A method for simultaneously detecting multiple antibiotic residues in pre-prepared dishes

Abstract

The present application relates to the technical field of food detection, and more particularly to a method for simultaneously detecting multiple antibiotic residues in prepared dishes. The method comprises the following steps: (1) mixing prepared dishes, a mixed internal standard solution and an extraction solution, ultrasonicating, centrifuging, taking supernatant, and obtaining a mixed solution; (2) purifying the mixed solution, drying, and obtaining a dried product; (3) mixing the dried product with a redissolution solution, centrifuging, taking supernatant, and obtaining a to-be-tested sample after membrane filtration; and (4) detecting the content of veterinary drug residues in the to-be-tested sample by high performance liquid chromatography-mass spectrometry. The method has simple sample processing steps, is convenient to operate, does not require complex derivatization treatment, has low detection cost, can realize rapid detection of batch samples, is suitable for both routine detection in laboratories and rapid screening on site by food supervision departments, and has important practical value for guaranteeing the safety of prepared dishes and standardizing the development of the industry.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, and in particular to a method for simultaneously detecting multiple antibiotic residues in prepared dishes. Background Technology

[0002] As a convenient and standardized food category, pre-cooked meals have become increasingly popular in the catering industry and in household consumption in recent years. Their ingredients often include livestock and poultry meat, aquatic products, and vegetables. During the breeding, processing, and storage processes, antibiotics may be overused to prevent diseases and extend shelf life, resulting in antibiotic residues in pre-cooked meals.

[0003] Enrofloxacin, norfloxacin, pefloxacin, ciprofloxacin, ofloxacin, and lomefloxacin belong to the quinolone class of antibiotics, while tetracycline, oxytetracycline, chlortetracycline, and doxycycline belong to the tetracycline class. Both classes of antibiotics are common food residues. Long-term consumption of prepared foods containing these antibiotic residues can pose potential health risks, such as disrupting gut microbiota balance, inducing bacterial resistance, and triggering allergic reactions. Some quinolone antibiotics may also affect bone development in minors, while tetracycline antibiotics may cause tooth discoloration and enamel hypoplasia. Therefore, establishing a rapid, accurate, and efficient method to simultaneously detect multiple antibiotic residues in prepared foods is of great significance for ensuring food safety and regulating the development of the prepared food industry.

[0004] Currently, the main methods for detecting antibiotic residues in food include enzyme-linked immunosorbent assay (ELISA), microbial detection, gas chromatography, liquid chromatography, and liquid chromatography-tandem mass spectrometry (LC-MS / MS). While enzyme-linked immunosorbent assay (ELISA) is simple to operate and low in cost, it has poor specificity, is easily affected by matrix interference, and is difficult to detect multiple antibiotics simultaneously. Microbial detection methods have low sensitivity and long detection cycles, failing to meet the needs of rapid detection. Gas chromatography requires derivatization of samples, which is cumbersome and unsuitable for detecting antibiotics with poor thermal stability. Although liquid chromatography can separate and detect multiple antibiotics, the complex pre-prepared vegetable matrix contains interfering substances such as oils, proteins, and pigments, resulting in insufficient detection sensitivity and accuracy, making it difficult to meet the requirements for detecting low concentrations of residues. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) combines the advantages of good separation, high sensitivity, and strong specificity, making it the mainstream technology for antibiotic residue detection. However, existing detection methods based on this technology are mostly for single-class antibiotics, or although they can detect multiple classes of antibiotics simultaneously, they suffer from problems such as cumbersome pretreatment steps, poor purification effects, and low detection efficiency. They are difficult to adapt to the complexity of pre-prepared vegetable matrices and cannot effectively eliminate errors in sample pretreatment and instrument analysis, making it difficult to guarantee the precision and accuracy of detection results.

[0005] Therefore, developing a simple and rapid detection method that can detect multiple antibiotics has become an urgent need in the field of veterinary drug residue detection in pre-cooked food. Summary of the Invention

[0006] The purpose of this invention is to provide a method for simultaneously detecting multiple antibiotic residues in prepared foods using an internal standard method. Specifically, it relates to a method for simultaneously detecting multiple antibiotic residues in prepared foods.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for simultaneously detecting multiple antibiotic residues in prepared dishes, comprising the following steps: (1) Mix the pre-cooked vegetables, mixed internal standard solution and extract, sonicate, centrifuge, and take the supernatant to obtain the mixture; (2) After purifying the mixture, dry it to obtain the dried product; (3) Mix the dried material with the reconstituted solution, centrifuge, take the supernatant, filter through a membrane, and obtain the sample to be tested; (4) High performance liquid chromatography-mass spectrometry was used to detect the content of veterinary drug residues in the sample. The antibiotics include quinolone antibiotics and tetracycline antibiotics; The quinolone antibiotics include enrofloxacin, difloxacin, pefloxacin, ciprofloxacin, ofloxacin, lomefloxacin, besifloxacin, and sarafloxacin; The tetracycline antibiotics include tetracycline, oxytetracycline, chlortetracycline, and doxycycline; The chromatographic conditions for the high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) detection were as follows: column: ACQUITY BEH C18 (2.1 mm × 100 mm, 1.7 μm), column temperature: 34~36℃, injection volume: 1.8~2.2 μL, mobile phase A was 0.08~0.12 vt% formic acid aqueous solution, mobile phase B was acetonitrile, and flow rate was 0.2~0.4 mL / min; The gradient elution program was as follows: 0–4 min, 10–40% mobile phase A, 90–60% mobile phase B; 4–8 min, 40–90% mobile phase A, 60–10% mobile phase B; 8–10 min, 90–99% mobile phase A, 10–1% mobile phase B; 10–10.1 min, 99–10% mobile phase A, 1–90% mobile phase B; 10.1–12 min, 10% mobile phase A, 90% mobile phase B. The mass spectrometry conditions for the high-performance liquid chromatography-mass spectrometry (HPLC-MS) detection are as follows: ionization mode: electrospray ionization positive ion mode (ESI). +The mass spectrometry scanning mode was: multiple reaction detection (MRM) scanning; capillary voltage was 1100~1200V; cone voltage was 25~30V; ion source temperature was 550~600℃; desolventizing temperature was 450~480℃; and desolventizing gas flow rate was 900~950L / h.

[0008] Preferably, the mixed internal standard solution comprises 20-30 ng / mL enrofloxacin-d5, 20-30 ng / mL difloxacin hydrochloride, 20-30 ng / mL pefloxacin-d5, 20-30 ng / mL ciprofloxacin hydrochloride-d8, 20-30 ng / mL ofloxacin-d3, 20-30 ng / mL lomefloxacin hydrochloride-d5, 20-30 ng / mL besifloxacin hydrochloride, 20-30 ng / mL sarafloxacin-d8, 20-30 ng / mL tetracycline-d6, 20-30 ng / mL oxytetracycline hydrochloride, 20-30 ng / mL chlortetracycline hydrochloride, and 20-30 ng / mL doxycycline-d3.

[0009] Preferably, the extract in step (1) is an aqueous solution of acetonitrile containing glacial acetic acid; The concentration of the glacial acetic acid is 0.5-1.5%; The concentration of the acetonitrile aqueous solution is 80-85%.

[0010] Preferably, the mass-to-volume ratio of the pre-prepared vegetable and the mixed internal standard solution in step (1) is 2~3g:100~150μL; The mass-to-volume ratio of the pre-prepared vegetables and the extract in step (1) is 2~3g:8~12mL.

[0011] Preferably, the power of the ultrasound in step (1) is 150~250W and the ultrasound time is 15~25min; The centrifugation speed in step (1) is 7000~9000 rpm, the centrifugation time is 8~12 min, and the centrifugation temperature is 2~6℃.

[0012] Preferably, the purification method in step (2) is through a solid-phase extraction column; the solid-phase extraction column is OasisPRiME HLB; The drying method described in step (2) is nitrogen drying.

[0013] Preferably, the reconstituted solution in step (3) is a 10% methanol aqueous solution.

[0014] Preferably, the centrifugation speed in step (3) is 4500~5500 rpm, the centrifugation time is 4~6 min, and the centrifugation temperature is 2~6℃.

[0015] Preferably, the pore size of the filter membrane in step (3) is 0.2~0.25μm.

[0016] The present invention also provides the application of the method in the rapid and simultaneous detection of quinolone antibiotic and tetracycline antibiotic residues in prepared dishes; The quinolone antibiotics include enrofloxacin, difloxacin, pefloxacin, ciprofloxacin, ofloxacin, lomefloxacin, besifloxacin, and sarafloxacin; The tetracycline antibiotics include tetracycline, oxytetracycline, chlortetracycline, and doxycycline.

[0017] Beneficial effects

[0018] This invention targets the physicochemical properties of quinolone and tetracycline antibiotics, selecting internal standards with similar structures and physicochemical properties to the target analytes. These standards maintain synchronous response during sample pretreatment and chromatographic-mass spectrometry analysis, effectively eliminating errors caused by sample loss, instrument fluctuations, matrix interference, and other factors, thus significantly improving the accuracy and precision of the detection results.

[0019] This invention rationally selects the extraction solution and solid-phase extraction column. The solid-phase extraction column selected in this invention can effectively remove interfering substances such as oils, proteins, and pigments from the sample, thereby improving the detection sensitivity.

[0020] The chromatographic-mass spectrometry conditions of this invention can achieve complete separation of multiple antibiotics within 12 minutes, with good separation effect, no overlapping peaks, and high detection efficiency.

[0021] The method of this invention is suitable for the detection of various types of pre-prepared food samples (pre-prepared meat and poultry dishes, pre-prepared aquatic dishes, pre-prepared vegetable dishes, mixed pre-prepared dishes, etc.). The sample processing steps are simple and convenient to operate, without the need for complex derivation processes, and the detection cost is low. It can realize the rapid detection of batch samples and is applicable to both routine laboratory testing and rapid on-site screening by food regulatory authorities. It has important practical value for ensuring the safety of pre-prepared food and standardizing the development of the industry.

[0022] The method of this invention exhibits good linearity, with a recovery rate ranging from 89.3% to 105.6%. Attached Figure Description

[0023] Figure 1 Ion chromatograms of quinolone antibiotics; Figure 2 This is the ion current spectrum of tetracycline antibiotics. Detailed Implementation

[0024] The technical effects provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] The standards for enrofloxacin, difloxacin, pefloxacin, ciprofloxacin, ofloxacin, lomefloxacin, besifloxacin, sarafloxacin, tetracycline, oxytetracycline, chlortetracycline, and doxycycline mentioned in the embodiments of the present invention are all commercially available. Enrofloxacin (CAS No.: 93106-60-6), Difloxacin (CAS No.: 98106-17-3), Pefloxacin (CAS No.: 70458-95-6), Ciprofloxacin (CAS No.: 85721-33-1), Ofloxacin (CAS No.: 82419-36-1), and Lomefloxacin (CAS No.: 98079-51-7) are reference standards. Besifloxacin (CAS No.: 141388-76-3), Sarafloxacin (CAS No.: 98105-99-8), Tetracycline (CAS No.: 73483-88-2), Oxytetracycline (CAS No.: 6153-64-6), Chlorotetracycline (CAS No.: 64-72-2), and Doxycycline (CAS No.: 24390-14-5) are all standards.

[0026] The internal isotope standard for enrofloxacin is enrofloxacin-d5 (CAS No.: 1173021-92-5), for difloxacin is difluoxacin hydrochloride (CAS No.: 91296-86-5), for pefloxacin is pefloxacin-d5 (CAS No.: 1228182-51-1), for ciprofloxacin is ciprofloxacin hydrochloride-d8 (CAS No.: 1216659-54-9), for ofloxacin is ofloxacin is ofloxacin-d3 (CAS No.: 1173147-91-5), and for lomefloxacin is lomefloxacin hydrochloride-d5. Lomefloxacin-D5 (CAS No.: 98079-52-8), besifloxacin (CAS No.: 405165-61-9), sarafloxacin (CAS No.: 1352879-52-7), tetracycline (CAS No.: 2373374-42-4), oxytetracycline (CAS No.: 2058-46-0), and chlortetracycline (CAS No.: 64-72-2) are all internally labeled with various chlortetracyclines. The internal isotope standard of doxycycline is doxycycline-d3 (CAS No.: 24390-14-5).

[0027] The high-performance liquid chromatography-mass spectrometer used in the embodiments of the present invention is a Xevo TQ-S Cronos ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer, purchased from Waters Corporation, USA; The nitrogen drying method used in this embodiment of the invention is the YT-QD12G fully automatic water bath nitrogen blower, brand name Yuntang.

[0028] The method for preparing the extract in this embodiment of the invention is as follows: after mixing 840 mL of acetonitrile with 150 mL of water, 10 mL of glacial acetic acid is added to obtain an acetonitrile aqueous solution containing 1% glacial acetic acid, which is the extract. The complex solution described in this embodiment of the invention is a 10% methanol aqueous solution.

[0029] Example 1

[0030] 1. Test Methods

[0031] (1) Preparation of mixed standard solution

[0032] Pipette 1 mL each of the standard solutions (100 μg / mL) of enrofloxacin, difloxacin, pefloxacin, ciprofloxacin, ofloxacin, lomefloxacin, besifloxacin, sarafloxacin, tetracycline, oxytetracycline, chlortetracycline, and doxycycline into a 100 mL volumetric flask, and dilute to the mark with methanol to prepare a mixed standard solution with a mass concentration of 1 μg / mL.

[0033] (2) Sample processing

[0034] Place 2g of the prepared vegetable sample into a 50mL centrifuge tube, add 100μL of mixed internal standard solution and 10mL of extraction solution, mix well, sonicate at 200W for 20min, then centrifuge at 8000rpm, 4℃ for 10min. Collect the supernatant and pass it through an Oasis PRiMEHLB solid-phase extraction column. Collect the filtrate, take 5mL of the filtrate, and vortex to mix. Dry the sample in a 45℃ water bath using a nitrogen evaporator. Dissolve the residue in 1mL of reconstitution solution, centrifuge at 5000rpm, 4℃ for 5min, collect the supernatant, filter through a 0.22μm filter membrane, and collect the filtrate to obtain the sample to be tested.

[0035] Preparation of the mixed internal standard solution: Enrofloxacin-d5, difloxacin hydrochloride-d5, pefloxacin-d5, ciprofloxacin hydrochloride-d8, ofloxacin-d3, lomefloxacin hydrochloride-d5, besifloxacin hydrochloride, sarafloxacin-d8, tetracycline-d6, oxytetracycline hydrochloride, chlortetracycline hydrochloride, and doxycycline-d3 were diluted with methanol and then mixed to prepare a mixed solution with an internal standard concentration of 200 ng / mL.

[0036] (3) Preparation of standard curve solution

[0037] Preparation of negative matrix: Take a blank sample and process it according to step (2) to obtain negative matrix.

[0038] Take the mixed standard solution and the mixed internal standard solution, dilute with negative matrix to obtain matrix standard working solutions with mass concentrations of 1 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL and 100 ng / mL, where the mass concentration of the internal standard is 5 ng / mL.

[0039] (4) Chromatographic conditions

[0040] Chromatographic column: ACQUITY BEH C18 (2.1 mm × 100 mm, 1.7 μm), column temperature: 35℃, injection volume: 2 μL, mobile phase A is 0.1 vt% formic acid aqueous solution, mobile phase B is acetonitrile, flow rate is 0.3 mL / min; The gradient elution procedure is shown in Table 1.

[0041] Table 1 Gradient elution program

[0042] (5) Mass spectrometry conditions

[0043] The ionization mode is: electrospray ionization positive ion mode (ESI). + The mass spectrometry scanning mode was: multiple reaction detection (MRM) scanning; capillary voltage was 1200V; cone voltage was 28V; ion source temperature was 600℃; desolventizing temperature was 460℃; and desolventizing gas flow rate was 920L / h.

[0044] 2. Results

[0045] (1) The ion chromatogram of the mixed standard solution is as follows: Figures 1-2 As shown.

[0046] The mass concentration of the mixed standard solutions was 5 ng / mL. Figures 1-2 It can be seen that the mixed standard solution exhibits good resolution under these chromatographic conditions, and the corresponding mass spectrometry values ​​meet the requirements. The upper chromatogram of each standard sample represents the quantitative ion pair, and the lower chromatogram represents the qualitative ion pair.

[0047] (2) Linear relationship

[0048] The standard curve was obtained by plotting the mass concentration of the antibiotic standard on the x-axis and the ratio of the peak area to the corresponding internal standard peak area on the y-axis, as shown in Table 2. The negative matrix for the standard curve was prepared using pre-cooked dishes with pork as the main component as blank samples.

[0049] Table 2 Standard Curve

[0050] Table 2 shows that the peak area and mass concentration of antibiotics in the range of 1 ng / mL to 100 ng / mL have a good linear relationship, and the correlation coefficients r are all above 0.99.

[0051] (3) Spiked recycling

[0052] Blank samples of pre-cooked dishes with pork as the main ingredient were taken, and internal standard solutions of 2 μg / kg, 5 μg / kg, and 10 μg / kg were added respectively. After sample processing, the samples were analyzed by chromatography and mass spectrometry according to the conditions described above. The average recovery rate and relative standard deviation (RSD) of each antibiotic under different spiking gradients were obtained. The results are shown in Table 3.

[0053] Table 3 Spike Recovery Results

[0054] Table 3 shows that the average recovery rate of antibiotics in pork-based prepared dishes was 89.3%–105.6%, with an RSD of 0.7%–3.4%, which meets the requirements.

[0055] Example 2

[0056] The method in Example 1 was used to detect antibiotic residues in the crispy pigeon pre-cooked dish produced by Shandong Zeyuan Food Co., Ltd. Only tetracycline residue was detected, with a concentration of 0.18 μg / kg, which is lower than the residue limit stipulated by the national food safety standards.

[0057] Example 3

[0058] The antibiotic residue in teriyaki chicken cutlet produced by Shandong Xiantanhong Food Co., Ltd. was detected using the method in Example 1. Only doxycycline residue with a concentration of 0.12 μg / kg was detected.

[0059] Example 4

[0060] Antibiotic residues in crispy pork and Kung Pao chicken produced by Shandong Huifa Food Co., Ltd. were tested using the method described in Example 1. Enrofloxacin residue of 0.08 μg / kg was detected in the crispy pork, while no antibiotic residues were detected in the Kung Pao chicken.

[0061] The enrofloxacin residue in the crispy pork was below the national food safety standard.

[0062] In summary, this invention provides a method for the simultaneous detection of multiple antibiotic residues in prepared foods. Targeting the physicochemical properties of quinolone and tetracycline antibiotics, this invention selects internal standards with structures and physicochemical properties similar to the target analytes, ensuring synchronous response during sample pretreatment and chromatographic-mass spectrometry analysis. This effectively eliminates errors caused by sample loss, instrument fluctuations, and matrix interference, significantly improving the accuracy and precision of the detection results. The method is suitable for the detection of various prepared food samples (prepared meat and poultry dishes, aquatic products, vegetables, and mixed dishes, etc.). The sample processing steps are simple and convenient, requiring no complex derivatization, resulting in low detection costs. It enables rapid detection of batches of samples and is applicable to both routine laboratory testing and rapid on-site screening by food regulatory authorities. It has significant practical value for ensuring the safety of prepared food and regulating the industry. Furthermore, the method exhibits good linearity, with a recovery rate ranging from 89.3% to 105.6%.

[0063] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for simultaneously detecting multiple antibiotic residues in prepared dishes, characterized in that, Includes the following steps: (1) Mix the pre-cooked vegetables, mixed internal standard solution and extract, sonicate, centrifuge, and take the supernatant to obtain the mixture; (2) After purifying the mixture, dry it to obtain the dried product; (3) Mix the dried material with the reconstituted solution, centrifuge, take the supernatant, filter through a membrane, and obtain the sample to be tested; (4) High performance liquid chromatography-mass spectrometry was used to detect the content of veterinary drug residues in the sample. The antibiotics include quinolone antibiotics and tetracycline antibiotics; The quinolone antibiotics include enrofloxacin, difloxacin, pefloxacin, ciprofloxacin, ofloxacin, lomefloxacin, besifloxacin, and sarafloxacin; The tetracycline antibiotics include tetracycline, oxytetracycline, chlortetracycline, and doxycycline; The chromatographic conditions for the high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) detection were as follows: column: ACQUITY BEH C18 (2.1 mm × 100 mm, 1.7 μm), column temperature: 34~36℃, injection volume: 1.8~2.2 μL, mobile phase A was 0.08~0.12 vt% formic acid aqueous solution, mobile phase B was acetonitrile, and flow rate was 0.2~0.4 mL / min; The gradient elution program was as follows: 0–4 min, 10–40% mobile phase A, 90–60% mobile phase B; 4–8 min, 40–90% mobile phase A, 60–10% mobile phase B; 8–10 min, 90–99% mobile phase A, 10–1% mobile phase B; 10–10.1 min, 99–10% mobile phase A, 1–90% mobile phase B. 10.1~12 min, 10% mobile phase A, 90% mobile phase B; The mass spectrometry conditions for the high-performance liquid chromatography-mass spectrometry (HPLC-MS) detection are as follows: ionization mode: electrospray ionization positive ion mode (ESI). + The mass spectrometry scanning mode was: multiple reaction detection (MRM) scanning; capillary voltage was 1100~1200V; cone voltage was 25~30V; ion source temperature was 550~600℃; desolventizing temperature was 450~480℃; and desolventizing gas flow rate was 900~950L / h.

2. The method according to claim 1, characterized in that, The mixed internal standard solution includes 20-30 ng / mL enrofloxacin-d5, 20-30 ng / mL difloxacin hydrochloride, 20-30 ng / mL pefloxacin-d5, 20-30 ng / mL ciprofloxacin hydrochloride-d8, 20-30 ng / mL ofloxacin-d3, 20-30 ng / mL lomefloxacin hydrochloride-d5, 20-30 ng / mL besifloxacin hydrochloride, 20-30 ng / mL sarafloxacin-d8, 20-30 ng / mL tetracycline-d6, 20-30 ng / mL oxytetracycline hydrochloride, 20-30 ng / mL chlortetracycline hydrochloride, and 20-30 ng / mL doxycycline-d3.

3. The method according to claim 1, characterized in that, The extract in step (1) is an acetonitrile aqueous solution containing glacial acetic acid; The concentration of the glacial acetic acid is 0.5-1.5%; The concentration of the acetonitrile aqueous solution is 80-85%.

4. The method according to claim 1, characterized in that, The mass-to-volume ratio of the pre-prepared vegetable and the mixed internal standard solution in step (1) is 2~3g:100~150μL; The mass-to-volume ratio of the pre-prepared vegetables and the extract in step (1) is 2~3g:8~12mL.

5. The method according to claim 1, characterized in that, The ultrasonic power in step (1) is 150~250W, and the ultrasonic time is 15~25min; The centrifugation speed in step (1) is 7000~9000 rpm, the centrifugation time is 8~12 min, and the centrifugation temperature is 2~6℃.

6. The method according to claim 1, characterized in that, The purification method described in step (2) is through a solid-phase extraction column; the solid-phase extraction column is an Oasis PRiME HLB; The drying method described in step (2) is nitrogen drying.

7. The method according to claim 1, characterized in that, The reconstituted solution in step (3) is a 10% methanol aqueous solution.

8. The method according to claim 1, characterized in that, In step (3), the centrifugation speed is 4500~5500 rpm, the centrifugation time is 4~6 min, and the centrifugation temperature is 2~6℃.

9. The method according to claim 1, characterized in that, The pore size of the filter membrane used in step (3) is 0.2~0.25μm.

10. The application of the method according to any one of claims 1 to 9 in the rapid and simultaneous detection of quinolone antibiotic and tetracycline antibiotic residues in prepared dishes; The quinolone antibiotics include enrofloxacin, difloxacin, pefloxacin, ciprofloxacin, ofloxacin, lomefloxacin, besifloxacin, and sarafloxacin; The tetracycline antibiotics include tetracycline, oxytetracycline, chlortetracycline, and doxycycline.

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