A method for quantitative detection of antibiotics in soil based on LC-MS / MS

Abstract

This invention discloses a method for quantitative detection of antibiotics in soil based on LC-MS / MS, comprising: freeze-drying a soil sample, weighing a trace amount of the freeze-dried powder, adding an extraction solution, extracting by vortexing and ultrasonication, centrifuging to collect the supernatant, concentrating to dryness by nitrogen blowing, redissolving and filtering, and then performing qualitative and quantitative analysis by high-performance liquid chromatography-tandem mass spectrometry. This invention eliminates the need for a solid-phase extraction column purification step, making the pretreatment operation simple and efficient. Only 0.1g of sample is required, and it exhibits high sensitivity, a low limit of quantitation, and uses an internal standard method to correct for matrix effects. The linear correlation coefficient R0 is high. 2 The spiked recovery rate is consistently between 80% and 120%, with a value >0.99, indicating that the method is accurate and reliable. This method can be used for the simultaneous or separate quantitative detection of more than 20 antibiotics, including chloramphenicol, tetracyclines, quinolones, and macrolides, in soil. It can also be directly extended to complex environmental matrices such as animal feces and sludge. It consumes little organic solvent and is environmentally friendly.

Description

Technical Field

[0001] This invention relates to the field of environmental analytical chemistry, and in particular to a method for the quantitative detection of antibiotics in soil based on LC-MS / MS. Background Technology

[0002] Antibiotics, as a class of chemical substances with antibacterial activity, are widely used globally in various fields such as human clinical treatment, disease prevention and control in livestock and aquaculture, and promotion of animal growth. However, most antibiotics, after being ingested by humans or animals, cannot be completely absorbed and metabolized by the body; approximately 30% to 90% are excreted in feces and urine in the form of the original drug or active metabolites. These excrements containing antibiotic residues continuously enter the farmland soil environment through agricultural fertilization, sewage irrigation, and sludge land application, making the soil a significant sink for antibiotic-related emerging organic pollutants.

[0003] Antibiotics entering the soil not only inhibit or alter the structure and function of soil microbial communities and induce antibiotic resistance in environmental bacteria, but also pollute surface water and groundwater through surface runoff and leaching, posing a potential threat to human health and ecosystem security through the food chain. Due to the highly complex composition of the soil matrix, containing large amounts of humic acid, minerals, and various coexisting organic matter, and the significant differences in the physicochemical properties (such as acid-base dissociation constants, hydrophobicity, and metal chelating ability) of different types of antibiotics, the simultaneous and accurate quantitative detection of multiple types of antibiotic residues in soil presents a significant technical challenge.

[0004] Currently, existing methods for detecting antibiotic residues mainly include microbial inhibition methods, enzyme-linked immunosorbent assay (ELISA), high-performance capillary electrophoresis, thin-layer chromatography, gas chromatography, gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), and liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0005] While microbiological and immunoassay methods are relatively simple to operate, they often suffer from drawbacks such as low sensitivity, poor specificity, the ability to detect only one or a few classes of antibiotics, and the inability to simultaneously distinguish and accurately quantify multi-component compounds. Capillary electrophoresis and thin-layer chromatography have limited reproducibility and sensitivity, making them unsuitable for detecting trace antibiotics in complex matrices. Gas chromatography and gas chromatography-mass spectrometry (GC-MS) methods typically require cumbersome derivatization of highly polar antibiotics, resulting in complex and time-consuming procedures, and the stability of these methods is difficult to guarantee.

[0006] In contrast, high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS / MS) have gradually become the mainstream techniques for antibiotic residue analysis due to their high separation efficiency and accurate qualitative and quantitative analysis. However, currently available methods for detecting antibiotics in soil based on liquid chromatography or liquid chromatography-mass spectrometry generally suffer from one or more of the following shortcomings: (1) The sample pretreatment process is cumbersome and complicated. Most existing methods require purification and enrichment steps using solid phase extraction columns (such as HLB columns, SAX columns, etc.) during the pretreatment process to remove complex soil matrix interferences. This not only significantly increases the operation time, organic solvent consumption and consumable costs, but also easily leads to unsatisfactory batch-to-batch reproducibility of analytical results due to operational errors.

[0007] (2) Large sample volume and low detection throughput. In order to ensure that the target substance at a low concentration can be detected, traditional methods usually require weighing several grams or even tens of grams of soil sample, which leads to a significant increase in the volume of extraction solvent and a substantial extension of the concentration time. This makes it difficult to meet the monitoring needs of rapid screening of large batches of environmental samples and also increases the environmental burden of laboratory waste discharge.

[0008] (3) Limited applicability to a single type of antibiotic, lacking versatility. Most existing technologies are developed only for a specific class of antibiotics (such as detecting only tetracyclines or only quinolones). Due to the significant differences in the chemical properties of different classes of antibiotics, when it is necessary to analyze multiple antibiotics with different physicochemical properties such as chloramphenicol, tetracyclines, quinolones, and macrolides in soil, it is often necessary to use multiple different pretreatment procedures, which greatly affects work efficiency and increases systematic errors between samples.

[0009] (4) Insufficient long-term stability of the method and insufficient control of matrix effect. In simplified methods that omit the solid-phase extraction purification step, how to effectively compensate for the ion inhibition or enhancement effect caused by the soil matrix and ensure the inter-batch and intra-batch stability of the analytical method in long-term operation remains a key problem that the existing technology has not fully solved.

[0010] In summary, developing a technical solution that is simple and quick to perform pretreatment, requires minimal organic solvents, involves small sample sizes, has high sensitivity, and can be applied to the simultaneous quantitative detection of multiple types of antibiotics in soil is of great practical significance for conducting large-scale soil antibiotic pollution surveys, assessing environmental risks, and guiding safe agricultural production. Summary of the Invention

[0011] The purpose of this invention is to provide a method for quantitative detection of antibiotics in soil based on LC-MS / MS, in order to solve at least some of the problems existing in the prior art.

[0012] Technical solution: A method for quantitative detection of antibiotics in soil based on LC-MS / MS, comprising the following steps: A method for quantitative detection of antibiotics in soil based on LC-MS / MS, characterized by comprising the following steps: Sample pretreatment: Soil samples were collected and freeze-dried to obtain freeze-dried soil powder samples; Extraction: Weigh a predetermined mass of freeze-dried soil powder sample, add extraction solution to it for extraction treatment, and collect the supernatant after centrifugation; the extraction treatment includes vortex mixing and ultrasonic-assisted extraction. Concentration and redissolution: The collected supernatant was concentrated by nitrogen blowing until dry to obtain the extract residue. Then, a redissolution solution was added for redissolution. After vortex mixing and centrifugation, the supernatant was taken to obtain the test solution. Detection and Analysis: High performance liquid chromatography-tandem mass spectrometry was used to analyze the test solution to detect the target antibiotic in the soil sample.

[0013] Preferably, when the target antibiotic is a tetracycline antibiotic, during the extraction process, the masking agent dipotassium ethylenediaminetetraacetate dihydrate is first added to the freeze-dried soil powder sample, and then the extraction solution is added for extraction; the extraction solution is prepared by mixing acetonitrile containing 2% ammonia, 0.1 mol / L sodium hydroxide solution and McIlvaine buffer solution in a volume ratio of 1:1:1.

[0014] Preferably, when the target antibiotic is a tetracycline antibiotic, the mass of the freeze-dried soil powder sample is 0.10 g, the amount of ethylenediaminetetraacetic acid dihydrate dipotassium added is 0.05 g, and the amount of extract added is 400 μL. The specific operation of the extraction process is as follows: vortex mixing for 5 minutes, followed by ultrasonic treatment at 100% power for 10 minutes; centrifugation conditions are 5000 rpm for 5 minutes; the extraction process and centrifugation and collection of supernatant are repeated 2-3 times, and the obtained supernatants are combined.

[0015] Preferably, when the target antibiotic is a quinolone antibiotic, during the extraction process, phosphoric acid solution is first added to the freeze-dried soil powder sample for acidification, and then EDTA masking agent and acidified acetonitrile are added as extraction solution for extraction.

[0016] Preferably, when the target antibiotic is a chloramphenicol antibiotic, the extract is composed of ammonia and ethyl acetate during the extraction process; and before the extraction process, a mixed standard solution and an internal standard solution are added to the freeze-dried soil powder sample.

[0017] Preferably, when the target antibiotic is a chloramphenicol antibiotic, the extract contains 15 μL of ammonia and 500 μL of ethyl acetate; the internal standard concentration is 10 ng / mL.

[0018] Preferably, when the target antibiotic is a macrolide antibiotic, the extractant is a methanol solution containing 1% ammonia during the extraction process; and before the extraction process, a mixed standard solution and an internal standard solution are added to the freeze-dried soil powder sample.

[0019] Preferably, during the sample pretreatment process, the collected soil samples are first frozen at -80℃ and then freeze-dried. The dried samples are then stored at -20℃ away from light. In step (3), the reconstitution solution is a 10% methanol aqueous solution or a 10% methanol aqueous solution containing 0.1% formic acid.

[0020] Preferably, during the concentration and reconstitution process, high performance liquid chromatography-tandem mass spectrometry uses an electrospray ionization source and performs positive or negative ion scanning in multiple reaction monitoring mode; during data processing, internal standard or external standard methods are used for quantification to ensure that the linear correlation coefficient R2 of each target antibiotic is greater than 0.99.

[0021] Beneficial effects: The pretreatment operation of this invention is simple and rapid, requiring no solid-phase extraction purification, and significantly shortening the processing time for a single sample; only 0.1g of trace sample is needed to achieve trace detection, with high sensitivity and low limit of quantitation; the matrix effect is corrected by the internal standard method, with a linear correlation coefficient R²>0.99, and the spiked recovery rate is stable at 80%~120%, ensuring accurate and reliable quantification; the method is applicable to the detection of more than 20 antibiotics in soil, including chloramphenicol, tetracyclines, quinolones, and macrolides, and can be extended to complex matrices such as animal feces and sludge; at the same time, the amount of organic solvent used is small, making it green and environmentally friendly. Attached Figure Description

[0022] Figure 1 This is a flowchart of the overall solution of the present invention. Detailed Implementation

[0023] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0024] Example 1: A general method for quantitative detection of antibiotics in soil based on LC-MS / MS like Figure 1 As shown, this embodiment provides a general method for detecting various types of antibiotics. Its core lies in establishing a standardized operating platform encompassing "low-temperature preservation - freeze-drying - micro-sampling - liquid chromatography - tandem mass spectrometry analysis." The specific steps are as follows: Step (1) Sample Pretreatment: Freshly collected soil samples were placed in 50 mL centrifuge tubes and frozen at -80°C. Before testing, the frozen samples were transferred to a freeze dryer and freeze-dried under vacuum for 48 hours to completely remove soil moisture and prevent moisture from affecting subsequent micro-weighing and extraction efficiency. After drying, the centrifuge tubes were sealed and stored at -20°C in the dark to obtain freeze-dried soil powder samples for testing.

[0025] Step (2) Target Extraction: Using a 0.001 g balance, accurately weigh 0.10 ± 0.001 g of the above freeze-dried soil powder sample and transfer it to a new 2 mL centrifuge tube. Add 400-500 μL of extraction buffer to the centrifuge tube (the specific composition of the extraction buffer is selected according to the target antibiotic category, see subsequent examples for details). Place the centrifuge tube on a vortex mixer and vortex at 2500 rpm for 5-10 minutes. Then place it in an ultrasonic cleaner and extract with ultrasonic assistance at 100% power (usually 40-100 kHz) for 10 minutes. After extraction, place the centrifuge tube in a high-speed centrifuge and centrifuge at 8000 rpm for 5 minutes. Carefully aspirate the supernatant with a pipette and transfer it to another clean 2 mL centrifuge tube. To ensure complete extraction, add an equal volume of extraction buffer to the soil precipitate again and repeat the above extraction and centrifugation steps 1-2 times, combining all supernatants into the same centrifuge tube.

[0026] Step (3) Concentration and Reconstitution: Place the centrifuge tube containing the combined supernatant under a nitrogen dryer, adjust the nitrogen flow rate to cause slight movement of the liquid surface, and slowly dry it at room temperature until the liquid in the tube is completely evaporated, obtaining a dry extract residue. Add 200 μL of reconstitution solution (10% methanol aqueous solution, or 10% methanol aqueous solution containing 0.1% formic acid) to the residue, and vortex for 5 minutes to fully dissolve it. Finally, centrifuge the reconstitution solution at 12000 rpm for 5 minutes, draw up the supernatant with a syringe, filter it through a 0.22 μm syringe filter into a brown vial, and obtain the final test solution.

[0027] Step (4) LC-MS / MS Detection and Analysis: The analyte was injected into the autosampler of a high-performance liquid chromatograph for analysis. The LC conditions were: reversed-phase C18 column (2.1×100mm, 1.7μm), column temperature 40℃, mobile phase A was an aqueous solution containing 0.1% formic acid, mobile phase B was acetonitrile, flow rate 0.3mL / min, gradient elution. The chromatographic effluent was directly introduced into a triple quadrupole mass spectrometer, and data was acquired using an electrospray ionization source in positive ion (ESI+) or negative ion (ESI-) multiple reaction monitoring mode. The abundance ratio of the analyte and the standard was qualitatively confirmed by comparing their chromatographic retention times and characteristic ions, and quantification was performed using the peak area method (internal standard method or external standard method).

[0028] Method validation results: Using this general method, all target antibiotics showed excellent linearity within their respective linear ranges, with linear correlation coefficients R0. 2 All values ​​were greater than 0.99. At all three spiking concentration levels, the recoveries were in the range of 80%–120%, indicating that the method is accurate and reliable.

[0029] Example 2: A quantitative detection method specifically for tetracycline antibiotics in soil This embodiment, within the general framework of Example 1, specifically optimizes the characteristics of tetracycline antibiotics (tetracycline, oxytetracycline, chlortetracycline, doxycycline) that readily chelate with metal ions and have low recovery rates in soil matrix.

[0030] The operation steps are as follows: Step (1): Same as in Example 1.

[0031] Step (2): Weigh 0.10g of freeze-dried soil powder sample into a 2mL centrifuge tube, and first add 0.05g of dipotassium ethylenediaminetetraacetate dihydrate (EDTA-DIK). The powder was used as a metal ion masking agent and gently shaken to mix. Then, 400 μL of a special extraction buffer was added, which was freshly prepared by mixing acetonitrile containing 2% ammonia, 0.1 mol / L sodium hydroxide solution, and McIlvaine buffer (pH 4.0) in a volume ratio of 1:1:1. The mixture was vortexed for 5 minutes, sonicated at 100% power for 10 minutes, and centrifuged at 5000 rpm for 5 minutes. The supernatant was collected. This extraction and centrifugation process was repeated three times, and the supernatants were combined.

[0032] Steps (3) and (4): Same as in Example 1, except the reconstitution solution is a 10% methanol aqueous solution, and the mass spectrometry is performed in ESI+ mode.

[0033] Detection performance: The limits of quantitation for this method are 0.5-2 ng / g for four tetracycline antibiotics. Linear correlation coefficient R0 2 >0.99, with recoveries of 85%–110% at three spiking levels of 1, 5, and 20 ng / g, and relative standard deviations (RSD) less than 10%. Compared with no spiking... Compared with the comparative experiments, the recovery rate increased by an average of 20%-35%, proving that the combination of the masking agent and the specific extract has a significant effect.

[0034] Example 3: A quantitative detection method specifically for quinolone antibiotics in soil This embodiment optimizes the extraction of quinolone antibiotics (including 13 types such as enrofloxacin, ciprofloxacin, and norfloxacin) within the general framework of Example 1, and adopts a "double acidification" extraction strategy to further improve extraction efficiency and reduce the amount of organic solvent used.

[0035] The operation steps are as follows: Step (1): Same as in Example 1.

[0036] Step (2): Weigh 0.10 g of freeze-dried soil powder sample into a 2 mL centrifuge tube. First, add 250 μL of phosphoric acid solution to initially acidify the soil matrix. Then, add 0.02 g of EDTA as a masking agent, followed by 250 μL of acidified acetonitrile for extraction. Vortex mix for 10 minutes, sonicate at 100% power for 10 minutes, centrifuge at 8000 rpm for 5 minutes, and collect the supernatant. Repeat the extraction 3 times and combine the supernatants.

[0037] Step (3): Same as in Example 1, except the reconstitution solution is a 10% methanol aqueous solution containing 0.1% formic acid.

[0038] Step (4): Same as in Example 1, mass spectrometry is performed in ESI+ mode.

[0039] Detection performance: This method enables simultaneous analysis of 13 quinolone drugs, with a total analysis time controlled within 15 minutes. The limits of quantitation for the 13 compounds range from 0.1 to 2 ng / g. The linear correlation coefficient R0... 2 All values ​​were greater than 0.99, and the spiked recoveries ranged from 90% to 130%. Compared with the conventional acetonitrile extraction method, this method reduces the amount of organic solvent (acetonitrile) used by about 50%, demonstrating its green and environmentally friendly characteristics.

[0040] Example 4: A quantitative detection method specifically for chloramphenicol antibiotics in soil This embodiment optimizes the use of chloramphenicol antibiotics (chloramphenicol, florfenicol, thiamphenicol) by employing specific alkaline organic extraction solvents and internal standard methods to ensure the stability of trace analysis.

[0041] The operation steps are as follows: Step (1): Same as in Example 1.

[0042] Step (2): Weigh 0.10 g of freeze-dried soil powder sample into a 2 mL centrifuge tube, add the mixed standard solution (for preparing the standard curve) and deuterated chloramphenicol internal standard solution to make the final internal standard concentration 10 ng / mL. Then, add 500 μL of freshly prepared extraction buffer, which is a mixture of 15 μL ammonia and 500 μL ethyl acetate. Vortex for 10 minutes, centrifuge at 8000 rpm for 5 minutes, and collect the supernatant. Repeat the extraction and centrifugation steps once, and combine the supernatants.

[0043] Step (3): Same as in Example 1, except the reconstitution solution is a 10% methanol aqueous solution.

[0044] Step (4): Same as in Example 1, mass spectrometry is performed using ESI-mode detection.

[0045] Detection performance: By introducing an internal standard, the matrix effect in the pretreatment and ionization processes was effectively corrected. The three chloramphenicol antibiotics showed good linearity in the concentration range of 2-40 ng / mL, with R... 2 >0.99. The limit of quantitation is as low as 0.1-0.5 ng / g, and the spiked recovery rate is stable between 80% and 120%.

[0046] Example 5: A quantitative detection method specifically for macrolide antibiotics in soil This embodiment simplifies and optimizes the extraction solution based on the chemical properties of macrolide antibiotics (such as erythromycin, roxithromycin, tylosin, etc., a total of 7 types).

[0047] The operation steps are as follows: Step (1): Same as in Example 1.

[0048] Step (2): Weigh 0.10 g of freeze-dried soil powder sample into a 2 mL centrifuge tube, add the mixed standard solution and internal standard solution. Then, add 500 μL of extraction buffer (1% ammoniated methanol, i.e., 99 mL methanol + 1 mL ammonia). Vortex to mix for 10 minutes, centrifuge at 8000 rpm for 5 minutes, and collect the supernatant. Repeat the extraction once and combine the supernatants.

[0049] Steps (3) and (4): Same as in Example 1, except the reconstitution solution is a 10% methanol aqueous solution, and the mass spectrometry is performed in ESI+ mode.

[0050] Detection performance: All seven macrolide compounds were well separated and showed good peak shapes within 10 minutes. All compounds exhibited excellent linearity in the concentration range of 0.1–50 ng / mL, with R0... 2 >0.99. The spiked recoveries were 80%–120%, and the limits of quantitation were 0.05–1 ng / g.

[0051] Example 6: Application Extension of this Method in Other Complex Matrices To verify the universality of this patented method, the detection methods of Examples 1-5 were applied to two complex matrices: animal feces (pig feces, chicken feces) and residual sludge from urban sewage treatment plants.

[0052] Sample sources: Pig manure and chicken manure were collected from large-scale farms, and residual sludge was collected from the dewatering room of a municipal wastewater treatment plant.

[0053] Experimental procedure: Weigh 0.10g of the freeze-dried fecal or sludge sample and follow the specific pretreatment and instrumental analysis steps in Example 2 (for tetracyclines), Example 3 (for quinolones), Example 4 (for chloramphenicols), and Example 5 (for macrolides).

[0054] Experimental results: In animal feces and sludge substrates, the linear correlation coefficient R of all target antibiotics was [missing information]. 2 The concentration remained above 0.99. In spiked recovery tests at three concentration levels, the recovery rate ranged from 75% to 125%. This indicates that the "lyophilized trace sample-simplified extraction-LC-MS / MS analysis" technical platform established in this invention successfully overcomes the matrix effect differences caused by various complex environmental matrices, demonstrating strong versatility and portability. It can be widely applied to the simultaneous detection of multiple antibiotics in various solid environmental samples such as soil, feces, and sludge.

[0055] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various equivalent transformations can be made to the technical solutions of the present invention, and these equivalent transformations all fall within the protection scope of the present invention.

Claims

1. A method for quantitative detection of antibiotics in soil based on LC-MS / MS, characterized in that, Includes the following steps: Sample pretreatment: Soil samples were collected and freeze-dried to obtain freeze-dried soil powder samples; Extraction: Weigh a predetermined mass of freeze-dried soil powder sample, add extraction solution to it for extraction treatment, and collect the supernatant after centrifugation; the extraction treatment includes vortex mixing and ultrasonic-assisted extraction. Concentration and redissolution: The collected supernatant was concentrated by nitrogen blowing until dry to obtain the extract residue. Then, a redissolution solution was added for redissolution. After vortex mixing and centrifugation, the supernatant was taken to obtain the test solution. Detection and Analysis: High performance liquid chromatography-tandem mass spectrometry was used to analyze the test solution to detect the target antibiotic in the soil sample.

2. The method according to claim 1, characterized in that, When the target antibiotic is a tetracycline antibiotic, during the extraction process, the masking agent dipotassium ethylenediaminetetraacetate dihydrate is first added to the freeze-dried soil powder sample, and then the extraction solution is added for extraction; the extraction solution is prepared by mixing acetonitrile containing 2% ammonia, 0.1 mol / L sodium hydroxide solution and McIlvaine buffer solution in a volume ratio of 1:1:

1.

3. The method according to claim 2, characterized in that, During the extraction process, the weight of the freeze-dried soil powder sample was 0.10 g, the amount of dipotassium ethylenediaminetetraacetate dihydrate added was 0.05 g, and the amount of extraction solution added was 400 μL. The specific extraction process was as follows: vortex mixing for 5 minutes, followed by ultrasonic treatment at 100% power for 10 minutes; centrifugation at 5000 rpm for 5 minutes; the extraction process and centrifugation and collection of supernatant were repeated 2-3 times, and the resulting supernatants were combined.

4. The method according to claim 1, characterized in that, When the target antibiotic is a quinolone antibiotic, during the extraction process, phosphoric acid solution is first added to the freeze-dried soil powder sample for acidification, and then EDTA masking agent and acidified acetonitrile are added as extraction solution for extraction.

5. The method according to claim 1, characterized in that, When the target antibiotic is chloramphenicol, the extraction solution consists of ammonia and ethyl acetate during the extraction process; and before the extraction process, a mixed standard solution and an internal standard solution are added to the freeze-dried soil powder sample.

6. The method according to claim 5, characterized in that, The extract contained 15 μL of ammonia and 500 μL of ethyl acetate; the internal standard concentration was 10 ng / mL.

7. The method according to claim 1, characterized in that, When the target antibiotic is a macrolide antibiotic, the extraction solution is a methanol solution containing 1% ammonia during the extraction process; and before the extraction process, a mixed standard solution and an internal standard solution are added to the freeze-dried soil powder sample.

8. The method according to claim 1, characterized in that, During the sample pretreatment process, the collected soil samples were first frozen at -80℃ and then freeze-dried. The dried samples were stored at -20℃ in the dark. In step (3), the reconstitution solution was a 10% methanol aqueous solution or a 10% methanol aqueous solution containing 0.1% formic acid.

9. The method according to claim 1, characterized in that, During concentration and reconstitution, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used with an electrospray ionization source, performing positive or negative ion scans in multiple reaction monitoring (MRM) mode. Data processing employed internal or external standard methods for quantification to ensure the linear correlation coefficient R of each target antibiotic. 2 >0.99.

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