高效液相色谱法测定比格犬血浆中的芍药苷浓度及其药物代谢动力学研究

HPLC-UV determination of paeoniflorin in Beagle dog plasma and its pharmacokinetic study

目的建立高效液相色谱法测定比格犬血浆中芍药苷的含量,并用于药物代谢动力学研究。方法血浆样品采用乙酸乙酯处理,以栀子苷作为内标,色谱柱为Phenomenex Luna C18（150 mm×4.6 mm,5μm）,流动相为水（含5 mmol·L^-1醋酸铵和0.05%H3PO4）-乙腈,梯度洗脱,流速为1.0 mL·min^-1,紫外检测波长232 nm。比格犬单剂量静脉注射3、6和12 mg·kg^-1的芍药苷,HPLC-UV法测定芍药苷的血药浓度,并采用DAS 2.0软件计算药物代谢动力学参数。结果结果表明内源性杂质不干扰芍药苷和内标的测定,线性范围0.125-16.0μg·m L^-1,定量限为0.125μg·m L^-1。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,适合比格犬血浆中芍药苷浓度的测定,可以应用该方法进行芍药苷的药物代谢动力学研究。比格犬单剂量静脉注射3、6和12 mg·kg^-1芍药苷后的血药浓度-时间曲线下面积（AUC0-τ）分别为（225.17±49.86）、（484.66±125.63）、（1 042.35±164.69）μg·min·mL^-1,不同剂量下AUC0-τ比为1：2.2：4.6,与剂量比1：2：4近似成比例,说明在研究剂量范围内,芍药苷在Beagle犬体内的消除过程是线性的。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芍药苷在比格犬体内的药物代谢动力学研究。

Objective To establish a simple, sensitive and specific high performance liquid chromatography method to determine paeoniflorin and to study its pharmacokinetics in Beagle dogs. Methods Paeoniflorin and jasminoidin（the internal standard, IS） were extracted by liquid-liquid extraction and separated on a Phenomenex Luna C18 column, with a gradient of acetonitrile and water containing 5 mmol·L^- 1 ammonium acetate and 0.05% phosphoric acid as the mobile phase. The flow rate was 1.0 m L·min^- 1 and the analytical wavelength was 232 nm. After validation, the developed method was used to evaluate the pharmacokinetics of paeoniflorin in Beagle dogs after intravenous administration of 3 doses of paeoniflorin. Results The method showed a good linearity of 0.125- 16.0 μg·mL^- 1 with a sensitivity of 0.125 μg·mL^- 1 as the limit of quantification. The precision, accuracy, stability and mean recoveries all met the requirements of biological sample measurement. The method was successfully applied in the pharmacokinetic study of paeoniflorin in Beagle dog plasma. The areas under the plasma concentration-time curves（AUC） of paeoniflorin after single intravenous doses of 3, 6 and 12 mg·kg^-1 were（225.17±49.86）,（484.66±125.63）, and（1 042.35±164.69） μg·min·mL^- 1. The relationship between dose and AUC0 - τ showed a good linearity. The elimination process of paeoniflorin was linear pharmacokinetics. Conclusion This HPLC-UV method for the determination of paeoniflorin has sufficient selectivity, sensitivity and reproducibility in the pharmacokinetic study of paeoniflorin in Beagle dogs.