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Clinical Chemistry 45: 1517-1522, 1999;
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(Clinical Chemistry. 1999;45:1517-1522.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Method for the Determination of Total Homocysteine in Plasma and Urine by Stable Isotope Dilution and Electrospray Tandem Mass Spectrometry

Mark J. Magera1, Jean M. Lacey1, Bruno Casetta2 and Piero Rinaldo1,a

1 Mayo Clinic, Department of Laboratory Medicine and Pathology, Biochemical Genetics Laboratory, 200 First St. SW, Rochester, MN 55905.

2 PE Biosystems, European Life Science Center, Langen 63225, Germany.
a Author for correspondence. Fax 507-266-4176; e-mail: rinaldo{at}mayo.edu

Background: Total homocysteine (tHcy) has emerged as an important independent 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 spectrometry (LC-MS/MS) method based on the analysis of 100 µL of either plasma or urine with homocystine-d8 (2 nmol) added as internal standard. After sample reduction and deproteinization, the analysis was performed in the multiple reaction monitoring mode in which tHcy and Hcy-d4 were detected through 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-d4 was 1.5 min in a 2.5-min analysis.

Results: Daily calibrations between 2.5 and 60 µmol/L exhibited consistent linearity and reproducibility. At a plasma concentration of 0.8 µmol/L, the signal-to-noise ratio for tHcy was 17:1. The regression equation for the comparison between our previous HPLC method (y) and the LC-MS/MS method (x) was y = 1.097x - 1.377 (r = 0.975; Sy|x =1.595 µmol/L; n = 367), and for comparison between a fluorescence polarization immunoassay (Abbott IMx; y) and LC-MS/MS (x) was y = 1.039x + 0.025 (r = 0.969; Sy|x =1.146 µmol/L; n = 367). Inter- and intraassay CVs were 2.9–5.9% and 3.6–5.3%, respectively, at mean concentrations of 3.9, 22.7, and 52.8 µmol/L. Mean recovery of tHcy was 94.2% (20 µmol/L) and 97.8% (50 µmol/L).

Conclusions: The sensitivity and specificity of tandem mass spectrometry are well suited to perform high-volume analysis of tHcy. Reagents are inexpensive and sample preparation of a batch of 40 specimens is completed in less than 1 h and is amenable to automation.




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