柱前衍生HPLC-UV同时测定人工牛黄中胆酸及猪去氧胆酸的含量

Simultaneous testing of bile acid and hyodeoxycholic acid in artificial calculus bovis using HPLC-UV with pre-column derivatization

目的建立柱前衍生HPLC-UV同时测定人工牛黄中胆酸及猪去氧胆酸的含量。方法以2-萘基溴甲基酮为衍生试剂,三乙胺为催化剂,60℃水浴条件下对胆酸及猪去氧胆酸进行衍生,考察衍生试剂用量、催化剂用量及反应时间等对衍生产物的影响,确定衍生反应的条件,然后考察建立HPLC-UV分离检测方法对人工牛黄中胆酸及猪去氧胆酸含量进行测定。结果当衍生试剂的摩尔量与胆酸及猪去氧胆酸的摩尔总量的比大于30∶1,催化剂的摩尔量与两者的摩尔总量的比在5∶1~60∶1范围内,在60℃水浴条件下反应50 min时,胆酸及猪去氧胆酸的衍生物反应完全。方法学验证:胆酸在0.4~5.0μg范围内线性关系良好(r=0.999 5);猪去氧胆酸在0.12~1.5μg范围内线性关系良好(r=0.999 8)。胆酸的平均回收率(n=6)为100%,RSD为2.40%;猪去氧胆酸的平均回收率(n=6)为101%,RSD为1.33%。此批次人工牛黄样品中胆酸的含量为5.55%,猪去氧胆酸的含量为2.55%。结论本方法灵敏度高、准确可靠、稳定性和重复性好,可用于测定人工牛黄中胆酸及猪去氧胆酸的含量。

Objective To establish a pre-column derivation of HPLC-UV and determine bile acid and hyodeoxycholic acid in artificial calculus bovis.Methods Bile acid and hyodeoxycholic acid were derived by 2-bromo-2＇-acetonaphthoneat using triethylamine as the catalyst in 60 ℃ water bath.The effects of derivatizing agent usage, catalyst and derivatizing time were investigated to identify the optimal parameters.HPLC-UV parameters were established to determine the content of hyodeoxycholic acid in artificial calculus bovis.Results When the ratio of molar amount of derivatizing agents to that of cholic acid and hyodeoxycholic was over 30∶1, and the ratio of molar amount of catalyst to that of cholic acid and hyodeoxycholic ranged from 5∶1 to 60∶1, the reaction was completed in 60℃ water bath for 50 minutes.The linear relationship was found with agent concentration （0.4 -5.0 μg） in cholic acid （r=0.999 5） and the linear relationship was also present in hyodeoxycholic （0.12-1.5 μg, r=0.999 8）.The average recovery rate of bile acid （n=6） was 100% and that of the RSD was 2.40%.The average recovery rate of hyodeoxycholic acid was 101% and that of the RSD was 1.33%.In this sample, the content of cholic acid was 5.55% and the content of hyodeoxycholic was 2.55%.Conclusion The method is replicable, reliable and accurate to determine bile acid and hyodeoxycholic acid in artificial calculus bovis.