# Analysis of Total-Forms of Cyanotoxins Microcystins in Biological Matrices: A Methodological Review
## 6 Conclusions  
As bound MCs can account for a significant part of the total MCs bioaccumulated in an organism, it is thus necessary to develop and optimize the methods for the extraction and analysis of total MCs. Among the four methods presented in this review (MMPB method, deconjugation at basic pH, ozonolysis, and LID), the MMPB method seems to be the one that has been applied the most for a variety of matrices and for which there have been the most attempts of improvement. Attempts to optimize each step of the MMPB method have been made, for example, during the pre-treatment of the sample, whether chemical or enzymatic, or whether it aims to lyse the cells and concentrate the sample. In the same way, different steps of Lemieux oxidation, extraction/purification, and analysis have evolved, as shown in Table 1, which summarizes the different protocols for the MMPB method implemented in the literature. However, the performances of this method remain very variable, and it is necessary to continue improving key steps, in particular, the purification step, the critical phase of the MMPB method. More recently, deconjugation at basic pH has shown comparable extraction yields to the MMPB method in its first application to cyanobacterial and animal matrices. It would be relevant to compare the different protocols but also to extend the tests to different matrices. The third method reviewed here, ozonolysis, presents good extraction yields for cyanobacteria and sediments but has never been applied to animal matrices where MCs could be bound to proteins. It would therefore be necessary to extend this method to these kinds of matrices. Few studies used ozonolysis, probably due to the specific equipment required to carry out this method. Finally, the LID method is the most recently developed method for the extraction of the total forms of MCs and presents yields higher than 85% on free forms without really demonstrating its efficiency on bound forms. Moreover, it would be necessary to extend it to other matrices, and unlike the MMPB and deconjugation approaches, this method requires specific equipment. Among the different methods reviewed in this article, the MMPB method seems to be the reference despite matrix- and protocol-dependent extraction yields and important matrix effects. Conversely, the other methods reviewed here (deconjugation, ozonolysis, and LID) could be interesting alternatives to the MMPB method but require more hindsight on their use and performance. Although these tests are not yet routinely applied to samples, they would allow a better understanding of the distribution of free/bound toxins, for example, during the accidental intoxication of animals with cyanobacteria, but it would also be a first step towards estimating the sanitary risk during the consumption of species contaminated by MCs.  
The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/toxins14080550/s1](https://www.mdpi.com/article/10.3390/toxins14080550/s1)]([https://www.mdpi.com/article/10.3390/toxins14080550/s1](https://www.mdpi.com/article/10.3390/toxins14080550/s1)), Table S1: Different genera of cyanobacteria known to produce microcystins classed by environment and morphotype (modified from [1], page 56).  
Bibliographic research, P.B.; Writing, P.B.; Review and editing, E.L., T.G. and R.B. All authors have read and agreed to the published version of the manuscript.  
This research received no external funding.  
Not applicable.  
The authors declare no conflict of interest.