高效液相色谱法测定大鼠血浆中安石榴苷的含量及其药动学研究

Development and validation of an HPLC method for determination of punicalagin in rat plasma and application to a pharmacokinetic study

目的建立高效液相色谱法测定大鼠血浆中安石榴苷的含量,并用于药代动力学研究。方法血浆样品采用色谱甲醇处理,以大黄素作为内标,Agilent XDB-C18(250 mm×4.6 mm,5μm)色谱柱,流动相为甲醇(A)-0.1%三氟乙酸水溶液(B)梯度洗脱,流速为1.0 ml/min,检测波长为260 nm,柱温为30℃。采用大鼠单剂量静注13 mg/kg的安石榴苷,HPLC法测定安石榴苷的血药浓度,并采用DAS 2.0软件计算药代动力学参数。结果方法学实验结果表明内源性杂质不干扰安石榴苷和内标的测定,线性范围0.02 016~1.00 800 mg/ml。方法精密度、准确度、稳定性和回收率均符合生物样品测定的要求。结论本方法操作简便、灵敏、专属性强,方法学考证符合生物样品测定的要求,并成功用于安石榴苷在大鼠体内的药代动力学研究。

Objective To establish the high- performance liquid chromatography method for determination of punicalagin in order to study the pharmacokinetics of punicalagin in rats. Methods Punicalagin and emodin（ internal standard,IS） were extracted by methanol（ for HPLC） extraction and separated on a Agilent XDB- C18（250 mm × 4. 6 mm,5 μm） column,with methanol and water（ containing 0. 1% trifluoroacetic acid）,gradient elution. The flow rate of 1. 0 ml / min and detected at 387 nm,the column temperature was 30 ℃. After validating,the developed method was used for evaluating pharmacokinetics of punicalagin in rats following intravenous administration of a dose（13 mg / kg） of punicalagin. The pharmacokinetic parameters were calculated by the software DAS 2. 0. Results The methodological study showed endogenous impurities did not interfere the determination of punicalagin,and the internal standard and a good linear relationship were found within 0. 02 016 ～ 1. 00 800 mg / ml. The precision,accuracy,stability and mean recoveries met the requirements of biological sample measurement. Conclusion This HPLC method for determination of punicalagin was proved to have sufficient selectivity,sensitivity and reproducibility and was successfully applied to the pharmacokinetic study of punicalagin in rats.