柱前衍生HPLC法测定体外培育牛黄中主要胆汁酸类成分

Quantification of main bile acids in in vitro cultivated Calculus bovis by high-performance liquid chromatography with pre-column derivatization

目的:建立体外培育牛黄样品溶液柱前衍生化处理后使用HPLC-UV进行测定的方法。方法:2-溴-4′-硝基苯乙酮和18-冠醚-6作为衍生化反应试剂,采用Agilent Zorbax SB-C_(18)(150 mm×4.6 mm,5μm)色谱柱,以水为流动相A,甲醇为流动相B,梯度洗脱,流速1 mL·min^(-1),在263 nm波长处进行测定,柱温为30℃。结果:胆酸和去氧胆酸进样量分别在0.25~4.00和0.20~3.21μg范围内线性关系良好;回收率分别为95.13%和94.87%,RSD(n=6)分别为2.09%和3.15%。3批样品的胆酸含量分别为7.3%、8.4%、8.1%,去氧胆酸含量分别为4.9%、5.6%、5.4%。结论:该法经方法学验证,适用于体外培育牛黄中主要胆汁酸类成分的含量测定。

Objective： To establish a method for quantification of cholic acid （ CA ） and deoxycholic acid （ DCA ） in in vitro cultivated Calculus bovis by high-performance liquid chromatography with pre-column derivatization. Methods： With 2-bromine-4＇ -nitroacetophenone and 18-crown ether-6 as derivatization agents, the analysis was carried out on an Agilent Zorbax SB-C18 column （ 150 mm × 4.6 mm, 5 μ m ） at a column temperature of 30 ℃, using water and methanol as mobile phase with gradient elution at a flow-rate of 1 mL. min-1. The detection wavelength was set at 263 rim. Results： The linear ranges of CA and DCA were 0.25-4.00 μ g and 0.20-3.21 μg, respectively. The corresponding average recovery rates were 95.13% and 94.87%, respectively, the relative standard deviations （n=6）were 2.09% and 3.15%, respectively. The contents of CA in three samples were 7.3%, 8.4%, 8.1% and the contents of DCA in the three samples were 4.9%, 5.6%, 5.4%, respectively. Conclusion： The method is proved by methodology validation to be applicable to the quantification of CA and DCA in in vitro cultivated Calculus bovis.