High-throughput semi-automated 96-well liquid/liquid extraction and liquid chromatography/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 developed for RAD001 and cyclosporin A (CsA) in human blood. After addition of internal standard and ammonium hydroxide, samples were extracted twice with methyl tert-butyl ether (MTBE). The organic extract was evaporated to dryness and reconstituted in mobile phase. Where possible, sample transfer and LLE steps were automated using a Tomtec Quadra 96 workstation. Samples were analyzed using ESI-LC/MS/MS employing the transitions of ([M + NH(4)](+) --> [M + H](+)) for CsA and ([M + NH(4)](+) --> [M + H-(CH(3)OH + H(2)O)](+)) for RAD001, under isocratic chromatographic conditions (75:25, (v/v), acetonitrile/20 mM ammonium acetate) with a run time of 3.6 min. A lower limit of quantitation (LLOQ) of 0.368 ng/mL and 5.23 ng/mL was achieved for RAD001 and 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 curves had a correlation coefficient >0.99 over the concentration range 0.368 to 409 ng/mL and 5.24 to 1748 ng/mL for RAD001 and CsA, respectively. The inter-day coefficient of variation (CV) was less than 15% at the LLOQ for both compounds. The method was applied to the analysis of clinical samples. Under 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 throughput efficiency was realized with this LLE method when compared to existing solid phase extraction (SPE) methods which deal with both RAD001 and CsA.