Method for detecting sulfonamide residues in milk

Abstract

The invention discloses a method for detecting sulfonamides residues in milk, which comprises the following steps: firstly, mixing to-be-detected milk containing sulfonamides with a deproteinization reagent, performing deproteinization treatment on the to-be-detected milk, then removing solids in the formed deproteinized mixture, and adjusting the obtained clear liquid to be acidic, obtaining a pretreated sample; then uniformly mixing the pretreated sample and a color developing agent to form a mixed sample; then, applying a medium-frequency oscillation electric field to the mixed sample, enabling the mixed sample to fully react to form a to-be-detected sample, continuously applying the medium-frequency oscillation electric field, and measuring the light absorption value of the to-be-detected sample at the ultraviolet wavelength; and finally, calculating the content of the sulfonamides in the milk to be detected according to the measured light absorption value and a pre-established standard curve relational expression. Compared with the prior art, the detection method is high in precision, sensitivity and accuracy and short in detection time.

Description

Technical Field

[0001] The present application relates to a method for detecting drug residues in milk, and specifically to a method for detecting sulfonamide drug residues in milk by combining electric field with spectrophotometry, and belongs to the technical field of food safety. Background Art

[0002] Milk is rich in nutrients such as protein (2.8%~3.3%), fat (2.8%~4%), vitamins and minerals (0.7%~0.75%, mainly Ca, K, Mg and P), which play a positive role in promoting human digestion, absorption and metabolism. It has become one of the daily nutritional diets of many families. However, with the rapid development of the dairy industry, the quality and safety of milk, especially the residues of antibiotics, has become one of the main safety hazards in the development of the industry. Among them, sulfonamides are synthetic drugs with antibacterial effects. Due to their high potency, low toxicity and broad antibacterial spectrum, they are widely used in the prevention and treatment of diseases such as dairy cow dysentery and mastitis. However, these drugs may remain in the body of dairy cows and enter the human body through milk. Long-term accumulation can easily lead to biological toxicity, drug resistance and allergic reactions, which in turn endangers human health. Therefore, it is necessary to detect the residues of sulfonamides in milk to avoid damage to the human body.

[0003] Currently, the commonly used methods for detecting sulfonamide residues in milk mainly include enzyme-linked immunosorbent assay, spectrophotometry, high-performance liquid chromatography and capillary electrophoresis. Among them, enzyme-linked immunosorbent assay has strong selectivity, but the detection accuracy is insufficient, and false positive or false negative results may occur; capillary electrophoresis has poor reproducibility; high-performance liquid chromatography can be used with detectors such as ultraviolet, fluorescence and diode array to achieve accurate and rapid detection of sulfonamides, but the detection throughput is low, and the instrument equipment cost and detection threshold are high. In contrast, ultraviolet spectrophotometry has the advantages of simple operation, low cost, high sensitivity and wide linear range, but the precision, accuracy and stability of detection still need to be improved. Summary of the invention

[0004] The main purpose of this application is to provide a method for detecting sulfonamide drug residues in milk, which is mainly based on spectrophotometry and combined with electric field processing, and can achieve high-precision, high-accuracy and high-stability detection, thereby overcoming the shortcomings of the prior art.

[0005] In order to achieve the aforementioned invention objectives, the technical solutions adopted in this application include: Some embodiments of the present application provide a method for detecting sulfonamide drug residues in milk, which comprises the following steps: S1, mixing the milk to be tested containing sulfonamide drugs with a deproteinizing agent to deproteinize the milk to be tested, then removing the solids in the deproteinized mixture, and adjusting the obtained clarified liquid to acidity, thereby obtaining a pretreated sample; S2, mixing the pretreated sample and the developer evenly to form a mixed sample; S3, applying a medium frequency oscillating electric field to the mixed sample, and allowing the mixed sample to react fully to form a sample to be tested, and keeping applying the medium frequency oscillating electric field to the sample to be tested, while measuring the absorbance value A of the sample to be tested at a wavelength of 454 nm, wherein the electric field strength of the medium frequency oscillating electric field is 1-5 V / cm, the frequency is 10-100 kHz, and the power is 1-5 W; S4. Calculate the content of sulfonamide drugs in the milk to be tested based on the relationship between A and the standard curve Y=50.723A+C, where the value of C is 0.205~11.035.

[0006] Among them, the sulfonamide drugs include sulfacetamide, sulfamethazine, sulfadimethoxine, sulfapyridine, sulfamethoxazole and the like, but are not limited thereto.

[0007] Compared with the prior art, the detection method provided in the present application applies a medium-frequency oscillating electric field during the condensation reaction of sulfonamide drugs with a color-developing substance, and uses the electric field to change the electron cloud distribution of the condensation reaction product (Schiff's base) of the sulfonamide drugs after reacting with dimethylaminobenzaldehyde under acidic conditions, so that the positive and negative electron cloud range approaches 454 nm, the absorption peak signal is enhanced, and the peak width is narrowed, thereby effectively improving the precision, sensitivity and accuracy of the detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 It is a schematic diagram of the principle of detecting sulfonamide drug residues in milk by using electric field combined with spectrophotometry in a typical implementation scheme of the present application.

[0009] Figure 2 It is the ultraviolet absorption spectrum of the condensation reaction product of sulfonamide drugs and color-developing substances in the range of 350-550 nm under the conditions of applying and not applying the medium frequency oscillating electric field in Example 1. DETAILED DESCRIPTION

[0010] The principle of using ultraviolet spectrophotometry to detect sulfonamide drug residues in the prior art is mainly that sulfonamide drugs can undergo condensation reactions with display substances such as p-dimethylaminobenzaldehyde to generate Schiff base condensation reaction products, which can absorb ultraviolet light with a wavelength in the range of 350 to 550 nm. Using this method, when sulfonamide drugs are qualitatively or semi-quantitatively detected, relatively accurate detection results can be obtained. However, if quantitative detection is to be performed, its precision, accuracy and stability still need to be improved. In response to these problems, the applicant was very surprised to find during the research that when sulfonamide drugs are detected by ultraviolet spectrophotometry, if a medium-frequency oscillating electric field is applied to the reaction system during the reaction and combination of sulfonamide drugs and display substances, it can not only promote the condensation reaction rate and improve the stability of the condensate structure, but also improve the sensitivity and accuracy of the detection. Based on such unexpected findings, the applicant was able to propose the technical solution of the present application, which relates to a method for detecting sulfonamide drug residues in milk. In some embodiments, the detection method comprises the following steps: S1. Mixing the milk to be tested containing sulfonamide drugs such as sulfacetamide, sulfamethazine, sulfadimethoxine, sulfapyridine, sulfamethoxazole or sulfathiazole with a deproteinizing reagent to deproteinize the milk to be tested, then removing the solids in the deproteinized mixture, and adjusting the obtained clarified liquid to acidity, thereby obtaining a pretreated sample; S2, mixing the pretreated sample and the developer evenly to form a mixed sample; S3, applying a medium frequency oscillating electric field to the mixed sample, and allowing the mixed sample to react fully to form a sample to be tested, and keeping applying the medium frequency oscillating electric field to the sample to be tested, while measuring the absorbance value A of the sample to be tested at a wavelength of 454 nm, wherein the electric field strength of the medium frequency oscillating electric field is 1-5 V / cm, the frequency is 10-100 kHz, and the power is 1-5 W; S4. Calculate the content of sulfonamides in the milk to be tested based on A and the standard curve relationship Y=50.723A+C, where the value of C is 0.205~11.035.

[0011] In one embodiment, the deproteinization reagent includes a clarifier, and the clarifier includes a combination of one or more of a potassium ferrocyanide solution with a concentration of 5wt% to 10wt% or a zinc acetate solution with a concentration of 10wt% to 30wt%, but is not limited thereto.

[0012] In one embodiment, the deproteinization reagent includes trichloroacetic acid solution or an organic solvent, such as a combination of one or more of trichloroacetic acid solution with a concentration of 10wt% to 30wt%, anhydrous ethanol, acidified acetonitrile or acetone, and is not limited thereto.

[0013] In one embodiment, the volume ratio of the milk to be tested to the deproteinized reagent is 1:1 to 5:1.

[0014] In one embodiment, the deproteinization treatment is performed at a temperature of 30-50° C. and for a time of 10-30 min.

[0015] In one embodiment, step S1 specifically comprises: removing solids in the deproteinized mixture by at least centrifugation, wherein the centrifugation speed is 4000-8000 rpm, the centrifugation time is 20-30 min, and collecting the clarified liquid.

[0016] In some cases, the solids in the deproteinized mixture can be removed by filtration, static sedimentation, etc. to obtain the clarified liquid.

[0017] In some cases, step S1 may further include: after the deproteinization process is completed, continuing to add an organic solvent for fat removal. Exemplarily, after the deproteinization process is completed and the solids in the deproteinized mixture are removed, an equal volume of n-hexane is added and mixed thoroughly (e.g., mixed for more than 5 minutes), and then centrifuged to remove fat (e.g., more than 8000 rpm, more than 5 minutes), and then the clarified liquid is collected.

[0018] In one embodiment, step S1 specifically includes: adjusting the pH value of the clarified liquid to 2-5, thereby obtaining the pretreated sample. The reagent used to adjust the pH value of the clarified liquid may be hydrochloric acid, phosphate buffer or other buffered salt solutions.

[0019] In one embodiment, the volume ratio of the pretreated sample to the developer in step S2 is 5:1-50:1, preferably 8:1-40:1, and more preferably 12.5:1-25:1, so as to achieve a better color development effect.

[0020] In one embodiment, the developer comprises a p-dimethylaminobenzaldehyde solution with a concentration of 1 wt % to 4 wt %.

[0021] In the present application, if the electric field strength of the medium frequency oscillating electric field is too large, some substances will be ionized and decomposed, affecting the stability of the color-developing substance, and the ion polarization layer on the electrode surface will be uneven, affecting the uniformity and stability of the color development. If the electric field strength of the medium frequency oscillating electric field is too small, the transfer between the charge and the molecular functional groups is insufficient, and the color development reaction cannot be effectively driven, the reaction rate slows down, the color development effect is not obvious, and a longer reaction time is required to achieve the expected color development effect.

[0022] In the present application, if the frequency of the medium frequency oscillating electric field is too high, the molecules or ions involved in the color development reaction will oscillate too fast in the electric field, causing the interaction between them to become complicated and unstable, destroying the selectivity of the color development reaction, that is, the substances of the color development reaction are disturbed. If the frequency of the medium frequency oscillating electric field is too low, the movement of the molecules and ions involved in the reaction and their interaction are restricted, the kinetic process of the reaction is slow, and the color development efficiency is reduced.

[0023] In the present application, if the power of the medium frequency oscillating electric field is too high, the excessive energy may destroy the structure of the substances involved in the reaction, affect the selectivity and accuracy of the reaction, cause some side reactions, and affect the color development degree and effect. If the power of the medium frequency oscillating electric field is too low, the interaction of molecules or ions involved in the color development reaction is also limited, some reaction substances cannot be fully combined with the color developer, less colored complexes are generated, and the absorbance is reduced, which affects the detection results.

[0024] In one embodiment, when detecting the absorbance value A in step S3, the transmission direction of the ultraviolet light acting on the sample to be tested and the electric field direction of the medium frequency oscillating electric field acting on the sample to be tested are perpendicular to each other.

[0025] In one embodiment, the detection method specifically includes: S2, adding the pretreated sample and the developer into a quartz cuvette with an optical path of 2 cm, and mixing them evenly to form the mixed sample; S3. Dispose two corrosion-resistant electrodes on the inner sides of two frosted side walls of the quartz cuvette containing the mixed sample, respectively, and arrange the two corrosion-resistant electrodes opposite to each other, and connect the two corrosion-resistant electrodes to an excitation power supply to generate the intermediate frequency oscillating electric field.

[0026] In the present application, the pre-treated sample and the color developer should be added to the cuvette in a certain order, otherwise it may lead to incomplete condensation reaction and unstable color development.

[0027] In the present application, while measuring the absorbance value A of the sample to be tested at a wavelength of 454 nm, the medium frequency oscillating electric field should be applied to the sample to be tested, otherwise the data during detection is prone to fluctuation and poor repeatability.

[0028] Further, the corrosion-resistant electrode includes one of a platinum electrode, a stainless steel electrode or a titanium alloy electrode, but is not limited thereto. The corrosion-resistant electrode may be in the form of a sheet or a plate, etc., and its size should at least meet the following requirements: the mixed sample and the sample to be tested are completely contained in the medium frequency oscillating electric field formed between the two corrosion-resistant electrodes.

[0029] Furthermore, the excitation power supply is a variable frequency AC power supply.

[0030] In one embodiment, step S3 is performed in an environment with a temperature of 20-30°C.

[0031] In one embodiment, the reaction time of the reaction in step S3 is 1 to 3 min.

[0032] In one embodiment, step S3 further includes: when detecting the absorbance value A, a blank sample is prepared with distilled water and a color developer for reference. The specific operation may include: replacing the pre-treated sample in step S2 with distilled water, and then performing step S3 to measure the absorbance value of the blank sample at a wavelength of 454nm, and then subtracting the absorbance value of the blank sample from the measured value of the absorbance value of the sample to be tested at a wavelength of 454nm to obtain A.

[0033] In one embodiment, the detection method specifically comprises: taking a series of sulfonamide drug solutions of different concentrations as standard samples, and detecting the absorbance values ​​of the samples to be tested formed by the reaction of different standard samples with a color developer at a wavelength of 454 nm according to the operations of steps S1 to S3, thereby establishing a relationship curve between the sulfonamide drug content of each standard sample and the corresponding absorbance value, and then obtaining the standard curve relationship. Each standard sample contains the same sulfonamide drug, and the concentration of the sulfonamide drug in each standard sample is known.

[0034] In the present application, the detection of blank samples, pre-treated samples and standard samples containing the same sulfonamide drug is carried out based on exactly the same detection conditions.

[0035] In one embodiment, the value of C may preferably be 0.295-10.855.

[0036] The above embodiments of the present application provide a method for detecting sulfonamide drug residues in milk. By combining electric field treatment on the basis of ultraviolet spectrophotometry, especially continuously applying medium-frequency oscillating electric field treatment during the condensation reaction of sulfonamide drugs and color-developing substances and during the process of detecting the absorbance of the condensation reaction product (Schiff's base), the electron cloud distribution of the condensation reaction product can be changed, so that the positive and negative electron cloud range is close to 454 nm, the absorption peak signal is enhanced, and the peak width is narrowed, thereby effectively improving the precision, sensitivity and accuracy of the detection method.

[0037] The technical solution, its implementation process and principle, etc. will be further explained in conjunction with several embodiments as follows. However, it should be understood that within the scope of this application, the above-mentioned technical features of this application and the technical features specifically described in the following (embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0038] In the following examples of the present application, unless otherwise specified, the various raw materials, reagents, reaction equipment, testing equipment and methods used can be obtained through commercial purchase and other channels.

[0039] In addition, in the following examples of the present application, the calculation methods of the spike recovery θ (%) and the relative deviation (%) of the detection results of different methods are respectively: Calculation of spike recovery θ (%): Add 0.05 mL of 0.5 g / L sulfonamide standard solution to 5 mL of pretreated sample (sulfonamide concentration is Y0), calculate the spike concentration Y1, and perform detection according to the steps described in S1~S3 to obtain the corresponding absorbance value A. Then, based on the relationship between A and the standard curve, calculate the measured value Y2 of the sulfonamide content in the spiked milk to be tested, then the spike recovery θ (%) = (Y2-Y0) / Y1×100.

[0040] Calculation of relative deviation (%) of the test results of different methods: Let the test result of this implementation method be Y a , the control method test result is Y b , then the relative deviation (%) = (Y a -Y b ) / Y c ×100, where Y c is the average of the two test results.

[0041] Stability test: within 10 minutes after the reaction, the sulfonamide content of the sample was tested every 2 minutes, and the relative standard deviation was calculated.

[0042] The following will further explain the technical solution, its implementation process and principle of the present application in conjunction with several embodiments. However, it should be understood that within the scope of the present application, the above-mentioned technical features of the present application and the technical features specifically described in the following (embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0043] In the following examples, unless otherwise specified, the various raw materials, reagents, reaction equipment, testing equipment and methods used can be obtained through commercial purchase or other channels.

[0044] Example 1 Please refer to Figure 1 A method for detecting sulfamethazine drug residues in pure milk comprises the following steps: S1. Take 10 mL of pure milk containing sulfamethazine, mix it with 1 mL of 8% (weight % unless otherwise specified) potassium ferrocyanide solution and 1 mL of 20% zinc acetate solution, and then let it stand in a 40 °C water bath for 20 min to fully precipitate the protein. Then centrifuge it at 8000 rpm for 20 min, collect the clarified liquid, and adjust the pH value to 3 with 0.1 mol / L HCl solution to obtain the pretreated sample.

[0045] S2, take 5 mL of the pre-treated sample 1 and add it to a quartz cuvette 2 with an optical path of 2 cm, then add 0.2 mL of a 3% p-dimethylaminobenzaldehyde solution, mix thoroughly, and obtain a mixed sample. At the same time, replace the pre-treated sample with 5 mL of distilled water, and prepare a blank sample according to the same operation. A platinum electrode is inserted into the inner side of each frosted side wall 21 of the quartz cuvette, and its smooth side wall 22 serves as a light-transmitting surface for ultraviolet light to pass through for detection.

[0046] S3. Place the quartz cuvette containing the mixed sample in a spectrophotometer, set the ambient temperature to 25°C, and connect the two platinum electrodes 3 to the excitation power supply 4 to generate a medium-frequency oscillating electric field and act on the mixed sample. The electric field strength of the medium-frequency oscillating electric field is 5 V / cm, the frequency is 100 kHz, and the power is 5 W. Allow to react for 2 minutes, then continue to apply the medium-frequency oscillating electric field to the formed sample to be tested, and use the blank sample as a reference to measure the absorbance value A of the sample to be tested at a wavelength of 454 nm.

[0047] Standard curve determination: Take a series of sulfamethazine solutions with concentrations of 0, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, and 65 μg / kg as standard samples, and determine the absorbance of these standard samples at a wavelength of 454 nm according to the methods described in S1~S3. By collecting the obtained data and building a model, the relationship between the sulfamethazine content Y in the standard sample and the absorbance value A is obtained, Y=50.723A+0.295, and the detection range is 4.75~49.50 μg / kg.

[0048] Figure 2 The ultraviolet absorption spectra of the Schiff base product formed by the reaction of sulfamethazine and p-dimethylaminobenzaldehyde in the wavelength range of 350-550 nm are shown when the standard sample with a concentration of 40 μg / kg is tested under the conditions of applying and not applying the medium frequency oscillating electric field in this embodiment.

[0049] For pure milk to be tested with unknown sulfamethazine content, the test is performed according to the steps described in S1 to S3 to obtain its corresponding absorbance value A, and then the sulfamethazine content in the pure milk to be tested can be calculated based on the relationship between A and the above standard curve.

[0050] The methods of this embodiment and GB / T 21316-2007 (national standard method) were used to detect the content of sulfamethazine in 8 commercially available pure milks. As shown in Table 1, the relative standard deviation of the detection results of the method of this embodiment was always less than 3%, the spike recovery rate fluctuated between 94.29% and 98.32%, and the relative standard deviation of the continuous detection results was always less than 2.33%, indicating that the method has high precision and stability. In addition, compared with the method described in GB / T 21316-2007, the relative deviation of the detection results was always less than ±4%, and the accuracy was high.

[0051] Table 1 Detection results of sulfamethazine content in 8 commercially available pure milks by the method of Example 1

[0052] Example 2 A method for detecting sulfacetamide drug residues in skim milk comprises the following steps: S1. Take 10 mL of skim milk containing sulfacetamide and 2 mL of 20% trichloroacetic acid solution and mix them thoroughly. Let it stand in a 50 ℃ water bath for 10 min to fully precipitate the protein. Then centrifuge it at 6000 rpm for 30 min, collect the clarified liquid, and adjust the pH value to 2 with 0.1 mol / L HCl solution to obtain the pretreated sample.

[0053] S2. Take 5 mL of the pretreated sample and add it to a quartz cuvette with an optical path of 2 cm, then add 0.4 mL of a 4% p-dimethylaminobenzaldehyde solution, mix thoroughly, and obtain a mixed sample. At the same time, replace the pretreated sample with 5 mL of distilled water, and prepare a blank sample according to the same operation. An electrode is inserted into the inner side of each frosted side wall of the quartz cuvette. The electrode is a titanium alloy electrode.

[0054] S3. Place the quartz cuvette containing the mixed sample in a spectrophotometer, set the ambient temperature to 20°C, and connect the two titanium alloy electrodes to an excitation power supply to generate a medium-frequency oscillating electric field and apply it to the mixed sample. The electric field strength of the medium-frequency oscillating electric field is 1 V / cm, the frequency is 10 kHz, and the power is 1 W. Allow the mixture to react for 3 minutes, then continue to apply the medium-frequency oscillating electric field to the formed sample to be tested, and use the blank sample as a reference to determine the absorbance value A of the sample to be tested at a wavelength of 454 nm.

[0055] Standard curve determination: Take a series of sulfacetamide solutions with concentrations of 0, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, and 65 μg / kg as standard samples, and determine the absorbance values ​​of these standard samples at a wavelength of 454 nm according to the steps described in S1-S3. By collecting the obtained data and building a model, the relationship between the sulfacetamide content Y in the standard sample and the absorbance value A is obtained, which is Y=50.723A+0.397, and the detection range is 4.47-36.89 μg / kg.

[0056] For the skim milk to be tested with unknown sulfacetamide content, the test is performed according to the steps described in S1 to S3 to obtain its corresponding absorbance value A, and then the sulfacetamide content in the skim milk to be tested is calculated based on the relationship between A and the above standard curve.

[0057] The methods of this embodiment and GB / T 21316-2007 (national standard method) were used to detect the sulfacetamide content in 8 commercial skim milks. As shown in Table 2, the relative standard deviation of the detection results of the method of this embodiment was always less than 4%, the spike recovery rate fluctuated between 92.12% and 96.55%, and the relative standard deviation of the continuous detection results was always below 3.55%, indicating that the method has high precision and stability. In addition, compared with the method described in GB / T 21316-2007, the relative deviation of the detection results was always less than ±4%, and the accuracy was high.

[0058] Table 2 Test results of sulfacetamide content in 8 commercial skim milks by the method of Example 2

[0059] Example 3 A method for detecting sulfadimethoxine drug residues in whole milk comprises the following steps: S1. Take 5 mL of whole milk containing sulfadimethoxine and 5 mL of 30% acidified acetonitrile and mix them thoroughly. Let it stand in a 30 ℃ water bath for 30 min to fully precipitate the protein. Then centrifuge it at 8000 rpm for 20 min, collect the clarified liquid, add 3 mL of n-hexane and mix it thoroughly for 5 min, then centrifuge it at 8000 rpm for 5 min, take the lower clear liquid, adjust the pH value to 5 with phosphate buffer solution, and obtain the pretreated sample.

[0060] S2. Take 5 mL of the pretreated sample and add it to a quartz cuvette with an optical path of 2 cm, then add 0.4 mL of 1% p-dimethylaminobenzaldehyde solution, mix thoroughly, and obtain a mixed sample. At the same time, replace the pretreated sample with 5 mL of distilled water, and prepare a blank sample according to the same operation. An electrode is inserted into the inner side of each frosted side wall of the quartz cuvette. The electrodes are stainless steel electrodes.

[0061] S3. Place the quartz cuvette containing the mixed sample in a spectrophotometer, set the ambient temperature to 30°C, and connect the two electrodes to an excitation power supply to generate a medium-frequency oscillating electric field and apply it to the mixed sample. The electric field strength of the medium-frequency oscillating electric field is 2 V / cm, the frequency is 60 kHz, and the power is 3 W. Allow it to react for 1 min, then continue to apply the medium-frequency oscillating electric field to the formed sample to be tested, and use the blank sample as a reference to measure the absorbance value A of the sample to be tested at a wavelength of 454 nm.

[0062] Standard curve determination: Take a series of sulfamethazine solutions with concentrations of 0, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, and 65 μg / kg as standard samples, and determine the absorbance of these standard samples at a wavelength of 454 nm according to the steps described in S1-S3. By collecting the obtained data and building a model, the relationship between the sulfamethazine content Y in the standard sample and the absorbance value A is obtained, which is Y=50.723A+10.855. The detection range is 11.86-50.50 μg / kg.

[0063] For the whole milk to be tested with unknown sulfadimethoxine content, the test is performed according to the steps described in S1 to S3 to obtain the corresponding absorbance value A, and then the sulfadimethoxine content in the whole milk to be tested is calculated based on A and the relationship between the above standard curve.

[0064] The methods of this embodiment and GB / T 21316-2007 (national standard method) were used to detect the content of sulfadimethoxine in 8 commercial whole milks. As shown in Table 3, the relative standard deviation of the detection results of the method of this embodiment was always less than 4%, the spike recovery rate fluctuated between 91.23% and 96.11%, and the relative standard deviation of the continuous detection results was always less than 3.16%, indicating that the method has high precision and stability. In addition, compared with the method described in GB / T 21316-2007, the relative deviation of the detection results was always less than ±3%, and the accuracy was high.

[0065] Table 3 Detection results of sulfadimethoxine content in 8 portions of commercially available whole milk using the method of Example 3

[0066] Comparative Example 1 The method for detecting sulfamethoxazole drug residues in pure milk provided in this comparative example is basically the same as that in Example 1, except that: in step S3, no medium frequency oscillating electric field treatment is applied.

[0067] Standard curve determination: Take a series of sulfamethazine solutions with concentrations of 0, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, and 65 μg / kg as standard samples (the same as in Example 1), and determine the absorbance values ​​of these standard samples at a wavelength of 454 nm according to the steps described in the comparative example. By collecting the obtained data and modeling, the relationship between the sulfamethazine content Y and the absorbance value A in the standard sample is obtained, Y=48.534A+2.461, and the detection range is 4.89~49.50 μg / kg.

[0068] For pure milk to be tested with unknown sulfamethazine content, the test is performed according to the steps described in this comparative example to obtain its corresponding absorbance value A, and then the sulfamethazine content in the pure milk to be tested is calculated based on A and the relationship between the above standard curve.

[0069] The content of sulfamethazine in 8 portions of commercially available pure milk (the same as Example 1) was detected by the method of this comparative example and the method of GB / T 21316-2007 (national standard method), respectively. As shown in Table 4, the relative standard deviation of the detection results of the comparative example method was always higher than 3%, and the maximum reached 7.75%, the spiked recovery rate fluctuated between 84.37% and 91.00%, and the relative standard deviation of the continuous detection results varied in the range of 3.75% to 6.83%, indicating that the precision and reproducibility of the method were poor. In addition, compared with the detection results of the method described in GB / T 21316-2007, the relative deviation was larger, up to 6.13%, indicating that the accuracy of the detection results was insufficient, which may be related to the fact that the medium-frequency oscillating electric field was not applied during the reaction process, resulting in a slowdown in the condensation rate and a decrease in the stability of the compound.

[0070] Table 4 Comparative Example 1 method for the detection results of sulfamethazine content in 8 commercially available pure milk

[0071] Comparative Example 2 The method for detecting sulfamethoxazole drug residues in pure milk provided in this comparative example is basically the same as that in Example 1, except that the electric field strength of the medium frequency oscillating electric field used in step S3 is 6 V / cm.

[0072] Standard curve determination: Take a series of sulfamethazine solutions with concentrations of 0, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, and 65 μg / kg as standard samples (the same as in Example 1), and determine the absorbance values ​​of these standard samples at a wavelength of 454 nm according to the steps described in the comparative example. By collecting the obtained data and modeling, the relationship between the sulfamethazine content Y and the absorbance value A in the standard sample is obtained, Y=42.174A+4.738, and the detection range is 5.75~45.85 μg / kg.

[0073] For pure milk to be tested with unknown sulfadiazine content, the test is performed according to the steps described in this comparative example to obtain its corresponding absorbance value A, and then the sulfadiazine sodium content in the pure milk to be tested is calculated based on A and the relationship between the above standard curve.

[0074] The content of sulfamethoxazole in 8 portions of commercially available pure milk (the same as Example 1) was detected by the method of this comparative example and the method of GB / T 21316-2007 (national standard method), respectively. As shown in Table 5, the relative standard deviation of the detection results of the comparative example method was always higher than 4%, and the maximum reached 6.62%, the spiked recovery rate fluctuated between 86.44% and 90.72%, and the relative standard deviation of the continuous detection results varied in the range of 3.03% to 5.21%, indicating that the precision and reproducibility of the method were poor. In addition, compared with the detection results of the method described in GB / T 21316-2007, the relative deviation was larger, up to 6.58%, indicating that the accuracy of the detection results was insufficient, which may be related to the high electric field strength of the medium-frequency oscillating electric field applied during the reaction process, resulting in rapid temperature rise in the reaction system and reduced stability of the compound.

[0075] Table 5 Comparative Example 2 Method for Testing the Content of Sulfadiazine in 8 Commercially Available Pure Milks

[0076] Comparative Example 3 The method for detecting sulfamethoxazole drug residues in pure milk provided in this comparative example is basically the same as that in Example 1, except that the electric field strength of the medium frequency oscillating electric field used in step S3 is 0.8 V / cm.

[0077] Standard curve determination: Take a series of sulfamethazine solutions with concentrations of 0, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, and 65 μg / kg as standard samples (the same as in Example 1), and determine the absorbance values ​​of these standard samples at a wavelength of 454 nm according to the steps described in the comparative example. By collecting the obtained data and modeling, the relationship between the sulfamethazine content Y and the absorbance value A in the standard sample is obtained, Y=48.126A+3.645, and the detection range is 4.85~46.90 μg / kg.

[0078] For pure milk to be tested with unknown sulfadiazine content, the test is performed according to the steps described in this comparative example to obtain its corresponding absorbance value A, and then the sulfadiazine sodium content in the pure milk to be tested is calculated based on A and the relationship between the above standard curve.

[0079] The content of sulfamethoxazole in 8 portions of commercially available pure milk (the same as Example 1) was detected by the method of this comparative example and the method of GB / T 21316-2007 (national standard method), respectively. As shown in Table 6, the relative standard deviation of the detection results of the comparative example method was always higher than 4%, and the maximum reached 6.49%, the spiked recovery rate fluctuated between 85.58% and 89.81%, and the relative standard deviation of the continuous detection results varied in the range of 3.01% to 5.16%, indicating that the precision and reproducibility of the method were poor. In addition, compared with the detection results of the method described in GB / T 21316-2007, the relative deviation was large, up to 6.77%, indicating that the accuracy of the detection results was insufficient, which may be related to the small electric field strength of the medium-frequency oscillating electric field applied during the reaction process, resulting in poor stability of the final product compound of the reaction system, and the weakened influence of the electric field on the electron cloud.

[0080] Table 6 Test results of sulfamethazine content in 8 commercially available pure milks by comparative example 3

[0081] Comparative Example 4 The method for detecting sulfamethoxazole drug residues in pure milk provided in this comparative example is basically the same as that in Example 1, the only difference is that in step S3, after the reaction is completed, the medium frequency oscillating electric field is removed, and the absorbance value A of the sample to be tested at a wavelength of 454 nm is measured in the absence of the medium frequency oscillating electric field.

[0082] Standard curve determination: Take a series of sulfamethazine solutions with concentrations of 0, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, and 65 μg / kg as standard samples (the same as in Example 1), and determine the absorbance values ​​of these standard samples at a wavelength of 454 nm according to the steps described in the comparative example. By collecting the obtained data and modeling, the relationship between the sulfamethazine content Y and the absorbance value A in the standard sample is obtained, Y=49.232A+0.477, and the detection range is 4.40~48.50 μg / kg.

[0083] For pure milk to be tested with unknown sulfadiazine content, the test is performed according to the steps described in this comparative example to obtain its corresponding absorbance value A, and then the sulfadiazine sodium content in the pure milk to be tested is calculated based on A and the relationship between the above standard curve.

[0084] The content of sulfamethoxazole in 8 portions of commercially available pure milk (the same as Example 1) was detected by the method of this comparative example and the method of GB / T 21316-2007 (national standard method), respectively. As shown in Table 7, the relative standard deviation of the detection results of the comparative example method fluctuated between 2.82% and 4.58%, the spiked recovery rate fluctuated between 89.16% and 92.86%, and the relative standard deviation of the continuous detection results varied in the range of 1.50% to 3.66%. In addition, compared with the detection results of the method described in GB / T 21316-2007, the relative deviation was larger, up to -4.67%, indicating that the accuracy and reproducibility of the detection results were poor. It may be that the medium frequency oscillating electric field was not applied during the detection process, and the final product after the system reaction was still unstable, resulting in a poor color development effect during the detection period and a decrease in detection accuracy.

[0085] Table 7 Comparative Example 4 Method for the detection of sulfamethazine content in 8 commercially available pure milk

[0086] In addition, the applicant also referred to Example 1 and verified the accuracy of detecting the sulfamethazine content in 8 portions of commercially available pure milk (the same as in Example 1) when the frequency of the medium-frequency oscillating electric field was lower than 10 kHz, higher than 100 kHz, lower than 1 W, and higher than 5 W, respectively, with the other conditions remaining unchanged. However, the accuracy and reproducibility of the test results were poor.

[0087] The above is a detailed description of the present application in combination with the embodiments, but the implementation methods of the present application are not limited to the above embodiments, and any other changes, replacements, combination simplifications, etc. made under the core guiding idea of ​​the present application are included in the protection scope of the patent of this application.

Claims

1. A method for detecting sulfonamide residues in milk, characterized in that: The steps include: S1, mixing the milk to be tested containing sulfonamide drugs with a deproteinizing agent to deproteinize the milk to be tested, then removing the solids in the deproteinized mixture, and adjusting the obtained clarified liquid to acidity, thereby obtaining a pretreated sample; S2, mixing the pretreated sample and the developer evenly to form a mixed sample; S3, applying a medium frequency oscillating electric field to the mixed sample, and allowing the mixed sample to react fully to form a sample to be tested, and keeping applying the medium frequency oscillating electric field to the sample to be tested, while measuring the absorbance value A of the sample to be tested at a wavelength of 454 nm, wherein the electric field strength of the medium frequency oscillating electric field is 1-5 V / cm, the frequency is 10-100 kHz, and the power is 1-5 W; S4. Calculate the content of sulfonamides in the milk to be tested based on A and the standard curve relationship Y=50.723A+C, where the value of C is 0.205~11.

035.

2. The detection method according to claim 1, characterized in that: The sulfonamide drug includes a combination of one or more of sulfacetamide, sulfamethazine, sulfadimethoxine, sulfapyridine, sulfamethoxazole or sulfathiazole; And / or, the deproteinizing agent includes a clarifier, the clarifier includes a combination of one or more of a potassium ferrocyanide solution with a concentration of 5wt% to 10wt% or a zinc acetate solution with a concentration of 10wt% to 30wt%, or the deproteinizing agent includes a combination of one or more of a trichloroacetic acid solution with a concentration of 10wt% to 30wt%, anhydrous ethanol, acidified acetonitrile or acetone; And / or, the volume ratio of the milk to be tested to the deproteinization reagent is 1:1 to 5:1; And / or, the deproteinization treatment is performed at a temperature of 30-50° C. and for a time of 10-30 min.

3. The detection method according to claim 1, characterized in that: Step S1 specifically includes: At least centrifugation is used to remove solids in the deproteinized mixture, wherein the centrifugation speed is 4000-8000 rpm, the centrifugation time is 20-30 min, and the clarified liquid is collected; And / or, the clarified liquid is adjusted to a pH value of 2-5, thereby obtaining the pretreated sample.

4. The detection method according to claim 1, characterized in that: In step S2, the volume ratio of the pretreated sample to the developer is 5:1 to 50:1; and / or, the developer comprises a p-dimethylaminobenzaldehyde solution with a concentration of 1 wt% to 4 wt%.

5. The detection method according to claim 1, characterized in that: When detecting the absorbance value A in step S3, the transmission direction of the ultraviolet light acting on the sample to be tested and the electric field direction of the medium frequency oscillating electric field acting on the sample to be tested are perpendicular to each other.

6. The detection method according to claim 5, characterized in that: The detection method specifically comprises: S2, adding the pretreated sample and the developer into a quartz cuvette with an optical path of 2 cm, and mixing them evenly to form the mixed sample; S3. Dispose two corrosion-resistant electrodes on the inner sides of two frosted side walls of the quartz cuvette containing the mixed sample, and arrange the two corrosion-resistant electrodes opposite to each other. Also, connect the two corrosion-resistant electrodes to an excitation power source to generate the intermediate frequency oscillating electric field.

7. The detection method according to claim 6, characterized in that: The corrosion-resistant electrode comprises one of a platinum electrode, a stainless steel electrode or a titanium alloy electrode; and / or the excitation power supply is a variable frequency AC power supply.

8. The detection method according to any one of claims 1, 5 and 6, characterized in that: Step S3 is performed in an environment with a temperature of 20-30° C.; and / or, the reaction time of the reaction in step S3 is 1-3 min.

9. The detection method according to any one of claims 1, 5 and 6, characterized in that: Step S3 also includes: when detecting the absorbance value A, a blank sample is prepared with distilled water and a color developer for reference.

10. The detection method according to claim 1, characterized in that: Specifically, the method comprises: taking a series of sulfonamide drug solutions of different concentrations as standard samples, and detecting the absorbance values ​​of the samples to be tested formed by the reaction of different standard samples with a color developer at a wavelength of 454 nm according to the operations of steps S1 to S3, thereby establishing a relationship curve between the sulfonamide drug content of each standard sample and the corresponding absorbance value, and then obtaining the standard curve relationship formula.

Images