## 5 Analytical Methods for Detection, Identification, and Quantification of C-CTXs  
Currently, there is no official method for CTX monitoring of food in Europe, although it is stated in European regulation that \"Controls shall take place to ensure that fishery products containing biotoxins such as ciguatera or other toxins dangerous to human health are not placed on the market\" (Commission Implementing Regulation (EU) 2019/627). In the Pacific, [PERSON] and [PERSON] [16; 182; 183] noted that mild ciguatera outbreaks occur after exposures to 0.001 ppb of P-CTX-1. Based on these estimates, a 10\(\times\) risk factor was applied to estimate that the concentration of a'safe' fish would contain no more than approximately 0.01 ppb P-CTX-1 [16]. The U. S. Food and Drug Administration set a  
Figure 4: Comparison of flow diagrams of ciguatoxin rapid extraction methods (CREM) developed by [PERSON] et al. [177] (red) and further modified by [PERSON] et al. [178] (in orange) and [PERSON] et al. [179] (in blue), and an alternative approach proposed by [PERSON] et al. [180] (in green). guidance value 10-fold below the lowest observed adverse effect level (LOAEL) determined by [PERSON] et al. [16], which was 0.01 ppb P-CTX-1 equivalent [184]. This concentration is just below the lowest concentration (0.022 ppb P-CTX1 eq.) seen in fish samples associated with CP cases in Guadeloupe [22]. However, in this article [22], the ciguatoxin content was evaluated using P-CTX-1 as reference material. Based on the ratio of LD\({}_{50}\) in IP injected mice between C-CTX-1 (LD\({}_{50}\) = 3.6 \(\mathrm{\SIUnitSymbolMicro g}\)/kg [38]) and P-CTX-1 (LD\({}_{50}\) = 0.25 \(\mathrm{\SIUnitSymbolMicro g}\)/kg [122; 171]), a guidance level of 0.1 ppb C-CTX-1 equivalent was proposed for C-CTX-1 [7; 184]. In 2010, the CONTAM Panel of the European Food Safety Authority (EFSA) also indicated that a concentration of 0.01 ppb P-CTX-1 eq. in fish is not expected to exert effects in sensitive individuals eating a significant amount of contaminated fish [185], although maximum CTX limits for fish still have not been established for regulatory purposes in Europe [26]. Ciguatoxin detection is a significant analytical challenge due to the low concentrations of toxins in complex matrices such as fish flesh and the diversity of toxin profiles encountered in cigarette fishes. The methodological approach for ciguatoxin monitoring usually involves a two-tier approach, with the first tier using a bioassay screen and the second tier using analytical methods. Screening can be carried out on fish extract or during chromatographic fractionation using the mouse bioassay (MBA), which has been used for decades [166; 167; 23; 38; 42; 156]. For example, [PERSON] et al. [186] used female mice, and [PERSON] [166] used male mice to specifically allow for the observation of characteristic penile symptoms associated with all CTXs, regardless of their geographic origin [19]. Alternatives to in vivo assays include cytotoxicity assays [170; 171; 172; 173; 174; 175; 176; 177; 178; 179; 39; 37; 180; 39; 170; 174; 176; 177; 178; 179; 37; 39; 108; 112; 143; 170; 171; 172; 173; 174; 175; 176; 177; 178; 179; 37; 39; 108; 112; 143; 170; 174; 176; 177; 178; 179; 37; 39; 108; 112; 143; 170; 177; 176; 188; 189], calcium influx assays [81], radio-ligand binding assays (RLB), currently known as competitive sodium channel receptor binding assays (RBA) with radiolabeled brevetoxin-3 (rRBA) [144; 172; 190; 191; 23], or recently developed fluorescence (fRBA) [173; 192] and chemiluminescence assays [193]. In the case of Caribbean ciguatoxin analysis, improved sensitivity was reported for cell-based assays (CBA) over RBAs [173; 194]. In the second tier, analytical methods typically employ reversed-phase liquid chromatography (LC) coupled to electrospray (ESI) mass spectrometry (MS) detection. These methods were initially developed for Pacific CTXs [171; 32; 33], and later for Caribbean CTXs [172; 173; 38; 41].