SEPARATION OF BILE-ACID METHYL-ESTERS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Conjugated bile acids, namely glyco- and tauro-3alpha,6alpha-dihydroxy-5beta-cholanoic acid (hyodeoxycholic acid), 3alpha,7alpha-dihydroxy-5beta-cholanoic acid (chenodeoxycholic acid), 3alpha,6alpha,7alpha-trihydroxy-5beta-cholanoic acid (hyocholic acid) and 3alpha-hydroxy-6-oxo-5beta-cholanoic acid (6-keto-litocholic acid) were isolated from pig bile, and subsequently transformed into the corresponding methyl esters. Separation of the methyl esters of the isolated bile acids by high-performance liquid chromatography (HPLC) was accomplished on a ZORBAX-CN column (Dupont, Boston, MA) with n-hexane/2-propanol/methylene chloride (89:6:5, by vol) as the mobile phase containing traces (almost-equal-to 1%) of amyl alcohol and water as moderators. HPLC analysis of the methyl esters also showed the presence of methyl 3alpha-hydroxy-6-oxo-5alpha-cholanoate, which was probably produced in the course of alkaline hydrolysis of the conjugated bile acids.