CN112557557A - Method for detecting residual quantity of sulfonamides, quinolones and tetracyclines in vegetables - Google Patents

Method for detecting residual quantity of sulfonamides, quinolones and tetracyclines in vegetables Download PDF

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CN112557557A
CN112557557A CN202011402903.4A CN202011402903A CN112557557A CN 112557557 A CN112557557 A CN 112557557A CN 202011402903 A CN202011402903 A CN 202011402903A CN 112557557 A CN112557557 A CN 112557557A
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quinolones
sulfonamides
tetracyclines
acetonitrile
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李曼
张嘉楠
曹秀梅
李宏娟
杨赵伟
张珍珍
王爱卿
段培姿
马珊珊
王狄
李博
侯桂双
陈军
王艳华
郭万加
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Qinhuangdao Agricultural Product Quality And Safety Supervision And Inspection Center Qinhuangdao Veterinary Drug Supervision Institute Qinhuangdao Feed Monitoring Station
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Qinhuangdao Agricultural Product Quality And Safety Supervision And Inspection Center Qinhuangdao Veterinary Drug Supervision Institute Qinhuangdao Feed Monitoring Station
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    • 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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86

Abstract

The invention relates to the technical field of antibiotic detection, and provides a method for detecting residual amounts of sulfonamides, quinolones and tetracyclines in vegetables, which comprises the following steps of S1, sample pretreatment: freeze-drying a vegetable sample, grinding and sieving to obtain a sieved sample; s2, crude extraction of a sample: adding an extracting agent into the screened sample to extract the sample; s3, secondary extraction; s4, concentrating, fixing the volume and filtering; and S5, carrying out high performance liquid chromatography-tandem mass spectrometry to obtain the residual amount. Through the technical scheme, the problems that the pretreatment means in the prior art is complex, better separation and extraction cannot be realized, and the types of antibiotics which can be detected are single are solved.

Description

Method for detecting residual quantity of sulfonamides, quinolones and tetracyclines in vegetables
Technical Field
The invention relates to the technical field of antibiotic detection, in particular to a method for detecting residual quantities of sulfonamides, quinolones and tetracyclines in vegetables.
Background
Antibiotics refer to secondary metabolites produced by microorganisms or higher animals and plants during life or analogs artificially synthesized according to the chemical structure of antibiotics. Antibiotics can be continuously accumulated along with the transmission of a food chain, and the antibiotics seriously harm human health after reaching a certain concentration. Along with the continuous improvement of the living standard of people, people pay more and more attention to the nutrition balance, the demand of vegetarian vegetables is gradually increased, and meanwhile, the food safety problem brought by antibiotics is also concerned by people. Therefore, there is a need to continuously optimize the analysis and detection technology of antibiotics in vegetables to provide support for food safety and provide a guarantee for people to eat safely.
The prior detection technology has complex pretreatment means, can not realize better separation and extraction, and can influence the subsequent spectrogram analysis and test results. The type of antibiotics which can be detected by the existing method is single, the residual quantity of various antibiotics cannot be detected at one time, and the existing method has higher detection cost for various antibiotics.
Disclosure of Invention
The invention provides a method for detecting residual quantities of sulfonamides, quinolones and tetracyclines in vegetables, and solves the problems of poor pretreatment separation and extraction effects and single type of antibiotics in the prior art.
The technical scheme of the invention is as follows:
the method for detecting the residual quantity of sulfonamides, quinolones and tetracyclines in vegetables comprises the following steps:
s1, sample pretreatment: freeze-drying a vegetable sample, grinding and sieving to obtain a sieved sample;
s2, crude extraction of a sample: adding an extracting agent into the screened sample to extract the sample;
s3, secondary extraction;
s4, concentrating, fixing the volume and filtering;
and S5, carrying out high performance liquid chromatography-tandem mass spectrometry to obtain the residual amount.
As a further technical scheme, the extracting agent comprises a formic acid acetonitrile solution, citric acid, disodium hydrogen phosphate and disodium ethylene diamine tetraacetate.
As a further technical scheme, S30 is also included between S3 and S4, and a compound adsorbent is added into an S3 sample for purification;
wherein the compound adsorbent comprises N-propyl ethylenediamine and C18The mass ratio of the adsorbent to the graphite carbon black is 10:10: 1.
As a further technical scheme, in the step S2, specifically, 10g of sample is weighed into a 50mL centrifuge tube, the sample is accurately weighed to 0.01g, 10mL of formic acid-acetonitrile solution, 0.12g of citric acid, 0.14g of disodium hydrogen phosphate and 0.34g of disodium ethylene diamine tetraacetate are added, homogeneous extraction is performed for 2min, centrifugation is performed at 8000r/min for 5min, and the supernatant is transferred into another centrifuge tube
Wherein the formic acid-acetonitrile solution is formic acid-acetonitrile solution with volume concentration of 0.1%.
As a further technical scheme, in the step S3, the extraction is repeated once with 10mL of 0.1% formic acid-acetonitrile solution with volume concentration, the obtained supernatant is combined with the supernatant obtained in the step S2, and the volume is adjusted to 30mL with acetonitrile.
As a further technical scheme, the step S30 specifically comprises the step of adding 150mg of N-propylethylenediamine and 150mg of C into 8mL of supernate18Adsorbent and 15mg graphite carbon black, vortex mixing for 30s, 8000r/min centrifuging for 5min, and taking 6mL supernatant in a graduated test tube.
As a further technical scheme, in the step S4, the supernatant obtained in S3 is concentrated to less than 0.5mL with nitrogen at 50 ℃, a formic acid-water solution with a volume concentration of 0.1% is added to make a volume of 2mL, and the volume is filtered through a 0.22 μm filter membrane to obtain the sample to be tested.
As a further technical solution, the chromatographic conditions of the high performance liquid chromatography-tandem mass spectrometry of step S5 are specifically:
a chromatographic column: phenomenex Kinetex F5, 3.0X 50mm, 2.6 μm;
mobile phase A: 0.1% strength by volume aqueous formic acid solution, B: acetonitrile;
gradient elution procedure: 0-0.25 min, 10% B; 0.25-6 min, 10% -30% of B; 6-9 min, 30% -50% B; 9-10 min, 50% -90% B; 10-11 min, 90% B; 11-13 min, 10% B;
flow rate: 0.3 mL/min; column temperature: 30 ℃; sample introduction amount: 5 μ L.
As a further technical solution, the mass spectrometry conditions of the high performance liquid chromatography-tandem mass spectrometry of step S5 are specifically:
electrospray ion source ESI, positive ion mode;
ion source temperature: 550 ℃;
spraying voltage: 5500V;
the air pressure of the air curtain is 206.8 kPa;
the pressure of the atomization gas is 413.7 kPa;
the auxiliary atomization gas pressure is 482.6 kPa;
Schedule-MRM set the condition as a time monitoring window of 40s, i.e. the acquisition time for each compound is its retention time ± 40 s.
The working principle and the beneficial effects of the invention are as follows:
1. the invention can realize simultaneous determination of the residual quantity of 34 antibiotics such as sulfonamides, quinolones, tetracyclines and the like in vegetables through one-time pretreatment.
2. According to the invention, the compound extracting solution is obtained by compounding the four components of the formic acid acetonitrile solution, the citric acid, the disodium hydrogen phosphate and the ethylene diamine tetraacetic acid, and the standard addition recovery rate is high. The tetracycline antibiotics are easy to be matched or chelated with the heavy metals to form stable complexes or chelates, so that the extraction efficiency is influenced, and the interference of the heavy metals on the tetracycline medicaments in the extraction process can be better reduced.
3. The invention adopts a Phenomenex Kinetex F5 chromatographic column, and the adopted pentafluorophenyl fixed phase is suitable for the analysis of halogenated compounds, conjugated compounds, isomeric or strongly polar compounds, and especially can realize better separation effect on isomeric isomers of a benzene ring substituent group ectopic position.
4. According to the method for preparing the matrix matching standard solution, 34 antibiotics generally have medium to strong matrix effects, and the experiment can well eliminate the influence of the matrix and meet the analysis requirements.
Drawings
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 is a total ion flow graph of 34 antibiotics at a concentration of 10 ug/L;
figure 2 is a matrix effect of 34 antibiotics in four vegetable matrices.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any inventive step, are intended to be within the scope of the present invention. The test methods described in the following examples are all conventional methods unless otherwise specified; the reagents, unless otherwise specified, are commercially available.
Reagents and raw materials:
acetonitrile, methanol (HPLC grade, TEDIA corporation, usa);
formic acid (HPLC grade, dima technology);
water (prepared from Millipore ultra pure water machine);
citric acid (analytically pure);
disodium hydrogen phosphate (analytically pure);
disodium ethylene diamine tetraacetate (Na)2EDTA) (premium grade pure, dimar technology);
QuEChERS disperse solid phase kit (150mg PSA, 150mg C18, 15mg GCB) available from Bora Elgilogy technologies, Inc.;
standards of 16 sulfonamides, 14 quinolones and 4 tetracycline antibiotics are purchased from dr. ehrenstorfer, and have purity higher than 94.6%, and the names of the drugs are shown in table 1.
Preparation of standard stock solution
Standard stock solutions (1000mg/L) were prepared from the standards in methanol and stored at-18 ℃.
Accurately sucking a proper amount of standard stock solution, diluting an initial mobile phase (0.1% formic acid solution and acetonitrile in a ratio of 8:2) step by step to prepare a series of standard working solutions, and preparing the working solutions on site.
Experiment 1: optimization of chromatographic conditions
Example 1
Filtering the mixed standard solution with the concentration of 10ug/L by a 0.22 mu m needle filter to a brown sample bottle, and simultaneously determining the content of 34 antibiotics in the mixed standard solution by using a high performance liquid chromatography-tandem mass spectrometry method, wherein the specific conditions are as follows:
chromatographic conditions:
a chromatographic column: phenomenex Kinetex F5 (3.0X 50mm, 2.6 μm);
mobile phase: a: 0.1% strength by volume aqueous formic acid solution, B: acetonitrile;
gradient elution procedure: 0-0.25 min, 10% B; 0.25-6 min, 10% -30% of B; 6-9 min, 30% -50% B; 9-10 min, 50% -90% B; 10-11 min, 90% B; 11.01-13min, 10% B;
flow rate: 0.3 mL/min; column temperature: 30 ℃; sample introduction amount: 5 μ L
Mass spectrum conditions:
electrospray ion source (ESI), positive ion mode;
ion source temperature: 550 ℃;
spraying voltage: 5500V;
the air pressure of the air curtain is 206.8 kPa;
the pressure of the atomization gas is 413.7 kPa;
the auxiliary atomization gas pressure is 482.6 kPa;
Schedule-MRM set the condition as a time monitoring window of 40s, i.e. the acquisition time for each compound is its retention time ± 40 s.
Example 2
The column used in example 1 was replaced by an ACQUITY UPLC BEH C18 (2.1X 100mm, 1.7 μm), the rest remaining the same as in example 1.
The result shows that the ACQUITY UPLC BEH C18 chromatographic column has good separation effect on most compounds, but the separation of sulfamethoxydiazine and sulfamethoxypyridazine is difficult to realize, the retention time is close, the chromatogram shows cross peaks, and the quantitative analysis is influenced mutually. The Phenomenex Kinetex F5 chromatographic column can better separate the two isomers, because the pentafluorophenyl fixed phase adopted by the Phenomenex Kinetex F5 chromatographic column is suitable for the analysis of halogenated compounds, conjugated compounds, isomeric or strongly polar compounds, and especially the isomer with the para-benzene ring substituent group ectopic position can realize better separation effect. Therefore, the invention finally selects a Phenomenex Kinetex F5 (3.0X 50mm, 2.6 μm) chromatographic column to separate 34 antibiotics.
Example 3
The mobile phase B of example 1 was replaced with methanol and the other operations were the same as in example 1.
Example 4
The same procedure as in example 1 was repeated except that the mobile phase a of example 1 was replaced with an aqueous solution of formic acid having a volume concentration of 0.2%.
Example 5
The same procedure as in example 1 was repeated except that the mobile phase A in example 1 was replaced with an aqueous solution of formic acid having a volume concentration of 0.2%, and the mobile phase B was replaced with methanol.
The results show that in the ESI (+) mode, the mobile phase composed of 0.1% formic acid aqueous solution-acetonitrile in volume concentration has better peak shape and higher response value among 34 antibiotics, so that the experiment finally selects 0.1% formic acid aqueous solution-acetonitrile in volume concentration as the mobile phase. The total ion current chromatogram (TIC) of the 34 compounds is shown in FIG. 1.
Experiment 2: optimization of mass spectrometry conditions
Example 6
By adopting the sample preparation method of example 1, 10 mug/kg, 20 mug/kg and 50 mug/kg of standard mixed liquor are respectively prepared, the chromatographic conditions are the same as those of example 1, in this example, the first-order mass spectrum scanning is carried out on each compound by adopting an injection pump continuous sampling mode under an ESI positive ion mode to obtain the excimer ion peak of 34 antibiotics; and then performing secondary mass spectrometry on the ion fragments to obtain fragment ion information and determine quantitative ions and qualitative ions. The strength of the excimer ions and the characteristic fragment ions of the 34 compounds is maximized by optimizing the Declustering Potential (Declustering Potential) and the collision Energy (Collison Energy) of each compound; the parameters of ion source temperature, desolvation gas temperature and flow, collision gas flow and the like are optimized, so that the ionization efficiency of each compound is optimized. According to the requirement of the European Union 2002/657/EC on the veterinary drug residue confirmation method, 4 identification points are required for confirmation detection. In the experiment, through optimization and screening, 2 monitoring ion pairs are finally selected for each compound so as to meet the requirements of the European Union animal residue detection confirmation method, and the mass spectrum conditions obtained through specific optimization are shown in table 1.
Table 134 antibiotic retention times, ion pair information, recoveries and relative standard deviations
Figure BDA0002817517250000051
Figure BDA0002817517250000061
Experiment 3: optimization of extraction conditions
Example 7
Freeze-drying the collected lettuce samples, grinding, filtering by a 2mm sieve, accurately weighing 10g of the ground samples, and mixing the antibiotic mixed standard solution in the lettuce samples to obtain 0.01mg/kg of standard lettuce samples. Placing a lettuce sample added with a standard in a 50mL centrifugal tube, adding an extracting solution (10mL of a formic acid-acetonitrile solution with the volume concentration of 0.1%, 0.12g of citric acid, 0.14g of disodium hydrogen phosphate and 0.34g of disodium ethylene diamine tetraacetate), homogenizing and extracting for 2min, centrifuging for 5min at 8000r/min, transferring a supernatant into another centrifugal tube, using 10mL of a formic acid-acetonitrile solution with the volume concentration of 0.1%, combining the supernatants obtained in the two steps, and fixing the volume to 30mL by using acetonitrile. 150mg of N-Propylethylenediamine (PSA) and 150mg of C were added to 8mL of the supernatant18Mixing the adsorbent and 15mg of Graphite Carbon Black (GCB) by vortex for 30s, centrifuging for 5min at 8000r/min, taking 6mL of supernatant fluid to be put in a graduated test tube, concentrating the supernatant fluid to be less than 0.5mL by nitrogen at 50 ℃, metering the volume to be 2mL by formic acid-water solution with volume concentration of 0.1 percent, and filtering the supernatant fluid by a 0.22 mu m filter membrane for UPLC-MS/MS measurement, wherein the test conditions are the same as the conditions of the example 1.
Example 8
The extract liquid in example 7 was replaced with acetonitrile, and a formic acid-acetonitrile solution having a volume concentration of 0.1% was replaced with acetonitrile, and the other operations were the same.
Example 9
The extract in example 7 was replaced with 10ml of acidified acetonitrile (1% formic acid in acetonitrile, v/v), and a 0.1% formic acid-acetonitrile solution by volume was replaced with acidified acetonitrile, and the other operations were the same.
Example 10
The extract of example 7 was replaced with 10ml of Na2EDTA-Mclvaine buffer solution-acetonitrile (1:1, v/v), volume concentration0.1% formic acid-acetonitrile solution was replaced with Na2EDTA-Mclvaine buffer-acetonitrile (1:1, v/v), other procedures are the same.
Example 11
The extract of example 7 was replaced with 10ml of Na2EDTA-Mclvaine buffer solution-acetonitrile (3:7, v/v), and the volume concentration of 0.1% formic acid-acetonitrile solution is replaced by Na2EDTA-Mclvaine buffer-acetonitrile (3:7, v/v), other procedures are the same.
Example 12
The extract of example 7 was replaced with 1.86g of Na2EDTA, 10ml of 0.1% formic acid acetonitrile (v/v), the other operations being identical.
The experiment shows that: when acetonitrile or acidified acetonitrile is used for extraction, the standard recovery rate of both the sulfonamides and the quinolones is lower than 20%, and the standard recovery rate of the tetracycline is lower than 10%, because the water content of a vegetable sample is too high (generally 70-98%), while the chemical properties of antibiotics are relatively complex, particularly, the quinolone antibiotics are zwitterionic compounds, and have high solubility under acidic or alkaline conditions, and the tetracycline has high water solubility under acidic conditions; with different proportions of Na2When 34 antibiotics in vegetables are extracted by the EDTA-Mclvaine buffer solution-acetonitrile mixed solvent, the lettuce cannot be completely separated from the extraction solution after being homogenized, extracted and centrifuged, and needs to be filtered and collected by filter paper, meanwhile, great difficulty is brought to concentration, and the standard recovery rate of the 34 antibiotics is lower than 30%. Considering the high water content of lettuce, the experiment converted the water content of 10g lettuce to 10mL by adding a certain amount of Na2Na with the concentration of 0.1mol/L formed by EDTA, citric acid, disodium hydrogen phosphate and other salts2EDTA and Na2EDTA-Mclvaine two buffer solution systems, and then adding acidified acetonitrile for extraction. Through experimental comparison, Na is adopted2The recovery rate of EDTA and acidified acetonitrile in the extraction of part of quinolone antibiotics such as sarafloxacin, norfloxacin and oxytetracycline is less than 30%; citric acid, disodium hydrogen phosphate and Na are adopted2Standard recovery rate of tetracycline antibiotics extracted from EDTA mixed salt and acidified acetonitrileUse of Na in proportion2The extraction of EDTA and acidified acetonitrile is increased by 30-40%, and the standard recovery rate of quinolone and sulfonamide antibiotics is 60-90%. This is because tetracycline antibiotics are easily complexed or chelated with heavy metals to form stable complexes or chelates, which affects the extraction efficiency, while Na affects the extraction efficiency2EDTA-Mclvaine buffer solution to Na2The EDTA buffer solution is used as a chelating agent, so that the interference of heavy metals on tetracycline medicaments in the extraction process can be better reduced. Therefore, 0.12g of citric acid, 0.14g of disodium hydrogen phosphate and 0.34g of Na were selected for the experiment2EDTA, and 10mL of 0.1% formic acid-acetonitrile (v/v) as extraction solvents.
Experiment 4: optimization of purification conditions
Example 13
Since the adsorbent can adsorb strong hydrophobic interferents such as fat, the purification is usually two-sided, and the target compound may be adsorbed while the purification is performed. In the experiment, Chinese cabbage is taken as a matrix, and the influence of adsorbents with different adding amounts on the recovery rate of the target compound is examined at the level of 0.01 mg/kg. The other operations were the same as in experiment 6 except that the amount of the adsorbent to be added was adjusted. The results of the experiments show that PSA and C18The addition amount of the adsorbent is within the range of 50-200 mg, and the adsorbent has no obvious adsorption effect on 34 antibiotics; when the addition amount of the Graphite Carbon Black (GCB) is not more than 15mg, the adsorption rate of the graphite carbon black to 34 antibiotics is less than 5 percent; when the addition amount of the Graphite Carbon Black (GCB) is within the range of 15-50 mg, the average adsorption rate of 34 antibiotics is 1.1-51.2%. Therefore, the experiment finally determined that the amounts of PSA, C18 and Graphite Carbon Black (GCB) added were 150mg, 150mg and 15mg, respectively, on the basis of ensuring the purification effect on the sample.
Experiment 5: evaluation of matrix Effect
Example 14
In the experiment, 4 matrixes, such as lettuce, cucumber, summer radish, Chinese cabbage and the like, are respectively selected, blank samples are processed according to the method of experiment 6, matrix blank liquid and a solvent are respectively used for preparing mixed standard solutions with the mass concentrations of 5 mug/L, 10 mug/L, 20 mug/L, 50 mug/L and 100 mug/L, standard curves are respectively made according to the mass concentrations corresponding to peak areas, and then the difference of the slopes of the two standard curves is compared, so that the strength of the matrix effect is judged.
The matrix effect is mainly due to the phenomenon that other components in the matrix compete with the target compound for ionization during ionization of the sample, so that the detection sensitivity and reproducibility of the target compound are influenced, and the matrix effect comprises a matrix enhancement effect and a matrix inhibition effect. The Matrix Effect (ME) is calculated by the following formula: ME (%) - (slope of substrate matching standard curve/slope of solvent standard curve-1) × 100%. A negative matrix effect indicates the presence of a matrix-inhibiting effect; positive values are indicated as matrix enhancement effects. The absolute value is 0-20%, which indicates that a weak matrix effect exists; the absolute value is 20-50%, which indicates that medium matrix effect exists; absolute > 50%, indicating a strong matrix effect.
The mean Matrix Effect (ME) of the 34 antibiotics in the 4 vegetable matrices is shown in figure 2. The experimental result shows that 3 (ME) antibiotics in the four vegetable matrixes have an average matrix effect of 0-20%, 15 antibiotics have an average matrix effect of 20-50% and 16 antibiotics have an average matrix effect of more than 50%, and the 34 antibiotics show a medium-to-strong matrix effect on the whole. Therefore, the method for preparing the matrix matching standard solution is adopted in the experiment, the influence of the matrix can be better eliminated, and the analysis requirement is met.
Experiment 6: linear range, detection limit and quantitation limit
Example 15
A series of 34 antibiotic mixed standard solutions with mass concentrations of 1, 2, 5, 10, 20, 50 and 100 mug/L are prepared by using a blank substrate, and are analyzed and measured under the optimal conditions of the above-mentioned examples 1 and 7, and a standard curve is drawn by the peak area (y) of each component to the mass concentration (x) of each component in the solution. The results show that 30 kinds of sulfanilamide and quinolone antibiotics form a good linear relationship in the range of 1-100 mu g/L, and the correlation coefficients are all larger than 0.997 (see table 2); the 4 tetracycline antibiotics form a good linear relation in the range of 2-100 mug/L, and the correlation coefficients of the four tetracycline antibiotics are all larger than 0.997 (see table 2). The detection Limit (LOD) and the quantification Limit (LOQ) of the compound were determined at 3-fold and 10-fold signal-to-noise ratios (S/N), with detection limit and quantification limit being 0.02-0.35. mu.g/kg and 0.2-0.90. mu.g/kg, respectively (see Table 2).
Table 234 antibiotics Linear Range in radish matrix, regression equation, correlation coefficient (r), detection Limit, lower quantitative Limit and average matrix Effect in lettuce, cucumber, summer radish and cabbage matrices
Figure BDA0002817517250000091
Experiment 7: recovery and precision
Example 16
Blank vegetable samples were taken and subjected to spiking recovery tests at 3 addition levels (10. mu.g/kg, 20. mu.g/kg, 50. mu.g/kg) low, medium and high, each level being measured in parallel 6 times, the results being shown in Table 1. The average standard recovery rate of the 34 antibiotics is 57.2-86.4%, and the relative standard deviation is 4.2-11.9%.
The present invention is not limited to the above preferred embodiments, but rather, any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (9)

1. The method for detecting the residual quantity of sulfonamides, quinolones and tetracyclines in vegetables is characterized by comprising the following steps of:
s1, sample pretreatment: freeze-drying a vegetable sample, grinding and sieving to obtain a sieved sample;
s2, crude extraction of a sample: adding an extracting agent into the screened sample to extract the sample;
s3, secondary extraction;
s4, concentrating, fixing the volume and filtering;
and S5, carrying out high performance liquid chromatography-tandem mass spectrometry to obtain the residual amount.
2. The method for detecting residual amounts of sulfonamides, quinolones and tetracyclines in vegetables according to claim 1, wherein said extracting agent comprises acetonitrile formate solution, citric acid, disodium hydrogen phosphate and disodium ethylenediaminetetraacetate.
3. The method for detecting the residual quantity of sulfonamides, quinolones and tetracyclines in vegetables according to claim 1, wherein S30 is further included between S3 and S4, and a compound adsorbent is added into the S3 sample for purification;
wherein the compound adsorbent comprises N-propyl ethylenediamine and C18The mass ratio of the adsorbent to the graphite carbon black is 10:10: 1.
4. The method for detecting residual amounts of sulfonamides, quinolones and tetracyclines in vegetables according to claim 1, wherein step S2 is specifically that 10g of sample is weighed into a 50mL centrifuge tube to an accuracy of 0.01g, 10mL of formic acid-acetonitrile solution, 0.12g of citric acid, 0.14g of disodium hydrogen phosphate and 0.34g of disodium ethylene diamine tetraacetic acid are added, homogeneous extraction is carried out for 2min, centrifugation is carried out for 5min at 8000r/min, and supernatant is transferred into another centrifuge tube;
wherein the formic acid-acetonitrile solution is formic acid-acetonitrile solution with volume concentration of 0.1%.
5. The method for detecting residual amounts of sulfonamides, quinolones and tetracyclines in vegetables according to claim 1, wherein said step S3 comprises repeating said extraction with 10mL of 0.1% formic acid-acetonitrile solution, combining the obtained supernatant with the supernatant obtained in step S2, and adding acetonitrile to a volume of 30 mL.
6. The method for detecting residual amounts of sulfonamides, quinolones and tetracyclines in vegetables according to claim 3, wherein said step S30 comprises adding 150mg of N-propylethylenediamine and 150mg of C to 8mL of supernatant18Adsorbent and 15mg graphite carbon black, vortex mixing for 30s, 8000r/min centrifuging for 5min, and taking 6mL supernatant in a graduated test tube.
7. The method for detecting residual amounts of sulfonamides, quinolones and tetracyclines in vegetables according to claim 1, wherein step S4 comprises concentrating the supernatant obtained in step S3 at 50 ℃ with nitrogen to less than 0.5mL, diluting to 2mL with 0.1% formic acid-water solution, and filtering with 0.22 μm filter membrane to obtain the sample to be detected.
8. The method for detecting residual amounts of sulfonamides, quinolones and tetracyclines in vegetables according to claim 1, wherein the chromatographic conditions of the step S5 hplc-tandem mass spectrometry are as follows:
a chromatographic column: phenomenex Kinetex F5, 3.0X 50mm, 2.6 μm;
mobile phase A: 0.1% strength by volume aqueous formic acid solution, B: acetonitrile;
gradient elution procedure: 0-0.25 min, 10% B; 0.25-6 min, 10% -30% of B; 6-9 min, 30% -50% B; 9-10 min, 50% -90% B; 10-11 min, 90% B; 11-13 min, 10% B;
flow rate: 0.3 mL/min; column temperature: 30 ℃; sample introduction amount: 5 μ L.
9. The method for detecting residual amounts of sulfonamides, quinolones and tetracyclines in vegetables according to claim 1, wherein the mass spectrometric conditions of the step S5 hplc-tandem mass spectrometry are as follows:
electrospray ion source ESI, positive ion mode;
ion source temperature: 550 ℃;
spraying voltage: 5500V;
the air pressure of the air curtain is 206.8 kPa;
the pressure of the atomization gas is 413.7 kPa;
the auxiliary atomization gas pressure is 482.6 kPa;
Schedule-MRM sets the conditions to a time monitoring window of 40s, i.e. the acquisition time for each compound is its retention time ± 40 s.
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Application publication date: 20210326