A method for testing the quality standard of compound ginkgo tongmai oral liquid

Abstract

The present invention discloses a method for detecting the quality standard of compound ginkgo tongmai oral liquid, including establishing a fingerprint of compound ginkgo tongmai oral liquid, wherein the fingerprint establishment includes: preparing a test solution 1, preparing a reference solution 1, and preparing a reference medicinal material solution 1; respectively determining the reference solution 1, the reference medicinal material solution 1, and multiple batches of test solution 1 by an HPLC liquid chromatograph; importing the chromatograms of multiple batches of test solution 1 into a similarity evaluation system of a Chinese medicine chromatographic fingerprint, using the chromatogram of one batch of test solution 1 as a reference spectrum, and obtaining the fingerprint of compound ginkgo tongmai oral liquid through multi-point correction and data analysis. The present invention may further include analysis of Ligustrum lucidum, Uncaria rhynchophylla, Cyathula officinalis, Eucommia ulmoides, and detection of the contents of total flavonol glycosides, salvianolic acid B, and stilbene glycosides. The present invention can fully reflect product quality information, improve the controllability of product quality, and further improve the quality standard of compound ginkgo tongmai oral liquid.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for detecting the quality standard of compound Ginkgo Tongmai oral liquid. Background Art

[0002] The Chinese medicine formula preparation compound ginkgo tongmai oral liquid contains 7 Chinese medicinal materials: ginkgo leaf, processed Polygonum multiflorum, Ligustrum lucidum, Salvia miltiorrhiza, Eucommia ulmoides, Cyathula officinalis, and Uncaria rhynchophylla. Its functions and indications are to nourish yin and tonify the kidney, soothe the liver and unclog the meridians. It is used for dizziness and headache, tinnitus and deafness, blurred vision, memory loss, soreness of waist and knees, numbness of limbs and other symptoms caused by mild cerebral arteriosclerosis in middle-aged and elderly people, which are caused by liver and kidney yin deficiency.

[0003] At present, the quality standard of compound ginkgo tongmai oral liquid only conducts qualitative identification of ginkgo leaves, processed polygonum multiflorum, and salvia miltiorrhiza, and controls the content of rhubarb in processed polygonum multiflorum by thin layer chromatography. However, compound ginkgo tongmai oral liquid contains many chemical components, and the content of each chemical component varies greatly. Only by measuring the content of a single component to detect and control the quality of compound ginkgo tongmai oral liquid, it cannot reflect the quality of compound ginkgo tongmai oral liquid as a whole. Therefore, in order to further optimize and improve the quality standard of compound ginkgo tongmai oral liquid, improve the controllability of product quality, ensure the effectiveness of drugs, and better control the overall quality of compound ginkgo tongmai oral liquid, it is urgent to establish a better and more comprehensive quality control method for evaluating compound ginkgo tongmai oral liquid. Summary of the invention

[0004] The technical problem solved by the present invention is to provide a method for detecting the quality of Ginkgo Tongmai Oral Liquid, which can comprehensively reflect the quality of the Ginkgo Tongmai Oral Liquid as a whole.

[0005] The technical problem solved by the present invention is achieved by adopting the following technical solutions:

[0006] A method for detecting the quality standard of compound ginkgo tongmai oral liquid, characterized in that it includes establishing a fingerprint spectrum of the compound ginkgo tongmai oral liquid, and the fingerprint spectrum establishment includes:

[0007] Preparation of test solution 1: Take an appropriate amount of compound Ginkgo Tongmai oral liquid, add methanol and mix, then perform ultrasonic treatment, and then filter the filtrate to obtain the test solution;

[0008] Preferably, during ultrasonic treatment, the ultrasonic power is 80-120W, the frequency is 40-60kHz, and the ultrasonic treatment time is 5-15min.

[0009] Preparation of reference solution 1: Dissolve the samples of diphenylethylene glycoside, privet glucoside, salvianolic acid B, quercetin, pinoresinol diglucoside, cyperasterone, isorhynchophylline, and oleanolic acid in water to prepare a mixed reference solution 1-a; dissolve the sample of potassium sorbate in methanol to prepare a reference solution 1-b.

[0010] Preferably, diphenylethylene glycosides, privet glycosides, salvianolic acid B, and quercetin are dissolved in water to serve as a mixed reference solution 1-a1, pinoresinol diglucoside, cyperasterone, isorhynchophylline, and oleanolic acid samples are dissolved in water to serve as a mixed reference solution 1-a2, and potassium sorbate samples are dissolved in methanol alone to serve as a reference solution 1-b, thereby enabling the key components to be detected to achieve a good separation effect and play a better control role.

[0011] Control medicinal material solution 1: pre-treat seven medicinal materials including Ginkgo biloba leaves, processed Polygonum multiflorum, Fructus Ligustri Lucidi, Salvia miltiorrhiza, Eucommia ulmoides, Cyathula officinalis and Uncaria rhynchophylla, and obtain control medicinal material solutions according to the preparation method of test solution 1;

[0012] The reference solution 1, the reference medicinal material solution 1 and multiple batches of test solution 1 were respectively measured by HPLC liquid chromatography;

[0013] The chromatograms of multiple batches of test solution 1 were introduced into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and the chromatogram of one batch of test solution 1 was used as a reference spectrum. After multi-point calibration and data analysis, the fingerprint spectrum of Compound Ginkgo Tongmai Oral Liquid was obtained;

[0014] Chromatographic conditions: C18 column, flow rate 1.0 ml / min, detection wavelength 220-230 nm, column temperature 20-30 °C; mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid, gradient elution is performed in the following order:

[0015]

[0016] Furthermore, the chromatographic conditions were: using a Waters xterra C18 model 250 mm×4.6 mm×5 μm chromatographic column, a flow rate of 1.0 ml / min, a detection wavelength of 223 nm, and a column temperature of 25°C.

[0017] Furthermore, the fingerprint of the compound Ginkgo Tongmai oral liquid includes 27 common peaks. Compared with the reference substance, it is confirmed that the common peaks include the peak corresponding to salvianolic acid B, the peak corresponding to potassium sorbate, the peak corresponding to diphenyl glycoside, the peak corresponding to cyperasterone, the peak corresponding to pinoresinol diglucoside and the peak corresponding to privet glycoside; compared with the reference medicinal materials, it is confirmed that the chromatographic peaks 4, 17 and 21 with retention times of 5.055, 21.376 and 27.294 respectively are from Ligustrum lucidum fruit, with retention times of 24.031, 25 Chromatographic peaks 18, 19 and 20 with retention times of .137 and 26.521 respectively come from Salvia miltiorrhiza, and chromatographic peak 10 with retention time of 12.576 comes from Uncaria rhynchophylla; chromatographic peaks 3, 11, 12 and 13 with retention times of 4.093, 13.549, 14.188 and 15.164 respectively come from Eucommia ulmoides, chromatographic peak 15 with retention time of 17.221 comes from processed Polygonum multiflorum as diphenylethylene, and chromatographic peaks 2, 7 and 14 with retention times of 3.021, 9.627 and 15.748 respectively come from Ginkgo biloba leaves.

[0018] Furthermore, thin layer chromatography is also used to identify the Fructus Ligustri Lucidi in the compound Ginkgo Tongmai oral liquid, and the method includes: taking an appropriate amount of the compound Ginkgo Tongmai oral liquid, adding an equal volume of ethyl acetate for extraction, taking the water layer after the extraction is completed, adding an equal volume of chloroform to continue extraction, recovering chloroform after the extraction is completed, and dissolving the residue in methanol as the test solution 2; taking oleanolic acid to prepare a reference solution 2; taking Fructus Ligustri Lucidi to prepare a reference medicinal material solution 2; taking an appropriate amount of a negative control sample lacking Fructus Ligustri Lucidi, and preparing a negative reference solution 1 according to the preparation method of the test solution 2; using a developing agent to develop the test solution 2, the reference solution 2, the reference medicinal material solution 2, and the negative reference solution 1 on a silica gel G thin layer plate, and the developing agent is composed of toluene, ethyl acetate, and glacial acetic acid; after the development is completed, take it out, dry it, spray it with 10% sulfuric acid ethanol solution, and heat it until the spots are clearly colored. During the process operation, the interference of other components can be effectively eliminated, the color development and cleaning are convenient to operate.

[0019] Furthermore, thin layer chromatography is also used to identify the Uncaria rhynchophylla in the compound Ginkgo Tongmai oral liquid, and the method includes: the method also includes thin layer chromatography to identify the Uncaria rhynchophylla in the compound Ginkgo Tongmai oral liquid, and the method includes: taking an appropriate amount of the compound Ginkgo Tongmai oral liquid, adding an appropriate amount of concentrated ammonia water to mix well, and then adding an equal volume of chloroform to extract, and dissolving the residue after recovering the solvent with methanol as the test solution 3; taking another Uncaria rhynchophylla medicinal material to prepare a control medicinal material solution 3; preparing a negative control solution 2 lacking Uncaria rhynchophylla; absorbing the test solution 3, the control medicinal material solution 3, the negative control solution 2, isorhynchophylline, and the Uncaria rhynchophylla reference solution respectively on the same silica gel G thin layer plate, developing with a developing agent, and then taking it out, drying it, and inspecting it under an ultraviolet light, and the developing agent is composed of acetone and petroleum ether in a volume ratio of 9:11. In the thin layer chromatography process, no color developer is required and the spot is washed, and the operation is convenient.

[0020] Furthermore, it also includes using thin layer chromatography to identify the Cyathula capitata in the compound Ginkgo Tongmai oral liquid, the method comprising: taking an appropriate amount of the compound Ginkgo Tongmai oral liquid, adding an equal volume of water to saturate n-butanol for extraction, recovering the n-butanol, dissolving the residue with methanol, and then passing it through a neutral alumina column, eluting the neutral alumina column with 40 to 60 mL of water, then eluting with 40 to 60 mL of 40 to 60% ethanol, discarding the eluent, and then eluting with 100 to 120 mL of 70 to 75% ethanol, collecting the eluent, and eluting The residue after recovering the solvent was dissolved in methanol as the test solution 4; amaranthone was taken to prepare a reference solution 4; a negative control solution 3 lacking Cyathulae Bidentatae was prepared; a reference medicinal material solution 4 was prepared by taking Cyathulae Bidentatae as a reference medicinal material; the test solution 4, the reference solution 4, the negative control solution 3, and the control medicinal material solution 4 were aspirated and spotted on a silica gel G thin layer plate, respectively, and developed with a developing agent composed of chloroform and methanol, then taken out, dried, sprayed with 10% ethanolic sulfuric acid solution as a color developer, and heated until the spots were clearly colored.

[0021] Furthermore, it also includes using thin layer chromatography to identify the Eucommia ulmoides in the compound Ginkgo Tongmai oral liquid. The method includes: taking an appropriate amount of the compound Ginkgo Tongmai oral liquid, adding an equal volume of water to saturate n-butanol for extraction, recovering the n-butanol, dissolving the residue with methanol and then passing it through a neutral alumina column, eluting the neutral alumina column with 40-60mL of water, then eluting with 40-60mL of 40-60% ethanol, discarding the eluent, and then eluting with 100-120mL of 70-75% ethanol, collecting the eluent, dissolving the residue after the eluent recovers the solvent with methanol as the test solution 5, and preparing a negative control solution 4 lacking Eucommia ulmoides; absorbing the test solution 5 and the negative control solution 4, respectively spotting them on the same silica gel G thin layer plate, developing them with a developing agent composed of chloroform, methanol and water, then taking them out, drying them, and inspecting them under an ultraviolet lamp.

[0022] Furthermore, the method further includes using HPLC liquid chromatography to analyze the total flavonol glycosides in the compound Ginkgo Tongmai oral liquid, the method comprising:

[0023] Preparation of test solution 6: Mix compound Ginkgo Tongmai oral liquid, methanol and hydrochloric acid, heat in a water bath first and then reflux, cool to room temperature quickly after heating, dilute with methanol and filter, and use the filtrate as the test solution;

[0024] Preferably, the reflux heating time is 20 to 40 minutes, and the reflux temperature is 70 to 90°C.

[0025] More preferably, the reflux heating time is 30 min and the reflux temperature is 80°C.

[0026] Preferably, the concentration of hydrochloric acid is 20-30%, and more preferably, the concentration of hydrochloric acid is 25%.

[0027] Preparation of reference solution 6: Take appropriate amount of quercetin, kaempferol and isorhamnetin samples, add methanol to dissolve and obtain a mixed solution as the reference solution;

[0028] Preparation of negative control solution 5: Prepare negative control solution by using compound Ginkgo Tongmai oral solution lacking Ginkgo biloba leaves according to the method for preparing test solution 6;

[0029] The test solution 6, the reference solution 6 and the negative control solution 5 were measured by HPLC liquid chromatography; the chromatographic conditions were as follows: the chromatographic column was filled with octadecylsilane bonded silica gel; the mobile phase A was methanol, and the mobile phase B was 0.4% phosphoric acid, and the elution was carried out in the range of a volume ratio of 50-55:45-50; the flow rate was 0.9-1.1 ml / min; the column temperature was 28-32°C; the detection wavelength was 340-380 nm; the injection volume was 10 μl, and the theoretical plate number was not less than 2500 based on the quercetin peak.

[0030] Furthermore, the method further includes using HPLC liquid chromatography to analyze salvianolic acid B in compound Ginkgo Tongmai oral liquid, the method comprising:

[0031] Preparation of test solution 7: Dilute the compound Ginkgo Tongmai oral solution with methanol and filter it. The filtrate is used as test solution 7;

[0032] Preparation of reference solution 7: Take an appropriate amount of salvianolic acid B sample, add methanol and water to prepare reference solution 7;

[0033] Preparation of negative control solution 6: prepare negative control solution 6 by using compound Ginkgo Tongmai oral solution lacking Danshen according to the preparation method of test solution;

[0034] The test solution 7, the reference solution 7 and the negative control solution 6 were measured by HPLC liquid chromatography; the chromatographic conditions were as follows: the chromatographic column was filled with octadecylsilane bonded silica gel; the mobile phase A was acetonitrile, and the mobile phase B was 0.1% phosphoric acid solution, and the elution was carried out in the range of a volume ratio of 20-25:75-80; the flow rate was 0.9-1.1 ml / min; the column temperature was 18-22°C; the detection wavelength was 280-290 nm; the injection volume was 20 μl.

[0035] Furthermore, the method also includes using HPLC liquid chromatography to analyze diphenylethylene glycosides in the compound ginkgo tongmai oral liquid, and the method includes:

[0036] Preparation of test solution 8: dilute the compound Ginkgo Tongmai oral solution with ethanol and filter it, and the filtrate is used as the test solution 8;

[0037] Preparation of reference solution 8: prepare the diphenylethylene glycoside sample into reference solution 8;

[0038] Preparation of negative control solution 7: prepare negative control solution 7 by using compound Ginkgo Tongmai oral solution lacking Polygonum multiflorum according to the method for preparing test solution 8;

[0039] The test solution 8, the reference solution 8 and the negative control solution 7 were measured by HPLC liquid chromatography; the chromatographic conditions were: the chromatographic column was filled with octadecylsilane bonded silica gel; the mobile phase A was acetonitrile, and the mobile phase B was water; the detection wavelength was 300-350 nm; the injection volume was 10 μl.

[0040] Beneficial effects:

[0041] 1) The detection method of the quality standard of the compound ginkgo tongmai oral liquid described in the present invention has good precision, repeatability and stability, and can accurately detect the composition and content of the sample. In the process of establishing the fingerprint spectrum, the method of ultrasonic extraction of methanol in the preparation of the test solution has a good extraction effect on the characteristic components, which can retain the characteristic components to the greatest extent. At the same time, through specific chromatographic conditions, the chromatographic peak separation is good, the peak time and peak area are appropriate, and the peak shape is good. The similarity between multiple batches of compound ginkgo tongmai oral liquid and the control spectrum is 0.985-1.00, indicating that the difference is small and the similarity is high, which can effectively characterize the quality of the compound ginkgo tongmai oral liquid.

[0042] 2) The detection method for the quality standard of the compound Ginkgo Tongmai oral liquid described in the present invention calibrates 27 common peaks by establishing a fingerprint spectrum, confirms that the common peaks include the peak corresponding to salvianolic acid B, the peak corresponding to potassium sorbate, the peak corresponding to diphenyl glycoside, and the peak corresponding to cyperasterone, and determines the medicinal ingredients to which most of the common peaks belong, and determines the retention time of the common peaks.

[0043] 3) The method for detecting the quality standard of the compound Ginkgo Tongmai oral liquid of the present invention further adopts thin layer chromatography to identify the Fructus Ligustri Lucidi, and / or Uncaria Rhynchophylla, and / or Cyathula officinalis, and / or Eucommia ulmoides in the compound Ginkgo Tongmai oral liquid, and adopts a targeted method for extraction and development, with clear color development, good separation, and no negative interference, so that the quality control of the compound Ginkgo Tongmai oral liquid is more scientific and reasonable, and the quality is more controllable.

[0044] 4) The method for detecting the quality standard of the compound Ginkgo Tongmai oral liquid of the present invention further adopts HPLC liquid chromatography to analyze the main characteristic total flavonol glycosides, and / or salvianolic acid B, and / or diphenylethylene glycosides in the compound Ginkgo Tongmai oral liquid. The total flavonol glycosides, salvianolic acid B and diphenylethylene glycosides have a good linear relationship within their respective linear ranges, and the average sample recovery rate is 93.24%-99.67%, thereby reflecting the intrinsic comprehensive quality of the compound Ginkgo Tongmai oral liquid in a more multi-dimensional and real manner, and better ensuring the medication safety and effectiveness of the compound Ginkgo Tongmai oral liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the HPLC chromatogram of the reference medicinal material Ligustrum lucidum.

[0046] Figure 2 The HPLC chromatogram of prepared Polygonum multiflorum is the reference medicinal material.

[0047] Figure 3 This is the HPLC chromatogram of the reference medicinal material Salvia miltiorrhiza.

[0048] Figure 4 This is the HPLC chromatogram of the reference medicinal material Eucommia ulmoides.

[0049] Figure 5 This is the HPLC chromatogram of the reference medicinal material Ginkgo biloba.

[0050] Figure 6 It is the HPLC chromatogram of the reference medicinal material Uncaria rhynchophylla.

[0051] Figure 7 This is the HPLC chromatogram of the reference medicinal material Rhizoma Cyathulae.

[0052] Figure 8 This is the fingerprint of Compound Ginkgo Tongmai Oral Liquid.

[0053] Fig. 9 HPLC chromatograms of test solution 1 (A), reference solution 1-a1 (B), reference solution 1-a2 (C) and blank solvent (D) in Compound Ginkgo Tongmai Oral Liquid.

[0054] Fig.10 This is the HPLC chromatogram of the test solution 1 using the InertSustain C18 column.

[0055] Fig.11 This is the HPLC chromatogram of the test solution 1 using a Waters XTERRA MS C18 column.

[0056] Fig.12 This is the chromatogram of the identification of Ligustrum lucidum fruit in Compound Ginkgo Tongmai Oral Liquid by thin layer chromatography.

[0057] Fig.13 This is the chromatogram of the identification of Cyathula capitata in Compound Ginkgo Tongmai Oral Liquid by thin layer chromatography.

[0058] Fig.14 This is the chromatogram of the identification of Uncaria rhynchophylla in Compound Ginkgo Tongmai Oral Liquid by thin layer chromatography.

[0059] Fig.15 This is the chromatogram of the identification of Eucommia ulmoides in Compound Ginkgo Tongmai Oral Liquid by thin layer chromatography.

[0060] Fig.16 The result diagram of the specificity test in Example 6 (where A is the negative control solution 5, B is the test solution 6, and C is the reference solution 6)

[0061] Fig.17 The result diagram of the specificity test in Example 7 (where A is the negative control solution 6, B is the test solution 7, and C is the reference solution 7)

[0062] Fig.18 The result diagram of the specificity test in Example 8 (where A is the negative control solution 7, B is the test solution 8, and C is the reference solution 8) DETAILED DESCRIPTION

[0063] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments.

[0064] The compound ginkgo tongmai oral liquid used in the embodiment is prepared as follows: ginkgo leaves, Ligustrum lucidum and salvia miltiorrhiza are heated and extracted three times with 70% ethanol, combined and filtered, and the filtrate is used for standby; the residues and the remaining four ingredients such as prepared Polygonum multiflorum are decocted twice with water, combined and filtered, and the filtrate is concentrated to a relative density of 1.12 (60-65°C) and precipitated with alcohol, and ethanol is recovered and combined with the concentrated solution of the above three ingredients such as ginkgo leaves, and appropriate amounts of honey, potassium sorbate and water are added to 1L, mixed, filled, sterilized, and obtained. Repeat the preparation process to prepare 10 batches of oral liquid preparations.

[0065] Example 1

[0066] A method for detecting the quality standard of compound ginkgo tongmai oral liquid, comprising establishing a fingerprint spectrum of the compound ginkgo tongmai oral liquid, wherein the fingerprint spectrum establishment includes:

[0067] Preparation of test solution 1: accurately measure 10 mL of compound Ginkgo Tongmai oral solution, place it in a 25 mL volumetric flask, add 10 mL of methanol, and ultrasonicate for 10 min at an ultrasonic power of 100 W and a frequency of 50 kHz. Then add methanol to make the volume to 25 mL, filter through a 0.45 μm microporous filter membrane, and take the filtrate as the test solution;

[0068] Preparation of reference solution 1: dissolve stilbene glycoside, privet glycoside, salvianolic acid B, and quercetin samples in water to prepare mixed reference solution 1-a1; dissolve pinoresinol diglucoside, cyasterone, isorhynchophylline, and oleanolic acid samples in water to prepare mixed reference solution 1-a2; dissolve potassium sorbate sample in methanol to prepare reference solution 1-b;

[0069] Control medicinal material solution 1: Ginkgo biloba, Ligustrum lucidum and Salvia miltiorrhiza were respectively extracted with 70% ethanol for 2 times, each time for 2 hours, the extracts were combined and filtered, and the filtrate was decompressed to recover ethanol until there was no alcohol taste, and then used for standby; the medicinal residues were decocted with water twice, each time for 2 hours, the decoctions were combined, filtered, and the filtrate was concentrated to the corresponding volume; the four flavors of Polygonum multiflorum, Eucommia ulmoides, Uncaria rhynchophylla and Cyathula officinalis were decocted with water twice, each time for 2 hours, the decoctions were combined, filtered, and the filtrate was concentrated to a relative density of 1.12, and then ethanol was added to make the alcohol content reach 60%, stirred, allowed to stand, filtered, and the filtrate was decompressed to recover ethanol until there was no alcohol taste. The pretreated solutions were respectively prepared according to the preparation method of the test solution 1 to obtain the control medicinal material solution.

[0070] Chromatographic conditions:

[0071] A Waters xterra C18 chromatographic column (250 mm×4.6 mm, 5 μm) was used; the mobile phase of the UV detector was acetonitrile (A)-0.1% phosphoric acid water (B), gradient elution, flow rate 1.0 ml / min, detection wavelength 224 nm, column temperature 25° C., injection volume 20 μl.

[0072] The conditions for gradient elution were:

[0073]

[0074] in Figure 1 to Figure 7 The following are the HPLC chromatograms of seven reference medicinal materials.

[0075] Take 10 batches of compound Ginkgo Tongmai oral liquid (batch numbers are 20220601, 20220602, 20220603, 20211010, 20220409, 20220302, 20220507, 20220509, 20220513, 20220514), inject and measure according to the above chromatographic conditions, and record the chromatogram. Using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" 2012.130723 version, the chromatogram of the sample (batch number: 20220602) was used as the reference spectrum. After multi-point correction and automatic matching, the control fingerprint spectrum was generated by the mean method, and 27 common peaks were determined, see Figure 8By comparing with the reference substance, five components were identified, namely, salvianolic acid B, potassium sorbate, diphenylethylene glycoside, terephthaloside, and pinoresinol diglucoside. By comparing with the reference medicinal materials, it was confirmed that the chromatographic peaks 4, 17, and 21 with retention times of 5.055, 21.376, and 27.294 were from Ligustrum lucidum, and the chromatographic peaks 18, 19, and 20 with retention times of 24.031, 25.137, and 26.521 were from From Danshen, the chromatographic peak No. 10 with a retention time of 12.576 comes from Uncaria rhynchophylla; the chromatographic peaks No. 3, 11, 12, and 13 with retention times of 4.093, 13.549, 14.188, and 15.164 come from Eucommia ulmoides, the chromatographic peak No. 15 with a retention time of 17.221 comes from Polygonum multiflorum processed as diphenylethylene, and the chromatographic peaks No. 2, 7, and 14 with retention times of 3.021, 9.627, and 15.748 come from Ginkgo biloba leaves. In addition, the chromatographic peak No. 21 is potassium sorbate, and the peaks No. 1, 2, and 4 are found in many medicinal materials. The chromatographic peaks No. 6, 10, 12, 15, 17, 18, and 27 were not found. The similarity between the 10 batches of compound Ginkgo Tongmai oral liquid and the control spectrum is between 0.985 and 1.000, indicating that the difference is small and the similarity is high.

[0076] The retention times of the common peaks are shown in Table 1.

[0077] Table 1

[0078] Common peak Retention time 1 2.825 2 3.021 3 4.093 4 5.055 5 6.532 6 8.312 7 9.627 8 10.479 9 10.949 10 12.576 11 13.549 12 14.188 13 15.164 14 15.748 15 17.221 16 19.732 17 21.376 18 24.031 19 25.137 20 26.521 21 27.294 22 30.442 23 31.146 24 32.985 25 33.731 26 34.284 27 34.993

[0079] (I) Specificity test: Accurately pipette 20 μL of reference solution 1, test solution 1 and blank solvent (methanol), respectively, and inject according to the above chromatographic conditions, and record the chromatogram. The blank solvent does not interfere with the components of the test solution, indicating good specificity. The results are as follows: Fig. 9 As shown in AD.

[0080] (II) Precision test: The compound Ginkgo Tongmai oral liquid test solution 1 was continuously sampled and measured 6 times according to the above chromatographic conditions, and the retention time and relative peak area of ​​each chromatographic peak were recorded. The chromatographic peak of No. 19 diphenylethylene glycoside was used as the reference peak (S). The RSD% of the relative retention time of each common peak was calculated to be 0.01% to 0.12%, and the RSD% of the peak area was 0.10% to 1.80%. The results are shown in Table 2, indicating that the instrument has good precision.

[0081] Table 2

[0082]

[0083]

[0084] (III) Repeatability test: Take the same batch of compound Ginkgo Tongmai oral liquid, prepare 6 test solutions 1 in parallel, inject and measure according to the chromatographic conditions as mentioned above, record the chromatogram, import it into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" 2012.130723 version, and calculate the RSD% of the relative retention time of each common peak to be between 0.01% and 0.21%, and the RSD% of the peak area to be between 0.06% and 3.40%. The results are shown in Table 3, indicating that the method has good repeatability.

[0085] Table 3

[0086]

[0087]

[0088] (IV) Stability test: Take the test solution 1 of the same batch of Compound Ginkgo Tongmai Oral Liquid, and inject and measure at 0, 2, 6, 9, 11, and 20 h according to the above chromatographic conditions, and use the chromatographic peak of No. 19 diphenylethylene glycoside as the reference peak (S). The RSD% of the relative retention time of each common peak is calculated to be between 0.01% and 2.42%. The results are shown in Table 4, indicating that the test solution has good stability within 20 h.

[0089] Table 4

[0090]

[0091]

[0092] In the present invention, the selection of chromatographic columns is extremely important. The same batch of compound Ginkgo Tongmai oral liquid was used to prepare the test solution 1, and Waters XTERRA MS C18 (250mm×4.6mm, 5μm) and InertSustain C18 (250mm×4.6mm, 5μm) chromatographic columns were used for injection and measurement according to the above chromatographic conditions. The results showed that the peak area of ​​the InertSustain C18 chromatographic column could not be fully covered, and the separation was poor. Fig.10 As shown, the separation is good using Waters XTERRA MS C18 (250 mm × 4.6 mm, 5 μm), as shown Fig.11 shown.

[0093] In the present invention, the detection wavelength needs to be controlled at 220-230 nm, among which the test solution 1 performs best at 223 nm, with more peaks, better peak shape, and appropriate peak area.

[0094] The selection of the mobile phase of the present invention has undergone long-term experiments. In the early stage, methanol and water were used as the mobile phases for comparative gradient elution. As a result, it was found that the separation effect of the chromatographic peak was poor and the peak elution time was late. In subsequent research and development, acetonitrile-0.1% phosphoric acid was used as the mobile phase, and when the elution speed and time were strictly controlled, it was shown that the chromatographic peak had a more ideal separation degree and a more appropriate peak elution time. The best performance was achieved by using acetonitrile-0.1% phosphoric acid as the mobile phase for gradient elution under given conditions.

[0095] In the present invention, the extraction method has a very critical influence on the detection result. During the extraction, characteristic components should be retained as much as possible, and the selected solvent should have a good extraction effect on most chemical components. The present invention adopts a method combining methanol and ultrasonic extraction, and under the given parameter conditions, effective ingredients such as Salvia miltiorrhiza, Ligustrum lucidum, and Polygonum multiflorum can be retained, and a good extraction effect is also achieved for most fingerprint components.

[0096] In the present invention, the chromatographic peak shape of diphenylethylene glycoside is good, and the peak time and peak area are moderate, so the No. 19 peak special privet glycoside is selected as the reference peak.

[0097] Example 2

[0098] A method for detecting the quality standard of compound ginkgo tongmai oral liquid, based on Example 1, further comprising using thin layer chromatography to identify the glossy privet component in the compound ginkgo tongmai oral liquid, the method comprising: taking 50 ml of the compound ginkgo tongmai oral liquid, adding an equal volume of ethyl acetate to extract twice, taking the water layer and adjusting the pH to about 10 with concentrated ammonia water, and then adding an equal volume of chloroform to extract twice, recovering the solvent, and adding 3 ml of methanol to dissolve the residue as a test solution 2; taking oleanolic acid and adding methanol to prepare a reference solution 2 with a concentration of 1.0 mg / ml; taking 2 g of glossy privet, according to Prepare the control medicinal material solution 2 according to the identification item of Ligustrum lucidum in the Chinese Pharmacopoeia; take 50 ml of the negative control sample lacking Ligustrum lucidum, and prepare the negative control solution 1 according to the preparation method of the test solution 2; use the developing agent to develop the test solution 2, the reference solution 2, the control medicinal material solution 2, and the negative control solution 1 on the silica gel G thin layer plate, respectively, and the developing agent is composed of toluene, ethyl acetate, and glacial acetic acid in a volume ratio of 12:4:0.5; after the development is completed, take it out, dry it, spray it with 10% sulfuric acid ethanol solution, and heat it at 100-110°C until the spots are clearly colored. The results are as follows Fig.12 As shown (where test samples 1, 2, and 3 are the results of three groups of test sample solutions 2 set up in parallel), the chromatogram of the compound ginkgo tongmai oral liquid shows spots of the same color at the corresponding positions of the control medicinal materials, with good separation and no negative interference, making the quality control of the compound ginkgo tongmai oral liquid more scientific and reasonable, and the quality more controllable.

[0099] In the traditional method, the preparation method of the test sample of Ligustrum lucidum includes: concentrating 50ml of compound Ginkgo Tongmai oral liquid into a paste, adding 100ml of anhydrous ethanol, stirring and filtering, and evaporating the filtrate to dryness. The residue is dissolved in 50ml of water, extracted twice with chloroform, and the chloroform is recovered. The residue is dissolved in anhydrous ethanol-chloroform to obtain the test sample, and the test sample is identified by thin layer chromatography. The results show that there is no corresponding point between the test sample and the medicinal material.

[0100] The present invention uses a unique process to make the chromatographic detection of the compound ginkgo tongmai oral liquid in the reference medicinal materials more controllable.

[0101] Example 3

[0102] A method for detecting the quality standard of compound ginkgo tongmai oral liquid, based on Example 1, further comprising using thin layer chromatography to identify the Uncaria rhynchophylla in the compound ginkgo tongmai oral liquid, the method comprising: taking 50 ml of the compound ginkgo tongmai oral liquid, adding concentrated ammonia water to adjust the pH to 10-11, mixing and then adding an equal volume of chloroform to extract twice, recovering the solvent and dissolving the residue in 2 mL of methanol as a test solution 3; taking another 2 g of Uncaria rhynchophylla medicinal material, adding 6 mL of concentrated ammonia water, standing for 30 min, adding 50 mL of chloroform, Heat to reflux for 1h, filter, evaporate the filtrate and dissolve the residue in 1.5mL methanol as control medicinal material solution 3; prepare negative control solution 2 lacking Uncaria rhynchophylla; absorb test solution 3, control medicinal material solution 3, negative control solution 2, isorhynchophylline and rhynchophylline reference solution and spot them on the same silica gel G thin layer plate, develop with developing agent, take out, dry, and observe under ultraviolet light at 365nm. The developing agent is composed of acetone and petroleum ether in a volume ratio of 9:11, and the developing temperature is about 75℃. The results are as follows Fig.13 As shown (where test samples 1, 2, and 3 are the results of three groups of test sample solutions 3 set up in parallel), it shows that the chromatogram of the compound ginkgo tongmai oral liquid shows spots of the same color at the corresponding positions of the control medicinal materials, with good separation and no negative interference, thus making the quality control of the compound ginkgo tongmai oral liquid more scientific and reasonable, and the quality more controllable.

[0103] The identification of Uncaria rhynchophylla has undergone a long period of research and development, and the preparation of the test solution 3 and the control of the conditions of the development process are extremely important. When the developing agent is composed of acetone-petroleum ether in a volume ratio of 4:6, the color development temperature is 45°C, and the color developer is a modified potassium bismuth iodide solution, it is heated until the spots are clearly colored, and it is found that the test spots are not obvious. When it is observed at 365nm, it is found that the two spots of the medicinal material are not completely separated. When the developing agent is composed of acetone-petroleum ether in a volume ratio of 9:11, it is observed at 365nm. The sample chromatogram shows the same fluorescent spot at the corresponding position of the control medicinal material, the separation is good, and there is no interference with the negative.

[0104] Example 4

[0105] A method for detecting the quality standard of compound ginkgo tongmai oral liquid, based on Example 1, further comprises using thin layer chromatography to identify cyathulae radix in compound ginkgo tongmai oral liquid, the method comprising: taking 50 ml of compound ginkgo tongmai oral liquid, adding an equal volume of water to saturated n-butanol to extract twice, recovering the n-butanol and dissolving the residue with 3 ml of methanol and then passing it through a neutral alumina column (filling height 7 cm), eluting the neutral alumina column with 50 ml of water first, then with 50 ml of 50% ethanol, discarding the eluent, and then eluting with 100 ml of 70% ethanol, collecting the eluent, and dissolving the residue after the eluent recovers the solvent with 1 ml of methanol as a test solution 4; taking cyathulae radix as a reference solution 4 made of 1.0 mg / ml by adding methanol; preparing a negative control lacking cyathulae radix Solution 3; take 2g of the reference medicinal material of Cyathula officinalis and place it in a conical flask, add 20ml of methanol and reflux for 1h, filter and take the filtrate, concentrate the filtrate to 1ml and pass it through a neutral alumina column. When eluting the neutral alumina column, first elute it with 50mL of water, then elute it with 50mL of 50% ethanol, discard the eluent, and then elute it with 100mL of 70% ethanol, collect the eluent, and dissolve the residue after recovering the solvent from the eluent with 3mL of methanol as the reference medicinal material solution 4; absorb the test solution 4, the reference solution 4, the negative control solution 3, and the reference medicinal material solution 4, and spot them on the silica gel G thin layer plate respectively, and develop it with a developing agent composed of chloroform and methanol in a volume ratio of 10:3, then take it out, dry it, spray it with 10% sulfuric acid ethanol solution as a color developer, and heat it until the spots are clearly colored. The results are as follows Fig.14 As shown (where test samples 1, 2, and 3 are the results of three groups of test sample solutions 4 set up in parallel), it shows that the chromatogram of the compound ginkgo tongmai oral liquid shows spots of the same color at the corresponding positions of the control medicinal materials, with good separation and no negative interference, thus making the quality control of the compound ginkgo tongmai oral liquid more scientific and reasonable, and the quality more controllable.

[0106] Example 5

[0107] A method for detecting the quality standard of compound ginkgo tongmai oral liquid, based on Example 1, further includes using thin layer chromatography to identify the eucommia in the compound ginkgo tongmai oral liquid, the method comprising: taking the test solution 4 in Example 4 as the test solution 5 in this example, preparing a negative control solution 4 lacking eucommia; absorbing the test solution 5 and the negative control solution 4, respectively spotting them on the same silica gel G thin layer plate, developing them with a developing agent composed of chloroform, methanol, and water in a volume ratio of 10:3:0.3, then taking them out, drying them, and inspecting them under a 365nm ultraviolet lamp. The results are as follows Fig.15As shown (where test samples 1, 2, and 3 are the results of three groups of test sample solutions 5 set up in parallel), it shows that the chromatogram of the compound ginkgo tongmai oral liquid shows spots of the same color at the corresponding positions of the control medicinal materials, with good separation and no negative interference, thus making the quality control of the compound ginkgo tongmai oral liquid more scientific and reasonable, and the quality more controllable.

[0108] Example 6

[0109] A method for detecting the quality standard of Compound Ginkgo Tongmai Oral Liquid, based on Example 1, further comprising using HPLC liquid chromatograph to analyze the total flavonol glycosides in the Compound Ginkgo Tongmai Oral Liquid, the method comprising:

[0110] Preparation of test solution 6:

[0111] An orthogonal test was conducted with reflux time, reflux temperature and hydrochloric acid concentration as factors, and the results of total flavonol glycoside extraction were shown in Table 5.

[0112] Table 5

[0113]

[0114] The optimal conditions were as follows: reflux time of 30 min, reflux temperature of 80°C, and hydrochloric acid concentration of 25%.

[0115] Prepare under the optimal conditions: take the compound ginkgo tongmai oral liquid under the filling volume difference item, mix well, accurately measure 10ml, add 16ml methanol and 6.0ml 25% hydrochloric acid, heat to 80℃ in a water bath and reflux for 3min, quickly cool to room temperature, transfer to a 50ml volumetric flask, add methanol to dilute to the scale, mix well, filter with a 0.45μm microporous filter membrane, and use the filtrate as the test solution;

[0116] Preparation of reference solution 6: Accurately weigh appropriate amounts of quercetin, kaempferol and isorhamnetin reference substances, add methanol to prepare a mixed solution containing 33.25 μg, 29.43 μg and 19.22 μg of each substance per ml as the reference solution;

[0117] Preparation of negative control solution 5: Prepare negative control solution by using compound Ginkgo Tongmai oral solution lacking Ginkgo biloba leaves according to the method for preparing test solution 6;

[0118] The test solution 6, the reference solution 6, and the negative control solution 5 were measured by HPLC liquid chromatography; the chromatographic conditions were as follows: the chromatographic column was filled with octadecylsilane bonded silica gel; the mobile phase A was methanol, and the mobile phase B was 0.4% phosphoric acid, and the elution was carried out in a volume ratio of 55:45; the flow rate was 1.0 ml / min; the column temperature was 30°C; the detection wavelength was 360 nm; the injection volume was 10 μl, and the theoretical plate number was not less than 2500 based on the quercetin peak.

[0119] The results of the methodological investigation are as follows:

[0120] Specificity test: 10 μL of reference solution 6, test solution 6 and negative control solution 5 were accurately pipetted and tested according to the above chromatographic conditions. Results: In the chromatogram of the test solution, there were chromatographic peaks at the positions corresponding to the chromatographic peaks of the reference solutions of quercetin, kaempferol and isorhamnetin, while there were no such peaks in the chromatogram of the negative control solution. The chromatogram is shown in the figure below. Fig.16 As shown, it means that there is no interference in the negative, control, test sample, and negative sample.

[0121] Linear relationship investigation: Precisely aspirate a mixed solution containing quercetin, kaempferol and isorhamnetin, with concentrations of (33.26, 13.30, 6.65, 3.32, 1.66), (29.43, 11.77, 5.89, 2.94, 1.47), (19.22, 7.69, 3.84, 1.92, 0.96) μg / ml, inject 10 μl of each solution, measure the peak area, and use the peak area as the ordinate (y) and the injection concentration as the abscissa (x) for linear regression (n=6). The regression equation for quercetin is y=44419x–11858 (R 2 =0.999), kaempferol regression equation y = 47153x-8220 (R 2 =0.999), isorhamnetin regression equation y = 43343x–8096 (R 2 =0.999), the test results showed that the quercetin injection amount showed a good linear relationship in the range of 0.0166-0.3326 μg, the kaempferol injection amount showed a good linear relationship in the range of 0.0147-0.2943 μg, and the isorhamnetin injection amount showed a good linear relationship in the range of 0.0096-0.1922 μg.

[0122] Precision test: Take the same batch of compound Ginkgo Tongmai oral liquid samples, prepare 6 test solutions according to the above method, and inject them continuously for 6 times. The results show that the RSD value of the quercetin peak area is 0.28%, the RSD value of the kaempferol peak area is 0.88%, and the RSD value of the isorhamnetin peak area is 2.2%, indicating that the instrument has good precision.

[0123] Stability test: Take the same batch of compound Ginkgo Tongmai oral liquid samples to prepare 6 portions of test solution. The samples were injected at 0, 1, 3, and 7 hours respectively. The results showed that the test solution was stable within 7 hours.

[0124] Repeatability test: Take the same batch of compound Ginkgo Tongmai oral liquid samples, prepare 6 test solutions, and measure according to the chromatographic conditions described in this example, inject 10 μl of each sample, inject into the chromatograph, and record the chromatogram. The results show that the RSD value of the quercetin peak area is 1.1%, the RSD value of the kaempferol peak area is 2.5%, and the RSD value of the isorhamnetin peak area is 3.2%. The test results show that the method has good repeatability.

[0125] Accuracy test: 2 ml of 6 samples with known contents (batch number: 20200604, contents: quercetin 6.95 μg / ml, kaempferol 5.95 μg / ml, isorhamnetin 3.084 μg / ml) were accurately measured, and appropriate amounts of mixed reference solution were accurately added to each sample. The test solution was prepared according to the method for preparing the test solution. The content was determined according to the above method and the recovery rate was calculated. 2 ml of 3 samples with known contents (batch number: 20200813, contents: quercetin 13.892 μg / ml, kaempferol 11.891 μg / ml, isorhamnetin 6.168 μg / ml) were accurately measured, and appropriate amounts of mixed reference solution were accurately added to each sample. The test solution was prepared according to the method for preparing the test solution. The content was determined according to the above method and the recovery rate was calculated. The results are shown in Tables 6-8.

[0126] Table 6 Quercetin sample recovery test results

[0127]

[0128] Table 7 Kaempferol sample recovery test results

[0129]

[0130] Table 8 Isorhamnetin sample recovery test results

[0131]

[0132] Example 7

[0133] A method for detecting the quality standard of Compound Ginkgo Tongmai Oral Liquid, based on Example 1, further comprising using HPLC liquid chromatograph to analyze the content of salvianolic acid B in Compound Ginkgo Tongmai Oral Liquid, the method comprising:

[0134] Preparation of test solution 7: Take the product under the item of volume difference, mix well, accurately measure 10 ml, dilute to 25 ml with methanol, mix well, filter with a 0.45 μm microporous membrane, and the filtrate is used as test solution 7;

[0135] Preparation of reference solution 7: Take an appropriate amount of salvianolic acid B reference substance, weigh accurately, and add methanol-water (8:2) mixture to make a solution containing 0.10 mg per 1 ml, as reference solution 7;

[0136] Preparation of negative control solution 6: prepare negative control solution 6 by using compound Ginkgo Tongmai oral solution lacking Danshen according to the preparation method of test solution 7;

[0137] The test solution 7, the reference solution 7 and the negative control solution 6 were measured by HPLC liquid chromatography; the chromatographic conditions were as follows: the chromatographic column was filled with octadecylsilane bonded silica gel; the mobile phase A was acetonitrile, and the mobile phase B was 0.1% phosphoric acid solution, eluted within the range of a volume ratio of 22:78; flow rate: 1.0 ml / min; column temperature: 20°C; detection wavelength: 286 nm; injection volume: 20 μl.

[0138] Accurately pipette 20 μL of reference solution and test solution respectively, inject into liquid chromatograph for determination.

[0139] The results of the methodological investigation are as follows:

[0140] Specificity test: 20 μL of reference solution 7, test solution 7 and negative reference solution 6 were accurately aspirated and tested according to the above chromatographic conditions. Results: There was a chromatographic peak of salvianolic acid B in the chromatogram of the test solution at the corresponding position of the chromatographic peak of the reference solution, but there was no such peak in the chromatogram of the negative reference solution, indicating that there was no interference with the negative solution. The results are shown in Fig.17 shown.

[0141] Linear range test: Accurately pipette reference solution 7, adjust the concentration to 0.045, 0.091, 0.105, 0.227, 0.378 mg / ml, inject 20 μl of each, measure the peak area, use the peak area as the ordinate (y), and the injection concentration as the abscissa (x), perform linear regression (n=6), linear regression equation: y=3E+07x-53207. R2=0.998. The test results show that the injection volume has a good linear relationship in the range of 0.045-0.378 mg.

[0142] Stability test: 7 portions of test solution were prepared from the same batch of compound Ginkgo Tongmai oral liquid samples, and the samples were injected at 0, 1, 3, 6, 9, and 18 hours respectively. The results showed that the test solution was stable within 18 hours.

[0143] Precision experiment: 7 portions of test solution were prepared from the same batch of Compound Ginkgo Tongmai Tablets samples, and the samples were injected 6 times continuously. The RSD value of the peak area was 0.46%, indicating that the instrument had good precision.

[0144] Repeatability test: Take the same batch of compound Ginkgo Tongmai oral liquid to prepare six test solutions, and measure according to the above chromatographic conditions. The RSD value of the peak area is 1.05%. The test results show that the method has good repeatability.

[0145] Accuracy test: Accurately measure 9 samples with known content (batch number: 20210825, content: 0.65 mg / ml) respectively, accurately add appropriate amount of mixed reference solution respectively, prepare test solution according to the preparation method of test solution, determine the content and calculate the recovery rate according to the above method. The results are shown in Table 9.

[0146] Table 9

[0147]

[0148] Example 8

[0149] A method for detecting the quality standard of compound ginkgo tongmai oral liquid, based on Example 1, further comprising using HPLC liquid chromatograph to analyze the content of 2,3,5,4-tetrahydroxystilbene-2-O-β-D glucoside (i.e., stilbene glycoside) in Polygonum multiflorum in compound ginkgo tongmai oral liquid, the method comprising:

[0150] Preparation of test solution 8: Take the product under the item of volume difference, mix well, accurately measure 5 ml, add 62% ethanol to make up to 25 ml, shake well, filter, and the filtrate is used as test solution 8;

[0151] Preparation of reference solution 8: Take an appropriate amount of stilbene glycoside sample and add dilute ethanol to prepare a solution with a concentration of 0.2 mg / ml as reference solution 8;

[0152] Preparation of negative control solution 7: prepare negative control solution 7 by using compound Ginkgo Tongmai oral solution lacking Polygonum multiflorum according to the method for preparing test solution 8;

[0153] The test solution 8, reference solution 8 and negative control solution 7 were measured by HPLC liquid chromatography; the chromatographic conditions were: the chromatographic column was filled with octadecylsilane bonded silica gel; the mobile phase A was acetonitrile, and the mobile phase B was water; the volume ratio was 20:80, and the detection wavelength was 320nm; the injection volume was 10μl.

[0154] Methodological investigation:

[0155] Specificity test: 10 μL of reference solution 8, test solution 8 and negative reference solution 7 were accurately aspirated and tested according to the above chromatographic conditions. Results: There was a chromatographic peak of diphenylethylene glycoside in the chromatogram of the test solution at the corresponding position of the chromatographic peak of the reference solution, but there was no such peak in the chromatogram of the negative reference solution, indicating that there was no interference with the negative solution. The results are shown in Fig.18 .

[0156] Linear relationship investigation: Accurately draw the reference solution 8, adjust the concentration to 0.186, 0.0928, 0.0464, 0.0232, 0.0116 mg / ml, inject 10 μl of each, measure the peak area, use the peak area as the ordinate (y), and the injection concentration as the abscissa (x), and perform linear regression (n=6). The linear regression equation is: y=38319878.11x-85539.83 (R2=0.9999). The test results show that the injection volume has a good linear relationship in the range of 0.116-1.856 μg.

[0157] Precision test: Take the same batch of compound Ginkgo Tongmai oral liquid samples, prepare 1 sample solution, and inject the sample 6 times continuously. The RSD value of the peak area of ​​stilbene glycoside is 1.06%, indicating that the precision of the instrument is good.

[0158] Stability test: Take the same batch of compound Ginkgo Tongmai oral liquid to prepare 8 portions of test solution, and inject samples at 0, 2, 4, 6, 10, and 23 hours respectively. The RSD value of the peak area is 0.29%. The results show that the test solution is stable within 23 hours.

[0159] Repeatability test: Take the same batch of compound Ginkgo Tongmai oral liquid to prepare 8 six test solutions. The peak area RSD of the results measured under the above chromatographic conditions is 1.27%, indicating that the method has good repeatability.

[0160] Accuracy: Accurately measure 9 portions of compound Ginkgo Tongmai oral solution (batch number: 20200604, content: 0.0699 mg / ml) with known content, accurately add appropriate amount of mixed reference solution 8, prepare the test solution according to the preparation method of test solution 8, determine the content and calculate the recovery rate according to the above method. The results are shown in Table 10.

[0161] Table 10

[0162]

[0163] Example 9

[0164] A method for detecting the quality standard of compound Ginkgo Tongmai oral liquid, based on Example 1, further comprises using HPLC liquid chromatograph to analyze the total flavonol glycoside content, salvianolic acid B content and stilbene glycoside content of the compound Ginkgo Tongmai oral liquid, the test sample solution preparation and chromatographic conditions are respectively as shown in Example 6, Example 7 and Example 8, and the test results of different batches of test samples are shown in Table 11, and the results can more intuitively show the differences in components and contents of different batches.

[0165]

[0166] Example 10

[0167] A method for testing the quality standard of compound Ginkgo Tongmai oral liquid includes establishing a fingerprint of compound Ginkgo Tongmai oral liquid; using thin layer chromatography to identify the four ingredients of Ginkgo biloba, Uncaria rhynchophylla, Cyathula officinalis and Eucommia ulmoides in compound Ginkgo Tongmai oral liquid; and using HPLC liquid chromatography to analyze the contents of total flavonol glycosides, salvianolic acid B and stilbene glycosides in compound Ginkgo Tongmai oral liquid. This testing method can fully reflect and control the overall quality of compound Ginkgo Tongmai oral liquid, improve the controllability of product quality, and ensure the effectiveness of the drug.

[0168] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for detecting the quality standard of compound ginkgo tongmai oral liquid, characterized in that: Including the establishment of the fingerprint spectrum of compound ginkgo tongmai oral liquid, the establishment of the fingerprint spectrum includes: Preparation of test solution 1: Take an appropriate amount of compound Ginkgo Tongmai oral liquid, add methanol and mix, then perform ultrasonic treatment, and then filter the filtrate to obtain the test solution; Preparation of reference solution 1: dissolve samples of stilbene glycoside, privet glycoside, salvianolic acid B, quercetin, pinoresinol diglucoside, cyasterone, isorhynchophylline, and oleanolic acid in water to prepare mixed reference solution 1-a; dissolve potassium sorbate sample in methanol to prepare reference solution 1-b; Control medicinal material solution 1: pre-treat seven medicinal materials including Ginkgo biloba leaves, processed Polygonum multiflorum, Fructus Ligustri Lucidi, Salvia miltiorrhiza, Eucommia ulmoides, Cyathula officinalis and Uncaria rhynchophylla, and obtain control medicinal material solutions according to the preparation method of test solution 1; The reference solution 1, the reference medicinal material solution 1 and multiple batches of test solution 1 were respectively measured by HPLC liquid chromatography; The chromatograms of multiple batches of test solution 1 were introduced into the Chinese medicine chromatographic fingerprint similarity evaluation system, and the chromatogram of one batch of test solution 1 was used as a reference spectrum. After multi-point calibration and data analysis, the fingerprint spectrum of the compound ginkgo tongmai oral liquid was obtained; it was confirmed that the common peaks included the peak corresponding to salvianolic acid B, the peak corresponding to potassium sorbate, the peak corresponding to diphenylethylene glycoside, the peak corresponding to pinoresinol diglucoside, and the peak corresponding to privet glycoside; Chromatographic conditions: C18 column, Waters xterra C18 model 250mm×4.6mm×5μm column; flow rate 1.0ml / min, detection wavelength 220-230nm, column temperature 20-30℃; mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid, gradient elution is performed in the following order:

2. The method for detecting the quality standard of Compound Ginkgo Tongmai Oral Liquid according to claim 1, characterized in that: The chromatographic conditions were: flow rate 1.0 ml / min, detection wavelength 223 nm, column temperature 25 °C.

3. The method for detecting the quality standard of Compound Ginkgo Tongmai Oral Liquid according to claim 2, characterized in that: The fingerprint spectrum of the compound Ginkgo Tongmai oral liquid includes 27 common peaks; compared with the reference medicinal materials, it is confirmed that among the common peaks, chromatographic peaks 4, 17, and 21 with retention times of 5.055, 21.376, and 27.294, respectively, are from Ligustrum lucidum, chromatographic peaks 18, 19, and 20 with retention times of 24.031, 25.137, and 26.521, respectively, are from Salvia miltiorrhiza, and chromatographic peak 10 with retention time of 12.576 is from Uncaria rhynchophylla; chromatographic peaks 3, 11, 12, and 13 with retention times of 4.093, 13.549, 14.188, and 15.164, respectively, are from Eucommia ulmoides, chromatographic peak 15 with retention time of 17.221 is from processed Polygonum multiflorum as diphenylethylene, and chromatographic peaks 2, 7, and 14 with retention times of 3.021, 9.627, and 15.748, respectively, are from Ginkgo biloba leaves.

4. The method for detecting the quality standard of compound Ginkgo Tongmai oral liquid according to claim 1, characterized in that: The invention also includes the use of thin layer chromatography to identify the Fructus Ligustri Lucidi in the Compound Ginkgo Tongmai Oral Liquid, and the method includes: taking an appropriate amount of the Compound Ginkgo Tongmai Oral Liquid, adding an equal volume of ethyl acetate for extraction, taking the water layer and adding an equal volume of chloroform for further extraction after the extraction is completed, recovering the chloroform after the extraction is completed, and dissolving the residue in methanol as the test solution 2; taking oleanolic acid to prepare a reference solution 2; taking Fructus Ligustri Lucidi to prepare a reference medicinal material solution 2; taking an appropriate amount of a negative control sample lacking Fructus Ligustri Lucidi, and preparing a negative control solution 1 according to the preparation method of the test solution 2; using a developing agent to develop the test solution 2, the reference solution 2, the reference medicinal material solution 2, and the negative control solution 1 on a silica gel G thin layer plate, respectively, and the developing agent is composed of toluene, ethyl acetate, and glacial acetic acid; taking out after the development is completed, drying, spraying with 10% sulfuric acid ethanol solution, and heating until the spots are clearly colored.

5. The method for detecting the quality standard of Compound Ginkgo Tongmai Oral Liquid according to claim 1, characterized in that: It also includes using thin layer chromatography to identify the Uncaria rhynchophylla in the compound Ginkgo Tongmai oral liquid. The method includes: taking an appropriate amount of the compound Ginkgo Tongmai oral liquid, adding an appropriate amount of concentrated ammonia water to mix well, and then adding an equal volume of chloroform to extract, and dissolving the residue after recovering the solvent with methanol as a test solution 3; taking another Uncaria rhynchophylla medicinal material to prepare a control medicinal material solution 3; preparing a negative control solution 2 without Uncaria rhynchophylla; absorbing the test solution 3, the control medicinal material solution 3, the negative control solution 2, isorhynchophylline, and the Uncaria rhynchophylla reference solution respectively on the same silica gel G thin layer plate, developing it with a developing agent, and then taking it out, drying it, and examining it under an ultraviolet lamp. The developing agent is composed of acetone and petroleum ether in a volume ratio of 9:

11.

6. The method for detecting the quality standard of Compound Ginkgo Tongmai Oral Liquid according to claim 1, characterized in that: The invention also includes the use of thin layer chromatography to identify the Cyathula capitata in the compound Ginkgo Tongmai oral liquid. The method includes: taking an appropriate amount of the compound Ginkgo Tongmai oral liquid, adding an equal volume of water to saturate n-butanol for extraction, recovering the n-butanol, dissolving the residue with methanol, and then passing it through a neutral alumina column, eluting the neutral alumina column with 40 to 60 mL of water, then eluting with 40 to 60 mL of 40 to 60% ethanol, discarding the eluent, and then eluting with 100 to 120 mL of 70 to 75% ethanol, collecting the eluent, and recovering the eluent. The residue after solvent removal was dissolved in methanol as test solution 4; amaranthone was taken to prepare reference solution 4; a negative control solution 3 lacking Cyathulae Bidentatae was prepared; a reference medicinal material solution 4 was prepared by taking Cyathulae Bidentatae reference material; the test solution 4, reference solution 4, negative control solution 3, and control medicinal material solution 4 were aspirated and spotted on a silica gel G thin layer plate, respectively, and developed with a developing agent composed of chloroform and methanol, then taken out, dried, sprayed with 10% ethanolic sulfuric acid solution as a color developer, and heated until the spots were clearly colored.

7. The method for detecting the quality standard of Compound Ginkgo Tongmai Oral Liquid according to claim 6, characterized in that: The invention also includes the use of thin layer chromatography to identify the Eucommia ulmoides in the compound Ginkgo Tongmai oral liquid. The method includes: taking an appropriate amount of the compound Ginkgo Tongmai oral liquid, adding an equal volume of water to saturate n-butanol for extraction, dissolving the residue after recovering the n-butanol with methanol and then passing it through a neutral alumina column, eluting the neutral alumina column with 40-60 mL of water, then eluting with 40-60 mL of 40-60% ethanol, discarding the eluent, and then eluting with 100-120 mL of 70-75% ethanol, collecting the eluent, dissolving the residue after the eluent recovers the solvent with methanol as a test solution 5, and preparing a negative control solution 4 lacking Eucommia ulmoides; absorbing the test solution 5 and the negative control solution 4, respectively spotting them on the same silica gel G thin layer plate, developing them with a developing agent composed of chloroform, methanol and water, then taking them out, drying them, and inspecting them under an ultraviolet lamp.

8. The method for detecting the quality standard of Compound Ginkgo Tongmai Oral Liquid according to claim 1, characterized in that: The invention also includes analyzing the total flavonol glycosides in the compound Ginkgo Tongmai oral liquid by using HPLC liquid chromatography, and the method includes: Preparation of test solution 6: Mix compound Ginkgo Tongmai oral liquid, methanol and hydrochloric acid, heat in a water bath first and then reflux, cool to room temperature quickly after heating, dilute with methanol and filter, and use the filtrate as the test solution; Preparation of reference solution 6: Take appropriate amount of quercetin, kaempferol and isorhamnetin samples, add methanol to dissolve and obtain a mixed solution as the reference solution; Preparation of negative control solution 5: Prepare negative control solution by using compound Ginkgo Tongmai oral solution lacking Ginkgo biloba leaves according to the method for preparing test solution 6; The test solution 6, the reference solution 6 and the negative control solution 5 were measured by HPLC liquid chromatography; the chromatographic conditions were as follows: the chromatographic column was filled with octadecylsilane bonded silica gel; the mobile phase A was methanol, and the mobile phase B was 0.4% phosphoric acid, and the elution was carried out in the range of a volume ratio of 50-55:45-50; the flow rate was 0.9-1.1 ml / min; the column temperature was 28-32°C; the detection wavelength was 340-380 nm; the injection volume was 10 μl, and the theoretical plate number was not less than 2500 based on the quercetin peak.

9. The method for detecting the quality standard of Compound Ginkgo Tongmai Oral Liquid according to claim 1, characterized in that: The invention also includes analyzing salvianolic acid B in compound ginkgo tongmai oral liquid by using HPLC liquid chromatography, and the method includes: Preparation of test solution 7: Dilute the compound Ginkgo Tongmai oral solution with methanol and filter it. The filtrate is used as test solution 7; Preparation of reference solution 7: Take an appropriate amount of salvianolic acid B sample, add methanol and water to prepare reference solution 7; Preparation of negative control solution 6: prepare negative control solution 6 by using compound Ginkgo Tongmai oral solution lacking Danshen according to the preparation method of test solution; The test solution 7, the reference solution 7 and the negative control solution 6 were measured by HPLC liquid chromatography; the chromatographic conditions were as follows: the chromatographic column was filled with octadecylsilane bonded silica gel; the mobile phase A was acetonitrile, and the mobile phase B was 0.1% phosphoric acid solution, and the elution was carried out in the range of a volume ratio of 20-25:75-80; the flow rate was 0.9-1.1 ml / min; the column temperature was 18-22°C; the detection wavelength was 280-290 nm; the injection volume was 20 μl.

10. The method for detecting the quality standard of Compound Ginkgo Tongmai Oral Liquid according to claim 1, characterized in that: The invention also includes using HPLC liquid chromatography to analyze diphenylethylene glycosides in compound ginkgo tongmai oral liquid, and the method includes: Preparation of test solution 8: dilute the compound Ginkgo Tongmai oral solution with ethanol and filter it, and the filtrate is used as the test solution 8; Preparation of reference solution 8: prepare the diphenylethylene glycoside sample into reference solution 8; Preparation of negative control solution 7: prepare negative control solution 7 by using compound Ginkgo Tongmai oral solution lacking Polygonum multiflorum according to the method for preparing test solution 8; The test solution 8, the reference solution 8 and the negative control solution 7 were measured by HPLC liquid chromatography; the chromatographic conditions were: the chromatographic column was filled with octadecylsilane bonded silica gel; the mobile phase A was acetonitrile, and the mobile phase B was water; the detection wavelength was 300-350nm; the injection volume was 10μl.

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