# Analysis of Total-Forms of Cyanotoxins Microcystins in Biological Matrices: A Methodological Review
## 2 Quantification of Total MC by the MMPB Method
### Extraction and Purification of the MMPB Product  
Purification is another essential part of the MMPB method, allowing the samples to be both cleaned from impurities coming from the matrix and to also be concentrated. Indeed, the use of a large number of salts (KMMO4, NaIO4, Na2 HPO4, or NaH2 PO4) during this method can negatively affect the performance of the analysis [79]. Furthermore, the volume of the extract (which can be more than 20 mL depending on the protocol) may necessitate a sample concentration. Two different purification methods are mainly used: liquid-liquid extraction (LLE) and solid-phase extraction (SPE). Although the majority of the studies reviewed here use SPE, LLE has been tested on various matrices such as water samples [65; 66] but also animal matrices (Patagonian pejerrey (_Odontesthes hatcher_); tiling fish; salmon Liver and Dunggeness Crab Larvae; saltwater mussels (_Mytitlus edulis_)) [5; 60; 71; 81]. Several organic solvents can be used during LLE, notably diethyl ether [60; 71; 81] and n-hexane [5], but ethyl acetate seems to be the most optimal [65; 66]. Indeed, a study has investigated different solvents according to their non-miscibility with water and their influence on the MMPB yield. Ethyl acetate was selected, and the yield was further improved by the addition of a solution of saturated NaCl in the aqueous phase allowing for a reduction in the solubility of the neutral compounds and consequently improving the extraction (salting-out effect) [66]. SPE, the main method for the purification of extracts after the oxidation phase, allows, similar to LLE, cleaning but also the concentration of the samples (with a lower elution volume than the loading one). Among the protocols gathered in Table 1, 16 different types of SPE cartridges were identified. These differences concern the sorbent used (for example, C18, Styrene-divinylbenzene (SDB)), its quantity (from 30 to 500 mg), or the volume of the cartridge. Each type of sorbent used in the cartridge phase has its own properties; the wash and elution parameters are generally optimized and may differ between different SPE protocols. Some studies compared SPE cartridges based on their recovery for a given matrix. [PERSON] and [PERSON] [72], for example, compared three SPE cartridges (3M C8-SD(\(\circledapprox\), 3M SDB-XC(\circledapprox\), and Waters Oasis HLB(\circledapprox\)) on chicken and rat liver matrices. Blank liver samples were extracted with each cartridge with the following procedure: conditioning with 1 mL of methanol (MeOH), 1 mL of double distilled water, loading the sample, washing with 0.5 mL of MeOH, and elution with 1 mL of MeOH. The highest signal strength of the Oasis extract indicated that the cartridge did not effectively clean the sample and was subsequently dismissed. To decide between the two remaining cartridges, the MMPB recovery rate was tested, and the SDB-XC cartridge showed a higher recovery (82.2%) compared to the C8 (62.5%) SPE cartridge. [PERSON] et al. [29] compared the use of two other cartridges, Oasis HLB(\(\circledapprox\) (200 mg) from Waters and Agilent SampliQOPT(r) (150 mg, Interchim, Montlucon, France) on MMPB standard solutions by varying the elution conditions (MeOH 80%, MeOH 100%, and MeOH 80%, with 0.05% acetic acid (AcOH), 100% AcOH). Whatever the elution conditions tested, the recovery rates obtained were better with the SampliQ OPT(r) cartridges than with the Oasis HLB(r). The highest yields among the SampliQ OPT(r) cartridge tests were obtained by eluting with 80% MeOH (average recovery rate: 80.9%) or with 100% MeOH (average recovery rate: 81.6%). The addition of 0.05% AcOH to 80% MeOH did not increase the recovery rate (average of 78.5%). These conditions were then applied to rainbow trout matrices spiked with 100 ng of MC-LR (equivalent (eq.) to 20.9 ng of MMPB) and 50 ng of MC-LR (eq. to 10.5 ng of MMPB) overnight at 4 \({}^{\circ}\)C. The SPE extraction efficiency was 25% for the intestines, 19% for the muscles, 28% for the liver, and 18% for the gills.