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Development and Validation of a U-HPLC-MS/MS Method for the Concurrent Measurement of four Immunosuppressants in Whole Blood by Cheonghwa Lee, Hyebin Choi, Jae Hyun Cha, Juyeon Kim, Myung-Hyun Nam, Minjeong Nam, Chang Kyu Lee, Yunjung Cho, Seung Gyu Yun

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 µL 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 within 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.

DOI: 10.7754/Clin.Lab.2022.220329