Fully automated determination of eserine N-oxide in human plasma using on-line solid-phase extraction with liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A sensitive and entirely automated solid-phase extraction/liquid chromatogr aphy/electrospray ionization tandem mass spectrometric (SPE/LC/ESI-MS/MS) m ethod was developed and validated for the determination of eserine N-oxide (ENO), a cholinesterase inhibitor-like physostigmine in human plasma, for u se in pharmacokinetic studies, ENO is light-sensitive and the use of a full y on-line process increased the reliability of the assay, Plasma samples pr eviously mixed with neostigmine bromide to prevent in vitro degradation, an d tacrine as internal standard (IS), were directly injected into the SPE/LC /ESI-MS/MS system. MS software piloted the overall system. MS/MS detection of ENO and the IS was performed in the positive ion ESI mode using multiple reaction monitoring. The linear calibration curve for ENO ranged from 25 p g ml(-1) to 12.5 ng ml(-1). The limit of quantitation was 25 pg ml(-1) with 250 mu l of plasma injected. Precision, accuracy and stability tests were within the acceptable range and just one analyst is required to analyze 50 unknown samples a day five days per week, from the preparation of the sampl es (i.e. thawing and centrifugation) to data processing. A pilot pharmacoki netic study in three healthy volunteers treated with 4.5 mg of ENO (Geneser ine3(R)) showed that the method was suitable for pharmacokinetic studies in humans. Copyright (C) 2000 John Wiley & Sons, Ltd.