Institute of Forensic Medicine; Jena University Hospital, Jena, Germany.
Institute of Forensic Medicine; Jena University Hospital, Jena, Germany.
Forensic Sci Int. 2022 Oct;339:111420. doi: 10.1016/j.forsciint.2022.111420. Epub 2022 Aug 10.
Analysis of endogenous biomolecules is an important aspect of many forensic investigations especially with focus on DNA analysis for perpetrator/victim identification and protein analysis for body fluid identification. Recently, small endogenous biomolecules have been used for differentiation of synthetic "fake" urine from authentic urine and might be also useful for biofluid identification. Therefore, the aim of this study was to adapt and optimize a method for analysis of small EBs and to investigate long time stability of 35 small endogenous biomolecules (including acylcarnitines with their isomers and metabolites as well as amino acids with their metabolites) in spotted urine samples. Urine samples were spotted on seven different surfaces (Whatman 903 Protein Saver Cards, cotton swabs, cotton glove, denim, underwear, and smooth and rough flagstone) and stored under six environmental conditions (reference condition, sunlight, LED light, 4 °C, 37 °C, humidity of 95%). At certain time points (d0, d7, d28 and d56) samples were analyzed in triplicates by an optimized extraction and LC-HRMS approach. In addition, the urine marker Tamm-Horsfall-Protein was determined on cotton swabs at the same time points using a commercial lateral flow test. Twenty-one of 35 small endogenous biomolecules were stable on most materials/surfaces and under most storage conditions. Significant lower endogenous biomolecule peak areas were found for rough flagstone and underwear as well as for high humidity storage. Kynurenic acid proved to be photo labile. While high long time stabilities were found for 19 of 28 acylcarnitines, nine acylcarnitines showed aberrant stability patterns without evident structural reason. For Tamm-Horsfall-Protein degradation within 28 days was observed even under reference conditions. The presented study demonstrated the value of sensitive LC-HRMS analysis for small endogenous biomolecules / pattern. However, further studies will be indispensable for unambiguous body fluid identification by small endogenous biomolecules.
分析内源性生物分子是许多法医学研究的一个重要方面，特别是在关注 DNA 分析以识别犯罪者/受害者身份以及蛋白质分析以识别体液身份时。最近，小内源性生物分子已被用于区分合成“假”尿液与真实尿液，并且可能也可用于生物体液识别。因此，本研究的目的是适应和优化一种分析小内源性生物分子的方法，并研究 35 种小内源性生物分子（包括酰基辅酶 A 及其异构体和代谢物以及氨基酸及其代谢物）在点样尿液样本中的长时间稳定性。尿液样本点在七种不同的表面上（Whatman 903 蛋白保存卡、棉签、棉手套、牛仔布、内衣以及光滑和粗糙的石板），并在六种环境条件下储存（参考条件、阳光、LED 光、4°C、37°C、95%湿度）。在特定的时间点（d0、d7、d28 和 d56），通过优化的提取和 LC-HRMS 方法对样本进行三重分析。此外，在同一时间点，使用商业侧向流动测试在棉签上测定尿液标志物 Tamm-Horsfall-Protein。在大多数材料/表面和大多数储存条件下，35 种小内源性生物分子中有 21 种稳定。在粗糙的石板和内衣以及高湿度储存条件下，发现内源性生物分子峰面积明显降低。犬尿氨酸被证明是光不稳定的。虽然 28 种酰基辅酶 A 中的 19 种具有较高的长时间稳定性，但没有明显结构原因的 9 种酰基辅酶 A 显示出异常的稳定性模式。即使在参考条件下，Tamm-Horsfall-Protein 在 28 天内也观察到降解。本研究表明，敏感的 LC-HRMS 分析对内源性生物小分子/模式具有重要价值。然而，为了通过小内源性生物分子进行明确的体液识别，还需要进一步的研究。
