A method for rapid detection of sulfonamides in meat using a modified QuEChERS combined with enzyme inhibition colorimetric method

Abstract

The present invention discloses a method for rapidly detecting sulfonamides in meat by combining a modified QuEChERS method with an enzyme inhibition colorimetric method. The method uses a natural deep eutectic solvent instead of a toxic organic solvent as an extractant, thereby reducing damage to the environment and human body, improving extraction efficiency, and protecting enzyme activity, making detection more sensitive and stable. A magnetic COF (TpPa-Cl)@Fe3O4, synthesized by a simple and rapid stirring method, not only has good chemical stability and can be reused multiple times, but also has excellent adsorption capacity and strong magnetism. Using it as an adsorbent for the modified QuEChERS method, a small amount can effectively remove interfering substances in the sample, shortening the pretreatment time and simplifying the purification process. During the enzyme inhibition colorimetric detection process, quantitative analysis is performed in combination with a smartphone digital image colorimetric method. The method has significant advantages such as being green, efficient, time-saving, low-cost, simple to operate, and highly sensitive.

Description

Technical Field

[0001] The invention belongs to the technical field of food safety detection, and specifically discloses a method for quickly detecting sulfonamides in meat by using an improved QuEChERS combined with enzyme inhibition colorimetry. Background Art

[0002] The QuEChERS method, proposed by Anastassiades et al. in 2003, has attracted considerable attention for its advantages, including high sample throughput, minimal solvent consumption, low cost, simplicity, effectiveness, and high recovery. Despite its significant advantages, the QuEChERS method still requires further improvement. The traditional QuEChERS method uses acetonitrile as the extractant and ethylenediamine-N-propylsilane (PSA), graphitized carbon black (GCB), octadecylsilane (C18), multi-walled carbon nanotubes (MWCNTs), and graphene as cleanup adsorbents. The primary toxicity of acetonitrile comes from its effects on the central nervous system, and its use violates the principles of green chemistry. Furthermore, traditional adsorbents are expensive and require additional centrifugation and filtration, which increases pretreatment time. Therefore, there is an urgent need to develop new, environmentally friendly, and low-cost extractants and adsorbents to improve extraction and cleanup efficiency. Summary of the Invention

[0003] The present invention proposes a method for rapidly detecting sulfonamides in meat by combining an improved QuEChERS method with enzyme inhibition colorimetry, so as to overcome the defects of the traditional QuEChERS method.

[0004] The method for rapid detection of sulfonamides in meat using the modified QuEChERS combined with enzyme inhibition colorimetry is targeted at one or more of sulfadiazine, sulfonamide, sulfathiazole, sulfamethoxazine, and sulfadimethoxine, and comprises the following steps:

[0005] S1, using any one of ethylene glycol, propylene glycol, octanoic acid, and butyric acid as a hydrogen bond donor and any one of choline chloride, betaine, thymol, vanillin, and tetrabutylammonium chloride as a hydrogen bond acceptor, heating in a water bath to form a clear and transparent liquid, to obtain six different natural deep eutectic solvents as extractants: ethylene glycol-choline chloride, ethylene glycol-betaine, propylene glycol-betaine, octanoic acid-thymol, butyric acid-vanillin, and butyric acid-tetrabutylammonium chloride;

[0006] S2, adding any one of the natural deep eutectic solvents prepared in step S1 to the meat sample, vortex-mixing, centrifuging, and extracting the liquid to obtain an extract a;

[0007] S3, adding an adsorbent to the extract a obtained in step S2, and obtaining an extract b after separation and impurity removal;

[0008] The adsorbent is any one of ethylenediamine-N-propylsilane, octadecylsilane, graphitized carbon black, graphene, multi-walled carbon nanotubes, Fe3O4 or COF (TpPa-Cl)@Fe3O4;

[0009] S4, the extract b obtained in step S3 is placed in a container and mixed with a freshly prepared carbonic anhydrase solution, phosphate buffer, and p-nitrophenyl acetate solution. After incubating the mixture, the container is placed in an opaque white box, the top of the white box is evenly illuminated by an LED light panel, and the color change of the mixture in the container is captured using a smartphone. Then, Adobe Photoshop CC software is used to read the channel values ​​of the red, green, and blue channels of the active site in RGB mode, and the intensity of the sulfonamide to be detected in the meat sample is calculated;

[0010] S5. Prepare standards of different sulfonamides, mix them with freshly prepared carbonic anhydrase solution, phosphate buffer, and p-nitrophenyl acetate solution in a container, incubate the mixture, and then place the container in an opaque white box. The top of the white box is evenly illuminated by an LED light panel. Use a smartphone to capture the color changes of the mixture in the container. Then use Adobe Photoshop CC software to read the channel values ​​of the red, green, and blue channels of the active site in RGB mode, and calculate the intensity of the standard. Draw a standard working curve for different sulfonamides with the standard concentration as the horizontal axis and the corresponding intensity as the vertical axis. Substitute the intensity of the sulfonamide to be detected in the meat sample obtained in step S4 into the standard working curve to obtain the concentration of the sulfonamide in the meat sample.

[0011] Specifically, in step S1, the mixing ratio of the hydrogen bond donor and the hydrogen bond acceptor is 1:1 to 1:6 in molar ratio, the water bath temperature is 80° C., and the water content of the natural deep eutectic solvent is 0%.

[0012] Preferably, in step S2, the natural deep eutectic solvent selected is propylene glycol-betaine, and the propylene glycol-betaine mixing ratio is 1:4 in molar ratio.

[0013] Specifically, in step S2, the meat sample and the natural deep eutectic solvent are mixed according to the ratio of 0.5-3 g: 200-800 μL; the vortex time is 0.5-2 min; the centrifugation time is 90 s, and the rotation speed is 5000 r / min.

[0014] Preferably, in step S3, the adsorbent selected is COF (TpPa-Cl)@Fe3O4, and the extract a and COF (TpPa-Cl)@Fe3O4 are mixed in a ratio of 1 mL:10 mg. After vortexing, the impurities are removed by adsorption in an external magnetic field to obtain the extract b.

[0015] Specifically, in step S3, COF (TpPa-Cl)@Fe3O4 is prepared at room temperature using a stirring method, comprising the following steps:

[0016] Fe3O4 was added to dimethyl sulfoxide at a ratio of 1.2 mg:1 mL and ultrasonically dispersed uniformly. Then, 1,3,5-triformylphthalenetriol and 4-chloro-m-phenylenediamine were added respectively, with 0.63 mg of 1,3,5-triformylphthalenetriol and 0.9626 mg of 4-chloro-m-phenylenediamine corresponding to each 1 mL of dimethyl sulfoxide. Ultrasonication was performed again, and then stirred and glacial acetic acid was added, with 0.02 mL of glacial acetic acid corresponding to each 1 mL of dimethyl sulfoxide. After the reaction, the mixture was allowed to stand to obtain a brown-yellow product. The supernatant was washed with methanol and water several times until it was colorless and dried in a vacuum drying oven to obtain COF (TpPa-Cl)@Fe3O4.

[0017] Specifically, in steps S4 and S5, the concentration of the carbonic anhydrase solution is 100-350 U·mL -1 The concentration of phosphate buffer was 10.0 mM, and the pH range was 6.5–9. The concentration of p-nitrophenyl acetate solution was 0.02–0.12 mg mL -1 ;

[0018] In step S4, the volume ratio of the extract b, carbonic anhydrase solution, phosphate buffer, and p-nitrophenyl acetate solution is 1:0.5:2:1;

[0019] In step S5, the volume ratio of the standard, carbonic anhydrase solution, phosphate buffer, and p-nitrophenyl acetate solution is 1:0.5:2:1.

[0020] Specifically, in steps S4 and S5, the incubation temperature is 20-70° C., and the incubation time is 1-6 min.

[0021] Specifically, in steps S4 and S5, the intensity I =280-G, where G is the channel value of the green channel.

[0022] Preferably, in step S2, the meat sample and propylene glycol-betaine are mixed at a ratio of 1 g:500 μL, vortexed for 1 min, and centrifuged for 90 s; in steps S4 and S5, the concentration of the carbonic anhydrase solution is 300 U·mL -1 The concentration of phosphate buffer was 10.0 mM and the pH was 8.5. The concentration of p-nitrophenyl acetate solution was 0.1 mg mL -1 ; The incubation temperature was 40 ℃ and the incubation time was 3 min.

[0023] Compared with the prior art, the present invention has the following beneficial effects.

[0024] 1. This invention provides a method for rapid detection of sulfonamides in meat using a modified QuEChERS-based enzyme inhibition colorimetric method. Based on a natural deep eutectic solvent and the adsorbent COF (TpPa-Cl)@Fe₃O₄, this method combines a modified QuEChERS-based method with carbonic anhydrase for smartphone-based colorimetric detection of sulfonamide residues in pork. This method is rapid, sensitive, simple, and accurate, providing a valuable tool for routine monitoring and laying the foundation for further food safety risk assessment.

[0025] 2. The improved QuEChERS combined with enzyme inhibition colorimetry provided by the present invention is a method for rapid detection of sulfonamides in meat. It improves the traditional QuEChERS method and uses a new green solvent, a natural deep eutectic solvent, instead of traditional organic solvents such as acetonitrile as an extractant. The natural deep eutectic solvent has the advantages of simple preparation, low melting point, high volatility, low cost, and strong solubility for both hydrophilic and hydrophobic substances. It can achieve green and environmentally friendly extraction of sulfonamides in pork. At the same time, the natural deep eutectic solvent is safe and stable, and can protect the activity of the enzyme during subsequent detection.

[0026] 3. The present invention uses COF (TpPa-Cl)@Fe3O4 instead of the traditional QuEChERS adsorbent. COF (TpPa-Cl)@Fe3O4 combines the advantages of covalent organic framework materials and magnetic nanoparticles of Fe3O4, making it an ideal adsorbent for QuEChERS. Its magnetic separation speed is 10 times faster than the centrifugation process of traditional QuEChERS adsorbents. COF (TpPa-Cl)@Fe3O4 is prepared at room temperature using a simple and convenient stirring method. It has the advantages of large specific surface area, strong magnetic properties, good stability, low cost, and reusability. It can achieve effective purification effects with a small amount. This improved QuEChERS can achieve rapid and environmentally friendly extraction and purification of sulfonamides in pork.

[0027] 4. The method provided by the present invention, based on enzyme inhibition colorimetry, can directly detect sulfonamides through color changes, eliminating the need for complex detection procedures. Directly utilizing natural carbonic anhydrase to detect sulfonamides offers advantages such as high sensitivity, short response time, simple operation, low cost, and good selectivity. Smartphones, due to their portability, diverse information transmission capabilities, and storage capacity, are the most convenient image acquisition tools and currently the most promising platform for on-site monitoring and portable testing. Combining enzyme inhibition colorimetric detection with smartphones significantly improves the accuracy of test results and shortens detection time.

[0028] 5. The method provided by the present invention can process a large number of samples at one time when testing, and the amount of reagents used to process the samples is small, which is green and environmentally friendly.

[0029] 6. The method provided by the present invention is time-saving, and the entire sample pretreatment and detection process only takes 5 minutes.

[0030] 7. The method provided by the present invention does not require large-scale instruments for detection. It is a simple, rapid and green method. It has been applied to the analysis of sulfonamides in pork samples, with a recovery rate of 98.42% to 101.6% and a relative standard deviation of less than 3.6%. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 Flowchart of the method for rapid detection of sulfonamides in meat by improving QuEChERS combined with enzyme inhibition colorimetric method;

[0033] Figure 2 This is a diagram showing the effect of natural deep eutectic solvent types on extraction results;

[0034] Figure 3 This is the effect of the molar ratio of natural deep eutectic solvent on the extraction effect;

[0035] Figure 4 This is a graph showing the effect of the volume of natural deep eutectic solvent on the extraction effect;

[0036] Figure 5 This is a diagram showing the effect of water content in natural deep eutectic solvents on extraction results;

[0037] Figure 6 This is the effect of adsorbent type on extraction effect;

[0038] Figure 7 This is the effect of adsorbent dosage on extraction effect;

[0039] Figure 8 is the carbonic anhydrase concentration versus colorimetric intensity ( I ) influence diagram;

[0040] Figure 9 is the substrate concentration versus colorimetric detection intensity ( I ) influence diagram;

[0041] Figure 10 is the pH of phosphate buffer solution versus colorimetric intensity ( I ) influence diagram;

[0042] Figure 11 is the reaction temperature versus colorimetric detection intensity ( I ) influence diagram;

[0043] Figure 12 is the reaction time versus colorimetric detection intensity ( I ) influence diagram. DETAILED DESCRIPTION

[0044] To date, a variety of methods have been used to detect sulfonamides, including high-performance liquid chromatography, spectroscopy, capillary electrophoresis, mass spectrometry, and electrochemical methods. Generally speaking, these traditional methods have limitations, including expensive equipment, specialized laboratory personnel, and time-consuming procedures. Furthermore, the bulky and difficult-to-port equipment precludes on-site testing. In contrast, carbonic anhydrase, which hydrolyzes p-nitrophenylacetate to p-nitrophenol, is inhibited by sulfonamide. This enzyme inhibition by sulfonamide allows for direct detection of sulfonamides through a color change, eliminating the need for complex detection procedures. Digital image colorimetry is an emerging colorimetric method that uses image acquisition tools to record color and analyze the colors in the captured image using image processing software. Smartphones, due to their portability, diverse information transmission capabilities, and storage capacity, are the most convenient image acquisition tools and currently offer the most promising platform for on-site monitoring and portable detection. They can be used for quantitative and qualitative analysis of a variety of samples, significantly improving the accuracy of test results and becoming a popular alternative to colorimetric detection of analytes. This example is the first to use a magnetic covalent organic framework material as an adsorbent and a natural deep eutectic solvent as an extraction solvent to modify QuEChERS and combine it with enzyme inhibition smartphone digital image colorimetry to detect sulfonamide residues in pork.

[0045] The present invention includes four parts: the first is the preparation of a low eutectic solvent and a magnetic adsorbent COF (TpPa-Cl)@Fe3O4; the second is the extraction and purification of samples using a modified QuEChERS method, and the conditions are optimized to obtain the optimal extraction conditions; the third is the detection of samples using enzyme inhibition colorimetry, and the conditions are optimized to obtain the optimal detection conditions; and the fourth is the determination of meat samples using the optimal extraction, purification, and detection conditions.

[0046] Example 1 Preparation of deep eutectic solvent and magnetic adsorbent COF(TpPa-Cl)@Fe3O4

[0047] Choline chloride, betaine, thymol, vanillin and tetrabutylammonium chloride were selected as hydrogen bond acceptors, and ethylene glycol, propylene glycol, octanoic acid and butyric acid were selected as hydrogen bond donors. The mixture was continuously heated in a water bath at 80°C until a clear and transparent liquid was formed. Six different natural deep eutectic solvents, namely extractants, were synthesized.

[0048] COF (TpPa-Cl)@Fe3O4 was prepared using a stirring method at room temperature. First, 120 mg of Fe3O4 was weighed and added to 100 mL of dimethyl sulfoxide (DMSO). Ultrasonication was performed for 10 minutes to achieve uniform dispersion. Then, 1,3,5-triformylm-benzenetriol (63 mg) and 4-chloro-m-phenylenediamine (96.26 mg) were added, followed by an additional 10 minutes of ultrasonication. Under electric stirring, 2 mL of glacial acetic acid was added, the mixture was allowed to react for 30 minutes, and the mixture was allowed to stand for 35 minutes to obtain a brownish-yellow product. The supernatant was rinsed multiple times with methanol and water until it became almost colorless. After drying in a vacuum oven for 12 hours, COF (TpPa-Cl)@Fe3O4 was obtained.

[0049] Example 2 Parameter Optimization

[0050] The present invention is influenced by numerous factors, requiring a series of optimization experiments. Therefore, single-factor optimization was performed for the type of natural deep eutectic solvent, the molar ratio of the natural deep eutectic solvent, the volume of the natural deep eutectic solvent, the water content of the natural deep eutectic solvent, the type and amount of the adsorbent, the carbonic anhydrase concentration, the substrate (p-nitrophenyl acetate) concentration, the pH of the phosphate buffer, the reaction temperature, and the reaction time. Samples were purchased from local supermarkets to verify the reliability of the test results of the present method in practical applications. Unless otherwise specified, the materials and reagents used in the examples are commercially available.

[0051] This embodiment uses pork as an example. The original amount of sulfonamides is 0. After adding sulfonamides according to a preset amount, it is used as a test sample, tested, and the recovery rate is calculated.

[0052] 1. Optimization of natural deep eutectic solvent types

[0053] Step 1: In a 5 mL centrifuge tube, 1 g of meat sample was vortexed with 0.5 mL of different natural deep eutectic solvents for 1 min and centrifuged for 90 s. The liquid was extracted to obtain extract a and placed in a centrifuge tube.

[0054] In step 2, 2 mg of COF (TpPa-Cl)@Fe3O4 was then added to the centrifuge tube containing 0.2 ml of extract a. After vortexing for 30 seconds, the extract b was quickly separated using an external magnet.

[0055] Step 3: 100 μL of extract b, 50 μL of freshly prepared carbonic anhydrase solution (300 units / mL), 200 μL of phosphate buffer (10.0 mM, pH 8.5), and 100 μL of p-nitrophenyl acetate solution (0.1 mg mL -1) were mixed in a 0.5 mL centrifuge tube. The mixture was then incubated at 40°C for 3 minutes. The centrifuge tube was placed in an opaque white box with LED light panels around the top of the box for uniform illumination. The color changes were captured using a smartphone, and then Adobe Photoshop CC software was used to read the red (R), green (G), and blue (B) channel values ​​of the active site in RGB mode and calculate the intensity ( I ),formula I =280-G.

[0056] Step 4: Substitute the strength of the sulfonamide drug to be detected obtained in step 3 into the standard working curve of sulfonamide drugs to obtain the concentration of sulfonamide drugs in the meat sample and calculate the recovery rate.

[0057] See the results Figure 1 The results showed that the natural deep eutectic solvent synthesized from propylene glycol and betaine had the highest extraction recovery rate, which may be due to its stronger hydrogen bonding and electrostatic interactions with sulfonamides than other deep eutectic solvents. Therefore, propylene glycol and betaine were selected as the optimal natural deep eutectic solvents.

[0058] 2. Optimization of the molar ratio of natural deep eutectic solvents

[0059] The molar ratio of hydrogen bond acceptors to hydrogen bond donors affects the polarity, solubility, and viscosity of the natural deep eutectic solvent (NDES), thereby affecting the extraction of sulfonamides. In this experiment, propylene glycol and betaine were selected at molar ratios of 1:1, 1:2, 1:3, 1:4, 1:5, and 1:6 to synthesize the NDEs. Other procedures were the same as those for optimizing the NDE species in step 1 above.

[0060] See the results Figure 2 The results showed that the extraction recovery increased when the molar ratio of propylene glycol to betaine was between 1:1 and 1:4, while the extraction recovery decreased when the molar ratio was between 1:4 and 1:6. This effect was attributed to the reduction and instability of hydrogen bonds in the natural deep eutectic solvent. Ultimately, the molar ratio of propylene glycol to betaine was 1:4.

[0061] 3. Optimization of natural deep eutectic solvent volume

[0062] Extractant volume is a crucial factor in QuEChERS extraction efficiency. Insufficient extractant volume results in inadequate extraction of target analytes, while excessive volume reduces sensitivity. Different extractant volumes ranging from 200 to 800 μL were used in this experiment. Other procedures were similar to those for optimizing the natural deep eutectic solvent in step 1 above.

[0063] See the results Figure 3The results showed that the extraction efficiency was the highest when the volume was 500 μL, but as the volume of the extractant increased, the extraction efficiency gradually decreased. Therefore, 500 μL was selected as the optimal volume of the extractant.

[0064] 4. Optimization of water content in natural deep eutectic solvents

[0065] Water content affects the viscosity and polarity of a natural deep eutectic solvent (NDE). The optimal water content also depends on the physical and chemical properties of the specific compound. Experiments were conducted using water contents of 0%, 20%, 40%, 60%, and 80%. Other procedures were the same as for optimizing the NDE type in step 1 above.

[0066] See the results Figure 4 As the water content in the natural deep eutectic solvent gradually increases, the extraction recovery rate gradually decreases. The addition of water may disrupt the hydrogen bonds between hydrogen bond donors and hydrogen bond acceptors, thereby affecting the extraction. Therefore, a natural deep eutectic solvent without water was used in the experiment.

[0067] 5. Optimization of adsorbent types

[0068] The use of adsorbents reduces matrix interference and improves selectivity for sulfonamides. Different materials were selected as adsorbents: ethylenediamine-N-propylsilane (PSA), octadecylsilane (C18), graphitized carbon black (GCB), graphene, multi-walled carbon nanotubes (MWCNTs), Fe₃O₄, and COF (TpPa-Cl)@Fe₃O₄. Other procedures were similar to those for optimizing the natural deep eutectic solvent in step 1 above. Fe₃O₄ was synthesized via a solvothermal reaction.

[0069] See the results Figure 5 Results showed that traditional adsorbents such as ethylenediamine-N-propylsilane (PSA), octadecylsilane (C18), graphitized carbon black (GCB), graphene, and multi-walled carbon nanotubes (MWCNTs) had reduced recovery rates for sulfonamides, while magnetic COF (TpPa-Cl)@Fe3O4 exhibited high recovery rates for sulfonamides. Fe3O4 has the disadvantage of easily agglomerating and possessing few surface functional groups, resulting in reduced adsorption capacity for impurities. COF (TpPa-Cl)@Fe3O4 not only has a large specific surface area but also possesses abundant benzene rings and a porous structure, providing numerous adsorption sites for impurities through π-π stacking and hydrophobic interactions. Using COF (TpPa-Cl)@Fe3O4, the extract and adsorbent can be successfully separated in seconds using only an external magnet. Compared to traditional centrifugal separation, magnetic separation is more convenient and faster.

[0070] 6. Optimization of adsorbent dosage

[0071] In the QuEChERS method, the adsorbent dosage plays a crucial role in achieving satisfactory analyte recoveries and enhanced purification performance. To optimize the adsorbent dosage, we experimentally investigated the adsorption performance of COF (TpPa-Cl)@Fe₃O₄ over a range of 1 to 5 mg. All other procedures were similar to those for optimizing the natural deep eutectic solvent (NUE) in step 1 above.

[0072] See the results Figure 6 The results showed that when the adsorbent dosage was 2 mg, the active sites were more accessible to interacting impurities, resulting in the highest analyte recovery. When the adsorbent mass exceeded 2 mg, the adsorbent particles aggregated, reducing the adsorption surface area and leading to lower recovery. Ultimately, 2 mg was determined to be the optimal adsorbent dosage for the experiment.

[0073] 7. Optimization of carbonic anhydrase concentration

[0074] The enzyme content is crucial to the construction of the colorimetric process and the detection sensitivity. In the experiment, the content of carbonic anhydrase was 100~350 U·mL -1 The other operations were the same as the optimization of the natural deep eutectic solvent type in the above 1. The results are shown in Figure 7 , the results showed that with the increase of carbonic anhydrase content, the strength ( I ) gradually increased. When the carbonic anhydrase concentration exceeded 300 U·mL -1 When the intensity ( I ) had no significant changes. Finally, 300U·mL -1 Carbonic anhydrase was the optimal enzyme content in the experiment. This may be because after the binding of carbonic anhydrase and p-nitrophenyl acetate reached saturation, increasing the amount of carbonic anhydrase did not produce a significant change.

[0075] 8. Optimization of substrate concentration

[0076] The substrate concentration is a key parameter affecting the precision of colorimetric detection. The experiment studied the concentration of p-nitrophenyl acetate in the range of 0.02~0.12 mg·mL -1 Regarding the effect on the colorimetric detection of sulfonamides, other operations were the same as the optimization of the natural deep eutectic solvent type in 1 above.

[0077] See the results Figure 8 , the results showed that with the increase of p-nitrophenyl acetate content, the strength ( I ) gradually increased. When the p-nitrophenyl acetate content exceeded 0.1 mg·mL -1 When the intensity ( I) no longer increased significantly. This phenomenon may be due to the binding of p-nitrophenylacetate to carbonic anhydrase, occupying some of the binding sites between carbonic anhydrase and sulfonamides, thus hindering the inhibitory effect of sulfonamides on carbonic anhydrase. -1 is the optimal substrate concentration in the experiment.

[0078] 9. Optimization of phosphate buffer pH

[0079] In order to study the detection performance of the colorimetric method under different buffer conditions, the pH value of the buffer was optimized. Other operations were the same as the optimization of the natural deep eutectic solvent type in the above 1.

[0080] Strength at different pH values ​​( I ) changes in the results can be seen in Figure 9 , the results show that with the increase of pH value, the intensity ( I ) gradually increases and reaches its maximum value at pH 8.5; the color of the solution also changes from yellow to colorless. When the pH value exceeds 8.5, the activity of carbonic anhydrase weakens and the intensity ( I ) gradually decreased. Ultimately, all experiments were performed at a pH of 8.5. This may be because an excessively high pH value affects the binding of carbonic anhydrase to the substrate, resulting in a weakened colorimetric detection effect.

[0081] 10. Optimization of reaction temperature

[0082] To investigate the effect of reaction temperature on colorimetric detection, sulfonamides were added to carbonic anhydrase and incubated at various temperatures for several minutes. Following the addition of buffer and substrate, the effects of incubation temperatures of 20, 30, 40, 50, 60, and 70°C on colorimetric detection were investigated. All other procedures were the same as those for optimizing the natural deep eutectic solvent in step 1 above.

[0083] See the results Figure 10 , the results showed that, with the increase of incubation temperature from 20 ℃ to 40 ℃, the intensity ( I ) also increased, then decreased after 40°C. This is because high temperatures inhibit the activity of carbonic anhydrase. Ultimately, the experiment selected 40°C as the optimal incubation temperature, at which sulfonamides exhibit a strong inhibitory effect on carbonic anhydrase, promoting the application of colorimetric sulfonamide detection in real-time on-site detection.

[0084] 11. Optimization of response time

[0085] Reaction time to intensity ( I The experiment studied reaction times ranging from 1 to 6 min, with other operations similar to the optimization of the natural deep eutectic solvent type in step 1 above.

[0086] See the results Figure 11, the results show that the intensity ( I ) steadily increased and reached saturation after 3 minutes. Over time, the yellow color turned colorless and remained stable. Ultimately, the optimal reaction time was 3 minutes.

[0087] Example 3 Effect Verification

[0088] In this example, untreated pork was used as the test sample, and the sulfonamides in meat were detected using a modified QuEChERS combined with enzyme inhibition colorimetric method.

[0089] (1) Preparation of natural deep eutectic solvents

[0090] Propylene glycol and betaine were mixed in a molar ratio of 1:4 and continuously heated in a water bath at 80°C until a clear and transparent liquid was formed, thereby obtaining a natural deep eutectic solvent, which was then stored at room temperature for future use.

[0091] (2) Preparation of adsorbent COF (TpPa-Cl)@Fe3O4.

[0092] (3) Preparation of sample solution

[0093] Accurately weigh 1 g of the pulverized and homogenized pork sample to be tested, add 0.5 mL of natural deep eutectic solvent, vortex for 1 min and centrifuge for 90 s for extraction to obtain extract a.

[0094] (4) Purification and collection

[0095] Take out 0.2 ml of extract a from step (3) above, add 2 mg of COF (TpPa-Cl)@Fe3O4, vortex for 30 seconds, and quickly separate with an external magnet to obtain extract b.

[0096] (5) Colorimetric detection of sulfonamides

[0097] According to Example 2, extract b, carbonic anhydrase solution, phosphate buffer, and p-nitrophenyl acetate solution were mixed in a 0.5 mL centrifuge tube. The mixture was then incubated at 40°C for 3 min. The centrifuge tube was placed in an opaque white box with LED light panels around the top of the box for uniform illumination. The color changes were captured using a smartphone, and then Adobe Photoshop CC software was used to read the red (R), green (G), and blue (B) channel values ​​of the active site in RGB mode and calculate the intensity ( I ),formula I =280-G.

[0098] (6) Testing of actual samples

[0099] This example evaluated the application of a modified QuEChERS-based enzyme inhibition colorimetric assay for the detection of sulfonamides in pork using the optimal conditions obtained in Example 2. Under the optimal conditions, the established method was validated in terms of linear range, limit of detection, limit of quantification, coefficient of determination, and precision. The limits of detection and quantification were estimated using the formulas 3σ / K and 10σ / K, where σ represents the standard deviation of 10 blank samples and K is the slope of the calibration curve. The limits of detection for all analytes ranged from 0.53 to 0.82 µg·kg. -1 The detection limit is in line with the EU MRL of 100 μg·kg for the total content of sulfonamides in animal food tissues. -1 The limit of quantification was 1.52 ~ 1.98µg·kg -1 . Intra-day and inter-day analysis were used to evaluate the precision, accuracy, and repeatability of the established method. Intra-day and inter-day precision were tested in parallel on one day and three days, respectively (n=3). The results are shown in Table 1. The linear correlation was good, and the determination coefficient was between 0.9956 and 0.9991. The intra-day and inter-day relative standard deviations were 1.96% to 3.42% and 1.32 to 3.58%, respectively. In order to investigate the reproducibility of the method, spiked experiments were carried out in three samples, and five sets of parallel experiments were performed and the relative standard deviations were calculated. The results are shown in Table 2. The results showed that the recovery was in the range of 98.42% to 101.60%, and the relative standard deviation was between 1.2% and 3.6%. The results showed that the established method has good reproducibility and is suitable for the detection of sulfonamides in food.

[0100]

[0101]

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for rapid detection of sulfonamides in meat using a modified QuEChERS combined with enzyme inhibition colorimetry, wherein the sulfonamides to be detected include one or more of sulfadiazine, sulfanilamide, sulfathiazole, sulfamethoxazine, and sulfadimethoxine, characterized in that: The steps include: S1, using propylene glycol as a hydrogen bond donor and betaine as a hydrogen bond acceptor, with a molar ratio of 1:1 to 1:6, heating in a water bath to form a clear and transparent liquid at a water bath temperature of 80°C, and obtaining propylene glycol-betaine as an extractant, with a water content of 0%; S2, adding the propylene glycol-betaine prepared in step S1 to the meat sample, mixing the meat sample and propylene glycol-betaine in a ratio of 0.5-3 g: 200-800 μL, vortexing to mix, and then centrifuging for 0.5-2 min, centrifuging for 90 s, and rotating at 5000 rpm to obtain extract a; S3, adding an adsorbent to the extract a obtained in step S2, wherein the adsorbent is COF (TpPa-Cl)@Fe3O4, and mixing the extract a and COF (TpPa-Cl)@Fe3O4 at a ratio of 1 mL:10 mg. After vortexing, the extract b is obtained after impurities are removed by adsorption using an external magnetic field; S4, the extract b obtained in step S3 is placed in a container and mixed with a freshly prepared carbonic anhydrase solution, phosphate buffer, and p-nitrophenyl acetate solution. After incubating the mixture, the container is placed in an opaque white box, the top of the white box is evenly illuminated by an LED light panel, and the color change of the mixture in the container is captured using a smartphone. Then, Adobe Photoshop CC software is used to read the channel values ​​of the red, green, and blue channels of the active site in RGB mode, and the intensity of the sulfonamide to be detected in the meat sample is calculated; S5. Prepare standards of different sulfonamides, mix them with freshly prepared carbonic anhydrase solution, phosphate buffer, and p-nitrophenyl acetate solution in a container, incubate the mixture, and then place the container in an opaque white box. The top of the white box is evenly illuminated by an LED light panel. Use a smartphone to capture the color change of the mixture in the container. Then use Adobe Photoshop CC software to read the channel values ​​of the red, green, and blue channels of the active site in RGB mode, and calculate the intensity of the standard. Draw a standard working curve for different sulfonamides with the standard concentration as the horizontal axis and the corresponding intensity as the vertical axis. Substitute the intensity of the sulfonamide to be detected in the meat sample obtained in step S4 into the standard working curve to obtain the concentration of the sulfonamide in the meat sample.

2. The method for rapid detection of sulfonamides in meat using the improved QuEChERS combined with enzyme inhibition colorimetry according to claim 1, characterized in that: The mixing ratio of propylene glycol and betaine is 1:4 by molar ratio.

3. The method for rapid detection of sulfonamides in meat using a modified QuEChERS combined with enzyme inhibition colorimetry according to claim 1, characterized in that: In step S3, COF (TpPa-Cl)@Fe3O4 is prepared at room temperature by a stirring method, comprising the following steps: Fe3O4 was added to dimethyl sulfoxide at a ratio of 1.2 mg:1 mL and ultrasonically dispersed uniformly. Then, 1,3,5-triformylphthalenetriol and 4-chloro-m-phenylenediamine were added respectively, with 0.63 mg of 1,3,5-triformylphthalenetriol and 0.9626 mg of 4-chloro-m-phenylenediamine corresponding to each 1 mL of dimethyl sulfoxide. Ultrasonication was performed again, and then stirred and glacial acetic acid was added, with 0.02 mL of glacial acetic acid corresponding to each 1 mL of dimethyl sulfoxide. After the reaction, the mixture was allowed to stand to obtain a brown-yellow product. The supernatant was washed with methanol and water several times until it was colorless and dried in a vacuum drying oven to obtain COF (TpPa-Cl)@Fe3O4.

4. The method for rapid detection of sulfonamides in meat using the improved QuEChERS combined with enzyme inhibition colorimetry according to claim 3, characterized in that: In steps S4 and S5, the concentration of carbonic anhydrase solution is 100~350 U·mL -1 ; The phosphate buffer concentration was 10.0 mM and the pH range was 6.5–9; The concentration of p-nitrophenyl acetate solution is 0.02~0.12 mg·mL -1 ; In step S4, the volume ratio of the extract b, carbonic anhydrase solution, phosphate buffer, and p-nitrophenyl acetate solution is 1:0.5:2:1; In step S5, the volume ratio of the standard, carbonic anhydrase solution, phosphate buffer, and p-nitrophenyl acetate solution is 1:0.5:2:

1.

5. The method for rapid detection of sulfonamides in meat using the improved QuEChERS combined with enzyme inhibition colorimetry according to claim 4, characterized in that: In steps S4 and S5, the incubation temperature is 20-70°C, and the incubation time is 1-6 minutes.

6. The method for rapid detection of sulfonamides in meat using the improved QuEChERS combined with enzyme inhibition colorimetry according to claim 5, characterized in that: In steps S4 and S5, the intensity I =280-G, where G is the channel value of the green channel.

7. The method for rapid detection of sulfonamides in meat using the improved QuEChERS combined with enzyme inhibition colorimetry according to claim 6, characterized in that: In step S2, meat sample and propylene glycol-betaine were mixed at a ratio of 1 g:500 μL, vortexed for 1 min, and centrifuged for 90 s; In steps S4 and S5, the concentration of carbonic anhydrase solution is 300 U·mL -1 ; Phosphate buffer concentration was 10.0 mM, pH 8.5; The concentration of p-nitrophenyl acetate solution is 0.1 mg mL -1 ; The incubation temperature was 40°C and the incubation time was 3 min.

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