A method for determining the bioavailability of antibiotics in soil using continuous solvent extraction-solid phase extraction-liquid chromatography-tandem mass spectrometry.

Abstract

This invention discloses a method for detecting the bioavailability of antibiotics in soil using continuous solvent extraction-solid phase extraction-liquid chromatography-tandem mass spectrometry. The method first uses continuous solvent extraction to extract the bioactive and extractable antibiotic components from the soil. Then, a solid phase extraction column is used for sample purification. Finally, multiple reaction monitoring (MRM) mode of liquid chromatography-tandem mass spectrometry is used to determine the concentrations of various antibiotics, including chlortetracycline, doxycycline, oxytetracycline, tetracycline, sulfadiazine, sulfadiazine, sulfamethoxazole, ciprofloxacin, enrofloxacin, norfloxacin, ofloxacin, azithromycin, erythromycin, roxithromycin, tylosin, trimethoprim, and lincomycin. This invention combines continuous solvent extraction with solid phase extraction, coupled with liquid chromatography-tandem mass spectrometry detection, to achieve accurate evaluation of trace antibiotic bioavailability under complex matrix conditions.

Description

Technical Field

[0001] This invention relates to the field of soil antibiotic detection technology, and in particular to a method for detecting the bioavailability of antibiotics in soil using a continuous solvent extraction-solid phase extraction-liquid chromatography-tandem mass spectrometry method. Background Technology

[0002] The widespread use and abuse of antibiotics cause serious environmental pollution problems. Antibiotics released from polluted sites such as livestock farms and pharmaceutical factories eventually enter the soil environment through various transmission routes, becoming a significant reservoir of antibiotics. Residual antibiotics in the environment lead to increased antibiotic resistance and the spread of antibiotic-resistant bacteria. The problem of bacterial resistance has become an imminent public crisis and a major environmental and health challenge that urgently needs to be addressed, seriously threatening human health. Therefore, antibiotic content detection is crucial for the effective management of antibiotics.

[0003] Recent studies have shown that using total antibiotic concentration to assess the effects of pollutants can overestimate the degree of environmental pollution, while their bioavailability, which can be directly absorbed and utilized by plants and animals, can provide more accurate scientific data for environmental and toxicity assessments. Currently, research on the bioavailability of organic pollutants is limited to non-polar organic targets, with a serious lack of discussion on polar organic pollutants such as antibiotics. The characterization methods for the bioavailability of antibiotics in environmental media urgently need further research; therefore, developing efficient and accurate characterization methods for the bioavailability of antibiotics in environmental media is particularly important.

[0004] The bioavailability of antibiotics in the environment depends on the environmental medium and interfacial behavior. Different soil environments and contact with antibiotics significantly affect the distribution between their bioavailable and extractable components. For example, tetracycline antibiotics are easily adsorbed by clay, macrolide antibiotics are easily adsorbed on sandy loam and clay soils, while sulfonamides are highly mobile and not easily adsorbed by soil. Antibiotics undergo physical, chemical, and biological processes such as adsorption / desorption, hydrolysis / degradation, and transmembrane / transport at the interfaces of particulate matter, colloids, and microorganisms, leading to changes in their concentration and bioavailability. Therefore, traditional analytical systems that use total antibiotic concentration to measure antibiotic dosage can lead to biases of tens or even hundreds of times, thus greatly underestimating the bioavailable utilization capacity of antibiotics in the environment. Therefore, it is urgent to quantify the bioavailability of antibiotics in soil to further understand the potential environmental and health risks posed by soil antibiotics.

[0005] Due to the complexity of soil matrix and the large interference in detection, it is urgent to develop an efficient, broad-spectrum, highly sensitive, and low-detection-limit analytical method for antibiotics in soil in order to quickly and accurately quantify the bioactive and extractable components of antibiotics in soil, and to provide a theoretical basis for assessing their health risks. Summary of the Invention

[0006] The purpose of this invention is to address the problems of low sensitivity, limited accuracy, and significant matrix interference in existing techniques for quantifying the bioavailability of antibiotics in soil. This invention proposes a continuous solvent extraction-solid phase extraction-liquid chromatography-tandem mass spectrometry method for detecting the bioavailability of antibiotics in soil. This invention has two core advantages: high sensitivity and low detection limit. It also features small sample volume required for pretreatment and convenient operation, enabling the analysis and detection of trace bioactive components and extractable components of antibiotics under complex matrix conditions.

[0007] The objective of this invention is achieved through the following technical solution: a method for detecting the bioavailability of antibiotics in soil using continuous solvent extraction-solid phase extraction-liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0008] (1) After collecting soil samples, place them in a freezer at -20℃ for storage;

[0009] (2) The soil sample to be tested was obtained by air drying the soil sample in the laboratory by natural ventilation, and then it was thoroughly ground with an agate mortar and filtered through a 200-mesh sieve.

[0010] (3) Weigh 1.9-2.1 g of the filtered soil sample obtained in step (2) and place it in a centrifuge tube. Add 3 mL of 0.01 M calcium chloride extraction solution and extract on a mixer for 4 hours. Then centrifuge at 3000×g for 5 minutes. Transfer the supernatant and add 3 mL of 0.01 M calcium chloride extraction solution to continue extraction for 4 hours. Centrifuge at 3000×g for 5 minutes again. Repeat once more and combine the extracts to obtain the effective components of the antibiotic.

[0011] (4) Add 5 mL of mixed extract to the remaining soil sample in step (3) and sonicate for 15 minutes. Then centrifuge at 8000×g for 5 minutes, remove the supernatant and add fresh mixed extract. Perform two more sonic extraction cycles. Combine the extracts to obtain the antibiotic extractable component. Dilute with ultrapure water to ensure that the organic solvent content in the solution is less than or equal to 5%.

[0012] (5) The HLB column required for solid-phase extraction was activated by 6 mL of methanol and 12 mL of ultrapure water respectively. The bioactive components of antibiotics were directly enriched by passing through the HLB column, while the extractable components of antibiotics were enriched by passing through the HLB column at a flow rate of 3 mL / min. Then, 10 mL of ultrapure water was used for washing, and the HLB column was dried under vacuum until the bottom turned white.

[0013] (6) The target substance retained on the HLB column was eluted with 12 mL of methanol containing 0.1% formic acid under standard atmospheric pressure to obtain the sample of the target substance to be tested.

[0014] (7) The sample of the target substance obtained in step (6) is concentrated to 0.05 mL by vacuum centrifugation, and then 1 mL of acetonitrile and water are added. The mixture is vortexed to obtain a mixed solution containing the target substance with acetonitrile / water as the solvent system, which is used for liquid chromatography-tandem mass spectrometry detection; wherein the volume ratio of acetonitrile / water is 90 / 10.

[0015] (8) Using an aqueous solution containing 0.1% formic acid and pure acetonitrile as the mobile phase for liquid chromatography-tandem mass spectrometry, gradient elution was performed for 16 minutes. The concentrations of various antibiotics were separated and quantified by multiple reaction monitoring to determine the concentrations of bioactive and extractable components of chlortetracycline, doxycycline, oxytetracycline, tetracycline, sulfadiazine, sulfadiazine, sulfamethoxazole, ciprofloxacin, enrofloxacin, norfloxacin, ofloxacin, azithromycin, erythromycin, roxithromycin, tylosin, trimethoprim, and lincomycin in the soil.

[0016] Further, in step (4), the mixed extract is formed by mixing acetonitrile-ethylenediaminetetraacetic acid-citric acid phosphate buffer and Mg(NO3)2-NH3·H2O in a volume ratio of 3:1.

[0017] Furthermore, in step (5), the HLB column has a specification of 6cc and 200mg.

[0018] Furthermore, in step (7), the working temperature of the vacuum centrifugal concentrator is 40°C and the rotation speed is 2000 r / min.

[0019] Further, in step (8), the mass ion source temperature of the liquid chromatography-tandem mass spectrometry is 650℃; the chromatographic column of the liquid chromatography-tandem mass spectrometry is an ACQUITY UPLC HSS T3 column, which has a column length of 100mm, an inner diameter of 2.1mm, and a packing particle size of 1.8μm.

[0020] Furthermore, in step (8), the specific conditions for gradient elution are as follows:

[0021] Within 0-12 minutes, the volume ratio of formic acid aqueous solution to pure acetonitrile solution decreased from 95:5 to 20:80;

[0022] Within 12-13 minutes, the volume ratio of formic acid aqueous solution to pure acetonitrile solution decreased from 20:80 to 5:95;

[0023] Within 13-14 minutes, the volume ratio of formic acid aqueous solution to pure acetonitrile solution decreased from 5:95 to 95:5;

[0024] Within 14-16 minutes, the volume ratio of formic acid aqueous solution to pure acetonitrile solution was 95:5.

[0025] Furthermore, in step (8), the specific conditions of the multiple reaction monitoring method are shown in the table below:

[0026]

[0027]

[0028] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention screens out extracts suitable for extracting both the bioactive components and extractable components of antibiotics, optimizes pretreatment parameters, enriches and concentrates the antibiotic extract using solid-phase extraction and vacuum centrifugation, improves the material recovery rate, reduces matrix interference, and accurately identifies antibiotic components in soil using multiple reaction monitoring (MRM) methods; this invention enables the extraction of bioactive components of antibiotics under complex matrix conditions using 0.01 mol / L calcium chloride and a mixed extract of acetonitrile-ethylenediaminetetraacetic acid-citric acid phosphate buffer-Mg(NO3)2-NH3·H2O. This invention extracts the extractable components of antibiotics and enriches them through solid-phase extraction. The use of methanol eluent containing formic acid improves elution efficiency, and the antibiotics are concentrated using a vacuum centrifuge, thereby increasing sample recovery and reducing detection limits and matrix interference. This invention constructs a quantitative antibiotic MRM method using characteristic fragment ions, establishing a method for the simultaneous detection of trace antibiotic bioactive components and extractable components in soil, enabling the detection of antibiotics down to the ng / kg level in soil. This invention has advantages such as small soil sample volume requirement, low detection limit, high sensitivity, broad spectrum, and short detection time, making it suitable for the detection of trace antibiotics under complex matrix conditions. Attached Figure Description

[0029] Figure 1 This is a flowchart of the method for detecting the bioavailability of antibiotics in soil using continuous solvent extraction-solid phase extraction-liquid chromatography-tandem mass spectrometry according to the present invention.

[0030] Figure 2 The effect of different volumes of calcium chloride extract on the recovery rates of 18 antibiotics is shown in the figure.

[0031] Figure 3 The effect of different extraction times of calcium chloride extract on the relative signal intensity of 18 antibiotics is shown in the figure.

[0032] Figure 4 The effect of different extraction cycles of calcium chloride extract on the recovery rate of 18 antibiotics is shown in the figure.

[0033] Figure 5 The effect of different types of organic solvents in the mixed extract on the recovery rate of 18 antibiotics is shown in the figure.

[0034] Figure 6 The effect of different pH values ​​in the mixed extract on the recovery rate of 18 antibiotics is shown in the figure.

[0035] Figure 7 The effect of different ratios of organic solvent and buffer in the mixed extract on the recovery rate of 18 antibiotics is shown in the figure.

[0036] Figure 8 Figure 1 shows the effect of different volumes of mixed extract on the recovery rates of 18 antibiotics.

[0037] Figure 9 The graph shows the effect of different volumes of elution solution on the recovery rates of 18 antibiotics in solid-phase extraction.

[0038] Figure 10 The graph shows the effect of different temperatures on the recovery rates of 18 antibiotics during vacuum centrifugation concentration. Detailed Implementation

[0039] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application. Rather, they are merely examples of apparatuses and methods consistent with some aspects of this application as detailed in the appended claims. It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit this application.

[0040] The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The singular forms “a,” “the,” and “the” used in this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0041] It should be understood that although the terms first, second, third, etc., may be used in this application to describe various information, such information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another. For example, without departing from the scope of this application, first information may also be referred to as second information, and similarly, second information may also be referred to as first information. Depending on the context, the word "if" as used herein may be interpreted as "when," "in response to determination," or "includes." Moreover, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process or method that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process or method. Without further limitations, an element defined by the phrase "comprising a..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0042] The present invention will now be described in detail with reference to the accompanying drawings. Unless otherwise specified, the features of the following embodiments and implementations can be combined with each other.

[0043] See Figure 1 The method for detecting the bioavailability of antibiotics in soil using continuous solvent extraction-solid phase extraction-liquid chromatography-tandem mass spectrometry of the present invention specifically includes the following steps:

[0044] (1) After collecting soil samples, they were frozen at -20°C for subsequent extraction of soil antibiotics.

[0045] (2) The soil sample to be tested was obtained by air-drying the soil sample in the laboratory by natural ventilation, and then it was thoroughly ground with an agate mortar and filtered through a 200-mesh sieve.

[0046] (3) Weigh 1.9-2.1 g of the filtered soil sample obtained in step (2) and place it in a centrifuge tube. Add 3 mL of 0.01 M calcium chloride extraction solution and extract on a mixer for 4 hours. Then centrifuge at 3000×g for 5 minutes, transfer the supernatant and add 3 mL of 0.01 M calcium chloride extraction solution to continue extraction for 4 hours. Centrifuge at 3000×g for 5 minutes again and repeat once more. The combined extracts are the active components of the antibiotic.

[0047] It should be noted that 0.01M calcium chloride was used to extract the bioactive components of soil antibiotics.

[0048] (4) Add 5 mL of mixed extract to the remaining soil sample from step (3) and sonicate for 15 minutes. Then centrifuge at 8000×g for 5 minutes, remove the supernatant and add fresh mixed extract. Perform two more sonic extraction cycles. Combine the extracts to obtain the antibiotic extractable component. Dilute with ultrapure water to ensure that the organic solvent content in the solution is less than or equal to 5%.

[0049] Furthermore, the mixed extract is formed by mixing acetonitrile-ethylenediaminetetraacetic acid-citric acid phosphate buffer and Mg(NO3)2-NH3·H2O at a volume ratio of 3:1. The mixed extract is used to extract extractable components of soil antibiotics.

[0050] In summary, the method of the present invention extracts both bioactive and extractable components of soil antibiotics, and subsequent steps simultaneously analyze both bioactive and extractable components.

[0051] (5) The HLB column required for solid-phase extraction was activated by 6 mL of methanol and 12 mL of ultrapure water respectively. The bioactive components of the antibiotic were directly enriched by passing through the HLB column, while the extractable components of the antibiotic were enriched by passing through the HLB column at a flow rate of 3 mL / min. Then, 10 mL of ultrapure water was used for washing, and the HLB column was dried under vacuum until the bottom turned white.

[0052] Furthermore, the HLB column specifications are 6cc, 200mg.

[0053] (6) The target substance retained on the HLB column was eluted with 12 mL of methanol containing 0.1% formic acid under standard atmospheric pressure to obtain the test sample of the target substance.

[0054] Furthermore, the standard atmospheric pressure is 101.325 kPa.

[0055] (7) The sample of the target substance obtained in step (6) is concentrated to 0.05 mL by vacuum centrifugation, and then 1 mL of acetonitrile and water are added. The mixture is vortexed to obtain a mixed solution containing the target substance with acetonitrile / water as the solvent system, which is used for liquid chromatography-tandem mass spectrometry detection; wherein the volume ratio of acetonitrile / water is 90 / 10.

[0056] Furthermore, the vacuum centrifugal concentrator operates at a temperature of 40℃ and a rotation speed of 2000 r / min.

[0057] (8) Using an aqueous solution containing 0.1% formic acid and pure acetonitrile as the mobile phase for liquid chromatography-tandem mass spectrometry, gradient elution was performed for 16 minutes. The concentrations of various antibiotics were separated and quantified by multiple reaction monitoring to determine the concentrations of bioactive and extractable components of chlortetracycline, doxycycline, oxytetracycline, tetracycline, sulfadiazine, sulfadiazine, sulfamethoxazole, ciprofloxacin, enrofloxacin, norfloxacin, ofloxacin, azithromycin, erythromycin, roxithromycin, tylosin, trimethoprim, and lincomycin in the soil.

[0058] Furthermore, the mass ion source temperature for liquid chromatography-tandem mass spectrometry is 650℃; the chromatographic column for liquid chromatography-tandem mass spectrometry is an ACQUITY UPLC HSS T3 column, which has a column length of 100 mm, an inner diameter of 2.1 mm, and a packing particle size of 1.8 μm.

[0059] Furthermore, the specific conditions for gradient elution are as follows:

[0060] Within 0-12 minutes, the volume ratio of formic acid aqueous solution to pure acetonitrile solution decreased from 95:5 to 20:80;

[0061] Within 12-13 minutes, the volume ratio of formic acid aqueous solution to pure acetonitrile solution decreased from 20:80 to 5:95;

[0062] Within 13-14 minutes, the volume ratio of formic acid aqueous solution to pure acetonitrile solution decreased from 5:95 to 95:5;

[0063] Within 14-16 minutes, the volume ratio of formic acid aqueous solution to pure acetonitrile solution was 95:5.

[0064] Furthermore, the specific conditions for the multiple reaction monitoring method are shown in Table 1:

[0065] Table 1: Setup conditions for multiple reaction monitoring methods

[0066]

[0067]

[0068] The following describes in detail, based on embodiments, the method for simultaneous detection of bioactive and extractable antibiotic components in soil using continuous solvent extraction-solid phase extraction-liquid chromatography-tandem mass spectrometry, which will make the objectives and effects of the present invention more apparent.

[0069] Example 1

[0070] This embodiment investigated the effect of different volumes of 0.01M calcium chloride extract on the recovery rates of 18 antibiotics, specifically including the following steps:

[0071] (1) Prepare simulated soil samples containing the same concentration of 18 antibiotics to obtain the test samples.

[0072] (2) Add 3, 6 and 9 mL of 0.01M calcium chloride extraction solution to the test samples obtained in step (1), respectively, and extract for 4 hours on a mixer. Then, centrifuge the samples at 3000×g for 5 minutes, transfer the supernatant and add 3, 6 and 9 mL of 0.01M calcium chloride extraction solution respectively for 4 hours of subsequent extraction. Centrifuge again under the same conditions, repeat once more, and then combine the extracts.

[0073] (3) Activate the HLB column required for solid phase extraction with 6 mL of methanol and 12 mL of ultrapure water respectively. Enrich the extract obtained in step (2) by passing it through the HLB column, then wash it with 10 mL of ultrapure water, and dry the HLB column under vacuum until the bottom turns white.

[0074] (4) The target substance retained on the HLB column was eluted with 12 mL of methanol containing 0.1% formic acid under standard atmospheric pressure to obtain the test sample of the target substance.

[0075] (5) The sample of the target substance obtained in step (4) is concentrated to 0.05 mL by vacuum centrifugation, and then 1 mL of acetonitrile and water are added. The mixture is vortexed to obtain a mixed solution containing the target substance with acetonitrile / water (volume ratio of 90 / 10) as the solvent system. This solution is used for detection by liquid chromatography-tandem mass spectrometry.

[0076] (6) A solution containing 0.1% formic acid and pure acetonitrile was used as the mobile phase for liquid chromatography-tandem mass spectrometry (LC-MS / MS). Gradient elution was performed for 16 minutes. The concentrations of various antibiotics were separated and quantified using multiple reaction monitoring (MRM) to determine the concentrations of chlortetracycline, doxycycline, oxytetracycline, tetracycline, sulfadiazine, sulfadiazine, sulfamethoxazole, ciprofloxacin, enrofloxacin, norfloxacin, ofloxacin, azithromycin, erythromycin, roxithromycin, tylosin, trimethoprim, and lincomycin in the soil. The mass ion source temperature for LC-MS / MS was 650℃. The chromatographic column for LC-MS / MS was an ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 mm × 100 mm).

[0077] The specific conditions for gradient elution are as follows:

[0078] Within 0-12 minutes, the volume ratio of formic acid aqueous solution to pure acetonitrile solution decreased from 95:5 to 20:80;

[0079] Within 12-13 minutes, the volume ratio of formic acid aqueous solution to pure acetonitrile solution decreased from 20:80 to 5:95;

[0080] Within 13-14 minutes, the volume ratio of formic acid aqueous solution to pure acetonitrile solution decreased from 5:95 to 95:5;

[0081] Within 14-16 minutes, the volume ratio of formic acid aqueous solution to pure acetonitrile solution was 95:5.

[0082] like Figure 2 As shown, the horizontal axis represents the volume of 0.01M calcium chloride extract, and the vertical axis represents the recovery rate of 18 antibiotics at different extract volumes. The effects of 3, 6, and 9 mL of 0.01M calcium chloride extract on the antibiotic recovery rate were tested. The results showed that calcium chloride only had an extraction effect on sulfonamides, lincomycin, and trimethoprim, but the volume of calcium chloride extract did not have a significant effect on the recovery rate of these antibiotics. Therefore, a volume of 3 mL of 0.01M calcium chloride extract was selected.

[0083] Example 2

[0084] This embodiment investigated the effect of extraction time of 0.01M calcium chloride extract on the relative signal intensity of 18 antibiotics, specifically including the following steps:

[0085] (1) Prepare simulated soil samples containing the same concentration of 18 antibiotics to obtain the test samples.

[0086] (2) Add 3 mL of 0.01 M calcium chloride extract to the sample obtained in step (1) and extract for 4, 8, 12 and 24 hours on a mixer. Then centrifuge the sample at 3000×g for 5 minutes, transfer the supernatant and add 3 mL of 0.01 M calcium chloride extract for subsequent extraction for the corresponding extraction time (hours). Centrifuge again under the same conditions and repeat once more before combining the extracts.

[0087] (3) Activate the HLB column required for solid phase extraction with 6 mL of methanol and 12 mL of ultrapure water respectively. Enrich the extract obtained in step (2) by passing it through the HLB column, then wash it with 10 mL of ultrapure water, and dry the HLB column under vacuum until the bottom turns white.

[0088] (4) The target substance retained on the HLB column was eluted with 12 mL of methanol containing 0.1% formic acid under standard atmospheric pressure to obtain the test sample of the target substance.

[0089] (5) The sample of the target substance obtained in step (4) is concentrated to 0.05 mL by vacuum centrifugation, and then 1 mL of acetonitrile and water are added. The mixture is vortexed to obtain a mixed solution containing the target substance in acetonitrile / water (volume ratio of 90 / 10) as the solvent system. This solution is used for detection by liquid chromatography-tandem mass spectrometry.

[0090] (6) A solution containing 0.1% formic acid and pure acetonitrile was used as the mobile phase for liquid chromatography-tandem mass spectrometry (LC-MS / MS). Gradient elution was performed for 16 minutes under the same conditions as in Example 1. The concentrations of various antibiotics were separated and quantified using multiple reaction monitoring (MRM) to determine the concentrations of chlortetracycline, doxycycline, oxytetracycline, tetracycline, sulfadiazine, sulfadiazine, sulfamethoxazole, ciprofloxacin, enrofloxacin, norfloxacin, ofloxacin, azithromycin, erythromycin, roxithromycin, tylosin, trimethoprim, and lincomycin in the soil. The mass ion source temperature for LC-MS / MS was 650°C; the chromatographic column for LC-MS / MS was an ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 mm × 100 mm).

[0091] like Figure 3 As shown, the horizontal axis represents extraction time, and the vertical axis represents the relative signal intensity of 18 antibiotics at different extraction times. The effects of extraction time with 0.01M calcium chloride extract for 4, 8, 12, and 24 hours on the relative signal intensity of antibiotics were tested. The experimental results showed that the extraction time had no significant effect on the relative signal intensity of these 18 antibiotics, so the extraction time of 0.01M calcium chloride extract for 4 hours was selected.

[0092] Example 3

[0093] This example investigated the effect of the number of extraction cycles of 0.01M calcium chloride extract on the recovery rate of 18 antibiotics, specifically including the following steps:

[0094] (1) Prepare simulated soil samples containing the same concentration of 18 antibiotics to obtain the test samples.

[0095] (2) Add 3 mL of 0.01 M calcium chloride extract to the sample obtained in step (1), extract for 4 hours on a mixer, then centrifuge the sample at 3000×g for 5 minutes, transfer the supernatant, and add 3 mL of 0.01 M calcium chloride extract for secondary, tertiary and quaternary extractions. After centrifugation under the same conditions, transfer the extract.

[0096] (3) Activate the HLB column required for solid phase extraction with 6 mL of methanol and 12 mL of ultrapure water respectively. Enrich the extract obtained in step (2) by passing it through the HLB column, then wash it with 10 mL of ultrapure water, and dry the HLB column under vacuum until the bottom turns white.

[0097] (4) The target substance retained on the HLB column was eluted with 12 mL of methanol containing 0.1% formic acid under standard atmospheric pressure to obtain the test sample of the target substance.

[0098] (5) The sample of the target substance obtained in step (4) is concentrated to 0.05 mL by vacuum centrifugation, and then 1 mL of acetonitrile and water are added. The mixture is vortexed to obtain a mixed solution containing the target substance in acetonitrile / water (volume ratio of 90 / 10) as the solvent system. This solution is used for detection by liquid chromatography-tandem mass spectrometry.

[0099] (6) A solution containing 0.1% formic acid and pure acetonitrile was used as the mobile phase for liquid chromatography-tandem mass spectrometry (LC-MS / MS). Gradient elution was performed for 16 minutes under the same conditions as in Example 1. The concentrations of various antibiotics were separated and quantified using multiple reaction monitoring (MRM) to determine the concentrations of chlortetracycline, doxycycline, oxytetracycline, tetracycline, sulfadiazine, sulfadiazine, sulfamethoxazole, ciprofloxacin, enrofloxacin, norfloxacin, ofloxacin, azithromycin, erythromycin, roxithromycin, tylosin, trimethoprim, and lincomycin in the soil. The mass ion source temperature for LC-MS / MS was 650°C; the chromatographic column for LC-MS / MS was an ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 mm × 100 mm).

[0100] like Figure 4 As shown, the horizontal axis represents the number of extraction cycles, and the vertical axis represents the recovery rate of 18 antibiotics at different extraction cycle numbers. The effect of the number of extraction cycles with 0.01M calcium chloride extract (one, two, three, and four cycles) on the antibiotic recovery rate was tested. The results showed that the recovery rate of the 18 antibiotics increased with the increase of the number of extraction cycles, but the third and fourth extraction cycles had no significant effect on the antibiotic recovery rate. Therefore, three extraction cycles with 0.01M calcium chloride were selected.

[0101] Example 4

[0102] This embodiment investigated the effect of the type of organic solvent in the mixed extract on the recovery rate of 18 antibiotics, specifically including the following steps:

[0103] (1) Prepare simulated soil samples containing the same concentration of 18 antibiotics to obtain the test samples.

[0104] (2) Add 5 ml of acetonitrile or methanol to the sample to be tested obtained in step (1) and perform ultrasonic extraction for 15 minutes. Then centrifuge at 8000×g for 5 minutes, remove the supernatant and add fresh mixed extract. Perform two more ultrasonic extraction cycles. After combining the extracts, dilute with ultrapure water to ensure that the organic solvent content in the solution is ≤5%.

[0105] (3) The HLB column required for solid-phase extraction was activated with 6 mL of methanol and 12 mL of ultrapure water respectively. The extract was enriched by passing it through the HLB column at a flow rate of about 3 mL per minute. Then, it was washed with 10 mL of ultrapure water and the HLB column was dried under vacuum until the bottom turned white.

[0106] (4) The target substance retained on the HLB column was eluted with 12 mL of methanol containing 0.1% formic acid under standard atmospheric pressure to obtain the test sample of the target substance.

[0107] (5) The sample of the target substance obtained in step (4) is concentrated to 0.05 mL by vacuum centrifugation concentrator, and then 1 mL of acetonitrile and water are added. The mixture is vortexed to obtain a mixed solution containing the target substance in acetonitrile / water (volume ratio of 90 / 10) solvent system, which is used for detection by liquid chromatography-tandem mass spectrometry.

[0108] (6) A solution containing 0.1% formic acid and pure acetonitrile was used as the mobile phase for liquid chromatography-tandem mass spectrometry (LC-MS / MS). Gradient elution was performed for 16 minutes under the same conditions as in Example 1. The concentrations of various antibiotics were separated and quantified using multiple reaction monitoring (MRM) to determine the concentrations of chlortetracycline, doxycycline, oxytetracycline, tetracycline, sulfadiazine, sulfadiazine, sulfamethoxazole, ciprofloxacin, enrofloxacin, norfloxacin, ofloxacin, azithromycin, erythromycin, roxithromycin, tylosin, trimethoprim, and lincomycin in the soil. The mass ion source temperature for LC-MS / MS was 650°C; the chromatographic column for LC-MS / MS was an ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 mm × 100 mm).

[0109] like Figure 5 As shown, the horizontal axis represents the types of organic solvents in the extract, and the vertical axis represents the recovery rates of 18 antibiotics under different extracts. The effects of acetonitrile and methanol extracts on the antibiotic recovery rates were tested separately. The experimental results showed that acetonitrile had high and stable recovery rates for all 18 antibiotics, especially for fluoroquinolone antibiotics. Therefore, acetonitrile was chosen as a suitable extract.

[0110] Example 5

[0111] This embodiment investigated the effect of pH value of the mixed extract on the recovery rate of 18 antibiotics, specifically including the following steps:

[0112] (1) Prepare simulated soil samples containing the same concentration of 18 antibiotics to obtain the test samples.

[0113] (2) Add 5 mL of acetonitrile-ethylenediaminetetraacetic acid-citric acid phosphate buffer-Mg(NO3)2-NH3·H2O mixed extraction solution to the sample to be tested obtained in step (1), perform ultrasonic extraction for 15 minutes, then centrifuge at 8000×g for 5 minutes, remove the supernatant and add fresh extraction solution, and perform two more ultrasonic extraction cycles. Dilute with ultrapure water to ensure that the organic solvent content in the solution is ≤5%.

[0114] (3) Activate the HLB column required for solid-phase extraction with 6 mL of methanol and 12 mL of ultrapure water respectively, pass it through the HLB column at a flow rate of about 3 mL per minute for enrichment, wash with 10 mL of ultrapure water, and dry the HLB column under vacuum until the bottom turns white.

[0115] (4) The target substance retained on the HLB column was eluted with 12 mL of methanol containing 0.1% formic acid under standard atmospheric pressure to obtain the test sample of the target substance.

[0116] (5) The sample of the target substance obtained in step (4) is concentrated to 0.05 mL by vacuum centrifugation, and then 1 mL of acetonitrile and water are added. The mixture is vortexed to obtain a mixed solution containing the target substance in acetonitrile / water (volume ratio of 90 / 10) as the solvent system. This solution is used for detection by liquid chromatography-tandem mass spectrometry.

[0117] (6) A solution containing 0.1% formic acid and pure acetonitrile was used as the mobile phase for liquid chromatography-tandem mass spectrometry (LC-MS / MS). Gradient elution was performed for 16 minutes under the same conditions as in Example 1. The concentrations of various antibiotics were separated and quantified using multiple reaction monitoring (MRM) to determine the concentrations of chlortetracycline, doxycycline, oxytetracycline, tetracycline, sulfadiazine, sulfadiazine, sulfamethoxazole, ciprofloxacin, enrofloxacin, norfloxacin, ofloxacin, azithromycin, erythromycin, roxithromycin, tylosin, trimethoprim, and lincomycin in the soil. The mass ion source temperature for LC-MS / MS was 650°C; the chromatographic column for LC-MS / MS was an ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 mm × 100 mm).

[0118] like Figure 6 As shown, the horizontal axis represents pH value, and the vertical axis represents the recovery rate of 18 antibiotics at different pH values ​​of the mixed extract. The effects of pH values ​​of 4 and 8 of the mixed extract on the antibiotic recovery rate were tested. The experimental results showed that the lower pH value of 4 significantly improved the extraction efficiency of tetracycline and fluoroquinolone antibiotics. Therefore, pH value of 4 of the mixed extract was selected as the most suitable extraction environment.

[0119] Example 6

[0120] This embodiment investigated the effect of the ratio of organic solvent to buffer solution in the mixed extract on the recovery rate of 18 antibiotics, specifically including the following steps:

[0121] (1) Prepare simulated soil samples containing the same concentration of 18 antibiotics to obtain the test samples.

[0122] (2) Add 3 mL of 0.01 M calcium chloride extract to the sample obtained in step (1), extract for 4 hours on a mixer, then centrifuge the sample at 3000×g for 5 minutes and remove the supernatant, add 3 mL of 0.01 M calcium chloride extract for two more extractions, centrifuge again under the same conditions and remove the extract, and combine the extracts to obtain the effective components of the antibiotic.

[0123] (3) Add 5 ml of acetonitrile-ethylenediaminetetraacetic acid-citric acid phosphate buffer: Mg(NO3)2-NH3·H2O mixed extraction solution (2:0, 2:1, 3:1) to the remaining soil sample from step (2), perform ultrasonic extraction for 15 minutes, then centrifuge at 8000×g for 5 minutes, remove the supernatant and add fresh extraction solution, and perform two more ultrasonic extraction cycles. Combine the extraction solutions to obtain the antibiotic extractable component. Dilute with ultrapure water to ensure that the organic solvent content in the solution is ≤5%.

[0124] (4) The HLB column required for solid-phase extraction was activated by 6 mL of methanol and 12 mL of ultrapure water respectively. The bioactive components of the antibiotic were directly enriched by passing through the HLB column, while the extractable components of the antibiotic were enriched by passing through the HLB column at a flow rate of about 3 mL per minute. Then, 10 mL of ultrapure water was used for washing, and the HLB column was dried under vacuum until the bottom turned white.

[0125] (5) The target substance retained on the HLB column was eluted with 12 mL of methanol containing 0.1% formic acid under standard atmospheric pressure to obtain the test sample of the target substance.

[0126] (6) The sample of the target substance obtained in step (5) is concentrated to 0.05 mL by vacuum centrifugation, and then 1 mL of acetonitrile and water are added. The mixture is vortexed to obtain a mixed solution containing the target substance in acetonitrile / water (volume ratio of 90 / 10) as the solvent system. This solution is used for detection by liquid chromatography-tandem mass spectrometry.

[0127] (7) A solution containing 0.1% formic acid and pure acetonitrile was used as the mobile phase for liquid chromatography-tandem mass spectrometry (LC-MS / MS). Gradient elution was performed for 16 minutes under the same conditions as in Example 1. The concentrations of various antibiotics were separated and quantified using multiple reaction monitoring (MRM) to determine the concentrations of chlortetracycline, doxycycline, oxytetracycline, tetracycline, sulfadiazine, sulfadiazine, sulfamethoxazole, ciprofloxacin, enrofloxacin, norfloxacin, ofloxacin, azithromycin, erythromycin, roxithromycin, tylosin, trimethoprim, and lincomycin in the soil. The mass ion source temperature for LC-MS / MS was 650°C; the chromatographic column for LC-MS / MS was an ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 mm × 100 mm).

[0128] like Figure 7 As shown, the horizontal axis represents 18 antibiotics, and the vertical axis represents the recovery rates of the 18 antibiotics under different mixed extraction solution ratios. The effects of the ratios of acetonitrile-ethylenediaminetetraacetic acid-citrate phosphate buffer-Mg(NO3)2-NH3·H2O mixed extraction solution (2:0, 2:1, 3:1) on the antibiotic recovery rates were tested. The results showed that the addition of Mg(NO3)2-NH3·H2O significantly improved the extraction efficiency of fluoroquinolone antibiotics, while the mixed extraction solution ratio of 3:1 showed the optimal recovery rate for all 18 antibiotics. Therefore, a ratio of acetonitrile-ethylenediaminetetraacetic acid-citrate phosphate buffer:Mg(NO3)2-NH3·H2O of 3:1 was selected as the most suitable mixed extraction solution ratio.

[0129] Example 7

[0130] This embodiment investigated the effect of the volume of the mixed extract on the recovery rate of 18 antibiotics, specifically including the following steps:

[0131] (1) Prepare simulated soil samples containing the same concentration of 18 antibiotics to obtain the test samples.

[0132] (2) Add 3 mL of 0.01 M calcium chloride extract to the sample obtained in step (1), extract for 4 hours on a mixer, then centrifuge the sample at 3000×g for 5 minutes and remove the supernatant, add 3 mL of 0.01 M calcium chloride extract for two more extractions, centrifuge again under the same conditions and remove the extract, and combine the extracts to obtain the effective components of the antibiotic.

[0133] (3) Add 5 ml of a 3:1 mixture of acetonitrile-ethylenediaminetetraacetic acid-citric acid phosphate buffer and Mg(NO3)2-NH3·H2O to the remaining soil sample from step (2), perform ultrasonic extraction for 15 minutes, then centrifuge at 8000×g for 5 minutes, remove the supernatant and add fresh extract, and perform two more ultrasonic extraction cycles. Combine the extracts to obtain the antibiotic extractable component. Dilute with ultrapure water to ensure that the organic solvent content in the solution is ≤5%.

[0134] (4) The HLB column required for solid-phase extraction was activated by 6 mL of methanol and 12 mL of ultrapure water respectively. The bioactive components of the antibiotic were directly enriched by passing through the HLB column, while the extractable components of the antibiotic were enriched by passing through the HLB column at a flow rate of about 3 mL per minute. Then, 10 mL of ultrapure water was used for washing, and the HLB column was dried under vacuum until the bottom turned white.

[0135] (5) The target substance retained on the HLB column was eluted with 12 mL of methanol containing 0.1% formic acid under standard atmospheric pressure to obtain the test sample of the target substance.

[0136] (6) The sample of the target substance obtained in step (5) is concentrated to 0.05 mL by vacuum centrifugation, and then 1 mL of acetonitrile and water are added. The mixture is vortexed to obtain a mixed solution containing the target substance in acetonitrile / water (volume ratio of 90 / 10) as the solvent system. This solution is used for detection by liquid chromatography-tandem mass spectrometry.

[0137] (7) A solution containing 0.1% formic acid and pure acetonitrile was used as the mobile phase for liquid chromatography-tandem mass spectrometry (LC-MS / MS). Gradient elution was performed for 16 minutes under the same conditions as in Example 1. The concentrations of various antibiotics were separated and quantified using multiple reaction monitoring (MRM) to determine the concentrations of chlortetracycline, doxycycline, oxytetracycline, tetracycline, sulfadiazine, sulfadiazine, sulfamethoxazole, ciprofloxacin, enrofloxacin, norfloxacin, ofloxacin, azithromycin, erythromycin, roxithromycin, tylosin, trimethoprim, and lincomycin in the soil. The mass ion source temperature for LC-MS / MS was 650°C; the chromatographic column for LC-MS / MS was an ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 mm × 100 mm).

[0138] like Figure 8As shown, the horizontal axis represents 18 antibiotics, and the vertical axis represents the recovery rate of the 18 antibiotics at different mixed extraction solution volumes. The recovery rate is the sum of the bioactive components and extractable components of the antibiotics. The effects of mixed extraction solution volumes of 5, 10, and 20 mL on the antibiotic recovery rate were tested. The experimental results showed that only 5 mL of mixed extraction solution had the best recovery rate for all 18 antibiotics. Therefore, 5 mL of acetonitrile-ethylenediaminetetraacetic acid-citric acid phosphate buffer-Mg(NO3)2-NH3·H2O mixed extraction solution was selected as the most suitable mixed extraction solution volume.

[0139] Example 8

[0140] This example investigated the effect of different volumes of elution solution on the recovery rate of 18 antibiotics, specifically including the following steps:

[0141] (1) Prepare simulated soil samples containing the same concentration of 18 antibiotics to obtain the test samples.

[0142] (2) Add 3 mL of 0.01 M calcium chloride extract to the sample obtained in step (1), extract for 4 hours on a mixer, then centrifuge the sample at 3000×g for 5 minutes and remove the supernatant, add 3 mL of 0.01 M calcium chloride extract for two more extractions, centrifuge again under the same conditions and remove the extract, and combine the extracts to obtain the effective components of the antibiotic.

[0143] (3) Add 5 ml of a 3:1 mixture of acetonitrile-ethylenediaminetetraacetic acid-citric acid phosphate buffer and Mg(NO3)2-NH3·H2O to the remaining soil sample from step (2), perform ultrasonic extraction for 15 minutes, then centrifuge at 8000×g for 5 minutes, remove the supernatant and add fresh extract, and perform two more ultrasonic extraction cycles. Combine the extracts to obtain the antibiotic extractable component. Dilute with ultrapure water to ensure that the organic solvent content in the solution is ≤5%.

[0144] (4) The HLB column required for solid-phase extraction was activated by 6 mL of methanol and 12 mL of ultrapure water respectively. The bioactive components of the antibiotic were directly enriched by passing through the HLB column, while the extractable components of the antibiotic were enriched by passing through the HLB column at a flow rate of about 3 mL per minute. Then, 10 mL of ultrapure water was used for washing, and the HLB column was dried under vacuum until the bottom turned white.

[0145] (5) Under standard atmospheric pressure, the target substance retained on the HLB column was eluted with 2, 4, 6, 8, 10, or 12 mL of methanol containing 0.1% formic acid to obtain the test sample of the target substance.

[0146] (6) The sample of the target substance obtained in step (5) is concentrated to 0.05 mL by vacuum centrifugation, and then 1 mL of acetonitrile and water are added. The mixture is vortexed to obtain a mixed solution containing the target substance in acetonitrile / water (volume ratio of 90 / 10) as the solvent system. This solution is used for detection by liquid chromatography-tandem mass spectrometry.

[0147] (7) A solution containing 0.1% formic acid and pure acetonitrile was used as the mobile phase for liquid chromatography-tandem mass spectrometry (LC-MS / MS). Gradient elution was performed for 16 minutes under the same conditions as in Example 1. The concentrations of various antibiotics were separated and quantified using multiple reaction monitoring (MRM) to determine the concentrations of chlortetracycline, doxycycline, oxytetracycline, tetracycline, sulfadiazine, sulfadiazine, sulfamethoxazole, ciprofloxacin, enrofloxacin, norfloxacin, ofloxacin, azithromycin, erythromycin, roxithromycin, tylosin, trimethoprim, and lincomycin in the soil. The mass ion source temperature for LC-MS / MS was 650°C; the chromatographic column for LC-MS / MS was an ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 mm × 100 mm).

[0148] like Figure 9 As shown, the horizontal axis represents 18 antibiotics, and the vertical axis represents the recovery rate of the 18 antibiotics under different volumes of elution solution. The recovery rate is the sum of the bioactive components and extractable components of the antibiotics. The effect of elution solution volumes of 2, 4, 6, 8, 10, and 12 mL on the antibiotic recovery rate was tested. The results showed that the recovery rate increased significantly when the elution volume increased from 2 mL to 8 mL. Although the recovery rate of some antibiotics did not change much when the elution solution volume increased to 10 mL, indicating that 10 mL of elution solution was sufficient to completely elute most antibiotics, 12 mL significantly improved the recovery rate of sulfonamides and chlortetracycline. Therefore, 12 mL was selected as the most suitable elution solution volume.

[0149] Example 9

[0150] This embodiment investigated the effect of different vacuum centrifugation concentration temperatures on the recovery rates of 18 antibiotics, specifically including the following steps:

[0151] (1) Prepare simulated soil samples containing the same concentration of 18 antibiotics to obtain the test samples.

[0152] (2) Add 3 mL of 0.01 M calcium chloride extract to the sample obtained in step (1), extract for 4 hours on a mixer, then centrifuge the sample at 3000×g for 5 minutes and remove the supernatant, add 3 mL of 0.01 M calcium chloride and perform two subsequent extractions, centrifuge again under the same conditions and remove the extract, and combine the extracts to obtain the effective components of the antibiotic.

[0153] (3) Add 5 ml of a 3:1 mixture of acetonitrile-ethylenediaminetetraacetic acid-citric acid phosphate buffer and Mg(NO3)2-NH3·H2O to the remaining soil sample from step (2), perform ultrasonic extraction for 15 minutes, then centrifuge at 8000×g for 5 minutes, remove the supernatant and add fresh extract, and perform two more ultrasonic extraction cycles. Combine the extracts to obtain the antibiotic extractable component. Dilute with ultrapure water to ensure that the organic solvent content in the solution is ≤5%.

[0154] (4) The HLB column required for solid-phase extraction was activated by 6 mL of methanol and 12 mL of ultrapure water respectively. The bioactive components of the antibiotic were directly enriched by passing through the HLB column, while the extractable components of the antibiotic were enriched by passing through the HLB column at a flow rate of about 3 mL per minute. Then, 10 mL of ultrapure water was used for washing, and the HLB column was dried under vacuum until the bottom turned white.

[0155] (5) The target substance retained on the HLB column was eluted with 12 mL of methanol containing 0.1% formic acid under standard atmospheric pressure to obtain the test sample of the target substance.

[0156] (6) The sample of the target substance obtained in step (5) is concentrated to 0.05 mL by vacuum centrifugation concentrator at 80, 60, 50, 40 and 30 °C. Then, 1 mL of acetonitrile and water are added and vortexed to obtain a mixed solution containing the target substance in acetonitrile / water (volume ratio of 90 / 10) solvent system, which is used for detection by liquid chromatography-tandem mass spectrometry.

[0157] (7) A solution containing 0.1% formic acid and pure acetonitrile was used as the mobile phase for liquid chromatography-tandem mass spectrometry (LC-MS / MS). Gradient elution was performed for 16 minutes under the same conditions as in Example 1. The concentrations of various antibiotics were separated and quantified using multiple reaction monitoring (MRM) to determine the concentrations of chlortetracycline, doxycycline, oxytetracycline, tetracycline, sulfadiazine, sulfadiazine, sulfamethoxazole, ciprofloxacin, enrofloxacin, norfloxacin, ofloxacin, azithromycin, erythromycin, roxithromycin, tylosin, trimethoprim, and lincomycin in the soil. The mass ion source temperature for LC-MS / MS was 650°C; the chromatographic column for LC-MS / MS was an ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 mm × 100 mm).

[0158] like Figure 10As shown, the horizontal axis represents the vacuum centrifugation concentration temperature, and the vertical axis represents the recovery rate of 18 antibiotics at different vacuum centrifugation concentration temperatures. The recovery rate is the sum of the bioactive components and extractable components of the antibiotics. The effects of vacuum centrifugation concentration temperatures of 80, 60, 50, 40, and 30℃ on the antibiotic recovery rate were tested. The experimental results show that the recovery rate significantly increases when the vacuum centrifugation concentration temperature decreases from 80℃ to 40℃. After decreasing the vacuum centrifugation concentration temperature to 30℃, the antibiotic recovery rate does not change. This indicates that vacuum centrifugation concentration at 40℃ effectively improves the concentration efficiency without affecting the recovery rate. Therefore, 40℃ was selected as the most suitable vacuum centrifugation concentration temperature.

[0159] For example, the method described in this invention was used to test 10 groups of soil samples, and the results are shown in Table 2. The test results showed that the extractable components of doxycycline (DTC) were detected in 100% of the 10 samples; while among all the bioactive components and extractable components of antibiotics, only sulfamethoxazole (SMT) and sulfamethoxazole (SMR) were not detected in these 10 groups of soil samples. Among the 10 soil samples, chlortetracycline (CTC) had the highest detection concentration, with 26.5 μg / kg detected in the antibiotic extractable fraction of soil sample one. This was followed by oxytetracycline (OTC), also detected in the antibiotic extractable fraction of soil sample one at 18.0 μg / kg. Ciprofloxacin (CIP) was detected in the antibiotic extractable fraction of soil sample two at 13.1 μg / kg, norfloxacin (NOR) in the antibiotic extractable fraction of soil sample eight at 10.9 μg / kg, and enrofloxacin (ENR) in the antibiotic extractable fraction of soil sample one at 10.4 μg / kg. This indicates that these antibiotics warrant attention.

[0160] Table 2: Simultaneous detection of bioavailable and extractable components of 18 antibiotics in soil

[0161]

[0162] In summary, this invention combines continuous solvent extraction with solid-phase extraction, along with liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection, to achieve accurate evaluation of the bioavailability of trace antibiotics under complex matrix conditions.

[0163] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the bioavailability of antibiotics in soil using a continuous solvent extraction-solid phase extraction-liquid chromatography-tandem mass spectrometry method, characterized in that, Includes the following steps: (1) After collecting soil samples, place them in a freezer at -20℃ for storage; (2) The soil sample to be tested was obtained by air drying the soil sample in the laboratory by natural ventilation, and then it was thoroughly ground with an agate mortar and filtered through a 200-mesh sieve. (3) Weigh 1.9-2.1 g of the filtered soil sample obtained in step (2) and place it in a centrifuge tube. Add 3 mL of 0.01 M calcium chloride extraction solution and extract on a mixer for 4 hours. Then centrifuge at 3000×g for 5 minutes. Transfer the supernatant and add 3 mL of 0.01 M calcium chloride extraction solution to continue extraction for 4 hours. Centrifuge at 3000×g for 5 minutes again. Repeat once more and combine the extracts to obtain the effective components of the antibiotic. (4) Add 5 mL of mixed extraction solution to the remaining soil sample in step (3) and sonicate for 15 minutes. Then centrifuge at 8000×g for 5 minutes, remove the supernatant and add fresh mixed extraction solution. Perform two more sonic extraction cycles. Combine the extracts to obtain the extractable components of the antibiotic. Dilute with ultrapure water to ensure that the organic solvent content in the solution is less than or equal to 5%. In step (4), the mixed extraction solution is formed by mixing acetonitrile-ethylenediaminetetraacetic acid-citric acid phosphate buffer and Mg(NO3)2-NH3•H2O in a volume ratio of 3:

1. (5) The HLB column required for solid-phase extraction was activated with 6 mL of methanol and 12 mL of ultrapure water respectively. The bioactive components of the antibiotic were directly enriched by passing through the HLB column, while the extractable components of the antibiotic were enriched by passing through the HLB column at a flow rate of 3 mL / min. Then, 10 mL of ultrapure water was used for washing, and the HLB column was dried under vacuum until the bottom turned white. (6) The target substance retained on the HLB column was eluted with 12 mL of methanol containing 0.1% formic acid under standard atmospheric pressure to obtain the sample of the target substance to be tested. (7) The sample of the target substance obtained in step (6) is concentrated to 0.05 mL by vacuum centrifugation, and then 1 mL of acetonitrile and water are added. The mixture is vortexed to obtain a mixed solution containing the target substance with acetonitrile / water as the solvent system, which is used for liquid chromatography-tandem mass spectrometry detection; wherein the volume ratio of acetonitrile / water is 90 / 10. (8) Using a 0.1% formic acid aqueous solution and pure acetonitrile solution as the mobile phase of liquid chromatography-tandem mass spectrometry, gradient elution for 16 minutes, the concentrations of multiple antibiotics were separated and quantified by multiple reaction monitoring method to determine the concentrations of bioactive and extractable components of chlortetracycline, doxycycline, oxytetracycline, tetracycline, sulfadiazine, sulfadiazine, sulfamethoxazole, ciprofloxacin, enrofloxacin, norfloxacin, ofloxacin, azithromycin, erythromycin, roxithromycin, tylosin, trimethoprim and lincomycin in soil.

2. The method for detecting the bioavailability of antibiotics in soil using continuous solvent extraction-solid phase extraction-liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, In step (5), the HLB column has a specification of 6cc and 200mg.

3. The method for detecting the bioavailability of antibiotics in soil using continuous solvent extraction-solid phase extraction-liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, In step (7), the vacuum centrifuge concentrator operates at a temperature of 40°C and a rotation speed of 2000 r / min.

4. The method for detecting the bioavailability of antibiotics in soil using continuous solvent extraction-solid phase extraction-liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, In step (8), the mass ion source temperature of the liquid chromatography-tandem mass spectrometry is 650℃; the chromatographic column of the liquid chromatography-tandem mass spectrometry is an ACQUITY UPLC HSS T3 column, which has a column length of 100mm, an inner diameter of 2.1mm, and a packing particle size of 1.8μm.

5. The method for detecting the bioavailability of antibiotics in soil using continuous solvent extraction-solid phase extraction-liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, In step (8), the specific conditions for gradient elution are as follows: Within 0-12 minutes, the volume ratio of formic acid aqueous solution to pure acetonitrile solution decreased from 95:5 to 20:80; Within 12-13 minutes, the volume ratio of formic acid aqueous solution to pure acetonitrile solution decreased from 20:80 to 5:95; Within 13-14 minutes, the volume ratio of formic acid aqueous solution to pure acetonitrile solution decreased from 5:95 to 95:5; Within 14-16 minutes, the volume ratio of formic acid aqueous solution to pure acetonitrile solution was 95:

5.

6. The method for detecting the bioavailability of antibiotics in soil using continuous solvent extraction-solid phase extraction-liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, In step (8), the specific conditions for the multiple reaction monitoring method are shown in the table below: 。

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