Application Of DTPA-derivatives-Eu Complexes In The Analysis And Dection Of The Food Additives

In recent years,food safety problems have occurred frequently in various parts of China.For example,“clenbuterol”poisoning,“Instant chicken”with antibiotics and hormones,melamine milk powder,sudan red egg yolk,olaquindox excessive in aquatic products,sulfadimidine excessive in poultry,yellow croaker and bean curd skin dyed with basic orange and so on.These events have brought great harm to people’s health.Long-term accumulation of melamine（Mel）can cause kidney stones,eventually lead to kidney failure.Long-term accumulation of clenbuterol（Clb）can lead to cardio-cerebrovascular disease and nervous system diseases.Basic orange（BO）can cause chronic poisoning,eventually cancer.Sulfamethylamine（SMZ）excessive will lead to abnormal reactions and allergic reactions,carcinogenic,teratogenic,mutagenic,hormone like effects.These food safety problems highlight the current situation of agricultural and veterinary drug abuse and illegal additives,lack of food market supervision and other problems.So,it is urgent to find an effective test method to supervise food safety.At present,there are many methods for detecting these additives,such as high performance liquid chromatography,gas chromatography,GC-MS,enzyme immunoassay,molecular imprinting,fluorescence spectroscopy,etc.But most of the methods require expensive instruments,high cost and complex operation.It is not suitable for online detection.So,it is urgent to find a simple,fast,sensitive and selective method to detect these food additives.According to the literature,amino polycarboxylic acid compound is a classic chelating agent.It has good coordination ability,which can form stable complex with almost all metal ions.The complex formed with certainlanthanide metal ions also has a long fluorescence life and can emit stable characteristic fluorescence.Besides,the bases on the DNA/RNA single strand are arranged in a certain sequence,such as-GTAACGG-.According to the principle of base complementary pairing,A（adenine）is paired with T（thymine）,G（guanine）is paired with C（cytosine）,and A（adenine）is paired with U（uracil）,to form DNA/RNA double helix structure according to a certain sequence.In view of the above two points,we take the amino polycarboxylic acid compound as the ligand.According to the chemical properties and structural composition of food additives,refer to DNA/RNA single chain sequencing rules,base or base analog which can recognize the additives were selected to modify ligands.The ligands were reacted with metal ions to synthesize a new type of rare earth amino polycarboxylic acid europium complex,as fluorescent probe,for the detection of food additives.In this paper,we synthesized four kinds of complex fluorescent probes,Eu^Ⅲ-dtpa-bis（melamine）,Eu^Ⅲ-dtpa-bis（3,5-dicchloroaniline）(Eu^Ⅲ-dtpa-bis（DCA）),Eu^Ⅲ-dtpa-bis（cytosine）and Eu^Ⅲ-dtpa-bis（adenine）,respectively.The feasibility of these complex fluorescent probes for detecting food additives,such as melamine（Mel）,clenbuterol（Clb）,basic orange（BO）and sulfadimidine（SMZ）were studied by fluorescence spectrophotometry and UV-vis spectrophotometry,according to their special structures and properties,the proportion of combination and function mechanism were also speculated.The findings are as follows:1)The complex Eu^Ⅲ-dtpa-bis（melamine）has better specificity in the detection of melamine,the presence of analogues such as cyanuric acid（Cya）,cyanuric chloride（Cyach）,trichloroisocyanuric acid（Isocya）and acetoguanamine（Methyla）,did not affect the detection of melamine.In the range of 5-100μmol/L,the concentration of melamine was linearly related to its corresponding fluorescence intensity.The linear equation was y=38.68x+568.32（R²=0.9972）,the detection limit（LOD）was 0.3743μmol/L（LOD=3σ/s）.When C^[Mel]=C^{[EuIII-dtpa-bis（melamine）]}=100μmol/L,the fluorescence intensity of the complex Eu^Ⅲ-dtpa-bis（melamine）was maximized and remained unchanged.It indicated that the ratio of Eu^Ⅲ-dtpa-bis（melamine）and melamine（Mel）is 1:1.The recovery of melamine in actual samples milk,yogurt and milk powder were between 99.8%-100.5%by standard addition method,the detection results were satisfactory.2)The complex Eu ^Ⅲ-dtpa-bis（DCA）has better specificity for detecting clenbuterol,the presence of co-existence,such as Urea（U）,Glucose（G）,Hippuric Acid（Ha）,L-phenylalanine（Pha）and Uric acid（Ua）does not affect the detection of clenbuterol.In the range of 5-300μmol/L,F₀/F(F₀ is the fluorescence strength of the complex Eu^Ⅲ-dtpa-bis（DCA）,F is the fluorescence strength of the complex in the presence of clenbuterol)and the concentration of clenbuterol(C_[Clb])showed a good linear relationship,the linear equation is y=0.0558x+1.0157（R²=0.9984）,the detection limit（LOD）was 0.3369μmol/L（LOD=3σ/s）.When C^[Clb]=3C^{[EuIII-dtpa-bis（DCA）]}=300μmol/L,the fluorescence intensity of the complex Eu^Ⅲ-dtpa-bis（DCA）was minimized and remained unchanged.It indicated that the ratio of Eu^Ⅲ-dtpa-bis（DCA）and clenbuterol（Clb）is 1:3.The recovery of clenbuterol in the actual sample urine was between 90.8%-96.2%by standard addition method,the detection results were satisfactory.3)The complex Eu^Ⅲ-dtpa-bis（cytosine）as a fluorescent probe,has good specificity in the detection of basic orange,the presence of basic orange co-existence,such as Glucose（G）,L-phenylalanine（Pha）,Histidine（His）,Ascorbic acid（Aa）and Creatinine（Cre）had little effect on fluorescence intensity,which does not affect the detection of basic orange.In the range of 5-100μmol/L,the concentration of basic orange and its corresponding fluorescence intensity showed a good relationship,and the linear equation was y=-32.3406x+561.9674（R²=0.9946）,the detection limit（LOD=3σ/s）was 0.1291μmol/L.When C^[BO]=3C^{[EuIII-dtpa-bis（cytosine）]}=300μmol/L,the fluorescence intensity was close to the minimum and remained unchanged.It indicated that the ratio of Eu^Ⅲ-dtpa-bis（cytosine）and basic orange（BO）is 1:3.The recovery of basic orange in the actual soybean milk film was between 80.8%-87.3%by standard addition method,and the detection results were satisfactory.4)The complex Eu^Ⅲ-dtpa-bis（adenine）as a fluorescent probe,has good specificity in the detection of sulfamethylamine,the presence of the co-existence,such as Glucose（G）,L-phenylalanine（Pha）,Histidine（His）,Ascorbic acid（Aa）and Glycine（Gly）did not affect sulfamethylamine detection.In the range of 5-100μmol/L,F₀/F(where F₀ is the fluorescence intensity of the probe Eu^Ⅲ-dtpa-bis（adenine）,F is the fluorescence intensity of Eu^Ⅲ-dtpa-bis（adenine）in the presence of sulfamethylamine)and the concentration of sulfamethylamine(C_[SMZ])shows a good linear relationship.The linear equation is y=0.2938x+0.8562（R²=0.9923）,the detection limit（LOD）was 0.4527×10^-77 mol/L（LOD=3σ/s）.When C^[SMZ]=C^{[EuIII-dtpa-bis（adenine）]}=1.00×10^-44 mol/L,the fluorescence intensity was close to the minimum and remained unchanged,which indicated that the complex ratio was 1:1.The recovery of sulfonamethylamine in honey was between 87.2%-92.5%by standard addition method,the detection results were satisfactory.