Telving Rasmus, Hasselstrøm Jørgen Bo, Andreasen Mette Findal
Section for Forensic Chemistry, Department of Forensic Medicine, Aarhus University, Palle Juul-Jensens Boulevard 99, DK-8200 Aarhus N, Denmark.
Section for Forensic Chemistry, Department of Forensic Medicine, Aarhus University, Palle Juul-Jensens Boulevard 99, DK-8200 Aarhus N, Denmark.
Forensic Sci Int. 2016 Sep;266:453-461. doi: 10.1016/j.forsciint.2016.07.004. Epub 2016 Jul 18.
A broad targeted screening method based on broadband collision-induced dissociation (bbCID) ultra-performance liquid chromatography high-resolution time-of-flight mass spectrometry (UPLC-HR-TOF-MS) was developed and evaluated for toxicological screening of whole blood samples. The acidic, neutral and basic substances covered by the method were identified in postmortem and antemortem whole blood samples from forensic autopsy cases, clinical forensic cases and driving under the influence of drugs (DUID) cases by a reverse target database search. The screening method covered 467 substances. Validation was performed on spiked whole blood samples and authentic postmortem and antemortem whole blood samples. For most of the basic drugs, the established cut-off limits were very low, ranging from 0.25ng/g to 50ng/g. The established cut-off limits for most neutral and acidic drugs, were in the range from 50ng/g to 500ng/g. Sample preparation was performed using simple protein precipitation of 300μL of whole blood with acetonitrile and methanol. Ten microliters of the reconstituted extract were injected and separated within a 13.5min UPLC gradient reverse-phase run. Positive electrospray ionization (ESI) was used to generate the ions in the m/z range of 50-1000. Fragment ions were generated by bbCID. Identification was based on retention time, accurate mass, fragment ion(s) and isotopic pattern. A very sensitive broad toxicological screening method using positive electrospray ionization UPLC-HR-TOF-MS was achieved in one injection. This method covered basic substances, substances traditionally analyzed in negative ESI (e.g., salicylic acid), small highly polar substances such as beta- and gamma-hydroxybutyric acid (BHB and GHB, respectively) and highly non-polar substances such as amiodarone. The new method was shown to combine high sensitivity with a very broad scope that has not previously been reported in toxicological whole blood screening when using only one injection.
开发了一种基于宽带碰撞诱导解离（bbCID）的超高效液相色谱高分辨率飞行时间质谱（UPLC-HR-TOF-MS）的广泛靶向筛查方法，并对全血样本进行毒理学筛查评估。通过反向目标数据库搜索，在法医尸检病例、临床法医病例和药物影响下驾驶（DUID）病例的死后和生前全血样本中鉴定了该方法涵盖的酸性、中性和碱性物质。该筛查方法涵盖467种物质。在加标全血样本以及真实的死后和生前全血样本上进行了验证。对于大多数碱性药物，设定的截断限非常低，范围为0.25ng/g至50ng/g。大多数中性和酸性药物设定的截断限在50ng/g至500ng/g范围内。使用乙腈和甲醇对300μL全血进行简单的蛋白质沉淀来进行样本制备。注入10μL重构提取物，并在13.5分钟的UPLC梯度反相运行中进行分离。使用正电喷雾电离（ESI）在m/z范围为50-1000内生成离子。通过bbCID生成碎片离子。基于保留时间、精确质量、碎片离子和同位素模式进行鉴定。通过一次进样实现了一种使用正电喷雾电离UPLC-HR-TOF-MS的非常灵敏的广泛毒理学筛查方法。该方法涵盖碱性物质、传统上在负ESI中分析的物质（如水杨酸）、小的高极性物质如β-和γ-羟基丁酸（分别为BHB和GHB）以及高非极性物质如胺碘酮。当仅使用一次进样时，新方法显示出高灵敏度与非常广泛的范围相结合，这在毒理学全血筛查中以前尚未报道过。
