## 5 Analytical Methods for Detection, Identification, and Quantification of C-CTXs
### Analysis of C-CTXs in Fish by HPLC-MS/MS (Low-Resolution)
#### 5.3.2 Contribution of HRMS to Study Gambierdiscus spp  
Toxins  
The HRMS technique has also been applied to study algal toxin profiles [43; 73; 86; 109; 206; 207]. Combining LC-MS/MS for rapid screening with a quantitative estimation and LC-HRMS for confirmation and characterisation, [PERSON] et al. showed that the toxin profiles of Gambierdiscus spp. dinoflagellates from the Mediterranean Sea and Northeast Atlantic were similar to those detected in CP endemic regions, except for the presence of CTXs, with the detection of 44-methylgambierone, gambieric acid C and D, gambierone analogue, and gambieroxide in Gambierdiscus australes strains (Balearic Islands, Spain) [207]. This study highlighted species and regional specificity of Gambierdiscus spp., with a 44-methylgambierone and gambierone-producing Gambierdiscus sp. strain from Crete (Greece) and an MTX-4 producing G. _excentricus_ strain from the Canary Islands (Spain) [207]. HRMS did not detect maitotoxin-1 (MTX1), desulfo-MTX1, and didehydro-desulfo-MTX1 above the detection limit in Mediterranean strains of G. _australes_[207]. Sulfo-gambierone and dihydrosulfo-gambierone, two new analogues of gambierone, were also characterised in two _G. excentricus_ strains initially isolated from the Bahamas and the Florida Keys [208], however, no algal ciguatoroxins were detected in these two strains, which was consistent with the low ciguatorsin-like content evaluated using the CBA-N2a assay [208]. An m-aminophenylboronic acid agarose gel was used to confirm the presence of gambierone and identify a novel isomer of 44-methylgambierone in an extract of _Gambierdiscus silvae_ from the U.S. Virgin Islands [206].  
Recently, a major breakthrough was achieved with the identification of an algal toxin C-CTX-5 that was extracted from a _Gambierdiscus silvae_ strain and two _Gambierdiscus caribaeus_ strains. The toxin was isolated from macroalgae collected from reefs surrounding St. Thomas (U. S. Virgin Islands) [43]. These _Gambierdiscus_ strains showed C-CTX-like activity on voltage-gated sodium channel using CBA-N2a, with a higher toxicity for _G. silvae_ compared to the two _G. caribaeus_ strains tested [43]. The toxins were screened for known CTXs using reference standards (C-CTX-1, gambierone and 44-methylgambierone) and unknown putative CTXs using LC-HRMS on a Q-Orbitrap (Q Exactive, Thermo Fischer Scientific) with a heated electrospray ionization probe (HESI-II) [43]. With a full-scan acquisition (\(m/z\) 1000-1250), product-ion spectra using targeted parallel reaction monitoring (PRM) scan mode, and chemical transformations, [PERSON] et al. identified a novel C-CTX analogue, C-CTX-5, for the first time from Caribbean _Gambierdiscus_ strains [43]. An initial assessment of CTX-like activity was made using in vitro CBA-N2a, which confirmed that C-CTX-5 was ciguotoxic, with an IC\({}_{50}\)-\(\frac{1}{2}\) IC\({}_{50}\) of C-CTX-1/-2. However, this remains to be quantitatively evaluated using quantified purified materials [43]. Moreover, C-CTX-1/-2 were also detected compared with a reference standard, and it was estimated at -7% of the level of C-CTX-5 in _G. silvae_. Therefore, it could also contribute directly to toxicity of fish that ingest _Gambierdiscus_ cells [43]. Gambierone was detected in all algal extracts, and 44-methylgambierone was detected in _G. silvae_ and one of the two _G. caribaeus_[43]. Interestingly, the transformation of C-CTX-5 into C-CTX-1/-2 occurred via a reduction step [43] rather than oxidation, as observed for Pacific toxins, CTX-4A, CTX-4B, and CTX-3C, which are oxidized to the corresponding CTXs found in fish from the Pacific Ocean [90].