HPLC-FLUORESCENCE DETERMINATION OF INDIVIDUAL FREE AND CONJUGATED BILE-ACIDS IN HUMAN SERUM

A method for the quantitative analysis of unconjugated and conjugated bile acids (BA) in serum of patients with primary biliary cirrhosis (P BC) before and after therapy with antibiotic or ursodeoxycholic acid ( UDCA) is described, After separation of the free, glycine and taurine conjugated (F, G and T conjugated) fractions by solid-phase extraction , the isolated T conjugates were hydrolysed enzymatically using cholyg lycine hydrolase. The BA fractions were derivatized using 2-bromoacety l-6-methoxynaphthalene (Br-AMN) and detected fluorimetrically (lambda( exc)=300 nm, lambda(em)=460 nm), The derivatization reaction was perfo rmed under mild conditions (10 min at 40 degrees C) in an aqueous medi um in the presence of tetrakis (decyl) ammonium bromide (TDeABr), The HPLC separation was achieved using an ODS column and with a mobile pha se gradient mixture of A-B, where A is water and B is acetonitrile:met hanol (60:40 v/v) for elution at a flow-rate of 1.2 mL/min, The reprod ucibility, recovery and separation of individual BA under gradient elu tion conditions were satisfactory, allowing a sensitive detection of e ach BA in serum samples with a detection limit of about 1-2 pmol.