Development and Single-Laboratory Validation of a Liquid Chromatography Tandem Mass Spectrometry Method for Quantitation of Tetrodotoxin in Mussels and Oysters

Abstract

Abstract In recent years, evidence has grown for the presence of tetrodotoxin (TTX) in bivalve mollusks, leading to the potential for consumers of contaminated products to be affected by Tetrodotoxin Shellfish Poisoning (TSP). A single-laboratory validation was conducted for the hydrophilic interaction LC (HILIC) tandem MS (MS/MS) analysis of TTX in common mussels andPacific oysters—the bivalve species that havebeen found to contain TTXs in the United Kingdom inrecent years. The method consists of a single-step dispersive extraction in 1% acetic acid, followed by acarbon SPE cleanup step before dilution and instrumental analysis. The full method was developed as a rapid tool for the quantitation of TTX, as well as forthe associated analogs 4-epi-TTX; 5,6,11-trideoxy TTX; 11-nor TTX-6-ol; 5-deoxy TTX; and 4,9-anhydro TTX. The method can also be run as the acquisition of TTX together with paralytic shellfish toxins. Resultsdemonstrated acceptable method performance characteristics for specificity, linearity, recovery, ruggedness, repeatability, matrix variability, and within-laboratory reproducibility for the analysis of TTX. The LOD and LOQ were fit-for-purpose in comparison tothe current action limit for TTX enforced in The Netherlands. In addition, aspects of method performance(LOD, LOQ, and within-laboratory reproducibility) were found to be satisfactory for three other TTX analogs (11-nor TTX-6-ol, 5-deoxy TTX, and 4,9-anhydro TTX). The method was found to be practical and suitable for use in regulatory testing, providing rapid turnaround of sample analysis. Plans currently underway on a full collaborative study to validate a HILIC-MS/MS method for paralytic shellfish poisoning toxins will be extended to include TTX in order to generate international acceptance, ultimately for use as an alternative official control testing method shouldregulatory controls be adopted.