Establishment of HPLC fingerprint of Changyile capsule (I)

Abstract

The application provides a method for establishing HPLC fingerprint of Changnyle capsule (I), which is characterized in that 16 active components including gastrodin, danshensu, quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiopicroside, 3'-hydroxy puerarin, puerarin, 3'-methoxy puerarin, puerarin apioside, paeoniflorin, daidzin, rosmarinic acid, shikonin, isoquercitrin, ononin, daidzein, salvianolic acid B and calycosin are used as reference substances, HPLC is adopted, methanol-acetonitrile-0.5vol% phosphoric acid aqueous solution is used as a mobile phase, different batches of samples are detected, and finally the fingerprint of Changnyle capsule (I) is obtained. The fingerprint can comprehensively represent the main components in Changnyle capsule (I), thereby being beneficial to the quality control of Changnyle capsule (I).

Description

Technical Field

[0001] This invention belongs to the field of quality control of traditional Chinese medicine preparations, and specifically relates to a method for establishing the HPLC fingerprint of Changmaile capsules (I). Background Technology

[0002] Changmaile Capsules (I) are made from Astragalus membranaceus, Salvia miltiorrhiza, Polygonum multiflorum, Gastrodia elata, Paeonia lactiflora, and Pueraria lobata. They have the effects of nourishing the kidneys, promoting blood circulation, dispelling wind, and unblocking the meridians, and are suitable for kidney deficiency and blood stasis syndrome in middle-aged and elderly people with arteriosclerosis or post-stroke sequelae. Regarding the quality control of Changmaile Capsules (I), only thin-layer chromatography (TLC) has been reported for qualitative identification of Astragalus membranaceus, Salvia miltiorrhiza, Polygonum multiflorum, Gastrodia elata, Paeonia lactiflora, and Pueraria lobata, and HPLC has been used to simultaneously determine the content of puerarin, paeoniflorin, and salvianolic acid B in Changmaile Capsules (I). However, a comprehensive fingerprint spectrum has not been established, therefore, a comprehensive quality assessment is impossible. Traditional Chinese medicine (TCM) components are complex and diverse, and their efficacy is often the result of the combined action of multiple components. Fingerprint spectra, with their large information content, specificity, integrity, and strong fuzziness, can fully reflect the chemical composition information of TCM, thereby enabling comprehensive evaluation and effective control of its intrinsic quality. Currently, no research on the fingerprint spectrum of Changmaile Capsules (I) has been reported. Summary of the Invention

[0003] In view of the current problem that the quality evaluation methods for Changmaile Capsules (I) are relatively simple and fail to fully characterize the main components in Changmaile Capsules (I), this invention provides a method for establishing HPLC fingerprint of Changmaile Capsules (I), which can classify 16 active ingredients in Changmaile Capsules (I) and provide a reference for the quality analysis and control of Changmaile Capsules (I).

[0004] To achieve the above objectives, the present invention adopts the following technical solution: A method for establishing an HPLC fingerprint of Changmaile capsules (I) includes the following steps: 1) Preparation of reference solutions: Accurately weigh the following reference standards: gastrodin, tanshinone, quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside, 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, puerarin apigenin, paeoniflorin, daidzein, rosmarinic acid, shikonin, isoquercitrin, gentiopicrin, daidzein, salvianolic acid B, and verbascoflavonoids. Place them separately in volumetric flasks, dissolve in an appropriate amount of methanol, and dilute to volume. Filter through a 0.22 μm microporous membrane to obtain the corresponding reference solutions with concentrations of 5.504 mg / mL, 1.154 mg / mL, 1.000 mg / mL, 1.180 mg / mL, 1.180 mg / mL, 0.526 mg / mL, 1.022 mg / mL, 0.878 mg / mL, and 1.260 mg / mL, respectively. mg / mL, 1.180 mg / mL, 1.180 mg / mL, 0.4700 mg / mL, 1.038 mg / mL, 1.040 mg / mL, 0.612 mg / mL, 1.240 mg / mL; 2) Preparation of test solution: Accurately weigh 1.0 g of the contents of the same batch of Changmaile capsules (I), place it in an Erlenmeyer flask, add 25 mL of 50 vol% methanol solution, let stand overnight, then weigh and record the Erlenmeyer flask, extract by ultrasonic extraction for 30 min, add weight after cooling to room temperature, filter the supernatant through a 0.22 μm microporous membrane to obtain the test solution; 3) Sample determination: Accurately pipette each reference solution and test solution, and detect them by HPLC / DAD to obtain the chromatograms of the reference standards and test samples; 4) Establishment of fingerprint spectrum: The chromatogram of the obtained test sample was imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012A Version", a reference spectrum was set, the time window was set to 0.5 min, the median method was used, and after multi-point correction and automatic matching, the fingerprint spectrum of Changmaile Capsules (I) was generated, and the characteristic components were assigned using the chromatogram of the reference standard.

[0005] Furthermore, the chromatographic conditions for the HPLC detection are: Thermo Scientific Accucore. TMXL C18 HPLC column, 4.6 mm × 250 mm, 4 μm; injection volume 10 μL; column temperature 35 ℃; mobile phase methanol-acetonitrile-0.5 vol% phosphoric acid aqueous solution, gradient elution: 0~2 min, methanol volume set to 1%, acetonitrile volume set to 2%; 2~5 min, methanol volume maintained at 1%, acetonitrile volume increased from 2% to 4%; 5~10 min, methanol volume maintained at 1%, acetonitrile volume increased from 4% to 9%; 10~15 min, methanol volume maintained at 1%, acetonitrile volume maintained at 9%; 15~20 min, methanol volume increased from 1% to 3%, acetonitrile volume increased from 9% to 15%; 20~45 min, methanol volume decreased from 3% to 2%, acetonitrile volume increased from 15% to 33%; 45~46 min, methanol volume decreased from 2% to 0%, acetonitrile volume increased from 33% to 60%; 46~60 min, methanol volume decreased from 2% to 0%, acetonitrile volume increased from 33% to 60%; 46~60 min, methanol volume decreased from 2% to 0%, acetonitrile volume increased from 33% to 60%; For min, the methanol volume was maintained at 0%, and the acetonitrile volume was maintained at 60%; the flow rate was 1 mL / min. -1 The detection wavelengths were 230 nm (for detecting paeoniflorin and isoquercitrin) and 280 nm (for detecting gastrodin, tanshinone, quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside, 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, puerarin apigenin, daidzein, rosmarinic acid, shikonin, gentiopicrin, daidzein, salvianolic acid B, and verbascoflavonoids).

[0006] The significant advantages of this invention are: (1) The present invention can simultaneously determine 16 components in Changmaile capsules (I) under the same elution program and dual wavelength mode, which enables it to more comprehensively characterize the main components of Changmaile capsules (I) and is beneficial to the quality control of Changmaile capsules (I).

[0007] (2) The method of the present invention has the characteristics of high precision and good reproducibility, which can effectively monitor the quality of Changmaile capsules (Ⅰ) between different batches and is conducive to the comprehensive evaluation of product quality. Attached Figure Description

[0008] Figure 1 The HPLC chromatograms of Changmaile capsules (Ⅰ) obtained by extraction with different solvents are shown.

[0009] Figure 2 The HPLC chromatograms of Changmaile capsules (Ⅰ) obtained using different extraction times are shown.

[0010] Figure 3 The HPLC chromatograms of Changmaile capsules (Ⅰ) obtained by separation using different chromatographic columns are shown.

[0011] Figure 4The HPLC chromatogram of Changmaile capsules (Ⅰ) obtained by detection using different absorption wavelengths is shown.

[0012] Figure 5 The fingerprint spectra of different batches of Changmaile capsules (Ⅰ) were determined at a wavelength of 230 nm, where R is the control spectrum.

[0013] Figure 6 The fingerprint spectra of different batches of Changmaile capsules (Ⅰ) were determined at a wavelength of 280 nm, where R is the control spectrum.

[0014] Figure 7 The chromatograms (A) of the reference standard and (B) of the test sample were obtained at a wavelength of 230 nm, where 1 is paeoniflorin and 2 isoquercitrin.

[0015] Figure 8 The chromatograms (A) of the reference standard and (B) of the test sample are shown below, where 1 is gastrodin, 2 is tanshinone, 3 is quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside, 4 is 3'-hydroxypuerarin, 5 is puerarin, 6 is 3'-methoxypuerarin, 7 is puerarin apigenin, 8 is daidzein, 9 is rosmarinic acid, 10 is shikonin, 11 is gentiopicrin, 12 is daidzeinogen, 13 is salvianolic acid B, and 14 is salvia miltiorrhizae. Detailed Implementation

[0016] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto. Example

[0017] 1. Instruments and Materials 1.1 Instruments: Dionex U3000 HPLC system (Thermo Fisher Scientific (China) Co., Ltd.); P680HPLC PUMP (lot number: 8075071); PDA-100 Photodiode Array Detector (lot number: 8076510); Thermostatted Column Compartment TTC-100 (lot number: 8076074); ASI-100 Automated Sample Injector (lot number: 8076542); Chromeleon 7.1 chromatography workstation; general analytical balance (model: YP502N, Shanghai Precision Scientific Instruments Co., Ltd.); ultrasonic cleaner (model: SB4200DT, Ningbo Xinzhi Biotechnology Co., Ltd.); electronic balance (Ohaus Instruments (Changzhou) Co., Ltd., lot number: C222133996).

[0018] 1.2 Materials: Reference standards gastrodin (batch number 110807-202010), paeoniflorin (batch number 110736-201943), isoquercitrin (batch number 111809-202205), rosmarinic acid (batch number 111871-202007), salvianolic acid B (111562-201917), and puerarin (batch number 110752-202217) were all purchased from the National Institutes for Food and Drug Control; reference standards tanshinone (batch number H09N10S102463) and quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside (batch number P10) were also purchased from the National Institutes for Food and Drug Control. A10U94985), 3'-hydroxypuerarin (batch number A10HB191367), 3'-methoxypuerarin (batch number J12HB173942), daidzein (batch number J11J12T137054), daidzeinogen (batch number C11D11Y134057), and shikonin (batch number Y30S7H22269) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.; puerarin apigenin (batch number PS000386), daidzein (batch number PS000671), and verbascoside (batch number PS010251) were all purchased from Chengdu Pusi Biotechnology Co., Ltd. Changmaile Capsules (I) were provided by the Pharmaceutical Preparation Center of the Second Affiliated Hospital of Fujian University of Traditional Chinese Medicine (batch numbers are 202112020, 202202002, 202203018, 202206005, 202208012, 202210014, 202211013, 202302005, 202303010, and 202305010, respectively).

[0019] Reagents: Methanol (HPLC grade, Sinopharm Chemical Reagent Co., Ltd., batch number: 20181203); Phosphoric acid (AR grade, Sinopharm Chemical Reagent Co., Ltd., batch number: 10015408); Acetonitrile (HPLC grade, Shanghai Xingke High Solvent Co., Ltd., batch number: 0113220702). Water was ultrapure water (resistivity 18.25Ω).

[0020] 2 Experimental methods and results 2.1 Investigation of extraction and chromatographic conditions 2.1.1 Selection of extraction solvent Accurately weigh six portions (1.0 g each) of the contents of the same batch of Changmaile capsules (I), place them in conical flasks, and add 25 mL each of 75% ethanol, 95% ethanol, 50% ethanol, water, 50% methanol, and 100% methanol, respectively. Let stand overnight. Weigh and record the weight of the conical flasks, extract using ultrasonic extraction for 30 min, and weigh again after cooling to room temperature. Filter the supernatant through a 0.22 μm microporous membrane and inject. Determine the optimal extraction solvent based on the total number of chromatographic peaks and peak area. The results are shown in [Figure number missing]. Figure 1 .

[0021] from Figure 1 It can be seen that the six solvents have a certain impact on the extraction effect of Changmaile capsules (Ⅰ). Taking all factors into consideration, 50% methanol was selected as the best extraction solvent.

[0022] 2.1.2 Selection of extraction time Accurately weigh three portions (1.0 g each) of the contents of the same batch of Changmaile capsules (I), place them in conical flasks, add 25 mL of 50% methanol to each flask, and let stand overnight. Weigh and record the weight of the conical flasks, and extract using ultrasonic extraction for 10 min, 30 min, and 60 min respectively. After cooling to room temperature, weigh the flasks again. Filter the supernatant through a 0.22 μm microporous membrane and inject it into the sample. Determine the optimal extraction time based on the total number of chromatographic peaks and peak area. The results are shown in [Figure number missing]. Figure 2 .

[0023] from Figure 2 It can be seen that different extraction times have a certain impact on the extraction effect of Changmaile capsules (Ⅰ). Taking all factors into consideration, 30 min was selected as the optimal extraction time.

[0024] 2.1.3 Selection of Chromatographic Column Accurately weigh three portions of the contents of the same batch of Changmaile capsules (I), 1.0 g each, and place them in conical flasks. Add 25 mL of 50% methanol to each flask and let them stand overnight. Weigh and record the weight of each flask. Extract using ultrasonic extraction for 30 min, and weigh again after cooling to room temperature. Filter the supernatant through a 0.22 μm microporous membrane. Inject the test solution using SinoChrom ODS-BP, Accucore XL C18, and Acclaim 120 C18 columns, respectively. Determine the optimal column based on the total number of chromatographic peaks. The results are shown in [Figure number missing]. Figure 3 .

[0025] from Figure 3 As can be seen, different chromatographic columns produce different chromatograms. Taking all factors into consideration, the Accucore XL C18 column was selected.

[0026] 2.1.4 Determination of Absorption Wavelength Accurately weigh three portions (1.0 g each) of the contents of the same batch of Changmaile capsules (I), place them in conical flasks, add 25 mL of 50% methanol to each flask, and let stand overnight. Weigh and record the weight of each flask, and extract using ultrasonic extraction for 30 min. After cooling to room temperature, weigh the flasks again. Filter the supernatant through a 0.22 μm microporous membrane. Inject the test solution and compare the chromatograms at wavelengths of 230, 254, 280, 320, and 360 nm. Determine the optimal chromatographic column based on the total number of chromatographic peaks. The results are shown in [Figure number missing]. Figure 4 .

[0027] from Figure 4 As can be seen, the number of peaks, peak area, response value, and resolution all differ under different absorption wavelengths. Taking all factors into consideration, 230 nm and 280 nm were selected as the dual wavelengths for detection.

[0028] 2.2 Solution Preparation 2.2.1 Preparation of reference solutions: Accurately weigh the following reference standards: gastrodin, tanshinone, quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside, 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, puerarin apigenin, paeoniflorin, daidzein, rosmarinic acid, shikonin, isoquercitrin, mangiferin, daidzein, salvianolic acid B, and verbascoside. Place them separately in volumetric flasks, dissolve in an appropriate amount of methanol, and dilute to volume. Filter through a 0.22 μm microporous membrane to obtain the corresponding reference solutions with concentrations of 5.504 mg / mL, 1.154 mg / mL, 1.000 mg / mL, 1.180 mg / mL, 1.180 mg / mL, 0.526 mg / mL, 1.022 mg / mL, 0.878 mg / mL, and 1.260 mg / mL, respectively. mg / mL, 1.180 mg / mL, 1.180 mg / mL, 0.4700 mg / mL, 1.038 mg / mL, 1.040 mg / mL, 0.612 mg / mL, 1.240 mg / mL.

[0029] 2.2.2 Preparation of test solution: Take 1.0 g of the contents (Ⅰ) of Changmaile capsules, place it in an Erlenmeyer flask, add 25 mL of 50% methanol and let it stand overnight. Then weigh the Erlenmeyer flask and record the weight. Extract using ultrasonic extraction for 30 min (power 250 W, frequency 50 kHz). After cooling to room temperature, add weight. Filter the supernatant through a 0.22 μm microporous membrane and collect the filtrate.

[0030] 2.3 Chromatographic conditions Column: Thermo Scientific Accucore TM XL C18 HPLC column (4.6 mm × 250 mm, 4 μm); mobile phase: methanol-acetonitrile-0.5 vol% phosphoric acid aqueous solution, gradient elution as shown in Table 1; flow rate: 1 mL·min -1Injection volume: 10 μL; Column temperature: 35 ℃; Detection wavelength: 230 nm (for detecting paeoniflorin and isoquercitrin), 280 nm (for detecting gastrodin, tanshinone, quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside, 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, puerarin apigenin, daidzein, rosmarinic acid, shikonin, gentiopicrin, daidzein, salvianolic acid B, and verbascoflavonoids).

[0031] Table 1 Gradient elution program (unit: %)

[0032] 2.4 Methodological Examination 2.4.1 System Suitability Test: 10 μL each of the test solution, reference solution, and blank solvent of Changmaile Capsules (I) were taken and analyzed under the chromatographic conditions described in section 2.3, and the chromatograms were recorded. The results showed that the chromatographic peaks of the 16 components were well separated from adjacent peaks, and there were no chromatographic peaks interfering with the target components at the corresponding positions in the blank solvent, indicating that the method has good specificity.

[0033] 2.4.2 Precision Test: Changmaile capsules (I) (batch number: 202302005) were used. The test solution was prepared according to the method described in section 2.2.2, and the chromatographic conditions described in section 2.3 were followed. Six consecutive injections were performed, and the chromatograms were recorded. The results showed that the retention time RSD was 0.04%–0.25%, and the peak area RSD was 0.21%–2.26%, indicating good instrument precision.

[0034] 2.4.3 Repeatability Test: Six parallel test solutions of Changmaile Capsules (I) (batch number: 202302005) were prepared according to the method described in section 2.2.2. The solutions were then analyzed under the chromatographic conditions described in section 2.3, and the chromatograms were recorded. The results showed that the average contents of gastrodin, tanshinone, quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside, 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, puerarin apigenin, daidzein, rosmarinic acid, shikonin, gentiopicrin, daidzein, salvianolic acid B, verbascoside, paeoniflorin, and isoquercitrin were 39.65 mg·g. -1 0.9993 mg·g -1 0.7711 mg·g -1 2.544 mg·g -1 5.399 mg·g -1 3.240 mg·g -1 3.430 mg·g -1 4.133 mg·g -1 0.1626 mg·g-1 0.5028 mg·g -1 0.1535 mg·g -1 0.2675 mg·g -1 3.297 mg·g -1 0.2984 mg·g -1 5.112 mg / g -1 13.34 mg·g -1 The RSD was between 0.20% and 3.29%, indicating that the method has good repeatability.

[0035] 2.4.3 Stability Test: The prepared Changmaile Capsule (I) (batch number 202302005) test solution as described in section 2.4.2 was injected in 10 μL at 0, 2, 4, 8, 12, and 24 h, and the chromatographic conditions described in section 2.3 were used for determination. The results showed that the RSD of the peak areas of the 16 target components were between 0.49% and 2.04%, indicating that the sample solution was stable within 24 h after preparation.

[0036] 2.5 Establishment of fingerprint map Accurately weigh different batches of Changmaile capsules (Ⅰ) (S1: 202112020, S2: 202202002, S3: 202203018, S4: 202206005, S5: 202208012, S6: 202210014, S7: 202211013, S8: 202302005, S9: 202303010, S10: 202305010), prepare the test solution according to the method in section 2.2.2, and inject the samples sequentially according to the chromatographic conditions in section 2.3, and record the chromatograms of the samples. The HPLC chromatograms of 10 batches of Changmaile Capsules (I) were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (Version 2012A). Using the S8 fingerprint chromatogram as a reference, with a time window of 0.5 min and the median method, a reference fingerprint chromatogram of Changmaile Capsules (I) was generated after automatic matching and multi-point correction. Figure 5 , 6 As shown.

[0037] 2.6 Chemical composition analysis of fingerprint chromatograms Sixteen reference solutions were injected sequentially under the chromatographic conditions described in section 2.3, and the chromatograms of the samples were recorded. The chromatographic peaks of each reference solution were compared with the fingerprint chromatogram of the test sample to assign their characteristic components. The HPLC fingerprint chromatograms of the reference solutions and Changmaile capsules (I) are shown below. Figure 7 , 8 .

[0038] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A method for establishing an HPLC fingerprint of Changmaile capsules (I), characterized in that, Includes the following steps: 1) Preparation of reference solutions: Accurately weigh the following reference standards: gastrodin, tanshinone, quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside, 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, puerarin apigenin, paeoniflorin, daidzein, rosmarinic acid, shikonin, isoquercitrin, mangosteenin, daidzein, salvianolic acid B, and verbascoflavonoids. Place them separately in volumetric flasks, dissolve in an appropriate amount of methanol, and dilute to volume. Filter through a 0.22 μm microporous membrane to obtain the corresponding reference solutions with concentrations of 5.504 mg / mL, 1.154 mg / mL, 1.000 mg / mL, 1.180 mg / mL, 1.180 mg / mL, 0.526 mg / mL, 1.022 mg / mL, 0.878 mg / mL, and 1.260 mg / mL, respectively. mg / mL, 1.180 mg / mL, 1.180 mg / mL, 0.4700 mg / mL, 1.038 mg / mL, 1.040 mg / mL, 0.612 mg / mL, 1.240 mg / mL; 2) Preparation of test solution: Accurately weigh 1.0 g of the contents of the same batch of Changmaile capsules (I), place it in an Erlenmeyer flask, add 25 mL of 50 vol% methanol solution, let stand overnight, then weigh and record the Erlenmeyer flask, extract by ultrasonic extraction for 30 min, add weight after cooling to room temperature, filter the supernatant through a 0.22 μm microporous membrane to obtain the test solution; 3) Sample determination: Accurately pipette each reference solution and test solution, and detect them by HPLC / DAD to obtain the chromatograms of the reference standards and test samples; 4) Establishment of fingerprint spectrum: The chromatogram of the obtained test sample is imported into the "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine 2012A" for evaluation to obtain the corresponding fingerprint spectrum, and the characteristic components are assigned using the chromatogram of the reference standard. The chromatographic conditions for the HPLC / DAD detection were: Thermo Scientific Accucore. TM The XL C18 HPLC column, with dimensions of 4.6 mm × 250 mm and a diameter of 4 μm, was used. The mobile phase was methanol-acetonitrile-0.5 vol% phosphoric acid aqueous solution, with the following gradient elution program: ； The detection wavelengths were 230 nm and 280 nm, respectively.

2. The method for establishing the HPLC fingerprint of Changmaile capsules (I) according to claim 1, characterized in that, The HPLC / DAD detection method used an injection volume of 10 μL; a column temperature of 35 ℃; and a mobile phase flow rate of 1 mL·min. -1 .

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