Analysis of Total-Forms of Cyanotoxins Microcystins in Biological Matrices: A Methodological Review

Abstract

Microcystins (MCs) are cyclic heptapeptidic toxins produced by many cyanobacteria. Microcystins can be accumulated in various matrices in two forms: a free cellular fraction and a covalently protein-bound form. To detect and quantify the concentration of microcystins, a panel of techniques on various matrices (water, sediments, and animal tissues) is available. The analysis of MCs can concern the free or the total (free plus covalently bound) fractions. Free-form analyses of MCs are the most common and easiest to detect, whereas total-form analyses are much less frequent and more complex to achieve. The objective of this review is to summarize the different methods of extraction and analysis that have been developed for total forms. Four extraction methods were identified: MMPB (2-methyl-3-methoxy-4-phenylbutyric acid) method, deconjugation at basic pH, ozonolysis, and laser irradiation desorption. The study of the bibliography on the methods of extraction and analysis of the total forms of MCs showed that the reference method for the subject remains the MMPB method even if alternative methods and, in particular, deconjugation at basic pH, showed results encouraging the continuation of the methodological development on different matrices and on naturally-contaminated samples.

Keywords: MMPB method; cyanotoxins; deconjugation at basic pH; laser irradiation desorption; method of analysis; microcystins; ozonolysis; total toxins content.

Figure 1

Topological representation of an MC with 1: D-Alanine, 2: X (variable), 3: D-erythro-b-methyl-D-aspartic acid, 4: Z (variable), 5: Adda, 6: D-Glutamate, 7: Mdha. The Adda fragment is a common part of all MC variants and nodularins, another cyanotoxins. The Mdha fragment plays a role in the binding of MCs to proteins. The variable amino acids (X and Z) as well as the possibility of substitution on 1, 3, 5, 6, 7 will define the identity of the MCs variant and participates in the diversity of the MCs. 279 MCs congeners have been listed in Bouaïcha et al. [9].

Figure 2

Structure of microcystin-LR (MC-LR) with its specific Adda moiety subject to Lemieux oxidation resulting in MMPB production (modified after Suchy and Berry [61]).

Figure 3

The different stages of MMPB method. These steps have been the subject of optimization work described in the following sections.

Figure 4

A schematic representation of (1) the mechanism of conjugation of the Mdha⁷/Dha⁷ moiety of MCs with different thiols to create MCs-thiols derivatives as model compound for (2) deconjugation reaction with base (modified after [114]).