Preparation Of Matrix Standard Samples Of Four Sulfonamides Residues In Grass Carp Powder

Sulfonamides（SAs）are synthetic antibiotics with p-aminobenzene sulfonamide as the basic structure.They are widely used in aquaculture to treat diseases because of their significant broad-spectrum antibacterial effect.In recent years,environmental pollution and food safety caused by sulfonamides residues have attracted more and more attention.Sulfonamides are the key items of quality and safety monitoring of aquatic products.According to the notice on supervision and sampling inspection of aquatic products issued by the Ministry of agriculture and rural areas and the State Administration of market supervision,sulfamethoxazole（SMZ）,sulfaisoxazole（SFZ）,sulfamethazine（SM₂）and sulfamethazine（SM₁）in aquatic products are still detected,Accurate determination of sulfonamides residues in aquatic products is the key to ensure the quality and safety of aquatic products.The standard sample is a tool for quality control in the detection and analysis of drug residues in the laboratory.It can be used to calibrate the detection instrument,effectively eliminate the interference of the matrix effect of aquatic products on the detection results in the analysis experiment,and improve the accuracy and traceability of the detection of drug residues in aquatic products.In the national standard sample management platform,the results of standard samples containing a variety of sulfonamides residues prepared with aquatic products as the matrix are limited,which is difficult to meet the needs of practical application.Based on this,in order to ensure the accuracy,reliability and traceability of the monitoring results of sulfonamides in aquatic products,it is necessary to develop corresponding standard samples.Taking grass carp as the research object,this study studied the preparation method of matrix standard samples containing SMZ,SFZ,SM₂ and SM₁.The research content mainly includes the following three parts:1.By optimizing the instrument detection conditions,sample extraction and purification conditions,a high accuracy determination method of four sulfonamides residues in grass carp powder,liquid chromatography tandem mass spectrometry,was established.The grass carp powder sample was extracted by ultrasonic with 0.1%formic acid acetonitrile.After freezing and centrifugation,the supernatant was evaporated and concentrated.After re dissolution,the sample was purified with 25 mg C₁₈ and 25 mg PSA.C₁₈ chromatographic column was used for separation,5.0 mmol/L ammonium acetate aqueous solution（containing 0.1%formic acid）and methanol were used as mobile phase,gradient elution was carried out for four sulfonamides,and internal standard method was used for quantification.The results showed that SMZ,SFZ,SM₂ and SM₁were in the range of 10～200 ng/ml,and the correlation coefficients were 0.9981、0.9989、0.9999 and 0.9999,respectively.The detection limit of SMZ,SFZ,SM₂ and SM₁ methods were all 0.5μg/kg and the quantitative limits were 1.0,1.5,1.0 and 1.0μg/kg,respectively.When the standard addition level in blank grass carp powder is 10,20 and 50μg/kg,the recoveries of SMZ,SFZ,SM₂ and SM₁ were 92.90%～102.84%,and the relative standard deviation was 1.70%～9.83%.The method has high sensitivity and reliable results.The precision of the method can meet the detection requirements of SMZ,SFZ,SM₂ and SM₁in grass carp powder samples,which lays a method foundation for the detection of SMZ,SFZ,SM₂ and SM₁ in matrix standard samples in grass carp powder in the later stage.2.Through the medicine bath experiment of grass carp in the culture pond,the culture conditions required to obtain positive samples of grass carp were studied,and the relationship between the residue of sulfonamides in grass carp muscle and the initial medicine bath concentration was established.According to the acquisition principle of natural pollution,the relationship between the residues of SMZ,SFZ,SM₂ and SM₁ in grass carp muscle and the water temperature of breeding environment,different medicine bath time and individual size was studied.The results showed that the absorption and metabolism levels of SMZ,SFZ,SM₂ and SM₁ in grass carp were significantly lower than those at（23±2）℃.When the temperature was（23±2）℃,grass carp was in good condition,and the absorption levels of SMZ,SFZ,SM₂ and SM₁ showed a slow upward trend.Finally,the breeding conditions of positive samples were determined as follows:the water temperature was（23±2）℃,the medicine bath time was 10 h,and the fish body specification was（1000±100）g.The initial drug bath concentrations x₁ of SMZ,SFZ,SM₂ and SM₁ were 2.72、3.75、1.05、2.49 mg/L.after 10 hours of drug bath,the content y₁ in grass carp muscle was 12.59、17.45、16.88、17.22μg/kg,respectively.Therefore,the relationship between the content y of SMZ,SFZ,SM₂ and SM₁ in grass carp muscle and the initial drug bath concentration x was determined as y=4.63 x;y=4.65 x;y=16.08 x;y=6.92 x,respectively.The expected target content y₂ in grass carp muscle is 5μg/kg,and the initial drug bath concentration x₂ is 1.08、1.08、0.31 and 0.72 mg/L,respectively.The relationship between Y and X is verified by drug bath for 6 times.The test results showed that the P value of single sample t-test of the content test value of four sulfonamides was greater than 0.05,indicating that there was no significant difference between the content test value and the expected target content,and the feasibility of the relationship was verified.3.By setting the expected target content,a batch of matrix standard samples of SMZ,SFZ,SM₂ and SM₁ drug residues in grass carp powder were developed.According to the determined initial drug bath concentration,the grass carp was given a drug bath.After homogenization and mixing,freeze-drying,crushing and screening,secondary mixing and sub packaging,the candidate of grass carp powder matrix standard sample was obtained.Liquid chromatography tandem mass spectrometry was used to detect the standard sample of grass carp powder matrix.The uniformity of standard samples was tested by sampling,and the statistical results of uniformity were analyzed by one-way variance test.The stability of grass carp powder samples was judged by t-test.The stability of SMZ,SFZ,SM₂ and SM₁ in the samples was tested by simulating the sample transportation environment;Taking﹣20℃as the long-term storage temperature of SMZ,SFZ,SM₂ and SM₁ samples,the long-term stability of the samples was investigated to evaluate the validity period of the matrix standard samples of grass fish meal drug residues.The characteristic quantity of grass carp powder matrix standard sample was determined by joint setting,and the setting results were completed together with 6qualified laboratories.The results show that the uniformity test results of the prepared grass carp powder standard sample are good,the stability is good after being stored at-20℃for 6 months,and the quantity value is still stable and reliable after being stored at50℃for 15 days.The samples of SMZ,SFZ,SM₂ and SM₁ can be transported at room temperature.This preparation method can provide a technical reference for the preparation of sulfonamides residue matrix standard samples in aquatic products matrix.Finally,the matrix standard samples of four sulfonamides residues in grass carp powder were quasi determined and their uncertainty was evaluated.The quantitative results of SMZ,SFZ,SM₂ and SM₁ in grass carp powder were 22.22±7.75、24.04±8.40、31.11±5.84、28.55±7.18μg/kg,respectively.