SIMULTANEOUS DETERMINATION OF COMPONENTS IN ARTIFICIAL BEZOAR BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH EVAPORATIVE LIGHT-SCATTERING DETECTOR

AIM: To establish a simple reverse phase high-performance liquid chromatographic method for the simultaneous determination of several components in artificial ox gallstone. METHODS: Chromatography was performed on a 5 μm Nova-Park C₁₈ steel column (250 mm×4.6 mm ID, Waters) at ambient temperature. The mobile phase composition was methanol — water — glacial acetic acid (80∶20∶0.01). The flow rate was 1.0 mL.min^-1. An evaporative light-scattering detector (ELSD) Model 500 (Alltech, USA) was used as detector, its parameters were set as follows: nitrogen carrier gas flow 2.05 SLPM; drift tube temperature 105℃. RESULTS: Complete resolution of cholesterol, free bile acids ［especially cholic acid (CA) and hyodeoxycholic acid (HDCA)］ and inorganic salts in artificial ox gallstone was obtained for the first time. The method fulfills all the standard requirements of precision, accuracy and linearity. The recoveries of components at three concentration levels were: cholic acid (CA) 98.88%～101.2%, hyodeoxycholic acid (HDCA) 99.02%～102.2%, chenodeoxycholic acid (CDCA) 98.56%～102.4%, deoxycholic acid (DCA) 98.32%～101.8%, cholesterol 98.41%～99.18%. The detection limits for 20 μL injected were CA and DCA 0.10 μg, CDCA 0.106 μg, HDCA 0.09 μg, cholesterol 0.054 μg, respectively. The detectivity of this method for bile acids is higher than that reported. CONCLUSION: HPLC, combined with ELSD, can give more accurate and sensitive results for the analyses of those compounds without chromophore. The new method established is more suitable for analyses of artificial ox gallstone.