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

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 and Pacific oysters-the bivalve species that have been found to contain TTXs in the United Kingdom in recent years. The method consists of a single-step dispersive extraction in 1% acetic acid, followed by a carbon 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 for the 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. Results demonstrated 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 to the 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 should regulatory controls be adopted.

Distribution of Tetrodotoxin in the New Zealand Clam, Paphies australis, Established Using Immunohistochemistry and Liquid Chromatography-Tandem Quadrupole Mass Spectrometry

“…Samples were then centrifuged (17,000× g , 1 min) and the supernatant cleaned with the GPC Solid Phase Extraction (SPE) method as described by Boundy et al [ 24 ] using Supelclean ENVI-Carb 250 mg/3 mL SPE cartridges (Sigma-Aldrich). Tetrodotoxin was analyzed and quantified by liquid chromatography tandem-mass spectrometry analysis as described by Turner et al [ 57 ].…”

Development and validation of a maleimide-based enzyme-linked immunosorbent assay for the detection of tetrodotoxin in oysters and mussels

“…Additionally, since multiple reaction monitoring (MRM) transitions for Arg have been found to suppress TTX response in the mass spectrometer source [42], the possible interference of this amino acid in the mELISA was also evaluated. As can be seen in Fig.…”

“…Although some of these immunochemical approaches have been applied to the analysis of puffer fish [32,33,36,38,[40][41], newts [26], caddisflies [27], terrestrial flatworms [29], sea snails [38], urine [36] and milk/apple juice [37], none of them has been applied to the analysis of bivalve mollusks. Indeed, up to date only the LC-MS/MS method has been applied to the detection of TTXs in mussels and oysters [42,43]. However, this technique requires trained personnel, sample pre-treatment and expensive equipment.…”

Temporal Variation of the Profile and Concentrations of Paralytic Shellfish Toxins and Tetrodotoxin in the Scallop, Patinopecten yessoensis, Cultured in a Bay of East Japan

“…First, the conditions for hydrophilic interaction chromatography (HILIC)-MS/MS in multiple reaction monitoring (MRM) mode, especially for MS conditions, were optimized for the simultaneous analysis of PSTs and TTXs based on the previously reported methods [47][48][49][50][51][52]. Liquid chromatography (LC) conditions reported by Thomas et al [52] were slightly modified for use in this study (see, Section 4.7).…”