## 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 first mass spectrum of C-CTX-1 acquired using an ESI-triple quadrupole mass spectrometer in 1995 suggested that C-CTX-1 has a molecular ion at \(m/z\) 1123 [102]. A triple quadrupole mass spectrometer was also used to confirm the presence of an ion at \(m/z\) 1123 in partially purified extract of the greater amberjack _Seriola dumerili_, which was implicated in an outbreak of ciguatera among US soldiers in Haiti [172]. However, subsequent MS analysis revealed this ion was associated with the first loss of water from the [M+H]\({}^{+}\) ion at \(m/z\) 1141 [38], which was later confirmed when the full structure of C-CTX-1 was elucidated using NMR [35]. Moreover, in the same study, two minor toxins that targeted receptor site 5 on the voltage-dependent sodium channel, one more polar and one less polar than C-CTX-1, were also found through HPLC-UV analysis [35]. The first characterisation using HPLC-MS with water/acetonitrile eluents modified with 0.1% trifluoroacetic acid (TFA) and an ionspray-triple quadrupole (PE-Sciex API III) was performed on pure Caribbean C-CTX-1 and -2 extracted from 51 kg of horse-eye jack (see Figures 2 and 3) [38]. This low energy MS approach revealed a series of positive ions corresponding to the pseudo-molecular ion [M+H]\({}^{+}\) at \(m/z\) 1141.6 and five successive water losses [M+H-nH\({}_{2}\)O]\({}^{+}\), as well as potassium [M+K]\({}^{+}\), sodium [M+Na]\({}^{+}\), and ammonium [M+NH\({}_{4}\)]\({}^{+}\) adducts associated with the parent ion [38]. HPLC-MS analysis of this extract revealed the presence of up to five C-CTXs, including C-CTX-1, C-CTX-2, and two coelutingcompounds that were less hydrophobic than C-CTX-1. These compounds were assigned [M+H]\({}^{+}\) ions of \(m/z\) 1157.6 and \(m/z\) 1143.6 Da, respectively. Another compound with a presumed [M+H]\({}^{+}\) ion of \(m/z\) 1107.6 Da was observed, but its relationship to C-CTX-1 remains unclear [38]. Indeed, during the purification of the lipid-soluble residue obtained from pooled horse-eye jacks, several fractions were toxic in mice [35; 38], as indicated in Figure 3 (see Section 4.1). 1.2 Combined Analysis of Toxin Profiles in Caribbean Fish Specimens (Horse-Eye Jacks, Grey Snapper, Grouper, Black Jack, and Barracuda) Using MBA, HPLC-MS, and RLB  
Based on their retention time and typical MS fragmentation compared to the C-CTX reference standards [35; 38], C-CTX-1 and C-CTX-2 were identified and quantified in flesh extracts (see Section 4.1 and Figure 2) of horse-eye jack from Saint-Barthelemy (F.W.I) [42]. Using a water/acetonitrile gradient, the detection of C-CTX-1 characteristic ions (adducts and water losses) and the signal/noise ratio were improved using formic acid-modified eleuents instead of trifluoracetic acid-modified eleuents [42]. Formic acid-modified eleuents enhanced the area of the base peak chromatogram by 2.7-fold and the peak height by 3.4-fold when the major ion [M+H-H\({}_{2}\)O]\({}^{+}\) was extracted [42]. Individual analysis of fish profiles using MBA [166] and HPLC-MS showed that, contrary to low-toxic C. _latus_ specimens (75-90% of C-CTX-1), highly ciguatoxic fish had C-CTX-1 levels \(<\) 50% that varied independently of mouse toxicity and were subjected to large losses of activity during purification, suggesting unstable ciguatoxins were also present [40; 42]. A search for CTX with characteristic patterns of ion formation in MS (for details, see Table 2 in [42]) revealed five C-CTX congeners with pseudo-molecular ions at \(m/z\) 1141.58, 1143.60, 1157.57, 1159.58, and 1127.57, and additional isomers at \(m/z\) 1141.58 (2 isomers), 1143.60 (1 isomer), and 1157.57 (2 isomers) were present only in some flesh extracts [42].