CN111638348B - Unified extraction pretreatment kit for veterinary drug residues in animal-derived foods and application of unified extraction pretreatment kit - Google Patents
Unified extraction pretreatment kit for veterinary drug residues in animal-derived foods and application of unified extraction pretreatment kit Download PDFInfo
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Abstract
The invention discloses a pretreatment kit for unified extraction of veterinary drug residues in animal-derived foods and application thereof, wherein the kit comprises a reagent 1, a reagent 2 and a reagent 3, wherein the reagent 1 is 10g trichloroacetic acid and 100mL50% ethanol solution; reagent 2 is 10g magnesium sulfate, 10g sodium chloride and 200mL sodium acetate; the reagent 3 is 0.01mol/L phosphate buffer (PH=7-7.5), 3% ovalbumin and 0.25g/L stabilizer. The kit is reasonable in design, and can solve the problems of long time consumption, low efficiency and the like in the prior art when detecting a large number of samples. Meanwhile, the detection is carried out by using various veterinary drug residue immunodetection products, the method has the advantages of simplicity and rapidness in operation, high sensitivity, strong specificity and strong accuracy, can meet the requirement of detecting veterinary drug residues in duck meat, chicken meat and pork samples, and has good application prospects.
Description
Technical Field
The invention belongs to the field of rapid detection of drug residues, and relates to a pretreatment kit for unified extraction of veterinary drug residues in animal-derived foods and application thereof.
Background
With the improvement of living standard, animal-derived foods have a larger proportion in the diet structure of people in China, and people have a higher and higher safety awareness on foods and a higher requirement on food quality. As a pharmaceutical feed additive, the veterinary drug plays a positive role in preventing and treating livestock and poultry diseases, promoting the growth and development of livestock and poultry and improving the feed benefit. However, due to lack of scientific knowledge and driving of economic benefits, the phenomenon of animal drug abuse in the livestock and poultry raising process is common, and the direct result is that animal drug residues in animal-derived foods exceed standards, and meanwhile, some poisoning reactions, anaphylactic reactions, teratogenic reactions and the like may occur, which also affects the development of the breeding industry and the normal order of the market in the long term. Therefore, effective measures are taken to actively detect and monitor veterinary drug residues, and animal-derived foods with residues exceeding standards are prevented from flowing into the market, so that the method is urgent.
Currently, the detection method of veterinary drug residues mainly comprises high performance liquid chromatography, mass spectrometry, liquid chromatography-mass spectrometry (HPLC-MS) and other instrument methods, and the recent immunodetection methods developed on the basis of antigen-antibody specific recognition include immunochromatography, enzyme-linked immunosorbent assay (ELISA) and the like. Although the instrument method has high accuracy, the pretreatment is complex and the detection cost is high, the instrument method is not suitable for high-throughput rapid screening, and the ELISA method can detect the samples at high throughput and has low cost, but only can detect the pretreatment samples singly, and the operation is complex, the time consumption is long and the efficiency is low when a plurality of samples are processed. Therefore, the development of a practical pretreatment kit for the unified extraction of veterinary drug residues in the animal-derived food is of great significance.
Disclosure of Invention
The invention aims to provide a pretreatment kit for uniformly extracting veterinary drug residues in animal-derived foods and application thereof, discloses a reagent formula of a plurality of novel pretreatment kits, and provides a pretreatment kit which is simple to operate, short in time consumption and capable of simultaneously extracting a plurality of veterinary drugs.
In order to achieve the above object, the technical scheme of the present invention is as follows:
a pretreatment kit for unified extraction of veterinary drug residues in animal-derived foods comprises a reagent 1, a reagent 2 and a reagent 3. Reagent 1 is 10g trichloroacetic acid and 100mL50% ethanol solution;
reagent 2 is 10g magnesium sulfate, 10g sodium chloride and 200mL sodium acetate;
the reagent 3 is 0.01mol/L phosphate buffer (PH=7-7.5), 3% ovalbumin and 0.25g/L stabilizer.
The veterinary medicine is any one or more of sulfonamides, tetracyclines, tilmicosin, trimethoprim, erythromycin, lincomycin, florfenicol, quinolones, amantadine, azithromycin, rimantadine, carboxin metabolites, olaquindox metabolites, clenbuterol, ractopamine, salbutamol, terbutaline, metronidazole, ceftiofur metabolites, nicarbazin, dezuril, tylosin and kitasamycin.
The animal-derived food samples are duck, chicken and pork samples.
A unified extraction pretreatment method for veterinary drug residues in animal-derived foods comprises the following steps:
1) Taking a certain animal tissue to remove fat, and mincing with a meat mincer;
2) Placing a sample to be tested 3g into a centrifuge tube, sequentially adding a reagent 1, a reagent 2 and acetonitrile, and centrifuging after vortex to obtain a supernatant;
3) Adding the supernatant obtained in the step 2) into a 4mL centrifuge tube, drying by nitrogen, adding the reagent 3, and centrifuging after vortex;
4) Taking supernatant to be measured;
the reagent 1 is 10g trichloroacetic acid, 100mL50% ethanol solution
The reagent 2 is 10g magnesium sulfate, 10g sodium chloride and 200mL sodium acetate
The reagent 3 is 0.01mol/L phosphate buffer solution with pH of 7-7.5, 3% ovalbumin and 0.25g/L stabilizer.
The application of the pretreatment kit for uniformly extracting veterinary drug residues in animal-derived foods is that after a sample to be detected is treated by the pretreatment kit method, the sample is detected by each veterinary drug residue immunodetection product.
The veterinary drug residue immunodetection products are provided by Beijing WeideWikang biotechnology Co.
The veterinary drug residue immunodetection products comprise veterinary drug detection ELISA plates, veterinary drug monoclonal antibodies, target veterinary drug monoclonal antibodies marked by horseradish peroxidase (HRP), washing liquid, substrate color development liquid, stop liquid and veterinary drug standard working liquid.
The detection step of the veterinary drug residue immunity detection product comprises the steps of adding standard working solution or solution of the sample into an ELISA plate; adding the specific antibody of the target veterinary drug; after incubation, washing and beating to dryness, adding the enzyme-labeled secondary antibody, developing color by using TMB color development liquid, stopping and measuring the absorbance value by using an enzyme-labeled instrument.
The analysis process of the detection result provided by the invention comprises the following steps:
the absorbance average value (B) of the standard working solution at each concentration obtained was divided by the absorbance value (B0) of the first standard solution (0 standard) and multiplied by 100%, i.e., the percent absorbance value. The calculation formula is as follows: percent absorbance (%) = (B/B0) ×100%.
And drawing a standard curve graph by taking half-logarithmic value of concentration (mug/L) of each veterinary drug standard working solution as an X axis and the percentage absorbance value as a Y axis. The percentage absorbance value of the sample solution is calculated by the same method, and the content of each veterinary drug in the sample can be read from the standard curve corresponding to the concentration of each sample.
Experiments prove that the detection limit of the residual immunity detection products for the veterinary drugs, which are disclosed by the invention, for detecting sulfonamides, tetracyclines, tilmicosin, trimethoprim, erythromycin, lincomycin and florfenicol in tissue samples is 10 mug/kg; the detection limit of quinolones, amantadine, azithromycin, rimantadine and carbazedox metabolites is 1 mug/kg; the detection limit of the olaquindox metabolite, the clenbuterol, the ractopamine, the salbutamol, the terbutaline and the metronidazole is 0.5 mug/kg; the detection limit of ceftiofur metabolite, nicarbazin and diclazuril is 50 mug/kg; the detection limit of tylosin and kitasamycin is 20 mug/kg, the inter-batch variation coefficient is less than 10%, and the intra-batch variation coefficient is less than 15%.
The unified extraction pretreatment kit provided by the invention can extract a plurality of medicines simultaneously, and can solve the defects of long time consumption, low efficiency and the like in the detection of a large number of samples in the prior art; meanwhile, veterinary drug residues in duck meat, chicken meat and pork tissue samples can be detected by using corresponding matched veterinary drug residue immunodetection products, and the method has the advantages of high accuracy, strong specificity and the like, and provides scientific basis.
Drawings
FIGS. 1-23 are standard graphs of the detection of veterinary drug residues.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Example 1, specific method of operating pretreatment kit for duck, carnis gallus Domesticus, carnis Sus Domestica sample
Taking duck, chicken and pork samples, accurately weighing 3+/-0.05 and g, mincing with a meat mincer, sequentially adding 50 mu L of reagent 1, 0.7 and g of reagent 2 and 6 mL of acetonitrile, whirling for 5 min, and centrifuging for 5 min at 4000 g; taking 3 mL supernatant, drying with nitrogen, adding 2 mL n-hexane, fully swirling 30 s, adding 1 mL reagent 3, and swirling 30 s at low speed; 4000 And g, centrifuging for 5 min, and taking 50 mu L of supernatant for analysis.
Reagent 1 is 10g trichloroacetic acid, 100mL50% ethanol solution;
reagent 2 was 10g magnesium sulfate, 10g sodium chloride and 200mL sodium acetate;
the reagent 3 is 0.01mol/L phosphate buffer (PH=7-7.5), 3% ovalbumin and 0.25g/L stabilizer.
Example 2 use of the kit
1. The duck meat, chicken and pork samples treated by the unified extraction pretreatment kit can be simultaneously used for corresponding matched veterinary drug residue immunity detection products, and the detection steps are as follows:
1. the ELISA plate is inserted into the ELISA plate frame and marked, and each sample is parallel to 3 samples;
2. adding the veterinary drug standard solution and the sample to be detected into the plate holes of the enzyme-labeled plate from low to high in concentration, wherein each hole is 50 mu L;
3. adding 50 mu L of antibody working solution into each plate hole, gently shaking for 30 s, covering a sealing plate film, and incubating at 25 ℃ for 30 min;
4. after pouring out the liquid in the plate holes, adding 200 mu L of washing working solution into each hole, and washing for 3 times each time for 3 min; inverting the ELISA plate on the absorbent paper, and drying;
5. adding 100 mu L of enzyme marker working solution into each plate hole; covering the cover plate film, lightly oscillating the ELISA plate 10 s, fully mixing, and carrying out light-shielding reaction for 30 min at 25 ℃;
6. repeating the step 4;
7. immediately adding 100 mu L of substrate developing solution A, B mixed solution (the substrate developing solution A and the substrate developing solution B are mixed according to the volume of 1:1) into each hole, covering a cover plate film, and carrying out light-proof reaction for 15 min;
8. uncovering the cover plate film, adding 50 mu L of stop solution into each plate hole, gently oscillating the ELISA plate 10 s, and fully and uniformly mixing;
9. the absorbance of the microplate was read with a microplate reader at wavelength 450 nm within 5 minutes after termination.
2. Analyzing the detection result
1. Calculation of the percent absorbance values
The average absorbance value of each standard (or sample to be measured) is divided by the absorbance value of the zero standard (standard with the concentration of 0 mug/L), and the absorbance value is multiplied by 100%, so that the percentage of the absorbance corresponding to each standard (or sample to be measured), namely the percentage absorbance value, can be obtained.
Absorbance percentage = B/B 0 ×100%
Wherein: average absorbance value of B-standard (or sample); b (B) 0 -average absorbance value of a standard at a concentration of 0 ppb.
2. Production of standard curve
The competition standard curve was fitted with four parameters in the origin8.0 (originLab Corp., northampton, mass., USA) software, with the percentage absorbance values of the standards as ordinate and the concentration of each veterinary drug in each standard working fluid (μg/kg) as abscissa:
y=(A-D)/[1+(x/C)B]+D
the standard curve equation and the correlation coefficient of each veterinary drug are shown in table 1 through the test data.
The standard graph is shown in figures 1-23.
Table 1 standard curve equation and correlation coefficient for detecting veterinary drug residues
3. Determination of detection limits of veterinary drugs
Duck, chicken and pork blank samples (negative in LC-MS/MS detection) were taken, detected according to the method of example 2, measured values were obtained according to a standard curve, the average value was calculated, and the minimum detection Limit (LOD) was obtained by adding 3 times of standard deviation. The results are shown in tables 2 to 4.
Table 2 determination results (mug/kg) of veterinary drug residue detection blank samples in duck meat
Table 3 determination results (mug/kg) of veterinary drug residue detection blank samples in chicken
Table 4 determination results (mug/kg) of veterinary drug residue detection blank samples in pork
The results show that, to prevent false positives, the detection limit of sulfonamides, tetracyclines, tilmicosin, trimethoprim, erythromycin, lincomycin and florfenicol can be defined as 10 mug/kg; the detection limit of quinolones, amantadine, azithromycin, rimantadine, carbazedox metabolites can be defined as 1 μg/kg; the detection limit of the olaquindox metabolite, clenbuterol, ractopamine, salbutamol, terbutaline and metronidazole can be defined as 0.5 mug/kg; the detection limit of ceftiofur metabolite, nicarbazin and diclazuril can be defined as 50 mug/kg; the detection limit of tylosin and kitasamycin can be defined as 20 mug/kg
4. Accuracy and precision test of residue detection of each veterinary drug
Duck, chicken, pork blank samples (negative for LC-MS/MS detection) were pre-treated as described in example 1 to final concentrations of 1 LOQ and 2 LOQ, 3 replicates were added for each concentration, and the addition recovery was calculated using the average. Additive recovery (%) = detection value/average value x 100%
The above test was repeated 3 times in different working days, and the daily coefficient of variation and the daytime coefficient of variation were counted, and the results are shown in tables 5 to 7, respectively.
Table 5 accuracy and precision of detection of residues of various veterinary drugs in duck meat
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TABLE 6 accuracy and precision of detection of residues of veterinary drugs in Carnis gallus Domesticus
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TABLE 7 accuracy and precision of detection of residues of veterinary drugs in pork
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The results show that the recovery rate of each addition concentration of all samples is between 80 and 120 percent. The intra-batch variation coefficient of each additive concentration is lower than 10% and the inter-batch variation coefficient is lower than 15%.
Claims (4)
1. A unified extraction pretreatment kit for veterinary drug residues in animal-derived foods is characterized in that: comprises a reagent 1, a reagent 2 and a reagent 3, wherein the reagent 1 is 10g of trichloroacetic acid and 100mL of 50% ethanol solution; reagent 2 is 10g of magnesium sulfate, 10g of sodium chloride and 200mL of sodium acetate; the reagent 3 is 0.01mol/L phosphate buffer solution with pH of 7-7.5, 3% ovalbumin and 0.25g/L stabilizer; the veterinary medicine is any one or more of sulfonamides, tetracyclines, tilmicosin, trimethoprim, erythromycin, lincomycin, florfenicol, quinolones, amantadine, azithromycin, rimantadine, carboxin metabolites, olaquindox metabolites, clenbuterol, ractopamine, salbutamol, terbutaline, metronidazole, ceftiofur metabolites, nicarbazin, dezuril, tylosin and kitasamycin.
2. The unified extraction pretreatment kit for veterinary drug residues in animal-derived foods according to claim 1, wherein the kit comprises: the animal-derived food is duck meat, chicken meat and pork.
3. A unified extraction pretreatment method for veterinary drug residues in animal-derived foods comprises the following steps:
1) Taking a certain animal tissue to remove fat, and mincing with a meat mincer;
2) Placing 3g of a sample to be tested into a centrifuge tube, sequentially adding a reagent 1, a reagent 2 and acetonitrile, and centrifuging after vortex to obtain a supernatant;
3) Adding the supernatant obtained in the step 2) into a 4mL centrifuge tube, drying by nitrogen, adding the reagent 3, and centrifuging after vortex;
4) Taking supernatant to be measured;
the reagent 1 is: 10g of trichloroacetic acid, 100mL of 50% ethanol solution;
the reagent 2 is as follows: 10g of magnesium sulfate, 10g of sodium chloride and 200mL of sodium acetate;
the reagent 3 is as follows: 0.01mol/L phosphate buffer solution with pH7-7.5, 3% ovalbumin and 0.25g/L stabilizer.
4. The application of the unified extraction pretreatment kit for veterinary drug residues in animal-derived foods is characterized in that after a sample to be detected is treated by the unified extraction pretreatment kit according to claims 1-2, the sample is detected by each veterinary drug residue immunodetection product.
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