A method for enriching and separating sulfonamide antibiotics in drinking water or milk

Abstract

The present invention discloses a method for enriching and separating sulfonamide antibiotics in drinking water or milk, comprising the following steps: sample processing: adding trichloroacetic acid solution to the sample, vortexing and then ultrasonically centrifuging, and collecting the supernatant; magnetic deep eutectic solvent synthesis: stirring choline chloride, phenol and ferric chloride at room temperature to form a magnetic deep eutectic solvent; magnetic deep eutectic solvent / salt two-phase aqueous system: adding the formed magnetic deep eutectic solvent to the supernatant, vortexing and oscillating, adding salt, vortexing and oscillating, to form a magnetic deep eutectic solvent / salt two-phase aqueous system; extraction: placing a magnet at the bottom of the solution to attract magnetic droplets, moving the magnet upward to achieve phase separation of the two-phase aqueous system, accurately measuring the volumes of the upper and lower phases, and using a high performance liquid chromatograph to determine the concentration of sulfonamide antibiotics in the lower inorganic salt phase. This method can efficiently separate and enrich sulfonamide antibiotics, and the selected solvent is non-toxic and pollution-free, providing a new idea for the separation and enrichment of trace sulfonamide antibiotics.

Description

Technical Field

[0001] The invention relates to the technical field of sulfonamide compound extraction, and in particular to a method for enriching and separating sulfonamide antibiotics in drinking water or milk. Background Art

[0002] Various antibiotics have played a significant role in treating infectious diseases, effectively safeguarding human life and health. However, their widespread use has led to increasing negative impacts on humans and the ecological environment, potentially jeopardizing human health and survival. In recent years, the environmental impacts of antibiotics have garnered increasing attention.

[0003] Sulfonamides are inexpensive, easy to use, have a broad antimicrobial spectrum, and are stable, making them an important anti-infective treatment for livestock and poultry. However, sulfonamide residues in drinking water and milk are extremely low, and the complex sample matrices make direct detection of sulfonamide antibiotics in these samples difficult. To date, commonly used methods for pre-concentration and extraction of sulfonamide antibiotic residues in food include liquid-liquid extraction, solid-phase extraction, matrix-based solid-phase dispersion, immunoaffinity chromatography, and supercritical fluid extraction. While these methods have their own advantages for drug residue analysis, they also have numerous drawbacks, such as the use of volatile organic solvents, difficulties with standardization, high processing costs, cumbersome procedures, and limited enrichment multiples. Therefore, there is a need for efficient, non-toxic, and pollution-free separation and enrichment methods. Summary of the Invention

[0004] The present invention discloses a method for enriching and separating sulfonamide antibiotics in drinking water or milk. The method can efficiently separate and enrich sulfonamide antibiotics, and the selected solvent is non-toxic and pollution-free, providing a new idea for the separation and enrichment of trace sulfonamide antibiotics.

[0005] The technical solutions of the present invention are as follows:

[0006] A method for enriching and separating sulfonamide antibiotics in drinking water or milk comprises the following steps:

[0007] (1) Sample preparation: Weigh the sample, add trichloroacetic acid solution, vortex and sonicate, centrifuge, and collect the supernatant;

[0008] (2) Synthesis of magnetic deep eutectic solvent: Choline chloride, phenol and ferric chloride are stirred at room temperature until a homogeneous transparent liquid is formed, i.e., a magnetic deep eutectic solvent [ChCl / Ph2][FeCl4] is formed;

[0009] (3) Magnetic deep eutectic solvent / salt aqueous two-phase system: the magnetic deep eutectic solvent is added to the supernatant, vortexed, and then salt is added and vortexed to form a magnetic deep eutectic solvent / salt aqueous two-phase system;

[0010] (4) Extraction: Place a magnet at the bottom of the solution to attract the magnetic droplets, move the magnet upward to separate the two-phase aqueous system, accurately measure the volumes of the upper and lower phases, and use high performance liquid chromatography to determine the concentration of sulfonamide antibiotics in the lower inorganic salt phase.

[0011] Furthermore, in step (1), 2 mL of 10% trichloroacetic acid solution was added to 8 mL of sample.

[0012] Furthermore, in step (2), the molar ratio of choline chloride, phenol and ferric chloride is 1:2:1.

[0013] Furthermore, the salt in step (3) is Na2SO4, (NH4)2SO4, NH4Cl, K2HPO4 or Na2HPO4.

[0014] Furthermore, the mass concentration of the magnetic deep eutectic solvent in step (3) is 7.5-17.5%. Preferably, the concentration of the magnetic deep eutectic solvent is 10%.

[0015] Furthermore, in step (3), the mass concentration of salt in the system is 20%-40%, preferably 30%.

[0016] Furthermore, the pH of the system is 4-6, preferably, the pH of the system is 4.5.

[0017] Beneficial effects

[0018] The magnetic deep eutectic solvent adopted in the present invention has a simple synthesis process, low cost, is non-toxic and pollution-free, and the product does not need to be purified; the entire extraction is carried out at room temperature, no solvent volatilizes during the extraction process, and there is no pollution to the environment; the magnetic separation technology does not require a centrifugation step, has low energy consumption, and is fast; under optimized conditions, the extraction efficiency is high and the enrichment multiple is high, and the four sulfonamide antibiotics, sulfadiazine, sulfamethoxazole, and sulfamethoxypyrimidine, can be effectively separated and enriched. It is a green pretreatment technology for the detection of sulfonamide antibiotics and provides a new idea for the separation and enrichment of trace sulfonamide antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The effect of salt types on the extraction rate of sulfonamide antibiotics;

[0020] Figure 2 The effect of salt concentration on the extraction rate of sulfonamide antibiotics;

[0021] Figure 3 The effect of magnetic deep eutectic solvent concentration on the extraction rate of sulfonamide antibiotics;

[0022] Figure 4This is the effect of system pH value on the extraction rate of sulfonamide antibiotics. DETAILED DESCRIPTION

[0023] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process. However, the protection scope of the present invention is not limited to the following embodiment.

[0024] Example 1

[0025] A method for enriching and separating sulfonamide antibiotics in drinking water or milk comprises the following steps:

[0026] (1) Sample treatment: Accurately measure 8 mL of spiked milk sample, add 2 mL of 10% trichloroacetic acid solution, vortex for 2 min, then ultrasonicate for 10 min to fully mix the trichloroacetic acid with the milk sample, and then rotate at 10000 r min. -1 The mixture was centrifuged for 10 min under the conditions of 40 °C and 80 °C, and all the supernatant (about 6 mL) was collected for the next extraction study.

[0027] (2) Synthesis of magnetic deep eutectic solvent (MDES): Choline chloride (ChCl), phenol (Ph) and ferric chloride were mixed in a molar ratio of 1:2:1 and stirred at room temperature until a transparent liquid was formed, i.e., a magnetic deep eutectic solvent [ChCl / Ph2][FeCl4] was formed, with the structural formula shown below.

[0028]

[0029] (3) Magnetic deep eutectic solvent / salt aqueous two-phase system: Take an appropriate amount of MDES in a 5 mL centrifuge tube, then add 2.5 mL of the treated sample solution to the centrifuge tube and vortex for 20 seconds to form a homogenous solution. Next, add a certain amount of salt to the solution and vortex for 1 minute. After the salt dissolves, the system becomes turbid, and a magnetic deep eutectic solvent / salt aqueous two-phase system is formed.

[0030] (4) Extraction step: Use a super-strong magnet to attract the magnetic droplets around the bottom of the centrifuge tube, and then slowly move the super-strong magnet upward to achieve phase separation of the two-phase aqueous system. Finally, use a pipette to accurately measure the volumes of the upper and lower phases and use a high-performance liquid chromatograph to determine the concentration of sulfonamide antibiotics in the lower inorganic salt phase. The extraction rate (E) of sulfonamide antibiotics in the upper phase of the magnetic deep eutectic solvent two-phase aqueous system is calculated according to the following formula:

[0031]

[0032] Where C0 is the original concentration of sulfonamide antibiotics in the spiked milk sample, C saltis the sulfonamide antibiotic in the lower phase of the extraction system, V0 is the volume of the sample added to the system, V b is the volume of the lower phase of the aqueous two-phase system.

[0033] (5) Preparation of standard solution: Four standard substances, sulfadiazine (SDE), sulfamethoxazole (SMR), sulfamethoxazole (SMX), and sulfamethoxypyrimidine (SDD), were accurately prepared into a standard stock solution with a concentration of 500 μg / mL using methanol as solvent and stored in the dark at 4°C for future use.

[0034] Taking the separation and enrichment process of four antibiotics in spiked milk as an example, the specific optimization plan is as follows:

[0035] Selection of salt types: The extraction of sulfonamide antibiotics by a two-phase aqueous system of a magnetic low eutectic solvent consisting of five salts, Na2SO4, (NH4)2SO4, NH4Cl, K2HPO4 and Na2HPO4, was investigated. 0.4g [ChCl / Ph2][FeCl4] was placed in a 5mL centrifuge tube, and then 2.5mL of the treated sample solution was added to the centrifuge tube, and the solution was vortexed for 20s to form a uniform solution. Next, 1g of different types of inorganic salts were added to form five different extraction systems, and the system was vortexed for 1min. After the salts were dissolved, the system was separated by a super strong magnet and the extraction rate was determined. The experimental results are shown in the attached figure ( Figure 1 ), the results showed that the magnetic low eutectic solvent system formed with the participation of (NH4)2SO4 had the highest extraction rate, reaching 85.42-87.44%.

[0036] Selection of salt concentration: [ChCl / Ph2][FeCl4] / (NH4)2SO4 aqueous two-phase system was used to extract sulfonamide antibiotics from the pretreated samples. 0.4g [ChCl / Ph2][FeCl4] was placed in a 5mL centrifuge tube, and then 2.5mL of the treated sample solution was added to the centrifuge tube. The solution was vortexed for 20s to form a uniform solution. Next, (NH4)2SO4 was added to make the mass concentrations in the five systems 20%, 25%, 30%, 35%, and 40%, respectively. The solution was vortexed for 1min. After the salt was dissolved, the system was separated by a super strong magnet and the extraction rate was determined. The experimental results are shown in the attached figure ( Figure 2 ), the results showed that when the (NH4)2SO4 concentration in the system was 30%, the extraction rate of the four sulfonamide antibiotics was the highest, reaching 90.55-93.78%.

[0037] Determination of the concentration of magnetic deep eutectic solvent: Using the above steps, the mass concentration of (NH4)2SO4 in the system was fixed at 30%, and the effect of the mass concentration of [ChCl / Ph2][FeCl4] in the system (7.5%, 10%, 12.5%, 15%, 17.5%) on the extraction and enrichment of sulfonamide antibiotics was investigated. The experimental results are shown in the attached figure ( Figure 3 ), the results showed that the extraction rate of sulfonamide antibiotics reached its highest value at a [ChCl / Ph2][FeCl4] concentration of 10% and remained unchanged with increasing [ChCl / Ph2][FeCl4] concentration. However, as the concentration of [ChCl / Ph2][FeCl4] in the system increased, the volume of the upper phase increased, and the enrichment factor of the target extract gradually decreased. Therefore, a 10% [ChCl / Ph2][FeCl4] / 30% (NH4)2SO4 aqueous two-phase system was selected for further research, and the extraction rates of the four sulfonamide antibiotics ranged from 90.55% to 93.78%.

[0038] System pH selection: Using the above system, the effect of system pH (4-6) on the extraction and enrichment of four sulfonamide antibiotics was investigated. The experimental results are shown in the attached figure ( Figure 4 ), the results showed that when the pH of the system was 4.5, the extraction rate of the four sulfonamide antibiotics was the highest, reaching 94.45-96.88%.

[0039] Optimal process conditions: When pH is 4.5, the extraction rate of four sulfonamide antibiotics in the 10% [ChCl / Ph2][FeCl4] / 30% (NH4)2SO4 aqueous two-phase system reaches 94.45-96.88%.

[0040] Under optimal operating conditions, the four sulfonamide antibiotics exhibited good linear relationships between peak area and concentration in the range of 0.5-60 mg / mL. The limits of detection for the four sulfonamide antibiotics were between 0.030-0.590 mg / mL, and recoveries ranged from 94.45% to 96.88%. This method is highly sensitive, selective, rapid, simple, and economical, and can be applied to the determination of sulfonamide antibiotics in water samples.

[0041] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for enriching and separating sulfonamide antibiotics in drinking water or milk, characterized in that: The following steps are involved: (1) Sample preparation: Weigh the sample, add trichloroacetic acid solution, vortex and sonicate, centrifuge, and collect the supernatant; (2) Synthesis of magnetic deep eutectic solvent: Choline chloride, phenol and ferric chloride are stirred at room temperature until a homogeneous transparent liquid is formed, i.e., a magnetic deep eutectic solvent [ChCl / Ph2][FeCl4] is formed; (3) Magnetic deep eutectic solvent / salt two-phase aqueous system: the magnetic deep eutectic solvent is added to the supernatant, vortexed and then salt is added, wherein the salt is Na2SO4, (NH4)2SO4, NH4Cl, K2HPO4 or Na2HPO4, and vortexed to form a magnetic deep eutectic solvent / salt two-phase aqueous system; (4) Extraction: Place a magnet at the bottom of the solution to attract the magnetic droplets, move the magnet upward to separate the two-phase aqueous system, accurately measure the volumes of the upper and lower phases, and use high performance liquid chromatography to determine the concentration of sulfonamide antibiotics in the lower inorganic salt phase.

2. The method for enriching and separating sulfonamide antibiotics in drinking water or milk according to claim 1, characterized in that: In step (1), 2 mL of 10% trichloroacetic acid solution was added to 8 mL of sample.

3. The method for enriching and separating sulfonamide antibiotics in drinking water or milk according to claim 1, characterized in that: In step (2), the molar ratio of choline chloride, phenol and ferric chloride is 1:2:

1.

4. The method for enriching and separating sulfonamide antibiotics in drinking water or milk according to claim 1, characterized in that: The mass concentration of the magnetic deep eutectic solvent in step (3) is 7.5-17.5%.

5. The method for enriching and separating sulfonamide antibiotics in drinking water or milk according to claim 4, characterized in that: The mass concentration of the magnetic deep eutectic solvent is 10%.

6. The method for enriching and separating sulfonamide antibiotics in drinking water or milk according to claim 1, characterized in that: In step (3), the mass concentration of salt in the system is 20%-40%.

7. The method for enriching and separating sulfonamide antibiotics in drinking water or milk according to claim 6, characterized in that: The mass concentration of salt in the system is 30%.

8. The method for enriching and separating sulfonamide antibiotics in drinking water or milk according to claim 1, characterized in that: The pH value of the magnetic deep eutectic solvent / salt aqueous two-phase system is 4-6.

9. The method for enriching and separating sulfonamide antibiotics in drinking water or milk according to claim 8, characterized in that: The pH value of the magnetic deep eutectic solvent / salt aqueous two-phase system is 4.5.

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