SEPARATION AND CHARACTERIZATION OF CARBOXYL-LINKED GLUCURONIDES OF BILE-ACIDS IN INCUBATION MIXTURE OF RAT-LIVER MICROSOMES

The carboxyl-linked 24-glucuronides of common bile acids have been ide ntified by means of liquid chromatography (LC)/atmospheric pressure ch emical ionization (APCI)-mass spectrometry (MS) in an incubation mixtu re with a male Wistar rut liver microsomal fraction. The authentic spe cimens of bile acid 24-glucuronide acetate-methyl esters were synthesi zed unequivocally using the Mitsunobu reaction, and the APCI-mass spec trometric properties of these glucuronide derivatives were also charac terized. After incubation of common unconjugated bile acids with hepat ic microsomes, glucuronides were extracted and purified with a Sep-Pak C-18 cartridge and lipophilic ion exchange gel, piperidino-hydroxypro pyl Sephadex LH-20, and then derivatized into the acetate-methyl ester s. Subsequent resolution into alpha- and beta-isomers at the glucurono syl linkage was attained by LC on Cosmosil 5C(8) and Sumichiral OA-250 0 columns using 200 mM ammonium acetate (pH 7.0)-methanol (1:4, v/v), where 24-glucuronides,were monitored with characteristic positive ions [M + NH4](+). The 24-glucuronides of lithocholic, chenodeoxycholic, d eoxycholic, ursodeoxycholic and cholic acid were definitely characteri zed, in contrast to no formation of corresponding 3-glucuronides, (C) 1998 by Elsevier Science Inc.