High-throughput semi-automated 96-well liquid/liquid extraction and liquidchromatography/mass spectrometric analysis of everolimus (RAD 001) and cyclosporin a (CsA) in whole blood

A semi-automated high-throughput liquid/liquid extraction (LLE) assay was d eveloped for RAD001 and cyclosporin A (CsA) in human blood. After addition of internal standard and ammonium hydroxide, samples were extracted twice w ith methyl tert-butyl ether (MTBE). The organic extract was evaporated to d ryness and reconstituted in mobile phase. Where possible, sample transfer a nd LLE steps were automated using a Tomtec Quadra 96 workstation. Samples w ere analyzed using ESI/LC/MS/MS employing the transitions of ([M + NH4](+) --> [M + H](+)) for CsA and ([M + NH4](+) --> [M + H-(CH3OH + H2O)](+)) for RAD001, under isocratic chromatographic conditions (75:25, (v/v), acetonit rile/20 mM ammonium acetate) with a run time of 3.6 min. A lower limit of q uantitation (LLOQ) of 0.368 ng/mL and 5.23 ng/mL was achieved for RAD001 an d CsA, respectively, using a sample volume of 0.3 mL for the analysis. The method was validated over a 3-day period and the resulting calibration curv es had a correlation coefficient >0.99 over the concentration range 0.368 t o 409 ng/mL and 5.24 to 1748 ng/mL for RAD001 and CsA, respectively. The in ter-day coefficient of variation (CV) was less than 15% at the LLOQ for bot h compounds. The method was applied to the analysis of clinical samples. Un der normal working conditions four 96-well plates could be extracted and LC /MS analysis completed in less than 28 h. A marked improvement in sample th roughput efficiency was realized with this LLE method when compared to exis ting solid phase extraction (SPE) methods which deal with both RAD001 and C sA. Copyright (C) 2001 John Wiley & Sons, Ltd.