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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 m L·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·m L-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 HPLC method for the determination of paeoniflorin in Beagle dog plasma and to study its pharmacokinetics. Methods The plasma samples were treated with ethyl acetate. Geniposide was used as internal standard. The chromatographic column was Phenomenex Luna C18 (150 mm × 4.6 mm, 5 μm). The mobile phase consisted of water containing 5 mmol·L -1 ammonium acetate and 0.05% H3PO4) -acetonitrile gradient elution at a flow rate of 1.0 m L · min-1 with UV detection at 232 nm. Paeoniflorin at a dose of 3, 6 and 12 mg · kg-1 was injected intravenously into beagle dogs. The plasma concentration of paeoniflorin was determined by HPLC-UV and the pharmacokinetic parameters were calculated by DAS 2.0 software. The results showed that endogenous impurities did not interfere with the determination of paeoniflorin and internal standard, the linear range of 0.125 ~ 16.0μg · m L-1, the limit of quantification was 0.125μg · m L-1. The precision, accuracy, stability and recovery of the method are in line with the requirements of the biological sample determination, suitable for the determination of paeoniflorin concentration in Beagle dog plasma, which can be used to study the pharmacokinetics of paeoniflorin. The area under the plasma concentration-time curve (AUC0 ~ τ) after single-dose intravenous injection of 3, 6 and 12 mg · kg-1 paeoniflorin in Beagle dogs was (225.17 ± 49.86), (484.66 ± 125.63) 042.35 ± 164.69) μg · min · m L-1. The AUC0 ~ τ ratios at different doses were 1: 2.2: 4.6 and were approximately proportional to the dose ratio of 1: 2: 4, indicating that within the study dose range, The elimination process in dogs is linear. Conclusion The method is simple, sensitive and specific. The methodological verification meets the requirements of biological samples and has been successfully applied to the pharmacokinetic study of paeoniflorin in Beagle dogs.