Abstract
Fifteen kinds of bile acids in serum are determined simultaneously by high-performance liquid chromatography (HPLC) using a new automated pretreatment system.The eluate from separation column (Bile pak; Nihon Bunko) was mixed with NAD and fluorescence intensity of NADH produced by enzymatic reaction with immobilized 3α-hydroxysteroid dehydrogenase was measured.The automated pretreatment system consists of reversedphase column (Serumout-25;Sekisui Chemical Co.), line filter, three ports valve for degas, five ports valve for injector, six ports valve for sampler and pump.With use of this system, most of interfering substances to determine bile acids by HPLC, that is water-soiuble protein, peptide, ion etc, are eliminated with in2-5min.The determination of individual bile acids can be done with 5-10μl of serum without loss or dilution.The recovery rates of cholic acid, glycocholic acid and taurocholic acid added to serum ranged101±2,100±3 and 102±3%, respectively and coefficients of variation ranged from 2.0 to 5.5% (n=5).
