CN105388234B - The detection method of content of taxol in Chinese yew branch - Google Patents
The detection method of content of taxol in Chinese yew branch Download PDFInfo
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- CN105388234B CN105388234B CN201511000519.0A CN201511000519A CN105388234B CN 105388234 B CN105388234 B CN 105388234B CN 201511000519 A CN201511000519 A CN 201511000519A CN 105388234 B CN105388234 B CN 105388234B
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- 229930012538 Paclitaxel Natural products 0.000 title claims abstract description 92
- 229960001592 paclitaxel Drugs 0.000 title claims abstract description 85
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 title claims abstract description 85
- 238000001514 detection method Methods 0.000 title claims abstract description 31
- 241001149649 Taxus wallichiana var. chinensis Species 0.000 title claims abstract description 29
- 239000000243 solution Substances 0.000 claims abstract description 79
- 238000012360 testing method Methods 0.000 claims abstract description 41
- 239000013558 reference substance Substances 0.000 claims abstract description 39
- 239000007788 liquid Substances 0.000 claims abstract description 16
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000002360 preparation method Methods 0.000 claims abstract description 9
- 150000004579 taxol derivatives Chemical class 0.000 claims abstract description 7
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 4
- 239000011259 mixed solution Substances 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 81
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 30
- 238000000605 extraction Methods 0.000 claims description 21
- 239000000047 product Substances 0.000 claims description 16
- 239000012071 phase Substances 0.000 claims description 15
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 12
- 239000007791 liquid phase Substances 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 10
- 238000005238 degreasing Methods 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 9
- 239000002904 solvent Substances 0.000 claims description 9
- 238000000638 solvent extraction Methods 0.000 claims description 8
- 239000012141 concentrate Substances 0.000 claims description 7
- 238000000622 liquid--liquid extraction Methods 0.000 claims description 7
- 238000005374 membrane filtration Methods 0.000 claims description 7
- 238000002137 ultrasound extraction Methods 0.000 claims description 7
- 239000003208 petroleum Substances 0.000 claims description 6
- 239000008346 aqueous phase Substances 0.000 claims description 4
- YDVNLQGCLLPHAH-UHFFFAOYSA-N dichloromethane;hydrate Chemical compound O.ClCCl YDVNLQGCLLPHAH-UHFFFAOYSA-N 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 244000050510 Cunninghamia lanceolata Species 0.000 claims description 2
- 240000001417 Vigna umbellata Species 0.000 claims description 2
- 235000011453 Vigna umbellata Nutrition 0.000 claims description 2
- 238000002604 ultrasonography Methods 0.000 claims description 2
- 239000002893 slag Substances 0.000 claims 1
- 241001116500 Taxus Species 0.000 abstract description 6
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 abstract description 6
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 3
- WSGYTJNNHPZFKR-UHFFFAOYSA-N 3-hydroxypropanenitrile Chemical compound OCCC#N WSGYTJNNHPZFKR-UHFFFAOYSA-N 0.000 abstract 1
- 238000000034 method Methods 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 239000000284 extract Substances 0.000 description 6
- WXYIONYJZVWSIJ-UHFFFAOYSA-N acetonitrile;methanol;hydrate Chemical compound O.OC.CC#N WXYIONYJZVWSIJ-UHFFFAOYSA-N 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 239000012488 sample solution Substances 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000000401 methanolic extract Substances 0.000 description 3
- 238000003672 processing method Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- YWLXLRUDGLRYDR-YLPZXOTFSA-N 10-dab iii Chemical compound O([C@H]1[C@@H]2[C@](C([C@H](O)C3=C(C)[C@@H](O)C[C@]1(O)C3(C)C)=O)(C)[C@H](O)C[C@H]1OC[C@@]12OC(=O)C)C(=O)C1=CC=CC=C1 YWLXLRUDGLRYDR-YLPZXOTFSA-N 0.000 description 1
- 235000016408 Podocarpus macrophyllus Nutrition 0.000 description 1
- 244000162450 Taxus cuspidata Species 0.000 description 1
- 235000009065 Taxus cuspidata Nutrition 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 239000003005 anticarcinogenic agent Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 125000003963 dichloro group Chemical group Cl* 0.000 description 1
- YJVCRVPZGVVAEQ-UHFFFAOYSA-N ethanol;methanol;propan-2-one Chemical compound OC.CCO.CC(C)=O YJVCRVPZGVVAEQ-UHFFFAOYSA-N 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- -1 is well mixed Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The present invention relates to a kind of detection method of content of taxol in Chinese yew branch, comprise the following steps：The preparation of need testing solution；The preparation of reference substance solution；High effective liquid chromatography for measuring：It is accurate respectively to draw reference substance solution and each 20 μ L of need testing solution, liquid chromatograph is injected, records collection of illustrative plates, calculates the peak area of taxol in the peak area of taxol and reference substance solution in need testing solution respectively；Chromatographic condition：By the use of C18 posts as chromatographic column, using methanol acetonitrile water, acetonitrile water or methanol water mixed solution as mobile phase, Detection wavelength 227nm, flow velocity 1.00mL/min, 30 DEG C of column temperature, theoretical cam curve is not less than 3000；Paclitaxel concentration calculates：C taxols=(C reference substances × A test samples)/A reference substances.For the present invention using the content of taxol in high performance liquid chromatography detection branches and leaves of yew, this detection method is simple to operate, accurate, reproducible, can preferably detect the content of taxol in branches and leaves of yew.
Description
Technical field
The present invention relates to a kind of detection method, the detection method of content of taxol in specifically a kind of Chinese yew branch.
Background technology
Chinese yew, also known as Japanese yew, it is natural rare plants for anticancer endangered in the world, category first-grade state protection is planted
Thing, existing tall and big arbor, there are short shrub, about tens kinds of the whole world again.It is to integrate to view and admire and medicinal treasure
Your seeds, the taxol extracted from plant is universally acknowledged anticarcinogen, expensive.Thus, Chinese yew also has " plant
The title of gold ".
Due to the performance chemical structure of taxol, the paclitaxel api of industrialized production is mainly from red bean at present
It is naturally being extracted in China fir or by extracting 10-DABIII from Chinese yew, then prepared by the way of semi-synthetic.
《Chinese Pharmacopoeia》There is standard to the detection method of paclitaxel api, but to the inspection of content of taxol in Chinese yew branch
Survey does not have standard but, and traditional detection method is excessively complicated, is unsuitable for Chinese yew raw material screening mistake in large-scale industrial production
Detection in journey.
The content of the invention
The purpose of the present invention is to be directed to above-mentioned present situation, there is provided a kind of simple, quick, accurate, precision is good branch of Ramulus et folium taxi cuspidatae
The detection method of content of taxol in bar.
The detection method of content of taxol in a kind of Chinese yew branch, it comprises the following steps：
S11：The preparation of need testing solution：Fresh Chinese yew branch washing, drying, 60~80 mesh are crushed to, precision weighs
Chinese yew branch powder 2.00g, is placed in centrifuge tube, and powder is molten through degreasing, extraction, liquid-liquid extraction step, concentrate methanol
Constant volume is solved, is well mixed, 0.45 μm of membrane filtration of solution, filtrate is as need testing solution；
S12：The preparation of reference substance solution：Precision weighs taxol control product 10mg and put in 10mL volumetric flasks, adds methanol
Dissolve and be settled to 10mL, after being well mixed, measure 1mL with liquid-transfering gun precision and put in 100mL volumetric flasks, add methanol to scale
Line, it is 10mg/L reference substance solutions to shake up and produce concentration, and -20 DEG C are in store for；
S13：High effective liquid chromatography for measuring：It is accurate respectively to draw reference substance solution and each 20 μ L of need testing solution, injection
Liquid chromatograph, collection of illustrative plates is recorded, calculate the peak area of taxol and taxol in reference substance solution in need testing solution respectively
Peak area；Chromatographic condition：By the use of C18 posts as chromatographic column, using methanol-acetonitrile-water, acetonitrile-water or methanol-water mixed solution as stream
Dynamic phase, Detection wavelength 227nm, flow velocity 1.00mL/min, 30 DEG C of column temperature, theoretical cam curve are not less than 3000；
S14：Paclitaxel concentration calculates：C taxols=(C reference substances × A test samples)/A reference substances；Wherein：A reference substances are
The peak area of taxol in reference substance solution；A test samples are the peak area of taxol in need testing solution；C reference substances are control
The concentration of taxol in product solution；C taxols are the concentration of taxol in need testing solution.
The present invention is ground using the content of taxol in high performance liquid chromatography detection branches and leaves of yew by detection method
Study carefully, optimize processing method, chromatographic condition, show that this detection method of content has the spy such as simple to operate, accurate, reproducible
Point, it can preferably detect the content of taxol in branches and leaves of yew.
Brief description of the drawings
Fig. 1 show the flow of the detection method of content of taxol in the Chinese yew branch of embodiment of the present invention offer
Figure.
Fig. 2 show mobile phase as taxol control product solution and branch of Ramulus et folium taxi cuspidatae bar sample under the conditions of methanol-water (60: 40)
Solution comparison diagram.
Fig. 3 show mobile phase as taxol control product solution and Chinese yew under the conditions of methanol-acetonitrile-water (25: 35: 40)
Branch sample solution comparison diagram.
Fig. 4 show mobile phase condition as taxol control product solution and Chinese yew branch under the conditions of acetonitrile-water (55: 45)
Sample solution comparison diagram.
Fig. 5 show taxol control product solution standard curve.
Embodiment
The principles and features of the present invention are described below, and the given examples are served only to explain the present invention, is not intended to limit
Determine the scope of the present invention.
As Figure 1-5, the detection side of content of taxol in a kind of Chinese yew branch provided for embodiment of the present invention
Method, it comprises the following steps：
S11：The preparation of need testing solution：Fresh Chinese yew branch washing, drying, 60~80 mesh are crushed to, precision weighs
Chinese yew branch powder 2.00g, is placed in centrifuge tube, and powder is molten through degreasing, extraction, liquid-liquid extraction step, concentrate methanol
Constant volume is solved, is well mixed, 0.45 μm of membrane filtration of solution, filtrate is as need testing solution；
S12：The preparation of reference substance solution：Precision weighs taxol control product 10mg and put in 10mL volumetric flasks, adds methanol
Dissolve and be settled to 10mL, after being well mixed, measure 1mL with liquid-transfering gun precision and put in 100mL volumetric flasks, add methanol to scale
Line, it is 10mg/L reference substance solutions to shake up and produce concentration, and -20 DEG C are in store for；
S13：High effective liquid chromatography for measuring：It is accurate respectively to draw reference substance solution and each 20 μ L of need testing solution, injection
Liquid chromatograph, collection of illustrative plates is recorded, calculate the peak area of taxol and taxol in reference substance solution in need testing solution respectively
Peak area；Chromatographic condition：By the use of C18 posts as chromatographic column, using methanol-acetonitrile-water, acetonitrile-water or methanol-water mixed solution as stream
Dynamic phase, Detection wavelength 227nm, flow velocity 1.00mL/min, 30 DEG C of column temperature, theoretical cam curve are not less than 3000；
S14：Paclitaxel concentration calculates：C taxols=(C reference substances × A test samples)/A reference substances；Wherein：A reference substances are
The peak area of taxol in reference substance solution；A test samples are the peak area of taxol in need testing solution；C reference substances are control
The concentration of taxol in product solution；C taxols are the concentration of taxol in need testing solution.
In present embodiment, degreasing solvent described in step S11 is petroleum ether, solvent volume 30mL, is repeated 2 times.
In present embodiment, Extraction solvent described in step S11 is methanol, ethanol, acetone.It is preferred that the Extraction solvent is
Methanol.
In present embodiment, extraction or ultrasonic extraction are extracted as described in step S11.It is preferred that the ultrasound that is extracted as carries
Take, extraction conditions are：Time 60min, power 250W, frequency 100kHz.
In present embodiment, the liquid-liquid extraction described in step S11 is dichloromethane-water (1: 1), aqueous phase dichloromethane
Extraction is three times.Concrete operation step is：By the methanol extract liquid after merging, it is concentrated under reduced pressure and is evaporated in 50 DEG C of water-baths, concentrate is used
Dichloromethane-aqueous solution that 60mL volume ratios are 1: 1 redissolves, and takes dichloromethane layer, and aqueous phase adds the extraction of 30mL dichloromethane
Twice, dichloromethane phase three times is merged；Dichloromethane solution, which is concentrated under reduced pressure, to be evaporated, and concentrate is dissolved with methanol, and is settled to
100mL。
In present embodiment, C18 chromatographic columns described in step S13 are TR-C18-X liquid-phase chromatographic columns, preferably 4.6 ×
The TR-C18-X liquid-phase chromatographic columns of 250mm × 5 μm.
In present embodiment, mobile phase described in step S13 be volume ratio be 25: 35: 40 methanol-acetonitrile-water, volume
Than the methanol-water that the acetonitrile-water for 55: 45 or volume ratio are 60: 40.
Test example
1st, instrument and reagent information：
Agilent1100 types high performance liquid chromatograph (Agilent companies of the U.S.), a ten thousandth balance (Changzhou day it
Flat experimental instruments and equipment limited), miiner ultrapure water machines, electric drying oven with forced convection (Wuhan Jin Baohua Science and Technology Ltd.), surpass
Sound wave cleaning machine (Jing Zuancheng ultrasonic equipments Co., Ltd of Shenzhen), (Hunan perseverance promise instrument and equipment has 5-5N low speed centrifuges
Limit company), GZY scientific researches and pulverizer (Shanghai Gao Zhi precision instruments Co., Ltd).
Taxol control product (lot number, Beijing Century AudioCodes Bioisystech Co., Ltd), hplc grade methanol, chromatographic grade second
Nitrile, analysis level methanol, analysis level ethanol analyze grade acetone, analysis level petroleum ether, and water is that ultra-pure water is made in laboratory by oneself.
2nd, method and result
The selection of 2.1 liquid phase chromatogram conditions
2.1.1 the selection of mobile phase
In the selection of mobile phase, to methanol-acetonitrile-water (25: 35: 40), acetonitrile-water (55: 45), methanol-water (60: 40)
Screened etc. different mobile phases, the results showed that the separation of gained chromatogram using methanol-water (60: 40) solution as mobile phase
Degree, symmetry are preferable, so from methanol-water (60: 40) solution as mobile phase.
2.1.2 the selection of wavelength is determined
Take concentration be 50mg/L reference substance solution tested with ultraviolet specrophotometer, measure taxol wavelength 227~
There is absorption maximum at 228nm, the result is consistent with result reported in the literature, so being measure wavelength from 227nm.
Comprehensive many reasons, it is determined that the optimal chromatographic condition of the present invention is：
Chromatographic column：The TR-C18-X of 4.6 × 250mm × 5 μm；Mobile phase: methanol-water (60: 40) solution；Wavelength：
227nm；Column temperature：30℃；Flow velocity：1.00mL/min.
The selection of 2.2 test sample processing methods
2.2.1 the selection of test sample Extraction solvent
Precision weighs tri- parts of Chinese yew branch powder 2.00g, is placed in centrifuge tube, adds petroleum ether 30mL, ultrasonic degreasing 2
It is secondary, each 30min, 50 DEG C of drying of residue after degreasing；Dried residue is separately added into 30mL methanol, ethanol, acetone, ultrasound
Extraction, each 60min, extract 3 times altogether, merge extract solution, wherein add first after the rotated evaporation drying of ethanol and acetone soln
Alcohol redissolves, and is placed in after methanol solution centrifugation in 100mL volumetric flasks, scale is added to methanol, be well mixed, 0.45 μm of filter of solution
Membrane filtration produces.The content results of taxol are determined, the results are shown in Table 1
The different solvents extraction effect of table 1 compares
| Extraction solvent | Methanol | Ethanol | Acetone |
| Taxol (%) | 0.021% | 0.019% | 0.018% |
As a result show, the extraction effect of methanol is good, therefore selects methanol as Extraction solvent.
2.2.2 the selection of sample extraction method
Precision weighs two parts of Chinese yew branch powder 2.00g, is placed in centrifuge tube, adds petroleum ether 30mL, ultrasonic degreasing 2
It is secondary, each 30min, 50 DEG C of drying of residue；Dried residue adds 30mL methanol, and portion is using ultrasonic (power 250W, frequency
60min 100kHz) is extracted, is extracted 3 times；Another uses Soakage extraction 24h, extracts 3 times, merges extract solution, is placed in after centrifugation
In 100mL volumetric flasks, scale is added to methanol, is well mixed, solution is produced with 0.45 μm of membrane filtration.Measure taxol contains
Result is measured, the results are shown in Table 2.
The Different Extraction Method extraction effect of table 2 compares
| Extracting method | Ultrasonic extraction | Soakage extraction |
| Taxol (%) | 0.022% | 0.021% |
As a result show, for ultrasonic extraction 60min compared with Soakage extraction 24h, measured content of taxol is almost suitable, real
The operating procedure tested, so from relatively simple ultrasonic extraction.
2.2.3 the selection of Sample Purification on Single method
Precision weighs two parts of Chinese yew branch powder 2.00g, is placed in centrifuge tube, adds petroleum ether 30mL, ultrasonic degreasing 2
It is secondary, each 30min, 50 DEG C of drying of residue；Dried residue adds 30mL methanol, ultrasonic extraction 3 times, each 60min, merges
Extract solution；Then, after a methanol extract liquid is centrifuged, with methanol constant volume to 100mL, 0.45 μm of membrane filtration, as trying
Product solution；By another methanol extract liquid, be concentrated under reduced pressure and be evaporated in 50 DEG C of water-baths, concentrate with 60mL volume ratios be 1: 1 two
Chloromethanes-aqueous solution redissolves, and takes dichloromethane layer, aqueous phase adds 30mL dichloromethane and is extracted twice, and merges dichloro three times
Methane phase；Dichloromethane solution, which is concentrated under reduced pressure, to be evaporated, and concentrate 100mL methanol redissolves, and redissolves 0.45 μm of membrane filtration of liquid.Cross
Solution after filter, liquid phase detection, determines content of taxol, the results are shown in Table 3.
The effect whether table 3 purifies compares
| Method | Do not purify | Liquid-liquid extraction |
| Taxol (%) | 0.025% | 0.019% |
As a result show, content of taxol is higher in the test sample solution not by purification process, and purification process uses
The method of dichloromethane-water liquid-liquid extraction, it is soluble in dichloromethane using taxol and is insoluble in the characteristic of water, but operated
Be concentrated under reduced pressure step and solution transfer several times is have passed through in journey, these steps can cause taxol loss.Extract without liquid liquid
Taxol target peak separates preferably with impurity peaks in the liquid chromatogram of the test sample solution taken, does not influence to detect, so choosing
With step, simple and taxol detects the higher method not purified.
The preparation of 2.3 reference substance solutions
Precision weighs taxol control product 10mg and put in 10mL volumetric flasks, adds methanol and dissolves and be settled to 10mL, mixes
After uniformly, measure 1mL with liquid-transfering gun precision and put in 100mL volumetric flasks, add methanol to graduation mark, shake up and produce concentration and be
10mg/L reference substance solutions, -20 DEG C are in store for.
2.4 high-efficient liquid phase chromatogram process measuring method：
It is accurate respectively to draw reference substance solution and each 20 μ L of need testing solution, liquid chromatograph is injected, records collection of illustrative plates, respectively
Calculate the peak area of the peak area of taxol and taxol in reference substance solution in need testing solution.
2.5 Method validation
2.5.1 taxol linear relationship is investigated
Take reference substance standard reserving solution, standard reserving solution is diluted to 2.0 successively, 5.0,10.0,20.0,50.0,
100.0th, 200.0 μ g/mL series standard concentration.It is accurate respectively to draw the above-mentioned μ L of solution 20, inject in liquid chromatograph, upper
State the peak area that taxol is determined under liquid phase chromatogram condition.Using reference substance solution concentration as abscissa, using taxol peak area as
Ordinate, standard curve is made, regression equation is y=3076.2x-901.46 (R=0.9998).
As a result show, for taxol in the range of 2.0~200 μ g/mL, peak area and sample size are in good linear relation.
2.5.2 precision test
It is the μ L of 10mg/L taxol control product solution 20 that precision, which draws above-mentioned concentration, is injected in liquid chromatograph, by above-mentioned
Chromatographic condition detects, parallel determination 5 times.It is that 30120.45, RSD is 0.87% (n=to measure 5 taxol peak area average values
5), show that instrument precision is good.
2.5.3 stability test
At 0,2,4,6,8,10,12 hour, draw respectively with crowd μ L of need testing solution (150908) 20, inject liquid chromatogram
In instrument, detected by above-mentioned liquid-phase condition, the RSD for measuring 5 taxol peak areas is 1.57%, shows need testing solution in 12h
It is interior that there is preferable stability.
2.5.4 replica test
Precision is weighed with batch (150912) 5 parts of Chinese yew branch powder, and every part is 2.0g, in described in embodiment 1
Method prepares need testing solution, and the content of taxol is determined by above-mentioned liquid-phase condition, as a result shows taxol detection with batch RSD
For 1.13% (n=5), show that this method reappearance is good.
2.5.5 it is loaded recovery experiment
Same batch Chinese yew branch powder (150921) (former content of taxol is 241.89mg/Kg) about 2.0g is taken, totally 5
Part, precision weighs, accurate respectively to add taxol control product (1000mg/L) 0.5mL, after carry sample is dried completely, by example 1
Described method prepares need testing solution, and content of taxol is determined by above-mentioned chromatographic condition.As a result show that the average of taxol adds
The sample rate of recovery is that 98.87%, RSD is 0.98% (n=5).The present invention is using in high performance liquid chromatography detection branches and leaves of yew
The content of taxol, studied by detection method, optimize processing method, chromatographic condition, show that this detection method of content has
There is the features such as simple to operate, accurate, reproducible, can preferably detect the content of taxol in branches and leaves of yew.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc., it should be included in the scope of the protection.
Claims (5)
1. the detection method of content of taxol in a kind of Chinese yew branch, it is characterised in that comprise the following steps：
S11：The preparation of need testing solution：Fresh Chinese yew branch washing, drying, 60~80 mesh are crushed to, precision weighs red bean
China fir branch powder 2.00g, is placed in centrifuge tube, adds petroleum ether 30mL, and ultrasonic degreasing 2 times, each 30min is residual after degreasing
50 DEG C of drying of slag；The extracted solvent extraction of dried residue, liquid-liquid extraction step, the Extraction solvent are methanol, concentrate
Constant volume is dissolved with methanol, is well mixed, 0.45 μm of membrane filtration of solution, filtrate is as need testing solution；
S12：The preparation of reference substance solution：Precision weighs taxol control product 10mg and put in 10mL volumetric flasks, adds methanol dissolving
And 10mL is settled to, and after being well mixed, measure 1mL with liquid-transfering gun precision and put in 100mL volumetric flasks, addition methanol to graduation mark,
It is 10mg/L reference substance solutions to shake up and produce concentration, and -20 DEG C are in store for；
S13：High effective liquid chromatography for measuring：It is accurate respectively to draw reference substance solution and each 20 μ L of need testing solution, inject liquid phase
Chromatograph, collection of illustrative plates is recorded, calculate the peak face of the peak area of taxol and taxol in reference substance solution in need testing solution respectively
Product；Chromatographic condition：By the use of C18 posts as chromatographic column, with methanol-water 60:40 mixed solutions are mobile phase, Detection wavelength 227nm,
Flow velocity is 1.00mL/min, and 30 DEG C of column temperature, theoretical cam curve is not less than 3000；
S14：Paclitaxel concentration calculates：C taxols=(C reference substances × A test samples)/A reference substances；Wherein：A reference substances are control
The peak area of taxol in product solution；A test samples are the peak area of taxol in need testing solution；C reference substances are that reference substance is molten
The concentration of taxol in liquid；C taxols are the concentration of taxol in need testing solution.
2. the detection method of content of taxol in Chinese yew branch as claimed in claim 1, it is characterised in that in step S11
It is described to be extracted as ultrasonic extraction or Soakage extraction.
3. the detection method of content of taxol in Chinese yew branch as claimed in claim 2, it is characterised in that the ultrasound carries
The condition taken is：Time 60min, power 250W, frequency 100kHz.
4. the detection method of content of taxol in Chinese yew branch as claimed in claim 1, it is characterised in that in step S11
Described liquid-liquid extraction is dichloromethane-water 1:1, aqueous phase is extracted three times with dichloromethane.
5. the detection method of content of taxol in Chinese yew branch as claimed in claim 1, it is characterised in that in step S13
The C18 chromatographic columns are the TR-C18-X liquid-phase chromatographic columns of 4.6 × 250mm × 5 μm.
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