Serum Bile Acids By Rp-hplc Analysis Of The Methodological Research And Clinical Applications

Objective:To establish a sensitive and reproducible analytical method for determination of bile acids in serum by RP-HPLC.Methods :1.Nonconjugated and glycine-conjugated bile acids are derived with 4-bromomethyl-7-methoxycoumarin in the presence of dicyclohexano-18-crown-6 as catalyst. Taurine conjungated bile acids was hydrolysed with cholylglycine hydrolase prior to derivatization.2. RP-HPLC method was applied to quantitative analysis.Derived bile acids were eluted using an acetonitrile/methanol/water gradient on a Waters Nova C18 column(3.9×150mm,5μm).The flow rate was 1.0ml/min.The column was kept at 25℃. The eluate was monitored by a fluorophotometer at330nm(λex) and 410nm(λem). Quantification is obtained using laurid acid (LA)as an internal standard.3.Free and glycine-conjugated bile acids were extracted from pretreated samples using Sep-Pak C18 cartridges. Results:The assay was linear in the range 50-400pmol.Correlation coeffients for linear regression ranged from 0.9988 to 0.9998.Rcoveries from serum was greater than 85%.Precision was within 8%.The analysis time was 24min.Conclusion: It is proved that the proposed method could be considered sufficiently sensitive and reliable for the routine analysis of bile acids in serum.