Determination of sirolimus concentration in human whole blood by UPLC-MS/MS and homogeneous enzyme immunoassay
Objective To quantify sirolimus concentration in human whole blood by UPLC-MS/MS and to evaluate the agreement with the homogeneous enzyme immunoassay.Methods Human whole blood(100 μL)was spiked with 10 μL sirolimus-D3(the internal standard,IS),100 μL 0.1 mol·L-1 ZnSO4 and 300 μL methanol.After the vortex and centrifugation(14 000 r·min-1),5 μL supernatant was injected.Water(containing 10 mmol·L-1 ammonium acetate and 0.1％formic acid,solvent A)and methanol(containing 0.1％formic acid,solvent B)were used as the eluent,with the gradient elution at 0.5 mL·min-1 in FastCore Super C18 column(2.1 mm×100 mm,2.6 μm).The multiple reaction monitoring(MRM)with electrospray ionization(ESI)in the positive-ion mode was used.m/z 931.6＞864.6 and m/z 934.6＞864.6 were used for sirolimus and the IS quantitation.Method validation,including selectivity,LLOQ,calibration curve,accuracy,precision,remaining-effect,matrix effect,stability and recovery was evaluated.Passing-Bablok regression analysis and Bland-Altman plot were used to assess the agreement between UPLC-MS/MS and homogeneous enzyme immunoassay.Results The calibration ranged 2.5～50 ng·mL-1for sirolimus.The relative error of intra-and inter-day accuracy of sirolimus ranges from-5.32％to 7.46％,and the coefficient of variation was less than 11.8％.The IS normalized recovery and matrix effect were 100.22％～108.56％and 95.45％～96.48％with coefficient of variation less than 7.0％.Sirolimus was stable in the test conditions.Comparison between UPLC-MS/MS with homogeneous enzyme immunoassay showed a deviation in proportion.Conclusion UPLC-MS/MS method for the determination of sirolimus in human whole blood is rapid,easy and reliable.A proportion deviation in the two methods is observed.
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