title: Glucose and glycerol concentrations and their tracer enrichment measurements using liquid chromatography tandem mass spectrometry.
abstract: 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 glycerol were derivatized using benzoyl chloride in order to form benzoylated derivatives via new simplified fast procedures. For glucose, two internal standards were evaluated, [U-(13) C(6)]glucose and [U-(13) C(6), D(7)]glucose, and for glycerol, [U-(13) C(3), D(8)]glycerol was used. The method was validated by means of calibration curves, quality control samples, and plasma samples spiked with [6,6-D(2)]glucose, [U-(13) C(6)]glucose, and [1,1,2,3,3-D(5)]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 r(2) = 0.9998 for [U-(13) C(6)]glucose and r(2) = 0.9996 for [U-(13) C(6), D(7)]glucose, and r(2) = 0.9995 for the glycerol concentration. Interday accuracy for glucose using [U-(13) C(6)]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 14 days. 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.
authors.affiliation: Clinical Metabolomics Core Facility (CMCF), Rigshospitalet, section 7652, Ole Maaløesvej 26, 2100, Copenhagen Ø, Denmark.
authors.forename: Andreas
authors.initials: A
authors.lastname: Bornø
authors.forename: Lene
authors.initials: L
authors.lastname: Foged
authors.forename: Gerrit
authors.initials: G
authors.lastname: van Hall
mesh_terms: D001786:Blood Glucose / Q000032:analysis / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods*; D005990:Glycerol / Q000097:blood / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D016014:Linear Models; D008066:Lipolysis; D055432:Metabolomics; D010949:Plasma / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D001786:Blood Glucose; D005990:Glycerol
keywords: glucose enrichment; glycerol enrichment; in vivo lipolytic rate; stable isotopes; tracers