Liquid chromatography–mass spectrometric method combined with derivatization for determination of 1α-hydroxyvitamin D3 in human plasma

A stable isotope dilution liquid chromatography–tandem mass spectrometric (LC–MS–MS) method for the determination of plasma 1α-hydroxyvitamin D3 [1α(OH)D3] 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)D3 with a detection limit of 6.3 fmol per injection. The plasma 1α(OH)D3 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)D3 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)D3 was also described.