LC-MS/MS法测定大鼠血浆中R/S-吗啉硝唑及其在药物-药物相互作用研究中的应用

Determination of R-morinidazole and S-morinidazole in rat plasma by LC-MS/MS method and its application in a pharmacokinetic interaction study

目的:建立LC-MS/MS分析方法分离、定量大鼠血浆中的R-吗啉硝唑与S-吗啉硝唑,并对吗啉硝唑与丙磺舒、西咪替丁、二甲双胍、替诺福韦联用后R-吗啉硝唑与S-吗啉硝唑的药代动力学变化进行研究。方法:以吗啉硝唑-d8为内标,25μL大鼠血浆样本与5μL内标混合后加75μL甲醇,处理后取上清进行LC-MS/MS定量分析。采用Lux cellulose-4手性柱(4.6 mm×250 mm,5μm),以水-甲醇(10∶90)为流动相,流速0.6 mL·min^-1,柱温30℃,进样量5μL;采用正离子模式,MRM扫描,吗啉硝唑m/z 271.3→m/z 144.3,吗啉硝唑-d8(内标)m/z 279.4→m/z 152.3。大鼠于给药后的不同时间点采集静脉血进行药代动力学分析。结果:R-吗啉硝唑和S-吗啉硝唑质量浓度在5~1000 ng·mL^-1范围内线性良好(r2=0.9983±0.0002),定量下限为5 ng·mL^-1,回收率在95.6%~106.6%之间,以RSD表示的日内与日间精密度均小于8.2%,所有稳定性考察项目结果均符合要求。吗啉硝唑与二甲双胍联用后,仅R-吗啉硝唑的tmax参数差异具有统计学意义,与其他药物联用后药代动力学参数无显著变化。结论:本方法可对大鼠血浆中的R-吗啉硝唑和S-吗啉硝唑进行手性分离及定量分析。吗啉硝唑分别与丙磺舒、西咪替丁、二甲双胍、替诺福韦联用后,大鼠血浆中R-吗啉硝唑、S-吗啉硝唑药代动力学未发生显著改变。

Objective:To establish an LC-MS/MS analytical method to separate and quantify R-morinidazole and S-morinidazole in rat plasma,and to study the pharmacokinetic interaction of morinidazole after combined with probenecid,cimetidine,metformin,and tenofovir.Methods:After mixed with morinidazole-d8(internal standard),25μL of rat plasma sample was mixed with 5μL of internal standard and precipitated by 75μL of methanol,and the supernatant was analyzed by a LC-MS/MS method.The analyte was separated on a Lux cellulose-4 chiral column(4.6 mm×250 mm,5μm)with water-methanol(10∶90)as mobile phase at the mobile phase of 0.6 mL·min^-1,and the column temperature was 30℃.5μL of analyte was injected into column.Detection was performed using negative MRM mode on a TurboIonSpray source.The mass transitions monitored were m/z 271.3→144.3 and m/z 279.4→152.3 for morinidazole and morinidazole-d8,respectively.Venous blood samples for pharmacokinetic measurements were collected from the rats after administration.Results:The linear range of the calibration curve(5^-1000 ng·mL^-1)was obtained with a good correlation coefficient(r2=0.9983±0.0002).The lower limit of quantification was 5 ng·mL^-1,the average recovery was 95.6%^-106.6%and the intra-and interday precision represented by RSD were less than 8.2%.Results for process efficiency test and all stability tests met the acceptance criteria.After the combination of morinidazole and metformin,the tmax of R-morinidazole was statistically significant,and the pharmacokinetic parameters were not significantly changed after combination with other drugs.Conclusion:This method can perform chiral separation and quantitative analysis of R-morinidazole and S-morinidazole in rat plasma.After morpholindazole is combined with probenecid,cimetidine,metformin and tenofovir respectively,the pharmacokinetics of R-morinidazole and S-morinidazole in rat plasma is not changed significantly.