CHARACTERIZATION OF THE METABOLIC PATHWAY OF 1,25-DIHYDROXY-16-ENE VITAMIN-D-3 IN RAT-KIDNEY BY ONLINE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY-ELECTROSPRAY TANDEM MASS-SPECTROMETRY

1,25-Dihydroxy-16-ene vitamin D-3 is a synthetic analog of 1,25-dihydroxyvitamin D-3, the most physiologically active metabolite of vitamin D-3. The renal metabolism of 1,25-dihydroxy-16-ene vitamin D-3 had been studied previously using a perfused rat kidney system [Reddy et al., Bioorg Med Chem Lett 3: 1879-1884, 1993], and its C-24 oxidative metabolic pathway had been found to be different from that of 1,25-dihydroxyvitamin D-3 by HPLC. To further delineate the differences between the C-24 oxidative metabolic pathways of 1,25-dihydroxyvitamin D-3 and 1,25-dihydroxy-16-ene vitamin D-3 in this present study we investigated the C-24 oxidation pathway of 1,25-dihydroxy-16-ene vitamin D-3 using a novel detection approach based on on-line capillary liquid chromatography coupled to electrospray tandem mass spectrometry. Two types of tandem mass spectrometric detection were employed to characterize the metabolites in the kidney perfusate: (a) the preliminary screening of metabolites by parent scan, which led to the tentative discovery of the production of 1,23,25-trihydroxy-24-oxo-16-ene vitamin D-3, a new metabolite of 1,25-dihydroxy-16-ene vitamin D-3, and (b) the pharmacokinetic studies of; the substrate, 1,25-dihydroxy-16-ene vitamin D-3 and its metabolites by multiple reaction monitoring. In the latter, the mass spectrometric sensitivity for quantification was found to be about 20-fold better than UV detection. The current work concluded that the C-24 oxidative metabolic pathway of 1,25-dihydroxy-16-ene vitamin D-3 closely mimicked that of its natural counterpart. Furthermore, the use of mass spectrometry permitted the clearance rate of the starting substrate to be studied at a more physiological level (ng/mL or submicromolar level), which had not been possible previously by HPLC-UV detection.