Abstract

Background: Liquid chromatography-tandem mass spectrometry (LC/MS/MS) is a powerful technique for the simultaneous determination of cyclosporin A, tacrolimus, sirolimus and everolimus in whole blood. LC/MS/MS has the benefits of lower detection limits, greater specificity and a broader dynamic range than commercial immunoassays. This paper evaluates the use of a commercially available kit from Recipe GmbH of whole blood calibrators and controls with a quick and simple sample preparation method for LC/MS/MS determination of four immunosuppressants.
Material/Methods: Samples were prepared by adding 200 μL of protein precipitation reagent containing internal standards to 100 μL of whole blood (unknown, calibrators and controls). Samples were then vortexed and centrifuged. Two-dimensional chromatography was performed on the supernatant, with detection by LC/MS/MS using multiple reaction monitoring (MRM) in positive mode. Mass spectrometer parameters were optimized for each analyte.
Results: The whole blood calibrator kit contained 7 calibrators including a blank. The LC/MS/MS assay showed excellent linearity over the concentration range with calibration regression coefficient ranging from 0.9994 to 0.9998. The intra-assay accuracy and precision was within the 20% cutoff, with the exception of the lowest concentration calibrator. Inter-assay accuracy ranged from 89-102% for the low concentration quality control, with % CVs ranging from 5.5-17%. Unknown sample results show good correlation with results obtained from a separate laboratory that prepares their own calibrators and controls in-house.
Conclusions: This study has demonstrated the successful use of a commercially available kit for whole blood calibrators and controls for the determination of four immunosuppressants in unknown samples by LC/MS/MS.

Keywords: Chromatography, immunosuppressants, Sirolimus