Simultaneous analysis and retention behavior of the glucuronide, glucoside, and N-acetylglucosaminide conjugates of bile acids in conventional and inclusion high-performance liquid chromatographic methods

Abstract

Simultaneous analysis and structure–retention correlation were examined for various glycosidic-conjugated bile acids in reversed-phase high-performance liquid chromatography (HPLC). The substrates examined included the β-glucuronide, β-glucoside, and β-N-acetylglucosaminide conjugates of C 24 bile acids related to lithocholic, hyodeoxycholic, chenodeoxycholic, ursodeoxycholic, deoxycholic, hyocholic, and cholic acids having an O-β- d-glycosidic linkage at the position C-3, C-6, or C-7 in the 5β-steroid nucleus. The bile acids as their fluorescence-sensitive C 24 pyrenacyl ester derivatives were measured on a C 18 reversed-phase column, eluting with methanol–water mixtures in the absence and presence of methyl-β-cyclodextrin (Me-β-CD). Changes in the retentions of these compounds depended significantly not only on the Me-β-CD concentration but rather on the methanol content, implying the possible formation of solute–Me-β-CD–methanol ternary complexes by hydrogen-bonding interactions. The combined use of conventional and inclusion HPLC retention data reported herewith provided important information for characterizing the structures of bile acid glycosides as well as inclusion complexes.