## 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
The main toxic fraction, FrA, produced a delayed onset of symptoms in IP injected mice, typical of slow-acting toxins (diarrhea, dyspnea, penile cyanosis, hypersalivation, shortest survival time of \(\sim\)30 min). The second unknown toxic fraction, FrB, induced a rapid onset of symptoms, typical of fast-acting toxins (irritability, hypersalivation, dyspnea, penile cyanosis, tail arching, hindquarters paralysis, ataxia of forelimbs, shortest survival time of \(\sim\)10 min). HPLC-MS analysis confirmed that FrA and FrB were distinct, with only a weak overlap of some compounds. C-CTX-1 accounted for 90% of the total toxicity of the barracuda flesh (49 ppb in FrA). C-CTX-2, C-CTX-1141b, C-CTX-1143, and C-CTX-1157a (traces) were also identified, but they did not show any significant activity in the RLB assay. Conversely, fast-acting toxins active on voltage-gated sodium channels with molecular masses \(<\)900 Da were suspected to contribute to the \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Retention Time** & **[M+H]\({}^{+}\) \(m/z\)** & **C-CTX-** & **RLB Peaks (Relative** **Intensity**)** \\ \hline  & 1159.58 & & \\
48.51 & 859.42 * & 1159 & A (+) \\  & 811.47 ** & & \\ \hline
51.55 & 1143.57 & 1143a & no peak \\ \hline
52.03 & 1143.60 & 1143 & no peak \\
52.17 & 1157.57 & 1157 & B (++) \\
53.20 & 1181.60 & 1181 & \\ \hline
54.47 & 1141.58 & 1141a & \\
54.84 & 1141.58 & 1 & C (+++) \\
55.26 & 1127.57 & 1127 & \\ \hline
58.99 & 1141.61 & 2 & D (+++) \\ \hline
60.32 & 1141.58 & 1141b & no peak \\ \hline
61.38 & 1141.58 & 1141c & E (+++) \\ \hline \hline \end{tabular}  
* compounds identified by ions [M+H]\({}^{+}\), [M+Na]\({}^{+}\), [M+K]\({}^{+}\), [M+H-nH\({}_{2}\)0]\({}^{+}\) (\(n=1\)–4). ** compound identified by ions [M+H]\({}^{+}\) and [M+H-nH\({}_{2}\)0]\({}^{+}\) (\(n=1\)–4).  
\end{table} 
Table 2: Retention time, pseudo-molecular ion \(m/z\), and name of corresponding C-CTX congeners detected using HPLC-MS [42] and radiolabeled-ligand binding (RLB) analysis (unpublished data reported from [40]) of a TSK extract from a Caribbean horse-eye jack (C. _latus_) known as CL5 [42]. Peaks A to E correspond to Na\({}^{+}\) channel binding of CTX analogues (intensity levels are indicated in brackets). LC-MS analyses were conducted using a Zorbax C3 300 SB (2.1 \(\times\) 150 mm) column using a water/acetonitrile gradient with 0.1% formic acid and a Quadrupole Time-of-Flight PE Sciex QStar Pulsar. overall toxicity. Indeed, despite important losses in toxicity during further purification of FrB (>60%), peak sodium channel activity was identified, and two pseudo-molecular ions at \(m/z\) 809.43 and \(m/z\) 857.42 were detected using LC-MS in the corresponding fraction, suggesting that one or both may account for the FAT effects in mice [40; 41]. The capacity of fish to detoxify ciquatoxins was already shown for _S. barracuda_ by [PERSON] et al. [199]; therefore, we hypothesized that these fast-acting toxins could be metabolites of C-CTXs or of other toxins with a molecular mass within the same range as the brevetoxins [41]. Figure 5: Extracted chromatograms (in cps) for C-CTX-1143 (referred to as C-CTX-3/-4 in [120]), C-CTX-1157, C-CTX-1127, C-CTX-1159, C-CTX-1181, and C-CTX-1141 from HPLC-MS [42] and radiolabeled-ligand binding (RLB in cpm) analysis (unpublished data from [40]) of a TSK extract from a Caribbean horse-eye jack (_C. lattus_) known as CL5 in [42]. Peaks A to E correspond to binding of CTX analogues to Na\({}^{+}\) channels. LC-MS analyses were conducted using a Zorbax C3 300 SB (2.1 \(\times\) 150 mm) column with a water/acetonitrile gradient containing 0.1% formic acid and a Quadrupole Time-of-Flight PE Sciex QStar Pulsar. In conclusion, the cited HPLC-MS studies carried out in the early 2000s on the toxin profiles of different species of fish from Saint Barthelemy and Guadeloupe were the first to highlight the multiplicity of ciguatoxins in the Caribbean.