Determination of tetrodotoxin in bivalve mollusks by hydrophilic interaction liquid chromatography-triple quadrupole mass spectrometry with internal standard calibration and its contamination in Zhejiang province, China

Abstract

A method was established for determining tetrodotoxin (TTX) in bivalve mollusks by hydrophilic interaction liquid chromatography-triple quadrupole mass spectrometry (HILIC-MS/MS) using kasugamycin as the internal standard for quantification. Samples were subjected to ultrasonic extraction with methanol–water (1:1, v/v) containing 0.5% acetic acid, protein precipitation with acetonitrile, clean-up using a cation exchange solid phase extraction cartridge, elution with acetonitrile:water (1:1, v/v) containing 0.3% hydrochloric acid, neutralization with ammonia before HILIC-MS/MS analysis. The average recovery of the samples spiked at 3 levels ranged in 84.6%-98.1% with the relative standard deviation less than 7.2%. Using this method, the contamination of TTX in 429 bivalve mollusk samples collected in the local markets during 2018 and 2020 was investigated. The detection rates were 12.0–18.8%, following the order of oyster > mussel > clam > scallop. High contaminated oysters and mussels with TTX were found in July to August. Moreover, TTX analogs found in bivalve mollusks included 4-epiTTX, 5,6,11-trideoxyTTX, 4,9-anhydroTTX, and 5-deoxyTTX/11-deoxyTTX.