Analysis method and system for detecting various antibiotic residues in edible parts of vegetables

Abstract

The invention relates to the technical field of food safety detection, and particularly provides an analysis method and system for detecting various antibiotic residues in edible parts of vegetables. According to the method, aiming at the problem that various antibiotics in vegetables are difficult to efficiently and accurately detect at the same time in the prior art, synchronous and efficient extraction and purification of sulfonamides, quinolones and tetracyclines antibiotics are realized by optimizing a sample pretreatment process; according to the method, specific ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry analysis conditions are combined, intelligent comparison is performed by utilizing a pre-constructed standard compound database, and rapid screening and accurate quantification of various target antibiotics in the complex vegetable matrix are completed. The invention provides a set of complete solution, has the advantages of universal and efficient pretreatment, high detection flux, good accuracy and the like, and provides a reliable technical means for daily monitoring and risk assessment of antibiotic residues in vegetables.

Description

Technical Field

[0001] This invention relates to the field of environmental pollutant and food safety analysis and detection technology, specifically to an analytical method and system for detecting multiple antibiotic residues in edible parts of vegetables. Background Technology

[0002] Antibiotics are widely used globally as important drugs for the prevention and treatment of infectious diseases in humans and livestock. my country is a major producer and user of antibiotics, and large quantities of incompletely metabolized antibiotics enter the agricultural environment through livestock manure and other routes. This manure containing antibiotic residues is used as organic fertilizer in farmland or introduced through irrigation water, leading to the continuous accumulation of antibiotics in farmland soil, becoming a new and concerning pollutant.

[0003] Vegetable cultivation systems are a crucial link in the migration of antibiotics from soil to the food chain. Due to their persistence in the environment, antibiotics can be absorbed by vegetable roots and transported to edible parts. Long-term dietary intake of vegetables containing low doses of antibiotics may disrupt gut microbiota homeostasis and potentially accelerate the development and spread of bacterial resistance, posing a potential threat to public health. Therefore, my country has included several antibiotics in its list of key controlled new pollutants, making it crucial to strengthen their monitoring in agricultural products.

[0004] Currently, methods for detecting antibiotics in the environment and agricultural products mostly focus on matrices such as water, soil, or sediment, and are usually only applicable to a single class (such as sulfonamides or quinolones) or a limited number (usually less than 20). However, the matrix of edible parts of vegetables is extremely complex, containing a large amount of endogenous interfering substances such as chlorophyll, pigments, organic acids, sugars, and oils. These substances not only severely interfere with the instrument's detection signal for trace antibiotics, but also pose a significant challenge in developing a pretreatment method that can simultaneously and efficiently extract multiple classes of antibiotics from complex vegetable matrices due to their vastly different binding abilities with antibiotics of different polarities and chemical properties. Furthermore, the significant differences in tissue structure and chemical composition between different types of vegetables (such as leafy vegetables like lettuce, and fruit vegetables like tomatoes and peppers) further increase the difficulty of establishing a universally applicable method for analyzing multiple classes of antibiotic residues.

[0005] Therefore, current technologies lack an integrated analytical method capable of rapidly, accurately, and simultaneously determining the residues of dozens of commonly used antibiotics in various types of vegetables. This technological bottleneck prevents us from comprehensively assessing the mixed contamination status of antibiotics in vegetables and the risk of dietary exposure. Developing an analytical method applicable to complex vegetable matrices and capable of simultaneously detecting 52 antibiotics from three major classes—sulfonamides, quinolones, and tetracyclines—has become a critical technical problem urgently needing to be solved in food safety supervision and risk assessment. Summary of the Invention

[0006] The present invention aims to solve or improve the technical problem in which it is impossible to simultaneously, efficiently and accurately detect multiple types of antibiotic residues in vegetables.

[0007] To solve the above-mentioned technical problems, the technical solution proposed in this application is as follows:

[0008] This invention provides an analytical method for detecting multiple antibiotic residues in edible parts of vegetables, comprising the following steps: S1: Vegetable samples are pretreated to obtain the analysis solution; S2: The test solution is analyzed using ultra-high performance liquid chromatography-tandem high resolution mass spectrometry to obtain the mass spectrometry information of the compounds in the test solution; wherein, the mass spectrometry information includes retention time, fragment ion information and precursor ion mass information; S3: Based on the mass spectrometry information of the compounds in the detection solution, compare it with the preset standard antibiotic compound information database to identify and quantify the sulfonamides, quinolones and tetracyclines present in the vegetable sample; The preset standard antibiotic compound information database contains standard mass spectrometry information for 52 target antibiotics, including 26 sulfonamide antibiotics, 20 quinolone antibiotics and 6 tetracycline antibiotics.

[0009] Further, step S1 includes: S11: Crush and pre-treat vegetable samples; S12: Add the isotope internal standard mixed solution and extraction solution to the pretreated sample, extract, and combine the extraction supernatants; S13: Concentrate and reconstitute the combined extract supernatant; S14: The target compound in the complex solution is enriched and purified using a solid-phase extraction column, and the analyte solution is obtained after elution.

[0010] Furthermore, the pretreatment in step S11 includes adding Na2EDTA, NaCl and anhydrous Na2SO4 to the vegetable sample; For every 2g of vegetable sample, 0.2g of Na2EDTA, 0.1g of NaCl, and 0.2g of anhydrous Na2SO4 were added.

[0011] Further, in step S12, the extract is a mixed solution of methanol, acetonitrile and hydrochloric acid in a volume ratio of 48:48:4.

[0012] Further, in step S12, the isotope internal standard mixed solution contains sulfamethazine- 13C6, sulfamethoxypyrimidine 13 C6, trimethoprim-d3, sulfadiazine-d4, sulfamethoxazole-d4, ofloxacin-d3, and doxycycline-d3.

[0013] Furthermore, in step S14, the solid-phase extraction column is an HLB solid-phase extraction column; The enrichment and purification process includes: activating the HLB solid-phase extraction column sequentially with methanol and ultrapure water; loading the reconstituted solution onto the column; rinsing with ultrapure water; and eluting the target compound with methanol after drying.

[0014] Furthermore, in step S2, the ultra-high performance liquid chromatography uses a C18 column, with mobile phase A being an aqueous solution containing 1 mM formic acid and mobile phase B being a methanol solution containing 1 mM formic acid, and gradient elution is performed. The gradient elution procedure is as follows: 0 min, mobile phase A 90%, mobile phase B 10%; 2 min, mobile phase A 80%, mobile phase B 20%; 8 min, mobile phase A 68%, mobile phase B 32%; 12 min, mobile phase A 65%, mobile phase B 35%; 17 min, mobile phase A 35%, mobile phase B 65%; 20 min, mobile phase A 30%, mobile phase B 70%; 21 min, mobile phase A 5%, mobile phase B 95%; 24 min, mobile phase A 5%, mobile phase B 95%; 25 min, mobile phase A 90%, mobile phase B 10%; 29 min, mobile phase A 90%, mobile phase B 10%.

[0015] Furthermore, in step S2, the tandem high-resolution mass spectrometry employs an electrospray ionization source, positive ion mode, and multiple reaction monitoring (MRM) scanning mode.

[0016] Furthermore, step S3 specifically includes: S31: Match the precursor ion mass information of the compound in the test solution with the precursor ion mass information of each standard antibiotic in the database, with an error range within ±5ppm, and screen out suspected target compounds; S32: Compare and confirm the retention time and fragment ion information of the suspected target compound with the retention time and fragment ion information of the corresponding standard antibiotics in the database; S33: Using the internal standard method, quantitative analysis is performed on the confirmed target compound based on the peak area ratio of the target compound and the corresponding isotopic internal standard and the standard curve.

[0017] On the other hand, this application also claims protection for a system for implementing the analytical method for detecting multiple antibiotic residues in edible parts of vegetables, comprising: The pretreatment module is used to pretreat vegetable samples to obtain the analytical solution. The mass spectrometry analysis module is used to analyze the test solution using an ultra-high performance liquid chromatography-tandem high resolution mass spectrometer to obtain the mass spectrometry information of the compounds in the test solution; The data processing module is used to compare the mass spectrometry information of the compounds in the detection solution with a preset standard antibiotic compound information database to identify and quantify the sulfonamides, quinolones and tetracyclines present in the vegetable sample. The preset standard antibiotic compound information database contains standard mass spectrometry information for 52 target antibiotics.

[0018] Compared with the prior art, the present invention achieves the following beneficial technical effects: This application establishes for the first time an integrated analytical method capable of simultaneously detecting 52 antibiotic residues from three major categories in vegetables. Through an optimized sample pretreatment process, this method achieves efficient and simultaneous extraction and purification of multiple target compounds from different types of vegetable matrices. Combined with specific chromatographic-mass spectrometry analysis conditions and a standard database, it effectively overcomes interference from complex matrices, achieving high-throughput and high-accuracy screening and quantification. This provides a reliable and efficient technical means for comprehensively assessing the dietary exposure risk of antibiotic residues in vegetables. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a flowchart of an analytical method for detecting multiple antibiotic residues in edible parts of vegetables according to the present invention.

[0021] Figure 2 shows the XIC chromatograms of 52 antibiotic compounds and 7 isotope-labeled compounds in one embodiment of the present invention. The compounds corresponding to each peak are as follows: Figure (A): 1. Sulfadiazine (SDZ) 2. Sulfamethazine (SMA) 3. Sulfathiazole (STZ) 4. Sulfapyridine (SPD) 5. Sulfamethazine (SMR) 6. Trimethoprim (TMP) 7. N-Ac-Sulfamethazine (N-AcSDZ) 8. Sulfamethoxypyridine (SME) 9. Sulfamethhidiazole (SMT) 10. Sulfamethisopyrimidine (SIM) 11. N-Ac-Sulfapyridine (N-AcSPD) 12. Sulfamethoxypyridazine (SMP) 13. Sulfachlorpyridazine (SCP) 14. N- 15. Sulfamethoxypyrimidine (N-AcSMA) 16. Sulfamethoxypyrimidine (SMX) 17. Sulfamethoxypyrimidine (SMM) 18. Carbadox (CBD) 19. Sulfamethoxypyrimidine (SDO) 20. Sulfaisoxazole (SIZ) 21. Benzoylsulfonamide (SBZ) 22. Sulfabenzylazole (SPZ) 23. Sulfachloropyrazine (SCZ) 24. N-AcSMX (N-AcSMA) 25. Sulfamethoxypyrimidine (SDM) 26. Sulfanitrobenzene (SNT) 27. Sulfaquinoxaline (SQX) Figure (B): 27. Piperidin (PPA) 28. Marbofloxacin (MBF) 29. Fleroxacin (FLX) 30. Ofloxacin (OFX) 31. Enoxacin (ENX) 32. Pefloxacin Mesylate (PEF) 33. Norfloxacin (NOR) 34. Ciprofloxacin (CIP) 35. Enrofloxacin (ENR) 36. Daflonaxyl-DAN 37. Lomefloxacin Hydrochloride (LOM) 38. Obifacin (ORB) 39. Difloxacin (DIF) 40. Sarafloxacin (SAR) 41. Gatifloxacin (GAT) 42. Moxifloxacin (MXF) 43. Sinoxacin (CNX) 44. Oxyquinic Acid (OXA) 45. Nasalidine Diethylcarboxylic Acid (NAL) 46. Flumethylquine (FLU) Figure (C): 47. Isochlortetracycline (ICTC) 48. Oxytetracycline (OTC) 49. 4-ECTC (4-Achromic Acid) 50. Doxycycline (DOX) 51. Acid-dehydrotetracycline (ATC) 52. Acid-dehydrotetracycline (4-EATC) Figure (D): 53. Sulfamethazine- 13 C654. Sulfamethoxypyrimidine 13 C655. Trimethoprim-d3 56. Sulfamethazine-d4 57. Sulfamethoxazole-d4 58. Ofloxacin-d3 59. Doxycycline-d3.

[0022] Figure 3 shows the chemical structural formulas of 52 target compounds for the analytical method of this application for detecting multiple antibiotic residues in edible parts of vegetables. Among them, (A) represents 26 sulfonamide antibiotics, (B) represents 20 quinolone antibiotics, and (C) represents 6 tetracycline antibiotics.

[0023] Figure 4 This is a structural block diagram of an analytical system for detecting multiple antibiotic residues in edible parts of vegetables according to the present invention. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] This application provides a complete solution for the simultaneous detection of multiple antibiotic residues in edible parts of vegetables. These vegetables include, but are not limited to, lettuce, tomatoes, green peppers, and red peppers. The core of this solution lies in combining an optimized sample pretreatment process, high-resolution mass spectrometry analysis conditions, and a database containing information on 52 target compounds to achieve high-throughput, high-accuracy screening and quantification.

[0026] This application provides an analytical method for detecting multiple antibiotic residues in edible parts of vegetables, capable of simultaneously detecting 52 antibiotic residues across three major categories. For example... Figure 1 As shown, this method mainly includes three stages: S1: Vegetable samples are pretreated to obtain the analysis solution; The purpose of this step is to efficiently extract and purify the target antibiotic from the complex vegetable matrix to prepare a detection solution suitable for instrumental analysis. Taking lettuce samples as an example, approximately 2.0 g of homogenized sample is first weighed. To ensure the accuracy of subsequent detection, a known amount of isotopic internal standard mixed solution is added at the beginning of sample processing to correct for recovery and matrix effects throughout the process. Then, through a series of physicochemical treatments (such as adding salting-out agents, using extraction solvents in specific ratios, vortexing, sonication, centrifugation, etc.), the antibiotic is transferred from the sample to an organic solvent. Next, the extract is concentrated to enrich the target analyte and purified using a solid-phase extraction (SPE) column to remove a large amount of interfering substances such as pigments, sugars, and organic acids from the sample, finally obtaining a clear detection solution for analysis. This series of steps ensures the method's universality and high recovery rate across different types of vegetable matrices.

[0027] S2: The test solution is analyzed using ultra-high performance liquid chromatography-tandem high resolution mass spectrometry to obtain the mass spectrometry information of the compounds in the test solution; wherein, the mass spectrometry information includes retention time, fragment ion information and precursor ion mass information; The pretreated detection solution was injected into an ultra-high performance liquid chromatography-tandem high resolution mass spectrometry (UPLC-HRMS) system for analysis. Preferably, chromatographic separation was performed on a C18 reversed-phase column using a methanol-water system containing formic acid as the mobile phase, and a specific gradient elution program was executed. This program was carefully optimized to achieve effective separation of 52 target antibiotics and their internal standards within approximately 30 minutes, particularly successfully separating several pairs of isomers (such as dihydrotetracycline). Mass spectrometry detection employed electrospray ionization (ESI) in positive ion mode. Based on the precise molecular weight of the target compound, information on its precursor ion and characteristic fragment ions was acquired using multiple reaction monitoring (MRM) or similar highly selective scanning modes. This mass spectrometry information (including retention time, precursor ion mass, fragment ion mass-to-charge ratio, and abundance) is crucial for subsequent compound identification.

[0028] S3: Based on the mass spectrometry information of the compounds in the detection solution, compare it with the preset standard antibiotic compound information database to identify and quantify the sulfonamides, quinolones and tetracyclines present in the vegetable sample; This application pre-constructs a "standard antibiotic compound information database," which includes standardized information for 52 target compounds (covering 26 sulfonamides, 20 quinolones, and 6 tetracyclines, see Figure 3), such as: Chinese name, English name, CAS number, molecular formula, protonated molecular ion (M+H), optimized chromatographic retention time, characteristic fragment ions, and their collision energies. When analyzing actual samples, the data processing software automatically compares the measured precise mass of each chromatographic peak in the sample solution with the theoretical mass in the database (with an allowable error range, e.g., ±5 ppm), quickly screening out "suspected target peaks." Then, it further compares the retention time and secondary mass spectrometry fragmentation map of these suspected peaks with the standard information in the database. Only compounds that match all three (protonated molecular ion, retention time, and fragmentation map) are finally confirmed. For confirmed compounds, the system automatically calculates their accurate concentration in the original vegetable sample based on the ratio of their chromatographic peak area to the corresponding isotopic internal standard, combined with a pre-established internal standard curve.

[0029] This application provides a further optimized and detailed process for step S1 described above. Specifically, it can be divided into the following four sub-steps: S11: Grinding and pretreatment of vegetable samples. Fresh or frozen vegetable samples (e.g., tomatoes) are thoroughly homogenized using a clean grinding device. A precise amount (e.g., 2.0 g) of the homogenized sample is placed in a centrifuge tube. Before solvent extraction, a pretreatment agent is added to the sample. Preferably, disodium ethylenediaminetetraacetate (Na2EDTA) is added, which can integrate metal ions and improve the extraction efficiency of certain antibiotics (e.g., tetracyclines). Simultaneously, sodium chloride (NaCl) and anhydrous sodium sulfate (Na2SO4) are added to utilize salting-out and absorb excess water, promoting the partitioning of the target analyte into the organic phase.

[0030] S12: Add the isotope internal standard mixed solution and extraction solvent to the pretreated sample, perform extraction, and combine the extraction supernatants. Add a mixed internal standard solution containing multiple stable isotope-labeled antibiotics to the sample from the previous step. After standing for a period of time to allow the internal standard to fully bind with the sample matrix, add the extraction solvent. This application has found that using a mixed solution composed of methanol, acetonitrile, and a certain proportion of hydrochloric acid as the extraction solvent can achieve efficient and simultaneous extraction of sulfonamides, quinolones, and tetracyclines with a wide polarity range. Extraction is fully carried out by vortexing and ultrasonic assistance, followed by centrifugation, and the supernatant is collected. This extraction process is usually repeated 2-3 times to ensure complete extraction, and all extraction supernatants are combined.

[0031] S13: Concentrate and redissolve the combined extract supernatant. The combined extract is concentrated to near dryness by purging with high-purity nitrogen under gentle heating (e.g., a 40°C water bath). Immediately redissolve the residue with a small amount of ultrapure water and vortex to mix. This step aims to transfer the target compound from a large volume of organic extraction solvent to a small volume of aqueous phase, preparing for subsequent solid-phase extraction purification steps.

[0032] S14: The target compound in the reconstituted solution is enriched and purified using a solid-phase extraction (SPE) column. After elution, the analytical solution is obtained. The aqueous reconstituted solution is loaded onto a pre-activated SPE column. The packing material of the SPE column is selected, preferably an HLB packing material of lipophilic-hydrophilic balance, which has good retention capacity for a broad spectrum of organic compounds. After loading, the column is rinsed with ultrapure water to remove some water-soluble interfering substances. After drying the column, the target antibiotic adsorbed on the column is eluted with pure methanol. The eluent is collected, dried again with nitrogen, and finally accurately reconstituted with a small amount of chromatographically pure methanol (e.g., 0.5 mL). After centrifugation, the supernatant is collected to obtain the clean final analytical solution, which is placed in a vial for instrument analysis.

[0033] Furthermore, the embodiments of this application provide preferred solutions for the reagents added in sample pretreatment step S11 and their dosages. Experiments show that for approximately 2.0g of vegetable sample, the simultaneous addition of 0.2g Na2EDTA, 0.1g NaCl, and 0.2g anhydrous Na2SO4 produces a synergistic effect, achieving stable and efficient extraction recoveries in various vegetable matrices. Na2EDTA primarily acts on tetracycline antibiotics, NaCl provides a salting-out effect, while anhydrous Na2SO4 effectively adsorbs residual moisture in the homogenized sample, preventing moisture from reducing the efficiency of the extraction solvent. This formulation is the result of systematic optimization and is well-suited for vegetable samples with high moisture content or complex compositions, such as lettuce and peppers.

[0034] Furthermore, the composition of the extraction solvent in step S12 of the embodiments of this application is specifically defined. Comparative experiments revealed that using a mixed solution of methanol, acetonitrile, and hydrochloric acid (concentration approximately 37%) in a volume ratio of 48:48:4 as the extraction solvent yielded significant results. The mixture of methanol and acetonitrile provides broad solubility for compounds of different polarities, while the addition of a small amount of hydrochloric acid creates a slightly acidic environment. This is crucial for improving the extraction efficiency of antibiotics that are more stable under acidic conditions (such as quinolones and tetracyclines), and also helps to better release the target analytes from the matrix in molecular form. This ratio is one of the keys to the method's ability to achieve "one-step extraction" of 52 antibiotics from three major classes.

[0035] Furthermore, embodiments of this application specifically specify the isotopic internal standard used in step S12. Preferably, the mixed internal standard solution comprises the following seven stable isotope-labeled compounds: sulfamethazine- 13 C6, sulfamethoxypyrimidine 13 C6, trimethoprim-d3, sulfadiazine-d4, sulfamethoxazole-d4, ofloxacin-d3, and doxycycline-d3. These internal standards correspond to representative compounds in the sulfonamide, quinolone, and tetracycline classes, respectively. Their chemical properties are extremely similar to the target analytes, and they experience essentially the same losses and matrix effects during sample pretreatment and instrumental analysis. By monitoring the responses of these internal standards, the determination results of corresponding categories or even specific compounds in each sample can be accurately corrected, thereby greatly improving the accuracy and precision of quantitative analysis, especially in the complex and variable vegetable matrix.

[0036] Furthermore, the embodiments of this application provide details of the solid-phase extraction purification step S14. Oasis HLB or an equivalent polymer adsorbent solid-phase extraction column (e.g., 200 mg packing material, 6 mL column volume) is preferably used. During operation, the packing material is first moistened and activated with an appropriate amount of methanol, then the methanol is replaced with ultrapure water to prepare for sample loading. The reconstituted sample aqueous solution is passed through the activated column at a controlled flow rate (e.g., approximately 3 mL / min), at which point the target antibiotic is retained on the column. Subsequently, sufficient ultrapure water is used for rinsing to remove residual salts and strongly polar interfering substances. After the water in the column is removed as much as possible using a vacuum pump or positive pressure, it is eluted with pure methanol, and the enriched and purified target analyte is collected in a test tube. This HLB column purification step effectively removes large amounts of interfering substances such as chlorophyll and acidic pigments from vegetable extracts, which is key to obtaining clean chromatograms and low background noise.

[0037] Furthermore, embodiments of this application provide a preferred procedure for ultra-high performance liquid chromatography gradient elution in step S2. A C18 column with an inner diameter of 2.1 mm, a length of 100 mm, and a packing particle size of 2.5 μm is used, and the column temperature is maintained at 30°C. Mobile phase A is an aqueous solution containing 1 mmol / L formic acid, and mobile phase B is a methanol solution containing 1 mmol / L formic acid. The flow rate is set to 0.25 mL / min. The specific settings of the gradient elution procedure are as follows: 0 min, mobile phase A 90%, mobile phase B 10%; 2 min, mobile phase A 80%, mobile phase B 20%; 8 min, mobile phase A 68%, mobile phase B 32%; 12 min, mobile phase A 65%, mobile phase B 35%; 17 min, mobile phase A 35%, mobile phase B 65%; 20 min, mobile phase A 30%, mobile phase B 70%; 21 min, mobile phase A 5%, mobile phase B 95%; 24 min, mobile phase A 5%, mobile phase B 95%; 25 min, mobile phase A 90%, mobile phase B 10%; 29 min, mobile phase A 90%, mobile phase B 10%.

[0038] The procedure begins with a high aqueous phase, gradually increasing the organic phase ratio to allow the more polar sulfonamides to elute first. In the middle stage, the organic phase ratio is rapidly increased to elute moderately polar quinolones and some sulfonamides. In the final stage, a high proportion of organic phase is used to wash away strongly retained tetracyclines and their isomers, and the initial conditions are quickly restored after the analysis to equilibrate the column. This gradient has been optimized to achieve good baseline separation of all 52 target compounds and the internal standard within a total run time of 29 minutes.

[0039] Furthermore, the embodiments of this application specify the mass spectrometry detection conditions for step S2. The mass spectrometry section preferably uses a high-resolution mass spectrometer (such as Orbitrap or Q-TOF) equipped with an electrospray ionization source. The ionization source operates in positive ion mode, and the source temperature is set to 320°C. Multiple reaction monitoring (MRM) mode is used for data acquisition based on the precursor ion characteristics of 52 target antibiotics. The scan mass range covers 50 to 750 m / z to ensure the capture of all precursor ions and characteristic fragment ions of the target analytes. For the collision-induced dissociation process, a stepped collision energy (e.g., 10 eV, 30 eV, and 50 eV respectively) is used to obtain abundant fragment ion information suitable for qualitative and quantitative analysis. These conditions collectively ensure the high sensitivity and high selectivity of the method.

[0040] Furthermore, the embodiments of this application refine the logical flow of data processing and quantification in step S3.

[0041] S31: Match the precursor ion mass information of the compound in the detection solution with the precursor ion mass information of each standard antibiotic in the database, with an error range within ±5 ppm, to screen out suspected target compounds. First, extract the precise precursor ion mass of all chromatographic peaks in the sample data and compare it with the theoretical precise mass of each antibiotic in the built-in database. Set a strict mass tolerance window (e.g., ±5 ppm). Chromatographic peaks falling within this window are marked as "suspected target compounds".

[0042] S32: The retention time and fragment ion information of the suspected target compound are compared with the retention time and fragment ion information of the corresponding standard antibiotics in the database for confirmation. For the suspected peaks initially screened, their chromatographic retention times are further compared to see if they are consistent with the retention times of the corresponding standards in the database (minor deviations are allowed, such as ±0.2 min). More importantly, the mass and relative abundance of the secondary mass spectrometry fragment ions (such as the 2-3 most intense characteristic fragments) generated at specific collision energies are compared to see if they match the standard fragment spectra in the database. Only compounds that pass the triple verification of accurate mass, retention time, and fragment spectra are finally confirmed as "detected".

[0043] S33: Using the internal standard method, quantitative analysis is performed on confirmed target compounds based on the peak area ratio of the target compound and its corresponding isotopic internal standard, as well as the standard curve. For each confirmed target compound, the system finds the chromatographic peak of its corresponding isotopic internal standard (or the internal standard with the closest chemical structure). The ratio of the target compound's peak area to the internal standard peak area is calculated. This ratio is then substituted into the internal standard curve equation (linear or nonlinear fitting) pre-established using a series of concentration standards for the compound to automatically calculate its concentration in the final detection solution. This concentration is then converted to the residue level (e.g., μg / kg) in the original vegetable sample based on the sample weight and final volume. This process minimizes matrix effects.

[0044] Furthermore, this application also discloses a system for implementing the analytical method for detecting multiple antibiotic residues in edible parts of vegetables, such as... Figure 4 As shown, it includes: The pretreatment module is used to pretreat vegetable samples to obtain the analytical solution. The mass spectrometry analysis module is used to analyze the test solution using an ultra-high performance liquid chromatography-tandem high resolution mass spectrometer to obtain the mass spectrometry information of the compounds in the test solution; The data processing module is used to compare the mass spectrometry information of the compounds in the detection solution with a preset standard antibiotic compound information database to identify and quantify the sulfonamides, quinolones and tetracyclines present in the vegetable sample. The preset standard antibiotic compound information database contains standard mass spectrometry information for 52 target antibiotics.

[0045] It is understood that the methods and systems of this application are executed using electronic devices. These devices include at least one processor and a memory. The memory stores computer program instructions and the aforementioned "standard antibiotic compound information database." When the processor executes the computer program instructions, it controls the electronic device to perform the following functions: receive or read raw mass spectrometry data files acquired by the UPLC-HRMS system; automatically execute the data processing flow as described in the embodiments (including mass number matching, retention time and fragmentation verification, internal standard quantification, etc.); and finally generate and output a detection report containing compound identification results and quantitative concentrations. This electronic device can be a dedicated data processing workstation connected to the mass spectrometer, or it can be a standalone server or computer. Its core function is to automate and intelligently implement the innovative analytical method flow of this application by running dedicated software.

[0046] To verify the effectiveness of the method in this application, detailed experimental data is provided, which are described below in the form of embodiments: 1. Establish standard curves for 52 antibiotic compounds. Standards of 26 sulfonamide antibiotics, 20 quinolone antibiotics, and 6 tetracycline antibiotics (as shown in Table 1) were diluted to 5 mg / L with methanol (HPLC-grade) as stock solutions. These stock solutions were then serially diluted to concentration gradients from 0.001 to 1000 μg / L (with a fixed isotope concentration of 20 μg / L in each gradient). The target compounds in the mixed standard solutions were analyzed using ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry (UPLC-HRMS). Standard curves were plotted based on the ratio of the peak area of ​​the target compound to the peak area of ​​the isotope and the corresponding concentration, which were used for subsequent calculation of the concentration in actual vegetable samples. The limits of detection (LOD) and limits of quantitation (LOQ) for these compounds were evaluated. The standard curve parameters are shown in Table 2.

[0047] Table 1: Basic Information on 52 Antibiotics and 7 Isotope Compounds in 3 Categories

[0048] Table 2: Parameters of Quantitative Standard Curves for 52 Antibiotic Compounds in 3 Classes

[0049] 2. Establishment of sample pretreatment methods (1) Preparation of recycled samples Clean, uncontaminated vegetable samples were selected. Clean 15mL plastic centrifuge tubes and 3mm steel balls were prepared. The samples were cleaned by sonication with purified water for 10 minutes, followed by sonication with methanol (chromatographic grade) for 10 minutes. A stainless steel grinder was cleaned with water and methanol, and the clean vegetable samples were ground using the grinder. The ground samples were then placed in an aluminum box and stored at -20℃. 2g of each of the four ground vegetable samples (lettuce, tomato, red pepper, and green pepper) were weighed out in triplicate. A blank (2g) was also prepared for each vegetable sample, resulting in a total of 12 samples and 4 blanks. All samples and blanks were weighed three times, for a total of 36 samples and 12 blanks. Each sample was placed into 48 15mL plastic centrifuge tubes and labeled. To ensure thorough mixing, three cleaned 3mm steel balls were added to each centrifuge tube for later recovery experiments.

[0050] (2) Recovery experiment of mixed standard solutions of different concentrations Prepare clean 50mL and 15mL plastic centrifuge tubes. Glassware used in the experiment was washed three times with ultrapure water, sonicated for 10 min, cleaned three times with methanol (chromatographic grade), sonicated for 10 min, and washed three times with ultrapure water. The glassware was then baked in a muffle furnace at 450℃ for 4 h to remove interference from impurities. The clean vegetable samples were divided into three groups, each containing 12 vegetable samples (4 types of vegetables, 3 replicates of each type) and 4 blanks (1 sample of each type of vegetable). 10 μL of a 1 mg / L or 2.5 mg / L isotope internal standard mixture (sulfamethazine-...) was added to each of the three groups. 13 C6, sulfamethoxypyrimidine 13 C6, trimethoprim-d3, sulfadiazine-d4, sulfamethoxazole-d4, ofloxacin-d3, and doxycycline-d3 (doxycycline concentration 2.5 mg / L, other isotopes concentration 1 mg / L), were then added to a mixed standard solution containing 52 antibiotics at concentrations of 1 mg / L, 5 mg / L, and 5 mg / L, respectively. This resulted in an isotope spike concentration of 20 μg / L (doxycycline-d3 spike concentration 50 μg / L), and non-isotope compound spike concentrations of 5 μg / L, 20 μg / L, and 100 μg / L, respectively. After spiking, the solutions were covered with aluminum foil and allowed to stand for 2 hours before further processing.

[0051] Add 0.2g Na2EDTA, 0.1g NaCl, and 0.2g anhydrous Na2SO4 (to prevent excess water from interfering with compound extraction) to the prepared vegetable sample centrifuge tube, and add 8ml of methanol / acetonitrile / hydrochloric acid (48%, 48%, and 4%) mixed extraction solution to extract the target compound. The samples were vortexed at 2500 rpm for 10 min using a Kylin-Bell vortex mixer, sonicated at 100 kHz for 10 min, and centrifuged at 8000 rpm for 5 min. The supernatant was then transferred to a 50 mL plastic centrifuge tube. For the vegetable samples, 0.2 g Na2EDTA and 8 mL of a methanol / acetonitrile / hydrochloric acid (48%, 48%, and 4%) mixed extraction solution were added. The samples were vortexed at 2500 rpm for 10 min, sonicated at 100 kHz for 10 min, and centrifuged at 8000 rpm for 5 min. The supernatant was then transferred to a 50 mL plastic centrifuge tube. For the third extraction of the vegetable samples, another 8 mL of the methanol / acetonitrile / hydrochloric acid (48%, 48%, and 4%) mixed extraction solution was added. The vortexing, sonication, and centrifugation steps were repeated, and the supernatant was transferred to a 50 mL plastic centrifuge tube. The combined three extraction solvents were collected in a 50 mL centrifuge tube and concentrated to near dryness using high-purity nitrogen (using a nitrogen blowdown apparatus heated in a 40°C water bath). The solvent was immediately redissolved in 4 mL of ultrapure water and vortexed until homogeneous (approximately 2 min). An HLB solid-phase extraction column (200 mg, 6 mL) was selected for enrichment and purification of the target compound. a. Activation: The solid-phase extraction column was activated by sequentially adding 6 mL of methanol and 6 mL of ultrapure water; b. Sample loading: Using a Supelco 24-tube SPE solid-phase extraction apparatus (USA), 4 mL of the redissolved sample was passed through the activated HLB solid-phase extraction column at a flow rate of approximately 3 mL / min to enrich the target compound. Then, 2 mL of ultrapure water was added to the centrifuge tube to redissolve any remaining target compound, and the mixture was vortexed until homogeneous. The mixture was then passed through the activated HLB solid-phase extraction column at a flow rate of approximately 3 mL / min. After sample loading, the HLB solid-phase extraction column was rinsed with 6 mL of ultrapure water. After rinsing, the aqueous solution in the solid-phase extraction column was dried by frequently changing the vacuum level using a YM-20 diaphragm vacuum pump. c. Elution: The target compound was eluted from the solid-phase extraction column using 12 mL of methanol solution, and the eluent was collected in a 15 mL plastic centrifuge tube. The eluent was dried completely under high-purity nitrogen gas in a 40°C water bath, and immediately reconstituted in 0.5 mL of methanol solution, vortexed until homogeneous. The reconstituted solution was transferred to a 1.5 mL plastic centrifuge tube and centrifuged at 7500 g for 10 min. The supernatant was immediately transferred to a 1.5 mL brown vial (with a 0.5 mL glass liner) and stored at -20°C for later analysis.

[0052] (3) Parameter optimization of ultra-high performance liquid chromatography tandem high resolution mass spectrometry The target compounds in the spiked recovery sample extracts were determined using ultra-high performance liquid chromatography (UPLC) tandem high resolution mass spectrometry (HRMS). The recovery rate of each target compound was calculated as (spike sample concentration - unspike (blank) sample concentration) / added standard concentration × 100%. The recovery rates of the three different concentrations of mixed standard solutions in the four vegetable samples are shown in Table 3-6.

[0053] Table 3 Recovery rates of lettuce at different spiked concentrations (5 μg / L, 20 μg / L, 100 μg / L)

[0054] Table 4 Recovery rates of tomatoes at different spiked concentrations (5 μg / L, 20 μg / L, 100 μg / L)

[0055] Table 5 Recovery rates of green peppers at different spiked concentrations (5 μg / L, 20 μg / L, 100 μg / L)

[0056] Table 6 Recovery rates of red chili peppers at different spiked concentrations (5 μg / L, 20 μg / L, 100 μg / L)

[0057] The method described in this application achieves recoveries ranging from 51% to 130% at the three spiked concentrations for each sample. The method's precision, expressed as relative standard deviation (RSD), is 0-14% in vegetable samples. According to the Japanese Ministry of Land, Infrastructure, Transport and Tourism's manual for the analysis of endocrine-disrupting compounds, recoveries between 50% and 120% and an RSD below 20% are considered acceptable. High recoveries of organic compounds are often due to differences in absolute recoveries between the target compound and its internal standard; therefore, an acceptable recovery rate for organic compounds is recommended to be between 50% and 150%. The recoveries of each target compound in Tables 3-6 indicate that the recoveries of all antibiotic compounds involved in this application in the four vegetable samples generally meet the quality control requirements. The linearity, sensitivity, recovery, and precision of this method all demonstrate its reliability and applicability for extracting antibiotic compounds with similar properties from vegetable samples.

[0058] The purpose of the recovery experiment by adding mixed standard solutions of three different concentrations (5 μg / L, 20 μg / L, and 100 μg / L) to four vegetable samples was to verify that the developed extraction method is reliable and can be used to extract actual samples, ensuring that the target compounds in most actual samples can be effectively extracted by this method.

[0059] The concentration of the internal standard mixture added to both the actual sample and the spiked recovery sample was 20 μg / L, as determined by the instrument. At this concentration, the UPLC-HRMS instrument was most stable and had the most sensitive response. Therefore, when extracting actual vegetable samples, a mixture of internal standard compounds labeled with isotopes of 20 μg / L was selected for quantification of antibiotic compounds in the actual samples.

[0060] 2. Quantitative analysis of antibiotic compounds in edible parts of vegetable samples (1) Sample collection and extraction Four types of vegetables were collected from the field: 7 lettuce, 11 tomatoes, 7 green peppers, and 7 red peppers, for a total of 32 samples. 72 clean 15mL centrifuge tubes, 36 clean 50mL centrifuge tubes, and 36 clean 1.5mL centrifuge tubes were prepared. All other glassware used was washed three times with ultrapure water and three times with methanol (chromatographic grade), then baked in a muffle oven at 450℃ for 4 hours to remove impurities. For each of the 32 vegetable samples, 2±0.02g was weighed and placed in a 15mL plastic centrifuge tube. A blank control group was also set up, with 2g of clean, uncontaminated lettuce, tomato, green pepper, and red pepper weighed and placed in a 15mL centrifuge tube as a blank control. 10μL of a 1mg / L / 2.5mg / L isotope internal standard mixed solution (sulfamethazine-) was added to each of the 32 actual samples and the 4 blanks. 13 C6, sulfamethoxypyrimidine 13 C6, trimethoprim-d3, sulfadiazine-d4, sulfamethoxazole-d4, ofloxacin-d3, and doxycycline-d3 (doxycycline concentration was 2.5 mg / L, and the concentrations of the other isotopes were 1 mg / L). After standing for 2 hours, the vegetable samples were processed: Add 0.2g Na2EDTA, 0.1g NaCl, and 0.2g anhydrous Na2SO4 (to prevent excess water from interfering with compound extraction) to the prepared vegetable samples and vegetable blank centrifuge tubes. Add 8ml of methanol / acetonitrile / hydrochloric acid (48%, 48%, and 4%) mixed extraction solution to extract the target compound. The samples were vortexed at 2500 rpm for 10 min using a Kylin-Bell vortex mixer, sonicated at 100 kHz for 10 min, and centrifuged at 8000 rpm for 5 min. The supernatant was transferred to a 50 mL plastic centrifuge tube. For the vegetable samples, 0.2 g Na₂EDTA was added to the centrifuge tube, followed by 8 mL of a methanol / acetonitrile / hydrochloric acid (48%, 48%, and 4%) mixed extraction solution. The above extraction steps were repeated, and the supernatant was transferred to a 50 mL plastic centrifuge tube. For the third extraction of the vegetable samples, another 8 mL of the methanol / acetonitrile / hydrochloric acid (48%, 48%, and 4%) mixed extraction solution was added, and the above extraction steps were repeated. The supernatant was transferred to a 50 mL plastic centrifuge tube. The combined extracts from the three extractions were collected in a 50 mL centrifuge tube and concentrated to near dryness using high-purity nitrogen (using a nitrogen blower heated in a 40°C water bath). The concentrated extract was immediately reconstituted in 4 mL of ultrapure water and vortexed until homogeneous (approximately 2 min). HLB solid-phase extraction (SPE) columns (200 mg, 6 mL) were used to enrich and purify the target compounds. a. Activation: The SPE column was activated by sequentially adding 6 mL of methanol and 6 mL of ultrapure water. b. Sample loading: Using a Supelco 24-tube SPE apparatus, 4 mL of the reconstituted sample was passed through the activated HLB SPE column at a flow rate of approximately 3 mL / min to enrich the target compounds. Then, 2 mL of ultrapure water was added to the centrifuge tube to reconstitute any remaining target compounds, and the mixture was vortexed until homogeneous. The mixture was then passed through the activated HLB SPE column at a flow rate of approximately 3 mL / min. After sample loading, the HLB SPE column was rinsed with 6 mL of ultrapure water. After rinsing, the aqueous solution in the SPE column was dried using a YM-20 diaphragm vacuum pump with frequent vacuum adjustments. c. Elution: The target compounds were eluted from the SPE column using 12 mL of methanol solution, and the eluent was collected in a 15 mL plastic centrifuge tube. The eluent was dried completely under high-purity nitrogen gas in a 40°C water bath, and immediately reconstituted in 0.5 mL of methanol solution. The solution was then vortexed until homogeneous. The reconstituted solution was transferred to a 1.5 mL plastic centrifuge tube and centrifuged at 7500 g for 10 min. The supernatant was immediately transferred to a 1.5 mL brown vial (with a 0.5 mL glass liner) and stored at -20°C for later analysis.

[0061] (2) Instrumental analysis Analytical instrument: Ultra-high performance liquid chromatography (UPLC) tandem high resolution mass spectrometry (HRMS) Column: Waters XBridge® BEH C18 column (2.5 μm, 2.1 × 100 mm) Column temperature: 30℃ Mobile phase: A: H2O (containing 1 mM formic acid), B: methanol (containing 1 mM formic acid) Flow rate: 0.25 ml / min Injection volume: 5 μL Washing solution for needles: Strong wash: methanol (100%), weak wash: methanol / water (85% / 15%) The mobile phase gradient is shown in Table 7: Table 7 Gradient elution parameters of the mobile phase

[0062] Sample signals were acquired using a midpoint spray ionization (ESI) source in positive ion mode on a mass spectrometer via Full scan mode. The MS scan range was 50-750 m / z, the ion source temperature was 320 °C, and the auxiliary gas flow rate was 10 arb. A stepped collision energy mode was employed, with collision energies of 10 eV, 30 eV, and 50 eV.

[0063] (3) Quantitative analysis of the target compound The internal standard method was used to quantitatively analyze the target compounds in vegetable samples. Xcalibur software was used to integrate the peak areas of the target compounds and isotopes in the samples, and the ratio of the peak area of ​​the target compound to that of the corresponding isotope was calculated. The results were compared with an existing standard curve to confirm the quantitative analysis of the target compounds in the vegetable samples using the internal standard method.

[0064] (4) Test results Taking vegetables as an example, quantitative analysis results showed that among the 52 antibiotics involved in this application, 3 sulfonamide antibiotics, 14 quinolone antibiotics, and 2 tetracycline antibiotics were detected in vegetable samples. The average concentrations of sulfonamides in vegetable samples ranged from -10.47 μg / kg (ND), quinolone antibiotics from -51.70 μg / kg (ND), and tetracycline antibiotics from -6.90 μg / kg (ND).

[0065] This application conducted spiked recovery experiments by adding a mixture of 52 antibiotic standards at low, medium, and high concentrations to four blank matrices: lettuce, tomato, green pepper, and red pepper. As shown in Tables 3 to 6 and Figure 2, the recoveries of most compounds in the four matrices ranged from 50% to 130%, with relative standard deviations (RSD) of less than 20%, demonstrating that the method is accurate, reliable, and applicable to different vegetables.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An analytical method for detecting multiple antibiotic residues in edible parts of vegetables, characterized in that, Includes the following steps: S1: Vegetable samples are pretreated to obtain the analysis solution; S2: The test solution is analyzed using ultra-high performance liquid chromatography-tandem high resolution mass spectrometry to obtain the mass spectrometry information of the compounds in the test solution; wherein, the mass spectrometry information includes retention time, fragment ion information and precursor ion mass information; S3: Based on the mass spectrometry information of the compounds in the detection solution, compare it with the preset standard antibiotic compound information database to identify and quantify the sulfonamides, quinolones and tetracyclines present in the vegetable sample; The preset standard antibiotic compound information database contains standard mass spectrometry information for 52 target antibiotics, including 26 sulfonamide antibiotics, 20 quinolone antibiotics and 6 tetracycline antibiotics.

2. The method according to claim 1, characterized in that, Step S1 includes: S11: Crush and pre-treat vegetable samples; S12: Add the isotope internal standard mixed solution and extraction solution to the pretreated sample, extract, and combine the extraction supernatants; S13: Concentrate and reconstitute the combined extract supernatant; S14: The target compound in the complex solution is enriched and purified using a solid-phase extraction column, and the analyte solution is obtained after elution.

3. The method according to claim 2, characterized in that, The pretreatment in step S11 includes adding Na2EDTA, NaCl and anhydrous Na2SO4 to the vegetable sample; Each 2g vegetable sample contained 0.2g Na2EDTA, 0.1g NaCl, and 0.2g anhydrous Na2SO4.

4. The method according to claim 2, characterized in that, In step S12, the extract is a mixed solution of methanol, acetonitrile and hydrochloric acid in a volume ratio of 48:48:

4.

5. The method according to claim 2, characterized in that, In step S12, the isotope internal standard mixed solution contains sulfamethazine- 13 C6, sulfamethoxypyrimidine 13 C6, trimethoprim-d3, sulfadiazine-d4, sulfamethoxazole-d4, ofloxacin-d3, and doxycycline-d3.

6. The method according to claim 2, characterized in that, In step S14, the solid-phase extraction column is an HLB solid-phase extraction column; The enrichment and purification process includes: activating the HLB solid-phase extraction column sequentially with methanol and ultrapure water; loading the reconstituted solution onto the column; rinsing with ultrapure water; and eluting the target compound with methanol after drying.

7. The method according to claim 1, characterized in that, In step S2, the ultra-high performance liquid chromatography uses a C18 column, with mobile phase A being an aqueous solution containing 1 mM formic acid and mobile phase B being a methanol solution containing 1 mM formic acid, and gradient elution is performed. The gradient elution procedure is as follows: 0 min, mobile phase A 90%, mobile phase B 10%; 2 min, mobile phase A 80%, mobile phase B 20%; 8 min, mobile phase A 68%, mobile phase B 32%; 12 min, mobile phase A 65%, mobile phase B 35%; 17 min, mobile phase A 35%, mobile phase B 65%; 20 min, mobile phase A 30%, mobile phase B 70%; 21 min, mobile phase A 5%, mobile phase B 95%; 24 min, mobile phase A 5%, mobile phase B 95%; 25 min, mobile phase A 90%, mobile phase B 10%; 29 min, mobile phase A 90%, mobile phase B 10%.

8. The method according to claim 1, characterized in that, In step S2, the tandem high-resolution mass spectrometry uses an electrospray ionization source, positive ion mode, and multiple reaction monitoring (MRM) scanning mode.

9. The method according to claim 1, characterized in that, Step S3 specifically includes: S31: Match the precursor ion mass information of the compound in the test solution with the precursor ion mass information of each standard antibiotic in the database, with an error range within ±5ppm, and screen out suspected target compounds; S32: Compare and confirm the retention time and fragment ion information of the suspected target compound with the retention time and fragment ion information of the corresponding standard antibiotics in the database; S33: Using the internal standard method, quantitative analysis is performed on the confirmed target compound based on the peak area ratio of the target compound and the corresponding isotopic internal standard and the standard curve.

10. A system for implementing the analytical method for detecting multiple antibiotic residues in edible parts of vegetables as described in claim 1, characterized in that, include: The pretreatment module is used to pretreat vegetable samples to obtain the analytical solution. The mass spectrometry analysis module is used to analyze the test solution using an ultra-high performance liquid chromatography-tandem high resolution mass spectrometer to obtain the mass spectrometry information of the compounds in the test solution; The data processing module is used to compare the mass spectrometry information of the compounds in the detection solution with a preset standard antibiotic compound information database to identify and quantify the sulfonamides, quinolones and tetracyclines present in the vegetable sample. The preset standard antibiotic compound information database contains standard mass spectrometry information for 52 target antibiotics.

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