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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 establish a LC-MS / MS method for the simultaneous determination of tetrahydropalmatine and imperatorin in plasma of Beagle dogs and to study the pharmacokinetics of Yuanhu Zhitong Tablet. Methods The sample was treated by liquid-liquid extraction. The extractant was methyl tert-butyl ether and the internal standard was phenoxybenzene. The column was chromatographed on a XDB-C18 column (50 mm × 4.6 mm, 5 μm) ℃, and the volume flow rate was 0.4 m L / min. The mobile phase consisted of methanol-0.1% formic acid aqueous solution with gradient elution. Electrospray ionization (ESI), positive ion scan and MRM were used to determine the content of tetrahydropalmatine : m / z 356.2 [M + H] +? m / z 192.1, imperatorin: m / z 271.1 [M + H] +? m / z 203.0, internal standard of phenaceous amine: m / z 258.1 [M + H] + → m / z 121.1. Pharmacokinetic parameters were calculated using Win Nonlin 6.3 software non-compartmental model statistical moment method. Results Linearity of tetrahydropalmatine was good at 0.05 ~ 20 ng / m L, and the linearity was 0.005 ~ 2 ng / m L for imperatorin. The lower limit of quantitation of tetrahydropalmatine and imperatorin were 0.05 ng / m L and 0.005 ng / m L; the intra and inter batch precision were less than 14.4%, the accuracy was -7.19% ~ 5.89%; extraction recovery was 82.8% ~ 108%, matrix effect was 88.2% ~ 109%. The pharmacokinetic parameters tmax, Cmax, AUC0 ~ t, AUC0 ~ ∞, MRT0 ~ t, MRT0 ~ ∞, Vd, CL and t1 / 2 of Tetrahydropalmatine were (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 ), (133.00 ± 63.00) L, (11.90 ± 5.54) L / h and (7.71 ± 1.07) h, respectively. The main pharmacokinetic parameters of maxium, Cmax, AUC0 ~ t, AUC0 ~ (1.17 ± 0.26) h, (0.063 4 ± 0.023 5) ng / m L, (0.176 ± 0.091 9) h · ng / m L, 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 and (2.390 ± 0.877) h. Conclusions This assay is specific and sensitive and can be used for simultaneous determination of tetrahydropalmatine and imperatorin in the plasma of dogs. It can be used to study pharmacokinetics in dogs after the administration of Yuanhu Zhitong Tablet.