Determination of sulfonamide antibiotics in water environment by ultra performance liquid chromatography tandem mass spectrometry

Abstract

The application discloses a method for detecting sulfonamide antibiotics in water environment by using ultra-high performance liquid chromatography tandem mass spectrometry, which comprises the following steps: (1) sampling; (2) dispersive solid phase extraction; (3) concentration and constant volume; (4) ultra-high performance liquid chromatography-tandem mass spectrometry detection; (5) drawing a standard curve; (6) according to the ion flow chromatogram of the sulfonamide antibiotics in the solution in the step (4), the type of the detected sulfonamide antibiotics is identified and determined, the quantitative ion integral peak area ratio of each sulfonamide antibiotic and the corresponding isotopic internal standard is obtained, the ratio is compared with the corresponding standard curve, and finally the actual concentration of the sulfonamide antibiotics in the sample can be obtained through conversion. The application has the advantages of simple operation, fast sample pretreatment, accurate detection result, high sensitivity, satisfactory recovery rate and reproducibility, and can be used for the content determination of the sulfonamide antibiotics in different matrix water environments such as fresh water and seawater.

Description

Technical Field

[0001] This invention relates to the field of water pollution detection technology, and in particular to a method for detecting sulfonamide antibiotics in the aquatic environment using ultra-high performance liquid chromatography-tandem mass spectrometry. Background Technology

[0002] Sulfonamide antibiotics are a class of synthetic antibacterial drugs with a p-aminobenzenesulfonamide structure, widely used in human and veterinary medicine to treat various bacterial, protozoan, and fungal infections. However, sulfonamide antibiotics are not completely metabolized in the body, and a large portion is excreted through urine or feces, ultimately releasing into the aquatic environment, posing a threat to human health and the ecological environment. To ensure food safety, the EU and China have set the maximum residue limit for total sulfonamide antibiotics in animal-derived foods at 100 μg / kg, with an acceptable daily intake of 50 μg / kg.

[0003] Currently, various techniques are available for the determination of sulfonamide antibiotics, such as colloidal gold immunochromatography, capillary electrophoresis, high-performance liquid chromatography (HPLC), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). Among these techniques, LC-MS / MS has become the most effective technique for analyzing sulfonamide antibiotics. Because sulfonamide antibiotic concentrations are relatively low and the aqueous matrix is ​​complex, sample pretreatment is crucial for establishing analytical methods for sulfonamide antibiotic residues in complex matrices.

[0004] When using liquid chromatography-tandem mass spectrometry (LC-MS / MS) to detect sulfonamide antibiotics, sample pretreatment techniques such as solid-phase extraction (SPE), solid-phase microextraction (SPE), magnetic solid-phase extraction (MS / MS), and dispersive liquid-liquid microextraction (DL / L / MS) are commonly used to enrich sulfonamide antibiotics in water samples. Among these, solid-phase extraction is the most widely used. The choice of adsorbent is crucial for SPE. Currently, commercially available HLB SPE columns are the most commonly used. For example, in the study "Determination of 21 Sulfonamide Antibiotic Residues in Aquaculture Water by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry" (Environmental Chemistry, Vol. 42, No. 2, February 2023), Oasis HLB SPE columns were used for water sample pretreatment. However, HLB SPE columns are relatively expensive; for example, a 500 mg / 6 mL Oasis HLB column costs approximately 90 RMB. Furthermore, it requires the use of a fully automated SPE instrument, resulting in high detection costs for sulfonamide antibiotics. Other classic adsorbents such as C8, C18, MAX, and MCX have poor enrichment effects.

[0005] Therefore, developing a novel, highly selective, inexpensive, and efficient adsorbent for the enrichment of sulfonamide antibiotics in aquatic environments, combined with highly sensitive and accurate isotope dilution-ultra-high performance liquid chromatography-tandem mass spectrometry detection technology, is expected to solve the current technical bottlenecks. Summary of the Invention

[0006] The purpose of this invention is to provide an ultra-high performance liquid chromatography-tandem mass spectrometry method for the detection of sulfonamide antibiotics in aquatic environments, which is low in detection cost, simple to operate, and has high sensitivity, accuracy and reproducibility.

[0007] To achieve the above objectives, the present invention employs the following technical solution: The method for detecting sulfonamide antibiotics in an aquatic environment using ultra-high performance liquid chromatography-tandem mass spectrometry of the present invention includes the following steps:

[0008] (1) Sampling

[0009] After filtering, the water sample should be refrigerated. The collected water sample should be placed in a brown glass bottle and transported back to the laboratory under refrigeration.

[0010] (2) Dispersive solid phase extraction

[0011] Take 500 mL of sample, adjust the pH of the water sample to 6-9, add disodium EDTA, vortex to dissolve, add internal standards of 17 sulfonamide antibiotics corresponding to their isotopes, mix well, add 5 mg of sericin-based carbon, shake, centrifuge, discard the supernatant, collect the precipitate and pack it into a sericin-based carbon solid-phase extraction column, rinse the sericin-based carbon solid-phase extraction column with deionized water, dry under nitrogen, and elute sequentially with acetone, methanol, and a 1:1 (v / v) acetone / methanol mixture. Combine the eluates; each isotope... The amount of isotope internal standard added was 20 ng. The 17 sulfonamide antibiotics were sulfaguanidine, sulfapyridine, sulfadiazine, sulfathiazole, sulfamethoxypyrimidine, sulfamethoxazole, sulfamethoxazole, sulfadimethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, and sulfamethoxazole. The corresponding isotope internal standards for these 17 sulfonamide antibiotics were sulfaguanidine-D4, sulfapyridine-D4, and sulfamethoxazole-D4. 13 C6, sulfadiazine- 13 C6, sulfathiazole-D4, sulfamethazine- 13 C6, sulfamethhidiazole 13 C6, Sulfamethoxazole 13 C6, Sulfamethoxazole 13 C6, sulfadimethazine-D4, sulfadimethylpyrimidine-D4, sulfamethoxypyrimidine-D4, sulfamethoxypyridazine-D3, sulfamethoxypyrimidine-D4, sulfachlorpyridazine- 13 C6, sulfaquinoxaline- 13 C6, sulfamethoxypyrimidine-D3, sulfamethoxypyrimidine-D6; the sericin-based carbon is prepared by the following method:

[0012] (a) Mix the sericin solution and carboxymethyl chitosan solution thoroughly and refrigerate.

[0013] (b) After refrigeration, add an appropriate amount of sodium chloride solution and prepare hydrogels using the time-current method with a three-electrode system of an electrochemical workstation.

[0014] (c) After freeze-drying the prepared hydrogel, high-temperature carbonization was carried out in a tube furnace to obtain sericin-based carbon. The specific steps of high-temperature carbonization were as follows: First, under a nitrogen flow rate of 150 mL / min, the tube furnace was heated from room temperature to 120-150 °C at a heating rate of 10 °C / min and held for 120 min to remove moisture. Then, the nitrogen gas was turned off, and the temperature was increased to 350 °C at a heating rate of 5 °C / min and held for 180 min. Finally, the temperature was increased to 1050 °C at a heating rate of 2 °C / min and held for 120 min. The high-temperature carbonization step in the preparation of sericin-based carbon is crucial. In the prior art (CN114288989A), an N2 atmosphere is maintained during the carbonization of sericin-based hydrogels, that is, nitrogen gas is continuously purged during carbonization. However, the inventors of this invention unexpectedly discovered that the nitrogen purging method during the high-temperature carbonization of hydrogels affects the adsorption capacity of the obtained sericin-based carbon for sulfonamide antibiotics. In this invention, when carbonizing hydrogels at high temperature, only by using a nitrogen purging method of "heating the tube furnace from room temperature to 120-150°C at a heating rate of 10°C / min, holding for 120 minutes, and then stopping the nitrogen purging" can the obtained sericin-based carbon exhibit better adsorption and elution capacity for sulfonamide antibiotics.

[0015] (d) Wash the sericin-based carbon with deionized water, freeze-dry it, and set it aside for later use.

[0016] This invention creatively employs self-made sericin-based carbon for the specific adsorption of sulfonamide antibiotics, and uses a specific eluent and elution method to ensure the adsorption and elution effect of the sericin-based carbon. Furthermore, the amount of sericin-based carbon used is significantly reduced compared to the amount of adsorbent (HLB solid-phase extraction column) required for solid-phase extraction in existing technologies (500 mg HLB solid-phase extraction column for 200 mL water sample), thus greatly reducing detection costs. When selecting dispersion solid-phase extraction conditions, the inventors investigated the effects of water sample pH and the amount of sericin-based carbon (adsorbent). The inventors found that pH values ​​of 6 and 7 were optimal for effective extraction. At pH 8 and 9, the recoveries of 17 sulfonamide antibiotics using the internal standard method were between 70% and 120% under different pH conditions. Therefore, the pH of the water sample needed to be adjusted to 6-9. When the amount of sericin-based carbon increased from 5 mg to 50 mg, the recoveries of the 17 sulfonamide antibiotics using the external standard method gradually decreased, from 7%-67% (average 49%) to 1%-25% (average 11%). Based on this, in order to obtain better recovery results, the amount of sericin-based carbon was selected as 5 mg. After isotope internal standard correction, the internal standard recovery rate was 82%-97% (average 88%), which met the requirements of the analytical method.

[0017] (3) Concentration and volume adjustment

[0018] Evaporate the eluent to dryness, dissolve the resulting solid in an appropriate amount of initial mobile phase, bring the volume to 1 mL, filter, and use the filtrate as the loading solution.

[0019] (4) Ultra-high performance liquid chromatography-tandem mass spectrometry detection

[0020] The sample solution was drawn using a syringe and loaded according to the set chromatographic-mass spectrometry conditions. The sample was then detected using ultra-high performance liquid chromatography-tandem mass spectrometry to obtain the ion chromatogram of sulfonamide antibiotics in the sample solution.

[0021] (5) Draw the standard curve

[0022] Qualitative analysis was performed using multiple reaction monitoring (MRM) mode and retention time: Seventeen sulfonamide antibiotics and their corresponding 17 isotopic internal standards were dissolved separately in the initial mobile phase to obtain sulfonamide antibiotic mixed standard solution and sulfonamide antibiotic isotopic internal standard mixed standard solution. In the sulfonamide antibiotic mixed standard solution, the concentration of each sulfonamide antibiotic was 1 mg / L; in the sulfonamide antibiotic isotopic internal standard solution, the concentration of each isotopic internal standard was 1 mg / L. 20 μL of each of these solutions was taken and diluted to 1 mL with the initial mobile phase to obtain the concentration of each sulfonamide antibiotic and its corresponding internal standard. The standard working solution should have a concentration of 20 μg / L for each isotope internal standard. The standard working solution is injected and detected according to the method in step (4) to obtain the total ion chromatogram of the standard working solution. The total ion chromatogram is compared with the ion chromatograms of each sulfonamide antibiotic and the corresponding isotope internal standard for qualitative and quantitative ions. Combined with the retention time, the 17 sulfonamide antibiotics in the total ion chromatogram of the standard working solution are identified. The qualitative ions, quantitative ions, retention times of the 17 sulfonamide antibiotics and the quantitative ions and retention times of the corresponding 17 isotope internal standards are used as the basis for identifying the chromatographic peaks of the 17 sulfonamide antibiotics when using the isotope-labeled internal standard method for quantification.

[0023] Quantification was performed using the isotope-labeled internal standard method: Seventeen sulfonamide antibiotics were dissolved in the initial mobile phase to prepare mixed standard solutions of six sulfonamide antibiotics at concentrations of 1 μg / L, 10 μg / L, 100 μg / L, and 1000 μg / L, respectively. 50 μL of each of the following solutions were taken: 100 μL of 10 μg / L, 50 μL of 100 μg / L, 100 μL of 100 μg / L, and 500 μL of the other solutions. Each sulfonamide antibiotic was prepared with 100 μg / L of mixed standard solution and 200 μL of mixed standard solution with 1000 μg / L of mixed standard solution. 20 μL of the corresponding 1 mg / L isotope internal standard solution was added to each solution, and then each solution was brought to 1 mL with the initial mobile phase. This yielded six standard curve series solutions with different spiking concentrations and an isotope internal standard concentration of 20 μg / L. Based on these six standard curve series solutions with different spiking concentrations, a standard curve for each sulfonamide antibiotic was established, with the concentration of each antibiotic as the X-axis and the ratio of the quantitative ion integrated peak area of ​​each antibiotic to the quantitative ion integrated peak area of ​​the corresponding isotope internal standard as the Y-axis. The order in which the standard curves are plotted is only required to be before step (6) and there are no particular restrictions.

[0024] (6) Based on the ion chromatogram of sulfonamide antibiotics in the solution in step (4), identify and determine the types of sulfonamide antibiotics detected, obtain the ratio of the quantitative ion integral peak area of ​​each sulfonamide antibiotic to the corresponding isotope internal standard, compare the ratio with the corresponding standard curve, and finally obtain the actual concentration of sulfonamide antibiotics in the sample by conversion.

[0025] Preferably, in step (1), the water sample is filtered through a glass fiber microporous membrane with a pore size of 0.45 μm and refrigerated at 4°C.

[0026] As a preferred embodiment, in step (2), 0.2 g of disodium ethylenediaminetetraacetate is added; the mixture is shaken in a constant temperature shaker at 25°C and 100 rpm for 8–24 h; centrifuged at 6000 rpm for 3–5 min; and eluted sequentially with 4 mL of acetone, 4 mL of methanol, and 4 mL of a 1:1 acetone / methanol mixed solvent.

[0027] Preferably, in step (a), the sericin solution contains 8-10% sericin by mass, the carboxymethyl chitosan solution contains 1% carboxymethyl chitosan by mass, and the aqueous carboxymethyl chitosan solution has a pH of 12; the sericin solution and the aqueous carboxymethyl chitosan solution are mixed and stirred evenly at a volume ratio of 1:1; the stirring speed is 500-700 rpm, the stirring time is 4-6 h; and the mixture is refrigerated at 3-5℃.

[0028] Preferably, the sericin solution is prepared by the following method: after filtering silk degumming wastewater, it is placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed with deionized water for 3 days.

[0029] Preferably, in step (b), the sodium chloride solution contains 0.5% sodium chloride by mass.

[0030] Preferably, in step (3), the eluent is evaporated to dryness at 50°C; the initial mobile phase is a mixture of formic acid / ammonium acetate aqueous solution and acetonitrile with a volume ratio of 9:1, wherein the concentration of ammonium acetate in the formic acid / ammonium acetate aqueous solution is 2 mmol / L and the volume percentage of formic acid is 0.1%; and filtration is performed using a 0.22 μm polytetrafluoroethylene filter membrane.

[0031] Preferably, in step (4), the chromatographic conditions are as follows: injection volume is 5 μL; a Waters BEHC18 column is used, with column dimensions of 2.1 mm × 100 mm and 1.7 μm; column temperature is 40 °C; binary gradient elution is used at 0.3 mL / min; mobile phase A is formic acid / ammonium acetate aqueous solution, in which the concentration of ammonium acetate is 2 mmol / L and the volume percentage of formic acid is 0.1%; mobile phase B is acetonitrile; gradient elution is used. Program: 0~1.5min, 10%B; 1.5~6.5min, 10%~12.5%B; 6.5~9.5min, 12.5%~30%B; 9.5~10.5min, 30%~40% B; 10.5~10.7min, 40%~90%B; 10.7~11.5min, 90%B; 11.5~11.8min, 90%~10%B; 11.8~15min, 10%B;

[0032] The mass spectrometry conditions were as follows: electrospray ionization in positron mode and multiple reaction monitoring (MRM); capillary voltage 3.0 kV; ion source temperature 150 °C; desolvation gas temperature 600 °C; cone gas flow rate 150 L / h; desolvation gas flow rate 800 L / h.

[0033] Preferably, in step (5), the initial mobile phase is a mixture of formic acid / ammonium acetate aqueous solution and acetonitrile with a volume ratio of 9:1, wherein the concentration of ammonium acetate in the formic acid / ammonium acetate aqueous solution is 2 mmol / L and the volume percentage of formic acid is 0.1%; therefore, the present invention has the following beneficial effects:

[0034] (1) Sulfonamide antibiotics are specifically adsorbed using self-made sericin-based carbon, and eluted with a specific eluent and elution method to ensure the adsorption and elution effect of sericin-based carbon. The amount of sericin-based carbon used is greatly reduced compared with the amount of adsorbent (HLB solid phase extraction column) required for solid phase extraction in the existing technology (500mg HLB solid phase extraction column is required for 200mL water sample), which can greatly reduce the detection cost.

[0035] (2) A method for detecting sulfonamide antibiotics in aquatic environments by ultra-high performance liquid chromatography-tandem mass spectrometry is provided. The method is simple to operate, can quickly complete sample pretreatment, and has accurate detection results. The detection limits for 17 sulfonamide antibiotics are 0.02-0.1 ng / L, the recovery rate is 70%-106%, and the relative standard deviation (n=5) is 1.9-13.5%. It has high sensitivity, satisfactory recovery rate and reproducibility, and can be used to determine the content of sulfonamide antibiotics in different substrate aquatic environments such as freshwater and seawater. Attached Figure Description

[0036] Figure 1 This is a photograph of the hydrogel prepared in Example 1.

[0037] Figure 2 This is a physical image of the sericin-based carbon in Example 1.

[0038] Figure 3 This is an electron microscope image of the sericin-based carbon in Example 1.

[0039] Figure 4 This is an ion chromatogram of sulfapyridine and sulfamethoxazole detected in the water sample of Example 2.

[0040] Figure 5 This is an ion chromatogram of sulfapyridine, sulfadiazine, sulfamethoxazole, sulfadimethylpyrimidine, and sulfamethoxypyrimidine detected in the water sample of Example 3. Detailed Implementation

[0041] The present invention will now be further described with reference to the accompanying drawings and specific embodiments.

[0042] Investigating the effect of different nitrogen purging methods on the adsorption capacity of sericin-based carbon

[0043] Three groups of sericin-based carbons were prepared, denoted as group A, group B, and group C, respectively.

[0044] Group A sericin-based carbon was prepared by the following method:

[0045] (a) Mix a sericin protein solution with a sericin protein content of 10% by weight and a carboxymethyl chitosan solution with a carboxymethyl chitosan content of 1% by weight at a volume ratio of 1:1 and stir evenly at a stirring speed of 700 rpm for 4 hours and refrigerate at 4°C.

[0046] (b) After refrigeration, add an appropriate amount of sodium chloride solution with a mass percentage of 5% and prepare hydrogels using the time-current method with a three-electrode system of an electrochemical workstation.

[0047] (c) After freeze-drying the prepared hydrogel, high-temperature carbonization was carried out in a tube furnace to obtain sericin-based carbon. The specific steps of high-temperature carbonization were as follows: First, under a nitrogen flow rate of 150 mL / min, the tube furnace was heated from room temperature to 120-150 °C at a heating rate of 10 °C / min and held for 120 min to remove moisture. Then, the nitrogen gas was turned off, and the temperature was increased to 350 °C at a heating rate of 5 °C / min and held for 180 min. Finally, the temperature was increased to 1050 °C at a heating rate of 2 °C / min and held for 120 min.

[0048] (d) Wash the sericin-based carbon with deionized water, freeze-dry it, and set it aside for later use.

[0049] The method for preparing sericin-based carbon in Group B differs from that in Group A in that, in step (c), the temperature is increased to 350°C at a rate of 5°C / min, held for 180 min, and then the nitrogen gas is turned off.

[0050] The method for preparing sericin-based carbon in group C differs from that in group A in that, in step (c), the temperature is increased to 1050℃ at a rate of 2℃ / min, held for 120min, and then the nitrogen gas is turned off.

[0051] The experiments showed that when using group A sericin-based carbon, the external standard recovery rates of 17 sulfonamide antibiotics ranged from 12% to 69%, with an average of 49%; when using group B sericin-based carbon, the external standard recovery rates ranged from 0.064% to 3.8%, with an average of 1.1%; and when using group C sericin-based carbon, the external standard recovery rates ranged from 0.15% to 8.0%, with an average of 1.6%. After internal standard correction, the internal standard recovery rate of group A was 84% ​​to 102%, with an average of 90%, meeting the analytical method requirements; while in groups B and C, the relative recoveries of several sulfonamide antibiotics, such as sulfaguanidine, sulfadiazine, sulfamethoxazole, and sulfadiazine, were <50%, failing to meet the analytical method requirements.

[0052] This indicates that only the sericin-based carbon obtained using the nitrogen-purging method of this invention has good adsorption and elution performance, which can meet the requirements of the analytical method.

[0053] Example 1

[0054] (1) Sampling

[0055] The water sample was commercially available Wahaha purified water. The water sample was filtered through a glass fiber microporous membrane with a pore size of 0.45μm and refrigerated at 4℃.

[0056] (2) Dispersive solid phase extraction

[0057] Take 500 mL of sample, adjust the pH of the water sample to 6-9, add 0.2 g of disodium ethylenediaminetetraacetate, vortex to dissolve, add isotopic internal standards corresponding to 17 sulfonamide antibiotics, mix well, add 5 mg of sericin-based carbon, shake in a constant temperature shaker at 25℃ and 100 rpm for 8 h, centrifuge at 6000 rpm for 3 min, discard the supernatant, collect the precipitate and pack it into a sericin-based carbon solid-phase extraction column, wash the sericin-based carbon solid-phase extraction column three times with 3 × 5 mL of deionized water, dry in nitrogen, and then wash successively with 4 mL of acetone, 4 mL of methanol, ... Elute with 4 mL of acetone / methanol mixed solvent (1:1 v / v), and combine the eluates; 20 ng of each isotope internal standard was added. The 17 sulfonamide antibiotics were sulfaguanidine, sulfapyridine, sulfadiazine, sulfathiazole, sulfamethylpyrimidine, sulfamethhidiazole, sulfamethoxazole, sulfadimethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, and sulfamethoxazole. The corresponding isotope internal standards were sulfaguanidine-D4, sulfapyridine-D4, and sulfamethoxazole-D4. 13 C6, sulfadiazine- 13 C6, sulfathiazole-D4, sulfamethazine- 13 C6, sulfamethhidiazole 13 C6, Sulfamethoxazole 13 C6, Sulfamethoxazole 13 C6, sulfadimethazine-D4, sulfadimethylpyrimidine-D4, sulfamethoxypyrimidine-D4, sulfamethoxypyridazine-D3, sulfamethoxypyrimidine-D4, sulfachlorpyridazine- 13 C6, sulfaquinoxaline- 13 C6, sulfamethoxypyrimidine-D3, sulfamethoxypyrimidine-D6; the sericin-based carbon is prepared by the following method:

[0058] (a) Mix a sericin protein solution with a sericin protein content of 10% by weight and a carboxymethyl chitosan solution with a carboxymethyl chitosan content of 1% by weight at a volume ratio of 1:1 and stir evenly at a stirring speed of 700 rpm for 4 hours and refrigerate at 4°C.

[0059] (b) After refrigeration, add an appropriate amount of sodium chloride solution with a mass percentage of 5%. Using a three-electrode system of an electrochemical workstation, prepare the hydrogel using the time-current method. The prepared hydrogel is shown in the figure. Figure 1 As shown;

[0060] (c) After freeze-drying the prepared hydrogel, it is subjected to high-temperature carbonization in a tube furnace to obtain sericin-based carbon, which is as follows: Figure 2 As shown, the carbon electron microscopy image of sericin-based protein is as follows. Figure 3 As shown, the specific steps of high-temperature carbonization are as follows: First, under a nitrogen flow rate of 150 mL / min, the tube furnace is heated from room temperature to 150°C at a heating rate of 10°C / min, held for 120 min to remove moisture, and then the nitrogen gas is turned off. Next, the temperature is increased to 350°C at a heating rate of 5°C / min and held for 180 min. Finally, the temperature is increased to 1050°C at a heating rate of 2°C / min and held for 120 min.

[0061] (d) Wash the sericin-based carbon with deionized water, freeze-dry it, and set it aside for later use.

[0062] (3) Concentration and volume adjustment

[0063] The eluent was evaporated to dryness under nitrogen at 50°C. The resulting solid was dissolved in an appropriate amount of the initial mobile phase and brought to a final volume of 1 mL. The solution was filtered through a 0.22 μm polytetrafluoroethylene (PTFE) membrane, and the filtrate was used as the loading solution. The initial mobile phase was a mixture of formic acid / ammonium acetate aqueous solution and acetonitrile in a volume ratio of 9:1. In the formic acid / ammonium acetate aqueous solution, the concentration of ammonium acetate was 2 mmol / L, and the volume percentage of formic acid was 0.1%.

[0064] (4) Ultra-high performance liquid chromatography-tandem mass spectrometry detection

[0065] The sample solution was drawn using a syringe and loaded according to the set chromatographic-mass spectrometry conditions. Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) was used for detection, yielding a total ion chromatogram of the sample solution. The chromatographic conditions were as follows: injection volume 5 μL; Waters BEHC18 column (2.1 mm × 100 mm, 1.7 μm); column temperature 40℃; binary gradient elution at 0.3 mL / min; mobile phase A was formic acid / ammonium acetate aqueous solution, with ammonium acetate concentration of 2 mmol / L. The cumulative percentage content is 0.1%, mobile phase B is acetonitrile, and the gradient elution program is as follows: 0–1.5 min, 10% B; 1.5–6.5 min, 10%–12.5% ​​B; 6.5–9.5 min, 12.5%–30% B; 9.5–10.5 min, 30%–40% B; 10.5–10.7 min, 40%–90% B; 10.7–11.5 min, 90% B; 11.5–11.8 min, 90%–10% B; 11.8–15 min, 10% B.

[0066] The mass spectrometry conditions were as follows: electrospray ionization in positron mode and multiple reaction monitoring (MRM); capillary voltage 3.0 kV; ion source temperature 150 °C; desolvation gas temperature 600 °C; cone gas flow rate 150 L / h; desolvation gas flow rate 800 L / h.

[0067] (5) Draw the standard curve

[0068] Qualitative analysis was performed using multiple reaction monitoring (MRM) mode and retention time: Seventeen sulfonamide antibiotics and their corresponding 17 isotope internal standards were dissolved in the initial mobile phase to obtain mixed standard working solutions for sulfonamide antibiotics and mixed standard working solutions for sulfonamide antibiotic isotope internal standards. In the mixed standard working solution for sulfonamide antibiotics, the concentration of each sulfonamide antibiotic was 1 mg / L. In the mixed standard working solution for sulfonamide antibiotics and the mixed standard working solution for sulfonamide antibiotic isotope internal standards, the concentration of each isotope internal standard was 1 mg / L. 20 μL of each mixed standard working solution for sulfonamide antibiotics and 20 μL of the mixed standard working solution for sulfonamide antibiotic isotope internal standards were taken and diluted to 1 mL with the initial mobile phase to obtain standard working solutions with a concentration of 20 μg / L for each sulfonamide antibiotic and the corresponding isotope internal standard. The initial mobile phase was a mixture of formic acid / ammonium acetate aqueous solution and acetonitrile in a volume ratio of 9:1. In the formic acid / ammonium acetate aqueous solution, the concentration of ammonium acetate was 2 mmol / L and the volume percentage of formic acid was 0.1%.

[0069] The standard working solution was injected and detected according to the method in step (4) to obtain the total ion chromatogram of the standard working solution. The total ion chromatogram was compared with the ion chromatograms of each sulfonamide antibiotic and the corresponding isotope internal standard for qualitative and quantitative ion analysis. Combined with the retention time, the 17 sulfonamide antibiotics in the total ion chromatogram of the standard working solution were identified. The qualitative ion, quantitative ion, retention time of the 17 sulfonamide antibiotics and the quantitative ion and retention time of the corresponding 17 isotope internal standards were used as the basis for identifying the chromatographic peaks of the 17 sulfonamide antibiotics when using the isotope-labeled internal standard method for quantification. The retention times of the 17 sulfonamide antibiotics and the corresponding 17 isotope internal standards and the multiple reaction monitoring conditions are shown in Table 1.

[0070] Table 1. Retention times and multiple reaction monitoring conditions of 17 sulfonamide antibiotics and their corresponding 17 isotopic internal standards.

[0071]

[0072]

[0073] Note: *Quantitative ions.

[0074] Quantification was performed using the isotope-labeled internal standard method: Seventeen sulfonamide antibiotics were dissolved in the initial mobile phase to prepare mixed standard solutions of six sulfonamide antibiotics at concentrations of 1 μg / L, 10 μg / L, 100 μg / L, and 1000 μg / L, respectively. 50 μL of each of the following solutions were taken: 10 μL of 10 μg / L, 50 μL of 100 μg / L, 100 μg / L, 500 μL of 100 μg / L, and 200 μL of each sulfonamide antibiotic at a concentration of 100 μg / L. A 1000 μg / L sulfonamide antibiotic mixed standard working solution was prepared by adding 20 μL of the corresponding 1 mg / L isotope internal standard mixed solution, and then bringing the volume to 1 mL with the initial mobile phase to obtain six standard curve series solutions with different spiking concentrations and an isotope internal standard concentration of 20 μg / L. The initial mobile phase was a mixture of formic acid / ammonium acetate aqueous solution and acetonitrile in a volume ratio of 9:1. In the formic acid / ammonium acetate aqueous solution, the concentration of ammonium acetate was 2 mmol / L and the volume percentage of formic acid was 0.1%. Based on the six standard curve series solutions with different spiking concentrations, a standard curve for each sulfonamide antibiotic was established with the concentration of each sulfonamide antibiotic as the X-axis and the ratio of the quantitative ion integrated peak area of ​​each sulfonamide antibiotic to the quantitative ion integrated peak area of ​​the corresponding isotope internal standard as the Y-axis. The linear regression equation, linear range, correlation coefficient and detection limit of the standard curve are shown in Table 2.

[0075] Table 2. Linear regression equations, linear ranges, correlation coefficients, and limits of detection for 17 sulfonamide antibiotics.

[0076]

[0077] Where, a: linear range represents the concentration of the analyte in the standard curve series solutions; b: y and x represent the ratio of the quantitative ion integrated peak area of ​​the analyte to the quantitative ion integrated peak area of ​​the corresponding isotopic internal standard and the theoretical concentration of the analyte in the initial mobile phase, respectively.

[0078] (6) Based on the ion chromatogram of sulfonamide antibiotics in the solution in step (4), identify and determine the types of sulfonamide antibiotics detected, obtain the ratio of the quantitative ion integral peak area of ​​each sulfonamide antibiotic to the corresponding isotope internal standard, compare the ratio with the corresponding standard curve, and finally obtain the actual concentration of sulfonamide antibiotics in the sample by conversion.

[0079] Using the Wahaha purified water described above, after processing according to the requirements of step (1), three 500mL water samples were taken. 20μL of a mixed standard solution of sulfonamide antibiotics with a concentration of 100μg / L for each type of sulfonamide antibiotic, 20μL of a mixed standard solution of sulfonamide antibiotics with a concentration of 1000μg / L for each type of sulfonamide antibiotic, and 200μL of a mixed standard solution of sulfonamide antibiotics with a concentration of 1000μg / L for each type of sulfonamide antibiotic were added to prepare spiked samples at three concentration levels: low (4ng / L), medium (40ng / L), and high (400ng / L). Then, 20μL of the corresponding 1mg / L sulfonamide antibiotic isotope internal standard solution was added to each sample. The mixture was vortexed and mixed thoroughly. Five parallel operations were performed according to the requirements of steps (1) to (4) above, and compared with the standard curve obtained in step (5) above. The final concentration of the 17 sulfonamides in the spiked samples was obtained through conversion. The recovery rate was calculated according to the following formula:

[0080]

[0081] Where: R—recovery rate, %;

[0082] C s —The concentration of 17 sulfonamide antibiotics in the spiked sample, ng / L;

[0083] C0—Concentration of 17 sulfonamide antibiotics in the actual sample, ng / L;

[0084] C – The theoretical spiking concentration of 17 sulfonamide antibiotics in the spiked sample, ng / L.

[0085] Tests showed that none of the 17 sulfonamide antibiotics in Wahaha purified water were detected. The results of the spiked recovery experiment at different spiked concentrations in Wahaha purified water are shown in Table 3.

[0086] Table 3. Background values, spiked recoveries, and method precision of 17 sulfonamide antibiotics in Wahaha purified water (n=5)

[0087]

[0088] Note: "ND" means Not Detected.

[0089] As shown in Table 3, the recovery rate of spiked Wahaha purified water is 70-106%, and the relative standard deviation (n=5) is 1.9-12.0%, which meets the requirements of the analytical method for recovery rate and reproducibility.

[0090] Example 2

[0091] The difference between Example 2 and Example 1 is that the water sample was river water collected from a river in Zhoushan. The carbonization heating procedure was "first, under a nitrogen flow rate of 150 mL / min, the tube furnace was heated from room temperature to 120°C at a heating rate of 10°C / min, and held for 120 min to remove moisture before the nitrogen was turned off". The rest was the same as in Example 1.

[0092] Tests revealed the presence of sulfapyridine and sulfamethoxazole in the river water, with concentrations of 2.668 and 18.051 ng / L, respectively. The ion chromatograms are shown below. Figure 4 As shown, the other 15 sulfonamide antibiotics were not detected. The results of the spiked recovery experiments at different spiked concentrations in the river water are shown in Table 4.

[0093] Table 4. Background values, spiked recoveries, and method precision of 17 sulfonamide antibiotics in river water (n=5)

[0094]

[0095] Note: "ND" means Not Detected.

[0096] As shown in Table 4, the recovery rate of the spiked river water was 76-104%, and the relative standard deviation (n=5) was 2.8-12.8%, which met the requirements of the analytical method for recovery rate and reproducibility.

[0097] Example 3

[0098] The difference between Example 3 and Example 1 is that the water sample is a seawater sample collected from the Wenzhou sea area, while the rest is the same as Example 1.

[0099] The seawater was tested and found to contain sulfapyridine, sulfadiazine, sulfamethoxazole, sulfadimethylpyrimidine, and sulfamethoxypyrimidine, with concentrations of 0.161, 0.148, 2.115, 0.172, and 0.524 ng / L, respectively. The ion chromatograms are shown below. Figure 5 As shown, the other 12 sulfonamide antibiotics were not detected. The results of the spiked recovery experiments at different spiked concentrations in the spiked seawater are shown in Table 5.

[0100] Table 5. Background values, spiked recoveries, and method precision of 17 sulfonamide antibiotics in seawater (n=5)

[0101]

[0102]

[0103] Note: "ND" means Not Detected.

[0104] As shown in Table 5, the recovery rate of spiked seawater was 75-105%, and the relative standard deviation (n=5) was 3.2-13.5%, which met the requirements of the analytical method for recovery rate and reproducibility.

[0105] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications may be made without departing from the technical solutions described in the claims.

Claims

1. A method for detecting sulfonamide antibiotics in water environment by ultra-high performance liquid chromatography tandem mass spectrometry, characterized in that, Comprising the following steps: (1) Sampling After filtering the water sample, store in cold storage; (2) Dispersive solid phase extraction Take 500 mL of sample, adjust the pH of the water sample to 6-9, add disodium EDTA, vortex to dissolve, add internal standards of 17 sulfonamide antibiotics corresponding to their isotopes, mix well, add 5 mg of sericin-based carbon, shake, centrifuge, discard the supernatant, collect the precipitate and pack it into a sericin-based carbon solid-phase extraction column, rinse the sericin-based carbon solid-phase extraction column with deionized water, dry under nitrogen, and elute sequentially with acetone, methanol, and a 1:1 (v / v) acetone / methanol mixture. Combine the eluates; each isotope... The amount of isotope internal standard added was 20 ng. The 17 sulfonamide antibiotics were sulfaguanidine, sulfapyridine, sulfadiazine, sulfathiazole, sulfamethoxypyrimidine, sulfamethoxazole, sulfamethoxazole, sulfadimethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, and sulfamethoxazole. The corresponding isotope internal standards for these 17 sulfonamide antibiotics were sulfaguanidine-D4, sulfapyridine-D4, and sulfamethoxazole-D4. 13 C6, sulfadiazine- 13 C6, sulfathiazole-D4, sulfamethazine- 13 C6, sulfamethhidiazole 13 C6, Sulfamethoxazole 13 C6, Sulfamethoxazole 13 C6, sulfadimethazine-D4, sulfadimethylpyrimidine-D4, sulfamethoxypyrimidine-D4, sulfamethoxypyridazine-D3, sulfamethoxypyrimidine-D4, sulfachlorpyridazine- 13 C6, sulfaquinoxaline- 13 C6, sulfamethoxypyrimidine-D3, sulfamethoxypyrimidine-D6; the sericin-based carbon is prepared by the following method: (a) Mix the sericin protein solution and the carboxymethyl chitosan solution and stir until uniform, and store in cold storage; (b) After cold storage, add sodium chloride solution, and use the three-electrode system of the electrochemical workstation to prepare the hydrogel by time-current method; (c) After freeze-drying the prepared hydrogel, use a tube furnace to perform high-temperature carbonization, and the specific steps are as follows: first, under the flow of nitrogen gas with a flow rate of 150 mL / min, increase the temperature of the tube furnace from room temperature to 120-150 DEG C at a rate of 10 DEG C / min, maintain for 120 min to remove water, then turn off the nitrogen gas, increase the temperature to 350 DEG C at a rate of 5 DEG C / min, maintain for 180 min, and finally increase the temperature to 1050 DEG C at a rate of 2 DEG C / min, maintain for 120 min; (d) After washing the sericin protein-based carbon with deionized water, freeze-dry, and wait for use; (3) Concentration and constant volume Evaporate the eluent to dryness, dissolve the obtained solid with the initial mobile phase, and then constant volume to 1 mL, filter, and the filtrate is used as the sample solution; (4) Ultra-high performance liquid chromatography-tandem mass spectrometry detection Use the sample needle to extract the sample solution, and load according to the set chromatography-mass spectrometry conditions, and use ultra-high performance liquid chromatography-tandem mass spectrometry for detection, and obtain the ion flow chromatogram of the 17 sulfonamide antibiotics in the sample solution; (5) Draw standard curve Use multiple reaction monitoring mode and retention time for qualitative analysis: dissolve the 17 sulfonamide antibiotics and the corresponding 17 isotopic internal standards in the initial mobile phase respectively, and obtain the sulfonamide antibiotic mixed standard solution and the sulfonamide antibiotic isotopic internal standard mixed standard solution, in the sulfonamide antibiotic mixed standard solution, the concentration of each sulfonamide antibiotic is 1 mg / L, in the sulfonamide antibiotic isotopic internal standard mixed standard solution, the concentration of each isotopic internal standard is 1 mg / L, take 20 μL of the sulfonamide antibiotic mixed standard solution and 20 μL of the sulfonamide antibiotic isotopic internal standard mixed standard solution respectively, constant volume to 1 mL with the initial mobile phase, and obtain the standard working solution with the concentration of each sulfonamide antibiotic and the corresponding each isotopic internal standard being 20 μg / L; detect the standard working solution by the method of step (4), and obtain the total ion flow chromatogram of the standard working solution; compare the total ion flow chromatogram with the ion flow chromatogram of each sulfonamide antibiotic and the corresponding isotopic internal standard, and combine the retention time to determine the 17 sulfonamide antibiotics in the total ion flow chromatogram of the standard working solution; and use the qualitative ion, the quantitative ion, the retention time of the 17 sulfonamide antibiotics, and the quantitative ion, the retention time of the corresponding 17 isotopic internal standards as the basis for identifying the chromatographic peaks of the 17 sulfonamide antibiotics when quantified by the isotopic labeling internal standard method; Quantify by the isotopic labeling internal standard method: dissolve the 17 sulfonamide antibiotics in the initial mobile phase to prepare 17 sulfonamide antibiotic mixed standard solutions with the concentration of each sulfonamide antibiotic being 1 μg / L, 10 μg / L, 100 μg / L, and 1000 μg / L respectively, Respectively take 50 muL each sulfonamides antibiotic concentration is 1 mu g / L sulfonamides antibiotic mixed standard solution, 100 muL each sulfonamides antibiotic concentration is 10 mu g / L sulfonamides antibiotic mixed standard solution, 50 muL each sulfonamides antibiotic concentration is 100 mu g / L sulfonamides antibiotic mixed standard solution, 100 muL each sulfonamides antibiotic concentration is 100 mu g / L sulfonamides antibiotic mixed standard solution, 500 muL each sulfonamides antibiotic concentration is 100 mu g / L sulfonamides antibiotic mixed standard solution and 200 muL each sulfonamides antibiotic concentration is 1000 mu g / L sulfonamides antibiotic mixed standard solution, respectively, add corresponding 1 mg / L isotope internal standard mixed solution 20 muL, respectively, with the initial mobile phase constant volume to 1 mL, obtain six kinds of standard curve series solution with different concentrations, isotope internal standard concentration is 20 mu g / L, according to six different standard curve series solution with different concentrations, each sulfonamides antibiotic concentration is X axis, each sulfonamides antibiotic quantitative ion integral peak area and corresponding isotope internal standard quantitative ion integral peak area ratio is Y axis, establish each sulfonamides antibiotic standard curve; (6) according to the ion flow chromatogram of sulfonamides antibiotic in step (4) on-line solution, identify and determine the type of detected sulfonamides antibiotic, obtain the quantitative ion integral peak area ratio of each sulfonamides antibiotic and corresponding isotope internal standard, then compare the ratio with the corresponding standard curve, finally through the conversion, the actual concentration of sulfonamides antibiotic in the sample can be obtained.

2. The method for detecting sulfonamide antibiotics in water environment by ultra-high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that, In step (1), the water sample is filtered through a glass fiber microporous filter with a pore size of 0.45 mu m and stored at 4 DEG C.

3. The method for detecting sulfonamide antibiotics in water environment by ultra-high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that, In step (2), 0.2 g of disodium ethylenediaminetetraacetate is added, and the mixture is shaken at 25 DEG C and 100 rpm for 8-24 h, then centrifuged at 6000 rpm for 3-5 min, and then eluted with 4 mL of acetone, 4 mL of methanol, and 4 mL of a mixture of acetone and methanol in a volume ratio of 1:

1.

4. The method for detecting sulfonamide antibiotics in water environment by ultra-high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that, In step (a), the silk fibroin solution has a silk fibroin mass percentage of 8-10%, the carboxymethyl chitosan solution has a carboxymethyl chitosan mass percentage of 1%, and the carboxymethyl chitosan solution has a pH of 12; the silk fibroin solution and the carboxymethyl chitosan solution are mixed in a volume ratio of 1:1 and stirred uniformly; the stirring speed is 500-700 rpm, and the stirring time is 4-6 h; and the mixture is stored at 3-5 DEG C.

5. The method for detecting sulfonamide antibiotics in water environment by ultra-high performance liquid chromatography tandem mass spectrometry according to claim 1 or 4, characterized in that, The silk fibroin solution is prepared by the following method: filtering the degumming wastewater of silk, and then dialyzing with deionized water flowing in a dialysis bag with a molecular weight cut-off of 3.5 kDa for 3 days.

6. The method for detecting sulfonamide antibiotics in water environment by ultra-high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that, In step (b), the sodium chloride solution has a sodium chloride mass percentage of 0.5%.

7. The method for detecting sulfonamide antibiotics in water environment by ultra-high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that, In step (3), the eluent was evaporated at 50℃ until dryness; the initial mobile phase was a mixture of formic acid / ammonium acetate aqueous solution and acetonitrile at a volume ratio of 9:1, the formic acid / ammonium acetate aqueous solution had an ammonium acetate concentration of 2 mmol / L and a formic acid volume percentage of 0.1%, and the mixture was filtered using a 0.22 μm polytetrafluoroethylene filter.

8. The method for detecting sulfonamide antibiotics in water environment by ultra-high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that, In step (4), the chromatographic conditions were as follows: the injection volume was 5 μL; a Waters BEH C18 column was used, the column specifications were 2.1 mm x 100 mm x 1.7 μm, and the column temperature was 40℃; a binary gradient elution was used at a flow rate of 0.3 mL / min, mobile phase A was formic acid / ammonium acetate aqueous solution, the formic acid / ammonium acetate aqueous solution had an ammonium acetate concentration of 2 mmol / L and a formic acid volume percentage of 0.1%, mobile phase B was acetonitrile, and the gradient elution program was as follows: 0-1.5 min, 10% B; 1.5-6.5 min, 10%-12.5% B; 6.5-9.5 min, 12.5%-30% B; 9.5-10.5 min, 30%-40% B; 10.5-10.7 min, 40%-90% B; 10.7-11.5 min, 90% B; 11.5-11.8 min, 90%-10% B; 11.8-15 min, 10% B. The mass spectrometry conditions were as follows: electrospray positive ion mode ionization, multiple reaction monitoring mode monitoring; capillary voltage 3.0 kV; ion source temperature 150℃; desolvation gas temperature 600℃; cone gas flow rate 150 L / h; desolvation gas flow rate 800 L / h. In step (5), the initial mobile phase was a mixture of formic acid / ammonium acetate aqueous solution and acetonitrile at a volume ratio of 9:1, the formic acid / ammonium acetate aqueous solution had an ammonium acetate concentration of 2 mmol / L and a formic acid volume percentage of 0.1%. ​ 9. The method for detecting sulfonamide antibiotics in water environment by ultra-high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that, ​

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