Establishment of characteristic spectrum and detection method of Dan'e Fukang decoction

Abstract

The present invention belongs to the technical field of quality detection of traditional Chinese medicine, and specifically relates to the establishment and detection method of the characteristic spectrum of Dan'e Fukang decoction. The method for establishing the characteristic spectrum is as follows: a. Accurately weigh chlorogenic acid, ferulic acid, and salvianolic acid B reference substances, mix them, and then add methanol solution to dissolve them to obtain a reference solution; b. Accurately weigh Dan'e Fukang decoction, add water for ultrasonic extraction, centrifuge, take the supernatant and pass it through a macroporous adsorption resin, elute with water and then with ethanol solution, collect the eluate, evaporate to dryness, add water, filter, and take the subsequent filtrate to obtain a test solution; c. Use ultra-high performance liquid chromatography to measure the test solution and the reference solution, and obtain a characteristic spectrum consisting of 11 common characteristic peaks. This method constructs the characteristic spectrum of Dan'e Fukang decoction, which provides a scientific experimental basis for the study of the quality evaluation system of Dan'e Fukang decoction; and also establishes a method for rapid and comprehensive detection of the effective ingredients of Dan'e Fukang decoction.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine quality detection, and particularly relates to a characteristic spectrum establishment and detection method of Dan'e Fukang decoction. Background Art

[0002] Dan'e Fukang Decoction is a traditional Chinese medicine preparation. It has the effects of promoting blood circulation and removing blood stasis, soothing the liver and regulating qi, regulating menstruation and relieving pain, softening and removing hard masses, etc. It is a commonly used medicine for the clinical treatment of endometriosis. Dan'e Fukang Decoction is dark brown and viscous semi-fluid. The whole formula is composed of 10 herbs: Salvia miltiorrhiza, Curcuma zedoaria, Bupleurum yunnanensis, Panax notoginseng, Paeonia lactiflora, Angelica sinensis, Trillium, Cyperus rotundus, Corydalis yanhusuo and Licorice.

[0003] Dan'e Fukang Decoction contains many prescription medicinal materials, so the ingredients are complex and diverse. In "Dan'e Fukang Decoction" (National Drug Standard of the State Food and Drug Administration), liquid chromatography was used to establish a content determination method for the four components of paeoniflorin, ginsenoside Rg1, ginsenoside Rb1 and notoginsenoside R1.

[0004] However, Dan'e Fukang Decoction is a compound preparation containing multiple ingredients such as phenylpropanoids, coumarins and phenolic acids, and has a complex material basis. Simply controlling its drug quality by testing four index components is obviously incomplete, and cannot reflect the overall characteristics of Chinese medicine compound preparations. Therefore, it is very necessary to develop and establish a simple, accurate, and repeatable detection method that can reflect as many ingredients as possible in compound preparations for the quality control and evaluation of Dan'e Fukang Decoction. Summary of the invention

[0005] The first object of the present invention is to provide a method for establishing a characteristic spectrum of Dan'e Fukang decoction which is more conducive to quality identification.

[0006] The method for establishing the characteristic spectrum of Dan'e Fukang decoction provided by the present invention comprises the following steps:

[0007] a. Accurately weigh the chlorogenic acid, ferulic acid, and salvianolic acid B reference substances, mix them, and dissolve them in methanol to obtain the reference solution;

[0008] b. Accurately weigh Dan'e Fukang Decoction, add water for ultrasonic extraction, centrifuge, take the supernatant and pass it through macroporous adsorption resin, elute with water and then with ethanol solution, collect the eluate, evaporate to dryness, add water, filter, and take the filtrate to obtain the test solution;

[0009] c. The test solution and the reference solution are measured by ultra-high performance liquid chromatography to obtain a characteristic spectrum consisting of 11 common characteristic peaks.

[0010] Among them, in the method for establishing the characteristic spectrum of Dan'e Fukang decoction, in step c, the mobile phase used in the ultra-high performance liquid chromatography is: a phosphoric acid solution with a volume concentration of 0.1% as mobile phase A, and acetonitrile as mobile phase B. When the chromatographic column used is Acclaim RSLC C120 C18 (2.1mm×100mm, 2.2μm), the gradient elution program is:

[0011] From 0 to 33 min, the volume fraction of mobile phase A decreased from 95% to 76%, and the volume fraction of mobile phase B increased from 5% to 24%;

[0012] From 33 to 33.1 min, the volume fraction of mobile phase A decreased from 76% to 50%, and the volume fraction of mobile phase B increased from 24% to 50%;

[0013] 33.1-36 min, maintain the volume fraction of mobile phase A at 50%, and the volume fraction of mobile phase B at 50%;

[0014] From 36 to 36.1 min, the volume fraction of mobile phase A increased from 50% to 95%, and the volume fraction of mobile phase B decreased from 50% to 5%;

[0015] 36.1~45min, the volume fraction of mobile phase A was maintained at 95%, and the volume fraction of mobile phase B was maintained at 5%.

[0016] When the chromatographic column used is Acclaim RSLC C120 C18 (2.1 mm×150 mm, 2.2 μm), the gradient elution program is:

[0017] From 0 to 60 min, the volume fraction of mobile phase A decreased from 95% to 70%, and the volume fraction of mobile phase B increased from 5% to 30%.

[0018] When the chromatographic column used is Agilent SB-Aq (2.1 mm×100 mm, 1.9 μm), the gradient elution procedure is:

[0019] From 0 to 60 min, the volume fraction of mobile phase A decreased from 95% to 70%, and the volume fraction of mobile phase B increased from 5% to 30%.

[0020] Preferably, the chromatographic column used in the present invention is Acclaim RSLC C120 C18 (2.1 mm×100 mm, 2.2 μm), with a phosphoric acid solution with a volume concentration of 0.1% as the mobile phase A, acetonitrile as the mobile phase B, and the gradient elution program is:

[0021] From 0 to 33 min, the volume fraction of mobile phase A decreased from 95% to 76%, and the volume fraction of mobile phase B increased from 5% to 24%;

[0022] From 33 to 33.1 min, the volume fraction of mobile phase A decreased from 76% to 50%, and the volume fraction of mobile phase B increased from 24% to 50%;

[0023] 33.1-36 min, maintain the volume fraction of mobile phase A at 50%, and the volume fraction of mobile phase B at 50%;

[0024] From 36 to 36.1 min, the volume fraction of mobile phase A increased from 50% to 95%, and the volume fraction of mobile phase B decreased from 50% to 5%;

[0025] 36.1~45min, the volume fraction of mobile phase A was maintained at 95%, and the volume fraction of mobile phase B was maintained at 5%.

[0026] Furthermore, in step c, the ultra-high performance liquid chromatography method also includes the following detection conditions: column temperature of 30-40° C.; detection wavelength of 200-400 nm; flow rate of 0.3-0.4 mL / min.

[0027] Preferably, the column temperature is 35°C; the detection wavelength is 320 nm; and the flow rate is 0.4 mL / min.

[0028] Wherein, in the method for establishing the characteristic spectrum of Dan'e Fukang decoction, in step a, each 1 mL of the reference solution contains 0.1 mg of chlorogenic acid, 0.1 mg of ferulic acid, and 0.5 mg of salvianolic acid B.

[0029] Among them, in the method for establishing the characteristic spectrum of Dan'e Fukang decoction, in step b, 5 g of Dan'e Fukang decoction is accurately weighed, and 50 mL of water is added for ultrasonic extraction.

[0030] The power of the ultrasonic extraction is 50-720W, the ultrasonic frequency is 10-60kHz, and the time is 5-120min.

[0031] Preferably, the power of the ultrasonic extraction is 500 W; the ultrasonic frequency is 40 kHz; and the time is 30 min.

[0032] The centrifugal speed is 2000-12000 r / min, and the time is 3-30 min.

[0033] Preferably, the centrifugal speed is 6000 r / min and the time is 10 min.

[0034] The inner diameter of the macroporous adsorption resin is 10-60 mm; the column height is 6-72 cm.

[0035] Preferably, the inner diameter of the macroporous adsorption resin is 24 mm; the column height is 20-22 cm.

[0036] The volume concentration of the ethanol solution is 50-95%.

[0037] Preferably, the volume concentration of the ethanol solution is 70%.

[0038] After the eluate was collected, it was evaporated to dryness, 25 mL of water was accurately added, and the mixture was filtered.

[0039] The characteristic spectrum of Dan'e Fukang decoction established by the present invention determines 11 common peaks, the peak separation is good, and the characteristic peak information of Dan'e Fukang decoction can be fully reflected. The method is stable, the precision is high, and the reproducibility is good.

[0040] The second purpose of the present invention is to provide a detection method for Dan'e Fukang decoction, which can quickly detect multiple chemical components and evaluate the quality of Dan'e Fukang decoction more comprehensively and scientifically.

[0041] The detection method of Dan'e Fukang decoction provided by the present invention comprises the following steps: accurately aspirating a test solution of Dan'e Fukang decoction, measuring by ultra-high performance liquid chromatography, and comparing the obtained ultra-high performance liquid chromatogram with the characteristic spectrum of Dan'e Fukang decoction obtained by the above-mentioned characteristic spectrum establishment method; the mobile phase used in the ultra-high performance liquid chromatography is: a phosphoric acid solution with a volume concentration of 0.1% as mobile phase A, and acetonitrile as mobile phase B. When the chromatographic column used is Acclaim RSLC C120 C18 (2.1 mm×100 mm, 2.2 μm), the gradient elution program is:

[0042] From 0 to 33 min, the volume fraction of mobile phase A decreased from 95% to 76%, and the volume fraction of mobile phase B increased from 5% to 24%;

[0043] From 33 to 33.1 min, the volume fraction of mobile phase A decreased from 76% to 50%, and the volume fraction of mobile phase B increased from 24% to 50%;

[0044] 33.1-36 min, maintain the volume fraction of mobile phase A at 50%, and the volume fraction of mobile phase B at 50%;

[0045] From 36 to 36.1 min, the volume fraction of mobile phase A increased from 50% to 95%, and the volume fraction of mobile phase B decreased from 50% to 5%;

[0046] 36.1-45 min, maintain the volume fraction of mobile phase A at 95% and the volume fraction of mobile phase B at 5%;

[0047] When the chromatographic column used is Acclaim RSLC C120 C18 (2.1 mm×150 mm, 2.2 μm), the gradient elution program is as follows: from 0 to 60 min, the volume fraction of mobile phase A decreases from 95% to 70%, and the volume fraction of mobile phase B increases from 5% to 30%.

[0048] When the chromatographic column used is Agilent SB-Aq (2.1 mm×100 mm, 1.9 μm), the gradient elution program is: 0 to 60 min, the volume fraction of mobile phase A decreases from 95% to 70%, and the volume fraction of mobile phase B increases from 5% to 30%.

[0049] Preferably, the mobile phase used in the ultra-high performance liquid chromatography method is: a phosphoric acid solution with a volume concentration of 0.1% as mobile phase A, acetonitrile as mobile phase B, and the chromatographic column used is Acclaim RSLC C120 C18 (2.1 mm×100 mm, 2.2 μm). The gradient elution procedure is:

[0050] From 0 to 33 min, the volume fraction of mobile phase A decreased from 95% to 76%, and the volume fraction of mobile phase B increased from 5% to 24%;

[0051] From 33 to 33.1 min, the volume fraction of mobile phase A decreased from 76% to 50%, and the volume fraction of mobile phase B increased from 24% to 50%;

[0052] 33.1-36 min, maintain the volume fraction of mobile phase A at 50%, and the volume fraction of mobile phase B at 50%;

[0053] From 36 to 36.1 min, the volume fraction of mobile phase A increased from 50% to 95%, and the volume fraction of mobile phase B decreased from 50% to 5%;

[0054] 36.1~45min, the volume fraction of mobile phase A was maintained at 95%, and the volume fraction of mobile phase B was maintained at 5%.

[0055] Furthermore, the ultra-high performance liquid chromatography method also includes the following detection conditions: column temperature of 30-40°C; detection wavelength of 200-400nm; flow rate of 0.3-0.4mL / min.

[0056] Preferably, the column temperature is 35°C; the detection wavelength is 320 nm; and the flow rate is 0.4 mL / min.

[0057] Furthermore, the preparation method of the Dan'e Fukang decoction test solution is: accurately weigh the Dan'e Fukang decoction, add water for ultrasonic extraction, centrifuge, take the supernatant and pass it through a macroporous adsorption resin, elute with water and then with ethanol solution, collect the eluate, evaporate to dryness, add water, filter, and take the filtrate to obtain the test solution.

[0058] Furthermore, 5 g of Dan'e Fukang decoction was accurately weighed and added with 50 mL of water for ultrasonic extraction.

[0059] The power of the ultrasonic extraction is 50-720W, the ultrasonic frequency is 10-60kHz, and the time is 5-120min.

[0060] Preferably, the power of the ultrasonic extraction is 500 W; the ultrasonic frequency is 40 kHz; and the time is 30 min.

[0061] The centrifugal speed is 2000-12000 r / min, and the time is 3-30 min.

[0062] Preferably, the centrifugal speed is 6000 r / min and the time is 10 min.

[0063] The inner diameter of the macroporous adsorption resin is 10-60 mm; the column height is 6-72 cm.

[0064] Preferably, the inner diameter of the macroporous adsorption resin is 24 mm; the column height is 20-22 cm.

[0065] The volume concentration of the ethanol solution is 50-95%.

[0066] Preferably, the volume concentration of the ethanol solution is 70%.

[0067] After the eluate was collected, it was evaporated to dryness, 25 mL of water was accurately added, and the mixture was filtered.

[0068] The beneficial effects of the present invention are:

[0069] The present invention adopts ultra-high performance liquid chromatography to establish a characteristic spectrum of Dan'e Fukang decoction, and determines 11 common peaks including neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, p-coumaric acid, ferulic acid, rutin, rosmarinic acid, a chromatographic peak belonging to Salvia miltiorrhiza, salvianolic acid B, salvianolic acid A, etc. The peaks have good separation, and can fully and comprehensively reflect the characteristic peak information of Dan'e Fukang decoction. The method of the present invention is stable, has high precision and good reproducibility, provides a rapid and comprehensive detection means for the quality control of Dan'e Fukang decoction, and provides a scientific experimental basis for the research on the quality evaluation system of Dan'e Fukang decoction.

[0070] The present invention also provides a detection method for Dan'e Fukang decoction. By optimizing the detection conditions of ultra-high performance liquid chromatography and the preparation of the test solution, the present invention can simultaneously detect 11 chemical components in Dan'e Fukang decoction, including neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, p-coumaric acid, ferulic acid, rutin, rosmarinic acid, a chromatographic peak belonging to Salvia miltiorrhiza, salvianolic acid B, and salvianolic acid A, thereby avoiding the one-sidedness of judging the overall quality of the preparation by only measuring one or two chemical components. The detection method of the present invention can evaluate the quality of Dan'e Fukang decoction more comprehensively and scientifically, thereby ensuring the quality and efficacy of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 The characteristic spectrum of Dan'e Fukang decoction established in the present invention (presenting 11 characteristic peaks, wherein peak 1 is neochlorogenic acid, peak 2 is protocatechuic aldehyde, peak 3 is chlorogenic acid, peak 4 is cryptochlorogenic acid, peak 5 is p-coumaric acid, peak 6 is ferulic acid, peak 7 is rutin, peak 8 is rosmarinic acid, peak 9 belongs to Salvia miltiorrhiza, peak 10 is salvianolic acid B, and peak 11 is salvianolic acid A).

[0072] Figure 2 This is the investigation spectrum of the mobile phase in Example 1.

[0073] Figure 3 This is the investigation spectrum of the wavelength of Example 1.

[0074] Figure 4 This is the investigation spectrum of the column temperature in Example 1.

[0075] Figure 5 This is an investigation chart of the preparation method of the test solution in Example 1.

[0076] Figure 6 This is the characteristic peak attribution spectrum of Dan'e Fukang decoction in Example 1.

[0077] Figure 7 This is the characteristic peak attribution spectrum of Dan'e Fukang decoction in Example 1.

[0078] Figure 8 These are the characteristic spectra of 10 batches of samples in Example 1 (sample batch numbers: 200802, 200901, 201101, 201103, 201104, 210201, 210202, 210303, 210401, 210404).

[0079] Fig. 9 It is the characteristic spectrum of 11 batches of samples in Example 1 (sample batch numbers: 200801, 200902, 200903, 201102, 210301, 210302, 210304, 210305, 210306, 210402, 210403).

[0080] Fig.10 This is a graph showing the stability of the test solution in Example 1.

[0081] Fig.11 This is the spectrum of the precision investigation of Example 1.

[0082] Fig.12 This is the spectrum of the repeatability study of Example 1. DETAILED DESCRIPTION

[0083] The present invention will be further described in detail below through specific examples.

[0084] Example 1

[0085] 1. Instruments and test drugs

[0086] 1.1 Instrument

[0087] ACQUITY H-Class Bio liquid chromatograph (Waters Technology (Shanghai) Co., Ltd.), KQ-500DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), Mettler-Toledo XS 105DU analytical balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.), TG16-WS desktop high-speed centrifuge (Changsha High-tech Industrial Development Zone Xiangyi Centrifuge Instrument Co., Ltd.), RE-52AA rotary evaporator (Shanghai Rongya Biochemical Instrument Factory).

[0088] 1.2 Reagents

[0089] Acetonitrile and phosphoric acid were of chromatographic grade; water was purified water; and anhydrous ethanol was of analytical grade.

[0090] 1.3 Reference substances

[0091] Neochlorogenic acid (batch number: N2205002, purity: 99.24%), cryptochlorogenic acid (batch number: C2205003, purity: 98.39%), p-coumaric acid (batch number: H2107001, purity: 99.32%), and salvianolic acid A (batch number: S2108002, purity: 98.81%) were all purchased from Chengdu Maiske Pharmaceutical Technology Co., Ltd.

[0092] Protocatechuic aldehyde (batch number: 110810-201909, purity: 99.6%), chlorogenic acid (batch number: 110753-202119, purity: 96.3%), ferulic acid (batch number: 110773-201915, purity: 99.4%), rosmarinic acid (batch number: 111871-202007, purity: 98.1%), salvianolic acid B (batch number: 111562-201917, purity: 96.6%), and rutin (batch number: 100080-201811, purity: 91.7%); all were purchased from China Food and Drug Inspection Institutes.

[0093] 2. Methods and Results

[0094] 2.1 Chromatographic conditions

[0095] Chromatographic column: Acclaim RSLC C120 C18 (2.1mm×100mm, 2.2μm); mobile phase: 0.1% volume concentration of phosphoric acid solution as mobile phase A, acetonitrile as mobile phase B, gradient elution according to Table 1, column temperature 35°C; flow rate 0.4mL / min; detection wavelength 320nm; injection volume 1μL. The theoretical plate number calculated based on the salvianolic acid B peak should not be less than 250000.

[0096] Table 1 Mobile phase gradient elution conditions

[0097] Time (minutes) Acetonitrile (%) Phosphoric acid solution with a volume concentration of 0.1% (%) 0～33 5→24 95→76 33～33.1 24→50 76→50 33.1～36 50 50 36～36.1 50→5 50→95 36.1～45 5 95

[0098] 2.2 Preparation of test solution:

[0099] Take 5g of Dan'e Fukang decoction, accurately weigh it, put it in a stoppered conical flask, add 50mL of water, ultrasonically treat (power 500W, frequency 40kHz) for 30 minutes, take it out, cool, centrifuge, take the supernatant, pass it through HPD-100 macroporous adsorption resin (inner diameter 24mm, column height 20-22cm), elute with 500mL of water, discard the water, and then elute with 300mL of 70% ethanol solution by volume concentration, collect the eluate, recover ethanol under reduced pressure to evaporate to dryness, accurately add 25ml of water, shake well, filter, and take the filtrate to obtain.

[0100] 2.3 Preparation of reference solution

[0101] Take appropriate amounts of chlorogenic acid reference substance, ferulic acid reference substance, and salvianolic acid B reference substance, accurately weigh them, and add a methanol solution with a volume concentration of 70% to prepare a mixed solution containing 0.1 mg of chlorogenic acid, 0.1 mg of ferulic acid, and 0.5 mg of salvianolic acid B per 1 mL.

[0102] 2.4 Determination method

[0103] Accurately pipette 1 μL of the test solution and the reference solution respectively, inject into the ultra-high performance liquid chromatograph, measure, record the chromatogram, and obtain the characteristic spectrum of Dan'e Fukang decoction, see Figure 1 .

[0104] 2.5 Screening of chromatographic conditions

[0105] 2.5.1 Choice of mobile phase

[0106] The present invention investigates the separation effects of a variety of different mobile phases, as follows:

[0107] (1) From 0 to 60 min, the volume fraction of acetonitrile increased from 5 to 30%, and the volume fraction of 0.1% phosphoric acid solution decreased from 95% to 70%.

[0108] (2) From 0 to 60 min, the volume fraction of acetonitrile increased from 5 to 30%, and the volume fraction of 0.5% glacial acetic acid solution decreased from 95% to 70%.

[0109] (3) From 0 to 60 min, the volume fraction of acetonitrile increased from 5 to 30%, and the volume fraction of 0.1% trifluoroacetic acid solution decreased from 95% to 70%.

[0110] (4) From 0 to 60 min, the volume fraction of acetonitrile increased from 5 to 30%, and the volume fraction of 0.5% formic acid solution decreased from 95% to 70%.

[0111] (5) From 0 to 60 min, the volume fraction of acetonitrile increased from 5 to 30%, and the volume fraction of 0.05 M ammonium acetate solution decreased from 95% to 70%.

[0112] (6) From 0 to 60 min, the volume fraction of acetonitrile increased from 5 to 30%, and the volume fraction of 0.05 M potassium dihydrogen phosphate solution decreased from 95% to 70%.

[0113] (7) From 0 to 60 min, the volume fraction of methanol increased from 5 to 40%, and the volume fraction of 0.1% phosphoric acid solution decreased from 95% to 60%.

[0114] (8) The mobile phase gradient of the method of the present invention is as follows: from 0 to 33 min, the volume fraction of the 0.1% phosphoric acid solution decreases from 95% to 76%, and the volume fraction of acetonitrile increases from 5% to 24%; from 33 to 33.1 min, the volume fraction of the 0.1% phosphoric acid solution decreases from 76% to 50%, and the volume fraction of acetonitrile increases from 24% to 50%; from 33.1 to 36 min, the volume fraction of the 0.1% phosphoric acid solution is maintained at 50%, and the volume fraction of acetonitrile is maintained at 50%; from 36 to 36.1 min, the volume fraction of the 0.1% phosphoric acid solution increases from 50% to 95%, and the volume fraction of acetonitrile decreases from 50% to 5%; from 36.1 to 45 min, the volume fraction of the 0.1% phosphoric acid solution is maintained at 95%, and the volume fraction of acetonitrile is maintained at 5%.

[0115] The chromatographic column used in methods (1) to (7) was Acclaim RSLC C120 C18 (2.1 mm × 150 mm, 2.2 μm); the chromatographic column used in method (8) was Acclaim RSLC C120 C18 (2.1 mm × 100 mm, 2.2 μm). The results of methods (1) to (7) are shown in Figure 2 , the results of method (8) are shown in Figure 1 The results show that the separation effect of the samples eluted using the mobile phase gradient conditions of the methods (1) and (8) of the present invention is the best.

[0116] 2.5.2 Wavelength selection

[0117] The detection conditions corresponding to the Acclaim RSLC C120 C18 (2.1mm×150mm, 2.2μm) chromatographic column were used, and the PDA detector was used to perform full wavelength scanning detection on the sample in the range of 200nm to 400nm. According to the principle of maximizing effective information, the wavelength that can best reflect the sample component information was selected. The results showed that there were multiple peaks with maximum absorption near the wavelength of 320nm. There were more chromatographic peaks, greater absorption, richer information, and stronger characteristics, so the detection wavelength was selected as 320nm. Figure 3 .

[0118] 2.5.3 Selection of column temperature

[0119] The detection conditions corresponding to the Acclaim RSLC C120 C18 (2.1mm×100mm, 2.2μm) column were investigated at three column temperatures of 30℃, 35℃, and 40℃. The chromatographic peak separation was better at 35℃, so the column temperature was selected as 35℃. Figure 4 .

[0120] 2.6 Investigation of the preparation method of the test solution

[0121] The chromatographic conditions corresponding to the Agilent SB-Aq (2.1mm×100mm, 1.9μm) column were used to investigate the preparation methods of five test products, namely:

[0122] Take 5 g of Dan'e Fukang decoction sample, add 50 mL of water for ultrasonic extraction, centrifuge, and take the supernatant as the test solution 1 for injection.

[0123] Take 5g of Dan'e Fukang decoction sample, add 50mL of water for ultrasonic extraction, centrifuge, take the supernatant and pass it through macroporous adsorption resin, elute with water, and discard the eluent. Elute with 15% ethanol solution, collect the 15% ethanol solution eluent; then elute with 30% ethanol solution, collect the 30% ethanol eluent; then elute with 70% ethanol solution, collect the 70% ethanol eluent. Take 15%, 30%, and 70% ethanol eluents respectively, recover ethanol under reduced pressure to evaporate to dryness, accurately add 25mL of water, shake well, filter, and take the filtrate as the test solution 2, 3, and 4 injections.

[0124] Take 5g of Dan'e Fukang decoction sample, add 50mL of water, perform ultrasonic extraction, centrifuge, take the supernatant and pass it through macroporous adsorption resin, elute with water, discard the eluate, elute with 70% ethanol solution, collect the 70% ethanol eluate, recover ethanol to dryness under reduced pressure, accurately add 25mL of water, shake well, filter, and take the filtrate as the test solution 5 for injection. The results show that the test solution 5 prepared by the method of the present invention can effectively remove most impurities and retain the target components. Figure 5 .

[0125] 2.7 Identification of characteristic peaks

[0126] Acclaim RSLC C120 C18 (2.1 mm × 100 mm, 2.2 μm) column was used for the detection conditions. The reference solutions of neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, cryptochlorogenic acid, p-coumaric acid, ferulic acid, rutin, rosmarinic acid, salvianolic acid B, and salvianolic acid A and the test solution of Dan'e Fukang decoction were injected respectively, and the chromatograms were recorded. The above reference substances were compared with the test samples, and the chemical components in the samples were identified according to the retention time. The results showed that peak 1 was neochlorogenic acid, peak 2 was protocatechuic aldehyde, peak 3 was chlorogenic acid, peak 4 was cryptochlorogenic acid, peak 5 was p-coumaric acid, peak 6 was ferulic acid, peak 7 was rutin, peak 8 was rosmarinic acid, peak 10 was salvianolic acid B, and peak 11 was salvianolic acid A. The results are shown in Table 1. Figure 6 , Figure 7 .

[0127] 2.8 Provisions for common peaks in characteristic spectra

[0128] Acclaim RSLC C120 C18 (2.1 mm × 100 mm, 2.2 μm) chromatographic column (batch number: 200802, 200901, 201101, 201103, 201104, 210201, 210202, 210303, 210401, 210404, 200801, 200902, 200903, 201102, 210301, 210302, 210304, 210305, 210306, 210402, 210403) were used to determine the retention time of each characteristic peak according to the method of the present invention, and the RSD values ​​of the retention time and the relative retention time were calculated. The results are shown in Tables 2 and 3. Figure 8 , Fig. 9 The RSD of retention time was between 0.30% and 0.86%, and the RSD of relative retention time was between 0.08% and 0.26%. The quality stability and batch-to-batch consistency of 21 batches of Dan'e Fukang decoction were good.

[0129] Table 2 Retention time of 21 batches of samples (min)

[0130]

[0131] Table 3 Relative retention time of 21 batches of samples (min)

[0132]

[0133]

[0134] There are 11 characteristic peaks in the chromatogram of the test solution, among which peaks 3, 6 and 10 should correspond to the retention times of the corresponding reference substances chlorogenic acid, ferulic acid and salvianolic acid B. Peak 6 corresponding to the ferulic acid reference substance is peak S. The relative retention times of the remaining characteristic peaks and peak S are calculated, and their relative retention times should be within ±10% of the specified value.

[0135] 3 Methodological investigation

[0136] 3.1 Investigation of injection precision

[0137] According to the detection conditions corresponding to the Acclaim RSLC C120 C18 (2.1mm×100mm, 2.2μm) chromatographic column, chlorogenic acid, ferulic acid, and salvianolic acid B were injected 5 times in succession, 1μL per injection, and the retention time and peak area were recorded. The RSD values ​​of retention time and peak area were calculated. The results are shown in Tables 4 and 5. The retention time RSD was between 0.19% and 0.81%, and the peak area RSD was between 1.32% and 1.44%, indicating that the injection precision was good.

[0138] Table 4 Injection precision-retention time (min)

[0139] serial number Chlorogenic acid Ferulic acid Salvianolic acid B 1 6.702 13.554 29.017 2 6.670 13.522 29.003 3 6.613 13.451 28.901 4 6.584 13.392 28.932 5 6.583 13.369 28.903 RSD(%) 0.81 0.59 0.19

[0140] Table 5 Injection precision-peak area

[0141] serial number Chlorogenic acid Ferulic acid Salvianolic acid B 1 592967 228998 1290027 2 597222 230004 1296464 3 614425 236955 1336575 4 598783 231650 1295609 5 599123 231823 1298434 RSD(%) 1.36 1.32 1.44

[0142] 3.2 Stability study of test solution

[0143] According to the detection conditions corresponding to the Acclaim RSLC C120 C18 (2.1mm×100mm, 2.2μm) chromatographic column, Dan'e Fukang Decoction (batch number: 200901) was taken, and the test solution was prepared according to the test solution preparation method of the present invention. The retention time and peak area were measured at 0h, 2h, 4h, 8h, 12h, 24h, 36h, and 48h, and the RSD values ​​of the retention time and peak area were calculated. The results are shown in Tables 6 and 7. Fig.10 The retention time RSD was between 0.43% and 2.06%, indicating good stability; except for the slightly larger peak area RSDs of characteristic peaks 5 and 11, the other peak area RSDs were between 0.51% and 1.40%, indicating that the test solution was relatively stable within 48 hours.

[0144] Table 6 Stability Study-Retention Time (min)

[0145]

[0146] Table 7 Stability Study-Peak Area

[0147]

[0148]

[0149] 3.3 Precision investigation

[0150] Take Dan'e Fukang Decoction (batch number: 200901), according to the detection conditions of Acclaim RSLC C120 C18 (2.1mm×100mm, 2.2μm) chromatographic column, inject 5 injections continuously, 1μL per injection, record the retention time and peak area, calculate the RSD value of retention time and peak area, the results are shown in Table 8 and Table 9. Fig.11 The retention time RSD was between 0.04% and 0.14%, and the peak area RSD was between 0.28% and 2.07%, indicating good precision.

[0151] Table 8 Precision-Retention Time (min)

[0152]

[0153] Table 9 Precision-Peak Area

[0154]

[0155] 3.4 Repeatability study

[0156] Take Dan'e Fukang Decoction (batch number: 200901) and prepare 10 test solutions according to the detection conditions corresponding to the Acclaim RSLC C120 C18 (2.1mm×100mm, 2.2μm) chromatographic column. Determine the retention time of each characteristic peak and calculate the RSD value of the retention time and relative retention time. The results are shown in Tables 10 and 11. Fig.12 The RSD of retention time was between 0.11% and 0.42%, and the RSD of relative retention time was between 0.08% and 0.35%, indicating that the method had good repeatability.

[0157] Table 10 Repeatability study - retention time (min)

[0158]

[0159] Table 11 Repeatability study - relative retention time (min)

[0160]

[0161] Embodiment 2 Detection method of Dan'e Fukang decoction

[0162] Take Dan'e Fukang decoction (batch number: 200802, 200901, 201101, 201103, 201104, 210201, 210202, 210303, 210401, 210404, 200801, 200902, 200903, 201102, 210301, 210302, 210304, 210305, 210306, 210402, 210403) a total of 21 batches of samples according to Example 1 "2.2 Preparation of test solution" to obtain the test sample solution, accurately draw the test sample solution, inject it into the ultra-high performance liquid chromatograph for determination, the chromatographic conditions of the ultra-high performance liquid chromatograph are the same as those in Example 1 "2.1 Chromatographic conditions". The detection spectrum of the sample to be tested is as follows Figure 8 and Fig. 9 The RSD values ​​of retention time and relative retention time are shown in Table 2 and Table 3.

Claims

1. A method for establishing a characteristic spectrum of Dan'e Fukang decoction, characterized in that: The steps include: a. Accurately weigh the chlorogenic acid, ferulic acid and salvianolic acid B reference substances, mix them and dissolve them in methanol to obtain the reference solution; b. Accurately weigh Dan'e Fukang decoction, add water for ultrasonic extraction, centrifuge, take the supernatant and pass it through HPD-100 macroporous adsorption resin, elute with water, discard the water and then elute with 70% ethanol solution by volume, collect the eluate, evaporate to dryness, add water, filter, and take the filtrate to obtain the test solution; c. The test solution and the reference solution are measured by ultra-high performance liquid chromatography to obtain a characteristic spectrum consisting of 11 common characteristic peaks; in step c, the mobile phase used in the ultra-high performance liquid chromatography is: a phosphoric acid solution with a volume concentration of 0.1% as mobile phase A, acetonitrile as mobile phase B, and gradient elution is adopted, and the procedure of the gradient elution is: From 0 to 33 min, the volume fraction of mobile phase A decreased from 95% to 76%, and the volume fraction of mobile phase B increased from 5% to 24%; From 33 to 33.1 min, the volume fraction of mobile phase A decreased from 76% to 50%, and the volume fraction of mobile phase B increased from 24% to 50%; 33.1-36 min, maintain the volume fraction of mobile phase A at 50%, and the volume fraction of mobile phase B at 50%; From 36 to 36.1 min, the volume fraction of mobile phase A increased from 50% to 95%, and the volume fraction of mobile phase B decreased from 50% to 5%; 36.1-45 min, maintain the volume fraction of mobile phase A at 95% and the volume fraction of mobile phase B at 5%; Or the following gradient elution program is used: from 0 to 60 min, the volume fraction of mobile phase A decreases from 95% to 70%, and the volume fraction of mobile phase B increases from 5% to 30%; The column temperature was 30-40°C; the detection wavelength was 320 nm; the flow rate was 0.3-0.4 mL / min; the chromatographic column was Acclaim RSLCC120 C18, 2.1 mm × 100 mm, 2.2 μm or Acclaim RSLC C120 C18, 2.1 mm × 150 mm, 2.2 μm.

2. The method for establishing the characteristic spectrum of Dan'e Fukang decoction according to claim 1, characterized in that: In step c, the column temperature was 35° C. and the flow rate was 0.4 mL / min.

3. The method for establishing the characteristic spectrum of Dan'e Fukang decoction according to claim 1, characterized in that: In step a, each 1 mL of the reference solution contains 0.1 mg of chlorogenic acid, 0.1 mg of ferulic acid and 0.5 mg of salvianolic acid B.

4. The method for establishing the characteristic spectrum of Dan'e Fukang decoction according to claim 1, characterized in that: In step b, at least one of the following is satisfied: Accurately weigh 5 g of Dan'e Fukang decoction, add 50 mL of water and perform ultrasonic extraction; The power of the ultrasonic extraction is 50-720W; the ultrasonic frequency is 10-60kHz; and the time is 5-120min. The centrifugal speed is 2000-12000 r / min; the time is 3-30 min; The inner diameter of the macroporous adsorption resin is 10 to 60 mm; the column height is 6 to 72 cm; After the eluate was collected, it was evaporated to dryness, 25 mL of water was accurately added, and the mixture was filtered.

5. The detection method of Dan'e Fukang decoction is characterized in that: The method comprises the following steps: accurately absorbing a Dan'e Fukang decoction test solution, measuring it by ultra-high performance liquid chromatography, and comparing the obtained ultra-high performance liquid chromatogram with the characteristic spectrum of Dan'e Fukang decoction obtained by the method for establishing the characteristic spectrum according to any one of claims 1 to 4; the mobile phase used in the ultra-high performance liquid chromatography is: a phosphoric acid solution with a volume concentration of 0.1% as mobile phase A, acetonitrile as mobile phase B, and gradient elution, and the procedure of the gradient elution is: From 0 to 33 min, the volume fraction of mobile phase A decreased from 95% to 76%, and the volume fraction of mobile phase B increased from 5% to 24%; From 33 to 33.1 min, the volume fraction of mobile phase A decreased from 76% to 50%, and the volume fraction of mobile phase B increased from 24% to 50%; 33.1-36 min, maintain the volume fraction of mobile phase A at 50%, and the volume fraction of mobile phase B at 50%; From 36 to 36.1 min, the volume fraction of mobile phase A increased from 50% to 95%, and the volume fraction of mobile phase B decreased from 50% to 5%; 36.1-45 min, maintain the volume fraction of mobile phase A at 95% and the volume fraction of mobile phase B at 5%; Or use the following gradient elution program: 0-60min, the volume fraction of mobile phase A decreased from 95% to 70%, and the volume fraction of mobile phase B increased from 5% to 30%; column temperature was 30-40°C; detection wavelength was 320nm; flow rate was 0.3-0.4mL / min; chromatographic column was Acclaim RSLCC120 C18, 2.1mm×100mm, 2.2μm or Acclaim RSLC C120 C18, 2.1mm×150mm, 2.2μm; The preparation method of the Dan'e Fukang decoction test solution is as follows: accurately weigh the Dan'e Fukang decoction, add water for ultrasonic extraction, centrifuge, take the supernatant and pass it through HPD-100 macroporous adsorption resin, elute with water, discard the water and then elute with 70% ethanol solution by volume, collect the eluate, evaporate to dryness, add water, filter, and take the filtrate to obtain the test solution.

6. The detection method of Dan'e Fukang decoction according to claim 5, characterized in that: The column temperature was 35°C; the flow rate was 0.4 mL / min.

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