Analysis of pyrrolizidine alkaloids in food – determination of the sum by high-performance thin-layer chromatography and identi-fication by planar solid phase extraction coupled with mass spectrometry

食品中吡咯利西啶类生物碱（PA）的两种分析方法有待开发。通过易于操作的方法，样品中总PA含量（sum参数）的测定以及对单个PA的快速和经济有效的鉴定和定量应该是可能的。在这种背景下，应该从根本上研究和理解PA的减少。此外，平面固相萃取与质谱（pSPE-MS）的液相耦合应自动化用于PA分析，并充分阐述其常规应用。此外，离子迁移率将在pSPE-MS方法中进行研究，以分离等压PA。用高效液相色谱-荧光检测（HPTLC-FLD）分析总PA时，将PA还原为necine碱并衍生为荧光产物。这种方法将适用于反曲霉素和太阳神定类型的PA，并将覆盖约90%的PA。通过pSPE-MS测定完整PA的方法，在MS检测之前使用直接在线耦合而不进行色谱分离，将以一种非常创新的方式在单个质谱中对所有PA进行鉴定和定量，因为在依赖数据库的评估方法中只需要几个点击。在第一步，将开发和优化具有代表性的PA的HPTLC-FLD和pSPE-MS筛选方法，随后将其应用于各种茶叶基质。在接下来的步骤中，这两种筛选方法将适用于经常被PA污染的食品，如香料、蜂蜜和谷物产品。不同的食物基质带来了巨大的挑战，因为基质负荷与PA含量相比是巨大的，而且基质非常复杂。有效和经济的筛选方法将提供可靠的PA含量结果，从而为PA污染的食品类别提供必要的数据。此外，两种方法的结合将代表一种非常有效的分析策略。这两种方法都可以快速提供PA污染的数据，因此对于估计PA暴露非常有价值，这是欧盟迫切需要和要求的。此外，还可以监测最近设置的最高水平。项目成果将服务于基于液体的pSPE-MS自动化耦合的基础研究，为耦合策略应用于后续的各种项目提供基础，并为实际分析问题提供创新的解决方案。因此，开发几种易于执行和有效的筛选方法将得到保证。因此，所获得的结果和知识不仅对PA分析有用，而且为现代食品分析提供了基础和广泛的利益。

Two analytical alternatives as screening methods for the analysis of pyrrolizidine alkaloids (PA) in food should be developed. By the easy to perform methods, the determination of the total PA content (sum parameter) in the sample and a fast and cost-effective identification and quantitation of single PA should be possible. In this context, the reduction of the PA should be fundamentally studied and understand. In addition, the liquid-based coupling of planar solid phase extraction with mass spectrometry (pSPE–MS) should be automated for the analysis of PA and completely elaborated for routine application. Furthermore, ion mobility will be investigated within the pSPE–MS approach for the separation of isobaric PA. For the analysis of the total PA by high-performance liquid chromatography with fluorescent detection (HPTLC–FLD), PA will be reduced to the necine base and derivatized to a fluorescent product. This approach will be applicable to PA of the retronecine and heliotridine type and will cover > 90% of the PA. The method for the determination of intact PA by pSPE–MS, which uses the direct online coupling without a chromatographic separation before MS detection, will offer the identification and quantitation of all PA in a single mass spectrum in a very innovative way, since only a few klicks within a database-dependent evaluation approach will be necessary. In the first step, the HPTLC–FLD and the pSPE–MS screening methods will be developed and optimized for representative PA, and subsequently, applied to various tea matrices. In following steps, both screening methods will be adapted to food that is frequently contaminated with PA, such as spices, honey, and cereal-based products. The different food matrices pose a great challenge, because the matrix load is immense compared to the PA content, and the matrices are highly complex. The efficient and cost-effective screening methods will deliver reliable results for the PA content, and thus, provide necessary data on the contamination of food categories with PA. Furthermore, the combination of both methods will represent a very efficient analysis strategy. Both methods will rapidly deliver data on the contamination with PA and, therefore, are very valuable for the estimation of the exposure to PA, which is urgently needed and claimed by the European Union. Additionally, monitoring of recently set maximum levels will be possible. The results of the project will moreover serve the fundamental research on the automated liquid-based pSPE–MS coupling, providing a basis for the coupling strategy to be applicable to various further projects, and offer innovative solutions for actual analytical questions. Thus, the development of several easy to perform and efficient screening methods will be guaranteed. Consequently, the obtained results and knowledge will not only be useful for the PA analysis, but provide fundamental and broad benefit for modern food analysis.