Development and characterization of tetrodotoxin reference materials

Tetrodotoxin (TTX) is a potent natural toxin commonly associated with puffer fish, originally discovered in Tetraodontidae spp., which primarily inhabit tropical and sub-tropical waters. The more recent detection of TTX in shellfish from temperate waters has increased the demand for analytical capabilities to test for TTX in a variety of sample types. Here, we describe the production of TTX reference materials (RMs) essential for the development, validation, and quality control for TTX analyses. Research to develop a TTX calibration solution considered the impact of solvent, pH, and temperature on stability. The certified reference material (CRM) was prepared in 1 mM acetic acid, value assigned (6.75 ± 0.24 µg/g TTX) using quantitative nuclear magnetic resonance, and was stable at temperatures up to 4 °C over a 2-year stability study. Feasibility studies were performed to produce shellfish matrix RMs by blending mussels (Mytilus edulis) with TTX-contaminated sea slug (Pleurobranchaea maculata). Separate wet tissue and lyophilized materials were evaluated, and good homogeneity was demonstrated for both. In the lyophilized material, TTX was stable at temperatures up to 40 °C over 28 days; however, in the wet homogenate, TTX degraded at temperatures of 4 °C and above over the same time period. Liquid chromatography and capillary electrophoresis methods with tandem mass spectrometry detection were developed for characterization of the materials and showed good agreement. The availability of a TTX calibration solution CRM, established processes for matrix RM preparation, and improved characterization methods provide the tools necessary for supporting TTX analyses in shellfish considering its increased occurrence globally.