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 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 microL 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 multiple reaction monitoring mode in which tHcy and Hcy-d(4) 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-d(4) was 1.5 min in a 2.5-min analysis.


RESULTS
Daily calibrations between 2.5 and 60 micromol/L exhibited consistent linearity and reproducibility. At a plasma concentration of 0.8 micromol/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; S(y|x) =1.595 micromol/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; S(y|x) =1.146 micromol/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 micromol/L. Mean recovery of tHcy was 94.2% (20 micromol/L) and 97.8% (50 micromol/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.
Magera
M J
MJ
Mayo Clinic, Department of Laboratory Medicine and Pathology, Biochemical Genetics Laboratory, 200 First St. SW, Rochester, MN 55905, USA.
Lacey
J M
JM
Casetta
B
B
Rinaldo
P
P
D002851:Chromatography, High Pressure Liquid; D016231:Fluorescence Polarization Immunoassay; D006710:Homocysteine / Q000097:blood* / Q000652:urine*; D006801:Humans; D013058:Mass Spectrometry; D011865:Radioisotope Dilution Technique; D012680:Sensitivity and Specificity
D006710:Homocysteine