A method for detecting sulfonamide antibiotics in liquid samples

Abstract

The present invention belongs to the field of chemical analysis and specifically discloses a method for detecting sulfonamide antibiotics in liquid samples. The present invention combines electromembrane extraction technology with liquid chromatography to establish a method for quickly and accurately detecting trace sulfonamide antibiotics in liquid samples. By developing a new, green, and selective organic extraction solvent and screening electromembrane extraction conditions, the selective separation, purification, and enrichment of trace sulfonamide antibiotics are achieved. At the same time, the mass transfer mechanism of the analyte is studied to complete efficient extraction. Combined with liquid chromatography technology, the detection of sulfonamide antibiotics in liquid samples can be achieved simply, quickly, and accurately. The linear range of the detection method of the present invention is 5 to 5000 μg / L, and the detection limit is 16 to 32 μg / L. It can be applied to the detection of trace sulfonamide antibiotics in complex sample systems such as milk, tap water, and lake water, and has important theoretical guidance significance for the trace detection of sulfonamide antibiotics.

Description

Technical Field

[0001] The present invention relates to the field of chemical analysis, and in particular to a method for detecting sulfonamide antibiotics in a liquid sample. Background Art

[0002] Sulfonamide antibiotics, due to their broad antimicrobial spectrum, stable properties, and ease of use, have become effective drugs for the prevention and treatment of infectious diseases. In livestock farming, sulfonamide antibiotics can be added to feed, or administered orally or through intravenous routes to treat bacterial infections and other diseases in poultry and livestock. However, long-term or excessive consumption or use of sulfonamide antibiotics by poultry and livestock can lead to their accumulation in the animals and subsequent residues in animal foods. Sulfonamide antibiotics have a long half-life and are not easily degraded in the natural environment. Long-term consumption of low-dose sulfonamide residues in food can cause serious side effects such as carcinogenesis and teratogenicity. Consequently, many countries, including China and Japan, have implemented strict regulations on the use of sulfonamide antibiotics and their residues in animal foods. my country's national food safety standard, GB 31650-2019, "Maximum Residue Limits of Veterinary Drugs in Food," stipulates that the total residue of sulfonamide antibiotics in animal foods cannot exceed 100 μg / kg. Therefore, in order to ensure food quality and safety and human health, it is particularly important to develop simple, accurate and highly sensitive methods for detecting sulfonamide antibiotic residues in food.

[0003] Domestic and international studies have shown that the main methods for detecting sulfonamide antibiotic residues include microbiological detection, immunological analysis, liquid chromatography, and liquid chromatography-mass spectrometry. Liquid chromatography, characterized by its ease of operation, high accuracy, and good reproducibility, is currently the most commonly used method for qualitative and quantitative detection of antibiotics in food. The matrix in animal-derived foods is relatively complex, and selecting a sample pretreatment technique with strong purification capabilities is key to ensuring accurate detection of trace sulfonamide antibiotic residues in complex matrix samples. Common sample pretreatment techniques include solid-phase extraction (SPE), liquid-liquid extraction (LIE), and liquid-phase microextraction (LPME). SPE primarily relies on the adsorption of analytes by adsorbents (such as activated carbon, graphene, and molecularly imprinted materials) to achieve separation and purification. However, the complex matrix in food competes with the analyte for the adsorbent's surface sites, thereby reducing the interaction between the adsorbent and the analyte. Furthermore, SPE is cumbersome to operate and consumes large amounts of organic solvents. Liquid-liquid extraction (LIFE) exploits the different solubilities of analytes in mutually incompatible solvents to extract them. However, this method suffers from limited selectivity, poor sample matrix cleanup, and high organic solvent consumption. Liquid-phase microextraction (LPME), developed by combining miniaturized LPME with membrane separation technology, can be used to separate and detect trace analytes in complex samples. However, mass transfer of analytes is a passive diffusion process, which requires a long time.

[0004] To effectively improve the mass transfer rate of analytes, Pedersen-Bjergaard et al. introduced the principles of electrophoresis into liquid-phase microextraction in 2006, innovatively proposing a novel sample pretreatment technique: electromembrane extraction. This technique relies on an electric field causing charged analytes in the sample phase solution to migrate toward the oppositely charged electrode, passing through a supported liquid membrane into the receiving phase for extraction and separation. Based on the selectivity of the supported liquid membrane and the principle of electrophoresis, electromembrane extraction offers excellent sample purification capabilities and rapid mass transfer rates, leading to its rapid development in the field of sample pretreatment. Summary of the Invention

[0005] In view of the problems and shortcomings in the prior art, the present invention aims to provide a method for detecting sulfonamide antibiotics in liquid samples.

[0006] To achieve the purpose of the invention, the technical solution adopted by the present invention is as follows:

[0007] A method for detecting sulfonamide antibiotics in a liquid sample comprises the following steps:

[0008] (1) taking a liquid sample, centrifuging, filtering, drying, and re-dissolving to obtain a liquid analysis sample;

[0009] (2) using the liquid analysis sample as a donor phase, extracting the sulfonamide antibiotics in the liquid analysis sample using an electromembrane extraction technique, and detecting the concentration of the sulfonamide antibiotics in the receiving phase of the electromembrane extraction device after the extraction is completed;

[0010] The membrane used in the electromembrane extraction technology is a porous polypropylene fiber membrane, and the porous polypropylene fiber membrane is loaded with an extraction solvent, which is a mixed solution of 2-nitrophenyl octyl ether and methyl trioctyl ammonium chloride.

[0011] According to the method for detecting sulfonamide antibiotics in a liquid sample, preferably, the mass percentage of methyltrioctylammonium chloride in the mixed solution is 0.1% to 2%, and most preferably 1%.

[0012] According to the method for detecting sulfonamide antibiotics in a liquid sample, preferably, the specific process of the electromembrane extraction is: selecting a receiving phase container with two ends open, fixing a porous polypropylene fiber membrane on one end of the receiving phase container and sealing it; coating an organic extraction solvent on the surface of the porous polypropylene fiber membrane, and then injecting the receiving phase into the receiving phase container; injecting the donor phase into the donor phase container; inserting the receiving phase container into the donor phase so that the porous polypropylene fiber membrane is just in contact with the donor phase; inserting a working electrode and a counter electrode into the donor phase and the receiving phase respectively, applying an electric field to the working electrode and the counter electrode, and extracting and enriching the sulfonamide antibiotics in the sample under the action of the electric field.

[0013] According to the method for detecting sulfonamide antibiotics in a liquid sample, preferably, the solvent used in the re-dissolution process in step (1) is an alkaline solution with a pH of 11 to 13, and most preferably a pH of 12.

[0014] According to the method for detecting sulfonamide antibiotics in a liquid sample, preferably, the receiving phase in step (2) is an alkaline solution with a pH of 8 to 13, and most preferably a pH of 12.

[0015] According to the method for detecting sulfonamide antibiotics in a liquid sample, preferably, the voltage of the electric field applied between the donor phase and the receiving phase in the electromembrane extraction technique in step (2) is 2 to 15 V, most preferably 10 V.

[0016] According to the method for detecting sulfonamide antibiotics in a liquid sample, preferably, the extraction time in step (2) is 5 to 60 minutes, and the most preferred extraction time is 40 minutes.

[0017] According to the method for detecting sulfonamide antibiotics in a liquid sample, preferably, the liquid sample is any one of milk, tap water, and lake water.

[0018] According to the method for detecting sulfonamide antibiotics in a liquid sample, preferably, when the liquid sample is milk, the specific operation of step (1) is: dissolving the milk sample in an extraction solvent, centrifuging, and filtering to obtain a supernatant; then adding an organic solvent to the supernatant, centrifuging again, and discarding the upper organic solvent layer to obtain a lower solution; drying the lower solution and then re-dissolving it to obtain a milk analysis sample. The extraction solvent is acetonitrile, the organic solvent is n-hexane, and the centrifugal speed is preferably 10,000 rpm.

[0019] According to the method for detecting sulfonamide antibiotics in a liquid sample, preferably, when the liquid sample is tap water or lake water, the specific operation of step (1) is: centrifuging and filtering the tap water or lake water to obtain a supernatant; blowing the supernatant dry with nitrogen and then redissolving it to obtain a tap water analysis sample or a lake water analysis sample.

[0020] According to the method for detecting sulfonamide antibiotics in a liquid sample, preferably, the sulfonamide antibiotic is a mixture of sulfadiazine, sulfathiazole, sulfamethazine, sulfamethoxazole and sulfabenzoyl.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The present invention uses electromembrane extraction technology to extract five sulfonamide antibiotics from food and environmental water samples before detecting sulfonamide antibiotics in liquid samples. By developing a new, selective organic extraction solvent and screening electromembrane extraction conditions, the five sulfonamide antibiotics can be selectively separated, purified and enriched, solving the problems of low residual concentration of sulfonamide antibiotics in food and environmental water samples and severe interference from the sample matrix in the prior art, and greatly improving the detection accuracy and precision of sulfonamide antibiotics in food and environmental water samples. At the same time, the mass transfer of sulfonamide antibiotics during the extraction process is mainly based on electrical migration, which greatly shortens the extraction time. The present invention combines electromembrane extraction technology with liquid chromatography to establish an accurate trace analysis technology. This method has not been publicly reported in domestic and foreign literature.

[0023] (2) Based on the structural properties of sulfonamide antibiotics, the present invention developed a new organic extraction solvent, a mixture of 2-nitrophenyl octyl ether and methyl trioctyl ammonium chloride, and systematically studied the mass transfer kinetics of the target analyte under different extraction conditions, explored the chemical mechanism by which the extraction conditions affect the mass transfer process of the analyte, clarified the influence rules, and achieved efficient extraction of trace target analytes. The present invention has important theoretical guiding significance for the trace detection of sulfonamide antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the device for electromembrane extraction of sulfonamide antibiotics in the present invention;

[0025] Figure 2 This is the standard curve of five sulfonamide antibiotics. DETAILED DESCRIPTION

[0026] The following examples are intended only to further illustrate the present invention. It should be noted that all technical and scientific terms used herein have the same meanings as in the art to which the present invention pertains, unless otherwise indicated. Experimental methods in the following examples, where specific conditions are not specified, were performed using conventional techniques in the art or in accordance with the conditions recommended by the manufacturer. Reagents and instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0027] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0028] (1) Optimization of electromembrane extraction conditions for sulfonamide antibiotics

[0029] Example 1: Screening experiment of organic extraction solvents

[0030] In order to study the effects of different organic extraction solvents on the electro-membrane extraction of five sulfonamide antibiotics, namely sulfadiazine, sulfathiazole, sulfamethazine, sulfamethoxazole and sulfabenzoyl, the electro-membrane extraction experiment was carried out using a standard solution of a mixture of five sulfonamide antibiotics. After the extraction, the concentrations of the five sulfonamide antibiotics in the receiving phase were detected by high performance liquid chromatography, and the extraction rates of sulfadiazine, sulfathiazole, sulfamethazine, sulfamethoxazole and sulfabenzoyl were calculated respectively.

[0031] The specific contents of the experiments of Example 1-1 to Example 1-5 are as follows:

[0032] Example 1-1:

[0033] A method for detecting sulfonamide antibiotics in a liquid sample, comprising the following steps:

[0034] (1) Prepare standard solutions containing sulfadiazine, sulfathiazole, sulfadimethazine, sulfamethoxazole and sulfabenzoyl:

[0035] Sulfadiazine, sulfathiazole, sulfamethazine, sulfamethoxazole and sulfabenzoyl were dissolved in a NaOH solution with a pH of 12 to prepare standard solutions of the five sulfonamide antibiotics with a concentration of 5 mg / L each, which were set aside.

[0036] (2) Construction of electromembrane extraction device

[0037] a) Cut a piece of porous polypropylene fiber membrane to an appropriate size and secure it to the wide end of a 1 mL pipette tip using heat bonding. Cut off a portion of the narrow end of the pipette tip to use as the receiving phase container.

[0038] b) coating the surface of the porous polypropylene fiber membrane with 10 μL of an organic extraction solvent, wherein the organic extraction solvent is a mixed solution of 2-nitrophenyl octyl ether and methyl trioctyl ammonium chloride, and the mass percentage of methyl trioctyl ammonium chloride in the mixed solution is 0.1%.

[0039] c) injecting 200 μL of receiving phase into the receiving phase container, wherein the receiving phase is a NaOH solution with a pH of 12; taking a 2 mL centrifuge tube as a donor phase container and adding 1 mL of donor phase thereto, wherein the donor phase is the standard solution prepared in step (1); inserting the receiving phase container into the donor phase container so that the porous polypropylene fiber membrane is just in contact with the liquid surface of the donor phase solution, inserting a working electrode and a counter electrode into the donor phase solution and the receiving phase solution respectively, connecting the working electrode to the negative electrode of the electrophoresis instrument power supply, and connecting the counter electrode to the positive electrode of the electrophoresis instrument power supply, thereby forming an electromembrane extraction device, the schematic diagram of which is shown in FIG. Figure 1 shown.

[0040] (3) Electromembrane extraction experiment:

[0041] The electromembrane extraction device was placed on an oscillator. Both the electrophoresis instrument and the oscillator were powered on simultaneously. Using an external voltage and vibration-assisted operation, a selective extraction experiment was conducted for five sulfonamide antibiotics. The electrophoresis instrument voltage was 10 V, the extraction time was 40 min, and the oscillator speed was 1000 rpm. After extraction, the receiving phase was collected from the receiving phase container and analyzed for the five sulfonamide antibiotics using high-performance liquid chromatography (HPLC). HPLC conditions were as follows: a ThermoHypersil GOLD C18 column (100 mm × 3 mm, 5 μm particle size), a gradient mobile phase consisting of a mixture of acetonitrile and an aqueous solution containing 0.1% formic acid, with the mobile phase ratios shown in Table 1. The flow rate was 0.3 mL / min, the injection volume was 10 μL, the detection wavelength was 265 nm, and the column oven temperature was 40°C.

[0042] Table 1 Gradient ratio of mobile phase

[0043] Time (min) Acetonitrile 0.1% formic acid aqueous solution 0 5 95 5 25 75 9 40 60 10-11 65 35 11.1-15 5 95

[0044] The extraction rates of the five sulfonamide antibiotics were calculated separately, and the results are shown in Table 2.

[0045] Example 1-2:

[0046] The content of Example 1-2 is basically the same as that of Example 1-1, except that:

[0047] The organic extraction solvent used in step (2) is a mixed solution of 2-nitrophenyl octyl ether and methyl trioctyl ammonium chloride, and the mass percentage of methyl trioctyl ammonium chloride in the mixed solution is 0.5%.

[0048] Example 1-3:

[0049] The content of Example 1-3 is basically the same as that of Example 1-1, except that:

[0050] The organic extraction solvent used in step (2) is a mixed solution of 2-nitrophenyl octyl ether and methyl trioctyl ammonium chloride, and the mass percentage of methyl trioctyl ammonium chloride in the mixed solution is 1%.

[0051] Example 1-4:

[0052] The content of Example 1-4 is basically the same as that of Example 1-1, except that:

[0053] The organic extraction solvent used in step (2) is a mixed solution of 2-nitrophenyl octyl ether and methyl trioctyl ammonium chloride, and the mass percentage of methyl trioctyl ammonium chloride in the mixed solution is 2%.

[0054] Example 1-5:

[0055] The content of Example 1-5 is basically the same as that of Example 1-1, except that:

[0056] The organic extraction solvent used in step (2) is 2-nitrophenyl octyl ether.

[0057] The specific extraction rate results of Examples 1-1 to 1-5 are shown in Table 2:

[0058] Table 2 Effects of organic extraction solvents on electromembrane extraction of five sulfonamide antibiotics

[0059]

[0060] As shown in Table 2, within the range of 2-nitrophenyl octyl ether: methyl trioctyl ammonium chloride = (99.9:0.1) to (98:2), as the mass percentage of methyl trioctyl ammonium chloride in the organic extraction solvent increases, the extraction effect of the five sulfonamide antibiotics is significantly improved. When the percentage of methyl trioctyl ammonium chloride in the organic extraction solvent mixed solution is 1%, the extraction rates of the five sulfonamide antibiotics are 75.03%, 86.43%, 89.11%, 93.63% and 81.14%, respectively, all of which meet the analysis requirements. If the proportion of methyl trioctyl ammonium chloride is further increased, the system current will be too high, resulting in a rapid decrease in the extraction rate. Therefore, the present invention uses a mixed solution of 2-nitrophenyl octyl ether and methyl trioctyl ammonium chloride with a mass ratio of 99:1 as the preferred organic extraction solvent.

[0061] Example 2: Optimization experiment of extraction voltage

[0062] To investigate the effect of extraction voltage on the electromembrane extraction of five sulfonamide antibiotics, including sulfadiazine, sulfathiazole, sulfadimethazine, sulfamethoxazole, and sulfabenzoyl, electromembrane extraction experiments were conducted using a standard solution of a mixture of the five sulfonamide antibiotics. After extraction, the concentrations of the five sulfonamide antibiotics in the receiving phase were measured using a high-performance liquid chromatograph, and the extraction yields of sulfadiazine, sulfathiazole, sulfadimethazine, sulfamethoxazole, and sulfabenzoyl were calculated. The specific details of Examples 2-1 to 2-4 are as follows:

[0063] Example 2-1:

[0064] A method for detecting sulfonamide antibiotics in a liquid sample, comprising the following steps:

[0065] (1) Prepare standard solutions containing sulfadiazine, sulfathiazole, sulfadimethazine, sulfamethoxazole and sulfabenzoyl:

[0066] Sulfadiazine, sulfathiazole, sulfamethazine, sulfamethoxazole and sulfabenzoyl were dissolved in a NaOH solution with a pH of 12 to prepare standard solutions of the five sulfonamide antibiotics with a concentration of 5 mg / L each, which were set aside.

[0067] (2) Construction of electromembrane extraction device:

[0068] a) Cut a piece of porous polypropylene fiber membrane to an appropriate size and secure it to the wide end of a 1 mL pipette tip using heat bonding. Cut off a portion of the narrow end of the pipette tip to use as the receiving phase container.

[0069] b) coating the surface of the porous polypropylene fiber membrane with 10 μL of an organic extraction solvent, wherein the organic extraction solvent is a mixed solution of 2-nitrophenyl octyl ether and methyl trioctyl ammonium chloride, and the mass percentage of methyl trioctyl ammonium chloride in the mixed solution is 1%.

[0070] c) injecting 200 μL of receiving phase into the receiving phase container, wherein the receiving phase is a NaOH solution with a pH of 12; taking a 2 mL centrifuge tube as a donor phase container and adding 1 mL of donor phase thereto, wherein the donor phase is the standard solution prepared in step (1); inserting the receiving phase container into the donor phase container so that the porous polypropylene fiber membrane is just in contact with the liquid surface of the donor phase solution, inserting a working electrode and a counter electrode into the donor phase solution and the receiving phase solution respectively, connecting the working electrode to the negative pole of the electrophoresis instrument power supply, and connecting the counter electrode to the positive pole of the electrophoresis instrument power supply, thereby forming an electromembrane extraction device, the schematic diagram of which is shown in FIG. Figure 1 shown.

[0071] (3) Electromembrane extraction experiment:

[0072] The electromembrane extraction device is placed on an oscillator, and the electrophoresis instrument power supply and oscillator power supply are turned on at the same time. Under the driving and vibration-assisted effect of the applied voltage, the selective extraction experiment of 5 kinds of sulfonamide antibiotics is carried out. Wherein, the electrophoresis instrument voltage is 2V, the extraction time is 40min, and the oscillator speed is 1000rpm. After the extraction is completed, the receiving phase in the receiving phase container is collected, and the 5 kinds of sulfonamide antibiotics in the receiving phase are detected using high performance liquid chromatography. The detection conditions of the high performance liquid chromatograph are the same as those in Example 1, and the extraction yields of the 5 kinds of sulfonamide antibiotics are calculated respectively. The specific extraction yield results are shown in Table 3.

[0073] Example 2-2:

[0074] The content of Example 2-2 is basically the same as that of Example 2-1, except that:

[0075] In step (3), the voltage of the electrophoresis apparatus is 0V.

[0076] Example 2-3:

[0077] The content of Example 2-3 is basically the same as that of Example 2-1, except that:

[0078] In step (3), the voltage of the electrophoresis apparatus is 5V.

[0079] Example 2-4:

[0080] The content of Example 2-4 is basically the same as that of Example 2-1, except that:

[0081] In step (3), the voltage of the electrophoresis apparatus is 15V.

[0082] The specific extraction rate results of Examples 2-1 to 2-4 are shown in Table 3:

[0083] Table 3 Effect of extraction voltage on electromembrane extraction of five sulfonamide antibiotics

[0084]

[0085] As shown in Table 3, when no electric field is applied to the system (extraction voltage is 0V), the extraction effect of the five sulfonamide antibiotics is poor, and there is basically no extraction. After applying an external electric field, as the extraction voltage gradually increases to 10V, the extraction efficiency of the five sulfonamide antibiotics is significantly improved. When the extraction voltage is further increased to 15V, the extraction efficiency of the five sulfonamide antibiotics decreases. This is mainly because the mass transfer process of the electromembrane extraction is the active migration of charged ions under electric field drive. Increasing the extraction voltage within a certain range can promote the extraction of sulfonamide antibiotics, but too high an extraction voltage can cause the system current to rise, and the system is unstable, which can suppress the extraction of sulfonamide antibiotics. Therefore, the extraction voltage is preferably 10V.

[0086] Example 3: Optimization experiment of extraction time

[0087] To investigate the effect of extraction time on the electro-membrane extraction of five sulfonamide antibiotics, including sulfadiazine, sulfathiazole, sulfadimethazine, sulfamethoxazole, and sulfabenzoyl, electro-membrane extraction experiments were conducted using a standard solution of a mixture of the five sulfonamide antibiotics. After extraction, the concentrations of the five sulfonamide antibiotics in the receiving phase were measured using a high-performance liquid chromatograph, and the extraction yields of sulfadiazine, sulfathiazole, sulfadimethazine, sulfamethoxazole, and sulfabenzoyl were calculated. The specific details of Examples 3-1 to 3-6 are as follows:

[0088] Example 3-1:

[0089] A method for detecting sulfonamide antibiotics in a liquid sample, comprising the following steps:

[0090] (1) Prepare standard solutions containing sulfadiazine, sulfathiazole, sulfadimethazine, sulfamethoxazole and sulfabenzoyl:

[0091] Sulfadiazine, sulfathiazole, sulfamethazine, sulfamethoxazole and sulfabenzoyl were dissolved in a NaOH solution with a pH of 12 to prepare standard solutions of the five sulfonamide antibiotics with a concentration of 5 mg / L each, which were set aside.

[0092] (2) Construction of electromembrane extraction device

[0093] a) Cut a piece of porous polypropylene fiber membrane to an appropriate size and secure it to the wide end of a 1 mL pipette tip using heat bonding. Cut off a portion of the narrow end of the pipette tip to use as the receiving phase container.

[0094] b) coating the surface of the porous polypropylene fiber membrane with 10 μL of an organic extraction solvent, wherein the organic extraction solvent is a mixed solution of 2-nitrophenyl octyl ether and methyl trioctyl ammonium chloride, and the mass percentage of methyl trioctyl ammonium chloride in the mixed solution is 1%.

[0095] c) injecting 200 μL of receiving phase into the receiving phase container, wherein the receiving phase is a NaOH solution with a pH of 12; taking a 2 mL centrifuge tube as a donor phase container and adding 1 mL of donor phase thereto, wherein the donor phase is the standard solution prepared in step (1); inserting the receiving phase container into the donor phase container so that the porous polypropylene fiber membrane is just in contact with the liquid surface of the donor phase solution, inserting a working electrode and a counter electrode into the donor phase solution and the receiving phase solution respectively, connecting the working electrode to the negative electrode of the electrophoresis instrument power supply, and connecting the counter electrode to the positive electrode of the electrophoresis instrument power supply, thereby forming an electromembrane extraction device, the schematic diagram of which is shown in FIG. Figure 1 shown.

[0096] (3) Electromembrane extraction experiment:

[0097] The electromembrane extraction device is placed on an oscillator, and the electrophoresis instrument power supply and the oscillator power supply are turned on simultaneously. Under the driving and vibration-assisted effect of the applied voltage, the selective extraction experiments of 5 kinds of sulfonamide antibiotics are carried out. Wherein, the voltage of the electrophoresis instrument is 10V, the extraction time is 5min, and the rotating speed of the oscillator is 1000rpm. After the extraction is completed, the receiving phase in the receiving phase container is collected, and the 5 kinds of sulfonamide antibiotics in the receiving phase are detected by high performance liquid chromatography. The testing conditions of the high performance liquid chromatograph are the same as in Example 1, and the extraction yields of 5 kinds of sulfonamide antibiotics are calculated respectively. The specific extraction yield results are shown in Table 4.

[0098] Example 3-2:

[0099] The content of Example 3-2 is basically the same as that of Example 3-1, except that the extraction time in step (3) is 10 minutes.

[0100] Example 3-3:

[0101] The content of Example 3-3 is basically the same as that of Example 3-1, except that the extraction time in step (3) is 20 minutes.

[0102] Example 3-4:

[0103] The content of Example 3-4 is basically the same as that of Example 3-1, except that the extraction time in step (3) is 30 minutes.

[0104] Example 3-5:

[0105] The content of Example 3-5 is basically the same as that of Example 3-1, except that the extraction time in step (3) is 50 minutes.

[0106] Example 3-6:

[0107] The content of Example 3-6 is basically the same as that of Example 3-1, except that the extraction time in step (3) is 60 minutes.

[0108] The specific extraction rate results of Examples 3-1 to 3-6 are shown in Table 4.

[0109] Table 4 Effect of extraction time on electromembrane extraction of five sulfonamide antibiotics

[0110]

[0111] As shown in Table 4, as the extraction time increases from 5 min to 40 min, the extraction rates of the five sulfonamide antibiotics all show a continuous upward trend. Further extension of the extraction time shows that although the extraction efficiency of sulfadiazine and sulfamethazine continues to improve, the extraction rates of sulfathiazole, sulfamethoxazole, and sulfabenzoyl instead show a downward trend. Comprehensive analysis shows that when the extraction time is 40 min, the extraction rates of the five sulfonamide antibiotics are 75.03%, 86.43%, 89.11%, 93.63%, and 81.14%, respectively, all above 75%, meeting the analytical testing requirements. Therefore, the present invention uses 40 min as the preferred extraction time.

[0112] Example 4: Optimization experiment of pH value of receiving phase

[0113] To investigate the effect of receiving phase pH on the electro-membrane extraction of five sulfonamide antibiotics (sulfadiazine, sulfathiazole, sulfadimethazine, sulfamethoxazole, and sulfabenzoyl), electro-membrane extraction experiments were conducted using a standard solution of a mixture of the five sulfonamide antibiotics. After extraction, the concentrations of the five sulfonamide antibiotics in the receiving phase were measured using a high-performance liquid chromatograph, and the extraction yields of sulfadiazine, sulfathiazole, sulfadimethazine, sulfamethoxazole, and sulfabenzoyl were calculated. The specific experimental details are shown in Examples 4-1 to 4-5.

[0114] Example 4-1:

[0115] A method for detecting sulfonamide antibiotics in a liquid sample, comprising the following steps:

[0116] (1) Prepare standard solutions containing sulfadiazine, sulfathiazole, sulfadimethazine, sulfamethoxazole and sulfabenzoyl:

[0117] Sulfadiazine, sulfathiazole, sulfamethazine, sulfamethoxazole and sulfabenzoyl were dissolved in a NaOH solution with a pH of 12 to prepare standard solutions of the five sulfonamide antibiotics with a concentration of 5 mg / L each, which were set aside.

[0118] (2) Construction of electromembrane extraction device

[0119] a) Cut a piece of porous polypropylene fiber membrane to an appropriate size and secure it to the wide end of a 1 mL pipette tip using heat bonding. Cut off a portion of the narrow end of the pipette tip to use as the receiving phase container.

[0120] b) coating the surface of the porous polypropylene fiber membrane with 10 μL of an organic extraction solvent, wherein the organic extraction solvent is a mixed solution of 2-nitrophenyl octyl ether and methyl trioctyl ammonium chloride, and the mass percentage of methyl trioctyl ammonium chloride in the mixed solution is 1%.

[0121] c) injecting 200 μL of receiving phase into the receiving phase container, wherein the receiving phase is a NaOH solution with a pH of 8; using a 2 mL centrifuge tube as a donor phase container and adding 1 mL of donor phase thereto, wherein the donor phase is the standard solution prepared in step (1); inserting the receiving phase container into the donor phase container so that the porous polypropylene fiber membrane is just in contact with the liquid surface of the donor phase solution, inserting a working electrode and a counter electrode into the donor phase solution and the receiving phase solution respectively, connecting the working electrode to the negative pole of the electrophoresis instrument power supply, and connecting the counter electrode to the positive pole of the electrophoresis instrument power supply, thereby forming an electromembrane extraction device, the schematic diagram of which is shown in FIG. Figure 1 shown.

[0122] (3) Electromembrane extraction experiment:

[0123] The electromembrane extraction device is placed on an oscillator, and the electrophoresis instrument power supply and the oscillator power supply are turned on simultaneously. Under the driving and vibration-assisted effect of the applied voltage, the selective extraction experiments of 5 kinds of sulfonamide antibiotics are carried out. Wherein, the voltage of the electrophoresis instrument is 10V, the extraction time is 40min, and the rotating speed of the oscillator is 1000rpm. After the extraction is completed, the receiving phase in the receiving phase container is collected and the 5 kinds of sulfonamide antibiotics in the receiving phase are detected by high performance liquid chromatography. The testing conditions of the high performance liquid chromatograph are the same as in Example 1, and the extraction yields of 5 kinds of sulfonamide antibiotics are calculated respectively. The specific extraction yield results are shown in Table 5.

[0124] Example 4-2:

[0125] The content of Example 4-2 is basically the same as that of Example 4-1, except that:

[0126] In step (2), the receiving phase used is a NaOH solution with a pH of 9.

[0127] Example 4-3:

[0128] The content of Example 4-3 is basically the same as that of Example 4-1, except that:

[0129] In step (2), the receiving phase used is a NaOH solution with a pH of 10.

[0130] Example 4-4:

[0131] The content of Example 4-4 is basically the same as that of Example 4-1, except that:

[0132] In step (2), the receiving phase used is a NaOH solution with a pH of 11.

[0133] Example 4-5:

[0134] The content of Example 4-5 is basically the same as that of Example 4-1, except that:

[0135] In step (2), the receiving phase used is a NaOH solution with a pH of 13.

[0136] The specific extraction rate results of Example 4-1 to Example 4-5 are shown in Table 5.

[0137] Table 5 Effect of receiving phase pH on electromembrane extraction of five sulfonamide antibiotics

[0138]

[0139] As shown in Table 5, the extraction rates of the five sulfonamide antibiotics first increase and then decrease with increasing pH values. When the pH value of the receiving phase is 12, the extraction rates of the five sulfonamide antibiotics are the highest, reaching 75.03%, 86.43%, 89.11%, 93.63%, and 81.14%, respectively. Therefore, the present invention uses 12 as the preferred pH value for the receiving phase solution.

[0140] Example 5: Optimization experiment of donor phase pH value

[0141] To investigate the effect of donor phase pH on the electro-membrane extraction of five sulfonamide antibiotics (sulfadiazine, sulfathiazole, sulfadimethazine, sulfamethoxazole, and sulfabenzoyl), electro-membrane extraction experiments were conducted using a standard solution of a mixture of the five sulfonamide antibiotics. After extraction, the concentrations of the five sulfonamide antibiotics in the receiving phase were measured using high-performance liquid chromatography, and the extraction yields of sulfadiazine, sulfathiazole, sulfadimethazine, sulfamethoxazole, and sulfabenzoyl were calculated. The details of Examples 5-1 and 5-2 are as follows.

[0142] Example 5-1:

[0143] A method for detecting sulfonamide antibiotics in a liquid sample, comprising the following steps:

[0144] (1) Prepare standard solutions containing sulfadiazine, sulfathiazole, sulfadimethazine, sulfamethoxazole and sulfabenzoyl:

[0145] Sulfadiazine, sulfathiazole, sulfamethazine, sulfamethoxazole and sulfabenzoyl were dissolved in a NaOH solution with a pH of 11 to prepare standard solutions of the five sulfonamide antibiotics with a concentration of 5 mg / L each, which were set aside.

[0146] (2) Construction of electromembrane extraction device

[0147] a) Cut a piece of porous polypropylene fiber membrane to an appropriate size and secure it to the wide end of a 1 mL pipette tip using heat bonding. Cut off a portion of the narrow end of the pipette tip to use as the receiving phase container.

[0148] b) coating the surface of the porous polypropylene fiber membrane with 10 μL of an organic extraction solvent, wherein the organic extraction solvent is a mixed solution of 2-nitrophenyl octyl ether and methyl trioctyl ammonium chloride, and the mass percentage of methyl trioctyl ammonium chloride in the mixed solution is 1%.

[0149] c) injecting 200 μL of receiving phase into the receiving phase container, wherein the receiving phase is a NaOH solution with a pH of 12; taking a 2 mL centrifuge tube as a donor phase container and adding 1 mL of donor phase thereto, wherein the donor phase is the standard solution prepared in step (1); inserting the receiving phase container into the donor phase container so that the porous polypropylene fiber membrane is just in contact with the liquid surface of the donor phase solution, inserting a working electrode and a counter electrode into the donor phase solution and the receiving phase solution respectively, connecting the working electrode to the negative electrode of the electrophoresis instrument power supply, and connecting the counter electrode to the positive electrode of the electrophoresis instrument power supply, thereby forming an electromembrane extraction device, the schematic diagram of which is shown in FIG. Figure 1 shown.

[0150] (3) Electromembrane extraction experiment:

[0151] The electromembrane extraction device is placed on an oscillator, and the electrophoresis instrument power supply and the oscillator power supply are turned on simultaneously. Under the driving and vibration-assisted effect of the applied voltage, the selective extraction experiments of 5 kinds of sulfonamide antibiotics are carried out. Wherein, the voltage of the electrophoresis instrument is 10V, the extraction time is 40min, and the rotating speed of the oscillator is 1000rpm. After the extraction is completed, the receiving phase in the receiving phase container is collected and the 5 kinds of sulfonamide antibiotics in the receiving phase are detected by high performance liquid chromatography. The testing conditions of the high performance liquid chromatograph are the same as in Example 1, and the extraction yields of 5 kinds of sulfonamide antibiotics are calculated respectively. The specific extraction yield results are shown in Table 6.

[0152] Example 5-2:

[0153] The content of Example 5-2 is basically the same as that of Example 5-1, except that:

[0154] In step (1), a NaOH solution with a pH of 13 is used to dissolve five sulfonamide antibiotics.

[0155] The specific extraction rate results of Example 5-1 to Example 5-2 are shown in Table 6.

[0156] Table 6 Effect of donor phase pH on electromembrane extraction of five sulfonamide antibiotics

[0157]

[0158] As shown in Table 6, as the pH value of the donor phase increases from 11 to 13, the extraction rates of the five sulfonamide antibiotics initially increase and then decrease. The highest extraction rates were achieved when the five sulfonamide antibiotics were dissolved in a NaOH solution at pH 12, reaching 75.03%, 86.43%, 89.11%, 93.63%, and 81.14%, respectively. Therefore, a pH value of 12 was selected as the optimal donor phase pH value for the present invention.

[0159] (II) Detection of trace amounts of five sulfonamide antibiotics in food and environmental water samples

[0160] To demonstrate the application effect of the present invention in the field of food and environmental analysis and detection, extraction experiments were conducted on actual sample solutions based on the optimal electro-membrane extraction conditions optimized in Examples 1 to 5. The specific experimental procedures are shown in Examples 6-1 to 6-3.

[0161] Example 6-1:

[0162] A method for detecting sulfonamide antibiotics in tap water comprises the following steps:

[0163] (1) Sample preparation:

[0164] Take 5 mL of tap water, centrifuge at 10,000 rpm for 20 min, remove the supernatant and transfer it to another centrifuge tube, centrifuge again for 20 min, take the supernatant and filter it with a filter head, then blow dry the supernatant with nitrogen gas, and then re-dissolve it with 5 mL of NaOH solution with pH = 12 to obtain a tap water sample solution.

[0165] (2) Construction of electromembrane extraction device:

[0166] a) Cut a piece of porous polypropylene fiber membrane to an appropriate size and secure it to the wide end of a 1 mL pipette tip using heat bonding. Cut off a portion of the narrow end of the pipette tip to use as the receiving phase container.

[0167] b) coating the surface of the porous polypropylene fiber membrane with 10 μL of an organic extraction solvent, wherein the organic extraction solvent is a mixed solution of 2-nitrophenyl octyl ether and methyl trioctyl ammonium chloride, and the mass percentage of methyl trioctyl ammonium chloride in the mixed solution is 1%.

[0168] c) injecting 200 μL of receiving phase into the receiving phase container, wherein the receiving phase is a NaOH solution with a pH of 12; taking a 2 mL centrifuge tube as a donor phase container and adding 1 mL of donor phase thereto, wherein the donor phase is the standard solution prepared in step (1); inserting the receiving phase container into the donor phase container so that the porous polypropylene fiber membrane is just in contact with the liquid surface of the donor phase solution, inserting a working electrode and a counter electrode into the donor phase solution and the receiving phase solution respectively, connecting the working electrode to the negative electrode of the electrophoresis instrument power supply, and connecting the counter electrode to the positive electrode of the electrophoresis instrument power supply, thereby forming an electromembrane extraction device, the schematic diagram of which is shown in FIG. Figure 1 shown.

[0169] (3) Electromembrane extraction experiment:

[0170] The electrophoresis device was placed on an oscillator, and both the electrophoresis instrument and oscillator power supplies were turned on simultaneously. Using an applied voltage and vibration as a driver, a selective extraction experiment was conducted on five sulfonamide antibiotics. The electrophoresis instrument voltage was 10V, the extraction time was 40 minutes, and the oscillator speed was 1000 rpm.

[0171] (4) After the extraction is completed, the concentrations of the five sulfonamide antibiotics in the receiving phase solution are detected by high performance liquid chromatography. When the concentrations of the five sulfonamide antibiotics in the receiving phase are determined by high performance liquid chromatography, it is necessary to first prepare a standard curve of the five sulfonamide antibiotics. The preparation process of the standard curve is as follows: sulfadiazine, sulfathiazole, sulfadimethazine, sulfamethoxazole and sulfabenzoyl are dissolved in a NaOH solution with a pH of 12, and a series of sulfonamide antibiotic mixed standards with a concentration gradient of 5 μg / L to 5000 μg / L are prepared. The sulfonamide antibiotic mixed standard solution is extracted according to the electromembrane extraction operation described in the above steps (2) and (3). After extraction, the chromatographic peak areas of the five sulfonamide antibiotics in the receiving phase solution are determined by high performance liquid chromatography, and the standard curves of the five sulfonamide antibiotics are drawn, and the relevant performance parameters of the detection method proposed by the present invention are obtained, including the linear range, linear correlation coefficient, detection limit and quantification limit. The standard curve diagram is as shown below. Figure 2 The performance parameter results are shown in Table 7.

[0172] Table 7 Parameters related to the detection method of mixed standard solutions of five sulfonamide antibiotics

[0173]

[0174] Table 7 shows that the five sulfonamide antibiotics exhibited good linear relationships within the concentration range of 5 to 5000 μg / L, with linear correlation coefficients greater than 0.99. The calculated limits of detection for the five sulfonamide antibiotics ranged from 16 μg / L to 32 μg / L, meeting the requirements for practical sample analysis.

[0175] (5) Tap water actual sample spike recovery experiment:

[0176] Three 5 mL tap water samples were taken and centrifuged at 10,000 rpm for 20 min. The supernatant was removed and transferred to another centrifuge tube and centrifuged again for 20 min. The supernatant was taken and filtered with a filter head. The supernatant was then blown dry with nitrogen and reconstituted with 5 mL of a mixed solution of 0.05 mg / L, 0.1 mg / L, and 0.2 mg / L of the five sulfonamide antibiotic standards to obtain tap water sample spiked solutions. The three tap water sample spiked solutions were extracted according to the electromembrane extraction operation described in steps (2) and (3). After extraction, the concentrations of the five sulfonamide antibiotics in the receiving phase solution were determined by high performance liquid chromatography. The spiked recoveries of the five sulfonamide antibiotics in the tap water spiked samples were calculated, as shown in Table 8.

[0177] Table 8 Recovery rates of five sulfonamide antibiotics in tap water spiked samples

[0178]

[0179] As shown in Table 8, the recoveries of the five sulfonamide antibiotics at low, medium and high spike concentrations (0.05 mg / L, 0.1 mg / L and 0.2 mg / L) in tap water were between 77.63% and 117.78%, meeting the analysis requirements.

[0180] Example 6-2:

[0181] A method for detecting sulfonamide antibiotics in lake water comprises the following steps:

[0182] The content of Example 6-2 is basically the same as that of Example 6-1, except that:

[0183] The sample in step (1) is lake water, and the sample pretreatment method is the same as that in Example 6-1.

[0184] The sample in step (5) was lake water. The actual sample spiked sample preparation method was the same as that in Example 6-1. The results of the spiked recoveries of the five sulfonamide antibiotics in the lake water spiked samples are shown in Table 9.

[0185] Table 9 Recovery rates of five sulfonamide antibiotics in lake water spiked samples

[0186]

[0187] As shown in Table 9, the recoveries of the five sulfonamide antibiotics at low, medium, and high spike concentrations (0.05 mg / L, 0.1 mg / L, and 0.2 mg / L) in lake water were between 81.85% and 118.31%, meeting the analytical requirements.

[0188] Example 6-3:

[0189] A method for detecting sulfonamide antibiotics in milk comprises the following steps:

[0190] The content of Example 6-3 is basically the same as that of Example 6-1, except that:

[0191] (1) Take 5 mL of milk and add it to 20 mL of acetonitrile solution. After vortexing for 5 minutes, centrifuge at 10,000 rpm for 10 minutes. Take out the supernatant and transfer it to another centrifuge tube. Add 12.5 mL of n-hexane. After vortexing for 5 minutes, centrifuge again for 5 minutes. After the centrifugation, discard the n-hexane layer. Filter the remaining solution with a filter head, blow dry the solution with nitrogen, and then re-dissolve it with 5 mL of NaOH solution with pH = 12 to obtain a milk sample solution.

[0192] (5) Milk actual sample spike recovery experiment: Three 5 mL portions of milk sample were taken and added to 20 mL of acetonitrile solution. After vortexing for 5 minutes, the sample was centrifuged at 10,000 rpm for 10 minutes. The supernatant was removed and transferred to another centrifuge tube. 12.5 mL of n-hexane was added. The sample was vortexed for 5 minutes and centrifuged for another 5 minutes. After the centrifugation, the n-hexane layer was discarded. The remaining solution was filtered with a filter head and dried with nitrogen. The solution was then re-dissolved with 5 mL of a mixed solution of 5 sulfonamide antibiotic standards at 0.05 mg / L, 0.1 mg / L, and 0.2 mg / L (prepared by dissolving the 5 sulfonamide antibiotics in a NaOH solution at pH = 12) to obtain the milk sample spike solution. The rotation speed of the centrifuge was 10,000 rpm. The three milk sample spiked solutions were extracted according to the electromembrane extraction operation described in steps (2) and (3) of Example 6-1. After extraction, the concentrations of the five sulfonamide antibiotics in the receiving phase solution were determined by high performance liquid chromatography, and the spiked recoveries of the five sulfonamide antibiotics in the milk spiked samples were calculated. The results are shown in Table 10.

[0193] Table 10 Recovery rates of five sulfonamide antibiotics in spiked milk samples

[0194]

[0195] As shown in Table 10, the recoveries of the five sulfonamide antibiotics in the milk samples at low, medium and high spike concentrations were between 81.06% and 121.07%, which met the analytical requirements.

[0196] The above embodiments are specific implementation methods of the present invention, but the implementation methods of the present invention are not limited to the above embodiments. Any other combination, change, modification, substitution, and simplification that does not exceed the design concept of the present invention shall fall within the scope of protection of the present invention.

Claims

1. A method for detecting sulfonamide antibiotics in a liquid sample, characterized in that: The following steps are involved: (1) Take a liquid sample, centrifuge, filter, dry and redissolve it to obtain a liquid analysis sample; the solvent used in the redissolution process is an alkaline solution with a pH of 12; (2) using the liquid analysis sample as a donor phase, extracting the sulfonamide antibiotics in the liquid analysis sample by electromembrane extraction technology, and detecting the concentration of the sulfonamide antibiotics in the receiving phase of the electromembrane extraction device after the extraction is completed, wherein the receiving phase is an alkaline solution with a pH of 12, and the sulfonamide antibiotics are a mixture of sulfadiazine, sulfathiazole, sulfamethazine, sulfamethoxazole and sulfabenzoyl; The membrane used in the electro-membrane extraction technology is a porous polypropylene fiber membrane, and the porous polypropylene fiber membrane is coated with an extraction solvent, which is a mixed solution of 2-nitrophenyl octyl ether and methyl trioctyl ammonium chloride, and the mass percentage of methyl trioctyl ammonium chloride in the mixed solution is 1% to 2%; The liquid sample is any one of milk, tap water, and lake water; In the electromembrane extraction technique in step (2), the voltage of the electric field applied between the donor phase and the receiving phase is 5V to 15V, and the extraction time is 30 to 60 minutes.

2. The method for detecting sulfonamide antibiotics in a liquid sample according to claim 1, wherein The specific process of the electromembrane extraction is as follows: selecting a receiving phase container with two ends open, fixing a porous polypropylene fiber membrane on one end of the receiving phase container and sealing it; coating an organic extraction solvent on the surface of the porous polypropylene fiber membrane, and then injecting a receiving phase into the receiving phase container; Inject the donor phase into the donor phase container; insert the receiving phase container into the donor phase so that the porous polypropylene fiber membrane is just in contact with the donor phase; A working electrode is inserted into the donor phase, and a counter electrode is inserted into the receiving phase respectively. The working electrode is connected to the negative pole of the electrophoresis apparatus power supply, and the counter electrode is connected to the positive pole of the electrophoresis apparatus power supply. An electric field is applied to the working electrode and the counter electrode to extract and enrich the sulfonamide antibiotics in the sample under the action of the electric field.

3. The method for detecting sulfonamide antibiotics in a liquid sample according to claim 1, wherein When the liquid sample is milk, the specific operation of step (1) is as follows: dissolving the milk sample in an extraction solvent, centrifuging, and filtering to obtain a supernatant; then adding an organic solvent to the supernatant, centrifuging again and discarding the upper organic solvent to obtain a lower solution; drying the lower solution and then redissolving it to obtain a milk analysis sample; the extraction solvent is acetonitrile, and the organic solvent is n-hexane.

4. The method for detecting sulfonamide antibiotics in a liquid sample according to claim 1, wherein When the liquid sample is tap water or lake water, the specific operation of step (1) is: centrifuging and filtering the tap water or lake water to obtain a supernatant; blowing the supernatant dry with nitrogen and then re-dissolving it to obtain a tap water analysis sample or a lake water analysis sample.

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