大鼠胆汁中酮洛芬Ⅱ相代谢物酮洛芬葡萄糖醛酸的测定方法

RP-HPLC determination of ketoprofen glucuronides in rat bile liquid

目的:建立 RP-HPLC 法测定胆汁中酮洛芬Ⅱ相代谢物酮洛芬葡萄糖醛酸(S-KPG、R-KPG)。方法:采用 ODS 柱(5μm,4.6 mm×250 mm);流动相为乙腈-水(35:65),内含0.05 mol·L^(-1)磷酸二氢钾和0.005 mol·L^(-1)的四丁基溴化胺,pH5.6;流速为1 mL·min^(-1);检测波长232 nm;柱温30℃;进样是20μL。内标为10 mg·L^(-1)的萘普生甲醇溶液。结果:在2～1000μmol·L^(-1)浓度范围内,胆汁中 S-KPG、R-KPG 回归方程分别为 Y=0.0039X-0.0256,r=0.9998和 Y=0.0039X-0.0229,r=0.9998;最低检测限分别为0.5 μmol·L^(-1)和1 μmol·L^(-1)。S-KPG 和 R-KPG 在室温和胆汁生理 pH 时不稳定,在 pH4时最稳定。结论:本文应用 RP-HPLC 检测胆汁 S-KPG、R-KPG,方法简便、易行、实用,可以作为肝脏葡萄糖醛酸转移酶活性和胆排泄实验研究的分析手段。

Objective： To establish an RP - HPLC method to determine ketoprofen glucuronides （ S - KPG and R - KPG） in bile liquid. Methods：The chromatographic separation was performed using an ODS analytical column. The mobile phase consisted of acetonitrile and 0.05 mol · L ^- 1 phosphate buffer （ pH 5.6） including 5 mmol · L ^- 1 tetrabutylammonium bromide （35： 65 ）. The detector wavelength was 232 nm, the flow rate was 1 mL ~ min - 1, the volume of injection was 20 I,~L, and the oven temperature was 30 ~C. The internal standard was naproxen dissolved by methanol. Results：For both kinds of KPGs, the linear range was 2 - 1000 μmol · L^-1. The linear equation of S - KPG was Y = 0. 0039X - 0. 0256, r = 0. 9998 ; the linear equation of R - KPG was Y = 0. 0039X - 0. 0229, r = 0. 9998. KPGs were easy to be hydrolyzed in the condition of room temperature and bile physiological pH. However, KPGs were stable in the condition of pH adjusted to 4. Conclusion：The method is simple and utility. And it can be used for the empirical study of the glueuronyltransferase activity and bile excretion function.