High-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of diazepam, atropine and pralidoxime in human plasma

A high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS) procedure for the simultaneous determination of diazepam from avizafone, atropine and pralidoxime in human plasma is described. Sample pretreatment consisted of protein precipitation from 100 mu l of plasma using acetonitrile containing the internal standard (diazepam D5). Chromatographic separation was performed on a X-Terra (R) MS C-8 column (100 mm x 2.1 mm, i.d. 3.5 mu m), with a quick step-wise gradient using a formate buffer (pH 3. 2 mM) and acetonitrile at a flow rate of 0.2 ml/min. The triple quadrupole mass spectrometer was operated in positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over the concentration ranges of 1-500 ng/ml for diazepam, 0.25-50 ng/ml for atropine and 5-1000 ng/ml for pralidoxime. The coefficients of variation were always <15% for both intra-day and inter-day precision for each analyte. Mean accuracies were also within +/- 15%. This method has been successfully applied to a pharmacokinetic study of the three compounds after intramuscular injection of an avizafone-atropine-pralidoxime combination. in healthy subjects. (C) 2008 Elsevier B.V. All rights reserved.