Determination of cyclosporine, tacrolimus, sirolimus and everolimus by liquid chromatography coupled to electrospray ionization and tandem mass spectrometry: Assessment of matrix effects and assay performance

Objective. The immunosuppressants cyclosporine, tacrolimus, sirolimus and everolimus are used in rejection prophylaxis after transplantation. Liquid chromatography tandem mass spectrometry (LC-MS/MS) has become a widely used methodology for monitoring of the drug levels to ensure therapeutic exposure. The main objective of the study was to evaluate the existence and potential influence of matrix effects on LC-MS/MS measurements of the immunosuppressants in clinical blood samples. Methods. The samples were prepared by protein precipitation and thereafter analysed by reversed-phase chromatography coupled to MS/MS via an electrospray interface. Assay performance including within-and between-series imprecision and deviations from external controls were examined. Elution of overall matrix components and glycerophosphocholines were investigated. The MS/MS signals were monitored in post-column infusion experiments, and post-precipitation addition of compounds provided a basis for quantification of the matrix effects. The influence of matrix effects on assay performance was investigated after dilution of quality controls with blood from multiple individuals. Results. Between-series coefficients of variation were <= 5.1, <= 6.6, <= 11.0 and <= 7.4 %, and the mean deviations from external controls were -10.3, -6.7, 15.6 and 4.3% for cyclosporine, tacrolimus, sirolimus and everolimus, respectively. The elution of matrix components including glycerophosphocholines overlapped to some extent with the target compounds, and the average ion suppression ranged from 8.5-21%. However, the drugs and internal standards were correspondingly influenced. Conclusion. The internal standards consistently corrected the between-individual variability of matrix effects. These findings consolidate the reliability of the assay.