A method for constructing UHPLC fingerprint of a traditional Chinese medicine composition for resolving dampness and detoxification and a quality control method thereof

Abstract

The application discloses a kind of Huo Shi Jie Du traditional Chinese medicine composition UHPLC fingerprint and its quality control method, which utilizes the characteristic spectrum of traditional Chinese medicine composition sample by ultra-high performance liquid chromatograph, acetonitrile and 0.1% phosphoric acid aqueous solution are selected as mobile phase when detecting, gradient elution is adopted, various material compositions in sample can be accurately separated, 25 obvious characteristic peaks are obtained, and the overall quality level of traditional Chinese medicine composition can be effectively characterized by the provision of relative retention time.By comparing the characteristic spectrum of the extract, concentrated liquid, dry paste and finished product granule sample of traditional Chinese medicine composition in preparation process of different batches, whether traditional Chinese medicine composition changes is determined, so as to control the quality of traditional Chinese medicine composition in preparation process, which is beneficial to realize the multi-component monitoring of traditional Chinese medicine composition quality.

Description

Technical Field

[0001] This invention belongs to the field of drug detection and analysis technology, specifically relating to a method for constructing a UHPLC fingerprint spectrum of a traditional Chinese medicine composition for resolving dampness and detoxifying, and its quality control method. Background Technology

[0002] This herbal composition for resolving dampness and detoxifying is derived from Yu Ping Feng San (Jade Screen Powder). The formula consists of Astragalus membranaceus, stir-fried Atractylodes macrocephala, Saposhnikovia divaricata, Dryopteris crassirhizoma, Lonicera japonica, Citrus reticulata peel, and Eupatorium fortunei. It has the effects of tonifying Qi and strengthening the exterior, resolving dampness and detoxifying. It is mainly used for syndromes of exterior deficiency and weakened defensive Yang with damp-heat. Symptoms include mild aversion to wind, fatigue, limb pain or throat discomfort, poor appetite, pale red or red tongue, and a thin white or greasy white coating.

[0003] Traditional Chinese medicine (TCM) has a complex composition, and a single component is insufficient to reflect its overall quality. TCM fingerprinting, a technology that has emerged in recent years, allows for comprehensive analysis of multiple components within TCM, effectively characterizing its overall quality level.

[0004] UHPLC fingerprinting is a more advanced chromatographic analysis method and an upgrade from HPLC technology. Its advantages in analyzing the chemical components of traditional Chinese medicine include high sensitivity, high efficiency, and a high degree of automation. The application of UHPLC fingerprinting can further improve the quality control level of traditional Chinese medicine. Summary of the Invention

[0005] This invention provides a method for constructing a UHPLC fingerprint spectrum of a traditional Chinese medicine composition for resolving dampness and detoxifying, and a method for quality control thereof. It can effectively separate the main chemical components of a traditional Chinese medicine composition for resolving dampness and detoxifying using an ultra-high performance liquid chromatograph and obtain its characteristic spectrum. By identifying reference standards and specifying relative retention times, the purpose of quality control can be achieved.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for constructing a UHPLC fingerprint of a traditional Chinese medicine composition for resolving dampness and detoxifying, which includes detecting the fingerprint of a sample of the traditional Chinese medicine composition for resolving dampness and detoxifying using an ultra-high performance liquid chromatograph.

[0008] The herbal composition for resolving dampness and detoxifying is made from raw materials including Astragalus membranaceus, stir-fried Atractylodes macrocephala, Saposhnikovia divaricata, Lonicera japonica, Eupatorium fortunei, Dryopteris crassirhizoma, and Citrus reticulata.

[0009] In the above technical solution, this application uses ultra-high performance liquid chromatography (UHPLC) to detect the fingerprint spectrum of the dampness-resolving and detoxifying traditional Chinese medicine composition. This composition is prepared from raw materials including Astragalus membranaceus, stir-fried Atractylodes macrocephala, Saposhnikovia divaricata, Lonicera japonica, Eupatorium fortunei, Dryopteris crassirhizoma, and Citrus reticulata peel. This composition contains various effective substances. This application uses acetonitrile and a 0.1% phosphoric acid aqueous solution as the mobile phase for gradient elution, which can accurately separate various substances in the dampness-resolving and detoxifying traditional Chinese medicine composition sample, obtaining a fingerprint spectrum with distinct and complete characteristic peaks. Twenty-five common peaks are identified, and by comparison with reference standards, six chromatographic peaks are identified: neochlorogenic acid (peak 3), chlorogenic acid (peak 6), cryptochlorogenic acid (peak 8), 3,5-O-dicaffeoylquinic acid (peak 21), hesperidin (peak 22), and 5-O-dicaffeoylquinic acid (peak 23). The relative retention time ranges of other characteristic peaks are specified using the chlorogenic acid peak as a reference.

[0010] In the above technical solution, the chromatographic column used for detection is a C18 water-resistant small-particle-size column. This column facilitates the accurate and complete separation of various substances in the detoxifying and dampness-resolving traditional Chinese medicine composition using ultra-high performance liquid chromatography (UHPLC), and displays them in the chromatogram.

[0011] In the above technical solution, the column temperature of the chromatographic column during detection is 25℃~35℃. This column temperature is beneficial for the ultra-high performance liquid chromatography (UHPLC) to accurately and completely separate the key components of the detoxifying and dampness-resolving traditional Chinese medicine composition and display them in the chromatogram.

[0012] In the above technical solution, the ultraviolet detector is set to a detection wavelength of 235nm to 255nm for liquid phase detection. Analysis and experiments have shown that this set wavelength is suitable for various effective components in the herbal composition for resolving dampness and detoxifying, meaning the absorption wavelength of these components is between 235nm and 255nm, ensuring that specific peaks appear in the spectrum.

[0013] In the above technical solution, acetonitrile and 0.1% phosphoric acid aqueous solution are used as the mobile phase for detection, and gradient elution is adopted. The flow rate of the mobile phase is 0.2-0.3 mL / min, and the volume ratio of acetonitrile in the mobile phase is 1%-35%.

[0014] In the above technical solution, gradient elution is performed using the following method:

[0015] Between 0 and 11 minutes, the volume percentage of acetonitrile in the mobile phase increased from 1% to 14%.

[0016] Between 11 and 21 minutes, the volume percentage of acetonitrile in the mobile phase increased from 14% to 35%.

[0017] In the above example, eluting the mobile phase using the above method is beneficial for separating various effective substances in the herbal composition for resolving dampness and detoxifying, so that they can be displayed completely and clearly in the subsequent chromatogram.

[0018] During the gradient elution described above, the flow rate of the mobile phase is 0.2–0.3 mL / min. Elution with the mobile phase at this flow rate facilitates the separation of various effective components from the herbal composition for resolving dampness and detoxifying, and avoids wasting the mobile phase. Preferably, the flow rate of the mobile phase is 0.25 mL / min.

[0019] In a second aspect, this application provides a quality control method for a traditional Chinese medicine composition for resolving dampness and detoxifying, which includes taking extract samples, concentrated liquid samples, dry paste samples and finished product granule samples from different batches of the traditional Chinese medicine composition for resolving dampness and detoxifying during the preparation process, and applying the above-mentioned fingerprint spectrum establishment method for the traditional Chinese medicine composition for resolving dampness and detoxifying to detect the fingerprint spectrum. All fingerprint spectra include at least 25 common chromatographic peaks, and the similarity of all fingerprint spectra is ≥0.97.

[0020] In the above technical solution, this application compares the fingerprint spectra of extracts, concentrates, dried pastes, and finished granules of different batches of the herbal composition for resolving dampness and detoxifying during the preparation process. By comparing the number and height of common peaks, it determines whether the various effective components in the extracts, concentrates, dried pastes, and finished granules of the herbal composition for resolving dampness and detoxifying are the same. This allows for the control of the quality of the herbal composition during preparation and for monitoring the quality of the herbal composition through multi-component analysis.

[0021] In the above technical solution, the dampness-resolving and detoxifying traditional Chinese medicine composition is prepared by the following method:

[0022] According to the weight proportions, 390-430 parts of Astragalus membranaceus, 350-450 parts of stir-fried Atractylodes macrocephala, 400-430 parts of Saposhnikovia divaricata, 400-450 parts of Lonicera japonica, 400-440 parts of Eupatorium fortunei, 200-300 parts of Dryopteris crassirhizoma, and 250-270 parts of Citrus reticulata peel are extracted with water and heated 1-3 times, each time for 0.5-2 hours, with a solvent ratio of 8-15 times. The extracts are combined, filtered, concentrated to obtain a concentrated liquid, dried to obtain a dry extract, and excipients are added to obtain the finished granular Chinese medicine preparation.

[0023] The dampness-resolving and detoxifying traditional Chinese medicine composition prepared by the above method can effectively preserve the main effective components of the medicinal materials without change, and the resulting dampness-resolving and detoxifying traditional Chinese medicine composition has good efficacy.

[0024] In the above technical solution, the extract sample, concentrated liquid sample, dry paste sample, and finished product granule sample are obtained through the following methods:

[0025] The extract was filtered to obtain the extract sample;

[0026] Take 1g of the concentrated solution and add 10mL of 70% methanol. Sonicate the solution for 30 minutes (power 250W, frequency 40kHz), shake well, and filter to obtain the concentrated solution sample.

[0027] Take 0.2g of the dry paste and add 10mL of 70% methanol. Sonicate the mixture (power 250W, frequency 40kHz) for 30 minutes, shake well, and filter to obtain the dry paste sample.

[0028] Take 0.2g of the finished product granules, add 10mL of 70% methanol, sonicate (power 250W, frequency 40kHz) for 30 minutes, shake well, and filter to obtain the finished product granule sample.

[0029] In the above example, the extract sample, concentrated sample, dry paste sample, and finished particle sample are prepared by the above method, ensuring that the concentrations of the extract sample, concentrated sample, dry paste sample, and finished particle sample are not significantly different, which is beneficial for subsequent comparison of fingerprint spectra.

[0030] Compared with the prior art, this application has the following advantages:

[0031] This application provides a method for establishing a fingerprint spectrum of a traditional Chinese medicine composition for resolving dampness and detoxifying. The method involves detecting the fingerprint spectrum of the composition sample using ultra-high performance liquid chromatography (UHPLC). The composition is prepared from raw materials including Astragalus membranaceus, stir-fried Atractylodes macrocephala, Saposhnikovia divaricata, Lonicera japonica, Eupatorium fortunei, Dryopteris crassirhizoma, and Citrus reticulata peel. This composition contains various effective components. By using UHPLC, this application significantly shortens the detection time and reduces the detection cost. The method employs a gradient elution of the composition sample with acetonitrile and a 0.1% phosphoric acid aqueous solution as the mobile phase, accurately separating various components and obtaining a fingerprint spectrum with clearly defined and complete characteristic peaks.

[0032] Furthermore, the quality control method for a dampness-resolving and detoxifying traditional Chinese medicine composition provided in this application compares the fingerprint spectra of extracts, concentrates, dried pastes, and finished granules of different batches of the dampness-resolving and detoxifying traditional Chinese medicine composition during the preparation process. By comparing the number and height of their common chromatographic peaks, it determines whether the various effective substances in the extracts, concentrates, dried pastes, and finished granules of the dampness-resolving and detoxifying traditional Chinese medicine composition during the preparation process are the same, and whether the various effective substances in the dampness-resolving and detoxifying traditional Chinese medicine composition have changed during the preparation process and between different batches, thereby achieving the control of the quality of the dampness-resolving and detoxifying traditional Chinese medicine composition during the preparation process. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 The images show the UHPLC fingerprint of the dry extract of the dampness-resolving and detoxifying traditional Chinese medicine composition obtained in Example 1 of this application and the ultra-high performance liquid chromatogram of the mixed reference solution, wherein S1 is the chromatogram of the dry extract of the dampness-resolving and detoxifying traditional Chinese medicine composition and S2 is the chromatogram of the mixed reference solution.

[0035] Figure 2 The reference fingerprint R is the traditional Chinese medicine composition for resolving dampness and detoxifying in Example 2, wherein peak 3 is neochlorogenic acid; peak 6 is chlorogenic acid; peak 8 is cryptochlorogenic acid; peak 21 is 3,5-O-dicaffeoylquinic acid; peak 22 is hesperidin; and peak 23 is 4,5-O-dicaffeoylquinic acid.

[0036] Figure 3 The above are the UHPLC superimposed fingerprint chromatograms and control fingerprint chromatograms R of the wet detoxifying traditional Chinese medicine composition obtained in Example 2 of this application, wherein S1 to S10 are batches 01 to 10 of the wet detoxifying traditional Chinese medicine composition respectively;

[0037] Figure 4 The UHPLC fingerprint spectrum and control fingerprint spectrum R of the wet detoxifying traditional Chinese medicine composition obtained in Example 3 of this application are shown, wherein S1 to S4 are fingerprint spectra of extract, concentrated liquid, dry extract, and finished product granules, respectively. Detailed Implementation

[0038] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this application. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0039] Ultra-high performance liquid chromatography (UHPLC) is a highly efficient separation method in liquid chromatography. It separates different substances by utilizing the difference in the partition coefficients between the stationary phase and the mobile phase. Through higher pressure, smaller packing particle size, and a more optimized mobile phase system, it achieves higher separation efficiency and sensitivity, thereby saving time and costs.

[0040] However, the methods used for detecting different mixed liquids using UHPLC vary. An inappropriate selection of the detection method can lead to incomplete liquid separation and unclear peaks in the detected chromatogram.

[0041] The following describes in detail a method for establishing a fingerprint spectrum of a traditional Chinese medicine composition for resolving dampness and detoxifying, and a method for quality control of the traditional Chinese medicine composition for resolving dampness and detoxifying, according to embodiments of this application.

[0042] This application provides a method for establishing a fingerprint spectrum of a traditional Chinese medicine composition for resolving dampness and detoxifying, which uses UHPLC to detect samples of the traditional Chinese medicine composition for resolving dampness and detoxifying.

[0043] The herbal composition for resolving dampness and detoxifying is made from raw materials including Astragalus membranaceus, stir-fried Atractylodes macrocephala, Saposhnikovia divaricata, Lonicera japonica, Eupatorium fortunei, Dryopteris crassirhizoma, and Citrus reticulata peel.

[0044] Based on the effective components of the above-mentioned traditional Chinese medicine ingredients, and considering the need to analyze and eliminate interference between different substances, selecting a suitable chromatographic column and mobile phase, as well as controlling the flow rate of the mobile phase, is beneficial for separating the various effective components of the dampness-resolving and detoxifying traditional Chinese medicine composition. This ensures that each effective component presents specific peaks in the chromatogram, resulting in accurate chromatographic data.

[0045] In this application, 0.5–3 μL of sample was taken for UHPLC detection. Acetonitrile and 0.1% phosphoric acid aqueous solution were used as the mobile phase, with the volume ratio of acetonitrile in the mobile phase being 1%–35%. Gradient elution was used, and the flow rate of the mobile phase was 0.2–0.3 mL / min.

[0046] Optionally, the measurement wavelength of the ultraviolet detector is set to 235 nm to 255 nm. This wavelength can be used for various effective components in the herbal composition for resolving dampness and detoxifying, that is, the absorption wavelength of a limited number of components in the herbal composition for resolving dampness and detoxifying is between 235 nm and 255 nm, which can ensure that the effective components in the herbal composition for resolving dampness and detoxifying can present specific peaks in the spectrum.

[0047] The ultraviolet detector can be either built into the ultra-high performance liquid chromatograph or external.

[0048] Optionally, the chromatographic column used in this application is a C18 water-resistant small-particle-size column, and the column temperature during detection is 25℃~35℃. The chromatographic column is selected based on the active ingredients and impurities in the sample, as well as their type, structure, dosage form, acidity / alkalinity, and molecular weight. Correct selection of the chromatographic column and setting of the detection column temperature facilitate the accurate and complete separation of various substances in the herbal composition for resolving dampness and detoxifying, and their display in the chromatogram.

[0049] When performing gradient elution, the following methods can be used:

[0050] Between 0 and 11 min, the volume percentage of acetonitrile in the mobile phase increased from 1% to 14%, the volume percentage of 0.1% phosphoric acid aqueous solution decreased from 99% to 86%, and the flow rate of the mobile phase was 0.2–0.3 mL / min.

[0051] Optionally, the flow rate of the mobile phase is 0.25 mL / min;

[0052] Between 11 and 21 minutes, the volume percentage of acetonitrile in the mobile phase increased from 14% to 35%, the volume percentage of 0.1% phosphoric acid aqueous solution decreased from 86% to 65%, and the flow rate of the mobile phase was 0.2–0.3 mL / min.

[0053] Optionally, the flow rate of the mobile phase is 0.25 mL / min;

[0054] This application also provides a quality control method for a traditional Chinese medicine composition for resolving dampness and detoxifying, which includes taking samples of the extract, concentrate, dry paste and finished granules of the traditional Chinese medicine composition for resolving dampness and detoxifying during the preparation process using the above-mentioned fingerprint spectrum establishment method, and detecting the fingerprint spectrum. All fingerprint spectra include at least 25 common chromatographic peaks, and the similarity of all fingerprint spectra is ≥0.97.

[0055] This application analyzes the number, height, and similarity of common chromatographic peaks in the fingerprint spectra of the extract, concentrate, dried paste, and finished granules of the traditional Chinese medicine composition for resolving dampness and detoxifying during the preparation process. By examining the differences in the spectra, it reflects the differences in various effective components of the traditional Chinese medicine composition for resolving dampness and detoxifying, thereby verifying whether the various effective substances in the traditional Chinese medicine composition for resolving dampness and detoxifying have changed during the preparation process and in different batches, thus achieving the goal of controlling the quality of the traditional Chinese medicine composition for resolving dampness and detoxifying during the preparation process.

[0056] It should be noted that all fingerprint similarity was obtained using the Traditional Chinese Medicine Chromatography Fingerprint Similarity Evaluation System Software (version 2012.130723). All fingerprints to be compared were imported into the Traditional Chinese Medicine Chromatography Fingerprint Similarity Evaluation System Software (version 2012.130723), and UHPLC fingerprints were established using the median method to generate a reference characteristic chromatogram consisting of 25 common chromatographic peaks with a similarity ≥ 0.97. Six chromatographic peaks were identified using reference standards: neochlorogenic acid (peak 3), chlorogenic acid (peak 6), cryptochlorogenic acid (peak 8), 3,5-O-dicaffeoylquinic acid (peak 21), hesperidin (peak 22), and 5-O-dicaffeoylquinic acid (peak 23). Using peak 6 (chlorogenic acid) as a reference, the relative retention times of the other characteristic peaks were within ±10% of the specified values. The specified relative retention times were: 0.55 (peak 1), 0.63 (peak 2), 0.80 (peak 3), 0.95 (peak 4), 0.97 (peak 5), and 1. 02 (peak 7), 1.05 (peak 8), 1.12 (peak 9), 1.15 (peak 10), 1.23 (peak 11), 1.26 (peak 12), 1.29 (peak 13), 1.35 (peak 14), 1.49 (peak 15), 1.56 (peak 16), 1.59 (peak 17), 1.61 (peak 18), 1.66 (peak 19), 1.69 (peak 20), 1.73 (peak 21), 1.78 (peak 22), 1.83 (peak 23), 1.92 (peak 24), 2.16 (peak 25).

[0057] The traditional Chinese medicine composition for resolving dampness and detoxifying is prepared by the following method:

[0058] 1. Raw material preparation

[0059] Weigh out the following ingredients by weight: Astragalus membranaceus 390–430 parts, stir-fried Atractylodes macrocephala 350–450 parts, Saposhnikovia divaricata 400–430 parts, Lonicera japonica 400–450 parts, Eupatorium fortunei 400–440 parts, Dryopteris crassirhizoma 200–300 parts, and Citrus reticulata 250–270 parts.

[0060] For the processing methods of the medicinal materials, please refer to the 2020 edition of the Chinese Pharmacopoeia.

[0061] Optionally, according to the weight parts, weigh out 420 parts of Astragalus membranaceus, 395 parts of stir-fried Atractylodes macrocephala, 415 parts of Saposhnikovia divaricata, 420 parts of Lonicera japonica, 405 parts of Eupatorium fortunei, 275 parts of Dryopteris crassirhizoma, and 260 parts of Citrus reticulata peel.

[0062] 2. Preparation of traditional Chinese medicine components for resolving dampness and detoxifying

[0063] The above-mentioned Chinese medicinal materials are boiled with water and extracted 1 to 3 times, each time for 0.5 to 2 hours. The amount of water added each time is 8 to 15 times the mass of the Chinese medicinal materials. The extracts are combined, filtered through a mesh larger than 200, and concentrated under reduced pressure at 60 to 80°C to a concentrate with a relative density of 1.05 to 1.20 (60 to 80°C). The concentrate is then dried to obtain a dry paste with a yield of 22% to 32%. After adding excipients, the finished granular Chinese medicine preparation is obtained.

[0064] The excipients include, but are not limited to, any one or more of maltodextrin, dextrin, powdered sugar, povidone, polyethylene glycol, xylitol, β-cyclodextrin, and silicon dioxide.

[0065] The extract sample is the extract described above.

[0066] The concentrated sample was prepared by adding 1g of the concentrated solution to 10mL of 70% methanol, ultrasonicating (power 250W, frequency 40kHz) for 30 minutes, shaking well, and filtering.

[0067] The dry paste sample was prepared by adding 0.2g of the dry paste to 10mL of 70% methanol, sonicating (power 250W, frequency 40kHz) for 30 minutes, shaking well, and filtering.

[0068] The finished particle sample was obtained by adding 0.2g of the finished particles to 10mL of 70% methanol, ultrasonicating (power 250W, frequency 40kHz) for 30 minutes, shaking well, and filtering.

[0069] The concentrations of the above-mentioned extract samples, concentrated liquid samples, dry paste samples, and finished product granule samples are not significantly different, which is beneficial for subsequent comparison of fingerprint spectra.

[0070] The following describes in further detail, with reference to embodiments, a method for establishing a fingerprint spectrum of a traditional Chinese medicine composition for resolving dampness and detoxifying, and a method for quality control of the traditional Chinese medicine composition for resolving dampness and detoxifying.

[0071] Example 1

[0072] This embodiment provides a method for establishing the fingerprint spectrum of a traditional Chinese medicine composition for resolving dampness and detoxifying, including the following steps:

[0073] 1. Preparation of a traditional Chinese medicine composition for resolving dampness and detoxifying

[0074] According to the weight proportions, 420 parts of Astragalus membranaceus, 395 parts of stir-fried Atractylodes macrocephala, 415 parts of Saposhnikovia divaricata, 420 parts of Lonicera japonica, 405 parts of Eupatorium fortunei, 275 parts of Dryopteris crassirhizoma, and 260 parts of Citrus reticulata were weighed and boiled twice with water, each time for 1 hour. The amount of water added each time was 10 times the weight of the Chinese medicinal materials. The extracts were combined, filtered, and concentrated under reduced pressure at 60℃ to obtain a concentrated liquid. The concentrate was dried to obtain a dry paste. After adding excipients, the finished granular Chinese medicine preparation was obtained.

[0075] 2. Preparation of dry extract samples and mixed control samples

[0076] Take 0.2g of the dry paste obtained above, add 10mL of 70% methanol, sonicate (power 250W, frequency 40kHz) for 30 minutes, shake well, and filter to obtain the dry paste sample;

[0077] The mixed reference solution consisted of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 3,5-O-dicaffeoylquinic acid, hesperidin, and 4,5-O-dicaffeoylquinic acid. Each reference standard was added to 70% methanol to prepare the mixed reference solution.

[0078] 3. Establish fingerprint spectrum of traditional Chinese medicine composition for resolving dampness and detoxifying

[0079] In this embodiment, 1 μL of the dry extract sample was subjected to UHPLC analysis. The detection wavelength of the ultraviolet detector was set to 235 nm–255 nm. A C18 water-resistant small-particle-size column was used, and the column temperature was set to 25 °C–35 °C during detection. Acetonitrile and 0.1% phosphoric acid aqueous solution were used as the mobile phase, and gradient elution was performed as shown in Table 1.

[0080] Table 1. Mobile phase and flow rate of Example 1 (UHPLC Detection)

[0081] 0～11 1→14 99→86 0.25 0～21 14→35 86→65 0.25

[0082] The obtained UHPLC fingerprint spectrum is as follows Figure 1 As shown, the peaks of various substances are obvious, and the separation degree and signal-to-noise ratio are high.

[0083] Example 2

[0084] This embodiment provides a method for quality control of a traditional Chinese medicine composition for resolving dampness and detoxifying, including the following steps:

[0085] 1. Sample preparation

[0086] Ten batches of the herbal composition for resolving dampness and detoxifying were prepared according to the method of Example 1, and ten batches of the dry extract were prepared into dry extract samples according to the method of Example 1.

[0087] 2. Establish quality control for traditional Chinese medicine compositions for resolving dampness and detoxifying.

[0088] UHPLC fingerprints of 10 batches of dry extract samples of the detoxifying and dampness-resolving traditional Chinese medicine composition were obtained according to the UHPLC detection method in Example 1. All fingerprints were imported into the Traditional Chinese Medicine Fingerprint Similarity Evaluation System software (version 2012.130723). UHPLC fingerprints were established using the median method, generating a control characteristic spectrum consisting of 25 common peaks. Figure 2 , 3 As shown. Of the 25 characteristic chromatograms, 6 were identified by comparison with a mixed control, with peak 6 (chlorogenic acid) as the reference. The relative retention times of the other characteristic peaks were within ±10% of the specified values. The specified relative retention times were: 0.55 (peak 1), 0.63 (peak 2), 0.80 (peak 3), 0.95 (peak 4), 0.97 (peak 5), 1.02 (peak 7), 1.05 (peak 8), 1.12 (peak 9), and 1.15 (peak 10). The relative retention times are shown in Table 2. (0), 1.23 (peak 11), 1.26 (peak 12), 1.29 (peak 13), 1.35 (peak 14), 1.49 (peak 15), 1.56 (peak 16), 1.59 (peak 17), 1.61 (peak 18), 1.66 (peak 19), 1.69 (peak 20), 1.73 (peak 21), 1.78 (peak 22), 1.83 (peak 23), 1.92 (peak 24), 2.16 (peak 25).

[0089] Table 2 Relative retention time table of 10 batches of dry paste samples

[0090]

[0091]

[0092] Example 3

[0093] This embodiment provides a method for quality control of a traditional Chinese medicine composition for resolving dampness and detoxifying, including the following steps:

[0094] 1. Sample preparation

[0095] Samples were prepared from the extract, concentrated solution, dried paste, and finished granules of the dampness-resolving and detoxifying traditional Chinese medicine composition obtained in Example 1 according to the following methods:

[0096] Extract sample and take the above extract;

[0097] The concentrated sample was prepared by adding 1g of the concentrated solution to 10mL of 70% methanol, ultrasonicating (power 250W, frequency 40kHz) for 30 minutes, shaking well, and filtering.

[0098] The dry paste sample was prepared by adding 0.2g of the dry paste to 10mL of 70% methanol, sonicating (power 250W, frequency 40kHz) for 30 minutes, shaking well, and filtering.

[0099] The finished particle sample was obtained by adding 0.2g of the finished particles to 10mL of 70% methanol, ultrasonicating (power 250W, frequency 40kHz) for 30 minutes, shaking well, and filtering.

[0100] 2. Establish a quality control system for traditional Chinese medicine compositions for resolving dampness and detoxifying.

[0101] The UHPLC fingerprint spectra of the above-mentioned extract sample, concentrated sample, dried paste sample, and finished product particle sample were obtained according to the UHPLC detection method of Example 1, as follows: Figure 3 As shown, each fingerprint spectrum has 25 identical chromatographic peaks at corresponding positions. The similarity and relative retention times were calculated, as shown in Tables 3 and 4.

[0102] Table 3 Fingerprint Similarity Calculation Table

[0103] Extract 1 0.999 0.997 0.997 0.997 Concentrate 0.999 1 0.997 0.997 0.997 Dry ointment 0.997 0.997 1 0.999 0.999 Finished granules 0.997 0.997 0.999 1 0.999 Comparison of fingerprint patterns 0.997 0.997 0.999 0.999 1

[0104] Table 4 Relative Retention Schedule

[0105]

[0106]

[0107] As shown in Table 3, the similarity of each chromatogram is ≥0.97; as shown in Table 4, the relative retention times of each chromatographic peak are all within the standard range.

[0108] The above description is merely a specific embodiment of this application and is not intended to limit this application. For those skilled in the art, this application can have various changes and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for constructing a UHPLC fingerprint of a traditional Chinese medicine composition for resolving dampness and detoxifying, characterized in that, The raw materials of the traditional Chinese medicine composition sample for resolving dampness and detoxifying are composed of Astragalus membranaceus, stir-fried Atractylodes macrocephala, Saposhnikovia divaricata, Lonicera japonica, Eupatorium fortunei, Dryopteris crassirhizoma, and Citrus reticulata peel; The construction method includes the following steps: 1) Preparation method of the test solution The method for preparing the test solution is to take the powder of the herbal composition for resolving dampness and detoxifying, add a 70% methanol solution, sonicate, and filter to obtain the solution. 2) Preparation method of mixed reference solution The mixed reference solution consists of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 3,5-O-dicaffeoylquinic acid, hesperidin, and 4,5-O-dicaffeoylquinic acid. Each reference standard is added to 70% methanol to prepare the mixed reference solution. 3) Take the test solution and the mixed reference solution separately and perform ultra-high performance liquid chromatography to obtain the fingerprint spectrum of the traditional Chinese medicine composition for resolving dampness and detoxifying; Acetonitrile and 0.1% phosphoric acid solution were used for detection; the flow rate of the mobile phase was 0.2–0.3 mL / min; the detection wavelength of the ultraviolet detector was set to 235 nm–255 nm for liquid chromatography detection; the elution method was gradient elution, specifically: from 0 to 11 min, the volume proportion of acetonitrile in the mobile phase increased from 1% to 14%; from 11 to 21 min, the volume proportion of acetonitrile in the mobile phase increased from 14% to 35%. The chromatographic column used for the test was a C18 water-resistant small particle size column.

2. The construction method according to claim 1, characterized in that, When performing liquid chromatography detection, the column temperature of the chromatographic column is 25℃~35℃.

3. The construction method according to claim 1, characterized in that, During gradient elution, the flow rate of the mobile phase is 0.25 mL / min.

4. The construction method according to claim 1, characterized in that, The dampness-resolving and detoxifying traditional Chinese medicine composition is prepared by the following method: According to the weight proportions, 390-430 parts of Astragalus membranaceus, 350-450 parts of stir-fried Atractylodes macrocephala, 400-430 parts of Saposhnikovia divaricata, 400-450 parts of Lonicera japonica, 400-440 parts of Eupatorium fortunei, 200-300 parts of Dryopteris crassirhizoma, and 250-270 parts of Citrus reticulata peel are extracted with water and heated 1-3 times, each time for 0.5-2 hours, with a solvent ratio of 8-15 times. The extracts are combined, filtered, concentrated to obtain a concentrated liquid, dried to obtain a dry extract, and excipients are added to obtain the finished granular Chinese medicine preparation.

5. The construction method according to claim 1, characterized in that, Using the chlorogenic acid chromatographic peak as a reference, the six reference peaks in the fingerprint spectrum are: peak 3: neochlorogenic acid, peak 6: chlorogenic acid, peak 8: cryptochlorogenic acid, peak 21: 3,5-O-dicaffeoylquinic acid, peak 22: hesperidin, and peak 23: 5-O-dicaffeoylquinic acid.

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