## 4 Extraction and Purification of C-CTXs
### Extraction and Purification Methods for C-CTXs Isolation (>1 kg of Fish Flesh)  
It is important to note that the extractive power of acetone is enhanced by the initial presence of water in flesh, and it is less efficient on dry flesh. Raw or cooked fish tissue can be used, as toxin profiles appear to be unchanged by cooking, which matches the cooked flesh typically consumed [23; 91; 104; 108]. Thus, after an initial extraction of wet flesh using acetone, a second extraction must be performed using acetone/water 80:20 (\(v/v\)) to achieve >90% recovered toxicity [38], and a third acetone extraction is unnecessary [91]. It is better to perform a single extraction, followed by rinsing the cake with 80% aqueous acetone, which has the advantage of significantly reducing the contaminant/ciguatoxin ratio [104]. Both suction chromatography and flash chromatography were shown to save time when using 5-25 kg of fish tissue [104; 167]. A summary of the extraction and purification methods for isolation of C-CTXs from large quantities of Caribbean fish (several kg) has been published in [38; 104]. A flow diagram of the isolation and purification of fish tissue used to study toxin profile in Caribbean fish [23; 40; 42] is provided in Figure 2. This protocol was adapted from the procedure developed for P-CTXs by [PERSON] et al. [122; 171], with the first preparative chromatography on the silica phase replaced with Florisil(r) to avoid acid catalysed spiroisomerisation of spirohaetal on silica gels [1]. This approach allowed for the isolation and structural characterisation of C-CTX-1 and its epimer C-CTX-2 from horse-eye jacks (_C. latus_) [35; 38]. The TSK (Toyoyeural(r) HW 40S)-purified extracts from _C. latus_ were then subjected to reversed-phase HPLC-UV (Figure 3) to isolate 90% pure C-CTX-1, C-CTX-2, and three other toxic fractions (using MBA) that accounted for <20% of the total toxicity: a sleep-inducing fraction (<1% of the total toxicity), a minor toxin (~1% of the total toxicity), and a fraction known as the fast-acting fraction (19% of the total toxicity) [35; 38]. At the time of these publications, their relationship to fish ciguatoxins had not been determined, but the lack of penile symptoms in mice indicates that their mode of action in vivo was likely different from the known ciguatoxins. From a highly toxic amberjack extract, [PERSON] et al. also used reverse chromatography to separate two minor fractions in addition to a fraction containing C-CTX-1 [172]. These two minor fractions, one more polar than C-CTX-1 and the other less polar, were active in the RLB assay [172]. Other fast-acting toxins with effects identical to CTXs but much more pronounced were later found in coarser extracts [41; 104] (see Section 5.1.2). Another protocol, adapted from the Dickey R.W. procedure (unpublished data) [102], was recently used to prepare C-CTX-1 reference materials from fish tissue (50 kg) and fish liver (6 kg) from the Canary Islands (Spain) and the Madeira archipelago (Portugal) [130]. The main differences from the previous protocol (Figure 2) lie in the sequencing of the extractions and the use of SPE cartridges (Florisil(r) and C8) for chromatographic purification. [PERSON] et al. compared the order of the liquid-liquid extraction (LLE) procedures and showed that when the diethyl ether extraction is performed prior to hexane cleaning, detection of C-CTX-like toxicity using CBA-N2a can be enhanced [113]. However, it remains to be determined if this change affects LC-MS detection. To study the diversity of ciguatoxins and control the presence of toxins in fish implicated in CP, the extracted lipid-soluble residue can be submitted to additional purification procedures to remove additional non-polar and polar compounds using solid phase extraction (SPE) on silica [39; 107; 108; 120], amino [173], aminopropyl [37; 174], or Florisil(r) [109; 110; 112; 175; 176] cartridges. In some studies, a C18 reversed-phase SPE clean-up step is added to further reduce matrix effects.