College of Food Science & Technology, Shanghai Ocean University, Shanghai 201306, China.
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, China.
Talanta. 2017 Aug 15;171:179-184. doi: 10.1016/j.talanta.2017.04.078. Epub 2017 May 1.
Tetrodotoxin (TTX) is one of the most toxic substances of non-protein in nature. In present study, a rapid and sensitive method based on in vivo solid-phase microextraction (SPME) coupled to liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed to detect the endogenous TTX in pufferfish (Fugu). Fiber evaluation experiments demonstrated that, compared with the commercial PDMS and PA fibers, the home-made electrospun PS@PDA-GA fibers exhibited much better extraction performance towards to TTX in water (120 times and 20 times, respectively), under the optimized conditions. Then, the home-made SPME fibers were employed to extract TTX in spiked homogeneous fish muscle samples, and a LC-MS-MS was used for the analysis. The reproducibilities (RSDs of inter and intra fiber were 12.1%, and 7.9% respectively), linear range (10-1000ngg, R=0.9963) and sensitivity (the LOD was 2.3ngg) of the method were found to be excellent and satisfactory for further in vivo experiments. Especially the LOD of the established method is lower than the National Standard Method of China (GB/T 23217-2008, LOD 50ngg). Subsequently, the method was successfully applied to detect the TTX in the dorsal muscle of living pufferfish, and the accuracy was verified with traditional liquid extraction (LE) method. In general, this is the first study to detect TTX in pufferfish by in vivo sampling method, which provided a promising alternative method for the studies of TTX, and also advanced the implementation of SPME for more in vivo studies.
河豚鱼（Fugu）体内毒素（TTX）是自然界中最毒的非蛋白质物质之一。本研究建立了一种基于体内固相微萃取（SPME）与液相色谱串联质谱（LC-MS/MS）联用的快速灵敏检测河豚鱼内源性 TTX 的方法。纤维评价实验表明，与商业 PDMS 和 PA 纤维相比，自制的静电纺丝 PS@PDA-GA 纤维对水中 TTX 的萃取性能更好（分别为 120 倍和 20 倍），在优化条件下。然后，将自制的 SPME 纤维用于提取加标均相鱼肌肉样品中的 TTX，并采用 LC-MS-MS 进行分析。该方法的重现性（纤维间和纤维内的 RSD 分别为 12.1%和 7.9%）、线性范围（10-1000ngg，R=0.9963）和灵敏度（LOD 为 2.3ngg）均表现出色，满足进一步体内实验的要求。特别是该方法的 LOD 低于中国国家标准方法（GB/T 23217-2008，LOD 为 50ngg）。随后，该方法成功应用于活体河豚鱼背部肌肉中 TTX 的检测，并与传统的液液萃取（LE）方法进行了准确性验证。总的来说，这是首次采用体内采样法检测河豚鱼中的 TTX，为 TTX 的研究提供了一种有前途的替代方法，也推动了 SPME 在更多体内研究中的应用。
