Development And Application Of The Detection Method For Melamine In Fish Samples

Melamine can go into the aquaculture water through various channels then absorbed by fishes, further through the food chain and endanger human body health. Therefore, it is necessary to explore the behavior and ecological risks of melamine residues, migration, and end-result in aquaculture environment. The detection method is a key problem to solve in first. Due to the low content of melamine in aquatic samples with many complex interfering substances, we must adopt an efficient pretreatment approach such as appropriate extraction, enrichment, separation and purification to detect the melamine. The aim of this work is to develop a rapid, sensitive and accurate analytical method for melamine residues in aquatic products, then the new method will be applied to study the accumulation of melamine in tilapia in polluted water and its elimination in clean water under the simulated aquatic ecosystem. The main work and conclusions are as follows:Chapter1:Clarified the background of this study, research ideas and main content.Chapter2:Summarized the recently developed techniques for analysis of melamine and research advances on toxicity of melamine and the residues and metabolism of melamine in animals in the past decade.Chapter3:A method for the rapid determination of melamine in fish samples by using microwave-assisted derivatization was proposed. Samples was extracted by methanol and purified by mixed cationic exchange solid-phase extraction column, then dried by nitrogen gas and rapid derivatized by N,O-Bis(trimethylsilyl)trifluoroacetamide with microwave, then detected by gas chromatography-mass spectrometry and quantified by the external standard method. Extraction solvent and some important parameters affecting the derivatization such as microwave heating time, pyridine volume and derivatization reagent volume were investigated. The study showed that methanol can extracted the melamine effectively in the aquatic products and precipitated the protein. Under the optimized conditions (pyridine volume:300uL, derivatization reagent volume:150μL, microwave heating time:1min with the microwave power of420W), the average recovery of melamine was89.65%～105.16%with the relative standard deviation was less than6.78%(n=4). The linear range was from0.02to50mg/kg with detection limit as low as0.0058mg/kg. The method is also suitable for the determination of melamine in many other aquatic products such as tilapia, grass carp, mantis shrimp, winkles and the corbicula fluminea samples.Chapter4:The accumulation and elimination of melamine in tilapia in polluted water and clean water were investigated with the new method. The results showed that melamine could be easily enriched and eliminated by tilapia, especially in its innards. The concentration of melamine in tilapia was enhanced rapidly in the polluted water. The accumulated amount of melamine in the innards and muscles almost rose linearly to the time, and its concentration was2-3times higher than that in the muscles. When the cumulative quantity of melamine in the innards was higher, tilapia showed obvious toxicity symptom. However, when the fish were transferred to clean water without melamine, the residues of melamine in its muscles and innards were decreased drastically, and the toxicity symptom of tilapia disappeared gradually and its physical activity was towards normal.Chapter5:The distribution and accumulation of melamine in tilapia (muscles, kidney, liver and other innards) and winkles (muscles and innards) in a simulation aquaculture system with the elements of water, soil, hornwort, tilapia and winkles were investigated. There were differences in the distribution and accumulation of melamine for different organisms. Winkles had obviously the accumulation of melamine, especially in visceral tissue, increasing continually during the experiment. For tilapia, the significant differences of melamine accumulation existed between kidney, liver, muscle and other viscera tissues. It is the highest in kidney than in liver and muscle, indicating that the melamine was easy to get enrichment in kidney. Later, the concentration of melamine in tilapia was decreased gradually with the decreasing concentration of melamine in water.