## 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
Similarly, elevated levels of C-CTX3/4 and the methylated compounds can potentially occur under acidic conditions [131]. C-CTX-1 was identified as responsible for more than 60% of the total toxin content, followed by putative 17-hydroxy-C-CTX-1, initially designated as C-CTX-1157 and identified as toxic using RBA (see Section 5.1.2) [42]. Its toxicity was confirmed using CBA-N2a [108]. The thermal stability of C-CTX-1 and 17-hydroxy-C-CTX-1 was also confirmed by cooking one of the samples. UHPLC-HRMS/MS can also contribute structural insights from identified MS fragment ions generated chemically, including those that modify the A and N rings of C-CTX-1. [PERSON] et al. reported that the fragmentation of the ladder-frame backbone of C-CTX-1/-2 followed a characteristic pattern and defined a generalized nomenclature for the MS/MS product ions [120]. This was aligned with previous studies that identified only three types of product ions that were formed during fragmentation of the polyether backbone [32; 33; 38; 42]. In addition, a MS-based methodology was proposed for the characterisation of new C-CTXs, with oxidation and reduction reactions used to assist the examination of the structural modifications of C-CTX-1 found in king fish (_Somberomorus cavalla_) and great barracuda (_Sphryena barracuda_) samples [120]. The reduction of C-CTX1/-2 using sodium borohydride (NaBH\({}_{4}\)) and borodeuteride (NaBD\({}_{4}\)) generated pairs of isomers with protonated molecules at \(m/z\) 1143.6463 and 1144.6535, respectively [120]. This led to the structural characterisation of two new analogues, C-CTX-3 and C-CTX-4, containing an open N-ring, and thus corresponding to C-CTX-1 and -2 reduced at C-56 [120]. Caribbean CTX congeners with a protonated molecule at \(m/z\) 1143.6 were identified earlier in several fish from the Caribbean [23; 38; 41; 42; 108], and more recently from the Eastern Atlantic Ocean [131]. However, these are an open (unstable) form of C-CTX-1 that are generated under acidic conditions and lack stereochemistry, making the assignment of the two isomers controversial. [PERSON] et al. also showed that a pentafluorophenyl-propyl UHPLC column (100\(\times\)2.1 mm, 1.7 \(\upmu\)m) significantly improved the shape of C-CTX-1 and -2 peaks and provided better chromatographic separation between C-CTX-1/-2 and C-CTX-3/-4. However, the octadecylsilane-based LC column (100 \(\times\) 2.1 mm, 1.5 \(\upmu\)m) allowed for the best anomer separation regardless of chromatographic conditions [120]. A comparison of the peak shapes of C-CTX-1 and -2 during LC-HRMS in acidic and neutral mobile phases on the C-18 column were indicative of a rapid on-column interchange between C-CTX-1 and -2 that was accelerated under acidic conditions, whereas the peak shapes of 3 and 4 were unaffected [120]. To improve the HRMS sensitivity compared to that of targeted LC-MS/MS, [PERSON] et al. also proposed a promising chemical derivatization approach employing a fast and simple one-pot derivatization with [PERSON]'s reagent T (GRT) [205]. The sensitivity of their LC-MS/MS method using a triple quadrupole (6495B, Agilent Technologies, Santa Clara, CA, USA) and the LC-HRMS method on a Q-Orbitrap (Q-Exactive, Thermo Fisher Scientific, Waltham, MA, USA) was improved for C-CTX-1 by \(\sim\)40- and 17-fold, respectively [205]. Derivatization with GRT improved method selectivity and ionization efficiency by inserting a quaternary ammonium ion onto C-CTX-1. However, the use of GRT derivatization for routine applications still requires validation [205]. UHPLC-HRMS methods in negative and positive ionisation modes on a Q-Orbitrap (Q-Exactive, Thermo Fisher Scientific) were also adapted for CYP and UGT metabolism experiments and chemical reduction by [PERSON] et al. They reported the production of phase II GlcA conjugates of C-CTX-1/-2 in vitro by liver microsomes of five fish species from the northern Gulf of Mexico and Atlantic salmon, which was the first time it was observed [165].