Determination of the vitamin D analog EB 1089 (seocalcitol) in human and pig serum using liquid chromatography–tandem mass spectrometry

A liquid chromatographic–tandem mass spectrometric assay in human and pig serum has been developed for quantitative analysis of EB 1089 (seocalcitol). EB 1089 is a novel vitamin D analog under development for the treatment of cancer. The analyte was extracted from serum 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 Symmetri 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. EB 1089 and the internal standard [d6]-EB 1089 were detected by using MS–MS. The ion source was operated in the positive electrospray ionisation (ESI) mode. The assay is specific, sensitive, and has a capacity of more than 100 samples per day, with a limit of quantitation of 10 pg ml−1 for a 1.0-ml sample aliquot. It is now used for routine analysis in connection with pharmacokinetic studies in humans and toxicokinetic studies in pigs.