A method for determining chemical components in a drug for treating vitiligo and a method for establishing a fingerprint spectrum

Abstract

The application discloses a method for determining chemical components in a medicine for treating vitiligo and a method for establishing a fingerprint spectrum of the medicine. The method comprises the following steps: S1, preparing a to-be-detected solution and a reference solution: finely grinding Baoling tablets, dissolving the Baoling tablets in a solvent, extracting, and obtaining the to-be-detected solution; dissolving paeoniflorin reference substances in a solvent to prepare the reference solution; wherein the extraction solvent is a methanol-water mixed solution; S2, under the same detection conditions, obtaining chromatograms of the to-be-detected solution and the reference solution by using an ultra-high performance liquid chromatography method, and performing qualitative analysis and / or quantitative analysis on the chemical components in the chromatogram of the to-be-detected solution; wherein the detection conditions comprise that the detector wavelength is 201±2 nm; the elution conditions comprise that acetonitrile is used as phase A, water is used as phase B, and elution is performed through a gradient elution program. The chemical components in the medicine for treating vitiligo can be accurately determined through the method, so that the fingerprint spectrum library of the medicine for treating vitiligo can be better established.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, specifically to a method for determining the chemical components in a drug for treating vitiligo and a method for establishing a fingerprint spectrum. Background Technology

[0002] Vitiligo is a chronic skin disease caused by the destruction of melanocytes. Existing treatments (such as the traditional Chinese medicine compound preparation Bailing tablets) typically rely on the synergistic effects of multiple components to regulate immunity and oxidative stress. However, due to differences in planting location, harvesting season, and processing techniques, the content of active ingredients (such as psoralen and astragaloside A) in Chinese medicinal herbs fluctuates significantly. This fluctuation can directly lead to inconsistencies in efficacy between batches. For example, the lack of quantitative control over key active ingredients can result in significant differences in the activation rate of tyrosinase activity between different batches of the drug. Bailing tablets are made from 11 Chinese medicinal herbs, including Angelica sinensis, Panax notoginseng, Carthamus tinctorius, Paeonia suffruticosa, Prunus persica, Saposhnikovia divaricata, Atractylodes lancea, Angelica dahurica, Portulaca oleracea, Paeonia lactiflora, and Scutellaria baicalensis. They have the effects of promoting blood circulation, removing blood stasis, and increasing photosensitivity, and are clinically used to treat vitiligo. The quality standard for this preparation is included in Volume 5 of the Ministry-issued Standards (standard number WS-B-0914-91). The standard includes items such as appearance, thin-layer chromatography identification of Panax notoginseng and Angelica sinensis. These identification methods can only perform qualitative analysis on specific components, and the accuracy of quantitative analysis is poor. Li Jianming et al. reported on the determination of paeoniflorin content and uncertainty evaluation in Bailing tablets by high performance liquid chromatography in "HPLC Determination of Paeoniflorin Content and Uncertainty Evaluation in Bailing Tablets" (China Pharmacy, 2015, 26(36):5152-5154). The method uses 230nm as the detection wavelength and paeoniflorin in Bailing tablets is eluted under isocratic conditions. Although this method can achieve relatively accurate quantification of paeoniflorin, at this detection wavelength, other effective components besides paeoniflorin have few peaks, making it difficult to detect multiple other components at the same time. Bailing tablets are made from multiple medicinal materials, and the chemical composition is relatively complex. Qualitative or quantitative analysis of a single component cannot fully reflect its overall quality characteristics.

[0003] Although fingerprinting technology has been used for quality evaluation of some traditional Chinese medicines, there is a significant technological gap in its application to Bailing tablets. Therefore, establishing a fingerprinting spectrum for Bailing tablets will provide a scientific basis for the quality evaluation of drugs used to treat vitiligo, as well as for their production and quality control. Summary of the Invention

[0004] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes a method for determining the chemical components in a drug for treating vitiligo. This method can simultaneously and accurately determine multiple chemical components in a vitiligo-treating tablet, thereby better achieving the quality evaluation of the drug for treating vitiligo.

[0005] This invention also proposes a method for establishing a drug fingerprint spectrum for treating vitiligo using the above-mentioned measurement method.

[0006] This invention also proposes the application of the above-described method for establishing drug fingerprint spectra for the treatment of vitiligo.

[0007] According to a first aspect of the present invention, a method for determining the chemical components in a drug for treating vitiligo, the drug for treating vitiligo including Bailing tablets, the determination method comprising the following steps:

[0008] S1. Preparation of test solution and reference solution: Grind Bai Ling tablets into a fine powder, dissolve in a solvent, extract, and prepare test solution; dissolve paeoniflorin reference standard in a solvent to prepare reference solution; wherein, the solvent is a methanol-water mixture;

[0009] S2. Using ultra-high performance liquid chromatography (UPLC) under the same detection conditions, chromatograms of the test solution and the reference solution are obtained separately. Qualitative and / or quantitative analysis of the chemical components in the chromatogram of the test solution is performed. The detection conditions include:

[0010] The detector wavelength is 201±2nm;

[0011] The chromatographic column is a C18 silica gel column;

[0012] The elution conditions include: using acetonitrile as phase A and water as phase B, elution is carried out through a gradient elution program, with a total gradient duration of not less than 24 min. During the first 21 min, the volume percentage of phase A increases non-linearly from 0% to 60%, and after 21 min, the volume percentage of phase A decreases from 60% to 0%.

[0013] The method for determining the chemical components in a drug for treating vitiligo according to embodiments of the present invention has at least the following beneficial effects: The present invention employs ultra-high performance liquid chromatography (UPLC) to conduct fingerprint studies on the drug for treating vitiligo. Analysis of multiple batches of Bailing tablets revealed at least 11 common peaks, and the method can accurately identify chromatographic peaks of multiple components such as paeoniflorin, amygdalin, cimicifugain, ginsenoside Rg1, and 5-O-methylvisamiloside. Simultaneously, it allows for quantitative analysis of paeoniflorin, cimicifugain, and 5-O-methylvisamiloside. This method can provide a reference for improving the quality standards of drugs for treating vitiligo, more comprehensively evaluate and control the quality of these drugs, and improve quality testing standards. When the detection wavelength is around 201 nm, the number of chromatographic peaks obtained during detection increases significantly. Using the detection method of this invention, only paeoniflorin needs to be used as a reference to accurately identify multiple components such as paeoniflorin, amygdalin, cimicifugain, ginsenoside Rg1, and 5-O-methylvisamidol. Not only are the results accurate and reliable with good identification effect, but the detection cost can also be greatly reduced, while achieving qualitative and accurate quantitative detection of multiple components.

[0014] When this assay was used, paeoniflorin showed good linearity (correlation coefficient ≥ 0.999) within the range of 4.994 μg / ml to 499.4 μg / ml. The RSD of the paeoniflorin peak area was 0.5% in the precision test, 0.5% in the stability test, and 0.4% in the repeatability test. The average recovery rate was 96% with an RSD of 1.2%. Using paeoniflorin as a quality control indicator for Bailing tablets and establishing its fingerprint spectrum is simple to operate and provides accurate and reliable identification results. This allows for better overall quality control of the product, providing effective assurance for drug production and clinical application.

[0015] According to some embodiments of the present invention, the gradient elution program is as follows: 0–3 min, the volume fraction of phase A increases from 0% to 5%; 3–6 min, the volume fraction of phase A increases from 5% to 10%; 6–9 min, the volume fraction of phase A increases from 10% to 15%; 9–12 min, the volume fraction of phase A increases from 15% to 20%; 12–15 min, the volume fraction of phase A is maintained at 20%; 15–18 min, the volume fraction of phase A increases from 20% to 40%; 18–21 min, the volume fraction of phase A increases from 40% to 60%; 21–24 min, the volume fraction of phase A decreases from 60% to 0%; 24–30 min, the volume fraction of phase A is maintained at 0%.

[0016] Using this elution gradient not only promotes the effective elution of more chemical components, but also significantly improves the separation effect, which plays a positive role in ensuring the accuracy and reliability of the detection results.

[0017] According to some embodiments of the present invention, the detection conditions further include the following: the mobile phase flow rate is 0.3 ± 0.05 min / mL. Choosing this flow rate level results in lower column pressure.

[0018] According to some embodiments of the present invention, the detection conditions further include the following: column temperature of 20–60°C, such as 20°C, 30°C, or 40°C.

[0019] According to some embodiments of the present invention, the detection conditions further include the following: column temperature of 20–45°C.

[0020] According to some embodiments of the present invention, the detection conditions further include the following condition: column temperature of 40±2℃.

[0021] Precise control of flow rate and column temperature can further improve the stability and accuracy of test results.

[0022] According to some embodiments of the present invention, the detection conditions further include the following condition: the injection volume is 2±1μL.

[0023] According to some embodiments of the present invention, the chromatographic column is Acquity. HSS T3.

[0024] According to some embodiments of the present invention, the chromatographic column has the following specifications: diameter of 2.1 mm; length of 100 mm; and average particle size of packing material of 1.8 μm.

[0025] According to some embodiments of the present invention, the ultra-high performance liquid chromatography (UPLC) method is implemented by an ultra-high performance liquid chromatograph, wherein the ultra-high performance liquid chromatograph is an Agilent LC1290 II high performance liquid chromatograph.

[0026] Other chromatographs may also be used.

[0027] According to some embodiments of the present invention, the volume percentage of methanol in the methanol-water mixture is less than 70%.

[0028] According to some embodiments of the present invention, the volume percentage of methanol in the methanol-water mixed solution is less than 50%. When the volume percentage of methanol is less than 50%, the peak shape and separation effect are better.

[0029] According to some embodiments of the present invention, the volume percentage of methanol in the methanol-water mixed solution is 30±5%.

[0030] When the test solution is prepared using this specific ratio of solvent, the measured chromatogram exhibits a larger characteristic fingerprint peak area. Simultaneously, it allows for the detection of more chemical components, thereby further ensuring the accuracy and reliability of the detection results. The test solution prepared using the solvent of this invention exhibits a chromatogram similarity higher than 0.994. This characteristic significantly enhances the stability and accuracy of the chromatogram, thus contributing to improved precision of the detection results.

[0031] According to some embodiments of the present invention, in step S1, the mass-to-volume ratio of Bailing tablets to the methanol-water mixed solution is 1g:(50±5)mL.

[0032] According to some embodiments of the present invention, in step S1, the extraction method is reflux extraction. The analyte obtained by reflux extraction exhibits a higher response in the liquid chromatography detection system during chromatographic analysis.

[0033] According to some embodiments of the present invention, in step S1, the heating reflux time is 30±10 min.

[0034] According to some embodiments of the present invention, the detector wavelength is 201±2 nm, and the volume percentage of methanol in the methanol-water solution is 30±5%. By using this wavelength and extraction conditions, the interference of detection can be significantly reduced, more peaks can be obtained, the peak separation effect is better, and the baseline is more stable.

[0035] According to some embodiments of the present invention, the qualitative analysis includes determining the chromatographic peak positions of other components based on the relative retention time of paeoniflorin, thereby performing a qualitative analysis of the chemical components in the test solution.

[0036] According to some embodiments of the present invention, the quantitative analysis includes obtaining chromatograms of paeoniflorin at different concentrations, preparing a standard curve based on the relationship between chromatographic peak area and concentration, and performing quantitative analysis of the paeoniflorin concentration in the test solution based on the standard curve.

[0037] According to some embodiments of the present invention, the quantitative analysis further includes obtaining quantitative standard curves of different concentrations of cimicifugain glycoside or 5-O-methylvisamiloside, and performing quantitative analysis of cimicifugain glycoside or 5-O-methylvisamiloside in the test solution according to the standard curves.

[0038] Cimicifugoside or 5-O-methylvisamiloside can be added to the reference solution for simultaneous detection, or cimicifugoside or 5-O-methylvisamiloside reference solutions can be prepared separately to obtain chromatograms at different concentrations. Based on the relationship between the concentrations and peak areas of paeoniflorin, cimicifugoside, and 5-O-methylvisamiloside, simultaneous quantitative analysis of the three components can be achieved.

[0039] According to some embodiments of the present invention, the determination method further includes the following steps: obtaining chromatograms of reference solutions containing different concentrations of paeoniflorin reference standards, plotting a standard curve with the peak area of ​​paeoniflorin as the ordinate (y) and the injection concentration of paeoniflorin as the abscissa (x), and fitting the standard curve with a linear regression equation to obtain the linear regression equation of paeoniflorin as y = 13.688x - 39.134.

[0040] According to some embodiments of the present invention, the determination method further includes the following steps: obtaining chromatograms of sample solutions containing different concentrations of cimicifugain reference standards; plotting a standard curve with the peak area of ​​cimicifugain as the ordinate (y2) and the injection concentration of cimicifugain as the abscissa (x2); fitting the standard curve with a linear regression equation to obtain the linear regression equation of cimicifugain as y2 = 12.14796x2 + 0.28342.

[0041] According to some embodiments of the present invention, the determination method further includes the following steps: obtaining chromatograms of sample solutions containing different concentrations of 5-O-methylvisamilol glycoside reference standards; plotting a standard curve with the peak area of ​​5-O-methylvisamilol glycoside as the ordinate (y3) and the injection concentration of 5-O-methylvisamilol glycoside as the abscissa (x3); fitting the standard curve with a linear regression equation to obtain the linear regression equation of 5-O-methylvisamilol glycoside as y3 = 11.82116x3 - 0.04101.

[0042] The above content regression equation can more accurately determine the specific contents of paeoniflorin, cimicifugoside, and 5-O-methylvisamidol.

[0043] According to some embodiments of the present invention, the concentration of paeoniflorin reference standard in the reference solution is 5, 10, 25, 50, 100, 250, or 500 μg / mL.

[0044] According to some embodiments of the present invention, the volume percentage of methanol in the methanol-water mixture is less than 70%.

[0045] According to some embodiments of the present invention, the volume percentage of methanol in the methanol-water mixed solution is less than 50%. When the volume percentage of methanol is less than 50%, the peak shape and separation effect are better.

[0046] According to some embodiments of the present invention, the volume percentage of methanol in the methanol-water mixed solution is 30±5%.

[0047] When the test solution is prepared using this specific ratio of solvent, the measured chromatogram exhibits a larger characteristic fingerprint peak area. Simultaneously, it allows for the detection of more chemical components, thereby further ensuring the accuracy and reliability of the detection results. The test solution prepared using the solvent of this invention exhibits a chromatogram similarity higher than 0.994. This characteristic significantly enhances the stability and accuracy of the chromatogram, thus contributing to improved precision of the detection results.

[0048] According to some embodiments of the present invention, in step S1, the mass-to-volume ratio of Bailing tablets to the methanol-water mixed solution is 1g:(50±5)mL.

[0049] According to some embodiments of the present invention, in step S1, the extraction method is reflux extraction. The analyte obtained by reflux extraction exhibits a higher response in the liquid chromatography detection system during chromatographic analysis.

[0050] According to some embodiments of the present invention, in step S1, the heating reflux time is 30±10 min.

[0051] According to some embodiments of the present invention, the detector wavelength is 201±2 nm, and the volume percentage of methanol in the methanol-water solution is 30±5%. By using this wavelength and extraction conditions, the interference of detection can be significantly reduced, more peaks can be obtained, the peak separation effect is better, and the baseline is more stable.

[0052] According to some embodiments of the present invention, the raw materials for preparing the drug for treating vitiligo include Angelica sinensis, Astragalus membranaceus, Panax notoginseng, Carthamus tinctorius, Paeonia lactiflora, Paeonia suffruticosa, Portulaca oleracea, Prunus persica, Saposhnikovia divaricata, Angelica dahurica, and Atractylodes lancea. It can be prepared into tablets or other dosage forms, all of which are applicable to the present invention.

[0053] The method for establishing a fingerprint spectrum according to a second aspect of the present invention includes the following steps:

[0054] The chromatographic data of the drug for treating vitiligo were obtained using the above-described determination method, and the fingerprint spectrum of the drug for treating vitiligo was constructed using the chromatographic data.

[0055] The fingerprint spectrum established using the method of this invention exhibits better separation of different components with less interference. When used for quality control of drugs for treating vitiligo, the established fingerprint spectrum shows at least 11 common peaks, with a relative retention time RSD ≤ 0.7% and a relative peak area RSD ≤ 3.5%, indicating that the method of this invention is stable and reliable. The traditional Chinese medicine fingerprint spectrum constructed by this invention can simultaneously detect and identify the characteristic components of 10 medicinal materials in Bailing tablets. Through the overall comparative analysis of multidimensional chromatographic characteristic peaks, it effectively overcomes the limitations of traditional quality control methods that only target single components. This technical solution, by systematically characterizing the complete chemical composition of compound preparations, significantly improves the comprehensiveness and reliability of quality evaluation of drugs for treating vitiligo, providing a more scientific and precise evaluation method for the quality control of traditional Chinese medicine compound preparations.

[0056] According to some embodiments of the present invention, the establishment method includes the following steps: importing the chromatographic data obtained by the determination method into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" for multi-point correction and peak matching to obtain a fingerprint spectrum.

[0057] The similarity evaluation results of the fingerprint spectra show that the similarity of the fingerprint spectra of Bai Ling tablets is above 0.994 (for example, the similarity of 16 batches can reach 0.997, 0.996, 0.994, 0.998, 0.997, 0.999, 0.997, 0.998, 0.999, 0.995, 0.999, 0.999, 0.998, 0.999, 0.995, and 0.996, all above 0.994), indicating that the determination method of the present invention has good precision.

[0058] According to some embodiments of the present invention, the fingerprint spectrum contains the following characteristic fingerprint peaks: paeoniflorin, amygdalin, cimicifugain, ginsenoside Rg1, and 5-O-methylvisamidol.

[0059] Common peaks are determined based on the relative retention times of chromatographic peaks in the UPLC fingerprint spectrum, and characteristic fingerprint peaks are selected from the common peaks.

[0060] The above-mentioned fingerprinting method is applied in drug quality testing and control.

[0061] The fingerprint of vitiligo drugs established by the present invention can be applied to the quality control or quality evaluation of vitiligo drugs. The present invention innovatively constructs a set of quality control and evaluation system for vitiligo drugs, which is centered on adopting UPLC-DAD spectrum analysis technology to successfully construct a high-precision fingerprint of vitiligo drugs. This method is easy to operate, has excellent precision, good repeatability, is stable and reliable, exhibits high sensitivity and long-term stability, and is an effective quality evaluation method for vitiligo drugs, which will provide a scientific basis for the production and quality control of vitiligo drugs. Through a systematic quality characterization system, the fingerprint technology can perform qualitative and quantitative analysis on the active ingredient group in vitiligo drugs, and provide a scientific basis for process control of pharmaceutical production, batch consistency evaluation and improvement of product quality standards. Compared with the traditional single-index quality control method, the solution of the present invention has technical advantages such as simultaneous monitoring of multiple components and digital comparison of characteristic spectra, which conforms to the current modern development trend of quality control of traditional Chinese medicines and natural medicines.

[0062] According to some embodiments of the present invention, the application comprises the following steps: performing similarity calculation on the chromatogram of the Bailing Tablet sample to be tested and the standard fingerprint, evaluating the quality of the sample to be tested according to the similarity calculation result, and if the similarity is greater than 0.9, the sample is qualified; otherwise, the sample is unqualified. It is generally considered that a similarity reaching 0.9 can be deemed as meeting the requirement, and the requirement can also be further improved as needed.

[0063] According to some embodiments of the present invention, the sample is qualified if the similarity is above 0.95.

[0064] According to some embodiments of the present invention, the sample is qualified if the similarity is above 0.99.

[0065] According to some embodiments of the present invention, the sample is qualified if the similarity is above 0.994. In view of the determination method of the solution of the present invention, a good sample separation effect is achieved, so that the similarity of the chromatogram obtained by the determination method of the solution of the present invention can reach above 0.994.

[0066] Additional aspects and advantages of the present invention will be partially set forth in the following description, and partially will become apparent from the following description, or be learned through the practice of the present invention. Description of Drawings

[0067] Figure 1 is the chromatogram of the test solution measured in Example 1 of the present invention;

[0068] Figure 2 is the chromatogram of the reference solution measured in Example 1 of the present invention;

[0069] Figure 3 is the fingerprint of Bailing Tablets constructed in Example 2 of the present invention;

[0070] Figure 4 This is the blank solvent chromatogram obtained in Verification Example 1 of this invention;

[0071] Figure 5 This is a chromatogram of the cimicifugin glycoside reference solution obtained in Verification Example 1 of this invention;

[0072] Figure 6 This is a chromatogram of the 5-O-methylvisamidol glycoside reference solution obtained in Verification Example 1 of this invention;

[0073] Figure 7 This is the chromatogram of the negative sample solution for Saposhnikovia divaricata obtained in Verification Example 1 of this invention;

[0074] Figure 8 This is the chromatogram of the test solution of the single negative sample of Paeonia lactiflora deficiency obtained in Verification Example 1 of this invention;

[0075] Figure 9 This is the chromatogram of the single negative sample solution lacking peony bark obtained in Verification Example 1 of this invention;

[0076] Figure 10 This is the chromatogram of the test solution of the double negative sample sample lacking both Paeonia lactiflora and Paeonia suffruticosa obtained in Verification Example 1 of this invention;

[0077] Figure 11 This is the standard fingerprint spectrum of Bai Ling tablets constructed in Verification Example 2 of this invention;

[0078] Figure 12 This is a comparison chart of the test results of a single medicinal material and a sample of Bai Ling in Verification Example 2 of this invention;

[0079] Figure 13 This is a comparison of the chromatogram effects of the test solutions obtained by different extraction solvents in Example 3 of the present invention;

[0080] Figure 14 This is the chromatogram of the test solution obtained in Example 4 of the present invention;

[0081] Figure 15 This is the chromatogram of the test solution obtained in Example 5 of the present invention;

[0082] Figure 16 This is the chromatogram of the test solution obtained in Example 6 of the present invention;

[0083] Figure 17 This is the chromatogram of the test solution measured at 210 nm in Comparative Example 1 of this invention;

[0084] Figure 18 This is the chromatogram of the test solution measured at 230 nm in Comparative Example 1 of this invention;

[0085] Figure 19 This is the chromatogram of the test solution measured at 270 nm in Comparative Example 1 of this invention;

[0086] Figure 20 This is the chromatogram of the test solution measured at 300 nm in Comparative Example 1 of this invention;

[0087] Figure 21 This is the chromatogram of the test solution obtained in Comparative Example 2 of this invention;

[0088] Figure 22 This is the chromatogram of the test solution obtained in Comparative Example 3 of this invention;

[0089] Figure 23 This is the chromatogram of the test solution obtained in Comparative Example 4 of this invention;

[0090] Figure 24 This is the chromatogram of the test solution obtained in Comparative Example 5 of this invention;

[0091] Figure 25 This is the chromatogram of the test solution obtained in Comparative Example 6 of this invention;

[0092] Figure 26 This is the chromatogram of the test solution obtained in Comparative Example 7 of this invention;

[0093] Figure 27 This is the chromatogram of the test solution obtained in Comparative Example 8 of this invention. Detailed Implementation

[0094] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available. Unless otherwise specified, the same parameter value is the same in all embodiments. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0095] In the description of this invention, references to terms such as "some embodiments" indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0096] The procurement information for the instruments, reagents, and medicinal materials used in the following examples and comparative examples is as follows:

[0097] 1) Instruments: Agilent LC1290 II high-performance liquid chromatograph; Waters HSS T3 (2.1mm×100mm, 1.8μm) chromatographic column, column number: 03133334128508; ML204T electronic analytical balance; KDM type temperature-controlled electric heating mantle.

[0098] 2) Reagents: Acetonitrile (chromatographic grade), methanol (analytical grade), methanol (chromatographic grade), water (ultrapure water), paeoniflorin reference standard (110736-202447, 98.1%), amygdalin reference standard (110820-20241, 94.9%), cimicifugin reference standard (111522-202214, 95.7%), 5-O-methylvisamidolol reference standard (111523-202212, 97.8%), ginsenoside Rg1 reference standard (110703-202436, 97.0%), all of which were obtained from the China National Institutes for Food and Drug Control.

[0099] 3) Medicinal Materials: Bailing Tablets (composed of Angelica sinensis, Astragalus membranaceus, Panax notoginseng, Carthamus tinctorius, Paeonia lactiflora, Paeonia suffruticosa, Portulaca oleracea, Prunus persica, Saposhnikovia divaricata, Angelica dahurica, and Atractylodes lancea), batch numbers A0002, A0009, A0028, A0044, A0051, A2068, B0003, B0020, B0035, B0066, B0083, C0001, C0003, 240018, C0053, C0056, and S0001, manufactured by Sinopharm Group Fenglexing (Foshan) Pharmaceutical Co., Ltd., and sequentially labeled as samples M1 to M16. Samples lacking Saposhnikovia divaricata (single negative), Paeonia lactiflora (single negative), Paeonia suffruticosa (single negative), and both Paeonia lactiflora and Paeonia suffruticosa (double negative) are identical to Bailing Tablets in their preparation process and other raw materials, differing only in the absence of one or two specific drugs. The specific preparation process can be referred to as follows: According to the same prescription amount as Bailing tablets, take 43% of Angelica sinensis and Panax notoginseng and grind them into coarse powder. Add water to the remaining Angelica sinensis, Saposhnikovia divaricata, Atractylodes lancea, and Angelica dahurica and distill for 2 hours at a gentle boil, collecting the aromatic oil. Add water to the residue with Carthamus tinctorius, Prunus persica, Portulaca oleracea, and Astragalus membranaceus and decoct twice. Add 5 times the amount of water for the first decoction and heat for 2 hours; add 4 times the amount of water for the second decoction and heat for 1.5 hours. Combine the decoctions, filter, and concentrate the filtrate to an extract with a relative density of 1.11–1.16 (65℃). Mix this extract with the coarse powder, dry, grind into fine powder, sieve, and granulate. Spray in the aromatic oil and mix well to obtain a double-negative sample simultaneously lacking Paeonia lactiflora and Paeonia suffruticosa. Prepare a double-negative sample control solution lacking Paeonia lactiflora and Paeonia suffruticosa according to the Bailing tablet test solution preparation method.

[0100] According to the same prescription dosage for Bailing tablets, 43% of Angelica sinensis and Panax notoginseng were pulverized into coarse powder. The remaining Angelica sinensis, Saposhnikovia divaricata, Atractylodes lancea, and Angelica dahurica were added to water and distilled at a gentle boil for 2 hours to collect the aromatic oil. The residue was decocted twice with Paeonia lactiflora, Carthamus tinctorius, Prunus persica, Portulaca oleracea, and Astragalus membranaceus. The first decoction was decocted twice with 5 times the amount of water and heated for 2 hours. The second decoction was decocted twice with 4 times the amount of water and heated for 1.5 hours. The decoctions were combined, filtered, and the filtrate was concentrated to an extract with a relative density of 1.11–1.16 (65℃). This extract was mixed with the coarse powder, dried, pulverized into fine powder, sieved, and granulated. The aromatic oil was sprayed into the granules, and after mixing, a single negative sample lacking Paeonia suffruticosa was obtained. A control solution for a single negative sample lacking Paeonia suffruticosa was prepared according to the preparation method of the Bailing tablet test solution.

[0101] According to the same prescription dosage for Bailing tablets, 43% of Angelica sinensis and Panax notoginseng were pulverized into coarse powder. The remaining Angelica sinensis, along with Saposhnikovia divaricata, Atractylodes lancea, Angelica dahurica, and Paeonia suffruticosa, were added to water and distilled at a gentle boil for 2 hours to collect the aromatic oil. The residue was decocted twice with Carthamus tinctorius, Prunus persica, Portulaca oleracea, and Astragalus membranaceus. For the first decoction, 5 times the amount of water was added and the mixture was heated for 2 hours. For the second decoction, 4 times the amount of water was added and the mixture was heated for 1.5 hours. The decoctions were combined, filtered, and the filtrate was concentrated to obtain an extract with a relative density of 1.11–1.16 (65°C). This extract was mixed with the coarse powder, dried, pulverized into fine powder, sieved, and granulated. The aromatic oil was then sprayed into the granules, and the mixture was stirred to obtain a negative sample for Paeonia lactiflora deficiency. A single negative control solution for Paeonia lactiflora deficiency was prepared according to the preparation method of the Bailing tablet test solution.

[0102] According to the same prescription dosage for Bailing tablets, 43% of Angelica sinensis and Panax notoginseng were pulverized into coarse powder. The remaining Angelica sinensis, Atractylodes lancea, Angelica dahurica, and Paeonia suffruticosa were added to water and distilled at a gentle boil for 2 hours to collect the aromatic oil. The residue was decocted twice with Paeonia lactiflora, Carthamus tinctorius, Prunus persica, Portulaca oleracea, and Astragalus membranaceus. The first decoction was decocted twice with 5 times the amount of water and heated for 2 hours. The second decoction was decocted twice with 4 times the amount of water and heated for 1.5 hours. The decoctions were combined, filtered, and the filtrate was concentrated to an extract with a relative density of 1.11–1.16 (65℃). This extract was mixed with the coarse powder, dried, pulverized into fine powder, sieved, and granulated. The aromatic oil was sprayed into the granules, and after mixing, a negative control solution for Paeonia lactiflora deficiency was obtained. A negative control solution for Saposhnikovia divaricata deficiency was prepared according to the same method as for Bailing tablets.

[0103] Angelica sinensis, Astragalus membranaceus, Panax notoginseng, Carthamus tinctorius, Paeonia lactiflora, Paeonia suffruticosa, Portulaca oleracea, Prunus persica, Saposhnikovia divaricata, Angelica dahurica, and Atractylodes lancea were all purchased by Sinopharm Group Feng Lexing (Foshan) Pharmaceutical Co., Ltd., and the preparation process of their test solution was the same as that of Bailing tablets.

[0104] Unless otherwise specified, instruments or reagents are common in this field.

[0105] Example 1

[0106] This example provides a method for determining the chemical components in drugs used to treat vitiligo. The specific procedure is as follows:

[0107] S1. Preparation of the test solution and reference solution: Take 20 tablets of a drug for treating vitiligo (Bai Ling tablets, batch number C0056), grind them into a fine powder, take about 1g, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of 30vol% methanol, accurately weigh it, heat under reflux for 30min, weigh it again, make up the weight loss, shake well, filter, and take the filtrate to obtain the test solution. Take paeoniflorin reference standard, accurately weigh it, and accurately add 100ml of 30vol% methanol to obtain the reference solution (concentration of 50μg / mL). In addition, take cimicifugain glycoside and 5-O-methylvisamidolol glycoside, respectively, and add 30% methanol to prepare reference solutions containing 8μg per ml.

[0108] S2. Using ultra-high performance liquid chromatography (UPLC), chromatograms of the test solution and the reference solution were obtained separately under the same detection conditions. The chromatographic conditions included: HSS T3 (2.1 mm × 100 mm, 1.8 μm) column was used; gradient elution was performed using acetonitrile (A)-water solution (B) as the mobile phase (gradient elution program as follows: 0–3 min, A phase volume fraction increased from 0% to 5%; 3–6 min, A phase volume fraction increased from 5% to 10%; 6–9 min, A phase volume fraction increased from 10% to 15%; 9–12 min, A phase volume fraction increased from 15% to 20%; 12– The volume fraction of phase A remained at 20% for 15 min; from 15 to 18 min, the volume fraction of phase A increased from 20% to 40%; from 18 to 21 min, the volume fraction of phase A increased from 40% to 60%; from 21 to 24 min, the volume fraction of phase A decreased from 60% to 0%; and from 24 to 30 min, the volume fraction of phase A remained at 0%. The flow rate was 0.3 mL / min; the detection wavelength was 201 nm; the column temperature was 40 °C; and the injection volume was 2 μL.

[0109] The chromatogram of the test solution is as follows: Figure 1 As shown, the chromatogram of the reference solution is as follows: Figure 2 As shown.

[0110] Depend on Figure 1 and 2 It was found that the chromatographic peaks of the test sample solution had the same retention time at the corresponding chromatographic positions as the reference solution. Furthermore, UV spectral scanning and peak purity detection of the paeoniflorin peak using a diode array detector revealed that the UV absorption spectrum of the test sample was completely identical to that of the reference standard, and the peak purity factor was greater than 998.

[0111] Example 2

[0112] This example provides a method for establishing a fingerprint spectrum of a drug for treating vitiligo, including the following steps: The chromatographic data obtained using the method described in Example 1 is imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" for multi-point correction and peak matching to obtain the fingerprint spectrum. Specifically, 16 batches of Bailing tablets (M1-M16) are taken and prepared into test solutions according to the operation steps in S1 of Example 1. Then, the test solutions are analyzed by chromatography according to step S2 to obtain the fingerprint spectrum of the test solutions. The fingerprint spectrum is then imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)". The chromatogram of sample M1 is set as a reference, the time window width is set to 0.1 min, and multi-point correction is performed using the mean method to generate the standard fingerprint spectrum R of Bailing tablets (see the constructed fingerprint spectrum). Figure 3 The similarity of chromatographic data from 16 batches of Bailing tablets was evaluated. The results are shown in Table 1 below. It shows that the fingerprint similarity of all 16 batches of Bailing tablets was ≥0.994.

[0113] Table 1

[0114] S1 0.997 S2 0.996 S3 0.994 S4 0.998 S5 0.997 S6 0.999 S7 0.997 S8 0.998 S9 0.999 S10 0.995 S11 0.999 S12 0.999 S13 0.998 S14 0.999 S15 0.995 S16 0.996

[0115] As shown in Table 1, the fingerprint similarity of the 16 batches of Bai Ling tablets were 0.997, 0.996, 0.994, 0.998, 0.997, 0.999, 0.997, 0.998, 0.999, 0.995, 0.999, 0.999, 0.998, 0.999, 0.995, and 0.996, respectively, all above 0.994. This indicates that the extraction and chromatographic conditions of the present invention resulted in good sample separation and detection response, and the detection effect was accurate and reliable.

[0116] Verification Example 1

[0117] This example demonstrates the specificity of the assay method of the present invention. The difference between this example and Example 1 is that the test subject is a blank solvent, other reference standards (citric acid glycoside and 5-O-methylvisamiloside, each prepared with 30% methanol to a reference solution containing 8 μg per ml), negative samples lacking *Saposhnikovia divaricata*, single negative samples lacking *Paeonia lactiflora*, single negative samples lacking *Paeonia suffruticosa*, and double negative samples lacking both *Paeonia lactiflora* and *Paeonia suffruticosa*. The negative samples lacking *Saposhnikovia divaricata*, single negative samples lacking *Paeonia lactiflora*, single negative samples lacking *Paeonia suffruticosa*, and double negative samples lacking both *Paeonia lactiflora* and *Paeonia suffruticosa* are prepared as test solutions using the same processing procedure as the test solution in Example 1. The chromatogram is determined by injection using the method described in this example. The results are as follows: Figure 4-10 As shown.

[0118] As shown in the figure, at the corresponding chromatographic positions of the reference or other standard solutions, the test solution and the single-negative test solution lacking Paeonia lactiflora and Paeonia suffruticosa showed peaks with the same retention times. The chromatograms of the double-negative test solutions lacking Paeonia lactiflora and Paeonia suffruticosa, the negative test solution lacking Saposhnikovia divaricata, and the blank solution did not show the corresponding detection components at the corresponding retention times (the double-negative test solutions lacking Paeonia lactiflora and Paeonia suffruticosa did not show paeoniflorin peaks, the negative test solution lacking Saposhnikovia divaricata did not show cimicifugoside and 5-O-methylvisamidolol peaks, and the blank solution showed no paeoniflorin, cimicifugoside, or 5-O-methylvisamidolol peaks). This indicates that other components and solvents do not interfere with the content determination, and the method has good specificity. Furthermore, UV spectral scanning and peak purity detection of the paeoniflorin, cimicifugoside, and 5-O-methylvisamidololol peaks using a diode array detector revealed that the UV absorption spectra of the test sample were completely consistent with the reference standard, and the peak purity factors were all greater than 998.

[0119] Verification Example 2

[0120] In this example, the attribution and confirmation of common peaks are verified: amygdalin, prim-O-glucosylcimifugin, ginsenoside Rg1, 5-O-methylvisammioside and paeoniflorin are prepared into reference substance solutions according to the operation of reference substance solution in step S1 of Example 1. Meanwhile, Bailing Tablets are prepared into test solution according to the operation in step S1 of Example 1, and injected for determination according to the chromatographic conditions under step S2. At the same time, taking the detection spectra of 16 batches of Bailing Tablets in Example 2 as the research object, relevant software is used for fingerprint similarity analysis, and different strategies such as data extraction, time matching and peak matching are used to determine common peaks. Through multi-point correction of chromatographic peaks, a control fingerprint is established. According to the relative retention time of each chromatographic peak in the chromatogram, 11 common components are identified, as Figure 11 shown. Qualitative identification is carried out by reference substances, single medicinal materials, retention time and ultraviolet absorption spectrum. The comparison diagram of test results of single medicinal materials and Bailing Tablet samples is shown in Figure 12 , where the numbers marked in the diagram represent the 11 confirmed common components. It can be seen from the diagram that Persicae Semen is the only source of amygdalin (peak 4); Saposhnikoviae Radix is the only source of prim-O-glucosylcimifugin (peak 6) and 5-O-methylvisammioside (peak 8); Notoginseng Radix et Rhizoma is the only source of ginsenoside Rg1 (peak 9); while paeoniflorin (peak 5) is a common peak of Paeoniae Radix Rubra and Moutan Cortex. Five peaks are selected as characteristic fingerprint peaks, among which peak 4 is amygdalin, peak 5 is paeoniflorin, peak 6 is prim-O-glucosylcimifugin, peak 8 is 5-O-methylvisammioside, and peak 9 is ginsenoside Rg1. Peak 5 has a good peak shape and a large peak area, so selecting paeoniflorin peak as the reference peak S can make the determination method more accurate and reliable.

[0121] Validation Example 3

[0122] In this example, to verify the precision of the determination method of the present invention, the test solution (sample batch number: C0056) is prepared with reference to step S1 in Example 1, and then injected for 6 consecutive times according to the conditions in step S2. Taking paeoniflorin peak as the reference peak (S), the relative retention time RSD and relative peak area RSD of each common peak are calculated, and the results are shown in Tables 2 and 3 below.

[0123] Table 2 Retention time of paeoniflorin peak and each common peak and RSD% of relative retention time

[0124]

[0125]

[0126] Table 3 Peak area of paeoniflorin peak and each common peak and RSD% of relative peak area

[0127] 1 125.50858 131.35385 131.80500 129.76319 128.37611 127.41407 2.0 2 85.63679 92.28551 91.92953 93.294 93.63894 93.69572 3.5 3 97.43948 96.84170 98.32223 100.05066 100.63379 100.90326 1.9 4 83.04736 82.58114 84.74582 85.58848 84.80892 84.94111 1.5 5(S) 820.43167 818.52373 818.02587 817.59946 817.88291 818.68919 / 6 118.46969 117.92116 117.67389 119.14488 120.07454 120.41157 1.0 7 76.95794 78.45200 79.77031 79.41198 79.00104 78.93213 1.4 8 128.00681 128.68941 130.38307 131.12439 131.79272 131.77742 1.4 9 97.07588 96.35930 96.23892 96.36374 96.50904 96.38585 0.3 10 69.31384 69.33144 69.09758 69.03812 68.64993 68.94742 0.4 11 155.14226 147.93346 148.05464 148.14048 148.31697 147.86004 1.9

[0128] As can be seen from the table above, the sample solutions measured using the method of this invention have a relative retention time RSD of ≤0.7% and a relative peak area RSD of ≤3.5% for each common peak. Using the fingerprint chromatogram obtained from the first injection as a reference, the similarity calculated using the traditional Chinese medicine chromatogram evaluation system is 1.000. This indicates that the method has good precision.

[0129] Verification Example 4

[0130] To demonstrate the repeatability of the determination method of the present invention, six test solutions (sample batch number C0056) were prepared according to step S1 in Example 1, and then analyzed according to the conditions in step S2. The paeoniflorin peak was used as the reference peak (S), and the relative retention time RSD and relative peak area RSD of each common peak were calculated. The results are shown in Tables 4 and 5 below.

[0131] Table 4. Retention times of paeoniflorin peak and common peaks, and relative retention times (RSD%).

[0132]

[0133]

[0134] Table 5. Peak area and relative peak area (RSD%) of paeoniflorin peak and each common peak.

[0135] 1 82.80231 86.78931 81.94400 82.53788 90.07157 87.92577 4.1 2 84.42195 80.51806 79.59428 76.40000 84.09166 82.75332 4.3 3 88.43015 88.93704 89.53357 88.94052 90.33544 88.99356 0.8 4 80.00940 80.12768 86.70523 85.28575 83.22662 84.78287 2.8 5(S) 793.73394 791.54222 803.21059 809.33897 808.18949 798.08323 / 6 111.05941 112.97348 112.41883 114.34646 113.58565 111.41068 0.9 7 75.05571 73.29257 69.43470 70.49353 74.69696 74.29569 3.8 8 129.67519 126.78491 128.81953 129.52916 129.94822 127.55459 0.9 9 98.27980 97.10695 95.33675 96.20635 94.66781 93.51602 2.4 10 68.48473 63.91317 64.81760 66.10617 63.72184 64.22369 3.2 11 157.27934 150.07143 153.63381 157.10741 150.96522 150.80164 2.2

[0136] As can be seen from the table above, the sample solutions measured using the method of this invention have a relative retention time RSD of ≤0.7% and a relative peak area RSD of ≤4.3% for each common peak. Using the fingerprint chromatogram obtained from the first injection as a reference, the similarity calculated using the traditional Chinese medicine chromatogram evaluation system is 1.000. This indicates that the method has good repeatability.

[0137] Verification Example 5

[0138] To demonstrate the stability of the test solution processed by the method of the present invention, the test solution (sample batch number C0056) was prepared according to step S1 in Example 1. The sample was injected and analyzed at 0h, 2h, 4h, 8h, 12h, 18h and 24h after the sample preparation was completed, respectively, according to the conditions of step S2. The paeoniflorin peak was used as the reference peak (S), and the relative retention time RSD and relative peak area RSD of each common peak were calculated. The results are shown in Tables 6 and 7 below.

[0139] Table 6. Retention times of paeoniflorin peak and common peaks, and relative retention times (RSD%).

[0140] 1 4.609 4.571 4.580 4.578 4.566 4.572 4.563 0.4 2 5.152 5.105 5.113 5.112 5.102 5.105 5.096 0.4 3 9.673 9.681 9.684 9.679 9.670 9.675 9.669 0.1 4 10.198 10.206 10.209 10.203 10.191 10.195 10.192 0.1 5(S) 12.390 12.403 12.403 12.397 12.388 12.394 12.392 / 6 12.918 12.934 12.930 12.926 12.918 12.923 12.925 0.1 7 13.888 13.903 13.899 13.895 13.890 13.894 13.895 0.1 8 15.993 16.014 16.004 15.998 16.004 16.000 16.013 0.1 9 19.205 19.207 19.203 19.201 19.197 19.199 19.211 0.1 10 19.403 19.409 19.406 19.404 19.400 19.403 19.413 0.1 11 20.517 20.518 20.515 20.514 20.508 20.512 20.517 0.1

[0141] Table 7 Peak area and relative peak area (RSD%) of paeoniflorin peak and each common peak.

[0142]

[0143]

[0144] As can be seen from the table above, the sample solutions measured using the method of this invention have a relative retention time RSD of ≤0.4% and a relative peak area RSD of ≤3.8% for each common peak. Using the fingerprint chromatogram obtained from the first injection as a reference, the similarity calculated using the traditional Chinese medicine chromatogram evaluation system is 1.000 for all samples. This indicates that the test solution prepared by the method of this invention remains stable within 24 hours.

[0145] Verification Example 6

[0146] This example verifies the linearity of the determination method of the present invention. Appropriate amounts of paeoniflorin, cimicifugain, and 5-O-methylvisamiloside reference standards were taken and added to 30 vol% methanol to prepare reference standard solutions of different mass concentrations. The concentrations of paeoniflorin reference standard were 5, 10, 25, 50, 100, 250, and 500 μg / ml, and the concentrations of cimicifugain and 5-O-methylvisamiloside reference standard solutions were 3.2, 4, 8, 16, 40, and 80 μg / ml. These solutions can be mixed to prepare a single solution or prepared separately. The conditions of step S2 in Example 1 were used for determination, and the peak areas were recorded. A linear regression was performed between the peak area (Y) of the analyte and its mass concentration (X), and the results are shown in Table 8.

[0147] Table 8. Linear relationships of each component

[0148] paeoniflorin Y = 13.688x + 39.134 0.999850 4.994～499.4 Cimicifugain Y = 12.14796x + 0.28342 0.999995 3.212～80.31 5-O-methylvisamidolol Y = 11.82116x - 0.04101 1.000000 3.191～79.79

[0149] As can be seen from the table above, the determination method of paeoniflorin using the embodiments of the present invention has a wider linear range.

[0150] To further verify the accuracy of the above linear equation, six test solutions from the same batch of Bailing tablets were analyzed under the chromatographic conditions of step S2 in Example 1, using the aforementioned linear relationship. The measured peak areas were converted into contents using the above linear equation. Specifically, the average contents of paeoniflorin, cimicifugoside, and 5-O-methylvisamidol were 2.6 mg / g, 0.48 mg / g, and 0.56 mg / g, respectively, with RSD% of 0.4%, 2.1%, and 2.2%, respectively. This indicates that the quantitative results obtained through the above linear equation are accurate and reliable, especially the quantitative results for paeoniflorin. This provides a basis for rapid determination of batch content results and improves the accuracy of content detection results.

[0151] Verification Example 7

[0152] This example verifies the precision of the determination method of the present invention. Paeoniflorin control solution (44.98 μg / ml), cimicifugoside control solution (8.039 μg / ml), and 5-O-methylvisamiloside control solution (7.700 μg / ml) were taken and injected six times consecutively under the chromatographic conditions of step S2 in Example 1. The peak areas were measured, and the RSD% of the peak areas of paeoniflorin, cimicifugoside, and 5-O-methylvisamiloside were calculated to be 0.5%, 0.3%, and 0.2%, respectively, indicating that the instrument precision meets the requirements.

[0153] Verification Example 8

[0154] This example verifies the accuracy of the determination method of the present invention. Six portions of each of the following 1 ml solutions were accurately measured: paeoniflorin reference solution (1.3 mg / ml), cimicifugoside reference solution (1.3 mg / ml), and 5-O-methylvisamidolol reference solution (1.3 mg / ml). These were placed in stoppered conical flasks and allowed to evaporate to dryness. Then, approximately 0.5 g of a known amount of Bailing tablets (sample batch number C0056, known paeoniflorin content 2.6 mg / g) were accurately weighed. The test solutions were prepared according to step S1 of Example 1, and the samples were injected and determined under the chromatographic conditions of step S2 of Example 1. The average recovery rate of each indicator component was calculated to be 96%–102%, and the RSD was 1.2%–2.9% (n=6), indicating that the method has good accuracy.

[0155] Content determination

[0156] Take 10 batches of Bailing tablets, prepare the test solution according to step S1 of Example 1, accurately pipette 2 μL of the test solution and the reference solution, and inject them for determination under the chromatographic conditions of step S2 of Example 1. The content determination results of each batch are shown in Table 9.

[0157] Table 9. Results of content determination (mg / g) of three indicator components in Bailing tablets.

[0158] A0009 0.446 0.461 4.0 A0044 0.346 0.512 3.7 A2068 0.313 0.547 3.1 B0003 0.284 0.528 3.2 B0035 0.367 0.454 3.2 B0083 0.347 0.476 3.1 C0001 0.359 0.496 2.9 C0003 0.346 0.459 2.4 C0018 0.375 0.546 3.3 C0056 0.463 0.551 2.6

[0159] As can be seen from the table above, the determination method of the present invention can accurately detect multiple components such as cimicifugain, 5-O-methylvisamilol, and paeoniflorin simultaneously. However, due to batch differences in raw materials, the contents of cimicifugain, 5-O-methylvisamilol, and paeoniflorin in the prepared Bailing tablets show significant fluctuations. This further illustrates the importance of constructing fingerprint spectra for them.

[0160] Example 3

[0161] This example provides a method for determining the chemical components in a drug for treating vitiligo. The only difference from Example 1 is that when using a methanol-water mixture as the solvent, the volume percentage of methanol is above 50%, specifically 70%. Furthermore, the effectiveness of pure methanol as the extraction solvent was compared and investigated. The extraction effects of methanol, 30% methanol, and 70% methanol are compared as follows: Figure 13 As shown in the figure, extraction with 30% methanol solution yields the best results in terms of the number of peaks, peak shape, and separation. Therefore, 30% methanol solution is the preferred extraction solvent for this preparation method. The extraction effects of ultrasound and reflux were also investigated. The number of chromatographic peaks in the resulting chromatograms was not significantly different. Considering that ultrasound is easily affected by power frequency, reflux is the preferred extraction method for this preparation method.

[0162] Example 4

[0163] This example provides a method for determining the chemical components in a drug for treating vitiligo. The only difference between this method and Example 1 is that the batch number of the Bailing tablet sample is S0001. The determination results are as follows: Figure 14 As shown.

[0164] Example 5

[0165] This example provides a method for determining the chemical components in a drug for treating vitiligo, which differs from Example 4 only in that the column temperature is 20°C. The determination results are as follows: Figure 15 As shown.

[0166] Example 6

[0167] This example provides a method for determining the chemical components in a drug for treating vitiligo, which differs from Example 4 only in that the column temperature is 30°C. The determination results are as follows: Figure 16 As shown. Comparison Figure 1 and Figure 15 and 16 It can be seen that multiple components can be detected at column temperatures of 20℃ or 30℃, but the chromatographic separation effect is better at a column temperature of 40℃.

[0168] Comparative Example 1

[0169] This example provides a method for determining the chemical components in a drug for treating vitiligo, which differs from Example 4 only in that the test wavelength is 210nm, 230nm, 270nm, or 300nm. The test results of the test solution prepared in Example 1 at 210nm, 230nm, 270nm, and 300nm are as follows: Figures 17 to 20 As shown. Figure 1 and Figures 17 to 20 A comparison shows that, using the paeoniflorin peak as a reference, Figure 1The peak area of ​​paeoniflorin was 945.74494, which was significantly larger than that under other conditions, indicating that the method of the present invention has a significant advantage in selecting a detection wavelength below 210nm.

[0170] Comparative Example 2

[0171] This example provides a method for determining the chemical components in a drug for treating vitiligo. The only difference from Example 1 is that the volume percentage of phase A in the elution gradient did not increase to 60% within 21 minutes. Specifically, the elution gradient is as follows: 0–6 min, 0% A → 5% A; 6–9 min, 5% A → 10% A; 9–12 min, 10% A → 15% A; 12–15 min, 15% A → 20% A; 15–18 min, 20% A; 18–21 min, 20% A → 40% A; 21–24 min, 40% A → 60% A; 24–27 min, 60% A → 0% A; 27–33 min, 0% A. The chromatogram of the test solution is shown below. Figure 21 As shown.

[0172] Comparative Example 3

[0173] This example provides a method for determining the chemical components in a drug for treating vitiligo. The only difference from Example 1 is that the volume percentage of phase A increases linearly to 60% within 21 minutes during the elution gradient. Specifically, from 0 to 21 minutes, 0% A → 60% A; from 21 to 24 minutes, 60% A → 0% A; from 24 to 30 minutes, 0% A. The chromatogram of the test solution is shown below. Figure 22 As shown.

[0174] Comparative Example 4

[0175] This example provides a method for determining the chemical components in a drug for treating vitiligo. The only difference from Example 1 is that the volume percentage of phase A in the elution gradient did not increase to 60% within 21 minutes. Specifically, the elution gradient is as follows: 0–3 min, 0% A → 5% A; 3–6 min, 5% A → 10% A; 6–9 min, 10% A → 15% A; 9–12 min, 15% A → 20% A; 12–15 min, 20% A; 15–18 min, 20% A → 40% A; 18–27 min, 40% A → 60% A; 27–30 min, 60% A → 0% A; 30–36 min, 0% A. The chromatogram of the test solution is shown below. Figure 23 As shown.

[0176] contrast Figure 1 and Figures 21 to 23 It can be seen that when the volume percentage of phase A does not increase from 0% to 60% in a non-linear gradient within the first 21 minutes of the elution program, the number of chromatographic peaks in the measured chromatogram is significantly reduced and the area of ​​each signal peak is also significantly reduced.

[0177] Comparative Example 5

[0178] This example provides a method for determining the chemical components in a drug for treating vitiligo. The only difference between this method and Example 1 is the mobile phase; mobile phase A is methanol. The determination results are as follows: Figure 24 As shown.

[0179] Comparative Example 6

[0180] This example provides a method for determining the chemical components in a drug for treating vitiligo. The only difference between this method and Example 1 is the mobile phase; mobile phase B is a 0.1 vol% aqueous phosphoric acid solution. The determination results are as follows: Figure 25 As shown.

[0181] Comparative Example 7

[0182] This example provides a method for determining the chemical components in a drug for treating vitiligo. The only difference between this method and Example 1 is the mobile phase; mobile phase B is a 0.1 vol% formic acid aqueous solution. The determination results are as follows: Figure 26 As shown.

[0183] Comparative Example 8

[0184] This example provides a method for determining the chemical components in a drug for treating vitiligo. The only difference between this method and Example 1 is the mobile phase; mobile phase B is a 0.1 vol% aqueous acetic acid solution. The determination results are as follows: Figure 27 As shown.

[0185] contrast Figure 1 and Figures 24 to 27 It can be seen that the baseline is more stable and the separation and peak shape of each chromatographic peak are the best when using acetonitrile-water chromatogram.

[0186] In summary, the method of this invention enables excellent separation and identification of chemical components in drugs for treating vitiligo. Furthermore, the synergistic effect of the specific detection parameters (especially the synergistic coordination between wavelength, solvent, and gradient parameters) better eliminates interference, resulting in more accurate and reliable results with good reproducibility and stability. This invention allows for the qualitative identification of at least 11 peaks, identifying 5 of them, and quantitative analysis of at least 3 peaks. It enables effective determination of chemical components in drugs for treating vitiligo and the construction of UPLC fingerprints for these drugs. This allows for a more comprehensive quality evaluation of vitiligo drugs, compensating for the shortcomings of existing quality evaluation methods and providing data support for improving the quality standards of vitiligo drugs.

[0187] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A method for determining the chemical components in a drug for treating vitiligo, characterized in that: The medication for treating vitiligo includes Bailing tablets, and the assay method includes the following steps: S1. Preparation of test solution and reference solution: Grind Bai Ling tablets into a fine powder, dissolve in a solvent, extract, and prepare test solution; dissolve paeoniflorin reference standard in a solvent to prepare reference solution; wherein, the solvent is a methanol-water mixture; S2. Using ultra-high performance liquid chromatography (UHPLC) under the same detection conditions, chromatograms of the test solution and the reference solution are obtained separately. Qualitative and / or quantitative analysis of the chemical components in the chromatogram of the test solution is then performed. The detection conditions include: The detector wavelength is 201±2 nm; The chromatographic column was an Acquity UPLC. ® HSS T3, model specifications are as follows: diameter 2.1mm; length 100mm; average particle size of filler 1.8μm; The mobile phase flow rate was 0.3 ± 0.05 mL / min; The column temperature is 40±2℃; The injection volume was 2 ± 1 μL; The ultra-high performance liquid chromatography method is achieved by an ultra-high performance liquid chromatograph, which is an Agilent LC1290 II high performance liquid chromatograph. The elution conditions included acetonitrile as phase A and water as phase B, eluted using a gradient elution program as follows: 0–3 min, phase A volume fraction increased from 0% to 5%; 3–6 min, phase A volume fraction increased from 5% to 10%; 6–9 min, phase A volume fraction increased from 10% to 15%; 9–12 min, phase A volume fraction increased from 15% to 20%; 12–15 min, phase A volume fraction remained at 20%; 15–18 min, phase A volume fraction increased from 20% to 40%; 18–21 min, phase A volume fraction increased from 40% to 60%; 21–24 min, phase A volume fraction decreased from 60% to 0%; 24–30 min, phase A volume fraction remained at 0%. Step S1 includes at least one of the following conditions: 1) the volume percentage of methanol in the methanol-water mixed solution is 30±5%; 2) the extraction is performed by heating and reflux extraction, and the heating and reflux time is 30±10 min. The qualitative analysis includes determining the chromatographic peak positions of other components based on the relative retention time of paeoniflorin, thereby performing qualitative analysis of the chemical components in the test solution; the quantitative analysis includes obtaining chromatographic peak diagrams of paeoniflorin, cimicifugain, and 5-O-methylvisamilol at different concentrations, constructing a standard curve based on the relationship between chromatographic peak area and concentration, and performing quantitative analysis of the concentrations of paeoniflorin, cimicifugain, and 5-O-methylvisamilol in the test solution based on the standard curve.

2. The method for determining the chemical components in the drug for treating vitiligo according to claim 1, characterized in that: Step S1 includes the following conditions: the mass-to-volume ratio of Bai Ling tablets to methanol-water mixed solution is 1g:(50±5)mL.

3. The method for determining the chemical components in the drug for treating vitiligo according to claim 1, characterized in that: The determination method further includes the following steps: obtaining chromatograms of paeoniflorin reference standards at different concentrations, plotting a standard curve with the peak area of ​​paeoniflorin as the ordinate (y1) and the injection concentration of paeoniflorin as the abscissa (x1), and fitting the standard curve with a linear regression equation to obtain the linear regression equation of paeoniflorin as y1 = 13.688x1 - 39.134; and / or, the determination method further includes the following steps: obtaining chromatograms of cimicifugain reference standards at different concentrations, plotting a standard curve with the peak area of ​​cimicifugain as the ordinate (y2) and the injection concentration of cimicifugain as the abscissa (x2), and fitting the standard curve with a linear regression equation to obtain the linear regression equation of paeoniflorin as y1 = 13.688x1 - 39.134; and / or, the determination method further includes the following steps: obtaining chromatograms of cimicifugain reference standards at different concentrations, plotting a standard curve with the peak area of ​​cimicifugain as the ordinate (y2) and the injection concentration of cimicifugain as the abscissa (x2), and fitting the standard curve with a linear regression equation to obtain the linear regression equation of cimicifuga ... The linear regression equation for the cimicifugain glycoside is obtained by fitting the regression equation as y2=12.14796x2+0.28342; and / or, the determination method further includes the following steps: obtaining chromatograms of 5-O-methylvisamilol glycoside reference standards at different concentrations, plotting a standard curve with the peak area of ​​5-O-methylvisamilol glycoside as the ordinate (y3) and the injection concentration of 5-O-methylvisamilol glycoside as the abscissa (x3), and fitting the standard curve with a linear regression equation as the result to obtain the linear regression equation for the 5-O-methylvisamilol glycoside as y3=11.82116x3-0.04101.

4. A method for establishing a drug fingerprint spectrum for treating vitiligo, characterized in that: Includes the following steps: Chromatographic data of a drug for treating vitiligo are obtained using the determination method described in any one of claims 1 to 3, and a fingerprint spectrum of the drug for treating vitiligo is constructed using the chromatographic data.

5. The method for establishing according to claim 4, characterized in that: The fingerprint spectrum contains the following characteristic fingerprint peaks: paeoniflorin, amygdalin, cimicifugain, ginsenoside Rg1, and 5-O-methylvisamidol.

6. The application of the fingerprint pattern establishment method according to any one of claims 4 to 5 in drug quality detection and control.

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