CN102675427B - High-pressure liquid-phase preparation method for enramycin standard sample - Google Patents
High-pressure liquid-phase preparation method for enramycin standard sample Download PDFInfo
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- CN102675427B CN102675427B CN 201210159394 CN201210159394A CN102675427B CN 102675427 B CN102675427 B CN 102675427B CN 201210159394 CN201210159394 CN 201210159394 CN 201210159394 A CN201210159394 A CN 201210159394A CN 102675427 B CN102675427 B CN 102675427B
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Abstract
The invention discloses a high-pressure liquid-phase preparation method for an enramycin standard sample, and the method comprises preparation of a fermented thallus test sample solution and preparation of a animal feed test sample solution, wherein the preparation of the fermented thallus test sample solution comprises the following steps of: suspending fermented thallus in an extraction solution, which is formed by mixing acetone, hydrochloric acid solution and water in a ratio of 20:1:21, in a ratio of 30-90 ml/g, disrupting cells by an ultrasonic disruptor or an ultrasonic machine to obtain an extraction solution containing enramycin, collecting a supernatant, filtering the supernatant by a filter membrane, and using the supernatant as the test sample solution for the future use; and the preparation of the animal feed test sample solution comprises the following steps of: performing reverse-phase high-performance liquid chromatography in corresponding conditions, collecting according to the retention time of the constituents, and then removing a solvent according to a conventional rotary evaporation process, thus obtaining the standard sample of the enramycin constituent. The high-pressure liquid-phase preparation method of the enramycin standard sample disclosed by the invention has the following advantages: 1, the pre-treatment is simple, fast, and easy to operate; 2, the collection method is simple; and 3, the purity of the prepared standard sample is high and up to more than 90%.
Description
Technical field
The present invention relates to a kind of high-pressure liquid phase preparation method of enramycin standard substance.
Background technology
Enramycin (enramycin) is by the polypeptide antibiotics that comprises that 13 different types of 17 amino acid moleculars and fatty acid molecule are formed, and wherein amino acid molecular constitutes the ring type polypeptide structure, and lipid acid is positioned at the polypeptide structure end.According to its terminal lipid acid kind difference, be divided into grace and draw mould A (C
107H
138C
L2N
26O
31) and enramycin B (C
108H
140C
L2N
26O
31), enramycin then is by these two kinds of mixtures that one-tenth is grouped into.The hydrochloride of enramycin is white crystalline powder, and molecular weight is about 2500, and fusing point is 238～245 ℃, is soluble in methyl-sulphoxide, dissolves in methyl alcohol, aqueous ethanol, is insoluble in acetone, is insoluble to benzene, chloroform.The hydrochloride of enramycin has fabulous stability to heat, illumination and humidity.Enramycin has very high stability in feed, standing storage is seldom degraded at ambient temperature, and is also highly stable in making the particulate material process, with after feed mixes at room temperature standing storage tire descend very little.Enramycin is not degraded in enteron aisle, can keep original anti-microbial activity.Do not find to relate to enramycin standard substance preparation method's report at present as yet.
Summary of the invention
Purpose of the present invention just provides a kind of simple and fast, easy handling, the high-pressure liquid phase preparation method of the enramycin standard substance that the standard substance purity of preparation is high.
The high-pressure liquid phase preparation method of enramycin standard substance of the present invention may further comprise the steps:
1, fermentation thalline sample solution preparation:
After zymophyte body and function washed with de-ionized water, ratio in 30-90ml/g is suspended in by acetone: hydrochloric acid soln: in the extracting solution of water=20:1:21 mixed, acetone is analytical pure concentration, the concentration of hydrochloric acid soln is 2mol/L, carry out cytoclasis 10min with Ultrasonic Cell Disruptor or ultrasonic machine, the mixture that obtains is at 20 ℃, 90 min are extracted in vibration under the condition of 180rpm, obtain containing the extracting solution of enramycin, centrifugal 10min under the rotating speed of 10000r/min then, collect supernatant liquor, standby as sample solution with 0.45 μ m membrane filtration, sample solution concentration is no more than 2000 μ g/ml;
The preparation of animal-feed sample solution:
Choose animal-feed sample 200g-500g by the regulation of GB/T 14699.1, be reduced to 100g with quartering, pulverize by the 0.45mm aperture sieve, mixing, be stored in the port grinding bottle standby, get above-mentioned animal-feed sample 5-10g, join 80ml by acetone: hydrochloric acid soln: in the extracting solution of water=20:1:21 mixed, acetone is analytical pure concentration, the concentration of hydrochloric acid soln is 2mol/L, at 20 ℃, 90min is extracted in vibration under the condition of 180rpm, obtains containing the extracting solution of enramycin, centrifugal 10min under the rotating speed of 10000r/min then, collect supernatant liquor, standby as sample solution with 0.45 μ m membrane filtration, sample solution concentration is no more than 2000 μ g/ml.
2, enramycin standard substance preparation
:Carry out anti-phase high pressure liquid chromatography by following condition:
Sample solution concentration: be no more than 2000 μ g/ml;
Sample size: 100 μ l;
Chromatographic column: C18 post half preparation type, long 250mm, internal diameter 10mm, granularity 5 μ m;
Moving phase: B: trifluoroacetic acid aqueous solution, A:0.2% acetic acid, method for making is that absorption 2ml chromatographically pure acetic acid is dissolved in the 1000ml pure water;
Flow velocity: 2.0ml/min;
Column temperature: 30 ℃;
Detect wavelength: 267nm;
Retention time according to component A and B component is collected;
Component A peak and the rotary evaporation technology desolventizing routinely of B component peak difference with collecting namely get enramycin component standard substance.
Described fermentation thalline refers to the mycelium of enramycin generation bacterium in the fermented liquid.
Described animal-feed refers to animal mixed feed, concentrated fodder, premixed feed or enramycin premix.
The high-pressure liquid phase preparation method of enramycin standard substance of the present invention has the following advantages: 1: pre-treatment simple and fast, easy handling; 2, collection method is simple, as long as collect respective components according to retention time; 3, Zhi Bei standard substance purity height, purity can reach more than 90%; 4, can be with component A and B component separate collection, for theoretical investigation in the future lays the foundation.
Embodiment
Embodiment 1: after zymophyte body and function washed with de-ionized water, ratio in 65ml/g is suspended in by acetone: hydrochloric acid soln: in the extracting solution of water=20:1:21 mixed, acetone is analytical pure concentration, the concentration of hydrochloric acid soln is 2mol/L, carry out cytoclasis 10min with Ultrasonic Cell Disruptor or ultrasonic machine, the mixture that obtains is at 20 ℃, 90 min are extracted in vibration under the condition of 180rpm, obtain containing the extracting solution of enramycin, centrifugal 10min under the rotating speed of 10000r/min then, collect supernatant liquor, standby as sample solution with 0.45 μ m membrane filtration.
With the sample handled well by following chromatographic condition sample introduction:
Sample size: 100 μ l;
Chromatographic column: Phenomenex C18 post half preparation type, long 250mm, internal diameter 10mm, granularity 5 μ m;
Moving phase: B: trifluoroacetic acid aqueous solution, A:0.2% acetic acid, method for making is that absorption 2ml chromatographically pure acetic acid is dissolved in the 1000ml pure water;
Flow velocity: 2.0ml/min;
Column temperature: 30 ℃;
Detect wavelength: 267nm;
The retention time of collecting component A and B component: component A and B component by the reference colour spectrogram respectively is respectively 14.176min and 21.070min, and to the collecting of component A and B component, component A and B component are collected 8.2 L and 4.5 L respectively according to retention time.
Component A and the rotary evaporation technology desolventizing routinely of B component difference with collecting namely get enramycin component standard substance.Carry out purity detecting, the purity of component A is 96.5%, and the purity of B component is 92.9%.
Embodiment 2: choose animal-feed sample 400g by the regulation of GB/T 14699.1, be reduced to 100g with quartering, pulverize by the 0.45mm aperture sieve, mixing, be stored in the port grinding bottle standby, get above-mentioned animal-feed sample 5-10g, join 80ml by acetone: hydrochloric acid soln: in the extracting solution of water=20:1:21 mixed, acetone is analytical pure concentration, the concentration of hydrochloric acid soln is 2mol/L, at 20 ℃, vibration was extracted 90min hour under the condition of 180rpm, obtained containing the extracting solution of enramycin, centrifugal 10min under the rotating speed of 10000r/min then, collect supernatant liquor, standby as sample solution with 0.45 μ m membrane filtration, sample solution concentration is no more than 2000 μ g/ml.
With the sample handled well by following chromatographic condition sample introduction:
Sample size: 100 μ l;
Chromatographic column: Phenomenex C18 post half preparation type, long 250mm, internal diameter 10mm, granularity 5 μ m;
Moving phase: B: trifluoroacetic acid aqueous solution, A:0.2% acetic acid, method for making is that absorption 2ml chromatographically pure acetic acid is dissolved in the 1000ml pure water;
Flow velocity: 2.0ml/min;
Column temperature: 30 ℃;
Detect wavelength: 267nm;
The retention time of collecting component A and B component: component A and B component by the reference colour spectrogram respectively is respectively 14.231min and 21.108min, and to the collecting of component A and B component, component A and B component are collected 8.7L and 4.9 L respectively according to retention time.
Component A and the rotary evaporation technology desolventizing routinely of B component difference with collecting namely get enramycin component standard substance.Carry out purity detecting, the purity of component A is 96.1%, and the purity of B component is 92.8%.
Claims (1)
1. the high-pressure liquid phase preparation method of enramycin standard substance, it is characterized in that: it may further comprise the steps:
(1), fermentation thalline sample solution preparation:
After zymophyte body and function washed with de-ionized water, ratio in 30-90ml/g is suspended in by acetone: hydrochloric acid soln: in the extracting solution of water=20:1:21 mixed, acetone is analytical pure concentration, the concentration of hydrochloric acid soln is 2mol/L, carry out cytoclasis 10min with Ultrasonic Cell Disruptor or ultrasonic machine, the mixture that obtains is at 20 ℃, 90 min are extracted in vibration under the condition of 180rpm, obtain containing the extracting solution of enramycin, centrifugal 10min under the rotating speed of 10000r/min then, collect supernatant liquor, standby as sample solution with 0.45 μ m membrane filtration, sample solution concentration is no more than 2000 μ g/ml;
The preparation of animal-feed sample solution:
Choose animal-feed sample 200g-500g by the regulation of GB/T 14699.1, be reduced to 100g with quartering, pulverize by the 0.45mm aperture sieve, mixing, be stored in the port grinding bottle standby, get above-mentioned animal-feed sample 5-10g, join 80ml by acetone: hydrochloric acid soln: in the extracting solution of water=20:1:21 mixed, acetone is analytical pure concentration, the concentration of hydrochloric acid soln is 2mol/L, at 20 ℃, 90min is extracted in vibration under the condition of 180rpm, obtains containing the extracting solution of enramycin, centrifugal 10min under the rotating speed of 10000r/min then, collect supernatant liquor, standby as sample solution with 0.45 μ m membrane filtration, sample solution concentration is no more than 2000 μ g/ml;
(2), enramycin standard substance preparation
:Carry out anti-phase high pressure liquid chromatography by following condition:
Sample solution concentration: be no more than 2000 μ g/ml;
Sample size: 100 μ l;
Chromatographic column: C18 post half preparation type, long 250mm, internal diameter 10mm, granularity 5 μ m;
Moving phase: B: trifluoroacetic acid aqueous solution, A:0.2% acetic acid, method for making is that absorption 2ml chromatographically pure acetic acid is dissolved in the 1000ml pure water;
Flow velocity: 2.0ml/min;
Column temperature: 30 ℃;
Detect wavelength: 267nm;
Retention time according to component A and B component is collected;
Component A peak and the rotary evaporation technology desolventizing routinely of B component peak difference with collecting namely get enramycin component standard substance;
Described fermentation thalline refers to the mycelium of enramycin generation bacterium in the fermented liquid;
Described animal-feed refers to animal mixed feed, concentrated fodder, premixed feed.
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| CN102898510B (en) * | 2012-11-04 | 2014-05-07 | 乐占线 | Method for separating enramycin A from enramycin B |
| CN103232530B (en) * | 2013-04-17 | 2014-10-29 | 南京工业大学 | Enramycin A and B separation method |
| CN104877010B (en) * | 2015-06-15 | 2018-05-29 | 华北制药集团新药研究开发有限责任公司 | A kind of preparation method of pristinamycin I A one pack systems |
| CN104892726B (en) * | 2015-06-15 | 2018-06-01 | 华北制药集团新药研究开发有限责任公司 | A kind of preparation method of pristinamycin I IA one pack systems |
| CN105131090A (en) * | 2015-10-15 | 2015-12-09 | 山西新源华康化工股份有限公司 | Dissolving solution for purifying enramycin and method for purifying enramycin |
| CN105687130A (en) * | 2016-03-12 | 2016-06-22 | 石家庄高科动物保健品有限公司 | Enramycin solution and preparation method thereof |
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Non-Patent Citations (2)
| Title |
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| Koichi Inoue，et al.An approach to on-line electrospray mass spectrometric detection of polypeptide antibiotics of enramycin for high-speed counter-current chromatographic separation.《Journal of Pharmaceutical and Biomedical Analysis》.2010,第51卷1154–1160. * |
| Xihou Yin,et al.Enduracidin Analogues with Altered Halogenation Patterns Produced by Genetically Engineered Strains of Streptomyces fungicidicus.《J. Nat. Prod》.2010,第73卷583–589. * |
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