## 6 Discussion and Perspectives
### Considerations for Analytical Approaches for C-CTX Detection and Quantification
Moreover, sub-2-um solid-core particles could enable large increases in efficiency. However, it is crucial to implement an efficient preanalytical step to eliminate interfering substances present in fish flesh, such as lipids, which can affect sample loading, MS sensitivity, and column life. The most commonly used stationary phase is reversed-phase C18 (octadecylsilane), but some authors have also used a pentafluorophenyl-propyl stationary phase to significantly improve the separation of C-CTX-1/-2 and C-CTX-3/-4 peaks [120]. Since the 2010s, three methodological approaches in low-resolution LC-MS/MS using single (SRM) or multiple reaction monitoring (MRM) modes have been distinguished (Table 6). These approaches depend on the type and sensitivity of the mass spectrometer, as well as the intended purpose, such as quantifying C-CTX-1 with a purified standard or identifying C-CTXs based on their MS characteristic pattern of ion formation. Currently, for sensitive C-CTX-1 quantification, a water/methanol gradient is combined with a SRM of the prominent adduct [M+Na]\({}^{+}\) as precursor and product ions, using an ESI-triple quadrupole [109; 110; 112; 155; 175; 176], which has been previously used for P-CTXs [30; 210]. Confirmation of C-CTX-1 and search for C-CTX analogues are performed using a water/acetonitrile gradient and MRM using the most prominent peak of each analogue [M+Na]\({}^{+}\), [M+H+H\({}_{2}\)O], [M+H]\({}^{+}\), or [M+H-CH\({}_{3}\)-H\({}_{2}\)O]\({}^{+}\), and serially dehydrated ions [110; 175; 176]. However, a comparison with the approach based on SRM of sodium adduct as precursor and product ions [30] showed that using an ESI-triple quadrupole/linear ion trap (API 4000, Sciex) and a methanol-based mobile phase, better selectivity through confirmatory transitions and better sensitivity with less matrix effects allowed for the detection of P-CTX-1 and P-CTX-3C by choosing [M+NH\({}_{4}\)]\({}^{+}\) and [M+H]\({}^{+}\) ion precursors, respectively, and [M+H-2H\({}_{2}\)O]\({}^{+}\) as product ions [211]. A third approach was developed for multi-analytes screening of CTXs sorted into four groups: CTX-4A and CTX3C groups (for P-CTXs), a C-CTX group, and an I-CTX, by monitoring sodium adducts separated with a water/methanol/acetonitrile gradient [180]. MS confirmation was achieved by monitoring four predicted MRM transitions per CTX and choosing the ammonium adduct [M+NH\({}_{4}\)]\({}^{+}\) as the precursor ion and the corresponding [M+H]\({}^{+}\) and [M+H-nH\({}_{2}\)O]\({}^{+}\) with \(n=1\)-3 as product ions [180]. This approach allowed for the relative contribution of the identified cigutoxins from the CTX3C group to be compared using LC-MS/MS in 52 tissue samples of _Lutjanus bohar_[34]. This approach could be extended to species-specific CTX metabolism and inter-ocean CTX toxin profile analyses. Indeed, analytical validation performed on three different fish genera (_Lutjanus, Scarus_, and _Epinelus_) also highlighted quantitative species-specific differences in MS responses, suggesting fish-specific matrix effects, which is a major problem for quantification from different matrices [180]. Thus, another promising perspective is the synthesis of the \({}^{18}\)O-labeled C-CTX-1/2, C-CTX-3/4, gambierone, and 44-methylgambierone quantified by [PERSON] et al. [132]. Importantly, isotope-labeled standards allow for isotope dilution mass spectrometry and gold-standard correction for matrix effects and analyte losses during sample preparation. In addition to the targeted mass spectrometry approach using (U)HPLC-MS/MS, high-resolution mass spectrometry (HRMS) represents a promising advance for the structural identification of marine biotoxins by improving the accuracy of assigning chemical formulas [212]. However, HRMS instruments such as the Quadrupole Time-of-Flight (QToF) and Q-Orbitrap are expensive and can generate large amounts of complex data, which is not always convenient for routine analysis.