CN112684093B - Quality detection method of eleven-prescription medicinal liquor - Google Patents
Quality detection method of eleven-prescription medicinal liquor Download PDFInfo
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Abstract
The invention discloses a quality detection method of eleven-prescription medicinal liquor, belonging to the technical field of quality detection of medicines. The invention firstly identifies three components of dragon's blood, angelica and rhizoma paridis in the medicinal liquor of the first ten prescription by using a thin-layer chromatography, and carries out qualitative analysis on the components; and then, the contents of the hydroxysafflor yellow A and the loureirin B in the medicinal liquor of the first ten are simultaneously detected by utilizing the high performance liquid chromatography, quantitative analysis is carried out, the operation condition is simple and easy to implement, and the detection result is accurate and feasible by adopting the conditions of the thin layer chromatography and the high performance liquid chromatography. The quality detection method of the invention has the advantages of stability, reliability, strong specificity and good reproducibility, and combines qualitative and quantitative analysis to ensure that the quality control and detection of the eleven-party medicinal liquor are safer and more effective.
Description
Technical Field
The invention belongs to the technical field of quality detection of medicines, and particularly relates to a quality detection method of eleven-prescription medicinal liquor.
Background
The ten-prescription medicinal liquor is a preparation which is independently researched and developed by Guangxi Chinese medicinal university and is worthy of being saved and has determined curative effect, and consists of multiple Chinese medicaments such as rhizoma paridis, pseudo-ginseng, safflower, calcined native copper, frankincense, large-leaved gentian, myrrh, teasel root, rhubarb, nux vomica and the like.
However, the previous quality control only involves the measurement of the components such as nux vomica, pseudo-ginseng, teasel root and the like, but no quality detection method suitable for the contents of the three components such as dragon's blood, angelica and rhizoma paridis, as well as the contents of hydroxysafflor yellow A and loureirin B exists, and an efficient and accurate detection method is urgently needed for further ensuring the stability and the curative effect of the medicinal liquor of the first ten prescriptions.
Disclosure of Invention
Aiming at the problems, the invention provides a quality detection method of a ten-party medicinal liquor, which is characterized in that three components of dragon blood, angelica and rhizoma paridis in the ten-party medicinal liquor are identified by thin-layer chromatography for qualitative analysis; and simultaneously detecting the contents of hydroxysafflor yellow A and dracaena B in the medicinal liquor of the tenth party by using a high performance liquid chromatography, and carrying out quantitative analysis, thereby ensuring the stability and effectiveness of the medicinal liquor of the tenth party.
The invention is realized by the following technical scheme:
a quality detection method of eleven-prescription medicinal liquor comprises the following steps:
carrying out qualitative analysis on three components of dragon's blood, angelica and rhizoma paridis in the eleven medicinal liquor by using a thin-layer chromatography;
detecting the content of hydroxysafflor yellow A and dracaena B in the medicinal liquor of the first ten by using a high performance liquid chromatography.
In the qualitative analysis of the components, the dracaena cochinchinensis, the angelica and the rhizoma paridis in the ten-party medicinal liquor are respectively extracted by n-butyl alcohol, ethyl ether and ethyl acetate.
As a further preferred aspect of the present invention, the thin layer chromatography further comprises a developing solvent, wherein the developing solvent used for the three components of the dragon's blood, the angelica and the rhizoma paridis is sequentially: a mixed solution of n-hexane-ethyl acetate-methanol at a volume ratio of 20-25, a mixed solution of toluene-acetone-ammonia water at a volume ratio of 1-3:1-4 of 0.2-0.5, and a lower layer solution of chloroform-ethyl acetate-methanol-water at a volume ratio of 8-10.
As a further preferred aspect of the present invention, the high performance liquid chromatography comprises:
when the content of the hydroxysafflor yellow A is detected, octadecylsilane chemically bonded silica is used as a filling agent, acetonitrile-0.1% phosphoric acid solution is used as a mobile phase, the detection wavelength is 285-403nm, the flow rate is 1.0mL/min, the column temperature is 20-30 ℃, gradient elution is respectively carried out on the hydroxysafflor yellow A reference solution, the eleventh formula medicinal liquor test solution and the negative reference solution, a hydroxysafflor yellow A reference chromatogram and an eleventh formula medicinal liquor test chromatogram are obtained, and the content of the hydroxysafflor yellow A is determined by calculating the corresponding peak area in the eleventh formula medicinal liquor test chromatogram;
when the content of the loureirin B is detected, acetonitrile-0.1% phosphoric acid solution is used as a mobile phase, the detection wavelength is 255-280nm, the flow rate is 1.0mL/min, the column temperature is 20-30 ℃, gradient elution is respectively carried out on loureirin B reference substance solution, eleven-party medicinal liquor test solution and negative reference solution, a loureirin B reference substance chromatogram and an eleven-party medicinal liquor test solution chromatogram are obtained, and the content of the loureirin B is determined by calculating the corresponding peak appearance area in the eleven-party medicinal liquor test solution chromatogram.
As a further preferred aspect of the present invention, the gradient elution specifically comprises:
detecting hydroxy safflower yellow A content in 0-10min,10-20% acetonitrile; 10-15min,20-26% acetonitrile; 15-20min,26% acetonitrile; 20-30min,26-35% acetonitrile; 30-40min,35-20% acetonitrile.
When the content of the loureirin B is detected, 20% acetonitrile is added for 0-10 min; 10-15min,20-30% acetonitrile; 15-20min,30% acetonitrile; 20-30min,30-38% acetonitrile; 30-40min,38-30% acetonitrile.
Compared with the prior art, the invention has the advantages and beneficial effects that:
1. the invention firstly identifies three components of dragon's blood, angelica and rhizoma paridis in the medicinal liquor of the first ten prescription by using a thin-layer chromatography for qualitative analysis; and then, the contents of the hydroxysafflor yellow A and the loureirin B in the medicinal liquor of the first ten are simultaneously detected by utilizing the high performance liquid chromatography, quantitative analysis is carried out, the operation condition is simple and easy to implement, and the detection result is accurate and feasible by adopting the conditions of the thin layer chromatography and the high performance liquid chromatography. The quality detection method of the invention has the advantages of stability, reliability, strong specificity and good reproducibility, and the qualitative and quantitative analysis of the invention is combined to lay a foundation for establishing the quality standard of the eleven-prescription medicinal liquor.
2. The method of the invention can effectively control the quality of the eleven medicinal liquor, is beneficial to stabilizing the quality of the product, ensures the safety and effectiveness of medication and better meets the requirements of medical treatment and market by identifying three components of dragon's blood, angelica and rhizoma paridis in the eleven medicinal liquor and detecting the contents of hydroxy safflower yellow A and dracaolin B in the eleven medicinal liquor.
Drawings
FIG. 1 is a thin-layer chromatogram of sanguis Draxonis, in which: 1 represents a dragon blood reference substance, 2 represents a dragon blood reference medicinal material, 3 represents a dragon blood test sample, and 4 represents a negative reference substance.
FIG. 2 is a thin layer chromatogram of Angelica sinensis, in which: 1 represents angelica sinensis control medicinal materials, 2 represents eleven-party medicinal liquor, and 3 represents angelica sinensis negative control.
FIG. 3 is a thin layer chromatogram of Paris polyphylla, in which: 1 represents the paris polyphylla reference medicinal material, 2 represents the paris polyphylla sample, and 3 represents the paris polyphylla negative control.
FIG. 4 is a chromatogram of hydroxysafflor yellow A reference and an eleventh medicinal wine sample.
FIG. 5 is a chromatogram of a loureirin B reference substance and an eleven-ingredient medicinal liquor test sample.
Detailed Description
The present invention is further illustrated by the following examples, which are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.
A quality detection method of eleven-prescription medicinal liquor comprises qualitatively analyzing three components of sanguis Draxonis, radix Angelicae sinensis, and rhizoma paridis in eleven-prescription medicinal liquor by thin layer chromatography; and detecting the contents of hydroxysafflor yellow A and dracaena B in the medicinal liquor of the first ten by using a high performance liquid chromatography for quantitative analysis.
Example 1
The qualitative analysis of three components of dragon's blood, angelica and rhizoma paridis in the eleven medicinal liquor by using the thin-layer chromatography comprises the following steps:
(1) Identifying dragon's blood by thin-layer chromatography:
1) Preparing a test solution: collecting 50mL of the eleven-prescription medicinal liquor, shaking and extracting with saturated n-butyl alcohol for 3 times, 50mL each time, combining n-butyl alcohol extracts, washing with ammonia water test solution for 3 times, 50mL each time, discarding alkali liquor, combining n-butyl alcohol solutions, washing with water saturated n-butyl alcohol until neutral, evaporating the n-butyl alcohol solution to dryness, adding 2mL of absolute ethyl alcohol into residues to dissolve and fix the volume to 5mL, and taking the residues as a test sample solution;
2) Negative control solution preparation: taking 50mL of a sample prepared from a dragon blood medicinal material, shaking and extracting with water-saturated n-butyl alcohol for 3 times, 50mL each time, combining n-butyl alcohol extracts, washing with ammonia water for 3 times, 50mL each time, discarding an alkali solution, combining n-butyl alcohol solutions, washing with water-saturated n-butyl alcohol until the solution is neutral, evaporating the n-butyl alcohol solution to dryness, adding 2mL of absolute ethyl alcohol into residues to dissolve and fix the volume to 5mL, and taking the residues as a negative control solution;
3) Preparation of a control solution: taking a loureirin B reference substance, adding absolute ethyl alcohol to prepare a solution containing 1mg of loureirin B per 1mL, and taking the solution as a reference substance solution;
4) Thin layer chromatography and scanning conditions: respectively sucking 5 mu L of the control solution, 10 mu L of the test solution and 10 mu L of the negative control solution, respectively dispensing on the same silica gel G thin-layer plate (10 multiplied by 10cm, qingdao oceanic factory), adopting dot-shaped spotting, taking out by taking a mixed solution of n-hexane, ethyl acetate and methanol with a volume ratio of 20. In the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution, while the negative reference solution has no interference. The results are shown in FIG. 1.
(2) Identification of angelica by thin-layer chromatography:
1) Preparing a test solution: taking 30mL of eleven-prescription medicinal liquor, extracting with diethyl ether for 3 times, 20mL each time, discarding the diethyl ether layer, extracting the aqueous solution with water-saturated n-butanol for 3 times, 20mL each time, combining the extracts, washing with water-saturated n-butanol to neutrality, evaporating the n-butanol solution to dryness, adding 2mL of methanol into the residue to dissolve and fix the volume to 5mL to obtain a sample solution;
2) Negative control solution preparation: extracting 30mL of a sample prepared from a Chinese angelica medicinal material with diethyl ether for 3 times, 20mL each time, discarding a diethyl ether layer, extracting an aqueous solution with water-saturated n-butyl alcohol for 3 times, 20mL each time, combining extract liquor, washing with water-saturated n-butyl alcohol to be neutral, evaporating the n-butyl alcohol solution to dryness, adding 2mL of methanol into residues to dissolve the residues and fixing the volume to 5mL to serve as a negative control solution;
3) Preparation of a control solution: taking ferulic acid reference substance, adding anhydrous alcohol to make into solution containing 1mg loureirin B per 1mL as reference substance solution;
4) Thin layer chromatography and scanning conditions: respectively sucking 5 mu L of the control solution, 10 mu L of the test solution and 10 mu L of the negative control solution, respectively spotting on the same silica gel G thin-layer plate (10 multiplied by 10cm, qingdao oceanic factory), adopting a dot spotting mode, taking toluene-acetone-ammonia water as a developing agent according to a volume ratio of 1. In the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the control solution, while the negative control solution has no interference. The results are shown in FIG. 2.
(3) Identifying rhizoma paridis by thin-layer chromatography:
1) Preparing a test solution: collecting 30mL of the eleven-prescription medicinal liquor, extracting with ethyl acetate for 3 times with shaking, 30mL each time, mixing the ethyl acetate extractive solutions, evaporating to dryness, adding 2mL of anhydrous ethanol into the residue to dissolve, and diluting to a constant volume of 5mL to obtain a sample solution;
2) Negative control solution preparation: taking 30mL of a sample prepared from the rhizoma paridis medicinal material, extracting with ethyl acetate for 3 times with shaking, 30mL each time, combining ethyl acetate extracting solutions, evaporating to dryness, adding 2mL of absolute ethanol into residues to dissolve and fix the volume to 5mL, and taking the residue as a negative control solution;
3) Preparation of a reference solution: taking a rhizoma paridis saponin A reference substance, adding anhydrous ethanol to obtain a solution containing 1mg of rhizoma paridis saponin A per 1mL as a reference substance solution;
4) Thin layer chromatography and scanning conditions: respectively sucking 5 mu L of the control solution, 10 mu L of the test solution and 10 mu L of the negative control solution, respectively dropping the control solution, the test solution and the negative control solution on the same silica gel G thin-layer plate (10 multiplied by 10cm, qingdao ocean chemical plant), adopting round-point spotting, taking a lower-layer solution of chloroform-ethyl acetate-methanol-water as a developing agent, wherein the volume ratio of the lower-layer solution is 8. In the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution, while the negative reference solution has no interference. The results are shown in FIG. 3.
Example 2
The qualitative analysis of three components of dragon's blood, angelica and rhizoma paridis in the eleven medicinal liquor by using the thin-layer chromatography comprises the following steps:
(1) Identifying dragon's blood by thin-layer chromatography:
1) Preparing a test solution: collecting 30mL of the eleven-prescription medicinal liquor, shaking and extracting with saturated n-butyl alcohol for 3 times, 30mL each time, combining n-butyl alcohol extracts, washing with ammonia water test solution for 2 times, 30mL each time, discarding alkali liquor, combining n-butyl alcohol solutions, washing with water saturated n-butyl alcohol until neutral, evaporating the n-butyl alcohol solution to dryness, adding 2mL of methanol into residues to dissolve and fix the volume to 5mL, and taking the residues as a test sample solution;
2) Negative control solution preparation: taking 30mL of a sample prepared from a dragon blood medicinal material, shaking and extracting with water saturated n-butyl alcohol for 3 times, 30mL each time, combining n-butyl alcohol extracts, washing with ammonia water for 3 times, 30mL each time, discarding an alkali solution, combining n-butyl alcohol solutions, washing with water saturated n-butyl alcohol until the n-butyl alcohol solutions are neutral, evaporating the n-butyl alcohol solutions to dryness, adding 2mL of methanol into residues to dissolve and fix the volume to 5mL, and taking the residues as a negative control solution;
3) Preparation of a reference solution: adding methanol into loureirin B reference substance to obtain 1mg loureirin B solution per 1mL as reference solution;
4) Thin layer chromatography and scanning conditions: respectively sucking 5 mu L of the control solution, 10 mu L of the test solution and 10 mu L of the negative control solution, respectively dropping the solutions on the same silica gel G thin-layer plate (10 multiplied by 10cm, qingdao ocean chemical plant), adopting a dot-shaped dropping sample, taking out the solution with a volume ratio of 25. In the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the control solution, while the negative control solution has no interference.
(2) Identification of angelica by thin-layer chromatography:
1) Preparing a test solution: taking 30mL of eleven-prescription medicinal liquor, extracting with diethyl ether for 3 times, 20mL each time, discarding the diethyl ether layer, extracting the aqueous solution with water-saturated n-butanol for 3 times, 20mL each time, combining the extracts, washing with water-saturated n-butanol to neutrality, evaporating the n-butanol solution to dryness, adding 2mL of methanol into the residue to dissolve and fix the volume to 5mL to obtain a sample solution;
2) Negative control solution preparation: extracting 30mL of a sample prepared from a Chinese angelica medicinal material with diethyl ether for 3 times, 20mL each time, discarding a diethyl ether layer, extracting an aqueous solution with water-saturated n-butyl alcohol for 3 times, 20mL each time, combining extract liquor, washing with water-saturated n-butyl alcohol to be neutral, evaporating the n-butyl alcohol solution to dryness, adding 2mL of methanol into residues to dissolve the residues and fixing the volume to 5mL to serve as a negative control solution;
3) Preparation of a control solution: taking ferulic acid reference substance, adding anhydrous alcohol to make into solution containing 1mg of loureirin B per 1mL as reference substance solution;
4) Thin layer chromatography and scanning conditions: respectively sucking 5 mu L of the reference substance solution, 10 mu L of the test substance solution and 10 mu L of the negative reference solution, respectively dropping the solutions on the same silica gel G thin-layer plate (10 multiplied by 10cm, qingdao ocean factory), adopting dot spotting, taking out the solution by taking toluene-acetone-ammonia water as a developing agent according to the volume ratio of 3. In the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the control solution, while the negative control solution has no interference.
(3) Identifying rhizoma paridis by thin-layer chromatography:
1) Preparing a test solution: collecting 30mL of the eleven-prescription medicinal liquor, extracting with ethyl acetate for 3 times with shaking, 30mL each time, mixing the ethyl acetate extractive solutions, evaporating to dryness, adding 2mL of anhydrous ethanol into the residue to dissolve, and diluting to a constant volume of 5mL to obtain a sample solution;
2) Negative control solution preparation: taking 30mL of a sample prepared from a rhizoma paridis medicinal material, extracting for 3 times by shaking with ethyl acetate, 30mL each time, combining ethyl acetate extracting solutions, evaporating to dryness, adding 2mL of absolute ethanol into residues to dissolve, and fixing the volume to 5mL to serve as a negative control solution;
3) Preparation of a reference solution: taking a rhizoma paridis saponin A reference substance, adding anhydrous ethanol to obtain a solution containing 1mg of rhizoma paridis saponin A per 1mL as a reference substance solution;
4) Thin layer chromatography and scanning conditions: respectively sucking 5 mu L of the control solution, 10 mu L of the test solution and 10 mu L of the negative control solution, respectively dropping the control solution, the test solution and the negative control solution on the same silica gel G thin-layer plate (10 multiplied by 10cm, qingdao ocean factory), adopting round-point spotting, taking a lower-layer solution of chloroform-ethyl acetate-methanol-water as a developing agent, spreading the solution at a spreading distance of 8cm, taking out, airing, spraying a 5% sulfuric acid ethanol solution, and heating at 110 ℃ for 5min until spots are clear. In the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the control solution, while the negative control solution has no interference.
Example 3
Simultaneously detecting the contents of hydroxysafflor yellow A and dracaena B in the medicinal liquor of the first ten by utilizing a high performance liquid chromatography, and carrying out quantitative analysis, wherein the quantitative analysis comprises the following steps:
(1) Detecting hydroxysafflor yellow A by high performance liquid chromatography:
1) Chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent, acetonitrile-0.1% phosphoric acid solution is used as a mobile phase, the detection wavelength is 403nm, the flow rate is 1.0mL/min, the column temperature is 30 ℃, and gradient elution is carried out;
TABLE 1 gradient elution Table
2) Preparation of a reference solution: taking a proper amount of a reference substance of the hydroxysafflor yellow A, precisely weighing, adding methanol to prepare a solution of the hydroxysafflor yellow A with the component mass concentration of 420 mu g/mL, filtering through a 0.45 mu m microporous membrane, and taking a subsequent filtrate to obtain a reference substance solution of the hydroxysafflor yellow A;
3) Preparing a test solution: precisely measuring 5mL of a ten-party medicinal liquor, evaporating to dryness in a water bath, adding 30mL of hot water for 3 times to dissolve, transferring to a separating funnel, adding 10mL of diethyl ether to extract for 1 time, standing, removing a diethyl ether layer, extracting a water layer with water-saturated n-butyl alcohol for 2 times, 10mL each time, mixing n-butyl alcohol, washing with water-saturated n-butyl alcohol for 2 times, 30mL each time, evaporating to dryness in a water bath of the n-butyl alcohol layer, adding a methanol solution into residues, fixing the volume to a 10mL measuring flask, shaking uniformly, filtering through a 0.45-micrometer microporous membrane, and taking a subsequent filtrate to obtain the medicinal liquor;
4) Negative control solution preparation: taking 5mL of a sample prepared from safflower, evaporating to dryness in a water bath, adding 30mL of hot water for 3 times to dissolve, transferring to a separating funnel, adding 10mL of diethyl ether to extract for 1 time, standing, removing a diethyl ether layer, extracting a water layer with water-saturated n-butyl alcohol for 2 times, 10mL each time, combining n-butyl alcohol, washing n-butyl alcohol-saturated ammonia test solution for 2 times, 30mL each time, evaporating to dryness in a water bath of the n-butyl alcohol layer, adding a methanol solution into residues, fixing the volume to a 10mL measuring flask, shaking uniformly, filtering through a 0.45 mu m microporous membrane, and taking a subsequent filtrate to obtain the safflower extract;
5) And (3) determination: precisely sucking 10 μ L of the mixed reference solution, the sample solution, and the negative reference solution, respectively, injecting into a liquid chromatograph, and measuring. The result is shown in figure 4, and the content of hydroxysafflor yellow A is determined by calculating the corresponding peak area in the chromatogram of the eleven medicinal liquor test sample.
(2) Detecting loureirin B by high performance liquid chromatography:
1) Chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent, acetonitrile-0.1% phosphoric acid solution is used as a mobile phase, the detection wavelength is 285nm, the flow rate is 1.0mL/min, the column temperature is 30 ℃, and gradient elution is carried out;
TABLE 2 gradient elution Table
2) Preparation of a reference solution: taking a proper amount of loureirin B reference substance, precisely weighing, adding methanol to prepare a loureirin B solution with the component mass concentration of 124 mu g/mL, filtering through a 0.45 mu m microporous membrane, and taking a subsequent filtrate to obtain a loureirin B reference substance solution;
3) Preparing a test solution: precisely measuring 5mL of a ten-party medicinal liquor, evaporating to dryness in a water bath, adding 0.5% hydrochloric acid, dissolving for 3 times, 10mL each time, filtering, adding 2mL of concentrated ammonia water into the filtrate to adjust the pH to 8, extracting for 3 times by using n-hexane, adding a methanol solution into the residue, metering to a 10mL measuring flask, shaking up, filtering through a 0.45-micrometer microporous membrane, and taking a subsequent filtrate to obtain the medicinal liquor;
4) Negative control solution preparation: taking 5mL of a sample prepared from safflower, evaporating to dryness in a water bath, adding 0.5% hydrochloric acid, dissolving for 3 times, 10mL each time, filtering, adding 2mL of concentrated ammonia water into the filtrate to adjust the pH value to 8, extracting for 3 times by using n-hexane, adding a methanol solution into the residue, metering to a 10mL measuring flask, shaking up, filtering through a 0.45-micrometer microporous membrane, and taking a subsequent filtrate to obtain the safflower extract;
5) And (3) determination: precisely sucking 10 μ L of the mixed reference solution, the sample solution, and the negative reference solution, respectively, injecting into a liquid chromatograph, and measuring. The result is shown in figure 5, and the content of the loureirin B is determined by calculating the corresponding peak area in the chromatogram of the test sample of the eleven medicinal liquor.
Example 4
Simultaneously detecting the contents of hydroxysafflor yellow A and dracaena B in the medicinal liquor of the first ten by utilizing a high performance liquid chromatography, and carrying out quantitative analysis, wherein the quantitative analysis comprises the following steps:
(1) Detecting hydroxysafflor yellow A by high performance liquid chromatography:
1) Chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent, acetonitrile-0.1% phosphoric acid solution is used as a mobile phase, the detection wavelength is 285nm, the flow rate is 1.0mL/min, the column temperature is 20 ℃, and gradient elution is carried out;
TABLE 1 gradient elution Table
2) Preparation of a reference solution: taking a proper amount of a reference substance of the hydroxysafflor yellow A, precisely weighing, adding methanol to prepare a solution of the hydroxysafflor yellow A with the component mass concentration of 420 mu g/mL, filtering through a 0.45 mu m microporous membrane, and taking a subsequent filtrate to obtain a reference substance solution of the hydroxysafflor yellow A;
3) Preparing a test solution: precisely measuring 5mL of a ten-party medicinal liquor, evaporating to dryness in a water bath, adding 30mL of hot water for 3 times to dissolve, transferring to a separating funnel, adding 10mL of diethyl ether to extract for 1 time, standing, removing a diethyl ether layer, extracting a water layer with water-saturated n-butyl alcohol for 2 times, 10mL each time, mixing n-butyl alcohol, washing with water-saturated n-butyl alcohol for 2 times, 30mL each time, evaporating to dryness in a water bath of the n-butyl alcohol layer, adding a methanol solution into residues, fixing the volume to a 10mL measuring flask, shaking uniformly, filtering through a 0.45-micrometer microporous membrane, and taking a subsequent filtrate to obtain the medicinal liquor;
4) Negative control solution preparation: taking 5mL of a sample prepared from safflower, evaporating to dryness in a water bath, adding 30mL of hot water for 3 times to dissolve, transferring to a separating funnel, adding 10mL of diethyl ether to extract for 1 time, standing, removing a diethyl ether layer, extracting a water layer with water-saturated n-butyl alcohol for 2 times, 10mL each time, combining n-butyl alcohol, washing n-butyl alcohol-saturated ammonia test solution for 2 times, 30mL each time, evaporating to dryness in a water bath of the n-butyl alcohol layer, adding a methanol solution into residues, fixing the volume to a 10mL measuring flask, shaking uniformly, filtering through a 0.45 mu m microporous membrane, and taking a subsequent filtrate to obtain the safflower extract;
5) And (3) determination: precisely sucking 10 μ L of the mixed reference solution, the sample solution, and the negative reference solution, respectively, injecting into a liquid chromatograph, and measuring. And determining the content of hydroxysafflor yellow A by calculating the corresponding peak area in the chromatogram of the test sample of the eleven medicinal liquor.
(2) Detecting loureirin B by high performance liquid chromatography:
1) Chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent, acetonitrile-0.1% phosphoric acid solution is used as a mobile phase, the detection wavelength is 255nm, the flow rate is 1.0mL/min, the column temperature is 20 ℃, and gradient elution is carried out;
TABLE 2 gradient elution Table
2) Preparation of a reference solution: taking a proper amount of loureirin B reference substance, precisely weighing, adding methanol to prepare a loureirin B solution with the component mass concentration of 124 mu g/mL, filtering through a 0.45 mu m microporous membrane, and taking a subsequent filtrate to obtain a loureirin B reference substance solution;
3) Preparing a test solution: precisely measuring 5mL of a ten-party medicinal liquor, evaporating to dryness in a water bath, adding 0.5% hydrochloric acid, dissolving for 3 times, 10mL each time, filtering, adding 2mL of concentrated ammonia water into the filtrate to adjust the pH to 8, extracting for 3 times by using n-hexane, adding a methanol solution into the residue, metering to a 10mL measuring flask, shaking up, filtering through a 0.45-micrometer microporous membrane, and taking a subsequent filtrate to obtain the medicinal liquor;
4) Negative control solution preparation: taking 5mL of a sample prepared from safflower, evaporating to dryness in a water bath, adding 0.5% hydrochloric acid, dissolving for 3 times, 10mL each time, filtering, adding 2mL of concentrated ammonia water into the filtrate to adjust the pH value to 8, extracting for 3 times by using n-hexane, adding a methanol solution into the residue, metering to a 10mL measuring flask, shaking up, filtering through a 0.45-micrometer microporous membrane, and taking a subsequent filtrate to obtain the safflower extract;
5) And (3) determination: precisely sucking 10 μ L of the mixed reference solution, the sample solution, and the negative reference solution, respectively, injecting into a liquid chromatograph, and measuring. And determining the content of loureirin B by calculating the corresponding peak area in the chromatogram of the eleven medicinal liquor test sample.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and the like that are within the spirit and principle of the present invention are included in the present invention.
Claims (3)
1. A quality detection method of eleven-ingredient medicinal liquor is characterized by comprising the following steps:
carrying out qualitative analysis on three components of dragon's blood, angelica and rhizoma paridis in the eleven medicinal liquor by using a thin-layer chromatography;
detecting the content of hydroxysafflor yellow A and loureirin B in the thirteen-party medicinal liquor by using a high performance liquid chromatography;
in the qualitative analysis of the components, the dragon blood, the angelica and the rhizoma paridis in the medicinal liquor of the tenth prescription of n-butyl alcohol, ethyl ether and ethyl acetate are respectively extracted; in the thin-layer chromatography, a developing solvent is also used, and the developing solvent used correspondingly for the dragon's blood, the angelica and the rhizoma paridis sequentially comprises the following components: a mixed solution of n-hexane-ethyl acetate-methanol at a volume ratio of 20-25, a mixed solution of toluene-acetone-ammonia water at a volume ratio of 1-3:1-4 of 0.2-0.5, and a lower layer solution of chloroform-ethyl acetate-methanol-water at a volume ratio of 8-10.
2. The quality detection method of the ten-party medicinal liquor according to claim 1, characterized in that the high performance liquid chromatography comprises the following steps:
when the content of the hydroxysafflor yellow A is detected, octadecylsilane chemically bonded silica is used as a filling agent, acetonitrile-0.1% phosphoric acid solution is used as a mobile phase, the detection wavelength is 285-403nm, the flow rate is 1.0mL/min, the column temperature is 20-30 ℃, gradient elution is respectively carried out on the hydroxysafflor yellow A reference substance solution, the eleventh formula medicinal liquor test solution and the negative reference solution to obtain a hydroxysafflor yellow A reference substance chromatogram and an eleventh formula medicinal liquor test solution chromatogram, and the content of the hydroxysafflor yellow A is determined by calculating the corresponding peak area in the eleven medicinal liquor test solution chromatogram;
when the content of the loureirin B is detected, acetonitrile-0.1% phosphoric acid solution is used as a mobile phase, the detection wavelength is 255-280nm, the flow rate is 1.0mL/min, the column temperature is 20-30 ℃, gradient elution is respectively carried out on loureirin B reference substance solution, eleven-party medicinal liquor test solution and negative reference solution, a loureirin B reference substance chromatogram and an eleven-party medicinal liquor test solution chromatogram are obtained, and the content of the loureirin B is determined by calculating the corresponding peak area in the eleven-party medicinal liquor test solution chromatogram.
3. The method for detecting the quality of an eleven-ingredient medicinal liquor according to claim 2, wherein the gradient elution is specifically as follows:
detecting content of hydroxysafflor yellow A with 10-20% acetonitrile for 0-10 min; 10-15min,20-26% acetonitrile; 15-20min,26% acetonitrile; 20-30min,26-35% acetonitrile; 30-40min,35-20% acetonitrile;
when the content of the loureirin B is detected, the content is 0-10min,20% acetonitrile; 10-15min,20-30% acetonitrile; 15-20min,30% acetonitrile; 20-30min,30-38% acetonitrile; 30-40min,38-30% acetonitrile.
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