CN107219310B - The screening method of 110 kinds of drugs in a kind of feed - Google Patents
The screening method of 110 kinds of drugs in a kind of feed Download PDFInfo
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Abstract
The invention discloses a kind of screening methods of 110 kinds of drugs in feed, specifically includes the following steps: the preparation of standard working solution, the analysis pre-treatment of sample to be tested, foundation and qualitative analysis of database etc., utilize liquid chromatography-tandem quadrupole rod time of-flight mass spectrometer, highly sensitive MSMS function, parent ion and daughter ion in drug can accurately be determined, quickly judge the drug ingedient of addition, establish the detection method of a variety of antibacterials in feed again simultaneously, adding a variety of antibacterials for China's feedstuff industry has efficiently, accurate accurate detection means, belong to domestically leading level at present.The screening method that the present invention establishes, substantially increase disorderly filled out in feedstuff industry disorderly plus the phenomenon that, be the perfect more accurate accurate detection method of detection department, save more times, most experiment consumptive material is saved, the life of food-safe and human health has immeasurable contribution.
Description
Technical field
The invention belongs to feed detection technique fields, and in particular to the screening method of 110 kinds of drugs in a kind of feed.
Background technique
It is reported for the first time from Moore etc. (1946), antibacterials is added in feed, the daily gain of broiler chicken can be significantly improved.
Successively there are more than 60 Antibiotics usages in animal husbandry since then, in animal diseases control, improves efficiency of feed utilization, promotion livestock and poultry life
It is long etc. to have played important function.But it with a large amount of uses of antibacterials, does not abuse scientifically especially, bacterial drug resistance
And the problems such as medicament residue, becomes increasingly conspicuous, and causes countries in the world government and the great attention of insider.2006, European Union was comprehensive
Prohibit the application of antibacterials feed addictive.China is Production of Livestock and Poultry big country, in China's feed preparation, using anti-
The phenomenon that bacterium drug, is very universal.Chemical industry association and pharmaceuticals industry association statistical data in 2005 show that China resists every year
Bacterium medicine material output is about 210,000 tons, wherein there is 9.7 ten thousand tons (account for gross annual output amount 46.1%) for animal husbandry.Make
It include the Amoxicillin of mono- lactams of B, the gentamicin of aminoglycoside and neomycin, in big ring with the type of antibacterials
The erythromycin of esters, woods can amine clindamycin etc..It is reported that 90% in the antibacterials total amount that the whole world consumes every year
Be used in edible animal, and wherein 90% be intended only to improve food conversion ratio and as feed addictive come using.
Currently, being mainly using antibacterials problem in feed: 1, blindness, random medication outstanding problem in feed.I
State raiser (field) lacks scientific culture technology and animal doctor's knowledge, only with feeling medication；2, excess, long-time service antimicrobial
Object；3, occur using when disabling antibacterials phenomenon；Since 2000, country discloses successively some to be prohibited in Production of Livestock and Poultry
Drug." veterinary drug and other compound inventories of food animal disabling " announced such as Ministry of Agriculture's in April, 2002.It is wherein bright
Text forbids chloramphenicol and its salt, ester (including Chloramphenicol Succinate) and preparation to make the use of all purposes in all food animals.But
When in veterinary drug supervision and check or farm and it can be seen that it is more than using still prohibiting.4, people's medicine phenomenon for animals is still deposited
?.It is for animals that " veterinary drug residues regulations " prohibite people's medicine.In production, due to the of less types or people of veterinary drug antibacterials
Worry the quality of veterinary drug, often uses medical.5, it is not used by the regulation off-drug period.The length and drug of off-drug period is in animal
Intracorporal elimination factor is related with residual quantity, and related with animal species, dosage and administration route.Country is to some veterinary drugs
Especially medicated feed additive all defines the off-drug period, but when part raiser (field) uses the feed of drug containing additive
Seldom executed by the regulation off-drug period.
Due to the above reasons, to increase the chance of bacterial drug resistance, the body of the mankind is directly or indirectly compromised
Health destroys ecological environment.Therefore, quality of the fodder is the basis of human food's safety safely, establishes anti-microbial type in feed
Quickly and effectively detection method is very important drug.Existing drug multi-residue determination method mainly has liquid both at home and abroad at present
The methods of phase chromatography, liquid chromatography tandem time-of-flight mass spectrometry (TOFMS) and liquid chromatography tandem mass spectrometry.Existing detection method
It is comparatively mature to single kind drug research, drug how remaining detection method when establishing factor in need of consideration compared with
More, the physical chemical differences of each drug, pre-treating method is complex, and that there are sensitivity is low, medicament categories covering surface is narrow
The disadvantages of.Remain the technology special monitoring and early warning advantage that have based on drug, in recent years, each R&D institution, China is also gradually more
Pay attention to the research of drug multi-residue determination technology.It is domestic at present to measure in livestock products more left drugs simultaneously and to its accurate quantitative analysis
Method more lack, therefore be badly in need of establishing detection technique that is a kind of while measuring a variety of medicament residues.
Summary of the invention
To solve the above problems, the present invention provides a kind of screening method of 110 kinds of drugs in feed, it is residual to be added in feed
It stays antibacterials to provide a kind of complete detection method, enriches national industry residue detection standard, increase antibacterials
Screening technological means provides safety guarantee for national feed product and green cultivation.
The purpose of the present invention is what is realized with following technical proposals:
The screening method of 110 kinds of drugs in a kind of feed, specifically includes the following steps:
110 kinds of antibacterials are as follows:
(a), macrolides: tylosin, Tilmicosin, Tiamulin, azithromycin, clarithromycin, erythromycin, sieve
Erythromycin, Natamycin, oleandomycin, Spiramycin I, kitasamycin；
(b), fluoroquinolones: Enrofloxacin, Ciprofloxacin, sarafloxacin, Difloxacin, Nadifloxacin, Sparfloxacin,
Norfloxacin, oxolinic acid, pefloxacin, marbofloxacin, Orbifloxacin, flumequine, acidum nalidixicum, cinoxacin, Lomefloxacin, oxygen
Flucloxacillin, fleraxacin；
(c), sulfamido: daimeton, sulfamethoxazole, madribon, sulfaquinoxaline, sulfanilamide (SN)
Diformazan pyrimidine, cistosulfa, sulfapryidine, bosporon, ayerlucil, domian, kynix
Piperazine, sulfaclozine, fanasil, sulphadiazine, sulfamerazine, bacteresulf, sulfabenzamide, sulfanilamide (SN) nitre
Benzene, 5-methoxysulfadiazine, sulphathiazole；
(d), quinoxaline class: quinocetone, carbadox, mequindox, olaquindox；
(e), beta-lactam: ampicillin, oxacillin, flucloxacillin, cefapirin, cefotaxime, cefalexin,
Cefradine, Ceftiofur；
(f), woods can amine: lincomycin, clindamycin；
(g), Tetracyclines: terramycin, tetracycline, aureomycin, Doxycycline；
(h), amphenicols: chloramphenicol, Thiamphenicol, Florfenicol；
(i), itrofurans: furazolidone, furaltadone；
(j), nitro glyoxaline: Ornidazole, Dimetridazole, ipronidazole, secnidazole, metronidazole；
(k), anticoccidial class: Robenidine, clopidol；
(l), benzimidazole: febantel, flubendazole, albendazole, Phenbendasol, oxibendazole, oxfendazole,
Thiabendazolum, Albendazole sulfone, mebendazol；
(m), beta-receptor agonist: Clenbuterol, Ractopamine, salbutamol, Clorprenaline, Rabat sieve, class's cloth
Special sieve, Bbu Tero, bromine Boot sieve；
(n), sedative class: chlorpromazine, droperidol, haloperidol, azaperone, fenazil, Xylazine, Ao Shaxi
It dissolves, nitrazepam, diazepam, perphenazine；
(o), antiviral class: amantadine, Rimantadine, famciclovir；
(p), other: trimethoprim, atropine；
(1) preparation of standard working solution:
Each standard items 10mg is accurately weighed, in 10mL measuring bottle, scale is dissolved and is diluted to methanol, is configured to 1mg/mL
Each Standard Reserving Solution；For insoluble medicine, a small amount of 0.1mol/LHCl solution is added in Enrofloxacin, Ciprofloxacin, Norfloxacin,
A small amount of 0.1mol/L NaOH solution is added in Lomefloxacin, and Cefquinome, olaquindox and quinocetone add appropriate dimethyl sulfoxide to make it
It is fixed to scale with methanol after dissolution；
Each Standard Reserving Solution 1mL is measured respectively, and in 100mL measuring bottle, with methanol dilution to scale, being configured to concentration is 10
Working solution among μ g/mL hybrid standard；
Working solution 0.1mL among hybrid standard is taken, is diluted with acetonitrile+water mixed liquid, the work of 1 μ g/mL hybrid standard is made
Liquid；
(2) the analysis pre-treatment of sample to be tested:
1) 2 ± 0.02g sample is weighed in 50mL centrifuge tube, addition 6mL extracting solution, vortex 30s, mechanical shaking extraction 10min,
It is centrifuged 5min with the speed of 8000r/min, pipettes supernatant in 10mL centrifuge tube；Residue repeats to extract with 4mL acetonitrile, centrifugation
After merge supernatant, as reserve liquid；
2) amino solid-phase extraction column is activated with 6mL acetonitrile, is pipetted reserve liquid and is crossed column, and efflux is collected, molten with 50% acetonitrile
Liquid is settled to 5mL, mixes, and crosses 0.22 μm of microporous Nylon filter membrane；
(3) Database:
The hybrid standard working solution of 110 kinds of drugs is carried out liquid phase color by the molecular formula and molecular weight for determining 110 kinds of drugs
Spectrum-tandem mass spectrum measurement, obtains the total ion current figure of hybrid standard working solution, then the chromatography of ions figure of every kind of drug is extracted
Come, determines the retention time of every kind of drug；
The molecular weight of drug and retention time are inputted into MSMS analysis system, MSMS points are carried out to hybrid standard working solution
Analysis obtains the daughter ion scanning mass spectrogram of each drug, and according to the fracture mechanism of the fragment of each drug and fracture difficulty, determines each
The fragments characteristic ion of drug；
The type of each drug, title, molecular formula, retention time and fragments characteristic ion information are summarized, 110 kinds of medicines are established
Object screening database, as shown in the table:
1 database of table
(4) qualitative analysis:
1) liquid chromatography-tandem mass spectrometry analysis is carried out to the sample to be tested by the processing of step (2) the method, will obtained
Result be compared with the data in database, blank solvent there can be no quasi-molecular ions identical with standard solution, sample
The accurate mass number of parent ion and fragments characteristic ion should be consistent with standard solution, deviation should all≤0.05Da, sample chromatography
The retention time at peak, should be consistent with the retention time of standard solution, and allowable deviation is ± 0.2min, parent ion and fragment ion color
The signal-to-noise ratio (S/N) of spectral peak is all in 3:1 or more, and signal-to-noise ratio is with peak to peak (PtP) calculating, when full scan spectrum records, in sample
The relative abundance of fragments characteristic ion must be over the 10% of the characteristic ion spectrum abundance of standard solution reference, filter out satisfaction
The drug of qualitative condition, as doubtful positive sample；
2) for doubtful positive sample, according to the retention time of drug and molecular weight editor's MSMS method, and data are acquired,
The daughter ion of obtained doubtful substance scans mass spectrogram, is compared with the daughter ion of standard solution scanning mass spectrogram, in phase
Under same condition and similar concentration, the permission of main fragment ion relative abundance and the relative abundance of standard solution is inclined in sample
Difference is no more than range as defined in following table, then can determine that as there are corresponding determinands in sample:
The tolerance of the main fragment ion relative abundance of table 2 and the relative abundance of standard solution
。
Liquid-phase chromatographic analysis condition in the step (3) and step (4) are as follows: use Waters UPLCTMBEH C18 color
Column is composed, 2.1mm × 100mm, 1.7 μm, organic Phase Proportion is slowly varying to 95% from initial 2%, to guarantee all drugs all
It can effectively detect, gradient elution program see the table below 3:
3 chromatography gradient elution program of table
Note: ESI+:A:0.1% formic acid solution；B: acetonitrile, gradient elution；
ESI-:A: water；B: acetonitrile, gradient elution；
1 is variation immediately, and 6 be linear change.
Mass spectral analysis condition in the step (3) and step (4) is as shown in the table:
4 source parameters of table
5 screening mass spectrometry method parameter of table
Note: leucine enkephalin real-time online molecular weight calibration, molecular weight are 556.2771 (ESI+)/554.2615
(ESI-), sweep time 0.5s, sweep spacing 10s.
The volume ratio of the acetonitrile of the mixed liquor of acetonitrile and water and water is 1:1 in the step (1).
Extracting solution in the step (2) is the mixed solution of acetonitrile and water, and the volume ratio of acetonitrile and water is 1:1.
Further include the measurement of the detection limit to 110 kinds of drugs, measuring method are as follows: addition 10 μ g/mL standard solution, 100 μ L in
In 2g blank feed, measured after extracted, the signal-to-noise ratio S/N > 3 of drug shows that the detection of each drug is limited to 0.5mg/kg.
The present invention utilizes liquid chromatography-tandem quadrupole rod time of-flight mass spectrometer, highly sensitive MSMS function, Neng Goujing
True determines parent ion and daughter ion in drug, quickly judges the drug ingedient of addition.This method has wide range of applications,
The detection method for establishing a variety of antibacterials in feed again simultaneously, adding a variety of antibacterials for China's feedstuff industry has
Efficiently, accurate accurate detection means, belongs to domestically leading level at present.The screening method that the present invention establishes, substantially increases
Disorderly filled out in feedstuff industry disorderly plus the phenomenon that, be the perfect more accurate accurate detection method of detection department, when saving more
Between, most experiment consumptive material is saved, the life of food-safe and human health has immeasurable contribution.
Detailed description of the invention
Fig. 1 is the total ion current figure (ESI+) of mixed standard solution.
Fig. 2 is the extraction chromatography of ions figure (ESI+) of Enrofloxacin.
Fig. 3 is Enrofloxacin standard solution daughter ion scanning mass spectrogram.
Fig. 4 is lincomycin standard solution fragments characteristic the ion extraction chromatogram.
Fig. 5 is lincomycin standard solution parent ion extraction chromatography figure.
Fig. 6 is lincomycin fragments characteristic the ion extraction chromatogram in sample 1.
Fig. 7 is lincomycin parent ion extraction chromatography figure in sample 1.
Fig. 8 is lincomycin standard solution secondary fragment ions mass spectrogram.
Fig. 9 is lincomycin secondary fragment ions mass spectrogram in sample 1.
Figure 10 is albendazole standard solution fragments characteristic the ion extraction chromatogram
Figure 11 is albendazole standard solution parent ion extraction chromatography figure.
Figure 12 is albendazole fragments characteristic the ion extraction chromatogram in sample 5.
Figure 13 is albendazole parent ion extraction chromatography figure in sample 5.
Figure 14 is albendazole standard solution secondary fragment ions mass spectrogram.
Figure 15 is albendazole secondary fragment ions mass spectrogram in sample 5.
Figure 16 is the schematic diagram for the thinking that two or more complementary solid-phase extraction columns are used cooperatively.
Figure 17 is Carb and NH2With the use of when flow diagram.
Specific embodiment
The feed is mixed feed, concentrated feed, premixed feed.
110 kinds of antibacterials are as follows:
(a), macrolides (11 kinds): tylosin, Tilmicosin, Tiamulin, azithromycin, clarithromycin, red
Mycin, roxithromycin, Natamycin, oleandomycin, Spiramycin I, kitasamycin；
(b), fluoroquinolones (17 kinds): Enrofloxacin, Ciprofloxacin, sarafloxacin, Difloxacin, Nadifloxacin, department
Pa sand star, Norfloxacin, oxolinic acid, pefloxacin, marbofloxacin, Orbifloxacin, flumequine, acidum nalidixicum, cinoxacin, Lome
Sha Xing, Ofloxacin, fleraxacin；
(c), sulfamido (20 kinds): daimeton, sulfamethoxazole, madribon, sulfanilamide (SN) quinoline are disliked
Quinoline, sulfadimidine, cistosulfa, sulfapryidine, bosporon, ayerlucil, domian, sulphur
Amine methoxy piperazine, sulfaclozine, fanasil, sulphadiazine, sulfamerazine, bacteresulf, sulfabenzamide,
Sulfanitran, 5-methoxysulfadiazine, sulphathiazole；
(d), quinoxaline class (4 kinds): quinocetone, carbadox, mequindox, olaquindox；
(e), beta-lactam (8 kinds): ampicillin, oxacillin, flucloxacillin, cefapirin, cefotaxime, cephalo
Ammonia benzyl, Cefradine, Ceftiofur；
(f), woods can amine (2 kinds): lincomycin, clindamycin；
(g), Tetracyclines (4 kinds): terramycin, tetracycline, aureomycin, Doxycycline；
(h), amphenicols (3 kinds): chloramphenicol, Thiamphenicol, Florfenicol；
(i), itrofurans (2 kinds): furazolidone, furaltadone；
(j), nitro glyoxaline (5 kinds): Ornidazole, Dimetridazole, ipronidazole, secnidazole, metronidazole；
(k), anticoccidial class (2 kinds): Robenidine, clopidol；
(l), benzimidazole (9 kinds): febantel, flubendazole, albendazole, Phenbendasol, oxibendazole, Ao Fen
It rattles away azoles, thiabendazolum, Albendazole sulfone, mebendazol；
(m), beta-receptor agonist (8 kinds): Clenbuterol, Ractopamine, salbutamol, Clorprenaline, Rabat sieve,
Bambuterol, Bbu Tero, bromine Boot sieve；
(n), sedative class (10 kinds): chlorpromazine, droperidol, haloperidol, azaperone, fenazil, Xylazine, Austria
Sha Xi dissolves, nitrazepam, diazepam, perphenazine；
(o), antiviral class (3 kinds): amantadine, Rimantadine, famciclovir；
(p), other (2 kinds): trimethoprim, atropine.
1. the preparation of standard working solution:
Each standard items 10mg is accurately weighed, in 10mL measuring bottle, scale is dissolved and is diluted to methanol, is configured to 1mg/mL
Each Standard Reserving Solution；For insoluble medicine, a small amount of 0.1mol/LHCl solution is added in Enrofloxacin, Ciprofloxacin, Norfloxacin,
A small amount of 0.1mol/L NaOH solution is added in Lomefloxacin, and Cefquinome, olaquindox and quinocetone add appropriate dimethyl sulfoxide to make it
It is fixed to scale with methanol after dissolution；
Each Standard Reserving Solution 1mL is measured respectively, and in 100mL measuring bottle, with methanol dilution to scale, being configured to concentration is 10
Working solution among μ g/mL hybrid standard；
Working solution 0.1mL among hybrid standard is taken, is diluted with acetonitrile+water mixed liquid, the work of 1 μ g/mL hybrid standard is made
Liquid.
2. the analysis pre-treatment of sample to be tested:
2.1 take smashed test sample, as sample of having a try.
The storage of 2.2 air dryings is spare.
The screening of 2.3 extracting solutions and sample extraction
2.3.1 the selection of Extraction solvent
With reference to pertinent literature, we have investigated these three more common solution of acetonitrile, first alcohol and water.It has been respectively compared acetonitrile
And the effect that methanol different proportion aqueous solution extracts, find acetonitrile+water and methanol+water as extracting solution, the extraction of drug
Effect is all more satisfactory.But methanol+water sedimentation effect is poor, the muddy difficult purification of extracting solution, so selecting extraction efficiency height simultaneously
And the good acetonitrile+water of sedimentation effect is as extracting solution.After the rate of recovery for comparing different proportion acetonitrile water, 6mL is finally used
Water+acetonitrile (50+50, v/v) extracts, and residue repeats to extract with 4mL acetonitrile.
2.3.2 2g sample (being accurate to 0.01g) is weighed in 50mL centrifuge tube, and 6mL water+acetonitrile (50+50, v/v) is added,
Vortex 30s, mechanical shaking extraction 10min are centrifuged 5min with the speed of 8000r/min, pipette supernatant in 10mL centrifuge tube.Residue
Repeat to extract primary, centrifugation, merging supernatant, as reserve liquid with 4mL acetonitrile.
2.3.3 the determination of purification condition
According to experimental conditions, we devise following several purification schemes:
1) extracting solution direct purification grease removal
a.NH2Column: acetonitrile activation → loading → collected column liquid → nitrogen and blows → constant volume.
B. carbon black column: methylene chloride, methanol activation → loading → collected column liquid → nitrogen and blow → constant volume.
C. alumina column (acid, neutral, alkalinity): acetonitrile activation → loading → collected column liquid → nitrogen and blows → constant volume.
D. n-hexane grease removal.
2) extracting solution is dried with nitrogen, and Solid Phase Extraction column purification is crossed after being dissolved in water, and eluent is dried with nitrogen rear constant volume mistake
Machine in filter investigates purification and retention.The common solids extraction column such as MAX, MCX, HLB, SCX, carbon black is mainly investigated.Processing
Method is as follows:
A.MAX: methanol, water activation → loading → water, methanol elute the elution of → 5% formic acid methanol.
B.MCX, SCX: methanol, water activation → loading → water, methanol elute the elution of → 5% ammoniated methanol.
C.HLB: methanol, water activation → loading → water elution → methanol elution.
D.Carb: methylene chloride, methanol, water activation → loading → methanol elution.
3) since these extraction columns selectivity is stronger, and the nature difference of drug is very big, and a kind of pillar can not be to all
Drug is withed a hook at the end, therefore it is contemplated that two or more complementary solid-phase extraction columns is used cooperatively, thinking such as 16 institute of attached drawing
Show.
For example, Carb and NH2With the use of when flow chart it is as shown in Fig. 17.
Investigated the methods of n-hexane grease removal and Solid Phase Extraction column purification respectively, due to n-hexane and acetonitrile have it is certain mutual
Dissolubility, therefore acetonitrile saturation n-hexane is used to carry out grease removal, the discovery rate of recovery is lower, and reason may be interference component removal in matrix
Not exclusively, still detection is interfered, or is that a part of object dissolves in n-hexane layer during grease removal, caused certain
Loss.Investigated SCX column, MCX column, MAX column and HLB column pair, since various kinds of drug active group is different, retention difference compared with
Greatly, the macrolides rate of recovery is not retained between 64%~92%, but to sulfa drugs such as SCX column.It examines respectively
Nh 2 column, aluminium oxide (neutral, alkalinity and acidity) column and carbon black column have been examined to the rate of recovery of each drug.The result shows that aluminium oxide
Pillar is extremely low to tetracycline and the quinolone drugs rate of recovery, and carbon black column is poor to quinolone drugs yield, and nh 2 column pair
Protein has certain reservation, has achieved the purpose that purification, while all drugs have preferable recycling, can satisfy experiment
It is required that.
It is final to determine purification method are as follows: amino solid-phase extraction column is activated with 6mL acetonitrile, is pipetted reserve liquid and is crossed column, collects stream
Liquid out is settled to 5mL with 50% acetonitrile solution, mixes, and crosses 0.22 μm of microporous Nylon filter membrane.
3. instrument parameter is set
3.1 liquid chromatogram reference conditions
Chromatographic column: Waters UPLCTM BEH C18Column, column length 100mm, column internal diameter 2.1mm, 1.7 μm or comparable of granularity
Analytical column.
Column temperature: 40 DEG C.
Mobile phase:
ESI+:A phase: 0.1% formic acid solution, B phase: acetonitrile, gradient elution.
ESI-:A phase: water；B phase: acetonitrile, gradient elution.
Gradient elution program is shown in Table 3-1.
Flow velocity: 0.45mL/min.
Sample volume: 2 μ L.
Table 3-1 chromatography gradient elution program
Note: 1 is variation immediately, and 6 be linear change.
3.2 mass spectral analysis conditions
Since the medicament categories being related to are more, and pharmaceutical properties difference is big, it is impossible to which every kind of drug all reaches optimum detection item
Part.It is final to determine source parameters screening mass spectrometry method parameter after optimization, respectively as shown in table 3-2, table 3-3, at this
Under part, most of drug has preferable response.
Table 3-2 source parameters
Table 3-3 flight time mass spectrum method parameter
Note: leucine enkephalin real-time online molecular weight calibration, molecular weight are 556.2771 (ESI+)/554.2615
(ESI-), sweep time 0.5s, sweep spacing 10s.
4. the foundation of database:
The hybrid standard working solution of each drug is carried out liquid phase color by the molecular formula and accurate molecular weight for determining 110 kinds of drugs
Spectrum-tandem mass spectrum measurement, obtains the total ion current figure of hybrid standard working solution, as shown in Figure 1；Again the ion color of every kind of drug
Spectrogram extracts, and determines the retention time of every kind of drug, and by taking Enrofloxacin as an example, it is as shown in Figure 2 to extract result；
The molecular weight of drug and retention time are inputted into MSMS analysis system, MSMS points are carried out to hybrid standard working solution
Analysis obtains the daughter ion scanning mass spectrogram of each drug, and by taking Enrofloxacin as an example, the daughter ion scanning mass spectrogram of Enrofloxacin is as schemed
Shown in 3, and according to the fracture mechanism of the fragment of each drug and fracture difficulty, the fragments characteristic ion of each drug is determined；
The type of each drug, title, molecular formula, retention time and fragments characteristic ion information are summarized, established non-in 122
Prescription drug screening database, as shown in the table:
Table 4-1 database
5. the sample to be tested progress liquid chromatography-tandem mass spectrometry analysis measurement pair handled by step 2 the method, sample introduction
Sequentially are as follows:
Solvent blank is into 1 needle；
Whether standard working solution determines system within allowable error into 1 needle, RSD < 10%；
Solvent blank is into 1 needle；
Sample 1 is into 1 needle；
Solvent blank is into 1 needle；
…………
Alternately；
…………
Reagent blank is into 1 needle；
Standard working solution is into 1 needle；
…………
Note: any adjustment of sequence should have adequate cause to prove its reasonability.
6. qualitative analysis and result confirmation
6.1 qualitative analysis
The result that step 5 obtains is compared with the data in database, blank solvent there can be no with standard solution
Identical quasi-molecular ions, the parent ion of sample and the accurate mass number of fragments characteristic ion should be consistent with standard solution, and deviation is equal
Answer≤0.05Da, the retention time of sample chromatographic peak should be consistent with the retention time of standard solution, allowable deviation be ±
The signal-to-noise ratio (S/N) of 0.2min, parent ion and fragment ion chromatographic peak is all in 3:1 or more, and signal-to-noise ratio is in terms of peak to peak (PtP)
Calculate, when full scan spectrum records, in sample the relative abundance of fragments characteristic ion must be over the feature of standard solution reference from
Sub-light composes the 10% of abundance, filters out the drug for meeting qualitative condition, as doubtful positive sample.
6.2 results confirmation
For doubtful positive sample, according to the retention time of drug and molecular weight editor's MS/MS method, and data are acquired,
The daughter ion of obtained doubtful substance scans mass spectrogram, is compared with the daughter ion of standard solution scanning mass spectrogram, in phase
Under same condition and similar concentration, the permission of main fragment ion relative abundance and the relative abundance of standard solution is inclined in sample
Difference is no more than range as defined in following table, then can determine that as there are corresponding determinands in sample:
The tolerance of the main fragment ion relative abundance of table 6-1 and the relative abundance of standard solution
| Relative ion abundance (%) | >50 | > 20~50 | > 10~20 | ≤10 |
| The maximum deviation (%) of permission | ±20 | ±25 | ±30 | ±50 |
7. actual sample detects
Sample to be tested is detected in aforementioned manners, as a result as shown in table 7-1, positive findings are shown in Fig. 4-15.
Table 7-1 actual sample testing result
| Sample number into spectrum | Feed title | Sample source | Detect over the counter ingredient |
| 1 | Chicken batch | Farm | Lincomycin |
| 2 | Chicken concentrate feed | Feed Enterprise | It is not detected |
| 3 | Pig premix | Farm | It is not detected |
| 4 | Pig concentrate feed | Farm | It is not detected |
| 5 | Pig batch | Feed Enterprise | Albendazole |
8. detecting the determination of limit
In order not to cause the pollution of instrument, on sample before machine measurement, measured after at least diluting 1000 times to sample.By
After injection, solution and the extracted liquid dilution of soluble powder, the solution of upper machine measurement and the solution of standard items are substantially equivalent.Cause
This, we limit the detection that the detection of various drug standards is limited to this method.Add 10 μ g/mL standard solution, 100 μ L in
In 2g blank feed, measured after extracted, the signal-to-noise ratio S/N > 3 of drug shows that the detection of each drug is limited to 0.5mg/kg.
9. the rate of recovery is tested
It selects blank mixed feed, concentrated feed and premixed feed sample as test object, carries out mark-on test.?
0.5mg/kg concentration carries out recovery of standard addition experiment, and 3 parallel laboratory tests calculate relative standard deviation.110 kinds of the average of drug return
Yield and relative standard deviation are shown in Table 9-1.
Table 9-1 TIANZHU XINGNAO Capsul test result
What has been described above is only a preferred embodiment of the present invention, it is noted that for those skilled in the art,
Without depart from that overall concept of the invention, several changes and improvements can also be made, these also should be considered as of the invention
Protection scope.
Claims (4)
1. the screening method of 110 kinds of drugs in a kind of feed, it is characterised in that: specifically includes the following steps:
110 kinds of antibacterials are as follows:
(a), macrolides: tylosin, Tilmicosin, Tiamulin, azithromycin, clarithromycin, erythromycin, Luo Hong are mould
Element, Natamycin, oleandomycin, Spiramycin I, kitasamycin；
(b), fluoroquinolones: Enrofloxacin, Ciprofloxacin, sarafloxacin, Difloxacin, Nadifloxacin, Sparfloxacin, promise fluorine
Sha Xing, oxolinic acid, pefloxacin, marbofloxacin, Orbifloxacin, flumequine, acidum nalidixicum, cinoxacin, Lomefloxacin, oxygen fluorine are husky
Star, fleraxacin；
(c), sulfamido: daimeton, sulfamethoxazole, madribon, sulfaquinoxaline, sulfanilamide (SN) diformazan
Pyrimidine, cistosulfa, sulfapryidine, bosporon, ayerlucil, domian, kynix, sulphur
Amine chloropyrazine, fanasil, sulphadiazine, sulfamerazine, bacteresulf, sulfabenzamide, sulfanitran, sulphur
Amine is to Sulfamonomethoxine, sulphathiazole；
(d), quinoxaline class: quinocetone, carbadox, mequindox, olaquindox；
(e), beta-lactam: ampicillin, oxacillin, flucloxacillin, cefapirin, cefotaxime, cefalexin, cephalo
Draw fixed, Ceftiofur；
(f), woods can amine: lincomycin, clindamycin；
(g), Tetracyclines: terramycin, tetracycline, aureomycin, Doxycycline；
(h), amphenicols: chloramphenicol, Thiamphenicol, Florfenicol；
(i), itrofurans: furazolidone, furaltadone；
(j), nitro glyoxaline: Ornidazole, Dimetridazole, ipronidazole, secnidazole, metronidazole；
(k), anticoccidial class: Robenidine, clopidol；
(l), benzimidazole: febantel, flubendazole, albendazole, Phenbendasol, oxibendazole, oxfendazole, thiophene benzene
Up to azoles, Albendazole sulfone, mebendazol；
(m), beta-receptor agonist: Clenbuterol, Ractopamine, salbutamol, Clorprenaline, Rabat sieve, bambuterol,
Bbu Tero, bromine Boot sieve；
(n), sedative class: chlorpromazine, droperidol, haloperidol, azaperone, fenazil, Xylazine, Oxazepam, nitre
West dissolves, diazepam, perphenazine；
(o), antiviral class: amantadine, Rimantadine, famciclovir；
(p), other: trimethoprim, atropine；
(1) preparation of standard working solution:
Each standard items 10mg is accurately weighed, in 10mL measuring bottle, scale is dissolved and be diluted to methanol, 1mg/mL is configured to and respectively marks
Quasi- stock solution；For insoluble medicine, a small amount of 0.1mol/L HCl solution, Lome is added in Enrofloxacin, Ciprofloxacin, Norfloxacin
A small amount of 0.1mol/L NaOH solution is added in Sha Xing, and Cefquinome, olaquindox and quinocetone add appropriate dimethyl sulfoxide to make it dissolve
Afterwards, fixed to scale with methanol；
Each Standard Reserving Solution 1mL is measured respectively, and in 100mL measuring bottle, with methanol dilution to scale, being configured to concentration is 10 μ g/
Working solution among mL hybrid standard；
Working solution 0.1mL among hybrid standard is taken, is diluted with acetonitrile+water mixed liquid, 1 μ g/mL hybrid standard working solution is made；
(2) the analysis pre-treatment of sample to be tested:
1) 2 ± 0.02g sample is weighed in 50mL centrifuge tube, addition 6mL extracting solution, vortex 30s, mechanical shaking extraction 10min, with
The speed of 8000r/min is centrifuged 5min, pipettes supernatant in 10mL centrifuge tube；Residue repeats to extract with 4mL acetonitrile, after centrifugation
Merge supernatant, as reserve liquid；
2) amino solid-phase extraction column is activated with 6mL acetonitrile, is pipetted reserve liquid and is crossed column, collects efflux, fixed with 50% acetonitrile solution
Hold to 5mL, mix, crosses 0.22 μm of microporous Nylon filter membrane；
(3) Database:
The hybrid standard working solution of 110 kinds of drugs is carried out liquid chromatogram-string by the molecular formula and molecular weight for determining 110 kinds of drugs
Join mass spectroscopy, obtains the total ion current figure of hybrid standard working solution, then the chromatography of ions figure of every kind of drug is extracted, really
The retention time of fixed every kind of drug；
The molecular weight of drug and retention time are inputted into MSMS analysis system, MSMS analysis is carried out to hybrid standard working solution, is obtained
Daughter ion to each drug scans mass spectrogram, and according to the fracture mechanism of the fragment of each drug and fracture difficulty, determines each drug
Fragments characteristic ion；
The type of each drug, title, molecular formula, retention time and fragments characteristic ion information are summarized, 110 kinds of drug sieves are established
Database is looked into, as shown in the table:
1 database of table
(4) qualitative analysis:
1) liquid chromatography-tandem mass spectrometry analysis, the knot that will be obtained are carried out to the sample to be tested by the processing of step (2) the method
Fruit is compared with the data in database, blank solvent there can be no quasi-molecular ions identical with standard solution, sample it is female from
Son and the accurate mass number of fragments characteristic ion should be consistent with standard solution, deviation should all≤0.05Da, sample chromatographic peak
Retention time, should be consistent with the retention time of standard solution, and allowable deviation is ± 0.2min, parent ion and fragment ion chromatographic peak
Signal-to-noise ratio (S/N) all in 3:1 or more, signal-to-noise ratio is with peak to peak (PtP) calculating, when full scan spectrum records, feature in sample
The relative abundance of fragment ion must be over the 10% of the characteristic ion spectrum abundance of standard solution reference, filter out meet it is qualitative
The drug of condition, as doubtful positive sample；
2) for doubtful positive sample, according to the retention time of drug and molecular weight editor's MSMS method, and data are acquired, gained
The daughter ion of the doubtful substance arrived scans mass spectrogram, is compared with the daughter ion of standard solution scanning mass spectrogram, identical
The tolerance of main fragment ion relative abundance and the relative abundance of standard solution is not under condition and similar concentration, in sample
More than range as defined in following table, then can determine that as there are corresponding determinands in sample:
The tolerance of the main fragment ion relative abundance of table 2 and the relative abundance of standard solution
；
Liquid-phase chromatographic analysis condition in the step (3) and step (4) are as follows: use Waters UPLCTMBEH C18 chromatography
Column, 2.1mm × 100mm, 1.7 μm, organic Phase Proportion is slowly varying to 95% from initial 2%, to guarantee that all drugs can
Effectively detection, gradient elution program see the table below 3:
3 chromatography gradient elution program of table
Note: ESI+:A:0.1% formic acid solution；B: acetonitrile, gradient elution；
ESI-:A: water；B: acetonitrile, gradient elution；
1 is variation immediately, and 6 be linear change；
Mass spectral analysis condition in the step (3) and step (4) is as shown in the table:
4 source parameters of table
5 screening mass spectrometry method parameter of table
Note: leucine enkephalin real-time online molecular weight calibration, molecular weight are 556.2771 (ESI+)/554.2615 (ESI-),
Sweep time 0.5s, sweep spacing 10s.
2. the screening method of 110 kinds of drugs in feed according to claim 1, it is characterised in that: second in the step (1)
The volume ratio of acetonitrile and water in the mixed liquor of nitrile and water is 1:1.
3. the screening method of 110 kinds of drugs in feed according to claim 1, it is characterised in that: in the step (2)
Extracting solution is the mixed solution of acetonitrile and water, and the volume ratio of acetonitrile and water is 1:1.
4. the screening method of 110 kinds of drugs in feed according to claim 1, it is characterised in that: further include to 110 kinds of medicines
The measurement of the detection limit of object, measuring method are as follows: 10 μ g/mL standard solution, 100 μ L is in 2g blank feed for addition, surveys after extracted
Fixed, the signal-to-noise ratio S/N > 3 of drug shows that the detection of each drug is limited to 0.5mg/kg.
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