Method for simultaneously detecting three veterinary drug residues

Abstract

The invention belongs to the technical field of food chemical analysis, and discloses a method for simultaneously detecting three veterinary drug residues, which comprises the following steps: adding water into a sample, shaking to disperse, then adding an extracting solution, fully oscillating, centrifuging, taking the supernate into a centrifugal tube with a plug, containing a QuEChERS purification tube (C18, PSA, anhydrous magnesium sulfate and neutral alumina powder in a weight ratio of 0.5 g: 0.1 g: 1g: 0.2 g) special for veterinary drug residues, oscillating, centrifuging, and collecting the supernatant; taking supernate, blowing the supernate in a water bath by using nitrogen until the supernate is nearly dry or rotationally evaporating the supernate until the supernate is nearly dry, then fixing the volume by using 0.02% formic acid water containing 0.5 mmol / L ammonium acetate, adding a proper amount of n-hexane, fully oscillating and centrifuging, taking a lower-layer solution, enabling the lower-layer solution to pass through a 0.22 mu m nylon membrane, and testing by using a liquid chromatograph-mass spectrometer. According to the method, azithromycin, rifampicin and acetaminophen in livestock and poultry meat can be pretreated at the same time and then detected at the same time, the detection cost is reduced, the detection efficiency is improved, meanwhile, steps are simplified, and personal errors are reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of food chemical analysis, and particularly relates to a method for simultaneously detecting three veterinary drug residues. Background Art

[0002] With the improvement of living standards, food safety issues have received more and more attention. However, modern breeding industry is developing rapidly with increasing intensification and scale. In order to pursue greater commercial interests and reduce the risks brought by high-density breeding, farmers often abuse veterinary drugs, resulting in excessive veterinary drug residues when livestock and poultry are released, which seriously threatens the health of consumers. Therefore, the use and residues of veterinary drugs have received more and more attention. Many countries and international organizations have formulated limit standards for drug residues in livestock and poultry products and developed relevant detection methods. At present, the main methods for detecting veterinary drug residues in livestock and poultry meat are enzyme-linked immunosorbent assay, high-performance liquid chromatography, gas chromatography-tandem mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS) and liquid chromatography-high-resolution mass spectrometry. However, basically only one veterinary drug can be detected at a time. If multiple veterinary drug items need to be detected, the pre-treatment process of each item is different, which increases labor and material costs, reduces detection efficiency, and multiple pre-treatments are not conducive to data stability. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a method for simultaneously detecting three veterinary drug residues, which overcomes the defects of the prior art and can simultaneously pre-treat azithromycin, rifampicin and acetaminophen in livestock and poultry meat and then simultaneously detect them, thereby reducing the detection cost, improving the detection efficiency, simplifying the steps and reducing human errors.

[0004] In order to solve the above technical problems, the technical solution of the present invention is:

[0005] A method for simultaneously detecting three veterinary drug residues comprises the following steps:

[0006] a. Accurately weigh 2g of sample (accurate to 0.01g) into a 50ml stoppered centrifuge tube, add 4ml of water, vortex oscillator for 1min to fully disperse the sample, add 0.5g of sodium chloride and 10ml of extract solution in sequence, vortex oscillator for 10min, and then centrifuge at 8000r / min for 1min;

[0007] b. Pipette 7 ml of the supernatant obtained in step a into a 15 ml stoppered centrifuge tube equipped with a QuEChERS purification tube for animal residues, vortex on a vortex oscillator for 1 min, and then centrifuge at 8000 r / min for 1 min;

[0008] c. Accurately transfer 5 ml of the supernatant from the centrifuge in step b to another 15 ml centrifuge tube, place in a 40°C water bath and blow with nitrogen until nearly dry, then make the volume up to 1.0 ml with 0.02% formic acid water (containing 0.5 mmol / L ammonium acetate), add 2 ml of n-hexane, vortex on a vortex oscillator for 10 seconds, then centrifuge at 10,000 rpm for 3 minutes, take the lower layer of solution and pass it through a 0.22 μm nylon membrane, and test it using a liquid chromatography-mass spectrometer.

[0009] Preferably, the extract in step a is methanol, acetonitrile or ethyl acetate.

[0010] Furthermore, the extract in step a is acetonitrile.

[0011] Preferably, the purification reagent in the QuEChERS purification tube for animal residues in step b is a mixture of C18, PSA, anhydrous magnesium sulfate and neutral alumina powder, and the weight ratio thereof is 0.1-0.5g:0.1-0.2g:1g:0.2g.

[0012] Furthermore, in the step b, the weight ratio of C18, PSA, anhydrous magnesium sulfate and neutral alumina powder in the purification reagent in the QuEChERS purification tube for animal residues is 0.5g:0.1g:1g:0.2g.

[0013] Preferably, the 0.02% formic acid water in step c is prepared by mixing formic acid and distilled water in a weight ratio of 0.02:100, and contains 0.5 mmol / L ammonium acetate.

[0014] Preferably, in step c, the concentration method is to blow nitrogen gas to near dryness. A nitrogen inlet pipe is inserted into the centrifuge tube to blow nitrogen gas, the water temperature is controlled at 40° C., the nitrogen flow rate is controlled, and the solution is blown to near dryness.

[0015] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0016] The present invention combines the pretreatment processes in the detection of azithromycin, rifampicin and acetaminophen animal residues by changing the extraction reagent, screening the QuEChERS purification tube for animal residues and selecting a specific constant volume reagent, thereby achieving the purpose of simultaneous processing and simultaneous detection of the above three veterinary drugs.

[0017] In summary, the present invention can reduce detection costs, improve detection efficiency, simplify steps and reduce human errors by pre-treating azithromycin, rifampicin and acetaminophen in livestock and poultry meat at the same time and then detecting them at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1The liquid phase mass spectrometry chromatogram detected in Example 1 of the present invention;

[0019] Figure 2 The liquid phase mass spectrometry chromatogram detected by Example 2 of the present invention;

[0020] Figure 3 The liquid phase mass spectrometry chromatogram detected by Example 3 of the present invention;

[0021] Figure 4 This is a liquid phase mass spectrometry chromatogram detected by Example 4 of the present invention;

[0022] Figure 5 This is a liquid phase mass spectrometry chromatogram detected in Example 5 of the present invention;

[0023] Figure 6 This is a liquid phase mass spectrometry chromatogram detected by Example 6 of the present invention;

[0024] Figure 7 is a liquid phase mass spectrometry chromatogram of the residual amount of azithromycin in the comparative example of the present invention;

[0025] Figure 8 is a liquid phase mass spectrometry chromatogram of the residual amount of rifampicin in the comparative example of the present invention;

[0026] Fig. 9 It is a liquid phase mass spectrometry chromatogram of the residual amount of acetaminophen in the comparative example of the present invention. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below in conjunction with embodiments:

[0028] The samples used in all the embodiments and comparative examples were the same piece of pork purchased from the market.

[0029] Embodiment 1:

[0030] a. Accurately weigh 2g of sample (accurate to 0.01g) into a 50ml stoppered centrifuge tube, add 4ml of water, vortex oscillator for 1min to fully disperse the sample, add 0.5g of sodium chloride and 10ml of ethyl acetate in sequence, continue vortex oscillator oscillation for 10min, and then centrifuge at 8000r / min for 1min;

[0031] b. Pipette 7 ml of the supernatant obtained in step a into a 15 ml stoppered centrifuge tube equipped with a QuEChERS purification tube for animal residues (the weight ratio of purification reagent C18, PSA, anhydrous magnesium sulfate and neutral alumina powder is 0.1 g: 0.1 g: 1 g: 0.2 g), vortex oscillator for 1 min, and then centrifuge at 8000 r / min for 1 min;

[0032] c. Accurately transfer 5 ml of the supernatant in the centrifuge in step b to another 15 ml centrifuge tube, place in a 40°C water bath and blow with nitrogen until nearly dry, then dilute to 1.0 ml with 0.5 mmol / L ammonium acetate and 0.02% formic acid water, add 2 ml of n-hexane, vortex oscillator for 10 seconds, then centrifuge at 10000 r / min for 3 minutes, take the lower layer of solution and pass it through a 0.22 μm nylon membrane and test it using a liquid chromatography-mass spectrometer. The test results are: the residual amounts of azithromycin, rifampicin and acetaminophen are 9.76 μg / kg, 9.27 μg / kg and 7.35 μg / kg, respectively.

[0033] Embodiment 2:

[0034] a. Accurately weigh 2g of sample (accurate to 0.01g) into a 50ml stoppered centrifuge tube, add 4ml of water, vortex oscillator for 1min to fully disperse the sample, add 0.5g of sodium chloride and 10ml of ethyl acetate in sequence, vortex oscillator for 10min, and then centrifuge at 8000r / min for 1min;

[0035] b. Pipette 7 ml of the supernatant obtained in step a into a 15 ml stoppered centrifuge tube equipped with a QuEChERS purification tube for animal residues (the weight ratio of purification reagent C18, PSA, anhydrous magnesium sulfate and neutral alumina powder is 0.1 g: 0.2 g: 1 g: 0.2 g), vortex oscillator oscillate for 1 min, and then centrifuge at 8000 r / min for 1 min;

[0036] c. Accurately transfer 5 ml of the supernatant from the centrifuge in step b to another 15 ml centrifuge tube, place in a 40°C water bath and blow with nitrogen until nearly dry, then dilute to 1.0 ml with 0.5 mmol / L ammonium acetate and 0.02% formic acid water, add 2 ml of n-hexane, vortex oscillator for 10 seconds, then centrifuge at 10000 r / min for 3 minutes, take the lower layer of solution and pass it through a 0.22 μm nylon membrane and test it using a liquid chromatography-mass spectrometer. The test results are: the residual amounts of azithromycin, rifampicin and acetaminophen are 8.03 μg / kg, 6.43 μg / kg and 6.91 μg / kg, respectively.

[0037] Embodiment 3:

[0038] a. Accurately weigh 2 g of sample (accurate to 0.01 g) into a 50 ml stoppered centrifuge tube, add 4 ml of water, vortex oscillator for 1 min to fully disperse the sample, add 0.5 g of sodium chloride and 10 ml of acetonitrile in sequence, vortex oscillator for 10 min, and then centrifuge at 8000 r / min for 1 min;

[0039] b. Pipette 7 ml of the supernatant obtained in step a into a 15 ml stoppered centrifuge tube equipped with a QuEChERS purification tube for animal residues (the weight ratio of purification reagent C18, PSA, anhydrous magnesium sulfate and neutral alumina powder is 0.5 g: 0.1 g: 1 g: 0.2 g), vortex oscillator oscillate for 1 min, and then centrifuge at 8000 r / min for 1 min;

[0040] c. Accurately transfer 5 ml of the supernatant from the centrifuge in step b to another 15 ml centrifuge tube, place in a 40°C water bath and blow with nitrogen until nearly dry, then dilute to 1.0 ml with 0.02% formic acid water containing 0.5 mmol / L ammonium acetate, add 2 ml of n-hexane, vortex oscillator for 10 seconds, then centrifuge at 10000 r / min for 3 minutes, take the lower layer of solution and pass it through a 0.22 μm nylon membrane and test it using a liquid chromatography-mass spectrometer. The test results are: the residual amounts of azithromycin, rifampicin and acetaminophen are 9.85 μg / kg, 9.48 μg / kg and 9.47 μg / kg, respectively.

[0041] Embodiment 4:

[0042] a. Accurately weigh 2 g of sample (accurate to 0.01 g) into a 50 ml stoppered centrifuge tube, add 4 ml of water, vortex oscillator for 1 min to fully disperse the sample, add 0.5 g of sodium chloride and 10 ml of acetonitrile in sequence, vortex oscillator for 10 min, and then centrifuge at 8000 r / min for 1 min;

[0043] b. Pipette 7 ml of the supernatant obtained in step a into a 15 ml stoppered centrifuge tube equipped with a QuEChERS purification tube for animal residues (the weight ratio of purification reagent C18, PSA, anhydrous magnesium sulfate and neutral alumina powder is 0.5 g: 0.2 g: 1 g: 0.2 g), vortex oscillator oscillate for 1 min, and then centrifuge at 8000 r / min for 1 min;

[0044] c. Accurately transfer 5 ml of the supernatant in the centrifuge in step b to another 15 ml centrifuge tube, place in a 40°C water bath and blow with nitrogen until nearly dry, then dilute to 1.0 ml with 0.02% formic acid water (containing 0.5 mmol / L ammonium acetate), add 2 ml of n-hexane, vortex oscillator for 10 seconds, then centrifuge at 10000 r / min for 3 minutes, take the lower layer of solution and pass it through a 0.22 μm nylon membrane and test it using a liquid chromatography-mass spectrometer. The test results are: the residual amounts of azithromycin, rifampicin and acetaminophen are 9.06 μg / kg, 8.78 μg / kg and 9.15 μg / kg, respectively.

[0045] Embodiment 5:

[0046] a. Accurately weigh 2g of sample (accurate to 0.01g) into a 50ml stoppered centrifuge tube, add 4ml of water, vortex oscillator for 1min to fully disperse the sample, add 0.5g of sodium chloride and 10ml of methanol in sequence, vortex oscillator for 10min, and then centrifuge at 8000r / min for 1min;

[0047] b. Pipette 7 ml of the supernatant obtained in step a into a 15 ml stoppered centrifuge tube equipped with a QuEChERS purification tube for animal residues (the weight ratio of purification reagent C18, PSA, anhydrous magnesium sulfate and neutral alumina powder is 0.25 g: 0.1 g: 1 g: 0.2 g), vortex oscillator oscillate for 1 min, and then centrifuge at 8000 r / min for 1 min;

[0048] c. Accurately transfer 5 ml of the supernatant from the centrifuge in step b to another 15 ml centrifuge tube, place in a 40°C water bath and blow with nitrogen until nearly dry, then dilute to 1.0 ml with 0.02% formic acid water (containing 0.5 mmol / L ammonium acetate), add 2 ml of n-hexane, vortex for 10 seconds, centrifuge at 10000 r / min for 3 minutes, take the lower layer of solution and pass it through a 0.22 μm nylon membrane and test it using a liquid chromatography-mass spectrometer. The test results are: the residual amounts of azithromycin, rifampicin and acetaminophen are 8.05 μg / kg, 9.88 μg / kg and 7.80 μg / kg, respectively.

[0049] Embodiment 6:

[0050] a. Accurately weigh 2g of sample (accurate to 0.01g) into a 50ml stoppered centrifuge tube, add 4ml of water, vortex for 1min to fully disperse the sample, add 0.5g of sodium chloride and 10ml of methanol in sequence, vortex for 10min, and then centrifuge at 8000r / min for 1min;

[0051] b. Pipette 7 ml of the supernatant obtained in step a into a 15 ml stoppered centrifuge tube equipped with a QuEChERS purification tube for animal residues (the weight ratio of purification reagent C18, PSA, anhydrous magnesium sulfate and neutral alumina powder is 0.25 g: 0.2 g: 1 g: 0.2 g), vortex oscillator oscillate for 1 min, and then centrifuge at 8000 r / min for 1 min;

[0052] c. Accurately transfer 5 ml of the supernatant from the centrifuge in step b to another 15 ml centrifuge tube, place in a 40°C water bath and blow with nitrogen until nearly dry, then dilute to 1.0 ml with 0.02% formic acid water (containing 0.5 mmol / L ammonium acetate), add 2 ml of n-hexane, vortex oscillator for 10 seconds, then centrifuge at 10000 r / min for 3 minutes, take the lower layer of solution and pass it through a 0.22 μm nylon membrane and test it using a liquid chromatography-mass spectrometer. The test results are: the residual amounts of azithromycin, rifampicin and acetaminophen are 9.05 μg / kg, 8.44 μg / kg and 8.07 μg / kg, respectively.

[0053] Comparative Example

[0054] Weigh 2g of sample accurately (accurate to 0.01g) and test the residues of azithromycin, rifampicin and acetaminophen according to the test methods of azithromycin "GB 31660.1-2019", rifampicin (enterprise standard test method) and acetaminophen "GB 29683-2013". The results are shown in Figures 7 to 9 The residues of azithromycin, rifampicin and acetaminophen were 9.98μg / kg, 9.36μg / kg and 9.17μg / kg respectively.

[0055] Result analysis:

[0056] The samples of Examples 1-6 were tested respectively by the method of the present invention and the comparative example azithromycin "GB31660.1-2019", rifampicin (enterprise standard test method), and acetaminophen "GB 29683-2013" test methods, and the test results are shown in Table 1.

[0058] Table 1 Test results of Examples 1-6 and Comparative Examples

[0059]

[0060]

[0061] It can be seen from the test data of Examples 1-6 that the residual amounts of azithromycin, rifampicin and acetaminophen are detected by the process method of the present invention, and different extractants, purifiers and concentration methods are used to extract samples and perform detection; among them, the process conditions of Example 3 are the best, and the results obtained by the detection methods of rifampicin and the enterprise standard, and azithromycin and acetaminophen and the national standard are not significantly different, and can be applied to actual detection.

[0062] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.

Claims

1. A method for simultaneously detecting three veterinary drug residues, characterized in that: The following steps are involved: a. Accurately weigh 2g of sample into a 50ml stoppered centrifuge tube, add 4ml of water, vortex oscillator for 1min to fully disperse the sample, add 0.5g of sodium chloride and 10ml of extract solution in sequence, vortex oscillator for 10min, and then centrifuge at 8000r / min for 1min; b. Pipette 7 ml of the supernatant obtained in step a into a 15 ml stoppered centrifuge tube equipped with a QuEChERS purification tube for animal residues, vortex on a vortex oscillator for 1 min, and then centrifuge at 8000 r / min for 1 min; c. Accurately transfer 5 ml of the supernatant from the centrifuge in step b to another 15 ml centrifuge tube, place in a 40°C water bath and blow with nitrogen until nearly dry, then dilute to 1.0 ml with 0.02% formic acid water containing 0.5 mmol / L ammonium acetate, add 2 ml of n-hexane, vortex on a vortex oscillator for 10 seconds, then centrifuge at 10,000 r / min for 3 minutes, remove the lower layer of solution, pass it through a 0.22 μm nylon membrane, and test it using a liquid chromatography-mass spectrometer.

2. The method for simultaneously detecting three veterinary drug residues as claimed in claim 1, characterized in that: The extract in step a is methanol, acetonitrile or ethyl acetate.

3. The method for simultaneously detecting three veterinary drug residues as claimed in claim 2, characterized in that: The extract in step a is acetonitrile.

4. The method for simultaneously detecting three veterinary drug residues as claimed in claim 1, characterized in that: The purification reagent in the QuEChERS purification tube for animal residues in step b is a mixture of C18, PSA, anhydrous magnesium sulfate, and neutral alumina powder, and the weight ratio thereof is 0.1-0.5g:0.1-0.2g:1g:0.2g.

5. The method for simultaneously detecting three veterinary drug residues as claimed in claim 4, characterized in that: In the step b, the purification reagents in the QuEChERS purification tube for animal residues are C18, PSA, anhydrous magnesium sulfate and neutral alumina powder in a weight ratio of 0.5g:0.1g:1g:0.2g.

6. The method for simultaneously detecting three veterinary drug residues as claimed in claim 1, characterized in that: The 0.02% formic acid water in step c is prepared by mixing formic acid and distilled water in a weight ratio of 0.02:100, and contains 0.5 mmol / L ammonium acetate.

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