Method for determining contents of 15 beta-receptor stimulants, 3 amido alcohols and 11 nitroimidazole antibiotics in agricultural products

Abstract

The invention relates to a method for determining the contents of 15 beta-receptor stimulants, 3 amido alcohols and 11 nitroimidazole antibiotics in agricultural products, and belongs to the technical field of detection. The invention relates to a method for determining the contents of 15 beta-receptor stimulants, 3 amido alcohols and 11 nitroimidazole antibiotics in agricultural products. The agricultural products are spinach, asparagus lettuce or peach. The 15 kinds of beta-receptor stimulant antibiotics are cimaterol, cibuterol, ritodrine, clenbuterol, hydroxymethyl clenbuterol, clenproterol, ractopamine, clorprenaline, clenbuterol, tubuterol, brombuterol, bambuterol, mabuterol, clenbupant and mappenterol, and the 15 kinds of beta-receptor stimulant antibiotics are selected from the group consisting of propamine, propamine, propamine, propamine, propamine, propamine, propamine, propamine, propamine, propamine, propamine, propamine, propamine and propamine. The three kinds of amide alcohol antibiotics are chloramphenicol, thiamphenicol and fluorine thiamphenicol. The method has the characteristics of simplicity, convenience, high efficiency, accuracy, sensitivity, economy and the like.

Description

Technical Field

[0001] The invention relates to a method for determining the contents of 15 beta-receptor agonists, 3 amide alcohols and 11 nitroimidazole antibiotics in agricultural products, belonging to the technical field of detection. Background Art

[0002] Antibiotics as anti-infective drugs have been widely used in human disease treatment, animal husbandry, veterinary medicine and agricultural planting. Among them, the abuse of antibiotics is common. If we often eat foods containing residual antibiotics, the antibiotics will gradually accumulate in the body and cause organ lesions. However, the main focus of antibiotic pollution in food is still animal-derived food. With the emergence of new antibiotic pollutants in the environment, antibiotics remaining in water, soil and animal feces can be absorbed and enriched by plants. The problem of antibiotic pollution in vegetables has become more prominent. Wu Xiaolian, Bao Yanping, Xiang Lei, et al. Simultaneous determination of 8 sulfonamide antibiotics in vegetables by solid phase extraction-high performance liquid chromatography-tandem mass spectrometry [J]. Environmental Chemistry, 2013, 32(6): 1038-1044. It is disclosed that when testing the residual amount of quinolone antibiotics in vegetables, quinolones were found in 9 vegetables such as potatoes. Among them, the total content of ciprofloxacin and enrofloxacin in bitter melon was 190μg / kg, exceeding The maximum residue limit in animal-derived foods (100 μg / kg) was set; Lu Jinlian, Du Minghui, Pan Yunan, et al. Contamination characteristics and risk assessment of quinolone antibiotics in organic vegetables [J]. Environmental Science and Technology, 2021, 44(3): 9. The results of the content analysis and health risk assessment of 4 quinolone antibiotics in 39 organic vegetables produced by 5 typical organic vegetable bases showed that the detection rate of 4 quinolone antibiotics in 39 organic vegetable samples was 52% to 100%, of which norfloxacin and enrofloxacin were as high as 100%, and 4 quinolone antibiotics were detected simultaneously in more than 50% of the samples. The daily intake of vegetables in the healthy diet of residents is more than twice that of animal-derived foods. It can be seen that conducting systematic work such as investigation and survey of antibiotic contamination in vegetables, risk assessment, setting limit standards, and pollution control has become a food safety issue that needs to be solved urgently. How to quickly, efficiently, comprehensively and accurately detect the types and contents of antibiotics in vegetables is an important premise and key. However, a standard system for detecting antibiotic content in plant-based foods such as vegetables has not yet been established. The only two relevant standards, DB34T 4335-2022 "Determination of quinolone residues in sprout vegetables by liquid chromatography-mass spectrometry / mass spectrometry" and BJS202310 "Determination of quinolones, sulfonamides, nitroimidazoles and tetracycline compounds in bean sprouts, soy products, hot pot and spicy hot pot bases", are only applicable to samples such as sprouts and soy products. Some literature has reported methods for the determination of antibiotics such as quinolones, sulfonamides, tetracyclines, etc. in vegetables with low chlorophyll content such as bean sprouts and wild rice stems. However, there are no reports on methods for the simultaneous detection of β-receptor agonists, amides and nitroimidazole antibiotics in complex matrix vegetables and fruit samples with rich nutrients and high chlorophyll content such as spinach, lettuce and peaches. Summary of the invention

[0003] The purpose of the present invention is to provide a new method for determining the contents of 15 beta-receptor agonists, 3 amide alcohols and 11 nitroimidazole antibiotics in agricultural products.

[0004] To achieve the above purpose, the agricultural product is spinach, lettuce or peach;

[0005] The 15 β-receptor agonist antibiotics are cimaterol, cibutrol, ritodrine, clenbuterol, hydroxymethyl clenbuterol, clenproperol, ractopamine, chlorprenaline, clenbuterol, tolbuterol, bromobuterol, bambuterol, mabuterol, clenpentol, and mapentol;

[0006] The three amide antibiotics are chloramphenicol, thiamphenicol, and florfenicol;

[0007] The 11 nitroimidazole antibiotics are 4-nitroimidazole, 2-methyl-5-nitroimidazole, hydroxymetronidazole, hydroxymethylmetronidazole, metronidazole, ronidazole, dimenamidazole, benznidazole, chlorometronidazole, isopronidazole, and hydroxyisopropranidazole;

[0008] The determination method comprises:

[0009] A. Sample pretreatment:

[0010] After homogenizing the agricultural product, add 0.1% ammonia water and acetonitrile to mix, ultrasonically treat for 30 minutes, cool to room temperature, add NaCl to mix, centrifuge at 8000rpm / min for 5 minutes, take 6mL of supernatant and mix with QuEChERS purification package, shake vigorously for 1min, centrifuge at 8000rpm / min for 5min, take 5.0mL of supernatant, blow nitrogen to nearly dryness in a 30℃ water bath, dissolve the residue with 1.0mL of re-dissolving solvent, filter through 0.22μm nylon filter membrane, and obtain the test solution, wherein the re-dissolving solvent is methanol: acetonitrile: water: formic acid mixed in a volume ratio of 10:10:80:0.1;

[0011] The mass volume ratio of the agricultural product, 0.1% ammonia acetonitrile, and NaCl is: 10 g: 25.0 mL: 5 g;

[0012] The QuEChERS purification package contains 500 mg of anhydrous magnesium sulfate, 50 mg of PSA, 50 mg to 100 mg of C18, and 50 mg of GCB;

[0013] B. measuring the solution to be tested in step A by liquid chromatography-tandem mass spectrometry;

[0014] The chromatographic conditions of the liquid chromatography are as follows: Chromatographic column: Agilent RRHD SB-C 18Chromatographic column; mobile phase: 0.1% formic acid water-0.1% formic acid acetonitrile containing 20% ​​methanol; flow rate: 0.2mL / min; column temperature: 30℃, injection volume: 5μL;

[0015] The mass spectrometry conditions of the tandem mass spectrometer are: electrospray ion source positive and negative ion mode detection, and the monitoring mode is a multi-reaction mode; specific instrument parameters: drying gas N2 temperature 300°C, drying gas N2 flow rate 10L / min, nebulizing gas pressure 40psi (276kPa), sheath gas N2 temperature 350°C, sheath gas N2 flow rate 12L / min, capillary voltage 3500V.

[0016] The volume percentage is 0.1% formic acid acetonitrile containing 20% ​​methanol. For example, take 200mL methanol, 1mL formic acid, and add acetonitrile to make up to 1L.

[0017] In a specific embodiment, the agricultural product is spinach.

[0018] In a specific embodiment, the method further comprises step C. subtracting the test result of a blank experiment from the test result of step B to obtain a final test result; the blank experiment does not add agricultural products, and the rest is the same as steps A and B.

[0019] In a specific embodiment, the Agilent RRHD SB-C 18 The specifications of the chromatographic column are 100 mm × 2.1 mm, 1.8 μm.

[0020] In a specific embodiment, the gradient elution conditions of the liquid chromatography are shown in Table 1:

[0021] Table 1 Gradient elution program

[0022]

[0023] In a specific embodiment, the retention time and MRM parameters of the mass spectra of the 15 β-receptor agonists, 3 amide alcohols and 11 nitroimidazole antibiotics are shown in Table 2:

[0024] Table 2 Mass spectral retention times and mass spectral parameters of 15 β-receptor agonists, 3 amide alcohols and 11 nitroimidazole antibiotics

[0025]

[0026] In a specific embodiment, the mass of the agricultural product is accurate to 0.01 g.

[0027] In a specific embodiment, among the 15 β-receptor agonists, 3 amides and 11 nitroimidazole antibiotics, the detection limits of thiamphenicol and fluorosulfamethoxazole are 0.25 μg / kg, and the quantification limits are 1.25 μg / kg, while the detection limits of the remaining β-receptor agonists, amides and nitroimidazole antibiotics are 0.05 μg / kg, and the quantification limits are 0.25 μg / kg.

[0028] In a specific embodiment, the average spiked recoveries of thiamphenicol and florfenicol in the spinach, lettuce or peach are 90.19% to 106.95%, and the average spiked recoveries of the remaining target substances are in the range of 60.95% to 118.55%.

[0029] In a specific embodiment, the relative standard deviation of thiamphenicol and florfenicol in the spinach, lettuce or peach is in the range of 0.1% to 5.4%, and the relative standard deviation of the other target substances is in the range of 0.1% to 14.9%.

[0030] Beneficial effects:

[0031] The method of the present invention has good linear relationships in the mass concentration range of 2.5 ng / mL to 125 ng / mL for thiamphenicol and florfenicol, and in the mass concentration range of 0.5 ng / mL to 25 ng / mL for other β-receptor agonists, amide alcohols and nitroimidazole antibiotics, and the correlation coefficients r are all ≥0.99;

[0032] The detection limits of thiamphenicol and fluorosulfamethoxazole were 0.25 μg / kg, the quantification limits were 1.25 μg / kg, the average spiked recoveries were 90.19% to 106.95%, and the relative standard deviations were 0.1% to 5.4%. The detection limits of the remaining β-receptor agonists, amides and nitroimidazole antibiotics were all 0.05 μg / kg, the quantification limits were all 0.25 μg / kg, the average spiked recoveries were 60.95% to 118.55%, and the relative standard deviations were 0.1% to 14.9%.

[0033] The method of the present invention has the characteristics of simplicity, high efficiency, accuracy, sensitivity and economy, and can be applied to the simultaneous and accurate determination of the contents of 15 beta-receptor agonists, 3 amide alcohols and 11 nitroimidazole substances in spinach, lettuce and peaches. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Example 2 Agilent RRHD SB-C 18 (2.1mm×100mm,1.8μm) separation of target substances.

[0035] Figure 2Comparative Example 1: Separation of target substances by Kinetex F5 (3.0 mm×50 mm, 2.7 μm). DETAILED DESCRIPTION

[0036] To achieve the above purpose, the agricultural product is spinach, lettuce or peach;

[0037] The 15 β-receptor agonist antibiotics are cimaterol, cibutrol, ritodrine, clenbuterol, hydroxymethyl clenbuterol, clenproperol, ractopamine, chlorprenaline, clenbuterol, tolbuterol, bromobuterol, bambuterol, mabuterol, clenpentol, and mapentol;

[0038] The three amide antibiotics are chloramphenicol, thiamphenicol, and florfenicol;

[0039] The 11 nitroimidazole antibiotics are 4-nitroimidazole, 2-methyl-5-nitroimidazole, hydroxymetronidazole, hydroxymethylmetronidazole, metronidazole, ronidazole, dimenamidazole, benznidazole, chlorometronidazole, isopronidazole, and hydroxyisopropranidazole;

[0040] The determination method comprises:

[0041] A. Sample pretreatment:

[0042] After homogenizing the agricultural product, add 0.1% ammonia water and acetonitrile to mix, ultrasonically treat for 30 minutes, cool to room temperature, add NaCl to mix, centrifuge at 8000rpm / min for 5 minutes, take 6mL of supernatant and mix with QuEChERS purification package, shake vigorously for 1min, centrifuge at 8000rpm / min for 5min, take 5.0mL of supernatant, blow nitrogen to nearly dryness in a 30℃ water bath, dissolve the residue with 1.0mL of re-dissolving solvent, filter through 0.22μm nylon filter membrane, and obtain the test solution, wherein the re-dissolving solvent is methanol: acetonitrile: water: formic acid mixed in a volume ratio of 10:10:80:0.1;

[0043] The mass volume ratio of the agricultural product, 0.1% ammonia acetonitrile, and NaCl is: 10 g: 25.0 mL: 5 g;

[0044] The QuEChERS purification package contains 500 mg of anhydrous magnesium sulfate, 50 mg of PSA, 50 mg to 100 mg of C18, and 50 mg of GCB;

[0045] B. measuring the solution to be tested in step A by liquid chromatography-tandem mass spectrometry;

[0046] The chromatographic conditions of the liquid chromatography are as follows: Chromatographic column: Agilent RRHD SB-C 18Chromatographic column; mobile phase: 0.1% formic acid water-0.1% formic acid acetonitrile containing 20% ​​methanol; flow rate: 0.2mL / min; column temperature: 30℃, injection volume: 5μL;

[0047] The mass spectrometry conditions of the tandem mass spectrometer are: electrospray ion source positive and negative ion mode detection, and the monitoring mode is a multi-reaction mode; specific instrument parameters: drying gas N2 temperature 300°C, drying gas N2 flow rate 10L / min, nebulizing gas pressure 40psi (276kPa), sheath gas N2 temperature 350°C, sheath gas N2 flow rate 12L / min, capillary voltage 3500V.

[0048] The volume percentage is 0.1% formic acid acetonitrile containing 20% ​​methanol. For example, take 200mL methanol, 1mL formic acid, and add acetonitrile to make up to 1L.

[0049] In a specific embodiment, the agricultural product is spinach.

[0050] In a specific embodiment, the method further comprises step C. subtracting the test result of a blank experiment from the test result of step B to obtain a final test result; the blank experiment does not add agricultural products, and the rest is the same as steps A and B.

[0051] In a specific embodiment, the Agilent RRHD SB-C 18 The specifications of the chromatographic column are 100 mm × 2.1 mm, 1.8 μm.

[0052] In a specific embodiment, the gradient elution conditions of the liquid chromatography are shown in Table 1:

[0053] Table 1 Gradient elution program

[0054]

[0055] In a specific embodiment, the retention time and MRM parameters of the mass spectra of the 15 β-receptor agonists, 3 amide alcohols and 11 nitroimidazole antibiotics are shown in Table 2:

[0056] Table 2 Mass spectral retention times and mass spectral parameters of 15 β-receptor agonists, 3 amide alcohols and 11 nitroimidazole antibiotics

[0057]

[0058] In a specific embodiment, the mass of the agricultural product is accurate to 0.01 g.

[0059] In a specific embodiment, among the 15 β-receptor agonists, 6 amides and 11 nitroimidazole antibiotics, the detection limits of thiamphenicol and fluorosulfamethoxazole are both 0.25 μg / kg, and the quantification limits are both 1.25 μg / kg, while the detection limits of the remaining β-receptor agonists, amides and nitroimidazole antibiotics are all 0.05 μg / kg, and the quantification limits are all 0.25 μg / kg.

[0060] In a specific embodiment, the average spiked recoveries of thiamphenicol and florfenicol in the spinach, lettuce or peach are 90.19% to 106.95%, and the average spiked recoveries of the remaining target substances are in the range of 60.95% to 118.55%.

[0061] In a specific embodiment, the relative standard deviation of thiamphenicol and florfenicol in the spinach, lettuce or peach is in the range of 0.1% to 5.4%, and the relative standard deviation of the other target substances is in the range of 0.1% to 14.9%.

[0062] The specific implementation modes of the present invention are further described below in conjunction with embodiments, but the present invention is not limited to the scope of the embodiments.

[0063] Example 1

[0064] 1 Materials and methods

[0065] 1.1 Main materials and reagents

[0066] Mixed standard solution of β-receptor agonists in methanol: the concentration is 100 mg / L, mixed standard of 3 amide antibiotics including chloramphenicol in methanol: chloramphenicol is 40 mg / L, the others are 200 mg / L, mixed standard solution of nitroimidazoles in methanol: the concentration is 1000 mg / L, standard solution of hydroxyisopropranidazole in methanol: the concentration is 1000 mg / L, all purchased from Shanghai Anpu Cuishi Standard Technology Service Co., Ltd. Ethylenediamine-N-propylsilane silica gel PSA: particle size is 40 μm to 60 μm, octadecylsilane modified silica gel C 18 : particle size is 40μm-60μm, graphitized carbon black GCB: particle size is 40μm-120μm; analytical grade anhydrous magnesium sulfate, sodium chloride, and ammonia water were purchased from Chengdu Kelong Chemical Reagent Factory. Chromatographic grade formic acid, acetonitrile, and methanol were purchased from Sigma-Aldrich, USA. Spinach, lettuce, and peaches were purchased from local farmers' markets.

[0067] 1.2 Main instruments and equipment

[0068] Agilent 1290 InfinityⅡ-G6460C high performance liquid chromatography-tandem mass spectrometer: equipped with electrospray ion source and Version 1.1 data processing system, Agilent Technologies, USA; BP 211D electronic balance: Sartorius, Germany; Milli-Q ultrapure water device: Millipore, USA; KH-500DE CNC ultrasonic cleaner: Kunshan Hechuang Ultrasonic Instrument Co., Ltd.; XW-80A vortex mixer: Shanghai Chitang Industrial Co., Ltd.; MPEva II fully automatic parallel concentrator: Ruilibo Instrument (Guangzhou) Co., Ltd.

[0069] 1.3 Experimental methods

[0070] 1.3.1 Chromatographic conditions Chromatographic column: Agilent RRHD SB-C 18 Chromatographic column (100 mm×2.1 mm, 1.8 μm); mobile phase: 0.1% formic acid water-0.1% formic acid acetonitrile containing 20% ​​methanol, gradient elution conditions are shown in Table 1 above; flow rate: 0.2 mL / min; column temperature: 30° C., injection volume: 5 μL.

[0071] 1.3.2 Mass spectrometry conditions Electrospray ionization source positive and negative ion modes (ESI + 、ESI - ) detection, and the monitoring mode is multiple reaction mode (MRM). Specific instrument parameters: drying gas (N2) temperature 300℃, drying gas (N2) flow rate 10L / min, nebulizer gas pressure 40psi (276kPa), sheath gas (N2) temperature 350℃, sheath gas (N2) flow rate 12L / min, capillary voltage 3500V, the retention time and MRM parameters of each target to be detected are detailed in Table 2 above.

[0072] 1.3.3 Sample pretreatment Weigh 10 g of the homogenized sample (accurate to 0.01 g), add 25.0 mL of 0.1% ammonia acetonitrile, vortex mix, ultrasonicate for 30 min, cool to room temperature, add 5 g of NaCl, vortex mix, centrifuge at 8000 rpm / min for 5 min, take 6 mL of the supernatant and place it in a QuEChERS cleanup tube (containing 500 mg of anhydrous magnesium sulfate, 50 mg of PSA, and C 18 50 mg, GCB 50 mg), shake vigorously for 1 min, centrifuge at 8000 rpm / min for 5 min, take 5.0 mL of supernatant, blow it to near dryness with nitrogen in a 30°C water bath, dissolve the residue with 1.0 mL of reconstitution solvent (the reconstitution solvent is methanol: acetonitrile: water: formic acid according to the volume ratio = 10:10:80:0.1), pass through a 0.22 μm nylon filter membrane, and provide for determination by liquid chromatography-tandem mass spectrometry.

[0073] 2 Results and analysis

[0074] 2.1 Method sensitivity verification

[0075] The detection limit (LOD) of the corresponding target was determined with a signal-to-noise ratio (S / N) of 3, and the quantification limit (LOQ) of the corresponding target was determined with an average signal-to-noise ratio (S / N) of 6 times of 10. The results are shown in Tables 3 to 5. As can be seen from the table, the detection limits of thiamphenicol and fluorothiamphenicol in spinach determined by this method are both 0.25 μg / kg, and the quantification limits are 1.25 μg / kg, while the detection limits of the remaining β-agonists, amide alcohols and nitroimidazole antibiotics are all 0.05 μg / kg, and the quantification limits are all 0.25 μg / kg, indicating that this method can well meet the needs of trace or even ultra-trace determination of 15 β-agonists, 3 amide alcohols and 11 nitroimidazole antibiotics in spinach, lettuce and peaches.

[0076] Table 3 Linear equations, correlation coefficients, detection limits and quantification limits of each target substance in spinach

[0077]

[0078] Table 4 Linear equations, correlation coefficients, detection limits and quantification limits of each analyte in lettuce

[0079]

[0080]

[0081] Table 5 Linear equations, correlation coefficients, detection limits and quantification limits of each analyte in peach

[0082]

[0083] 2.2 Method linearity and range verification Accurately pipette 0.1 mL of 100 mg / L β-receptor agonist mixed standard solution, 0.25 mL of amide antibiotic mixed standard solution, 0.01 mL of 1000 mg / L nitroimidazole mixed standard solution and 0.01 mL of 1000 mg / L hydroxyisoproprazole standard solution into the same 1 mL volumetric flask, add methanol to the mark, prepare thiamphenicol and florfenicol mass concentration of 50.00 mg / L, and the mass concentration of the remaining analytes is 10.00 mg / L mixed standard stock solution, sealed and stored at -18 ° C, with a shelf life of 3 months. Before use, pipette 0.10 mL of the mixed standard stock solution into a 10 mL volumetric flask, add methanol to the mark, prepare thiamphenicol and florfenicol mass concentration of 500 μg / L. The mass concentration of the remaining target substances to be measured is 100 μg / L mixed standard intermediate solution.

[0084] Take 6 portions of blank sample and prepare 6 portions of blank matrix solution according to the steps of 1.3.3. Then accurately add appropriate amount of each mixed standard stock solution, blow to nearly dry with nitrogen gas, add 1.0mL of reconstitution solvent (the reconstitution solvent is a mixture of methanol: acetonitrile: water: formic acid in a volume ratio of 10:10:80:0.1) to dissolve the residue, filter through 0.22μm nylon filter membrane, and prepare a series of matrix mixed standard working solutions with mass concentrations of 2.5ng / mL, 5.0ng / mL, 12.5ng / mL, 25ng / mL, 50ng / mL and 125ng / mL for thiamphenicol and 0.5μg / L, 1.0μg / L, 2.5μg / L, 5.0μg / L, 10.0μg / L and 25.0μg / L for the other analytes. The standard working curve was drawn with the mass concentration of each analyte (x) as the horizontal axis and the quantitative ion peak area (y) as the vertical axis. The results are shown in Tables 3 to 5. It can be seen from the table that this method has good linearity and range for the determination of 15 β-receptor agonists, 3 amide alcohols and 11 nitroimidazole antibiotics in spinach, lettuce and peach.

[0085] 2.3 Verification of the accuracy and precision of the method Eighteen blank samples were taken and different amounts of 100 μg / L mixed standard intermediate solution were added to make the concentrations of thiamphenicol and fluorosulfamethoxazole in the spiked samples 2.5 μg / kg (low spike), 12.5 μg / kg (medium spike), and 25 μg / kg (high spike), respectively, while the mass concentrations of the remaining target substances were 0.5 μg / kg (low spike), 2.5 μg / kg (medium spike), and 5.0 μg / kg (high spike). Six parallel samples were prepared for each spiked concentration level, and the pretreatment and determination were carried out according to the method in 1.3 to investigate the accuracy and precision of this method. The determination results are shown in Tables 6 to 8. As can be seen from the table, the average recoveries of thiamphenicol and fluoroquinol in spinach, lettuce and peach at different spike levels were between 90.19% and 106.95%, and the relative standard deviations were between 0.1% and 5.4%. The average recoveries of the other target substances at different spike levels were between 60.95% and 118.55%, and the relative standard deviations were between 0.1% and 14.9%, which fully met the requirements of GB5009.295-2023 "General Rules for Validation of Chemical Analysis Methods for National Food Safety Standards" and GB / T 27404-2008 "Laboratory Quality Control Specifications for Physical and Chemical Detection of Food" and other relevant standards for method recovery and precision, indicating that this method can well meet the needs of simultaneous and accurate determination of 15 β-receptor agonists, 3 amide alcohols and 11 nitroimidazole antibiotics in spinach.

[0086] Table 6 Spinach spike recovery results

[0087]

[0088]

[0089] Table 7 Lettuce spike recovery results

[0090]

[0091]

[0092] Table 8 peach spike recovery results

[0093]

[0094] Example 2 and Comparative Example 1

[0095] The chromatographic columns of Example 2 and Comparative Example 1 were Agilent RRHD SB-C 18 (2.1mm×100mm, 1.8μm, the best chromatographic conditions are detailed in Table 1, flow rate: 0.2mL / min; column temperature: 30℃, injection volume: 5μL) and Kinetex F5 (3.0mm×50mm, 2.7μm, the best chromatographic conditions are detailed in Table 9). The effects of the two types of chromatographic columns on the effective separation of each target in the same mixed standard working solution are shown in Figure 1 and Figure 2 As can be seen from the figure, under the optimal separation conditions of each chromatographic column, each target compound is 18 The separation effect on the chromatographic column is better, and the peak shape is sharp and symmetrical.

[0096] Table 9 F5 column mobile phase conditions

[0097]

[0098]

[0099] Comparative Example 2

[0100] The purification effect of MCX column (60 mg / 3 mL) on each target substance to be tested in spinach was compared. The specific method is as follows: 10 g of spinach sample was weighed, 10.0 mL of extraction solution was added, ultrasonicated for 30 min, vortexed and mixed, centrifuged at 8000 r / min for 5 min, 5.0 mL of supernatant was passed through an MCX column pre-activated with 5 mL of methanol and 5 mL of water, the effluent was discarded, 3 mL of water and 3 mL of methanol were added for elution, the eluent was discarded, 5 mL of 5% ammonia methanol solution was used for elution, the eluent was collected, and nitrogen was blown to near dryness at 30°C. 1.0 mL of re-dissolution solvent (the re-dissolution solvent is methanol: acetonitrile: water: formic acid mixed in a volume ratio of 10:10:80:0.1) was used to dissolve the residue, vortexed and mixed, and filtered through a 0.22 μm nylon filter membrane to obtain a solution to be tested. The other instrument detection conditions were the same as those in Example 1. The comparison results are shown in Table 10. From the results, it can be seen that the pH value is The MCX solid phase extraction column was used for extraction with 2.0 M phosphoric acid solution, 0.5 M sulfuric acid solution and pH 2.0 methanol phosphoric acid solution (1:1, V:V). Amide alcohols and nitroimidazole antibiotics were not retained or basically not retained on the MCX column. The recovery rate of β-receptor agonist antibiotics was also low. The recovery rate of most of the target substances was less than 70%, indicating that the MCX solid phase extraction column is not suitable for the enrichment and purification of the three types of target substances in spinach.

[0101] Table 10 Investigation of the enrichment and purification effect of MCX solid phase extraction cartridges (in terms of recovery rate, n=2)

[0102]

[0103]

[0104] Comparative Example 3

[0105] The differences in the recovery rates of the target substances in spinach when different amounts of PSA, C18 and GCB adsorbents were added were compared. The comparative example and the example only differed in the amount of PSA, C18 and GCB adsorbents added, and the rest were completely consistent. The comparative experimental results are shown in Table 11. It can be seen from the table that when PSA, C18 and GCB adsorbents were added, the recovery rates of the target substances in spinach were different. 18 When the added amount of and GCB is 50 mg, that is, the QuEChERS combination of Example 1 and the combination of 50 mg PSA, 100 mg C18, and 50 mg GCB, the recovery rate of each target substance to be tested meets the requirements of GB 5009.295-2023 "General Rules for Validation of Chemical Analysis Methods for Food Safety National Standard" and GB / T 27404-2008 "Laboratory Quality Control Specification for Physical and Chemical Detection of Food".

[0106] Table 11 Effects of different QuEChERS combinations on the purification of target compounds (in terms of recovery, n=2)

[0107]

Claims

1. A method for determining the contents of 15 β-receptor agonists, 3 amide alcohols and 11 nitroimidazole antibiotics in agricultural products, characterized in that: The agricultural product is spinach, lettuce or peach; The 15 β-receptor agonist antibiotics are cimaterol, cibutrol, ritodrine, clenbuterol, hydroxymethyl clenbuterol, clenproperol, ractopamine, chlorprenaline, clenbuterol, tolbuterol, bromobuterol, bambuterol, mabuterol, clenpentol, and mapentol; The three amide antibiotics are chloramphenicol, thiamphenicol, and florfenicol; The 11 nitroimidazole antibiotics are 4-nitroimidazole, 2-methyl-5-nitroimidazole, hydroxymetronidazole, hydroxymethylmetronidazole, metronidazole, ronidazole, dimenamidazole, benznidazole, chlorometronidazole, isopronidazole, and hydroxyisopropranidazole; The determination method comprises: A. Sample pretreatment: After homogenizing the agricultural product, add 0.1% ammonia water and acetonitrile to mix, ultrasonically treat for 30 minutes, cool to room temperature, add NaCl to mix, centrifuge at 8000rpm / min for 5 minutes, take 6mL of supernatant and mix with QuEChERS purification package, shake vigorously for 1min, centrifuge at 8000rpm / min for 5min, take 5.0mL of supernatant, blow nitrogen to nearly dryness in a 30℃ water bath, dissolve the residue with 1.0mL of re-dissolving solvent, filter through 0.22μm nylon filter membrane, and obtain the test solution, wherein the re-dissolving solvent is methanol: acetonitrile: water: formic acid mixed in a volume ratio of 10:10:80:0.1; The mass volume ratio of the agricultural product, 0.1% ammonia acetonitrile, and NaCl is: 10 g: 25.0 mL: 5 g; The QuEChERS purification package contains 500 mg of anhydrous magnesium sulfate, 50 mg of PSA, 50 mg to 100 mg of C18, and 50 mg of GCB; B. measuring the solution to be tested in step A by liquid chromatography-tandem mass spectrometry; The chromatographic conditions of the liquid chromatography are as follows: Chromatographic column: Agilent RRHD SB-C 18 Column; mobile phase: 0.1% formic acid water-0.1% formic acid acetonitrile containing 20% ​​methanol; flow rate: 0.2mL / min; column temperature: 30℃, injection volume: 5μL; The mass spectrometry conditions of the tandem mass spectrometer are: electrospray ion source positive and negative ion mode detection, and the monitoring mode is a multi-reaction mode; specific instrument parameters: drying gas N2 temperature 300°C, drying gas N2 flow rate 10L / min, nebulizing gas pressure 40psi, sheath gas N2 temperature 350°C, sheath gas N2 flow rate 12L / min, and capillary voltage 3500V.

2. The method for determining the contents of 15 β-receptor agonists, 3 amide alcohols and 11 nitroimidazole antibiotics in agricultural products according to claim 1, characterized in that: The agricultural product is spinach.

3. The method for determining the contents of 15 β-receptor agonists, 3 amide alcohols and 11 nitroimidazole antibiotics in agricultural products according to claim 1 or 2, characterized in that: The method further comprises step C. subtracting the test result of the blank experiment from the test result of step B to obtain the final test result; the blank experiment does not add agricultural products, and the rest is the same as steps A and B.

4. The method for determining the contents of 15 β-receptor agonists, 3 amide alcohols and 11 nitroimidazole antibiotics in agricultural products according to claim 1 or 2, characterized in that: The AgilentRRHD SB-C 18 The specifications of the chromatographic column are 100 mm × 2.1 mm, 1.8 μm.

5. The method for determining the contents of 15 β-receptor agonists, 3 amide alcohols and 11 nitroimidazole antibiotics in agricultural products according to claim 1 or 2, characterized in that: The gradient elution conditions of the liquid chromatography are shown in Table 1: Table 1 Gradient elution program 6. The method for determining the contents of 15 β-receptor agonists, 3 amide alcohols and 11 nitroimidazole antibiotics in agricultural products according to claim 1 or 2, characterized in that: The retention time and MRM parameters of the mass spectra of the 15 β-receptor agonists, 3 amide alcohols and 11 nitroimidazole antibiotics are shown in Table 2: Table 2 Mass spectral retention times and mass spectral parameters of 15 β-receptor agonists, 3 amide alcohols and 11 nitroimidazole antibiotics 7. The method for determining the contents of 15 β-receptor agonists, 3 amide alcohols and 11 nitroimidazole antibiotics in agricultural products according to claim 1 or 2, characterized in that: The mass of the agricultural product is accurate to 0.01 g.

8. The method for determining the contents of 15 β-receptor agonists, 3 amide alcohols and 11 nitroimidazole antibiotics in agricultural products according to claim 1 or 2, characterized in that: The detection limits of thiamphenicol and fluorosulfamethoxazole are 0.25 μg / kg, and the quantification limits are 1.25 μg / kg, while the detection limits of the remaining beta-receptor agonists, amide alcohols and nitroimidazole antibiotics are all 0.05 μg / kg, and the quantification limits are all 0.25 μg / kg.

9. The method for determining the contents of 15 β-receptor agonists, 3 amide alcohols and 11 nitroimidazole antibiotics in agricultural products according to claim 1 or 2, characterized in that: The average recovery rates of thiamphenicol and florfenicol in the spinach, lettuce or peach are 90.19% to 106.95%, and the average recovery rates of the other target substances are 60.95% to 118.55%.

10. The method for determining the contents of 15 β-receptor agonists, 3 amide alcohols and 11 nitroimidazole antibiotics in agricultural products according to claim 1 or 2, characterized in that: The relative standard deviation range of thiamphenicol and florfenicol in the spinach, lettuce or peach is 0.1% to 5.4%, and the relative standard deviation range of other target objects is 0.1% to 14.9%.

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