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 1 alpha ,25-dihydroxyvitamin D-3 [1 alpha ,25(OH)(2)D-3] 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 C-18 cartridge. The analytical chromatography was performed using a Symmetry C-8 50x2.1 mm, 3.5 mum column. The mobile phase was a linear gradient from 75 to 99% methanol with a constant concentration of 2 mM ammonium acetate, 1 alpha ,25(OH)(2)D-3 and the internal standard [H-2(6)]1 alpha ,25(OH)(2)D-3 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 1 alpha ,25(OH)(2)D-3 in serum from rats and pigs simultaneously with the analysis of the vitamin D analog seocalcitol. (C) 2001 Elsevier Science B.V. All rights reserved.