Background In recent years, there have been frequent incidents of tetrodotoxin (TTX) poisoning caused by consumption of the Nassarius spp. However, the current national standard (GB 5009.206—2016) uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) which is better for detecting TTX in pufferfish. Therefore, there is an urgent need to establish an efficient and accurate method suitable for detecting TTX content in Nassarius spp.
Objective The aim of the study is to establish a highly efficient and accurate method for determining TTX content in the Nassarius spp. using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).
Methods By comparing the effects of different pretreatment methods (extraction solution, extraction method, freezing time) on TTX recovery rate, along with the effects of different chromatographic conditions (mobile phases and injection volume) on chromatographic peaks, the optimal experimental conditions were obtained. In addition, the method was evaluated for accuracy and reliability by comparing the results obtained using the optimized method with those obtained using the current national standard method for detecting 9 samples collected from Putian City and Ningde City in Fujian Province.
Results The optimal experimental conditions were as follows: firstly, the TTX in samples was extracted by 0.1% aqueous acetic acid solution combined with ultrasonication at 60 ℃ for 20 min, and then diluted with 80% (V/V) 0.1% methanol acetic acid solution and centrifuged after freezing at −18 ℃ for 30 min, and finally the supernatant was taken through the membrane and then analyzed by UPLC-MS/MS. The UPLC-MS/MS conditions were as follows: 0.1% formic acid solution containing 5 mmol·L−1 ammonium acetate and acetonitrile were used as mobile phases, and after gradient elution on an Amide-80 column, the electrospray positive ion selective reaction monitoring mode was used, and the matrix standard curve was quantified by the external standard method. The matrix effect of TTX in textured snails was 68.9%, and the linear relationship was good within the matrix standard curve range of 1.0 to 100 ng·mL−1. The linear equation correlation coefficient of TTX was more than 0.99. The detection limit and quantification limit of this method were 10 μg·kg−1 and 25 μg·kg−1, respectively, which could meet the requirements of China (2 200 μg·kg−1) and the European Food Safety Commission (44 μg·kg−1) requirements for safety limits of TTX in aquatic products. In addition, at three TTX spiked concentrations (50, 500, and 4 000 μg·kg−1), the average recovery rates were 74.8% to 104%, and the relative standard deviations (RSD) were all less than 15% (n=6). The optimized experimental method and the current national standard method were used to detect the 9 Nassarius spp. samples, respectively. The TTX content range detected by the two methods were <0.025 0−22.8, 0.012 0−23.5 mg·kg−1, and the relative deviations were 1.51%−14.6%, indicating the accuracy and reliability of this detection method.
Conclusion This study optimized and established a UPLC-MS/MS suitable for the determination of TTX content in the Nassarius spp. It showed the advantages of simple pre-treatment, fast and accurate and low cost. This study will lay a good foundation for the rapid batch detection of TTX in the Nassarius spp.
DownLoad: