Extraction and LC-MS/MS Analysis of Ciguatoxins: A Semi-Targeted Approach Designed for Fish of Unknown Origin

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.

Gambierdiscus and Its Associated Toxins: A Minireview

“…Cell-based assays can help detect CTXs [ 116 ]. Regardless of the shortcomings of this method ( Table 2 ), it is often employed in combination with other CTX detection methods [ 80 , 117 , 118 ]. Immunoassays such as radioimmunoassays [ 119 ], enzyme immunoassays [ 120 ], antibody-based immunoassays [ 121 , 122 ], membrane immunobead assays [ 123 ], enzyme-linked immunosorbent assays [ 124 ], and capillary electrophoresis-based immunoassays [ 125 ] are also utilized to detect CTXs.…”

“…To date, more than 30 CTX congeners have been identified. They are classified into CTX3C, Caribbean Sea CTXs (C-CTXs) [ 78 ], Pacific Ocean CTXs (P-CTXs/CTX4A) [ 12 ], and Indian Ocean CTXs (I-CTXs) [ 79 ] based on the make-up of the structural backbone of each molecule [ 80 ]. CTXs are lipophilic, ladder-shaped polyethers with 13–14 cyclic consecutively connected rings ( Figure 4 ) [ 81 ] and have similar structures to yessotoxins and brevetoxins.…”

Identical Ciguatoxin-3C group profiles in Lutjanus bohar from the Pacific and Indian Oceans - indicating the need to re-evaluate geographical CTX classifications

“…Fish samples were extracted according to Spielmeyer et al (2021). In brief, 5 g fish tissue was enzymatically degraded using papain.…”

“…The cleaned-up samples were analyzed by LC-MS/MS. Details concerning the analytical methods and setups are provided in Spielmeyer et al (2021) and in the Supplementary Material. In brief, low-resolution analyses were performed on an Agilent 1290 Infinity II UHPLC (Agilent, Waldbronn, Germany) connected to a Sciex QTrap 6500+ mass spectrometer (Sciex, Darmstadt, Germany) using the selected reaction monitoring mode (SRM).…”

“…All sample extracts revealed the presence of several CTX3Cgroup congeners. Congeners from other groups were not detected (detection limit for CTX1B, 52-epi-54deoxyCTX1B and 54-deoxyCTX1B: 0.02 µg kg -1 (Spielmeyer et al, 2021); see also Supplementary Figure 2). In total, ten different congener peaks were identified, most likely belonging to 2,3,51-trihydroxyCTX3C, 2,3-dihydroxyCTX3C and its 49-epimer, M-seco-CTX3C and its 49-epimer, 2-hydroxyCTX3C and its 49-epimer, 51-hydroxyCTX3C, and CTX3C and its 49-epimer.…”