In therapy with immunosuppressant drugs, therapeutic drug monitoring (TDM) is essential to treatment success. Therefore, analytical methods with higher precision, accuracy, sensitivity, selectivity and speed are demanding. This project aims to develop an improved sample-preparation protocol for simultaneous quantification of sirolimus, everolimus, tacrolimus and ciclosporin in whole blood by ultra high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS). The sample preparation will be performed by protein precipitation with ZnSO4 and acetonitrile, and liquid-liquid extraction using methanol. The drugs separation will be performed with a Waters UPLC BEH C18 (50 mm × 2.1 mm) 1.7-m column maintained at 45 æC using the mobile phase A consisted of 2 mM ammonium acetate with 0.1% formic acid (v/v) in water, and mobile phase B consisted of 2 mM ammonium acetate with 0.1% formic acid (v/v) in methanol. The mass spectrometer will operate in multiple reaction monitoring (MRM) in positive electrospray ionization mode. The method validation will be performed according to European Medicines Agency validation guideline. Comparison of the results obtained with the previous routine LC-MS/MS method and new rapid LC-MS/MS method will be done using a set of proficiency testing samples and real patient samples.
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