Science.gov

Sample records for quantitative mass spectrometry

  1. Quantitative mass spectrometry: an overview.

    PubMed

    Urban, Pawel L

    2016-10-28

    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

  2. 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'.

  3. Quantitative mass spectrometry: an overview.

    PubMed

    Urban, Pawel L

    2016-10-28

    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'.

  4. 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

  5. Quantitative mass spectrometry methods for pharmaceutical analysis.

    PubMed

    Loos, Glenn; Van Schepdael, Ann; Cabooter, Deirdre

    2016-10-28

    Quantitative pharmaceutical analysis is nowadays frequently executed using mass spectrometry. Electrospray ionization coupled to a (hybrid) triple quadrupole mass spectrometer is generally used in combination with solid-phase extraction and liquid chromatography. Furthermore, isotopically labelled standards are often used to correct for ion suppression. The challenges in producing sensitive but reliable quantitative data depend on the instrumentation, sample preparation and hyphenated techniques. In this contribution, different approaches to enhance the ionization efficiencies using modified source geometries and improved ion guidance are provided. Furthermore, possibilities to minimize, assess and correct for matrix interferences caused by co-eluting substances are described. With the focus on pharmaceuticals in the environment and bioanalysis, different separation techniques, trends in liquid chromatography and sample preparation methods to minimize matrix effects and increase sensitivity are discussed. Although highly sensitive methods are generally aimed for to provide automated multi-residue analysis, (less sensitive) miniaturized set-ups have a great potential due to their ability for in-field usage.This article is part of the themed issue 'Quantitative 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 matrix-assisted laser desorption/ionization mass spectrometry

    PubMed Central

    Roder, Heinrich; Hunsucker, Stephen W.

    2008-01-01

    This review summarizes the essential characteristics of matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS), especially as they relate to its applications in quantitative analysis. Approaches to quantification by MALDI-TOF MS are presented and published applications are critically reviewed. PMID:19106161

  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'. PMID:27644971

  9. 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'.

  10. Mass spectrometry as a quantitative tool in plant metabolomics.

    PubMed

    Jorge, Tiago F; Mata, Ana T; António, Carla

    2016-10-28

    Metabolomics is a research field used to acquire comprehensive information on the composition of a metabolite pool to provide a functional screen of the cellular state. Studies of the plant metabolome include the analysis of a wide range of chemical species with very diverse physico-chemical properties, and therefore powerful analytical tools are required for the separation, characterization and quantification of this vast compound diversity present in plant matrices. In this review, challenges in the use of mass spectrometry (MS) as a quantitative tool in plant metabolomics experiments are discussed, and important criteria for the development and validation of MS-based analytical methods provided.This article is part of the themed issue 'Quantitative mass spectrometry'.

  11. Mass spectrometry as a quantitative tool in plant metabolomics.

    PubMed

    Jorge, Tiago F; Mata, Ana T; António, Carla

    2016-10-28

    Metabolomics is a research field used to acquire comprehensive information on the composition of a metabolite pool to provide a functional screen of the cellular state. Studies of the plant metabolome include the analysis of a wide range of chemical species with very diverse physico-chemical properties, and therefore powerful analytical tools are required for the separation, characterization and quantification of this vast compound diversity present in plant matrices. In this review, challenges in the use of mass spectrometry (MS) as a quantitative tool in plant metabolomics experiments are discussed, and important criteria for the development and validation of MS-based analytical methods provided.This article is part of the themed issue 'Quantitative mass spectrometry'. PMID:27644967

  12. Mass spectrometry-based quantitative analysis and biomarker discovery.

    PubMed

    Suzuki, Naoto

    2011-01-01

      Mass spectrometry-based quantitative analysis and biomarker discovery using metabolomics approach represent one of the major platforms in clinical fields including for the prognosis or diagnosis, assessment of severity and response to therapy in a number of clinical disease states as well as therapeutic drug monitoring (TDM). This review first summarizes our mass spectrometry-based research strategy and some results on relationship between cysteinyl leukotriene (cysLT), thromboxane (TX), 12-hydroxyeicosatetraenoic acid (12-HETE) and other metabolites of arachidonic acid and diseases such as atopic dermatitis, rheumatoid arthritis and diabetes mellitus. For the purpose of evaluating the role of these metabolites of arachidonic acid in disease status, we have developed sensitive determination methods with simple solid-phase extraction and applied in clinical settings. In addition to these endogenous compounds, using mass spectrometry, we have developed actually applicable quantitative methods for TDM. Representative example was a method of TDM for sirolimus, one of the immunosuppressant agents for a recipient of organ transplant, which requires rigorous monitoring of blood level. As we recognized great potential in mass spectrometry during these researches, we have become interested in metabolomics as the non-targeted analysis of metabolites. Now, established strategy for the metabolomics investigation applies to samples from cells, animals and humans to separate groups based on altered patterns of metabolites in biological fluids and to identify metabolites as potential biomarkers discriminating groups. We would be honored if our research using mass spectrometry would contribute to provide useful information in the field of medical pharmacy. PMID:21881303

  13. 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.

  14. Quantitative mass spectrometry of unconventional human biological matrices.

    PubMed

    Dutkiewicz, Ewelina P; Urban, Pawel L

    2016-10-28

    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'. PMID:27644966

  15. 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'.

  16. Methods and algorithms for quantitative proteomics by mass spectrometry.

    PubMed

    Matthiesen, Rune; Carvalho, Ana Sofia

    2013-01-01

    Protein quantitation by mass spectrometry (MS) is attractive since it is possible to obtain both identification and quantitative values of proteins and their posttranslational modifications in a single experiment. In contrast, protein arrays only provide quantitative values of targeted proteins and their modifications. There are an overwhelming number of quantitative MS methods for protein and peptide quantitation. The aim here is to provide an overview of the most common MS methods and algorithms used in quantitative proteomics and discuss the computational algorithms needed to reliably quantitate proteins, peptides, and their posttranslational modifications. One of the main challenges in data analysis of many experimental projects is to pipe together a number of software solutions that are either commercial or freely available. The aim of this chapter is to provide a good set of algorithms, ideas, and resources that can easily be implemented in scripting language like R, Python, or Perl. By understanding the algorithmic ideas presented here, data from any instrument or modified experimental protocol can be analyzed and is therefore in the authors' opinion more valuable than a black box concept.

  17. 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.

  18. 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

  19. Peptide quantitation with methyl iodide isotopic tags and mass spectrometry.

    PubMed

    Blagojevic, Voislav; Zhidkov, Nickholas; Tharmaratnam, Samuel; Pham, Van Thong; Kaplan, Harvey; Bohme, Diethard K

    2010-06-01

    A novel method is presented for the quantitation of peptides based on their methylation by in vacuo chemical reaction with methyl iodide. Samples of two small peptides, hexaglycine and pentaalanine, were labeled with CH(3)I and CD(3)I, representing the "unknown" and "standard" respectively, and then subjected to a series of tests using mass spectrometry to ascertain the suitability of the isotopic labels for peptide quantitation. The experiments show methyl iodide to be a very quantitative label, exhibiting a linear relationship in concentration over the dynamic range of the mass spectrometer used in the analysis (up to 4 orders of magnitude) both as pure samples and in a complex mixture of peptides. The tendency of trimethylated peptides to preferentially form a(2) fragment ions in MS(2) produces a significant increase in sensitivity, especially when the mass spectrometer is used in the MRM mode. Tests were also performed to verify the stability of the label against H/D exchange and its suitability for long-term storage, showing little degradation while in solution and during subsequent chemical processing.

  20. 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

  1. Quantitative Fourier transform ion cyclotron resonance mass spectrometry--the determination of creatinine by isotope dilution mass spectrometry.

    PubMed

    Bristow, Tony; Stokes, Peter; O'Connor, Gavin

    2005-01-01

    Accurate quantitation has been demonstrated on many different types of mass spectrometer. However, quantitative applications of Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) have been limited. In this study, the quantitative potential of FTICRMS has been investigated using an exact matching isotope dilution method for the determination of creatinine in serum. Creatinine is an important clinical biomarker and its measurement is used as an assessment of renal function. The quantitation of creatinine was selected because a high-accuracy high-performance liquid chromatography/mass spectrometry (HPLC/MS) determination using a triple quadrupole mass spectrometer has already been successfully developed in-house. Therefore, a direct comparison of the quantitative capability of FTICRMS could be made against an established method. The accuracy of the quantitation of creatinine was found to be equivalent to that obtained using LC/MS. However, the expanded measurement uncertainty (k = 2) was larger, at 6%, when using FTICRMS compared with 1% when using HPLC/MS with the triple quadrupole mass spectrometer.

  2. Investigating quantitation of phosphorylation using MALDI-TOF mass spectrometry

    PubMed Central

    Parker, Laurie; Engel-Hall, Aaron; Drew, Kevin; Steinhardt, George; Helseth, Donald L.; Jabon, David; McMurry, Timothy; Angulo, David S.; Kron, Stephen J.

    2010-01-01

    Despite advances in methods and instrumentation for analysis of phosphopeptides using mass spectrometry, it is still difficult to quantify the extent of phosphorylation of a substrate due to physiochemical differences between unphosphorylated and phosphorylated peptides. Here we report experiments to investigate those differences using MALDI-TOF mass spectrometry for a set of synthetic peptides by creating calibration curves of known input ratios of peptides/phosphopeptides and analyzing their resulting signal intensity ratios. These calibration curves reveal subtleties in sequence-dependent differences for relative desorption/ionization efficiencies that cannot be seen from single-point calibrations. We found that the behaviors were reproducible with a variability of 5–10% for observed phosphopeptide signal. Although these data allow us to begin addressing the issues related to modeling these properties and predicting relative signal strengths for other peptide sequences, it is clear this behavior is highly complex and needs to be further explored. PMID:18064576

  3. 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.

  4. Quantitative mass spectrometry of posttranslational modifications: keys to confidence.

    PubMed

    Hennrich, Marco Leo; Gavin, Anne-Claude

    2015-04-07

    Posttranslational modifications (PTMs) of proteins represent an important level of cellular control. They participate in the efficient transduction of signals and form the basis of long-term cellular memory, allowing cells to adapt to a rapidly changing environment. More than 200 different PTMs have been described that affect many aspects of protein functions, and the importance of these modifications is evident from the number of diseases that arise from their deregulation. The proteome-wide analysis of certain PTMs, such as phosphorylation, acetylation, glycosylation, methylation, ubiquitination, and sumoylation, has become a standard procedure in many laboratories. We highlight and discuss some important aspects of systems-wide PTM analyses using mass spectrometry-based methods.

  5. Qualitative and quantitative mass spectrometry imaging of drugs and metabolites

    PubMed Central

    Lietz, Christopher B.; Gemperline, Erin; Li, Lingjun

    2013-01-01

    Mass spectrometric imaging (MSI) has rapidly increased its presence in the pharmaceutical sciences. While quantitative whole-body autoradiography and microautoradiography are the traditional techniques for molecular imaging of drug delivery and metabolism, MSI provides advantageous specificity that can distinguish the parent drug from metabolites and modified endogenous molecules. This review begins with the fundamentals of MSI sample preparation/ionization, and then moves on to both qualitative and quantitative applications with special emphasis on drug discovery and delivery. Cutting-edge investigations on sub-cellular imaging and endogenous signaling peptides are also highlighted, followed by perspectives on emerging technology and the path for MSI to become a routine analysis technique. PMID:23603211

  6. Quantitative and confirmative performance of liquid chromatography coupled to high-resolution mass spectrometry compared to tandem mass spectrometry.

    PubMed

    Kaufmann, Anton; Butcher, Patrick; Maden, Kathryn; Walker, Stephan; Widmer, Miryam

    2011-04-15

    The quantitative and confirmative performance of two different mass spectrometry (MS) techniques (high-resolution MS and tandem MS) was critically compared. Evaluated was a new extraction and clean-up protocol which was developed to cover more than 100 different veterinary drugs at trace levels in a number of animal tissues and honey matrices. Both detection techniques, high-resolution mass spectrometry (HRMS) (single-stage Orbitrap instrument operated at 50 000 full width at half maximum) and tandem mass spectrometry (MS/MS) (quadrupole technology) were used to validate the method according to the EU Commission Decision 2002/657/EEC. Equal or even a slightly better quantitative performance was observed for the HRMS-based approach. Sensitivity is higher for unit mass resolution MS/MS if only a subset of the 100 compounds has to be monitored. Confirmation of suspected positive findings can be done by evaluating the intensity ratio between different MS/MS transitions, or by accurate mass based product ion traces (no precursor selection applied). MS/MS relies on compound-specific optimized transitions; hence the second, confirmatory transition generally shows relatively high ion abundance (fragmentation efficacy). This is often not the case in single-stage HRMS, since a generic (not compound-optimized) collision energy is applied. Hence, confirmation of analytes present at low levels is superior when performed by MS/MS. Slightly better precision, but poorer accuracy (fortified matrix extracts versus pure standard solution) of ion ratios were observed when comparing data obtained by HRMS versus MS/MS. PMID:21416536

  7. Quantitative peptide and protein profiling by mass spectrometry.

    PubMed

    Schmidt, Alexander; Bisle, Birgit; Kislinger, Thomas

    2009-01-01

    Proteomics may be defined as the systematic analysis of proteins expressed in a given organism (Electrophoresis 16:1090-1094, 1995). Important technical innovations in mass spectrometry (MS), protein identification methods, and database annotation, over the past decade, now make it possible to routinely identify thousands of proteins in complex biological samples (Nature 422:198-207, 2003). However, to gain new insights regarding fundamental biological questions, accurate protein quantification is also required. In this chapter, we present methods for the biochemical separation of different cellular compartments, two-dimensional chromatographic separation of the constituent peptide populations, and the recently published Spectral Counting Strategy, a label-free MS-based protein quantification technology (Cell 125:173-186, 2006; Anal Chem 76:4193-4201, 2004; Mol Cell Proteomics 4:1487-1502, 2005; Cell 125:1003-1013, 2006; Methods 40:135-142, 2006; Anal Chem 77:6218-6224, 2005; J Proteome Res 5:2339-2347, 2006). Additionally, highly accurate protein quantification based on isotope dilution, describing the isotope coded protein label (ICPL) -- method shall be explained in detail (Mol Cell Proteomics 5:1543-1558, 2006; Proteomics 5:4-15, 2005).

  8. Sample preparation for quantitation of tritium by accelerator mass spectrometry.

    PubMed

    Chiarappa-Zucca, Marina L; Dingley, Karen H; Roberts, Mark L; Velsko, Carol A; Love, Adam H

    2002-12-15

    The capability to prepare samples accurately and reproducibly for analysis of tritium (3H) content by accelerator mass spectrometry (AMS) greatly facilitates isotopic tracer studies in which attomole levels of 3H can be measured in milligram-sized samples. A method has been developed to convert the hydrogen of organic samples to a solid, titanium hydride, which can be analyzed by AMS. Using a two-step process, the sample is first oxidized to carbon dioxide and water. In the second step, the water is transferred within a heated manifold into a quartz tube, reduced to hydrogen gas using zinc, and reacted with titanium powder. The 3H/1H ratio of the titanium hydride is measured by AMS and normalized to standards whose ratios were determined by decay counting to calculate the amount of 3H in the original sample. Water, organic compounds, and biological samples with 3H activities measured by liquid scintillation counting were utilized to develop and validate the method. The 3H/1H ratios were quantified in samples that spanned 5 orders of magnitude, from 10(-10) to 10(-15), with a detection limit of 3.0 x 10(-15), which is equivalent to 0.02 dpm tritium/mg of material. Samples smaller than 2 mg were analyzed following addition of 2 mg of a tritium-free-hydrogen carrier. Preparation of organic standards containing both 14C and 3H in 2-mg organic samples demonstrated that this sample preparation methodology can also be applied to quantify both of these isotopes from a single sample. PMID:12510750

  9. Comprehensive evaluation of direct injection mass spectrometry for the quantitative profiling of volatiles in food samples.

    PubMed

    Lebrón-Aguilar, R; Soria, A C; Quintanilla-López, J E

    2016-10-28

    Although qualitative strategies based on direct injection mass spectrometry (DIMS) have recently emerged as an alternative for the rapid classification of food samples, the potential of these approaches in quantitative tasks has scarcely been addressed to date. In this paper, the applicability of different multivariate regression procedures to data collected by DIMS from simulated mixtures has been evaluated. The most relevant factors affecting quantitation, such as random noise, the number of calibration samples, type of validation, mixture complexity and similarity of mass spectra, were also considered and comprehensively discussed. Based on the conclusions drawn from simulated data, and as an example of application, experimental mass spectral fingerprints collected by direct thermal desorption coupled to mass spectrometry were used for the quantitation of major volatiles in Thymus zygis subsp. zygis chemotypes. The results obtained, validated with the direct thermal desorption coupled to gas chromatography-mass spectrometry method here used as a reference, show the potential of DIMS approaches for the fast and precise quantitative profiling of volatiles in foods.This article is part of the themed issue 'Quantitative mass spectrometry'. PMID:27644978

  10. Comprehensive evaluation of direct injection mass spectrometry for the quantitative profiling of volatiles in food samples.

    PubMed

    Lebrón-Aguilar, R; Soria, A C; Quintanilla-López, J E

    2016-10-28

    Although qualitative strategies based on direct injection mass spectrometry (DIMS) have recently emerged as an alternative for the rapid classification of food samples, the potential of these approaches in quantitative tasks has scarcely been addressed to date. In this paper, the applicability of different multivariate regression procedures to data collected by DIMS from simulated mixtures has been evaluated. The most relevant factors affecting quantitation, such as random noise, the number of calibration samples, type of validation, mixture complexity and similarity of mass spectra, were also considered and comprehensively discussed. Based on the conclusions drawn from simulated data, and as an example of application, experimental mass spectral fingerprints collected by direct thermal desorption coupled to mass spectrometry were used for the quantitation of major volatiles in Thymus zygis subsp. zygis chemotypes. The results obtained, validated with the direct thermal desorption coupled to gas chromatography-mass spectrometry method here used as a reference, show the potential of DIMS approaches for the fast and precise quantitative profiling of volatiles in foods.This article is part of the themed issue 'Quantitative mass spectrometry'.

  11. Quantitative liquid chromatography/mass spectrometry/mass spectrometry warfarin assay for in vitro cytochrome P450 studies.

    PubMed

    Zhang, Z Y; King, B M; Wong, Y N

    2001-11-01

    A sensitive assay using high-performance liquid chromatography tandem mass spectrometry (MS/MS) has been established for the quantitative analysis of cytochrome P450 form-specific activities using warfarin as a probe substrate. Four metabolites, 6-, 7-, 8-, and 10-hydroxywarfarin, were chromatographically resolved within 10 min using gradient mobile phases. The mass spectrometry was operated under negative ionization mode. The MS/MS product ion spectra of warfarin and the metabolites were generated using collision-activated dissociation and interpreted. The abundant product ions of the metabolites were selected for quantification applying multiple reaction monitoring. Quantification was based on a quadratic or power curve of the peak area ratio of the metabolite over the internal standard against the respective concentration of the metabolite. This assay has been validated from 2 to 1000 nM for 10-hydroxywarfarin and from 2 to 5000 nM for 6-, 7-, and 8-hydroxywarfarin and successfully applied to evaluate cytochrome P450-mediated drug-drug interactions in vitro using human hepatocytes and liver microsomal preparations. PMID:11673893

  12. Development of rapid methodologies for the isolation and quantitation of drug metabolites by differential mobility spectrometry - mass spectrometry.

    PubMed

    Hall, Adam B; Coy, Stephen L; Nazarov, Erkinjon; Vouros, Paul

    2012-09-01

    Clinical and forensic toxicology laboratories are inundated with thousands of samples requiring lengthy chromatographic separations prior to mass spectrometry. Here, we employ differential mobility spectrometry (DMS) interfaced to nano-electrospray ionization-mass spectrometry to provide a rapid ion filtration technique for the separation of ions in gas phase media prior to mass spectral analysis on a DMS-integrated AB SCIEX API 3000 triple-quadrupole mass spectrometer. DMS is efficient at the rapid separation of ions under ambient conditions and provides many advantages when used as an ion filtration technique in tandem with mass spectrometry (MS) and MS/MS. Our studies evaluated DMS-MS/MS as a rapid, quantitative platform for the analysis of drug metabolites isolated from urine samples. In targeted applications, five metabolites of common drugs of abuse were effectively and rapidly separated using isopropanol and ethyl acetate as transport gas modifiers, eliminating the gas chromatography or liquid chromatography-based separations commonly employed in clinical and forensic toxicology laboratories. Calibration curves were prepared for the selected drug metabolites utilizing deuterated internal standards for quantitative purposes. The feasibility of separating and quantitating drug metabolites in a rapid fashion was evaluated by compensation voltage stepping followed by multiple reaction monitoring (MRM) detection. Rapid profiling of clinical and forensic toxicology samples could help to address an urgent need within the scientific community by developing high-throughput analytical methodologies, which could reduce significant case backlogs present within these laboratories.

  13. 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.

  14. Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry

    PubMed Central

    Gillette, Michael A; Carr, Steven A

    2014-01-01

    Targeted mass spectrometry (MS) is becoming widely used in academia and in pharmaceutical and biotechnology industries for sensitive and quantitative detection of proteins, peptides and post-translational modifications. Here we describe the increasing importance of targeted MS technologies in clinical proteomics and the potential key roles these techniques will have in bridging biomedical discovery and clinical implementation. PMID:23269374

  15. 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.

  16. Evolution of an open-access quantitative bioanalytical mass spectrometry service in a drug discovery environment.

    PubMed

    Wright, Patricia; Chassaing, Christophe; Cussans, Nigel; Gibson, Drew; Green, Caroline; Gleave, Michelle; Jones, Russell; Macrae, Paul; Saunders, Kenneth

    2006-01-01

    Increased demand for assays for compounds at the early stages of drug discovery within the pharmaceutical industry has led to the need for open-access mass spectrometry systems for performing quantitative analysis in a variety of biological matrices. The open-access mass spectrometers described here are LC/MS/MS systems operated in 'multiple reaction monitoring' (MRM) mode to obtain the sensitivity and specificity required to quantitate low levels of pharmaceutical compounds in an excess of biological matrix. Instigation of these open-access systems has resulted in mass spectrometers becoming the detectors of choice for non-expert users, drastically reducing analytical method development time and allowing drug discovery scientists to concentrate on their core expertise of pharmacokinetics and drug metabolism. Setting up an open-access facility that effectively allows a user with minimal mass spectral knowledge to exploit the MS/MS capability of triple quadrupole mass spectrometers presents a significantly different challenge from setting up qualitative single stage mass spectrometry systems. Evolution of quantitative open access mass spectrometry within a pharmaceutical drug metabolism and pharmacokinetics group, from its beginnings as a single generic system to a series of specialist fully integrated walk-up facilities, is described.

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

    PubMed

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

    2014-01-01

    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.

  18. Quantitation of Parathyroid Hormone in Serum or Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

    PubMed

    Ketha, Hemamalini; Singh, Ravinder J

    2016-01-01

    Parathyroid hormone (PTH), an 84 amino acid peptide hormone, is an important regulator of calcium homeostasis. Quantitation of PTH in serum is useful for the diagnosis of primary hyperparathyroidism, hypoparathyroidism, and for monitoring osteodystrophy in patients with renal failure. The biological activity of PTH arises from binding of PTH (N terminus) to its target receptor (D'Amour et al., Kidney Int 68: 998-1007, 2005). Several C-terminal and N-terminal fragments circulate in normal subjects. Recent studies have demonstrated that accurate quantitation of PTH fragments may be of clinical value. In this chapter a mass spectrometry based method for quantitation of PTH(1-84) is described. This method involves immunoaffinity capture of PTH followed by trypsinization and quantitation of PTH-specific tryptic peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The N-terminal tryptic peptide, PTH(1-13) as surrogate of 1-84 PTH, is used for quantitation. PMID:26602132

  19. 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.

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

    PubMed

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

    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

  1. 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

  2. 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.

  3. 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.

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

    PubMed Central

    Steinhauser, Matthew L.; Lechene, Claude P.

    2014-01-01

    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. PMID:23660233

  5. Absolute Quantitative MALDI Imaging Mass Spectrometry: A Case of Rifampicin in Liver Tissues.

    PubMed

    Chumbley, Chad W; Reyzer, Michelle L; Allen, Jamie L; Marriner, Gwendolyn A; Via, Laura E; Barry, Clifton E; Caprioli, Richard M

    2016-02-16

    Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) elucidates molecular distributions in thin tissue sections. Absolute pixel-to-pixel quantitation has remained a challenge, primarily lacking validation of the appropriate analytical methods. In the present work, isotopically labeled internal standards are applied to tissue sections to maximize quantitative reproducibility and yield accurate quantitative results. We have developed a tissue model for rifampicin (RIF), an antibiotic used to treat tuberculosis, and have tested different methods of applying an isotopically labeled internal standard for MALDI IMS analysis. The application of the standard and subsequently the matrix onto tissue sections resulted in quantitation that was not statistically significantly different from results obtained using HPLC-MS/MS of tissue extracts. Quantitative IMS experiments were performed on liver tissue from an animal dosed in vivo. Each microspot in the quantitative images measures the local concentration of RIF in the thin tissue section. Lower concentrations were detected from the blood vessels and around the portal tracts. The quantitative values obtained from these measurements were comparable (>90% similarity) to HPLC-MS/MS results obtained from extracts of the same tissue.

  6. Detecting outlier peptides in quantitative high-throughput mass spectrometry data.

    PubMed

    Erhard, Florian; Zimmer, Ralf

    2012-06-18

    Quantitative high-throughput mass spectrometry has become an established tool to measure relative gene expression proteome-wide. The output of such an experiment usually consists of a list of expression ratios (fold changes) for several thousand proteins between two conditions. However, we observed that individual peptide fold changes may show a significantly different behavior than other peptides from the same protein and that these differences cannot be explained by imprecise measurements. Such outlier peptides can be the consequence of several technical (misidentifications, misquantifications) or biological (post-translational modifications, differential regulation of isoforms) reasons. We developed a method to detect outlier peptides in mass spectrometry data which is able to delineate imprecise measurements from real outlier peptides with high accuracy when the true difference is as small as 1.4 fold. We applied our method to experimental data and investigated the different technical and biological effects that result in outlier peptides. Our method will assist future research to reduce technical bias and can help to identify genes with differentially regulated protein isoforms in high throughput mass spectrometry data. PMID:22483996

  7. Minimizing technical variation during sample preparation prior to label-free quantitative mass spectrometry.

    PubMed

    Scheerlinck, E; Dhaenens, M; Van Soom, A; Peelman, L; De Sutter, P; Van Steendam, K; Deforce, D

    2015-12-01

    Sample preparation is the crucial starting point to obtain high-quality mass spectrometry data and can be divided into two main steps in a bottom-up proteomics approach: cell/tissue lysis with or without detergents and a(n) (in-solution) digest comprising denaturation, reduction, alkylation, and digesting of the proteins. Here, some important considerations, among others, are that the reagents used for sample preparation can inhibit the digestion enzyme (e.g., 0.1% sodium dodecyl sulfate [SDS] and 0.5 M guanidine HCl), give rise to ion suppression (e.g., polyethylene glycol [PEG]), be incompatible with liquid chromatography-tandem mass spectrometry (LC-MS/MS) (e.g., SDS), and can induce additional modifications (e.g., urea). Taken together, all of these irreproducible effects are gradually becoming a problem when label-free quantitation of the samples is envisioned such as during the increasingly popular high-definition mass spectrometry (HDMS(E)) and sequential window acquisition of all theoretical fragment ion spectra (SWATH) data-independent acquisition strategies. Here, we describe the detailed validation of a reproducible method with sufficient protein yield for sample preparation without any known LC-MS/MS interfering substances by using 1% sodium deoxycholate (SDC) during both cell lysis and in-solution digest. PMID:26302362

  8. Minimizing technical variation during sample preparation prior to label-free quantitative mass spectrometry.

    PubMed

    Scheerlinck, E; Dhaenens, M; Van Soom, A; Peelman, L; De Sutter, P; Van Steendam, K; Deforce, D

    2015-12-01

    Sample preparation is the crucial starting point to obtain high-quality mass spectrometry data and can be divided into two main steps in a bottom-up proteomics approach: cell/tissue lysis with or without detergents and a(n) (in-solution) digest comprising denaturation, reduction, alkylation, and digesting of the proteins. Here, some important considerations, among others, are that the reagents used for sample preparation can inhibit the digestion enzyme (e.g., 0.1% sodium dodecyl sulfate [SDS] and 0.5 M guanidine HCl), give rise to ion suppression (e.g., polyethylene glycol [PEG]), be incompatible with liquid chromatography-tandem mass spectrometry (LC-MS/MS) (e.g., SDS), and can induce additional modifications (e.g., urea). Taken together, all of these irreproducible effects are gradually becoming a problem when label-free quantitation of the samples is envisioned such as during the increasingly popular high-definition mass spectrometry (HDMS(E)) and sequential window acquisition of all theoretical fragment ion spectra (SWATH) data-independent acquisition strategies. Here, we describe the detailed validation of a reproducible method with sufficient protein yield for sample preparation without any known LC-MS/MS interfering substances by using 1% sodium deoxycholate (SDC) during both cell lysis and in-solution digest.

  9. 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

  10. 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

  11. Reference map for liquid chromatography-mass spectrometry-based quantitative proteomics.

    PubMed

    Kim, Yeoun Jin; Feild, Brian; Fitzhugh, William; Heidbrink, Jenny L; Duff, James W; Heil, Jeremy; Ruben, Steven M; He, Tao

    2009-10-15

    The accurate mass and time (AMT) tag strategy has been recognized as a powerful tool for high-throughput analysis in liquid chromatography-mass spectrometry (LC-MS)-based proteomics. Due to the complexity of the human proteome, this strategy requires highly accurate mass measurements for confident identifications. We have developed a method of building a reference map that allows relaxed criteria for mass errors yet delivers high confidence for peptide identifications. The samples used for generating the peptide database were produced by collecting cysteine-containing peptides from T47D cells and then fractionating the peptides using strong cationic exchange chromatography (SCX). LC-tandem mass spectrometry (MS/MS) data from the SCX fractions were combined to create a comprehensive reference map. After the reference map was built, it was possible to skip the SCX step in further proteomic analyses. We found that the reference-driven identification increases the overall throughput and proteomic coverage by identifying peptides with low intensity or complex interference. The use of the reference map also facilitates the quantitation process by allowing extraction of peptide intensities of interest and incorporating models of theoretical isotope distribution.

  12. 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

  13. Visualizing spatial distribution of alectinib in murine brain using quantitative mass spectrometry imaging

    PubMed Central

    Aikawa, Hiroaki; Hayashi, Mitsuhiro; Ryu, Shoraku; Yamashita, Makiko; Ohtsuka, Naoto; Nishidate, Masanobu; Fujiwara, Yasuhiro; Hamada, Akinobu

    2016-01-01

    In the development of anticancer drugs, drug concentration measurements in the target tissue have been thought to be crucial for predicting drug efficacy and safety. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is commonly used for determination of average drug concentrations; however, complete loss of spatial information in the target tissue occurs. Mass spectrometry imaging (MSI) has been recently applied as an innovative tool for detection of molecular distribution of pharmacological agents in heterogeneous targets. This study examined the intra-brain transitivity of alectinib, a novel anaplastic lymphoma kinase inhibitor, using a combination of matrix-assisted laser desorption ionization–MSI and LC-MS/MS techniques. We first analyzed the pharmacokinetic profiles in FVB mice and then examined the effect of the multidrug resistance protein-1 (MDR1) using Mdr1a/b knockout mice including quantitative distribution of alectinib in the brain. While no differences were observed between the mice for the plasma alectinib concentrations, diffuse alectinib distributions were found in the brain of the Mdr1a/b knockout versus FVB mice. These results indicate the potential for using quantitative MSI for clarifying drug distribution in the brain on a microscopic level, in addition to suggesting a possible use in designing studies for anticancer drug development and translational research. PMID:27026287

  14. 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.

  15. Quantitative determination of tilmicosin in canine serum by high performance liquid chromatography-tandem mass spectrometry.

    PubMed

    Herrera, Michael; Ding, Haiqing; McClanahan, Robert; Owens, Jane G; Hunter, Robert P

    2007-09-15

    A highly sensitive and quantitative LC/MS/MS assay for the determination of tilmicosin in serum has been developed and validated. For sample preparation, 0.2 mL of canine serum was extracted with 3 mL of methyl tert-butyl ether. The organic layer was transferred to a new vessel and dried under nitrogen. The sample was then reconstituted for analysis by high performance liquid chromatography-tandem mass spectrometry. A Phenomenex Luna C8(2) analytical column was used for the chromatographic separation. The eluent was subsequently introduced to the mass spectrometer by electrospray ionization. A single range was validated for 50-5000 ng/mL for support of toxicokinetic studies. The inter-day relative error (inaccuracy) for the LLOQ samples ranged from -5.5% to 0.3%. The inter-day relative standard deviations (imprecision) at the respective LLOQ levels were < or =10.1%.

  16. 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. PMID:26138893

  17. The mzQuantML data standard for mass spectrometry-based quantitative studies in proteomics.

    PubMed

    Walzer, Mathias; Qi, Da; Mayer, Gerhard; Uszkoreit, Julian; Eisenacher, Martin; Sachsenberg, Timo; Gonzalez-Galarza, Faviel F; Fan, Jun; Bessant, Conrad; Deutsch, Eric W; Reisinger, Florian; Vizcaíno, Juan Antonio; Medina-Aunon, J Alberto; Albar, Juan Pablo; Kohlbacher, Oliver; Jones, Andrew R

    2013-08-01

    The range of heterogeneous approaches available for quantifying protein abundance via mass spectrometry (MS)(1) leads to considerable challenges in modeling, archiving, exchanging, or submitting experimental data sets as supplemental material to journals. To date, there has been no widely accepted format for capturing the evidence trail of how quantitative analysis has been performed by software, for transferring data between software packages, or for submitting to public databases. In the context of the Proteomics Standards Initiative, we have developed the mzQuantML data standard. The standard can represent quantitative data about regions in two-dimensional retention time versus mass/charge space (called features), peptides, and proteins and protein groups (where there is ambiguity regarding peptide-to-protein inference), and it offers limited support for small molecule (metabolomic) data. The format has structures for representing replicate MS runs, grouping of replicates (for example, as study variables), and capturing the parameters used by software packages to arrive at these values. The format has the capability to reference other standards such as mzML and mzIdentML, and thus the evidence trail for the MS workflow as a whole can now be described. Several software implementations are available, and we encourage other bioinformatics groups to use mzQuantML as an input, internal, or output format for quantitative software and for structuring local repositories. All project resources are available in the public domain from the HUPO Proteomics Standards Initiative http://www.psidev.info/mzquantml.

  18. 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.

  19. New simultaneous thermogravimetry and modulated molecular beam mass spectrometry apparatus for quantitative thermal decomposition studies

    SciTech Connect

    Behrens, R. Jr.

    1987-03-01

    A new type of instrument has been designed and constructed to measure quantitatively the gas phase species evolving during thermal decompositions. These measurements can be used for understanding the kinetics of thermal decomposition, determining the heats of formation and vaporization of high-temperature materials, and analyzing sample contaminants. The new design allows measurements to be made on the same time scale as the rates of the reactions being studied, provides a universal detection technique to study a wide range of compounds, gives quantitative measurements of decomposition products, and minimizes interference from the instrument on the measurements. The instrument design is based on a unique combination of thermogravimetric analysis (TGA), differential thermal analysis (DTA), and modulated beam mass spectroscopy (MBMS) which are brought together into a symbiotic relationship through the use of differentially pumped vacuum systems, modulated molecular beam techniques, and computer control and data-acquisition systems. A data analysis technique that calculates partial pressures in the reaction cell from the simultaneous microbalance force measurements and the modulated mass spectrometry measurements has been developed. This eliminates the need to know the ionization cross section, the ion dissociation channels, the quadrupole transmission, and the ion detector sensitivity for each thermal decomposition product prior to quantifying the mass spectral data. The operation of the instrument and the data analysis technique are illustrated with the thermal decomposition of contaminants from a precipitated palladium powder.

  20. Improving quantitative precision and throughput by reducing calibrator use in liquid chromatography-tandem mass spectrometry.

    PubMed

    Rule, Geoffrey S; Rockwood, Alan L

    2016-05-01

    To improve efficiency in our mass spectrometry laboratories we have made efforts to reduce the number of calibration standards utilized for quantitation over time. We often analyze three or more batches of 96 samples per day, on a single instrument, for a number of assays. With a conventional calibration scheme at six concentration levels this amounts to more than 5000 calibration points per year. Modern LC-tandem mass spectrometric instrumentation is extremely rugged however, and isotopically labelled internal standards are widely available. This made us consider whether alternative calibration strategies could be utilized to reduce the number of calibration standards analyzed while still retaining high precision and accurate quantitation. Here we demonstrate how, by utilizing a single calibration point in each sample batch, and using the resulting response factor (RF) to update an existing, historical response factor (HRF), we are able to obtain improved precision over a conventional multipoint calibration approach, as judged by quality control samples. The laboratory component of this study was conducted with an existing LC tandem mass spectrometric method for three androgen analytes in our production laboratory. Using examples from both simulated and laboratory data we illustrate several aspects of our single point alternative calibration strategy and compare it with a conventional, multipoint calibration approach. We conclude that both the cost and burden of preparing multiple calibration standards with every batch of samples can be reduced while at the same time maintaining, or even improving, analytical quality. PMID:27086099

  1. 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.

  2. 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.

  3. 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).

  4. 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.

  5. 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. PMID:16013850

  6. Quantitative imaging mass spectrometry of renal sulfatides: validation by classical mass spectrometric methods1[S

    PubMed Central

    Marsching, Christian; Jennemann, Richard; Heilig, Raphael; Gröne, Hermann-Josef; Hopf, Carsten; Sandhoff, Roger

    2014-01-01

    Owing to its capability of discriminating subtle mass-altering structural differences such as double bonds or elongated acyl chains, MALDI-based imaging MS (IMS) has emerged as a powerful technique for analysis of lipid distribution in tissue at moderate spatial resolution of about 50 μm. However, it is still unknown if MS1-signals and ion intensity images correlate with the corresponding apparent lipid concentrations. Analyzing renal sulfated glycosphingolipids, sulfatides, we validate for the first time IMS-signal identities using corresponding sulfatide-deficient kidneys. To evaluate the extent of signal quenching effects interfering with lipid quantification, we surgically dissected the three major renal regions (papillae, medulla, and cortex) and systematically compared MALDI IMS of renal sulfatides with quantitative analyses of corresponding lipid extracts by on-target MALDI TOF-MS and by ultra-performance LC-ESI-(triple-quadrupole)tandem MS. Our results demonstrate a generally strong correlation (R2 > 0.9) between the local relative sulfatide signal intensity in MALDI IMS and absolute sulfatide quantities determined by the other two methods. However, high concentrations of sulfatides in the papillae and medulla result in an up to 4-fold signal suppression. In conclusion, our study suggests that MALDI IMS is useful for semi-quantitative dissection of relative local changes of sulfatides and possibly other lipids in tissue. PMID:25274613

  7. xTract: software for characterizing conformational changes of protein complexes by quantitative cross-linking mass spectrometry.

    PubMed

    Walzthoeni, Thomas; Joachimiak, Lukasz A; Rosenberger, George; Röst, Hannes L; Malmström, Lars; Leitner, Alexander; Frydman, Judith; Aebersold, Ruedi

    2015-12-01

    Chemical cross-linking in combination with mass spectrometry generates distance restraints of amino acid pairs in close proximity on the surface of native proteins and protein complexes. In this study we used quantitative mass spectrometry and chemical cross-linking to quantify differences in cross-linked peptides obtained from complexes in spatially discrete states. We describe a generic computational pipeline for quantitative cross-linking mass spectrometry consisting of modules for quantitative data extraction and statistical assessment of the obtained results. We used the method to detect conformational changes in two model systems: firefly luciferase and the bovine TRiC complex. Our method discovers and explains the structural heterogeneity of protein complexes using only sparse structural information. PMID:26501516

  8. xTract: software for characterizing conformational changes of protein complexes by quantitative cross-linking mass spectrometry

    PubMed Central

    Walzthoeni, Thomas; Joachimiak, Lukasz A; Rosenberger, George; Röst, Hannes L; Malmström, Lars; Leitner, Alexander; Frydman, Judith; Aebersold, Ruedi

    2016-01-01

    Chemical cross-linking in combination with mass spectrometry generates distance restraints of amino acid pairs in close proximity on the surface of native proteins and protein complexes. In this study we used quantitative mass spectrometry and chemical cross-linking to quantify differences in cross-linked peptides obtained from complexes in spatially discrete states. We describe a generic computational pipeline for quantitative cross-linking mass spectrometry consisting of modules for quantitative data extraction and statistical assessment of the obtained results. We used the method to detect conformational changes in two model systems: firefly luciferase and the bovine TRiC complex. Our method discovers and explains the structural heterogeneity of protein complexes using only sparse structural information. PMID:26501516

  9. Nonlinear calibration curves in secondary ion mass spectrometry for quantitative analysis of gesi heterostructures with nanoclusters

    NASA Astrophysics Data System (ADS)

    Drozdov, M. N.; Drozdov, Yu. N.; Novikov, A. V.; Yunin, P. A.; Yurasov, D. V.

    2016-03-01

    For the first time in the practice of secondary ion mass spectrometry, we obtained a nonlinear calibration curve for the ratio of the cluster and elementary secondary ions of germanium Ge2/Ge without secondary ions of silicon, which enables the quantification of germanium in Ge x Si1- x heterostructures in the entire range of 0 < x ≤ 1. We developed a method for quantitative lateral analysis based on the plotting of a lateral map of x. An algorithm to identify and analyze the lateral heterogeneity of x in Ge x Si1- x heterostructures with 3D clusters by comparing the results of depth profiling analysis, obtained using linear and nonlinear calibration curves, is developed, and concentration x in the self-assembled nanoislands is determined.

  10. Epiproteomics: quantitative analysis of histone marks and codes by mass spectrometry.

    PubMed

    Zheng, Yupeng; Huang, Xiaoxiao; Kelleher, Neil L

    2016-08-01

    Histones are a group of proteins with a high number of post-translational modifications, including methylation, acetylation, phosphorylation, and monoubiquitination, which play critical roles in every chromatin-templated activity. The quantitative analysis of these modifications using mass spectrometry (MS) has seen significant improvements over the last decade. It is now possible to perform large-scale surveys of dozens of histone marks and hundreds of their combinations on global chromatin. Here, we review the development of three MS strategies for analyzing histone modifications that have come to be known as Bottom Up, Middle Down, and Top Down. We also discuss challenges and innovative solutions for characterizing and quantifying complicated isobaric species arising from multiple modifications on the same histone molecule. PMID:27371874

  11. 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.

  12. Quantitative Composition Analysis of Lipid Membranes by High-Resolution Secondary Ion Mass Spectrometry

    SciTech Connect

    Kraft, M L; Weber, P K; Lin, W C; Blanchette, C D; Longo, M L; Hutcheon, I D; Boxer, S G

    2005-04-29

    The lateral organization and interactions of lipid and protein components within biological membranes are essential for their functions. Investigations of the lateral organization within membranes hinge upon the ability to differentiate one component of interest from another. Typically, fluorophores are conjugated to specific components, and the organization is probed with fluorescence microscopy. However, bulky fluorophores may change the physical properties of the components they label, only the labeled components can be visualized, and the diffraction limit of light restricts the lateral resolution. Here we present a method to image microdomains within supported lipid membranes using isotopic labels and high-resolution secondary ion mass spectrometry (SIMS) performed with the NanoSIMS 50 (Cameca). Lateral resolution of 100 nm is achieved with high sensitivity. Quantitative information on the lipid composition within each domain was determined using calibration curves constructed from homogeneous lipid bilayer samples that systematically varied in the isotopically labeled lipid content.

  13. Quantitative profiling and pattern analysis of triacylglycerol species in Arabidopsis seeds by electrospray ionization mass spectrometry.

    PubMed

    Li, Maoyin; Baughman, Ethan; Roth, Mary R; Han, Xianlin; Welti, Ruth; Wang, Xuemin

    2014-01-01

    Plant triacylglycerols (TAGs), or vegetable oils, provide approximately 25% of dietary calories to humans and are becoming an increasingly important source of renewable bioenergy and industrial feedstocks. TAGs are assembled by multiple enzymes in the endoplasmic reticulum from building blocks that include an invariable glycerol backbone and variable fatty acyl chains. It remains a challenge to elucidate the mechanism of synthesis of hundreds of different TAG species in planta. One reason is the lack of an efficient analytical approach quantifying individual molecular species. Here we report a rapid and quantitative TAG profiling approach for Arabidopsis seeds based on electrospray ionization tandem mass spectrometry with direct infusion and multiple neutral loss scans. The levels of 93 TAG molecular species, identified by their acyl components, were determined in Arabidopsis seeds. Quantitative TAG pattern analyses revealed that the TAG assembly machinery preferentially produces TAGs with one elongated fatty acid. The importance of the selectivity in oil synthesis was consistent with an observation that an Arabidopsis mutant overexpressing a patatin-like phospholipase had enhanced seed oil content with elongated fatty acids. This quantitative TAG profiling approach should facilitate investigations aimed at understanding the biochemical mechanisms of TAG metabolism in plants.

  14. Quantitative analysis of clenbuterol in meat products using liquid chromatography-electrospray ionisation tandem mass spectrometry.

    PubMed

    Guy, P A; Savoy, M C; Stadler, R H

    1999-12-24

    A method is presented that allows quantitation of clenbuterol in meat and liver products at the ng/kg level by liquid chromatography-electrospray ionisation tandem mass spectrometry (LC-ESIMS-MS) using a stable isotopically labeled internal standard. The practical procedure involves acid extraction followed by two solid-phase clean-up steps with C18 and strong cation-exchange (SCX) resins. The typical recovery of the analyte spiked at 0.4 microg/kg in meat and liver samples was at 63+/-7%. Mass spectral acquisition was done in multiple reaction monitoring (MRM) to provide a high degree of sensitivity, achieving a limit of detection and quantitation at 10 and 15 ng/kg, respectively. Two precursor ions at m/z 277 and 279, corresponding to the characteristic isotopic cluster of the two chlorine atoms of clenbuterol, were monitored by LC-ESIMS-MS to provide unambiguous identity of the analyte. Samples of meat and liver of various origins with either incurred residues or spiked with known amounts of clenbuterol were used to validate the method.

  15. 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.

  16. A single-vial analytical and quantitative gas chromatography-mass spectrometry assay for terpene synthases.

    PubMed

    O'Maille, Paul E; Chappell, Joe; Noel, Joseph P

    2004-12-15

    A quantitative assay for the analysis of sesquiterpene synthases, wherein each reaction mixture is formulated in glass gas chromatography vials, overlaid with organic solvent such as ethyl acetate, and subsequently vortexed to extract hydrocarbon reaction products into the organic phase after a suitable incubation period, was developed. The product-enriched organic phase is then sampled in an automated fashion and injected directly into a gas chromatograph-mass spectrometer without further workup for analysis and quantification of hydrocarbon products. Application of the vial assay to the analysis of amorpha-4,11-diene synthase (ADS), a sesquiterpene synthase, demonstrated the sensitivity of the assay for detection of major and minor reaction products and most notably for the identification of several sesquiterpene products that had escaped previous detection. A steady-state kinetic analysis of tobacco 5-epi-aristolochene synthase (TEAS), another sesquiterpene synthase, validated the quantitative nature of the assay, providing an alternative means to the established method of using radiolabeled substrate, extraction, and scintillation counting. This simplified assay provides a standardized method to facilitate analysis of terpene synthases and diverse mutant enzyme libraries by supplanting the common practice of using larger scale reactions, multiple extractions, and evaporative concentration of the organic phase prior to gas chromatography-mass spectrometry (GC-MS) analysis. PMID:15556559

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

    PubMed

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

    2016-09-20

    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.

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

    PubMed

    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

  19. 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.

  20. Quantitative, Multidrug Pain Medication Testing by Liquid Chromatography: Tandem Mass Spectrometry (LC-MS/MS).

    PubMed

    Bodor, Geza S

    2016-01-01

    Chronic pain is often treated with narcotic analgesics. The most commonly used narcotic analgesics are the opiates (natural or modified compounds of the poppy plant) or opioids (synthetic chemicals that act on opiate receptors). While opiates and opioids are excellent analgesics, they can also have significant side effects that include respiratory depression, coma, or death. Tolerance, physical dependence, and addiction (psychological dependence) are other severe side effects of opioid use. Patients who develop dependence or addiction often times abuse other, non-opioid narcotics and may trade their prescription medication for illegal street drugs (called "diversion"). In order to minimize side effects, detect possible multidrug abuse and prove diversion, simultaneous monitoring of numerous prescription and illicit drugs is required. The method described in this chapter is for the quantitative measurement of 43 different drugs in urine. The panel includes narcotic pain medications, benzodiazepines, NIDA drugs, and other, commonly abused medications. The analytes of interests are injected in the presence of deuterated internal standards to correct for possible extraction inefficiencies, ion suppression, or other interferences. The sample is prepared by adding dilution buffer with the deuterated internal standards to the sample, followed by reversed-phase, gradient HPLC separation on a Phenyl-Hexyl column using water and methanol as mobile phases. Detection of the analytes of interest is done by isotope-dilution mass spectrometry on a triple-quadrupole tandem mass spectrometer following electrospray ionization in the positive mode. Mass spectrometric (MS) data are collected in the scheduled MRM (sMRM) mode. Two MRM transitions are monitored for each analyte and one MRM transition is monitored for each IS. Quantitation of the unknown analytes is achieved by comparing the peak area ratios of the analytes to that of the internal standards and reading the unknown

  1. Quantitative and qualitative analyses of human salivary NEFA with gas-chromatography and mass spectrometry

    PubMed Central

    Kulkarni, Bhushan V.; Wood, Karl V.; Mattes, Richard D.

    2012-01-01

    Salivary non-esterified fatty acids (NEFA) are proposed to play a role in oral health, oral fat detection, and they may hold diagnostic and prognostic potential. Yet, little is known about the array and concentrations of NEFA in saliva. The aim of the study was to conduct qualitative and quantitative analyses of salivary NEFA in healthy humans and to present a new, efficient protocol to perform such analyses. Resting saliva samples from fifteen participants were collected. The salivary lipids were extracted using a modified Folch extraction. The NEFA in the extracted lipids were selectively subjected to pentafluorobenzyl bromide (PFB) derivatization and qualitatively and quantitatively analyzed using gas chromatography–mass spectrometry (GC–MS). A total of 16 NEFA were identified in resting saliva. The four major NEFA were palmitic, linoleic, oleic, and stearic acids. Their concentrations ranged from 2 to 9 μM. This is the first study to characterize individual human salivary NEFA and their respective concentrations. The method used in the study is sensitive, precise, and accurate. It is specific to fatty acids in non-esterified form and hence enables analysis of NEFA without their separation from other lipid classes. Thus, it saves time, reagents and prevents loss of sample. These properties make it suitable for large scale analysis of salivary NEFA. PMID:22934076

  2. 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; Aebersold, Ruedi

    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, to generate data sets that are comparable between experiments and laboratories, and have high throughput to support statistical studies. In this paper, 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, no de-glycosylated peptides by LC-ESI-MS, 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 to their control littermates.

  3. Quantitative analysis of buprenorphine and norbuprenorphine in urine using liquid chromatography tandem mass spectrometry.

    PubMed

    Fox, E J; Tetlow, V A; Allen, K R

    2006-05-01

    Buprenorphine is an opioid analgesic drug that is used as an alternative to methadone to treat heroin addiction. Established methods for the analysis of buprenorphine and its metabolites in urine such as gas chromatography-mass spectrometry (GC-MS) involve complicated sample extraction procedures. The aim of the present study was to develop a sensitive yet straightforward method for the simultaneous analysis of buprenorphine and norbuprenorphine in urine using liquid chromatography-MS-MS. The method comprised an enzymatic hydrolysis using Patella vulgata b-glucuronidase, followed by centrifugation and direct analysis of the supernatant. The limits of detection and quantitation were < 1 microg/L for buprenorphine and < 1 and 4 microg/L, respectively, for norbuprenorphine. Assay coefficients of variation (CVs) were < 15%, with the exception of concentrations close to the limit of quantitation, where CVs were below 20%. In direct comparison with an established GC-MS protocol, the method showed minimal negative bias (8.7% for buprenorphine and 1.8% for norbuprenorphine) and was less susceptible to sample carryover. The extent of conjugation in unhydrolyzed urine was investigated and found to be highly variable, with proportions of unconjugated buprenorphine and norbuprenorphine of 6.4% [range 0% to 67%; standard deviation (SD) 9.7%] and 34% (range 0% to 100%; SD 23.8%), respectively. PMID:16803661

  4. 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.

    2016-10-01

    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.

  5. 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

  6. 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…

  7. Tools and procedures for quantitative microbeam isotope ratio imaging by secondary ion mass spectrometry.

    PubMed

    Gillen, Greg; Bright, David

    2003-01-01

    In this work we demonstrate the use of secondary ion mass spectrometry (SIMS) combined with the Lispix image processing program (Bright 1995) to generate quantitative isotope ratio images from a test sample of a calcium-aluminum rich inclusion from the Allende meteorite that is known to contain discrete mineral grains with perturbed Mg isotopic ratios. Using 19.5 keV impact O- primary ion bombardment and detection of positive secondary ions, microbeam imaging SIMS has allowed us to identify, from the isotope ratio images, enrichments in the 26Mg/24Mg isotope ratio of approximately 5-15% in selected mineral grains. Using custom image processing software, each isotopic ratio image is corrected on an individual pixel basis for a number of factors including detector dead-time, mass bias effects, and isobaric interferences. We have developed procedures for correlating the isotopic images with polarized optical microscopy so that targeted mineral grains could be identified for further SIMS analysis. Finally, additional image processing tools have been developed to allow for pixel-by-pixel evaluation of the influence of detector dead-time and count rate errors on the isotopic ratio images and for correlation of the isotopic images with elemental distribution maps.

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

    PubMed

    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

    2016-03-01

    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 Lys(27) 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

  9. Evaluation of Large Scale Quantitative Proteomic Assay Development Using Peptide Affinity-based Mass Spectrometry*

    PubMed Central

    Whiteaker, Jeffrey R.; Zhao, Lei; Abbatiello, Susan E.; Burgess, Michael; Kuhn, Eric; Lin, ChenWei; Pope, Matthew E.; Razavi, Morteza; Anderson, N. Leigh; Pearson, Terry W.; Carr, Steven A.; Paulovich, Amanda G.

    2011-01-01

    Stable isotope standards and capture by antipeptide antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution and detection by multiple reaction monitoring mass spectrometry to provide quantitative measurement of peptides as surrogates for their respective proteins. In this report, we describe a feasibility study to determine the success rate for production of suitable antibodies for SISCAPA assays in order to inform strategies for large-scale assay development. A workflow was designed that included a multiplex immunization strategy in which up to five proteotypic peptides from a single protein target were used to immunize individual rabbits. A total of 403 proteotypic tryptic peptides representing 89 protein targets were used as immunogens. Antipeptide antibody titers were measured by ELISA and 220 antipeptide antibodies representing 89 proteins were chosen for affinity purification. These antibodies were characterized with respect to their performance in SISCAPA-multiple reaction monitoring assays using trypsin-digested human plasma matrix. More than half of the assays generated were capable of detecting the target peptide at concentrations of less than 0.5 fmol/μl in human plasma, corresponding to protein concentrations of less than 100 ng/ml. The strategy of multiplexing five peptide immunogens was successful in generating a working assay for 100% of the targeted proteins in this evaluation study. These results indicate it is feasible for a single laboratory to develop hundreds of assays per year and allow planning for cost-effective generation of SISCAPA assays. PMID:21245105

  10. Quantitative measurements of small molecule mixtures using laser electrospray mass spectrometry.

    PubMed

    Flanigan, Paul M; Perez, Johnny J; Karki, Santosh; Levis, Robert J

    2013-04-01

    Quantitative measurements of atenolol, tioconazole, tetraethylammonium bromide, and tetrabutylammonium iodide using laser electrospray mass spectrometry (LEMS) reveal monotonic signal response as a function of concentration for single analytes, two- and four-component equimolar mixtures, and two-component variable molarity mixtures. LEMS analyses of single analytes as a function of concentration were linear over ~2.5 orders of magnitude for all four analytes and displayed no sign of saturation. Corresponding electrospray ionization (ESI) measurements displayed a nonmonotonic increase as saturation occurred at higher concentrations. In contrast to the LEMS experiments, the intensity ratios from control experiments using conventional ESI-MS deviated from expected values for the equimolar mixture measurements due to ion suppression of less surface active analytes, particularly in the analysis of the four-component mixture. In the analyses of two-component nonequimolar mixtures, both techniques were able to determine the concentration ratios after adjustment with response factors although conventional ESI-MS was subject to a greater degree of saturation and ion suppression at higher analyte concentrations.

  11. Identification of Cell Cycle Dependent Interaction Partners of the Septins by Quantitative Mass Spectrometry

    PubMed Central

    Renz, Christian; Oeljeklaus, Silke; Grinhagens, Sören; Warscheid, Bettina; Johnsson, Nils; Gronemeyer, Thomas

    2016-01-01

    The septins are a conserved family of GTP-binding proteins that, in the baker's yeast, assemble into a highly ordered array of filaments at the mother bud neck. These filaments undergo significant structural rearrangements during the cell cycle. We aimed at identifying key components that are involved in or regulate the transitions of the septins. By combining cell synchronization and quantitative affinity-purification mass-spectrometry, we performed a screen for specific interaction partners of the septins at three distinct stages of the cell cycle. A total of 83 interaction partners of the septins were assigned. Surprisingly, we detected DNA-interacting/nuclear proteins and proteins involved in ribosome biogenesis and protein synthesis predominantly present in alpha-factor arrested that do not display an assembled septin structure. Furthermore, two distinct sets of regulatory proteins that are specific for cells at S-phase with a stable septin collar or at mitosis with split septin rings were identified. Complementary methods like SPLIFF and immunoprecipitation allowed us to more exactly define the spatial and temporal characteristics of selected hits of the AP-MS screen. PMID:26871441

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

    PubMed Central

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

    2013-01-01

    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. PMID:23242554

  13. 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.

  14. Quantitation of Thioprolines in Grape Wine by Isotope Dilution-Liquid Chromatography-Tandem Mass Spectrometry.

    PubMed

    Liu, Jingjing; Meng, Xiangpeng; Chan, Wan

    2016-02-17

    Cysteine reacts with reactive carbonyls to form thioprolines, which have been demonstrated to possess various pharmaceutical properties. Therefore, thioproline formation is considered as a major detoxification pathway for carcinogenic reactive carbonyls. In this study, we report the initial identification of thiazolidine-4-carboxylic acid (1) and 2-methylthiazolidine-4-carboxylic acid (2), two very common thioprolines, formed by reacting formaldehyde and acetaldehyde with cysteine in grape wine samples. We have developed an isotope dilution-liquid chromatography-tandem mass spectrometry method featuring high sensitivity (limit of detection of ≤1.5 ng/mL) and selectivity to quantitate compounds 1 and 2. The method after validated to be highly accurate (recovery of ≥92%) and precise [intraday relative standard deviation (RSD) of ≤4.1% and interday RSD of ≤9.7%] was applied to determine the varying compound 1 and 2 contents in grape wine samples. Results revealed the grape type and storage duration-dependent formation of thioprolines in grape wines. Overall, the results are expected to facilitate compound-dependent investigations of the health benefits of grape wine, and our findings could be adopted to predict the age of grape wine.

  15. Elution profile analysis of SDS-induced subcomplexes by quantitative mass spectrometry.

    PubMed

    Texier, Yves; Toedt, Grischa; Gorza, Matteo; Mans, Dorus A; van Reeuwijk, Jeroen; Horn, Nicola; Willer, Jason; Katsanis, Nicholas; Roepman, Ronald; Gibson, Toby J; Ueffing, Marius; Boldt, Karsten

    2014-05-01

    Analyzing the molecular architecture of native multiprotein complexes via biochemical methods has so far been difficult and error prone. Protein complex isolation by affinity purification can define the protein repertoire of a given complex, yet, it remains difficult to gain knowledge of its substructure or modular composition. Here, we introduce SDS concentration gradient induced decomposition of protein complexes coupled to quantitative mass spectrometry and in silico elution profile distance analysis. By applying this new method to a cellular transport module, the IFT/lebercilin complex, we demonstrate its ability to determine modular composition as well as sensitively detect known and novel complex components. We show that the IFT/lebercilin complex can be separated into at least five submodules, the IFT complex A, the IFT complex B, the 14-3-3 protein complex and the CTLH complex, as well as the dynein light chain complex. Furthermore, we identify the protein TULP3 as a potential new member of the IFT complex A and showed that several proteins, classified as IFT complex B-associated, are integral parts of this complex. To further demonstrate EPASIS general applicability, we analyzed the modular substructure of two additional complexes, that of B-RAF and of 14-3-3-ε. The results show, that EPASIS provides a robust as well as sensitive strategy to dissect the substructure of large multiprotein complexes in a highly time- as well as cost-effective manner. PMID:24563533

  16. Quantitative analysis of tRNA modifications by HPLC-coupled mass spectrometry

    PubMed Central

    Su, Dan; Chan, Clement T.Y.; Gu, Chen; Lim, Kok Seong; Chionh, Yok Hian; McBee, Megan E.; Russell, Brandon S.; Babu, I. Ramesh; Begley, Thomas J.; Dedon, Peter C.

    2015-01-01

    Post-transcriptional modification of RNA is an important determinant of RNA quality control, translational efficiency, RNA-protein interactions, and stress response. This is illustrated by the observation of toxicant-specific changes in the spectrum of tRNA modifications in a stress response mechanism involving selective translation of codon-biased mRNA for critical proteins. To facilitate systems-level studies of RNA modifications, we developed a liquid chromatography-coupled mass spectrometry (LC-MS) technique for the quantitative analysis of modified ribonucleosides in tRNA or other RNA species. The protocol includes tRNA purification by HPLC, enzymatic hydrolysis, reversed-phase HPLC resolution of the ribonucleosides, and identification and quantification of individual ribonucleosides by LC-MS using dynamic multiple reaction monitoring. This approach enables quantification of modified ribonucleosides in several micrograms of tRNA, or other RNA, in a 15-minute LC-MS run. By comparison, traditional methods for detecting modified ribonucleosides are labor and time intensive, require larger RNA quantities, are modification-specific, or require radioactive labeling. PMID:24625781

  17. 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.

  18. Characterization of the Ribosome Biogenesis Landscape in E. coli using Quantitative Mass Spectrometry

    PubMed Central

    Chen, Stephen S.; Williamson, James R.

    2012-01-01

    The ribosome is an essential and highly complex biological system in all living cells. A large body of literature is available on the assembly of the ribosome in vitro, but a clear picture of this process inside the cell has yet to emerge. Here, we directly characterized in vivo ribosome assembly intermediates and associated assembly factors from wild-type E. coli cells using a general quantitative mass spectrometry (qMS) approach. The presence of distinct populations of ribosome assembly intermediates was verified using an in vivo stable isotope pulse-labeling approach, and their exact ribosomal protein (r-protein) contents were characterized against an isotopically labeled standard. The model-free clustering analysis of the resultant protein levels for the different ribosomal particles produced four 30S assembly groups that correlate very well with previous in vitro assembly studies of the small ribosomal subunit, and six 50S assembly groups that clearly define an in vivo assembly landscape for the larger ribosomal subunit. In addition, de novo proteomics identified a total of 21 known and potentially new ribosome assembly factors co-localized with various ribosomal particles. These results represent new in vivo assembly maps of the E. coli 30S and 50S subunits, and the general qMS approach should be a solid platform for future studies of ribosome biogenesis across a host of model organisms. PMID:23228329

  19. Measuring the dynamics of E. coli ribosome biogenesis using pulse-labeling and quantitative mass spectrometry

    PubMed Central

    Chen, Stephen S.; Sperling, Edit; Silverman, Joshua M.; Davis, Joseph H.; Williamson, James R.

    2012-01-01

    The ribosome is an essential organelle responsible for cellular protein synthesis. Until recently, the study of ribosome assembly has been largely limited to in vitro assays, with few attempts to reconcile these results with the more complex in vivo ribosome biogenesis process. Here, we characterize the ribosome synthesis and assembly pathway for each E. coli ribosomal protein (r-protein) in vivo using a stable isotope pulse-labeling timecourse. Isotope incorporation into assembled ribosomes was measured by quantitative mass spectrometry (qMS) and fit using steady-state flux models. Most r-proteins exhibit precursor pools ranging in size from 0% to 7% of completed ribosomes, and that the sizes of these individual r-protein pools correlate well with the order of r-protein binding in vitro. Additionally, we observe anomalously large precursor pools for specific r-proteins with known extra-ribosomal functions and we have detected three r-proteins with significant turnover during steady-state growth. Taken together, this highly precise, time-dependent proteomic qMS approach should prove useful in future studies of ribosome biogenesis and could be easily extended to explore other complex biological processes in a cellular context. PMID:23090316

  20. Identification of Cell Cycle Dependent Interaction Partners of the Septins by Quantitative Mass Spectrometry.

    PubMed

    Renz, Christian; Oeljeklaus, Silke; Grinhagens, Sören; Warscheid, Bettina; Johnsson, Nils; Gronemeyer, Thomas

    2016-01-01

    The septins are a conserved family of GTP-binding proteins that, in the baker's yeast, assemble into a highly ordered array of filaments at the mother bud neck. These filaments undergo significant structural rearrangements during the cell cycle. We aimed at identifying key components that are involved in or regulate the transitions of the septins. By combining cell synchronization and quantitative affinity-purification mass-spectrometry, we performed a screen for specific interaction partners of the septins at three distinct stages of the cell cycle. A total of 83 interaction partners of the septins were assigned. Surprisingly, we detected DNA-interacting/nuclear proteins and proteins involved in ribosome biogenesis and protein synthesis predominantly present in alpha-factor arrested that do not display an assembled septin structure. Furthermore, two distinct sets of regulatory proteins that are specific for cells at S-phase with a stable septin collar or at mitosis with split septin rings were identified. Complementary methods like SPLIFF and immunoprecipitation allowed us to more exactly define the spatial and temporal characteristics of selected hits of the AP-MS screen. PMID:26871441

  1. 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

    2016-01-01

    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.

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

    PubMed

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

    2015-06-01

    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.

  3. 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

    2016-01-01

    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

  4. 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

  5. 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.

  6. Quantitative Mass Spectrometry Reveals Plasticity of Metabolic Networks in Mycobacterium smegmatis *

    PubMed Central

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

    2014-01-01

    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. PMID:24997995

  7. 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.

  8. 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.

  9. Quantitative determination of terbutaline and orciprenaline in human plasma by gas chromatography/negative ion chemical ionization/mass spectrometry.

    PubMed

    Leis, H J; Gleispach, H; Nitsche, V; Malle, E

    1990-06-01

    A method for the determination of unconjugated terbutaline and orciprenaline in human plasma is described. The assay is based on stable isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometry. An inexpensive and rapid method for preparation of stable isotope labelled analogues as well as their use in quantitative gas chromatography/mass spectrometry is shown. A highly efficient sample work-up procedure with product recoveries of more than 95% is presented. The method developed permits quantitative measurement of terbutaline and orciprenaline in human plasma down to 100 pg ml-1, using 1 ml of sample. Plasma levels of terbutaline after oral administration of 5 mg of terbutaline sulphate were estimated. PMID:2357489

  10. 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.

  11. Potential of monitoring isotopologues by quantitative gas chromatography with time-of-flight mass spectrometry for metabolomic assay.

    PubMed

    Wang, Yi; Hu, Haiyan; Su, Yue; Zhang, Fang; Guo, Yinlong

    2016-03-01

    Because of the extreme complexity of metabolomic samples, the effectiveness of quantitative gas chromatography with time-of-flight mass spectrometry depends substantially on the expansion of the linear dynamic range. Facing the existence of numerous saturated detector signals, a data processing method based on monitoring isotopologues has been developed. The monoisotopic ion kept the high mass spectrometry sensitivity, and the less abundant isotopologue ions extended the linear dynamic range. This alternative method was proved to extend the linear dynamic range to five orders of magnitude successfully and overcome the quantitative problems induced by the ion detector saturation. Finally, to validate the applicability, the method was applied to a metabolomic assay of Alzheimer's disease. Comparing with the traditional monoisotopic method, the use of monitoring isotopologues helped us to discover an additional eight metabolites with significant difference and to conduct a more reliable principal component analysis as well. The results demonstrated that monitoring isotopologues in quantitative gas chromatography with time-of-flight mass spectrometry could improve the authenticity of metabolomic analysis. PMID:26763370

  12. Simplified quantitative glycomics using the stable isotope label Girard's reagent p by electrospray ionization mass spectrometry.

    PubMed

    Wang, Chengjian; Wu, Zhiyu; Yuan, Jiangbei; Wang, Bo; Zhang, Ping; Zhang, Ying; Wang, Zhongfu; Huang, Linjuan

    2014-02-01

    Fast, sensitive, and simple methods for quantitative analysis of disparities in glycan expression between different biological samples are essential for studies of protein glycosylation patterns (glycomics) and the search for disease glycan biomarkers. Relative quantitation of glycans based on stable isotope labeling combined with mass spectrometric detection represents an emerging and promising technique. However, this technique is undermined by the complexity of mass spectra of isotope-labeled glycans caused by the presence of multiple metal ion adduct signals, which result in a decrease of detection sensitivity and an increase of difficulties in data interpretation. Herein we report a simplified quantitative glycomics strategy, which features nonreductive isotopic labeling of reducing glycans with either nondeuterated (d0-) or deuterated (d5-) Girard's reagent P (GP) without salts introduced and simplified mass spectrometric profiles of d0- and d5-GP derivatives of neutral glycans as molecular ions without complex metal ion adducts, allowing rapid and sensitive quantitative comparison between different glycan samples. We have obtained optimized GP-labeling conditions and good quantitation linearity, reproducibility, and accuracy of data by the method. Its excellent applicability was validated by comparatively quantitative analysis of the neutral N-glycans released from bovine and porcine immunoglobulin G as well as of those from mouse and rat sera. Additionally, we have revealed the potential of this strategy for the high-sensitivity analysis of sialylated glycans as GP derivatives, which involves neutralization of the carboxyl group of sialic acid by chemical derivatization.

  13. 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.

  14. 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.

  15. 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)

  16. 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.

  17. Quantitative mass spectrometry-based assay development and validation: from small molecules to proteins.

    PubMed

    Božović, Andrea; Kulasingam, Vathany

    2013-04-01

    Mass spectrometry (MS) has emerged as a powerful analytical tool for the identification, characterization and quantification of various biomolecules (small molecules, drug metabolites and proteins) in biological specimens. The use of mass spectrometers in the clinical diagnostic laboratories have gained popularity due to its ease of development of new assays, ability to measure multiple analytes in a single analytical run, low volume requirements and low reagent costs. Novel technological advancements in ionization sources, instrumentation and software have increased the popularity of these platforms. Consequently, a number of home-brew assays, utilizing the power of MS, are being developed and validated for clinical diagnostic use. In this review, we will discuss the two phases that precede method implementation: method development and validation for both small molecule analysis and protein quantification using liquid chromatography tandem mass spectrometry (LC-MS/MS). Some of the challenges facing protein quantification will be highlighted and an outlook for the future of laboratory medicine and MS will be provided. PMID:23041077

  18. A multiplex lectin-channel monitoring method for human serum glycoproteins by quantitative mass spectrometry.

    PubMed

    Ahn, Yeong Hee; Ji, Eun Sun; Shin, Park Min; Kim, Kwang Hoe; Kim, Yong-Sam; Ko, Jeong Heon; Yoo, Jong Shin

    2012-02-01

    A mass profiling method and multiple reaction monitoring (MRM)-based quantitative approach were used to analyze multiple lectin-captured fractions of human serum using different lectins such as aleuria aurantia lectin (AAL), phytohemagglutinin-L(4) (L-PHA), concanavalin A (Con A), and Datura stramonium agglutinin (DSA) to quantitatively monitor protein glycosylation diversity. Each fraction, prepared by multiple lectin-fractionation and tryptic digestion, was analyzed by 1-D LC-MS/MS. Semi-quantitative profiling showed that the list of glycoproteins identified from each lectin-captured fraction is significantly different according to the used lectin. Thus, it was confirmed that the multiplex lectin-channel monitoring (LCM) using multiple lectins is useful for investigating protein glycosylation diversity in a proteome sample. Based on the semi-quantitative mass profiling, target proteins showing lectin-specificity among each lectin-captured fraction were selected and analyzed by the MRM-based method in triplicate using each lectin-captured fraction (average CV 7.9%). The MRM-based analysis for each lectin-captured fraction was similar to those obtained by the profiling experiments. The abundance of each target protein measured varied dramatically, based on the lectin-specificity. The multiplex LCM approach using MRM-based analyses is useful for quantitatively monitoring target protein glycoforms selectively fractionated by multiple lectins. Thus through multiplex LCM rather than single, we could inquire minutely into protein glycosylation states. PMID:22158852

  19. 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.

  20. 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

  1. 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

  2. Peak intensity prediction in MALDI-TOF mass spectrometry: A machine learning study to support quantitative proteomics

    PubMed Central

    Timm, Wiebke; Scherbart, Alexandra; Böcker, Sebastian; Kohlbacher, Oliver; Nattkemper, Tim W

    2008-01-01

    Background Mass spectrometry is a key technique in proteomics and can be used to analyze complex samples quickly. One key problem with the mass spectrometric analysis of peptides and proteins, however, is the fact that absolute quantification is severely hampered by the unclear relationship between the observed peak intensity and the peptide concentration in the sample. While there are numerous approaches to circumvent this problem experimentally (e.g. labeling techniques), reliable prediction of the peak intensities from peptide sequences could provide a peptide-specific correction factor. Thus, it would be a valuable tool towards label-free absolute quantification. Results In this work we present machine learning techniques for peak intensity prediction for MALDI mass spectra. Features encoding the peptides' physico-chemical properties as well as string-based features were extracted. A feature subset was obtained from multiple forward feature selections on the extracted features. Based on these features, two advanced machine learning methods (support vector regression and local linear maps) are shown to yield good results for this problem (Pearson correlation of 0.68 in a ten-fold cross validation). Conclusion The techniques presented here are a useful first step going beyond the binary prediction of proteotypic peptides towards a more quantitative prediction of peak intensities. These predictions in turn will turn out to be beneficial for mass spectrometry-based quantitative proteomics. PMID:18937839

  3. 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

  4. Direct and quantitative analysis of underivatized acylcarnitines in serum and whole blood using paper spray mass spectrometry

    PubMed Central

    Yang, Qian; Manicke, Nicholas E.; Wang, He; Petucci, Christopher; Cooks, R. Graham

    2013-01-01

    A simple protocol for rapid quantitation of acylcarnitines in serum and whole blood has been developed using paper spray mass spectrometry. Dried serum and whole blood containing a mixture of ten acylcarnitines at various concentrations were analyzed as spots from paper directly without any sample pretreatment, separation, or derivatization. The composition of the spray solvent was found to be a critical factor: for serum samples, spray solvent of methanol/water/formic acid (80:20:0.1) gave the best signal intensity while for blood samples which contain more matrix components, acetonitrile/water (90:10) was a much more suitable spray solvent. For the paper type and size used, 0.5 μL of sample provided an optimal signal for both serum and whole blood samples. For quantitative profiling, the limits of quantitation obtained from both serum and blood were much lower than the clinically validated cutoff values for diagnosis of fatty acid oxidation disorders in newborn screening. Linearity (R2>0.95) and reproducibility (RSD ~10 %) were achieved in the concentration ranges from 100 nM to 5 μM for the C2 acylcarnitine, and for other acylcarnitines, these values were from 10 to 500 nM. Acylcarnitine profiles offer an effective demonstration of the fact that paper spray mass spectrometry is an appropriate, simple, rapid method with high sensitivity and high reproducibility applicable to newborn screening tests. PMID:22760507

  5. [Progress in quantitative methods based on liquid chromatography-mass spectrometry for drug metabolizing enzymes in human liver microsomes].

    PubMed

    Wang, Huanhuan; Lu, Yayao; Peng, Bo; Qian, Xiaohong; Zhang, Yangjun

    2015-06-01

    Cytochrome P450 (CYP) enzymes and uridine 5-diphospho-glucuronosyltransferase (UGT) enzymes are critical enzymes for drug metabolism. Both chemical drugs and traditional Chinese medicines are converted to more readily excreted compounds by drug metabolizing enzymes in human livers. Because of the disparate expression of CYP and UGT enzymes among different individuals, accurate quantification of these enzymes is essential for drug pharmacology, drug-drug interactions and drug clinical applications. The research progress in quantitative methods based on liquid chromatography-mass spectrometry for drug metabolizing enzymes in human liver microsomes in the recent decade is reviewed. PMID:26536756

  6. [Progress in quantitative methods based on liquid chromatography-mass spectrometry for drug metabolizing enzymes in human liver microsomes].

    PubMed

    Wang, Huanhuan; Lu, Yayao; Peng, Bo; Qian, Xiaohong; Zhang, Yangjun

    2015-06-01

    Cytochrome P450 (CYP) enzymes and uridine 5-diphospho-glucuronosyltransferase (UGT) enzymes are critical enzymes for drug metabolism. Both chemical drugs and traditional Chinese medicines are converted to more readily excreted compounds by drug metabolizing enzymes in human livers. Because of the disparate expression of CYP and UGT enzymes among different individuals, accurate quantification of these enzymes is essential for drug pharmacology, drug-drug interactions and drug clinical applications. The research progress in quantitative methods based on liquid chromatography-mass spectrometry for drug metabolizing enzymes in human liver microsomes in the recent decade is reviewed.

  7. 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.

  8. 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. PMID:19939702

  9. 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. PMID:27237773

  10. 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.

  11. Quantitative determination of nebivolol from human plasma using liquid chromatography-tandem mass spectrometry.

    PubMed

    Nandania, Jatin; Rajput, S J; Contractor, Pritesh; Vasava, Pragnesh; Solanki, Bhavik; Vohra, Mohsin

    2013-04-01

    In the present work, a rapid, sensitive, specific, precise and accurate liquid chromatography-tandem mass spectrometry method for determination of nebivolol in human plasma was developed and validated with a large calibration curve range (50-5000 pg/mL) which can be used for routine drug analysis and bioequivalence studies. Liquid-liquid extraction method was used to extract the analyte from the human plasma. The separation was achieved using Waters symmetry, C18, 4.6 × 150 mm, 5 μm column with formic acid in water, 0.01%, v/v: Acetonitrile (40:60) as a mobile phase. A flow rate of 0.8 mL/min, no splitting and run time 2.00 min was used for the chromatographic analysis of nebivolol. Sensitivity of this method was found to be 30 pg/mL. The analyte was analyzed by mass spectrometry in the multiple reaction monitoring mode. A Turbo-Ion spray source was interfaced between the HPLC and triple quadrupole mass spectrometer (MDS Sciex API 4000). The precursor-product ion m/z 406.00-151.00 for nebivolol and m/z 410.20-151.00 for nebivolol-D4 were used for quantification of an analyte and its IS. The method was validated in terms of accuracy, precision, selectivity, absolute recovery, freeze-thaw stability, bench-top stability, dry extract stability, short and long term stock solution stability, wet extract stability and re-injection reproducibility. The within- and between-batch accuracy was found to lie within the range of 87.00-100.40% and within- and between-batch precision was obtained within the range 0.33-8.67%. The mean recovery of all three concentration levels for drug was obtained 67.67% where as the mean recovery of IS was 68.74%. The %RSD value at higher concentration and lower concentration in all stability experiments was within 15%. This method is free from ion suppression, ion enhancement and any type of abnormal ionization.

  12. Quantitative analysis of mitragynine in human urine by high performance liquid chromatography-tandem mass spectrometry.

    PubMed

    Lu, Shijun; Tran, Buu N; Nelsen, Jamie L; Aldous, Kenneth M

    2009-08-15

    Mitragynine is the primary active alkaloid extracted from the leaves of Mitragyna speciosa Korth, a plant that originates in South-East Asia and is commonly known as kratom in Thailand. Kratom has been used for many centuries for their medicinal and psychoactive qualities, which are comparable to that of opiate-based drugs. Kratom abuse can lead to a detectable content of mitragynine residue in urine. Ultra trace amount of mitragynine in human urine was determined by a high performance liquid chromatography coupled to an electrospray tandem mass spectrometry (HPLC-ESI/MS/MS). Mitragynine was extracted by methyl t-butyl ether (MTBE) and separated on a HILIC column. The ESI/MS/MS was accomplished using a triple quadrupole mass spectrometer in positive ion detection and multiple reactions monitoring (MRM) mode. Ajmalicine, a mitragynine's structure analog was selected as internal standard (IS) for method development. Quality control (QC) performed at three levels 0.1, 1 and 5 ng/ml of mitragynine in urine gave mean recoveries of 90, 109, and 98% with average relative standard deviation of 22, 12 and 16%, respectively. The regression linearity of mitragynine calibration ranged from 0.01 to 5.0 ng/ml was achieved with correlation coefficient greater than 0.995. A detection limit of 0.02 ng/ml and high precision data within-day and between days analysis were obtained. PMID:19577523

  13. Liquid chromatography quadrupole linear ion trap mass spectrometry for quantitative steroid hormone analysis in plasma, urine, saliva and hair.

    PubMed

    Gaudl, Alexander; Kratzsch, Juergen; Bae, Yoon Ju; Kiess, Wieland; Thiery, Joachim; Ceglarek, Uta

    2016-09-16

    Steroid analysis is being conquered by liquid chromatography tandem mass spectrometry (LC-MS/MS) benefiting from higher standardization, selectivity and diversity. Regarding high throughput in routine diagnostics rapid chromatography is mandatory. Introducing MS(3) (MS/MS/MS), specificity of mass spectrometric detection can be enhanced without sacrificing analysis time. 100mL of human plasma/serum, saliva, urine and 10-20mg of hair are used for the simultaneous quantification of 17α-hydroxyprogesterone, aldosterone, androstenedione, cortisol, cortisone, dehydroepiandrosterone sulfate (DHEAS), estradiol, progesterone, and testosterone using online solid phase extraction (SPE) LC-MS/MS or LC-MS(3). Steroids can be analyzed in 4min after a single manual dilution and protein precipitation step. In complex sample matrices like hair MS(3) detection was found to be appropriate for quantitation. Lower limits of quantitation ranged from 37pmol/L (estradiol) up to 3.1nmol/L (DHEAS). General accuracy was 89-107% with between-run imprecision ≤10%. Comparison to immunoassays revealed significant differences in quantitation for urinary cortisol (-71% mean), aldosterone (-40% mean) and plasma aldosterone (-45% mean). The comparison of MS(2) and MS(3) quantitation of hair cortisol also revealed significant differences. In general, quantitation via MS(3) was not applicable for a long time. But with the current generation of mass spectrometers quantitation via MS(3) can be superior to MS(2) regarding specificity and accuracy when dealing with matrix issues. However, drawbacks regarding flexibility and precision have to be taken into account. Concludingly, simple protein precipitation combined with rapid online SPE LC-MS/MS/MS allows us to quantify over broad, essential concentration ranges in human serum, saliva, urine and hair. PMID:27554022

  14. Quantitative mass spectrometry imaging of emtricitabine in cervical tissue model using infrared matrix-assisted laser desorption electrospray ionization

    PubMed Central

    Bokhart, Mark T.; Rosen, Elias; Thompson, Corbin; Sykes, Craig; Kashuba, Angela D. M.; Muddiman, David C.

    2015-01-01

    A quantitative mass spectrometry imaging (QMSI) technique using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is demonstrated for the antiretroviral (ARV) drug emtricitabine in incubated human cervical tissue. Method development of the QMSI technique leads to a gain in sensitivity and removal of interferences for several ARV drugs. Analyte response was significantly improved by a detailed evaluation of several cationization agents. Increased sensitivity and removal of an isobaric interference was demonstrated with sodium chloride in the electrospray solvent. Voxel-to-voxel variability was improved for the MSI experiments by normalizing analyte abundance to a uniformly applied compound with similar characteristics to the drug of interest. Finally, emtricitabine was quantified in tissue with a calibration curve generated from the stable isotope-labeled analog of emtricitabine followed by cross-validation using liquid chromatography tandem mass spectrometry (LC-MS/MS). The quantitative IR-MALDESI analysis proved to be reproducible with an emtricitabine concentration of 17.2±1.8 μg/gtissue. This amount corresponds to the detection of 7 fmol/voxel in the IR-MALDESI QMSI experiment. Adjacent tissue slices were analyzed using LC-MS/MS which resulted in an emtricitabine concentration of 28.4±2.8 μg/gtissue. PMID:25318460

  15. Quantitative Cross-linking/Mass Spectrometry Using Isotope-labeled Cross-linkers and MaxQuant.

    PubMed

    Chen, Zhuo A; Fischer, Lutz; Cox, Jürgen; Rappsilber, Juri

    2016-08-01

    The conceptually simple step from cross-linking/mass spectrometry (CLMS) to quantitative cross-linking/mass spectrometry (QCLMS) is compounded by technical challenges. Currently, quantitative proteomics software is tightly integrated with the protein identification workflow. This prevents automatically quantifying other m/z features in a targeted manner including those associated with cross-linked peptides. Here we present a new release of MaxQuant that permits starting the quantification process from an m/z feature list. Comparing the automated quantification to a carefully manually curated test set of cross-linked peptides obtained by cross-linking C3 and C3b with BS(3) and isotope-labeled BS(3)-d4 revealed a number of observations: (1) Fully automated process using MaxQuant can quantify cross-links in our reference data set with 68% recall rate and 88% accuracy. (2) Hidden quantification errors can be converted into exposed failures by label-swap replica, which makes label-swap replica an essential part of QCLMS. (3) Cross-links that failed during automated quantification can be recovered by semi-automated re-quantification. The integrated workflow of MaxQuant and semi-automated assessment provides the maximum of quantified cross-links. In contrast, work on larger data sets or by less experienced users will benefit from full automation in MaxQuant. PMID:27302889

  16. Direct and rapid quantitation of ephedrines in human urine by paper spray ionization/high resolution mass spectrometry.

    PubMed

    Jeong, Eun Sook; Kim, Ki Hun; Cha, Eunju; Kwon, Oh-Seung; Cha, Sangwon; Lee, Jaeick

    2016-08-15

    A rapid and direct paper spray ionization/mass spectrometry (PSI/MS) method was developed for quantitative analysis of ephedrine, pseudoephedrine, norpseudoephedrine, and methylephedrine in human urine. This method involves the use of a triangular filter paper and high-resolution mass spectrometry, where the molecular ions of ephedrines were generated by applying high voltage after loading the spray solvent to the paper which urine sample was pre-loaded. Small amounts (2μL) of urine spiked with an internal standard were directly analyzed for ephedrines. The PSI/MS method was validated for linearity, within- and between-run precision, accuracy, and limit of detection. The results showed good linearity (R(2)≥0.9928) and acceptable precision and accuracy. Furthermore, the accuracy of the method was assessed by analyzing a blind urine sample from World Anti-Doping Agency and comparing the measured concentrations with the nominal concentrations. This test resulted in accuracies ranging from 96.4 to 106.1%, indicating that the PSI/MS method has the potential to be an alternative technique for the fast quantitation of ephedrines in doping control analysis. PMID:27393909

  17. Quantitative Cross-linking/Mass Spectrometry Using Isotope-labeled Cross-linkers and MaxQuant*

    PubMed Central

    Cox, Jürgen

    2016-01-01

    The conceptually simple step from cross-linking/mass spectrometry (CLMS) to quantitative cross-linking/mass spectrometry (QCLMS) is compounded by technical challenges. Currently, quantitative proteomics software is tightly integrated with the protein identification workflow. This prevents automatically quantifying other m/z features in a targeted manner including those associated with cross-linked peptides. Here we present a new release of MaxQuant that permits starting the quantification process from an m/z feature list. Comparing the automated quantification to a carefully manually curated test set of cross-linked peptides obtained by cross-linking C3 and C3b with BS3 and isotope-labeled BS3-d4 revealed a number of observations: (1) Fully automated process using MaxQuant can quantify cross-links in our reference data set with 68% recall rate and 88% accuracy. (2) Hidden quantification errors can be converted into exposed failures by label-swap replica, which makes label-swap replica an essential part of QCLMS. (3) Cross-links that failed during automated quantification can be recovered by semi-automated re-quantification. The integrated workflow of MaxQuant and semi-automated assessment provides the maximum of quantified cross-links. In contrast, work on larger data sets or by less experienced users will benefit from full automation in MaxQuant. PMID:27302889

  18. Quantitative mass spectrometry imaging of emtricitabine in cervical tissue model using infrared matrix-assisted laser desorption electrospray ionization.

    PubMed

    Bokhart, Mark T; Rosen, Elias; Thompson, Corbin; Sykes, Craig; Kashuba, Angela D M; Muddiman, David C

    2015-03-01

    A quantitative mass spectrometry imaging (QMSI) technique using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is demonstrated for the antiretroviral (ARV) drug emtricitabine in incubated human cervical tissue. Method development of the QMSI technique leads to a gain in sensitivity and removal of interferences for several ARV drugs. Analyte response was significantly improved by a detailed evaluation of several cationization agents. Increased sensitivity and removal of an isobaric interference was demonstrated with sodium chloride in the electrospray solvent. Voxel-to-voxel variability was improved for the MSI experiments by normalizing analyte abundance to a uniformly applied compound with similar characteristics to the drug of interest. Finally, emtricitabine was quantified in tissue with a calibration curve generated from the stable isotope-labeled analog of emtricitabine followed by cross-validation using liquid chromatography tandem mass spectrometry (LC-MS/MS). The quantitative IR-MALDESI analysis proved to be reproducible with an emtricitabine concentration of 17.2 ± 1.8 μg/gtissue. This amount corresponds to the detection of 7 fmol/voxel in the IR-MALDESI QMSI experiment. Adjacent tissue slices were analyzed using LC-MS/MS which resulted in an emtricitabine concentration of 28.4 ± 2.8 μg/gtissue.

  19. The quantitative analysis of inhalational anaesthetics in forensic samples by gas chromatography/mass spectrometry/selected ion monitoring.

    PubMed

    Maruyama, K; Takatsu, A; Obata, T

    1995-01-01

    The quantitative analysis of volatile anaesthetics for biomedical applications by means of gas chromatograph/mass spectrometry/selected ion monitoring (GC/MS/SIM) was studied. Xenon gas was selected as an internal standard for the assay by adding to a closed system, because of its stability and inactivity. In the assay of inhalational anaesthetics, isoflurane and nitrous oxide (laughing gas), in forensic samples (serum and cerebrospinal fluid), the calibration of the anaesthetic was linear from 0.12 to 12 nmol/mL in isoflurane and from 30 to 300 nmol/mL in nitrous oxide. Our results suggest that this new method is suitable for the quantitative analysis of inhalational anaesthetics in the biomedical field. PMID:8520207

  20. Quantitative analysis of positional isomers of triacylglycerols via electrospray ionization tandem mass spectrometry of sodiated adducts.

    PubMed

    Herrera, Lisandra Cubero; Potvin, Michael A; Melanson, Jeremy E

    2010-09-01

    Herein we report a reversed-phase high-performance liquid chromatography tandem mass spectrometry (RP-HPLC/MS/MS) method for the analysis of positional isomers of triacylglycerols (TAGs) in vegetable oils. The fragmentation behavior of [M + X](+) ions (X = NH(4), Li, Na or Ag) was studied on a quadrupole-time-of-flight (Q-TOF) mass spectrometer under low-energy collision-induced dissociation (CID) conditions. Mass spectra that were dependent on the X(+) ion and the nature and position of the acyl substituents were observed for four pairs of 'AAB/ABA'-type TAGs, namely PPO/POP, OOP/OPO, LLO/LOL and OOL/OLO (where P is 16:0, palmitic acid; O is 18:1, oleic acid; and L is 18:2, linoleic acid). For the majority of [M + X](+) adducts, the loss of the fatty acid in the outer positions (sn-1 or sn-3) was favored over the loss in the central position (sn-2), which enabled the determination of the fractional abundance of the isomers. Ratios of the intensity of fragment ions at various AAB/ABA compositions produced linear calibration curves with positive slopes, comparable to those obtained traditionally by ESI-MS/MS of [M + NH(4)](+) adducts. The only exceptions were the [M + Ag](+) adducts of the PPO/POP system, which produced calibration curves with negative slopes. Sodium adducts provided the most consistent level of isomeric discrimination for the TAGs studied and also offered the most convenience in that they required no additive to the mobile phase. Therefore, calibration curve data derived from [M + Na](+) adducts were applied to the quantification of TAG regioisomers in sunflower and olive oils. The regiospecific analysis showed that palmitic acid was typically located at positions sn-1 or sn-3, whereas unsaturated fatty acids, oleic and linoleic acids were mostly found at the sn-2 position. PMID:20814981

  1. Multiplexed isobaric tagging protocols for quantitative mass spectrometry approaches to auditory research.

    PubMed

    Vetter, Douglas E; Basappa, Johnvesly; Turcan, Sevin

    2009-01-01

    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 other 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 dependant 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.

  2. 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. PMID:27259924

  3. 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

  4. High resolution mass spectrometry for quantitative analysis and untargeted screening of algal toxins in mussels and passive samplers.

    PubMed

    Zendong, Zita; McCarron, Pearse; Herrenknecht, Christine; Sibat, Manoella; Amzil, Zouher; Cole, Richard B; Hess, Philipp

    2015-10-16

    Measurement of marine algal toxins has traditionally focussed on shellfish monitoring while, over the last decade, passive sampling has been introduced as a complementary tool for exploratory studies. Since 2011, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been adopted as the EU reference method (No. 15/2011) for detection and quantitation of lipophilic toxins. Traditional LC-MS approaches have been based on low-resolution mass spectrometry (LRMS), however, advances in instrument platforms have led to a heightened interest in the use of high-resolution mass spectrometry (HRMS) for toxin detection. This work describes the use of HRMS in combination with passive sampling as a progressive approach to marine algal toxin surveys. Experiments focused on comparison of LRMS and HRMS for determination of a broad range of toxins in shellfish and passive samplers. Matrix effects are an important issue to address in LC-MS; therefore, this phenomenon was evaluated for mussels (Mytilus galloprovincialis) and passive samplers using LRMS (triple quadrupole) and HRMS (quadrupole time-of-flight and Orbitrap) instruments. Matrix-matched calibration solutions containing okadaic acid and dinophysistoxins, pectenotoxin, azaspiracids, yessotoxins, domoic acid, pinnatoxins, gymnodimine A and 13-desmethyl spirolide C were prepared. Similar matrix effects were observed on all instruments types. Most notably, there was ion enhancement for pectenotoxins, okadaic acid/dinophysistoxins on one hand, and ion suppression for yessotoxins on the other. Interestingly, the ion selected for quantitation of PTX2 also influenced the magnitude of matrix effects, with the sodium adduct typically exhibiting less susceptibility to matrix effects than the ammonium adduct. As expected, mussel as a biological matrix, quantitatively produced significantly more matrix effects than passive sampler extracts, irrespective of toxin. Sample dilution was demonstrated as an effective measure to reduce

  5. An automated gas chromatography time-of-flight mass spectrometry instrument for the quantitative analysis of halocarbons in air

    NASA Astrophysics Data System (ADS)

    Obersteiner, F.; Bönisch, H.; Engel, A.

    2015-09-01

    We present the characterization and application of a new gas chromatography-time-of-flight mass spectrometry instrument (GC-TOFMS) for the quantitative analysis of halocarbons in air samples. The setup comprises three fundamental enhancements compared to our earlier work (Hoker et al., 2015): (1) full automation, (2) a mass resolving power R = m/Δ m of the TOFMS (Tofwerk AG, Switzerland) increased up to 4000 Th/Th and (3) a fully accessible data format of the mass spectrometric data. Automation in combination with the accessible data allowed an in-depth characterization of the instrument. Mass accuracy was found around 5 ppm after automatic recalibration of the mass axis in each measurement. A TOFMS configuration giving R = 3500 was chosen to provide an R-to-sensitivity ratio suitable for our purpose. Calculated detection limits were as low as a few femtograms as mass traces could be made highly specific for selected molecule fragments with the accurate mass information. The precision for substance quantification was 0.15 % at the best for an individual measurement and in general mainly determined by the signal-to-noise ratio of the chromatographic peak. The TOFMS was found to be linear within a concentration range from about 1 pg to 1 ng of analyte per Liter of air. At higher concentrations, non-linearities of a few percent were observed (precision level: 0.2 %) but could be attributed to a potential source within the detection system. A straight-forward correction for those non-linearities was applied in data processing, again by exploiting the accurate mass information. Based on the overall characterization results, the GC-TOFMS instrument was found to be very well-suited for the task of quantitative halocarbon trace gas observation and a big step forward compared to scanning, low resolution quadrupole MS and a TOFMS technique reported to be non-linear and restricted by a small dynamical range.

  6. An automated gas chromatography time-of-flight mass spectrometry instrument for the quantitative analysis of halocarbons in air

    NASA Astrophysics Data System (ADS)

    Obersteiner, F.; Bönisch, H.; Engel, A.

    2016-01-01

    We present the characterization and application of a new gas chromatography time-of-flight mass spectrometry instrument (GC-TOFMS) for the quantitative analysis of halocarbons in air samples. The setup comprises three fundamental enhancements compared to our earlier work (Hoker et al., 2015): (1) full automation, (2) a mass resolving power R = m/Δm of the TOFMS (Tofwerk AG, Switzerland) increased up to 4000 and (3) a fully accessible data format of the mass spectrometric data. Automation in combination with the accessible data allowed an in-depth characterization of the instrument. Mass accuracy was found to be approximately 5 ppm in mean after automatic recalibration of the mass axis in each measurement. A TOFMS configuration giving R = 3500 was chosen to provide an R-to-sensitivity ratio suitable for our purpose. Calculated detection limits are as low as a few femtograms by means of the accurate mass information. The precision for substance quantification was 0.15 % at the best for an individual measurement and in general mainly determined by the signal-to-noise ratio of the chromatographic peak. Detector non-linearity was found to be insignificant up to a mixing ratio of roughly 150 ppt at 0.5 L sampled volume. At higher concentrations, non-linearities of a few percent were observed (precision level: 0.2 %) but could be attributed to a potential source within the detection system. A straightforward correction for those non-linearities was applied in data processing, again by exploiting the accurate mass information. Based on the overall characterization results, the GC-TOFMS instrument was found to be very well suited for the task of quantitative halocarbon trace gas observation and a big step forward compared to scanning, quadrupole MS with low mass resolving power and a TOFMS technique reported to be non-linear and restricted by a small dynamical range.

  7. 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.

  8. Label-Free Quantitative Mass Spectrometry Reveals a Panel of Differentially Expressed Proteins in Colorectal Cancer

    PubMed Central

    Fan, Nai-Jun; Gao, Jiang-Ling; Liu, Yan; Song, Wei; Zhang, Zhan-Yang; Gao, Chun-Fang

    2015-01-01

    To identify potential biomarkers involved in CRC, a shotgun proteomic method was applied to identify soluble proteins in three CRCs and matched normal mucosal tissues using high-performance liquid chromatography and mass spectrometry. Label-free protein profiling of three CRCs and matched normal mucosal tissues were then conducted to quantify and compare proteins. Results showed that 67 of the 784 identified proteins were linked to CRC (28 upregulated and 39 downregulated). Gene Ontology and DAVID databases were searched to identify the location and function of differential proteins that were related to the biological processes of binding, cell structure, signal transduction, cell adhesion, and so on. Among the differentially expressed proteins, tropomyosin-3 (TPM3), endoplasmic reticulum resident protein 29 (ERp29), 18 kDa cationic antimicrobial protein (CAMP), and heat shock 70 kDa protein 8 (HSPA8) were verified to be upregulated in CRC tissue and seven cell lines through western blot analysis. Furthermore, the upregulation of TPM3, ERp29, CAMP, and HSPA8 was validated in 69 CRCs byimmunohistochemistry (IHC) analysis. Combination of TPM3, ERp29, CAMP, and HSPA8 can identify CRC from matched normal mucosal achieving an accuracy of 73.2% using IHC score. These results suggest that TPM3, ERp29, CAMP, and HSPA8 are great potential IHC diagnostic biomarkers for CRC. PMID:25699276

  9. 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

  10. DetectTLC: Automated Reaction Mixture Screening Utilizing Quantitative Mass Spectrometry Image Feature

    PubMed Central

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

    2016-01-01

    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. PMID:26508443

  11. 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.

  12. 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.

  13. 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. PMID:26508443

  14. msIQuant--Quantitation Software for Mass Spectrometry Imaging Enabling Fast Access, Visualization, and Analysis of Large Data Sets.

    PubMed

    Källback, Patrik; Nilsson, Anna; Shariatgorji, Mohammadreza; Andrén, Per E

    2016-04-19

    This paper presents msIQuant, a novel instrument- and manufacturer-independent quantitative mass spectrometry imaging software suite that uses the standardized open access data format imzML. Its data processing structure enables rapid image display and the analysis of very large data sets (>50 GB) without any data reduction. In addition, msIQuant provides many tools for image visualization including multiple interpolation methods, low intensity transparency display, and image fusion. It also has a quantitation function that automatically generates calibration standard curves from series of standards that can be used to determine the concentrations of specific analytes. Regions-of-interest in a tissue section can be analyzed based on a number of quantities including the number of pixels, average intensity, standard deviation of intensity, and median and quartile intensities. Moreover, the suite's export functions enable simplified postprocessing of data and report creation. We demonstrate its potential through several applications including the quantitation of small molecules such as drugs and neurotransmitters. The msIQuant suite is a powerful tool for accessing and evaluating very large data sets, quantifying drugs and endogenous compounds in tissue areas of interest, and for processing mass spectra and images.

  15. 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

    2013-11-01

    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.

  16. 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.

  17. 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

    2016-07-01

    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.

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

    PubMed

    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. Graphical Abstract ᅟ. PMID:27392730

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

    PubMed

    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. Graphical Abstract ᅟ.

  20. Quantitative Mass Spectrometry Reveals Partial Translational Regulation for Dosage Compensation in Chicken

    PubMed Central

    Uebbing, Severin; Konzer, Anne; Xu, Luohao; Backström, Niclas; Brunström, Björn; Bergquist, Jonas; Ellegren, Hans

    2015-01-01

    There is increasing evidence that dosage compensation is not a ubiquitous feature following sex chromosome evolution, especially not in organisms where females are the heterogametic sex, like in birds. Even when it occurs, compensation can be incomplete and limited to dosage-sensitive genes. However, previous work has mainly studied transcriptional regulation of sex-linked genes, which may not reflect expression at the protein level. Here, we used liquid chromatography–tandem mass spectrometry to detect and quantify expressed levels of more than 2,400 proteins in ten different tissues of male and female chicken embryos. For comparison, transcriptome sequencing was performed in the same individuals, five of each sex. The proteomic analysis revealed that dosage compensation was incomplete, with a mean male-to-female (M:F) expression ratio of Z-linked genes of 1.32 across tissues, similar to that at the RNA level (1.29). The mean Z chromosome-to-autosome expression ratio was close to 1 in males and lower than 1 in females, consistent with partly reduced Z chromosome expression in females. Although our results exclude a general mechanism for chromosome-wide dosage compensation at translation, 30% of all proteins encoded from Z-linked genes showed a significant change in the M:F ratio compared with the corresponding ratio at the RNA level. This resulted in a pattern where some genes showed balanced expression between sexes and some close to 2-fold higher expression in males. This suggests that proteomic analyses will be necessary to reveal a more complete picture of gene regulation and sex chromosome evolution. PMID:26108680

  1. Quantitative determination of sarsasapogenin in rat plasma using liquid chromatography-tandem mass spectrometry.

    PubMed

    Yang, Bo; Liu, Zhirui; Hu, Jing; Lai, Xiaodan; Xia, Peiyuan

    2016-06-01

    Sarsasapogenin, a natural compound from Chinese medical herb Anemarrhena asphodeloides Bge., has recently received a great deal of attention due to its various bioactivities. In this study, an easy and applicable liquid chromatography tandem mass spectrometry method for the quantification of sarsasapogenin in rat plasma was developed. Sample preparation was accomplished through a simple one-step protein precipitation procedure with methanol. Negative electrospray ionization was performed using multiple reactions monitoring (MRM) mode with transitions of m/z 417.4/273.2 for sarsasapogenin, and 415.2/271.4 for diosgenin (internal standard). The calibration curve was linear over the range of 0.5-500ng/mL (r=0.9994), with a lower limit of quantification at 0.5ng/mL. The RSD of intra- and inter-day precision was below 6.41%, and accuracy ranged from 87.60% to 99.20%. The RSD of matrix effect and recovery yield was within ±15% of nominal concentrations and sarsasapogenin was stable during stability tests. This validated method had been successfully applied to the preclinical pharmacokinetic studies of sarsasapogenin in rats. The half-life (t1/2) was (15.1±2.3), (16.1±3.0) and (15.4±3.9) h after single intragastric administration of 25, 50 and 100mg/kg sarsasapogenin, respectively. And it was found that, the area under the plasma concentration versus time curve (AUC0-72h) and the maximum plasma concentration (Cmax) were linearly related to dose. PMID:27107248

  2. Quantitative Mass Spectrometry Reveals Partial Translational Regulation for Dosage Compensation in Chicken.

    PubMed

    Uebbing, Severin; Konzer, Anne; Xu, Luohao; Backström, Niclas; Brunström, Björn; Bergquist, Jonas; Ellegren, Hans

    2015-10-01

    There is increasing evidence that dosage compensation is not a ubiquitous feature following sex chromosome evolution, especially not in organisms where females are the heterogametic sex, like in birds. Even when it occurs, compensation can be incomplete and limited to dosage-sensitive genes. However, previous work has mainly studied transcriptional regulation of sex-linked genes, which may not reflect expression at the protein level. Here, we used liquid chromatography-tandem mass spectrometry to detect and quantify expressed levels of more than 2,400 proteins in ten different tissues of male and female chicken embryos. For comparison, transcriptome sequencing was performed in the same individuals, five of each sex. The proteomic analysis revealed that dosage compensation was incomplete, with a mean male-to-female (M:F) expression ratio of Z-linked genes of 1.32 across tissues, similar to that at the RNA level (1.29). The mean Z chromosome-to-autosome expression ratio was close to 1 in males and lower than 1 in females, consistent with partly reduced Z chromosome expression in females. Although our results exclude a general mechanism for chromosome-wide dosage compensation at translation, 30% of all proteins encoded from Z-linked genes showed a significant change in the M:F ratio compared with the corresponding ratio at the RNA level. This resulted in a pattern where some genes showed balanced expression between sexes and some close to 2-fold higher expression in males. This suggests that proteomic analyses will be necessary to reveal a more complete picture of gene regulation and sex chromosome evolution. PMID:26108680

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

    PubMed Central

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

    2016-01-01

    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. PMID:26463340

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

    SciTech Connect

    Leach, J.

    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.

  5. A new approach for the comparative analysis of multiprotein complexes based on 15N metabolic labeling and quantitative mass spectrometry.

    PubMed

    Trompelt, Kerstin; Steinbeck, Janina; Terashima, Mia; Hippler, Michael

    2014-01-01

    The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling ((14)N/(15)N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions. PMID:24686495

  6. 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

    2010-02-01

    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.

  7. Development and evaluation of a liquid chromatography-mass spectrometry method for rapid, accurate quantitation of malondialdehyde in human plasma.

    PubMed

    Sobsey, Constance A; Han, Jun; Lin, Karen; Swardfager, Walter; Levitt, Anthony; Borchers, Christoph H

    2016-09-01

    Malondialdhyde (MDA) is a commonly used marker of lipid peroxidation in oxidative stress. To provide a sensitive analytical method that is compatible with high throughput, we developed a multiple reaction monitoring-mass spectrometry (MRM-MS) approach using 3-nitrophenylhydrazine chemical derivatization, isotope-labeling, and liquid chromatography (LC) with electrospray ionization (ESI)-tandem mass spectrometry assay to accurately quantify MDA in human plasma. A stable isotope-labeled internal standard was used to compensate for ESI matrix effects. The assay is linear (R(2)=0.9999) over a 20,000-fold concentration range with a lower limit of quantitation of 30fmol (on-column). Intra- and inter-run coefficients of variation (CVs) were <2% and ∼10% respectively. The derivative was stable for >36h at 5°C. Standards spiked into plasma had recoveries of 92-98%. When compared to a common LC-UV method, the LC-MS method found near-identical MDA concentrations. A pilot project to quantify MDA in patient plasma samples (n=26) in a study of major depressive disorder with winter-type seasonal pattern (MDD-s) confirmed known associations between MDA concentrations and obesity (p<0.02). The LC-MS method provides high sensitivity and high reproducibility for quantifying MDA in human plasma. The simple sample preparation and rapid analysis time (5x faster than LC-UV) offers high throughput for large-scale clinical applications. PMID:27437618

  8. Impacts of CD44 knockdown in cancer cells on tumor and host metabolic systems revealed by quantitative imaging mass spectrometry.

    PubMed

    Ohmura, Mitsuyo; Hishiki, Takako; Yamamoto, Takehiro; Nakanishi, Tsuyoshi; Kubo, Akiko; Tsuchihashi, Kenji; Tamada, Mayumi; Toue, Sakino; Kabe, Yasuaki; Saya, Hideyuki; Suematsu, Makoto

    2015-04-30

    CD44 expressed in cancer cells was shown to stabilize cystine transporter (xCT) that uptakes cystine and excretes glutamate to supply cysteine as a substrate for reduced glutathione (GSH) for survival. While targeting CD44 serves as a potentially therapeutic stratagem to attack cancer growth and chemoresistance, the impact of CD44 targeting in cancer cells on metabolic systems of tumors and host tissues in vivo remains to be fully determined. This study aimed to reveal effects of CD44 silencing on alterations in energy metabolism and sulfur-containing metabolites in vitro and in vivo using capillary electrophoresis-mass spectrometry and quantitative imaging mass spectrometry (Q-IMS), respectively. In an experimental model of xenograft transplantation of human colon cancer HCT116 cells in superimmunodeficient NOG mice, snap-frozen liver tissues containing metastatic tumors were examined by Q-IMS. As reported previously, short hairpin CD44 RNA interference (shCD44) in cancer cells caused significant regression of tumor growth in the host liver. Under these circumstances, the CD44 knockdown suppressed polyamines, GSH and energy charges not only in metastatic tumors but also in the host liver. In culture, HCT116 cells treated with shCD44 decreased total amounts of methionine-pool metabolites including spermidine and spermine, and reactive cysteine persulfides, suggesting roles of these metabolites for cancer growth. Collectively, these results suggest that CD44 expressed in cancer accounts for a key regulator of metabolic interplay between tumor and the host tissue. PMID:25461272

  9. Quantitative mass spectrometry reveals a role for the GTPase Rho1p in actin organization on the peroxisome membrane.

    PubMed

    Marelli, Marcello; Smith, Jennifer J; Jung, Sunhee; Yi, Eugene; Nesvizhskii, Alexey I; Christmas, Rowan H; Saleem, Ramsey A; Tam, Yuen Yi C; Fagarasanu, Andrei; Goodlett, David R; Aebersold, Ruedi; Rachubinski, Richard A; Aitchison, John D

    2004-12-20

    We have combined classical subcellular fractionation with large-scale quantitative mass spectrometry to identify proteins that enrich specifically with peroxisomes of Saccharomyces cerevisiae. In two complementary experiments, isotope-coded affinity tags and tandem mass spectrometry were used to quantify the relative enrichment of proteins during the purification of peroxisomes. Mathematical modeling of the data from 306 quantified proteins led to a prioritized list of 70 candidates whose enrichment scores indicated a high likelihood of them being peroxisomal. Among these proteins, eight novel peroxisome-associated proteins were identified. The top novel peroxisomal candidate was the small GTPase Rho1p. Although Rho1p has been shown to be tethered to membranes of the secretory pathway, we show that it is specifically recruited to peroxisomes upon their induction in a process dependent on its interaction with the peroxisome membrane protein Pex25p. Rho1p regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis.

  10. Metal-tag labeling coupled with multiple reaction monitoring-mass spectrometry for absolute quantitation of proteins.

    PubMed

    Wang, Xueying; Wang, Xin; Qin, Weijie; Lin, Hongjun; Wang, Jifeng; Wei, Junying; Zhang, Yangjun; Qian, Xiaohong

    2013-09-21

    Mass spectrometry-based quantitative proteomics, consisting of relative and absolute parts, has been used to discover and validate proteins with key functions related to physiological and pathological processes. Currently, stable isotope dilution-multiple reaction monitoring-mass spectrometry (SID-MRM-MS) is the most commonly used method for the absolute determination of proteins in a biological sample. A prerequisite for this method is obtaining internal standards with isotope labels. Although many approaches have been developed for the labeling and preparation of internal peptides, expensive stable isotope labeling coupled with SID-MRM-MS has limited the application and development of an absolute quantitative method. Recently, a low-cost strategy using metal-tag labeling and MS has been developed for relative quantification of peptides or proteins. The introduction of labeling using metal tags has the merits of allowing multiple labeling and enlarging the mass shift to overcome the overlap of adjacent isotope clusters. However, most papers described MRM-MS for protein absolute quantification based on the metal in its peptides labelled with metal by inductively coupled plasma mass spectrometry (ICP MS) but not on its peptides labelled with metal. In this work, a novel approach based on metal-tag labeling coupled with MRM-MS was established for the absolute quantification of peptides or proteins. The principle of the method is that a bifunctional chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid bearing an N-hydroxysuccinimide ester (DOTA-NHS ester), is used to modify the N-termini of signature peptides from a target protein, and the modified peptides then chelate a certain metal, such as thulium, to form metal-tagged peptides (Tm-DOTA-P). Internal peptides are chemically synthesized and labeled with another metal, such as terbium (Tb-DOTA-P), as the internal standard. Both the Tb-DOTA- and Tm-DOTA-labeled peptides in samples can be analysed via

  11. 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.

  12. 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. PMID:24054645

  13. 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.

  14. New HDAC6-mediated deacetylation sites of tubulin in the mouse brain identified by quantitative mass spectrometry.

    PubMed

    Liu, Ningning; Xiong, Yun; Li, Shanshan; Ren, Yiran; He, Qianqian; Gao, Siqi; Zhou, Jun; Shui, Wenqing

    2015-11-19

    The post-translational modifications (PTMs) occurring on microtubules have been implicated in the regulation of microtubule properties and functions. Acetylated K40 of α-tubulin, a hallmark of long-lived stable microtubules, is known to be negatively controlled by histone deacetylase 6 (HDAC6). However, the vital roles of HDAC6 in microtubule-related processes such as cell motility and cell division cannot be fully explained by the only known target site on tubulin. Here, we attempt to comprehensively map lysine acetylation sites on tubulin purified from mouse brain tissues. Furthermore, mass spectrometry-based quantitative comparison of acetylated peptides from wild-type vs HDAC6 knockout mice allowed us to identify six new deacetylation sites possibly mediated by HDAC6. Thus, adding new sites to the repertoire of HDAC6-mediated tubulin deacetylation events would further our understanding of the multi-faceted roles of HDAC6 in regulating microtubule stability and cellular functions.

  15. 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.

  16. 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. PMID:25914245

  17. 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.

  18. 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.

  19. ARiBo pull-down for riboproteomic studies based on label-free quantitative mass spectrometry

    PubMed Central

    Di Tomasso, Geneviève; Miller Jenkins, Lisa M.

    2016-01-01

    As part of their normal life cycle, most RNA molecules associate with several proteins that direct their fate and regulate their function. Here, we describe a novel method for identifying proteins that associate with a target RNA. The procedure is based on the ARiBo method for affinity purification of RNA, which was originally developed to quickly purify RNA with high yields and purity under native conditions. The ARiBo method was further optimized using in vitro transcribed RNA to capture RNA-associating proteins from cellular extracts with high yields and low background protein contamination. For these RNA pull-downs, stem–loops present in the immature forms of let-7 miRNAs (miRNA stem–loops) were used as the target RNAs. Label-free quantitative mass spectrometry analysis allowed for the reliable identification of proteins that are specific to the stem–loops present in the immature forms of two miRNAs, let-7a-1 and let-7g. Several proteins known to bind immature forms of these let-7 miRNAs were identified, but with an improved coverage compared to previous studies. In addition, several novel proteins were identified that better define the protein interactome of the let-7 miRNA stem–loops and further link let-7 biogenesis to important biological processes such as development and tumorigenesis. Thus, combining the ARiBo pull-down method with label-free quantitative mass spectrometry provides an effective proteomic approach for identification of proteins that associate with a target RNA. PMID:27659051

  20. Mass spectrometry in India.

    PubMed

    Vairamani, M; Prabhakar, S

    2012-01-01

    This review emphasizes the mass spectrometry research being performed at academic and established research institutions in India. It consists of three main parts covering the work done in organic, atomic and biological mass spectrometry. The review reveals that the use of mass spectrometry techniques started in the middle of the 20th century and was applied to research in the fields of organic, nuclear, geographical and atomic chemistry. Later, with the advent of soft and atmospheric ionization techniques it has been applied to pharmaceutical and biological research. In due course, several research centers with advanced mass spectrometry facilities have been established for specific areas of research such as gas-phase ion chemistry, ion-molecule reactions, proscribed chemicals, pesticide residues, pharmacokinetics, protein/peptide chemistry, nuclear chemistry, geochronological studies, archeology, petroleum industry, proteomics, lipidomics and metabolomics. Day-by-day the mass spectrometry centers/facilities in India have attracted young students for their doctoral research and other advanced research applications.

  1. 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. PMID:26396078

  2. A Simultaneous Metabolic Profiling and Quantitative Multimetabolite Metabolomic Method for Human Plasma Using Gas-Chromatography Tandem Mass Spectrometry.

    PubMed

    Savolainen, Otto I; Sandberg, Ann-Sofie; Ross, Alastair B

    2016-01-01

    For the first time it is possible to simultaneously collect targeted and nontargeted metabolomics data from plasma based on GC with high scan speed tandem mass spectrometry (GC-MS/MS). To address the challenge of getting broad metabolome coverage while quantifying known biomarker compounds in high-throughput GC-MS metabolomics, we developed a novel GC-MS/MS metabolomics method using a high scan speed (20 000 Da/second) GC-MS/MS that enables simultaneous data acquisition of both nontargeted full scan and targeted quantitative tandem mass spectrometry data. The combination of these two approaches has hitherto not been demonstrated in metabolomics. This method allows reproducible quantification of at least 37 metabolites using multiple reaction monitoring (MRM) and full mass spectral scan-based detection of 601 reproducible metabolic features from human plasma. The method showed good linearity over normal concentrations in plasma (0.06-343 to 0.86-4800 μM depending on the metabolite) and good intra- and interbatch precision (0.9-16.6 and 2.6-29.6% relative standard deviation). Based on the parameters determined for this method, targeted quantification using MRM can be expanded to cover at least 508 metabolites while still collecting full scan data. The new simultaneous targeted and nontargeted metabolomics method enables more sensitive and accurate detection of predetermined metabolites and biomarkers of interest, while still allowing detection and identification of unknown metabolites. This is the first validated GC-MS/MS metabolomics method with simultaneous full scan and MRM data collection, and clearly demonstrates the utility of GC-MS/MS with high scanning rates for complex analyses.

  3. 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...

  4. 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

  5. Investigation of Pokemon-regulated proteins in hepatocellular carcinoma using mass spectrometry-based multiplex quantitative proteomics.

    PubMed

    Bi, Xin; Jin, Yibao; Gao, Xiang; Liu, Feng; Gao, Dan; Jiang, Yuyang; Liu, Hongxia

    2013-01-01

    Pokemon is a transcription regulator involved in embryonic development, cellular differentiation and oncogenesis. It is aberrantly overexpressed in multiple human cancers including Hepatocellular carcinoma (HCC) and is considered as a promising biomarker for HCC. In this work, the isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics strategy was used to investigate the proteomic profile associated with Pokemon in human HCC cell line QGY7703 and human hepatocyte line HL7702. Samples were labeled with four-plex iTRAQ reagents followed by two-dimensional liquid chromatography coupled with tandem mass spectrometry analysis. A total of 24 differentially expressed proteins were selected as significant. Nine proteins were potentially up-regulated by Pokemon while 15 proteins were potentially down-regulated and many proteins were previously identified as potential biomarkers for HCC. Gene ontology (GO) term enrichment revealed that the listed proteins were mainly involved in DNA metabolism and biosynthesis process. The changes of glucose-6-phosphate 1-dehydrogenase (G6PD, up-regulated) and ribonucleoside-diphosphate reductase large sub-unit (RIM1, down-regulated) were validated by Western blotting analysis and denoted as Pokemon's function of oncogenesis. We also found that Pokemon potentially repressed the expression of highly clustered proteins (MCM3, MCM5, MCM6, MCM7) which played key roles in promoting DNA replication. Altogether, our results may help better understand the role of Pokemon in HCC and promote the clinical applications. PMID:24261083

  6. 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.

  7. LaCyTools: A Targeted Liquid Chromatography-Mass Spectrometry Data Processing Package for Relative Quantitation of Glycopeptides.

    PubMed

    Jansen, Bas C; Falck, David; de Haan, Noortje; Hipgrave Ederveen, Agnes L; Razdorov, Genadij; Lauc, Gordan; Wuhrer, Manfred

    2016-07-01

    Bottom-up glycoproteomics by liquid chromatography-mass spectrometry (LC-MS) is an established approach for assessing glycosylation in a protein- and site-specific manner. Consequently, tools are needed to automatically align, calibrate, and integrate LC-MS glycoproteomics data. We developed a modular software package designed to tackle the individual aspects of an LC-MS experiment, called LaCyTools. Targeted alignment is performed using user defined m/z and retention time (tr) combinations. Subsequently, sum spectra are created for each user defined analyte group. Quantitation is performed on the sum spectra, where each user defined analyte can have its own tr, minimum, and maximum charge states. Consequently, LaCyTools deals with multiple charge states, which gives an output per charge state if desired, and offers various analyte and spectra quality criteria. We compared throughput and performance of LaCyTools to combinations of available tools that deal with individual processing steps. LaCyTools yielded relative quantitation of equal precision (relative standard deviation <0.5%) and higher trueness due to the use of MS peak area instead of MS peak intensity. In conclusion, LaCyTools is an accurate automated data processing tool for high-throughput analysis of LC-MS glycoproteomics data. Released under the Apache 2.0 license, it is freely available on GitHub ( https://github.com/Tarskin/LaCyTools ).

  8. Investigation of Pokemon-regulated proteins in hepatocellular carcinoma using mass spectrometry-based multiplex quantitative proteomics.

    PubMed

    Bi, Xin; Jin, Yibao; Gao, Xiang; Liu, Feng; Gao, Dan; Jiang, Yuyang; Liu, Hongxia

    2013-01-01

    Pokemon is a transcription regulator involved in embryonic development, cellular differentiation and oncogenesis. It is aberrantly overexpressed in multiple human cancers including Hepatocellular carcinoma (HCC) and is considered as a promising biomarker for HCC. In this work, the isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics strategy was used to investigate the proteomic profile associated with Pokemon in human HCC cell line QGY7703 and human hepatocyte line HL7702. Samples were labeled with four-plex iTRAQ reagents followed by two-dimensional liquid chromatography coupled with tandem mass spectrometry analysis. A total of 24 differentially expressed proteins were selected as significant. Nine proteins were potentially up-regulated by Pokemon while 15 proteins were potentially down-regulated and many proteins were previously identified as potential biomarkers for HCC. Gene ontology (GO) term enrichment revealed that the listed proteins were mainly involved in DNA metabolism and biosynthesis process. The changes of glucose-6-phosphate 1-dehydrogenase (G6PD, up-regulated) and ribonucleoside-diphosphate reductase large sub-unit (RIM1, down-regulated) were validated by Western blotting analysis and denoted as Pokemon's function of oncogenesis. We also found that Pokemon potentially repressed the expression of highly clustered proteins (MCM3, MCM5, MCM6, MCM7) which played key roles in promoting DNA replication. Altogether, our results may help better understand the role of Pokemon in HCC and promote the clinical applications.

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

    DOE PAGES

    O'Maille, Grace; Go, Eden P.; Hoang, Linh; Want, Elizabeth J.; Smith, Colin; O'Maille, Paul; NordstrÖm, Anders; Morita, Hirotoshi; Qin, Chuan; Uritboonthai, Wilasinee; et al

    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.

  10. Fourier transform mass spectrometry.

    PubMed

    Scigelova, Michaela; Hornshaw, Martin; Giannakopulos, Anastassios; Makarov, Alexander

    2011-07-01

    This article provides an introduction to Fourier transform-based mass spectrometry. The key performance characteristics of Fourier transform-based mass spectrometry, mass accuracy and resolution, are presented in the view of how they impact the interpretation of measurements in proteomic applications. The theory and principles of operation of two types of mass analyzer, Fourier transform ion cyclotron resonance and Orbitrap, are described. Major benefits as well as limitations of Fourier transform-based mass spectrometry technology are discussed in the context of practical sample analysis, and illustrated with examples included as figures in this text and in the accompanying slide set. Comparisons highlighting the performance differences between the two mass analyzers are made where deemed useful in assisting the user with choosing the most appropriate technology for an application. Recent developments of these high-performing mass spectrometers are mentioned to provide a future outlook.

  11. Fourier Transform Mass Spectrometry

    PubMed Central

    Scigelova, Michaela; Hornshaw, Martin; Giannakopulos, Anastassios; Makarov, Alexander

    2011-01-01

    This article provides an introduction to Fourier transform-based mass spectrometry. The key performance characteristics of Fourier transform-based mass spectrometry, mass accuracy and resolution, are presented in the view of how they impact the interpretation of measurements in proteomic applications. The theory and principles of operation of two types of mass analyzer, Fourier transform ion cyclotron resonance and Orbitrap, are described. Major benefits as well as limitations of Fourier transform-based mass spectrometry technology are discussed in the context of practical sample analysis, and illustrated with examples included as figures in this text and in the accompanying slide set. Comparisons highlighting the performance differences between the two mass analyzers are made where deemed useful in assisting the user with choosing the most appropriate technology for an application. Recent developments of these high-performing mass spectrometers are mentioned to provide a future outlook. PMID:21742802

  12. Clinical protein mass spectrometry.

    PubMed

    Scherl, Alexander

    2015-06-15

    Quantitative protein analysis is routinely performed in clinical chemistry laboratories for diagnosis, therapeutic monitoring, and prognosis. Today, protein assays are mostly performed either with non-specific detection methods or immunoassays. Mass spectrometry (MS) is a very specific analytical method potentially very well suited for clinical laboratories. Its unique advantage relies in the high specificity of the detection. Any protein sequence variant, the presence of a post-translational modification or degradation will differ in mass and structure, and these differences will appear in the mass spectrum of the protein. On the other hand, protein MS is a relatively young technique, demanding specialized personnel and expensive instrumentation. Many scientists and opinion leaders predict MS to replace immunoassays for routine protein analysis, but there are only few protein MS applications routinely used in clinical chemistry laboratories today. The present review consists of a didactical introduction summarizing the pros and cons of MS assays compared to immunoassays, the different instrumentations, and various MS protein assays that have been proposed and/or are used in clinical laboratories. An important distinction is made between full length protein analysis (top-down method) and peptide analysis after enzymatic digestion of the proteins (bottom-up method) and its implication for the protein assay. The document ends with an outlook on what type of analyses could be used in the future, and for what type of applications MS has a clear advantage compared to immunoassays.

  13. 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…

  14. Identification and quantitative analysis of beta-sitosterol oxides in vegetable oils by capillary gas chromatography-mass spectrometry.

    PubMed

    Zhang, Xin; Julien-David, Diane; Miesch, Michel; Geoffroy, Philippe; Raul, Francis; Roussi, Stamatiki; Aoudé-Werner, Dalal; Marchioni, Eric

    2005-12-01

    As vegetable oils and phytosterol-enriched spreads are marketed for frying food or cooking purposes, temperature is one of the most important factors leading to the formation of phytosterol oxides in food matrix. A methodology based on saponification, organic solvent extraction, solid-phase extraction (SPE), followed by mass spectrometric identification and quantitation of beta-sitosterol oxides using capillary gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode was developed and characterized. Relative response factors of six beta-sitosterol oxides, including 7alpha-hydroxy, 7beta-hydroxy, 5,6alpha-epoxy, 5,6beta-epoxy, 7-keto, and 5alpha,6beta-dihydroxysitosterol, were calculated against authentic standards of 19-hydroxycholesterol or cholestanol. Linear calibration data, limit of detection, and sample recoveries during analytical process. Recoveries of these oxidation compounds in spiked samples ranged from 88 to 115%, while relative standard derivation (R.S.D.) values were below 10% in most cases. The analytical method was applied to quantify beta-sitosterol oxides formed in thermal-oxidized vegetable oils which were heated at different temperatures and for varying time periods. Sitosterol oxidation is strikingly higher in sunflower oil relative to olive oil under all conditions of temperature and heating time.

  15. Identification and quantitative analysis of beta-sitosterol oxides in vegetable oils by capillary gas chromatography-mass spectrometry.

    PubMed

    Zhang, Xin; Julien-David, Diane; Miesch, Michel; Geoffroy, Philippe; Raul, Francis; Roussi, Stamatiki; Aoudé-Werner, Dalal; Marchioni, Eric

    2005-12-01

    As vegetable oils and phytosterol-enriched spreads are marketed for frying food or cooking purposes, temperature is one of the most important factors leading to the formation of phytosterol oxides in food matrix. A methodology based on saponification, organic solvent extraction, solid-phase extraction (SPE), followed by mass spectrometric identification and quantitation of beta-sitosterol oxides using capillary gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode was developed and characterized. Relative response factors of six beta-sitosterol oxides, including 7alpha-hydroxy, 7beta-hydroxy, 5,6alpha-epoxy, 5,6beta-epoxy, 7-keto, and 5alpha,6beta-dihydroxysitosterol, were calculated against authentic standards of 19-hydroxycholesterol or cholestanol. Linear calibration data, limit of detection, and sample recoveries during analytical process. Recoveries of these oxidation compounds in spiked samples ranged from 88 to 115%, while relative standard derivation (R.S.D.) values were below 10% in most cases. The analytical method was applied to quantify beta-sitosterol oxides formed in thermal-oxidized vegetable oils which were heated at different temperatures and for varying time periods. Sitosterol oxidation is strikingly higher in sunflower oil relative to olive oil under all conditions of temperature and heating time. PMID:16038955

  16. 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.

  17. Environmental Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Lebedev, Albert T.

    2013-06-01

    Environmental mass spectrometry is an important branch of science because it provides many of the data that underlie policy decisions that can directly influence the health of people and ecosystems. Environmental mass spectrometry is currently undergoing rapid development. Among the most relevant directions are a significant broadening of the lists of formally targeted compounds; a parallel interest in nontarget chemicals; an increase in the reliability of analyses involving accurate mass measurements, tandem mass spectrometry, and isotopically labeled standards; and a shift toward faster high-throughput analysis, with minimal sample preparation, involving various approaches, including ambient ionization techniques and miniature instruments. A real revolution in analytical chemistry could be triggered with the appearance of robust, simple, and sensitive portable mass spectrometers that can utilize ambient ionization techniques. If the cost of such instruments is reduced to a reasonable level, mass spectrometers could become valuable household devices.

  18. 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.

  19. 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. PMID:19296591

  20. 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. PMID:18398847

  1. Rapid and simple solid-phase esterification of sialic acid residues for quantitative glycomics by mass spectrometry.

    PubMed

    Miura, Yoshiaki; Shinohara, Yasuro; Furukawa, Jun-ichi; Nagahori, Noriko; Nishimura, Shin-Ichiro

    2007-01-01

    A rapid and quantitative method for solid-phase methyl esterification of carboxy groups of various sialylated oligosaccharides has been established. The method employed a triazene derivative, 3-methyl-1-p-tolyltriazene, for facile derivatization of oligosaccharides immobilized onto general solid supports such as Affi-Gel Hz and gold colloidal nanoparticles in a multiwell plate. The workflow protocol was optimized for the solid-phase processing of captured sialylated/unsialylated oligosaccharides separated from crude sample mixtures by chemical ligation. From tryptic and/or PNGase F-digest mixtures of glycoproteins, purification by chemoselective immobilization, esterification and recovery were achieved in the same well of the filter plate within three hours when used in conjunction with "glycoblotting technology" (S.-I. Nishimura, K. Niikura, M. Kurogochi, T. Matsushita, M. Fumoto, H. Hinou, R. Kamitani, H. Nakagawa, K. Deguchi, N. Miura, K. Monde, H. Kondo, High-throughput protein glycomics: Combined use of chemoselective glycoblotting and MALDI-TOF/TOF mass spectrometry: Angew. Chem. 2005, 117, 93-98; Angew. Chem. Int. Ed. 2005, 44, 91-96). The recovered materials were directly applicable to subsequent characterization by mass spectrometric techniques such as MALDI-TOF for large-scale glycomics of both neutral and sialylated oligosaccharides. On-bead/on-gold nanoparticle derivatization of glycans containing sialic acids allowed rapid and quantitative glycoform profiling by MALDI-TOF MS with reflector and positive ion mode. In addition to its simplicity and speed, the method eliminates the use of unfavorable halogenated solvents such as chloroform and dichloromethane or volatile solvents such as diethyl ether and hexane, resulting in a practical and green chemical method for automated robotic adaptation.

  2. Calibration strategy for semi-quantitative direct gas analysis using inductively coupled plasma mass spectrometry

    NASA Astrophysics Data System (ADS)

    Gerdes, Kirk; Carter, Kimberly E.

    2011-09-01

    A process is described by which an ICP-MS equipped with an Octopole Reaction System (ORS) is calibrated using liquid phase standards to facilitate direct analysis of gas phase samples. The instrument response to liquid phase standards is analyzed to produce empirical factors relating ion generation and transmission efficiencies to standard operating parameters. Empirical factors generated for liquid phase samples are then used to produce semi-quantitative analysis of both mixed liquid/gas samples and pure gas samples. The method developed is similar to the semi-quantitative analysis algorithms in the commercial software, which have here been expanded to include gas phase elements such as Xe and Kr. Equations for prediction of relative ionization efficiencies and isotopic transmission are developed for several combinations of plasma operating conditions, which allows adjustment of limited parameters between liquid and gas injection modes. In particular, the plasma temperature and electron density are calculated from comparison of experimental results to the predictions of the Saha equation. Comparisons between operating configurations are made to determine the robustness of the analysis to plasma conditions and instrument operating parameters. Using the methods described in this research, the elemental concentrations in a liquid standard containing 45 analytes and treated as an unknown sample were quantified accurately to ± 50% for most elements using 133Cs as a single internal reference. The method is used to predict liquid phase mercury within 12% of the actual concentration and gas phase mercury within 28% of the actual concentration. The results verify that the calibration method facilitates accurate semi-quantitative, gas phase analysis of metal species with sufficient sensitivity to quantify metal concentrations lower than 1 ppb for many metallic analytes.

  3. Phenolic compounds from Bursera simaruba Sarg. bark: phytochemical investigation and quantitative analysis by tandem mass spectrometry.

    PubMed

    Maldini, Mariateresa; Montoro, Paola; Piacente, Sonia; Pizza, Cosimo

    2009-03-01

    Phytochemical investigation of the methanolic extract of Bursera simaruba bark led to the isolation of 11 compounds, including lignans yatein, beta-peltatin-O-beta-D-glucopyranoside, hinokinin and bursehernin, and three natural compounds namely 3,4-dimetoxyphenyl-1-O-beta-D-(6-sulpho)-glucopyranoside, 3,4,5-trimetoxyphenyl 1-O-beta-D-(6-sulpho)-glucopyranoside and 3,4-diidroxyphenylethanol-1-O-beta-D-(6-sulpho)-glucopyranoside. Their structures were established by NMR and ESI/MS experiments. Additionally, an LC-ESI/MS qualitative study on the phenolic compounds and an LC-ESI/MS/MS quantitative study on the lignans found in the methanolic extract of B. simaruba bark were performed to give value to the plant as source of these biological active compounds. Quantitative analyses results confirmed that compounds yatein, beta-peltatin-O-beta-D-glucopyranoside, hinokinin and bursehernin are major compounds in the bark and, in particular, beta-peltatin-O-beta-D-glucopyranoside appears to be the most abundant.

  4. Quantitative Analysis of rRNA Modifications Using Stable Isotope Labeling and Mass Spectrometry

    PubMed Central

    2015-01-01

    Post-transcriptional RNA modifications that are introduced during the multistep ribosome biogenesis process are essential for protein synthesis. The current lack of a comprehensive method for a fast quantitative analysis of rRNA modifications significantly limits our understanding of how individual modification steps are coordinated during biogenesis inside the cell. Here, an LC-MS approach has been developed and successfully applied for quantitative monitoring of 29 out of 36 modified residues in the 16S and 23S rRNA from Escherichia coli. An isotope labeling strategy is described for efficient identification of ribose and base methylations, and a novel metabolic labeling approach is presented to allow identification of MS-silent pseudouridine modifications. The method was used to measure relative abundances of modified residues in incomplete ribosomal subunits compared to a mature 15N-labeled rRNA standard, and a number of modifications in both 16S and 23S rRNA were present in substoichiometric amounts in the preribosomal particles. The RNA modification levels correlate well with previously obtained profiles for the ribosomal proteins, suggesting that RNA is modified in a schedule comparable to the association of the ribosomal proteins. Importantly, this study establishes an efficient workflow for a global monitoring of ribosomal modifications that will contribute to a better understanding of mechanisms of RNA modifications and their impact on intracellular processes in the future. PMID:24422502

  5. 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…

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

    PubMed Central

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

    2015-01-01

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

  7. 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.

  8. 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. PMID:27373704

  9. 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

  10. Identification and quantitation of unsaturated fatty acid isomers by electrospray ionization tandem mass spectrometry: a shotgun lipidomics approach.

    PubMed

    Yang, Kui; Zhao, Zhongdan; Gross, Richard W; Han, Xianlin

    2011-06-01

    Identification and quantification of unsaturated fatty acid (FA) isomers in a biological system are significant in the study of lipid metabolism and catabolism, membrane biophysics, and pathogenesis of diseases but are challenging in lipidomics. We developed a novel approach for identification and quantitation of unsaturated FA isomers by exploiting two facts: (1) unsaturated FA anions yield fragment ion(s) from loss of CO(2) or H(2)O from the anions upon collision-induced dissociation; and (2) the fragment ions yielded from discrete FA isomers have distinct profiles of the fragment ion intensity vs. collision conditions. These distinct profiles likely result from the differential interactions of the negative charge of the fragment ion with the electron clouds of the double bonds due to their different distances in discrete FA isomers. The novel approach was also extended to analyze the double bond isomers of FA chains present in phospholipids by multistage tandem mass spectrometry. Collectively, we developed a new approach for identification and quantification of the double bond isomers of endogenous FA species or FA chains present in intact phospholipid species. We believe that this approach should further advance the lipidomic power for identification of the biochemical mechanisms underlying metabolic diseases.

  11. Validation of Direct Analysis Real Time source/Time-of-Flight Mass Spectrometry for organophosphate quantitation on wafer surface.

    PubMed

    Hayeck, Nathalie; Ravier, Sylvain; Gemayel, Rachel; Gligorovski, Sasho; Poulet, Irène; Maalouly, Jacqueline; Wortham, Henri

    2015-11-01

    Microelectronic wafers are exposed to airborne molecular contamination (AMC) during the fabrication process of microelectronic components. The organophosphate compounds belonging to the dopant group are one of the most harmful groups. Once adsorbed on the wafer surface these compounds hardly desorb and could diffuse in the bulk of the wafer and invert the wafer from p-type to n-type. The presence of these compounds on wafer surface could have electrical effect on the microelectronic components. For these reasons, it is of importance to control the amount of these compounds on the surface of the wafer. As a result, a fast quantitative and qualitative analytical method, nondestructive for the wafers, is needed to be able to adjust the process and avoid the loss of an important quantity of processed wafers due to the contamination by organophosphate compounds. Here we developed and validated an analytical method for the determination of organic compounds adsorbed on the surface of microelectronic wafers using the Direct Analysis in Real Time-Time of Flight-Mass Spectrometry (DART-ToF-MS) system. Specifically, the developed methodology concerns the organophosphate group.

  12. Capillary zone electrophoresis and capillary electrophoresis-mass spectrometry for analyzing qualitative and quantitative variations in therapeutic albumin.

    PubMed

    Marie, Anne-Lise; Przybylski, Cédric; Gonnet, Florence; Daniel, Régis; Urbain, Rémi; Chevreux, Guillaume; Jorieux, Sylvie; Taverna, Myriam

    2013-10-24

    The present study describes a reproducible and quantitative capillary zone electrophoresis (CZE) method, which leads to the separation of nine forms (native, oxidized and glycated) of human serum albumin (HSA). In an attempt to identify the different species separated by this CZE method, the capillary electrophoresis was coupled to mass spectrometry using a sheath liquid interface, an optimized capillary coating and a suitable CE running buffer. CE-MS analyses confirmed the heterogeneity of albumin preparation and revealed new truncated and modified forms such as Advanced Glycation End products (AGEs). Assignment of the CZE peaks was carried out using specific antibodies, carboxypeptidase A or sample reduction before or during the CE separation. Thus, five HSA forms were unambiguously identified. Using this CZE method several albumin batches produced by slightly different fractionation ways could be discriminated. Furthermore, analyses of HSA preparations marketed by five pharmaceutical industries revealed that two therapeutic albumins, including that marketed by LFB, contained the highest proportion of native form and lower levels of oxidized forms. PMID:24120174

  13. Optimisation of the quantitative determination of chlormequat by matrix-assisted laser desorption/ionisation mass spectrometry.

    PubMed

    Horak, J; Werther, W; Schmid, E R

    2001-01-01

    The plant growth regulator chlormequat, an involatile quaternary ammonium salt, has been quantified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). Restrictions for quantitative MALDI-TOFMS analysis, such as irreproducible crystallisation and unsatisfactory laser stability, have been overcome by the application of two synthesised isotopically labelled standards and the optimisation of the measurement protocol. Data acquisition at constant laser power was compared to data acquisition at approximately constant ion abundance of the relevant ions (analyte and internal standards). Data acquisition at constant ion abundance performed better and enabled a high number of consecutive firings to the same sample deposition area. Furthermore an increased sample-to-sample repeatability and a high reproducibility over several weeks without re-calibration have been attained by this method. Linearity over three orders of magnitude (0.05 to 30 ng/microL chlormequat), with a correlation coefficient of 0.9997, was achieved using [13C3]-chlormequat as internal standard. Limit of detection and limit of determination were determined to be in the low pg/microL range for pure standard solutions. Thin-layer chromatography was applied for the removal of high amounts of choline, which is often present in plant tissue extracts and can adversely affect the ionisation and detection of chlormequat by MALDI-TOFMS. The use of two internal standards ([13C3]- and [2H9]-chlormequat) enabled direct quantification and simultaneous control of the recovery. PMID:11223954

  14. Development of a method for rapid quantitation of amino acids by liquid chromatography-tandem mass spectrometry (LC-MSMS) in plasma.

    PubMed

    Casetta, B; Tagliacozzi, D; Shushan, B; Federici, G

    2000-05-01

    A new analytical method has been developed and is proposed for the rapid determination of eighteen common amino acids, including tryptophan, in plasma and dried blood spots, by liquid chromatography coupled with ionspray tandem mass spectrometry. Potentially the method can include other amino acids and can be used for the diagnosis of metabolic disease. The use of the ionspray tandem mass spectrometry approach permits extremely rapid chromatographic separation of all amino acids requiring less than four minutes for the analysis of each sample, after a simple sample preparation procedure. The chromatographic separation of the analytes was achieved using a CN normal phase column and a water/acetonitrile/trifluoroacetic acid mobile phase at flow rate of 1 ml/min. The mass spectrometer was operated in multiple reaction monitoring mode, where each analyte had its own unique precursor and product ion setting. The quantitative analysis of amino acids was achieved using as internal standards just two representative isotopically labeled amino acids: D4-Ala and D5-Phe. Calibration is made externally by using aqueous solutions with the same labelled amino acids as internal standards. The high specificity of tandem mass spectrometry coupled with a fast chromatographic process is suitable for the rapid and reliable assay of metabolically significant amino acids. The liquid chromatography-tandem mass spectrometry method is more effective than other published tandem mass spectrometry methods at distinguishing isobaric amino acids like Leu, Ile and HO-Pro and certainly far more rapid than HPLC or ion-exchange chromatographic methods.

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

    PubMed

    Wang, Hailong; Li, Yan; Yang, Lijuan; Yu, Baofeng; Yan, Ping; Pang, Min; Li, Xiaobing; Yang, Hong; Zheng, Guoping; Xie, Jun; Guo, Rui

    2014-10-01

    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

  16. Protein composition of wheat gluten polymer fractions determined by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry

    PubMed Central

    2014-01-01

    Background Certain wheat gluten proteins form large protein polymers that are extractable in 0.5% SDS only after sonication. Although there is a strong relationship between the amounts of these polymers in the flour and bread-making quality, the protein components of these polymers have not been thoroughly investigated. Results Flour proteins from the US bread wheat Butte 86 were extracted in 0.5% SDS using a two-step procedure with and without sonication. Proteins were further separated by size exclusion chromatography (SEC) into monomeric and polymeric fractions and analyzed by quantitative two-dimensional gel electrophoresis (2-DE). When proteins in select 2-DE spots were identified by tandem mass spectrometry (MS/MS), overlapping spots from the different protein fractions often yielded different identifications. Most high-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) partitioned into the polymer fractions, while most gliadins were found in the monomer fractions. The exceptions were alpha, gamma and omega gliadins containing odd numbers of cysteine residues. These proteins were detected in all fractions, but comprised the largest proportion of the SDS-extractable polymer fraction. Several types of non-gluten proteins also were found in the polymer fractions, including serpins, triticins and globulins. All three types were found in the largest proportions in the SDS-extractable polymer fraction. Conclusions This is the first study to report the accumulation of gliadins containing odd numbers of cysteine residues in the SDS-extractable glutenin polymer fraction, supporting the hypothesis that these gliadins serve as chain terminators of the polymer chains. These data make it possible to formulate hypotheses about how protein composition influences polymer size and structure and provide a foundation for future experiments aimed at determining how environment affects glutenin polymer distribution. In addition, the

  17. Development of a liquid chromatography-electrospray-tandem mass spectrometry method for the quantitative determination of benzoxazinone derivatives in plants.

    PubMed

    Bonnington, Lea; Eljarrat, Ethel; Guillamón, Miriam; Eichhorn, Peter; Taberner, Andreu; Barceló, Damià

    2003-07-01

    A new method for the quantification of benzoxazinone derivatives in extracts of wheat foliage and root samples using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) is described. Using this method, the characterization, separation, and quantitative detection of a mixture of six naturally occurring 1,4-benzoxazin-3(4H)-one derivatives, including the hydroxamic acids (DIMBOA, DIBOA), lactams (HBOA, and HMBOA), benzoxazilinones (BOA, MBOA), and two synthetic methoxylated variations of DIBOA and HBOA, was achieved. The application of a novel, highly modified reversed-phase LC column, the dodecyl (C12) TMS end-capped Synergi MAX-RP, enhanced the on-line chromatographic separation through improvements to component resolution, analyte stability and peak shape and also to the column lifetime. The complete ESI-MS-MS precursor-product ion fragmentation pathways for the benzoxazinone derivatives are described for the first time and used to deduce a generic fragmentation pattern for the compound class. Characteristic transitions for the benzoxazinones were thus used in the developed analytical method enabling reliable quantification with simultaneous screening for other potentially present derivatives, while eliminating interferences from other coeluting contaminants from the complex plant extract matrix. Quantitative analysis was done in the multiple reaction monitoring mode, using two specific combinations of a precursor-product ion transitions for each compound. The ESI-MS-MS detection method offered improvements to the sensitivity and selectivity, as compared with previously applied LC methods, with detection limits down to 0.002-0.023 ng/microL. The developed method was demonstrated by analyzing foliages and roots of six different wheat cultivars using pressurized liquid extraction-solid-phase extraction cleanup-LC-ESI-MS-MS. The analytes were detected in the range of 0.7-207 microg/g of dry weight.

  18. Stable isotope labeling assisted liquid chromatography-electrospray tandem mass spectrometry for quantitative analysis of endogenous gibberellins.

    PubMed

    Hao, Yan-Hong; Zhang, Zheng; Wang, Lu; Liu, Chao; Lei, Ai-Wen; Yuan, Bi-Feng; Feng, Yu-Qi

    2015-11-01

    In the current study, we developed a stable isotope labeling strategy for the absolute quantification of gibberellins (GAs) by high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). N,N-dimethyl ethylenediamine (DMED) and its deuterated counterpart d(4)-DMED were used to derivatize GAs extracted from plant tissue samples and GA standards respectively. The both derivatives of GAs were mixed and then subjected to HPLC-ESI-MS/MS analysis. The absolute quantification of GAs in plant tissues could be achieved by calculating the peak area ratios of DMED labeled GAs/d(4)-DMED labeled GAs. In the proposed strategy, the derivatization reaction of the labeling reagents with GAs could be completed rapidly (within 5 min) with high efficiency (>99%) under mild conditions. The resulting derivatives could produce specific fragments in collision induced dissociation (CID), leading to high selectivity in multiple-reaction monitoring (MRM) mode, thus enhanced the reliability of the LC-MS/MS method. Furthermore, the limits of quantitation (LOQs) of GAs were considerably decreased (2-32 folds) due to incorporating easily ionized moieties into GAs, and the quantification of GAs in plant tissue could be achieved without isotopically labeled GA standards. Good linearity was obtained with correlation coefficients R(2) values of >0.99. The limits of detection (LODs) and quantitation (LOQs) ranged from 0.02 to 0.74 pg and 0.07 to 2.45 pg, respectively. Eleven GAs could be successfully determined in spiked sample with 72-128% recoveries and the relative standard deviations (RSDs) were between 1.0% and 13.9%. Finally, the developed method was successfully applied for the detection of GAs in 50mg (fresh weight) Oryza sativa leaves. PMID:26452831

  19. Stable isotope labeling assisted liquid chromatography-electrospray tandem mass spectrometry for quantitative analysis of endogenous gibberellins.

    PubMed

    Hao, Yan-Hong; Zhang, Zheng; Wang, Lu; Liu, Chao; Lei, Ai-Wen; Yuan, Bi-Feng; Feng, Yu-Qi

    2015-11-01

    In the current study, we developed a stable isotope labeling strategy for the absolute quantification of gibberellins (GAs) by high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). N,N-dimethyl ethylenediamine (DMED) and its deuterated counterpart d(4)-DMED were used to derivatize GAs extracted from plant tissue samples and GA standards respectively. The both derivatives of GAs were mixed and then subjected to HPLC-ESI-MS/MS analysis. The absolute quantification of GAs in plant tissues could be achieved by calculating the peak area ratios of DMED labeled GAs/d(4)-DMED labeled GAs. In the proposed strategy, the derivatization reaction of the labeling reagents with GAs could be completed rapidly (within 5 min) with high efficiency (>99%) under mild conditions. The resulting derivatives could produce specific fragments in collision induced dissociation (CID), leading to high selectivity in multiple-reaction monitoring (MRM) mode, thus enhanced the reliability of the LC-MS/MS method. Furthermore, the limits of quantitation (LOQs) of GAs were considerably decreased (2-32 folds) due to incorporating easily ionized moieties into GAs, and the quantification of GAs in plant tissue could be achieved without isotopically labeled GA standards. Good linearity was obtained with correlation coefficients R(2) values of >0.99. The limits of detection (LODs) and quantitation (LOQs) ranged from 0.02 to 0.74 pg and 0.07 to 2.45 pg, respectively. Eleven GAs could be successfully determined in spiked sample with 72-128% recoveries and the relative standard deviations (RSDs) were between 1.0% and 13.9%. Finally, the developed method was successfully applied for the detection of GAs in 50mg (fresh weight) Oryza sativa leaves.

  20. 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

  1. Application of liquid chromatography-tandem mass spectrometry in quantitative bioanalyses of organic molecules in aquatic environment and organisms.

    PubMed

    Bussy, Ugo; Li, Ke; Li, Weiming

    2016-05-01

    Analytical methods using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of metabolites or contaminants (or both) in various tissues of aquatic organisms and in the aquatic environment have received increasing attention in the last few years. This review discusses the findings relevant to such procedures published between 2005 and 2015. The aim is to evaluate the advantages, restrictions, and performances of the procedures from sample preparation to mass spectrometry measurement. To support these discussions, a general knowledge on LC-MS/MS is also provided. PMID:26996906

  2. Quantitative analysis of pesticide residues in vegetables and fruits by liquid chromatography quadrupole time-of-flight mass spectrometry.

    PubMed

    Saito-Shida, Shizuka; Nemoto, Satoru; Teshima, Reiko; Akiyama, Hiroshi

    2016-01-01

    The applicability of liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) for the quantitative analysis of pesticide residues in vegetables and fruits was demonstrated. The LC-QTOF-MS parameters, such as cone voltage, capillary voltage, collision energy and mass extraction window, were carefully optimised for the analysis of pesticide residues. The LC-QTOF-MS method was validated for 149 pesticides in four vegetables and fruits, i.e. apple, potato, cabbage and spinach, at a spiking level of 0.01 mg kg(-1). The samples were prepared according to the Japanese official multi-residue method with a modification to the column clean-up procedure. Of the 149 pesticides, recoveries in the range of 70-120% were achieved for 147 pesticides in apple, 145 in potato, 141 in cabbage and 131 in spinach, with intra-day precisions (RSDs) of < 25% and inter-day precisions (RSDs) of < 30%, which are within the acceptable range given in the Japanese method validation guideline. Matrix effects were negligible for the majority of the target pesticides. Except for spiroxamine in spinach, no interfering peaks were observed in the blank samples. The target pesticides, except those with low sensitivity, achieved calibration curves with satisfactory linearity, with correlation coefficients (r) greater than 0.995 in the concentration range of 0.002-0.1 μg ml(-1). Furthermore, the majority of the target pesticides provided more than one fragment ion or isotope ion that could be used for confirmation. The overall results suggest that LC-QTOF-MS is a powerful tool for the quantification of pesticide residues in vegetables and fruits at the level of 0.01 mg kg(-1).

  3. Quantitation of bentysrepinine (Y101) in rat plasma by liquid chromatography tandem mass spectrometry: application to pharmacokinetic study.

    PubMed

    Fan, Huirong; Li, Ruixing; Gu, Yuan; Si, Duanyun; Liu, Changxiao

    2012-03-15

    A simple, accurate and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitation of bentysrepinine (Y101) in rat plasma. After the addition of diphenhydramine (internal standard, IS), plasma samples were pretreated by protein precipitation. Chromatographic separation was carried out on an Atlantis(®) analytical column (4.6 mm × 100 mm, 5 μm, Waters) with methanol: 20 mM ammonium formate consisting of 1.0% formic acid (65:35, v/v) as the mobile phase at an isocratic flow rate of 0.4 mL/min for 7.5 min. The multiple reaction monitoring (MRM) transitions were performed at m/z 490.2→339.5 for Y101 and m/z 256.0→167.0 for IS on a SCIEX API 4000 mass spectrometer in the positive ion mode with electrospray ionization (ESI) source. Good linearity was achieved over the concentration range of 1-2500 ng/mL. The intra- and inter-day precisions were less than 8.3%, and the accuracy ranged from -4.0% to 2.8%. Y101 was stable during the analysis and the storage period. The pharmacokinetic profiles of Y101 at three dose levels were successfully studied for the first time in rats by this method. After single intra-gastric administration of Y101 at the doses of 25, 50 and 100 mg/kg, C(max) and AUC(0-t) were proportional to the doses given. PMID:22366283

  4. 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.

  5. Key issues in the acquisition and analysis of qualitative and quantitative mass spectrometry data for peptide-centric proteomic experiments.

    PubMed

    Thompson, Andrew J; Abu, Mika; Hanger, Diane P

    2012-09-01

    Proteomic technologies have matured to a level enabling accurate and reproducible quantitation of peptides and proteins from complex biological matrices. Analysis of samples as diverse as assembled protein complexes, whole cell lysates or sub-cellular proteomes from cell cultures, and direct analysis of animal and human tissues and fluids demonstrate the incredible versatility of the fundamental nature of the technique that forms the basis of most proteomic applications today (mass spectrometry). Determining the mass of biomolecules and their fragments or related products with high accuracy can convey a highly specific assay for detection and identification. Importantly, ion currents representative of these specifically identified analytes can be accurately quantified with the correct application of smart isobaric tagging chemistries, heavy and light isotopically derivatised samples or standards, or by careful application of workflows to compare unlabelled samples in so-called 'label-free' and targeted selected reaction monitoring experiments. In terms of exploring biology, a myriad of protein changes and modifications are being increasingly probed and quantified, including diverse chemical changes from relatively decisive modifications such as protein splicing and truncation, to more transient dynamic modifications such as phosphorylation, acetylation and ubiquitination. Proteomic workflows can be complex beasts and several key considerations to ensure effective applications have been outlined in the recent literature. The past year has witnessed the publication of several excellent reviews that thoroughly describe the fundamental principles underlying the state of the art. This review further elaborates on specific critical issues introduced by these publications and raises other important unaddressed considerations and new developments that directly impact on the effectiveness of proteomic technologies, in particular for, but not necessarily exclusive to peptide

  6. 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

    2015-09-01

    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.

  7. 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

    2015-09-01

    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. PMID:26388380

  8. Quantitative Characterization of Gold Nanoparticles by Coupling Thin Layer Chromatography with Laser Ablation Inductively Coupled Plasma Mass Spectrometry.

    PubMed

    Yan, Neng; Zhu, Zhenli; Jin, Lanlan; Guo, Wei; Gan, Yiqun; Hu, Shenghong

    2015-06-16

    Metal nanoparticles (NPs) determination has recently attracted considerable attention because of the continuing boom of nanotechnology. In this study, a novel method for separation and quantitative characterization of NPs in aqueous suspension was established by coupling thin layer chromatography (TLC) with laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Gold nanoparticles (AuNPs) of various sizes were used as the model system. It was demonstrated that TLC not only allowed separation of gold nanoparticles from ionic gold species by using acetyl acetone/butyl alcohol/triethylamine (6:3:1, v/v) as the mobile phase, but it also achieved the separation of differently sized gold nanoparticles (13, 34, and 47 nm) by using phosphate buffer (0.2 M, pH = 6.8), Triton X-114 (0.4%, w/v), and EDTA (10 mM) as the mobile phase. Various experimental parameters that affecting TLC separation of AuNPs, such as the pH of the phosphate buffer, the coating of AuNPs, the concentrations of EDTA and Triton X-114, were investigated and optimized. It was found that separations of AuNPs by TLC displayed size dependent retention behavior with good reproducibility, and the retardation factors (R(f) value) increased linearly with decreasing nanoparticle size. The analytical performance of the present method was evaluated under optimized conditions. The limits of detection were in the tens of pg range, and repeatability (RSD, n = 7) was 6.3%, 5.9%, and 8.3% for 30 ng of 13 nm AuNPs, 34 nm AuNPs, and 47 nm AuNPs, respectively. The developed TLC-LA-ICP-MS method has also been applied to the analysis of spiked AuNPs in lake water, river water, and tap water samples.

  9. 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

  10. Human aflatoxin albumin adducts quantitatively compared by ELISA, HPLC with fluorescence detection, and HPLC with isotope dilution mass spectrometry.

    PubMed

    McCoy, Leslie F; Scholl, Peter F; Sutcliffe, Anne E; Kieszak, Stephanie M; Powers, Carissa D; Rogers, Helen S; Gong, Yun Yun; Groopman, John D; Wild, Christopher P; Schleicher, Rosemary L

    2008-07-01

    Essential to the conduct of epidemiologic studies examining aflatoxin exposure and the risk of heptocellular carcinoma, impaired growth, and acute toxicity has been the development of quantitative biomarkers of exposure to aflatoxins, particularly aflatoxin B(1). In this study, identical serum sample sets were analyzed for aflatoxin-albumin adducts by ELISA, high-performance liquid chromatography (HPLC) with fluorescence detection (HPLC-f), and HPLC with isotope dilution mass spectrometry (IDMS). The human samples analyzed were from an acute aflatoxicosis outbreak in Kenya in 2004 (n = 102) and the measured values ranged from 0.018 to 67.0, nondetectable to 13.6, and 0.002 to 17.7 ng/mg albumin for the respective methods. The Deming regression slopes for the HPLC-f and ELISA concentrations as a function of the IDMS concentrations were 0.71 (r(2) = 0.95) and 3.3 (r(2) = 0.96), respectively. When the samples were classified as cases or controls, based on clinical diagnosis, all methods were predictive of outcome (P < 0.01). Further, to evaluate assay precision, duplicate samples were prepared at three levels by dilution of an exposed human sample and were analyzed on three separate days. Excluding one assay value by ELISA and one assay by HPLC-f, the overall relative SD were 8.7%, 10.5%, and 9.4% for IDMS, HPLC-f, and ELISA, respectively. IDMS was the most sensitive technique and HPLC-f was the least sensitive method. Overall, this study shows an excellent correlation between three independent methodologies conducted in different laboratories and supports the validation of these technologies for assessment of human exposure to this environmental toxin and carcinogen.

  11. 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)

  12. 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)

  13. 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

  14. Selective quantitative bioanalysis of proteins in biological fluids by on-line immunoaffinity chromatography-protein digestion-liquid chromatography-mass spectrometry.

    PubMed

    Hoos, Johannes S; Sudergat, Hilke; Hoelck, Jens-Peter; Stahl, Mark; de Vlieger, Jon S B; Niessen, Wilfried M A; Lingeman, Henk; Irth, Hubertus

    2006-01-18

    A quantitative method for the determination of proteins in complex biological matrices has been developed based on the selectivity of antibodies for sample purification followed by proteolytic digestion and quantitative mass spectrometry. An immunosorbent of polyclonal anti-bovine serum albumin (BSA) antibodies immobilized on CNBR agarose is used in the on-line mode for selective sample pretreatment. Next, the purified sample is trypsin digested to obtain protein specific peptide markers. Subsequent analysis of the peptide mixture using a desalination procedure and a separation step coupled, on-line to an ion-trap mass spectrometer, reveals that this method enables selective determination of proteins in biological matrices like diluted human plasma. This approach enhances substantially the selectivity compared to common quantitative analysis executed with immunoassays and colorimetry, fluorimetry or luminescence detection. Hyphenation of the immunoaffinity chromatography with on-line digestion and chromatography-mass spectrometry is performed and a completely on-line quantification of the model protein BSA in bovine and human urine was established. A detection limit of 170 nmol/l and a quantification limit of 280 nmol/l is obtained using 50 microl of either standard or spiked biological matrix. The model system allows fully automated absolute quantitative mass spectrometric analysis of intact proteins in biological matrices without time-consuming labeling procedures.

  15. 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

  16. 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...

  17. Hydrogen Exchange Mass Spectrometry.

    PubMed

    Mayne, Leland

    2016-01-01

    Hydrogen exchange (HX) methods can reveal much about the structure, energetics, and dynamics of proteins. The addition of mass spectrometry (MS) to an earlier fragmentation-separation HX analysis now extends HX studies to larger proteins at high structural resolution and can provide information not available before. This chapter discusses experimental aspects of HX labeling, especially with respect to the use of MS and the analysis of MS data.

  18. 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

  19. 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.

  20. [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.

  1. 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.

  2. Tissue-specific metabolite profiling of Cyperus rotundus L. rhizomes and (+)-nootkatone quantitation by laser microdissection, ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry, and gas chromatography-mass spectrometry techniques.

    PubMed

    Jaiswal, Yogini; Liang, Zhitao; Guo, Ping; Ho, Hing-Man; Chen, Hubiao; Zhao, Zhongzhen

    2014-07-23

    Cyperus rotundus L. is a plant species commonly found in both India and China. The caused destruction of this plant is of critical concern for agricultural produce. Nevertheless, it can serve as a potential source of the commercially important sesquiterpenoid (+)-nootkatone. The present work describes comparative metabolite profiling and (+)-nootkatone content determination in rhizome samples collected from these two countries. Laser dissected tissues, namely, the cortex, hypodermal fiber bundles, endodermis, amphivasal vascular bundles, and whole rhizomes were analyzed by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS). Gas chromatography-mass spectrometry (GC-MS) analysis was used for profiling of essential oil constituents and quantitation of (+)-nootkatone. The content of (+)-nootkatone was found to be higher in samples from India (30.47 μg/10 g) compared to samples from China (21.72 μg/10 g). The method was validated as per International Conference on Harmonisation (ICH) guidelines (Q2 R1). The results from this study can be applied for quality control and efficient utilization of this terpenoid-rich plant for several applications in food-based industries. PMID:24938835

  3. "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. PMID:26486514

  4. "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.

  5. "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.

  6. Quantitative determination of dimethyl fumarate in silica gel by solid-phase microextraction/gas chromatography/mass spectrometry and ultrasound-assisted extraction/gas chromatography/mass spectrometry.

    PubMed

    Bocchini, Paola; Pinelli, Francesca; Pozzi, Romina; Ghetti, Federica; Galletti, Guido C

    2015-06-01

    Dimethyl fumarate (DMF) is a chemical compound which has been added to silica gel bags used for preserving leather products during shipment. DMF has recently been singled out due to its ability to induce a number of medical problems in people which touch products contaminated by it. Its use as a biocide has been recently made illegal in Europe. Two different extraction techniques, namely ultrasound-assisted extraction (UAE) and solid-phase microextraction (SPME), both coupled with gas chromatography/mass spectrometry were applied to the quantitative determination of DMF in silica gel. Linearity of the methods, reproducibility and detection limits were determined. The two methods were applied to the quantification of DMF in thirty-four silica gel samples used as anti-mould agents in different leather products sold in Italy, and the obtained results were statistically compared. PMID:25939646

  7. 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]. PMID:27358907

  8. 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].

  9. Qualitative and Quantitative Analysis of Major Triterpenoids in Alismatis Rhizoma by High Performance Liquid Chromatography/Diode-Array Detector/Quadrupole-Time-of-Flight Mass Spectrometry and Ultra-Performance Liquid Chromatography/Triple Quadrupole Mass Spectrometry.

    PubMed

    Zhao, Wanli; Huang, Xiaoqiang; Li, Xiaoyan; Zhang, Fangfang; Chen, Sainan; Ye, Miao; Huang, Mingqing; Xu, Wen; Wu, Shuisheng

    2015-07-31

    Alismatis Rhizoma (AMR) is a well-known natural medicine with a long history in Chinese medicine and has been commonly used for treating a wide range of ailments related to dysuria, edema, nephropathy, hyperlipidaemia, diabetes, inflammation as well as tumors in clinical applications. Most beneficial effects of AMR are attributed to the presence of protostane terpenoids, the major active ingredients of Alismatis Rhizoma (AMR). In this study, a systematic high performance liquid chromatography/diode-array detector/quadrupole-time-of-flight mass spectrometry (HPLC-DAD-Q-TOF MS) and ultra-performance liquid chromatography/triple quadrupole mass spectrometry (UPLC-QqQ MS) method was developed for qualitative and quantitative analyses of the major AMR triterpenoids. First, a total of 25 triterpenoid components, including 24 known compounds and one new compound were identified by comparison with UV spectra, molecular ions and fragmentation behaviors of reference standards or the literature. Second, an efficient method was established for the rapid simultaneous determination of 14 representative triterpenoids by UPLC-QqQ MS. Forty-three batches of AMR were analyzed with linearity (r, 0.9980-0.9999), intra-day precision (RSD, 1.18%-3.79%), inter-day precision (RSD, 1.53%-3.96%), stability (RSD, 1.32%-3.97%), repeatability (RSD, 2.21%-4.25%), and recovery (98.11%-103.8%). These results indicated that new approaches combining HPLC-DAD-Q-TOF MS and UPLC-QqQ MS are applicable in the qualitative and quantitative analysis of AMR.

  10. 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.

  11. Qualitative and quantitative analysis of the anthelmintic fenbendazole and its metabolites in biological matrices by direct exposure probe mass spectrometry.

    PubMed

    Barker, S A; Hsieh, L C; McDowell, T R; Short, C R

    1987-04-01

    Methodology for the qualitative and quantitative analysis of the anthelmintic fenbendazole and its metabolites in goat feces using electron impact (EI)/direct exposure probe (DEP)/mass spectrometric (MS) and tandem mass spectrometric (MS/MS) techniques is presented. Analyses were conducted on extracts from spiked feces and feces from animals treated per os with 5 mg fenbendazole/kg, with samples being collected at zero time and at twelve hour intervals for 144 h. The results of the EI/DEP/MS quantitation of these samples are compared to those for the same samples analysed by high pressure liquid chromatography (HPLC). Mass spectral data for fenbendazole and its metabolites are presented and the advantages of the use of EI/DEP/MS and/or DEP/MS/MS over HPLC are discussed. This methodology may be used as a confirmatory method for the HPLC analysis of fenbendazole and its metabolites or may be used as a method in its own right for the rapid qualitative and quantitative analysis of these compounds.

  12. 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.

  13. Identification and quantitation of amphetamine, methamphetamine, MDMA, pseudoephedrine, and ephedrine in blood, plasma, and serum using gas chromatography-mass spectrometry (GC/MS).

    PubMed

    Gunn, Josh; Kriger, Scott; Terrell, Andrea R

    2010-01-01

    Amphetamine, methamphetamine, MDMA, pseudoephedrine, and ephedrine are measured in blood, serum, and plasma using gas chromatography coupled to mass spectrometry (GC/MS). Following a simple liquid-liquid extraction, analytes are derivatized with heptafluorobutyric anhydride (HFBA) and 1 microL injected onto a HP-5MS 15-meter capillary column. Quantitation of each analyte is accomplished using a multi-point calibration curve and deuterated internal standards. The method provides a simple, robust, and reliable means to identify and measure these analytes.

  14. Screening and quantitation of multiclass drugs of abuse and pharmaceuticals in hair by fast liquid chromatography electrospray time-of-flight mass spectrometry.

    PubMed

    Domínguez-Romero, Juan C; García-Reyes, Juan F; Molina-Díaz, Antonio

    2011-07-15

    In this work, an automated screening method for the simultaneous identification and quantitation of 30 representative multiclass drugs (including opiates, cocaine and its main metabolite, cannabinoids, amphetamines and other stimulants in hair samples) has been developed using fast liquid-chromatography time-of-flight mass spectrometry (LC-TOFMS). The identification and quantitation of the drugs were carried out by liquid chromatography using a C(18) column (4.6×50 mm) with 1.8 μm particle size. Accurate mass measurements of ions of interest (typically [M+H](+)) by electrospray time-of-flight mass spectrometry in the positive ionization mode were used for unambiguous confirmation of the targeted species. Three sample preparation methodologies were evaluated: (a) direct methanolic extraction by sonication, (b) acidic extraction, and (c) alkaline digestion. Direct methanolic extraction showed better recoveries and cleaner extracts. The limits of detection obtained in hair matrix were as low as 5 pg mg(-1) for cocaine and cannabidiol, ranging from 5 to 75 pg mg(-1) for the studied species while the LOQ ranged from 15 to 250 pg mg(-1). The method has been applied to six hair samples from drug consumer volunteers, where the presence of at least one drug was confirmed by accurate mass measurements within 2 ppm (mass error) in most cases. The present study demonstrates the usefulness of LC-TOFMS for both screening and quantitation purposes in drug testing in hair. In addition, the possibility of non-target or a posteriori data analysis of samples or the extension of the procedure for testing for additional compounds offers interesting features for forensic analysis.

  15. 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.

  16. Comprehensive characterization of glioblastoma tumor tissues for biomarker identification using mass spectrometry-based label-free quantitative proteomics

    PubMed Central

    Heroux, Maxime S.; Chesnik, Marla A.; Halligan, Brian D.; Al-Gizawiy, Mona; Connelly, Jennifer M.; Mueller, Wade M.; Rand, Scott D.; Cochran, Elizabeth J.; LaViolette, Peter S.; Malkin, Mark G.; Schmainda, Kathleen M.

    2014-01-01

    Cancer is a complex disease; glioblastoma (GBM) is no exception. Short survival, poor prognosis, and very limited treatment options make it imperative to unravel the disease pathophysiology. The critically important identification of proteins that mediate various cellular events during disease is made possible with advancements in mass spectrometry (MS)-based proteomics. The objective of our study is to identify and characterize proteins that are differentially expressed in GBM to better understand their interactions and functions that lead to the disease condition. Further identification of upstream regulators will provide new potential therapeutic targets. We analyzed GBM tumors by SDS-PAGE fractionation with internal DNA markers followed by liquid chromatography-tandem mass spectrometry (MS). Brain tissue specimens obtained for clinical purposes during epilepsy surgeries were used as controls, and the quantification of MS data was performed by label-free spectral counting. The differentially expressed proteins were further characterized by Ingenuity Pathway Analysis (IPA) to identify protein interactions, functions, and upstream regulators. Our study identified several important proteins that are involved in GBM progression. The IPA revealed glioma activation with z score 2.236 during unbiased core analysis. Upstream regulators STAT3 and SP1 were activated and CTNNα was inhibited. We verified overexpression of several proteins by immunoblot to complement the MS data. This work represents an important step towards the identification of GBM biomarkers, which could open avenues to identify therapeutic targets for better treatment of GBM patients. The workflow developed represents a powerful and efficient method to identify biomarkers in GBM. PMID:24803679

  17. Rapid-resolution liquid chromatography/mass spectrometry for determination and quantitation of polyphenols in grape berries.

    PubMed

    Cavaliere, Chiara; Foglia, Patrizia; Gubbiotti, Riccardo; Sacchetti, Paolo; Samperi, Roberto; Laganà, Aldo

    2008-10-01

    A rapid-resolution liquid chromatography/mass spectrometric (RRLC/MS) method for detection and quantitation of polyphenols in grape berry skins and seeds has been developed. Pulp-free berry skins were treated with liquid nitrogen and ground; seeds were also ground. Then, 3 g of samples were extracted with 30 mL of a mixture of methanol/water/formic acid 70:30:1 (v/v/v) under sonication and 1 microL of the final extract was injected into two 100 x 2.1 mm i.d., 1.8 microm Zorbax Eclipse plus C18 columns connected in series. Compounds were fractionated using a gradient elution of acidified acetonitrile/methanol 50:50 (v/v)/water. Columns were thermostatted at 70 degrees C. MS was carried out on an Agilent 6410 QqQ instrument equipped with an electrospray ionization source. Positive and negative MS/MS product ion scans were used for compound identification, whereas positive full scan MS in the m/z range 200-1400 was used for quantitation. By means of mass spectra comparison, various flavonols, flavan-3-ols, anthocyanins and stilbenes were identified. Quantitation was performed by external calibration, and concentration values were corrected for matrix effect that was evaluated in separate experiments. Semi-quantitative estimation was performed for compounds for which standards were not commercially available. Recoveries ranged from 90-102% with relative standard deviation (RSD) <5%, whereas the between samples RSD was in the range 4-12%. Two surrogate standards were used for quality control. The developed method was applied to analyze the polyphenol content of three Vitis vinifera table cultivars at physiological maturity and after proper preservation for 6 weeks. Results demonstrated that during preservation about half of the polyphenol content was lost.

  18. Rapid-resolution liquid chromatography/mass spectrometry for determination and quantitation of polyphenols in grape berries.

    PubMed

    Cavaliere, Chiara; Foglia, Patrizia; Gubbiotti, Riccardo; Sacchetti, Paolo; Samperi, Roberto; Laganà, Aldo

    2008-10-01

    A rapid-resolution liquid chromatography/mass spectrometric (RRLC/MS) method for detection and quantitation of polyphenols in grape berry skins and seeds has been developed. Pulp-free berry skins were treated with liquid nitrogen and ground; seeds were also ground. Then, 3 g of samples were extracted with 30 mL of a mixture of methanol/water/formic acid 70:30:1 (v/v/v) under sonication and 1 microL of the final extract was injected into two 100 x 2.1 mm i.d., 1.8 microm Zorbax Eclipse plus C18 columns connected in series. Compounds were fractionated using a gradient elution of acidified acetonitrile/methanol 50:50 (v/v)/water. Columns were thermostatted at 70 degrees C. MS was carried out on an Agilent 6410 QqQ instrument equipped with an electrospray ionization source. Positive and negative MS/MS product ion scans were used for compound identification, whereas positive full scan MS in the m/z range 200-1400 was used for quantitation. By means of mass spectra comparison, various flavonols, flavan-3-ols, anthocyanins and stilbenes were identified. Quantitation was performed by external calibration, and concentration values were corrected for matrix effect that was evaluated in separate experiments. Semi-quantitative estimation was performed for compounds for which standards were not commercially available. Recoveries ranged from 90-102% with relative standard deviation (RSD) <5%, whereas the between samples RSD was in the range 4-12%. Two surrogate standards were used for quality control. The developed method was applied to analyze the polyphenol content of three Vitis vinifera table cultivars at physiological maturity and after proper preservation for 6 weeks. Results demonstrated that during preservation about half of the polyphenol content was lost. PMID:18819110

  19. 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.

  20. Quantitative determination of acetylcholine in microdialysis samples using liquid chromatography/atmospheric pressure spray ionization mass spectrometry.

    PubMed

    Keski-Rahkonen, Pekka; Lehtonen, Marko; Ihalainen, Jouni; Sarajärvi, Timo; Auriola, Seppo

    2007-01-01

    A fast, simple and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the determination of acetylcholine in rat brain microdialysis samples. The chromatographic separation was achieved in 3 min on a reversed-phase column with isocratic conditions using a mobile phase containing 2% (v/v) of acetonitrile and 0.05% (v/v) of trifluoroacetic acid (TFA). A stable isotope-labeled internal standard was included in the analysis and detection was carried out with a linear ion trap mass spectrometer using selected reaction monitoring (SRM). Analyte ionization was performed with an atmospheric pressure chemical ionization (APCI) source without applying discharge current (atmospheric pressure spray ionization). This special ionization technique offered significant advantages over electrospray ionization for the analysis of acetylcholine with reversed-phase ion-pairing chromatography. The lower limit of quantification was 0.15 nM (1.5 fmol on-column) and linearity was maintained over the range of 0.15-73 nM, providing a concentration range that is significantly wider than that of the existing LC/MS methods. Good accuracy and precision were obtained for concentrations within the standard curve range. The method was validated and has been used extensively for the determination of acetylcholine in rat brain microdialysis samples.

  1. On-line double isotope dilution laser ablation inductively coupled plasma mass spectrometry for the quantitative analysis of solid materials.

    PubMed

    Fernández, Beatriz; Rodríguez-González, Pablo; García Alonso, J Ignacio; Malherbe, Julien; García-Fonseca, Sergio; Pereiro, Rosario; Sanz-Medel, Alfredo

    2014-12-01

    We report on the determination of trace elements in solid samples by the combination of on-line double isotope dilution and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The proposed method requires the sequential analysis of the sample and a certified natural abundance standard by on-line IDMS using the same isotopically-enriched spike solution. In this way, the mass fraction of the analyte in the sample can be directly referred to the certified standard so the previous characterization of the spike solution is not required. To validate the procedure, Sr, Rb and Pb were determined in certified reference materials with different matrices, including silicate glasses (SRM 610, 612 and 614) and powdered samples (PACS-2, SRM 2710a, SRM 1944, SRM 2702 and SRM 2780). The analysis of powdered samples was carried out both by the preparation of pressed pellets and by lithium borate fusion. Experimental results for the analysis of powdered samples were in agreement with the certified values for all materials. Relative standard deviations in the range of 6-21% for pressed pellets and 3-21% for fused solids were obtained from n=3 independent measurements. Minimal sample preparation, data treatment and consumption of the isotopically-enriched isotopes are the main advantages of the method over previously reported approaches.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

    2015-12-01

    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.

  8. 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

  9. 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. PMID:24070492

  10. 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.

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

    PubMed

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

    2015-12-16

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

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

    PubMed

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

    2015-12-16

    Undeclared food allergens account for 30-40% of food recalls in the United States. Compliance with ingredient labeling regulations and the implementation of effective manufacturing allergen control plans require the use of reliable methods for allergen detection and quantitation in complex food products. The objectives of this work were to (1) produce industry-processed model foods incurred with egg, milk, and peanut allergens, (2) compare analytical method performance for allergen quantitation in thermally processed bakery products, and (3) determine the effects of thermal treatment on allergen detection. Control and allergen-incurred cereal bars and muffins were formulated in a pilot-scale industry processing facility. Quantitation of egg, milk, and peanut in incurred baked goods was compared at various processing stages using commercial enzyme-linked immunosorbent assay (ELISA) kits and a novel multi-allergen liquid chromatography (LC)-tandem mass spectrometry (MS/MS) multiple-reaction monitoring (MRM) method. Thermal processing was determined to negatively affect the recovery and quantitation of egg, milk, and peanut to different extents depending on the allergen, matrix, and analytical test method. The Morinaga ELISA and LC-MS/MS quantitative methods reported the highest recovery across all monitored allergens, whereas the ELISA Systems, Neogen BioKits, Neogen Veratox, and R-Biopharm ELISA Kits underperformed in the determination of allergen content of industry-processed bakery products. PMID:26595064

  13. 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.

  14. 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.

  15. 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

    2013-01-01

    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.

  16. Tandem Mass Spectrometry Quantitation of Lyso-Gb3 and Six Related Analogs in Plasma for Fabry Disease Patients.

    PubMed

    Boutin, Michel; Lavoie, Pamela; Abaoui, Mona; Auray-Blais, Christiane

    2016-01-01

    Fabry disease is an X-linked lysosomal storage disorder, caused by a deficit in α-galactosidase A enzyme activity, leading to the storage of sphingolipids such as globotriaosylsphingosine (lyso-Gb3 ), globotriaosylceramide (Gb3 ), and galabiosylceramide (Ga2 ) in organs, tissues and biological fluids. A recent metabolomic study performed in plasma revealed lyso-Gb3 analogs as novel Fabry disease biomarkers. These molecules correspond to lyso-Gb3 with different chemical modifications on the sphingosine chain (-C2 H4 , -H2 , +O, +H2 O, +H2 O2, and +H2 O3 ). An ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the multiplex analysis of lyso-Gb3 and its 6 analogs in plasma. The samples are prepared by solid phase extraction using mixed-mode strong cation exchange (MCX) cartridges. An in-house synthesized N-glycinated lyso-Gb3 derivative was used for the internal standard. The limits of detection (LODs) measured for lyso-Gb3 and its analogs ranged from 0.06 to 0.29 nM. © 2016 by John Wiley & Sons, Inc.

  17. 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.

  18. 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. PMID:26230053

  19. Quantitative mass spectrometry imaging of small-molecule neurotransmitters in rat brain tissue sections using nanospray desorption electrospray ionization.

    PubMed

    Bergman, Hilde-Marléne; Lundin, Erik; Andersson, Malin; Lanekoff, Ingela

    2016-06-01

    Small molecule neurotransmitters are essential for the function of the nervous system, and neurotransmitter imbalances are often connected to neurological disorders. The ability to quantify such imbalances is important to provide insights into the biochemical mechanisms underlying the disorder. This proof-of-principle study presents online quantification of small molecule neurotransmitters, specifically acetylcholine, γ-aminobutyric acid (GABA) and glutamate, in rat brain tissue sections using nanospray desorption electrospray ionization (nano-DESI) mass spectrometry imaging. By incorporating deuterated internal standards in the nano-DESI solvent we show identification, accurate mapping, and quantification of these small neurotransmitters in rat brain tissue without introducing any additional sample preparation steps. We find that GABA is about twice as abundant in the medial septum-diagonal band complex (MSDB) as in the cortex, while glutamate is about twice as abundant in the cortex as compared to the MSDB. The study shows that nano-DESI is well suited for imaging of small molecule neurotransmitters in health and disease. PMID:26859000

  20. Tandem Mass Spectrometry Quantitation of Lyso-Gb3 and Six Related Analogs in Plasma for Fabry Disease Patients.

    PubMed

    Boutin, Michel; Lavoie, Pamela; Abaoui, Mona; Auray-Blais, Christiane

    2016-01-01

    Fabry disease is an X-linked lysosomal storage disorder, caused by a deficit in α-galactosidase A enzyme activity, leading to the storage of sphingolipids such as globotriaosylsphingosine (lyso-Gb3 ), globotriaosylceramide (Gb3 ), and galabiosylceramide (Ga2 ) in organs, tissues and biological fluids. A recent metabolomic study performed in plasma revealed lyso-Gb3 analogs as novel Fabry disease biomarkers. These molecules correspond to lyso-Gb3 with different chemical modifications on the sphingosine chain (-C2 H4 , -H2 , +O, +H2 O, +H2 O2, and +H2 O3 ). An ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the multiplex analysis of lyso-Gb3 and its 6 analogs in plasma. The samples are prepared by solid phase extraction using mixed-mode strong cation exchange (MCX) cartridges. An in-house synthesized N-glycinated lyso-Gb3 derivative was used for the internal standard. The limits of detection (LODs) measured for lyso-Gb3 and its analogs ranged from 0.06 to 0.29 nM. © 2016 by John Wiley & Sons, Inc. PMID:27367163

  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 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. PMID:18570431

  3. 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. PMID:26077745

  4. Array of Chemically Etched Fused Silica Emitters for Improving the Sensitivity and Quantitation of Electrospray Ionization Mass Spectrometry

    SciTech Connect

    Kelly, Ryan T.; Page, Jason S.; Tang, Keqi; Smith, Richard D.

    2007-06-01

    An array of emitters has been developed for increasing the sensitivity of electrospray ionization mass spectrometry (ESI-MS). The linear array consists of 19 chemically etched fused silica capillaries arranged with 500 µm (center-to-center) spacing. The multi-emitter device has a low dead volume to facilitate coupling to capillary liquid chromatography (LC) separations. The high aspect ratio of the emitters enables operation at flow rates as low as 20 nL/min/emitter, effectively extending the benefits of nanoelectrospray to higher flow rate analyses. To accommodate the larger ion current produced by the emitter array, a multi-capillary inlet to the mass spectrometer was also constructed. The inlet, which matched the dimensions of the emitter array, effectively preserved ion transmission efficiency. Standard reserpine solutions of varying concentration were electrosprayed at 1 µL/min using the multi-emitter/multi-inlet combination, and compared to a standard, single emitter configuration. A nine-fold sensitivity enhancement was observed for the multi-emitter relative to the single emitter. A bovine serum albumin tryptic digest was also analyzed and resulted in a sensitivity increase ranging from 2.4 to 12.3-fold for the detected tryptic peptides; the varying response was attributed to reduced ion suppression under the nano-ESI conditions afforded by the emitter array. An equimolar mixture of leucine enkephalin and maltopentaose was studied to verify that ion suppression is indeed reduced for the multi-ESI array relative to a single emitter over a range of flow rates.

  5. International Mass Spectrometry Society (IMSS).

    PubMed

    Cooks, R G; Gelpi, E; Nibbering, N M

    2001-02-01

    This paper gives a brief description of the recently formalized International Mass Spectrometry Society (IMSS). It is presented here in order to increase awareness of the opportunities for collaboration in mass spectrometry in an international context. It also describes the recent 15th International Mass Spectrometry Conference, held August/September 2000, in Barcelona. Each of the authors is associated with the IMSS. The 15th Conference, which covers all of mass spectrometry on a triennial basis, was chaired by Professor Emilio Gelpi of the Instituto de Investigaciones Biomedicas, Barcelona. The outgoing and founding President of the IMSS is Professor Graham Cooks, Purdue University, and the incoming President is Professor Nico Nibbering, University of Amsterdam. Similar material has been provided to the Editors of other journals that cover mass spectrometry.

  6. Quantitative determination of ɛ-N-carboxymethyl-L-lysine in human plasma by liquid chromatography-tandem mass spectrometry.

    PubMed

    Kuang, Liqing; Jing, Zhiqiang; Wang, Jing; Ma, Liyuan; Liu, Xiaoqiang; Yang, Jin

    2014-03-01

    ɛ-N-carboxymethyl-L-lysine (CML) is a stable chemical modification of protein lysine residues resulting from glycation and oxidation reactions and a potential biomarker of oxidative stress caused by sugar and lipid oxidation. In this study, a rapid, simple and sensitive method based on liquid chromatography-tandem spectrometry (LC-MS/MS) for the determination of CML in human plasma has been developed and validated. Sample preparation involved protein precipitation using trichloroacetic acid after addition of deuterated CML as internal standard. Chromatography was performed on an amino column by gradient-elution with a mobile phase containing acetonitrile:ultrapure water (80:20, v/v). CML and CML-d2 were detected by multiple reaction monitoring mode with ion pairs 205.0/130.1 and 207.2/84.1 respectively. The assay was linear in the range 10-1000 ng/mL with a lower limit of quantitation (LLOQ) of 10 ng/mL and recovery >90%. Assay validation showed that inter- and intra-day precision and accuracy were satisfactory. The method was applied to compare plasma CML levels in healthy Chinese subjects and patients with diabetes and uremia. In healthy subjects CML concentration (mean±SD) was 16.6±7.8 ng/mL. CML level in diabetic patients was not significantly different from healthy subjects whereas the level in patients with uremia was significantly higher than both healthy subjects and diabetic patients (P<0.001). The method will be useful to assess the value of CML as a biomarker of diabetic vascular complications resulting from elevated oxidative stress. PMID:24317023

  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. Method for screening and quantitative determination of serum levels of salicylic Acid, acetaminophen, theophylline, phenobarbital, bromvalerylurea, pentobarbital, and amobarbital using liquid chromatography/electrospray mass spectrometry.

    PubMed

    Hori, Yasushi; Fujisawa, Manami; Shimada, Kenji; Hirose, Yasuo; Yoshioka, Toshiharu

    2006-01-01

    We investigated a method for the simultaneous screening, identification, and quantitative determination of salicylic acid, acetaminophen, theophylline, barbiturates, and bromvalerylurea, drugs that frequently cause acute poisoning in Japan and therefore require rapid analysis for effective treatment in the clinical setting. The method employs liquid chromatography/electrospray mass spectrometry (LC/MS) of solid-phase extracted serum samples. For LC/MS ionization, the electrospray-ionization method was used, with acetaminophen in the positive-ion mode, and salicylic acid, theophylline, phenobarbital, bromvalerylurea, pentobarbital, amobarbital, and o-acetamidophenol (internal standard) in the negative-ion mode, the base ions were used in each case for quantitative analysis. Quantitation was possible for the following sample concentration ranges: salicylic acid and acetaminophen, 100 to 5 microg/ml; theophylline, 100 to 0.5 microg/ml; and phenobarbital, bromvalerylurea, pentobarbital, and amobarbital, 100 to 1 microg/ml. Using full-scan mass spectrometry, the lower detection limits of 1 microg/ml for salicylic acid and acetaminophen, 0.1 microg/ml for theophylline, and 0.5 microg/ml for phenobarbital, bromvalerylurea, pentobarbital, and amobarbital were adequate for identifying acute poisoning. When each compound was added to serum to a final concentration of 5 microg/ml and solid-phase extraction was performed using Oasis HLB 1-cc (30-mg), the mean recovery rate of each compound was 89.2 to 96.1% (n=5), and the coefficients of variation of the intraday and interday assays were 3.55 to 6.05% (n=5) and 3.68 to 6.38% (n=5), respectively, which are acceptable. When this method of analysis was applied in testing the sera of a female patient who had consumed a large amount of an unknown commercial drug, salicylic acid and bromvalerylurea were identified, and the treatment strategy could be determined in accordance with the serum concentration of those drugs.

  9. Identification and quantitation of MHC class II-bound peptides from mouse spleen dendritic cells by immunoprecipitation and mass spectrometry analysis

    PubMed Central

    Bozzacco, Leonia; Yu, Haiqiang

    2014-01-01

    Summary Advances in immunology and immune therapies require knowledge of antigenic peptide sequences that are presented on MHC class II and class I molecules of antigen presenting cells. The most specialized antigen presenting cells are dendritic cells (DCs). In the past, the small number of DCs that can be isolated from mouse spleen prevented direct analysis of the MHC II peptide repertoire presented by DCs. Here we describe a protocol that integrates immunological methods (in vivo enrichment of mouse spleen DCs by Flt3L treatment and immunoprecipation of MHC II-peptide complexes), mass spectrometry analysis and peptide synthesis (LC-MS/MS and quantitation analysis for non tryptic peptides) to identify and quantitate the endogenous peptides that are bound to MHC II molecules on DCs. The described method produces quantitative data that are reproducible and reliable enough to cover a wide range of peptide copy numbers. We propose the application of this method in future studies to quantitatively investigate the MHC II repertoire on DCs presented during viral infections or different immunizations in vaccine development research. PMID:23963941

  10. 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.

  11. 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.).

  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.). PMID:26444340

  13. 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.

  14. 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.

  15. Analysis of protein adduction kinetics by quantitative mass spectrometry: competing adduction reactions of glutathione-S-transferase P1-1 with electrophiles.

    PubMed

    Orton, Christopher R; Liebler, Daniel C

    2007-06-30

    Defining the mechanisms and consequences of protein adduction is crucial to understanding the toxicity of reactive electrophiles. Application of tandem mass spectrometry and data analysis algorithms enables detection and mapping of chemical adducts at the level of amino acid sequence. Nevertheless, detection of adducts does not indicate relative reactivity of different sites. Here, we describe a method to measure the kinetics of competing adduction reactions at different sites on the same protein. Adducts are formed by electrophiles at Cys14 and Cys47 on the metabolic enzyme glutathione-S-transferase P1-1 and modification is accompanied by a loss of enzymatic activity. Relative quantitation of protein adducts was done by tagging N-termini of peptide digests with isotopically labeled phenyl isocyanate and tracking the ratio of light-tagged peptide adducts to heavy-tagged reference samples in liquid chromatography-tandem mass spectrometry analyses using a multiple reaction monitoring method. This approach was used to measure rate constants for adduction at both positions with two different model electrophiles, N-iodoacetyl-N-biotinylhexylenediamine and 1-biotinamido-4-(4'-[maleimidoethyl-cyclohexane]-carboxamido)butane. The results indicate that Cys47 was approximately two- to three-fold more reactive toward both electrophiles than was Cys14. This result was consistent with the relative reactivity of these electrophiles in a complex proteome system and with previously reported trends in reactivity of these sites. Kinetic analyses of protein modification reactions provide a means of evaluating the selectivity of reactive mediators of chemical toxicity.

  16. 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…

  17. 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.

  18. Analytical Aspects of Hydrogen Exchange Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Engen, John R.; Wales, Thomas E.

    2015-07-01

    This article reviews the analytical aspects of measuring hydrogen exchange by mass spectrometry (HX MS). We describe the nature of analytical selectivity in hydrogen exchange, then review 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 HX MS 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 can 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.

  19. Stable isotope-labeled collagen: a novel and versatile tool for quantitative collagen analyses using mass spectrometry.

    PubMed

    Taga, Yuki; Kusubata, Masashi; Ogawa-Goto, Kiyoko; Hattori, Shunji

    2014-08-01

    Collagens are the most abundant proteins in animals and are involved in many physiological/pathological events. Although various methods have been used to quantify collagen and its post-translational modifications (PTMs) over the years, it is still difficult to accurately quantify type-specific collagen and minor collagen PTMs. We report a novel quantitative method targeting collagen using stable isotope-labeled collagen named "SI-collagen", which was labeled with isotopically heavy lysine, arginine, and proline in fibroblasts culture. We prepared highly labeled and purified SI-collagen for use as an internal standard in mass spectrometric analysis, particularly for a new approach using amino acid hydrolysis. Our method enabled accurate collagen analyses, including quantification of (1) type-specific collagen (types I and III in this paper), (2) total collagen, and (3) collagen PTMs by LC-MS with high sensitivity. SI-collagen is also applicable to other diverse analyses of collagen and can be a powerful tool for various studies, such as detailed investigation of collagen-related disorders.

  20. 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.

  1. 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. PMID:25722141

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

    PubMed Central

    Liu, Xin; Hummon, Amanda B.

    2015-01-01

    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 hours 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. PMID:25604392

  3. Quantitation of cocaine, benzoylecgonine, ecgonine methyl ester, and cocaethylene in urine and blood using gas chromatography-mass spectrometry (GC-MS).

    PubMed

    Fleming, Steven W; Dasgupta, Amitava; Garg, Uttam

    2010-01-01

    Cocaine, a stimulant, is a commonly abused drug. Cocaine and its metabolites are measured in various biological specimens for clinical and forensic purposes. Urine or plasma or serum is spiked with deuterated internal standards cocaine-d3, benzoylecgonine-d3, ecgonine methyl ester-d3, and cocaethylene-d3 and buffered with phosphate buffer. The drugs in the sample are extracted by cation-exchange solid phase extraction. The drugs from the solid phase cartridge are eluted and the eluent is dried under the stream of nitrogen. The residue is incubated with pentafluoropropionic acid anhydride and pentafluoropropanol to form pentafluoropropionyl derivatives of ecgonine methyl ester and benzoylecgonine. Cocaine and cocaethylene are refractory to derivatization. The extract is dried, reconstituted in ethyl acetate, and injected into gas chromatography mass-spectrometry analyzer. Quantitation of the drugs in the samples is made, using selected ion monitoring, from a 3-point calibration curve. PMID:20077067

  4. 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.

  5. Analytical method for the quantitative determination of cyanuric acid as the degradation product of sodium dichloroisocyanurate in urine by liquid chromatography mass spectrometry.

    PubMed

    Patel, Katan; Jones, Kate

    2007-06-15

    A simple and selective analytical method for the quantitative determination of cyanuric acid, the degradation product of sodium dichloroisocyanurate (NaDCC), in human urine is reported herein. The sample preparation involved the use of diatomaceous earth extraction columns. Quantification was achieved by liquid chromatography mass spectrometry using negative ion electrospray with a cyano (CN) column. Between day relative standard deviation less than 10% (n=6) was obtained at the 5 mg L(-1) level. The assay was linear over the investigated range 0-20 mg L(-1) and the limit of detection (LOD) was confirmed to be 0.1 mg L(-1). The method was applied to monitoring levels of cyanuric acid in healthcare workers using disinfectants products containing NaDCC. PMID:17409034

  6. Quantitative analysis of glycosaminoglycans, chondroitin/dermatan sulfate, hyaluronic acid, heparan sulfate, and keratan sulfate by liquid chromatography-electrospray ionization-tandem mass spectrometry.

    PubMed

    Osago, Harumi; Shibata, Tomoko; Hara, Nobumasa; Kuwata, Suguru; Kono, Michihaya; Uchio, Yuji; Tsuchiya, Mikako

    2014-12-15

    We developed a method using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) with a selected reaction monitoring (SRM) mode for simultaneous quantitative analysis of glycosaminoglycans (GAGs). Using one-shot analysis with our MS/MS method, we demonstrated the simultaneous quantification of a total of 23 variously sulfated disaccharides of four GAG classes (8 chondroitin/dermatan sulfates, 1 hyaluronic acid, 12 heparan sulfates, and 2 keratan sulfates) with a sensitivity of less than 0.5 pmol within 20 min. We showed the differences in the composition of GAG classes and the sulfation patterns between porcine articular cartilage and yellow ligament. In addition to the internal disaccharides described above, some saccharides derived from the nonreducing terminal were detected simultaneously. The simultaneous quantification of both internal and nonreducing terminal saccharides could be useful to estimate the chain length of GAGs. This method would help to establish comprehensive "GAGomic" analysis of biological tissues.

  7. Intercomparison of inductively coupled plasma mass spectrometry, quantitative neutron capture radiography, and prompt gamma activation analysis for the determination of boron in biological samples.

    PubMed

    Schütz, C L; Brochhausen, C; Hampel, G; Iffland, D; Kuczewski, B; Otto, G; Schmitz, T; Stieghorst, C; Kratz, J V

    2012-10-01

    Boron determination in blood and tissue samples is a crucial task especially for treatment planning, preclinical research, and clinical application of boron neutron capture therapy (BNCT). Comparison of clinical findings remains difficult due to a variety of analytical methods, protocols, and standard reference materials in use. This paper addresses the comparability of inductively coupled plasma mass spectrometry, quantitative neutron capture radiography, and prompt gamma activation analysis for the determination of boron in biological samples. It was possible to demonstrate that three different methods relying on three different principles of sample preparation and boron detection can be validated against each other and yield consistent results for both blood and tissue samples. The samples were obtained during a clinical study for the application of BNCT for liver malignancies and therefore represent a realistic situation for boron analysis.

  8. Deciphering the complexities of the wheat flour proteome using quantitative two-dimensional electrophoresis, three proteases and tandem mass spectrometry

    PubMed Central

    2011-01-01

    Background Wheat flour is one of the world's major food ingredients, in part because of the unique end-use qualities conferred by the abundant glutamine- and proline-rich gluten proteins. Many wheat flour proteins also present dietary problems for consumers with celiac disease or wheat allergies. Despite the importance of these proteins it has been particularly challenging to use MS/MS to distinguish the many proteins in a flour sample and relate them to gene sequences. Results Grain from the extensively characterized spring wheat cultivar Triticum aestivum 'Butte 86' was milled to white flour from which proteins were extracted, then separated and quantified by 2-DE. Protein spots were identified by separate digestions with three proteases, followed by tandem mass spectrometry analysis of the peptides. The spectra were used to interrogate an improved protein sequence database and results were integrated using the Scaffold program. Inclusion of cultivar specific sequences in the database greatly improved the results, and 233 spots were identified, accounting for 93.1% of normalized spot volume. Identified proteins were assigned to 157 wheat sequences, many for proteins unique to wheat and nearly 40% from Butte 86. Alpha-gliadins accounted for 20.4% of flour protein, low molecular weight glutenin subunits 18.0%, high molecular weight glutenin subunits 17.1%, gamma-gliadins 12.2%, omega-gliadins 10.5%, amylase/protease inhibitors 4.1%, triticins 1.6%, serpins 1.6%, purinins 0.9%, farinins 0.8%, beta-amylase 0.5%, globulins 0.4%, other enzymes and factors 1.9%, and all other 3%. Conclusions This is the first successful effort to identify the majority of abundant flour proteins for a single wheat cultivar, relate them to individual gene sequences and estimate their relative levels. Many genes for wheat flour proteins are not expressed, so this study represents further progress in describing the expressed wheat genome. Use of cultivar-specific contigs helped to overcome

  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. 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. PMID:26942737

  11. Stable isotope dilution ultra-high performance liquid chromatography-tandem mass spectrometry quantitative profiling of tryptophan-related neuroactive substances in human serum and cerebrospinal fluid.

    PubMed

    Hényková, Eva; Vránová, Hana Přikrylová; Amakorová, Petra; Pospíšil, Tomáš; Žukauskaitė, Asta; Vlčková, Magdaléna; Urbánek, Lubor; Novák, Ondřej; Mareš, Jan; Kaňovský, Petr; Strnad, Miroslav

    2016-03-11

    Many compounds related to L-tryptophan (L-TRP) have interesting biological or pharmacological activity, and their abnormal neurotransmission seems to be linked to a wide range of neurodegenerative and psychiatric diseases. A high-throughput method based on ultra-high performance liquid chromatography connected to electrospray tandem mass spectrometry (UHPLC-ESI-MS/MS) was developed for the quantitative analysis of L-TRP and 16 of its metabolites in human serum and cerebrospinal fluid (CSF), representing both major and minor routes of L-TRP catabolism. The combination of a fast LC gradient with selective tandem mass spectrometry enabled accurate analysis of almost 100 samples in 24h. The standard isotope dilution method was used for quantitative determination. The method's lower limits of quantification for serum and cerebrospinal fluid ranged from 0.05 to 15nmol/L and 0.3 to 45nmol/L, respectively. Analytical recoveries ranged from 10.4 to 218.1% for serum and 22.1 to 370.0% for CSF. The method's accuracy ranged from 82.4 to 128.5% for serum matrix and 90.7 to 127.7% for CSF matrix. All intra- and inter-day coefficients of variation were below 15%. These results demonstrate that the new method is capable of quantifying endogenous serum and CSF levels of a heterogeneous group of compounds spanning a wide range of concentrations. The method was used to determine the physiological levels of target analytes in serum and CSF samples from 18 individuals, demonstrating its reliability and potential usefulness in large-scale epidemiological studies.

  12. Qualitative and quantitative two-dimensional thin-layer chromatography/high performance liquid chromatography/diode-array/electrospray-ionization-time-of-flight mass spectrometry of cholinesterase inhibitors.

    PubMed

    Mroczek, Tomasz

    2016-09-10

    Recently launched thin-layer chromatography-mass spectrometry (TLC-MS) interface enabling extraction of compounds directly from TLC plates into MS ion source was unusually extended into two-dimensional thin-layer chromatography/high performance liquid chromatography (2D, TLC/HPLC) system by its a direct connection to a rapid resolution 50×2.1mm, I.D. C18 column compartment followed by detection by diode array (DAD) and electrospray ionisation time-of-flight mass spectrometry (ESI-TOF-MS). In this way, even not separated bands of complicated mixtures of natural compounds could be analysed structurally, only within 1-2min after development of TLC plates. In comparison to typically applied TLC-MS interface, no ion suppression for acidic mobile phases was observed. Also, substantial increase in ESI-TOF-MS sensitivities and quality of spectra, were noticed. It has been utilised in combination with TLC- based bioautographic approaches of acetylcholinesterase (AChE) inhibitors, However, it can be also applied in any other procedures related to bioactivity (e.g. 2,2-Diphenyl-1-picryl-hydrazyl-DPPH screen test for radicals). This system has been also used for determination of half maximal inhibitory concentration (IC50 values) of the active inhibitor-galanthamine, as an example. Moreover, AChE inhibitory potencies of some of purified plant extracts, never studied before, have been quantitatively measured. This is first report of usage such the 2D TLC/HPLC/MS system both for qualitative and quantitative evaluation of cholinesterase inhibitors in biological matrices.

  13. Assessment of capillary anion exchange ion chromatography tandem mass spectrometry for the quantitative profiling of the phosphometabolome and organic acids in biological extracts.

    PubMed

    Kvitvang, Hans F N; Kristiansen, Kåre A; Bruheim, Per

    2014-11-28

    Metabolic profiling has become an important tool in biological research, and the chromatographic separation of metabolites coupled with mass spectrometric detection is the most frequently used approach for such studies. The establishment of robust chromatographic methods for comprehensive coverage of the anionic metabolite pool is especially challenging. In this study, the development of a capillary ion exchange chromatography (capIC) - negative ESI tandem mass spectrometry (MS/MS) workflow for the quantitative profiling of the phosphometabolome (e.g., sugar phosphates and nucleotides) is presented. The chromatographic separation and MS/MS conditions were optimized, and the precision of repetitive injections and accuracy in terms of error percentage to true concentration were assessed. The precision is excellent for a capillary flow system with an average CV% of 8.5% for a 50-fmol standard injection and in the lower 2.4-4.4% range for higher concentrations (500-7,500 fmol). The limit of detection (LOD) ranges from 1 to 100 nM (5-500 fmol injected on column), and the limit of quantitation (LOQ) ranges from 1 to 500 nM (5-2,500 fmol injected on column). A fast gradient method with the injection of 50% methanol in water between analytical samples is needed to eliminate carry-over and ensure optimal re-equilibration of the column. Finally, the quantitative applicability of the system was tested on real biological matrices using the constant-volume standard addition method (SAM). Extracts of the human kidney Hek293 cell line were spiked with increasing concentrations of standards to determine the concentration of each metabolite in the sample. Forty-four metabolites were detected with an average uncertainty of 4.1%. Thus, the capIC-MS/MS method exhibits excellent selectivity, sensitivity and precision for the quantitative profiling of the phosphometabolome.

  14. A new method for rapid and quantitative detection of the Bacillus cereus emetic toxin cereulide in food products by liquid chromatography-tandem mass spectrometry analysis.

    PubMed

    Yamaguchi, Mizuka; Kawai, Takao; Kitagawa, Mikiya; Kumeda, Yuko

    2013-05-01

    The Bacillus cereus emetic toxin cereulide causes foodborne intoxication, which may occasionally result in severe disease, and even death. To differentially diagnose the emetic-type of foodborne disease caused by B. cereus and assess the safety of commercial food, we developed a rapid method to quantitate cereulide. This method was combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for the extraction of cereulide from food using a normal-phase silica gel cartridge. The limits of detection and quantification were 0.1 and 0.5 ng of cereulide ml(-1), respectively. Spiked cereulide was reproducibly recovered with over 67% efficiency from nine diverse foods implicated in cereulide food poisoning. The recovery rate, reproducibility, and intermediate precision for this single laboratory validation using boiled rice were 87.1%, 4.4%, and 7.0%, respectively. Further, we detected a wide range of cereulide concentrations in leftover food and vomitus samples from two emetic foodborne outbreaks. LC-MS/MS analysis correlated closely with those acquired using the HEp-2 cell assay, and quantitated cereulide from 10 food samples at least five times faster than the bioassay. This new method will provide clinicians with an improved tool for more rapidly and quantitatively determining the presence of cereulide in food and diagnosing food poisoning caused by cereulide.

  15. 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

  16. 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. PMID:26228790

  17. 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.

  18. Development and Validation of a Liquid Chromatography-Tandem Mass Spectrometry Method for the Quantitation of Microcystins in Blue-Green Algal Dietary Supplements.

    PubMed

    Parker, Christine H; Stutts, Whitney L; DeGrasse, Stacey L

    2015-12-01

    A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous detection and quantitation of seven microcystin congeners (1-7) and nodularin-R (8) in blue-green algal dietary supplements. Single-laboratory method validation data were collected in four supplement matrices (capsule, liquid, powder, and tablet) fortified at toxin concentrations from 0.25-2.00 μg/g (ppm). Average recoveries and relative standard deviations (RSD) using matrix-corrected solvent calibration curves were 101% (6% RSD) for all congeners and supplements investigated. Limits of detection (0.006-0.028 μg/g) and quantitation (0.018-0.084 μg/g) were sufficient to confirm the presence of microcystin contamination at the Oregon-mandated guidance concentration of 1.0 μg of microcystin-LReq/g. Quantitated concentrations of microcystin contamination in market-available Aphanizomenon flos-aquae blue-green algal supplements ranged from 0.18-1.87 μg of microcystin-LReq/g for detected congeners microcystin-LR, microcystin-LA, and microcystin-LY (3-5). Microcystin-RR, -YR, -LW, and -LF and nodularin-R (1, 2, and 6-8) were not detected in the supplements examined.

  19. 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.

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

    PubMed Central

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

    2015-01-01

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

  1. Method for Detection and Quantitation of Fathead Minnow Vitellogenin (Vtg) by Liquid Chromatography and Matrix Assisted Laser Desorption/ Ionization Mass Spectrometry

    SciTech Connect

    Wunschel, David S.; Schultz, Irv R.; Skillman, Ann D.; Wahl, Karen L.

    2005-03-11

    Vitellogenin (Vtg) is a well recognized biomarker of estrogen exposure in many species, particularly fish. This large protein shares a high degree of sequence homology across a large number of species. Quantitative measurement is currently done using antibody-based assays. These assays frequently require purification of Vtg and antibody production from each species because there is poor cross reactivity between antibodies for different fish. Therefore, complementary methods of measuring Vtg are desirable. Mass spectrometric (MS) analysis coupled to database searching offers the promise of a general method for protein identification. In this study we used the well characterized Vtg from rainbow trout (O. mykiss) to evaluate the analytical parameters for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of the intact and tryptic digested protein. An analytical scale HPLC separation combined with MALDI-MS was used to measure and confirm the identity of Vtg from the plasma of an important species for regulatory agencies, fathead minnow (Pimephales promelas). The small volume requirement of this method (< 10 uL) was found to be compatible with the plasma volume obtained from a few minnows. A semi quantitative measurement of Vtg from minnows exposed to estradiol was achieved, which was similar to previously obtained ELISA data.

  2. Capture of the volatile carbonyl metabolite of flecainide on 2,4-dinitrophenylhydrazine cartridge for quantitation by stable-isotope dilution mass spectrometry coupled with chromatography.

    PubMed

    Prokai, Laszlo; Szarka, Szabolcs; Wang, Xiaoli; Prokai-Tatrai, Katalin

    2012-04-01

    Carbonyl compounds are common byproducts of many metabolic processes. These volatile chemicals are usually derivatized before mass spectrometric analysis to enhance the sensitivity of their detections. The classically used reagent for this purpose is 2,4-dinitrophenylhydrazine (DNPH) that forms the corresponding hydrazones. When DNPH is immobilized on specific cartridges it permits solvent-free collection and simultaneous derivatization of aldehydes and ketones from gaseous samples. The utility of this approach was tested by assembling a simple apparatus for the in vitro generation of trifluoroacetaldehyde (TFAA) and its subsequent capture on the attached DNPH cartridge. TFAA was generated via cytochrome P450-catalyzed dealkylation of flecainide, an antiarrhythmic agent, in pooled human liver microsomes. Stable-isotope dilution mass spectrometry coupled with GC and LC using negative chemical ionization (NCI) and electrospray ionization (ESI) was evaluated for quantitative analyses. To eliminate isotope effects observed with the use of deuterium-labeled DNPH, we selected its (15)N(4)-labeled analog to synthesize the appropriate TFAA adduct, as internal standard. Quantitation by GC-NCI-MS using selected-ion monitoring outperformed LC-ESI-MS methods considering limits of detection and linearity of the assays. The microsomal metabolism of 1.5 μmol of flecainide for 1.5h resulted in 2.6 ± 0.5 μg TFAA-DNPH, corresponding to 9.3 ± 1.7 nmol TFAA, captured by the cartridge. PMID:22342210

  3. Capture of the volatile carbonyl metabolite of flecainide on 2,4-dinitrophenylhydrazine cartridge for quantitation by stable-isotope dilution mass spectrometry coupled with chromatography

    PubMed Central

    Prokai, Laszlo; Szarka, Szabolcs; Wang, Xiaoli; Prokai-Tatrai, Katalin

    2012-01-01

    Carbonyl compounds are common byproducts of many metabolic processes. These volatile chemical entities are usually derivatized before mass spectrometric analysis to enhance the sensitivity of their detections. The classically used reagent for this purpose is 2,4-dinitrophenylhydrazine (DNPH) that forms the corresponding hydrazones. When DNPH is immobilized on specific cartridges it permits solvent-free collection and simultaneous derivatization of aldehydes and ketones from gaseous samples. The utility of this approach was tested by assembling a simple apparatus for the in vitro generation of trifluoroacetaldehyde (TFAA) and its subsequent capture on the attached DNPH cartridge. TFAA was generated via cytochrome P450-catalyzed dealkylation of flecainide, an antiarrhythmic agent, in pooled human liver microsomes. Stable-isotope dilution mass spectrometry coupled with GC and LC using negative chemical ionization (NCI) and electrospray ionization (ESI) was evaluated for quantitative analyses. To eliminate isotope effects observed with the use of deuterium-labeled DNPH, we selected its 15N4-labeled analog to synthesize the appropriate TFAA adduct, as internal standard. Quantitation by GC–NCI-MS using selected-ion monitoring outperformed LC–ESI-MS methods considering limits of detection and linearity of the assays. The microsomal metabolism of 1.5 μmol of flecainide for 1.5 h resulted in 2.6 ± 0.5 μg TFAA-DNPH, corresponding to 9.3 ± 1.7 nmol TFAA, captured by the cartridge. PMID:22342210

  4. Quantitative determination of trimebutine maleate and its three metabolites in human plasma by liquid chromatography-tandem mass spectrometry.

    PubMed

    Wang, Hongyun; Zhou, Hui; Horimoto, Shingo; Jiang, Ji; Mayumi, Tsuyoshi; Hu, Pei

    2002-11-01

    A sensitive and selective HPLC-MS-MS method was developed for the determination of trimebutine maleate (TM) and its major metabolites N-monodemethyltrimebutine (TM-MPB), N-didemethyltrimebutine (APB) and 3,4,5-trimethoxybenzoic acid (TMBA) in human plasma. The analytes were extracted from plasma samples by liquid-liquid extraction and chromatographed on a YMC J'sphere C(18) column. The mobile phase consisted of 2 mM ammonium acetate buffer (pH 6.5)-methanol (20:80, v/v), and at a flow-rate of 0.2 ml/min. Detection was carried out on a triple quadrupole tandem mass spectrometer in multiple reactions monitoring (MRM) mode using positive-negative switching electrospray ionization (ESI). The method was validated over the concentration range of 1-100 ng/ml for trimebutine maleate and APB, 1-500 ng/ml for MPB, and 50-10,000 ng/ml for TMBA. Inter- and intra-day precision (RSD%) for trimebutine maleate and its three metabolites were all within +/-15% and the accuracy was within 85-115%. The limit of quantitation was 1 ng/ml for trimebutine maleate, TM-MPB and APB, and 50 ng/ml for TMBA. The extraction recovery was on average 58.2% for trimebutine maleate, 69.6% for MPB, 51.2% for APB and 62.5% for TMBA. The method was applied to the pharmacokinetic study of trimebutine maleate and its metabolites in healthy Chinese volunteers. PMID:12361732

  5. 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.

  6. 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.

  7. Quantitative Imaging of Chemical Composition in Single Cells by Secondary Ion Mass Spectrometry: Cisplatin Affects Calcium Stores in Renal Epithelial Cells

    PubMed Central

    Chandra, Subhash

    2010-01-01

    A detailed protocol for quantitative single cell mass spectrometry imaging (MSI) analysis is described in this chapter with examples of the treatment of cells with anticancer drug, cisplatin. Cisplatin, cis-diamminedichloridoplatinum (ii) (CDDP), is widely used for the treatment of many malignancies, including testicular, ovarian, bladder, cervical, head and neck, and small cell and non-small cell lung cancers. The possibility of renal injury by cisplatin treatment is a major dose-limiting factor in this cancer therapy. At present, the mechanisms of cisplatin-induced renal cytotoxicity are poorly understood. In this work, secondary ion mass spectrometry (SIMS) was used for investigating cisplatin-induced alterations in intracellular chemical composition in a well established model (LLC-PK1 cell line) for studying renal injury. The cells were cryogenically prepared by the sandwich freeze-fracture method for subcellular imaging analysis of chemical composition (total concentrations of K+, Na+ and Ca2+) in individual cells. The single cell analysis of these diffusible ions necessitates the use of reliable cryogenic sample preparations for SIMS. The sandwich freeze-fracture method offers a simple approach for cryogenically preserving diffusible ions and molecules inside the cells for SIMS analysis. A CAMECA IMS 3f SIMS ion microscope instrument capable of producing chemical images of single cells with 500 nm spatial resolution was used in the study. In cisplatin treated cells, SIMS imaging showed the presence of detectable amount of platinum at mass 195, as 195Pt+ secondary ions in individual cells. SIMS observations also revealed that individual cells differed in their response to cisplatin. While the chemical composition of some cells was unaffected by cisplatin, others showed a reduction in cytoplasmic calcium stores that was not associated with changes in their intracellular K or Na concentrations. Another population of cells displayed an increased in cytoplasmic

  8. [Rapid screening and quantitative detection of 11 illegally added antidiabetics in health care products by ultra performance liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry].

    PubMed

    Du, Yanshan; Li, Qiang; Wu, Chunmin; Zhang, Yan

    2015-04-01

    A method for rapid screening and quantification of 11 antidiabetics (nateglinide, pioglitazone hydrochloride, gliquidone, gliclazide, glipizide, glibenclamide, metformin hydrochloride, repaglinide, phenformin hydrochloride, rosiglitazone hydrochloride, glimepiride) illegally added in health care products by ultra performance liquid chromatography (UPLC)-quadrupole/ electrostatic field orbitrap mass spectrometry was established. The samples were extracted with methanol, and separated on an Agilent Poroshell 120 SB-C18 column (100 mm x 4.6 mm, 2.7 µm) with acetonitrile-10 mmol/L ammonium acetate solution as mobile phases by gradient elution. The positive mode was used in the MS detection. The resolution of the precursor mass was 70,000, while the resolution of the product mass was 17,500. The results indicated that the linearity of all the 11 antidiabetics ranged from 0.005 mg/L to 0.5 mg/L with the correlation coefficients greater than 0.99. The limits of detection were confirmed by spiked samples, and were between 2.7 and 5.1 µg/kg for the 11 antidiabetics. The recoveries were in the range of 87.3% to 98.3%, with the relative standard deviations in the range of 2.18%-5.21%. This method is accurate, simple and rapid, and can be used in rapid screening and quantitative analysis of the 11 illegally added antidiabetics in health care products. PMID:26292406

  9. 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.

  10. Mass Spectrometry Based Identification of Geometric Isomers during Metabolic Stability Study of a New Cytotoxic Sulfonamide Derivatives Supported by Quantitative Structure-Retention Relationships

    PubMed Central

    Belka, Mariusz; Hewelt-Belka, Weronika; Sławiński, Jarosław; Bączek, Tomasz

    2014-01-01

    A set of 15 new sulphonamide derivatives, presenting antitumor activity have been subjected to a metabolic stability study. The results showed that besides products of biotransformation, some additional peaks occurred in chromatograms. Tandem mass spectrometry revealed the same mass and fragmentation pathway, suggesting that geometric isomerization occurred. Thus, to support this hypothesis, quantitative structure-retention relationships were applied. Human liver microsomes were used as an in vitro model of metabolism. The biotransformation reactions were tracked by liquid chromatography assay and additionally, fragmentation mass spectra were recorded. In silico molecular modeling at a semi-empirical level was conducted as a starting point for molecular descriptor calculations. A quantitative structure-retention relationship model was built applying multiple linear regression based on selected three-dimensional descriptors. The studied compounds revealed high metabolic stability, with a tendency to form hydroxylated biotransformation products. However, significant chemical instability in conditions simulating human body fluids was noticed. According to literature and MS data geometrical isomerization was suggested. The developed in sillico model was able to describe the relationship between the geometry of isomer pairs and their chromatographic retention properties, thus it supported the hypothesis that the observed pairs of peaks are most likely geometric isomers. However, extensive structural investigations are needed to fully identify isomers’ geometry. An effort to describe MS fragmentation pathways of novel chemical structures is often not enough to propose structures of potent metabolites and products of other chemical reactions that can be observed in compound solutions at early drug discovery studies. The results indicate that the relatively non-expensive and not time- and labor-consuming in sillico approach could be a good supportive tool assisting the

  11. 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.

  12. 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.

  13. 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).

  14. 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.

  15. Automation of dimethylation after guanidination labeling chemistry and its compatibility with common buffers and surfactants for mass spectrometry-based shotgun quantitative proteome analysis.

    PubMed

    Lo, Andy; Tang, Yanan; Chen, Lu; Li, Liang

    2013-07-25

    Isotope labeling liquid chromatography-mass spectrometry (LC-MS) is a major analytical platform for quantitative proteome analysis. Incorporation of isotopes used to distinguish samples plays a critical role in the success of this strategy. In this work, we optimized and automated a chemical derivatization protocol (dimethylation after guanidination, 2MEGA) to increase the labeling reproducibility and reduce human intervention. We also evaluated the reagent compatibility of this protocol to handle biological samples in different types of buffers and surfactants. A commercially available liquid handler was used for reagent dispensation to minimize analyst intervention and at least twenty protein digest samples could be prepared in a single run. Different front-end sample preparation methods for protein solubilization (SDS, urea, Rapigest™, and ProteaseMAX™) and two commercially available cell lysis buffers were evaluated for compatibility with the automated protocol. It was found that better than 94% desired labeling could be obtained in all conditions studied except urea, where the rate was reduced to about 92% due to carbamylation on the peptide amines. This work illustrates the automated 2MEGA labeling process can be used to handle a wide range of protein samples containing various reagents that are often encountered in protein sample preparation for quantitative proteome analysis.

  16. Development of a multi-variate calibration approach for quantitative analysis of oxidation resistant Mo-Si-B coatings using laser ablation inductively coupled plasma mass spectrometry

    NASA Astrophysics Data System (ADS)

    Cakara, Anja; Bonta, Maximilian; Riedl, Helmut; Mayrhofer, Paul H.; Limbeck, Andreas

    2016-06-01

    Nowadays, for the production of oxidation protection coatings in ultrahigh temperature environments, alloys of Mo-Si-B are employed. The properties of the material, mainly the oxidation resistance, are strongly influenced by the Si to B ratio; thus reliable analytical methods are needed to assure exact determination of the material composition for the respective applications. For analysis of such coatings, laser ablation inductively coupled mass spectrometry (LA-ICP-MS) has been reported as a versatile method with no specific requirements on the nature of the sample. However, matrix effects represent the main limitation of laser-based solid sampling techniques and usually the use of matrix-matched standards for quantitative analysis is required. In this work, LA-ICP-MS analysis of samples with known composition and varying Mo, Si and B content was carried out. Between known analyte concentrations and derived LA-ICP-MS signal intensities no linear correlation could be found. In order to allow quantitative analysis independent of matrix effects, a multiple linear regression model was developed. Besides the three target analytes also the signals of possible argides (40Ar36Ar and 98Mo40Ar) as well as detected impurities of the Mo-Si-B coatings (108Pd) were considered. Applicability of the model to unknown samples was confirmed using external validation. Relative deviations from the values determined using conventional liquid analysis after sample digestion between 5 and 10% for the main components Mo and Si were observed.

  17. 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.

  18. Quantitative measurement of intact alpha-synuclein proteoforms from post-mortem control and Parkinson's disease brain tissue by intact protein mass spectrometry.

    PubMed

    Kellie, John F; Higgs, Richard E; Ryder, John W; Major, Anthony; Beach, Thomas G; Adler, Charles H; Merchant, Kalpana; Knierman, Michael D

    2014-01-01

    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). PMID:25052239

  19. 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

    2013-09-01

    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. PMID:23837631

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. Quantitative isomer-specific N-glycan fingerprinting using isotope coded labeling and high performance liquid chromatography-electrospray ionization-mass spectrometry with graphitic carbon stationary phase.

    PubMed

    Michael, Claudia; Rizzi, Andreas M

    2015-02-27

    Glycan reductive isotope labeling (GRIL) using (12)C6-/(13)C6-aniline as labeling reagent is reported with the aim of quantitative N-glycan fingerprinting. Porous graphitized carbon (PGC) as stationary phase in capillary scale HPLC coupled to electrospray mass spectrometry with time of flight analyzer was applied for the determination of labeled N-glycans released from glycoproteins. The main benefit of using stable isotope-coding in the context of comparative glycomics lies in the improved accuracy and precision of the quantitative analysis in combined samples and in the potential of correcting for structure-dependent incomplete enzymatic release of oligosaccharides when comparing identical target proteins. The method was validated with respect to mobile phase parameters, reproducibility, accuracy, linearity and limit of detection/quantification (LOD/LOQ) using test glycoproteins. It is shown that the developed method is capable of determining relative amounts of N-glycans (including isomers) comparing two samples in one single HPLC-MS run. The analytical potential and usefulness of GRIL in combination with PGC-ESI-TOF-MS is demonstrated comparing glycosylation in human monoclonal antibodies produced in Chinese hamster ovary cells (CHO) and hybridoma cell lines.

  5. Distinct energy metabolism of auditory and vestibular sensory epithelia revealed by quantitative mass spectrometry using MS2 intensity.

    PubMed

    Spinelli, Kateri J; Klimek, John E; Wilmarth, Phillip A; Shin, Jung-Bum; Choi, Dongseok; David, Larry L; Gillespie, Peter G

    2012-01-31

    Measuring the abundance of many proteins over a broad dynamic range requires accurate quantitation. We show empirically that, in MS experiments, relative quantitation using summed dissociation-product ion-current intensities is accurate, albeit variable from protein to protein, and outperforms spectral counting. By applying intensities to quantify proteins in two complex but related tissues, chick auditory and vestibular sensory epithelia, we find that glycolytic enzymes are enriched threefold in auditory epithelia, whereas enzymes responsible for oxidative phosphorylation are increased at least fourfold in vestibular epithelia. This striking difference in relative use of the two ATP-production pathways likely reflects the isolation of the auditory epithelium from its blood supply, necessary to prevent heartbeat-induced mechanical disruptions. The global view of protein expression afforded by label-free quantitation with a wide dynamic range reveals molecular specialization at a tissue or cellular level.

  6. 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...

  7. A New Strategy of Using O18-Labeled Iodoacetic Acid for Mass Spectrometry-Based Protein Quantitation

    NASA Astrophysics Data System (ADS)

    Wang, Shunhai; Kaltashov, Igor A.

    2012-07-01

    A new O18 labeling protocol is designed to assist quantitation of cysteine-containing proteins using LC/MS. Unlike other O18 labeling strategies, the labeling is carried out at the intact protein level (prior to its digestion) during reduction/alkylation of cysteine side chains using O18-labeled iodoacetic acid (IAA). The latter can be easily prepared by exchanging carboxylic oxygen atoms of commercially available IAA in O18-enriched water at low pH. Since incorporation of the O18 label in the protein occurs at the whole protein, rather than peptide level, the quantitation results are not peptide-dependent. The excellent stability of the label in mild pH conditions provides flexibility and robustness needed of sample processing steps following the labeling. In contrast to generally costly isotope labeling reagents, this approach uses only two relatively inexpensive commercially available reagents (IAA and H2O18). The feasibility of the new method is demonstrated using an 80 kDa human serum transferrin (hTf) as a model, where linear quantitation is achieved across a dynamic range spanning three orders of magnitude. The new approach can be used in quantitative proteomics applications and is particularly suitable for a variety of tasks in the biopharmaceutical sector, ranging from pharmacokinetic studies to quality control of protein therapeutics.

  8. Digital Imaging Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Bamberger, Casimir; Renz, Uwe; Bamberger, Andreas

    2011-06-01

    Methods to visualize the two-dimensional (2D) distribution of molecules by mass spectrometric imaging evolve rapidly and yield novel applications in biology, medicine, and material surface sciences. Most mass spectrometric imagers acquire high mass resolution spectra spot-by-spot and thereby scan the object's surface. Thus, imaging is slow and image reconstruction remains cumbersome. Here we describe an imaging mass spectrometer that exploits the true imaging capabilities by ion optical means for the time of flight mass separation. The mass spectrometer is equipped with the ASIC Timepix chip as an array detector to acquire the position, mass, and intensity of ions that are imaged by matrix-assisted laser desorption/ionization (MALDI) directly from the target sample onto the detector. This imaging mass spectrometer has a spatial resolving power at the specimen of (84 ± 35) μm with a mass resolution of 45 and locates atoms or organic compounds on a surface area up to ~2 cm2. Extended laser spots of ~5 mm2 on structured specimens allows parallel imaging of selected masses. The digital imaging mass spectrometer proves high hit-multiplicity, straightforward image reconstruction, and potential for high-speed readout at 4 kHz or more. This device demonstrates a simple way of true image acquisition like a digital photographic camera. The technology may enable a fast analysis of biomolecular samples in near future.

  9. Quantitative analysis of poly- and perfluoroalkyl compounds in water matrices using high resolution mass spectrometry: optimization for a laser diode thermal desorption method.

    PubMed

    Munoz, Gabriel; Vo Duy, Sung; Budzinski, Hélène; Labadie, Pierre; Liu, Jinxia; Sauvé, Sébastien

    2015-06-30

    An alternative analysis technique for the quantitation of 15 poly- and perfluoroalkyl substances (PFASs) in water matrices is reported. Analysis time between each sample was reduced to less than 20s, all target molecules being analyzed in a single run with the use of laser diode thermal desorption atmospheric pressure chemical ionization (LDTD/APCI) coupled with high resolution accurate mass (HRMS) orbitrap mass spectrometry. LDTD optimal settings were investigated using either one-factor-at-a-time or experimental design methodologies, while orbitrap parameters were optimized simultaneously by means of a Box-Behnken design. Following selection of an adequate sample concentration and purification procedure based on solid-phase extraction and graphite clean-up, the method was validated in an influent wastewater matrix. Environmentally significant limits of detection were reported (0.3-4ngL(-1) in wastewater and 0.03-0.2ngL(-1) in surface water) and out of the 15 target analytes, 11 showed excellent accuracies (±20% of the target values) and recovery rates (75-125%). The method was successfully applied to a selection of environmental samples, including wastewater samples in 7 locations across Canada, as well as surface and tap water samples from the Montreal region, providing insights into the degree of PFAS contamination in this area. PMID:26041525

  10. Rapid environmental organic analysis by direct sampling Glow Discharge Mass Spectrometry and Ion Trap Mass Spectrometry: Summary of pilot studies

    SciTech Connect

    Wise, M.B.; Buchanan, M.V.; Guerin, M.R.

    1990-03-01

    Direct Sampling Mass Spectrometry (DSMS) techniques employing both Glow Discharge Mass Spectrometry and Ion Trap Mass Spectrometry are being developed to quantitatively determine preselected organics in water, soil, and air samples at part per billion levels in less than five minutes. Direct sampling requires little or no sample preparation and no prior chromatographic separation and is applicable to both volatile and semivolatile organics. 25 figs., 3 tabs.

  11. 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

  12. 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.

  13. 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

    2016-06-01

    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. PMID:27131804

  14. 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).

  15. 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

    2016-06-01

    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.

  16. 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). PMID:24548435

  17. 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.

  18. 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

  19. Statistical Model to Analyze Quantitative Proteomics Data Obtained by 18O/16O Labeling and Linear Ion Trap Mass Spectrometry

    PubMed Central

    Jorge, Inmaculada; Navarro, Pedro; Martínez-Acedo, Pablo; Núñez, Estefanía; Serrano, Horacio; Alfranca, Arántzazu; Redondo, Juan Miguel; Vázquez, Jesús

    2009-01-01

    Statistical models for the analysis of protein expression changes by stable isotope labeling are still poorly developed, particularly for data obtained by 16O/18O labeling. Besides large scale test experiments to validate the null hypothesis are lacking. Although the study of mechanisms underlying biological actions promoted by vascular endothelial growth factor (VEGF) on endothelial cells is of considerable interest, quantitative proteomics studies on this subject are scarce and have been performed after exposing cells to the factor for long periods of time. In this work we present the largest quantitative proteomics study to date on the short term effects of VEGF on human umbilical vein endothelial cells by 18O/16O labeling. Current statistical models based on normality and variance homogeneity were found unsuitable to describe the null hypothesis in a large scale test experiment performed on these cells, producing false expression changes. A random effects model was developed including four different sources of variance at the spectrum-fitting, scan, peptide, and protein levels. With the new model the number of outliers at scan and peptide levels was negligible in three large scale experiments, and only one false protein expression change was observed in the test experiment among more than 1000 proteins. The new model allowed the detection of significant protein expression changes upon VEGF stimulation for 4 and 8 h. The consistency of the changes observed at 4 h was confirmed by a replica at a smaller scale and further validated by Western blot analysis of some proteins. Most of the observed changes have not been described previously and are consistent with a pattern of protein expression that dynamically changes over time following the evolution of the angiogenic response. With this statistical model the 18O labeling approach emerges as a very promising and robust alternative to perform quantitative proteomics studies at a depth of several thousand proteins

  20. A validated UHPLC-tandem mass spectrometry method for quantitative analysis of flavonolignans in milk thistle (Silybum marianum) extracts.

    PubMed

    Graf, Tyler N; Cech, Nadja B; Polyak, Stephen J; Oberlies, Nicholas H

    2016-07-15

    Validated methods are needed for the analysis of natural product secondary metabolites. These methods are particularly important to translate in vitro observations to in vivo studies. Herein, a method is reported for the analysis of the key secondary metabolites, a series of flavonolignans and a flavonoid, from an extract prepared from the seeds of milk thistle [Silybum marianum (L.) Gaertn. (Asteraceae)]. This report represents the first UHPLC MS-MS method validated for quantitative analysis of these compounds. The method takes advantage of the excellent resolution achievable with UHPLC to provide a complete analysis in less than 7min. The method is validated using both UV and MS detectors, making it applicable in laboratories with different types of analytical instrumentation available. Lower limits of quantitation achieved with this method range from 0.0400μM to 0.160μM with UV and from 0.0800μM to 0.160μM with MS. The new method is employed to evaluate variability in constituent composition in various commercial S. marianum extracts, and to show that storage of the milk thistle compounds in DMSO leads to degradation.

  1. Mass spectrometry for biomarker development

    SciTech Connect

    Wu, Chaochao; Liu, Tao; Baker, Erin Shammel; Rodland, Karin D.; Smith, Richard D.

    2015-06-19

    Biomarkers potentially play a crucial role in early disease diagnosis, prognosis and targeted therapy. In the past decade, mass spectrometry based proteomics has become increasingly important in biomarker development due to large advances in technology and associated methods. This chapter mainly focuses on the application of broad (e.g. shotgun) proteomics in biomarker discovery and the utility of targeted proteomics in biomarker verification and validation. A range of mass spectrometry methodologies are discussed emphasizing their efficacy in the different stages in biomarker development, with a particular emphasis on blood biomarker development.

  2. Quantitation of orotic acid in urine using isotope dilution-selected ion gas chromatography-mass spectrometry.

    PubMed

    Chen, Jie; Bennett, Michael J

    2010-01-01

    The measurement of urinary orotic acid excretion is an important test for establishing a diagnosis of hereditary orotic aciduria, a genetic defect of pyrimidine biosynthesis. Measurement of secondary urinary orotic acid elevation is also an important clinical test for the differential diagnosis of hyperammonemia due to some of the primary disorders of the urea cycle including ornithine transcarbamylase (OTC) deficiency, and the hyperornithinemia-hyperammonemia-homocitrullinemia (HHH) syndrome. Low levels of orotic acid are observed in carbamylphosphate synthetase (CPS) defects. This method utilizes a stable-isotope labeled internal standard (1, 3-(15)N-orotic acid), which is added to the standards, controls, and patient samples prior to extraction. Interference from urea is removed by incubation of samples with urease and the orotic acid is derivatized by trimethylsilylation. Quantitation is made against an eight-point standard curve using specific selected ions from both the labeled and unlabeled orotic acid. PMID:20077096

  3. The interplay of light and oxygen in the reactive oxygen stress response of Chlamydomonas reinhardtii dissected by quantitative mass spectrometry.

    PubMed

    Barth, Johannes; Bergner, Sonja Verena; Jaeger, Daniel; Niehues, Anna; Schulze, Stefan; Scholz, Martin; Fufezan, Christian

    2014-04-01

    Light and oxygen are factors that are very much entangled in the reactive oxygen species (ROS) stress response network in plants, algae and cyanobacteria. The first obligatory step in understanding the ROS network is to separate these responses. In this study, a LC-MS/MS based quantitative proteomic approach was used to dissect the responses of Chlamydomonas reinhardtii to ROS, light and oxygen employing an interlinked experimental setup. Application of novel bioinformatics tools allow high quality retention time alignment to be performed on all LC-MS/MS runs increasing confidence in protein quantification, overall sequence coverage and coverage of all treatments measured. Finally advanced hierarchical clustering yielded 30 communities of co-regulated proteins permitting separation of ROS related effects from pure light effects (induction and repression). A community termed redox(II) was identified that shows additive effects of light and oxygen with light as the first obligatory step. Another community termed 4-down was identified that shows repression as an effect of light but only in the absence of oxygen indicating ROS regulation, for example, possibly via product feedback inhibition because no ROS damage is occurring. In summary the data demonstrate the importance of separating light, O₂ and ROS responses to define marker genes for ROS responses. As revealed in this study, an excellent candidate is DHAR with strong ROS dependent induction profiles.

  4. 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%.

  5. 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

    2013-08-01

    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). PMID:23841685

  6. 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

  7. 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.

  8. 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.

  9. A multiplexed targeted assay for high-throughput quantitative analysis of serum methylamines by ultra performance liquid chromatography coupled to high resolution mass spectrometry.

    PubMed

    Kadar, Hanane; Dubus, Justine; Dutot, Jérémie; Hedjazi, Lyamine; Srinivasa, Suman; Fitch, Kathleen V; Grinspoon, Steven K; Nicholson, Jeremy K; Dumas, Marc-Emmanuel; Gauguier, Dominique

    2016-05-01

    Methylamines are biologically-active metabolites present in serum and urine samples, which play complex roles in metabolic diseases. Methylamines can be detected by proton nuclear magnetic resonance (NMR), but specific methods remain to be developed for their routine assay in human serum in clinical settings. Here we developed and validated a novel reliable "methylamine panel" method for simultaneous quantitative analysis of trimethylamine (TMA), its major detoxification metabolite trimethylamine-N-oxide (TMAO), and precursors choline, betaine and l-carnitine in human serum using Ultra Performance Liquid Chromatography (UPLC) coupled to High Resolution Mass Spectrometry (HRMS). Metabolite separation was carried out on a HILIC stationary phase. For all metabolites, the assay was linear in the range of 0.25-12.5 μmol/L and enabled to reach limit of detection of about 0.10 μmol/L. Relative standard deviations were below 16% for the three levels of concentrations. We demonstrated the strong reliability and robustness of the method, which was applied to serum samples from healthy individuals to establish the range of concentrations of the metabolites and their correlation relationships and detect gender differences. Our data provide original information for implementing in a clinical environment a MS-based diagnostic method with potential for targeted metabolic screening of patients at risk of cardiometabolic diseases.

  10. Quantitative analysis of biologically active ingredients of Five Seeds Combo by liquid chromatography-quadrupole time-of-flight mass spectrometry for quality control of commercial herbal product.

    PubMed

    Chen, Meng-Li; Miao, Lan; Cao, Jin; Ip, Siu-Po; Che, Chun-Tao

    2012-07-01

    Five Seeds Combo (wu zi yan zong wan) is a traditional Chinese herbal formula composed of fructus Lycii, semen Cuscutae, fructus Rubi, semen Plantaginis, and fructus Schisandrae. This herbal prescription has been developed into herbal products by many pharmaceutical manufacturers for treating age-related symptoms. The present study aims to develop an analytical method for the quality control of this herbal drug. Nine active ingredients including schisantherin A, schisandrin B, schisandrin, schisandrin A, quercitrin, betaine, verbascoside, hyperoside, and kaempferol were selected as the targeted analytes for the analysis. By using liquid chromatogram/quadrupole time-of-flight mass spectrometry (MS), the nine chemical compounds were determined simultaneously from the chromatogram. The parameters for MS were optimized by orthogonal array testing and the best condition of the MS for the determination of the nine marker compounds was found to be 175, 75, and 700 V for fragmentor, skimmer, and voltage of capillary, respectively. The method validation showed that this analytical method had high precision and sensitivity (limit of quantitation was smaller than 10 ng/mL for most of the analytes). The method was found to be able to demonstrate the quality of Five Seeds Combo from different manufacturers. PMID:22761139

  11. A multiplexed targeted assay for high-throughput quantitative analysis of serum methylamines by ultra performance liquid chromatography coupled to high resolution mass spectrometry.

    PubMed

    Kadar, Hanane; Dubus, Justine; Dutot, Jérémie; Hedjazi, Lyamine; Srinivasa, Suman; Fitch, Kathleen V; Grinspoon, Steven K; Nicholson, Jeremy K; Dumas, Marc-Emmanuel; Gauguier, Dominique

    2016-05-01

    Methylamines are biologically-active metabolites present in serum and urine samples, which play complex roles in metabolic diseases. Methylamines can be detected by proton nuclear magnetic resonance (NMR), but specific methods remain to be developed for their routine assay in human serum in clinical settings. Here we developed and validated a novel reliable "methylamine panel" method for simultaneous quantitative analysis of trimethylamine (TMA), its major detoxification metabolite trimethylamine-N-oxide (TMAO), and precursors choline, betaine and l-carnitine in human serum using Ultra Performance Liquid Chromatography (UPLC) coupled to High Resolution Mass Spectrometry (HRMS). Metabolite separation was carried out on a HILIC stationary phase. For all metabolites, the assay was linear in the range of 0.25-12.5 μmol/L and enabled to reach limit of detection of about 0.10 μmol/L. Relative standard deviations were below 16% for the three levels of concentrations. We demonstrated the strong reliability and robustness of the method, which was applied to serum samples from healthy individuals to establish the range of concentrations of the metabolites and their correlation relationships and detect gender differences. Our data provide original information for implementing in a clinical environment a MS-based diagnostic method with potential for targeted metabolic screening of patients at risk of cardiometabolic diseases. PMID:27036856

  12. 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). PMID:23106487

  13. Development and Validation of an Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Method for Quantitative Analysis of Platinum in Plasma, Urine, and Tissues.

    PubMed

    Zhang, Ti; Cai, Shuang; Forrest, Wai Chee; Mohr, Eva; Yang, Qiuhong; Forrest, M Laird

    2016-09-01

    Cisplatin, a platinum chemotherapeutic, is one of the most commonly used chemotherapeutic agents for many solid tumors. In this work, we developed and validated an inductively coupled plasma mass spectrometry (ICP-MS) method for quantitative determination of platinum levels in rat urine, plasma, and tissue matrices including liver, brain, lungs, kidney, muscle, heart, spleen, bladder, and lymph nodes. The tissues were processed using a microwave accelerated reaction system (MARS) system prior to analysis on an Agilent 7500 ICP-MS. According to the Food and Drug Administration guidance for industry, bioanalytical validation parameters of the method, such as selectivity, accuracy, precision, recovery, and stability were evaluated in rat biological samples. Our data suggested that the method was selective for platinum without interferences caused by other presenting elements, and the lower limit of quantification was 0.5 ppb. The accuracy and precision of the method were within 15% variation and the recoveries of platinum for all tissue matrices examined were determined to be 85-115% of the theoretical values. The stability of the platinum-containing solutions, including calibration standards, stock solutions, and processed samples in rat biological matrices was investigated. Results indicated that the samples were stable after three cycles of freeze-thaw and for up to three months. PMID:27527103

  14. A novel quantitation approach for maximizing detectable targets for offensive/volatile odorants with diverse functional groups by thermal desorption-gas chromatography-mass spectrometry.

    PubMed

    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

  15. 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.

  16. 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.

  17. 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

  18. Qualitative and Quantitative Drug residue analyses: Florfenicol in white-tailed deer (Odocoileus virginianus) and supermarket meat by liquid chromatography tandem-mass spectrometry.

    PubMed

    Anderson, Shanoy C; Subbiah, Seenivasan; Gentles, Angella; Austin, Galen; Stonum, Paul; Brooks, Tiffanie A; Brooks, Chance; Smith, Ernest E

    2016-10-15

    A method for confirmation and detection of Florfenicol amine residues in white-tailed deer tissues was developed and validated in our laboratory. Tissue samples were extracted with ethyl acetate and cleaned up on sorbent (Chem-elut) cartridges. Liguid chromatography (LC) separation was achieved on a Zorbax Eclipse plus C18 column with gradient elution using a mobile phase composed of ammonium acetate in water and methanol at a flow rate of 300μL/min. Qualitative and quantitative analyses were carried out using liquid chromatography - heated electrospray ionization(HESI) and atmospheric pressure chemical ionization (APCI)-tandem mass spectrometry in the multiple reaction monitoring (MRM) interface. The limits of detection (LODs) for HESI and APCI probe were 1.8ng/g and 1.4ng/g respectively. Limits of quantitation (LOQs) for HESI and APCI probe were 5.8ng/g and 3.4ng/g respectively. Mean recovery values ranged from 79% to 111% for APCI and 30% to 60% for HESI. The validated method was used to determine white-tailed deer florfenicol tissue residue concentration 10-days after exposure. Florfenicol tissue residues concentration ranged from 0.4 to 0.6μg/g for liver and 0.02-0.05μg/g for muscle and a trace in blood samples. The concentration found in the tested edible tissues were lower than the maximum residual limit (MRL) values established by the federal drug administration (FDA) for bovine tissues. In summary, the resulting optimization procedures using the sensitivity of HESI and APCI probes in the determination of florfenicol in white-tailed deer tissue are the most compelling conclusions in this study, to the extent that we have applied this method in the evaluation of supermarket samples drug residue levels as a proof of principle.

  19. Quantitative determination of zidovudine diaryl phosphate triester pro-drugs in rat plasma by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.

    PubMed

    Zhu, Meixiang; Xie, Baoying; Tang, Guo; Hu, Anfu; Fang, Meijuan; Wu, Zhen; Zhao, Yufen

    2008-12-15

    A rapid, simple, and sensitive high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) method was developed and validated for quantitative analysis of 3'-azido-3'-deoxythymidine (zidovudine, AZT) diaryl phosphate triester pro-drugs, in rat plasma using 2',3'-dideoxy-2'3'-didehydrothymidine (d4T) as internal standard (IS). The analytes were extracted from rat plasma with methanol after protein precipitation. The compounds were separated by HPLC with gradient elution (on a Shim-pack VP-ODS C(18) analytical column using a mobile phase of methanol/10mM ammonium acetate). All the analytes were detected in positive ion mode using multiple reaction monitoring (MRM). The method was validated and the specificity, linearity, lower limit of quantitation (LLOQ), precision, accuracy, recoveries and stability were determined. LLOQs were 10 ng mL(-1) for M1, M2, M3, M4, and M5, respectively. Correlation coefficient (r) values for the linear range of 10-10,000 ng mL(-1) were greater than 0.999 for all the analytes. The intra-day and inter-day precision and accuracy were higher than 7.13%. The relative and absolute recovery was above 72% and no matrix effects were observed for all the analytes. This validated method provides a modern, rapid, and robust procedure for the pharmacokinetic studies of the pro-drugs after intravenous administration to rats. Some important results of AZT diaryl phosphate triester pro-drugs concerning chemical effect on pharmacokinetic performance are also studied. PMID:19118970

  20. Qualitative and Quantitative Drug residue analyses: Florfenicol in white-tailed deer (Odocoileus virginianus) and supermarket meat by liquid chromatography tandem-mass spectrometry.

    PubMed

    Anderson, Shanoy C; Subbiah, Seenivasan; Gentles, Angella; Austin, Galen; Stonum, Paul; Brooks, Tiffanie A; Brooks, Chance; Smith, Ernest E

    2016-10-15

    A method for confirmation and detection of Florfenicol amine residues in white-tailed deer tissues was developed and validated in our laboratory. Tissue samples were extracted with ethyl acetate and cleaned up on sorbent (Chem-elut) cartridges. Liguid chromatography (LC) separation was achieved on a Zorbax Eclipse plus C18 column with gradient elution using a mobile phase composed of ammonium acetate in water and methanol at a flow rate of 300μL/min. Qualitative and quantitative analyses were carried out using liquid chromatography - heated electrospray ionization(HESI) and atmospheric pressure chemical ionization (APCI)-tandem mass spectrometry in the multiple reaction monitoring (MRM) interface. The limits of detection (LODs) for HESI and APCI probe were 1.8ng/g and 1.4ng/g respectively. Limits of quantitation (LOQs) for HESI and APCI probe were 5.8ng/g and 3.4ng/g respectively. Mean recovery values ranged from 79% to 111% for APCI and 30% to 60% for HESI. The validated method was used to determine white-tailed deer florfenicol tissue residue concentration 10-days after exposure. Florfenicol tissue residues concentration ranged from 0.4 to 0.6μg/g for liver and 0.02-0.05μg/g for muscle and a trace in blood samples. The concentration found in the tested edible tissues were lower than the maximum residual limit (MRL) values established by the federal drug administration (FDA) for bovine tissues. In summary, the resulting optimization procedures using the sensitivity of HESI and APCI probes in the determination of florfenicol in white-tailed deer tissue are the most compelling conclusions in this study, to the extent that we have applied this method in the evaluation of supermarket samples drug residue levels as a proof of principle. PMID:27529828

  1. 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.

  2. Qualitative and quantitative determination of 15 main active constituents in Fructus Sophorae pill by liquid chromatography tandem mass spectrometry

    PubMed Central

    Zhi, Xu-ran; Zhang, Zhi-yong; Jia, Pei-pei; Zhang, Xiao-xu; Yuan, Lin; Sheng, Ning; Zhang, Lan-tong

    2015-01-01

    Background: Fructus Sophorae pill, one of the traditional Chinese medicine, was widely used for hemorrhoids, hypertension and odontalgia. This paper describes a sensitive and specific assay for the determination of the 15 active constituents (sophoricoside, genistin, genistein, rutin, quercetin, kaempferol, baicalein, baicalin, naringin, naringenin, hesperidin, neohesperidin, wogonin and cimifugin, prim-O-glucosylcimifugin) in Fructus Sophorae pill. Materials and Methods: Chromatographic separation was performed on a C18 column with acidified aqueous methanol gradients at a flow rate of 0.8 mL/min. The identification and quantification of the analytes were achieved by use of a hybrid quadrupole linear ion trap mass spectrometer. Multiple-reaction monitoring scanning was applied to quantification with switching electrospray ion source polarity between positive and negative modes. Results: The proposed method was used to analyze 40 batches of samples with good linearity (r, 0.9990-0.9999), intraday precisions (RSD, 0.14-2.55%), interday precisions (RSD, 0.51-2.81%), stability (RSD, 0.31-2.65%), and recovery (RSD, 1.29-2.95%) of the 15 compounds. In addition, the hierarchical cluster analysis, including a method called furthest neighbor and nearest neighbor, was employed to classify samples according to characteristics of the 15 constituents. Conclusion: The results indicated that the analytical method was rapid, reliable, simple and suitable for the quality evaluation of Fructus Sophorae pill. PMID:25709233

  3. Quantitation of the Oral Anticoagulants Dabigatran, Rivaroxaban, Apixaban, and Warfarin in Plasma Using Ultra-Performance Liquid Chromatography with Tandem Mass Spectrometry (UPLC-MS/MS).

    PubMed

    Noguez, Jaime H; Ritchie, James C

    2016-01-01

    This chapter describes a method to measure the oral anticoagulants dabigatran, rivaroxaban, apixaban, and warfarin in plasma samples using ultra-performance liquid chromatography combined with tandem mass spectrometry (UPLC-MS/MS). The instrument is operated in multiple reaction monitoring (MRM) mode with an electrospray ionization (ESI) source in positive ionization mode. Samples are extracted with a 90:10 methanol/0.1 N hydrochloric acid solution containing stable isotope-labeled internal standards for each analyte. After centrifugation the supernatant is transferred to a mass spectrometry vial, injected onto the UPLC-ESI-MS/MS, and quantified using an eight-point calibration curve.

  4. 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

  5. [Determination of the interaction kinetics between meloxicam and β-cyclodextrin using the quantitative high-performance affinity chromatography coupled with mass spectrometry].

    PubMed

    Wang, Cai-fen; Li, Zhuo; Wang, Xiao-bo; Li, Hai-yan; Zhang, Ji-wen; Sun, Li-xin

    2015-09-01

    The association rate constant and dissociation rate constant are important parameters of the drug-cyclodextrin supermolecule systems, which determine the dissociation of drugs from the complex and the further in vivo absorption of drugs. However, the current studies of drug-cyclodextrin interactions mostly focus on the thermodynamic parameter of equilibrium constants (K). In this paper, a method based on quantitative high performance affinity chromatography coupled with mass spectrometry was developed to determine the apparent dissociation rate constant (k(off,app)) of drug-cyclodextrin supermolecule systems. This method was employed to measure the k(off,app) of meloxicam and acetaminophen. Firstly, chromatographic peaks of drugs and non-retained solute (uracil) on β-cyclodextrin column at different flow rates were acquired, and the retention time and variance values were obtained via the fitting the peaks. Then, the plate heights of drugs (H(R)) and uracil (H(M,C)) were calculated. The plate height of theoretical non-retained solute (H(M,T)) was calculated based on the differences of diffusion coefficient and the stagnant mobile phase mass transfer between drugs and uracil. Finally, the k(off,app) was calculated from the slope of the regression equation between (H(R)-H(M,T)) and uk/(1+k)2, (0.13 ± 0.00) s(-1) and (4.83 ± 0.10) s(-1) for meloxicam and acetaminophen (control drug), respectively. In addition, the apparent association rate constant (k(on,app)) was also calculated through the product of K (12.53 L x mol(-1)) and k(off,app). In summary, it has been proved that the method established in our study was simple, efficiently fast and reproducible for investigation on the kinetics of drug-cyclodextrin interactions. PMID:26757555

  6. Accelerator mass spectrometry

    SciTech Connect

    Vogel, J.S.; Turteltaub, K.W.; Finkel, R.; Nelson, D.E.

    1995-06-01

    Accelerator mass spectroscopy (AMS) can be used for efficient detection of long-lived isotopes at part-per-quadrillion sensitivities with good precision. In this article we present an overview of AMS and its recent use in archaeology, geochemistry and biomolecular tracing. All AMS systems use cesium sputter ion sources to produce negative ions from a small button of a solid sample containing the element of interest, such as graphite, metal halide, or metal oxide, often mixed with a metal powder as binder and thermal conductor. Experience shows that both natural and biomedical samples are compatible in a single AMS system, but few other AMS sites make routine {sup 14}C measurements for both dating and tracing. AMS is, in one sense, just `a very sensitive decay counter`, but if AMS sensitivity is creatively coupled to analytical chemistry of certain isotopes, whole new areas of geosciences, archaeology, and life sciences can be explored. 29 refs., 2 figs., 1 tab.

  7. Trends in mass spectrometry instrumentation for proteomics.

    PubMed

    Smith, Richard D

    2002-12-01

    Mass spectrometry has become a primary tool for proteomics because of its capabilities for rapid and sensitive protein identification and quantitation. It is now possible to identify thousands of proteins from microgram sample quantities in a single day and to quantify relative protein abundances. However, the need for increased capabilities for proteome measurements is immense and is now driving both new strategies and instrument advances. These developments include those based on integration with multi-dimensional liquid separations and high accuracy mass measurements and promise more than order of magnitude improvements in sensitivity, dynamic range and throughput for proteomic analyses in the near future.

  8. 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.

  9. 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

  10. Quantitative imaging of selenium, copper, and zinc in thin sections of biological tissues (slugs-genus arion) measured by laser ablation inductively coupled plasma mass spectrometry.

    PubMed

    Becker, J S; Matusch, A; Depboylu, C; Dobrowolska, J; Zoriy, M V

    2007-08-15

    Quantitative imaging analysis of endogenous an exogenous elements throughout entire organisms is required for studies of bioavailability, transport processes, distribution, contamination and to monitor environmental risks using indicator organisms. An imaging mass spectrometric technique using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) was developed to analyze selenium and metal distributions in longitudinal sections (thickness, 100 microm) of entire slugs (genus arion). Slugs were fed with either a placebo or solutions containing 1000 microg mL(-1) Se. Samples (raster area, 25 mmx45 mm) were scanned together with synthetic matrix-matched standards with a focused beam of a Nd:YAG laser (wavelength, 266 nm; diameter of laser crater, 50 microm; laser power density, 3x10(9) W cm(-2)) in a large laser ablation chamber. The ablated material was transported with argon as carrier gas to the ICP ion source at a double focusing sector field ICPMS. Ion intensities of selenium (78Se+, 82Se+) were measured together with 13C+, 63Cu+, and 64Zn+ within the entire tissue section. The regression coefficient of the calibration curve was 0.998. Inhomogeneous distributions for Se but also for C, Cu, and Zn were found. Selenium was enriched in the kidney (150 microg g(-1) in Se-treated animals versus 15 microg g(-1) in the placebo-treated animal, respectively) and in the digestive gland (200 microg g(-1) versus 25 microg g(-1)). Highest Se concentrations were detected in the gut of Se-treated slugs (250 microg g(-1)), and additional Se occurred in the skin of these animals. Cu was enriched in the heart and the mucous ventral skin. Interestingly, in addition to the localization in the digestive gland, Zn was detected only in the dorsal skin but not the ventral skin. The developed analytical technique allows the quantitative imaging of selenium together with selected metals in thin sections of biological tissue with limits of detection at the submicrogram per

  11. 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…

  12. Mass spectrometry imaging for biomedical applications

    PubMed Central

    Liu, Jiangjiang; Ouyang, Zheng

    2013-01-01

    The development of mass spectrometry imaging technologies is of significant current research interest. Mass spectrometry potentially is capable of providing highly specific information about the distribution of chemical compounds on tissues at highly sensitive levels. The required in-situ analysis for the tissue imaging forced MS analysis being performed off the traditional conditions optimized in pharmaceutical applications with intense sample preparation. This critical review seeks to present an overview of the current status of the MS imaging with different sampling ionization methods and to discuss the 3D imaging and quantitative imaging capabilities needed to be further developed, the importance of the multi-modal imaging, and a balance between the pursuit of the high imaging resolution and the practical application of MS imaging in biomedicine. PMID:23539099

  13. 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

  14. 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

  15. 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.

  16. 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). PMID:27634210

  17. 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

  18. Quantitation of the sulfur mustard metabolites 1,1'-sulfonylbis[2-(methylthio)ethane] and thiodiglycol in urine using isotope-dilution Gas chromatography-tandem mass spectrometry.

    PubMed

    Boyer, Anne E; Ash, Doris; Barr, Dana B; Young, Carrie L; Driskell, W J; Whitehead, Ralph D; Ospina, Maria; Preston, Kerry E; Woolfitt, Adrian R; Martinez, Rodolfo A; Silks, L A Pete; Barr, John R

    2004-01-01

    Sulfur mustard (HD), or bis(2-chloroethyl)sulfide, has several urinary metabolites that can be measured to assess human exposure. These metabolites include the simple hydrolysis product thiodiglycol (TDG) and its oxidative analogue, TDG-sulfoxide, as well as metabolites of the glutathione/b-lyase pathway 1,1'-sulfonylbis[2-(methyl-sulfinyl)ethane] (SBMSE) and 1-methyl-sulfinyl-2-[(methylthio)ethyl-sulfonyl]ethane (MSMTESE). Current methods focus on either the TDG or the b-lyase metabolites. We have developed a single method that measures products of both metabolic branches, with the reduced compound of SBMSE and MSMTESE, 1,1'-sulfonylbis [2(methylthio)ethane] (SBMTE), as the definitive analyte and TDG as a confirmation analyte. Sample preparation included b-glucuronidase hydrolysis for TDG-glucuronide conjugates, titanium trichloride reduction of sulfoxides to SBMTE and TDG, solid-phase extraction, and a chemical derivatization. We analyzed samples using gas chromatography-tandem mass spectrometry with quantitation using isotope-dilution calibration. The method limits of detection for TDG and SBMTE were 0.5 ng/mL and 0.25 ng/mL, respectively, with relative standard deviations of less than 10%. Urine samples from individuals with no known exposure to mustard agent HD had measurable concentrations of TDG, but no SBMTE was detected. The geometric mean concentration of TDG was 3.43 ng/mL, with concentrations ranging from < 0.5 ng/mL to 20 ng/mL. PMID:15239851

  19. 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

  20. 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. PMID:25997845

  1. [Qualitative and quantitative analysis of the amino acids in Rhizoma Arisaematis by ultra high performance liquid chromatography-tandem mass spectrometry and high performance liquid chromatography].

    PubMed

    Wang, Xing; Chi, Yumei; Kang, An

    2014-12-01

    A method for the identification and determination of the polar amino components without ultraviolet activity in traditional Chinese medicines was developed. With Rhizoma Arisaematis as the object of this study, using pre-column derivatization with phenyl isothiocyanate (PITC) as the derivatization reagent, compounds were separated and identified on a C18 column (100 mm x 2.1 mm, 3.5 µm) by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). A total of 20 components, including 18 amino acids and 2 amine compounds were identified. Furthermore, after the optimization of the derivatization conditions, 15 amino acids were determined by high performance liquid chromatography (HPLC) on Diamonsil C18 column (250 mm x 4.6 mm, 5 µm), detected at 254 nm and gradiently eluted by acetonitrile and 0. 05 mol/L ammonium acetate-acetic acid (pH 6. 5) as the mobile phases. The results of methodological study demonstrated that the method can meet the requirements of the determination. All calibration curves expressed good linearity: Glu, Try in the range of 2-100 mg/L, Arg in the range of 6-300 mg/L, others in the range of 0. 8-40 µg/L, with the correlation coefficients ≥ 0. 999 5. The average recovery of this method was among 95%-105% and the RSD was less than 3%. The developed method was successfully applied to quantitative determination of amino compounds in 12 batches of Rhizoma Arisaematis samples. The method is simple, sensitive, accurate, and can be used for rapid identification and determination of amino components in traditional Chinese medicines.

  2. 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.

  3. Quantitative profiling of bile acids in rat bile using ultrahigh-performance liquid chromatography-orbitrap mass spectrometry: Alteration of the bile acid composition with aging.

    PubMed

    Lee, Gakyung; Lee, Hyunbeom; Hong, Jongki; Lee, Soo Hyun; Jung, Byung Hwa

    2016-09-15

    Bile acids (BAs) play important roles in physiological functions, including the homeostasis of cholesterol and lipids and as ligands for G protein-coupled receptors (GPCRs). With the increasing importance of BAs, analytical methods for their quantification and screening have been developed. However, due to the diverse range and variety of BAs with different activation potency, a simple, effective, and sensitive method is required to screen BAs for accurate quantification and identification. This paper presents an application of ultrahigh-performance liquid chromatography-orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS) for profiling BAs in bile. Using this method, along with the accurate quantification of 19 targeted BAs, 22 unknown BAs were detected and characterized by their fragmentation patterns. The method is beneficial for screening most of the BAs (quantitatively and qualitatively) in rat bile with simple preparation in a single run. The sample dilution ranges of each BA were optimized depending on the concentration of BAs in the bile to obtain good peak separation and accurate data. The method validation was performed successfully using charcoal-treated bile and the intra and inter-day coefficients of variation were less than 20% for all BAs while the recovery were above 88.5% except for the lithocholic acid. The method was applied to profile the age-dependent changes in the contents of rat BAs. Through statistical analysis, we found that as the rats aged, unconjugated BAs and glycine-conjugated BAs decreased or were unaffected, while taurine-conjugated BAs were increased in general. Among the unknown BAs, 5 of the taurine-conjugated BAs increased, while a glycine-conjugated BA decreased, in agreement with the trends of the targeted BAs. PMID:27450898

  4. 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

  5. Application of screening experimental designs to assess chromatographic isotope effect upon isotope-coded derivatization for quantitative liquid chromatography-mass spectrometry.

    PubMed

    Szarka, Szabolcs; Prokai-Tatrai, Katalin; Prokai, Laszlo

    2014-07-15

    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-, (13)C6-, (15)N2-, or (15)N4-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 (15)N or (13)C labeling instead of deuterium labeling, while chromatographic parameters had no general influence. Comparison of the alternate isotope-coded derivatization assay (AIDA) using deuterium versus (15)N 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 (15)N- or (13)C-labeled derivatizing reagent to avoid possible unfavorable consequences of chromatographic isotope effects. PMID:24922593

  6. 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).

  7. 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. PMID:27240302

  8. 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.

  9. 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

  10. 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

  11. Quantitative measurement of full-length and C-terminal proteolyzed RBP4 in serum of normal and insulin-resistant humans using a novel mass spectrometry immunoassay.

    PubMed

    Yang, Qin; Eskurza, Iratxe; Kiernan, Urban A; Phillips, David A; Blüher, Matthias; Graham, Timothy E; Kahn, Barbara B

    2012-03-01

    Serum retinol-binding protein 4 (RBP4) levels are increased in insulin-resistant humans and correlate with severity of insulin resistance in metabolic syndrome. Quantitative Western blotting (qWestern) has been the most accurate method for serum RBP4 measurements, but qWestern is technically complex and labor intensive. The lack of a reliable, high-throughput method for RBP4 measurements has resulted in variability in findings in insulin-resistant humans. Many commonly used ELISAs have limited dynamic range. Neither the current ELISAs nor qWestern distinguish among full-length and carboxyl terminus proteolyzed forms of circulating RBP4 that are altered in different medical conditions. Here, we report the development of a novel quantitative mass spectrometry immunoaffinity assay (qMSIA) to measure full-length and proteolyzed forms of RBP4. qMSIA and qWestern of RBP4 were performed in identical serum aliquots from insulin-sensitive/normoglycemic or insulin-resistant humans with impaired glucose tolerance or type 2 diabetes. Total RBP4 qMSIA measurements were highly similar to qWestern and correlated equally well with clinical severity of insulin resistance (assessed by clamp glucose disposal rate, r = -0.74), hemoglobin A1c (r = 0.63), triglyceride/high-density lipoprotein (r = 0.55), waist/hip (r = 0.61), and systolic blood pressure (r = 0.53, all P < 0.001). Proteolyzed forms of RBP4 accounted for up to 50% of total RBP4 in insulin-resistant subjects, and des(Leu)-RBP4 (cleavage of last leucine) correlated highly with insulin resistance (assessed by glucose disposal rate, r = -0.69). In multiple regression analysis, insulin resistance but not glomerular filtration rate was the strongest, independent predictor of serum RBP4 levels. Thus, qMSIA provides a novel tool for accurately measuring serum RBP4 levels as a biomarker for severity of insulin resistance and risk for type 2 diabetes and metabolic syndrome. PMID:22253430

  12. 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

  13. 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

  14. Quantitation of cocaine, benzoylecgonine, cocaethylene, methylecgonine, and norcocaine in human hair by positive ion chemical ionization (PICI) gas chromatography-tandem mass spectrometry.

    PubMed

    Bourland, J A; Hayes, E F; Kelly, R C; Sweeney, S A; Hatab, M M

    2000-10-01

    A total of 30 human head-hair samples were analyzed for cocaine (COC), cocaethylene (CE), benzoylecgonine (BE), methylecgonine (EME), and norcocaine (NCOC) using a sensitive positive ion chemical ionization gas chromatography-tandem mass spectrometry (GC-MS-MS) method. All 30 hair samples had been previously submitted to the laboratory and had confirmed positive for cocaine. Hair samples (20 mg each) were cut into small segments (2-5 mm) and incubated overnight at 45 degrees C in 0.1 N HCl after the addition of 50 microL of an internal standard mix of COC-d3 (1.0 ng/mg), BE-d3 (0.5 ng/mg), EME-d3 (0.25 ng/mg), and NCOC-d3 (0.25 ng/mg). The samples were then extracted with Clean Screen extraction columns from United Chemical Technologies, Inc. The final extract was evaporated to dryness and derivatized with 50 microL of 1,1,1,3,3,3-hexafluoro-2-propanol and 50 microL of trifluoroacetic anhydride at 90 degrees C for 15 min. The derivatized samples were allowed to cool to room temperature, evaporated to dryness, and then reconstituted in 50 microL of ethyl acetate. Parent set masses (unbolded ions) and product ions were m/z 304 and m/z 182 and 82 (COC), m/z 307 and m/z 185 and 85 (COC-d3), m/z 318 and m/z 196 and 82 (CE), m/z 440 and m/z 318 and 105 (BE), m/z 443 and m/z 321 and 105 (BE-d3), m/z 296 and m/z 182, and 82 (EME), m/z 299 and m/z 185 and 85 (EME-d3), m/z 403 and m/z 386 and 105 (NCOC), m/z 406 and m/z 389 and 105 (NCOC-d3). Quantitation was accomplished by calculating the area ratio of the higher mass product ion (underlined ions) of analyte to the respective internal standard based on multilevel calibrations from 0.01 to 10.0 ng/mg. The GC-MS-MS method had a limit of detection of 0.01 ng/mg and a limit of quantitation of 0.05 ng/mg for all five analytes. COC, BE, and EME were detected in all 30 samples, and CE and NCOC were found in 19 and 29 samples, respectively. The average relative percentages of each metabolite normalized to the cocaine

  15. 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. PMID:27283655

  16. 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.

  17. 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.

  18. 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.

  19. An integrated strategy to quantitatively differentiate chemome between Cistanche deserticola and C. tubulosa using high performance liquid chromatography-hybrid triple quadrupole-linear ion trap mass spectrometry.

    PubMed

    Song, Yuelin; Song, Qingqing; Li, Jun; Zhang, Na; Zhao, Yunfang; Liu, Xiao; Jiang, Yong; Tu, Pengfei

    2016-01-15

    It is important to conduct large-scale detection, identification, and quantitation of metabolites in a given sample. Herein, a practical strategy was proposed to quantitatively compare the chemome between Cistanche deserticola (CD) and C. tubulosa (CT), which have been widely believed as the ideal edible and medicinal plants for conquering the deserts. The entire workflow was implemented on high performance liquid chromatography-hybrid triple quadrupole-linear ion trap mass spectrometer and consisted of three primary steps: (1) component detection and identification, various mass spectrometric approaches were applied to globally screen the chemical constituents, and structural elucidation was achieved by comparing with authentic compounds, analyzing MS(2) spectra, and referring to the literature along with accessible databases; (2) comprehensive relative quantitation, scheduled multiple reaction monitoring algorithm was introduced for relative quantitation of all detected ingredients; and (3) chemome comparison, the quantitative dataset was subjected for multivariate statistical analysis to carry out comparative study. A total of 513 metabolites were detected and relatively quantitated, and 379 ones were annotated. Betaine, Krebs cycle intermediates, phenylethanoid glycosides, and iridoids were picked out as the chemical markers being responsible for the discrimination of the chemical profiles between CD and CT. Above all, the quantitative chemome of CD and CT were exhaustively characterized and compared, which could advance their values concerning drug development, economics, and desertification control. The proposed strategy is expected as a reliable choice for widely targeted metabolomics of plants.

  20. An integrated strategy to quantitatively differentiate chemome between Cistanche deserticola and C. tubulosa using high performance liquid chromatography-hybrid triple quadrupole-linear ion trap mass spectrometry.

    PubMed

    Song, Yuelin; Song, Qingqing; Li, Jun; Zhang, Na; Zhao, Yunfang; Liu, Xiao; Jiang, Yong; Tu, Pengfei

    2016-01-15

    It is important to conduct large-scale detection, identification, and quantitation of metabolites in a given sample. Herein, a practical strategy was proposed to quantitatively compare the chemome between Cistanche deserticola (CD) and C. tubulosa (CT), which have been widely believed as the ideal edible and medicinal plants for conquering the deserts. The entire workflow was implemented on high performance liquid chromatography-hybrid triple quadrupole-linear ion trap mass spectrometer and consisted of three primary steps: (1) component detection and identification, various mass spectrometric approaches were applied to globally screen the chemical constituents, and structural elucidation was achieved by comparing with authentic compounds, analyzing MS(2) spectra, and referring to the literature along with accessible databases; (2) comprehensive relative quantitation, scheduled multiple reaction monitoring algorithm was introduced for relative quantitation of all detected ingredients; and (3) chemome comparison, the quantitative dataset was subjected for multivariate statistical analysis to carry out comparative study. A total of 513 metabolites were detected and relatively quantitated, and 379 ones were annotated. Betaine, Krebs cycle intermediates, phenylethanoid glycosides, and iridoids were picked out as the chemical markers being responsible for the discrimination of the chemical profiles between CD and CT. Above all, the quantitative chemome of CD and CT were exhaustively characterized and compared, which could advance their values concerning drug development, economics, and desertification control. The proposed strategy is expected as a reliable choice for widely targeted metabolomics of plants. PMID:26742897

  1. Understanding the Mechanisms Enabling an Ultra-high Efficiency Moving Wire Interface for Real-time Carbon 14 Accelerator Mass Spectrometry Quantitation of Samples Suspended in Solvent

    NASA Astrophysics Data System (ADS)

    Thomas, Avraham Thaler

    Carbon 14 (14C) quantitation by accelerator mass spectrometry (AMS) is a powerfully sensitive and uniquely quantitative tool for tracking labeled carbonaceous molecules in biological systems. This is due to 14C's low natural abundance of 1 ppt, the nominal difference in biological activity between an unlabeled and a 14C-labeled molecule, and the ability of AMS to measure isotopic ratios independently of a sample's other characteristics. To make AMS more broadly accessible, a moving wire interface for real-time coupling of high pressure liquid chromatography (HPLC) to AMS and high throughput AMS quantitation of minute single samples has been developed. Prior to this work, samples needed to be converted to solid carbon before measurement. This conversion process has many steps and requires that the sample size be large enough to allow precise handling of the resulting graphite. These factors make the process susceptible to error and time consuming, as well as requiring 0.5 ug of carbon. Samples which do not contain enough carbon, such as HPLC fractions, must be bulked up. This adds background and increases effort. The moving wire interface overcomes these limitations by automating sample processing. Samples placed on the wire are transported through a solvent removal stage followed by a combustion stage after which the combustion products are directed to a gas accepting ion source. The ion source converts the carbon from the CO2 combustion product into C ions, from which an isotopic ratio can be determined by AMS. Although moving wire interfaces have been implemented for various tasks since 1964, the efficiency of these systems at transferring fluid from an HPLC to the wire was only 3%, the efficiency of transferring combustion products from the combustion oven to ion source was only 30%, the flow and composition of the carrier gas from the combustion oven to the ion source needed to be optimized for coupling to an AMS gas accepting ion source and the drying ovens

  2. 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

    2013-06-01

    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.

  3. 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.

  4. 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.

  5. 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.

  6. Quantitation of Flecainide, Mexiletine, Propafenone, and Amiodarone in Serum or Plasma Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

    PubMed

    Slawson, Matthew H; Johnson-Davis, Kamisha L

    2016-01-01

    Flecainide, mexiletine, propafenone, and amiodarone are antiarrhythmic drugs that are used primarily in the treatment of cardiac arrhythmias. The monitoring of the use of these drugs has applications in therapeutic drug monitoring and overdose situations. LC-MS/MS is used to analyze plasma/serum extracts with loxapine as the internal standard to ensure accurate quantitation and control for any potential matrix effects. Positive ion electrospray is used to introduce the analytes into the mass spectrometer. Selected reaction monitoring of two product ions for each analyte allows for the calculation of ion ratios which ensures correct identification of each analyte, while a matrix matched calibration curve is used for quantitation. PMID:26660169

  7. 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

  8. 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.

  9. Simple quantitative determination of potent thiols at ultratrace levels in wine by derivatization and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis.

    PubMed

    Capone, Dimitra L; Ristic, Renata; Pardon, Kevin H; Jeffery, David W

    2015-01-20

    Volatile sulfur compounds contribute characteristic aromas to foods and beverages and are widely studied, because of their impact on sensory properties. Certain thiols are particularly important to the aromas of roasted coffee, cooked meat, passion fruit, grapefruit, and guava. These same thiols enhance the aroma profiles of different wine styles, imparting pleasant aromas reminiscent of citrus and tropical fruits (due to 3-mercaptohexan-1-ol, 3-mercaptohexyl acetate, 4-mercapto-4-methylpentan-2-one), roasted coffee (2-furfurylthiol), and struck flint (benzyl mercaptan), at nanogram-per-liter levels. In contrast to the usual gas chromatography (GC) approaches, a simple and unique high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for routine analysis of five wine thiols, using 4,4'-dithiodipyridine (DTDP) as a derivatizing agent and polydeuterated internal standards for maximum accuracy and precision. DTDP reacted rapidly with thiols at wine pH and provided stable derivatives, which were enriched by solid-phase extraction (SPE) prior to analysis by HPLC-MS/MS. All steps were optimized and the method was validated in different wine matrices, with method performance being comparable to a well-optimized but more cumbersome gas chromatography-mass spectrometry (GC-MS) method. A range of commercial wines was analyzed with the new method, revealing the distribution of the five thiols in white, red, rosé, and sparkling wine styles.

  10. Counting Molecules by Desorption Ionization and Mass Spectrometry/Mass Spectrometry.

    ERIC Educational Resources Information Center

    Cooks, R. G.; Busch, K. L.

    1982-01-01

    Discusses two newer methods in mass spectrometry and shows how they can increase signal and signal-to-noise ratios, respectively. The first method, desorption ionization (DI), increases sensitivity while the second method, mass spectrometry/mass spectrometry (MS/MS), increases specificity. Together, the two methods offer improved analytical…

  11. Nanotip Ambient Ionization Mass Spectrometry.

    PubMed

    Zhou, Zhenpeng; Lee, Jae Kyoo; Kim, Samuel C; Zare, Richard N

    2016-05-17

    A method called nanotip ambient ionization mass spectrometry (NAIMS) is described, which applies high voltage between a tungsten nanotip and a metal plate to generate a plasma in which ionized analytes on the surface of the metal plate are directed to the inlet and analyzed by a mass spectrometer. The dependence of signal intensity is investigated as a function of the tip-to-plate distance, the tip size, the voltage applied at the tip, and the current. These parameters are separately optimized to achieve sensitivity or high spatial resolution. A partially observable Markov decision process is used to achieve a stabilized plasma as well as high ionization efficiency. As a proof of concept, the NAIMS technique has been applied to phenanthrene and caffeine samples for which the limits of detection were determined to be 0.14 fmol for phenanthrene and 4 amol for caffeine and to a printed caffeine pattern for which a spatial resolution of 8 ± 2 μm, and the best resolution of 5 μm, was demonstrated. The limitations of NAIMS are also discussed. PMID:27087600

  12. Identification and quantitation of cocaine, benzoylecgonine, and cocaethylene in blood, serum, and plasma using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS).

    PubMed

    Kriger, Scott; Gunn, Josh; Terrell, Andrea R

    2010-01-01

    Cocaine is a widely abused stimulant. Numerous methods exist for the identification of the drug, or more commonly, one of its metabolites in urine. Urine testing is useful for most cases, but it is necessary to use other matrices in forensic situations and when subjects are anuric. We describe a novel method for the analysis of cocaine, benzoylecgonine, and cocaethylene in blood, serum, and plasma utilizing ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Sample preparation has been minimized to a simple deproteinization step in which each specimen is mixed with an acetonitrile-internal standard mixture. The method has excellent precision across the linear range of 25-2,000 ng/mL for each analyte. With a run-time of 4 min, this method provides a significant improvement over traditional GC/MS methods. PMID:20077068

  13. 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).

  14. Isotope dilution for quantitation of steroid estrogens and nonylphenols by gas chromatography with tandem mass spectrometry in septic, soil, and groundwater matrices.

    PubMed

    Stanford, Benjamin D; Weinberg, Howard S

    2007-12-28

    A simplified, in-line solid-phase extraction and clean-up process is presented for rapid extraction and analysis of steroid estrogens and nonylphenols by gas chromatography with tandem mass spectrometry. Isotope dilution with multiple deuterated standards has been developed, evaluated, and verified using this method against standard addition for the analysis of estrone, estradiol, estriol, ethinylestradiol, and 4-nonylphenols in septic, soil, and groundwater matrices. The accuracy and precision of isotope dilution was comparable to standard addition yet was able to quantify analytes with the use of fewer samples by including [(2)H(4)]estradiol, [(2)H(4)]ethinylestradiol, [(2)H(4)]nonylphenol, and estradiol-17-acetate as internal standards. Specific details and validation of the method are presented as are data showing the occurrence of estrogens and nonylphenols in an on-site wastewater treatment (septic) system with advanced stages of pretreatment and recirculation to an office building for non-potable reuse.

  15. 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. PMID:26925555

  16. 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.

  17. Quantitative liquid chromatography/tandem mass spectrometry determination of chloramphenicol residues in food using sub-2 microm particulate high-performance liquid chromatography columns for sensitivity and speed.

    PubMed

    Kaufmann, Anton; Butcher, Patrick

    2005-01-01

    The use of chloramphenicol (CAP)--a highly effective broad-spectrum antibiotic used in animal husbandry--is banned in many countries. Therefore, a very low minimum required performance limit (MRPL) of 0.3 microg/kg CAP in meat for human consumption has been defined. Analytical methods capable of quantifying and confirming such low residue levels require sophisticated instrumentation. Preferably sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) or gas chromatography/mass spectrometry (GC/MS) methods have been used. This paper suggests the use of sub-2 microm particulate high-performance liquid chromatography (HPLC) columns to gain additional sensitivity and improve resolution as well as speed. Depending on the operating conditions, higher chromatographic resolution and speed can be obtained at the price of a significantly increased operating pressure, requiring dedicated LC equipment. A 3-4-fold overall improvement of the signal-to-noise ratio for CAP was obtained compared to more classical 5 microm particulate HPLC columns. The proposed analytical methodology includes an enzymatic digestion, which liberates glucuronide-bound CAP from kidney tissue. The extracts obtained after an Extrelut clean-up are sufficiently pure to permit routine injection of biological samples into the sub-2 microm particulate HPLC column, without observing rapid deterioration of peak shape or column clogging problems. The time for one chromatographic run was 4.2 min. The described method was validated for two particularly difficult matrices (kidney and honey). Decision limits (CC alpha) were 0.007 microg/kg (honey) und 0.011 microg/kg (kidney), which are significantly below the current MRPL. PMID:16299695

  18. 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. PMID:26708481

  19. 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.

  20. Developments in ion mobility spectrometry-mass spectrometry.

    PubMed

    Collins, D C; Lee, M L

    2002-01-01

    Ion mobility spectrometry (IMS) has been used for over 30 years as a sensitive detector of organic compounds. The following is a brief review of IMS and its principles with an emphasis on its usage when coupled to mass spectrometry. Since its inception, IMS has been interfaced with quadrupole, time-of-flight, and Fourier-transform ion cyclotron resonance mass spectrometry. These hybrid instruments have been employed for the analysis of a variety of target analytes, including biomolecules, explosives, chemical warfare degradation products, and illicit drugs. PMID:11939214

  1. 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

  2. Nonaqueous Capillary Electrophoresis Mass Spectrometry.

    PubMed

    Klampfl, Christian W; Himmelsbach, Markus

    2016-01-01

    The term nonaqueous capillary electrophoresis (NACE) commonly refers to capillary electrophoresis with purely nonaqueous background electrolytes (BGE). Main advantages of NACE are the possibility to analyze substances with very low solubility in aqueous media as well as separation selectivity that can be quite different in organic solvents (compared to water)-a property that can be employed for manipulation of separation selectivities. Mass spectrometry (MS) has become more and more popular as a detector in CE a fact that applies also for NACE. In the present chapter, the development of NACE-MS since 2004 is reviewed. Relevant parameters like composition of BGE and its influence on separation and detection in NACE as well as sheath liquid for NACE-MS are discussed. Finally, an overview of the papers published in the field of NACE-MS between 2004 and 2014 is given. Applications are grouped according to the field (analysis of natural products, biomedical analysis, food analysis, analysis of industrial products, and fundamental investigations). PMID:27645734

  3. Mass Spectrometry Imaging Quick View

    SciTech Connect

    2013-01-24

    MSI QuickView is a software designed to provide a graphical user interface (GUI) for greatly speeding up experimental feedback (visualization and analysis) of mass spectrometry imaging (MSI or IMS) data during data acquisition. Often different software loads the entire data set, i.e., all lines of data into computer memory (RAM). This causes out of memory errors for larger datasets. We solved this in MSI QuickView by reading in the data one line at a time. Only the required information (e.g. the final pixel values for that line of heat map) is maintained in RAM. Interim analysis options include the mean intensity vs. m/z spectrum, intensity vs. time spectrums for up to 6 different m/z values or ranges chosen by the user and heat maps for each line. This assists in validating the usefulness of the particular experiment after scanning the first few lines. In addition, the tool facilitates further processing and analysis of the massive datasets. The user can manually pick different m/z values, time ranges, scroll through the spectra for any line in the data without having to load it in manually, save multiple images, change aspect ratios for the heat maps, and process the heat maps in multiple ways including overlaying images at different m/z values, displaying up to 9 different heat maps, alignment of scans along each line etc. There is no manipulation of the data required by the user to visualize the data.

  4. A validated assay for the quantitative analysis of vatalanib in human EDTA plasma by liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

    PubMed

    Lankheet, A G; Hillebrand, M J X; Langenberg, M H G; Rosing, H; Huitema, A D R; Voest, E E; Schellens, J H M; Beijnen, J H

    2009-11-01

    A sensitive and accurate method for the determination of vatalanib in human EDTA plasma was developed using high-performance liquid chromatography and detection with tandem mass spectrometry. Stable isotopically labeled imatinib was used as internal standard. Plasma proteins were precipitated and an aliquot of the supernatant was directly injected onto a Phenomenex Gemini C18 analytical column (50 mm x 2.0 mm ID, 5.0 microm particle size) and then compounds were eluted with a linear gradient. The outlet of the column was connected to a Sciex API 365 triple quadrupole mass spectrometer and ions were detected in positive multiple reaction monitoring mode. The lower limit of quantification was 10 ng/mL (S/N approximately 10, CV < or = 8.4%). This method was validated over a linear range from 10 to 2500 ng/mL, and results from the validation study demonstrated a good intra- and inter-assay accuracy (inaccuracy < or = 9.57%) and precision (CV < or = 8.81%). This method has been used to determine plasma vatalanib concentrations in patients with advanced solid tumor, enrolled in a phase I pharmacokinetic trial with the drug. PMID:19762293

  5. Qualitative and quantitative DNA and RNA analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

    PubMed

    Ding, Chunming

    2006-01-01

    Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) gives extremely precise reading of mass-to-charge ratios (two analytes differ by 1 Da can be distinguished) and provides high sensitivity (less than 1 fmole of a DNA oligonucleotide can be detected), allowing DNA quantifications with single base specificity and single DNA molecule sensitivity (coupled with polymerase chain reaction [PCR]). To quantify a DNA sequence of interest, a competitive synthetic (60-80 bases) oligonucleotide standard with an artificial single base mutation in the middle is introduced, and these two virtually identical sequences are co-amplified by PCR. A third primer (extension primer) is designed to anneal to the region immediately upstream of the mutation site. Depending on the specific mutation introduced and the ddNTP/dNTP mixtures used, either one or two bases are added to the extension primer to produce two extension products from the two templates. Last, the two extension products are detected and quantified by high-throughput MALDI-TOF MS. In addition, with an improved primer extension method called single allele base extension reaction (SABER), rare mutant DNA can be robustly detected even when normal DNA is present at 50-fold or more than the DNA mutants.

  6. Quantitative analysis of the major constituents in Chinese medicinal preparation SuoQuan formulae by ultra fast high performance liquid chromatography/quadrupole tandem mass spectrometry

    PubMed Central

    2013-01-01

    Background The SuoQuan formulae containing Fructus Alpiniae Oxyphyllae has been used to combat the urinary incontinence symptoms including frequency, urgency and nocturia for hundreds of years in China. However, the chemical information was not well characterized. The quality control marker constituent only focused on one single compound in the current Chinese Pharmacopeia. Hence it is prudent to identify and quantify the main constituents in this herbal product. This study aimed to analyze the main constituents using ultra-fast performance liquid chromatography coupled to tandem mass spectrometry (UFLC-MS/MS). Results Fourteen phytochemicals originated from five chemical classes constituents were identified by comparing the molecular mass, fragmentation pattern and retention time with those of the reference standards. A newly developed UFLC-MS/MS was validated demonstrating that the new assay was valid, reproducible and reliable. This method was successfully applied to simultaneously quantify the fourteen phytochemicals. Notably, the content of these constituents showed significant differences in three pharmaceutical preparations. The major constituent originated from each of chemical class was isolinderalactone, norisoboldine, nootkatone, yakuchinone A and apigenin-4’,7-dimethylther, respectively. The variation among these compounds was more than 1000 times. Furthermore, the significant content variation between the two different Suoquan pills was also observed. Conclusion The proposed method is sensitive and reliable; hence it can be used to analyze a variety of SuoQuan formulae products produced by different pharmaceutical manufacturers. PMID:23899222

  7. QUANTITATION OF ESTROGENS IN GROUND WATER AND SWINE LAGOON SAMPLE USING SOLID PHASE EXTRACTION, PENTAFLUROBENZYL/TRIMETHYLSILYL DERIVATIZATIONS AND GAS CHROMATOGRAPHY NEGATIVE ION CHEMICAL IONIZATION/MASS SPECTROMETRY/MASS SPECTROMETRY

    EPA Science Inventory

    A method was developed for the confirmed identification and quantitation of 17B-estradiol, estrone, 17B-ethynylestrodial and 16a-hydroxy-17B-estradiol (estriol) in ground water and swine lagoon samples. Centrifuged and filtered samples were extracted using solid phase extraction...

  8. Metabolite profiling of soy sauce using gas chromatography with time-of-flight mass spectrometry and analysis of correlation with quantitative descriptive analysis.

    PubMed

    Yamamoto, Shinya; Bamba, Takeshi; Sano, Atsushi; Kodama, Yukako; Imamura, Miho; Obata, Akio; Fukusaki, Eiichiro

    2012-08-01

    Soy sauces, produced from different ingredients and brewing processes, have variations in components and quality. Therefore, it is extremely important to comprehend the relationship between components and the sensory attributes of soy sauces. The current study sought to perform metabolite profiling in order to devise a method of assessing the attributes of soy sauces. Quantitative descriptive analysis (QDA) data for 24 soy sauce samples were obtained from well selected sensory panelists. Metabolite profiles primarily concerning low-molecular-weight hydrophilic components were based on gas chromatography with time-of-flightmass spectrometry (GC/TOFMS). QDA data for soy sauces were accurately predicted by projection to latent structure (PLS), with metabolite profiles serving as explanatory variables and QDA data set serving as a response variable. Moreover, analysis of correlation between matrices of metabolite profiles and QDA data indicated contributing compounds that were highly correlated with QDA data. Especially, it was indicated that sugars are important components of the tastes of soy sauces. This new approach which combines metabolite profiling with QDA is applicable to analysis of sensory attributes of food as a result of the complex interaction between its components. This approach is effective to search important compounds that contribute to the attributes.

  9. 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.

  10. Utility of mass spectrometry in the diagnosis of prion diseases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We developed a sensitive mass spectrometry-based method of quantitating the prions present in a variety of mammalian species. Calibration curves relating the area ratios of the selected analyte peptides and their oxidized analogs to their homologous stable isotope labeled internal standards were pre...

  11. 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...

  12. 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...

  13. Mass Spectrometry Imaging Quick View

    2013-01-24

    MSI QuickView is a software designed to provide a graphical user interface (GUI) for greatly speeding up experimental feedback (visualization and analysis) of mass spectrometry imaging (MSI or IMS) data during data acquisition. Often different software loads the entire data set, i.e., all lines of data into computer memory (RAM). This causes out of memory errors for larger datasets. We solved this in MSI QuickView by reading in the data one line at a time.more » Only the required information (e.g. the final pixel values for that line of heat map) is maintained in RAM. Interim analysis options include the mean intensity vs. m/z spectrum, intensity vs. time spectrums for up to 6 different m/z values or ranges chosen by the user and heat maps for each line. This assists in validating the usefulness of the particular experiment after scanning the first few lines. In addition, the tool facilitates further processing and analysis of the massive datasets. The user can manually pick different m/z values, time ranges, scroll through the spectra for any line in the data without having to load it in manually, save multiple images, change aspect ratios for the heat maps, and process the heat maps in multiple ways including overlaying images at different m/z values, displaying up to 9 different heat maps, alignment of scans along each line etc. There is no manipulation of the data required by the user to visualize the data.« less

  14. Quantitative analysis of phenylalanine, tyrosine, tryptophan and kynurenine in rat model for tauopathies by ultra-high performance liquid chromatography with fluorescence and mass spectrometry detection.

    PubMed

    Galba, Jaroslav; Michalicova, Alena; Parrak, Vojtech; Novak, Michal; Kovac, Andrej

    2016-01-01

    We developed and validated a simple and sensitive ultra-high performance liquid chromatography (UHPLC) method for the analysis of phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp) and kynurenine (Kyn) in rat plasma. Analytes were separated on Acquity UPLC HSS T3 column (2.1 mm×50 mm, 1.8 μm particle size) using a 4 min ammonium acetate (pH 5) gradient and detected by fluorescence and positive ESI mass spectrometry. Sample preparation involved dilution of plasma, deproteinization by trichloroacetic acid and centrifugation. The procedure was validated in compliance with the FDA guideline. The limits of quantification (LOQ) were 0.3 μM for Kyn and from 1.5 to 3 μM for Phe, Tyr, Trp. The method showed excellent linearity with regression coefficients higher than 0.99. The accuracy was within the range of 86-108%. The inter-day precision (n=5 days), expressed as % RSD, was in the range 1-13%. The benefit of using UHPLC is a short analysis period and thus, a very good sample throughput. Using this method, we analyzed plasma samples and detected significant changes of Kyn and Phe in transgenic rat model for tauopathies.

  15. Qualitative and Quantitative Analysis of Volatile Components of Zhengtian Pills Using Gas Chromatography Mass Spectrometry and Ultra-High Performance Liquid Chromatography

    PubMed Central

    Liu, Cui-ting; Zhang, Min; Yan, Ping; Liu, Hai-chan; Liu, Xing-yun; Zhan, Ruo-ting

    2016-01-01

    Zhengtian pills (ZTPs) are traditional Chinese medicine (TCM) which have been commonly used to treat headaches. Volatile components of ZTPs extracted by ethyl acetate with an ultrasonic method were analyzed by gas chromatography mass spectrometry (GC-MS). Twenty-two components were identified, accounting for 78.884% of the total components of volatile oil. The three main volatile components including protocatechuic acid, ferulic acid, and ligustilide were simultaneously determined using ultra-high performance liquid chromatography coupled with diode array detection (UHPLC-DAD). Baseline separation was achieved on an XB-C18 column with linear gradient elution of methanol-0.2% acetic acid aqueous solution. The UHPLC-DAD method provided good linearity (R2 ≥ 0.9992), precision (RSD < 3%), accuracy (100.68–102.69%), and robustness. The UHPLC-DAD/GC-MS method was successfully utilized to analyze volatile components, protocatechuic acid, ferulic acid, and ligustilide, in 13 batches of ZTPs, which is suitable for discrimination and quality assessment of ZTPs. PMID:26904360

  16. Quantitative Determination and Subcellular Imaging of Cu in Single Cells via Laser Ablation-ICP-Mass Spectrometry Using High-Density Microarray Gelatin Standards.

    PubMed

    Van Malderen, Stijn J M; Vergucht, Eva; De Rijcke, Maarten; Janssen, Colin; Vincze, Laszlo; Vanhaecke, Frank

    2016-06-01

    This manuscript describes the development and characterization of a high-density microarray calibration standard, manufactured in-house and designed to overcome the limitations in precision, accuracy, and throughput of current calibration approaches for the quantification of elemental concentrations on the cellular level using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICPMS). As a case study, the accumulation of Cu in the model organism Scrippsiella trochoidea resulting from transition metal exposure (ranging from 0.5 to 100 μg/L) was evaluated. After the Cu exposure, cells of this photosynthetic dinoflagellate were treated with a critical point drying protocol, transferred to a carbon stub, and sputter-coated with a Au layer for scanning electron microscopy (SEM) analysis. In subsequent LA-ICPMS analysis, approximately 100 cells of each population were individually ablated. This approach permitted the evaluation of the mean concentration of Cu in the cell population across different exposure levels and also allowed the examination of the cellular distribution of Cu within the populations. In a cross-validation exercise, subcellular LA-ICPMS imaging was demonstrated to corroborate synchrotron radiation confocal X-ray fluorescence (SR-XRF) microimaging of single cells investigated under in vivo conditions. PMID:27149342

  17. 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.

  18. 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.

  19. 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

    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.

  20. 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 Central

    Schofield, Ryan C.; Ramanathan, Lakshmi V.; Murata, Kazunori; Grace, Marie; Fleisher, Martin; Pessin, Melissa S.; Carlow, Dean C.

    2016-01-01

    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-N10-methylpteroic acid (DAMPA) concentrations in serum was developed. MTX was isolated from serum samples (100 μL) 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 1000 nmol/L and for 7-OH MTX from 20 to 2000 nmol/L. Dilutions of 10, 100 and 1000-fold were validated giving a clinically reportable range of 10 nmol/L to 5 × 105 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. PMID:26322588

  1. 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. PMID:27492594

  2. Development of a low-density-solvent dispersive liquid-liquid microextraction with gas chromatography and mass spectrometry method for the quantitation of tetrabromobisphenol-A from dust.

    PubMed

    Barrett, Christopher A; Orban, David A; Seebeck, Shannon E; Lowe, Luis E; Owens, Janel E

    2015-07-01

    The development of an alternative dispersive liquid-liquid microextraction protocol utilizing a low-density extraction solvent, toluene, is described here for the extraction of the brominated flame retardant, tetrabromobisphenol-A, from dust prior to selected ion monitoring analysis by gas chromatography with mass spectrometry. Method parameters of dispersive solvent type and extraction solvent type were optimized. Excellent recovery (88.9%; n = 5 spike replicates) with good precision was achieved in a spike and recovery study. This developed method was utilized to survey tetrabromobisphenol-A concentrations in dust sampled from a local electronics recycling facility from the ambient environment and 20 computer towers undergoing recycling. Concentrations of tetrabromobisphenol-A from dust in computer towers ranged from not detected (n = 2) up to 64 μg/g with a mean value of 11 μg/g and median of 4.1 μg/g tetrabromobisphenol-A. A composite sample of dust collected from the ambient indoor environment was analyzed with a resulting concentration of 36 μg/g. This is the first application of this novel green method for pre-concentrating flame retardants from dust and the first report of tetrabromobisphenol-A concentrations at a U.S.-based electronics recycling facility.

  3. 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.

  4. 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).

  5. Development and validation of a liquid chromatography-tandem mass spectrometry assay for the simultaneous quantitation of microcystin-RR and its metabolites in fish liver.

    PubMed

    Wu, Laiyan; Xie, Ping; Chen, Jun; Zhang, Dawen; Liang, Gaodao

    2010-02-26

    A novel method for identification and quantification of microcystin-RR (MC-RR) and its metabolites (MC-RR-GSH and MC-RR-Cys) in the fish liver was developed and validated. These analytes were simultaneously extracted from fish liver using water containing EDTA with 5% acetic acid, followed by a mixed-mode cation-exchange SPE (Oasis MCX) and subsequently determined by liquid chromatography-electrospray ionization ion trap mass spectrometry (LC-ESI-ITMS). Extraction parameters including volume and pH of eluting solvents, were optimized. Best recoveries were obtained by using 10 mL of 15% ammonia solution in methanol. The mean recoveries at three concentrations (0.2, 1.0, and 5.0 microg g(-1) dry weight [DW]) for MC-RR, MC-RR-GSH and MC-RR-Cys were 93.6-99%, 68.1-73.6% and 90.0-95.2%, respectively. Method detection limit (MDL) were 4, 7 and 5 ng g(-1) DW for MC-RR, MC-RR-GSH and MC-RR-Cys, respectively. Limits of quantification (LOQs) for MC-RR, MC-RR-GSH and MC-RR-Cys were calculated to be 10, 18 and 13 ng g(-1) DW, respectively. Finally, this method was successfully applied to the identification and quantification of MC-RR, MC-RR-GSH and MC-RR-Cys in the liver of bighead carp with acute exposure of MCs. PMID:20060532

  6. 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 is