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.
Brignol
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Drug Metabolism and Pharmacokinetics Department, Novartis Institute for Biomedical Research East Hanover, NJ 07936-1080, USA.
McMahon
L M
LM
Luo
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S
Tse
F L
FL
D002853:Chromatography, Liquid / Q000379:methods*; D016572:Cyclosporine / Q000097:blood*; D004359:Drug Therapy, Combination; D000068338:Everolimus; D008401:Gas Chromatography-Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020123:Sirolimus / Q000031:analogs & derivatives / Q000097:blood*; D013997:Time Factors
D007166:Immunosuppressive Agents; D016572:Cyclosporine; D000068338:Everolimus; D020123:Sirolimus