Method for constructing HPLC fingerprint of compound pearl acne tablets sample and control fingerprint

Abstract

The present invention discloses a method for constructing a sample HPLC fingerprint of a compound pearl acne tablet and a control fingerprint, comprising: injecting 15 batches of samples of the compound pearl acne tablet into a liquid chromatograph for single-needle fingerprint determination, recording the fingerprint, substituting the fingerprint into a similarity software for fitting to obtain a control fingerprint; injecting a test solution into a liquid chromatograph for determination and recording the chromatographic peak to obtain a sample fingerprint; the chromatographic conditions adopted include: using octadecylsilane bonded silica gel as a filler, acetonitrile as a mobile phase A, and phosphoric acid solution as a mobile phase B for gradient elution, and the detection wavelength is 230nm. The method has good specificity, and the characteristic peaks are not interfered by impurity peaks; the precision, repeatability, and stability all meet the requirements, and have good adaptability. The constructed fingerprint can fully reflect the quality information of the compound pearl acne tablet, has the advantages of being fast, stable, high in precision, and strong in reproducibility, and can be used for the quality control of the compound pearl acne tablet.

Description

Technical Field

[0001] The invention relates to a method for constructing a fingerprint of a Chinese patent medicine, and in particular to a method for constructing an HPLC fingerprint of a compound pearl acne tablet sample and a control fingerprint, belonging to the field of constructing a fingerprint of a compound pearl acne tablet and a control fingerprint. Background Art

[0002] Chinese patent medicines are made from Chinese medicinal materials. Under the guidance of traditional Chinese medicine theory, they are processed into Chinese medicine products of certain dosage forms according to prescribed prescriptions and preparation processes for the purpose of preventing and treating diseases. However, due to the complexity of the Chinese patent medicine system and the diversity and complexity of its ingredients, its quality should be evaluated by a detection method that is suitable for it and can provide rich identification information. However, the current methods such as microscopic identification, physical and chemical identification and content determination are not sufficient to solve this problem.

[0003] At present, fingerprint is internationally recognized as one of the most suitable means for controlling the quality of traditional Chinese medicine or natural medicine. The fingerprint of traditional Chinese medicine refers to the chromatogram or spectrum that can indicate the chemical characteristics of certain traditional Chinese medicines or traditional Chinese medicine preparations after proper treatment and certain analytical methods. It is a comprehensive and quantifiable identification method. The establishment of the fingerprint of traditional Chinese medicine will be able to more comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine and its preparations, and then make an overall description and evaluation of the quality of the medicine, which is also in line with the holistic theory of traditional Chinese medicine. Therefore, the comprehensive use of fingerprints combined with computer intelligent information processing can make a more comprehensive evaluation of the quality of traditional Chinese medicine.

[0004] Compound Pearl Acne Tablets are medicines developed by the applicant for the treatment of facial acne (commonly known as acne) and skin eczema, dermatitis and other diseases in young people. They are made of medicinal materials such as honeysuckle, dandelion, scutellaria, phellodendron, wine rhubarb, red peony root and other medicinal materials according to the conventional process of Chinese patent medicine tablets, and have the effects of clearing heat, drying dampness, detoxifying, purging fire and cooling blood. The legal quality standard of this product only has the qualitative identification of red peony root and paeoniflorin, rhubarb and berberine hydrochloride by thin layer chromatography, and the content of baicalin, the main component of scutellaria, is determined by high performance liquid chromatography. Compound Pearl Acne Tablets are a traditional Chinese medicine compound with a large number of medicinal flavors and very complex chemical components. This standard is difficult to fully reflect the overall quality characteristics of the product and cannot control the quality of the product as a whole. Summary of the invention

[0005] The main purpose of the present invention is to provide a method for constructing an HPLC fingerprint of the compound pearl acne tablets. The constructed fingerprint can comprehensively reflect the quality information of the compound pearl acne tablets, has the advantages of being fast, stable, high in precision, strong in reproducibility, etc., and can be used for the quality control of the compound pearl acne tablets.

[0006] A method for constructing a HPLC fingerprint of a compound pearl acne tablet, including a method for constructing a HPLC control fingerprint of a compound pearl acne tablet and a method for constructing a HPLC fingerprint of a compound pearl acne tablet sample to be tested.

[0007] The method for constructing the HPLC reference fingerprint of the compound pearl acne tablets comprises:

[0008] Inject 15 batches of samples of Compound Pearl Acne Tablets into a liquid chromatograph for single-needle fingerprint determination, record the fingerprint, and substitute it into the similarity software to obtain the control fingerprint of Compound Pearl Acne Tablets;

[0009] The method for constructing the HPLC fingerprint of the compound pearl acne tablet sample to be tested comprises:

[0010] (1) Preparation of the test solution: Remove the coating of the compound pearl acne tablet to be tested, grind it into powder, use 10-100% methanol as the extraction solvent, extract it by ultrasonic or reflux to obtain an extract, filter it, and take the filtrate to obtain the test solution;

[0011] (2) aspirating the test sample solution and injecting it into a liquid chromatograph for determination, and recording the chromatographic peaks within 110 minutes to obtain the fingerprint of the compound pearl acne tablet sample to be tested;

[0012] The chromatographic conditions for determining the control fingerprint of the compound pearl acne tablet or the fingerprint of the compound pearl acne tablet sample to be tested by liquid chromatography include: using octadecylsilane bonded silica gel as a filler, a chromatographic column of Welch Ultimate AQ-C18, Waters Xselect HSS T3 or Welch Ultimate LP C18; using acetonitrile as mobile phase A, using 0.05-0.2% phosphoric acid (V / V) solution as mobile phase B, and performing gradient elution according to the following procedure:

[0013]

[0014] The flow rate is 0.5-1.5 ml / min, the detection wavelength is 230 nm, and the column temperature is 20-35°C; the theoretical plate number calculated based on the baicalin peak should be no less than 10,000, and the separation degree between the baicalin peak and the berberine peak should be no less than 1.5.

[0015] As a preferred specific embodiment of the present invention, the method for constructing the HPLC reference fingerprint of the compound pearl acne tablets also includes: preparing a reference solution, aspirating the reference solution and injecting it into a liquid chromatograph for measurement, and recording the chromatographic peaks within 110 minutes; the role of the reference in the reference fingerprint is to confirm which compound the 5 peaks among the 28 common peaks are.

[0016] As a preferred specific embodiment of the present invention, the reference solution is prepared by taking gallic acid reference, chlorogenic acid reference, paeoniflorin reference, baicalin reference, and berberine hydrochloride reference, respectively, and adding 10-100% methanol to prepare a mixed solution; more preferably, the reference solution is prepared by taking gallic acid reference, chlorogenic acid reference, paeoniflorin reference, baicalin reference, and berberine hydrochloride reference, respectively, and adding 50% methanol to prepare a mixed solution containing 30 μg of each reference substance per 1 ml.

[0017] As a preferred specific embodiment of the present invention, the preparation method of the test solution is preferably: take the compound pearl acne tablets, remove the coating, grind them into powder, use 50% methanol as the extraction solvent, extract them by ultrasonic means for 10-45 minutes (preferably 30 minutes), cool the extract, filter, take the filtrate, and obtain the test solution.

[0018] Since the material composition of the compound pearl acne tablets covers components of large and small polarity, it is necessary to select a solvent that takes into account the extraction of water-soluble and alcohol-soluble components. The present invention takes the compound pearl acne tablets, removes the coating, grinds it into powder, accurately weighs 3 parts of powder, and adds 30% methanol, 50% methanol, and 100% methanol for ultrasonic extraction. From the results, it can be found that the sample extracted by methanol has a solvent effect, the peak shape is poor, and the peak area of ​​large polar components such as gallic acid is significantly smaller than the peak area of ​​samples extracted by 30% methanol and 50% methanol, and the peak area of ​​baicalin characteristic peak is also the smallest. The characteristic peaks extracted by 30% methanol and 50% methanol are basically corresponding, but the peak area of ​​the characteristic component peak of Scutellaria baicalensis extracted by 50% methanol is significantly larger than that of the sample extracted by 30% methanol, indicating that the 50% methanol extraction is more complete. Therefore, the present invention preferably uses 50% methanol as the extraction solvent.

[0019] In order to determine the appropriate wavelength in the chromatographic detection conditions, the present invention screened within the wavelength scanning range of 190nm-400nm. The results showed that the characteristic chromatographic peaks of medicinal materials such as red peony root and phellodendron chinense in the prescription of Compound Pearl Acne Tablets only had obvious ultraviolet absorption near 230nm. The fingerprint information of this wavelength band was obviously richer than that of other wavelength bands, and the baseline of this wavelength band was relatively stable with less impurity interference. Therefore, the present invention finally determined 230nm as the detection wavelength of the fingerprint spectrum of Compound Pearl Acne Tablets.

[0020] The formula and process of the compound pearl acne tablets include two components: water-extracted extract powder and crushed medicinal materials. Therefore, its components include water-soluble polar components and water-soluble and fat-soluble components in the original medicinal powder, with large polarity differences. Therefore, in the present invention, it is necessary to first use a low proportion of organic phase and a high proportion of water phase to elute the water-soluble polar characteristic components, and then gradually increase the proportion of organic phase to elute the small polar characteristic components. In the mobile phase composition of the present invention, when the organic phase is adjusted to an initial ratio of 3%, the highly polar components can be effectively separated, which is different from the conventional C18 reverse phase chromatography elution (Appendix 0512 High Performance Liquid Chromatography of Part IV of the 2020 Pharmacopoeia mentions: The organic solvent in the mobile phase should generally not be less than 5%, otherwise it is easy to cause a decrease in column efficiency and instability of the chromatographic system). Therefore, in terms of chromatographic column selection, it is necessary to select a chromatographic column that has been specially treated and can tolerate a pure water mobile phase. Therefore, this study examined three chromatographic columns that met the requirements under the same gradient, namely Waters Xselect HSS T3 (4.6×250mm, 5μm), Welch Ultimate LP C18 (4.6×250mm, 5μm), and Welch Ultimate AQ C18 (4.6×250mm, 5μm). From the investigation results, it can be found that when the Welch Ultimate AQ C18 (4.6×250 mm, 5 μm) column is used, the overall separation effect of the chromatographic peak is the best. Therefore, the present invention preferably uses Welch Ultimate AQ C18 as the liquid chromatography column for the fingerprint of the compound pearl acne tablets.

[0021] The invention investigates the influence of acetonitrile-water, acetonitrile-phosphoric acid solution with a volume fraction of 0.1%, acetonitrile-glacial acetic acid solution with a volume fraction of 0.5%, acetonitrile-glacial acetic acid solution with a volume fraction of 0.1%, acetonitrile-formic acid solution with a volume fraction of 0.5%, and methanol-phosphoric acid solution with a volume fraction of 0.1% on chromatographic peaks. The results show that when acetonitrile-water is used as the mobile phase system, the peak shapes of most chromatographic peaks are poor and tailing occurs to varying degrees; when glacial acetic acid and formic acid with different concentrations are used as the mobile phase, the baselines all have large drifts, which affects the quality of the spectrum; when methanol-phosphoric acid is used as the mobile phase system, the separation between the chromatographic peaks is not ideal; when the mobile phase system is acetonitrile-phosphoric acid solution with a volume fraction of 0.1%, the overall baseline is relatively stable, the chromatographic peak separation is good, the number of chromatographic peaks is large, the impurity peaks are small, and the peak appearance time is relatively fast.

[0022] The present invention investigates the influence of different volume fractions of phosphoric acid solution on the chromatographic peak, using acetonitrile-0.05% phosphoric acid, acetonitrile-0.1% phosphoric acid, acetonitrile-0.2% phosphoric acid, elution gradient and column temperature and other chromatographic conditions remain the same, the results show that the retention time of berberine hydrochloride peak is greatly affected by the phosphoric acid concentration, the higher the phosphoric acid concentration, the longer the retention time, and the separation of the other chromatographic characteristic peaks has no obvious difference. When the volume fraction of phosphoric acid is 0.1%, berberine hydrochloride is better separated from adjacent characteristic peaks, and there is no interference from impurities, so a phosphoric acid solution with a volume fraction of 0.1% is preferred.

[0023] The present invention investigates the effects of column temperatures of 25°C, 30°C, and 35°C on chromatogram peaks. The results show that when the column temperature is 30°C, each chromatographic peak can achieve better separation. Therefore, a column temperature of 30°C is preferably used in the chromatographic detection conditions of the present invention.

[0024] The present invention investigates the influence of mobile phase flow rates of 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min on the chromatogram peaks. The results show that when the flow rate is 1 ml / min, each chromatographic peak can achieve good separation. Therefore, the chromatographic detection condition of the present invention preferably adopts a flow rate of 1 ml / min.

[0025] The HPLC reference fingerprint of the compound pearl acne tablets constructed by the present invention presents chromatographic peaks with the same retention time as the chromatographic peaks of gallic acid, chlorogenic acid, paeoniflorin, baicalin and berberine hydrochloride reference substances, and 28 common peaks appear. By comparing with the reference substances, 5 characteristic peaks were finally identified based on the complete consistency of retention time and ultraviolet characteristic spectrum, namely, peak 1: gallic acid; peak 4: chlorogenic acid; peak 6: paeoniflorin; peak 16: baicalin; peak 17: berberine hydrochloride.

[0026] Substitute the recorded HPLC fingerprint of the compound pearl acne tablet sample to be tested into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" for similarity calculation and compare it with the control fingerprint. The similarity shall not be less than 0.90.

[0027] Compound Pearl Acne Tablets are film-coated tablets that are administered orally. Water is used to extract and concentrate during the extraction process, so the main active ingredients in the product are all water-soluble substances with high polarity. Combined with the properties of its own active ingredients, this study mainly uses high-performance liquid chromatography technology to investigate the fingerprint characteristics of the highly polar components in Compound Pearl Acne Tablets to better reflect the relevance of its pharmacological and pharmacodynamic basis. According to the prescription medicinal materials data collected in the previous work, the honeysuckle in the prescription contains the characteristic component of chlorogenic acid, red peony root and wine rhubarb contain the characteristic component of gallic acid, red peony root contains the characteristic component of paeoniflorin, scutellaria contains the characteristic component of baicalin, and phellodendron contains berberine and other characteristic components. According to the pharmacodynamic components and their main chemical properties of each medicinal flavor, the corresponding reference solution is prepared for positioning and comparison, so that the constructed standard fingerprint can fully reflect the quality information of Compound Pearl Acne Tablets, avoiding the shortcomings of the existing single and one-sided quality control standards, and can be used for the comprehensive quality evaluation and control of Compound Pearl Acne Tablets.

[0028] The present invention establishes the fingerprint of the compound pearl acne tablets by high performance liquid chromatography, and verifies the methodologically. The method has good specificity, and the characteristic peaks are not interfered by impurity peaks; the precision, repeatability and stability all meet the requirements, indicating that the method is stable and reliable, and has good adaptability. The fingerprints of 15 batches of compound pearl acne tablets were measured, and the fingerprints of 15 batches of compound pearl acne tablets were substituted into the "Chinese Medicine Fingerprint Similarity Evaluation System" 2012 version, and the mark peaks were matched to fit the reference fingerprints, and a CDF format file was generated, which can provide a reference fingerprint for the determination of other compound pearl acne tablets. After calculation, the similarity between the fingerprints of 15 batches of compound pearl acne tablets and the fitted reference maps is above 0.99, indicating that the fingerprints of 15 batches of compound pearl acne tablets have a high degree of quality consistency, and the production process is stable and controllable.

[0029] The present invention uses a mixed reference solution to identify the common peaks of gallic acid, chlorogenic acid, paeoniflorin, baicalin, and berberine hydrochloride. The constructed standard fingerprint can fully reflect the quality information of the compound pearl acne tablets, has the advantages of being fast, stable, high precision, and strong reproducibility, and avoids the shortcomings of the existing quality control standards being single and one-sided, and can be used for the comprehensive quality evaluation and control of the compound pearl acne tablets. The newly established fingerprint was used to conduct a comprehensive quality comparison of 15 batches of compound pearl acne tablets samples to prove the quality consistency of the product, and the conclusion was relatively objective and accurate. The "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" recommended by the National Pharmacopoeia Committee was used to identify the fingerprint, which is simple and fast to operate. The similarity results obtained in this way were used to evaluate the fingerprint of the compound pearl acne tablets, and the conclusion was relatively objective and accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1Fingerprints at different detection wavelengths.

[0031] Figure 2 The fingerprints of different chromatographic columns.

[0032] Figure 3 Fingerprints of different types of mobile phases.

[0033] Figure 4 The fingerprints of mobile phases with different phosphoric acid concentrations.

[0034] Figure 5 The fingerprints of different chromatographic column temperatures.

[0035] Figure 6 Fingerprints of different flow rates.

[0036] Figure 7 The fingerprints of different extraction solvents.

[0037] Figure 8 Fingerprints of different extraction methods.

[0038] Fig. 9 Fingerprints of different extraction times.

[0039] Fig.10 This is the chromatogram of the precision test.

[0040] Fig.11 This is the chromatogram of the repeatability test.

[0041] Fig.12 Fingerprints for different operators.

[0042] Fig.13 Fingerprints of different liquid chromatographs.

[0043] Fig.14 This is the chromatogram of the stability test.

[0044] Fig.15 The fingerprints of the compound pearl acne tablets and the reference solution are shown in Figure 1, where the unit of the horizontal axis is min.

[0045] Fig.16 These are the fingerprint maps of 15 batches of Compound Pearl Acne Tablets.

[0046] Fig.17 This is the control fingerprint of Compound Pearl Acne Tablets (sample concentration: each milliliter is equivalent to 0.1 grams of Compound Pearl Acne Tablets), among which, Peak 1: gallic acid, Peak 4: chlorogenic acid, Peak 6: paeoniflorin, Peak 16 (S): baicalin, Peak 17: berberine hydrochloride.

[0047] Fig.18 This is a mixed control map. DETAILED DESCRIPTION

[0048] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0049] Experimental Example 1 Establishment and Methodological Verification of the HPLC Fingerprint Construction Method of Compound Pearl Acne Tablets

[0050] 1 Establishment of fingerprint chromatography method

[0051] 1.1 Selection of detection wavelength

[0052] Method: Mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid (V / V) solution, linear gradient elution is performed according to the established procedure; flow rate is 1ml / min, column temperature is 30℃, PDA monitoring, wavelength scanning range is 190nm-400nm. Figure 1 As shown. Figure 1 It can be seen that the characteristic chromatographic peaks of medicinal materials such as red peony root and phellodendron chinense in the prescription of Compound Pearl Acne Tablets have obvious ultraviolet absorption only near 230nm. The fingerprint information of this wavelength band is obviously richer than that of other wavelength bands, and the baseline of this wavelength band is relatively stable with less impurity interference. Therefore, the present invention determines 230nm as the detection wavelength of the fingerprint spectrum of Compound Pearl Acne Tablets.

[0053] 1.2 Selection of chromatographic columns

[0054] The formula and process of the compound pearl acne tablets include two components: water-extracted extract powder and crushed medicinal materials. Therefore, its components include water-soluble polar components and water-soluble and fat-soluble components in the original medicinal powder, with large polarity differences. Therefore, in the present invention, it is necessary to first use a low proportion of organic phase and a high proportion of water phase to elute the water-soluble polar characteristic components, and then gradually increase the proportion of organic phase to elute the small polar characteristic components. In the mobile phase composition of the present invention, when the organic phase is adjusted to an initial ratio of 3%, the highly polar components can be effectively separated, which is different from the conventional C18 reverse phase chromatography elution (Appendix 0512 High Performance Liquid Chromatography of Part IV of the Pharmacopoeia 2020 Edition mentions: The organic solvent in the mobile phase should generally not be less than 5%, otherwise it will easily lead to a decrease in column efficiency and instability of the chromatographic system). Therefore, in terms of chromatographic column selection, it is necessary to select a chromatographic column that has been specially treated and can tolerate a pure water mobile phase. Therefore, this study examined three chromatographic columns that met the requirements under the same gradient, namely Waters Xselect HSS T3 (4.6×250mm, 5μm), Welch Ultimate LP C18 (4.6×250mm, 5μm), and Welch Ultimate AQ C18 (4.6×250mm, 5μm). The results are as follows Figure 2 As shown. Figure 2 It can be found that when using Welch Ultimate AQ C18 (4.6×250mm, 5μm) column, the overall separation effect of chromatographic peaks is the best, so Welch Ultimate AQ C18 is selected as the liquid chromatography column for the fingerprint spectrum of Compound Pearl Acne Tablets.

[0055] 1.3 Selection of mobile phase

[0056] The effects of acetonitrile-water, acetonitrile-0.1% phosphoric acid solution, acetonitrile-0.5% glacial acetic acid solution, acetonitrile-0.1% glacial acetic acid solution, acetonitrile-0.5% formic acid solution, and methanol-0.1% phosphoric acid solution as mobile phase systems on the chromatographic peaks were investigated. The results are as follows: Figure 3 As shown. The results show that when acetonitrile-water is used as the mobile phase system, the peak shape of most chromatographic peaks is poor, and tailing occurs to varying degrees; when different concentrations of glacial acetic acid and formic acid are used as mobile phases, the baseline drifts greatly, affecting the quality of the spectrum; when methanol-phosphoric acid is used as the mobile phase system, the separation between the chromatographic peaks is not ideal; when the mobile phase system is acetonitrile-phosphoric acid solution with a volume fraction of 0.1%, the overall baseline is relatively stable, the chromatographic peak separation is better, the number of chromatographic peaks is large, the impurity peaks are small, and the peak elution time is fast, so acetonitrile-phosphoric acid solution is selected as the final mobile phase.

[0057] 1.4 Selection of phosphoric acid concentration

[0058] The effect of different volume fractions of phosphoric acid solution on the chromatographic peak was investigated. Acetonitrile-0.05% phosphoric acid, acetonitrile-0.1% phosphoric acid, and acetonitrile-0.2% phosphoric acid were used. Other chromatographic conditions such as elution gradient and column temperature were kept the same. Figure 4 shown.

[0059] The results showed that the retention time of berberine hydrochloride peak was greatly affected by phosphoric acid concentration. The higher the phosphoric acid concentration, the longer the retention time. There was no significant difference in the separation of other chromatographic characteristic peaks. When the volume fraction of phosphoric acid was 0.1%, berberine hydrochloride was better separated from adjacent characteristic peaks, and there was no interference from impurities, so a phosphoric acid solution with a volume fraction of 0.1% was preferred.

[0060] 1.5 Selection of column temperature

[0061] The effects of column temperatures of 25°C, 30°C, and 35°C on the chromatogram peaks were investigated. Figure 5 ,according to Figure 5 The results show that when the column temperature is 30℃, each chromatographic peak can achieve good separation, so the column temperature is 30℃.

[0062] 1.6 Selection of different flow rates

[0063] The influence of mobile phase flow rate on chromatogram peak was investigated at 0.9ml / min, 1.0ml / min and 1.1ml / min respectively. Figure 6 .according to Figure 6 It can be seen that when the flow rate is 1 ml / min, each chromatographic peak can achieve good separation, so the flow rate of 1 ml / min is adopted.

[0064] 1.7 Determination of chromatographic conditions

[0065] The chromatographic column was Welch Ultimate AQ C18 (4.6×250 mm, 5 μm), A was acetonitrile, B was 0.1% phosphoric acid solution (V / V), the flow rate was 1 ml / min, the detection wavelength was 230 nm, and the column temperature was 30° C. The gradient elution was as shown in Table 1 below.

[0066] Table 1 Fingerprint elution gradient

[0067]

[0068] 2 Establishment of the test solution preparation method

[0069] 2.1 Selection of extraction solvent

[0070] Take the compound pearl acne tablets, remove the coating, grind them into powder, accurately weigh 3 portions of powder, 0.5g each, put them into a 50ml volumetric bottle, add 30% methanol, 50% methanol, and methanol to the mark, respectively, extract by ultrasonic for 30 minutes, cool, add the corresponding solvent to the mark, shake well, filter, take the filtrate, and measure. The results are as follows Figure 7 shown.

[0071] Since the material composition of the Compound Pearl Acne Tablets covers components of both large and small polarity, it is necessary to select a solvent that takes into account the extraction of both water-soluble and alcohol-soluble components. From the results, it can be found that the samples extracted with methanol have a solvent effect, the peak shape is poor, and the peak area of ​​large polar components such as gallic acid is significantly smaller than the peak area of ​​samples extracted with 30% methanol and 50% methanol, and the peak area of ​​the characteristic peak of baicalin is also the smallest. The characteristic peaks extracted with 30% methanol and 50% methanol are basically corresponding, but the peak area of ​​the characteristic component peak of Scutellaria baicalensis extracted with 50% methanol is significantly larger than that of the sample extracted with 30% methanol, indicating that the 50% methanol extraction is more complete, so 50% methanol is preferred as the extraction solvent.

[0072] 2.2 Selection of extraction method

[0073] Take the compound pearl acne tablets, remove the coating, grind them into powder, accurately weigh 2 portions of powder, 0.5g each, divide them into two groups, A and B, accurately add 50mL of 50% methanol to each, weigh the weight, ultrasonicate group A for 30 minutes, and reflux group B in a water bath for 30 minutes, cool to room temperature, weigh, make up the lost weight with 50% methanol, shake well, filter, take the filtrate, and measure. The results are as follows Figure 8 The results showed that there was no significant difference between the spectra of ultrasonic and reflux extraction. Considering that ultrasonic extraction is simpler and more convenient, ultrasonic extraction was selected.

[0074] 2.3 Selection of extraction time

[0075] Take the compound pearl acne tablets, remove the coating, grind them into powder, accurately weigh 3 portions of powder, 0.5g each, put them into a 50ml volumetric bottle, add 50% methanol to the mark, ultrasonically extract for 15min, 30min, and 45min respectively, let cool, add 50% methanol to the mark, shake well, filter, take the filtrate, and measure. The results are as follows Fig. 9 The results show that the peak area of ​​the sample with 15 minutes of ultrasound is smaller, and there is no significant difference between the extraction effect of 30 minutes and 45 minutes. Therefore, the preferred extraction time is 30 minutes.

[0076] 2.4 Determination of the preparation method of the test solution

[0077] Take the compound pearl acne tablets, remove the coating, grind them into powder, accurately weigh 0.5g, put them in a 50ml volumetric flask, add 50% methanol to the scale, plug it tightly, extract with ultrasound for 30 minutes, cool it, add 50% methanol to the scale, shake it well, filter it, and take the filtrate to obtain the product.

[0078] 3 Methodological validation

[0079] 3.1 Precision test

[0080] Take the test solution (batch number: 20014, provided by Sinopharm Dezhong (Foshan) Pharmaceutical Co., Ltd.) and measure it 6 times continuously according to the proposed test conditions. Record the fingerprint spectrum and compare it with the control fingerprint spectrum generated by it to calculate the similarity. The similarity results are all 1, the RSD of the common peak retention time is less than 1%, and the RSD of the relative peak area of ​​the common peak is less than 5% ( Fig.10 ), indicating that the precision of the method is good.

[0081] Table 2 Precision test results - characteristic peak relative peak area ratio

[0082]

[0083] Table 3 Precision test results - characteristic peak relative retention time ratio

[0084]

[0085]

[0086] 3.2 Repeatability test

[0087] Experimenter A took the same batch of test samples (batch number: 20014, provided by Sinopharm Dezhong (Foshan) Pharmaceutical Co., Ltd.), prepared 6 test sample solutions according to the "Preparation of Test Sample Solutions", accurately aspirated 10 μL of the test sample solution, injected it into the liquid chromatograph, measured it according to the "Chromatographic Conditions", recorded the fingerprint spectrum, compared it with the generated control fingerprint spectrum, calculated the similarity, and the similarity results were all 1, the common peak retention time RSD was less than 1%, and the common peak relative peak area RSD was less than 5% ( Fig.11 ), indicating that the method has good repeatability.

[0088] Table 4 Repeatability test results - characteristic peak relative peak area ratio

[0089]

[0090]

[0091] Table 5 Repeatability test results - characteristic peak relative retention time ratio

[0092]

[0093]

[0094] 3.3 Intermediate precision test

[0095] 3.3.1 Comparison of different operators

[0096] According to the requirements of the "repeatability test", the experimenter B prepared 6 samples of the test sample (batch number: 20014, provided by Sinopharm Dezhong (Foshan) Pharmaceutical Co., Ltd.) and measured them under the established chromatographic conditions. The fingerprint spectrum was recorded and substituted into the similarity software. Each fingerprint spectrum should have 28 characteristic peaks, and the similarity with the control fingerprint spectrum was calculated respectively. The results showed that under the operation of two different personnel, the similarity between each fingerprint spectrum and the control fingerprint spectrum was 1, the RSD of the common peak retention time was less than 1%, and the RSD of the relative peak area of ​​the common peak was less than 5% ( Fig.12 ), indicating that the intermediate precision of this method is good.

[0097]

[0098]

[0099] 3.3.2 Comparison of different brands and models of liquid chromatographs

[0100] The same test solution was taken and tested on Waters e2695 liquid chromatograph, Thermo Ultimate 3000 liquid chromatograph and Waters AQUITY Arc liquid chromatograph according to the specified chromatographic conditions. The results are shown in Fig.13 .

[0101] The results showed that the fingerprints obtained by Waters e2695 liquid chromatograph, Thermo Ultimate 3000 liquid chromatograph and Waters AQUITY Arc liquid chromatograph were analyzed by the similarity evaluation system, and the similarities were 1, 0.996 and 1, respectively, indicating that there was no significant difference in the results of liquid chromatographs of different brands or models, and the intermediate precision of the method was good.

[0102] 3.4 Stability test

[0103] Take a portion of the test solution of the intermediate precision item and measure it at 0, 2, 4, 6, 8, 10, 22, 38, and 51 hours. Record the fingerprint spectrum and substitute it into the similarity software. Each fingerprint spectrum should have 28 characteristic peaks. The results show that in the results of the measurement results at different time periods, the similarity of each fingerprint spectrum with the fingerprint spectrum at 0 hours is 1, the RSD of the common peak retention time is less than 1%, and the RSD of the relative peak area of ​​the common peak is less than 5%, indicating that the sample measurement results are stable within 51 hours ( Fig.14).

[0104]

[0105]

[0106] 4. Establishment of reference fingerprint

[0107] 4.1 Sources of medicinal materials for major chromatographic peaks

[0108] Referring to the process of Compound Pearl Acne Tablets and "Preparation of Test Solution", the test solutions of the single original medicinal materials in the prescription were prepared respectively, 10μl was accurately drawn respectively, injected into the liquid chromatograph, and the injection test was performed according to the "Chromatographic Conditions". The results showed that the 28 common peaks in the standard fingerprint of Compound Pearl Acne Tablets were basically clearly identified in the fingerprint of the prescription medicinal materials, among which: Peak 1 was assigned to red peony root and wine rhubarb medicinal materials; Peaks 2, 4, 5, 7, 13, 14, and 15 were assigned to honeysuckle medicinal materials; Peak 3 was assigned to dandelion medicinal materials; Peaks 6 and 10 were assigned to red peony root medicinal materials; Peaks 8, 9, and 17 were assigned to phellodendron medicinal materials; Peaks 11, 12, 16, 18, 19, 20, 21, 22, 23, 25, 26, 27, and 28 were assigned to scutellaria medicinal materials; Peak 24 was assigned to wine rhubarb medicinal materials.

[0109] 4.2 Identification of characteristic peaks

[0110] The high performance liquid chromatography method was used to perform full wavelength scanning, and the characteristic peak compounds were confirmed by comparing the retention time and UV absorption spectrum with those of the reference substance. The results showed that peak 1 was gallic acid, peak 4 was chlorogenic acid, peak 6 was paeoniflorin, peak 16 was baicalin, and peak 17 was berberine hydrochloride ( Fig.15 ).

[0111] 4.3 Fitting of reference spectrum

[0112] The proposed method was used to measure the single-needle fingerprint of 15 batches of samples of Compound Pearl Acne Tablets, record the fingerprint, substitute it into the similarity software, and generate a reference map ( Figure 16-Figure 17 ), and calculated the similarity between the fingerprints of 15 batches of test products and the control. The results are shown in Fig.18 and Table 10.

[0113] Table 10 Similarity of fingerprint spectra of 15 batches of Compound Pearl Acne Tablets

[0114]

[0115]

[0116] The test results in Table 10 above show that the similarities of the fingerprint spectra of 15 batches of Compound Pearl Acne Tablets are all above 0.99.

Claims

1. A method for constructing a HPLC fingerprint of compound pearl acne tablets, characterized in that: Including the construction of the HPLC control fingerprint of the compound pearl acne tablets and the construction of the HPLC fingerprint of the compound pearl acne tablets samples to be tested; The method for constructing the HPLC reference fingerprint of the compound pearl acne tablets comprises: Inject 15 batches of samples of Compound Pearl Acne Tablets into a liquid chromatograph for single-needle fingerprint determination, record the fingerprint, substitute it into the similarity software, and fit the control fingerprint of Compound Pearl Acne Tablets; The method for constructing the HPLC fingerprint of the compound pearl acne tablet sample to be tested comprises: (1) Preparation of test solution: remove the coating of the compound pearl acne tablets to be tested, grind them into powder, use 10-100% methanol as the extraction solvent, extract by ultrasonic or reflux to obtain an extract, filter, and take the filtrate to obtain the test solution; (2) Pipette the test solution and inject it into the liquid chromatograph for determination, and record the chromatographic peak within 110 minutes to obtain the fingerprint of the Compound Pearl Acne Tablet sample to be tested; The chromatographic conditions for determining the control fingerprint of the compound pearl acne tablet or the fingerprint of the compound pearl acne tablet sample to be tested by liquid chromatography include: using octadecylsilane bonded silica gel as a filler, and using Welch Ultimate AQ-C18, Waters Xselect HSS T3 or Welch Ultimate LP C18 as a chromatographic column; using acetonitrile as mobile phase A, and using a 0.05-0.2% by volume phosphoric acid solution as mobile phase B, and performing gradient elution according to the following procedure: The detection wavelength is 230 nm.

2. The construction method according to claim 1, characterized in that: The method for constructing the HPLC reference fingerprint of the compound pearl acne tablets also includes: preparing a reference solution, aspirating the reference solution and injecting it into a liquid chromatograph for determination, and recording the chromatographic peaks within 110 minutes; wherein, the method for preparing the reference solution includes: taking gallic acid reference substance, chlorogenic acid reference substance, paeoniflorin reference substance, baicalin reference substance, and berberine hydrochloride reference substance respectively, adding 10-100% methanol to prepare a mixed solution, and obtaining the result.

3. The construction method according to claim 2, characterized in that: The preparation method of the reference solution comprises: taking gallic acid reference substance, chlorogenic acid reference substance, paeoniflorin reference substance, baicalin reference substance, and berberine hydrochloride reference substance respectively, and adding 50% methanol to prepare a mixed solution containing 30 μg of each reference substance per 1 ml.

4. The construction method according to claim 1, characterized in that: The preparation method of the test solution comprises: taking the compound pearl acne tablets, removing the coating, grinding, using 50% methanol as an extraction solvent, extracting for 10-45 minutes by ultrasonic extraction, cooling the extract, filtering, taking the filtrate, and obtaining the test solution.

5. The construction method according to claim 4, characterized in that: Ultrasonic extraction was used for 30 minutes.

6. The construction method according to claim 1, characterized in that: 0.1% phosphoric acid solution was used as mobile phase B.

7. The construction method according to claim 1, characterized in that: The column temperature in the chromatographic detection condition was 20-35°C.

8. The construction method according to claim 7, characterized in that: The column temperature in the chromatographic detection condition was 30°C.

9. The construction method according to claim 1, characterized in that: The flow rate in the chromatographic detection conditions was 0.5-1.5 ml / min.

10. The construction method according to claim 9, characterized in that: The flow rate in the chromatographic detection condition was 1 ml / min.

11. The construction method according to claim 1, characterized in that: The constructed HPLC reference fingerprint of the compound pearl acne tablets showed chromatographic peaks with consistent retention times with those of the chromatographic peaks of gallic acid, chlorogenic acid, paeoniflorin, baicalin, and berberine hydrochloride reference substances, and 28 common peaks appeared.

12. The construction method according to claim 1, characterized in that: The recorded HPLC fingerprint of the compound pearl acne tablet sample to be tested was substituted into the Chinese medicine chromatographic fingerprint similarity evaluation system for similarity calculation, and compared with the control fingerprint, the similarity was not less than 0.90.

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