Separation and Characterization of Carboxyl-linked Glucuronides of Bile Acids in Incubation Mixture of Rat Liver Microsomes

Abstract

The carboxyl-linked 24-glucuronides of common bile acids have been identified by means of liquid chromatography (LC)/atmospheric pressure chemical ionization (APCI)-mass spectrometry (MS) in an incubation mixture with a male Wistar rat liver microsomal fraction. The authentic specimens of bile acid 24-glucuronide acetate-methyl esters were synthesized unequivocally using the Mitsunobu reaction, and the APCI-mass spectrometric properties of these glucuronide derivatives were also characterized. After incubation of common unconjugated bile acids with hepatic microsomes, glucuronides were extracted and purified with a Sep-Pak C 18 cartridge and lipophilic ion exchange gel, piperidino-hydroxypropyl Sephadex LH-20, and then derivatized into the acetate-methyl esters. Subsequent resolution into α- and β-isomers at the glucuronosyl linkage was attained by LC on Cosmosil 5C 8 and Sumichiral OA-2500 columns using 200 mM ammonium acetate (pH 7.0)-methanol (1:4, v/v), where 24-glucuronides were monitored with characteristic positive ions [M + NH 4] +. The 24-glucuronides of lithocholic, chenodeoxycholic, deoxycholic, ursodeoxycholic and cholic acid were definitely characterized, in contrast to no formation of corresponding 3-glucuronides.