Abstract
Tetrodotoxin （TTX） is a powerful small-molecule neurotoxin primarily produced by specific marine endosymbiotic bacteria and can be enriched during symbiosis with aquatic organisms such as pufferfish， gastropods， and blue-ringed octopuses. TTX prevents sodium ions from entering nerve cells， which affects neuromuscular conduction and leads to progressive paralysis and even death due to respiratory failure. Poisoning ascribable to the ingestion of TTX-containing seafood has occurred occasionally in some coastal areas of China. The early identification of toxins and the administration of symptomatic detoxification therapies can improve the resuscitation success rates of poisoned patients. The concentration of TTX in clinical biological samples reflects the degree of patient poisoning and their prognosis. A method was established for the determination of the TTX in poisoned biological samples by two-dimensional liquid chromatography-tandem mass spectrometry （2D-LC-MS/MS）. A human plasma or urine sample （100 μL） was accurately pipetted into a 2-mL centrifuge tube， sequentially added a 10 mg/L kasugamycin solution （10 μL； internal standard）， ultrapure water （150 μL）， and 0.5% （v/v） acetic acid in acetonitrile （250 μL） as the extraction solvent， after which the mixture was subjected to vortex mixing at 2 200 r/min for 10 min and centrifugation for 10 min at 15 000 r/min and 4 ℃. The supernatant was roughly separated using a first-dimensional reverse-phase C(18) column （Hypersil Gold C(18)， 50 mm×2.1 mm， 1.9 μm）. The target fraction was then transferred to a hydrophilic liquid chromatography column （Acquity UPLC BEH Amide， 150 mm×3.0 mm， 1.7 μm） via a six-way switching valve for second-dimensional separation and analysis using positive electrospray ionization and selected reaction monitoring （SRM） modes. Kasugamycin served as the internal standard for TTX quantitation， using matrix-matched calibration combined with the internal standard method. TTX exhibited good linearity in the 0.2-40.0 μg/L range （equivalent to 1.0-200.0 μg/L in biological samples）， with a correlation coefficient exceeding 0.999 4. The TTX in human plasma and urine samples exhibited matrix effects of 80.9% and 98.9%， respectively， with LODs and LOQs of 0.3 and 1.0 μg/L， respectively， determined for both sample types， based on three- and ten-times signal-to-noise ratios， respectively. The TTX in human plasma and urine exhibited intra-day recoveries of 84.4%-98.4% and 84.4%-96.9%， respectively， with inter-day recoveries of 87.7%-96.2% and 84.8%-95.7%， respectively， at spiked levels of 2.0， 10.0， 50.0， and 200.0 μg/L. Intra-day relative standard deviations （RSDs） of 3.2%-7.2% and 2.9%-5.7% were recorded for TTX in human plasma and urine， respectively， with inter-day RSDs of 2.3%-3.2% and 1.0%-7.5%， respectively. The intra-day and inter-day RSDs of both sample types were determined to be lower than 7.5%. The method is accurate， fast， avoids complicated pretreatment steps， and was successfully used to detect TTX in food-poisoning scenarios.