Abstract
A stable isotope dilution liquid chromatography–tandem mass spectrometric (LC–MS–MS) method for the determination of plasma 1α-hydroxyvitamin D 3 [1α(OH)D 3] has been developed. The method employed derivatization, the reaction with 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione and acetylation, which significantly improved the ionization efficiency of 1α(OH)D 3 with a detection limit of 6.3 fmol per injection. The plasma 1α(OH)D 3 was extracted with acetonitrile, purified with disposable cartridges, derivatized and subjected to LC–MS–MS analysis using atmospheric pressure chemical ionization. The intra- and inter-assay coefficients of variation were below 10.6 and 4.7%, respectively, and the analytical recovery of 1α(OH)D 3 was quantitative. The limit of quantitation was 25 pg/ml for a 1.0-ml plasma aliquot. The application of the developed method to the sample of a volunteer orally given 1α(OH)D 3 was also described.
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