CN116718699A - Milk and product thereof, and quantitative detection method for eight tetracycline drugs and three metabolite residues in chicken and pork - Google Patents
Milk and product thereof, and quantitative detection method for eight tetracycline drugs and three metabolite residues in chicken and pork Download PDFInfo
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- CN116718699A CN116718699A CN202310719560.1A CN202310719560A CN116718699A CN 116718699 A CN116718699 A CN 116718699A CN 202310719560 A CN202310719560 A CN 202310719560A CN 116718699 A CN116718699 A CN 116718699A
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- 235000013336 milk Nutrition 0.000 title claims abstract description 111
- 239000008267 milk Substances 0.000 title claims abstract description 111
- 210000004080 milk Anatomy 0.000 title claims abstract description 111
- 239000004098 Tetracycline Substances 0.000 title claims abstract description 93
- 235000019364 tetracycline Nutrition 0.000 title claims abstract description 93
- 150000003522 tetracyclines Chemical class 0.000 title claims abstract description 84
- 239000002207 metabolite Substances 0.000 title claims abstract description 74
- 241000287828 Gallus gallus Species 0.000 title claims abstract description 67
- 235000015277 pork Nutrition 0.000 title claims abstract description 62
- 229960002180 tetracycline Drugs 0.000 title claims abstract description 52
- 229930101283 tetracycline Natural products 0.000 title claims abstract description 52
- 238000001514 detection method Methods 0.000 title claims abstract description 47
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- SOVUOXKZCCAWOJ-HJYUBDRYSA-N (4s,4as,5ar,12ar)-9-[[2-(tert-butylamino)acetyl]amino]-4,7-bis(dimethylamino)-1,10,11,12a-tetrahydroxy-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide Chemical compound C1C2=C(N(C)C)C=C(NC(=O)CNC(C)(C)C)C(O)=C2C(O)=C2[C@@H]1C[C@H]1[C@H](N(C)C)C(=O)C(C(N)=O)=C(O)[C@@]1(O)C2=O SOVUOXKZCCAWOJ-HJYUBDRYSA-N 0.000 claims description 12
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- CYDMQBQPVICBEU-XRNKAMNCSA-N chlortetracycline Chemical compound C1=CC(Cl)=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O CYDMQBQPVICBEU-XRNKAMNCSA-N 0.000 claims description 7
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- FMTDIUIBLCQGJB-UHFFFAOYSA-N Demethylchlortetracyclin Natural products C1C2C(O)C3=C(Cl)C=CC(O)=C3C(=O)C2=C(O)C2(O)C1C(N(C)C)C(O)=C(C(N)=O)C2=O FMTDIUIBLCQGJB-UHFFFAOYSA-N 0.000 claims description 2
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
- G01N2030/146—Preparation by elimination of some components using membranes
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the field of veterinary drug residue detection, and relates to a quantitative detection method for eight tetracycline drugs and three metabolite residues in milk and products thereof, chickens and pork, which comprises the following steps: by Na 2 EDTA-Mclvaine is used as an extraction reagent for milk and products thereof, and Na is used as an extraction reagent 2 EDTA-Mclvaine buffer solution, 1% formic acid aqueous solution and acetonitrile are used as extracting reagents of chicken and pork, milk and products thereof and chicken and pork samples are extracted, waters Oasis PRiME HLB small column is adopted to purify the samples, and after purificationDrying and concentrating the sample liquid of the sample liquid under nitrogen flow; re-dissolving the concentrated and blow-dried sample by adopting an initial mobile phase, filtering by adopting a filter membrane, and detecting by adopting UPLC-MS/MS; the method can realize accurate qualitative and quantitative detection, has high recovery rate and accuracy and good sensitivity, and meets the requirements of veterinary drug residue detection.
Description
Technical Field
The invention belongs to the field of veterinary drug residue detection, and particularly relates to an ultra-high performance liquid chromatography-tandem mass spectrometry (HPLC-MS) method for detecting multi-residue of milk and products thereof (raw milk, pasteurized milk, 75 ℃ fresh milk and milk powder), and chicken and pork aureomycin (CTC), oxytetracycline (OTC), tetracycline (TC), doxycycline (DOC), dimetracycline (DMC), metacycline (MTC), minocycline (MNC), tigecycline (TGC) and three metabolites (differential aureomycin (ECTC), differential oxytetracycline (EOTC) and differential tetracycline (ETC)).
Background
At present, pretreatment methods related to multi-residue tetracycline drugs in animal foods at home and abroad mainly comprise a liquid-liquid extraction method and a solid-phase extraction method, and extraction reagents mainly comprise acetonitrile, formic acid and Na 2 EDTA-Mclvaine, etc., the solid phase extraction column adopts HLB or C 18 . Up to now, research on simultaneous extraction of aureomycin, oxytetracycline, tetracycline, doxycycline, dimycycline, metacycline, minocycline, tigecycline and three metabolites (doxycycline, doxycycline and doxycycline) from animal tissues has not been reported at home and abroad.
The detection method for multi-residue of tetracycline in domestic and foreign countries mainly comprises a high performance liquid chromatography-ultraviolet detection method, a high performance liquid chromatography-diode array detection method, a high performance liquid chromatography-fluorescence detection method and a high performance liquid chromatography-tandem mass spectrometry method, and the method for simultaneously detecting multi-residue of eight kinds of tetracycline in milk and products thereof, chickens and pork and three kinds of metabolites by using the ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) has not been reported at home and abroad.
Disclosure of Invention
In order to solve the problems of extracting and purifying eight kinds of tetracycline drugs and three kinds of metabolites from milk and products thereof and chicken and pork. The invention provides a quantitative detection method for eight tetracycline drugs and three metabolite residues in milk and products thereof, chickens and pork, which adopts a liquid-liquid extraction combined with solid phase extraction method to extract eight tetracycline drugs and three metabolite residues in raw fresh milk, pasteurized milk, 75 ℃ fresh milk, milk powder, chickens and pork, and has better accuracy and sensitivity.
In order to be able to apply UPLC-MS/MS to detect eight tetracyclines and three kinds of metabolite multi-residues in milk and products thereof, chickens and pork simultaneously, the invention provides a UPLC-MS/MS detection method. Through methodological parameter verification, the method can realize accurate qualitative and quantitative detection, and has the advantages of high recovery rate, high accuracy and good sensitivity, and meets the requirements of veterinary drug residue detection.
The technical scheme provided by the invention is as follows:
a quantitative detection method for eight kinds of tetracycline drugs and three kinds of metabolite residues in milk and products thereof, chickens and pork is characterized in that Na is used for 2 EDTA-Mclvaine is used as an extraction reagent for milk and products thereof, and Na is used as an extraction reagent 2 EDTA-Mclvaine buffer solution, 1% formic acid aqueous solution and acetonitrile are taken as extraction reagents of chicken and pork, milk and products thereof and chicken and pork samples are extracted, watersOasis PRiME HLB small columns are adopted for purifying the samples, and the purified sample liquid is dried and concentrated under nitrogen flow; re-dissolving the concentrated and dried sample by adopting an initial mobile phase, filtering by adopting a filter membrane, and detecting by adopting UPLC-MS/MS; the initial mobile phase is a mixed solution of 1% trifluoroacetic acid aqueous solution and methanol, wherein the volume ratio of the 1% trifluoroacetic acid aqueous solution to the methanol is 7:3.
Further, the milk and its products include raw milk, pasteurized milk, 75 ℃ fresh milk and milk powder.
Further, the eight tetracycline drugs and the three metabolites include aureomycin, oxytetracycline, tetracycline, doxycycline, demeclocycline, metacycline, minocycline, tigecycline, doxycycline and doxycycline.
Further, gradient elution is adopted, and the elution procedure is as follows:
a quantitative detection method for multi-residue of eight tetracycline drugs and three metabolites in milk and products thereof, chickens and pork comprises the following steps:
accurately weighing 1 (+ -0.01) g of raw milk, pasteurized milk and 75 ℃ fresh milk into different 50mL conical polypropylene centrifuge tubes with plugs, adding 15mL of 0.1mol/L Na into the raw milk 2 EDTA-Mclvaine solution, pasteurized milk and fresh milk at 75deg.C with 10mL of 0.1mol/L Na 2 EDTA-Mclvaine solution. Vortex mixing for 2min, ultrasonic treating in room temperature water bath for 10min, centrifuging at 4deg.C for 10min at 10000r/min, filtering the supernatant with rapid filter paper, collecting filtrate in 50mL centrifuge tube with plug, and storing in dark place for purification.
Accurately weighing 0.1 (+ -0.001) g of milk powder sample into a 50mL centrifuge tube, adding 0.9 (+ -0.01) mL of ultrapure water to dissolve the milk powder, and enabling the whole weight to be 1 (+ -0.01) g, and vortex shaking for 1min until the milk powder is completely dissolved. 15mL of 0.1mol/LNa was added to the centrifuge tube 2 EDTA-Mclvaine solution, vortex mixing for 2min, ultrasonic treatment in room temperature water bath for 10min, centrifuging at 4deg.C for 10min at 10000r/min, filtering the supernatant with rapid filter paper, collecting filtrate in 50mL centrifuge tube with plug, and storing in dark for purification.
Raw milk, pasteurized milk, 75 ℃ fresh milk and milk powder: sample purification is carried out by adopting a Waters Oasis PRiME HLB small column, 3mL of methanol and 3mL of ultrapure water are sequentially used for activating and balancing the small column, a proper amount of sample to be purified is added, liquid drops pass through the small column at the speed of 1 drop per second, after all sample liquid flows out, 3mL of 5% methanol water is used for leaching, effluent liquid is discarded, the small column is pumped and dried under negative pressure by a vacuum pump, 2mL of methanol is used for eluting, and eluent is collected. 1mL of the eluent is placed in a 15mL centrifuge tube, dried and concentrated to dryness by dry bath nitrogen at 40 ℃, redissolved by 2mL of initial mobile phase, vortexed and oscillated for 2min, and detected by UPLC-MS/MS after passing through a 0.22 mu m nylon organic filter membrane.
Chicken and pork: accurately weigh 1 (+ -0.01) g blank sample into a 50mL conical centrifuge tube, add 2mL 0.1mol/LNa 2 EDTA-Mclvaine buffer solution, vortex oscillation for 2min, centrifugation at 4 ℃ for 5min at 5000r/min, and supernatant taking for later use; 2mL of 1% formic acid aqueous solution was added to the residue, 6mL of acetonitrile was added thereto, the mixture was vortexed and mixed for 5 minutes, sonicated in a room temperature water bath for 15 minutes, and centrifuged at 10000r/min at 4℃for 10 minutesAnd (3) taking the supernatant, combining and storing the two parts of supernatant in a 50mL polypropylene centrifuge tube, and storing the supernatant in a dark place after passing through the rapid filter paper for purification.
And (3) carrying out through purification on the sample by adopting a Waters Oasis PRiME HLB small column, wherein the small column does not need activation balance, the sample liquid is directly injected into the SPE small column, the flow rate of one drop per second is kept, and the middle filtrate is collected after the front-stage effluent is discarded. 1mL of effluent is taken into a 15mL polypropylene centrifuge tube, dried bath nitrogen is concentrated to dryness at 40 ℃,2mL of initial mobile phase is used for redissolution, vortex shaking is carried out for 2min, and after passing through a 0.22 mu m nylon organic filter membrane, UPLC-MS/MS detection is carried out.
Further, the liquid chromatography conditions for UPLC-MS/MS detection are: WATERS ACQUITY UPLC BEH C 18 (1.7 μm,100 mm. Times.2.1 mm), column temperature: 33 ℃; mobile phase a:1% aqueous trifluoroacetic acid, mobile phase B: methanol; flow rate: 0.3mL/min; sample injection amount: 10 mu L.
Further, mass spectrum conditions of UPLC-MS/MS detection are as follows: an electrospray ion source; scanning positive ions; monitoring multiple reaction ions; air curtain gas: 36psi; electrospray ionization voltage: 5500V; ion source temperature: 550 ℃; ion source spray gas: 45psi; auxiliary heating gas: 45psi; collision gas: 8psi; injection voltage: 10V; residence time: 100ms.
Further, the peak areas detected by UPLC-MS/MS are used for calculating the residual amounts of aureomycin, oxytetracycline, tetracycline, doxycycline, dimeticycline, metacycline, minocycline, tigecycline, doxycycline and doxycycline in raw milk, pasteurized milk, fresh milk at 75 ℃, milk powder, chicken and pork.
Milk and its products (fresh milk, pasteurized milk, fresh milk at 75deg.C and milk powder) and chicken and pork samples were extracted and purified by solid phase extraction and detected by UPLC-MS/MS. Ultra-high performance liquid chromatography conditions: chromatographic column: WATERS ACQUITY UPLC BEH C18 (1.7 μm,100 mm. Times.2.1 mm), column temperature: 33 ℃; mobile phase a:1% aqueous trifluoroacetic acid, mobile phase B: methanol; flow rate: 0.3mL/min; sample injection amount: 10. Mu.L; gradient elution. Mass spectrometry conditions: the triple quadrupole tandem mass spectrometer is provided with an electrospray ion source; scanning positive ions; monitoring multiple reaction ions; air curtain gas: 36psi; electrospray ionization voltage: 5500V; ion source temperature: 550 ℃; ion source spray gas: 45psi; auxiliary heating gas: 45psi; collision gas: 8psi; injection voltage: 10V; residence time: 100ms.
A process for extracting and purifying eight kinds of tetracycline medicines and three kinds of metabolites from milk and its products, chicken and pork includes liquid-liquid extraction, solid-phase extraction and purification, nitrogen concentration and re-dissolution.
The extraction steps of raw milk, pasteurized milk and fresh milk at 75 ℃ are as follows: accurately weighing 1 (+ -0.01) g of raw milk, pasteurized milk and 75 ℃ fresh milk into different 50mL conical polypropylene centrifuge tubes with plugs, adding 15mL of 0.1mol/L Na into the raw milk 2 EDTA-Mclvaine solution, pasteurized milk and fresh milk at 75deg.C 10mL of 0.1mol/L Na 2 EDTA-Mclvaine solution. Vortex mixing for 2min, ultrasonic treating in room temperature water bath for 10min, centrifuging at 4deg.C for 10min at 10000r/min, filtering the supernatant with rapid filter paper, collecting filtrate in 50mL centrifuge tube with plug, and storing in dark place for purification.
The extraction steps of the milk powder are as follows: accurately weighing 0.1 (+ -0.001) g milk powder sample into a 50mL centrifuge tube, adding 1mL ultrapure water, and vortex shaking for 1min until complete dissolution. 15mL of 0.1mol/L Na was added to the centrifuge tube 2 EDTA-Mclvaine solution, vortex mixing for 2min, ultrasonic treatment in room temperature water bath for 10min, centrifuging at 4deg.C for 10min at 10000r/min, filtering the supernatant with rapid filter paper, collecting filtrate in 50mL centrifuge tube with plug, and storing in dark for purification.
The purification steps of raw milk, pasteurized milk, fresh milk at 75 ℃ and milk powder are as follows: sequentially activating and balancing Waters Oasis PRiME HLB small column with 3mL of methanol and 3mL of ultrapure water, adding a proper amount of sample to be purified, allowing liquid drops to pass through the small column at a speed of 1 drop per second, eluting with 3mL of 5% methanol water after the sample liquid completely flows out, discarding the effluent, pumping the small column by vacuum pump under negative pressure, eluting with 2mL of methanol, and collecting the eluent. 1mL of eluent is placed in a 15mL centrifuge tube, dried by nitrogen blowing at 40 ℃, redissolved by 2mL of initial mobile phase, vortexed for 2min, and detected by UPLC-MS/MS after passing through a 0.22 mu m nylon organic filter membrane.
Chicken meatThe pork extraction steps are as follows: accurately weighing 1 (+ -0.01) g blank sample into a 50mL conical centrifuge tube, adding 2mL of 0.1mol/L Na 2 EDTA-Mclvaine buffer solution, vortex oscillation for 2min, centrifugation at 4 ℃ for 5min at 5000r/min, and supernatant taking for later use; 2mL of 1% formic acid aqueous solution is added into the residue, 6mL of acetonitrile is added, vortex mixing is carried out for 5min, room temperature water bath ultrasonic treatment is carried out for 15 min, 10000r/min centrifugation is carried out for 10min at 4 ℃, supernatant is taken, two parts of supernatant are combined and stored in a 50mL polypropylene centrifuge tube, and after fast filter paper is adopted, the mixture is stored in a dark place for purification.
The chicken and pork purification steps are as follows: and (3) carrying out through purification on the sample by adopting a Waters Oasis PRiME HLB small column, wherein the small column does not need activation balance, the sample liquid is directly injected into the SPE small column, the flow rate of one second is kept, and the liquid is collected after the effluent liquid of the front stage is discarded. Taking 1mL of effluent liquid into a 15mL polypropylene centrifuge tube, carrying out dry bath nitrogen blowing concentration at 40 ℃ until the effluent liquid is dry, carrying out redissolution by using 2mL of initial mobile phase, carrying out vortex vibration for 2min, and detecting by UPLC-MS/MS after passing through a 0.22 mu m nylon organic filter membrane.
Advantageous effects
The invention compares the effect of the combination of liquid-liquid extraction and solid-phase extraction on the result. As a result, it was found that the compound obtained by the extraction method combining liquid-liquid extraction and solid phase extraction was excellent in peak shape, less in interfering impurity peak, and high in sample response value. Meanwhile, the influence of different QuEChERS methods on the extraction effect of target substances in milk and products thereof (raw milk, pasteurized milk, 75 ℃ fresh milk and milk powder) is compared, and the result shows that the QuEChERS method cannot meet the method establishment requirement. Comparison of Na 2 EDTA-Mcllvaine、Na 2 EDTA, ethylenediamine tetraacetic acid (EDTA) and EDTA-Mclvaine as buffer, and the results show Na 2 EDTA-Mclvaine works best. The purification effect and recovery rate of the four solid phase extraction columns of WATERS Oasis PRiME HLB, WATERS Oasis HLB, CNW HLB and CNW HLB Pro are compared, and the results show that: WATERS Oasis PRiME HLB the purifying effect of the small column is best. Finally, the effect of formic acid with different concentrations on the extraction effect of target compounds of chicken and pork species is compared, and the result shows that 2mL of 0.1mol/L Na 2 After one extraction of EDTA-Mclvaine buffer, 2m was addedThe repeated extraction of the L1% formic acid aqueous solution and 6mL acetonitrile is better. Thus, the test was finally performed with Na 2 EDTA-Mclvaine is used as an extraction reagent for milk and products thereof, and Na is used as an extraction reagent 2 EDTA-Mclvaine buffer solution, 1% formic acid aqueous solution and acetonitrile are used as extraction reagents of chicken and pork, and eight kinds of tetracycline drugs and three kinds of metabolite residues in livestock and poultry tissues are extracted and purified by a method combining liquid-liquid extraction and solid phase extraction.
According to the invention, a WATERS ACQUITY UPLC BEH C (1.7 mu m,100mm multiplied by 2.1 mm) chromatographic column is adopted, positive ion scanning and multi-reaction ion monitoring are adopted, and finally, the obtained 11 target compounds have good chromatographic peak shapes (high sensitivity), moderate analyte retention time and no other impurity peak interference.
The invention provides a method for detecting multi-residues of eight tetracyclines and three metabolites by utilizing ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The method has high recovery rate, precision and sensitivity and good repeatability, and is suitable for application and popularization in batch sample analysis. Through methodological parameter verification, the method can realize accurate qualitative and quantitative detection, and has the advantages of high recovery rate, high accuracy and good sensitivity, and meets the requirements of veterinary drug residue detection.
Compared with the reported analysis method, the pretreatment method for the test is simple to operate, quick and effective and good in purifying effect. The detection method has the advantages of quick analysis time, more accurate qualitative and quantitative detection, good accuracy and sensitivity, high recovery rate and good repeatability, can finish the detection of 11 target compounds in 9 minutes, provides a new technical support for the multi-residue detection of eight tetracycline drugs and three metabolites in milk and products thereof, chickens and pork, and provides a scientific basis for the establishment of UPLC-MS/MS detection standards of the multi-residue tetracycline drugs in animal-derived foods.
Drawings
FIG. 1 total ion flow chromatogram (TIC) and extracted ion chromatogram (XICs) of eight tetracyclines and three metabolites in blank raw milk sample;
FIG. 2 Multi-reaction monitoring (MRM) and extraction ion chromatograms (XICs) of blank raw milk sample with 100.0 μg/kg of eight tetracyclines and three metabolite standards;
FIG. 3 total ion flow chromatogram (TIC) and extracted ion chromatogram (XICs) of eight tetracyclines and three metabolites in blank pasteurized milk samples;
FIG. 4 Multiple Reaction Monitoring (MRM) and extraction ion chromatograms (XICs) of blank pasteurized milk samples with addition of 100.0 μg/kg of eight tetracyclines and three metabolite standards;
FIG. 5 total ion flow chromatogram (TIC) and extracted ion chromatogram (XICs) of eight tetracyclines and three metabolites in a blank 75deg.C fresh milk sample;
FIG. 6 Multi-reaction monitoring (MRM) and extraction ion chromatograms (XICs) of blank 75℃fresh milk sample with 100.0 μg/kg of eight tetracyclines and three metabolite standards;
FIG. 7 total ion flow chromatogram (TIC) and extracted ion chromatogram (XICs) of eight tetracyclines and three metabolites in a blank milk powder sample;
FIG. 8 Multi-reaction monitoring (MRM) and extraction ion chromatograms (XICs) of a blank milk powder sample with 100.0 μg/kg of eight tetracyclines and three metabolite standards added;
FIG. 9 total ion flow chromatogram (TIC) and extracted ion chromatogram (XICs) of eight tetracyclines and three metabolites in a blank chicken sample;
FIG. 10 Multi-reaction monitoring (MRM) and extract ion chromatograms (XICs) of blank chicken sample with 100.0 μg/kg of eight tetracyclines and three metabolite standards;
FIG. 11 total ion flow chromatogram (TIC) and extracted ion chromatogram (XICs) of eight tetracyclines and three metabolites in a blank pork sample;
FIG. 12 Multi-reaction monitoring (MRM) and extraction ion chromatograms (XICs) of eight tetracyclines added to a blank pork sample at 100.0 μg/kg and three metabolite standards.
Detailed Description
The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art unless otherwise indicated.
The invention will be described in further detail below in connection with specific embodiments and with reference to the data. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
In the following examples, various processes and methods, which are not described in detail, are conventional methods well known in the art. The sources of the reagents used, the trade names and the necessary list the constituents are all indicated at the first occurrence, and the same reagents used thereafter, unless otherwise indicated, are all the same as the first indicated.
Example 1
Sample collection of Carnis gallus Domesticus and Carnis Sus Domestica
Animal feeding and sample collection in this experiment were approved by the ethical committee of the university of Yangzhou, xianglong poultry industry development Co., yangzhou and Changzhou Kangle farming Co., ltd.
10 Shoujies with the age of 100 days are randomly selected from the development limited company of the Yangzhou Xianglong poultry industry, and the complete feed without any medicine provided by the Yangzhou Yangda feed factory is fed during the whole test period in a mode of single cage feeding and free drinking. After 20 days of raising, slaughtering, respectively collecting pectoral muscle meat of each chicken, cutting, homogenizing, and storing in a freezer at-34 ℃ as blank sample for use.
6-head 6-month-old ternary hybrid pigs (Du X Long X big) are randomly selected from Changzhou Kangle farm and animal husbandry limited company, and the complete feed without any drugs provided by YangDafeed factories in YangZhou City is fed in a mode of single-circle feeding and free drinking. After 20 days of feeding, slaughtering, respectively collecting, chopping and homogenizing the longest back muscle of each pig, and storing in a freezer at-34 ℃ as a blank sample for later use.
(II) the steps of extraction, purification, concentration and re-dissolution of the invention
(1) Raw milk, pasteurized milk, 75 ℃ fresh milk: accurately weighing 1 (+ -0.01) g of raw milk, pasteurized milk and 75 ℃ fresh milk into different 50mL conical polypropylene centrifuge tubes with plugs, adding 15mL of 0.1mol/L Na into the raw milk 2 EDTA-Mclvaine solution, pasteurized milk and fresh milk at 75deg.C10mL of 0.1mol/L Na was added 2 EDTA-Mclvaine solution. Vortex mixing for 2min, ultrasonic treating in room temperature water bath for 10min, centrifuging at 4deg.C for 10min at 10000r/min, filtering the supernatant with rapid filter paper, collecting filtrate in 50mL centrifuge tube with plug, and storing in dark place for purification.
Milk powder: accurately weighing 0.1 (+ -0.001) g of milk powder sample into a 50mL centrifuge tube, adding 0.9 (+ -0.01) mL of ultrapure water to dissolve the milk powder, and enabling the whole weight to be 1 (+ -0.01) g, and vortex shaking for 1min until the milk powder is completely dissolved. 15mL of 0.1mol/L Na was added to the centrifuge tube 2 EDTA-Mclvaine solution, vortex mixing for 2min, ultrasonic treatment in room temperature water bath for 10min, centrifuging at 4deg.C for 10min at 10000r/min, filtering the supernatant with rapid filter paper, collecting filtrate in 50mL centrifuge tube with plug, and storing in dark for purification.
Chicken and pork: accurately weigh 1 (+ -0.01) g blank sample into a 50mL conical centrifuge tube, add 2mL 0.1mol/LNa 2 EDTA-Mclvaine buffer solution, vortex oscillation for 2min, centrifugation at 4 ℃ for 5min at 5000r/min, and supernatant taking for later use; 2mL of 1% formic acid aqueous solution is added into the residue, 6mL of acetonitrile is added, vortex mixing is carried out for 5min, room temperature water bath ultrasonic treatment is carried out for 15 min, 10000r/min centrifugation is carried out for 10min at 4 ℃, supernatant is taken, two parts of supernatant are combined and stored in a 50mL polypropylene centrifuge tube, and after fast filter paper is adopted, the mixture is stored in a dark place for purification.
(2) Raw milk, pasteurized milk, 75 ℃ fresh milk and milk powder: sample purification is carried out by adopting a Waters Oasis PRiME HLB small column, 3mL of methanol and 3mL of ultrapure water are sequentially used for activating and balancing the small column, a proper amount of sample to be purified is added, liquid drops pass through the small column at the speed of 1 drop per second, after all sample liquid flows out, 3mL of 5% methanol water is used for leaching, effluent liquid is discarded, the small column is pumped and dried under negative pressure by a vacuum pump, 2mL of methanol is used for eluting, and eluent is collected.
Chicken and pork: adopting Waters Oasis PRiME HLB small column to carry out pass-through purification on the sample, the small column does not need activation balance, the sample liquid is directly injected into the SPE small column, the flow rate of one second and one drop is kept, and the middle filtrate is collected after the front-stage effluent is discarded
(3) 1mL of the purified sample solution is placed in a 15mL centrifuge tube, dried by dry bath nitrogen at 40 ℃, redissolved by 2mL of an initial mobile phase (1% trifluoroacetic acid aqueous solution: methanol, 70:30, V/V), vortexed for 2min, passed through a 0.22 μm nylon organic filter membrane, and detected by UPLC-MS/MS.
(III) UPLC-MS/MS analysis conditions
1. Ultra-high performance liquid chromatography conditions
Chromatographic column: WATERS ACQUITY UPLC BEH C18 (1.7 μm,100 mm. Times.2.1 mm), column temperature: 33 ℃; mobile phase a:1% aqueous trifluoroacetic acid, mobile phase B: methanol; flow rate: 0.3mL/min; sample injection amount: 10. Mu.L; gradient elution, elution procedure is shown in table 1.
TABLE 1 gradient elution procedure
2. Mass spectrometry conditions
The triple quadrupole tandem mass spectrometer is provided with an electrospray ion source; scanning positive ions; monitoring multiple reaction ions; air curtain gas: 36psi; electrospray ionization voltage: 5500V; ion source temperature: 550 ℃; ion source spray gas: 45psi; auxiliary heating gas: 45psi; collision gas: 8psi; injection voltage: 10V; residence time: 100ms. The main mass spectrum parameters are shown in Table 2. The collection and processing of data, qualitative and quantitative analysis was done by AB SCIEX analysis (Version 1.6.3) software.
Table 2 mass spectrum optimization parameters for eight tetracyclines and three metabolites thereof
Note that: "*": quantitative ion pairs
(IV) quantitative method
1. Drawing of a Standard Curve
Accurately weighing 20 parts of 1 (+ -0.01) g of homogenized blank raw fresh milk, pasteurized milk, 75 ℃ fresh milk, chicken, pork and 0.1 (+ -0.001) g of milk powder respectively, preprocessing the blank samples according to the method (II), respectively preparing six blank matrix extracting solutions, and storing the six blank matrix extracting solutions in a refrigerator at the temperature of minus 34 ℃ for later use. The blank matrix extracting solution is utilized to dilute 11 mixed standard stock solutions (40.0 mug/mL) step by step from high concentration to low concentration, eight kinds of tetracycline medicaments and three kinds of metabolite serial matrix matching mixed standard solutions with the concentration of LOQ, 10, 20, 50, 100, 200 and 400ng/mL are respectively prepared (wherein, the LOQ mixed concentration point is obtained by diluting a single standard working solution with the blank matrix extracting solution).
The above series of matrix-matched mixed standard solutions were quantitatively analyzed under established and optimized UPLC-MS/MS conditions, 6 times per concentration point in parallel. And (3) drawing substrate matching standard curves of 11 target compounds in blank raw milk, pasteurized milk, 75 ℃ fresh milk, milk powder, chicken and pork by taking the concentration of serial substrate matching mixed standard solutions (CTC, OTC, TC, DOC, DMC, MTC, MNC, TGC, ECTC, EOTC and ETC) as independent variables (x) and the quantitative sub-ion peak area of corresponding concentrations as dependent variables (y), wherein the curves are used as quantitative curves of samples to be tested.
The concentration of the mixed standard solution of the matrix matching CTC, OTC, TC, DOC, DMC, MTC, MNC, TGC, ECTC, EOTC of blank milk and the products (raw milk, pasteurized milk, 75 ℃ fresh milk and milk powder) thereof and ETC is in the range of LOQ-400.0ng/mL, the mass chromatographic peak area (y) of characteristic ions (quantitative sub-ions) of each target compound and the concentration (x) of the corresponding matrix matching standard solution are respectively in good linear relation (R2 is more than or equal to 0.9990), and the linear regression equation, the determination coefficient and the linear range are shown in Table 3; when the concentration of the mixed standard solution of the blank chicken and pork matrix matching CTC, OTC, TC, DOC, DMC, MTC, MNC, TGC, ECTC, EOTC and ETC is in the LOQ-400.0ng/mL range, the characteristic ion mass chromatographic peak area (y) and the concentration (x) of the matrix matching standard solution are in good linear relation, the determination coefficients are higher than 0.9987, and the linear regression equation, the determination coefficients and the linear range are shown in Table 4. If the analyzed concentration exceeds the linear range of the sample, the analyzed concentration is diluted to the range, and the detected result is multiplied by the dilution multiple to obtain the concentration of the original sample.
Table 3 milk and its product matrix match the linear regression equation, determination coefficients and linear range of eight TCs and three metabolites (n=6)
Table 4 chicken and pork matrices matched linear regression equations, determination coefficients and linear ranges for eight TCs and three metabolites (n=6)
2. Determination of recovery and precision
Accurately weighing (1.0+/-0.01) g of a homogenized blank sample, processing the blank sample according to the method of (II), adding a proper amount of mixed standard stock solution of eight tetracycline drugs and three metabolites, so that the final addition concentration of each blank sample is LOQ, 0.5MRL, 1.0MRL and 2.0MRL, wherein each addition concentration is set to be 6 parallel, respectively carrying out sample pretreatment on the blank sample by the method of (II), obtaining filtrate for UPLC-MS/MS detection, and then carrying the detection result into a blank matrix standard curve to obtain the concentration, and obtaining the sample standard recovery rate compared with the concentration of the analyte actually added.
The total ion flow chromatogram (TIC) and the extracted ion chromatogram (XICs) of eight tetracyclines and three metabolites in blank raw milk sample are shown in figure 1; the multi-reaction monitoring chart (MRM) and the extraction ion chromatogram (XICs) of the blank raw fresh milk sample added with 100.0 mug/kg eight tetracycline drugs and three metabolite standard substances are shown in FIG. 2; the total ion flow chromatogram (TIC) and the extracted ion chromatogram (XICs) of the eight tetracyclines and the three metabolites in the blank pasteurized milk sample are shown in figure 3; the multi-reaction monitoring chart (MRM) and the extraction ion chromatogram (XICs) of the blank pasteurized milk sample added with 100.0 mug/kg of eight tetracyclines and three metabolite standards are shown in FIG. 4; the total ion flow chromatogram (TIC) and the extracted ion chromatogram (XICs) of eight tetracyclines and three metabolites in fresh milk sample at 75deg.C are shown in figure 5; a multi-reaction monitoring chart (MRM) and an extracted ion chromatogram (XICs) of a blank 75 ℃ fresh milk sample added with 100.0 mug/kg eight tetracycline drugs and three metabolite standards are shown in FIG. 6; the total ion flow chromatogram (TIC) and the extracted ion chromatogram (XICs) of the eight tetracyclines and the three metabolites in the blank milk powder sample are shown in figure 7; the multi-reaction monitoring chart (MRM) and the extraction ion chromatogram (XICs) of the blank milk powder sample added with 100.0 mug/kg eight tetracycline drugs and three metabolite standards are shown in FIG. 8; the total ion flow chromatogram (TIC) and the extracted ion chromatogram (XICs) of the eight tetracyclines and the three metabolites in the blank chicken sample are shown in figure 9; the multi-reaction monitoring chart (MRM) and the extraction ion chromatogram (XICs) of the blank chicken sample added with 100.0 mug/kg eight tetracycline drugs and three metabolite standards are shown in FIG. 10; the total ion flow chromatogram (TIC) and the extracted ion chromatogram (XICs) of eight tetracyclines and three metabolites in the blank pork sample are shown in figure 11; a multi-reaction monitoring chart (MRM) and an extraction ion chromatogram (XICs) of a blank pork sample added with 100.0 mug/kg of eight tetracyclines and three metabolite standards are shown in FIG. 12.
Intra-day (intra-batch) precision: samples of 4 additive concentrations (LOQ, 0.5MRL, 1.0MRL, 2.0 MRL) at different time points on the same day were assayed using the same instrument and the same standard curve, and each additive concentration was repeatedly tested for RSD 6 times.
Daytime (inter-batch) precision: samples of 4 additive concentrations (LOQ, 0.5MRL, 1.0MRL, 2.0 MRL) were assayed on different days of the week using the same instrument and different standard curves (standard curves were drawn every day), and RSD was repeatedly measured 6 times for each additive concentration.
Under the condition, the recovery rate and the precision of the addition of eight tetracycline drugs and three metabolites in the livestock and poultry tissues extracted by the method are shown in tables 5-10.
Table 5 recovery and precision of addition of eight tetracyclines and three metabolites in fresh milk (n=6)
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Note that: "a": maximum residual limit, the following is the same
Table 6 recovery and precision of addition of eight tetracyclines and three metabolites in pasteurized milk (n=6)
Table 7 recovery and precision of addition of eight tetracyclines and three metabolites in 75 ℃ fresh milk (n=6)
Recovery and precision of addition of eight tetracyclines and three metabolites s in milk powder of table 8 (n=6)
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Table 9 recovery and precision of addition of eight tetracyclines and three metabolites in chicken (n=6)
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Table 10 recovery and precision of addition of eight tetracyclines and three metabolites in pork (n=6)
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3. Determination of detection limit and quantitative limit
Accurately weighing (1.0+/-0.01) g of a homogenized blank sample, and preparing blank matrix extract according to the method in six (two). The mixed standard stock solution of 11 target compounds is diluted step by using the blank matrix extracting solution, and detection is carried out according to the established UPLC-MS/MS method. Each concentration was analyzed 6 times in duplicate and the average signal to noise ratio (S/N) was calculated. When S/N is more than or equal to 3, the corresponding target compound concentration is the detection Limit (LOD); when S/N is more than or equal to 10, the corresponding target compound concentration is the limit of quantification (LOQ).
According to the above-mentioned 6-parallel blank sample addition recovery test, LODs and LOQs in milk and its products (raw milk, pasteurized milk, 75 ℃ fresh milk, milk powder) and chicken, pork under the existing conditions were obtained for aureomycin, terramycin, tetracycline, doxycycline, dimetocycline, metacycline, minocycline, tigecycline, duromycin and durycline.
Table 11 milk and eight tetracyclines in its products, LODs and LOQs for three metabolites (n=6)
Table 12 LODs and LOQs for eight tetracyclines and three metabolites in chicken and pork (n=6)
4. Determination of determination limits and determination of detection capacities
The determined limits (CC alpha) and the tested capacities (CC beta) are shown in tables 13 and 14 by analyzing 20 parts of blank milk and its products (raw milk, pasteurized milk, 75 ℃ fresh milk and milk powder) and standard samples of chicken, pork (CTC, OTC, TC, ECTC, EOTC and ETC added at MRL, DOC, DMC, MTC, MNC and TGC added at LOQ).
Table 13 milk and eight tetracyclines and three metabolites in its products CC alpha and CC beta (n=20)
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Table 14 eight tetracyclines and CC of three metabolites in chicken and pork (n=20)
5. Matrix effect assessment
Matrix Effects (ME) are evaluated by comparing the slope of the Matrix matching standard curve to the slope of the solvent standard curve.
Drawing a solvent standard curve: the 11 mixed standard stock solutions (40.0 mug/mL) were taken in appropriate amounts and serially diluted with the initial mobile phase from high to low concentration to obtain 11 series of solvent mixed standard solutions of 0.1, 1, 10, 20, 50, 100, 200 and 400 ng/mL. The series of solvent mixed standard solutions were quantitatively analyzed under established and optimized UPLC-MS/MS conditions, and each concentration point was analyzed in parallel for 6 times. And (3) drawing solvent standard curves of 11 target compounds by taking the concentration of the serial solvent mixed standard solution (CTC, OTC, TC, DOC, DMC, MTC, MNC, TGC, ECTC, EOTC and ETC) as an independent variable (x) and the quantitative sub-ion peak area of the corresponding concentration as a dependent variable (y).
ME is calculated according to the following formula.
Wherein:
ME-matrix effect (%);
slope a-matrix matches the Slope of the calibration curve;
slope B-Slope of solvent standard curve.
The matrix effect evaluation of eight tetracyclines and three metabolites in blank milk and its products (raw milk, pasteurized milk, 75 ℃ fresh milk and milk powder) and chicken, pork are shown in Table 15.
Table 15 matrix effects of eight Tetracycline drugs and three metabolites in milk and its products and chickens, pork (n=6,%)
(fifth) method comparison
At present, a plurality of researches on a liquid chromatography and a liquid chromatography-mass spectrometry (LC-MS/MS) method for simultaneously detecting tetracycline residues in dairy products exist, and analysis results of different sample pretreatment conditions and detection conditions for detecting the tetracycline residues in different dairy products are compared in Table 16.
The detection method of tetracycline medicine residues in meat products mainly comprises U/HPLC-MS/MS, U/HPLC-UVD, U/HPLC-PDAD, U/HPLC-FLD methods and the like. Su et al (2014) reported an HPLC-MS/MS analysis method for simultaneous determination of CTC, OTC and TC in pork and chicken, the sample extractant was EDTA-Mcllvaine buffer, and a Agala Cleanert PEP-2 column was used to purify the extract. The liquid phase separation is carried out on a Agela Durashell C chromatographic column, the mobile phase is 0.3% acetonitrile formate-0.3% formic acid water, the LODs are measured to be 10-40 mug/kg, and the recovery rate is 80% -100%. Su Zhi (2021) develops a UPLC-MS/MS method for detecting residues of CTC, OTC and TC in animal source foods such as chicken and pork, and the like, ultrasonic extraction is carried out after acetonitrile is added into a sample, a C18 SPE small column is purified, a chromatographic column is RP18, a mobile phase is methanol-0.1% formic acid aqueous solution, gradient elution time is 13min, sample standard adding recovery rate is about 75%, LOQs is 10 mug/kg, RSDs are lower than 10%, and monitoring requirements are met. Lan et al (2020) analyze 37 veterinary drug residues including tetracycline in chicken by HPLC-MS/MS, wherein the extracting agent is 2% of formic acid water and acetonitrile, PRiME HLB column is adopted for passing purification, the chromatographic column is HSS T3, wherein LODs of the tetracycline is 2.5 mug/kg, LOQs is 5.0 mug/kg, the average recovery rate is 81.5% -88.2%, and the optimized method is successfully applied to detection of actual chicken samples. Li Jige et al (2019) simultaneously detect CTC, OTC, TC and DOC in chicken by SPE-UPLC-MS/MS, add EDTA-Mclvaine buffer with pH of 4.0 into the sample, ultrasonically extract for 10min, purify by HLB small column after centrifugation, chromatographic column is BEH C18, mobile phase is 0.1% formic acid acetonitrile-0.1% formic acid aqueous solution, gradient elution time is 11min. After optimizing the experimental conditions, the LODs of 4 TCs are 0.05-0.32 mug/kg, the adding recovery rate is 71.4% -124.4%, and the RSDs are 1.33% -7.56%. The test establishes an SPE-UPLC-MS/MS method for determining eight TCs and three metabolite residues in chicken and pork, and 0.1mol/L Na is adopted for sample extraction 2 EDTA-Mclvaine buffer solution, 1% formic acid aqueous solution and acetonitrile, the sample purification adopts PRiME HLB small column, and the high-flux sample purification can be rapidly completed by adopting a through type purification mode. The detection and verification of chicken and pork samples by an optimized UPLC-MS/MS method show that the quantitative ion chromatographic peak areas of 11 target compounds and the corresponding matrix matching standard solution concentrations of the target compounds are in good linear relation in the concentration range of LOQ-400ng/mL, the addition recovery rate of eight TCs and three metabolites in all samples is 84.21% -107.50%, the RSDs are not more than 6.59%, the LODs and LOQs in chicken are 0.05-0.48 mug/kg and 0.16-1.61 mug/kg respectively, the LODs and LOQs in pork are 0.05-0.52 mug/kg and 0.16-1.75 mug/kg respectively, and the detection time is 9min. Compared with other research reports, the analytical method established by the test is convenient and quick in pretreatment operation, short in instrument detection time, higher in recovery rate, precision and sensitivity than some methods reported in the test, and can be used for obviously improving the working efficiency and providing references for detection of eight tetracycline drugs and three metabolite residues in chicken and pork.
Table 16 comparison of processing method and detection result of different sample for detecting tetracycline drug residues in dairy products
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Claims (10)
1. The quantitative detection method for eight tetracycline drugs and three metabolite residues in milk and products thereof and chicken and pork is characterized by comprising the following steps:
by Na 2 EDTA-Mclvaine is used as an extraction reagent for milk and products thereof, and Na is used as an extraction reagent 2 EDTA-Mclvaine buffer solution, 1% formic acid aqueous solution and acetonitrile are used as extracting reagent for chicken and pork, milk and products thereof and chicken and pork samples are extracted, and Water is usedPurifying the sample by a s Oasis PRiME HLB small column, and drying and concentrating the purified sample liquid under nitrogen flow; re-dissolving the concentrated and blow-dried sample by adopting an initial mobile phase, filtering by adopting a filter membrane, and detecting by adopting UPLC-MS/MS; the initial mobile phase is a mixed solution of 1% trifluoroacetic acid aqueous solution and methanol, wherein the volume ratio of the 1% trifluoroacetic acid aqueous solution to the methanol is 7:3.
2. The method for quantitatively detecting the residues of eight tetracyclines and three metabolites in milk and products thereof and chicken and pork according to claim 1, wherein the milk and products thereof comprise raw milk, pasteurized milk, 75 ℃ fresh milk and milk powder.
3. The method for quantitatively detecting the residues of eight kinds of tetracyclines and three kinds of metabolites in milk and products thereof and chickens and pork according to claim 1, wherein the eight kinds of tetracyclines and the three kinds of metabolites comprise aureomycin, oxytetracycline, tetracycline, doxycycline, demeclocycline, metacycline, minocycline, tigecycline, doxycycline and doxycycline.
4. The method for quantitatively detecting the residues of eight tetracyclines and three metabolites in milk and products thereof and chicken and pork according to claim 1, wherein the extracting step comprises the following steps: adding fresh milk, pasteurized milk, 75 deg.C fresh milk and milk powder water solution into 0.1mol/L Na 2 EDTA-Mclvaine solution, vortex mixing for 2min, ultrasonic treating in room temperature water bath for 10min, centrifuging at 4deg.C for 10min at 10000r/min, filtering the supernatant with rapid filter paper, and collecting filtrate;
adding chicken and pork samples into 2mL of 0.1mol/L Na 2 EDTA-Mclvaine buffer solution, vortex oscillation for 2min, centrifugation at 4 ℃ for 5min at 5000r/min, and supernatant taking for later use; 2mL of 1% formic acid aqueous solution and 6mL of acetonitrile are added to the residue, vortex mixing is carried out for 5min, ultrasonic treatment is carried out in a room temperature water bath for 15 min, centrifugation is carried out for 10min at 10000r/min at 4 ℃, supernatant is taken, two parts of supernatant are combined and stored, filter paper is used for rapid filtration, and filtrate is collected.
5. The method for quantitatively detecting the residues of eight tetracyclines and three metabolites in milk and products thereof and chicken and pork according to claim 1, wherein the purifying step comprises:
raw milk, pasteurized milk, 75 ℃ fresh milk and milk powder: sample purification is carried out by adopting a Waters Oasis PRiME HLB small column, the small column is activated and balanced by using 3mL of methanol and 3mL of ultrapure water in sequence, a sample to be purified is added, liquid drops pass through the small column at the speed of 1 drop per second, after all sample liquid flows out, the small column is washed by using 3mL of 5% methanol water, effluent liquid is discarded, the small column is pumped to be dry by using a vacuum pump under negative pressure, 2mL of methanol is used for eluting, and eluent is collected. Placing 1mL of eluent into a 15mL centrifuge tube, concentrating to dryness by dry bath nitrogen at 40 ℃, re-dissolving by using 2mL of initial mobile phase, vortex oscillating for 2min, passing through a 0.22 mu m nylon organic filter membrane, and detecting by UPLC-MS/MS;
chicken and pork: adopting a Waters Oasis PRiME HLB small column to carry out through purification on a sample, directly injecting sample liquid into an SPE small column without activating and balancing the small column, keeping the flow rate of one second and one drop, discarding effluent of a front section, and collecting filtrate of a middle section; 1mL of effluent is taken into a 15mL polypropylene centrifuge tube, dried bath nitrogen is concentrated to dryness at 40 ℃,2mL of initial mobile phase is used for redissolution, vortex shaking is carried out for 2min, and after passing through a 0.22 mu m nylon organic filter membrane, UPLC-MS/MS detection is carried out.
6. The quantitative detection method for eight tetracycline drugs and three metabolite residues in milk and products thereof as well as chickens and pork according to claim 1, wherein the purified eluent is concentrated to dryness by dry bath nitrogen at 40 ℃, the initial mobile phase is adopted for redissolution, vortex oscillation is carried out for 2min, and after passing through a 0.22 mu m nylon organic filter membrane, the detection is carried out by UPLC-MS/MS.
7. The quantitative detection method for eight tetracycline drugs and three metabolite residues in milk and products thereof and chickens and pork according to claim 1, wherein the ultra-high performance liquid chromatography conditions are as follows: chromatographic column: WATERS ACQUITY UPLC BEH C18; column temperature: 33 ℃; mobile phase a:1% aqueous trifluoroacetic acid, mobile phase B: methanol; flow rate: 0.3mL/min; sample injection amount: 10 mu L.
8. The quantitative detection method for eight tetracycline drugs and three metabolite residues in milk and products thereof and chickens and pork according to claim 1, wherein mass spectrometry conditions are as follows: the triple quadrupole tandem mass spectrometer is provided with an electrospray ion source; scanning positive ions; monitoring multiple reaction ions; air curtain gas: 36psi; electrospray ionization voltage: 5500V; ion source temperature: 550 ℃; ion source spray gas: 45psi; auxiliary heating gas: 45psi; collision gas: 8psi; injection voltage: 10V; residence time: 100ms.
9. The quantitative detection method for eight tetracycline drugs and three metabolite residues in milk and products thereof and chickens and pork according to claim 1, wherein gradient elution is adopted, and the elution procedure is as follows:
10. the quantitative detection method for the residues of eight tetracyclines and three metabolites in milk and products thereof and chickens and pork according to claim 1, wherein the peak areas detected by UPLC-MS/MS are used for calculating the residues of eight tetracyclines and three metabolites in milk and products thereof and chickens and pork.
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