Abstract
Ciguatoxins (CTXs) are polyether marine biotoxins that can cause ciguatera poisoning (CP) after the consumption of fish or invertebrates containing sub ppb levels; concentrations that present a challenge for current extraction and analysis methods. Here, a newly developed and (partly) validated single-day extraction protocol is presented. First, the fish sample is broken-down by enzymatic digestion, followed by extraction and extract clean-up by defatting and two solid-phase extractions. Final extracts were investigated using two different CTX-analysis methods; an in vitro cytotoxicity assay (N2a-assay) and by LC-MS/MS. Validation was performed for both fillet and freeze-dried samples of snapper, parrotfish, and grouper spiked with CTX1B, 52-epi-54-deoxyCTX1B, 54-deoxyCTX1B, and CTX3C. Based on recovery rates (35–88%) and matrix effects (66–116%) determined by LC-MS/MS, the enzyme protocol is applicable to various matrices. The protocol was applied to naturally contaminated fish tissue (Lutjanus bohar) obtained during a CP incident in Germany. Several potential CTX congeners were identified by a two-tier LC-MS/MS approach (screening of sodium adducts, high-resolution or low-resolution confirmation via ammonium adducts). Inclusion of >30 known CTX congeners into the LC-MS/MS methods and single-day sample preparation make the method suitable for analysis of ciguatera suspect samples at sub ppb levels also with undisclosed CTX profiles.
Highlights
Ciguatoxins (CTXs) are marine biotoxins produced by dinoflagellates in the generaGambierdiscus and Fukuyoa [1,2,3,4]
Most methods utilize acetone or aqueous methanol for extraction. Both solvents are mixable with water leading to a transition of water and water-soluble matrix constituents from the fish sample into the extract
Due to the enzymatic treatment of the fish tissue, the hydrolyzed sample was expected to contain more polar protein fragments that can be extracted by polar solvents than solely mechanical treated samples
Summary
Ciguatoxins (CTXs) are marine biotoxins produced by dinoflagellates in the generaGambierdiscus and Fukuyoa [1,2,3,4]. CTXs are lipophilic polyether molecules (Figure 1) with (computed) logP values of, e.g., 2.5 for CTX1B [5] and 4.7 for CTX3C [6]. Over 30 different CTX congeners (including epimers) are known, differing in the number of condensed rings and in the presence/absence of side chains [7]. Based on their skeletal structure and the first region of isolation, four groups of CTXs are differentiated, namely CTX3C, CTX4A (both Pacific CTX and P-CTX), Caribbean CTX (C-CTX), and Indian Ocean CTX (I-CTX; Table 1). The structural elucidation of I-CTXs has not been accomplished so far [8,9,10]
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