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Borno A.,Clinical Metabolomics Core Facility CMCF | Foged L.,Clinical Metabolomics Core Facility CMCF | Van Hall G.,Clinical Metabolomics Core Facility CMCF | Van Hall G.,Copenhagen University
Journal of Mass Spectrometry | Year: 2014

The present study describes a new liquid chromatography tandem mass spectrometry method for high-throughput quantification of glucose and glycerol in human plasma using stable isotopically labeled internal standards and is suitable for simultaneous measurements of glucose and glycerol enrichments in connection to in vivo metabolic studies investigating glucose turnover and lipolytic rate.Moreover, in order to keep up with this new fast analysis, simple derivatization procedures have been developed. Prior to analysis, glucose and glycerolwere derivatized using benzoyl chloride in order to form benzoylated derivatives via new simplified fast procedures. For glucose, two internal standards were evaluated, [U-13C6]glucose and [U-13C6, D7]glucose, and for glycerol, [U-13C3, D8]glycerol was used. The method was validated by means of calibration curves, quality control samples, and plasma samples spiked with [6,6-D2]glucose, [U-13C6]glucose, and [1,1,2,3,3-D5]glycerol in order to test accuracy, precision, and recovery of the method. Moreover, post preparative and freeze-thaw sample stability were tested. The correlation of calibration curves for the glucose concentration were r2 = 0.9998 for [U-13C6]glucose and r2 = 0.9996 for [U-13C6, D7]glucose, and r2 = 0.9995 for the glycerol concentration. Interday accuracy for glucose using [U-13C6]glucose and glycerol determined in spiked plasma were respectively 103.5% and 106.0%, and the coefficients of variation were 2.0% and 9.7%, respectively. After derivatization, plasma samples were stable for at least 14days. In conclusion, we have developed and validated a novel, accurate, and sensitive high-throughput liquid chromatography tandem mass spectrometry method for simultaneous determination of glucose and glycerol concentrations and enrichment of infused tracers most commonly used in human metabolic kinetic studies. Copyright © 2014 John Wiley & Sons, Ltd. © 2014 John Wiley & Sons, Ltd. Source


Borno A.,Clinical Metabolomics Core Facility CMCF | Van Hall G.,Clinical Metabolomics Core Facility CMCF | Van Hall G.,Copenhagen University
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2014

An important area within clinical functional metabolomics is in vivo amino acid metabolism and protein turnover measurements for which accurate amino acid concentrations and stable isotopically labeled amino acid enrichments are mandatory not the least when tissue metabolomics is determined. The present study describes a new sensitive liquid chromatography tandem mass-spectrometry method quantifying 20 amino acids and their tracer(s) ([ring-13C6]/D5Phenylalanine) in human plasma and skeletal muscle specimens. Before analysis amino acids were extracted and purified via deprotonization/ion exchange, derivatized using a phenylisothiocyanate reagent and each amino acid was quantitated with its own stable isotopically labeled internal standard (uniformly labeled-13C/15N). The method was validated according to general recommendations for chromatographic analytical methods. The calibration curve correlations for amino acids were on average; r2=0.998. Interday accuracy for amino acids determined in spiked plasma was on average 97.3% and the coefficient of variation (CV) was 2.6%. The ([ring-13C6]/D5Phenylalanine) enrichment CV's for machine reproducibility in muscle tissue fluid and plasma were 4.4 and 0.8%, and the interday variability was 3.4% and the recovery was 90.5%, respectively. In conclusion, we have developed and validated a method for quantitative amino acid profiling that meets the requirements for systemic and tissue human in vivo amino acid and protein turnover kinetics measurements. Moreover, citrulline, ornithine, π-methyl-histidine, τ-methyl- l-histidine, hydroxy-proline and carnitine were analysed but when similar precision and accuray are required an additional stable istopically labeled internal standard for these meatablites should be be added. © 2014 Elsevier B.V. Source

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