ALTERNATIVE USE OF FLOW-INJECTION ANALYSIS AND THE COMBINATION OF LIQUID-CHROMATOGRAPHY AND FLOW-INJECTION ANALYSIS FOR THE DETERMINATION OF TOTAL AND INDIVIDUAL BILE-ACID CONCENTRATIONS IN SERUM

A hybrid liquid chromatographic (LC)-flow-injection system for the determination of bile acid concentrations in human fluids is reported. It was used to develop a method based on the prior use of the flow-injection manifold as a routine screening for the total concentration of the analytes, followed by individual determination by LC of only those samples with total concentrations close to or above the normal level in healthy individuals. The proposed method, which was applied to taurocholic, glycocholic, cholic and chenodeoxycholic acid, relies on the oxidation of these analytes, catalysed by 3-alpha-hydroxysteroid dehydrogenase with the aid of the coenzyme NAD+, the reduced form of which is monitored fluorimetrically. The features of the method (linearity in the range 1.0-10.0-mu-g ml-1, R.S.D. smaller than 3% in all instances and sampling frequency 30 and 4 h-1 for the flow-injection and subsystems, respectively) and the results obtained on application to serum samples show its usefulness.