# Determination of Cyanotoxins and Prymnesins in Water, Fish Tissue, and Other Matrices: A Review
## 5 Discussion
For example, the use of 75:25 water:acetonitrile for the extraction and use of chemically similar isotopically labeled surrogates, such as ANA-a-\({}^{13}\)C\({}_{4}\) or CYN-\({}^{15}\)N\({}_{5}\), which have similar extraction recovery, column retention, and ionization efficiency, can help with improved compensation for recovery bias and matrix effects [71; 114]. These highly sensitive methods provide method detection limits (MDLs) as low as 0.14 ng/g for ANA-a and 0.12 ng/g for CYN [71]. The methods for the analysis of only BMAA in tissue were also limited in scope. Because it is a small amino acid and highly hydrophilic, studies that concurrently analyze BMAA with these other toxins are limited because of the need for either chemical derivatization or specialized HILIC LC-MS/MS methods, as seen in [60; 61; 62]. In general, the extraction of BMAA from tissue has been accomplished with an acidic solvent, but the need for hydrolysis, derivatization, and clean-up steps to reduce matrix interferences have made the process lengthy and potentially incompatible with other toxins. Following extraction, analysis was performed using HPLC-FLD or LC-MS/MS, with measurements ranging from micrograms/gram to milligrams/gram. The data from both analysis methods are generally comparable, indicating that the use of HPLC-FLD could be a beneficial first step or screening method. Studies support the use of LC-MS/MS as a necessary confirmation tool [115; 116]. Most methods for STX detection in complex matrices usually involve extraction under acidic conditions (with hydrochloric acid or acetic acid, for instance) and often at an elevated temperature [55; 56; 57; 58]. Detection was accomplished with ELISA, HPLC-FLD, LC-MS/MS, or LC-qTOF MS, with DLs mostly in the micrograms/kilogram range. The ELISA method is sensitive and allows for rapid screening of a large number of samples, and LC-MS/MS serves as a useful confirmation tool. STX is stable at high temperatures, which is an important consideration for food safety; it also plays a role in many extraction procedures that require the sample to be boiled [55; 56; 57; 58; 59; 117]. STX is known to persist for over 90 days and is considered extremely stable even at a high temperature and low pH [38]. Although it can be stored in acid without loss for many decades, some studies suggest that it may not be stable at pH \(>\) 8, even at ambient temperature [118]. This suggests that STX may not be stable in water and bivalves unless it is stabilized with acid. The stability also depends on the chemical structure of specific compounds; GTX1, GTX4, and NEO variants are less stable at acidic pH than GTX2, GTX3, and STX [119]. Overall, the stability of various STX variants is an unsolved analytical problem that is in need of continued study. Significant questions and uncertainties remain with regard to the toxins associated with _P. parvum_ at this time. Sequential extraction of cultures with various solvents in [47]identified material with cytotoxic properties, including fatty acids and fatty acid amides, but no pyrmnesins. However, in [63], two toxins classified as pyrmnesins were observed by LC-MS/MS, following multiple preparative and clean-up steps, including chlorophyll removal and post-extraction solid-phase extraction. At this point, it remains unclear which toxins are the specific causative agents for _P. parvum_ toxicity, a problem exacerbated by a lack of commercially available standards from which to perform toxicity assessments. One of the emerging needs in analytical laboratories is for methods suitable for quantifying multiple cyanotoxins in a single workflow. As cyanobacterial blooms become more common and severe, the need for rapid analysis is only going to increase and methods suitable for screening and quantifying multiple classes of toxin at once will greatly improve sample throughput. In Table 3 are listed 20 studies relying on multi-toxin methods.