A method for constructing HPLC characteristic spectrum of Fuke Duanhongyin capsule

Abstract

The present invention belongs to the technical field of quality detection of traditional Chinese medicine, and specifically relates to a method for constructing an HPLC characteristic spectrum of Fuke Duanhongyin capsule. The method for constructing an HPLC characteristic spectrum of Fuke Duanhongyin capsule of the present invention has good precision and repeatability for the instrument, and the prepared test solution has good stability and durability; the characteristic spectrum of Fuke Duanhongyin capsule constructed by the method of the present invention should have 11 characteristic peaks, 6 chromatographic peaks are identified, and 10 characteristic peaks are attributed; the peaks of 1.2.3.4.6-O-pentagalloylglucose are used as reference peaks, and the relative retention time of the characteristic peaks relative to the reference peaks is within ±5% of the specified value, which can provide a basis for the quality control of Fuke Duanhongyin capsule.

Description

Technical Field

[0001] The invention belongs to the technical field of quality detection of traditional Chinese medicines, and particularly relates to a method for constructing an HPLC characteristic spectrum of Fuke Duanhongyin capsules. Background Art

[0002] Fuke Duanhongyin Capsule is a pure Chinese medicine compound preparation composed of six medicinal materials, including red peony root, motherwort, notoginseng, herbaceous agrimony, charcoal from Sanguisorba officinalis, and charcoal from Pollen Typhae. It is produced by Zhuzhou Qianjin Pharmaceutical Co., Ltd. This preparation has the effects of cooling blood, removing blood stasis, and stopping bleeding. It is used for dysfunctional uterine bleeding, which is manifested as excessive menstruation and prolonged menstruation, diagnosed as "leakage syndrome" in traditional Chinese medicine, and syndrome differentiation belongs to blood heat. The symptoms are heavy menstrual blood, or dripping and unclean, dark red or purple-red, viscous, with a small amount of blood clots, accompanied by red face and dizziness, irritability, dry mouth and thirst, constipation and red urine. At present, there are not many quality inspection methods for Fuke Duanhongyin Capsules. Among them, the method for inspecting the six components of the preparation is also an invention patent of Zhuzhou Qianjin Pharmaceutical Co., Ltd. The inspection method of this patent is: six components are identified separately by thin layer chromatography, and the six components are identified separately by different solution preparation methods and chromatographic conditions. This method not only requires more test samples, but also the detection conditions for each component are different, and the inspection steps are cumbersome. Therefore, for the quality inspection of Fuke Duanhongyin Capsules, it is necessary to find a more convenient method.

[0003] The characteristic spectrum of traditional Chinese medicine refers to the spectrum of common peaks that can indicate the characteristics of various component groups in the traditional Chinese medicine after proper treatment and certain analytical methods. It is a new quality control mode for traditional Chinese medicine. The characteristic spectrum of traditional Chinese medicine is a multi-index quality control mode that can comprehensively reflect the types and quantities of chemical components contained, so as to comprehensively and comprehensively reflect and control the quality of traditional Chinese medicine (Qin Honglin, Li Yongshen. Application of characteristic spectrum technology in quality analysis of traditional Chinese medicine [J]. China Practical Medicine, 2014, 9(07): 253-255.); High performance liquid chromatography is not limited by the volatility and thermal stability of the sample. The entire separation process is in a relatively closed environment and is less affected by the outside world. It has high sensitivity and precision, and has a good separation effect. It is currently the most widely used characteristic spectrum identification method for traditional Chinese medicine (Lv Dongfang. Application of characteristic spectrum technology of traditional Chinese medicine in identification of traditional Chinese medicine [J]. Electronic Journal of Clinical Medical Literature, 2018, 5(99): 198.).

[0004] At present, there are studies on the characteristic spectra of each component in Fuke Duanhongyin Capsule. For example, the Chinese invention patent discloses the detection method of Yimucao granules fingerprint spectrum; the establishment method of Zhongjiang Shaoyao HPLC fingerprint spectrum; the construction method of HPLC fingerprint spectrum of Panax notoginseng medicinal materials and Panax notoginseng total saponins; the identification method of characteristic spectrum of Herba Agrimoniae formula granules; the identification method of characteristic spectrum of Radix Diyutan formula granules, etc. However, these characteristic spectrum methods are all for their individual components, and their specific characteristics are different from each other. They are not established for Fuke Duanhongyin Capsule. The existing HPLC characteristic spectrum detection method is not suitable for the establishment of HPLC characteristic spectrum of Fuke Duanhongyin Capsule. In order to better control and evaluate the quality of Fuke Duanhongyin Capsule, it is very necessary to establish an HPLC characteristic spectrum for the entire Fuke Duanhongyin Capsule. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies in the existing quality control and evaluation of Fuke Duanhongyin Capsules, and to provide a method for constructing an HPLC characteristic spectrum of Fuke Duanhongyin Capsules. The method has good precision, stability, repeatability and durability, and there are 11 characteristic peaks in the detected characteristic spectrum, 6 chromatographic peaks are identified, 10 characteristic peaks are attributed, and the relative retention time of the characteristic peaks to the reference peaks is within ±5% of the specified value; the method can provide a basis for the quality control of Fuke Duanhongyin Capsules.

[0006] The object of the present invention is to provide a method for pre-treating a HPLC test solution of Fuke Duanhongyin capsules.

[0007] The present invention also aims to provide a method for constructing an HPLC characteristic spectrum of Fuke Duanhongyin Capsule.

[0008] The present invention also aims to provide a quality detection method for Fuke Duanhongyin capsules.

[0009] The above-mentioned purpose of the present invention is achieved by the following technical means:

[0010] The invention provides a pretreatment method for a HPLC test sample solution of Fuke Duanhongyin capsules, comprising the steps of extracting a test sample with water to obtain a water extract of the test sample; performing solid-liquid separation on the water extract of the test sample to obtain a supernatant; fully extracting the supernatant with ether; taking an ether layer, removing the solvent ether of the ether layer, and fully drying to obtain a solid; dissolving the solid with a 60-90wt% ethanol solution, filtering through a 0.45μm filter membrane, and obtaining a subsequent filtrate to obtain a test sample solution.

[0011] Preferably, the step of extracting the test sample with water to obtain the water extract of the test sample comprises: fully dissolving the test sample in water, ultrasonically treating the sample, cooling the sample to room temperature, shaking the sample evenly, and centrifuging the sample.

[0012] Preferably, 70-80 wt % ethanol solution is used to dissolve the solid.

[0013] More preferably, the solid is dissolved using a 75 wt % ethanol solution.

[0014] Further preferably, the ultrasonic treatment is performed at a power of 100 to 500 W for 5 to 30 min; and the centrifugation is performed at 10,000 rpm for 1 to 10 min.

[0015] Further preferably, the ultrasonic treatment is performed at a power of 200 to 300 W for 8 to 12 min; and the centrifugation is performed at 10,000 rpm for 1 to 3 min.

[0016] Most preferably, the ultrasonic treatment is performed at a power of 300 W and a frequency of 40 Hz for 10 min; and the centrifugation is performed at 10,000 rpm for 2 min.

[0017] Preferably, the mass ratio of the test sample to water is 1-5:10-40; the volume ratio of the supernatant of the water extract, ether and ethanol is 5-30:10-40:5-10.

[0018] Further preferably, the mass ratio of the test sample to water is 1-2:20-30; the volume ratio of the supernatant of the water extract, ether and ethanol is 10-20:25-35:5-10.

[0019] Most preferably, the mass ratio of the test sample to water is 2:25; the volume ratio of the supernatant of the water extract, ether and ethanol is 15:30:5.

[0020] The application of the pretreatment method in constructing the HPLC characteristic spectrum of Fuke Duanhongyin Capsules is also within the protection scope of the present invention.

[0021] The present invention provides a method for constructing an HPLC characteristic spectrum of Fuke Duanhongyin Capsule, comprising the following steps:

[0022] S1. Pre-treat the sample according to the pre-treatment method of Fuke Duanhongyin Capsule HPLC sample solution; use 1.2.3.4.6-O-pentagalloyl glucose solution with a solvent of 60-90wt% ethanol and a concentration of 0.4-0.8mg / mL as the reference solution.

[0023] S2.HPLC detection

[0024] Acetonitrile was used as mobile phase A, 0.2 wt % phosphoric acid aqueous solution was used as mobile phase B, gradient elution was performed, the flow rate was 0.8-1.2 mL / min, the column temperature was 33-36° C., the injection volume was 5 μL, and the detection wavelength was 200-204 nm for HPLC detection.

[0025] Preferably, in step S1, extracting the test sample with water to obtain the water extract of the test sample comprises fully dissolving the test sample in water, ultrasonically treating the sample, cooling the sample to room temperature, shaking the sample evenly, and centrifuging the sample.

[0026] Preferably, in step S1, 70-80 wt % ethanol solution is used to dissolve the solid.

[0027] Further preferably, in step S1, 75 wt % ethanol solution is used to dissolve the solid.

[0028] Further preferably, the ultrasonic treatment is performed at a power of 100 to 500 W for 5 to 30 min; and the centrifugation is performed at 10,000 rpm for 1 to 10 min.

[0029] Further preferably, the ultrasonic treatment is performed at a power of 200 to 300 W for 8 to 12 min; and the centrifugation is performed at 10,000 rpm for 1 to 3 min.

[0030] Most preferably, the ultrasonic treatment in step S1 is performed at a power of 300 W and a frequency of 40 Hz for 10 min; and the centrifugation is performed at 10,000 rpm for 2 min.

[0031] Preferably, the mass ratio of the test sample to water is 1-5:10-40; the volume ratio of the supernatant of the water extract, ether and ethanol is 5-30:10-40:5-10.

[0032] Further preferably, the mass ratio of the test sample to water is 1-2:20-30; the volume ratio of the supernatant of the water extract, ether and ethanol is 10-20:25-35:5-10.

[0033] Most preferably, in step S1, the mass ratio of the test sample to water is 2:25; and the volume ratio of the supernatant of the water extract, ether and ethanol is 15:30:5.

[0034] Preferably, the concentration of the 1.2.3.4.6-O-pentagalloylglucose reference solution in step S1 is 0.5-0.6 mg / mL.

[0035] Further preferably, the concentration of the 1.2.3.4.6-O-pentagalloylglucose reference solution in step S1 is 0.5 mg / mL.

[0036] Preferably, in step S2, Shimadzu Shim-pack GISS (4.6 mm*250 mm, 5 μm) is used as the chromatographic column.

[0037] Preferably, the gradient elution procedure in step S2 is: mobile phase A: acetonitrile: 0-17 min: 14-15%; 17-47 min: 14-15% → 30%; 47-55 min: 30% → 40%; 55-60 min: 40% → 15-16%.

[0038] Further preferably, the gradient elution procedure in step S2 is: mobile phase A acetonitrile 0-17 min 14%; 17-47 min 14% → 30%; 47-55 min 30% → 40%; 55-60 min 40% → 14%.

[0039] Preferably, the detection wavelength in step S2 is 203 nm.

[0040] Preferably, step S3 uses acetonitrile as mobile phase A and 0.2 wt % phosphoric acid aqueous solution as mobile phase B for gradient elution; flow rate is 1.0 mL / min; column temperature is 35° C.; injection volume is 5 μL; detection wavelength is 200-204 nm, and HPLC detection is performed.

[0041] The characteristic spectrum constructed by the method for constructing the HPLC characteristic spectrum of Fuke Duanhongyin capsule of the present invention takes the peaks of 1.2.3.4.6-O-pentagalloylglucose as reference peaks, and calculates the relative retention time of the characteristic peaks; the characteristic spectrum should have 11 characteristic peaks; the relative retention time of the characteristic peaks of the characteristic spectrum is within ±5% of the specified value; the specified values ​​of the relative retention time of the characteristic peaks of the characteristic spectrum are: peak 1 is 0.126, peak 2 is 0.148, peak 3 is 0.241, peak 4 is 0.316, peak 5 is 0.327, peak 6 is 0.455, peak 7 is 0.526, peak 8 is 0.740, peak 10 is 1.22, and peak 11 is 1.59.

[0042] A method for quality inspection of Fuke Duanhongyin capsules is to use the method for constructing an HPLC characteristic spectrum of Fuke Duanhongyin capsules to construct a characteristic spectrum for quality inspection.

[0043] The application of the method for constructing the HPLC characteristic spectrum of Fuke Duanhongyin Capsules in the quality inspection of Fuke Duanhongyin Capsules is also within the protection scope of the present invention.

[0044] In the present invention, HPLC refers to high performance liquid chromatography; the subsequent filtrate refers to the subsequent filtrate obtained by a general method, usually referring to the filtrate obtained after filtering 1 mL.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] The present invention provides a method for constructing an HPLC characteristic spectrum of Fuke Duanhongyin capsules. The method has good precision and repeatability for instruments, and the prepared test solution has good stability and durability. Meanwhile, the characteristic spectrum of Fuke Duanhongyin capsules constructed by the method of the present invention should have 11 characteristic peaks, 6 chromatographic peaks are identified, 10 characteristic peaks are attributed, and the relative retention time of the characteristic peaks relative to the reference peaks is within ±5% of the specified value, which can provide a basis for the quality control of Fuke Duanhongyin capsules. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 These are the chromatograms of the test sample and 6 negative samples; the chromatograms from top to bottom are the negative samples of Fuke Duanhongyin Capsules lacking Panax notoginseng, lacking Pollen Typhae charcoal, lacking Sanguisorba officinalis charcoal, lacking Leonurus japonicus, lacking Paeonia lactiflora, and lacking Agrimonia sinensis, and the test sample Fuke Duanhongyin Capsules 20200704.

[0048] Figure 2 The chromatograms are for the test sample and 5 reference samples.

[0049] Figure 3 This is the UV absorption diagram of the absorption peaks of 5 reference substances.

[0050] Figure 4 It is the UV absorption diagram of the absorption peak of the test sample.

[0051] Figure 5 This is the HPLC characteristic spectrum of Fuke Duanhongyin Capsule in Example 3; Peak 1: gallic acid; Peak 4: syringic acid; Peak 6: ethyl gallate; Peak 7: 3,4-dihydroxy-5-methoxy-benzoic acid methyl ester; Peak 9 (S): 1.2.3.4.6-O-pentagalloylglucose; Peak 11: benzoylpeonidin.

[0052] Figure 6 These are the HPLC characteristic spectra of 15 batches of Fuke Duanhongyin Capsules.

[0053] Figure 7 This is the chromatogram of the test sample in Comparative Example 1 with the mobile phase being Gradient 1.

[0054] Figure 8 This is the chromatogram of the test sample in Comparative Example 1 with the mobile phase being Gradient 2.

[0055] Fig. 9 This is the chromatogram of the test sample in Comparative Example 1 with the mobile phase being gradient 3.

[0056] Fig.10 This is the chromatogram of the test product in Comparative Example 2 using Shimadzu Shim-pack GISS as the chromatographic column.

[0057] Fig.11This is the chromatogram of the test sample in Comparative Example 2 using Inertsil ODS-3 as the chromatographic column.

[0058] Fig.12 This is the chromatogram of the test sample in Comparative Example 2 using SHIMSEN Ankylo C18-AQ as the chromatographic column.

[0059] Fig.13 This is the chromatogram of the test sample prepared by Comparative Example 3 Method 1.

[0060] Fig.14 The chromatogram is of the test sample prepared by method 2 of comparative example 3.

[0061] Fig.15 The chromatogram is of the test sample prepared by method 3 of comparative example 3.

[0062] Fig.16 The chromatogram is of the test sample prepared by method 4 of comparative example 3. DETAILED DESCRIPTION

[0063] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0064] Unless otherwise specified, the reagents and materials used in the following examples were commercially available. The ether used was analytical grade.

[0065] Example 1 Attribution of Chromatographic Peaks in HLPC Chromatogram of Fuke Duanhongyin Capsule

[0066] 1. Methods

[0067] The chromatographic peaks of the characteristic spectrum were assigned by comparing the chromatographic peaks of the test solution and the chromatographic peaks of the negative sample solution.

[0068] 1.1 Preparation of test solution:

[0069] Accurately weigh 2g of Fuke Duanhongyin capsule powder, place it in a stoppered conical flask, accurately add 25mL of water to dissolve, ultrasonically treat (power 300W, frequency 40Hz) for 10min, cool, shake well, centrifuge at 10000rpm for 2min, accurately measure 15mL of the supernatant of the water extract after centrifugation, extract twice with ether, 15mL each time, combine the ether layers after extraction, evaporate to dryness, dissolve the dried solid residue with an appropriate amount of 75wt% ethanol, transfer to a 5mL volumetric flask, add 75wt% ethanol to dilute to the scale, shake well, filter, and take the filtrate to obtain the test solution.

[0070] 1.2 Preparation of negative sample solution:

[0071] Fuke Duanhongyin capsules lacking Sanguisorba officinalis charcoal, Panax notoginseng, Herba Agrimoniae, Paeonia lactiflora, Leonurus japonicus and Pollen Typhae charcoal were used as negative samples, and 6 negative sample solutions were prepared according to the preparation method of the test sample solution.

[0072] 1.3HPLC detection

[0073] Shimadzu Shim-pack GISS (4.6mm*250mm, 5μm) was used as the chromatographic column, acetonitrile was used as the mobile phase A, 0.2wt% phosphoric acid aqueous solution was used as the mobile phase B, and gradient elution was performed according to the mobile phase gradient elution program in Table 1; the flow rate was 1.0mL / min; the column temperature was 35°C; the injection volume was 5μL; the detection wavelength was 203nm, and the chromatogram was recorded for 60 minutes.

[0074] Table 1 Mobile phase gradient elution program

[0075]

[0076] 2. Information on test samples and reference materials

[0077]

[0078]

[0079] 3. Results

[0080] The chromatograms of the test sample and 6 negative samples are as follows: Figure 1 As shown. Figure 1 It can be seen that the chromatogram of the negative sample lacking Sanguisorba officinalis charcoal has no characteristic peak 2 and characteristic peak 7, so characteristic peak 2 and characteristic peak 7 can be attributed to Sanguisorba officinalis charcoal; the chromatogram of the negative sample lacking Pollen Typhae charcoal has no peak 5, so characteristic peak 5 can be attributed to Pollen Typhae charcoal; the chromatogram of the negative sample lacking Leonurus japonicus has no characteristic peak 4, so characteristic peak 4 can be attributed to Leonurus japonicus; the chromatogram of the negative sample lacking Paeonia lactiflora has no characteristic peak 3, peak 6, peak 8, peak 9, peak 10 and peak 11, so these six characteristic peaks can be attributed to Paeonia lactiflora.

[0081] Example 2 Identification of Chromatographic Peaks in the HLPC Chromatogram of Fuke Duanhongyin Capsules

[0082] 1. Methods

[0083] The main chemical components and key quality attributes of the relevant medicinal flavors were understood by consulting the literature. The Shimadzu LCMS-9030 high-resolution Q-TOF liquid-mass spectrometer was used to conduct qualitative analysis of Fuke Duanhongyin Capsules, providing a basis for the identification of chromatographic peaks in the characteristic spectrum. The DAD detector was used to perform full-wavelength scanning of the relevant compound control solution and the test solution at 190-400nm to obtain the ultraviolet absorption graph of the absorption peaks of each compound and the test sample. The main characteristic peaks of the characteristic spectrum were identified by comparing the retention time and ultraviolet absorption graph of the peaks of the compound and the test solution.

[0084] 1.1 Preparation of test solution: Same as Example 1.

[0085] 1.2 Preparation of reference solution:

[0086] Take an appropriate amount of 1.2.3.4.6-O-pentagalloylglucose reference substance, weigh accurately, and add 75wt% ethanol to make a solution containing about 0.5mg of 1.2.3.4.6-O-pentagalloylglucose per 1mL;

[0087] Take an appropriate amount of syringic acid reference substance, weigh accurately, and add ethanol to make a solution containing about 0.6 mg of syringic acid per 1 mL;

[0088] Take appropriate amount of ethyl gallate and benzoylpeonidin reference substances, weigh accurately, and add methanol to prepare a solution containing about 65 μg of benzoylpeonidin and 0.5 mg of ethyl gallate per 1 mL respectively;

[0089] Take an appropriate amount of gallic acid reference substance, weigh accurately, and add water to make a solution containing approximately 1.0 mg of gallic acid per 1 mL.

[0090] 1.3 Take the test solution and 5 reference solution and inject them according to the chromatographic conditions of Example 1. The test results are as follows: Figure 2 As shown; the DAD detector was used to perform full wavelength scanning of the reference solution and the test solution at 190-400 nm, and the UV absorption diagram of each reference absorption peak is shown in Figure 3 As shown, the UV absorption diagram of the absorption peak of the test sample is as follows Figure 4 shown.

[0091] 2.2. Information on test samples and reference materials

[0092]

[0093] 3. Results

[0094] The results of mass spectrometry analysis of Fuke Duanhongyin Capsules are shown in Table 2.

[0095] Table 2 Mass spectrometry analysis results of Fuke Duanhongyin Capsules

[0096]

[0097]

[0098] According to Table 2, by comparing the primary mass spectrum and secondary ion mass spectrum information, the molecular formulas of the main components of the specific 6 chromatographic peaks were predicted. Combined with the relevant information such as their elemental composition, the structural formulas of the 6 unknown substances in the active ingredients of traditional Chinese medicine were inferred to be gallic acid, syringic acid, ethyl gallate, 3,4-dihydroxy-5-methoxy-benzoic acid methyl ester, 1,2,3,4,6-O-pentagalloylglucose and benzoylpeonidin.

[0099] Retention time and UV absorption spectrum of the combined compound and test solution (see Figures 2 to 4 ), showing that the retention times of compounds gallic acid, syringic acid, ethyl gallate, 3,4-dihydroxy-5-methoxy-benzoic acid methyl ester, 1,2,3,4,6-O-pentagalloyl glucose and benzoylpeonidin were consistent with peak 1 (4.15 min), peak 4 (10.4 min), peak 6 (15.0 min), peak 7 (17.4 min), peak 9 (33.0 min) and peak 11 (52.7 min) in the chromatogram of the test sample. Therefore, peak 1, peak 4, peak 6, peak 7, peak 9 and peak 11 were assigned to compounds gallic acid, syringic acid, ethyl gallate, 3,4-dihydroxy-5-methoxy-benzoic acid methyl ester, 1.2.3.4.6-O-pentagalloyl glucose and benzoylpeonidin, respectively.

[0100] In view of the fact that the chromatographic peak retention time of 1,2,3,4,6-O-pentagalloylglucose is in the middle, the separation is good, and the response value is high, 1,2,3,4,6-O-pentagalloylglucose (peak 9) is selected as the reference peak of the reference substance, marked as peak S, and the 1,2,3,4,6-O-pentagalloylglucose reference substance is used as a reference substance.

[0101] Example 3 Detection method of HPLC characteristic spectrum of Fuke Duanhongyin capsule

[0102] 1. Methods

[0103] 1.1 Preparation of test solution

[0104] Accurately weigh 2g of Fuke Duanhongyin capsule powder, place it in a stoppered conical flask, accurately add 25mL of water to dissolve, ultrasonically treat (power 300W, frequency 40Hz) for 10min, cool, shake well, centrifuge at 10000rpm for 2min, accurately measure 15mL of the supernatant of the water extract after centrifugation, extract twice with ether, 15mL each time, combine the ether layers after extraction, evaporate to dryness, dissolve the dried solid residue with an appropriate amount of 75wt% ethanol, transfer to a 5mL volumetric flask, add 75% ethanol to dilute to the scale, shake well, filter, and take the filtrate to obtain the test solution.

[0105] 1.2 Preparation of reference solution

[0106] Accurately weigh 1.2.3.4.6-O-pentagalloyl glucose and add 75 wt% ethanol to prepare a solution containing 0.5 mg of 1.2.3.4.6-O-pentagalloyl glucose per 1 mL to obtain a reference solution.

[0107] 1.3HPLC detection

[0108] Shimadzu Shim-pack GISS (4.6mm*250mm, 5μm) was used as the chromatographic column, acetonitrile was used as the mobile phase A, and 0.2wt% phosphoric acid aqueous solution was used as the mobile phase B. Gradient elution was performed according to the mobile phase gradient elution program in Table 1 of Example 1; flow rate was 1.0mL / min; column temperature was 35°C; injection volume was 5μL; detection wavelength was 203nm, and HPLC detection was performed. The theoretical plate number was calculated based on the 1.2.3.4.6-O-pentagalloylglucose peak and should not be less than 3000. 5μL of the reference solution and the test solution were accurately aspirated respectively, injected into the high performance liquid chromatograph, measured, and the chromatogram was recorded for 60 minutes.

[0109] 2. Information on test samples and reference materials

[0110]

[0111] 3. Results

[0112] The characteristic spectrum of the Fuke Duanhongyin capsule test product is as follows Figure 5 As shown, from Figure 5It can be seen that there are 11 characteristic peaks in the characteristic spectrum of the Fuke Duanhongyin capsule test product. The peaks of 1.2.3.4.6-O-pentagalloylglucose are used as reference peaks (S peaks), and the relative retention times of characteristic peaks 1, 2, 3, 4, 5, 6, 7, 8, 10, and 11 are calculated. The relative retention times are within ±5% of the specified values. The specified values ​​are 0.126 (peak 1), 0.148 (peak 2), 0.241 (peak 3), 0.316 (peak 4), 0.327 (peak 5), 0.455 (peak 6), 0.526 (peak 7), 0.740 (peak 8), 1.22 (peak 10), and 1.59 (peak 11).

[0113] The present invention also uses different proportions of Fuke Duanhongyin capsules and water, the supernatant of the water extract, ether, and ethanol to prepare the test sample solution and different concentrations of 1.2.3.4.6-O-pentagalloylglucose reference solution as samples, and uses HPLC for detection. The specific conditions and proportions are as follows:

[0114]

[0115] Conditions 1 and 2 only changed the amounts of Fuke Duanhongyin capsules, water, the supernatant of the water extract, ether, ethanol, and 1.2.3.4.6-O-pentagalloylglucose;

[0116] Condition 3 only changes the mobile phase gradient to

[0117]

[0118] Under conditions 1, 2 and 3, there are 11 characteristic peaks in the characteristic spectrum of the Fuke Duanhongyin capsule test product. The peaks of 1.2.3.4.6-O-pentagalloylglucose are used as reference peaks (S peaks), and the relative retention times of characteristic peaks 1, 2, 3, 4, 5, 6, 7, 8, 10 and 11 are calculated. The relative retention times are within ±5% of the specified values. The specified values ​​are 0.126 (peak 1), 0.148 (peak 2), 0.241 (peak 3), 0.316 (peak 4), 0.327 (peak 5), 0.455 (peak 6), 0.526 (peak 7), 0.740 (peak 8), 1.22 (peak 10) and 1.59 (peak 11).

[0119] Example 4 Detection wavelength of HPLC characteristic spectrum of Fuke Duanhongyin Capsule

[0120] 1. Methods

[0121] The test solution was prepared by the method of Example 3, and the HPLC detection was performed under the chromatographic conditions of Example 3. The DAD detector was used to perform full wavelength scanning at 190-400 nm to obtain the ultraviolet absorption graph of each absorption peak. The ultraviolet absorption graph of each absorption peak is shown in FIG. Figure 4 .

[0122] 2. Results

[0123] from Figure 4 It can be seen that each absorption peak has different degrees of response in the range of 190-400nm, that is, different degrees of absorption. The detection wavelength is around 203-204nm. Each absorption peak has higher absorption, and the intersection of each absorption curve is closer to 203nm. Therefore, 203nm is suitable as the detection wavelength.

[0124] Experimental Example 1: Precision Test

[0125] 1. Methods

[0126] Take a sample of the contents of Fuke Duanhongyin capsules, prepare a test solution according to the method of Example 3, repeat the injection 6 times according to the chromatographic conditions of Example 3, and record the chromatogram. Take the peaks of 1.2.3.4.6-O-pentagalloylglucose as the reference peaks, and calculate the relative retention time and relative peak area of ​​the characteristic peaks in the chromatogram.

[0127] 2. Results

[0128] The relative retention time and relative peak area of ​​the characteristic peaks in the chromatograms detected with different injection times are shown in Tables 3 and 4.

[0129] Table 3 Relative retention time of characteristic peaks in the chromatograms with different injection times

[0130]

[0131] Table 4 Relative peak areas of characteristic peaks in chromatograms with different injection times

[0132]

[0133]

[0134] It can be seen from Tables 3 and 4 that the RSD of the relative retention time of the characteristic peaks in the chromatograms detected with different injection times is 0.00-0.32%, and the RSD of the relative peak area of ​​the characteristic peaks is 0.00-3.62%, indicating that the method of Example 3 has good precision for the HPLC instrument.

[0135] Experimental Example 2 Repeatability Test

[0136] 1. Methods

[0137] Take the same batch of Fuke Duanhongyin capsule samples, prepare 6 test solutions according to the method of Example 3, and inject all 6 test solutions according to the chromatographic conditions of Example 3, and record the chromatogram. Take the peak of 1.2.3.4.6-O-pentagalloylglucose as the reference peak, and calculate the relative retention time and relative peak area of ​​the characteristic peak in the chromatogram.

[0138] 2. Results

[0139] The relative retention times and relative peak areas of the characteristic peaks in the chromatograms of the six test samples are shown in Tables 5 and 6.

[0140] Table 5 Relative retention time of common peaks in the chromatograms of 6 test solutions

[0141]

[0142] Table 6 Relative peak areas of common peaks in the chromatograms of the 6 test solutions

[0143]

[0144]

[0145] It can be seen from Tables 5 and 6 that the RSD of the relative retention time of the characteristic peaks in the chromatograms of the six test solutions is 0.00-0.32%, and the RSD of the relative peak area of ​​the characteristic peaks is <3.32%, indicating that the method of Example 3 has good repeatability.

[0146] Experimental Example 3: Stability Test

[0147] 1. Methods

[0148] Take a sample of Fuke Duanhongyin capsules, prepare a test solution according to the method of Example 3, inject the test solution at 0h, 2h, 4h, 6h, 8h, and 12h according to the chromatographic conditions of Example 3, and record the chromatogram. Take the peak of 1.2.3.4.6-O-pentagalloylglucose as the reference peak, and calculate the relative retention time and relative peak area of ​​the characteristic peak in the chromatogram.

[0149] 2. Results

[0150] The relative retention time and relative peak area of ​​the characteristic peaks in the chromatograms injected at different times are shown in Tables 7 and 8.

[0151] Table 7 Relative retention time of characteristic peaks in the chromatograms injected at different times

[0152]

[0153]

[0154] Table 8 Relative peak areas of characteristic peaks in the chromatograms injected at different times

[0155]

[0156] It can be seen from Tables 7 and 8 that the RSD of the relative retention time of the characteristic peaks in the chromatograms injected at different times is 0.00% to 0.32%, and the RSD of the relative peak area of ​​the characteristic peaks is <3.44%, indicating that the test solution prepared by the method of Example 3 is stable for at least 12 hours.

[0157] Experimental Example 4 Instrument Durability Test

[0158] 1. Methods

[0159] Take a sample of Fuke Duanhongyin Capsule, prepare a test solution according to the method of Example 3, inject the sample on three high performance liquid chromatographs, Agilent 1260Ⅰ, Agilent 1260Ⅱ and Waters 2695, according to the chromatographic conditions of Example 3, record the chromatogram, and calculate the relative retention time of the characteristic peaks in the chromatogram.

[0160] 2. Results

[0161] The relative retention times of the characteristic peaks in the chromatograms detected by different HPLC are shown in Table 9.

[0162] Table 9 Relative retention time of characteristic peaks in chromatograms detected by different HPLC

[0163]

[0164] As can be seen from Table 9, the RSD of the relative retention time of characteristic peaks in the chromatograms detected by different HPLC instruments is <2.91%, indicating that the method of Example 3 has good durability on different instruments.

[0165] Experimental Example 5: Multiple batches of sample testing

[0166] 1. Methods

[0167] Take 15 batches of Fuke Duanhongyin Capsule samples, prepare the test solution according to the method of Example 3, inject the sample according to the chromatographic conditions of Example 3, and record the chromatogram. The HPLC characteristic spectrum of 15 batches of Fuke Duanhongyin Capsule is as follows: Figure 6 As shown. Taking 1.2.3.4.6-O-pentagalloylglucose (peak 9) as the reference peak, the ratio of the retention time of the remaining characteristic peaks to the reference peak is defined as the relative retention time of the remaining peaks. The relative retention times of 11 characteristic peaks of 15 batches of Fuke Duanhongyin Capsules were calculated according to the above method.

[0168] 2. Results

[0169] The relative retention times of the characteristic peaks in the chromatograms of 15 batches of Fuke Duanhongyin Capsules are shown in Table 10.

[0170] Table 10 Relative retention time of characteristic peaks in the chromatograms of 15 batches of Fuke Duanhongyin Capsules

[0171]

[0172]

[0173] As can be seen from Table 10, the RSD of the relative retention time of the characteristic peaks in the chromatograms of 15 batches of Fuke Duanhongyin Capsules is less than 0.24%. According to the average value of the relative retention time of the characteristic peaks of 15 batches of samples, the relative retention time of each peak is determined to be 0.126 (peak 1), 0.148 (peak 2), 0.241 (peak 3), 0.316 (peak 4), 0.327 (peak 5), 0.455 (peak 6), 0.526 (peak 7), 0.740 (peak 8), 1.22 (peak 10), and 1.59 (peak 11).

[0174] Comparative Example 1 Mobile Phase Gradient for HPLC Characteristic Spectrum Detection of Fuke Duanhongyin Capsules

[0175] 1. Methods

[0176] The test solution was prepared by the method of Example 3, and the HPLC detection was performed under the chromatographic conditions of Example 3, except that the mobile phase gradient was different. The different gradient programs of the mobile phase are shown in Table 11. (Gradient 1 is the mobile phase gradient of Example 3)

[0177] Table 11 Different gradient programs of mobile phase

[0178]

[0179]

[0180] 2. Results

[0181] The chromatograms detected by mobile phase gradient 1 to 3 programs are as follows: Figure 7 to Figure 9 From the chromatogram Figure 7 to Figure 9 It can be seen that gradients 2 and 3 have problems such as poor peak separation effect and too early peak elution time. The chromatogram detected by gradient 1 has a clear separation peak, good separation effect, and reasonable peak elution time. Therefore, gradient 1 has the best peak elution time and separation effect, that is, the mobile phase gradient of Example 3.

[0182] Comparative Example 2 Chromatographic column for HPLC characteristic spectrum detection of Fuke Duanhongyin Capsule

[0183] 1. Methods

[0184] The test solution was prepared by the method of Example 3, and the HPLC detection was carried out under the chromatographic conditions of Example 3, except that the chromatographic columns were different. The specific chromatographic columns were Shimadzu Shim-pack GISS (i.e., the chromatographic column of Example 3), Inertsil ODS-3, and SHIMSEN Ankylo C18-AQ.

[0185] 2. Results

[0186] The chromatograms of Shimadzu Shim-pack GISS, Inertsil ODS-3, and SHIMSEN Ankylo C18-AQ columns are shown below: Figure 10 to Figure 12 .

[0187] Comparison of chromatograms of the three columns for the test samples Figure 10 to Figure 12 It can be seen that all three chromatographic columns can separate the test samples, but only the chromatographic column Shimadzu Shim-pack GISS has the best separation effect, that is, the chromatographic column in Example 3.

[0188] Comparative Example 3 Effects of different test sample preparation methods on the HPLC characteristic spectrum of Fuke Duanhongyin Capsules

[0189] 1. Methods

[0190] The HPLC detection was carried out under the chromatographic conditions of Example 3, except that the preparation method of the test solution was different. The test solution was prepared by the method shown in Table 12.

[0191] Table 12 Preparation method of test solution

[0192]

[0193] 2. Results

[0194] The chromatograms of the test solutions prepared by methods 1 to 4 are as follows: Figure 13 to Figure 16 ,contrast Figure 13 to Figure 16 It can be seen that the chromatograms of the test solutions prepared by different methods are significantly different. The chromatograms of the test solutions prepared by methods 1, 3 and 4 have overlapping chromatographic peaks and poor separation effects. The chromatogram of the test solution prepared by method 2 (i.e., the method of Example 3) has the best chromatographic peak shape and separation effect.

[0195] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for pre-treating a test solution of Fuke Duanhongyin Capsule HPLC, characterized in that: Extracting the test sample with water to obtain a water extract of the test sample; solid-liquid separation of the water extract of the test sample, and taking the supernatant; The supernatant is fully extracted with ether; the ether layer is taken, the solvent ether of the ether layer is removed, and the solid is fully dried to obtain a solid; the solid is dissolved with a 60-90wt% ethanol solution, and the subsequent filtrate is taken as the test solution; The mass volume ratio of the test sample to water is 2-5 g:10-40 mL; the volume ratio of the supernatant of the water extract, ether and ethanol is 5-30 mL:10-40 mL:5-10 mL.

2. The pre-treatment method according to claim 1, characterized in that: The method of extracting the test sample with water to obtain the water extract of the test sample specifically comprises: fully dissolving the test sample in water, ultrasonically treating the sample, cooling the sample to room temperature, shaking the sample evenly, and centrifuging the sample.

3. The pre-treatment method according to claim 2, characterized in that: The ultrasonic treatment is performed at a power of 100 to 500 W for 5 to 30 minutes; the centrifugal treatment is performed at 10,000 rpm for 1 to 10 minutes.

4. Use of the pretreatment method according to any one of claims 1 to 3 in constructing the HPLC characteristic spectrum of Fuke Duanhongyin Capsule.

5. A method for constructing an HPLC characteristic spectrum of Fuke Duanhongyin Capsule, characterized in that: The steps include: S1. Pre-treat the sample according to any of the pre-treatment methods of claims 1 to 3; use a 1.2.3.4.6-O-pentagalloyl glucose solution with a solvent of 60 to 90 wt% ethanol and a concentration of 0.4 to 0.8 mg / mL as a reference solution; S2.HPLC detection Acetonitrile is used as mobile phase A, 0.2wt% phosphoric acid aqueous solution is used as mobile phase B, gradient elution is performed; flow rate is 0.8-1.2mL / min; column temperature is 33-36°C; injection volume is 5μL; detection wavelength is 200-204nm, HPLC detection is performed, and the gradient elution program is: mobile phase A acetonitrile 0-17min 14-15%; 17-47min 14-15%→30%; 47-55min 30%→40%; 55-60min 40%→15-16%; HPLC detection was performed using Shimadzu Shim-pack GISS, Inertsil ODS-3 or SHIMSEN Ankylo C18-AQ as chromatographic columns.

6. The method according to claim 5, characterized in that: In step S3, Shimadzu Shim-pack GISS was used as the chromatographic column.

7. Application of the method according to any one of claims 5 to 6 in quality inspection of Fuke Duanhongyin Capsules.

8. A quality inspection method for Fuke Duanhongyin capsules, characterized in that: The quality detection is performed using the characteristic spectrum constructed by the method described in any one of claims 5 to 6.

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