UPLC-MS/MS法检测大鼠血浆中新利司他杂质A的含量及其在药代动力学中的应用

UPLC-MS/MS method to detect the content of cetilistat impurity A in rat plasma and its application in pharmacokinetics

目的:建立超高效液相色谱-串联质谱法(UPLC-MS/MS)检测大鼠血浆内新利司他杂质A(2-[[(十六烷氧基)羰基]氨基]-5-甲基苯甲酸)含量的方法以及该方法在药代动力学中的应用。方法:大鼠含药血浆以奥利司他为内标,用乙腈沉淀蛋白,10000 r·min^(-1)离心10 min,吸取上清液150μL,加入四氢呋喃10μL,混匀后进样分析。采用Hypersil GOLD(50 mm×2.1 mm,1.9μm)色谱柱,以0.1%甲酸乙腈-0.1%甲酸水(82∶18)为流动相,流速为0.5 mL·min^(-1),柱温为30℃;质量检测器使用ESI离子源,正离子模式检测,待测物杂质A及内标奥利司他用于定量的母→子离子对分别为m/z 420.3→160.3、m/z 496.4→319.3。结果:新利司他杂质A质量浓度在0.0200~10.0 mg·L^(-1)范围内呈良好的线性关系。稳定性、准确度的RE均在0.1%~6.3%范围内,精密度、回收率、基质效应的RSD均在3.8%以下。在大鼠灌胃低(20 mg·kg^(-1))、中(80 mg·kg^(-1))、高(320 mg·kg^(-1))3个剂量新利司他杂质A混悬液的药代动力学实验中,血浆中新利司他杂质A的最高血药浓度(C_(max))仅为12.1 mg·L^(-1),3个剂量组的AUC分别为22.2、64.9、85.0 mg·L^(-1)·h;血浆暴露量虽随剂量增加而增加,但是增加倍数小于剂量增加倍数。3个剂量组的达峰时间(T_(max))在2.6~3.0 h,消除半衰期(t_(1/2z))在3.08~3.85 h,吸收、消除过程类似。结论:本文建立了超高效液相色谱-串联质谱法测定大鼠血浆内新利司他杂质A含量的方法。大鼠灌胃新利司他杂质A混悬液,在20~320 mg·kg^(-1)剂量范围内吸收入血的量比较少,血浆暴露量小于剂量相关线性增加,口服带来的安全性问题有限。

Objective:To establish an ultra-high performance liquid-tandem mass spectrometry(UPLC-MS/MS)detection method for the determination of cetilistat impurity A(2-[[(hexadecyloxy)carbonyl]amino]-5-methylb enzoic acid)in rat plasma and to apply it in a pharmacokinetic study.Methods:Orlistat was used as the internal standard in rat plasma,and the protein was precipitated by acetonitrile,centrifuged at 10000 r·min^(-1)for 10 min,150μL of supernatant was added to 10μL of tetrahydrofuran and mixed well,and then sampled for analysis.Using Hypersil GOLD(50 mm×2.1 mm,1.9μm)chromatographic column,and the column temperature was 30℃,with 0.1%formic acid acetonitrile-0.1%formic acid water(82∶18)as fluidity,the flow rate was 0.5 mL·min^(-1).ESI source was used as mass detector in positive ion mode.The precursor→product ion pairs of cetilistat impurity A and olistat were m/z 420.3→160.3 and m/z 496.4→319.3,respectively.Results:The mass concentration of cetilistat impurity A shows a good linear relationship in the concentration range of 0.0200-10.0 mg·L^(-1).The REs of stability and accuracy were in the range of 0.1%-6.3%,and the RSDs of precision,recovery,and matrix effect were all below 3.8%.In the pharmacokinetic experiment of three doses of cetilistat impurity A suspension in rats with low(20 mg·kg^(-1)),medium(80 mg·kg^(-1))and high(320 mg·kg^(-1))gavage,the peak concentration(C_(max))of cetilistat impurity A was as low as 12.1 mg·L^(-1)in rat plasma in high dose group.The AUC of the low,medium,and high-dose groups were 22.2,64.9,and 85.0 mg·L^(-1)·h,respectively.The plasma exposure increased with the dose,but the increase rate was lower than the dose increase rate.The peak time(T_(max))of the three-dose group was between 2.6 and 3.0 h,and the elimination half-life(t_(1/2z))was between 3.08 and 3.85 h.The three dose groups showed similar absorption and elimination processes.Conclusion:A method for the determination of cetilistat impurity A in rat plasma by ultra-high performance liquid phase-tandem mass spectrometry was established.And the rat plasma exposure is less than the dose-related linear increase in the dose range of 20-320 mg·kg^(-1)after gavaged with cetilistat-impurity A suspension,and the amount absorbed into blood is relatively small,and the satety problems caused by oral administration are limited.