HPLC-MS/MS法同时测定大鼠血浆中山楂叶提取物3种黄酮类成分及其药代动力学研究

Simultaneous determination of three flavonoids in hawthorn leaves extract by HPLC-MS/MS method and analysis of their pharmacokinetics in plasma of rats

关键词:  药代动力学  高效液相色谱质谱联用法  山楂叶提取物

Key words:  pharmacokinetics  HPLC-MS/MS  hawthorn leaves extract

摘要:

[目的] 建立高效液相色谱-串联质谱法（HPLC-MS/MS）同时测定大鼠灌胃给予山楂叶提取物后牡荆素、牡荆素-4“-O-葡萄糖苷、牡荆素-2”-O-鼠李糖苷的药代动力学。[方法] 大鼠给予山楂叶提取物（1、2、4 g/kg）后采集不同时间点血浆样品，经甲醇处理后，以乙腈-水（含0.1%甲酸）为流动相，通过Agilent ZORBAX XDB-C₁₈（3.5 μm，2.1×50 mm）色谱柱梯度洗脱，采用负离子电喷雾离子化源，多反应监测（MRM）模式进行测定。[结果] 牡荆素、牡荆素-4“-O-葡萄糖苷、牡荆素-2”-O-鼠李糖苷分别在0.5~500.0 ng/mL（r=0.998）、1.0~1 000.0 ng/mL（r=0.999）、1.0~1 000.0 ng/mL（r=0.998）范围内呈良好的线性关系，方法精密度、准确度、回收率和稳定性均符合生物样品的测定要求。[结论] 本方法简便、快速、专属性强，可用于大鼠血浆中牡荆素、牡荆素-4“-O-葡萄糖苷、牡荆素-2”-O-鼠李糖苷的同时测定及其药动学研究。

Abstract:

[Objective] To establish a HPLC-MS/MS method for the stimultancous pharmacokinetic study of vitexin-4“-O-glucoside(VOG), vitexin-2”-O-rhamnoside(VOR) and vitexin(VIT) in rat plasma after administration of hawthorn leaves extract orally. [Methods] Samples of plasma were obtained at different time after rats were administrated with three doses of hawthorn leaves extract (1, 2, 4 g/kg). After the deproteinization by methanol, extract of plasma samples were separated on a Agilent ZORBAX XDB-C₁₈ column (3.5 μm,2.1×50 mm) with gradient elution using methanol and water (containing 0.1% formic acid) as mobile phase. Mass spectrometric detection was carried out by multiple reaction monitoring (MRM) using electrospray ionization in negative ion mode. [Results] A good linearity of VIT, VOG and VOR was shown in the ranges of 0.50~500.0 ng/mL (r=0.998), 1.0~1000.0 ng/mL(r=0.999) and 1.0~1000.0 ng/mL(r=0.998), respectively. The methods of precision, accuracy, recovery and stability are in line with the requirements of determination of biological samples. [Conclusion] The method is proved to be simple, rapid, and specific, and to be suitable for the simultaneous determination of VIT, VOG and VOR in plasma of rats and the pharmacokinetic study.