Fully Automated Dried Blood Spot Extraction coupled to Liquid Chromatography-tandem Mass Spectrometry for Therapeutic Drug Monitoring of Immunosuppressants

Abstract

Patients receiving immunosuppressant therapy, require intensive follow-up via therapeutic drug monitoring (TDM). This puts quite a burden on the patient involving frequent hospital visits and venipunctures and could (partially) be resolved by the use of dried blood microsamples (e.g. dried blood spots, DBS). One of the drawbacks of the use of DBS is the requirement for a dedicated, manual sample preparation. Fully automated DBS extraction systems, online coupled to standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) configurations, could provide a solution for that. The aim of this study was to evaluate the use of the DBS-MS 500, online coupled to an LC-MS/MS system, for the TDM of immunosuppressants using DBS. Two methods for the quantification of tacrolimus, sirolimus, everolimus and cyclosporin A, in both DBS and whole blood, were developed and validated based on international guidelines. For the DBS method also DBS-specific parameters were taken into account. Both methods proved to be accurate and reproducible with biases below 11% (20% for the LLOQ) and CVs (%) below 14% (with a single exception) (20% for the LLOQ) over a calibration range from 1 to 50 ng/mL for tacrolimus, sirolimus and everolimus and 20 to 1500 ng/mL for cyclosporin A. Reproducible (CV < 15%) IS-compensated relative recovery values were obtained. However, a hematocrit-dependent relative recovery was observed for DBS, with lower hematocrit values yielding higher relative recoveries (and vice versa). Relative to the reference hematocrit of 0.37, this difference exceeded 15% at hematocrit extremes (0.18 and 0.60). Application on venous left-over patient samples showed reasonable agreement between the results of both methodologies (8,6,9 and 9/10 mean DBS results within 20% of the mean whole blood result for tacrolimus, sirolimus, everolimus and cyclosporin A, respectively), although also here an impact of the hematocrit could be discerned. As a next step, larger patient sets are needed to allow a better insight on how (correction for) the hct effect affects the quantification of immunosuppressants via fully automated DBS analysis.