UPLC-MS/MS快速测定大鼠血浆中盐酸美沙酮及其代谢产物含量

Rapid Determination of Methadone Hydrochloride and Its Metabolites in Rat Plasma by UPLC-MS/MS

目的建立超高效液相色谱-串联质谱(UPLC-MS/MS)测定大鼠血浆中盐酸美沙酮及其代谢产物乙二胺二甲基次磷酸(2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine,EDDP)的含量。方法以地西泮为内标,大鼠血浆用乙腈沉淀蛋白,运用UPLC-MS/MS检测大鼠灌胃盐酸美沙酮6 mg·kg-1后血浆中美沙酮和EDDP的血药浓度,并对数据进行分析。结果大鼠血浆中盐酸美沙酮和EDDP的定量限均为0.5 ng·m L^(-1);线性相关系数r>0.999;日内精密度和日间精密度的RSD均<15%;绝对回收率均>85%,相对回收率在90%~110%之间;基质效应在92.80%~97.71%之间。主要药动学参数:美沙酮、EDDP在大鼠血浆中的消除半衰期(t1/2)为(1.479±0.839),(2.590±0.760)h;最高血药浓度(Cmax)为(48.562±10.501),(25.542±3.706)mg·L^(-1);平均滞留时间(MRT0-t)为(3.476±0.310),(3.883±0.514)h;曲线下面积(AUC0-t)为(204.036±61.145),(86.031±15.287)mg·h·L^(-1);清除率(CL)为(32.338±14.343)、(70.137±10.786)L·h-1·kg-1,各项药动学参数比较差异均有统计学意义。结论该法专属性强、分离完全、快速灵敏,适用于大鼠血浆中盐酸美沙酮及其代谢产物EDDP的含量测定和药动学研究。

OBJECTIVE To propose a UPLC-MS/MS method for the determination of methadone hydrochloride and its metabolites 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine（EDDP） in rat plasma. METHODS Diazepam was used as internal standard, rat plasma was precipitated with acetonitrile, the concentration of methadone hydrochloride and EDDP in the plasma of rats after intragastric administration methadone 6 mg·kg^（-1）, determined by UPLC-MS/MS, and the data were analyzed. RESULTS The limit of quantitation was 0.5 ng·m L^（-1） for both methadone hydrochloride and EDDP in rat plasma; the linear correlation coefficient r0.999; the intra-day and inter-day RSDs were all 15%; Absolute recovery rates were above 85%. The relative recoveries ranged from 90% to 110%; the matrix effect ranged from 92.80% to 97.71%. The main pharmacokinetic parameters of methadone and EDDP were as follow： t1/2 of methadone and EDDP were（1.479±0.839）,（2.590±0.760） h; Cmax were（48.562±10.501）,（25.542±3.706） mg·L^（-1）; MRT0-t were（3.476±0.310）,（3.883±0.514） h; AUC0-t were（204.036±61.145）,（86.031±15.287） mg·h·L^（-1）; CL were（32.338±14.343）,（70.137±10.786） L·h^（-1）·kg^（-1）, respectively, and there were significant differences in the parameters between the rats before and after being administrated with methadone and EDDP. CONCLUSION The method is proved to be highly sensitive, selective, and has been successfully applied to the determination of methadone hydrochloride and its metabolite EDDP, and can be used for pharmacokinetic studies in rats.