CN108267529A - The method of quality control of Trauma Yellow-water preparation - Google Patents

The method of quality control of Trauma Yellow-water preparation Download PDF

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Publication number
CN108267529A
CN108267529A CN201810240689.3A CN201810240689A CN108267529A CN 108267529 A CN108267529 A CN 108267529A CN 201810240689 A CN201810240689 A CN 201810240689A CN 108267529 A CN108267529 A CN 108267529A
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China
Prior art keywords
solution
trauma
yellow
cape jasmine
coptis
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Chinese (zh)
Inventor
刘效仿
郑芳昊
陈韵姿
余俊文
李子鸿
李怀国
刘东文
杨雄辉
冼少华
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FOSHAN TRADITIONAL CHINESE MEDICINE HOSPITAL
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FOSHAN TRADITIONAL CHINESE MEDICINE HOSPITAL
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Priority to CN201810240689.3A priority Critical patent/CN108267529A/en
Publication of CN108267529A publication Critical patent/CN108267529A/en
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/04Preparation or injection of sample to be analysed
    • G01N30/06Preparation
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/88Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90Plate chromatography, e.g. thin layer or paper chromatography
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/88Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample

Abstract

The method of quality control of Trauma Yellow-water preparation, includes the following steps:(1) hydrochloric acid phellodendrine, Jatrorrhizine chloride, hydrochloric acid epiberberine, hydrochloric acid coptisine, palmatin hydrochloride, Berberine hydrochloride, Gardenoside and chlorogenic acid are weighed, adds absolute methanol or methanol aqueous solution, reference substance solution is made;(2) content of Trauma Yellow-water preparation to be determined is taken, absolute methanol or methanol aqueous solution dissolving is added in, filters and take subsequent filtrate as test solution;(3) reference substance solution and 1~5 μ L of test solution are taken respectively, the combination instrument of injection liquid chromatography mass is detected, and the content of phellodendrine, jateorrhizine, epiberberine, coptisine, palmatine, jamaicin, Gardenoside and chlorogenic acid is included by calculating the acquisition of quantitative chromatographic figure;The present invention enables the quantitative assessing index being short of originally with primary quantitative determination to complete;The quantitative chromatographic figure of this method can be shown that wave crest separation is good, thus method differentiates, is quick, is accurate.

Description

The method of quality control of Trauma Yellow-water preparation
Technical field
The present invention relates to Quality Control Technology field more particularly to the method for quality control of Trauma Yellow-water preparation.
Background technology
Trauma Yellow-water is the product of applicant's exploitation, and related patents (patent announcement number was obtained in 2000: 96123078.9), but because it is clinical proved recipe, the research work all the time in relation to variety and quality evaluation is not opened deeply Exhibition, leads to preparation production technique relative poor, quality standard is relatively low, and clinical efficacy is difficult to control.It it is particularly noteworthy that should Preparation compound composition is complicated, and material base is unknown, and still perception official judges in addition at present in terms of quality standard and Qualitive test is (thin Layer chromatography) based on, still lack the specific index ingredient of feature, quality evaluation system demands perfection urgently.
We combine pharmacological action and physicochemical property of Trauma Yellow-water etc., using technologies such as liquid chromatogram, mass spectrums to traumatology The main constituents and content of yellow water are studied.Pass through liquid chromatography tandem Fourier Transform Mass Spctrometry (LC-FTICR- MS) technology, we analyze the first mass spectrometric and second order ms of Trauma Yellow-water main component, according to level-one parent ion molecular weight (very high resolution 0.0001Da) and corresponding two level fragmentation data are compared with reference substance, database or document, to the system Agent has carried out qualitative detection.As a result it finds altogether and confirms 20 kinds of ingredients in compound, also big quantity of material parent ion molecular weight energy Enough matchings, but since chromatogram is not ideal enough, do not include into result.
Invention content
It is an object of the invention to propose using LC-MS and HPLC technologies, establish jamaicin, epiberberine, palmatine, Jateorrhizine, coptisine, phellodendrine, chlorogenic acid and Gardenoside ingredient content assaying method, and carried out methodological study.By Compare repeatedly, it is found that LC-MS can measure the jamaicin in Trauma Yellow-water, epiberberine, palmatine, jateorrhizine, the coptis simultaneously Totally 8 kinds of component contents such as alkali, phellodendrine, chlorogenic acid, Gardenoside, it is as a result reliable and stable, it can refer to as the Quality Control of Trauma Yellow-water Mark
For this purpose, the present invention uses following technical scheme:
The method of quality control of Trauma Yellow-water preparation, the composition of Trauma Yellow-water preparation include:The coptis, Cortex Phellodendri, cape jasmine, purple Grass, peppermint and alum;The method of quality control includes the following steps:
(1) hydrochloric acid phellodendrine, Jatrorrhizine chloride, hydrochloric acid epiberberine, hydrochloric acid coptisine, palmatin hydrochloride, hydrochloric acid are weighed Jamaicin, Gardenoside and chlorogenic acid add absolute methanol or methanol aqueous solution, reference substance solution are made;
(2) content of Trauma Yellow-water preparation to be determined is taken, adds in absolute methanol or methanol aqueous solution dissolving, filtering is simultaneously Subsequent filtrate is taken as test solution;
(3) reference substance solution and 1~5 μ L of test solution are taken respectively, inject the combination of liquid chromatography-mass spectrography Instrument is detected, and is obtained respectively to phellodendrine, jateorrhizine, table barberry in the reference substance solution and the test solution The quantitative chromatographic figure of alkali, coptisine, palmatine, jamaicin, Gardenoside and chlorogenic acid totally 8 kinds of compositions;The reference substance solution with Test solution appearance on identical position;And pass through and be calculated including phellodendrine, jateorrhizine, epiberberine, Huang The even content of alkali, palmatine, jamaicin, Gardenoside and chlorogenic acid.
Illustrate further, in mass spectrographic condition, phellodendrine mass-to-charge ratio be 342.3 → 192.1, wherein go cluster voltage 80~ 120, impact energy 30~50;
The mass-to-charge ratio of jateorrhizine is 339.3 → 323.2 or 339.3 → 295.2, wherein going cluster voltage 80~120, impact energy 30~50;
The mass-to-charge ratio of epiberberine is 336.1 → 320.2 or 336.1 → 292.1, wherein removing cluster voltage 80~120, is collided Energy 30~50;
The mass-to-charge ratio of coptisine is 320.2 → 292.0, wherein cluster voltage 80~120 is removed, impact energy 30~50;
The mass-to-charge ratio of palmatine is 352. → 336.1 or 352. → 308.2, wherein cluster voltage 80~120 is removed, impact energy 30 ~50;
The mass-to-charge ratio of jamaicin is 336.1 → 320.1 or 336.1 → 292.1, wherein going cluster voltage 80~120, impact energy 30~50;
The mass-to-charge ratio of Gardenoside is 411.2 → 217.4, wherein cluster voltage 80~120 is removed, impact energy 30~50;
The mass-to-charge ratio of chlorogenic acid is 355.2 → 163.2, wherein cluster voltage 80~120 is removed, impact energy 30~50.
It illustrates further, the condition of liquid chromatogram is:Using octadecylsilane chemically bonded silica as stationary phase;Mobile phase A Including aqueous formic acid;Mobile phase B includes methanol;
Gradient elution is carried out using the mobile phase A and the Mobile phase B:
0~15min, the mobile phase A:70%, the Mobile phase B:30%;
15~20min, the mobile phase A:60~70%, the Mobile phase B:30~40%;
After 20min, the mobile phase A:60~85%, the Mobile phase B:15~40%;
The aqueous formic acid concentration of volume percent is:0.1~0.4%;The flow rate of mobile phase condition of liquid chromatogram is: 0.1~1.0mL/min, the condition of liquid chromatogram column temperature are:25~35 DEG C.
It illustrates further, electric spray ion source (ESI) temperature condition in mass spectrum is 300~380 DEG C;It is atomized in mass spectrum Gas N2Flow conditions are 7.0~11.0L/min.
It illustrates further, hydrochloric acid phellodendrine, Jatrorrhizine chloride, hydrochloric acid epiberberine, hydrochloric acid in the reference substance solution Coptisine, palmatin hydrochloride, Berberine hydrochloride, Gardenoside, chlorogenic acid concentration are 100 μ g/mL.
It illustrates further, the concentration of volume percent of the methanol aqueous solution is:75%~100%.
It illustrates further, which is characterized in that further include the thin-layer identification method of the coptis;The thin-layer identification method packet of the coptis Include following steps:
(1) Trauma Yellow-water 10mL is taken, adds n-butanol shaking extraction 2 times, each 15mL, merges n-butanol liquid, put in water-bath It is evaporated;Ethyl alcohol 10mL is added in residue and makes its dissolving, as indentification by TLC test sample;
(2) it takes the control medicinal material of the coptis and adds water;It is heated to reflux;It cools down and filters;In filtrate plus n-butanol dilution is molten Solution, as coptis control sample;
(3) Cortex Phellodendri, cape jasmine, Asian puccoon, peppermint and alum, heating extraction 1.5 hours are weighed by the proportioning of Trauma Yellow-water preparation After filter;Concentration, constant volume is to get liquid;10ml liquids are taken, adds n-butanol shaking extraction 2 times, each 15mL, merges n-butanol Solution is put and is evaporated in water-bath;Ethyl alcohol 10mL is added in residue and makes its dissolving, is prepared into coptis negative control sample;
(4) it is tested according to thin-layered chromatography, the coptis control of aspiration step (1) indentification by TLC test sample, step (2) Sample and each 2 μ L of step (3) coptis negative control sample are put respectively on same silica gel g thin-layer plate, with toluene-acetic acid Ethyl ester-isopropanol-methanol-liquor ammoniae fortis is solvent, is put in the pre-saturated expansion cylinder of ammonia steam, treats indentification by TLC for examination Product, coptis control sample and coptis negative control sample are unfolded in silica gel g thin-layer plate, open up and are taken out away from 10cm, are dried;
(5) silica gel g thin-layer plate of step (4) is placed under the ultraviolet lamp of 365nm and inspected;
On silica gel g thin-layer plate, indentification by TLC test sample is compared respectively in position corresponding with control medicinal material chromatography On, the fluorescence spot of aobvious same color;In coptis negative control sample chromatography, in the chromatography corresponding position of coptis control sample On, it has no corresponding spot, shows to contain coptis ingredient in indentification by TLC test sample.
It illustrates further, the proportionate relationship of the toluene-ethyl acetate-isopropanol-methanol-liquor ammoniae fortis is 6:3: 1.5:1.5:0.6。
It illustrates further, further includes the thin-layer identification method of cape jasmine and Gardenoside;The thin layer of cape jasmine and Gardenoside differentiates Method includes the following steps:
(1) Trauma Yellow-water 10mL is taken, adds n-butanol shaking extraction 2 times, each 15mL, merges n-butanol liquid, put in water-bath It is evaporated;Ethyl alcohol 10mL is added in residue and makes its dissolving, as indentification by TLC test sample;
(2) control medicinal material of cape jasmine is taken to add water;It is heated to reflux;It cools down and filters;It is molten that n-butanol dilution is added in filtrate Solution, as cape jasmine control medicinal material solution;
(3) reference substance of Gardenoside is taken, ethyl alcohol is added to dissolve, as Gardenoside reference substance solution;
(4) coptis, Cortex Phellodendri, Asian puccoon, peppermint and alum are taken by the proportioning of Trauma Yellow-water preparation, heating extraction is after 1.5 hours Filtering;Concentration, constant volume is to get liquid;10ml liquids are taken, adds n-butanol shaking extraction 2 times, each 15mL, it is molten to merge n-butanol Liquid is put and is evaporated in water-bath;Ethyl alcohol 10mL is added in residue and makes its dissolving, and the cape jasmine negative control sample without cape jasmine is made;
(5) it is tested according to thin-layered chromatography, draws above-mentioned indentification by TLC test sample, cape jasmine control medicinal material solution, cape jasmine Glycosides reference substance solution and each 5~10 μ L of cape jasmine negative control sample are put respectively on same silica gel g thin-layer plate, with ethyl acetate- The upper solution of n-butanol-water is solvent, is put in the pre-saturated expansion cylinder of ammonia steam, treats above-mentioned indentification by TLC for examination Product, cape jasmine control medicinal material solution, Gardenoside reference substance solution and cape jasmine negative control sample are unfolded on silica gel g thin-layer plate, exhibition Away from about 10cm, take out, dry;
(6) silica gel g thin-layer plate of step (5) is sprayed with 10% ethanol solution of sulfuric acid, it is clear is heated to spot development at 110 DEG C It is clear;
Wherein, on silica gel g thin-layer plate, indentification by TLC test sample, compare respectively with cape jasmine control medicinal material solution And the position of Gardenoside reference substance solution, the spot of aobvious same color;And in cape jasmine negative control sample chromatography, it is compareed in cape jasmine On the chromatography corresponding position of medicinal material solution and Gardenoside reference substance solution, corresponding spot is had no, show that indentification by TLC supplies Contain Compositions within Gardenia Jasminoides in test product.
It illustrates further, the proportionate relationship of the ethyl acetate-n-butanol-water is 1:2:1.
Beneficial effects of the present invention:
1st, the present invention makes the quantitative assessing index being short of originally, can be completed with primary quantitative determination;
2nd, the present invention in reference substance solution in the chromatogram of test solution on identical position appearance;It detects respectively With the quantitative chromatographic figure for calculating 8 kinds of ingredients of gained, show that wave crest separation is good, thus method differentiates, is quick, is accurate, reappearing, It is easy to universal grasp, effectively increases detection efficiency
Description of the drawings
Fig. 1 is the chromatogram of the thin-layer identification method of the coptis of the present invention;
Wherein, 1~3 is indentification by TLC test sample;4 be coptis reference substance solution;5 be coptis negative control sample;
Fig. 2 is the chromatogram of the thin-layer identification method of cape jasmine of the present invention and Gardenoside;
Wherein 1~3 is indentification by TLC test sample;4 be cape jasmine control medicinal material solution;5 be Gardenoside reference substance solution; 6 be cape jasmine negative control sample;
Fig. 3 a test the chromatogram of phellodendrine;
Fig. 3 b test the chromatogram of jateorrhizine;
Fig. 3 c test the chromatogram of epiberberine and jamaicin;
Fig. 3 d test the chromatogram of coptisine;
Fig. 3 e test the chromatogram of palmatine;
Fig. 3 f test the chromatogram of Gardenoside;
Fig. 3 g test the chromatogram of chlorogenic acid;
Wherein, in 3a~3g, A negative controls;B reference substance solutions;C test solutions.
J solvent fronts.
Specific embodiment
Technical solution to further illustrate the present invention below with reference to the accompanying drawings and specific embodiments.
The method of quality control of Trauma Yellow-water preparation, the composition of Trauma Yellow-water preparation include:The coptis, Cortex Phellodendri, cape jasmine, purple Grass, peppermint and alum;The method of quality control includes the following steps:
(1) hydrochloric acid phellodendrine, Jatrorrhizine chloride, hydrochloric acid epiberberine, hydrochloric acid coptisine, palmatin hydrochloride, hydrochloric acid are weighed Jamaicin, Gardenoside and chlorogenic acid add absolute methanol or methanol aqueous solution, reference substance solution are made;
(2) content of Trauma Yellow-water preparation to be determined is taken, adds in absolute methanol or methanol aqueous solution dissolving, filtering is simultaneously Subsequent filtrate is taken as test solution;
(3) reference substance solution and 1~5 μ L of test solution are taken respectively, inject the combination of liquid chromatography-mass spectrography Instrument is detected, and is obtained respectively to phellodendrine, jateorrhizine, table barberry in the reference substance solution and the test solution The quantitative chromatographic figure of alkali, coptisine, palmatine, jamaicin, Gardenoside and chlorogenic acid totally 8 kinds of compositions;The reference substance solution with Test solution appearance on identical position;And pass through and be calculated including phellodendrine, jateorrhizine, epiberberine, Huang The even content of alkali, palmatine, jamaicin, Gardenoside and chlorogenic acid.
It illustrates further, as figure this method by LC-MS technology (LC-MS) while measures the reference substance solution With the test solution Berberine, epiberberine, palmatine, jateorrhizine, coptisine, phellodendrine, chlorogenic acid and Gardenoside Totally 8 kinds of component contents make the quantitative assessing index being short of originally, can be completed with primary quantitative determination;The experiment of this method As a result it is:The reference substance solution and appearance, detection and calculating on identical position in the chromatogram of the test solution The quantitative chromatographic figure of 8 kinds of ingredients of gained shows that wave crest separation is good, thus method differentiates, is quick, is accurate, reappearing, and is easy to general And grasp, effectively increase detection efficiency.
Illustrate further, in mass spectrographic condition, phellodendrine mass-to-charge ratio be 342.3 → 192.1, wherein go cluster voltage 80~ 120, impact energy 30~50;
The mass-to-charge ratio of jateorrhizine is 339.3 → 323.2 or 339.3 → 295.2, wherein going cluster voltage 80~120, impact energy 30~50;
The mass-to-charge ratio of epiberberine is 336.1 → 320.2 or 336.1 → 292.1, wherein removing cluster voltage 80~120, is collided Energy 30~50;
The mass-to-charge ratio of coptisine is 320.2 → 292.0, wherein cluster voltage 80~120 is removed, impact energy 30~50;
The mass-to-charge ratio of palmatine is 352. → 336.1 or 352. → 308.2, wherein cluster voltage 80~120 is removed, impact energy 30 ~50;
The mass-to-charge ratio of jamaicin is 336.1 → 320.1 or 336.1 → 292.1, wherein going cluster voltage 80~120, impact energy 30~50;
The mass-to-charge ratio of Gardenoside is 411.2 → 217.4, wherein cluster voltage 80~120 is removed, impact energy 30~50;
The mass-to-charge ratio of chlorogenic acid is 355.2 → 163.2, wherein cluster voltage 80~120 is removed, impact energy 30~50.
It illustrates further, wherein the value change sign of " → " for the mass-to-charge ratio of determinand in mass spectrum.
Above-mentioned parameter can influence the abundance of molecular ion, because during ion transmits, with the decline of temperature, The ion ionized may be gathered in once again, we will can't detect in this way, so as to reduced sensitivity;Wherein, it goes Cluster voltage cannot be excessive, excessive to cause to crack in source, can equally lower the abundance of molecular ion peak.
It illustrates further, the condition of liquid chromatogram is:Using octadecylsilane chemically bonded silica as stationary phase;Mobile phase A Including aqueous formic acid;Mobile phase B includes methanol;
Gradient elution is carried out using the mobile phase A and the Mobile phase B:
0~15min, the mobile phase A:70%, the Mobile phase B:30%;
15~20min, the mobile phase A:60~70%, the Mobile phase B:30~40%;
After 20min, the mobile phase A:60~85%, the Mobile phase B:15~40%;
The aqueous formic acid concentration of volume percent is:0.1~0.4%;The flow rate of mobile phase condition of liquid chromatogram is: 0.1~1.0mL/min, the condition of liquid chromatogram column temperature are:25~35 DEG C.
It illustrates further, carries out separating degree and detection sensitivity that gradient elution is conducive to improve liquid chromatogram.
It illustrates further, electric spray ion source (ESI) temperature condition in mass spectrum is 300~380 DEG C;It is atomized in mass spectrum Gas N2Flow conditions are 7.0~11.0L/min.
It illustrates further, hydrochloric acid phellodendrine, Jatrorrhizine chloride, hydrochloric acid epiberberine, hydrochloric acid in the reference substance solution Coptisine, palmatin hydrochloride, Berberine hydrochloride, Gardenoside, chlorogenic acid concentration are 100 μ g/mL.
It illustrates further, the concentration of volume percent of the methanol aqueous solution is:75%~100%.
It illustrates further, which is characterized in that further include the thin-layer identification method of the coptis;The thin-layer identification method packet of the coptis Include following steps:
(1) Trauma Yellow-water 10mL is taken, adds n-butanol shaking extraction 2 times, each 15mL, merges n-butanol liquid, put in water-bath It is evaporated;Ethyl alcohol 10mL is added in residue and makes its dissolving, as indentification by TLC test sample;
(2) it takes the control medicinal material of the coptis and adds water;It is heated to reflux;It cools down and filters;In filtrate plus n-butanol dilution is molten Solution, as coptis control sample;
(3) Cortex Phellodendri, cape jasmine, Asian puccoon, peppermint and alum, heating extraction 1.5 hours are weighed by the proportioning of Trauma Yellow-water preparation After filter;Concentration, constant volume is to get liquid;10ml liquids are taken, adds n-butanol shaking extraction 2 times, each 15mL, merges n-butanol Solution is put and is evaporated in water-bath;Ethyl alcohol 10mL is added in residue and makes its dissolving, is prepared into coptis negative control sample;
(4) it is tested according to thin-layered chromatography, the coptis control of aspiration step (1) indentification by TLC test sample, step (2) Sample and each 2 μ L of step (3) coptis negative control sample are put respectively on same silica gel g thin-layer plate, with toluene-acetic acid Ethyl ester-isopropanol-methanol-liquor ammoniae fortis is solvent, is put in the pre-saturated expansion cylinder of ammonia steam, treats indentification by TLC for examination Product, coptis control sample and coptis negative control sample are unfolded in silica gel g thin-layer plate, open up and are taken out away from 10cm, are dried;
(5) silica gel g thin-layer plate of step (4) is placed under the ultraviolet lamp of 365nm and inspected;
On silica gel g thin-layer plate, indentification by TLC test sample is compared respectively in position corresponding with control medicinal material chromatography On, the fluorescence spot of aobvious same color;In coptis negative control sample chromatography, in the chromatography corresponding position of coptis control sample On, it has no corresponding spot, shows in indentification by TLC test sample containing coptis ingredient, such as Fig. 1.
It illustrates further, has been carried out in step (1) plus n-butanol shakes and extracts the operation of 2 times;Because Trauma Yellow-water Preparation is impure hybrid medicine, adds in n-butanol and extracts and is conducive to the impurity of Trauma Yellow-water being dissolved in n-butanol simultaneously for 2 times Separation.
It illustrates further, the proportionate relationship of the toluene-ethyl acetate-isopropanol-methanol-liquor ammoniae fortis is 6:3: 1.5:1.5:0.6。
It illustrates further, further includes the thin-layer identification method of cape jasmine and Gardenoside;The thin layer of cape jasmine and Gardenoside differentiates Method includes the following steps:
(1) Trauma Yellow-water 10mL is taken, adds n-butanol shaking extraction 2 times, each 15mL, merges n-butanol liquid, put in water-bath It is evaporated;Ethyl alcohol 10mL is added in residue and makes its dissolving, as indentification by TLC test sample;
(2) control medicinal material of cape jasmine is taken to add water;It is heated to reflux;It cools down and filters;It is molten that n-butanol dilution is added in filtrate Solution, as cape jasmine control medicinal material solution;
(3) reference substance of Gardenoside is taken, ethyl alcohol is added to dissolve, as Gardenoside reference substance solution;
(4) coptis, Cortex Phellodendri, Asian puccoon, peppermint and alum are taken by the proportioning of Trauma Yellow-water preparation, heating extraction is after 1.5 hours Filtering;Concentration, constant volume is to get liquid;10ml liquids are taken, adds n-butanol shaking extraction 2 times, each 15mL, it is molten to merge n-butanol Liquid is put and is evaporated in water-bath;Ethyl alcohol 10mL is added in residue and makes its dissolving, and the cape jasmine negative control sample without cape jasmine is made;
(5) it is tested according to thin-layered chromatography, draws above-mentioned indentification by TLC test sample, cape jasmine control medicinal material solution, cape jasmine Glycosides reference substance solution and each 5~10 μ L of cape jasmine negative control sample are put respectively on same silica gel g thin-layer plate, with ethyl acetate- The upper solution of n-butanol-water is solvent, is put in the pre-saturated expansion cylinder of ammonia steam, treats above-mentioned indentification by TLC for examination Product, cape jasmine control medicinal material solution, Gardenoside reference substance solution and cape jasmine negative control sample are unfolded on silica gel g thin-layer plate, exhibition Away from about 10cm, take out, dry;
(6) silica gel g thin-layer plate of step (5) is sprayed with 10% ethanol solution of sulfuric acid, it is clear is heated to spot development at 110 DEG C It is clear;
Wherein, on silica gel g thin-layer plate, indentification by TLC test sample, compare respectively with cape jasmine control medicinal material solution And the position of Gardenoside reference substance solution, the spot of aobvious same color;And in cape jasmine negative control sample chromatography, it is compareed in cape jasmine On the chromatography corresponding position of medicinal material solution and Gardenoside reference substance solution, corresponding spot is had no, show that indentification by TLC supplies Contain Compositions within Gardenia Jasminoides, such as Fig. 2 in test product.
It illustrates further, the proportionate relationship of the ethyl acetate-n-butanol-water is 1:2:1.
Embodiment 1:
It is prepared by sample
(1):Weigh hydrochloric acid phellodendrine, Jatrorrhizine chloride, hydrochloric acid epiberberine, hydrochloric acid coptisine, palmatin hydrochloride, hydrochloric acid Jamaicin, Gardenoside and chlorogenic acid add absolute methanol or methanol aqueous solution, reference substance solution are made.
(2):Trauma Yellow-water dosage contents are taken, absolute methanol or methanol aqueous solution dissolving is added in, filters and take subsequent filtrate As test solution.
Verification experimental verification
(1) specificity is tested
1 part of (2) test solution of the step of taking same concentration (being equivalent to 100% concentration level), step (1) reference substance are molten 1 part of 1 part of liquid and negative control solution are measured.Record chromatogram, the specificity of illustration method.As a result such as Fig. 3 a~3g institutes Show, under same testing conditions, for negative control without significantly interfering with, specificity is strong.Reference substance is with test sample in same appearance time Occur respectively phellodendrine, jateorrhizine, epiberberine, coptisine, palmatine, jamaicin, Gardenoside, chlorogenic acid etc. totally 8 kinds into The chromatographic peak divided, appearance time is suitable, and peak shape is symmetrical, and separating degree is high.
(2) linear relationship is investigated
Take the reference substance solution, be prepared into 1 through accurate dilution, 10,50,100,200,500, the series of 1000ng/mL Reference substance working solution.With a concentration of abscissa of reference substance, reference substance peak area is ordinate, carries out linear regression.Experiment knot Fruit is as shown in table 1, and each alkaloid concentrations are good in 1~1000ng/mL linear relationships, R2(coefficient of determination) is all higher than 0.999, can For the assay of 8 kinds of active ingredient such as phellodendrine in sample.
The standard curve table of 8 kinds of ingredients such as 1 phellodendrine of table
(3) accuracy test
The test solution of same concentration (being equivalent to 100% concentration level) is taken, with the knot at least measuring 6 parts of samples Fruit is evaluated, and the addition of the reference substance solution and the ratio between component amount to be determined in test sample is taken to control 1:1 or so. Test solution is prepared, records the peak area value of each ingredient to be measured.Experimental result is as shown in table 2,6 parts of each ingredients of test sample plus For the sample rate of recovery between 95~115%, RSD% is respectively less than 5%, illustrates that sample handling processes influence smaller, accuracy of measurement It is higher.
2 accuracy test result of table
(4) precision test
A repeatability:
Under the same conditions, it is taken by same analysis personnel with a uniformly test sample (being equivalent to 100% concentration level), It is evaluated with 6 parts of result is at least measured, reference substance addition and the ratio between component amount to be determined is taken in test sample to control exist 1:1 or so.Test solution is prepared, records the peak area value of each ingredient to be measured.Experimental result is as shown in table 3, same test sample For the sample recovery rate that each ingredient METHOD FOR CONTINUOUS DETERMINATION is 6 times between 95~115%, RSD% is respectively less than 5%, illustrates the sample of this research Product processing method and the instrument parameter repeatability established are good.
3 repetitive test result of table
B Intermediate precisions:
In same laboratory, two different analysis personnel, respectively according to aforementioned " repeatability " experimental implementation, with same Instrument carries out assay.Experimental result is as shown in table 4, different operation personnel each ingredient METHOD FOR CONTINUOUS DETERMINATION 6 times to same test sample Sample recovery rate between 95~115%, RSD% is respectively less than 5%, illustrate this research sample treatment and establish Instrument parameter Intermediate precision is higher.
4 Intermediate precision result of the test of table
(5) durability is investigated:
A stability tests:
With portion test solution (being equivalent to 100% concentration level), same analysis personnel prepare test solution, Respectively 0,2,4,8,12, sample introduction for 24 hours, record the peak area value of each ingredient to be measured.Experimental result is as shown in table 5,8 in test sample Kind ingredient is stablized in interior property for 24 hours, and RSD% is respectively less than 5%, illustrates in the LC-MS analysis methods that can be established with this research interior for 24 hours It measures.
5 stability test result of table
The influence of b chromatographic columns
The test sample of same concentration (being equivalent to 100% concentration level) is taken, XBridge C18, Welch is respectively adopted XtimateTM C18、Waters MS C18 chromatographic columns are measured, and record the peak area value of each ingredient to be measured.It is real Test that the results are shown in Table 6, different manufacturers same class can be used in sample treatment and the chromatographic condition established using this research The chromatographic column of type is carried out at the same time 8 kinds of ingredients in Trauma Yellow-water measure, and RSD% is less than 5%, and system suitability is good.
The influence of 6 chromatographic column of table
(6) LC-MS measures the content of 8 kinds of ingredients in 3 batch Trauma Yellow-water samples simultaneously
Using the LC-MS content assaying methods of above-mentioned foundation, 3 different batches (lot numbers of Trauma Yellow-water are measured:1721506、 1721614th, 1721724) test sample, each sample is 3 parts parallel, records the peak area value of each ingredient to be measured.Experimental result is such as Shown in table 7, the measurement result of 3 each ingredients of different batches sample is more consistent, and RSD% values are respectively less than 5%.
The measurement result of 8 kinds of ingredients in 73 batch Trauma Yellow-water sample of table
The technical principle of the present invention is described above in association with specific embodiment.These descriptions are intended merely to explain the present invention's Principle, and it cannot be construed to limiting the scope of the invention in any way.Based on explanation herein, the technology of this field Personnel would not require any inventive effort the other specific embodiments that can associate the present invention, these modes are fallen within Within protection scope of the present invention.

Claims (10)

1. the method for quality control of Trauma Yellow-water preparation, Trauma Yellow-water preparation includes:The coptis, Cortex Phellodendri, cape jasmine, Asian puccoon, peppermint and Alum;It is characterized in that, the method for quality control includes the following steps:
(1) hydrochloric acid phellodendrine, Jatrorrhizine chloride, hydrochloric acid epiberberine, hydrochloric acid coptisine, palmatin hydrochloride, hydrochloric acid barberry are weighed Alkali, Gardenoside and chlorogenic acid add absolute methanol or methanol aqueous solution, reference substance solution are made;
(2) content of Trauma Yellow-water preparation to be determined is taken, absolute methanol or methanol aqueous solution dissolving is added in, filters and take continuous Filtrate is as test solution;
(3) reference substance solution and 1~5 μ L of test solution are taken respectively, inject the combination instrument of liquid chromatography-mass spectrography It is detected, is obtained respectively to phellodendrine, jateorrhizine, epiberberine, Huang in the reference substance solution and the test solution The even quantitative chromatographic figure of alkali, palmatine, jamaicin, Gardenoside and chlorogenic acid totally 8 kinds of compositions;The reference substance solution and the confession Test sample solution appearance on identical position;And pass through be calculated including phellodendrine, jateorrhizine, epiberberine, coptisine, bar Ma Ting, jamaicin, Gardenoside and chlorogenic acid content.
2. the method for quality control of Trauma Yellow-water preparation according to claim 1, which is characterized in that in mass spectrographic condition, Phellodendrine mass-to-charge ratio is 342.3 → 192.1, wherein cluster voltage 80~120 is removed, impact energy 30~50;
The mass-to-charge ratio of jateorrhizine is 339.3 → 323.2 or 339.3 → 295.2, wherein cluster voltage 80~120 is removed, impact energy 30~ 50;
The mass-to-charge ratio of epiberberine is 336.1 → 320.2 or 336.1 → 292.1, wherein cluster voltage 80~120 is removed, impact energy 30 ~50;
The mass-to-charge ratio of coptisine is 320.2 → 292.0, wherein cluster voltage 80~120 is removed, impact energy 30~50;
The mass-to-charge ratio of palmatine is 352. → 336.1 or 352. → 308.2, wherein cluster voltage 80~120 is removed, impact energy 30~ 50;
The mass-to-charge ratio of jamaicin is 336.1 → 320.1 or 336.1 → 292.1, wherein cluster voltage 80~120 is removed, impact energy 30~ 50;
The mass-to-charge ratio of Gardenoside is 411.2 → 217.4, wherein cluster voltage 80~120 is removed, impact energy 30~50;
The mass-to-charge ratio of chlorogenic acid is 355.2 → 163.2, wherein cluster voltage 80~120 is removed, impact energy 30~50.
3. the method for quality control of Trauma Yellow-water preparation according to claim 1, which is characterized in that the condition of liquid chromatogram For:Using octadecylsilane chemically bonded silica as stationary phase;Mobile phase A includes aqueous formic acid;Mobile phase B includes methanol;
Gradient elution is carried out using the mobile phase A and the Mobile phase B:
0~15min, the mobile phase A:70%, the Mobile phase B:30%;
15~20min, the mobile phase A:60~70%, the Mobile phase B:30~40%;
After 20min, the mobile phase A:60~85%, the Mobile phase B:15~40%;
The aqueous formic acid concentration of volume percent is:0.1~0.4%;The flow rate of mobile phase condition of liquid chromatogram is:0.1 ~1.0mL/min, the condition of liquid chromatogram column temperature are:25~35 DEG C.
4. the method for quality control of Trauma Yellow-water preparation according to claim 1, which is characterized in that the electron spray in mass spectrum Ion source (ESI) temperature condition is 300~380 DEG C;Atomization gas N in mass spectrum2Flow conditions are 7.0~11.0L/min.
5. the method for quality control of Trauma Yellow-water preparation according to claim 1, which is characterized in that the reference substance solution Middle hydrochloric acid phellodendrine, Jatrorrhizine chloride, hydrochloric acid epiberberine, hydrochloric acid coptisine, palmatin hydrochloride, Berberine hydrochloride, Gardenoside, Chlorogenic acid concentration is 100 μ g/mL.
6. the method for quality control of Trauma Yellow-water preparation according to claim 1, which is characterized in that the methanol aqueous solution Concentration of volume percent be:75%~100%.
7. the method for quality control of Trauma Yellow-water preparation according to claim 1, which is characterized in that further include the thin of the coptis Layer discrimination method;The thin-layer identification method of the coptis includes the following steps:
(1) Trauma Yellow-water 10mL is taken, adds n-butanol shaking extraction 2 times, each 15mL, merges n-butanol liquid, put and be evaporated in water-bath; Ethyl alcohol 10mL is added in residue and makes its dissolving, as indentification by TLC test sample;
(2) it takes the control medicinal material of the coptis and adds water;It is heated to reflux;It cools down and filters;In filtrate plus n-butanol dilution is dissolved, and is made For coptis control sample;
(3) Cortex Phellodendri, cape jasmine, Asian puccoon, peppermint and alum are weighed by the proportioning of Trauma Yellow-water preparation, mistake after heating extraction 1.5 hours Filter;Concentration, constant volume is to get liquid;10ml liquids are taken, adds n-butanol shaking extraction 2 times, each 15mL, merges butanol solution, It puts and is evaporated in water-bath;Ethyl alcohol 10mL is added in residue and makes its dissolving, is prepared into coptis negative control sample;
(4) it is tested according to thin-layered chromatography, aspiration step (1) indentification by TLC test sample, step (2) coptis control sample With step (3) each 2 μ L of the coptis negative control sample, put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate- Isopropanol-methanol-liquor ammoniae fortis is solvent, puts in the pre-saturated expansion cylinder of ammonia steam, treats indentification by TLC test sample, Huang Even control sample and coptis negative control sample are unfolded in silica gel g thin-layer plate, open up and are taken out away from 10cm, are dried;
(5) silica gel g thin-layer plate of step (4) is placed under the ultraviolet lamp of 365nm and inspected;
On silica gel g thin-layer plate, indentification by TLC test sample is compared respectively on position corresponding with control medicinal material chromatography, The fluorescence spot of aobvious same color;In coptis negative control sample chromatography, on the chromatography corresponding position of coptis control sample, not See corresponding spot, show to contain coptis ingredient in indentification by TLC test sample.
8. the method for quality control of Trauma Yellow-water preparation according to claim 7, which is characterized in that the toluene-acetic acid The proportionate relationship of ethyl ester-isopropanol-methanol-liquor ammoniae fortis is 6:3:1.5:1.5:0.6.
9. the method for quality control of Trauma Yellow-water preparation according to claim 1, which is characterized in that further include cape jasmine and Cape jasmine The thin-layer identification method of sub- glycosides;The thin-layer identification method of cape jasmine and Gardenoside includes the following steps:
(1) Trauma Yellow-water 10mL is taken, adds n-butanol shaking extraction 2 times, each 15mL, merges n-butanol liquid, put and be evaporated in water-bath; Ethyl alcohol 10mL is added in residue and makes its dissolving, as indentification by TLC test sample;
(2) control medicinal material of cape jasmine is taken to add water;It is heated to reflux;It cools down and filters;N-butanol dilution dissolving is added in filtrate, is made For cape jasmine control medicinal material solution;
(3) reference substance of Gardenoside is taken, ethyl alcohol is added to dissolve, as Gardenoside reference substance solution;
(4) coptis, Cortex Phellodendri, Asian puccoon, peppermint and alum are taken by the proportioning of Trauma Yellow-water preparation, heating extraction filters after 1.5 hours; Concentration, constant volume is to get liquid;10ml liquids are taken, adds n-butanol shaking extraction 2 times, each 15mL, merges butanol solution, put It is evaporated in water-bath;Ethyl alcohol 10mL is added in residue and makes its dissolving, and the cape jasmine negative control sample without cape jasmine is made;
(5) it is tested according to thin-layered chromatography, draws above-mentioned indentification by TLC test sample, cape jasmine control medicinal material solution, Gardenoside pair According to product solution and each 5~10 μ L of cape jasmine negative control sample, put respectively on same silica gel g thin-layer plate, with ethyl acetate-positive fourth The upper solution of alcohol-water is solvent, puts in the pre-saturated expansion cylinder of ammonia steam, treats above-mentioned indentification by TLC test sample, Cape jasmine Sub- control medicinal material solution, Gardenoside reference substance solution and cape jasmine negative control sample are unfolded on silica gel g thin-layer plate, open up away from about 10cm takes out, dries;
(6) silica gel g thin-layer plate of step (5) is sprayed with 10% ethanol solution of sulfuric acid, it is clear is heated to spot development at 110 DEG C;
Wherein, on silica gel g thin-layer plate, indentification by TLC test sample, compare respectively with cape jasmine control medicinal material solution and Cape jasmine The position of sub- glycosides reference substance solution shows the spot of same color;And in cape jasmine negative control sample chromatography, in cape jasmine control medicinal material On the chromatography corresponding position of solution and Gardenoside reference substance solution, have no corresponding spot, show indentification by TLC test sample In contain Compositions within Gardenia Jasminoides.
10. the method for quality control of Trauma Yellow-water preparation according to claim 9, which is characterized in that the ethyl acetate- The proportionate relationship of n-butanol-water is 1:2:1.
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CN113759065A (en) * 2021-02-24 2021-12-07 北京康仁堂药业有限公司 Method for simultaneously identifying components of coptis chinensis and phellodendron amurense and application of method
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