CN111443075A - Rapid detection method for sulfamethazine - Google Patents

Abstract

The application provides a rapid detection method of sulfadimidine in an animal body, which utilizes a density functional theory to determine a Raman characteristic peak of the sulfadimidine, establishes a quantitative analysis curve by using the characteristic peak intensity with high Raman peak intensity and good peak shape, and indicates that the accuracy and precision of the method are better by a standard recovery rate experiment.

Description

Rapid detection method for sulfamethazine

technical field

The invention relates to the field of rapid biological detection, in particular to a rapid detection method for sulfamethazine.

Background technique

Residues of veterinary drugs refer to the parent compounds of veterinary drugs and/or their metabolites, as well as impurities related to veterinary drugs, contained in any edible part of animal products. These include antibiotics, anthelmintics, antiprotozoal drugs, trypanosomes, etc.

Residual veterinary drugs and feed additives in animal-derived foods, along with the food chain entering the human body, pose a potential threat to human health, and this threat has attracted more and more attention. With the change of people's demand for animal-derived foods, the international requirements for drug residues in animal-derived foods are getting higher and higher, and drug residues in animal-derived foods have gradually become a focus of attention around the world. great attention. Large and frequent use of antibiotics can make drug-resistant pathogens in animals easily infect humans; and antibiotic drug residues can cause bacteria in humans to develop drug resistance, disrupt human microecology and produce various side effects. Veterinary drugs and additives remaining in animal food, along with the food chain entering the human body, pose a potential threat to human health, and this threat has attracted more and more attention. Therefore, simple, fast and convenient on-site trace detection of antibiotics can play a key role in the effective supervision of drugs, ensuring public health and protecting the environment.

The detection methods of sulfonamide antibiotics in animal tissues and urine mainly include high performance liquid chromatography, liquid chromatography, and enzyme-linked immunosorbent assay. Chromatography and mass spectrometry generally have defects such as long pretreatment time, complicated and highly technical operation, and expensive equipment, which make it difficult to meet the requirements of rapid screening of a large number of samples in the field. The ELISA method has a large sample size, low cost, and simple and portable instruments. However, there are many factors affecting the ELISA method, and the sensitivity and stability need to be improved. It is of great significance to develop a fast, simple, real-time and accurate detection method in order to improve the work efficiency of the inspectors while ensuring the precision of the inspection.

SUMMARY OF THE INVENTION

Aiming at the deficiencies in the above-mentioned technologies, the present invention provides a surface-enhanced Raman spectroscopy technique for rapidly analyzing sulfamethazine in animals, using silver glue as an enhanced A method for qualitative and quantitative analysis of sulfamethazine by surface-enhanced Raman spectroscopy is proposed.

In order to achieve the above object, the present invention provides a rapid detection method for sulfamethazine, which adopts surface-enhanced Raman spectroscopy to quickly analyze and detect the sample, including the following steps:

S1: preparation of Raman-enhanced base fluid;

S2: preparation of standard samples;

S3: Rapid detection of samples;

S4: Data result analysis.

Preferably, in step S1, a silver nano-enhanced base solution is prepared, and trisodium citron is added to the boiling silver nitrate solution, and a silver sol is obtained after stirring.

Preferably, dissolve 20ml of silver nitrate solution in 100ml of ultrapure water for heating, add 1.85ml of sodium citrate with a mass concentration of 0.01g/ml after boiling, heat and continue to stir until the color of the solution changes from transparent to light brown Finally, it is cooled and stored in gray-green color, and mixed with 1% sodium chloride solution when using.

Preferably, in step S2, include the following sub-steps:

S21: dissolve the sulfamethazine standard, and then gradually dilute it into a solution with a certain gradient concentration;

S22: process the negative sample of animal urine, then add the sulfamethazine solution obtained in S21 to obtain a positive sample;

S23: Add an extractant to the positive sample for extraction, and use a Raman spectrometer for detection.

Preferably, in step S21, weigh the sulfamethazine standard product, dissolve it in an organic solvent, put it into a brown volumetric flask, and then use the same organic solvent to gradually dilute it into a standard working solution, and store it at 4°C for later use; the organic solvent It is one of methanol, ethanol or ethylene glycol.

Preferably, in step S22, a negative sample of animal urine is taken in a centrifuge tube, high-speed centrifugation is performed to remove the precipitate, and the supernatant is taken and added to a standard working solution to form a positive sample.

Preferably, in step S23, a positive sample is taken and added with ethyl acetate or acetonitrile as an extractant, vortexed and left to stand for nitrogen blowing, and then a corresponding extractant is added to the volume for detection.

Preferably, before step S1, it is also necessary to perform data simulation calculation on sulfamethazine, and calculate its theoretical Raman spectrum peak, so as to determine the qualitative spectrum peak of the standard product.

Preferably, in step S3, when using a surface Raman instrument for detection, the parameters used are: wavenumber range 400 to 2000cm-1, laser wavelength 785nm, power 400mW, resolution 2cm-1, integration time 10s.

Preferably, when performing on-machine detection, 500 μL of silver glue, 40 μL of the solution to be tested, and 100 μL of sodium chloride solution are sequentially added to the injection bottle, and the Raman signal is collected by the instrument within 5s after shaking.

The beneficial effects of the present invention are as follows: the present application conducts preliminary research on the rapid detection method of surface-enhanced Raman spectroscopy of sulfonamide antibiotics in animal excrement. Using density functional theory, the Raman characteristic peaks of sulfamethazine were identified. Quantitative analysis curves were established based on characteristic peak intensities with high Raman peak intensity and good peak shape. The spike recovery experiments showed that the method had good accuracy and precision. The minimum detection concentration of sulfamethazine by this method is 2mg/L. This method is used to qualitatively and quantitatively analyze the residues of sulfonamide antibiotics in pig urine. The detection of a single sample is completed within 2 minutes, and the detection speed is fast.

Description of drawings

Fig. 1 is the experimental spectrogram of sulfamethazine of the present invention;

Fig. 2 is the sulfamethazine surface-enhanced Raman spectrum of the present invention;

Fig. 3 is the concentration spectrum of sulfamethazine standard solution;

Figure 4 is the quantitative analysis curve of sulfamethazine.

Detailed ways

In order to express the present invention more clearly, the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

Surface-enhanced Raman spectroscopy technology refers to the use of gold and silver nanoparticles as carriers. When the molecules to be tested are adsorbed to the rough gold and silver surfaces, the surface area of light and molecules will be greatly increased, and the electromagnetic field of the metal surface will also be large. The amplitude is enhanced, and the obtained Raman signal strength is more than 10 ⁶ times of the ordinary one. Surface-enhanced Raman spectroscopy has the advantages of simple sample preparation, fast detection of a single sample, and high detection sensitivity. Therefore, it is widely used in the rapid monitoring of trace pesticide residues, antibiotic drug residues and other quality and safety issues in food and agricultural products. in the state of the art.

First, prepare the nano-silver glue, accurately weigh 0.100g of trisodium citrate solid, dissolve it in a beaker with deionized water, transfer it to a 10ml volumetric flask, and set the volume to the mark to obtain a lemon with a concentration of 0.01g/mL. Trisodium citrate solution; measure 20.00ml of silver nitrate solution in 100ml of ultrapure water for rapid heating, add 0.01g/mL trisodium citrate solution dropwise within 1 minute, stir at a speed of 500r/min, and wait for the solution to change from The transparent turns light brown and finally turns grey-green, then the heating is stopped, and after cooling to room temperature, the nano-silver glue is obtained, that is, the silver nano-enhanced base liquid.

Accurately weigh 0.5g of solid sodium chloride, dissolve it in a beaker with deionized water and transfer it to a 50mL volumetric flask, and set the volume to the mark to obtain a 1% sodium chloride solution; 500 μL of silver glue, 40 μL of test solution, and 100 μL of sodium chloride solution were added in sequence, and the Raman signal was collected after shaking. Acquisition parameters: wavenumber range 400 to 2000cm-1, laser wavelength 785nm, power 400mW, resolution 2cm ^-1 , integration time 10s. When adding the sample, when measuring the sample with the pipette, it is necessary to extend the sample below the surface of the silver glue, drop the sodium chloride solution above the surface of the silver glue, shake it well, and test it on the machine within 5s. Therefore, while effectively enhancing the surface Raman, it is ensured that the detection will not be affected by the precipitation of silver chloride. After the detection is completed, the generated precipitate wraps the sample, which is more convenient for processing. The collected data was planned and processed with MATLAB-R2012b.

Weigh 0.0100g of standard sulfamethazine, dissolve it in a beaker with methanol and transfer it to a 100ml brown volumetric flask to obtain a standard solution of 100mg/L, and then gradually dilute it with methanol to a concentration of 20, 18, 16, 14, 12 , 10, 8, 6, 4, 2, and 1 mg/L standard working solution, stored at 4°C for later use; take 100 ml of pig urine or bovine urine negative sample in a centrifuge tube, centrifuge at 4500 r/min for 5 min, remove the precipitate, and take the upper layer. 5 mL of clear liquid was prepared into pig urine samples containing sulfamethazine at concentrations of 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, and 1 mg/L, respectively, and stored at 4°C for later use.

Since sulfonamide antibiotics are almost insoluble in water and can be dissolved in organic solvents, most of the water removed from pig urine or cow urine is mainly composed of urea, uric acid, hippuric acid and electrolytes, which are mostly water-soluble substances, so organic solvents are used for extraction. In this application, ethyl acetate is used as an extractant for extraction and extraction; more specifically, 1 mL of pig urine or cow urine sample is taken, 3 mL of ethyl acetate is added, vortex extraction is performed for 2 min, and the layers are left to stand for stratification, and the upper layer of acetic acid is taken. The ethyl ester layer was blown to dryness under nitrogen at 40°C, dissolved in ethyl acetate to a volume of 1 mL, and detected on the machine; on the one hand, the use of ethyl acetate in this application can effectively solve the sulfonamide antibiotics, and also uses ethyl acetate as a solution. The unique odor of esters can effectively cover the bad odor in animal urine, so that the testing personnel can obtain a more comfortable sensory experience during the testing process.

Before the relevant detection, the theoretical spectrum calculation of the corresponding sulfamethazine needs to be carried out, and the relevant software is used to construct the sulfamethazine molecular model. According to the density functional theory, using the B3LYP method, the relevant software is used to optimize the structure and calculate Its theoretical Raman peaks.

GaussView 5.0 software was used to build a molecular model of sulfamethazine (sulfamethazine, chemical formula C12H14N4O2S, mainly composed of benzene ring, pyrimidine group, sulfonamide group, etc.), according to density functional theory (DFT), using B3LYP method, with Guassian09w software performs structural optimization and calculates its theoretical Raman peaks. As shown in Figure 1, (a) is the experimental spectrum of sulfamethazine, and (b) is its theoretically calculated spectrum. In the range of 400～1800cm ^-1 , the experimental spectra at 1587, 1531, 819 and 662cm ^-1 deviate slightly from the theoretical spectra but are basically consistent with the theoretically calculated main Raman peaks 1456 and 756cm ^-1 which are not found in the experimental spectra. However, the main Raman peaks 1004 and 580 cm ^-1 in the experimental spectrum did not appear in the theoretical calculation. The main reason is that there is molecular interaction between the molecule to be tested and the solvent system, which leads to different enhancement effects of the reinforcing substrate on different groups, thus affecting the displacement of molecular groups. The theoretical simulation calculation is only ideal for a single molecule. Not affected by intermolecular forces. By comparison, it is found that the spectral peaks of the main characteristic peaks are assigned to the pyrimidine ring stretching vibration at 1531 cm ^-1 (1541 cm ^-1 ), the para ^- disubstituted benzene ring breathing vibration at 819 cm -1 (832 cm ^-1 ), and the breathing vibration of the para-disubstituted benzene ring at 662 cm ^-1 . (670cm ^-1 ) characterizes para-disubstituted benzene ring deformation vibration and so on.

The following table shows the assignment of the main spectral peaks of sulfamethazine

In Figure 2, (a) is the surface-enhanced Raman spectrum of the standard solution with a concentration of 25 mg/L sulfamethazine, (b) is the surface-enhanced Raman spectrum of ethyl acetate, and (c) is the negative pre-treatment method in 2.7. Surface-enhanced Raman spectrum of pig urine sample, (d) is the Raman spectrum of silver nano-enhanced substrate. It can be seen from Figure 2(d) that the silver nano-enhanced substrate has no Raman peak, indicating that the silver sol used will not affect the solution Raman signal of the target; compare Figure 2(a), (b) and (c) It can be seen that the spectral peaks of sulfamethazine in the range of 1000cm ^-1 to 1600cm ^-1 partially overlap with those of ethyl acetate and negative swine urine samples, so the surface-enhanced Raman spectrum of sulfamethazine standard solution The peaks at 552, 580 and 662 cm ^-1 of sulfamethazine were used as the qualitative peaks of sulfamethazine.

The configured sulfamethazine standard solution was diluted to a standard working solution with gradient concentrations of 5, 2, 1, 0.5, 0.4, and 0.3 mg/L. The surface-enhanced Raman spectrum collected after the on-board detection is shown in Figure 3, a ~f concentrations were 5, 2, 1, 0.5, 0.4, and 0.3 mg/L, respectively. It can be observed from Figure 3 that the spectral peak at 662cm ^-1 can be distinguished when the concentration is 0.4mg/L, but it cannot be distinguished when the concentration is 0.3 mg/L, so the minimum detection concentration of sulfamethazine standard solution is 0.4mg /L.

The prepared pig urine samples containing sulfamethazine antibiotic gradient concentrations of 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, 1 mg/L were pre-treated, tested on the machine, and the surface was collected. Enhanced Raman spectroscopy. The peak intensity and concentration at 662cm ^-1 that can be resolved when the concentration of the sulfamethazine standard solution is the lowest detection concentration, establish a quantitative analysis curve, as shown in Figure 4, in the range of 2 ~ 20mg/L, the linear equation is y = 0.0047x-0.0758, the correlation coefficient R ² =0.9915, the lowest detection concentration was 2mg/L. The standard solution of sulfamethazine was added to pig urine to prepare pig urine samples with concentrations of 5, 11, and 17 mg/L. Three parallel samples were prepared for each concentration to test the accuracy and precision of this method. The average recovery rate of samples was between 95.93% and 104.16%, and the relative standard deviation was between 1.18% and 7.79%. Another 6 pig urine samples containing sulfamethazine of unknown concentration were prepared. After pre-processing, the data was planned and processed after collecting 3 Raman signals and averaging, and the calculation results were compared with the results of the chemical detection method of high performance liquid chromatography. In contrast, the relative error is between 1.16% and 6.34%.

The advantage of the present invention is that the standard curve and linear regression equation of sulfamethazine are established, and in the actual measurement process, the specific content of sulfamethazine can be calculated only by measuring the relevant values in the sample, which greatly improves the detection rate. efficiency.

Claims (10)

1. a fast detection method of sulfamethazine, is characterized in that, adopts surface-enhanced Raman spectroscopy to carry out fast analysis and detection to sample, comprises the following steps: S1: preparation of Raman-enhanced base fluid; S2: preparation of standard samples; S3: Rapid detection of samples; S4: Data result analysis. 2. the rapid detection method of sulfamethazine according to claim 1, is characterized in that, in step S1, prepare silver nano-enhanced base liquid, join trisodium citrate in the boiling silver nitrate solution, after stirring, obtain silver sol. 3. the rapid detection method of sulfamethazine according to claim 2 is characterized in that, it is characterized in that, 20ml silver nitrate solution is dissolved in 100ml ultrapure water and is heated, and adding mass concentration after boiling is 0.01g /ml of sodium citrate 1.85ml, heat and continue to stir, when the color of the solution changes from transparent to light brown and finally to gray-green, it is cooled and stored, and mixed with 1% sodium chloride solution when using. 4. the rapid detection method of sulfamethazine according to claim 1, is characterized in that, in step S2, comprises following substep: S21: dissolve the sulfamethazine standard, and then gradually dilute it into a solution with a certain gradient concentration; S22: process the negative sample of animal urine, then add the sulfamethazine solution obtained in S21 to obtain a positive sample; S23: Add an extractant to the positive sample for extraction, and use a Raman spectrometer for detection. 5. the rapid detection method of sulfamethazine according to claim 4, is characterized in that, in step S21, take by weighing sulfamethazine standard substance, adopt organic solvent to carry out dissolving and put into brown volumetric flask, and then put into brown volumetric flask. Use the same organic solvent to gradually dilute to a standard working solution, and store at 4 °C for future use; the organic solvent is one of methanol, ethanol or ethylene glycol. 6. The method for rapid detection of sulfamethazine according to claim 4, characterized in that, in step S22, a negative sample of animal urine is taken in a centrifuge tube, high-speed centrifugation is performed to remove the precipitation, and the supernatant is taken and added to the standard work The solution was prepared as a positive sample. 7. The method for rapid detection of sulfamethazine according to claim 4, characterized in that, in step S23, a positive sample is taken and ethyl acetate or acetonitrile is added as an extractant, and after vortexing, nitrogen blowing is performed, and then Add the corresponding extractant to the volume for detection. 8. sulfamethazine rapid detection method according to claim 1, is characterized in that, before step S1, also needs to carry out data simulation calculation to sulfamethazine, calculate its theoretical Raman spectrum peak, thereby determine this standard The qualitative peaks of the product. 9. sulfamethazine rapid detection method according to claim 1, is characterized in that, in step S3, when adopting surface Raman instrument to detect, adopting parameter is: wave number range 400 to 2000 cm-1, laser wavelength 785 nm, power 400 mW, resolution 2 cm-1, integration time 10 s. 10. The method for rapid detection of sulfamethazine according to claim 1, characterized in that, when carrying out on-board detection, 500 μL of silver glue, 40 μL of liquid to be tested, 100 μL of chlorinated chlorination were added to the sampling bottle in turn. Sodium solution, shake up and collect Raman signal on the instrument within 5s.

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