CN109932459A - A kind of method of avilamycin in quantitative detection chicken alimentary canal - Google Patents
A kind of method of avilamycin in quantitative detection chicken alimentary canal Download PDFInfo
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Abstract
The present invention provides a kind of method of avilamycin content in quantitative detection chicken alimentary canal, carry out that standard curve is drawn out in detection, acquisition chicken alimentary canal sample extracts and purify preparation sample detection liquid, draws avilamycin in the working curve in chicken alimentary canal, test sample liquid record peak area, the content of avilamycin in calculating sample including preparing avilamycin standard items；Eluent gradient elution protocol is adjusted by improving the concentration of ammonium acetate and flow velocity, improves separating degree between each peak, and mobile phase does not need to adjust pH value, operation is simple by the present invention.The extractant of avilamycin selects acetone in chicken alimentary canal, selects methylene chloride to carry out second extraction after grease removal, is finally extracted with silica gel extraction column, reduces impurity in sample, the accuracy and high sensitivity of detection, the rate of recovery reach testing requirements.
Description
Technical field
The present invention relates to substance detection technique fields in animal alimentary canal, and in particular in a kind of quantitative detection chicken alimentary canal
The method of avilamycin.
Background technique
Avilamycin is a kind of growth-promoting class antibiotic, gram-positive bacteria is mainly acted on, frequently as feed addition
Agent is applied to clinic to promote growth of animal, prophylactic treatment disease.The growth promotion mechanism of avilamycin mainly passes through saving
The consumption of glucose in alimentary canal, improves the generation of volatile fatty acid, to reduce alimentary canal intracellular metabolite product.Can additionally it increase
Add the deposition of nitrogen in animal body so that the utilization rate of protein and the increase of partial amino-acid digestibility in feed, reach promotion with this
The effect of growth.The antibacterial action mechanism of avilamycin is mainly to interfere the synthesis of bacterio protein, studies have shown that Avila
Mycin can reduce the adhesive type of Escherichia coli in alimentary canal, by inhibiting the growth of bacterial flagellum and the adherency of bacterium, to reach
Bacteriostasis.Pertinent literature is research shows that avilamycin effectively can control and treat the necrosis of chicken caused by C.perfringens
Property enteritis.Therefore avilamycin can be used as antimicrobial for preventing and treating the gangrenosum acne intestines that C.perfringens causes poultry
Inflammation has good development prospect.
Animal derived bacterial drug resistance has been the major issue of global concern, for reinforce antimicrobial for animals treatment level,
Reduce bacterial drug resistance, ensure the good development of the healthy and safe and animal husbandry of livestock and poultry, establish and improve antimicrobial for animals to
Prescription case is most important.The dosage regimen of foundation will make the distributed density of drug in chicken body be higher than in drug resistance selection mutation window,
Therefore the present invention establishes the quantitative detecting method of avilamycin in a breeder alimentary canal, judges that avilamycin is digested in chicken
Whether the content in road has optimum efficiency.
Early period is mainly liquid chromatography-mass spectrography serial connection technology and high performance liquid chromatography about the detection method of avilamycin
Method-evaporative light scattering detector (ELSD) is used in series, and the expense of operation scheme is costly, complicated for operation, and at present
Extracting method about avilamycin is confined to the muscle of pig and rat, fat, the blood plasma and tissue of carp, and disappears for chicken
Change the avilamycin in road and detects a kind of simple effective method not yet.
Summary of the invention
The present invention in order to overcome the deficiencies of the prior art, provides the detection side HPLC of avilamycin in a breeder alimentary canal
Method can effectively reduce the interference of impurity in alimentary canal, realize accurate quantitative analysis using reasonable extracting method and quantitative approach, build
Vertical one kind easy to operate, quick, high sensitivity, accurately and reliably avilamycin detection method can accurately detect that chicken digests
The content of avilamycin in road.
In order to achieve the above objectives, The technical solution adopted by the invention is as follows:
The method of avilamycin, includes the following steps: in a kind of quantitative detection chicken alimentary canal
(1) prepare avilamycin standard solution and sample extracting solution to be detected；
(2) draw standard curve: the standard solution of 3-5 parts of various concentrations, which is respectively used to HPLC measurement type of elution, is
Gradient elution, record standard product chromatogram and peak area carry out linear regression analysis；
(3) accuracy and precision: accuracy and precision are indicated with the rate of recovery and the coefficient of variation respectively；Blank chicken disappears
Change the avilamycin that high, medium and low three concentration is added in road content, according to the extracting method system of sample extracting solution to be detected
Standby sample purification liquid is detected for HPLC；Each concentration is repeated 5 times on the same day, is averaged, and is calculated in a few days relative error and is
The in a few days coefficient of variation；Each concentration repeats 5 days；Average value and relative error are calculated, the coefficient of variation in the daytime is obtained；The rate of recovery uses
Single-point calibration method calculates, and formula is as follows:
X=AV/ASW × 100% (a)
In formula: the determinand rate of recovery in X- sample；The peak area of A- sample；V- sample extracts constant volume after drying, single
Position mL；The peak area of As- standard working solution；
W: volume of sample, Unit/mL；
(4) it draws working curve: taking blank chicken alimentary canal content, in the Avila that 3-5 parts of various concentrations are wherein added
Mycin is extracted according to the identical extracting method of sample extracting solution, HPLC detection；In triplicate afterwards by gained peak area with it is right
Answer concentration to make linear regression, draw working curve and calculate regression equation of the avilamycin in chicken alimentary canal content and
Related coefficient；
(5) actual sample measurement and result calculate: after chicken single oral gavage avilamycin, acquire its alimentary canal content into
It goes and extracts with HPLC detection, record chromatogram and peak area, and list is calculated separately out with step (4) resulting equation of linear regression
After secondary administration in different time points chicken alimentary canal avilamycin content.
As a further improvement of the present invention, HPLC testing conditions are as follows:
Chromatographic column: Agilent SB-Aq, 250 × 4.6mm (i.d.), 5 μm；
Mobile phase: acetonitrile (B) -100mM ammonium acetate (A)；
Ultraviolet detection wavelength: 295nm；
Sample volume: 35 μ L；
Flow velocity: 0.5mL/min；
Column temperature: 30 DEG C；
Mobile phase elution protocol is as follows:
Further, the solvent of the avilamycin standard solution is methanol, and concentration is 0.1~20 μ g/mL.
Further, the concentration of acetonitrile is 100% in the mobile phase, and the concentration of ammonium acetate is 0.1mol/L.
As a further improvement of the present invention, sample extracting solution to be detected the preparation method is as follows:
(1) into ileal contents to be detected plus acetone be used as extracting solution, be vortexed, be centrifuged, take supernatant, thereto according to
The secondary n-hexane progress grease removal that NaCl weak solution and saturation is added；It using methylene chloride as extractant, is vortexed, is centrifuged, in removing
The n-hexane layer of layer, retains the dichloromethane layer of lower layer, and dry up under nitrogen atmosphere；Solid phase residue is redissolved with chloroform,
It is to be clean；
(2) Solid phase extraction: selecting silica gel extraction column, first uses methanol, chloroform activation, with chloroform and acetone
Mixed liquor eluted and eluted, collect eluent, and dried up under nitrogen atmosphere, with initial liquid phase (i.e. 100mM acetic acid
Ammonium: acetonitrile=57:43) dissolved residue, obtain sample extracting solution to be detected.
Preferably, the concentration of the NaCl weak solution is 10wt%.
Preferably, the nitrogen atmosphere is 50 DEG C of nitrogen atmospheres.
Preferably, the leacheate is the chloroform and acetone mixture that volume ratio is 4:1, and eluent is volume ratio 3:
7 chloroform and acetone mixture.
Compared with prior art, the technical effect that the present invention generates is as follows:
Method accuracy of the invention is high, and accuracy and sensitivity are good, and impurity is less, mould as Avila in chicken alimentary canal
The detection method of content of element provides good directive significance in the intracorporal metabolism distribution detection of chicken to avilamycin；This hair
It is bright to be improved for conventional HPLC method, flow velocity is become into 0.5mL/min by 1mL/min, flows decrease makes drug target
The retention time of absorption peak increases, and delays appearance time, increases the separating degree between each peak, so that the spirit at drug target peak
Sensitivity increases, and readjusts to leacheate and eluent, and compared with conventional eluent, the rate of recovery is improved.In the present invention
In eluent, chloroform is added, enhances in silica gel extraction column with the binding force of drug, improves the rate of recovery.Chicken alimentary canal
The extractant of middle avilamycin selects acetone, selects methylene chloride to carry out second extraction after grease removal, finally uses silica silica gel
Extraction column is extracted, and reduces impurity in sample, the accuracy and high sensitivity of detection, the rate of recovery reach testing requirements.
Detailed description of the invention
Fig. 1 is the canonical plotting and equation of avilamycin standard items；
Fig. 2 is the working curve diagram and regression equation of avilamycin after chicken alimentary canal content sample treatment；
Fig. 3 is blank sample solution chromatogram in embodiment 1；
Fig. 4 is sample solution chromatogram in embodiment 1 (concentration is 0.4 μ g/mL)；
Fig. 5 is the standard chromatogram of comparative example 1；
Fig. 6 is the blank sample chromatogram of comparative example 1；
Fig. 7 is the blank sample chromatogram of comparative example 2；
Fig. 8 is sample solution chromatogram in comparative example 2 (concentration is 0.4 μ g/mL).
Specific embodiment
It is clearly and completely described below in conjunction with the technical solution in the embodiment of the present invention, it is clear that described reality
Applying example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is general
Logical technical staff every other embodiment obtained without making creative work belongs to what the present invention protected
Range.
Embodiment 1:
Liquid chromatograph: Shimadzu LC-20AD；
Detector: Shimadzu SPD-20A ultraviolet-visible detector；
Chromatographic column: Agilent SB-Aq, 250 × 4.6mm (i.d.), 5 μm；
Mobile phase A: 0.1mol/L ammonium acetate；
Mobile phase B: 100% acetonitrile；
Ultraviolet detection wavelength: 295nm；
Sample volume: 35 μ L；
Flow velocity: 0.5mL/min；
Column temperature: 30 DEG C.
Mobile phase elution protocol see the table below
Specific detection method is as follows:
(1) prepare avilamycin standard items stock solution: precision weighs avilamycin standard items 12.5mg, is dissolved in 10mL
In methanol, Ultrasonic Heating dissolution as avilamycin standard items stock solution one, takes stock solution one, uses methanol dilution 50 respectively
Again, 100 times, as stock solution two and three；
(2) prepare avilamycin standard items working solution: stock solution three is diluted respectively 2 times, 10 times, 20 times, 50 times,
100 times of working solutions that 5 parts of various concentrations are made wait for that HPLC is detected；
(3) it draws standard curve: opening liquid chromatograph and detector, after instrument parameter reaches requirement, balance baseline
20min, by above-mentioned 5 parts of working solutions successively sample introduction, each solution is 3 times parallel, records chromatogram, such as Fig. 3, with peak area with it is corresponding
Concentration carries out linear regression analysis, obtains standard curve and equation, as shown in Figure 1；
(4) in chicken alimentary canal avilamycin extraction: accurately weigh 1g chicken intestinal contents in 10mL centrifuge tube pipe, add
Acetone 3mL, vortex 5min, 10000r/min centrifugation 10min. take supernatant in 100mL centrifuge tube, acetone are added in residue
3mL extracts once according still further to the above method, merges supernatant twice.6mL 10wt%NaCl, vortex 5min, 5000r/ is added
Min is centrifuged 4min, and the n-hexane of 3mL 10wt% saturation is then added, and vortex 5min, 5000r/min are centrifuged 4min, go just oneself
Alkane layer (upper layer), to achieve the purpose that grease removal, then is added 15ml twice respectively, 10mL methylene chloride, vortex 5min, 5000r/
Min is centrifuged 4min, takes dichloromethane layer (lower layer) in 50mL centrifuge tube after stratification, merge twice dichloromethane layer in 50
It DEG C is dried with nitrogen, residue 3mL chloroform redissolves, to be clean；
SPE purification: silica gel extraction column (Silica) 5mL methanol, the activation of 5mL chloroform take liquid to be clean to cross column, use
5mL chloroform: the elution of acetone (4:1) mixed liquor, 5mL chloroform: eluent, 50 DEG C of nitrogen are collected in acetone (3:7) elution
It blows, with 1mL initial liquid phase dissolved residue, is vortexed and mixes, 0.22 μm of filter membrane obtains sample extracting solution to be detected；
(5) working curve: corresponding avilamycin standard solution is added in the chicken alimentary canal sample of blank, makes
Drug concentration in sample reach 0.2 μ g/mL, 0.5 μ g/mL, 2 μ g/mL, 5 μ g/mL, 10 μ g/mL according to above-mentioned (4) side
Method step process obtains sample extracting solution, then opens liquid chromatograph and detector, after instrument parameter reaches requirement, balance
Baseline 20min, by this 5 parts of samples successively sample introduction, each solution is 3 times parallel, carries out linear regression with peak area and corresponding concentration
Analysis, obtains working curve and regression equation, as shown in Figure 2；
(6) accuracy and precision: accuracy and precision are indicated with the rate of recovery and the coefficient of variation respectively；In blank chicken
Alimentary canal content be added 0.2 μ g/mL, 0.4 μ g/mL, 2 tri- concentration of μ g/mL avilamycin, according to sample to be detected
The extracting method preparation sample purification liquid of extracting solution is detected for HPLC；Each concentration is repeated 5 times on the same day, is averaged, and is counted
Calculating in a few days relative error is the in a few days coefficient of variation；Each concentration repeats 5 days；Average value and relative error are calculated, is obtained in the daytime
The coefficient of variation；The rate of recovery is calculated using single-point calibration method, formula are as follows: X=AV/ASW × 100%, in formula: determinand in X- sample
The rate of recovery；The peak area of A- sample；V- sample extracts constant volume after drying, Unit/mL；The peak area of As- standard working solution；
W: volume of sample, Unit/mL；The rate of recovery that avilamycin in ileal contents is calculated is 78.4%~90%, is become in the daytime
Different coefficient is 2.8%~7.8%.
(7) sample detection and result calculate: after chicken single oral gavage 2.5mg/kg.b.w. avilamycin, acquiring its digestion
Road content is extracted to be detected with HPLC, chromatogram and peak area as shown in Figure 4 is recorded, according to the peak of actually detected sample
Area brings the regression equation y=14737x+1410.6 (wherein y indicates that peak area, x indicate drug concentration) of step (5), meter into
Corresponding drug concentration is calculated, the content of avilamycin in the chicken alimentary canal that different time points acquire after being thus administered,
Concrete outcome sees attached list 1；
The appearance time for obtaining avilamycin be can be seen that by Fig. 3-4 in 18.879min, retention time relatively lags,
It can avoid the interference of other impurity peaks.
The sample detection and calculated result of 1 embodiment 1 of table
Comparative example 1:
This comparative example the difference from embodiment 1 is that, HPLC detect when flow velocity be 1mL/min.Remaining measurement method and to
Test sample is identical, and HPLC testing result is shown, target peak retention time is in 15min or so, but alimentary canal sample exists
There are apparent impurity peaks at 15min, the results are shown in attached figure 5, Fig. 6.
Comparative example 2:
This comparative example the difference from embodiment 1 is that, eluent is conventional eluent, i.e. acetone.Remaining measurement method
It is identical with sample to be tested.It can be seen that acetone as extracting solution according to the chromatogram of Fig. 7 and Fig. 8, protected in target peak
It stays and is not separated at time point with impurity peaks.
Embodiment described above is only that preferred embodiment of the invention is described, and is not carried out to the scope of the present invention
It limits, without departing from the spirit of the design of the present invention, those of ordinary skill in the art make technical solution of the present invention
Various changes and improvements, should all fall into claims of the present invention determine protection scope in.
Claims (6)
1. a kind of method of avilamycin in quantitative detection chicken alimentary canal, which comprises the steps of:
(1) prepare avilamycin standard solution and sample extracting solution to be detected；
(2) it draws standard curve: the avilamycin standard solution of 3-5 parts of various concentrations being respectively used to HPLC measurement, is washed
Off-square formula is gradient elution, record standard product chromatogram and peak area, carries out linear regression analysis, and drafting obtains standard curve；
(3) accuracy and precision: accuracy and precision are indicated with the rate of recovery and the coefficient of variation respectively；It is digested in blank chicken
The avilamycin of high, medium and low three concentration is added in road content, prepares according to the extracting method of sample extracting solution to be detected
Sample purification liquid is detected for HPLC；Each concentration is repeated 5 times on the same day, is averaged, and calculating in a few days relative error is day
The interior coefficient of variation；Each concentration repeats 5 days；Average value and relative error are calculated, the coefficient of variation in the daytime is obtained；
The rate of recovery is calculated using single-point calibration method, and formula is as follows:
X=AV/ASW × 100% (a)
In formula: the determinand rate of recovery in X- sample；The peak area of A- sample；V- sample extracts constant volume after drying, Unit/mL；
The peak area of As- standard working solution；W: volume of sample, Unit/mL；
(4) draw working curve: take blank chicken alimentary canal content, wherein be added 3-5 parts of various concentrations avilamycin,
It is extracted according to the identical extracting method of sample extracting solution, HPLC detection；In triplicate afterwards by gained peak area with it is corresponding dense
Degree makees linear regression, draws working curve and calculates regression equation and correlation of the avilamycin in chicken alimentary canal content
Coefficient；
(5) actual sample measurement and result calculate: after chicken single oral gavage avilamycin, acquiring its alimentary canal content and mentioned
Take and detected with HPLC, record chromatogram and peak area, and with step (4) resulting equation of linear regression calculate separately out single to
After medicine in different time points chicken alimentary canal avilamycin content.
2. the method for avilamycin in a kind of quantitative detection chicken alimentary canal according to claim 1, which is characterized in that
HPLC testing conditions are as follows:
Chromatographic column: Agilent SB-Aq, 250 × 4.6mm (i.d.), 5 μm；
Mobile phase: A-100mM ammonium acetate, B- acetonitrile；
Ultraviolet detection wavelength: 295nm；
Sample volume: 35 μ L；
Flow velocity: 0.5mL/min；
Column temperature: 30 DEG C；
Mobile phase elution protocol is as follows:
3. the method for avilamycin in a kind of quantitative detection chicken alimentary canal according to claim 1, which is characterized in that
The sample extracting solution of detection the preparation method is as follows:
(1) add acetone as extracting solution into ileal contents to be detected, be vortexed, centrifugation takes supernatant, successively adds thereto
The n-hexane for entering NaCl weak solution and saturation carries out grease removal；It using methylene chloride as extractant, is vortexed, centrifugation removes upper layer
N-hexane layer retains the dichloromethane layer of lower layer, and dries up under nitrogen atmosphere；Solid phase residue is redissolved with chloroform, to net
Change；
(2) Solid phase extraction: selecting silica gel extraction column, first uses methanol, chloroform activation, mixed with chloroform and acetone
Close liquid to be eluted and eluted, collect eluent, and dried up under nitrogen atmosphere, with initial liquid phase dissolved residue, obtain to
The sample extracting solution of detection.
4. the method for avilamycin in a kind of quantitative detection chicken alimentary canal according to claim 3, which is characterized in that institute
The concentration for stating NaCl weak solution is 10wt%.
5. the method for avilamycin in a kind of quantitative detection chicken alimentary canal according to claim 3, which is characterized in that institute
Stating nitrogen atmosphere is 50 DEG C of nitrogen atmospheres.
6. the method for avilamycin in a kind of quantitative detection chicken alimentary canal according to claim 3, which is characterized in that institute
The chloroform and acetone mixture that leacheate is volume ratio 4:1 are stated, the chloroform and acetone that eluent is volume ratio 3:7 are mixed
Close liquid.
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| CN110643578A (en) * | 2019-10-25 | 2020-01-03 | 浙江工商大学 | Anti-avilamycin A monoclonal antibody hybridoma cell strain and application thereof |
| RU2783284C1 (en) * | 2021-11-29 | 2022-11-11 | Федеральное государственное бюджетное учреждение "Всероссийский государственный Центр качества и стандартизации лекарственных средств для животных и кормов" (ФГБУ "ВГНКИ") | Method for determining residual amounts of avilamycin in biological tissues of animal origin |
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| CN107164362A (en) * | 2017-07-04 | 2017-09-15 | 广东容大生物股份有限公司 | A kind of screening technique of avilamycin superior strain |
| CN108152414A (en) * | 2017-12-22 | 2018-06-12 | 河北圣雪大成制药有限责任公司 | A kind of method of HPLC-ELSD detections avilamycin pre-mixing agent |
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| CN110643578A (en) * | 2019-10-25 | 2020-01-03 | 浙江工商大学 | Anti-avilamycin A monoclonal antibody hybridoma cell strain and application thereof |
| RU2783284C1 (en) * | 2021-11-29 | 2022-11-11 | Федеральное государственное бюджетное учреждение "Всероссийский государственный Центр качества и стандартизации лекарственных средств для животных и кормов" (ФГБУ "ВГНКИ") | Method for determining residual amounts of avilamycin in biological tissues of animal origin |
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Application publication date: 20190625 |