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.
McMahon
L M
LM
Drug Metabolism and Pharmacokinetics Department, Novartis Institute for Biomedical Research, East Hanover, NJ 07936-1080, USA. louis.mcmahon@pharma.novartis.com
Luo
S
S
Hayes
M
M
Tse
F L
FL
D001331:Automation / Q000295:instrumentation / Q000379:methods; D002138:Calibration; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D016572:Cyclosporine / Q000097:blood* / Q000737:chemistry; D000068338:Everolimus; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood / Q000737:chemistry; D016030:Kidney Transplantation / Q000276:immunology; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D015394:Molecular Structure; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020123:Sirolimus / Q000031:analogs & derivatives* / Q000097:blood / Q000737:chemistry
D007166:Immunosuppressive Agents; D016572:Cyclosporine; D000068338:Everolimus; D020123:Sirolimus