Development and Validation of a U-HPLC-MS/MS Method for the Concurrent Measurement of four Immunosuppressants in Whole Blood

초록

Background: This study aimed to develop and validate a U-HPLC-MS/MS method for simultaneous determination of four immunosuppressants in human whole blood.Methods: The method was based on the injection of 20 mu L of calibrators and controls pretreated with the liquid phase extraction method for chromatography separation and mass spectrometry determination. LPE offline was performed by adding 0.1 mol/L ZnSO4 and acetonitrile, while separation of target compounds was achieved with-in 2.5 minutes by a Zorbax Eclipse XDB-C8 column using ammonium acetate and ACN mixed with formic acid as solvents. Results: The assay offers ng/mL detection limits (from 1.1 to 12.4 ng/mL), accuracy (% deviation from-4.4% to 5.6%), precision (CV less than 15% at all QC levels), and linearity (from 23.4 to 948 ng/mL for CsA, from 2.11 to 45.5 ng/mL for TAC, SIR and EVR). The recovery and matrix results were acceptable, and the carryover was less than 1%. The results of method comparison show that IA-based methods overestimated the concentration of drugs compared with the MS-based method. Comparing our MS-based method with external LC-MS/MS showed that the results were within 2 SDs.Conclusions: We have developed a reliable assay for the analysis of CsA, TAC, SIR and EVR in whole blood using U-HPLC-MS/MS.

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