Method for the determination of total homocysteine in plasma and urine by stable isotope dilution and electrospray tandem mass spectrometry

Background: Total homocysteine (tHcy) has emerged as an important independe nt risk factor for cardiovascular disease. Analytical methods are needed to accommodate the high testing volumes for tHcy and provide rapid turnaround . Methods: We developed liquid chromatography electrospray tandem mass spectr ometry (LC-MS/MS) method based on the analysis of 100 mu L of either plasma or urine with homocystine-d(8) (2 nmol) added as internal standard. After sample reduction and deproteinization, the analysis was performed in the mu ltiple reaction monitoring mode in which tHcy and Hcy-d(4) were detected th rough the transition from the precursor to the product ion (m/z 136 to m/z 90 and m/z 140 to m/z 94, respectively). The retention time of tHcy and Hcy -d(4) was 1.5 min in a 2.5-min analysis. Results: Daily calibrations between 2.5 and 60 mu mol/L exhibited consisten t linearity and reproducibility. At a plasma concentration of 0.8 mu mol/L, the signal-to-noise ratio for tHcy was 17:1. The regression equation for t he comparison between our previous HPLC method (y) and the LC-MS/MS method (x) was y = 1.097x - 1.377 (r = 0.975; S-y/x = 1.595 mu mol/L; n = 367), an d for comparison between a fluorescence polarization immunoassay (Abbott IM x; y) and LC-MS/MS (x) was y = 1.039x + 0.025 (r = 0.969; S-y/x = 1.146 mu mol/L; n = 367). Inter- and intraassay CVs were 2.9-5.9% and 3.6-5.3%, resp ectively, at mean concentrations of 3.9, 22.7, and 52.8 mu mol/L. Mean reco very of tHcy was 94.2% (20 mu mol/L) and 97.8% (50 mu mol/L). Conclusions: The sensitivity and specificity of tandem mass spectrometry ar e well suited to perform high-volume analysis of tHcy. Reagents are inexpen sive and sample preparation of a batch of 40 specimens is completed in less than 1 h and is amenable to automation. (C) 1999 American Association for Clinical Chemistry.