# Determination of Cyanotoxins and Prymnesins in Water, Fish Tissue, and Other Matrices: A Review
## 5 Discussion  
Methods for the extraction and measurement of toxins from complex matrices are influenced by the chemistry of both the toxins and the composition of various matrices. MCs are a particularly diverse class of cyanotoxins, with more than 248 structurally diverse congeners identified in the environment, with chemical properties (including hydrophobicity) varying accordingly. Analysis of MCs in tissue and organs primarily relies on the extraction of homogenized tissue with various fractions of methanol:water, typically 75-80%, or conversion of MCs to MMPB to enable measurements of a single species rather than an ensemble [78; 106; 107; 108]. In both pathways, the extracts are often cleaned via centrifugation [106], hexane wash [108], or SPE [107]. The methanol:water extracts are compatible with LC-MS/MS or other chromatographic methods directly or with ELISA analysis after dilution or solvent evaporation and reconstitution (to reduce the organics in the final sample) to avoid matrix effects [109]. While only a limited number of individual congeners can be quantified using LC-MS/MS methods due to the need for matching analytical standards (in limited availability) and difficulty with isomer separation, both ELISA and the MMPB method can be used to measure total MCs, with the latter potentially also including bound, conjugated, or partially degraded MCs. DLs for ELISA and LC-MS/MS methods for tissue matrices in the literature are generally as low as a few nanograms/gram. DLs for PPIA [106] in water matrices were higher, on the order of 1 \(\mu\)g/L in water. LC-PDA methods also generally have higher detections for MCs in water, with 2.9 \(\mu\)g/L as the lowest level detected, in [97]. Total MCs quantified using the MMPB method, as a proxy for total MCs, have been shown to have DLs of 2.18 ng/g wet weight [51]. One concern for anti-adda-based ELISA methods in the measurement of MCs is the potential for false positives, discussed above; confirmation of ELISA results with LC-MS/MS or direct monoclonal antibodies has been found to be effective at confirming or refuting such results [49]. Due to their structural similarity to MCs, NODs are also frequently extracted using methanol or methanol:water mixtures. If only pure methanol is used, as in [110], if em ployed for a simultaneous analysis for MCs, it would be expected to be susceptible to the same under-extraction of more hydrophilic MCs, as seen in [72]. More broadly, there is cross-reactivity in ELISA analysis between NODs and MCs, which can lead to difficulties differentiating the specific toxins at a site. LC-MS/MS methods should be used to confirm the specific toxins present if this information is needed. In the reports discussed herein, detection of NODs in tissue varied significantly from organ to organ, from 21 ug/kg to 1.4 mg/kg, with ELISA being the predominant detection method used for tissue samples. In this review, methods for the analysis of solely ANA-a or CYN were limited and some are more than a decade old. These studies employed extraction of ANA-a using solvents (ethanol:acetic acid, 80:20), immunoaffinity beads, or SPE, followed by analysis using ELISA, IMS, or HPLC-UV [52; 53; 54]. While DLs for these methods were comparable to extraction/analysis procedures reviewed for other toxins (25 ng/L in water and 4 ug/g in tissue), they are rarely used. Similarly, the extraction of only CYN was achieved using methanol or water, followed by analysis using ELISA, LC-MS/MS, or LC-UV methods, achieving detection varying from 2.7 ng/g for the highly sensitive MS/MS methods to 2.5 ug/g using LC-UV [111; 112; 113]. LC-MS/MS is the most common methodology currently employed to detect ANA-a and CYN concurrently in the studies reviewed. Since most recent studies optimize the extraction of multiple toxins in one workflow, the multi-toxin methods reviewed herein provide the path forward and best practices for ANA-a and CYN detection in complex matrices.