CN107132304A - 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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- CN107132304A CN107132304A CN201710496283.7A CN201710496283A CN107132304A CN 107132304 A CN107132304 A CN 107132304A CN 201710496283 A CN201710496283 A CN 201710496283A CN 107132304 A CN107132304 A CN 107132304A
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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
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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
Abstract
The present invention relates to a kind of Polygala tenuifolia finger-print and index component content method for measuring, comprise 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 set up；Step 2：The chromatographic peak of finger-print in step 1 is pointed out using LC-MS technology, ultraviolet detection shared fingerprint peakses and non-shared peak are determined；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 clear and definite index composition of structure carries out assay.
Description
Technical field
The invention belongs to Analysis of Chinese Traditional Medicine and Chinese medicine assay field, and in particular to a kind of Polygala tenuifolia finger-print and
Index component content method for measuring.
Background technology
Polygala 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, the effects such as with tranquilize the mind and promote the intelligence, eliminating phlegm and diminishing swelling.Found through modern study, Polygala tenuifolia
Principle active component includes saponins, sugar esters, mouth mountain ketone and alkaloids etc., and with antibacterial, anti-inflammatory, raising is cognitive, change
Kind memory capability, anti-senile dementia, antidepression, antitumor, anti-ageing pharmacological action of waiting for a long time.
The power of Chinese medicine drug effect is good and bad closely related with quality of medicinal material, 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
Act on Mutiple Targets and play its pharmacological action.Qualitative analysis on Polygala tenuifolia, predominantly fingerprint map analyzing at present, but it
The method of preceding foundation is time-consuming longer, and loss reagent is more, is unfavorable for high throughput analysis, is not also filled for pointing out for complicated ingredient
Point；Quantitative analysis to chemical composition in Polygala tenuifolia, focuses primarily upon the measure that individual components are index, single index components
Be difficult to the inherent quality for accurately and comprehensively embodying Chinese medicine, the quality control of Chinese medicine multi objective and evaluation model need it is various into
Point reference substance, but part Chinese medicine reference substance separating difficulty is big, monomer is unstable, be difficult to supply or the problems such as supply price is high
In the presence of the demand in terms of preventing it from meeting traditional Chinese medicine quality monitoring and new drug development has determined content in current Polygala tenuifolia
Compound amounts it is less.Therefore point out the defects, this hair such as insufficient and quantitative chemical composition is less for above-mentioned complicated ingredient
Bright to provide a kind of Polygala tenuifolia finger-print and index component content method for measuring, this method can be more fully to polygala
Compound carries out qualitative and quantitative analysis in medicinal material.
The content of the invention
, should it is an object of the invention to provide a kind of Polygala tenuifolia finger-print and index component content method for measuring
Method more fully can carry out qualitative and quantitative analysis to compound in Polygala tenuifolia.
To achieve the above object, the technical scheme that provides of the present invention is：
A kind of content assaying method of Polygala tenuifolia finger-print and index composition, step is 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, sets up 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, plus methanol dissolving, it 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
Concentration is 300-500 μ g/mL, shakes up, produces mixed reference substance solution, is placed in standby in 4 DEG C of refrigerators；
The preparation of need testing solution：Take Polygala tenuifolia to crush and cross No. three sieves of pharmacopeia, take Polygala tenuifolia powder about 0.2g, essence
It is close weighed, put in conical flask with cover, precision pipettes 70% methanol solution 20mL, weighed weight, 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, filters, takes
Subsequent filtrate, crosses 0.22 μm of miillpore filter, produces；
The measure of finger-print：Accurate respectively to draw mixed reference substance solution and each 2-10 μ L of need testing solution, injection is super
High performance liquid chromatograph, is recorded 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 C18Post or T3Post；
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, ultraviolet detection is determined
Shared fingerprint peakses and non-shared peak, wherein liquid phase chromatogram condition is with step 1, and Mass Spectrometry Conditions are as follows：
Ion gun：Electric spray ion source (ESI)；Scan mode：Negative ions are scanned 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 composition (sibiricose A are filtered out in the 23 shared peaks identified from 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 composition (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 compositions；
Assay method is：Accurate respectively to draw above-mentioned mixed reference substance solution and each 2-10 μ L of need testing solution, injection is super
High performance liquid chromatograph, determines the chromatographic peak of each composition to be measured, and mixed reference substance solution is diluted step by step, sets up 10 indexs
Property composition equation of linear regression, respectively calculate need testing solution in 10 compounds as described above content；
Wherein, chromatographic condition is with step 1.
Further, Mobile phase B is the aqueous formic acid that volumetric concentration is 0.1% in step 1 and 3；Sample size：2μL；Post
Temperature：40℃；Flow velocity：0.4mL/min；Detection wavelength：320nm.
Compared to prior art, the present invention is by way of UPLC " Qualitative fingerprint+index component quantifying ", from whole
Body and the aspect of key component two realize the lifting of quality control standard jointly, not only the quality that product is determined of specific quantitative comprehensively
Controllability, and due to the introducing of UPLC technologies, substantially reduces the existing minute for generally using HPLC technologies so that
The complexity of Polygala tenuifolia finger-print is characterized and is achieved.
Brief description of the drawings
7 batches of Polygala tenuifolia finger-prints and standard finger-print that Fig. 1 provides for the present invention；Wherein sample 1-7 numberings are shown in
Table 1, R is reference fingerprint；
Fig. 2 is the liquid matter chromatogram of polygala 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 need testing 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)
Embodiment
Following examples are used to illustrate this method, but are not limited to the scope of the present invention.
Content, makees furtherly with specific embodiment to the present invention below in conjunction with the accompanying drawings for a better understanding of 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 set up；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 pumps core, obtains RADIX POLYGALAE, is crushed, and crosses No. three sieves of pharmacopeia, standby.Test material specifying information is shown in Table 1.
The polygala sample message table of table 1
Instrument：Ultra performance liquid chromatography system (Waters companies ACQUITY UPLC, including vacuum degassing machine, binary ladder
Spend pump, automatic sampler, 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
Weigh each reference substance respectively appropriate, it is accurately weighed, plus methanol dissolving, be settled to lancerin,
Sibiricaxanthone B, glomeratose A, 7-O-methyl mangiferin, polygalaxanthone III are whole
Concentration is 1-100 μ g/mL, sibiricose A5、sibiricose A6, the final concentration of 100-300 μ g/ of tenuifoliside C
The final concentration of 300-500 μ g/mL of mL, 3,6 '-disinapoyl sucrose, tenuifoliside A mixing reference substance is molten
Liquid, shakes up, and produces, and is placed in standby in 4 DEG C of refrigerators；
3. the preparation of need testing solution
Take Polygala tenuifolia to crush and cross the sieve of pharmacopeia three, take Polygala tenuifolia powder about 0.2g, it is accurately weighed, put tool plug taper
In bottle, precision pipettes 70% methanol solution 20mL, weighed weight, 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, filtration, takes subsequent filtrate, crosses 0.22 μm of micropore
Filter membrane, is produced；
4. the measure of finger-print
Precision draws the μ L of need testing solution 2, injects Ultra Performance Liquid Chromatography instrument, records chromatogram, produces 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 scope：190-400nm.
Using " similarity evaluation 2008A editions " software to 7 batches of Polygala tenuifolia UPLC fingerprints
Collection of illustrative plates carries out data processing, it is determined that shared 32, peak (as shown in Figure 1), because 3,6 '-disinapoyl sucrose are at each batch
Occurred in secondary polygala, separating degree is good and peak area is larger, therefore 3,6 '-disinapoyl sucrose of selection (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, fingerprint spectrum method is carried out
Learn and investigate, assay method is identical with step 1.As a result show, same sample solution continuous sample introduction 6 times, each shared peak is relative to be protected
Time RSD ＜ 0.15%, relative peak area RSD ＜ 0.98% are stayed, precision is good；Each shared peak of study on the stability is relative 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 polygala 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 Measure 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, ultraviolet detection is determined
Shared fingerprint peakses；
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 with step 1.
2. Mass Spectrometry Conditions are as follows：
Ion gun：Electric spray ion source (ESI)；Scan mode：Negative ions are scanned 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 to 7 batches of Polygala tenuifolias with chromatographic condition in step 1.32 of each sample are had
Peak ultraviolet spectrogram is analyzed, 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 designated as No. 1-32 successively.In finger-print, with 3,6 '-
Disinapoyl sucrose (No. 9 peaks) be with reference to peak S peaks, wherein 1,2,3,4,5,9,11, No. 14 peaks use reference substance chromatogram
And mass spectrum retention behavior is pointed out, sibiricose A are followed successively by5、sibiricose A6、sibiricaxanthone B、
Glomeratose A, polygalaxanthone III, 3,6 '-disinapoyl sucrose, tenuifoliside A,
Tenuifoliside C, because compound lancerin and 7-O-methyl mangiferin content are relatively low, in fingerprint image
There is no the shared peak of the two compounds in spectrum, can be by reference substance chromatogram 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, it respectively has higher into swarming under the conditions of positive and negative ion
Response, it is and corresponding with ultraviolet chromatogram.
Foundation reference substance chromatogram and mass spectrum retention behavior, and data and chromatogram retention behavior (chromatogram in bibliography
See Fig. 2), 23 in 32 shared peaks are identified and (are shown in Table 3), including 14 sugar ester constituents, 6 saponinses into
Point, and 3 mouth mountain ketones components.With reference to the data in document and chromatogram retention behavior to other 15 in polygala 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,.
The polygala finger-print of table 3 has the ownership (negative ion mode) at peak
The ownership (negative ion mode) at non-shared peak in the polygala finger-print that table 4 is pointed out based on liquid matter
Step 3：8 index composition (sibiricose A are filtered out in the 23 shared peaks pointed out from 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 composition (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 compositions.
1. test material, instrument and reagent are with step 1.
2. the preparation of mixed reference substance solution and need testing solution is with step 1.
3. the assay of index composition
It is accurate respectively to draw above-mentioned mixed reference substance solution and each 2 μ L of need testing solution, Ultra Performance Liquid Chromatography instrument is injected,
Mixed reference substance solution and need testing solution are measured respectively by chromatographic condition in step 1, the color of each composition to be measured is determined
Spectral peak, and mixed reference substance solution is diluted step by step, the equation of linear regression of 10 index compositions is set up, is calculated respectively for examination
Sibiricose A in product solution5、sibiricose A6、lancerin、sibiricaxanthone B、glomeratose A、
7-O-methyl mangiferin, polygalaxanthone III, 3,6 '-disinapoyl sucrose,
Tenuifoliside A, 10 index compositions of tenuifoliside C content, wherein, the line of 10 index compositions
Property regression equation is shown in Table 5.
The equation of linear regression and the range of linearity of 10 kinds of index compositions in the Polygala tenuifolia of table 5
Wherein, X is that corresponding composition detects obtained absorbance at corresponding Detection wavelength
4. precision test precision measures the μ L of mixed reference substance solution 2 in step 1, carried out by the chromatographic condition in step 1
Determine, continuous sample introduction 6 times, record 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%th, 0.29%, 0.26%, 0.33%；The RSD values of peak area be respectively 0.41%, 0.59%, 0.84%, 0.61%,
0.87%th, 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, room temperature decentralization are prepared by sampling method in step 1 is parallel
Put, by chromatographic condition in step 1, in 0,2,4,8,12,18,24h, sample introduction is analyzed respectively, calculates 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%th, 0.36%, 0.21%, 0.17%, 0.16%；The RSD values of peak area be respectively 0.86%, 0.95%, 1.63%,
0.60%th, 1.50%, 0.48%, 0.48%, 0.45%, 0.84%, 0.34%, show that sample is good in 24h internal stabilities.
6. replica test takes same batch of sample (No. 3), 6 parts are prepared by sampling method in step 1 is parallel, by step 1
Chromatographic condition sample introduction is analyzed, and calculates 10 Compound Retention times and mass fraction RSD values, and the RSD values of retention time are respectively
0.57%th, 0.56%, 0.47%, 0.55%, 0.49%, 0.50%, 0.46%, 0.30%, 0.28%, 0.33%；Quality point
Several RSD values be respectively 1.43%, 1.22%, 2.00%, 0.70%, 1.99%, 1.67%, 2.99%, 1.08%,
0.88%th, 1.22%, show that experimental repeatability is good.
7. average recovery experiment takes 6 parts of No. 3 samples determined, every part is 0.1g, is divided into 3 groups, every group 3 parts, precision
It is weighed, it is placed in conical flask with cover, respectively the accurate each reference substance added equivalent to sample composition amount 50%, 100%, 150%
Solution, need testing solution is prepared by sampling method in step 1, is analyzed by chromatographic condition sample introduction in step 1,10 kinds of compositions of calculating
Average recovery, average recovery rate is respectively 98.45%, 99.17%, 95.76%, 95.72%, 95.44%, 92.29%,
91.85%th, 99.18%, 96.11%, 94.91%；RSD is respectively 0.96%, 1.78%, 1.68%, 1.77%, 1.72%,
1.70%th, 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, which is determined, presses 7 batches of need testing solutions of sampling method preparation in step 1, by chromatographic condition in step 1 point
Not Ce Ding 7 batches of polygala samples 10 kinds of compositions content, and calculate the total content of 10 kinds of compositions 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, the chromatogram of polygala sample
Figure is as shown in Figure 4.
The content of 10 kinds of chemical compositions in 67 batches of polygalas 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, it is characterised in that step is 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 set up；
The preparation of mixed reference substance solution：It is appropriate that each reference substance is weighed respectively, it is accurately weighed, plus methanol dissolving, it 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
Concentration is 300-500 μ g/mL, shakes up, produces mixed reference substance solution, is placed in standby in 4 DEG C of refrigerators；
The preparation of need testing solution：Take Polygala tenuifolia to crush and cross No. three sieves of pharmacopeia, take Polygala tenuifolia powder about 0.2g, precision claims
It is fixed, put in conical flask with cover, precision pipettes 70% methanol solution 20mL, weighed weight, ultrasonically treated 30min (power 500W, frequency
Rate 40kHz), put to room temperature, then weighed weight, the weight of less loss is supplied with 70% methanol solution, is shaken up, filters, takes subsequent filtrate,
0.22 μm of miillpore filter is crossed, is produced；
The measure of finger-print：It is accurate respectively to draw each 2-10 μ L of need testing solution, Ultra Performance Liquid Chromatography instrument is injected, color is recorded
Spectrogram, is recommended using Chinese Pharmacopoeia Commission《Similarity evaluation》Generate Polygala tenuifolia fingerprint
Collection of illustrative plates；
Wherein, chromatographic condition is as follows：
Chromatographic column：UPLC C18Post or T3Post；
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 has
Fingerprint peakses and non-shared peak, wherein liquid phase chromatogram condition is with step 1, and Mass Spectrometry Conditions are as follows：
Ion gun：Electric spray ion source (ESI)；Scan mode：Negative ions are scanned 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 composition (sibiricose A are filtered out in the 23 shared peaks identified from 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 composition (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 compositions；
Assay method is：It is accurate respectively to draw above-mentioned mixed reference substance solution and each 2-10 μ L of need testing solution, inject ultra high efficiency
Liquid chromatograph, determines the chromatographic peak of each composition to be measured, and mixed reference substance solution is diluted step by step, set up 10 indexs into
The equation of linear regression divided, calculates the content of 10 compounds as described above in need testing solution respectively；
Wherein, chromatographic condition is with step 1.
2. a kind of Polygala tenuifolia finger-print as claimed in claim 1 and index component content method for measuring, its 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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| CN201710496283.7A CN107132304B (en) | 2017-06-26 | 2017-06-26 | A kind of Polygala tenuifolia finger-print and index component content method for measuring |
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| CN109633003A (en) * | 2018-12-26 | 2019-04-16 | 成都普思生物科技股份有限公司 | Radix Polygalae mouth mountain ketone III and 3,6 in a kind of measurement Radix Polygalae, the method for-two mustard acyl cane sugar contents |
| CN113917004A (en) * | 2021-08-31 | 2022-01-11 | 湖北瑞华制药有限责任公司 | Quality detection method for polygala tenuifolia medicinal material |
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| CN109580829A (en) * | 2018-12-28 | 2019-04-05 | 成都康美药业生产有限公司 | The detection method of bis- mustard acyl sucrose of Radix Polygalae mountain ketone III and 3,6- in a kind of Radix Polygalae |
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