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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
Authors:Abbara Chadi  Bardot Isabelle  Cailleux Annie  Lallement Guy  Le Bouil Anne  Turcant Alain  Clair Pascal  Diquet Bertrand
Institution:Université d'Angers, UFR médecine, Angers cedex F-49045, France. chadi.abbara@gmail.com
Abstract: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 100microl of plasma using acetonitrile containing the internal standard (diazepam D5). Chromatographic separation was performed on a X-Terra MS C8 column (100mmx2.1mm, i.d. 3.5microm), with a quick stepwise gradient using a formate buffer (pH 3, 2mM) and acetonitrile at a flow rate of 0.2ml/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 1-500ng/ml for diazepam, 0.25-50ng/ml for atropine and 5-1000ng/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.
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