Research On The Correlation Between Amantadine Residue And Safety Of Algae

As an antiviral drug used to kill flu virus,amantadine is a prohibitive veterinary medicine.However,amantadine was illegally used in animal feeding in recent years.In 2005,the ministry of agriculture announced that it was prohibited in animal husbandry because of the adverse for health in poultry products.The marine would be polluted by wastewater containing amantadine.However,in the current study,more attention was paid to the detection of amantadine residues in animal-derived foods and feeds.Its distribution in the marine environment and impact on Laminaria japonica have rarely been reported domestic and overseas.Therefore,it is of great importance to the healthy and sustainable development of algae by clarifying the source of amantadine in algae,protecting the environment in which it depends,and proposing practical control technology.In this dissertation,a high performance liquid chromatography tandem mass spectrometry method(HPLC-MS/MS)for determination of amantadine was established in seawater and algae.The Laminaria japonica in Yantai Changdao Aquaculture Zone was monitored continuously to analyze the relationship between the content in Laminaria japonica and seawater at different growth stages,and evaluate the impact on food quality and safety.Meanwhile,the regular pattern of the reduction was analyzed under different processing methods.The accumulation and eliminate was analyzed in semi-static water in Gracilaria lichenoides.It was hoped that this thesis can be a valuable research to provide certain foundational data on harmful level degree and mechanism constipation.The main results were summarized as follows:1.Establishment of the method of amantadine in seawater and algaeIn this dissertation,the pretreatment,liquid chromatography and mass spectrometry conditions were optimized,and HPLC-MS/MS method for detection of amantadine was established and verified.The results show that:(1)The algae sample were extracted ultrasonically with 1% acetic acid in acetonitrile/water at 20? for 20 min,and the rocovery was 117.75%(internal standard method).The enrichment and purification were carried out by solid phase extraction.The optimal of purification fillers was 500 mg PSA,500 mg C18 and 10 g anhydrous sodium sulfate,and the recovery was 106.02%.After adding 10 mL 0.20 mol/L hydrochloric acid in seawater,MCX column was used for enrichment and purification,and the recovery was 59.76%.(2)The mobile phase was optimized,and when 0.1% formic acid in acetonitrile/water,the peak shape was better and the response value higher,and the impurities could be separated with amantadine.The detection was improved,and matrix was reduced.Retention time and ion abundance ratio were uesd for qualitative analysis.(3)The detection limits were 1.00 ?g/kg and 5.00 ng/L in algae and saewater,respectively.The quantitative limits were 2.00 ?g/kg and 10.00 ng/L.The method above was evaluated and proved to be highly sensitive and reproducible and of good efficient in separation,qualitative and quantitative accurately.2.Variation of amantadine content in Laminaria japonicaTaking rafts culture areas as objects,three areas in Daqin Island,Yantai,Shandong province.The accumulation and elimination in Laminaria japonica were studied by the content of amantadine in different varieties,different growth stages and parts of the same variety.Food safety indexes and risk coefficient were used to evaluate the potential risk of the amantadine residues.The results show that:(1)The content of amantadine in seawater of long Island was stable at 1.72 ng/L~2.15 ng/L,and the content was significantly different in different sampling points(P<0.05).There was no significant difference between different months(P>0.05).(2)The enrichment rates were different in different species,and showing No.3>No.1>No.2 in the same part with the growth of Laminaria japonica.(3)For the same species,there was no significant difference in the amount of enrichment in different parts at the initial stage of growth.With the growth,the enrichment amount showed the tendency of concave and convex part of leaf body > straight part of leaf body > root and stalk of knot.(4)According to the safety index and risk coefficient,the Laminaria japonica monitored was safe and low risk in the experiment month.3.Effects of different processing methods on amantadine content in Laminaria japonica.The effects of soaking,blanching,boiling,salting and baking on the residue in Laminaria japonica were investigated.The results showed that: the five methods above can reduce the content differently,and amantadine can be removed by salting for 3 h completely.57.6% amantadine can be removed when boiling for 15 min;The boiled process can eliminate more amantadine in a shorter period of time than the 65.07% reduction in soaking for 2 hours and the reduction of 33.90% in 1 min.When baked at 150°C for 7 min,amantadine can be reduced by 76.05%,but increased with the baking time and temperature.4.The rules of accumulation and residue elimination of amantadine in Gracilaria lichenoidesThe accumulation and eliminate in Gracilaria lichenoides was analyzed in semi-static water contact and the pharmacokinetic characteristics were also studied.The results show that:(1)There was a positive correlation between the content of amantadine and exposure concentration in Gracilaria lichenoides,and increased gradually with the exposure time.(2)The enrichment coefficient of amantadine was larger when the exposure concentration lower in Gracilaria lichenoides.The enrichment coefficient,exposure concentration and time of amantadine were different significantly(P<0.05).The enrichment rate in the early stage was significantly faster than that in the later and decreased with the prolongation of time gradually,then stabilized.Compared with persistent organic pollutants,amantadine has weak enrichment ability in Gracilaria lichenoides.Even at low concentrations,the residues in algae may be absorbed by the human body through the food chain,resulting in toxic effects.(3)In the early stage of the elimination,the rate was relatively fast,decreased slowly after reaching a certain degree,and maintaining at the low concentration level for a long time.In the end of the elimination experiment,amantadine was decreased 89.33%,92.38% and 97.60% in 5.00 ?g/L,25.00 ?g/L and 100.00 ?g/L groups,respectively.The results showed that the elimination rate was faster and the content in Gracilaria lichenoides was decreased rapidly when removed from the exposed environment.(4)The dynamic parameter derivation of amantadine showed that the content in Gracilaria lichenoides was correlated with the exposure concentration positively,indicating that when the content of amantadine higher in the elimination,the elimination rate was faster.