Modular Point-of-Need Tropane Alkaloid Detection at Regulatory Levels: Combining Solid-Liquid Extraction from Buckwheat with a Paper-Immobilized Liquid-Phase Microextraction and Immuno-Detection in Interconnectable 3D-Printed Devices.

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 ₅₀ = 0.56 ng mL ^-1 in standard solutions). The modular workflow was validated by analyzing 24 blank and spiked buckwheat cereal samples with 5 and 10 μg kg ^-1 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.