Sensitive analysis of 1α,25-dihydroxyvitamin D3 in biological fluids by liquid chromatography–tandem mass spectrometry

A liquid chromatographic–tandem mass spectrometric assay using 5% bovine serum albumin as the calibration matrix has been developed for the quantitative analysis of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] in biological fluids. The analyte was extracted from the matrix after protein precipitation using an automated solid-phase extraction procedure involving both a reversed-phase and normal-phase procedure on a single C18 cartridge. The analytical chromatography was performed using a Symmetry C8 50×2.1 mm, 3.5 μm column. The mobile phase was a linear gradient from 75 to 99% methanol with a constant concentration of 2 mM ammonium acetate. 1α,25(OH)2D3 and the internal standard [2H6]1α,25(OH)2D3 were detected by using MS–MS. The ion source was operated in the positive electrospray ionisation mode. The assay is specific, sensitive, and has a capacity of more than 100 samples per day, with a limit of quantitation of 20 pg ml−1 for a 1.0-ml sample aliquot. The assay has been used for the analysis of 1α,25(OH)2D3 in serum from rats and pigs simultaneously with the analysis of the vitamin D analog seocalcitol.