# Analysis of Total-Forms of Cyanotoxins Microcystins in Biological Matrices: A Methodological Review
## 4 Quantification of Total MC after Base-Catalyzed Deconjugation
### Miles' Approach of Based-Catalyzed Deconjugation
#### 4.2.2 Sulfone/Sulfoxide Mediated Deconjugation  
Another hypothesis considered was that the deconjugation reaction could be improved by first converting the sulfide group of the MC to a more easily-leaving group, such as sulfoxide or sulfone [113]. This strategy allows closeness to a more-known reaction: the base-catalyzed \(\beta\)-sulfone elimination reaction [117; 118]. As in the previous study, MCs conjugated to thiol derivatives (e.g., mercaptoethanol) were used as a model compound. These compounds with a dialkyl sulfide linkage were oxidized to either sulfoxide or sulfone, which upon basic treatment removed the thiol adduct to its sulfenate or sulfinate to recover a deconjugated MC upon \(\beta\)-elimination with the \(\alpha\), \(\beta\)-unsaturated carbonyl group intact. Sulfone-mediated deconjugation was performed on the mercaptoethanol conjugate of MC-RY with oxone in a methanolic carbonate buffer. The rapid and complete conversion of this conjugate to its free form MC-RY via the sulfone form was achieved, and after 26 min of reaction, the conjugate form was no longer detachable. On the other hand, after one night, a drop by half in the intensity of the MC-RY peak was observed and attributed to the instability of MC with oxone. The possibilities put forward to remedy this would be either to quench the reaction after the formation of the sulfones or to extract the products by SPE to remove excess oxidant, for example, by adding a sacrificial sulfoxide such as Me\({}_{2}\)SO [119]. In this study, the other hypothesis tested was sulfoxide-mediated deconjugation. H\({}_{2}\)O\({}_{2}\), which is known to oxidize the Met (methionine) residue of MCs to their sulfoxide, was used to oxidize the methanethiol conjugate of [Asp\({}^{3}\)]MC-RY and the mercaptoethanol conjugate of eight other MC variants. All of these conjugates were oxidized and then separated by SPE from the excess oxidant before being deconjugated in a carbonate buffer at pH 10.7. Between these two mediated deconjugations, the study of the reaction kinetics between sulfones and sulfoxide-mediated deconjugation showed that the sulfones were thus deconjugated twice as fast as their corresponding sulfoxide. Thus, an optimized sulfoxide-mediated deconjugation procedure using Me\({}_{2}\)SO was developed and applied to a mixture of eight MCs conjugated to a mercaptoethanol residue. This experiment was performed as a one-pot reaction in an LC vial, first by adding Me\({}_{2}\)SO and then oxone, and one to two hours later, a carbonate buffer was added to start the deconjugation. The  
Figure 4: A schematic representation of (1) the mechanism of conjugation of the Mdha\({}^{7}\)/Dha\({}^{7}\) moiety of MCs with different thiols to create MCs-thiols derivatives as model compound for (2) deconjugation reaction with base (modified after [114]).  
deconjugation process was almost complete after 2 h and totally finished after 5 h. Further studies showed that MCs containing both Arg\({}^{2}\) and Arg\({}^{4}\) residues underwent a faster deconjugation process than MCs with only one Arg residue, which in turn underwent a faster deconjugation process than MCs with no Arg residue. Similarly, this method was tested on various other MC conjugates: cys-conjugate of [Dha\({}^{7}\)]MC-LR, MeSH conjugate of [Asp\({}^{3}\)]MC-RY, GSH-conjugate of [Dha\({}^{7}\)]MC-LR, and [Asp3]MC-RY yielded a complete reaction within minutes at pH 10.7 and within a few hours at pH 9.2 [113].