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 (PBC) 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 cholyglycine hydrolase. The BA fractions were derivatized using 2-bromoacetyl-6-methoxynaphthalene (Br-AMN) and detected fluorimetrically (lambda exc = 300 nm, lambda em = 460 nm). The derivatization reaction was performed under mild conditions (10 min at 40 degrees C) in an aqueous medium in the presence of tetrakis (decyl) ammonium bromide (TDeABr). The HPLC separation was achieved using an ODS column and with a mobile phase gradient mixture of A-B, where A is water and B is acetonitrile:methanol (60:40 v/v) for elution at a flow-rate of 1.2 mL/min. The reproducibility, recovery and separation of individual BA under gradient elution conditions were satisfactory, allowing a sensitive detection of each BA in serum samples with a detection limit of about 1-2 pmol.