高灵敏度HPLC测定大鼠血浆中益母草碱浓度及其药动学研究

Determination and Pharmacokinetic Study of Leonurine in Rat Plasma by Sensitive HPLC

关键词:  益母草碱  高效液相色谱法  血浆浓度  药动学

Key words:  Leonurine  HPLC  plasma concentration  pharmacokinetics

摘要:

目的 建立一种高灵敏度HPLC测定大鼠血浆中益母草碱浓度，并研究益母草碱在大鼠体内的药动学特征。方法 大鼠口服益母草碱混悬溶液（50 mg·kg^-1）后，不同时间点尾静脉采血，以苯甲酰精氨酸乙酯为内标，血浆样品经酸化后乙酸乙酯萃取，采用HPLC进行测定。色谱条件：采用Diamonsil C₁₈（250 mm×4.6 mm，5 μm）为色谱柱，以乙腈-0.02 mol·L^-1磷酸二氢钾缓冲溶液（pH 3.0）（22：78）为流动相，流速1.0 mL·min^-1，柱温35℃，检测波长277 nm。并利用PKS 1.0软件计算药动学参数。结果 益母草碱血浆浓度在0.05~1.5 μg·mL^-1内线性关系良好（r=0.999 1）。方法的定量下限（LLOQ）为0.05 μg·mL^-1（RSD=12.8%，n=5）；提取回收率为76.5%~82.5%；批内、批间准确度为96.9%~104.9%；日内、日间精密度均<10%；质控样品经反复冻融3次及-20℃放置1个月后均较稳定。大鼠口服益母草碱后，药-时曲线符合二室开放模型，主要药动学参数为t_max=0.95 h，C_max=0.51 μg·mL^-1，t_1/2=3.64 h，AUC_0-t=1.56 μg·mL^-1·h^-1，AUC_0-∞=1.78 μg·mL^-1·h^-1。结论 该方法准确度、灵敏度高，重复性好，可用于生物样品中益母草碱浓度的测定。

Abstract:

OBJECTIVE To establish an HPLC method for determination of plasma concentration of leonurine (LE) and investigate its pharmacokinetics in rats. METHODS After oral administration of LE suspension (50 mg·kg^-1), plasma samples were collected at different points. After extracted from plasma by ethyl acetate, the plasma concentrations of LE and its internal standard (IS) n-benzoyl-L-arginine ethyl ester (BAEE) were determined by HPLC. Chromatographic separation was performed on a Diamonsil C₁₈(250 mm×4.6 mm, 5 μm) with UV detection at 277 nm, using acetonitrile-0.02 mol·L^-1 monopotassium phosphate (pH 3.0) (22:78) as mobile phase at 1.0 mL·min^-1 flow rate, and the column temperature was 35℃. The pharmacokinetic parameters were calculated by PKS 1.0 software program. RESULTS The linear calibration curve of LE was obtained in the concentration range of 0.05-1.5 μg·mL^-1(r=0.999 1), and the lower limit of quantitation(LLOQ) was 0.05 μg·mL^-1(RSD=12.8%, n=5); The absolute recovery in plasma was 76.5%-82.5%; Intra-day and inter-day relative standard deviations were both below 10%, with accuracy in the range 96.9%-104.9%. The QC plasma samples were stable through repeated three freeze/thaw cycles and under the frozen condition at -20℃ for 30 d. The process of LE in rat fit two-compartment open model, and the main pharmacokinetic parameters obtained were t_max=0.95 h, C_max=0.51 μg·mL^-1, t_1/2=3.64 h, AUC_0-t=1.56 μg·mL^-1·h^-1, AUC_0-∞=1.78 μg·mL^-1·h^-1. CONCLUSION The assay method is proved to be simple, sensitive, precise and reliable enough for the pharmacokinetics study of LE in rat after oral administration.