Fingerprint spectrum of Qixuekang oral liquid as well as construction method and application of fingerprint spectrum

Abstract

The invention discloses a fingerprint spectrum of Qixuekang oral liquid as well as a construction method and application thereof, and belongs to the technical field of pharmaceutical analysis. The construction method comprises the following steps: preparing a Qixuekang oral liquid test solution; the method comprises the following steps: respectively preparing reference substance solutions of ginsenoside Rg1, Rb1, Re, Rf and Rd, notoginsenoside R1, astragaloside, calycosin-7-glucoside and puerarin; respectively and precisely sucking the test solution and the reference solution and injecting the test solution and the reference solution into a high performance liquid chromatograph for determination to obtain a test fingerprint spectrum and a reference spectrum; performing chromatographic positioning and attribution by adopting a reference substance; and carrying out similarity analysis on the fingerprints of the multiple batches of test products to obtain the fingerprint of the Qixuekang oral liquid. The invention also discloses the fingerprint spectrum of the Qixuekang oral liquid obtained by the method and application of the Qixuekang oral liquid in quality monitoring. According to the invention, the fingerprint spectrum of the Qixuekang oral liquid is constructed for the first time, the product quality can be comprehensively and accurately reflected and controlled, and the medicine safety and effectiveness are guaranteed.

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 the effects of lowering blood lipids, anti-oxidation, anti-anxiety and immune regulation. 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 effects 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. It was inferred that a specific proportion of ginsenosides Rg1, Re, and Rb1, as well as ginsenosides Rb2, Rb3, Re, Rd, Rh2, and Rg3 in Panax notoginseng and notoginseng saponin R1, notoginseng extract, 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 membranaceus The dried root of (Fisch.) Bge. is sweet and slightly warm in nature. It enters the lung and spleen meridians. It has the effects of replenishing qi and raising yang, consolidating the exterior and stopping sweating, promoting diuresis and reducing swelling, promoting fluid and nourishing blood, relieving stagnation and relieving arthritis, expelling toxins and draining pus, and healing sores and promoting tissue regeneration. Flavonoids and saponins are the main active ingredients of Astragalus. In the Chinese Pharmacopoeia (2020 edition, Volume 1), the content of Astragaloside I and Calycosin glucoside is determined as the content of Astragalus.

[0005] Panax ginseng Panax ginseng The dried root and rhizome of CA Mey. are sweet, slightly bitter, slightly warm in nature, and are good for the spleen, lung, heart, and kidney meridians; they have the effects of greatly replenishing vital energy, restoring pulse and strengthening deficiency, replenishing spleen and lung, promoting body fluid and nourishing blood, and calming the mind and improving intelligence. The literature "Research Progress on Ginseng Regulating Immune System Diseases and Prediction and Analysis of Quality Markers" (Xu Hui et al., Chinese Journal of Traditional Chinese Medicine, 1-20 [2025-03-10]. http: / / kns.cnki.net / kcms / detail / 21.1546.R.20240805.1506.016.html.) summarizes the chemical components and pharmacological effects of ginseng in regulating immune function, and based on the principle of quality markers, predictive analysis is conducted from the aspects of plant affinity, chemical component effectiveness, uniqueness, and compound compatibility. The major components are ginsenosides, polysaccharides, proteins, and volatile oils as quality markers for ginseng regulating immune function. Ginsenoside Rg l 、Re、Rb l , Rg3, Rh1, Rh2, Rd, CK, Ro, Rg5, ginseng D-glucose, galactose, arabinose, and ginsenoside were selected as alternative substances.

[0006] Traditional Chinese medicine fingerprint is an overall quality evaluation method that systematically characterizes the complex components of traditional Chinese medicine through modern analytical technology. Its core is to obtain spectra or chromatograms that can reflect the characteristics of multiple components of traditional Chinese medicine through specific analytical means, so as to achieve overall identification and quality control of the "chemical component group" of traditional Chinese medicine. The establishment of traditional Chinese medicine fingerprint detection method has broken through the limitations of single component determination, especially in solving key problems such as complex component characterization, batch stability control, and authenticity identification. However, there is no research on the fingerprint of Qixuekang oral liquid in the prior art. Therefore, providing a fingerprint detection method for Qixuekang oral liquid, which can locate key components and is simple to operate, has become a problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0007] One of the purposes of the present invention is to provide a method for constructing a fingerprint of Qixuekang oral liquid, which can efficiently separate and accurately detect multiple components in Qixuekang oral liquid, and has simple operation and low safety risks.

[0008] The second object of the present invention is to provide a fingerprint of Qixuekang oral liquid, which is obtained by the above-mentioned construction method.

[0009] The third object of the present invention is to provide an application of the fingerprint spectrum of the Qixuekang oral liquid.

[0010] To achieve the above purpose, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides a method for constructing a fingerprint of Qixuekang oral liquid, the method comprising the following steps: Preparation of test solution: Take Qixuekang oral solution and dilute it with 40-60% methanol; Preparation of reference solution: take ginsenoside Rg1 reference substance, add methanol to prepare 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, 18~20%A, 80~82%B; 12~32 min, 18~20%A→23~25%A, 80~82%B→75~77%B; 32~75 min, 23~25%A→30~34%A, 75~77%B→66~70%B; 75~84 min, 30~34%A→34~38%A, 66~70%B→62~66%B; 84~85 min, 34~38%A→18~20%A, 62~66%B→80~82%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; 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.

[0011] In some embodiments of the present invention, the gradient elution is as follows: 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.

[0012] In some embodiments of the present invention, Qixuekang oral liquid is taken and diluted 5 to 15 times with 40 to 60% methanol to obtain a test solution; Take an appropriate amount of ginsenoside Rg1 reference substance, accurately weigh it, add methanol to make a solution containing 0.1-0.4 mg per 1 ml, and obtain the ginsenoside Rg1 reference substance solution; prepare 9 reference substance solutions of ginsenoside Rb1, ginsenoside Re, ginsenoside Rf, ginsenoside Rd, notoginseng saponin R1, astragaloside I, calycosin isoflavone glucoside, and puerarin in the same way. In some embodiments of the present invention, take Qixuekang oral liquid, add 50% methanol to dilute it 10 times, and 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.

[0013] In some embodiments of the present invention, the chromatogram is collected within 95 minutes; in the similarity evaluation step, 10 common peaks are determined according to the similarity ≥ 0.90, and are numbered as peaks 1 to 10. In some embodiments of the present invention, 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.

[0014] In some embodiments of the present invention, with peak No. 2 as the reference peak, the relative retention time of each peak is within ±8% of a first specified value; 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.

[0015] The "Technical Guidelines for the Study of Characteristic Spectra of Traditional Chinese Medicine Preparations (Trial)" issued by the Drug Review Center of the National Medical Products Administration in February 2024 pointed out that when evaluating by characteristic peak retention time, the relevant chromatographic peaks of the characteristic spectrum of the test sample should be the same as the retention time of the corresponding reference chromatographic peak. The specified value range of the relative retention time of the characteristic peak should generally not exceed ±10%. Based on the research results of multiple batches of Qixuekang oral liquid, the present invention uses Peak No. 2 as the reference peak to determine the relative retention time of each peak, and limits its relative retention time to within the first specified value range of ±8%. The range value includes two endpoint values.

[0016] The second aspect of the present invention provides a fingerprint of Qixuekang oral liquid, which is obtained by the above-mentioned construction method.

[0017] The third aspect of the present invention provides an application of the fingerprint spectrum of Qixuekang oral liquid, which is an application as a control fingerprint spectrum in the quality monitoring of Qixuekang oral liquid.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention constructs the fingerprint spectrum of Qixuekang oral liquid for the first time, which changes the problem of using a single ingredient as an indicator for quality control in the prior art and cannot fully and accurately reflect and control product quality, thereby further ensuring the safety and effectiveness of patients' medication.

[0019] The method of the present invention is simple, easy to operate, and has accurate 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 key components such as ginsenoside Rg1, ginsenoside Rb1, and ginsenoside Re, which can reach baseline separation with adjacent peaks. The method of the present invention ensures the stability and reproducibility of the test results through comprehensive methodological verification (including specificity, precision, repeatability, solution stability, and durability), and meets the quality control requirements. The quality evaluation method is established based on fingerprint technology, which can effectively identify the impact of production links such as packaging changes on the composition of drug ingredients and ensure quality stability. The present invention adopts a direct dilution method to prepare the test solution, simplifies the operation steps, reduces the use of organic solvents, reduces the difficulty of operation and safety hazards, and improves the work efficiency of the laboratory.

[0020] The present invention reduces the testing cost of enterprises and improves production efficiency by improving detection efficiency and simplifying operation procedures, while reducing the amount of organic solvents used and reducing laboratory operating costs. The present invention helps to improve the quality control level of drugs and ensure the safety of patients' medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The sample chromatogram before the gradient elution program optimization in Example 1; Figure 2 The sample chromatogram after the gradient elution program is optimized in Example 1; Figure 3 The chromatograms of samples using different solvents as diluents; Figure 4 is the chromatogram of the labeled sample; Figure 5 This is the result diagram of the specificity investigation; Figure 6 This is the result diagram of precision investigation; Figure 7This is the result diagram of repeatability investigation; Figure 8 This is the result of the solution stability investigation; Fig. 9 This is the synthetic control fingerprint. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0023] The high performance liquid chromatograph used in the present invention is Agilent 1260 produced by Agilent Technologies of the United States.

[0024] Example 1 This embodiment discloses the methodological study of the fingerprint of the present invention.

[0025] Chromatographic conditions and system suitability test: Chromatographic column: SB-C18, 250mm×4.6mm, 5μm; acetonitrile as mobile phase A, water as mobile phase B, gradient elution as specified in Table 1; flow rate of 1.2ml / min; detection wavelength of 203nm; column temperature of 20℃.

[0026] Table 1

[0027] Preparation of test solution: Accurately measure 10 ml of Qixuekang oral liquid, extract it with water-saturated n-butanol by shaking for 4 times (20 ml, 20 ml, 15 ml, 15 ml), combine the n-butanol extracts, wash it with ammonia test solution twice, 20 ml each time, discard the ammonia test solution, recover the solvent with 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 filtrate to obtain.

[0028] Accurately pipette 10 μl of the test solution and inject it into the liquid chromatograph for determination. The results are as shown in the attached figure. Figure 1 The results show that the main peak of the chromatogram has not been effectively separated.

[0029] The gradient program was optimized and the post-run time was increased. The specific gradient elution program is shown in the following table: Table 2

[0030] Accurately pipette 10 μl of the test solution, inject it into the liquid chromatograph, and measure it using the gradient elution program shown in Table 2. The results are shown in the attached figure. Figure 2The results show that each chromatographic peak is basically effectively separated.

[0031] Example 2 This example discloses an investigation of the method for preparing the test solution of the present invention.

[0032] Qixuekang oral solution 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 performed after dilution with water, anhydrous methanol, and 50% methanol.

[0033] The specific method for preparing 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 filtrate to obtain the solution.

[0034] Accurately pipette 10 μl of each test solution, inject it into the liquid chromatograph, and measure it using the gradient elution program shown in Table 2. The results are shown in the attached figure. Figure 3 The results show that the chromatograms obtained using different diluents are basically the same. In order to reduce the harm of using organic solvents to testers and protect the instrument at the same time, 50% methanol was finally selected as the diluent.

[0035] Example 3 This embodiment discloses an experiment for determining the information collection time according to the present invention.

[0036] The chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as filler (chromatographic column: Agilent ZORBAX SB-C18, 250mm×4.6mm, 5μm); acetonitrile is used as mobile phase A, water is used as mobile phase B, and gradient elution is performed as specified in the following table; flow rate is 1.2ml / min; detection wavelength is 203nm; column temperature is 20℃. The number of theoretical plates calculated based on the ginsenoside Rg1 peak should not be less than 4000. It can be seen from the collection of chromatographic information within 95min that there is no absorption peak after 85min, so the chromatographic information within 95min is collected.

[0037] Table 3 Chromatographic information acquisition time gradient table

[0038] Example 4 This embodiment discloses an investigation test of the attribution of chromatographic peaks of the present invention.

[0039] 1. Preparation of reference solution: Take ginsenoside Rg1 reference substance, dissolve and dilute it with 50% methanol as diluent to prepare a reference solution containing 0.2 mg of ginsenoside Rg1 per 1 ml.

[0040] The same method was used to prepare ginsenoside Rb1 reference solution, ginsenoside Re reference solution, ginsenoside Rf reference solution, ginsenoside Rd reference solution, notoginsenoside R1 reference solution, astragaloside IV reference solution, calycosin isoflavone glucoside reference solution, and puerarin reference solution.

[0041] 2. The test solution is prepared as follows: take 1 ml of Qixuekang oral solution and place it in a 10 ml volumetric flask, dilute it to the mark with 50% methanol, shake well, filter, and take the filtrate to obtain the solution.

[0042] 3. Accurately pipette 10 μl of each reference solution and test solution, inject into the liquid chromatograph, and measure according to the chromatographic conditions of Example 3 to obtain the chromatograms of each reference solution and the test solution, respectively. According to the chromatograms of each reference solution, the chromatographic peaks in the chromatogram of the test solution were assigned and located, and finally 5 chromatographic peaks were assigned and located: Panax notoginseng saponin R1 (peak 1), ginsenoside Rg1 (peak 2), ginsenoside Re (peak 3), ginsenoside Rb1 (peak 9), ginsenoside Rd (peak 10), among which the ginsenoside Rg1 reference solution had the largest peak response value, and finally the ginsenoside Rg1 reference solution was used as the reference peak. For details, see the attached Figure 4 shown.

[0043] Example 5 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: octadecylsilane bonded silica gel is used as the filler (chromatographic column: SB-C18, 250mm×4.6mm, 5μm); the flow rate is 1.2ml / min; the detection wavelength is 203nm; the column temperature is 20℃. The theoretical plate number calculated based on the ginsenoside Rg1 peak should be no less than 4000.

[0044] Mobile phase: Use acetonitrile as mobile phase A and water as mobile phase B, and perform gradient elution as specified in the table below. Table 4 Mobile phase gradient

[0045] Preparation of reference solution: Take an appropriate amount of ginsenoside Rg1 reference substance, weigh it accurately, and add methanol to make a solution containing 0.2 mg per 1 ml.

[0046] Preparation of test solution: Take 1 ml of Qixuekang oral liquid and place it in a 10 ml volumetric flask, add 50% methanol to dilute to the scale, shake well, filter, and take the filtrate to obtain the solution.

[0047] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, measure, and record the chromatographic peak within 95 minutes.

[0048] According to the guidelines for validation of analytical methods in General Chapter 9101 of the "Chinese Pharmacopoeia" (2020 edition), the analytical method is validated, and the items include: specificity, precision, repeatability, solution stability, and durability.

[0049] 5.1 Specificity inspection Preparation of 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 filtrate to obtain the solution.

[0050] Take another 50% methanol as the blank solution.

[0051] Accurately pipette 10 μl of blank solution and test solution respectively, inject into liquid chromatograph, measure according to the above chromatographic conditions, and record the chromatogram for 95 minutes. Figure 5 The results show that the blank solution has no chromatographic peak at the peak position of the reference solution, and the test solution has no chromatographic peak at the corresponding peak position, indicating that the blank solution has no interference with the fingerprint spectrum and has good specificity.

[0052] 5.2 Precision test Preparation of 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 filtrate to obtain the solution.

[0053] Take an appropriate amount of ginsenoside Rg1 reference substance, weigh it accurately, and add methanol to make a solution containing 0.2 mg per 1 ml to obtain the reference substance solution.

[0054] Accurately pipette 10 μl of the test solution and inject it into the liquid chromatograph. Perform the measurement according to the above chromatographic conditions, record the chromatogram for 95 minutes, and inject the sample 6 times continuously.

[0055] Accurately aspirate 10 μl of the reference solution, inject it into the liquid chromatograph, measure according to the above chromatographic conditions, and record the chromatogram.

[0056] Taking ginsenoside Rg1, peak 2, as the reference, the relative retention time and relative peak area of ​​each main chromatographic peak were calculated. The results are shown in Table 5, Table 6 and Appendix. Figure 6 shown.

[0057] Table 5 Relative retention time of precision test of Qixuekang oral liquid

[0058] Table 6 Relative peak area of ​​precision test of Qixuekang oral liquid

[0059] The above results show that the relative retention times of the 10 common peaks in 6 injections are stable, with RSDs less than 5%, and the relative peak areas are relatively stable, with RSDs less than 5%, indicating that the instrument has good precision.

[0060] 5.3 Repeatability test Preparation of test solution: Take 6 portions of Qixuekang oral liquid from the same batch, take 1 ml of each portion and place it in a 10 ml volumetric flask, dilute to the mark with 50% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0061] Preparation of reference solution: Take an appropriate amount of ginsenoside Rg1 reference substance, weigh it accurately, and add methanol to make a solution containing 0.2 mg per 1 ml.

[0062] Accurately pipette 10 μl of ginsenoside Rg1 reference solution and 10 μl of each test solution into the liquid chromatograph, respectively, and measure according to the above chromatographic conditions, and record the chromatogram for 95 minutes. Using the No. 2 peak ginsenoside Rg1 as the reference, 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 shown.

[0063] Table 7 Relative retention time of Qixuekang oral liquid repeatability test

[0064] Table 8 Relative peak area of ​​Qixuekang oral liquid repeatability test

[0065] The results showed that the relative retention times of the 10 common peaks in the 6 test solutions were stable, with RSDs less than 5%, and the relative peak areas were relatively stable, with RSDs less than 5%, indicating that the method had good repeatability.

[0066] 5.4 Solution stability study Preparation of 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 filtrate to obtain the solution.

[0067] Preparation of reference solution: Take an appropriate amount of ginsenoside Rg1 reference substance, weigh it accurately, and add methanol to make a solution containing 0.2 mg per 1 ml.

[0068] Accurately pipette 10 μl of ginsenoside Rg1 reference solution and inject it into the liquid chromatograph, perform the determination according to the above chromatographic conditions, and record the chromatogram.

[0069] Accurately pipette 10 μl of the test solution, and inject and test according to the above chromatographic conditions at 0h, 5h, 17h, 29h, 39h, and 49h, and record the chromatogram for 95 minutes. Using the No. 2 peak ginsenoside Rg1 as the reference, calculate the relative retention time and relative peak area of ​​each main chromatographic peak. The results are shown in the attached figure. Figure 8 shown.

[0070] Conclusion: Qixuekang oral liquid was tested at different time points, and the results showed that the relative retention time of the 10 common peaks was relatively stable, with RSDs less than 5%. Only the RSD values ​​of peaks 4 and 6 were between 6% and 8% for the relative peak areas, and the RSDs of the relative peak areas of the other 8 common peaks were all less than 5%, indicating that except for peaks 4 and 6, the other common peaks of the test solution were relatively stable within 49 hours. In order to better obtain its chromatographic behavior, the test solution should be prepared fresh (within 5 hours).

[0071] 5.5 Durability test 5.5.1 Durability of different instruments According to the proposed fingerprint determination method, different instruments were used to detect the same batch of Qixuekang oral liquid, and the Agilent instrument spectrum was used as a reference to calculate the similarity of the spectrum between different instruments. The results showed that the determination results between different instruments were roughly the same, and the fingerprint spectrum similarity was greater than 0.95, indicating that the different instruments of high performance liquid chromatography had little effect on this fingerprint method. The results are shown in Table 9.

[0072] Table 9 Instrument durability test results

[0073] 5.5.2 Durability of different chromatographic columns According to the proposed fingerprint determination method, different serial number chromatographic columns were used to detect the same batch of Qixuekang oral liquid, and the chromatographic column (serial number QCHPLC2111-138) was used as a reference to calculate the similarity. The results showed that the determination results between different chromatographic columns were roughly the same, and the fingerprint similarity was greater than 0.95, indicating that the use of chromatographic columns with different serial numbers had little effect on this method. The results are shown in Table 10.

[0074] Table 10 Results of the batch durability test of the chromatographic column for Qixuekang oral liquid

[0075] in conclusion: (1) The fingerprint similarities of the same batch of Qixuekang oral liquid tested by different instruments were 1.000 and both were greater than 0.95, indicating that the different instruments of HPLC had little effect on the fingerprint method.

[0076] (2) The same batch of Qixuekang oral liquid was tested using chromatographic columns with different serial numbers. The similarities of the fingerprints were 1.000 and both were greater than 0.95, indicating that the use of chromatographic columns with different serial numbers had little effect on this method.

[0077] 5.6 Reference map establishment 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 tested according to the fingerprint determination method established above, and the reference fingerprint was generated by fitting the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" (2012.0 version) of the National Pharmacopoeia Committee, as shown in the attached figure. Fig. 9 shown.

[0078] The above description is only a 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 modifications and improvements made by ordinary technicians in this field to the technical solution of the present invention should fall within the scope of protection 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 with 40-60% methanol; Preparation of reference solution: take ginsenoside Rg1 reference substance, add methanol to dissolve to prepare 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, 18~20%A, 80~82%B; 12~32 min, 18~20%A→23~25%A, 80~82%B→75~77%B; 32~75 min, 23~25%A→30~34%A, 75~77%B→66~70%B; 75~84 min, 30~34%A→34~38%A, 66~70%B→62~66%B; 84~85 min, 34~38%A→18~20%A, 62~66%B→80~82%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; 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: The gradient elution was as follows: 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.

3. The method for constructing the fingerprint of Qixuekang oral liquid according to claim 1, characterized in that: Take Qixuekang oral solution and dilute it 5 to 15 times with 40 to 60% 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 0.1~0.4 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.

4. 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.

5. The method for constructing the fingerprint of Qixuekang oral liquid according to any one of claims 1 to 4, characterized in that: The chromatogram was collected within 95 minutes; in the similarity evaluation step, 10 common peaks were determined according to the similarity ≥ 0.90, and they were numbered 1 to 10.

6. The method for constructing the fingerprint of Qixuekang oral liquid according to claim 5, 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.

7. The method for constructing the fingerprint of Qixuekang oral liquid according to claim 6, 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.

8. A fingerprint spectrum of Qixuekang oral liquid, characterized in that: Obtained by the construction method described in any one of claims 1 to 7.

9. The application of the fingerprint spectrum of Qixuekang oral liquid according to claim 8, characterized in that: Application of reference fingerprint in quality monitoring of Qixuekang oral liquid.

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