CN108107119B - Method for detecting chloramphenicol residues in aquatic products - Google Patents
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- 229960005091 chloramphenicol Drugs 0.000 title claims abstract description 50
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical group ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 41
- 238000001514 detection method Methods 0.000 claims abstract description 25
- 239000007791 liquid phase Substances 0.000 claims abstract description 5
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- 239000000047 product Substances 0.000 claims description 38
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 33
- 238000000605 extraction Methods 0.000 claims description 33
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 29
- 238000001035 drying Methods 0.000 claims description 26
- 239000007788 liquid Substances 0.000 claims description 21
- 239000002904 solvent Substances 0.000 claims description 21
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 20
- 238000009210 therapy by ultrasound Methods 0.000 claims description 20
- 239000007787 solid Substances 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 12
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- 238000005119 centrifugation Methods 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 10
- 238000005303 weighing Methods 0.000 claims description 8
- 239000007789 gas Substances 0.000 claims description 7
- 238000004949 mass spectrometry Methods 0.000 claims description 7
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 6
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
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- 238000000132 electrospray ionisation Methods 0.000 claims description 4
- 238000010828 elution Methods 0.000 claims description 4
- 150000002500 ions Chemical class 0.000 claims description 4
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 4
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- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 238000011084 recovery Methods 0.000 abstract description 12
- 238000000746 purification Methods 0.000 abstract description 10
- 230000000694 effects Effects 0.000 abstract description 4
- 238000002414 normal-phase solid-phase extraction Methods 0.000 abstract description 3
- 241001107116 Castanospermum australe Species 0.000 description 7
- 235000021279 black bean Nutrition 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 5
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- AYIRNRDRBQJXIF-NXEZZACHSA-N (-)-Florfenicol Chemical compound CS(=O)(=O)C1=CC=C([C@@H](O)[C@@H](CF)NC(=O)C(Cl)Cl)C=C1 AYIRNRDRBQJXIF-NXEZZACHSA-N 0.000 description 2
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- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 2
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- 229960003053 thiamphenicol Drugs 0.000 description 2
- OTVAEFIXJLOWRX-NXEZZACHSA-N thiamphenicol Chemical compound CS(=O)(=O)C1=CC=C([C@@H](O)[C@@H](CO)NC(=O)C(Cl)Cl)C=C1 OTVAEFIXJLOWRX-NXEZZACHSA-N 0.000 description 2
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Classifications
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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
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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
- G01N30/14—Preparation by elimination of some components
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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Abstract
The invention belongs to the technical field of aquatic product detection, and particularly relates to a method for detecting chloramphenicol residues in aquatic products, wherein a liquid phase color-tandem mass spectrometer is used for measuring the content of chloramphenicol in aquatic products, the linear range is 0.5-10ng/mL, the average recovery rate of addition is 92.3% -97.5%, and the relative standard deviation is 2.2% -6.4%. The method solves the problem of detecting the chloramphenicol in the deep-processed aquatic products, and simultaneously can meet the requirement of detecting the chloramphenicol residue in the fresh and live aquatic products. The HLB solid-phase extraction column purifies a sample, improves the purification effect and the recovery rate, reduces the pollution to a chromatographic column and the environment, not only meets the limit requirement of chloramphenicol residues in aquatic products at home and abroad, but also can be used for detecting and confirming the chloramphenicol in the processed aquatic products.
Description
Technical Field
The invention belongs to the technical field of aquatic product detection, and particularly relates to a method for detecting chloramphenicol residues in aquatic products.
Background
The aquatic products are various in variety and rich in nutrition, and are widely popular with consumers. In recent years, with the continuous expansion of the culture scale, diseases of aquaculture are increasingly serious. The phenomena of drug abuse such as blind drug use, human drug use, overdose and the like are common, which easily causes the worsening pollution of the culture environment and can also cause the residue of the drug in aquatic products, thereby affecting the health of eaters.
Chloramphenicol is a broad-spectrum antibacterial antibiotic, and has been widely used for bacterial disease treatment in aquaculture industry at home and abroad for a long time. Chloramphenicol in an edible aquatic product can be accumulated in a human body through a food chain, so that the health of the human body is threatened, the chloramphenicol has certain toxic and side effects on the hematopoietic function of the human body, and aplastic anemia, thrombocytopenia and the like can be caused (Wan translation, Deng Guo, Liuli, and the like. high performance liquid chromatography-tandem mass spectrometry is used for simultaneously measuring the residual quantities [ J ] of the chloramphenicol, thiamphenicol and florfenicol in the aquatic product, an analysis laboratory is 2013,32(5): 84-87). The use of chloramphenicol has been banned in many countries and regions such as European Union, and chloramphenicol cannot be detected in aquatic products specified in the limit of fishery drug residues in pollution-free food and aquatic products in China; no. 235 bulletin "maximum residual limit of veterinary drug in animal food" of Ministry of agriculture stipulates that chloramphenicol cannot be detected in animal food.
A plurality of methods are available for detecting chloramphenicol in aquatic products, one method is a gas-mass spectrometry technology, and the detection limit can reach 1 mug/kg. And GB/T20756-2006 determination of residual amounts of chloramphenicol, thiamphenicol and florfenicol in edible animal muscles, livers and aquatic products, and liquid chromatography-tandem mass spectrometry are adopted, the detection limit can reach 0.1 mu g/kg, and the content of chloramphenicol in the aquatic products is more than or equal to 0.1 mu g/kg and is considered to be detected. In addition, there are high performance liquid chromatography, enzyme linked immunosorbent assay, etc. Among these quantitative detection methods, the chromatography is internationally acknowledged, and mainly includes gas chromatography and liquid chromatography, which have the advantages of good repeatability, high sensitivity, more accurate result, and the disadvantages of long time for pretreatment, high cost, slow detection speed and very complex test process. The aim of the national center for quality supervision and inspection of aquatic products is to adopt an enzyme-linked immunosorbent assay to qualitatively and primarily screen a sample, then confirm and quantitatively analyze the sample by using a gas chromatograph and a GC-MS combined technology, and the two methods are combined, so that the labor intensity can be effectively reduced, and the accuracy of a detection result can be ensured.
Therefore, the method for establishing a sensitive, efficient and good-selectivity analysis method for detecting chloramphenicol in aquatic products has important practical significance.
Disclosure of Invention
In order to solve the problems, the invention provides a method for detecting chloramphenicol residues in aquatic products.
The method is realized by the following technical scheme:
a method for detecting chloramphenicol residues in an aquatic product comprises the following steps:
1) crushing: taking a product to be detected, and crushing the product for later use;
2) extraction: weighing the sample treated in the step 1), adding an extraction solvent with the mass 2 times that of the sample into a centrifugal tube, carrying out ultrasonic treatment, centrifuging, taking the supernatant, adding the extraction solvent into the precipitate for secondary extraction, centrifuging, combining the two supernatants, fixing the volume, taking 1/4-1/5 liquid, and carrying out rotary drying to obtain a solid 1; performing a matrix labeling experiment before extracting a sample, weighing 5g of the sample without chloramphenicol residues in a centrifuge tube, adding 500 mu L of chloramphenicol internal standard solution with the mass concentration of 10.0ng/mL and 10mL of extraction solvent, performing ultrasonic treatment, centrifuging, taking supernatant, adding the extraction solvent in the precipitate for re-extraction, centrifuging, combining the supernatant obtained twice, performing constant volume, taking 1/4-1/5 liquid, and performing rotary drying to obtain a solid 2;
3) purifying: adding clear water with the same mass as that before drying into a rotary drying bottle filled with the solid 1 and the solid 2, carrying out vortex mixing, pouring into a cylindrical funnel connected with an HLB column below, passing through the column, rinsing the rotary drying bottle with the clear water with the same volume after the solution is completely drained, passing all the washing liquid through the HLB column, eluting with 5mL ethyl acetate after the solution is completely drained, collecting the eluent into a 10mL centrifuge tube, drying with nitrogen, and fixing the volume to 1mL with pure water to obtain a purified liquid;
4) and (3) detection: detecting by liquid phase-mass spectrometry;
5) drawing a standard curve: respectively adding 5.0ng of chloramphenicol-D5 internal standard into 0, 0.5, 1.0, 2.0, 4.0, 8.0 and 10.0ng/mL gradient standard solutions, performing automatic sample injection analysis, and drawing a standard curve by taking the peak area ratio of a target substance to the corresponding internal standard as a ordinate and the concentration of the target substance as an abscissa.
Further, the extraction solvent is formed by mixing ethyl acetate, ammonia water and a sodium chloride solution according to a volume ratio of 22.5:1:2, wherein the mass fraction of the sodium chloride solution is 5-7%.
Further, the ultrasonic treatment is carried out for 5-10min under the ultrasonic frequency of 20-30KHz, the ultrasonic treatment is suspended for 5-7min and is placed in the environment with the temperature of 35-40 ℃, and the operation is repeated for 1 time.
Further, the centrifugation in the step 2) is performed for 5-10min at the rotating speed of 5500-6000 r/min.
Further, step 3) purification, the HLB column was activated with 5mL of methanol before use and 10mL of pure water.
Further, the detection in the step 4) is carried out, wherein the liquid phase detection conditions are that a Shim-PackX R-ODS chromatographic column is adopted, the column temperature is 40 ℃, the mobile phase A is water, the mobile phase B is methanol, the flow rate is 0.30mL/min, the sample injection amount is 5 mu L, and gradient elution is adopted.
Further, in the gradient elution, the volume fraction of water is 90% within 0-1.5min, the volume fraction of methanol is 10%, the volume fraction of water is 23% within 1.5-4.0min, the volume fraction of methanol is 77%, the volume fraction of water is 92% within 4.00-7.0min, and the volume fraction of methanol is 8%.
Further, step 4), detecting, wherein the mass spectrum condition is electrospray ionization (ESI); multiple Reaction Monitoring (MRM) negative ion mode; the electrospray voltage is-4500V; the gas curtain gas is 20 Ps; the auxiliary gas is 50 Psi; the declustering voltage is-80V, the collision voltage is-10V, the ion source temperature is 500 ℃, and the related parameters of the mass spectrum are shown in Table 1.
TABLE 1 Mass Spectrometry related parameters
The column used was a Shim-PackXR-ODS column (75 mm. times.2.0 mm, 5 μm); 4000QTrap mass/liquid chromatography/mass spectrometer (AB bio);
the content of the chloramphenicol standard substance is more than or equal to 98 percent (Dr. Ehrenstontorfer company); content of chloramphenicol-D5 internal standard substance is more than or equal to 98% (Dr. Ehrenstontorfer company); methanol, ethyl acetate and n-hexane as chromatographic purities (Merck, Germany); ammonia water is analytically pure (national pharmaceutical company); the experimental water was ultrapure water.
Accurately weighing a proper amount of standard chloramphenicol, dissolving with methanol to a constant volume, and preparing into a standard stock solution of 100 μ g/mL; accurately weighing appropriate amount of standard chloramphenicol-D5, dissolving with methanol to desired volume, making into 100 μ g/mL standard stock solution, and storing at-20 deg.C. An appropriate amount of 100 mug/mL chloramphenicol standard stock solution is removed, diluted by methanol to prepare a series of standard working solutions of 0, 0.5, 1.0, 2.0, 4.0, 8.0 and 10.0ng/mL respectively, and stored at 4 ℃ for later use.
Has the advantages that: through the analysis, the method establishes that the content of the chloramphenicol in the aquatic product is measured by using a liquid phase color-tandem mass spectrometer, the sample is processed, a mixed extraction solvent is adopted, ultrasonic treatment is carried out twice, the linear range of the method is 0.5-10ng/mL, the detection limit of the method is 0.1 mug/kg calculated according to 3 times of signal to noise ratio, and the quantitative limit of the method is 0.3 mug/kg calculated according to 10 times of signal to noise ratio.
The average recovery of 3 spiked levels of chloramphenicol (0.5, 1.0, and 5.0 μ g/kg) ranged from 92.3% to 97.5%, with a relative standard deviation of 2.2% to 6.4%.
The method solves the problem of detecting the chloramphenicol in the deep-processed aquatic products, and simultaneously can meet the requirement of detecting the chloramphenicol residue in the fresh and live aquatic products. The HLB solid-phase extraction column purifies a sample, improves the purification effect and the recovery rate, reduces the pollution to a chromatographic column and the environment, not only meets the limit requirement of chloramphenicol residues in aquatic products at home and abroad, but also can be used for detecting and confirming the chloramphenicol in the processed aquatic products.
Detailed Description
The following is a detailed description of the embodiments of the present invention, but the present invention is not limited to these embodiments, and any modifications or substitutions in the basic spirit of the embodiments are included in the scope of the present invention as claimed in the claims.
Example 1
A method for detecting chloramphenicol residues in an aquatic product comprises the following steps:
1) crushing: taking the canned crucian with black beans as a product to be detected, and crushing the canned crucian with black beans for later use;
2) extraction: weighing 5g of the sample treated in the step 1), adding 10ml of extraction solvent into a centrifuge tube, carrying out ultrasonic treatment and centrifugation, taking the supernatant, adding the extraction solvent into the precipitate for secondary extraction, carrying out centrifugation, combining the two supernatants, fixing the volume, taking 5ml of liquid, and carrying out rotary drying to obtain a solid 1;
3) purifying: adding clear water with the same mass as that before drying into a rotary drying bottle filled with the solid 1, carrying out vortex mixing, pouring into a cylindrical funnel connected with an HLB column below, passing through the column, rinsing the rotary drying bottle with the same volume of clear water after the solution is completely drained, passing all the washing liquid through the HLB column, eluting with 5mL ethyl acetate after the solution is completely drained, collecting the eluent into a 10mL centrifugal tube, drying with nitrogen, and carrying out constant volume treatment to 1mL with pure water to obtain a purified liquid;
4) and (3) detection: detecting by liquid phase-mass spectrometry;
the extraction solvent is formed by mixing ethyl acetate, ammonia water and a sodium chloride solution according to the volume ratio of 22.5:1:2, wherein the mass fraction of the sodium chloride solution is 5%.
The ultrasonic treatment is carried out for 5min under the ultrasonic frequency of 20KHz, the ultrasonic treatment is suspended for 5min and is placed in the environment with the temperature of 35 ℃, and the operation is repeated for 1 time.
The centrifugation in the step 2) is carried out for 5min at the rotating speed of 5500 r/min.
Step 3) purification, HLB column activated with 5mL methanol before use and 10mL pure water.
Experimental example 2
A method for detecting chloramphenicol residues in an aquatic product comprises the following steps:
1) crushing: taking the canned crucian with black beans as a product to be detected, and crushing the canned crucian with black beans for later use;
2) extraction: weighing 5g of the sample treated in the step 1), adding 10ml of extraction solvent into a centrifuge tube, carrying out ultrasonic treatment and centrifugation, taking the supernatant, adding the extraction solvent into the precipitate for secondary extraction, carrying out centrifugation, combining the two supernatants, fixing the volume, taking 5ml of liquid, and carrying out rotary drying to obtain a solid 1;
3) purifying: adding clear water with the same mass as that before drying into a rotary drying bottle filled with the solid 1, carrying out vortex mixing, pouring into a cylindrical funnel connected with an HLB column below, passing through the column, rinsing the rotary drying bottle with the same volume of clear water after the solution is completely drained, passing all the washing liquid through the HLB column, eluting with 5mL ethyl acetate after the solution is completely drained, collecting the eluent into a 10mL centrifugal tube, drying with nitrogen, and carrying out constant volume treatment to 1mL with pure water to obtain a purified liquid;
4) and (3) detection: and detecting by liquid phase-mass spectrometry.
The extraction solvent is formed by mixing ethyl acetate, ammonia water and a sodium chloride solution according to the volume ratio of 22.5:1:2, wherein the mass fraction of the sodium chloride solution is 6%.
The ultrasonic treatment is carried out for 7min under the ultrasonic frequency of 25KHz, the ultrasonic treatment is suspended for 6min and placed in the environment with the temperature of 37 ℃, and the operation is repeated for 1 time.
The centrifugation in the step 2) is carried out for 7min at the rotating speed of 5800 r/min.
Step 3) purification, HLB column activated with 5mL methanol before use and 10mL pure water.
Example 3
A method for detecting chloramphenicol residues in an aquatic product comprises the following steps:
1) crushing: taking the canned crucian with black beans as a product to be detected, and crushing the canned crucian with black beans for later use;
2) extraction: weighing 5g of the sample treated in the step 1), adding 10ml of extraction solvent into a centrifuge tube, carrying out ultrasonic treatment and centrifugation, taking the supernatant, adding the extraction solvent into the precipitate for secondary extraction, carrying out centrifugation, combining the two supernatants, fixing the volume, taking 5ml of liquid, and carrying out rotary drying to obtain a solid 1;
3) purifying: adding clear water with the same mass as that before drying into a rotary drying bottle filled with the solid 1, carrying out vortex mixing, pouring into a cylindrical funnel connected with an HLB column below, passing through the column, rinsing the rotary drying bottle with the same volume of clear water after the solution is completely drained, passing all the washing liquid through the HLB column, eluting with 5mL ethyl acetate after the solution is completely drained, collecting the eluent into a 10mL centrifugal tube, drying with nitrogen, and carrying out constant volume treatment to 1mL with pure water to obtain a purified liquid;
4) and (3) detection: and detecting by liquid phase-mass spectrometry.
The extraction solvent is formed by mixing ethyl acetate, ammonia water and a sodium chloride solution according to a volume ratio of 22.5:1:2, wherein the mass fraction of the sodium chloride solution is 7%.
The ultrasonic treatment is carried out for 10min under the ultrasonic frequency of 30KHz, the ultrasonic treatment is suspended for 7min and is placed in the environment of 40 ℃, and the operation is repeated for 1 time.
And 2) centrifuging at the rotating speed of 6000r/min for 10 min.
Step 3) purification, HLB column activated with 5mL methanol before use and 10mL pure water.
Examples 4-8 the extraction solvent is shown in Table 2, and the other steps are the same as in example 2.
TABLE 2 extraction solvents used in examples 4-8
Example 9
The ultrasonic treatment described in example 9 was carried out at an ultrasonic frequency of 25KHz for 7min, and the other detection steps were the same as in example 2.
Examples of the experiments
1. Linearity, detection limit and quantification limit of the method
In the measurement under the instrument conditions described in the specification, a standard curve is drawn by taking the concentration of chloramphenicol and its internal standard substance as the abscissa and the ratio of its corresponding peak areas as the ordinate, to obtain a linear equation, and the detection limit and the quantitative limit of the method are shown in table 3.
TABLE 3 Linear range, Standard Curve, detection Limit and quantitation Limit for Chloramphenicol residue detection in aquatic products
The linear range of the method is 0.5-10ng/mL, the detection limit of the method is 0.1 mug/kg calculated according to 3 times of signal to noise ratio, and the quantification limit of the method is 0.3 mug/kg calculated according to 10 times of signal to noise ratio.
2. Recovery and precision of the process
The purification according to the purification method of examples 1-9, 3 additive recovery experiments were carried out on canned black bean carp without chloramphenicol residues, and the average recovery of the chloramphenicol at 3 spiked levels (0.5, 1.0 and 5.0. mu.g/kg) was 92.3% -97.5%, with a relative standard deviation of 2.2% -6.4%.
TABLE 4 recovery of chloramphenicol addition and relative standard deviation in the samples
3. Other sample detection
The same extraction and purification method is adopted, and the method has good addition recovery rate due to the content of chloramphenicol in crayfish, live fresh water crab, fish meal and dried fish. The linear range of the method is 0.5-10ng/mL, the detection limit of the method is 0.1 mug/kg calculated according to 3 times of signal to noise ratio, and the quantification limit of the method is 0.3 mug/kg calculated according to 10 times of signal to noise ratio. The average recovery of 3 spiked levels of chloramphenicol (0.5, 1.0, and 5.0. mu.g/kg) ranged from 93.5% to 98.5%, with a relative standard deviation of 2.7% to 7.4%.
Through the analysis, the method establishes that the content of the chloramphenicol in the aquatic product is measured by using a liquid phase color-tandem mass spectrometer, the sample is processed, a mixed extraction solvent is adopted, ultrasonic treatment is carried out twice, the linear range of the method is 0.5-10ng/mL, the detection limit of the method is 0.1 mug/kg calculated according to 3 times of signal to noise ratio, and the quantitative limit of the method is 0.3 mug/kg calculated according to 10 times of signal to noise ratio.
The average recovery of 3 spiked levels of chloramphenicol (0.5, 1.0, and 5.0 μ g/kg) ranged from 92.3% to 97.5%, with a relative standard deviation of 2.2% to 6.4%.
The method solves the problem of detecting the chloramphenicol in the deep-processed aquatic products, and simultaneously can meet the requirement of detecting the chloramphenicol residue in the fresh and live aquatic products. The HLB solid-phase extraction column purifies a sample, improves the purification effect and the recovery rate, reduces the pollution to a chromatographic column and the environment, not only meets the limit requirement of chloramphenicol residues in aquatic products at home and abroad, but also can be used for detecting and confirming the chloramphenicol in the processed aquatic products.
Claims (3)
1. A method for detecting chloramphenicol residues in an aquatic product is characterized by comprising the following steps:
1) crushing: taking a product to be detected, and crushing the product for later use;
2) extraction: weighing the sample treated in the step 1), adding an extraction solvent with the mass 2 times that of the sample into a centrifugal tube, carrying out ultrasonic treatment, centrifuging, taking the supernatant, adding the extraction solvent into the precipitate for secondary extraction, centrifuging, combining the two supernatants, fixing the volume, taking 1/4-1/5 liquid, and carrying out rotary drying to obtain a solid 1; the ultrasonic treatment is carried out for 5-10min under the ultrasonic frequency of 20-30KHz, the ultrasonic treatment is suspended for 5-7min, and the ultrasonic treatment is placed in the environment with the temperature of 35-40 ℃ and repeated for 1 time; the centrifugation is carried out for 5-10min at the rotating speed of 5500-6000 r/min;
3) purifying: adding clear water with the same mass as that before drying into a rotary drying bottle filled with the solid 1, carrying out vortex mixing, pouring into a cylindrical funnel connected with an HLB column below, passing through the column, rinsing the rotary drying bottle with the same volume of clear water after the solution is completely drained, passing all the washing liquid through the HLB column, eluting with 5mL ethyl acetate after the solution is completely drained, collecting the eluent into a 10mL centrifugal tube, drying with nitrogen, and carrying out constant volume treatment to 1mL with pure water to obtain a purified liquid; the HLB column was activated with 5mL methanol and then 10mL pure water before use;
4) and (3) detection: detecting by liquid phase-mass spectrometry; the detection conditions of the liquid phase are that a Shim-Pack XR-ODS chromatographic column is adopted, the column temperature is 40 ℃, the mobile phase A is water, the mobile phase B is methanol, the flow rate is 0.30mL/min, the sample injection amount is 5 mu L, and gradient elution is adopted;
the extraction solvent is formed by mixing ethyl acetate, ammonia water and a sodium chloride solution according to a volume ratio of 22.5:1:2, wherein the mass fraction of the sodium chloride solution is 5-7%.
2. The method of claim 1, wherein the gradient elution comprises 90% water, 10% methanol, 23% water, 77% methanol, 92% water, and 8% methanol within 0-1.5 min.
3. The method of claim 1, wherein the mass spectrometric conditions of step 4) are electrospray ionization (ESI); multiple Reaction Monitoring (MRM) negative ion mode; the electrospray voltage is-4500V; the gas curtain gas is 20 Ps; the auxiliary gas is 50 Psi; the declustering voltage is-80V, the collision voltage is-10V, and the ion source temperature is 500 ℃.
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