## 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
Among these, only 13 samples belonging to two fish species, red hind _Epinephelus guttatus_ (\(n=12\)) and white grunt _Haemulon plumierii_ (\(n=1\)), contained levels of C-CTX-1 detectable using LC-MS/MS. Although queen trigferfish (_Balistes vetula_) exhibited the highest cytotoxicity using CBA-N2a (0.01 to 0.11 ppb C-CTX-1 eq. ), surprisingly, the presence of C-CTX-1 could not be confirmed using LC-MS/MS [37]. This suggests the possibility of potential false positive results from the CBA-N2a assay, or the presence of uncharacterised CTXs, such as the recently identified C-CTX-5 produced by the Caribbean _Gambierdiscus_ species [43]. * Confirmation of C-CTX-1 and identification of C-CTX congeners in Fish from the Eastern Atlantic  
In 2010, the presence of C-CTX-1 in several amberjacks (_Serola_ spp.) responsible for CP in the Eastern Atlantic (Canary and Madeira islands) was confirmed using CBA-N2a and HPLC-MS/MS, with reference to a standard of C-CTX-1 [106]. [PERSON] et al. analysed fish implicated in several intoxications captured in the waters of the Selvagens Islands (Madeira archipelago) using ultra-high-performance liquid chromatography mass spectrometry (UHPLC-MS). The presence of ciguatoxins in a 70 kg greater amberjack _Serola dumerili_ and a 20 kg lesser amberjack _Serola fasciata_ was confirmed [107]. The biological activity of these samples was checked using an electrophysiological assay (perforated patch-clamp recording), and an analytical UHPLC-MS method was optimized using a Waters BEH C18 column (100 \(\times\) 2.1 mm, 1.7 \(\upmu\)m) eluted with a water/acetonitrile gradient containing 50 mM of formic acid and 2 mM of ammonium formate. CTX identification was achieved using an ESI-triple quadripole (Xevo TQ MS, Waters), first using a scan mass mode (\(m/z\) 1000-1500), followed by selected ion monitoring (SIM) of the two more prominent ions [M+H]\({}^{+}\) with a typical fragmentation pattern of CTXs (sodium, ammonium, and proton adducts, and loss of water molecules). In this study [107], six congeners of CTX were recovered, with the three most abundant peaks detected at \(m/z\) 1040.6 (two putative CTX-3C group toxins, e.g., 51-OH-CTX-3) and at \(m/z\) 1141.6 (C-CTX-1/-2 or I-CTX-1/2). Low levels of P-CTX-1, CTX-3C, and CTX-4A/B, or an analogue at \(m/z\) 1061.0, were reported. However, in this study, references to CTXs were only available to confirm the presence of P-CTX-1 and CTX-3C [107]. Single reaction monitoring of C-CTX-1 sodium adducts as parent and product ions used for C-CTX-1 quantification (see Section 5.2.2) achieved high sensitivity in MS detection but compromised the confirmation of diagnostic fragment ions in a multiple reaction monitoring (MRM) mode [201]. Therefore, as part of the European project \"EuroCigua\", further LC-MS/MS analysis was needed, using a mobile phase of aqueous acetonitrile with ammonium formate and formic acid modifiers to enhance C-CTXs precursor ions [M+H-H\({}_{2}\)O]\({}^{+}\) and generate additional fragment ions with the aim of confirming the presence of C-CTX-1 and identifying potential CTX analogues in fish from Macaronesia [110; 175; 176]. The use of ammonium formate significantly reduced matrix effects, allowing for larger injection volumes without compromising detection using an ESI-triple quadrupole (6495 Agilent) [110]. C-CTX-1 was confirmed in several fish species from the Atlantic coasts of Spain and Portugal, along with C-CTX analogues (Table 5), using multiple reaction monitoring (MRM) from different types of precursor ions depending on C-CTX and corresponding to [M+H-nH2O]+, [M+H]+, [M+Na]+, or [M+H-CH3-H2O]+[110,175,176]. [PERSON] et al. identified four C-CTX analogues showing CTX-like activity using CBA-N2a in the HPLC-purified fractions of a _Seriola fasciata_ implicated in CP [175]. In this amberjack, the C-CTX congeners detected in order of increasing polarity were: C-CTX-1157, C-CTX-1 (the main analogue responsible for the CBA-N2a toxicity), C-CTX-1127, and a C-CTX-1 isomer (presumed to be C-CTX-2) [175].