CN106565732A - Method for separating camptothecin and 10-hydroxycamptothecin by adoption of rosin-based macromolecules - Google Patents
Method for separating camptothecin and 10-hydroxycamptothecin by adoption of rosin-based macromolecules Download PDFInfo
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- CN106565732A CN106565732A CN201611001162.2A CN201611001162A CN106565732A CN 106565732 A CN106565732 A CN 106565732A CN 201611001162 A CN201611001162 A CN 201611001162A CN 106565732 A CN106565732 A CN 106565732A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F2/00—Processes of polymerisation
- C08F2/12—Polymerisation in non-solvents
- C08F2/16—Aqueous medium
- C08F2/20—Aqueous medium with the aid of macromolecular dispersing agents
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F289/00—Macromolecular compounds obtained by polymerising monomers on to macromolecular compounds not provided for in groups C08F251/00 - C08F287/00
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Abstract
The invention discloses a method for separating camptothecin and 10-hydroxycamptothecin by the adoption of rosin-based macromolecules. According to the method, alpha-methacrylic acid (or methyl methacrylate) is used as a monomer, maleated rosin-[(2-acryloyloxy) ethyl] ester is used as a cross-linking agent, and an micro-suspension free radical polymerization method is adopted to prepare rosin-based macromolecule microspheres, wherein the microspheres are a spherical porous material, the article size distribution of the rosin-based macromolecule microspheres is 3-10 microns, the average pore size of the rosin-based macromolecule microspheres is 10-15 nm, the specific surface area of the rosin-based macromolecule microspheres is 90-120 m<2>/g, and the acid value of the rosin-based macromolecule microspheres is 50-150 mgKOH/g; wet column packing is conducted on the rosin-based macromolecule microspheres by the adoption of a column packing machine, so that a chromatographic column is prepared; HPLC separation is conducted on the camptothecin and the 10-hydroxycamptothecin, wherein the detecting wave length is 230-290 nm, the temperature is 30+/-10 DEG C, the flow speed is 0.3-1.0 mL/min, and the separation degree of the camptothecin and the 10-hydroxycamptothecin is 1.80-2.15. By the adoption of the method for separating the camptothecin and the 10-hydroxycamptothecin by the adoption of the rosin-based macromolecules, a high separation degree of the camptothecin and the 10-hydroxycamptothecin is achieved, the selectivity is high, the method is high in sensitivity, easy to operate, rapid and efficient, and no secondary pollution to medicine, health care products and food is caused.
Description
Technical field
The invention belongs to high performance liquid chromatography field, particularly a kind of to apply Colophonium based high molecular chromatographic column high efficiency separation
The method of camptothecine and 10-hydroxycamptothecine.
Background technology
Camptothecine, chemical structural formula：
Camptothecine is indoles alkaloid contained in Fructus seu radix camptothecae acuminatae (Fructus Camptothecae Acuminatae), is the 3rd of plant resourceses the with significant active anticancer
Big natural anti-cancer drugs, camptothecine has preferable curative effect to intestines and stomach and incidence cancer etc., is played by suppressing topoisomerase I
Cytotoxicity；Structure is similar with 10-hydroxycamptothecine, but pharmacological action is different.
10-hydroxycamptothecine, chemical structural formula：
10-hydroxycamptothecine is the less indoles alkaloid of content in Fructus seu radix camptothecae acuminatae (Fructus Camptothecae Acuminatae), it is also possible to synthesized by camptothecine.It is not
Only isolated than Fructus seu radix camptothecae acuminatae (Fructus Camptothecae Acuminatae) alkaloids have higher pharmacologically active, and produce less toxicity, structure and camptothecin
Seemingly, but pharmacological action be higher than camptothecine.
Liquid-phase chromatographic analysis refer to the chromatographic technique that mobile phase is liquid, by the granularity and post pressure of improving filler, in Jing
The plate theory of gas chromatogram is introduced on the basis of the liquid column chromatography of allusion quotation, high pressure pump is technically employed, efficiently
Fixing phase and highly sensitive detector, realize that analyze speed is fast, separation efficiency is high and operation automation, this chromatographic technique
It is referred to as high performance liquid chromatography (High performance liquid chromatography, abbreviation HPLC).HPLC has
Following characteristics：(1) analyze speed it is fast-generally one sample of analysis in 15～30min, some samples are even in 5min
Complete；(2) selectivity it is high-separation analysis can be carried out to such as chiral material to the material of diversity very little；(3) detection sensitivity
Up to 0.01ng, fluorescence and electrochemical detector are up to 0.1pg for height-UV-detector；Pillar Reusability, with a chromatograph
The separable different compound of post；Sample size is few, easily reclaims；Sample is not destroyed after chromatographic column, can collect single
It is prepared by component.
At present, with regard to camptothecine and the detached method report of 10-hydroxycamptothecine, we find the following document：
1. Liu Wen is wise, Qin Haiyan, Suo Zhirong. the HPLC analyses of camptothecine and 10-hydroxycamptothecine in Fructus Camptothecae Acuminatae. medicine point
Analysis magazine .2005,25 (2)：168-170；It is mentioned that the liquid phase chromatogram condition of camptothecine and 10-hydroxycamptothecine, uses
HypersilODS chromatographic columns (250mm × 4.0mm), by mobile phase of first alcohol and water gradient elution is carried out, flow velocity 1.0mL/min,
Detection wavelength 254nm, 25 DEG C of column temperature.
2. Shi Wei states, Zu YuanGang, Yang Lei. 10-hydroxycamptothecine and vincoside-lactam grinds in polyamide separation and purification Fructus Camptothecae Acuminatae
Study carefully. CHINA JOURNAL OF CHINESE MATERIA MEDICA .2008,33 (21):2486-2489；It is mentioned that the detection side of 10-hydroxycamptothecine and vincoside-lactam
Method, using C18Chromatographic column (250mm × 4.6mm), with acetonitrile and water as mobile phase, flow velocity 1.0mL/min, Detection wavelength 254nm,
35 DEG C of column temperature.
3. application number：200510024487.8, denomination of invention：A kind of production method of 10-hydroxycamptothecine, it is mentioned that
The separation of camptothecine and 10-hydroxycamptothecine, (filler is little particle spherical silica gel, and particle diameter is 10 to adopt high-pressure liquid phase to prepare post
～16 μm) the sample introduction upper prop under conditions of 10～30 DEG C, flow velocity is 250～350mL/min, sets ultraviolet detection wavelength 254nm.
With regard to using Colophonium based high molecular chromatographic column separating common camptotheca fruit alkali and 10-hydroxycamptothecine, up to the present not appearing in the newspapers
Road..
The content of the invention
It is an object of the invention to provide a kind of apply Colophonium based high molecular chromatographic column high efficiency separation camptothecine and 10- hydroxyls
The method of camptothecine, the method sensitivity is high, simple to operate, rapidly and efficiently, will not cause secondary to medicine, health product, food
Pollution.
For achieving the above object, the present invention provides technical scheme below：
A kind of method of application Colophonium based high molecular chromatographic column high efficiency separation camptothecine and 10-hydroxycamptothecine, by Colophonium
Based high molecular microsphere prepares Colophonium based high molecular chromatographic column as fixed phase stuffing packing column machine wet method dress post；By abietyl
Macromolecule chromatographic column access chromatograph of liquid, arrange chromatograph of liquid flow rate of mobile phase be 0.3-1.0mL/min, Detection wavelength
230-290nm, 30 ± 10 DEG C of column oven；Starting injection valve makes mobile phase by camptothecine and the mixed solution of 10-hydroxycamptothecine
In bringing Colophonium based high molecular chromatographic column into, the separation of camptothecine and 10-hydroxycamptothecine is realized.
Rosinyl polymer microsphere separates the mechanism of two kinds of materials：In camptothecine and 10-hydroxycamptothecine molecule, itself band
There is C=O ,-OH group, can interact with-the COOH in rosinyl polymer microsphere, form stable associated complex, Fructus seu radix camptothecae acuminatae (Fructus Camptothecae Acuminatae)
Alkali can dissociate H with 10-hydroxycamptothecine in mobile phase+, its intermolecular forces with fixing phase can be weakened, therefore can be eluted
Get off.But the hydroxyl more than camptothecine of No. 10 positions in 10-hydroxycamptothecine molecule, it is different from fixing phase active force, in stream
The H dissociateed in dynamic phase+It is more, it is weaker with the active force of fixing phase, so in separation process, 10-hydroxycamptothecine can be first
Appearance.
Used as the further improvement of technical scheme, above-described rosinyl polymer microsphere particle diameter distribution is 3-10 μm,
Average pore size is 10-15nm, and specific surface area is 90-120m2/ g, acid number 50-150mgKOH/g.
Used as the further improvement of technical scheme, above-described packing column machine wet method dress post is with constant pressure pump that abietyl is high
Molecule microsphere is filled in sky chromatographic column, and 120-180min is loaded under 3000psi pressure, continues to increase pressure to 3500psi
Filling 10-60min, after post flattens weighing apparatus, chromatographic column is removed from packing column machine, loads onto stigma, you can prepare abietyl
Macromolecule chromatographic column.
Used as the further improvement of technical scheme, the preparation method of above-described rosinyl polymer microsphere is：With α-
Methacrylic acid or methyl methacrylate are monomer, and maleic rosin acrylic acid glycol ester is cross-linking agent, using micro suspension certainly
Rosinyl polymer microsphere is prepared by base polymerization.
Used as the further improvement of technical scheme, the above loose micro suspension free radical polymerisation process is：By ionized water, ten
Sodium dialkyl sulfate, the water phase of polyvinyl alcohol composition and α-methacrylic acid or methyl methacrylate, cross-linking agent maleic rosin
Acrylic acid glycol ester, solvent ethyl acetate, porogen isobutyltrimethylmethane., the oil phase mixing of initiator azodiisobutyronitrile composition, use
Dispersion machine is disperseed, and it is 3000-5000rpm that dispersion machine arranges rotating speed, and jitter time is 5-20min, then enters line program liter
Temperature reaction is obtained described rosinyl polymer microsphere.α-methacrylic acid or methyl methacrylate are present invention separation
The function monomer of camptothecine and 10-hydroxycamptothecine.
It is 1 as the mass ratio of the further improvement of technical scheme, above-described oil phase and water phase：3-6.
Used as the further improvement of technical scheme, routine described above temperature reaction is：70 DEG C of reaction 10- of temperature programming
80min, 80 DEG C of reaction 20-100min, 95 DEG C of reaction 30-60min, you can obtain described rosinyl polymer microsphere.
As the further improvement of technical scheme, deionized water, sodium lauryl sulphate, poly- second in above-described water phase
The mass ratio of enol is 100：0.1-0.5：1-3.
As the further improvement of technical scheme, α-methacrylic acid or methyl methacrylate in above-described oil phase
Ester, cross-linking agent, solvent, porogen, initiator quality ratio are 0.5-3：3-12.：50-100：1-5：0.1-2.
Compared with prior art, beneficial effects of the present invention are：
1. the Colophonium based high molecular chromatographic column that the present invention is prepared, its fixed phase stuffing swelling degree is little, specific surface area big, can
Effective ingredient in for extracting plant, and can use in organic solvent, the network knot of polymer will not be destroyed because of expansion
Structure, by filler of rosinyl polymer microsphere Colophonium based high molecular chromatographic column is prepared, because rich in pore structure not of uniform size, having
Permeability is good, back pressure is low, efficiently and the advantages of high flux, under higher flow velocity and pressure, the phenomenon of be collapsed do not occur, and
And good stability, reusable, after long-time use, the filler in chromatographic column is still without destruction, dissolving.
2. chromatographic column used in the present invention, has preferable separating effect, best result to camptothecine and 10-hydroxycamptothecine
From degree up to more than 2.15, with existing commodity post C18Post is compared, C18The separating degree of post is 1.74, and separating effect can improve
More than 24%.
3. rosinyl polymer microsphere is cheap and easy to get with the derivant of product of natural product rosin as raw material in the present invention, machinery
Intensity is high, and safety non-toxic can be used for the separation of food stage.
4. the present invention prepares Colophonium based high molecular chromatographic column using wet method dress post, and post effect is high, and crushing resistance is good.
5. the HPLC sensitivity of the present invention is high, simple to operate, rapidly and efficiently.
Compared with prior art, beneficial effects of the present invention are：
1. in Detection wavelength：230-290nm, mobile phase be 0.1% acetic acid methanol, 30 DEG C of temperature, flow velocity 0.5mL/min's
Under the conditions of, the separating degree of patent of the present invention compares C18The separating degree of post is big.
2., using Colophonium based high molecular chromatographic column separating common camptotheca fruit alkali and 10-hydroxycamptothecine, mobile phase is single, no for the present invention
Need plus buffer solution, be conducive to the preparation of future drugs, reduce the pollution again of medicine.
3. the cost and C of Colophonium based high molecular chromatographic column fixed phase of the present invention18The fixing phase cost of post compares, the present invention
Fixing phase cost it is relatively low.
Description of the drawings
Fig. 1 is C18Post is to camptothecine and the analysis chart of 10-hydroxycamptothecine mixed solution；
Fig. 2 is the embodiment of the present invention 4 to camptothecine and the analysis chart of 10-hydroxycamptothecine mixed solution；
Fig. 3 is the embodiment of the present invention 5 to camptothecine and the analysis chart of 10-hydroxycamptothecine mixed solution；
Fig. 4 is the embodiment of the present invention 6 to camptothecine and the analysis chart of 10-hydroxycamptothecine mixed solution；
Fig. 5 is the embodiment of the present invention 7 to camptothecine and the analysis chart of 10-hydroxycamptothecine mixed solution.
Specific embodiment
With reference to embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited to
The scope that embodiment is represented.
The preparation of rosinyl polymer microsphere：
Embodiment 1：
200g deionized waters, 0.4g sodium lauryl sulphates, 4.0g polyvinyl alcohol (ions are added in the beaker of 250mL
Water, sodium lauryl sulphate, the mass ratio of polyvinyl alcohol are 100：0.2：2), being heated to 95 DEG C makes polyvinyl alcohol and dodecyl
Sodium sulfate is completely dissolved, and obtains water phase, and being transferred in water-bath makes temperature be down to 55 DEG C of constant temperature.
3.00g maleic rosin acrylic acid glycol esters are weighed, in being dissolved in 50.0g ethyl acetate, using ultrasound wave dissolution solution,
After to be crosslinked dose is completely dissolved, 0.50g α-methacrylic acids, 1.00g isobutyltrimethylmethane .s, 0.10g azodiisobutyronitriles are sequentially added
(function monomer, cross-linking agent, solvent, porogen, initiator quality ratio are 0.5：3：50：1：0.1), sonic oscillation 10min, dispersion
Uniformly, oil phase is obtained.
The oil phase for preparing is added in water phase (mass ratio of oil phase and water phase is 1：3.75), carried out with dispersion machine point
Dissipate, dispersing speed is 3000rpm, and jitter time is 5min, obtains emulsion, after the completion of dispersion, emulsion is moved to into tri- mouthfuls of 500mL
In flask, heat up polymerization under the mixing speed of 100rad/min, constant temperature 10min when temperature rises to 70 DEG C, perseverance when rising to 80 DEG C
Warm 20min, constant temperature 30min when rising to 95 DEG C.
After reaction terminates, product is sieved, the sieve aperture of sieve is 100 mesh, the polymer Jing 5000rad/min's after screening
Rotating speed is centrifuged 5min, and polymer is transferred in 500mL beakers with distilled water wash for several times to remove unnecessary dispersant, will produce
Thing uses successively ethyl acetate, ethanol, methanol solution surname extraction.Finally remove the acetic acid second in polymer with steam distillation
Ester, ethanol, methanol, obtain rosinyl polymer microsphere.
Analyze after testing, the rosinyl polymer microsphere particle diameter distribution that the present embodiment is obtained is 3-10 μm, average pore size is
10-15nm, specific surface area is 90-120m2/ g, acid number 50-150mgKOH/g.
Embodiment 2：
To in the beaker of 500mL add 400g deionized waters, 2.00g sodium lauryl sulphates, 12.00g polyvinyl alcohol (from
Sub- water, sodium lauryl sulphate, the mass ratio of polyvinyl alcohol are 100：0.5：3), being heated to 95 DEG C makes polyvinyl alcohol and dodecane
Base sodium sulfate is completely dissolved, and obtains water phase, and being transferred in water-bath makes temperature be down to 55 DEG C of constant temperature.
12.00g maleic rosin acrylic acid glycol esters are weighed, in being dissolved in 100.00g ethyl acetate, using ultrasound wave dissolution
Solution, after to be crosslinked dose is completely dissolved, sequentially adds 3.00g methyl methacrylates, 5.00g isobutyltrimethylmethane .s, 2.00g azos two different
(function monomer, cross-linking agent, solvent, porogen, initiator quality ratio are 3 to butyronitrile：12：100：5：2), sonic oscillation 10min, point
Dissipate uniform, oil phase is obtained.
The oil phase for preparing is added in water phase (mass ratio of oil phase and water phase is 1：3.4), disperseed with dispersion machine,
Dispersing speed is 4000rpm, and jitter time is 10min, obtains emulsion, after the completion of dispersion, emulsion is moved to into tri- mouthfuls of 1000mL
In flask, heat up polymerization under the mixing speed of 120rad/min, constant temperature 50min when temperature rises to 70 DEG C, perseverance when rising to 80 DEG C
Warm 60min, constant temperature 60min when rising to 95 DEG C.
After reaction terminates, product is sieved, the sieve aperture of sieve is 100 mesh, the polymer Jing 5000rad/min's after screening
Rotating speed is centrifuged 10min, and polymer is transferred in 500mL beakers with distilled water wash for several times to remove unnecessary dispersant, will
Product uses successively ethyl acetate, ethanol, methanol solution surname extraction.Finally remove the acetic acid in polymer with steam distillation
Ethyl ester, ethanol, methanol, obtain rosinyl polymer microsphere.
Analyze after testing, the rosinyl polymer microsphere particle diameter distribution that the present embodiment is obtained is 3-10 μm, average pore size is
10-15nm, specific surface area is 90-120m2/ g, acid number 50-150mgKOH/g.
Embodiment 3：
300g deionized waters, 3.0g polyvinyl alcohol, 0.30g sodium lauryl sulphate (ions are added in the beaker of 400mL
Water, sodium lauryl sulphate, the mass ratio of polyvinyl alcohol are 100：0.1：1), being heated to 95 DEG C makes polyvinyl alcohol and dodecyl
Sodium sulfate is completely dissolved, and obtains water phase, and being transferred in water-bath makes temperature be down to 55 DEG C of constant temperature.
3.25g maleic rosin acrylic acid glycol esters are weighed, in being dissolved in 80.0g ethyl acetate, using ultrasound wave dissolution solution,
After to be crosslinked dose is completely dissolved, 1.55g α-methacrylic acids, 3.0g isobutyltrimethylmethane .s, 1.0g azodiisobutyronitrile (work(are sequentially added
Energy monomer, cross-linking agent, solvent, porogen, initiator quality ratio are 1.55：3.25：80：3：1), sonic oscillation 10min, dispersion
Uniformly, oil phase is obtained.
The oil phase for preparing is added in water phase (mass ratio of oil phase and water phase is 1：3.42), carried out with dispersion machine point
Dissipate, dispersing speed is 5000rpm, and jitter time is 20min, obtains emulsion, after the completion of dispersion, emulsion is moved to into 500mL tri-
In mouth flask, heat up polymerization under the mixing speed of 100rad/min, constant temperature 80min when temperature rises to 70 DEG C, when rising to 80 DEG C
Constant temperature 100min, constant temperature 45min when rising to 95 DEG C.
After reaction terminates, product is sieved, the sieve aperture of sieve is 100 mesh, the polymer Jing 5000rad/min's after screening
Rotating speed is centrifuged 5min, and polymer is transferred in 500mL beakers with distilled water wash for several times to remove unnecessary dispersant, will produce
Thing uses successively ethyl acetate, ethanol, methanol solution surname extraction.Finally remove the acetic acid second in polymer with steam distillation
Ester, ethanol, methanol, obtain rosinyl polymer microsphere.
Analyze after testing, the rosinyl polymer microsphere particle diameter distribution that the present embodiment is obtained is 3-10 μm, average pore size is
10-15nm, specific surface area is 90-120m2/ g, acid number 50-150mgKOH/g.
Using Colophonium based high molecular chromatographic column high efficiency separation camptothecine and the method for 10-hydroxycamptothecine
Embodiment 4：
The rosinyl polymer microsphere of the gained of embodiment 2 is adopted into wet method dress post preparative hplc post, specially：Packing column machine is wet
Method dress post is that rosinyl polymer microsphere is filled in sky chromatographic column with constant pressure pump, and under 3000psi pressure 140min is loaded,
Continue to increase pressure to 3500psi filling 30min, after post flattens weighing apparatus, chromatographic column removed from packing column machine, load onto stigma,
Colophonium based high molecular chromatographic column can be prepared, sample introduction by balancing is rinsed to baseline with 0.10% acetic acid methanol solution.
A kind of method of application Colophonium based high molecular chromatographic column high efficiency separation camptothecine and 10-hydroxycamptothecine, according to such as
Lower step is carried out：
(1) sample solution is prepared：Appropriate camptothecine, 10-hydroxycamptothecine are taken, is dissolved with methanol, be configured to every 1L containing 2.5
×10-4The Fructus seu radix camptothecae acuminatae (Fructus Camptothecae Acuminatae) of mol and 10-hydroxycamptothecine mixed solution, sample introduction；
(2) setup parameter：Colophonium based high molecular chromatographic column is accessed into chromatograph of liquid, the mobile phase of chromatograph of liquid is set
Flow velocity is 0.5mL/min, Detection wavelength 230nm, 30 DEG C of column oven；
(3) separate：Starting injection valve makes methanol bring sample in Colophonium based high molecular chromatographic column into, and sample size is 20 μ L,
The separation of camptothecine and 10-hydroxycamptothecine is realized, acquired results in retention time, 24.68min as shown in figure 1, occur
10-hydroxycamptothecine peak, occurs camptothecine peak in retention time 33.61min, and separating degree is 2.15.
Embodiment 5：
The rosinyl polymer microsphere of the gained of embodiment 1 is adopted into wet method dress post preparative hplc post, specially：Packing column machine is wet
Method dress post is that rosinyl polymer microsphere is filled in sky chromatographic column with constant pressure pump, and under 3000psi pressure 120min is loaded,
Continue to increase pressure to 3500psi filling 10min, after post flattens weighing apparatus, chromatographic column removed from packing column machine, load onto stigma,
Colophonium based high molecular chromatographic column can be prepared, sample introduction by balancing is rinsed to baseline with methanol.
A kind of method of application Colophonium based high molecular chromatographic column high efficiency separation camptothecine and 10-hydroxycamptothecine, according to such as
Lower step is carried out：
(1) sample solution is prepared：Appropriate camptothecine, 10-hydroxycamptothecine are taken, is dissolved with methanol, be configured to every 1L containing 2.5
×10-4The camptothecine of mol and 10-hydroxycamptothecine mixed solution, sample introduction；
(2) setup parameter：Colophonium based high molecular chromatographic column is accessed into chromatograph of liquid, the mobile phase of chromatograph of liquid is set
Flow velocity is 0.3mL/min, Detection wavelength 254nm, 25 DEG C of column oven；
(3) separate：Starting injection valve makes methanol bring sample in Colophonium based high molecular chromatographic column into, and sample size is 20 μ L,
The separation of camptothecine and 10-hydroxycamptothecine is realized, acquired results in retention time, 30.69min as shown in Fig. 2 occur
10-hydroxycamptothecine peak, occurs camptothecine peak in retention time 39.38min, and separating degree is 1.93.
Embodiment 6：
The rosinyl polymer microsphere of the gained of embodiment 3 is adopted into wet method dress post preparative hplc post, specially：Packing column machine is wet
Method dress post is that rosinyl polymer microsphere is filled in sky chromatographic column with constant pressure pump, and under 3000psi pressure 160min is loaded,
Continue to increase pressure to 3500psi filling 50min, after post flattens weighing apparatus, chromatographic column removed from packing column machine, load onto stigma,
Colophonium based high molecular chromatographic column can be prepared, sample introduction by balancing is rinsed to baseline with methanol solution.
A kind of application Colophonium based high molecular chromatographic column high efficiency separation Fructus seu radix camptothecae acuminatae (Fructus Camptothecae Acuminatae) and the method for 10-hydroxycamptothecine, according to as follows
Step is carried out：
(1) sample solution is prepared：Appropriate camptothecine, 10-hydroxycamptothecine are taken, is dissolved with methanol, be configured to every 1L containing 2.5
×10-4The camptothecine of mol and 10-hydroxycamptothecine mixed solution, sample introduction；
(2) setup parameter：Colophonium based high molecular chromatographic column is accessed into chromatograph of liquid, the mobile phase of chromatograph of liquid is set
Flow velocity is 0.8mL/min, Detection wavelength 290nm, 35 DEG C of column oven；
(3) separate：Starting injection valve makes methanol bring sample in Colophonium based high molecular chromatographic column into, and sample size is 20 μ L,
The separation of camptothecine and 10-hydroxycamptothecine is realized, acquired results in retention time, 28.55min as shown in figure 3, occur
10-hydroxycamptothecine peak, occurs camptothecine peak in retention time 36.61min, and separating degree is 1.99.
Embodiment 7：
The rosinyl polymer microsphere of the gained of embodiment 2 is adopted into wet method dress post preparative hplc post, specially：Packing column machine is wet
Method dress post is that rosinyl polymer microsphere is filled in sky chromatographic column with constant pressure pump, and under 3000psi pressure 180min is loaded,
Continue to increase pressure to 3500psi filling 60min, after post flattens weighing apparatus, chromatographic column removed from packing column machine, load onto stigma,
Colophonium based high molecular chromatographic column can be prepared, sample introduction by balancing is rinsed to baseline with 0.10% acetic acid methanol solution.
A kind of method of application Colophonium based high molecular chromatographic column high efficiency separation camptothecine and 10-hydroxycamptothecine, according to such as
Lower step is carried out：
(1) sample solution is prepared：Appropriate camptothecine, 10-hydroxycamptothecine are taken, is dissolved with methanol, be configured to every 1L containing 2.5
×10-4The camptothecine of mol and 10-hydroxycamptothecine mixed solution, sample introduction；
(2) setup parameter：Colophonium based high molecular chromatographic column is accessed into chromatograph of liquid, the mobile phase of chromatograph of liquid is set
Flow velocity is 1.0mL/min, Detection wavelength 254nm, 30 DEG C of column oven；
(3) separate：Starting injection valve makes methanol bring sample in Colophonium based high molecular chromatographic column into, and sample size is 20 μ L,
The separation of camptothecine and 10-hydroxycamptothecine is realized, acquired results in retention time 26.96min as shown in figure 4,10- occur
Hydroxy camptothecin peak, occurs camptothecine peak in retention time 37.27min, and separating degree is 2.10.
Claims (9)
1. a kind of method of application Colophonium based high molecular chromatographic column high efficiency separation camptothecine and 10-hydroxycamptothecine, its feature exists
In：Colophonium based high molecular chromatograph is prepared using rosinyl polymer microsphere as fixed phase stuffing packing column machine wet method dress post
Post；Colophonium based high molecular chromatographic column is accessed into chromatograph of liquid, the flow rate of mobile phase for arranging chromatograph of liquid is 0.3-1.0mL/
Min, Detection wavelength 230-290nm, 30 ± 10 DEG C of column oven；Starting injection valve makes mobile phase by camptothecine and 10- hydroxy-camptothecins
The mixed solution of alkali is brought in Colophonium based high molecular chromatographic column, realizes the separation of camptothecine and 10-hydroxycamptothecine.
2. one kind according to claim 1 is liked using Colophonium based high molecular chromatographic column high efficiency separation camptothecine and 10- hydroxyls
The method of tree alkali, it is characterised in that：Described rosinyl polymer microsphere particle diameter distribution is 3-10 μm, and average pore size is 10-
15nm, specific surface area is 90-120m2/ g, acid number 50-150mgKOH/g.
3. one kind according to claim 1 and 2 is using Colophonium based high molecular chromatographic column high efficiency separation camptothecine and 10- hydroxyls
The method of camptothecine, it is characterised in that：Described packing column machine wet method dress post is to be filled rosinyl polymer microsphere with constant pressure pump
In empty chromatographic column, 120-180min is loaded under 3000psi pressure, continues to increase pressure to 3500psi filling 10-60min,
After post flattens weighing apparatus, chromatographic column is removed from packing column machine, load onto stigma, you can prepare Colophonium based high molecular chromatographic column.
4. one kind according to claim 1 and 2 is using Colophonium based high molecular chromatographic column high efficiency separation camptothecine and 10- hydroxyls
The method of camptothecine, it is characterised in that：The preparation method of described rosinyl polymer microsphere is：With α-methacrylic acid or first
Base acrylic acid methyl ester. is monomer, and maleic rosin acrylic acid glycol ester is cross-linking agent, using micro suspension free radical polymerisation process system
Standby rosinyl polymer microsphere.
5. one kind according to claim 4 is liked using Colophonium based high molecular chromatographic column high efficiency separation camptothecine and 10- hydroxyls
The method of tree alkali, it is characterised in that：The loose micro suspension free radical polymerisation process is：By ionized water, sodium lauryl sulphate, gather
The water phase and α-methacrylic acid or methyl methacrylate of vinyl alcohol composition, cross-linking agent maleic rosin acrylic acid glycol ester,
Solvent ethyl acetate, porogen isobutyltrimethylmethane., the oil phase mixing of initiator azodiisobutyronitrile composition, are disperseed with dispersion machine,
It is 3000-5000rpm that dispersion machine arranges rotating speed, and jitter time is 5-20min, then carries out temperature-programmed reaction and institute is obtained
The rosinyl polymer microsphere stated.
6. one kind according to claim 5 is liked using Colophonium based high molecular chromatographic column high efficiency separation camptothecine and 10- hydroxyls
The method of tree alkali, it is characterised in that：Described oil phase and the mass ratio of water phase is 1：3-6.
7. one kind according to claim 5 is liked using Colophonium based high molecular chromatographic column high efficiency separation camptothecine and 10- hydroxyls
The method of tree alkali, it is characterised in that：Described temperature-programmed reaction is：70 DEG C of reaction 10-80min of temperature programming, 80 DEG C of reactions
20-100min, 95 DEG C of reaction 30-60min, you can obtain described rosinyl polymer microsphere.
8. one kind according to claim 5 is liked using Colophonium based high molecular chromatographic column high efficiency separation camptothecine and 10- hydroxyls
The method of tree alkali, it is characterised in that：Deionized water, sodium lauryl sulphate, the mass ratio of polyvinyl alcohol are in described water phase
100：0.1-0.5：1-3.
9. according to the arbitrary described one kind application Colophonium based high molecular chromatographic column high efficiency separation camptothecine of claim 5-8 and 10-
The method of hydroxy camptothecin, it is characterised in that：α-methacrylic acid or methyl methacrylate in described oil phase, cross-linking agent,
Solvent, porogen, initiator quality ratio are 0.5-3：3-12：50-100：1-5：0.1-2.
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