## 5 Analytical Methods for Detection, Identification, and Quantification of C-CTXs
### Analysis of C-CTXs in Fish by HPLC-MS/MS (Low-Resolution)
#### 5.3.1 Contribution of HRMS to Study Fish C-CTXs  
HRMS provides an opportunity for targeted analysis of C-CTX-1 and known putative analogues, as well as untargeted analysis to determine ciguatoxin diversity and new potential metabolites [113; 120; 131; 204; 205]. Acquisition in full-scan MS mode and targeted MS/MS mode using a Q-TOF 6550 iFunnel (Agilent Technologies) of C-CTX-1 reference material showed a predominant ion [M+H-H2O]\({}^{+}\) at \(m/z\) 1123.6184 (\(-\)4.5 ppm), followed by the sodium adduct [M+Na]\({}^{+}\) at \(m/z\) 1163.6106 (\(-\)1.6 ppm) [204]. The protonated C-CTX-1 and its ammonium and potassium adducts were also present, but with a lower intensity and with higher mass differences (>10 ppm) [204]. The presence of C-CTX-1 was confirmed in a naturally contaminated barred hogfish (_Bodianus scrofa_) caught near the Selvagens Islands (Portugal) due to its retention time, the detection of the first water loss [M+H-H\({}_{2}\)O]\({}^{+}\), and the sodium adduct [M+Na]\({}^{+}\). However, the sensitivity was not sufficient to obtain MS/MS spectra in this sample [204]. In another work on fish specimens from the Madeira and Selvagens Archipelagos, a HRMS full-scan methodology was used to detect putative CTXs using four criteria: \(m/z\) ratio, mass accuracy (ppm), the ring double-bond equivalent (RDBE), and the mono-isotopic pattern (M + 1 ion) of the main, with a M + 1/M ion ratio = 0.6-0.7 [113]. This method identified C-CTX-1 in the flesh of moray eels (_M. helena, M. augusti_ and _G. unicolor_) and their corresponding livers, which exhibited composite toxicity up to six-times higher than that of the flesh. C-CTX-1 was also identified in four liver samples, with an exact mass for the [M+H]\({}^{+}\) ion of 1141.6305 [113]. [PERSON] et al. also reported the presence of a possible CTX analogue in some flesh and/or liver samples corresponding to putative dihydro-CTX2 (C\({}_{60}\)H\({}_{84}\)O\({}_{18}\) MW 1092.5652 Da) or a structural isomer, a putative new C-CTX-1109 identified by its ammonium adduct at \(m/z\) 1127.6023, as well as gambieric acid A [113]. However, further studies on the CTX-like activity of C-CTX-1109 are required to confirm its contribution to ciquatera risk. Recently, [PERSON] et al. developed an analytical approach using capillary LC-HRMS to determine the ciquatoxins present in contaminated fish [131]. Full MS-data dependent acquisition mode was used for identification and quantitation of CTXs, with parallel reaction monitoring (PRM) used to obtain additional structural inferences with reference to C-CTX-1. The analyses confirmed that C-CTX-1 was the principal ciquatoxin present in four fish originating from the Canary Islands (Spain) or the Selvagen Islands (Madeira archipelago, Portugal): an amberjack (_Seriola_ sp. ), a cubera snapper (_Lutjanus cyanoterus_), a barred hogfish (_Bodianus scrofa_), and a dusky grouper (_Epinepleus marginatus_). Minor C-CTX analogues were also identified using full MS mode from \(m/z\) 1000-1200 Da, looking for ion patterns matching cyclic polyethers including the ciquatoxins ([M+H+nH\({}_{2}\)O]\({}^{+}\), [M+H]\({}^{+}\), [M+NH\({}_{4}\)]\({}^{+}\), [M+Na]\({}^{+}\), and [M+K]\({}^{+}\). Methoxylation with formic acid was also used to assess the possible transformation in their methoxylated derivatives and to confirm the structure of their N-ring [121]. The putative structures of C-CTX analogues characterised by MS include [131]:  
* 17-hydroxy-C-CTX-1 ([M+H]\({}^{+}\)\(m/z\) 1157.6255) with a possible location of the -OH group in the E-ring,
* two N-seco- forms of C-CTX-1 ([M+H]\({}^{+}\)\(m/z\) 1143.6462) corresponding to C-CTX-1-reduced forms, previously known as C-CTX-3/-4 [120],
* 50,51-didehydro-C-CTX-3 ([M+H]\({}^{+}\)\(m/z\) 1141.6306),
* 17-hydroxy-50,51-didehydro-C-CTX-3 ([M+H]\({}^{+}\)\(m/z\) 1157.6255). The methylated analogues of C-CTX-1 and C-CTX-3/-4 were likely artefacts arising from the use of methanol and formic acid during the HPLC fractionation [121; 130].