Total Plasma Homocysteine Measured by Liquid Chromatography–Tandem Mass Spectrometry with Use of 96-Well Plates

Abstract

Increased total plasma or serum homocysteine is regarded as a risk factor for occlusive arterial or venous disease [reviewed in Refs. (1), (2)]. Homocysteine is measured by HPLC (3), immunoassays (4), or liquid chromatography–mass spectrometry (5)(6)(7)(8)(9). We were faced with a large workload of ∼200 samples/day, plus high material costs for commercially available immuno- and HPLC assays and laborious sample pretreatment for our HPLC application. We thus wished to establish a high-throughput liquid chromatography–tandem mass spectrometry (LC-MS/MS) method. We aimed at a 96-well plate format for sample pretreatment and autosampling without protein precipitation, centrifugation, or derivatization steps. We pipetted 50 μL of NaF-plasma, calibrator, or quality-control sample into each well of a 96-well plate (well volume, 2.2-mL) and added to each well 1000 μL of 0.4 μmol/L homocystine-d8 (3,3,3′,3′,4,4,4′,4′-d8-homocystine; Cambridge Isotope Laboratories). The plate was sealed with Parafilm (the tightness was tested with colored solutions) and mixed by vigorous shaking. Of this mixture, 50 μL was transferred to each well of a 96-well plate (well volume, 1.2 mL), diluted with 500 μL of dilute NaOH (50 μL of 6 mol/L NaOH in 500 mL of water), and reduced by the addition of 25 μL of 200 mmol/L dithiothreitol (threo-1,4-mercapto-2,3-butandiol; Aldrich). The plate was sealed (see above), shaken to mix the contents of the wells, left at room …