CN107632090A - A kind of method of multiple antibiotic residues in quick detection feces of livestock and poultry - Google Patents
A kind of method of multiple antibiotic residues in quick detection feces of livestock and poultry Download PDFInfo
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Abstract
The invention provides a kind of method of multiple antibiotic residues in quick detection feces of livestock and poultry, using improved QuEchERs technologies as sample pre-treatments means, using the highly sensitive triple level Four bar linear ion hydrazine tandem mass spectrometers of Waters Acquity UPLC AB5500 liquid chromatograies as detection means, establish the rapid screening and confirmation technology for detecting a variety of common antibiotics medicament residues in feces of livestock and poultry simultaneously, it is short with detection cycle, the advantages that detection efficiency is high, good reliability.
Description
Technical field
The present invention disclose is related to antibiotic detect technical field, more particularly in a kind of quick detection feces of livestock and poultry it is a variety of
The method of antibiotic residue.
Background technology
The development of intensive animal husbandry causes veterinary drug largely to use, Tetracyclines, macrolides, sulfamido, quinolone
The antibiotic medicines such as class are because it has diseases prevention, cures the disease, promotes growth of animal, improves food conversion ratio and other effects and raised in milk animal fowl
Support extensive use in industry.Said medicine is generally difficult to be absorbed by animal stomach, and about 30%~90% can pass through animal dung with prototype
Just it is directly released into environment.Because livestock and poultry breeding industry is scattered in around China rural area and cities and towns mostly, feces of livestock and poultry fail into
Row effectively disposal, causes a large amount of residual animal medicines to enter in environment, may bring bio-toxicity or cause pathogenic bacteria to develop immunity to drugs
The environment such as gene, ecological risk, and harm can be produced to population health.
Residual animal medicine can be entered in soil and groundwater environment by the use of breeding wastewater and excrement, turned into and received much concern
Novel environmental organic pollution.Barnes etc. (2008) has carried out sampling detection to underground water at 18, U.S. state 47,
Organic pollution materials are detected in 81% water sample, wherein, because polluter caused by livestock and poultry medication accounts for total detected level
23%.China produces as veterinary drug and using big country, feces of livestock and poultry veterinary drug residue correlation study is not also very comprehensive, pertinent literature
Report it is less, and the analysis determining method for establishing a variety of residues of veterinary drug in feces of livestock and poultry be carry out residue of veterinary drug migrate in the environment
Accumulation, and the basis of Evaluation Environment risk.
An important means being monitored to the residue of veterinary drug in feces of livestock and poultry is exactly that foundation can quickly and accurately be entered
The method of row residual analyte detection.At present, the medicament residue detection method reported both at home and abroad mainly has liquid chromatography (HPLC), hair
Cons electrophoresis-MS (CE-MS), Liquid Chromatography-tandem Mass method (LC-MS/MS), ELISA etc..I
State's detection of veterinary drugs in food technology is quickly grown, and detection technique research is more, such as：β-interior in SN/T3256-2012 outlets milk
In the ROSA methods of amide-type and tetracycline medication residual quick detection, SN/T1777.2-2007 animal derived foods in big ring
Esters antibiotic residue assay method part 2-high performance liquid chromatography tandem mass spectrum method, GB29692-2013 food securities country
QNS measure-high performance liquid chromatography of residual, the animal derived foods of GB/T 21316-2007 more in standard-milk
The measure of sulfa drug residue in product.In existing HPLC-MS/MS methods, some methods are that pretreatment process is complex,
Some methods are that the scope of application is small, the detection limit for height of some methods, can not meet newest limitation requirement.Although LC-MS/MS exists
The application of multi-medicament residue detection is current study hotspot, but can only realize the inspection of single classification antibiotic in the prior art
Survey, detected while plurality of classes antibiotic can not be realized, detection cycle length be present, the problems such as detection efficiency is low.
The content of the invention
In consideration of it, the present disclosure provides a kind of method of multiple antibiotic residues in quick detection feces of livestock and poultry, with
At least solve to detect the problems such as species is single, detection cycle is long, detection efficiency is low present in current detection technique.
Technical scheme provided by the invention, specifically, in a kind of quick detection feces of livestock and poultry multiple antibiotic residues side
Method, it is characterised in that comprise the following steps：
1) internal standard compound and various antibiotic samples are configured to standard reserving solution；
2) using standard reserving solution configuration hybrid standard working solution；
3) parameter setting of liquid chromatogram-triple level Four bar-linear ion hydrazine tandem mass spectrometer is carried out；
4) internal standard compound and each antibiotic mark are generated in liquid chromatogram-triple level Four bar-linear ion hydrazine tandem mass spectrometer
Directrix curve model；
5) feces of livestock and poultry to be detected is subjected to sample pre-treatments, it is standby；
6) liquid chromatogram-triple level Four bar-linear ion hydrazine tandem mass spectrum instrument parameter set according to step 3), passes through step
It is rapid 4) in the internal standard compound that is generated in liquid chromatogram-triple level Four bar-linear ion hydrazine tandem mass spectrometer and each antibiotic standard
Curve model, the feces of livestock and poultry to be detected after sample pre-treatments will be carried out in step 5) in liquid chromatogram-triple level Four bar-linear
Analyzed to obtain testing result in ion trap tandem mass spectrometry instrument.
It is preferred that the antibiotic is in tetracycline antibiotics, carbostyril antibiotic, sulfa antibiotics and big ring
Esters antibiotic；
The internal standard compound is D5- Enrofloxacins.
Further preferably, the tetracycline antibiotics are the one or more in terramycin, tetracycline and aureomycin；
The carbostyril antibiotic is Enrofloxacin, Ofloxacin, Ciprofloxacin, Norfloxacin, oxolinic acids and fluorine
One or more in first quinoline；
The sulfa antibiotics are sulfadoxine, sulphadiazine, radonil, sulphathiazole, sulfonamidoxazole, sulfanilamide (SN) two
First pyrimidine, N'-phenylsulfanilamide are than azoles, sulfadimethoxine, sulfamethyldiazine, sulfamethoxypyridazine, daimeton, sulphur
One or more in amine chlorine pyridazine, sulfabenzamide and sulfaquinoxaline；
The macrolide antibiotics be lincomycin, spiramvcin, kitasamycin, tylosin, clindamycin with
And the one or more in josamycin.
Further preferably, the sample pre-treatments of feces of livestock and poultry to be detected are specially in the step 5)：
5.1st, feces of livestock and poultry to be detected is weighed in centrifuge tube, adds Na2EDTA and 1% formic acid acetonitrile solution, shakes
Swing；
5.2nd, anhydrous sodium sulfate and sodium chloride are added in the centrifuge tube after vibration, is vortexed and mixes, centrifuges, takes after ultrasound
Clear liquid；
5.3rd, anhydrous sodium sulfate and C18 adsorbents are added in the supernatant, is centrifuged after horizontal oscillations, staticly settles egg
Bai Hou, supernatant is taken in test tube；
5.4th, after the supernatant test tube is concentrated to dryness under nitrogen, after adding 0.1% formic acid acetonitrile constant volume liquid, whirlpool
Rotation mixes, and crosses 0.22 μm of filter membrane, standby.
Further preferably, liquid chromatogram-triple level Four bar-linear ion hydrazine tandem mass spectrometer is specifically pressed in the step 3)
It is configured according to following parameter,
Liquid phase chromatogram condition is：
Chromatographic column：ACQUITY UPLCTMBEH C18 (100mm × 2.1mm i.d., 1.7 μm)；
Column temperature：40℃；
Sample room temperature：Room temperature；
Sampling volume：10μL；
Flow velocity：0.2mL/min；
Mobile phase：Methanol (A) and 0.1% aqueous formic acid (B), gradient elution program：0min, 5%A, 2min, 5%A,
8min, 25%A, 14min, 42%A, 30min, 95%A, 32min, 95%A, 35min, 5%A；
Mass Spectrometry Conditions are：
Ion gun：ESI+；
Ion source temperature：550℃；
Ion spray voltage：5500V；
Entrance potential：10V；
Atomization gas 50Psi；
Auxiliary heating gas：50Psi；
Gas curtain gas：30Psi；
Monitoring pattern：Using MRM patterns.
Further preferably, in the step 6) by step 4) in liquid chromatogram-triple level Four bar-linear ion hydrazine string
The internal standard compound and each antibiotic standard curve model generated in connection mass spectrograph, will be to be checked after progress sample pre-treatments in step 5)
Feces of livestock and poultry is surveyed to be analyzed to obtain testing result tool in liquid chromatogram-triple level Four bar-linear ion hydrazine tandem mass spectrometer
Body is,
6.1st, qualitative analysis：
Each antibiotic selects 1 parent ion, and 2 daughter ions carry out qualitative analyses, wherein, will be to be detected during qualitative analysis
Antibiotic and internal standard compound in the ratio between retention time of determinand and internal standard compound, with each hybrid standard working solution in feces of livestock and poultry sample
The ratio between retention time be respectively compared, deviation is within ± 2.5%, and each component is qualitative in feces of livestock and poultry sample to be detected
The relative abundance of ion and the relative abundance of the qualitative ion of antibiotic in each hybrid standard working solution are respectively compared, if partially
Difference is no more than defined scope, then is determined as corresponding determinand in feces of livestock and poultry sample to be detected being present；
6.2nd, quantitative determine：
Each antibiotic selects 1 parent ion, and 1 daughter ion is quantitative determined by internal standard method.
Further preferably, the specific formula for calculation of the quantitative determination in the step 6.2 is：
In formula：
X is measured object residual quantity in feces of livestock and poultry sample to be detected, and unit is ng/kg (μ g/kg)；
Cs is the concentration of antibiotic in corresponding hybrid standard working solution, and unit is nanograms per milliliter (ng/mL)；
A is the chromatographic peak area of measured object in feces of livestock and poultry sample solution to be detected；
As is the chromatographic peak area of antibiotic in corresponding hybrid standard working solution；
Ci is the concentration of internal standard compound in feces of livestock and poultry sample solution to be detected, and unit is nanograms per milliliter (ng/mL)；
Csi is the concentration of internal standard compound in corresponding hybrid standard working solution, and unit is nanograms per milliliter (ng/mL)；
Asi is the chromatographic peak area of internal standard compound in corresponding hybrid standard working solution；
Ai is the chromatographic peak area of internal standard compound in feces of livestock and poultry sample solution to be detected；
V is the final constant volume of feces of livestock and poultry sample solution to be detected, and unit is milliliter (mL)；
M is the quality of feces of livestock and poultry sample to be detected, and unit is gram (g).
The method of multiple antibiotic residues in quick detection feces of livestock and poultry provided by the invention, with improved QuEchERs skills
Art is sample pre-treatments means, with highly sensitive Waters Acquity UPLC-AB5500 liquid chromatograies-triple level Four bar-
Linear ion hydrazine tandem mass spectrometer is detection means, establishes and detects a variety of common antibiotics medicament residues in feces of livestock and poultry simultaneously
Rapid screening and confirmation technology.
The method of multiple antibiotic residues, specifically there is following characteristics in quick detection feces of livestock and poultry provided by the invention：
1st, detectable antibiotic kind substantially covers antibiotic and the China for having residue limits requirement both at home and abroad at present
In feces of livestock and poultry common antibiotics residual species, this method detection limit is low, fully meet both at home and abroad in environment to antibiotic
The requirement of residue limits.
2nd, linearly added with tandem mass spectrum system Waters Acquity UPLC-AB5500 liquid chromatograies-triple level Four bar-
Fast ion trap tandem mass spectrometry instrument is detection means, can be achieved, to Multiple Classes of Antibiotics while quick detection, to save time (current mark
Quasi- detection method is mostly the detection of single classification antibiotic, and efficiency is low, time length)；Further, since the instrumental sensitivity is high, it is fixed
Property it is quantitatively accurate, the detection limit of method is greatly lowered, is fully able to reach the international rule to antibiotic residue in farm environment
Threshold quantity.Therefore, the drawbacks of existing detection method is present is overcome completely, is a kind of high efficiency, the analytical technology of high reliability.
3rd, improved QuEChERS quick pretreatments technology is used in sample pre-treatments, extraction is abundant, good purification,
On the basis of the automaticity and reappearance that improve pre-treating method, antibiotic retention analysis in feces of livestock and poultry enormously simplify
Pretreatment process, detected while realizing multiple antibiotic residues in feces of livestock and poultry.
Brief description of the drawings
Accompanying drawing herein is merged in specification and forms the part of this specification, shows the implementation for meeting the present invention
Example, and for explaining principle of the invention together with specification.
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the required accompanying drawing used in technology description to be briefly described, it should be apparent that, for those of ordinary skill in the art
Speech, on the premise of not paying creative work, other accompanying drawings can also be obtained according to these accompanying drawings.
Fig. 1 discloses the life in liquid chromatogram-triple level Four bar-linear ion hydrazine tandem mass spectrometer in embodiment for the present invention
Into the total ion current graph model of 29 kinds of antibiotic mixed standard solutions, wherein, 1- sulphadiazines, 2- sulphathiazole, 3- sulfalenes
Yl pyrimidines, 4- terramycin, 5- tetracyclines, 6- oxygen Norfloxacin, 7- Norfloxacins, 8- sulfadimidines, 9- kynixs are rattled away
Piperazine, 10- cistosulfas, 11- lincomycins, 12- aureomycin, 13- radonils, 14- daimetons, 15- rings third are husky
Star, 16- Enrofloxacins, 17- sulfadoxines, 18- sulfonamidoxazoles, 19- sulfabenzamides, 20- sulfaphenazolums, 21- sulfanilamide (SN) two
The safe happy bacterium of Sulfamonomethoxine, 22- oxolinic acids, 23- sulfaquinoxalines, 24- spiramvcins, 25- flumequines, 26- clindamycins, 27-
Element, 28- kitasamycins, 29- josamycins；
Fig. 2~Figure 28 is disclosed in embodiment in liquid chromatogram-triple level Four bar-linear ion hydrazine tandem mass spectrum for the present invention
The 29 kinds of veterinary drugs and the multiple-reaction monitoring chromatogram graph model of internal standard compound generated in instrument.
Embodiment
Here exemplary embodiment will be illustrated in detail, its example is illustrated in the accompanying drawings.Following description is related to
During accompanying drawing, unless otherwise indicated, the same numbers in different accompanying drawings represent same or analogous key element.Following exemplary embodiment
Described in embodiment do not represent and the consistent all embodiments of the present invention.On the contrary, they be only with it is such as appended
The example of the consistent method of some aspects being described in detail in claims, of the invention.
According to both at home and abroad to each antibiotics residue limitation requirement in feces of livestock and poultry, and make with reference to current antibioticses veterinary drug
With present situation, the technical scheme that the present embodiment provides is applied under the conditions of test in laboratory, simultaneously qualitative and fixed in feces of livestock and poultry
The method of amount detection multiple antibiotic residues, its detection process are specific as follows：
1st, instrument：
Liquid Chromatography-Tandem Mass Spectrometry instrument (Waters Acquity/AB5500), Waters, US/American AB company；
Refrigerated centrifuge (Beckman Coulter Inc. of the U.S.)；The turbula shakers of Vortex-Genie 2 (U.S. Scientific
Industries companies)；Electronic analytical balance MP200, Shanghai balance factory of precision scientific instrument company；Electronic analytical balance
AE163, Mettler companies of Switzerland；Nitrogen blows concentrating instrument (Organomation Associates, EVAP 112)；Milli-Q surpasses
Water purification machine (Millipore).
2nd, reagent and material：
Methanol, acetonitrile are chromatographically pure；
Formic acid, disodium ethylene diamine tetraacetate (Na2EDTA), anhydrous sodium sulfate, anhydrous magnesium sulfate, sodium chloride, sodium acetate are equal
It is pure to analyze；
C18, PAX, PSA, NH2 Tianjin Bonna-Agela companies.
3rd, standard items：
Tetracyclines (3 kinds)：Terramycin, tetracycline, aureomycin；Quinolones (6 kinds)：Enrofloxacin, Ofloxacin, ring
Third husky star, Norfloxacin, oxolinic acids, flumequine；Sulfamido (14 kinds)：Sulfadoxine, sulphadiazine, radonil, sulphathiazole,
Sulfonamidoxazole, sulfadimidine, N'-phenylsulfanilamide than azoles, sulfadimethoxine, sulfamethyldiazine, sulfamethoxypyridazine,
Daimeton, cistosulfa, sulfabenzamide, sulfaquinoxaline；Macrolides (6 kinds)：Lincomycin, spiral are mould
Element, kitasamycin, tylosin, clindamycin, josamycin, purity >=95.0%, German Dr.Ehrenstorfer.
Internal standard compound：D5- Enrofloxacins, purity >=95.0%, German Dr.Ehrenstorfer.
4th, Standard Stock solutions：
Appropriate each Antibiotic standard and reference substance is weighed, selects methanol dilution to be made according to the dissolubility of each Antibiotic standard and reference substance
Standard reserving solution, 0~4 DEG C stored refrigerated.
5th, hybrid standard working solution：
According to response condition of each antibiotic on instrument, different amounts of storing solution is drawn, mixing is made with methanol dilution
Standard working solution, it is stored refrigerated in 0~4 DEG C.
6th, sample pre-treatments：
2.0g feces of livestock and poultry to be detected is weighed in 50mL plastic centrifuge tubes, adds 0.1g Na2EDTA's and 10mL
1.0% (v/v) formic acid acetonitrile solution, 1min is vibrated, wherein, extract solution is optimized the step, traditional QuEChERS
The conventional extract solution of method is acetonitrile, but in view of containing substantial amounts of organic matter, fat, while consulting literatures point in feces of livestock and poultry
Analysis, design Na2The extract solution of EDTA acid acetonitrile (containing 1% formic acid) is tested, and is carried out pair with simple acetonitrile extract solution
Than, the results showed that when using only acetonitrile being Extraction solvent, the rate of recovery of tetracycline antibiotics is relatively low, and this may be with such material
Easily chelated with nitrogen, phosphorus, potassium ion in feces of livestock and poultry to be formed indissoluble salt it is relevant, use add Na2EDTA acid acetonitrile (contains
1% formic acid) when being used as extract solution, suppress the chelating of such compound and cation, the rate of recovery is higher, therefore, from Na2EDTA
Acid acetonitrile (containing 1% formic acid) be used as extract solution；
4.0g anhydrous sodium sulfates and 1.0g sodium chloride are added in centrifuge tube after vibration, is vortexed and mixes 15s, ultrasound
9000r/min centrifuges 5min after 15min, takes 6mL supernatants, wherein, due to containing substantial amounts of moisture in feces of livestock and poultry, therefore go
Except processing of the moisture therein for sample is extremely important.Conventional water absorbing agent has anhydrous magnesium sulfate and anhydrous sodium sulfate, experiment
This is compared.As a result show, anhydrous magnesium sulfate adds the different degrees of reduction quinolones of meeting, sulfamido, Fourth Ring
The rate of recovery of plain class and macrolides veterinary drug, wherein, the reduction of tetracycline medication is the most notable.This is due to MgSO4In
Mg2+Chelate is combined to form with Tetracyclines veterinary drug, so as to have impact on the extraction of Tetracyclines veterinary drug.Therefore, finally determine
Water absorbing agent is anhydrous sodium sulfate；
Add 900mg anhydrous sodium sulfates and 150mg C18 adsorbents in the supernatant, after horizontal oscillations 5min, make
Adsorbent and extract solution fully contact, with 9000r/min centrifuge 5min, stand 10min protein precipitations after, take 3mL supernatants in
In 10mL test tubes, wherein, inventor's experimental selection C18, NH2, PSA and PAX 4 kinds of adsorbents, compare its clean-up effect and its
Influence to the compound rate of recovery.As a result find, influence of 4 kinds of adsorbents for the most antibiotics rate of recovery does not have obvious area
Not, PSA adsorbents are preferable to the clean-up effect of most of target compounds, but the larger tetracycline compound of polarity is reclaimed
Rate is relatively low, thus it is speculated that and this is relevant with absorption of the PSA to tetracycline antibiotics, and this absorption is probably the result of hydrogen bond action, and
Influenceed by factors such as water content of substrate, pH value.Compared with PSA, C18 adsorbents are to all including tetracycline compound
Target compound has the preferable rate of recovery, and substrate inhibition improves substantially, therefore is purified from C18 adsorbents；
By the supernatant test tube after nitrogen is concentrated to dryness at 40 DEG C, the formic acid acetonitrile (9: 1) for adding 1mL0.1% is fixed
After holding liquid, it is vortexed and mixes, crosses 0.22 μm of filter membrane, it is standby, wherein, qualitative and quantitative analysis while a variety of veterinary drug antibiotic residues
In, response and peak shape of the constant volume liquid with target veterinary drug are closely related, it is therefore desirable to preferably go out blanket for all medicines
Constant volume liquid.Respectively select different volumes than 0.1% formic acid:Acetonitrile (10:0,9:1,8:2,7:3,6:4,5:5,4:6,3:7,2:
8,1:9,0:10) it is compared experiment for constant volume liquid.Calculate 29 kinds of antibiotic response average value in a certain ratio constant volume liquid.Pass through
The average value for analyzing each ratio constant volume liquid medicine response is found, when 0.1% formic acid:Acetonitrile is 10:0 and 9:When 1, average response is most
It is high.By observing compound peak shape, except 9:Outside 1 ratio constant volume liquid, other ratio constant volume liquid produce to the peak shape of part veterinary drug
Influence.Comprehensive two aspects, it is final to determine that constant volume liquid is 0.1% formic acid：Acetonitrile (9:1, V/V).
7th, instrument detects：
1) liquid phase chromatogram condition：
Chromatographic column：ACQUITY UPLCTM BEH C18 (100mm × 2.1mm i.d., 1.7 μm)；Column temperature：40℃；Sample
Room temperature：Room temperature；Sampling volume：10μL；Flow velocity：0.2mL/min；Mobile phase：Methanol (A) and 0.1% aqueous formic acid (B),
Gradient elution, condition are shown in Table 1.
The condition of gradient elution of table 1.
2) Mass Spectrometry Conditions：
Ion gun：ESI+；Ion source temperature：550℃；Ion spray voltage：5500V；Entrance potential：10V；Atomization gas
50Psi；Auxiliary heating gas：50Psi；Gas curtain gas：30Psi；Monitoring pattern：Using MRM patterns.The 29 kinds of veterinary drug antibiosis determined
The detection ion of element, the parameters such as cluster voltage and collision energy are gone to refer to table 2.
The Mass Spectrometry Conditions optimum results of 2 29 kinds of veterinary drug antibiotic of table and internal standard compound
Note：* it is quota ion
Wherein, Fig. 1 is that 29 kinds of antibiotic are generated in liquid chromatogram-triple level Four bar-linear ion hydrazine tandem mass spectrometer
The total ion current graph model of mixed standard solution；Fig. 2~Figure 28 is in liquid chromatogram-triple level Four bar-linear ion hydrazine series connection
The 29 kinds of veterinary drugs and the multiple-reaction monitoring chromatogram graph model of internal standard compound generated in mass spectrograph.
8th, qualitative and quantitative analysis
With reference to european union directive 2002/657/EC, each antibiotic selects 1 parent ion, and 2 daughter ions progress are qualitative, its
In 1 parent ion, 1 daughter ion quantifies.When qualitative, the retention time of determinand and internal standard compound in feces of livestock and poultry sample to be detected
The ratio between, with the ratio between retention time of antibiotic and internal standard compound in each hybrid standard working solution, deviation is treated within ± 2.5%
Detect in feces of livestock and poultry sample antibiotic in relative abundance and each hybrid standard working solution of the qualitative ion of each component it is qualitative from
The relative abundance of son is respectively compared, if deviation is no more than defined scope, can determine that as feces of livestock and poultry sample to be detected
Determinand corresponding to middle presence.Maximum allowable offset during qualitative confirmation with respect to abundance of ions is shown in Table 3.
With respect to the maximum allowable offset of abundance of ions during 3 qualitative confirmation of table
Determined using inner mark method ration.Internal standard compound is D5- Enrofloxacins.Treated according in feces of livestock and poultry sample solution to be detected
Survey antibiotic and estimate content, select the hybrid standard working solution of concentration comparable, it is desirable to hybrid standard working solution and to be detected
The response of antibiotic all should be in the range of linearity that instrument detects, to hybrid standard working solution in feces of livestock and poultry sample solution
Join in equal volume with feces of livestock and poultry sample solution to be detected and inject sample measure, inner mark method ration.Calculation formula is as follows：
In formula：
X is measured object residual quantity in feces of livestock and poultry sample to be detected, and unit is ng/kg (μ g/kg)；
Cs is the concentration of antibiotic in corresponding hybrid standard working solution, and unit is nanograms per milliliter (ng/mL)；
A is the chromatographic peak area of measured object in feces of livestock and poultry sample solution to be detected；
As is the chromatographic peak area of antibiotic in corresponding hybrid standard working solution；
Ci is the concentration of internal standard compound in feces of livestock and poultry sample solution to be detected, and unit is nanograms per milliliter (ng/mL)；
Csi is the concentration of internal standard compound in corresponding hybrid standard working solution, and unit is nanograms per milliliter (ng/mL)；
Asi is the chromatographic peak area of internal standard compound in corresponding hybrid standard working solution；
Ai is the chromatographic peak area of internal standard compound in feces of livestock and poultry sample solution to be detected；
V is the final constant volume of feces of livestock and poultry sample solution to be detected, and unit is milliliter (mL)；
M is the quality of feces of livestock and poultry sample to be detected, and unit is gram (g).
9th, actual sample detects
Inventor applies above-mentioned detection method, and cow dung, pig manure, horsehit, chicken manure are randomly selected from Shenyang and surrounding area
Detected, tens of remaining batches of detection batch, as a result detect Ciprofloxacin in feces of livestock and poultry of classifying, Ofloxacin, Norfloxacin,
Aureomycin and lincomycin, detection situation see the table below.
Detection situation (mg/kg) of the table .29 kinds antibiotic in each feces of livestock and poultry
Actual sample testing result in upper table, it may be possible to which part antibiotic is during animal feeding using causing it in excrement
Just remain, and then have influence on the quality of product.The foundation of this method and application can be a variety of present in rapid screening feces of livestock and poultry
Antibiotic, so as to effectively be supervised to the residual condition of antibiotic during livestock and poultry cultivation and the residual of Sichuan thing
Survey.
Those skilled in the art will readily occur to the present invention its after considering specification and putting into practice invention disclosed herein
Its embodiment.The application be intended to the present invention any modification, purposes or adaptations, these modifications, purposes or
Person's adaptations follow the general principle of the present invention and including undocumented common knowledges in the art of the invention
Or conventional techniques.Description and embodiments be considered only as it is exemplary, true scope and spirit of the invention by right will
Ask and point out.
It should be appreciated that the invention is not limited in the precision architecture for being described above and being shown in the drawings, and
And various modifications and changes can be being carried out without departing from the scope.The scope of the present invention is only limited by appended claim.
Claims (7)
1. a kind of method of multiple antibiotic residues in quick detection feces of livestock and poultry, it is characterised in that comprise the following steps：
1) internal standard compound and various antibiotic samples are configured to standard reserving solution；
2) using standard reserving solution configuration hybrid standard working solution；
3) parameter setting of liquid chromatogram-triple level Four bar-linear ion hydrazine tandem mass spectrometer is carried out；
4) internal standard compound is generated in liquid chromatogram-triple level Four bar-linear ion hydrazine tandem mass spectrometer and each antibiotic standard is bent
Line model；
5) feces of livestock and poultry to be detected is subjected to sample pre-treatments, it is standby；
6) liquid chromatogram-triple level Four bar-linear ion hydrazine tandem mass spectrum instrument parameter set according to step 3), by step 4)
In the internal standard compound that is generated in liquid chromatogram-triple level Four bar-linear ion hydrazine tandem mass spectrometer and each antibiotic standard curve
Model, by the feces of livestock and poultry to be detected after progress sample pre-treatments in step 5) in liquid chromatogram-triple level Four bar-linear ion
Analyzed to obtain testing result in trap tandem mass spectrometer.
2. according to claim 1 in quick detection feces of livestock and poultry multiple antibiotic residues method, it is characterised in that it is described
Antibiotic is tetracycline antibiotics, carbostyril antibiotic, sulfa antibiotics and macrolide antibiotics；
The internal standard compound is D5- Enrofloxacins.
3. according to claim 2 in quick detection feces of livestock and poultry multiple antibiotic residues method, it is characterised in that it is described
Tetracycline antibiotics are the one or more in terramycin, tetracycline and aureomycin；
The carbostyril antibiotic is Enrofloxacin, Ofloxacin, Ciprofloxacin, Norfloxacin, oxolinic acids and flumequine
In one or more；
The sulfa antibiotics are that sulfadoxine, sulphadiazine, radonil, sulphathiazole, sulfonamidoxazole, sulfanilamide (SN) diformazan are phonetic
Pyridine, N'-phenylsulfanilamide are than azoles, sulfadimethoxine, sulfamethyldiazine, sulfamethoxypyridazine, daimeton, nefrosulfin
One or more in pyridazine, sulfabenzamide and sulfaquinoxaline；
The macrolide antibiotics is lincomycin, spiramvcin, kitasamycin, tylosin, clindamycin and friendship
One or more in arenomycin.
4. according to claim 1 in quick detection feces of livestock and poultry multiple antibiotic residues method, it is characterised in that it is described
The sample pre-treatments of feces of livestock and poultry to be detected are specially in step 5)：
5.1st, feces of livestock and poultry to be detected is weighed in centrifuge tube, adds Na2EDTA and 1% formic acid acetonitrile solution, vibration；
5.2nd, anhydrous sodium sulfate and sodium chloride are added in the centrifuge tube after vibration, is vortexed and mixes, is centrifuged after ultrasound, takes supernatant
Liquid；
5.3rd, anhydrous sodium sulfate and C18 adsorbents are added in the supernatant, is centrifuged after horizontal oscillations, staticly settles albumen
Afterwards, supernatant is taken in test tube；
5.4th, after the supernatant test tube is concentrated to dryness under nitrogen, after adding 0.1% formic acid acetonitrile constant volume liquid, it is vortexed mixed
It is even, 0.22 μm of filter membrane is crossed, it is standby.
5. according to claim 1 in quick detection feces of livestock and poultry multiple antibiotic residues method, it is characterised in that it is described
Liquid chromatogram-triple level Four bar-linear ion hydrazine tandem mass spectrometer is specifically configured according to following parameter in step 3),
Liquid phase chromatogram condition is：
Chromatographic column：ACQUITY UPLCTMBEH C18 (100mm × 2.1mm i.d., 1.7 μm)；
Column temperature：40℃；
Sample room temperature：Room temperature；
Sampling volume：10μL；
Flow velocity：0.2mL/min；
Mobile phase：Methanol (A) and 0.1% aqueous formic acid (B), gradient elution program：0min, 5%A, 2min, 5%A, 8min,
25%A, 14min, 42%A, 30min, 95%A, 32min, 95%A, 35min, 5%A；
Mass Spectrometry Conditions are：
Ion gun：ESI+；
Ion source temperature：550℃；
Ion spray voltage：5500V；
Entrance potential：10V；
Atomization gas 50Psi；
Auxiliary heating gas：50Psi；
Gas curtain gas：30Psi；
Monitoring pattern：Using MRM patterns.
6. according to claim 1 in quick detection feces of livestock and poultry multiple antibiotic residues method, it is characterised in that it is described
Internal standard compound in step 6) by being generated in step 4) in liquid chromatogram-triple level Four bar-linear ion hydrazine tandem mass spectrometer
With each antibiotic standard curve model, will carry out in step 5) feces of livestock and poultry to be detected after sample pre-treatments liquid chromatogram-
Analyzed to obtain testing result in triple level Four bar-linear ion hydrazine tandem mass spectrometers specifically,
6.1st, qualitative analysis：
Each antibiotic selects 1 parent ion, and 2 daughter ions carry out qualitative analyses, wherein, during qualitative analysis, by livestock and poultry to be detected
The guarantor of antibiotic and internal standard compound in the ratio between retention time of determinand and internal standard compound, with each hybrid standard working solution in fecal specimens
The ratio between time is stayed to be respectively compared, deviation is within ± 2.5%, and the qualitative ion of each component in feces of livestock and poultry sample to be detected
Relative abundance and the relative abundance of the qualitative ion of antibiotic in each hybrid standard working solution be respectively compared, if deviation is not
More than defined scope, then it is determined as corresponding determinand in feces of livestock and poultry sample to be detected being present；
6.2nd, quantitative determine：
Each antibiotic selects 1 parent ion, and 1 daughter ion is quantitative determined by internal standard method.
7. according to claim 6 in quick detection feces of livestock and poultry multiple antibiotic residues method, it is characterised in that it is described
The specific formula for calculation quantitative determined in step 6.2 is：
<mrow>
<mi>X</mi>
<mo>=</mo>
<msub>
<mi>C</mi>
<mi>S</mi>
</msub>
<mo>&times;</mo>
<mfrac>
<mi>A</mi>
<msub>
<mi>A</mi>
<mi>S</mi>
</msub>
</mfrac>
<mo>&times;</mo>
<mfrac>
<msub>
<mi>C</mi>
<mi>i</mi>
</msub>
<msub>
<mi>C</mi>
<mrow>
<mi>s</mi>
<mi>i</mi>
</mrow>
</msub>
</mfrac>
<mo>&times;</mo>
<mfrac>
<msub>
<mi>A</mi>
<mrow>
<mi>s</mi>
<mi>i</mi>
</mrow>
</msub>
<msub>
<mi>A</mi>
<mi>i</mi>
</msub>
</mfrac>
<mo>&times;</mo>
<mfrac>
<mi>V</mi>
<mi>m</mi>
</mfrac>
<mo>&times;</mo>
<mfrac>
<mn>1000</mn>
<mn>1000</mn>
</mfrac>
</mrow>
In formula：
X is measured object residual quantity in feces of livestock and poultry sample to be detected, and unit is ng/kg (μ g/kg)；
Cs is the concentration of antibiotic in corresponding hybrid standard working solution, and unit is nanograms per milliliter (ng/mL)；
A is the chromatographic peak area of measured object in feces of livestock and poultry sample solution to be detected；
As is the chromatographic peak area of antibiotic in corresponding hybrid standard working solution；
Ci is the concentration of internal standard compound in feces of livestock and poultry sample solution to be detected, and unit is nanograms per milliliter (ng/mL)；
Csi is the concentration of internal standard compound in corresponding hybrid standard working solution, and unit is nanograms per milliliter (ng/mL)；
Asi is the chromatographic peak area of internal standard compound in corresponding hybrid standard working solution；
Ai is the chromatographic peak area of internal standard compound in feces of livestock and poultry sample solution to be detected；
V is the final constant volume of feces of livestock and poultry sample solution to be detected, and unit is milliliter (mL)；
M is the quality of feces of livestock and poultry sample to be detected, and unit is gram (g).
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