CN102323121A - A kind of from egg, the extraction simultaneously and the method for derivatization Amoxicillin and ampicillin - Google Patents
A kind of from egg, the extraction simultaneously and the method for derivatization Amoxicillin and ampicillin Download PDFInfo
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- CN102323121A CN102323121A CN201110175198A CN201110175198A CN102323121A CN 102323121 A CN102323121 A CN 102323121A CN 201110175198 A CN201110175198 A CN 201110175198A CN 201110175198 A CN201110175198 A CN 201110175198A CN 102323121 A CN102323121 A CN 102323121A
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Abstract
The present invention relates to the detection of veterinary drugs in food field, be specifically related to Amoxicillin in a kind of egg and extract simultaneously the ampicillin, the method for purification and derivatization.This method is after egg is processed homogenate, and the vibration of adding acetonitrile, mixing directly extract, and be centrifugal, and supernatant is shifted out, and repeats to extract 1-2 time, merges supernatant, promptly gets extract; In extract, add through ultrapure water saturated methylene chloride, fully vibration, mixing, centrifugal shift out upper water mutually, repeat to extract 1-2 time, merge water; At last salicylide is added in the aqueous phase extracted, carries out derivative reaction.This method simply, easily and fast, the recovery is high, average recovery rate is all more than 77.6%, in a few days and in the daytime RSD is lower than 9.6% and 14.8% respectively, meets the residual mensuration requirement of AOAC.
Description
Technical field
The present invention relates to the detection of veterinary drugs in food field, be specifically related to Amoxicillin in a kind of egg and extract simultaneously the ampicillin, the method for purification and derivatization.
Background technology
When the food residue thing is detected, if can detect multiple material simultaneously, will increase work efficiency greatly so, can multiple residue be extracted from sample simultaneously simultaneously but have only, just possibly set up detection method efficiently.Amoxicillin and ampicillin are white or off-white color crystalline powder, mildly bitter flavor, and slightly soluble in water, almost insoluble in ethanol.But sodium salt of the two or trihydrate are prone to dissolve in water, and the ethanol part omitted is dissolved.Method for distilling about Amoxicillin and ampicillin has many reports both at home and abroad.Extraction agent is generally potassium dihydrogen phosphate buffer solution, acetonitrile, methanol aqueous solution etc.The method of up to the present, both at home and abroad these two kinds of medicines in the egg being extracted simultaneously, deriving is not appeared in the newspapers as yet.
Summary of the invention
Can not be simultaneously from egg, extract the problem of Amoxicillin and ampicillin in order to solve prior art, the invention provides a kind ofly extract simultaneously, the method for purification and derivatization, this method simply, easily and fast, recovery height.
The technical scheme that the present invention adopted is: a kind of method of extraction and derivatization Amoxicillin and ampicillin from egg simultaneously; Be after egg is processed homogenate, the vibration of adding acetonitrile, mixing directly extract, and be centrifugal; Supernatant is shifted out; Repeat to extract 1-2 time, merge supernatant, promptly get extract;
In extract, add through ultrapure water saturated methylene chloride, fully vibration, mixing, centrifugal, through this liquid-liquid extraction step, medicine is transferred to the upper strata aqueous phase, and upper water is shifted out mutually, repeats to extract 1-2 time, merges water.At last salicylide is added in the aqueous phase extracted, carries out derivative reaction.Wherein the addition of salicylide depends on the content of sample Chinese traditional medicine, because salicylide only and sample Chinese traditional medicine generation derivative reaction; Through a large amount of experiments, the salicylide addition 25 μ L that filter out must be oversaturated, be fit to all egg samples, but the amount of water are advisable with 1.0-1.5 mL.
In the said method, acetonitrile is the chromatographically pure level, and addition is preferably 4 times of volumes of sample size.The methylene chloride addition equates with the addition of acetonitrile.
Adopt the inventive method can Amoxicillin in the egg and ampicillin be extracted simultaneously, and can detect usefulness for HPLC through the solution that salicylide derivatization step obtains.
The present invention adopts acetonitrile as extraction agent, and the extraction efficiency of two kinds of medicines is all very high, all reaches more than 77.4%.In addition, acetonitrile can effectively be removed protein, can reduce endogenous impurity with dichloromethane extraction again, helps sample purification.
Pass through optimization in this test to the derivatization condition; The best derivatization condition that filters out is: heat 60min in 25% trichloroacetic acid, 200 μ L (for derivative reaction provides acid environment, under the prerequisite of adding water volume 1.0-1.5mL, filtering out the concentration of trichloroacetic acid), salicylide 25 μ L, 100 ℃ of water-baths
Filter out best pre-treatment, the deriving method of Amoxicillin and ampicillin residues in the egg.This method simply, easily and fast, the recovery is high, average recovery rate is all more than 77.6%, in a few days and in the daytime RSD is lower than 9.6% and 14.8% respectively, meets the residual mensuration requirement of AOAC.
Description of drawings
Fig. 1 is the derivative reagent chromatogram;
Fig. 2 is egg blank sample chromatogram figure;
Fig. 3 is AMO (20ngmL
-1) and AMP (20ngmL
-1) standard solution chromatogram (100 μ L sample introduction);
Fig. 4 is AMO (25ngg
-1) and AMP (25ngg
-1) blank egg interpolation chromatogram (200 μ L sample introduction);
Fig. 5 is oral administration sample chromatogram figure.
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 is divided into groups
Choose 120 of the yellow chicken laying hens in 28 seas, capital in age in week, body weight 1.75 ± 0.20kg is divided into 4 groups at random, is respectively Amoxicillin test group, ampicillin test group, Amoxicillin and ampicillin bulk testing group, blank group, every group of 30 laying hens.The all single cage of laying hen is raised, and weighs respectively before the administration, numbers.Raised in advance for 1 week before the test, feeding does not contain the complete feed of any antibacterials, freely drinks water.
Dosage, approach and sample collecting
The former powder of former powder in Amoxicillin and ampicillin is made into 50 mgmL with aseptic ultrapure water
-1, 90mgmL
-1The WS, then soup directly is injected in the chicken stomach with the syringe that has a thin emulsion tube respectively.Each test group laying hen dosage is seen table 1:
Each test group laying hen dosage of table 1.
| Group | Amoxicillin (AMO) group | Ampicillin (AMP) group | Combined group | Control group |
| Dosage (mg/kg.b.w) | 25 | 60 | AMO 25+AMP 60 | 0.0 |
Test group administration every day 1 time, administration time be all at 8:00～9:00 in the morning, continuously 5d; Rise self administration of medication day to the drug withdrawal in the 20d; 17:30～18:00 collects each test group egg every day, and marked respectively writes down each test group egg number of every day; Can gather 13～16 eggs every group of every day, and egg sample is kept in the low temperature egg storehouse.
2, extraction of the present invention, extraction, purification and derivatization step:
1. with refiner egg is processed homogenate;
2. accurately take by weighing 5.0-6.0 g egg homogenate sample, be put in the 30-50 mL tool plug centrifuge tube;
3. add the 20-25mL acetonitrile solution and put mixing on the eddy mixer;
4. vortex oscillation mixing 2-3min, the centrifugal 10-15min of 8000-10000 * g, temperature is set to 10-15 ℃;
5. supernatant is transferred in another 50 mL tool plug centrifuge tube;
6. add 20-25mL and put mixing on the eddy mixer (make medicine be dissolved in the upper strata water, acetonitrile is dissolved in the dichloromethane solution) with ultrapure water saturated dichloromethane solution;
7. vortex oscillation mixing 2-3min, the centrifugal 10-15min of 6500-8000 * g, temperature is set to 20-25 ℃;
8. getting 1.0-1.5mL upper strata water has in the glass centrifuge tube of scale tool plug to 10-15mL;
9. in above-mentioned glass centrifuge tube, add trichloroacetic acid solution, the 25 μ L salicylide (see figure 1)s of 200 μ L25% respectively, vortex oscillation mixing 1-2min puts it in the boiling water bath, and derivatization reaction continues 60min;
10. sample is taken out in the reaction back, is cooled to room temperature, and sample transfer in the 2mL centrifuge tube, is washed 10mL glass centrifuge tube 1-2 time with 200-400 μ L 50% acetonitrile solution respectively, and merged in the 2mL centrifuge tube, is settled to the 2mL scale with 50% acetonitrile solution at last;
With the centrifugal 15min of 18000-20000 * g, cross 0.22 μ m filter membrane before the last machine, filtrating supplies HPLC to detect (seeing Fig. 2, Fig. 3, Fig. 5).
.The mensuration of the recovery accurately takes by weighing the blank egg homogenate after the 5.0g homogenate; Place the 50mL polypropylene centrifuge tube; Add the standard operation liquid of basic, normal, high three kinds of concentration simultaneously, make and contain Amoxicillin 5.0,25,125 ngg simultaneously respectively in the blank egg liquid
-1With ampicillin 5.0,25,125 ngg
-1Each addition establish 6 parallel, behind extraction, purification and the derivatization of above-mentioned sample, sample introduction 200 μ L make HPLC and detect (see figure 4).The HPLC testing conditions: chromatographic column is C
18, 5 μ m, 250mm * 4.6mm; Moving phase is: A is 0.01 molL
-1Potassium dihydrogen phosphate, transfer pH to 5.5 with 2M hydrochloric acid; B is a chromatographically pure level acetonitrile solution; Fluorescence detector, excitation wavelength 354 nm, emission wavelength 445 nm; Column temperature: 35 ~ 40 ℃; The gradient elution program is seen table 2.Add the recovery according to calculated by peak area.The AMO and the interpolation recovery of AMP and the result of the coefficient of variation see table 3 in the inventive method extraction egg.
Table 2. gradient elution program
| Sequence number | Time (min) | Flow velocity (mLmin -1) | A liquid (%) | B liquid (%) | The gradient curve shape |
| 1 | 0 | 1.0 | 65 | 35 | The linear gradient curve |
| 2 | 10 | 1.0 | 65 | 35 | The |
| 3 | 11 | 1.0 | 40 | 60 | The linear gradient curve |
| 4 | 18 | 1.0 | 40 | 60 | The linear gradient curve |
| 5 | 19 | 1.0 | 65 | 35 | The linear gradient curve |
| 6 | 22 | 1.0 | 65 | 35 | The linear gradient curve |
The interpolation recovery of AMO and AMP and the coefficient of variation (n=6) in table 3 egg
4.The mensuration of precision
Withinday precision is measured: will detect the remaining sample of the recovery and be mixed into 3 parts respectively, the sample that contains basic, normal, high 3 kinds of drug concentrations that the amount of obtaining is more by the drug concentration height.With the sample of same typical curve and 6 basic, normal, high 3 kinds of drug concentrations of replication of same HPLC, try to achieve in a few days (in batch) precision at same different time in a few days.
Day to day precision is measured: will detect the remaining sample of the recovery and be mixed into 3 parts respectively, the sample that contains basic, normal, high 3 kinds of drug concentrations that the amount of obtaining is more by the drug concentration height.The different daily different typical curves and the sample of 6 basic, normal, high 3 kinds of drug concentrations of replication of same HPLC in a week are tried to achieve in the daytime (between batch) precision.
Claims (3)
1. one kind is extracted from egg and the method for derivatization Amoxicillin and ampicillin simultaneously, is after egg is processed homogenate, adds the acetonitrile vibration, mixing directly extracts, and is centrifugal, and supernatant is shifted out, and repeats to extract 1-2 time, and the merging supernatant promptly gets extract;
In extract, add through ultrapure water saturated methylene chloride, fully vibration, mixing, centrifugal shift out upper water mutually, repeat to extract 1-2 time, merge water; At last salicylide is added in the aqueous phase extracted, carries out derivative reaction.
2. the method for extraction and derivatization Amoxicillin and ampicillin from egg simultaneously according to claim 1 is characterized in that said acetonitrile consumption is 4 times of volumes of sample size, and the methylene chloride consumption equates with the consumption of acetonitrile.
3. the method for extraction and derivatization Amoxicillin and ampicillin from egg simultaneously according to claim 1 and 2 is characterized in that the condition of described derivative reaction is: heat 60min in 25% trichloroacetic acid, 200 μ L, salicylide 25 μ L, 100 ℃ of water-baths.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102706991A (en) * | 2012-07-06 | 2012-10-03 | 扬州大学 | Method for simultaneously detecting residual amoxicillin, amoxicillin acid, diketopiperazine amoxicillin and ampicillin in egg |
| CN102721772A (en) * | 2012-07-06 | 2012-10-10 | 扬州大学 | Method for simultaneously extracting amoxicillin, amoxicillin acid, diketopiperazine amoxicillin and ampicillin from eggs |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5854390A (en) * | 1995-02-07 | 1998-12-29 | Lek, Tovarna Farmacevtskih In Kemicnih Izdelkov, D.D. | Chromatographic purification of vancomycin hydrochloride by use of preparative HPLC |
| CN101980012A (en) * | 2010-10-22 | 2011-02-23 | 舟山市质量技术监督检测院 | Method for performing high performance liquid chromatography with fluorescence detection on residues of ampicillin and amoxicillin in aqueous product |
| CN102072892A (en) * | 2010-11-05 | 2011-05-25 | 云南健牛生物科技有限公司 | Method for detecting amoxicillin residual quantity in milk and meat product |
-
2011
- 2011-06-27 CN CN201110175198A patent/CN102323121A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5854390A (en) * | 1995-02-07 | 1998-12-29 | Lek, Tovarna Farmacevtskih In Kemicnih Izdelkov, D.D. | Chromatographic purification of vancomycin hydrochloride by use of preparative HPLC |
| CN101980012A (en) * | 2010-10-22 | 2011-02-23 | 舟山市质量技术监督检测院 | Method for performing high performance liquid chromatography with fluorescence detection on residues of ampicillin and amoxicillin in aqueous product |
| CN102072892A (en) * | 2010-11-05 | 2011-05-25 | 云南健牛生物科技有限公司 | Method for detecting amoxicillin residual quantity in milk and meat product |
Non-Patent Citations (2)
| Title |
|---|
| B. LUGOBONI ET. AL: "Development and validation of a liquid chromatography/tandem mass spectrometry method for quantitative determination of amoxicillin in bovine muscle", 《JOURNAL OF CHROMATOGRAPHY B》 * |
| WENHONG LUO ET. AL: "Simultaneous Determination of Amoxicillin and Ampicillin in Bovine Milk by HPLC with Fluorescence Detection", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102706991A (en) * | 2012-07-06 | 2012-10-03 | 扬州大学 | Method for simultaneously detecting residual amoxicillin, amoxicillin acid, diketopiperazine amoxicillin and ampicillin in egg |
| CN102721772A (en) * | 2012-07-06 | 2012-10-10 | 扬州大学 | Method for simultaneously extracting amoxicillin, amoxicillin acid, diketopiperazine amoxicillin and ampicillin from eggs |
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Application publication date: 20120118 |