# Determination of Cyanotoxins and Prymnesins in Water, Fish Tissue, and Other Matrices: A Review
## 6 Conclusions and Recommendations  
This review documents the methodologies currently used to measure cyanotoxins and pyrmnesins in complex matrices and to assess the advantages and limitations of the various techniques summarized in this paper. Due to the diversity in chemical structures and properties among cyanotoxins, it is a significant challenge to develop and validate procedures for consistent and high-yield extraction from various matrices, including tissues. In individual toxin methods, optimum conditions can typically be achieved through validation experiments, such as the consensus for extractions using 75-90% methanol:water for MCs/NODs consistently seen in the literature, although variations in matrix constituents such as lipid or protein content could potentially result in varied outcomes. The use of matrix spikes, where known amounts of toxins are fortified to sample matrices, can help further quantify the various matrix effects and interferences in extraction and analysis of cyanotoxins and add confidence to an analytical workflow. This is particularly important in cases where ambient detection of toxins was negative, as in studies such as [55], where only spiked samples showed detectable levels of toxins to allow for evaluation of recovery efficiencies. As the breadth of cyanotoxins known to be present in the environment continues to increase, it is clear that methods for quantifying single classes of toxin may eventually overwhelm analytical facilities. A single method capable of extracting and detecting many or all cyanotoxins would be an ideal goal for the future. Several publications describe such approaches using mass spectrometric detectors for a subset of contaminants of interest, but as expected from a group of compounds with diverse chemistry, there are significant limitations in recoveries during sample processing, chromatographic performance, and sensitivity [104; 105; 106; 107; 108; 109; 110; 111; 112; 113]. For example, in [69], MCs, NOD, ANA-a, CYN, and STXs were all analyzed using LC-MS/MS following extraction, with sequential extraction first with methanol, then a mixture of acetonitrile:water (45:55), which were pooled for analysis. Method recoveries in this study varied between 80 and 200% and the reported detection limits were high, most likely due to matrix interferences. In other cases, multiple complementary workflows for hydrophilic and hydrophobic toxins were developed, as in [71], and showed improved performance. To control for variation in extraction efficiencies, one approach is to add isotopically labeled internal standards to the sample prior to extraction, allowing for quantification of recovery percentages through the extraction and analysis procedures. This can help compensate for recovery bias and matrix suppression/enhancement and is a recommended best practice but is limited in scope to those compounds for which isotopically labeled materials are readily available and when analytical methods can differentiate the native and labeled material (e.g., mass spectrometric methods). Recently, labeled materials have become available for a broader set of cyanotoxins, including MCs, STX, ANA-a, and CYN, which has made this approach feasible. Examples of this as applied to toxin measurement can be found in reference [71], where recoveries were assessed and corrected for both extraction efficiency and matrix effects using labeled toxin analogs. Analysis with LC-MS/MS has become an essential tool for cyanotoxin detection and it can potentially be used for the concurrent analysis of multiple classes of toxins due to its rapid scan rate and ability to cycle polarity from positive to negative ion modes. LC-MS/MS is best employed for targeted screening for toxins, particularly where both native and isotopically labeled standards are available. In contrast, LC coupled with HRMS or TOF is better suited to detect unknown toxins or those for which standards are not available, but a more complete discussion is beyond the scope of this article [82].