HPLC法测定大鼠尿液中瑞香素的浓度

HPLC determination of the concentration of daphnetin in rat urine

目的:建立简单、灵敏、准确的大鼠尿液中瑞香素浓度的HPLC测定方法。方法:尿样经乙酸乙酯萃取,采用HPLC法检测,色谱柱为Agilent Zorbax SB-C18(250 mm×4.6 mm,5μm)柱,流动相为甲醇-0.5%醋酸水(22∶78),流速1 mL·min-1,检测波长327 nm。大鼠灌胃不同剂量的瑞香素固体分散体溶液(5,25,125 mg·kg-1),收集0～2,2～6,6～12,12～24,24～48 h的尿液并分析,计算瑞香素在尿液中的累积排泄率。结果:瑞香素尿液的线性范围为0.155～396μg.mL-1(r=0.9991),定量限为0.155μg·mL-1。大鼠灌胃给药瑞香素后2 h即能检测到原型药物,同时发现其代谢产物,24 h内经尿液排泄基本完全,累积排泄率不到6%。结论:所建立的方法准确可靠,可用于大鼠灌胃瑞香素后原型药物尿样浓度的测定及其尿排泄研究。

Objective：To develop a rapid and sensitive HPLC method to determine daphnetin in rat urine.Methods： The analytes were extracted with ethyl acetate.The separation was performed on an Agilent Zorbax SB-C18 column（250 mm×4.6 mm,5 μm） with methanol-0.5% acetic acid（22∶78） as mobile phase at a flow rate of 1 mL·min-1 and a wavelength of 327 nm.Solid dispersion solution of daphnetin was administered intragastrically to rats at doses of 5,25 and 125 mg·kg-1,and the cumulative excretion rate of daphnetin in urine were determined at different time（0-2,2-6,6-12,12-24,24-48 h）.Results： The calibration curve was linear in the range of 0.155-396 μg·mL-1（r=0.9991） with LOQ of 0.155 μg·mL-1.Daphnetin and its metabolites were detected in the urine collected within 2 h after intragastric administration and almost completely excreted within 24 h.Cumulative urinary excretion of 24 h was not more than 6% of administration dosage.Conclusion：The method is accurate,reliable and suitable for determination of daphnetin in rat urine and the study of cumulative excretion.