A whole quality control method of compound eucommia ulmoides waist strengthening capsule

Abstract

A kind of overall quality control method of compound eucommia ulmacea waist capsule, including: psoralen and isopsoralen content determination: with hydrochloric acid-methanol solution as extraction solvent, heating reflux extraction, with methanol-phosphoric acid aqueous solution as mobile phase isocratic elution;Macon glycoside, paeoniflorin and paeonol glycoside content determination: with 50% ethanol ultrasonic extraction, with acetonitrile-0.1% phosphoric acid solution as mobile phase gradient elution;HPLC fingerprint is established: 21 common peaks are calibrated, 6 characteristic peaks are identified, and the similarity is not less than 0.992;Thin layer chromatography identification: establish white peony root and cornus officinalis synchronous identification, ephedra identification and radix rehmanniae praeparata identification method.The present application is based on the identification of 92 kinds of compounds by LC-MS technology, and a multi-dimensional quality control system combining multi-component quantitative, fingerprint overall evaluation and exclusive identification is constructed, which overcomes the shortcomings of single existing standard index and lack of overall evaluation, and can comprehensively and accurately control the quality of compound eucommia ulmacea waist capsule.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine quality control technology, specifically to a comprehensive quality control method for Compound Eucommia Ulmoides Strengthening Capsules, including the determination of the content of multiple indicator components, thin-layer chromatography identification, and the establishment and evaluation of high-performance liquid chromatography fingerprint chromatograms. Background Technology

[0002] Compound Eucommia ulmoides waist-strengthening capsules are a commonly used traditional Chinese medicine preparation in clinical practice. They are composed of deer antler, sheep tibia, Cibotium barometz, Cuscuta chinensis, Achyranthes bidentata, Cornus officinalis, Allium tuberosum seed, Paeonia lactiflora, Lycium barbarum, Eucommia ulmoides, Rehmannia glutinosa, Ephedra sinica, Asarum heterotropoides, Artemisia argyi, Corydalis yanhusuo, and Codonopsis pilosula. They have the effects of tonifying the kidney and strengthening the waist, clearing the meridians and relieving pain. They are widely used to treat symptoms such as lower back and knee pain, and rheumatic bone pain.

[0003] Due to the large formulation and numerous ingredients in this compound preparation, along with the diverse chemical components and properties of each herb, quality control is extremely difficult. Currently, the existing quality standards for Compound Eucommia Ulmoides Strengthening Capsules have the following significant shortcomings: Quantitative indicators are often limited and fail to reflect overall efficacy: Current standards typically only quantitatively control one or two components (such as psoralen or paeoniflorin alone). However, the efficacy of compound traditional Chinese medicines is the result of the synergistic effect of multiple components. For example, loganin in Cornus officinalis, paeoniflorin and paeoniflorin lactone in Paeonia lactiflora, and psoralen and isopsoralen in Psoralea corylifolia are all key active ingredients. Controlling only a single indicator not only fails to comprehensively reflect drug quality but also easily leaves room for unscrupulous individuals to falsify results by adding a single, inexpensive extract to "meet the required standards."

[0004] Lack of comprehensive quality evaluation methods: Current standards lack comprehensive quality control technologies such as fingerprinting. While different batches of drugs may meet the standards for the content of key components, the proportions of other components may fluctuate significantly, leading to unstable clinical efficacy. For Compound Eucommia Ulmoides Strengthening Capsules, which contain more than 90 complex compounds, establishing a fingerprint spectrum that reflects the overall chemical characteristics is urgently needed.

[0005] Inadequate or inefficient identification methods: Thin-layer chromatography (TLC) identification methods are lacking for some medicinal materials, or suffer from significant negative interference and unclear spots. Furthermore, traditional methods often employ a "one drug, one plate" identification model, requiring separate preparation of the test solution and separate development for each medicinal material. This results in high reagent consumption, low detection efficiency, and difficulty in meeting the demands of modern rapid testing in production.

[0006] The detection method failed to overcome matrix interference: the compound components are extremely complex, and conventional liquid chromatography conditions (such as isocratic elution) often fail to completely separate the target components (especially loganin, paeoniflorin, etc. with large polarity differences) from impurity peaks, resulting in inaccurate quantitative results; or the pretreatment method is simple and crude, with low extraction efficiency and many impurities.

[0007] Therefore, it is urgent to develop a comprehensive quality control method that integrates multi-component quantification, overall fingerprint evaluation, and specific identification, based on a thorough understanding of the material basis of Compound Eucommia Ulmoides Strengthening Capsules, in order to improve the quality standards of this preparation and ensure the safety and efficacy of clinical use. This invention is based on the above needs. Building upon the identification of 92 chemical components using liquid chromatography-mass spectrometry (LC-MS), it has specifically established an overall quality control system that includes quantification of 5 indicator components, fingerprint spectroscopy with 21 common peaks, and identification of multiple medicinal materials. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a comprehensive quality control method for Compound Eucommia Ulmoides Strengthening Capsules. Based on in-depth research into the material basis of the compound, this method constructs a multi-dimensional quality evaluation system that includes multi-component content determination, HPLC fingerprinting, and TLC-specific identification, enabling comprehensive, accurate, and efficient quality control of Compound Eucommia Ulmoides Strengthening Capsules.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for determining the content of psoralen and isopsoralen in compound Eucommia ulmoides waist-strengthening capsules.

[0010] To address the challenges and interferences associated with the extraction of psoralen compounds from complex matrices, this invention creatively employs an acidic alcohol solution as the extraction solvent. The specific steps are as follows: (1) Preparation of test solution: Take the contents of compound Eucommia ulmoides capsules, add hydrochloric acid-methanol mixed solution (volume ratio 1:4), heat and reflux to extract, cool, filter, discard 2 ml of the initial filtrate, take 1 ml of the subsequent filtrate and put it into a sample vial as the test solution.

[0011] (2) Chromatographic conditions: Octadecylsilane bonded silica gel was used as the packing material; isocratic elution was performed using methanol-0.4% phosphoric acid solution (45:55) as the mobile phase; the detection wavelength was 246 nm; and the column temperature was 35 °C.

[0012] (3) Determination: Inject the reference solution and the test solution into the liquid chromatograph, respectively, measure the peak area, and calculate the content.

[0013] This invention reveals that using hydrochloric acid:methanol (1:4) for reflux extraction significantly improves the extraction rate of psoralen and isopsoralen compared to extraction with pure methanol or ordinary ethanol. At the same time, it effectively reduces the interference of impurity peaks on the target peak, resulting in good chromatographic peak separation and a stable baseline.

[0014] Secondly, the present invention provides a method for determining the content of loganin, paeoniflorin and paeoniflorin in compound Eucommia ulmoides waist-strengthening capsules.

[0015] To address the challenge of separating these three components under the same isocratic conditions due to their significant polarity differences (loganin is highly polar, paeoniflorin is low polarity), this invention designs a specific gradient elution procedure, the specific steps of which are as follows: (1) Preparation of test solution: Take the contents of this product, add 50% ethanol solution, sonicate, filter, discard 2 ml of the initial filtrate, take 1 ml of the subsequent filtrate and put it into a sample vial as the test solution.

[0016] (2) Chromatographic conditions: Octadecylsilane bonded silica gel was used as the packing material; mobile phase A was 0.1% phosphoric acid solution and mobile phase B was acetonitrile; gradient elution was used, the detection wavelength was 235 nm and the column temperature was 35 °C.

[0017] The gradient elution program is as follows: at 0 min, phase A 95% and phase B 5%; at 30 min, phase A 65% and phase B 35%; at 45 min, phase A 10% and phase B 90%; at 50 min, phase A 95% and phase B 5%; at 50–60 min, phase A 95% and phase B 5% (equilibration column).

[0018] This method is the first to achieve the simultaneous determination of loganin from Cornus officinalis and paeoniflorin and paeoniflorin lactone from Paeonia lactiflora in a single injection of Compound Eucommia ulmoides and Waist-Strengthening Capsules, which greatly improves the detection efficiency.

[0019] Thirdly, this invention provides a method for establishing and evaluating the quality of compound Eucommia ulmoides waist-strengthening capsules using high-performance liquid chromatography fingerprinting.

[0020] Under the chromatographic conditions for the determination of loganin, paeoniflorin, and paeoniflorin lactone, the present invention establishes a standard fingerprint spectrum containing 21 common peaks.

[0021] Specific methods include: (1) Prepare test solutions for more than 10 batches of samples and record the chromatograms; (2) A reference fingerprint (R) was generated using the “Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System”, and 21 common peaks were identified; (3) Identify the six characteristic peaks, namely protocatechuic acid (peak 6), loganin (peak 9), paeoniflorin (peak 10), paeoniflorin (peak 12), psoralen (peak 18), and isopsoralen (peak 19). (4) Quality evaluation standard: The similarity between the fingerprint spectrum of the test sample and the fingerprint spectrum of the control should not be less than 0.992.

[0022] This fingerprint spectrum covers the main active ingredient groups in the compound, and can sensitively reflect the quality fluctuations of raw materials and the stability of the production process, filling the gap in the overall quality evaluation of this preparation.

[0023] Fourthly, this invention provides a thin-layer chromatography (TLC) identification method for multiple medicinal materials in Compound Eucommia Ulmoides Strengthening Capsules.

[0024] (1) Simultaneous identification of white peony and cornus officinalis: The "one plate double detection" technique was adopted. The same test solution was spotted, and dichloromethane-methanol-water (14:3:0.2) was used as the developing solvent. 5% vanillin sulfuric acid was used for color development. Paeoniflorin (blue-purple) and loganin (red-purple) spots could be clearly detected at the same time, and the negative results were not interfered with.

[0025] (2) Identification of ephedra: alkalization-dichloromethane extraction was used, followed by development with dichloromethane-methanol-concentrated ammonia solution (4:1:0.1), and ninhydrin was used for color development, resulting in clear spots.

[0026] (3) Identification of Rehmannia glutinosa: Water extraction-n-butanol extraction, xylene-ethyl acetate (1:1) development, 2,4-dinitrophenylhydrazine color development, with strong specificity.

[0027] The present invention provides a comprehensive quality control method for compound Eucommia ulmoides waist-strengthening capsules, which, based on detailed experimental data and methodological verification, has the following significant beneficial effects: 1. A scientific quality control system based on profound material foundation research was established: This invention did not blindly select indicator components, but first used liquid chromatography-mass spectrometry (LC-MS) to comprehensively analyze the material basis of Compound Eucommia Ulmoides Strengthening Capsules in both positive and negative ion modes. A total of 92 compounds were identified, clarifying the chemical composition of 24 flavonoids, 22 phenylpropanoids, 17 terpenoids, 12 organic acids, 8 alkaloids, and 9 other types of compounds. This research provides a solid scientific basis for the subsequent selection of the most representative quantitative indicators (psoralen, loganin, etc.) and fingerprint spectrum common peaks, ensuring the targeted and comprehensive nature of quality control; 2. Optimized extraction and separation conditions for psoralen and isopsoralen: For the determination of psoralen and isopsoralen content, this invention, by comparing different extraction solvents, mobile phases, and chromatographic column conditions, finally determined an optimized scheme using "hydrochloric acid:methanol (1:4)" as the solvent for heating and reflux extraction, combined with "methanol-0.4% phosphoric acid solution (45:55)" for elution. Experimental results show that under these conditions, the chromatographic peak separation effect is optimal. Methodological investigation shows that the linearity, stability, repeatability, specificity, precision, and recovery rate of this method are all "good" (based on the original material, the results are "good"), and it can be accurately and reliably used for the quantitative control of psoralen and isopsoralen in this formulation. 3. Overcame the separation challenge of simultaneous determination of multiple components: For the simultaneous determination of loganin (a cyclohexene glycoside), paeoniflorin, and paeoniflorin lactone (a monoterpene glycoside), this invention, through investigation of extraction solvents and elution procedures, determined a technical scheme of ultrasonic extraction with 50% ethanol followed by gradient elution with acetonitrile-0.1% phosphoric acid solution. This method successfully solved the separation challenge of multiple polarity-differentiated components in complex matrices. Validation data showed that the method exhibited good linearity, stability, repeatability, specificity, precision, and recovery, achieving simultaneous and accurate determination of three key components derived from Cornus officinalis and Paeonia lactiflora. 4. A high-similarity fingerprint spectrum was established, enabling overall quality evaluation: Based on content determination, this invention established a high-performance liquid chromatography (HPLC) fingerprint spectrum for Compound Eucommia Ulmoides Strengthening Capsules containing 21 common peaks, and clearly identified the chemical composition of 6 characteristic peaks (protocatechuic acid, loganin, paeoniflorin, paeoniflorin, psoralen, and isopsoralen). Analysis of 10 batches of samples showed a similarity range of 0.992-1.000. This extremely high data consistency indicates that the fingerprint spectrum method established in this invention has good stability and can objectively reflect the uniformity of product quality produced by existing manufacturing processes, providing a quantifiable standard tool for the overall quality consistency evaluation of Compound Eucommia Ulmoides Strengthening Capsules. 5. Screening for Highly Specific and Interference-Free Thin-Layer Chromatography Identification Methods: This invention conducted a systematic thin-layer chromatography study on seven medicinal materials in the compound formula. Through numerous negative control experiments, this invention eliminated the identification methods for Codonopsis pilosula, Eucommia ulmoides, and Cibotium barometz, which suffered from severe interference and unclear spots. It then optimized and established specific identification methods for Paeonia lactiflora, Ephedra sinica, Cornus officinalis, and Rehmannia glutinosa. Paeonia lactiflora and Cornus officinalis: Using the same developing system "dichloromethane-methanol-water (14:3:0.2)" and the same colorimetric reagent "5% vanillin-sulfuric acid," both showed clear spots with no negative interference. This allows for the identification of the two medicinal materials using the same reagent system in actual testing, simplifying the operation. Ephedra sinica: Using a special pretreatment and developing system of "concentrated ammonia solution + dichloromethane," interference was effectively overcome, and the spots were clearly displayed. Rehmannia glutinosa: Using a pretreatment of "water boiling + n-butanol extraction," clear identification of the characteristic spots of Rehmannia glutinosa was achieved, with no interference from the negative control. The above method has been experimentally verified to produce clear spots, good separation, and no interference from negative results, significantly improving the reliability and practicality of the identification standard. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0029] Figure 1 This is the test quality spectrum of the Compound Eucommia Ulmoides Strengthening Capsule in this application; Figure 2 These are the quality spectra of the test sample and the control sample of Compound Eucommia Ulmoides Strengthening Capsules in this application. Figure 3 Mass spectrometry identification information of some compounds in the Compound Eucommia Ulmoides Strengthening Capsule in this application; Figure 4 This is a mass spectrum of the fragmentation pathway of kaempferol and its secondary fragments in the scheme of this application; Figure 5 The fragmentation pathway of psoralen in this application and its secondary fragment mass spectra are shown. Figure 6 The fragmentation pathway of catechin in this application and its secondary fragment mass spectra are shown. Figure 7 The fragmentation pathway of ephedrine and its secondary fragment mass spectra in this application are shown. Figure 8 The fragmentation pathway of paeoniflorin and its secondary fragment mass spectra in this application are shown. Figure 9 The HPLC chromatogram of Compound Eucommia Bark Strengthening Capsules in this application is shown. Figure 10 The HPLC chromatograms of loganin, paeoniflorin, and paeoniflorin lactone in the compound Eucommia ulmoides capsules of this application are shown. Figure 11 This is the HPLC specificity verification chromatogram for loganin, paeoniflorin, and paeoniflorin lactone in the compound Eucommia ulmoides and waist-strengthening capsules of this application; Figure 12 The fingerprint spectrum of Compound Eucommia Ulmoides Strengthening Capsules in this application scheme; Figure 13 The superimposed fingerprint spectrum of Compound Eucommia Ulmoides Strengthening Capsules in this application scheme; Figure 14 Comparison of HPLC chromatograms of the single herb in the Compound Eucommia Bark Strengthening Capsule in this application with the test sample; Figure 15 TLC image of white peony root in Compound Eucommia ulmoides and Waist-Strengthening Capsules of this application; Figure 16 TLC image of Ephedra in Compound Eucommia Ulmoides and Waist-Strengthening Capsules of this application; Figure 17 TLC image of Cornus officinalis in Compound Eucommia ulmoides and Waist-Strengthening Capsules of this application; Figure 18 The image shows the TLC diagram of Rehmannia glutinosa in the Compound Eucommia ulmoides and Waist-Strengthening Capsule of this application. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] This invention provides a method for determining the content of psoralen and isopsoralen in Compound Eucommia Ulmoides Strengthening Capsules.

[0032] To address the challenges and interferences associated with the extraction of psoralen compounds from complex matrices, this invention creatively employs an acidic alcohol solution as the extraction solvent. The specific steps are as follows: (1) Preparation of test solution: Take the contents of compound Eucommia ulmoides capsules, add hydrochloric acid-methanol mixed solution (volume ratio 1:4), heat and reflux to extract, cool, filter, discard 2 ml of the initial filtrate, take 1 ml of the subsequent filtrate and put it into a sample vial as the test solution.

[0033] (2) Chromatographic conditions: Octadecylsilane bonded silica gel was used as the packing material; isocratic elution was performed using methanol-0.4% phosphoric acid solution (45:55) as the mobile phase; the detection wavelength was 246 nm; and the column temperature was 35 °C.

[0034] (3) Determination: Inject the reference solution and the test solution into the liquid chromatograph, respectively, measure the peak area, and calculate the content.

[0035] This invention reveals that using hydrochloric acid:methanol (1:4) for reflux extraction significantly improves the extraction rate of psoralen and isopsoralen compared to extraction with pure methanol or ordinary ethanol. At the same time, it effectively reduces the interference of impurity peaks on the target peak, resulting in good chromatographic peak separation and a stable baseline.

[0036] This invention provides a method for determining the content of loganin, paeoniflorin and paeoniflorin in Compound Eucommia Ulmoides Strengthening Capsules.

[0037] To address the challenge of separating these three components under the same isocratic conditions due to their significant polarity differences (loganin is highly polar, paeoniflorin is low polarity), this invention designs a specific gradient elution procedure, the specific steps of which are as follows: (1) Preparation of test solution: Take the contents of this product, add 50% ethanol solution, sonicate, filter, filter again, discard 2 ml of the initial filtrate, take 1 ml of the subsequent filtrate and place it in a sample vial as the test solution.

[0038] (2) Chromatographic conditions: Octadecylsilane bonded silica gel was used as the packing material; mobile phase A was 0.1% phosphoric acid solution and mobile phase B was acetonitrile; gradient elution was used, the detection wavelength was 235 nm and the column temperature was 35 °C.

[0039] The gradient elution program is as follows: at 0 min, phase A 95% and phase B 5%; at 30 min, phase A 65% and phase B 35%; at 45 min, phase A 10% and phase B 90%; at 50 min, phase A 95% and phase B 5%; at 50–60 min, phase A 95% and phase B 5% (equilibration column).

[0040] This method is the first to achieve the simultaneous determination of loganin from Cornus officinalis and paeoniflorin and paeoniflorin lactone from Paeonia lactiflora in a single injection of Compound Eucommia ulmoides and Waist-Strengthening Capsules, which greatly improves the detection efficiency.

[0041] This invention provides a method for establishing high-performance liquid chromatography fingerprints and evaluating the quality of Compound Eucommia Ulmoides Strengthening Capsules.

[0042] Under the chromatographic conditions for the determination of loganin, paeoniflorin, and paeoniflorin lactone, the present invention establishes a standard fingerprint spectrum containing 21 common peaks.

[0043] Specific methods include: (1) Prepare test solutions for more than 10 batches of samples and record the chromatograms; (2) A reference fingerprint (R) was generated using the “Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System”, and 21 common peaks were identified; (3) Identify the six characteristic peaks, namely protocatechuic acid (peak 6), loganin (peak 9), paeoniflorin (peak 10), paeoniflorin (peak 12), psoralen (peak 18), and isopsoralen (peak 19). (4) Quality evaluation standard: The similarity between the fingerprint spectrum of the test sample and the fingerprint spectrum of the control should not be less than 0.992.

[0044] This fingerprint spectrum covers the main active ingredient groups in the compound, and can sensitively reflect the quality fluctuations of raw materials and the stability of the production process, filling the gap in the overall quality evaluation of this preparation.

[0045] This invention provides a thin-layer chromatography (TLC) method for identifying multiple medicinal materials in Compound Eucommia Ulmoides Strengthening Capsules.

[0046] (1) Simultaneous identification of white peony and cornus officinalis: The "one plate double detection" technique was adopted. The same test solution was spotted, and dichloromethane-methanol-water (14:3:0.2) was used as the developing solvent. 5% vanillin sulfuric acid was used for color development. Paeoniflorin (blue-purple) and loganin (red-purple) spots could be clearly detected at the same time, and the negative results were not interfered with.

[0047] (2) Identification of ephedra: alkalization-dichloromethane extraction was used, followed by development with dichloromethane-methanol-concentrated ammonia solution (4:1:0.1), and ninhydrin was used for color development, resulting in clear spots.

[0048] (3) Identification of Rehmannia glutinosa: Water extraction-n-butanol extraction, xylene-ethyl acetate (1:1) development, 2,4-dinitrophenylhydrazine color development, with strong specificity.

[0049] The following section, with reference to the accompanying drawings, will explain the material basis analysis of this application. Grind 0.5g of the contents of Compound Eucommia Ulmoides Strengthening Capsules, dissolve in 10ml of methanol, sonicate for 40min, filter, and obtain the test solution. Take an appropriate amount of the reference standard, dissolve in methanol, and prepare a 10μg / ml mixed reference solution. Column temperature 35℃, mobile phase 0.1% formic acid water (A) - acetonitrile (B), gradient elution program as shown in Table 1: Table 1. LC-MS Material Basis Analysis - Gradient Elution Conditions

[0050] Electrospray ionization (ESI) was used for analysis in both positive and negative ion modes. Mass spectrometry data were analyzed using DataAnalysis software, and compounds in the sample solution were identified based on their relative molecular mass, ion fragmentation information, reference standards, and relevant literature reports.

[0051] A total of 92 compounds were identified, including 24 flavonoids, 22 phenylpropanoids, 17 terpenoids, 12 organic acids, 8 alkaloids, and 9 other chemical types.

[0052] like Figure 1 As shown, this invention employs ultra-high performance liquid chromatography-electrospray ionization mass spectrometry (UPLC-ESI-MS) to analyze the test solution. The mobile phase is 0.1% formic acid-acetonitrile, and elution is performed according to the gradient elution program in Table 1. The column temperature is 35℃. Base peak chromatograms (BPC) in positive ion mode (A) and negative ion mode (B) are collected respectively. In option A (positive ion mode BPC+AllMS): gradient elution was performed using 0.1% formic acid-water-acetonitrile as the mobile phase at a column temperature of 35℃. In positive ion mode, multiple characteristic chromatographic peaks (such as peaks 6, 10, 13, 60, and 64) were detected within a retention time range of 0–60 min. This mode exhibits good ionization response for compounds containing basic groups (such as alkaloids and some flavonoid aglycones), providing direct chromatographic retention behavior and mass spectrometry information for the identification of these compounds.

[0053] Among them, B (negative ion mode BPC-AllMS): Under the same chromatographic conditions, multiple characteristic chromatographic peaks (such as peaks 1, 2, 3, 25, 27, etc.) were detected in negative ion mode. This mode has a better ionization response to compounds containing acidic groups (such as organic acids, flavonoid glycosides, and phenylpropanoids), and complements the positive ion mode.

[0054] The above-mentioned positive and negative ion mode base peak chromatograms comprehensively characterized the chemical composition spectrum of Compound Eucommia ulmoides Strengthening Capsules. Combined with the molecular weight, fragment ion information and reference standard verification of mass spectrometry, this invention identified a total of 92 compounds, including flavonoids, phenylpropanoids, terpenoids, organic acids, alkaloids and other types, laying a material foundation for improving the quality standard of this preparation.

[0055] like Figure 2 As shown, this invention uses UPLC-ESI-MS to analyze the test sample and mixed reference solution. The chromatographic conditions are the same as in Table 1, and the baseline chromatograms are obtained in positive and negative ion modes, respectively: Positive ion mode (a, b) a is the positive ion mode chromatogram of the test solution, and b is the positive ion mode chromatogram of the mixed reference solution.

[0056] In the chromatogram of the test sample, characteristic peaks (peak 1: ephedrine hydrochloride, peak 2: pseudoephedrine hydrochloride, peak 3: psoralen, peak 4: isopsoralen) appear at the same retention time as the reference sample, indicating that the test sample contains these components and that the method has good specificity for basic target components in positive ion mode.

[0057] Negative ion mode (c, d) c is the negative ion mode chromatogram of the test solution, and d is the negative ion mode chromatogram of the mixed reference solution.

[0058] In the chromatogram of the test sample, characteristic peaks appeared at the same retention times as the reference sample (peak 5: protocatechuic acid, peak 6: monoglucoside, peak 7: loganin, peak 8: pinoresinol diglucoside, peak 9: paeoniflorin, peak 10: paeoniflorin), indicating that the test sample contains these components and that the method has good specificity for acidic / neutral target components in negative ion mode.

[0059] The above comparison results show that the UPLC-MS analysis method used in this invention can accurately identify the key quality control components in Compound Eucommia Ulmoides Capsules, providing a reliable technical basis for subsequent content determination, fingerprint spectrum establishment and thin-layer identification.

[0060] like Figure 3As shown, this invention employs ultra-high performance liquid chromatography-electrospray ionization mass spectrometry (UPLC-ESI-MS) to analyze the test solution. The mass spectrometry data is then analyzed using DataAnalysis software and compared with reference standards and precise mass information reported in relevant literature. This process identified 92 compounds in Compound Eucommia Ulmoides Strengthening Capsules. Figure 3 Detailed mass spectrometry information for some representative compounds is listed below: Compound: The name of the compound identified; Formula: The molecular formula of a compound; Retention time (min): The retention time of a compound on a chromatographic column; Ionmode / Fragmentor: The ion mode (e.g., [MH]⁻ is the negative ion mode) and fragmentation voltage for mass spectrometry detection; Calculatedmass(m / z): Theoretical exact molecular weight; Measuredmass (m / z): The experimentally measured molecular weight; Mass deviation (m / m): The deviation between the measured mass and the theoretical mass. All deviations are within ±5ppm, indicating that the identification results are accurate and reliable. Origin: The source of the compound in the prescription medicine (abbreviations represent different medicines, such as AY for mugwort, GL for licorice, etc.).

[0061] The above mass spectrometry information combined Figure 1 The baseline chromatograms of the two compounds jointly verified the identification results of 92 compounds in Compound Eucommia Ulmoides Strengthening Capsules, providing key data support for the elucidation of the material basis of the preparation and the improvement of its quality standards.

[0062] like Figure 4 As shown, to verify the accuracy of flavonoid identification, kaempferol (a representative flavonoid) was analyzed by secondary mass spectrometry (MS / MS) to obtain fragmentation pathways and characteristic fragment information. Figure 4 A: Fragmentation pathway The main fragmentation mechanisms of kaempferol under electrospray ionization (ESI) negative ion mode were demonstrated: the parent ion [MH]⁻ (m / z 285.0416) generated characteristic fragment ions through different fragmentation pathways, including m / z 241.0522 (loss of CO), m / z 271.0864 (loss of CH2O), m / z 151.0032 (A ring fragment), m / z 133.0299 (B ring fragment), etc. These fragmentation patterns are consistent with the typical fragmentation characteristics of flavonoids.

[0063] Figure 4B: Secondary fragment mass spectrum The image shows the secondary mass spectrum of the kaempferol precursor ion (m / z 285.0416). The fragment ion peaks detected in the image (such as m / z 241.0522, 151.0032, 133.0299, etc.) are in complete agreement with the theoretical fragmentation pathway, verifying the rationality of the fragmentation pathway and the accuracy of the structure identification.

[0064] The above results demonstrate that the UPLC-MS / MS technology used in this invention can not only identify the molecular weight of compounds, but also verify their structure through characteristic fragmentation patterns, providing a reliable method for the accurate identification of complex components such as flavonoids in Compound Eucommia Ulmoides Capsules.

[0065] like Figure 5 As shown, to verify the accuracy of the identification of key quality control components of coumarins, psoralen was used as an example, and secondary mass spectrometry (MS / MS) analysis was performed to obtain fragmentation pathways and characteristic fragment information: Figure 5 A: Fragmentation pathway The main fragmentation mechanisms of psoralen in electrospray ionization (ESI) positive ion mode were demonstrated: the parent ion [M+H]⁺ (m / z 187.0385) generated characteristic fragment ions through different fragmentation pathways, including m / z 159.0454 (loss of CO), m / z 143.0489 (loss of CO2), m / z 131.0488, m / z 115.0539, etc. These fragmentation patterns are consistent with the typical fragmentation characteristics of coumarin compounds.

[0066] Figure 5 B: Secondary fragment mass spectrum The image shows the secondary mass spectrum of the psoralen precursor ion (m / z 187.0385). The fragment ion peaks detected in the image (such as m / z 159.0454, 143.0489, 131.0488, etc.) are in complete agreement with the theoretical fragmentation pathway, verifying the rationality of the fragmentation pathway and the accuracy of the structure identification.

[0067] The above results demonstrate that the UPLC-MS / MS technology used in this invention can accurately resolve the structure of psoralen, providing a reliable technical basis for the content determination of this component and the quality control of Compound Eucommia Ulmoides Capsules.

[0068] like Figure 6 As shown, to verify the accuracy of organic acid compound identification, catechin (a representative organic acid component) was analyzed by secondary mass spectrometry (MS / MS) to obtain fragmentation pathway and characteristic fragment information. Figure 6 A: Fragmentation pathway The main fragmentation mechanism of catechin in electrospray ionization (ESI) negative ion mode is shown: the parent ion [MH]⁻ (m / z 153.0195) generates characteristic fragment ions m / z 109.0302 (loss of CO2) and m / z 91.0207 (further loss of H2O) by continuously losing CO2 and H2O. These fragmentation patterns are consistent with the typical fragmentation characteristics of phenolic acid compounds.

[0069] Figure 6 B: Secondary fragment mass spectrum The image shows the secondary mass spectrum of the catechin precursor ion (m / z 153.0195). The fragment ion peaks detected in the image (m / z 109.0302, 91.0207, etc.) are in complete agreement with the theoretical fragmentation pathway, verifying the rationality of the fragmentation pathway and the accuracy of the structure identification.

[0070] The above results demonstrate that the UPLC-MS / MS technology used in this invention can accurately resolve the structure of organic acid compounds, providing a reliable method for the comprehensive identification of multiple types of components in Compound Eucommia Ulmoides Strengthening Capsules.

[0071] like Figure 7 As shown, to verify the accuracy of the identification of key quality control components of alkaloids, ephedrine was used as an example, and secondary mass spectrometry (MS / MS) analysis was performed to obtain fragmentation pathways and characteristic fragment information: Figure 7 A: Fragmentation pathway The main fragmentation mechanism of ephedrine in electrospray ionization (ESI) positive ion mode was demonstrated: the parent ion [M+H]⁺ (m / z 166.1226) generates fragment ions with m / z 148.1122 by losing NH3, which further breaks down to obtain characteristic fragments with m / z 133.0882, 117.0699, etc. These fragmentation patterns are consistent with the typical fragmentation characteristics of alkaloid compounds.

[0072] Figure 7 B: Secondary fragment mass spectrum The image shows the secondary mass spectrum of the ephedrine precursor ion (m / z 166.1226). The fragment ion peaks detected in the image (m / z 148.1122, 133.0882, 117.0699, etc.) are in complete agreement with the theoretical fragmentation pathway, verifying the rationality of the fragmentation pathway and the accuracy of the structure identification.

[0073] The above results demonstrate that the UPLC-MS / MS technology used in this invention can accurately resolve the structure of alkaloid compounds, providing a reliable basis for the identification and quality control of key quality control components in Compound Eucommia Ulmoides Capsules.

[0074] like Figure 8As shown, to verify the accuracy of the identification of key quality control components of iridoid glycosides, loganin was used as an example, and secondary mass spectrometry (MS / MS) analysis was performed to obtain fragmentation pathway and characteristic fragment information: Figure 8 A: Fragmentation pathway The main fragmentation pathways of loganin in the electrospray ionization (ESI) negative ion mode were demonstrated: the parent ion [MH]⁻ (m / z 433.1625) generates an aglycone fragment of m / z 271.1057 by losing a glucose unit (162 Da), and further loses small molecules such as CO2 and CH2O to generate characteristic fragments such as m / z 227.1097 and 121.0297; at the same time, there is another fragmentation pathway, which loses a sugar ring fragment to generate a fragment of m / z 409.1462, and finally obtains an aglycone-related fragment of m / z 165.0641. These fragmentation patterns are consistent with the typical fragmentation characteristics of iridoid glycosides.

[0075] Figure 8 B: Secondary fragment mass spectrum The image shows the secondary mass spectrum of the loganin precursor ion (m / z 433.1625). The fragment ion peaks detected in the image (m / z 271.1057, 227.1097, 121.0297, etc.) are in complete agreement with the theoretical fragmentation pathway, verifying the rationality of the fragmentation pathway and the accuracy of the structural identification.

[0076] The above results demonstrate that the UPLC-MS / MS technology used in this invention can accurately resolve the structure of glycoside compounds, providing a reliable basis for the identification and quality control of key quality control components in Compound Eucommia Ulmoides Capsules.

[0077] like Figure 9 As shown, this invention uses high performance liquid chromatography (HPLC) to determine the content of psoralen and isopsoralen. After investigating the extraction solvent, mobile phase ratio, chromatographic column, and other conditions, the optimal method was determined as follows: 0.5 g of the contents were taken and extracted with 50 ml of hydrochloric acid-methanol (1:4) as the extraction solvent, and the mixture was heated under reflux for 1 h; the chromatographic conditions were: octadecylsilane-bonded silica column, mobile phase: methanol-0.4% phosphoric acid solution (45:55), detection wavelength: 246 nm, flow rate: 1.0 mL / min, injection volume: 10 μL, and column temperature: 35 °C. Figure 9 To validate the specificity of this method, the following chromatograms were used: Figure 9 A (test solution): Under the above chromatographic conditions, the test solution showed characteristic peaks (peak 1: psoralen, peak 2: isopsoralen) at retention times of approximately 14.0 min and 16.0 min, respectively. The peaks were symmetrical and the separation was good, indicating that the target component could be effectively detected in the test sample.

[0078] Figure 9Solution B (psoralen and isopsoralen reference solutions): Two clear chromatographic peaks appeared at the exact same retention time as the test sample, and the retention time and peak shape were completely consistent with the test sample peak, verifying the peak correspondence.

[0079] Figure 9 C (negative sample solution lacking psoralen): No interfering peaks appeared within the retention time range of psoralen and isopsoralen, indicating that other medicinal materials in the prescription did not interfere with the determination of the target components, and the method has good specificity.

[0080] The linearity, stability, repeatability, precision, and recovery of this method are all good, indicating that the HPLC content determination method established in this invention can accurately and stably determine the content of psoralen and isopsoralen in Compound Eucommia Ulmoides Capsules, providing a reliable technical means for the quality control of this preparation.

[0081] like Figure 10 As shown, this invention uses high-performance liquid chromatography (HPLC) to determine the contents of loganin, paeoniflorin, and paeoniflorin lactone. After investigating the extraction solvent, mobile phase gradient, and chromatographic column conditions, the optimal method was determined: 0.5 g of the contents were extracted with 50% ethanol solution as the extraction solvent, and ultrasonically extracted for 30 min; the chromatographic conditions were an octadecylsilane-bonded silica column, an acetonitrile-0.1% phosphoric acid solution as the mobile phase, and the gradient elution program as follows: 0 min: Phase A (0.1% phosphoric acid solution) 95%, Phase B (acetonitrile) 5% 30 min: Phase A 65%, Phase B 35% 45 min: Phase A 10%, Phase B 90% 50 min: Phase A 95%, Phase B 5% 50–60 min: Phase A 95%, Phase B 5% (equilibrium column) The detection wavelength was 235 nm, the flow rate was 1.0 mL / min, the injection volume was 10 μL, and the column temperature was 35 °C.

[0082] In the picture: The test solution showed three clear characteristic peaks at the characteristic retention time: loganin (peak 1), paeoniflorin (peak 2), and paeoniflorin (peak 3). The peaks were symmetrical and well separated, indicating that the target components could be effectively detected in the test sample.

[0083] The linearity, stability, repeatability, specificity, precision, and recovery of this method are all good, indicating that the HPLC content determination method established in this invention can accurately and stably determine the content of loganin, paeoniflorin, and paeoniflorin lactone in Compound Eucommia ulmoides Strengthening Capsules, providing a reliable technical means for the multi-index quality control of this preparation.

[0084] like Figure 11 As shown, this invention uses high-performance liquid chromatography (HPLC) to determine the contents of loganin, paeoniflorin, and paeoniflorin lactone. The optimal method has been determined as follows: 0.5 g of the contents are extracted with 50% ethanol solution as the extraction solvent by ultrasonic extraction for 30 min; the chromatographic conditions are: octadecylsilane-bonded silica column, mobile phase is acetonitrile-0.1% phosphoric acid solution, and the gradient elution program is as follows: 0 min: Phase A (0.1% phosphoric acid solution) 95%, Phase B (acetonitrile) 5% 30 min: Phase A 65%, Phase B 35% 45 min: Phase A 10%, Phase B 90% 50 min: Phase A 95%, Phase B 5% 50–60 min: Phase A 95%, Phase B 5% (equilibrium column) The detection wavelength was 235 nm, the flow rate was 1.0 mL / min, the injection volume was 10 μL, and the column temperature was 35 °C. Figure 11 The specificity verification results for this method are as follows: Figure a (test solution): Three clear characteristic peaks appear at the characteristic retention time, namely loganin (peak 1), paeoniflorin (peak 2), and paeoniflorin (peak 3). The peaks are symmetrical and well separated, indicating that the target components can be effectively detected in the test sample.

[0085] Figure b (reference solution): Three characteristic peaks appear at the exact same retention time as the test sample, and the retention time and peak shape are completely consistent with the peaks of the test sample, verifying the correspondence of the peaks.

[0086] Figure c (negative sample solution lacking white peony root): No interfering peaks appeared within the retention time range of paeoniflorin (peak 2) and paeoniflorin (peak 3), indicating that other medicinal materials in the prescription, except for white peony root, did not interfere with the determination of these two components.

[0087] In the figure, d (negative sample solution lacking Cornus officinalis): no interfering peaks appeared within the retention time range of loganin (peak 1), indicating that other medicinal materials in the prescription, except for Cornus officinalis, did not interfere with the determination of loganin.

[0088] The linearity, stability, repeatability, precision, and recovery of this method are all good, indicating that the HPLC content determination method established in this invention can accurately and stably determine the content of loganin, paeoniflorin, and paeoniflorin lactone in Compound Eucommia ulmoides Strengthening Capsules, providing a reliable technical means for the multi-index quality control of this preparation.

[0089] like Figure 12As shown, the present invention uses the same method as the determination of loganin, paeoniflorin, and paeoniflorin content to prepare the test solution (take 0.5 g of the contents, use 50% ethanol solution as the extraction solvent, and ultrasonically extract for 30 min), and under the same chromatographic conditions: octadecylsilane-bonded silica column, acetonitrile-0.1% phosphoric acid solution as the mobile phase, and the gradient elution program is as follows: 0 min: Phase A (0.1% phosphoric acid solution) 95%, Phase B (acetonitrile) 5% 30 min: Phase A 65%, Phase B 35% 45 min: Phase A 10%, Phase B 90% 50 min: Phase A 95%, Phase B 5% 50–60 min: Phase A 95%, Phase B 5% (equilibrium column) The fingerprint analysis of 10 batches of Compound Eucommia ulmoides Strengthening Capsules was performed using a detection wavelength of 235 nm, a flow rate of 1.0 mL / min, an injection volume of 10 μL, and a column temperature of 35 ℃. Figure a (fingerprint spectrum of the test sample): A total of 21 common peaks were identified, of which 6 characteristic peaks were confirmed by reference standards, namely protocatechuic acid (peak 6), loganin (peak 9), paeoniflorin (peak 10), paeoniflorin (peak 12), psoralen (peak 18), and isopsoralen (peak 19). The retention time and peak area of ​​each common peak showed good repeatability.

[0090] Figure b (fingerprint of reference standard): Six characteristic peaks appear at the same retention time as the test sample, which are completely consistent with the retention time of the peaks in the test sample, verifying the component attribution of the common peaks.

[0091] The chromatographic data of 10 batches of test samples were imported into the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine. The results showed that the similarity ranged from 0.992 to 1.000, indicating that the compound Eucommia ulmoides strengthening waist capsules had uniform quality and stable process. This fingerprint can be used for the overall quality control of the preparation and, together with the previous content determination method, constitutes a multi-dimensional quality evaluation system.

[0092] like Figure 13 As shown, the present invention uses the same method as the determination of loganin, paeoniflorin, and paeoniflorin content to prepare the test solution (take 0.5 g of the contents, use 50% ethanol solution as the extraction solvent, and ultrasonically extract for 30 min), and under the same chromatographic conditions: octadecylsilane-bonded silica column, acetonitrile-0.1% phosphoric acid solution as the mobile phase, and the gradient elution program is as follows: 0 min: Phase A (0.1% phosphoric acid solution) 95%, Phase B (acetonitrile) 5% 30 min: Phase A 65%, Phase B 35% 45 min: Phase A 10%, Phase B 90% 50 min: Phase A 95%, Phase B 5% 50–60 min: Phase A 95%, Phase B 5% (equilibrium column) The detection wavelength was 235 nm, the flow rate was 1.0 mL / min, the injection volume was 10 μL, and the column temperature was 35 ℃. Fingerprint analysis was performed on 10 batches of Compound Eucommia Ulmoides Strengthening Capsules, and the superimposed fingerprint spectra were obtained. R (reference fingerprint): A reference fingerprint was generated based on the common pattern of 10 batches of test samples. A total of 21 common peaks were identified, of which 6 characteristic peaks were identified by reference standards (peak 3: protocatechuic acid, peak 7: loganin, peak 10: paeoniflorin, peak 12: paeoniflorin, peak 18: psoralen, peak 19: isopsoralen).

[0093] S1–S10 (Fingerprints of 10 batches of test samples): After the fingerprints of 10 batches of test samples are superimposed with the control fingerprint (R), the retention times and peak heights of all 21 common peaks are consistent, and there is no obvious peak position shift or peak area difference, which intuitively reflects the consistency of composition of different batches of samples.

[0094] The chromatographic data of 10 batches of test samples were imported into the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine. The results showed that the similarity ranged from 0.992 to 1.000, indicating that the compound Eucommia ulmoides strengthening waist capsules had uniform quality and stable process. This superimposed fingerprint spectrum can be used for the overall quality control of the preparation, and together with the previous content determination method, it constitutes a multi-dimensional quality evaluation system.

[0095] like Figure 14 As shown, this invention uses the same method as the determination of loganin, paeoniflorin, and paeoniflorin content (0.5g sample, 50% ethanol solution as extraction solvent, ultrasonic extraction for 30min) to prepare the test sample and solutions of 17 single medicinal materials, and under the same chromatographic conditions: octadecylsilane-bonded silica column, acetonitrile-0.1% phosphoric acid solution as the mobile phase, and the gradient elution program is as follows: 0 min: Phase A (0.1% phosphoric acid solution) 95%, Phase B (acetonitrile) 5% 30 min: Phase A 65%, Phase B 35% 45 min: Phase A 10%, Phase B 90% 50 min: Phase A 95%, Phase B 5% 50–60 min: Phase A 95%, Phase B 5% (equilibrium column) The analysis was performed at a detection wavelength of 235 nm, a flow rate of 1.0 mL / min, an injection volume of 10 μL, and a column temperature of 35 °C. a–q (chromatograms of single medicinal materials): These are chromatograms of Artemisia argyi, Paeonia lactiflora, Psoralea corylifolia, Codonopsis pilosula, Eucommia ulmoides, Cibotium barometz, Lycium barbarum, Allium tuberosum seed, Cervi cornu, Ephedra sinica, Achyranthes bidentata, Cornus officinalis, Rehmannia glutinosa, Cuscuta chinensis, Asarum heterotropoides, Corydalis yanhusuo, and sheep tibia, respectively, showing the characteristic chromatographic peaks of each medicinal material.

[0096] r (Chromatogram of the test sample): The chromatographic peaks of the test sample correspond to the characteristic peaks of each single herb, such as the paeoniflorin and paeoniflorin-lactone peaks of white peony root, the loganin peak of cornus officinalis, and the psoralen and isopsoralen peaks of psoralea corylifolia, etc., which verify the herb attribution of the common peaks in the fingerprint spectrum.

[0097] The results show that the common peaks of the fingerprint spectrum established in this invention correspond highly with the characteristic peaks of the prescription medicinal materials, providing a reliable basis for the quality traceability of Compound Eucommia ulmoides and other medicinal materials. Together with the previous fingerprint spectrum and content determination method, it constitutes a multi-dimensional quality control system.

[0098] like Figure 15 As shown, this invention establishes a thin-layer chromatography identification method for Paeonia lactiflora, as detailed below: Solution preparation: Take 4g (8 capsules) of the contents of this product, grind them into a fine powder, add 50ml of ethanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of ethanol, and use it as the test solution; take paeoniflorin reference standard, add ethanol to prepare a solution containing 1mg per 1ml, and use it as the reference solution; prepare negative samples lacking white peony root according to the prescription process, and prepare negative sample solutions in the same way.

[0099] Identification method: Take 5 μL each of the test solution, negative sample solution, and control solution and spot them separately on the same silica gel G thin layer plate. Use dichloromethane-methanol-water (14:3:0.2) as the developing solvent, develop, remove and air dry, spray with 5% vanillin sulfuric acid solution, and heat at 105℃ until the spots are clearly visible.

[0100] result( Figure 15 In the chromatogram of the test solution (points 1 and 2), blue-purple spots appear at the same positions as those in the chromatogram of the paeoniflorin reference solution (point 4); in the chromatogram of the negative sample solution (point 3) lacking white peony root, no interfering spots appear at the corresponding positions, indicating that the method has good specificity and can be used for the identification of white peony root in Compound Eucommia Ulmoides Capsules.

[0101] like Figure 16 As shown, this invention establishes a thin-layer chromatography identification method for ephedra, as detailed below: Solution preparation: Take 4g (8 tablets) of the contents of this product, grind them into a fine powder, add 5mL of concentrated ammonia test solution and 50mL of dichloromethane, sonicate for 50 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1mL of methanol to prepare the test solution; take ephedrine reference standard, add methanol to prepare a solution containing 1mg per 1ml to prepare the reference solution; prepare a negative sample lacking ephedrine according to the prescription process, and prepare the negative sample solution in the same way.

[0102] Identification method: Take 5 μL each of the test solution, negative sample solution, and control solution and spot them separately on the same silica gel G thin layer plate. Use dichloromethane-methanol-concentrated ammonia solution (4:1:0.1) as the developing solvent, develop, remove and air dry, spray with ninhydrin solution, and heat at 105℃ until the spots are clearly visible.

[0103] result( Figure 16 In the chromatogram of the test solution (points 1 and 2), reddish-purple spots appear at the same positions as those in the chromatogram of the ephedrine reference solution (point 4); in the chromatogram of the negative sample solution (point 3) lacking ephedrine, no interfering spots appear at the corresponding positions, indicating that the method has good specificity and can be used for the identification of ephedrine in Compound Eucommia Ulmoides Capsules.

[0104] like Figure 17 As shown, this invention establishes a thin-layer chromatographic identification method for Cornus officinalis, as detailed below: Solution preparation: Take 4g (8 capsules) of the contents of this product, grind them into a fine powder, add 50ml of ethanol, sonicate for 20 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of ethanol to prepare the test solution; take loganin reference standard, add ethanol to prepare a solution containing 1mg per 1ml to prepare the reference solution; prepare a negative sample without Cornus officinalis according to the prescription process, and prepare the negative sample solution in the same way.

[0105] Identification method: Take 5 μL each of the test solution, negative sample solution, and control solution and spot them separately on the same silica gel G thin layer plate. Use dichloromethane-methanol-water (14:3:0.2) as the developing solvent, develop, remove and air dry, spray with 5% vanillin sulfuric acid solution, and heat at 105℃ until the spots are clearly visible.

[0106] result( Figure 17 In the chromatogram of the test solution (points 1 and 2), reddish-purple spots appear at the same positions as those in the chromatogram of the loganin reference solution (point 4); in the chromatogram of the negative sample solution lacking Cornus officinalis (point 3), no interfering spots appear at the corresponding positions, indicating that the method has good specificity and can be used for the identification of Cornus officinalis in Compound Eucommia ulmoides Strengthening Capsules.

[0107] like Figure 18 As shown, this invention establishes a thin-layer chromatographic identification method for Rehmannia glutinosa, as detailed below: Solution preparation: Take 4g (8 capsules) of the contents of this product, add 100mL of water, heat to boiling for 30 minutes, cool and centrifuge, take the supernatant and extract twice with water-saturated n-butanol (20mL each time), combine the n-butanol solutions and evaporate to dryness, dissolve the residue in 1mL of methanol to obtain the test solution; take 5g of prepared Rehmannia glutinosa reference material to prepare the reference material solution in the same way; prepare negative samples lacking prepared Rehmannia glutinosa according to the prescription process, and prepare negative sample solutions in the same way.

[0108] Identification method: Take 5 μL each of the test solution, negative sample solution, and control medicinal material solution and spot them separately on the same silica gel G thin layer plate. Use xylene-ethyl acetate (1:1) as the developing solvent, develop, remove and air dry, spray with 2,4-dinitrophenylhydrazine ethanol test solution, heat at 105℃ until the spots are clearly visible, and examine under sunlight.

[0109] result( Figure 18 In the chromatograms of the test solution (points 1 and 2), green spots appear at the same positions as those in the chromatogram of the prepared Rehmannia glutinosa reference solution (point 4); in the chromatogram of the negative sample solution lacking prepared Rehmannia glutinosa (point 3), no interfering spots appear at the corresponding positions, indicating that the method has good specificity and can be used for the identification of prepared Rehmannia glutinosa in Compound Eucommia ulmoides Strengthening Capsules.

[0110] It should be noted that this invention relates to two different chromatographic analysis methods: (1) LC-MS material basis analysis: 0.1% formic acid water (A)-acetonitrile (B) was used as the mobile phase, and the gradient elution program was as follows: 0–5 min A phase 90%→90%, 5–45 min A phase 90%→30%, 45–50 min A phase 30%→5%, 50–60 min A phase 5%→5%, 60–61 min A phase 5%→90%, 61–70 min A phase 90%→90%. (2) HPLC content determination and fingerprint chromatogram: 0.1% phosphoric acid solution (A)-acetonitrile (B) was used as the mobile phase, and the gradient elution program is shown in the table below: Table 2 HPLC content determination and fingerprint chromatogram - gradient elution procedure

[0111] The two methods use different mobile phase acid additives and gradient programs, and are used for compound identification and quantitative analysis, respectively.

[0112] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for overall quality control of Compound Eucommia Ulmoides Strengthening Capsules, characterized in that, The method includes the determination of the contents of psoralen and isopsoralen, as well as the determination of the contents of loganin, paeoniflorin and paeoniflorin lactone. The determination of the contents of psoralen and isopsoralen includes: preparing a test solution by heating and reflux extraction with hydrochloric acid-methanol solution as the extraction solvent; and performing isocratic elution with octadecylsilane-bonded silica gel as the packing material and methanol-phosphoric acid aqueous solution as the mobile phase. The determination of the contents of loganin, paeoniflorin and paeoniflorin lactone includes: preparing the test solution by ultrasonic extraction using ethanol aqueous solution as the extraction solvent; performing gradient elution using octadecylsilane-bonded silica gel as the packing material and acetonitrile-phosphoric acid aqueous solution as the mobile phase, with a detection wavelength of 235 nm.

2. The overall quality control method for Compound Eucommia Ulmoides Strengthening Capsules according to claim 1, characterized in that, In the determination of the contents of psoralen and isopsoralen, the extraction solvent was a mixed solution of hydrochloric acid and methanol in a volume ratio of 1:4; the mobile phase was methanol-0.4% phosphoric acid solution in a volume ratio of 45:55; the detection wavelength was 246 nm; and the column temperature was 35 °C.

3. The overall quality control method for Compound Eucommia Ulmoides Strengthening Capsules according to claim 1, characterized in that, In the determination of the contents of loganin, paeoniflorin and paeoniflorin lactone, the extraction solvent was 50% ethanol solution; mobile phase A was 0.1% phosphoric acid solution, and mobile phase B was acetonitrile; the gradient elution program was as follows: at 0 min, phase A 95% and phase B 5%; at 30 min, phase A 65% and phase B 35%; at 45 min, phase A 10% and phase B 90%; at 50 min, phase A 95% and phase B 5%; at 50–60 min, phase A 95% and phase B 5%.

4. The overall quality control method for Compound Eucommia Ulmoides Strengthening Capsules according to claim 1 or 3, characterized in that, The method also includes establishing a fingerprint spectrum for Compound Eucommia Ulmoides Strengthening Capsules: The same chromatographic conditions as those used for the determination of the contents of loganin, paeoniflorin and paeoniflorin lactone were used to record the chromatogram and identify 21 common peaks. Six characteristic peaks were identified as protocatechuic acid, loganin, paeoniflorin, psoralen, and isopsoralen. Calculate the similarity between the fingerprint chromatogram of the test sample and the fingerprint chromatogram of the control sample; the similarity should not be less than 0.

992.

5. The overall quality control method for Compound Eucommia Ulmoides Strengthening Capsules according to claim 1, characterized in that, The method also includes using thin-layer chromatography to simultaneously identify white peony root and cornus fruit in Compound Eucommia ulmoides and Waist-Strengthening Capsules: Preparation of test solution: Take the contents and extract with ethanol by ultrasonication; Development and color development: Take the test solution and the reference solution and spot them on the same silica gel G thin layer plate. Develop the plate with dichloromethane:methanol:water = 14:3:0.2 as the developing solvent. Spray with 5% vanillin sulfuric acid solution and heat until the spots are clearly visible. Result interpretation: In the chromatogram of the test sample, blue-purple spots and red-purple spots appeared at the corresponding positions as paeoniflorin reference standard and loganin reference standard, respectively.

6. The overall quality control method for Compound Eucommia Ulmoides Strengthening Capsules according to claim 1, characterized in that, The method also includes using thin-layer chromatography to identify ephedra in Compound Eucommia Ulmoides Strengthening Capsules: Preparation of test solution: Take the contents, add concentrated ammonia test solution and dichloromethane, and extract by ultrasonication; Development and color development: Using dichloromethane:methanol:concentrated ammonia solution = 4:1:0.1 as the developing solvent, spray with ninhydrin solution and heat to develop color.

7. The overall quality control method for Compound Eucommia Ulmoides Strengthening Capsules according to claim 1, characterized in that, The method also includes using thin-layer chromatography to identify Rehmannia glutinosa in Compound Eucommia ulmoides and Waist-Strengthening Capsules: Preparation of test solution: Boil the contents in water, and extract the supernatant with water-saturated n-butanol; Development and color development: Using xylene:ethyl acetate = 1:1 as the developing solvent, spray with 2,4-dinitrophenylhydrazine ethanol solution and heat to develop color.

8. The overall quality control method for Compound Eucommia Ulmoides Strengthening Capsules according to claim 4, characterized in that, In the construction of the fingerprint spectrum, the test solution is prepared as follows: take 0.5g of the contents, add 50ml of 50% ethanol solution, extract by ultrasonication for 30min, filter, discard 2ml of the initial filtrate, take 1ml of the subsequent filtrate, and place it in a sample vial to obtain the fingerprint spectrum.

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