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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
Author(s) -
Brignol Nastry,
McMahon Louis M.,
Luo Suyi,
Tse Francis L. S.
Publication year - 2001
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.323
Subject(s) - chemistry , chromatography , extraction (chemistry) , liquid chromatography–mass spectrometry , ammonium hydroxide , liquid–liquid extraction , detection limit , ether , mass spectrometry , ammonium acetate , sample preparation , quantitative analysis (chemistry) , coefficient of variation , analytical chemistry (journal) , calibration curve , high performance liquid chromatography , organic chemistry
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. Copyright © 2001 John Wiley & Sons, Ltd.