Validation and application of a fast semi-automated whole blood extraction for LC-MS/MS simultaneous quantification of cyclosporine A, tacrolimus, sirolimus and everolimus - application to high throughput routine therapeutic drug monitoring

Numerous laboratories carried out simultaneous whole blood quantitation of everolimus, sirolimus, tacrolimus and cyclosporine-A on a liquid chromatography-tandem mass-spectrometry (LC-MS/MS) system with online solid phase extraction (SPE) after manual deproteinization and centrifugation. This method had a relatively low sample throughput due to labor intensive and time-consuming sample preparation. We develop a fast and robust method of semi-automated whole blood sample pretreatment able to support over 120 samples daily. Whole blood samples were mixed in a V-bottom plate with 2 volumes of ZnSO4/methanol containing deuterated internal standards. After centrifugation, the supernatant was loaded on an online-SPE before being transferred and monitored on the LC-MS/MS system. Assay performance included inter-and intra-day accuracy and imprecision, relative and absolute matrix effects, extraction recovery and selectivity. Intra-and inter-day imprecision was <15% for all tested concentrations. The lower limit of quantification of cyclosporine-A, everolimus, sirolimus and tacrolimus was respectively 26.6, 1.13, 1.57 and 1.28 ng mL(-1), with an accuracy between -0.63% and 11.48% and a precision <12% of the nominal concentration. Standard line slope imprecision did not exceed 20% and ion enhancement or suppression was negligible. The comparison methods were performed by Passing and Bablok regression analysis: a high correlation was found between the two pre-treatment methods for tacrolimus, everolimus and sirolimus and between LC-MS/MS vs. radio-immuno-assay for cyclosporine-A. We developed a fast, sound and less misleading analytical method for high throughput (over 120 samples daily) therapeutic drug monitoring of multiple immunosuppressive drugs in routine practice.