Method for determining residual quantity of 16 veterinary drugs in animal-derived food by combining QuEChERS with high performance liquid chromatography-tandem mass spectrometry

Abstract

The invention relates to a method for determining the residual quantity of 16 veterinary drugs in animal-derived food by QuEChERS combined with high performance liquid chromatography-tandem mass spectrometry, which comprises the following steps: dispersing an animal-derived food sample in water, extracting with a formic acid acetonitrile solution and an extraction salt package, the extraction salt package being anhydrous sodium sulfate and sodium chloride, and obtaining an extracting solution; a QuEChERS purifying agent is adopted to purify the extracting solution, the QuEChERS purifying agent is PSA, C18 and anhydrous magnesium sulfate, and supernate is taken after centrifugation; sequentially carrying out concentration, redissolution, acetonitrile saturated normal hexane purification and filtration treatment on the supernate to obtain a to-be-detected solution; and detecting the residual quantity of 16 veterinary drugs in the to-be-detected liquid by using a high performance liquid chromatography-tandem mass spectrometry method. According to the method, QuEChERS is combined with high performance liquid chromatography-tandem mass spectrometry to measure the residual quantity of 16 common veterinary drugs such as sulfonamides, quinolones and macrolides, methodological investigation and tests confirm that the method is high in detection speed, high in sensitivity and good in reproducibility, and the method is a rapid and effective means for simultaneously measuring the residual quantity of various veterinary drugs.

Description

Technical Field

[0001] The invention belongs to the field of veterinary drug residue detection, and in particular relates to a method for determining 16 veterinary drug residues in animal-derived foods by combining QuEChERS with high performance liquid chromatography tandem mass spectrometry. Background Art

[0002] As the world's major pork producer and consumer, veterinary drugs play a vital role in animal husbandry and play a key role in preventing animal diseases and ensuring food production. However, for economic benefits, some farmers may abuse veterinary drugs or fail to comply with drug use regulations, resulting in veterinary drug residues in pork exceeding the prescribed limits, which in turn causes food safety issues. If people consume these animal-derived foods with excessive veterinary residues for a long time, the accumulation of drugs in the body may induce toxic reactions and endanger health. Therefore, it is particularly important to ensure that commercially available pork meets quality and safety standards and strengthen the detection and research of veterinary drug residues in pork. Current national standards GB 31650-2019 "Maximum Residue Limits of Veterinary Drugs in Foods" and GB 31650.1-2022 "Maximum Residue Limits of 41 Veterinary Drugs in Foods" and other standards and regulations have set clear limit standards for veterinary drug residues in animal-derived foods such as meat, eggs, and dairy products. In order to protect the health of consumers and ensure the quality and safety of animal-derived foods, it is of great significance to develop a fast, efficient and accurate analytical method that can simultaneously detect multiple veterinary drug residues.

[0003] Animal-derived foods have a complex matrix and many potential interferences, which poses a challenge to the detection of veterinary drug residues. Since the content of veterinary drug residues in samples is very low, the high selectivity and high sensitivity of the instrument have become the key factors in promoting the development of veterinary drug residue detection technology. High-performance liquid chromatography tandem mass spectrometry (HPLC-MS / MS) combines the advantages of liquid chromatography's efficient separation capability and mass spectrometry's high sensitivity and selectivity, making it the preferred method for veterinary drug residue detection. The current national standard GB 31658.17-2021 "Determination of tetracycline, sulfonamide and quinolone drug residues in animal foods by liquid chromatography-tandem mass spectrometry" has provided a detection and analysis method for detecting 36 veterinary drug residues in pork. However, the sample pretreatment process in this standard is relatively cumbersome, the processing efficiency is not high, the solvent usage is large, and the cost is relatively high, which cannot meet the needs of rapid detection of veterinary drug residues. Therefore, the development of more efficient sample pretreatment methods has become the focus of the current field of veterinary drug residue analysis.

[0004] The QuEChERS technique is to pre-treat the sample by extractants, water absorbents and purifiers. The purifiers such as octadecylsilane bonded silica gel (C18), N-propylethylenediamine (PSA), graphitized carbon black (GCB) are directly added to the sample extract, which can effectively adsorb and remove most impurities to achieve the purpose of purifying the sample. The QuEChERS technique is widely used in the field of veterinary drug residue analysis due to its advantages such as high efficiency, time saving, convenient operation, low cost, strong stability and good safety. Therefore, this study combined the QuEChERS pre-treatment method with LC-MS / MS technology, optimized the pre-treatment conditions, chromatography, mass spectrometry conditions and other parameters, and established a simple, efficient and highly sensitive detection method for the simultaneous detection of 16 common veterinary drug residues in pork, including sulfonamides, quinolones, macrolides, etc., in order to provide a reference for the future detection of veterinary drug residues. Summary of the invention

[0005] In view of the problems existing in the prior art, the object of the present invention is to provide a method for determining 16 veterinary drug residues in animal-derived foods by combining QuEChERS with high performance liquid chromatography tandem mass spectrometry.

[0006] The objective of the present invention is achieved through the following technical solutions: A method for determining 16 veterinary drug residues in animal-derived foods by QuEChERS combined with high performance liquid chromatography tandem mass spectrometry comprises the following steps: S1: Animal-derived food samples are dispersed in water, extracted with formic acid acetonitrile solution and extraction salt package, the extraction salt package is anhydrous sodium sulfate and sodium chloride in a mass ratio, to obtain an extract; S2: The extract is purified by using a QuEChERS purifier, which is PSA, C18 and anhydrous magnesium sulfate. The supernatant is collected after centrifugation. The supernatant is sequentially concentrated, redissolved, purified by acetonitrile-saturated n-hexane and filtered to obtain a test solution. S3: Detect the residues of 16 veterinary drugs in the test solution using high performance liquid chromatography-tandem mass spectrometry.

[0007] Furthermore, the above-mentioned animal-derived foods include poultry and livestock meat and offal.

[0008] Furthermore, the above step S1 is specifically as follows: mince the animal-derived food sample, take 5 g and put it into a centrifuge tube, add 3 mL of ultrapure water and an appropriate amount of ceramic homogenizer, vortex mix for 1 min, then add 20 mL of 0.1% formic acid acetonitrile solution, shake thoroughly for 1 min, vortex and extract for 5 min, then add 4 g of anhydrous sodium sulfate and 1 g of sodium chloride, vortex for 5 min, and centrifuge at 4°C and 5500 rpm for 10 min. The obtained supernatant is the extract.

[0009] Further, the above step S2 is specifically as follows: the extract is transferred to a centrifuge tube containing 50 mg PSA powder, 200 mg C18 powder and 1 g anhydrous sodium sulfate, vortexed for 2 min, and then centrifuged at 6000 rpm for 10 min to collect the supernatant; 5 mL of the supernatant is blown to dryness with nitrogen at 40 ° C, 1 mL of 25% acetonitrile aqueous solution is added to dissolve the residue, and then 1 mL of acetonitrile-saturated n-hexane is added, vortexed to mix, centrifuged at 5000 rpm for 5 min, separated into two layers, and the lower layer solution is filtered through a microporous filter membrane with a pore size of 0.22 μm to obtain a test solution.

[0010] Further, the chromatographic conditions in the above step S3 are: chromatographic column: Agilent Eclipse Plus C18 chromatographic column, 2.1mm×50mm×1.8μm; column temperature: 40°C; mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile; flow rate: 0.4mL / min; injection volume: 2μL; gradient elution program: 0~3.60min, 5% B; 3.60~3.80min, 5%~12% B; 3.80~7.00min, 12% B; 7.00~7.10min, 12%~26% B; 7.10~9.00min, 26%~55% B; 9.00~9.10min, 55%~100% B; 9.10~11.50min, 100% B; 11.50~11.60min, 100%~5% B; 11.60~14.00min, 5% B.

[0011] Furthermore, the mass spectrometry conditions in the above step S3 are: electrospray ion source positive ion mode, multiple reaction ion monitoring mode detection; sheath gas temperature 250°C, flow rate 11.0L / min; nebulizer gas pressure 45psi; capillary voltage 3.5kV; drying gas temperature 300°C, flow rate 8.0L / min.

[0012] Furthermore, the above 16 veterinary drugs are sulfamethoxazole, sulfamethoxazole, sulfadimethoxazole, sulfaquinoxaline, sulfadiazine, sulfachloropyridazine, sulfamethoxazole, lincomycin, roxithromycin, tilmicosin, rimantadine hydrochloride, levamisole, metoclopramide, lomefloxacin, ofloxacin and sarafloxacin.

[0013] Furthermore, the mass spectrometry detection parameters of the above 16 veterinary drugs are:

[0014] * is the quantitative ion.

[0015] The significant advantages of the present invention are: Compared with the prior art, the present invention adopts the QuEChERS pretreatment method for extraction and purification, which improves the recovery rate and sensitivity of the analyte compared with the method in the national standard; after separation by reverse phase chromatography column, 16 veterinary drugs in livestock and poultry meat and viscera samples are simultaneously detected by ultra-high performance liquid chromatography tandem mass spectrometry. The detection method of the present invention is sensitive, accurate and rapid, and can simultaneously perform qualitative and quantitative analysis of 16 veterinary drugs within 9 minutes, with a detection limit (LOD) of 0.01μg / kg~0.25μg / kg. The detection method of the present invention also has a certain reference value for the detection of other veterinary drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 :Total ion current chromatogram of mixed standard solution of 16 veterinary drugs. 1. SDZ; 2. LEV; 3. SMR; 4.LIN; 5. SDM; 6. OFL; 7. MET; 8.LOM; 9. SMM; 10. SCP; 11. SMZ; 12.SAR; 13. RIM; 14.TIL; 15. SQX; 16.ROX.

[0017] Figure 2 : Chemical structures and MRM chromatograms of SMR, SDM, SMM, SDZ, LOM, OFL, LIN, LEV, and MET.

[0018] Figure 3 : Chemical structures and MRM chromatograms of SMZ, SQX, SCP, SAR, ROX, TIL, and RIM.

[0019] Figure 4 : Comparison of the on-machine responses of 16 veterinary drugs in pork samples spiked with 10µg / kg water and without water in the extraction solvent. 1. SDZ; 2. LEV; 3. SMR; 4. LIN; 5. SDM; 6. OFL; 7. MET; 8. LOM; 9. SMM; 10. SCP; 11. SMZ; 12. SAR; 13. RIM; 14. TIL; 15. SQX; 16. ROX.

[0020] Figure 5 : Comparison of the recoveries of 16 veterinary drugs in pork samples spiked at 20 µg / kg using different QuEChERS cleanup reagent combinations. DETAILED DESCRIPTION

[0021] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0022] The 16 veterinary drugs described in the present invention are sulfamethoxazole, sulfamethoxazole, sulfadimethoxine, sulfaquinoxaline, sulfadiazine, sulfachloropyridazine, sulfamethoxazole, lincomycin, roxithromycin, tilmicosin, rimantadine hydrochloride, levamisole, metoclopramide, lomefloxacin, ofloxacin and sarafloxacin.

[0023] Embodiment 1: This embodiment provides a method for determining 16 veterinary drug residues in animal-derived foods using QuEChERS combined with high performance liquid chromatography tandem mass spectrometry.

[0024] S1: Extraction: The pork to be tested was minced into minced meat by a meat grinder, and 5 g of minced meat was accurately weighed and placed in a 50 mL centrifuge tube. Subsequently, 3 mL of ultrapure water and 1 ceramic homogenizer were added. The specifications of the ceramic homogenizer were 10 mm in diameter × 20 mm in length. Vortex mixing was performed for 1 min to ensure that the meat was evenly dispersed. Then, 20 mL of 0.1% (v / v) formic acid acetonitrile solution was added, the tube lid was tightly closed and shaken thoroughly for 1 min, and then vortexed and extracted for 5 min. Then, 4 g of sodium sulfate and 1 g of sodium chloride were added. After vortexing for 5 min, the tube was centrifuged at 5500 rpm for 10 min at a low temperature of 4 °C. The obtained supernatant was the extract.

[0025] S2: Purification: The extract obtained from S1 was transferred to a 15 mL plastic centrifuge tube containing 50 mg PSA powder, 200 mg C18 powder, and 1 g anhydrous sodium sulfate, vortexed for 2 min, and then centrifuged at 6000 rpm for 10 min to collect the supernatant; 5 mL of the supernatant was quantitatively transferred and blown to dryness with nitrogen at 40 °C, 1 mL of 25% (v / v) acetonitrile aqueous solution was added to dissolve the residue, and then 1 mL of acetonitrile-saturated n-hexane was added, vortexed to mix, centrifuged at 5000 rpm for 5 min, separated into two layers, and the lower layer solution was extracted with a 1 mL syringe and filtered through a microporous filter membrane with a pore size of 0.22 μm to obtain the test solution.

[0026] S3: Sample analysis: The residues of various veterinary drugs in the test solution obtained from S2 were detected by high performance liquid chromatography tandem mass spectrometry; Chromatographic conditions: chromatographic column: Agilent Eclipse Plus C18 column (2.1mm×50mm, 1.8μm); flow rate: 0.4mL / min; injection volume: 2μL; column temperature: 40℃; mobile phase A: 0.1% (v / v) formic acid aqueous solution; mobile phase B: acetonitrile; gradient elution program: 0~3.60min, 5% B; 3.60~3.80min, 5%~12% B; 3.80~7.00min, 12% B; 7.00~7.10min, 12%~26% B; 7.10~9.00min, 26%~55% B; 9.00~9.10min, 55%~100% B; 9.10~11.50min, 100% B; 11.50~11.60min, 100%~5% B; 11.60~14.00min, 5% B; Mass spectrometry conditions: electrospray ion source positive ion mode (ESI+); sheath gas (nitrogen) temperature 250°C, flow rate 11.0 L / min; nebulizer gas (nitrogen) pressure 45 psi; capillary voltage 3.5 kV; drying gas (nitrogen) temperature 300°C, flow rate 8.0 L / min; scanning mode: multiple reaction ion monitoring. The mass spectrometry detection parameters of 16 veterinary drugs are shown in Table 1.

[0027] Table 1 Veterinary drug mass spectrometry detection parameters

[0028] Note: ions marked with * are quantitative ions; the dwell time is 50 ms; the acceleration voltage of the collision cell is 4 V.

[0029] S4: Establishment of quantitative equation Make a matrix standard quantitative curve, and use the external standard method for quantitative calculation: take a blank pork matrix sample and add 16 kinds of veterinary drugs at a concentration of 2, 5, 10, 20, 50, and 100 μg / kg. Then extract, purify, and analyze the sample according to the above method, and determine the linear equation (with the quantitative ion peak area (Y) as the ordinate axis and the spike concentration (X) as the abscissa axis), linear range, and correlation coefficient of the 16 kinds of veterinary drugs. The results are shown in Table 2. From Table 2, it can be seen that the concentration of each component is in the linear range of 2.0~100.0μg / kg. Further reduction of the concentration will lead to a weakening of the mass spectrometry signal, making it difficult to obtain a peak with relatively good stability, resulting in inaccurate qualitative and quantitative analysis. Increasing the upper limit of the concentration linearity will lead to ion suppression and poor linear correlation. For high concentration points, they can be diluted for testing. Generally, it is not recommended to directly load high concentrations for liquid mass spectrometry, which is easy to cause residues. When the concentration of the component measured in the sample is within the linear range, the sample concentration is calculated according to the linear equation; when the concentration of the component measured in the sample is outside the linear range, the sample is diluted to within the linear range and then the sample concentration is calculated according to the linear equation. The total ion flow chromatogram obtained by the above method is as follows Figure 1 As shown, the MRM chromatogram is Figure 2~3 shown.

[0030] Table 2 Working curve equations, detection limits and quantification limits of 16 veterinary drugs

[0031] Method Spiked recovery rate: A mixed spiked recovery test of 16 veterinary drugs was carried out with blank pork matrix samples as samples, with spiked levels of 2.0 μg / kg, 4.0 μg / kg, and 20.0 μg / kg. Six parallel experiments were conducted for each concentration group, and the average spiked recovery rate and RSD of the experiment were calculated. The results are shown in Table 3. The spiked recovery rate ranged from 70.4% to 118.1%, and the relative standard deviation RSD was 1.1% to 9.0%, which met the requirements for recovery and precision in GB / T 27404-2008 "Laboratory Quality Control Specifications for Physical and Chemical Testing of Foods".

[0032] Table 3 Recovery and precision of the method (n= 6)

[0033] Embodiment 2: This example studies the effect of adding water or not adding water to the extraction solvent on the detection effect.

[0034] ① Take a blank pork matrix sample and add 16 kinds of veterinary drugs at a concentration of 10 μg / kg, and then extract, purify and analyze the sample according to the method in Example 1.

[0035] ② Take a blank pork matrix sample and add 16 kinds of veterinary drugs at a concentration of 10μg / kg. Grind it into minced meat with a meat grinder. Accurately weigh 5g of minced meat and put it into a 50mL centrifuge tube. Then add 1 ceramic homogenizer with a diameter of 10mm×length of 20mm. Vortex mix for 1min to ensure that the meat is evenly dispersed. Then add 20mL of 0.1% (v / v) formic acid acetonitrile solution, cover the tube tightly and shake it thoroughly for 1min, then vortex and extract for 5min, then add 4g of sodium sulfate and 1g of sodium chloride. After vortexing for 5min, centrifuge at 5500rpm for 10min at a low temperature of 4℃. The obtained supernatant is the extract, and then the purification and sample analysis are carried out according to the method in Example 1.

[0036] The comparison results are as follows Figure 4 As shown. Figure 4 It can be seen that the response of the veterinary drugs in the blank pork matrix sample spiked with 10µg / kg as the test sample was higher after adding water dispersion and then extracted with organic solvent, indicating that the extraction efficiency of this method is better than that of directly adding organic solvent. Therefore, 0.1% formic acid acetonitrile / water mixed solvent was used for extraction, and stratification was achieved by salting out centrifugation separation technology.

[0037] Embodiment 3: This example studies the effects of different QuEChERS purification agent combinations on the detection effect.

[0038] ① Take a blank pork matrix sample and add 16 kinds of veterinary drugs at a concentration of 20 μg / kg, and then extract, purify and analyze the sample according to the method in Example 1.

[0039] ② Take a blank pork matrix sample and add 16 kinds of veterinary drugs at a concentration of 20μg / kg, and then extract according to the method in Example 1. The obtained extract was transferred to a 15mL plastic centrifuge tube containing 100mg PSA powder, 100mg C18 powder, and 1g anhydrous sodium sulfate, vortexed for 2min, and then centrifuged at 6000rpm for 10min to collect the supernatant; quantitatively transfer 5mL of the supernatant to dryness with nitrogen at 40℃, add 1mL of 25% (v / v) acetonitrile aqueous solution to dissolve the residue, and then add 1mL of acetonitrile saturated n-hexane, vortexed and mixed, centrifuged at 5000rpm for 5min, separated into two layers, and the lower layer solution was extracted with a 1mL syringe and filtered through a microporous filter membrane with a pore size of 0.22μm to obtain the test solution, and then the sample was analyzed according to the method in Example 1.

[0040] ③ Take a blank pork matrix sample for mixing and spiked, the spiked concentration is 20μg / kg, and then extract according to the method in Example 1. The obtained extract is transferred to a 15mL plastic centrifuge tube containing 200mg PSA powder, 50mg C18 powder, and 1g anhydrous sodium sulfate, vortexed for 2min, and then centrifuged at 6000rpm for 10min to collect the supernatant; quantitatively transfer 5mL of the supernatant to dryness with nitrogen at 40℃, add 1mL of 25% (v / v) acetonitrile aqueous solution to dissolve the residue, and then add 1mL of acetonitrile saturated n-hexane, vortexed and mixed, centrifuged at 5000rpm for 5min, separated into two layers, and the lower layer solution is extracted with a 1mL syringe and filtered through a microporous filter membrane with a pore size of 0.22μm to obtain the test solution, and then the sample analysis is carried out according to the method in Example 1.

[0041] The comparison results are as follows Figure 5 As shown. Figure 5 It can be seen that by comparing the recoveries of the three adsorbent combinations, it can be found that the recovery rate when the dosage of PSA is 50 mg and the dosage of C18 is 200 mg is generally better than that of other groups. Therefore, 1.0 g of anhydrous sodium sulfate, 50 mg of PSA and 200 mg of C18 were used as the purification agent combination for sample pretreatment.

[0042] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for determining 16 veterinary drug residues in animal-derived foods by combining QuEChERS with high performance liquid chromatography tandem mass spectrometry, characterized in that: The following steps are involved: S1: Animal-derived food samples are dispersed in water, extracted with formic acid acetonitrile solution and extraction salt package, the extraction salt package is anhydrous sodium sulfate and sodium chloride, to obtain an extract; S2: The extract is purified by using a QuEChERS purifier, which is PSA, C18 and anhydrous magnesium sulfate. The supernatant is collected after centrifugation. The supernatant is sequentially concentrated, redissolved, purified by acetonitrile-saturated n-hexane and filtered to obtain a test solution. S3: Detect the residues of 16 veterinary drugs in the test solution using high performance liquid chromatography-tandem mass spectrometry.

2. The method according to claim 1, characterized in that: The animal-derived food includes poultry and livestock meat and offal.

3. The method according to claim 1, characterized in that: The step S1 is specifically as follows: mince the animal-derived food sample, take 5 g and put it into a centrifuge tube, add 3 mL of ultrapure water and an appropriate amount of ceramic homogenizer, vortex mix for 1 min, then add 20 mL of 0.1% formic acid acetonitrile solution, shake thoroughly for 1 min, vortex extract for 5 min, then add 4 g of anhydrous sodium sulfate and 1 g of sodium chloride, vortex for 5 min, and centrifuge at 4°C and 5500 rpm for 10 min. The obtained supernatant is the extract.

4. The method according to claim 1, characterized in that: The step S2 is specifically as follows: transferring the extract to a centrifuge tube containing 50 mg PSA powder, 200 mg C18 powder and 1 g anhydrous sodium sulfate, vortexing for 2 minutes, centrifuging at 6000 rpm for 10 minutes, and collecting the supernatant; taking 5 mL of the supernatant and blowing it to dryness with nitrogen at 40° C., adding 1 mL of 25% acetonitrile aqueous solution to dissolve the residue, and then adding 1 mL of acetonitrile-saturated n-hexane, vortexing to mix, centrifuging at 5000 rpm for 5 minutes, separating into two layers, and filtering the lower layer solution through a microporous filter membrane with a pore size of 0.22 μm to obtain a test solution.

5. The method according to claim 1, characterized in that: The chromatographic conditions in step S3 are as follows: chromatographic column: Agilent Eclipse Plus C18 chromatographic column, 2.1 mm×50 mm×1.8 μm; Column temperature: 40°C; mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile; Flow rate: 0.4mL / min; injection volume: 2μL; gradient elution program: 0~3.60min, 5% B; 3.60~3.80min, 5%~12% B; 3.80~7.00min, 12% B; 7.00~7.10min, 12%~26% B; 7.10~9.00min, 26%~55% B; 9.00~9.10min, 55%~100% B; 9.10~11.50min, 100% B; 11.50~11.60min, 100%~5% B; 11.60~14.00min, 5% B.

6. The method according to claim 1, characterized in that: The mass spectrometry conditions in step S3 are: electrospray ion source positive ion mode, multiple reaction ion monitoring mode detection; sheath gas temperature 250°C, flow rate 11.0 L / min; nebulizer gas pressure 45 psi; capillary voltage 3.5 kV; drying gas temperature 300°C, flow rate 8.0 L / min.

7. The method according to claim 1, characterized in that: The 16 veterinary drugs are sulfamethoxazole, sulfamethoxazole, sulfadimethoxazole, sulfaquinoxaline, sulfadiazine, sulfachloropyridazine, sulfamethoxazole, lincomycin, roxithromycin, tilmicosin, rimantadine hydrochloride, levamisole, metoclopramide, lomefloxacin, ofloxacin and sarafloxacin.

8. The method according to claim 7, characterized in that: The mass spectrometry detection parameters of the 16 veterinary drugs are: * is the quantitative ion.

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