Liquid chromatography--mass spectrometric method combined with derivatization for determination of 1 alpha-hydroxyvitamin D(3) in human plasma.

A stable isotope dilution liquid chromatography-tandem mass spectrometric (LC-MS-MS) method for the determination of plasma 1 alpha-hydroxyvitamin D(3) [1 alpha(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 1 alpha(OH)D(3) with a detection limit of 6.3 fmol per injection. The plasma 1 alpha(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 1 alpha(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 1 alpha(OH)D(3) was also described.