CN107228912A - The screening method of 79 kinds of antibacterials in a kind of animal food - Google Patents
The screening method of 79 kinds of antibacterials in a kind of animal food Download PDFInfo
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Abstract
The invention discloses a kind of screening method of 79 kinds of antibacterials in animal food, following steps are specifically included：The preparation of standard working solution, the analysis pre-treatment of testing sample：The drafting of standard curve, qualitative analysis and quantitative analysis.High flux examination and quantitative detection platform that the present invention is built, so that developing in the art from detection single compound to the direction of detection multiple compounds simultaneously, compared with original technology, testing cost is only original 1/2nd or so, the detection cycle of sample shorten to original half, greatly improves operating efficiency.The present invention sets up examination technology and has played effective effect in the activities such as the residue of veterinary drug Supervisory Surveillance Program that the Ministry of Agriculture implements, publicly harmless animal product detection plan and the customary detection plan of national livestock products.
Description
Technical field
The invention belongs to technical field of biological, and in particular to the examination of 79 kinds of antibacterials in a kind of animal food
Method.
Background technology
In recent years, the animal husbandry of China suddenly becomes the important column support type industry of agricultural, achieves huge achievement, this with
The development of veterinary drug and reasonable employment are undivided.Veterinary drug is prevention, diagnosis, the important substance for treating Animal diseases, is rationally made
Used time, it is possible to decrease incidence and mortality, promotion growth of animal, improvement animal product quality and the raising feed conversion of livestock and poultry
Rate.Clinically conventional veterinary drug has fluoroquinolones, sulfamido, macrolides, Tetracyclines, woods can amine etc., but mesh
It is preceding treat and prevent Animal diseases during drug abuse situation it is commonplace, some drug doses considerably beyond
Measured the need for treating and preventing Animal diseases.Its toxic action is caused by long term accumulation mostly, can also pass through food
Chain is transmitted, and the exceeded animal food of long-term consumption residue of veterinary drug can produce allergic reaction and bacterial drug resistance in human body,
With potential carcinogenicity, so as to endanger human health.The Ministry of Agriculture of the People's Republic of China, MOA is with announcing No. 235 clear stipulaties western
Dissolve, Dimetridazole, metronidazole allow treatment to use, but must not be detected in animal food；Chloramphenicol, dapsone etc. are forbidden
Use, must not be detected in animal food.And in issue in 2005 on checking the urgent of the antiviral drugs such as amantadine
Notify, production to antivirus veterinary medicine and using sternly being investigated and prosecuted.
It is a kind of very universal phenomenon that veterinary drug, which abuses phenomenon in cultivating link, and medicament residue also leverages food peace
Entirely.Beta receptor stimulant medicine cultivating link application it is even more a kind of very important the problem of, beta-receptor activator (β-
Agonists) be a class chemical synthesis phenyl ethyl amine material, such medicine is added in feed to having after detoxification
Repartitioning function is acted on, and can significantly improve the lean meat percentage of animal, but people have eaten the animal products of these medicament residues
After the adverse reaction symptom such as flushed face, headache, dizzy, uncomfortable in chest, palpitaition, numb limb occurs, serious possibility jeopardizes life
Life.Therefore the successively legislation of many countries is forbidden using such medicine in Production of Livestock and Poultry in the world, and the Chinese government also prohibites
It is used.At present, European Union and China all pig is urinated, in the animal food such as pork liver, beef and mutton the detection of such medicine judge limit
It is set to 1 μ g/L (μ g/kg).
Domestic and international existing medicine multi-residue determination method mainly has liquid chromatography, liquid chromatography tandem flight time matter
The method such as spectrometry and liquid chromatography tandem mass spectrometry.Existing detection method to single kind drug research comparatively into
Ripe, the factor that needs consider when the detection method remained medicine is set up more is more, and the physicochemical properties of each medicine are totally different, preceding
Processing method is complex, there is the shortcomings of sensitivity is low, medicament categories coverage rate is narrow.Had based on the technology of residual more than medicine
Special monitoring and early warning advantage, in recent years, China each R＆D institution also gradually pay attention to medicine multi-residue determination technology and grind
Study carefully.It is domestic at present to determine in livestock products many left drugs simultaneously and the method to its accurate quantitative analysis more lacks, therefore need badly and build
Stand a kind of while determining the detection technique of multi-medicament residual.
The content of the invention
It is an object of the invention to：It is livestock products there is provided a kind of screening method of 79 kinds of antibacterials in animal food
Product and animal food addition residual fungistatic medicine provide a kind of complete detection method, are filled with national industry residual inspection
Mark is accurate, adds antibacterials examination technological means, is that state food has safely provided safety guarantee.
The purpose of the present invention is realized in the following manner：
The screening method of 79 kinds of antibacterials, specifically includes following steps in a kind of animal food：
79 kinds of antibacterials are：
(a) sulfamido：Daimeton, sulfamethoxazole, madribon, sulfaquinoxaline, sulfanilamide (SN) diformazan
Pyrimidine, cistosulfa, sulfapryidine, bosporon, ayerlucil, domian, sulfaclozine,
Kynix, fanasil, sulphadiazine, sulfamerazine, sulfonamidoxazole, sulfabenzamide, sulfanilamide (SN) are to first
Oxygen pyrimidine, sulphathiazole；
(b) cephalo-type：Cefquinome；
(c) Tetracyclines：Terramycin, tetracycline, aureomycin；
(d) amphenicols：Chloramphenicol, Florfenicol, Thiamphenicol；
(e) macrolides：Tylosin, Tilmicosin, azithromycin, CLA, ROX, kitasamycin A5；
(f) woods can amine：Lincomycin, clindamycin；
(g) fluoroquinolones：Enrofloxacin, Ciprofloxacin, sarafloxacin, Difloxacin, Nadifloxacin, Norfloxacin, training fluorine
Sha Xing, acidum nalidixicum, cinoxacin, Lomefloxacin, Ofloxacin, oxolinic acid, flumequine, Enoxacin；
(h) nitro glyoxaline：Ornidazole, Tinidazole, Dimetridazole, Ronidazole, metronidazole, carnidazole；
(i) antiviral class：Amantadine, Rimantadine；
(j) tranquillizer class：Droperidol, haloperole, azaperone, fenazil, diazepam；
(k) antipyretic-antalgic class：Paracetamol, antipyrine, aminopyrine；
(l) beta-receptor activator class：Ractopamine, Clenbuterol, salbutamol, bromine Boot sieve, Formoterol, Rabat
Sieve, Bbu Tero, Tulobuterol, Cimaterol, Zilpaterol, Clorprenaline；
(m) other classes：Atropine, Tiamulin, dapsone, TMP；
(1) preparation of standard working solution：
Each standard items 10mg is accurately weighed, in 10mL measuring bottles, is dissolved with methanol and is diluted to scale, be configured to 1mg/mL and respectively mark
Quasi- stock solution；For insoluble medicine, Enrofloxacin, Ciprofloxacin, Norfloxacin add a small amount of 0.1mol/L HCl solutions, Lip river
U.S. sand star adds a small amount of 0.1mol/L NaOH solutions, and Cefquinome adds appropriate dimethyl sulfoxide (DMSO) to make after its dissolving, fixed with methanol
To scale；
Each Standard Reserving Solution is measured into 100mL measuring bottles, with methanol dilution to scale, hybrid standard intermediate solution is made；Wherein
Sulfamido, fluoroquinolones, woods can amine, dapsone, Tiamulin and TMP respectively take 0.4mL；Antipyretic-antalgic class and gold
Firm ethamine respectively takes 2mL；Cefquinome takes 4mL；Tetracyclines, tylosin, Tilmicosin, kitasamycin A5, Ornidazole and gold
Firm ethamine respectively takes 1mL；Beta-receptor activator class, amphenicols, atropine, droperidol and haloperole respectively take 0.2mL；Ah
Zha Pailong, fenazil and diazepam respectively take 0.1mL；
Hybrid standard intermediate solution 0.1mL, 0.2mL, 0.5mL are pipetted respectively into 10mL measuring bottles, with methanol dilution to scale, system
Obtain LOQ concentration hybrid standards working solution, 2LOQ concentration hybrid standard working solutions and the work of 5LOQ concentration hybrid standard molten
Liquid；
(2) the analysis pre-treatment of testing sample：
1) extract：2 ± 0.02g of homogeneous sample is weighed in 50mL polypropylene centrifuge tubes, Na is successively added2EDTA solution and second
Nitrile, methanol, ethyl acetate solution, vortex 10-30s are mixed, and vibrate 10-30min；Centrifuged with 5000-8000r/min rotating speeds
5min, supernatant is transferred in 50mL polypropylene centrifuge tubes；Add extract solution residue is carried out to repeat extraction, merge extract solution；
And it is blown to solution residue about 1-2mL in nitrogen at 40-50 DEG C；It is used as reserve liquid；
2) purify：Activated respectively with 5-8mL methanol and 3-6mL water using Agela Cleanert PEP-2 solid-phase extraction columns, will
Reserve liquid all crosses post, and coutroi velocity is about 1-3 drops/s；Eluted with leacheate, vacuum drains 3-5min；With elution,
Eluent is dried up in nitrogen at 30-40 DEG C；Accurate to add 20% acetonitrile solution 1-3mL dissolving constant volumes, be vortexed mistake after mixing
0.22 μm of microporous Nylon filter membrane；
(3) drafting of standard curve：Precision measures 1 μ g/mL mixed standard solutions in right amount, and blank sample matrix solution is configured to dense
Spend for 1,2,5,10,20,50,100ng/mL series standard solution, determined for liquid chromatography tandom mass spectrometry determination.With characteristic ion
Mass chromatography peak area is ordinate, and corresponding concentration of standard solution is abscissa, draws standard curve, obtains 79 kinds and resists
The qualitative analysis chromatogram picture library of bacterium medicine；
(4) mass spectral analysis is carried out to the sample of the 2LOQ concentration containing each medicine quantitative limit LOQ described in step (3), obtained
The quasi-molecular ion of each medicine, and two characteristic ions：Qualitative ion and quota ion, it is as shown in the table：
(5) qualitative analysis：The testing sample handled by step (2) methods described is selected 1 parent ion and 2 features from
Son, carries out the retention time of determinand in disposable liquid analysis of hplc, sample corresponding with spectrogram storehouse obtained by step (3)
Retention time deviation within ± 2.5%, and in sample the qualitative ion of each component relative abundance it is close with concentration standard it is molten
The relative abundance of corresponding qualitative ion is compared in liquid, and deviation is no more than scope as defined in following table, then can determine that as sample
In there is corresponding determinand：
| Relative ion abundance (%) | >50 | >20~50 | >10~20 | ≤10 |
| The maximum deviation (%) of permission | ±20 | ±25 | ±30 | ±50 |
(6) quantitative analysis：Under the best conditions of instrument, the testing sample and step of the processing of step of learning from else's experience (2) methods described
Suddenly the hybrid standard working solution prepared by (1) carries out mass spectral analysis, obtains chromatographic peak area response, makees single-point or multiple spot school
Standard, uses quantified by external standard method；The feature of each determinand during the response of determinand and hybrid standard work also in testing sample solution
The response of ion if testing sample solution concentration exceedes the range of linearity, all should use second in the range of linearity of Instrument measuring
Nitrile/water (V/V, 20:80) redeterminated after solution dilution；
Each medicament contg X is in terms of mass fraction in sample, and unit is milligrams per kilogram (mg/kg), is calculated by formula (1)：
In formula：
The peak area of determinand in A-testing sample solution；
The peak area of corresponding determinand in As-hybrid standard working solution；
Testing concentration in Cs-hybrid standard working solution, unit is micrograms per millilitre (μ g/mL)；
The volume of V-dissolving residue, unit is milliliter (mL)；
The quality of m-sample, unit is gram (g)；
The extracting liquid volume of V1-mistake solid-phase extraction column, unit is milliliter (mL)；
V2-total extracting liquid volume, unit is milliliter (mL)；
N-extension rate.
Measurement result is represented with the arithmetic mean of instantaneous value of parallel determination, as a result retains three effective digitals.
The animal food is animal muscle based food, animal's liver, animal kidney, egg and milk fish.
Liquid-phase chromatographic analysis condition in the step (3) and step (5) is：Chromatographic column：Waters UPLC
C18,100 mm × 2.1mm id, 1.7 μm；Mobile phase A：The aqueous solution, Mobile phase B：Acetonitrile solution, mobile phase C：0.1% formic acid
The aqueous solution, mobile phase D：Methanol solution；Following table is positive and negative ion gradient elution mode；Flow velocity：0.1-0.3mL/min；Sample introduction
Amount：2μL；Column temperature：25- 40℃；
Note：6 be linear change, and 1 is change immediately.
Mass spectral analysis condition in the step (4) and step (6) is：
Duration of oscillation in the step (2) is 20min, and extract solution is the mixed solution of acetonitrile and water, and volume ratio is 15:
2；Leacheate is water 1-3mL, and eluent is acetonitrile solution and each 2-5mL of methanol solution.
Na in the step (2)2The concentration of EDTA solution is 0.1mol/L.
This method is directed to the anti-microbial type commonly used in China's livestock products breeding process, antiviral class, antipyretic-antalgic class, tranquillizer
The medicines such as class, beta-receptor activator class, the efficiently separating property being had based on ultra performance liquid chromatography tandem mass spectrum and can be simultaneously accurate
The characteristics of certainty is quantitative, establishes while determining sulfamido, fluoroquinolones, Tetracyclines, nitro miaow in beef, chicken
13 class, 79 kinds of medicines such as azole, cephalo-type, amphenicols, macrolides, beta-receptor activator class and Lin Ke amines
UPLC-MS/MS methods, the livestock products that can contain exceeded veterinary drug and forbidden drug for main pipe portion gatekeeper pipe in the market provide skill
Art ensures that a weight pound blow is given in the behavior of illegal, Misuse medicine in being raised to livestock products.
The high flux examination and quantitative detection platform built relative to prior art, the present invention so that in the art
From detection single compound to detecting that the direction of multiple compounds is developed simultaneously, compared with original technology, testing cost is only
Original 1/2nd or so, the detection cycle of sample shorten to original half, greatly improves operating efficiency.The present invention
Set up residue of veterinary drug Supervisory Surveillance Program, publicly harmless animal product detection plan and national livestock products that examination technology is implemented in the Ministry of Agriculture
Effective effect has been played in the activities such as product routine detection plan.In China, provincial livestock products Product Quality Verification Centers have carried out sample inspection
Survey, according to incompletely statistics, popularization and application sample size amounts to more than 20,000 parts.For experimental cost, high flux examination side is used
Method, greatly reduces testing cost, shortens the detection time of half, and economic benefit can save about 8,000,000 yuan every year.This hair
Bright application provides strong technical guarantee for the quality safety of China's animal and animal's products；For the animal and animal of China
Product enters international market, and the limitation for breaking through " Green Trade Barrier " provides safeguard；Improve competition of the China in international trade
Status, it is ensured that the extra exit of agricultural products in China, increases foreign exchange earnings, creates good economic benefit；Simultaneously to ensureing food
The sound development of production industry, promotes economic sustainable development, maintains social stability, and produces great social profit.
Brief description of the drawings
Fig. 1 is the influence to each medicine rate of recovery of different extract solutions.
Fig. 2 is influence of the different solid-phase extraction columns to each medicine rate of recovery.
Fig. 3 is influence of the different eluents to each medicine rate of recovery.
Fig. 4 is the chromatogram of sulfapryidine.
Fig. 5 is the chromatogram of sulphadiazine.
Fig. 6 is the chromatogram of sulfamethoxazole.
Fig. 7 is the chromatogram of sulphathiazole.
Fig. 8 is the chromatogram of sulfamerazine.
Fig. 9 is bosporon and the chromatogram of sulfonamidoxazole, wherein left side peak is bosporon, right side peak is that sulfanilamide (SN) is different
Oxazole.
Figure 10 is the chromatogram of ayerlucil.
Figure 11 is the chromatogram of sulfabenzamide.
Figure 12 is the chromatogram of domian.
Figure 13 is the chromatogram of sulfadimidine.
Figure 14 is the chromatogram of 5-methoxysulfadiazine, kynix and daimeton, wherein left side peak is sulfanilamide (SN)
To Sulfamonomethoxine, middle crest is kynix, and right side peak is daimeton.
Figure 15 is the chromatogram of cistosulfa and sulfaclozine, wherein left side peak is cistosulfa, right side peak is sulfanilamide (SN)
Chloropyrazine.
Figure 16 is the chromatogram of TMP.
Figure 17 is the chromatogram of fanasil and madribon, wherein left side peak is the adjacent dimethoxy of sulfanilamide (SN)
Pyrimidine, right side peak is madribon.
Figure 18 is the chromatogram of sulfaquinoxaline.
Figure 19 is the chromatogram of diazepam.
Figure 20 is the chromatogram of Dimetridazole.
Figure 21 is the chromatogram of Cefquinome.
Figure 22 is the chromatogram of metronidazole.
Figure 23 is the chromatogram of Ronidazole.
Figure 24 is the chromatogram of Ornidazole.
Figure 25 is the chromatogram of Tinidazole.
Figure 26 is the chromatogram of amantadine.
Figure 27 is the chromatogram of Rimantadine.
Figure 28 is the chromatogram of paracetamol.
Figure 29 is the chromatogram of aminopyrine.
Figure 30 is the chromatogram of antipyrine.
Figure 31 is the chromatogram of dapsone.
Figure 32 is the chromatogram of atropine.
Figure 33 is the chromatogram of Tiamulin.
Figure 34 is the chromatogram of acidum nalidixicum.
Figure 35 is the chromatogram of oxolinic acid.
Figure 36 is the chromatogram of flumequine.
Figure 37 is the chromatogram of cinoxacin.
Figure 38 is the chromatogram of Norfloxacin.
Figure 39 is the chromatogram of enoxacin.
Figure 40 is the chromatogram of Ciprofloxacin.
Figure 41 is the chromatogram of Pefloxacin.
Figure 42 is the chromatogram of Lomefloxacin.
Figure 43 is the chromatogram of Enrofloxacin.
Figure 44 is the chromatogram of Nadifloxacin.
Figure 45 is the chromatogram of Ofloxacin.
Figure 46 is the chromatogram of sarafloxacin.
Figure 47 is the chromatogram of Difloxacin.
Figure 48 is the chromatogram of fenazil.
Figure 49 is the chromatogram of lincomycin.
Figure 50 is the chromatogram of clindamycin.
Figure 51 is the chromatogram of terramycin.
Figure 52 is the chromatogram of aureomycin.
Figure 53 is the chromatogram of tetracycline.
Figure 54 is the chromatogram of CLA.
Figure 55 is the chromatogram of azithromycin.
Figure 56 is the chromatogram of kitasamycin.
Figure 57 is the chromatogram of ROX.
Figure 58 is the chromatogram of Tilmicosin.
Figure 59 is the color vomiting of tylosin.
Figure 60 is the chromatogram of haloperole.
Figure 61 is the chromatogram of droperidol.
Figure 62 is the chromatogram of azaperone.
Figure 63 is the chromatogram of carnidazole.
Figure 64 is the chromatogram of chloramphenicol.
Figure 65 is the chromatogram of Thiamphenicol.
Figure 66 is the chromatogram of Florfenicol.
Figure 67 is the chromatogram of Clenbuterol.
Figure 68 is the chromatogram of salbutamol.
Figure 69 is the chromatogram of Bbu Tero.
Figure 70 is the chromatogram of Tulobuterol.
Figure 71 is the chromatogram of Rabat sieve.
Figure 72 is the chromatogram of Formoterol.
Figure 73 is the chromatogram of Cimaterol.
Figure 74 is the chromatogram of Clorprenaline.
Figure 75 is the chromatogram of bromine Boot sieve.
Figure 76 is the chromatogram of Ractopamine.
Figure 77 is the chromatogram of Zilpaterol.
Embodiment
Examined medicine 79 kinds of antibacterials of totally 13 class, be respectively：
(a) sulfamido：Daimeton, sulfamethoxazole, madribon, sulfaquinoxaline, sulfanilamide (SN) diformazan
Pyrimidine, cistosulfa, sulfapryidine, bosporon, ayerlucil, domian, sulfaclozine,
Kynix, fanasil, sulphadiazine, sulfamerazine, sulfonamidoxazole, sulfabenzamide, sulfanilamide (SN) are to first
Oxygen pyrimidine, sulphathiazole；
(b) cephalo-type：Cefquinome；
(c) Tetracyclines：Terramycin, tetracycline, aureomycin；
(d) amphenicols：Chloramphenicol, Florfenicol, Thiamphenicol；
(e) macrolides：Tylosin, Tilmicosin, azithromycin, CLA, ROX, kitasamycin A5；
(f) woods can amine：Lincomycin, clindamycin；
(g) fluoroquinolones：Enrofloxacin, Ciprofloxacin, sarafloxacin, Difloxacin, Nadifloxacin, Norfloxacin, training fluorine
Sha Xing, acidum nalidixicum, cinoxacin, Lomefloxacin, Ofloxacin, oxolinic acid, flumequine, Enoxacin；
(h) nitro glyoxaline：Ornidazole, Tinidazole, Dimetridazole, Ronidazole, metronidazole, carnidazole；
(i) antiviral class：Amantadine, Rimantadine；
(j) tranquillizer class：Droperidol, haloperole, azaperone, fenazil, diazepam；
(k) antipyretic-antalgic class：Paracetamol, antipyrine, aminopyrine；
(l) beta-receptor activator class：Ractopamine, Clenbuterol, salbutamol, bromine Boot sieve, Formoterol, Rabat
Sieve, Bbu Tero, Tulobuterol, Cimaterol, Zilpaterol, Clorprenaline；
(m) other classes：Atropine, Tiamulin, dapsone, TMP.
1. the preparation of standard working solution：
Each standard items 10mg is accurately weighed, in 10mL measuring bottles, is dissolved with methanol and is diluted to scale, be configured to 1mg/mL and respectively mark
Quasi- stock solution.Preserve, be valid for three months at -20 DEG C；For insoluble medicine, Enrofloxacin, Ciprofloxacin, Norfloxacin add
Enter a small amount of 0.1 mol/L HCl solutions, Lomefloxacin adds a small amount of 0.1mol/L NaOH solutions, and Cefquinome adds appropriate diformazan
Base sulfoxide makes after its dissolving, fixed to scale with methanol；
Each Standard Reserving Solution is measured into 100mL measuring bottles, with methanol dilution to scale, hybrid standard intermediate solution is made；Wherein
Sulfamido, fluoroquinolones, woods can amine, dapsone, Tiamulin and TMP respectively take 0.4mL；Antipyretic-antalgic class and gold
Firm ethamine respectively takes 2mL；Cefquinome takes 4mL；Tetracyclines, tylosin, Tilmicosin, kitasamycin A5, Ornidazole and gold
Firm ethamine respectively takes 1mL；Beta-receptor activator class, amphenicols, atropine, droperidol and haloperole respectively take 0.2mL；Ah
Zha Pailong, fenazil and diazepam respectively take 0.1mL；
Hybrid standard intermediate solution 0.1mL, 0.2mL, 0.5mL are pipetted respectively into 10mL measuring bottles, with methanol dilution to scale, system
Obtain LOQ concentration hybrid standards working solution, 2LOQ concentration hybrid standard working solutions and the work of 5LOQ concentration hybrid standard molten
Liquid, 4 DEG C are kept in dark place, the term of validity 1 week.
2. the analysis pre-treatment of testing sample：
2.1 prepare：The chopping in advance of beef, chicken in tissue refiner after being homogenized, as ready sample, by ready sample in-
Preserved at 20 DEG C.
2.2 extract：Muscle：Weigh 2 ± 0.02g of homogeneous sample and 200 μ L 0.1 are added in 50mL polypropylene centrifuge tubes, successively
Mol/LNa2EDTA solution and 10mL acetonitrile/methanols (V/V, 95:5) extract solution, vortex 10-30s is mixed, and vibrates 10-30min；
5min is centrifuged with 5000-8000r/min rotating speeds, supernatant is transferred in 50mL polypropylene centrifuge tubes；Add 10mL acetonitrile/waters
(V/V, 15:2) solution carries out repeating extraction, merges extract solution；And it is blown to solution residue about 0.5-1mL in nitrogen at 40-50 DEG C；
Cleaned respectively with 0.5mL acetonitriles and 7.5 mL water, be used as reserve liquid.
Liver, kidney, egg, milk：2g samples (± 0.02g) are weighed in 50mL centrifuge tubes, 200 μ L are separately added into
0.1mol/L EDTA solution and 10mL acetonitriles, are vortexed after mixing, and vibrate 20min.5min is centrifuged in 5000r/min on centrifuge,
Supernatant is transferred in 50mL centrifuge tubes.10mL acetonitrile/waters (V/V, 15 are added into residue:2) solution carries out repeating to carry
Take, merge extract solution twice；And it is blown to solution residue about 0.5-1mL in nitrogen at 40-50 DEG C；Respectively with 0.5mL acetonitriles and
7.5mL water is cleaned, and is used as reserve liquid.
The present invention has investigated the extraction effect of acetonitrile, methanol, ethyl acetate solution respectively, then and respectively to acetonitrile/methanol
(95:5, v/v), acetonitrile/water (15:2, v/v), acetonitrile/water (60:40, v/v) and acetonitrile/water (50:50, v/v) solution etc. is carried
Liquid is taken to be investigated, Fig. 1 is the influence to each medicine rate of recovery of different extract solutions.Test result indicates that：Methanol and acetic acid second
Ester has preferable extraction efficiency to most of medicine, but can amine, antiviral class extraction efficiency to macrolides, woods
It is poor；Preferably, added in acetonitrile makes extraction efficiency more preferably to the extraction effect of acetonitrile after a small amount of methanol；Four kinds of pure organisms of the above
System is to tetracycline medication recovery rate between 10%~26%.Acetonitrile/water (15:2, v/v) have preferably to each medicine
Extraction efficiency, it is particularly of a relatively high to the extraction effect of tetracycline medication, but to chloramphenicol and partial Stabilization agent class medicine
The extraction efficiency of thing is than acetonitrile/methanol (95:5, v/v) it is slightly lower；Acetonitrile/water (60:40, v/v) and acetonitrile/water (50:50, v/v)
Solution can extract out more polar impurity, and Tetracyclines, macrolides, Antipyretics thing have higher matrix
Suppress, inhibiting rate is up to 80%.Acetonitrile/methanol (95:5, v/v) and acetonitrile/water (15:2, v/v) there is preferable complementation,
There is the of a relatively high rate of recovery using each medicine during both solution respectively, and with relatively low matrix effect, miscellaneous peak interference
It is few.Water content is excessively unfavorable for the concentration of extract solution, but adds a small amount of Na in extract solution2EDTA solution and a small amount of water can be carried substantially
The rate of recovery of high macrolides and tetracycline medication, it may be possible to because this two classes medicine in the solution easily formed divalence or
Tricationic in the extraction stage so as to cause damage, Na2EDTA can form complex compound with this two classes medicine, thus can keep away
Exempt from because of loss and caused by the rate of recovery it is relatively low.
The present invention is also investigated to the concussion time in extraction process, compares the extraction of concussion each medicines of 10min and 20min
As a result.As a result show after concussion time lengthening, in addition to Dimetridazole and the paracetamol rate of recovery have 10% reduction, other
The rate of recovery of medicine has 0~25% raising, therefore is used as the concussion time in the stage of extraction selection 20min.
2.3 purification：Using Oasis HLB, Agela Cleanert PEP-2, Varian Bond Elut C18 and Waters
Sep-pak C18 solid-phase extraction columns are activated with 5mL methanol and 5mL water respectively, reserve liquid are all crossed into post, coutroi velocity is about
1-3 drops/s；Eluted with 2mL leacheates, vacuum drains 3-5min；Eluted respectively with 3mL acetonitriles and 3mL methanol, collect eluent
Dried up in nitrogen at 40 DEG C；It is accurate to add 20% acetonitrile solution 0.5mL dissolving constant volumes, it is vortexed after mixing and crosses 0.22 μm of micropore
Nylon leaching film；
By the screening to solid-phase extraction column, by Fig. 2 it was found that recovery of the C18 posts to sulfamido, Macrocyclolactone lactone kind medicine
Effect is slightly worse, and the dry post bed that easily causes of post bed is collapsed, it is impossible to ensure the stability of result；HLB posts and PEP-2 posts are to each medicine
Thing, which has, preferably to be retained and detergent power, with similar recovering effect.HLB solid-phase extraction columns are expensive, PEP-2 posts
Price is relatively low, under conditions of with identical clean-up effect, therefore selection Agela Cleanert PEP-2 solid-phase extraction columns
For the decontaminating column of the present invention.
The species of eluent directly affects the clean-up effect of solid-phase extraction column, and the present invention is investigated to eluent, made respectively
With acetonitrile, methanol, acetonitrile/formic acid water (pH 3.5) (2:1, v/v) and acetonitrile/methanol (1:1, v/v) it is eluent, Fig. 3 selections
Sulphathiazole, ayerlucil etc. are represented to eluent species more sensitive medicine, show the elution of each eluent
Ability.As a result show, the elution effect of acetonitrile is not so good as methanol on the whole, but methanol is to metronidazole, Dimetridazole and Lin Ke
The elution effect of mycin is not so good as acetonitrile；Acetonitrile/formic acid water (pH 3.5) (2:1, v/v) when being eluent, the recovery of sulfabenzamide
Rate is only 30%；When being eluted respectively as eluent using 3mL acetonitriles and 3mL methanol, the rate of recovery of each medicine is optimal,
And with preferable peak shape.
The present invention have also been made investigation to leacheate, and cephalosporins medicine is more sensitive to organic solvents such as acetonitrile, methanol, low concentration
Acetonitrile solution and methanol aqueous solution can be washed out, therefore use 2mL water to be leacheate.
3. liquid phase chromatogram condition:
Chromatographic column：Waters UPLCC18 (100mm × 2.1mm id, 1.7 μm)；Mobile phase A：The aqueous solution, mobile phase
B：Acetonitrile solution, mobile phase C：0.1% aqueous formic acid, mobile phase D：Methanol solution；Table 1 is positive and negative ion gradient elution side
Formula；Flow velocity： 0.1-0.3mL/min；Sample size：2μL；Column temperature：25-40℃.
The gradient elution program of table 1
Note：6 be linear change, and 1 is change immediately.
By the optimization repeatedly of liquid-phase condition, when being analyzed while various ingredients, in order to increase the separating degree between each medicine,
Frequently with gradient elution mode.This method for chromatographic column, is investigated with Waters C18 (100mm × 2.1mm id., 1.7 μm)
Four kinds of flow visualizings that methanol, acetonitrile, water and 0.1% aqueous formic acid are constituted, ring by the chromatographic peak for contrasting each medicine
Answer and peak type, the S/N of each medicine is higher when finding to use methanol under positive ion mode, and 0.1% formic acid water can make most of medicine
The Ionization Efficiency of thing increases, partial nitro imidazoles when methanol-water is mobile phase with higher effect and
Preferable peak type [12], is the first of methanol -0.1% to take into account the mobile phase used under the measure of all medicines, positive ion mode
Aqueous acid；Use acetonitrile-water equal as the chromatogram peak type and response of amphenicols medicine during mobile phase under negative ion mode
To be optimal.
4. Mass Spectrometry Conditions source parameters, mass spectrometry method parameter are shown in Table 2：
The source parameters of table 2
By the screening and optimization of Mass Spectrometry Conditions, method is diluted using 20% acetonitrile solution to each standard liquid, is made
The standard liquid of each medicine of 200ng/mL, under ESI positive and negative ion patterns, is entered using flow injection pump continuous sample introduction mode
Row full scan, determines the parent ion of each medicine.And to the daughter ion full scan under the parent ion, to its mass spectrum under MRM patterns
Condition is optimized.Finally choose two stronger daughter ions of abundance and be used as qualitative, quota ion.Wherein sulfamido, fluorine quinoline promise
Ketone, woods can the medicine such as amine, Tetracyclines, nitro glyoxaline, macrolides at [M+H]+Sensitivity is higher under pattern,
Therefore selection is measured in the positive-ion mode；Amphenicols medicine is at [M- H]-There is higher response under pattern, therefore select
It is measured in the negative ion mode.
5. the foundation of chromatogram picture library：
It is appropriate that precision measures 1 μ g/mL mixed standard solutions, blank sample matrix solution be configured to concentration for 1,2,5,10,20,
50th, 100ng/mL series standard solution, is determined for liquid chromatography tandom mass spectrometry determination.Using characteristic ion mass chromatography peak area as
Ordinate, corresponding concentration of standard solution is abscissa, draws standard curve, obtains the qualitative analysis of 79 kinds of antibacterials
Chromatogram picture library, as shown in Fig. 4-Figure 77；
Mass spectral analysis, ion gun ginseng are carried out to the sample of the 2LOQ concentration containing each medicine quantitative limit LOQ described in step 5
Number, mass spectrometry method parameter are shown in Table 2, obtain the quasi-molecular ion of each medicine, and two characteristic ions：Qualitative ion and fixed
Ion and corresponding collision energy reference value are measured, as shown in table 3：
The qualitative of the medicine of table 3, quota ion and taper hole voltage, impact energy
Qualitative analysis：1 parent ion and 2 characteristic ions are selected to the testing sample handled by step 2 methods described,
Carry out disposable liquid analysis of hplc, in sample the retention time of determinand with step 5 gained spectrogram storehouse during corresponding reservation
Between deviation it is right in the relative abundance standard liquid close with concentration of the qualitative ion of each component within ± 2.5%, and in sample
The relative abundance for the qualitative ion answered is compared, and deviation is no more than scope as defined in table 4, then can determine that to exist in sample
Corresponding determinand：
With respect to the maximum allowable offset of abundance of ions during 4 qualitative confirmation of table
| Relative ion abundance (%) | >50 | >20~50 | >10~20 | ≤10 |
| The maximum deviation (%) of permission | ±20 | ±25 | ±30 | ±50 |
Quantitative analysis：Hybrid standard prepared by the testing sample and step (1) of the processing of step of learning from else's experience (2) methods described
Working solution carries out mass spectral analysis, obtains chromatographic peak area response, makees single-point or multiple spot calibration, uses quantified by external standard method；Treat test sample
The response of determinand all should be in the range of linearity of Instrument measuring, if testing sample solution concentration exceedes linearly in product solution
During scope, with acetonitrile/water (V/V, 20:80) redeterminated after solution dilution；
Each medicament contg X is in terms of mass fraction in sample, and unit is milligrams per kilogram (mg/kg), is calculated by formula (1)：
In formula：
The peak area of determinand in A-testing sample solution；
The peak area of corresponding determinand in As-hybrid standard working solution；
Testing concentration in Cs-hybrid standard working solution, unit is micrograms per millilitre (μ g/mL)；
The volume of V-dissolving residue, unit is milliliter (mL)；
The quality of m-sample, unit is gram (g)；
V1The extracting liquid volume of-mistake solid-phase extraction column, unit is milliliter (mL)；
V2- total extracting liquid volume, unit is milliliter (mL)；
N-extension rate.
Measurement result is represented with the arithmetic mean of instantaneous value of parallel determination, as a result retains three effective digitals.
Method validation
8.1. it is linear：Blank sample is weighed, is handled according to step 2 methods described, eluent is accurately pipetted mixed before nitrogen drying
Standardization solution is appropriate, adds in eluent, be made each medicine quantitative limit LOQ 0.5LOQ, LOQ, 2LOQ, 5LOQ,
10LOQ, 20 LOQ concentration matrix matching standard liquid, each medicine are related in good linear in the range of related concentrations, such as table
5-6, coefficient correlation is all higher than 0.99.
8.2. test limit and quantitative limit
Test limit：A certain amount of mixed standard solution is added in 2g blank samples, by extraction and cleaning and is determined, the letter of medicine
When making an uproar than S/N >=3, you can obtain the test limit of medicine, it the results are shown in Table 5-6.
Quantitative limit：A certain amount of mixed standard solution is added in 2g blank samples, by extraction and cleaning and is determined, the letter of medicine
When making an uproar than S/N >=10, you can obtain the quantitative limit of medicine, it the results are shown in Table 5-6.
8.3. method precision and the degree of accuracy
Add tri- concentration mixed standard solutions of LOQ, 2LOQ, 5LOQ respectively in ox, chicken muscle blank tissue, each concentration is done
5 parallel.As a result show, the average recovery rate of 79 kinds of medicines, the coefficient of variation are as shown in table 5 in beef.As can be seen that 79 kinds
The average recovery rate of medicine is between 45%-125%, and relative standard deviation is respectively less than 20% in batch.79 kinds of medicines in chicken
Average recovery rate, the coefficient of variation are as shown in table 6.As can be seen that the average recovery rate of 79 kinds of medicines is between 43%-118%,
Relative standard deviation is respectively less than 20% in batch.
The range of linearity, coefficient correlation, average recovery rate and the RSD of 79 kinds of medicines in the beef of table 5
The range of linearity, coefficient correlation, average recovery rate and the RSD of 79 kinds of medicines in the chicken of table 6
9. brief summary
9.1 method sensitivity
The present invention A Zhapailong, fenazil and diazepam in pig, ox, sheep, the musculature of chicken, egg, milk quantitative limit
For 0.5 μ g/kg；Beta-receptor activator class, amphenicols, atropine, droperidol and quantifying for haloperole are limited to 1 μ g/
kg；Sulfamido, fluoroquinolones, woods can amine, dapsone, Tiamulin and quantifying for TMP be limited to 2 μ g/kg；Fourth Ring
Plain class, tylosin, Tilmicosin, kitasamycin A5, Ornidazole and quantifying for Rimantadine are limited to 5 μ g/kg；Antipyretic-antalgic
Class and quantifying for amantadine are limited to 10 μ g/kg；Quantifying for Cefquinome is limited to 20 μ g/kg.
A Zhapailong, fenazil and quantifying for diazepam are limited to 1 μ g/ in pig, ox, sheep, the liver organization of chicken, renal tissue
kg；Beta-receptor activator class, amphenicols, atropine, droperidol and quantifying for haloperole are limited to 2 μ g/kg；Sulfamido,
Fluoroquinolones, woods can amine, dapsone, Tiamulin and quantifying for TMP be limited to 4 μ g/kg；Tetracyclines, safe pleasure
Rhzomorph, Tilmicosin, kitasamycin A5, Ornidazole and quantifying for Rimantadine are limited to 10 μ g/kg；Antipyretic-antalgic class and Buddha's warrior attendant
Quantifying for alkanamine is limited to 20 μ g/kg；Quantifying for Cefquinome is limited to 40 μ g/kg.
9.2 the degree of accuracy
The present invention 0.5~20 μ g/kg in pig, ox, sheep, the musculature of chicken, egg, milk add the recovery on concentration level
Rate is 30%~120%, and the rate of recovery that 1~40 μ g/kg add on concentration level in liver organization, renal tissue is 30%
~120%.
9.3 precision
Batch interior relative standard deviation≤20%, relative standard deviation≤20% between batch of the present invention.
10. actual sample detection is as shown in table 7：
The actual sample testing result of table 7
Detection data establish in pig, ox, sheep, the musculature of chicken, egg, milk 79 kinds using UPLC-MS/MS technologies above
The rapid screening analysis method of medicament residue.This method is compared with single medicine species detection method in traditional livestock products, detection
Efficiency can improve more than 5 times, 45 batches of samples can be handled daily, and accurate qualitative, quantitative, corresponding detection can be carried out to testing result
Cost can reduce by 60% or so, and have the features such as quick, accurate, sensitivity is high, examination scope is wide, be suitable in livestock products
The rapid screening analysis of residue of veterinary drug, carries out Risk Monitoring for livestock products and provides a kind of strong technology.
Claims (6)
1. the screening method of 79 kinds of antibacterials in a kind of animal food, it is characterised in that：Specifically include following steps：
79 kinds of antibacterials are：
(a) sulfamido：Daimeton, sulfamethoxazole, madribon, sulfaquinoxaline, sulfanilamide (SN) diformazan
Pyrimidine, cistosulfa, sulfapryidine, bosporon, ayerlucil, domian, sulfaclozine, sulphur
Amine methoxy piperazine, fanasil, sulphadiazine, sulfamerazine, sulfonamidoxazole, sulfabenzamide, sulfanilamide (SN) are to methoxy
Pyrimidine, sulphathiazole；
(b) cephalo-type：Cefquinome；
(c) Tetracyclines：Terramycin, tetracycline, aureomycin；
(d) amphenicols：Chloramphenicol, Florfenicol, Thiamphenicol；
(e) macrolides：Tylosin, Tilmicosin, azithromycin, CLA, ROX, kitasamycin A5；
(f) woods can amine：Lincomycin, clindamycin；
(g) fluoroquinolones：Enrofloxacin, Ciprofloxacin, sarafloxacin, Difloxacin, Nadifloxacin, Norfloxacin, training fluorine
Sha Xing, acidum nalidixicum, cinoxacin, Lomefloxacin, Ofloxacin, oxolinic acid, flumequine, Enoxacin；
(h) nitro glyoxaline：Ornidazole, Tinidazole, Dimetridazole, Ronidazole, metronidazole, carnidazole；
(i) antiviral class：Amantadine, Rimantadine；
(j) tranquillizer class：Droperidol, haloperole, azaperone, fenazil, diazepam；
(k) antipyretic-antalgic class：Paracetamol, antipyrine, aminopyrine；
(l) beta-receptor activator class：Ractopamine, Clenbuterol, salbutamol, bromine Boot sieve, Formoterol, Rabat
Sieve, Bbu Tero, Tulobuterol, Cimaterol, Zilpaterol, Clorprenaline；
(m) other classes：Atropine, Tiamulin, dapsone, TMP；
(1) preparation of standard working solution：
Each standard items 10mg is accurately weighed, in 10mL measuring bottles, is dissolved with methanol and is diluted to scale, be configured to 1mg/mL and respectively mark
Quasi- stock solution；For insoluble medicine, Enrofloxacin, Ciprofloxacin, Norfloxacin add a small amount of 0.1mol/L HCl solutions, Lome
Sha Xing adds a small amount of 0.1mol/L NaOH solutions, and Cefquinome adds appropriate dimethyl sulfoxide (DMSO) to make after its dissolving, with methanol surely to quarter
Degree；
Each Standard Reserving Solution is measured into 100mL measuring bottles, with methanol dilution to scale, hybrid standard intermediate solution is made；Wherein
Sulfamido, fluoroquinolones, woods can amine, dapsone, Tiamulin and TMP respectively take 0.4mL；Antipyretic-antalgic class and gold
Firm ethamine respectively takes 2mL；Cefquinome takes 4mL；Tetracyclines, tylosin, Tilmicosin, kitasamycin A5, Ornidazole and gold
Firm ethamine respectively takes 1mL；Beta-receptor activator class, amphenicols, atropine, droperidol and haloperole respectively take 0.2mL；A Zha
Grand, fenazil and diazepam is sent respectively to take 0.1mL；
Hybrid standard intermediate solution 0.1mL, 0.2mL, 0.5mL are pipetted respectively into 10mL measuring bottles, with methanol dilution to scale, system
Obtain LOQ concentration hybrid standards working solution, 2LOQ concentration hybrid standard working solutions and 5LOQ concentration hybrid standard working solutions；
(2) the analysis pre-treatment of testing sample：
1) extract：2 ± 0.02g of homogeneous sample is weighed in 50mL polypropylene centrifuge tubes, Na is successively added2EDTA solution and acetonitrile,
Methanol, ethyl acetate solution, vortex 10-30s are mixed, and vibrate 10-30min；5min is centrifuged with 5000-8000r/min rotating speeds, on
Clear liquid is transferred in 50mL polypropylene centrifuge tubes；Add extract solution residue is carried out to repeat extraction, merge extract solution；And in 40-
Nitrogen is blown to solution residue about 1-2mL at 50 DEG C；It is used as reserve liquid；
2) purify：Activated respectively with 5-8mL methanol and 3-6mL water using Agela Cleanert PEP-2 solid-phase extraction columns, will
Reserve liquid all crosses post, and coutroi velocity is about 1-3 drops/s；Eluted with leacheate, vacuum drains 3-5min；With elution,
Eluent is dried up in nitrogen at 30-40 DEG C；Accurate to add 20% acetonitrile solution 1-3mL dissolving constant volumes, be vortexed mistake after mixing
0.22 μm of microporous Nylon filter membrane；
(3) drafting of standard curve：Precision measures 1 μ g/mL mixed standard solutions in right amount, and blank sample matrix solution is configured to dense
Spend for 1,2,5,10,20,50,100ng/mL series standard solution, determined for liquid chromatography tandom mass spectrometry determination；With characteristic ion
Mass chromatography peak area is ordinate, and corresponding concentration of standard solution is abscissa, draws standard curve, obtains 79 kinds of antibacterials
The qualitative analysis chromatogram picture library of medicine；
(4) mass spectral analysis is carried out to the sample of the 2LOQ concentration containing each medicine quantitative limit LOQ described in step (3), obtained
The quasi-molecular ion of each medicine, and two characteristic ions：Qualitative ion and quota ion, it is as shown in the table：
(5) qualitative analysis：To passing through step 2) testing sample of methods described processing selects 1 parent ion and 2 characteristic ions,
Carry out disposable liquid analysis of hplc, the retention time and step 3 of determinand in sample) obtained by spectrogram storehouse during corresponding reservation
Between deviation it is right in the relative abundance standard liquid close with concentration of the qualitative ion of each component within ± 2.5%, and in sample
The relative abundance for the qualitative ion answered is compared, and deviation is no more than scope as defined in following table, then can determine that to exist in sample
Corresponding determinand：
(6) quantitative analysis：Under the best conditions of instrument, step 2 of learning from else's experience) methods described processing testing sample and step
1) the hybrid standard working solution prepared by carries out mass spectral analysis, obtains chromatographic peak area response, makees single-point or multiple spot calibration, uses
Quantified by external standard method；The characteristic ion of each determinand during the response of determinand and hybrid standard work also in testing sample solution
Response if testing sample solution concentration exceedes the range of linearity, all should use acetonitrile/water in the range of linearity of Instrument measuring
(V/V, 20:80) redeterminated after solution dilution；
Each medicament contg X is in terms of mass fraction in sample, and unit is milligrams per kilogram (mg/kg), is calculated by formula (1)：
<mrow>
<mi>X</mi>
<mo>=</mo>
<mfrac>
<mrow>
<mi>A</mi>
<mo>&times;</mo>
<mi>C</mi>
<mi>s</mi>
<mo>&times;</mo>
<mi>V</mi>
<mo>&times;</mo>
<msub>
<mi>V</mi>
<mn>2</mn>
</msub>
</mrow>
<mrow>
<mi>A</mi>
<mi>s</mi>
<mo>&times;</mo>
<mi>m</mi>
<mo>&times;</mo>
<msub>
<mi>V</mi>
<mn>1</mn>
</msub>
</mrow>
</mfrac>
<mo>&times;</mo>
<mi>n</mi>
<mo>-</mo>
<mo>-</mo>
<mo>-</mo>
<mrow>
<mo>(</mo>
<mn>1</mn>
<mo>)</mo>
</mrow>
</mrow>
In formula：
The peak area of determinand in A-testing sample solution；
The peak area of corresponding determinand in As-hybrid standard working solution；
Testing concentration in Cs-hybrid standard working solution, unit is micrograms per millilitre (μ g/mL)；
The volume of V-dissolving residue, unit is milliliter (mL)；
The quality of m-sample, unit is gram (g)；
V1The extracting liquid volume of-mistake solid-phase extraction column, unit is milliliter (mL)；
V2- total extracting liquid volume, unit is milliliter (mL)；
N-extension rate.
Measurement result is represented with the arithmetic mean of instantaneous value of parallel determination, as a result retains three effective digitals.
2. the screening method of 79 kinds of antibacterials in animal food according to claim 1, it is characterised in that：It is described dynamic
Physical property food is animal muscle based food, animal's liver, animal kidney, egg and milk fish.
3. the screening method of 79 kinds of antibacterials in animal food according to claim 1, it is characterised in that：The step
Suddenly the liquid-phase chromatographic analysis condition in (3) and step (5) is：Chromatographic column：Waters UPLCC18,100mm × 2.1mm
Id, 1.7 μm；Mobile phase A：The aqueous solution, Mobile phase B：Acetonitrile solution, mobile phase C：0.1% aqueous formic acid, mobile phase D：Methanol
Solution；Following table is positive and negative ion gradient elution mode；Flow velocity：0.1-0.3mL/min；Sample size：2μL；Column temperature：25-40℃；
Note：6 be linear change, and 1 is change immediately.
4. the screening method of 79 kinds of antibacterials in animal food according to claim 1, it is characterised in that：The step
Suddenly the mass spectral analysis condition in (4) and step (6) is：
5. the screening method of 79 kinds of antibacterials in animal food according to claim 1, it is characterised in that：The step
Suddenly the duration of oscillation in (2) is 30min, and extract solution is the mixed solution of acetonitrile and water, and volume ratio is 15:2；Leacheate is water 2-
5mL, eluent is acetonitrile solution and each 3-5mL of methanol solution.
6. the screening method of 79 kinds of antibacterials in animal food according to claim 1, it is characterised in that：The step
Suddenly Na in (2)2The concentration of EDTA solution is 0.1mol/L.
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