quantitative liquid chromatographic-tandem mass spectrometric determiantion of reserpine in FVB/N mouse plasma using a 'chelating'agent (disodium EDTA) for releasing protein-bound analytes during 96-well liquid-liquid extraction

摘要

A sensitive,specitic. accurate and reproducible aRalytk;aI method employing adivalent'cation chelating agent (disodium :OTA) for sample treatment was developed to quantitate reserpine in FVB!N mouse plasma. Samples pretreated with 40 fJ.I )f 2% disodium EDTA in water were extracted by a semi-automated 96-well liquid-liquid extraction (LLE) procedure to ;1 solate reserpine and a structural analog internal standard (l.S.), rescinnamine, from mouse p.lasma. The extracts werec;- malyzed by turbo ionspray liquid chromatography-tandem ma.~s spectrometry (LC-MS-MS) in the po!;itive ion mode. ;ample preparation time for conventional LLE was dramatically reduced by the semi-automated 96-well LLE approach. The. lssay demonstrated a lower limit of quantitation of 0.02 ngfml using O.I-ml plasma sample aliquots. The calibration curves liere linear from 0.02 to 10 ng!ml for reseI:Pine. The intra- and inter-assay precision of quality control (QC) samples ranged rom 1.75 to 10.9% for reserpine. The intra- and inter-assay accuracy of QC samples ranged from -8.11 to 8.610/c,. ~eserpine and the I.S. were found to be highly bound to FVB!N mouse plasma protein. This is the first report of disodium' :OT A employed as a special protein-bound release agent' to recover protein-bound analytes from plasma, These matrix- :ffects and the effects of pH in the HPLC mobile phase on the sensitivities of LC-MS-MS are discussed in this paper.