Objectives: Although, due to its high specificity and sensitivity, LC-MS/MS is an efficient technique for the routine determination of immunosuppressants in whole blood, it involves time-consuming manual sample preparation. The aim of the present study was therefore to develop an automated sample-preparation protocol for the quantification of sirolimus, everolimus and tacrolimus by LC-MS/MS using a liquid handling platform.
Methods: Six-level commercially available blood calibrators were used for assay development, while four quality control materials and three blood samples from patients under immunosuppressant treatment were employed for the evaluation of imprecision. Barcode reading, sample re-suspension, transfer of whole blood samples into 96-well plates, addition of internal standard solution, mixing, and protein precipitation were performed with a liquid handling platform. After plate filtration, the deproteinised supernatants were submitted for SPE on-line. The only manual steps in the entire process were de-capping of the tubes, and transfer of the well plates to the HPLC autosampler.
Results: Calibration curves were linear throughout the selected ranges. The imprecision and accuracy data for all analytes were highly satisfactory. The agreement between the results obtained with manual and those obtained with automated sample preparation was optimal (n=390, r=0.96). In daily routine (100 patient samples) the typical overall total turnaround time was less than 6h.
Conclusions: Our findings indicate that the proposed analytical system is suitable for routine analysis, since it is straightforward and precise. Furthermore, it incurs less manual workload and less risk of error in the quantification of whole blood immunosuppressant concentrations than conventional methods.
Keywords: Automation; CV; ESI; EVE; FK-506; HPLC; IS; Immunosuppressants; LC-MS/MS; LLOQ; LOD; MRM; MS; MS/MS; QC; Routine application; SD; SIR; SPE; TDM; Whole blood; coefficient of variation; electrospray ionization interface; everolimus; high performance liquid chromatography; internal standard; limit of detection; liquid chromatography-tandem mass spectrometry; lower limit of quantification; mass spectrometry; multiple-reaction monitoring; quality control; sirolimus; solid phase extraction; standard deviation; tacrolimus; tandem mass; therapeutic drug monitoring.
© 2013.