六味调更片10种成分的大鼠血浆药动学研究

Pharmacokinetics Study of Ten Constituents of Liuwei Tiaogeng Tablet in Rats

关键词:  六味调更片  高效液相色谱-串联质谱  有机酸  黄酮  药动学

Key words:  Liuwei Tiaogeng tablets  UHPLC-MS/MS  organic acid  flavone  pharmacokinetics

摘要:

目的 建立超高效液相色谱-串联质谱法(UHPLC-MS/MS)测定六味调更片中10种成分(阿魏酸、山柰酚、齐墩果酸、藁本内酯、槲皮素、黄芪甲苷、甜菜碱、毛蕊异黄酮、毛蕊异黄酮苷、茯苓酸)在大鼠中的血药浓度，并研究10种成分在大鼠体内的药动学特征。方法 血浆样品的处理采用乙酸乙酯液-液萃取方法，以格列苯脲为内标。采用Thermo Hypersil Gold色谱柱(2.1 mm×100 mm，1.9 μm)；以0.1%甲酸水(A)-甲醇(B)为流动相；梯度洗脱，流速为0.3 mL·min^?1；柱温为40 ℃；进样体积2 μL；采用电喷雾电离源(ESI离子源)；平行离子检测模式进行正负离子检测。灌胃给药后，测定大鼠血浆10种成分的浓度，并用DAS 2.0软件计算药动学参数。结果 血浆中10种成分线性关系均良好(r>0.991)，各成分日内和日间精密度RSD均<14%，血浆中各待测成分低、中、高3个浓度的提取回收率为79.41%~93.63%，基质效应为83.29%~106.29%，符合生物样品测定的要求。大鼠灌胃六味调更片后，10种成分的血药浓度达峰时间(T_max)为0.25~4.96 h，血药浓度-时间曲线下面积(AUC_0-∞)为12.19~3 488.29 ng·mL^?1·h，血药达峰浓度(C_max)为0.81~143.33 ng·mL^?1。结论 该检测方法专属性强，重复性好，可用于六味调更片10种成分的大鼠血浆药动学研究。

Abstract:

OBJECTIVE To establish an UHPLC-MS/MS method to determine the concentration of ten constituents(ferulic acid, kaempferol, oleanolic acid, ligustilide, quercetin, astragaloside, betaine, calycosin, calycosin glucoside, poriacolic acid) in rat plasma, and to study the pharmacokinetic characteristics of them in rat in vivo. METHODS The plasma sample was treated with a liquid-liquid extraction of ethyl acetate, and glibenclamide was used as an internal standard. The determination was carried out with a Thermo Hypersil Gold column(2.1 mm×100 mm, 1.9 μm) and the mobile phase comprised of water containing 0.1% formic acid(A) and methanol(B). The flow rate, column temperature and injection volume were 0.3 mL·min^–1, 40 ℃ and 2 μL. The electrospray ionization source(ESI) and parallel reaction detection mode were used for quantitative analysis. After oral administration, the concentrations of 10 constituents in rat plasma were measured and pharmacokinetic parameters were calculated using DAS 2.0 software. RESULTS The linear relationship of 10 components in plasma was good(r>0.991), and the RSD of intra-day and inter-day precision of each component was <14%. The extraction recoveries of low, medium and high concentrations of each component in plasma were 79.41%?93.63%, and the matrix effect was 83.29%?106.29%, which met the requirements of biological sample determination. After rats were intragastrically administrated with Liuwei Tiaogeng tablets, the blood concentration peak time(T_max) of the 10 constituents was 0.25–4.96 h, the area under the blood concentration-time curve(AUC_0–∞) was 12.19–3 488.29 ng·mL^?1·h, and the blood concentration peak concentration(C_max) was 0.81–143.33 ng·mL^?1. CONCLUSION The method is specific and repeatable, and suitable for determination of ten constituents of Liuwei Tiaogeng tablets in rat plasma for pharmacokinetic study.