A validated UPLC-MS/MS method for the quantification of immunosuppressive drugs in peripheral blood mononuclear cells using liquid-liquid extraction with low temperature purification without complex pretreatment steps

Immunosuppressive drugs (ISDs) are given to avoid the allograft rejection after transplantation. The concentrations of ISDs should be closely monitored owing to their wide inter-individual variability in its pharmacokinetics and narrow therapeutic window. Currently, the whole blood concentration measurement is the major approach of therapeutic drug monitoring of clinical ISDs in organ transplantation. Its correlation with the efficacy of ISDs remains elusive. While the acute rejection after transplantation may occur even when whole-blood ISDs concentrations are within the target range. Since the site of action of ISDs are within the lymphocyte, direct measurement of drug exposure in target cells may more accurately reflect the clinical efficacy of ISDs. Although several methods have been developed for the peripheral blood mononuclear cells (PBMCs) extraction and drug concentration measurement, the complex pre-processing has limited the study of the relationship between intracellular ISDs concentrations and the occurrence of rejection. In this study, the extraction of ISDs in PBMCs was carried out by the liquid-liquid extraction with low temperature purification, without centrifugation. The lower limit of quantitation were 0.2 ng/mL for cyclosporine A, tacrolimus and sirolimus, 1.0 ng/mL for mycophenolic acid, and the within-run and between-run coefficient of variations were both less than 12.4 %. The calibration curves of mycophenolic acid had a linear range (ng/mL): 1.0-128.0 (r(2) = 0.9992). The calibration curves of other three ISDs had a linear range (ng/mL): 0.2-20.48 (r(2) > 0.9956). A total of 157 clinical samples were analyzed by the UPLC-MS/MS for ISDs concentration in blood or plasma ([ISD](blood or plasma)) and the concentration within PBMCs ([ISD](PBMC)). Although there was strong association between [ISD](PBMC) and [ISD](blood or plasma), the large discrepancies between concentration within [ISD](blood or plasma) and [ISD](PBMC) were observed in a small proportion of clinical samples. The developed method with short analysis time and little amounts of blood sample can be successfully applied to therapeutic drug monitoring of ISDs in PBMCs for analysis of large numbers of clinical samples and is helpful to explore the clinical value of ISDs concentration in PBMCs.