## 5 Analytical Methods for Detection, Identification, and Quantification of C-CTXs
### Analysis of C-CTXs in Caribbean Fish Using HPLC-MS (Low-Resolution)
#### 5.1.1 First Characterisation of C-CTX-1 and C-CTX-2 in Caribbean Fish
A nomenclature based on [M+H] ions was defined to identify these new C-CTX analogues, and a hypothetical structural assessment was proposed for C-CTX-1143 and its isomers (putative reduced forms of C-CTX-1/-2, later named C-CTX-3/-4 in [120]), C-CTX-1157 and its isomers (putative oxidized forms of C-CTX-1/-2, later named 17-hydroxy-C-CTX-1 in [131]), C-CTX-1159 (possible addition of water to a C-CTX-1 double bond), and C-CTX-1127 (possible loss of a -CH\({}_{2}\) group or loss of a methyl group from C-CTX-1). A comparative analysis of raw and cooked flesh in two specimens showed that cooking did not modify toxicity in mice and that the toxin profiles were identical, with the relative proportion of each C-CTX class insignificantly changed by cooking [42]. Any conversion of C-CTX-1 into its closely eluting isomer C-CTX-1141a after cooking did not change toxicity, suggesting that both CTX had similar toxicity [42]. Given the lack of standards for most C-CTX congeners, their relative contribution to toxicity was assessed by grouping the LC-MS peak areas of C-CTXs of the same mass, as a function of the toxin concentration in the flesh determined using the mouse bioassay for 12 _C. latus_ specimens. These included C-CTX-1 (4 isomers), C-CTX-1143 (2 isomers), C-CTX-1157 (3 isomers), and C-CTX-1127. While no quantitative correlation with flesh toxicity was found, the C-CTX-1157 isomers increased from \(\sim\)2 to 20% with increasing fish toxicity [42]. The putative C-CTX analogues identified in the horse-eye jacks study [42] were also identified in other fish specimens implicated in CP in Guadeloupe (F.W.I. ), including a grey snapper (_Lutijanus griseus_), an unidentified grouper (_Sernanidae_ sp. ), and a black jack (_Caranx lugubris_) [23]. Specifically, C-CTX-1 was quantified using pure C-CTX-1 standard, at 0.24, 0.90, and 13.8 ppb in the snapper, the grouper, and the black jack, respectively. However, this only partially contributed to the overall toxicity determined using the MBA [23]. Other identified toxins were C-CTX-2, three additional isomers of C-CTX-1 or -2, and five C-CTX congeners (C-CTX-1127, C-CTX-1143 and its isomer C-CTX-1143a, and C-CTX-1157 and its isomer C-CTX-1157b). Putative hydroxy-polyether-like compounds were also detected in the grouper flesh, with [M+H]\({}^{+}\) ions at \(m/z\) 851.51, 857.50, 875.51, 875.49, and 895.54 Da [23]. Further HPLC-MS and RLB analyses of the TSK-purified extract of black jack (C. _lugubris_) identified C-CTX-1 and C-CTX-1127. However, due to their close elution, it was not possible to confirm whether C-CTX-1127 has an affinity for sodium channels (unpublished data from [40], pp. 197-198). Subsequently, an extract of an inedible horse-eye jack, known as CL5 [42], was analysed using HPLC-MS and radiolabeled-ligand binding assay (RLB) simultaneously (unpublished data from [40]). This additional combined analysis revealed five peaks of binding to sodium channels (A to E) (Figure 5 and Table 2). Unfortunately, some peaks co-eluted (peaks A, B and C), which did not allow for sodium channel affinity to be assigned to specific congeners (Table 2). Moreover, in the toxic fraction A containing C-CTX-1159, two other pseudomolecular ions with unusual masses for CTX were detected: [M+H]\({}^{+}\) at \(m/z\) 859.42 with sodium and potassium adducts and four water losses, and [M+H]\({}^{+}\) at \(m/z\) 811.47 with only four water losses (Table 2). In one highly ciguotoxic specimen of barracuda (_Sphynena barracuda_), as determined by MBA [41], two distinct fractions (FrA and FrB) were separated during the first fractionation stage on Sephadex LH20 (gel filtration chromatography), resulting in two different response profiles in IP injected mice, as previously observed with _S. barracuda_ and _G. func fibrils_[104]. It should be noticed that the Florisil\({}^{\otimes}\) chromatography (Figure 2) was bypassed because barracuda giguatoxins have previously shown a lack of stability during this purification stage [104].