Determination of four immunosuppressive drugs in whole blood using MEPS and LC–MS/MS allowing automated sample work-up and analysis

► New method for immunosuppressive quantification utilizing MEPS as sample preparation. ► Fully automated method. ► 50μl sample volume was used. ► Good precision and accuracy results. ► Compassion was made with protein precipitation. In treatment with immunosuppressive drugs, monitoring of blood drug concentration is needed. The aim of this work was to explore micro extraction by packed sorbent (MEPS) as a possible on-line sample preparation method in combination with liquid chromatography–tandem mass spectrometry (LC–MS/MS) for quantification of cyclosporine, everolimus, sirolimus and tacrolimus in whole blood. An automated on-line MEPS system connected with a LC–MS/MS instrument was set up. A C8 sorbent was used for the MEPS extraction. Subsequent analysis was performed with a gradient LC system. The adduct ions [M+NH4]+ of the analytes were monitored in SRM mode for quantification. Ascomycin and cyclosporine D were used as internal standards. The chromatographic run time 2.5min and the quantification ranges were 3–1500ng/mL (r2≥0.999, n=6) for cyclosporine and 0.5–50ng/mL for everolimus, sirolimus and tacrolimus (r2≥0.998, 0.994 and 0.993, respectively, n=6). Precision and accuracy were documented at three levels. Accuracy results were between 102% and 109% with precision between 2% and 13% and carry over