Fingerprint spectrum and determination method of multi-component content of Xiangsha Pingwei granules

Abstract

The application provides a Xiangsha Pingwei granules fingerprint spectrum and a multi-component content determination method, adopts optimized pretreatment and a high performance liquid chromatography method, realizes qualitative analysis of 20 components of 6 medicinal materials in the Xiangsha Pingwei granules under the same chromatographic condition, and identifies 8 chemical components including chlorogenic acid, magnolianoside B, magnolianoside A, hesperidin, quercetin, glycyrrhizic acid, honokiol and magnolol; meanwhile, quantitative analysis is conducted on 7 chemical components including chlorogenic acid, magnolianoside B, magnolianoside A, hesperidin, glycyrrhizic acid (taking ammonium glycyrrhizinate as a control, conversion: glycyrrhizic acid weight = ammonium glycyrrhizinate / 1.0207), honokiol and magnolol. The application solves the technical problems that the chemical components of the Xiangsha Pingwei granules are complex, the chromatographic peaks of the components are prone to mutual interference, and it is difficult to comprehensively control the quality of the medicine, and the quality of the Xiangsha Pingwei granules can be more effectively controlled.

Description

Technical Field

[0001] This invention relates to the field of quality control of traditional Chinese medicine, specifically to a detection method for Xiangsha Pingwei granules. Background Technology

[0002] The Xiangsha Pingwei Granules of this invention are granules derived from the modified "Pingwei Powder" in "Jianyao Jizhong Fang" compiled by Zhou Ying of the Northern Song Dynasty (adding Cyperus rotundus and Amomum villosum, and reducing ginger and jujube). It is a traditional Chinese medicine compound preparation made from stir-fried Atractylodes lancea, ginger-processed Magnolia officinalis, Citrus reticulata peel, vinegar-processed Cyperus rotundus, Glycyrrhiza uralensis, and Amomum villosum. It has the effects of strengthening the spleen, warming the middle jiao, and drying dampness. It is used to treat irregular diet, food and dampness stagnation, stomach distension and pain, and indigestion.

[0003] The quality standard for Xiangsha Pingwei Granules is included in Volume 12 of the "Drug Standards of the Ministry of Health" for Traditional Chinese Medicine Compound Preparations, WS3-B-2369-97. The current quality standard only controls the quality of the appearance and the inspection items under the granule section. The quality standard is low and cannot comprehensively control Xiangsha Pingwei Granules.

[0004] A few studies have reported on the quality control improvement of Xiangsha Pingwei Granules. Among them, the literature "Simultaneous determination of hesperidin, magnolol and honokiol in Xiangsha Pingwei Granules by HPLC [J]. West China Journal of Pharmaceutical Sciences, 2017, 32(5):531-533" uses reversed-phase high-performance liquid chromatography with a diode array detector and methanol-water gradient elution as the mobile phase to determine the content of hesperidin, magnolol and honokiol. This method can only perform quantitative analysis of three components and lacks fingerprint spectroscopy determination. The literature "Quality evaluation of Xiangsha Pingwei Granules based on fingerprint spectroscopy combined with chemometrics [J]. China Pharmaceutical Industry, 2022, 31(23): 88-91” established an HPLC fingerprint analysis method and combined it with chemometrics for the quality control of Xiangsha Pingwei Granules. This method only uses fingerprint analysis for qualitative research and lacks quantitative determination of indicator components. The literature “Determination of Atractylodes, Magnolol, Honokiol, Hesperidin, Rutin, and Glycyrrhizin in Xiangsha Pingwei Granules by UPLC-MS / MS [J]. Modern Drugs and Clinical, 2023, 38(2): 330-334” established a UPLC-MS / MS method to simultaneously determine atractylodes, honokiol, honokiol, hesperidin, rutin, and glycyrrhizin in Xiangsha Pingwei Granules. This method requires UPLC-MS / MS, which has high requirements for instruments and operators, high operating costs, and can only perform quantitative analysis of 6 components, lacking fingerprint analysis. The methods in the above three literatures cannot fully reflect the product quality.

[0005] Currently, there are no literature or patent reports on controlling the quality of Xiangsha Pingwei Granules by simultaneously measuring the content of multiple components using fingerprint spectroscopy. Existing technologies cannot achieve comprehensive quality control of Xiangsha Pingwei Granules.

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a fingerprint spectrum and multi-component content determination method for Xiangsha Pingwei granules. Using optimized pretreatment and high-performance liquid chromatography (HPLC) methods, qualitative analysis of 20 components from 6 medicinal herbs in Xiangsha Pingwei granules can be achieved under the same chromatographic conditions, identifying 8 chemical components: chlorogenic acid, magnologenin B, magnologenin A, hesperidin, quercetin, glycyrrhizic acid, and magnolol. Simultaneously, quantitative analysis of 7 chemical components in Xiangsha Pingwei granules—chlorogenic acid, magnologenin B, magnologenin A, hesperidin, glycyrrhizic acid, and magnolol—is performed. This invention solves the technical problems of the complex chemical composition of Xiangsha Pingwei granules, the easy interference between chromatographic peaks of various components, and the difficulty in comprehensively controlling the quality of the drug, thus enabling more effective quality control of Xiangsha Pingwei granules. Summary of the Invention

[0007] This invention provides a fingerprint spectrum of Xiangsha Pingwei granules and a method for determining the content of various components, comprising the following steps:

[0008] (1) Preparation of reference solution: Take chlorogenic acid, magnolol B, magnolol A, hesperidin, quercetin, ammonium glycyrrhizate, and magnolol and magnolol reference standards, dissolve them in solvent and make up to volume to obtain the solution;

[0009] (2) Preparation of test solution: Take Xiangsha Pingwei granules sample, add solvent to extract, filter, and take the filtrate to obtain the test solution;

[0010] (3) Preparation of single herbal medicine sample solution: Take a single herbal medicine sample, add solvent to extract, filter, and take the filtrate to obtain the solution;

[0011] (4) Preparation of negative test solution: Take each negative sample, add solvent to extract, filter, and take the filtrate to obtain the solution;

[0012] (5) Fingerprint chromatogram establishment: High performance liquid chromatography was used to inject the reference solution from step (1), the test solution from step (2), the single herb test solution from step (3), and the negative test solution from step (4) into the high performance liquid chromatograph, record the chromatogram, and construct the fingerprint chromatogram of Xiangsha Pingwei Granules.

[0013] (6) Content determination: Using the same chromatographic conditions as in step (5), take the reference solution from step (1) and the test solution from step (2), inject them into the high performance liquid chromatograph, record the chromatogram, and calculate the content of chlorogenic acid, magnolol A, magnolol B, hesperidin, glycyrrhizic acid, honokiol and honokiol in the test solution using the external standard method.

[0014] In the chromatographic conditions of the high performance liquid chromatography, mobile phase A is methanol and mobile phase B is 0.1% phosphoric acid aqueous solution, with gradient elution.

[0015] Preferably, the gradient elution program is as follows: 0~25 min, 10%→25% A; 25~50 min, 25%→40% A; 50~60 min, 40%→60% A; 60~70 min, 60%→80% A; 70~80 min, 80%→90% A; 80~85 min, 90% A.

[0016] Preferably, the chromatographic column used in the high-performance liquid chromatography is a C18 column. More preferably, the chromatographic column is a Shim-pack CLC-ODS, 150 × 6.0 mm, 5 μm.

[0017] Preferably, the flow rate of the high-performance liquid chromatography (HPLC) is 0.95~1.00 mL / min. More preferably, the flow rate of the HPLC is 1.00 mL / min.

[0018] Preferably, the column temperature of the high-performance liquid chromatography (HPLC) is 20-30°C. More preferably, the column temperature of the HPLC is 25°C.

[0019] Preferably, the injection volume of the high-performance liquid chromatography (HPLC) is 5-20 μL. More preferably, the injection volume of the HPLC is 10 μL.

[0020] Preferably, the detection wavelength of the high-performance liquid chromatography (HPLC) is selected from one or more combinations of 220 nm, 250 nm, and 330 nm. More preferably, the detection wavelength of the HPLC varies with the time of the gradient elution program, specifically as follows: 0~20 min, detection wavelength is 330 nm; 20~65 min, detection wavelength is 220 nm; 65~75 min, detection wavelength is 250 nm; 75~85 min, detection wavelength is 220 nm.

[0021] Preferably, the solvent used in steps (1) and (2) is a 50% to 90% methanol solution, more preferably, the solvent used is an 80% methanol solution.

[0022] Preferably, the extraction method in step (2) is reflux extraction, cold soaking extraction, or ultrasonic extraction. More preferably, the extraction method is ultrasonic extraction.

[0023] More preferably, the ultrasonic extraction time in step (2) is 20-50 min, preferably 30 min. The ultrasonic extraction power is 200-400 W, preferably 250 W. The ultrasonic extraction frequency is 30-50 kHz, preferably 40 kHz.

[0024] Preferably, the weight-to-volume ratio of the Xiangsha Pingwei granules sample to be tested in step (2) to the 50%~90% methanol solution is equivalent to 3.0~10g of medicinal slices: 50mL, and more preferably equivalent to 4.5~5.4g of medicinal slices: 50mL.

[0025] This invention provides a fingerprint pattern construction method for Xiangsha Pingwei granules, which includes the following steps:

[0026] (1) Ten batches of test sample solutions were injected sequentially to obtain chromatograms of ten batches of test samples. The chromatograms of the ten batches of test samples were then imported into the Chinese medicine chromatographic fingerprint similarity evaluation system. Twenty common peaks were identified and a reference fingerprint chromatogram of Xiangsha Pingwei Granules was generated.

[0027] (2) Compare the chromatograms of the common peaks with those of the single herbal medicine test sample, negative test sample and reference sample, assign each common peak to a specific group, and identify the chemical components of some common peaks. Peak 3 is identified as chlorogenic acid, peak 5 as magnolol B, peak 6 as magnolol A, peak 12 as hesperidin, peak 15 as quercetin, peak 18 as glycyrrhizic acid, peak 19 as honokiol, and peak 20 as honokiol.

[0028] (3) The quality of Xiangsha Pingwei Granules was evaluated using the reference fingerprint chromatogram. According to the similarity evaluation system of Chinese medicine chromatographic fingerprint chromatogram, the similarity between the fingerprint chromatogram of each batch of test sample and the reference fingerprint chromatogram was not less than 0.9.

[0029] The beneficial effects of this invention are as follows:

[0030] 1. The content detection method provided by this invention has passed the verification of precision, repeatability, stability, linearity and recovery rate. The detection results are accurate and reliable. It can simultaneously and accurately quantify the content of 7 chemical components in Xiangsha Pingwei Granules, which is of great significance for promoting the inspection, control and evaluation of the quality of Xiangsha Pingwei Granules.

[0031] 2. The fingerprint spectrum obtained by applying the mobile phase, elution conditions, and detection wavelength provided by this invention has a clean background, good peak separation, stable baseline, good precision, good repeatability, and high stability, making it suitable for establishing fingerprint spectra of Xiangsha Pingwei granules.

[0032] 3. This invention uses a combination of chemical composition content determination and fingerprinting to control the quality of Xiangsha Pingwei Granules, which can achieve effective and comprehensive control of Xiangsha Pingwei Granules and provide a basis for the quality control standards of Xiangsha Pingwei Granules. Attached Figure Description

[0033] Figure 1 This is a chromatogram of the mobile phase and gradient condition 1 in Example 1 of the present invention.

[0034] Figure 2 This is a chromatogram of the mobile phase and gradient condition 2 in Example 1 of the present invention.

[0035] Figure 3 This is a chromatogram of the mobile phase and gradient condition 3 in Example 1 of the present invention.

[0036] Figure 4 This is a chromatogram of the mobile phase and gradient condition 4 in Example 1 of the present invention.

[0037] Figure 5 This is a chromatogram of the mobile phase and gradient condition 5 in Example 1 of the present invention.

[0038] Figure 6 This is a chromatogram of the mobile phase and gradient condition 6 in Example 1 of the present invention.

[0039] Figure 7 This is a chromatogram of the mobile phase and gradient condition 7 in Example 1 of the present invention.

[0040] Figure 8 This is a chromatogram of the wavelength switching in Embodiment 1 of the present invention.

[0041] Figure 9 This is a chromatogram of phosphoric acid at different concentrations in Example 1 of the present invention.

[0042] Figure 10 The images show chromatograms of different column temperatures in Example 1 of this invention.

[0043] Figure 11 These are chromatograms of different flow rates in Example 1 of the present invention.

[0044] Figure 12 The figures shown are the determination diagram and reference fingerprint chromatogram of common peaks in Example 2 of the present invention. A is a superimposed high performance liquid chromatogram of 10 batches of Xiangsha Pingwei Granules; B is the reference fingerprint chromatogram of Xiangsha Pingwei Granules, with common peaks 1 to 20 from left to right.

[0045] Figure 13 The figure shows the common peaks of Xiangsha Pingwei Granules in Example 2 of the present invention (Ginger Magnolia officinalis). The numbers in the figure indicate the chromatographic peaks of Xiangsha Pingwei Granules that belong to Ginger Magnolia officinalis.

[0046] Figure 14 The common peaks of Xiangsha Pingwei Granules in Example 2 of the present invention are attributed to Chenpi (dried tangerine peel). The numbers in the figure indicate the chromatographic peaks of Xiangsha Pingwei Granules that belong to Chenpi.

[0047] Figure 15 The common peaks of Xiangsha Pingwei Granules in Example 2 of this invention are attributed to licorice. The numbers in the figure indicate the chromatographic peaks of Xiangsha Pingwei Granules that belong to licorice.

[0048] Figure 16 The figures show the common peaks (Atractylodes lancea and Magnolia officinalis) of Xiangsha Pingwei Granules in Example 2 of this invention. The numbers in the figure indicate the chromatographic peaks of Xiangsha Pingwei Granules that belong to Atractylodes lancea and Magnolia officinalis.

[0049] Figure 17 The figures in Example 2 of this invention show the common peaks of Xiangsha Pingwei Granules (Atractylodes lancea, Citrus reticulata peel, Amomum villosum and Cyperus rotundus in vinegar). The numbers in the figure indicate the chromatographic peaks of Xiangsha Pingwei Granules that belong to Atractylodes lancea, Citrus reticulata peel, Amomum villosum and Cyperus rotundus in vinegar.

[0050] Figure 18 The specificity chromatogram for content determination in Example 3 of the present invention is shown below, where A is blank solvent; B is blank excipient; C is Xiangsha Pingwei granules test solution; and D is mixed reference solution (1. chlorogenic acid; 2. magnolol B; 3. magnolol A; 4. hesperidin; 5. quercetin; 6. glycyrrhizic acid; 7. magnolol; 8. magnolol). Detailed Implementation

[0051] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0052] The instruments and reagents used in this invention are as follows:

[0053] Instruments: Agilent 1260 high performance liquid chromatograph (Agilent Technologies, Inc., USA), XP205 analytical balance (d=0.0001g, Mettler Toledo GmbH, Switzerland), PL3002 analytical balance (d=0.01g, Mettler Toledo GmbH, Switzerland), ultrasonic cleaner (Shanghai Kedao Ultrasonic Instrument Co., Ltd.).

[0054] Reagents: Methanol and phosphoric acid were of chromatographic grade, all other reagents were of analytical grade, and water was Wahaha purified water.

[0055] Reference standards: chlorogenic acid (batch number 110753-202119, content 96.3%), hesperidin (batch number 110721-201617, content 97.9%), quercetin (batch number 100081-201610, content 99.1%), ammonium glycyrrhizate (batch number 110731-202122, content 94.4%), and magnolol (batch number 110729-20231). 6. Magnoliin (batch number 110730-202416, purity 99.2%), all reference standards were purchased from the China National Institutes for Food and Drug Control; Magnolanin A (batch number 130205-202504, purity 97.03%) and Magnolanin B (batch number 130206-202504, purity 97.62%), all reference standards were purchased from Kunming Senlan Technology Co., Ltd.

[0056] Samples: Multiple batches of Xiangsha Pingwei Granules were provided by Yunnan Baiyao Group Co., Ltd. Sample information is shown in Table 1.

[0057]

[0058] Example 1: Determination of Chromatographic Conditions

[0059] 1. Preparation of the test solution

[0060] Take an appropriate amount of Xiangsha Pingwei granules, grind them into a fine powder, take an appropriate amount (about equivalent to 5g of medicinal slices), accurately weigh it, place it in a stoppered conical flask, add 50mL of 80% methanol solution, seal tightly, weigh it, sonicate for 30min, cool, weigh it again, replenish the lost weight with 80% methanol, shake well, filter through a 0.22μm microporous membrane, and take the filtrate to obtain the test solution.

[0061] 2. Chromatographic conditions

[0062] Chromatographic column: Shim-pack CLC-ODS (150mm×6.0mm, 5μm); Mobile phase: Methanol (A)-0.1% phosphoric acid aqueous solution (B), gradient elution: 0~25min, 10%→25% A; 25~50min, 25%→40% A; 50~60min, 40%→60% A; 60~70min, 60%→80% A; 70~80min, 80%→90% A; 80~85min, 90% A. Flow rate: 1.0mL / min; Column temperature: 25℃; Injection volume: 10μL; Detection wavelength: 0~20min, 330nm; 20~65min, 220nm; 65~75min, 250nm; 75~85min, 220nm.

[0063] 3. Selection of mobile phase and elution gradient

[0064] Except for the gradient, mobile phase, and detection wavelength, the other chromatographic conditions are the same as those described above. Detection wavelengths of 220, 250, and 330 nm are used. The gradient program and mobile phase are used as variables, and multiple different mobile phases and elution gradients are used to determine the same sample solution.

[0065] 3.1 Mobile Phase and Gradient Conditions 1

[0066] Mobile phase: Acetonitrile (A) - 0.05% phosphoric acid (B), gradient elution: 0~5 min, 5% A; 5~10 min, 5%→10% A; 10~35 min, 10%→30% A; 35~50 min, 30%→50% A; 50~60 min, 50%→70% A; 60~70 min, 70%→90% A; 70~75 min, 90%→5% A.

[0067] The results are as follows Figure 1 As shown, the mobile phase and elution gradient can detect relatively few chromatographic peaks, and most of the chromatographic peaks have poor resolution and peak shape, so the elution gradient needs to be further adjusted.

[0068] 3.2 Mobile Phase and Gradient Conditions 2

[0069] Mobile phase: Acetonitrile (A) - 0.05% phosphoric acid (B), gradient elution: 0~5 min, 5% A; 5~10 min, 5%→10% A; 10~50 min, 10%→35% A; 50~60 min, 35%→70% A; 60~70 min, 70%→90% A; 70~75 min, 90%→5% A; 75~80 min, 5% A.

[0070] The results are as follows Figure 2 As shown, after adjusting the elution gradient, the resolution of chromatographic peaks was significantly improved, but the number of detectable chromatographic peaks was still relatively small. Therefore, the mobile phase and elution gradient were further adjusted.

[0071] 3.3 Mobile Phase and Gradient Conditions

[0072] Mobile phase: Methanol (A) - 0.05% phosphoric acid (B), gradient elution: 0~5 min, 5% A; 5~10 min, 5%→10% A; 10~20 min, 10%→30% A; 20~50 min, 30%→70% A; 50~60 min, 70%→90% A; 60~70 min, 90% A; 70~75 min, 90%→5% A; 75~80 min, 5% A.

[0073] The results are as follows Figure 3As shown, after adjusting the mobile phase to methanol-0.05% phosphoric acid and the elution gradient, the number of detected chromatographic peaks increased, but the chromatographic peaks with retention times of 30-40 min (originating from Magnolia officinalis and Citrus reticulata) had poor separation. Therefore, the elution gradient was further adjusted.

[0074] 3.4 Mobile Phase and Gradient Conditions 4

[0075] Mobile phase: Methanol (A) - 0.05% phosphoric acid (B), gradient elution: 0~20 min, 5%→20% A; 20~50 min, 20%→40% A; 50~60 min, 40%→90% A; 60~70 min, 90% A; 70~75 min, 90%→5% A; 75~80 min, 5% A.

[0076] The results are as follows Figure 4 As shown, after adjusting the elution gradient, the separation of the chromatographic peaks from 20 to 40 min (derived from Magnolia officinalis) was significantly improved, but the separation of the chromatographic peaks from 55 to 70 min (derived from Citrus reticulata and Glycyrrhiza uralensis) was still poor. Therefore, the elution gradient was further adjusted.

[0077] 3.5 Mobile Phase and Gradient Conditions 5

[0078] Mobile phase: Methanol (A) - 0.05% phosphoric acid (B), gradient elution: 0~25 min, 10%→25% A; 25~50 min, 25%→40% A; 50~60 min, 40%→60% A; 60~70 min, 60%→90% A; 70~80 min, 90% A; 80~85 min, 90%→10% A; 85~90 min, 10% A.

[0079] The results are as follows Figure 5 As shown, after adjusting the elution gradient, the separation of the chromatographic peaks at 55-70 min (derived from tangerine peel and licorice) was significantly improved, but the separation of the chromatographic peaks at retention times of 71-75 min (derived from licorice) from adjacent chromatographic peaks was poor, so the elution gradient was further adjusted.

[0080] 3.6 Mobile Phase and Gradient Conditions 6

[0081] Mobile phase: Methanol (A) - 0.05% phosphoric acid (B), gradient elution: 0~25 min, 10%→25%A; 25~50 min, 25%→40%A; 50~60 min, 40%→60%A; 60~70 min, 60%→80%A; 70~80 min, 80%→90%A; 80~85 min, 90%A; 85~90 min, 90%→10%A; 90~95 min, 10%A.

[0082] The results are as follows Figure 6 As shown, after adjusting the elution gradient, the separation between the chromatographic peak with a retention time of 71-75 min (derived from licorice) and the adjacent chromatographic peak was improved to some extent. However, the chromatographic peak with a retention time of 74.649 min (glycyrrhizic acid) still failed to achieve baseline separation with the adjacent chromatographic peak. Therefore, the mobile phase was further adjusted.

[0083] 3.7 Mobile Phase and Gradient Conditions 7

[0084] Mobile phase: Methanol (A) - 0.1% phosphoric acid (B), gradient elution: 0~25 min, 10%→25%A; 25~50 min, 25%→40%A; 50~60 min, 40%→60%A; 60~70 min, 60%→80%A; 70~80 min, 80%→90%A; 80~85 min, 90%A; 85~90 min, 90%→10%A; 90~95 min, 10%A.

[0085] The results are as follows Figure 7 As shown, after adjusting the mobile phase to methanol-0.1% phosphoric acid, the chromatographic peak (glycyrrhizic acid) with a retention time of 74.162 min can achieve baseline separation with the adjacent chromatographic peaks. The peak shape and resolution of each chromatographic peak are good. Therefore, this mobile phase and elution gradient are determined to be the final mobile phase and elution gradient.

[0086] 4. Selection of detection wavelength

[0087] Except for the detection wavelength, all other chromatographic conditions were the same as those described above. The elution of peaks at single wavelengths of 220, 250, and 330 nm was examined. Wavelength switching at different times was performed to maximize the retention of peaks for each active ingredient, thus determining the final detection wavelength. Results are as follows: Figure 7 As shown. Based on the results of single wavelength, the wavelengths were determined as follows: 0~20min, 330nm; 20~65min, 220nm; 65~75min, 250nm; 75~85min, 220nm.

[0088] The results are as follows Figure 8 As shown, switching the wavelength allows for the simultaneous detection of multiple component peaks in a single chromatogram, significantly improving the sensitivity and quantitative accuracy of each component, while also enhancing detection efficiency. Therefore, this wavelength was chosen as the final detection wavelength.

[0089] 5. Selection of phosphoric acid aqueous solution concentration

[0090] Except for the different phosphoric acid concentrations, all other chromatographic conditions were the same as those described above. The same sample solution was measured using 0.1% phosphoric acid and 0.2% phosphoric acid as mobile phase B, and the chromatograms were recorded.

[0091] The results are as follows Figure 9 As shown, using different concentrations of phosphoric acid as mobile phase B, all common peaks were well separated and exhibited good peak shapes. 0.1% phosphoric acid was preferred as mobile phase B for the determination.

[0092] 6. Column Temperature Selection

[0093] Except for the column temperature, all other chromatographic conditions were the same as those described above. The same sample solution was measured at column temperatures of 20, 25 and 30 °C, and the chromatograms were recorded.

[0094] The results are as follows Figure 10 As shown, at 20℃, the resolution of some chromatographic peaks was poor, and at 30℃, due to wavelength switching, some chromatographic peaks could not be eluted normally, resulting in fewer detected components. At a column temperature of 25℃, the resolution and peak shape of each chromatographic peak were optimal, and more components could be detected. Therefore, a column temperature of 25℃ is preferred for the determination.

[0095] 7. Selection of Flow Rate

[0096] Except for the flow rate, all other chromatographic conditions were the same as those described above. The same sample solution was measured at flow rates of 0.8, 1.0 and 1.1 mL / min, and the chromatograms were recorded.

[0097] The results are as follows Figure 11 As shown, at flow rates of 0.8 and 1.1 mL / min, the resolution of some chromatographic peaks was poor, and due to wavelength switching, some peaks failed to elute properly, resulting in fewer detected components. Therefore, at a flow rate of 1.0 mL / min, the resolution and peak shape of all chromatographic peaks were optimal, and more components could be detected. A flow rate of 1.0 mL / min is preferred for the determination.

[0098] Example 2: Construction of fingerprint map

[0099] 1. Preparation of reference solution and test solution

[0100] 1.1 Reference solution

[0101] Take appropriate amounts of chlorogenic acid, magnologenin B, magnologenin A, hesperidin, quercetin, ammonium glycyrrhizate, and honokiol and honokiol reference standards, respectively, accurately weigh them, and add 80% methanol to prepare a mixed reference solution containing approximately 10 μg of chlorogenic acid, 20 μg of magnologenin B, 20 μg of magnologenin A, 40 μg of hesperidin, 2 μg of quercetin, 15 μg of ammonium glycyrrhizate, and 1 μg of honokiol and honokiol per 1 ml.

[0102] 1.2 Test solution

[0103] Take appropriate amounts of Xiangsha Pingwei granules from different batches, grind them into fine powder, take an appropriate amount (approximately equivalent to 5g of medicinal slices), accurately weigh it, place it in a stoppered conical flask, add 50mL of 80% methanol solution, seal tightly, weigh it, sonicate for 30 minutes, cool, weigh it again, replenish the lost weight with 80% methanol, shake well, filter through a 0.22μm microporous membrane, and take the filtrate to obtain the test solution.

[0104] 1.3 Negative test solution

[0105] Take negative test samples of Atractylodes lancea, Magnolia officinalis, Citrus reticulata, Cyperus rotundus, Glycyrrhiza uralensis, and Amomum villosum, respectively, grind them into fine powder, take an appropriate amount (about equivalent to 5g of the decoction pieces), accurately weigh them, place them in a stoppered conical flask, add 50mL of 80% methanol solution, seal tightly, weigh, sonicate for 30 minutes, cool, weigh again, make up the lost mass with 80% methanol solution, shake well, filter through a 0.22μm microporous membrane, and take the filtrate to obtain the solutions of each negative test sample.

[0106] 1.4 Single-herb medicinal slices test solution

[0107] Take approximately 0.7g of each herbal extract sample for testing, accurately weigh it, place it in a stoppered conical flask, add 50mL of 80% methanol, seal tightly, weigh it, sonicate for 30 minutes, cool, weigh it again, replenish the lost mass with 80% methanol solution, shake well, filter through a 0.22μm microporous membrane, and collect the filtrate to obtain the test solution of each herbal extract.

[0108] 2. Chromatographic conditions

[0109] The chromatographic conditions are the same as in Example 1.

[0110] 3. Measurement

[0111] The reference standard and the test sample from Example 2 were injected and analyzed under the chromatographic conditions described above, and the chromatograms were recorded.

[0112] 4. Determination of common peaks

[0113] Ten batches of Xiangsha Pingwei granules were taken, and test solutions were prepared according to the above method and analyzed. The resulting high-performance liquid chromatograms of the ten batches of Xiangsha Pingwei granules were overlaid, as shown in the figure below. Figure 12 As shown in (A), the high performance liquid chromatographic fingerprint spectra of the 10 batches of Xiangsha Pingwei granules obtained above were compared with those of the "Similarity Evaluation System for Chromatographic Fingerprint Spectra of Traditional Chinese Medicine" (2012 edition) to identify 20 common peaks and obtain reference fingerprint spectra. Figure 12 (B) is the comparative fingerprint spectrum of Xiangsha Pingwei Granules of the present invention, with common peaks 1 to 20 from left to right.

[0114] 5. Attribution and identification of common peaks

[0115] By comparing with single-herb decoction pieces, reference solutions, and negative test solutions, the peak assignments and identifications are as follows:

[0116] The common peaks belonging to Magnolia officinalis are peaks 2, 4-7, 10, 13, 15, 19 and 20. Among them, peak 5 is the chromatographic peak of magnolol B, peak 6 is the chromatographic peak of magnolol A, peak 15 is the chromatographic peak of quercetin, peak 19 is the chromatographic peak of magnolol, and peak 20 is the chromatographic peak of magnolol. Figure 13 The chromatograms are of Xiangsha Pingwei Granules, Quercetin (reference), Magnolanin A (reference), Magnolanin B (reference), Magnoliol (reference), Magnolanin (reference), Magnolanin-negative, and Magnolanin-processed slices.

[0117] The common peaks belonging to dried tangerine peel are peaks 11, 12, 14 and 16, among which peak 12 is the hesperidin chromatographic peak; Figure 14 These are chromatograms of Xiangsha Pingwei Granules, hesperidin control, tangerine peel negative, and tangerine peel slices.

[0118] The common peaks belonging to licorice are peaks 8, 9, 17 and 18, among which peak 18 is the glycyrrhizic acid chromatographic peak; Figure 15 These are chromatograms of Xiangsha Pingwei Granules, ammonium glycyrrhizate control, licorice negative control, and licorice slices.

[0119] The common peak attributed to Atractylodes lancea and Magnolia officinalis is peak 3, which is the chromatographic peak of chlorogenic acid. Figure 16 The chromatograms are of Xiangsha Pingwei Granules, chlorogenic acid control, and negative results for Atractylodes lancea and Magnolia officinalis.

[0120] The common peak belonging to Atractylodes lancea, Citrus reticulata peel, Amomum villosum and Cyperus rotundus is peak No. 1; Figure 17 These are chromatograms of Xiangsha Pingwei Granules, Atractylodes lancea slices, Citrus reticulata peel slices, Amomum villosum slices, and Cyperus rotundus slices (processed with vinegar).

[0121] 6. Methodological Research

[0122] 6.1 Precision

[0123] Take a sample of Xiangsha Pingwei granules and proceed according to the method described in Example 2, under the preparation of the test solution. Inject the sample six times consecutively under the chromatographic conditions described above, detect the fingerprint chromatogram, and record the chromatogram. Using hesperidin as a reference peak, calculate the RSD values ​​of the relative retention time and relative peak area of ​​each common peak. The results are shown in Tables 2 and 3, respectively.

[0124]

[0125]

[0126] As shown in Tables 2 and 3, after six consecutive injections of Xiangsha Pingwei Granules, the RSD values ​​of the relative retention times of all common peaks were less than 1%, and the RSD values ​​of the relative peak areas were less than 3%, indicating good precision.

[0127] 6.2 Repeatability

[0128] Six portions of the same batch of Xiangsha Pingwei granules were accurately weighed and injected according to the method described in Example 2, under the preparation of the test solution. The fingerprint chromatograms were detected and the chromatograms were recorded. Using hesperidin as a reference peak, the RSD values ​​of the relative retention time and relative peak area of ​​each common peak were calculated. The results are shown in Tables 4 and 5, respectively.

[0129]

[0130]

[0131] As shown in Tables 4 and 5, the RSD values ​​of the relative retention times of the common peaks in the six test solutions were all less than 1%, and the RSD values ​​of the relative peak areas were all less than 3%, indicating good repeatability.

[0132] 6.4 Solution stability

[0133] Accurately weigh the Xiangsha Pingwei granules sample and proceed according to the method described in Example 2, Test Solution Preparation. Inject the sample at 0, 3, 6, 9, 12, and 24 hours to detect the fingerprint chromatogram. Record the chromatograms, using hesperidin as the reference peak, and calculate the RSD values ​​of the relative retention time and relative peak area of ​​each common peak. The results are shown in Tables 6 and 7, respectively.

[0134]

[0135]

[0136] Results: As shown in Tables 6 and 7, the RSD values ​​of the relative retention times of all common peaks within 24 hours were all less than 1%, and the RSD values ​​of the relative peak areas were all less than 3%. This indicates that the test solution had good stability within 24 hours.

[0137] In summary, through methodological research, the fingerprint spectrum detection method of the present invention has good repeatability, stability and precision, and can comprehensively control the quality of Xiangsha Pingwei granules.

[0138] 7. Similarity results of fingerprint spectrum of Xiangsha Pingwei Granules

[0139] Ten batches of Xiangsha Pingwei Granules were taken and processed according to the method described in Example 2, under the preparation of the test solution. The chromatograms were recorded after injection under the aforementioned chromatographic conditions. The chromatograms were then imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 edition) for similarity calculation. The similarity results between the fingerprint chromatograms of each batch of test samples and the control fingerprint chromatogram are shown in Table 8.

[0140]

[0141] Results: The similarity between the fingerprint chromatograms of each batch of samples and the control fingerprint chromatograms was greater than 0.9, indicating good similarity.

[0142] Example 3: Determination of the content of index components in Xiangsha Pingwei Granules

[0143] 1. Preparation of reference solution and test solution

[0144] 1.1 Mixed reference solution

[0145] Take appropriate amounts of chlorogenic acid, magnologenin B, magnologenin A, hesperidin, quercetin, ammonium glycyrrhizate, and honokiol and honokiol reference standards, respectively, accurately weigh them, and add 80% methanol to prepare a mixed reference solution containing approximately 10 μg of chlorogenic acid, 20 μg of magnologenin B, 20 μg of magnologenin A, 40 μg of hesperidin, 2 μg of quercetin, 15 μg of ammonium glycyrrhizate, and 1 μg of honokiol and honokiol per 1 ml.

[0146] 1.2 Test solution

[0147] Take appropriate amounts of Xiangsha Pingwei granules from different batches, grind them into fine powder, take an appropriate amount (approximately equivalent to 5g of medicinal slices), accurately weigh it, place it in a stoppered conical flask, add 50mL of 80% methanol solution, seal tightly, weigh it, sonicate for 30 minutes, cool, weigh it again, replenish the lost weight with 80% methanol, shake well, filter through a 0.22μm microporous membrane, and take the filtrate to obtain the test solution.

[0148] 1.3 Blank excipient test solution

[0149] Take about 2g of sucrose and place it in a stoppered conical flask. Accurately add 50ml of 80% methanol solution, seal tightly, weigh, and sonicate (power 350W, frequency 35kHz) for 30 minutes. Let it cool, weigh it again, and make up the weight loss with 80% methanol solution. Shake well, filter, and take the filtrate to obtain the blank excipient test solution.

[0150] 2. Chromatographic conditions

[0151] The chromatographic conditions are the same as in Example 1.

[0152] 3. Measurement

[0153] The reference solution and the test solution from Example 3 were injected and analyzed under the chromatographic conditions described above, and the chromatograms were recorded.

[0154] 4. Methodological Research

[0155] 4.1 Specificity

[0156] Take 80% methanol blank solvent, blank excipient test solution, mixed reference solution and test solution respectively, inject them into high performance liquid chromatograph, determine and record the chromatogram (e.g. Figure 18 As shown in the figure, the blank solvent and blank excipient did not interfere with the detection of any component. This indicates that the method has good specificity.

[0157] 4.2 Linearity and Range

[0158] Chlorogenic acid reference standards were prepared at concentrations of 0.605, 1.210, 3.025, 6.050, 9.074, 15.124, and 30.248 μg / ml, respectively, and injected under the chromatographic conditions described above. A standard curve was plotted with the concentration of the reference standard as the abscissa (X) and the peak area as the ordinate (Y), and regression calculations were performed. The results showed that chlorogenic acid exhibited good linearity, and the regression equation is shown in Table 9.

[0159]

[0160] Magnolanein B reference standards were prepared at concentrations of 0.990, 1.980, 4.949, 9.899, 14.848, 24.747, and 49.493 μg / ml, respectively, and injected under the chromatographic conditions described above. A standard curve was plotted with the concentration of the reference standard as the x-axis and the peak area as the y-axis, and regression calculations were performed. The results showed that magnolanlane B exhibited good linearity, and the regression equation is shown in Table 10.

[0161]

[0162] Magnolanein A reference standard was prepared at concentrations of 0.997, 1.995, 4.987, 9.975, 14.962, 24.937, and 49.873 μg / ml, respectively, and injected under the chromatographic conditions described above. A standard curve was plotted with the concentration of the reference standard as the abscissa (X) and the peak area as the ordinate (Y), and regression calculations were performed. The results showed that magnolanlanein A exhibited good linearity, and the regression equation is shown in Table 11.

[0163]

[0164] Hesperidin reference standards at concentrations of 1.894, 9.470, 18.940, 37.880, 56.820, 75.760, and 113.640 μg / ml were injected under the chromatographic conditions described above. A standard curve was plotted with the concentration of the reference standard as the x-axis and the peak area as the y-axis, and regression calculations were performed. The results showed that hesperidin exhibited good linearity, and the regression equation is shown in Table 12.

[0165]

[0166] Quercetin reference standard concentrations of 0.082, 0.164, 0.411, 0.822, 1.233, 2.055, and 4.111 μg / ml were taken and injected under the above chromatographic conditions. A standard curve was plotted with the concentration of the reference standard as the abscissa (X) and the peak area as the ordinate (Y), and regression calculations were performed. The results showed that quercetin exhibited good linearity, and the regression equation is shown in Table 13.

[0167]

[0168] Ammonium glycyrrhizate reference standards were prepared at concentrations of 0.775, 1.551, 3.877, 7.754, 11.631, 19.385, and 38.770 μg / ml, respectively, and injected under the chromatographic conditions described above. A standard curve was plotted with the concentration of the reference standard as the abscissa (X) and the peak area as the ordinate (Y), and regression calculations were performed. The results showed that glycyrrhizic acid exhibited good linearity, and the regression equation is shown in Table 14.

[0169]

[0170] Concentrations of magnolol reference standards of 0.202, 0.303, 0.403, 1.008, 3.781, 6.302, and 10.084 μg / ml were taken and injected under the chromatographic conditions described above. A standard curve was plotted with the concentration of the reference standard as the abscissa (X) and the peak area as the ordinate (Y), and regression calculations were performed. The results showed a good linear relationship between magnolol and the standard. The regression equation is shown in Table 15.

[0171]

[0172] Magnolol reference standards were prepared at concentrations of 0.030, 0.061, 0.152, 0.304, 0.455, 0.759, and 1.518 μg / ml, respectively, and injected under the chromatographic conditions described above. A standard curve was plotted with the concentration of the reference standard as the abscissa (X) and the peak area as the ordinate (Y), and regression calculations were performed. The results showed that magnolol exhibited good linearity, and the regression equation is shown in Table 16.

[0173]

[0174] 4.4 Precision

[0175] A mixed reference solution of chlorogenic acid, magnoside B, magnoside A, hesperidin, quercetin, ammonium glycyrrhizate, and honokiol was injected six times consecutively according to the above method. The chromatograms were recorded, and the RSD values ​​of the peak areas of chlorogenic acid, magnoside B, magnoside A, hesperidin, quercetin, glycyrrhizic acid, and honokiol were calculated to be 0.14%, 0.13%, 0.25%, 0.29%, 0.27%, 0.25%, 1.04%, and 0.62%, respectively. This indicates that the precision of this method is good.

[0176] 4.5 Repeatability Test

[0177] Six portions of the same batch of Xiangsha Pingwei granules were prepared according to the test solution preparation method. The samples were injected and analyzed under the chromatographic conditions described above. The RSD values ​​for the contents of chlorogenic acid, magnolol B, magnolol A, hesperidin, quercetin, glycyrrhizic acid, and honokiol were calculated to be 2.01%, 1.49%, 2.16%, 1.49%, 1.44%, 1.67%, 1.98%, and 1.67%, respectively. This indicates that the method has good repeatability.

[0178] 4.6 Recovery Test

[0179] Seven portions of Xiangsha Pingwei granules, each approximately equivalent to 2.5g of medicinal slices, were accurately weighed. The first portion was prepared according to the method for preparing the test solution. Appropriate amounts of each reference standard were added to portions 2-7, and the solutions were prepared according to the method for preparing the test solution. The samples were injected and determined under the chromatographic conditions described above. The average recoveries of chlorogenic acid, magnolol B, magnolol A, hesperidin, quercetin, glycyrrhizic acid, magnolol, and honokiol were calculated to be 86.43%, 97.72%, 97.44%, 97.12%, 81.56%, 98.59%, 97.58%, and 102.42%, respectively, with RSD values ​​of 3.38%, 2.10%, 1.49%, 1.03%, 2.21%, 1.67%, 3.59%, and 1.71%, respectively. The recovery results showed that quercetin exhibited poor peak separation in the chromatographic recovery experiment, failing to meet the methodological requirement of 85-110%. Therefore, quercetin was excluded from the quantitative analysis of the content determination. The recovery of the other seven components met the requirements for validation, indicating that the method has good accuracy.

[0180] 5. Results of Xiangsha Pingwei Granule Content Determination

[0181] Ten batches of Xiangsha Pingwei granules were prepared according to the test solution preparation method. The samples were injected and determined under the above chromatographic conditions. The contents of seven components in the samples, namely chlorogenic acid, magnolol B, magnolol A, hesperidin, glycyrrhizic acid, honokiol, and magnolol, were calculated. The results are shown in Table 17.

[0182]

[0183] In summary, this invention provides a fingerprint spectrum and a method for determining the content of multiple components in Xiangsha Pingwei granules. Utilizing optimized pretreatment and high-performance liquid chromatography (HPLC) methods, qualitative analysis of 20 components from six medicinal herbs in Xiangsha Pingwei granules can be achieved under the same chromatographic conditions, identifying eight chemical components: chlorogenic acid, magnologin B, magnologin A, hesperidin, quercetin, glycyrrhizic acid, and magnolol. Simultaneously, quantitative analysis of seven other chemical components in Xiangsha Pingwei granules—chlorogenic acid, magnologin B, magnologin A, hesperidin, glycyrrhizic acid, and magnolol—was performed. This comprehensive testing of Xiangsha Pingwei granules allows for more comprehensive and effective quality control, providing a scientific basis for quality control of this medicine.

Claims

1. A method for determining the fingerprint spectrum and multi-component content of Xiangsha Pingwei granules, characterized in that, The method includes the following steps: (1) Preparation of reference solution: Take chlorogenic acid, magnolol B, magnolol A, hesperidin, quercetin, ammonium glycyrrhizate, and magnolol and magnolol reference standards, dissolve them in solvent and make up to volume to obtain the solution; (2) Preparation of test solution: Take Xiangsha Pingwei granules sample, add solvent to extract, filter, and take the filtrate to obtain the test solution; (3) Preparation of single herbal medicine sample solution: Take a single herbal medicine sample, add solvent to extract, filter, and take the filtrate to obtain the solution; (4) Preparation of negative test solution: Take each negative sample, add solvent to extract, filter, and take the filtrate to obtain the solution; (5) Chromatographic conditions: The chromatographic column was a C18 column, mobile phase A was methanol, mobile phase B was an aqueous solution containing phosphoric acid, gradient elution was used, the elution gradient was 0~25 min, 10%→25% A; 25~50 min, 25%→40% A; 50~60 min, 40%→60% A; 60~70 min, 60%→80% A; 70~80 min, 80%→90% A; 80~85 min, 90% A, the flow rate was 1.0 mL / min, the column temperature was 25℃, the injection volume was 5~20 μL, and the detection wavelength changed with the time of the gradient elution program, specifically: 0~20 min, detection wavelength was 330 nm; 20~65 min, detection wavelength was 220 nm; 65~75 min, detection wavelength was 250 nm; 75~85 min, detection wavelength was 220 nm. (6) Fingerprint chromatogram establishment: High performance liquid chromatography was used. The reference solution of step (1), the test solution of step (2), the single herb test solution of step (3) and the negative test solution of step (4) were injected into the high performance liquid chromatograph in sequence, the chromatograms were recorded, and the fingerprint chromatogram of Xiangsha Pingwei Granules was constructed. (7) Content determination: Using the same chromatographic conditions as in step (5), take the reference solution from step (1) and the test solution from step (2), inject them into the high performance liquid chromatograph, record the chromatogram, and calculate the content of 7 components in the test solution using the external standard method: chlorogenic acid, magnolol A, magnolol B, hesperidin, glycyrrhizic acid, honokiol and magnolol. The solvent used in steps (1)-(4) is 50%~90% methanol solution.

2. The method for determining the fingerprint spectrum and multi-component content of Xiangsha Pingwei granules according to claim 1, characterized in that, The C18 column used in step (5) was a Shim-pack CLC-ODS column, 150×6.0mm, 5μm.

3. The method for determining the fingerprint spectrum and multi-component content of Xiangsha Pingwei granules according to claim 1, characterized in that, In step (5), the volume percentage of phosphoric acid in the aqueous solution containing phosphoric acid is 0.1% to 0.2%.

4. The method for determining the fingerprint spectrum and multi-component content of Xiangsha Pingwei granules according to claim 1, characterized in that, The extraction methods described in steps (2)-(4) are heating reflux method, cold soaking method or ultrasonic extraction method.

5. The method for determining the fingerprint spectrum and multi-component content of Xiangsha Pingwei granules according to claim 1, characterized in that, The extraction method described in steps (2)-(4) is ultrasonic extraction, wherein the ultrasonic power is 200~400W, the ultrasonic frequency is 30~50kHz, and the ultrasonic time is 20~50min.

6. The method for determining the fingerprint spectrum and multi-component content of Xiangsha Pingwei granules according to claim 1, characterized in that, The weight-to-volume ratio of the test sample to the 50%~90% methanol solution in steps (2)-(4) is 3.0~10g of medicinal slices: 50mL.

7. The method for determining the fingerprint spectrum and multi-component content of Xiangsha Pingwei granules according to claim 1, characterized in that, Step (6) fingerprint mapping includes the following steps: (1) Ten batches of test sample solutions were injected sequentially to obtain chromatograms of ten batches of test samples. The chromatograms of the ten batches of test samples were imported into the Chinese medicine chromatographic fingerprint similarity evaluation system, and 20 common peaks were identified and a reference fingerprint spectrum of Xiangsha Pingwei Granules was generated. (2) Compare the chromatograms of the common peaks with those of the single herbal medicine test sample, negative test sample and reference sample, assign each common peak to a specific group, and identify the chemical components of some common peaks. Peak 3 is identified as chlorogenic acid, peak 5 as magnolol B, peak 6 as magnolol A, peak 12 as hesperidin, peak 15 as quercetin, peak 18 as glycyrrhizic acid, peak 19 as honokiol, and peak 20 as honokiol. (3) The quality of Xiangsha Pingwei Granules was evaluated using the reference fingerprint chromatogram. According to the similarity evaluation system of Chinese medicine chromatographic fingerprint chromatogram, the similarity between the fingerprint chromatogram of each batch of test sample and the reference fingerprint chromatogram was not less than 0.9.

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