Improved and simplified LC–ESI-MS/MS method for homocysteine determination in human plasma: Application to the study of cardiovascular diseases

A liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) method was developed and validated for the determination of human plasma homocysteine (Hcy), an important independent risk factor for cardiovascular disease, with a simplified sample pretreatment procedure and a zero blank free of endogenous Hcy for calibrator/QC preparation. Following protein precipitation, chromatographic separation was performed on Hypersil Aquasil C18 column (50 mm × 2.1 mm, 5 μm, Thermo) using mobile phase of aqueous 10% methanol containing 0.02% formic acid at 0.25 mL/min. Hcy and deuterated internal standard were detected in the multiple reaction monitoring mode with precursor to product ion transitions of m/z 136.1/90.0 and 140.1/94.0, respectively. The retention time was 1.2 min, and the total run time was 2 min per injection. A streamlined three-point calibration curve and one-point QC was used. Excellent linearity was observed with correlation coefficient (r) > 0.99. The intra- and inter-batch were ≤3.24% and ≤4.04%, and accuracy was within ±10%. Method comparison between the proposed method (y) and FPIA assay (x) demonstrated a correlation equation of y = 1.003x + 0.4318 (r = 0.9589). The developed method, improved for automation with cost-effective reagents, was proven to be suitable for high-throughput quantitative determination of Hcy in clinical practice by successfully applying it to the cardiovascular disease study.