LC-MS/MS法测定Beagle犬全血中他克莫司的浓度

Determination of Tacrolimus in Beagle Dog Blood with LC-MS / MS

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 establish an LC-MS /MS method for determination of tacrolimus in Beagle dog blood. Methods Tacrolimus and internal standard ascomycin extracted from whole blood of Beagle dogs by methanol which was used as the deproteinated solvent were separated on an Agilent C_（18）（ 125 mm × 4. 6 mm,5 μm） maintained at 60 ℃. The mobile phase consisted of methanol and 10 mmol·L^-1 ammonium acetate by gradient elution at a flow rate of 0. 4 ml·min^-1. Results The standard curve was linear in the range of 0. 625- 50 ng·ml^-1. The limit of quantification was 0. 625 ng·ml^-1. The recovery of tacrolimus was 89. 8%~101. 4%,matrix effect was 11. 8%and the within-day and between-day RSDs were less than 10. 3%. Conclusion This method has proved to be suitablefor determination of tacrolimus in Beagle dog blood after oral administration of low dosage,which is much more sensitive than previously reported methods.