CN102253148A - Method for detecting residues of five kinds of sulfa veterinary medicines in animal-based functional food - Google Patents
Method for detecting residues of five kinds of sulfa veterinary medicines in animal-based functional food Download PDFInfo
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- CN102253148A CN102253148A CN2010106049371A CN201010604937A CN102253148A CN 102253148 A CN102253148 A CN 102253148A CN 2010106049371 A CN2010106049371 A CN 2010106049371A CN 201010604937 A CN201010604937 A CN 201010604937A CN 102253148 A CN102253148 A CN 102253148A
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Abstract
The invention discloses a method for detecting residues of five kinds of sulfa veterinary medicines in an animal-based functional food. The method comprises the following steps of: (1) extracting a sample: weighing a proper amount of sample, extracting for 2 to 4 times by using acetonitrile as a solvent in a vortex oscillation way, combining acetonitrile solution and placing in a liquid separation funnel for later use; (2) purifying the sample: extracting the acetonitrile solution obtained in the step (1) by using normal hexane, concentrating the acetonitrile solution on the bottom layer and dissolving by using 10 percent acetonitrile water, and filtering; and (3) testing the residual amount of five kinds of sulfa veterinary medicines in the sample by using a high performance liquid chromatography-mass spectrometry-mass spectrometry (HPLC-MS-MS) system. By the test method, the separability of the five kinds of the sulfa veterinary medicine residues in the functional food is high, the sensitivity is high, and the selectivity and the specificity are high; matrix interference and false positive can be eliminated effectively, and the minimum detection concentration is low; furthermore, in a sample treatment process, the degressive amount of acetonitrile is used for extracting, so that the recovery rate of targets is guaranteed, the using amount of organic solvents is reduced, the emission of waste liquid is reduced and the detection cost is saved.
Description
Technical field
The present invention relates to a kind of animal derived functional food veterinary drug residue detection method, particularly the detection method of sulfamido residue of veterinary drug.
Background technology
Reform and opening-up is over 30 years, the functional food industry more and more presents vitality as the sunrise industry of 21 century, functional food has also been come into broad masses of the people's life gradually, has brought into play vital role to promoting aspects such as body health, breakthrough inferior health, dispelling disease.
Animal derived functional food as: preparation such as the production of calcium powder, fish oil and honey etc. is many to be raw material with herding, aquatic livestock and semi-manufacture, and sulfa drugs is cheap because of it, disease resisting effect good, be widely used in the bacterial infection disease of prevention and treatment domestic animal and aquatic products, it is residual that this just might produce multiple sulfamido.The sulfamido residue of veterinary drug mainly comprises three kinds to the direct toxic action of human body: 1. to the influence of human gi-tract microorganism, in recent years many both at home and abroad researchs are thought, the animal product that the antibiotic property medicament residue is arranged, can human intestine's normal flora be exerted an adverse impact, the part sensitive bacteria is suppressed or is killed, and makes that normal microbial environment is destroyed, and some conditionality pathogenic bacteria (as Escherichia coli etc.) may breed in a large number, or external pathogen is invaded, the infringement human health.2. cause the chemical sproof increase of human pathogen, use the feed contain antibacterials such as microbiotic after, antibacterials are the residual medicine that may make human pathogen contact these low concentrations for a long time in animal product, thereby produces drug resistance.In October, 1997 is " microbiotic is applied to behind the food animal human medical treatment be influenced " international symposium first, the expert is consistent to think that food animal long-term low dose use microbiotic can increase drug-fast bacteria, and the drug resistant gene of bacterium can be in the crowd transmits between bacterium and the ecosystem produce in bacterium, the fauna the bacterium mutually, can cause pathogenic bacteria to produce drug resistance thus and causes the failure of human and animal's infectious diseases treatment.3. directly cause human diseases, have many antibiotic residues can directly cause human body to produce disease.Sulfa drugs is residual in milk, can not only suppress the fermentation of milk product, but also may make the people produce allergy and allergic reaction.Therefore, the sulfamido total amount must not surpass 100 μ g/kg in the international food code council (CAC), European Union and U.S.'s regulation food and the feed, and the residual limit standard of fishing medicine (NY5070-2002) has also been stipulated same limit value in the aquatic products in the nuisanceless aquatic products of China.Stipulated the LC-MS-MS method of 16 kinds of sulfa drug residue amounts in the livestock and poultry meat among the same GB/T 20759-2006, various sulfa drugs method detection limits are as follows: sulphadiazine, sulfamethyldiazine are 5.0 μ g/kg; Sulfamethazine is 20.0 μ g/kg; The arnosulfan yl pyrimidines is 10.0 μ g/kg; And the present temporary endless standard of sulfaquinoxaline.Be defined among the GB/T221316-2007 that 23 kinds of residual quantitative limit of sulfonamide are 50 μ g/kg in animal liver, kidney, musculature and the milk; 23 kinds of sulfonamides are residual in aquatic products quantitatively is limited to 10 μ g/kg.
At present, the detection method of sulfamido residue of veterinary drug amount mainly contains thin-layered chromatography, capillary electrophoresis, immunoassay, high performance liquid chromatography and ultraviolet, fluorescent technique, electrochemical measuring technique coupling etc. in the animal derived food.These methods all have the concentration limit height, can not effectively get rid of matrix interference, occur shortcomings such as false positive and organic solvent use amount are big easily.Composition more complicated in the functional food contains multiple heterogeneity mostly, and this brings difficulty just for the sulfamido residue of veterinary drug that detects wherein.At present, the sulfamido detection of veterinary drugs in food does not have bibliographical information as yet in the functional food.
Summary of the invention
In order to overcome the deficiency of prior art, the method that the purpose of this invention is to provide sulphadiazine (SDZ) in a kind of effectively detection functionality food, sulfamethyldiazine (SMR), sulfamethazine (SDM), sulfaquinoxaline (SQX) and five kinds of sulfamido residues of veterinary drug of madribon (SDT), to solve concentration limit height in the prior art, can not effectively get rid of matrix interference, occur shortcomings such as false positive and organic solvent use amount are big easily.
The present invention is achieved by the following technical solutions: the HPLC-MS-MS detection method of five kinds of sulfamido residues of veterinary drug in a kind of functional food comprises the steps:
(1) sample extraction: take by weighing an amount of sample, acetonitrile merges acetonitrile liquid as solvent vortex vibration extraction 2-4 time, puts in the separating funnel stand-by; Sample weighting amount is 0.5 gram-10.0 grams, and each solvent extraction acetonitrile consumption is 5mL-100mL.
(2) sample purification: with gained acetonitrile liquid in the n-hexane extraction step (1), bottom acetonitrile liquid is separated with 10% acetonitrile is water-soluble after concentrating, and filters;
(3) take five kinds of sulfamido residue of veterinary drug amounts in the HPLC-MS-MS system measurement sample.
Described five kinds of sulfamido veterinary drugs comprise sulphadiazine, sulfamethyldiazine, sulfamethazine, sulfaquinoxaline and madribon.
Described step (3) is taked five kinds of sulfamido residue of veterinary drug amounts in the HPLC-MS-MS system measurement sample, and testing conditions is:
Chromatographic condition:
Moving phase is:
Acetonitrile 0-90 parts by volume
0.05%-0.2% aqueous formic acid 5-95 parts by volume
Flow velocity is 0.3mL/min; Sample size: 5 μ L, column temperature: 35 ℃;
The mass spectrum condition:
The ionization mode is an electron spray ionisation, positive ion (ESI+);
Capillary voltage: 3.50kV;
Ion source temperature: 110 ℃;
Taper hole gas: nitrogen, 50L/Hr;
Desolventizing gas: nitrogen, 500L/Hr, 400 ℃ of temperature;
Collision gas: argon gas, 0.14mL/min.
Described moving phase aqueous formic acid is ammonium acetate solution or ammonium formate solution, and concentration is 0.05mM-10mM.
Described sample is the functional food that animal derived raw material is made, and for example is the class functional food of replenishing the calcium that raw material is made with bone meal, is the functional food that raw material is made with the egg yolk powder, perhaps is functional food of making of raw material etc. with honey.
The invention has the beneficial effects as follows: assay method of the present invention, to sulphadiazine in the functional food made from animal derived raw material, sulfamethyldiazine, sulfamethazine, the separation property of sulfaquinoxaline and five kinds of sulfamido residues of veterinary drug of madribon is good, highly sensitive, selectivity and specificity are good, can effectively get rid of matrix interference and false positive, concentration limit is low, reach 20ng/ml, and in the sample preparation process, adopt the decrement acetonitrile to extract, promptly guaranteed the recovery of object, also reduce the use amount of organic solvent, reduced discharging of waste liquid, saved the detection cost.
Compare with additive method, HPLC-MS-MS (liquid chromatography-mass spectrography/mass spectrum) systems technology is highly sensitive, selectivity and specificity are good, not only can carry out qualitative affirmation to the multiple medicines thing of low concentration in the sample is residual, overcoming sample substrate disturbs, and can effectively get rid of false positive, carry out residue accurate quantitative analysis in the animal derived functional food of making for raw material.The present invention has set up the HPLC-MS-MS technology that is applicable to that the sulfamido residue of veterinary drug is measured in the functional food, be effectively to fulfil one of important content of HACCP management system in the animal derived functional food production run, it is the powerful measure of security function food security, also be the healthy important assurances of consumers in general, for functional food veterinary drug residue monitoring later on provides the otherwise effective technique support.
Description of drawings
Fig. 1 is the TIC spectrogram of five kinds of sulfamido veterinary drugs of the present invention.
Embodiment
Below will the invention will be further described by example, these descriptions will help those skilled in the art further to understand the present invention, but not limit the present invention in any form.
1. sample extraction: get the sample that mixes, take by weighing 2g (being accurate to 0.01g) sample and be loaded in the tool plug spiral cover tetrafluoroethene test tube, add the 30mL acetonitrile, vortex vibration 1min, 30min is extracted in vibration, in the centrifugal 5min of 10000r/min, acetonitrile layer is moved in the 150mL separating funnel.Add the 20mL acetonitrile again to sediment and shake up, 30min is extracted in vibration, in the centrifugal 5min of 10000r/min, merges the acetonitrile extract.The centrifuged deposit thing adds the 10mL acetonitrile again, and is centrifugal according to the aforesaid operations vibration, merges the acetonitrile extract, to be clean.
2. sample purification: add the 25mL normal hexane in the extract, jolting 5min moves into the bottom acetonitrile solution in the 150mL heart bottle, adds the 15mL isopropyl alcohol, does near with rotary evaporator reduction vaporization in 45 ℃ of water-baths.Add the 5mL acetonitrile in the heart bottle, ultrasonic 30s dissolved residue moves into lysate in the 15mL centrifuge tube; Add the 3mL acetonitrile again in the heart bottle, ultrasonic 30s merges lysate.Lysate nitrogen on Nitrogen evaporator dries up.The dissolving of 10% acetonitrile solution, whirlpool 1min crosses 0.22 μ m miillpore filter, feed flow phase chromatography-mass spectroscopy/mass spectroscopy.
3. chromatogram mass spectrum condition: flow velocity is 0.3mL/min; Sample size: 5 μ L, column temperature: 35 ℃, moving phase and condition of gradient elution see Table 1.The MS/MS condition determination is: the ionization mode is an electron spray ionisation, positive ion (ESI+); Capillary voltage: 3.50kV; Ion source temperature: 110 ℃; Taper hole gas: nitrogen, 50L/Hr; Desolventizing gas: nitrogen, 500L/Hr, 400 ℃ of temperature; Collision gas: argon gas, 0.14mL/min.Experimentize with multiple-reaction monitoring (MRM) pattern.All cpds ion pair and voltage see Table 2.
Table 1 moving phase and condition of gradient elution
The ion pair of table 2 all cpds and voltage
*Be used for quantitatively
The equation of linear regression of 5 kinds of sulfamido veterinary drugs and the coefficient of determination in table 3 functional food
The different concentration recovery and the repeated results of adding of five kinds of sulfamido veterinary drugs of table 4
Claims (5)
1. the detection method of five kinds of sulfamido residues of veterinary drug in the animal derived functional food is characterized in that, comprises the steps:
(1) sample extraction: take by weighing sample, acetonitrile merges acetonitrile liquid as solvent vortex vibration extraction 2-4 time, puts in the separating funnel stand-by;
(2) sample purification: with gained acetonitrile liquid in the n-hexane extraction step (1), bottom acetonitrile liquid is separated with 10% acetonitrile is water-soluble after concentrating, and filters;
(3) take five kinds of sulfamido residue of veterinary drug amounts in the HPLC-MS-MS system measurement sample.
2. detection method according to claim 1 is characterized in that, described five kinds of sulfamido veterinary drugs comprise sulphadiazine, sulfamethyldiazine, sulfamethazine, sulfaquinoxaline and madribon.
3. detection method according to claim 1 is characterized in that, described step (3) is taked five kinds of sulfamido residue of veterinary drug amounts in the HPLC-MS-MS system measurement sample, and testing conditions is:
Chromatographic condition:
Moving phase is:
Acetonitrile 0-90 parts by volume
0.05%-0.2% aqueous formic acid 5-95 parts by volume
Flow velocity is 0.3mL/min; Sample size: 5 μ L, column temperature: 35 ℃;
The mass spectrum condition:
The ionization mode is an electron spray ionisation, positive ion (ESI+);
Capillary voltage: 3.50kV;
Ion source temperature: 110 ℃;
Taper hole gas: nitrogen, 50L/Hr;
Desolventizing gas: nitrogen, 500L/Hr, 400 ℃ of temperature;
Collision gas: argon gas, 0.14mL/min.
4. detection method according to claim 3 is characterized in that, described aqueous formic acid is ammonium acetate solution or ammonium formate solution, and concentration is 0.05mM-10mM.
5. detection method according to claim 1 is characterized in that, described step (1) sample weighting amount is 0.5 gram-10.0 grams, and each solvent extraction acetonitrile consumption is 5mL-100mL.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104931610A (en) * | 2015-05-28 | 2015-09-23 | 衢州出入境检验检疫局综合技术服务中心 | Method for simultaneously measuring 16 sulfonamides in beewax by high performance liquid chromatography tandem mass spectrometry |
| CN105891389A (en) * | 2016-04-08 | 2016-08-24 | 广东省农业科学院农产品公共监测中心 | Pretreatment method for improving accuracy of measuring sulfonamide residues in meat |
| CN106018602A (en) * | 2016-05-23 | 2016-10-12 | 青岛蓥康食品检测技术服务有限公司 | Compound detection reagent for detecting sulfonamide compound and detection method thereof |
| CN106770863A (en) * | 2015-11-19 | 2017-05-31 | 内蒙古蒙牛乳业（集团）股份有限公司 | The method of remains of pesticide content in detection liquid diary product |
| CN107202837A (en) * | 2017-04-12 | 2017-09-26 | 华南农业大学 | A kind of analysis method for being used to detect animal muscle veterinary drug residue thing |
| CN108181403A (en) * | 2018-01-31 | 2018-06-19 | 安徽泓顺源生物科技有限公司 | A kind of efficient liquid-phase chromatography method of rapidly quantifying honey quality |
| CN111340642A (en) * | 2020-03-18 | 2020-06-26 | 浙江省动物疫病预防控制中心 | Antibacterial drug use reduction measurement and calculation method in livestock and poultry breeding process |
| CN112526024A (en) * | 2020-12-08 | 2021-03-19 | 河海大学 | Method for detecting sulfonamide antibiotics and acetylated metabolites thereof in aquatic products |
-
2010
- 2010-12-24 CN CN2010106049371A patent/CN102253148A/en active Pending
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| MARCELA SEIFRTOVÁ，ET AL: "An overview of analytical methodologies for the determination of antibiotics in environmental waters", 《ANALYTICA CHIMICA ACTA》 * |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104931610A (en) * | 2015-05-28 | 2015-09-23 | 衢州出入境检验检疫局综合技术服务中心 | Method for simultaneously measuring 16 sulfonamides in beewax by high performance liquid chromatography tandem mass spectrometry |
| CN106770863A (en) * | 2015-11-19 | 2017-05-31 | 内蒙古蒙牛乳业（集团）股份有限公司 | The method of remains of pesticide content in detection liquid diary product |
| CN106770863B (en) * | 2015-11-19 | 2018-07-13 | 内蒙古蒙牛乳业（集团）股份有限公司 | The method for detecting remains of pesticide content in liquid diary product |
| CN105891389A (en) * | 2016-04-08 | 2016-08-24 | 广东省农业科学院农产品公共监测中心 | Pretreatment method for improving accuracy of measuring sulfonamide residues in meat |
| CN106018602A (en) * | 2016-05-23 | 2016-10-12 | 青岛蓥康食品检测技术服务有限公司 | Compound detection reagent for detecting sulfonamide compound and detection method thereof |
| CN106018602B (en) * | 2016-05-23 | 2017-05-31 | 青岛蓥康食品检测技术服务有限公司 | Combine detection reagent and its detection method for detecting sulfonamides compound |
| CN107202837A (en) * | 2017-04-12 | 2017-09-26 | 华南农业大学 | A kind of analysis method for being used to detect animal muscle veterinary drug residue thing |
| CN108181403A (en) * | 2018-01-31 | 2018-06-19 | 安徽泓顺源生物科技有限公司 | A kind of efficient liquid-phase chromatography method of rapidly quantifying honey quality |
| CN111340642A (en) * | 2020-03-18 | 2020-06-26 | 浙江省动物疫病预防控制中心 | Antibacterial drug use reduction measurement and calculation method in livestock and poultry breeding process |
| CN111340642B (en) * | 2020-03-18 | 2023-06-30 | 浙江省动物疫病预防控制中心 | Method for measuring and calculating use reduction of antibacterial agent in livestock and poultry breeding process |
| CN112526024A (en) * | 2020-12-08 | 2021-03-19 | 河海大学 | Method for detecting sulfonamide antibiotics and acetylated metabolites thereof in aquatic products |
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Application publication date: 20111123 |