## 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

Table 3: Summary of analytical approaches and results of C-CTX-1 quantification using MRM with an ESI-triple quadrupole (6495 Agilent) in five studies on fish from the Macaronesia Islands. Quantitation was done using a P-CTX-1 pure standard, and LOD and LOQ were 0.0045 ppb and 0.015 ppb P-CTX-1 eq., respectively [109; 110; 112; 175; 176]. scrofa_ (0.11 ppb); and a 4.5-kg island grouper, _Myteroperca fusca_ (0.25 ppb) [109]. No CTXs were detected in any of the fish samples from Madeira Island [109]. C-CTX-1 levels above the FDA guidance levels of 0.1 ppb C-CTX-1 eq. were also quantified using HPLC-MS/MS in four fish implicated in CP and with a positive CTX-like toxicity (by CBA-N2a): a dusky grouper, _Epinephrine marginatus_ (0.12 ppb); an amberjack, _Seriola_ spp. (0.37 ppb); a cubera snapper, _Lutianus cyanobacteria_ (0.49 ppb) from the Canary Islands (Spain); and a red Porgy, _Pagrus Pagrus_ (0.76 ppb) from the Selvagens Islands (Portugal) [110]. In a 37-kg amberjack, _Seriola fasciata_, implicated in a CP and captured near the Selvagens Islands (Portugal), the C-CTX-1 level was 0.84 ppb C-CTX-1 eq. using LC-MS/MS, whereas its composite toxicity was evaluated at 1.4 ppb C-CTX-1 eq. using the CBA-N2a [175]. [PERSON] et al. reported that C-CTX-1 was the only CTX analogue identified using LC-MS/MS in all the species evaluated, with concentrations of C-CTX-1 of up to 0.48 ppb in barred hogfish (_B. scrofa_), a species with an intermediate position in the food web [176]. Finally, when [PERSON] et al. compared the CTX content of the flesh and liver of 109 specimens from the Canary Islands (Spain) [112], CTX-like toxicity was detected using CBA-N2a in 107 livers out of 109. A total of 93 of the fish (85.3%) also showed toxicity in their flesh, with large differences in CTX concentrations found between tissues of the same fish, in particular in black moray eel (_Muraena helena_) specimens [112]. Of the 62 fish specimens further analysed using LC-MS/MS, the presence of C-CTX-1 in flesh was confirmed for only 30 fish samples at levels ranging from 0.018 to 0.270 ppb of C-CTX-1. C-CTX1 was the only CTX analogue identified among the monitored CTXs [112]. In all these studies on fish from the East Atlantic Ocean [109; 110; 112; 175; 176; 177], no identified Pacific CTXs were detected, and discrepancies between toxicity determined using CBA-N2a and C-CTX-1 levels quantified using LC-MS/MS, although this difference was not evaluated for statistical significance. This may suggest the presence of other toxic analogues in the fish from this area and/or a toxicity overestimation arising from the cell-based assays and/or that matrix suppression was not adequately assessed across species. 2.3 Identification of C-CTX-1 and Identification of C-CTX Congeners in Fish Using HPLC-MS/MS with Water/Acetonitrile LC Gradients  
[PERSON] and [PERSON] [33] first demonstrated that acetonitrile-water gradients buffered with 1 mM of ammonium acetate improved the separation and detection of Pacific ciquatoxins using HPLC-MS compared to a water/acetonitrile gradient modified with 0.1% trifluoroacetic acid (TFA). Subsequently, acetonitrile-water gradients modified with formic acid, ammonium acetate, or ammonium formate were used to enhance the detection of the major ion [M+H-H\({}_{2}\)O]+ of C-CTX-1 in LC-MS [42] and LC-MS/MS [37; 39; 108; 174]. The LC-MS/MS methods that have been developed since the 2010s either on ESI-quadrupole-linear ion trap instrument [37; 39; 108; 174] or ESI-triple quadrupole [110; 175; 176] confirmed C-CTX-1 presence in fish and tentatively identified new C-CTXs congeners. This approach also allowed for links between ciquatoxins prevalence and regional fish species distribution to be established in the Caribbean Sea and in the Eastern Atlantic Ocean, as detailed below.