title: Aqueous normal phase chromatography improves quantification and qualification of homocysteine, cysteine and methionine by liquid chromatography-tandem mass spectrometry.
abstract: Elevation of plasma homocysteine concentration is recognized as an independent predictor of cardiovascular disease risk. Therefore, quantification of homocysteine and related sulphur amino acids cysteine and methionine from plasma samples is routinely performed in clinical laboratories. Due to the highly hydrophilic character of these amino acids, previously reported LC-MS methods often suffered from very short chromatographic retention resulting in inadequate separation from matrix background and possible co-eluents. In the present method, aqueous normal phase (ANP) chromatography was introduced to improve chromatographic separation for liquid chromatography-electrospray ionization tandem mass spectrometry. Selective qualification of analytes and internal standards was achieved by qualifier ion monitoring. Using this enhanced selectivity, spurious co-eluents were identified and separated from the analyte signal by optimization of chromatographic conditions. Method validation proved high precision and accuracy (intra-assay reproducibility 1.2-4.3% CV, inter-assay reproducibility 3.4-6.1% CV, accuracy 91.3-105.9%). Total cycle time of 7 min and low costs per sample allow high-throughput application in clinical diagnostics and research trials.
authors.affiliation: Division of Metabolism and Nutritional Medicine, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-University of Munich, Lindwurmstr. 4, D-80337 Munich, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Hellmuth
authors.forename: Berthold
authors.initials: B
authors.lastname: Koletzko
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Peissner
mesh_terms: D002318:Cardiovascular Diseases; D002853:Chromatography, Liquid / Q000379:methods*; D003545:Cysteine / Q000097:blood* / Q000737:chemistry; D006710:Homocysteine / Q000097:blood* / Q000737:chemistry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007118:Immunoassay; D016014:Linear Models; D008715:Methionine / Q000097:blood* / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D006710:Homocysteine; D008715:Methionine; D003545:Cysteine