在200 μL人全血样品中加入20 μL混合内标溶液（环孢素A-d₄和他克莫司-¹³C，d₂质量浓度分别为5，0.5 mg·L^-1），混匀后加入300 mmol·L^-1硫酸锌溶液200 μL，涡旋60 s。再加入600 μL乙腈，涡旋120 s，静置5 min，离心10 min。分取上清液约1 mL，注入PRiME HLB通过式固相萃取柱，收集流出液，注入高效液相色谱仪，在InfinityLab Poroshell 120 EC-C₁₈色谱柱（50 mm×4.6 mm，2.7 μm）上用不同体积比的含0.1%（体积分数，下同）甲酸的0.01 mol·L^-1乙酸铵溶液和含0.1%甲酸的乙腈溶液为流动相进行梯度洗脱，所得目标物在串联质谱仪的电喷雾离子源正离子模式下电离、多反应监测模式下检测，以基质匹配法和内标法定量。结果显示：环孢素A和他克莫司工作曲线的线性范围分别为10~2 000 μg·L^-1和0.5~100 μg·L^-1，检出限分别为1.24，0.05 μg·L^-1；加标回收率分别为92.2%~106%和94.1%~108%，测定值的相对标准偏差（n=5）分别为2.9%~7.8%，4.7%~7.6%（日内精密度试验）和4.5%~6.7%，5.1%~8.2%（日间精密度试验）；方法用于10个实际样品的分析，环孢素A和他克莫司的检出量分别为100.6~135.0 μg·L^-1和8.5~13.8 μg·L^-1。

An aliquot (20 μL) of the mixed internal standard solution containing 5 mg·L^-1 cyclosporine A-d₄ and 0.5 mg·L^-1 tacrolimus-¹³C,d₂ was added into 200 μL of human whole blood sample. After blending, 200 μL of 300 mmol·L^-1 zinc sulfate solution and 600 μL of acetonitrile were added in sequence, and the mixture was vortexed for 60 s and 120 s, respectively. The solution obtained was settled for 5 min, and centrifuged for 10 min. An aliquot (about 1 mL) of the supernatant was loaded on the PRiME HLB pass-through solid phase extraction column, and the effluent was collected and introduced into the high performance liquid chromatograph. The targets were separated on the InfinityLab Poroshell 120 EC-C₁₈ column (50 mm×4.6 mm, 2.7 μm) by gradient elution using mixed solutions composed of 0.01 mol·L^-1 ammonium acetate solution containing 0.1% (volume fraction, the same below) formic acid and acetonitrile solution containing 0.1% formic acid at various volume ratios. The obtained targets were ionized in positive ion mode of electrospray ion source, detected in multiple reaction monitoring mode in the tandem mass spectrometer, and quantified by matrix matching method and internal standard method. As found by results that the linear ranges of the working curves of cyclosporine A and tacrolimus were 10-2 000 μg·L^-1 and 0.5-100 μg·L^-1, with detection limits of 1.24, 0.05 μg·L^-1. Test for recovery by standard addition method was made, giving recoveries in the ranges of 92.2%-106% and 94.1%-108%, and RSDs (n=5) of the determined values were in the ranges of 2.9%-7.8%, 4.7%-7.6% (for intra-day precision test) and 4.5%-6.7%, 5.1%-8.2% (for inter-day precision test). The proposed method was applied to the analysis of 10 actual samples, and the detected amounts of cyclosporine A and tacrolimus were in the ranges of 100.6-135.0 μg·L^-1 and 8.5-13.8 μg·L^-1, respectively.