CN101104631A - Method for preparing erythromycin A, erythromycin B and erythromycin C by high speed adverse current chromatogram - Google Patents
Method for preparing erythromycin A, erythromycin B and erythromycin C by high speed adverse current chromatogram Download PDFInfo
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- CN101104631A CN101104631A CNA2007100435179A CN200710043517A CN101104631A CN 101104631 A CN101104631 A CN 101104631A CN A2007100435179 A CNA2007100435179 A CN A2007100435179A CN 200710043517 A CN200710043517 A CN 200710043517A CN 101104631 A CN101104631 A CN 101104631A
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- berythromycin
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Abstract
The invention relates to a method for prepararing highly purified erythromycin A, erythromycin B, and erythromycin C simultaneously or separately from erythromycin raw material with high-speed countercurrent chromatography, comprising that a solvent system composed of stationary phases and mobile phases is prepared, the stationary phases are filled in a countercurrent chromatograph column, and then the mobile phases are pumped into the column. The raw material containing components of the erythromycin A, B, C is dissolved in lower phase solvent. Sample is injected through a sample injection valve, component peaks of the erythromycin A, the erythromycin B, and the erythromycin C are respectively received according to a UV atlas of a detector. The solvent system is composed of n-hexane, ethyl acetate, methanol and water, the amount and volume ratio is 0.5-1.3: 0.5-2.0: 2.0-1. The method is characterized in high efficient and fast separation, large quantity of separation amount, no lost of the sample, high recovery rate, mild separation environment and solvent saving.
Description
Technical field
The present invention relates to a kind of method that from the erythromycin raw material, prepares high purity Erythromycin A, berythromycin, Erythromycin C, particularly relate to a kind of method that adopts high speed adverse current chromatogram to prepare high purity Erythromycin A, berythromycin, Erythromycin C.
Background technology
Erythromycin (Erythromycin is called for short EM) is a kind of Macrolide Broad spectrum antibiotics.The present known six kinds of isomer that wherein have are respectively Erythromycin A, B, and C, D, E and F, A are main active ingredient.The physico-chemical property of berythromycin and Erythromycin C and antimicrobial spectrum are similar to Erythromycin A, but their anti-microbial activity generally has only 30%～60% of Erythromycin A.Therefore, the major part of using in the medical treatment at present is an Erythromycin A.
The sample of highly purified erythromycin component all has crucial meaning for the research work of various relevant erythromycin.
The similar performance of Erythromycin A and berythromycin, Erythromycin C, general separation means are difficult to it is separated.High speed adverse current chromatogram (High Speed Counter Current Chromatography, HSCCC) be a kind of efficient liquid liquid distribution chromatography isolation technique that need not the solid support thing of releasing in 1980, successfully apply to fields such as biological chemistry, biotechnology, medical science, pharmacy, natural product chemistry, organic synthesis, chemical industry, environment, agricultural, material with its high-recovery, high preparative capacibility and high accumulation ability.High-speed countercurrent chromatography is gentle and effectively separate and make it obtain using widely separating antibiotic field, is especially seeming very potential aspect the preparation microbiotic standard substance.
Adopting high speed adverse current chromatogram to separate in the research work of preparation erythromycin component, people such as Booth adopt high speed adverse current chromatogram at solvent systems to be: normal hexane: ethyl acetate: methyl alcohol: water=1.4: 2.0: 2.0: 1.0, theoretical plate number is not higher than separates the preparation Erythromycin A under 1219 the situation, obtained purity at the Erythromycin A more than 97%
[1], [2], [3]([1]A.J.Booth，G.J.Lye.Optimization?of?the?fractionation?and?recovery?ofpolyketide?antibiotics?by?countercurrent?chromatography.J.Liq.Chromatogr.Rel.Technol.，2001，24(11&12)：1841-1861
[2]A.J.Booth，I.A.Sutherland，G.J.Lye.Modeling?the?performance?of?pilot-scalecountercurrent?chromatography：Scale-up?predictions?and?experimental?verificationof?erythromycin?separation.Biotechnology?and?Bioengineering，2003，81(6)：640-649
[3]A.J.Booth，S.H.Ngiam，G.J.Lye.Antibiotic?purification?from?fermentation?brothsby?counter-current?chromatography：analysis?of?product?purity?and?yield?trade-offs.Bioprocess?and?Biosystems?Engineering，2004，27：51-61)。
And have not yet to see the research report that adopts high speed adverse current chromatogram to separate preparation high purity berythromycin, C, domestic do not have highly purified berythromycin and Erythromycin C sample to provide as yet yet.
Summary of the invention
The purpose of this invention is to provide a kind of method that from the erythromycin raw material, prepares high purity Erythromycin A, berythromycin and Erythromycin C.Present method can prepare Erythromycin A, berythromycin and Erythromycin C at the same time or separately.
The present invention takes following technical scheme: a kind of method that adopts high speed adverse current chromatogram to prepare high purity Erythromycin A, berythromycin, Erythromycin C, comprise: the solvent system that constitutes stationary phase, moving phase by normal hexane, ethyl acetate, methyl alcohol, water preparation, on be stationary phase mutually, be moving phase mutually down, make in the whole cylinder of counter current chromatograph to be full of stationary phase, again moving phase is pumped in the post; The erythromycin raw material that will contain erythromycin component A, B, C is dissolved in the following phase solvent, by the sampling valve sample introduction, uv atlas difference receiving target component Erythromycin A, berythromycin, Erythromycin C according to detector, and carry out lyophilize, it is characterized in that the consumption volume ratio of normal hexane, ethyl acetate, methyl alcohol, water is 0.5～1.3: 0.5～2.0 in the described solvent system: 0.5～2.0: 1.
Wherein, said erythromycin raw material is the erythromycin that Yueyang produces with connection pharmaceutcal corporation, Ltd, and product batch number is 20060710.
In above-mentioned solvent system, the consumption volume ratio of normal hexane, ethyl acetate, methyl alcohol, water is 0.6～1.2: 1.0～2.0 in the described solvent system: 0.8～1.6: 1 o'clock, the purity of the Erythromycin A that obtains was up to 90%～100%;
The consumption volume ratio of normal hexane, ethyl acetate, methyl alcohol, water is 0.6～1.2: 0.8～2.0 in the described solvent system: 0.8～1.6: 1 o'clock, the purity of the berythromycin that obtains was up to 80%～100%;
The consumption volume ratio of normal hexane, ethyl acetate, methyl alcohol, water is 0.6～1.2: 1.0～2.0 in the described solvent system: 0.8～1.2: 1 o'clock, the purity of the Erythromycin C that obtains was up to 70%～95%.
The invention has the advantages that: 1. adopted the high speed adverse current chromatogram separation method in present method, high-speed countercurrent chromatography has been avoided the chemical modification because of the sample loss that adsorption causes, sample component, separation efficiency can be compared with preparation HPLC with capacity, and does not generally have the conditions of streaking at peak.2. when under described solvent systems, adopting high speed adverse current chromatogram to separate preparation Erythromycin A, berythromycin, Erythromycin C in the method, can prepare Erythromycin A, berythromycin, Erythromycin C as required at the same time or separately; 3, present method is separated preparation Erythromycin A, berythromycin, Erythromycin C, number of effective theoretical plates be 1300～2500, have separate efficient, fast, fractional dose big, sample free of losses, rate of recovery height, isolating environment gentleness, save characteristics such as solvent.
Embodiment
For better understanding the present invention, the invention will be further described below by embodiment, but for embodiment do not limit protection scope of the present invention:
Embodiment 1
Use half countercurrent chromatography instrument, be furnished with constant flow pump, the 15ml sampling valve, tetrafluoroethylene post, column volume are 200ml, the UV UV-detector.With volume ratio is 1.2: 2: 1.2: 1 normal hexane: ethyl acetate: methyl alcohol: water is miscible in separating funnel, shake up the back standing demix, get its upper solution (going up phase) and be stationary phase, lower floor's solution (following phase) is moving phase, behind the ultrasonic degas, earlier be full of whole cylinder with stationary phase, open high-speed counter-current chromatograph then, the adjustment engine speed is 900rpm, flow velocity with 1.3ml/min pumps into moving phase in the post, after treating that whole system is set up running balance, with the erythromycin raw material of 100mg be dissolved in 3ml following mutually in, by the sampling valve sample introduction, then according to the detector uv atlas, collect Erythromycin A respectively, berythromycin, the Erythromycin C component peaks, blow out organic solvent wherein, lyophilize, obtain white erythromycin solid, analyze through HPLC, Erythromycin A purity is 99.87%, and berythromycin purity is 92.09%, Erythromycin C purity is 90.24%, the number of effective theoretical plates that wherein separates Erythromycin A is 2116, and the number of effective theoretical plates that separates berythromycin is 1898, and the number of effective theoretical plates that separates Erythromycin C is 1764.
Embodiment 2
Use half countercurrent chromatography instrument, be furnished with constant flow pump, the 15ml sampling valve, tetrafluoroethylene post, column volume are 200ml, the UV UV-detector.With volume ratio is 0.6: 1.2: 1: 1 normal hexane: ethyl acetate: methyl alcohol: water is miscible in separating funnel, shake up the back standing demix, get its upper solution (going up phase) and be stationary phase, lower floor's solution (following phase) is moving phase, behind the ultrasonic degas, earlier be full of whole cylinder with stationary phase, open high-speed counter-current chromatograph then, the adjustment engine speed is 900rpm, flow velocity with 1.3ml/min pumps into moving phase in the post, after treating that whole system is set up running balance, with the erythromycin raw material of 100mg be dissolved in 3ml following mutually in, by the sampling valve sample introduction, then according to the detector uv atlas, collect Erythromycin A respectively, berythromycin, the Erythromycin C component peaks, blow out organic solvent wherein, lyophilize, obtain white erythromycin solid, analyze through HPLC, Erythromycin A purity is 99.61%, and berythromycin purity is 99.03%, Erythromycin C purity is 81.20%, the number of effective theoretical plates that wherein separates Erythromycin A is 1393, and the number of effective theoretical plates that separates berythromycin is 2401, and the number of effective theoretical plates that separates Erythromycin C is 1600.
Claims (4)
1. method that adopts high speed adverse current chromatogram to prepare high purity Erythromycin A, berythromycin, Erythromycin C, comprise: the solvent system that constitutes stationary phase, moving phase by normal hexane, ethyl acetate, methyl alcohol, water preparation, on be stationary phase mutually, be moving phase mutually down, make in the whole cylinder of counter current chromatograph to be full of stationary phase, again moving phase is pumped in the post; The erythromycin raw material that will contain erythromycin component A, B, C is dissolved in the following phase solvent, by the sampling valve sample introduction, uv atlas difference receiving target component Erythromycin A, berythromycin, Erythromycin C according to detector, and carry out lyophilize, it is characterized in that the consumption volume ratio of normal hexane, ethyl acetate, methyl alcohol, water is 0.5～1.3: 0.5～2.0 in the described solvent system: 0.5～2.0: 1.
2. the method for preparing high purity Erythromycin A, berythromycin, Erythromycin C as claimed in claim 1, it is characterized in that the consumption volume ratio of normal hexane, ethyl acetate, methyl alcohol, water is 0.6～1.2: 1.0～2.0 in the described solvent system: 0.8～1.6: 1.
3. the method for preparing high purity Erythromycin A, berythromycin, Erythromycin C as claimed in claim 1, it is characterized in that the consumption volume ratio of normal hexane, ethyl acetate, methyl alcohol, water is 0.6～1.2: 0.8～2.0 in the described solvent system: 0.8～1.6: 1.
4. the method for preparing high purity Erythromycin A, berythromycin, Erythromycin C as claimed in claim 1, it is characterized in that the consumption volume ratio of normal hexane, ethyl acetate, methyl alcohol, water is 0.6～1.2: 1.0～2.0 in the described solvent system: 0.8～1.2: 1.
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| CNB2007100435179A CN100509835C (en) | 2007-07-06 | 2007-07-06 | Method for preparing erythromycin A, erythromycin B and erythromycin C by high speed adverse current chromatogram |
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| CNB2007100435179A CN100509835C (en) | 2007-07-06 | 2007-07-06 | Method for preparing erythromycin A, erythromycin B and erythromycin C by high speed adverse current chromatogram |
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| CN100509835C CN100509835C (en) | 2009-07-08 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102093379A (en) * | 2010-12-30 | 2011-06-15 | 上海同田生物技术有限公司 | Preparation method of high-purity bergapten and psoralen |
| CN102127132A (en) * | 2010-12-08 | 2011-07-20 | 华东理工大学 | Method for preparing isovalerylspiramycin II and isovalerylspiramycin III |
| CN102863458A (en) * | 2012-09-20 | 2013-01-09 | 福建省微生物研究所 | Purification method of tacrolimus crude products |
| CN102924547A (en) * | 2012-11-28 | 2013-02-13 | 宁夏启元药业有限公司 | Purification method for erythromycin B pure product |
| CN103145722A (en) * | 2013-03-05 | 2013-06-12 | 福建省微生物研究所 | Method for separating and purifying epothilone by high-speed counter-current chromatography |
-
2007
- 2007-07-06 CN CNB2007100435179A patent/CN100509835C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102127132A (en) * | 2010-12-08 | 2011-07-20 | 华东理工大学 | Method for preparing isovalerylspiramycin II and isovalerylspiramycin III |
| CN102093379A (en) * | 2010-12-30 | 2011-06-15 | 上海同田生物技术有限公司 | Preparation method of high-purity bergapten and psoralen |
| CN102093379B (en) * | 2010-12-30 | 2015-08-05 | 上海同田生物技术股份有限公司 | The preparation method of a kind of high purity bergapton and psoralene |
| CN102863458A (en) * | 2012-09-20 | 2013-01-09 | 福建省微生物研究所 | Purification method of tacrolimus crude products |
| CN102863458B (en) * | 2012-09-20 | 2015-05-13 | 福建省微生物研究所 | Purification method of tacrolimus crude products |
| CN102924547A (en) * | 2012-11-28 | 2013-02-13 | 宁夏启元药业有限公司 | Purification method for erythromycin B pure product |
| CN103145722A (en) * | 2013-03-05 | 2013-06-12 | 福建省微生物研究所 | Method for separating and purifying epothilone by high-speed counter-current chromatography |
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| CN100509835C (en) | 2009-07-08 |
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Granted publication date: 20090708 Termination date: 20130706 |