Qualitative and Quantitative Analysis of Clonazepam and its Metabolite 7-aminoclonazepam in Blood by LC-tandem QTOF/MS and LC-MS/MS

Objective A highly sensitive and specifi c analytical method was developed for the qualitative and quantitative analysis of clonazepam (CLOZ) and its metabolite 7-aminoclonazepam (7-AC) in blood. The analytes were identifi ed by automated solid phase extraction-liquid chromatography-tandem quadrupole time-of-fl ight mass spectrometry (ASPELC-QTOF/MS) and quantitatively determined by automated solid phase extraction-liquid chromatography-tandem triple quadrupole mass spectrometry (ASPE-LC-MS/MS). Methods After purifi ed by automated solid-phase extraction (ASPE), the qualitative analysis of CLOZ and 7-AC in blood was performed by separation on an Agilent Eclipse Plus C18 HPLC column (50 mm×2.1 mm, 1.8 μm) using methanol-0.1%(v/v) formic acid aqueous solution. The detection was performed on a quadrupole time-of-fl ight mass spectrometer. The quantitative analysis of CLOZ and 7-AC in blood was carried out on LC-MS/MS. The chromatography separation was performed on a Waters Atlantis TM dC18 column (150mm×3.9mm,5.0μm) using methanol-water with 0.1%(v/v) formic acid as mobile phase. The detection was carried out on a triple quadrupole mass spectrometer under electrospray ion source (ESI) in positive ionization mode and multiple reaction monitoring (MRM). Results The result screening database of antidepressants was built using Agilent Mass Hunter Personal Compound Database Library (PCDL) Manager software and then used for the analysis of spiked samples. The results showed that both CLOZ and 7-AC could be correctly identifi ed with low deviation of retention time (<0.1 min), mass (<1 mDa) and MS matching scores, isotopic abundance matching scores, isotope spacing matching scores (all> 95 points). Furthermore, good linear responses were obtained between the concentration range, 2-1000 ng/mL with a regression coeffi cient (r) higher than 0.99 for both CLOZ and 7-AC. The LOD values for CLOZ and 7-AC were 0.5 ng/mL and 0.2 ng/mL, respectively. The LOQ values for CLOZ and 7-AC were 2 ng/mL and 1 ng/mL, respectively. The RSD values obtained from intra-day and inter-day experiments ranged from 4.2 to 10.3%. The recovery ranges for CLOZ and 7-AC were all above 70% for three spiked levels. Conclusion The developed method was further applied for the analysis of poisoning case, both CLOZ and 7-AC in blood were confi rmed and quantifi ed. The combination of ASPE-LC-QTOF/MS and ASPE-LC-MS/MS is suitable for the identifi cation and quantifi cation of benzodiazepines and their metabolites in forensic toxicology.