Quantitation of Clonazepam and Its Major Metabolite 7-Aminoclonazepam in Blood by Automated Solid Phase Extraction and Liquid Chromatography-Tandem Mass Spectrometry

KeyWord:clonazepam  7-aminoclonazepam  liquid chromatography-tandem mass spectrometry  automated solid phase extraction  blood

Abstract:

      An automated solid phase extraction/liquid chromatography-tandem mass spectrometric£¨ASPE/LC-MS/MS£© method for the quantitative determination of clonazepam and its major metabolite 7-aminoclonazepam in blood.The samples were cleaned up with a C18 solid phase extraction cartridge using methanol as elute solution,and separated on a Waters AtlantisTM dC18 column£¨150 mm¡Á3.9 mm,5.0 ¦Ìm£© by gradient elution using methanol and water£¨containing 0.1% formic acid£© as mobile phase.The analysis of target compounds were carried out by LC-MS/MS under multiple reaction monitoring£¨MRM£© in positive ionization mode.The calibration curves showed good linearities in the concentration range of 2-1 000 ¦Ìg/L with correlation coefficients more than 0.995 9.The detection limits of the method were in the range of 0.2-0.5 ¦Ìg/L.The mean recoveries at three spiked levels of 50,200,1 000 ¦Ìg/L were in the range of 72.6%-96.3%,with relative standard deviations of 4.2%-10.3%.The proposed method was rapid,sensitive,simple and accurate,and could be used to detect clonazepam and 7-aminoclonazepam in forensic toxicology£®