Method for detecting cephalosporin in atmospheric environment medium and application thereof

Abstract

The application belongs to the technical field of pollutant detection and environmental protection, and discloses a detection method and application of cephalosporin in atmospheric environmental medium, which comprises the following steps: S1, determining key environmental indicators; S2, sampling atmospheric particulates; S3, extracting cephalosporin; S4, batch detection and data analysis. The application also discloses the application of the detection method in environmental and health risk assessment. The application proposes a specific and feasible key detection scheme with low cost through a whole-process strategy of'screening key environmental indicators, optimizing sampling network, sample pretreatment enrichment and purification, high-efficiency separation, high-sensitivity detection, method verification and risk assessment', develops and standardizes the detection method, and qualitatively and quantitatively detects different generations of structural characteristics, so as to improve the detection efficiency, reduce the detection cost, improve the overall monitoring capability of atmospheric environmental safety, and be applicable to activities such as atmospheric environmental health risk assessment.

Description

Technical Field

[0001] This invention belongs to the field of pollutant detection and environmental protection technology, specifically relating to a rapid detection method for multiple cephalosporins in atmospheric media and its application in environmental health risk assessment. Background Technology

[0002] Antibiotics, as one of the 14 key new pollutants listed in the "List of Key Controlled New Pollutants (2023 Edition)," have become a focus of attention for both society and the scientific research field due to the severity and uniqueness of their environmental risks. Cephalosporins belong to the β-lactam antibiotic class and are currently widely detected in the environment. However, the occurrence characteristics of atmospheric particulate matter, one of the important mediators of antibiotics in the environment, are often overlooked. Cephalosporins are a collective term for a class of antibiotics, not a single chemical component. Their core is the cephalosporin nucleus structure. Different types of cephalosporins are formed by attaching different side chain groups to the nucleus. Commonly used clinical varieties are: First generation: cefadroxil, cephalexin (side chains are mostly simple aliphatic or aromatic groups, with a narrower antibacterial spectrum); Second generation: cefuroxime, cefaclor (side chains introduce groups such as aminothiazole, extending the antibacterial spectrum to some Gram-negative bacteria); Third generation: ceftriaxone, ceftazidime (side chains are more complex, such as triazine rings and carboxyl groups, with stronger effects against Gram-negative bacteria, and some can cross the blood-brain barrier); Fourth generation: cefepime (side chains contain quaternary ammonium salt structures, with broader antibacterial activity and better efficacy against drug-resistant bacteria).

[0003] Cephalosporins in the environment are mainly associated with emissions from human medical treatment, animal husbandry, and pharmaceutical production activities, with industrial wastewater significantly contributing to their presence in the environment. Currently, cephalosporins are widely detected in various environmental media, including water, sediments, and soil. With the continued large-scale emissions from the pharmaceutical industry, the accumulation load of cephalosporins in aquatic environments has significantly increased, becoming a major anthropogenic source of this type of antibiotic in water bodies. Similar to water, soil, and sediment, atmospheric particulate matter, such as dust and settling dust, can also serve as a medium for antibiotic accumulation. Previous studies have shown that residues of various antibiotics can be detected in atmospheric particulate matter in specific locations such as pharmaceutical plants, pig farms, livestock farms, composting plants, and urban environments. The high surface area of ​​atmospheric particulate matter allows it to adsorb antibiotics, enabling them to remain suspended in the air for longer periods. Furthermore, bacteria in particulate-polluted air carry more antibiotic resistance genes (ARGs) than those in chemically polluted air. Therefore, cephalosporins released into the air have a significant and far-reaching impact on the spread of antibiotic resistance. Existing research indicates that antibiotics present in the lungs can cause dysbiosis of the lung and gut microbiota in aged rats exposed to air pollution. Pharmaceutical manufacturing is one of the main production processes for cephalosporins, and physical processes such as pulverization, tableting, granulation, filling, and packaging release dust-laden waste gases into the environment. Considering the cephalosporin production output of pharmaceutical companies and based on their production processes, cephalosporins are highly likely to be emitted into the surrounding ambient air, potentially impacting the environment and population. Therefore, it is necessary to conduct qualitative and quantitative detection and environmental impact assessment of cephalosporins in atmospheric particulate matter in specific locations such as pharmaceutical manufacturing sites. This will support environmental monitoring and governance, facilitate the survey and screening of new pollutants, trace pollution sources, evaluate the effectiveness of environmental governance, and provide a scientific basis for formulating antibiotic emission standards and environmental limits, and for the precise control of pollution sources (including pharmaceutical waste gas and medical waste).

[0004] However, current methods for detecting cephalosporins in the environment mostly focus on media such as water and soil (e.g., the liquid chromatography-mass spectrometry method for detecting cephalosporin antibiotics in water disclosed in CN111537633A). There is no clear method for detecting cephalosporins in atmospheric particulate matter. Furthermore, due to the β-lactam ring in the cephalosporin structure, they are easily hydrolyzed and biodegraded, resulting in low detection concentrations in the environment. To achieve qualitative and quantitative detection of cephalosporins in atmospheric particulate matter, the core is to address the three major challenges of "extremely small concentrations," "matrix complexity," and "generational structural differences" by constructing a comprehensive strategy encompassing "sample pretreatment enrichment and purification → efficient separation → high-sensitivity detection → method validation," while also taking into account the structural differences of different generations of cephalosporins. Although the standard T / PIAC 00002-2021 "Detection Method of Cephalosporins in Antibiotic Residue, Organic Fertilizer Substrate, Crops and Environmental Media" introduces the detection method of cephalosporin C in the atmosphere as absorption liquid sampling → centrifugation and membrane filtration → UPLC-MS / MS, with a detection limit of 0.02 μg / m³, it does not provide a feasible, complete and highly accurate specific practical detection method. The recommended UPLC-MS / MS (ultra-high performance liquid chromatography-tandem mass spectrometry) method is costly and difficult to promote and apply in practice.

[0005] The current pretreatment process for cephalosporin-contaminated samples in soil, sediment, and water can be summarized as follows: ultrasonic extraction, organic solvent concentration and dilution, SPE purification, nitrogen blowing concentration, and instrumental detection and analysis. In the extraction stage, existing studies widely use organic solvent systems, specifically acetonitrile-phosphate buffer, acetonitrile-citric acid buffer, methanol-acetonitrile mixture, and methanol-acetone mixture. Because these extraction solvents have a high organic solvent content, their concentration must be reduced during pretreatment, followed by dilution with pure water. The core purpose of this operation is to meet the requirements of the subsequent SPE purification step, ensuring effective retention and purification of the target analyte while minimizing matrix interference. However, this process has certain limitations: on the one hand, the operation is time-consuming, and cephalosporins are easily degraded in the environment due to their chemical structure (containing an unstable β-lactam ring), so excessive pretreatment time may lead to an underestimation of the actual content in the sample; on the other hand, to maintain the pH stability of the extraction system and optimize extraction efficiency through acid-base adjustment, buffer solutions are often added to the extraction solvent, but the preparation of buffer solutions is cumbersome and usually needs to be freshly prepared, which is inconvenient for processing large batches of samples. In the purification and enrichment stage, hydrophilic-lipophilic balance (HLB) solid-phase extraction columns are commonly used enrichment materials in cephalosporin detection, and their activation process often involves sequential treatment with methanol and water; the choice of elution solvent varies depending on the type of target analyte, including methanol, ethyl acetate, 5% formic acid aqueous solution / methanol solution (V / V), etc., with methanol being the most commonly used eluent.

[0006] In summary, existing technologies have the following shortcomings: They lack low-cost, practical detection methods for cephalosporin contamination in atmospheric particulate matter in key areas; existing conventional technologies cannot achieve qualitative and quantitative detection of cephalosporins in atmospheric particulate matter, cannot simultaneously address technical issues such as trace concentrations, matrix complexity, generational structural differences, complex processes, and high costs, cannot simultaneously consider the structural characteristics differences of different generations of cephalosporins, and do not consider the synergistic monitoring of cephalosporin resistance genes. They cannot meet the needs of supporting environmental monitoring and governance, providing technical support for unified and standardized environmental monitoring, or providing scientific basis for environmental health risk assessment, the formulation of antibiotic emission standards and environmental limits, and the precise control of pollution sources (such as pharmaceutical exhaust and medical waste). Summary of the Invention

[0007] To address the aforementioned shortcomings of existing technologies, this invention provides a method and application for detecting cephalosporins in atmospheric environmental media. By constructing a comprehensive strategy encompassing "screening key environmental indicators → optimizing the sampling network → sample pretreatment enrichment and purification → efficient separation → high-sensitivity detection → method validation → risk assessment," this invention proposes a specific, feasible, and low-cost key detection scheme, develops and standardizes the detection method, and while ensuring the confidence level of the assessment results, also considers the structural differences of different generations of cephalosporins, improving detection efficiency and reducing detection costs. This enhances the overall monitoring capability of atmospheric environmental safety, enabling its application in atmospheric environmental health risk assessment and supporting environmental monitoring and governance.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A method for detecting cephalosporins in atmospheric environmental media, comprising the following steps:

[0010] S1. Identify key environmental indicators

[0011] Cephalexin, cefradine, ceftriaxone and cefuroxime were selected as key environmental indicators to monitor the presence of cephalosporins in the atmospheric environmental media of the key areas of concern and the surrounding airspace, with atmospheric particulate matter as the atmospheric environmental presence medium.

[0012] S2, Atmospheric particulate matter sampling

[0013] Each monitoring point used a high-flow-rate atmospheric particulate matter sampler and a quartz fiber filter membrane to collect samples in batches. Each sampling time was 24 hours, and multiple samples were collected continuously or at intervals to obtain multiple batches of samples.

[0014] S3, cephalosporin extract

[0015] The collected samples were then processed through sample pretreatment and solid-phase extraction to extract cephalosporins from the samples, enriching the target analytes and removing interfering substances to obtain the sample extract.

[0016] After collecting the sample filter membranes, the membrane samples were further divided uniformly under aseptic conditions. One portion was used for cephalosporin extraction and chemical analysis, and the other portion was used for microbial DNA extraction and subsequent gene analysis. The first portion of the divided filter membrane was then placed into sterile sealed bags and numbered, and stored at -20°C. The second portion of the filter membrane was immediately placed into a sterile centrifuge tube containing a DNA protectant (such as TE buffer containing EDTA to inhibit nuclease activity) and frozen at -80°C to prevent DNA degradation.

[0017] S4. Batch testing and data analysis

[0018] HPLC-MS / MS and internal standard method were used to detect the chemical components and analyze the data of the sample extract. The results of the detection of cephalosporin components and concentrations in the sample extract were output for qualitative and quantitative monitoring of cephalosporin pollutant emissions.

[0019] Step S5: Co-monitoring of cephalosporin resistance genes

[0020] Microbial DNA was extracted from the sample extract and gene analysis was performed to obtain cephalosporin resistance gene data, which was used for the synergistic monitoring of cephalosporin resistance genes. The cephalosporin concentration data (ng / m³) was combined with the abundance data of resistance genes (copies / m³) to assess the correlation between the two and the potential environmental and health risks.

[0021] The application of the aforementioned detection methods for cephalosporins in atmospheric environmental media in environmental and health risk assessment.

[0022] The present invention provides a method and application, which, compared with the prior art, have at least the following beneficial effects:

[0023] 1. This invention focuses on scientific research and method development, resulting in a standardized detection method. Specifically, it involves constructing a process of "screening key environmental indicators → optimizing the sampling network → sample pretreatment enrichment and purification → efficient separation → high-sensitivity detection → method validation → risk assessment". This invention proposes a comprehensive, feasible, and low-cost key detection scheme, develops and standardizes detection methods, and, while ensuring the confidence level of the assessment results, takes into account the structural differences of different generations of cephalosporins, improves detection efficiency, and reduces detection costs, thereby enhancing the overall monitoring capability for atmospheric environmental safety. This invention can detect 14 cephalosporins (cephalosporins, cefprozil, cefotiam, cefoxitin, cefradine, cefuroxime, cefixime, cefpodoxime proxetil, cefixime methyl ester, cefotaxime sodium, cefoperazone sodium, cefuroxime axetil, cefpirome, and cefnicotinic acid), and achieves good recoveries for all of them. The recoveries for matrix and blank spiked samples range from 33.9% to 169% and 38.6% to 121%, respectively, with RSD < 9.48%, while the recoveries of conventional techniques are typically between 64.3% and 119%. This invention can provide technical support for unified and standardized environmental monitoring and improve overall monitoring capabilities.

[0024] 2. This invention simplifies the detection process and reduces costs by screening key environmental indicators. Cephalexin, cefadroxil, ceftriaxone, and cefuroxime are used as key environmental indicators to monitor the presence of cephalosporins in the atmospheric environment of key areas of concern (such as pharmaceutical factory areas) and surrounding airspace. Atmospheric particulate matter is used as the atmospheric environment presence medium. This approach comprehensively considers the generational and structural differences of these indicators, as well as their respective focuses in production and use, environmental detection, resistance induction potential, and detection feasibility. The combined monitoring of these key environmental indicators can comprehensively reflect the pollution status and support atmospheric environmental health risk assessment, as well as environmental monitoring and governance. It can provide a scientific basis for conducting new pollutant surveys and screenings, tracing pollution sources, evaluating the effectiveness of environmental governance, and formulating antibiotic emission standards and environmental limits, as well as for precise control of pollution sources (such as pharmaceutical exhaust gas and medical waste).

[0025] 3. The detection method provided by this invention combines chemical analysis with gene analysis. By combining cephalosporin concentration data (ng / m³) with drug resistance gene abundance data (copies / m³), the correlation between the two and the potential environmental and health risks are assessed, which improves the scope of data application and enhances the confidence and comprehensiveness of the assessment method and results.

[0026] 4. The pretreatment and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) used in this invention for qualitative and quantitative analysis can detect a wide variety of target analytes with low detection limits. Furthermore, the extraction solvent is not diluted, shortening the experimental time. Formic acid is used to maintain the pH of the extraction system, which is simple to operate, avoiding the cumbersome preparation of traditional buffer solutions and saving costs. Acetonitrile is used as the elution solvent, which can reduce the degradation of β-lactam compounds in the concentration step and improve the accuracy of the detection results. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the main process of the detection method and application of cephalosporins in atmospheric environmental media according to an embodiment of the present invention;

[0028] Figure 2 This is a schematic diagram illustrating the low-spiking (L) and high-spiking (H) recoveries of cephalosporins in blank and matrix samples in embodiments of the present invention.

[0029] Figure 3 This is a schematic diagram of the chromatogram of the target compound and the internal standard in an embodiment of the present invention.

[0030] Figure 4 This is a schematic diagram showing the total cephalosporin content in samples at different locations (left), the cephalosporin content in samples at different times (middle), and the difference in cephalosporin content in samples at different locations (right) in embodiments of the present invention. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the following description is provided in conjunction with the appendix. Figures 1 to 4 The present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of the invention described below can be combined with each other as long as they do not conflict with each other.

[0032] Basic Implementation

[0033] See appendix Figure 1 The method for detecting cephalosporins in atmospheric environmental media provided in this embodiment includes the following steps:

[0034] S1. Screening key environmental indicators

[0035] Cephalexin, cefadroxil, ceftriaxone, and cefuroxime were selected as key environmental indicators to monitor the presence of cephalosporins in the atmospheric environment of key areas of concern and surrounding airspace, with atmospheric particulate matter as the atmospheric environment presence medium. The specific steps include the following:

[0036] S1-1: Select representative drugs as key environmental indicators based on generational data.

[0037] The first generation includes: Cefazolin, Cefalexin, Cefradine, and Cefadroxil;

[0038] The second generation includes: Cefuroxime, Cefamandole, Cefotiam, and Cefaclor;

[0039] The third generation includes: cefotaxime, ceftriaxone, ceftazidime, and cefoperazone.

[0040] The fourth generation includes: cefepime and cefpirome;

[0041] S1-2: Based on production and use, environmental detection, drug resistance induction potential and testing feasibility, four key environmental indicators were finally selected: cephalexin, cefadroxil, ceftriaxone and cefuroxime. Atmospheric particulate matter was used as the main carrier for combined monitoring to more comprehensively reflect the status of environmental pollutants.

[0042] S2, Atmospheric particulate matter sampling

[0043] At each monitoring point, samples were collected in batches using high-flow-rate atmospheric particulate matter samplers and quartz fiber filter membranes. Each sampling session lasted 24 hours, with multiple samplings conducted continuously or at intervals to obtain multiple batches of samples. The specific steps included the following:

[0044] S2-1 Constructing a Monitoring Network

[0045] The key areas of concern and the surrounding airspace are delineated, and multiple monitoring points are set up in and within these areas. Each monitoring point is equipped with at least one high-flow-rate atmospheric particulate matter sampler, and samples are collected simultaneously during monitoring to form a monitoring network.

[0046] The monitoring points of the monitoring network should cover: sensitive areas within the region (including production workshops, sewage treatment facilities, and areas near exhaust gas outlets), upwind and downwind areas outside the region, and sensitive protected targets downwind (such as residential areas, schools, etc.).

[0047] S2-2 Deployment of sampling equipment

[0048] At each monitoring point, at least one high-flow-rate atmospheric particulate matter sampler should be deployed, with the sampler 1.5 m above the ground;

[0049] S2-3 Atmospheric particulate matter sampling

[0050] Each monitoring station collected samples in batches using high-flow-rate atmospheric particulate matter samplers and quartz fiber filter membranes. Each sampling session lasted 24 hours, with a sampling flow rate of 1 m³ / h. 3 / min, collected multiple times continuously or at intervals;

[0051] S3, cephalosporin extract

[0052] The collected samples were then processed through sample pretreatment and solid-phase extraction to extract cephalosporins, enriching the target analytes and removing interfering substances to obtain the sample extract. The specific steps included were as follows:

[0053] S3-1, Filter membrane collection

[0054] The collected sample membranes were collected and uniformly divided under aseptic conditions. One portion was used for cephalosporin extraction and chemical analysis, and the other portion was used for microbial DNA extraction and subsequent gene analysis. The divided membranes were then placed into sterile sealed bags, numbered, and stored in a low-temperature freezer.

[0055] After collecting the sample filter membranes, the membrane samples were further divided uniformly under aseptic conditions. One portion was used for cephalosporin extraction and chemical analysis, and the other portion was used for microbial DNA extraction and subsequent gene analysis. The first portion of the divided filter membrane was then placed into sterile sealed bags and numbered, and stored at -20°C. The second portion of the filter membrane was immediately placed into a sterile centrifuge tube containing a DNA protectant (such as TE buffer containing EDTA to inhibit nuclease activity) and frozen at -80°C to prevent DNA degradation.

[0056] S3-2, Sample Pretreatment for Chemical Analysis

[0057] Cut the filter membrane into small pieces with clean scissors and place them in a 50 mL centrifuge tube. Add 4.5 g sodium chloride, 10 mg ascorbic acid, and 100 mg Na2EDTA (disodium ethylenediaminetetraacetate). Add 40 mL of ultrapure water adjusted to pH 3 with formic acid. Add 100 µl of internal standard (cefotaxime-D3 concentration of 100 µg·mL). -1 Vortex for 10 min, sonicate at room temperature for 20 min (avoid high temperature water bath), and centrifuge at 3500 r·min for 15 min. -1 Collect the supernatant into a clean brown glass bottle. Repeat this process twice and combine the three supernatants.

[0058] S3-3, Solid-phase extraction of cephalosporins for chemical analysis

[0059] The HLB solid-phase extraction column was pre-activated with 5 mL of methanol, 5 mL of 0.1% formic acid acetonitrile, and 5 mL of ultrapure water. The sample flow rate was controlled at ≤2 drops / second through the extraction column, and the liquid level was kept above the top of the column packing throughout the process. The column was dried by vacuum pump for 30 min. Subsequently, it was eluted with 10 mL of 0.1% formic acid-acetonitrile solution, and the eluent was transferred to a 15 mL centrifuge tube and concentrated to near dryness under a gentle nitrogen flow. The solution was then diluted to volume with 500 μL of acetonitrile / water (1:1, V / V), filtered through a 0.22 μm organic phase filter membrane, and transferred to a sample vial. It was stored at -20 ℃ to obtain the sample extract for later use. The entire process was carried out in the dark.

[0060] S4. Batch testing and data analysis of chemical analysis

[0061] HPLC-MS / MS and internal standard method were used to detect the chemical components and analyze the data of the sample extract, and the detection results of the components and concentrations of cephalosporins in the sample extract were output for qualitative and quantitative monitoring of cephalosporin pollutant emissions.

[0062] Specifically, the steps include the following:

[0063] S4-1 Prepare testing instruments and reagents

[0064] HPLC-MS / MS was used for analysis. Mixed standard solutions with mass concentrations of 5.0, 10.0, 20.0, 50.0, 100.0, 200.0, 500.0, 1000.0, 2000.0, and 5000.0 ng / mL were prepared, and corresponding isotope-labeled mixtures were added as internal standards for quantification. The concentration of the isotope internal standards was 2000 ng / mL for all of them.

[0065] S4-2 Upper-level Inspection

[0066] Electrospray ionization (ESI) was used, with positive ion mode and multiple reaction monitoring (MRM) mode. The ion source temperature was 300℃. The instrument was stabilized before detection. All target analytes showed good linearity with a correlation coefficient (R) greater than 0.995.

[0067] The mobile phase consisted of 0.1% formic acid water (A) and 0.1% formic acid-acetonitrile (B).

[0068] A Shim-pack GIST C18-AQ HP (50 mm × 2.1 mm, 1.9 μm) column was used, with an injection volume of 2 μL and a flow rate of 300 μL / min.

[0069] S4-3 Data Analysis

[0070] Recovery rates were assessed: Good recoveries were achieved for 14 cephalosporins (cephalosporins, cefprozil, cefotiam, cefoxitin, cefradine, cefuroxime, cefixime, cefpodoxime proxetil, cefixime methyl ester, cefotaxime sodium, cefoperazone sodium, cefuroxime axetil, cefpirome, and cefnicotinic acid). The recoveries for matrix and blank spiked samples ranged from 33.9% to 169% and 38.6% to 121%, respectively, with RSD < 9.48%.

[0071] Limit of detection: The limit of detection is 0.06 pg / m³. 3 ~1.74 pg / m 3 ;

[0072] Output results: The analysis yields and outputs the detection data of the specific components and concentrations of cephalosporins in the sample extract.

[0073] Step S5: Co-monitoring of cephalosporin resistance genes

[0074] Microbial DNA was extracted from the sample extract and gene analysis was performed to obtain cephalosporin resistance gene data. This data was used for the synergistic monitoring of cephalosporin resistance genes. The cephalosporin concentration data (ng / m³) was combined with the abundance data of resistance genes (copies / m³) to assess the correlation between the two and the potential environmental and health risks. The specific steps included are as follows:

[0075] S5-1 Separation of particulate matter components (chemical and microbial separation to avoid mutual interference)

[0076] Because atmospheric particulate matter has a complex matrix (containing carbon black, heavy metals, and organic impurities), it is necessary to first separate "cephalosporins (chemical components)" from "microorganisms (biological components)" and then process them separately.

[0077] Particulate matter elution: Take the filter membrane for drug resistance gene detection, place it in a sterile centrifuge tube, add sterile phosphate buffer (PBS, pH 7.2), and use a combination of "ultrasonic elution + oscillation" (ultrasonic power 300W, time 10 minutes, avoid excessive sonication to avoid damaging microbial cells) to elute the particulate matter from the filter membrane to obtain a "microorganism-particulate matter mixed suspension".

[0078] Centrifugation: Centrifuge the mixed suspension at 4°C and 5000×g for 10 minutes. Transfer the supernatant (containing dissolved cephalosporins, which can be used to supplement chemical detection) to a new tube. The precipitate (containing microbial cells and particulate residue) is used for subsequent DNA extraction.

[0079] Residue purification: Add sterile physiological saline to the precipitate, gently blow it and centrifuge again (4℃, 3000×g, 5 minutes), discard the supernatant, repeat twice to remove residual particulate impurities (such as heavy metals, humic acid, to avoid inhibiting the activity of subsequent DNA extraction enzymes).

[0080] S5-2 Microbial DNA Extraction and Purification (High Extraction Yield, Removal of Inhibitors)

[0081] Atmospheric particulate matter contains low levels of microorganisms (usually ≤10). 4 For samples containing cells / m³ and DNA inhibitors (such as humic acid and metal ions), extraction methods suitable for "low-biomass environmental samples" should be selected, including:

[0082] Cell lysis (key: complete cell wall disruption and DNA release): Add sterile lysis buffer (containing lysozyme and proteinase K to lyse bacterial cell walls and degrade proteins, respectively) to the purified microbial pellet, and incubate at 37°C for 1 hour; then add SDS (sodium dodecyl sulfate) and incubate at 65°C for 30 minutes to further disrupt the cell membrane and ensure complete DNA release.

[0083] DNA Extraction and Purification: The extraction method uses a "column-based DNA extraction kit" (preferably an environmental sample kit such as the Qiagen PowerSoil® DNA Isolation Kit), which contains an "inhibitor removal column" that can efficiently adsorb impurities such as humic acid and heavy metals. The procedure is as follows: add ethanol to precipitate DNA according to the kit instructions, centrifuge to bind the DNA to the silica gel column, remove residual impurities with washing buffer, and finally wash the DNA with sterile, enzyme-free water to obtain purified total microbial DNA.

[0084] DNA quality and concentration testing: Purity testing was performed using Nanodrop to determine the A260 / A280 ratio (1.8-2.0 is acceptable, indicating low protein contamination) and the A260 / A230 ratio (≥2.0 is acceptable, indicating sufficient inhibitor removal). Concentration and integrity testing were performed using a Qubit real-time fluorescence analyzer to determine DNA concentration (≥10 ng / μL is required to meet the needs of subsequent gene analysis). DNA integrity was assessed using 1% agarose gel electrophoresis (the appearance of clear high molecular weight bands with no obvious degradation).

[0085] S5-3: Qualitative and quantitative analysis of cephalosporin resistance genes (targeted detection, matching resistance mechanisms)

[0086] Since cephalosporin resistance genes are mainly β-lactamase genes (such as blaTEM, blaCTX-M, and blaKPC, corresponding to different generations of cephalosporin resistance), it is necessary to combine "qualitative screening (to determine gene types)" with "quantitative analysis (to determine gene abundance)," specifically:

[0087] Qualitative analysis employed PCR amplification and gene identification, including:

[0088] Targeted primer design: Design specific primers for conserved regions of cephalosporin resistance genes (such as blaCTX-M gene primers: F-5'-ATGTGCAGYACCAGTAARGTKATGGC-3', R-5'-TTACAAACCGTCGGTGACGGT-3') to ensure coverage of major resistance genotypes.

[0089] Conventional PCR amplification: Using purified DNA as a template, add primers, Taq DNA polymerase, dNTPs, etc., and set the amplification program (94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 55-60℃ annealing for 30 seconds, 72℃ extension for 30 seconds, 35 cycles; 72℃ final extension for 10 minutes).

[0090] Results identification: The PCR products were subjected to agarose gel electrophoresis. If a band of the same size as the target gene fragment appeared (e.g., blaCTX-M was 593 bp), the presence of the drug resistance gene was preliminarily determined. The gene sequence was further verified by Sanger sequencing to confirm the genotype (to avoid false positives caused by non-specific primer binding).

[0091] Quantitative analysis uses real-time quantitative PCR (qPCR), including:

[0092] Construction of standard curve: Dilute a known concentration of drug resistance gene plasmid (such as a recombinant plasmid containing blaTEM) to 10¹⁰-10⁻⁶. 7 Gradient standards of copies / μL were amplified by qPCR. A standard curve was constructed with "Ct value" as the ordinate and "plasmid copy number log" as the abscissa (R² ≥ 0.99, amplification efficiency 90%-110%).

[0093] Sample quantification: Using purified DNA as a template, qPCR was performed using the SYBR Green or TaqMan probe method. The absolute copy number of the target drug resistance gene in the sample was calculated using a standard curve. Combined with the sampling volume, the result was converted to "gene copy number / m³ air" to reflect the environmental abundance of the drug resistance gene.

[0094] Quality control: Each batch of experiments includes a "blank control (enzyme-free water)," a "negative control (E. coli DNA without drug resistance genes)," and a "positive control (plasmids with known concentrations of drug resistance genes)" to avoid false positives / false negatives caused by contamination.

[0095] S5-4: Data Association and Environmental Health Risk Assessment (Collaborative Analysis, Establishing "Chemical-Gene" Associations)

[0096] The association and potential risks between cephalosporin concentration data (ng / m³) and resistance gene abundance data (copies / m³) were assessed by combining the two data.

[0097] Data correlation analysis includes:

[0098] Statistical analysis: Pearson correlation analysis or principal component analysis was used to determine the correlation between cephalosporin concentration and the abundance of resistance genes (e.g., whether the concentration of third-generation cephalosporins is positively correlated with the abundance of the blaCTX-M gene) to verify whether "environmental cephalosporin exposure drives the spread of resistance genes".

[0099] Spatial difference analysis: If monitoring is conducted at multiple sampling points, the "cephalosporin-drug resistance gene" combination characteristics of different areas (such as the area around hospitals, industrial areas, and residential areas) can be compared to identify high-risk areas.

[0100] Environmental health risk assessment includes:

[0101] Environmental risk assessment: Assess the potential for the spread of drug-resistant genes (e.g., when the abundance is ≥10³ copies / m³, it is necessary to be vigilant about the spread of drug-resistant genes to surrounding water bodies / soil through atmospheric particulate matter, causing cross-media transmission of drug-resistant genes).

[0102] Health risk assessment: Based on the amount of atmospheric particulate matter inhaled (approximately 10 m³ of air per day for adults), estimate the number of drug resistance gene copies inhaled by the human body daily; if there is long-term exposure to an environment with high abundance of drug resistance genes, it suggests that "drug resistance genes enter the human body through the respiratory tract, which may increase the potential risk of drug resistance in the human flora," and further verification with epidemiological data is necessary if needed.

[0103] The aforementioned method for detecting cephalosporins in atmospheric environmental media is applied to environmental and health risk assessment by combining cephalosporin concentration data (ng / m³) with drug resistance gene abundance data (copies / m³) to assess the correlation and potential risks between the two, including both environmental risk assessment and health risk assessment.

[0104] Example 1

[0105] See Figures 2-4The method and application for detecting cephalosporins in atmospheric environmental media provided in this embodiment are based on the basic embodiment, and specifically carry out chemical analysis of cephalosporins in atmospheric environmental media.

[0106] To overcome the problem that atmospheric particulate matter, as one of the important media for antibiotics in the environment, often has its occurrence characteristics overlooked, this embodiment establishes a standardized method system for the detection of 14 cephalosporins in atmospheric particulate matter samples. This system involves sample collection, pretreatment, and further qualitative and quantitative analysis using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). By optimizing the extraction solvent, a pretreatment and analysis method for simultaneously detecting 14 cephalosporins in atmospheric particulate matter filter membrane samples was established. The method is illustrated in detail using an example of a cephalosporin pharmaceutical company that continuously collected and analyzed atmospheric particulate matter samples for four days during its production process.

[0107] 1. Testing Materials and Methods

[0108] 1.1 Materials, Reagents and Instruments

[0109] Materials and reagents: 15mL and 50mL centrifuge tubes, sodium chloride (analytical grade, Shanghai Aladdin Biochemical Technology Co., Ltd.), L-ascorbic acid (analytical grade, Shanghai Maclean Biotechnology Co., Ltd.), disodium ethylenediaminetetraacetate (analytical grade, Shanghai Maclean Biotechnology Co., Ltd.), chromatographic grade formic acid (HCOOH, Shanghai Anpu Experimental Technology Co., Ltd.), acetonitrile (ACN, chromatographic grade, Shanghai Maclean Biotechnology Co., Ltd.), dichloromethane (DCM, chromatographic grade) and isooctane (ISO, chromatographic grade), CoPurify OMNI-HLB (6 mL, 200 mg; Waters), quartz fiber filter membrane (20.3×25.4cm, EPM 2000, 136 Whatman, UK).

[0110] Instruments: Shimadzu high-performance liquid chromatograph, AB SCIEX ZenoTOF 7600 triple quadrupole mass spectrometer (ABSCIEX, USA), Shim-pack GIST C18-AQ HP (50 mm*2.1 mm, 1.9 μm) (Shimadzu, Japan), analytical balance (Shanghai Anting Electronic Instrument Factory, China), vortex mixer (Scientific Industries, USA), L535-1 centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd., China), nitrogen blower (Organomation, USA), KQ-500DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., China), 0.22-μm organic phase (nylon) needle filter membrane (Tianjin Jinteng Experimental Equipment, China), solid-phase extraction device, vacuum pump, intelligent high-flow-rate atmospheric particulate matter sampler (1m). 3 ·min -1 (YI-1000, Qingdao Jingcheng, China).

[0111] 1.2 Standard Products

[0112] Cephalexin, cefprozil, cefotiam, cefoxitin, cefradine, cefuroxime, cefixime, cefpodoxime proxetil, cefixime methyl ester, cefotaxime sodium, cefoperazone sodium, cefuroxime axetil, cefpirome, cefnicillin, and the internal standard cefotaxime-d3 were all purchased from Tianjin Alta Technology Co., Ltd. Detailed information is shown in Table 1 (Information Table of Target Compounds and Internal Standards).

[0113] Table 1

[0114] Table 1 The information of target chemicals and internal standards

[0115]

[0116] Note: a Sourced from Pub Chem. Note: a obtained from PubChem

[0117] 1.3 Sample Collection and Preprocessing

[0118] 1.3.1 Sample Collection

[0119] Atmospheric particulate matter samples were collected near a wastewater treatment plant at locations S1 (50 m from the wastewater treatment plant), S2 (5 m from the wastewater treatment plant), and S3 (near the wastewater treatment plant's equalization tank) during cephalosporin production at a pharmaceutical company in Guangzhou. A high-flow-rate particulate matter sampler and a quartz fiber filter membrane were used for sampling, with a sampling flow rate of 1 m³ / s. 3 ·min -1 The sampling time was 24 hours, and the samples were collected continuously for 4 days. The sampling rack was 1.5 m above the ground, wrapped in aluminum foil, and then baked in a muffle oven at 450°C for 4 hours.

[0120] During sampling, the temperature, air pressure and other meteorological conditions were recorded. The collected filter membranes were placed in sterile sealed bags and numbered, and stored in a refrigerator (-20℃).

[0121] 1.3.2 Sample Pretreatment

[0122] Cut the filter membrane into small pieces with clean scissors and place them in a 50 mL centrifuge tube. Simultaneously add 4.5 g of pre-weighed sodium chloride, 10 mg of ascorbic acid, and 100 mg of Na2EDTA (disodium ethylenediaminetetraacetate). Add 40 mL of ultrapure water adjusted to pH 3 with formic acid. Add 100 µL of internal standard (cefotaxime-d3 concentration of 100 µg·mL). -1 Vortex for 10 min, sonicate at room temperature for 20 min (avoid high temperature water bath), and centrifuge at 3500 r·min for 15 min. -1 Collect the supernatant into a clean brown glass bottle. Repeat this process twice and combine the supernatants from all three times.

[0123] The HLB solid-phase extraction column was pre-activated with 5 mL of methanol, 5 mL of 0.1% formic acid-acetonitrile, and 5 mL of ultrapure water. The sample flow rate was controlled to ≤2 drops / second through the extraction column, and the liquid level was maintained above the top of the column packing throughout the process. The column was dried under vacuum for 30 min. Subsequently, it was eluted with 10 mL of 0.1% formic acid-acetonitrile solution, and the eluent was transferred to a 15 mL centrifuge tube and concentrated to near dryness under a gentle nitrogen flow. The eluent was then diluted with 500 μL of acetonitrile / water (1:1). V / V After bringing the volume to a final depth, filter through a 0.22 μm organic phase membrane, transfer the solution to a sample vial, and store at -20 ℃ for analysis. The entire process should be conducted in the dark.

[0124] 1.4 Instrumental Analysis

[0125] HPLC-MS / MS was used for analysis. The mobile phase was 0.1% formic acid in water (A) and 0.1% formic acid-acetonitrile (B). The elution gradient was as follows: 0–0.5 min: 95% A, 0.5–5 min: 95%–40% A, 5–13 min: 40%–2% A, 13–17 min: 2% A, 17–17.1 min: 0–95% A, 17.1–20 min: 95% A. A Shim-pack GIST C18-AQ HP column (50 mm × 2.1 mm, 1.9 μm) was used, with an injection volume of 2 μL and a flow rate of 300 μL·min. -1 Electrospray ionization (ESI) was used in positive ion mode. Multiple reaction monitoring (MRM) was employed with an ion source temperature of 300 °C. The parent ion, daughter ion, collision energy (CE), and declustering voltage (DP) are shown in Table 2 (MRM analysis parameters for all target compounds).

[0126] Table 2

[0127]

[0128] Note: a For quantitative ions, b These are qualitative ions. Note: a It is a quantitative ion. b is a qualitative ion

[0129] 1.5 Quality Assurance and Quality Control (QA / QC)

[0130] All glassware used was rinsed with tap water and ultrapure water and dried in an oven before use, then calcined in a muffle furnace at 400 °C for 4 h, and rinsed and dried with dichloromethane and isooctane before use. During sample testing, acetonitrile was injected as a solvent blank for every 10 samples.

[0131] The limit of quantification (LOQ) and limit of detection (LOD) are the mean concentration of the target compound in the blank sample plus 10 or 3 times the standard deviation. If the target compound is not detected in the blank, the limit of detection is defined as 10 or 3 times the signal-to-noise ratio. Three blank and three matrix samples, three low-spiked blank samples, three low-spiked matrix samples, and three high-spiked blank samples and three high-spiked matrix samples were prepared. Samples were pretreated according to the method in 1.3.2. The target compound content was then detected using the instrument. The internal standard method was used for quantification. The recovery rate and relative standard deviation were calculated by subtracting the average concentration of the blank and matrix from the spiked concentrations of the blank and matrix samples.

[0132] 2. Comparative verification and effect analysis

[0133] 2.1 Selection of Extraction Solvent

[0134] The extraction solvents designed for cephalosporins include acetonitrile-phosphate buffer solution, acetonitrile-citric acid buffer solution, and methanol-acetonitrile (1:3). V / V ), methanol-acetone (1:2, V / V Multiple extraction methods were employed, including pure water. This embodiment compared and verified the effectiveness of three extraction solvents for cephalosporin extraction from the sample: Scheme M1: 0.1% formic acid acetonitrile; Scheme M2: ultrapure water; Scheme M3: ultrapure water with pH adjusted to 3 by formic acid. Ascorbic acid and Na2EDTA were added to each scheme. Ascorbic acid, as a reducing agent, prevents the antibiotic from being oxidized and degraded, while Na2EDTA, as a stabilizer, prevents the complexation of antibiotics with metal ions. The recovery rates of the three schemes are shown below. Figure 2 As shown.

[0135] First, using scheme M1, ultrasonic extraction was performed according to method 1.3.2. The extracts were then combined and diluted with ultrapure water to 200 mL. The sample solution was then pumped at a rate of 1 mL / min. -1HLB column extraction was performed. After treatment, the recoveries of the target analytes in the matrix and in the blank spike ranged from 13.1% to 154% and 0.19% to 100%, respectively, with an RSD of <9.36%. The recoveries of some target analytes were below 40%, indicating significant losses. This may be because the high proportion of organic solvent in the sample solution affected the binding ability of the target analytes on the HLB column, causing them to pass through directly and fail to be enriched. In scheme M2, pure water was used as the extraction solvent. After treatment, the recoveries of the target analytes in the matrix and in the blank spike ranged from 6.23% to 116% and 5.00% to 79.6%, respectively, with an RSD of <17.3%. The overall recovery rate of the target analytes was significantly improved compared to the previous method, but the recovery rate of some target analytes was still below 40%. The ionic strength of the sample solution affects the solid-phase extraction effect. In addition, the pH value of the sample solution can also change the ionization or protonation degree of the target analytes and adsorbents, affecting the solid-phase extraction effect and thus the recovery rate of the target compounds. Studies have shown that the optimal recovery rate of β-lactam drugs is achieved when 4.5 g of sodium chloride is added to the solution and the solution pH is 3. To obtain better spiked recovery rates, this embodiment uses scheme M3, which involves first cutting the filter membrane into small pieces and placing it in a centrifuge tube. Then, 10 mg of pre-weighed ascorbic acid, 100 mg (Na2EDTA), and 4.5 g of sodium chloride are simultaneously added to the centrifuge tube, followed by ultrapure water adjusted to pH 3 with formic acid as the extraction solvent. After the above treatment, the recoveries of the target analytes in the matrix and in the blank were 33.9%–169% and 38.6%–121%, respectively, with relative standard deviations (RSD) of less than 9.48%. In scheme M3, the recovery rates of some target analytes were still relatively low, such as cefixime (recovery rate between 33.9% and 48.0%), possibly because the β-lactam ring in its structure may still undergo slow hydrolysis or ring-opening degradation during extraction and concentration steps, and its Log Kow= -0.7, exhibiting strong polarity and hydrophilicity, may result in very weak affinity for the hydrophobic portion of the HLB packing material, leading to partial breakthrough loss. In scheme M3, the recovery rate of cefotaxime was high and varied (96.7%–169%), which may be attributed to the following two reasons: 1) its Log... Kow= 0.5, exhibiting a certain lipophilic tendency. In Scheme M3, NaCl significantly increased the ionic strength of the extract, reducing the solubility of cefotaxime in the aqueous phase and greatly enhancing its affinity for the hydrophobic portion of the HLB packing material, thus ensuring strong and stable retention during the loading step; 2) The low and high spiked recoveries of cefotaxime both showed matrix > blank, indicating the presence of co-extractants in the matrix that enhance the ionization efficiency of cefotaxime, resulting in a stronger signal response during detection. Therefore, the extraction solvent used in Scheme M3 can achieve good recoveries for the 14 target compounds in this example, with a relatively wide recovery range.

[0136] 2.2 Methodological Validation

[0137] 2.2.1 Standard curve and limit of detection

[0138] Prepare solutions with mass concentrations of 5.0, 10.0, 20.0, 50.0, 100.0, 200.0, 500.0, 1000.0, 2000.0, and 5000.0 ng·mL. -1 A mixed standard solution was prepared, and corresponding isotope-labeled mixed solutions were added as quantitative internal standards. The concentration of each isotope internal standard was 2000 ng·mL. -1 The instrument was tested under stable conditions. All target objects showed a good linear relationship, with a correlation coefficient ( ). R The limit of detection (LOD) is greater than 0.995. The LOD for each compound was calculated according to the method in section 1.5; the LOD for the target analyte ranged from 0.10 to 4.60 pg·m⁻¹. -3 For details, please refer to Table 3 (correlation coefficients of target compounds ( ) R ), Limit of Quantitation (LOQ) and Limit of Detection (LOD) (pg·m -3 )).

[0139] Table 3

[0140]

[0141] 2.2.2 Recovery rate and precision

[0142] Recovery rates were determined by adding target analyte standards (low spike: 1 µg; high spike: 100 µg) to blank and matrix samples, with three replicates for each group. The spiked recoveries and internal standard chromatograms of the target analyte are shown in Tables 4 and 5, respectively. Figure 3 As shown in Table 4, the recoveries of the target compounds in the blank samples ranged from 38.6% to 121%, with RSDs ranging from 0.07% to 9.36%; the recoveries of the target compounds in the matrix samples ranged from 33.9% to 169%, with RSDs ranging from 0.09% to 9.48% (Table 4, Results of Target Compound Spike Recovery). The results indicate that the pretreatment method meets the detection requirements.

[0143] Table 4

[0144]

[0145] 2.3 Analysis of Atmospheric Particulate Matter Samples

[0146] The preprocessing method established in this paper was used to detect cephalosporin content in atmospheric particulate matter collected within the factory area. Three cephalosporins were detected in samples collected from three sampling sites, with total content ranging from 292 to 469 pg·m³. -3 Between, the median value was 370 pg·m -3The maximum total pollutant concentration was higher than that in Beijing's urban area (∑Antibiotics: 0.9–276.3 pg·m³). -3 Median: 27.8 pg·m -3 The levels were lower than those in the urban area of ​​Shijiazhuang (∑Antibiotics: 2.8–774.7 pg·m). -3 Median: 41.3 pg·m -3 The compounds with the highest detection rates were cephalexin and cefadroxil, with a detection rate of 100% and detection concentrations of 9.09–61.1 pg·m⁻¹, respectively. -3 (Median: 38.5 pg·m) -3 ), 209-408 pg·m -3 (Median: 315 pg·m) -3 The second most common antibiotic was cefuroxime, with a detection rate of 66.7% and a detection concentration of nd—8.18 pg·m³. -3 (Median: 2.69 pg·m) -3 The compound detected at the highest concentration was cefadroxil (209–408 pg·m). -3 ).

[0147] The detection of cephalosporin spectra in the samples was consistent with the products manufactured by the company, which can be attributed to continuous pharmaceutical activities during the sampling period: on the one hand, process exhaust gas emissions; on the other hand, pharmaceutical and rinsing wastewater from the workshop flowed into an open-air wastewater treatment unit, where water droplets carrying cephalosporins were dispersed into the atmosphere via aerosols and collected. Figure 3 (Left) It can be seen that the total detected concentrations show a pattern of S2 > S1 > S3. Figure 3 (Right) This shows that there was no statistically significant difference in the total concentration of pollutants detected among S1, S2, and S3, indicating that the sampling location (S1 / S2 / S3) was not a significant factor affecting the pollutant concentration. This phenomenon is consistent with... McEachran Similar results indicate that particulate matter in open-air environments disperses unevenly under the influence of wind energy, resulting in no significant decreasing or increasing trend between different locations. From Figure 3(The middle section shows that trace amounts of cefuroxime were detected in the first day's samples, but production of this type of drug had ceased on the day of sampling, possibly due to production occurring two days prior. Given that cefuroxime has a photodegradation half-life of only 2.8 days, it indicates that it can remain in environmental particulate matter for a short period. Only cefadroxil and cefalexin were detected on the second day, consistent with the drugs produced by the pharmaceutical company that day. The half-lives of cefadroxil under dark and light conditions were 6.6 days and 5 days, respectively, while cefalexin relied on a specific chemical degradation mechanism. Furthermore, photodegradation was negligible for both cefadroxil and cefalexin, indicating that cefadroxil and cefalexin released into the ambient air are not degraded by light and can remain in atmospheric particulate matter. In addition, this study found that the detection concentration of cefadroxil was higher than that of cefalexin, which may be related to the difference in degradation efficiency of the two compounds in the environment and the different pollutant contents in the emission sources.) No pollutants were detected at any of the sampling sites on the third day. This is likely because of continuous heavy rainfall during the sampling period, which may have cleared or diluted the concentration of atmospheric particulate matter, thus reducing the amount of pollutants that could be collected. Simultaneously, the increased moisture could also promote microbial activity, thereby accelerating the degradation of antibiotics through microbial metabolism or hydrolysis.

[47] Preliminary monitoring results indicate that although cephalosporins have a short half-life of only 2-6 days, wastewater treatment plants at pharmaceutical factories can still serve as a source of pollution, potentially leading to continuous exposure of airborne microorganisms and workers to cephalosporins, resulting in drug resistance or other environmental health risks. The specific test results in this embodiment only reflect the actual situation of samples collected within the study area, and the results may differ from those of samples collected at other times due to changes in weather conditions (such as unstable weather) or differences in enterprise production emissions.

[0148] The above embodiments of the present invention have at least the following advantages:

[0149] 1. Good recovery rate was achieved, with good recovery rates for all 14 cephalosporins. The recoveries for matrix and blank spiked samples ranged from 33.9% to 169% and 38.6% to 121%, respectively, with RSD < 9.48%.

[0150] 2. The detection limit is low, and the actual detection limit is low (0.06 pg / m3—1.74 pg / m3), which is lower than the 0.02 μg / m3 required in T / PIAC 00002-2021 "Detection Method of Cephalosporins in Antibiotic Residue, Organic Fertilizer Base Material, Crops and Environmental Media".

[0151] 3. Do not dilute the extraction solvent to shorten the experimental time.

[0152] The time efficiency advantage of the detection method in this embodiment is mainly aimed at the pretreatment process of similar environmental particulate matter samples (such as sediments, soil, and sludge), especially in scenarios involving large-scale sample analysis. In previous studies, organic solvents (such as acetonitrile) were often used to extract cephalosporins from particulate matter. Since a high proportion of organic solvent can affect the retention capacity of the target analyte on an HLB column, the extract usually needs to be concentrated and then diluted. This operation not only prolongs the pretreatment time but also often involves heating during rotary evaporation concentration, and cephalosporin compounds generally have poor thermal stability, which may lead to degradation or loss of the target analyte during concentration. In contrast, the detection method in this embodiment uses ultrapure water adjusted to pH = 3 with formic acid as the extraction solvent, avoiding the concentration-dilution step required after the introduction of organic solvents, thus significantly simplifying the operation process. In large-scale sample processing, this embodiment can effectively save the additional time spent on organic solvent treatment. In practical applications, the overall time for pretreatment (including extraction, HLB solid-phase extraction, and nitrogen blowing concentration) in this embodiment is approximately 3-4 hours.

[0153] 4. Using formic acid to maintain pH is simple to operate and low in cost.

[0154] Existing pretreatment methods often use buffer solutions (such as phosphate buffer, citrate buffer, borate buffer, Na2EDTA-Mcllvaine standard buffer, etc.) to assist in the extraction of cephalosporins from particulate matter (such as sediment, soil, sludge). In this embodiment, formic acid used to maintain pH is a single reagent, eliminating the need for precise preparation of acid-salt buffer pairs (such as sodium dihydrogen phosphate-disodium hydrogen phosphate) as with buffer solutions, simplifying the preparation process. Furthermore, formic acid is less expensive than some specialized buffer solutions (such as Mcllvaine buffer, which requires a mixture of citrate and disodium hydrogen phosphate) and is easy to store (stable at room temperature), making it suitable for large-scale sample processing. Additionally, while some pretreatment methods do not use buffer solutions, they limit the number of target analytes they can detect (typically 1–3). This embodiment, however, simultaneously detects 14 cephalosporins (with a focus on 4 of them), covering a broader range of target analytes and highlighting key areas, thus overcoming the limitations of existing technologies.

[0155] 5. Using acetonitrile as the elution solvent can reduce degradation during the concentration step of β-lactam compounds.

[0156] Existing technologies primarily limit the sample range to environmental particulate matter (such as soil, sediment, sludge, etc.). For cephalosporin drugs in such media, methanol or formic acid-containing methanol solutions are the main eluents. However, under a mild nitrogen flow, methanol in the presence of the cephalosporin may degrade β-lactams. The β-lactam ring is highly susceptible to nucleophilic attack, leading to ring-opening reactions. Methanol, a polar organic solvent with strong nucleophilicity, will act as a nucleophile to attack the carbonyl carbon of the β-lactam ring, resulting in a nucleophilic addition-elimination reaction. In this embodiment, acetonitrile is used as the eluent. Acetonitrile's nucleophilicity is much weaker than methanol, making it difficult to initiate β-lactam ring opening. Acetonitrile is a more suitable solvent for handling β-lactam compounds in the concentration step. Furthermore, the addition of a certain amount of formic acid to acetonitrile improves the stability of cephalosporins.

[0157] Therefore, this invention provides a complete standardized chemical analysis and detection scheme, focusing on atmospheric particulate matter—a special and often overlooked antibiotic-bearing medium. It designs pretreatment and analytical methods applicable to 14 cephalosporins, as well as monitoring methods targeting four of them. This embodiment uses ultrapure water adjusted to pH 3 with formic acid as the extraction solvent, combining ultrasonic extraction and HLB solid-phase extraction purification. This eliminates the cumbersome concentration-dilution steps required after organic solvent extraction in previous studies, effectively saving pretreatment time. Method validation shows that the 14 target compounds achieved good recoveries and low limits of detection, greatly improving the detection capability of trace cephalosporins in atmospheric particulate matter. Applied to the analysis of atmospheric particulate matter samples from around pharmaceutical companies, it successfully detected cefadroxil, cephalexin, and cefuroxime, with total concentrations ranging from 292 to 469 pg·m³. -3 The practicality and stability of the method were verified. The detection method in this embodiment is highly targeted and practical for the detection of trace cephalosporins in atmospheric particulate matter, and can provide a scientific reference for regional cephalosporin pollution tracing and environmental risk assessment.

[0158] Example 2

[0159] The method and application for detecting cephalosporins in atmospheric environmental media provided in this embodiment are based on the basic embodiment and Example 1, and further specifically involve gene analysis of cephalosporins in atmospheric environmental media. This embodiment uses high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) technology to detect cephalosporin antibiotics in atmospheric particulate matter, and simultaneously employs molecular biology methods (PCR / qPCR) to detect related drug resistance genes, achieving synergistic monitoring of chemical pollutants and microbial drug resistance.

[0160] The overall experimental procedure is as follows: sample collection → sample preservation → particulate elution → component separation → cephalosporin detection → microbial DNA extraction → drug resistance gene PCR detection → drug resistance gene qPCR quantification → data correlation analysis.

[0161] I. Main Instruments and Equipment

[0162] 1.1 Sampling equipment

[0163] Atmospheric particulate matter sampler: TH-150CIII medium-flow atmospheric sampler (Wuhan Tianhong Instrument Co., Ltd.)

[0164] PM2.5 cutter: For use with TH-150CIII

[0165] Sampling pump: Flow rate range 50-150 L / min, accuracy ±2%

[0166] 1.2 Laboratory Equipment

[0167] Ultra-high performance liquid chromatography-tandem mass spectrometry: Agilent 1290 UPLC + 6460 Triple QuadMS (Agilent Technologies)

[0168] Real-time quantitative PCR instrument: Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific)

[0169] Standard PCR instrument: Applied Biosystems Veriti 96-Well Thermal Cycler

[0170] High-speed refrigerated centrifuge: Eppendorf 5810R (cools down to 4°C, maximum speed 15,000 x g)

[0171] Ultrasonic Cleaner: KQ-500DE CNC Ultrasonic Cleaner (Kunshan Ultrasonic Instrument Co., Ltd.)

[0172] Ultrapure water system: Milli-Q Integral 3 (Merck Milli-Q)

[0173] Aseptic Clean Bench: SW-CJ-2FD Model (Suzhou Purification Equipment Co., Ltd.)

[0174] Incubator: DHP-9052 (Shanghai Yiheng Scientific Instruments Co., Ltd.)

[0175] Vortex Oscillator: Vortex-Genie 2 (Scientific Industries)

[0176] Nitrogen Evaporator: TurboVap LV (Caliper Life Sciences)

[0177] II. Reagents and Materials

[0178] 2.1 The cephalosporin standards and chemical reagents used were the same as those in the examples, and were all commercially available products.

[0179] 2.2 DNA Extraction and PCR Reagents

[0180] DNA Extraction Kit: PowerSoil DNA Isolation Kit (Qiagen, catalog number 12888-100)

[0181] PCR Kit: TaKaRa Taq™ DNA Polymerase (Takara Bio Inc., Catalog No. R001A)

[0182] qPCR kit: TaKaRa TB Green® Premix Ex Taq™ II (Takara Bio Inc., catalog number RR820A)

[0183] DNA Marker: TaKaRa DL2000 DNA Marker (Takara Bio Inc., Product No. D501A)

[0184] Primers: Synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0185] Sterile water: Nuclease-free water, Sigma-Aldrich, catalog number W4502

[0186] 2.3 Consumables

[0187] Quartz fiber filter membrane: Whatman QMA, diameter 47mm, part number 1851-047

[0188] Solid phase extraction column: Oasis HLB (6cc / 200mg, Waters, catalog number 186000115)

[0189] Sterile centrifuge tubes: 15mL and 50mL Falcon centrifuge tubes (Corning)

[0190] Sterile microcentrifuge tubes: 1.5mL and 2.0mL Eppendorf centrifuge tubes

[0191] PCR reaction tube: 0.2mL PCR tube (Axygen)

[0192] qPCR reaction plate: 96-well optical reaction plate (Applied Biosystems)

[0193] Disposable sterile syringe: 10mL (BD)

[0194] Needle-type filter: 0.22μm nylon membrane (Millipore)

[0195] III. Detailed Experimental Procedure

[0196] 3.1 Sample Collection and Preservation

[0197] 3.1.1 Preparations before sampling

[0198] a. Membrane pretreatment:

[0199] The quartz fiber filter membrane was calcined in a muffle furnace at 450°C for 4 hours to remove organic impurities.

[0200] Cool to room temperature in a sterile laminar flow hood.

[0201] Use sterile forceps to place the filter membrane into a sterile petri dish and seal it for preservation.

[0202] b. Sampler calibration:

[0203] The sampling pump flow rate was calibrated using a soap film flow meter before sampling.

[0204] The sampling flow rate is set to 1000 L / min.

[0205] 3.1.2 Sample Collection

[0206] In a sterile laminar flow hood, sterile forceps are used to install the pretreated filter membrane into the sampler.

[0207] The sampling time is set to 24 hours, ensuring a sampling volume ≥ 860 m³.

[0208] Maintain the sampler temperature at 4-8℃ during the sampling process.

[0209] After sampling, carefully remove the filter membrane with sterile forceps to avoid contamination.

[0210] Cut the filter membrane in half along the center line:

[0211] a. Half is used for cephalosporin testing; place it in a clean aluminum foil bag.

[0212] b. The other half is used for drug resistance gene detection and is placed in a sterile centrifuge tube containing 5 mL of DNA protection solution (0.05 M EDTA, pH 8.0).

[0213] 3.1.3 Sample Preservation

[0214] Cephalosporin test samples: Store at -20℃ protected from light, and complete analysis within 2 weeks.

[0215] Antimicrobial resistance gene testing samples: Store at -80℃ and complete analysis within 1 month.

[0216] 3.2 Particulate matter elution and component separation

[0217] 3.2.1 Particulate matter elution

[0218] Remove the filter membrane sample for drug resistance gene detection from the -80℃ freezer.

[0219] Thaw at room temperature, then vortex for 5 minutes.

[0220] Transfer the suspension to a 50 mL sterile centrifuge tube.

[0221] Add 5 mL of sterile PBS buffer (pH 7.2) to the original centrifuge tube and vortex for 2 minutes.

[0222] Combine the two eluents, the total volume is approximately 10 mL.

[0223] 3.2.2 Component Separation

[0224] Centrifuge the eluent at 4°C and 5,000 × g for 10 minutes.

[0225] Carefully aspirate the supernatant into a new 50 mL centrifuge tube (for supplementing cephalosporin testing).

[0226] Add 10 mL of sterile physiological saline to the precipitate and gently resuspend by pipetting.

[0227] Centrifuge at 4℃ and 3,000×g for 5 minutes, then discard the supernatant.

[0228] Repeat steps 3-4 twice to remove residual impurities.

[0229] The final precipitate is used for microbial DNA extraction.

[0230] 3.3 Cephalosporin Detection

[0231] 3.3.1 Sample Preprocessing

[0232] A. Sample extraction for cephalosporin testing:

[0233] Take the cephalosporin detection filter membrane out of the -20°C freezer.

[0234] Place the filter membrane into a 50 mL centrifuge tube and add 10 mL of a methanol-water mixture (1:1, v / v).

[0235] Ultrasonic extraction for 30 minutes (300W power, 20-25℃ temperature).

[0236] Centrifuge at 4℃, 8,000×g for 10 minutes, and collect the supernatant.

[0237] Repeat the extraction twice and combine the supernatants.

[0238] B. Solid-phase extraction purification:

[0239] Activation of Oasis HLB solid-phase extraction column: Activate sequentially with 5 mL of methanol and 5 mL of ultrapure water.

[0240] Sample loading: Pass the extract through the solid-phase extraction column at a flow rate of 1 mL / min.

[0241] Eluting: Elute the column with 5 mL of 5% methanol aqueous solution, then discard the eluent.

[0242] Elution: Elute the target compound with 10 mL of methanol and collect the eluent.

[0243] Concentration: Eluent was purged with nitrogen at 40°C until nearly dry, and then reconstituted with 1 mL of mobile phase.

[0244] Filtration: Filtered through a 0.22μm nylon membrane, to be tested

[0245] 3.3.2 HPLC-MS / MS detection

[0246] A. Chromatographic conditions:

[0247] Chromatographic column: Agilent ZORBAX Eclipse Plus C18 column (2.1×150mm, 1.8μm)

[0248] Column temperature: 40℃

[0249] Mobile phase: Phase A (0.1% formic acid aqueous solution), Phase B (acetonitrile)

[0250] Gradient elution procedure:

[0251] 0-2min: 5% B

[0252] 2-10 min: 5%-30% B

[0253] 10-12 min: 30%-95% B

[0254] 12-15 min: 95% B

[0255] 15-16 min: 95%-5% B

[0256] 16-20 min: 5% B

[0257] Flow rate: 0.3 mL / min

[0258] Injection volume: 5 μL

[0259] B. Mass spectrometry conditions:

[0260] Ion source: Electrospray ionization source (ESI+)

[0261] Scanning mode: Multiple Response Monitoring (MRM)

[0262] Drying gas temperature: 350℃

[0263] Drying airflow rate: 10L / min

[0264] Atomizer pressure: 45psi

[0265] Capillary voltage: 4000V

[0266] MRM parameters for each compound are shown in Appendix B.

[0267] C. Plotting the standard curve:

[0268] Prepare mixed standard solutions with concentrations of 0.1, 0.5, 1, 5, 10, 50, and 100 ng / mL.

[0269] Inject and analyze the sample under the chromatographic and mass spectrometric conditions described above.

[0270] Plot a standard curve with peak area on the ordinate and concentration on the abscissa.

[0271] 3.4 Microbial DNA Extraction and Purification

[0272] 3.4.1 Cell lysis

[0273] Resuspend the microbial precipitate obtained in step 5.2.2 in 1 mL of sterile physiological saline.

[0274] Transfer to a 2mL sterile centrifuge tube and centrifuge at 4°C and 12,000×g for 5 minutes.

[0275] Discard the supernatant, add 60 μL of sterile deionized water and 30 μL of lysozyme solution (10 mg / mL) to the precipitate, vortex to mix, and incubate at 37°C for 30 minutes. Add 30 μL of proteinase K solution (20 mg / mL) and 150 μL of PowerSoil buffer C1, vortex to mix, and incubate at 65°C for 30 minutes, vortexing once every 10 minutes during this period.

[0276] 3.4.2 DNA extraction and purification (follow the instructions for the PowerSoil DNA Isolation Kit)

[0277] 1. Add 250 μL of PowerSoil buffer C2 and vortex for 10 seconds.

[0278] Centrifuge at 2.4℃, 10,000×g for 1 minute, then transfer the supernatant to a new tube.

[0279] 3. Add 200 μL of PowerSoil buffer C3 and vortex for 10 seconds.

[0280] Centrifuge at 4.4℃ and 10,000×g for 1 minute, then transfer the supernatant to a new tube.

[0281] 5. Add 1.2 mL of PowerSoil buffer C4 and vortex to mix.

[0282] 6. Transfer the mixture to a Spin Filter and centrifuge at 6,000 × g for 1 minute.

[0283] 7. Discard the filtrate, add 500 μL of PowerSoil buffer C5, and centrifuge at 6,000 × g for 1 minute.

[0284] 8. Repeat step 7 once.

[0285] 9. Centrifuge empty tubes at 10,000×g for 2 minutes to remove residual buffer solution.

[0286] 10. Transfer the Spin Filter to a new collection tube and add 100 μL of sterile deionized water.

[0287] 11. Let stand at room temperature for 5 minutes, then centrifuge at 10,000 × g for 1 minute.

[0288] 12. Collect the eluent, which is the purified microbial DNA.

[0289] 3.4.3 DNA Quality Testing

[0290] 1. Concentration detection: Use Nanodrop to determine DNA concentration, ensuring a concentration ≥10 ng / μL.

[0291] 2. Purity testing: Determine the A260 / A280 ratio (1.8-2.0 is acceptable) and the A260 / A230 ratio (≥2.0 is acceptable).

[0292] 3. Integrity testing: 1% agarose gel electrophoresis was performed to test DNA integrity.

[0293] 3.5 Drug resistance gene detection

[0294] 3.5.1 Qualitative PCR Detection

[0295] A. PCR reaction system (25 μL):

[0296] 1.10×PCR Buffer: 2.5μL

[0297] 2.dNTP Mixture (2.5mM each): 2μL

[0298] 3. Upstream primer (10 μM): 1 μL

[0299] 4. Downstream primer (10μM): 1μL

[0300] 5.Taq DNA Polymerase (5U / μL): 0.2μL

[0301] 6. DNA template: 2μL

[0302] 7. Sterile water: 16.3 μL

[0303] B. PCR reaction procedure:

[0304] Pre-denaturation at 8.94℃ for 5 minutes

[0305] 9.35 cycles:

[0306] Denaturation at 1.94℃: 30 seconds

[0307] 2. Annealing: 30 seconds (see Appendix A for annealing temperature)

[0308] 3.72℃ extension: 30 seconds

[0309] Final extension at 10.72℃: 10 minutes

[0310] Insulation at 11.4℃: ∞

[0311] C. PCR product detection:

[0312] 12. Preparation of 1.5% agarose gel

[0313] 13. Mix 5 μL of PCR product with 1 μL of Loading Buffer.

[0314] 14. Electrophoretic analysis, voltage 120V, time 30 minutes

[0315] 15. Observation results of the ultraviolet gel imaging system

[0316] 3.5.2 qPCR Quantitative Detection

[0317] A. qPCR reaction system (20 μL):

[0318] 16.TB Green Premix Ex Taq II: 10μL

[0319] 17. Upstream primer (10 μM): 0.8 μL

[0320] 18. Downstream primer (10 μM): 0.8 μL

[0321] 19.ROX Reference Dye II: 0.4μL

[0322] 20. DNA template: 2μL

[0323] 21. Sterile water: 6 μL

[0324] B. qPCR reaction procedure:

[0325] Pre-denaturation at 22.95℃ for 30 seconds

[0326] 23.40 cycles:

[0327] Denaturation at 1.95℃: 5 seconds

[0328] 2. Annealing: 34 seconds (see Appendix A for annealing temperature)

[0329] 24. Melting curve analysis:

[0330] 1.95℃: 15 seconds

[0331] 2.60℃: 1 minute

[0332] 3.95℃: 15 seconds

[0333] C. Construction of the standard curve:

[0334] 25. Perform 10-fold serial dilutions (10¹-10⁻¹⁰) on recombinant plasmids of known concentrations. 7 copies / μL)

[0335] 26. Perform 3 replicates for each dilution.

[0336] 27. Plot a standard curve with Ct values ​​on the ordinate and the logarithm of plasmid copy number on the abscissa.

[0337] 4. Data Processing and Analysis

[0338] 4.1 Calculation of cephalosporin concentration

[0339] A. Calculation formula for WEI:

[0340] C = (C1 × V1 × V2) / V

[0341] In the formula:

[0342] 1.C: Atmospheric cephalosporin concentration (ng / m³)

[0343] 2.C1: Detected concentration (ng / mL)

[0344] 3. V1: Reconstituted volume (mL)

[0345] 4. V2: Total volume of extract / Volume of eluent

[0346] 5. V: Sampling volume (m³)

[0347] B. Data Correction: Correct the detection results based on the matrix spike recovery rate.

[0348] 4.2 Calculation of drug resistance gene abundance

[0349] A. Gene copy number calculation:

[0350] Gene copy number = 10 ((Ct - b) / a)

[0351] Where: a: slope of the standard curve, b: intercept of the standard curve

[0352] B. Unit conversion:

[0353] Gene abundance (copies / m³) = gene copy number × dilution factor / sampling volume

[0354] 4.3 Data Correlation Analysis

[0355] A. Correlation Analysis: Pearson correlation analysis was used to assess the correlation between cephalosporin concentration and the abundance of resistance genes.

[0356] B. Principal Component Analysis: Analyzing the impact of different environmental factors on the distribution of drug resistance genes.

[0357] C. Risk Assessment: Assess environmental health risks by combining exposure dose and toxicological data.

[0358] The key focus of the above embodiments of this invention is the establishment of methods for the detection, analysis, and application of four key cephalosporins and all 14 cephalosporins in atmospheric particulate matter samples. In the chemical analysis, ultrapure water extraction with formic acid adjusted to pH 3 was used, followed by purification using an HLB solid-phase extraction column. This method showed good recoveries for all target compounds. The feasibility and stability of this method were verified by analyzing atmospheric particulate matter samples collected from the environment of a pharmaceutical company. The results showed that cefadroxil, cephalexin, and cefuroxime, produced by the pharmaceutical company, were detected in the samples, with total contents ranging from 292 to 469 pg·m³. -3 Cephalosporins present in atmospheric particulate matter may cause allergic reactions in surrounding populations and may also induce drug resistance in bacteria in the air, carrying more ARGs. Therefore, this invention uses PCR amplification and gene identification, real-time quantitative PCR (qPCR) to calculate the abundance of drug resistance genes, and further uses Pearson correlation analysis to evaluate the correlation between cephalosporin concentration and drug resistance gene abundance, greatly expanding the scope of the above methods and their applications.

[0359] The methods and applications provided by this invention are applicable not only to the detection and analysis of multiple cephalosporins in ambient atmospheric particulate matter, but also to the detection and analysis of multiple cephalosporins in water, soil and other samples, and can provide a reference for the formulation of regional pollution control and antibiotic emission policies.

[0360] It should be noted that in other embodiments of the present invention, other different solutions obtained by making specific selections within the scope of the steps, components, processes, formulas, parameters and testing instruments described in the present invention can all achieve the technical effects described in the present invention. Therefore, the present invention will not list them one by one.

[0361] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solution of the present invention using the methods and techniques disclosed above, or modify it into equivalent embodiments with equivalent changes, without departing from the scope of the present invention. All equivalent changes made to the components, proportions, and processes of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for detecting cephalosporins in atmospheric environmental media, characterized in that, It includes the following steps: S1. Screening key environmental indicators Cephalexin, cefadroxil, ceftriaxone, and cefuroxime were selected as key environmental indicators to monitor the presence of cephalosporins in the atmospheric environment of key areas of concern and surrounding airspace, using particulate matter as the atmospheric environment presence medium. The specific steps included are as follows: S1-1. Based on generational preliminary selection, representative drugs are used as key environmental indicators. The first generation includes: cefazolin, cephalexin, cefadroxil, and cefadroxil; The second generation includes: cefuroxime, cefamandole, cefotiam, and cefaclor; The third generation includes: cefotaxime, ceftriaxone, ceftazidime, and cefoperazone; The fourth generation includes: cefepime and cefpirome; S1-2. Based on production and use, environmental detection, drug resistance induction potential and testing feasibility, four key environmental indicators were finally selected: cephalexin, cefadroxil, ceftriaxone and cefuroxime. Atmospheric particulate matter was used as the main carrier for combined monitoring to more comprehensively reflect the status of environmental pollutants. S2, Atmospheric particulate matter sampling Each monitoring point used a high-flow-rate atmospheric particulate matter sampler and a quartz fiber filter membrane to collect samples in batches. Each sampling time was 24 hours, and multiple samples were collected continuously or at intervals to obtain multiple batches of samples. S3, cephalosporin extract The collected samples were then processed through sample pretreatment and solid-phase extraction to extract cephalosporins from the samples, enriching the target analytes and removing interfering substances to obtain the sample extract. The process involves collecting the sample filter membranes and then, under aseptic conditions, uniformly dividing the membrane samples. One portion is used for cephalosporin extraction and chemical analysis, while the other is used for microbial DNA extraction and subsequent gene analysis. The first portion of the divided filter membrane is then placed into sterile sealed bags, numbered, and stored at -20°C. The second portion is immediately placed into sterile centrifuge tubes containing a DNA protectant and frozen at -80°C to prevent DNA degradation. The specific steps include: S3-1, Filter membrane collection The collected sample membranes were collected and uniformly divided under aseptic conditions. One portion was used for cephalosporin extraction and chemical analysis, and the other portion was used for microbial DNA extraction and subsequent gene analysis. The divided membranes were then placed into sterile sealed bags, numbered, and stored in a low-temperature freezer. S3-2, Sample Pretreatment Cut the filter membrane into small pieces with clean scissors and place them in a 50 mL centrifuge tube. Add 4.5 g sodium chloride, 10 mg ascorbic acid, and 100 mg Na2EDTA. Add 40 mL of ultrapure water adjusted to pH=3 with formic acid. Add 100 µl of internal standard. Vortex for 10 min, sonicate at room temperature for 20 min, and centrifuge at 3500 r·min for 15 min. -1 Collect the supernatant into a clean brown glass bottle. Repeat this process twice and combine the three supernatants. S3-3, Cephalosporin Solid Phase Extraction The HLB solid-phase extraction column was pre-activated with 5 mL methanol, 5 mL 0.1% formic acid acetonitrile, and 5 mL ultrapure water. The sample flow rate was controlled at ≤2 drops / second through the extraction column, and the liquid level was kept above the top of the column packing throughout the process. The column was dried by vacuum pump for 30 min. Then, it was eluted with 10 mL 0.1% formic acid-acetonitrile solution, and the eluent was transferred to a 15 mL centrifuge tube and concentrated to near dryness under a gentle nitrogen flow. The solution was then diluted to volume with 500 μL acetonitrile / water, filtered through a 0.22 μm organic phase filter membrane, and transferred to a sample vial. It was stored at -20 ℃ to obtain the sample extract for later use. The entire process was conducted in the dark. S4. Batch testing and data analysis HPLC-MS / MS and internal standard method were used to detect the chemical components and analyze the data of the sample extract, and the detection results of the components and concentrations of cephalosporins in the sample extract were output for qualitative and quantitative monitoring of cephalosporin pollutant emissions. S5, Co-monitoring of Cephalosporin Resistance Genes Microbial DNA was extracted from the sample extract and gene analysis was performed to obtain cephalosporin resistance gene data. This data was used for the synergistic monitoring of cephalosporin resistance genes. The cephalosporin concentration data was combined with the abundance data of resistance genes to assess the correlation between the two and the potential environmental and health risks.

2. The method for detecting cephalosporins in atmospheric environmental media according to claim 1, characterized in that, Step S2 specifically includes the following steps: S2-1. Construct a monitoring network The key areas of concern and the surrounding airspace are delineated, and multiple monitoring points are set up in and within these areas. Each monitoring point is equipped with at least one high-flow-rate atmospheric particulate matter sampler, and samples are collected simultaneously during monitoring to form a monitoring network. The monitoring points of the monitoring network should cover: sensitive areas within the region, upwind and downwind areas outside the region, and sensitive protected targets downwind. S2-2, Deploy sampling equipment At each monitoring point, at least one high-flow-rate atmospheric particulate matter sampler should be deployed, with the sampler 1.5 m above the ground; S2-3, Atmospheric particulate matter sampling Each monitoring station collected samples in batches using high-flow-rate atmospheric particulate matter samplers and quartz fiber filter membranes. Each sampling session lasted 24 hours, with a sampling flow rate of 1 m³ / h. 3 / min, collected multiple times continuously or at intervals.

3. The method for detecting cephalosporins in atmospheric environmental media according to claim 1, characterized in that, Step S4 specifically includes the following steps: S4-1. Prepare testing instruments and reagents. HPLC-MS / MS was used for analysis. Mixed standard solutions with mass concentrations of 5.0, 10.0, 20.0, 50.0, 100.0, 200.0, 500.0, 1000.0, 2000.0, and 5000.0 ng / mL were prepared, and corresponding isotope-labeled mixtures were added as internal standards for quantification. The concentration of the isotope internal standards was 2000 ng / mL for all of them. S4-2, Higher-level inspection When the instrument is in a stable state, all target objects show a good linear relationship during testing, with a correlation coefficient R greater than 0.

995. A Shim-pack GIST C18-AQ HP column was used, with an injection volume of 2 μL and a flow rate of 300 μL / min. The mobile phase is 0.1% formic acid in water and 0.1% formic acid-acetonitrile; An electrospray ionization source was used, with the scanning mode being positive ion mode and multiple reaction monitoring mode, and the ion source temperature being 300℃. S4-3, Data Analysis Recovery rates were assessed: Good recoveries were achieved for 14 cephalosporins, including cephalexin, cefprozil, cefotiam, cefoxitin, cefradine, cefuroxime, cefixime, cefpodoxime proxetil, cefixime methyl ester, cefotaxime sodium, cefoperazone sodium, cefuroxime axetil, cefpirome, and cefnicotinic acid. The recoveries for matrix and blank spiked samples ranged from 33.9% to 169% and 38.6% to 121%, respectively, with RSD < 9.48%. Limit of detection: The limit of detection is 0.06 pg / m³. 3 ~1.74 pg / m 3 ; Output results: The analysis yields and outputs the detection data of the specific components and concentrations of cephalosporins in the sample extract.

4. The method for detecting cephalosporins in atmospheric environmental media according to claim 1, characterized in that, Step S5 specifically includes the following steps: S5-1, Particulate matter component separation Particulate matter elution: Take the filter membrane used for drug resistance gene detection, put it into a sterile centrifuge tube, add sterile phosphate buffer, and use a combination of ultrasonic elution and shaking to elute the particulate matter from the filter membrane to obtain a microbial-particulate matter mixed suspension; Centrifugation: Centrifuge the mixed suspension at 4°C and 5000×g for 10 minutes, and transfer the supernatant to a new tube; the precipitate is used for subsequent DNA extraction. Residue purification: Add sterile saline to the precipitate, gently blow it and centrifuge again, discard the supernatant, repeat 2 times to remove residual particulate impurities; S5-2, Microbial DNA Extraction and Purification Cell lysis: Add sterile lysis buffer to the purified microbial pellet and incubate at 37°C for 1 hour; then add SDS and incubate at 65°C for 30 minutes to further disrupt the cell membrane and ensure complete DNA release. DNA Extraction and Purification: The extraction method uses a column-based DNA extraction kit to efficiently adsorb humic acid and heavy metal impurities. The procedure is as follows: add ethanol to precipitate DNA according to the kit instructions, centrifuge to bind DNA to the silica gel column, remove residual impurities with washing buffer, and finally wash the DNA with sterile enzyme-free water to obtain purified total microbial DNA. DNA quality and concentration testing: Purity testing uses Nanodrop to determine the A260 / A280 ratio and A260 / A230 ratio of DNA; concentration and integrity testing uses a Qubit fluorescence quantitative quantitation system to determine DNA concentration. DNA integrity was detected by 1% agarose gel electrophoresis; S5-3 Qualitative and Quantitative Analysis of Cephalosporin Resistance Genes Qualitative analysis employed PCR amplification and gene identification, including: Targeted primer design: Design specific primers for conserved regions of cephalosporin resistance genes to cover major resistance genotypes; Conventional PCR amplification: Using purified DNA as a template, add primers, Taq DNA polymerase, and dNTPs. Set the amplification program as follows: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 55-60℃ annealing for 30 seconds, 72℃ extension for 30 seconds, 35 cycles; 72℃ final extension for 10 minutes. Results identification: The PCR products were subjected to agarose gel electrophoresis. If a band of the same size as the target gene fragment appeared, it was preliminarily determined that the drug resistance gene was present. Further verification of the gene sequence and confirmation of the genotype were performed by Sanger sequencing to avoid false positives caused by non-specific primer binding. Quantitative analysis uses real-time quantitative PCR, including: Construction of standard curve: Dilute the known concentration of drug resistance gene plasmid to 10¹⁻¹⁰. 7 Gradient standards of copies / μL were amplified by qPCR, and a standard curve was constructed with "Ct value" as the ordinate and "plasmid copy number log" as the abscissa. Sample quantification: Using purified DNA as a template, qPCR was performed using the SYBR Green or TaqMan probe method. The absolute copy number of the target drug resistance gene in the sample was calculated using a standard curve. Combined with the sampling volume, the result was converted to gene copy number / m³ air to reflect the environmental abundance of the drug resistance gene. Quality control: Each batch of experiments includes a "blank control", "negative control" and "positive control" to avoid false positives / false negatives caused by contamination.

5. The method for detecting cephalosporins in atmospheric environmental media according to claim 4, characterized in that, Step S5 further includes the following steps: S5-4, Data Linkage and Environmental Health Risk Assessment By combining cephalosporin concentration data with drug resistance gene abundance data, the correlation and potential risks between the two can be assessed. Data correlation analysis includes: Statistical analysis: Pearson correlation analysis or principal component analysis was used to determine the correlation between cephalosporin concentration and drug resistance gene abundance, including whether the concentration of third-generation cephalosporins is positively correlated with the abundance of the blaCTX-M gene, and to verify whether environmental cephalosporin exposure drives the spread of drug resistance genes. Spatial difference analysis: If monitoring is conducted at multiple sampling points, the cephalosporin-resistance gene combination characteristics of different regions can be compared to identify high-risk areas; Environmental health risk assessment includes: Environmental risk assessment: Assess the potential for the spread of drug-resistant genes, including being alert to the possibility of their spread to surrounding water bodies / soil via atmospheric particulate matter when the abundance is ≥10³ copies / m³, which could lead to the cross-media transmission of drug-resistant genes. Health risk assessment: Based on the amount of atmospheric particulate matter inhaled, estimate the number of drug resistance gene copies inhaled by the human body daily; if there is long-term exposure to an environment with high abundance of drug resistance genes, it suggests that drug resistance genes may enter the human body through the respiratory tract, which may increase the potential risk of drug resistance in the human flora.

6. The application of the method for detecting cephalosporins in atmospheric environmental media as described in any one of claims 1 to 5 in environmental and health risk assessment.

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