CN113791150B - Method for detecting residual quantity of cephalosporin drugs in animal-derived food - Google Patents
Method for detecting residual quantity of cephalosporin drugs in animal-derived food Download PDFInfo
- Publication number
- CN113791150B CN113791150B CN202111055413.6A CN202111055413A CN113791150B CN 113791150 B CN113791150 B CN 113791150B CN 202111055413 A CN202111055413 A CN 202111055413A CN 113791150 B CN113791150 B CN 113791150B
- Authority
- CN
- China
- Prior art keywords
- graphene
- magnetic
- sample
- solution
- mixed solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
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
- G01N30/26—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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
-
- 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
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the field of food safety detection. The technical proposal is as follows: the method for detecting the cephalosporin drug residue in the animal-derived product comprises the following steps: 1) Sample pretreatment: weighing 1-2g of sample into a 50mL centrifuge tube, adding 10mL of ethylenediamine tetraacetic acid (EDTA) -phosphate buffer solution, 50 mu L of choline chloride o-cresol mixed solution and 0.05-0.1g of magnetic graphene molecularly imprinted polymer, and sucking out the magnetic graphene molecularly imprinted polymer by using a magnetic gun after vortex and ultrasonic extraction; adding 2mL of 0.2% methanolic formate eluent to analyze the cephalosporin drug residue in the sample, drying by nitrogen, re-dissolving by 1.0mL of water, and filtering by a 0.22 mu m filter membrane to obtain a liquid to be detected; 2) Detecting a liquid to be detected: determination was performed in positive ion mode under mass spectrum ESI source using HPLC-MS/MS. The method has good selectivity and high sensitivity.
Description
Technical Field
The invention belongs to the field of food safety detection, and particularly relates to a detection method for cephalosporin drug residues in animal-derived foods.
Background
The cephalosporin medicine belongs to broad-spectrum antibiotics and has strong bactericidal power, however, excessive use of the cephalosporin medicine can bring potential harm to human health, has larger influence on children, old people, particularly fetuses, and can cause fetal lesions, deformity and even death. And the residual quantity of the cephalosporin drugs in partial animal-derived products is limited by the reasons of drug resistance, anaphylactic reaction and the like generated by bacteria in various countries around the world. The national standard now issues a series of determination methods for detecting cephalosporin compounds in animal-derived foods. However, the operation is complicated, the time consumption is long, and a detection method for simultaneously measuring the 6 cephalosporin drugs is not established.
The molecular imprinting technique (molecularly imprinted technique, MIT) is a technique for preparing a polymer-molecularly imprinted polymer having a specific selective recognition ability for a template molecule. The technology is widely focused due to strong selectivity, good stability and easy preparation. The graphene material has large specific surface area and excellent adsorption capacity, atoms of the graphene material are arranged in a shape similar to a hexagonal honeycomb, and a special structure can form a stronger pi-pi conjugated system with other organic compounds, so that the graphene material has excellent acid and alkali resistance and chemical stability. The graphene has wider application by modifying the chemical property of graphene. If graphene is combined with the magnetic nano material, the strong adsorption performance and convenient magnetic separation are combined to form a novel magnetic graphene composite material, and finally a molecular imprinting shell layer is synthesized on the surface of the graphene magnetic nano particle. The magnetic molecularly imprinted polymer not only has the advantage of molecular imprinting, can specifically identify and adsorb target molecules, but also can be rapidly separated under the action of a magnetic field, and has a large specific surface area and large adsorption capacity due to small particle size of graphene, thereby having wide application prospects in veterinary drug residue detection.
Disclosure of Invention
The invention aims to overcome the defect of complex pretreatment means for detecting cephalosporin substances in animal-derived products and provides a simple and convenient method for detecting cephalosporin drug residues. The method has the characteristics of good selectivity, high sensitivity and short detection time, so as to improve the detection efficiency.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the method for detecting the cephalosporin drug residue in the animal-derived product comprises the following steps:
1) Sample pretreatment: weighing 1-2g of sample into a 50mL centrifuge tube, adding 10mL of ethylenediamine tetraacetic acid (EDTA) -phosphate buffer solution, 50 mu L of choline chloride o-cresol mixed solution and 0.05-0.1g of magnetic graphene molecularly imprinted polymer, and sucking out the magnetic graphene molecularly imprinted polymer by using a magnetic gun after vortex and ultrasonic extraction; adding 2mL of 0.2% methanolic formate eluent to analyze the cephalosporin drug residue in the sample, drying by nitrogen, re-dissolving by 1.0mL of water, and filtering by a 0.22 mu m filter membrane to obtain a liquid to be detected;
2) Detecting a liquid to be detected: the HPLC-MS/MS was used to determine the positive ion mode under mass spectrum ESI source, and the external standard was quantitative.
The ethylenediamine tetraacetic acid (EDTA) -phosphate buffer solution is prepared according to the following method: 30.25g of disodium ethylenediamine tetraacetate (Na) 2 EDTA·2H 2 O), 6.0g of sodium dihydrogen phosphate (NaH) 2 PO 4 ) Dissolving in ultrapure water, diluting to 1,000 mL, and dissolving in disodium hydrogen phosphate (Na 2 HPO 4 ) The pH was adjusted to 8.5 to 0.1.
The choline chloride o-cresol mixed solution is prepared by the following steps: choline chloride, o-cresol, methyl tertiary butyl ether according to 0.1: weighing a proper amount according to the molar ratio of 0.2:1, and heating under constant stirring at 50 ℃ until stable, uniform and transparent liquid is formed; the prepared mixed solution was stored in a sealed vial and kept in a desiccator.
The magnetic graphene molecularly imprinted polymer is prepared by the following method:
(1) Modification of graphene: the graphene powder is placed in a refrigerator at the temperature of minus 18 ℃ for freezing for 1 hour, 3g of the graphene powder is weighed, and 200mL of concentrated sulfuric acid H mixed according to the proportion of 3:1:0.1 is slowly added 2 SO 4 Concentrated nitric acid HNO 3 Carrying out ultrasonic reaction for 1h in an isopropyl alcohol mixed solution, heating and stirring at 80 ℃ for 2h, carrying out suction filtration, washing with distilled water to be neutral, carrying out vacuum drying at 50 ℃ to be constant weight, grinding and sieving to obtain modified graphene for later use;
(2) Preparing the magnetic carbon nano tube by a solvothermal method: 5.410g of ferric trichloride (FeCl) was weighed 3 ·6H 2 O) and 3.475g of ferrous sulfate (FeSO) 4 ·7H 2 Placing the mixture into a 250mL flat-bottom flask, adding 100mL of water, magnetically stirring at a constant temperature of 60 ℃, after all the mixture is dissolved, rapidly adding 25mL of ammonia water, 2g of modified graphene prepared in the step (1) and 5mL of ethylene glycol-polyethylene glycol mixed solvent formed according to a ratio of 1:1, magnetically stirring at a constant temperature of 60 ℃ for 2 hours to obtain a black mixed solution, washing with ethanol and water for several times, and vacuum drying at 50 ℃ to constant weight to obtain the magnetic carbon nanotube for later use;
(3) Vortex mixing the magnetic carbon nanotube obtained in the step (2), 1mmol of 7-amino deacetoxycephalosporanic acid, 5mmol of methacrylic acid, 5mmol of acrylic acid, 1mL of dimethyl sulfoxide and 50mL of ethanol-polyethylene glycol mixed solution formed by the proportion of 9:1 for 1min, adding 10mmol of ethylene glycol dimethacrylate, 0.2g of polyvinylpyrrolidone and 0.010g of initiator azo diiso Ding Qi (AIBN), introducing nitrogen for sealing, and performing water bath ultrasonic treatment at 80 ℃ for 5h; after the reaction is finished, separating the supernatant by a magnetic field, wherein the volume ratio of the rest materials is 9:1, repeatedly performing ultrasonic washing on the methanol-formic acid mixed solution until the supernatant cannot detect the template molecules by a differential detector, and performing vacuum drying at 50 ℃ until the weight is constant, thus obtaining the magnetic graphene molecularly imprinted polymer.
The chromatographic conditions of the ultra-high performance liquid chromatography-triple quadrupole mass spectrometer are as follows:
chromatographic column: an Agilent RRHD SB-C18 chromatographic column (2.1X100 mm,1.8 μm)); flow rate: 0.25mL/min; sample injection amount: 2. Mu.L; column temperature: 35 ℃. Solution a-0.1% formic acid in water, B-acetonitrile (0.1% formic acid), gradient elution procedure: 0min,10% B%,0min-0.20min,11% B%,0.20min-3.00min,11% -90% B%,3.00min-4.00min,90% B%,4.10min-11.00min,11% B%.
The mass spectrum conditions of the ultra-high performance liquid chromatography-triple quadrupole mass spectrometer are as follows:
ion source: electrospray ion source (ESI source).
Spray pressure: 40psi.
Drying gas flow rate: 11L/min.
Drying gas temperature: 300 ℃.
Capillary voltage: 3500V.
Monitoring mode: positive ion monitoring mode. The monitoring ion pairs and the associated voltage parameter settings are shown in table 1.
TABLE 1 triple quadrupole ion pairs and related voltage parameter set table
Compared with the prior art, the invention has the beneficial effects that:
1) EDTA-phosphate buffer solution and hydrophobic eutectic solvent (choline chloride o-cresol mixed solution) are combined to be used as extraction solvent, so that the extraction rate of cephalosporin substances is greatly improved; comparing the extracts of different extracts
Experiments were performed with the effects and the data are shown in table 2;
TABLE 2 comparison of the extraction effects of different extracts
Therefore, the extraction solution provided by the invention has good extraction selectivity for cefpiralin, cefalexin, cefalonine, cefquinome, cefazolin and ceftiofur in animal-derived products, and the extraction rate is obviously higher than that of phosphate buffer solution, EDTA buffer solution and acetonitrile. Meanwhile, the extraction rate of the six medicines can reach more than 90%, the dosage of the extraction solvent is only 10mL, the use of organic solvents is reduced, and the method is more environment-friendly.
2) The modified magnetic graphene molecularly imprinted polymer is used as a purification means, so that the steps of purifying a solid phase extraction column, nitrogen blowing concentration and the like are reduced, the operation is simpler and more convenient, most matrix interferents can be removed, the pretreatment step is simplified, the pretreatment time is greatly shortened (the pretreatment time is shortened from a few hours to 5 minutes), the method is suitable for simultaneously treating multiple batches of samples, and the detection efficiency is improved. The magnetic graphene molecularly imprinted polymer can be recycled, so that the detection cost is reduced.
3) The quantitative limit of 6 compounds is 1.0 mug/kg through optimization of chromatographic and mass spectrum conditions, the sensitivity is high, the Relative Standard Deviation (RSD) is less than 5%, the recovery rate is 90-110%, and the result shows that the method is suitable for detecting cefpirome, cefalexin, cefalotin, cefquinome, cefazolin and ceftiofur drug residues in animal-derived products by using the method with the sensitivity, precision and accuracy standard method.
Detailed Description
The invention will be described in detail below with reference to the drawings and to specific embodiments.
Example 1
1) Sample pretreatment: 2.0g of pork sample is weighed into a 50mL centrifuge tube, 10mL of ethylenediamine tetraacetic acid-phosphate buffer solution, 50 mu L of choline chloride o-cresol mixed solution and 0.05g of magnetic graphene molecularly imprinted polymer are added, and after vortex and ultrasonic extraction, the magnetic graphene molecularly imprinted polymer is sucked out by a magnetic gun. Adding 2mL of eluent (0.2% methanol formate) to analyze the cephalosporin drug residue in the sample, drying by nitrogen, re-dissolving by 1.0mL of water, and filtering with a 0.22 mu m filter membrane to obtain a liquid to be detected;
2) Detecting a liquid to be detected: the HPLC-MS/MS was used to determine the positive ion mode under mass spectrum ESI source, and the external standard was quantitative.
The chromatographic conditions of the ultra-high performance liquid chromatography-triple quadrupole mass spectrometer are as follows:
chromatographic column: an Agilent RRHD SB-C18 chromatographic column (2.1X100 mm,1.8 μm)); flow rate: 0.25mL/min; sample injection amount: 2. Mu.L; column temperature: 35 ℃. Solution a-0.1% formic acid in water, B-acetonitrile (0.1% formic acid), gradient elution procedure: 0min,10% B%,0min-0.20min,11% B%,0.20min-3.00min,11% -90% B%,3.00min-4.00min,90% B%,4.10min-11.00min,11% B%.
The mass spectrum conditions of the ultra-high performance liquid chromatography-triple quadrupole mass spectrometer are as follows:
ion source: electrospray ion source (ESI source).
Spray pressure: 40psi.
Drying gas flow rate: 11L/min.
Drying gas temperature: 300 ℃.
Capillary voltage: 3500V.
Monitoring mode: positive ion monitoring mode. The monitoring ion pairs and the associated voltage parameter settings are shown in table 3.
TABLE 3 triple quadrupole ion pairs and related voltage parameter set table
3) The sample detection results were omitted.
The accuracy of the detection data of the samples is verified as follows:
1) Preparation of standard stock solution: respectively weighing 10mg of cefpirome, cefalexin, cefalonine, cefquinome, cefazolin and ceftiofur, respectively dissolving with ultrapure water to constant volume of 100.0mL to obtain six standard stock solutions with the concentration of 100 mug/mL;
2) Preparation of mixed standard stock solution: respectively sucking 1.00mL of cefpirome, cefalexin, cefalonine, cefquinome, cefazolin and ceftiofur standard stock solution into the same 100mL volumetric flask, uniformly mixing, and then fixing the volume by using ultrapure water to obtain a 1.00 mug/mL mixed standard solution;
3) Preparing a matrix working solution: after the negative pork sample is treated according to the sample pretreatment method, the obtained negative pork sample matrix solution is used for diluting the mixed standard stock solution to prepare six mixed standard working solutions with different concentrations of 1.0ng/mL, 2.0ng/mL, 5.0ng/mL, 10.0ng/mL, 15.0ng/mL and 25.0 ng/mL;
4) Detecting on the machine; the HPLC-MS/MS was used to determine the positive ion mode under mass spectrum ESI source, and the external standard was quantitative.
The chromatographic conditions, mass spectrum conditions and monitoring ion pairs and related voltage parameters of the mass spectrometer are the same as those of the detection of the liquid to be detected.
Sampling the mixed standard working solution from low to high concentration, measuring under the instrument condition, and performing linear regression by taking the concentration of the mixed standard working solution as an abscissa and the peak area ratio of the target compound (six cephalosporin drugs) as an ordinate; the regression equation, the correlation coefficient, the detection limit and the linear range are shown in Table 4;
TABLE 4 regression equation, correlation coefficient, detection limit and linear range of cephalosporin drugs in pork
From the results in Table 4, the correlation coefficient of regression equation of 6 kinds of cephalosporin drugs in pig liver matrix is over 0.999; quantitative limit: 1.0 μg/kg; linear range 1.0-25ng/mL;
three negative pork samples (2.0 g each) were weighed, and after the sample pretreatment method of step 1) in example 1, three concentrations of mixed standard working solutions of 1.0 μg/kg, 5.0 μg/kg and 10.0 μg/kg were added respectively, and the recovery rate and precision were examined by a standard addition recovery test, and the results are shown in Table 5:
TABLE 5 recovery and precision test in pork samples
As is clear from Table 5, the average recovery rate of the 6 cephalosporin drugs of the invention in pork matrix is 92.4-101.5% and RSD is 2.0-3.4%. From the above results, the recovery rate and the precision of the present example satisfy the detection requirements.
Example 2
1) Sample pretreatment: 2.0g of egg sample is weighed into a 50mL centrifuge tube, 10mL of ethylenediamine tetraacetic acid-phosphate buffer solution, 50 mu L of choline chloride o-cresol mixed solution and 0.1g of magnetic graphene molecularly imprinted polymer are added, and after vortex and ultrasonic extraction, the magnetic graphene molecularly imprinted polymer is sucked out by a magnetic gun. Adding 1mL of eluent (0.2% methanol formate) to analyze the cephalosporin drug residue in the sample, drying by nitrogen, re-dissolving by 1.0mL of water, and filtering with a 0.22 mu m filter membrane to obtain a liquid to be detected;
2) Detecting a liquid to be detected: the HPLC-MS/MS was used to determine the positive ion mode under mass spectrum ESI source, and the external standard was quantitative.
The chromatographic conditions of the ultra-high performance liquid chromatography-triple quadrupole mass spectrometer are as follows:
chromatographic column: an Agilent RRHD SB-C18 chromatographic column (2.1X100 mm,1.8 μm)); flow rate: 0.25mL/min; sample injection amount: 2. Mu.L; column temperature: 35 ℃. Solution a-0.1% formic acid in water, B-acetonitrile (0.1% formic acid), gradient elution procedure: 0min,10% B%,0min-0.20min,11% B%,0.20min-3.00min,11% -90% B%,3.00min-4.00min,90% B%,4.10min-11.00min,11% B%.
The mass spectrum conditions of the ultra-high performance liquid chromatography-triple quadrupole mass spectrometer are as follows:
ion source: electrospray ion source (ESI source).
Spray pressure: 40psi.
Drying gas flow rate: 11L/min.
Drying gas temperature: 300 ℃.
Capillary voltage: 3500V.
Monitoring mode: positive ion monitoring mode. The monitoring ion pairs and the associated voltage parameter settings are shown in table 6.
TABLE 6 triple quadrupole ion pairs and related voltage parameter set tables
3) The sample detection results were omitted.
The accuracy of the detection data of the samples is verified as follows:
1) Preparation of standard stock solution: respectively weighing 10mg of cefpirome, cefalexin, cefalonine, cefquinome, cefazolin and ceftiofur, respectively dissolving with ultrapure water to constant volume of 100.0mL to obtain six standard stock solutions with the concentration of 100 mug/mL;
2) Preparation of mixed standard stock solution: respectively sucking 1.00mL of cefpirome, cefalexin, cefalonine, cefquinome, cefazolin and ceftiofur stock solution into the same 100mL volumetric flask, uniformly mixing, and then fixing the volume by using ultrapure water to obtain a mixed standard solution with the concentration of 1.00 mug/mL;
3) Preparing a matrix working solution: and (2) after the negative egg sample is treated according to the sample pretreatment method in the step (1), diluting the mixed standard stock solution with the obtained negative egg sample matrix solution to prepare six mixed standard working solutions with different concentrations of 1.0ng/mL, 2.0ng/mL, 5.0ng/mL, 10.0ng/mL, 15.0ng/mL and 25.0 ng/mL.
4) Detecting on the machine; determining under positive ion mode under mass spectrum ESI source by HPLC-MS/MS, and quantifying by external standard; the chromatographic conditions, mass spectrum conditions and monitoring ion pairs and related voltage parameters of the mass spectrometer are the same as those of the detection of the liquid to be detected.
And (3) sampling the mixed standard working solution from low to high concentration, measuring under the instrument condition, and carrying out linear regression by taking the concentration of the mixed standard working solution as an abscissa and the peak area ratio of the target compound (six cephalosporin drugs) as an ordinate. The regression equation, correlation coefficient, detection limit and linear range are shown in Table 7.
TABLE 7 regression equation, correlation coefficient, detection limit and linear range of cephalosporin drugs in eggs
As can be seen from the results in Table 7, the correlation coefficient of the regression equation of 6 kinds of cephalosporins in egg matrix is more than 0.999; quantitative limit: 1.0 μg/kg; the linear range is 1.0-25ng/mL.
Three negative egg samples (2.0 g each) were weighed and treated according to the sample pretreatment method of step 1) in example 2, and then three concentrations of 1.0. Mu.g/kg, 5.0. Mu.g/kg and 10. Mu.g/kg of mixed standard working solution were added respectively, and the recovery rate and precision were examined by a standard addition recovery test, and the results are shown in Table 8:
table 8 recovery and precision test in egg samples
As is clear from Table 8, the average recovery rate of the 6 cephalosporins of the invention in the egg matrix is 92.5 to 102.5% and the RSD is 2.1 to 4.1%. From the above results, the recovery rate and the precision of the present example satisfy the detection requirements.
Example 3
1) Sample pretreatment: 2.0g of milk sample is weighed into a 50mL centrifuge tube, 10mL of ethylenediamine tetraacetic acid-phosphate buffer solution, 50 mu L of choline chloride o-cresol mixed solution and 0.05g of magnetic graphene molecularly imprinted polymer are added, and after vortex and ultrasonic extraction, the magnetic graphene molecularly imprinted polymer is sucked out by a magnetic gun. Adding 1mL of eluent (0.2% methanol formate) to analyze the cephalosporin drug residue in the sample, drying by nitrogen, re-dissolving by 1.0mL of water, and filtering with a 0.22 mu m filter membrane to obtain a liquid to be detected;
2) Detecting a liquid to be detected: high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was used to determine in positive ion mode under mass spectrum ESI source, external standard method.
The chromatographic conditions of the ultra-high performance liquid chromatography-triple quadrupole mass spectrometer are as follows:
chromatographic column: an Agilent RRHD SB-C18 chromatographic column (2.1X100 mm,1.8 μm)); flow rate: 0.25mL/min; sample injection amount: 2. Mu.L; column temperature: 35 ℃. Solution a-0.1% formic acid in water, B-acetonitrile (0.1% formic acid), gradient elution procedure: 0min,10% B%,0min-0.20min,11% B%,0.20min-3.00min,11% -90% B%,3.00min-4.00min,90% B%,4.10min-11.00min,11% B%.
The mass spectrum conditions of the ultra-high performance liquid chromatography-triple quadrupole mass spectrometer are as follows:
ion source: electrospray ion source (ESI source).
Spray pressure: 40psi.
Drying gas flow rate: 11L/min.
Drying gas temperature: 300 ℃.
Capillary voltage: 3500V.
Monitoring mode: positive ion monitoring mode. The monitoring ion pairs and associated voltage parameter settings are shown in table 9.
Table 9 triple quadrupole ion pair and related voltage parameter set table
3) The sample detection results were omitted.
The accuracy of the detection data of the samples is verified as follows:
1) Preparation of standard stock solution: respectively weighing 10mg of cefpirome, cefalexin, cefalonine, cefquinome, cefazolin and ceftiofur, respectively dissolving with ultrapure water to constant volume of 100.0mL to obtain six standard stock solutions with the concentration of 100 mug/mL;
2) Preparation of mixed standard stock solution: respectively sucking 1.00mL of cefpirome, cefalexin, cefalonine, cefquinome, cefazolin and ceftiofur standard stock solution into the same 100mL volumetric flask, uniformly mixing, and then fixing the volume by using ultrapure water to obtain a 1.00 mug/mL mixed standard solution;
3) Preparing a matrix working solution: and (3) after the negative milk sample is treated according to a sample pretreatment method, diluting the mixed standard stock solution with the obtained negative milk sample matrix solution to prepare six mixed standard working solutions with different concentrations of 1.0ng/mL, 2.0ng/mL, 5.0ng/mL, 10.0ng/mL, 15.0ng/mL and 25.0 ng/mL.
4) Detecting on the machine; determining under positive ion mode under mass spectrum ESI source by HPLC-MS/MS, and quantifying by external standard; the chromatographic conditions, mass spectrum conditions and monitoring ion pairs and related voltage parameters of the mass spectrometer are the same as those of the detection of the liquid to be detected.
And (3) sampling the mixed standard working solution from low to high concentration, measuring under the instrument condition, and carrying out linear regression by taking the concentration of the mixed standard working solution as an abscissa and the peak area ratio of the target compound as an ordinate. The regression equation, correlation coefficient, detection limit and linear range are shown in Table 10.
TABLE 10 regression equation, correlation coefficient, detection limit and linear range of cephalosporin drugs in milk
As can be seen from the results in Table 10, the correlation coefficient of the regression equation of 6 kinds of cephalosporins in milk matrix is more than 0.999; quantitative limit: 1.0 μg/kg; the linear range is 1.0-25ng/mL.
Three negative milk samples (2.0 g each) were treated according to the sample pretreatment method of step 1) in example 3, and then three concentrations of 1.0. Mu.g/kg, 5.0. Mu.g/kg and 10. Mu.g/kg of mixed standard working solution were added respectively, and the recovery rate and precision were examined by a standard addition recovery test, and the results are shown in Table 11:
TABLE 11 recovery and precision test in milk samples
As is clear from Table 11, the recovery rate of the 6 kinds of cephalosporins of the invention in a milk matrix was 93.2 to 100.9%, and the RSD was 2.1 to 3.9%. From the above results, the recovery rate and the precision of the present example satisfy the detection requirements.
Claims (3)
1. The method for detecting the cephalosporin drug residue in the animal-derived product comprises the following steps:
sample pretreatment: weighing 1-2g samples in a 50mL centrifuge tube, adding 10mL of ethylenediamine tetraacetic acid-phosphate buffer solution, 50 mu L of choline chloride-o-cresol mixed solution and 0.05-0.1g of magnetic graphene molecularly imprinted polymer, and sucking out the magnetic graphene molecularly imprinted polymer by a magnetic gun after vortex and ultrasonic extraction; adding 2mL of 0.2% methanolic formate eluent to analyze the cephalosporin drug residue in the sample, drying by nitrogen, re-dissolving by 1.0mL water, and filtering with a 0.22 mu m filter membrane to obtain a liquid to be detected;
detecting a liquid to be detected: by using high performance liquid chromatography-tandem mass spectrometry, under the positive ion mode under the ESI source of mass spectrum,
quantifying by an external standard method;
the choline chloride-o-cresol mixed solution is prepared by the following steps: choline chloride, o-cresol, methyl tertiary butyl ether according to 0.1: weighing a proper amount according to the molar ratio of 0.2:1, and heating under constant stirring at 50 ℃ until stable, uniform and transparent liquid is formed; storing the prepared mixed solution in a sealed vial and in a desiccator;
the magnetic graphene molecularly imprinted polymer is prepared by the following method:
(1) Modification of graphene: freezing graphene powder in a refrigerator at the temperature of minus 18 ℃ for 1 hour, weighing 3g of graphene powder, slowly adding the graphene powder into a concentrated sulfuric acid-concentrated nitric acid-isopropanol mixed solution mixed by 200mL according to the ratio of 3:1:0.1, carrying out ultrasonic reaction for 1 hour, heating and stirring at the temperature of 80 ℃ for reaction for 2h, carrying out suction filtration, washing with distilled water to be neutral, carrying out vacuum drying at the temperature of 50 ℃ to constant weight, grinding and sieving to obtain modified graphene, and standing by;
(2) Preparing the magnetic carbon nano tube by a solvothermal method: weighing 5.410g ferric trichloride and 3.475g ferrous sulfate, placing the materials into a 250mL flat-bottomed flask, adding 100mL water, magnetically stirring at a constant temperature of 60 ℃, completely dissolving, and rapidly adding 25mL of ammonia water 2 g; the modified graphene prepared in the step (1) and 5mL of glycol-polyethylene glycol mixed solvent which is formed by the ratio of 1:1 are magnetically stirred for 2 hours at the constant temperature of 60 ℃ to obtain black mixed solution, ethanol and water are used for a plurality of times, and the mixed solution is dried to constant weight in vacuum at the temperature of 50 ℃ to obtain the magnetic carbon nanotube for standby;
(3) Mixing the magnetic carbon nanotube obtained in the step (2), 1mmol of 7-amino deacetyl-cephalosporanic acid, 5mmol of methacrylic acid, 5mmol of acrylic acid, 1mL of dimethyl sulfoxide and 50mL in a ratio of 9:1 by vortex for 1min, adding 10mmol of ethylene glycol dimethacrylate, 0.2g of polyvinylpyrrolidone and 0.010g of azo diiso Ding Qi initiator, introducing nitrogen for sealing, and performing water bath ultrasonic treatment at 80 ℃ for 5h; after the reaction is finished, separating the supernatant by a magnetic field, wherein the volume ratio of the rest materials is 9:1, repeatedly performing ultrasonic washing on the methanol-formic acid mixed solution until the supernatant cannot detect the template molecules through a differential detector, and performing vacuum drying at 50 ℃ until the weight is constant to obtain the magnetic graphene molecularly imprinted polymer;
the medicine is cefpirome, cefalexin, cefalonine, cefquinome, cefazolin or ceftiofur;
the sample is pork, egg or milk.
2. The method for detecting cephalosporin drug residues in animal derived products according to claim 1, wherein the method comprises the steps of: the ethylenediamine tetraacetic acid-phosphate buffer solution is prepared according to the following method: 30.25g of disodium ethylenediamine tetraacetate, 6.0. 6.0g of disodium hydrogen phosphate and ultrapure water were weighed and dissolved, diluted to 1,000 mL, and the pH was adjusted to 0.1 with sodium dihydrogen phosphate.
3. The method for detecting cephalosporin drug residues in animal derived products according to claim 2, wherein: the chromatographic conditions of the ultra-high performance liquid chromatography-triple quadrupole mass spectrometer are as follows:
chromatographic column: an Agilent RRHD SB-C18 chromatographic column, 2.1X100 mm,1.8 μm; flow rate: 0.25mL/min; sample injection amount: 2. mu L; column temperature: 35. at the temperature, the solution A is 0.1 percent formic acid aqueous solution, and the solution B is acetonitrile, and contains 0.1 percent formic acid; gradient elution procedure: 0min,10% B, 0min-0.20min,11% B,0.20 min-3.00min,11% -90% B,3.00min-4.00 min,90% B,90% -11% B,4.10 min-11.00min,11% B;
the mass spectrum conditions of the ultra-high performance liquid chromatography-triple quadrupole mass spectrometer are as follows:
ion source: electrospray ion source (ESI source);
spray pressure: 40 psi;
drying gas flow rate: 11L/min;
drying gas temperature: 300 ℃;
capillary voltage: 3500 and V;
monitoring mode: positive ion monitoring mode.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111055413.6A CN113791150B (en) | 2021-09-09 | 2021-09-09 | Method for detecting residual quantity of cephalosporin drugs in animal-derived food |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111055413.6A CN113791150B (en) | 2021-09-09 | 2021-09-09 | Method for detecting residual quantity of cephalosporin drugs in animal-derived food |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113791150A CN113791150A (en) | 2021-12-14 |
| CN113791150B true CN113791150B (en) | 2023-08-11 |
Family
ID=78879762
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111055413.6A Active CN113791150B (en) | 2021-09-09 | 2021-09-09 | Method for detecting residual quantity of cephalosporin drugs in animal-derived food |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113791150B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| PL334427A1 (en) * | 1999-07-16 | 2001-01-29 | Inst Biotechnologii I Antybiot | Method of determining isomers in beta-lactamic antibiotics of cephalosporin group |
| WO2010138899A2 (en) * | 2009-05-28 | 2010-12-02 | The Cleveland Clinic Foundation | Trimethylamine-containing compounds for diagnosis and prediction of disease |
| CN106501405A (en) * | 2016-11-01 | 2017-03-15 | 中国检验检疫科学研究院 | Cephalosporins residual quick screening method in a kind of animal muscle tissue |
| CN107037140A (en) * | 2016-10-21 | 2017-08-11 | 浙江省海洋水产研究所 | A kind of method of cynnematin residual quantity in efficient detection aquatic products |
| CN113061216A (en) * | 2021-03-10 | 2021-07-02 | 大理州质量技术监督综合检测中心 | Magnetic molecularly imprinted polymer for simultaneously extracting multiple cephalosporin antibiotics and preparation method and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2678367C (en) * | 2007-03-02 | 2014-07-08 | Farnam Companies, Inc. | Sustained release compositions using wax-like materials |
-
2021
- 2021-09-09 CN CN202111055413.6A patent/CN113791150B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| PL334427A1 (en) * | 1999-07-16 | 2001-01-29 | Inst Biotechnologii I Antybiot | Method of determining isomers in beta-lactamic antibiotics of cephalosporin group |
| WO2010138899A2 (en) * | 2009-05-28 | 2010-12-02 | The Cleveland Clinic Foundation | Trimethylamine-containing compounds for diagnosis and prediction of disease |
| CN107037140A (en) * | 2016-10-21 | 2017-08-11 | 浙江省海洋水产研究所 | A kind of method of cynnematin residual quantity in efficient detection aquatic products |
| CN106501405A (en) * | 2016-11-01 | 2017-03-15 | 中国检验检疫科学研究院 | Cephalosporins residual quick screening method in a kind of animal muscle tissue |
| CN113061216A (en) * | 2021-03-10 | 2021-07-02 | 大理州质量技术监督综合检测中心 | Magnetic molecularly imprinted polymer for simultaneously extracting multiple cephalosporin antibiotics and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 汪雪雁等.鸡肉中头孢类抗生素的MISPE-HPCE检测.《江苏农药学报》.2012,第28卷(第28期),193-197. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113791150A (en) | 2021-12-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cui et al. | Restricted accessed material-copper (II) ion imprinted polymer solid phase extraction combined with inductively coupled plasma-optical emission spectrometry for the determination of free Cu (II) in urine and serum samples | |
| WO2019196233A1 (en) | Method for measuring contents of various endocrine disruptors in estuary sediment | |
| CN106268707B (en) | A kind of phosphoeptide enrichment new method based on magnetic porous material | |
| CN106872617B (en) | Rapid extraction and LC-MS-MS detection method of benzimidazole and thiazole residual drugs in aquatic products | |
| CN108276584A (en) | The detection method of aromatic amine compound in a kind of human urine | |
| Wei et al. | A new hydride generation system applied in determination of arsenic species with ion chromatography–hydride generation-atomic fluorescence spectrometry (IC–HG-AFS) | |
| CN111650320B (en) | Detection method and kit for quinolone veterinary drugs based on magnetic solid-phase extraction | |
| CN110514774B (en) | Method for analyzing phenolic compounds in water | |
| Liu et al. | The determination of pesticides in tea samples followed by magnetic multiwalled carbon nanotube-based magnetic solid-phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry | |
| CN112684069A (en) | Method for measuring methylnaphthalene compound in water | |
| CN109459506B (en) | Rapid sample pretreatment method for detecting polychlorinated biphenyl in tea | |
| CN113791150B (en) | Method for detecting residual quantity of cephalosporin drugs in animal-derived food | |
| CN103396512B (en) | The preparation method and application of hybrid template molecularly imprinted polymer and solid-phase extraction column thereof | |
| Fan et al. | Application of core–satellite polydopamine-coated Fe 3 O 4 nanoparticles–hollow porous molecularly imprinted polymer combined with HPLC-MS/MS for the quantification of macrolide antibiotics | |
| CN113061216A (en) | Magnetic molecularly imprinted polymer for simultaneously extracting multiple cephalosporin antibiotics and preparation method and application thereof | |
| CN102944635B (en) | Method for determining tris (2,3-dibromopropyl) phosphate content of water | |
| CN113533548B (en) | Method for detecting 1-vinyl imidazole in chemical product | |
| Chen et al. | Ethylenediamine-functionalized superparamagnetic carbon nanotubes for magnetic molecularly imprinted polymer matrix solid-phase dispersion extraction of 12 fluoroquinolones in river water | |
| CN113640429B (en) | Method for detecting quinolone drug residues in animal-derived food | |
| CN113655153B (en) | Method for detecting adamantanamine drug residues in animal-derived products | |
| CN103344726B (en) | Method for extraction of dicyandiamide component in dairy product | |
| CN112198249A (en) | Detection method of ethanolamine compound in soil | |
| CN106749903B (en) | A kind of amphiphilic Acetochlor magnetic molecularly imprinted polymer and its preparation method and application | |
| CN111413422B (en) | Choline proline ionic liquid modified magnetic nano material and detection method of epristeride by combining with HPLC | |
| CN116943624B (en) | Magnetic solid phase extraction material, preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |