Sensitive analysis of 1alpha,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 1alpha,25-dihydroxyvitamin D3 [1alpha,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 x 2.1 mm, 3.5 microm column. The mobile phase was a linear gradient from 75 to 99% methanol with a constant concentration of 2 mM ammonium acetate. 1alpha,25(OH)2D3 and the internal standard [2H6]1alpha,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 1alpha,25(OH)2D3 in serum from rats and pigs simultaneously with the analysis of the vitamin D analog seocalcitol.
Kissmeyer
A M
AM
Department of Pharmacokinetics and Metabolism, Leo Pharmaceutical Products, Ballerup, Denmark. anne-marie.kissmeyer@leo-pharma.com
Sonne
K
K
D000818:Animals; D002117:Calcitriol / Q000031:analogs & derivatives / Q000097:blood*; D002138:Calibration; D006801:Humans; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D013552:Swine
D002117:Calcitriol; C078903:seocalcitol