CN113252416A - Method for extracting sulfonamides from pork - Google Patents
Method for extracting sulfonamides from pork Download PDFInfo
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- CN113252416A CN113252416A CN202110504365.8A CN202110504365A CN113252416A CN 113252416 A CN113252416 A CN 113252416A CN 202110504365 A CN202110504365 A CN 202110504365A CN 113252416 A CN113252416 A CN 113252416A
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- C07D237/02—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings
- C07D237/06—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
- C07D237/10—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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- C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
- C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
- C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
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- C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
- C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
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- C07D261/10—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D261/14—Nitrogen atoms
- C07D261/16—Benzene-sulfonamido isoxazoles
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- C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D277/38—Nitrogen atoms
- C07D277/50—Nitrogen atoms bound to hetero atoms
- C07D277/52—Nitrogen atoms bound to hetero atoms to sulfur atoms, e.g. sulfonamides
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- C07D285/04—Thiadiazoles; Hydrogenated thiadiazoles not condensed with other rings
- C07D285/12—1,3,4-Thiadiazoles; Hydrogenated 1,3,4-thiadiazoles
- C07D285/125—1,3,4-Thiadiazoles; Hydrogenated 1,3,4-thiadiazoles with oxygen, sulfur or nitrogen atoms, directly attached to ring carbon atoms, the nitrogen atoms not forming part of a nitro radical
- C07D285/135—Nitrogen atoms
Abstract
The invention discloses a method for extracting sulfonamides from pork, which comprises the following steps: step one, homogenizing and uniformly mixing pork injected with sulfonamides by a tissue triturator, filling the mixture into a clean container, sealing and marking; step two, freezing and storing the sample at the temperature of 18-20 ℃ below zero to obtain a homogeneous sample for later use; and step three, weighing 4-5 g of the homogeneous sample in a beaker, adding 20-25 mL of phosphate buffer solution, performing ultrasonic centrifugation, and taking the supernatant to perform content determination on the sulfonamide. The invention provides a method for extracting sulfonamide residues in pork, which has the advantages of high sensitivity, good precision and high accuracy; compared with a homogeneous extraction method and an oscillation extraction method, the method has the advantages of simple and convenient operation and high extraction efficiency, and can effectively avoid the experimental results of false positive and false negative; through ultrasonic treatment of the pork sample, pork cells can be fully broken, and intracellular sulfonamides are released and dissolved in the extracting solution, so that the aim of fully extracting is fulfilled.
Description
Technical Field
The invention belongs to a sample extraction method, and particularly relates to a method for extracting sulfonamides from pork.
Background
Sulfanilamide medicines are artificially synthesized medicines with sulfanilamide as a basic structure. Has the characteristics of wide antimicrobial spectrum, stable drug effect, low price and the like, can be used for preventing and treating animal diseases and promoting the growth of animals, and has wide application in the fields of animal husbandry and the like. Improper use of the medicine can cause high-concentration medicine residue in animal muscle, milk and egg, and harm to human health.
In order to ensure human health, the residual limit of sulfonamides in animal food has been strictly regulated in many countries and organizations. For example, the European Union specifies a maximum residual limit of 100 μ g/kg of sulfonamides in foods of animal origin. The method is specified in GB 31650 plus 2019 (maximum residual limit of veterinary drugs in national food safety standards) in China, the residual quantity of the sulfonamides is counted by the sum of prototype veterinary drugs, the maximum residual limit of muscles (all food animals) is 100 mu g/kg, and the daily allowable intake is 0-50 mu g/(kg bw d).
Food safety supervision is an important ring in ensuring food safety, and a feasible supervision technology cannot be separated from a high-efficiency and accurate detection technology. At present, enzyme-linked immunosorbent assay is mainly selected for on-site monitoring and rapid screening of a large number of samples. The pork sample has complex matrix and more interference substances, and is not easy to separate and extract sulfonamide residues from the sample. Therefore, the sample extraction method is an important part for ensuring the accuracy of the detection result. The extraction method in the existing standard adopts a homogeneous extraction method and an oscillation extraction method, so that the extraction efficiency is low, and false positive and false negative experimental results are easy to appear.
Disclosure of Invention
The purpose of the invention is as follows: in order to overcome the defects in the prior art, the invention aims to provide a simple, convenient and efficient method for extracting sulfonamides from pork.
The technical scheme is as follows: the invention relates to a method for extracting sulfonamides from pork, which comprises the following steps:
step one, sample preparation: homogenizing and mixing pork with tissue mashing machine, placing into a clean container, sealing and marking;
step two, sample preservation: freezing and storing the sample at-18-20 ℃ to obtain a homogeneous sample for later use;
step three, extraction: weighing 4-5 g of a homogeneous sample in a beaker, adding 20-25 mL of phosphate buffer solution, performing ultrasonic centrifugation, and taking supernatant to perform sulfonamide content determination.
Further, in the first step, 400-600 g of pork is taken. The rotating speed of the tissue triturator is 8000-12000 r/min, and the time for homogenizing and mixing is 30-60 s. The pork is divided into two parts, one part is used for subsequent detection, and the other part is sealed and stored for recheck.
Further, in the second step, the freezing time is not more than 72 h. The freezing time exceeds 72h, and the sulfanilamide medicine can be degraded. The sample should be protected from contamination or from changes in the residue content during operation.
Further, in the third step, the concentration of the phosphate buffer solution is 0.05-0.15 mol/L; the concentration of the phosphate buffer solution is lower than 0.05mol/L, so that the sulfonamides are not fully extracted; phosphate buffer concentrations above 0.15mol/L increase background interference during detection. The pH value of the phosphate buffer solution is 7.2-7.4; the pH value of the phosphate buffer solution is lower than 7.2, and the sulfonamides are not fully extracted; the pH of the phosphate buffer is higher than 7.4, and the sulfonamides are not fully dissolved in the extracting solution. The phosphate buffer solution comprises 1.4-4.2 parts of sodium dihydrogen phosphate, 15.4-46.2 parts of disodium hydrogen phosphate, 4.25-12.75 parts of sodium chloride and 970-990 parts of water. Compared with organic reagents such as acetonitrile and the like, the phosphate buffer solution has the advantage of environmental friendliness.
Furthermore, in the third step, the power of the ultrasound is 150-250W, the time is 8-12 min, and the temperature is 15-25 ℃. The ultrasonic power is lower than 150W, the ultrasonic effect is not obvious, and the extraction of the sulfonamides is insufficient; the power of the ultrasonic is higher than 250W, the extracting solution and the sample matrix are not easy to separate, and the extracting solution cannot be completely recovered, so that errors are caused. When the temperature is lower than 15 ℃, the sulfanilamide medicines can not be fully dissolved in the extracting solution; temperatures above 25 ℃ may cause decomposition of the sulfonamide. The rotating speed of the centrifugation is 3000-4000 r/min, and the time is 10-15 min. The centrifugal rotating speed is lower than 3000r/min, so that the sample matrix and the extracting solution are not completely separated; the centrifugal rotating speed is higher than 4000r/min, which causes energy waste.
Further, the sulfanilamide drug is one or more of sulfamethoxazole, sulfathiazole, sulfamethoxypyrimidine, sulfamethoxyzine, sulfapyridine, sulfamethyldiazole and sulfachlorpyridazine.
The working principle is as follows: based on cavitation, mechanical effect and thermal effect of ultrasonic treatment, release, diffusion and dissolution of intracellular effective substances are accelerated, and trace sulfonamide residues in pork are effectively extracted by combining the dissolution effect of the extracting solution.
Has the advantages that: compared with the prior art, the invention has the following remarkable characteristics:
1. the method for extracting sulfonamide residues in pork is high in sensitivity, good in precision and high in accuracy; compared with a homogeneous extraction method and an oscillation extraction method specified in the existing standard, the method is simple and convenient to operate and high in extraction efficiency, and can effectively avoid false positive and false negative experimental results;
2. through ultrasonic treatment of a pork sample, pork cells can be fully crushed, and intracellular sulfonamides are released and dissolved in an extracting solution, so that the aim of fully extracting the sulfonamides is fulfilled;
3. the extracting solution used in the invention is phosphate buffer solution, and has the characteristic of environmental friendliness compared with organic reagent extracting solutions such as acetonitrile and the like in the prior art.
Drawings
FIG. 1 is a standard curve diagram of ELISA for sulfonamides according to the present invention.
Detailed Description
The raw materials and the apparatus used in the following examples were purchased and used as received. The pork is commercially available pork. The sulfonamide medicine is one or more of sulfamethoxazole, sulfathiazole, sulfamethoxydiazine, sulfamethoxyzine, sulfapyridine, sulfamethizole and sulfachlorpyridazine.
Example 1
A method for extracting sulfonamides from pork comprises the following steps:
(a) sample preparation:
injecting sulfanilamide medicine into 400g pork to make its final concentration 50 μ g/Kg, standing for 12 hr, homogenizing and mixing for 60s with tissue triturator at rotation speed of 8000r/min, dividing into two parts, placing into clean container, sealing and marking;
(b) and (3) sample preservation:
freezing and storing the sample at-18 ℃ for 4h to obtain a homogeneous sample for later use, wherein the sample is prevented from being polluted or the content of residues is prevented from changing in the operation process;
(c) extraction:
weighing 4.00g of a homogeneous sample, placing the homogeneous sample in a beaker, adding 20mL of 0.05mol/L phosphate buffer solution with the pH of 7.2, wherein the phosphate buffer solution comprises 1.4 parts of sodium dihydrogen phosphate, 15.4 parts of disodium hydrogen phosphate, 4.25 parts of sodium chloride and 990 parts of water, uniformly mixing by vortex, carrying out ultrasonic treatment at 150W and 15 ℃ for 8min, centrifuging at 3000r/min for 15min, and then taking the supernatant for measuring the content of the sulfonamide.
And (3) determination:
sequentially adding 50 mu L of a sample or a standard solution into corresponding micropores of a sulfonamide enzyme-linked immunosorbent assay kit, adding 50 mu L of sulfonamide antibody enzyme conjugate into all the micropores, gently mixing, and incubating for 30min at 37 ℃. Spin-drying the liquid in the wells, and washing the microplate 5 times with washing solution. 50 mul of color developing solution A, B was added to each well, the microplate was shaken slightly to mix well, and incubated at 37 ℃ for 10min in the dark. Add 50. mu.L of stop solution to each well, mix well, and measure absorbance at 450nm wavelength. And calculating the content of the sulfonamides in the sample extracting solution according to the absorbance by using a standard curve.
The results show that, as shown in FIG. 1, in the concentration range of 0-500. mu.g/kg, the standard curve equation is: y 44.29x +19.22, R2The inhibition rate of the sulfonamides and the logarithm of the concentration form a good linear relation, and the requirement of detection can be met.
Example 2
A method for extracting sulfonamides from pork comprises the following steps:
(a) sample preparation:
injecting sulfanilamide medicine into 600g pork to make its final concentration 50 μ g/Kg, standing for 12 hr, homogenizing and mixing uniformly for 30s with tissue triturator at 12000r/min, dividing into two parts, placing into clean container, sealing and marking;
(b) and (3) sample preservation:
freezing and storing the sample at-20 ℃ for 72h to obtain a homogeneous sample for later use, wherein the sample is prevented from being polluted or the content of residues is prevented from changing in the operation process;
(c) extraction:
weighing 5.00g of a homogeneous sample, placing the homogeneous sample in a beaker, adding 25mL of 0.15mol/L phosphate buffer solution with the pH of 7.4, wherein the phosphate buffer solution comprises 4.2 parts of sodium dihydrogen phosphate, 46.2 parts of disodium hydrogen phosphate, 12.75 parts of sodium chloride and 970 parts of water, uniformly mixing by vortex, carrying out ultrasonic treatment at 250W and 25 ℃ for 12min, centrifuging at 4000r/min for 10min, and taking the supernatant for measuring the content of the sulfonamide.
And (3) determination:
sequentially adding 50 mu L of a sample or a standard solution into corresponding micropores of a sulfonamide enzyme-linked immunosorbent assay kit, adding 50 mu L of sulfonamide antibody enzyme conjugate into all the micropores, gently mixing, and incubating for 30min at 37 ℃. Spin-drying the liquid in the wells, and washing the microplate 5 times with washing solution. 50 mul of color developing solution A, B was added to each well, the microplate was shaken slightly to mix well, and incubated at 37 ℃ for 10min in the dark. Add 50. mu.L of stop solution to each well, mix well, and measure absorbance at 450nm wavelength. And calculating the content of the sulfonamides in the sample extracting solution according to the absorbance by using a standard curve.
Example 3
A method for extracting sulfonamides from pork comprises the following steps:
(a) sample preparation:
injecting sulfanilamide medicine into 500g pork to make its final concentration 50 μ g/Kg, standing for 12 hr, homogenizing and mixing for 45s with tissue triturator with rotation speed of 10000r/min, dividing into two parts, placing into clean container, sealing and marking;
(b) and (3) sample preservation:
freezing and storing the sample at-19 ℃ for 36h to obtain a homogeneous sample for later use, wherein the sample is prevented from being polluted or the content of residues is prevented from changing in the operation process;
(c) extraction:
weighing 4.50g of a homogeneous sample, placing the homogeneous sample in a beaker, adding 23mL of 0.10mol/L phosphate buffer solution with the pH of 7.3, wherein the phosphate buffer solution comprises 2.8 parts of sodium dihydrogen phosphate, 30.8 parts of disodium hydrogen phosphate, 8.50 parts of sodium chloride and 980 parts of water, uniformly mixing by vortex, carrying out ultrasonic treatment at 200W and 20 ℃ for 10min, centrifuging at 3500r/min for 13min, and taking the supernatant for measuring the content of the sulfonamide.
And (3) determination:
sequentially adding 50 mu L of a sample or a standard solution into corresponding micropores of a sulfonamide enzyme-linked immunosorbent assay kit, adding 50 mu L of sulfonamide antibody enzyme conjugate into all the micropores, gently mixing, and incubating for 30min at 37 ℃. Spin-drying the liquid in the wells, and washing the microplate 5 times with washing solution. 50 mul of color developing solution A, B was added to each well, the microplate was shaken slightly to mix well, and incubated at 37 ℃ for 10min in the dark. Add 50. mu.L of stop solution to each well, mix well, and measure absorbance at 450nm wavelength. And calculating the content of the sulfonamides in the sample extracting solution according to the absorbance by using a standard curve.
Example 4
A method for extracting sulfonamides from pork comprises the following steps:
(a) sample preparation:
injecting sulfanilamide medicine into 450g pork to make its final concentration 50 μ g/Kg, standing for 12h, homogenizing and mixing for 52s with tissue triturator with rotation speed 9000r/min, dividing into two parts, placing into clean container, sealing and marking;
(b) and (3) sample preservation:
freezing and storing the sample at-18 ℃ for 10h to obtain a homogeneous sample for later use, wherein the sample is prevented from being polluted or the content of residues is prevented from changing in the operation process;
(c) extraction:
weighing 4.20g of a homogeneous sample, adding 21mL of 0.07mol/L phosphate buffer solution with the pH of 7.2, wherein the phosphate buffer solution comprises 1.96 parts of sodium dihydrogen phosphate, 21.56 parts of disodium hydrogen phosphate, 5.95 parts of sodium chloride and 974 parts of water, uniformly mixing by vortex, carrying out ultrasonic treatment at 170W and 18 ℃ for 9min, centrifuging at 3200r/min for 14min, and taking the supernatant for measuring the content of the sulfonamide.
And (3) determination:
sequentially adding 50 mu L of a sample or a standard solution into corresponding micropores of a sulfonamide enzyme-linked immunosorbent assay kit, adding 50 mu L of sulfonamide antibody enzyme conjugate into all the micropores, gently mixing, and incubating for 30min at 37 ℃. Spin-drying the liquid in the wells, and washing the microplate 5 times with washing solution. 50 mul of color developing solution A, B was added to each well, the microplate was shaken slightly to mix well, and incubated at 37 ℃ for 10min in the dark. Add 50. mu.L of stop solution to each well, mix well, and measure absorbance at 450nm wavelength. And calculating the content of the sulfonamides in the sample extracting solution according to the absorbance by using a standard curve.
Example 5
A method for extracting sulfonamides from pork comprises the following steps:
(a) sample preparation:
injecting sulfanilamide medicine into 550g pork to make its final concentration 50 μ g/Kg, standing for 12 hr, homogenizing and mixing for 38s with tissue triturator at rotation speed of 11000r/min, dividing into two parts, placing into clean container, sealing and marking;
(b) and (3) sample preservation:
freezing and storing the sample at-20 ℃ for 60h to obtain a homogeneous sample for later use, wherein the sample is prevented from being polluted or the content of residues is prevented from changing in the operation process;
(c) extraction:
weighing 4.80g of a homogeneous sample, adding 24mL of 0.12mol/L phosphate buffer solution with the pH of 7.4 into a beaker, uniformly mixing the mixture by vortex, performing ultrasonic treatment at 230W and 23 ℃ for 11min, centrifuging at 3900r/min for 11min, and taking the supernatant for measuring the content of the sulfonamide, wherein the phosphate buffer solution comprises 3.36 parts of sodium dihydrogen phosphate, 36.96 parts of disodium hydrogen phosphate, 10.2 parts of sodium chloride and 984 parts of water.
And (3) determination:
sequentially adding 50 mu L of a sample or a standard solution into corresponding micropores of a sulfonamide enzyme-linked immunosorbent assay kit, adding 50 mu L of sulfonamide antibody enzyme conjugate into all the micropores, gently mixing, and incubating for 30min at 37 ℃. Spin-drying the liquid in the wells, and washing the microplate 5 times with washing solution. 50 mul of color developing solution A, B was added to each well, the microplate was shaken slightly to mix well, and incubated at 37 ℃ for 10min in the dark. Add 50. mu.L of stop solution to each well, mix well, and measure absorbance at 450nm wavelength. And calculating the content of the sulfonamides in the sample extracting solution according to the absorbance by using a standard curve.
Example 6
A method for extracting sulfonamides from pork comprises the following steps:
(a) sample preparation:
injecting sulfanilamide medicines into 500g pork to make the final concentration of the sulfanilamide medicines be 1 microgram/Kg, 50 microgram/Kg, 100 microgram/Kg and 150 microgram/Kg, and standing for 12 hours for measuring the accuracy; taking 500g of blank pork for sensitivity measurement; another 500g pork was used for precision determination. Homogenizing and mixing uniformly for 52s with a tissue masher with a rotation speed of 9000r/min, dividing into two parts, putting into a clean container, sealing and marking;
(b) and (3) sample preservation:
freezing and storing the sample at-18 ℃ for 6h to obtain a homogeneous sample for later use, wherein the sample is prevented from being polluted or the content of residues is prevented from changing in the operation process;
(c) extraction:
weighing 4.00g of a homogeneous sample, placing the homogeneous sample in a beaker, adding 20mL of 0.10mol/L phosphate buffer solution with the pH of 7.4, wherein the phosphate buffer solution comprises 2.8 parts of sodium dihydrogen phosphate, 30.8 parts of disodium hydrogen phosphate, 8.5 parts of sodium chloride and 980 parts of water, uniformly mixing by vortex, carrying out ultrasonic treatment at 150W and 20 ℃ for 10min, centrifuging at 4000r/min for 10min, and taking the supernatant for measuring the content of the sulfonamide.
And (3) determination:
sequentially adding 50 mu L of a sample or a standard solution into corresponding micropores of a sulfonamide enzyme-linked immunosorbent assay kit, adding 50 mu L of sulfonamide antibody enzyme conjugate into all the micropores, gently mixing, and incubating for 30min at 37 ℃. Spin-drying the liquid in the wells, and washing the microplate 5 times with washing solution. 50 mul of color developing solution A, B was added to each well, the microplate was shaken slightly to mix well, and incubated at 37 ℃ for 10min in the dark. Add 50. mu.L of stop solution to each well, mix well, and measure absorbance at 450nm wavelength. And calculating the content of the sulfonamides in the sample extracting solution according to the absorbance by using a standard curve.
Comparative example 1
A method for extracting sulfonamides from pork comprises the following steps:
(a) sample preparation:
injecting sulfanilamide medicine into 500g pork to make its final concentration be 50 μ g/kg, standing for 12 hr, homogenizing and mixing uniformly for 45s with tissue triturator with rotation speed of 10000r/min, dividing into two parts, placing into clean container, sealing and marking;
(b) and (3) sample preservation:
freezing and storing the sample at-19 ℃ for 36h to obtain a homogeneous sample for later use, wherein the sample is prevented from being polluted or the content of residues is prevented from changing in the operation process;
(c) extraction:
weighing 4.50g of a homogeneous sample in a beaker, adding 23mL of acetonitrile, uniformly mixing by vortex, carrying out ultrasonic treatment at 200W and 20 ℃ for 10min, centrifuging at 3500r/min for 13min, and taking the supernatant for measuring the content of the sulfonamide.
And (3) determination:
sequentially adding 50 mu L of a sample or a standard solution into corresponding micropores of a sulfonamide enzyme-linked immunosorbent assay kit, adding 50 mu L of sulfonamide antibody enzyme conjugate into all the micropores, gently mixing, and incubating for 30min at 37 ℃. Spin-drying the liquid in the wells, and washing the microplate 5 times with washing solution. 50 mul of color developing solution A, B was added to each well, the microplate was shaken slightly to mix well, and incubated at 37 ℃ for 10min in the dark. Add 50. mu.L of stop solution to each well, mix well, and measure absorbance at 450nm wavelength. And calculating the content of the sulfonamides in the sample extracting solution according to the absorbance by using a standard curve.
The results show that, at an addition concentration of 50. mu.g/kg,
comparative example 2
A method for extracting sulfonamides from pork comprises the following steps:
(a) sample preparation:
injecting sulfanilamide medicine into 500g pork to make its final concentration 50 μ g/Kg, standing for 12 hr, homogenizing and mixing for 45s with tissue triturator with rotation speed of 10000r/min, dividing into two parts, placing into clean container, sealing and marking;
(b) and (3) sample preservation:
freezing and storing the sample at-19 ℃ for 36h to obtain a homogeneous sample for later use, wherein the sample is prevented from being polluted or the content of residues is prevented from changing in the operation process;
(c) extraction:
weighing 4.50g of a homogeneous sample, placing the homogeneous sample in a beaker, adding 23mL of 0.10mol/L phosphate buffer solution with the pH of 7.3, wherein the phosphate buffer solution comprises 2.8 parts of sodium dihydrogen phosphate, 30.8 parts of disodium hydrogen phosphate, 8.50 parts of sodium chloride and 980 parts of water, uniformly mixing by vortex, carrying out ultrasonic treatment at 100W and 20 ℃ for 10min, centrifuging at 3500r/min for 13min, and taking the supernatant for measuring the content of the sulfonamide.
And (3) determination:
sequentially adding 50 mu L of a sample or a standard solution into corresponding micropores of a sulfonamide enzyme-linked immunosorbent assay kit, adding 50 mu L of sulfonamide antibody enzyme conjugate into all the micropores, gently mixing, and incubating for 30min at 37 ℃. Spin-drying the liquid in the wells, and washing the microplate 5 times with washing solution. 50 mul of color developing solution A, B was added to each well, the microplate was shaken slightly to mix well, and incubated at 37 ℃ for 10min in the dark. Add 50. mu.L of stop solution to each well, mix well, and measure absorbance at 450nm wavelength. And calculating the content of the sulfonamides in the sample extracting solution according to the absorbance by using a standard curve.
The results showed that the average recovery of the pork samples was 69.42% at an additive concentration of 50. mu.g/kg.
Comparative example 3
A method for extracting sulfonamides from pork comprises the following steps:
(a) sample preparation:
injecting sulfanilamide medicine into 500g pork to make its final concentration 50 μ g/Kg, standing for 12 hr, homogenizing and mixing for 45s with tissue triturator with rotation speed of 10000r/min, dividing into two parts, placing into clean container, sealing and marking;
(b) and (3) sample preservation:
freezing and storing the sample at-19 ℃ for 36h to obtain a homogeneous sample for later use, wherein the sample is prevented from being polluted or the content of residues is prevented from changing in the operation process;
(c) extraction:
weighing 4.50g of a homogeneous sample, placing the homogeneous sample in a beaker, adding 23mL of 0.10mol/L phosphate buffer solution with the pH of 7.3, wherein the phosphate buffer solution comprises 2.8 parts of sodium dihydrogen phosphate, 30.8 parts of disodium hydrogen phosphate, 8.50 parts of sodium chloride and 980 parts of water, uniformly mixing by vortex, carrying out ultrasonic treatment at 300W and 20 ℃ for 10min, centrifuging at 3500r/min for 13min, and taking the supernatant for measuring the content of the sulfonamide.
And (3) determination:
sequentially adding 50 mu L of a sample or a standard solution into corresponding micropores of a sulfonamide enzyme-linked immunosorbent assay kit, adding 50 mu L of sulfonamide antibody enzyme conjugate into all the micropores, gently mixing, and incubating for 30min at 37 ℃. Spin-drying the liquid in the wells, and washing the microplate 5 times with washing solution. 50 mul of color developing solution A, B was added to each well, the microplate was shaken slightly to mix well, and incubated at 37 ℃ for 10min in the dark. Add 50. mu.L of stop solution to each well, mix well, and measure absorbance at 450nm wavelength. And calculating the content of the sulfonamides in the sample extracting solution according to the absorbance by using a standard curve.
The results show that the average recovery rate of the pork sample is 69.23% at an additive concentration of 50. mu.g/kg.
Comparative example 4
A method for extracting sulfonamides from pork comprises the following steps:
(a) sample preparation:
injecting sulfanilamide medicines into 500g pork to make the final concentration of the sulfanilamide medicines be 1 microgram/Kg, 50 microgram/Kg, 100 microgram/Kg and 150 microgram/Kg, and standing for 12 hours for measuring the accuracy; taking 500g of blank pork for sensitivity measurement; another 500g pork was used for precision determination. Homogenizing and mixing uniformly for 52s with a tissue masher with a rotation speed of 9000r/min, dividing into two parts, putting into a clean container, sealing and marking;
(b) and (3) sample preservation:
freezing and storing the sample at-18 ℃ for 6h to obtain a homogeneous sample for later use, wherein the sample is prevented from being polluted or the content of residues is prevented from changing in the operation process;
(c) extraction:
weighing 4.00g of a homogenized sample, adding 20mL of 0.10mol/L phosphate buffer solution with the pH of 7.4, wherein the phosphate buffer solution comprises 2.8 parts of sodium dihydrogen phosphate, 30.8 parts of disodium hydrogen phosphate, 8.5 parts of sodium chloride and 980 parts of water, uniformly mixing by vortex, homogenizing at 10000r/min for 3min, and taking the supernatant for measuring the content of sulfonamides.
And (3) determination:
sequentially adding 50 mu L of a sample or a standard solution into corresponding micropores of a sulfonamide enzyme-linked immunosorbent assay kit, adding 50 mu L of sulfonamide antibody enzyme conjugate into all the micropores, gently mixing, and incubating for 30min at 37 ℃. Spin-drying the liquid in the wells, and washing the microplate 5 times with washing solution. 50 mul of color developing solution A, B was added to each well, the microplate was shaken slightly to mix well, and incubated at 37 ℃ for 10min in the dark. Add 50. mu.L of stop solution to each well, mix well, and measure absorbance at 450nm wavelength. And calculating the content of the sulfonamides in the sample extracting solution according to the absorbance by using a standard curve.
Comparative example 5
A method for extracting sulfonamides from pork comprises the following steps:
(a) sample preparation:
injecting sulfanilamide medicines into 500g pork to make the final concentration of the sulfanilamide medicines be 1 microgram/Kg, 50 microgram/Kg, 100 microgram/Kg and 150 microgram/Kg, and standing for 12 hours for measuring the accuracy; taking 500g of blank pork for sensitivity measurement; another 500g pork was used for precision determination. Homogenizing and mixing uniformly for 52s with a tissue masher with a rotation speed of 9000r/min, dividing into two parts, putting into a clean container, sealing and marking;
(b) and (3) sample preservation:
freezing and storing the sample at-18 ℃ for 6h to obtain a homogeneous sample for later use, wherein the sample is prevented from being polluted or the content of residues is prevented from changing in the operation process;
(c) extraction:
weighing 4.00g of a homogeneous sample, placing the homogeneous sample in a beaker, adding 20mL of 0.10mol/L phosphate buffer solution with the pH of 7.4, wherein the phosphate buffer solution comprises 2.8 parts of sodium dihydrogen phosphate, 30.8 parts of disodium hydrogen phosphate, 8.5 parts of sodium chloride and 980 parts of water, uniformly mixing by vortex, carrying out oscillation treatment for 10min, and taking the supernatant for measuring the content of the sulfonamides.
And (3) determination:
sequentially adding 50 mu L of a sample or a standard solution into corresponding micropores of a sulfonamide enzyme-linked immunosorbent assay kit, adding 50 mu L of sulfonamide antibody enzyme conjugate into all the micropores, gently mixing, and incubating for 30min at 37 ℃. Spin-drying the liquid in the wells, and washing the microplate 5 times with washing solution. 50 mul of color developing solution A, B was added to each well, the microplate was shaken slightly to mix well, and incubated at 37 ℃ for 10min in the dark. Add 50. mu.L of stop solution to each well, mix well, and measure absorbance at 450nm wavelength. And calculating the content of the sulfonamides in the sample extracting solution according to the absorbance by using a standard curve.
TABLE 1 evaluation of methodology
Note: "-" indicates that data was not determined
As can be seen from the above table 1, the extraction methods of the embodiments 1 to 6 of the present invention have high extraction efficiency, high detection sensitivity, good precision, and high recovery rate. Combining comparative example 1 and comparative example 3, the average recovery when acetonitrile was used as the buffer in step (c) was not as high as the average recovery when phosphate buffered solution was used in example 3; combining comparative examples 2 and 3 and example 3, when the power of step (c) is 100W and 300W, the average recovery rate is much lower than that of example 3, so the ultrasonic power is preferably 150-250W; in combination with comparative examples 4 and 5 and example 6, when the extraction method in step (c) is an ultrasonic extraction method, the detection limit and the quantification limit are higher than those in example 6, and the recovery rate of the commercial pork sample at the same addition concentration is significantly lower than that in example 6, which shows that the extraction method of the present invention has the advantages of simple operation, high extraction efficiency, and effective avoidance of false positive and false negative experimental results.
Claims (10)
1. A method for extracting sulfonamides from pork is characterized by comprising the following steps:
step one, homogenizing and mixing pork uniformly by using a tissue triturator, putting the pork into a clean container, sealing and marking;
step two, freezing and storing the sample at the temperature of 18-20 ℃ below zero to obtain a homogeneous sample for later use;
and step three, weighing 4-5 g of the homogeneous sample in a beaker, adding 20-25 mL of phosphate buffer solution, performing ultrasonic centrifugation, and taking the supernatant to perform content determination on the sulfonamide.
2. The method for extracting sulfonamides from pork according to claim 1, wherein the method comprises the following steps: in the first step, 400-600 g of pork is taken.
3. The method for extracting sulfonamides from pork according to claim 1, wherein the method comprises the following steps: in the first step, the rotating speed of the tissue triturator is 8000-12000 r/min, and the time for homogenizing and mixing is 30-60 s.
4. The method for extracting sulfonamides from pork according to claim 1, wherein the method comprises the following steps: in the second step, the freezing time is not more than 72 h.
5. The method for extracting sulfonamides from pork according to claim 1, wherein the method comprises the following steps: in the third step, the concentration of the phosphate buffer solution is 0.05-0.15 mol/L.
6. The method for extracting sulfonamides from pork according to claim 1, wherein the method comprises the following steps: in the third step, the pH value of the phosphate buffer solution is 7.2-7.4.
7. The method for extracting sulfonamides from pork according to claim 1, wherein the method comprises the following steps: in the third step, the phosphate buffer solution comprises 1.4-4.2 parts of sodium dihydrogen phosphate, 15.4-46.2 parts of disodium hydrogen phosphate, 4.25-12.75 parts of sodium chloride and 970-990 parts of water.
8. The method for extracting sulfonamides from pork according to claim 1, wherein the method comprises the following steps: in the third step, the power of the ultrasound is 150-250W, the time is 8-12 min, and the temperature is 15-25 ℃.
9. The method for extracting sulfonamides from pork according to claim 1, wherein the method comprises the following steps: in the third step, the rotating speed of the centrifugation is 3000-4000 r/min, and the time is 10-15 min.
10. The method for extracting sulfonamides from pork according to claim 1, wherein the method comprises the following steps: the sulfanilamide drug is one or more of sulfamethoxazole, sulfathiazole, sulfamethoxydiazine, sulfamethoxyzine, sulfapyridine, sulfamethizole and sulfachlorpyridazine.
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