# Analysis of Total-Forms of Cyanotoxins Microcystins in Biological Matrices: A Methodological Review
## 3 Quantitation of Total MC after Ozonolysis  
First described by [PERSON] et al. [96], ozonolysis has historically been the second method for screening total MCs in cyanobacteria. Ozonolysis results can be associated with MMPB method products as ozonolysis produces the same fragment. The reaction of MC oxidation by ozone in a bloom sample from a Japanese lake was completed within 30 min, and the products were analyzed by thermospray-liquid chromatography/mass spectrometry (TSP-LC/MS) or electron ionization-gas chromatography/mass spectrometry (EI-GC/MS). The ozonolysis method has also been applied to sediments [97]. On this matrix, MCs tend to be adsorbed [98], causing the analysis to be more complex than in water samples; a liquid-liquid extraction step (three successive partitions with n-hexane) was added. A methylation step (with 14% boron trifluoride-methanol (BF3-MeOH) at 70 \({}^{\circ}\)C for one hour) that made the MMPB volatile for GC-MS analysis was also added. The detection limit of this method was evaluated at the pmol levels with yields of 90.5% for MC-LR and -RR and 91.7% for MC-YR. In the most recent application of ozonolysis on MCs, lake water samples spiked with MCs were used to test a slightly different analytical method [99]. After ozonolysis with an ozone concentration of 2.8 mg/L at a flow rate of 100 mL/min for 1 to 20 min, the excess ozone was removed with a nitrogen stream for 5 min. As in the method of [PERSON] et al. [97], LLE steps were used. Unlike the sediment ozonolysis method, methyl esterification was performed with MeOH, pyridine, and methylchloroformate (MCF), while the LLE was performed with chloroform in a saturated sodium chloride solution followed by GC/MS analysis. When compared to the previous method using 14% BF\({}_{3}\)-MeOH at a high temperature for at least an hour, the [PERSON] et al. [99] method showed yields between 77.7 and 80.2%, depending on the spike level (300 and 100 \(\upmu\)g/L, respectively). The LOD of this method was 0.34 \(\upmu\)g/L for a shorter reaction time (10 min), at room temperature and under mild conditions. Although this method can be performed at room temperature, [PERSON] et al. [99] showed that the most optimal conditions for this ozonolysis protocol were a reaction with an ozone concentration of 2.8 mg/L for 10 min at an acidic pH (pH \(\leq\) 3) and with a temperature of 35 \({}^{\circ}\)C. To our knowledge, these are the only publications using ozone for an analysis of the total forms of MCs. Subsequently, the use of ozone on water containing MCs was performed in the context of treating micropollutants in water by ozonation reaction for remediation purposes [100; 101; 102; 103; 104; 105]. Although this method yielded good results for blooms (77.7-80.2%) [99] and sediments (between 90.5 and 91.7 depending on the MCs variants) [97], to our knowledge, it has never been applied to more complex matrices, such as animal matrices. As mentioned previously regarding the MMPB method, the transition to such matrices can lead to a significant decrease in the yields of total MCs due to more significant matrix effects. Another point of vigilance concerns the term \"total\" form. Indeed, this term, depending on the matrix to which it refers, does not necessarily refer to the same type of binding between the MCs and the matrix. In the study by [PERSON] et al. [96], using ozonolysis on cyanobacterial blooms only refers to intra/extracellular MCs, whereas in the other case, dealing with MCs adsorbed to sediment, the binding does not correspond to MCs covalently bound as in animal matrices. While being an interesting and fast method to obtain good recoveries of the total MCs on bloom and sediment matrices, more experience is needed in the application of ozonolysis on animal matrices to better appreciate the performances of this method, which nevertheless requires adapted equipment in the laboratory.