Characteristic chromatogram of platelet increasing capsule as well as construction method and application thereof

Abstract

The invention discloses a platelet increasing capsule characteristic chromatogram and a construction method and application thereof, and belongs to the technical field of quality control of traditional Chinese medicine compound preparations. According to the method, 13 control components including gallic acid, catechin, paeoniflorin, forsythiaside B, benzoic acid, astragalus smicus glycoside, forsythin, arctiin, liquiritigenin, benzoyl paeoniflorin, paeonol, arctigenin and indirubin are selected, a high performance liquid chromatography-mass spectrometry technology is applied, multiple batches of preparation samples are analyzed, and the content of the components in the preparation samples is determined. And establishing a characteristic spectrum of the chemical components. Compared with a traditional method, the technology has good precision and reproducibility, the data result is accurate and reliable, meanwhile, the technology can identify the 13 components at the same time under the same chromatographic condition, the analysis efficiency is remarkably improved, synchronous monitoring of multiple effective components is achieved, and the consistency and stability of the preparation quality can be guaranteed.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of quality control of traditional Chinese medicine compound preparation, and particularly relates to a characteristic spectrum of Shengxueplatelet Capsules and a construction method and application thereof. BACKGROUND

[0002] Shengxueplatelet Capsules (Guojiazhunzi Z20025029) is a compound preparation composed of five traditional Chinese medicines, namely, indigo, forsythia, henonitis, peony root and licorice, which plays a synergistic role in clearing heat and resolving toxins, cooling blood and stopping bleeding, dispersing blood stasis and removing spots, etc. through scientific compatibility. It is mainly used for treating primary thrombocytopenic purpura, improving systemic petechiae or ecchymosis, fever, polydipsia, short red urine, constipation, epistaxis or gingival bleeding, red tongue with yellow fur, and pulse slippery, rapid or stringy, etc.

[0003] At present, the quality control system of Shengxueplatelet Capsules is not perfect, and existing researches are mainly focused on clinical efficacy observation, and lack of in-depth exploration and effective monitoring of the internal material basis. Although the Chinese patent application with the publication number CN119688899A provides a preliminary basis for the quality control of Shengxueplatelet Capsules, the method has obvious limitations: on the one hand, the detection time of the method is long, and the long running time cannot meet the demand of rapid and real-time quality monitoring of a large number of samples in modern drug production process, which increases the time and labor cost; on the other hand, the number of characteristic peaks calibrated by the method is limited, and the method cannot fully cover the numerous known active ingredients in the traditional Chinese medicine compound, and it is difficult to fully reflect the complex chemical composition of the preparation. Therefore, a new type of characteristic spectrum with lower cost and higher detection efficiency is needed. SUMMARY

[0004] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a characteristic spectrum of Shengxueplatelet Capsules and a construction method and application thereof, so as to solve the technical problem that the existing quality control method of Shengxueplatelet Capsules is high in cost and difficult to fully reflect the complex chemical composition of the preparation.

[0005] In order to achieve the above-mentioned purpose, the following technical solutions are adopted in the present application: In a first aspect, the present application discloses a construction method of a characteristic spectrum of Shengxueplatelet Capsules, comprising the following steps: S1, preparing a test solution: different batches of Shengxueplatelet Capsules fine powder are weighed, and pure methanol solution is added for ultrasonic extraction to obtain a test solution; S2, preparing a single reference solution with gallic acid, catechin, paeoniflorin, forsythia ester glycoside B, benzoic acid, astragalin, forsythia glycoside, thistle glycoside, licorice, benzoyl paeoniflorin, peony phenol, thistle glycoside and indigoferin as reference substances; S3. Perform chromatographic analysis on the test solution obtained in S1 and the reference solution in S2, and record the corresponding chromatograms; S4. Perform similarity analysis on the chromatograms obtained in S3 to confirm the reliability of the results; S5. Perform high-resolution mass spectrometry analysis on the test solution to obtain the total ion chromatogram; based on the total ion chromatogram and the mass spectrometry results of the chemical components, and combined with the chromatogram of the single reference solution obtained in S3, determine the chemical components of each peak in the characteristic spectrum to obtain the characteristic spectrum of the platelet-raising capsule.

[0006] Preferably, in S1, ultrasonic extraction is performed for 45 minutes.

[0007] Preferably, in S1, the ratio of platelet-enhancing capsule powder to pure methanol solution is 0.04 g / mL.

[0008] Preferably, the preparation method of the platelet-raising capsule test solution is as follows: weigh 1.0 g of fine powder of platelet-raising capsules from different batches, add 25 mL of pure methanol solution, weigh the solution, sonicate for 45 min, cool, weigh the solution, replenish the lost weight with pure methanol, shake well, filter, and obtain the platelet-raising capsule test solution.

[0009] Preferably, in S2, the preparation method of the reference solution is as follows: each reference standard is dissolved in pure methanol to prepare a single reference solution containing 38 μg gallic acid, 28 μg catechin, 30 μg paeoniflorin, 38 μg forsythoside B, 25 μg benzoic acid, 54 μg astragaloside, 29 μg forsythoside, 49 μg arctiin, 33 μg glycyrrhizin, 25 μg benzoyl paeoniflorin, 60 μg paeonol, 30 μg arctigenin, and 69 μg indirubin per 1 mL.

[0010] Preferably, in S3, the chromatographic conditions are as follows: column type Hedera ODS-2-C18, 4.6×250 mm×5 µm; mobile phase is acetonitrile-0.05% phosphoric acid aqueous solution; UV-Vis absorption detector is used, detection wavelength is 205 nm; flow rate: 1.0 mL / min; injection volume: 10 μL; column temperature: 30℃.

[0011] Preferably, in S5, the high-resolution mass spectrometry detection conditions are: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300℃, auxiliary gas temperature 300℃, full scan mode, and mass-to-charge ratio scan range m / z of 100. 1500.

[0012] Preferably, in step S5, based on the total ion chromatogram and the mass spectrometry results of the chemical components, combined with the reference chromatogram obtained in step S3, the chemical components of each peak in the chromatogram of the test sample are determined as follows: Peak 1 is gallic acid, retention time 7.880 min; Peak 5 is catechin, retention time 18.640 min; Peak 9 is paeoniflorin, retention time 27.470 min; Peak 13 is forsythoside B, retention time 34.620 min; Peak 16 is benzoic acid, retention time 40.483 min; Peak 19 is astragaloside, retention time 44.697 min; Peak 23 is forsythoside, retention time 51.937 min; Peak 24 is arctiin, retention time 52.770 min; Peak 26 is glycyrrhizin, retention time 54.933 min; Peak 28 is benzoylpaeoniflorin, retention time 59.007 min. Peak 31 is paeonol, retention time 64.353 min; peak 32 is arctigenin, retention time 65.727 min; peak 33 is indirubin, retention time 75.270 min.

[0013] Preferably, after obtaining the characteristic spectrum of the platelet-raising capsules, an attribution analysis is performed on the characteristic spectrum results of the platelet-raising capsules. Glycyrrhizin is derived from licorice; paeoniflorin, paeonol, benzoic acid, and benzoylpaeoniflorin are derived from peony bark; indigo is derived from indigo naturalis; forsythoside, forsythoside B, astragaloside, arctiin, and arctigenin are derived from forsythia; and gallic acid and catechin are derived from peony bark and agrimony.

[0014] In a second aspect, the present invention discloses the characteristic map of platelet-enhancing capsules obtained by the above-described construction method.

[0015] A third aspect of the present invention discloses the application of the above-described construction method in constructing quality standards for platelet-boosting capsules or in detecting drugs with the same name and formula as platelet-boosting capsules.

[0016] Preferably, the quality of the platelet-raising capsule or its equivalent drug is detected using the aforementioned characteristic spectrum of platelet-raising capsule, with one or more of the following as quality markers: gallic acid, catechin, paeoniflorin, forsythoside B, benzoic acid, astragaloside, forsythoside, arctiin, glycyrrhizin, benzoylpaeoniflorin, paeonol, arctigenin, and indirubin.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The method for constructing the characteristic chromatogram of platelet-raising capsules provided by this invention can accurately, clearly, and objectively assess the quality of platelet-raising capsules, and has significant practical value for effectively controlling the quality of platelet-raising capsules and ensuring clinical efficacy. By systematically optimizing chromatographic conditions, analytical efficiency is greatly improved, effectively overcoming the bottleneck of long running time in existing methods, and significantly reducing the time and labor costs of quality control. Simultaneously, by identifying more characteristic peaks and successfully identifying multiple active ingredients related to efficacy using high-resolution mass spectrometry, a "multi-component synchronous monitoring" system containing more characteristic peaks and richer information can be constructed, achieving comprehensive coverage of the chemical composition of the formulation. This allows for a more scientific and accurate assessment of the uniformity and stability of quality between different batches of products, ensuring that its chemical composition is consistent with clinically validated effective batches, and controlling the overall quality evaluation. The characteristic chromatogram of platelet-raising capsules established by this method identified 33 common peaks, of which 13 characteristic peaks were clearly identified, significantly improving the resolution of the chromatogram. This characteristic chromatogram has significant specificity and can comprehensively reflect the chemical composition of the formulation. The chromatograms obtained by this invention exhibit stable baselines and good peak shapes, providing technical support for improving the quality standards of the formulation. By examining the stability, repeatability, and precision of retention time and peak area, the results show that the RSD values ​​are all below 3%, confirming the good reproducibility and reliable data of the method. Furthermore, based on systematic analysis of multiple batches of samples, it is demonstrated that the quality evaluation system established by this invention is more comprehensive and objective, capable of accurately assessing the quality uniformity and stability between different batches of products, ensuring that the chemical composition of each batch of products remains highly consistent with clinically effective batches, thereby guaranteeing the reliability of clinical efficacy and medication safety from the source. This technological breakthrough not only lays a solid foundation for improving the quality standards of platelet-raising capsules but also provides a referable technical path for the modernization of quality control of traditional Chinese medicine, which is of positive significance for promoting high-quality development of the industry. This invention constructs a characteristic chromatographic quality evaluation system for platelet-raising capsules, which can systematically analyze the types and contents of the main chemical components contained in the formulation, providing a rapid, accurate, and comprehensive detection method for the quality assessment of traditional Chinese medicine compound preparations. This method is simple to operate, has good stability, and possesses high precision and reproducibility, making it suitable for the quality control of platelet-raising capsules.

[0018] Furthermore, by systematically optimizing chromatographic analysis conditions, including key parameters such as extraction solvent selection, extraction method evaluation, mobile phase composition, column temperature control, and flow rate adjustment, an octadecylsilane-bonded silica column was employed with gradient elution using acetonitrile-phosphoric acid aqueous solution as the mobile phase. This method significantly improved the separation efficiency of various active ingredients, enabling the characteristic chromatograms to present richer peak information. Combined with high-resolution mass spectrometry, 13 characteristic chemical components were successfully identified, achieving comprehensive quality control of the platelet-enhancing capsules. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0020] Figure 1 The chromatogram (A) and 3D image (B) are obtained in Example 1 during the preparation of the platelet-raising capsule test solution of the present invention. Figure 2 This is a chromatogram obtained by optimizing the mobile phase composition under chromatographic conditions according to the present invention; Figure 3 This is a chromatogram obtained by optimizing the extraction method during the preparation of the test sample solution according to the present invention; Figure 4 This is a chromatogram obtained by optimizing the extraction solvent during the preparation of the test sample solution according to the present invention; Figure 5 The chromatograms obtained by optimizing different concentrations of the extraction solvent under chromatographic conditions according to the present invention are shown below. Figure 6 This is a chromatogram obtained by optimizing column temperature under chromatographic conditions according to the present invention; Figure 7 This is a chromatogram obtained by optimizing the flow rate under chromatographic conditions according to the present invention; Figure 8 This is a chromatogram obtained by optimizing the feed-to-liquid ratio under chromatographic conditions according to the present invention; Figure 9 This is a chromatogram obtained by optimizing the elution procedure under chromatographic conditions according to the present invention; Figure 10 This is the total ion chromatogram of negative ions for the platelet-boosting capsules of this invention. Figure 11 This is the total positive ion chromatogram of the platelet-boosting capsules of the present invention. Figure 12 The images show the chromatogram (A), 3D image (B), and UV spectrum (C) of the gallic acid standard of this invention. Figure 13 The chromatogram (A), 3D image (B), and UV spectrum (C) of the catechin standard of the present invention are shown. Figure 14 The chromatogram (A), 3D image (B), and UV spectrum (C) of paeoniflorin standard of the present invention are shown. Figure 15 The chromatogram (A), 3D chromatogram (B), and UV spectrum (C) of forsythoside B standard of the present invention are shown. Figure 16The chromatogram (A), 3D image (B), and UV spectrum (C) of the benzoic acid standard of this invention are shown. Figure 17 The chromatogram (A), 3D chromatogram (B), and UV spectrum (C) of astragalin standard of the present invention are shown. Figure 18 The chromatogram (A), 3D image (B), and UV spectrum (C) of the forsythoside standard of the present invention are shown. Figure 19 The chromatogram (A), 3D image (B), and UV spectrum (C) of arctiin standard are shown in the figures for this invention. Figure 20 The chromatogram (A), 3D image (B), and UV spectrum (C) of glycyrrhizin standard are shown in the present invention. Figure 21 The chromatogram (A), 3D image (B), and UV spectrum (C) of benzoylpaeoniflorin standard are shown in the figure. Figure 22 The chromatogram (A), 3D chromatogram (B), and UV spectrum (C) of the paeonol standard of this invention are shown. Figure 23 The chromatogram (A), 3D image (B), and UV spectrum (C) of arctigenin standard are shown in the figures for this invention. Figure 24 The chromatogram (A), 3D image (B), and UV spectrum (C) of the indirubin standard of this invention are shown. Figure 25 This is the gallic acid mass spectrum of the present invention; Figure 26 This is a catechin mass spectrum of the present invention; Figure 27 This is the mass spectrum of astragaloside in this invention; Figure 28 This is a spectral image of licorice root from the present invention; Figure 29 This is the paeonol mass spectrum of the present invention; Figure 30 This is the mass spectrum of indigo red in this invention; Figure 31 The characteristic chromatograms of 15 batches of platelet-boosting capsules tested according to this invention are shown. Detailed Implementation

[0021] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0022] This invention provides a method for constructing a characteristic map of platelet-boosting capsules, comprising the following steps: S1. Preparation of test solution: Take 1.0 g of fine powder from different batches of platelet-raising capsules, place them in a stoppered conical flask, add 25 mL of pure methanol solution, stopper tightly, weigh, sonicate for 45 min, cool, weigh, replenish the lost weight with pure methanol, shake well, filter, and filter the filtrate through a 0.45 µm microporous membrane to obtain the platelet-raising capsule test solution.

[0023] S2. Preparation of single reference solutions: Accurately weigh each reference standard and place it in a stoppered conical flask. Add pure methanol solution to prepare a single reference solution containing 38 μg gallic acid, 28 μg catechin, 30 μg paeoniflorin, 38 μg forsythoside B, 25 μg benzoic acid, 54 μg astragaloside, 29 μg forsythoside, 49 μg arctiin, 33 μg glycyrrhizin, 25 μg benzoyl paeoniflorin, 60 μg paeonol, 30 μg arctigenin, and 69 μg indirubin per 1 mL.

[0024] S3. Inject the test solution obtained in S1 and the reference solution obtained in S2 into a high-performance liquid chromatograph (HPLC) for chromatographic analysis and record the corresponding chromatograms. The HPLC conditions are as follows: column: Hedera ODS-2-C18 (4.6 × 250 mm × 5 µm) column; detector: UV-Vis detector, detection wavelength: 205 nm; flow rate: 1.0 mL / min; injection volume: 10 μL; column temperature: 30℃; mobile phase: acetonitrile-0.05% phosphoric acid aqueous solution, gradient elution. S4. Import the chromatograms obtained in S3 into the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine. Select the chromatographic peaks that are present in the chromatograms of different batches of platelet-raising capsules as common peaks. Perform similarity analysis on the chromatograms of the test solution after data import, multi-point correction and data matching to confirm the reliability of the results. S5. Perform high-resolution mass spectrometry analysis on the above test solution to obtain the total ion chromatogram; based on the total ion chromatogram and the mass spectrometry results of the chemical components, import the detection data into Xcalibur software, enter the Qual Browser interface, and determine the chemical components of each peak in the chromatogram of the test sample by combining the total ion chromatogram and the high-resolution mass spectrometry results of the chemical components with the chromatogram of the single reference standard, and obtain the characteristic chromatogram of the platelet-raising capsule.

[0025] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading this description, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0026] The instruments used in the following examples are shown in Table 1. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. The 15 batches of platelet-boosting capsules used in the following examples were all purchased commercially and supplied by Shaanxi Haoqijun Pharmaceutical Co., Ltd.; the reference standards used were: gallic acid (batch number: P1426143, purity: 98%) purchased from Shanghai Titan Technology Co., Ltd.; catechin (batch number: 877-200001, purity: 98%) and paeonol (batch number: 110708-200505, purity: 98%) purchased from the National Institutes for Food and Drug Control; paeoniflorin (batch number: 20030203, purity: 98.11%) purchased from Chengdu Pufeide Biotechnology Co., Ltd.; forsythoside B (batch number: AF20052702, purity: 98%) and glycyrrhizin (batch number: AFBH3102, purity: 98%) purchased from Chengdu Efa. The following reagents were purchased from Chengdu Pusi Biotechnology Co., Ltd.: Benzoic acid (batch number: PS012647, purity: 98%), astragaloside (batch number: PS010127, purity: 98%), forsythoside (batch number: PS000579, purity: 98%), arctiin (batch number: DSTDN000201, purity: 98%), benzoylpaeoniflorin (batch number: PS000157, purity: 98%), arctigenin (batch number: PS010438, purity: 98%), and indirubin (batch number: AF21021205, purity: 97%). The reagents used are shown in Table 2. Unless otherwise specified, all other raw materials used were conventional commercially available products with specifications conforming to the general practice in this field.

[0027] Table 1 Instruments

[0028] Table 2 Drugs and Reagents

[0029] Example 1 The method for constructing the characteristic map of platelet-boosting capsules includes the following steps: S1. Preparation of the test solution: Accurately weigh 1.0 g of 15 batches of platelet-enhancing capsule powder, place it in a stoppered conical flask, add 25 mL of pure methanol solution, seal tightly, weigh, sonicate for 45 min, cool, weigh again, replenish the lost weight with pure methanol, shake well, filter, and filter the filtrate through a 0.45 µm microporous membrane to obtain the platelet-enhancing capsule test solution.

[0030] S2. Preparation of single reference solutions: Accurately weigh each reference standard and place it in a stoppered conical flask. Add pure methanol solution to prepare a single reference solution containing 38 μg gallic acid, 28 μg catechin, 30 μg paeoniflorin, 38 μg forsythoside B, 25 μg benzoic acid, 54 μg astragaloside, 29 μg forsythoside, 49 μg arctiin, 33 μg glycyrrhizin, 25 μg benzoyl paeoniflorin, 60 μg paeonol, 30 μg arctigenin, and 69 μg indirubin per 1 mL.

[0031] S3. Accurately pipette 10 µL each of the test solution obtained in S1 and the reference solution in S2, and inject them into the high-performance liquid chromatograph for chromatographic analysis. Record the corresponding chromatograms. Figure 1 , Figures 12-24 ).

[0032] The liquid chromatography conditions were as follows: column: Hedera ODS-2-C18 (4.6 × 250 mm × 5 µm) column; detector: UV-Vis absorption detector, detection wavelength: 205 nm; flow rate: 1.0 mL / min; injection volume: 10 μL; column temperature: 30℃; mobile phase: acetonitrile-0.05% phosphoric acid aqueous solution, gradient elution, the elution program is shown in Table 3. Table 3 Elution Procedure 5

[0033] S4. Import the chromatograms obtained in S3 into the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine. Select the chromatographic peaks that are present in the chromatograms of different batches of platelet-raising capsules as common peaks. Perform similarity analysis on the chromatograms of the test solution after data import, multi-point correction and data matching to confirm the reliability of the results (Table 4).

[0034] Table 4. Similarity between different batches of platelet-enhancing capsules and common chromatographic peak patterns.

[0035] S5. To determine the chemical composition in the characteristic spectrum, the above-mentioned test solution was subjected to high-resolution mass spectrometry analysis. The high-resolution mass spectrometry detection conditions were: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300℃, auxiliary gas temperature 300℃, full scan mode, and mass-to-charge ratio scan range m / z of 100. 1500, import the detection data into Xcalibur software, enter the Qual Browser interface, and perform data analysis based on the peak distribution of chemical components to obtain the total ion chromatogram ( Figure 10 and 11 Based on the obtained total ion chromatogram and the peak elution of the test sample obtained in S3, data analysis was performed to obtain high-resolution mass spectrometry results of each chemical component. Figures 25-30 ); S6. Based on the total ion chromatogram and the high-resolution mass spectrometry results of the chemical components, combined with the reference standard chromatogram obtained in S3, determine the chemical components of each peak in the chromatogram of the test sample, and obtain the characteristic chromatogram of the platelet-raising capsules. Figure 31 Among them, peak 1 is gallic acid, retention time 7.880 min; peak 5 is catechin, retention time 18.640 min; peak 9 is paeoniflorin, retention time 27.470 min; peak 13 is forsythoside B, retention time 34.620 min; peak 16 is benzoic acid, retention time 40.483 min; peak 19 is astragaloside, retention time 44.697 min; peak 23 is forsythoside, retention time 51.937 min; peak 24 is arctiin, retention time 52.770 min; peak 26 is glycyrrhizin, retention time 54.933 min; peak 28 is benzoylpaeoniflorin, retention time 59.007 min; peak 31 is paeonol, retention time 64.353 min; peak 32 is arctigenin, retention time 65.727 min. Peak 33 was indirubin, with a retention time of 75.270 min. Attribution analysis of the chemical components in the platelet-raising capsules showed that glycyrrhizin was derived from glycyrrhiza uralensis; paeoniflorin, paeonol, benzoic acid, and benzoylpaeoniflorin were derived from Paeonia suffruticosa; indirubin was derived from indigo naturalis; forsythoside, forsythoside B, astragaloside, arctiin, and arctigenin were derived from Forsythia suspensa; and gallic acid and catechins were derived from Paeonia suffruticosa and Agrimonia pilosa.

[0036] S7. Methodological Investigation 1. Precision test The test solution obtained in S1 was injected 6 times in parallel under the chromatographic conditions in S3, with an injection volume of 10 μL. The retention time and peak area were analyzed and the RSD value was calculated. The results showed that the RSD values ​​of retention time and peak area were both less than 3%, indicating that the instrument has good precision.

[0037] Table 5 Peak area and retention time in precision studies

[0038] 2. Stability test The test solution obtained in S1 was taken and analyzed at 0 h, 2 h, 4 h, 8 h, 12 h and 24 h according to the chromatographic conditions in S3. The injection volume was 10 μL. The retention time and peak area of ​​the common peaks in the HPLC characteristic chromatogram of the sample were analyzed and the RSD value was calculated. The results showed that the RSD values ​​of retention time and peak area were both less than 3%, indicating that the platelet-raising capsule test solution had good stability within 24 h.

[0039] Table 6 Peak area and retention time in stability studies

[0040] 3. Repeatability test Six portions of platelet-boosting capsule test solution were taken and injected for analysis according to the chromatographic conditions in S3. The retention time and peak area of ​​the common peaks in the HPLC characteristic chromatograms of the samples were analyzed and the RSD value was calculated. The results showed that the RSD values ​​of retention time and peak area were both less than 3%, indicating that the method had good repeatability.

[0041] Table 7 Peak area and retention time in repeatability studies

[0042] Example 2 The purpose of this embodiment is to investigate the effect of different mobile phases (acetonitrile-0.1% phosphoric acid, acetonitrile-water, methanol-water, acetonitrile-0.1% acetic acid, methanol-0.1% acetic acid, acetonitrile-0.03% phosphoric acid, methanol-0.05% phosphoric acid, acetonitrile-0.05% phosphoric acid) on the detection of platelet-raising capsule test solution. The test solution was prepared using the method in Example 1. The detection results are as follows: Figure 2 As shown. (Through) Figure 2 It can be seen that when the mobile phase is acetonitrile-0.05% phosphoric acid, the number of peaks is greater and the peak shape and separation effect are better.

[0043] Example 3 The purpose of this embodiment is to investigate the effects of different extraction methods (ultrasound for 30 min, ultrasound for 45 min, ultrasound for 60 min, reflux, and maceration) on the detection of platelet-raising capsule test solutions. The chromatographic conditions were the same as in Example 1, and the preparation conditions for all other test solutions were the same as in Example 1. The detection results are as follows: Figure 3 Shown. Through Figure 3It is known that the number of peaks and response values ​​are not significantly different when using ultrasonic and reflux extraction methods. However, ultrasonic extraction is more cost-effective and efficient than reflux extraction. Therefore, ultrasonic extraction is chosen as the extraction method. Figure 3 It can be seen that the peak response value and peak shape of ultrasound detection results after 45 min are better than those after 30 min and 60 min. Therefore, ultrasound for 45 min is selected as the best extraction method.

[0044] Example 4 The purpose of this embodiment is to investigate the effect of different extraction solvents (anhydrous ethanol, acetonitrile, methanol, water, 0.05% phosphoric acid) on the detection of platelet-raising capsule test solutions. The chromatographic conditions are the same as in Example 1, and the preparation conditions of the remaining test solutions are also the same as in Example 1. The detection results are as follows: Figure 4 As shown. (Through) Figure 4 It is known that methanol is the extraction solvent, which produces the highest content of components, the best peak shape, and the highest separation in the chromatogram. Therefore, methanol is chosen as the extraction solvent.

[0045] Example 5 The purpose of this embodiment is to investigate the effect of different concentrations of the optimal extraction solvent (30% methanol, 50% methanol, 80% methanol, and pure methanol) on the detection of platelet-enhancing capsule test solution. The test solution was prepared using the method of Example 1, and all other chromatographic conditions were the same as in Example 1. The detection results are as follows: Figure 5 As shown. (Through) Figure 5 It can be seen that the chromatogram contains the most comprehensive information and has the most peaks when the extraction solvent is pure methanol. Therefore, pure methanol is selected as the best extraction solvent.

[0046] Example 6 The purpose of this embodiment is to investigate the effect of different column temperatures (25℃, 30℃, 35℃) on the detection of platelet-enhancing capsule test solution. The test solution was prepared using the method of Example 1, and all other chromatographic conditions were the same as in Example 1. The detection results are as follows: Figure 6 As shown. (Through) Figure 6 It can be seen that the separation effect of each component is better and more peaks are produced when the column temperature is 30℃. Therefore, 30℃ is selected as the column temperature condition.

[0047] Example 7 The purpose of this embodiment is to investigate the effect of different flow rates (0.6 mL / min, 0.8 mL / min, 1.0 mL / min) on the detection of platelet-enhancing capsule test solution. The test solution was prepared using the method of Example 1, and all other chromatographic conditions were the same as in Example 1. The detection results are as follows: Figure 7 As shown. (Through) Figure 7It can be seen that the peak elution is optimal and the separation of each component is better when the flow rate is kept at 1.0 mL / min. Therefore, the flow rate of 1.0 mL / min was finally selected.

[0048] Example 8 The purpose of this embodiment is to investigate the effect of different solid-liquid ratios (0.04 g / mL, 0.06 g / mL, 0.08 g / mL, 0.10 g / mL, and 0.12 g / mL) on the detection of platelet-raising capsule test solution. The test solution was prepared using the method in Example 1, and all other chromatographic conditions were the same as in Example 1. The detection results are as follows: Figure 8 As shown. (Through) Figure 8 As shown, the baseline was most stable and the separation effect of each component was better when the material-to-liquid ratio was 0.04 g / mL. Therefore, the material-to-liquid ratio of 0.04 g / mL was finally selected.

[0049] Example 9 The purpose of this embodiment is to investigate the effect of different elution programs on the detection of platelet-raising capsule test solution. The test solution was prepared using the method of Example 1, and elution programs were set for each solution. All other chromatographic conditions were the same as in Example 1. Some elution programs are shown in Tables 3 and 8-11. Table 8 Elution Procedure 1

[0050] Table 9 Washing Procedure 2

[0051] Table 10 Elution Procedure 3

[0052] Table 11 Elution Procedure 4

[0053] Test results as follows Figure 9 As shown. (Through) Figure 9 It can be seen that elution program 5 has good separation, high peak height and complete chromatographic information. Therefore, elution program 5 is selected as the optimal elution program.

[0054] The above experimental results show that the method for constructing the characteristic chromatogram of platelet-raising capsules by high performance liquid chromatography combined with high resolution mass spectrometry provided by the present invention has the characteristics of good stability, high precision and good repeatability, and can comprehensively and objectively evaluate the quality of platelet-raising capsules, providing quality assurance for clinical efficacy.

[0055] The above description is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made in accordance with the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for constructing a characteristic map of platelet-raising capsules, characterized by, It comprises the following steps: S1, preparing a test sample solution: different batches of Shengxuetong capsules are weighed, and then dissolved in pure methanol solution, and ultrasonic extraction is performed to obtain the test sample solution; S2, using gallic acid, catechin, paeonol, forsythoside B, benzoic acid, astrin, forsythoside, arctiin, glycyrrhizin, benzoylpaeonol, paeonol, arctiinogen, and indigo naturalis as reference substances, single reference substance solutions are prepared; S3, performing chromatographic analysis on the test sample solution obtained in S1 and the single reference substance solution in S2, and recording the corresponding chromatograms; S4, performing similarity analysis on the chromatograms obtained in S3 to confirm the reliability of the results; S5, performing high-resolution mass spectrometry analysis on the test sample solution to obtain a total ion current chromatogram; According to the total ion current chromatogram and the mass spectrometry result chart of the chemical components, combined with the single reference substance solution chromatogram obtained in S3, the chemical components of each peak in the characteristic chromatogram are determined, and the characteristic chromatogram of Shengxuetong capsules is obtained.

2. The method of claim 1, wherein the platelet-raising capsule feature map is constructed by: In S1, the ultrasonic extraction is performed for 45 min.

3. The method of claim 1, wherein the platelet-raising capsule feature map is constructed by: In S1, the ratio of Shengxuetong capsule powder to pure methanol solution is 0.04 g / mL.

4. The method of claim 1, wherein the platelet-raising capsule feature map is constructed by: In S3, the chromatographic conditions are as follows: the chromatographic column type is Hedera ODS-2-C18, 4.6*250 mm*5 μm; the mobile phase is acetonitrile-0.05% phosphoric acid aqueous solution; the ultraviolet-visible absorption detector is used, the detection wavelength is 205 nm; the flow rate is 1.0 mL / min; the injection volume is 10 μL; the column temperature is 30℃; the gradient elution program is as follows: 。 5. The method of claim 1, wherein the platelet-raising capsule feature map is constructed by: In S5, the high resolution mass spectrometry detection conditions are: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300 °C, auxiliary gas temperature 300 °C, and the scan mode is full scan mode, and the mass-to-charge ratio scan range m / z is 100 1500.

6. The method of claim 1, wherein the platelet-raising capsule feature map is constructed by: In S5, according to the total ion current chromatogram and the mass spectrometry result chart of the chemical components, combined with the reference substance chromatogram obtained in S3, the chemical components of each peak in the test sample chromatogram are determined, which are as follows: peak No. 1 is gallic acid, the retention time is 7.880 min; peak No. 5 is catechin, the retention time is 18.640 min; peak No. 9 is paeonol, the retention time is 27.470 min; peak No. 13 is forsythoside B, the retention time is 34.620 min; peak No. 16 is benzoic acid, the retention time is 40.483 min; peak No. 19 is astrin, the retention time is 44.697 min; peak No. 23 is forsythoside, the retention time is 51.937 min; peak No. 24 is arctiin, the retention time is 52.770 min; peak No. 26 is glycyrrhizin, the retention time is 54.933 min; peak No. 28 is benzoylpaeonol, the retention time is 59.007 min; peak No. 31 is paeonol, the retention time is 64.353 min; peak No. 32 is arctiinogen, the retention time is 65.727 min; peak No. 33 is indigo naturalis, the retention time is 75.270 min.

7. The method of claim 1, wherein the platelet-raising capsule feature map is constructed by: After obtaining the characteristic chromatogram of Shengxuetong capsules, attribute analysis is performed on the characteristic chromatogram results of Shengxuetong capsules, glycyrrhizin is derived from licorice, paeonol, benzoic acid, benzoylpaeonol are derived from peony root, indigo naturalis is derived from indigo, forsythoside, forsythoside B, astrin, arctiin, arctiinogen are derived from forsythia, gallic acid, catechin are derived from peony root and agrimonia.

8. The characteristic spectrum of the platelet-raising capsule obtained by the construction method of any one of claims 1-7.

9. A method of detecting the quality of a thrombocyte-raising capsule, characterized in that The quality of the platelet-raising capsule or its homonymic and homopharmic drugs is detected by the HPLC characteristic spectrum of the platelet-raising capsule of claim 8, taking one or more of gallic acid, catechin, paeoniflorin, forsythoside B, benzoic acid, astragalin, forsythoside, arctiin, glycyrrhizin, benzoylpaeoniflorin, paeonol, arctiinogen, and indigo naturalis as quality markers.

10. The method of claim 9, wherein the platelet raising capsule is a capsule containing a mixture of a plurality of ingredients, and the method comprises detecting the quality of the capsule by detecting the presence or absence of the mixture of the plurality of ingredients in the capsule. The substance basis of the platelet-raising capsule or its homonymic and homopharmic drugs is detected for the components gallic acid, catechin, paeoniflorin, forsythoside B, benzoic acid, astragalin, forsythoside, arctiin, glycyrrhizin, benzoylpaeoniflorin, paeonol, arctiinogen, and indigo naturalis.

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