Determination of Cyclosporin A and Tacrolimus in Human Whole Blood by High Performance Liquid Chromatography-Tandem Mass Spectrometry after Purification with Pass-Through Solid Phase Extraction Column

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.