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目的采用LC-MS/MS法测定Beagle犬全血中他克莫司的浓度。方法 Beagle犬全血样品加0.05 mol·L~(-1)ZnSO_4溶液100μl,甲醇直接沉淀蛋白,混匀后13 000 r·min~(-1),离心10 min。取上清液进样20μl分析。使用Agilent C_(18)(125 mm×4.6 mm,5μm)色谱柱,以甲醇~(-1)0 mmol·L~(-1)醋酸铵为流动相,梯度洗脱,流速0.4 ml·min~(-1)。ESI离子源,多反应离子监测。用于定量和定性分析的离子对他克莫司分别为m/z 821.9→768.5,821.9→576.4;内标子囊霉素为809.8→756.5,809.8→564.8。结果线性范围为0.625~50 ng·ml~(-1),最低定量限为0.625 ng·ml~(-1),方法回收率为89.8%~101.4%,内标归一化基质效应因子的变异系数为11.8%,日内和日间RSD均小于10.3%。结论本方法快速、简便、灵敏度高,适用于Beagle犬全血中他克莫司浓度的测定及药代动力学和生物等效性的研究。
Objective To determine the concentration of tacrolimus in whole blood of Beagle dogs by LC-MS / MS. Methods Beagle dog whole blood samples were added with 100 μl of 0.05 mol·L -1 ZnSO 4 solution, and the protein was directly precipitated by methanol. After centrifugation for 13 000 r · min -1, centrifugation was performed for 10 min. Take the supernatant injection 20μl analysis. The mobile phase was eluted with ammonium C_ (18) (125 mm × 4.6 mm, 5 μm) column using methanol (-1) 0 mmol·L -1 ammonium acetate as the mobile phase at a flow rate of 0.4 ml · min ~ (-1). ESI source, multi-reactive ion monitoring. The ion pair tacrolimus used for quantitative and qualitative analysis was m / z 821.9 → 768.5, 821.9 → 576.4, respectively; the internal standard ascomycin 809.8 → 756.5,809.8 → 564.8. Results The linear range was 0.625 ~ 50 ng · ml ~ (-1), the lowest limit of quantification was 0.625 ng · ml ~ (-1), and the recovery was 89.8% ~ 101.4%. The normalized matrix effect factor The coefficient is 11.8% with daily and daytime RSDs less than 10.3%. Conclusion The method is rapid, simple and sensitive. It is suitable for the determination of tacrolimus concentration in whole blood of Beagle dogs and its pharmacokinetics and bioequivalence.