Determination of conjugated bile acids using HPLC and immobilized choloylglycine hydrolase

Abstract

A new HPLC method for the determination of glycine and taurine conjugated bile acids was investigated. The system was based on a postcolumn enzyme reactor using immobilized choloylglycine hydrolase (CGH) linked to fluorescence detection. Conjugated bile acids were separated using HPLC and then hydrolzyed by the immobilized CGH. The hydrolysis yielded free bile acids and the amino acids glycine and taurine.The liberated amino acids were subsequently derivatized to fluorescent o-phthalaldehyde derivative that was monitored with a high degree of specificity and sensitivity by a fluorescence detector. The separation of 8 conjugated bile acids was performed by using this system. The quantitation of glycocholate and taurocholate was demonstrated. Detection limits for these two bile acids were determined.