UPLC-MS法同时测定大鼠血浆中曲马多及其3个代谢物和异丙嗪的质量浓度

Simultaneous determination of tramadol,three metabolites of tramadol and promethazine in rat plasma using an UPLC-MS method

目的建立同时测定大鼠血浆中曲马多、O-去甲基曲马多、N-去甲基曲马多、N,O-二去甲基曲马多和异丙嗪的UPLC-MS方法,并将其应用于复方盐酸曲马多和盐酸异丙嗪注射液在大鼠体内的药动学研究。方法采用沉淀蛋白法进行样品预处理,色谱柱为Acquity UPLC BEH C18(50 mm×2.1 mm,1.7μm)柱,流动相采用乙腈-5 mmol·L^(-1)醋酸铵(含体积分数0.02%甲酸溶液)梯度洗脱,以正离子选择离子监测模式进行检测。结果血浆中曲马多、O-去甲基曲马多、N-去甲基曲马多、N,O-二去甲基曲马多和异丙嗪分别在质量浓度为1.84~368.0μg·L^(-1)、0.21~42.00μg·L^(-1)、0.49~98.00μg·L^(-1)、0.48~96.00μg·L^(-1)和1.90~380.0μg·L^(-1)内线性关系良好,日内和日间精密度RSD均不超过10.8%,提取回收率为93.2%~109.5%,基质效应为93.3%~104.6%,5个分析物的血浆样品在所考察的储存条件下均可保持稳定。结论该方法适用于复方盐酸曲马多和盐酸异丙嗪注射液在大鼠体内的药动学研究。与单独给药后的药动学行为进行比较,复方同时给药后,曲马多及其3个代谢物的ρmax和AUC均增加,特别是在雌性大鼠体内;而复方给药后,曲马多对异丙嗪的药动学行为无明显影响。

Objective To establish an UPLC-MS method for the simultaneous determination of tramadol and its three metabolites, and promethazine in rat plasma, and to study the pharmacokinetic behavior of tramadol and promethazine after co-administration of tramadol and promethazine in rats. Methods After the plasma samples were treated by protein precipitation method with acetonitrile chromatographic separation was performed on an Acquity UPLC BEH C18 column using a gradient elution with mobile phase consisting of acetonitrile（ B）and 5 mmol.L-1 NHnAC containing 0.02% formic acid（A）. The samples were detected by single ion monitoring in positive ion mode. Results The method was linear over the range of 1.84 - 368.0 μg.L-1 for tramadol,0.21 -42.00μg.L-1 for O-desmethyltramadol,0. 49 -98.00 μg.L-1 for N- desmethyltramadol,0. 48 - 96. 00 μg . L-1 for N, O-didesmethyltramadol, 1.90 - 380. 0 μg . L-1 for promethazine with the intra-and inter-day precision below 10.8%. The extraction recovery was ranged from 93.2% to 109.5% and the matrix effect was ranged from 93.3% to 104.6%. The five analytes were found to be stable in plasma samples under the different conditions investigated in this study. Conclusions The developed method was feasible for the pharmacokinetic study of tramadol and promethazine. The results showed that the Pmax and AUC of tramadol and its three metabolites were increased after co-administration with promethazine, especially in female rats; whereas co-administration with tramadol has no significant influence on the pharmacokinetic parameters of promethazine.