A pretreatment method for improving the analysis efficiency of multiple antibiotics in organic fertilizers

Abstract

The present invention discloses a pretreatment method for improving the analysis efficiency of multiple antibiotics in organic fertilizers, comprising: Step 1, freeze-dry and pulverize the organic fertilizer and place it in a centrifuge tube, add Na2EDTA-Mcllvaine buffer solution and 1% formic acid acetonitrile solution, vortex once and perform ultrasonic treatment, add extraction salt, vortex twice and then centrifuge to obtain the upper organic phase; Step 2, take the upper organic phase into a centrifuge tube, add N-propylethylenediamine, high-efficiency matrix fat adsorbent and carbon-based adsorbent for purification, vortex and mix evenly, centrifuge and take 1 mL of the supernatant to pass through an organic filter membrane to obtain a filtrate; Step 3, load the filtrate onto the machine and analyze multiple antibiotics by high performance liquid chromatography-mass spectrometry technology. The method of the present invention has high sensitivity, good selectivity and simple operation, and at the same time has the characteristics of safety, high efficiency and high throughput; it can provide technical support for the risk assessment of antibiotic drug residues, quality and safety supervision, and organic fertilizer quality evaluation in organic fertilizers.

Description

Technical Field

[0001] The present invention relates to the technical field of antibiotic detection, and particularly to a pretreatment method for improving the analysis efficiency of multiple antibiotics in organic fertilizers. Background Art

[0002] Livestock and poultry manure can be used as organic fertilizer for farmland after fermentation and processing. However, the phenomenon of over-dosage and over-range use of antibiotics is common, resulting in about 40% - 90% of the drugs being excreted through feces and urine because they cannot be completely absorbed or metabolized by animals, and then entering the soil and water environment. Currently, the antibiotics widely used in the livestock and poultry breeding industry can be classified into sulfonamides, quinolones, tetracyclines, macrolides, lincosamides, etc. according to their chemical structures.

[0003] In organic fertilizers, antibiotics are very stable and even their content increases over time. Antibiotics can reach the soil environment through agricultural application of organic fertilizers, further affecting the structure and function of soil bacterial communities. As a fertilizer, organic fertilizers entering the farmland system will cause antibiotic residues in agricultural products, seriously affecting quality and safety, and then endangering human health. At the same time, drug residues in livestock and poultry manure are also a key indicator for evaluating its quality. Therefore, it is very necessary and significant to establish a method for detecting and analyzing antibiotic residues in organic fertilizers.

[0004] At present, the analytical methods for determining the content of veterinary drugs in food and environmental matrices (water, soil, and sewage sludge) have been well developed, but few people pay attention to the analysis of veterinary drug residues in organic fertilizers (manure). The matrix of organic fertilizers is rich in complex matrices such as pigments, lipids, and organic acids. Therefore, the sample purification method is crucial, and the QuEChERS method is widely used in the analysis and detection of drug residues due to its advantages of rapidity, high efficiency, and safety. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a pretreatment method for improving the analysis efficiency of multiple antibiotics in organic fertilizers.

[0006] The technical solution of the present invention is: a pretreatment method for improving the analysis efficiency of multiple antibiotics in organic fertilizers, comprising the following steps:

[0007] Step 1: Freeze-dry and pulverize the organic fertilizer and place it in a centrifuge tube. Add 3 - 7 mL of Na 2 EDTA-Mcllvaine buffer solution and 15 - 25 mL of 1% formic acid acetonitrile solution to each gram of the organic fertilizer, vortex and ultrasonically treat it once and add extraction salts, then vortex it a second time and centrifuge to obtain the upper organic phase;

[0008] Step 2: Transfer the upper organic phase to a centrifuge tube, add 5 - 15 mg of N-propylethylenediamine, 5 - 15 mg of EMR-Lipid, and 2 - 6 mg of carbon-based adsorbent to each mL of the upper organic phase for purification. Vortex thoroughly and centrifuge. Take 1 mL of the supernatant and filter it through an organic filter membrane to obtain the filtrate.

[0009] Step 3: Inject the filtrate into the instrument and analyze multiple antibiotics using high performance liquid chromatography - mass spectrometry technology.

[0010] Further, in Step 1, the first vortex time is 1 - 2 min and the second vortex time is 2 - 5 min.

[0011] Note: Since the homogenization mixing of the Na 2 EDTA-Mcllvaine buffer solution and 1% formic acid acetonitrile solution with the organic fertilizer is relatively fast, the above first vortex time can meet the effects of homogenization mixing and stirring; while the mixing of the upper organic phase with N-propylethylenediamine, EMR-Lipid, and carbon-based adsorbent is relatively slow, and the above second vortex time can meet the effects of homogenization mixing and stirring.

[0012] Further, the time of the ultrasonic treatment is 8 - 15 min.

[0013] Note: Using the above ultrasonic treatment time can effectively decompose the inorganic and organic substances in the organic fertilizer, making the sample easier to perform subsequent extraction salt treatment, helping to shorten the time, improve the quality of the obtained upper organic phase, and promoting the dissolution and extraction of organic and inorganic substances in the organic fertilizer, contributing to comprehensively obtaining information on various antibiotics in the sample and improving the comprehensiveness of the organic fertilizer analysis.

[0014] Even further, the specific steps of the ultrasonic treatment are as follows:

[0015] 1) After ultrasonic treatment for 2 - 5 min, add extraction salt to the solution system;

[0016] 2) After 1 - 2 min of adding the extraction salt, perform ultrasonic treatment on the solution system again until the ultrasonic treatment ends;

[0017] Among them, the frequency of the ultrasonic treatment is 20 - 30 kHz and the intensity is 5 - 10 W / cm 2 .

[0018] Note: By adding the extraction salt to the solution system in advance during the ultrasonic treatment and supplemented with ultrasonic treatment, the recovery rate of 33 kinds of antibiotic drugs from the organic fertilizer can be significantly improved.

[0019] Furthermore, the carbon-based adsorbent is a hollow carbon sphere and its surface is etched. N-propylethylenediamine is attached to one hemispherical surface of the hollow carbon sphere, and EMR-Lipid is attached to the other hemispherical surface, forming a biconvex shell-shaped hollow carbon sphere.

[0020] Explanation: By using the carbon-based adsorbent as a carrier, N-propylethylenediamine and EMR-Lipid are attached to one side of the spherical surface. Through the structure of the biconvex shell-shaped hollow carbon sphere, the convex shell structures (N-propylethylenediamine or EMR-Lipid) attached to the two poles of the carbon-based adsorbent do not affect each other.

[0021] And under the influence of the convex shell structures at the two poles of the carbon-based adsorbent, the problem that the carbon-based adsorbent has a strong adsorption effect on drugs with a planar structure can be reduced, so as to avoid affecting the number of antibiotic drugs within the recovery rate range of 80% - 110% on the premise of removing the pigment of organic fertilizer.

[0022] Furthermore, the extraction salt is composed of anhydrous sodium sulfate and sodium chloride in a mass ratio of 4 - 6:3, and 7 - 9 g of the extraction salt is added to each g of organic fertilizer.

[0023] Explanation: Compared with using the combination of anhydrous magnesium sulfate and sodium chloride as the purification extraction agent, the extraction salt composed of anhydrous sodium sulfate and sodium chloride with the above ratio can effectively improve the recovery rate of 33 kinds of antibiotic drugs, so as to more comprehensively obtain information on various antibiotics in the sample and improve the comprehensiveness of the analysis of antibiotics in organic fertilizer.

[0024] Furthermore, the centrifuge tube is a polypropylene centrifuge tube, and the organic filter membrane is 0.22 μm.

[0025] Explanation: The chemical properties of the polypropylene centrifuge tube are stable, and it does not react with most chemical drugs, and it meets the requirements of medical and laboratory for the purity of materials, ensuring the accuracy and reliability of the experimental analysis results; selecting an organic filter membrane of 0.22 μm can make the obtained filtrate meet the usage requirements of the liquid chromatography-tandem mass spectrometer.

[0026] Furthermore, in steps 1 and 2, the centrifugation is carried out at 7000 - 9000 r / min for 4 - 8 min.

[0027] Explanation: Using the above centrifugation parameters basically meets the usage requirements of the analysis method of the present invention. Of course, theoretically, the higher the centrifugation speed, the better, but considering the actual equipment conditions and other issues, choosing the above centrifugation parameters can have better condition adaptability.

[0028] Furthermore, 5 mL of Na 2 EDTA-Mcllvaine buffer solution and 20 mL of 1% formic acid acetonitrile solution are added to each g of organic fertilizer.

[0029] Note: When methanol is used as the mobile phase, the chromatographic peaks of components are prone to broadening, and the resolution of components is low. Using Na with the above dosage 2 Gradient elution is carried out using EDTA-Mcllvaine buffer solution and 1% formic acid acetonitrile solution as the mobile phase. Using acetonitrile as the mobile phase can significantly improve the peak shape of acid-base compounds. Adding formic acid to the mobile phase can enhance the retention of compounds, and the separation degree of chromatographic peaks is improved.

[0030] Furthermore, 10 mg of N-propylethylenediamine (PSA), 10 mg of EMR-Lipid, and 5 mg of carbon-based adsorbent are added to each mL of the upper organic phase for purification.

[0031] Note: Since organic fertilizers contain various complex matrices such as pigments, organic acids, and crude fat, purification treatment is required during the detection and analysis process. Carbon-based adsorbent (CarbonS) can effectively adsorb pigments, but it also has a strong adsorption effect on drugs with planar structures. EMR-Lipid and N-propylethylenediamine (PSA) can effectively adsorb components such as fatty acids and organic acids in the organic fertilizer matrix. At the above dosages, the dosing and use effects of N-propylethylenediamine (PSA), EMR-Lipid, and carbon-based adsorbent are the best.

[0032] The beneficial effects of the present invention are as follows:

[0033] (1) The present invention adopts ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) technology and QuEChERS pretreatment and purification method to establish an analytical method for the residues of 33 kinds of antibiotic drugs in organic fertilizers, providing technical support for the work of veterinary drug residue risk assessment, quality and safety supervision, and quality evaluation in organic fertilizers;

[0034] (2) The present invention pretreats the complex matrix samples of organic fertilizers through QuEChERS pretreatment technology, effectively reducing the interference of sample matrix, and combines with high performance liquid chromatography-mass spectrometry technology to establish an analytical method for simultaneously determining the residues of 33 kinds of antibiotic drugs in organic fertilizers.

[0035] (3) The method of the present invention has high sensitivity, good selectivity, and simple operation, and at the same time has the characteristics of safety, high efficiency, and high throughput; it can provide technical support for the risk assessment of antibiotic drug residues, quality and safety supervision, and quality evaluation of organic fertilizers. Description of the Drawings

[0036] Figure 1 It is a diagram showing the influence of different buffers of the present invention on the recovery rate of antibiotics.

[0037] Figure 2It is a graph of the extraction recovery rates of three adsorbents, PSA, EMR-Lipid, and CarbonS.

[0038] Figure 3 It is a matrix effect graph of treating organic fertilizers by the method of the present invention and detecting. Detailed implementation manners

[0039] The present invention will be further described in detail below in conjunction with the specific implementation manners to better reflect the advantages of the present invention.

[0040] Example 1: A pretreatment method for improving the analysis efficiency of multiple antibiotics in organic fertilizers, comprising the following steps:

[0041] Step 1: After freeze-drying and pulverizing the organic fertilizer, accurately weigh 1.0 g and place it in a 50 mL polypropylene centrifuge tube. Add 5 mL of Na 2 EDTA-Mcllvaine buffer solution (pH = 4.0) and 20 mL of 1% formic acid acetonitrile solution. After vortexing for 1 min and ultrasonic treatment for 10 min once, add 5 g of anhydrous sodium sulfate and 3 g of sodium chloride. After vortexing for 2 min for mixing twice, centrifuge at 8000 r / min for 5 min to obtain the upper organic phase;

[0042] Among them, the frequency of the ultrasonic treatment is 25 kHz, and the intensity is 8 W / cm 2 ;

[0043] Step 2: Take 2 mL of the upper organic phase into a 15 mL polypropylene centrifuge tube, add 20 mg of N-propylethylenediamine, 20 mg of EMR-Lipid, and 10 mg of CarbonS for purification. Vortex and mix evenly, centrifuge at 8000 r / min for 5 min, take 1 mL of the supernatant and pass it through a 0.22 μm organic filter membrane to obtain a filtrate;

[0044] Step 3: Inject the filtrate into the instrument and analyze 33 antibiotics by high performance liquid chromatography-mass spectrometry technology. The 33 antibiotics are divided into 9 sulfonamides, 9 quinolones, 9 macrolides, 4 tetracyclines, and 2 lincosamides.

[0045] To verify the detection effect of the above analysis method, the following application tests are now carried out:

[0046] I. Instruments, reagents, and materials are as follows:

[0047] 1) Instruments: 1290-6495 ultra-high performance liquid chromatography tandem mass spectrometer (Agilent Technologies, USA); Milli-Q ultrapure water purifier (Millipore Corporation, USA); MS205DU electronic analytical balance (Mettler Toledo, Switzerland); 0.22 μm nylon filter membrane (i.e., organic filter membrane) (Agilent Technologies, USA); Coulter Avanti J-26XP ultra-high speed refrigerated centrifuge (Bechman, Germany); Vortex3 vortex oscillator (IKA, Germany).

[0048] 2) Reagents and materials: 9 sulfonamides, 9 quinolones, 9 macrolides, 4 tetracyclines and 2 lincosamides were purchased from Dr. Ehrenstorfer, Germany, with a purity higher than 96%. The drug names and abbreviations are shown in Table 2; formic acid, acetonitrile and methanol (chromatographic grade, Shanghai Aladdin); ethylenediamine-N-propyl silane (PSA) with a particle size of 40 μm (Welchrom, USA); CarbonS, EMR-Lipid (Agilent Technologies, USA); anhydrous sodium sulfate, sodium chloride (analytical grade, Guangzhou Chemical Reagent Factory). The ultrapure water used in the experiment was prepared by a Milli-Q water treatment system.

[0049] II. Preparation of standard solutions is as follows:

[0050] Standard stock solution (1.0 mg / mL): Weigh 50.00 mg of each antibiotic standard into a 50 mL volumetric flask and make up to the mark with methanol to obtain a single standard stock solution with a concentration of 1.0 mg / mL.

[0051] Standard intermediate solution (20.0 mg / L): Pipette 1 mL of each of the above standard stock solutions into a 50 mL volumetric flask and make up to the mark with methanol to obtain a single standard intermediate solution with a concentration of 20.0 mg / L.

[0052] Mixed standard working solution (1.0 mg / L): Pipette 0.5 mL of each of the above standard intermediate solutions into a 10 mL volumetric flask and make up to the mark with methanol to obtain a mixed standard working solution with a concentration of 1.0 mg / L; all the above standard solutions were stored in the dark at -18 °C in a refrigerator.

[0053] III. Sample collection is as follows:

[0054] The samples were commercially available organic fertilizers. Samples were collected from 5 different positions inside each organic fertilizer packaging bag, mixed well, and 0.5 kg was taken as the test sample according to the quartering method. After air drying, pulverizing and passing through a 1 mm sieve, it was ready for use.

[0055] IV. Sample treatment is as follows:

[0056] Using the treatment steps for organic fertilizers in Example 1, the filtrate was loaded onto the machine, and 33 antibiotics were analyzed using high performance liquid chromatography-mass spectrometry technology.

[0057] V. The UPLC-MS / MS conditions are as follows:

[0058] Chromatographic column: ACQUITY UPLC BEH C18 chromatographic column, column temperature: 40 °C; flow rate: 0.3 mL / min; injection volume: 5 μL; mobile phase: A is formic acid aqueous solution (containing 1% formic acid), B is acetonitrile; the gradient elution program is shown in Table 1:

[0059] Table 1 Gradient elution program

[0060]

[0061] (Mobile phase A: 0.1% (v / v) formic acid solution.; mobile phase B: acetonitrile)

[0062] VI. Conclusions and analysis:

[0063] 1) Analysis of chromatographic and mass spectrometry conditions: The mobile phase has a great influence on the ionization efficiency, retention time, peak shape, etc. of electrospray mass spectrometry. When methanol is used as the mobile phase, the chromatographic peak shape of the components is prone to broadening and the component resolution is low. When acetonitrile is used as the mobile phase, the peak shape of acid-base compounds can be significantly improved. Adding formic acid to the mobile phase can enhance the retention of compounds and the separation degree of chromatographic peaks is also improved. Therefore, in this experiment, acetonitrile and 1% formic acid aqueous solution were selected as the mobile phase for gradient elution.

[0064] To obtain the best sensitivity and separation effect of the target compounds, single standard solutions (100.00 μg / L) of each compound were used to perform parent ion scanning (MS2 Scan) in the continuous injection mode of the syringe pump in turn to obtain the parent ions of the target compounds, and their mass-to-charge ratios (m / z) were recorded; the scanning range covering the possible daughter ion masses was set, and the daughter ions of the target compounds were obtained through the product ion scanning mode (Product Scan), and the 2 daughter ions with the strongest response were respectively selected as the qualitative and quantitative ions, and the m / z was recorded. The MRM mode was selected to optimize the mass spectrometry parameters such as the collision energy, declustering voltage, and capillary voltage of each target compound. The results are shown in Table 2:

[0065] Table 2 Mass spectrometry parameters of 33 antibiotic drugs

[0066]

[0067]

[0068] 2) Analysis of extraction conditions

[0069] 1. Selection of the constant-volume solution: To achieve better responses of 33 compounds in the organic fertilizer on the mass spectrometer, the extraction effects of methanol, acetonitrile, 1% formic acid in acetonitrile, and 1% formic acid in methanol as extraction solvents at a concentration of 100 μg / kg were compared by quantitative addition. The experimental results are shown in Table 3:

[0070] Table 3 Analysis table of the constant-volume solution

[0071]

[0072] As shown in Table 3, the average instrument responses when using methanol, acetonitrile, and 1% formic acid in methanol as the constant-volume solutions are lower than that of 1% formic acid in acetonitrile. This is because acetonitrile has a wide polarity range and strong tissue penetration ability, resulting in better solubility and extraction efficiency for most target compounds. Adding formic acid can inhibit the dissociation of carboxyl groups in the target compounds, thereby improving the instrument response. Therefore, 1% formic acid in acetonitrile was finally selected as the extraction solvent.

[0073] 2. Analysis of the buffer solution during sample extraction

[0074] Na 2 EDTA-Mcllvaine buffer salt can chelate with metal ions. In this experiment, Na 2 EDTA-Mcllvaine buffer solution was selected to be added in advance to improve the recovery rate of the target substances. The pH value may affect the efficiency of the buffer solution in extracting antibiotics from the organic fertilizer. Therefore, on the basis of adding Na 2 EDTA-Mcllvaine buffer solution in advance, three pH values (unadjusted pH, pH = 4.0, pH = 10.0) were investigated; the effects of Na 2 EDTA-Mcllvaine buffer solution on the recovery rate were studied. When the pH value was 4.0, the number of antibiotic drugs with a recovery rate between 80% - 110% was significantly higher than that of other pH value buffer solutions. The results are shown in Figure 1 . Therefore, in this experiment, Na 2 EDTA-Mcllvaine buffer solution with pH = 4.0 was selected to be added in advance to improve the recovery rate of the target substances.

[0075] 3. Analysis of the extraction salts

[0076] To make the target substances more easily enter the organic phase, MgSO 4 , Na 2 SO 4 and NaCl are commonly used as extraction salts in the QuEChERS method. In this study, the recovery rates of 33 antibiotic drugs were investigated when using anhydrous sodium sulfate and sodium chloride, and anhydrous magnesium sulfate and sodium chloride as purification extraction agents.

[0077] The results showed that when anhydrous magnesium sulfate and sodium chloride were selected as the purification extraction agents, due to the strong adsorption of anhydrous magnesium sulfate on the target compounds, the recovery rates of 25 drugs were less than 65%, and MgSO 4 When used as the extraction salt, the extraction recovery rates of quinolone and tetracycline drugs were both low, which might be related to the fact that the carboxyl and carbonyl groups contained in the molecular structures of antibiotic drugs were easily chelated with metal ions such as Mg + etc. When anhydrous sodium sulfate and sodium chloride were selected as the purification extraction agents, the recovery rates of 33 drugs were all higher than 65%. Therefore, Na 2 SO 4 and NaCl were selected as the extraction salts.

[0078] 4. Analysis of the dosage of QuEChERS adsorbents

[0079] Since organic fertilizers contain various complex matrices such as pigments, organic acids, and crude fat, purification treatment is required during the detection process. PSA, CarbonS, EMR-Lipid, etc. are new and efficient adsorbents for the QuEChERS method. CarbonS can effectively adsorb pigments, but at the same time has a strong adsorption effect on drugs with planar structures; EMR-Lipid and PSA can effectively adsorb components such as fatty acids and organic acids in the organic fertilizer matrix, and the removal of water by anhydrous Na 2 SO 4 helps with purification.

[0080] In this experiment, blank organic fertilizer samples were selected as the experimental materials, a mixed standard solution with a content of 100 μg / kg was added, each sample was measured in parallel 3 times, and the external standard method was used for quantification to optimize the usage amounts of PSA, CarbonS, and EMR-Lipid. The extraction recovery rates of five dosages of adsorbents were compared: 20 mg PSA, 20 mg EMR-Lipid, 10 mg CarbonS; 20 mg PSA, 20 mg EMR-Lipid, 20 mg CarbonS; 20 mg PSA, 30 mg EMR-Lipid, 10 mg CarbonS; 30 mg PSA, 20 mg EMR-Lipid, 10 mg CarbonS; 30 mg PSA, 30 mg EMR-Lipid, 10 mg CarbonS

[0081] The results are as Figure 2As shown, it was found that the purification effect of 20 mg PSA, 20 mg EMR-Lipid, and 10 mg CarbonS was the best, and the recovery rates of 29 antibiotic drugs were all in the range of 80% - 110%. After increasing the amount of CarbonS used, the number of antibiotic drugs within the 80% - 110% recovery range decreased significantly. Therefore, 20 mg PSA, 20 mg EMR-Lipid, and 10 mg CarbonS were selected as the purification adsorbents and their appropriate dosages.

[0082] 3. Analysis of matrix effect

[0083] The complexity of matrix components can bring unpredictable interference to the analysis of antibiotics, and there may be a relatively high matrix effect, seriously affecting the accuracy of the analysis method. The blank organic fertilizer samples were processed according to the optimized pretreatment method. Under the optimized pretreatment conditions, when the average matrix effect was between 80% - 120%, it indicated that the matrix effect could be ignored. When the matrix effect was between 50% - 80% and 120% - 150%, it indicated that there was a slight non-negligible matrix effect. The matrix effect (ME) was calculated by the following formula: ME(%) = A 2 / A 1 ×100%. In the formula, A 1 was the average peak area of the antibiotic in pure solvent at a specific concentration; A 2 was the average peak area of the antibiotic in the blank organic fertilizer extract at the same concentration. The organic fertilizer samples were processed and detected using the improved pretreatment method, and the results were as Figure 3 follows: Only the matrix effects of 5 drugs in the organic fertilizer samples processed by the unimproved pretreatment method could be ignored; among the organic fertilizer samples processed by the improved pretreatment method, the matrix effects of 29 drugs could be ignored, and the matrix enhancement or inhibition effects of 4 antibiotics (JOS, FL, TL, SNM) were slightly lower than 80%. For these four drugs, matrix-matched calibration curves were used for quantification to reduce the influence of matrix interference on the results.

[0084] 4. Methodological verification

[0085] 1) Linear range, detection limit, and quantification limit

[0086] A series of standard solutions with mass concentrations of 0.5, 1, 2, 5, 10, 20, 50, and 100 μg / L were prepared by spiking blank matrix, and standard curves were plotted with concentration - peak area. The correlation coefficient results are shown in Table 3;

[0087] The results showed that within the range of 0.5 - 100 μg / L, the correlation coefficients (R 2 ) of the standard curves of 33 antibiotic drugs were all greater than 0.9950, indicating good linearity.

[0088] 2) Recovery rate and sensitivity

[0089] A blank organic fertilizer sample was taken and spiked with a mixed standard solution of 33 antibiotics at three levels of low, medium, and high (5, 20, and 100 μg / kg), and the spiked recovery experiment was repeated 6 times. The recovery rate and relative standard deviation (RSD) were calculated, and the results are shown in Table 3.

[0090] Table 3 Results of correlation coefficient, detection limit, quantification limit, spiked recovery rate, and relative standard deviation (n = 6)

[0091]

[0092]

[0093] A blank organic fertilizer sample was taken and spiked with a mixed standard solution of 33 antibiotics at three levels of low, medium, and high (5, 20, and 100 μg / kg), and the spiked recovery experiment was repeated 6 times. The recovery rate and relative standard deviation (RSD) were calculated. The results showed that the recovery rate was 78.38% - 107.57%, and the RSD was 0.14% - 9.6%, which could meet the requirements for the detection of antibiotic residues in organic fertilizers. In the prior art, the QuEChERS-HPLC-MS / MS method was used to detect the residues of 10 fluoroquinolone drugs in organic fertilizers. The linear correlation coefficients of the 10 fluoroquinolone drugs were all greater than 0.9930 in the range of 10 - 500 μg / kg. The method detection limit was 0.5 - 2.5 μg / kg, and the quantification limit was 1.7 - 8.3 μg / kg. The average spiked recovery rate of the samples was 82.5% - 117.1%, and the relative standard deviation was 3.4% - 10.2%. It can be seen that the results of this experiment are similar to those of the prior art and meet the requirements for the detection of organic fertilizer samples. It can be seen that the optimal values are: the recovery rate is 78.38% - 107.57%, and the relative standard deviation is 0.14% - 9.6%.

[0094] Example 2: The difference between this example and Example 1 is that 3 mL of Na 2 EDTA-Mcllvaine buffer solution (pH = 4.0) and 20 mL of 1% formic acid acetonitrile solution were added.

[0095] Example 3: The difference between this example and Example 1 is that 7 mL of Na 2 EDTA-Mcllvaine buffer solution (pH = 4.0) and 25 mL of 1% formic acid acetonitrile solution were added.

[0096] Example 4: The difference between this example and Example 1 is that 4 g of anhydrous sodium sulfate and 3 g of sodium chloride were added.

[0097] Example 5: The difference between this example and Example 1 is that 6 g of anhydrous sodium sulfate and 3 g of sodium chloride were added.

[0098] Example 6: The difference between this example and Example 1 is that 10 mg of N-propylethylenediamine, 10 mg of EMR-Lipid, and 4 mg of CarbonS are added for purification.

[0099] Example 7: The difference between this example and Example 1 is that 30 mg of N-propylethylenediamine, 30 mg of EMR-Lipid, and 12 mg of CarbonS are added for purification.

[0100] Example 8: The difference between this example and Example 1 is that the ultrasonic treatment time is 8 min.

[0101] Example 9: The difference between this example and Example 1 is that the ultrasonic treatment time is 15 min.

[0102] To verify the effects of the dosages of various agents and parameters on this method, the experimental method of Example 1 is now used to measure the effects of Examples 2 - 9, and the results are as follows:

[0103] The recovery rate of Example 2 is 42.07% - 98.74%, and the relative standard deviation is 0.6% - 15.6%;

[0104] The recovery rate of Example 3 is 52.21% - 110.06%, and the relative standard deviation is 1.5% - 14.3%;

[0105] By comparing the optimal values of Example 2, Example 3 with Example 1, it is found that by adjusting the ratio and dosage of the added Na 2 EDTA-Mcllvaine buffer (pH = 4.0) and 1% formic acid acetonitrile solution, it has a certain impact on the antibiotic detection and analysis effect of this method. Among them, the ratio and dosage of the Na 2 EDTA-Mcllvaine buffer (pH = 4.0) and 1% formic acid acetonitrile solution in Example 1 are relatively optimal.

[0106] The recovery rate of Example 4 is 54.34% - 93.87%, and the relative standard deviation is 2.5% - 9.3%;

[0107] The recovery rate of Example 5 is 72.74% - 106.65%, and the relative standard deviation is 0.5% - 12.36%;

[0108] By comparing the optimal values of Example 4, Example 5 with Example 1, it is found that by adjusting the ratio and dosage of anhydrous sodium sulfate and sodium chloride in the extraction salt, it has a certain impact on the antibiotic detection and analysis effect of this method. Among them, the ratio and dosage of anhydrous sodium sulfate and sodium chloride in Example 1 are relatively optimal.

[0109] The recovery rate of Example 6 was 39.08% - 109.33%, and the relative standard deviation was 2.53% - 22.36%;

[0110] The recovery rate of Example 7 was 65.76% - 105.43%, and the relative standard deviation was 0.54% - 10.43%;

[0111] By comparing the optimal values of Example 6, Example 7 and Example 1, it was found that by adjusting the addition doses of N-propylethylenediamine, EMR-Lipid and CarbonS, it had a certain influence on the antibiotic detection and analysis effect of this method. Among them, the addition doses of N-propylethylenediamine, EMR-Lipid and CarbonS in Example 1 were relatively optimal.

[0112] The recovery rate of Example 8 was 65.63% - 108.36%, and the relative standard deviation was 0.5% - 12.3%;

[0113] The recovery rate of Example 9 was 72.22% - 120.65%, and the relative standard deviation was 1.5% - 9.3%;

[0114] By comparing the optimal values of Example 8, Example 9 and Example 1, it was found that by controlling the duration of ultrasonic treatment, it had a certain influence on the antibiotic detection and analysis effect of this method. Among them, the duration of ultrasonic treatment in Example 1 was relatively optimal.

[0115] Example 10: The difference between this example and Example 1 was that the specific steps of the ultrasonic treatment were as follows:

[0116] 1) After ultrasonic treatment for 4 min, extraction salt was added to the solution system;

[0117] 2) 85 s after the addition of the extraction salt, the solution system was ultrasonically treated again until the ultrasonic treatment ended;

[0118] Among them, the frequency of the ultrasonic treatment was 25 kHz, and the intensity was 8 W / cm 2 .

[0119] Example 11: The difference between this example and Example 10 was that extraction salt was added to the solution system after ultrasonic treatment for 2 min.

[0120] Example 12: The difference between this example and Example 10 was that extraction salt was added to the solution system after ultrasonic treatment for 5 min.

[0121] Example 13: The difference between this example and Example 10 was that it was 1 min after the addition of the extraction salt.

[0122] Example 14: The difference between this example and Example 10 is that it is 2 minutes after the addition of the extraction salt.

[0123] Example 15: The difference between this example and Example 10 is that the frequency of the ultrasonic treatment is 20 kHz and the intensity is 5 W / cm 2 。

[0124] Example 16: The difference between this example and Example 10 is that the frequency of the ultrasonic treatment is 30 kHz and the intensity is 10 W / cm 2 。

[0125] To verify the influence of different ultrasonic treatment methods and parameters on this method, the experimental method of Example 1 was used to measure the effects of Examples 10 - 18, and the results are as follows:

[0126] The recovery rate of Example 10 is 82.96% - 111.29%, and the relative standard deviation is 0.12% - 8.5%;

[0127] By comparing the optimal values of Example 10 and Example 1, it is found that by optimizing the ultrasonic treatment method, the detection and analysis effect of antibiotics is improved to a certain extent compared with the method of Example 1.

[0128] The recovery rate of Example 11 is 81.48% - 109.86%, and the relative standard deviation is 0.14% - 9.1%;

[0129] The recovery rate of Example 12 is 82.84% - 111.43%, and the relative standard deviation is 0.12% - 8.5%;

[0130] By comparing the optimal values of Example 11, Example 12 and Example 1, it is found that by adjusting the addition time of the extraction salt, it has a certain influence on the detection and analysis effect of antibiotics of this method. Among them, the addition time of the extraction salt in Example 10 is relatively optimal.

[0131] The recovery rate of Example 13 is 80.39% - 109.14%, and the relative standard deviation is 0.14% - 9.4%;

[0132] The recovery rate of Example 14 is 81.76% - 110.32%, and the relative standard deviation is 0.13% - 8.9%;

[0133] By comparing the optimal values of Example 13, Example 14 and Example 1, it is found that by adjusting the ultrasonic treatment time, it has a certain influence on the detection and analysis effect of antibiotics of this method. Among them, the ultrasonic treatment time in Example 10 is relatively optimal.

[0134] The recovery rate of Example 15 was 81.86% - 110.47%, and the relative standard deviation was 0.13% - 9.0%;

[0135] The recovery rate of Example 16 was 82.13% - 120.65%, and the relative standard deviation was 0.12% - 8.8%;

[0136] By comparing the optimal values of Example 15, Example 16 and Example 1, it was found that by adjusting the frequency and intensity of ultrasonic treatment, it had a certain impact on the antibiotic detection and analysis effect of this method. Among them, the frequency and intensity of ultrasonic treatment in Example 10 were relatively optimal.

[0137] Example 17: The difference between this example and Example 1 is that the carbon-based adsorbent is a hollow carbon sphere and its surface is etched. N-propylethylenediamine is attached to one hemispherical surface of the hollow carbon sphere, and EMR-Lipid is attached to the other hemispherical surface to form a biconvex shell-shaped hollow carbon sphere;

[0138] Specifically, N-propylethylenediamine and EMR-Lipid were each mixed with deionized water at a mass ratio of 1:20 to obtain a mixed solution A and a mixed solution B; the hollow carbon spheres with a particle size of 1 mm were soaked in 25% dilute nitric acid for 1 minute, then rinsed with deionized water, dried and spread on a well plate. Subsequently, the mixed solution A was evenly sprayed on this side hemispherical surface of the hollow carbon spheres on the upper side of the well plate, and then pushed into an oven to be dried at 60 °C, and the treatment was repeated 5 times;

[0139] Subsequently, the mixed solution B was evenly sprayed on this side hemispherical surface of the hollow carbon spheres on the lower side of the well plate, and then pushed into an oven to be dried at 60 °C, and the treatment was repeated 5 times; after the treatment was completed, the hollow carbon spheres were shaken off and collected to obtain the finished product, and the residual N-propylethylenediamine and EMR-Lipid on the well plate were rinsed and collected with deionized water to measure the loss amount, and the dosage of the added N-propylethylenediamine and EMR-Lipid was calculated;

[0140] It can be understood that when the dosage of N-propylethylenediamine and EMR-Lipid is significantly greater than that of the hollow carbon spheres, it is difficult to completely attach N-propylethylenediamine and EMR-Lipid on both hemispherical surfaces of the hollow carbon spheres, and the remaining N-propylethylenediamine and EMR-Lipid are added to the upper organic phase by mixing with the biconvex shell-shaped hollow carbon spheres.

[0141] To verify the influence of different carbon-based adsorbents on this method, the experimental method of Example 1 was used to measure the effect of Example 17, and the results are as follows:

[0142] The recovery rate of Example 17 was 84.47% - 112.35%, and the relative standard deviation was 0.1% - 8.1%. By comparing with the optimal value of Example 1, it was found that the recovery rate of Example 17 was significantly higher than that of Example 1, and the recovery efficiency was more stable. At the same time, the relative standard deviation was also smaller. Therefore, after optimizing and adjusting the carbon-based adsorbent, the detection accuracy of this analytical method was effectively improved.

Claims

1. A pretreatment method for improving the analysis efficiency of multiple antibiotics in organic fertilizers, characterized in that: The following steps are involved: Step 1, freeze-drying and crushing the organic fertilizer and placing it in a centrifuge tube, adding 5 mL of Na2EDTA-Mcllvaine buffer and 20 mL of 1% formic acid acetonitrile solution to each gram of organic fertilizer, the pH value of the Na2EDTA-Mcllvaine buffer is 4.0, vortexing and ultrasonic treatment are performed once, and extraction salt is added, and vortexing is performed twice and then centrifuged to obtain an upper organic phase; the extraction salt is composed of anhydrous sodium sulfate and sodium chloride in a mass ratio of 5:3, and 8 g of extraction salt is added to each gram of organic fertilizer; Step 2: Take the upper organic phase to a centrifuge tube, add 10 mg of N-propylethylenediamine, 10 mg of EMR-Lipid and 5 mg of carbon-based adsorbent per mL of the upper organic phase for purification, vortex mix, centrifuge and take 1 mL of the supernatant to pass through an organic filter membrane to obtain a filtrate; Step 3, the filtrate is put into the machine and a high performance liquid chromatography-mass spectrometry technique is used to analyze a variety of antibiotics; The ultrasonic treatment time was 10 min, the frequency of the ultrasonic treatment was 25 kHz, and the intensity was 8 W / cm 2 ; The specific steps of the ultrasonic treatment are as follows: 1) After ultrasonic treatment for 4 min, adding extraction salt to the solution system; 2) 85 seconds after the extraction salt is added, the solution system is subjected to ultrasonic treatment again until the ultrasonic treatment is completed; The carbon-based adsorbent is a hollow carbon sphere, and the surface of the hollow carbon sphere is etched, and N-propylethylenediamine is attached to one hemispherical surface of the hollow carbon sphere, and EMR-Lipid is attached to the other hemispherical surface, forming a double convex shell-shaped hollow carbon sphere; The 33 antibiotics are tylosin, clarithromycin, erythromycin, josamycin, roxithromycin, azithromycin, spiramycin, tilmecocin, kitasamycin, clindamycin, lincomycin, enrofloxacin, fleroxacin, sarafloxacin, ciprofloxacin, ofloxacin, lomefloxacin, norfloxacin, mabofloxacin, pefloxacin, tetracycline, oxytetracycline, chlortetracycline, doxycycline, sulfamethoxazole, sulfadimethoxine, sulfadiazine, sulfadiazine, sulfapyridine standard, sulfachloropyridazine, sulfathiazole, sulfadimethoxine, and trimethoprim.

2. A pretreatment method for improving the analysis efficiency of multiple antibiotics in organic fertilizers as claimed in claim 1, characterized in that: In the step 1, the first vortex time is 1 to 2 minutes, and the second vortex time is 2 to 5 minutes.

3. A pre-treatment method for improving the analysis efficiency of multiple antibiotics in organic fertilizers as claimed in claim 1, characterized in that: The centrifuge tube is a polypropylene centrifuge tube, and the organic filter membrane is 0.22 μm.

4. A pre-treatment method for improving the analysis efficiency of multiple antibiotics in organic fertilizers as claimed in claim 1, characterized in that: In step 1 and step 2, the centrifugation is performed at 7000-9000 r / min for 4-8 min.

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