CN112415109A - HPLC (high performance liquid chromatography) detection method for simultaneously determining different characteristic components in acne-removing composition - Google Patents

HPLC (high performance liquid chromatography) detection method for simultaneously determining different characteristic components in acne-removing composition Download PDF

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CN112415109A
CN112415109A CN202011209945.6A CN202011209945A CN112415109A CN 112415109 A CN112415109 A CN 112415109A CN 202011209945 A CN202011209945 A CN 202011209945A CN 112415109 A CN112415109 A CN 112415109A
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acne
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刘林峰
吴知情
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Guangzhou Nabion Biotechnology Co ltd
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
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Abstract

The invention provides an HPLC detection method for simultaneously determining different characteristic components in an acne-removing composition, which comprises the following steps: preparing the acne-removing composition into a sample, detecting by HPLC (high performance liquid chromatography) for gradient elution to obtain a detection result, and calculating the content of the characteristic component by using a standard curve; the characteristic component comprises one or the combination of at least two of chlorogenic acid, caffeic acid, gentiopicroside, berberine, baicalin or emodin. The detection method provided by the invention is rapid, sensitive and accurate, has low cost, and solves the problem that the existing instrument cannot simultaneously detect and analyze the analysis method of single medicinal material in the acne-removing composition and various characteristic components in the compound composition.

Description

HPLC (high performance liquid chromatography) detection method for simultaneously determining different characteristic components in acne-removing composition
Technical Field
The invention belongs to the field of cosmetic raw material detection, particularly relates to an HPLC (high performance liquid chromatography) detection method for simultaneously determining different characteristic components in an acne-removing composition, and particularly relates to an HPLC detection method for rapidly and sensitively simultaneously determining different characteristic components in an acne-removing composition.
Background
Acne is a multiple disease in adolescence and is also one of clinical common diseases and multiple diseases of dermatology. The disease causes are complex, and no specific therapy is available in western medicine. Honeysuckle, dandelion, rhubarb, phellodendron bark and the like have the functions of clearing heat, removing toxicity and drying dampness, which is consistent with the result that the propionibacterium acnes such as rhubarb, phellodendron bark and the like are highly sensitive in the research of the bacteriostatic action of the propionibacterium acnes in modern medicine.
Honeysuckle, dandelion, gentian, phellodendron, scutellaria, rhubarb and the like are common extracts with antibacterial effect, and the acne removing composition compounded by the extracts according to a certain proportion is popular in the market as a core functional component applied to cosmetics. However, the acne-removing composition has many medicinal herbs and complex components, and the quality of the whole compound and the quality of the Chinese herbal compound are difficult to be reflected and monitored by single chemical component measurement during quality control. At present, the research on the control of the acne-removing composition substance is still in a relatively deficient state. The development of the HPLC-DAD method provides a technical basis for simultaneously measuring various components in a complex Chinese medicinal compound, and the proposal of a Chinese medicinal quality marker (quality marker) provides a theoretical basis for the effective and quality control of Chinese medicaments.
The establishment of the analysis method is beneficial to accelerating the further research of the effective components of the acne-removing composition, and provides technical support for the quality control of products related to the acne-removing composition in the future. However, due to the complex components and the high separation difficulty in the acne-removing composition, most of efficacy characteristic components and other impurities cannot be well separated by the existing detection method. Therefore, the precision is not high, and errors are easy to occur in the actual operation process.
CN110646537A discloses a method for simultaneously determining the contents of various components in a three-yellow tablet based on an HPLC wavelength switching technology, relating to the technical field of medicine detection methods, and carrying out qualitative and quantitative analysis on 7 components of baicalin, berberine hydrochloride, aloe-emodin, rhein, emodin, chrysophanol and physcion simultaneously according to the following steps: preparing a mixed reference substance solution; preparing a test solution; separating and detecting by high performance liquid chromatography; qualitative and quantitative analysis of the test sample. By using HPLC wavelength switching technology, baicalin wavelength switching is detected at 280nm, berberine hydrochloride long switching is detected at 264nm, and aloe-emodin, rhein, emodin, chrysophanol and physcion wavelength switching is detected at 254 nm; elution conditions: mobile phase A acetonitrile, mobile phase B phosphoric acid water solution, 0-10 min: 15% A, 85% B; 20 min: 25% a, 75% B; 30 min: 40% A, 60% B; 42 min: 55% A, 45% B; 50-60 min: 80% A, 20% B. Simple operation, high detection sensitivity and more accurate detection result. But it is undetectable for other acne-removing composition ingredients.
CN101324546 discloses a method for simultaneously determining the content of chlorogenic acid and caffeic acid in a dandelion preparation by an HPLC method, which can effectively solve the problem of laying a foundation for solving the quality standard of the dandelion preparation by taking chlorogenic acid and caffeic acid as index components and simultaneously performing content determination, and the specific technical scheme for realizing the invention is as follows: the method is simple, reliable and rapid, has high sensitivity, good reproducibility and high recovery rate, establishes a foundation for establishing the quality standard of the dandelion and the preparation thereof, and provides a more scientific basis by taking the chlorogenic acid and the caffeic acid as index components. But the components such as gentiopicroside, berberine, baicalin, emodin and the like have no detection effect.
CN104897787B discloses a method for simultaneously determining the contents of six active ingredients, namely chrysophanol, emodin, liquiritin, phillyrin, baicalin and berberine hydrochloride in Niuhuang Gong tablets by an HPLC method, wherein the chromatographic conditions adopted by the method are as follows: a chromatographic column: TC-C18(4.6 mm. times.250 mm, 5 μm); detection wavelength: 280 nm; mobile phase: methanol, 0.05% phosphoric acid; gradient elution: 0-35 min: 10% -80% methanol; 35-50 min: 80% methanol; 50-60 min: 80% -10% methanol; flow rate: 1.0 mL/min; column temperature: 25 ℃; sample introduction amount: 10 μ L. Under the chromatographic condition, chromatographic peak separation is good, and the concentrations of chrysophanol, emodin, liquiritin, phillyrin, baicalin and berberine hydrochloride and peak areas show good linear relation. The method is simple, rapid, accurate, and good in reproducibility, and can provide quality basis for overall evaluation and control of NIU HUANG NING GONG tablet.
At present, no HPLC detection method capable of effectively separating and detecting components in the acne-removing composition exists, so that how to provide a rapid and sensitive HPLC detection method for different characteristic components in the acne-removing composition becomes a problem to be solved urgently.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a High Performance Liquid Chromatography (HPLC) detection method for simultaneously determining different characteristic components in an acne-removing composition, and particularly provides a rapid and sensitive HPLC detection method for simultaneously determining different characteristic components in the acne-removing composition. The detection method provided by the invention is rapid, sensitive and accurate, has low cost, and solves the problem that the existing instrument cannot simultaneously detect and analyze the analysis method of single medicinal material in the acne-removing composition and various characteristic components in the compound composition.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an HPLC detection method for simultaneously determining different characteristic components in an acne-removing composition, which comprises the following steps: preparing the acne-removing composition into a sample, detecting by HPLC (high performance liquid chromatography) for gradient elution to obtain a detection result, and calculating the content of the characteristic component by using a standard curve.
The characteristic component includes any one or combination of at least two of chlorogenic acid, caffeic acid, gentiopicroside, berberine, baicalin or emodin, such as combinations of chlorogenic acid and caffeic acid, gentiopicroside and baicalin or emodin and chlorogenic acid, but is not limited to the listed combinations, and other combinations not listed in the above combination range are also applicable.
The detection method can simultaneously detect the contents of chlorogenic acid, caffeic acid, gentiopicroside, berberine, baicalin and emodin in the sample at one time, and has the advantages of rapidness, sensitivity, accuracy and low cost.
Preferably, the acne-removing composition can be purchased from the market, and can also be obtained by compounding honeysuckle, dandelion, gentian, phellodendron, scutellaria and rheum officinale.
Preferably, the solvent for gradient elution is an ultra-pure aqueous solution of methanol and phosphoric acid.
Preferably, the phosphoric acid ultrapure water solution is 0.03 to 0.08% by mass, for example, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, or 0.08%, and the like, but is not limited to the recited values, and other values not recited in the above range of values are also applicable.
Preferably, the gradient elution is performed according to the following procedure: the volume of the methanol in the eluent is 10% at 0min, the volume of the phosphoric acid ultrapure water solution is 90%, the volume of the methanol is 25% at the time of uniform speed change to 6min, the volume of the phosphoric acid ultrapure water solution is 75%, the volume of the methanol is 30% at the time of uniform speed change to 15min, the volume of the phosphoric acid ultrapure water solution is 70%, the volume of the methanol is 40% at the time of uniform speed change to 21min, the volume of the phosphoric acid ultrapure water solution is 60%, the volume of the methanol is 55% at the time of uniform speed change to 27min, the volume of the phosphoric acid ultrapure water solution is 45%, the volume of the methanol is 90% at the time of uniform speed change to 40min, and the volume of the phosphoric acid ultrapure water solution is 10%.
The specific gradient elution procedure can completely separate chlorogenic acid, caffeic acid, gentiopicroside, berberine, baicalin and emodin in the acne-removing composition, and is convenient for detection.
Preferably, the preparation of the acne-removing composition into a sample comprises the following steps: extracting the acne-removing composition with purified water under reflux, filtering, adsorbing and eluting the filtrate with macroporous adsorbent resin, evaporating the eluate, concentrating, diluting to constant volume with methanol, and filtering to obtain the sample.
Preferably, the temperature of the reflux is 75-85 ℃.
Preferably, the refluxing time is 50-70 min.
The temperature may be 75 ℃, 76 ℃, 77 ℃, 78 ℃, 79 ℃, 80 ℃, 81 ℃, 82 ℃, 83 ℃, 84 ℃ or 85 ℃ and the time may be 50min, 52min, 54min, 56min, 58min, 60min, 62min, 64min, 66min, 68min or 70min, but is not limited to the enumerated values, and other non-enumerated values within the above numerical range are also applicable.
The combination of the specific conditions can completely extract chlorogenic acid, caffeic acid, gentiopicroside, berberine, baicalin and emodin in the acne-removing composition, thereby improving the detection accuracy.
Preferably, the first and second electrodes are formed of a metal,the HPLC uses Agilent InfinityLab Poroshell 120SB-C18The specification of the chromatographic column is 150mm multiplied by 4.60mm, and the grain diameter of the filler is 2.8 mu m.
Preferably, the column temperature for HPLC detection is 35-45 ℃.
Preferably, the flow rate of the gradient elution is 0.8-1.2 mL/min.
Preferably, the sample injection volume for HPLC detection is 8-12 μ L.
Preferably, the wavelength of the HPLC detection is 260-300 nm.
Wherein the column temperature may be 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃ or 45 ℃, the flow rate may be 0.8mL/min, 0.9mL/min, 1.0mL/min, 1.1mL/min or 1.2mL/min, the sample volume may be 8. mu.L, 9. mu.L, 10. mu.L, 11. mu.L or 12. mu.L, the wavelength may be 260nm, 270nm, 280nm, 290nm or 300nm, but not limited to the enumerated values, and other values not enumerated within the above-mentioned value range are also applicable.
The combination of the specific conditions can completely separate and detect chlorogenic acid, caffeic acid, gentiopicroside, berberine, baicalin and emodin in the acne-removing composition.
Preferably, the standard curve is obtained by the following steps: and (3) dissolving and diluting the standard sample of the characteristic component with methanol, then carrying out HPLC detection according to the gradient elution program, and calculating a standard curve according to the detection result.
The standard curve can be used for carrying out quantitative analysis on the detection result, and powerful experimental data are provided for further researching and developing the pharmacological activity and spectral efficiency relationship of natural components of the raw materials of the medicinal materials such as honeysuckle, dandelion, gentian, phellodendron, scutellaria baicalensis, rheum officinale and the like.
Compared with the prior art, the invention has the following beneficial effects:
the HPLC detection method for simultaneously determining different characteristic components in the acne-removing composition provided by the invention is rapid, sensitive, accurate and low in cost, the precision RSD reaches within 3%, and good precision is displayed; through a specific elution procedure, chlorogenic acid, caffeic acid, gentiopicroside, berberine, baicalin and emodin in the pox composition can be completely separated, so that the detection is convenient; by optimizing the detection conditions, chlorogenic acid, caffeic acid, gentiopicroside, berberine, baicalin and emodin in the acne-removing composition can be completely separated and detected; the detection result can be quantitatively analyzed by utilizing the standard curve, and powerful experimental data is provided for further researching and developing the pharmacological activity and spectral efficiency relationship of natural components of the raw materials of the medicinal materials such as honeysuckle, dandelion, gentian, phellodendron, scutellaria baicalensis, rheum officinale and the like.
Drawings
FIG. 1 shows HPLC detection results of flos Lonicerae extract, chlorogenic acid standard solution and mixed standard solution, wherein 1-chlorogenic acid, 2-caffeic acid, 3-gentiopicroside, 4-berberine, 5-baicalin, and 6-emodin;
FIG. 2 shows HPLC detection results of herba Taraxaci extract, caffeic acid standard solution and mixed standard solution, wherein 1-chlorogenic acid, 2-caffeic acid, 3-gentiopicroside, 4-berberine, 5-baicalin, and 6-emodin;
FIG. 3 shows HPLC detection results of radix Gentianae extract, gentiopicrin standard solution and mixed standard solution, wherein 1-chlorogenic acid, 2-caffeic acid, 3-gentiopicrin, 4-berberine, 5-baicalin, and 6-emodin;
FIG. 4 shows HPLC detection results of cortex Phellodendri extract, berberine standard solution and mixed standard solution, wherein 1-chlorogenic acid, 2-caffeic acid, 3-gentiopicroside, 4-berberine, 5-baicalin and 6-emodin are contained;
FIG. 5 shows HPLC detection results of Scutellariae radix extract, baicalin standard solution and mixed standard solution, wherein 1-chlorogenic acid, 2-caffeic acid, 3-gentiopicroside, 4-berberine, 5-baicalin, and 6-emodin;
FIG. 6 shows HPLC detection results of flos Rhei extract, emodin standard solution and mixed standard solution, wherein 1-chlorogenic acid, 2-caffeic acid, 3-gentiopicroside, 4-berberine, 5-baicalin, and 6-emodin;
fig. 7 shows HPLC detection results of the acne-removing composition and the mixed standard solution provided in example 1, wherein 1-chlorogenic acid, 2-caffeic acid, 3-gentiopicroside, 4-berberine, 5-baicalin, and 6-emodin are provided.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
In the following preparations and examples, HPLC was from Agilent, model 1260 (DAD detector);
the chromatographic column is Agilent InfinityLab Poroshell 120SB-C18The specification of the chromatographic column is 150mm multiplied by 4.60mm, and the grain diameter of the filler is 2.8 mu m.
Preparation example 1
Respectively dissolving 10.0mg of 6 characteristic component standard substances (chlorogenic acid, caffeic acid, gentiopicroside, berberine, baicalin and emodin) with methanol, and diluting to 100mL with methanol to obtain each characteristic component standard substance solution; and (3) sucking 1mL of each standard solution, mixing, metering the volume to 10mL by using methanol, and uniformly mixing to obtain a mixed standard solution.
Preparation example 2
Respectively taking honeysuckle, dandelion, gentian, phellodendron, scutellaria baicalensis and rhubarb, and purified water, refluxing for 60min at 80 ℃ according to the material-liquid ratio of 1g to 10mL, then filtering, eluting the filtrate for 2BV by using 70% ethanol at the flow rate of 4BV/h, collecting the eluent, carrying out rotary evaporation and concentration at low temperature, fixing the volume to 2mL by using methanol, and carrying out membrane filtration by using an organic filter membrane of 0.45 mu L to obtain the honeysuckle, dandelion, gentian, phellodendron, scutellaria baicalensis and rhubarb extracts.
Example 1
The embodiment provides an HPLC (high performance liquid chromatography) detection method for simultaneously determining different characteristic components in an acne-removing composition, wherein the acne-removing composition is prepared by compounding 30 parts by weight of honeysuckle, 30 parts by weight of dandelion, 10 parts by weight of gentian, 10 parts by weight of golden cypress, 10 parts by weight of scutellaria baicalensis and 20 parts by weight of rheum officinale.
Refluxing the acne-removing composition and purified water at a feed-liquid ratio of 1g:10mL for 50min at 85 ℃, filtering, eluting the filtrate for 2BV with 70% ethanol at a flow rate of 4BV/h, collecting the eluent, performing low-temperature rotary evaporation and concentration, fixing the volume to 2mL with methanol, filtering with 0.45 muL of organic filter membrane, and performing HPLC detection at a column temperature of 40 ℃, an elution flow rate of 1.0mL/min, a detection wavelength of 280nm and a sample injection volume of 10 muL, wherein the gradient elution procedure is as follows: the volume of the methanol in the eluent is 10% at 0min, the volume of the phosphoric acid ultrapure water solution is 90%, the volume of the methanol is 25% at the time of uniform speed change to 6min, the volume of the phosphoric acid ultrapure water solution is 75%, the volume of the methanol is 30% at the time of uniform speed change to 15min, the volume of the phosphoric acid ultrapure water solution is 70%, the volume of the methanol is 40% at the time of uniform speed change to 21min, the volume of the phosphoric acid ultrapure water solution is 60%, the volume of the methanol is 55% at the time of uniform speed change to 27min, the volume of the phosphoric acid ultrapure water solution is 45%, the volume of the methanol is 90% at the time of uniform speed change to 40min, and the volume of the phosphoric acid ultrapure water solution is 10%.
And (3) preparing a standard curve:
respectively taking 10mg of standard samples of chlorogenic acid, caffeic acid, gentiopicrin, berberine, baicalin and emodin, dissolving with methanol to a constant volume of 100mL, respectively injecting 5 muL, 10 muL, 15 muL, 20 muL and 25 muL according to the detection conditions in the embodiment 1, calculating a standard curve according to the detection result, and taking the concentration as the x axis of a horizontal coordinate and the peak area as the y axis, wherein the results are shown in the following table 1:
TABLE 1 results of standard curves
Figure BDA0002758362770000081
Figure BDA0002758362770000091
The data in table 1 show that the detection method provided by the invention has a good linear relationship in the concentration range for the detection of chlorogenic acid, caffeic acid, gentiopicroside, berberine, baicalin and emodin.
And (3) standard substance detection:
the characteristic component standard solution and the mixed standard solution in the preparation example 1 and the extracts of honeysuckle, dandelion, gentian, phellodendron, scutellaria and rheum officinale in the preparation example 2 are subjected to HPLC detection according to the HPLC detection conditions in the example 1, and the detection results are shown in a figure 1-figure 6, wherein 1-chlorogenic acid, 2-caffeic acid, 3-gentiopicroside, 4-berberine, 5-baicalin and 6-emodin are shown in the figure, and the detection method provided by the invention has a good separation effect on the simultaneous detection of chlorogenic acid, caffeic acid, gentiopicroside, berberine, baicalin and emodin.
Precision analysis:
preparing 6 characteristic component standards, respectively, performing sample injection analysis by the analysis method provided in example 1 at 5 different time points in the same day under the above chromatographic conditions, calculating standard deviation and Relative Standard Deviation (RSD) from the measured areas, and obtaining day precision density values, the results are shown in table 2:
TABLE 2 results of precision analysis
Figure BDA0002758362770000092
As can be seen from the table, the precision RSD of the 6 characteristic component standards is within 3 percent, which shows that the detection method provided by the invention has good precision.
And (3) repeatability analysis:
according to the detection method provided by the embodiment 1, 6 parts of the extract of the acne-removing composition prepared by compounding the same batch of Chinese medicinal materials is subjected to sample injection under the chromatographic conditions, the Relative Standard Deviation (RSD) is calculated according to the areas of the 6 measured characteristic components, the repeatability is analyzed, and the results are shown in Table 3:
TABLE 3 results of repeatability analysis
Figure BDA0002758362770000101
The table shows that the repeatability RSD of the 6 characteristic component standards is within 2.76 percent, which indicates that the detection method provided by the invention has good repeatability.
And (3) stability analysis:
the mixed standard solution provided in preparation example 1 was taken, left to stand at 25 ℃, injected at 0, 2, 4, 6, 8, 12 and 24 hours, and the solution stability was analyzed by measuring the area of each characteristic component, and the results are shown in table 4:
TABLE 4 stability analysis results
Figure BDA0002758362770000102
As can be seen from Table 4, the stability RSD of the standard substance of the 6 characteristic components is less than or equal to 2.07% when the standard substance is kept at a temperature of 25 ℃, which indicates that the detection method provided by the invention has good stability.
And (3) analysis of recovery rate:
weighing the acne-removing composition, preparing a test solution according to the method of example 1 according to the formula of example 1, adding 1 μ L of the mixed standard solution provided in preparation example 1, injecting the sample under the detection conditions provided in example 1, repeating the sample for 6 times, and analyzing the sample recovery rate, wherein the results are shown in Table 5:
TABLE 5 results of recovery analysis
Figure BDA0002758362770000111
As can be seen from Table 5, the standard substance of 6 characteristic components has a recovery rate of 95% -105% under the detection method provided by the invention, and reaches the requirement of chromatographic analysis, thus the detection method provided by the invention has reliable results.
Example detection:
the mixed standard solution of preparation example 1, the extracts of honeysuckle, dandelion, gentian, phellodendron, scutellaria and rheum officinale of preparation example 2 were tested by the test method of example 1, and the test results of example 1 were collected, with the results shown in fig. 7 and table 6:
TABLE 6 characteristic component content table
Figure BDA0002758362770000112
Figure BDA0002758362770000121
The results show that the detection method provided by the invention can fully separate and quantitatively detect chlorogenic acid, caffeic acid, gentiopicroside, berberine, baicalin and emodin in the acne-removing composition.
The applicant states that the present invention is illustrated by the above examples of the HPLC detection method for simultaneously measuring different characteristic components in the acne-removing composition, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must rely on the above examples to be implemented. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Claims (10)

1. An HPLC detection method for simultaneously determining different characteristic components in an acne-removing composition, which is characterized by comprising the following steps: preparing the acne-removing composition into a sample, detecting by HPLC (high performance liquid chromatography) for gradient elution to obtain a detection result, and calculating the content of the characteristic component by using a standard curve;
the characteristic component comprises one or the combination of at least two of chlorogenic acid, caffeic acid, gentiopicroside, berberine, baicalin or emodin.
2. The HPLC detection method for simultaneously determining different characteristic components in an acne-removing composition according to claim 1, wherein the solvent for gradient elution is an ultra-pure aqueous solution of methanol and phosphoric acid;
preferably, the mass fraction of the phosphoric acid ultrapure water solution is 0.03-0.08%.
3. The HPLC detection method for simultaneously determining different characteristic components in an acne-removing composition according to claim 1 or 2, wherein the gradient elution is performed according to the following procedure: the volume of the methanol in the eluent is 10% at 0min, the volume of the phosphoric acid ultrapure water solution is 90%, the volume of the methanol is 25% at the time of uniform speed change to 6min, the volume of the phosphoric acid ultrapure water solution is 75%, the volume of the methanol is 30% at the time of uniform speed change to 15min, the volume of the phosphoric acid ultrapure water solution is 70%, the volume of the methanol is 40% at the time of uniform speed change to 21min, the volume of the phosphoric acid ultrapure water solution is 60%, the volume of the methanol is 55% at the time of uniform speed change to 27min, the volume of the phosphoric acid ultrapure water solution is 45%, the volume of the methanol is 90% at the time of uniform speed change to 40min, and the volume of the phosphoric acid ultrapure water solution is 10%.
4. An HPLC detection method for simultaneously determining different characteristic components in an acne-removing composition according to any one of claims 1-3, wherein the preparation of the acne-removing composition into a sample comprises the following steps: extracting the acne-removing composition with purified water under reflux, filtering, adsorbing and eluting the filtrate with macroporous adsorbent resin, evaporating the eluate, concentrating, diluting to constant volume with methanol, and filtering to obtain the sample;
preferably, the temperature of the reflux is 75-85 ℃;
preferably, the refluxing time is 50-70 min.
5. The simultaneous assay according to any one of claims 1-4The HPLC detection method for different characteristic components in the acne-removing composition is characterized in that Agilent InfinityLab Poroshell 120SB-C is adopted by HPLC18The specification of the chromatographic column is 150mm multiplied by 4.60mm, and the grain diameter of the filler is 2.8 mu m.
6. The HPLC detection method for simultaneously determining different characteristic ingredients in an acne-removing composition according to any one of claims 1-5, wherein the column temperature of the HPLC detection is 35-45 ℃.
7. HPLC detection method for simultaneous determination of different characteristic ingredients in acne-removing composition according to any of claims 1-6, characterized in that the flow rate of the gradient elution is 0.8-1.2 mL/min.
8. The HPLC detection method for simultaneously determining different characteristic ingredients in the acne-removing composition according to any one of claims 1 to 6, wherein the sample injection volume of the HPLC detection is 8-12 μ L.
9. The HPLC detection method for simultaneously determining different characteristic components in an acne-removing composition according to any one of claims 1-6, wherein the wavelength of the HPLC detection is 260-300 nm.
10. HPLC detection method for simultaneous determination of different characteristic components in an acne-removing composition according to any of claims 1 to 6, characterized in that the standard curve is obtained by the following steps: and (3) dissolving and diluting the standard sample of the characteristic component with methanol, then carrying out HPLC detection according to the gradient elution program, and calculating a standard curve according to the detection result.
CN202011209945.6A 2020-11-03 2020-11-03 HPLC (high performance liquid chromatography) detection method for simultaneously determining different characteristic components in acne-removing composition Withdrawn CN112415109A (en)

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CN113495110A (en) * 2021-08-05 2021-10-12 魏秀丽 Method for simultaneously measuring 4 effective components in dandelion bluish green blue particles

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