Chromatographic determination of bile acids in biological fluids with sensitive and selective detection

Separation and determination of bile acids in biological fluids by high-performance liquid chromatography are reviewed. The capacity ratios of bile acids on an ODS column were affected by the number, position and configuration of the hydroxyl group on the steroid nucleus, and the chromatographic behavior was markedly influenced by the pH of a mobile phase according to the conjugated form at C-24. A new pre-column derivatization reagent, 1-anthroyl nitrile, was developed and applied to the analysis of bile acids in biological fluids. Bile acids were derivatized through the 3 alpha-hydroxyl group into the corresponding esters, separated on an ODS column, and monitored by a fluorescence detector with detection limit of 20 fmol. The sensitive method for the determination of bile acids in biological materials by gas chromatography (GC) in combination with negative ion chemical ionization (NICI) mass spectrometry is also described. Of various derivatives for the carboxyl group, the pentafluorobenzyl (PFB) ester provided the highest value of the ratio of the negative to positive ion current. A characteristic carboxylate anion M-181]- was produced as the most abundant ion by the loss of the PFB group in NICI. PFB esters of bile acids were further derivatized into the dimethylethylsilyl ethers and then separated by GC. The detection limit was 2 fg when the characteristic anion was monitored in the NICI mode. The preparation of 18O-labelled bile acids, as the internal standard for the trace analysis or the tracer for the metabolic study, was developed. Finally, the clean-up procedure for bile acids in biological fluids was investigated. The combined use of solid-phase extraction with a Sep-pak C18 or Bond Elut cartridge and group separation on a lipophilic ion-exchange gel, piperidinohydroxypropyl Sephadex LH-20, was found most effective for this purpose.