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Clinical Chemistry 50: 755-757, 2004; 10.1373/clinchem.2003.029686
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(Clinical Chemistry. 2004;50:755-757.)
© 2004 American Association for Clinical Chemistry, Inc.

Technical Briefs

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

Torsten Arndt1,a, Brunhilde Guessregen1, Axel Hohl1 and Bernd Heicke1

1 Bioscientia GmbH, Konrad-Adenauer-Strasse 17, D-55218 Ingelheim, Germany

aauthor for correspondence: fax 49-6132-781-428, e-mail torsten.arndt@bioscientia.de

The first 20% of the full text of this article appears below.

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 . . . [Full Text of this Article]




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