SEPARATION AND CHARACTERIZATION OF MONOGLUCURONIDES OF VITAMIN-D-3 AND 25-HYDROXYVITAMIN-D-3 IN RAT BILE BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

The separation and characterization of vitamin D-3- and 25-hydroxyvita min D-3-monoglucuronides, biliary metabolites obtained from rats dosed with D-3 and 25-hydroxyvitamin D-3 per os, respectively, were carried out by HPLC. The glucuronide fractions were obtained from bile specim ens by the combined use of a Bond Elut C18 cartridge, for solid phase extraction, and a lipophilic gel (piperidinohydroxypropyl Sephadex LH- 20), for ion-exchange chromatography. Each glucuronide was identified by comparison with an authentic sample in three ways: its chromatograp hic behavior, that of its fluorescent labeled derivative using -2-benz oxazolyl)phenyl]-1,2,4-triazoline-3,5-dione and data obtained followin g enzymatic hydrolysis using beta-glucuronidase.