CN103389356A - Method for detecting polyether antibiotics in feed - Google Patents
Method for detecting polyether antibiotics in feed Download PDFInfo
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- CN103389356A CN103389356A CN2013103411699A CN201310341169A CN103389356A CN 103389356 A CN103389356 A CN 103389356A CN 2013103411699 A CN2013103411699 A CN 2013103411699A CN 201310341169 A CN201310341169 A CN 201310341169A CN 103389356 A CN103389356 A CN 103389356A
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Abstract
The invention relates to a method for detecting polyether antibiotics in feed. The method comprises the following steps of: passing through a mixed acidic mobile phase, and simultaneously separating lasalocid, monensin, salinomycin and narasin on a chromatographic separation column, namely extracting sample filtrate of the feed; removing air in the mixed acidic mobile phase by a degassing machine, then passing through a liquid phase pump, flowing into a high performance liquid chromatographic instrument via a safety switch, and adding the sample filtrate through a sample adding device; performing fluorescence detection on substances after separation by the chromatographic column to obtain a detection result of the lasalocid; introducing a mixed solution after detection into a reactor A through a backflow protector, and adding a derivative reagent A into the reactor A, wherein the reaction temperature is room temperature; then leading the mixed solution after reaction into a reactor B, and adding a derivative reagent B, wherein the reaction temperature is 90 DEG C; and detecting the solution after reaction so as to obtain the detection results of the monensin, the salinomycin and the narasin.
Description
Technical field
The invention belongs to antibiotic detection field, particularly the method for the multiple polyether antibiotics of a kind of disposable Simultaneous Determination.
Background technology
Lasalocid sodium, coban, salinomycin and NARASIN belong to polyether antibiotics together, are the ionophore class microbiotic that is produced by actinomycetes fermentation liquor.Be most popular coccidiostat in present poultry farming, mainly make an addition in feed and use, can increase efficiency of feed utilization, prevention global-worm illness.But along with its application in the animal diseases prevention deepens continuously, the coccidia drug resistance constantly strengthens, so also constantly increase of dosage, causes the residual continuous increase in animal products.Therefore be necessary the addition of polyether antibiotics medicine in feed is monitored, the assurance people's is healthy.
Generally adopting at present liquid phase chromatography to carry out microbiotic detects.In polyether antibiotics, lasalocid sodium self contains fluorophor, can directly with fluorescence detector, detect in neutral mobile phase.And the equal acomia color of polyether antibiotics coban, salinomycin and NARASIN group is end to absorb in ultra-violet absorption spectrum, can not direct-detection.But contain carboxyl and hydroxyl in their structures, can with aromatic aldehyde generation aldol reaction, detect with UV-detector after generating colored compound.But this reaction all need to be carried out under the environment of high temperature peracid, and at present the mobile phase that adopts of bibliographical information is all: the acidic mixed phase solution of methyl alcohol or acetonitrile and formic acid or glacial acetic acid, phosphoric acid solution.And lasalocid sodium can produce decompose and can not react and obtains desirable chromophoric group under this reaction environment, causes and can not detect.Have no at present the disposable pertinent literature report that detects simultaneously polyether antibiotics lasalocid sodium in Feed Sample, coban, salinomycin and NARASIN content, in practical operation, these four kinds of polyether antibioticses are to adopt distinct methods that sample is detected respectively, the method complex steps and increase testing amount.
Summary of the invention
The present invention has determined a kind of new acid mobile phase, and connected high performance liquid chromatography device and post-column derivation consersion unit, fluorescence detector, UV-detector, the content of lasalocid sodium, coban, salinomycin and NARASIN in disposable analyzing and testing feed simultaneously.This detection method is highly sensitive, high specificity, quick easy operating, can be used for trace detection.
In a kind of feed, the detection method of polyether antibiotics, is characterized in that, by mixing acid mobile phase, lasalocid sodium, coban, salinomycin and NARASIN separated on chromatography column; Concrete detecting step is as follows:
1) be 10% methanol solution to adding the 10ml volume ratio in the Powdered Feed Sample of 2g, shake 5min on turbine mixer, after standing 10min with the centrifugal 10min of 5200r/min rotating speed, get supernatant, repeat the said extracted step once, twice gained supernatant dried up to obtain residue with nitrogen, cross 0.22 μ m miillpore filter after dissolving described residue with the mixed liquor that the 1ml volume ratio is the methyl alcohol of 9:1 and water, obtain sample filtrate;
2) mix acid mobile phase and flow into high performance liquid chromatograph by liquid phase pump through safety switch after degasser is removed air, flow velocity is 0.7ml/min, and 40 ℃ of column temperatures add the described sample filtrate 10 μ l of step 1 by sample injector;
3) material after chromatographic column is separated in step 2 is carried out fluoroscopic examination, setting the fluorescence detector excitation wavelength is 310nm, and emission wavelength is 420nm, obtains the testing result of lasalocid sodium;
4) mixed liquor after fluoroscopic examination imports in the reactor A that volume is 0.1ml by reversed current protector with step 3;
5) in reactor A, add derivative reagent A with the speed of 0.3ml/min by derivative pump A in the described mixed liquor of step 3, temperature of reaction is room temperature, described derivating agent A is the concentrated sulphuric acid and methyl alcohol 4:96 mixing by volume gained;
6) be to add derivating agent B by derivative pump B in described mixed liquor to step 5 with the speed of 0.3ml/min in the reactor B of 1.4 ml at volume, temperature of reaction is 90 ℃, described derivating agent B be 4-hydroxyl-3-methoxylbenxaldehyde with methyl alcohol by weight/volume ratio 6:95 mixing gained;
7) the UV-detector wavelength is made as 520nm, solution after the described reaction of step 6 is detected, obtain coban, salinomycin and NARASIN testing result, the mixed liquor after detection, through back pressure regulator, flows into the waste liquid receiving flask.
It is methyl alcohol with 5% acetic acid take volume ratio as 9:1 mixing gained that the present invention is preferably the acid mobile phase of described mixing.
Being preferably the chromatography column that step 2 adopts is Z0RBAX SB-C18 type packed column.
Be preferably when safety switch is arranged on 510psi and open.
Preferred described back pressure regulator is set as 100psi.
The invention has the advantages that:
1) the acid mobile phase of the mixing of the present invention's use makes lasalocid sodium, coban, salinomycin and NARASIN separate on chromatography column simultaneously, meet multiple polyether antibiotics content requirement in disposable simultaneous quantitative qualitative determination Feed Sample, reduce and adopt different detection methods, separately process the workload of test sample, save time, testing result accurately and reliably;
2) adopt safety feature protective coloration spectrometer, described safety switch only when chromatographic analytical column forefront pressure reaches certain pressure, just allows the past column reaction system start-up operation; Reversed current protector is retaining valve, prevents from occurring the reagent siphon infringement chromatograph that backflows when the pump closed condition;
3) connect back pressure regulator after UV-detector, prevent mobile phase bumping or generation bubble in the flow cell of detecting device that in reactor B, temperature is higher, guarantee to react safety.
Description of drawings
Fig. 1 is the testing process block diagram of the embodiment of the present invention.
Fig. 2 is the mixed mark of coban, salinomycin, NARASIN and lasalocid sodium chromatographic resolution testing result figure in embodiment 1.
Embodiment
, below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are not used in and limit the scope of the invention for explanation the present invention.
The foundation of embodiment 1 standard working curve
(1) preparation of reaction reagent
Coban standard inventory solution preparation: accurately take a certain amount of coban standard specimen, dissolve also constant volume with methyl alcohol and mix, making coban concentration is 1mg/ml;
Salinomycin standard inventory solution preparation: accurately take a certain amount of salinomycin standard specimen, dissolve also constant volume with methyl alcohol and mix, making salinomycin concentration is 1mg/ml;
NARASIN standard inventory solution preparation: accurately take a certain amount of NARASIN standard specimen, dissolve also constant volume with methyl alcohol and mix, making NARASIN concentration is 1mg/ml;
Lasalocid sodium standard inventory solution preparation: directly using the concentration of purchasing is 100 μ g/ml standard items, perhaps has other concentration standard reference substances also can use;
The preparation of hybrid standard working fluid: draw respectively appropriate above-mentioned four kinds of standard inventory solution, adding volume ratio is the methanol/water solution of 9:1, dilution mixes, make coban in the hybrid standard product working fluid that is made into, the concentration of salinomycin and NARASIN is respectively 5,10,20,30,50,60,100 μ g/ml, and the concentration of lasalocid sodium corresponds to 5,10,20,30,40,60,80 μ g/ml;
Mix the preparation of acid mobile phase: get 5ml chromatographic grade acetic acid water and be settled to 100ml, then get the above-mentioned acetic acid aqueous solution of 100ml and be added in the 1000ml volumetric flask and use methanol constant volume, mix;
The preparation of derivative reagent A: get the 40ml concentrated sulphuric acid and slowly add in the volumetric flask that methyl alcohol is housed, with methanol constant volume,, to 1000ml, mix;
?The preparation of derivative reagent B: take the 60g 4-hydroxyl-3-methoxylbenxaldehyde, with 950ml methyl alcohol, dissolve, mix.
(2) experimental procedure
A) open the high performance liquid chromatograph device, will mix acid mobile phase and remove mobile phase solution Air through degasser, the liquid phase pump flow velocity is set as 0.7ml/min.When the analytical column forefront pressure of high performance liquid chromatograph reached 510psi, safety switch allowed to start the operation of past column reaction system.By injector with the hybrid standard working fluid sample introduction 10 μ l that prepare.Adopt Z0RBAX SB-C18 chromatography column (4.6mmX250mm, i.d, 5 μ m), column temperature is set as 40 ℃;
B) component substance after the chromatographic column separation of flowing through is carried out fluoroscopic examination, setting the fluorescence detector excitation wavelength is 310nm, and emission wavelength is 420nm, obtains the testing result of lasalocid sodium;
C) mixed liquor of step b after fluoroscopic examination imported in the reactor A that volume is 0.1ml by reversed current protector;
D) in reactor A, add derivative reagent A with the speed of 0.3ml/min by derivative pump A in the described mixed liquor of step b, temperature of reaction is room temperature;
E) be to add derivating agent B by derivative pump B in described mixed liquor to steps d with the speed of 0.3ml/min in the reactor B of 1.4 ml at volume, temperature of reaction is 90 ℃;
F) ultraviolet-visible detector being detected wavelength set is 520nm, and solution after the described reaction of step e is detected, and obtains coban, salinomycin and NARASIN testing result; Mixed liquor after testing, by the 100psi back pressure regulator, flows into the waste liquid receiving flask.
(3) experimental result
According to the described method of step (2), hybrid standard product solution is measured (each concentration triplicate), its average peak area value is as shown in table 1:
Table 1 variable concentrations polyether antibiotics average peak area (n=3)
Concern the drawing standard working curve with average peak area (Y) and concentration (X).The coban standard working curve is: Y=10.013X+2.1019, correlation coefficient r=0.9968; The salinomycin standard working curve is: Y=1.9833X+0.6064, correlation coefficient r=0.9997; The NARASIN standard working curve is: Y=1.8796X+1.6667, correlation coefficient r=0.9996; The lasalocid sodium standard working curve is: Y=0.6966X+0.318, correlation coefficient r=0.9996.
The mixed mark of coban, salinomycin, NARASIN and lasalocid sodium chromatogram as shown in Figure 2, four kinds of medicines can thoroughly separate in analyzing and testing, explanation is under the inventive method testing conditions, and four kinds of polyether antibiotics medicines can disposablely obtain effectively separating and detecting simultaneously.
Test experience is reclaimed in embodiment 2 checkings, interpolation
, as blank sample, be ground into Powdered the Feed Sample that do not contain coban, salinomycin, NARASIN and lasalocid sodium.Take the 2g sample in the 50ml centrifuge tube, add respectively suitable coban, salinomycin, NARASIN and lasalocid sodium standard solution and make the mixed fodder sample, make the concentration of four kinds of microbiotic in the final filtrate of mixed fodder sample be respectively 10,20,60 μ g/ml.Be converted into concentration in the mixed fodder sample and be respectively 5,10,30 mg/kg.
Be in 10% methanol solution to adding the 10ml volume ratio in above-mentioned mixed fodder sample, shake 5min on turbine mixer,, with the centrifugal 10min of 5200r/min rotating speed, get supernatant after standing 10min, then repeat the said extracted step once.Merge twice supernatant and dry up to obtain residue with nitrogen, after dissolving described residue with the mixed liquor that the 1ml volume ratio is the methyl alcohol of 9:1 and water, cross 0.22 μ m miillpore filter, obtain sample filtrate.Press machine testing on the described method of embodiment 1, acquired results is as shown in table 2:
Table 2 polyether antibiotics adds recovery experimental result
Experimental result shows: when interpolation concentration is three kinds of concentration of 10,20,60 μ g/ml in blank Feed Sample.The coban recovery is 73.41-75.51%, and the salinomycin recovery is 75.85-87.01%, and the NARASIN recovery is 78.29-98.38%, and the lasalocid sodium recovery is 79.24-99.87%.Can meet the technical requirement of feed Chinese traditional medicine content detection.
Claims (5)
1. the detection method of polyether antibiotics in a feed, is characterized in that, by mixing acid mobile phase, La Shaluo rhzomorph, coban, salinomycin and NARASIN separated on chromatography column; Concrete detecting step is as follows:
1) be 10% methanol solution to adding the 10ml volume ratio in the Powdered Feed Sample of 2g, shake 5min on turbine mixer,, with the centrifugal 10min of 5200r/min rotating speed, get supernatant after standing 10min, repeat the said extracted step once; Twice gained supernatant dried up to obtain residue with nitrogen, cross 0.22 μ m miillpore filter after dissolving described residue with the mixed liquor that the 1ml volume ratio is the methyl alcohol of 9:1 and water, obtain sample filtrate;
2) mix acid mobile phase and flow into high performance liquid chromatograph by liquid phase pump through safety switch after degasser is removed air, flow velocity is 0.7ml/min, and 40 ℃ of column temperatures add the described sample filtrate 10 μ l of step 1 by sample injector;
3) material after chromatographic column is separated in step 2 is carried out fluoroscopic examination, setting the fluorescence detector excitation wavelength is 310nm, and emission wavelength is 420nm, obtains the testing result of La Shaluo rhzomorph;
4) mixed liquor after fluoroscopic examination in step 3 is imported in the reactor A that volume is 0.1ml by reversed current protector;
5) in reactor A, add derivative reagent A with the speed of 0.3ml/min by derivative pump A in the described mixed liquor of step 3, temperature of reaction is room temperature, described derivating agent A is the concentrated sulphuric acid and methyl alcohol 4:96 mixing by volume gained;
6) be to add derivating agent B by derivative pump B in described mixed liquor to step 5 with the speed of 0.3ml/min in the reactor B of 1.4 ml at volume, temperature of reaction is 90 ℃, described derivating agent B be 4-hydroxyl-3-methoxylbenxaldehyde with methyl alcohol by weight/volume ratio 6:95 mixing gained;
7) the UV-detector wavelength is made as 520nm, solution after the described reaction of step 6 is detected, obtain coban, salinomycin and NARASIN testing result, the mixed liquor after detection, through back pressure regulator, flows into the waste liquid receiving flask.
2. a kind of polyether antibiotics detection method according to claim 1, is characterized in that the acid mobile phase of described mixing is methyl alcohol with 5% acetic acid take volume ratio as 9:1 mixing gained.
3. a kind of polyether antibiotics detection method according to claim 1 is characterized in that: the chromatography column that step 2 adopts is Z0RBAX SB-C18 type packed column.
4. according to claim 1-3 described a kind of polyether antibiotics detection methods, is characterized in that when described safety switch is arranged on 510psi opening.
5. a kind of polyether antibiotics detection method according to claim 4, is characterized in that described back pressure regulator is set as 100psi.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105527366A (en) * | 2015-12-01 | 2016-04-27 | 赵林萍 | Detection method for antibiotic residues in feeds |
| CN112505167A (en) * | 2021-02-01 | 2021-03-16 | 安徽省公众检验研究院有限公司 | HPLC method for detecting purity of lasalocid sodium |
| CN113896798A (en) * | 2021-09-29 | 2022-01-07 | 中国农业大学 | Lasacosin and salinomycin single-chain antibody and bispecific single-chain antibody and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0290998A2 (en) * | 1987-05-11 | 1988-11-17 | F. Hoffmann-La Roche Ag | Anticoccidial composition |
| CN1677107A (en) * | 2005-03-14 | 2005-10-05 | 江苏省农业科学院 | Immune antibody for testing residual of polyether-like antibiotic and use thereof |
-
2013
- 2013-08-07 CN CN2013103411699A patent/CN103389356A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0290998A2 (en) * | 1987-05-11 | 1988-11-17 | F. Hoffmann-La Roche Ag | Anticoccidial composition |
| CN1677107A (en) * | 2005-03-14 | 2005-10-05 | 江苏省农业科学院 | Immune antibody for testing residual of polyether-like antibiotic and use thereof |
Non-Patent Citations (4)
| Title |
|---|
| ELLIOTT C T 等: "Methods for the detection of polyether ionophore residues in poultry", 《ANALYST》, vol. 123, 30 June 1998 (1998-06-30), pages 45 - 56 * |
| GERHARDT G C 等: "Determination of ionophores in the tissues of food animals by liquid chromatography", 《FOOD ADDITIVES & CONTAMINANTS》, vol. 12, no. 6, 10 January 2009 (2009-01-10), pages 731 - 737 * |
| 李娜 等: "聚醚类抗生素残留检测方法的研究进展", 《家禽科学》, 31 May 2007 (2007-05-31), pages 38 - 40 * |
| 陈明 等: "饲料中聚醚类抗生素的柱后衍生同时测定方法研究", 《中国饲料》, no. 20, 31 October 2005 (2005-10-31) * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105527366A (en) * | 2015-12-01 | 2016-04-27 | 赵林萍 | Detection method for antibiotic residues in feeds |
| CN112505167A (en) * | 2021-02-01 | 2021-03-16 | 安徽省公众检验研究院有限公司 | HPLC method for detecting purity of lasalocid sodium |
| CN113896798A (en) * | 2021-09-29 | 2022-01-07 | 中国农业大学 | Lasacosin and salinomycin single-chain antibody and bispecific single-chain antibody and application thereof |
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Application publication date: 20131113 |