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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 establish a method for the determination of tacrolimus in human whole blood by high performance liquid chromatography-mass spectrometry. Methods: Shim-pack VP-ODS column (5 μm, 150 mm × 4.6 mm) was used with acetonitrile-10 mmol·L-1 ammonium acetate as mobile phase and separated by gradient elution. The flow rate was 0.8 mL · min -1; column temperature: 25 ℃; injection volume: 20μL. The samples were submitted to protein precipitation with acetonitrile and selected for multiplex reaction monitoring (MRM) scanning using a 3200QTRAP mass spectrometer. Results: The linear range of tacrolimus was 0.1-25.0 ng · mL-1. The limits of quantification (LODs) and the lowest limits of detection were 0.1 ng · mL-1 and 0.05 ng · mL-1, respectively. The results of accuracy and precision showed that the methods were both less than 15% and the relative deviation was -6.5% -4.3% during the day and the day, and the recoveries of the methods were all close to 100.0% with good stability. Conclusion: The method established in this study is fast, sensitive, specific and reproducible, and is suitable for the bioequivalence test of tacrolimus.