CN101747246A - Method for simultaneously extracting thiamphenicol, florfenicol and metabolite florfenicol amine from eggs - Google Patents
Method for simultaneously extracting thiamphenicol, florfenicol and metabolite florfenicol amine from eggs Download PDFInfo
- Publication number
- CN101747246A CN101747246A CN200910264442A CN200910264442A CN101747246A CN 101747246 A CN101747246 A CN 101747246A CN 200910264442 A CN200910264442 A CN 200910264442A CN 200910264442 A CN200910264442 A CN 200910264442A CN 101747246 A CN101747246 A CN 101747246A
- Authority
- CN
- China
- Prior art keywords
- florfenicol
- acetonitrile
- thiamphenicol
- egg
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Extraction Or Liquid Replacement (AREA)
Abstract
The invention relates to a method for simultaneously extracting thiamphenicol, florfenicol and metabolite florfenicol amine from eggs. The method comprises the following concrete steps: preparing eggs into homogenate; adding the homogenate to 30 to 50 percent acetonitrile solution and mixing the two well; performing extraction by use of ethyl acetate, acetonitrile and ammonia water in a volume ratio of 49:49:2; degreasing extract solution through saturated n-hexane; and blowing and drying the obtained product through a nitrogen blowing instrument. The method can be adopted to simultaneously extract thiamphenicol, florfenicol and metabolite florfenicol amine from eggs, thereby having the advantages of simplicity, convenience, quickness and high recovery rate.
Description
Technical field
The present invention relates to the detection of veterinary drugs in food field, be specifically related to that thiamphenicol, florfenicol and metabolite florfenicol amine extract simultaneously in a kind of egg, purification and spissated method.
Background technology
When the food residue thing is detected, if can detect multiple material simultaneously, will increase work efficiency greatly so, multiple residue can be extracted from sample simultaneously simultaneously but have only, just may set up detection method efficiently.Florfenicol is slightly soluble in water, is dissolved in organic solvent.Metabolite florfenicol amine polarity is strong, and is soluble in water, and the extracting method about thiamphenicol, florfenicol and florfenicol amine has many reports both at home and abroad.Extraction agent is generally ethyl acetate, acetonitrile, acetone etc.The method that these three kinds of medicines in the egg are extracted is not simultaneously appeared in the newspapers both at home and abroad.
Summary of the invention
Can not from egg, extract the problem of thiamphenicol, florfenicol and metabolite florfenicol amine simultaneously in order to solve prior art, the invention provides a kind ofly extract simultaneously, purification and concentration method, this method simply, easily and fast, rate of recovery height.
The technical solution adopted in the present invention is: a kind of method of extracting thiamphenicol, florfenicol and metabolite florfenicol amine simultaneously from egg, be after egg is made homogenate, add mixing in the 30%-50% acetonitrile solution, use ethyl acetate-acetonitrile of 49: 49: 2 of volume ratio-ammonia extraction again.
Said ethyl acetate-acetonitrile-ammonia extraction is with ethyl acetate-acetonitrile-ammoniacal liquor oscillation extraction, and is centrifugal, and supernatant liquor is shifted out, and re-extract 3-4 time merges supernatant liquor, promptly gets extracting solution.
Above-mentioned said extracting solution dries up on Nitrogen evaporator, uses the acetonitrile dissolved residue, uses through the saturated normal hexane of pure acetonitrile and repeats degreasing twice, dries up once more on the Nitrogen evaporator then, and is centrifugal with 30%-35% acetonitrile solution dissolved residue, filters, and gets filtrate, i.e. concentrated solution.
Adopt the inventive method the thiamphenicol in the egg, florfenicol and metabolite florfenicol amine can be extracted simultaneously, and can detect usefulness for HPLC through the concentrated solution that purifies, enrichment step obtains.
The present invention has compared the influence of different extraction agents to the rate of recovery, the result shows acetonitrile, acetone extraction efficient is relatively poor, the rate of recovery is lower, all less than 40%, though ethyl acetate extraction efficient is high, but that extracts organizes endogenous material more, simultaneously can not effectively extract the florfenicol metabolite florfenicol amine again, because florfenicol amine only just can be dissolved in organic solvent under alkaline condition, so this experiment adopts ethyl acetate-acetonitrile-ammoniacal liquor (49: 49: 2) as extraction agent, the extraction efficiency of three kinds of medicines is all very high, all reaches more than 86%, ethyl acetate-the acetonitrile of alkalescence can effectively extract florfenicol amine, and does not influence the extraction of other two kinds of medicines; Acetonitrile can effectively be removed partial protein, reduces endogenous impurity, helps sample purification.
Filter out the best pre-treating process of thiamphenicol in the egg, florfenicol and residues of metabolite florfenicol amine.This method simply, easily and fast, rate of recovery height, average recovery rate be all more than 86%, in a few days and in the daytime RSD is lower than 5.7% and 9.8% respectively, meets the residual mensuration requirement of AOAC.
Embodiment
The percentage concentration of related reagent among the present invention all refers to mass percentage concentration except that specifying.
1. the raising of experimental chicken, administration and sample collecting
1.1 experimental chicken is selected and grouping
Choose 175 of the yellow chickens in 28 seas, capital in age in week, body weight 1.75 ± 0.20kg is divided into 7 groups at random, and 25 every group, single cage is raised.A, B, C group are the thiamphenicol test group, and D, E, F group are the florfenicol test group, and the G group is the blank group.Weigh respectively before the administration, number.Raised in advance for 1 week before the test, feeding does not contain the complete feed of any antibacterials, freely drinks water.
1.2 dosage, approach and sample collecting
Each test group laying hen dispensing dosage is as shown in table 1.Earlier thiamphenicol and florfenicol are poured in the Capsules before the administration, directly put into respectively in the crop of chicken then.Test group is administered once every day, and administration time is all at 8:00-9:00 in the morning, and 5d continuously plays the egg of collecting every day to the drug withdrawal in the 15d self administration of medication day.
Table 1 dosage regimen
2 (1) extractions of the present invention, purification and enrichment step:
1. with refiner egg is made homogenate;
2. accurately take by weighing 2.0-3.0g egg homogenate sample, be put in the 30-50mL tool plug centrifuge tube;
3. add 1-1.5mL 30%-50% acetonitrile solution and put mixing on the eddy mixer;
4. add 4-6mL ethyl acetate-acetonitrile-ammoniacal liquor (49: 49: 2, V/V), vortex vibration mixing 1-2min extracts 15min with ultra-sonic oscillation again;
5. the centrifugal 10-15min of 6000-8000r/min, supernatant liquor is transferred in the 20-25mL Glass tubing;
6. repeat to extract 3-4 time, merge supernatant liquor;
7. in 35-55 ℃ of water-bath, dry up with nitrogen;
8. add the dissolving of 1.0mL-1.5mL acetonitrile, vortex vibration mixing 1-2min;
9. add the saturated normal hexane of 3.0-5.0mL (saturated) degreasing, discard the upper strata normal hexane, repeat degreasing 2 times with pure acetonitrile;
10. in 35-55 ℃ of water-bath, dry up with nitrogen; Freezing preservation;
With the dissolving of 1.0mL 30%-35% acetonitrile, be transferred in the 1.5mL centrifuge tube before the last machine;
(2) acetonitrile extraction, purification and enrichment step:
1. with refiner egg is made homogenate;
2. accurately take by weighing 2.0g egg homogenate sample, be put in the 50mL tool plug centrifuge tube;
3. add 1mL 35% acetonitrile solution and put mixing on the eddy mixer;
4. add the 4mL acetonitrile, vortex vibration mixing 1-2min extracts 15min with ultra-sonic oscillation again;
5. the centrifugal 10-15min of 6000-8000r/min, supernatant liquor is transferred in the 25mL Glass tubing;
6. repeat to extract 3-4 time, merge supernatant liquor;
7. in 35-55 ℃ of water-bath, dry up with nitrogen;
8. add the dissolving of 1.0mL acetonitrile, vortex vibration mixing 1-2min;
9. add the saturated normal hexane of 3.0mL (saturated) degreasing, discard the upper strata normal hexane, repeat degreasing 2 times with pure acetonitrile;
10. in 35-55 ℃ of water-bath, dry up with nitrogen; Freezing preservation;
(3) acetone extraction, purification and enrichment step:
1. with refiner egg is made homogenate;
2. with refiner egg is made homogenate;
3. accurately take by weighing 2.0g egg homogenate sample, be put in the 50mL tool plug centrifuge tube;
4. the phosphate buffered saline buffer that adds 2mL pH7.0, vortex vibration mixing 1-2min;
5. add 4mL acetone vortex vibration mixing 1-2min, extract 15min with ultra-sonic oscillation again;
6. the centrifugal 5min of 6000-8000r/min;
7. solution is transferred in another 15mL centrifuge tube;
8. add the 3mL methylene dichloride again in solution, vortex vibration mixing 1min extracts 15min with ultra-sonic oscillation again;
9. the centrifugal 5min of 6000-8000r/min;
10. discard the upper strata water, put on 45 ℃ of Nitrogen evaporators and dry up;
(11) with 1mL 35% acetonitrile dissolved residue, vortex vibration mixing 1min;
(12) add 3mL normal hexane (saturated with pure acetone) vortex vibration mixing 1min, degreasing discards the upper strata normal hexane, repeats degreasing 2 times;
(13) water is transferred to the 1.5mL centrifuge tube, and supernatant liquor is through 0.22 μ m membrane filtration, and filtrate is measured for HPLC.
Blank egg homogenate after the mensuration 3.0g homogenate of 3 rate of recovery is put in the 50mL polypropylene centrifuge tube, adds the standard operation liquid of different concns, makes that the thiamphenicol addition is respectively 0.015mg/kg, 0.05mg/kg, 0.50mg/kg in the blank egg liquid; The florfenicol addition is respectively 0.015mg/kg, 0.05mg/kg, 0.50mg/kg; The florfenicol amine addition is respectively 0.005mg/kg, 0.05mg/kg, 0.50mg/kg.Each addition establish 6 parallel, after the extraction of above-mentioned sample, purification and concentrating, sample introduction 80 μ L make HPLC and detect.The HPLC testing conditions: chromatographic column is C18,5 μ m, 250mm * 4.6mm; Moving phase is acetonitrile: NaH
2PO
4Solution (0.01mol/L contains 0.005mol/L sodium lauryl sulphate and 0.10% triethylamine)=30～35: 70～65 (V/V) is used 85%H
3PO
4Transfer pH to 4.5-4.8; Fluorimetric detector, excitation wavelength 225nm, emission wavelength 285nm; Flow velocity: 1.0～1.2mL/min; Column temperature: 25～30 ℃.Add the rate of recovery according to calculated by peak area.What the inventive method was extracted sample the results are shown in Table 2.Acetonitrile, acetone extraction sample the results are shown in Table 3, table 4.
The interpolation rate of recovery of thiamphenicol in table 2 egg (TAP), florfenicol (FF) and florfenicol (FFA) and the variation coefficient (n=6)
The interpolation rate of recovery of thiamphenicol in table 3 egg (TAP), florfenicol (FF) and florfenicol (FFA) and the variation coefficient (n=6)
The interpolation rate of recovery of thiamphenicol in table 4 egg (TAP), florfenicol (FF) and florfenicol (FFA) and the variation coefficient (n=6)
The mensuration of 4 precision will detect the remaining sample of the rate of recovery and be mixed into 3 parts respectively by the drug level height, obtain the sample of high, medium and low three kinds of drug levels, at the same sample of 6 high, medium and low three kinds of drug levels of replication in a few days, try to achieve in a few days (in batch) precision.The sample of 6 high, medium and low three kinds of drug levels of replication on the same day not in a week is tried to achieve in the daytime (between batch) precision.It the results are shown in Table 2.
Claims (3)
1. method of from egg, extracting thiamphenicol, florfenicol and metabolite florfenicol amine simultaneously, it is characterized in that, after egg made homogenate, add mixing in the 30%-50% acetonitrile solution, use ethyl acetate-acetonitrile of 49: 49: 2 of volume ratio-ammonia extraction again.
2. method according to claim 1 is characterized in that, said is with ethyl acetate-acetonitrile-ammoniacal liquor oscillation extraction with ethyl acetate-acetonitrile-ammonia extraction, centrifugal, and supernatant liquor is shifted out, and re-extract 3-4 time merges supernatant liquor, promptly gets extracting solution.
3. method according to claim 1, it is characterized in that, extracting solution dries up on Nitrogen evaporator, uses the acetonitrile dissolved residue, uses through the saturated normal hexane of pure acetonitrile again and repeats degrease, dry up once more on the Nitrogen evaporator then, with 30%-35% acetonitrile solution dissolved residue, centrifugal, filter, get filtrate, i.e. concentrated solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910264442A CN101747246A (en) | 2009-12-22 | 2009-12-22 | Method for simultaneously extracting thiamphenicol, florfenicol and metabolite florfenicol amine from eggs |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910264442A CN101747246A (en) | 2009-12-22 | 2009-12-22 | Method for simultaneously extracting thiamphenicol, florfenicol and metabolite florfenicol amine from eggs |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101747246A true CN101747246A (en) | 2010-06-23 |
Family
ID=42474932
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910264442A Pending CN101747246A (en) | 2009-12-22 | 2009-12-22 | Method for simultaneously extracting thiamphenicol, florfenicol and metabolite florfenicol amine from eggs |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101747246A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102565269A (en) * | 2012-03-05 | 2012-07-11 | 扬州大学 | Method for simultaneously detecting chloramphenicol, thiamphenicol, florfenicol and florfenicol amine residues in eggs |
| CN103242450A (en) * | 2013-05-30 | 2013-08-14 | 西北农林科技大学 | Preparation method of anti-florfenicol amine IgY (egg yolk immunoglobulin) |
| CN106645477A (en) * | 2016-12-21 | 2017-05-10 | 青岛海润农大检测有限公司 | Method for detecting florfenicol amine residue and application |
-
2009
- 2009-12-22 CN CN200910264442A patent/CN101747246A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102565269A (en) * | 2012-03-05 | 2012-07-11 | 扬州大学 | Method for simultaneously detecting chloramphenicol, thiamphenicol, florfenicol and florfenicol amine residues in eggs |
| CN103242450A (en) * | 2013-05-30 | 2013-08-14 | 西北农林科技大学 | Preparation method of anti-florfenicol amine IgY (egg yolk immunoglobulin) |
| CN106645477A (en) * | 2016-12-21 | 2017-05-10 | 青岛海润农大检测有限公司 | Method for detecting florfenicol amine residue and application |
| CN106645477B (en) * | 2016-12-21 | 2018-11-06 | 青岛海润农大检测有限公司 | A kind of remaining method of detection florfenicol amine and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101726553B (en) | Method for detecting thiamphenicol, florfenicol and residues of metabolite florfenicol amine in egg simultaneously | |
| US20220273014A1 (en) | Plant extraction method | |
| CN101747246A (en) | Method for simultaneously extracting thiamphenicol, florfenicol and metabolite florfenicol amine from eggs | |
| CN102645499A (en) | Method for simultaneously extracting chloramphenicol, thiamphenicol, florfenicol and florfenicol amine residues from eggs | |
| CN101396381A (en) | Valerian extract and preparation method and use thereof | |
| CN104777243B (en) | It is a kind of at the same determine the tuber of pinellia in organic acid, nucleosides and ephedrine HPLC methods | |
| CN105287682B (en) | preparation method of dandelion extract | |
| HAJ et al. | Effects of Otostegia persica (Burm.) Boiss on morphine withdrawal syndrome in mice | |
| KR101592251B1 (en) | Purification method of Phlorotannins from brown algae | |
| Schaneberg et al. | Quantitative and qualitative HPLC analysis of thermogenic weight loss products | |
| CN105250236B (en) | The preparation method of fish catechin preparation | |
| CN103217498A (en) | Method for detecting dicyandiamide in milk powder with LC-MS (liquid chromatography/mass spectrometry) and sample preparation method | |
| Göker et al. | Chromatographic separation and identification of sildenafil and yohimbine analogues illegally added in herbal supplements | |
| CN104666383B (en) | A kind of technique of microwave radiation exaraction Dysosma versipellis flavone compound | |
| CN109001362A (en) | Dispersive solid-phase extraction-liquid chromatography-mass spectrography/mass spectrography measures the method for Amitraz and metabolite residual quantity in royal jelly simultaneously | |
| CN108822228A (en) | A kind of method for quickly purifying of lichee polysaccharide | |
| CN102269741B (en) | Method for simultaneously detecting amoxicillin and ampicillin residues in eggs | |
| CN101988917A (en) | Extraction method applied to liquid-phase chip and for detecting multiple harmful substances contained in foods | |
| CN101618053B (en) | Preparation process of isatis root medical solution | |
| CN101108214B (en) | Method of separating and extracting natural base from coptis chinensis with latex membrane | |
| CN105037311B (en) | A kind of compound liquid phase method for extraction and purification of the online two dimension of apiolin | |
| CN104292226A (en) | Paliperidone amino acid derivatives and application thereof | |
| CN103962007B (en) | A kind of automatic programme control supersonic jet uniform filtration device | |
| Shahe et al. | Compatibility studies between propafenone and selected excipients used in the development of controlled release formulations | |
| CN102600378A (en) | Method for preparing medicament composition by adopting ultrafiltration process |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20100623 |