LC-MS/MS法同时测定犬血浆中延胡索乙素和欧前胡素及其在元胡止痛片药动学研究中的应用

Simultaneous determination of tetrahydropalmatine and imperatorin in plasma of dog by LC-MS/MS method and pharmacokinetic study on application of Yuanhu Zhitong Tablets

目的 建立同时测定Beagle犬血浆中的延胡索乙素和欧前胡素的LC-MS/MS分析方法,并应用于元胡止痛片的药动学研究。方法 采用液-液萃取方式进行样品处理,萃取剂为甲基叔丁基醚,内标为乙氧苯柳胺;采用XDB-C18（50 mm×4.6 mm,5μm）柱色谱分离,柱温40℃,体积流量0.4 m L/min;流动相为甲醇-0.1%甲酸水溶液,梯度洗脱;采用电喷雾离子源（ESI）、正离子扫描、多反应离子监测（MRM）进行测定,延胡索乙素：m/z 356.2[M＋H]＋→m/z 192.1,欧前胡素：m/z 271.1[M＋H]＋→m/z 203.0,内标乙氧苯柳胺：m/z 258.1[M＋H]＋→m/z 121.1。应用Win Nonlin 6.3软件非房室模型统计矩方法计算药动学参数。结果 延胡索乙素在0.05~20 ng/m L线性良好,欧前胡素在0.005~2 ng/m L线性良好,延胡索乙素和欧前胡素的定量下限分别为0.05 ng/m L和0.005 ng/m L;批内和批间精密度均小于14.4%,准确度均在–7.19%~5.89%;提取回收率在82.8%~108%,基质效应在88.2%~109%。Beagle犬po给予元胡止痛片后,延胡索乙素的主要药动学参数tmax、Cmax、AUC0~t、AUC0~∞、MRT0~t、MRT0~∞、Vd、CL和t1/2分别为（1.08±0.20）h、（48.70±18.10）ng/m L、（170.00±75.70）h·ng/m L、（178.00±77.50）h·ng/m L、（6.41±1.13）h、（8.09±1.85）h、（133.00±63.00）L、（11.90±5.54）L/h和（7.71±1.07）h;欧前胡素的主要药动学参数tmax、Cmax、AUC0~t、AUC0~∞、MRT0~t、MRT0~∞、Vd、CL和t1/2分别为（1.17±0.26）h、（0.063 4±0.023 5）ng/m L、（0.176 0±0.091 9）h·ng/m L、（0.204 0±0.097 3）h·ng/m L、（2.550±0.669）h、（3.640±0.818）h、（22 351±7 990）L、（7 917±6 030）L/h和（2.390±0.877）h。结论 该分析方法专属性强,灵敏度高,能同时测定犬血浆中延胡索乙素和欧前胡素,可用于元胡止痛片给药后犬的药动学研究。

Objective To describe the pharmacokinetic profile ofYuanhu Zhitong Tablet （YZT） when administered to Beagle＇s dogs, a simple and sensitive method for determining tetrahydropalmatine and imperatorin in plasma of dogs was developed and validated by liquid chromatography tandem mass spectrometry （LC-MS/MS）. Methods Plasma samples were treated by a liquid-liquid extraction method with methyl tertiary butyl ether. Etofesalamide was used as internal standard （IS）. The separation was performed on a ZORBAX Eclipse XDB-CI8 column （50 mm × 4.6 mm, 5 μm） at 40 ℃ with a flow rate of 0.4 mL/min using gradient mobile phase. The mobile phase was consisted with methanol and 0.1% formic acid in water. After separated by liquid chromatography, analytes and IS were detected in electrospray ionization source （ESI） positive ion mode at the specific multiple reaction monitoring （MRM） transitions of m/z 356.2 [M ＋ H]^＋→m/z 192.1 for tetrahydropalmatine, m/z 271.1 [M ＋ H]^＋→m/z 203.0 for imperatorin, and m/z 258.1 [M ＋ H]^＋→/z 121.1 for IS. The pharmacokinetic parameters for tetrahydropalmatine and imperatorin were calculated by WinNonlin 6.3 pharmacokinetic program using non-compartmental analysis. Results Good linearity was achieved over the concentration ranges of 0.05 - 20 ng/mL for tetrahydropalmatine and 0.005-2 ng/mL for imperatorin, with the lower quantification limit of 0.05 and 0.005 ng/mL, respectively. The intra- and inter-batch precisions were less than 14.4%, and the accuracies were within ＋ 7.19% for all analytes. The mean extraction recoveries of analytes were between 88.2%-107%, and the matrix effects of analytes were between 82.2% -108%. The pharmacokinetic parameters oftetrahydropalmatine after ig administration of YZT including tmax, Cmax, AUC0-t, AUC0~∞,MRT0~t,MRT0~∞,Vd,CL, and t1/2 were as follows： （1.08 ±0.20） h, （48.70 ±18.10） ng/mL, （170.00 ± 75.70） h.ng/mL, （178.00± 77.50） h.ng/mL, （6.41 ±1.13） h, （8.09 ± 1.85） h, （133.00 ± 63.00） L, （11.90 ± 5.54） L/h, and （7.71± 1.07） h; The pharmacokinetic parameters of imperatorin were （1.17 ± 0.26） h, （0.063 4 ± 0.023 5） ng/mL, （0.1760 ± 0.091 9） h＇ng/mL, （0.204 0 ± 0.097 3） h.ng/mL, （2.550 ±0.669） h, （3.640 ±0.818） h, （22 351 ± 7 990） L, （7 917± 6 030） L/h, and （2.390 ± 0.877） h. Conclusion The fully validated LC-MS/MS method is successfully applied to a pharmacokinetic study on tetrahydropalmatine and imperatorin in Beagle＇s dogs following ig administration of YZT.