Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping, Sweden.
Anal Bioanal Chem. 2013 May;405(12):4107-25. doi: 10.1007/s00216-013-6798-0. Epub 2013 Mar 1.
A liquid chromatography/time-of-flight mass spectrometry (LC-TOF-MS) method for targeted toxicological screening in human postmortem blood samples from forensic autopsy cases has been developed, validated and compared with a previously used method using gas chromatography with nitrogen-phosphorus detection (GC-NPD). Separation was achieved within 12 min by high-resolution gradient chromatography. Ions were generated in positive and negative electrospray ionization mode and were detected in 2-GHz single mass spectrometry mode, m/z range 50-1,000. Before injection, 0.25 g blood was prepared by protein precipitation with 500 μL of a mixture of acetonitrile and ethanol containing deuterated internal standards. An in-house database comprising 240 drugs and metabolites was built by analysing solutions from certified standards or other documented reference material available. Identification was based on scoring of retention time, accurate mass measurement and isotopic pattern. Validation was performed on spiked blood samples and authentic postmortem blood samples. The thresholds defined as minimum required performance levels were for most compounds in the range from 0.01 to 0.10 μg/g. Typically, a mass error of less than 2 ppm and a precision of area measurements of less than 5 % coefficient of variation were achieved. Positive identification was confirmed at concentrations up to 500 μg/g. Most compounds were determined in positive ionization mode, but for a limited number of compounds (fewer than 4 %) negative ionization was needed and a few early-eluted compounds could not be identified owing to substantial influence of interferences from the matrix and were thus not included in the screening. A robust and valid toxicological screening by LC-TOF-MS for postmortem blood samples, covering 50 % more compounds, and with higher precision and sensitivity than the previously used screening by GC-NPD was achieved.
建立、验证并比较了一种用于法医尸检死后血样中毒物靶向筛选的液相色谱/飞行时间质谱（LC-TOF-MS）方法和先前使用的气相色谱/氮磷检测（GC-NPD）方法。通过高分辨梯度色谱在 12 分钟内实现分离。正、负离子电喷雾电离模式下产生离子，以 2GHz 单质谱模式检测，质荷比（m/z）范围为 50-1000。进样前，通过 500μL 乙腈和乙醇混合液沉淀 0.25g 血液中的蛋白质，加入氘代内标。通过分析认证标准品或其他有文件证明的参考物质溶液建立了包含 240 种药物和代谢物的内部数据库。基于保留时间、精确质量测量和同位素模式评分进行鉴定。在加标血样和真实死后血样中进行验证。定义的最低要求性能水平的阈值为大多数化合物在 0.01-0.10μg/g 范围内。通常可以实现质量误差小于 2ppm 和面积测量精度小于 5%变异系数。在高达 500μg/g 的浓度下可以确认阳性鉴定。大多数化合物在正离子化模式下测定，但对于少数化合物（少于 4%）需要负离子化，并且由于基质干扰的重大影响，一些早期洗脱的化合物无法鉴定，因此未包含在筛选中。与先前使用的 GC-NPD 筛选相比，LC-TOF-MS 对死后血样进行了更具鲁棒性和有效性的毒理学筛选，涵盖了 50%更多的化合物，具有更高的精度和灵敏度。
