CN108169384A - A kind of method for building up of lichee nuclear particle HPLC finger-prints - Google Patents
A kind of method for building up of lichee nuclear particle HPLC finger-prints Download PDFInfo
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- CN108169384A CN108169384A CN201810188665.8A CN201810188665A CN108169384A CN 108169384 A CN108169384 A CN 108169384A CN 201810188665 A CN201810188665 A CN 201810188665A CN 108169384 A CN108169384 A CN 108169384A
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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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Abstract
The present invention proposes a kind of method for building up of lichee nuclear particle HPLC finger-prints, preparation, chromatographic condition including test sample determine, it is measured using high performance liquid chromatograph, obtain the HPLC finger-prints of lichee nuclear particle, the comprehensive and accurate chemical information for expressing lichee nuclear particle, the finger-print has the features such as separating degree at shared peak and preferable, reproducible, operability is strong peak type, can efficiently control the quality of lichee nuclear particle.
Description
Technical field
The present invention relates to Chinese medicine preparation field of quality control, and in particular to a kind of lichee nuclear particle HPLC finger-prints are built
Cube method.
Background technology
Semen litchi is the dry mature seed of sapindaceous plant lichee.Its sweet in flavor, slight bitter, it is warm-natured.Return liver and kidney channel, have
There are promoting the circulation of qi dissipating bind, the function of cold-dispelling pain-relieving, for cold hernia stomachache, testiclar gall.Root is it was found that the main chemical compositions of semen litchi
For lychee seed saponin, flavonoids, volatile oil, organic acid and ester, amino acid, carbohydrate and trace element etc..
Although Chinese medicine now is fast-developing, but still there is the effective elements of the medicine in major part not clear and definite.Existing
The quality control standard of lichee nuclear particle cannot really reflect the interior quality and product matter between batches of lichee nuclear particle
The consistency of amount.Therefore, for lichee nuclear particle presently, there are deficiency, the present invention with HPLC gradient elution method to semen litchi
Grain finger-print is studied, and the lichee nuclear particle of different batches is selected to complete finger-print applicability and is examined, the finger established
Line collection of illustrative plates can more fully embody the overall chemical information of lichee nuclear particle；It is each in the lichee nuclear particle HPLC finger-prints of foundation
Chemical composition share peak separating degree and peak type it is preferable and reproducible, operability is strong, and one kind is provided for lichee nuclear particle
It is completely new, be generally applicable in, convenient-to-running method of quality control.
Invention content
The object of the invention for lichee nuclear particle HPLC finger-prints provides a kind of construction method and thus obtained by method
Finger-print.
The lichee nuclear particle HPLC fingerprints of the present invention are as follows：
A kind of method for building up of lichee nuclear particle HPLC finger-prints, which is characterized in that include the following steps：
(1) preparation of test solution：It is appropriate to weigh lichee nuclear particle, it is finely ground, it is accurately weighed to take about 0.2g~1.0g, add
Water dissolution, after boiling water bath heats 10~20min, cooling, centrifugation take supernatant and with after filtering with microporous membrane, are made for examination
Product solution；
(2) preparation of contrast solution：Precision weighs semen litchi 0.2~1.0g of control medicinal material, is dissolved in water, boiling water bath heating
After handling 10~20min, cooling, centrifugation take supernatant and with after filtering with microporous membrane, control comparisons solution are made；
(3) it measures：It is accurate respectively to draw test solution and each 10~20 μ L of control comparisons solution, inject liquid chromatogram
Instrument is measured according to high performance liquid chromatography, is recorded the chromatogram in 30 minutes, is handled, obtained with finger-print software
To the finger-print of lichee nuclear particle.
Chromatographic condition：Chromatographic column HypersilTMODS C18, mobile phase A are methanol；Mobile phase B is 0.5~1.0%
Phosphate aqueous solution；Detection wavelength：240~280nm；Column temperature：20-30℃；Flow velocity：1.0~1.5mLmin- 1；Elution program is
Gradient elution program.
The relationship of the volume ratio of mobile phase A and Mobile phase B at any time is in gradient elution program：0min：5%:95%；
6min：10%:90%；12min：30%:70%；18min：50%:50%；24min：70%:30%；30min：100%:
0%.
As further improvement of the invention, the specification of chromatographic column is 100mm × 4.6mm, and 5 μm, filler is octadecane
Base silane bonded silica gel
As further improvement of the invention, the aperture of octadecylsilane chemically bonded silica is
As further improvement of the invention, Detection wavelength：260nm；Column temperature：32℃；Flow velocity：1.2mL·min- 1。
As further improvement of the invention, miillpore filter is 0.45 μm.
As further improvement of the invention, centrifugation is under 2000~40000r/min, handles 5~10min
10 features, which are shared, in the lichee nuclear particle HPLC finger-prints that the present invention is established using the above method shares peak.
The present invention has the advantages that：
1st, the present invention provides a kind of method for building up of lichee nuclear particle HPLC finger-prints, the finger-print of foundation can be with
The comprehensive overall chemical information for embodying lichee nuclear particle；
2nd, chemical composition shares peak separating degree in the finger-print that the present invention establishes and peak type is good, the repetition of the finger-print
Property it is good, operability is strong, available for evaluating and controlling the quality of lichee nuclear particle, it is ensured that the safety of lichee nuclear particle and effectively
Property.
Description of the drawings
Fig. 1 is 10 crowdes of different batches lichee nuclear particle finger-print stacking charts.
Fig. 2 is the finger-print chromatogram of the semen litchi control medicinal material hydrotrope.
Specific embodiment
Embodiment 1
Instrument：Agilent high performance liquid chromatograph.
Medicinal material：The lichee nuclear particle that Hepu of Guangxi fruity garden Food Co., Ltd provides；Control medicinal material semen litchi is purchased from extensively
Western Yulin Xin Kang medicinal materials Co., Ltd.
Reagent：Mobile phase A methanol is chromatographically pure, and water is ultra-pure water, remaining reagent is pure to analyze.
It is prepared by test solution：It is appropriate to weigh lichee nuclear particle, it is finely ground, it is accurately weighed to take about 1.0g, it is dissolved in water, boiling water
After bath heat treatment 20min, cooling, centrifugation take supernatant and with after filtering with microporous membrane, test solution are made.
It is prepared by reference substance solution：Precision weighs semen litchi control medicinal material 1.0g, is dissolved in water, boiling water bath heat treatment
After 20min, cooling, centrifugation take supernatant and with after filtering with microporous membrane, control comparisons solution are made.
High-efficient liquid phase chromatogram condition：Chromatographic column HypersilTM ODS C18 (100mm × 4.6mm, 5 μm), mobile phase A is
Methanol；Mobile phase B is 0.5~1.0% phosphate aqueous solution；Detection wavelength：240~280nm；Column temperature：20-30℃；Flow velocity：
1.0~1.5mLmin- 1；Elution program is gradient elution program.
The volume by volume concentration of gradient elution program is：
| Time (min) | Methanol (%) | 0.7% phosphate aqueous solution (%) |
| 0 | 5 | 95 |
| 6 | 10 | 90 |
| 12 | 30 | 70 |
| 18 | 50 | 50 |
| 24 | 70 | 30 |
| 30 | 100 | 0 |
Linear relationship is investigated：
It is accurate respectively to draw test solution (0.04048gmL-1) 0,2,4,6,8,10 μ L, liquid chromatograph is injected, is pressed
Above-mentioned chromatographic condition measures, and is horizontal stroke with the sample size (X, μ g) of test solution with the peak area (Y) at the 10th peak for ordinate
Coordinate draws standard curve, acquires regression equation:Y=4.7752X+2.1127, r=0.9997 show semen litchi particle solution
Sample size is in good linear relationship in 0~404.8 μ g and peak area.
Precision test：
Precision draws test solution (0.04048gmL-1) 10 μ L, by above-mentioned chromatographic condition continuous sample introduction 5 times, as a result
Show the relative standard deviation (RSD) of the main chromatographic peak retention time of 5 collection of illustrative plates between 0.12%~0.30%, peak area
RSD values within 2.7%, the retention time of main peaks and the precision of peak area in liquid chromatograms obtained by 5 continuous sample introductions
Degree is higher, it was demonstrated that the precision of instrument well shows that instrument precision is good.
Stability test：
It takes with a batch of test solution (0.04048gmL-1), 0 after preparation, 4,8, l2, carry out for 24 hours
HPLC detects to obtain corresponding chromatogram, is analyzed by traditional Chinese medicine fingerprint similarity evaluation system.The result shows that 5
The RSD values of the main chromatographic peak retention time of collection of illustrative plates are between 0.12%~0.27%, and the RSD values of peak area are within 3.0%, examination
It is higher to test the retention time of main peaks and the precision of peak area in gained liquid chromatogram, it was demonstrated that in interior sample solution for 24 hours
It has good stability.
Repetitive test：
It takes with a batch of test solution (0.04048gmL-1) carry out HPLC detect to obtain corresponding chromatogram, lead to
Traditional Chinese medicine fingerprint similarity evaluation system is crossed to be analyzed.The result shows that the RSD of the main chromatographic peak retention time of 5 collection of illustrative plates
Value is between 0.15%~0.32%, and the RSD values of peak area are within 3.0%, main peaks in experiment gained liquid chromatogram
Retention time and the precision of peak area are higher, it was demonstrated that the repeatability that this method measures is good.
It is loaded recovery test：
The lichee nuclear particle for taking same lot number is appropriate, finely ground, takes about 0.20g, parallel 6 parts, accurately weighed, accurate respectively to add
Enter the test solution (0.04048gmL prepared-1) 5mL, test solution is made by test solution preparation method, presses
Above-mentioned chromatographic condition measures the area at the tenth peak, calculates the rate of recovery, as a result such as the following table 1：
1 semen litchi particle recovery of table is tested
Measurement result：As stated above, HPLC finger-prints are established simultaneously by the lichee nuclear particle to 10 batches of different batches
Analysis comparison is carried out, finds out its common characteristic peaks (totally 10 peaks), obtains the finger-print of lichee nuclear particle, 10 shared peaks
Retention time is respectively：5.0 minutes, 5.5 minutes, 6.2 minutes, 6.7 minutes, 7.3 minutes, 8.0 minutes, 9.6 minutes, 9.8 points
Clock, 10.7 minutes, 24.8 minutes.
Those skilled in the art is not under conditions of the spirit and scope of the present invention that claims determine are departed from, also
Various modifications can be carried out to more than content.Therefore the scope of the present invention be not limited in more than explanation, but by
The range of claims is come determining.
Claims (9)
1. a kind of method for building up of lichee nuclear particle HPLC finger-prints, which is characterized in that include the following steps：
(1) preparation of test solution：It is appropriate to weigh lichee nuclear particle, it is finely ground, it is accurately weighed to take about 0.2g~1.0g, add water-soluble
Solution, after boiling water bath heats 10~20min, cooling, centrifugation take supernatant and with after filtering with microporous membrane, it is molten that test sample are made
Liquid；
(2) preparation of contrast solution：Precision weighs semen litchi 0.2~1.0g of control medicinal material, is dissolved in water, boiling water bath heat treatment
After 10~20min, cooling, centrifugation take supernatant and with after filtering with microporous membrane, control comparisons solution are made；
(3) it measures：It is accurate respectively to draw test solution and each 10~20 μ L of control comparisons solution, liquid chromatograph is injected, according to
It is measured according to high performance liquid chromatography, records the chromatogram in 30 minutes, handled with finger-print software, obtain lichee
The finger-print of nuclear particle.
2. the method for building up of lichee nuclear particle HPLC finger-prints as described in claim 1, which is characterized in that high performance liquid chromatography
Condition is：Chromatographic column HypersilTM ODS C18, mobile phase A is methanol；The phosphoric acid that Mobile phase B is 0.5~1.0% is water-soluble
Liquid；Detection wavelength：240~280nm；Column temperature：20-30℃；Flow velocity：1.0~1.5mLmin-1；Elution program is washed for gradient
De- program.
3. the method for building up of lichee nuclear particle HPLC finger-prints as claimed in claim 2, which is characterized in that the chromatographic column
Specification is 100mm × 4.6mm, and 5 μm, filler is octadecylsilane chemically bonded silica.
4. the method for building up of lichee nuclear particle HPLC finger-prints as claimed in claim 1 or 2, which is characterized in that the ladder
The relationship of the volume ratio of mobile phase A and Mobile phase B at any time is in degree elution program：0min：5%:95%；6min：10%:
90%；12min：30%:70%；18min：50%:50%；24min：70%:30%；30min：100%:0%.
5. the method for building up of lichee nuclear particle HPLC finger-prints according to claim 2, which is characterized in that described 18
The aperture of alkyl silane bonded silica gel is
6. the method for building up of lichee nuclear particle HPLC finger-prints as claimed in claim 2, which is characterized in that Detection wavelength：
260nm；Column temperature：32℃；Flow velocity：1.2mL·min- 1。
7. a kind of method for building up of lichee nuclear particle HPLC finger-prints as described in claim 1, which is characterized in that described micro-
Hole filter membrane is 0.45 μm.
A kind of 8. method for building up of lichee nuclear particle HPLC finger-prints as described in claim 1, which is characterized in that the litchi
10 features, which are shared, in branch nuclear particle finger-print shares peak.
9. a kind of method for building up of lichee nuclear particle HPLC finger-prints as described in claim 1, which is characterized in that it is described from
The heart is under 2000~40000r/min, handles 5~10min.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810188665.8A CN108169384A (en) | 2018-03-07 | 2018-03-07 | A kind of method for building up of lichee nuclear particle HPLC finger-prints |
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| CN201810188665.8A CN108169384A (en) | 2018-03-07 | 2018-03-07 | A kind of method for building up of lichee nuclear particle HPLC finger-prints |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104820026A (en) * | 2014-05-09 | 2015-08-05 | 广州市中医医院 | Litchi seed dripping pill quality detection method |
| CN105353065A (en) * | 2015-12-22 | 2016-02-24 | 黑龙江大学 | Establishing method of HPLC (high-performance liquid chromatography) fingerprint spectrum of lychee seeds, standard fingerprint spectrum obtained with method and application of standard fingerprint spectrum |
-
2018
- 2018-03-07 CN CN201810188665.8A patent/CN108169384A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104820026A (en) * | 2014-05-09 | 2015-08-05 | 广州市中医医院 | Litchi seed dripping pill quality detection method |
| CN105353065A (en) * | 2015-12-22 | 2016-02-24 | 黑龙江大学 | Establishing method of HPLC (high-performance liquid chromatography) fingerprint spectrum of lychee seeds, standard fingerprint spectrum obtained with method and application of standard fingerprint spectrum |
Non-Patent Citations (2)
| Title |
|---|
| 曲世为等: "HPLC 法测定荔枝核方中丹参素", 《现代药物与临床》 * |
| 浦香兰等: "荔枝核配方颗粒质量标准研究", 《中国现代中药》 * |
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Application publication date: 20180615 |