Liquid-liquid Extraction of Everolimus an Immunosuppressant from Human Whole Blood and its Sensitive Determination by UHPLC-MS/MS

A sensitive and precise ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method has been developed and fully validated for therapeutic drug monitoring of everolimus in human whole blood. Sample preparation involved liquid-liquid extraction of everolimus and its deuterated internal standard (IS, everolimus-d4) from 100 µL blood sample using diethyl ether: ethyl acetate (30:70, v/v) solvent system under alkaline conditions. The chromatography was conducted on a COSMOSIL 2.5C18-MS-II (50 mm × 2.0 mm, 2.5 µm) analytical column. The analyte and  IS were eluted within 2.5 min under isocratic conditions using 10mM ammonium acetate, pH 6.00  adjusted with formic acid and acetonitrile (20:80, v/v). Multiple reaction monitoring was used for the quantitation of everolimus (m/z 975.5 → 908.5) and everolimus-d4 (m/z 979.6 → 912.6) in the positive ionization mode. The method was shown to be linear over the entire concentration range from 0.10-50.0 ng/mL. The recovery ranged from 90.9-94.8 % for everolimus and 91.4-95.6 % for everolimus-d4. The selectivity of the method is demonstrated in six different sources of blank human blood. The method is free from matrix effect as apparent from the post-column analyte infusion experiment, absolute and relative matrix effect results. The stability of everolimus in whole blood was thoroughly established under different storage conditions.