Comparison of Different Solid-Phase Cleanup Methods Prior to the Detection of Ciguatoxins in Fish by Cell-Based Assay and LC-MS/MS

Abstract

Ciguatera poisoning (CP) is the most reported food poisoning associated with fish consumption. Ciguatoxins (CTXs) are produced by microalgae and metabolized in fish; even low levels of these toxins in fish can lead to CP. To date, there is no unique validated methodology for their study, and demonstrating their presence in fish tissues is an analytical challenge. The main techniques used are cell-based assay and liquid chromatography, which may present different matrix effect interferences; thus, purification protocols are necessary. Six cleanup strategies for fish extracts, assessing the principal analogues found in fish in different parts of the world (CTX1B/CTX3C/C-CTX1), are compared here. Cleaned-up extracts are evaluated by cell-based assay and chromatography. All protocols are suitable for recovering the analogues of CTXs. Two of them, those that used polystyrene-divinylbenzene and silica cartridges, achieve the most adequate results showing toxicity in their fractions over 53% and chromatography efficiencies over 79% for CTX1B/CTX3C, proving to be the most versatile clean-ups for the study of the different CTX analogues.

Keywords: LC-MS/MS; cell-based assay; ciguatoxins; cleanup; fish; solid phase extraction.

1

Dose–response curves of CTX1B standard solution, raw flesh extract (F0), and each SPE fraction (F _n) with measurable toxicity. The superimposed graphs correspond to analyses of the same sample on different days.

2

Recovery (RE), matrix effect (ME) and efficiency for CTX1B and CTX3C in the different SPEs spiked at 10 ng/mL expressed as % in the different fractions (for further details, see Table S3).

3

Extracted ion chromatograms (XICs) of the ciguatoxins (CTXs) involved in the present study and used as a reference for retention times, and ion ration confirmation, compared with a TIC of SPE fraction where C-CTX1 was detected. (A) 17-OH-C-CTX1 from a fish flesh (outbreak associated sample cleaned-up with second strategy (different Florisil batch)); (B) CTX1B solution standard from ILM; (C) C-CTX1 from a fish flesh (outbreak associated sample cleaned-up with second strategy (different Florisil batch)); (D) CTX3C solution standard from ILM; (E) sample example: F4 Flo-C18 SPE.