CN111443075A - Rapid detection method of sulfadimidine - Google Patents
Rapid detection method of sulfadimidine Download PDFInfo
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- CN111443075A CN111443075A CN202010479204.3A CN202010479204A CN111443075A CN 111443075 A CN111443075 A CN 111443075A CN 202010479204 A CN202010479204 A CN 202010479204A CN 111443075 A CN111443075 A CN 111443075A
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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
Technical Field
The invention relates to the field of biological rapid detection, in particular to a rapid detection method of sulfadimidine.
Background
Veterinary residue refers to the parent compound of the veterinary drug and/or metabolites thereof, as well as veterinary-related impurities, contained in any edible part of the animal product. Including antibiotics, anthelmintics, antiprotozoals, trypanosomimetics, and the like.
Veterinary drugs and feed additives remaining in animal-derived foods pose a potential threat to human health as the food chain enters the human body, and the threat has attracted more and more attention of people. With the change of the demand of people on animal-derived food, the international requirement on the drug residue of the animal-derived food is higher and higher, the drug residue in the animal-derived food gradually becomes a focus of worldwide attention, and the antibiotic residue is greatly concerned. A large amount of antibiotics are frequently used, so that drug-resistant pathogenic bacteria in animal bodies can easily infect humans; and antibiotic drug residues can cause bacteria in the human body to generate drug resistance, disturb the micro-ecology of the human body and generate various toxic and side effects. Veterinary drugs and additives remaining in animal food pose a potential threat to human health as the food chain enters the human body, and the threat has attracted more and more attention of people. Therefore, the antibiotic is simply, quickly and conveniently detected on site in trace amount, and the method can play a key role in effectively supervising the medicine, ensuring the public life health and protecting the environment.
The detection method of sulfonamide antibiotics in animal tissues and urine mainly comprises a high performance liquid chromatography, a liquid chromatography-mass spectrometry, an enzyme linked immunosorbent assay and the like. The defects of long pretreatment process time, complex operation, strong operation technology, expensive instrument and equipment and the like of the chromatography and mass spectrometry generally exist, and the requirement of rapidly screening a large number of samples on site is difficult to meet. The ELISA method has the advantages of large sample amount, low cost, simple and portable instrument, but the ELISA method has more influencing factors and needs to improve the sensitivity and the stability. The working efficiency of detection personnel is improved while the detection precision is ensured, and the research of a rapid, simple, real-time and accurate detection method has very important significance.
Disclosure of Invention
Aiming at the defects in the technology, the invention provides a method for rapidly analyzing sulfadimidine in an animal body by using a surface-enhanced Raman spectroscopy technology, and provides a method for qualitatively and quantitatively analyzing the surface-enhanced Raman spectroscopy of the sulfadimidine by using silver colloid as an enhanced substrate.
In order to achieve the purpose, the invention provides a rapid detection method of sulfadimidine, which adopts surface enhanced Raman spectroscopy to perform rapid analysis and detection on a sample, and comprises the following steps:
s1: preparing a Raman enhancement base solution;
s2, preparing a standard sample;
s3: rapidly detecting a sample;
s4: and (6) analyzing data results.
Preferably, in step S1, a silver nano-reinforcing base solution is prepared, trisodium citrate is added to a boiling silver nitrate solution, and a silver sol is obtained after stirring.
Preferably, 20ml of silver nitrate solution is dissolved in 100ml of ultrapure water for heating, 1.85ml of sodium citrate with the mass concentration of 0.01g/ml is added after boiling, heating and continuous stirring are carried out, cooling and storing are carried out after the solution color is changed from transparent to light brown and finally to grey green, and 1% of sodium chloride solution is mixed for use.
Preferably, in step S2, the method includes the following substeps:
s21: dissolving a sulfadimidine standard substance, and then gradually diluting the sulfadimidine standard substance into a solution with a certain gradient concentration;
s22, processing the negative sample of the animal urine, and then adding the sulfadimidine solution obtained in the step S21 to obtain a positive sample;
and S23, adding an extracting agent into the positive sample for extraction, and detecting by using a Raman spectrometer.
Preferably, in step S21, the sulfadimidine standard is weighed, dissolved by an organic solvent, put into a brown volumetric flask, gradually diluted by the same organic solvent into a standard working solution, and stored at 4 ℃ for later use; the organic solvent is one of methanol, ethanol or ethylene glycol.
Preferably, in step S22, a negative sample of animal urine is taken out of the centrifuge tube, high-speed centrifugation is performed to remove the precipitate, and the supernatant is taken out and added to a standard working solution to prepare a positive sample.
Preferably, in step S23, the positive sample is taken, ethyl acetate or acetonitrile is added as an extractant, the mixture is vortexed and left to stand, nitrogen is blown, and then the corresponding extractant is added to a constant volume for detection.
Preferably, before step S1, a data simulation calculation is performed on sulfadimidine to calculate a theoretical raman spectrum peak thereof, so as to determine a qualitative spectrum peak of the standard.
Preferably, in step S3, when the surface raman spectroscopy is used for the detection, the following parameters are used: wave number range 400 to 2000cm-1, laser wavelength 785nm, power 400mW, resolution 2cm-1, integration time 10 s.
Preferably, in the on-machine detection, 500. mu. L of silver colloid, 40. mu. L of test solution and 100. mu. L of sodium chloride solution are added to a sample bottle in this order, shaken well and then subjected to on-machine acquisition of Raman signals within 5 seconds.
The method has the advantages that the method is preliminarily researched by adopting a surface enhanced Raman spectroscopy rapid detection method for the sulfa antibiotics in the animal excrement, a characteristic peak of sulfadimidine is determined by using a density functional theory, a quantitative analysis curve is established by using the characteristic peak intensity with high Raman peak intensity and good peak shape, and a standard addition recovery rate experiment shows that the method has better accuracy and precision, the lowest concentration detected by the method is respectively the lowest detection concentration of the sulfadimidine of 2 mg/L, the sulfa antibiotics residue in the pig urine is qualitatively and quantitatively analyzed by the method, the detection of a single sample is completed within 2min, and the detection speed is high.
Drawings
FIG. 1 is a spectrum diagram of an experiment on sulfadimidine according to the invention;
FIG. 2 is a surface enhanced Raman spectrum of sulfadimidine of the present invention;
FIG. 3 is a concentration spectrum of a standard solution of sulfadimidine;
figure 4 is a sulfadimidine quantitative analysis curve.
Detailed Description
In order to more clearly describe the present invention, the present invention is further described below with reference to the accompanying drawings and specific embodiments.
The surface enhanced Raman spectroscopy technology is characterized in that gold and silver nanoparticles are used as carriers, when molecules to be detected are adsorbed to rough gold and silver surfaces, the action surface area of light and the molecules can be greatly increased, the electromagnetic field of the metal surface can be greatly enhanced, and the intensity of the obtained Raman signal is 10 common6More than twice. The surface enhanced Raman spectroscopy technology has the advantages of simple pretreatment of experimental samples, high detection speed of single sample, high detection sensitivity and the like, so that the surface enhanced Raman spectroscopy technology can be widely applied to rapid monitoring of quality safety problems such as trace pesticide residues, antibiotic drug residues and the like in food and agricultural products, and is compared with the surface enhanced Raman spectroscopy technologyThe state of the art.
Firstly, preparing nano-silver colloid, accurately weighing 0.100g of trisodium citrate solid, dissolving the trisodium citrate solid in a beaker by using deionized water, transferring the beaker into a 10ml volumetric flask, fixing the volume to a scale mark to obtain an L trisodium citrate solution with the concentration of 0.01g/m, weighing 20.00ml of silver nitrate solution, quickly heating the silver nitrate solution in 100ml of ultrapure water, dropwise adding 0.01g/m L trisodium citrate solution within 1 minute, stirring the solution at the rotating speed of 500r/min, stopping heating when the solution is changed from transparent light brown to gray green, and cooling the solution to room temperature to obtain the nano-silver colloid, namely the silver nano-reinforcing base solution.
Accurately weighing 0.5g of sodium chloride solid, dissolving the sodium chloride solid in a beaker by using deionized water, transferring the beaker to a 50m L volumetric flask, fixing the volume to a scale mark to obtain a 1% sodium chloride solution, adding 500 mu L of silver colloid, 40 mu L to-be-detected liquid and 100 mu L of sodium chloride solution into a sample bottle in sequence during detection, shaking the solution uniformly and collecting Raman signals, wherein the collection parameters comprise a wave number range of 400-2000 cm < -1 >, a laser wavelength of 785nm, a power of 400mW and a resolution of 2cm-1When the sample is added, the liquid-moving gun measures the sample, the sample needs to be stretched below the liquid level of the silver colloid, the sodium chloride solution is dripped above the liquid level of the silver colloid, and the liquid-moving gun is used for detecting in 5s after shaking up, so that the surface Raman is effectively enhanced, and meanwhile, the detection is not influenced by the deposition of the silver chloride, after the detection is finished, the generated precipitate wraps the sample, and the processing is more convenient.
Weighing 0.0100g of sulfadimidine standard, dissolving in a beaker with methanol, transferring to a 100ml brown volumetric flask to obtain a 100 mg/L standard solution, gradually diluting with methanol to obtain a standard working solution with the concentration of 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, 1 mg/L, storing at 4 ℃ for later use, taking 100ml of a pig urine or cow urine negative sample in a centrifuge tube, centrifuging at 4500r/min for 5min, removing precipitates, taking 5m L of a supernatant to prepare a pig urine sample containing sulfadimidine with the concentration of 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, 1 mg/L respectively, and storing at 4 ℃ for later use.
The method comprises the following steps of extracting and extracting the pig urine or the cow urine by using an organic solvent, wherein the main components of water removed from the pig urine or the cow urine are urea, uric acid, hippuric acid and electrolyte, and the most of water is water-soluble substances, and the organic solvent is adopted, wherein the ethyl acetate is taken as an extracting agent for extraction and extraction, more specifically, 1m L of a pig urine or cow urine sample is taken, 3m L of ethyl acetate is added, vortex extraction is carried out for 2min, standing and layering are carried out, an upper ethyl acetate layer is taken and is blown to be dry at 40 ℃, ethyl acetate is used for dissolving to a constant volume of 1m L, and detection is carried out on a machine.
Before carrying out related detection, theoretical spectral calculation needs to be carried out on corresponding sulfadimidine, related software is adopted to construct a sulfadimidine molecular model, according to a density functional theory, a B3L YP method is used, and related software is used to carry out structure optimization and calculate a theoretical Raman spectral peak.
Constructing a sulfadimidine molecular model (sulfadimidine, the chemical formula of which is C12H14N4O2S and mainly comprises a benzene ring, a pyrimidine base, a sulfonamide group and the like) by using GaussView 5.0 software, performing structure optimization and calculating theoretical Raman spectrum peaks by using Guassian09w software by using a B3L YP method according to a Density Functional Theory (DFT), wherein (a) is an experimental spectrum of the sulfadimidine, (B) is a theoretical calculated spectrum of the sulfadimidine, and the theoretical Raman spectrum peaks are 400-1800 cm-1In the range of 1587, 1531, 819 and 662cm-1The experimental spectrum is slightly deviated from the theoretical spectrum but basically coincided with the theoretical spectrum, and the main Raman spectrum peaks 1456 and 756cm calculated by theory-1No peak was found in the experimental spectrum, while the main raman spectrum peaks 1004 and 580cm in the experimental spectrum-1And so on, are not present in the theoretical calculations. The main reason is that the molecular acting force exists between the molecule to be measured and the solvent system, so that the strengthening effect of the strengthening substrate on different groups is different, the displacement deviation of the molecular groups is influenced, and the theoretical simulation calculation only carries out ideal calculation on a single molecule and does not receive the acting force between moleculesInfluence. The spectral peak attribution of the main characteristic peaks is found to be 1531 cm respectively by comparison-1(1541cm-1) The mark shows the stretching vibration of the pyrimidine ring, 819cm-1(832cm-1) The department represents the para-position disubstituted benzene ring breath vibration absorption of 662cm-1(670cm-1) The point represents deformation vibration of the para-disubstituted benzene ring and the like.
The table below shows the assignment of the main peaks of sulfadimidine
In FIG. 2, (a) is surface enhanced Raman spectrum of 25 mg/L sulfadimidine standard solution, (b) is surface enhanced Raman spectrum of ethyl acetate, (c) is surface enhanced Raman spectrum of negative pig urine sample pretreated by the method in 2.7, and (d) is Raman spectrum of silver nano enhanced substrate, from FIG. 2(d), the silver nano enhanced substrate has no Raman peak, which shows that the used silver sol does not affect the Raman signal of the target solution, comparing FIGS. 2(a), (b) and (c), 1000cm-1～1600cm-1The spectral peaks of sulfadimidine in the range partially overlap with the spectral peaks of ethyl acetate and negative pig urine samples, so that the standard sulfadimidine solution is subjected to surface enhanced Raman spectroscopy at 552, 580 and 662cm-1The peak at (A) is taken as the peak of the qualitative spectrum of sulfadimidine.
The prepared standard sulfadimidine solution is diluted into standard working solution with gradient concentration of 5, 2, 1, 0.5, 0.4 and 0.3 mg/L, the surface enhanced Raman spectrum collected after on-machine detection is shown in figure 3, the concentrations of a to f are respectively 5, 2, 1, 0.5, 0.4 and 0.3 mg/L, the observation of figure 3 shows that 662cm is obtained when the concentration is 0.4 mg/L-1The peak can be resolved, and the peak can not be resolved when the concentration reaches 0.3 mg/L, so the lowest detected concentration of the sulfadimidine standard solution is 0.4 mg/L.
Pre-treating the pig urine sample containing sulfadimidine antibiotic with gradient concentration of 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, 1 mg/L, detecting on the machine, collecting surface enhanced Raman spectrum, and collecting sulfadimidine standard solution concentration662cm which can be distinguished at the lowest detection concentration-1A quantitative analysis curve is established between the peak intensity and the concentration, as shown in figure 4, in the range of 2-20 mg/L, the linear equation is that y is 0.0047x-0.0758, and the correlation coefficient R is20.9915, the minimum detection concentration is 2 mg/L, adding a sulfadimidine standard solution into pig urine to prepare pig urine samples with the concentrations of 5, 11 and 17 mg/L, and configuring 3 parallel samples for each concentration so as to detect the accuracy and precision of the method, wherein the average recovery rate of the samples is between 95.93 and 104.16 percent, and the relative standard deviation is between 1.18 and 7.79 percent, and after pretreatment, 6 pig urine samples with unknown concentration containing sulfadimidine are prepared, and after 3 times of Raman signal averaging, planning and processing data, comparing the calculation result with the result of the chemical detection method of high performance liquid chromatography, and the relative error is between 1.16 and 6.34 percent.
The method has the advantages that a standard curve and a linear regression equation of the sulfadimidine are established, and the specific content of the sulfadimidine can be calculated by measuring the correlation value in the sample in the actual measurement process, so that the detection efficiency is greatly improved.
Claims (10)
1. A rapid detection method of sulfadimidine is characterized in that a sample is rapidly analyzed and detected by adopting surface enhanced Raman spectroscopy, and the method comprises the following steps:
s1: preparing a Raman enhancement base solution;
s2, preparing a standard sample;
s3: rapidly detecting a sample;
s4: and (6) analyzing data results.
2. The method for rapidly detecting sulfadimidine as claimed in claim 1, wherein in step S1, preparing silver nano enhancing base solution, adding trisodium citrate into boiling silver nitrate solution, and stirring to obtain silver sol.
3. The method for rapidly detecting sulfadimidine as claimed in claim 2, wherein 20ml of silver nitrate solution is dissolved in 100ml of ultrapure water and heated, 1.85ml of sodium citrate with mass concentration of 0.01g/ml is added after boiling, heating and continuous stirring are carried out, cooling and storing are carried out after the solution color is changed from transparent to light brown to gray green, and 1% sodium chloride solution is mixed for use.
4. The method for rapidly detecting sulfadimidine as claimed in claim 1, wherein in step S2, the method comprises the following substeps:
s21: dissolving a sulfadimidine standard substance, and then gradually diluting the sulfadimidine standard substance into a solution with a certain gradient concentration;
s22, processing the negative sample of the animal urine, and then adding the sulfadimidine solution obtained in the step S21 to obtain a positive sample;
and S23, adding an extracting agent into the positive sample for extraction, and detecting by using a Raman spectrometer.
5. The method for rapidly detecting sulfadimidine as claimed in claim 4, wherein in step S21, the sulfadimidine standard is weighed, dissolved by organic solvent and then placed into a brown volumetric flask, and then gradually diluted by the same organic solvent into standard working solution, and stored at 4 ℃ for later use; the organic solvent is one of methanol, ethanol or ethylene glycol.
6. The method for rapid detection of sulfadimidine as claimed in claim 4, wherein in step S22, negative samples of animal urine are taken and put into a centrifuge tube, high speed centrifugation is carried out to remove precipitates, and supernatant is taken and added into standard working solution to prepare positive samples.
7. The method for rapidly detecting sulfadimidine as claimed in claim 4, wherein in step S23, ethyl acetate or acetonitrile is added to the positive sample as an extractant, nitrogen blowing is performed after vortex standing, and then a corresponding extractant is added for constant volume for detection.
8. The method for rapidly detecting sulfadimidine as claimed in claim 1, wherein before step S1, the method further comprises performing data simulation calculation on sulfadimidine, and calculating theoretical raman spectrum peak thereof, so as to determine qualitative spectrum peak of the standard.
9. The method for rapidly detecting sulfadimidine as claimed in claim 1, wherein in step S3, when the surface raman spectroscopy is used for detection, the parameters are as follows: wave number range 400 to 2000cm-1, laser wavelength 785nm, power 400mW, resolution 2cm-1, integration time 10 s.
10. The method for rapidly detecting sulfadimidine as claimed in claim 1, wherein during the machine-up detection, 500 μ L silver colloid, 40 μ L solution to be detected and 100 μ L sodium chloride solution are sequentially added into a sample bottle, and after shaking up, the raman signal is collected by an instrument within 5 s.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112945931A (en) * | 2021-02-03 | 2021-06-11 | 上海如海光电科技有限公司 | Raman rapid detection method of sulfamonomethoxine in dairy product |
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| CN102353665A (en) * | 2011-07-06 | 2012-02-15 | 上海海洋大学 | Surface enhanced raman spectroscopy detection method for sulfanilamide medicines |
| CN103105387A (en) * | 2013-01-29 | 2013-05-15 | 福州大学 | Method for detecting sulfamethazine in animal-derived food |
| CN107345911A (en) * | 2017-06-06 | 2017-11-14 | 浙江大学 | A kind of method of histamine in SERS qualitative and quantitative analysis rice fish tissue |
Non-Patent Citations (1)
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| CN112945931A (en) * | 2021-02-03 | 2021-06-11 | 上海如海光电科技有限公司 | Raman rapid detection method of sulfamonomethoxine in dairy product |
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