# Analysis of Total-Forms of Cyanotoxins Microcystins in Biological Matrices: A Methodological Review
## 2 Quantification of Total MC by the MMPB Method  
Since the 1990s and the development of the first ELISA methods for the analysis of MCs, the need to have a specific antibody for each MC variant caused the analysis to be  
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 [PERSON] et al. [9].  
restricted to a small number of MCs or even only to MC-LR [59; 60]. The methodological challenge was thus to develop a method that would be independent of the structural diversity of MC variants. The existence of a unique part common to all MC variants and nodularins, Adda moiety, was the basis of the strategy first developed by [PERSON] et al. [61] for use on cyanobacteria samples (Figure 1). It consists of the oxidation of the Adda part of MCs with potassium permanganate (KMnO4) and either sodium-iodate (NaIO3) or sodium-periodate (NaIO4) to form the corresponding carboxylic acid derivative: the 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB; Figure 2). The oxidation product (MMPB) was analyzed by GC coupled to a flame-ionization detector (FID) or by HPLC coupled to a spectro-fluorimeter. Concerns related to MC accumulation in animal tissues, in particular in aquatic species potentially exposed to cyanobacteria, have led to the identification of a protein-bound fraction of MCs in these biological matrices [16]. The use of the term \"bound\" toxin usually remains ambivalent as it can sometimes be applied to matrices such as sediments in which MCs are adsorbed on inorganic or organic particles, but also to MCs covalently bound to proteins, in particular PP1 and PP2, in animal matrices [62; 63]. However, these covalently-bound forms are not extractable as easily as free (i.e., not protein-bound) MCs in water, in bloom, or in the tissues of an organism. The use of the MMPB method (also called Lemieux oxidation) allows for the extraction of the total (i.e., protein-bound plus free) fraction of MCs. This method was first applied to mussels (_Mytilus edulis_), salmon (_Salmo salar_) (liver tissue), and Dungeness crab larvae (_Cancer magister_) [58; 64]. There is a strong disparity in the MMPB method protocols used, as shown in Table 1. Many different matrices were analyzed with a high proportion of matrices of aquatic origin. Other studies have used the MMPB method following animal mortalities in an effort to determine the cause of mortality [57; 65; 66]. Many steps of the protocol used can possibly influence the final performance of the method, beginning with the pre-treatment of the matrix (Figure 3).  
Figure 2: Structure of microcystin-LR (MC-LR) with its specific Adda moiety subject to Lemieux oxidation resulting in MMPB production (modified after [PERSON] and [PERSON] [61]).