High-throughput analysis of everolimus (RAD001) and cyclosporin A (CsA) in whole blood by liquid chromatography/mass spectrometry using a semi-automated 96-well solid-phase extraction system

A semi-automated solid-phase extraction (SPE) liquid chromatography/mass spectrometry (LC/MS) procedure was validated for the simultaneous determination of everolimus (RAD001) and cyclosporin A (CsA) in human blood. Whole blood samples (350microL) were pretreated with acetonitrile/zinc sulfate mixture to precipitate the sample proteins. The samples were centrifuged and the resulting supernatants were manually transferred to a 96-well plate format. All subsequent sample transfer and solid phase extraction was automated using a Tomtec Quadra 96 workstation. Samples were analyzed by LC/MS using an atmospheric pressure chemical ionization (APcI) interface. In order to enhance sensitivity, the MS method used negative ion mode for RAD001 (M]-) and its internal standard and positive ion mode for CsA (M + H]+) and its internal standard. The lower limit of quantitation was 0.375 ng.ml(-1) for RAD001 and 6.95 ng.ml(-1) for CsA. The reproducibility of the method was evaluated by analyzing six replicates at five or more quality control (QC) levels over the nominal concentration range 0.375 to 253 ng.ml(-1) for RAD001 and 6.95 to 1,530 ng.ml(-1) for CsA. The inter- and intra-day accuracy was found to range from 89.7 to 114% with precision (% CV) of less than 12% for both compounds. The sensitivity, small sample volume needed and high sample throughput of this method make it an attractive option for pharmacokinetic studies in pediatric patients.