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-hydroxyvitamin 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 specimens 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 chromatographic behavior, that of its fluorescent labeled derivative using 4-[4-(6-methoxy-2-benzoxazolyl)phenyl]-1,2,4-triazoline-3,5-dione and data obtained following enzymatic hydrolysis using beta-glucuronidase.