Pre-column derivatization of free bile acids for high-performance liquid chromatographic and gas chromatographic-mass spectrometric analysis

Pentachlorophenyl (PCP) esters of five free bile acids (FBA) were obtained by reacting the FBA and Kovacs' complex (KC) in a 1:8 molar ratio in acetone at 65 degrees C, and were purified by column chromatography on silica gel. The esters were crystallized from benzene-hexane, derivatized as trimethylsilyl ethers for gas chromatography on a DB-1 capillary column and for gas chromatography-mass spectrometry with a DB-5 column, and mass spectrometry (MS) in the electron-impact (EI) positive-ion mode at 70 eV. The reaction is specific for FBA even in the presence of glycine and taurine conjugates of bile acids. The PCP esters were treated with benzylamine in chloroform or methanol to produce N-benzyl derivatives of FBA. The N-benzylamides were separated by high-performance liquid chromatography (HPLC) on a 4-microns Nova-Pak C18 column, studied by thermospray-LC-MS, and in the direct insertion probe-EI positive-ion mode.