CN116265939A - Method for constructing HPLC fingerprint and contrast fingerprint of compound pearl acne tablet sample - Google Patents
Method for constructing HPLC fingerprint and contrast fingerprint of compound pearl acne tablet sample Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 43
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 28
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- 238000001514 detection method Methods 0.000 claims abstract description 16
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- BMRSEYFENKXDIS-KLZCAUPSSA-N cis-3-O-p-coumaroylquinic acid Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)cc2)[C@@H]1O)C(=O)O BMRSEYFENKXDIS-KLZCAUPSSA-N 0.000 claims description 11
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- 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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Analytical Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The invention discloses a method for constructing HPLC fingerprint and contrast fingerprint of compound pearl acne tablet sample, which comprises the following steps: injecting 15 batches of samples of the compound pearl acne tablet into a liquid chromatograph for single-needle fingerprint measurement, recording the fingerprint, substituting similarity software for fitting to obtain a control fingerprint; injecting the sample solution into a liquid chromatograph for measurement and recording chromatographic peaks to obtain a sample fingerprint; the chromatographic conditions used include: octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, a phosphoric acid solution is used as a mobile phase B for gradient elution, and the detection wavelength is 230nm. The method has good specificity, and the characteristic peak has no impurity peak interference; the precision, repeatability and stability meet the requirements, the fingerprint constructed by the method has better adaptability, can comprehensively reflect the quality information of the compound pearl acne tablet, has the advantages of rapidness, stability, high precision, strong reproducibility and the like, and can be used for quality control of the compound pearl acne tablet.
Description
Technical Field
The invention relates to a method for constructing a fingerprint of a Chinese patent medicine, in particular to a method for constructing an HPLC fingerprint and a reference fingerprint of a compound pearl acne tablet sample, belonging to the field of construction of compound pearl acne tablet fingerprints and reference fingerprints.
Background
The Chinese patent medicine is a Chinese medicinal product which is prepared from Chinese medicinal materials by a prescribed prescription and preparation process under the guidance of Chinese medicinal theory and aims at preventing and treating diseases. However, because of the complex system of the Chinese patent medicine and the diversity and complexity of the components, the quality of the Chinese patent medicine is evaluated by adopting a detection method which is suitable for the Chinese patent medicine and can provide rich identification information, but the existing methods of microscopic identification, physicochemical identification, content measurement and the like are not enough to solve the problem.
At present, the fingerprint is one of internationally recognized means which is most suitable for controlling the quality of traditional Chinese medicines or natural medicines, and the traditional Chinese medicine fingerprint refers to a chromatogram or a spectrogram which is obtained by adopting a certain analysis means and can mark the chemical characteristics of certain traditional Chinese medicines or traditional Chinese medicine preparations after being properly processed, and is a comprehensive and quantifiable identification means. The establishment of the fingerprint spectrum of the traditional Chinese medicine can reflect the types and the amounts of chemical components contained in the traditional Chinese medicine and the preparation thereof more comprehensively, so that the quality of the medicine is integrally described and evaluated, and the fingerprint spectrum of the traditional Chinese medicine exactly accords with the general theory of the traditional Chinese medicine. Therefore, the fingerprint spectrum is comprehensively utilized, and the quality of the Chinese patent medicine can be comprehensively evaluated by combining with the intelligent information processing of a computer.
The compound pearl acne tablet is a medicine developed by the applicant for treating young face acnes (commonly known as acne), skin eczema, dermatitis and the like, is prepared from medicinal materials such as honeysuckle, dandelion, radix scutellariae, phellodendron, wine rheum officinale, red paeony root and the like according to the conventional process of the Chinese patent medicine tablet, and has the effects of clearing heat, drying dampness, detoxifying, purging pathogenic fire and cooling blood. The quality standard of the method only has qualitative identification of the thin-layer chromatography of red paeony root, paeoniflorin, rheum officinale and berberine hydrochloride, the high performance liquid chromatography is used for measuring the content of baicalin which is the main component of the baicalin, the compound pearl acne tablet is a traditional Chinese medicine compound, the quantity of the medicine ingredients is large, the contained chemical components are very complex, the standard is difficult to comprehensively reflect the integral quality characteristics of the product, and the quality of the product cannot be controlled on the whole.
Disclosure of Invention
The invention mainly aims to provide a method for constructing the HPLC fingerprint spectrum of the compound pearl acne tablet, and the constructed fingerprint spectrum can comprehensively reflect the quality information of the compound pearl acne tablet, has the advantages of rapidness, stability, high precision, strong reproducibility and the like, and can be used for quality control of the compound pearl acne tablet.
A method for constructing HPLC fingerprint of compound pearl acne tablet, which comprises a method for constructing HPLC reference fingerprint of compound pearl acne tablet and a method for constructing HPLC fingerprint of sample of compound pearl acne tablet to be detected,
the method for constructing the HPLC control fingerprint of the compound pearl acne tablet comprises the following steps:
injecting 15 batches of samples of the compound pearl acne tablet into a liquid chromatograph for single-needle fingerprint measurement, recording the fingerprint, substituting similarity software for fitting to obtain a control fingerprint of the compound pearl acne tablet;
the construction method of the HPLC fingerprint of the compound pearl acne tablet sample to be detected comprises the following steps:
(1) Preparation of test solution: removing coating from compound pearl acne tablet to be detected, grinding, extracting with 10-100% methanol as extraction solvent by ultrasonic or reflux method to obtain extractive solution, filtering, and collecting filtrate to obtain sample solution;
(2) Sucking the solution of the sample to be detected, injecting the solution into a liquid chromatograph for measurement, and recording chromatographic peaks within 110 minutes to obtain the fingerprint of the compound pearl acne tablet sample to be detected;
wherein, the chromatographic conditions for measuring the control fingerprint spectrum of the compound pearl acne tablet or the fingerprint spectrum of the compound pearl acne tablet sample to be detected by adopting a liquid chromatograph comprise: octadecylsilane chemically bonded silica is used as a filler, and the chromatographic column is Welch Ultimate AQ-C18, waters Xselect HSS T3 or Welch Ultimate LP C18; acetonitrile is taken as a mobile phase A, 0.05-0.2% phosphoric acid (V/V) solution is taken as a mobile phase B, and gradient elution is carried out according to the following procedure:
the flow rate is 0.5-1.5ml/min, the detection wavelength is 230nm, and the column temperature is 20-35 ℃; the theoretical plate number is not lower than 10000 calculated according to baicalin peak, and the separation degree of baicalin peak and berberine peak is not less than 1.5.
As a preferred specific implementation scheme of the invention, the construction method of the HPLC control fingerprint of the compound pearl acne tablet further comprises the following steps: preparing a reference solution, sucking the reference solution, injecting the reference solution into a liquid chromatograph for measurement, and recording chromatographic peaks within 110 minutes; the effect of the reference in the control fingerprint was to confirm 5 peaks out of 28 common peaks, specifically that of the compound.
As a preferred embodiment of the present invention, the reference solution is prepared by: respectively taking gallic acid reference substance, chlorogenic acid reference substance, paeoniflorin reference substance, baicalin reference substance, berberine hydrochloride reference substance, and adding 10-100% methanol to obtain mixed solution; more preferably, the reference solution is prepared by the following steps: respectively taking gallic acid reference substance, chlorogenic acid reference substance, paeoniflorin reference substance, baicalin reference substance, berberine hydrochloride reference substance, and adding 50% methanol to obtain mixed solution containing 30 μg of each reference substance per 1 ml.
As a preferred embodiment of the present invention, the preparation method of the sample solution is preferably as follows: taking compound pearl acne tablet, removing coating, grinding, extracting with 50% methanol as extraction solvent by ultrasonic method for 10-45 min (preferably 30 min), cooling the extractive solution, filtering, and collecting filtrate to obtain sample solution.
Because the material composition of the compound pearl acne tablet covers the components with the polarity of the size, a solvent which combines water solubility and alcohol solubility component extraction needs to be selected. According to the invention, the compound pearl acne tablet is taken, the coating is removed, the tablet is ground, 3 parts of powder is precisely weighed, 30% of methanol, 50% of methanol and 100% of methanol are respectively added for ultrasonic extraction, and the result shows that the solvent effect exists in a sample extracted by the methanol, the peak area is poor, the peak area of the large polar components such as gallic acid is obviously smaller than the peak area of the sample extracted by the 30% of methanol and the 50% of methanol, and the characteristic peak area of baicalin is also minimum. The characteristic peaks of 30% methanol and 50% methanol extraction basically correspond, but the peak-to-peak area of the characteristic component of the baical skullcap root extracted by 50% methanol is obviously larger than that of a sample extracted by 30% methanol, which indicates that 50% methanol extraction is more complete, and therefore, 50% methanol is preferably used as an extraction solvent in the invention.
In order to determine the proper wavelength in the chromatographic detection condition, the invention screens in the wavelength scanning range of 190nm-400nm, and the result shows that the characteristic chromatographic peaks of medicinal materials such as red paeony root, amur corktree bark and the like in the prescription of the compound pearl acne tablet have obvious ultraviolet absorption only near 230nm, the fingerprint information of the wavelength band is obviously richer than that of other wavelength bands, the baseline of the wavelength band is smoother, and the impurity interference is less, so the invention finally determines 230nm as the detection wavelength of the fingerprint spectrum of the compound pearl acne tablet.
In the prescription process of the compound pearl acne tablet, the components of the compound pearl acne tablet comprise water extract powder and medicinal material crushing and feeding, so that the components comprise water-soluble large-polarity components and water-soluble and fat-soluble components in raw medicinal material powder, and the polarity difference is large. Therefore, the invention needs to use the low proportion of organic phase and the high proportion of water phase to elute the water-soluble large polar characteristic component, and then gradually improves the proportion of the organic phase to elute the small polar characteristic component. In the composition of the mobile phase, when the initial proportion of the organic phase is adjusted to be 3%, the large polar component can be effectively separated, unlike the conventional C18 reversed phase chromatographic elution (four appendices 0512 high performance liquid chromatography of pharmacopoeia 2020 edition, the organic solvent in the mobile phase is generally not lower than 5%, otherwise, the column efficiency is easily reduced, and the chromatographic system is unstable), therefore, in the aspect of chromatographic column selection, a chromatographic column which can resist the pure water phase mobile phase is required to be selected, and three chromatographic columns meeting the requirements are respectively examined under the same gradient in the study, namely Waters Xselect HSS T (4.6X250 mm,5 μm), welch Ultimate LP C18 (4.6X250 mm,5 μm) and Welch Ultimate AQ C18 (4.6X250 mm,5 μm). As can be seen from the examination result, when a Welch Ultimate AQ C (4.6X250 mm,5 μm) column is used, the whole chromatographic peak separation effect is the best, so the invention preferably adopts a liquid chromatographic column with Welch Ultimate AQ C as a fingerprint of the compound pearl acne tablet.
According to the invention, the influence on chromatographic peaks is examined respectively when acetonitrile-water, phosphoric acid solution with acetonitrile-volume fraction of 0.1%, glacial acetic acid solution with acetonitrile-volume fraction of 0.5%, glacial acetic acid solution with acetonitrile-volume fraction of 0.1%, formic acid solution with acetonitrile-volume fraction of 0.5% and phosphoric acid solution with methanol-volume fraction of 0.1% are used as a mobile phase system, and as a result, it is found that most of chromatographic peaks have poor peak types and tailing of different degrees when acetonitrile-water is used as the mobile phase system; glacial acetic acid and formic acid with different concentrations are adopted as mobile phases, and baseline drift is large, so that the quality of the map is affected; when methanol-phosphoric acid is adopted as a mobile phase system, the separation among the chromatographic peaks is not ideal; when the mobile phase system is phosphoric acid solution with acetonitrile-volume fraction of 0.1%, the whole baseline is stable, the chromatographic peak separation degree is good, the chromatographic peak number is more, the impurity peak is less, and the peak outlet time is faster.
According to the invention, the influence of phosphoric acid solutions with different volume fractions on chromatographic peaks is examined, acetonitrile-0.05% phosphoric acid, acetonitrile-0.1% phosphoric acid, acetonitrile-0.2% phosphoric acid, elution gradient, column temperature and other chromatographic conditions are kept consistent, and the result shows that the retention time of berberine hydrochloride peaks is greatly influenced by the concentration of phosphoric acid, the retention time is longer as the concentration of phosphoric acid is larger, and the separation conditions of other chromatographic characteristic peaks are not obviously different. When the volume fraction of phosphoric acid is 0.1%, berberine hydrochloride is well separated from adjacent characteristic peaks and has no interference of impurities, so that a phosphoric acid solution with the volume fraction of 0.1% is preferable.
The invention examines the influence of the column temperature at 25 ℃ and 30 ℃ and 35 ℃ on the chromatogram peaks, and discovers that the column temperature at 30 ℃ can achieve better separation of each chromatogram peak, therefore, the column temperature is preferably 30 ℃ in the chromatographic detection conditions.
The invention respectively examines the influences on the chromatogram peaks when the flow rates of the mobile phases are 0.9ml/min, 1.0ml/min and 1.1 ml/min. As a result, it was found that at a flow rate of 1ml/min, each chromatographic peak was well separated, and therefore, the chromatographic detection conditions of the present invention preferably used a flow rate of 1ml/min.
The HPLC control fingerprint of the compound pearl acne tablet constructed by the invention shows chromatographic peaks with consistent retention time with chromatographic peaks of gallic acid, chlorogenic acid, paeoniflorin, baicalin and berberine hydrochloride control substances, and 28 common peaks appear, and 5 characteristic peaks are finally identified as peak 1 respectively by comparing with the control substances on the basis of complete coincidence of the retention time and the ultraviolet characteristic spectrum: gallic acid; peak 4: chlorogenic acid; peak 6: paeoniflorin; peak 16: baicalin; peak 17: berberine hydrochloride.
Substituting the recorded HPLC fingerprint of the compound pearl acne tablet sample to be detected into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to perform similarity calculation, and comparing the recorded HPLC fingerprint with a control fingerprint, wherein the similarity is not lower than 0.90.
The compound pearl acne tablet is film coated tablet and is orally taken, and water is used in extracting and concentrating the compound pearl acne tablet in the technological extraction process, so that the main effective components in the compound pearl acne tablet are water soluble matters with great polarity. By combining the properties of the active ingredients, the research mainly adopts the high performance liquid chromatography technology to examine the fingerprint characteristics of the large polar ingredients in the compound pearl acne tablet so as to better reflect the correlation of the pharmacological and pharmacodynamic bases of the compound pearl acne tablet. According to the prescription medicinal material data collected in the earlier stage work, the lonicera japonica in the prescription contains chlorogenic acid characteristic components, the red paeony root and the rheum officinale contain gallic acid characteristic components, the red paeony root contains paeoniflorin characteristic components, the scutellaria baicalensis contains baicalin characteristic components, huang Baihan berberine and other characteristic components, and corresponding reference substance solutions are prepared for positioning comparison according to the medicinal effect components of each medicinal flavor and main chemical properties thereof, so that the constructed standard fingerprint can comprehensively reflect the quality information of the compound pearl acne tablet, the defects of single and one-sided existing quality control standard are avoided, and the method can be used for comprehensively evaluating and controlling the quality of the compound pearl acne tablet.
The fingerprint spectrum of the compound pearl acne tablet is established by high performance liquid chromatography, and the fingerprint spectrum is verified by methodology, and the method has good specificity and no interference of characteristic peaks and impurity peaks; the precision, repeatability and stability meet the requirements, and the method is stable and reliable and has good adaptability. 15 batches of compound pearl acne tablet fingerprints are measured, 15 batches of compound pearl acne tablet fingerprints are substituted into 2012 edition of traditional Chinese medicine fingerprint similarity evaluation System, mark peaks are used for matching, a comparison fingerprint is obtained through fitting, and a CDF format file is generated, so that the comparison fingerprint can be provided for measurement of other compound pearl acne tablet fingerprints. The calculated similarity between the fingerprint of 15 batches of compound pearl acne tablets and the fitted control spectrum is above 0.99, which shows that the fingerprint quality consistency degree of 15 batches of compound pearl acne tablets is high, and the production process is stable and controllable.
The invention utilizes the mixed reference substance solution to identify the common peaks of gallic acid, chlorogenic acid, paeoniflorin, baicalin and berberine hydrochloride, the constructed standard fingerprint can comprehensively reflect the quality information of the compound pearl acne tablet, has the advantages of rapidness, stability, high precision, strong reproducibility and the like, avoids the defects of single and one-sided existing quality control standard, and can be used for comprehensive quality evaluation and control of the compound pearl acne tablet. And the quality consistency of the product is proved to be objective and accurate by carrying out comprehensive quality comparison on 15 batches of compound pearl acne tablet samples by using the newly established fingerprint. The fingerprint is identified by adopting the Chinese medicine chromatographic fingerprint similarity evaluation system recommended by the national formulary Committee, the operation is simple, convenient and quick, the obtained similarity result is used for evaluating the fingerprint of the compound pearl acne tablet, and the conclusion is objective and accurate.
Drawings
Fig. 1 is a fingerprint spectrum at different detection wavelengths.
FIG. 2 is a fingerprint of different chromatographic columns.
Fig. 3 is a fingerprint of different types of mobile phases.
FIG. 4 is a fingerprint of mobile phases with different phosphoric acid concentrations.
FIG. 5 is a fingerprint of different column temperatures.
Fig. 6 is a fingerprint of different flow rates.
Fig. 7 is a fingerprint of different extraction solvents.
Fig. 8 is a fingerprint of different extraction modes.
Fig. 9 is a fingerprint of different extraction times.
FIG. 10 is a chromatogram of the precision test.
FIG. 11 is a repetitive test chromatogram.
Fig. 12 is a finger print of different operators.
Fig. 13 different liquid chromatograph finger prints.
FIG. 14 is a stability test chromatogram.
Fig. 15 is a fingerprint of a compound pearl acne tablet and a control solution, wherein the unit of the abscissa is min.
Fig. 16 is a fingerprint of 15 batches of compound pearl acne tablets.
Fig. 17 is a compound pearl acne tablet control fingerprint (sample concentration: equivalent to 0.1 gram of compound pearl acne tablet per milliliter), wherein peak 1: gallic acid, peak 4: chlorogenic acid, peak 6: paeoniflorin, peak 16 (S): baicalin, peak 17: berberine hydrochloride.
Fig. 18 is a mixed control profile.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the invention without departing from the spirit and scope of the invention, but these modifications and substitutions are intended to be within the scope of the invention.
Test example 1 establishment of HPLC fingerprint construction method of Compound Pearl acne tablet and methodological verification
1 establishment of fingerprint chromatography method
1.1 selection of detection wavelength
The method comprises the following steps: the mobile phase A is acetonitrile, the mobile phase B is 0.1% phosphoric acid (V/V) solution, and linear gradient elution is carried out according to a preset program; the flow rate is 1ml/min, the column temperature is 30 ℃, the PDA is used for monitoring, and the wavelength scanning range is 190nm-400nm. The results are shown in FIG. 1. As can be seen from the graph in fig. 1, the characteristic chromatographic peaks of medicinal materials such as red paeony root and amur corktree bark in the prescription of the compound pearl acne tablet have more obvious ultraviolet absorption only near 230nm, fingerprint information of the wavelength band is obviously richer than that of other wavelength bands, the baseline of the wavelength band is smoother, and the interference of impurities is less, so that the invention determines 230nm as the detection wavelength of the fingerprint spectrum of the compound pearl acne tablet.
1.2 selection of chromatographic columns
In the prescription process of the compound pearl acne tablet, the components of the compound pearl acne tablet comprise water extract powder and medicinal material crushing and feeding, so that the components comprise water-soluble large-polarity components and water-soluble and fat-soluble components in raw medicinal material powder, and the polarity difference is large. Therefore, the invention needs to use the low proportion of organic phase and the high proportion of water phase to elute the water-soluble large polar characteristic component, and then gradually improves the proportion of the organic phase to elute the small polar characteristic component. In the composition of the mobile phase, when the initial proportion of the organic phase is adjusted to be 3%, the large polar component can be effectively separated, unlike the conventional C18 reversed phase chromatographic elution (four appendices 0512 high performance liquid chromatography of pharmacopoeia 2020 edition, the organic solvent in the mobile phase is generally not lower than 5%, otherwise, the column efficiency is easily reduced, and the chromatographic system is unstable), therefore, in the aspect of chromatographic column selection, a chromatographic column which can resist the pure water phase mobile phase is required to be selected, and three chromatographic columns meeting the requirements are respectively examined under the same gradient in the study, namely Waters Xselect HSS T (4.6X250 mm,5 μm), welch Ultimate LP C18 (4.6X250 mm,5 μm) and Welch Ultimate AQ C18 (4.6X250 mm,5 μm). The results are shown in FIG. 2. As can be seen from the graph of FIG. 2, the chromatographic column of Welch Ultimate AQ C (4.6X105 mm,5 μm) has the best overall separation effect, so Welch Ultimate AQ C is selected as the liquid chromatographic column of the fingerprint of the compound pearl acne tablet.
1.3 selection of mobile phase species
The effect on chromatographic peaks when acetonitrile-water, a phosphoric acid solution with acetonitrile-volume fraction of 0.1%, a glacial acetic acid solution with acetonitrile-volume fraction of 0.5%, a glacial acetic acid solution with acetonitrile-volume fraction of 0.1%, a formic acid solution with acetonitrile-volume fraction of 0.5% and a phosphoric acid solution with methanol-volume fraction of 0.1% were examined as mobile phase systems, respectively, and the results are shown in fig. 3. The results show that when acetonitrile-water is used as a mobile phase system, most chromatographic peaks are poor in peak type, and tailing occurs to different degrees; glacial acetic acid and formic acid with different concentrations are adopted as mobile phases, and baseline drift is large, so that the quality of the map is affected; when methanol-phosphoric acid is adopted as a mobile phase system, the separation among the chromatographic peaks is not ideal; when the mobile phase system is phosphoric acid solution with acetonitrile-volume fraction of 0.1%, the whole baseline is stable, the chromatographic peak separation degree is good, the number of chromatographic peaks is large, the impurity peaks are few, and the peak outlet time is quick, so that the acetonitrile-phosphoric acid solution is selected as the final mobile phase.
1.4 selection of phosphoric acid concentration
The effect of different volume fractions of phosphoric acid solution on chromatographic peaks was examined, and other chromatographic conditions such as acetonitrile-0.05% phosphoric acid, acetonitrile-0.1% phosphoric acid, acetonitrile-0.2% phosphoric acid, elution gradient and column temperature were kept consistent, as shown in FIG. 4.
The result shows that the retention time of the berberine hydrochloride peak is greatly influenced by the concentration of phosphoric acid, the retention time is longer as the concentration of phosphoric acid is higher, and the separation conditions of the other chromatographic characteristic peaks are not obviously different. When the volume fraction of phosphoric acid is 0.1%, berberine hydrochloride is well separated from adjacent characteristic peaks and has no interference of impurities, so that a phosphoric acid solution with the volume fraction of 0.1% is preferable.
1.5 selection of column temperature
The effect on the chromatogram peaks was examined at a column temperature of 25℃and 30℃and 35℃respectively. As a result, according to FIG. 5, it was found that the column temperature was 30℃because each chromatographic peak was well separated at 30 ℃.
1.6 selection of different flow rates
The effects on the chromatogram peaks were examined at flow rates of 0.9ml/min, 1.0ml/min, and 1.1ml/min for the mobile phases, respectively. The results are shown in FIG. 6. As can be seen from FIG. 6, at a flow rate of 1ml/min, each chromatographic peak was well separated, so a flow rate of 1ml/min was used.
1.7 determination of chromatographic conditions
Column Welch Ultimate AQ C (4.6X250 mm,5 μm), A was acetonitrile and B was 0.1% phosphoric acid solution (V/V). The flow rate was 1ml/min, the detection wavelength was 230nm, and the column temperature was 30 ℃. The elution was performed according to the gradient of table 1 below.
TABLE 1 fingerprint elution gradient
2 establishment of test sample solution preparation method
2.1 extraction solvent selection
Taking compound pearl acne tablet, removing coating, grinding, precisely weighing 3 parts of powder, each part of powder is 0.5g, placing into a 50ml measuring flask, respectively adding 30% methanol, 50% methanol and methanol to the near scale, ultrasonically extracting for 30min, cooling, adding corresponding solvent to the scale, shaking uniformly, filtering, collecting filtrate, and measuring. The results are shown in FIG. 7.
Because the material composition of the compound pearl acne tablet covers the components with the polarity of the size, a solvent which combines water solubility and alcohol solubility component extraction needs to be selected. The results show that the samples extracted by the methanol have the solvent effect and poor peak type, and the peak areas of the large polar components such as the gallic acid and the like are obviously smaller than the peak areas of the samples extracted by 30% of methanol and 50% of methanol, and the characteristic peak areas of the baicalin are also minimum. Characteristic peaks of 30% methanol and 50% methanol extraction basically correspond, but peak-to-peak areas of characteristic components of the baikal skullcap root extracted by 50% methanol are obviously larger than those of a sample extracted by 30% methanol, which indicates that 50% methanol extraction is more complete, and therefore 50% methanol is preferable as an extraction solvent.
2.2 selection of extraction modes
Taking compound pearl acne tablet, removing coating, grinding, precisely weighing 2 parts of powder, dividing each part into two groups of A, B, precisely adding 50mL of 50% methanol respectively, weighing, performing ultrasonic treatment on group A for 30 minutes, performing water bath reflux on group B for 30 minutes, cooling to room temperature respectively, weighing, supplementing the lost weight with 50% methanol, shaking uniformly, filtering, taking subsequent filtrate, and measuring. The results are shown in FIG. 8. The result shows that the ultrasonic and reflux extraction maps have no obvious difference, and the ultrasonic extraction is selected in consideration of the simpler and more convenient ultrasonic extraction operation.
2.3 selection of extraction time
Taking compound pearl acne tablet, removing coating, grinding, precisely weighing 3 parts of powder, each part of powder is 0.5g, placing into a 50ml measuring flask, adding 50% methanol to the near scale, respectively extracting by ultrasonic for 15min, 30min and 45min, cooling, adding 50% methanol to the scale, shaking uniformly, filtering, taking the subsequent filtrate, and measuring. The results are shown in FIG. 9. The results show that the peak area of the sample after 15 minutes of ultrasonic treatment is smaller, and the extraction effect after 30 minutes is not obviously different from the extraction effect after 45 minutes. Therefore, the extraction time is preferably 30 minutes.
2.4 determination of the method for preparing the sample solution
Taking compound pearl acne tablet, removing coating, grinding, precisely weighing 0.5g, placing in a 50ml volumetric flask, adding 50% methanol to near scale, sealing, ultrasonic extracting for 30min, cooling, adding 50% methanol to scale, shaking, filtering, and collecting the subsequent filtrate.
3 methodological verification
3.1 precision test
Sample solutions (lot number: 20014, supplied by Dezhong (Buddha) pharmaceutical Co., ltd.) were taken and continuously measured 6 times according to the established test conditions. Recording the fingerprint, comparing the obtained fingerprint with the generated reference fingerprint, calculating the similarity, wherein the similarity is 1, the retention time RSD of the common peak is less than 1%, and the relative peak area RSD of the common peak is less than 5% (figure 10), which shows that the precision of the method is good.
TABLE 2 results of precision test-characteristic peak to peak area ratio
TABLE 3 results of precision test-characteristic peak to retention time ratio
3.2 repeatability test
The experimenter A takes the same batch of test articles (batch number: 20014, provided by the pharmaceutical industry Co., ltd., by the national pharmaceutical sciences), prepares 6 parts of test article solutions by referring to the preparation of the test article solutions, precisely absorbs 10 mu L of the test article solutions, injects the test article solutions into a liquid chromatograph, carries out sample injection measurement by referring to the chromatographic condition, records the fingerprint, compares the fingerprint with the generated reference fingerprint, calculates the similarity, and the result similarity is 1, the retention time RSD of the common peak is less than 1%, and the relative peak area RSD of the common peak is less than 5% (figure 11), thus showing that the repeatability of the method is good.
TABLE 4 repeatability test results-characteristic peak to peak area ratio
TABLE 5 repeatability test results-characteristic peak to retention time ratio
3.3 intermediate precision test
3.3.1 comparison of different operators
The laboratory technician B prepares 6 parts of test sample (batch number: 20014, supplied by Dezhong (Buddha) pharmaceutical Co., ltd.) solution according to the requirement of the repeatability test, and determines the test sample according to the established chromatographic condition. Recording fingerprints, substituting the fingerprints into similarity software, wherein each fingerprint should have 28 characteristic peaks, and respectively calculating the similarity with the reference fingerprint. The results show that under the operation of two different persons, the similarity between each fingerprint and the control fingerprint is 1, the retention time RSD of the common peak is less than 1%, and the relative peak area RSD of the common peak is less than 5% (figure 12), which shows that the method has good intermediate precision.
3.3.2 comparison of different brands and models by liquid chromatography
The same sample solution was taken and tested on Waters 2695 liquid chromatograph, thermo Ultimate 3000 liquid chromatograph, waters AQUITY Arc liquid chromatograph, respectively, according to the prescribed chromatographic conditions, and the results are shown in fig. 13.
The results show that the fingerprints obtained by the Waters 2695 liquid chromatograph, the Thermo Ultimate 3000 liquid chromatograph and the Waters AQUITY Arc liquid chromatograph are analyzed by a similarity evaluation system, the similarity is sequentially 1, 0.996 and 1, the results of the liquid chromatographs with different brands or models are not obviously different, and the intermediate precision of the method is good.
3.4 stability test
Taking one part of test solution of the intermediate precision item, and measuring at 0, 2, 4, 6, 8, 10, 22, 38 and 51 hours respectively. And recording fingerprints, substituting the fingerprints into similarity software, wherein each fingerprint has 28 characteristic peaks, and the result shows that in the measurement results of different time periods, the similarity between each fingerprint and the 0-hour fingerprint is 1, the retention time RSD of the common peak is less than 1%, the relative peak area RSD of the common peak is less than 5%, and the measurement results of the sample are stable within 51 hours (fig. 14).
4 establishment of reference fingerprint
4.1 sources of the medicinal materials of the main chromatographic peaks
Referring to the compound pearl acne tablet technology and the preparation of the test sample solution, respectively preparing the to-be-tested solutions of single raw medicinal materials in the prescription, respectively precisely sucking 10 μl, injecting into a liquid chromatograph, and referring to the chromatographic condition for sample injection test. The result shows that 28 common peaks in the standard fingerprint of the compound pearl acne tablet basically find definite attribution in the fingerprint of the prescription medicinal material, wherein: the No. 1 peak is the medicinal materials of red paeony root and rhubarb; 2. the No. 4, 5, 7, 13, 14 and 15 peaks belong to the lonicera japonica medicinal material; the No. 3 peak is attributed to dandelion herb; 6. the No. 10 peak belongs to red paeony root medicinal materials; 8. the No. 9 and No. 17 peaks belong to cortex phellodendri medicinal materials; 11. peaks 12, 16, 18, 19, 20, 21, 22, 23, 25, 26, 27 and 28 belong to baical skullcap root medicinal materials; the No. 24 peak belongs to the medicinal material of rheum officinale.
4.2 identification of characteristic peaks
And (3) performing full-wavelength scanning by adopting a high-efficiency liquid phase method, and comparing the full-wavelength scanning with the retention time and the ultraviolet absorption spectrum of a reference substance to confirm the characteristic peak compound. The results showed that peak 1 is gallic acid, peak 4 is chlorogenic acid, peak 6 is paeoniflorin, peak 16 is baicalin, and peak 17 is berberine hydrochloride (fig. 15).
4.3 fitting of control atlas
And (3) measuring single needle fingerprints of 15 batches of samples of the compound pearl acne tablet by adopting a formulated method, recording the fingerprints, substituting similarity software to generate a control spectrum (fig. 16-17), and calculating the similarity between 15 batches of samples and the control fingerprint, wherein the results are shown in fig. 18 and table 10.
Table 10 similarity of compound pearl acne tablet fingerprint of 15 batches
The test results in the table 10 show that the fingerprint similarity of the compound pearl acne tablets of 15 batches is above 0.99.
Claims (10)
1. The method for constructing the HPLC fingerprint of the compound pearl acne tablet is characterized by comprising the construction of an HPLC control fingerprint of the compound pearl acne tablet and the construction of an HPLC fingerprint of a sample of the compound pearl acne tablet to be detected;
the construction method of the HPLC control fingerprint of the compound pearl acne tablet comprises the following steps:
injecting 15 batches of samples of the compound pearl acne tablet into a liquid chromatograph for single-needle fingerprint measurement, recording the fingerprint, substituting similarity software, and fitting to obtain a control fingerprint of the compound pearl acne tablet;
the construction method of the HPLC fingerprint of the compound pearl acne tablet sample to be detected comprises the following steps:
(1) Preparation of test solution: removing coating from compound pearl acne tablet to be detected, grinding, extracting with 10-100% methanol as extraction solvent by ultrasonic or reflux method to obtain extractive solution, filtering, and collecting filtrate to obtain sample solution;
(2) Sucking the sample solution, injecting into a liquid chromatograph for measurement, and recording chromatographic peaks within 110 minutes to obtain the fingerprint of the compound pearl acne tablet sample to be detected;
wherein, the chromatographic conditions for measuring the fingerprint spectrum of the compound pearl acne tablet reference fingerprint spectrum or the fingerprint spectrum of the compound pearl acne tablet sample to be detected by adopting a liquid chromatograph comprise: octadecylsilane chemically bonded silica is used as a filler, and the chromatographic column is Welch Ultimate AQ-C18, waters Xselect HSS T3 or Welch Ultimate LP C18; acetonitrile is taken as a mobile phase A, a phosphoric acid solution with the volume ratio of 0.05-0.2% is taken as a mobile phase B, and gradient elution is carried out according to the following procedures:
the detection wavelength was 230nm.
2. The construction method of claim 1, wherein the construction method of the compound pearl acne tablet HPLC contrast fingerprint further comprises the following steps: preparing a reference solution, sucking the reference solution, injecting the reference solution into a liquid chromatograph for measurement, and recording chromatographic peaks within 110 minutes; the preparation method of the reference substance solution comprises the following steps: respectively taking gallic acid reference substance, chlorogenic acid reference substance, paeoniflorin reference substance, baicalin reference substance, berberine hydrochloride reference substance, and adding 10-100% methanol to obtain mixed solution;
preferably, the preparation method of the reference solution comprises the following steps: respectively taking gallic acid reference substance, chlorogenic acid reference substance, paeoniflorin reference substance, baicalin reference substance, berberine hydrochloride reference substance, and adding 50% methanol to obtain mixed solution containing 30 μg of each reference substance per 1 ml.
3. The method of claim 1, wherein the method of preparing the test solution comprises: taking compound pearl acne tablet, removing coating, grinding, extracting with 50% methanol as extraction solvent by ultrasonic extraction for 10-45 min, cooling the extractive solution, filtering, and collecting filtrate to obtain sample solution; preferably, the extraction is carried out by ultrasonic extraction for 30 minutes.
4. The method according to claim 1, wherein the mobile phase B is a 0.1% phosphoric acid solution.
5. The method of claim 1, wherein the column temperature is 20-35 ℃ in chromatographic detection conditions.
6. The method according to claim 5, wherein the column temperature is 30℃under chromatographic detection conditions.
7. The method of claim 1, wherein the flow rate in chromatographic detection conditions is 0.5-1.5ml/min
8. The method of claim 7, wherein the flow rate in chromatographic detection conditions is 1ml/min.
9. The construction method according to claim 1, wherein the HPLC control fingerprint of the constructed compound pearl acne tablet shows chromatographic peaks with retention time consistent with that of gallic acid, chlorogenic acid, paeoniflorin, baicalin and berberine hydrochloride control substances, and 28 common peaks appear.
10. The construction method according to claim 1, wherein the recorded HPLC fingerprint of the compound pearl acne tablet sample to be detected is substituted into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to perform similarity calculation, and compared with a control fingerprint, the similarity is not lower than 0.90.
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