Determination of serum metabolic profiles of bile acids by microcolumn liquid chromatography/laser‐induced fluorescence

Abstract

Abstract A method for the determination of individual free and conjugated bile acids in serum using microcolumn liquid chromatography coupled with a laser‐induced fluorescence detector is described. Bile acids are separated into free/glycine‐conjugate and taurine‐conjugate fractions using a Sep‐Pak SIL cartridge. The taurine‐conjugated bile acid fraction is subjected to enzymatic hydrolysis. Subsequently, free and conjugated bile acids are labeled using 4‐(bromomethyl)‐7‐methoxycoumarin as a fluorogenic reagent, producing stable derivatives that can be excited by the 325 nm line of a He/Cd laser. Prior to their fluorimetric detection, the individual components of a bile acid serum profile are separated by reversed‐phase microcolumn liquid chromatography.