CN111537630A - Method for establishing HPLC fingerprint spectrum and multi-index quantification of gynecological reconstruction pill - Google Patents
Method for establishing HPLC fingerprint spectrum and multi-index quantification of gynecological reconstruction pill Download PDFInfo
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Abstract
The invention discloses an HPLC fingerprint spectrum and multi-index quantitative establishment method for a gynecological reconstruction pill. The method establishes an HPLC fingerprint spectrum of the whole formula, calibrates 35 characteristic chromatographic peaks with good separation degree in a chromatogram, identifies 15 components, and establishes a multi-index method for 11 target components. The invention can rapidly and accurately identify the types and the quantities of main components in products of different manufacturers and different batches, and carry out quantitative analysis on multi-index components, thereby identifying the authenticity of the products.
Description
Technical Field
The invention relates to an HPLC fingerprint and multi-index quantitative establishment method, in particular to an HPLC fingerprint and multi-index quantitative establishment method for a gynecological reconstruction pill.
Background
The gynecological reconstruction pill is derived from an empirical formula of Mr. first Wang Xian (1895-1964) of four major medical doctors in Guizhou, comprises 42 traditional Chinese medicines such as codonopsis pilosula, white paeony root, rhizoma cyperi (stir-fried with vinegar), astragalus mongholicus, prepared rehmannia root, poria cocos, angelica sinensis (stir-fried with wine) and the like, and has the effects of tonifying liver and kidney, nourishing blood, regulating menstruation, warming uterus and relieving pain. Can be used for treating gynecological diseases such as irregular menstruation, long-term menstruation, dysmenorrhea, leukorrhagia, etc., and has been clinically applied for more than 80 years, with definite therapeutic effect. The preparation process of the gynecological reconstruction pill comprises the following steps: firstly, 8 Chinese herbal medicines of astragalus, eucommia, prepared rehmannia root, teasel root, large-leaved gentian, desertliving cistanche, twotooth achyranthes root and Chinese wolfberry root-bark are decocted by adding water to obtain filtrate, then the filtrate is concentrated, donkey-hide gelatin is melted by adding water and then is mixed with the prepared concentrated solution to be concentrated into clear paste, then the other 33 Chinese herbal medicine fine powder and the clear paste are mixed to be prepared into pills, and the pills are dried and coated to obtain the traditional Chinese medicine preparation, which is currently collected in the national Chinese patent medicine standard compilation-surgical gynecological booklet issued by the State drug administration in 2002.
The gynecological reconstruction pill is a large compound composed of 42 medicinal materials, comprises plant medicines, animal medicines and mineral medicines, has complex components, and reports on the existing standards and documents about the quality evaluation of the gynecological reconstruction pill, such as microscopic identification, thin-layer chromatography (TLC) identification, single-component content measurement and the like. In the national Chinese patent medicine standard, TLC method is adopted to identify nutgrass galingale rhizome, white paeony root, tangerine peel and baical skullcap root, special thin layer identification of tanshinone and danshen root, and high liquid chromatography (HPLC) method is adopted to determine the content of paeoniflorin. The chemical components of traditional Chinese medicines, particularly traditional Chinese medicine compound preparations are very complex, the internal quality of the traditional Chinese medicine compound preparations cannot be reflected by simply measuring the content of one or more compounds, the existing fingerprint spectrum and multi-index quantification become effective methods for evaluating the traditional Chinese medicines and the compound preparations thereof, the overall quality of the traditional Chinese medicines and the compound preparations thereof can be reflected, and the requirement of effectively controlling the quality stability of the traditional Chinese medicines is met. At present, Chinese patent (application number: 201711083060.4) discloses a detection method of gynecological reconstruction pills, which adopts HPLC to establish a determination method of the contents of paeoniflorin, baicalin and salvianolic acid B in a product, adopts different chromatographic conditions to detect 3 components, and has the defects of single detection of 3 components, complicated sample treatment and long detection period; the detection indexes are few, and the quality of the gynecological reconstruction pill cannot be comprehensively reflected. Chinese patent (application number: 201610304060.1) discloses a method for establishing a UPLC fingerprint of a gynecological reconstruction pill, wherein UPLC is adopted to establish the fingerprint of the gynecological reconstruction pill, 15 common peaks are determined, however, the components of the common peaks are not identified, quantitative analysis of index components is carried out, detection equipment is expensive, the common peaks are few, quantitative analysis of the index components is not carried out, and the quality of the gynecological reconstruction pill product cannot be comprehensively reflected. Xujian and the like establish a UPLC fingerprint of the gynecological reconstruction pill, mark 15 common peaks, and measure the contents of index components of hesperidin and baicalin, but the components are not determined, the quality of the gynecological reconstruction pill cannot be fully fed back due to too few multi-index components, an ultra-high performance liquid chromatograph (UPLC) is expensive and has higher requirements on chromatographic columns, and the UPLC is not popular in Chinese pharmaceutical enterprises and is not a main method for controlling the quality of traditional Chinese medicines.
Therefore, the method for establishing the HPLC fingerprint spectrum and multi-index quantification of the gynecological reconstruction pill can comprehensively embody the quality of the gynecological reconstruction pill only by carrying out multi-index quantitative analysis on a plurality of common peaks and identified components.
Disclosure of Invention
The invention aims to provide a method for establishing an HPLC fingerprint spectrum and multi-index quantification of a gynecological reconstruction pill. The invention can rapidly and accurately identify the types and the quantities of main components in products of different manufacturers and different batches, and carry out quantitative analysis on multi-index components, thereby identifying the authenticity of the products.
The technical scheme of the invention is as follows: an establishment method of HPLC fingerprint and multi-index quantification of a gynecological reconstruction pill comprises the following steps:
(1) formulation linearity of control solutions:
precisely weighing appropriate amount of each reference substance, respectively placing in 5mL volumetric flask, dissolving with methanol, diluting to scale, shaking, and making into reference substance solution containing gallic acid 1.10-1.40mg/mL, chlorogenic acid 0.30-0.55mg/mL, gentiopicroside 0.45-0.60mg/mL, hesperidin 0.45-0.65mg/mL, baicalin 1.00-1.25mg/mL, salvianolic acid B0.95-1.25mg/mL, wogonin 0.25-0.50mg/mL, baicalein 0.65-0.85mg/mL, wogonin 0.40-0.65mg/mL, senkyunolide A2.50-4.50mg/mL, and Z-ligustilide 1.85-2.10 mg/mL;
precisely absorbing a proper amount of each reference substance solution into a 10ml volumetric flask, adding methanol to a constant volume to a scale to obtain a mixed reference substance solution, absorbing a proper amount of the mixed reference substance solution, respectively placing the mixed reference substance solution into corresponding volumetric flasks, adding methanol to a constant volume, respectively diluting by different times to obtain 7 mixed reference substance solutions with different concentration levels, and filtering by using a 0.22 mu m microporous membrane; determining peak areas according to the following chromatographic conditions, and performing linear regression by taking the sample injection concentration X of each reference substance, mu g/mL as a horizontal coordinate and the corresponding compound peak area Y as a vertical coordinate;
(2) preparation of a test solution:
taking a proper amount of the gynecological reconstruction pill, grinding, precisely weighing about 3g, placing into a 50mL conical flask, precisely adding 15-30mL of 45-55% methanol, weighing, extracting for 30-90min in a heating reflux manner, extracting for 1 time, cooling, weighing again, complementing the reduced amount with 45-55% methanol, mixing uniformly, and filtering with a 0.22 μm microporous membrane to obtain the final product;
(3) chromatographic conditions are as follows:
a chromatographic column: cosmos il C18(3.0mm × 150mm, 2.6 μm), gradient elution with acetonitrile (A) -0.4% acetic acid (36%) water solution (B) at flow rate of 0.3-0.5mL/min, column temperature of 25 deg.C, detection wavelength of 280nm, and sample amount of 4 μ L;
(4) establishing a fingerprint spectrum:
preparing a test solution and a reference solution according to the method, injecting sample under the chromatographic condition, and recording chromatogram to obtain HPLC fingerprint of the gynecological reconstruction pill and HPLC chart of the mixed reference solution;
(5) confirming the fingerprint spectrum:
preparing a plurality of batches of gynecological reconstruction pill test sample solutions according to the test sample solution preparation method, and determining according to the chromatographic conditions; 35 common peaks are determined through analysis and comparison, the common peaks form a fingerprint of the gynecological reconstruction pill, and 15 components are identified in the fingerprint;
(6) multi-index component quantification:
preparing 12 batches of gynecological reconstruction pill finished products according to the preparation method of the test solution, detecting according to the chromatographic conditions, measuring each sample twice in parallel, recording peak areas, calculating the content according to a standard curve, calculating the average content of 11 compounds in the 12 batches of samples respectively, and comparing the content percentage of each component in the samples.
In the aforementioned method for establishing HPLC fingerprint and multi-index quantification of gynecological reconstruction pill, in step (1), the reference solution is prepared as follows: accurately weighing appropriate amount of each reference substance, respectively placing in 5mL volumetric flask, adding methanol to dissolve, diluting to scale, shaking, and making into reference substance solution of gallic acid 1.270mg/mL, chlorogenic acid 0.410mg/mL, gentiopicrin 0.502mg/mL, hesperidin 0.5600mg/mL, baicalin 1.1133mg/mL, salvianolic acid B1.055mg/mL, wogonin 0.383mg/mL, baicalein 0.770mg/mL, wogonin 0.5133mg/mL, senkyunolide A3.573 mg/mL, and Z-ligustilide 1.998 mg/mL.
In the aforementioned method for establishing HPLC fingerprint and multi-index quantification of gynecological reconstruction pill, in step (2), the sample solution is prepared as follows: taking a proper amount of the gynecological reconstruction pill, grinding, precisely weighing about 3g, placing into a 50mL conical flask, precisely adding 20mL of 50% methanol, weighing, extracting for 60min in a heating reflux manner, extracting for 1 time, cooling, weighing again, complementing the reduced amount with 50% methanol, mixing uniformly, filtering with a 0.22 μm microporous membrane before sample injection, and taking the subsequent filtrate for HPLC detection and analysis.
In the aforementioned method for establishing HPLC fingerprints and multi-index quantification of the gynaecological reforger pill, in the step (3), the gradient elution conditions are as follows: 0-12.5min, 2-16% of phase A, 12.5-19min, 16% of phase A, 24-30min, 22-30% of phase A, 30-33min, 30-32% of phase A, 33-36min, 32-50% of phase A, 36-47min, 50-60% of phase A, 47-49.5min, 60-83% of phase A, 49.5-52min, 83-95% of phase A, 52-55min and 95% of phase A.
In the aforementioned method for establishing HPLC fingerprint and multi-index quantification of gynecological reconstruction pill, in the step (3), the flow rate is 0.4 mL/min.
In the aforementioned method for establishing HPLC fingerprint and multi-index quantification of gynecological reconstruction pill, in step (5), 15 ingredients are identified as follows: gallic acid, chlorogenic acid, gentiopicrin, albiflorin, ferulic acid, hesperidin, baicalin, danshenic acid B, wogonoside, baicalein, wogonin, senkyunolide A, butylphthalide, Z-ligustilide and atractylenolide II.
In the aforementioned method for establishing HPLC fingerprints and multi-index quantification of the gynecological reconstruction pill, in the step (6), the multi-index quantification comprises the following components: gallic acid, chlorogenic acid, gentiopicrin, hesperidin, baicalin, danshenic acid B, wogonin, baicalein, wogonin, senkyunolide A and Z-ligustilide.
Compared with the prior art, the invention has the following beneficial effects:
the invention establishes an all-around HPLC fingerprint, calibrates 35 characteristic chromatographic peaks with good separation degree in a chromatogram, identifies 15 components, and establishes a multi-index method for 11 target components. The method can quickly and accurately identify the types and the quantities of main components in products of different manufacturers and different batches, and quantitatively analyze the multi-index components, so as to identify the authenticity of the product, has the advantages of simplicity, high efficiency, accuracy, reliability, good repeatability and the like, and can be used for quality control of gynecological reconstruction pills of enterprises and identification of the quality of the products of different manufacturers. Solves the problems that the prior art has expensive detection equipment, fewer common peaks of fingerprint spectra, less multi-index quantitative components, and can not comprehensively reflect the types and the quantities of the components contained in the gynecological reconstruction pill.
The inventors conducted a number of experiments, and the following are partial experimental studies
Example 1
1. Apparatus and materials
1.1 Experimental instruments
ULtiMate model 3000 high performance liquid chromatography (DAD-3000 uv detector) (seimer feishell science); electronic analytical balance (model: XS205 DuaLRange; manufacturer: Mettler-Torledo instruments, Inc.); an ultrasonic cleaning machine (model: HN 1006; manufacturer: southern China ultrasonic equipment, Inc., Guangzhou); constant temperature water bath (Guangzhou Kangheng instruments Co., Ltd.).
1.2 materials of the experiment
Gynecological reconstruction pill (available from Deshang Xiang pharmaceutical factory of pharmaceutical industry Co., Ltd., Han prescription, Guizhou, under the trade designation of 11717013(S1), 11717015(S2), 11717016(S3), 11717019(S4), 11717021(S5), 11717022(S6), 11717023(S7), 11717026(S8), 11717033(S9), 11717034(S10), 11717035(S11), 11717040 (S12).
Acetonitrile (chromatographically pure, Merck, germany); 36% acetic acid, methanol (analytical grade, Guangzhou chemical Co., Ltd.); purified water (Shenzhen Wawa Harha Rong Tai Shi Co., Ltd.).
2. Method and results
2.1 chromatographic conditions
A chromatographic column: cosmos il C18(3.0mm × 150mm, 2.6 μm), a mobile phase of acetonitrile (A) -0.4% acetic acid (36%) aqueous solution (B) is subjected to gradient elution with the flow rate of 0.3-0.5mL/min, the column temperature of 25 ℃, the detection wavelength of 280nm and the sample injection amount of 4 μ L, the gradient elution conditions are 0-12.5min, 2-16% of A phase, 12.5-19min, 16% of A phase, 24-30min, 22-30% of A phase, 30-33min, 30-32% of A phase, 33-36min, 32-50% of A phase, 36-47min, 50-60% of A phase, 47-49.5min, 60-83% of A phase, 49.5-52min, 83-95% of A phase, 52-55min and 95% of A phase.
2.2 preparation of control solutions
Accurately weighing appropriate amount of each reference substance, respectively placing in 5mL volumetric flask, adding methanol to dissolve, diluting to scale, shaking, and making into reference substance solution of gallic acid 1.270mg/mL, chlorogenic acid 0.410mg/mL, gentiopicrin 0.502mg/mL, hesperidin 0.5600mg/mL, baicalin 1.1133mg/mL, salvianolic acid B1.055mg/mL, wogonin 0.383mg/mL, baicalein 0.770mg/mL, wogonin 0.5133mg/mL, senkyunolide A3.573 mg/mL, and Z-ligustilide 1.998 mg/mL.
2.3 preparation of test solutions
Taking a proper amount of the gynecological reconstruction pill, grinding, precisely weighing about 3g, placing into a 50mL conical flask, precisely adding 20mL of 50% methanol, weighing, extracting in a heating reflux manner for 60min, extracting for 1 time, cooling, weighing again, complementing the reduced amount with 50% methanol, mixing uniformly, and filtering with a 0.22 μm microporous membrane before sample injection to obtain the final product.
2.4 Linear relationship
Precisely sucking a proper amount of each reference substance solution into a 10ml volumetric flask, adding methanol to a constant volume to reach a scale, and obtaining a mixed reference substance solution. Sucking appropriate amount of mixed reference solution, placing in corresponding volumetric flasks respectively, adding methanol to desired volume, diluting with different times to obtain 7 mixed reference solutions with different concentration levels, and filtering with 0.22 μm microporous membrane. The peak area was determined according to the chromatographic conditions, and linear regression was performed with the sample concentration (X,. mu.g/mL) of each control as abscissa and the corresponding compound peak area (Y) as ordinate. See table 1-11 chemical composition standard curve determination results, table 2-11 compounds linear relationship, detection limit and quantitative limit investigation; FIG. 1 is an HPLC chromatogram of a mixed standard for gynecological rebeaming; FIG. 2 is an HPLC chromatogram of a sample of the gynecological reconstruction pill.
TABLE 111 Standard Curve measurements of chemical Components
Remarking: a represents the peak area (mAU. min); c represents concentration (. mu.g/mL);
linear relationship, detection Limit and quantitation Limit Studies of the 211 Compounds in Table
2.5 HPLC fingerprint determination of gynecological reconstruction pill
2.5.1 methodology investigation
2.5.1.1 precision test
Sampling the same gynecological reconstruction pill sample solution (batch number: 11717035) for 6 times according to the chromatographic conditions, taking the 22 th peak as a reference peak, and calculating to obtain the relative peak area RSD of each common peak between 0.35% and 2.15% and the relative retention time RSD between 0.01% and 0.27%, wherein the result shows that the precision of the instrument is good. See Table 3 for precision (common peak relative retention time), Table 4 for precision (common peak relative peak area).
TABLE 3 results of precision tests (common peak relative retention time)
TABLE 4 precision test results (common peak relative peak area)
2.5.1.2 repeatability test
Weighing the same batch of gynecological reconstruction pills (batch number: 11717035), preparing 6 parts of test sample solution in parallel according to the test sample preparation method, detecting according to the chromatographic conditions, taking the No. 22 peak as a reference peak, and calculating to obtain the relative peak area RSD of each common peak between 0.63% and 2.79% and the relative retention time RSD between 0.00% and 0.30%, which indicates that the method has good repeatability. See table 5 for reproducibility test results (common peak relative retention time), table 6 for reproducibility test results (common peak relative peak area).
TABLE 5 repeatability test results (common peak relative retention time)
TABLE 6 repeatability test results (common peak relative peak area)
2.5.1.2 stability test
Taking the same batch of gynecological reconstruction pill test sample solution (batch number: 11717035), determining at 0h, 2h, 4h, 6h, 8h, 12h and 24h according to the chromatographic conditions, recording the peak area and retention time of each chromatographic peak, calculating the relative peak area and the relative retention time of each common peak by taking the No. 22 peak as a reference peak, wherein the RSD of the relative peak area is 0.85-2.90%, and the RSD of the relative retention time is 0.00-0.30%, which indicates that the test sample solution is stable within 24 h. See table 7 stability test results (common peak relative retention time), table 8 stability test results (common peak relative peak area).
TABLE 7 stability test results (common Peak relative Retention time)
TABLE 8 stability test results (common peak relative peak area)
2.5.2 establishment of HPLC fingerprint of gynecological reconstruction pill
Preparing 12 batches (serial numbers S1-S12) of gynecological reconstruction pill test sample solutions according to the test sample solution preparation method, measuring according to the chromatographic conditions, and recording chromatograms. The fingerprint of 12 batches of gynecological reconstruction pills measured by HPLC is used as the original data, a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2004A edition) is adopted for data generation, and the comparison fingerprint is generated by using the principle of average number fitting. And calculating the similarity of the fingerprints of each sample respectively to be 0.991, 0.998, 0.996, 0.997, 0.999, 0.995, 0.996, 0.998, 0.996 and 0.997 by taking the comparison fingerprint as a reference.
The tests show that the determination method is stable and reliable, the fingerprint spectrum is clear and stable, and the quality control of the gynecological reconstruction pill can be used. See figure 3 gynaecological reconstruction pill HPLC fingerprint and common peak, figure 4 gynaecological reconstruction pill contrast fingerprint and figure 5 gynaecological reconstruction pill 12 batches of sample fingerprints.
2.6 determination of multiple index components of gynecological reconstruction pill
2.6.1 methodology investigation
2.6.1.1 precision test
And taking the same mixed reference substance solution, carrying out parallel sample injection for 6 times according to the chromatographic conditions, and calculating to obtain the peak area RSD of each compound to be 1.13-1.99%, wherein the result shows that the precision of the instrument is good. See table 9 for precision (n-6).
Table 9 precision test results (n ═ 6)
2.6.1.2 repeatability test
Taking a sample of lot number 11717035, preparing 6 parts of a sample according to the sample preparation method, injecting samples according to the chromatographic conditions, and calculating the RSD of the content of each compound to be between 0.63% and 2.63%, thereby indicating that the method has good repeatability. See table 10 for the results of the repeatability tests (n ═ 6).
Table 10 repeatability test results (n ═ 6)
2.6.1.3 stability test
Taking a sample of lot number 11717035, preparing 1 part of the sample according to the sample preparation method, respectively carrying out sample injection analysis for 0h, 2h, 4h, 6h, 8h, 12h and 24h according to the chromatographic conditions, and calculating the peak area RSD of each component to be between 0.31% and 2.98%, thereby indicating that the sample solution is stable within 24 h. See table 11 for stability test results.
TABLE 11 stability test results
2.6.1.4 sample recovery test
Calculating the formula:
taking 6 parts of finished gynecological reconstruction pill products (batch number: 11717035) with known content, adding about 1.5g of each part of the finished gynecological reconstruction pill products into each compound mixed reference solution, preparing a test solution according to a test method, detecting according to the chromatographic conditions, and carrying out sample injection analysis. The calculation shows that the average sample adding recovery rate of each compound is in the range of 95.58-104.63%, and the RSD is between 1.30-2.80%, which indicates that the method has good accuracy and meets the requirement of measuring the content of the index component. See table 12 for the recovery rate test results of gynecological reconstruction pill (n ═ 6).
TABLE 12 recovery test results of gynecological reconstruction pill (n ═ 6)
2.6.2 Multi-index component quantitation
Taking 12 batches of gynecological reconstruction pill finished products, respectively preparing the finished products according to the preparation method of the test solution, detecting according to the chromatographic conditions, measuring each sample twice in parallel, recording peak areas, calculating the content according to a standard curve, and obtaining specific results shown in the following table 13 and fig. 6, wherein the content difference of each component among the batches is small. And then respectively calculating the average content of 11 compounds in 12 batches of samples, and comparing the content percentage of each component in the samples, wherein the result shows that the content of baicalin is highest, and next, the contents of gallic acid, gentiopicrin, wogonin and Z-ligustilide are lower, and the contents of ferulic acid and wogonin are lower. See table 13 for the content determination of the samples and fig. 6 for the comparison of the average content of 11 compounds in the samples of 12 batches of the gynecological reconstruction pill.
TABLE 13 determination of sample content
In conclusion, the invention can rapidly and accurately identify the types and the quantities of the main components in the products of different manufacturers and different batches, and quantitatively analyze the multi-index components, thereby identifying the authenticity of the products, has the advantages of simplicity, high efficiency, accuracy, reliability and good repeatability, and can be used for the quality control of the gynecological reconstruction pills of enterprises and the beneficial effects of identifying the quality of the products of different manufacturers.
Drawings
FIG. 1 is a HPLC chromatogram of a mixed standard substance of the gynecological reconstruction pill in a linear relationship according to the present invention;
FIG. 2 is the HPLC chromatogram of the test sample of the gynecological reconstruction pill in the linear relationship of the present invention;
FIG. 3 is the HPLC fingerprint and common peak of the gynecological reconstruction pill during the establishment of the HPLC fingerprint of the gynecological reconstruction pill of the present invention;
FIG. 4 is a comparison fingerprint of the gynecological reconstruction pill during the establishment of the HPLC fingerprint of the gynecological reconstruction pill of the present invention;
FIG. 5 is fingerprint of 12 batches of samples of the gynecological reconstruction pill in the establishment of the HPLC fingerprint of the gynecological reconstruction pill of the present invention;
FIG. 6 is a graph comparing the average content of 11 compounds in 12 batches of samples of the gynecological reconstruction pill in the multi-index component quantification of the present invention.
Detailed Description
The invention is further illustrated by the following figures and examples, which are not to be construed as limiting the invention.
Examples are given. An establishment method of HPLC fingerprint and multi-index quantification of a gynecological reconstruction pill comprises the following steps:
(1) formulation linearity of control solutions:
precisely weighing appropriate amount of each reference substance, respectively placing in 5mL volumetric flask, dissolving with methanol, diluting to scale, shaking, and making into reference substance solution containing gallic acid 1.10-1.40mg/mL, chlorogenic acid 0.30-0.55mg/mL, gentiopicroside 0.45-0.60mg/mL, hesperidin 0.45-0.65mg/mL, baicalin 1.00-1.25mg/mL, salvianolic acid B0.95-1.25mg/mL, wogonin 0.25-0.50mg/mL, baicalein 0.65-0.85mg/mL, wogonin 0.40-0.65mg/mL, senkyunolide A2.50-4.50mg/mL, and Z-ligustilide 1.85-2.10 mg/mL;
precisely absorbing a proper amount of each reference substance solution into a 10ml volumetric flask, adding methanol to a constant volume to a scale to obtain a mixed reference substance solution, absorbing a proper amount of the mixed reference substance solution, respectively placing the mixed reference substance solution into corresponding volumetric flasks, adding methanol to a constant volume, respectively diluting by different times to obtain 7 mixed reference substance solutions with different concentration levels, and filtering by using a 0.22 mu m microporous membrane; determining peak areas according to the following chromatographic conditions, and performing linear regression by taking the sample injection concentration X of each reference substance, mu g/mL as a horizontal coordinate and the corresponding compound peak area Y as a vertical coordinate;
(2) preparation of a test solution:
taking a proper amount of the gynecological reconstruction pill, grinding, precisely weighing about 3g, placing into a 50mL conical flask, precisely adding 15-30mL of 45-55% methanol, weighing, extracting for 30-90min in a heating reflux manner, extracting for 1 time, cooling, weighing again, complementing the reduced amount with 45-55% methanol, mixing uniformly, and filtering with a 0.22 μm microporous membrane to obtain the final product;
(3) chromatographic conditions are as follows:
a chromatographic column: cosmos il C18(3.0mm × 150mm, 2.6 μm), a mobile phase of acetonitrile (A) -0.4% acetic acid (36%) aqueous solution (B) is subjected to gradient elution with the flow rate of 0.3-0.5mL/min, the column temperature of 25 ℃, the detection wavelength of 280nm and the sample injection amount of 4 μ L, wherein the gradient elution conditions are 0-12.5min, 2-16% of A phase, 12.5-19min, 16% of A phase, 24-30min, 22-30% of A phase, 30-33min, 30-32% of A phase, 33-36min, 32-50% of A phase, 36-47min, 50-60% of A phase, 47-49.5min, 60-83% of A phase, 49.5-52min, 83-95% of A phase, 52-55min and 95% of A phase;
(4) establishing a fingerprint spectrum:
preparing a test solution and a reference solution according to the method, injecting sample under the chromatographic condition, and recording chromatogram to obtain HPLC fingerprint of the gynecological reconstruction pill and HPLC chart of the mixed reference solution;
(5) confirming the fingerprint spectrum:
preparing a plurality of batches of gynecological reconstruction pill test sample solutions according to the test sample solution preparation method, and determining according to the chromatographic conditions; 35 common peaks are determined through analysis and comparison, the common peaks form a fingerprint of the gynecological reconstruction pill, and 15 components are identified in the fingerprint; the 15 components are assigned to be: gallic acid, chlorogenic acid, gentiopicrin, albiflorin, ferulic acid, hesperidin, baicalin, danshenic acid B, wogonoside, baicalein, wogonin, senkyunolide A, butylphthalide, Z-ligustilide and atractylenolide II;
(6) multi-index component quantification:
preparing 12 batches of gynecological reconstruction pill finished products according to the preparation method of the test sample solution, detecting according to the chromatographic conditions, measuring each sample twice in parallel, recording peak areas, calculating the content according to a standard curve, calculating the average content of 11 compounds in the 12 batches of samples respectively, and comparing the content percentage of each component in the samples; the multi-index quantitative composition is: gallic acid, chlorogenic acid, gentiopicrin, hesperidin, baicalin, danshenic acid B, wogonin, baicalein, wogonin, senkyunolide A, and Z-ligustilide.
Claims (7)
1. An establishment method of HPLC fingerprint and multi-index quantification of a gynecological reconstruction pill is characterized by comprising the following steps: the method comprises the following steps:
(1) formulation linearity of control solutions:
precisely weighing appropriate amount of each reference substance, respectively placing in 5mL volumetric flask, dissolving with methanol, diluting to scale, shaking, and making into reference substance solution containing gallic acid 1.10-1.40mg/mL, chlorogenic acid 0.30-0.55mg/mL, gentiopicroside 0.45-0.60mg/mL, hesperidin 0.45-0.65mg/mL, baicalin 1.00-1.25mg/mL, salvianolic acid B0.95-1.25mg/mL, wogonin 0.25-0.50mg/mL, baicalein 0.65-0.85mg/mL, wogonin 0.40-0.65mg/mL, senkyunolide A2.50-4.50mg/mL, and Z-ligustilide 1.85-2.10 mg/mL;
precisely absorbing a proper amount of each reference substance solution into a 10ml volumetric flask, adding methanol to a constant volume to a scale to obtain a mixed reference substance solution, absorbing a proper amount of the mixed reference substance solution, respectively placing the mixed reference substance solution into corresponding volumetric flasks, adding methanol to a constant volume, respectively diluting by different times to obtain 7 mixed reference substance solutions with different concentration levels, and filtering by using a 0.22 mu m microporous membrane; determining peak areas according to the following chromatographic conditions, and performing linear regression by taking the sample injection concentration X of each reference substance, mu g/mL as a horizontal coordinate and the corresponding compound peak area Y as a vertical coordinate;
(2) preparation of a test solution:
taking a proper amount of the gynecological reconstruction pill, grinding, precisely weighing about 3g, placing into a 50mL conical flask, precisely adding 15-30mL of 45-55% methanol, weighing, extracting for 30-90min in a heating reflux manner, extracting for 1 time, cooling, weighing again, complementing the reduced amount with 45-55% methanol, mixing uniformly, and filtering with a 0.22 μm microporous membrane to obtain the final product;
(3) chromatographic conditions are as follows:
a chromatographic column: cosmos il C183.0mm × 150mm, 2.6 μm, gradient elution with acetonitrile (A) -0.4% acetic acid (36%) water solution (B) at flow rate of 0.3-0.5mL/min, column temperature of 25 deg.C, detection wavelength of 280nm, and sample injection amount of 4 μ L;
(4) establishing a fingerprint spectrum:
preparing a test solution and a reference solution according to the method, injecting sample under the chromatographic condition, and recording chromatogram to obtain HPLC fingerprint of the gynecological reconstruction pill and HPLC chart of the mixed reference solution;
(5) confirming the fingerprint spectrum:
preparing a plurality of batches of gynecological reconstruction pill test sample solutions according to the test sample solution preparation method, and determining according to the chromatographic conditions; 35 common peaks are determined through analysis and comparison, the common peaks form a fingerprint of the gynecological reconstruction pill, and 15 components are identified in the fingerprint;
(6) multi-index component quantification:
preparing 12 batches of gynecological reconstruction pill finished products according to the preparation method of the test solution, detecting according to the chromatographic conditions, measuring each sample twice in parallel, recording peak areas, calculating the content according to a standard curve, calculating the average content of 11 compounds in the 12 batches of samples respectively, and comparing the content percentage of each component in the samples.
2. The method for establishing the HPLC fingerprint and multi-index quantification of the gynecological reconstruction pill according to claim 1, which is characterized in that: in the step (1), the reference solution is prepared by: accurately weighing appropriate amount of each reference substance, respectively placing in 5mL volumetric flask, adding methanol to dissolve, diluting to scale, shaking, and making into reference substance solution of gallic acid 1.270mg/mL, chlorogenic acid 0.410mg/mL, gentiopicrin 0.502mg/mL, hesperidin 0.5600mg/mL, baicalin 1.1133mg/mL, salvianolic acid B1.055mg/mL, wogonin 0.383mg/mL, baicalein 0.770mg/mL, wogonin 0.5133mg/mL, senkyunolide A3.573 mg/mL, and Z-ligustilide 1.998 mg/mL.
3. The method for establishing the HPLC fingerprint and multi-index quantification of the gynecological reconstruction pill according to claim 1, which is characterized in that: in the step (2), the sample solution is prepared by: taking a proper amount of the gynecological reconstruction pill, grinding, precisely weighing about 3g, placing into a 50mL conical flask, precisely adding 20mL of 50% methanol, weighing, extracting for 60min in a heating reflux manner, extracting for 1 time, cooling, weighing again, complementing the reduced amount with 50% methanol, mixing uniformly, filtering with a 0.22 μm microporous membrane before sample injection, and taking the subsequent filtrate for HPLC detection and analysis.
4. The method for establishing the HPLC fingerprint and multi-index quantification of the gynecological reconstruction pill according to claim 1, which is characterized in that: in the step (3), the gradient elution conditions are as follows: 0-12.5min, 2-16% of phase A, 12.5-19min, 16% of phase A, 24-30min, 22-30% of phase A, 30-33min, 30-32% of phase A, 33-36min, 32-50% of phase A, 36-47min, 50-60% of phase A, 47-49.5min, 60-83% of phase A, 49.5-52min, 83-95% of phase A, 52-55min and 95% of phase A.
5. The method for establishing the HPLC fingerprint and multi-index quantification of the gynecological reconstruction pill according to claim 1, which is characterized in that: in the step (3), the flow rate is 0.4 mL/min.
6. The method for establishing the HPLC fingerprint and multi-index quantification of the gynecological reconstruction pill according to claim 1, which is characterized in that: in the step (5), 15 components are identified as follows: gallic acid, chlorogenic acid, gentiopicrin, albiflorin, ferulic acid, hesperidin, baicalin, danshenic acid B, wogonoside, baicalein, wogonin, senkyunolide A, butylphthalide, Z-ligustilide and atractylenolide II.
7. The method for establishing the HPLC fingerprint and multi-index quantification of the gynecological reconstruction pill according to claim 1, which is characterized in that: in the step (6), the multi-index quantitative components are as follows: gallic acid, chlorogenic acid, gentiopicrin, hesperidin, baicalin, danshenic acid B, wogonin, baicalein, wogonin, senkyunolide A and Z-ligustilide.
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