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Liquid Chromatography Mass Spectrometry (liquid + chromatography_mass_spectrometry)
Selected AbstractsCellular/intramuscular myxoma and grade I myxofibrosarcoma are characterized by distinct genetic alterations and specific composition of their extracellular matrixJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 7 2009Stefan M. Willems Abstract Cellular myxoma and grade I myxofibrosarcoma are mesenchymal tumours that are characterized by their abundant myxoid extracellular matrix (ECM). Despite their histological overlap, they differ clinically. Diagnosis is therefore difficult though important. We investigated their (cyto) genetics and ECM. GNAS1 -activating mutations have been described in intramuscular myxoma, and lead to downstream activation of cFos. KRAS and TP53 mutations are commonly involved in sarcomagenesis whereby KRAS subsequently activates c-Fos. A well-documented series of intramuscular myxoma (three typical cases and seven cases of the more challenging cellular variant) and grade I myxofibrosarcoma (n= 10) cases were karyotyped, analyzed for GNAS1, KRAS and TP53 mutations and downstream activation of c-Fos mRNA and protein expression. ECM was studied by liquid chromatography mass spectrometry and expression of proteins identified was validated by immunohistochemistry and qPCR. Grade I myxofibrosarcoma showed variable, non-specific cyto-genetic aberrations in 83,5% of cases (n= 6) whereas karyotypes of intramuscular myxoma were all normal (n= 7). GNAS1 -activating mutations were exclusively found in 50% of intramuscular myxoma. Both tumour types showed over-expression of c-Fos mRNA and protein. No mutations in KRAS codon 12/13 or in TP53 were detected. Liquid chromatography mass spectrometry revealed structural proteins (collagen types I, VI, XII, XIV and decorin) in grade I myxofibrosarcoma lacking in intramuscular myxoma. This was confirmed by immunohistochemistry and qPCR. Intramuscular/cellular myxoma and grade I myxofibrosarcoma show different molecular genetic aberrations and different composition of their ECM that probably contribute to their diverse clinical behaviour. GNAS1 mutation analysis can be helpful to distinguish intramuscular myxoma from grade I myxofibrosarcoma in selected cases. [source] Determination of glycyrrhetic acid in human plasma by HPLC-MS method and investigation of its pharmacokineticsJOURNAL OF CLINICAL PHARMACY & THERAPEUTICS, Issue 3 2008W.-J. Zhao PhD Summary Objective:, To develop a high performance liquid chromatography mass spectrometry (HPLC-MS) method for the determination of the glycyrrhetic acid (GA) in human plasma and for the investigation of its pharmacokinetics after the oral administration of 150 mg diammonium glycyrrhizinate test and reference capsule formulations. Methods:, The GA in plasma was extracted with ethyl acetate, separated on a C18 column with a mobile phase of methanol (5 mmol/L ammonium acetate),water (85 : 15, V/V) and analysed using a MS detector. Ursolic acid (UA) was used as internal standard. The target ions were m/z 469·5 for GA and m/z 455·6 for UA, the fragment voltages were 200 V and 100 V for GA and UA respectively. Results:, The calibration curve was linear over the range of 0·5,200 ng/mL (r = 0·9974). The limit of quantification for GA in plasma was 0·5 ng/mL, the recovery was 76·0,80·0%, and the inter- and intra-day relative standard deviations (RSD) were <12%. The pharmacokinetic parameters of GA after a single dose of 150 mg diammonium glycyrrhizinate test and reference were as follows: the half life (t1/2) 9·65 ± 3·54 h and 9·46 ± 2·85 h, the time to peak concentration (Tmax) 10·95 ± 1·32 h and 11·00 ± 1·30 h, the peak concentration (Cmax) 95·57 ± 43·06 ng/mL and 103·89 ± 49·24 ng/mL; the area under time-concentration curve (AUC0,48 and AUC0,,) 1281·84 ± 527·11 ng·h/mL and 1367·74 ± 563·27 ng·h/mL, 1314·32 ± 566·40 ng·h/mL and 1396·97 ± 630·06 ng·h/mL. The relative bioavailability of diammonium glycyrrhizinate capsule was 98·88 ± 12·98%. Conclusion:, The assay was sensitive, accurate and convenient, and can be used for the determination of GA in human plasma. Comparison of the bioavailability and pharmacokinetic profile of GA indicated that the test and reference capsules were bioequivalent. [source] A Fatal Case of Suspected Anaphylaxis with Cefoperazone and Sulbactam: LC-MS AnalysisJOURNAL OF FORENSIC SCIENCES, Issue 1 2008Kenji Tsujikawa M.S. Abstract: Cefoperazone and sublactam are prescribed in combination and used in the treatment of moderate to severe bacterial infections. Serious anaphylaxis is a rare side effect. This report describes a fatal case of suspected anaphylaxis after intravenous administration of a combination of the two drugs. Heart blood was analyzed for cefoperazone by protein precipitation with acetonitrile and by liquid-liquid precipitation for sublactam after protein precipitation with aqueous acetonitrile, followed by tandem mass spectrometry in the product ion scan mode for identification and by liquid chromatography mass spectrometry in the selected ion monitoring mode for quantitation. Calibration curves for cefoperazone and sublactam were linear over the range 0.07 to 1.93 and 0.046 to 0.914 ,g/ml respectively. The decedent's blood concentrations of cefoperazone and sublactam were 0.368 and 0.143 ,g/ml respectively. As these concentrations were below concentrations reported after single dosing studies and below those considered to be minimally inhibitory, death was presumed to have been caused by hypersensitivity and not an overdose. In conclusion, this procedure is useful for detecting and quantitating cefoperazone and sublactam in postmortem blood and may be useful in the evaluation of anaphylaxis. [source] Regulation of insulin action by an extract of Artemisia dracunculus L. in primary human skeletal muscle culture: A proteomics approach,PHYTOTHERAPY RESEARCH, Issue 9 2010Indu Kheterpal Abstract An ethanolic extract of Artemisia dracunculus L. (PMI 5011) has been observed to decrease glucose and insulin levels in animal models and enhance cellular signaling in cultured cells. To determine the mechanism of action of PMI-5011, we have measured changes in protein expression in human primary skeletal muscle culture (HSMC) from subjects with Type 2 diabetes. After obtaining skeletal muscle biopsies, HSMCs were initiated, grown to confluence, and exposed to 10,µg/mL PMI 5011 overnight. Two-dimensional difference in-gel electrophoresis was used to separate proteins, and liquid chromatography mass spectrometry was used to identify differentially regulated proteins. Additionally, real-time polymerase chain reaction (PCR) was used to confirm candidate proteins identified. These data demonstrate that a well characterized botanical extract of Artemisia dracunculus L. significantly modulates proteins involved in regulating inflammatory pathways, particularly the NF,B complex system. Copyright © 2010 John Wiley & Sons, Ltd. [source] Proteomics of the rat gut: Analysis of the myenteric plexus-longitudinal muscle preparationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2005Laure Marvin-Guy Abstract The enteric nervous system (ENS) , present all along the gastrointestinal tract , is the largest and most complicated division of the peripheral nervous system that can function independently of the brain. The peripheral nerve cells are organized in two separate but interconnected meshworks, called the myenteric and submucous plexus. The nervous control of intestinal motility is primarily governed by the myenteric plexus (MP), which lies in-between the longitudinal- (LM) and circular-muscle layers and regulates their functions. To determine whether the proteomic technology is adapted to the analysis of specific gut tissues, we dissected the MP-LM layers from the jejunum, ileum, and colon of Long Evans rats, homogenized them, and separated the proteins using two-dimensional gel electrophoresis. A subset of all the visualized protein spots, covering the entire range of molecular weights and isoelectric points, was then selected and further analyzed by matrix-assisted laser desorption/ionization-time of flight and liquid chromatography mass spectrometry. We identified around 80 proteins in each gut segment, and among those, five were segment-specific. Most of the proteins identified were derived from muscle cells, but we also detected some neuron-specific proteins. This study represents, to our knowledge, the first extensive protein catalog of a neuromuscular layer of the rat intestine and it may constitute the basis to understand pathophysiological mechanisms related to the ENS. [source] Comparison of gas chromatography and liquid chromatography mass spectrometric measurements for high accuracy analysis of cholesterol in human serum by isotope dilution mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 9 2002Céline S. J. Wolff Briche Cholesterol measurements are of vital clinical importance and reliable reference materials are essential for method validation. Gas chromatography with mass spectrometry (GC/MS) is usually used for the high accuracy analysis of cholesterol by isotope dilution. A certified reference material for cholesterol content in human serum was analysed by isotope dilution utilising GC/MS and liquid chromatography mass spectrometry (LC/MS). The use of LC/MS avoided the need for a derivatisation step. Both LC/MS and GC/MS produced results on the measurement of cholesterol that agreed within 0.5% of the certified value. Moreover, the precision obtained for ratio measurement using both techniques are comparable and lead to relative expanded standard uncertainties (with a coverage factor of 2) varying between 0.2 and 0.5%. Copyright © 2002 John Wiley & Sons, Ltd. [source] Methotrexate catabolism to 7-hydroxymethotrexate in rheumatoid arthritis alters drug efficacy and retention and is reduced by folic acid supplementationARTHRITIS & RHEUMATISM, Issue 8 2009Joseph E. Baggott Objective To assess the catabolism of methotrexate (MTX) to 7-hydroxy-MTX (7-OH-MTX) in patients with rheumatoid arthritis as well as the effect of folic acid and folinic acid on this catabolism. Methods Urinary excretion of MTX and its catabolite, 7-OH-MTX, was measured in 2 24-hour urine specimens collected after MTX therapy. Urine samples were collected from patients after the sixth and seventh weekly doses of MTX. MTX and 7-OH-MTX concentrations were determined by high-performance liquid chromatography mass spectrometry. Swelling and pain/tenderness indices were used to measure symptoms before and at 6 and 7 weeks of therapy. Patients received either folic acid or folinic acid supplements (1 mg/day) from week 6 to week 7. Results Folic acid inhibited aldehyde oxidase (AO), the enzyme that produces 7-OH-MTX, but folinic acid did not. Excretion of 7-OH-MTX (determined as a percentage of the dose of MTX or as mg 7-OH-MTX/gm creatinine) was not normally distributed (n = 39). Patients with marked improvement in swelling and pain/tenderness indices had a lower mean 7-OH-MTX excretion level (P < 0.05). Patients who received folic acid supplements had decreased 7-OH-MTX excretion (P = 0.03). Relatively high 7-OH-MTX excretion was correlated with relatively high MTX excretion and with relatively low MTX retention in vivo (P < 0.05) (n = 35). Conclusion Our findings of a non-normal distribution of 7-OH-MTX excretion suggest that there are at least 2 phenotypes for this catabolism. Decreased 7-OH-MTX formation suggests folic acid inhibition of AO and a better clinical response, while increased 7-OH-MTX formation may interfere with MTX polyglutamylation and binding to enzymes and, therefore, may increase MTX excretion and decrease MTX retention and efficacy in vivo. [source] Quantification of acetylcholine, an essential neurotransmitter, in brain microdialysis samples by liquid chromatography mass spectrometryBIOMEDICAL CHROMATOGRAPHY, Issue 1 2010Ramakrishna Nirogi Abstract Chemical neurotransmission has been the subject of intensive investigations in recent years. Acetylcholine is an essential neurotransmitter in the central nervous system as it has an effect on alertness, memory and learning. Enzymatic hydrolysis of acetylcholine in the synaptic cleft is fast and quickly metabolizes to choline and acetate by acetylcholinesterase. Hence the concentration in the extracellular fluid of the brain is low (0.1,6,nm). Techniques such as microdialysis are routinely employed to measure acetylcholine levels in living brain systems and the microdialysis sample volumes are usually less than 50,µL. In order to develop medicine for the diseases associated with cognitive dysfunction like mild cognitive impairment, Alzheimer's disease, schizophrenia and Parkinson's disease, or to study the mechanism of the illness, it is important to measure the concentration of acetylcholine in the extracellular fluid of the brain. Recently considerable attention has been focused on the development of chromatographic,mass spectrometric techniques to provide more sensitive and accurate quantification of acetylcholine collected from in-vivo brain microdialysis experiments. This review will provide a brief overview of acetylcholine biosynthesis, microdialysis technique and liquid chromatography mass spectrometry, which is being used to quantitate extracellular levels of acetylcholine. Copyright © 2009 John Wiley & Sons, Ltd. [source] Derivatisation for liquid chromatography/electrospray mass spectrometry: synthesis of pyridinium compounds and their amine and carboxylic acid derivativesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 6 2003Samantha J. Barry A simple method has been developed for the pre-column derivatisation of low molecular weight primary and secondary amines and carboxylic acids using quaternary nitrogen compounds to enhance their detection by liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS). The synthesis of seven novel quaternary nitrogen reagents is described. The derivatives are designed to be relatively small molecules to avoid some of the steric hindrance problems that may be associated with larger derivatisation reagents. The compounds have amine and carboxylic acid functional groups with which to derivatise carboxylic acids and amines, respectively. Two of the compounds contain a bromine atom in order to assess the advantages of a bromine isotope pattern in the mass spectra. This acts as a simple marker for derivatisation and enables data processing by cluster analysis. Activation of the carboxylic acid group was achieved by the use of either 1-chloro-4-methylpyridinium iodide (CMPI) or the more reactive 1-fluoro-4-methylpyridinium p -toluenesulphonate (FMP).1 Using both of these active reagents, the degree of nucleophilic substitution was investigated for the derivatisation of a variety of small molecules. Whilst giving some increase in the ESI-MS response for the derivatised compounds, the FMP itself acted as a derivatising reagent in a competing reaction. In the light of this finding, FMP was reacted with the test compounds separately and gave positive results as a derivatising reagent. Detection of the ,pre-charged' derivatives of amines and carboxylic acids by LC/ESI-MS was investigated with respect to their ESI response and chromatography. Copyright © 2003 John Wiley & Sons, Ltd. [source] Derivatization for liquid chromatography/electrospray mass spectrometry: synthesis of tris(trimethoxyphenyl)phosphonium compounds and their derivatives of amine and carboxylic acidsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 5 2002William J Leavens A simple method for the derivatization of primary amines and carboxylic acids with tris(trimethoxyphenyl)phosphonium (TMPP) reagents to enhance their detection by electrospray mass spectrometry (ESI-MS) has been developed. The synthesis of novel TMPP reagents and their stable isotopically labelled analogues is described. Through the use of stable isotopically labelled TMPP ,tags', incorporation of a doublet (1:1, 1H/2H or 12C/13C) into the target molecule can be achieved, enabling the use of isotopic target analysis to detect compounds of unknown molecular weight but with a characteristic isotope pattern and accurate mass difference. Copyright © 2002 John Wiley & Sons, Ltd. [source] Improved detection of reactive metabolites with a bromine-containing glutathione analog using mass defect and isotope pattern matchingRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 9 2010André LeBlanc Drug bioactivation leading to the formation of reactive species capable of covalent binding to proteins represents an important cause of drug-induced toxicity. Reactive metabolite detection using invitro microsomal incubations is a crucial step in assessing potential toxicity of pharmaceutical compounds. The most common method for screening the formation of these unstable, electrophilic species is by trapping them with glutathione (GSH) followed by liquid chromatography/mass spectrometry (LC/MS) analysis. The present work describes the use of a brominated analog of glutathione, N -(2-bromocarbobenzyloxy)-GSH (GSH-Br), for the invitro screening of reactive metabolites by LC/MS. This novel trapping agent was tested with four drug compounds known to form reactive metabolites, acetaminophen, fipexide, trimethoprim and clozapine. Invitro rat microsomal incubations were performed with GSH and GSH-Br for each drug with subsequent analysis by liquid chromatography/high-resolution mass spectrometry on an electrospray time-of-flight (ESI-TOF) instrument. A generic LC/MS method was used for data acquisition, followed by drug-specific processing of accurate mass data based on mass defect filtering and isotope pattern matching. GSH and GSH-Br incubations were compared to control samples using differential analysis (Mass Profiler) software to identify adducts formed via the formation of reactive metabolites. In all four cases, GSH-Br yielded improved results, with a decreased false positive rate, increased sensitivity and new adducts being identified in contrast to GSH alone. The combination of using this novel trapping agent with powerful processing routines for filtering accurate mass data and differential analysis represents a very reliable method for the identification of reactive metabolites formed in microsomal incubations. Copyright © 2010 John Wiley & Sons, Ltd. [source] Characterization of atenolol transformation products on light-activated TiO2 surface by high-performance liquid chromatography/high-resolution mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 1 2009Claudio Medana No abstract is available for this article. [source] High-throughput analysis in drug discovery: application of liquid chromatography/ion-trap mass spectrometry for simultaneous cassette analysis of ,-1a antagonists and their metabolites in mouse plasmaRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 8 2001Zongwei Cai The application of liquid chromatography/ion-trap mass spectrometry for simultaneous quantification of multiple drugs and detection of their metabolites for in vitro experiments was reported recently. In the current study, the use of these techniques was extended to in vivo pharmacokinetic (PK) studies of ,-1a antagonists. In combination with limited time-point PK, greatly increased throughput was demonstrated for the in vivo screening and investigation of in vivo,in vitro correlation. In addition to quantitative analyses, the technique allowed simultaneous detection of major in vivo metabolites without having to reanalyze the plasma samples. The drugs were individually dosed in mice intravenously via tail vein injection and the blood samples were collected 5,min and 2,h after dosing. After the plasma samples for the different drugs had been prepared separately, they were pooled for cassette analysis. The concentrations of five test compounds in the plasma samples at 2,h ranged from 36,1062,ng/mL, whereas their 5-min plasma levels were similar. From the same cassette analysis, major metabolites in the samples were also detected simultaneously through the interpretation of full-scan mass spectra. The metabolite identification confirmed the results from a previous report that the major sites of metabolism are hydroxylation of the phenyl ring not bearing the alkylsulfonamide substitutent, piperidine N-dealkylation, and N-demethylation of the alkylsulfonamide group. Copyright © 2001 John Wiley & Sons, Ltd. [source] Identification of lysophosphatidylcholine (LPC) and platelet activating factor (PAF) from PC12 cells and mouse cortex using liquid chromatography/multi-stage mass spectrometry (LC/MS3)RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 22 2008Jeffrey C. Smith Lipids play essential roles in cellular structural support, energy storage and signal transduction. Recently, mass spectrometry (MS) has been used to produce three-dimensional maps that elucidate the lipid composition of complex cellular lysates. The identification of individual lipids within these maps is slow and requires the synthesis and spiking of each candidate lipid. We present a novel MS-based technique that rapidly elucidates the atomic connectivity of the fatty acid/alcohol substituent on the sn -1 position of several different families of glycerophosphocholine-containing lipids within the confines of a chromatographic separation. Sodiated lipid species were fragmented to produce radical cations which lost successive methylene groups upon further collisional activation to reveal the identity of the parent molecule. This approach was demonstrated to be effective on isobaric members of the lysophosphatidylcholine (LPC) and platelet activating factor (PAF) families of glycerophospholipids. We demonstrate the application of this technique to unambiguously identify these species within complex cellular lysates and tissue extracts. Copyright © 2008 John Wiley & Sons, Ltd. [source] Rapid detection and characterization of reactive drug metabolites in vitro using several isotope-labeled trapping agents and ultra-performance liquid chromatography/time-of-flight mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 6 2009Timo Rousu Reactive metabolites are believed to be one of the main reasons for unexpected drug-induced toxicity issues, by forming covalent adducts with cell proteins or DNA. Due to their high reactivity and short lifespan they are not directly detected by traditional analytical methods, but are most traditionally analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) after chemical trapping with nucleophilic agents such as glutathione. Here, a simple but very efficient assay was built up for screening reactive drug metabolites, utilizing stable isotope labeled glutathione, potassium cyanide and semicarbazide as trapping agents and highly sensitive ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOFMS) as an analytical tool. A group of twelve structurally different compounds was used as a test set, and a large number of trapped metabolites were detected for most of them, including many conjugates not reported previously. Glutathione-trapped metabolites were detected for nine of the twelve test compounds, whereas cyanide-trapped metabolites were found for eight and semicarbazide-trapped for three test compounds. The high mass accuracy of TOFMS provided unambiguous identification of change in molecular formula by formation of a reactive metabolite. In addition, use of a mass defect filter was found to be a usable tool when mining the trapped conjugates from the acquired data. The approach was shown to provide superior detection sensitivity in comparison to traditional methods based on neutral loss or precursor ion scanning with a triple quadrupole mass spectrometer, and clearly more efficient detection and characterization of reactive drug metabolites with a simpler test setup. Copyright © 2009 John Wiley & Sons, Ltd. [source] Isotopic pattern and accurate mass determination in urine drug screening by liquid chromatography/time-of-flight mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 7 2006Suvi Ojanperä An efficient method was developed for toxicological drug screening in urine by liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry. The method relies on a large target database of exact monoisotopic masses representing the elemental formulae of reference drugs and their metabolites. Mass spectral identification is based on matching measured accurate mass and isotopic pattern (SigmaFitÔ) of a sample component with those in the database. Data post-processing software was developed for automated reporting of findings in an easily interpretable form. The mean and median of SigmaFitÔ for true-positive findings were 0.0066 and 0.0051, respectively. The mean and median of mass error absolute values for true-positive findings were 2.51 and 2.17,ppm, respectively, corresponding to 0.65 and 0.60,mTh. For routine screening practice, a SigmaFitÔ tolerance of 0.03 and a mass tolerance of 10,ppm were chosen. Ion abundance differences from urine extracts did not affect the accuracy of the automatically acquired SigmaFitÔ or mass values. The results show that isotopic pattern matching by SigmaFitÔ is a powerful means of identification in addition to accurate mass measurement. Copyright © 2006 John Wiley & Sons, Ltd. [source] | |||||||||||||