Analysis and structure determination of unsaturated 5β‐cholanoic acids

Abstract

Unsaturated cholanoic acids are known to arise as artifacts of chemical transformation processes and during storage and high-temperature gas liquid chromatography (GLC) of various derivatives of saturated bile acids. Nevertheless, there is evidence for their natural occurrence and isolation under conditions where artifactual formation of unsaturated bile acids would be unlikely. Since structural identification of such compounds is often complicated by a lack of knowledge of their analytical properties, a representative series of monounsaturated cholanoic acids with double bonds in rings A, B and C were prepared by POCl₃ and ZnCl₂ dehydration of saturated bile acids with selectively blocked hydroxyl functions. The cholenoic acids were indistinguishable from their saturated analogs by thin layer chromatography (TLC) on plain silica gel, but those compounds with sterically exposed double bonds were resolved by AgNO₃-TLC, using chloroform/methanol solvent systems. The synthetic 5β-cholenoic acids obeyed the general rules of GLC mobility based on the overall shape of the molecule and the number and configuration of the functional groups. Constant retention factors attributable to the double bond were observed for all of the double bond types on several GLC phases, and theoretical retention times could be calculated for combinations of double bonds and functional groups not specifically represented among the synthetic standards. With gas chromatography-mass spectrometry (GC-MS), the unsaturated bile acids gave several characteristic fragments, which, in conjunction with the chromatographic properties of the parent compounds, permitted an unambiguous distinction among different unsaturated acids, and between unsaturated and saturated bile acids of the same number and configuration of functional groups. For complete structural identification of saturated and unsaturated bile acids, capillary GC-MS represents the ideal state of the art, but the less expensive combination of AgNO₃-TLC and GLC also can yield much useful information concerning the structure of natural and synthetic 5β-cholenoic acids. This study emphasizes the need for special precautions in the isolation and derivatization of bile acids intended for studies of unsaturated components.