CN106596759B - A kind of HPLC analysis method of Ramulus et folium taxi cuspidatae extract and its preparation - Google Patents
A kind of HPLC analysis method of Ramulus et folium taxi cuspidatae extract and its preparation Download PDFInfo
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- CN106596759B CN106596759B CN201611105309.2A CN201611105309A CN106596759B CN 106596759 B CN106596759 B CN 106596759B CN 201611105309 A CN201611105309 A CN 201611105309A CN 106596759 B CN106596759 B CN 106596759B
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- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 55
- 238000002360 preparation method Methods 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 28
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 42
- 239000012085 test solution Substances 0.000 claims abstract description 14
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- YWLXLRUDGLRYDR-ZHPRIASZSA-N 5beta,20-epoxy-1,7beta,10beta,13alpha-tetrahydroxy-9-oxotax-11-ene-2alpha,4alpha-diyl 4-acetate 2-benzoate 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-ZHPRIASZSA-N 0.000 description 2
- DBXFAPJCZABTDR-KUEXGRMWSA-N Cephalomannine Natural products O=C(O[C@@H]1C(C)=C2[C@@H](OC(=O)C)C(=O)[C@]3(C)[C@@H](O)C[C@@H]4[C@](OC(=O)C)([C@H]3[C@H](OC(=O)c3ccccc3)[C@@](O)(C2(C)C)C1)CO4)[C@@H](O)[C@H](NC(=O)/C(=C\C)/C)c1ccccc1 DBXFAPJCZABTDR-KUEXGRMWSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 229930012538 Paclitaxel Natural products 0.000 description 2
- 229940123237 Taxane Drugs 0.000 description 2
- -1 alkanes diterpene Chemical class 0.000 description 2
- 229930014667 baccatin III Natural products 0.000 description 2
- DBXFAPJCZABTDR-WBYYIXQISA-N cephalomannine Chemical compound O([C@@H]1[C@]2(O)C[C@@H](C(=C([C@@H](OC(C)=O)C(=O)[C@]3(C)[C@@H](O)C[C@H]4OC[C@]4([C@H]31)OC(C)=O)C2(C)C)C)OC(=O)[C@H](O)[C@@H](NC(=O)C(/C)=C/C)C=1C=CC=CC=1)C(=O)C1=CC=CC=C1 DBXFAPJCZABTDR-WBYYIXQISA-N 0.000 description 2
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- 229940108949 paclitaxel injection Drugs 0.000 description 2
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- HHUWBBVYZCIXQS-UHFFFAOYSA-N taxinine M Natural products CC(=O)OC1CC(O)C(=C)C2C(OC(=O)C)C3CC(=O)C4(C)OCC3(C)C4(O)C(OC(=O)C)C(OC(=O)c5ccccc5)C12COC(=O)c6ccccc6 HHUWBBVYZCIXQS-UHFFFAOYSA-N 0.000 description 2
- 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 description 2
- XUMIEQQAVQWNBJ-UHFFFAOYSA-N taxumairol R Natural products C1C(=O)C(C2(O)C(OC(C)=O)C3OC(C)=O)(C)OCC2(C)C1C(OC(C)=O)C1C(=C)C(OC(=O)C)CC(OC(C)=O)C31COC(=O)C1=CC=CC=C1 XUMIEQQAVQWNBJ-UHFFFAOYSA-N 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- RSWGJHLUYNHPMX-UHFFFAOYSA-N Abietic-Saeure Natural products C12CCC(C(C)C)=CC2=CCC2C1(C)CCCC2(C)C(O)=O RSWGJHLUYNHPMX-UHFFFAOYSA-N 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 235000011615 Pinus koraiensis Nutrition 0.000 description 1
- 240000007263 Pinus koraiensis Species 0.000 description 1
- 208000004880 Polyuria Diseases 0.000 description 1
- KHPCPRHQVVSZAH-HUOMCSJISA-N Rosin Natural products O(C/C=C/c1ccccc1)[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 KHPCPRHQVVSZAH-HUOMCSJISA-N 0.000 description 1
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- 230000035619 diuresis Effects 0.000 description 1
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- KHPCPRHQVVSZAH-UHFFFAOYSA-N trans-cinnamyl beta-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OCC=CC1=CC=CC=C1 KHPCPRHQVVSZAH-UHFFFAOYSA-N 0.000 description 1
Classifications
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- 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
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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)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses the HPLC analysis methods of a kind of Ramulus et folium taxi cuspidatae extract and its preparation.First, weigh the Ramulus et folium taxi cuspidatae extract sample or its formulation samples of at least six qualification, it mixes, filter with methanol respectively, obtain the test solution of each sample, carry out HPLC analysis, the HPLC finger-print of each sample is obtained, Ramulus et folium taxi cuspidatae extract or the HPLC reference fingerprint of its preparation are established with this；Then, sample to be tested mixed with methanol, filtered, obtain the test solution of sample to be tested, then carried out HPLC analysis, obtain the HPLC finger-print of the sample to be tested；The finger-print is compared with corresponding HPLC reference fingerprint, the superiority and inferiority of Ramulus et folium taxi cuspidatae extract and its quality of the pharmaceutical preparations is judged according to comparison result.The present invention can control the quality of Ramulus et folium taxi cuspidatae extract and its preparation well.
Description
Technical field
The invention belongs to Pharmaceutical Analysis technical fields.More particularly, to a kind of Ramulus et folium taxi cuspidatae extract and its preparation
HPLC analysis method.
Background technique
Branches and leaves of yew be taxaceae plant yew dried leaf, branches and leaves of yew mainly contain two ring taxanes,
Various tricyclic taxanes, rosin alkanes and Korean pine alkanes diterpene and lignanoids, flavonoids and phenolic compound etc. at
Point.Branches and leaves of yew has the effects that inducing diuresis to remove edema, warm kidney promoting menstruation, antitumor, inhibition diabetes, thus can be used as drug
It utilizes.
Since branches and leaves of yew has above-mentioned drug effect, thus in the prior art, the extraction of branches and leaves of yew obtains more
Side's research.Ramulus et folium taxi cuspidatae extract refers to through intermediate extract made from legal technique, process route are as follows: takes dry Chinese yew
Branches and leaves, add water to cook secondary, 2 hours first times, second 1 hour, collecting decoction, filtration, and filtrate is concentrated into relative density
1.12~1.15(60 DEG C), add ethyl alcohol to alcohol content up to 85%, stands overnight, leaching supernatant, 65% ethyl alcohol of spare sediment
It washes twice, merges cleaning solution, stand overnight, leaching supernatant merges with spare supernatant, recycles ethyl alcohol, is added suitable
Water mixes, and filtration, filtrate is extremely extracted four times with acetic acid second, merges the cruel liquid of acetic acid second, and recycling acetic acid second is cruel and is condensed into thick paste,
Spray drying to get.
Ramulus et folium taxi cuspidatae extract, which is currently used primarily in, prepares paclitaxel injection, due to do not have to the place of production, Different climate and
Extraction process can all influence Ramulus et folium taxi cuspidatae extract active constituent content, so that the quality of Ramulus et folium taxi cuspidatae extract is influenced,
And the quality of Ramulus et folium taxi cuspidatae extract determines the quality of paclitaxel injection, it is therefore desirable to branches and leaves of yew
The main component of extract and its preparation is analyzed, and Ramulus et folium taxi cuspidatae extract and its quality of the pharmaceutical preparations quality and each batch are investigated
Quality difference between product.In the prior art, there is only to different sources branches and leaves of yew carry out quality analysis method,
Specifically: precision weighs 2 g sample powders, is ultrasonically treated 2 h at room temperature plus after 20 mL volume fraction, 95% ethyl alcohol immersion, 24 h;
4000rmin-1 is centrifuged 5 min, supernatant liquid filtering, and precipitating extracts 2 times with 95% EtOH Sonicate of volume fraction respectively, and every time 30
Min is centrifuged 5 min, supernatant liquid filtering through 4000 rmin-1;Merge 3 filtrates, is placed in Rotary Evaporators and is evaporated;
Then with V (chloroform): 10 mL of V (water)=1: 1 mixed solvent is extracted, and repeats extraction 3 times, merges three chloromethanes
Alkane phase extract liquor is evaporated with Rotary Evaporators in 35 DEG C；Medicinal extract methanol dissolves and is settled to 10 mL, with 0.45 μm of micropore
Membrane filtration, filtrate are for test liquid.HPLC analysis, 5 μ L of sample volume are carried out referring to above-mentioned chromatographic condition.Using following color
Spectral condition is eluted: Chinese nation C18 chromatographic column (4.6 mm × 250 mm, 5 μm)；227 nm of Detection wavelength；Column temperature is room temperature；
0.8 mLmin-1 of flow velocity；5 μ L of sample volume.Mobile phase be one aqueous solution of acetonitrile, binary gradient elutes, 0~20 min, 15%
Acetonitrile is changed to 50%, 20~50 min, and 50% acetonitrile is changed to 100%, 50~60 min, 100% acetonitrile.
In conclusion having the following disadvantages: (1) mainly the mass analysis technique of Ramulus et folium taxi cuspidatae extract at present
Fingerprint map analyzing is carried out to the branches and leaves of yew medicinal material of different sources, the quality for investigating different sources branches and leaves of yew is stablized
Property；Influence without considering the factors such as Different climate and extraction process.(2) contain in the resulting finger-print of the above-mentioned prior art
There are 12 Characteristic chromatographic peaks, only 3 therein is pointed out, the information content of reflection is small.Therefore, up to the present, still without
Preferable method analyzes main chemical compositions in Ramulus et folium taxi cuspidatae extract and its preparation comprehensively and investigates their quality
Stability.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the defect of the above-mentioned prior art and technical deficiencies, provide a kind of utilization
The HPLC analysis method of a kind of Ramulus et folium taxi cuspidatae extract and its preparation that high performance liquid chromatography carries out, mentions branches and leaves of yew
The main component of object and its preparation is taken to carry out comprehensive analysis, the fingerprint of Ramulus et folium taxi cuspidatae extract and its preparation to different batches
Map is mutually tested, and the HPLC reference fingerprint of Ramulus et folium taxi cuspidatae extract and its preparation is established, by testing Chinese yew
The similarity of the finger-print and reference fingerprint of branches and leaves extract and its formulation samples, to identify Ramulus et folium taxi cuspidatae extract
And its superiority and inferiority degree of the quality of the pharmaceutical preparations, achieve the purpose that control Ramulus et folium taxi cuspidatae extract and its quality of the pharmaceutical preparations.
The object of the present invention is to provide the HPLC analysis methods of a kind of Ramulus et folium taxi cuspidatae extract and its preparation.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The HPLC analysis method of a kind of Ramulus et folium taxi cuspidatae extract and its preparation, includes the following steps:
S1. HPLC reference fingerprint is established
S11. the Ramulus et folium taxi cuspidatae extract sample or Ramulus et folium taxi cuspidatae extract formulation samples of at least six qualification are weighed,
It mixes, filter with methanol respectively, obtain the test solution of each sample；
S12. HPLC analysis is carried out to the test solution of each sample of step S11, obtains the HPLC fingerprint image of each sample
Spectrum, establishes HPLC pairs of Ramulus et folium taxi cuspidatae extract or Ramulus et folium taxi cuspidatae extract preparation according to the HPLC finger-print of each sample
According to finger-print；
S2. sample to be tested detects
S21. sample to be tested mixed with methanol, filtered, obtain the test solution of sample to be tested, then carry out HPLC points
Analysis, obtains the HPLC finger-print of the sample to be tested；
S22. the finger-print is compared with corresponding HPLC reference fingerprint, judges red bean according to comparison result
The superiority and inferiority of China fir branches and leaves extract and its quality of the pharmaceutical preparations.
This method can achieve the purpose that control Ramulus et folium taxi cuspidatae extract and its quality of the pharmaceutical preparations.
Wherein it is preferred to which the chromatographic condition of the analysis of HPLC described in step S12 and mobile phase setting are as follows:
Acetonitrile-aqueous solution, binary gradient elutes: elution time is 0~10 min, the acetonitrile that concentration is 30%；
Elution time be 10~70 min, concentration from 30% be changed to 45% acetonitrile；
Elution time be 70~99 min, concentration from 45% be changed to 70% acetonitrile；
Elution time be 99~114 min, concentration from 70% be changed to 90% acetonitrile；
Elution time be 114~120 min, concentration from 90% be changed to 30% acetonitrile；
Elution time is 120~130 min, the acetonitrile that concentration is 30%.
Preferably, wherein the aqueous solution is pure water.
Preferably, Ramulus et folium taxi cuspidatae extract preparation described in step S11 need to first be crushed into powder, and branches and leaves of yew extraction is made
Object powder formulation sample.
Preferably, it before being filtered described in step S11, will first be cooled after the mixture of sample and methanol ultrasound.
It is highly preferred that the power of the ultrasound is 40~60kHz.
It is highly preferred that the power of the ultrasound is 60kHz.
It is highly preferred that the time of ultrasound is 20~40min.
It is highly preferred that the time of the ultrasound is 30min.
Preferably, the concentration of methanol described in step S11 or S21 is 100%.
Preferably, filtering described in step S11 or S21 is carried out using miillpore filter.
It is highly preferred that the aperture of the miillpore filter is 0.35~0.45 μm.
It is highly preferred that the aperture of the miillpore filter is 0.45 μm.
Preferably, in the analysis of HPLC described in step S21, equilibration time is the min of 10 min~30.
Preferably, in the analysis of HPLC described in step S21, wavelength, acquisition range are acquired using diode array detector are as follows:
The nm from the nm of 190 nm~210 to 390 nm~410；227 nm of Detection wavelength.
In addition, specifically, the HPLC finger-print described in step S12 according to each sample establish Ramulus et folium taxi cuspidatae extract or
The method of the HPLC reference fingerprint of Ramulus et folium taxi cuspidatae extract preparation are as follows: installed in Chinese Pharmacopoeia Commission on chromatograph
Medicine chromatographic fingerprinting similarity evaluation system, establishes Ramulus et folium taxi cuspidatae extract reference fingerprint, red bean by the system
China fir branches and leaves extract should have 7 common characteristic peaks.
The standard of the superiority and inferiority of Ramulus et folium taxi cuspidatae extract and its quality of the pharmaceutical preparations is judged described in step S22 according to comparison result
Are as follows: if sample to be tested has 7 characteristic peaks corresponding with reference fingerprint, and similar by chromatographic fingerprints of Chinese materia medica
Degree evaluation system show that the similarity of itself and reference fingerprint is greater than or equal to 0.90, then the quality of the sample to be tested is preferable.
Specifically: using similarity evaluation by the finger-print and reference fingerprint into
Row compares, and obtains the similarity of the finger-print and reference fingerprint；Branches and leaves of yew is being mentioned using reference fingerprint
When object sample being taken to be identified, in the finger-print of sample, Ying Chengxian 7 characteristic peaks corresponding with reference fingerprint, together
When, the finger-print of sample should be not less than 0.90 with the similarity for compareing map；If the sample to be tested have 7 with compare
The corresponding characteristic peak of finger-print, and by similarity evaluation obtain its with compare fingerprint image
The similarity of spectrum is greater than or equal to 0.90, it is believed that the quality of the sample to be tested is preferable.
The present invention is by a large amount of research and explores, and summarizes the HPLC for obtaining above-mentioned Ramulus et folium taxi cuspidatae extract and its preparation
Analysis method, basic conception are: establish the HPLC analysis method of Ramulus et folium taxi cuspidatae extract and its preparation, to different manufacturers,
The Ramulus et folium taxi cuspidatae extract and preparation HPLC fingerprint pattern of different batches compare research, establish Ramulus et folium taxi cuspidatae extract
And the HPLC reference fingerprint of preparation, the finger-print of sample to be tested is compared with the reference fingerprint then, is reflected
Not, quality, the superiority and inferiority for distinguishing Ramulus et folium taxi cuspidatae extract and its quality of the pharmaceutical preparations reach control Ramulus et folium taxi cuspidatae extract and its system
The purpose of agent quality.
The invention has the following advantages:
Compared with prior art, the present invention is due to having carried out reasonable setting to chromatographic condition, so that using of the invention
Contain 7 characteristic peaks in the resulting finger-print of method, and 7 therein are pointed out, compared with the prior art middle institute
Contain 12 characteristic peaks in the finger-print obtained, only 3 therein is pointed out, what the present invention reflected contains much information, can be more
Achieve the purpose that well to control Ramulus et folium taxi cuspidatae extract and its quality of the pharmaceutical preparations.
The HPLC analysis method of Ramulus et folium taxi cuspidatae extract and its preparation that the present invention establishes, to different manufacturers, different batches
Secondary Ramulus et folium taxi cuspidatae extract and preparation HPLC fingerprint pattern compares research, establishes Ramulus et folium taxi cuspidatae extract and preparation
HPLC reference fingerprint can distinguish Chinese yew to analyze the quality of Ramulus et folium taxi cuspidatae extract and its preparation
The quality of branches and leaves extract and its quality of the pharmaceutical preparations, superiority and inferiority realize the purpose of control Ramulus et folium taxi cuspidatae extract and its quality of the pharmaceutical preparations.
Detailed description of the invention
Fig. 1 is the reference fingerprint for the Ramulus et folium taxi cuspidatae extract that the embodiment of the present invention 1 is established.
Fig. 2 is Ramulus et folium taxi cuspidatae extract preparation-Chinese yew piece reference fingerprint that the embodiment of the present invention 2 is established.
Specific embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, agents useful for same and material of the present invention are commercially available.
Above-described HPLC is the abbreviation of high performance liquid chromatography.
Embodiment 1
1, reference fingerprint is established:
(1) 30 mg of Ramulus et folium taxi cuspidatae extract is taken, it is accurately weighed, it sets in stuffed conical flask, proper amount of methanol is added in precision,
It shakes up, is settled to 5 mL, (0.45 μm) of miillpore filter filtering is to get test solution.
(2) accurate to draw 10 μ L sample introduction of test solution, it is eluted using following chromatographic condition:
Chromatographic column (Phenomenex Luna C18 chromatographic column (4.6 mm × 250 mm, 5 μm))；
Acetonitrile-aqueous solution, binary gradient elutes: elution time is 0~10min, the acetonitrile that concentration is 30%；Elution time
For 10~70 min, concentration from 30% be changed to 45% acetonitrile；Elution time is 70~99 min, and concentration is changed to 70% from 45%
Acetonitrile；Elution time be 99~114 min, concentration from 70% be changed to 90% acetonitrile；Elution time is 114~120min,
Concentration from 90% be changed to 30% acetonitrile；Elution time is 120~130min, the acetonitrile that concentration is 30%；Equilibration time 20min；
1 mLmin-1 of flow velocity；10 μ L of sample volume；35 DEG C of column temperature；PDA detector acquisition range: 200 nm~400nm；Detection wavelength
227 nm。
(3) determining fingerprint pattern, chromatography are carried out by Ramulus et folium taxi cuspidatae extract sample of the above method to ten batches
Chinese Pharmacopoeia Commission's similarity evaluation is installed on instrument, branches and leaves of yew is established by the system
Extract reference fingerprint, Ramulus et folium taxi cuspidatae extract should have 7 common characteristic peaks (wherein to have 7 characteristic peaks to be referred to
Recognize), as a result as shown in Figure 1, in which:
Peak 1:10-DAB III；Peak 2:Baccatin III；Peak 3:Taxinine M；Peak 4:10-DAT；Peak 5:
Cephalomannine；Peak 6:7- table -10-DAT；Peak 7:Paclitaxel.
2, sample to be tested detects
(1) 30 mg of Ramulus et folium taxi cuspidatae extract to be measured is taken, it is accurately weighed, it sets in stuffed conical flask, appropriate first is added in precision
Alcohol shakes up, and is settled to 5 mL, (0.45 μm) of the miillpore filter test solution filtered to get sample to be tested.
(2) accurate to draw 10 μ L sample introduction of test solution, it is eluted using following chromatographic condition:
Chromatographic column (Phenomenex Luna C18 chromatographic column (4.6mm × 250mm, 5 μm))；
Acetonitrile-aqueous solution, binary gradient elutes: elution time is 0~10 min, the acetonitrile that concentration is 30%；Elution time
For 10~70 min, concentration from 30% be changed to 45% acetonitrile；Elution time is 70~99 min, and concentration is changed to 70% from 45%
Acetonitrile；Elution time be 99~114 min, concentration from 70% be changed to 90% acetonitrile；Elution time is 114~120 min,
Concentration from 90% be changed to 30% acetonitrile；Elution time is 120~130 min, the acetonitrile that concentration is 30%；Equilibration time 20
min；1 mLmin-1 of flow velocity；10 μ L of sample volume；35 DEG C of column temperature；PDA detector acquisition range: the nm of 200 nm~400；Inspection
Survey 227 nm of wavelength.
(3) after obtaining the finger-print of the Ramulus et folium taxi cuspidatae extract sample to be measured with above-mentioned test method, in recycling
The finger-print is compared by medicine chromatographic fingerprinting similarity evaluation system with reference fingerprint, obtains the finger-print
With the similarity of reference fingerprint.When being identified using reference fingerprint to Ramulus et folium taxi cuspidatae extract sample, sample
In the finger-print of product, Ying Chengxian 7 characteristic peaks corresponding with reference fingerprint, meanwhile, the finger-print of sample with it is right
0.90 should be not less than according to the similarity of map.If the sample to be tested has 7 features corresponding with reference fingerprint
Peak, and show that the similarity of itself and reference fingerprint is greater than or equal to by similarity evaluation
0.90, it is believed that the quality of the sample to be tested is preferable.It is red that control can be properly arrived at using reference fingerprint of the invention
The purpose of beans China fir branches and leaves extract quality.
Embodiment 2
1, reference fingerprint is established
(1) Ramulus et folium taxi cuspidatae extract preparation-Chinese yew piece 6 is taken, is pulverized, 30 mg of powder is weighed, sets stuffed conical flask
In, proper amount of methanol is added, 30 min of ultrasound let cool, are settled to 5 mL.(0.45 μm) filtering of miillpore filter is molten to get test sample
Liquid.
(2) accurate to draw 10 μ L sample introduction of test solution, it is eluted using following chromatographic condition:
Chromatographic column (Phenomenex Luna C18 chromatographic column (4.6 mm × 250 mm, 5 μm))；
Acetonitrile-aqueous solution, binary gradient elutes: elution time is 0~10 min, the acetonitrile that concentration is 30%；Elution time
For 10~70 min, concentration from 30% be changed to 45% acetonitrile；Elution time is 70~99 min, and concentration is changed to 70% from 45%
Acetonitrile；Elution time be 99~114 min, concentration from 70% be changed to 90% acetonitrile；Elution time is 114~120 min,
Concentration from 90% be changed to 30% acetonitrile；Elution time is 120~130 min, the acetonitrile that concentration is 30%；Equilibration time 20
min；1 mLmin-1 of flow velocity；10 μ L of sample volume；35 DEG C of column temperature；PDA detector acquisition range: the nm of 200 nm~400；Inspection
Survey 227 nm of wavelength.
(3) finger-print is carried out by Ramulus et folium taxi cuspidatae extract preparation-Chinese yew piece of the above method to ten batches
It is measured, Chinese Pharmacopoeia Commission's similarity evaluation is installed on chromatograph, passes through the system
Chinese yew piece reference fingerprint is established, Chinese yew piece reference fingerprint there should be 7 common characteristic peaks (wherein there are 7 spies
Pointed out at sign peak), as a result as shown in Figure 2, in which:
Peak 1:10-DAB III；Peak 2:Baccatin III；Peak 3:Taxinine M；Peak 4:10-DAT；Peak 5:
Cephalomannine；Peak 6:7- table -10-DAT；Peak 7:Paclitaxel.
2, sample to be tested detects
(1) Ramulus et folium taxi cuspidatae extract preparation-Chinese yew piece 6 is taken, is pulverized, 30 mg of powder is weighed, sets stuffed conical flask
In, proper amount of methanol is added, 30 min of ultrasound let cool, are settled to 5 mL.(0.45 μm) filtering of miillpore filter is molten to get test sample
Liquid.
(2) accurate to draw 10 μ L sample introduction of test solution, it is eluted using following chromatographic condition:
Chromatographic column (Phenomenex Luna C18 chromatographic column (4.6 mm × 250 mm, 5 μm))；
Acetonitrile-aqueous solution, binary gradient elutes: elution time is 0~10 min, the acetonitrile that concentration is 30%；Elution time
For 10~70 min, concentration from 30% be changed to 45% acetonitrile；Elution time is 70~99 min, and concentration is changed to 70% from 45%
Acetonitrile；Elution time be 99~114 min, concentration from 70% be changed to 90% acetonitrile；Elution time is 114~120 min,
Concentration from 90% be changed to 30% acetonitrile；Elution time is 120~130 min, the acetonitrile that concentration is 30%；Equilibration time 20
min；1 mLmin-1 of flow velocity；10 μ L of sample volume；35 DEG C of column temperature；PDA detector acquisition range: the nm of 200 nm~400；Inspection
Survey 227 nm of wavelength.
(3) Ramulus et folium taxi cuspidatae extract preparation to be measured-Chinese yew piece sample fingerprint image is obtained with above-mentioned test method
Spectrum recycles similarity evaluation to be compared the finger-print with characteristic spectrum, obtains this
The similarity of finger-print and reference fingerprint, while the corresponding characteristic peak number of the finger-print is observed, finally evaluate
Ramulus et folium taxi cuspidatae extract preparation-Chinese yew piece sample quality.In use reference fingerprint to Ramulus et folium taxi cuspidatae extract
When preparation-Chinese yew piece sample is identified, in the finger-print of sample, Ying Chengxian 7 corresponding with reference fingerprint
Characteristic peak, meanwhile, the finger-print of sample and the similarity of reference fingerprint should be not less than 0.90.If the sample to be tested
With 7 characteristic peaks corresponding with reference fingerprint, and obtained by similarity evaluation
The similarity of itself and reference fingerprint is greater than or equal to 0.90, it is believed that the quality of the sample to be tested is preferable.Use this hair
Bright reference fingerprint can be properly arrived at control Ramulus et folium taxi cuspidatae extract preparation-Chinese yew tablet quality purpose.
Claims (6)
1. the HPLC analysis method of a kind of Ramulus et folium taxi cuspidatae extract and its preparation, which comprises the steps of:
S1. HPLC reference fingerprint is established
S11. the Ramulus et folium taxi cuspidatae extract sample or Ramulus et folium taxi cuspidatae extract formulation samples of at least six qualification are weighed, respectively
It mixes, filter with methanol, obtain the test solution of each sample；
S12. HPLC analysis is carried out to the test solution of each sample of step S11, obtains the HPLC finger-print of each sample, root
Refer to according to the HPLC control that the HPLC finger-print of each sample establishes Ramulus et folium taxi cuspidatae extract or Ramulus et folium taxi cuspidatae extract preparation
Line map；
S2. sample to be tested detects
S21. sample to be tested mixed with methanol, filtered, obtain the test solution of sample to be tested, then carry out HPLC analysis,
Obtain the HPLC finger-print of the sample to be tested；
S22. the finger-print is compared with corresponding HPLC reference fingerprint, judges branch of Ramulus et folium taxi cuspidatae according to comparison result
The superiority and inferiority of leaf extract and its quality of the pharmaceutical preparations；
The chromatographic condition of the analysis of HPLC described in step S12 and mobile phase setting are as follows:
Acetonitrile-aqueous solution, binary gradient elutes: elution time is 0~10 min, the acetonitrile that concentration is 30%；
Elution time be 10~70 min, concentration from 30% be changed to 45% acetonitrile；
Elution time be 70~99 min, concentration from 45% be changed to 70% acetonitrile；
Elution time be 99~114 min, concentration from 70% be changed to 90% acetonitrile；
Elution time be 114~120 min, concentration from 90% be changed to 30% acetonitrile；
Elution time is 120~130 min, the acetonitrile that concentration is 30%；
Before being filtered described in step S11, it will first be cooled after the mixture of sample and methanol ultrasound；The power of the ultrasound be 40~
60kHz, ultrasonic time are 20~40min；
The standard of the superiority and inferiority of Ramulus et folium taxi cuspidatae extract and its quality of the pharmaceutical preparations is judged described in step S22 according to comparison result are as follows: such as
Fruit sample to be tested has 7 characteristic peaks corresponding with reference fingerprint, and is commented by chromatographic fingerprints of Chinese materia medica similarity
Valence system show that the similarity of itself and reference fingerprint is greater than or equal to 0.90, then the quality of the sample to be tested is preferable.
2. HPLC analysis method according to claim 1, which is characterized in that Ramulus et folium taxi cuspidatae extract described in step S11
Preparation need to first be crushed into powder, and Ramulus et folium taxi cuspidatae extract powder formulation sample is made.
3. HPLC analysis method according to claim 1, which is characterized in that filtering described in step S11 or S21 uses micropore
Filter membrane carries out.
4. HPLC analysis method according to claim 3, which is characterized in that the aperture of the miillpore filter be 0.35~
0.45μm。
5. HPLC analysis method according to claim 1, which is characterized in that according to the HPLC of each sample described in step S12
The method that finger-print establishes the HPLC reference fingerprint of Ramulus et folium taxi cuspidatae extract or Ramulus et folium taxi cuspidatae extract preparation are as follows:
Chinese Pharmacopoeia Commission's similarity evaluation is installed on chromatograph, red bean is established by the system
China fir branches and leaves extract reference fingerprint, Ramulus et folium taxi cuspidatae extract should have 7 common characteristic peaks.
6. HPLC analysis method according to claim 1, which is characterized in that the concentration of methanol described in step S11 or S21 is
100%。
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| CN101477102A (en) * | 2008-12-10 | 2009-07-08 | 上海康桥中药饮片有限公司 | Quality standard of south yew decoction pieces and its detection method |
| CN105388234A (en) * | 2015-12-24 | 2016-03-09 | 湖北祥瑞丰生态果业有限公司 | Detection method for content of paclitaxel in taxus chinensis branch |
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| CN101477102A (en) * | 2008-12-10 | 2009-07-08 | 上海康桥中药饮片有限公司 | Quality standard of south yew decoction pieces and its detection method |
| CN105388234A (en) * | 2015-12-24 | 2016-03-09 | 湖北祥瑞丰生态果业有限公司 | Detection method for content of paclitaxel in taxus chinensis branch |
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