High performance liquid chromatographic determination of free and conjugated bile acids in serum, liver biopsies, bile, gastric juice and feces by fluorescence labeling.

ABSTRACT

Separation and measurement of commonly occurring free and conjugated bile acids in serum, liver biopsies, bile, gastric juice and feces have been successfully accomplished using high performance liquid chromatography with fluorescence labeling. Free and conjugated bile acids were extracted from pretreated samples using Sep-Pak C18 cartridges and then fractionated on a piperidinohydroxypropyl Sephadex LH-20 column. Free and glycine conjugated bile acids were labelled with 4-bromomethyl-7-methoxycoumarin. Taurine conjugated bile acids were hydrolysed with cholylglycine hydrolase prior to derivatization with 4-bromomethyl-7-methoxycoumarin. Labelled bile acids were eluted using an acetonitrile/methanol/water gradient on an ultrasphere ODS column. The eluate was monitored by a fluorophotometer at 360 nm (excitation) and 410 nm (emission). Linearity was obtained between 50 and 400 nmol. Recoveries from serum, gastric juice and feces were greater than 87%. This is therefore a sensitive method for the precise quantification of bile acids in serum, liver biopsies, bile, gastric juice and feces.