in wine and grape juice samples.

  11. Mass spectrometry-based sequencing and SRM-based quantitation of two novel vitellogenin isoforms in the leatherback sea turtle (Dermochelys coriacea).

    PubMed

    Plumel, Marine I; Wasselin, Thierry; Plot, Virginie; Strub, Jean-Marc; Van Dorsselaer, Alain; Carapito, Christine; Georges, Jean-Yves; Bertile, Fabrice

    2013-09-06

    No biomarker has yet been discovered to identify the reproductive status of the endangered leatherback sea turtle (Dermochelys coriacea). Although vitellogenin (VTG) could be used for this, its sequence is not known in D. coriacea and no quantitative assay has been carried out in this species to date. Using de novo sequencing-based proteomics, we unambiguously characterized sequences of two different VTG isoforms that we named Dc-VTG1 and Dc-VTG2. To our knowledge, this is the first clear evidence of different VTG isoforms and the structural characterization of derived yolk proteins in reptiles. This work illustrates how massive de novo sequencing can characterize novel sequences when working on "exotic" nonmodel species in which even nucleotide sequences are not available. We developed assays for absolute quantitation of these two isoforms using selected reaction monitoring (SRM) mass spectrometry, thus providing the first SRM assays developed specifically for a nonsequenced species. Plasma levels of Dc-VTG1 and Dc-VTG2 decreased as the nesting season proceeded, and were closely related to the increased levels of reproductive effort. The SRM assays developed here therefore provide an original and efficient approach for the reliable monitoring of reproduction cycles not only in D. coriacea, but potentially in other turtle species.

  12. Qualitative and quantitative analysis of an alkaloid fraction from Piper longum L. using ultra-high performance liquid chromatography-diode array detector-electrospray ionization mass spectrometry.

    PubMed

    Li, Kuiyong; Fan, Yunpeng; Wang, Hui; Fu, Qing; Jin, Yu; Liang, Xinmiao

    2015-05-10

    In a previous research, an alkaloid fraction and 18 alkaloid compounds were prepared from Piper longum L. by series of purification process. In this paper, a qualitative and quantitative analysis method using ultra-high performance liquid chromatography-diode array detector-mass spectrometry (UHPLC-DAD-MS) was developed to evaluate the alkaloid fraction. Qualitative analysis of the alkaloid fraction was firstly completed by UHPLC-DAD method and 18 amide alkaloid compounds were identified. A further qualitative analysis of the alkaloid fraction was accomplished by UHPLC-MS/MS method. Another 25 amide alkaloids were identified according to their characteristic ions and neutral losses. At last, a quantitative method for the alkaloid fraction was established using four marker compounds including piperine, pipernonatine, guineensine and N-isobutyl-2E,4E-octadecadienamide. After the validation of this method, the contents of above four marker compounds in the alkaloid fraction were 57.5mg/g, 65.6mg/g, 17.7mg/g and 23.9mg/g, respectively. Moreover, the relative response factors of other three compounds to piperine were calculated. A comparative study between external standard quantification and relative response factor quantification proved no remarkable difference. UHPLC-DAD-MS method was demonstrated to be a powerful tool for the characterization of the alkaloid fraction from P. longum L. and the result proved that the quality of alkaloid fraction was efficiently improved after appropriate purification.

  13. Mass spectrometry assay as an alternative to the enzyme-linked immunosorbent assay test for biomarker quantitation in ecotoxicology: application to vitellogenin in Crustacea (Gammarus fossarum).

    PubMed

    Simon, Romain; Jubeaux, Guillaume; Chaumot, Arnaud; Lemoine, Jérôme; Geffard, Olivier; Salvador, Arnaud

    2010-07-30

    Vitellogenin (Vg) is a widespread biomarker for measuring exposure to endocrine disruptors. Vg quantification is usually done by using the ELISA test (enzyme-linked immunosorbent assay). Since this test is specific to a target protein, it can rarely be used with other species due to low cross-reactivity across species. Therefore alternative analytical methods have to be considered as the development of a specific and sensitive ELISA test for each new target is time-consuming and may prove unsuccessful. This paper focuses on the development of a quantitative assay by liquid chromatography tandem mass spectrometry (LC-MS/MS) of vitelogenin in an invertebrate (Gammarus fossarum) for which no ELISA kit is available. The linearity of the method was within the concentration range 2.5-25,000pg/mL and the limit of detection was estimated at 0.75pg/mL of Vg. This method has been demonstrated to be an alternative to existing immunological methods for quantifying Vg in invertebrates due to its greater sensitivity, specificity and reproducibility (intra- and inter-assay below 15%). This assay was applied for Vg determination in female G. fossarum following exposure to a known endocrine disruptor, methyl farnesoate, in crustaceans. The availability of a quantitative G. fossarum LC-MS/MS assay should open the way for further studies to evaluate xenoestrogen effects in aquatic male G. fossarum.

  14. Quantitative analysis of amoxicillin, its major metabolites and ampicillin in eggs by liquid chromatography combined with electrospray ionization tandem mass spectrometry.

    PubMed

    Sun, Lirui; Jia, Longfei; Xie, Xing; Xie, Kaizhou; Wang, Jianfeng; Liu, Jianyu; Cui, Lulu; Zhang, Genxi; Dai, Guojun; Wang, Jinyu

    2016-02-01

    In this present study, we developed a simple, rapid and specific method for the quantitative analysis of the contents of amoxicillin (AMO), AMO metabolites and ampicillin (AMP) in eggs. This method uses a simple liquid-liquid extraction with acetonitrile followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The optimized method has been validated according to requirements defined by the European Union and Food and Drug Administration. Extraction recoveries of the target compounds from the egg at 5, 10 and 25 μg/kg were all higher than 80%, with relative standard deviations not exceeding 10.00%. The limits of quantification in eggs were below the maximum residue limits (MRLs). The decision limits (CCα) ranged between 11.1 and 11.5 μg/kg, while detection capabilities (CCβ) from 12.1 to 13.0 μg/kg. These values were very close to the corresponding MRLs. Finally, the new approach was successfully verified for the quantitative determination of these analytes in 40 commercial eggs from local supermarkets.

  15. The detection and quantitative analysis of the psychoactive component of Salvia divinorum, salvinorin A, in human biological fluids using liquid chromatography-mass spectrometry.

    PubMed

    McDonough, Pamela C; Holler, Justin M; Vorce, Shawn P; Bosy, Thomas Z; Magluilo, Joseph; Past, Marilyn R

    2008-01-01

    Salvia divinorum, a member of the mint plant family, has hallucinogenic properties that have become increasingly sought after by recreational drug users. The main psychoactive component, salvinorin A, has potency comparable to lysergic acid diethylamide. Though still legal to possess in most of the United States and much of Europe, little is known regarding the compound's long-term health effects, addiction liability, and pharmacokinetics. Limited data are available in the scientific literature, and few analytical methods are published for the detection in human biological fluids. These factors contribute to the unfamiliarity of the compound and complicate the method development process necessary to accommodate special requested testing for salvinorin A. A sensitive analytical method for the detection and quantitation of salvinorin A in human biological fluids was developed and validated to resolve analytical shortcomings. The method utilizes a solid-phase extraction technique coupled with liquid chromatography-electrospray ionization mass spectrometry operated in selected ion monitoring mode. The assay has a linear range of 5.0-100 ng/mL with a correlation coefficient of 0.997. The limit of detection and limit of quantitation were experimentally determined as 2.5 and 5.0 ng/mL, respectively. The method has been applied to blood and urine samples successfully and can be used to detect the presence of salvinorin A in forensic testing.

  16. Immunoaffinity enrichment and liquid chromatography-selected reaction monitoring mass spectrometry for quantitation of carbonic anhydrase 12 in cultured renal carcinoma cells

    PubMed Central

    Rafalko, Agnes; Iliopoulos, Othon; Fusaro, Vincent A.; Hancock, William; Hincapie, Marina

    2010-01-01

    Liquid chromatography-selected reaction monitoring (LC-SRM) is a highly specific and sensitive mass spectrometry (MS) technique that is widely being applied to selectively qualify and validate candidate markers within complex biological samples. However, in order for LC-SRM methods to take on these attributes, target-specific optimization of sample processing is required, in order to reduce analyte complexity, prior to LC-SRM. In this study, we have developed a targeted platform consisting of protein immunoaffinity enrichment on magnetic beads and LC-SRM for measuring carbonic anhydrase 12 (CA12) protein in a renal cell carcinoma (RCC) cell line (PRC3), a candidate biomarker for RCC whose expression at the protein level has not been previously reported. Sample processing and LC-SRM assay were optimized for signature peptides selected as surrogate markers of CA12 protein. Using LC-SRM coupled with stable isotope dilution, we achieved limits of quantitation in the low fmol range sufficient for measuring clinically relevant biomarkers with good intra- and inter-assay accuracy and precision (≤17%). Our results show that using a quantitative immunoaffinity capture approach provides specific, accurate, and robust assays amenable to high-throughput verification of potential biomarkers. PMID:20936840

  17. Quantitative analysis of fatty-acid-based biofuels produced by wild-type and genetically engineered cyanobacteria by gas chromatography-mass spectrometry.

    PubMed

    Guan, Wenna; Zhao, Hui; Lu, Xuefeng; Wang, Cong; Yang, Menglong; Bai, Fali

    2011-11-11

    Simple and rapid quantitative determination of fatty-acid-based biofuels is greatly important for the study of genetic engineering progress for biofuels production by microalgae. Ideal biofuels produced from biological systems should be chemically similar to petroleum, like fatty-acid-based molecules including free fatty acids, fatty acid methyl esters, fatty acid ethyl esters, fatty alcohols and fatty alkanes. This study founded a gas chromatography-mass spectrometry (GC-MS) method for simultaneous quantification of seven free fatty acids, nine fatty acid methyl esters, five fatty acid ethyl esters, five fatty alcohols and three fatty alkanes produced by wild-type Synechocystis PCC 6803 and its genetically engineered strain. Data obtained from GC-MS analyses were quantified using internal standard peak area comparisons. The linearity, limit of detection (LOD) and precision (RSD) of the method were evaluated. The results demonstrated that fatty-acid-based biofuels can be directly determined by GC-MS without derivation. Therefore, rapid and reliable quantitative analysis of fatty-acid-based biofuels produced by wild-type and genetically engineered cyanobacteria can be achieved using the GC-MS method founded in this work.

  18. Simultaneous chemical fingerprint and quantitative analysis of Rhizoma Smilacis Glabrae by accelerated solvent extraction and high-performance liquid chromatography with tandem mass spectrometry.

    PubMed

    Dai, Weiquan; Zhao, Weiquan; Gao, Fangyuan; Shen, Jingjing; Lv, Diya; Qi, Yunpeng; Fan, Guorong

    2015-05-01

    Rhizoma Smilacis Glabrae (RSG) is a well-known herbal medicine with the homology of medicine and food. In this study, simultaneous chemical fingerprint and quantitative analysis of the bioactive flavonoid components of RSG were developed using accelerated solvent extraction and high-performance liquid chromatography coupled with ion trap tandem mass spectrometry. The operational parameters of accelerated solvent extraction including extraction solvent, extraction temperature, static extraction time, solid-to-liquid ratio, and extraction cycles were optimized. Hierarchical cluster analysis, similarity analysis, and principal component analysis were performed to evaluate the similarity and variation of the samples collected from several provinces in China. Subsequently, high-performance liquid chromatography fingerprints were established for the discrimination of 16 batches of RSG samples, and the major six flavonoids, namely, toxifolin, neoastilbin, astilbin, neoisoastilbin, isoastilbin, and engeletin were then quantitatively determined. The calibration curves for all the six analytes showed good linearity (r(2) > 0.999), and the limits of detection and quantification were less than 0.10 and 0.27 μg·mL(-1) , respectively. Therefore, the proposed extraction and determination methods were proved to be robust and reliable for the quality control of RSG.

  19. Development of a quantitation method to assay both lyoniresinol enantiomers in wines, spirits, and oak wood by liquid chromatography-high resolution mass spectrometry.

    PubMed

    Cretin, Blandine N; Dubourdieu, Denis; Marchal, Axel

    2016-05-01

    Wine taste balance evolves during oak aging by the release of volatile and non-volatile compounds from wood. Among them, an enantiomer of lyoniresinol, (+)-lyoniresinol, has been shown to exhibit bitterness. To evaluate the impact of (+)-lyoniresinol on wine taste, a two-step quantitation method was developed and validated. First, (±)-lyoniresinol was assayed in wines, spirits, and oak wood macerates by C-18 liquid chromatography-high resolution mass spectrometry (LC-HRMS). Then, the lyoniresinol enantiomeric ratio was determined by chiral LC-HRMS in order to calculate the (+)-lyoniresinol content. In red and white wines, the average concentrations of (+)-lyoniresinol were 1.9 and 0.8 mg/L, respectively. The enantiomer proportions were not affected by bottle aging, and lyoniresinol appeared to remain stable over time. The sensory study of (+)-lyoniresinol established its perception threshold at 0.46 mg/L in wine. All the commercial wines quantitated were above this perception threshold, demonstrating its impact on wine taste by an increase in bitterness. In spirits, (+)-lyoniresinol ranged from 2.0 to 10.0 mg/L and was found to be released continuously during oak aging. Finally, neither botanical origin nor toasting was found to significantly affect the (+)-lyoniresinol content of oak wood. Graphical abstract From oak wood to wine: evaluation of the influence of (+)-lyoniresinol on the bitterness of wines and spirits.

  20. More sensitive and quantitative proteomic measurements using very low flow rate porous silica monolithic LC columns with electrospray ionization-mass spectrometry

    SciTech Connect

    Luo, Quanzhou; Tang, Keqi; Yang, Feng; Elias, Ayesha; Shen, Yufeng; Moore, Ronald J.; Zhao, Rui; Hixson, Kim K.; Rossie, Sandra S.; Smith, Richard D.

    2006-05-01

    The sensitivity of proteomics measurements using liquid chromatography (LC) separations interfaced with electrospray ionization-mass spectrometry (ESI-MS) improves approximately inversely with liquid flow rate, making attractive the use of smaller inner diameter LC columns. We report the development and initial application of 10 µm i.d. silica-based monolithic LC columns providing more sensitive proteomics measurements. The implementation provides robust performance and suitability for automated proteome analyses due to integration with a micro solid phase extraction pre-column for ease of sample injection and clean-up prior to the reversed phased LC separation. Greater than 10-fold improvement in sensitivity was obtained compared to analyses using more conventional capillary LC, enabling e.g. the identification of >5000 different peptides by MS/MS from 100-ng of a Shewanella oneidensis tryptic digest using an ion trap MS. The low nL/min LC flow rates provide more uniform signal intensities for different peptides, and provided improved quantitative measurements compared to conventional separation systems without the use of internal standards or isotopic labeling. The improved sensitivity allowed LC-MS measurements of immunopurified protein phosphatase 5 that were in good agreement with quantitative western blot analyses.

  1. Quantitative Measurement of Intact Alpha-Synuclein Proteoforms from Post-Mortem Control and Parkinson's Disease Brain Tissue by Intact Protein Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Kellie, John F.; Higgs, Richard E.; Ryder, John W.; Major, Anthony; Beach, Thomas G.; Adler, Charles H.; Merchant, Kalpana; Knierman, Michael D.

    2014-07-01

    A robust top down proteomics method is presented for profiling alpha-synuclein species from autopsied human frontal cortex brain tissue from Parkinson's cases and controls. The method was used to test the hypothesis that pathology associated brain tissue will have a different profile of post-translationally modified alpha-synuclein than the control samples. Validation of the sample processing steps, mass spectrometry based measurements, and data processing steps were performed. The intact protein quantitation method features extraction and integration of m/z data from each charge state of a detected alpha-synuclein species and fitting of the data to a simple linear model which accounts for concentration and charge state variability. The quantitation method was validated with serial dilutions of intact protein standards. Using the method on the human brain samples, several previously unreported modifications in alpha-synuclein were identified. Low levels of phosphorylated alpha synuclein were detected in brain tissue fractions enriched for Lewy body pathology and were marginally significant between PD cases and controls (p = 0.03).

  2. Development and Validation of a Multiplexed Protein Quantitation Assay for the Determination of Three Recombinant Proteins in Soybean Tissues by Liquid Chromatography with Tandem Mass Spectrometry.

    PubMed

    Hill, Ryan C; Oman, Trent J; Shan, Guomin; Schafer, Barry; Eble, Julie; Chen, Cynthia

    2015-08-26

    Currently, traditional immunochemistry technologies such as enzyme-linked immunosorbent assays (ELISA) are the predominant analytical tool used to measure levels of recombinant proteins expressed in genetically engineered (GE) plants. Recent advances in agricultural biotechnology have created a need to develop methods capable of selectively detecting and quantifying multiple proteins in complex matrices because of increasing numbers of transgenic proteins being coexpressed or "stacked" to achieve tolerance to multiple herbicides or to provide multiple modes of action for insect control. A multiplexing analytical method utilizing liquid chromatography with tandem mass spectrometry (LC-MS/MS) has been developed and validated to quantify three herbicide-tolerant proteins in soybean tissues: aryloxyalkanoate dioxygenase (AAD-12), 5-enol-pyruvylshikimate-3-phosphate synthase (2mEPSPS), and phosphinothricin acetyltransferase (PAT). Results from the validation showed high recovery and precision over multiple analysts and laboratories. Results from this method were comparable to those obtained with ELISA with respect to protein quantitation, and the described method was demonstrated to be suitable for multiplex quantitation of transgenic proteins in GE crops.

  3. Quantitation of low molecular weight sugars by chemical derivatization-liquid chromatography/multiple reaction monitoring/mass spectrometry.

    PubMed

    Han, Jun; Lin, Karen; Sequria, Carita; Yang, Juncong; Borchers, Christoph H

    2016-07-01

    A new method for the separation and quantitation of 13 mono- and disaccharides has been developed by chemical derivatization/ultra-HPLC/negative-ion ESI-multiple-reaction monitoring MS. 3-Nitrophenylhydrazine (at 50°C for 60 min) was shown to be able to quantitatively derivatize low-molecular weight (LMW) reducing sugars. The nonreducing sugar, sucrose, was not derivatized. A pentafluorophenyl-bonded phase column was used for the chromatographic separation of the derivatized sugars. This method exhibits femtomole-level sensitivity, high precision (CVs of ≤ 4.6%) and high accuracy for the quantitation of LMW sugars in wine. Excellent linearity (R(2) ≥ 0.9993) and linear ranges of ∼500-fold for disaccharides and ∼1000-4000-fold for monosaccharides were achieved. With internal calibration ((13) C-labeled internal standards), recoveries were between 93.6% ± 1.6% (xylose) and 104.8% ± 5.2% (glucose). With external calibration, recoveries ranged from 82.5% ± 0.8% (ribulose) to 105.2% ± 2.1% (xylulose). Quantitation of sugars in two red wines and two white wines was performed using this method; quantitation of the central carbon metabolism-related carboxylic acids and tartaric acid was carried out using a previously established derivatization procedure with 3-nitrophenylhydrazine as well. The results showed that these two classes of compounds-both of which have important organoleptic properties-had different compositions in red and white wines.

  4. Mass spectrometry guided structural biology.

    PubMed

    Liko, Idlir; Allison, Timothy M; Hopper, Jonathan Ts; Robinson, Carol V

    2016-10-01

    With the convergence of breakthroughs in structural biology, specifically breaking the resolution barriers in cryo-electron microscopy and with continuing developments in crystallography, novel interfaces with other biophysical methods are emerging. Here we consider how mass spectrometry can inform these techniques by providing unambiguous definition of subunit stoichiometry. Moreover recent developments that increase mass spectral resolution enable molecular details to be ascribed to unassigned density within high-resolution maps of membrane and soluble protein complexes. Importantly we also show how developments in mass spectrometry can define optimal solution conditions to guide downstream structure determination, particularly of challenging biomolecules that refuse to crystallise.

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

    PubMed

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

    2016-02-01

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

  6. Liquid chromatography tandem mass spectrometry method for the quantitation of mycophenolate mofetil in human plasma: Application to a bioequivalence study and metabolite identification.

    PubMed

    Partani, Pankaj; Verma, Saurabh Manaswita; Monif, Tausif

    2015-10-01

    We established a sensitive, selective, and rapid analytical method for the quantitation and pharmacokinetic investigation of mycophenolate mofetil in human plasma. To our knowledge, this is the first method that characterizes presence of mycophenolate mofetil glucuronide in clinical samples through tandem mass spectrometry detection and resolves mycophenolate mofetil from its glucuronide metabolite. Liquid chromatography coupled to tandem mass spectrometry detection in positive ion mode was selected to provide optimal selectivity and sensitivity. Due to the ionizable characteristics of the mycophenolate mofetil, a mixed-mode cation-exchange disposable extraction cartridge was prudently chosen. The chromatographic separation was achieved on Luna(®) C18(2) (100×4.60 mm) column using mobile phase consisting of a mixture of 1±0.05 mM ammonium formate in water, titrated to pH 3.1±0.1 with formic acid, and methanol (20:80, v/v), at a flow rate of 0.7 mL/min. The detection was led at m/z ratios of 434.4→ 114.2 and 438.4→ 118.3, for mycophenolate mofetil and mycophenolate mofetil-D4, respectively. The developed method was linear between 40.2-4986.0 pg/mL. All validation parameters were within the defined limits. The validated method was then successfully applied for the evaluation of bioequivalence parameters of mycophenolate mofetil after an oral administration of 500 mg mycophenolate mofetil tablet to healthy male Indian volunteers.

  7. Quantitative analysis of methadone and two major metabolites in hair by positive chemical ionization ion trap mass spectrometry.

    PubMed

    Wilkins, D G; Nagasawa, P R; Gygi, S P; Foltz, R L; Rollins, D E

    1996-10-01

    A sensitive and specific method for the quantitative determination of D,L-methadone (MD) and its metabolites, D,L-2-ethyl-1,5-dimethyl-3, 3-diphenylpyrrolinium (EDDP) and D,L-2-ethyl-5-methyl-3, 3-diphenyl-1-pyrroline (EMDP), in hair has been developed. Deuterated internal standards of MD, EMDP, and EDDP were added to 20-mg hair samples and digested overnight at room temperature with 1N sodium hydroxide. Calibration standards containing known concentrations of MD, EMDP, and EDDP dried onto human hair were also digested. Digest solutions were extracted by a liquid-liquid extraction procedure and analyzed with splitless injection on a Finnigan MagnumTM ion trap mass spectrometer. Chromatographic separation was achieved with helium carrier gas on a DB-5MS-30M-0.25-micron capillary column. Positive chemicaionization was used with acetone as the reagent gas. The assay was linear from 0.5 ng/mg (MD and EDDP) or 1.0 ng/mg (EMDP) to 50.0 ng/mg of human hair with correlation coefficients greater than 0.99. Intra-assay and interassay coefficients of variation were determined to be less than 20% for all three analytes at 2.0 and 10.0 ng/mg of hair. Recovery was estimated to be greater than 70% (MD and EDDP) and 53% (EMDP) at 2.0 and 10.0 ng/mg of hair. The method has been applied to the analysis of both human and rat hair. Male long-Evans rats were shaved prior to dosing to obtain their drug-free hair. Animals were then administered 15 mg/kg MD by intraperitoneal injection daily for five days. Fourteen days after the first dose, hair was collected and analyzed for MD, EMDP, and EDDP. The mean plus standard error of the mean (SEM; n = 3) concentrations of MD and EDDP in pigmented hair were 31.1 ng/mg +/- 9.6 ng/mg and 8.6 +/- 2.4 ng/mg, respectively. EMDP was detected in the hair of one of three rats. In another experiment, hair was collected from two human subjects who had received long-term methadone therapy for the treatment of heroin addiction. Subject A received 60 mg of

  8. Protein composition of wheat gluten polymer fractions determined by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flour proteins from the US bread wheat Butte 86 were extracted in 0.5% SDS using a two-step procedure with and without sonication and further separated by size exclusion chromatography into monomeric and polymeric fractions. Proteins in each fraction were analyzed by quantitative two-dimensional gel...

  9. Rapid quantitative analysis of carnitine and acylcarnitines by ultra-high performance-hydrophilic interaction liquid chromatography-tandem mass spectrometry.

    PubMed

    Kivilompolo, Maarit; Öhrnberg, Leena; Orešič, Matej; Hyötyläinen, Tuulia

    2013-05-31

    l-Carnitine and its acyl esters (acylcarnitines) play an important role in the metabolism of fatty acids. However, most of the present methods for the quantitative analysis of acylcarnitines have restrictions both in sample preparation and in chromatographic separation. Herein we present a validated method for determination of carnitine and eleven acylcarnitines in human serum and rat tissue biopsies by using ultra-high performance-hydrophilic interaction liquid chromatography-tandem mass spectrometry (UHP-HILIC-MS/MS). The procedure uses minimal sample preparation including only addition of organic solvent, labeled internal standard, incubation and centrifugation. The separation is performed without derivatization or addition of ion-pairing reagent within 7min on a hydrophilic interaction liquid chromatographic column with mass spectrometric detection. The method is linear in response over the concentration range from 20 to 600ng/ml for carnitine and acetylcarnitine and 5-200ng/ml for the other acylcarnitines, with correlation coefficients higher than 0.994. Recoveries were higher than 88% for most of the compounds. Limits of detection were 5ng/ml for carnitine and acetylcarnitine and approximately 0.5ng/ml for other acylcarnitines. The method was applied to the analysis of serum and tissue samples.

  10. Quantitative determination of total L-carnitine in infant formula, follow-up formula, and raw materials by liquid chromatography with tandem mass spectrometry.

    PubMed

    Nadaoka, Isao; Hatakeyama, Emi; Tanada, Chihiro; Sakamoto, Tasuku; Fukaya, Shinichi; Akiba, Takashi; Inoue, Koichi; Yamano, Yutaka; Toyo'oka, Toshimasa

    2014-01-01

    We developed a rapid and useful routine screening assay for total L-carnitine content in various infant formulas and materials by liquid chromatography coupled with a tandem mass spectrometric method (LC-MS/MS) and alkaline hydrolysis. For separation of L-carnitine, a multi-mode octadecylsilane (ODS) column was used that contained ODS ligands, anion ligands, and cation ligands to avoid using ion-pairing agents. The stable isotope L-carnitine-d3 (m/z 165 → 103/85) was used in electrospray MS/MS in the multiple reaction monitoring mode with the ion transitions of m/z 162 → 103/85 for detection and quantitation of L-carnitine. Alkaline hydrolysis of short/medium chain (C2 - C15) acyl-L-carnitines in infant formula was analyzed by LC with quadrupole time-of-flight mass spectrometry (QTOF-MS). The majority of short/medium chain acyl-L-carnitines were hydrolyzed to free L-carnitine. The overall standard deviations for L-carnitine in infant formula, follow-up formula and raw materials ranged from 2.1 to 4.0. The overall mean recoveries ranged from 90.2 to 94.2%.

  11. Quantitative analysis of a novel HIV fusion inhibitor (sifuvirtide) in HIV infected human plasma using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.

    PubMed

    Che, Jinjing; Meng, Qingfang; Chen, Zhihang; Hou, Yunan; Shan, Chengqi; Cheng, Yuanguo

    2010-03-11

    A sensitive method for measuring sifuvirtide, a novel HIV fusion inhibitor peptide drug in HIV-1(+) human plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The plasma samples were treated by solvent/detergent (S/D) method to inactivate viral activity before analysis. After protein precipitation sifuvirtide was determined by LC-MS/MS. A structure analog was used as internal standard (IS). The mass spectrometer was operated in positive ion and multiple reaction monitoring mode with transitions m/z 946.3-->159.0 for sifuvirtide and 951.7-->159.2 for IS. The intra-day precision ranged from 2.74% to 7.57% with accuracy from 91.63% to 102.53%. The inter-day precision ranged from 2.65% to 3.58% and the accuracy from 95.53% to 105.28%. Stability studies showed that sifuvirtide was stable both during the assay procedure and long-term storage. The lower limit of quantitation (LLOQ) was 9.75ngml(-1). The method was used for analyzing samples from phase IIa clinical study of sifuvirtide in China.

  12. Profiling the iron, copper and zinc content in primary neuron and astrocyte cultures by rapid online quantitative size exclusion chromatography-inductively coupled plasma-mass spectrometry.

    PubMed

    Hare, Dominic J; Grubman, Alexandra; Ryan, Timothy M; Lothian, Amber; Liddell, Jeffrey R; Grimm, Rudolf; Matsuda, Toshiaki; Doble, Philip A; Cherny, Robert A; Bush, Ashley I; White, Anthony R; Masters, Colin L; Roberts, Blaine R

    2013-12-01

    Metals often determine the chemical reactivity of the proteins to which they are bound. Each cell in the body tightly maintains a unique metalloproteomic profile, mostly dependent on function. This paper describes an analytical online flow injection quantitative size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) method, which was applied to profiling the metal-binding proteins found in primary cultures of neurons and astrocytes. This method can be conducted using similar amounts of sample to those used for Western blotting (20-150 μg protein), and has a turnaround time of <15 minutes. Metalloprotein standards for Fe (as ferritin), Cu and Zn (as superoxide dismutase-1) were used to construct multi-point calibration curves for online quantification of metalloproteins by SEC-ICP-MS. Homogenates of primary neuron and astrocyte cultures were analysed by SEC-ICP-MS. Online quantification by external calibration with metalloprotein standards determined the mass of metal eluting from the column relative to time (as pg s(-1)). Total on-column Fe, Cu and Zn detection limits ranged from 0.825 ± 0.005 ng to 13.6 ± 0.7 pg. Neurons and astrocytes exhibited distinct metalloprotein profiles, featuring both ubiquitous and unique metalloprotein species. Separation and detection by SEC-ICP-MS allows appraisal of these metalloproteins in their native state, and online quantification was achieved using this relatively simple external calibration process.

  13. Effect of mobile phase additives on qualitative and quantitative analysis of ginsenosides by liquid chromatography hybrid quadrupole-time of flight mass spectrometry.

    PubMed

    Liang, Yan; Guan, Tianye; Zhou, Yuanyuan; Liu, Yanna; Xing, Lu; Zheng, Xiao; Dai, Chen; Du, Ping; Rao, Tai; Zhou, Lijun; Yu, Xiaoyi; Hao, Kun; Xie, Lin; Wang, Guangji

    2013-07-05

    This study was to systematically investigate the effect of mobile phase additives, including ammonia water, formic acid, acetic acid, ammonium chloride and water (as a control), on qualitative and quantitative analysis of fifteen representative ginsenosides based on liquid chromatography hybrid quadrupole-time of flight mass spectrometry (LC-Q-TOF/MS). To evaluate the influence of mobile phase additives on qualitative performance, the quality of the negative mode MS/MS spectra of ginsenosides produced by online LC-Q-TOF/MS analyses, particularly the numbers and intensities of fragment ions, were compared under different adduct ion states, and found to be strongly affected by the mobile phase additives. When 0.02% acetic acid was added in the mobile phase, the deprotonated ginsenosides ions produced the most abundant product ions, while almost no product ion was observed for the chlorinated ginsenoside ions when 0.1mM ammonium chloride was used as the mobile phase additive. On the other hand, sensitivity, linear range and precision were adopted to investigate the quantitative performance affected by different mobile phase additives. Validation results of the LC-Q-TOF/MS-based quantitative performance for ginsenosides showed that ammonium chloride not only provided the highest sensitivity for all the target analytes, but also dramatically improved the linear ranges, the intra-day and inter-day precisions comparing to the results obtained using other mobile phase additives. Importantly, the validated method, using 0.1mM ammonium chloride as the mobile phase additive, was successfully applied to the quantitative analysis of ginsenosides in rat plasma after intragastric administration of Ginsenoside Extract at 200mg/kg. In conclusion, 0.02% acetic acid was deemed to be the most suitable mobile phase additive for qualitative analysis of ginsenosides, and 0.1mM ammonium chloride in mobile phase could lead to the best quantitative performance. Our results reveal that

  14. Quantitative imaging of magnesium distribution at single-cell resolution in brain tumors and infiltrating tumor cells with secondary ion mass spectrometry (SIMS)

    PubMed Central

    Chandra, Subhash; Parker, Dylan J.; Barth, Rolf F.; Pannullo, Susan C.

    2016-01-01

    Glioblastoma multiforme (GBM) is one of the deadliest forms of human brain tumors. The infiltrative pattern of growth of these tumors includes the spread of individual and/or clusters of tumor cells at some distance from the main tumor mass in parts of the brain protected by an intact blood-brain-barrier. Pathophysiological studies of GBM could be greatly enhanced by analytical techniques capable of in situ single-cell resolution measurements of infiltrating tumor cells. Magnesium homeostasis is an area of active investigation in high grade gliomas. In the present study, we have used the F98 rat glioma as a model of human GBM and an elemental/isotopic imaging technique of secondary ion mass spectrometry (SIMS), a CAMECA IMS-3f ion microscope, for studying Mg distributions with single-cell resolution in freeze-dried brain tissue cryosections. Quantitative observations were made on tumor cells in the main tumor mass, contiguous brain tissue, and infiltrating tumor cells in adjacent normal brain. The brain tissue contained a significantly lower total Mg concentration of 4.70 ± 0.93 mmol/Kg wet weight (mean ± SD) in comparison to 11.64 ± 1.96 mmol/Kg wet weight in tumor cells of the main tumor mass and 10.72 ± 1.76 mmol/Kg wet weight in infiltrating tumor cells (p<0.05). The nucleus of individual tumor cells contained elevated levels of bound Mg. These observations demonstrate enhanced Mg-influx and increased binding of Mg in tumor cells and provide strong support for further investigation of GBMs for altered Mg homeostasis and activation of Mg-transporting channels as possible therapeutic targets. PMID:26703785

  15. Proteome-wide measurement of non-canonical bacterial mistranslation by quantitative mass spectrometry of protein modifications

    PubMed Central

    Cvetesic, Nevena; Semanjski, Maja; Soufi, Boumediene; Krug, Karsten; Gruic-Sovulj, Ita; Macek, Boris

    2016-01-01

    The genetic code is virtually universal in biology and was likely established before the advent of cellular life. The extent to which mistranslation occurs is poorly understood and presents a fundamental question in basic research and production of recombinant proteins. Here we used shotgun proteomics combined with unbiased protein modification analysis to quantitatively analyze in vivo mistranslation in an E. coli strain with a defect in the editing mechanism of leucyl-tRNA synthetase. We detected the misincorporation of a non-proteinogenic amino acid norvaline on 10% of all measured leucine residues under microaerobic conditions and revealed preferential deployment of a tRNALeu(CAG) isoacceptor during norvaline misincorporation. The strain with the norvalylated proteome demonstrated a substantial reduction in cell fitness under both prolonged aerobic and microaerobic cultivation. Unlike norvaline, isoleucine did not substitute for leucine even under harsh error-prone conditions. Our study introduces shotgun proteomics as a powerful tool in quantitative analysis of mistranslation. PMID:27377007

  16. Relative quantitation of protein nitration by liquid chromatography–mass spectrometry using isotope-coded dimethyl labeling and chemoprecipitation

    PubMed Central

    Guo, Jia; Prokai-Tatrai, Katalin; Prokai, Laszlo

    2012-01-01

    Protein nitration has been recognized as an important biomarker for nitroxidative stress associated with various diseases. While identification of protein targets for nitration is important, its quantitative profiling also is necessary to understand the biological impact of this low-abundance posttranslational modification. We have previously reported an efficient and straightforward enrichment method for nitropeptides to reduce sample complexity and permit unambiguous site-specific identifications by LC–MS analyses. This approach relies on two chemical derivatization steps: specifically reductive methylation of aliphatic amines and, then, conversion of nitrotyrosines to the corresponding aminotyrosines before their selective capture by a solid-phase reagent we introduced previously. Hence, the method inherently offers the opportunity for relative quantitation of nitropeptides by using isotopic variants of formaldehyde for reductive methylation. This simple method was tested via LC–MS analyses of differently N-methylated nitropeptides and nitroubiquitin as a model nitroprotein enriched from human serum albumin digest and from human plasma, respectively. PMID:22285050

  17. Use of SWATH mass spectrometry for quantitative proteomic investigation of Shewanella oneidensis MR-1 biofilms grown on graphite cloth electrodes.

    PubMed

    Grobbler, Christy; Virdis, Bernardino; Nouwens, Amanda; Harnisch, Falk; Rabaey, Korneel; Bond, Philip L

    2015-03-01

    Quantitative proteomics from low biomass, biofilm samples is not well documented. In this study we show successful use of SWATH-MS for quantitative proteomic analysis of a microbial electrochemically active biofilm. Shewanella oneidensis MR-1 was grown on carbon cloth electrodes under continuous anodic electrochemical polarizations in a bioelectrochemical system (BES). Using lactate as the electron donor, anodes serving as terminal microbial electron acceptors were operated at three different electrode potentials (+0.71 V, +0.21 V & -0.19 V vs. SHE) and the development of catalytic activity was monitored by measuring the current traces over time. Once maximum current was reached (usually within 21-29 h) the electrochemical systems were shut off and biofilm proteins were extracted from the electrodes for proteomic assessment. SWATH-MS analysis identified 704 proteins, and quantitative comparison was made of those associated with tricarboxcylic acid (TCA) cycle. Metabolic differences detected between the biofilms suggested a branching of the S. oneidensis TCA cycle when grown at the different electrode potentials. In addition, the higher abundance of enzymes involved in the TCA cycle at higher potential indicates an increase in metabolic activity, which is expected given the assumed higher energy gains. This study demonstrates high numbers of identifications on BES biofilm samples can be achieved in comparison to what is currently reported. This is most likely due to the minimal preparation steps required for SWATH-MS.

  18. Comprehensive quantitative analysis of Chinese patent drug YinHuang drop pill by ultra high-performance liquid chromatography quadrupole time of flight mass spectrometry.

    PubMed

    Wong, Tin-Long; An, Ya-Qi; Yan, Bing-Chao; Yue, Rui-Qi; Zhang, Tian-Bo; Ho, Hing-Man; Ren, Tian-Jing; Fung, Hau-Yee; Ma, Dik-Lung; Leung, Chung-Hang; Liu, Zhong-Liang; Pu, Jian-Xin; Han, Quan-Bin; Sun, Han-Dong

    2016-06-05

    YinHuang drop pill (YHDP) is a new preparation, derived from the traditional YinHuang (YH) decoction. Since drop pills are one of the newly developed forms of Chinese patent drugs, not much research has been done regarding the quality and efficacy. This study aims to establish a comprehensive quantitative analysis of the chemical profile of YHDP. ultra high-performance liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-Q-TOF-MS/MS) was used to identify 34 non-sugar small molecules including 15 flavonoids, 9 phenolic acids, 5 saponins, 1 iridoid, and 4 iridoid glycosides in YHDP samples, and 26 of them were quantitatively determined. Sugar composition of YHDP in terms of fructose, glucose and sucrose was examined via a high performance liquid chromatography-evaporative light scattering detector on an amide column (HPLC-NH2P-ELSD). Macromolecules were examined by high performance gel permeation chromatography coupled with ELSD (HPGPC-ELSD). The content of the drop pill's skeleton component PEG-4000 was also quantified via ultra-high performance liquid chromatography coupled with charged aerosol detector (UHPLC-CAD). The results showed that up to 73% (w/w) of YHDP could be quantitatively determined. Small molecules accounted for approximately 5%, PEG-4000 represented 68%, while no sugars or macromolecules were found. Furthermore, YHDP showed no significant differences in terms of daily dosage, compared to YinHuang granules and YinHuang oral liquid; however, it has a higher small molecules content compared to YinHuang lozenge.

  19. Quantitative analysis of fragrance in selectable one dimensional or two dimensional gas chromatography-mass spectrometry with simultaneous detection of multiple detectors in single injection.

    PubMed

    Tan, Hui Peng; Wan, Tow Shi; Min, Christina Liew Shu; Osborne, Murray; Ng, Khim Hui

    2014-03-14

    A selectable one-dimensional ((1)D) or two-dimensional ((2)D) gas chromatography-mass spectrometry (GC-MS) system coupled with flame ionization detector (FID) and olfactory detection port (ODP) was employed in this study to analyze perfume oil and fragrance in shower gel. A split/splitless (SSL) injector and a programmable temperature vaporization (PTV) injector are connected via a 2-way splitter of capillary flow technology (CFT) in this selectable (1)D/(2)D GC-MS/FID/ODP system to facilitate liquid sample injections and thermal desorption (TD) for stir bar sorptive extraction (SBSE) technique, respectively. The dual-linked injectors set-up enable the use of two different injector ports (one at a time) in single sequence run without having to relocate the (1)D capillary column from one inlet to another. Target analytes were separated in (1)D GC-MS/FID/ODP and followed by further separation of co-elution mixture from (1)D in (2)D GC-MS/FID/ODP in single injection without any instrumental reconfiguration. A (1)D/(2)D quantitative analysis method was developed and validated for its repeatability - tR; calculated linear retention indices (LRI); response ratio in both MS and FID signal, limit of detection (LOD), limit of quantitation (LOQ), as well as linearity over a concentration range. The method was successfully applied in quantitative analysis of perfume solution at different concentration level (RSD≤0.01%, n=5) and shower gel spiked with perfume at different dosages (RSD≤0.04%, n=5) with good recovery (96-103% for SSL injection; 94-107% for stir bar sorptive extraction-thermal desorption (SBSE-TD).

  20. Imaging mass spectrometry in microbiology

    PubMed Central

    Watrous, Jeramie D.; Dorrestein, Pieter C.

    2013-01-01

    Mass spectrometry tools which allow for the 2-D visualization of the distribution of trace metals, metabolites, surface lipids, peptides and proteins directly from biological samples without the need for chemical tagging or antibodies are becoming increasingly useful for microbiology applications. These tools, comprised of different imaging mass spectrometry techniques, are ushering in an exciting new era of discovery by allowing for the generation of chemical hypotheses based on of the spatial mapping of atoms and molecules that can correlate to or transcend observed phenotypes. In this review, we explore the wide range of imaging mass spectrometry techniques available to microbiologists and describe their unique applications to microbiology with respect to the types of microbiology samples to be investigated. PMID:21822293

  1. Symposium on accelerator mass spectrometry

    SciTech Connect

    1981-01-01

    The area of accelerator mass spectrometry has expanded considerably over the past few years and established itself as an independent and interdisciplinary research field. Three years have passed since the first meeting was held at Rochester. A Symposium on Accelerator Mass Spectrometry was held at Argonne on May 11-13, 1981. In attendance were 96 scientists of whom 26 were from outside the United States. The present proceedings document the program and excitement of the field. Papers are arranged according to the original program. A few papers not presented at the meeting have been added to complete the information on the status of accelerator mass spectrometry. Individual papers were prepared separately for the data base.

  2. Laser diode thermal desorption atmospheric pressure chemical ionization tandem mass spectrometry applied for the ultra-fast quantitative analysis of BKM120 in human plasma.

    PubMed

    Lanshoeft, Christian; Heudi, Olivier; Leuthold, Luc Alexis; Schlotterbeck, Götz; Elbast, Walid; Picard, Franck; Kretz, Olivier

    2014-09-01

    A sensitive and ultra-fast method utilizing the laser diode thermal desorption ion source using atmospheric pressure chemical ionization coupled to tandem mass spectrometry (LDTD-APCI-MS/MS) was developed for the quantitative analysis of BKM120, an investigational anticancer drug in human plasma. Samples originating from protein precipitation (PP) followed by salting-out assisted liquid-liquid extraction (SALLE) were spotted onto the LazWell™ plate prior to their thermal desorption and detection by tandem mass spectrometry in positive mode. The validated method described in this paper presents a high absolute extraction recovery (>90 %) for BKM120 and its internal standard (ISTD) [D8]BKM120, with precision and accuracy meeting the acceptance criteria. Standard curves were linear over the range of 5.00 to 2000 ng mL(-1) with a coefficient of determination (R (2)) >0.995. The method specificity was demonstrated in six different batches of human plasma. Intra- and inter-run precision as well as accuracy within ±20 % at the lower limit of quantification (LLOQ) and ±15 % (other levels) were achieved during a three-run validation for quality control (QC) samples. The post-preparative stability on the LazWell™ plate at room temperature was 72 h and a 200-fold dilution of spiked samples was demonstrated. The method was applied successfully to three clinical studies (n = 847) and cross-checked with the validated LC-ESI-MS/MS reference method. The sample analysis run time was 10 s as compared to 4.5 min for the current validated LC-ESI-MS/MS method. The resultant data were in agreement with the results obtained using the validated reference LC-ESI-MS/MS assay and the same pharmacokinetic (PK) parameters were calculated for both analytical assays. This work demonstrates that LDTD-APCI-MS/MS is a reliable method for the ultra-fast quantitative analysis of BKM120 which can be used to speed-up and support its bioanalysis in the frame of the clinical trials.

  3. Identification and Quantitation of Various Inositols and O-methylinositols Present in Plant Roots Using Gas Chromatograpghy/Mass Spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many inositols and O-methylinositols serve important roles in medicine and plant biology. A simple method was developed for the identification of these compounds in plant roots by extracting with 80% ethanol, derivatizing with trimethylsilyl imidazole, and analyzing by gas chromatography/mass spect...

  4. Novel mass spectrometry imaging software assisting labeled normalization and quantitation of drugs and neuropeptides directly in tissue sections.

    PubMed

    Källback, Patrik; Shariatgorji, Mohammadreza; Nilsson, Anna; Andrén, Per E

    2012-08-30

    MALDI MS imaging has been extensively used to produce qualitative distribution maps of proteins, peptides, lipids, small molecule pharmaceuticals and their metabolites directly in biological tissue sections. There is growing demand to quantify the amount of target compounds in the tissue sections of different organs. We present a novel MS imaging software including protocol for the quantitation of drugs, and for the first time, an endogenous neuropeptide directly in tissue sections. After selecting regions of interest on the tissue section, data is read and processed by the software using several available methods for baseline corrections, subtractions, denoising, smoothing, recalibration and normalization. The concentrations of in vivo administered drugs or endogenous compounds are then determined semi-automatically using either external standard curves, or by using labeled compounds, i.e., isotope labeled analogs as standards. As model systems, we have quantified the distribution of imipramine and tiotropium in the brain and lung of dosed rats. Substance P was quantified in different mouse brain structures, which correlated well with previously reported peptide levels. Our approach facilitates quantitative data processing and labeled standards provide better reproducibility and may be considered as an efficient tool to quantify drugs and endogenous compounds in tissue regions of interest.

  5. Quantitative mass barcode-like image of nicotine in single longitudinally sliced hair sections from long-term smokers by matrix-assisted laser desorption time-of-flight mass spectrometry imaging.

    PubMed

    Nakanishi, Toyofumi; Nirasawa, Takashi; Takubo, Takayuki

    2014-01-01

    The matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometric technique (IMS) offered a new breakthrough perspective in the analysis of drug abuse in forensic science; however, it only produced barcode-like images, semi-quantitative analysis. In order to develop intermittent monitoring by this IMS for forensic and medical sciences, it is important to quantitatively measure the contents of longitudinally sliced hair sections. We developed quantitative imaging mass spectrometry (QIMS) of nicotine (NC) in longitudinally sliced hairs by MALDI-IMS with the selected reaction monitoring mode using a labeled NC ((13)C3-NC) standard for the serially chronological monitoring and traceability of NC intake in heavy smokers. The calibration curve of NC/(13)C3-NC was virtually a linear equation at ranges from 1 to 50 ng/mL, the slope was 0.020, and the intercept was almost 0.023 and the R(2) was 0.9965. The limit of quantitation of NC was calculated as 1.6 ng/mg hair (an average weight of the hair would be assumed 0.06 mg/cm) by QIMS. Moreover, NC concentrations in two separate heavy smokers (n = 3) were 8.5 ± 1.2 and 34.5 ± 2.8 ng/mg hair, respectively, and covariations were ∼10% using a single hair. Quantitative mass barcode-like image of sliced section of hair allowed for the quantitative assessment of NC concentrations in long-term smokers similar to drugs and medicines during drug histories.

  6. Quantitation of tacrolimus in whole blood using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS).

    PubMed

    Donaldson, Keri J; Shaw, Leslie M

    2010-01-01

    We describe a multiple reaction monitoring positive ion HPLC/tandem mass spectrometric method for quantification of tacrolimus in human whole blood with online extraction and cleanup. Included in this procedure: API 2000 triple quadrupole mass spectrometer with turbo-ion spray source (Applied Biosystems, Foster City, CA); 10-port diverter/switching valve (Valco, Houston, TX); HPLC system (Agilent Technologies series 1100, Wilmington, DE); 10 mm (C(18)) guard cartridge (Perkin Elmer, Norwalk, CT) used as an extraction column; a Nova-Pak C18 analytical column (2.1 x 150 mm I.D., 4 microm, Waters Corp, Milford, MA); washing solution, methanol: 30 mM ammonium acetate pH 5.1 (80:20); eluting solution, methanol:30 mM ammonium acetate pH 5.1 (97:3); flow rate 0.8 mL/min; and a run-time of 2.8 min. The first and third quadrupoles were set to detect the ammonium adduct ion and a high mass fragment of tacrolimus (m/z 821.5-->768.3), and of an internal standard (ascomycin) (m/z 901.8-->834.4). The lower limit of quantification of this method is 3.75 mg/L. The concentration of drug is determined by comparing peak-area ratios for tacrolimus and internal standard to a standard curve constructed using non-weighted linear through zero regression.

  7. Liquid chromatography tandem mass spectrometric quantitation of sulfamethazine and its metabolites: direct analysis of swine urine by triple quadrupole and by ion trap mass spectrometry.

    PubMed

    Bartolucci, G; Pieraccini, G; Villanelli, F; Moneti, G; Triolo, A

    2000-01-01

    This work describes a new method for the quantitation of trace amounts of sulfamethazine (SMZ) and its main metabolite, N4-acetylsulfamethazine (Ac-SMZ), in swine urine, using high-performance liquid chromatography (HPLC) tandem mass spectrometric analysis of crude urine after addition of internal standard and simple dilution with water. The aim was to determine whether residues of this sulfamidic drug, normally administered to swine in order to prevent infectious diseases, were present in urine at levels lower than those permitted by regulatory authorities before human consumption (EU Project SMT, contract number CT 96-2092). A 10 microL volume of diluted urine was injected into a very short, narrow-bore chromatographic column (Zorbax SB-C18 2.1 i. d. x30 mm length, 3.5 microm pore size). Elution of the analytes of interest was achieved in less than seven minutes using a rapid gradient (from 20 to 80% methanol in 3 minutes). Either a PE Sciex API 365 triple quadrupole (QqQ), operated in the selected reaction monitoring (SRM) mode, or a Finnigan LCQ ion trap (IT) mass spectrometer, operated in narrow-range product ion scan, was used as the final detector. Electrospray (ESI) was used as the ionization technique. A comparison of the two tandem mass spectrometers was performed by analyzing the same set of test samples, at three concentration levels, on three different days. Linearity of responses of the calibration standards, intra- and inter-assay precision of the samples, specificity and limits of detection were evaluated for both systems. Both the QqQ and the IT instrument was suitable for rapid, sensitive and specific determination of the analytes, although the overall performance of the QqQ was slightly superior in terms of linearity, precision and sensitivity.

  8. Ion Mobility Spectrometry (IMS) and Mass Spectrometry

    SciTech Connect

    Shvartsburg, Alexandre A.

    2010-04-20

    In a media of finite viscosity, the Coulomb force of external electric field moves ions with some terminal speed. This dynamics is controlled by “mobility” - a property of the interaction potential between ions and media molecules. This fact has been used to separate and characterize gas-phase ions in various modes of ion mobility spectrometry (IMS) developed since 1970. Commercial IMS devices were introduced in 1980-s for field detection of volatile traces such as explosives and chemical warfare agents. Coupling to soft-ionization sources, mass spectrometry (MS), and chromatographic methods in 1990-s had allowed IMS to handle complex samples, enabling new applications in biological and environmental analyses, nanoscience, and other areas. Since 2003, the introduction of commercial systems by major instrument vendors started bringing the IMS/MS capability to broad user community. The other major development of last decade has been the differential IMS or “field asymmetric waveform IMS” (FAIMS) that employs asymmetric time-dependent electric field to sort ions not by mobility itself, but by the difference between its values in strong and weak electric fields. Coupling of FAIMS to conventional IMS and stacking of conventional IMS stages have enabled two-dimensional separations that dramatically expand the power of ion mobility methods.

  9. Detection and quantitation of trace phenolphthalein (in pharmaceutical preparations and in forensic exhibits) by liquid chromatography-tandem mass spectrometry, a sensitive and accurate method.

    PubMed

    Sharma, Kakali; Sharma, Shiba P; Lahiri, Sujit C

    2013-01-01

    Phenolphthalein, an acid-base indicator and laxative, is important as a constituent of widely used weight-reducing multicomponent food formulations. Phenolphthalein is an useful reagent in forensic science for the identification of blood stains of suspected victims and for apprehending erring officials accepting bribes in graft or trap cases. The pink-colored alkaline hand washes originating from the phenolphthalein-smeared notes can easily be determined spectrophotometrically. But in many cases, colored solution turns colorless with time, which renders the genuineness of bribe cases doubtful to the judiciary. No method is known till now for the detection and identification of phenolphthalein in colorless forensic exhibits with positive proof. Liquid chromatography-tandem mass spectrometry had been found to be most sensitive, accurate method capable of detection and quantitation of trace phenolphthalein in commercial formulations and colorless forensic exhibits with positive proof. The detection limit of phenolphthalein was found to be 1.66 pg/L or ng/mL, and the calibration curve shows good linearity (r(2) = 0.9974).

  10. Quantitative imaging of 2 nm monolayer-protected gold nanoparticle distributions in tissues using laser ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS).

    PubMed

    Elci, S Gokhan; Yan, Bo; Kim, Sung Tae; Saha, Krishnendu; Jiang, Ying; Klemmer, Gunnar A; Moyano, Daniel F; Tonga, Gulen Yesilbag; Rotello, Vincent M; Vachet, Richard W

    2016-04-21

    Functionalized gold nanoparticles (AuNPs) have unique properties that make them important biomedical materials. Optimal use of these materials, though, requires an understanding of their fate in vivo. Here we describe the use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to image the biodistributions of AuNPs in tissues from mice intravenously injected with AuNPs. We demonstrate for the first time that the distributions of very small (∼2 nm core) monolayer-protected AuNPs can be imaged in animal tissues at concentrations in the low parts-per-billion range. Moreover, the LA-ICP-MS images reveal that the monolayer coatings on the injected AuNPs influence their distributions, suggesting that the AuNPs remain intact in vivo and their surface chemistry influences how they interact with different organs. We also demonstrate that quantitative images of the AuNPs can be generated when the appropriate tissue homogenates are chosen for matrix matching. Overall, these results demonstrate the utility of LA-ICP-MS for tracking the fate of biomedically-relevant AuNPs in vivo, facilitating the design of improved AuNP-based therapeutics.

  11. Quantitative label-free mass spectrometry analysis of formalin-fixed, paraffin-embedded tissue representing the invasive cutaneous malignant melanoma proteome

    PubMed Central

    Dowling, Paul; Moran, Benvon; McAuley, Edel; Meleady, Paula; Henry, Michael; Clynes, Martin; McMenamin, Mairin; Leonard, Niamh; Monks, Mary; Wynne, Bairbre; Ormond, Patrick; Larkin, Annemarie

    2016-01-01

    Understanding the events at a protein level that govern the progression from melanoma in situ to invasive melanoma are important areas of current research to be developed. Recent advances in the analysis of formalin-fixed, paraffin-embedded tissue by proteomics, particularly using the filter-aided sample preparation protocol, has opened up the possibility of studying vast archives of clinical material and associated medical records. In the present study, quantitative protein profiling was performed using tandem mass spectrometry, and the proteome differences between melanoma in situ and invasive melanoma were compared. Biological pathway analyses revealed several signalling pathways differing between melanoma in situ and invasive melanoma, including metabolic pathways and the phosphoinositide 3-kinase-Akt signalling pathway. Selected proteins of interest (14–3-3ε and fatty acid synthase) were subsequently investigated using immunohistochemical analysis of tissue microarrays. Identifying the key proteins that play significant roles in the establishment of a more invasive phenotype in melanoma may ultimately aid diagnosis and treatment decisions. PMID:27899996

  12. A novel quantitation approach for maximizing detectable targets for offensive/volatile odorants with diverse functional groups by thermal desorption-gas chromatography-mass spectrometry

    PubMed Central

    Kim, Yong-Hyun; Kim, Ki-Hyun

    2016-01-01

    A multitude of analytical systems are needed to analyze diverse odorants with various functionalities. In this study, an experimental method was developed to assess the maximum covering range of odorants using a single experimental setup consisting of a thermal desorber-gas chromatography-mass spectrometry system. To this end, a total of 20 offensive odorants (aldehyde, ketone, ester, alcohol, aromatic, sulfide, amine, and carboxyl) were selected and tested by a single system. The analytical results of standards and environmental samples were evaluated in a number of respects. In the analysis of the standards, all targets were quantified via Carbopack (C + B + X) tube sampling while operating the thermal desorber at −25 °C. The method detection limits of 18 targets (exception of 2 out of the 20 targets: acetaldehyde and methanethiol) were excellent (mean 0.04 ± 0.03 ppb) in terms of their odor threshold values (74.7 ± 140 ~ 624 ± 1,729 ppb). The analysis of organic fertilizer plant samples at a pig farm (slurry treatment facility, compost facility, and ambient air) confirmed the presence of 18 odorants from 0.03 ppb (dimethyldisulfide, ambient sample) to 522 ppb (methyl ethyl ketone, slurry treatment facility). As such, our method allowed simultaneous quantitation of most key odorants with sufficient reliability and sensitivity. PMID:27404037

  13. Differential quantitative proteomics of Porphyromonas gingivalis by linear ion trap mass spectrometry: Non-label methods comparison, q-values and LOWESS curve fitting

    NASA Astrophysics Data System (ADS)

    Xia, Qiangwei; Wang, Tiansong; Park, Yoonsuk; Lamont, Richard J.; Hackett, Murray

    2007-01-01

    Differential analysis of whole cell proteomes by mass spectrometry has largely been applied using various forms of stable isotope labeling. While metabolic stable isotope labeling has been the method of choice, it is often not possible to apply such an approach. Four different label free ways of calculating expression ratios in a classic "two-state" experiment are compared: signal intensity at the peptide level, signal intensity at the protein level, spectral counting at the peptide level, and spectral counting at the protein level. The quantitative data were mined from a dataset of 1245 qualitatively identified proteins, about 56% of the protein encoding open reading frames from Porphyromonas gingivalis, a Gram-negative intracellular pathogen being studied under extracellular and intracellular conditions. Two different control populations were compared against P. gingivalis internalized within a model human target cell line. The q-value statistic, a measure of false discovery rate previously applied to transcription microarrays, was applied to proteomics data. For spectral counting, the most logically consistent estimate of random error came from applying the locally weighted scatter plot smoothing procedure (LOWESS) to the most extreme ratios generated from a control technical replicate, thus setting upper and lower bounds for the region of experimentally observed random error.

  14. Quantitative profiling of PrP(Sc) peptides by high-performance liquid chromatography mass spectrometry to investigate the diversity of prions.

    PubMed

    Gielbert, Adriana; Davis, Linda A; Sayers, A Robin; Tang, Yue; Hope, James; Sauer, Maurice J

    2013-05-01

    Prions are proteins that can exist in two (or more) folding states, a normal or cellular form and a series of infectious or prion forms, which are prone to aggregate. The prion form can induce conversion of the cellular form and so transmit phenotypic effects of this structural rearrangement within and between cells and organisms. The conversion of PrP(C), the mammalian prion glycoprotein, to its prion form, PrP(Sc), in the brain is a precursor to progressive neurological degeneration, and the various folded forms of PrP(Sc) (defined by the size and glycosylation of protease-resistant core peptides of the PrP aggregates, PrP(res)) are characteristic of a particular neurodegenerative phenotype or prion disease. Here, quantitative multiplex mass spectrometry was used for N-terminal amino acid profiling (N-TAAP) of PrP(res) from sheep affected by scrapie, the prion disease of small ruminants, to rapidly assess the diversity of prions within particular flocks. In 29 cases, PrP(res) concentrations varied from below the limit of detection (350 fmol/g) to 15 pmol/g wet brain. Although most had a single N-TAAP profile, two novel variants were identified: one common to the ARH/ARQ animals in this study and one in an animal of the wild-type sheep PrP genotype (ARQ/ARQ).

  15. An online automatic sample cleanup system for the quantitative detection of the benzene exposure biomarker S-phenylmercapturic acid in human urine by electrospray ionization tandem mass spectrometry.

    PubMed

    Liao, Pao-Chi; Li, Chien-Ming; Lin, Lung-Cheng; Hung, Chien-Wen; Shih, Tung-Sheng

    2002-01-01

    An online automatic sample cleanup system was developed for use with electrospray ionization tandem mass spectrometry (ESI-MS-MS) for the quantitative detection of the benzene exposure biomarker S-phenylmercapturic acid (S-PMA) in human urine. The sample clean-up system was constructed with an autosampling device, a reversed-phase C18 trap cartridge, a two-position switching valve, and controlling computer software and hardware. The sample cleanup system was interfaced directly with the ESI source of a triple-stage-quadrupole MS using multiple reaction monitoring of negative product ions derived from S-PMA and the internal standard as the detection mode. The calibration curve was linear using human urine spiked at concentrations from 0.23 to 100 mg/L S-PMA (R2 = 0.997). The detection limit of the analytical system for neat S-PMA standard solution was 0.04 microg/L, whereas the detection limit was estimated to be lower than 0.35 microg/L for a urine matrix containing trace amounts of S-PMA. Without tedious manual sample cleanup procedures, the analytical system is fully automatic and therefore useful for high-throughput urinary S-PMA determination. With the selectivity and the sensitivity provided by MS-MS detection, the analytical system can be used for high-throughput and accurate determination of S-PMA levels in human urinary samples as a biomarker for benzene exposure.

  16. Study on quantitative analysis of Ti, Al and V in clinical soft tissues after placing the dental implants by laser ablation inductively coupled plasma mass spectrometry

    NASA Astrophysics Data System (ADS)

    Sajnóg, Adam; Hanć, Anetta; Makuch, Krzysztof; Koczorowski, Ryszard; Barałkiewicz, Danuta

    2016-11-01

    Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used for in-situ quantitative analysis of oral mucosa of patients before and after implantation with titanium implants and a closing screw based on Ti6Al4V alloy. Two calibration strategies were applied, both were based on matrix matched solid standards with analytes addition. A novel approach was the application of powdered egg white proteins as a matrix material which have a similar composition to the examined tissue. In the another approach, certified reference material Bovine Muscle ERM-BB184 was used. The isotope 34S was found to be the most appropriate as an internal standard since it is homogenously distributed in the examined tissues and resulted in lower relative standard deviation values of signal of analytes of interest. Other isotopes (13C, 26Mg, 43Ca) were also evaluated as potential internal standards. The analytical performance parameters and microwave digestion of solid standards followed by solution nebulization ICP-MS analysis proved that both calibration methods are fit for their intended purpose. The LA-ICP-MS analysis on the surface of tissues after the implantation process revealed an elevated content of elements in comparison to the control group. Analytes are distributed inhomogeneously and display local maximal content of Ti up to ca. 900 μg g- 1, Al up to ca. 760 μg g- 1 and for V up to 160 μg g- 1.

  17. A novel quantitation approach for maximizing detectable targets for offensive/volatile odorants with diverse functional groups by thermal desorption-gas chromatography-mass spectrometry

    NASA Astrophysics Data System (ADS)

    Kim, Yong-Hyun; Kim, Ki-Hyun

    2016-07-01

    A multitude of analytical systems are needed to analyze diverse odorants with various functionalities. In this study, an experimental method was developed to assess the maximum covering range of odorants using a single experimental setup consisting of a thermal desorber-gas chromatography-mass spectrometry system. To this end, a total of 20 offensive odorants (aldehyde, ketone, ester, alcohol, aromatic, sulfide, amine, and carboxyl) were selected and tested by a single system. The analytical results of standards and environmental samples were evaluated in a number of respects. In the analysis of the standards, all targets were quantified via Carbopack (C + B + X) tube sampling while operating the thermal desorber at ‑25 °C. The method detection limits of 18 targets (exception of 2 out of the 20 targets: acetaldehyde and methanethiol) were excellent (mean 0.04 ± 0.03 ppb) in terms of their odor threshold values (74.7 ± 140 ~ 624 ± 1,729 ppb). The analysis of organic fertilizer plant samples at a pig farm (slurry treatment facility, compost facility, and ambient air) confirmed the presence of 18 odorants from 0.03 ppb (dimethyldisulfide, ambient sample) to 522 ppb (methyl ethyl ketone, slurry treatment facility). As such, our method allowed simultaneous quantitation of most key odorants with sufficient reliability and sensitivity.

  18. Progress toward the Quantitative Analysis of PAHs Adsorbed on Soot by Laser Desorption/Laser Ionization/Time-of-Flight Mass Spectrometry.

    PubMed

    Faccinetto, Alessandro; Focsa, Cristian; Desgroux, Pascale; Ziskind, Michael

    2015-09-01

    Ex situ analyses of substances extracted from flames provide useful albeit mostly qualitative information on the formation process of soot and on the impact of exhausts on the environment. An experimental setup based on the coupling of laser desorption, laser ionization and time-of-flight mass spectrometry (LD/LI/ToF-MS) is presented in past works as an alternative means to more traditional techniques like gas chromatography (GC) to characterize the polycyclic aromatic hydrocarbons (PAHs) content of soot. In this paper, we go one step further in the understanding of the laser desorption/laser ionization dynamics and propose a combined experimental/simulation approach: we estimate the limit of detection of LD/LI/ToF-MS as low as [0.2, 2.8] fmol per laser pulse and we make quantitative predictions on the concentration of PAHs desorbed from soot. In particular, external calibration with model samples where PAHs are adsorbed on black carbon at known concentrations allows us to link the concentration of PAHs desorbed and detected by photoionization ToF-MS to the concentration of PAHs adsorbed on soot. The comparison of data obtained from the analysis of flame sampled soot with standard commercial GC-MS run in parallel validates the approach and defines limits and potentialities of both techniques.

  19. Quantitative comparison of structure and dynamics of elastin following three isolation schemes by 13C solid state NMR and MALDI mass spectrometry.

    PubMed

    Papaioannou, A; Louis, M; Dhital, B; Ho, H P; Chang, E J; Boutis, G S

    2015-05-01

    Methods for isolating elastin from fat, collagen, and muscle, commonly used in the design of artificial elastin based biomaterials, rely on exposing tissue to harsh pH levels and temperatures that usually denature many proteins. At present, a quantitative measurement of the modifications to elastin following isolation from other extracellular matrix constituents has not been reported. Using magic angle spinning (13)C NMR spectroscopy and relaxation methodologies, we have measured the modification in structure and dynamics following three known purification protocols. Our experimental data reveal that the (13)C spectra of the hydrated samples appear remarkably similar across the various purification methods. Subtle differences in the half maximum widths were observed in the backbone carbonyl suggesting possible structural heterogeneity across the different methods of purification. Additionally, small differences in the relative signal intensities were observed between purified samples. Lyophilizing the samples results in a reduction of backbone motion and reveals additional differences across the purification methods studied. These differences were most notable in the alanine motifs indicating possible changes in cross-linking or structural rigidity. The measured correlation times of glycine and proline moieties are observed to also vary considerably across the different purification methods, which may be related to peptide bond cleavage. Lastly, the relative concentration of desmosine cross-links in the samples quantified by MALDI mass spectrometry is reported.

  20. A general approach for the quantitative analysis of bisphosphonates in human serum and urine by high-performance liquid chromatography/tandem mass spectrometry.

    PubMed

    Zhu, Lee S; Lapko, Veniamin N; Lee, Jean W; Basir, Yousef J; Kafonek, Chris; Olsen, Richard; Briscoe, Chad

    2006-01-01

    Bisphosphonates are extremely hydrophilic and structurally similar to many endogenous phosphorylated compounds, making their selective extraction from serum or urine very challenging. Many bisphosphonates lack strong chromophores for sensitive UV or fluorescence detection. We report here the first general approach to enable sensitive and selective quantitation of N-containing bisphosphonates by liquid chromatography/tandem mass spectrometry (LC/MS/MS) following derivatization with diazomethane. The novelty of the strategy lies in performing the derivatization on silica-based anion-exchange sorbents as an integrated step in the sample purification by solid-phase extraction (SPE). The 'on-cartridge' reaction with diazomethane not only led to higher efficiency of derivatization, but also enabled a more discriminatory recovery of the drug's derivatives. The derivatized bisphosphonates demonstrated improved chromatographic separation and increased sensitivity of the detection. The general applicability of the approach was demonstrated by validation of bioanalytical methods for risedronate and alendronate in human serum and urine. Sensitivity was achieved at the pg/mL level with merely 100-200 microL of sample.

  1. Quantitative determination of antidepressants and their select degradates by liquid chromatography/electrospray ionization tandem mass spectrometry in biosolids destined for land application.

    PubMed

    Niemi, Lydia M; Stencel, Katherine A; Murphy, Madigan J; Schultz, Melissa M

    2013-08-06

    Antidepressants are one of the most widely dispensed classes of pharmaceuticals in the United States. As wastewater treatment plants are a primary source of pharmaceuticals in the environment, the use of biosolids as fertilizer is a potential route for antidepressants to enter the terrestrial environment. A microsolvent extraction method, utilizing green chemistry, was developed for extraction of the target antidepressants and degradation products from biosolids, or more specifically lagoon biosolids. Liquid chromatography/tandem mass spectrometry was used for quantitative determination of antidepressants in the lagoon biosolid extracts. Recoveries from matrix spiking experiments for the individual antidepressants had an average of 96%. The limits of detection for antidepressant pharmaceuticals and degradates ranged from 0.36 to 8.0 ng/kg wet weight. The method was applied to biosolids destined for land application. A suite of antidepressants was consistently detected in the lagoon biosolid samples, and thus antidepressants are being introduced to terrestrial environments through the land application of these biosolids. Sertraline and norsertraline were the most abundant antidepressant and degradation product detected in the biosolid samples. Detected, individual antidepressant concentrations ranged from 8.5 ng/kg (norfluoxetine) to 420 ng/kg wet weight (norsertraline).

  2. A method for quantitating the intracellular metabolism of AZT amino acid phosphoramidate pronucleotides by capillary high-performance liquid chromatography-electrospray ionization mass spectrometry.

    PubMed

    Kim, Jisook; Park, Soobong; Tretyakova, Natalia Y; Wagner, Carston R

    2005-01-01

    A methodology has been developed for the analysis of the intracellular metabolism of 3'-azido-3'-deoxythymidine (AZT) amino acid phosphoramidates utilizing reverse-phase high-performance liquid chromatography interfaced with negative ion electrospray ionization mass spectrometry (LC/ESI(-) -MS). The presented work demonstrates the potential of capillary LC/MS and LC/MS/MS to identify and quantitate the cellular uptake and metabolism of nucleoside phosphoramidate. Significant intracellular amounts of D- and L-phenylalanine methyl ester or D- and L-tryptophan methyl ester AZT phosphoramidates were observed for human T-lymphoblastoid leukemia (CEM) cells incubated for 2 and 4 h with the prodrugs. AZT-MP was the primary metabolite observed for human T-lymphoblastoid leukemia (CEM) cells. In this paper, the details of using LC/MS to analyze AZT amino acid phosphoramidates in biological samples are discussed. LC/MS is an efficient method for analyzing multiple samples containing several analytes in a short period of time. The method also provides high selectivity and sensitivity, and requires minimal sample preparation. This approach should be broadly applicable for the analysis of the intracellular metabolism of nucleoside prodrugs and pronucleotides.

  3. Study of sample preparation for quantitative analysis of amino acids in human sweat by liquid chromatography-tandem mass spectrometry.

    PubMed

    Delgado-Povedano, M M; Calderón-Santiago, M; Priego-Capote, F; Luque de Castro, M D

    2016-01-01

    The determination of physiological levels of amino acids is important to aid in the diagnosis and treatment of several diseases and nutritional status of individuals. Amino acids are frequently determined in biofluids such as blood (serum or plasma) and urine; however, there are less common biofluids with different concentration profiles of amino acids that could be of interest. One of these biofluids is sweat that can be obtained in a non-invasive manner and is characterized by low complex composition. The analysis of amino acids in human sweat requires the development of sample preparation strategies according to the sample matrix and small collected volume. The influence of sample preparation on the quantitative analysis of amino acids in sweat by LC-MS/MS has been assessed through a comparison between two strategies: dilution of sweat and centrifugal microsolid-phase extraction (c-μSPE). In both cases, several dilution factors were assayed for in-depth knowledge of the matrix effects, and the use of c-μSPE provided the best results in terms of accuracy. The behavior of the target analytes was a function of the dilution factor, thus providing a pattern for sample preparation that depended on the amino acid to be determined. The concentration of amino acids in sweat ranges between 6.20 ng mL(-1) (for homocysteine) and 259.77 µg mL(-1) (for serine) with precision, expressed as relative standard deviation, within 1.1-21.4%.

  4. "EMERGING" POLLUTANTS, MASS SPECTROMETRY, AND ...

    EPA Pesticide Factsheets

    A foundation for Environmental Science - Mass Spectrometry: Historically fundamental to amassing our understanding of environmental processes and chemical pollution is the realm of mass spectrometry - the mainstay of analytical chemistry - the workhorse that supplies much of the definitive data that environmental scientists rely upon for identifying the molecular compositions (and ultimately the structures) of chemicals. This is not to ignore the complementary, critical roles played by the adjunct practices of sample enrichment (via any of various means of selective extraction) and analyte separation (via the myriad forms of chromatography and electrophoresis).While the power of mass spectrometry has long been highly visible to the practicing environmental chemist, it borders on continued obscurity to the lay public and most non-chemists. Even though mass spectrometry has played a long, historic (and largely invisible) role in establishing or undergirdidng our existing knowledge about environmental processes and pollution, what recognition it does enjoy is usually relegated to that of a tool. It is ususally the relevance of ssignificance of the knowledge acquired from the application of the tool that has ultimate meaning to the public and science at large - not how the knowledge was acquired. The research focused on in the subtasks is the development and application of state-of the-art technologies to meet the needs of the public, Office of Water, and ORD in

  5. Electrospray Ionization Mass Spectrometry

    SciTech Connect

    Kelly, Ryan T.; Marginean, Ioan; Tang, Keqi

    2014-06-13

    Electrospray Ionization (ESI) is a process whereby gas phase ions are created from molecules in solution. As a solution exits a narrow tube in the presence of a strong electric field, an aerosol of charged droplets are is formed that produces gas phase ions as they it desolvates. ESI-MS comprises the creation of ions by ESI and the determination of their mass to charge ratio (m/z) by MS.

  6. Liquid chromatography-tandem mass spectrometry for the simultaneous quantitation of artemether and lumefantrine in human plasma: application for a pharmacokinetic study.

    PubMed

    César, Isabela Costa; Ribeiro, José Antônio de Aquino; Teixeira, Leonardo de Souza; Bellorio, Karini Bruno; de Abreu, Fernanda Crunivel; Moreira, Josianny Mesquita; Chellini, Paula Rocha; Pianetti, Gerson Antônio

    2011-01-05

    A liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method for the simultaneous quantitation of artemether and lumefantrine in human plasma was developed and validated. Artesunate was used as an internal standard (IS). The analytes were extracted by a protein precipitation procedure and separated on a reversed-phase Zorbax SB-Ciano column with a mobile phase composed of methanol and 10mM aqueous ammonium acetate containing 0.2% (v/v) acetic acid and 0.1% (v/v) formic acid. Multiple reaction monitoring was performed using the transitions m/z 316 → m/z 267, m/z 530 → m/z 348 and m/z 402 → m/z 267 to quantify artemether, lumefantrine and artesunate, respectively. Calibration curves were constructed over the range of 10-1000 ng/mL for artemether and 10-18,000 ng/mL for lumefantrine. The lower limit of quantitation was 10 ng/mL for both drugs. The mean R.S.D. values for the intra-run precision were 2.6% and 3.0% and for the inter-run precision were 3.6% and 4.6% for artemether and lumefantrine, respectively. The mean accuracy values were 102.0% and 101.2% for artemether and lumefantrine, respectively. No matrix effect was detected in the samples. The validated method was successfully applied to determine the plasma concentrations of artemether and lumefantrine in healthy volunteers, in a one-dose pharmacokinetic study, over the course of 11 days.

  7. Mass spectrometry analysis and quantitation of peptides presented on the MHC II molecules of mouse spleen dendritic cells

    PubMed Central

    Bozzacco, Leonia; Yu, Haiqiang; Zebroski, Henry A.; Dengjel, Jörn; Deng, Haiteng; Mojsov, Svetlana; Steinman, Ralph M.

    2011-01-01

    Major histocompatibility complex class II (MHC II) molecules are expressed on the surface of antigen presenting cells and display short bound peptide fragments derived from self and nonself antigens. These peptide-MHC complexes function to maintain immunological tolerance in the case of self antigens and initiate the CD4+ T cell response in the case of foreign proteins. Here we report the application of LC-MS/MS analysis to identify MHC II peptides derived from endogenous proteins expressed in freshly isolated murine splenic DCs. The cell number was enriched in vivo upon treatment with Flt3L-B16 melanoma cells. In a typical experiment, starting with about 5× 108 splenic DCs, we were able to reliably identify a repertoire of over 100 MHC II peptides originating from about 55 proteins localized in membrane (23%), intracellular (26%), endo-lysosomal (12%), nuclear (14%) and extracellular (25%) compartments. Using synthetic isotopically labeled peptides corresponding to the sequences of representative bound MHC II peptides, we quantified by LC-MS relative peptide abundance. In a single experiment, peptides were detected in a wide concentration range spanning from 2.5 fmol/μL to 12 pmol/μL or from approximately 13 copies to 2×105 copies per DC. These peptides were found in similar amounts on B cells where we detected about 80 peptides originating from 55 proteins distributed homogenously within the same cellular compartments as in DCs. About 90 different binding motifs predicted by the epitope prediction algorithm were found within the sequences of the identified MHC II peptides. These results set a foundation for future studies to quantitatively investigate the MHC II repertoire on DCs generated under different immunization conditions. PMID:21913724

  8. Quantitative determination of BMS-378806 in human plasma and urine by high-performance liquid chromatography/tandem mass spectrometry.

    PubMed

    Xue, Y J; Yan, Jing-He; Arnold, Mark; Grasela, Dennis; Unger, Steve

    2007-06-01

    BMS-378806 is a human immunodeficiency virus (HIV) entry inhibitor that is being developed for the oral treatment of HIV infection. Human plasma and urine LC/MS/ MS methods have been developed and validated for the quantitation of BMS-378806. For human plasma method, methyl t-butyl ether was used to extract BMS-378806 from plasma in a 96-well format, and the organic layers were dried down and then reconstituted for the injection, while a dilute-and-shoot approach was used for human urine method in a 96-well format. Chromatographic separation was achieved isocratically on a Phenomenex C18 (2) Luna column (2 x 50 mm2, 5 microm). The mobile phase contained 60:40 v/v of 0.1% formic acid in water and ACN. Detection was by positive ion electrospray MS/MS. The standard curves ranged from 1.25 to 1000 ng/mL for the plasma assay and from 10 to 5000 ng/mL for the urine assay. The curves were fitted to a 1/x2 weighted quadratic regression model for both methods. The validation results demonstrated that both methods had satisfactory precision and accuracy across the calibration ranges. The methods were applied to the analysis of human plasma and urine samples from a single ascending dose clinical study to assess the pharmacokinetics of the drug. The pharmacokinetic analysis results indicated the absorption and disposition of the drug was rapid. The systemic exposure of BMS-378806 was generally dose proportional among the doses from 100 to 1200 mg, but not dose proportional to 1600 mg. There were modest increases in the systemic exposure when the drug was given with food or given as a solution formulation. Renal excretion was not a substantial elimination pathway of the drug. BMS378806 was safe and well tolerated over a dose range of 100-1600 mg administered as a single oral dose.

  9. Simultaneous qualitative and quantitative method using liquid chromatography selected reaction monitoring-triggered quantitation-enhanced data-dependent tandem mass spectrometry for the identification and classification of amphetamine-type stimulant abusers in human urine.

    PubMed

    Lee, Sang Kyu; Kim, So-Hee; Kim, Ho Jun; Yoo, Hye Hyun; Kwon, Oh Seung; In, Moon Kyo; Jin, Changbae; Kim, Dong Hyun; Lee, Jaeick

    2010-11-15

    Amphetamine (AP) and amphetamine-type stimulants, methamphetamine (MA) and N,N-dimethylamphetamine (DMA), are known as central nervous system stimulants, and their abuse throughout the world has recently increased. Since it is difficult to physically distinguish among AP, MA and DMA, analysts may not be aware of what abusers have administered. In this study, following the detection of specific metabolites of AP, MA and DMA as biomarkers in abuser urines, a rapid and sensitive method was developed for the identification and classification of AP-type stimulants abusers. After the simple filtration of the urine samples, the samples were directly analyzed using a liquid chromatography/tandem mass spectrometry system with selected reaction monitoring (SRM)-triggered quantitation-enhanced data-dependent MS/MS (QED-MS/MS) for the simultaneous qualitative and quantitative analysis of p-hydroxy AP, p-hydroxy MA, p-hydroxy DMA, AP, MA, DMA and DMA N-oxide. The determination of p-hydroxy AP, p-hydroxy MA, AP, MA, DMA and DMA N-oxide was accurate and reproducible, with the limits of quantitation of 5 ng/mL in urine. When applied to the urine samples of suspected AP-type stimulants abusers, the abused drugs were precisely identified between MA and DMA abusers.

  10. Simultaneous quantitative analysis of dextromethorphan, dextrorphan and chlorphenamine in human plasma by liquid chromatography-electrospray tandem mass spectrometry.

    PubMed

    Ding, Ying; Huang, Kai; Chen, Lan; Yang, Jie; Xu, Wen-Yan; Xu, Xue-Jiao; Duan, Ru; Zhang, Jing; He, Qing

    2014-03-01

    A sensitive and accurate HPLC-MS/MS method was developed for the simultaneous determination of dextromethorphan, dextrorphan and chlorphenamine in human plasma. Three analytes were extracted from plasma by liquid-liquid extraction using ethyl acetate and separated on a Kromasil 60-5CN column (3 µm, 2.1 × 150 mm) with mobile phase of acetonitrile-water (containing 0.1% formic acid; 50:50, v/v) at a flow rate of 0.2 mL/min. Quantification was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode using positive electrospray ionization. The calibration curve was linear over the range of 0.01-5 ng/mL for dextromethorphan, 0.02-5 ng/mL for dextrorphan and 0.025-20 ng/mL for chlorphenamine. The lower limits of quantification for dextromethorphan, dextrorphan and chlorphenamine were 0.01, 0.02 and 0.025 ng/mL, respectively. The intra- and inter-day precisions were within 11% and accuracies were in the range of 92.9-102.5%. All analytes were proved to be stable during sample storage, preparation and analytic procedures. This method was first applied to the pharmacokinetic study in healthy Chinese volunteers after a single oral dose of the formulation containing dextromethorphan hydrobromide (18 mg) and chlorpheniramine malaeate (8 mg).

  11. Qualitative and quantitative analysis of the chemical constituents in Mahuang-Fuzi-Xixin decoction based on high performance liquid chromatography combined with time-of-flight mass spectrometry and triple quadrupole mass spectrometers.

    PubMed

    Sun, Qihui; Cao, Hongjie; Zhou, Yanyan; Wang, Xu; Jiang, Haiqiang; Gong, Lili; Yang, Yong; Rong, Rong

    2016-11-01

    High-performance liquid chromatography coupled with time-of-flight mass spectrometry (HPLC-TOF/MS) and high-performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-QQQ/MS/MS) were utilized to clarify the chemical constituents of Mahuang-Fuzi-Xixin Decoction. There are 52 compounds, including alkaloids, amino acids and organic acids were identified or tentatively characterized by their characteristic high resolution mass data by HPLC-QQQ/MS/MS. In the subsequent quantitative analysis, 10 constituents, including methyl ephedrine, aconine, songrine, fuziline, neoline, talatisamine, chasmanine, benzoylmesaconine, benzoylaconine and benzoylhypaconine were simultaneously determined by HPLC-QQQ/MS/MS with multiple reaction monitoring mode. Satisfactory linearity was achieved with wide linear range and fine determination coefficient (r > 0.9992). The relative standard deviations (RSD) of inter- and intra-day precisions were <3%. This method was also validated by repeatability, stability and recovery with RSD <3% respectively. A highly sensitive and efficient method was established for chemical constituents studying, including identification and quantification of Mahuang-Fuzi-Xixin decoction.

  12. A flow-injection mass spectrometry fingerprinting scaffold for feature selection and quantitation of Cordyceps and Ganoderma extracts in beverage: a predictive artificial neural network modelling strategy

    PubMed Central

    2012-01-01

    Flow-injection mass spectrometry (FI/MS) represents a powerful analytical tool for the quality assessment of herbal formula in dietary supplements. In this study, we described a scaffold (proof-of-concept) adapted from spectroscopy to quantify Cordyceps sinensis and Ganoderma lucidum in a popular Cordyceps sinensis /Ganoderma lucidum -enriched health beverage by utilizing flow-injection/mass spectrometry/artificial neural network (FI/MS/ANN) model fingerprinting method with feature selection capability. Equal proportion of 0.1% formic acid and methanol (v/v) were used to convert extracts of Cordyceps sinensis and Ganoderma lucidum into their respective ions under positive MS polarity condition. No chromatographic separation was performed. The principal m/z values of Cordyceps sinensis and Ganoderma lucidum were identified as: 104.2, 116.2, 120.2, 175.2, 236.3, 248.3, 266.3, 366.6 and 498.6; 439.7, 469.7, 511.7, 551.6, 623.6, 637.7 and 653.6, respectively. ANN models representing Cordyceps sinensis and Ganoderma lucidum were individually trained and validated using three independent sets of matrix-free and matrix-matched calibration curves at concentration levels of 2, 20, 50, 100, 200 and 400 μg mL-1. Five repeat analyses provided a total of 180 spectra for herbal extracts of Cordyceps sinensis and Ganoderma lucidum. Root-mean-square-deviation (RMSE) were highly satisfactory at <4% for both training and validation models. Correlation coefficient (r2) values of between 0.9994 and 0.9997 were reported. Matrix blanks comprised of complex mixture of Lingzhi fermentation solution and collagen. Recovery assessment was performed over two days using six sets of matrix blank (n = 6) spiked at three concentration levels of approximately 83, 166 and 333 mg kg-1. Extraction using acetonitrile provided good overall recovery range of 92-118%. A quantitation limit of 0.2 mg L-1 was reported for both Cordyceps sinensis and Ganoderma lucidum. Intra-day and inter-day RMSE

  13. A quantitative method for acylcarnitines and amino acids using high resolution chromatography and tandem mass spectrometry in newborn screening dried blood spot analysis.

    PubMed

    Miller, John H; Poston, Philip A; Karnes, H Thomas

    2012-08-15

    We have developed a high resolution liquid chromatographic separation with electrospray ionization (ESI) mass spectrometry detection for the combined analysis of twelve acylcarnitines and seven amino acids commonly measured in newborn screening heritable metabolic disorders. Samples were prepared by punching 3.2 mm disks out of dried blood spots and extracting with a mixture of methanol and 0.1% formic acid containing stable isotopically labeled internal standards. Analysis was performed on an UHPLC system using a HILIC amide, 2.1 mm × 50 mm, 1.7 μm column. A normal phase gradient, employing 10mM ammonium acetate in 90:10 acetonitrile/water for mobile phase B and 0.1% formic acid in water for mobile phase A, was used. Optimized multiple reaction monitoring (MRM) was used for detection of amino acids and acylcarnitines on a Waters Premier mass spectrometer. Quantification of analytes was performed using internal calibration by fortification of sodium heparin whole blood with analytes at appropriate levels to encompass the range around the reported cut-off values. The method was fully validated with respect to precision, accuracy, recovery, linearity, matrix suppression and extraction robustness. Precision and accuracy were evaluated over 3 days and determined to be acceptable with an overall precision within 10% and accuracy within 15% of theoretical for all analytes except for acetylcarnitne at one fortified level, which quantitated 21.8% lower than the expected value. This method is suitable as a second-tier test for newborn screening of specific disorders associated with abnormal levels of acylcarnitines and amino acids, potentially reducing false positive cases and shortening the time to diagnosis.

  14. Mass spectrometry of aerospace materials

    NASA Technical Reports Server (NTRS)

    Colony, J. A.

    1976-01-01

    Mass spectrometry is used for chemical analysis of aerospace materials and contaminants. Years of analytical aerospace experience have resulted in the development of specialized techniques of sampling and analysis which are required in order to optimize results. This work has resulted in the evolution of a hybrid method of indexing mass spectra which include both the largest peaks and the structurally significant peaks in a concise format. With this system, a library of mass spectra of aerospace materials was assembled, including the materials responsible for 80 to 90 percent of the contamination problems at Goddard Space Flight Center during the past several years.

  15. Mass spectrometry and tandem mass spectrometry of citrus limonoids.

    PubMed

    Tian, Qingguo; Schwartz, Steven J

    2003-10-15

    Methods for atmospheric pressure chemical ionization tandem mass spectrometry (APCI-MS/MS) of citrus limonoid aglycones and electrospray ionization tandem mass spectrometry (ESI-MS/MS) of limonoid glucosides are reported. The fragmentation patterns of four citrus limonoid aglycones (limonin, nomilin, obacunone, and deacetylnomilin) and six limonoid glucosides, that is, limonin 17-beta-D-glucopyranoside (LG), nomilin 17-beta-D-glucopyranoside (NG), nomilinic acid 17-beta-D-glucopyranoside (NAG), deacetyl nomilinic acid 17-beta-D-glucopyranoside (DNAG), obacunone 17-beta-D-glucopyranoside (OG), and obacunoic acid 17-beta-D-glucopyranoside (OAG) were investigated using a quadruple mass spectrometer in low-energy collisionally activated dissociation (CAD). The four limonoid aglycones and four limonoid glucosides (LG, OG, NAG, and DNAG) were purified from citrus seeds; the other two limonoid glucosides (NG and OAG) were tentatively identified in the crude extract of grapefruit seeds by ESI mass spectrometry in both positive and negative ion analysis. Ammonium hydroxide or acetic acid was added to the mobile phase to facilitate ionization. During positive ion APCI analysis of limonoid aglycones, protonated molecular ion, [M + H]+, or adduct ion, [M + NH3 + H]-, was formed as base peaks when ammonium hydroxide was added to the mobile phase. Molecular anions or adduct ions with acetic acid ([M + HOAc - H] and [M + HOAc]-) or a deprotonated molecular ion were produced during negative ion APCI analysis of limonoid aglycones, depending on the mobile-phase modifier used. Positive ion ESI-MS of limonoid glucosides produced adduct ions of [M + H + NH3]+, [M + Na]+, and [M + K]+ when ammonium hydroxide was added to the mobile phase. After collisionally activated dissociation (CAD) of the limonoid aglycone molecular ions in negative ion APCI analysis, fragment ions indicated structural information of the precursor ions, showing the presence of methyl, carboxyl, and oxygenated ring

  16. Absolute Quantitation of Met Using Mass Spectrometry for Clinical Application: Assay Precision, Stability, and Correlation with MET Gene Amplification in FFPE Tumor Tissue

    PubMed Central

    Catenacci, Daniel V. T.; Liao, Wei-Li; Thyparambil, Sheeno; Henderson, Les; Xu, Peng; Zhao, Lei; Rambo, Brittany; Hart, John; Xiao, Shu-Yuan; Bengali, Kathleen; Uzzell, Jamar; Darfler, Marlene; Krizman, David B.; Cecchi, Fabiola; Bottaro, Donald P.; Karrison, Theodore; Veenstra, Timothy D.; Hembrough, Todd; Burrows, Jon

    2014-01-01

    Background Overexpression of Met tyrosine kinase receptor is associated with poor prognosis. Overexpression, and particularly MET amplification, are predictive of response to Met-specific therapy in preclinical models. Immunohistochemistry (IHC) of formalin-fixed paraffin-embedded (FFPE) tissues is currently used to select for ‘high Met’ expressing tumors for Met inhibitor trials. IHC suffers from antibody non-specificity, lack of quantitative resolution, and, when quantifying multiple proteins, inefficient use of scarce tissue. Methods After describing the development of the Liquid-Tissue-Selected Reaction Monitoring-mass spectrometry (LT-SRM-MS) Met assay, we evaluated the expression level of Met in 130 FFPE gastroesophageal cancer (GEC) tissues. We assessed the correlation of SRM Met expression to IHC and mean MET gene copy number (GCN)/nucleus or MET/CEP7 ratio by fluorescence in situ hybridization (FISH). Results Proteomic mapping of recombinant Met identified 418TEFTTALQR426 as the optimal SRM peptide. Limits of detection (LOD) and quantitation (LOQ) for this peptide were 150 and 200 amol/µg tumor protein, respectively. The assay demonstrated excellent precision and temporal stability of measurements in serial sections analyzed one year apart. Expression levels of 130 GEC tissues ranged (<150 amol/µg to 4669.5 amol/µg. High correlation was observed between SRM Met expression and both MET GCN and MET/CEP7 ratio as determined by FISH (n = 30; R2 = 0.898). IHC did not correlate well with SRM (n = 44; R2 = 0.537) nor FISH GCN (n = 31; R2 = 0.509). A Met SRM level of ≥1500 amol/µg was 100% sensitive (95% CI 0.69–1) and 100% specific (95% CI 0.92–1) for MET amplification. Conclusions The Met SRM assay measured the absolute Met levels in clinical tissues with high precision. Compared to IHC, SRM provided a quantitative and linear measurement of Met expression, reliably distinguishing between non-amplified and amplified MET

  17. Quantitative Analysis of Bisphenol A Leached from Household Plastics by Solid-Phase Microextraction and Gas Chromatography-Mass Spectrometry (SPME-GC-MS)

    ERIC Educational Resources Information Center

    Johnson, Bettie Obi; Burke, Fernanda M.; Harrison, Rebecca; Burdette, Samantha

    2012-01-01

    The measurement of trace levels of bisphenol A (BPA) leached out of household plastics using solid-phase microextraction (SPME) with gas chromatography-mass spectrometry (GC-MS) is reported here. BPA is an endocrine-disrupting compound used in the industrial manufacture of polycarbonate plastic bottles and epoxy resin can liners. This experiment…

  18. Quantitative measurement of cysteinyl leukotrienes and leukotriene B₄ in human sputum using ultra high pressure liquid chromatography-tandem mass spectrometry.

    PubMed

    Chappell, Gloria Paola; Xiao, Xiaoyao; Pica-Mendez, Arnaldo; Varnell, Tracey; Green, Stuart; Tanaka, Wesley K; Laterza, Omar

    2011-02-01

    The role of leukotrienes (LTs) in airway inflammatory diseases, such as asthma, has been extensively reported. The measurement of LTs in sputum supernatants, which is commonly done via enzyme immunoassays (EIAs), may prove to be useful for assessing airway inflammation. Despite the many advantages of EIA, these methods suffer from a lack of selectivity. Therefore, a selective and reliable method for the analysis of LTs in human sputum is needed. In this study we developed and validated a sensitive and specific method using ultra high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), to measure simultaneously cysteinyl leukotrienes (CysLTs) and leukotriene B₄ (LTB₄) in human sputum. Sputum supernatants obtained by ultracentrifugation were stabilized by protease inhibitors, spiked with stable isotopic internal standards, and subjected to solid phase extraction (SPE) and UHPLC separation. Multiple reaction monitoring (MRM) transitions were optimized and measured on a mass spectrometer. The limit of detection (LOD) for LTE₄ and LTB₄ was 9.8 and 19.5 pg/mL, respectively. The lower limit of quantitation (LLOQ) for LTE₄ and LTB₄ was 19.5 and 39.0 pg/mL, respectively. The dynamic range of the LTE₄ assay was from 9.8 to 5000 pg/mL, whereas for the LTB₄ assay was from 19.5 to 10,000 pg/mL. The intra- and inter-day % coefficient of variation (%CV) was <6.5% and <10%, for both LTE₄ and LTB₄, respectively. Spike recovery ranged from 105% to 111% for both analytes. In addition, twenty-two sputum samples were analyzed for cysLTs and LTB₄. Fourteen of these samples were purchased commercially and eight were collected during the course of a clinical trial. LTB₄ was detectable in all samples tested and it ranged from 79 to 7220 pg/mL. LTE₄ was detectable in most of the sputum samples (12.3-891 pg/mL), whereas LTC₄ and LTD₄ were below limit of detection for majority of sputum samples. The in vitro conversion of LTC₄ and LTD₄ into

  19. Advantageous Uses of Mass Spectrometry for the Quantification of Proteins

    PubMed Central

    Hale, John E.

    2013-01-01

    Quantitative protein measurements by mass spectrometry have gained wide acceptance in research settings. However, clinical uptake of mass spectrometric protein assays has not followed suit. In part, this is due to the long-standing acceptance by regulatory agencies of immunological assays such as ELISA assays. In most cases, ELISAs provide highly accurate, sensitive, relatively inexpensive, and simple assays for many analytes. The barrier to acceptance of mass spectrometry in these situations will remain high. However, mass spectrometry provides solutions to certain protein measurements that are difficult, if not impossible, to accomplish by immunological methods. Cases where mass spectrometry can provide solutions to difficult assay development include distinguishing between very closely related protein species and monitoring biological and analytical variability due to sample handling and very high multiplexing capacity. This paper will highlight several examples where mass spectrometry has made certain protein measurements possible where immunological techniques have had a great difficulty. PMID:23365751

  20. EMERGING POLLUTANTS, MASS SPECTROMETRY, AND ...

    EPA Pesticide Factsheets

    Historically fundamental to amassing our understanding of environmental processes and chemical pollution is the realm of mass spectrometry (MS) - the mainstay of analytical chemistry - the workhorse that supplies definitive data that environmental scientists and engineers reply upon for identifying molecular compositions (and ultimately structures) of chemicals. While the power of MS has long been visible to the practicing environmental chemist, it borders on obscurity to the lay public and many scientists. While MS has played a long, historic (and largely invisible) role in establishing our knowledge of environmental processes and pollution, what recognition it does enjoy is usually relegated to that of a tool. It is usually the relevance or significance of the knowledge acquired from the application of the tool that has ultimate meaning to the public and science at large - not how the data were acquired. Methods (736/800): Mass Spectrometry and the

  1. Imaging Mass Spectrometry in Neuroscience

    PubMed Central

    2013-01-01

    Imaging mass spectrometry is an emerging technique of great potential for investigating the chemical architecture in biological matrices. Although the potential for studying neurobiological systems is evident, the relevance of the technique for application in neuroscience is still in its infancy. In the present Review, a principal overview of the different approaches, including matrix assisted laser desorption ionization and secondary ion mass spectrometry, is provided with particular focus on their strengths and limitations for studying different neurochemical species in situ and in vitro. The potential of the various approaches is discussed based on both fundamental and biomedical neuroscience research. This Review aims to serve as a general guide to familiarize the neuroscience community and other biomedical researchers with the technique, highlighting its great potential and suitability for comprehensive and specific chemical imaging. PMID:23530951

  2. Hydrophilic interaction liquid chromatography-tandem mass spectrometry quantitative method for the cellular analysis of varying structures of gemini surfactants designed as nanomaterial drug carriers.

    PubMed

    Donkuru, McDonald; Michel, Deborah; Awad, Hanan; Katselis, George; El-Aneed, Anas

    2016-05-13

    Diquaternary gemini surfactants have successfully been used to form lipid-based nanoparticles that are able to compact, protect, and deliver genetic materials into cells. However, what happens to the gemini surfactants after they have released their therapeutic cargo is unknown. Such knowledge is critical to assess the quality, safety, and efficacy of gemini surfactant nanoparticles. We have developed a simple and rapid liquid chromatography electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the quantitative determination of various structures of gemini surfactants in cells. Hydrophilic interaction liquid chromatography (HILIC) was employed allowing for a short simple isocratic run of only 4min. The lower limit of detection (LLOD) was 3ng/mL. The method was valid to 18 structures of gemini surfactants belonging to two different structural families. A full method validation was performed for two lead compounds according to USFDA guidelines. The HILIC-MS/MS method was compatible with the physicochemical properties of gemini surfactants that bear a permanent positive charge with both hydrophilic and hydrophobic elements within their molecular structure. In addition, an effective liquid-liquid extraction method (98% recovery) was employed surpassing previously used extraction methods. The analysis of nanoparticle-treated cells showed an initial rise in the analyte intracellular concentration followed by a maximum and a somewhat more gradual decrease of the intracellular concentration. The observed intracellular depletion of the gemini surfactants may be attributable to their bio-transformation into metabolites and exocytosis from the host cells. Obtained cellular data showed a pattern that grants additional investigations, evaluating metabolite formation and assessing the subcellular distribution of tested compounds.

  3. Liquid chromatography tandem mass spectrometry method for the quantitative analysis of ceritinib in human plasma and its application to pharmacokinetic studies.

    PubMed

    Heudi, Olivier; Vogel, Denise; Lau, Yvonne Y; Picard, Franck; Kretz, Olivier

    2014-11-01

    Ceritinib is a highly selective inhibitor of an important cancer target, anaplastic lymphoma kinase (ALK). Because it is an investigational compound, there is a need to develop a robust and reliable analytical method for its quantitative determination in human plasma. Here, we report the validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the rapid quantification of ceritinib in human plasma. The method consists of protein precipitation with acetonitrile, and salting-out assisted liquid-liquid extraction (SALLE) using a saturated solution of sodium chloride prior to analysis by LC-MS/MS with electrospray ionization (ESI) technique in positive mode. Samples were eluted at 0.800 mL min(-1) on Ascentis Express® C18 column (50 mm × 2.1 mm, 2.7 μm) with a mobile phase made of 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B). The method run time was 3.6 min and the low limit of quantification (LLOQ) was estimated at 1.00 ng mL(-1) when using 0.100 mL of human plasma. The assay was fully validated and the method exhibited sufficient specificity, accuracy, precision, and sensitivity. In addition, recovery data and matrix factor (MF) in normal and in hemolyzed plasmas were assessed, while incurred samples stability (ISS) for ceritinib was demonstrated for at least 21 months at a storage temperature of -65 °C or below. The method was successfully applied to the measurement of ceritinib in clinical samples and the data obtained on incurred samples reanalysis (ISR) showed that our method was reliable and suitable to support the analysis of samples from the clinical studies.

  4. Multivariate analytical figures of merit as a metric for evaluation of quantitative measurements using comprehensive two-dimensional gas chromatography-mass spectrometry.

    PubMed

    Eftekhari, Ali; Parastar, Hadi

    2016-09-30

    The present contribution is devoted to develop multivariate analytical figures of merit (AFOMs) as a new metric for evaluation of quantitative measurements using comprehensive two-dimensional gas chromatography-mass spectrometry (GC×GC-MS). In this regard, new definition of sensitivity (SEN) is extended to GC×GC-MS data and then, other multivariate AFOMs including analytical SEN (γ), selectivity (SEL) and limit of detection (LOD) are calculated. Also, two frequently used second- and third-order calibration algorithms of multivariate curve resolution-alternating least squares (MCR-ALS) as representative of multi-set methods and parallel factor analysis (PARAFAC) as representative of multi-way methods are discussed to exploit pure component profiles and to calculate multivariate AFOMs. Different GC×GC-MS data sets with different number of components along with various levels of artifacts are simulated and analyzed. Noise, elution time shifts in both chromatographic dimensions, peak overlap and interferences are considered as the main artifacts in this work. Additionally, a new strategy is developed to estimate the noise level using variance-covariance matrix of residuals which is very important to calculate multivariate AFOMs. Finally, determination of polycyclic aromatic hydrocarbons (PAHs) in aromatic fraction of heavy fuel oil (HFO) analyzed by GC×GC-MS is considered as real case to confirm applicability of the proposed metric in real samples. It should be pointed out that the proposed strategy in this work can be used for other types of comprehensive two-dimensional chromatographic (CTDC) techniques like comprehensive two dimensional liquid chromatography (LC×LC).

  5. Simultaneous quantitative determination of melamine and cyanuric acid in cow's milk and milk-based infant formula by liquid chromatography-electrospray ionization tandem mass spectrometry.

    PubMed

    Desmarchelier, Aurélien; Guillamon Cuadra, Miriam; Delatour, Thierry; Mottier, Pascal

    2009-08-26

    An isotope dilution liquid chromatography-electrospray ionization tandem mass spectrometry method for the simultaneous determination of melamine and cyanuric acid in cow's milk (range of 0-0.3 mg/kg) and milk-based infant formulas (ranges of 0-0.3 and 0-2.0 mg/kg) is described. This quantitative method entails simple sample preparation, limited to a protein precipitation in acetonitrile/water followed by a centrifugation and direct injection of the supernatant. Selected reaction monitoring of two diagnostic transition reactions for each analyte and each corresponding ((13)C(3),(15)N(3))-labeled compound enables selective and confirmatory detection. Acquisition was performed sequentially in the negative ion mode for cyanuric acid, while in the positive mode for melamine within the same run. Validation of the method was conducted according to European Union criteria (CD 2002/657/EC). Internal standard-corrected recoveries were within the 99-116% range for both analytes in the two matrix types, along with repeatability and intermediate reproducibility values of

  6. Quantitative Analysis of Staphylococcal Enterotoxins A and B in Food Matrices Using Ultra High-Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS)

    PubMed Central

    Zuberovic Muratovic, Aida; Hagström, Thomas; Rosén, Johan; Granelli, Kristina; Hellenäs, Karl-Erik

    2015-01-01

    A method that uses mass spectrometry (MS) for identification and quantification of protein toxins, staphylococcal enterotoxins A and B (SEA and SEB), in milk and shrimp is described. The analysis was performed using a tryptic peptide, from each of the toxins, as the target analyte together with the corresponding 13C-labeled synthetic internal standard peptide. The performance of the method was evaluated by analyzing spiked samples in the quantification range 2.5–30 ng/g (R2 = 0.92–0.99). The limit of quantification (LOQ) in milk and the limit of detection (LOD) in shrimp was 2.5 ng/g, for both SEA and SEB toxins. The in-house reproducibility (RSD) was 8%–30% and 5%–41% at different concentrations for milk and shrimp, respectively. The method was compared to the ELISA method, used at the EU-RL (France), for milk samples spiked with SEA at low levels, in the quantification range of 2.5 to 5 ng/g. The comparison showed good coherence for the two methods: 2.9 (MS)/1.8 (ELISA) and 3.6 (MS)/3.8 (ELISA) ng/g. The major advantage of the developed method is that it allows direct confirmation of the molecular identity and quantitative analysis of SEA and SEB at low nanogram levels using a label and antibody free approach. Therefore, this method is an important step in the development of alternatives to the immune-assay tests currently used for staphylococcal enterotoxin analysis. PMID:26378579

  7. Application of Screening Experimental Designs to Assess Chromatographic Isotope Effect upon Isotope-Coded Derivatization for Quantitative Liquid Chromatography–Mass Spectrometry

    PubMed Central

    2015-01-01

    Isotope effect may cause partial chromatographic separation of labeled (heavy) and unlabeled (light) isotopologue pairs. Together with a simultaneous matrix effect, this could lead to unacceptable accuracy in quantitative liquid chromatography–mass spectrometry assays, especially when electrospray ionization is used. Four biologically relevant reactive aldehydes (acrolein, malondialdehyde, 4-hydroxy-2-nonenal, and 4-oxo-2-nonenal) were derivatized with light or heavy (d3-, 13C6-, 15N2-, or 15N4-labeled) 2,4-dinitrophenylhydrazine and used as model compounds to evaluate chromatographic isotope effects. For comprehensive assessment of retention time differences between light/heavy pairs under various gradient reversed-phase liquid chromatography conditions, major chromatographic parameters (stationary phase, mobile phase pH, temperature, organic solvent, and gradient slope) and different isotope labelings were addressed by multiple-factor screening using experimental designs that included both asymmetrical (Addelman) and Plackett–Burman schemes followed by statistical evaluations. Results confirmed that the most effective approach to avoid chromatographic isotope effect is the use of 15N or 13C labeling instead of deuterium labeling, while chromatographic parameters had no general influence. Comparison of the alternate isotope-coded derivatization assay (AIDA) using deuterium versus 15N labeling gave unacceptable differences (>15%) upon quantifying some of the model aldehydes from biological matrixes. On the basis of our results, we recommend the modification of the AIDA protocol by replacing d3-2,4-dinitrophenylhydrazine with 15N- or 13C-labeled derivatizing reagent to avoid possible unfavorable consequences of chromatographic isotope effects. PMID:24922593

  8. Quantitative analysis of amino acids and acylcarnitines combined with untargeted metabolomics using ultra-high performance liquid chromatography and quadrupole time-of-flight mass spectrometry.

    PubMed

    Roy, Cynthia; Tremblay, Pierre-Yves; Bienvenu, Jean-François; Ayotte, Pierre

    2016-08-01

    Metabolomics is an "omic" technique being increasingly used in epidemiological and clinical studies. We developed a method combining untargeted metabolomics with the quantitative determination of eight amino acids (AA) and eight acylcarnitines (AC) in plasma using ultra-high pressure liquid chromatography (UHPLC), electrospray ionization (ESI) and quadrupole time-of-flight mass spectrometry (QTOFMS). Separation of metabolites is performed by ion-pair reverse phase UHPLC using a HSS T3 column (2.1×100mm, 100Å, 1.8μm particle size) and formic acid-ammonium acetate-heptafluorobutyric acid in water and formic acid-ammonium acetate in methanol as mobile phases. Metabolite identification and quantification are achieved using a QTOFMS operating in ESI-positive and full-scan mode along with MS(E) acquisition of fragmentation patterns. Targeted metabolites are quantified using the appropriate labeled standards and include branched-chain AA (leucine, isoleucine, valine), aromatic AA (phenylalanine, tyrosine) as well as acetylcarnitine and propionylcarnitine, which have been identified as biomarkers of future cardiometabolic disease risk. The inter-day precision (relative standard deviation) for the targeted method was <15% for all but one metabolite and accuracy (bias) of amino acids ranged from 0.5% to 13.9% using SRM 1950 as the external standard. Untargeted metabolomics in 30 plasma samples from the general Canadian population revealed 5018 features, of which 48 metabolites were identified using the MZmine 2.19 software including 23 by our in-house library that comprises 671 annotated metabolites. SRM 1950 analysis revealed 11,684 features, among which 154 metabolites were identified. Our method is currently applied in several epidemiological studies to better characterize cardiometabolic diseases and identify new biomarkers for disease prevention.

  9. Quantitative determination of 13 organophosphorous flame retardants and plasticizers in a wastewater treatment system by high performance liquid chromatography tandem mass spectrometry.

    PubMed

    Woudneh, Million B; Benskin, Jonathan P; Wang, Guanghui; Grace, Richard; Hamilton, M Coreen; Cosgrove, John R

    2015-06-26

    A method for quantitative determination of 13 organophosphorous compounds (OPs) was developed and applied to influent, primary sludge, activated sludge, biosolids, primary effluent and final effluent from a wastewater treatment plant (WWTP). The method involved solvent extraction followed by solid phase clean-up and analysis by high performance liquid chromatography positive electrospray ionization-tandem mass spectrometry (HPLC(+ESI)MS/MS). Replicate spike/recovery experiments revealed the method to have good accuracy (70-132%) and precision (<19% RSD) in all matrices. Detection limits of 0.1-5 ng/L for aqueous samples and 0.01-0.5 ng/g for solid samples were achieved. In the liquid waste stream ∑OP concentrations were highest in influent (5764 ng/L) followed by primary effluent (4642 ng/L), and final effluent (2328 ng/L). In the solid waste stream, the highest ∑OP concentrations were observed in biosolids (3167 ng/g dw), followed by waste activated sludge (2294 ng/g dw), and primary sludge (2128 ng/g dw). These concentrations are nearly 30-fold higher than ∑polybrominated diphenyl ether (BDE) concentrations in influents and nearly 200-fold higher than ∑BDE concentrations in effluents from other sites in Canada. Tetrekis(2-chlorethyl)dichloroisopentyldiphosphate (V6), tripropylphosphate (TnPrP), and Tris(2,3-dibromopropyl)phosphate (TDBPP) are investigated for the first time in a WWTP. While TnPrP and TDBB were not detected, V6 was observed at concentrations up to 7.9 ng/g in solid waste streams and up to 40.7 ng/L in liquid waste streams. The lack of removal of OPs during wastewater treatment is a concern due to their release into the aquatic environment.

  10. Quantitative profiling of bacteriocins present in dairy-free probiotic preparations of Lactobacillus acidophilus by nanoliquid chromatography-tandem mass spectrometry.

    PubMed

    Nandakumar, Renu; Talapatra, Kesh

    2014-01-01

    Bacteriocins are a heterogeneous group of ribosomally synthesized peptides or proteins with antimicrobial activity, produced predominantly by lactic acid bacteria, with potential applications as biopreservatives and probiotics. We describe here a novel strategy based on a bottom-up, shotgun proteomic approach using nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) with multiple fragmentation techniques for the quantitative profiling of bacteriocins present in the probiotic preparations of Lactobacillus acidophilus. A direct LC-MS/MS analysis with alternate collision-induced dissociation, high-energy collision dissociation, and electron-transfer dissociation fragmentation following a filter-assisted size-exclusion sample prefractionation has resulted in the identification of peptides belonging to 37 bacteriocins or related proteins. Peptides from lactacin F, helveticin J, lysin, avicin A, acidocin M, curvaticin FS47, and carocin D were predominant. The process of freeze drying under vacuum was observed to affect both the diversity and abundance of bacteriocins. Data acquisition using alternating complementary peptide fragmentation modes, especially electron-transfer dissociation, has significantly enhanced the peptide sequence coverage and number of bacteriocin peptides identified. Multi-enzyme proteolytic digestion was observed to increase the sample complexity and dynamic range, lowering the chances of detection of low-abundant bacteriocin peptides by LC-MS/MS. An analytical platform integrating size exclusion prefractionation, nanoLC-MS/MS analysis with multiple fragmentation techniques, and data-dependent decision tree-driven bioinformatic data analysis is novel in bacteriocin research and suitable for the comprehensive bioanalysis of diverse, low-abundant bacteriocins in complex samples.

  11. Quantitative monitoring of tamoxifen in human plasma extended to 40 metabolites using liquid-chromatography high-resolution mass spectrometry: new investigation capabilities for clinical pharmacology.

    PubMed

    Dahmane, Elyes; Boccard, Julien; Csajka, Chantal; Rudaz, Serge; Décosterd, Laurent; Genin, Eric; Duretz, Bénédicte; Bromirski, Maciej; Zaman, Khalil; Testa, Bernard; Rochat, Bertrand

    2014-04-01

    Liquid-chromatography (LC) high-resolution (HR) mass spectrometry (MS) analysis can record HR full scans, a technique of detection that shows comparable selectivity and sensitivity to ion transitions (SRM) performed with triple-quadrupole (TQ)-MS but that allows de facto determination of "all" ions including drug metabolites. This could be of potential utility in in vivo drug metabolism and pharmacovigilance studies in order to have a more comprehensive insight in drug biotransformation profile differences in patients. This simultaneous quantitative and qualitative (Quan/Qual) approach has been tested with 20 patients chronically treated with tamoxifen (TAM). The absolute quantification of TAM and three metabolites in plasma was realized using HR- and TQ-MS and compared. The same LC-HR-MS analysis allowed the identification and relative quantification of 37 additional TAM metabolites. A number of new metabolites were detected in patients' plasma including metabolites identified as didemethyl-trihydroxy-TAM-glucoside and didemethyl-tetrahydroxy-TAM-glucoside conjugates corresponding to TAM with six and seven biotransformation steps, respectively. Multivariate analysis allowed relevant patterns of metabolites and ratios to be associated with TAM administration and CYP2D6 genotype. Two hydroxylated metabolites, α-OH-TAM and 4'-OH-TAM, were newly identified as putative CYP2D6 substrates. The relative quantification was precise (<20 %), and the semiquantitative estimation suggests that metabolite levels are non-negligible. Metabolites could play an important role in drug toxicity, but their impact on drug-related side effects has been partially neglected due to the tremendous effort needed with previous MS technologies. Using present HR-MS, this situation should evolve with the straightforward determination of drug metabolites, enlarging the possibilities in studying inter- and intra-patients drug metabolism variability and related effects.

  12. Qualitative and quantitative determination of polyacetylenes in different Bupleurum species by high performance liquid chromatography with diode array detector and mass spectrometry.

    PubMed

    Huang, Hai-Qiang; Su, Juan; Zhang, Xi; Shan, Lei; Zhang, Wei-Dong

    2011-02-25

    Polyacetylenes are main toxic ingredients in Bupleurum longiradiatum, a poisonous plant that has ever been misused as substitutes for Chaihu (Bupleuri Radix). For the first time, a high-performance liquid chromatography method coupled with diode array detector and mass spectrometry (HPLC-DAD-MS) was developed for qualitative and quantitative analysis of nine polyacetylenes in Bupleurum species. All references, including two new polyacetylenes, were isolated from B. longiradiatum and purified using a semi-preparation HPLC chromatography. The analysis was performed on a TSKgel ODS-100V C18 column (3 μm, 150 mm x 4.6 mm i.d.) using a gradient system of acetonitrile and water, with diode array detection (254 nm). The method was validated for linearity, precision, accuracy, limit of detection and quantification. A total of 27 Bupleurum samples were examined with this method, which showed a great variety in the distribution and contents of the polyacetylenes. It was found that polyacetylenes (1-8) were the main ingredients in B. longiradiatum, while a few kinds of polyacetylenes (5-8) were also identified in B. smithii, B. smithii var. parvifolium, B. bicaule and B. angustissimum. However, no polyacetylenes (1-9) were detected in the authentic Chaihu samples and the other Bupleurum species. The results indicated that the toxic B. longiradiatum could readily be distinguished from other medicinal Bupleurum species based on the polyacetylene profiles, and the guidelines and quality control of polyacetylenes for Chaihu are useful. The acute toxicity of the ethanol extract of B. longiradiatum and its fractions was also investigated.

  13. Quantitative Analysis of Staphylococcal Enterotoxins A and B in Food Matrices Using Ultra High-Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS).

    PubMed

    Muratovic, Aida Zuberovic; Hagström, Thomas; Rosén, Johan; Granelli, Kristina; Hellenäs, Karl-Erik

    2015-09-11

    A method that uses mass spectrometry (MS) for identification and quantification of protein toxins, staphylococcal enterotoxins A and B (SEA and SEB), in milk and shrimp is described. The analysis was performed using a tryptic peptide, from each of the toxins, as the target analyte together with the corresponding (13)C-labeled synthetic internal standard peptide. The performance of the method was evaluated by analyzing spiked samples in the quantification range 2.5-30 ng/g (R² = 0.92-0.99). The limit of quantification (LOQ) in milk and the limit of detection (LOD) in shrimp was 2.5 ng/g, for both SEA and SEB toxins. The in-house reproducibility (RSD) was 8%-30% and 5%-41% at different concentrations for milk and shrimp, respectively. The method was compared to the ELISA method, used at the EU-RL (France), for milk samples spiked with SEA at low levels, in the quantification range of 2.5 to 5 ng/g. The comparison showed good coherence for the two methods: 2.9 (MS)/1.8 (ELISA) and 3.6 (MS)/3.8 (ELISA) ng/g. The major advantage of the developed method is that it allows direct confirmation of the molecular identity and quantitative analysis of SEA and SEB at low nanogram levels using a label and antibody free approach. Therefore, this method is an important step in the development of alternatives to the immune-assay tests currently used for staphylococcal enterotoxin analysis.

  14. Mass spectrometry. [review of techniques

    NASA Technical Reports Server (NTRS)

    Burlingame, A. L.; Kimble, B. J.; Derrick, P. J.

    1976-01-01

    Advances in mass spectrometry (MS) and its applications over the past decade are reviewed in depth, with annotated literature references. New instrumentation and techniques surveyed include: modulated-beam MS, chromatographic MS on-line computer techniques, digital computer-compatible quadrupole MS, selected ion monitoring (mass fragmentography), and computer-aided management of MS data and interpretation. Areas of application surveyed include: organic MS and electron impact MS, field ionization kinetics, appearance potentials, translational energy release, studies of metastable species, photoionization, calculations of molecular orbitals, chemical kinetics, field desorption MS, high pressure MS, ion cyclotron resonance, biochemistry, medical/clinical chemistry, pharmacology, and environmental chemistry and pollution studies.

  15. Glycosaminoglycan Glycomics Using Mass Spectrometry*

    PubMed Central

    Zaia, Joseph

    2013-01-01

    The fact that sulfated glycosaminoglycans (GAGs) are necessary for the functioning of all animal physiological systems drives the need to understand their biology. This understanding is limited, however, by the heterogeneous nature of GAG chains and their dynamic spatial and temporal expression patterns. GAGs have a regulated structure overlaid by heterogeneity but lack the detail necessary to build structure/function relationships. In order to provide this information, we need glycomics platforms that are sensitive, robust, high throughput, and information rich. This review summarizes progress on mass-spectrometry-based GAG glycomics methods. The areas covered include disaccharide analysis, oligosaccharide profiling, and tandem mass spectrometric sequencing. PMID:23325770

  16. Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantitative determination of gamithromycin in animal plasma, lung tissue and pulmonary epithelial lining fluid.

    PubMed

    De Baere, Siegrid; Devreese, Mathias; Watteyn, Anneleen; Wyns, Heidi; Plessers, Elke; De Backer, Patrick; Croubels, Siska

    2015-06-12

    A sensitive and specific method for the quantitative determination of gamithromycin in animal plasma, lung tissue and pulmonary epithelial lining fluid (PELF) using liquid chromatography combined with heated electrospray ionization tandem mass spectrometry (LC-MS/MS) was developed. The sample preparation was rapid, straightforward and consisted of a deproteinization and phospholipid removal step using an Oasis(®) Ostro™ 96-well plate (chicken, turkey and calf plasma) or HybridSPE(®)-Phospholipid SPE cartridges (pig plasma and turkey lung tissue), while a liquid-liquid extraction with diethyl ether in alkaline medium was used for PELF of turkey poults. Chromatography was performed on a C18 Hypersil GOLD column using 0.01M ammonium acetate in water with a pH of 9, and acetonitrile as mobile phases. The MS/MS instrument was operated in the positive electrospray ionization mode and the following selected reaction monitoring transitions were monitored for gamithromycin (protonated molecule>product ion): m/z 777.45>619.35 and m/z 777.45>157.80 for quantification and identification, respectively. The method was validated in-house: matrix-matched calibration graphs were prepared and good linearity (r≥0.99) was achieved over the concentration ranges tested (2.5-10,000ngmL(-1) for chicken, pig and calf plasma; 5.0-2500ngmL(-1) for turkey plasma; 50-10,000ngg(-1) for turkey lung tissue and 20-1000ngmL(-1) for turkey PELF). Limits of quantification (LOQ) were 2.5ngmL(-1) for chicken, pig and calf plasma and 5.0ngmL(-1) for turkey plasma, while the limits of detection (LOD) ranged between 0.007 and 0.07ngmL(-1). For lung tissue and PELF, respective LOQ and LOD values of 50ngg(-1) and 0.76ngg(-1) (lung tissue) and 20ngmL(-1) and 0.1ngmL(-1) (PELF) were obtained. The results for the within-day and between-day precision, expressed as relative standard deviation (RSD), fell within the maximal RSD values. The accuracy fell within -30% to +10% (concentrations 1-10ngmL(-1)) or

  17. Ammonium chloride salting out extraction/cleanup for trace-level quantitative analysis in food and biological matrices by flow injection tandem mass spectrometry.

    PubMed

    Nanita, Sergio C; Padivitage, Nilusha L T

    2013-03-20

    A sample extraction and purification procedure that uses ammonium-salt-induced acetonitrile/water phase separation was developed and demonstrated to be compatible with the recently reported method for pesticide residue analysis based on fast extraction and dilution flow injection mass spectrometry (FED-FI-MS). The ammonium salts evaluated were chloride, acetate, formate, carbonate, and sulfate. A mixture of NaCl and MgSO4, salts used in the well-known QuEChERS method, was also tested for comparison. With thermal decomposition/evaporation temperature of <350°C, ammonium salts resulted in negligible ion source residual under typical electrospray conditions, leading to consistent method performance and less instrument cleaning. Although all ammonium salts tested induced acetonitrile/water phase separation, NH4Cl yielded the best performance, thus it was the preferred salting out agent. The NH4Cl salting out method was successfully coupled with FI/MS/MS and tested for fourteen pesticide active ingredients: chlorantraniliprole, cyantraniliprole, chlorimuron ethyl, oxamyl, methomyl, sulfometuron methyl, chlorsulfuron, triflusulfuron methyl, azimsulfuron, flupyrsulfuron methyl, aminocyclopyrachlor, aminocyclopyrachlor methyl, diuron and hexazinone. A validation study was conducted with nine complex matrices: sorghum, rice, grapefruit, canola, milk, eggs, beef, urine and blood plasma. The method is applicable to all analytes, except aminocyclopyrachlor. The method was deemed appropriate for quantitative analysis in 114 out of 126 analyte/matrix cases tested (applicability rate=0.90). The NH4Cl salting out extraction/cleanup allowed expansion of FI/MS/MS for analysis in food of plant and animal origin, and body fluids with increased ruggedness and sensitivity, while maintaining high-throughput (run time=30s/sample). Limits of quantitation (LOQs) of 0.01mgkg(-1) (ppm), the 'well-accepted standard' in pesticide residue analysis, were achieved in >80% of cases tested; while

  18. A mass spectrometry primer for mass spectrometry imaging

    PubMed Central

    Rubakhin, Stanislav S.; Sweedler, Jonathan V.

    2011-01-01

    Mass spectrometry imaging (MSI), a rapidly growing subfield of chemical imaging, employs mass spectrometry (MS) technologies to create single- and multi-dimensional localization maps for a variety of atoms and molecules. Complimentary to other imaging approaches, MSI provides high chemical specificity and broad analyte coverage. This powerful analytical toolset is capable of measuring the distribution of many classes of inorganics, metabolites, proteins and pharmaceuticals in chemically and structurally complex biological specimens in vivo, in vitro, and in situ. The MSI approaches highlighted in this Methods in Molecular Biology volume provide flexibility of detection, characterization, and identification of multiple known and unknown analytes. The goal of this chapter is to introduce investigators who may be unfamiliar with MS to the basic principles of the mass spectrometric approaches as used in MSI. In addition to guidelines for choosing the most suitable MSI method for specific investigations, cross-references are provided to the chapters in this volume that describe the appropriate experimental protocols. PMID:20680583

  19. A novel approach to the quantitative detection of anabolic steroids in bovine muscle tissue by means of a hybrid quadrupole time-of-flight-mass spectrometry instrument.

    PubMed

    Bussche, Julie Vanden; Decloedt, Anneleen; Van Meulebroek, Lieven; De Clercq, Nathalie; Lock, Stephen; Stahl-Zeng, Jianru; Vanhaecke, Lynn

    2014-09-19

    In recent years, the analysis of veterinary drugs and growth-promoting agents has shifted from target-oriented procedures, mainly based on liquid chromatography coupled to triple-quadrupole mass spectrometry (LC-QqQ-MS), towards accurate mass full scan MS (such as Time-of-Flight (ToF) and Fourier Transform (FT)-MS). In this study, the performance of a hybrid analysis instrument (i.e. UHPLC-QuadrupoleTime-of-Flight-MS (QqToF-MS)), able to exploit both full scan HR and MS/MS capabilities within a single analytical platform, was evaluated for confirmatory analysis of anabolic steroids (gestagens, estrogens including stilbenes and androgens) in meat. The validation data was compared to previously obtained results (CD 2002/657/EC) for QqQ-MS and single stage Orbitrap-MS. Additionally, a fractional factorial design was used to shorten and optimize the sample extraction. Validation according to CD 2002/657/EC demonstrated that steroid analysis using QqToF has a higher competing value towards QqQ-MS in terms of selectivity/specificity, compared to single stage Orbitrap-MS. While providing excellent linearity, based on lack-of-fit calculations (F-test, α=0.05 for all steroids except 17β-ethinylestradiol: α=0.01), the sensitivity of QqToF-MS proved for 61.8% and 85.3% of the compounds more sensitive compared to QqQ-MS and Orbitrap-MS, respectively. Indeed, the CCα values, obtained upon ToF-MS/MS detection, ranged from 0.02 to 1.74μgkg(-1) for the 34 anabolic steroids, while for QqQ-MS and Orbitrap-MS values ranged from 0.04 to 0.88μgkg(-1) and from 0.07 to 2.50μgkg(-1), respectively. Using QqToF-MS and QqQ-MS, adequate precision was obtained as relative standard deviations for repeatability and within-laboratory reproducibility, were below 20%. In case of Orbitrap-MS, some compounds (i.e. some estrogens) displayed poor precision, which was possibly caused by some lack of sensitivity at lower concentrations and the absence of MRM-like experiments. Overall, it can be

  20. Simultaneous quantitation of parabens, triclosan, and methyl triclosan in indoor house dust using solid phase extraction and gas chromatography-mass spectrometry.

    PubMed

    Fan, Xinghua; Kubwabo, Cariton; Rasmussen, Pat; Jones-Otazo, Heather

    2010-10-06

    An integrated analytical method for the simultaneous determination of five parabens (methyl-, ethyl-, propyl-, butyl-, and benzyl-), triclosan, and methyl triclosan in indoor house dust was developed based on gas chromatographic-mass spectrometric technique (GC/MS). Analytes were extracted from dust samples by sonication. After sample cleanup by solid-phase extraction (SPE), the extracts were derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and then analyzed by gas chromatography coupled with ion trap mass spectrometry operated in multiple reaction monitoring (MRM) mode. For quantitation, isotope-labelled internal standards were used for each corresponding target analyte. Only 0.05 g of dust sample was needed for the analysis. Method detection limits ranged from 6.5 to 10 ng/g, and absolute recoveries from 74% to 92%. The developed method demonstrated good repeatability and reproducibility, with relative standard deviations (RSDs) less than 16% for all the analytes. The analytes were determined in dust samples collected using two vacuum sampling methods from 63 Canadian homes: a sample of fresh or "active" dust (FD) collected using a Pullman-Holt vacuum sampler, and a composite sample taken from the household vacuum cleaner (HD). Methyl paraben, propyl paraben, and triclosan were detected in all HD and FD samples. HD samples yielded median values for methyl paraben, propyl paraben, and triclosan of 1080, 463, and 378 ng/g, respectively, which were comparable to the FD sample medians of 1120, 618 and 571 ng/g. Ethyl paraben was detected at frequencies of 89% in FD and 73% in HD samples, with median values of 52 and 25 ng/g, respectively. Butyl paraben was detected at frequencies of 44% in FD and 75% in HD samples, with median values of <10 and 59 ng/g, respectively. Benzyl paraben and methyl triclosan were not detected in any of the samples collected by either method. Samples collected according to the fresh dust protocol agreed with the

  1. Quantitative profiling of retinyl esters in milk from different ruminant species by using high performance liquid chromatography-diode array detection-tandem mass spectrometry.

    PubMed

    Rocchi, Silvia; Caretti, Fulvia; Gentili, Alessandra; Curini, Roberta; Perret, Daniela; Pérez-Fernández, Virginia

    2016-11-15

    An effective high performance liquid chromatography-diode array detection-tandem mass spectrometry (HPLC-DAD-MS/MS) analytical approach was developed for retinoid profiling in raw milk samples (cow, buffalo, ewe, and goat). The analytes were isolated by means of liquid-liquid extraction, including a "lipid freezing" step, with yields exceeding 66%. Since the positive atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) detection is not completely selective, a reliable identification has been accomplished by fully separating the analytes on a tandem C18/C30 column system under non-aqueous reversed phase (NARP) chromatography conditions. After validation, different milk varieties obtained from pasture-fed animals were analysed, providing, for the first time, the retinoid composition of both buffalo's and ewe's milk. According to the literature, retinyl palmitate has been found to be the most abundant vitamin A vitamer, but retinyl oleate is the prevalent form in the caprine milk.

  2. The quantitation of 2-oxo-3-hydroxy lysergic acid diethylamide (O-H-LSD) in human urine specimens, a metabolite of LSD: comparative analysis using liquid chromatography-selected ion monitoring mass spectrometry and liquid chromatography-ion trap mass spectrometry.

    PubMed

    Poch, G K; Klette, K L; Anderson, C

    2000-04-01

    This paper compares the potential forensic application of two sensitive and rapid procedures (liquid chromatography-mass spectrometry and liquid chromatography-ion trap mass spectrometry) for the detection and quantitation of 2-oxo-3-hydroxy lysergic acid diethylamide (O-H-LSD) a major LSD metabolite. O-H-LSD calibration curves for both procedures were linear over the concentration range 0-8,000 pg/mL with correlation coefficients (r2) greater than 0.99. The observed limit of detection (LOD) and limit of quantitation (LOQ) for O-H-LSD in both procedures was 400 pg/mL. Sixty-eight human urine specimens that had previously been found to contain LSD by gas chromatography-mass spectrometry were reanalyzed by both procedures for LSD and O-H-LSD. These specimens contained a mean concentration of O-H-LSD approximately 16 times higher than the LSD concentration. Because both LC methods produce similar results, either procedure can be readily adapted to O-H-LSD analysis for use in high-volume drug-testing laboratories. In addition, the possibility of significantly increasing the LSD detection time window by targeting this major LSD metabolite for analysis may influence other drug-free workplace programs to test for LSD.

  3. Affinity membrane introduction mass spectrometry

    SciTech Connect

    Xu, C.; Patrick, J.S.; Cooks, R.G. )

    1995-02-15

    A new technique, affinity membrane introduction mass spectrometry, is described. In this method, a chemically modified membrane is used to selectively adsorb analytes bearing a particular functional group and concentrate them from solution. Release of the bound analyte results in its transfer across the membrane and allows it to be monitored mass spectrometrically, using, in the present case, a benchtop ion trap instrument. Alkylamine-modified cellulose membranes are used to bind substituted benzaldehydes through imine formation at high pH. Release of the bound aldehyde is achieved by acid hydrolysis of the surface-bound imine. Benzaldehyde is detected with excellent specificity at 10 ppm in a complex mixture using this method. Using the enrichment capability of the membrane, a full mass spectrum of benzaldehyde can be measured at a concentration of 10 ppb. The behavior of a variety of other aldehydes is also discussed to illustrate the capabilities of the method. 21 refs., 5 figs., 2 tabs.

  4. Electrophoresis-mass spectrometry probe

    DOEpatents

    Andresen, Brian D.; Fought, Eric R.

    1987-01-01

    The invention involves a new technique for the separation of complex mixtures of chemicals, which utilizes a unique interface probe for conventional mass spectrometers which allows the electrophoretically separated compounds to be analyzed in real-time by a mass spectrometer. This new chemical analysis interface, which couples electrophoresis with mass spectrometry, allows complex mixtures to be analyzed very rapidly, with much greater specificity, and with greater sensitivity. The interface or probe provides a means whereby large and/or polar molecules in complex mixtures to be completely characterized. The preferred embodiment of the probe utilizes a double capillary tip which allows the probe tip to be continually wetted by the buffer, which provides for increased heat dissipation, and results in a continually operating interface which is more durable and electronically stable than the illustrated single capillary tip probe interface.

  5. Electrophoresis-mass spectrometry probe

    DOEpatents

    Andresen, B.D.; Fought, E.R.

    1987-11-10

    The invention involves a new technique for the separation of complex mixtures of chemicals, which utilizes a unique interface probe for conventional mass spectrometers which allows the electrophoretically separated compounds to be analyzed in real-time by a mass spectrometer. This new chemical analysis interface, which couples electrophoresis with mass spectrometry, allows complex mixtures to be analyzed very rapidly, with much greater specificity, and with greater sensitivity. The interface or probe provides a means whereby large and/or polar molecules in complex mixtures to be completely characterized. The preferred embodiment of the probe utilizes a double capillary tip which allows the probe tip to be continually wetted by the buffer, which provides for increased heat dissipation, and results in a continually operating interface which is more durable and electronically stable than the illustrated single capillary tip probe interface. 8 figs.

  6. Toward Single-Molecule Nanomechanical Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Roukes, Michael

    2009-03-01

    Mass spectrometry (MS) has become a preeminent methodology of proteomics since it provides rapid and quantitative identification of protein species with relatively low sample consumption. Yet with the trend toward biological analysis at increasingly smaller scales, ultimately down to the volume of an individual cell, MS with few-to-single molecule resolution will be required. We report the first realization of MS based on single-biological-molecule detection with nanoelectromechanical systems (NEMS). NEMS provide unparalleled mass resolution, now sufficient for detection of individual molecular species in real time. However, high sensitivity is only one of several components required for MS. We demonstrate a first complete prototype NEMS-MS system for single-molecule mass spectrometry providing proof-of-principle for this new technique. Nanoparticles and protein species are introduced by electrospray injection from the fluid phase in ambient conditions into vacuum and subsequently delivered to the NEMS detector by hexapole ion optics . Mass measurements are then recorded in real-time as analytes adsorb, one-by-one, onto a phase-locked, ultrahigh frequency (UHF) NEMS resonator. These first NEMS-MS spectra, obtained with modest resolution from only several hundred mass adsorption events, presage the future capabilities of this methodology. We outline the substantial improvements feasible in near term, through recent advances and technological avenues that are unique to NEMS-MS.

  7. High Technology Mass Spectrometry Laboratory

    DTIC Science & Technology

    2010-08-01

    GSH, hemoglobin beta-Cys93 ( Hb -C93-AN) were monitored. The second order rate constants in M-ls-1 were: disappe 0.0806; appearance of GS-AN in whole...blood, 0.0776, appearance of Hb -C9 appearance of AbC34-AN in plasma, 0.224. The data indicate that the mos blood is Cys34 of albumin. This site...than Hb -C93 15. SUBJECT TERMS acrylonitrile, adduct, mass spectrometry, biomarker, toxic industrial chemicals 16. SECURITY CLASSIFICATION OF: a

  8. A novel derivatization-based liquid chromatography tandem mass spectrometry method for quantitative characterization of naphthenic acid isomer profiles in environmental waters.

    PubMed

    Woudneh, Million B; Coreen Hamilton, M; Benskin, Jonathan P; Wang, Guanghui; McEachern, Preston; Cosgrove, John R

    2013-06-07

    A method for quantitative characterization of naphthenic acid (NA) isomer groups by carbon number and extent of cyclization was developed and validated with water samples from northern Alberta. Following solid phase extraction, NAs undergo derivatization with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) allowing detection by positive electrospray ionization tandem mass spectrometry (+ESI)-MS/MS. NA-EDC derivatives produce a common product ion by MS/MS, regardless of structure of the starting NA. Thus, approximately constant relative response factors (RRFs) were assumed for the various isomer groups that elute at a given point in the elution gradient (supported by calculated RRFs for individual model NAs), facilitating quantification using a single standard (1-pyrenebutyric acid). To reduce the impact of major background fatty acids on NA data, the method employed an optimized liquid chromatography method that separated straight chain (Z=0) analytes from other NAs. Method validation was performed at two spiking levels (7.72μg and 38.6μg total refined Merichem per 500mL of reagent water) and good accuracy (mean recoveries of 82.4±2.5% and 93.0±2.6%, respectively; range ~50-130%) and precision (<17% RSD) were achieved at both spiking levels for all 60 NA isomer groups. The method also performed well in an independent method comparison study in which method accuracy values of 107%, 120%, and 121% were obtained for 2 spiked reagent waters (1mg/L and 50mg/L NAs) and spiked Athabasca River water (0.035mg/L NAs), respectively. Application of the method to samples from northern Alberta revealed that NA concentrations decreased in the order: process water (52.8mg/L)>tailings pond water (30.6mg/L)>well water (0.086mg/L)>surface water (0.007mg/L), and that samples were distinguishable by NA isomer profile using Principal components analysis.

  9. Quantitation of the Minor Tobacco Alkaloids Nornicotine, Anatabine, and Anabasine in Smokers' Urine by High Throughput Liquid Chromatography-Mass Spectrometry.

    PubMed

    von Weymarn, Linda B; Thomson, Nicole M; Donny, Eric C; Hatsukami, Dorothy K; Murphy, Sharon E

    2016-03-21

    Nicotine is the most abundant alkaloid in tobacco accounting for 95% of the alkaloid content. There are also several minor tobacco alkaloids; among these are nornicotine, anatabine, and anabasine. We developed and applied a 96 well plate-based capillary LC-tandem mass spectrometry method for the analysis of nornicotine, anatabine, and anabasine in urine. The method was validated with regard to accuracy and precision. Anabasine was quantifiable to low levels with a limit of quantitation (LOQ) of 0.2 ng/mL even when nicotine, which is isobaric, is present at concentrations >2500-fold higher than anabasine. This attribute of the method is important since anatabine and anabasine in urine have been proposed as biomarkers of tobacco use for individuals using nicotine replacement therapies. In the present study, we analyzed the three minor tobacco alkaloids in urine from 827 smokers with a wide range of tobacco exposures. Nornicotine (LOQ 0.6 ng/mL) was detected in all samples, and anatabine (LOQ, 0.15 ng/mL) and anabasine were detected in 97.7% of the samples. The median urinary concentrations of nornicotine, anatabine, and anabasine were 98.9, 4.02, and 5.53 ng/mL. Total nicotine equivalents (TNE) were well correlated with anatabine (r(2) = 0.714) and anabasine (r(2) = 0.760). TNE was most highly correlated with nornicotine, which is also a metabolite of nicotine. Urine samples from a subset of subjects (n = 110) were analyzed for the presence of glucuronide conjugates by quantifying any increase in anatabine and anabasine concentrations after β-glucuronidase treatment. The median ratio of the glucuronidated to free anatabine was 0.74 (range, 0.1 to 10.9), and the median ratio of glucuronidated to free anabasine was 0.3 (range, 0.1 to 2.9). To our knowledge, this is the largest population of smokers for whom the urinary concentrations of these three tobacco alkaloids has been reported.

  10. Detection and Quantitation of Acrolein-Derived 1,N2-Propanodeoxyguanosine Adducts in Human Lung by Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry

    PubMed Central

    Zhang, Siyi; Villalta, Peter W.; Wang, Mingyao; Hecht, Stephen S.

    2008-01-01

    Acrolein, a widely distributed environmental pollutant, reacts with dGuo in DNA to form two pairs of 1,N2-propano-dGuo adducts: (6R/S)-3-(2′-deoxyribos-1′-yl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-10(3H)one (α-OH-Acr-dGuo) and (8R/S)-3-(2′-deoxyribos-1′-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)one (γ-OH-Acr-dGuo). α-OH-Acr-dGuo is the more mutagenic and induces mainly G→T transversions. A recent study demonstrated that acrolein DNA adducts are preferentially formed in p53 mutational hotspots in human lung cancer, but there are no reports on the presence of these adducts in human lung. To directly investigate this question, we have developed a sensitive and specific liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for quantitative analysis of Acr-dGuo adducts in DNA. Our method is based on the enzymatic hydrolysis of DNA isolated from human lung in the presence of [13C10,15N5]Acr-dGuo as internal standards. Acr-dGuo adducts are enriched from the hydrolysates by solid phase extraction and analyzed by LC-ESI-MS/MS using selected reaction monitoring. The method is accurate and precise, and the identity of the adducts was confirmed by monitoring different transitions from the same parent ion, and by carrying out reactions with NaOH and NaBH4, which produced N2-(3-hydroxypropyl)dGuo or 1,N2-(1,3-propano)dGuo from γ-OH-Acr-dGuo and α-OH-Acr-dGuo, respectively. Thirty DNA samples from lung tissue were analyzed and Acr-dGuo adducts were detected in all samples. Both α-OH- and γ-OH-Acr-dGuo were observed in most of the samples; total adduct concentrations ranged from 16 – 209 adducts/109 nucleotides. These results demonstrate for the first time that both types of Acr-dGuo adducts are present in human lung DNA. There was no difference in adduct levels between current and ex-smokers. Collectively, the results support a plausible role for acrolein as one cause of p53 mutations in human

  11. Neuroscience and Accelerator Mass Spectrometry

    SciTech Connect

    Palmblad, M N; Buchholz, B A; Hillegonds, D J; Vogel, J S

    2004-08-02

    Accelerator mass spectrometry (AMS) is a mass spectrometric method for quantifying rare isotopes. It has had great impact in geochronology and archaeology and is now being applied in biomedicine. AMS measures radioisotopes such as {sup 3}H, {sup 14}C, {sup 26}Al, {sup 36}Cl and {sup 41}Ca, with zepto- or attomole sensitivity and high precision and throughput, enabling safe human pharmacokinetic studies involving: microgram doses, agents having low bioavailability, or toxicology studies where administered doses must be kept low (<1 {micro}g/kg). It is used to study long-term pharmacokinetics, to identify biomolecular interactions, to determine chronic and low-dose effects or molecular targets of neurotoxic substances, to quantify transport across the blood-brain barrier and to resolve molecular turnover rates in the human brain on the timescale of decades. We will here review how AMS is applied in neurotoxicology and neuroscience.

  12. Neuroscience and accelerator mass spectrometry.

    PubMed

    Palmblad, Magnus; Buchholz, Bruce A; Hillegonds, Darren J; Vogel, John S

    2005-02-01

    Accelerator mass spectrometry (AMS) is a mass spectrometric method for quantifying rare isotopes. It has had a great impact in geochronology and archaeology and is now being applied in biomedicine. AMS measures radioisotopes such as 3H, 14C, 26Al, 36Cl and 41Ca, with zepto- or attomole sensitivity and high precision and throughput, allowing safe human pharmacokinetic studies involving microgram doses, agents having low bioavailability or toxicology studies where administered doses must be kept low (<1 microg kg(-1)). It is used to study long-term pharmacokinetics, to identify biomolecular interactions, to determine chronic and low-dose effects or molecular targets of neurotoxic substances, to quantify transport across the blood-brain barrier and to resolve molecular turnover rates in the human brain on the time-scale of decades. We review here how AMS is applied in neurotoxicology and neuroscience.

  13. Mass spectrometry in combinatorial chemistry.

    PubMed

    Enjalbal, C; Martinez, J; Aubagnac, J L

    2000-01-01

    In the fast expanding field of combinatorial chemistry, profiling libraries has always been a matter of concern--as illustrated by the buoyant literature over the past seven years. Spectroscopic methods, including especially mass spectrometry and to a lesser extent IR and NMR, have been applied at different levels of combinatorial library synthesis: in the rehearsal phase to optimize the chemistry prior to library generation, to confirm library composition, and to characterize after screening each structure that exhibits positive response. Most of the efforts have been concentrated on library composition assessment. The difficulties of such analyses have evolved from the infancy of the combinatorial concept, where large mixtures were prepared, to the recent parallel syntheses of collections of discrete compounds. Whereas the complexity of the analyses has diminished, an increased degree of automation was simultaneously required to achieve efficient library component identification and quantification. In this respect, mass spectrometry has been found to be the method of choice, providing rapid, sensitive, and informative analyses, especially when coupled to chromatographic separation. Fully automated workstations able to cope with several hundreds of compounds per day have been designed. After a brief introduction to describe the combinatorial approach, library characterization will be discussed in detail, considering first the solution-based methodologies and secondly the support-bound material analyses.

  14. Triple Quadrupole Versus High Resolution Quadrupole-Time-of-Flight Mass Spectrometry for Quantitative LC-MS/MS Analysis of 25-Hydroxyvitamin D in Human Serum

    NASA Astrophysics Data System (ADS)

    Geib, Timon; Sleno, Lekha; Hall, Rabea A.; Stokes, Caroline S.; Volmer, Dietrich A.

    2016-08-01

    We describe a systematic comparison of high and low resolution LC-MS/MS assays for quantification of 25-hydroxyvitamin D3 in human serum. Identical sample preparation, chromatography separations, electrospray ionization sources, precursor ion selection, and ion activation were used; the two assays differed only in the implemented final mass analyzer stage; viz. high resolution quadrupole-quadrupole-time-of-flight (QqTOF) versus low resolution triple quadrupole instruments. The results were assessed against measured concentration levels from a routine clinical chemiluminescence immunoassay. Isobaric interferences prevented the simple use of TOF-MS spectra for extraction of accurate masses and necessitated the application of collision-induced dissociation on the QqTOF platform. The two mass spectrometry assays provided very similar analytical figures of merit, reflecting the lack of relevant isobaric interferences in the MS/MS domain, and were successfully applied to determine the levels of 25-hydroxyvitamin D for patients with chronic liver disease.

  15. Triple Quadrupole Versus High Resolution Quadrupole-Time-of-Flight Mass Spectrometry for Quantitative LC-MS/MS Analysis of 25-Hydroxyvitamin D in Human Serum.

    PubMed

    Geib, Timon; Sleno, Lekha; Hall, Rabea A; Stokes, Caroline S; Volmer, Dietrich A

    2016-08-01

    We describe a systematic comparison of high and low resolution LC-MS/MS assays for quantification of 25-hydroxyvitamin D3 in human serum. Identical sample preparation, chromatography separations, electrospray ionization sources, precursor ion selection, and ion activation were used; the two assays differed only in the implemented final mass analyzer stage; viz. high resolution quadrupole-quadrupole-time-of-flight (QqTOF) versus low resolution triple quadrupole instruments. The results were assessed against measured concentration levels from a routine clinical chemiluminescence immunoassay. Isobaric interferences prevented the simple use of TOF-MS spectra for extraction of accurate masses and necessitated the application of collision-induced dissociation on the QqTOF platform. The two mass spectrometry assays provided very similar analytical figures of merit, reflecting the lack of relevant isobaric interferences in the MS/MS domain, and were successfully applied to determine the levels of 25-hydroxyvitamin D for patients with chronic liver disease. Graphical Abstract ᅟ.

  16. Clinical Application of Ambient Ionization Mass Spectrometry

    PubMed Central

    Li, Li-Hua; Hsieh, Hua-Yi; Hsu, Cheng-Chih

    2017-01-01

    Ambient ionization allows mass spectrometry analysis directly on the sample surface under atmospheric pressure with almost zero sample pretreatment. Since the development of desorption electrospray ionization (DESI) in 2004, many other ambient ionization techniques were developed. Due to their simplicity and low operation cost, rapid and on-site clinical mass spectrometry analysis becomes real. In this review, we will highlight some of the most widely used ambient ionization mass spectrometry approaches and their applications in clinical study. PMID:28337399

  17. Simultaneous qualitative and quantitative analysis of flavonoids and alkaloids from the leaves of Nelumbo nucifera Gaertn. using high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry.

    PubMed

    Guo, Yujie; Chen, Xi; Qi, Jin; Yu, Boyang

    2016-07-01

    A reliable method, combining qualitative analysis by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry and quantitative assessment by high-performance liquid chromatography with photodiode array detection, has been developed to simultaneously analyze flavonoids and alkaloids in lotus leaf extracts. In the qualitative analysis, a total of 30 compounds, including 12 flavonoids, 16 alkaloids, and two proanthocyanidins, were identified. The fragmentation behaviors of four types of flavone glycoside and three types of alkaloid are summarized. The mass spectra of four representative components, quercetin 3-O-glucuronide, norcoclaurine, nuciferine, and neferine, are shown to illustrate their fragmentation pathways. Five pairs of isomers were detected and three of them were distinguished by comparing the elution order with reference substances and the mass spectrometry data with reported data. In the quantitative analysis, 30 lotus leaf samples from different regions were analyzed to investigate the proportion of eight representative compounds. Quercetin 3-O-glucuronide was found to be the predominant constituent of lotus leaf extracts. For further discrimination among the samples, hierarchical cluster analysis, and principal component analysis, based on the areas of the eight quantitative peaks, were carried out.

  18. Development and full validation of a sensitive quantitative assay for the determination of clemastine in human plasma by liquid chromatography-tandem mass spectrometry.

    PubMed

    Xie, Zhiyong; Liao, Qiongfeng; Li, Zuojun; Zhu, Chenchen; Zeng, Yuaner; Liu, Shikun

    2007-08-15

    A sensitive high-performance liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of clemastine in human plasma. After having been extracted from plasma samples by ethyl acetate, clemastine and internal standard, diphenhydramine, were separated on a C(18) column. Detection was performed on Thermo Finnigan TSQ Quantum triple quadrupole mass spectrometer by selected reaction monitoring (SRM) mode via electrospray ionization (ESI) source. The method was linear in the concentration range of 5.0-1000.0 pg/ml for clemastine. The intra- and inter-day precisions were within 13.4% and the deviations were between -1.1% and 5.6%. The fully validated LC/ESI-MS/MS method has been successfully applied to the preliminary pharmacokinetic study in healthy male Chinese volunteers.

  19. Simultaneous quantitation of metformin and sitagliptin from mouse and human dried blood spots using laser diode thermal desorption tandem mass spectrometry.

    PubMed

    Swales, John G; Gallagher, Richard T; Denn, Mark; Peter, Raimund M

    2011-06-01

    A simple, rapid and robust high-throughput assay for the simultaneous analysis of metformin and sitagliptin from mouse and human dried blood spot samples using laser diode thermal desorption interfaced with atmospheric pressure chemical ionization tandem mass spectrometry (LDTD-APCI-MS/MS) was developed for use in a pharmaceutical discovery environment as an alternative to traditional plasma analysis. Analytes were extracted from dried blood spots using a simple punch disc and solvent extract procedure. Details of the method development and optimization of the instrumental parameters are presented. The method was successfully applied to spiked mouse and human dried blood spot samples. Analyte stability was determined in dried blood spots on FTA cards and as extracts of dried blood spots. The method was subsequently used to determine the oral pharmacokinetics of metformin and sitagliptin after dosing to male mice. Metformin and Sitagliptin results are compared to data generated by more traditional liquid chromatography-mass spectrometry methods. Intra-assay and inter-assay accuracy and precision across the analytes and species deviated by less than 30% at all calibration levels and less than 20% at all quality control levels.

  20. Evaluation of Laser Ablation Inductively Coupled Plasma Mass Spectrometry for the Quantitative Determination of Lead in Different Parts of Archeological Human Teeth

    PubMed Central

    Bellis, David J.; Parsons, Patrick J.; Jones, Joseph; Amarasiriwardena, Dula

    2011-01-01

    The lead content of teeth or tooth-parts has been used as a biomarker of cumulative lead exposure in clinical, epidemiological, environmental, and archaeological studies. Through the application of laser ablation inductively coupled plasma mass spectrometry, a pilot study of the micrometer-scale distribution and quantification of lead was conducted for two human teeth obtained from an archeological burial site in Manhattan, New York, USA. Lead was highly localized within each tooth, with accumulation in circumpulpal dentine and cementum. The maximum localized lead content in circumpulpal dentine was remarkably high, almost 2000 μg g-1, compared to the mean enamel and dentine content of about 5 μg g-1. The maximum lead content in cementum was approximately 700 μg g-1. The large quantity of cementum found in the teeth suggested that the subjects had hypercementosis (excess cementum formation) of the root, a condition reported to have been prevalent among African-American slave populations. The distribution of lead in these human teeth was remarkably similar to the distribution that we previously reported in the teeth of present-day lead-dosed goats. The data shown demonstrate the feasibility of using laser ablation inductively coupled plasma mass spectrometry to examine lead exposure in archaeological studies. PMID:22467976

  1. Identification and quantitative analysis of physalin D and its metabolites in rat urine and feces by liquid chromatography with triple quadrupole time-of-flight mass spectrometry.

    PubMed

    Zheng, Yunliang; Cao, Cong; Lin, Meihua; Zhai, You; Ge, Zhiwei; ShenTu, Jianzhong; Wu, Lihua; Hu, Xingjiang

    2017-04-07

    Physalin D is known to show extensive bioactivities. However, no excretion study has elucidated the excretion of physalin D and its metabolites. This study investigates the excretion of physalin D and its metabolites in rats. Metabolites in rat urine and feces were separated and identified by liquid chromatography with triple quadrupole time-of-flight mass spectrometry. Furthermore, a validated high-performance liquid chromatography with tandem mass spectrometry method was developed to quantify physalin D, physalin D glucuronide, and physalin D sulfate in rats feces and urine after the intragastric administration of physalin D. The analyte showed good linearity over a wide concentration range (r > 0.995), and the lower limit of quantification was 0.0532 μg/mL and 0.226 μg/g for urine and feces, respectively. Nine metabolites, including five phase I and four phase II metabolites, were identified and clarified after dosing in vivo. Only 4.0% of the gavaged dose, including physalin D and its phase II metabolites, was excreted in urine, whereas 10.8% was found in feces in the unchanged form. The results indicate that the extensive and rapid metabolism may be the main factors leading to the short half-life of physalin D. These results can provide a basis for further studies on the structural modification and pharmacology of physalin D. This article is protected by copyright. All rights reserved.

  2. Global identification and quantitative analysis of chemical constituents in traditional Chinese medicinal formula Qi-Fu-Yin by ultra-high performance liquid chromatography coupled with mass spectrometry.

    PubMed

    Li, Meng-Ning; Dong, Xin; Gao, Wen; Liu, Xin-Guang; Wang, Rui; Li, Ping; Yang, Hua

    2015-10-10

    Qi-Fu-Yin (QFY), a classical traditional Chinese medicine formula, is proven to have significant neuroprotective effects by modern pharmacological studies. However, the chemical constituents of QFY have not been fully explored. In this study, an ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF MS) was developed for comprehensive analysis of chemical constituents in QFY. By using characteristic ions and fragmentation rules, a reliable identification of 156 compounds was described here, including 69 triterpene saponins, 23 oligosaccharide esters, 22 flavanoids, 9 alkaloids, 9 phenolic acids, 8 phthalides, 7 phenylethanoid glycosides, 3 xanthones, 3 sesquiterpene lactones, 2 ionones and 1 iridoid glycoside. Twenty-six major compounds were then determined in a single run by UHPLC coupled with triple quadrupole tandem mass spectrometry (QQQ MS) with fast positive/negative polarity switching. It allows for the acquisition of MS data in both ionization modes from a single run. The proposed method was then validated in terms of linearity, accuracy, precision and recovery. The overall recoveries for 26 analytes ranged from 91.35% to 109.58%, with RSDs ranging from 0.82% to 4.83%. In addition, the content of 26 analytes in QFY prepared by five batches of herbal materials was also analyzed. These results demonstrated that our present method was effective and reliable for comprehensive quality evaluation of QFY. Meanwhile, the study might provide the chemical evidence for revealing the material basis of its therapeutic effects.

  3. High-throughput and sensitive quantitation of plasma catecholamines by ultraperformance liquid chromatography-tandem mass spectrometry using a solid phase microwell extraction plate.

    PubMed

    Dunand, Marielle; Gubian, Danilo; Stauffer, Maxime; Abid, Karim; Grouzmann, Eric

    2013-04-02

    Plasma catecholamines provide a reliable biomarker of sympathetic activity. The low circulating concentrations of catecholamines and analytical interferences require tedious sample preparation and long chromatographic runs to ensure their accurate quantification by HPLC with electrochemical detection. Published or commercially available methods relying on solid phase extraction technology lack sensitivity or require derivatization of catecholamine by hazardous reagents prior to tandem mass spectrometry (MS) analysis. Here, we manufactured a novel 96-well microplate device specifically designed to extract plasma catecholamines prior to their quantification by a new and highly sensitive ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. Processing time, which included sample purification on activated aluminum oxide and elution, is less than 1 h per 96-well microplate. The UPLC-MS/MS analysis run time is 2.0 min per sample. This UPLC-MS/MS method does not require a derivatization step, reduces the turnaround time by 10-fold compared to conventional methods used for routine application, and allows catecholamine quantification in reduced plasma sample volumes (50-250 μL, e.g., from children and mice).

  4. Quantitative subcellular imaging of boron compounds in individual mitotic and interphase human glioblastoma cells with imaging secondary ion mass spectrometry (SIMS).

    PubMed

    Chandra, S; Tjarks, W; Lorey, D R; Barth, R F

    2008-01-01

    Boron measurements at subcellular scale are essential in boron neutron capture therapy (BNCT) of cancer as the nuclear localization of boron-10 atoms can enhance the effectiveness of killing individual tumour cells. Since tumours contain a heterogeneous population of cells in interphase as well as in the M phase (mitotic division) of the cell cycle, it is important to evaluate the subcellular distribution of boron in both phases. In this work, the secondary ion mass spectrometry (SIMS) based imaging technique of ion microscopy was used to quantitatively image boron from two BNCT agents, clinically used p-boronophenylalanine (BPA) and 3-[4-(o-carboran-1-yl)butyl]thymidine (N4), in mitotic metaphase and interphase human glioblastoma T98G cells. N4 belongs to a class of experimental BNCT agents, designated 3-carboranyl thymidine analogues (3CTAs), which presumably accumulate selectively in cancer cells due to a process referred to as kinase-mediated trapping (KMT). The cells were exposed to BPA for 1 h and N4 for 2 h. A CAMECA IMS-3f SIMS ion microscope instrument capable of producing isotopic images with 500 nm spatial resolution was used in the study. Observations were made in cryogenically prepared fast frozen, and freeze-fractured, freeze-dried cells. Three discernible subcellular regions were studied: the nucleus, a characteristic mitochondria-rich perinuclear cytoplasmic region, and the remaining cytoplasm in interphase T98G cells. In metaphase cells, the chromosomes and the cytoplasm were studied for boron localization. Intracellular concentrations of potassium and sodium also were measured in each cell in which the subcellular boron concentrations were imaged. Since the healthy cells maintain a K/Na ratio of approximately 10 due to the presence of Na-K-ATPase in the plasma membrane of mammalian cells, these measurements provided validation for cryogenic sample preparation and indicated the analysis healthy, well preserved cells. The BPA-treated interphase

  5. Propolis specimens from different locations of central Italy: chemical profiling and gas chromatography-mass spectrometry (GC-MS) quantitative analysis of the allergenic esters benzyl cinnamate and benzyl salicylate.

    PubMed

    Aliboni, Andrea; D'Andrea, Armando; Massanisso, Paolo

    2011-01-12

    Propolis is a beehive product popular in natural medicine thanks to its noteworthy properties. Propolis is non-toxic but is responsible for allergic reactions in sensitive individuals. In this paper, we propose a new gas chromatography-mass spectrometry (GC-MS) analytical methodology for the quantitative analysis of two allergenic esters in propolis specimens, benzyl salicylate and benzyl cinnamate, and test it on specimens from different locations of central Italy. We also present the results obtained in the chemical characterization of the same specimens. The characterization showed that the resin fractions of all of the specimens are of poplar origin.

  6. 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.

  7. Quantitation of Cotinine and its Metabolites in Rat Plasma and Brain Tissue by Hydrophilic Interaction Chromatography Tandem Mass Spectrometry (HILIC-MS/MS)

    PubMed Central

    Li, Pei; Beck, Wayne D.; Callahan, Patrick M.; Terry, Alvin V.; Bartlett, Michael G.

    2014-01-01

    In this work, we developed a sensitive method to quantify cotinine (COT), norcotinine (NCOT), trans-3′-hydroxycotinine (OHCOT) and cotinine-N-oxide (COTNO) in rat plasma and brain tissue, using solid phase extraction (SPE), hydrophilic interaction liquid chromatography (HILIC) and tandem mass spectrometry (MS/MS). The linear range was 1–100 ng/ml for each analyte in rat plasma and brain homogenate (3–300 ng/g brain tissue). The method was validated with precision within 15% relative standard deviation (RSD) and accuracy within 15% relative error (RE). Stable isotope-labeled internal standards (IS) were used for all the analytes to achieve good reproducibility, minimizing the influence of recovery and matrix effects. This method can be used in future studies to simultaneously determine the concentrations of COT and three major metabolites in rat plasma and brain tissue. PMID:23022114

  8. Detection and quantitation of benzo(a)pyrene-derived DNA adducts in mouse liver by liquid chromatography - tandem mass spectrometry: comparison with P-32-postlabeling

    SciTech Connect

    Singh, R.; Gaskell, M.; Le Pla, R.C.; Kaur, B.; Azim-Araghi, A.; Roach, J.; Koukouves, G.; Souliotis, V.L.; Kyrtopoulos, S.A.; Farmer, P.B.

    2006-06-19

    The polycyclic aromatic hydrocarbon, benzo(a)pyrene (B(a)P) is a proven animal carcinogen that is potentially carcinogenic to humans. B( a)P is an ubiquitous environmental pollutant and is also present in tobacco smoke, coal tar, automobile exhaust emissions, and charred food. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using electrospray ionization and selected reaction monitoring (SRM) has been developed for the detection of 10-(deoxyguanosin-N{sub 2}-yl)-7,8,9-trihydroxy-7,8,9,10- tetrahydrobenzo(a)pyrene (B(a)PDE-N{sub 2}dG) adducts formed in DNA following the metabolic activation of B(a)P to benzo(a) pyrene-7,8-dihydrodiol-9,10-epoxide (B(a)PDE).

  9. Validation of a headspace trap gas chromatography and mass spectrometry method for the quantitative analysis of volatile compounds from degraded rapeseed oil.

    PubMed

    Sghaier, Lilia; Cordella, Christophe B Y; Rutledge, Douglas N; Watiez, Mickaël; Breton, Sylvie; Sassiat, Patrick; Thiebaut, Didier; Vial, Jérôme

    2016-05-01

    Due to lipid oxidation, off-flavors, characterized by a fishy odor, are emitted during the heating of rapeseed oil in a fryer and affect the flavor of rapeseed oil even at low concentrations. Thus, there is a need for analytical methods to identify and quantify these products. To study the headspace composition of degraded rapeseed oil, and more specifically the compounds responsible for the fishy odor, a headspace trap gas chromatography with mass spectrometry method was developed and validated. Six volatile compounds formed during the degradation of rapeseed oil were quantified: 1-penten-3-one, (Z)-4-heptenal, hexanal, nonanal, (E,E)-heptadienal, and (E)-2-heptenal. Validation using accuracy profiles allowed us to determine the valid ranges of concentrations for each compound, with acceptance limits of 40% and tolerance limits of 80%. This method was then successfully applied to real samples of degraded oils.

  10. Quantitative Analysis of Bioactive Compounds from Aromatic Plants by Means of Dynamic Headspace Extraction and Multiple Headspace Extraction-Gas Chromatography-Mass Spectrometry.

    PubMed

    Omar, Jone; Olivares, Maitane; Alonso, Ibone; Vallejo, Asier; Aizpurua-Olaizola, Oier; Etxebarria, Nestor

    2016-04-01

    Seven monoterpenes in 4 aromatic plants (sage, cardamom, lavender, and rosemary) were quantified in liquid extracts and directly in solid samples by means of dynamic headspace-gas chromatography-mass spectrometry (DHS-GC-MS) and multiple headspace extraction-gas chromatography-mass spectrometry (MHSE), respectively. The monoterpenes were 1st extracted by means of supercritical fluid extraction (SFE) and analyzed by an optimized DHS-GC-MS. The optimization of the dynamic extraction step and the desorption/cryo-focusing step were tackled independently by experimental design assays. The best working conditions were set at 30 °C for the incubation temperature, 5 min of incubation time, and 40 mL of purge volume for the dynamic extraction step of these bioactive molecules. The conditions of the desorption/cryo-trapping step from the Tenax TA trap were set at follows: the temperature was increased from 30 to 300 °C at 150 °C/min, although the cryo-trapping was maintained at -70 °C. In order to estimate the efficiency of the SFE process, the analysis of monoterpenes in the 4 aromatic plants was directly carried out by means of MHSE because it did not require any sample preparation. Good linearity (r2) > 0.99) and reproducibility (relative standard deviation % <12) was obtained for solid and liquid quantification approaches, in the ranges of 0.5 to 200 ng and 10 to 500 ng/mL, respectively. The developed methods were applied to analyze the concentration of 7 monoterpenes in aromatic plants obtaining concentrations in the range of 2 to 6000 ng/g and 0.25 to 110 μg/mg, respectively.

  11. Inorganic trace analysis by mass spectrometry

    NASA Astrophysics Data System (ADS)

    Becker, Johanna Sabine; Dietze, Hans-Joachim

    1998-10-01

    Mass spectrometric methods for the trace analysis of inorganic materials with their ability to provide a very sensitive multielemental analysis have been established for the determination of trace and ultratrace elements in high-purity materials (metals, semiconductors and insulators), in different technical samples (e.g. alloys, pure chemicals, ceramics, thin films, ion-implanted semiconductors), in environmental samples (waters, soils, biological and medical materials) and geological samples. Whereas such techniques as spark source mass spectrometry (SSMS), laser ionization mass spectrometry (LIMS), laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), glow discharge mass spectrometry (GDMS), secondary ion mass spectrometry (SIMS) and inductively coupled plasma mass spectrometry (ICP-MS) have multielemental capability, other methods such as thermal ionization mass spectrometry (TIMS), accelerator mass spectrometry (AMS) and resonance ionization mass spectrometry (RIMS) have been used for sensitive mono- or oligoelemental ultratrace analysis (and precise determination of isotopic ratios) in solid samples. The limits of detection for chemical elements using these mass spectrometric techniques are in the low ng g -1 concentration range. The quantification of the analytical results of mass spectrometric methods is sometimes difficult due to a lack of matrix-fitted multielement standard reference materials (SRMs) for many solid samples. Therefore, owing to the simple quantification procedure of the aqueous solution, inductively coupled plasma mass spectrometry (ICP-MS) is being increasingly used for the characterization of solid samples after sample dissolution. ICP-MS is often combined with special sample introduction equipment (e.g. flow injection, hydride generation, high performance liquid chromatography (HPLC) or electrothermal vaporization) or an off-line matrix separation and enrichment of trace impurities (especially for characterization of

  12. A quantitative evaluation of ethylene production in the recombinant cyanobacterium Synechocystis sp. PCC 6803 harboring the ethylene-forming enzyme by membrane inlet mass spectrometry.

    PubMed

    Zavřel, Tomáš; Knoop, Henning; Steuer, Ralf; Jones, Patrik R; Červený, Jan; Trtílek, Martin

    2016-02-01

    The prediction of the world's future energy consumption and global climate change makes it desirable to identify new technologies to replace or augment fossil fuels by environmentally sustainable alternatives. One appealing sustainable energy concept is harvesting solar energy via photosynthesis coupled to conversion of CO2 into chemical feedstock and fuel. In this work, the production of ethylene, the most widely used petrochemical produced exclusively from fossil fuels, in the model cyanobacterium Synechocystis sp. PCC 6803 is studied. A novel instrumentation setup for quantitative monitoring of ethylene production using a combination of flat-panel photobioreactor coupled to a membrane-inlet mass spectrometer is introduced. Carbon partitioning is estimated using a quantitative model of cyanobacterial metabolism. The results show that ethylene is produced under a wide range of light intensities with an optimum at modest irradiances. The results allow production conditions to be optimized in a highly controlled setup.

  13. Quantitation of metabolites of the nerve agents sarin, soman, cyclohexylsarin, VX, and Russian VX in human urine using isotope-dilution gas chromatography-tandem mass spectrometry.

    PubMed

    Barr, John R; Driskell, W J; Aston, Linda S; Martinez, Rodolfo A

    2004-01-01

    Organophosphorus nerve agents are among the most toxic organic compounds known and continue to be a threat for both military and terrorist use. We have developed an isotope-dilution gas chromatography-tandem mass spectrometric (GC-MS-MS) method for quantitating the urinary metabolites of the organophosphorus nerve agents sarin (GB), soman (GD), VX, Russian VX (RVX), and cyclohexylsarin (GF). Urine samples were acidified, extracted into ether-acetonitrile, derivatized by methylation with diazomethane, and analyzed by GC-MS-MS. The limits of detection were less than 1 micro g/L for all analytes.

  14. A Method Revealing Bacterial Cell-wall Architecture by Time-dependent Isotope Labeling and Quantitative Liquid Chromatography/Mass Spectrometry

    PubMed Central

    Patti, Gary J.; Chen, Jiawei; Gross, Michael L.

    2009-01-01

    The molecular details of the biosynthesis and resulting architecture of the bacterial cell wall remain unclear but are essential to understanding the activity of glycopeptide antibiotics, the recognition of pathogens by hosts, and the processes of bacterial growth and division. Here we report a new strategy to elucidate bacterial cell-wall architecture based on time-dependent isotope labeling of bacterial cells quantified by liquid chromatography/accurate mass measurement mass spectrometry. The results allow us to track the fate of cell-wall precursors (which contain the vancomycin-binding site) in Enterococcus faecium, a leading antibiotic-resistant pathogen. By comparing isotopic enrichments of post-insertionally modified cell-wall precursors, we find that tripeptides and species without Asx bridges are specific to mature cell wall. Additionally, we find that the sequence of cell-wall maturation varies throughout a cell cycle. We suggest that actively dividing E. faecium cells have three zones of unique peptidoglycan processing. Our results reveal new organizational characteristics of the bacterial cell wall that are important to understanding tertiary structure and designing novel drugs for antibiotic-resistant pathogens. PMID:19281243

  15. Quantitative determination of un-derivatised amino acids in artistic mural paintings using high-performance liquid chromatography/electrospray ionization triple quadrupole mass spectrometry.

    PubMed

    Zangrando, Roberta; Piazza, Rossano; Cairns, Warren R L; Izzo, Francesca C; Vianello, Alvise; Zendri, Elisabetta; Gambaro, Andrea

    2010-08-18

    The tempera painting technique is one of the most common methods used throughout art history. Tempera is defined by the type of binders used and in this work we study protein-based temperas. Proteinaceous binders can be characterized by the chromatographic determination of the amino acids present where techniques are either based on gas chromatography or high-performance liquid chromatography (HPLC) coupled to mass spectrometry. The objective of this work was to develop a derivatisation-free HPLC method with triple quadrupole tandem mass spectrometric detection (HPLC/ESI-MS/MS) of 21 amino acids contained in the protein-based binders of tempera paints. The analytical method identifies the painting techniques of two contemporary artists: Sironi and DeLuigi. The sample data are compared to painting material standards. The results show that the samples from works by DeLuigi contain mainly animal glue binders, while the samples from Sironi paintings contain binders that are an amino acid mixture with an overall composition between that of eggs and casein.

  16. Development of a thermal desorption gas chromatography-mass spectrometry method for quantitative determination of haloanisoles and halophenols in wineries' ambient air.

    PubMed

    Camino-Sánchez, F J; Ruiz-García, J; Zafra-Gómez, A

    2013-08-30

    An analytical method for the detection and quantification of haloanisoles and their corresponding halophenols in wineries' ambient air was developed. The target analytes were haloanisoles and halophenols, reported by previous scientific literature as responsible for wine taint. A calibrated pump and active tubes filled with Tenax GR™ were used for sampling. These tubes were thermally desorbed and analyzed using gas chromatography-triple quadrupole mass spectrometry in the selected reaction monitoring mode. The adsorption efficiencies of five commercial sampling tubes filled with different materials were evaluated. The efficiencies of the selected adsorbent were close to 100% for all sampled compounds. Desorption, chromatographic and mass spectrometric conditions were accurately optimized allowing very low limits of quantification and wide linear ranges. The limits of quantification in ambient air ranged from 0.8pgtube(-1) for 2,4,6-trichlorophenol, to 28pgtube(-1) for pentachlorophenol. These results are of great importance because human sensory threshold for haloanisoles is very low. The chromatographic method was also validated and the instrumental precision and trueness were established, a maximum RSD of 9% and a mean recovery of 91-106% were obtained. The proposed method involves an easy and sensitive technique for the early detection of haloanisoles and their precursor halophenols in ambient air avoiding contamination of wine or winery facilities.

  17. Broadband Analysis of Bioagents by Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Fenselau, Catherine; Wynne, Colin; Edwards, Nathan

    Mass spectrometry was first reported to provide analysis of intact metabolite biomarkers from whole cells in 1975.1 Since then advances in ionization techniques have extended our capabilities to polar lipids and, eventually, to proteins.2, 3 Mass spectrometry provides a broadband detection system, which, however, has great specificity. Bioinformatics plays an important role in providing flexible and rapid characterization of species, based on protein and peptide mass spectra collected in the field.

  18. Mass spectrometry-based proteomics: principles, perspectives, and challenges.

    PubMed

    Wiśniewski, Jacek R

    2008-10-01

    Mass spectrometry-based proteomics is a modern and rapidly developing methodology for qualitative and quantitative characterization of proteins and their posttranslational modification, subcellular localization, and interaction partners. It enables characterization of entire proteomes with unprecedented sensitivity and precision, providing platforms for identification of biomarkers and drug targets.

  19. Diagnosing Prion Diseases: Mass Spectrometry-Based Approaches

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mass spectrometry is an established means of quantitating the prions present in infected hamsters. Calibration curves relating the area ratios of the selected analyte peptides and their oxidized analogs to stable isotope labeled internal standards were prepared. The limit of detection (LOD) and limi...

  20. Analysis of proteins using DIGE and MALDI mass spectrometry

    EPA Science Inventory

    In this work the sensitivity of the quantitative proteomics approach 2D-DIGE/MS (twoDimensional Difference Gel Electrophoresis / Mass Spectrometry) was tested by detecting decreasing amounts of a specific protein at the low picomole and sub-picomole range. Sensitivity of the 2D-D...

  1. Quantitative analysis of aberrant fatty acid composition of zebrafish hepatic lipids induced by organochlorine pesticide using stable isotope-coded transmethylation and gas chromatography-mass spectrometry.

    PubMed

    Zhong, Hongying; Dong, Linjie; Dong, Qingjian; Ke, Changshu; Fu, Jieying; Wang, Xiaoli; Liu, Cong; Dai, Ling

    2012-07-01

    Organochlorine pesticides have been extensively used worldwide for agricultural purposes. Due to their resistance to metabolism, a major public health concern has been raised. Aberrant hepatic lipid composition has been a hallmark of many liver diseases associated with exposure to various toxins and chemicals. And thus lots of efforts have been focused on the development of analytical techniques that can rapidly and quantitatively determine the changes in fatty acid composition of hepatic lipids. In this work, changes in fatty acid composition of hepatic lipids in response to DDT (dichlorodiphenyltrichloroethane) exposure were quantitatively analyzed by a gas chromatography-mass spectrometric approach based on stable isotope-coded transmethylation. It has been quantitatively demonstrated that polyunsaturated fatty acids including C20:3n3, C20:4n6, and C22:6n3 decrease in response to DDT exposure. However, saturated long chain fatty acids including C16:0, C18:0, as well as monounsaturated long chain fatty acid C18:1n9 consistently increase in a DDT-concentration-dependent manner. In particular, much higher changes in the level of hepatic C16:0 and C18:0 for male fish were observed than that for female fish. These experimental results are in accordance with qualitative histopathological analysis that revealed liver morphological alterations. The stable isotope-coded mass spectrometric approach provides a reliable means for investigating hepatotoxicity associated with fatty acid synthesis, desaturation, mitochondrial beta-oxidation, and lipid mobilization. It should be useful in elucidation of hepatotoxic mechanisms and safety assessment of environmental toxins.

  2. Quantitation of amphetamine, methamphetamine, and their methylenedioxy derivatives in urine by solid-phase microextraction coupled with electrospray ionization-high-field asymmetric waveform ion mobility spectrometry-mass spectrometry.

    PubMed

    McCooeye, Margaret A; Mester, Zoltán; Ells, Barbara; Barnett, David A; Purves, Randy W; Guevremont, Roger

    2002-07-01

    Amphetamine, methamphetamine, and their methylenedioxy derivatives have been identified and measured in a human urine matrix using solid-phase microextraction (SPME) and high-field asymmetric waveform ion mobility spectrometry (FAIMS) in combination with electrospray ionization (ESI) and mass spectrometric detection (MS). Limits of detection in human urine between 200 pg/mL and 7.5 ng/mL have been achieved. The use of a simple extraction method, SPME, combined with the high sensitivity and selectivity of ESI-FAIMS-MS eliminates the need for chromatographic separation and allows for very rapid sample processing.

  3. Forensic applications of ambient ionization mass spectrometry.

    PubMed

    Ifa, Demian R; Jackson, Ayanna U; Paglia, Giuseppe; Cooks, R Graham

    2009-08-01

    This review highlights and critically assesses forensic applications in the developing field of ambient ionization mass spectrometry. Ambient ionization methods permit the ionization of samples outside the mass spectrometer in the ordinary atmosphere, with minimal sample preparation. Several ambient ionization methods have been created since 2004 and they utilize different mechanisms to create ions for mass-spectrometric analysis. Forensic applications of these techniques--to the analysis of toxic industrial compounds, chemical warfare agents, illicit drugs and formulations, explosives, foodstuff, inks, fingerprints, and skin--are reviewed. The minimal sample pretreatment needed is illustrated with examples of analysis from complex matrices (e.g., food) on various substrates (e.g., paper). The low limits of detection achieved by most of the ambient ionization methods for compounds of forensic interest readily offer qualitative confirmation of chemical identity; in some cases quantitative data are also available. The forensic applications of ambient ionization methods are a growing research field and there are still many types of applications which remain to be explored, particularly those involving on-site analysis. Aspects of ambient ionization currently undergoing rapid development include molecular imaging and increased detection specificity through simultaneous chemical reaction and ionization by addition of appropriate chemical reagents.

  4. Optimization of solid-phase-extraction cleanup and validation of quantitative determination of eugenol in fish samples by gas chromatography-tandem mass spectrometry.

    PubMed

    Li, Jincheng; Zhang, Jing; Liu, Yang

    2015-08-01

    This paper describes a rapid and sensitive method for the determination of eugenol in fish samples, based on solid-phase extraction (SPE) and gas chromatography-tandem mass spectrometry (GC-MS-MS). Samples were extracted with acetonitrile, and then cleanup was performed using C18 solid-phase extraction (SPE). The determination of eugenol was achieved using an electron-ionization source (EI) in multiple-reaction-monitoring (MRM) mode. Under optimized conditions, the average recoveries of eugenol were in the range 94.85-103.61 % and the relative standard deviation (RSD) was lower than 12.0 %. The limit of detection (LOD) was 2.5 μg kg(-1) and the limit of quantification (LOQ) was 5.0 μg kg(-1). This method was applied to an exposure study of eugenol residue in carp muscle tissues. The results revealed that eugenol was nearly totally eliminated within 96 h. Graphical Abstract Flow diagram for sample pretreatment.

  5. A column-switching liquid chromatography-tandem mass spectrometry method for quantitation of 2-cyanoethylmercapturic acid and 2-hydroxyethylmercapturic acid in Chinese smokers.

    PubMed

    Hou, Hongwei; Xiong, Wei; Gao, Na; Zhang, Xiaotao; Tang, Gangling; Hu, Qingyuan

    2012-11-01

    The acrylonitrile metabolites 2-cyanoethylmercapturic acid (CEMA) and 2-hydroxyethylmercapturic acid (HEMA) have been determined in human urine using an automated column-switching procedure. A diluted sample was centrifuged just prior to being injected into a reusable precolumn packed with a restricted access material and coupled to a liquid chromatography-tandem mass spectrometry system. This method achieved satisfactory reproducibility and accuracy. Average intra- and interday variations (% relative standard deviations) ranged from 2.4 to 3.8% for CEMA and from 2.7 to 10.5% for HEMA. The limits of quantification were 0.003 and 0.099ng/ml for CEMA and HEMA, respectively. It was used to study the uptake of acrylonitrile from smoke constituents by both nonsmokers and smokers of different tar yield cigarettes under ISO 3308 smoking condition. Metabolite concentrations in smoker urine samples were approximately 12 times higher compared with those in nonsmokers for CEMA and 3 times higher for HEMA. Urinary CEMA levels show a clear dose-response relationship with daily cigarette consumption and urinary cotinine. CEMA can also discriminate between smokers of different ISO cigarettes. Because HEMA is not specific, it is only slightly related to smoking and acrylonitrile exposure. The validated biomarker CEMA will continue to be useful for studies of acrylonitrile uptake by smokers.

  6. Quantitation of total 11-nor-9-carboxy-delta 9-tetrahydrocannabinol in urine and blood using gas chromatography-mass spectrometry (GC-MS).

    PubMed

    Frazee, C Clinton; Kiscoan, Michael; Garg, Uttam

    2010-01-01

    Marijuana, which is made from crushing the leaves, flowers, and sometimes the stems of the plant Cannabis sativa, contains more than 30 cannabinoids. The major psychoactive cannabinoid is delta-9-tetrahydrocannabinol (THC). The major metabolite of THC, 11-nor-delta 9-carboxy-tetrahydrocannabionol (THC-COOH), is excreted in the urine primarily as a glucuronide conjugate and is commonly analyzed in biological specimens for detecting marijuana usage. The procedure described here involves the addition of deuterated internal standard THC-COOH-d9 into the sample followed by hydrolysis of conjugated THC-COOH by alkali. THC-COOH is extracted from urine or blood using liquid-liquid extraction followed by preparation of its trimethylsilyl derivatives. The analysis of derivatized THC-COOH is performed using gas-chromatography/mass spectrometry (GC/MS). Quantification of the drug in a sample is achieved by comparing the responses of the unknown sample to the responses of the calibrators using selected ion monitoring.

  7. Quantitative on-line preconcentration-liquid chromatography coupled with tandem mass spectrometry method for the determination of pharmaceutical compounds in water.

    PubMed

    Idder, Salima; Ley, Laurent; Mazellier, Patrick; Budzinski, Hélène

    2013-12-17

    One of the current environmental issues concerns the presence and fate of pharmaceuticals in water bodies as these compounds may represent a potential environmental problem. The characterization of pharmaceutical contamination requires powerful analytical method able to quantify these pollutants at very low concentration (few ng L(-1)). In this work, a multi-residue analytical methodology (on-line solid phase extraction-liquid chromatography-triple quadrupole mass spectrometry using positive and negative electrospray ionization) has been developed and validated for 40 multi-class pharmaceuticals and metabolites for tap and surface waters. This on-line SPE method was very convenient and efficient compared to classical off-line SPE method because of its shorter total run time including sample preparation and smaller sample volume (1 mL vs up to 1 L). The optimized method included several therapeutic classes as lipid regulators, antibiotics, beta-blockers, non-steroidal anti-inflammatories, antineoplastic, etc., with various physicochemical properties. Quantification has been achieved with the internal standards. The limits of detection are between 0.7 and 15 ng L(-1) for drinking waters and 2-15 ng L(-1) for surface waters. The inter-day precision values are below 20% for each studied level. The improvement and strength of the analytical method has been verified along a monitoring of these 40 pharmaceuticals in Isle River, a French stream located in the South West of France. During this survey, 16 pharmaceutical compounds have been detected.

  8. Liquid chromatography and tandem mass spectrometry method for quantitative determination of pioglitazone and its metabolite 5-hydroxy pioglitazone in human plasma.

    PubMed

    Chinnalalaiah, R; Pigili, R; Avanapu, S R

    2017-03-01

    A liquid chromatography tandem mass spectrometry (LC-MS/MS) based method was developed for the simultaneous estimation of pioglitazone and its active metabolites in human plasma for applicability to pharmacokinetic studies. The chromatographic separation was carried on the reversed phase Peerless Basic C18, column (100×4.6mm, 5μm) at column temperature of 40°C using a binary mobile phase consisting of methanol: 5mM ammonium acetate in 0.1% formic acid (80:20, v/v). The mobile phase was run at a flow rate of 1mL/min and the sample injection was 10μL. The method utilized pioglitazone D4 (IS1) and 5-hydroxyl pioglitazone M-IV D4 (IS2) as an internal standard. The linearity of the method was validated over the range of 6.04-1503.21ng/mL for pioglitazone and 6.01-1496.28ng/mL for 5-hydroxyl pioglitazone. The mean extraction recovery of PIO & HPIO from the spiked plasma was found to be 94.92% for pioglitazone and 96.13% for 5-hydroxy pioglitazone. The developed method can be successfully employed in healthy human volunteers to monitor the pharmacokinetics profile of pioglitazone.

  9. Quantitative determination of a synthetic amide derivative of gallic acid, SG-HQ2, using liquid chromatography tandem mass spectrometry, and its pharmacokinetics in rats.

    PubMed

    Seo, Seung-Yong; Kang, Wonku

    2016-11-30

    An amide derivative of gallic acid (GA), 3,4,5-trihydroxy-N-(8-hydroxyquinolin-2-yl)benzamide) (SG-HQ2) was recently synthesized, and its inhibitory actions were previously shown on histamine release and pro-inflammatory cytokine expression. In this study, a simultaneous quantification method was developed for the determination of SG-HQ2 and its possible metabolite, GA, in rat plasma using liquid chromatography with a tandem mass spectrometry (LC-MS/MS). After simple protein precipitation with acetonitrile including diclofenac (internal standard, IS), the analytes were chromatographed on a reversed phased column with a mobile phase of acetonitrile and water (60:40, v/v, including 0.1% formic acid). The ion transitions of the precursor to the product ion were principally protonated ion [M+H](+) at m/z 313.2→160.6 for SG-HQ2, and deprotonated ions [M-H](-) at m/z 168.7→124.9 for GA and 296.0→251.6 for the IS. The accuracy and precision of the assay were in accordance with FDA regulations for the validation of bioanalytical methods. This method was successfully applied to a pharmacokinetic study of SG-HQ2 after intravenous administration in rats.

  10. Quantitation of the glutathione in human peripheral blood by matrix-assisted laser desorption ionization time-of-flight mass spectrometry coupled with micro-scale derivatization.

    PubMed

    Feng, Chia-Hsien; Huang, Hao-Yi; Lu, Chi-Yu

    2011-04-01

    Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been broadly applied to analyze high-molecular-weight compound (such as polymer or proteomic research) but seldom used for low-molecular-weight compound analysis. The objective of this study is the development of a simple analytical method for the determination of the concentration of tripeptide glutathione (GSH) by MALDI-TOF MS. Unfortunately, GSH could not be detected directly by MALDI-TOF MS. Our method is based on the derivatization of GSH with 4-bromomethyl-6,7-dimethoxycoumarin (BrDMC) in acetonitrile using potassium hydroxide (KOH) as a base catalyst. After simple extraction step, the supernatant is spotted on a target plate, mixed with matrix α-cyano-4-hydroxycinnamic acid (CHCA) and then detected by MALDI-TOF MS. Some parameters affecting the derivatization of GSH were investigated, such as the concentration of BrDMC, KOH, different base catalyst, and reaction time, etc. The regression equations of GSH derivative possessed good linearity (r≧0.995) over the range of 1.0-100.0 μM. The relative standard deviation (R.S.D.) and relative error (R.E.) values in intra- and inter-day assays were below 13%, which showed good precision and accuracy. This proposed method was successfully applied to monitor the concentration of GSH in human blood at micro-scale level.

  11. Quantitative determination of corticosteroids in bovine milk using mixed-mode polymeric strong cation exchange solid-phase extraction and liquid chromatography-tandem mass spectrometry.

    PubMed

    Tölgyesi, Adám; Tölgyesi, László; Sharma, Virender K; Sohn, Mary; Fekete, Jeno

    2010-12-01

    A new method was developed to identify and quantify corticosteroids (prednisolone, methylprednisone, flumetasone, dexamethasone, and methylprednisolone) in raw bovine milk by liquid chromatography-tandem mass spectrometry (LC-MS/MS) utilizing mixed-mode polymeric strong cation exchange and reversed-phase (MCX) solid-phase extraction (SPE) to reduce ion effects in a multimode ion (MMI) source. The main advantage of this method over other commonly used methods includes the use of a single SPE cartridge with a low volume for sample preparation and fast separation on the HPLC system with reduced ion suppression. This study is the first to report the determination of methylprednisone, a metabolite of methylprednisolone, in bovine milk. This method was validated in accordance with the European Union (EU) Commission Decision 2002/657/EC. The recoveries vary between 90% and 105%. The within-laboratory reproducibility (precision) is less than 30%. The decision limits and detection capabilities were calculated along with LODs, which ranged from 0.02 to 0.07 microg/kg. The method was further enhanced by its successful adaptation to other LC-MS/MS systems equipped with the newly developed ion source, Agilent Jet Stream (AJS). After optimization of the AJS ion source and MS parameters, even lower LOD values were achieved (0.001-0.006 microg/kg) for the corticosteroids. Analytical results obtained with the AJS were characterized by an enhanced area response and similar noise level comparable to those obtained with conventional orthogonal atmospheric ionization (API).

  12. Development and validation of a turbulent flow chromatography and tandem mass spectrometry method for the quantitation of methotrexate and its metabolites 7-hydroxy methotrexate and DAMPA in serum.

    PubMed

    Schofield, Ryan C; Ramanathan, Lakshmi V; Murata, Kazunori; Grace, Marie; Fleisher, Martin; Pessin, Melissa S; Carlow, Dean C

    2015-10-01

    A rapid and simple turbulent flow liquid chromatography (TFC-LC) method implementing positive heated electrospray ionization (HESI) for the accurate and precise determination of methotrexate (MTX), 7-hydroxy methotrexate (7-OH MTX), and 4-amino-4-deoxy-N(10)-methylpteroic acid (DAMPA) concentrations in serum was developed. MTX was isolated from serum samples (100μL) after protein precipitation with methanol containing formic acid and internal standard (MTX-D3) followed by centrifugation. The supernatant was injected into the turbulent flow liquid chromatography which is followed by electrospray positive ionization tandem mass spectrometry (TFC-LC-MS/MS) and quantified using a six-point calibration curve. For MTX and DAMPA the assays were linear from 10 to 1000nmol/L and for 7-OH MTX from 20 to 2000nmol/L. Dilutions of 10, 100 and 1000-fold were validated giving a clinically reportable range of 10nmol/L to 5×10(5)nmol/L. Within-day and between-day precisions at concentrations spanning the analytical measurement ranges were less than 10% for all three analytes. MTX, DAMPA and 7-OH MTX were sufficiently stable under all relevant analytical conditions. No significant matrix effect was observed during the method validation. The TFC-LC-MS/MS MTX method was also compared with three other clinically validated MTX assays: a dihydrofolate reductase (DHFR) inhibition assay, an immunoassay based on fluorescence polarization and a previously developed LC-MS/MS assay.

  13. Optimisation and validation of ultra-high performance liquid chromatographic-tandem mass spectrometry method for qualitative and quantitative analysis of potato steroidal alkaloids.

    PubMed

    Hossain, Mohammad B; Rai, Dilip K; Brunton, Nigel P

    2015-08-01

    An ultra-high performance liquid chromatographic-tandem mass spectrometry (UHPLC-MS/MS) method for quantification of potato steroidal alkaloids, namely α-solanine, α-chaconine, solanidine and demissidine was developed and validated. Three different column chemistries, i.e. ethylene bridged hybrid (BEH) C18, hydrophilic lipophilic interaction and amide columns, were assessed. The BEH C18 column showed best separation and sensitivity for the alkaloids. Validation data (inter-day and intra-day combined) for accuracy and recovery ranged from 94.3 to 107.7% and 97.0 to 103.5%, respectively. The accuracy data were within the acceptable range of 15% as outlined in the United States Food and Drug Administration (USFDA) guidelines. The recovery data were consistent and reproducible with a coefficient of variation (CV) ranging from 6.2 to 9.7%. In addition, precision of the method also met the criteria of the USFDA with CV values lower than 15% even at lower limit of quantification (LLOQ), while the permissible variation is considered acceptable below 20%. The limit of detection and LLOQ of the four alkaloids were in the range of 0.001-0.004μg/mL whereas the linearities of the standard curves were between 0.980 and 0.995.

  14. Quantitative determination of isoquinoline alkaloids and chlorogenic acid in Berberis species using ultra high performance liquid chromatography with hybrid triple quadrupole linear ion trap mass spectrometry.

    PubMed

    Singh, Awantika; Bajpai, Vikas; Kumar, Sunil; Arya, Kamal Ram; Sharma, Kulwant Rai; Kumar, Brijesh

    2015-06-01

    Berberis species are well known and used extensively as medicinal plants in traditional medicine. They have many medicinal values attributable to the presence of alkaloids having different pharmacological activities. In this study, a method was developed and validated as per international conference on harmonization guidelines using ultra high performance liquid chromatography with hybrid triple quadrupole-linear ion trap mass spectrometry operated in the multiple reaction monitoring mode for nine bioactive compounds, including protoberberine alkaloids, aporphine alkaloids and chlorogenic acid. This method was applied in different plant parts of eight Berberis species to determine variations in content of nine bioactive compounds. The separation was achieved on an ACQUITY UPLC CSH™ C18 column using a gradient mobile phase at flow rate 0.3 mL/min. Calibration curves for all the nine analytes provided optimum linear detector response (with R(2) ≥0.9989) over the concentration range of 0.5-1000 ng/mL. The precision and accuracy were within RSDs ≤2.4 and ≤2.3%, respectively. The results indicated significant variation in the total contents of the nine compounds in Berberis species.

  15. A Residue Resolved Bayesian Approach to Quantitative Interpretation of Hydrogen Deuterium Exchange from Mass Spectrometry: Application to Characterizing Protein-Ligand Interactions.

    PubMed

    Saltzberg, Daniel John; Broughton, Howard B; Pellarin, Riccardo; Chalmers, Michael J; Espada, Alfonso; Dodge, Jeffrey A; Pascal, Bruce D; Griffin, Patrick R; Humblet, Christine; Sali, Andrej

    2016-11-03

    Characterization of interactions between proteins and other molecules is crucial for understanding the mechanisms of action of biological systems and, thus, drug discovery. An increasingly useful approach to mapping these interactions is measurement of hydrogen/deuterium exchange (HDX) using mass spectrometry (HDX-MS), which measures the time-resolved deuterium incorporation of peptides obtained by enzymatic digestion of the protein. Comparison of exchange rates between apo- and ligand-bound conditions results in a mapping of the differential HDX (ΔHDX) of the ligand. Residue-level analysis of these data, however, must account for experimental error, sparseness and ambiguity due to overlapping peptides. Here, we propose a Bayesian method consisting of a forward model, noise model, prior probabilities, and a Monte Carlo sampling scheme. This method exploits a residue-resolved exponential rate model of HDX-MS data obtained from all peptides simultaneously, and explicitly models experimental error. The result is the best possible estimate of ΔHDX magnitude and significance for each residue given the data. We demonstrate the method by revealing richer structural interpretation of ΔHDX data on two nuclear receptors: vitamin D-receptor (VDR) and retinoic acid receptor gamma (RORγ). The method is implemented in HDX Workbench and as a standalone module of the open source Integrative Modeling Platform.

  16. Development of immobilized-pepsin microreactors coupled to nano liquid chromatography and tandem mass spectrometry for the quantitative analysis of human butyrylcholinesterase.

    PubMed

    Bonichon, Maud; Combès, Audrey; Desoubries, Charlotte; Bossée, Anne; Pichon, Valérie

    2016-08-26

    Human butyrylcholinesterase is a serine hydrolase that reacts with organophosphorus compounds (OP) to form stable adducts. These adducts are valuable biomarkers for OP exposure and can be analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after a preliminary digestion step in solution. However, this digestion step is time-consuming and cannot be directly coupled with LC-MS set ups. Therefore, the aim of this work was to develop pepsin-based immobilized enzyme microreactors (IMERs) for the rapid digestion of human butyrylcholinesterase (HuBuChE). Various IMERs were synthesized by grafting different amounts of pepsin on a CNBr-sepharose gel and the grafting yield was measured by a bicinchoninic acid assay (BCA). A sensitive nanoLC-MS/MS method was developed to evaluate the digestion yields of HuBuChE on IMERs which was made possible by a synthetic peptide which was used as a calibrant. The digestion was optimized by studying the impact of different parameters such as the digestion time, the temperature and the amount of pepsin grafted on IMER. This optimization allowed HuBuChE to be digested with-in 20min without pretreatment and with digestion yields up to 20%. The repeatability of the IMER synthesis and HuBuChE digestion was highlighted with the characterization of 3 similar IMERs. Finally, the digestion yields of HuBuChE were higher with IMERs when compared to a typical in solution digestion.

  17. Liquid Chromatography with Electrospray Ionization and Tandem Mass Spectrometry Applied in the Quantitative Analysis of Chitin-Derived Glucosamine for a Rapid Estimation of Fungal Biomass in Soil

    PubMed Central

    Olofsson, Madelen A.; Bylund, Dan

    2016-01-01

    This method employs liquid chromatography-tandem mass spectrometry to rapidly quantify chitin-derived glucosamine for estimating fungal biomass. Analyte retention was achieved using hydrophilic interaction liquid chromatography, with a zwitter-ionic stationary phase (ZIC-HILIC), and isocratic elution using 60% 5 mM ammonium formate buffer (pH 3.0) and 40% ACN. Inclusion of muramic acid and its chromatographic separation from glucosamine enabled calculation of the bacterial contribution to the latter. Galactosamine, an isobaric isomer to glucosamine, found in significant amounts in soil samples, was also investigated. The two isomers form the same precursor and product ions and could not be chromatographically separated using this rapid method. Instead, glucosamine and galactosamine were distinguished mathematically, using the linear relationships describing the differences in product ion intensities for the two analytes. The m/z transitions of 180 → 72 and 180 → 84 were applied for the detection of glucosamine and galactosamine and that of 252 → 126 for muramic acid. Limits of detection were in the nanomolar range for all included analytes. The total analysis time was 6 min, providing a high sample throughput method. PMID:26977151

  18. Quantitative determination of octylphenol, nonylphenol, alkylphenol ethoxylates and alcohol ethoxylates by pressurized liquid extraction and liquid chromatography-mass spectrometry in soils treated with sewage sludges.

    PubMed

    Andreu, Vicente; Ferrer, Emilia; Rubio, José Luís; Font, Guillermina; Picó, Yolanda

    2007-05-25

    Surfactants have one of the highest production rates of all organic chemicals. Non-ionic surfactants, especially alkylphenol ethoxylates, received most attention as precursors of estrogenic metabolic products generated during wastewater treatment. Alkylphenols (octyl and nonylphenol), alkylphenol polyethoxylates (APEOs), and alcohol ethoxylates (AEOs) have been determined in a Mediterranean forest soil (Mediterranean Rendzic Leptosol) amended with sludges from six waste water treatment plants (WWTPs) located in the Valencian Community. These compounds were isolated from soil by pressurized liquid extraction (PLE) using a mixture acetone-hexane (50:50 v/v), the extracts were cleaned up by solid-phase extraction (SPE) with C(18), and determined by liquid chromatography atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) using analytical standards for quantification. The method enabled high-reliable identification by monitoring the corresponding ammonium adduct [M+NH(3)](+) for AEOs and APEOs, and the deprotonated molecule [M-H](-) for octyl and nonylphenol. Recoveries, determined spiking soil samples at different concentrations, ranged from 89 to 94%, with limits of quantification from 1 to 100 microg kg(-1). Data obtained from a soil sample mixed with biosolids in the laboratory showed that these compounds are present at concentrations ranging from 0.02 to 5 mg kg(-1). According to these concentrations, levels of possible risk can be concluded for the presence of non-ionic surfactants in soil. However, further assessment will be necessary to establish the relationship between exposure and effect findings.

  19. Simultaneous qualitative assessment and quantitative analysis of flavonoids in various tissues of lotus (Nelumbo nucifera) using high performance liquid chromatography coupled with triple quad mass spectrometry.

    PubMed

    Chen, Sha; Fang, Linchuan; Xi, Huifen; Guan, Le; Fang, Jinbao; Liu, Yanling; Wu, Benhong; Li, Shaohua

    2012-04-29

    Flavonoid composition and concentration were investigated in 12 different tissues of 'Ti-1' lotus (Nelumbo nucifera) by high performance liquid chromatography equipped with photodiode array detection tandem electrospray ionization mass spectrometry (HPLC-DAD-ESI-MS(n)). A total of 20 flavonoids belonging to six groups (myricetin, quercetin, kaempferol, isohamnetin, diosmetin derivatives) were separated and identified. Myricetin 3-O-galactoside, myricetin 3-O-glucuronide, isorhamnetin 3-O-glucuronide and free aglycone diometin (3',5,7-trihydroxy-4'-methoxyflavone) were first reported in lotus. Flavonoid composition varied largely with tissue type, and diverse compounds (5-15) were found in leaf and flower stalks, flower pistils, seed coats and embryos. Flower tissues including flower petals, stamens, pistils, and, especially, reproductive tissue fruit coats had more flavonoid compounds (15-17) than leaves (12), while no flavonoids were detectable in seed kernels. The flavonoid content of seed embryos was high, 730.95 mg 100g(-1) DW (dry weight). As regards the other tissues, mature leaf pulp (771.79 mg 100 g(-1) FW (fresh weight)) and young leaves (650.67 mg 100 g(-1) FW) had higher total flavonoid amount than flower stamens (359.45 mg 100 g(-1) FW) and flower petals (342.97 mg 100g(-1) FW), while leaf stalks, flower stalks and seed coats had much less total flavonoid (less than 40 mg 100 g(-1) FW).

  20. Quantitative determination of β-hydroxymethylbutyrate and leucine in culture media and microdialysates from rat brain by UHPLC-tandem mass spectrometry.

    PubMed

    Santos-Fandila, A; Zafra-Gómez, A; Barranco, A; Navalón, A; Rueda, R; Ramírez, M

    2014-05-01

    The main objective of the present work was to develop a method to determine β-hydroxymethylbutyrate (HMB) and leucine (Leu) in culture media and brain microdialysates. An accurate, selective, and cost-effective method, based on the use of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), was developed for the identification and quantification of both compounds. The method consisted of sample dilution, direct injection onto the chromatographic equipment, and quantification with a triple quadrupole mass spectrometer using an electrospray ionization interface in positive mode. The procedure and the UHPLC-MS/MS parameters were accurately optimized to achieve the highest recoveries and to enhance the analytical characteristics of the method. For chromatographic separation, an Acquity UPLC BEH Hilic column using acetonitrile-water gradient with formic acid as additive was employed. The total run time was 4 min. The limits of detection (LODs) obtained ranged from 0.01 to 0.04 μg mL(-1), and the limits of quantification (LOQs) ranged from 0.04 to 0.12 μg mL(-1). Precision (expressed as relative standard deviation) was lower than 15 %, and the determination coefficient (R (2)) was higher than 99.0 % with a residual deviation for each calibration point lower than ±25 %. Mean recoveries were between 85 and 115 %. The method was successfully applied to the analysis of both compounds, HMB and Leu, in samples obtained from an experiment of blood-brain barrier (BBB) passage in vitro and to an experiment of brain microdialysis in rats in vivo after an oral challenge with HMB to detect its appearance in the brain.

  1. Methods for recalibration of mass spectrometry data

    DOEpatents

    Tolmachev, Aleksey V.; Smith, Richard D.

    2009-03-03

    Disclosed are methods for recalibrating mass spectrometry data that provide improvement in both mass accuracy and precision by adjusting for experimental variance in parameters that have a substantial impact on mass measurement accuracy. Optimal coefficients are determined using correlated pairs of mass values compiled by matching sets of measured and putative mass values that minimize overall effective mass error and mass error spread. Coefficients are subsequently used to correct mass values for peaks detected in the measured dataset, providing recalibration thereof. Sub-ppm mass measurement accuracy has been demonstrated on a complex fungal proteome after recalibration, providing improved confidence for peptide identifications.

  2. Identification and semi-quantitative determination of anti-oxidants in lubricants employing thin-layer chromatography-spray mass spectrometry.

    PubMed

    Kreisberger, Georg; Himmelsbach, Markus; Buchberger, Wolfgang; Klampfl, Christian W

    2015-02-27

    A quick and simple method for identification and semi-quantitative determination of nine antioxidants commonly used in lubricants is presented. A dual step thin-layer chromatography (TLC) separation, removes in a first step the oil matrix whereas in a second step the antioxidants are separated. Cutting the spots out of the TLC-plate in the form of triangles allows direct-spray mass spectrometric (MS) measurements, providing MS and MS(n) spectra (if an appropriate MS instrument is employed) of the antioxidants, allowing their identification but also giving information about potential oxidation or degradation of these additives. Calibration curves within the concentration range relevant for the analysis of real oil samples (0.2-1.2gL(-1)) were constructed with R(2) values above 0.98 (when using an appropriate internal standard). This allowed the semi-quantitative determination of the selected antioxidants in real oils samples. Comparison with results from HPLC-UV measurement showed acceptable agreement for all analytes.

  3. Plasma Desorption Mass Spectrometry: Coming of Age.

    ERIC Educational Resources Information Center

    Cotter, Robert J.

    1988-01-01

    Discusses the history and development of Plasma Desorption Mass Spectrometry to determine molecular weights and structures of proteins and polymers. Outlines theory, instrumentation, and sample preparation commonly used. Gives several examples of resulting spectra. (ML)

  4. DMS-prefiltered mass spectrometry for the detection of biomarkers

    NASA Astrophysics Data System (ADS)

    Coy, Stephen L.; Krylov, Evgeny V.; Nazarov, Erkinjon G.

    2008-04-01

    Technologies based on Differential Mobility Spectrometry (DMS) are ideally matched to rapid, sensitive, and selective detection of chemicals like biomarkers. Biomarkers linked to exposure to radiation, exposure to CWA's, exposure to toxic materials (TICs and TIMs) and to specific diseases are being examined in a number of laboratories. Screening for these types of exposure can be improved in accuracy and greatly speeded up by using DMS-MS instead of slower techniques like LC-MS and GC-MS. We have performed an extensive series of tests with nanospray-DMS-mass spectroscopy and standalone nanospray-DMS obtaining extensive information on chemistry and detectivity. DMS-MS systems implemented with low-resolution, low-cost, portable mass-spectrometry systems are very promising. Lowresolution mass spectrometry alone would be inadequate for the task, but with DMS pre-filtration to suppress interferences, can be quite effective, even for quantitative measurement. Bio-fluids and digests are well suited to ionization by electrospray and detection by mass-spectrometry, but signals from critical markers are overwhelmed by chemical noise from unrelated species, making essential quantitative analysis impossible. Sionex and collaborators have presented data using DMS to suppress chemical noise, allowing detection of cancer biomarkers in 10,000-fold excess of normal products 1,2. In addition, a linear dynamic range of approximately 2,000 has been demonstrated with accurate quantitation 3. We will review the range of possible applications and present new data on DMS-MS biomarker detection.

  5. Quantitative chemical imaging of element diffusion into heterogeneous media using laser ablation inductively coupled plasma mass spectrometry, synchrotron micro-X-ray fluorescence, and extended X-ray absorption fine structure spectroscopy.

    PubMed

    Wang, H A O; Grolimund, D; Van Loon, L R; Barmettler, K; Borca, C N; Aeschlimann, B; Günther, D

    2011-08-15

    Quantitative chemical imaging of trace elements in heterogeneous media is important for the fundamental understanding of a broad range of chemical and physical processes. The primary aim of this study was to develop an analytical methodology for quantitative high spatial resolution chemical imaging based on the complementary use of independent microanalytical techniques. The selected scientific case study is focused on high spatially resolved quantitative imaging of major elements, minor elements, and a trace element (Cs) in Opalinus clay, which has been proposed as the host rock for high-level radioactive waste repositories. Laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS), providing quantitative chemical information, and synchrotron radiation based micro-X-ray fluorescence (SR-microXRF), providing high spatial resolution images, were applied to study Cs migration into Opalinus clay rock. The results indicate that combining the outputs achievable by the two independent techniques enhances the imaging capabilities significantly. The qualitative high resolution image of SR-microXRF is in good agreement with the quantitative image recorded with lower spatial resolution by LA-ICPMS. Combining both techniques, it was possible to determine that the Opalinus clay sample contains two distinct domains: (i) a clay mineral rich domain and (ii) a calcium carbonate dominated domain. The two domains are separated by sharp boundaries. The spatial Cs distribution is highly correlated to the distribution of the clay. Furthermore, extended X-ray absorption fine structure analysis indicates that the trace element Cs preferentially migrates into clay interlayers rather than into the calcite domain, which complements the results acquired by LA-ICPMS and SR-microXRF. By using complementary techniques, the quantification robustness was improved to quantitative micrometer spatial resolution. Such quantitative, microscale chemical images allow a more detailed

  6. Enantioselectivity of mass spectrometry: challenges and promises.

    PubMed

    Awad, Hanan; El-Aneed, Anas

    2013-01-01

    With the fast growing market of pure enantiomer drugs and bioactive molecules, new chiral-selective analytical tools have been instigated including the use of mass spectrometry (MS). Even though MS is one of the best analytical tools that has efficiently been used in several pharmaceutical and biological applications, traditionally MS is considered as a "chiral-blind" technique. This limitation is due to the MS inability to differentiate between two enantiomers of a chiral molecule based merely on their masses. Several approaches have been explored to assess the potential role of MS in chiral analysis. The first approach depends on the use of MS-hyphenated techniques utilizing fast and sensitive chiral separation tools such as liquid chromatography (LC), gas chromatography (GC), and capillary electrophoresis (CE) coupled to MS detector. More recently, several alternative separation techniques have been evaluated such as supercritical fluid chromatography (SFC) and capillary electrochromatography (CEC); the latter being a hybrid technique that combines the efficiency of CE with the selectivity of LC. The second approach is based on using the MS instrument solely for the chiral recognition. This method depends on the behavioral differences between enantiomers towards a foreign molecule and the ability of MS to monitor such differences. These behavioral differences can be divided into three types: (i) differences in the enantiomeric affinity for association with the chiral selector, (ii) differences of the enantiomeric exchange rate with a foreign reagent, and (iii) differences in the complex MS dissociation behaviors of the enantiomers. Most recently, ion mobility spectrometry was introduced to qualitatively and quantitatively evaluate chiral compounds. This article provides an overview of MS role in chiral analysis by discussing MS based methodologies and presenting the challenges and promises associated with each approach.

  7. Quantitative targeted and retrospective data analysis of relevant pesticides, antibiotics and mycotoxins in bakery products by liquid chromatography-single-stage Orbitrap mass spectrometry.

    PubMed

    De Dominicis, Emiliano; Commissati, Italo; Gritti, Elisa; Catellani, Dante; Suman, Michele

    2015-01-01

    In addition to 'traditional' multi-residue and multi-contaminant multiple reaction monitoring (MRM) mass spectrometric techniques devoted to quantifying a list of targeted compounds, the global food industry requires non-targeted methods capable of detecting other possible potentially hazardous compounds. Ultra-high-performance liquid chromatography combined with a single-stage Orbitrap high-resolution mass spectrometer (UHPLC-HRMS Exactive™-Orbitrap Technology) was successfully exploited for the complete selective and quantitative determination of 33 target compounds within three major cross categories (pesticides, antibiotics and mycotoxins) in bakery matrices (specifically milk, wheat flour and mini-cakes). Resolution was set at 50 000 full width at half maximum (FWHM) to achieve the right compromise between an adequate scan speed and selectivity, allowing for the limitations related to the necessary generic sample preparation approach. An exact mass with tolerance of 5 ppm and minimum peak threshold of 10 000 units were fixed as the main identification conditions, including retention time and isotopic pattern as additional criteria devoted to greatly reducing the risk of false-positive findings. The full validation for all the target analytes was performed: linearity, intermediate repeatability and recovery (28 analytes within 70-120%) were positively assessed; furthermore, limits of quantification between 5 and 100 µg kg(-1) (with most of the analytes having a limit of detection below 6 µg kg(-1)) indicate good performance, which is compatible with almost all the regulatory needs. Naturally contaminated and fortified mini-cakes, prepared through combined use of industrial and pilot plant production lines, were analysed at two different concentration levels, obtaining good overall quantitative results and providing preliminary indications of the potential of full-scan HRMS cluster analysis. The effectiveness of this analytical approach was also tested in

  8. Rapid simultaneous quantitative determination of different small pharmaceutical drugs using a conventional matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system.

    PubMed

    Persike, Markus; Karas, Michael

    2009-11-01

    The present study establishes a simple, rapid and sensitive method for the simultaneous quantification of different small pharmaceutical drugs using a matrix-assisted laser desorption/ionization source (MALDI) coupled with a time-of-flight (TOF) mass analyzer. Neither time-consuming sample preparation, nor special target plates, isotopically labelled internal standards or other extra equipment are necessary. A simple standard dried-droplet preparation with the common matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) was used. The background signals of CHCA in the low-mass region did not pose the presumed problem, because the sensitivity, resolution and mass accuracy of a modern MALDI-TOF MS system is sufficient to overcome this difficulty. Four experiments were performed in order to verify the quantification method. First, ten different phenothiazines were quantified in the range of 5-2000 nM (1-880 ng/mL). A good precision (relative standard deviation (RSD) 4.4-9.3%), linearity (R2 >0.99) and accuracy (error 4.7-11%) was obtained in all cases. Additionally, simultaneous quantification of these ten phenothiazines was carried out in human plasma without prior chromatographic separation in the range of 2-1750 ng/mL yielding good linearity, precision and accuracy (mean RSD 7.6%; R2 >0.99, mean error 8.0%). Accordingly, a quantitative analysis of ten chemically and pharmaceutically unrelated drugs was performed in the same way. A comparable linearity (R2 >0.99), precision (mean RSD 7.6%) and accuracy (mean error 8.3%) was obtained in the range of 5-2000 nM. Finally, the prazosin content of a commercial tablet was directly determined without further purification steps.

  9. Mass spectrometry in the home and garden.

    PubMed

    Pulliam, Christopher J; Bain, Ryan M; Wiley, Joshua S; Ouyang, Zheng; Cooks, R Graham

    2015-02-01

    Identification of active components in a variety of chemical products used directly by consumers is described at both trace and bulk levels using mass spectrometry. The combination of external ambient ionization with a portable mass spectrometer capable of tandem mass spectrometry provides high chemical specificity and sensitivity as well as allowing on-site monitoring. These experiments were done using a custom-built portable ion trap mass spectrometer in combination with the ambient ionization methods of paper spray, leaf spray, and low temperature plasma ionization. Bactericides, garden chemicals, air fresheners, and other products were examined. Herbicide applied to suburban lawns was detected in situ on single leaves 5 d after application.

  10. Mass Spectrometry in the Home and Garden

    NASA Astrophysics Data System (ADS)

    Pulliam, Christopher J.; Bain, Ryan M.; Wiley, Joshua S.; Ouyang, Zheng; Cooks, R. Graham

    2015-02-01

    Identification of active components in a variety of chemical products used directly by consumers is described at both trace and bulk levels using mass spectrometry. The combination of external ambient ionization with a portable mass spectrometer capable of tandem mass spectrometry provides high chemical specificity and sensitivity as well as allowing on-site monitoring. These experiments were done using a custom-built portable ion trap mass spectrometer in combination with the ambient ionization methods of paper spray, leaf spray, and low temperature plasma ionization. Bactericides, garden chemicals, air fresheners, and other products were examined. Herbicide applied to suburban lawns was detected in situ on single leaves 5 d after application.

  11. Quantitative proteomic analysis of Pseudomonas pseudoalcaligenes CECT5344 in response to industrial cyanide-containing wastewaters using Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS)

    PubMed Central

    Ibáñez, María Isabel; Cabello, Purificación; Luque-Almagro, Víctor Manuel; Sáez, Lara P.; Olaya, Alfonso; Sánchez de Medina, Verónica; Luque de Castro, María Dolores; Moreno-Vivián, Conrado; Roldán, María Dolores

    2017-01-01

    Biological treatments to degrade cyanide are a powerful technology for cyanide removal from industrial wastewaters. It has been previously demonstrated that the alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 is able to use free cyanide and several metal−cyanide complexes as the sole nitrogen source. In this work, the strain CECT5344 has been used for detoxification of the different chemical forms of cyanide that are present in alkaline wastewaters from the jewelry industry. This liquid residue also contains large concentrations of metals like iron, copper and zinc, making this wastewater even more toxic. To elucidate the molecular mechanisms involved in the bioremediation process, a quantitative proteomic analysis by LC-MS/MS has been carried out in P. pseudoalcaligenes CECT5344 cells grown with the jewelry residue as sole nitrogen source. Different proteins related to cyanide and cyanate assimilation, as well as other proteins involved in transport and resistance to metals were induced by the cyanide-containing jewelry residue. GntR-like regulatory proteins were also induced by this industrial residue and mutational analysis revealed that GntR-like regulatory proteins may play a role in the regulation of cyanide assimilation in P. pseudoalcaligenes CECT5344. The strain CECT5344 has been used in a batch reactor to remove at pH 9 the different forms of cyanide present in industrial wastewaters from the jewelry industry (0.3 g/L, ca. 12 mM total cyanide, including both free cyanide and metal−cyanide complexes). This is the first report describing the biological removal at alkaline pH of such as elevated concentration of cyanide present in a heterogeneous mixture from an industrial source. PMID:28253357

  12. Quantitative proteomic analysis of Pseudomonas pseudoalcaligenes CECT5344 in response to industrial cyanide-containing wastewaters using Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS).

    PubMed

    Ibáñez, María Isabel; Cabello, Purificación; Luque-Almagro, Víctor Manuel; Sáez, Lara P; Olaya, Alfonso; Sánchez de Medina, Verónica; Luque de Castro, María Dolores; Moreno-Vivián, Conrado; Roldán, María Dolores

    2017-01-01

    Biological treatments to degrade cyanide are a powerful technology for cyanide removal from industrial wastewaters. It has been previously demonstrated that the alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 is able to use free cyanide and several metal-cyanide complexes as the sole nitrogen source. In this work, the strain CECT5344 has been used for detoxification of the different chemical forms of cyanide that are present in alkaline wastewaters from the jewelry industry. This liquid residue also contains large concentrations of metals like iron, copper and zinc, making this wastewater even more toxic. To elucidate the molecular mechanisms involved in the bioremediation process, a quantitative proteomic analysis by LC-MS/MS has been carried out in P. pseudoalcaligenes CECT5344 cells grown with the jewelry residue as sole nitrogen source. Different proteins related to cyanide and cyanate assimilation, as well as other proteins involved in transport and resistance to metals were induced by the cyanide-containing jewelry residue. GntR-like regulatory proteins were also induced by this industrial residue and mutational analysis revealed that GntR-like regulatory proteins may play a role in the regulation of cyanide assimilation in P. pseudoalcaligenes CECT5344. The strain CECT5344 has been used in a batch reactor to remove at pH 9 the different forms of cyanide present in industrial wastewaters from the jewelry industry (0.3 g/L, ca. 12 mM total cyanide, including both free cyanide and metal-cyanide complexes). This is the first report describing the biological removal at alkaline pH of such as elevated concentration of cyanide present in a heterogeneous mixture from an industrial source.

  13. Quantitative Analysis of Mixed Halogen Dioxins and Furans in Fire Debris Utilizing Atmospheric Pressure Ionization Gas Chromatography-Triple Quadrupole Mass Spectrometry.

    PubMed

    Organtini, Kari L; Myers, Anne L; Jobst, Karl J; Reiner, Eric J; Ross, Brian; Ladak, Adam; Mullin, Lauren; Stevens, Douglas; Dorman, Frank L

    2015-10-20

    Residential and commercial fires generate a complex mixture of volatile, semivolatile, and nonvolatile compounds. This study focused on the semi/nonvolatile components of fire debris to better understand firefighter exposure risks. Using the enhanced sensitivity of gas chromatography coupled to atmospheric pressure ionization-tandem mass spectrometry (APGC-MS/MS), complex fire debris samples collected from simulation fires were analyzed for the presence of potentially toxic polyhalogenated dibenzo-p-dioxins and dibenzofurans (PXDD/Fs and PBDD/Fs). Extensive method development was performed to create multiple reaction monitoring (MRM) methods for a wide range of PXDD/Fs from dihalogenated through hexa-halogenated homologue groups. Higher halogenated compounds were not observed due to difficulty eluting them off the long column used for analysis. This methodology was able to identify both polyhalogenated (mixed bromo-/chloro- and polybromo-) dibenzo-p-dioxins and dibenzofurans in the simulated burn study samples collected, with the dibenzofuran species being the dominant compounds in the samples. Levels of these compounds were quantified as total homologue groups due to the limitations of commercial congener availability. Concentration ranges in household simulation debris were observed at 0.01-5.32 ppb (PXDFs) and 0.18-82.11 ppb (PBDFs). Concentration ranges in electronics simulation debris were observed at 0.10-175.26 ppb (PXDFs) and 0.33-9254.41 ppb (PBDFs). Samples taken from the particulate matter coating the firefighters' helmets contained some of the highest levels of dibenzofurans, ranging from 4.10 ppb to 2.35 ppm. The data suggest that firefighters and first responders at fire scenes are exposed to a complex mixture of potentially hundreds to thousands of different polyhalogenated dibenzo-p-dioxins and dibenzofurans that could negatively impact their health.

  14. Qualitative and quantitative spectro-chemical analysis of dates using UV-pulsed laser induced breakdown spectroscopy and inductively coupled plasma mass spectrometry.

    PubMed

    Mehder, A O; Habibullah, Y B; Gondal, M A; Baig, Umair

    2016-08-01

    Laser Induced Breakdown Spectroscopy (LIBS) is demonstrated for the spectral analysis of nutritional and toxic elements present in several varieties of date fruit samples available in the Saudi Arabia market. The method analyzes the optical emission of a test sample when subjected to pulsed laser ablation. In this demonstration, our primary focus is on calcium (Ca) and magnesium (Mg), as nutritional elements, and on chromium (Cr), as a toxic element. The local thermodynamic equilibrium (LTE) condition was confirmed prior to the elemental characterization of date samples to ensure accuracy of the LIBS analysis. This was achieved by measuring parameters associated with the plasma, such as the electron temperature and the electron number density. These plasma parameters aid interpretation of processes such as ionization, dissociation, and excitation occurring in the plasma plume formed by ablating the date palm sample. The minimum detection limit was established from calibration curves that involved plotting the LIBS signal intensity as a function of standard date samples with known concentrations. The concentration of Ca and Mg detected in different varieties of date samples was between 187 and 515 and 35-196mgL(-1) respectively, while Cr concentration measured between 1.72 and 7.76mgL(-1). In order to optimize our LIBS system, we have studied how the LIBS signal intensity depends on the incident laser energy and the del