LC-MS／MS法测定人全血中他克莫司的浓度及其在生物等效性试验中的应用

LC-MS/MS determination of tacrolimus in human blood and application in its bioequivalence study

目的:建立高效液相色谱-质谱联用法测定人全血中他克莫司浓度。方法:选用Shim-pack VP-ODS柱色谱柱(5μm,150 mm×4.6 mm),以乙腈-10 mmol·L-1醋酸铵为流动相,采用梯度洗脱进行分离,流速:0.8 mL·min-1;柱温:25℃;进样量:20μL。样品用乙腈进行蛋白沉淀后进样,选用3200QTRAP型质谱仪的多重反应监测(MRM)扫描方式进行检测。结果:他克莫司的线性范围为0.1～25.0 ng·mL-1,定量下限和最低检测限分别为0.1 ng·mL-1和0.05 ng·mL-1。准确度与精密度结果显示方法日间、日内变异均小于15%,相对偏差为-6.5%～4.3%,方法提取回收率均接近100.0%,稳定性较好。结论:本研究所建立的方法快速、灵敏,专属性强,重复性好,适用于他克莫司人体生物等效性试验。

Objective： To develop an LC - MS/MS method for the determination of the tacrolimus （FKS06） in hu- man whole blood. Method： Tacrolimus and the internal standard were extracted from whole blood by acetonitrile which was used as deproteinated solvent,and separated on a Shim -pack VP -ODS column （5 μm, 150 mm × 4. 6 mm） maintained at 25℃ with acetonitrile and 10 mmol · L^-1 ammonium acetate as mobile phase by gradient elution. The flow rate was 0. 8 mL· min^-1 and 20 μL aliquot of residues were injected into the LC - MS/MS system. Detection was carried out by multiple reaction monitoring on a 3200QTRAP LC - MS/MS system. Results：The as- say was linear over the range 0. 1-25.0 ng·mL^-1 with a limit of quantitation of 0. 1 ng · mL^-1 and a limit of detection of 0.05 ng·mL^-1. Intra -and inter - day precision were less than 15 %, respectively. The relative deviation was from - 6. 5% to 4. 3%. The recoveries of tacrolimus were almost 100. 0% and stabilities were good. Conclusion ：The method is a rapid, sensitive, selective and reliable for the determination of tacrolimus in human whole blood. The assay is applied to a bioequivalence study of tacrolimus to healthy volunteers.