# Determination of Cyanotoxins and Prymnesins in Water, Fish Tissue, and Other Matrices: A Review
## 5 Discussion
One approach for extracting multiple toxins is to attempt a compromise extraction mixture that is suitable for both hydrophilic and more hydrophobic toxins, as described in [68; 69; 70]. In these studies, the percentage of organic solvent in the extraction mixture was reduced to ~50%, rather than the 75-90% range typical for MCs, to improve the recovery of the more hydrophilic classes of contaminants, including CYNs, ANA-a, and STXs. In [68], an acidic methanol:water (50:50) mixture was used to extract benthic algae and analysis was performed using LC-MS/MS and LC-qTOF. In this study, two STX variants were observed in field samples at 209-270 ng/g, but no detections were seen for MCs, NODs, CYN, or ANA-a. The matrix fortification of dried tissue prior to extraction found that all five classes of contaminant were recovered with 80-105% efficiency, with the caveat that the only MC congeners used were MC-LR and MC-RR, two of the most hydrophilic MCs. In [69], in contrast to a single ~50% organic extraction, sequential extractions were performed in fish and shellfish, first with 100% methanol, followed by an acidic water:acetonitrile (55:45) mixture, with the two pooled before further processing. This study was designed to be as broadly suitable as possible for screening purposes, with two chromatographic methods used, HILIC for hydrophilic toxins and reverse phase for the more lipophilic toxins. As a part of method validation, an extensive matrix spike evaluation was performed for numerous MCs, STX, and other toxins outside the scope of this review, such as domoic acid, and okadaic acid. The authors found that recoveries ranged significantly, with many within an 80-120% window but others, particularly ANA-a, showing around 200% recovery of the spiked amount. According to them, this was mostly likely the result of matrix interferences in the analysis procedure, which is not surprising given the exhaustive extraction procedure and tissue matrix, but without isotopically labeled materials, confirmation of this hypothesis is impossible. In addition, the DLs in this study were somewhat higher than typical, with DLs of 150 ng/g for MCs and 600 ng/g for the more hydrophilic toxins. In another multi-toxin study, [71], a method to measure MCs, NOD, ANA-a, CYN, and STX in tissue using LC-MS/MS was reported. Because of differences in chemical properties, the study relied on two separate extraction procedures, one for ANA-a, CYN, and STX and the other for NOD and MCs (Table 3). Once prepared, the samples were subject to one of two LC-MS/MS methods to separate and quantify the compounds, with C18-based chromatography for NOD/MCs and HILIC for ANA/CYN/STX. As described above, the technique of isotope dilution was used in this procedure to track recovery and quantification of ANA-a, CYN, and a subset of MCs through extraction and analysis. Matrix effects for MCs were seen in fish tissue, with magnitudes ranging from \(-\)26% to \(-\)58%, which were corrected through the internal standards to \(-\)16% to 10%. Comparable matrix suppressions of \(-\)44% to \(-\)50%, corrected to 1.4 to 3.4%, were observed for ANA-a and CYN, respectively. Similar magnitudes were seen in water extraction. Extensive method validation was performed using matrix spikes to determine DLs, from which MDLs were determined to be 0.12 to 0.70 \(\mathrm{\SIUnitSymbolMicro g}\)/kg in tissue and 4 to 80 ng/L in water. However, validation of this procedure in fish exposed to exogenous MCs in [71] was not successful in producing positive results, which the authors attributed to the short duration of exposure, but MCs were detected in environmental samples. Overall, this study illustrates the utility of the use of isotopically labeled materials in these multi-toxin methods to improve the tracking of interferences and extraction efficiencies.