A method for simultaneously detecting three veterinary drug residues

Abstract

This invention belongs to the field of food chemical analysis technology and discloses a method for simultaneously detecting three veterinary drug residues. The method involves adding water to the sample and shaking it to disperse the residue, then adding an extract, shaking thoroughly, centrifuging, and transferring the supernatant to a stoppered centrifuge tube containing a QuEChERS purification tube (C18, PSA, anhydrous magnesium sulfate, and neutral alumina powder in a weight ratio of 0.5g:0.1g:1g:0.2g). The tube is then shaken, centrifuged, and the supernatant is collected and dried under nitrogen in a water bath or by rotary evaporation until nearly dry. The solution is then diluted to volume with 0.02% formic acid water containing 0.5 mmol / L ammonium acetate, and an appropriate amount of n-hexane is added. The mixture is shaken thoroughly, centrifuged, and the lower layer is filtered through a 0.22 μm nylon membrane and analyzed using liquid chromatography-mass spectrometry (LC-MS). This invention enables simultaneous pretreatment of azithromycin, rifampin, and acetaminophen in livestock and poultry meat, followed by simultaneous detection, reducing detection costs, improving detection efficiency, and simplifying procedures to minimize human error.

Description

Technical Field

[0001] This invention belongs to the field of food chemical analysis technology, specifically relating to a method for simultaneously detecting residues of three veterinary drugs. Background Technology

[0002] With the improvement of living standards, food safety issues are receiving increasing attention. However, the rapid development of modern animal husbandry, characterized by intensification and large-scale operations, often leads to the overuse of veterinary drugs by farmers in pursuit of greater commercial profits and to reduce the risks associated with high-density farming. This results in excessive veterinary drug residues in livestock and poultry at slaughter, seriously threatening consumer health. Therefore, the use and residues of veterinary drugs are receiving widespread attention. Many countries and international organizations have established limits for drug residues in livestock and poultry products and developed relevant detection methods. Currently, the main methods for detecting veterinary drug residues in livestock and poultry meat include enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), gas chromatography-tandem mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), and liquid chromatography-high resolution mass spectrometry (HPLC-HDMS). However, these methods generally only allow for the detection of one veterinary drug at a time. If multiple veterinary drug items need to be detected, the pretreatment process for each item is different, leading to increased labor and material costs, reduced detection efficiency, and decreased data stability due to multiple pretreatment processes. 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. It can simultaneously pretreat azithromycin, rifampin and acetaminophen in livestock and poultry meat and then detect them simultaneously, thereby reducing detection costs, improving detection efficiency, and simplifying the steps to reduce human error.

[0004] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0005] A method for simultaneously detecting residues of three veterinary drugs includes 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 for 1min to fully disperse the sample, add 0.5g of sodium chloride and 10ml of extraction solution in sequence, vortex for 10min, and then centrifuge at 8000r / min for 1min.

[0007] b. Transfer 7 ml of the supernatant obtained in step a to a 15 ml stoppered centrifuge tube containing a QuEChERS purification tube for veterinary waste, vortex for 1 min, and then centrifuge at 8000 r / min for 1 min.

[0008] c. Accurately transfer 5 ml of the supernatant from step b to another 15 ml centrifuge tube, incubate in a 40°C water bath and purge with nitrogen until nearly dry, then bring the volume to 1.0 ml with 0.02% formic acid solution (containing 0.5 mmol / L ammonium acetate), add 2 ml of n-hexane, vortex for 10 s, then centrifuge at 10000 r / min for 3 min. Take the lower layer solution, pass it through a 0.22 μm nylon membrane, and test it using liquid chromatography-mass spectrometry.

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

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

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

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

[0013] Preferably, the 0.02% formic acid solution 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 involves blowing nitrogen gas until the solution is nearly dry. A nitrogen inlet tube is inserted into a centrifuge tube to blow nitrogen gas in, the water temperature is controlled at 40°C, and the nitrogen flow rate is controlled to blow the solution until it is nearly dry.

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

[0016] This invention combines the pretreatment processes for detecting azithromycin, rifampin, and acetaminophen by changing the extraction reagent, screening QuEChERS purification tubes specifically for veterinary drug residues, and selecting specific volume-adjusting reagents. This achieves the goal of simultaneous processing and detection of the above three veterinary drugs.

[0017] In summary, this invention enables simultaneous pretreatment of azithromycin, rifampin, and acetaminophen in livestock and poultry meat, followed by simultaneous detection, thereby reducing detection costs, improving detection efficiency, simplifying procedures, and reducing human error. Attached Figure Description

[0018] Figure 1This is a liquid chromatography-mass spectrometry chromatogram detected in Example 1 of the present invention;

[0019] Figure 2 This is the liquid chromatography-mass spectrometry chromatogram detected in Example 2 of the present invention;

[0020] Figure 3 This is the liquid chromatography-mass spectrometry chromatogram detected in Example 3 of the present invention;

[0021] Figure 4 This is the liquid chromatography-mass spectrometry chromatogram detected in Example 4 of the present invention;

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

[0023] Figure 6 This is the liquid chromatography-mass spectrometry chromatogram detected in Example 6 of the present invention;

[0024] Figure 7 This is a liquid chromatography-mass spectrometry chromatogram of the residual azithromycin in the comparative example of this invention;

[0025] Figure 8 This is a liquid chromatography-mass spectrometry chromatogram of the residual amount of rifampicin in the comparative example of this invention;

[0026] Figure 9 This is a liquid chromatography-mass spectrometry chromatogram of the residual amount of acetaminophen in the comparative example of this invention. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to embodiments:

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

[0029] Example 1:

[0030] 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 ethyl acetate in sequence, continue vortexing for 10min, and then centrifuge at 8000r / min for 1min.

[0031] b. Transfer 7 ml of the supernatant obtained in step a to a 15 ml stoppered centrifuge tube containing a QuEChERS purification tube for veterinary waste (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 for 1 min, and then centrifuge at 8000 r / min for 1 min.

[0032] c. Accurately transfer 5 ml of the supernatant from step b to another 15 ml centrifuge tube. Incubate in a 40°C water bath and purge with nitrogen until nearly dry. Then, bring the volume to 1.0 ml with a solution containing 0.5 mmol / L ammonium acetate and 0.02% formic acid. Add 2 ml of n-hexane, vortex for 10 s, and centrifuge at 10000 rpm for 3 min. Filter the lower layer solution through a 0.22 μm nylon membrane and analyze using liquid chromatography-mass spectrometry. The results showed that the residual amounts of azithromycin, rifampin, and acetaminophen were 9.76 μg / kg, 9.27 μg / kg, and 7.35 μg / kg, respectively.

[0033] Example 2:

[0034] 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 ethyl acetate in sequence, vortex for 10min, and then centrifuge at 8000r / min for 1min.

[0035] b. Transfer 7 ml of the supernatant obtained in step a to a 15 ml stoppered centrifuge tube containing a QuEChERS purification tube for veterinary waste (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 for 1 min, and then centrifuge at 8000 r / min for 1 min.

[0036] c. Accurately transfer 5 ml of the supernatant from step b to another 15 ml centrifuge tube, incubate in a 40°C water bath and purge with nitrogen until nearly dry, then bring the volume to 1.0 ml with a solution containing 0.5 mmol / L ammonium acetate and 0.02% formic acid. Add 2 ml of n-hexane, vortex for 10 s, then centrifuge at 10000 rpm for 3 min. Filter the lower layer solution through a 0.22 μm nylon membrane and analyze using liquid chromatography-mass spectrometry. The results showed that the residual amounts of azithromycin, rifampin, and acetaminophen were 8.03 μg / kg, 6.43 μg / kg, and 6.91 μg / kg, respectively.

[0037] Example 3:

[0038] 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 acetonitrile in sequence, vortex for 10min, and then centrifuge at 8000r / min for 1min.

[0039] b. Transfer 7 ml of the supernatant obtained in step a to a 15 ml stoppered centrifuge tube containing a QuEChERS purification tube for veterinary waste (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 for 1 min, and then centrifuge at 8000 r / min for 1 min.

[0040] c. Accurately transfer 5 ml of the supernatant from step b to another 15 ml centrifuge tube. Incubate in a 40°C water bath and purge with nitrogen until nearly dry. Then, bring the volume to 1.0 ml with 0.02% formic acid solution containing 0.5 mmol / L ammonium acetate. Add 2 ml of n-hexane, vortex for 10 s, and centrifuge at 10000 rpm for 3 min. Filter the lower layer solution through a 0.22 μm nylon membrane and analyze using liquid chromatography-mass spectrometry. The results showed that the residual amounts of azithromycin, rifampin, and acetaminophen were 9.85 μg / kg, 9.48 μg / kg, and 9.47 μg / kg, respectively.

[0041] Example 4:

[0042] 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 acetonitrile in sequence, vortex for 10min, and then centrifuge at 8000r / min for 1min.

[0043] b. Transfer 7 ml of the supernatant obtained in step a to a 15 ml stoppered centrifuge tube containing a QuEChERS purification tube for veterinary waste (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 for 1 min, and then centrifuge at 8000 r / min for 1 min.

[0044] c. Accurately transfer 5 ml of the supernatant from step b to another 15 ml centrifuge tube. Incubate in a 40°C water bath and purge with nitrogen until nearly dry. Then, bring the volume to 1.0 ml with 0.02% formic acid solution (containing 0.5 mmol / L ammonium acetate). Add 2 ml of n-hexane, vortex for 10 s, and centrifuge at 10000 rpm for 3 min. Filter the lower layer solution through a 0.22 μm nylon membrane and analyze using liquid chromatography-mass spectrometry. The results showed that the residual amounts of azithromycin, rifampin, and acetaminophen were 9.06 μg / kg, 8.78 μg / kg, and 9.15 μg / kg, respectively.

[0045] Example 5:

[0046] 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.

[0047] b. Transfer 7 ml of the supernatant obtained in step a to a 15 ml stoppered centrifuge tube containing a QuEChERS purification tube for veterinary waste (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 for 1 min, and then centrifuge at 8000 r / min for 1 min.

[0048] c. Accurately transfer 5 ml of the supernatant from step b to another 15 ml centrifuge tube, incubate in a 40°C water bath and purge with nitrogen until nearly dry, then bring the volume to 1.0 ml with 0.02% formic acid solution (containing 0.5 mmol / L ammonium acetate), add 2 ml of n-hexane, vortex for 10 s, centrifuge at 10000 r / min for 3 min, and pass the lower layer solution through a 0.22 μm nylon membrane for analysis using liquid chromatography-mass spectrometry. The results showed that the residual amounts of azithromycin, rifampin, and acetaminophen were 8.05 μg / kg, 9.88 μg / kg, and 7.80 μg / kg, respectively.

[0049] Example 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. Transfer 7 ml of the supernatant obtained in step a to a 15 ml stoppered centrifuge tube containing a QuEChERS purification tube for veterinary waste (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 for 1 min, and then centrifuge at 8000 r / min for 1 min.

[0052] c. Accurately transfer 5 ml of the supernatant from step b to another 15 ml centrifuge tube. Incubate in a 40°C water bath and purge with nitrogen until nearly dry. Then, bring the volume to 1.0 ml with 0.02% formic acid solution (containing 0.5 mmol / L ammonium acetate). Add 2 ml of n-hexane, vortex for 10 s, and centrifuge at 10000 rpm for 3 min. Filter the lower layer solution through a 0.22 μm nylon membrane and analyze using liquid chromatography-mass spectrometry. The results showed that the residual amounts of azithromycin, rifampin, and acetaminophen were 9.05 μg / kg, 8.44 μg / kg, and 8.07 μg / kg, respectively.

[0053] Comparative Example

[0054] Accurately weigh 2g of each sample (accurate to 0.01g) and test them according to the detection methods for azithromycin (GB 31660.1-2019), rifampin (enterprise standard test method), and acetaminophen (GB 29683-2013); the residues of azithromycin, rifampin, and acetaminophen are determined respectively. The results are shown in [Figure 1]. Figures 7 to 9 The chromatograms show that the residues of azithromycin, rifampin, and acetaminophen were 9.98 μg / kg, 9.36 μg / kg, and 9.17 μg / kg, respectively.

[0055] Results analysis:

[0056] The samples from Examples 1-6 were tested using the method of this invention, and the comparative samples were tested using the methods for azithromycin (GB31660.1-2019), rifampin (enterprise standard test method), and acetaminophen (GB 29683-2013). The test results are shown in Table 1.

[0057] Table 1. Detection results of Examples 1-6 and Comparative Examples

[0058]

[0059]

[0060] The test data from Examples 1-6 show that the residual amounts of azithromycin, rifampicin, and acetaminophen can be detected using the process method of the present invention. Different extractants, purification agents, and concentration methods are used for sample extraction and testing. Among them, the process conditions in Example 3 are the best. The results obtained by rifampicin and the enterprise standard test method, and by azithromycin and acetaminophen and the national standard test method are not significantly different, and can be applied to actual testing.

[0061] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for simultaneous detection of three veterinary drug residues, characterized in that: Includes the following steps: a. Accurately weigh 2g of the sample containing azithromycin, rifampin and acetaminophen 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 acetonitrile in sequence, vortex for 10min, and then centrifuge at 8000r / min for 1min. b. Transfer 7 ml of the supernatant obtained in step a to a 15 ml stoppered centrifuge tube containing a QuEChERS purification tube for veterinary residues. Vortex for 1 min, then centrifuge at 8000 r / min for 1 min. The purification reagent in the QuEChERS purification tube for veterinary residues is C18, PSA, anhydrous magnesium sulfate and neutral alumina powder in a weight ratio of 0.5 g:0.1 g:1 g:0.2 g. c. Accurately transfer 5 ml of the supernatant from step b to another 15 ml centrifuge tube, incubate in a 40°C water bath and purge with nitrogen until nearly dry, then bring the volume to 1.0 ml with 0.02% formic acid solution containing 0.5 mmol / L ammonium acetate, add 2 ml of n-hexane, vortex for 10 s, then centrifuge at 10000 r / min for 3 min. Take the lower layer solution, pass it through a 0.22 µm nylon membrane, and test it using liquid chromatography-mass spectrometry; the 0.02% formic acid solution is prepared by formic acid and distilled water in a weight ratio of 0.02:100, and contains 0.5 mmol / L ammonium acetate.

Images