Université de Reims Champagne-Ardenne, UMR-I 02 INERIS-URCA-ULH SEBIO, Unité Stress Environnementaux et BIOsurveillance des Milieux Aquatiques (SEBIO), BP 1039 F, 51687 Reims Cedex, France; ANSES, Laboratory for Food Safety, F-94701 Maisons-Alfort, France.
ANSES, Laboratory for Food Safety, F-94701 Maisons-Alfort, France.
Sci Total Environ. 2024 Nov 10;950:175339. doi: 10.1016/j.scitotenv.2024.175339. Epub 2024 Aug 6.
Microcystins (MCs) can be detected in various matrices in two forms: a freely extractable fraction and a total (free and covalently protein-bound) fraction. Although the majority of MCs analyses are limited to the free fraction, they do not allow the analysis of all MCs variants or protein-bound forms. Other methods, known as total MCs analysis methods, enable simultaneous analysis of all MCs variants, as well as bound forms, which may be a major form of toxin accumulation in organisms. Among these techniques, the chemical oxidation method (e.g. Lemieux) allows the detection of total forms of MC (and nodularins) by oxidizing the common part to all MC and nodularins, and analyzing the resultant MMPB product (2-methyl-3-methoxy-4-phenylbutyric acid). However, the execution of this method in the context of health monitoring is challenging due to the variability of the protocols, the recoveries obtained with these protocols, and the important matrix effects associated with the method. The objectives of this study were i) to optimize an existing protocol of chemical oxidation "Lemieux1" on fresh fish fillet matrices, ii) to compare two existing protocols ("Lemieux1" and "Lemieux2"), and iii) apply Lemieux oxidation to fish fillets and livers naturally contaminated with MCs-producing cyanobacteria and to freshwater mussels contaminated with MCs in laboratories. Optimization of the "Lemieux1" protocol, in particular in the oxidation and SPE (solid phase extraction) steps improved the method's yields on the fresh fish fillet matrix (from <5 % to around 40 %). Moreover, several quantification methods have been compared through various calibration techniques (solvent calibration curve, matrix-matched calibration curve, oxidized MC-LR calibration curve and also by testing the addition of d-MMPB as an internal standard). Comparison with the "Lemieux2" protocol showed the best results on the same matrix, with yields of around 65 %. MMPB was analyzed using this "Lemieux 2" protocol, in livers of carps sampled during an episode of cyanobacteria proliferation, at concentrations ranging from 17.9 to 27.5 μg/kg MMPB and at concentrations ranging from 50 to 2890 μg/kg MMPB in freshwater mussels laboratory contaminated to MCs.
微囊藻毒素（MCs）可以以两种形式存在于各种基质中：可自由提取的部分和总（自由和共价结合的蛋白质）部分。尽管大多数 MCs 分析仅限于游离部分，但它们不能分析所有 MCs 变体或蛋白质结合形式。其他方法，称为总 MCs 分析方法，可以同时分析所有 MCs 变体以及结合形式，这可能是生物体中毒素积累的主要形式。在这些技术中，化学氧化法（例如 Lemieux 法）通过氧化所有 MC 和 nodularins 的共同部分，并分析所得的 MMPB 产物（2-甲基-3-甲氧基-4-苯基丁酸）来检测总形式的 MC（和 nodularins）。然而，由于协议的可变性、这些协议获得的回收率以及与该方法相关的重要基质效应，该方法在健康监测方面的执行具有挑战性。本研究的目的是：i）优化化学氧化“Lemieux1”在新鲜鱼片基质上的现有协议，ii）比较两个现有协议（“Lemieux1”和“Lemieux2”），iii）将 Lemieux 氧化应用于自然受产 MC 蓝藻污染的鱼片和肝脏以及实验室受 MC 污染的淡水贻贝。“Lemieux1”协议的优化，特别是在氧化和 SPE（固相萃取）步骤，提高了该方法在新鲜鱼片基质上的产率（从<5%提高到约 40%）。此外，还通过各种校准技术（溶剂校准曲线、基质匹配校准曲线、氧化 MC-LR 校准曲线以及测试添加 d-MMPB 作为内标）比较了几种定量方法。与“Lemieux2”协议的比较表明，在相同的基质上得到了最好的结果，产率约为 65%。使用该“Lemieux 2”协议分析了鲤鱼肝脏中的 MMPB，鲤鱼肝脏是在蓝藻大量繁殖期间采样的，其浓度范围为 17.9 至 27.5μg/kg MMPB，在实验室受 MCs 污染的淡水贻贝中浓度范围为 50 至 2890μg/kg MMPB。
