CN106290599A - A kind of content assaying method of Chinese medicine composition - Google Patents
A kind of content assaying method of Chinese medicine composition Download PDFInfo
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Abstract
Chinese medicine composition of the present invention uses UPLC method, measure effective ingredient calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride and the content of rosmarinic acid contained by it simultaneously, method is simple and quick, it is applicable to the quality control of Chinese medicine composition of the present invention, provides reference for the monitoring the most constantly in Chinese medicine composition continuous prodution of the present invention.
Description
Technical field
The present invention relates to the content assaying method of a kind of Chinese medicine composition, belong to Analysis of Chinese Traditional Medicine field, be specifically related to the content assaying method of each composition in a kind of Chinese medicine composition.
Background technology
Chinese medicine composition of the present invention is mainly by the Radix Astragali, Cortex Phellodendri, Cortex Cinnamomi, Spica Prunellae, the Chinese medicine compositions such as Fructus Ligustri Lucidi, with tonifying speen and tonifying kidney, diuretic eliminating stagnation is Therapeutic Principle, the clinical main disease for benign prostatic hyperplasia is that patient provides a kind of determined curative effect and takes safe new product of Chinese medicine [Su Pingju, Liu Ying, Li Song, Gao Peng, Zhao Shaohua, Liu Minyan. in Chinese medicine composition of the present invention, the Ultra Performance Liquid Chromatography of 3 kinds of active component measures [A]. China Association of Traditional Chinese Medicine. network disease basic and clinic studies (10) [C]. China Association of Traditional Chinese Medicine:, 2014:3, He Lijuan, Ma Guiyun, Guo Jinzhi, Liu Wenbo, Ren Xiaoming. Chinese medicine composition microbial limit tests of the present invention research [J]. China's Chinese medicine modern distance education, 2014, 01:154-155.].nullIn prescription, the content assaying method of main component has thin layer chromatography [Lin Chaozhan at present,Zhu Chen,Huang Lin,Zeng Xiuhua. the content [J] of astragaloside in the Radix Astragali commodity medical material of tlc scanning determination Guangzhou. Guangdong pharmacy,2002,The refined English of Feng 05:26-28.,Bai Ming,Xie Qinghai. the research [J] of Cortex Phellodendri content assaying method. Shenyang Pharmacy College is learned,1992,03:212-214.]，High performance liquid chromatography [side sieve,Lin Nengming,Wu Yongjiang. high performance liquid chromatography measures the content [J] of 4 kinds of active component in Spica Prunellae medical material simultaneously. CHINA JOURNAL OF CHINESE MATERIA MEDICA,2010,05:616-619. Huang cloud tints,Fructus Perillae core,Bi Wenchuan,Li Jun,Shi Liqiong,Wen Yingqiang,Zhan Huaqiang. HPLC method measures the content [J] of nuezhenoside in Fructus Ligustri Lucidi. pharmaceutical analysis magazine,2009,05:824-826.]、Gas chromatography [Du Qingpeng,Tian Jingai,Shi Shangmei. GC method measures the content [J] of cinnamic aldehyde in import Cortex Cinnamomi. Chinese Pharmaceutical Affairs,2002,01:37-38.] etc..Traditional analytical speed is slow, the longest, consumes substantial amounts of organic reagent simultaneously, adds environmental pollution.The advantages such as the separating degree sensitivity that Ultra Performance Liquid Chromatography (UPLC) is efficient with it, quick and superpower are widely used in the Analysis of Chinese Traditional Medicine field of complex chemical composition [Liu Minyan, Zhao Shaohua, Wang Yufeng, Wang Hongtao, Tu Pengfei. ultra-performance liquid chromatography measures the content [J] of 8 kinds of active component in Shen Song Yang Xin Capsule simultaneously. Journal of Chinese Hospital Pharmacy, 2014,06:435-438.
Ren Weiguang, Wang Dongmei, Huang Linfang. UPLC method measures the content [J] of 8 compositions in Radix Et Rhizoma Rhei simultaneously. pharmaceutical analysis magazine, 2014,09:1565-1570.].
In Chinese medicine composition, comparison of ingredients is many, the most conventional detection method cycle is long, cost is high, therefore to product quality can effectively be controlled, ensure the drug safety of the public, this Chinese medicine composition is carried out quality control, the content that this research uses UPLC method to measure in this Chinese medicine composition 5 kinds of principle active component calycosin glucosides, specnuezhenside, palmatine hydrochloride, berberine hydrochloride and rosmarinic acid simultaneously is determined, method is simple and quick, provides reference for the monitoring the most constantly in Chinese medicine composition continuous prodution of the present invention.
Summary of the invention
It is an object of the invention to provide a kind of detection cycle short, highly sensitive, the method simultaneously detecting five kinds of component contents.
For solving above-mentioned technical problem, the technical solution adopted in the present invention has:
This Chinese medicine composition is made up of the crude drug of following weight ratio: Radix Astragali 30-150 part, Talcum 7-28 part, Spica Prunellae 10-40 part, Fructus Ligustri Lucidi 7-28 part, Semen Litchi 10-40 part, succinum 1-4 part, Cortex Cinnamomi 1.5-6 part, Cortex Phellodendri 3-15 part, and described content assaying method is the content using UPLC method simultaneously to measure five kinds of compositions.
Content assaying method is preferably:
A, the preparation method of need testing solution: weigh described Chinese medicine composition content, take 0.5-3 g, accurately weighed, puts in tool plug conical flask, accurate addition 50-100% methanol 10-75
Ml, weighed weight, supersound process 10-60 minute, let cool, weigh, supply the weight of less loss with 50-90% methanol, shake up, to obtain final product；
B, the preparation method of reference substance solution: weigh calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, rosmarinic acid reference substance in right amount, accurately weighed, methanol dissolves, and obtains concentration and is respectively 0.396
mg•ml-1、1.156 mg•ml-1、0.572 mg•ml-1、0.74 mg•ml-1、1.578 mg•ml-1Mixing reference substance stock solution, to obtain final product；
C, chromatographic condition: chromatographic column: C18 post, column temperature: 35 DEG C-45 DEG C；Mobile phase A is acetonitrile or methanol, and Mobile phase B is: phosphate aqueous solution or aqueous formic acid, and gradient elution ratio is: 0 ~ 2min
Mobile phase A is by 15% to 20%, and Mobile phase B is by 85% to 80%；2 ~ 7min mobile phase A is by 20% to 30%, and Mobile phase B is by 80% to 70%；7 ~ 10min mobile phase A is by 30% ~ 32%, and Mobile phase B is by 70% to 68%；10 ~ 12min mobile phase A is by 32% to 35%, and Mobile phase B is by 68% to 65%；12 ~ 14min mobile phase A is 35%, and Mobile phase B is 65%；Detection wavelength: 240 nm；Column temperature: 35 DEG C；Flow velocity: 0.2ml min-1；
D, mensuration: suck need testing solution, reference substance solution 0.2-5 L injects in UPLC liquid phase, with calculated by peak area sample size.
Content assaying method is preferably: the preparation method of step A need testing solution is: weigh described Chinese medicine composition content, takes 1 g, accurately weighed, put in tool plug conical flask, accurate addition 80% methanol 25 ml, weighed weight, 30 minutes (power 250w of supersound process, frequency 40kHz), let cool, weigh, supply the weight of less loss with 80% methanol, shake up, to obtain final product.
Content assaying method is preferably: chromatographic column model described in step C is Phenomenex UPLC
Kinetex C18, specification 2.1 × 100
Mm, 2.6 μm；Or Waters Acquity UPLC zero R
Shield RP18, specification 2.1 × 100
Mm, 1.7 μm；Or ACQUITY BEH C18, specification is 2.1 × 100mm, 1.7 μm.
Content assaying method is preferably: mobile phase A described in step C is methanol, and Mobile phase B is 0.1% phosphate aqueous solution；Or mobile phase A is methanol, Mobile phase B is 0.2% aqueous formic acid；Or mobile phase A is acetonitrile, Mobile phase B is 0.2% aqueous formic acid；Or mobile phase A is acetonitrile, Mobile phase B is 0.1% phosphate aqueous solution.
The weight ratio of the traditional Chinese medicinal composition raw materials of content assaying method is preferably:
The Radix Astragali 70 parts
14 parts of Talcum
Spica Prunellae 21 parts
Fructus Ligustri Lucidi 14 parts
Semen Litchi 21 parts
Succinum 2.1 parts
Cortex Cinnamomi 2.8 parts
Cortex Phellodendri 7 parts.
The weight ratio of the traditional Chinese medicinal composition raw materials of content assaying method is preferably:
The Radix Astragali 30 parts
7 parts of Talcum
Spica Prunellae 10 parts
Fructus Ligustri Lucidi 7 parts
Semen Litchi 10 parts
Succinum 1 part
Cortex Cinnamomi 1.5 parts
Cortex Phellodendri 3 parts.
The active component of the Chinese medicine composition of content assaying method is made up of following steps:
(1) weigh Chinese crude drug by crude drug part by weight, clean respectively, pulverize；
(2) taking the Radix Astragali, Cortex Phellodendri, add 50-70% ethanol, heating and refluxing extraction 1-3 time, each 0.5-2 hour, extracting solution filtered, and merges, after decompression recycling ethanol, is condensed into the clear paste that relative density is 1.20-1.25, standby；
(3) taking Fructus Ligustri Lucidi, add 6-10 times amount 70-90% ethanol, heating and refluxing extraction 1-3 time, the time is each 1-3 hour, and extracting solution filters, and merges, after decompression recycling ethanol, is condensed into the clear paste that relative density is 1.20-1.25, standby；
(4) take Cortex Cinnamomi, add 4-8 times amount water soaking after 1-2 hour, extract volatile oil 2-6 hour,
The another device of volatile oil is collected, and aqueous extract is collected by filtration, and residue is standby；
(5) take Spica Prunellae, Semen Litchi and step (4) residue obtained, add 8-10 times amount water, decoct twice, 1-3 hour for the first time, 1-2 hour for the second time, extracting solution filtered, and merged, add step (4) obtained aqueous solution, be condensed into the clear paste that relative density is 1.20-1.25, standby；
(6) take Talcum, succinum, be ground into 100 mesh fine powders, standby；
Step (2) gained clear paste, step (3) gained clear paste, step (4) gained volatile oil, step (5) gained clear paste and step (6) gained fine powder collectively form the active component of Chinese medicine composition of the present invention.
The preparation formulation of the Chinese medicine composition of content assaying method is capsule, tablet, oral liquid or pill.
The Chinese medicinal composition capsules agent of content assaying method is to comprise the steps of:
(1) taking above-mentioned Chinese crude drug, clean respectively, pulverize, amount weighs in proportion；
(2) Radix Astragali, Cortex Phellodendri are taken, add the 50-70% ethanol of 6-12 times, heating and refluxing extraction 1-3 time, extract 1-3 hour every time, extracting solution filters, merge, after decompression recycling ethanol, be condensed into the clear paste that relative density is 1.20-1.25, put into vacuum drying oven, drying under reduced pressure under the conditions of temperature 60-70 DEG C, vacuum 0.04-0.06Mpa, dry cream is standby；
(3) taking Fructus Ligustri Lucidi, add 6-10 times amount 70-90% ethanol, heating and refluxing extraction 1-3 time, the time is each 1-3 hour, extracting solution filters, and merges, after decompression recycling ethanol, is condensed into the clear paste that relative density is 1.20-1.25, put into vacuum drying oven, in temperature 60-70 DEG C, vacuum 0.04-0.06
Drying under reduced pressure under the conditions of Mpa, dry cream is standby；
(4) take Cortex Cinnamomi, add 4-8 times amount water soaking after 1-2 hour, extract volatile oil 2-6 hour,
The another device of volatile oil is collected, and oil yield must not be less than l.0%, and aqueous extract is collected by filtration, and residue is standby；
(5) take the residue after Spica Prunellae, Semen Litchi and Cortex Cinnamomi carry oil, add 8-10 times amount water, decoct twice, 1-3 hour for the first time, 1-2 hour for the second time, extracting solution filtered, and merged, add the aqueous solution after Cortex Cinnamomi carries oil, it is condensed into the clear paste that relative density is 1.20-1.25, puts into vacuum drying oven, in temperature 60-70 DEG C, drying under reduced pressure under the conditions of vacuum 0.04-0.06Mpa, it is standby that dry cream and the Radix Astragali, Cortex Phellodendri, Fructus Ligustri Lucidi dried cream powder are broken into 100 mesh powder；
(6) take Talcum, succinum, be ground into 100 mesh powder, standby；
(7) take dried cream powder, former medicated powder and starch, be mixed evenly, be binding agent with 80% ethanol, High Shear Mixer Granulator, 60-70 DEG C of drying, granulate；
(8) sift out fine powder, spray into volatile oil, mix homogeneously with granule, airtight, encapsulated, to obtain final product.
The selection of detection wavelength: use PDA detector that 5 kinds of compounds have been carried out spectral scan, calycosin glucoside presents three absworption peaks in PDA detection spectrogram: 220nm, 249nm, 290nm, response value size order is: 220nm > 249nm > 290nm, the a length of 226nm of specnuezhenside maximum absorption wave, except in addition to end absorption peak is relatively big, at 280-350nm absworption peak substantially in mild state, change is little；All there is absworption peak at about 228nm, 264nm, 347nm in berberine hydrochloride, palmatine hydrochloride, and response value is the most suitable；Rosmarinic acid presents two absworption peaks in PDA detection spectrogram: 220nm, 329nm, and response value is the most suitable.Five kinds of compounds all have absorption maximum at 220nm, but due near ultraviolet end, in whole chromatogram, impurity peaks increases, affect the accurate quantitative analysis of each composition, consider the spectrum characteristic of five kinds of compounds, at 240nm, the response of five kinds of compounds is of a relatively high and impurity peaks interference is less, finally determines the detection wavelength using 240nm as five kinds of compounds.
The selection of flowing phase: acetonitrile-0.1% phosphoric acid water, acetonitrile-0.2% phosphoric acid water, acetonitrile-0.1% formic acid water, the different combined investigation result that flows such as acetonitrile-0.2% formic acid water system shows, four kinds of flowings the most all can make each Component seperation to be measured, and each one-tenth swarming type is preferable, and separating degree all reaches more than 1.5, and disengaging time is short, the beneficially analysis of high flux sample.When flowing is acetonitrile-0.2% formic acid water gradient elution mutually, the Component seperation degree each to be measured in sample is good compared with other three kinds, and particularly with berberine hydrochloride and the palmatine hydrochloride of more difficult separation, separating degree is more than 2.5.
Extracting method selects: this experiment compares 50% methanol, 70% methanol, 80% methanol and the impact on constituents extraction rate to be measured of the 4 kinds of solvents of 100% methanol, result shows that 50% methanol, 70% methanol, 80% methanol and 4 kinds of solvents of 100% methanol are the highest to constituents extraction rate to be measured, so that 80% methanol extraction efficiency is the highest and impurity is the fewest；Having carried out preferably to the supersound extraction time (20min, 30min, 40min, 60min), result is extracted each component content of 30min and all can be extracted completely simultaneously.
" Chinese medicine composition content " of the present invention is the test substance of pharmaceutical preparation, such as: the content of capsule, tablet remove the label of coating, pill, oral liquid etc..
In order to verify feasibility and the accuracy of the method for the present invention, make following Method validation with the capsule in embodiment 1 and tested:
1
Instrument and material
1.1
Instrument
Ultra Performance Liquid Chromatography system (Waters Acquity-H-Class is furnished with quaternary geopressure gradient pump, vacuum degassing machine, automatic sampler, column oven, diode array detector, Empower 3 chromatographic work station)；Ultrasonic cleaner (KQ-300VDB, power 250W, frequency 40kHz, Kunshan Ultrasonic Instruments Co., Ltd.)；Analytical balance (AT201, METTLER TOLEDO)；Milli-Q
Advantage A10 ultrapure water system (Millipore).
Material
Calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, rosmarinic acid (lot number is respectively as follows: 111920-201102,111926-201102,110732-200506,110713-201212,111871-201001,110781-200613) are purchased from National Institute for Food and Drugs Control；
Chinese medicine composition of the present invention (lot number is respectively 130501,111001,131001,110601,110301), Shijiazhuang Yiling Pharmaceutical Co., Ltd produces.Acetonitrile, methanol (chromatographically pure, Merk company of Germany), formic acid (chromatographically pure, Fisher Scientific company of the U.S.).Other reagent is analytical pure.
Method and result
2.1 chromatographic condition
Chromatographic column: Waters Acquity UPLC
Shield RP18 (2.1 × 100 mm, 1.7μM), column temperature: 40 DEG C；Flowing phase: acetonitrile (A)-0.2% formic acid (B) solution, gradient elution (table 1), detection wavelength: 240 nm；Flow velocity: 0.2ml min-1；Sample size: 1μl。
TableEluent gradient
The preparation of 2.2 need testing solutions
Weigh Chinese medicine composition content of the present invention, take about 1 g, accurately weighed, put in tool plug conical flask, accurate addition 80% methanol 25 ml, weighed weight, supersound process 30 minutes (power 250w, frequency 40kHz), let cool, weigh, supply the weight of less loss with 80% methanol, shake up, to obtain final product.
2.3
The preparation of reference substance solution
Weighing calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, rosmarinic acid reference substance in right amount, accurately weighed, methanol dissolves, and obtains concentration and is respectively 0.396
mg·ml-1、1.156 mg·ml-1、0.572 mg·ml-1、0.74 mg·ml-1、1.578 mg·ml-1Mixing reference substance stock solution, to obtain final product.
2.4 linear relationships are investigated
A certain amount of reference substance stock solution of accurate absorption, is configured to Working Control product solution.Accurate Working Control product solution of drawing, in injection Ultra Performance Liquid Chromatography instrument, is measured.With concentration as abscissa, integrating peak areas value is vertical coordinate, draws standard curve, the results are shown in Table 2.
Table 2 linear relationship is investigated
2.5 precision tests and stability test
Precision draws Working Control product solution respectively, continuous sample introduction 5 times, measuring peak area, result calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, rosmarinic acid RSD are respectively 1.46%, 1.29%, 1.18%, 1.45%, 1.41%, show that instrument precision is good.
The same need testing solution of accurate absorption, respectively at 0,2,4,8,12,24h sample introduction measure peak area, result is in 24 hours measured, calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, the RSD of 5 kinds of compound peaks areas of rosmarinic acid are respectively 0.961%, 1.49%, 1.29%, 1.02%, 1.31%, show that need testing solution is basicly stable in 24 hours.
2.6
Replica test
Take with a collection of Chinese medicine composition sample of the present invention, 9 parts are processed by the preparation method of need testing solution under " 2.2 " item, inject Ultra Performance Liquid Chromatography instrument to measure, in results sample, calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, the RSD value of rosmarinic acid contents are respectively 2.35%, 1.71%, 2.33%, 2.33%, 2.20%, and illustration method repeatability is good.
2.7
Recovery test
Use sample-adding recovery test method, precision weighs the Chinese medicine composition sample 0.5g same of the present invention of known content, parallel 9 parts, precision adds a certain amount of reference substance mixed solution respectively, by the preparation method of need testing solution, carry out supersound extraction respectively, filter, inject Ultra Performance Liquid Chromatography instrument to measure, calculate the response rate, calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, rosmarinic acid is respectively 100.2%, 99.3%, 100.8%, 100.4%, 101.1%, RSD value is respectively 1.49%, 1.13%, 1.40%, 1.46%, 0.684%, show that method is accurately and reliably.
2.8 sample determination
Take Chinese medicine composition sample of the present invention, by the preparation method of above-mentioned need testing solution, carry out supersound extraction respectively, filter, inject Ultra Performance Liquid Chromatography instrument and measure, record chromatogram；Calculating calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, the content of rosmarinic acid under 240nm, assay the results are shown in Table 3.
Table 3 assay result (mg g -1)
Result shows, this analysis method specificity is strong, and precision is high, and result is accurate, stable, reproducible, sensitivity high, it is adaptable to the quality control of Chinese medicine composition of the present invention.
Detailed description of the invention
Embodiment
1
: the preparation of medicine capsule of the present invention
Prescription:
The Radix Astragali 70 grams
Talcum 21 grams of Fructus Ligustri Lucidi of 14 grams of Spica Prunellaes 14 grams
Semen Litchi 21 grams
Succinum 2.1 grams
Cortex Cinnamomi 2.8 grams
Cortex Phellodendri 7 grams
Preparation method:
(1), weigh Chinese crude drug by recipe quantity, clean respectively, pulverize；
(2), take the Radix Astragali, Cortex Phellodendri, add 60% ethanol, heating and refluxing extraction 2 times, adding 10 times amount for the first time, extract 2 hours, second time adds 8 times amount, extracting 1 hour, extracting solution filters, and merges, after decompression recycling ethanol, it is condensed into the clear paste that relative density is 1.20-1.25, puts into vacuum drying oven, temperature 65 DEG C, drying under reduced pressure under the conditions of vacuum 0.05Mpa, dry cream is standby；
(3), Fructus Ligustri Lucidi is taken, adding 8 times amount 80% ethanol, heating and refluxing extraction 2 times, the time is each 2 hours, extracting solution filters, merge, after decompression recycling ethanol, be condensed into the clear paste that relative density is 1.20-1.25, put into vacuum drying oven, at temperature 65 DEG C, drying under reduced pressure under the conditions of vacuum 0.05Mpa, dry cream is standby；
(4), taking Cortex Cinnamomi, add 6 times amount water soaking after 1 hour, extract volatile oil 4 hours, the another device of volatile oil is collected, and aqueous extract is collected by filtration, and residue is standby；
(5), to take Spica Prunellae, Semen Litchi and step (4) residue obtained, adds 9 times amount water, decocts twice, 2 hours for the first time, 1.5 hours for the second time, extracting solution filtered, and merged, add step (4) obtained aqueous solution, it is condensed into the clear paste that relative density is 1.20-1.25, puts into vacuum drying oven, temperature 65 DEG C, drying under reduced pressure under the conditions of vacuum 0.05Mpa, got dry extract got dry extract with step (2), that step (3) got dry extract mixed powder is broken into 100 mesh powder is standby；
(6), take Talcum, succinum, be ground into 100 mesh powder, standby；
(7), by step (5) gained dried cream powder, step (6) gained fine powder and appropriate amount of auxiliary materials mix homogeneously, it is binding agent with 80% ethanol, pelletizes, dry, granulate；
(8), sift out fine powder, spray into step (4) gained volatile oil, mix homogeneously with granule, airtight, encapsulated, to obtain final product.
Content assaying method comprises the steps of:
A, the preparation of need testing solution: weigh Chinese medicine composition content of the present invention, take about 1 g, accurately weighed, put in tool plug conical flask, accurate addition 80% methanol 25 ml, weighed weight, 30 minutes (power 250w of supersound process, frequency 40kHz), let cool, weigh, supply the weight of less loss with 80% methanol, shake up, to obtain final product.
B, the preparation of reference substance solution: weigh calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, rosmarinic acid reference substance in right amount, accurately weighed, methanol dissolves, and obtains concentration and is respectively 0.396
mg·ml-1、1.156 mg·ml-1、0.572 mg·ml-1、0.74 mg·ml-1、1.578 mg·ml-1Mixing reference substance stock solution, to obtain final product.
C, chromatographic condition: chromatographic column: Waters Acquity UPLCShield RP18 (2.1 × 100 mm, 1.7μM), column temperature: 40 DEG C；Flowing phase: acetonitrile (A)-0.2% formic acid (B) solution, gradient elution (table 4), detection wavelength: 240 nm；Flow velocity: 0.2ml min-1；Sample size: 1μl。
Experimental result:
1, linear relationship is investigated
A certain amount of reference substance stock solution of accurate absorption, is configured to Working Control product solution.Accurate Working Control product solution of drawing, in injection Ultra Performance Liquid Chromatography instrument, is measured.With concentration as abscissa, integrating peak areas value is vertical coordinate, draws standard curve, the results are shown in Table 5.
Table 5 linear relationship is investigated
2, precision test and stability test:
Precision draws Working Control product solution respectively, continuous sample introduction 5 times, measuring peak area, result calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, rosmarinic acid RSD are respectively 1.46%, 1.29%, 1.18%, 1.45%, 1.41%, show that instrument precision is good.
The same need testing solution of accurate absorption, respectively at 0,2,4,8,12,24h sample introduction measure peak area, result is in 24 hours measured, calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, the RSD of 5 kinds of compound peaks areas of rosmarinic acid are respectively 0.961%, 1.49%, 1.29%, 1.02%, 1.31%, show that need testing solution is basicly stable in 24 hours.
3, replica test
Take with a collection of Chinese medicine composition sample of the present invention, 9 parts are processed by the preparation method of need testing solution under " 2.2 " item, inject Ultra Performance Liquid Chromatography instrument to measure, in results sample, calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, the RSD value of rosmarinic acid contents are respectively 2.35%, 1.71%, 2.33%, 2.33%, 2.20%, and illustration method repeatability is good.
4, recovery test
Use sample-adding recovery test method, precision weighs the Chinese medicine composition sample 0.5g same of the present invention of known content, parallel 9 parts, precision adds a certain amount of reference substance mixed solution respectively, by the preparation method of need testing solution, carry out supersound extraction respectively, filter, inject Ultra Performance Liquid Chromatography instrument to measure, calculate the response rate, calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, rosmarinic acid is respectively 100.2%, 99.3%, 100.8%, 100.4%, 101.1%, RSD value is respectively 1.49%, 1.13%, 1.40%, 1.46%, 0.684%, show that method is accurately and reliably.
5, sample determination
Take Chinese medicine composition sample of the present invention, by the preparation method of above-mentioned need testing solution, carry out supersound extraction respectively, filter, inject Ultra Performance Liquid Chromatography instrument and measure, record chromatogram；Calculating calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, the content of rosmarinic acid under 240nm, assay the results are shown in Table 6.
Result shows, this analysis method specificity is strong, and precision is high, and result is accurate, stable, reproducible, sensitivity high, it is adaptable to the quality control of Chinese medicine composition of the present invention.
Embodiment
2
: the preparation of medicinal tablet of the present invention
Prescription:
The Radix Astragali 30 grams
Talcum 10 grams of Fructus Ligustri Lucidi of 7 grams of Spica Prunellaes 7 grams
Semen Litchi 10 grams
Succinum 1 gram
1.5 grams of Cortex Phellodendris of Cortex Cinnamomi 3 grams
Preparation method:
(1), weigh Chinese crude drug by recipe quantity, clean respectively, pulverize；
(2), take the Radix Astragali, Cortex Phellodendri, add 60% ethanol, heating and refluxing extraction 2 times, add 9 times amount for the first time, extracting 3 hours, second time adds 8 times amount, extracts 1.5 hours, and extracting solution filters, merge, after decompression recycling ethanol, be condensed into the clear paste that relative density is 1.20-1.25, standby；
(3), taking Fructus Ligustri Lucidi, add 7 times amount 80% ethanol, heating and refluxing extraction 2 times, the time is each 2 hours, and extracting solution filters, and merges, after decompression recycling ethanol, is condensed into the clear paste that relative density is 1.20-1.25, standby；
(4), taking Cortex Cinnamomi, add 6 times amount water soaking after 1.5 hours, extract volatile oil 5 hours, the another device of volatile oil is collected, and aqueous extract is collected by filtration, and residue is standby；
(5), to take Spica Prunellae, Semen Litchi and step (4) residue obtained, adds 9 times amount water, decocts twice, 2 hours for the first time, 1.5 hours for the second time, extracting solution filtered, and merged, add step (4) obtained aqueous solution, be condensed into the clear paste that relative density is 1.20-1.25, standby；
(6), take Talcum, succinum, be ground into 100 mesh fine powders, standby；
(7), formulation method makes tablet routinely.
Content assaying method comprises the steps of:
A, the preparation of need testing solution: weigh Chinese medicine composition content of the present invention, take about 1 g, accurately weighed, put in tool plug conical flask, accurate addition 80% methanol 50ml, weighed weight, 30 minutes (power 250w of supersound process, frequency 40kHz), let cool, weigh, supply the weight of less loss with 80% methanol, shake up, to obtain final product.
B, the preparation of reference substance solution: weigh calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, rosmarinic acid reference substance in right amount, accurately weighed, methanol dissolves, and obtains concentration and is respectively 0.396
mg·ml-1、1.156 mg·ml-1、0.572 mg·ml-1、0.74 mg·ml-1、1.578 mg·ml-1Mixing reference substance stock solution, to obtain final product.
C, chromatographic condition: chromatographic column: Waters Acquity UPLCShield RP18 (2.1 × 100 mm, 1.7μM), column temperature: 40 DEG C；Flowing phase: acetonitrile (A)-0.1% phosphoric acid (B) aqueous solution, gradient elution (table 7), detection wavelength: 240 nm；Flow velocity: 0.2ml min-1；Sample size: 0.5μl。
Experimental result:
1, precision test: precision draws Working Control product solution respectively, continuous sample introduction 5 times, measure peak area, result calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, rosmarinic acid RSD are respectively 1.38%, 1.07%, 1.25%, 1.37%, 1.18%, show that instrument precision is good.
2, stability test: the same need testing solution of accurate absorption, respectively at 0,2,4,8,12,24h sample introduction measure peak area, result is in 24 hours measured, calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, the RSD of 5 kinds of compound peaks areas of rosmarinic acid are respectively 0.87%, 1.17%, 1.42%, 1.12%, 1.53%, show that need testing solution is basicly stable in 24 hours.
3, replica test: take with a collection of Chinese medicine composition sample of the present invention, 9 parts are processed by the preparation method of need testing solution under " 2.2 " item, inject Ultra Performance Liquid Chromatography instrument to measure, in results sample, calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, the RSD value of rosmarinic acid contents are respectively 1.85%, 1.72%, 2.17%, 2.32%, 2.18%, and illustration method repeatability is good.
4, recovery test
Use sample-adding recovery test method, precision weighs the Chinese medicine composition sample 0.5g same of the present invention of known content, parallel 9 parts, precision adds a certain amount of reference substance mixed solution respectively, by the preparation method of need testing solution, carry out supersound extraction respectively, filter, inject Ultra Performance Liquid Chromatography instrument to measure, calculate the response rate, calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, rosmarinic acid is respectively 101.0%, 99.7%, 100.3%, 99.1%, 100.1%, RSD value is respectively 1.40%, 1.21%, 1.28%, 1.51%, 0.97%, show that method is accurately and reliably.
5, sample determination
Take Chinese medicine composition sample of the present invention, by the preparation method of above-mentioned need testing solution, carry out supersound extraction respectively, filter, inject Ultra Performance Liquid Chromatography instrument and measure, record chromatogram；Calculating calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, the content of rosmarinic acid under 240nm, assay the results are shown in Table 8.
Result shows, this analysis method specificity is strong, and precision is high, and result is accurate, stable, reproducible, sensitivity high, it is adaptable to the quality control of Chinese medicine composition of the present invention.
Embodiment
3
: the preparation of medicine capsule of the present invention
Prescription:
The Radix Astragali 150 parts
28 parts of Talcum
Spica Prunellae 40 parts
Fructus Ligustri Lucidi 28 parts
Semen Litchi 40 parts
Succinum 4 parts
Cortex Cinnamomi 6 parts
Cortex Phellodendri 15 parts.
Preparation method: formulation method makes capsule routinely.
Content assaying method comprises the steps of:
A, the preparation of need testing solution: weigh Chinese medicine composition content of the present invention, take about 3g, accurately weighed, puts in tool plug conical flask, accurate addition 50% methanol 75ml, weighed weight, supersound process 60 minutes, let cool, weigh, supply the weight of less loss with 50% methanol, shake up, to obtain final product.
B, the preparation of reference substance solution: weigh calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, rosmarinic acid reference substance in right amount, accurately weighed, methanol dissolves, and obtains concentration and is respectively 0.396
mg·ml-1、1.156 mg·ml-1、0.572 mg·ml-1、0.74 mg·ml-1、1.578 mg·ml-1Mixing reference substance stock solution, to obtain final product.
C, chromatographic condition: chromatographic column is Phenomenex UPLC Kinetex C18, specification 2.1 × 100 mm, 2.6 μm, column temperature: 35 DEG C；Flowing phase: methanol (A)-0.1% phosphoric acid (B) solution, gradient elution (table 9), detection wavelength: 240 nm；；Flow velocity: 0.2ml min-1；Sample size: 3μl。
Table 9 eluent gradient
Experimental result:
1, precision test: precision draws Working Control product solution respectively, continuous sample introduction 5 times, measure peak area, result calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, rosmarinic acid RSD are respectively 1.53%, 1.27%, 1.14%, 1.62%, 1.33%, show that instrument precision is good.
2, stability test: the same need testing solution of accurate absorption, respectively at 0,2,4,8,12,24h sample introduction measure peak area, result is in 24 hours measured, calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, the RSD of 5 kinds of compound peaks areas of rosmarinic acid are respectively 1.72%, 1.32%, 1.69%, 1.58%, 1.13%, show that need testing solution is basicly stable in 24 hours.
3, replica test: take with a collection of Chinese medicine composition sample of the present invention, 9 parts are processed by the preparation method of need testing solution under " 2.2 " item, inject Ultra Performance Liquid Chromatography instrument to measure, in results sample, calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, the RSD value of rosmarinic acid contents are respectively 1.72%, 2.31%, 2.18%, 2.51%, 2.10%, and illustration method repeatability is good.
4, recovery test
Use sample-adding recovery test method, precision weighs the Chinese medicine composition sample 1.5g same of the present invention of known content, parallel 9 parts, precision adds a certain amount of reference substance mixed solution respectively, by the preparation method of need testing solution, carry out supersound extraction respectively, filter, inject Ultra Performance Liquid Chromatography instrument to measure, calculate the response rate, calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, rosmarinic acid is respectively 100.8%, 98.9%, 101.0%, 99.6%, 100.1%, RSD value is respectively 1.84%, 1.35%, 1.57%, 1.49%, 1.92%, show that method is accurately and reliably.
5, sample determination
Take Chinese medicine composition sample of the present invention, by the preparation method of above-mentioned need testing solution, carry out supersound extraction respectively, filter, inject Ultra Performance Liquid Chromatography instrument and measure, record chromatogram；Calculating calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, the content of rosmarinic acid under 240nm, assay the results are shown in Table 10.
Result shows, this analysis method specificity is strong, and precision is high, and result is accurate, stable, reproducible, sensitivity high, it is adaptable to the quality control of Chinese medicine composition of the present invention.
Embodiment 4: the preparation of bolus of drug of the present invention
Prescription:
The Radix Astragali 30 grams
28 grams of Talcum
Spica Prunellae 10 grams
Fructus Ligustri Lucidi 28 grams
Semen Litchi 10 grams
Succinum 4 grams
Cortex Cinnamomi 1.5 grams
Cortex Phellodendri 15 grams
Preparation method: formulation method makes pill routinely.
Content assaying method comprises the steps of:
A, the preparation of need testing solution: weigh Chinese medicine composition content of the present invention, take about 0.5g, accurately weighed, puts in tool plug conical flask, accurate addition methanol 10ml, weighed weight, supersound process 10 minutes, let cool, weigh, supply the weight of less loss with methanol, shake up, to obtain final product.
B, the preparation of reference substance solution: weigh calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, rosmarinic acid reference substance in right amount, accurately weighed, methanol dissolves, and obtains concentration and is respectively 0.396
mg·ml-1、1.156 mg·ml-1、0.572 mg·ml-1、0.74 mg·ml-1、1.578 mg·ml-1Mixing reference substance stock solution, to obtain final product.
C, chromatographic condition: chromatographic column is ACQUITY BEH C18, specification is 2.1 × 100mm, 1.7 μm, column temperature: 45 DEG C；Flowing phase: methanol (A)-0.2% phosphoric acid (B) solution, gradient elution (table 11), detection wavelength: 240 nm；Flow velocity: 0.2ml min-1；Sample size: 1μl。
Experimental result:
1, precision test: precision draws Working Control product solution respectively, continuous sample introduction 5 times, measure peak area, result calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, rosmarinic acid RSD are respectively 1.05%, 1.32%, 1.28%, 1.47%, 1.55%, show that instrument precision is good.
2, stability test: the same need testing solution of accurate absorption, respectively at 0,2,4,8,12,24h sample introduction measure peak area, result is in 24 hours measured, calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, the RSD of 5 kinds of compound peaks areas of rosmarinic acid are respectively 1.35%, 1.39%, 1.78%, 1.66%, 1.57%, show that need testing solution is basicly stable in 24 hours.
3, replica test: take with a collection of Chinese medicine composition sample of the present invention, 9 parts are processed by the preparation method of need testing solution under " 2.2 " item, inject Ultra Performance Liquid Chromatography instrument to measure, in results sample, calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, the RSD value of rosmarinic acid contents are respectively 1.75%, 1.81%, 2.03%, 2.58%, 2.24%, and illustration method repeatability is good.
4, recovery test
Use sample-adding recovery test method, precision weighs the Chinese medicine composition sample 0.25g same of the present invention of known content, parallel 9 parts, precision adds a certain amount of reference substance mixed solution respectively, by the preparation method of need testing solution, carry out supersound extraction respectively, filter, inject Ultra Performance Liquid Chromatography instrument to measure, calculate the response rate, calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, rosmarinic acid is respectively 99.8%, 99.9%, 101.3%, 98.6%, 100.0%, RSD value is respectively 1.56%, 1.29%, 1.36%, 1.44%, 1.85%, show that method is accurately and reliably.
5, sample determination
Take Chinese medicine composition sample of the present invention, by the preparation method of above-mentioned need testing solution, carry out supersound extraction respectively, filter, inject Ultra Performance Liquid Chromatography instrument and measure, record chromatogram；Calculating calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, the content of rosmarinic acid under 240nm, assay the results are shown in Table 12.
Result shows, this analysis method specificity is strong, and precision is high, and result is accurate, stable, reproducible, sensitivity high, it is adaptable to the quality control of Chinese medicine composition of the present invention.
Claims (10)
1. the content assaying method of a Chinese medicine composition, this Chinese medicine composition is made up of the crude drug of following weight ratio: Radix Astragali 30-150 part, Talcum 7-28 part, Spica Prunellae 10-40 part, Fructus Ligustri Lucidi 7-28 part, Semen Litchi 10-40 part, succinum 1-4 part, Cortex Cinnamomi 1.5-6 part, Cortex Phellodendri 3-15 part, it is characterized in that, described content assaying method is to use UPLC method to measure calycosin glucoside in this Chinese medicine composition, specnuezhenside, palmatine hydrochloride, berberine hydrochloride and the content of five kinds of compositions of rosmarinic acid simultaneously.
Content assaying method the most according to claim 1, it is characterised in that described content assaying method comprises the steps of:
A, the preparation method of need testing solution: weigh described Chinese medicine composition content, finely ground, take 0.5-3 g, accurately weighed, put in tool plug conical flask, accurate addition 50-100% methanol 10-75 ml, weighed weight, supersound process 10-60 minute, let cool, weigh, supply the weight of less loss with 50-100% methanol, shake up, to obtain final product；
B, the preparation method of reference substance solution: weigh calycosin glucoside, specnuezhenside, palmatine hydrochloride, berberine hydrochloride, rosmarinic acid reference substance in right amount, accurately weighed, methanol dissolves, and must mix reference substance stock solution；
C, chromatographic condition: chromatographic column: C18Post, column temperature: 35 DEG C-45 DEG C；Mobile phase A is acetonitrile or methanol, and Mobile phase B is: phosphate aqueous solution or aqueous formic acid, and gradient elution ratio is: 0 ~ 2min mobile phase A is by 15% to 20%, and Mobile phase B is by 85% to 80%；2 ~ 7min mobile phase A is by 20% to 30%, and Mobile phase B is by 80% to 70%；7 ~ 10min mobile phase A is by 30% ~ 32%, and Mobile phase B is by 70% to 68%；10 ~ 12min mobile phase A is by 32% to 35%, and Mobile phase B is by 68% to 65%；12 ~ 14min mobile phase A is 35%, and Mobile phase B is 65%；Detection wavelength: 240 nm；Flow velocity: 0.2ml min-1；
D, mensuration: draw need testing solution respectively, reference substance solution each 0.2-5 L injects in UPLC liquid phase, with calculated by peak area sample size.
Content assaying method the most according to claim 2, it is characterised in that:
The preparation method of step A need testing solution is: weigh described Chinese medicine composition content, finely ground, takes 1 g, accurately weighed, puts in tool plug conical flask, accurate addition 80% methanol 25 ml, weighed weight, supersound process 30 minutes, let cool, weigh, supply the weight of less loss with 80% methanol, shake up, to obtain final product.
4. according to the content assaying method described in Claims 2 or 3, it is characterised in that: chromatographic column model described in step C is Phenomenex
UPLC Kinetex C18, specification 2.1 × 100 mm, 2.6 μm；Or Waters
Acquity UPLC
Shield RP18, specification 2.1 × 100 mm, 1.7μm；Or ACQUITY BEH C18 , specification is 2.1 × 100mm, 1.7 μm.
5. according to the content assaying method described in Claims 2 or 3, it is characterised in that: mobile phase A described in step C is methanol, and Mobile phase B is 0.1% phosphate aqueous solution；Or mobile phase A is methanol, Mobile phase B is 0.2% aqueous formic acid；Or mobile phase A is acetonitrile, Mobile phase B is 0.2% aqueous formic acid；Or mobile phase A is acetonitrile, Mobile phase B is 0.1% phosphate aqueous solution.
6. according to the content assaying method described in any one of claim 1-3, it is characterised in that the weight ratio of described traditional Chinese medicinal composition raw materials is: the Radix Astragali 70 parts, 14 parts of Talcum, Spica Prunellae 21 parts, Fructus Ligustri Lucidi 14 parts, Semen Litchi 21 parts, succinum 2.1 parts, Cortex Cinnamomi 2.8 parts, Cortex Phellodendri 7 parts.
7. according to the content assaying method described in any one of claim 1-3, it is characterised in that the weight ratio of described traditional Chinese medicinal composition raw materials is: the Radix Astragali 30 parts, 7 parts of Talcum, Spica Prunellae 10 parts, Fructus Ligustri Lucidi 7 parts, Semen Litchi 10 parts, succinum 1 part, Cortex Cinnamomi 1.5 parts, Cortex Phellodendri 3 parts.
8. according to the content assaying method described in any one of claim 1-3, it is characterised in that the active component of described Chinese medicine composition is made up of following steps:
(1) weigh Chinese crude drug by crude drug part by weight, clean respectively, pulverize；
(2) taking the Radix Astragali, Cortex Phellodendri, add 50-70% ethanol, heating and refluxing extraction 1-3 time, each 0.5-2 hour, extracting solution filtered, and merges, after decompression recycling ethanol, is condensed into the clear paste that relative density is 1.20-1.25, standby；
(3) taking Fructus Ligustri Lucidi, add 6-10 times amount 70-90% ethanol, heating and refluxing extraction 1-3 time, the time is each 1-3 hour, and extracting solution filters, and merges, after decompression recycling ethanol, is condensed into the clear paste that relative density is 1.20-1.25, standby；
(4) take Cortex Cinnamomi, add 4-8 times amount water soaking after 1-2 hour, extract volatile oil 2-6 hour,
The another device of volatile oil is collected, and aqueous extract is collected by filtration, and residue is standby；
(5) take Spica Prunellae, Semen Litchi and step (4) residue obtained, add 8-10 times amount water, decoct twice, 1-3 hour for the first time, 1-2 hour for the second time, extracting solution filtered, and merged, add step (4) obtained aqueous solution, be condensed into the clear paste that relative density is 1.20-1.25, standby；
(6) take Talcum, succinum, be ground into 100 mesh fine powders, standby；
Step (2) gained clear paste, step (3) gained clear paste, step (4) gained volatile oil, step (5) gained clear paste and step (6) gained fine powder collectively form the active component of Chinese medicine composition of the present invention.
9. according to the content assaying method described in any one of claim 1-3, it is characterised in that the preparation formulation of described Chinese medicine composition is capsule, tablet, oral liquid or pill.
Content assaying method the most according to claim 9, it is characterised in that described capsule is to comprise the steps of:
(1) taking above-mentioned Chinese crude drug, clean respectively, pulverize, amount weighs in proportion；
(2) Radix Astragali, Cortex Phellodendri are taken, add the 50-70% ethanol of 6-12 times, heating and refluxing extraction 1-3 time, extract 1-3 hour every time, extracting solution filters, merge, after decompression recycling ethanol, be condensed into the clear paste that relative density is 1.20-1.25, put into vacuum drying oven, drying under reduced pressure under the conditions of temperature 60-70 DEG C, vacuum 0.04-0.06Mpa, dry cream is standby；
(3) Fructus Ligustri Lucidi is taken, adding 6-10 times amount 70-90% ethanol, heating and refluxing extraction 1-3 time, the time is each 1-3 hour, extracting solution filters, merge, after decompression recycling ethanol, be condensed into the clear paste that relative density is 1.20-1.25, put into vacuum drying oven, drying under reduced pressure under the conditions of temperature 60-70 DEG C, vacuum 0.04-0.06 Mpa, dry cream is standby；
(4) take Cortex Cinnamomi, add 4-8 times amount water soaking after 1-2 hour, extract volatile oil 2-6 hour,
The another device of volatile oil is collected, and oil yield must not be less than l.0%, and aqueous extract is collected by filtration, and residue is standby；
(5) taking the residue after Spica Prunellae, Semen Litchi and Cortex Cinnamomi carry oil, add 8-10 times amount water, decoct twice, 1-3 hour for the first time, 1-2 hour for the second time, extracting solution filtered, and merged, and adds Cortex Cinnamomi and carries
Aqueous solution after oil, is condensed into the clear paste that relative density is 1.20-1.25, puts into vacuum drying oven, drying under reduced pressure under the conditions of temperature 60-70 DEG C, vacuum 0.04-0.06Mpa, and it is standby that dry cream and the Radix Astragali, Cortex Phellodendri, Fructus Ligustri Lucidi dried cream powder are broken into 100 mesh powder；
(6) take Talcum, succinum, be ground into 100 mesh powder, standby；
(7) take dried cream powder, former medicated powder and starch, be mixed evenly, be binding agent with 80% ethanol, High Shear Mixer Granulator, 60-70 DEG C of drying, granulate；
(8) sift out fine powder, spray into volatile oil, mix homogeneously with granule, airtight, encapsulated, to obtain final product.
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