Abstract：
Haff disease typically develops after eating contaminated marine or freshwater species, especially fish. Despite still having an unknown etiology, recent reports have suggested its possible correlation with palytoxins. Therefore, the present work aimed to optimize and perform a validation of a sensitive method using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the analysis of palytoxin and some of its analogs, with the main purpose of investigating their presence in marine and freshwater food samples associated with Haff disease in Brazil. The method optimization was performed using a central composite rotatable design and fish samples fortified with the palytoxin standard. Then, the optimized method was validated for different food matrices, including freshwater and marine fish, mollusks, and crustaceans. The sample preparation involved a solid-liquid extraction using methanol and water, solid-phase extraction using Strata-X cartridges, and on-column palytoxin oxidation. The detection of the main oxidized fragments (amino and amide aldehydes) was achieved by LC-MS/MS with electrospray ionization in positive mode, using a C18 column, as well as acetonitrile and water as mobile phases, both acidified with 0.1 % of formic acid. After optimization and validation, the etiological investigation involved the analysis of 16 Brazilian Haff disease-related food samples (in natura and leftover meals) from 2022. The method was demonstrated to be appropriate for quantitative analysis of freshwater and marine species. So far, it has proven to be one of the most sensitive methods related to palytoxin detection (LOD 10 μg/kg), being able to work in a range that includes the provisional ingestion limit (30 μg/kg). Regarding the Haff disease-related samples analysis, there is a strong indication of palytoxin contamination since the amino aldehyde (common fragment for all palytoxins) was detected in 15 of the 16 samples. Selected results were confirmed using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS).
摘要：
哈夫病通常在食用受污染的海洋或淡水物种后发展，尤其是鱼。尽管病因仍然未知，最近的报道表明它可能与palytinos相关。因此,本工作旨在优化和执行使用液相色谱与串联质谱联用（LC-MS/MS）的敏感方法的验证，用于分析palytoxin及其一些类似物，主要目的是调查它们在与巴西哈夫疾病相关的海洋和淡水食品样本中的存在。使用中央复合可旋转设计和用palytoxin标准品强化的鱼样品进行方法优化。然后，优化后的方法对不同的食物基质进行了验证，包括淡水鱼和海鱼，软体动物,和甲壳类动物.样品制备涉及使用甲醇和水的固液萃取，使用Strata-X柱进行固相萃取，和柱上的孢粉毒素氧化。通过LC-MS/MS以正模式电喷雾电离实现了主要氧化片段（氨基和酰胺醛）的检测，使用C18色谱柱，以及乙腈和水作为流动相，都用0.1%的甲酸酸化。经过优化和验证，病原学调查涉及从2022年开始分析16份与巴西哈夫疾病相关的食物样本(天然食物和剩余食物).该方法被证明适用于淡水和海洋物种的定量分析。到目前为止,它已被证明是与palytoxin检测相关的最灵敏的方法之一（LOD10μg/kg），能够在包括临时摄入限制（30μg/kg）的范围内工作。关于哈夫疾病相关样本分析，由于在16个样本中的15个样本中检测到了氨基醛（所有palytons的共同片段），因此有强烈的palytoxin污染的迹象。使用液相色谱与高分辨率质谱联用(LC-HRMS)确认所选择的结果。
