## 5 Analytical Methods for Detection, Identification, and Quantification of C-CTXs
### Analysis of C-CTXs in Fish by HPLC-MS/MS (Low-Resolution)
#### 5.2.1 First Quantification of C-CTX-1 in Caribbean Fish Using HPLC-MS/MS
Interestingly, some of these CTX analogues had [M+H]\({}^{+}\)\(m/z\) and characteristic fragments identical to those previously found in fish from the Caribbean Sea, including ions [M+H]\({}^{+}\) at \(m/z\) 1157 [23; 41; 108] and [M+H]\({}^{+}\) at \(m/z\) 1127 [23; 41]. In fractions found to have CTX-like activity (CBA-N2a) but devoid of the ion corresponding to C-CTX-1 sodium adduct [M+Na]\({}^{+}\) at \(m/z\) 1163.7, a new putative hydroxyl metabolite of C-CTX-1 was identified by its prominent sodium adduct at \(m/z\) 1181.7, in a study of fish from Macaroneis [110]. In addition, a product ion analysis at high collision energies of [M+H-H2O]\({}^{+}\)\(m/z\) 1123.6 ion of C-CTX-1 showed the presence of prominent ions at \(m/z\) 108.9 and \(m/z\) 191.1 (specific fragments) [110]. Based on this result, [PERSON] et al. suggested a revised molecular structure of C-CTX-1 [110], that was first described in 1998 [35], arguing that the N-ring of C-CTX-1 is more likely a seven-membered ring rather than a six-membered ring (see Figure 1). However, this proposed structure was based on inconclusive MS data and not supported by NMR [35] and chemical evidence [35; 43; 120; 202; 203]. 2.4 Multianalytes Screening (P-CTXs, I-CTXs, and C-CTXs) Using HPLC-MS/MS with MRM on Precursor Ions [M+Na]\({}^{+}\) and Confirmation Using MRM on Precursor Ions [M+NH4]\({}^{+}\) (Methanol/Acetonitrile Elution)  
[PERSON] et al. [180] adopted an analytical strategy based on a partially validated new extraction protocol (see Section 4.2. and Figure 4) coupled to CBA-N2a, LC-MS/MS with a QTrap 6500+ (Sciex), and confirmation using low-resolution or high-resolution LC-MS/MS via ammonium adducts. For the two types of analyses, liquid chromatography was performed using a Gemini NX-C18 (150\(\times\)2 mm, 3 \(\mu\)m; Phenomenex), and the eluents were (A) 1 mM ammonium acetate and 0.5% formic acid in water, and (B) methanol/acetonitrile (3:1). The multianalytes screening was developed to monitor the sodium adducts [M+Na]\({}^{+}\) of >30 CTX congeners classified into four groups, CTX-4A and CTX3C groups (for P-CTXs), a C-CTX group, and an I-CTX group, but using only P-CTX-1, P-CTX-2, P-CTX-3 and P-CTX-3C standards [180]. MS confirmation was achieved by monitoring four predicted MRM transitions per congener, choosing the ammonium adduct [M+NH4]\({}^{+}\) as the precursor ion and the corresponding [M+H]\({}^{+}\) and [M+H-nH2O]\({}^{+}\) with \(n\) = 1-3 as product ions [180]. Using this semi-targeted LC-MS/MS approach, 52 tissue samples of _Lutjanus bohar_ all tested positive within the CBA-N2a, which revealed the presence of a complex CTX contaminant profile with several congeners of the CTX3C-group [34]. Comparing only the peak areas of the detected CTXs (without quantification), the toxin profile of the Western Indian Ocean samples (seven samples associated with an outbreak in Europe in 2020, declared as captured off the southwest coast of India) was indistinguishable from the CTX profile of the Western Pacific Ocean samples (45 samples associated with an outbreak in Europe in 2017) [34]. However, additional studies are required to confirm that the challenges in independently certifying the location of the captured fish did not contribute to the surprising disparity in the type of CTX identified. Contribution of High-Resolution Mass Spectrometry for Identification and Chemical Characterisation of C-CTXs