Fingerprint spectrum of Qixuekang Oral Liquid, its construction method and application

Abstract

The present invention discloses a fingerprint of Qixuekang Oral Liquid, a construction method thereof and an application thereof, belonging to the technical field of pharmaceutical analysis. The construction method of the present invention includes: preparing a test solution of Qixuekang Oral Liquid; respectively preparing reference substance solutions of ginsenoside Rg1, Rb1, Re, Rf, Rd, notoginsenoside R1, astragaloside IV, calycosin-7-O-β-D-glucoside, and puerarin; precisely and respectively sucking the test solution and the reference substance solutions and injecting them into a high performance liquid chromatograph for determination to obtain a test fingerprint and a reference substance fingerprint; using the reference substances for chromatographic positioning and attribution; performing similarity analysis on the fingerprints of multiple batches of test solutions to obtain the fingerprint of Qixuekang Oral Liquid. The present invention also discloses the fingerprint of Qixuekang Oral Liquid obtained by this method and its application in quality control. The present invention constructs the fingerprint of Qixuekang Oral Liquid for the first time, which can comprehensively and accurately reflect and control the product quality and ensure the safety and effectiveness of the drug.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a fingerprint spectrum of Qixuekang oral liquid, a construction method and application thereof. Background Art

[0002] Qixuekang oral liquid is made of fresh Panax notoginseng, Astragalus, Ginseng and Pueraria root. It has the effects of strengthening the spleen and strengthening the foundation, nourishing yin and moistening dryness, promoting fluid and relieving cough. It is used for fatigue, shortness of breath and palpitations, yin deficiency and lack of fluid, dry mouth and tongue. In the existing technology, the analytical method for the quality inspection of Qixuekang oral liquid only uses high performance liquid chromatography (HPLC) to detect the total saponin content, which is difficult to fully reflect the material basis of the efficacy, ignores the overall characteristics of traditional Chinese medicine, and does not conform to the quality control trend of modern traditional Chinese medicine. In the preparation of test samples, the existing methods mostly use complex steps such as water-saturated n-butanol extraction, which is cumbersome to operate and uses a large amount of organic solvents, which increases the difficulty of operation and safety hazards.

[0003] Panax notoginseng Panax notoginseng The dried root and rhizome of (Burk.) FH Chen are warm in nature, sweet and slightly bitter in taste, and enter the liver and stomach meridians. They have the effects of dispersing blood stasis, stopping bleeding, reducing swelling and relieving pain. Modern pharmacological studies have shown that Panax notoginseng has lipid-lowering, anti-oxidation, anti-anxiety and immunomodulatory effects. The saponin compounds in Panax notoginseng are considered to be the main active ingredients of Panax notoginseng. The literature "Overview of Research on Chemical Composition and Pharmacological Actions of Panax notoginseng and Prediction of Quality Markers" (Shi Liping et al., Chinese Journal of Traditional Chinese Medicine, 2023, 48(8):2059-2067.DOI:10.19540 / j.cnki.cjcmm.20230213.201.) Based on the theory of quality markers (Q-markers) of traditional Chinese medicine, the quality markers of Panax notoginseng were predicted and analyzed from the aspects of plant kinship, efficacy, medicinal properties, and measurability of chemical components, and the specific proportion of ginsenoside Rg in Panax notoginseng was inferred. 1 、Re、Rb 1 , and ginsenoside Rb 2 , Rb 3 、Re、Rd、Rh 2 , Rg 3 and notoginsenoside R 1 , notoginseng and quercetin can be used as potential quality markers of Panax notoginseng.

[0004] Astragalus mongolica Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus Astragalus membranaceusThe dried root of Astragalus membranaceus (Fisch.) Bge. is sweet in taste and slightly warm in nature; it belongs to the lung and spleen meridians; it has the functions of invigorating qi and ascending yang, consolidating the exterior to stop sweating, promoting diuresis to alleviate edema, promoting the production of body fluid and nourishing blood, promoting qi and activating collaterals, expelling toxins and discharging pus, and astringing sores and promoting granulation. Flavonoids and saponins are the main active components of Astragalus membranaceus. In the Chinese Pharmacopoeia (2020 Edition, Volume I), the content determination indicators for Astragalus membranaceus medicinal materials are astragaloside IV and calycosin-7-O-β-D-glucoside.

[0005] Ginseng is the dried root and rhizome of Panax ginseng C. A. Mey. of the Araliaceae family. Panax ginseng It is sweet and slightly bitter in taste, slightly warm in nature, and belongs to the spleen, lung, heart, and kidney meridians; it has the functions of greatly tonifying primordial qi, restoring pulse and arresting collapse, invigorating the spleen and benefiting the lung, promoting the production of body fluid and nourishing blood, and calming the mind and improving intelligence. In the literature "Research Progress on the Regulation of Immune System Diseases by Ginseng and Prediction Analysis of Quality Markers" (Xu Hui et al., Chinese Archives of Traditional Chinese Medicine, 1 - 20 [2025 - 03 - 10]. http: / / kns.cnki.net / kcms / detail / 21.1546.R.20240805.1506.016.html.), based on summarizing the chemical components and pharmacological effects of ginseng in regulating immune function, prediction analysis was carried out from aspects such as plant affinity, chemical component effectiveness, uniqueness, and compound compatibility based on the principle of quality markers. The major components such as ginsenosides, polysaccharides, proteins, and volatile oils are used as quality markers for ginseng to regulate immune function. Single components that can be selected include ginsenoside Rg l , Re, Rb l , Rg 3 , Rh 1 , Rh 2 , Rd, CK, Ro, Rg 5 , ginseng D - glucose, galactose, arabinose, and panaxynol are used as alternative substances.

[0006] Traditional Chinese medicine fingerprint is a method for the overall quality evaluation of systematically characterizing the complex components of traditional Chinese medicine through modern analytical techniques. Its core is to obtain spectral or chromatographic maps that can reflect the characteristics of multiple components of traditional Chinese medicine through specific analytical means, so as to achieve the overall identification and quality control of the "chemical component group" of traditional Chinese medicine. The establishment of the detection method of traditional Chinese medicine fingerprint breaks through the limitations of single - component determination, and has significant advantages especially in solving key problems such as the characterization of complex components, batch stability control, and authenticity identification. However, there is no research on the fingerprint of Qixuekang Oral Liquid in the existing technology. Therefore, providing a fingerprint detection method for Qixuekang Oral Liquid, which can locate key components and is simple to operate, has become an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0007] One of the objectives of the present invention is to provide a method for constructing a fingerprint of Qixuekang Oral Liquid, which can efficiently separate and accurately detect various components in Qixuekang Oral Liquid, and has simple operation and low safety hazards.

[0008] Another objective of the present invention is to provide a fingerprint of Qixuekang Oral Liquid obtained by using the above construction method.

[0009] A third objective of the present invention is to provide the application of the fingerprint of Qixuekang Oral Liquid.

[0010] To achieve the above objectives, the technical solutions adopted by the present invention are as follows:

[0011] The present invention provides a method for constructing a fingerprint of Qixuekang Oral Liquid in the first aspect, and the method includes the following steps:

[0012] Preparation of the test solution: Take Qixuekang Oral Liquid and dilute it with 40 - 60% methanol to obtain the solution.

[0013] Preparation of the reference substance solution: Take ginsenoside Rg 1 reference substance, and dissolve it in methanol to prepare a ginsenoside Rg 1 reference substance solution; prepare reference substance solutions of ginsenoside Rb 1 , ginsenoside Re, ginsenoside Rf, ginsenoside Rd, notoginsenoside R 1 , astragaloside IV, calycosin - 7 - O - glucoside, puerarin in the same way;

[0014] Determination: Precisely pipette the test solution and the reference substance solutions respectively, inject them into a high - performance liquid chromatograph for determination to obtain the test solution fingerprint and the reference substance fingerprints; the chromatographic conditions include: using octadecylsilane chemically bonded silica as the filler, using acetonitrile as mobile phase A and water as mobile phase B, and performing gradient elution according to the following regulations:

[0015] 0 - 12 min, 18 - 20%A, 80 - 82%B;

[0016] 12 - 32 min, 18 - 20%A → 23 - 25%A, 80 - 82%B → 75 - 77%B;

[0017] 32 - 75 min, 23 - 25%A → 30 - 34%A, 75 - 77%B → 66 - 70%B;

[0018] 75 - 84 min, 30 - 34%A → 34 - 38%A, 66 - 70%B → 62 - 66%B;

[0019] 84 - 85 min, 34 - 38%A → 18 - 20%A, 62 - 66%B → 80 - 82%B;

[0020] The mobile phase flow rate is 1.0 - 1.4 ml / min; the detection wavelength is 203 nm; the column temperature is 15 - 25 °C;

[0021] Similarity evaluation: Locate and assign the chromatographic peaks in the test sample fingerprint spectrum according to the reference substance spectrum; Import the fingerprint spectra of 10 - 20 batches of Qixuekang Oral Liquid test samples into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System for analysis to obtain the fingerprint spectrum of Qixuekang Oral Liquid.

[0022] In some embodiments of the present invention, gradient elution is carried out according to the following regulations:

[0023] 0 - 12 min, 19% A, 81% B;

[0024] 12 - 32 min, 19% A → 23.8% A, 81% B → 76.2% B;

[0025] 32 - 75 min, 23.8% A → 32% A, 76.2% B → 68% B;

[0026] 75 - 84 min, 32% A → 36% A, 68% B → 64% B;

[0027] 84 - 85 min, 36% A → 19% A, 64% B → 81% B.

[0028] In some embodiments of the present invention, take Qixuekang Oral Liquid, dilute it 5 - 15 times with 40 - 60% methanol to obtain the test sample solution;

[0029] Take an appropriate amount of ginsenoside Rg 1 reference substance, accurately weigh it, dissolve it in methanol to prepare a solution containing 0.1 - 0.4 mg per 1 ml to obtain the ginsenoside Rg 1 reference substance solution; Prepare the ginsenoside Rb 1 , ginsenoside Re, ginsenoside Rf, ginsenoside Rd, notoginsenoside R 1 , astragaloside IV, calycosin - 7 - O - glucoside, puerarin 9 reference substance solutions in the same way. In some embodiments of the present invention, take Qixuekang Oral Liquid, dilute it 10 times with 50% methanol to obtain the test sample solution;

[0030] Take an appropriate amount of ginsenoside Rg 1 reference substance, accurately weigh it, dissolve it in methanol to prepare a solution containing 0.2 mg per 1 ml to obtain the ginsenoside Rg 1 reference substance solution; Prepare the ginsenoside Rb 1 , ginsenoside Re, ginsenoside Rf, ginsenoside Rd, notoginsenoside R 1, solutions of 9 reference substances including astragaloside IV, calycosin-7-O-β-D-glucoside, and puerarin.

[0031] In some embodiments of the present invention, the chromatography within 95 min is collected; in the similarity evaluation step, 10 common peaks are determined with a similarity ≥ 0.90, and the numbers are successively numbered as peaks 1 to 10. In some embodiments of the present invention, peak 1 is notoginsenoside R 1 , peak 2 is ginsenoside Rg 1 , peak 3 is ginsenoside Re, and peak 9 is ginsenoside Rb 1 , and peak 10 is ginsenoside Rd.

[0032] In some embodiments of the present invention, with peak 2 as the reference peak, the relative retention time of each peak is within ±8% of the first specified value; the first specified value is:

[0033] Peak 1: 0.7867, peak 3: 1.0411, peak 4: 1.2421, peak 5: 1.2885, peak 6: 1.4878, peak 7: 2.2905, peak 8: 2.4972, peak 9: 2.6657, peak 10: 3.2582.

[0034] The "Technical Guidelines for the Study of Characteristic Chromatograms of Traditional Chinese Medicine Preparations (Trial)" issued by the Center for Drug Evaluation of the National Medical Products Administration in February 2024 states that when evaluating using the retention time of characteristic peaks, the relevant chromatographic peaks of the test sample's characteristic chromatogram should be the same as those of the corresponding reference substance chromatogram. The specified value range of the relative retention time of characteristic peaks generally should not exceed ±10%. Based on the research results of multiple batches of Qixuekang Oral Liquid, the present invention uses peak 2 as the reference peak to determine the relative retention time of each peak and limits its relative retention time within the range of ±8% of the first specified value. This range value includes the two endpoint values.

[0035] The second aspect of the present invention provides a fingerprint of Qixuekang Oral Liquid obtained by using the above construction method.

[0036] The third aspect of the present invention provides the application of the fingerprint of Qixuekang Oral Liquid, and the application is as a control fingerprint in the quality control of Qixuekang Oral Liquid.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The present invention constructs the fingerprint of Qixuekang Oral Liquid for the first time, changing the problem in the prior art of using a single component as an index for quality control, which cannot comprehensively and accurately reflect and control the product quality, thereby further ensuring the safety and effectiveness of patients' medication.

[0039] The method of the present invention is simple, easy to operate, and accurate in results, providing strong technical support for product quality control. The method of the present invention improves the separation effect of each component in Qixuekang Oral Liquid, especially ginsenoside Rg 1 , ginsenoside Rb 1 , ginsenoside Re and other key components, which can achieve baseline separation from adjacent peaks. The method of the present invention ensures the stability and reproducibility of the detection results and meets the quality control requirements through comprehensive methodological verification (including specificity, precision, repeatability, solution stability, and durability). Based on the fingerprint technology to establish a quality evaluation method can effectively identify the impact of production links such as packaging changes on the composition of drug components and ensure quality stability. The present invention uses the direct dilution method to prepare the test solution, simplifies the operation steps, reduces the usage amount of organic solvents, reduces the operation difficulty and potential safety hazards, and improves the work efficiency of the laboratory at the same time.

[0040] The present invention reduces the detection cost of enterprises, improves production efficiency, reduces the usage amount of organic solvents, and reduces the operating cost of the laboratory by improving the detection efficiency and simplifying the operation process. The present invention helps to improve the level of drug quality control and ensure the safety of patients' medication. Description of the Drawings

[0041] Figure 1 It is the sample chromatogram before the optimization of the gradient elution program in Example 1;

[0042] Figure 2 It is the sample chromatogram after the optimization of the gradient elution program in Example 1;

[0043] Figure 3 It is the sample chromatogram using different solvents as diluents;

[0044] Figure 4 It is the sample chromatogram after marking;

[0045] Figure 5 It is the result chart of specificity investigation;

[0046] Figure 6 It is the result chart of precision investigation;

[0047] Figure 7 It is the result chart of repeatability investigation;

[0048] Figure 8 It is the result chart of solution stability investigation;

[0049] Figure 9 It is the synthesized control fingerprint. Detailed Embodiments

[0050] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. For those not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0051] The high-performance liquid chromatograph used in the present invention is Agilent 1260 produced by Agilent Technologies, USA.

[0052] Example 1

[0053] This example discloses the methodological study of the fingerprint of the present invention.

[0054] Chromatographic conditions and system suitability test: Chromatographic column: SB-C18, 250 mm × 4.6 mm, 5 μm; acetonitrile as mobile phase A and water as mobile phase B, gradient elution was carried out according to the provisions in Table 1; flow rate was 1.2 ml / min; detection wavelength was 203 nm; column temperature was 20 °C.

[0055] Table 1

[0056]

[0057] Preparation of test solution: Accurately measure 10 ml of Qixuekang Oral Liquid, shake and extract 4 times with water-saturated n-butanol (20 ml, 20 ml, 15 ml, 15 ml), combine the n-butanol extracts, wash with ammonia test solution 2 times, 20 ml each time, discard the ammonia test solution, recover the solvent of the n-butanol solution to dryness, dissolve the residue with methanol and transfer it to a 10 ml volumetric flask, add methanol to the scale, shake well, filter, and take the continuous filtrate, that is, obtain.

[0058] Precisely absorb 10 μl of the test solution, inject it into the liquid chromatograph for determination, and the results are as shown in the appendix Figure 1 It shows that the chromatographic main peaks have not achieved effective separation.

[0059] Optimize the gradient program and increase the post-run time at the same time. The specific gradient elution program is shown in the following table:

[0060] Table 2

[0061]

[0062] Precisely absorb 10 μl of the test solution, inject it into the liquid chromatograph, and carry out determination using the gradient elution program shown in Table 2. The results are as shown in the appendix Figure 2 It shows that each chromatographic peak has basically achieved effective separation.

[0063] Example 2

[0064] This example discloses the investigation of the preparation method of the test solution of the present invention.

[0065] Qixuekang Oral Liquid is a liquid preparation. In order to retain the chromatographic peaks of each component as much as possible, the preparation method of the test solution was optimized, and the direct dilution method was proposed to prepare the test solution. Instrumental analysis was carried out after dilution with water, anhydrous methanol, and 50% methanol respectively.

[0066] The specific preparation method of the test solution is as follows: Take 1 ml of Qixuekang Oral Liquid and place it in a 10-ml volumetric flask. Dilute it to the scale with water, anhydrous methanol, and 50% methanol respectively, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0067] Precisely pipette 10 μl of each test solution and inject it into the liquid chromatograph. The gradient elution program shown in Table 2 was used for determination, and the results are as shown in the appendix. Figure 3 The results show that the chromatograms obtained with different diluents are basically the same. In order to reduce the harm to the testers caused by the use of organic solvents and at the same time protect the instrument, 50% methanol was finally selected as the diluent.

[0068] Example 3

[0069] This example discloses the determination test of the information collection time of the present invention.

[0070] The chromatographic conditions are as follows: Octadecylsilane chemically bonded silica gel is used as the filler (chromatographic column: Agilent ZORBAX SB-C18, 250 mm × 4.6 mm, 5 μm); acetonitrile is used as mobile phase A and water is used as mobile phase B, and gradient elution is carried out according to the regulations in the following table; the flow rate is 1.2 ml / min; the detection wavelength is 203 nm; the column temperature is 20 °C. The number of theoretical plates calculated based on the ginsenoside Rg 1 peak should be not less than 4000. By collecting the chromatographic information within 95 min, it can be seen that there is no absorption peak after 85 min, so the chromatographic information within 95 min was collected.

[0071] Table 3 Chromatographic Information Collection Time Gradient Table

[0072]

[0073] Example 4

[0074] This example discloses the investigation test of the attribution of chromatographic peaks of the present invention.

[0075] 1. Preparation of the reference solution: Take ginsenoside Rg 1 reference substance, use 50% methanol as the diluent, dissolve and dilute it to prepare a reference solution containing 0.2 mg of ginsenoside Rg 1 per 1 ml.

[0076] Ginsenoside Rb prepared by the same method 1 Reference substance solution, ginsenoside Re reference substance solution, ginsenoside Rf reference substance solution, ginsenoside Rd reference substance solution, notoginsenoside R 1 Reference substance solution, astragaloside IV reference substance solution, calycosin-7-O-β-D-glucoside reference substance solution, puerarin reference substance solution.

[0077] 2. Preparation method of test solution: Take 1 ml of Qixuekang Oral Liquid and place it in a 10-ml volumetric flask, dilute it to the scale with 50% methanol, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0078] 3. Accurately pipette 10 μl of each reference substance solution and test solution respectively, inject them into the liquid chromatograph, and determine according to the chromatographic conditions in Example 3 to obtain the chromatograms of each reference substance and the test solution respectively. According to the chromatograms of each reference substance, the chromatographic peaks in the test solution chromatogram are assigned and located. Finally, 5 chromatographic peaks are assigned and located: notoginsenoside R 1 (peak No. 1), ginsenoside Rg 1 (peak No. 2), ginsenoside Re (peak No. 3), ginsenoside Rb 1 (peak No. 9), ginsenoside Rd (peak No. 10), among which the peak response value of ginsenoside Rg 1 reference substance is the largest. Finally, ginsenoside Rg 1 reference substance is used as the reference peak. Specifically, as shown in the appendix Figure 4 shown.

[0079] Example 5

[0080] This example discloses the methodological verification of the fingerprint detection method of the present invention. The chromatographic conditions and system suitability test of the fingerprint detection method of the present invention are as follows: Using octadecylsilane chemically bonded silica gel as the filler (chromatographic column: SB-C18, 250 mm × 4.6 mm, 5 μm); the flow rate is 1.2 ml / min; the detection wavelength is 203 nm; the column temperature is 20 °C. The number of theoretical plates calculated based on the peak of ginsenoside Rg 1 should not be less than 4000.

[0081] Mobile phase: Using acetonitrile as mobile phase A and water as mobile phase B, perform gradient elution according to the regulations in the following table.

[0082] Table 4 Mobile phase gradient

[0083]

[0084] Preparation of reference substance solution: Take an appropriate amount of ginsenoside Rg 1 reference substance, accurately weigh it, and dissolve it in methanol to make a solution containing 0.2 mg per 1 ml to obtain the reference substance solution.

[0085] Preparation of the test solution: Take 1 ml of Qixuekang Oral Liquid and place it in a 10-ml volumetric flask. Dilute it to the mark with 50% methanol, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0086] Assay method: Precisely pipette 10 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, conduct the determination, and record the chromatographic peaks within 95 minutes to obtain the results.

[0087] According to the General Principles for Validation of Analytical Methods 9101 in the Chinese Pharmacopoeia (2020 Edition), the analytical method was validated, and the items included: specificity, precision, repeatability, solution stability, and durability.

[0088] 5.1 Specificity investigation

[0089] Preparation of the test solution: Take 1 ml of Qixuekang Oral Liquid and place it in a 10-ml volumetric flask. Dilute it to the mark with 50% methanol, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0090] Take 50% methanol as the blank solution.

[0091] Precisely pipette 10 μl each of the blank solution and the test solution, inject them into the liquid chromatograph, conduct the determination according to the above chromatographic conditions, and record the chromatogram within 95 minutes. If as attached Figure 5 shown. The results indicate that there are no chromatographic peaks in the blank solution at the elution position of the reference substance, and there are no chromatographic peaks at the corresponding elution positions in the test solution, indicating that the blank solution has no interference on the fingerprint, and the specificity is good.

[0092] 5.2 Precision test

[0093] Preparation of the test solution: Take 1 ml of Qixuekang Oral Liquid and place it in a 10-ml volumetric flask. Dilute it to the mark with 50% methanol, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0094] Take an appropriate amount of ginsenoside Rg 1 reference substance, accurately weigh it, and dissolve it in methanol to prepare a solution containing 0.2 mg per 1 ml to obtain the reference solution.

[0095] Precisely pipette 10 μl of the test solution, inject it into the liquid chromatograph, conduct the determination according to the above chromatographic conditions, and record the chromatogram within 95 minutes. Inject the sample continuously for 6 times.

[0096] Precisely pipette 10 μl of the reference solution, inject it into the liquid chromatograph, conduct the determination according to the above chromatographic conditions, and record the chromatogram.

[0097] Taking ginsenoside Rg 1 in peak 2 as the reference substance, calculate the relative retention time and relative peak area of each main chromatographic peak. The results are shown in Table 5, Table 6, and attached Figure 6 as shown.

[0098] Table 5 Relative retention time of Jingmikang Oral Liquid precision test

[0099]

[0100] Table 6 Relative peak area of Jingmikang Oral Liquid precision test

[0101]

[0102] The above results show that the relative retention times of 10 common peaks in 6 injections are stable, with RSDs all less than 5%, and the relative peak areas are relatively stable, with RSDs all less than 5%; indicating that the precision of this instrument is good.

[0103] 5.3 Repeatability test

[0104] Preparation of test solution: Take 6 portions of Jingmikang Oral Liquid from the same batch, each take 1 ml and place it in a 10-ml volumetric flask, dilute and make up to the mark with 50% methanol respectively, shake well, filter, and take the subsequent filtrate to obtain.

[0105] Preparation of reference solution: Take an appropriate amount of reference substance of ginsenoside Rg 1 Accurately weigh an appropriate amount, dissolve it in methanol to make a solution containing 0.2 mg per 1 ml, and obtain.

[0106] Precisely pipette 10 μl of the reference solution of ginsenoside Rg 1 and 10 μl of each test solution, inject them into the liquid chromatograph respectively, and carry out the determination according to the above chromatographic conditions, and record the chromatogram for 95 minutes. Using the 2nd peak of ginsenoside Rg 1 as the reference substance, calculate the relative retention time and relative peak area of each main chromatographic peak. The results are shown in Table 7, Table 8 and Appendix Figure 7 as shown.

[0107] Table 7 Relative retention time of Jingmikang Oral Liquid repeatability test

[0108]

[0109] Table 8 Relative peak area of Jingmikang Oral Liquid repeatability test

[0110]

[0111] The results show that: the relative retention times of 10 common peaks in 6 test solutions are stable, with RSDs all less than 5%, and the relative peak areas are relatively stable, with RSDs all less than 5%; indicating that the repeatability of this method is good.

[0112] 5.4 Investigation on solution stability

[0113] Preparation of the test solution: Take 1 ml of Qixuekang Oral Liquid and place it in a 10-ml volumetric flask. Dilute it to the mark with 50% methanol, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0114] Preparation of the reference solution: Take an appropriate amount of ginsenoside Rg 1 reference substance, accurately weigh it, and dissolve it in methanol to prepare a solution containing 0.2 mg per 1 ml to obtain the reference solution.

[0115] Precisely pipette 10 μl of the ginsenoside Rg 1 reference solution and inject it into the liquid chromatograph, and carry out the determination according to the above chromatographic conditions, and record the chromatogram.

[0116] Precisely pipette 10 μl of the test solution, and inject it for detection according to the above chromatographic conditions at 0 h, 5 h, 17 h, 29 h, 39 h, and 49 h respectively, and record the chromatogram for 95 minutes. Using the ginsenoside Rg 1 at the 2nd peak as the reference substance, calculate the relative retention time and relative peak area of each main chromatographic peak. The results are shown as follows Figure 8 shown.

[0117] Conclusion: Qixuekang Oral Liquid was detected at different time points. The results showed that the relative retention times of 10 common peaks were relatively stable, with RSD values all less than 5%. Among the relative peak areas, only the RSD values of the 4th and 6th peaks were between 6% and 8%, and the RSD values of the relative peak areas of the remaining 8 common peaks were all less than 5%. This indicates that except for the 4th and 6th peaks in the test solution, the remaining common peaks were relatively stable within 49 hours. To better obtain its chromatographic behavior, the test solution should be freshly prepared before use (within 5 hours).

[0118] 5.5 Durability investigation

[0119] 5.5.1 Durability of different instruments

[0120] According to the established fingerprint determination method, use different instruments to detect the same batch of Qixuekang Oral Liquid. Taking the Agilent instrument chromatogram as the reference, calculate the similarity of the chromatograms between different instruments. The results show that the determination results between different instruments are roughly the same, and the fingerprint similarity is greater than 0.95, indicating that different instruments of the high-performance liquid chromatograph have little influence on this fingerprint method. The results are shown in Table 9.

[0121] Table 9 Results of the instrument durability test

[0122]

[0123] 5.5.2 Durability of different chromatographic columns

[0124] According to the established fingerprint determination method, the same batch of Qixuekang Oral Liquid was detected using chromatographic columns with different serial numbers. Taking the chromatogram of the chromatographic column (serial number QCHPLC2111 - 138) as a reference, the similarity was calculated. The results showed that the determination results were roughly the same among different chromatographic columns, and the fingerprint similarity was greater than 0.95, indicating that using chromatographic columns with different serial numbers had little impact on this method. The results are shown in Table 10.

[0125] Table 10 Results of the durability test of chromatographic column batches for Qixuekang Oral Liquid

[0126]

[0127] Conclusion:

[0128] (1) When the same batch of Qixuekang Oral Liquid was detected using different instruments, the fingerprint similarities were 1.000 respectively, all greater than 0.95, indicating that different instruments of high - performance liquid chromatographs had little impact on this fingerprint method.

[0129] (2) When the same batch of Qixuekang Oral Liquid was detected using chromatographic columns with different serial numbers, the fingerprint similarities were 1.000 respectively, all greater than 0.95, indicating that using chromatographic columns with different serial numbers had little impact on this method.

[0130] 5.6 Establishment of the reference chromatogram

[0131] Fifteen batches of Qixuekang Oral Liquid samples (batch numbers are 210171, 210372, 210573, 210774, 211072, 211076, 211174, 211275, 220171, 220272, 220274, 220372, 220374, 220377, 220271) were respectively detected according to the established fingerprint determination method above, and the reference fingerprint chromatogram was generated by fitting using the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints" (version 2012.0) of the Pharmacopoeia Commission of the People's Republic of China, as shown in the appendix Figure 9 as follows.

[0132] The above - mentioned is only the preferred embodiment of the present invention, which is merely illustrative of the present invention and does not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solution of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing a fingerprint of Qixuekang oral liquid, characterized in that: The steps include: Preparation of test solution: Take Qixuekang oral solution and dilute it 5 to 15 times with 40 to 60% methanol; Preparation of reference solution: Take ginsenoside Rg1 reference substance, add methanol to dissolve to prepare a solution containing 0.1~0.4 mg per 1 ml, that is, ginsenoside Rg1 reference solution; prepare 9 reference solutions of ginsenoside Rb1, ginsenoside Re, ginsenoside Rf, ginsenoside Rd, notoginsenoside R1, astragaloside IV, calycosin isoflavone glucoside, and puerarin in the same way; Determination: Accurately aspirate the test solution and the reference solution respectively, inject them into the high performance liquid chromatograph for determination, and obtain the fingerprint of the test sample and the fingerprint of the reference; the chromatographic conditions include: using octadecylsilane bonded silica gel as the filler, acetonitrile as the mobile phase A, water as the mobile phase B, and eluting according to the following gradient: 0-12 min, 19%A, 81%B; 12~32 min, 19% A→23.8%A, 81% B→76.2%B; 32~75 min, 23.8% A→32%A, 76.2% B→68%B; 75~84 min, 32% A→36%A, 68% B→64%B; 84~85 min, 36% A→19%A, 64% B→81%B The flow rate of the mobile phase was 1.0~1.4 ml / min; the detection wavelength was 203 nm; the column temperature was 15~25°C; Collect chromatograms within 95 minutes; Similarity evaluation: The chromatographic peaks in the fingerprint of the test sample were located and attributed according to the reference sample spectrum; the fingerprints of 10 to 20 batches of Qixuekang oral liquid test samples were imported into the Chinese medicine chromatographic fingerprint similarity evaluation system for analysis to obtain the fingerprint of Qixuekang oral liquid.

2. The method for constructing the fingerprint of Qixuekang oral liquid according to claim 1, characterized in that: Take Qixuekang oral solution and dilute it 10 times with 50% methanol to obtain the test solution; Take an appropriate amount of ginsenoside Rg1 reference substance, accurately weigh it, and add methanol to make a solution containing 0.2 mg per 1 ml to obtain the ginsenoside Rg1 reference substance solution. Use the same method to prepare 9 reference substance solutions of ginsenoside Rb1, ginsenoside Re, ginsenoside Rf, ginsenoside Rd, notoginsenoside R1, astragaloside I, calycosin isoflavone glucoside, and puerarin.

3. The method for constructing the fingerprint of Qixuekang oral liquid according to claim 1 or 2, characterized in that: In the similarity evaluation step, 10 common peaks were determined according to similarity ≥ 0.90, and they were numbered 1 to 10.

4. The method for constructing the fingerprint of Qixuekang oral liquid according to claim 3, characterized in that: Peak 1 is notoginsenoside R1, peak 2 is ginsenoside Rg1, peak 3 is ginsenoside Re, peak 9 is ginsenoside Rb1, and peak 10 is ginsenoside Rd.

5. The method for constructing the fingerprint of Qixuekang oral liquid according to claim 4, characterized in that: Taking peak 2 as the reference peak, the relative retention time of each peak is within ±8% of the first specified value, and the first specified value is: Peak 1: 0.7867, Peak 3: 1.0411, Peak 4: 1.2421, Peak 5: 1.2885, Peak 6: 1.4878, Peak 7: 2.2905, Peak 8: 2.4972, Peak 9: 2.6657, Peak 10: 3.2582.

6. A fingerprint spectrum of Qixuekang oral liquid, characterized in that: Obtained by the construction method described in any one of claims 1 to 5.

7. The application of the fingerprint spectrum of Qixuekang oral liquid according to claim 6, characterized in that: Application of reference fingerprint in quality monitoring of Qixuekang oral liquid.

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