Analysis of stereoisomeric C27-bile acids by high performance liquid chromatography with fluorescence detection

A method for differentially measuring the 24-hydroxylated stereoisomeric intermediates (3 alpha,7 alpha,12 alpha,24-tetrahydroxy- and 3 alpha,7 alpha,24-trihydroxy-5 beta-cholestan-26-oic acids) and related C27-bile acids in beta-oxidation of bile acid biosynthesis has been developed by high performance liquid chromatography with fluorescence detection. The method involved the derivatization of the above intermediable C27-bile acids into fluorescent esters with 3-(4-bromomethylphenyl)-7-diethylaminocoumarin, a newly synthesized labeling reagent for carboxylic acids. The fluorescent derivatives were subjected to a short silica gel column to eliminate interfering products prior to analysis by high performance liquid chromatography. The separation of the 16 kinds of bile acids containing stereoisomers was carried out using a reversed-phase Inertsil C8-column by gradient elution and detected with a fluorometer (Ex. 400 nm, Em. 475 nm). The linearity of calibration curve for each bile acid was from 0.5 to 250 pmol (r = 0.999) and the detection limits were about 15 fmol at a signal-to-noise ratio of 3. The method was applied to the determination of intermediates in beta-oxidation of bile acid biosynthesis using rat liver homogenate. The results showed that two stereoisomers of 24-hydroxylated C27-bile acids were predominantly produced, indicating the formation of the isomers by the cis-hydration with water.