Rapid determination of tacrolimus and sirolimus in whole human blood by direct coupling of solid-phase microextraction to mass spectrometry via microfluidic open interface

Abstract

Immunosuppressive drugs are administered to decrease immune system activity (e.g. of patients undergoing solid organ transplant). Concentrations of immunosuppressive drugs (ISDs) in circulating blood must be closely monitored during the period of immunosuppression therapy due to adverse effects that take place when concentration levels fall outside of the very narrow therapeutic concentration range of these drugs. This study presents the rapid determination of four relevant immunosuppressive drugs (tacrolimus, sirolimus, everolimus, and cyclosporine A) in whole human blood by directly coupling solid-phase microextraction to mass spectrometry via the microfluidic open interface (Bio-SPME-MOI-MS/MS). The BioSPME-MOI-MS/MS method offers ≤ 10% imprecision of in-house prepared quality controls over a 10-day period, ≤ 10% imprecision of ClinCal® Recipe calibrators over a three-day period, and single total turnaround time of ∼ 60 min (4.5 min for high throughput). The limits of quantification were determined to be 0.8 ng mL−1 for tacrolimus, 0.7 ng mL−1 sirolimus, 1.0 ng mL−1 for everolimus, and 0.8 ng mL−1 for cyclosporine. The limits of detection were determined to be 0.3 ng mL−1 for tacrolimus, 0.2 ng mL−1 for sirolimus, 0.3 ng mL−1 for everolimus, and 0.3 ng mL−1 for cyclosporine A. The R2 values for all analytes were above 0.9992 with linear dynamic range from 1.0 mL−1 to 50.0 ng mL−1 for tacrolimus, sirolimus, and everolimus while from 2.5 ng mL−1 to 500.0 ng mL−1 for cyclosporine A. To further evaluate the performance of the present method, 95 residual whole blood samples of tacrolimus and sirolimus from patients undergoing immunosuppression therapy were used to compare the Bio-SPME-MOI-MS/MS method against a clinically validated reference method based on chemiluminescent microparticle immunoassay, showing acceptable results. Our results demonstrated that Bio-SPME-MOI-MS/MS can be considered as a suitable alternative to existing methods for the determination of immunosuppressive drugs in whole blood providing faster analysis, better selectivity and sensitivity, and a wider dynamic range than current existing approaches.