# Analysis of Total-Forms of Cyanotoxins Microcystins in Biological Matrices: A Methodological Review
## 5 Quantification of Total MC after Laser Irradiation Desorption  
A recent method developed by [PERSON] et al. [120] used laser irradiation desorption (LID) as a means to break the covalent bond between MCs and proteins, including PP1 and PP2A. The tests were conducted during the development of the LID protocol to study the relationship between extraction efficiency and laser power density. Among the criteria studied, the time of exposure of the sample to the laser between 1 to 9 min was compared, and the results indicate that the best exposure time is 5 min. The second criterion studied was the sample volume. An excessive volume would lead to the dilution of the laser energy, while a volume that is too low would lead to an energy that is too high. Five extraction volumes were tested (1, 2, 3, 5, and 8 mL). Similar extraction yields were obtained with volumes of 1, 2, 3, and 5 mL, while a decrease in the yield of 15 to 20% was found with volumes exceeding 8 mL. The volume selected after these tests was 5 mL. The method was validated by studying different parameters such as selectivity, linearity, sensitivity (LOD and LOQ of the method were lower than 0.16 and 0.52 \(\upmu\)g/kg,respectively), accuracy, stability, and recovery (relative recoveries of MC ranging from 83.4 to 95.2%). Thus, the optimized protocol corresponds to a 2 g portion of fresh fish tissue (here surimi) crushed and then spiked with 10 uL of a mixture of MCs (MC-RR, -YR, and -LR). The tissues were extracted with 10 mL of MeOH:water:trifluoroacetic acid (TFA) (90:19.9:0.1) and then exposed to visible beam laser irradiation at 450 nm and 8 W for 5 min. After centrifugation at 10,000\(\times\)\(g\) for 10 min, the extract was evaporated to 1 mL and analyzed by LC-MS/MS. Once optimized, this method was compared to two other methods: one method in which the tissues were extracted by 4 mL of acidified water at pH = 2 and heated to 80 \({}^{\circ}\)C before LC-MS/MS analysis, and another method in which 50-100 ng of MC standard were analyzed by LC-UV after extraction with 75% MeOH and a clean-up of the sample using an immune-affinity column. The LID method (whose recoveries of -RR, -YR, -LR were 90.9%, 88.5%, and 86.3%, respectively) showed better yields than the method using acidified water (whose recoveries of -RR, -YR, -LR were 62%, 76%, and 71%, respectively) but slightly lower yields than the third method on MC standards (whose recoveries of -RR, -YR, -LR were 94%, 92%, and 95%, respectively). However, it should be noted that the third method does not seem to be really comparable to the other two because of the absence of a matrix and, therefore, no possibility of binding of MCs to proteins, which could complicate the analysis. The development of the method having been carried out on samples of fish tissues spiked with MCs, it was necessary to test the LID method on tissues naturally contaminated with cyanobacteria which can contain both free and bound forms of MCs. The LID method was finally tested on 18 fishes from five different species (_Carassius auratus_, _Ctenopharyngealgodon idellus_, _Aristicththys nobilis_, _Myloplurnggodon piccus_, and _Hypophthalmichtlthys molitrix_). Almost all of the fish species were MC positive, except _Aristicthys nobilis_ and MCs were detected in six different fish and quantified in four with MC values between 1.36 and 6.78 ug/kg in _Ctenopharyngealgodon idellus_ I and _Mylopharngodon piccus_ II, respectively. Yet, there is no information in this study about the total MCs naturally present in the samples, determined using a different method (e.g., Lemieux oxidation) to prove the extraction efficiency of the LID method regarding free and bound MCs. Furthermore, it is possible that during the methodological development, the samples are spiked with MCs but that the reported extraction yields are only related to the free forms of toxins. This method should be compared to another \"reference\" method for the extraction of the total toxins to demonstrate its ability to extract both free and bound forms of MCs.