Simultaneous quantitation of homocysteine, cysteine and methionine in plasma and urine by liquid chromatography-tandem mass spectrometry

Abstract

BACKGROUND: A method utilizing liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) has been developed and evaluated for the determination of total homocysteine, cysteine and methionine in plasma and urine. The simultaneous measurement of homocysteine and methionine concentrations may help explain the underlying mechanism responsible for hyperhomo-cysteinaemia. METHODS: Samples were prepared by simple protein precipitation after reduction of disulphides by dithiothreitol. Reduced analyte signal caused by ionization suppression effects, seen with plasma samples, was compensated for with matrix-matched standards, and the use of isotopically labelled internal standards. Recovery for each analyte was better than 94%. RESULTS: Concentrations of plasma homocysteine determined by LC-MS/MS were compared with those obtained by two automated commercially available FDA-approved procedures: (i) high-performance liquid chromatography (HPLC) with pre-column derivatization and fluorescence detection and (ii) by fluorescence polarization immunoassay (FPIA). Agreement with the LC-MS/MS method is given by the Deming regression equations LC-MS/MS = 1.062 HPLC-0.01 and LC-MS/MS = 1.104 FPIA-0.43. CONCLUSION: Low reagent costs together with the relative simplicity of sample preparation make the LC-MS/MS method well suited, not only for research work but also in those laboratories with a tandem mass spectrometer, for the measurement of routine clinical samples.