ASPE-LC-Q-TOF/MS检测血液中氯硝西泮和7-氨基氯硝西泮

目的: 建立了血液中氯硝西泮及其代谢产物7-氨基氯硝西泮的自动固相萃取-液相色谱-串联四极杆飞行时间质谱(ASPE-LC-Q-TOF/MS)的快速筛查方法。方法: 血液中的氯硝西泮和7-氨基氯硝西泮用固相萃取,然后用LC-Q-TOF/MS分析。采用Eclipse Plus C₁₈色谱柱(50 mm×2.1 mm,1.8 μm),柱温35 ℃,流动相为甲醇-0.2%甲酸水溶液(含5 mmol·L^-1甲酸铵)(20: 80),流速0.3 mL·min^-1,进样量2 μL;电喷雾电离源,正离子检测;在50~1 000 Da 质量范围内进行一级和二级质谱全扫描;通过MS匹配得分、保留时间偏差、实测质荷比、同位素丰度匹配得分、同位素间距匹配得分对血液中的目标物进行快速筛查与确证。结果: 目标物的线性范围为20~1 000 ng·mL^-1,相关系数为0.998 9~0.999 3,检出限为2~10 ng·mL^-1。添加浓度水平为50、200、800 ng·mL^-1时,平均回收率为70.6%~91.5%,RSD为4.7%~9.8%。利用Agilent Mass Hunter PCDL Manager软件建立目标物数据库,并应用于实际样品的筛查分析,保留时间偏差全部小于0.1 min,质量偏差小于1 mDa,同位素丰度匹配得分、同位素间距得分和MS匹配得分均大于95,检出氯硝西泮、7-氨基氯硝西泮。结论: 本文的方法经方法验证,可用于法庭与临床毒物分析。

Objective: To develop a method for rapid screening of clonazepam and 7-aminoclonazepine in blood by automated solid phase extraction-liquid chromatography-tandem quadrupole time of flight mass spectrometry(ASPE-LC-TOF/MS). Methods: The samples were cleaned up by C₁₈ solid phase extraction cartridge,the analyses were carried out by LC-Q-TOF/MS.The chromatographic separation was performed on Agilent Eclipse Plus C₁₈(50 mm×2.1 mm,1.8 μm)with gradient elution by using 20% methanol and 80% water(containing 0.1% formic acid)as mobile phases at the flow rate of 0.3 mL·min^-1 and the column temperature of 35 ℃ with the injection volume of 2 μL,and the samples were analyzed under positive electrospray ionization(ESI)mode.Full scan experiments(TOF/MS and product ion)were performed over the m/z range of 50-1 000 Da.Rapid screening and confirmation of target objects in blood were achieved according to MS matching scores,deviation of retention time,measured mass charge ratio,isotopic abundance matching scores,isotope spacing matching scores and MS/MS matching scores. Results: The linearity was over the range of 20-1 000 ng·mL^-1 for the target object and the correlation coefficient was 0.998 9-0.999 3.The detection limit of the method was 2-10 ng·mL^-1.The recoveries of the target compounds were 70.6%-91.5% and the relative standard deviations were 4.7%-9.8% at the addition concentration of 50 ng·mL^-1,200 ng·mL^-1,and 800 ng·mL^-1.The database of screening results built with Agilent MassHunter PCDL Manager software showed the analytes could be identified with low deviation of retention time(<0.1 min)and mass(<1 mDa)and with MS matching scores,isotopic abundance matching scores,isotope spacing matching scores being >95 points.Clonazepam and 7-aminoclonazepine were detected. Conclusion: The proposed method was suitable for the qualitative and confirmation of anslytes in forensic toxicology by methodological validation.