LC-HRMS and Chemical Derivatization Strategies for the Structure Elucidation of Caribbean Ciguatoxins: Identification of C-CTX-3 and -4

Abstract

Ciguatera poisoning is linked to the ingestion of seafood that is contaminated with ciguatoxins (CTXs). The structural variability of these polyether toxins in nature remains poorly understood due to the low concentrations present even in highly toxic fish, which makes isolation and chemical characterization difficult. We studied the mass spectrometric fragmentation of Caribbean CTXs, i.e., the epimers C-CTX-1 and -2 (1 and 2), using a sensitive UHPLC-HRMS/MS approach in order to identify product ions of diagnostic value. We found that the fragmentation of the ladder-frame backbone follows a characteristic pattern and propose a generalized nomenclature for the ions formed. These data were applied to the structural characterization of a pair of so far poorly characterized isomers, C-CTX-3 and -4 (3 and 4), which we found to be reduced at C-56 relative to 1 and 2. Furthermore, we tested and applied reduction and oxidation reactions, monitored by LC-HRMS, in order to confirm the structures of 3 and 4. Reduction of 1 and 2 with NaBH₄ afforded 3 and 4, thereby unambiguously confirming the identities of 3 and 4. In summary, this work provides a foundation for mass spectrometry-based characterization of new C-CTXs, including a suite of simple chemical reactions to assist the examination of structural modifications.

Keywords: Gambierdiscus; HRMS; LC–MS; Scomberomorus cavalla; Sphyraena barracuda; ciguatera; ciguatoxin; fragmentation pathways; ladder-frame molecule; polyether toxin.

Figure 1

Chemical structures of the C-CTX-1 and -2 (1 and 2) and their reduced analogues, C-CTX-3 and C-CTX-4 (3 and 4). Structures of 1 and 2 are shown in accordance with Lewis et al. [14]. Note that it is not possible from LC–MS data alone to determine which of the two chromatographic peaks corresponds to 3, and which to 4.

Figure 2

General nomenclature for MS/MS product ions of polyether ladder-frame molecules used in this study. Product ions are assigned by a letter and number. The letter indicates which carbon-carbon bond is broken during MS fragmentation, while the number represents the number of rings that are contained in the product ion (either as an intact ring or ring-fragment). The “prime” symbol (′) is used to distinguish between the two different ends of the molecule.

Figure 3

LC–HRMS/MS product ion spectrum from HCD of the [M − H₂O + H]⁺ ions of the chromatographically unresolved epimeric pair 1 and 2 (m/z 1123.6, t_R 6.3–6.5 min) and a scheme showing the proposed origin of major diagnostic product ions. Mass regions m/z 100–400 and m/z 400–700 are expanded by approximately 5- and 30-fold, respectively, in their vertical axes.

Figure 4

LC–HRMS/MS product ion spectrum from HCD of the [M + H]⁺ ions of the major epimer of 3/4 (m/z 1143.6, t_R 3.6–3.8 min), and a scheme showing the proposed origin of major diagnostic product ions. Mass regions m/z 100–400 and m/z 400–700 are expanded approximately 5- and 30-fold, respectively, in their vertical axes.

Figure 5

Scheme summarizing the reduction and oxidation reactions at the N-ring of C-CTX-1/2 (1/2) and -3/4 (3/4) following borohydride and periodate treatment. In the case where borodeuteride was used for reduction, the corresponding 56-D isotopologues of 3 and 4 (i.e., 5 and 6) were produced.

Figure 6

Total ion chromatogram (TIC), LC–HRMS extracted positive ion chromatograms (± 5 ppm) (left) and corresponding LC-HRMS spectra. (A) Methanolic fish extract containing predominantly 1, 2, 3 and 4; (B) reaction mixture containing predominantly 7 and 8 as the products after treatment of 1 and 2 with periodate, and; (C) reaction mixture containing predominantly 9 after treatment of 3 and 4 with periodate. TICs are used to illustrate the purity of fraction and mixtures.