CN107132304B - A kind of Polygala tenuifolia finger-print and index component content method for measuring - Google Patents
A kind of Polygala tenuifolia finger-print and index component content method for measuring Download PDFInfo
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- CN107132304B CN107132304B CN201710496283.7A CN201710496283A CN107132304B CN 107132304 B CN107132304 B CN 107132304B CN 201710496283 A CN201710496283 A CN 201710496283A CN 107132304 B CN107132304 B CN 107132304B
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- 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
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
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Abstract
The present invention relates to a kind of Polygala tenuifolia finger-print and index component content methods for measuring, include the following steps：Step 1：Using the visible uv detection method of ultra high efficiency liquid phase all-wave length, the finger-print of Polygala tenuifolia is established；Step 2：The chromatographic peak of finger-print in step 1 is pointed out using LC-MS technology, determines the shared fingerprint peaks of ultraviolet detection and non-shared peak；Step 3：There is neuroprotection, tranquilizing and allaying excitement, antidepression or anti-senile dementia isoreactivity to 10 identified in step 2 using the visible uv detection method of ultra high efficiency liquid phase all-wave length, and the specific index ingredient of structure carries out assay.
Description
Technical field
The invention belongs to Analysis of Chinese Traditional Medicine and Chinese medicine assay fields, and in particular to a kind of Polygala tenuifolia finger-print and
Index component content method for measuring.
Background technology
Radix Polygalae Polygala tenuifolia Willd., are recorded in earliest《Sheng Nong's herbal classic》, be classified as top grade, and by regarding
To support life key medicine.It is warm-natured, it is bitter, pungent, there is tranquilize the mind and promote the intelligence, eliminating phlegm and diminishing swelling and other effects.It is found through modern study, Polygala tenuifolia
Principle active component includes saponins, sugar esters, mouth mountain ketone and alkaloids etc., and there is antibacterial, anti-inflammatory, raising to recognize, change
The pharmacological actions such as kind memory capability, anti-senile dementia, antidepression, antitumor, anti-aging.
The power of Chinese medicine drug effect and the quality of quality of medicinal material are closely related, and contained in the quality and medicinal material of quality of medicinal material
The height of main chemical compositions content is closely related.A variety of known or unknown chemical compositions, pass through a plurality of approach in Chinese medicine
It acts on multiple target point and plays its pharmacological action.At present about the qualitative analysis of Polygala tenuifolia, predominantly fingerprint map analyzing, but it
The method of preceding foundation is time-consuming longer, and loss reagent is more, is unfavorable for high throughput analysis, pointing out for complicated ingredient is not also filled
Point；Quantitative analysis to chemical composition in Polygala tenuifolia focuses primarily upon the measurement that individual components are index, single index components
Be difficult to accurately and comprehensively embody the inherent quality of Chinese medicine, the quality control of Chinese medicine multi objective and evaluation model need it is various at
Point reference substance, but part Chinese medicine reference substance separating difficulty is big, monomer is unstable, is difficult to supply or the problems such as supply price is high
In the presence of preventing it from meeting the needs of traditional Chinese medicine quality monitoring and new drug development etc., measured content in Polygala tenuifolia at present
Compound amounts it is less.Therefore point out the defects of insufficient and quantitative chemical ingredient is less, this hair for above-mentioned complicated ingredient
It is bright to provide a kind of Polygala tenuifolia finger-print and index component content method for measuring, this method be more fully to Radix Polygalaes
Compound carries out qualitative and quantitative analysis in medicinal material.
Invention content
The purpose of the present invention is to provide a kind of Polygala tenuifolia finger-print and index component content methods for measuring, should
Method can more fully carry out qualitative and quantitative analysis to compound in Polygala tenuifolia.
To achieve the above object, technical solution provided by the invention is：
The content assaying method of a kind of Polygala tenuifolia finger-print and index ingredient, steps are as follows：
Step 1：It is complete using ultra high efficiency liquid phase (Ultra Performance Liquid Chromatography, UPLC)
The visible uv detection method of wavelength, establishes the finger-print of Polygala tenuifolia；
The preparation of mixed reference substance solution：It is appropriate that each reference substance is weighed respectively, it is accurately weighed, add methanol to dissolve, is settled to
lancerin、sibiricaxanthone B、glomeratose A、7-O-methyl mangiferin、
Final concentration of 1-100 μ g/mL, the sibiricose A of polygalaxanthone III5、sibiricose A6、
Tenuifoliside C final concentration of 100-300 μ g/mL, 3,6 '-disinapoyl sucrose, tenuifoliside A are whole
A concentration of 300-500 μ g/mL, shake up to get mixed reference substance solution, are placed in spare in 4 DEG C of refrigerators；
The preparation of test solution：It takes Polygala tenuifolia to crush and crosses No. three sieves of pharmacopeia, take Polygala tenuifolia powder about 0.2g, essence
It is close weighed, it sets in conical flask with cover, precision pipettes 70% methanol solution 20mL, and weighed weight is ultrasonically treated 30min (power
500W, frequency 40kHz), it puts to room temperature, then weighed weight, the weight of less loss is supplied with 70% methanol solution, is shaken up, filter, take
Subsequent filtrate, cross 0.22 μm of miillpore filter to get；
The measurement of finger-print：Accurate respectively to draw mixed reference substance solution and each 2-10 μ L of test solution, injection is super
High performance liquid chromatograph records chromatogram, is recommended using Chinese Pharmacopoeia Commission《Chromatographic fingerprints of Chinese materia medica similarity evaluation
System》Generate Polygala tenuifolia finger-print；
Wherein, chromatographic condition is as follows：
Chromatographic column：UPLC C18Column or T3Column；
Mobile phase：Mobile phase A is acetonitrile, and Mobile phase B is the aqueous formic acid that volumetric concentration is 0.1-1.0%；
Gradient：0-3min, 5-10%A；3-6min, 10-12%A；6-16min, 12-22%A；16-17min,
22%A；17-22min, 22-27%A；22-25min, 27-32%A；25-27min, 32-35%A；27-30min, 35%A；
30-36min, 35-40%A；36-37min, 40-100%A；37-39min, 100-5%A.
Sample size：2-10μL；
Column temperature：20-40℃；
Flow velocity：0.2-0.5mL/min；
Detection wavelength：290-330nm；
Step 2：The chromatographic peak of finger-print in step 1 is pointed out using LC-MS technology, determines ultraviolet detection
With step 1, Mass Spectrometry Conditions are as follows for shared fingerprint peaks and non-shared peak, wherein liquid phase chromatogram condition：
Ion source：Electric spray ion source (ESI)；Scan mode：Negative ions scan simultaneously；Spray voltage：3.5kV；Sheath
Gas velocity：35psi(1psi≈6.9kPa)；Secondary air speed：10psi；Capillary temperature：320℃；Probe warmer temperature：
300℃；Maximum spraying electric current：100A；S-Lens resolution ratio：55；Scanning range：m/z 100-2000；Mass resolution：
70000；
Step 3：8 index ingredient (sibiricose A are filtered out from the 23 shared peaks identified in step 25、
sibiricose A6, sibiricaxanthone B, glomeratose A, polygalaxanthone III, 3,6 '-
Disinapoyl sucrose, tenuifoliside A, tenuifoliside C), 2 are filtered out from 15 non-shared peaks
Index ingredient (lancerin, 7-O-methyl mangiferin), using the visible uv detection method of ultra high efficiency liquid phase all-wave length
Assay is carried out to above-mentioned 10 index ingredients；
Assay method is：Accurate respectively to draw above-mentioned mixed reference substance solution and each 2-10 μ L of test solution, injection is super
High performance liquid chromatograph determines the chromatographic peak of each ingredient to be measured, and mixed reference substance solution is diluted step by step, establishes 10 indexs
The equation of linear regression of property ingredient calculates separately the content of 10 compounds as described above in test solution；
Wherein, chromatographic condition is the same as step 1.
Further, Mobile phase B is aqueous formic acid that volumetric concentration is 0.1% in step 1 and 3；Sample size：2μL；Column
Temperature：40℃；Flow velocity：0.4mL/min；Detection wavelength：320nm.
Compared with the prior art, the present invention is by way of UPLC " Qualitative fingerprint+index component quantifying ", from whole
Body and the common promotion for realizing quality control standard of the aspect of key component two, the not only quality that product is determined of specific quantitative comprehensively
Controllability, and due to the introducing of UPLC technologies, substantially reduce the existing minute for generally using HPLC technologies so that
The complexity characterization of Polygala tenuifolia finger-print is achieved.
Description of the drawings
Fig. 1 is 7 batches of Polygala tenuifolia finger-prints provided by the invention and standard finger-print；Wherein sample 1-7 numbers are shown in
Table 1, R are reference fingerprint；
Fig. 2 is the liquid matter chromatogram of Radix Polygalae sample；In figure：A：Ultraviolet chromatogram；B：Total ion current color under positive ion mode
Spectrogram；C：Total ion chromatogram under negative ion mode；
Fig. 3 is the UPLC chromatograms of mixed reference substance solution；
Fig. 4 is the UPLC chromatograms of Polygala tenuifolia test solution；
In Fig. 3, Fig. 4：1 is sibiricose A5, 2 be sibiricose A6, 3 be lancerin, and 4 are
Sibiricaxanthone B, 5 be glomeratose A, and 6 be 7-O-methyl mangiferin, and 7 are
Polygalaxanthone III, 8 be 3,6 '-disinapoyl sucrose, and 9 be tenuifoliside A, and 10 are
tenuifoliside C)
Specific implementation mode
Following embodiment is not limited to the scope of the present invention for illustrating this method.
Content for a better understanding of the present invention in the following with reference to the drawings and specific embodiments makees furtherly the present invention
It is bright.
Embodiment 1
Step 1：Using the visible uv detection method of ultra high efficiency liquid phase all-wave length, the finger-print of Polygala tenuifolia is established；1. examination
Test material
Material, instrument and reagent
Test material：Polygala tenuifolia sample used is 7 batches of full roots of Polygala tenuifolia being collected into from Shanxi Province's different sources, is used
Hand is rubbed, and is pumped core, is obtained RADIX POLYGALAE, is crushed, and No. three sieves of pharmacopeia are crossed, spare.Test material specifying information is shown in Table 1.
1 Radix Polygalae sample message table of table
Instrument：Ultra performance liquid chromatography system (Waters companies ACQUITY UPLC, including vacuum degassing machine, binary ladder
Spend pump, autosampler, column oven, TUV detectors), electronic balance (CPA225D, German Sartorius companies), Milli-Q
Ultrapure water system (Millipore companies of the U.S.), ultrasonic cleaner (KQ-500DV, Kunshan Ultrasonic Instruments Co., Ltd.),
Similarity evaluation 2008A editions.
Reagent：Acetonitrile (chromatographically pure, Fisher companies), formic acid (chromatographically pure, Fisher companies), ultra-pure water (Milli-Q systems
It is standby), methanol (domestic analysis is pure).
2. the preparation of mixed reference substance solution
It is appropriate to weigh each reference substance respectively, it is accurately weighed, add methanol to dissolve, be settled to lancerin,
Sibiricaxanthone B, glomeratose A, 7-O-methyl mangiferin, polygalaxanthone III are whole
A concentration of 1-100 μ g/mL, sibiricose A5、sibiricose A6, the final concentration of 100-300 μ g/ of tenuifoliside C
The mixing reference substance of the final concentration of 300-500 μ g/mL of mL, 3,6 '-disinapoyl sucrose, tenuifoliside A is molten
Liquid shakes up to get being placed in spare in 4 DEG C of refrigerators；
3. the preparation of test solution
It takes Polygala tenuifolia to crush and crosses the sieve of pharmacopeia three, take Polygala tenuifolia powder about 0.2g, it is accurately weighed, set tool plug taper
In bottle, precision pipettes 70% methanol solution 20mL, weighed weight, is ultrasonically treated 30min (power 500W, frequency 40kHz), put to
Room temperature, then weighed weight, the weight of less loss is supplied with 70% methanol solution, is shaken up, and filtration takes subsequent filtrate, crosses 0.22 μm of micropore
Filter membrane to get；
4. the measurement of finger-print
Precision draws 2 μ L of test solution, injects Ultra Performance Liquid Chromatography instrument, record chromatogram is to get finger-print；
Wherein, chromatographic condition is as follows：
Chromatographic column：Phenomenex Kinetex C18(100 × 2.1mm, 2.6 μm) chromatographic column
Mobile phase：A is acetonitrile, and Mobile phase B is the aqueous formic acid that volumetric concentration is 0.1%；
Gradient elution：0-3min, 5-10%A；3-6min, 10-12%A；6-16min, 12-22%A；16-17min,
22%A；17-22min, 22-27%A；22-25min, 27-32%A；25-27min, 32-35%A；27-30min, 35%A；
30-36min, 35-40%A；36-37min, 40-100%A；37-39min, 100-5%A；
Temperature：40℃；
Flow velocity：0.4mL/min；
Chromatogram spectra collection range：190-400nm.
Using 7 batches of Polygala tenuifolia UPLC fingerprints of " similarity evaluation 2008A editions " software pair
Collection of illustrative plates carries out data processing, shared peak 32 (as shown in Figure 1) is determined, since 3,6 '-disinapoyl sucrose are at each batch
It is occurred in secondary Radix Polygalae, separating degree is good and peak area is larger, therefore selects 3,6 '-disinapoyl sucrose (No. 9 peaks)
As with reference to peak (S), each shared peak relative retention time and relative peak area are calculated with the reference peak, carry out fingerprint spectrum method
It learns and investigates, assay method is identical with step 1.The results show that same sample solution continuous sample introduction 6 times, each shared peak is opposite to be protected
Time RSD ＜ 0.15% is stayed, relative peak area RSD ＜ 0.98%, precision is good；Each shared peak of study on the stability is opposite to be retained
Time RSD ＜ 0.42%, relative peak area RSD ＜ 1.83%；Repeatability investigates each shared peak relative retention time RSD ＜
0.35%, relative peak area RSD ＜ 2.10%.
The UPLC finger-prints of 7 batches of Radix Polygalae samples are shown in Fig. 1, each place of production Polygala tenuifolia finger-print and reference fingerprint ratio
Right, similarity the results are shown in Table 2 between 0.927~0.971.
Similarity calculation result between 27 batches of Polygala tenuifolias of table (R is reference fingerprint)
Step 2：The chromatographic peak of finger-print in step 1 is pointed out using LC-MS technology, determines ultraviolet detection
Shared fingerprint peaks；
1. test material, instrument and reagent
Test material and reagent：With step 1.
Instrument：Thermo Scientific Q Exactive LC-MS, U.S. Thermo；Other instruments are the same as step 1.
2. Mass Spectrometry Conditions are as follows：
Ion source：Electric spray ion source (ESI)；Scan mode：Negative ions scan simultaneously；Spray voltage：3.5kV；Sheath
Gas velocity：35psi(1psi≈6.9kPa)；Secondary air speed：10psi；Capillary temperature：320℃；Probe warmer temperature：
300℃；Maximum spraying electric current：100A；S-Lens resolution ratio：55；Scanning range：m/z 100-2000；Mass resolution：
70000；
UPLC fingerprint map analyzings are carried out with 7 batches of Polygala tenuifolias of chromatographic condition pair in step 1.It is shared to 32 of each sample
Peak ultraviolet spectrogram compares and analyzes, and gained uv-spectrogram is as shown in Figure 1.
Recommended using Chinese Pharmacopoeia Commission《Similarity evaluation》Generate Polygala tenuifolia
Standard finger-print, wherein there is 32 shared peaks, peak number is denoted as No. 1-32 successively.In finger-print, with 3,6 '-
Disinapoyl sucrose (No. 9 peaks) are with reference to the peak peaks S, wherein 1,2,3,4,5,9,11, No. 14 peak uses reference substance chromatography
And mass spectrum retention behavior is pointed out, and sibiricose A are followed successively by5、sibiricose A6、sibiricaxanthone B、
Glomeratose A, polygalaxanthone III, 3,6 '-disinapoyl sucrose, tenuifoliside A,
Tenuifoliside C, since the content of compound lancerin and 7-O-methyl mangiferin are relatively low, in fingerprint image
There is no the shared peak of the two compounds in spectrum, it can be by reference substance chromatography and mass spectrum retention behavior, and according to bibliography pair
Two compounds are pointed out.
(Fig. 2) is can be seen that from Polygala tenuifolia total ion current figure, is respectively had under the conditions of positive and negative ion at swarming higher
Response, and it is corresponding with ultraviolet chromatogram.
Foundation reference substance chromatography and mass spectrum retention behavior, and the data in bibliography and chromatography retention behavior (chromatogram
See Fig. 2), 23 in 32 shared peaks are identified and (are shown in Table 3), including 14 sugar ester constituents, 6 saponins at
Point and 3 mouth mountain ketones components.With reference in document data and chromatography retention behavior to other 15 in Radix Polygalae sample
Non-shared peak is belonged to, including 9 sugar ester constituents and 6 mouth mountain ketones components, the results are shown in Table 4,.
3 Radix Polygalae finger-print of table shares the ownership (negative ion mode) at peak
The ownership (negative ion mode) at non-shared peak in the Radix Polygalae finger-print that table 4 is pointed out based on liquid matter
Step 3：8 index ingredient (sibiricose A are filtered out from the 23 shared peaks pointed out in step 25、
sibiricose A6, sibiricaxanthone B, glomeratose A, polygalaxanthone III, 3,6 '-
Disinapoyl sucrose, tenuifoliside A, tenuifoliside C), 2 are filtered out from 15 non-shared peaks
Index ingredient (lancerin, 7-O-methyl mangiferin), using the visible uv detection method of ultra high efficiency liquid phase all-wave length
Assay is carried out to above-mentioned 10 index ingredients.
1. test material, instrument and reagent are the same as step 1.
2. the preparation of mixed reference substance solution and test solution is the same as step 1.
3. the assay of index ingredient
It is accurate respectively to draw above-mentioned mixed reference substance solution and each 2 μ L of test solution, Ultra Performance Liquid Chromatography instrument is injected,
Mixed reference substance solution and test solution are measured respectively by chromatographic condition in step 1, determine the color of each ingredient to be measured
Spectral peak, and mixed reference substance solution is diluted step by step, the equation of linear regression of 10 index ingredients is established, is calculated separately for examination
Sibiricose A in product solution5、sibiricose A6、lancerin、sibiricaxanthone B、glomeratose A、
7-O-methyl mangiferin, polygalaxanthone III, 3,6 '-disinapoyl sucrose,
The content of 10 tenuifoliside A, tenuifoliside C index ingredients, wherein the line of 10 index ingredients
Property regression equation is shown in Table 5.
The equation of linear regression and the range of linearity of 10 kinds of index ingredients in 5 Polygala tenuifolia of table
Wherein, X is the absorbance that corresponding ingredient detects at corresponding Detection wavelength
4. precision test precision measures 2 μ L of mixed reference substance solution in step 1, carried out by the chromatographic condition in step 1
It measures, continuous sample introduction 6 times, records the chromatographic peak area of above-mentioned 10 compounds, and calculate 10 Compound Retention times and peak
Area RSD values, the RSD values of retention time are respectively 0.20%, 0.26%, 0.30%, 0.33%, 0.35%, 0.32%,
0.34%, 0.29%, 0.26%, 0.33%；The RSD values of peak area are respectively 0.41%, 0.59%, 0.84%, 0.61%,
0.87%, 0.80%, 0.38%, 0.70%, 0.91%, 0.55%, show that instrument precision is good.
5. stability test takes same batch of sample (No. 3), 6 parts are prepared by sampling method in step 1 is parallel, room temperature decentralization
Set, by chromatographic condition in step 1 respectively 0,2,4,8,12,18, for 24 hours when sample introduction is analyzed, calculate 10 Compound Retention times
And the RSD values of peak area, the RSD values of retention time are respectively 0.62%, 0.60%, 0.52%, 0.43%, 0.42%,
0.41%, 0.36%, 0.21%, 0.17%, 0.16%；The RSD values of peak area are respectively 0.86%, 0.95%, 1.63%,
0.60%, 1.50%, 0.48%, 0.48%, 0.45%, 0.84%, 0.34%, show that sample is good in internal stability for 24 hours.
6. repetitive test takes same batch of sample (No. 3), 6 parts are prepared by sampling method in step 1 is parallel, by step 1
Sample introduction is analyzed for chromatographic condition, calculates 10 Compound Retention times and mass fraction RSD values, the RSD values of retention time are respectively
0.57%, 0.56%, 0.47%, 0.55%, 0.49%, 0.50%, 0.46%, 0.30%, 0.28%, 0.33%；Quality point
Several RSD values are respectively 1.43%, 1.22%, 2.00%, 0.70%, 1.99%, 1.67%, 2.99%, 1.08%,
0.88%, 1.22%, show that experimental repeatability is good.
7. sample recovery rate experiment takes 6 parts of No. 3 samples measured, every part is 0.1g, is divided into 3 groups, every group 3 parts, accurate
It is weighed, it is placed in conical flask with cover, it is accurate respectively that each reference substance for being equivalent to sample composition amount 50%, 100%, 150% is added
Solution prepares test solution by sampling method in step 1, and by chromatographic condition in step 1, sample introduction is analyzed, 10 kinds of ingredients of calculating
Sample recovery rate, average recovery rate is respectively 98.45%, 99.17%, 95.76%, 95.72%, 95.44%, 92.29%,
91.85%, 99.18%, 96.11%, 94.91%；RSD is respectively 0.96%, 1.78%, 1.68%, 1.77%, 1.72%,
1.70%, 0.82%, 1.38%, 1.16%, 1.82%, meet 2015 editions《Chinese Pharmacopoeia》In《Drug standard analysis side
Method verification guide principle》Requirement.
8. sample size is measured prepares 7 batches of test solutions by sampling method in step 1, by chromatographic condition in step 1 point
Not Ce Ding 7 batches of Radix Polygalae samples 10 kinds of ingredients content, and calculate the total content of 10 kinds of ingredients of every batch of sample.7 batches of Polygala tenuifolias
In 10 index component content measurement results be shown in Table 6, mix the chromatogram of reference substance as shown in figure 3, Radix Polygalae sample chromatography
Figure is as shown in Figure 4.
The content of 10 kinds of chemical compositions in 67 batches of Radix Polygalaes of table
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (2)
1. a kind of Polygala tenuifolia finger-print and index component content method for measuring, which is characterized in that steps are as follows：
Step 1：Using the visible uv detection method of ultra high efficiency liquid phase all-wave length, the finger-print of Polygala tenuifolia is established；
The preparation of mixed reference substance solution：It is appropriate that each reference substance is weighed respectively, it is accurately weighed, add methanol to dissolve, is settled to
lancerin、sibiricaxanthone B、glomeratose A、7-O-methyl mangiferin、
Polygalaxanthone III final concentration of 1-100 μ g/mL, sibiricose A5, sibiricose A6,
Tenuifoliside C final concentration of 100-300 μ g/mL, 3,6 '-disinapoyl sucrose, tenuifoliside A are whole
A concentration of 300-500 μ g/mL, shake up to get mixed reference substance solution, are placed in spare in 4 DEG C of refrigerators；
The preparation of test solution：It takes Polygala tenuifolia to crush and crosses No. three sieves of pharmacopeia, Polygala tenuifolia powder about 0.2g, precision is taken to claim
It is fixed, it sets in conical flask with cover, precision pipettes 70% methanol solution 20mL, and weighed weight is ultrasonically treated 30min, the ultrasound work(
Rate 500W, frequency 40kHz, put to room temperature, then weighed weight, and the weight of less loss is supplied with 70% methanol solution, is shaken up, filtration,
Take subsequent filtrate, cross 0.22 μm of miillpore filter to get；
The measurement of finger-print：It is accurate respectively to draw mixed reference substance solution and each 2-10 μ L of test solution, inject ultra high efficiency
Liquid chromatograph records chromatogram, is recommended using Chinese Pharmacopoeia Commission《Chromatographic fingerprints of Chinese materia medica similarity evaluation system
System》Generate Polygala tenuifolia finger-print；
Wherein, chromatographic condition is as follows：
Chromatographic column：UPLC C18Column；
Mobile phase：Mobile phase A is acetonitrile, and Mobile phase B is the aqueous formic acid that volumetric concentration is 0.1-1.0%；
Gradient：0-3min, 5-10%A；3-6min, 10-12%A；6-16min, 12-22%A；16-17min, 22%A；
17-22min, 22-27%A；22-25min, 27-32%A；25-27min, 32-35%A；27-30min, 35%A；30-
36min, 35-40%A；36-37min, 40-100%A；37-39min, 100-5%A.
Sample size：2-10μL；
Column temperature：20-40℃；
Flow velocity：0.2-0.5mL/min；
Detection wavelength：290-330nm；
Step 2：The chromatographic peak of finger-print in step 1 is pointed out using LC-MS technology, determines that ultraviolet detection is shared
Fingerprint peaks and non-shared peak, for wherein liquid phase chromatogram condition with step 1, Mass Spectrometry Conditions are as follows：
Ion source：Electric spray ion source (ESI)；Scan mode：Negative ions scan simultaneously；Spray voltage：3.5kV；Sheath air-flow
Speed：35psi 1psi≈6.9kPa；Secondary air speed：10psi；Capillary temperature：320℃；Probe warmer temperature：300℃；
Maximum spraying electric current：100A；S-Lens resolution ratio：55；Scanning range：m/z 100-2000；Mass resolution：70000；
Step 3：8 index ingredient sibiricose A are filtered out from the 23 shared peaks identified in step 25、
sibiricose A6, sibiricaxanthone B, glomeratose A, polygalaxanthone III, 3,6 '-
Disinapoyl sucrose, tenuifoliside A, tenuifoliside C, filter out 2 from 15 non-shared peaks
Index ingredient lancerin, 7-O-methyl mangiferin, using the visible uv detection method pair of ultra high efficiency liquid phase all-wave length
Above-mentioned 10 index ingredients carry out assay；
Assay method is：It is accurate respectively to draw above-mentioned mixed reference substance solution and each 2-10 μ L of test solution, inject ultra high efficiency
Liquid chromatograph determines the chromatographic peak of each ingredient to be measured, and mixed reference substance solution diluted step by step, establish 10 indexs at
Point equation of linear regression, calculate separately the content of 10 compounds as described above in test solution；
Wherein, chromatographic condition is the same as step 1.
2. a kind of Polygala tenuifolia finger-print as described in claim 1 and index component content method for measuring, feature
It is, Mobile phase B is the aqueous formic acid of volumetric concentration 0.1% in the step 1 and 3；Sample size：2μL；Column temperature：40℃；Stream
Speed：0.4mL/min；Detection wavelength：320nm.
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