CN104422749A - Preparation method for separating and purifying paclitaxel and cephalomannine - Google Patents
Preparation method for separating and purifying paclitaxel and cephalomannine Download PDFInfo
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- CN104422749A CN104422749A CN201310395925.6A CN201310395925A CN104422749A CN 104422749 A CN104422749 A CN 104422749A CN 201310395925 A CN201310395925 A CN 201310395925A CN 104422749 A CN104422749 A CN 104422749A
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Abstract
本发明涉及一种分离、纯化紫杉醇与三尖杉宁碱的制备方法。本发明采用高效液相制备色谱法，色谱条件为：聚苯乙烯-二乙烯基苯共聚物色谱柱：MKF-EP-RP，5～12μm，(150～600)×(7.0～20.0)mm(I.D.)，流动相组成为：乙腈∶水＝45～60∶55～40(体积比)，流速为：1.0～3mL／min，检测波长：229nm；进样量：20～100μL；本发明克服了硅胶等介质的不可逆吸附以及工艺流程复杂的缺点，经一步分离即可得到98％以上的紫杉醇纯品。The invention relates to a preparation method for separating and purifying paclitaxel and cephalomannine. The present invention adopts high performance liquid phase preparative chromatography, and the chromatographic conditions are: polystyrene-divinylbenzene copolymer chromatographic column: MKF-EP-RP, 5-12 μ m, (150-600) × (7.0-20.0) mm ( I.D.), mobile phase is composed of: acetonitrile: water=45～60: 55～40 (volume ratio), flow velocity is: 1.0～3mL/min, detection wavelength: 229nm; Injection volume: 20～100 μ L; The present invention has overcome Due to the irreversible adsorption of silica gel and other media and the disadvantages of complicated process, more than 98% of pure paclitaxel can be obtained after one-step separation.
Description
(1) technical field
The present invention relates to a kind of high performance liquid preparative chromatography (HPLC) separation method, be specifically related to a kind of with the preparation method of the separation that is carrier of Aquapak A-440 chromatographic media, purification of paclitaxel and Cephalomannine.
(2) background technology
Taxol be separated from Chinese yew genus plants obtain and there is the diterpene-kind compound of broad spectrum anticancer activity.It has unique anticancer mechanism, all has good curative effect to kinds cancers such as treatment oophoroma, melanoma, lung cancer, breast cancer.In recent years, it has become new drug the most popular in natural medicinal plants field, and demand grows with each passing day.Because taxol is mainly derived from the bark of Chinese yew, scarcity of resources, content are humble, and higher to the purity requirement of taxol in clinical practice, and therefore separation and Extraction obtains highly purified taxol will be a great and engineering for complexity.Taxol to be carried out to a problem that separation and purification can encounter often be exactly taxol and being separated of Cephalomannine.Because the structure of these two kinds of materials is closely similar, only have C-13 side chain terminal structure different, thus both chromatographic behavior difference are very little, are difficult to both to separate with the column chromatography of routine and thin-layer chromatography.
For the Analyze & separate of taxol and Cephalomannine class bearing taxanes, do large quantity research both at home and abroad, and achieve certain effect, but what great majority all adopted is is that the high performance liquid chromatography (HPLC) of chromatographic column is [see Tian Guilian with silica matrix, Zhang Zhiqiang, Su Zhiguo. the synthesis of Phenyl-Silica Gel and the application in Purification of Taxol thereof. chromatogram .2001,19 (1): 47-50].Although this chromatographic column is through two step modifications and functional amido and phenyl ring derivatization, but still need through multistep be separated also only obtain purity less than 80% pure product of paclitaxel.Along with the development of chromatographic media, macroreticular resin [Li Yongchao, Yang Jing, duty star, Deng the research [J] of the taxol in .D4020 macroreticular resin purification fermentation liquor. food and biotechnology journal .2009,29 (5): 710-714] become focus, it is a kind of organic high molecular polymer adsorbent, and has a wide range of applications in the separation and purification of natural products.The superfine people of Li Yong uses taxol in this resin purification fermentation liquor, the recovery can reach 87%, this resin energy attached taxol of absorption-desorption is preferably described, but use the methyl alcohol of variable concentrations and ethanol water to carry out adsorption and desorption process to resin respectively in this technological process, and only illustrate the method and have positive effect to removal pigment impurity, do not specifically note the separation of the similar bearing taxanes of taxol and purity.Chinese patent CN1054382C (application number 96102442.9) discloses a kind of with the method for macroreticular resin isolation of taxol, namely first bearing taxanes is enriched on solid adsorbent, the aqueous solution re-using organic solvent carries out wash-out, Fraction collection cut, carries out post separation after concentrated again.The method technological process is loaded down with trivial details, and separation condition is not optimized.Find a kind of simple to operate and method of the extraction taxol that separation condition is perfect for this reason, for the preparation of taxol and commercial production, there is profound significance.
Therefore, the present invention adopts polystyrene one divinylbenzene polymer microsphere that is even, withstand voltage and acid and alkali-resistance to be filler, optimizes mobile phase kind and ratio, and taxol can be reached with Cephalomannine through a step chromatography and be separated, method of operating is easy, efficient.
(3) summary of the invention
The object of the invention is the shortcomings such as the complex process overcoming prior art, describe the preparation method of a kind of simple separation, purification of paclitaxel and Cephalomannine.
Technical scheme of the present invention is as follows, the preparation method of i.e. a kind of separation, purification of paclitaxel and Cephalomannine, its feature is to adopt a kind of even, withstand voltage, superpolymer preparative chromatography post that porous type polystyrene-divinylbenzene multipolymer is Stationary liquid, and high performance liquid preparative chromatography operating conditions is:
A. Aquapak A-440 chromatographic column: MKF-EP-RP, 5 ~ 12 μm, column length: 150 ~ 600, post footpath 7.0 ~ 20mm (I.D.);
B. mobile phase: acetonitrile/water=40 ~ 75/60 ~ 25 (volume ratio); Ethanol/water=50 ~ 75/50 ~ 25;
C. flow velocity: 1.0 ~ 3.0mL/min, sample size: 10 ~ 200 μ L, column temperature: room temperature;
D. adopt UV detecting device, determined wavelength is 229nm.
Compared with prior art, the advantage that has of the present invention and effect as follows:
1, whole analytical cycle is short, and chromatographic resolution overall process is about 40min only.
2, be separated the taxol that can obtain 99.1% purity through a step, and method of operating is simple.
3, the present invention can be not limited in costliness at the enterprising line operate of conventional high-performance liquid chromatograph equally, low pressure preparative chromatography, therefore low to the requirement of instrument, method popularization is good.
4, on AKTA explorer100 preparative scale chromatography instrument, taxol is prepared in amplification, can obtain the taxol of more than 98% purity, method good stability.
(4) accompanying drawing illustrates:
Fig. 1 is one of exemplary method design sketch of the present invention, i.e. actual conditions: taxol and Cephalomannine are in Aquapak A-440 chromatographic column: (8 μm, MKF-EP-RP type preparative chromatography post, 300 × 7.8m (I.D.)) in HPLC figure, mobile phase: acetonitrile/water=60:40 (volume ratio); Flow velocity: 1mL/min; Sample introduction material to be respectively in test sample-1 ,-3 and 4, Fig. 11,2 and to represent Cephalomannine and taxol respectively.Fig. 1 is one of exemplary method design sketch of the present invention, and other exemplary method figure does not provide one by one.
Fig. 1 liquid chromatogram
(5) embodiment
Experiment equipment and reagent:
(1) experimental apparatus:
Wear peace Summit type highly effective liquid phase chromatographic system (Dai An company of the U.S.); AB SCIEXAPI2000LC-MS/MS liquid matter instrument; AKTA explorer100 preparative scale chromatography system (general electronic corporation Medical Group);
(2) chromatographic column:
MKF-EP-RP type preparative column: 300mm × 7.8mm (i.d.), 8 μm, two; 200mm × 8.0mm (i.d.), 8 μm; 150mm × 20mm (i.d.), 10 μm; 300mm × 7.8mm (i.d.), 12 μm, two; 300mm × 7.8mm (i.d.), 10 μm; MKF-DK-RP preparative column: 150mm × 10mm (i.d.), 12 μm; 300mm × 10mm (i.d.), 12 μm; 150mm × 20mm (i.d.), 8 μm; 300mm × 20mm (i.d.), 12 μm; 150mm × 20mm (i.d.), 12 μm.(the efficient carrier of separating company limited of Nanjing Mai Kefei)
(3) experiment reagent:
Taxol standard specimen (purity is 93.6%); Cephalomannine standard specimen (purity 82.9%); (content of taxol is 33.9% to fermentation liquor; Cephalomannine content is 33.3%); Chromatographically pure ethanol; Trifluoroacetic acid aqueous solution; Efficient liquid phase deionized water.
The preparation of test sample:
Test sample-1: get in the volumetric flask of fermentation liquor 0.5ml to 20ml, by methanol constant volume to 20ml, (matching while using) to be measured
Test sample-2: get Taxol Standard and be about 10mg, accurately weighed, be settled to 10ml with methanol solvate, (matching while using) to be measured
Test sample-3: get Cephalomannine standard items and be about 10mg, accurately weighed, be settled to 10ml with methanol solvate, (matching while using) to be measured
Test sample-4: get in the volumetric flask of fermentation liquor 1ml to 20ml, by methanol constant volume to 20ml, (matching while using) to be measured
Embodiment 1
Chromatographic condition: detect machine: wear peace Summit type highly effective liquid phase chromatographic system;
Chromatographic column: MKF-EP-RP (300mm × 7.8mm, 8 μm); Mobile phase: acetonitrile (B)/water (A)=75:25 (volume ratio, on as follows); Flow velocity: 1mL/min; Determined wavelength: 229nm; Sample size: 20 μ L; Sample introduction material: test sample-1, the degree of separation R recording taxol and Cephalomannine is 0.84.
Embodiment 2
Chromatographic condition: detect machine: wear peace Summit type highly effective liquid phase chromatographic system;
Chromatographic column: MKF-EP-RP (200mm × 8.0mm, 8 μm); Mobile phase: acetonitrile (B)/water (A)=60:40 (volume ratio, on as follows); Flow velocity: 1mL/min; Determined wavelength: 229nm; Sample size: 20 μ L; Sample introduction material: test sample-2, the degree of separation R recording taxol and Cephalomannine is 1.04.
Embodiment 3
Chromatographic condition: detect machine: wear peace Summit type highly effective liquid phase chromatographic system;
Chromatographic column: MKF-EP-RP (600mm × 7.8mm, 12 μm); Mobile phase: acetonitrile (B)/water (A)=50:50 (volume ratio, on as follows); Flow velocity: 1.5mL/min; Determined wavelength: 229nm; Sample size: 20 μ L; Sample introduction material: test sample-3, the degree of separation R recording taxol and Cephalomannine is 1.27.
Embodiment 4
Chromatographic condition: detect machine: wear peace Summit type highly effective liquid phase chromatographic system;
Chromatographic column: MKF-EP-RP (300mm × 7.8mm, 10 μm); Mobile phase: acetonitrile (B)/water (A)=40:60; Flow velocity: 1.5mL/min; Determined wavelength: 254nm; Sample size: 10 μ L; Sample introduction material: test sample-1, the degree of separation R recording taxol and Cephalomannine is 1.52.
Embodiment 5
Chromatographic condition: detect machine: wear peace Summit type highly effective liquid phase chromatographic system;
Chromatographic column: MKF-EP-RP (600mm × 7.8mm, 8 μm); Mobile phase: ethanol (B)/water (A)=75:25; Flow velocity: 1.0mL/min; Determined wavelength: 254nm; Sample size: 10 μ L; Sample introduction material: test sample-1, the degree of separation R recording taxol and Cephalomannine is 0.92.
Embodiment 6
Chromatographic condition: detect machine: wear peace Summit type highly effective liquid phase chromatographic system;
Chromatographic column: MKF-EP-RP (300mm × 7.8mm, 8 μm); Mobile phase: ethanol (B)/water (A)=55:45; Flow velocity: 1.0mL/min; Determined wavelength: 254nm; Sample size: 10 μ L; Sample introduction material: test sample-1, the degree of separation R recording taxol and Cephalomannine is 1.36.
Embodiment 7
Chromatographic condition: detect machine: AKTA explorer100 preparative scale chromatography system;
Chromatographic column: MKF-DK-RP (150mm × 20mm, 8 μm); Mobile phase: acetonitrile (B)/water (A)=60:40; Flow velocity: 1mL/min; Determined wavelength: 229nm; Sample size: i00 μ L; Sample introduction material: test sample-4, the degree of separation R recording taxol and Cephalomannine is 1.45.
Embodiment 8
Chromatographic condition: detect machine: AKTA explorer100 preparative scale chromatography system;
Chromatographic column: MKF-DK-RP (450mm × 10mm, 12 μm); Mobile phase: acetonitrile (B)/water (A)=65:35; Flow velocity: 2mL/min; Determined wavelength: 229nm; Sample size: 100 μ L; Sample introduction material: test sample-4, the degree of separation R recording taxol and Cephalomannine is 1.17.
Embodiment 9
Chromatographic condition: detect machine: AKTA explorer100 preparative scale chromatography system;
Chromatographic column: MKF-DK-RP (450mm × 20mm, 12 μm); Mobile phase: acetonitrile (B)/water (A)=60:40; Flow velocity: 3mL/min; Determined wavelength: 254nm; Sample size: 200 μ L; Sample introduction material: test sample-4, the degree of separation R recording taxol and Cephalomannine is 1.32.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113512015A (en) * | 2021-05-26 | 2021-10-19 | 上海卓鼎生物技术有限公司 | Method for industrially purifying cephalomannine |
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| CN1140170A (en) * | 1996-03-05 | 1997-01-15 | 核工业北京化工冶金研究院 | A preparative liquid chromatography method for extracting and purifying paclitaxel and other taxane derivatives |
| US5969165A (en) * | 1999-01-07 | 1999-10-19 | 508037 (Nb) Inc. | Isolation and purification of paclitaxel and other related taxanes by industrial preparative low pressure chromatography on a polymeric resin column |
| CA2428654A1 (en) * | 2000-11-13 | 2002-05-16 | Lahu Saiji | Isolation of taxanes |
| US6452024B1 (en) * | 2000-02-22 | 2002-09-17 | Chaichem Pharmaceuticals International | Process for extraction and purification of paclitaxel from natural sources |
| CN1377882A (en) * | 2001-04-02 | 2002-11-06 | 北京普瑞孚天然药物现代纯化和分离研究所有限公司 | Process for coproducing high purity taxol cephalomtanine, and 10-deacetyl Bakating III |
| CN1442413A (en) * | 2003-04-18 | 2003-09-17 | 北京怡禾生物工程有限公司 | Method of extracting medical taxadol and its derivative using regenerable resources |
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2013
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Patent Citations (7)
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| CN1140170A (en) * | 1996-03-05 | 1997-01-15 | 核工业北京化工冶金研究院 | A preparative liquid chromatography method for extracting and purifying paclitaxel and other taxane derivatives |
| US5969165A (en) * | 1999-01-07 | 1999-10-19 | 508037 (Nb) Inc. | Isolation and purification of paclitaxel and other related taxanes by industrial preparative low pressure chromatography on a polymeric resin column |
| US6452024B1 (en) * | 2000-02-22 | 2002-09-17 | Chaichem Pharmaceuticals International | Process for extraction and purification of paclitaxel from natural sources |
| CA2428654A1 (en) * | 2000-11-13 | 2002-05-16 | Lahu Saiji | Isolation of taxanes |
| CN1377882A (en) * | 2001-04-02 | 2002-11-06 | 北京普瑞孚天然药物现代纯化和分离研究所有限公司 | Process for coproducing high purity taxol cephalomtanine, and 10-deacetyl Bakating III |
| CN1442413A (en) * | 2003-04-18 | 2003-09-17 | 北京怡禾生物工程有限公司 | Method of extracting medical taxadol and its derivative using regenerable resources |
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Non-Patent Citations (1)
| Title |
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| 雒丽娜等: "固相萃取高效液相色谱法快速测定红豆杉枝叶中的3种紫杉烷类化合物", 《分析试验室》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113512015A (en) * | 2021-05-26 | 2021-10-19 | 上海卓鼎生物技术有限公司 | Method for industrially purifying cephalomannine |
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Application publication date: 20150318 |