Intra- and inter-laboratory validation of a dipstick immunoassay for the detection of tropane alkaloids hyoscyamine and scopolamine in animal feed

Tropane alkaloids (TAs) are toxic secondary metabolites produced by plants of, <i>inter alia</i>, the genera <i>Datura</i> (thorn apple) and <i>Atropa</i> (deadly nightshade). The most relevant TAs are (–)-L-hyoscyamine and (–)-L-scopolamine, which act as antagonists of acetylcholine muscarinic receptors and can induce a variety of distinct toxic syndromes in mammals (anti-cholinergic poisoning). The European Union has regulated the presence of seeds of <i>Datura</i> sp. in animal feeds, specifying that the content should not exceed 1000 mg kg^–1 (Directive 2002/32/EC). For materials that have not been ground, visual screening methods are often used to comply with these regulations, but these cannot be used for ground materials and compound feeds. Immunological assays, preferably in dipstick format, can be a simple and cost-effective approach to monitor feedstuffs in an HACCP setting in control laboratories. So far no reports have been published on immunoassays that are capable of detecting both hyoscyamine and scopolamine with equal sensitivity and that can be used, preferably in dipstick format, for application as a fast screening tool in feed analysis. This study presents the results obtained for the in-house and inter-laboratory validation of a dipstick immunoassay for the detection of hyoscyamine and scopolamine in animal feed. The target level was set at 800 µg kg^–1 for the sum of both alkaloids. By using a representative set of compound feeds during validation and a robust study design, a reliable impression of the relevant characteristics of the assay could be obtained. The dipstick test displayed similar sensitivity towards the two alkaloids and it could be concluded that the test has a very low probability of producing a false-positive result at blank level or a false-negative result at target level. The assay can be used for monitoring of TAs in feedstuffs, but has also potential as a quick screening tool in food- or feed-related poisonings.

莨菪烷类生物碱（Tropane alkaloids, TAs）是植物产生的有毒次生代谢产物，尤其包括曼陀罗属（Datura，刺苹果）和颠茄属（Atropa，致命茄）等类群。其中最具代表性的莨菪烷类生物碱为(–)-L-莨菪碱与(–)-L-东莨菪碱，二者可作为乙酰胆碱毒蕈碱受体的拮抗剂，能够在哺乳动物体内引发多种特征鲜明的中毒综合征（抗胆碱能中毒）。欧盟已针对动物饲料中曼陀罗属（Datura sp.）种子的含量作出监管规定，明确要求其含量不得超过1000 mg·kg⁻¹（指令2002/32/EC）。对于未粉碎的物料，通常可采用目视筛查法以符合上述监管要求，但该方法不适用于粉碎物料及配合饲料。免疫测定法——优选试纸条形式——可作为危害分析与关键控制点（Hazard Analysis and Critical Control Points, HACCP）体系下，控制实验室中监测饲料原料的简便且经济高效的技术手段。截至目前，尚无研究报道开发出可同时以同等灵敏度检测莨菪碱与东莨菪碱、且优选采用试纸条形式的免疫测定法，用作饲料分析中的快速筛查工具。本研究报道了一种用于检测动物饲料中莨菪碱与东莨菪碱的试纸条免疫测定法的室内及实验室间验证结果。该方法的目标阈值设定为两种生物碱总含量800 µg·kg⁻¹。验证过程中，研究团队使用了具有代表性的配合饲料样本集，并采用严谨的研究设计，从而可对该测定方法的相关性能特性获得可靠评估。该试纸条检测对两种生物碱展现出相近的灵敏度，且可得出结论：在空白水平下出现假阳性结果、或在目标水平下出现假阴性结果的概率极低。该测定方法既可用于饲料原料中莨菪烷类生物碱的监测，同时也有望作为食品或饲料相关中毒事件中的快速筛查工具。