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
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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,5μm)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0.05～1.5μg·mL~(-1)内线性关系良好(r=0.999 1)。方法的定量下限(LLOQ)为0.05μg·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tmax=0.95 h,Cmax=0.51μg·mL~(-1),t1/2=3.64 h,AUC0-t=1.56μg·mL~(-1)·h~(-1),AUC0-∞=1.78μg·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。
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) was0.05 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%~(-1)04.9%. 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 tmax=0.95 h,Cmax=0.51 μg·mL~(-1),t1/2=3.64 h, AUC0-t=1.56 μg·mL~(-1)·h~(-1), AUC0-∞=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.
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) was0.05 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%~(-1)04.9%. 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 tmax=0.95 h,Cmax=0.51 μg·mL~(-1),t1/2=3.64 h, AUC0-t=1.56 μg·mL~(-1)·h~(-1), AUC0-∞=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.
引文
[1]CHAO Z,MA L L,ZHOU X J.The diuretic effect of alkaloids from yimu cao(herba leonuri)on rat[J].Lishizhen Med Mater Med Res(时珍国医国药),2005,16(1):11-12.
[2]LIU X H,XIN H,HOU A J,et al.Protective effects of leonurine in neonatal rat hypoxic cardiomyocytes and rat infarcted heart[J].Clin Exp Pharmacol Physiol,2009,36(7):696-703.
[3]LIU X H,CHEN P F,PAN L L,et a1.4-Guanidino-n-butylsyringate(leonurine,SCM l98)protects H9c2 rat ventricular cells from hypoxia-induced apoptosis[J].J Cardiovasc Pharmacol,2009,54(5):437-444.
[4]QI J,HONG Z Y,XIN H,et a1.Neuroprotective effects of leonurine on ischemia/reperfusion-induced mitochondrial dysfunctions in rat cerebral cortex[J].Biol Pharm Bull,2010,33(12):1958-1964.
[5]ZHANG J L,LIU H R,MAO Y,et al.Toxicokinetics of leonurine and its metabolite in rats[J].Chin J Pharm(中国医药工业杂志),2012,43(6):451-454,474.
[6]CHAO Z.Pharmacokinetics of leonurine in rats[J].Chin Pharm(中国药学杂志),2002,37(1):39-41.
[7]LIB H,WUJ D,LI X L.Simultaneous determination and pharmacokinetic study of stachydrine and leonurine in rat plasma after oral administration of Herba Leonuri extract by LC-MS/MS[J].J Pharm Biomed Anal,2013,76(25):192-199.
[8]ZHANG Y W,YIN L N,LIANG Z H,et al.Study on pharmacokinetics of Asiatic acid in rats[J].Chin J Mod Appl Pharm(中国现代应用药学),2015,32(3):314-317.
[2]LIU X H,XIN H,HOU A J,et al.Protective effects of leonurine in neonatal rat hypoxic cardiomyocytes and rat infarcted heart[J].Clin Exp Pharmacol Physiol,2009,36(7):696-703.
[3]LIU X H,CHEN P F,PAN L L,et a1.4-Guanidino-n-butylsyringate(leonurine,SCM l98)protects H9c2 rat ventricular cells from hypoxia-induced apoptosis[J].J Cardiovasc Pharmacol,2009,54(5):437-444.
[4]QI J,HONG Z Y,XIN H,et a1.Neuroprotective effects of leonurine on ischemia/reperfusion-induced mitochondrial dysfunctions in rat cerebral cortex[J].Biol Pharm Bull,2010,33(12):1958-1964.
[5]ZHANG J L,LIU H R,MAO Y,et al.Toxicokinetics of leonurine and its metabolite in rats[J].Chin J Pharm(中国医药工业杂志),2012,43(6):451-454,474.
[6]CHAO Z.Pharmacokinetics of leonurine in rats[J].Chin Pharm(中国药学杂志),2002,37(1):39-41.
[7]LIB H,WUJ D,LI X L.Simultaneous determination and pharmacokinetic study of stachydrine and leonurine in rat plasma after oral administration of Herba Leonuri extract by LC-MS/MS[J].J Pharm Biomed Anal,2013,76(25):192-199.
[8]ZHANG Y W,YIN L N,LIANG Z H,et al.Study on pharmacokinetics of Asiatic acid in rats[J].Chin J Mod Appl Pharm(中国现代应用药学),2015,32(3):314-317.