CN112051345A - High performance liquid chromatography detection method for sulfonamides in raw fresh milk - Google Patents
High performance liquid chromatography detection method for sulfonamides in raw fresh milk Download PDFInfo
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- 229940124530 sulfonamide Drugs 0.000 title claims abstract description 34
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 26
- 150000003456 sulfonamides Chemical class 0.000 title claims abstract description 26
- 238000001514 detection method Methods 0.000 title claims abstract description 20
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 66
- 239000003480 eluent Substances 0.000 claims abstract description 35
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 28
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 22
- 239000012071 phase Substances 0.000 claims abstract description 15
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims abstract description 14
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000000605 extraction Methods 0.000 claims abstract description 13
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- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 claims description 7
- 238000002386 leaching Methods 0.000 claims description 6
- 238000004885 tandem mass spectrometry Methods 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 23
- 239000000523 sample Substances 0.000 description 20
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- 229960002135 sulfadimidine Drugs 0.000 description 6
- 229960005404 sulfamethoxazole Drugs 0.000 description 6
- FDDDEECHVMSUSB-UHFFFAOYSA-N sulfanilamide Chemical compound NC1=CC=C(S(N)(=O)=O)C=C1 FDDDEECHVMSUSB-UHFFFAOYSA-N 0.000 description 6
- 235000013365 dairy product Nutrition 0.000 description 5
- 241000283690 Bos taurus Species 0.000 description 4
- 229910001873 dinitrogen Inorganic materials 0.000 description 4
- ASWVTGNCAZCNNR-UHFFFAOYSA-N sulfamethazine Chemical compound CC1=CC(C)=NC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 ASWVTGNCAZCNNR-UHFFFAOYSA-N 0.000 description 4
- 229960004936 sulfamethoxypyridazine Drugs 0.000 description 4
- JLKIGFTWXXRPMT-UHFFFAOYSA-N sulphamethoxazole Chemical compound O1C(C)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 JLKIGFTWXXRPMT-UHFFFAOYSA-N 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 3
- 238000004811 liquid chromatography Methods 0.000 description 3
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 229960000654 sulfafurazole Drugs 0.000 description 3
- VLYWMPOKSSWJAL-UHFFFAOYSA-N sulfamethoxypyridazine Chemical compound N1=NC(OC)=CC=C1NS(=O)(=O)C1=CC=C(N)C=C1 VLYWMPOKSSWJAL-UHFFFAOYSA-N 0.000 description 3
- GECHUMIMRBOMGK-UHFFFAOYSA-N sulfapyridine Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=CC=CC=N1 GECHUMIMRBOMGK-UHFFFAOYSA-N 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
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- NHUHCSRWZMLRLA-UHFFFAOYSA-N Sulfisoxazole Chemical compound CC1=NOC(NS(=O)(=O)C=2C=CC(N)=CC=2)=C1C NHUHCSRWZMLRLA-UHFFFAOYSA-N 0.000 description 2
- 238000010811 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods 0.000 description 2
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- SKIVFJLNDNKQPD-UHFFFAOYSA-N sulfacetamide Chemical compound CC(=O)NS(=O)(=O)C1=CC=C(N)C=C1 SKIVFJLNDNKQPD-UHFFFAOYSA-N 0.000 description 2
- 229960002673 sulfacetamide Drugs 0.000 description 2
- 229950008831 sulfachlorpyridazine Drugs 0.000 description 2
- 229960000973 sulfadimethoxine Drugs 0.000 description 2
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- 229960002211 sulfapyridine Drugs 0.000 description 2
- 125000000565 sulfonamide group Chemical group 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- OEDUIFSDODUDRK-UHFFFAOYSA-N 5-phenyl-1h-pyrazole Chemical compound N1N=CC=C1C1=CC=CC=C1 OEDUIFSDODUDRK-UHFFFAOYSA-N 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
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- 210000005228 liver tissue Anatomy 0.000 description 1
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- 231100000417 nephrotoxicity Toxicity 0.000 description 1
- 238000005220 pharmaceutical analysis Methods 0.000 description 1
- 235000020185 raw untreated milk Nutrition 0.000 description 1
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- 238000006467 substitution reaction Methods 0.000 description 1
- XOXHILFPRYWFOD-UHFFFAOYSA-N sulfachloropyridazine Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=CC=C(Cl)N=N1 XOXHILFPRYWFOD-UHFFFAOYSA-N 0.000 description 1
- ZZORFUFYDOWNEF-UHFFFAOYSA-N sulfadimethoxine Chemical compound COC1=NC(OC)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 ZZORFUFYDOWNEF-UHFFFAOYSA-N 0.000 description 1
- NVBFHJWHLNUMCV-UHFFFAOYSA-N sulfamide Chemical compound NS(N)(=O)=O NVBFHJWHLNUMCV-UHFFFAOYSA-N 0.000 description 1
- 229960004818 sulfaphenazole Drugs 0.000 description 1
- QWCJHSGMANYXCW-UHFFFAOYSA-N sulfaphenazole Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=CC=NN1C1=CC=CC=C1 QWCJHSGMANYXCW-UHFFFAOYSA-N 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
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- 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
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- 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
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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
- G01N30/26—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
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- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
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- 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
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Abstract
The invention discloses a high performance liquid chromatography detection method of sulfonamides in raw fresh milk, which comprises the following steps: s1, weighing 4-6 g of the uniformly mixed sample, and placing the sample in a centrifugal tube; s2, adding 15mL of perchloric acid solution into the centrifugal tube, and performing vortex oscillation extraction; s3, installing the MCX solid phase extraction column on a solid phase extraction device, sequentially passing 3mL of methanol and 3mL of 0.1mol/L hydrochloric acid solution through the column, taking the standby liquid to pass through the column S4, sequentially rinsing with 3mL of 0.1mol/L hydrochloric acid solution and 2mL of methanol, eluting with 5mL of 5% ammoniated methanol, and collecting eluent; s5, drying the eluent by using nitrogen, redissolving the eluent by using 1mL of initial mobile phase, filtering the eluent by using a filter membrane, and determining the eluent by using high performance liquid chromatography. The invention simplifies the steps of sample pretreatment, overcomes the problems of high cost, expensive instruments and low popularization rate of high performance liquid chromatography tandem mass spectrometry detection, provides more method choices for the detection of sulfonamides in fresh milk, and meets the requirements of different laboratories.
Description
Technical Field
The invention relates to the technical field of detection of sulfonamides in dairy products, in particular to a high-efficiency liquid chromatography detection method of sulfonamides in fresh milk.
Background
Sulfonamides are artificially synthesized spectrum antibacterial drugs, and the drugs are derivatives taking basic sulfamide of ammonia as a basic structure, and have the characteristics of low price, simple use, stable property and the like. Sulfonamides are widely used for preventing and treating infectious diseases of dairy cows, and feed production enterprises add the sulfonamides as additives into feed for preventing secondary infection caused by viral diseases of the dairy cows or improving the disease resistance of the dairy cows and improving the production performance of the dairy cows. With the improvement of living standard of people, the consumption of milk is gradually increased, and the safety of the milk is more and more concerned. After people eat milk containing sulfonamide residues for a long time, the drugs are continuously accumulated in the human body, and after the drugs are accumulated to a certain degree, toxic and side effects are generated on the human body, which are mainly reflected in allergy, nephrotoxicity, hematopoietic disorder, bacterial drug resistance, carcinogenesis and the like.
At present, methods for detecting sulfonamide residues mainly include a liquid chromatography-tandem mass spectrometry, a liquid chromatography, a capillary electrophoresis method, an immunoassay method, a microbiological method and the like. The liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and the liquid chromatography (HPLC) have the advantages of high sensitivity, rapidness, accuracy, colleges and universities, strong specificity and the like, so that the analysis capability and the analysis efficiency of the drug residue are greatly improved. Because UPLC-MS/MS and HPLC are widely used in pharmaceutical analysis.
At present, the high performance liquid chromatography method of the sulfanilamide medicines in the animal food is available, but the method is only suitable for detecting the sulfanilamide medicines in the muscle and liver tissues of pigs and chickens, but the detection of the sulfanilamide medicines in the fresh animals only adopts a liquid chromatography tandem mass spectrometry method, and does not adopt the high performance liquid chromatography method.
Disclosure of Invention
In view of the above, the present invention provides a high performance liquid chromatography detection method for sulfonamides in raw milk, which overcomes the disadvantages of tedious and time-consuming pretreatment and improves the efficiency of sample pretreatment.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a high performance liquid chromatography detection method for sulfonamides in raw fresh milk comprises the following steps:
s1, weighing 4-6 g of the uniformly mixed sample, and placing the sample in a centrifugal tube;
s2, adding 15mL of perchloric acid solution into the centrifugal tube, and performing vortex oscillation extraction;
s3, installing the MCX solid phase extraction column on a solid phase extraction device, sequentially passing through the column by using 3mL of methanol and 3mL of 0.1mol/L hydrochloric acid solution, taking the standby solution, and passing through the column
S4, sequentially leaching with 3mL of 0.1mol/L hydrochloric acid solution and 2mL of methanol, eluting with 5mL of 5% ammoniated methanol, and collecting eluent;
s5, drying the eluent by using nitrogen, redissolving the eluent by using 1mL of initial mobile phase, filtering the eluent by using a filter membrane, and determining the eluent by using high performance liquid chromatography.
Further, in step S1, the vortex oscillation extraction time is 5 to 15 minutes, and the rotation speed is 5000 to 10000 revolutions per minute.
Further, in step S5, the temperature of the nitrogen is 40-50 ℃.
Further, the chromatographic conditions are column: c18, specification of 4.6X 150mm,5 μm, flow rate: 1 mL/min; column temperature: 30 ℃; sample introduction volume: 10 μ L mobile phase: a is 0.1% formic acid water, B: and (3) acetonitrile.
The technical scheme of the invention has the following beneficial effects:
the invention can detect thirteen kinds of sulfanilamide drugs, including sulfacetamide, sulfapyridine, sulfamethazine, sulfamethoxazole, sulfamethazine, sulfamethoxypyridazine, metamitropyridazine, sulfamethoxypyridazine, sulfamethoxazole, sulfisoxazole, sulfamdimethoxypyridazine and sulfamtopyrazole.
The invention simplifies the steps of sample pretreatment, overcomes the problems of high cost, expensive instruments and low popularization rate of high performance liquid chromatography tandem mass spectrometry detection, provides more method choices for the detection of sulfonamides in fresh milk, and meets the requirements of different laboratories.
Drawings
FIG. 1 is a chromatogram of a mixed standard solution of 13 sulfonamides according to the invention.
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 creative effort, shall fall within the protection scope of the present invention.
Example one
The embodiment provides a high performance liquid chromatography detection method for sulfonamides in raw fresh milk, which comprises the following steps:
s1, weighing 5g of the uniformly mixed sample, and placing the sample in a centrifugal tube;
s2, adding 15mL of perchloric acid solution into the centrifugal tube, and performing vortex oscillation extraction;
s3, installing the MCX solid phase extraction column on a solid phase extraction device, sequentially passing through the column by using 3mL of methanol and 3mL of 0.1mol/L hydrochloric acid solution, taking the standby solution, and passing through the column
S4, sequentially leaching with 3mL of 0.1mol/L hydrochloric acid solution and 2mL of methanol, eluting with 5mL of 5% ammoniated methanol, and collecting eluent;
s5, drying the eluent by using nitrogen, redissolving the eluent by using 1mL of initial mobile phase, filtering the eluent by using a filter membrane, and determining the eluent by using high performance liquid chromatography.
In step S1, the vortex extraction time was 6 minutes and the rotation speed was 6000 rpm.
In step S5, the temperature of the nitrogen gas was 42 ℃.
The chromatographic conditions are chromatographic column: c18, specification of 4.6X 150mm,5 μm, flow rate: 1 mL/min; column temperature: 30 ℃; sample introduction volume: 10 μ L mobile phase: a is 0.1% formic acid water, B: and (3) acetonitrile.
Example two
The embodiment provides a high performance liquid chromatography detection method for sulfonamides in raw fresh milk, which comprises the following steps:
s1, weighing 5g of the uniformly mixed sample, and placing the sample in a centrifugal tube;
s2, adding 15mL of perchloric acid solution into the centrifugal tube, and performing vortex oscillation extraction;
s3, installing the MCX solid phase extraction column on a solid phase extraction device, sequentially passing through the column by using 3mL of methanol and 3mL of 0.1mol/L hydrochloric acid solution, taking the standby solution, and passing through the column
S4, sequentially leaching with 3mL of 0.1mol/L hydrochloric acid solution and 2mL of methanol, eluting with 5mL of 5% ammoniated methanol, and collecting eluent;
s5, drying the eluent by using nitrogen, redissolving the eluent by using 1mL of initial mobile phase, filtering the eluent by using a filter membrane, and determining the eluent by using high performance liquid chromatography.
In step S1, the vortex extraction time is 9 minutes and the rotation speed is 7000 rpm.
In step S5, the temperature of the nitrogen gas was 45 ℃.
The chromatographic conditions are chromatographic column: c18, specification of 4.6X 150mm,5 μm, flow rate: 1 mL/min; column temperature: 30 ℃; sample introduction volume: 10 μ L mobile phase: a is 0.1% formic acid water, B: and (3) acetonitrile.
EXAMPLE III
The embodiment provides a high performance liquid chromatography detection method for sulfonamides in raw fresh milk, which comprises the following steps:
s1, weighing 5g of the uniformly mixed sample, and placing the sample in a centrifugal tube;
s2, adding 15mL of perchloric acid solution into the centrifugal tube, and performing vortex oscillation extraction;
s3, installing the MCX solid phase extraction column on a solid phase extraction device, sequentially passing through the column by using 3mL of methanol and 3mL of 0.1mol/L hydrochloric acid solution, taking the standby solution, and passing through the column
S4, sequentially leaching with 3mL of 0.1mol/L hydrochloric acid solution and 2mL of methanol, eluting with 5mL of 5% ammoniated methanol, and collecting eluent;
s5, drying the eluent by using nitrogen, redissolving the eluent by using 1mL of initial mobile phase, filtering the eluent by using a filter membrane, and determining the eluent by using high performance liquid chromatography.
In step S1, vortex extraction time is 11 minutes, and rotation speed is 8000 rpm.
In step S5, the temperature of the nitrogen gas was 47 ℃.
The chromatographic conditions are chromatographic column: c18, specification of 4.6X 150mm,5 μm, flow rate: 1 mL/min; column temperature: 30 ℃; sample introduction volume: 10 μ L mobile phase: a is 0.1% formic acid water, B: and (3) acetonitrile.
Example four
The embodiment provides a high performance liquid chromatography detection method for sulfonamides in raw fresh milk, which comprises the following steps:
s1, weighing 5g of the uniformly mixed sample, and placing the sample in a centrifugal tube;
s2, adding 15mL of perchloric acid solution into the centrifugal tube, and performing vortex oscillation extraction;
s3, installing the MCX solid phase extraction column on a solid phase extraction device, sequentially passing through the column by using 3mL of methanol and 3mL of 0.1mol/L hydrochloric acid solution, taking the standby solution, and passing through the column
S4, sequentially leaching with 3mL of 0.1mol/L hydrochloric acid solution and 2mL of methanol, eluting with 5mL of 5% ammoniated methanol, and collecting eluent;
s5, drying the eluent by using nitrogen, redissolving the eluent by using 1mL of initial mobile phase, filtering the eluent by using a filter membrane, and determining the eluent by using high performance liquid chromatography.
In step S1, the vortex extraction time was 13 minutes and the rotation speed was 9000 rpm.
In step S5, the temperature of the nitrogen gas was 49 ℃.
The chromatographic conditions are chromatographic column: c18, specification of 4.6X 150mm,5 μm, flow rate: 1 mL/min; column temperature: 30 ℃; sample introduction volume: 10 μ L mobile phase: a is 0.1% formic acid water, B: and (3) acetonitrile.
Reagent
Comparison products: sulfonamides with purity of more than 97%; the methanol, the acetonitrile and the formic acid are all chromatographically pure;
preparing a standard stock solution: accurately weighing 13 sulfanilamide drug (sulfacetamide, sulfapyridine, sulfamethazine, sulfamethoxazole, sulfamethazine, sulfamethoxypyridazine, -metamiformis, -sulfamonomethoxine, sulfachloropyridazine, sulfamethoxazole, sulfisoxazole, sulfadimethoxine and sulfaphenazole) standard substances, diluting to 10mL with acetonitrile to obtain standard stock solution with the concentration of 1mg/mL, and storing at-20 ℃.
Preparing a standard intermediate solution: accurately transferring 1.0 mL of standard stock solution, placing in a 10mL volumetric flask, adding acetonitrile to constant volume to scale, preparing into mixed intermediate solution with concentration of 100 μ g/mL, and storing at-20 deg.C.
Chromatographic conditions
A chromatographic column: c18 (4.6X 150mm,5 μm), flow rate: 1 mL/min; column temperature: 30 ℃; sample introduction volume: 10 μ L mobile phase: a is 0.1% formic acid water, B: acetonitrile, gradient elution conditions see table below
Liquid phase mobile phase gradient elution condition
After pretreatment of the sample, the results are as follows:
linear range
The 13 sulfonamides present good linear relation in the concentration range of 0.01-5.00 mu g/mL, and the correlation coefficient R2The regression equation and the correlation coefficient of the 13 sulfonamides which are all more than or equal to 0.998 are shown in the following table,
regression equation and correlation coefficient of 13 sulfonamides
The chromatogram of the 13 sulfanilamide mixed standard solution is shown in figure 1, wherein 1-sulfanilamide is shown in the figure; 2-sulfapyridine; 3-sulfamethazine; 4-sulfamethoxazole; 5-sulfamethazine; 6-sulfamethoxypyridazine; 7-benzoylsulfonamide; 8-sulfamonomethoxine; 9-sulfachlorpyridazine; 10-sulfamethoxazole; 11-sulfisoxazole; 12-sulfadimethoxine; 13-sulfanilamide phenyl pyrazole.
Accuracy and precision experiment
Adding mixed standard solutions of 13 sulfonamides with different concentrations into a blank sample, wherein the adding amount is respectively 20 mug/kg, 50 mug/kg and 100 mug/kg, each adding standard amount simultaneously performs six parallel experiments, sample treatment is performed according to the experiment conditions, and the recovery rate and the relative standard deviation RSD are calculated by data processing, and the results are shown in the following table. Under the condition of four different scalar adding quantity levels, the recovery rate is in the range of 67.2% -81.2%, RSD is less than or equal to 4.1% (n =6), and the experimental result shows that the established method has good accuracy and precision.
Blank sample adding standard recovery rate and RSD (n =6) in fresh milk
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (4)
1. A high performance liquid chromatography detection method for sulfonamides in raw fresh milk is characterized by comprising the following steps: the method comprises the following steps:
s1, weighing 4-6 g of the uniformly mixed sample, and placing the sample in a centrifugal tube;
s2, adding 15mL of perchloric acid solution into the centrifugal tube, and performing vortex oscillation extraction;
s3, installing the MCX solid phase extraction column on a solid phase extraction device, sequentially passing through the column by using 3mL of methanol and 3mL of 0.1mol/L hydrochloric acid solution, taking the standby solution, and passing through the column
S4, sequentially leaching with 3mL of 0.1mol/L hydrochloric acid solution and 2mL of methanol, eluting with 5mL of 5% ammoniated methanol, and collecting eluent;
s5, drying the eluent by using nitrogen, redissolving the eluent by using 1mL of initial mobile phase, filtering the eluent by using a filter membrane, and determining the eluent by using high performance liquid chromatography.
2. The high performance liquid chromatography detection method of sulfonamides in fresh milk according to claim 1, wherein the method comprises: in step S1, the vortex oscillation extraction time is 5-15 minutes, and the rotation speed is 5000-10000 rpm.
3. The high performance liquid chromatography detection method of sulfonamides in fresh milk according to claim 1, wherein the method comprises: in step S5, the temperature of the nitrogen is 40-50 ℃.
4. The high performance liquid chromatography detection method of sulfonamides in fresh milk according to claim 1, wherein the method comprises: the chromatographic conditions are chromatographic column: c18, specification of 4.6X 150mm,5 μm, flow rate: 1 mL/min; column temperature: 30 ℃; sample introduction volume: 10 μ L mobile phase: a is 0.1% formic acid water, B: and (3) acetonitrile.
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