## 4 Extraction and Purification of C-CTXs
### Rapid Extraction Methods for CTXs (<5 g of Fish Flesh)  
To improve the efficiency of ciguatoxin extraction and HPLC-MS/MS detection, ciguatoxin rapid extraction methods (CREM) sampling small amounts of flesh (<5.0 g), have beenapplied to P-CTXs analysis [177; 178; 179; 180] (Figure 4). [PERSON] et al. [177] developed the first CREM that used a novel approach to CTX extraction, and the clean-up and was optimized to:  
1. Reduce the quantity of fish flesh extracted (\(\leq\)2 g). 2. Replace the initial acetone extraction and filtration step with a one-step extraction and hexane cleanup. 3. Reduce the number transfer and drying steps. 4. Use centrifugation to speed the separation of phases. 5. Consolidate LLE to a single step. 6. Incorporate two orthogonal SPE cleanup steps on C18 and silica. Figure 2: Flow diagram of the C-CTX extraction and purification stages used to study Caribbean fish toxin profiles using LC-MS-radiolabeled-ligand binding [23; 40; 42] and to isolate C-CTX-1 and -2 [35; 38]. (**a**) Extraction stage, (**b**) purification stage. * Toxicity was assessed using the mouse bioassay (MBA) on the fractions bordering toxic peaks. The fractions were evaporated under nitrogen, and 1/100 or 1/50 of each fraction was dissolved in 0.6 mL of 1% Tween 60 for IP (intraperitoneal) injection in mice. VB = bed volume, Ve = elution volume. CREM allowed for the quantification of P-CTX-1 in Australian fish at levels of \(\geq\) 0.1 ppb [177] and the screening of Pacific and Caribbean isolates of _Gambirediscus_ for MTX-like and CTX-like activity [81]. [PERSON] et al. then successfully adapted the CREM approach in an analytical laboratory setting for routine screening of suspect cigarette fishes in Queensland (Australia) using P-CTXs standards supplied by [PERSON] [178]. [PERSON] et al. later introduced a hydrophilic interaction liquid chromatography (HILIC) SPE cleanup instead of the C18 SPE cleanup, which improved P-CTX-1, -2, and -3 extraction efficiency but required an additional 85 min per batch of 12 samples [179]. To our knowledge, none of these CREM-LC-MS/MS methods have been used to detect and quantify clinically relevant levels of C-CTXs in fish flesh from the Atlantic. However, given their chemical similarities, these approaches are expected to be useful for the analysis of fish containing C-CTXs. In 2021, [PERSON] et al. developed and partially validated a new extraction protocol for multiple CTXs from all regions by introducing an enzymatic digestion for the first stage, followed by acetone extraction, defatting with modified n-hexane (with either sodium carbonate or 5% citric acid), a polar PS/DVB WAX SPE (polystyrene-divinylbenzene copolymer with a weak anion exchanger) and normal-phase SPE on silica [180]. However, due to the lack of reference material for C-CTX-1 and the absence of any standard for I-CTX-1, the recovery rates (35-88%) and matrix effects (66-116%) were determined using LC-MS/MS for only P-CTX-1, P-CTX-2, P-CTX-3, and P-CTX-3C. This method was applied to fish presumed to have been caught from the southwest coast of India, but with CTX profiles identical to those of fish from the Pacific Ocean (see Section 5.2.4). Additionally,  
Figure 3: Flow diagram of C-CTX purification using HPLC-UV for the isolation and characterisation of C-CTX-1 and -2 standards from TSK (Toyopearl\({}^{\text{\textregistered}}\) HW 40S) extracts of horse-eye jack tissues [35; 38]. * Fractions were evaporated under nitrogen, and up to 10% of the collected fractions was dissolved in 0.6 mL 1% Tween 60 for IP injection in mice (MBA). Ve: elution volume, RT: retention time. this protocol [180] needs to be validated on Atlantic fish to ensure that the use of acidified solutions is not detrimental to C-CTXs extraction efficiency. [PERSON] et al. also validated a rapid extraction method using accelerated solvent extraction (ASE) for the detection and quantification of P-CTX-1 in fish flesh (5.0 g) using HPLC-MS/MS.