Response Surface Methodology for the Optimization of Ultrasound-Assisted Extraction of Tetrodotoxin from the Liver of Takifugu pseudommus

Abstract

Tetrodotoxin (TTX) is a marine biotoxin that has high scientific value. However, the lack of efficient TTX extraction and preparation methods has led to a scarcity of TTX samples for clinical application. In this study, TTX from the liver of Takifugu pseudommus was ultrasound-assisted extracted with acidified organic solvents. The extraction process was analyzed and optimized by single factor method and response surface methodology (RSM). The optimal extraction conditions predicted by a response surface model were as follows: liquid:material ratio, 2.8:1; extraction temperature, 60 °C; extraction time, 23.3 min. Under these conditions, the extraction of TTX had a yield of 89.65%, and the results were further verified by experimental extraction, and analyzed by ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). It was found that the extracts of T. pseudommus liver contained TTX and its four analogues at certain proportions (TTX: 10.4%; 5,6,11-trideoxyTTX: 83.3%; 5,11-dideoxyTTX:2.4%; 4,9-anhydro TTX:2.6%; 5-deoxyTTX:1.3%). This study demonstrates a stable and efficient extraction process of TTX from pufferfish liver, which can be helpful for further research and analysis, as well as the utilization of TTX from pufferfish.

Tetrodotoxin and Its Analogues in Food: Recent Updates on Sample Preparation and Analytical Methods Since 2012

“…Selecting a proper extractant is important, and the most common one is acetic acid (AA). ,, To further clean up the sample mixtures, trichloroacetic acid or acetonitrile is often used for protein precipitation, while hexane or dichloromethane is used for fat removal. In recent years, ultrasonic-assisted extraction (UAE) and accelerated solvent extraction (ASE) have been more applied to sample preparation, since they overcome the disadvantages of traditional extraction techniques, such as low extraction rates and large solvent consumption. ,, …”

“…It utilizes ultrasound energy that can generate mixing, crushing, vibration, and cavitation effects in liquid media to increase the moving speed and the penetration of medium molecules. This method has been more attained in TTX extraction by virtue of its high extraction yield and short extraction time. ,,, Zhang et al developed a UAE to extract TTX from pufferfish and optimized it by response surface methodology. At the optimal conditions (liquid:material ratio = 2.8:1, extraction temperature = 60 °C, process time = 23.3 min), satisfactory extraction recovery of 89.65% was obtained.…”

Dispersive Liquid–Liquid Microextraction (DLLME) and LC-MS/MS Analysis for Multi-Mycotoxin in Rice Bran: Method Development, Optimization and Validation

“…It investigates the interaction effects between several factors at different levels. In this study, RSM was employed in the DLLME method optimization, as it offers several advantages, including a minimum number of experiments, saves time and cost, and minimal use of chemicals [ 24 , 25 , 26 ].…”

Tetrodotoxin binding protein in the marine puffer fish

“…TTX is a weak base organic compound that is non-protein, colorless, odorless, thermostable, and acid-stable (Figure 1). The TTX molecule is formed by a moiety of guanidinium connected to a highly oxygenated skeleton of carbon which has a portion of 2,4-dioxa adamantane with five groups of hydroxyl (Isbister & Kiernan, 2005;Zhang et al, 2018;Pinto et al, 2019). TTX's guanidinium moiety is essential for its toxicity and initially serves as an excellent target to predict the molecule's biosynthesis because of its secondary metabolite rarity.…”