Department of Safety in the Food Chain, National Reference Laboratory for the Monitoring of Marine Biotoxins, German Federal Institute for Risk Assessment, Max-Dohrn-Str. 8-10, 10589, Berlin, Germany.
Sci Rep. 2024 Sep 17;14(1):21715. doi: 10.1038/s41598-024-72708-1.
Ciguatera poisoning (CP) is a severe global public health problem caused by the consumption of seafood products contaminated with ciguatoxins (CTXs). The growing demand for seafood products requires high-throughput testing for CTX-susceptible seafood, however complex extraction and slow cleanup methods inhibit this goal. Herein, several methods for extracting CTXs from fish tissue were established and compared; these methods are sensitive, specific, and valid while achieving higher sample extraction throughput than currently established protocols. The trial fish material was generated from multiple species, with different physical conditions (wet and freeze-dried tissue), and naturally contaminated with various CTXs (i.e., CTX-1B, CTX-3C, and C-CTX-1), thus ensuring these methods are robust and broadly applicable. The extraction methods used were based on mechanical maceration with acetone or methanol or enzymatic digestion followed by acetone and ethyl acetate extraction. Crude extracts were investigated for CTX-like toxicity using an in vitro mouse neuroblastoma (N2a) cell-based assay (CBA). Among the three methods, there was no significant difference in toxin estimates (p = 0.219, two-way ANOVA), indicating their interchangeability. For speed (> 16 samples/day), accuracy (100%), and CTX analog retention confirmation by liquid chromatography-tandem mass spectrometry (LC‒MS/MS), the preferred extraction methods were both methanol and enzyme-based. All extraction methods post hoc confirmation of CTX analogs successfully met international seafood market-based CTX contaminant guidance. These methods can drastically increase global CTX screening capabilities and subsequently relieve sample processing bottlenecks, inhibiting environmental and human health-based CTX analysis.
雪卡毒素中毒（CP）是一种严重的全球公共卫生问题，由食用受雪卡毒素（CTXs）污染的海鲜产品引起。对海鲜产品的需求不断增长，需要对 CTX 敏感的海鲜进行高通量检测，但复杂的提取和缓慢的净化方法阻碍了这一目标的实现。本文建立并比较了几种从鱼肉组织中提取 CTX 的方法；这些方法具有较高的灵敏度、特异性和有效性，同时比目前建立的方法实现了更高的样品提取通量。试验鱼材料来自多个物种，具有不同的物理条件（湿和冻干组织），并且天然受到各种 CTX 的污染（即 CTX-1B、CTX-3C 和 C-CTX-1），从而确保这些方法具有稳健性和广泛适用性。所使用的提取方法基于丙酮或甲醇的机械捣碎或酶消化，然后用丙酮和乙酸乙酯提取。使用体外小鼠神经母细胞瘤（N2a）细胞基于测定法（CBA）研究粗提取物中的 CTX 样毒性。在这三种方法中，毒素估计值没有显著差异（p=0.219，双因素方差分析），表明它们可以互换。对于速度（>16 个样本/天）、准确性（100%）和通过液相色谱-串联质谱（LC-MS/MS）保留确认 CTX 类似物，首选的提取方法是甲醇和酶基。所有提取方法在后处理确认 CTX 类似物方面均成功符合国际海鲜市场基于 CTX 污染物的指南。这些方法可以极大地提高全球 CTX 筛选能力，从而缓解样品处理瓶颈，抑制基于环境和人类健康的 CTX 分析。
