Determination method of chemical components in medicine for treating vitiligo and establishment method of fingerprint spectrum

Abstract

The invention discloses a method for determining chemical components in a medicine for treating vitiligo and a method for establishing a fingerprint spectrum. Comprising the following steps: S1, preparing a to-be-detected solution and a reference substance solution: grinding Bailing tablets, dissolving the Bailing tablets in a solvent, and extracting to prepare the to-be-detected solution; dissolving a paeoniflorin reference substance in a solvent to prepare a reference substance solution; wherein the extraction solvent is a methanol-water mixed solution; s2, respectively obtaining chromatograms of the to-be-detected solution and the reference substance solution under the same detection conditions by adopting ultra-high performance liquid chromatography, and carrying out qualitative analysis and / or quantitative analysis on chemical components in the chromatograms of the to-be-detected solution; wherein the detection conditions are as follows: the wavelength of a detector is 201 + / -2nm; the elution conditions are as follows: acetonitrile is used as a phase A, water is used as a phase B, and elution is performed through a gradient elution procedure. According to the method, the chemical components in the medicine for treating the leucoderma can be accurately determined, so that the establishment of the fingerprint spectrum database of the medicine for treating the leucoderma can be better realized.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis, and particularly relates to a method for determining chemical components in a drug for treating vitiligo and a method for establishing a fingerprint. Background Art

[0002] Vitiligo is a chronic skin disease caused by the destruction of melanocytes. Existing therapeutic drugs (such as traditional Chinese medicine compound preparation Bailin Tablets, etc.) usually rely on the synergistic effect of multiple components to regulate immunity and oxidative stress. However, due to differences in planting regions, harvesting seasons, and processing techniques of traditional Chinese medicinal materials, the contents of active ingredients (such as psoralen, astragaloside IV, etc.) fluctuate significantly. This fluctuation may directly lead to inconsistent curative effects between batches. For example, the lack of quantitative control of key pharmacodynamic components results in obvious differences in the activation rate of tyrosinase activity among drugs of different batches. Bailin Tablets are made from 11 traditional Chinese medicinal materials including Angelica sinensis, Panax notoginseng, Carthamus tinctorius, Paeonia suffruticosa Andr., Prunus persica (L.) Batsch, Saposhnikovia divaricata (Turcz.) Schischk., Atractylodes lancea (Thunb.) DC., Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. f. ex Franch. et Sav., Portulaca oleracea L., Paeonia lactiflora Pall., and Phellodendron amurense Rupr., and have the effects of promoting blood circulation to remove blood stasis and increasing photosensitivity, and are clinically used for treating vitiligo. The quality standard of this preparation is included in the fifth volume of the "Ministry-issued Standards" (standard number: WS-B-0914-91). The standard includes items such as the character, thin-layer chromatography identification of Panax notoginseng and Angelica sinensis, etc. These identification methods can only perform qualitative analysis on specific components, and the accuracy of quantitative analysis is poor. Li Jianming et al. reported the determination of the content of paeoniflorin in Bailin Tablets and the evaluation of uncertainty by high performance liquid chromatography in "Determination of the content of paeoniflorin in Bailin Tablets by HPLC and evaluation of uncertainty" (China Pharmacy, 2015, 26(36): 5152-5154). This method selects 230 nm as the detection wavelength and elutes paeoniflorin in Bailin Tablets under isocratic elution. Although this method can achieve relatively accurate quantification of paeoniflorin, at this detection wavelength, few other active ingredients elute, and it is difficult to simultaneously detect multiple other components. Bailin Tablets are made from multiple medicinal materials, and the chemical components are relatively complex. The qualitative or quantitative analysis of a single component cannot comprehensively reflect its overall quality characteristics.

[0003] Although fingerprint technology has been used in the quality evaluation of some traditional Chinese medicines, there is an obvious technical gap in the application of Bailin Tablets. Therefore, establishing a fingerprint for Bailin Tablets will provide a scientific basis for the quality evaluation of drugs for treating vitiligo, as well as for their production and quality control. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. For this purpose, the present invention provides a method for determining chemical components in a drug for treating vitiligo. Through this method, multiple chemical components in Bailin Tablets can be accurately determined simultaneously, so as to better realize the quality evaluation of drugs for treating vitiligo.

[0005] The present invention also provides a method for establishing a fingerprint of drugs for treating vitiligo by using the above-mentioned determination method.

[0006] The present invention also provides an application of the method for establishing a fingerprint of drugs for treating vitiligo.

[0007] According to the method for determining chemical components in drugs for treating vitiligo according to the first aspect embodiment of the present invention, the drugs for treating vitiligo include Bailing tablets, and the determination method includes the following steps:

[0008] S1. Prepare a test solution and a reference solution: Grind Bailing tablets and dissolve them in a solvent, extract to obtain a test solution; dissolve paeoniflorin reference substance in a solvent to prepare a reference solution; wherein, the solvent is a methanol-water mixed solution;

[0009] S2. Use ultra-high performance liquid chromatography (UPLC) method to obtain chromatograms of the test solution and the reference solution respectively under the same detection conditions, and perform qualitative analysis and / or quantitative analysis on the chemical components in the chromatogram of the test solution; wherein, the detection conditions include:

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

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

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

[0013] The method for determining the chemical components in the medicine for treating vitiligo according to the embodiments of the present invention has at least the following beneficial effects: The solution of the present invention uses ultra-high performance liquid chromatography (UPLC) to study the fingerprint of the medicine for treating vitiligo. When analyzing multiple batches of Bailin tablets, there are at least 11 common peaks, and the chromatographic peaks of various components such as paeoniflorin, amygdalin, prim-O-glucosylcimifugin, ginsenoside Rg1, and 5-O-methylvisammioside can be accurately identified. At the same time, quantitative analysis is carried out on components such as paeoniflorin, prim-O-glucosylcimifugin, and 5-O-methylvisammioside. This method can provide a reference for improving the quality standard of the medicine for treating vitiligo, more comprehensively evaluate and control the quality of the medicine for treating vitiligo, and improve the quality detection standard. When the detection wavelength is about 201 nm, the number of chromatographic peaks obtained during detection increases significantly. Using the detection method of the solution of the present invention, only one component, paeoniflorin, is needed as a reference substance to accurately identify various components such as paeoniflorin, amygdalin, prim-O-glucosylcimifugin, ginsenoside Rg1, and 5-O-methylvisammioside. Not only are the results accurate and reliable, and the identification effect is good, but also the detection cost can be greatly reduced, and qualitative detection of multiple components and accurate quantitative detection of multiple components can be achieved simultaneously.

[0014] When using this determination method for detection, in the range of 4.994 μg / ml to 499.4 μg / ml of paeoniflorin, the linear relationship is good (correlation coefficient ≥ 0.999); the RSD of the peak area of paeoniflorin measured in the precision test is 0.5%; the RSD during the stability test is 0.5%; during the repeatability experiment, the RSD is 0.4%; in the spiked recovery experiment, the average recovery rate of the results is 96%, and the RSD is 1.2%. Using paeoniflorin as the quality control index component of Bailin tablets and establishing a fingerprint map based on this for quality control is simple to operate, and the identification result is accurate and reliable, which can better control the quality of the product as a whole and provide an effective guarantee for the production and clinical application of the medicine.

[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 rises from 0% A to 5%; 3 - 6 min, the volume fraction of phase A rises from 5% to 10%; 6 - 9 min, the volume fraction of phase A rises from 10% to 15%; 9 - 12 min, the volume fraction of phase A rises from 15% to 20%; 12 - 15 min, the volume fraction of phase A remains at 20%; 15 - 18 min, the volume fraction of phase A rises from 20% to 40%; 18 - 21 min, the volume fraction of phase A rises from 40% to 60%; 21 - 24 min, the volume fraction of phase A drops from 60% to 0%; 24 - 30 min, the volume fraction of phase A remains at 0%.

[0016] Using this elution gradient can not only promote the effective elution of more chemical components, but also significantly improve the separation effect, which has a positive effect on 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 conditions: the flow rate of the mobile phase is 0.3 ± 0.05 min / mL. Selecting this flow rate level results in a lower column pressure.

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

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

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

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

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

[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 model specifications of the chromatographic column are as follows: the diameter is 2.1 mm; the length is 100 mm; the average particle size of the packing is 1.8 μm.

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

[0026] Other chromatographs can also be used.

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

[0028] According to some embodiments of the present invention, the volume ratio of methanol in the methanol - water mixed solution is less than 50%. When the volume ratio 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 ratio of methanol in the methanol - water mixed solution is 30 ± 5%.

[0030] When preparing the test solution with the solvent in a specific ratio, the obtained chromatogram shows a larger characteristic fingerprint chromatographic peak area. Meanwhile, more chemical components can be detected, further ensuring the accuracy and reliability of the test results. The similarity of the chromatogram of the test solution prepared with the solvent according to the solution of the present invention is higher than 0.994. This characteristic significantly enhances the stability and accuracy of the chromatogram, thus facilitating the improvement of the precision of the test results.

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

[0032] According to some embodiments of the present invention, in the step S1, the extraction method is extraction by heating under reflux. The test solution obtained by extraction by heating under reflux has a higher response in the liquid phase detection system during chromatographic analysis.

[0033] According to some embodiments of the present invention, in the 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-aqueous solution is 30±5%. By using this wavelength and extraction conditions simultaneously, the interference in detection can be greatly reduced, more peaks can be obtained, the separation effect of the peaks 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, so as to perform qualitative analysis on 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, making a standard curve based on the relationship between the chromatographic peak area and the concentration, and performing quantitative analysis on the concentration of paeoniflorin in the test solution according to the standard curve.

[0037] According to some embodiments of the present invention, the quantitative analysis further includes obtaining the quantitative standard curves of prim-O-glucosylcimifugin or 5-O-methylvisamminol glycoside, and performing quantitative analysis on prim-O-glucosylcimifugin or 5-O-methylvisamminol glycoside in the test solution according to the standard curves.

[0038] Prim-O-glucosylcimifugin or 5-O-methylvisamminol glycoside can be added to the reference solution for synchronous detection, or a reference solution of prim-O-glucosylcimifugin or 5-O-methylvisamminol glycoside can be separately prepared. Chromatograms at different concentrations are obtained respectively, and quantitative analysis of the three components can be achieved simultaneously according to the relationships between the concentrations of paeoniflorin, prim-O-glucosylcimifugin, and 5-O-methylvisamminol glycoside and the peak areas.

[0039] According to some embodiments of the present invention, the determination method further includes the following steps: obtaining a chromatogram of a reference solution containing paeoniflorin reference substances with different concentrations, taking the peak area of paeoniflorin as the ordinate y, and the injection concentration of paeoniflorin as the abscissa x to plot a standard curve, fitting a linear regression equation to the standard curve, and obtaining 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 a chromatogram of a sample solution containing prim-O-glucosylcimifugin reference substances with different concentrations, taking the peak area of prim-O-glucosylcimifugin as the ordinate y2, and the injection concentration of prim-O-glucosylcimifugin as the abscissa x2 to plot a standard curve, fitting a linear regression equation to the standard curve, and obtaining the linear regression equation of prim-O-glucosylcimifugin as y2 = 12.14796x2 + 0.28342.

[0041] According to some embodiments of the present invention, the determination method further includes the following steps: obtaining a chromatogram of a sample solution containing 5-O-methylvisammioside reference substances with different concentrations, taking the peak area of 5-O-methylvisammioside as the ordinate y3, and the injection concentration of 5-O-methylvisammioside as the abscissa x3 to plot a standard curve, fitting a linear regression equation to the standard curve, and obtaining the linear regression equation of 5-O-methylvisammioside as y3 = 11.82116x3 - 0.04101.

[0042] Through the above content regression equations, the specific contents of paeoniflorin, prim-O-glucosylcimifugin, and 5-O-methylvisammioside can be obtained more accurately.

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

[0044] According to some embodiments of the present invention, the volume ratio of methanol in the methanol-water mixed solution is below 70%.

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

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

[0047] When preparing the test solution using the solvent with the specific ratio, the measured chromatogram exhibits a larger characteristic fingerprint chromatographic peak area. At the same time, more chemical components can be detected, thus further ensuring the accuracy and reliability of the test results. The similarity of the chromatogram of the test solution obtained using the solvent prepared according to the solution of the present invention is higher than 0.994. This characteristic significantly enhances the stability and accuracy of the chromatogram, and thus is beneficial to improving the precision of the test results.

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

[0049] According to some embodiments of the present invention, in the step S1, the extraction method is extraction by heating under reflux. The test solution obtained by extraction by heating under reflux has a higher response in the liquid phase detection system when performing chromatographic analysis.

[0050] According to some embodiments of the present invention, in the 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 ratio of methanol in the methanol-aqueous solution is 30±5%. By using this wavelength and extraction conditions simultaneously, the interference of the detection can be greatly reduced, more peaks can be obtained, the separation effect of the peaks 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, astragalus, notoginseng, safflower, red peony root, moutan bark, purslane, peach kernel, ledebouriella root, dahurian angelica root and atractylodes rhizome. It can be prepared into tablets or other dosage forms, and all can be applicable to the solution of the present invention.

[0053] According to the method for establishing a fingerprint spectrum according to the second aspect embodiment of the present invention, the following steps are included:

[0054] Obtain the chromatographic data of the drug for treating vitiligo using the above measurement method, and construct the fingerprint spectrum of the drug for treating vitiligo using the chromatographic data.

[0055] The fingerprint spectrum established by the determination method of the scheme of the present invention has better separation effect between different components and less interference. When the established fingerprint spectrum is used for quality control of vitiligo drugs, there are at least 11 common peaks, and the relative retention time RSD of the common peaks is ≤0.7%, and the relative peak area RSD is ≤3.5%, indicating that the method of the present invention is stable and reliable; the traditional Chinese medicine fingerprint spectrum constructed by the present invention can realize the simultaneous detection and identification of the characteristic components of 10 medicinal materials in Bailing Tablets, and through the overall comparison and analysis of multi-dimensional chromatographic characteristic peaks, it effectively overcomes the limitation of traditional quality control methods that only target a single component. This technical solution significantly improves the comprehensiveness and reliability of the quality evaluation of vitiligo treatment drugs by systematically characterizing the overall picture of the chemical composition of the compound preparation, and provides a more scientific and accurate 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)", performing multi-point correction and peak matching to obtain a fingerprint spectrum.

[0057] The similarity evaluation results of the fingerprint maps show that the similarity of the Bailing tablets fingerprint maps 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, 0.996, 0.994, 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 comprises the following characteristic fingerprint peaks: paeoniflorin, amygdalin, cimicifuga glycoside, ginsenoside Rg1 and 5-O-methylvisamidoside.

[0059] The common peaks are determined according to the relative retention times of the chromatographic peaks in the UPLC fingerprint, and characteristic fingerprint peaks are selected from the common peaks.

[0060] The application of the above fingerprint establishment method in drug quality detection 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 systems for vitiligo drugs. The core is to use UPLC-DAD spectrum analysis technology to successfully construct a high-precision vitiligo drug fingerprint. The method is easy to operate, has excellent precision, excellent repeatability, and is stable and reliable. It shows high sensitivity and long-term stability. It is an effective quality evaluation method for vitiligo drugs and will provide a scientific basis for the production and quality control of vitiligo drugs. The fingerprint technology can perform qualitative and quantitative analysis on the active ingredient groups in vitiligo drugs through a systematic quality characterization system, providing a scientific basis for process control, batch consistency evaluation and product quality standard improvement of drug production. Compared with the traditional single-indicator quality control method, the scheme of the present invention has technical advantages such as multi-component synchronous monitoring and digital comparison of characteristic spectra, which is in line with the current modernization development trend of quality control of traditional Chinese medicine and natural medicine.

[0062] According to some embodiments of the present invention, the application includes the following steps: calculating the similarity between the chromatogram of the Bailing Tablet sample to be tested and the standard fingerprint spectrum, and evaluating the quality of the sample to be tested according to the similarity calculation result, if the similarity is greater than 0.9, it is qualified, otherwise it is unqualified. It is generally believed that a similarity of 0.9 is considered to meet the requirements, and the requirements can be further increased as needed.

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

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

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

[0066] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 is the chromatogram of the test liquid obtained by measurement in Example 1 of the present invention;

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

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

[0070] Figure 4 is the chromatogram of the blank solvent measured in Verification Example 1 of the present invention;

[0071] Figure 5 is the chromatogram of the prim-O-glucosylcimifugin reference substance solution measured in Verification Example 1 of the present invention;

[0072] Figure 6 is the chromatogram of the 5-O-methylvisammioside reference substance solution measured in Verification Example 1 of the present invention;

[0073] Figure 7 is the chromatogram of the test solution of the negative sample without Saposhnikoviae Radix measured in Verification Example 1 of the present invention;

[0074] Figure 8 is the chromatogram of the test solution of the single negative sample without Paeoniae Radix Rubra measured in Verification Example 1 of the present invention;

[0075] Figure 9 is the chromatogram of the test solution of the single negative sample without Moutan Cortex measured in Verification Example 1 of the present invention;

[0076] Figure 10 is the chromatogram of the test solution of the double negative sample without Paeoniae Radix Rubra and without Moutan Cortex measured in Verification Example 1 of the present invention;

[0077] Figure 11 is the standard fingerprint of Bailing Tablets constructed in Verification Example 2 of the present invention;

[0078] Figure 12 is the comparison chart of the test results of single herbs and Bailing Tablets samples in Verification Example 2 of the present invention;

[0079] Figure 13 is the comparison of the chromatogram effects of the test solutions extracted with different extraction solvents in Example 3 of the present invention;

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

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

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

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

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

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

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

[0087] Figure 21 is the chromatogram of the test solution measured in Comparative Example 2 of the present invention;

[0088] Figure 22 is the chromatogram of the test solution measured in Comparative Example 3 of the present invention;

[0089] Figure 23 is the chromatogram of the test solution measured in Comparative Example 4 of the present invention;

[0090] Figure 24 is the chromatogram of the test solution measured in Comparative Example 5 of the present invention;

[0091] Figure 25 is the chromatogram of the test solution measured in Comparative Example 6 of the present invention;

[0092] Figure 26 is the chromatogram of the test solution measured in Comparative Example 7 of the present invention;

[0093] Figure 27 is the chromatogram of the test solution measured in Comparative Example 8 of the present invention. Detailed Embodiments

[0094] The following will clearly and completely describe the concept and technical effects generated by the present invention in combination with the embodiments 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, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention. The test methods used in the embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, are all reagents and materials that can be obtained from commercial channels. Unless otherwise specified, the same parameter values are taken in each embodiment. The embodiments described below are exemplary and are only used to explain the present invention and should not be construed as a limitation of the present invention.

[0095] In the description of the present invention, the description referring to terms such as "some embodiments" means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0096] The information on 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 (2.1 mm × 100 mm, 1.8 μm) 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 substance (110736 - 202447, 98.1%), amygdalin reference substance (110820 - 20241, 94.9%), prim-O-glucosylcimifugin reference substance (111522 - 202214, 95.7%), 5-O-methylvisammioside reference substance (111523 - 202212, 97.8%), ginsenoside Rg1 reference substance (110703 - 202436, 97.0%), all from the National Institutes for Food and Drug Control.

[0099] 3) Medicinal materials: Bailin 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), with batch numbers A0002, A0009, A0028, A0044, A0051, A2068, B0003, B0020, B0035, B0066, B0083, C0001, C0003, 240018, C0053, C0056, and S0001, produced by Guoyao Group Fengliao (Foshan) Pharmaceutical Co., Ltd., and sequentially labeled as samples M1 to M16. Single-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, their preparation processes and other raw materials are the same as those of Bailin Tablets, except that they lack a specific one or two drugs. The specific preparation process can refer to the following steps: According to the same prescription amount of Bailin Tablets, take 43% of Angelica sinensis and Panax notoginseng and crush them into coarse powder. The remaining Angelica sinensis, Saposhnikovia divaricata, Atractylodes lancea, and Angelica dahurica are distilled with water at a gentle boil for 2 hours to collect the aromatic oil. The residue is decocted twice with Carthamus tinctorius, Prunus persica, Portulaca oleracea, and Astragalus membranaceus. For the first time, add 5 times the amount of water and heat for 2 hours; for the second time, add 4 times the amount of water and heat for 1.5 hours. Combine the decoction, filter it, and concentrate the filtrate into an extract with a relative density of 1.11 - 1.16 (65°C). Mix it with the above-mentioned coarse powder, dry it, crush it into fine powder, sieve it, make granules, spray the above-mentioned aromatic oil into it, and mix well to obtain the double-negative sample lacking both Paeonia lactiflora and Paeonia suffruticosa. Prepare the control solution of the double-negative sample lacking both Paeonia lactiflora and Paeonia suffruticosa according to the preparation method of the test solution of Bailin Tablets.

[0100] According to the same prescription amount of Bailin Tablets, take 43% of Angelica sinensis and Panax notoginseng and crush them into coarse powder. The remaining Angelica sinensis, Saposhnikovia divaricata, Atractylodes lancea, and Angelica dahurica are distilled with water at a gentle boil for 2 hours to collect the aromatic oil. The residue is decocted twice with Paeonia lactiflora, Carthamus tinctorius, Prunus persica, Portulaca oleracea, and Astragalus membranaceus. For the first time, add 5 times the amount of water and heat for 2 hours; for the second time, add 4 times the amount of water and heat for 1.5 hours. Combine the decoction, filter it, and concentrate the filtrate into an extract with a relative density of 1.11 - 1.16 (65°C). Mix it with the above-mentioned coarse powder, dry it, crush it into fine powder, sieve it, make granules, spray the above-mentioned aromatic oil into it, and mix well to obtain the single-negative sample lacking Paeonia suffruticosa. Prepare the control solution of the single-negative sample lacking Paeonia suffruticosa according to the preparation method of the test solution of Bailin Tablets.

[0101] According to the same prescription amount of Bailin Tablets, take 43% of Angelica sinensis and Panax notoginseng and crush them into coarse powder. The remaining Angelica sinensis, Saposhnikovia divaricata, Atractylodes lancea, Angelica dahurica, and Paeonia suffruticosa are distilled with water at a gentle boil for 2 hours to collect the aromatic oil. The residue is decocted twice with Carthamus tinctorius, Prunus persica, Portulaca oleracea, and Astragalus membranaceus. For the first time, add 5 times the amount of water and heat for 2 hours; for the second time, add 4 times the amount of water and heat for 1.5 hours. Combine the decoction, filter it, and concentrate the filtrate into an extract with a relative density of 1.11 - 1.16 (65°C). Mix it with the above-mentioned coarse powder, dry it, crush it into fine powder, sieve it, make granules, spray the above-mentioned aromatic oil into it, and mix well to obtain the single-negative sample lacking Paeonia lactiflora. Prepare the control solution of the single-negative sample lacking Paeonia lactiflora according to the preparation method of the test solution of Bailin Tablets.

[0102] According to the same prescription amount of Bailing Tablets, take 43% of Angelica sinensis and Panax notoginseng and crush them into coarse powder. The remaining Angelica sinensis, Atractylodes lancea, Angelica dahurica, and Paeonia suffruticosa are distilled with water at a gentle boil for 2 hours to collect the aromatic oil. The residue is decocted twice with water together with Paeonia lactiflora, Carthamus tinctorius, Prunus persica, Portulaca oleracea, and Astragalus membranaceus. For the first time, add 5 times the amount of water and heat for 2 hours; for the second time, add 4 times the amount of water and heat for 1.5 hours. Combine the decoction liquids, filter, and concentrate the filtrate into an extract with a relative density of 1.11 - 1.16 (65 °C). Mix it with the above-mentioned coarse powder, dry, crush it into fine powder, sieve it, make granules, spray the above-mentioned aromatic oil into it, and mix evenly to obtain the negative sample without Paeonia lactiflora. Prepare the single negative control solution without Saposhnikovia divaricata according to the preparation method of the test solution of 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 from Guoyao Group Fengliaoxing (Foshan) Pharmaceutical Co., Ltd., and the preparation process of their test solutions is the same as that of Bailing Tablets.

[0104] Instruments or reagents not specifically stated are common instruments or reagents in this field.

[0105] Example 1

[0106] This example provides a method for determining the chemical components in a drug for treating vitiligo. The specific operation process is as follows:

[0107] S1. Prepare the test solution and the reference substance solution: Take 20 tablets of the drug for treating vitiligo (the sample batch number of Bailing Tablets is C0056), grind them finely, take about 1 g, weigh it precisely, place it in a stoppered conical flask, precisely add 50 ml of 30 vol% methanol, weigh it precisely, heat under reflux for 30 min, weigh it again, make up for the lost weight, shake well, filter, and take the subsequent filtrate to obtain the test solution. Take paeoniflorin reference substance, weigh it precisely, precisely add 100 ml of 30 vol% methanol to obtain the reference substance solution (concentration is 50 μg / mL). In addition, also take prim-O-glucosylcimifugin and 5-O-methylvisammioside, and prepare reference substance solutions containing 8 μg per 1 ml with 30% methanol respectively.

[0108] S2. Use the ultra-high performance liquid chromatography (UPLC) method to obtain the chromatograms of the test solution and the reference substance solution respectively under the same detection conditions. Among them, the chromatographic conditions include: using The HSS T3 (2.1 mm × 100 mm, 1.8 μm) was used as the chromatographic column; acetonitrile (A)-aqueous solution (B) was used as the mobile phase for gradient elution (the gradient elution program was as follows: 0 - 3 min, the volume fraction of phase A increased from 0% A to 5%; 3 - 6 min, the volume fraction of phase A increased from 5% to 10%; 6 - 9 min, the volume fraction of phase A increased from 10% to 15%; 9 - 12 min, the volume fraction of phase A increased from 15% to 20%; 12 - 15 min, the volume fraction of phase A remained at 20%; 15 - 18 min, the volume fraction of phase A increased from 20% to 40%; 18 - 21 min, the volume fraction of phase A increased from 40% to 60%; 21 - 24 min, the volume fraction of phase A decreased from 60% to 0%; 24 - 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; the injection volume was 2 μL.

[0109] The chromatogram of the test solution was as Figure 1 shown, and the chromatogram of the reference solution was as Figure 2 shown.

[0110] It can be seen from Figure 1 and 2 that at the chromatogram position corresponding to the reference solution, there were chromatographic peaks with the same retention time in the chromatogram of the test solution. At the same time, ultraviolet spectral scanning and peak purity detection were carried out on the paeoniflorin peak using a diode array detector, and it was found that the ultraviolet absorption spectra of the test solution and the reference were completely consistent, and the peak purity factors were all greater than 998.

[0111] Example 2

[0112] This example provides a method for establishing a fingerprint of a drug for treating vitiligo, including the following steps: Import the chromatographic data obtained by the determination method of Example 1 into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints" for multi-point calibration and peak matching to obtain the fingerprint. Specifically, take 16 batches of Bailin tablets (M1 - M16), prepare the test solutions respectively according to the operation steps of S1 in Example 1, and then obtain the fingerprints of the above test solutions through chromatographic analysis according to the operation of step S2. Import the above fingerprints into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)", set the chromatogram of sample M1 as the reference, set the time window width to 0.1 min, and use the average method for multi-point calibration to generate the standard fingerprint R of Bailin tablets (the obtained fingerprint is shown in Figure 3 ). The similarity evaluation was carried out on the chromatographic data of 16 batches of Bailin tablets. The results are shown in Table 1 below. It shows that the similarity of the fingerprints of 16 batches of Bailin tablets was ≥ 0.994.

[0113] Table 1

[0114] Batch number Similarity 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 can be seen from Table 1, the similarity degrees of the fingerprint spectra of 16 batches of Bailing Tablets are 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 in sequence, all of which are above 0.994. This indicates that under the extraction conditions and chromatographic conditions of the present invention's solution, a good sample separation effect and detection response degree are formed, and the detection effect is accurate and reliable.

[0116] Verification Example 1

[0117] This example is to prove the specificity of the determination method of the present invention. The difference from Example 1 is that the test objects are blank solvent, other reference substances (prim-O-glucosylcimifugin and 5-O-methylvisammioside, respectively made into reference substance solutions containing 8 μg per 1 ml with 30% methanol), negative samples lacking Saposhnikoviae Radix, single negative samples lacking Paeoniae Radix Rubra, single negative samples lacking Moutan Cortex, and double negative samples lacking both Paeoniae Radix Rubra and Moutan Cortex. Among them, the negative samples lacking Saposhnikoviae Radix, single negative samples lacking Paeoniae Radix Rubra, single negative samples lacking Moutan Cortex, and double negative samples lacking both Paeoniae Radix Rubra and Moutan Cortex are processed according to the same processing flow of the test solution in Example 1 to prepare test samples; the samples are injected through an injection needle using the method of Example 1, and their chromatograms are measured. The results are as Figures 4 - 10 shown.

[0118] As can be seen from the figure, at the chromatogram positions corresponding to the reference substance or other reference substance solutions, there are chromatographic peaks with the same retention time in the chromatograms of the test solution and the single negative test samples lacking Paeoniae Radix Rubra and Moutan Cortex; no corresponding detected components are seen in the chromatograms of the double negative test sample lacking both Paeoniae Radix Rubra and Moutan Cortex, the negative test sample lacking Saposhnikoviae Radix, and the blank solution (the double negative test sample lacking both Paeoniae Radix Rubra and Moutan Cortex has no paeoniflorin peak, the negative test sample lacking Saposhnikoviae Radix has no prim-O-glucosylcimifugin and 5-O-methylvisammioside peaks, and the blank solution has no paeoniflorin peak, prim-O-glucosylcimifugin, and 5-O-methylvisammioside peaks), indicating that the remaining components and solvents do not interfere with the content determination, and the specificity of this method is good. At the same time, ultraviolet spectral scanning and peak purity detection are carried out on the paeoniflorin peak, prim-O-glucosylcimifugin peak, and 5-O-methylvisammioside peak using a diode array detector, and it is found that the ultraviolet absorption spectra of the test samples are completely consistent with those of the reference substances, and the peak purity factors are all greater than 998.

[0119] Verification Example 2

[0120] This example is for verifying the attribution and confirmation of common peaks: Prepare a reference substance solution according to the operation of the reference substance solution in step S1 of Example 1 with amygdalin, prim-O-glucosylcimifugin, ginsenoside Rg1, 5-O-methylvisammioside, and paeoniflorin, and at the same time prepare a test solution for Bailing tablets according to the operation in step S1 of Example 1, and inject samples for determination under the chromatographic conditions in step S2. At the same time, take the detection chromatograms of 16 batches of Bailing tablets in Example 2 as the research object, use relevant software for fingerprint similarity analysis, and use different strategies such as data extraction, time matching, and peak matching to determine the common peaks, and establish a reference fingerprint by multi-point calibration of chromatographic peaks. According to the relative retention times of each chromatographic peak in the chromatogram, a total of 11 common components are confirmed, as Figure 11 shown. Qualitatively analyze by reference substances, single herbs, retention time, and ultraviolet absorption spectrum. The comparison chart of the test results of single herbs and Bailing tablets samples is as Figure 12 shown. The numbers marked in the figure represent the 11 common components confirmed. It can be seen from the figure that peach kernels are the only source of amygdalin (peak No. 4); Saposhnikovia divaricata is the only source of prim-O-glucosylcimifugin (peak No. 6) and 5-O-methylvisammioside (peak No. 8); Notoginseng is the only source of ginsenoside Rg1 (peak No. 9); while paeoniflorin (peak No. 5) is a common peak of Paeonia lactiflora Pall. and Cortex Moutan. Select 5 peaks as characteristic fingerprint peaks, among which peak No. 4 is amygdalin, peak No. 5 is paeoniflorin, peak No. 6 is prim-O-glucosylcimifugin, peak No. 8 is 5-O-methylvisammioside, and peak No. 9 is ginsenoside Rg1. The shape of chromatographic peak No. 5 is good and the peak area is large, so selecting the paeoniflorin peak as the reference peak S can make the determination method more accurate and reliable.

[0121] Verification Example 3

[0122] This example is to prove the precision of the determination method of the present invention. Refer to the preparation of the test solution in step S1 of Example 1 (sample batch number is C0056), and then inject samples continuously for 6 times under the conditions of step S2. Take the paeoniflorin peak as the reference peak (S), and calculate the relative retention time RSD and relative peak area RSD of each common peak. The results are shown in Tables 2 and 3 below.

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

[0124]

[0125]

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

[0127] Number S1 S2 S3 S4 S5 S6 Relative peak area RSD% 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 above table, for the sample solution measured by the measurement method of the embodiment of the present invention, the RSD of the relative retention time of each common peak is ≤0.7%, and the RSD of the relative peak area is ≤3.5%. Taking the fingerprint 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] In this example, to prove the repeatability of the measurement method of the present invention, 6 portions of the test solution (sample batch number C0056) were prepared with reference to the steps in Step S1 of Example 1, and then injected and analyzed under the conditions of Step S2 respectively. Taking the paeoniflorin peak as the reference peak (S), the RSD of the relative retention time and the RSD of the relative peak area of each common peak were calculated. The results are shown in Tables 4 and 5 below.

[0131] Table 4 Retention time of paeoniflorin peak and each common peak, and RSD% of relative retention time

[0132]

[0133]

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

[0135] Number S1 S2 S3 S4 S5 S6 Relative peak area RSD% 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 above table, for the sample solution measured by the measurement method of the embodiment of the present invention, the RSD of the relative retention time of each common peak is ≤0.7%, and the RSD of the relative peak area is ≤4.3%. Taking the fingerprint 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] In this example, to prove the stability of the test solution processed by the measurement method of the present invention, the test solution (sample batch number C0056) was prepared with reference to the steps in Step S1 of Example 1, and injected and analyzed under the conditions of Step S2 at 0h, 2h, 4h, 8h, 12h, 18h, and 24h after the sample preparation was completed respectively. Taking the paeoniflorin peak as the reference peak (S), the RSD of the relative retention time and the RSD of the relative peak area of each common peak were calculated. The results are shown in Tables 6 and 7 below.

[0139] Table 6 Retention time of paeoniflorin peak and each common peak, and RSD% of relative retention time

[0140] Number S1 S2 S3 S4 S5 S6 S7 Relative retention time RSD% 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 Paeoniflorin Peak, Peak Areas of Each Common Peak, and RSD% of Relative Peak Areas

[0142]

[0143]

[0144] As can be seen from the above table, for the sample solution measured by the determination method of the embodiment of the present invention, the RSD of the relative retention time of each common peak is ≤0.4%, and the RSD of the relative peak area is ≤3.8%. Taking the fingerprint 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 test solution prepared by the determination method of the present invention remains stable within 24 hours.

[0145] Verification Example 6

[0146] This example is to verify the linear relationship of the determination method of the embodiment of the present invention. Appropriate amounts of paeoniflorin, prim-O-glucosylcimifugin, and 5-O-methylvisammioside reference substances were taken and added to 30 vol% methanol to prepare reference substance solutions with different mass concentrations. Among them, the concentrations of the paeoniflorin reference substance were 5, 10, 25, 50, 100, 250, 500 μg / ml, and the concentrations of the reference substance solutions of prim-O-glucosylcimifugin and 5-O-methylvisammioside were 3.2, 4, 8, 16, 40, 80 μg / ml. They can be mixed to make one solution or prepared separately. The conditions in step S2 of Example 1 were used for determination in rows, and the peak areas were recorded. The linear regression was performed with the peak area (Y) of the component to be measured against the mass concentration (X), and the results are shown in Table 8.

[0147] Table 8 Linear Relationships of Each Component

[0148] Component Regression equation r Linear range (μg / ml) Paeoniflorin Y = 13.688x + 39.134 0.999850 4.994～499.4 Prim-O-glucosylcimifugin Y = 12.14796x + 0.28342 0.999995 3.212～80.31 5-O-methylvisammioside Y = 11.82116x - 0.04101 1.000000 3.191～79.79

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

[0150] To further verify the accuracy of the above linear equation, using the above linear relationship, 6 test solutions of the same batch of Bailing Tablets were measured according to the chromatographic conditions of step S2 of Example 1, and the measured peak areas were converted into contents according to the above linear equation. Specifically, the average contents of paeoniflorin, prim-O-glucosylcimifugin, and 5-O-methylvisammioside were 2.6 mg / g, 0.48 mg / g, and 0.56 mg / g respectively, and the RSD% were 0.4%, 2.1%, and 2.2% respectively, indicating that the quantitative results obtained through the above linear equation are accurate and reliable, especially the quantitative result of paeoniflorin is more accurate and reliable. It provides a basis for quickly obtaining the results for the detection of batch content results and improves the accuracy of the content detection results.

[0151] Verification Example 7

[0152] This example is to verify the precision of the determination method of the embodiments of the present invention. Take paeoniflorin control solution (44.98 μg / ml), prim-O-glucosylcimifugin control solution (8.039 μg / ml), and 5-O-methylvisammioside control solution (7.700 μg / ml), and according to the chromatographic conditions in step S2 of Example 1, inject samples continuously for 6 times to measure the peak areas. Calculate the RSD% of the peak areas of paeoniflorin, prim-O-glucosylcimifugin, and 5-O-methylvisammioside, which are 0.5%, 0.3%, and 0.2% respectively, indicating that the instrument precision meets the requirements.

[0153] Verification Example 8

[0154] This example is to verify the accuracy of the determination method of the embodiments of the present invention. Precisely measure 1 ml of paeoniflorin reference substance solution (1.3 mg / ml), prim-O-glucosylcimifugin reference substance solution (1.3 mg / ml), and 5-O-methylvisammioside reference substance solution (1.3 mg / ml), each in 6 portions, place them in a stoppered conical flask, evaporate to dryness, and then precisely weigh about 0.5 g of Baoling tablets with known content (sample batch number C0056, known paeoniflorin content 2.6 mg / g). Prepare the test solution according to step S1 of Example 1, and then inject and measure according to the chromatographic conditions in step S2 of Example 1. Calculate that the average recovery rates of each index component are 96% - 102%, and the RSD is 1.2% - 2.9% (n = 6), indicating that the accuracy of this method is good.

[0155] Content determination

[0156] Take 10 batches of Baoling tablets, prepare the test solution according to step S1 of Example 1, precisely absorb 2 μL of the test solution and the reference substance solution respectively, and inject and measure under the chromatographic conditions in step S2 of Example 1. The content determination results of each batch are shown in Table 9.

[0157] Table 9 Content determination results of 3 index components in Baoling tablets (mg / g)

[0158] Batch number Prim-O-glucosylcimifugin 5-O-methylvisammioside Paeoniflorin 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] It can be seen from the above table that through the determination method of the present invention's solution, multiple components such as prim-O-glucosylcimifugin, 5-O-methylvisammioside, and paeoniflorin can be accurately detected simultaneously. However, due to batch differences in raw materials, the contents of prim-O-glucosylcimifugin, 5-O-methylvisammioside, and paeoniflorin in the prepared Baoling tablets have relatively obvious fluctuations, which further illustrates the significance of constructing a fingerprint for it.

[0160] Example 3

[0161] This example provides a method for determining the chemical components in a drug for treating vitiligo. The difference from Example 1 is only that when using a methanol-water mixed solution as the solvent, the volume ratio of methanol is above 50%, specifically 70%. In addition, the effect of using pure methanol as the extraction solvent was also investigated for comparison. The extraction effects of methanol, 30% methanol, and 70% methanol are as Figure 13 shown. As can be seen from the figure, when using a 30% methanol solution for extraction, the number of peaks in the test solution prepared is large, the peak shape is good, and the separation effect is the best. Therefore, a 30% methanol solution is preferentially selected as the extraction solvent for this preparation method. At the same time, the extraction effects of ultrasonic and heating reflux on the test sample were also investigated. The difference in the number of chromatographic peaks in the obtained chromatograms is not significant. Considering that the ultrasonic method is easily affected by power frequency, heating reflux is preferentially selected as the extraction method for preparing this test solution.

[0162] Example 4

[0163] This example provides a method for determining the chemical components in a drug for treating vitiligo. The difference from Example 1 is only that the batch number of the Bailin tablets sample is S0001. The determination results are as Figure 14 shown.

[0164] Example 5

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

[0166] Example 6

[0167] This example provides a method for determining the chemical components in a drug for treating vitiligo. The difference from Example 4 is only that the column temperature is 30 °C. The determination results are as Figure 16 shown. Comparing Figure 1 with Figure 15 and 16 it can be seen that when the column temperature is 20 °C or 30 °C, various components can also be detected, but when the column temperature is 40 °C, the chromatographic separation effect is better.

[0168] Comparative Example 1

[0169] This example provides a method for determining the chemical components in a drug for treating vitiligo. The difference from Example 4 is only that the test wavelengths are 210 nm, 230 nm, 270 nm, or 300 nm. The test results of the test solution prepared in Example 1 at 210 nm, 230 nm, 270 nm, and 300 nm are successively as Figures 17 to 20 shown. Comparing Figure 1 with Figures 17 to 20 it can be seen that taking the paeoniflorin peak as a reference, Figure 1The peak area of paeoniflorin was 945.74494, which was significantly larger than that under other conditions. This indicates that the selection of a detection wavelength below 210 nm in the solution of the present invention has significant advantages.

[0170] Comparative Example 2

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

[0172] Comparative Example 3

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

[0174] Comparative Example 4

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

[0176] Comparison Figure 1 with Figures 21 to 23 It can be seen that when in the elution program, the volume ratio of phase A did not increase non-linearly from 0% to 60% within the first 21 min, in the measured chromatogram, the number of chromatographic peaks decreased significantly and the area of each signal peak also decreased significantly.

[0177] Comparative Example 5

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

[0179] Comparative Example 6

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

[0181] Comparative Example 7

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

[0183] Comparative Example 8

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

[0185] Comparison Figure 1 with Figures 24 to 27 It can be seen that the baseline in the acetonitrile - water chromatogram is relatively stable, and the separation and peak shape of each chromatographic peak are the best.

[0186] In summary, through the determination method of the present invention, good separation and identification of the chemical components in the drug for treating vitiligo can be achieved. At the same time, by utilizing the synergistic effect between the specific detection parameters of the present invention (especially the synergistic cooperation between parameters such as wavelength, solvent, and gradient), interference can be better eliminated, the results are more accurate and reliable, and it has good reproducibility and stability. Through the solution of the present invention, qualitative identification of at least 11 peaks can be achieved, and 5 of them can be identified. At the same time, quantitative analysis can be performed on at least 3 of the peaks. It can effectively determine the chemical components in the drug for treating vitiligo and construct a UPLC fingerprint of the drug for treating vitiligo, and can comprehensively evaluate the quality of the drug for treating vitiligo, making up for the deficiencies of its existing quality evaluation methods, and providing data support for the improvement of the quality standard of the drug for treating vitiligo.

[0187] The above has made a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various changes can also be made without departing from the gist of the present invention.

Claims

1. A method for determining the chemical components in a drug for treating vitiligo, characterized in that: The vitiligo treatment drug includes Bailing tablets, and the determination method includes the following steps: S1. Prepare a test solution and a reference solution: grind the Bailing tablets into powder and dissolve them in a solvent, extract them, and prepare a test solution; dissolve the paeoniflorin reference substance in a solvent to prepare a reference solution; wherein the solvent is a methanol-water mixed solution; S2. Using ultra-high performance liquid chromatography, obtain chromatograms of the test solution and the reference solution respectively under the same detection conditions, and perform qualitative and / or quantitative analysis on the chemical components in the chromatogram of the test solution; wherein the detection conditions include: The detector wavelength is 201±2nm; The chromatographic column is a C18 silica gel column; The elution conditions include: using acetonitrile as phase A and water as phase B, elution is performed through a gradient elution program, the total gradient time is not less than 24 minutes, the volume proportion of phase A in the first 21 minutes increases from 0% to 60% in a nonlinear gradient, and after 21 minutes, the volume proportion of phase A decreases from 60% to 0%.

2. The method for determining the chemical components in the vitiligo treatment drug according to claim 1, characterized in that: The gradient elution procedure 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%; From 15 to 18 minutes, the volume fraction of phase A increased from 20% to 40%; from 18 to 21 minutes, the volume fraction of phase A increased from 40% to 60%; from 21 to 24 minutes, the volume fraction of phase A decreased from 60% to 0%; from 24 to 30 minutes, the volume fraction of phase A remained at 0%.

3. The method for determining the chemical components in the vitiligo treatment drug according to claim 1, characterized in that: The detection conditions also include at least one of the following conditions: 1) the mobile phase flow rate is 0.3±0.05min / mL; 2) the column temperature is 40±2°C; 3) the injection volume is 2±1μL; 4) the chromatographic column is Acquity HSS T3; 5) The model specifications of the chromatographic column are as follows: diameter 2.1 mm; length 100 mm; average particle size of the filler is 1.8 μm; 6) The ultra-high performance liquid chromatography is implemented by an ultra-high performance liquid chromatograph, and the ultra-high performance liquid chromatograph is an Agilent LC1290 II high performance liquid chromatograph.

4. The method for determining the chemical components in the vitiligo treatment drug according to claim 1, characterized in that: The step S1 includes at least one of the following conditions: 1) the volume proportion of methanol in the methanol-water mixed solution is less than 50%; 2) the mass volume ratio of Bailing tablets to the methanol-water mixed solution is 1g: (50±5)mL; 3) the extraction method is heating reflux extraction.

5. The method for determining the chemical components in the vitiligo treatment drug according to claim 1, characterized in that: The step S1 includes at least one of the following conditions: 1) the volume proportion of methanol in the methanol-water mixed solution is 30±5%; 2) the extraction is performed by heating reflux method, and the heating reflux time during extraction is 30±10min.

6. The method for determining the chemical components in the vitiligo treatment drug according to claim 1, characterized in that: The qualitative analysis includes determining the chromatographic peak positions of other components according to the relative retention time of paeoniflorin, thereby performing qualitative analysis on the chemical components in the test solution; the quantitative analysis includes obtaining chromatographic peak graphs of paeoniflorin, cimicifuga glycoside or 5-O-methylvisamin glycoside at different concentrations, preparing a standard curve according to the relationship between the chromatographic peak area and the concentration, and performing quantitative analysis on the concentration of paeoniflorin, cimicifuga glycoside or 5-O-methylvisamin glycoside in the test solution according to the standard curve.

7. The method for determining the chemical components in the vitiligo treatment drug according to claim 6, characterized in that: The determination method further comprises the following steps: obtaining chromatograms of paeoniflorin reference substances with different concentrations, taking the peak area of ​​paeoniflorin as the ordinate y1 and the injection concentration of paeoniflorin as the abscissa x1, drawing a standard curve, and fitting the standard curve with a linear regression equation to obtain a linear regression equation of paeoniflorin of y1=13.688x1-39.134; and / or, the determination method further comprises the following steps: obtaining chromatograms of paeoniflorin reference substances with different concentrations, taking the peak area of ​​paeoniflorin as the ordinate y2 and the injection concentration of paeoniflorin as the abscissa x2, drawing a standard curve, and fitting the standard curve with a linear regression equation to obtain a linear regression equation of paeoniflorin of y1=13.688x1-39.

134. The linear regression equation is fitted to obtain the linear regression equation of the cimicifuga glycosides as y2=12.14796x2+0.28342; and / or, the determination method further comprises the following steps: obtaining the chromatograms of the 5-O-methylvisaminol glycoside reference substance with different concentrations, taking the peak area of ​​5-O-methylvisaminol glycoside as the ordinate y3, and the injection concentration of 5-O-methylvisaminol glycoside as the abscissa x3, drawing a standard curve, and fitting the standard curve with a linear regression equation to obtain the linear regression equation of the 5-O-methylvisaminol glycosides as y3=11.82116x3-0.04101.

8. A method for establishing a fingerprint spectrum of drugs for treating vitiligo, characterized in that: The steps include: The chromatographic data of the drug for treating vitiligo is obtained using the determination method as described in any one of claims 1 to 7, and the fingerprint of the drug for treating vitiligo is constructed using the chromatographic data.

9. The establishment method according to claim 8, characterized in that: The fingerprint spectrum contains the following characteristic fingerprint peaks: paeoniflorin, amygdalin, cimicifuga glycoside, ginsenoside Rg1 and 5-O-methylvisamidoside.

10. Application of the fingerprint establishment method according to any one of claims 8 to 9 in drug quality detection and control.

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