Institute of Legal Medicine, University Hospital Charité, Turmstraße 21, Building N, D-10559 Berlin, Germany.
Forensic Sci Int. 2011 Oct 10;212(1-3):215-26. doi: 10.1016/j.forsciint.2011.06.014. Epub 2011 Jul 18.
Time of flight mass spectrometry provides new possibilities of substance identification by determination of the molecular formula from accurate molecular mass and isotope pattern. However, the huge number of possible isomers requires additional evidence. As a suitable way for routine performance of systematic toxicological analysis, a method for combined use of liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) and high performance liquid chromatography with diode array detector (HPLC-DAD) was developed and applied to blood samples from 77 death cases. The blood samples were prepared by extraction with CH(2)Cl(2) and by protein precipitation with acetonitrile (1:4 (v/v)). The evaporated extracts were reconstituted in 35% acetonitril/0.1% formic acid/H(2)O and aliquots were injected for analysis by LC-QTOF-MS (Agilent 6530) and HPLC-DAD (Agilent 1200). A valve switching system enabled simultaneous operation of both separated chromatographic lines under their respective optimal conditions using the same autosampler. The ESI-QTOF-MS instrument was run in data dependent acquisition mode with switching between MS and MS/MS (cycle time 1.1s) and measuring the full mass spectra and the collision induced dissociation (CID) fragment spectra of all essential M+H ions. Libraries of accurate mass CID spectra (2500 substances) and of DAD-UV spectra (3300 substances) of the authors were used for substance identification. The application of this procedure is demonstrated in detail at four examples with multiple drug intake or administration. In the 77 cases altogether 198 substances were identified (87 by DAD and 195 by QTOF-MS) with a frequency between 1 and 20. In practical application, the sample preparation proved to be suitable for both techniques and for a wide variety of substances with different polarity. The automatic performance of the measurements was efficient and robust. Mutual confirmation, decrease of false positive and false negative identifications, and the semi-quantitative estimation of the concentrations by HPLC-DAD for a first assessment of the toxicological relevance of the qualitative result were shown to be the main advantages of the method combination.
飞行时间质谱法通过准确的分子量和同位素模式来确定分子式，为物质鉴定提供了新的可能性。然而，大量可能的异构体需要额外的证据。作为系统毒理学分析常规性能的合适方法，开发了一种液相色谱-混合四极杆飞行时间质谱（LC-QTOF-MS）和高效液相色谱与二极管阵列检测器（HPLC-DAD）联合使用的方法，并应用于 77 例死亡病例的血样。血样用 CH(2)Cl(2)提取，用乙腈（1:4（v/v））沉淀蛋白。蒸发后的提取物用 35%乙腈/0.1%甲酸/H(2)O 重新配制，取一部分进行 LC-QTOF-MS（Agilent 6530）和 HPLC-DAD（Agilent 1200）分析。通过切换系统，在使用相同自动进样器的情况下，在各自的最佳条件下同时操作两条分离的色谱线。ESI-QTOF-MS 仪器以数据依赖采集模式运行，在 MS 和 MS/MS 之间切换（循环时间 1.1s），测量所有主要 M+H 离子的全质谱和碰撞诱导解离（CID）碎片谱。作者的准确质量 CID 谱库（2500 种物质）和 DAD-UV 谱库（3300 种物质）用于物质鉴定。该程序的应用在四个具有多种药物摄入或给药的例子中详细演示。在 77 例中，总共鉴定了 198 种物质（87 种用 DAD 鉴定，195 种用 QTOF-MS 鉴定），频率为 1 到 20 不等。在实际应用中，样品制备被证明适用于两种技术和不同极性的各种物质。测量的自动性能高效且稳健。互证、减少假阳性和假阴性鉴定，以及 HPLC-DAD 对定性结果进行的半定量浓度评估，被证明是该方法组合的主要优势。
