Full-automatic accelerated solvent extraction method for extracting antibiotics in soil

Abstract

The invention provides a full-automatic accelerated solvent extraction method for extracting antibiotics in soil, and belongs to the technical field of chemical detection. The extraction method provided by the invention comprises the following steps: mixing a soil sample with a mixed antibiotic internal standard solution, a purifying agent and methanol to obtain a to-be-extracted soil sample; the soil sample to be extracted is extracted in an accelerated solvent extraction evaporator ExtrevaASE, and a liquid to be detected is obtained; the extraction solvent is a mixed solution of a citric acid-trisodium citrate buffer solution and acetonitrile; the pH value of the citric acid-trisodium citrate buffer solution is 3 to 4. According to the method, an accelerated solvent extraction evaporator ExtrevaASE is adopted for extraction, so that the extraction process is fully automatic, manpower resources are saved, meanwhile, the extraction pressure is low (200 psi), and a mixed solution of a citric acid-trisodium citrate buffer solution and acetonitrile is adopted as an extraction solvent, so that the extraction effect is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical detection, and particularly relates to a fully automatic accelerated solvent extraction method for extracting antibiotics from soil. Background Art

[0002] With the widespread use of antibiotics in human medicine, livestock and poultry farming, and agricultural planting, they enter the soil environment in large quantities through manure discharge, irrigation and fertilization, resulting in widespread residues and accumulation of antibiotics in the soil environment, which not only affects the function of the soil ecosystem, but may also affect groundwater safety and human health through migration and bioaccumulation. Therefore, accurate and efficient extraction and analysis of antibiotics in soil is the basic work of environmental risk assessment and pollution control.

[0003] Currently, the main methods for extracting antibiotics from soil are ultrasonic extraction and accelerated solvent extraction. Ultrasonic extraction suffers from high solvent consumption, a time-consuming extraction process, and low recoveries of some antibiotics, limiting its application in large-scale, high-throughput testing. Existing accelerated solvent extraction methods are automated only during the soil extraction phase; subsequent solid-phase extraction, evaporation, and reconstitution still require manual work. Furthermore, the extraction pressure of existing accelerated solvent extraction instruments is typically high, ranging from 1500 to 2000 psi, and acetonitrile or acetonitrile-McIlvain buffer is typically used as the extraction solvent. Consequently, extraction efficiency remains to be improved. Summary of the Invention

[0004] The present invention aims to provide a fully automated accelerated solvent extraction method for extracting antibiotics from soil. The method provided by the present invention can be performed fully automatically and has a better extraction effect.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a fully automatic accelerated solvent extraction method for extracting antibiotics from soil, comprising the following steps:

[0007] (1) mixing a soil sample with a mixed antibiotic internal standard solution, a scavenger, and methanol to obtain a soil sample to be extracted;

[0008] (2) The soil sample to be extracted obtained in step (1) is extracted in an accelerated solvent extraction evaporator Extreva ASE to obtain a test solution; the extraction solvent is a mixed solution of citric acid-trisodium citrate buffer and acetonitrile; the pH value of the citric acid-trisodium citrate buffer is 3-4.

[0009] Preferably, the mesh size of the soil sample in step (1) is ≥100 mesh.

[0010] Preferably, the purifier in step (1) is graphitized non-porous carbon or graphitized carbon black.

[0011] Preferably, the mass ratio of the soil sample to the purifier in step (1) is 1:(0.05-0.1).

[0012] Preferably, in step (1), the volume ratio of the soil sample to methanol is 2 g: (0.5-1) mL.

[0013] Preferably, the mixing time in step (1) is 10 to 15 hours.

[0014] Preferably, the volume ratio of citric acid-trisodium citrate buffer to acetonitrile in the extraction solvent of step (2) is 1: (0.9-1.1).

[0015] Preferably, the instrument parameters of the accelerated solvent extraction evaporator ExtrevaASE in step (2) include: extraction cell 5 mL, extraction temperature of room temperature, purge time of 58 to 62 s, extraction solvent pre-filling of 48 to 52%, extraction solvent flow rate of 0.2 to 0.3 mL / min, extraction time of 12 min, evaporation temperature of 40 to 80° C., total nitrogen flow rate of 190 to 210 mL / min, and vacuum degree of 2 to 8 psi.

[0016] Preferably, the method further comprises subjecting the test liquid to ultra-high performance liquid chromatography-tandem mass spectrometry detection.

[0017] Preferably, the parameters of the ultra-high performance liquid chromatography-tandem mass spectrometry detection include: column temperature of 25-28°C, mobile phase A is 0.1% formic acid solution by volume, mobile phase B is acetonitrile, gradient elution, ESI temperature of 340-360°C, carrier gas flow rate of 8-10 L / min, and atomization voltage of 34-36 psi.

[0018] The present invention provides a fully automated accelerated solvent extraction method for extracting antibiotics from soil, comprising the following steps: (1) mixing a soil sample with a mixed antibiotic internal standard solution, a scavenger, and methanol to obtain a soil sample to be extracted; (2) extracting the soil sample to be extracted obtained in step (1) in an accelerated solvent extraction evaporator ExtrevaASE to obtain a test solution; the extraction solvent is a mixed solution of citric acid-trisodium citrate buffer and acetonitrile; the pH value of the citric acid-trisodium citrate buffer is 3-4. The present invention uses the accelerated solvent extraction evaporator ExtrevaASE for extraction, making the extraction process fully automated and saving human resources. At the same time, the extraction pressure is low (200 psi), and the use of a mixed solution of citric acid-trisodium citrate buffer and acetonitrile as the extraction solvent improves the extraction effect. The results of the examples show that when the method of the present invention is used for extraction and detection, the average matrix spike recovery rate of each antibiotic is between 70% and 130%. DETAILED DESCRIPTION

[0019] The present invention provides a fully automatic accelerated solvent extraction method for extracting antibiotics from soil, comprising the following steps:

[0020] (1) mixing a soil sample with a mixed antibiotic internal standard solution, a scavenger, and methanol to obtain a soil sample to be extracted;

[0021] (2) The soil sample to be extracted obtained in step (1) is extracted in an accelerated solvent extraction evaporator Extreva ASE to obtain a test solution; the extraction solvent is a mixed solution of citric acid-trisodium citrate buffer and acetonitrile; the pH value of the citric acid-trisodium citrate buffer is 3-4.

[0022] Unless otherwise specified, the present invention has no particular limitation on the sources of the raw materials, and commercially available products known to those skilled in the art may be used.

[0023] The present invention mixes a soil sample with a mixed antibiotic internal standard solution, a purifier and methanol to obtain a soil sample to be extracted.

[0024] In the present invention, the soil sample is preferably dried, ground and sieved in sequence before use.

[0025] In the present invention, the drying is preferably freeze drying; the freeze drying temperature is preferably -50 to -40°C; and the freeze drying time is preferably 12 to 24 hours.

[0026] The present invention has no particular limitation on the grinding and sieving operations. Grinding and sieving techniques well known to those skilled in the art may be used to ensure that the mesh size of the soil sample is within the required range.

[0027] In the present invention, the mesh size of the soil sample is preferably ≥100 mesh.

[0028] In the present invention, the antibiotics preferably include sulfadiazine, sulfamethazine, ciprofloxacin, norfloxacin, tetracycline, chlortetracycline, clarithromycin and roxithromycin.

[0029] In the present invention, the antibiotic internal standards in the mixed antibiotic internal standard solution include sulfamethoxazole-d4, ciprofloxacin-d8, tetracycline-d6 and roxithromycin-d7.

[0030] In the present invention, the concentration of each antibiotic internal standard in the mixed antibiotic internal standard solution is preferably 0.9-1.1 mg / L, more preferably 1 mg / L.

[0031] In the present invention, the volume ratio of the mass of the soil sample to the mixed antibiotic internal standard solution is preferably 2 g: (48-52) μL, more preferably 2 g: 50 μL.

[0032] In the present invention, the purifier is preferably graphitized nonporous carbon or graphitized carbon black, more preferably graphitized nonporous carbon powder; the particle size of the graphitized nonporous carbon powder is preferably 38 to 125 μm. As an embodiment, the purifier can specifically be Envi-carb graphitized nonporous carbon powder.

[0033] In the present invention, the mass ratio of the soil sample to the purifier is preferably 1:(0.05-0.1). As an embodiment, the mass ratio of the soil sample to the purifier can be specifically 1:0.05, 1:0.06, 1:0.07, 1:0.08, 1:0.09, or 1:0.1. By adding the purifier and controlling its dosage, the present invention can fully remove impurities after extraction.

[0034] In the present invention, the mass ratio of the soil sample to the volume ratio of methanol is preferably 2g:(0.5-1)mL. As an embodiment, the mass ratio of the soil sample to the volume ratio of methanol can be specifically 2g:0.5mL, 2g:0.6mL, 2g:0.7mL, 2g:0.8mL, 2g:0.9mL or 2g:1mL. The present invention adds methanol and controls its amount to fully dissolve the mixed antibiotic internal standard solution, increase the contact area between the mixed antibiotic internal standard and the soil sample, and allow the mixed antibiotic internal standard to be adsorbed on the soil sample.

[0035] In the present invention, the mixing of the soil sample with the mixed antibiotic internal standard solution, the scavenger and methanol is preferably performed by adding the mixed antibiotic internal standard solution to the soil sample, then adding the scavenger, and finally adding methanol.

[0036] In the present invention, the mixing time is preferably 10 to 15 hours, more preferably 12 hours; the mixing is preferably performed in a fume hood and protected from light. The present invention controls the mixing time to allow the mixed antibiotic internal standard to be fully adsorbed on the soil sample.

[0037] After obtaining the soil sample to be extracted, the present invention extracts the soil sample to be extracted in an accelerated solvent extraction evaporator ExtrevaASE to obtain a test solution.

[0038] In the present invention, the extraction solvent is a mixed solution of citric acid-trisodium citrate buffer and acetonitrile.

[0039] In the present invention, the pH value of the citric acid-trisodium citrate buffer solution is 3-4; the volume ratio of the citric acid-trisodium citrate buffer solution to acetonitrile is preferably 1:(0.9-1.1), more preferably 1:1. The present invention uses the above-mentioned extraction solvent to adjust the pH value of the system, so that the antibiotics exist in a molecular state, inhibiting the electrostatic adsorption between the antibiotics and the soil. At the same time, the stability under high pressure conditions is better, and a stable pH environment can be continuously maintained, which can further improve the extraction effect.

[0040] In the present invention, the accelerated solvent extraction evaporator ExtrevaASE is preferably from Thermo Fisher Scientific (China) Co., Ltd.

[0041] In the present invention, the soil sample to be extracted is preferably placed in a sample pool.

[0042] In the present invention, the instrument parameters of the accelerated solvent extraction evaporator ExtrevaASE preferably include: an extraction cell of 5 mL, an extraction temperature of room temperature, a purge time of 58 to 62 s, an extraction solvent pre-filled with 48 to 52% (volume percentage), an extraction solvent flow rate of 0.2 to 0.3 mL / min, an extraction time of 12 min, an evaporation temperature of 40 to 80°C, a total nitrogen flow rate of 190 to 210 mL / min, and a vacuum degree of 2 to 8 psi; more preferably: a purge time of 60 s, an extraction solvent pre-filled with 50%, an extraction solvent flow rate of 0.25 mL / min, and a total nitrogen flow rate of 200 mL / min.

[0043] The present invention uses an accelerated solvent extraction evaporator, the ExtrevaASE, for extraction, integrating extraction, purification, evaporation, and volume determination steps on the same platform. This fully automates the soil antibiotic extraction process for the first time, significantly reducing extraction solvent consumption and greatly shortening extraction time. It also effectively improves the extraction recovery rate of multiple antibiotics, resolving technical issues with existing methods such as complex manual operations, poor repeatability, long extraction times, and low recovery rates. Furthermore, the method features a low extraction pressure (200 psi, the instrument's default pressure, which cannot be modified), and controls the evaporation temperature and vacuum (pressure), allowing extraction solvents containing a large amount of aqueous phase to be compatible with the instrument.

[0044] After obtaining the test liquid, the present invention preferably performs ultra-high performance liquid chromatography-tandem mass spectrometry detection on the test liquid.

[0045] In the present invention, the instrument model for ultra-high performance liquid chromatography-tandem mass spectrometry detection is preferably Agilent UHPLC-MS / MS (1290-6475).

[0046] In the present invention, the chromatographic parameters of the ultra-high performance liquid chromatography-tandem mass spectrometry detection preferably include: ACQUITYBEH C18 chromatographic column (2.1×100 mm, 1.7 μm, Waters), chromatographic column temperature of 25-28° C., mobile phase A of 0.1% formic acid solution by volume, mobile phase B of acetonitrile, gradient elution, ESI temperature of 340-360° C., carrier gas flow rate of 8-10 L / min, nebulizer voltage of 34-36 psi, and capillary voltage of 3900-4100 V (ESI). + More preferably, the column temperature is 25°C, the ESI temperature is 350°C, the carrier gas flow rate is 9 L / min, the atomization voltage is 35 psi, and the capillary voltage is 4000 V (ESI) + .

[0047] In the present invention, the gradient elution conditions are preferably: the volume percentage of mobile phase B from 0 to 1 min is 10%, the volume percentage of mobile phase B from 10% to 15% from 1 to 5 min, the volume percentage of mobile phase B from 15% to 90% from 5 to 20.1 min, the volume percentage of mobile phase B from 90% to 10% from 20.1 to 22.1 min, and the volume percentage of mobile phase B from 22.1 to 23 min is 10%, and the flow rate is 0.2 to 0.3 mL / min, more preferably 0.25 mL / min.

[0048] In the present invention, the mass spectrometry parameters of the ultra-high performance liquid chromatography-tandem mass spectrometry detection are preferably: sulfadiazine parent ion (m / z) 251, daughter ion (m / z) 156 / 108, fragmentation voltage is 100V, collision energy is 11 / 21V, and internal standard is sulfamethoxazole-d4; sulfadimethoxine parent ion (m / z) 279, daughter ion (m / z) 186 / 156, fragmentation voltage is 90V, collision energy is 15 / 15V, and internal standard is sulfamethoxazole-d4; norfloxacin parent ion (m / z) 320, daughter ion (m / z) 302 / 276, The fragmentation voltage was 70 V, the collision energy was 15 / 15 V, and the internal standard was ciprofloxacin-d8; the parent ion (m / z) of ciprofloxacin was 332, the daughter ion (m / z) was 314 / 231, the fragmentation voltage was 110 V, the collision energy was 20 / 35 V, and the internal standard was ciprofloxacin-d8; the parent ion (m / z) of chlortetracycline was 479, the daughter ion (m / z) was 462 / 444, the fragmentation voltage was 128 V, the collision energy was 18 / 30 V, and the internal standard was tetracycline-d6; the parent ion (m / z) of tetracycline was 445, the daughter ion (m / z) was 427 / 409.9, the fragmentation voltage was The fragmentation voltage was 125 V, the collision energy was 10 / 15 V, and the internal standard was tetracycline-d6; the parent ion (m / z) of clarithromycin was 748.8, the daughter ion (m / z) was 590 / 158, the fragmentation voltage was 110 V, the collision energy was 15 / 25 V, and the internal standard was roxithromycin-d7; the parent ion (m / z) of roxithromycin was 837.5, the daughter ion (m / z) was 679 / 158.2, the fragmentation voltage was 130 V, the collision energy was 15 / 35 V, and the internal standard was roxithromycin-d7; the parent ion (m / z) of sulfamethoxazole-d4 was 258.1, the daughter ion (m / z) was 160 , fragmentation voltage is 100 V, collision energy is 18 V; ciprofloxacin-d8 parent ion (m / z) 340.2, daughter ion (m / z) 322.2 / 296.2, fragmentation voltage is 130 V, collision energy is 20 / 16 V; tetracycline-d6 parent ion (m / z) 451.2, daughter ion (m / z) 416.2 / 160.1, fragmentation voltage is 120 V, collision energy is 16 / 28 V; roxithromycin-d7 parent ion (m / z) 844.6, daughter ion (m / z) 686.7, fragmentation voltage is 170 V, collision energy is 20 V.

[0049] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0050] The antibiotic standards and antibiotic internal standards in the examples and comparative examples were purchased from Alta Technology Co., Ltd.

[0051] Example 1

[0052] A fully automated accelerated solvent extraction method for extracting antibiotics from soil is as follows: (1) freeze-dry the soil sample at -50°C for 24 h, grind it, and pass it through a 100-mesh sieve. Accurately weigh 2 g of the sieved soil sample into a sample cell, add 50 μL of a 1 mg / L mixed antibiotic internal standard solution, then add 100 mg of Envi-carb graphitized non-porous carbon powder, and finally add 1 mL of methanol. Place the sample in a fume hood in the dark for 12 h to obtain the soil sample to be extracted.

[0053] (2) The soil sample to be extracted in step (1) is extracted in an accelerated solvent extraction evaporator ExtrevaASE, the extraction solvent is a mixed solution of citric acid-trisodium citrate buffer and acetonitrile, the pH value of the citric acid-trisodium citrate buffer is 3, the volume ratio of the citric acid-trisodium citrate buffer and acetonitrile is 1:1, the instrument parameters are: extraction cell 5mL, extraction temperature is room temperature, purge time is 60s, extraction solvent pre-filling 50%, extraction solvent flow rate is 0.25mL / min, extraction time is 12min, evaporation temperature is 40°C, total nitrogen flow rate is 200mL / min, vacuum degree is 2psi, and the test solution is obtained;

[0054] (3) The test solution was detected using an Agilent UHPLC-MS / MS (1290-6475) high performance liquid chromatography tandem mass spectrometry analyzer to obtain the content of antibiotics in the soil sample. The chromatographic parameters during the detection are shown in Table 1, and the mass spectrometry parameters are shown in Table 2.

[0055] Table 1 Chromatographic parameters during detection in Example 1

[0056]

[0057]

[0058] Table 2 Mass spectrometry parameters during detection in Example 1

[0059]

[0060] The contents of various antibiotics in the soil samples detected in Example 1 are shown in Table 3.

[0061] Table 3 The content of each antibiotic in the soil samples detected in Example 1

[0062]

[0063]

[0064] Test Example 1

[0065] A fully automated accelerated solvent extraction method for extracting antibiotics from soil is as follows: (1) freeze-dry the soil sample at -50°C for 24 h, grind it, and pass it through a 100-mesh sieve. Accurately weigh 2 g of the sieved soil sample into a sample cell, add 20 μL of a 1 mg / L mixed antibiotic standard solution and 50 μL of a 1 mg / L mixed antibiotic internal standard solution, then add 100 mg of Envi-carb graphitized non-porous carbon powder, and finally add 1 mL of methanol. Place the sample in a fume hood in the dark for 12 h to obtain a soil sample to be extracted.

[0066] (2) The soil sample to be extracted in step (1) is extracted in an accelerated solvent extraction evaporator ExtrevaASE, the extraction solvent is a mixed solution of citric acid-trisodium citrate buffer and acetonitrile, the pH value of the citric acid-trisodium citrate buffer is 3, the volume ratio of the citric acid-trisodium citrate buffer and acetonitrile is 1:1, the instrument parameters are: extraction cell 5mL, extraction temperature is room temperature, purge time is 60s, extraction solvent pre-filling 50%, extraction solvent flow rate is 0.25mL / min, extraction time is 12min, evaporation temperature is 40°C, total nitrogen flow rate is 200mL / min, vacuum degree is 2psi, and the test solution is obtained;

[0067] (3) The test solution was detected using an Agilent UHPLC-MS / MS (1290-6475) high performance liquid chromatography tandem mass spectrometer to obtain the content of antibiotics in the soil sample. The chromatographic parameters and mass spectrometry parameters during the detection were the same as those in Example 1.

[0068] The average matrix spiked recoveries and standard deviations of the antibiotics calculated after deducting the matrix background value in Test Example 1 are shown in Table 4.

[0069] Table 4 Average matrix spiked recoveries and standard deviations of each antibiotic calculated after deducting matrix background values ​​in Test Example 1

[0070] antibiotic name Average spike recovery (%) Standard deviation (%) Sulfadiazine 96.63 23.82 Sulfadiazine 85.19 6.09 Ciprofloxacin 84.93 3.35 Norfloxacin 81.82 2.85 tetracycline 107.84 5.21 Chlortetracycline 93.34 5.02 Clarithromycin 90.78 5.19 Roxithromycin 87.54 6.98

[0071] Test Example 2

[0072] The evaporation temperature in step (2) of Test Example 1 was replaced with 70° C., and the other parameters were the same as those of Test Example 1.

[0073] The average matrix spiked recoveries and standard deviations of the antibiotics calculated after deducting the matrix background value in Test Example 2 are shown in Table 5.

[0074] Table 5 Average matrix spiked recoveries and standard deviations of each antibiotic calculated after deducting matrix background values ​​in Test Example 2

[0075] antibiotic name Average spike recovery (%) Standard deviation (%) Sulfadiazine 90.72 1.86 Sulfadiazine 85.56 20.20 Ciprofloxacin 102.15 3.46 Norfloxacin 90.92 0.85 tetracycline 118.13 21.15 Chlortetracycline 125.72 15.02 Clarithromycin 84.49 7.23 Roxithromycin 98.66 6.33

[0076] Test Example 3

[0077] The vacuum degree in step (2) of Test Example 1 was replaced with 8 psi, and the other parameters were the same as those of Test Example 1.

[0078] The average matrix spiked recoveries and standard deviations of the antibiotics calculated after deducting the matrix background value in Test Example 3 are shown in Table 6.

[0079] Table 6 Average matrix spiked recoveries and standard deviations of each antibiotic calculated after deducting matrix background values ​​in Test Example 3

[0080] antibiotic name Average spike recovery (%) Standard deviation (%) Sulfadiazine 128.27 1.41 Sulfadiazine 102.59 10.96 Ciprofloxacin 88.47 4.54 Norfloxacin 90.04 3.47 tetracycline 85.81 7.97 Chlortetracycline 73.21 3.61 Clarithromycin 102.40 0.59 Roxithromycin 96.14 13.23

[0081] Comparative Example 1

[0082] An ultrasonic extraction method for extracting antibiotics in soil comprises the following steps: freeze-drying a soil sample at -50°C for 24 hours, grinding the soil sample and passing it through a 100-mesh sieve, accurately weighing 2 g of the sieved soil sample into a glass centrifuge tube, adding 20 μL of a 1 mg / L mixed antibiotic standard solution and 50 μL of a 1 mg / L mixed antibiotic internal standard solution, and then adding 1 mL of methanol. The mixture is kept in a fume hood away from light for 12 hours, adding 5 mL of citric acid / trisodium citrate buffer (pH = 3) to the centrifuge tube, vortexing for 10 seconds, ultrasonically extracting for 20 minutes, and then centrifuging at 2900 rpm for 8 minutes. The supernatant is removed and transferred to a clean glass beaker through a 0.45 μm organic nylon filter membrane using a 10 mL syringe. The above extraction steps are repeated twice, and the supernatants obtained from the three extractions are mixed and poured into the above beaker. 300 mL of ultrapure water is added and stirred, and 0.2 g of Na2EDTA is added and stirred until dissolved. The mixture is then filtered using Waters OasisHLB (6 mL, 500 mg) solid-phase extraction cartridges were used for solid-phase extraction. UHPLC-MS / MS was used for analysis, detection, and quantification. After deducting the matrix background value, the average matrix spike recovery and standard deviation of each antibiotic were calculated and shown in Table 7.

[0083] Table 7 Average matrix spiked recoveries and standard deviations of each antibiotic calculated after deducting matrix background values ​​in Test Example 1 and Comparative Example 1

[0084] antibiotic name Comparative Example 1 Recovery (%) Test Example 1 Recovery Rate (%) Sulfadiazine 66.54±3.25 96.63±23.82 Sulfadiazine 64.52±4.85 85.19±6.09 Ciprofloxacin 68.32±2.85 84.93±3.35 Norfloxacin 65.32±1.98 81.82±2.85 tetracycline 65.66±3.33 107.84±5.21 Chlortetracycline 118.63±11.32 93.34±5.02 Clarithromycin 84.33±2.31 90.78±5.19 Roxithromycin 80.45±3.52 87.54±6.98

[0085] It can be seen from Table 7 that the method of the present invention effectively improves the extraction recovery rate of antibiotics in soil and has good method stability.

[0086] Comparative Example 2

[0087] The extraction solvent in step (2) of Test Example 1 was replaced with acetonitrile, and the other parameters were the same as those of Test Example 1.

[0088] Comparative Example 3

[0089] The extraction solvent in step (2) of Test Example 1 was replaced with a mixed solution of McIlvain buffer (pH 4) and acetonitrile (acetonitrile containing 1% (volume concentration) acetic acid), the volume ratio of McIlvain buffer to acetonitrile was 1:1, and the other parameters were the same as those in Test Example 1.

[0090] The average matrix spiked recoveries of each antibiotic calculated after deducting the matrix background value in Test Example 1 and Comparative Examples 2-3 are shown in Table 8.

[0091] Table 8 Average matrix spiked recoveries of each antibiotic calculated after deducting matrix background values ​​in Test Example 1 and Comparative Examples 2-3

[0092]

[0093] As shown in Table 8, the recoveries of all eight antibiotics using a 1:1 mixture of citric acid / trisodium citrate buffer (pH = 3) and acetonitrile as an extractant ranged from 80% to 110%. Using a 1:1 mixture of McIlvain buffer (pH = 4) and acetonitrile (containing 1% acetic acid) resulted in better recoveries for ciprofloxacin, norfloxacin, tetracycline, chlortetracycline, and roxithromycin. However, the recoveries for sulfadiazine and sulfamethazine exceeded 200%, likely due to the significant matrix effect of this extractant, which enhanced the signal response of sulfonamide antibiotics. When acetonitrile was used as an extractant, the recoveries for ciprofloxacin and norfloxacin were less than 60%. Therefore, a 1:1 mixture of citric acid / trisodium citrate buffer (pH = 3) and acetonitrile as an extractant was superior to a 1:1 (volume ratio) mixture of acetonitrile, McIlvain buffer (pH = 4), and acetonitrile (containing 1% acetic acid) as an extractant.

[0094] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A fully automated accelerated solvent extraction method for extracting antibiotics from soil, comprising the following steps: (1) mixing a soil sample with a mixed antibiotic internal standard solution, a scavenger, and methanol to obtain a soil sample to be extracted; (2) The soil sample to be extracted obtained in step (1) is extracted in an accelerated solvent extraction evaporator Extreva ASE to obtain a test solution; the extraction solvent is a mixed solution of citric acid-trisodium citrate buffer and acetonitrile; the pH value of the citric acid-trisodium citrate buffer is 3-4.

2. The method according to claim 1, characterized in that The mesh size of the soil sample in step (1) is ≥100 mesh.

3. The method according to claim 1, characterized in that The purifier in step (1) is graphitized non-porous carbon or graphitized carbon black.

4. The method according to claim 1 or 3, characterized in that The mass ratio of the soil sample to the purifier in step (1) is 1:(0.05-0.1).

5. The method according to claim 1, wherein In the step (1), the mass ratio of the soil sample to the volume of methanol is 2 g: (0.5-1) mL.

6. The method according to claim 1, characterized in that The mixing time in step (1) is 10 to 15 hours.

7. The method according to claim 1, characterized in that The volume ratio of citric acid-trisodium citrate buffer solution to acetonitrile in the extraction solvent of step (2) is 1: (0.9-1.1).

8. The method according to claim 1, characterized in that The instrument parameters of the accelerated solvent extraction evaporator ExtrevaASE in step (2) include: an extraction cell of 5 mL, an extraction temperature of room temperature, a purge time of 58 to 62 s, an extraction solvent pre-fill of 48 to 52%, an extraction solvent flow rate of 0.2 to 0.3 mL / min, an extraction time of 12 min, an evaporation temperature of 40 to 80° C., a total nitrogen flow rate of 190 to 210 mL / min, and a vacuum degree of 2 to 8 psi.

9. The method according to claim 1, characterized in that The method also includes subjecting the test liquid to ultra-high performance liquid chromatography-tandem mass spectrometry detection.

10. The method according to claim 9, characterized in that The parameters of the ultra-high performance liquid chromatography-tandem mass spectrometry detection include: a chromatographic column temperature of 25-28°C, mobile phase A of 0.1% by volume formic acid solution, mobile phase B of acetonitrile, gradient elution, ESI temperature of 340-360°C, a carrier gas flow rate of 8-10 L / min, and a nebulizer voltage of 34-36 psi.

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