CN109596765B - Detection method of Liangfu dripping pills - Google Patents
Detection method of Liangfu dripping pills Download PDFInfo
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Abstract
The invention discloses a detection method of galangin dripping pills, which are prepared from galangin and rhizoma cyperi, and comprises a detection method for the contents of galangin, rhamnitrin, galangin-3-O-methyl ether, cypermethene, nootkatone and alpha-cypermethene in the galangin dripping pills and a fingerprint detection method for the galangin dripping pills, wherein the contents of the galangin, the rhamnitrin, the galangin-3-O-methyl ether, the cypermethene, the nootkatone and the alpha-cypermethene can be detected simultaneously. The quantitative analysis method can simultaneously determine the content of the main component in 6 of the galangal rhizome and aconite root, the qualitative detection in the invention can detect the specific chemical components in the galangal rhizome and the aconite root, the invention can effectively control the product quality, and can meet the stable and controllable requirements of the quality control of the galangal rhizome and aconite root.
Description
Technical Field
The invention relates to the technical field of quality control of Liangfu dripping pills, in particular to a detection method of Liangfu dripping pills.
Background
The Liangfu dripping pill is a Chinese patent medicine produced by Guizhou Huangguoshuang medicine industry Limited company and consists of two Chinese medicines of galangal and nutgrass galingale rhizome. The prescription of Liangfu dripping pill is a famous traditional Chinese medicine prescription, and is first recorded in Liangfu Jiaxilla, which is a famous prescription of Xiyuan Qing physician, and is mainly used for treating acute and chronic gastritis, gastric ulcer, gastrospasm and the like with the symptoms of congealing cold, qi stagnation, abdominal distension, pain, acid regurgitation and the like. The galangal mainly contains volatile oil, flavonoids, diaryl heptanes, phenylpropanoids, glycosides, sterols and the like, wherein the flavonoids have the effects of resisting digestive tract ulcer, relieving gastrointestinal spasm and preventing vomiting, and also have the effects of relieving pain and inflammation, resisting thrombosis, resisting oxidation and preventing chemically. The rhizoma cyperi mainly contains volatile oil, including various compounds such as monoterpenes, sesquiterpenes and oxides thereof, flavonoids, alkaloids, saccharides and triterpenes, and the like, and the complex chemical components enable the rhizoma cyperi to have wide pharmacological activity and medicinal value.
The existing detection method of the galangin dripping pill is simple, the quantitative analysis method can only determine the content of the galangin, the galangin dripping pill has complex components, the product quality is difficult to effectively control only by detecting a single index component, and the stable and controllable requirements of the quality control of the galangin dripping pill cannot be met; the qualitative detection method can only detect the existence of galangal and rhizoma cyperi in the galangal and rhizoma cyperi dropping pills, but cannot detect specific chemical components in the galangal and the rhizoma cyperi.
Therefore, the existing detection method of the galangal dripping pill can only determine the content of galangin serving as a single index component by a quantitative analysis method, cannot detect specific chemical components in galangal and rhizoma cyperi by qualitative detection, is difficult to effectively control the product quality by the existing detection method of the galangal dripping pill, and cannot meet the requirements of stability and controllability of quality control of the galangal dripping pill.
Disclosure of Invention
The invention aims to provide a detection method of Liangfu dripping pills. The quantitative analysis method can simultaneously determine the content of the main component in 6 of the galangal rhizome and aconite root, the qualitative detection in the invention can detect the specific chemical components in the galangal rhizome and the aconite root, the invention can effectively control the product quality, and can meet the stable and controllable requirements of the quality control of the galangal rhizome and aconite root.
The technical scheme of the invention is as follows: a detection method of rhizoma Alpiniae Officinarum dripping pill comprises rhizoma Alpiniae Officinarum and rhizoma Cyperi, wherein the rhizoma Alpiniae Officinarum dripping pill comprises galangin, rhamnitrin, galangin-3-O-methyl ether, cyperenone, nootkatone and alpha-cyperone,
preparation of mixed control solution: weighing galangin reference substance, rhamnan reference substance, galangin-3-O-methyl ether reference substance, cyperenone reference substance, nootkatone reference substance and alpha-cyperone reference substance, adding methanol to obtain mixed reference substance solution, and calculating the concentration of each reference substance solution;
preparation of a test solution: weighing Liangfu dripping pill, placing into conical bottle with plug, adding methanol or ethanol or methanol/ethanol solution, weighing, quantifying, ultrasonic treating, taking out, cooling, adding methanol or ethanol or methanol/ethanol solution to complement the weight loss, filtering, and collecting filtrate to obtain test solution;
content determination chromatographic conditions: a high performance liquid chromatograph; a PFP chromatography column; the mobile phase is acetonitrile A-water B or acetonitrile A-phosphoric acid aqueous solution B or methanol A-water B or methanol A-phosphoric acid aqueous solution B; the column temperature is 35-45 ℃; the flow rate is 0.6-1.4 ml/min-1(ii) a The detection wavelength is 220-280 nm;
content determination: respectively sucking the mixed reference solution and the test solution according to content determination chromatographic conditions, injecting into a high performance liquid chromatograph, and determining the content of galangin, rhamnocitrin, galangin-3-O-methyl ether, cyperenone, nootkatone and alpha-cyperone.
In the detection method of the galangal dripping pills, the mixed reference solution is prepared by weighing 10-15mg of galangin reference, 5-10mg of rhamnan citrate reference, 5-10mg of galangin-3-O-methyl ether reference, 5-10mg of cyperenone reference, 3-6mg of nocardone reference and 3-6mg of alpha-cyperone reference, adding methanol to prepare 20ml of mixed reference solution, and calculating the concentration of each reference solution.
In the detection method of Liangfu dripping pills, the test solution is prepared by weighing Liangfu dripping pills 0.5-1.5g, placing in a conical flask with a plug, adding methanol to prepare 5% Liangfu dripping pill solution, weighing, performing ultrasonic treatment for 30min, taking out, cooling, supplementing the weight loss with methanol, filtering, and collecting the filtrate to obtain the test solution.
In the detection method of the galangal dripping pill, the chromatographic condition for content determination is a high performance liquid chromatograph; the chromatographic column is Luna PFP2100A, 250X 4.60mm, 5 μm; the mobile phase is acetonitrile A-0.1 percent phosphoric acid water solution B, 40: 60; the column temperature was 40 ℃; the flow rate was 1.000 ml/min-1(ii) a The detection wavelength is 254 nm; the amount of the sample was 10. mu.L.
The detection method of the galangal rhizome dripping pill also comprises a fingerprint spectrum measuring method of the galangal rhizome dripping pill, the fingerprint spectrum measuring method of the galangal rhizome dripping pill comprises the following steps,
preparation of a test solution: weighing Liangfu dripping pill, placing into conical bottle with plug, adding methanol or ethanol or methanol/ethanol solution, weighing, quantifying, ultrasonic treating, taking out, cooling, adding methanol or ethanol or methanol/ethanol solution to complement the weight loss, filtering, and collecting filtrate to obtain test solution;
fingerprint chromatogram conditions: a high performance liquid chromatograph; a PFP chromatography column; mobile phase acetonitrile A-water B or acetonitrile A-phosphoric acid water solution B or methanol A-water B or methanol A-phosphoric acid water solution B, gradient elution: 20-52% (A) in 0-20 min, 52-70% (A) in 20-40 min, and 70-95% (A) in 40-50 min; the column temperature is 35-45 ℃; the flow rate is 0.6-1.4 ml/min-1(ii) a The detection wavelength is 160-220 nm;
fingerprint spectrum determination: and (3) absorbing the test solution according to the fingerprint chromatogram condition, injecting the test solution into a high performance liquid chromatograph, and measuring the fingerprint of the Liangfu dripping pill.
In the detection method of the Liangfu dripping pills, in the determination method of the fingerprint spectrum of the Liangfu dripping pills, the sample solution is prepared by weighing 0.5-1.5g of Liangfu dripping pills, placing the Liangfu dripping pills in a conical flask with a plug, adding methanol to prepare 5% Liangfu dripping pill solution, weighing the weight, carrying out ultrasonic treatment for 30min, taking out, cooling, complementing the weight loss with methanol, filtering, and taking filtrate to obtain the sample solution.
In the detection method of the galangal dripping pill, the fingerprint chromatogram condition is high performance liquid chromatograph; the chromatographic column is Luna PFP2100A, 250X 4.60mm, 5 μm; the mobile phase is acetonitrile A-0.1 percent phosphoric acid water solution B, and the gradient elution is as follows: 20-52% of A in 0-20 min, 52-70% of A in 20-40 min, and 70-95% of A in 40-50 min; the column temperature was 40 ℃; the flow rate was 1.000 ml/min-1(ii) a The detection wavelength is 190 nm; the amount of the sample was 10. mu.L.
In the detection method of the galangal pill, the similarity evaluation method of the fingerprint spectrum comprises the steps of measuring the fingerprint spectrums of a plurality of batches of galangal pill samples, carrying out similarity evaluation by adopting a 2012 edition of the national pharmacopoeia committee 'traditional Chinese medicine chromatogram fingerprint spectrum similarity evaluation system', carrying out multipoint correction by adopting a median method and taking any batch of fingerprint spectrum samples as reference spectrums, generating a reference spectrum, and calculating the similarity among the batches.
In the detection method of the galangal rhizome dripping pills, the fingerprint spectrums of the plurality of galangal rhizome dripping pills comprise 21 common peaks, wherein 6 characteristic peaks are galangin, rhamnocitrin, galangin-3-O-methyl ether, cyperenone, nootkatone and alpha-cyperone, 14 peaks in the common peaks are from galangal rhizome medicinal materials, 9 peaks are from cyperus rotundus medicinal materials, and 2 peaks exist in the galangal rhizome medicinal materials and the cyperus rotundus medicinal materials at the same time.
In the detection method of the galangal rhizome dripping pill, the galangal rhizome dripping pill is prepared from galangal rhizome and rhizoma cyperi according to the mass ratio of 1: 1.
Compared with the prior art, the content detection method of the galangin dripping pill can simultaneously detect the content of 6 main components of galangin, rhamnitridin, galangin-3-O-methyl ether, cyperenone, nootkatone and alpha-cyperone, the separation degree of each peak under the chromatographic condition of content detection is more than 1.5, the correlation coefficient of a linear regression equation of each reference substance is more than 0.9998, the precision of the content detection method is good, the repeatability is good, the stability in 24h is good, the accuracy is good, the difference among batches is small, the detection speed is high, and the content of 6 components can be detected simultaneously; the fingerprint spectrum batch similarity measured by the fingerprint spectrum measuring method of the galangal pills is greater than 0.99, a 15-batch fingerprint spectrum common mode of the galangal pills is established through a traditional Chinese medicine chromatogram fingerprint spectrum similarity evaluation system, 21 common peaks are determined, 6 characteristic peaks are identified and are galangin, rhamnitrin, galangin-3-O-methyl ether, cypermethenone, nootkatone and alpha-cyperone respectively, 14 peaks in the 21 common peaks are from galangal medicinal materials, 9 peaks are from cyperus rotundus medicinal materials, two peaks exist in the galangal medicinal materials and the cyperus rotundus medicinal materials at the same time, and the fingerprint spectrum detecting method has good precision, good stability in 24 hours and good repeatability. The quantitative analysis method can simultaneously determine the content of the main component in 6 of the galangal rhizome and aconite root, the qualitative detection in the invention can detect the specific chemical components in the galangal rhizome and the aconite root, the invention can effectively control the product quality, and can meet the stable and controllable requirements of the quality control of the galangal rhizome and aconite root.
Drawings
FIG. 1 shows HPLC fingerprints of 15 batches of Liangfu dripping pills (in the figure, R21 is the fingerprint of the mixed reference solution, and the other is the fingerprint of Liangfu dripping pill samples);
FIG. 2 shows characteristic peaks of Liangfu dripping pills;
FIG. 3 shows characteristic peaks of Alpinia officinarum Hance;
FIG. 4 shows characteristic peaks of rhizoma Cyperi (processed with vinegar);
FIG. 5 is a diagram of a systematic applicability of Liangfu dripping pill samples (in the figure, peak 1 is galangin, peak 2 is rhamnocitrin, peak 3 is galangin-3-O-methyl ether, peak 4 is cyperenone, peak 5 is nootkatone, and peak 6 is alpha-cyperone);
FIG. 6 is a chart showing the system applicability of the control solution (in the figure, peak 1 is galangin, peak 2 is rhamnocitrin, peak 3 is galangin-3-O-methyl ether, peak 4 is cyperenone, peak 5 is nootkatone, and peak 6 is alpha-cyperone).
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.
Apparatus and materials
1 apparatus
LC-2040C high performance liquid chromatograph (shimadzu, japan), one hundred thousand balance (mettler-toledo instruments shanghai ltd), one ten thousand balance (mettler-toledo instruments shanghai ltd), ultrasonic cleaner (nigbuck jagaku ltd), ultra pure water machine (wawter technologies ltd).
2 materials
15 batches of Liangfu pills (the Liangfu pills comprise galangal and rhizoma cyperi prepared in a mass ratio of 1: 1) are provided by Huangguoshuang medicine industry Co., Ltd, Guizhou (batch numbers 011123-011137, respectively expressed as S1-S15); galangin reference (batch No. wkq 16032503; HPLC ≥ 98%), rhamnocitrin reference (batch No. wkq 17081112; HPLC ≥ 98%), nootkatone reference (batch No. wkq 18080103; GC ≥ 97%), alpha-cyperone reference (batch No. wkq 16072701; HPLC ≥ 98%), all purchased from Szechwan Vickqi Biotech Co., Ltd; galangin-3-O-methyl ether control (batch number: 18042321; purity: HPLC ≥ 98%; purchased from Shanghai Hotan Biotechnology Ltd.); cyperenone reference (lot number: 17101605; purity: HPLC ≥ 95%; available from Dougeli Biotech, Inc.); the methanol and the acetonitrile are chromatographically pure, and the rest are analytically pure; ultrapure water.
Method and results
1 chromatographic conditions
1.1 assay chromatographic conditions
The chromatographic column is Luna PFP (2)100A (250X 4.60mm, 5 μm); the mobile phase is acetonitrile-0.1% phosphoric acid water solution (40: 60); the column temperature was 40 ℃; the flow rate was 1.000 ml/min-1(ii) a The detection wavelength is 254 nm; the amount of the sample was 10. mu.L.
1.2 fingerprint chromatogram conditions
The mobile phase was acetonitrile (a) -0.1% aqueous phosphoric acid (B), gradient elution: 20-52% (A) in 0-20 min, 52-70% (A) in 20-40 min, and 70-95% (A) in 40-50 min; the detection wavelength is 190 nm; the rest conditions are consistent with the chromatographic conditions of content measurement.
2 preparation of Mixed control solutions
Preparing galangin reference substance 13mg, rhamnocitrin reference substance 8mg, galangin-3-O-methyl ether reference substance 8mg, cyperenone reference substance 8mg, nootkatone reference substance 5mg, and alpha-cyperone reference substance 5mg with methanol to obtain 20ml mixed reference substance solution, and calculating the concentration of each reference substance.
3 preparation of test solutions
Weighing 1.0g of the product, precisely weighing, placing in a conical flask with a plug, adding methanol to prepare 5% solution, weighing, ultrasonically treating for 30min (power: 300W; frequency: 40KHz), taking out, cooling, supplementing the lost weight with methanol, filtering, and collecting filtrate.
Preparation of 4 negative control solution
Preparing negative samples of lesser galangal rhizome and nutgrass galingale rhizome according to the prescription respectively, and preparing negative control solution according to the preparation method of the test solution under item 2.3.
5 methodological investigation of HPLC fingerprinting
5.1 precision investigation
Taking the same galangal dripping pill sample (S1), preparing a test solution according to a test solution preparation method under item 3, carrying out continuous sample injection for 6 times under the chromatographic condition under item 1.2, carrying out similarity evaluation by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition) of the national pharmacopoeia committee, establishing a fingerprint sharing mode of 15 batches of galangal dripping pills, determining 21 shared peaks, calculating the relative retention time and the relative peak area of each shared peak by taking galangin as a reference peak S, and indicating that the relative retention time and the relative peak area RSD value of each peak are less than 3.0 percent, thereby indicating that the instrument has good precision.
5.2 stability Studies
Taking the same Liangfu dripping pill sample (S1), preparing a test solution according to the test solution preparation method under the item '3', respectively carrying out sample injection determination under the chromatographic condition under the item '1.2' after preparation for 0 hour, 2 hours, 4 hours, 8 hours, 12 hours and 24 hours, taking galangin as a reference peak S, calculating the relative retention time and the relative peak area of each common peak, and indicating that the RSD value of the relative retention time and the relative peak area of each peak is less than 3.0 percent, which indicates that the stability of the test solution is good within 24 hours.
5.3 repeatability test
Taking 6 parts of the same Liangfu dripping pill sample (S1), preparing a test solution according to the test solution preparation method under the item '3', carrying out sample injection measurement under the chromatographic condition under the item '1.2', taking galangin as a reference peak S, calculating the relative retention time and the relative peak area of each common peak, and indicating that the relative retention time and the relative peak area RSD value of each peak are both less than 3.0 percent.
6 establishment of fingerprint and peak attribution
6.1 creation of fingerprint
Taking 15 batches of rhizoma cyperi dropping pill samples (S1-S15), preparing a sample solution according to a preparation method of a test solution under the item 3, carrying out sample injection determination according to the chromatographic condition under the item 1.2, carrying out similarity evaluation by adopting a traditional Chinese medicine chromatography fingerprint similarity evaluation system (2012 edition) of the national pharmacopoeia committee, correcting multiple points by using a median method and taking the sample No. S1 as a reference map, generating a reference map R, and calculating a similarity value. The result shows that the similarity values of the sample spectrum and the reference spectrum R of 15 batches of the Liangfu dripping pills are all larger than 0.9, which indicates that the difference between the 15 batches of Liangfu dripping pills is smaller, and the suggested similarity value is not smaller than 0.9, and is shown in figure 1 and table 1.
Table 115 sample similarity results
6.2 common Peak assignment analysis
Respectively absorbing 10 μ L of test solution under item "3" and negative control solution under item "4", respectively, injecting sample under chromatographic conditions under item "1.2" (see figure 2-figure 4), establishing 15 batches of rhizoma Alpiniae Officinarum dripping pill fingerprint chromatogram common mode by "Chinese medicine chromatogram fingerprint chromatogram similarity evaluation system" published by the State pharmacopoeia Committee, determining 21 common peaks, and identifying 6 characteristic peaks, namely galangin (11 peak), rhamnocitrin (12 peak), galangin-3-O-methyl ether (15 peak), cyperenone (14 peak), nootkatone (16 peak), and alpha-cyperone (17 peak), wherein 1, 3, 4, 6, 8, 9, 10, 11, 12, 13, 15, 18 peaks are derived from rhizoma Alpiniae Officinarum, 2, 5, 7, 14, 16, 17, 21 peaks are derived from rhizoma Cyperi, 19. the 20 th peak exists in galangal and nutgrass galingale rhizome simultaneously. The galangin (11 peak) is used as a reference peak S, the relative retention time and the relative peak area of each common peak in 15 batches of samples are calculated, and the results show that the relative retention time and the relative peak area RSD value of each common peak are respectively 0.02-0.69% and 0.7-2.69%, which indicates that the quality consistency of 15 batches of galangal dropping pills is good.
Content determination of 6 ingredients in 7 Liangfu dripping pills
7.1 System suitability test
Respectively and precisely sucking 10 μ L of the reference solution under item "2" and the test solution under item "3", injecting into a liquid chromatograph, measuring according to the chromatographic condition under item "1.1", and recording chromatogram (see fig. 5-6), with the result that the separation degree of each peak is greater than 1.5.
7.2 Linear relationship investigation
Precisely measuring a proper amount of the mixed reference substance solution under the item 2, and gradually diluting the mixed reference substance solution into a series of reference substance solutions with different concentrations by adding methanol. Measuring according to chromatographic condition under the term of '1.1', drawing a working curve by taking the concentration as a horizontal coordinate X and the peak area as a vertical coordinate Y, and calculating a regression equation. The results are shown in Table 2, which shows that the index components have a good linear relationship within a certain mass concentration range.
Table 26 linear regression equations and ranges for the index constituents
7.3 precision test
Precisely absorbing the same sample solution (S1), continuously injecting sample for 6 times according to the content determination chromatographic condition under item '1.1', calculating the average content RSD values of galangin, rhamnitrin, galangin-3-O-methyl ether, cyperenone, nootkatone and alpha-cyperone in the Liangfu dripping pill to be 2.1%, 1.5%, 2.2%, 2.4%, 2.1% and 1.5%, respectively. Indicating that the precision of the instrument is good.
7.4 repeatability test
Respectively weighing 0.5 part, 1.0 part and 1.5g of the same batch of samples (S1), respectively 3 parts (S1), adding 20ml of methanol, preparing a test solution according to a test solution preparation method under the item '3', and performing sample injection measurement according to a content measurement chromatographic condition under the item '1.1' to calculate the average content of galangin, rhamnocitrate, galangin-3-O-methyl ether, cyperenone, nootkatone and alpha-cyperone in the galangal pill to be 9.3048, 3.1143, 1.4514, 2.3942, 0.8612 and 2.1909mg/g respectively, and the RSD value is respectively 2.3%, 2.7%, 2.3%, 2.4%, 2.8% and 1.9%. Indicating that the method has good repeatability.
7.5 stability test
The same sample solution (S1) is respectively placed for 0, 1, 2, 4, 8, 12 and 24 hours and then is subjected to sample injection determination according to the content determination chromatographic condition under the item of '1.1', and the results show that the content RSD of the galangin, the rhamnocitrin, the galangin-3-O-methyl ether, the cyperenone, the nocardone and the alpha-cyperone are respectively 1.2%, 1.0%, 1.4%, 1.8% and 2.1%. The stability of the test solution in 24 hours is good.
7.6 sample recovery test
Taking 0.5g of Liangfu dripping pill sample (S1) with known content, 9 parts in total, precisely weighing, respectively adding appropriate amount of galangin, rhamnocitrin, galangin-3-O-methyl ether, cyperenone, nootkatone and alpha-cyperone reference substances according to 50%, 100% and 150% of the content of 6 components, preparing a test solution according to the preparation method of the test solution under item '3', carrying out sample injection measurement according to the content measurement chromatographic condition under item '1.1', and calculating the sample addition recovery rate of the 6 components. As a result, the average sample recovery rates (n ═ 9) of galangin, rhamnitrin, galangin-3-O-methyl ether, cyperenone, nootkatone, and α -cyperone were 100.6%, 100.7%, 100.5%, 101.2%, 102.0%, and 102.0%, respectively, and the RSD values were 1.9%, 1.7%, 1.0%, 1.7%, 1.5%, and 1.6%, respectively. Indicating that the method has good accuracy.
7.7 sample determination
Taking 15 batches of Liangfu dripping pill samples, preparing sample solutions according to a method for preparing a test solution under the item 3, carrying out sample injection determination according to a chromatographic condition for content determination under the item 1.1, and calculating the content of 6 components in the 15 batches of Liangfu dripping pills, wherein the result shows that the RSD value of the content of 6 components in the 15 batches of Liangfu dripping pill samples is less than 3.0 percent, which indicates that the difference among the Liangfu dripping pill products is small and the uniformity is high. See table 3.
TABLE 315 Liangfu drop pills with 6 ingredient contents (mg/g, n ═ 3)
Discussion of the related Art
1 chromatographic column selection in the present study, a C18 column was selected for chromatographic peak separation at the beginning of the experiment, and as a result, it was found that galangin and rhamnitrin are both in an overlapping state under different chromatographic conditions. Because the galangin and the rhamnitrin have very similar structures, a PFP chromatographic column for separating the position isomers of the aromatic compounds is selected, and finally the galangin and the rhamnitrin can be completely separated.
2 wavelength selection the present study performed full wavelength scanning by hplc, found that the peak absorption of flavonoid and volatile oil components is maximized at 190nm, so 190nm was selected as fingerprint absorption wavelength of liangfuwan. In order to more accurately measure the content of 6 index components and reduce the interference of each miscellaneous peak on the index components, the content measurement research is finally carried out at 254 nm.
3 extraction method investigation this study respectively with 40% methanol, 70% methanol, ethanol, water ultrasonic treatment for 30min, methanol, water temperature bath for 30min, the Liangfu drop pill was subjected to extraction investigation, and the result is that the extraction rate of methanol ultrasonic treatment for 30min is higher than that of other extraction methods.
4 optimization of HPLC chromatographic conditions this study investigated 6 mobile phase systems of acetonitrile-water, acetonitrile-0.05% phosphoric acid aqueous solution, acetonitrile-0.1% phosphoric acid aqueous solution, acetonitrile-0.2% phosphoric acid aqueous solution, methanol-water, methanol-0.1% phosphoric acid aqueous solution, and as a result, the peak shape and the resolution of each chromatographic peak were better in the acetonitrile-0.1% phosphoric acid aqueous solution mobile phase system; in addition, different column temperatures and different flow rates were compared, resulting in a temperature of 40 ℃ and a flow rate of 1.0 ml/min-1Under the condition, the chromatographic peaks can be better separated.
In the research process, galangin and rhamnocitrin with very similar structures are found, the conventional octadecylsilane chemically bonded silica gel column is not easy to effectively separate, the galangin and the rhamnocitrin are well separated through a PFP chromatographic column, and a Liangfu dropping pill HPLC fingerprint spectrum and a method for simultaneously measuring the content of multiple index components are established.
The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and it should be understood that various changes and modifications can be effected therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. The detection method of the galangal rhizome dripping pill comprises the following steps of preparing galangal rhizome and rhizoma cyperi as raw materials, and is characterized in that: the detection method of the contents of galangin, rhamnocitrin, galangin-3-O-methyl ether, cyperenone, nootkatone and alpha-cyperone in the galangal pill is as follows,
preparation of mixed control solution: weighing galangin reference substance, rhamnan reference substance, galangin-3-O-methyl ether reference substance, cyperenone reference substance, nootkatone reference substance and alpha-cyperone reference substance, adding methanol to obtain mixed reference substance solution, and calculating the concentration of each reference substance solution;
preparation of a test solution: weighing Liangfu dripping pill, placing into conical bottle with plug, adding methanol or ethanol or methanol/ethanol solution, weighing, quantifying, ultrasonic treating, taking out, cooling, adding methanol or ethanol or methanol/ethanol solution to complement the weight loss, filtering, and collecting filtrate to obtain test solution;
content determination chromatographic conditions: a high performance liquid chromatograph; a PFP chromatography column; the mobile phase is acetonitrile A-0.1 percent phosphoric acid water solution B, 40: 60; the column temperature is 35-45 ℃; the flow rate is 0.6-1.4 ml/min-1(ii) a The detection wavelength is 220-280 nm;
content determination: respectively sucking the mixed reference solution and the test solution according to content determination chromatographic conditions, injecting into a high performance liquid chromatograph, and determining the content of galangin, rhamnocitrin, galangin-3-O-methyl ether, cyperenone, nootkatone and alpha-cyperone.
2. The method for detecting galangal dripping pills according to claim 1, wherein the method comprises the following steps: the mixed reference substance solution is prepared by weighing galangin reference substance 10-15mg, rhamnan citrate reference substance 5-10mg, galangin-3-O-methyl ether reference substance 5-10mg, cypermethene reference substance 5-10mg, nootkatone reference substance 3-6mg and alpha-cypermethene reference substance 3-6mg, adding methanol to prepare 20ml mixed reference substance solution, and calculating the concentration of each reference substance solution.
3. The method for detecting galangal dripping pills according to claim 1, wherein the method comprises the following steps: the sample solution is prepared by weighing Liangfu dripping pill 0.5-1.5g, placing in conical flask with plug, adding methanol to obtain 5% Liangfu dripping pill solution, weighing, ultrasonic treating for 30min, taking out, cooling, supplementing the weight loss with methanol, filtering, and collecting filtrate to obtain sample solution.
4. The method for detecting galangal dripping pills according to claim 1, wherein the method comprises the following steps: the chromatographic condition for content determination is a high performance liquid chromatograph; the chromatographic column is Luna PFP2100A, 250X 4.60mm, 5 μm; the mobile phase is acetonitrile A-0.1 percent phosphoric acid water solution B, 40: 60; the column temperature was 40 ℃; the flow rate was 1.000 ml/min-1(ii) a The detection wavelength is 254 nm; the amount of the sample was 10. mu.L.
5. A method for detecting Liangfu dripping pills according to any one of claims 1 to 4, wherein: also comprises a method for measuring the fingerprint spectrum of the Liangfu dripping pill, the method for measuring the fingerprint spectrum of the Liangfu dripping pill comprises the following steps,
preparation of a test solution: weighing Liangfu dripping pill, placing into conical bottle with plug, adding methanol or ethanol or methanol/ethanol solution, weighing, quantifying, ultrasonic treating, taking out, cooling, adding methanol or ethanol or methanol/ethanol solution to complement the weight loss, filtering, and collecting filtrate to obtain test solution;
fingerprint chromatogram conditions: a high performance liquid chromatograph; a PFP chromatography column; the mobile phase is acetonitrile A-0.1 percent phosphoric acid water solution B; gradient elution: 20-52% of A in 0-20 min, 52-70% of A in 20-40 min, and 70-95% of A in 40-50 min; the column temperature is 35-45 ℃; the flow rate is 0.6-1.4 ml/min-1(ii) a The detection wavelength is 160-220 nm;
fingerprint spectrum determination: and (3) absorbing the test solution according to the fingerprint chromatogram condition, injecting the test solution into a high performance liquid chromatograph, and measuring the fingerprint of the Liangfu dripping pill.
6. The method for detecting an galangal dripping pill according to claim 5, wherein the method comprises the following steps: in the fingerprint spectrum measuring method of Liangfu dripping pills, the sample solution is prepared by weighing Liangfu dripping pills 0.5-1.5g, placing in a conical flask with a plug, adding methanol to prepare Liangfu dripping pill solution with concentration of 5%, weighing, performing ultrasonic treatment for 30min, taking out, cooling, complementing the weight loss with methanol, filtering, and collecting the filtrate to obtain the sample solution.
7. The method for detecting an galangal dripping pill according to claim 5, wherein the method comprises the following steps: the fingerprint chromatogram condition is high performance liquid chromatograph; the chromatographic column is Luna PFP2100A, 250X 4.60mm, 5 μm; the mobile phase is acetonitrile A-0.1 percent phosphoric acid water solution B, and the gradient elution is as follows: 20-52% of A in 0-20 min, 52-70% of A in 20-40 min, and 70-95% of A in 40-50 min; the column temperature was 40 ℃; the flow rate was 1.000 ml/min-1(ii) a The detection wavelength is 190 nm; the amount of the sample was 10. mu.L.
8. The method for detecting an galangal dripping pill according to claim 5, wherein the method comprises the following steps: the similarity evaluation method of the fingerprint spectrum comprises the steps of measuring the fingerprint spectrums of a plurality of batches of Liangfu pill samples, carrying out similarity evaluation by adopting a 2012 edition of a Chinese medicine chromatography fingerprint spectrum similarity evaluation system of the State pharmacopoeia Committee, carrying out multipoint correction by using any one batch of fingerprint spectrum samples as a reference spectrum by adopting a median method, generating a reference spectrum, and calculating the similarity among the batches.
9. The method for detecting an galangal dripping pill according to claim 8, wherein the method comprises the following steps: the fingerprint spectrum of the multiple batches of galangal dripping pills comprises 21 common peaks, wherein 6 characteristic peaks are galangin, rhamnitrin, galangin-3-O-methyl ether, cypermethene, nootkatone and alpha-cypermethene respectively, 14 peaks in the common peaks are from galangal medicinal materials, 9 peaks are from cypermethene medicinal materials, and 2 peaks are simultaneously present in the galangin and the cypermethene medicinal materials.
10. A method for detecting an galangal dripping pill according to any one of claims 1 to 4 and 6 to 9, wherein the method comprises the steps of: the rhizoma galangae dropping pill is a dropping pill prepared from rhizoma galangae and rhizoma cyperi according to the mass ratio of 1: 1.
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