CN115436498B - Method for measuring multiple evaluation contents of cinnabar root medicinal material - Google Patents
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Abstract
The invention discloses a method for measuring the content of one-measurement-multiple-evaluation of a cinnabar root medicinal material, which adopts a high performance liquid chromatography to establish a method for measuring the content of gallic acid, gallocatechin, bergenin, baibiaojingsaponin A and baibiaojingsaponin B in the cinnabar root and taking bergenin as an internal standard substance. The detection result is accurate and stable, and can be used for quality control of the cinnabar root medicinal material; meanwhile, the method can make up for the defects of high price of the cinnabar root part reference substances (the bailiang gold saponin A and the bailiang gold saponin B), unstable part reference substances (gallocatechin) and the like. The invention can reduce the detection cost and the detection time, reduce the workload, improve the efficiency and lay a foundation for improving the quality standard of the cinnabar root.
Description
Technical Field
The invention relates to a medicinal material quality detection method, in particular to a method for measuring one-measurement-multiple-evaluation content of a cinnabar root medicinal material and application thereof.
Background
The radix Ardisiae Crenatae is dry root of Ardisiae Crenatae Ardisia crenata Sims of Ardisiaceae, has effects of removing toxic substance, relieving swelling, promoting blood circulation, relieving pain, dispelling pathogenic wind, and removing dampness, and can be used for treating laryngopharynx swelling and pain, rheumatalgia, traumatic injury, etc., and is used as laryngology holy medicine by Miao nationality in Guizhou. The existing quality control of the vermilion root mainly follows the Chinese pharmacopoeia (2020 edition), bergenin is used as a content measurement index component, and the quality control index is single and the specificity is poor. Through literature research, the cinnabar root mainly contains a plurality of compounds such as triterpenoid saponin, coumarin, phenols and the like, and the active ingredients of the cinnabar root not only can relieve cough and asthma and resist inflammation and bacteria, but also have the functions of resisting tumor activity, resisting fertility, inhibiting platelet aggregation, reducing blood pressure, contracting uterus, inhibiting cAMP phosphodiesterase and the like. Therefore, in order to solve the problems of single quality control index of the radix cinnabar medicinal material, a high performance liquid chromatography method is adopted, and bergenin is used as an internal standard to establish a content determination method of the radix cinnabar gallic acid, gallocatechin, bergenin, baibiasaponin A and baibiasaponin B. On the basis, in order to overcome the problems of high price of part of the control substances of the cinnabar root (the baibian saponin A and the baibian saponin B), unstable part of the control substances (gallocatechin) and the like, the method for determining the 5 components by one-test-multiple evaluation is researched and established.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for measuring the content of the Cinnabaris root by one measurement and multiple evaluations, which provides a basis for improving the quality standard of the Cinnabaris root. In order to achieve the above object, the present invention is achieved by the following technical scheme.
The invention provides a method for measuring the content of multiple evaluation of a cinnabar root medicinal material, which comprises the following steps:
(1) Preparing a reference substance solution;
(2) Preparing a sample solution;
(3) Establishing a high performance liquid chromatography for simultaneously measuring the contents of gallic acid, gallocatechin, bergenin, and baibian saponin A and baibian saponin B;
(4) Determining relative correction factors of gallic acid, gallocatechin, bailiangchin A and bailiangchin B by taking bergenin as an internal reference substance;
(5) And calculating bergenin content of the measured sample solution chromatogram, and calculating the contents of gallic acid, gallocatechin, baibiasaponin A and baibiasaponin B according to the relative correction factors.
According to the method of the present invention, in the step (1), a control solution containing gallic acid control, gallocatechin control, bergenin control, baibianjin saponin a control and baibianjin saponin B control is prepared using 50% methanol as a solvent.
According to the method, in the step (1), when a reference substance solution is prepared, 50% methanol is used as a solvent to prepare a reference substance solution containing gallic acid reference substance, gallocatechin reference substance, bergenin reference substance, hundred-two-gold-saponin-A reference substance and hundred-two-gold-saponin-B reference substance, wherein the concentrations of gallic acid, gallocatechin, bergenin, hundred-two-gold-saponin-A and hundred-two-gold-saponin-B are respectively 2-4 mug/mL, 15-25 mug/mL, 0.3-0.7 mg/mL, 0.6-1.0 mg/mL and 0.5-1.0 mg/mL.
According to the method, in the step (1), when a reference substance solution is prepared, 50% methanol is used as a solvent to prepare a reference substance solution containing gallic acid reference substance, gallocatechin reference substance, bergenin reference substance, hundred-two-gold-saponin-A reference substance and hundred-two-gold-saponin-B reference substance, wherein the concentrations of gallic acid, gallocatechin, bergenin, hundred-two-gold-saponin-A and hundred-two-gold-saponin-B are respectively 2.5-3.5 mug/mL, 18-24 mug/mL, 0.4-0.6 mg/mL, 0.8-0.9 mg/mL and 0.6-0.8 mg/mL.
The method according to the invention, wherein in step (2), the preparation method of the sample solution comprises the following steps: precisely weighing radix Ardisiae Crenatae sample 0.5g, precisely adding methanol 25mL, weighing, heating and refluxing for 40min, cooling, supplementing the reduced weight with methanol, shaking, and filtering with 0.22 μm microporous membrane.
The method according to the present invention, wherein in step (3), the chromatographic conditions of the high performance liquid chromatography are: c18 column (e.g. column size 250mm 4.6mm,5 μm, e.g. Diamond-C18 column); acetonitrile (A) and 0.1 percent phosphoric acid aqueous solution (B) are taken as mobile phases, and gradient elution is carried out (0 to 22min,8 percent A;22 to 30min,8 to 30 percent A;30 to 40min,30 to 30 percent A;40 to 60min,30 to 40 percent A;60 to 65min,40 to 100 percent A); the flow rate is 1.0mL/min; column temperature is 30 ℃; the detection wavelength is 203nm; the sample injection amount is 10 mu L; the theoretical plate number is not less than 5000 based on bergenin.
The method according to the present invention, wherein in step (4), the relative correction factors between gallic acid, gallocatechin, baibian saponin a, baibian saponin B and the reference bergenin are in the range of 0.48 to 0.52, 0.42 to 0.46, 83.5 to 86.5, 94.0 to 104.0, respectively; for example, the relative correction factors between gallic acid, gallocatechin, baibiasaponin a, baibiasaponin B and the reference bergenin are 0.4900, 0.4340, 84.8548, 98.3806, respectively.
The method according to the invention also comprises methodology investigation of the high performance liquid chromatography used, including for example linear relationship investigation, precision investigation, repeatability investigation, stability investigation and sample recovery verification.
The methods of the present invention exhibit many of the benefits detailed herein and in the context.
The invention has the beneficial effects that:
(1) The invention establishes a multi-index content determination method for the radix cinnabar, which is based on a high performance liquid chromatography technology and combines a multi-evaluation method, is accurate and reliable, is suitable for determining the content of multiple components in radix cinnabar, is favorable for the comprehensive quality control of radix cinnabar and related preparations, and has the advantages of high detection sensitivity, good stability, simple and convenient operation, easy grasp and convenient further popularization.
(2) The invention relates to a method for evaluating a cinnabar root medicinal material by a one-test-multiple-evaluation method, which takes bergenin as an internal reference substance, calculates relative correction factors of gallic acid, gallocatechin, baibianjin saponin A and baibianjin saponin B in the cinnabar root, and calculates the contents of gallic acid, gallocatechin, baibianjin saponin A and baibianjin saponin B by using the correction factors. In the method, two reference substances of the bailiang saponin A and the bailiang saponin B are expensive and scarce, the stability of the gallocatechin is poor, the existing preparation is needed, the detection cost and the detection time can be greatly reduced by adopting a one-measurement-multiple-evaluation content measuring method aiming at the problems, the workload is reduced, the efficiency is improved, and the method has important significance for quality control of the cinnabar root and guarantee of clinical curative effect.
Drawings
FIG. 1 is an HPLC chart of the mixed control (A) and the Cinnabaris sample (B). Wherein, 1: gallic acid; 2: gallocatechin; 3: bergenin; 4: bailiang of gold saponins a;5: bailiang of gold saponins B.
Detailed Description
In order that those skilled in the art will better understand the technical solution of the present invention, the present invention will be further described with reference to specific examples, but the examples are not intended to limit the present invention.
Example 1: content determination of Cinnabaris root by one-test-multiple-evaluation method
1. Instrument and reagent
1.1 instruments
Agilent-1260 type high performance liquid chromatograph (Agilent Co., USA); an analytical balance model XS-205 ten-thousandth (Mettler Toledo company, switzerland); HH-S6A water bath (Beijing Uyoemerging instruments Co., ltd.); FW-177 type pulverizer for Chinese herbal medicine (Teste instruments Co., tianjin).
1.2, reagent
Gallic acid (lot number: PS000688, purity. Gtoreq.98%), gallocatechin (lot number: PS 01955, purity. Gtoreq.98%), bergenin (lot number: PS000991, purity. Gtoreq.98%), baibiangjingsaponine A (lot number: PS011511, purity. Gtoreq.98%), baibiangjingsaponine B (lot number: PS011507, purity. Gtoreq.98%) controls were all purchased from Chengdu biosciences; acetonitrile is chromatographic purity; phosphoric acid (GR, the company MIEuro chemical reagent, inc., tianjin, inc.); dropen drinking water (AR, dropen food and beverage limited, guangzhou).
10 batches of Ardisia crenata medicinal materials were collected and identified as dried roots and rhizomes of Ardisia crenata Ardisia crenata Sims of the Philippinensis family of the Philippine, guizhou university of Chinese medicine, by the professor Wei sublimation.
2. Method and results
2.1 preparation of control solution
And respectively precisely weighing appropriate amounts of gallic acid, gallocatechin, bergenin, baijingsaponin A and baijingsaponin B as reference substances, adding 50% methanol for dissolving, and preparing into reference substance solutions containing 1.045mg/ml, 2.034mg/ml, 2.108mg/ml, 2.118mg/ml and 2.068mg/ml of gallic acid, gallocatechin, bergenin, baijingsaponin A and baijingsaponin B. And respectively precisely measuring appropriate amounts of the corresponding reference substance solutions, placing the reference substance solutions into a same 10mL volumetric flask, and fixing the volume by using 50% methanol to prepare mixed reference substance solutions with the concentrations of 3.135 mug/mL, 19.12 mug/mL, 0.4848mg/mL, 0.8472mg/mL and 0.7238mg/mL in sequence for later use.
2.2 preparation of sample solution
Weighing about 0.5g of radix Ardisiae Crenatae sample, precisely weighing, placing into a flat-bottom flask, precisely adding 25mL of methanol, weighing, heating and refluxing for 40min, cooling, supplementing the lost weight with methanol, shaking, and filtering with 0.22 μm microporous membrane.
2.3 chromatographic conditions
By means of Diamond-C 18 (250 mm. Times.4.6 mm,5 μm) column; the mobile phase is acetonitrile (A) -0.1% phosphoric acid aqueous solution (B), gradient elution (0-22 min,8% A, 22-30 min, 8-30% A, 30-40 min, 30-30% A, 40-60 min, 30-40% A, 60-65 min, 40-100% A); the flow rate is 1.0mL/min; column temperature is 30 ℃; the detection wavelength is 203nm; the sample injection amount was 10. Mu.L.
2.4 methodological investigation
2.4.1 linear relationship investigation
Respectively precisely sucking a proper amount of the mixed reference substance solution under the item "2.1", respectively placing the mixed reference substance solutions into a 5mL volumetric flask, adding 50% methanol to a fixed volume to scale, shaking uniformly to prepare a series of mixed reference substance solutions, carrying out sample injection measurement under the chromatographic condition of the item "2.1", taking the concentration of the reference substance solution as an abscissa and the peak area of a target peak as an ordinate, and calculating a regression equation, wherein the result is shown in Table 1.
Table 1: examination result of linear relation of 5 components in cinnabar root
| Composition of the components | Regression equation | R | Linear range (μg/mL) |
| Gallic acid | Y＝44.591X-2.7961 | 0.9998 | 0.2508～2.088 |
| Gallocatechin | Y＝30.088X+121.03 | 0.9990 | 1.530～12.73 |
| Bergenin | Y＝25.992X-69.812 | 0.9993 | 38.79～322.9 |
| Bailiang gold saponin A | Y＝0.2587X-4.1042 | 0.9991 | 67.78～762.5 |
| Bailiang gold saponin B | Y＝0.257X+2.0934 | 0.9995 | 57.90～482.1 |
2.4.2 precision test
Taking a proper amount of mixed reference substance solution, continuously sampling and measuring for 6 times according to the chromatographic condition under the item "2.3", recording peak areas, and calculating to obtain the RSD of the peak areas of gallic acid, gallocatechin, bergenin, baibianjin saponin A and baibianjin saponin B, wherein the RSD is respectively 0.42%, 2.11%, 0.53%, 2.60% and 2.14%, which indicates that the instrument precision is good.
2.4.3 stability test
Taking a test sample solution of the cinnabar root, and respectively carrying out sample injection at 0, 2, 4, 6, 8, 12, 24 and 36 hours after preparation to obtain gallic acid, gallocatechin, bergenin, baibiaojingsaponin A and baibiaojingsaponin B with RSD of peak areas of 2.66%, 2.57%, 2.50%, 2.73% and 1.67%, which indicates that the test sample solution has good stability in 36 hours.
2.4.4 repeatability test
Taking the same batch of test samples of the cinnabar roots, preparing 6 parts of test sample solutions in parallel according to the method under the item of 2.2, and carrying out sample injection measurement according to the chromatographic condition under the item of 2.1, so as to obtain the gallic acid, gallocatechin, bergenin, and the average content of the baibian saponin A and the baibian saponin B which are 38.94 mug/g, 249.76 mug/g, 15.02mg/g, 32.25mg/g and 24.05mg/g respectively, wherein RSD is 3.53%, 1.65%, 1.29%, 2.04% and 1.34% respectively, which indicates that the method has good repeatability.
2.4.5 sample recovery test
6 parts of test samples of cinnabar roots with known component content are precisely weighed, 0.25g of each test sample is precisely weighed, a proper amount of mixed reference substances are respectively added, test sample solutions are prepared according to the method under the item of 2.2, and sample injection measurement is carried out according to the chromatographic condition under the item of 2.3, so that the average sample injection recovery rates of gallic acid, gallocatechin, bergenin, baibian saponin A and baibian saponin B are respectively 100.10%, 102.38%, 103.42%, 97.82%, 99.50% and RSD are respectively 3.84%, 1.97%, 1.22%, 1.29% and 1.14%, and the method is proved to have good accuracy. The specific results are shown in Table 2 below.
Table 2: sample recovery rate test result of 5 components in cinnabar root
2.5 determination of correction factors
Taking mixed reference solution, respectively injecting 2, 4, 6, 8, and 10 μl under chromatographic conditions of "2.3", and calculating relative correction factors (f) of 4 components including gallic acid (a), gallocatechin (B), baibian saponin A (c), and baibian saponin B (d) with bergenin as internal reference substance(s) i/s) Taking the average value as f for quantification i/s [f i/s ＝f s /f i ＝A s C i /A i C s Wherein A is s Peak area, C, of bergenin control s A is the mass concentration of bergenin reference substance i For the peak area of the component to be measured, C i For the mass concentration of the component to be measured]. Calculating f of the components to be detected, namely gallic acid, gallocatechin, bailiang gold saponin A and bailiang gold saponin B i/s The results are shown in Table 3. The results show that the ratio (f) between gallic acid, gallocatechin, bailiangchin A, bailiangchin B and bergenin as an internal reference i/s) 0.4900, 0.4340, 84.8548, 98.3806, rsd values of 2.73%, 3.08%, 1.14%, 3.76%, respectively. The specific results are shown in Table 3.
Table 3: relative correction factor of 4 components in cinnabar root
| Sample volume/. Mu.L | f a/s | f b/s | f c/s | f d/s |
| 2 | 0.4825 | 0.4286 | 86.4276 | 103.6502 |
| 4 | 0.4810 | 0.4271 | 84.4292 | 100.1328 |
| 6 | 0.4839 | 0.4218 | 83.8495 | 94.0689 |
| 8 | 0.4893 | 0.4365 | 84.6330 | 96.1188 |
| 10 | 0.5133 | 0.4560 | 84.9345 | 97.9323 |
| Average value of | 0.4900 | 0.4340 | 84.8548 | 98.3806 |
| RSD/％ | 2.73 | 3.08 | 1.14 | 3.76 |
Note that: s is bergenin, and a-d are gallic acid, gallocatechin, and bailiangchin A and bailiangchin B respectively
2.6 durability and System applicability study
2.6.1 influence of instruments and columns on the relative correction factor
Agilent 1260 and Waters e2695 HPLC system and Diamond-C respectively 18 、Platisil-C 18 、Agilent5TC-C 18 (all specifications were 250 mm. Times.4.6 mm,5 μm) 3 columns, the effect on the relative correction factor was examined. The results show that different instruments and chromatographic columns have no obvious influence on the relative correction factors of the internal reference substances and the components to be detected, namely gallic acid, gallocatechin and bailiang of ginsenosides A (RSD)<5.00%) of the hundred-two gold saponins B has a slightly larger RSD value (RSD)<7.00%). The results are shown in Table 4.
Table 4: influence of different instruments and chromatographic columns on relative correction factors of 4 components in radix Ardisiae Crenatae
Note that: s is bergenin, and a-d are gallic acid, gallocatechin, and bailiangchin A and bailiangchin B respectively
2.6.2 influence of column temperature, flow Rate on relative correction factor
Under the same conditions, an Agilent 1260 high performance liquid chromatography system and a Diamond-C are adopted 18 The chromatographic column was examined for the effect of different column temperatures (25, 30, 35 ℃) and different flow rates (0.8, 1.0, 1.2 mL/min) on the relative correction factors, respectively. The results show its RSD<3.00% shows that the temperature and the flow rate of different columns have no obvious effect on the relative correction factor, and the method has better system tolerance. The results are shown in Table 5.
Table 5: influence of different column temperatures and flow rates on the relative correction factors of 4 components in cinnabar root
Note that: s is bergenin, and a-d are gallic acid, gallocatechin, and bailiangchin A and bailiangchin B respectively
2.6.3 positioning of chromatographic peaks of components to be measured
Using relative retention value method, using relative retention value (RRT) between internal reference and component to be detected as positioning standard, positioning each chromatographic peak, using bergenin as internal reference(s), calculating relative retention time (t) of 4 components of gallic acid (a), gallocatechin (B), baibian saponin A (c), baibian saponin B (d) i/s ) The relative retention times (t i/s ) The results show that the influence of different instruments and chromatographic columns on the relative retention values of internal reference substances and the components to be tested, namely gallic acid and gallocatechin, is smaller (RSD)<5.00 percent) can realize the accurate positioning of chromatographic peaks, but the RSD values of the bailiang gold saponin A and the bailiang gold saponin B are both more than 5.00 percent and reach more than 11 percent, so the positioning of the bailiang gold saponin A and the bailiang gold saponin B can only be performed by adopting qualitative reference substances, and the results are shown in Table 6.
Table 6: relative retention values of 4 components in cinnabar root under different instruments and chromatographic columns
Note that: s is bergenin, and a-d are gallic acid, gallocatechin, and bailiangchin A and bailiangchin B respectively
2.7 comparison of the measurement results of the one-test-multiple-evaluation method and the external standard method
The external standard method is to calculate the content of each component in the cinnabar root by using the control of each component, and the one-measurement multi-evaluation method is to calculate the relative correction factors between gallic acid, gallocatechin, baibiaojingsaponin A and baibiaojingsaponin B by using the relative correction factors between the gallic acid, gallocatechin, baibiaojingsaponin A and bergenin.
0.5g of sample powder is taken, 10 parts of sample powder is precisely weighed, a sample solution is prepared according to the method under the item "2.2", sample injection is carried out according to the chromatographic condition under the item "2.3", the peak area is recorded, and 5 component contents in 10 batch number cinnabar root samples are calculated by an External Standard Method (ESM) and a multi-evaluation method (QAMS) respectively, and the results are shown in Table 7.
Table 7: measurement of 5 ingredients in Cinnabaris root (mg/g)
As shown in the above table, there is no obvious difference between the External Standard Method (ESM) and the one-test multiple-evaluation method (QAMS) measurement results of the four components; in general, for both gallic acid and gallocatechin, the external standard method results are slightly higher than the one-test-multiple-evaluation method results, for example, each sample gallic acid external standard method result is 14.5% -15% higher than the one-test-multiple-evaluation method results, for example, the Z01 sample gallic acid external standard method results are 14.55% higher than the one-test-multiple-evaluation method results, each sample gallocatechin external standard method result is 9% -9.5% higher than the one-test-multiple-evaluation method results, for example, the Z01 sample gallocatechin external standard method results are 9.26% higher than the one-test-multiple-evaluation method results; however, for both of the baibian saponin a and the baibian saponin B, the one-test multiple-evaluation method result is slightly higher than the external standard method result, for example, the one-test multiple-evaluation method result of each sample baibian saponin a is 2% -2.2% higher than the external standard method result, for example, the one-test multiple-evaluation method result of the Z01 sample baibian saponin a is 2.06% higher than the external standard method result, the one-test multiple-evaluation method result of each sample baibian saponin B is 3.7% -3.8% higher than the external standard method result, for example, the one-test multiple-evaluation method result of the Z01 sample baibian saponin B is 3.73% higher than the external standard method result.
3. Discussion of the invention
3.1 selection of sample preparation conditions
The sample extraction amount (0.5 g, 0.8 g, 1.0 g and 1.2 g) is examined, the extraction method (ultrasonic and reflux) and the extraction time (40, 60, 80 and 100 min) are adopted, the detection components are more, the peak shape is good as an index, and finally the preparation method of the cinnabar root sample solution is determined to be 0.5g of the cinnabar root sample, 25mL of methanol is precisely added, the mixture is weighed, the extraction method is reflux extraction, the reflux time is 40min, the mixture is cooled, the methanol is supplemented, the mixture is shaken uniformly, filtered, and the subsequent filtrate is filtered through a microporous filter membrane of 0.22 mu m, so that the cinnabar root sample solution is obtained.
3.2 selection of chromatographic conditions
When the mobile phase is selected, acetonitrile-water and acetonitrile-0.1% phosphoric acid aqueous solution are respectively examined as a mobile phase system, the separation effect of 5 components measured in cinnabar root is used as an index, the acetonitrile-0.1% phosphoric acid aqueous solution of the mobile phase is finally determined, the separation conditions of chromatographic peaks by different column temperatures (25, 30 and 35 ℃) are examined, and the column temperature is finally selected to be 30 ℃ under the premise of ensuring the separation degree of each chromatographic peak.
3.3 selection of detection wavelength
In this example 1, the DAD detector was used to perform spectral scanning at 190-400 nm, and the detection wavelength was determined to be 203nm because the UV absorption response values of the bailiang of ginsenosides A and B were relatively low and belonging to terminal absorption.
3.4 selection of internal reference
The selected internal reference is the key for establishing a multi-evaluation method, and when the internal reference is selected, the components with large content, good stability, simple preparation and simple detection are generally selected. Bergenin is easy to obtain, low in price, stable in peak area and retention time, high in content in a test sample, and good in separation degree in an established chromatographic system, is positioned in the middle position of 5 components to be detected in the cinnabar root, and is beneficial to the stability of relative retention values in peak positioning, so that the bergenin is selected as an internal reference in the embodiment 1.
The embodiments described herein are only for illustrating the composition and the effect of the present invention and are not limited to the scope of the present invention, so that those skilled in the art can make similar modifications without departing from the structure of the present invention, and they are all included in the scope of the patent of the present invention. These should also be construed as protecting the invention, which does not affect the effect of the practice of the invention and the utility of the patent.
Claims (5)
1. The method for measuring the content of the cinnabar root medicine by one measurement and multiple evaluation comprises the following steps:
(1) Preparing a reference substance solution: when the reference substance solution is prepared, 50% methanol is used as a solvent to prepare the reference substance solution containing gallic acid reference substance, gallocatechin reference substance, bergenin reference substance, hundred-two-gold saponin A reference substance and hundred-two-gold saponin B reference substance, wherein the concentrations of gallic acid, gallocatechin, bergenin, hundred-two-gold saponin A and hundred-two-gold saponin B are respectively 2-4 mug/mL, 30-40 mug/mL, 0.3-0.7 mg/mL, 0.6-1.0 mg/mL and 0.5-1.0 mg/mL;
(2) Preparation of test solution: precisely weighing radix Ardisiae Crenatae sample 0.5g, precisely adding methanol 25mL, weighing, heating and refluxing for 40min, cooling, supplementing the reduced weight with methanol, shaking, and filtering with 0.22 μm microporous membrane;
(3) Establishing a high performance liquid chromatography for simultaneously measuring the contents of gallic acid, gallocatechin, bergenin, baibiao saponin A and baibiao saponin B, wherein the chromatographic conditions of the high performance liquid chromatography are as follows:
c18 column, column size 250mm x 4.6mm,5 μm;
mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid aqueous solution;
the gradient elution procedure was: 0-22 min,8% A; 22-30 min, 8-30% of A; 30-40 min, 30-30% of A; 40-60 min, 30-40% of A; 60-65 min, 40-100% of A;
the flow rate is 1.0mL/min;
column temperature is 30 ℃;
the detection wavelength is 203nm;
the sample injection amount is 10 mu L;
the theoretical plate number is not less than 5000 based on bergenin;
(4) Determining the relative correction factors between gallic acid, gallocatechin, baijingsaponin A and baijingsaponin B and the bergenin serving as an internal reference substance to be respectively in the range of 0.48-0.52, the range of 0.42-0.46, the range of 83.5-86.5 and the range of 94.0-104.0;
(5) And calculating bergenin content of the measured sample solution chromatogram, and calculating the contents of gallic acid, gallocatechin, baibiasaponin A and baibiasaponin B according to the relative correction factors.
2. The method according to claim 1, wherein in the step (1), when preparing the control solution, a control solution comprising gallic acid control, gallocatechin control, bergenin control, baibiasaponin a control and baibiasaponin B control is prepared by using 50% methanol as a solvent, wherein the concentrations of gallic acid, gallocatechin, bergenin, baibiasaponin a and baibiasaponin B are respectively 2.5 to 3.5 μg/mL, 32 to 38 μg/mL, 0.4 to 0.6mg/mL, 0.8 to 0.9mg/mL, and 0.6 to 0.8mg/mL.
3. The method according to claim 1, wherein in step (3), the C18 chromatographic column is a diamondsil-C18 column.
4. The method of claim 1 wherein in step (4) the relative correction factors between gallic acid, gallocatechin, baibian saponin a, baibian saponin B and the reference bergenin are 0.4900, 0.4340, 84.8548, 98.3806, respectively.
5. The method according to claim 1, further comprising performing methodology investigation on the high performance liquid chromatography used, including linear relationship investigation, precision investigation, repeatability investigation, stability investigation and sample recovery verification.
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Inventor after: Zhang Hai Inventor after: Wang Jueben Inventor after: Zhou Ying Inventor after: Beng Tingting Inventor after: Zhang Liyan Inventor after: Shi Hui Inventor after: Dong Xiu Inventor before: Zhang Hai Inventor before: Wang Jueben Inventor before: Zhou Ying Inventor before: Beng Tingting Inventor before: Zhang Liyan Inventor before: Zhou Shihui Inventor before: Dong Xiu |