## 6 Discussion and Perspectives
### Considerations for Analytical Approaches for C-CTX Detection and Quantification  
Significant impediments to the study of ciquatera include the scarcity of pure ciquatox-ins standards [129; 130] and the number of structural analogues contributing to the toxicity of hazardous fish [6; 114; 118; 197]. The lack of sufficient quantities of pure CTX standards impedes the development and harmonization of methods for their detection and public health protection. These difficulties are compounded by the commensurately low toxin thresholds for human poisoning and a sufficient sample pretreatment to reduce matrix interferences. Typically, ultra-trace analysis of lipophilic analytes (<1 ppb) is heavily influenced by the matrix involved, with biological matrices such as fish tissues often the most challenging. Although it is time-consuming and expensive, the only way to ensure potential biological activity of new C-CTX congeners is to carry out accurate chromatographic fractionation and to test for biological activity using adapted bioassays [118; 196; 198; 195; 196]. The selection of extraction and purification procedures depends on the specific objectives of the study. Given these diverse needs, a wide range of accelerated and comprehensive protocols have been developed, which are tailored to address specific matrix issues (e.g., fish tissue, microalgae, or blood). The desired outcomes include isolating reference materials, investigating fish toxin profiles, ensuring fish safety, or facilitating clinical diagnosis. Currently, no extraction protocol from biological matrix has been validated for CTXs, regardless of their origin. Various extraction and purification methodologies (see Section 4) are used for C-CTXs [118]. Currently, extraction procedures have been adapted from the method of [PERSON] [195] and the FDA method developed by [PERSON] [209]. Studies comparing the extraction of cooked and raw samples of flesh of C-CTX-containing fish have revealed only slight differences in toxin profiles and contents [23; 91; 108; 131; 104]. Therefore, within the fish flesh matrix, both slow and fast-acting ciquatoxins appear to be heat stable. However, particular attention should be paid during sample purification of certain fish species, such as moray eel and barracuda, due to the possible presence of fast-acting toxins (FATs). Some FATs have been characterised by specific symptoms in mice, and generally include more polar compounds than C-CTX-1 [38; 41; 104]. Potential FATs with molecular masses <900 Da, which are more comparable to the brevetoxins (850-900 Da) than known ciquatoxins (1100-1200 Da), were suspected to contribute to the total toxicity of a highly ciquatoxic barracuda [40; 41]. For this particular fish species, purification using Florisil(r) could be deleterious [104]. Additionally, losses of toxicity in the mouse bioassay observed during TSK gel filtration suggested that these losses were due to the disappearance of FATs [41]. The chemistry of the various chromatographic resins or solvents used is crucial for FAT stability. A consensus method should be established for the extraction and purification of C-CTXs from fish samples, considering the evaluation of pre-analytic efficiency and fish species-dependent matrix effects in mass spectrometry analyses. This will enable the comparison of studies on toxin profiles in ciquatoxic fish. Currently, several scientific groups in Europe and the USA are working on C-CTXs. Thus, an interlaboratory study could be useful in validating a unique protocol, or at least for the main stages of extraction and purification (e.g., the order of extraction and defatting or the type of SPE sorbents). The chromatographic separation stage must be optimized to minimize the co-elution of the molecules of interest. The use of UHPLC systems and the availability of numerous stationary phases contributes to improving the resolution between analytes peaks, detection sensitivity, shorter analytical run times, and reduced solvent consumption [83].