A simple reverse-phase high pressure liquid chromatographic determination of conjugated bile acids in serum and bile using a novel radial compression separation system

Abstract

A new HPLC method for analyzing conjugated bile acids in bile and especially in serum is presented. For separation of novel radial compression system with a 10-cm flexible-walled reverse phase column (RCM-100 Module, Waters Ass.) packed with micro-Bondapak C18 was used. Without any laborious hydrolysis and derivatization steps, 10 conjugated bile acids are separated easily with a mobile phase of methanol/0.01 mol/1 KH2PO4 (150:50, v/v; pH 6.0) in about 30 min, determined with a variable UV detector at 200 nm and a flow rate of only 0.5 ml/min. Dexamethasone was used as internal standard. Conjugated bile acids are extracted from serum with Sep-pak C18 cartridges after a 1:8 dilution with a mixture of the mobile phase and 0.2 mol/l NaOH (6:8, v/v). Extraction from bile was also performed using Sep-pak cartridges after a 1:20 dilution with 0.5 mol/l phosphate buffer (pH 7.0). The complete analysis time of the method, with Sep-pak extraction, is less than one hour. Recoveries for the different conjugated bile acid varied from 89.4% to 96.7% in serum. The HPLC method for serum and bile was validated for the primary bile acids cholate and chenodeoxycholate against the previously described GLC method. Agreement between the two methods was excellent. Duplicate analyses in serum and bile samples also showed that the reproducibility was good.