Laboratory of Organic Chemistry, Wageningen University & Research, Stippeneng 4, 6708 WE, Wageningen, The Netherlands.
Wageningen Food Safety Research, Wageningen University & Research, Akkermaalsbos 2, 6708 WB Wageningen, The Netherlands.
Anal Chem. 2024 Oct 15;96(41):16462-16468. doi: 10.1021/acs.analchem.4c04811. Epub 2024 Oct 4.
Contamination with tropane alkaloids in cereals is expected to increase globally. However, current identification tools (e.g., liquid chromatography-mass spectrometry) for tropane alkaloids are time-consuming and expensive. Furthermore, their miniaturized alternatives lack sensitivity and robustness. Therefore, there is a pressing need for inexpensive and effective screening methods. Here, an on-site applicable modular workflow for tropane alkaloid detection in buckwheat is presented. The modular workflow combines paper microfluidics and interconnectable 3D-printed sample preparation tools and was evaluated for different tropane alkaloids, including atropine and scopolamine. Furthermore, integration with an indirect competitive lateral flow immunoassay (icLFIA) for atropine detection at relevant levels was demonstrated. In the modular workflow, to minimize matrix coextraction, tropane alkaloids were extracted from the milled buckwheat cereals by a mixture of alkaline aqueous and immiscible organic solvents (extraction recoveries: 66-79%). The tropane alkaloids were subsequently concentrated with a newly developed paper-immobilized liquid-phase microextraction (PI-LPME, extraction recoveries: 34-60%, concentration factor to immobilized solution in paper: 60-108×). After the PI-LPME, with an integrated 3D-printed setup, the tropane alkaloids were directly eluted (elution recoveries: 83-93%) and detected with the icLFIA. Digital read-out of the icLFIA, by employing a hand-held reader, enabled semiquantification of atropine (IC = 0.56 ng mL in standard solutions). The modular workflow was validated by analyzing 24 blank and spiked buckwheat cereal samples with 5 and 10 μg kg atropine. A cutoff value was established with an estimated false negative rate of 1% and estimated false positive rate of 0.68%. Therefore, the modular workflow can aid in fast, inexpensive, and on-site atropine detection by nonexperts, and when integrated with a scopolamine-specific icLFIA expanded toward scopolamine detection. Moreover, the developed sample extraction and concentration method (PI-LPME) is suitable for the analysis of many other compounds with pH-dependent polarity.
预计全球范围内谷物中莨菪烷生物碱的污染将会增加。然而，目前用于检测莨菪烷生物碱的鉴定工具（例如液相色谱-质谱联用）既耗时又昂贵。此外，其微型化替代品缺乏灵敏度和稳健性。因此，迫切需要廉价且有效的筛选方法。在此，提出了一种用于检测荞麦中莨菪烷生物碱的现场适用的模块化工作流程。该模块化工作流程结合了纸微流控技术和可互连成一体的 3D 打印样品制备工具，并针对不同的莨菪烷生物碱（包括阿托品和东莨菪碱）进行了评估。此外，还展示了与间接竞争侧向流动免疫分析（icLFIA）集成用于检测相关水平的阿托品的能力。在模块化工作流程中，为了最大程度地减少基质共萃取，通过碱性水相和不混溶有机溶剂的混合物从研磨的荞麦谷物中提取莨菪烷生物碱（提取回收率：66-79%）。随后，使用新开发的纸固定相液相微萃取（PI-LPME）对莨菪烷生物碱进行浓缩（提取回收率：34-60%，固定在纸上的溶液的浓缩因子：60-108×）。PI-LPME 后，通过集成的 3D 打印装置，直接洗脱莨菪烷生物碱（洗脱回收率：83-93%），并使用 icLFIA 进行检测。通过手持式读取器对 icLFIA 进行数字读取，实现了对阿托品的半定量分析（标准溶液中 IC = 0.56ngmL）。通过分析 24 个空白和加标荞麦谷物样品（加标量为 5 和 10μgkg 的阿托品）对模块化工作流程进行了验证。建立了一个截止值，其假阴性率估计为 1%，假阳性率估计为 0.68%。因此，该模块化工作流程可以帮助非专业人员快速、廉价且在现场检测阿托品，并且当与针对东莨菪碱的特异性 icLFIA 集成时，可以扩展到东莨菪碱的检测。此外，所开发的样品提取和浓缩方法（PI-LPME）适用于分析许多具有 pH 依赖性极性的其他化合物。
