Pretreatment methods for the detection of multiple veterinary drug residues in animal samples

Abstract

The pretreatment method for detecting multiple veterinary drug residues in animal-derived samples of the present invention belongs to the technical field of drug residue determination. It solves the problem that multiple veterinary drug residues cannot be detected using the same pretreated animal-derived sample. The pretreatment method for detecting multiple veterinary drug residues in animal-derived samples includes the following steps: weighing the animal-derived sample to be detected, adding an internal standard solution and mixing it evenly with the animal-derived sample to be detected, then adding o-nitrobenzaldehyde solution and hydrochloric acid solution for derivatization, adding water-saturated n-hexane and a complex solution to obtain the test object, and finally testing. The pretreatment method of the present invention meets the demand that nitrofuran drug metabolites and amide alcohol drugs can be detected simultaneously through a single pretreatment process, avoids the process of multiple weighing, derivatization and extraction of animal-derived samples during the detection process, better meets the needs of large-scale detection of animal-derived samples, and improves detection efficiency.

Description

Technical Field

[0001] The invention discloses a pre-treatment method for detecting multiple veterinary drug residues in animal-derived samples, and belongs to the technical field of drug residue determination. Background Art

[0002] Animal-derived samples can come from different kinds of animals, including fish, seafood, insects, mammals, and birds. Drug metabolites in animal-derived samples refer to byproducts produced by the metabolism of drugs in animals. These metabolites are formed after the drug undergoes chemical changes in the animal body, including drug decomposition products or other substances into which the drug is "metabolized". After entering the animal body, most drugs will undergo chemical changes in various systems of the body to produce compounds that are more easily excreted from the body. These compounds are drug metabolites.

[0003] The detection of various veterinary drug residues is crucial for evaluating the safety of animal-derived foods, generally including nitrofuran drug metabolites and amide alcohol drugs, among which nitrofuran drug metabolites include furazolidone metabolites (AOZ), furatadone metabolites (AMOZ), nitrofurazone metabolites (SEM), and nitrofurantoin metabolites (AHD); amide alcohol drugs include chloramphenicol (CPA), thiamphenicol, and florfenicol. These drugs are often used in aquaculture to promote growth and prevent and treat diseases. By detecting the residues of nitrofuran drug metabolites, amide alcohol drugs, etc. in animal-derived samples, we can understand the metabolism of these drugs in animals and then evaluate their potential risks to human health. In addition, samples treated by pretreatment methods are detected by ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS / MS), etc., and various nitrofuran drug metabolites and amide alcohol drugs can be determined in animal-derived samples.

[0004] In the current national standard documents, HPLC-tandem mass spectrometry is used to detect nitrofuran drug metabolites and amide alcohol drugs in animal-derived foods. Samples must be taken from animal-derived foods before testing, and the samples must be pre-treated to obtain qualified samples for testing. In the current national standard documents, each time a class of drug metabolites is tested, the corresponding samples to be tested must be pre-treated separately, and the derivatization time of the samples to be tested during the pre-treatment is generally more than 15 hours. Therefore, when the samples to be tested are pre-treated by the methods in the current national standard documents, the processing process is cumbersome, and more organic reagents are consumed, resulting in waste of organic reagents. The derivatization time is too long, and nitrofuran drug metabolites and amide alcohol drugs cannot be detected at the same time, resulting in high testing costs, long pre-treatment time, and low testing efficiency.

[0005] The existing Chinese patent CN107677524A (application date: 2017.09.09) discloses a pretreatment method for the determination of animal-derived samples containing nitrofuran metabolites, wherein a derivatization reagent and an internal standard solution are added during the pretreatment process, and then ethyl acetate or acetonitrile is added to extract the sample to be extracted, and then the sample is dried and filtered to obtain a liquid to be detected. The liquid to be detected obtained by pretreating the animal-derived sample using this method can only detect the nitrofuran metabolites in the animal-derived sample, and other types of metabolites in the animal-derived sample cannot be detected after the above-mentioned pretreatment method is used. Therefore, the pretreatment process of this method has limitations, and the efficiency of the pretreatment is low, which cannot meet the requirements of simultaneously detecting nitrofuran drug metabolites and amide alcohol drugs by pretreating the animal-derived sample. Therefore, in view of the above problems, it is urgent to propose a pretreatment method for animal-derived samples. Summary of the invention

[0006] In view of the shortcomings of the existing technology, in order to avoid the high cost, long derivatization time and large amount of organic waste liquid generated for the pre-treatment of animal-derived samples using the current national standard documents, and at the same time, to address the problems that multiple veterinary drug residues cannot be detected using the same pre-treated animal-derived samples, a pre-treatment method for the detection of multiple veterinary drug residues in animal-derived samples is proposed.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The pretreatment method for detecting multiple veterinary drug residues in animal-derived samples is characterized by comprising the following steps:

[0009] S1. Weigh 2.0 g to 5.0 g of the animal-derived sample to be tested and mix well, and place it in a 50 mL centrifuge tube;

[0010] S2. Add the internal standard mixed solution to the centrifuge tube containing the mixed animal-derived sample to be tested, use a vortex mixer at a speed of 2500r / min to fully mix the animal-derived sample to be tested and the internal standard mixed solution and let it stand for 10 minutes;

[0011] S3, add 0.05 mol / L o-nitrobenzaldehyde solution and 0.2 mol / L hydrochloric acid solution to the above solution in sequence, mix well with a vortex mixer and place in a constant temperature light-proof water bath for shaking, take out the centrifuge tube and cool to room temperature, add 4.0 mL to 6.0 mL of 0.1 mol / L dipotassium hydrogen phosphate solution and 0.5 mL to 0.8 mL of 1 mol / L sodium hydroxide solution, adjust the pH value of the solution in the centrifuge tube to 7.0 to 7.5, and then vortex mix well;

[0012] S4. Add 10.0 mL of ethyl acetate to the above solution, mix evenly with a vortex mixer, centrifuge at a speed of 8000 r / min for 5 min in a high-speed centrifuge, draw the supernatant, put it in a nitrogen blowpipe, and blow dry it with nitrogen at a water temperature of 35° C. using a nitrogen blower to obtain an extract; then add 3.0 mL to 5.0 mL of water-saturated n-hexane and 0.5 mL of the complex solution to the extract, mix evenly with a vortex mixer to fully dissolve the extract, and then filter the mixed extract solution through a 0.22 μm water filter membrane to obtain the analyte;

[0013] S5. The obtained analytes are tested using a liquid chromatography-triple quadrupole mass spectrometer, the content of drug metabolites in animal-derived samples is detected using an internal standard method, and the test results are calculated.

[0014] Further, in step S2, the internal standard mixture includes AOZ-D4, AMOZ-D5, AHD- 13 C3, SEM.HCL- 13 C, 15 N2 and chloramphenicol-D5.

[0015] Furthermore, in step S2, the mass of the added internal standard mixed solution is 1.0-5.0 μg / kg.

[0016] Furthermore, in step S3, the volume of the o-nitrobenzaldehyde solution added is 0.2 mL to 0.4 mL, and the volume of the hydrochloric acid solution added is 3.0 mL to 6.0 mL.

[0017] Furthermore, in step S3, the temperature of the constant temperature light-proof oscillator is 60°C to 70°C.

[0018] More preferably, in step S3, the temperature of the constant temperature light-proof oscillator is 70°C.

[0019] Furthermore, in step S3, the uniformly mixed solution is oscillated in a constant temperature, light-proof oscillator for 2 h to 4 h.

[0020] More preferably, in step S3, the oscillation time of the uniformly mixed solution in a constant temperature and light-proof oscillator is 3 hours.

[0021] Furthermore, in step S4, the re-solution is deionized water.

[0022] Beneficial effects of the present invention: In the present invention, an internal standard mixed solution is added to a uniformly mixed animal-derived sample to be tested, and after sufficient mixing, an o-nitrobenzaldehyde solution is added. Since the nitrofuran drug metabolites have a small molecular weight, a simple structure, and a low ionization efficiency, their detection sensitivity during the detection process is very low. By adding o-nitrobenzaldehyde, the animal-derived sample to be tested is shaken in a constant temperature, light-proof water area of ​​60-70°C for 2-4 hours for derivatization, which can increase the molecular weight of the metabolite, facilitate its ionization, and be more conducive to the detection of nitrofuran drug metabolites, while improving the detection sensitivity; the analyte obtained by pre-treating the animal-derived sample by the present invention can use a liquid chromatography-triple quadrupole mass spectrometer using different mobile phases to respectively detect nitrofuran drug metabolites and amide alcohol drugs, which satisfies the requirement that nitrofuran drug metabolites and amide alcohol drugs can be separated by a single pre-treatment process. The need for detecting amide alcohol drugs at the same time is met, which avoids the multiple weighing, derivatization and extraction processes of animal-derived samples during the detection process, thereby reducing the loss of organic reagents, which is safe and environmentally friendly, saves time and cost, better meets the needs of large-scale detection of animal-derived samples, and improves detection efficiency; hydrochloric acid, dipotassium hydrogen phosphate and sodium hydroxide are added to the solution to keep the pH of the solution within a reasonable range, and the mixed solution is placed in a constant temperature and light-proof oscillator at 60°C to 70°C for 2h to 4h to ensure the efficient derivatization reaction, while also greatly shortening the derivatization time in the prior art, which can improve the sensitivity and accuracy of detection, thereby improving detection efficiency; water-saturated n-hexane and a re-dissolution solution are added to the obtained extract, which can remove oils and fats in the animal-derived samples on the one hand, and on the other hand, can avoid the emulsification of the re-dissolved extract, thereby reducing the influence of oils and fats and emulsification on the detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the mass spectrum of the nitrofuran drug metabolites in Example 1 of the present invention.

[0024] Figure 2 This is the mass spectrum of the amide alcohol drug in Example 1 of the present invention. DETAILED DESCRIPTION Example 1

[0025] S1. Weigh 2.0 g of mixed animal liver tissue and place it in a 50 mL centrifuge tube;

[0026] S2. Add 1.25 μg / kg of the internal standard mixture to the centrifuge tube containing the mixed animal-derived sample to be tested, use a vortex mixer at a speed of 2500 r / min to fully mix the animal-derived sample to be tested and the internal standard mixture and let it stand for 10 minutes;

[0027] S3, add 0.05 mol / L o-nitrobenzaldehyde solution 0.3 mL and 0.2 mol / L hydrochloric acid solution 5 mL to the above solution, mix well with a vortex mixer, and place in a constant temperature, light-proof oscillator at 70° C. for 3 h, take out the centrifuge tube and cool to room temperature, add 0.1 mol / L potassium dihydrogen phosphate solution 5.0 mL and 1 mol / L sodium hydroxide solution 0.6 mL, adjust the pH value of the solution in the centrifuge tube to 7.0, and then mix well with a vortex mixer;

[0028] S4. Add 10.0 mL of ethyl acetate to the above solution, mix evenly with a vortex mixer, centrifuge at a speed of 8000 r / min for 5 min in a high-speed centrifuge, draw the supernatant, put it in a nitrogen blowpipe, and blow dry it with nitrogen at a water temperature of 35° C. using a nitrogen blowpipe to obtain an extract; then add 3.0 mL of water-saturated n-hexane and 0.5 mL of water to the extract, mix evenly with a vortex mixer to fully dissolve the extract, and then filter the mixed extract solution through a 0.22 μm water filter membrane to obtain the analyte;

[0029] S5. The obtained analytes are tested using a liquid chromatography-triple quadrupole mass spectrometer, wherein the model of the liquid chromatography-triple quadrupole mass spectrometer is EXPEC 5210 from Puyu. The internal standard method is used to detect the content of drug metabolites in animal-derived samples, and the test results are calculated.

[0030] The detection conditions of nitrofuran drug metabolites in the analyte by liquid chromatography-triple quadrupole mass spectrometry are as follows:

[0031] ACQUITY UPLC BEH C18 separation and analysis column (1.7 μm, 2.1 mm × 100 mm); column temperature: 40 °C; injection volume: 10 μL;

[0032] Mobile phase: A is 0.1% formic acid aqueous solution; B is acetonitrile; the relationship between mobile phase, running time and column flow rate is shown in Table 1 below:

[0033] Table 1:

[0034]

[0035] The detection conditions of amide alcohol drugs in the analyte using liquid chromatography-triple quadrupole mass spectrometry are as follows:

[0036] ACQUITY UPLC BEH C18 separation and analysis column (1.7 μm, 2.1 mm × 100 mm); column temperature: 40 °C; injection volume: 10 μL;

[0037] Mobile phase: A is water; B is acetonitrile; the relationship between mobile phase, running time and column flow rate is shown in Table 2 below:

[0038] Table 2:

[0039]

[0040] This method was used to detect nitrofuran drug metabolites and amide alcohol drugs in animal-derived samples, and each experiment was performed in parallel with 6 groups. Example 2

[0041] Example 2 was carried out according to the same steps and conditions as described in Example 1, but in step S1 of this example, fish meat was weighed, and 6 groups of parallel experiments were also performed. Example 3

[0042] Example 3 was carried out according to the same steps and conditions as described in Example 1, but in step S1 of this example, pork was weighed, and 6 groups of parallel experiments were also performed.

[0043] Comparative Example 1

[0044] Comparative Example 1 was carried out according to the same steps and conditions as described in Example 1, but in step S1 of this example, the target substances were added, that is, the first addition level: 0.25 μg / kg of furazolidone metabolite (AOZ), 0.25 μg / kg of furaltadone metabolite (AMOZ), 0.25 μg / kg of nitrofurazone metabolite (SEM), 0.25 μg / kg of nitrofurantoin metabolite (AHD), 0.25 μg / kg of chloramphenicol, 0.12 μg / kg of thiamphenicol, and 0.6 μg / kg of florfenicol, and 6 groups of parallel experiments were also performed; the second addition level: 0.5 μg / kg of furazolidone metabolite (AOZ), 0.5 μg / kg of furaltadone metabolite (AMOZ), and 0.5 μg / kg of nitrofurazone metabolite (SEM). g / kg, nitrofurazone metabolite (SEM) 0.5μg / kg, nitrofurantoin metabolite (AHD) 0.5μg / kg, chloramphenicol 1.2μg / kg, thiamphenicol 1.2μg / kg and florfenicol 1.2μg / kg, and 6 groups of parallel experiments were also performed; the third addition level: furazolidone metabolite (AOZ) 1.0μg / kg, furazolidone metabolite (AMOZ) 1.0μg / kg, nitrofurazone metabolite (SEM) 1.0μg / kg, nitrofurantoin metabolite (AHD) 1.0μg / kg, chloramphenicol 2.5μg / kg, thiamphenicol 2.5μg / kg and florfenicol 2.5μg / kg, and 6 groups of parallel experiments were also performed.

[0045] Comparative Example 2

[0046] Comparative Example 2 was carried out according to the same steps and conditions as described in Example 2, but in step S1 of this example, the target substances were added, that is, the first addition level: furazolidone metabolite (AOZ) 0.25 μg / kg, furaltadone metabolite (AMOZ) 0.5 μg / kg, nitrofurazone metabolite (SEM) 0.5 μg / kg, nitrofurantoin metabolite (AHD) 0.5 μg / kg, chloramphenicol 0.12 μg / kg, thiamphenicol 0.6 μg / kg and florfenicol 0.6 μg / kg, and 6 groups of parallel experiments were also performed; the second addition level: furazolidone metabolite (AOZ) 2.5 μg / kg, furaltadone metabolite (AMOZ) 2.5 μg / kg kg, nitrofurazone metabolite (SEM) 2.5μg / kg, nitrofurazone metabolite (AHD) 2.5μg / kg, chloramphenicol 1.2μg / kg, thiamphenicol 1.2μg / kg and florfenicol 1.2μg / kg, and 6 groups of parallel experiments were also performed; the third addition level: furazolidone metabolite (AOZ) 5.0μg / kg, furazolidone metabolite (AMOZ) 5.0μg / kg, nitrofurazone metabolite (SEM) 5.0μg / kg, nitrofurazone metabolite (AHD) 5.0μg / kg, chloramphenicol 2.5μg / kg, thiamphenicol 2.5μg / kg and florfenicol 2.5μg / kg, and 6 groups of parallel experiments were also performed.

[0047] Comparative Example 3

[0048] Comparative Example 3 was carried out according to the same steps and conditions as described in Example 3, but in step S1 of this example, the target substances were added, that is, the first addition level: 0.25 μg / kg of furazolidone metabolite (AOZ), 0.25 μg / kg of furaltadone metabolite (AMOZ), 0.25 μg / kg of nitrofurazone metabolite (SEM), 0.25 μg / kg of nitrofurantoin metabolite (AHD), 0.25 μg / kg of chloramphenicol, 0.12 μg / kg of thiamphenicol, and 0.6 μg / kg of florfenicol, and 6 groups of parallel experiments were also performed; the second addition level: 0.5 μg / kg of furazolidone metabolite (AOZ), 0.5 μg / kg of furaltadone metabolite (AMOZ), and 0.5 μg / kg of nitrofurazone metabolite (SEM). g / kg, nitrofurazone metabolite (SEM) 0.5μg / kg, nitrofurantoin metabolite (AHD) 0.5μg / kg, chloramphenicol 1.2μg / kg, thiamphenicol 1.2μg / kg and florfenicol 1.2μg / kg, and 6 groups of parallel experiments were also performed; the third addition level: furazolidone metabolite (AOZ) 5.0μg / kg, furazolidone metabolite (AMOZ) 5.0μg / kg, nitrofurazone metabolite (SEM) 5.0μg / kg, nitrofurantoin metabolite (AHD) 5.0μg / kg, chloramphenicol 2.5μg / kg, thiamphenicol 2.5μg / kg and florfenicol 2.5μg / kg, and 6 groups of parallel experiments were also performed.

[0049] The above examples and comparative examples were tested by a liquid chromatography-triple quadrupole mass spectrometer of model PUYU EXPEC 5210, and the test results are shown in Tables 3 to 8 below:

[0050] Table 3: Linear regression equation, correlation coefficient, LOD and LOQ

[0051]

[0052] Table 4: MRM parameters

[0053]

[0054] Note: MRM parameters are a series of parameters used to guide the acquisition of mass spectrometry signals, which together determine the specificity and sensitivity of mass spectrometry analysis.

[0055] Through the relevant parameters of the instrument, it can be found that this method has a good response in the linear range of 0.1 ng / mL to 50.0 ng / mL, the correlation coefficients are all >0.99, the quantitative detection limits are all lower than 0.09μg / kg, and the precision does not exceed 10%, which meets the detection standard requirements.

[0056] Table 5: Results of recovery and relative precision obtained from the tests in Examples 1 to 3

[0057]

[0058]

[0059] It can be seen from Table 5 that no nitrofuran drug metabolites and amide alcohol drugs were detected in the animal liver and kidney tissues, fish meat and pork samples in aquatic products detected by the pretreatment method of the present invention.

[0060] Table 6: Results of recovery and relative precision obtained by detection in Comparative Example 1

[0061]

[0062] Table 7: Results of recovery and relative precision obtained by detection in Comparative Example 2

[0063]

[0064] Table 8: Results of recovery and relative precision obtained by detection in Comparative Example 3

[0065]

[0066] pass Figure 1 and Figure 2It can be seen that the nitrofuran drug metabolites and amide alcohol drugs detected after the pretreatment method of the present invention are consistent with our detection target, as shown in Table 4. It can be seen from Tables 6 to 8 that the recovery rate of animal liver and kidney tissues at three spiked levels ranges from 88.5 to 107.0%, and the precision does not exceed 10%; the recovery rate of fish meat in aquatic products ranges from 79.2 to 113.9% at three spiked levels, and the precision does not exceed 10%; the recovery rate of pork samples at three spiked levels ranges from 82.0 to 103.7%, and the precision does not exceed 10%. By adding the target object and calculating the recovery rate and relative precision through testing, the accuracy of the method is further confirmed, and it is shown that the derivatization time of animal-derived samples can be greatly shortened by increasing the temperature. The data of Examples 1 to 3 and Comparative Examples 1 to 3 prove that the temperature and derivatization time used in the pretreatment method of the present invention can fully derivatize animal-derived samples, meet the test requirements, and ensure the accuracy of the test.

Claims

1. A pretreatment method for detecting multiple veterinary drug residues in animal-derived samples, characterized in that: The steps include: S1. Weigh 2.0 g to 5.0 g of the animal-derived sample to be tested and mix well, and place it in a 50 mL centrifuge tube; S2. Add the internal standard mixed solution to the centrifuge tube containing the mixed animal-derived sample to be tested, use a vortex mixer at a speed of 2500r / min to fully mix the animal-derived sample to be tested and the internal standard mixed solution and let it stand for 10 minutes; Wherein, the internal standard mixed solution includes AOZ-D4, AMOZ-D5, AHD-13C3, SEM.HCL-13C, 15N2 and Chloramphenicol-D5; S3, to the solution obtained in step S2, add a 0.05 mol / L o-nitrobenzaldehyde solution and a 0.2 mol / L hydrochloric acid solution in sequence, mix well with a vortex mixer, and then oscillate in a constant temperature, light-proof oscillator, take out the centrifuge tube and cool it to room temperature, add 4.0 mL to 6.0 mL of 0.1 mol / L dipotassium hydrogen phosphate solution and 0.5 mL to 0.8 mL of 1 mol / L sodium hydroxide solution, adjust the pH value of the solution in the centrifuge tube to 7.0 to 7.5, and then mix well with a vortex mixer; The temperature of the constant temperature and light-proof oscillator is 60°C to 70°C; the oscillation time of the mixed solution in the constant temperature and light-proof oscillator is 2h to 4h; S4. Add 10.0 mL of ethyl acetate to the above solution, mix evenly with a vortex mixer, centrifuge at a speed of 8000 r / min for 5 min in a high-speed centrifuge, draw the supernatant, put it in a nitrogen blowpipe, and blow dry it with nitrogen at a water temperature of 35° C. using a nitrogen blowpipe to obtain an extract; then add 3.0 mL to 5.0 mL of water-saturated n-hexane and 0.5 mL of the complex solution to the extract, mix evenly with a vortex mixer to fully dissolve the extract, and then filter the mixed extract solution through a 0.22 μm water filter membrane to obtain the analyte; S5. Testing the obtained analyte using a triple quadrupole tandem mass spectrometer, detecting the content of drug metabolites in the animal-derived sample using an internal standard method, and calculating the test results; The detection conditions for nitrofuran drug metabolites in the analyte are as follows: ACQUITY UPLC BEH C18 separation and analysis column; column temperature: 40°C; injection volume: 10 μL; column flow rate: 0.3 mL / min; mobile phase: A is 0.1% formic acid aqueous solution, B is acetonitrile; elution conditions: 0.0 min, phase A 95%, phase B 5%; 0.0 min~5.0 min, phase A decreases from 95% to 5%, phase B increases from 5% to 95%; 5.0 min~6.0 min, phase A 5%, phase B 95%, hold for 1.0 min; 6.0 min~6.1 min, phase A increases from 5% to 95%, phase B decreases from 95% to 5%; 6.1 min~8.0 min, phase A 95%, phase B 5%, hold for 1.9 min; The detection conditions for amide alcohol drugs in the analytes are as follows: ACQUITY UPLC BEH C18 separation and analysis column; column temperature: 40°C; injection volume: 10 μL; column flow rate: 0.4 mL / min; mobile phase: A is water, B is acetonitrile; elution conditions: 0.0 min, phase A 90%, phase B 10%; 0.0 min~4.0 min, phase A decreases from 90% to 0%, phase B increases from 10% to 100%; 4.0 min~4.4 min, phase A 0%, phase B 100%, hold for 0.4 min; 4.4 min~4.5 min, phase A increases from 0% to 90%, phase B decreases from 100% to 10%; 4.5 min~6.0 min, phase A 90%, phase B 10%, hold for 1.5 min.

2. The pretreatment method for detecting multiple veterinary drug residues in animal-derived samples according to claim 1, characterized in that: In step S2, the mass of the added internal standard mixed solution is 1.0-5.0 μg / kg.

3. The pretreatment method for detecting multiple veterinary drug residues in animal-derived samples according to claim 1, characterized in that: In step S3, the volume of the o-nitrobenzaldehyde solution added is 0.2 mL to 0.4 mL; the volume of the hydrochloric acid solution added is 3.0 mL to 6.0 mL.

4. The pretreatment method for detecting multiple veterinary drug residues in animal-derived samples according to claim 1, characterized in that: In step S3, the temperature of the constant temperature light-proof oscillator is 70°C.

5. The pretreatment method for detecting multiple veterinary drug residues in animal-derived samples according to claim 1, characterized in that: In step S3, the uniformly mixed solution is oscillated in a constant temperature, light-proof oscillator for 3 hours.

6. The pretreatment method for detecting multiple veterinary drug residues in animal-derived samples according to claim 1, characterized in that: In step S4, the rehydration solution is deionized water.

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