CN107132304A - A kind of Polygala tenuifolia finger-print and index component content method for measuring - Google Patents

Abstract

The present invention relates to a method for measuring the fingerprint of Polygala medicinal material and the content of index components, comprising the following steps: Step 1: adopting the ultra-high performance liquid phase full-wavelength visible ultraviolet detection method to establish the fingerprint of Polygala medicinal material; Step 2: using liquid quality Identify the chromatographic peaks of the fingerprints in step 1 with the combined technology, and determine the common fingerprint peaks and non-common peaks of the ultraviolet detection; Content determination of index components with neuroprotective, sedative, antidepressant or anti-senile dementia activities and clear structure.

Description

A method for determining the fingerprint and index component content of polygala medicinal material

technical field

The invention belongs to the field of traditional Chinese medicine analysis and content determination of traditional Chinese medicinal materials, and in particular relates to a method for determining the fingerprint of Polygala medicinal material and the content of index components.

Background technique

Polygala tenuifolia Willd., first recorded in "Shen Nong's Materia Medica", listed as top grade, and regarded as vital medicine. Warm in nature, bitter in taste, pungent, has the effects of calming the nerves, improving intelligence, eliminating phlegm and reducing swelling. Modern research has found that the main active ingredients of polygala medicinal materials include saponins, sugar esters, ketones and alkaloids, etc., which have antibacterial, anti-inflammatory, improve cognition, improve memory, anti-senile dementia, anti-depression , anti-tumor, anti-aging and other pharmacological effects.

The efficacy of Chinese medicinal materials is closely related to the quality of the medicinal materials, and the quality of the medicinal materials is closely related to the content of the main chemical components contained in the medicinal materials. A variety of known or unknown chemical components in Chinese herbal medicines exert their pharmacological effects on multiple targets through multiple pathways. At present, the qualitative analysis of Polygala medicinal materials is mainly fingerprint analysis, but the previously established method takes a long time, consumes more reagents, is not conducive to high-throughput analysis, and is not sufficient for the identification of complex components; Quantitative analysis of components mainly focuses on the determination of individual components as indicators. It is difficult for a single indicator component to accurately and comprehensively reflect the internal quality of Chinese medicinal materials. The existence of problems such as difficult separation of reference substances, unstable monomers, difficulty in supply or high supply price makes it unable to meet the needs of quality control of traditional Chinese medicine and research and development of new drugs. Therefore, aiming at the shortcomings of insufficient identification of the above-mentioned complex components and fewer quantitative chemical components, the present invention provides a method for determining the fingerprint and index component content of Polygala medicinal materials. This method can more comprehensively characterize and quantify the compounds in Polygala medicinal materials. analyze.

Contents of the invention

The object of the present invention is to provide a method for determining the fingerprints of Polygala medicinal materials and the content of index components, which can perform qualitative and quantitative analysis on the compounds in Polygala medicinal materials more comprehensively.

To achieve the above object, the technical solution provided by the invention is:

A method for determining the fingerprints and index components of polygala medicinal materials, the steps are as follows:

Step 1: Using Ultra Performance Liquid Chromatography (UPLC) full-wavelength visible ultraviolet detection method to establish the fingerprint of Polygala medicinal material;

Preparation of mixed reference substance solution: Weigh an appropriate amount of each reference substance, accurately weigh, add methanol to dissolve, and dilute to the final concentration of lancerin, sibiricaxanthone B, glomeratose A, 7-O-methyl mangiferin, polygalaxanthone III to 1-100 μg/ mL, the final concentration of sibiricose A ₅ , sibiricose A ₆ , and tenuifoliside C is 100-300 μg/mL, and the final concentration of 3,6′-disinapoyl sucrose and tenuifoliside A is 300-500 μg/mL. Shake well to obtain a mixed reference solution. And put it in a refrigerator at 4°C for later use;

Preparation of the test solution: take Polygala medicinal material and crush it and pass it through the Pharmacopoeia No. 3 sieve, take about 0.2 g of Polygala medicinal material powder, accurately weigh it, put it in a stoppered Erlenmeyer flask, accurately pipette 20 mL of 70% methanol solution, and weigh it , sonicate for 30 minutes (power 500W, frequency 40kHz), put it to room temperature, weigh again, make up the lost weight with 70% methanol solution, shake well, filter, take the filtrate, pass through a 0.22 μm microporous membrane, instant;

Determination of fingerprints: Precisely draw 2-10 μL each of the mixed reference solution and the test solution, inject them into an ultra-high performance liquid chromatograph, record the chromatograms, and adopt the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" recommended by the National Pharmacopoeia Committee Generating the fingerprints of polygala medicinal materials;

Wherein, the chromatographic conditions are as follows:

Chromatographic column: UPLC C ₁₈ column or T ₃ column;

Mobile phase: mobile phase A is acetonitrile, and mobile phase B is an aqueous formic acid solution with a volume concentration of 0.1-1.0%;

Elution 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.

Injection volume: 2-10μL;

Column temperature: 20-40°C;

Flow rate: 0.2-0.5mL/min;

Detection wavelength: 290-330nm;

Step 2: use liquid chromatography-mass spectrometry to identify the chromatographic peaks of the fingerprints in step 1, and determine the common fingerprint peaks and non-common peaks of the ultraviolet detection, wherein the liquid chromatography conditions are the same as step 1, and the mass spectrometry conditions are as follows:

Ion source: electrospray ion source (ESI); scanning method: simultaneous positive and negative ion scanning; spray voltage: 3.5kV; sheath gas flow rate: 35psi (1psi≈6.9kPa); auxiliary gas flow rate: 10psi; capillary temperature: 320°C; probe Heater temperature: 300°C; maximum spray current: 100A; S-Lens resolution: 55; scanning range: m/z 100-2000; mass resolution: 70000;

Step 3: Screen out 8 index components (sibiricose A ₅ , sibiricose A ₆ , sibiricaxanthone B, glomeratose A, polygalaxanthone III, 3,6′-disinapoyl sucrose, tenuifoliside A , tenuifoliside C), 2 index components (lancerin, 7-O-methyl mangiferin) were screened out from 15 non-common peaks, and the content of the above 10 index components was determined by ultra-high performance liquid phase full-wavelength visible ultraviolet detection method. Determination;

The determination method is: respectively accurately draw 2-10 μL of the above-mentioned mixed reference substance solution and the test solution, inject into the ultra-high performance liquid chromatograph, determine the chromatographic peaks of each component to be tested, and dilute the mixed reference substance solution step by step to establish The linear regression equation of 10 index components calculates respectively the content of the above-mentioned 10 compounds in the need testing solution;

Wherein, the chromatographic conditions are the same as step 1.

Further, the mobile phase B in steps 1 and 3 is formic acid aqueous solution with a volume concentration of 0.1%; injection volume: 2 μL; column temperature: 40° C.; flow rate: 0.4 mL/min; detection wavelength: 320 nm.

Compared with the prior art, the present invention realizes the improvement of the quality control standard from the two aspects of the whole and key components through UPLC "fingerprint qualitative + index component quantification", and not only comprehensively but also specifically and quantitatively determines the quality of the product. Quality controllability, and due to the introduction of UPLC technology, the measurement time of the existing HPLC technology is greatly shortened, so that the complex characterization of the fingerprints of Polygala medicinal materials can be realized.

Description of drawings

Fig. 1 is the fingerprints and standard fingerprints of 7 batches of polygala medicinal materials provided by the present invention; wherein the numbers of samples 1-7 are shown in Table 1, and R is the control fingerprint;

Figure 2 is the liquid chromatogram of Polygala sample; in the figure: A: UV chromatogram; B: total ion flow chromatogram in positive ion mode; C: total ion flow chromatogram in negative ion mode;

Fig. 3 is the UPLC chromatogram of mixed reference substance solution;

Fig. 4 is the UPLC chromatogram of Polygalae medicinal material need testing solution;

In Figure 3 and Figure 4: 1 is sibiricose A ₅ , 2 is sibiricose A ₆ , 3 is lancerin, 4 is sibiricaxanthone B, 5 is glomeratose A, 6 is 7-O-methyl mangiferin, 7 is polygalaxanthone III, 8 is 3 , 6′-disinapoyl sucrose, 9 is tenuifoliside A, 10 is tenuifoliside C)

detailed description

The following examples are used to illustrate the method, but are not intended to limit the scope of the invention.

In order to better understand the contents of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

Example 1

Step 1: Using ultra-high performance liquid phase full-wavelength visible ultraviolet detection method to establish the fingerprint of Polygala medicinal material; 1. Test material

Materials, instruments and reagents

Test materials: The samples of Polygala medicinal materials used were 7 batches of whole roots of Polygala medicinal materials collected from different producing areas in Shanxi Province, kneaded by hand, and the wood core was removed to obtain Polygala tubes, crushed, passed through a No. 3 pharmacopoeia sieve, and set aside. The details of the test materials are listed in Table 1.

Table 1 Polygala sample information table

Instruments: ultra-high performance liquid chromatography system (ACQUITY UPLC from Waters, including vacuum degasser, binary gradient pump, autosampler, column oven, TUV detector), electronic balance (CPA225D, Sartorius, Germany), Milli -Q ultrapure water system (Millipore, USA), ultrasonic cleaner (KQ-500DV, Kunshan Ultrasonic Instrument Co., Ltd.), traditional Chinese medicine chromatographic fingerprint similarity evaluation system version 2008A.

Reagents: acetonitrile (chromatographically pure, Fisher Company), formic acid (chromatographically pure, Fisher Company), ultrapure water (Milli-Q), methanol (domestic analytical grade).

2. Preparation of mixed reference solution

Weigh an appropriate amount of each reference substance, accurately weigh it, add methanol to dissolve, and dilute to the final concentration of lancerin, sibiricaxanthone B, glomeratose A, 7-O-methyl mangiferin, polygalaxanthone III to 1-100 μg/mL, sibiricose A ₅ , sibiricose A _6. The final concentration of tenuifoliside C is 100-300 μg/mL, and the mixed reference solution with the final concentration of 3,6′-disinapoyl sucrose and tenuifoliside A is 300-500 μg/mL. in standby;

3. Preparation of the test solution

Take polygala medicinal material and crush it and pass through the pharmacopoeia No. 3 sieve, take about 0.2g of polygala medicinal material powder, weigh it accurately, put it in a stoppered Erlenmeyer flask, accurately pipette 20mL of 70% methanol solution, weigh it, and ultrasonically treat it for 30min (power 500W , frequency 40kHz), put it to room temperature, weigh again, make up the lost weight with 70% methanol solution, shake well, filter, take the continued filtrate, and pass through a 0.22 μm microporous membrane to obtain final product;

4. Determination of Fingerprint

Precisely draw 2 μL of the test solution, inject it into the ultra-high performance liquid chromatograph, record the chromatogram, and obtain the fingerprint;

Wherein, the chromatographic conditions are as follows:

Chromatographic column: Phenomenex Kinetex C ₁₈ (100×2.1mm, 2.6μm) chromatographic column

Mobile phase: A is acetonitrile, and mobile phase B is an aqueous formic acid solution with a volume concentration of 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°C;

Flow rate: 0.4mL/min;

Chromatogram spectrum acquisition range: 190-400nm.

Using the software "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2008A" to process the data of the UPLC fingerprints of seven batches of Polygala medicinal materials, it was determined that there were 32 peaks in total (as shown in Figure 1). All of them appeared in Polygala, with good resolution and large peak area, so 3,6′-disinapoyl sucrose (peak No. 9) was selected as the reference peak (S), and the relative retention time and relative retention time of each common peak were calculated based on this reference peak. For the peak area, carry out the methodological investigation of the fingerprint chromatogram, and the determination method is the same as in step 1. The results show that the same sample solution was injected continuously for 6 times, the relative retention time RSD of each common peak was less than 0.15%, the relative peak area RSD was less than 0.98%, and the precision was good; the relative retention time RSD of each common peak in the stability inspection was less than 0.42%. The peak area RSD<1.83%; the relative retention time RSD<0.35% and the relative peak area RSD<2.10% of the common peaks in repeatability inspection.

The UPLC fingerprints of the 7 batches of Polygala samples are shown in Figure 1. The fingerprints of the Polygala medicinal materials in each production area were compared with the control fingerprints, and the similarity ranged from 0.927 to 0.971. The results are shown in Table 2.

Table 2 Calculation results of similarity between 7 batches of Polygala medicinal materials (R is the control fingerprint)

Step 2: identify the chromatographic peaks of the fingerprints in step 1 by liquid chromatography-mass spectrometry, and determine the common fingerprint peaks of the ultraviolet detection;

1. Test materials, instruments and reagents

Test materials and reagents: same as step 1.

Instrument: Thermo Scientific Q Exactive LC-MS, Thermo, USA; other instruments are the same as step 1.

2. The mass spectrometry conditions are as follows:

Ion source: electrospray ion source (ESI); scanning method: simultaneous positive and negative ion scanning; spray voltage: 3.5kV; sheath gas flow rate: 35psi (1psi≈6.9kPa); auxiliary gas flow rate: 10psi; capillary temperature: 320°C; probe Heater temperature: 300°C; maximum spray current: 100A; S-Lens resolution: 55; scanning range: m/z 100-2000; mass resolution: 70000;

Using the chromatographic conditions in step 1, UPLC fingerprint analysis was carried out on 7 batches of Polygala medicinal materials. The UV spectra of 32 common peaks of each sample were compared and analyzed, and the obtained UV spectra are shown in Figure 1.

The standard fingerprint of Polygala medicinal materials was generated by using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" recommended by the National Pharmacopoeia Committee, in which there are 32 common peaks, and the peak numbers are recorded as 1-32 in sequence. In the fingerprint spectrum, 3,6'-disinapoyl sucrose (Peak No. 9) is used as the reference peak S peak, and peaks 1, 2, 3, 4, 5, 9, 11, and 14 use the reference substance chromatography and mass spectrometry retention behavior For identification, sibiricose A ₅ , sibiricose A ₆ , sibiricaxanthone B, glomeratose A, polygalaxanthone III, 3,6′-disinapoyl sucrose, tenuifoliside A, tenuifoliside C, due to the low content of compounds lancerin and 7-O-methyl mangiferin , there are no common peaks of these two compounds in the fingerprint spectrum, the two compounds can be identified according to the retention behavior of the reference substance chromatography and mass spectrum, and according to the references.

It can be seen from the total ion chromatogram of Polygala medicinal material (Fig. 2) that its component peaks have higher responses under positive and negative ion conditions, and correspond to the ultraviolet chromatogram.

Based on the retention behavior of the reference substance chromatography and mass spectrometry, and the data and chromatographic retention behavior in the reference literature (see Figure 2 for the chromatogram), 23 of the 32 common peaks were identified (see Table 3), including 14 sugar esters Class ingredients, 6 saponin-like ingredients, and 3 ketone-like ingredients. At the same time, the other 15 non-common peaks in Polygala samples were assigned based on the data and chromatographic retention behavior in the reference literature, including 9 sugar ester components and 6 mountain ketone components. The results are shown in Table 4.

Table 3 The attribution of common peaks in Polygala fingerprints (negative ion mode)

Table 4 Assignment of non-common peaks in Polygala fingerprints based on liquid mass assignment (negative ion mode)

Step 3: Screen out 8 index components (sibiricose A ₅ , sibiricose A ₆ , sibiricaxanthone B, glomeratose A, polygalaxanthone III, 3,6′-disinapoyl sucrose, tenuifoliside A, tenuifoliside C), 2 index components (lancerin, 7-O-methyl mangiferin) were screened out from 15 non-common peaks, and the content of the above 10 index components was determined by ultra-high performance liquid phase full-wavelength visible ultraviolet detection method .

1. Test materials, instruments and reagents are the same as step 1.

2. The preparation of the mixed reference solution and the test solution is the same as step 1.

3. Content determination of index components

Accurately draw 2 μL each of the above-mentioned mixed reference substance solution and need testing solution respectively, inject into the ultra-high performance liquid chromatograph, measure the mixed reference substance solution and need testing solution respectively according to the chromatographic conditions in step 1, and determine the content of each component to be tested. chromatographic peak, and the mixed reference substance solution was diluted step by step, and the linear regression equation of 10 index components was established, and the sibiricose A ₅ , sibiricose A ₆ , lancerin, sibiricaxanthone B, glomeratose A, 7-O in the test solution were calculated respectively. -methyl mangiferin, polygalaxanthone III, 3,6′-disinapoyl sucrose, tenuifoliside A, tenuifoliside C content of 10 index components, and the linear regression equations of the 10 index components are shown in Table 5.

Table 5 Linear regression equation and linear range of 10 index components in Polygala medicinal materials

Among them, X is the absorbance of the corresponding component detected at the corresponding detection wavelength

4. Precision test Take 2 μL of the mixed reference solution in step 1, measure it according to the chromatographic conditions in step 1, inject 6 times continuously, record the chromatographic peak areas of the above 10 compounds, and calculate the retention time of the 10 compounds And peak area RSD value, the RSD value of retention time is respectively 0.20%, 0.26%, 0.30%, 0.33%, 0.35%, 0.32%, 0.34%, 0.29%, 0.26%, 0.33%; The RSD value of peak area is respectively 0.41%, 0.59%, 0.84%, 0.61%, 0.87%, 0.80%, 0.38%, 0.70%, 0.91%, 0.55%, indicating that the precision of the instrument is good.

5. Stability test Take the same batch of samples (No. 3), prepare 6 copies in parallel according to the sample preparation method in step 1, place them at room temperature, and set the chromatographic conditions in step 1 at 0, 2, 4, 8, 12, 18, Inject analysis at 24 hours, calculate the RSD values of retention time and peak area of 10 compounds, the RSD values of retention time are 0.62%, 0.60%, 0.52%, 0.43%, 0.42%, 0.41%, 0.36%, 0.21%, 0.17%, 0.16%; RSD values of peak area were 0.86%, 0.95%, 1.63%, 0.60%, 1.50%, 0.48%, 0.48%, 0.45%, 0.84%, 0.34%, indicating that the sample was stable within 24h good.

6. Repeatability test Take the same batch of samples (No. 3), prepare 6 copies in parallel according to the sample preparation method in step 1, inject and analyze according to the chromatographic conditions in step 1, calculate the retention time and mass fraction RSD value of 10 compounds, and the retention time The RSD values are 0.57%, 0.56%, 0.47%, 0.55%, 0.49%, 0.50%, 0.46%, 0.30%, 0.28%, 0.33% respectively; the RSD values of mass fraction are 1.43%, 1.22%, 2.00% , 0.70%, 1.99%, 1.67%, 2.99%, 1.08%, 0.88%, 1.22%, indicating good repeatability of the experiment.

7. Sample addition recovery rate test Take 6 parts of the measured No. 3 sample, each part is 0.1g, divide into 3 groups, each group has 3 parts, weigh them accurately, place them in a stoppered conical flask, and accurately add the equivalent of For each reference substance solution of 50%, 100%, and 150% of the sample component amount, the sample preparation method in step 1 is used to prepare the test solution, and the chromatographic conditions in step 1 are used for sample introduction analysis, and the sample recovery rate of 10 kinds of components is calculated. The average recoveries were 98.45%, 99.17%, 95.76%, 95.72%, 95.44%, 92.29%, 91.85%, 99.18%, 96.11%, 94.91%; RSD were 0.96%, 1.78%, 1.68%, 1.77%, respectively. 1.72%, 1.70%, 0.82%, 1.38%, 1.16%, 1.82%, in line with the requirements of the "Guiding Principles for the Validation of Drug Quality Standards Analysis Methods" in the 2015 edition of "Chinese Pharmacopoeia".

8. Determination of sample content Prepare 7 batches of test solution according to the sample preparation method in step 1, measure the contents of 10 components of 7 batches of Polygala samples according to the chromatographic conditions in step 1, and calculate the total content of 10 components in each batch of samples . The determination results of 10 index components in 7 batches of Polygala medicinal materials are shown in Table 6, the chromatogram of the mixed reference substance is shown in Figure 3, and the chromatogram of Polygala sample is shown in Figure 4.

Table 6 Contents of 10 chemical components in 7 batches of Polygala

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope 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.