A high-throughput LC-MS/MS method for the quantification of four immunosu- ppressants drugs in whole blood

Background: Immunoassays and liquid chromatography tandem mass spectrometry (LC-MS/MS) are two major methods for therapeutic drug monitoring (TDM) of immunosuppressant drugs. Compared to the relatively limited analytical performance and cross reactivities of immunoassays, the LC-MS/MS method is considered as a gold standard; however, the lack of systematic evaluation and standardization needs to be addressed. Methods: A LC-MS/MS method for the determination of cyclosporine A, sirolimus, tacrolimus, and everolimus was developed. One-step protein precipitation was used to prepare blood samples. The newly developed method was systematically evaluated and validated according to the standard guidelines. Results: The quantitative method for four immunosuppressant drugs in human whole blood was validated according to the guidelines. The lower limits of the measuring interval (LLMI) for cyclosporine A, sirolimus, tacrolimus, and everolimus were 5, 0.5, 0.5, and 0.5 ng/mL, respectively. Linear correlation coefficients were all >0.999. Internal standard-normalized (IS-normalized) matrix correction factor was within the range 0.88-1.17. The average spiked recoveries of five replicates for the four immunosuppressant drugs were in the range 87.4-109.6%. Conclusion: An LC-MS/MS method combined with one-step protein precipitation was developed, providing short sample preparation and chromatographic run time, thus allowing easy clinical diagnosis.