Method for determining antibiotics in environmental soil

Abstract

The invention discloses a method for determining antibiotics in environmental soil, which comprises the following steps: S1, collecting fresh soil, crushing, sieving with a 0.1 mm-0. 2mm sieve, weighing 3 g-5 g of sample, and placing in a centrifuge tube; s2, adding 50-80 mL of a methanol-acetic acid-aqueous solution into the centrifuge tube, oscillating for 3-5 min, then carrying out normal-temperature ultrasonic treatment for 25-30 min, carrying out high-speed centrifugation for 10-15 min at a low temperature of 4-6 DEG C, taking a supernatant, repeatedly extracting residues twice, merging the obtained extract and the supernatant, and concentrating to 10-15 mL to obtain a concentrated solution A; s3, adding 20-25 mL of dichloromethane into the concentrated solution, repeating the extraction operation in the S2 to obtain a concentrated solution B, and adding ultrapure water into the concentrated solution B to dilute the concentrated solution B to 200 mL; and S4, adding 1-1.5 g of disodium ethylene diamine tetraacetate into the diluent obtained in S3, adjusting the pH value to 3.5, enabling the diluent to pass through a small solid-phase extraction column, eluting the small solid-phase extraction column with methanol, blow-drying under nitrogen flow, fixing the volume, passing through a filter membrane, and transferring into a sample bottle for UPLC-MS / MS determination. According to the method, the antibiotic extraction rate and the content determination accuracy are improved.

Description

Technical Field

[0001] The invention relates to the technical field of antibiotic detection, and in particular to a method for determining antibiotics in environmental soil. Background Art

[0002] The sources of antibiotics in environmental soil mainly include medical wastewater, livestock and poultry excrement, agricultural fertilization and pharmaceutical industry emissions, etc. Antibiotics from these sources enter the soil and water bodies through surface runoff, seepage and atmospheric deposition, forming a complex pollution pattern.

[0003] Antibiotic residues in soil may have a negative impact on soil microorganisms and destroy the balance of the soil ecosystem. Timely and accurate detection of antibiotic content in soil helps protect the ecological environment. Antibiotics may enter the human body through the food chain, posing a potential threat to human health. Therefore, detecting the content of antibiotics in soil is of great significance for protecting human health.

[0004] Since there are many types of antibiotics and their concentrations in the environment are usually low, highly sensitive detection technologies are needed to monitor and evaluate antibiotic contamination in soil-water systems. Currently, commonly used detection methods include high performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), gas chromatography-mass spectrometry (GC-MS), biosensor methods, etc. These technologies can detect antibiotic residues as low as nanogram levels, providing a powerful tool for environmental regulation. When determining antibiotics, the main problem of existing technologies is the low extraction rate of antibiotics, which has a certain impact on the determination results of antibiotic content in the soil environment. Therefore, it is necessary to improve the design of related technologies. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a method for determining antibiotics in environmental soil.

[0006] The above technical purpose of the present invention is achieved through the following technical scheme: A method for determining antibiotics in environmental soil, comprising the following steps:

[0007] S1. Collect fresh soil, crush it through a 0.1mm-0.2mm sieve, weigh 3g-5g of sample, and place it in a centrifuge tube;

[0008] S2. Add 50mL-80mL of methanol-acetic acid-water solution into a centrifuge tube, shake for 3min-5min, then ultrasonicate at room temperature for 25min-30min, centrifuge at high speed for 10min-15min at a low temperature of 4℃-6℃, take the supernatant, extract the residue repeatedly twice, combine the obtained extract with the supernatant, and then concentrate on a rotary concentrator to 10mL-15mL to obtain a concentrated solution A;

[0009] S3, add 20mL-25mL of dichloromethane to the concentrated solution, repeat the relevant extraction operations in S2 to obtain concentrated solution B, and add ultrapure water to the concentrated solution B to dilute it to 200mL;

[0010] S4. Add 1g-1.5g of disodium ethylenediaminetetraacetate to the diluted solution obtained in S3, adjust the pH to 3.5, pass the diluted solution through a solid phase extraction column, then elute the solid phase extraction column with methanol, blow dry under a weak inert gas flow, make up to volume, filter the membrane, and transfer to a sample bottle for UPLC-MS / MS determination of antibiotic content in soil.

[0011] Furthermore, in step S2, the speed of high-speed centrifugation is 4500 r / min-5000 r / min.

[0012] Furthermore, in step S4, the solid phase extraction column is: 5 mL of methanol, 5 mL of 5% pH 3.5 disodium ethylenediaminetetraacetic acid aqueous solution.

[0013] Further, in step S4, the chromatographic conditions are: symmetry C 18 Chromatographic column, column temperature 35℃-40℃, flow rate 0.3mL / min-0.5mL / min, mobile phase: A is ultrapure water with 0.2% formic acid, B is 0.2% acetonitrile aqueous solution; gradient program: 0min-5min, 5%-20%; 5min-10min, 20%-30%; 10min-15min, 30%-45%; 15min-20min, 45%-60%.

[0014] Furthermore, in step S4, the mass spectrometry conditions are: electrospray ion source, positive ion detection, capillary voltage of 3 kV, cone voltage of 5 V, lens voltage of 0.5 V, desolvation gas flow rate of 600 L / h, cone gas flow rate of 50 L / h, nitrogen source temperature of 100°C, and desolvation gas temperature of 350°C.

[0015] Furthermore, in step S4, the inert gas flow is nitrogen.

[0016] Furthermore, in step S2, the oscillation method is vortex oscillation.

[0017] Furthermore, in step S4, a formic acid aqueous solution with a volume fraction of 0.3% is used to fix the volume.

[0018] In summary, the present invention has the following beneficial effects:

[0019] 1. In this application, different chromatographic columns have different retention times for the same sample. Among antibiotics, since the tertiary amine and carboxyl functional groups of quinolone compounds can dissociate in water, the residual silanol groups and metal ions in the fillers of ordinary chromatographic columns can strongly adsorb quinolone compounds through hydrogen bonding and ion exchange, resulting in tailing of chromatographic peaks, unstable retention time and decreased separation. 18 The chromatographic column can achieve good separation of tetracycline compounds, quinolone compounds, and sulfadimethoxine antibiotics in the gradient elution procedure, and has good reproducibility, which is beneficial to improve the determination accuracy of the content of such antibiotics in the soil environment;

[0020] 2. In this application, quinolone compounds are acid-base amphoteric compounds, and their polar structure determines that they are soluble in dilute acid aqueous solutions and glacial acetic acid of organic solvents. Using methanol-acetic acid-water solution as the extraction solution, quinolone compounds can achieve better results. Adding disodium ethylenediaminetetraacetic acid buffer to the diluent significantly improves the extraction effect. Methanol is conducive to extracting other forms of antibiotics in the soil environment, and the extraction effect is relatively good. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present application will be described clearly and completely below; obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments, and all other embodiments obtained by ordinary technicians in this field based on the embodiments in the present application without making creative work are within the scope of protection of the present application.

[0022] Example 1

[0023] The present application discloses a method for determining antibiotics in environmental soil, comprising the following steps:

[0024] S1. Collect fresh soil, crush it through a 0.1 mm sieve, weigh 3 g of sample, and place it in a centrifuge tube;

[0025] S2. Add 50 mL of methanol-acetic acid-water solution into a centrifuge tube, vortex and shake for 3 minutes, then perform ultrasonic treatment at room temperature for 25 minutes, centrifuge at 4500 r / min for 10 minutes at a low temperature of 4°C, take the supernatant, repeat the extraction of the residue twice, combine the obtained extract with the supernatant, and then place it on a rotary concentrator to concentrate to 10 mL to obtain a concentrated solution A;

[0026] S3, add 20 mL of dichloromethane to the concentrated solution, repeat the relevant extraction operations in S2 to obtain concentrated solution B, and add ultrapure water to the concentrated solution B to dilute it to 200 mL;

[0027] S4, add 1 g of disodium ethylenediaminetetraacetate to the dilution obtained in S3, adjust the pH to 3.5, pass the dilution through a solid phase extraction column, then elute the solid phase extraction column with methanol (5 mL methanol, 5 mL 5% pH 3.5 disodium ethylenediaminetetraacetate aqueous solution), blow dry under a weak nitrogen flow, make up to volume with a 0.3% volume fraction of formic acid aqueous solution, filter the membrane, transfer to a sample bottle, and use it for UPLC-MS / MS to determine the content of antibiotics in soil;

[0028] Among them, the chromatographic conditions are: symmetry C 18 Chromatographic column, column temperature 35°C, flow rate 0.3 mL / min, mobile phase: A is ultrapure water with 0.2% formic acid, B is 0.2% acetonitrile aqueous solution; gradient program: 0 min, 5%; 5 min, 20%; 10 min, 30%; 15 min, 45%;

[0029] The mass spectrometry conditions were as follows: electrospray ion source, positive ion detection, capillary voltage of 3 kV, cone voltage of 5 V, lens voltage of 0.5 V, desolvation gas flow rate of 600 L / h, cone gas flow rate of 50 L / h, nitrogen source temperature of 100 °C, and desolvation gas temperature of 350 °C.

[0030] Example 2

[0031] The present application discloses a method for determining antibiotics in environmental soil, comprising the following steps:

[0032] S1. Collect fresh soil, crush it through a 0.2 mm sieve, weigh 5 g of sample, and place it in a centrifuge tube;

[0033] S2. Add 80 mL of methanol-acetic acid-water solution into a centrifuge tube, vortex and shake for 5 minutes, then perform ultrasonic treatment at room temperature for 30 minutes, centrifuge at 5000 r / min for 15 minutes at a low temperature of 6°C, take the supernatant, extract the residue repeatedly twice, combine the obtained extract with the supernatant, and then concentrate to 15 mL on a rotary concentrator to obtain a concentrated solution A;

[0034] S3, add 25 mL of dichloromethane to the concentrated solution, repeat the relevant extraction operations in S2 to obtain concentrated solution B, and add ultrapure water to the concentrated solution B to dilute it to 200 mL;

[0035] S4, add 1.5g of disodium ethylenediaminetetraacetate to the dilution obtained in S3, adjust the pH to 3.5, pass the dilution through a solid phase extraction column, then elute the solid phase extraction column with methanol (5mL methanol, 5mL 5% pH3.5 disodium ethylenediaminetetraacetate aqueous solution), blow dry under a weak nitrogen flow, make up to volume with a 0.3% volume fraction of formic acid aqueous solution, filter the membrane, transfer to a sample bottle, and use it for UPLC-MS / MS to determine the content of antibiotics in soil;

[0036] Among them, the chromatographic conditions are: symmetry C 18 Chromatographic column, column temperature 40°C, flow rate 0.5 mL / min, mobile phase: A is ultrapure water with 0.2% formic acid, B is 0.2% acetonitrile aqueous solution; gradient program: 5 min, 20%; 10 min, 30%; 15 min, 45%; 20 min, 60%;

[0037] The mass spectrometry conditions were as follows: electrospray ion source, positive ion detection, capillary voltage of 3 kV, cone voltage of 5 V, lens voltage of 0.5 V, desolvation gas flow rate of 600 L / h, cone gas flow rate of 50 L / h, nitrogen source temperature of 100 °C, and desolvation gas temperature of 350 °C.

[0038] Example 3

[0039] The present application discloses a method for determining antibiotics in environmental soil, comprising the following steps:

[0040] S1. Collect fresh soil, crush it through a 0.15 mm sieve, weigh 4 g of sample, and place it in a centrifuge tube;

[0041] S2. Add 60 mL of methanol-acetic acid-water solution into a centrifuge tube, vortex and shake for 4 minutes, then perform ultrasonic treatment at room temperature for 30 minutes, centrifuge at 4800 r / min for 15 minutes at a low temperature of 5°C, take the supernatant, repeat the extraction of the residue twice, combine the obtained extract with the supernatant, and then place it on a rotary concentrator to concentrate to 15 mL to obtain a concentrated solution A;

[0042] S3, add 20 mL of dichloromethane to the concentrated solution, repeat the relevant extraction operations in S2 to obtain concentrated solution B, and add ultrapure water to the concentrated solution B to dilute it to 200 mL;

[0043] S4, add 1g-1.5g of disodium ethylenediaminetetraacetate to the dilution obtained in S3, adjust the pH to 3.5, pass the dilution through a solid phase extraction column, then elute the solid phase extraction column with methanol (5mL methanol, 5mL 5% pH3.5 disodium ethylenediaminetetraacetate aqueous solution), blow dry under a weak nitrogen flow, make up to volume with a 0.3% volume fraction of formic acid aqueous solution, filter the membrane, transfer to a sample bottle, and use it for UPLC-MS / MS to determine the content of antibiotics in soil;

[0044] Among them, the chromatographic conditions are: symmetry C 18 Chromatographic column, column temperature 40°C, flow rate 0.5 mL / min, mobile phase: A is ultrapure water with 0.2% formic acid, B is 0.2% acetonitrile aqueous solution; gradient program: 5 min, 10%; 10 min, 25%; 15 min, 45%; 20 min, 50%;

[0045] The mass spectrometry conditions were as follows: electrospray ion source, positive ion detection, capillary voltage of 3 kV, cone voltage of 5 V, lens voltage of 0.5 V, desolvation gas flow rate of 600 L / h, cone gas flow rate of 50 L / h, nitrogen source temperature of 100 °C, and desolvation gas temperature of 350 °C.

[0046] The recovery rate test was conducted on the method for determining antibiotics in environmental soil of the above Examples 1-3 and the method for determining antibiotics in environmental soil commonly used in the prior art. The results are shown in the following table:

[0047]

[0048]

[0049] In summary, the method for determining antibiotics in the environment provided by the present application can improve the extraction efficiency of multiple antibiotics and improve the accuracy of the determination results compared with the commonly used determination methods in the prior art.

[0050] Quinolone compounds are acid-base compounds. Their polar structure determines that they are soluble in dilute acid aqueous solutions and glacial acetic acid in organic solvents. Using methanol-acetic acid-water solution as the extraction solution, quinolone compounds can achieve better results. Adding disodium ethylenediaminetetraacetic acid buffer to the diluent significantly improves the extraction effect. Methanol is conducive to extracting other forms of antibiotics in the soil environment, and the extraction effect is relatively good.

[0051] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A method for determining antibiotics in environmental soil, characterized in that: The steps include: S1. Collect fresh soil, crush it through a 0.1mm-0.2mm sieve, weigh 3g-5g of sample, and place it in a centrifuge tube; S2. Add 50mL-80mL of methanol-acetic acid-water solution into a centrifuge tube, shake for 3min-5min, then ultrasonicate at room temperature for 25min-30min, centrifuge at high speed for 10min-15min at a low temperature of 4℃-6℃, take the supernatant, extract the residue repeatedly twice, combine the obtained extract with the supernatant, and then concentrate on a rotary concentrator to 10mL-15mL to obtain a concentrated solution A; S3, add 20mL-25mL of dichloromethane to the concentrated solution, repeat the relevant extraction operations in S2 to obtain concentrated solution B, and add ultrapure water to the concentrated solution B to dilute it to 200mL; S4. Add 1g-1.5g of disodium ethylenediaminetetraacetate to the diluted solution obtained in S3, adjust the pH to 3.5, pass the diluted solution through a solid phase extraction column, then elute the solid phase extraction column with methanol, blow dry under a weak inert gas flow, make up to volume, filter the membrane, and transfer to a sample bottle for UPLC-MS / MS determination of antibiotic content in soil.

2. The method for determining antibiotics in environmental soil according to claim 1, characterized in that: In step S2, the speed of high-speed centrifugation is 4500r / min-5000r / min.

3. The method for determining antibiotics in environmental soil according to claim 1, characterized in that: In step S4, the solid phase extraction column is: 5 mL of methanol, 5 mL of 5% pH 3.5 disodium ethylenediaminetetraacetic acid aqueous solution.

4. The method for determining antibiotics in environmental soil according to claim 3, characterized in that: In step S4, the chromatographic conditions are: symmetry C 18 Chromatographic column, column temperature 35℃-40℃, flow rate 0.3mL / min-0.5mL / min, mobile phase: A is ultrapure water with 0.2% formic acid, B is 0.2% acetonitrile aqueous solution; gradient program: 0min-5min, 5%-20%; 5min-10min, 20%-30%; 10min-15min, 30%-45%; 15min-20min, 45%-60%.

5. The method for determining antibiotics in environmental soil according to claim 4, characterized in that: In step S4, the mass spectrometry conditions are: electrospray ion source, positive ion detection, capillary voltage of 3 kV, cone voltage of 5 V, lens voltage of 0.5 V, desolvation gas flow rate of 600 L / h, cone gas flow rate of 50 L / h, nitrogen source temperature of 100°C, and desolvation gas temperature of 350°C.

6. The method for determining antibiotics in environmental soil according to claim 5, characterized in that: In step S4, the inert gas flow is nitrogen.

7. The method for determining antibiotics in environmental soil according to claim 5, characterized in that: In step S2, the oscillation method is vortex oscillation.

8. The method for determining antibiotics in environmental soil according to claim 5, characterized in that: In step S4, a formic acid aqueous solution with a volume fraction of 0.3% is used to fix the volume.

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