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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 establish a simple, sensitive and accurate HPLC method for the determination of daphnetin in rat urine. Methods: The urine samples were extracted with ethyl acetate and detected by HPLC. The column was Agilent Zorbax SB-C18 (250 mm × 4.6 mm, 5 μm) with methanol-0.5% acetic acid water (22:78) 1 mL · min-1, detection wavelength 327 nm. Rats were dosed with different doses of daphnetin solid dispersion solution (5,25,125 mg · kg-1), collected 0 ~ 2, 2 ~ 6, 6 ~ 12, 12 ~ 24, 24 ~ 48 h of urine and analyzed , Calculate the cumulative excretion rate of daphnetin in the urine. Results: The linear range of daphnetin was 0.155 ~ 396μg.mL-1 (r = 0.9991) and the limit of quantification was 0.155μg · mL-1. After 2 h of intragastric administration of daphnetin, the prototype drugs could be detected and the metabolites were found. After 24 h, urine excretion was basically complete and the cumulative excretion rate was less than 6%. Conclusion: The established method is accurate and reliable, which can be used for the determination of urinary concentration of arginine in rats and its urinary excretion.