Construction method of gushengbao tablet fingerprint spectrum, fingerprint spectrum and application thereof

Abstract

The present application belongs to the technical field of Chinese medicine detection, and particularly relates to a construction method of a Gushenbao tablet fingerprint spectrum, the fingerprint spectrum and application thereof. The Gushenbao tablet is detected by using high performance liquid chromatography and mass spectrometry. By systematically and reasonably controlling chromatographic conditions, 39 common peaks can be simultaneously identified and analyzed, and 15 characteristic components can be identified, which are used to represent 6 medicinal materials, i.e. Herba Epimedii, Dipsacus asper, Radix Paeoniae Rubra, Rhizoma Chuanxiong, Rhizoma Anemarrhenae and Rhizoma Sparganii in the Gushenbao tablet. The established fingerprint spectrum covers main effective components in the Gushenbao tablet, greatly improves the detection precision of the chemical components in the Gushenbao tablet, and comprehensively reflects the types and quantities of the chemical components, which is beneficial to comprehensively control the product quality and help to ensure the quality consistency of each batch of products.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a construction method of a Gushangbao tablet fingerprint, the fingerprint and application thereof. BACKGROUND

[0002] Gushangbao tablet, with a State Drug Supervision Department Number: Z20150020, can effectively treat bone fracture, bone pain, osteoarthritis caused by bone flaccidity and prevent menopausal osteoporosis. The prescription composition comprises nine medicinal materials, i.e. Herba Epimedii, Rhizoma Dispori, Radix Paeoniae Rubra, Rhizoma Chuanxiong, Rhizoma Anemarrhenae, Rhizoma Curcumae, Rhizoma Sparganii, Radix Rehmanniae and Oyster Shell Calcined. Among them, Herba Epimedii, Radix Rehmanniae and Rhizoma Dispori have significant effects of promoting bone formation and inhibiting bone destruction, and can strengthen calcium absorption, thereby promoting bone health, in addition to improving body immunity and enhancing bone density to prevent osteoporosis. Rhizoma Chuanxiong, Rhizoma Curcumae, Radix Paeoniae Rubra and Rhizoma Sparganii are helpful to improve the microcirculation of the body, promote blood circulation, eliminate inflammatory reactions caused by osteoporosis, thereby relieving bone pain and discomfort. Oyster Shell Calcined as a natural biological calcium source can supplement calcium and effectively promote mineral supplement of the skeleton to enhance bone strength. Rhizoma Anemarrhenae can further improve bone health by inhibiting the synthesis and release of prostaglandin E to reduce inflammatory reactions. The combined use of the above-mentioned medicinal materials has the effects of activating blood and relieving pain, promoting blood circulation and removing blood stasis, and provides comprehensive protection for the skeleton to help maintain the health and stability of the skeleton.

[0003] At present, there are relatively few studies on Gushangbao tablet in China, especially the research on component analysis and quality control is not sufficient. The existing detection methods mainly focus on the detection of single components such as Herba Epimedii, which lacks comprehensiveness and is difficult to comprehensively evaluate the overall chemical component characteristics of Gushangbao tablet. Such single component detection method cannot effectively reflect the combined effect of Gushangbao tablet and cannot provide sufficient data support for its quality control. SUMMARY

[0004] In order to solve the above technical problems, the application provides a construction method of a Gushangbao tablet fingerprint, the fingerprint and application thereof, which can comprehensively detect various chemical components in Gushangbao tablet, and further draw a fingerprint with uniqueness and identifiability. Through the fingerprint, the chemical component characteristics of Gushangbao tablet can be intuitively understood, and the quality consistency of products of different batches and different sources can be analyzed. This not only provides real-time monitoring means for the production process of Gushangbao tablet, but also provides guarantee for the stability and consistency of drug quality.

[0005] The application is implemented by the following technical solutions.

[0006] The application provides a construction method of a Gushangbao tablet fingerprint, comprising the following steps:

[0007] Different batches of Gushenbao tablets are removed from coating, ground into powder, and the powder is added into methanol for ultrasonic extraction, filtration, and continuous filtration to prepare different batches of test sample solutions.

[0008] Gallic acid, protocatechuic acid, catechin, loganic acid, mangiferin, paeonolactone glycoside, paeonoside, vanillin, p-coumaric acid, ferulic acid, isochlorogenic acid B, damianol B, damianol C, icariin and baohuoglycoside I are used as reference substances, and are dissolved in methanol to prepare reference substance solutions of corresponding single components.

[0009] The test sample solutions and the reference substance solutions are injected into a high performance liquid chromatograph for analysis under the same high performance liquid chromatography conditions, and the chromatograms of the test sample solutions and the reference substance solutions are recorded.

[0010] The chromatograms of the test sample solutions are subjected to similarity analysis to confirm the reliability of the results.

[0011] One batch of test sample solution is subjected to high resolution mass spectrometry analysis to obtain a total ion current diagram, and the total ion current diagram is imported into Xcalibur software for data analysis according to the peak conditions of the chromatogram of the test sample solution to obtain mass spectrometry result diagrams of the chemical components.

[0012] According to the total ion current diagram and the mass spectrometry result diagrams of the chemical components, and in combination with the chromatograms of the reference substance solutions, the chemical components of each peak in the chromatogram of the test sample solution are determined to obtain a fingerprint spectrum.

[0013] The present application can simultaneously identify and analyze 39 common peaks and identify 15 characteristic components in the peaks by systematically and reasonably controlling the high performance liquid chromatography conditions, and the 6 medicinal materials of Herba Epimedii, Radix Curculiginis, Radix Paeoniae Rubra, Rhizoma Chuanxiong, Radix Anemarrhenae and Rhizoma Sparganii in Gushenbao tablets are characterized.

[0014] Preferably, in the test sample solution, the ratio of the powder and methanol is 1.5g:20mL, the volume fraction of methanol is ≥99.5%, the ultrasonic power is 250W, the frequency is 40kHz, the ultrasonic extraction time is 30min, and the continuous filtrate is filtered through a 0.45µm microporous filter membrane.

[0015] When preparing the control solution of the corresponding single component, the volume fraction of methanol is ≥99.5%, the dosage ratio of gallic acid and methanol is 0.57 mg:1 mL, the dosage ratio of protocatechuic acid and methanol is 0.63 mg:1 mL, the dosage ratio of catechin and methanol is 0.47 mg:1 mL, the dosage ratio of loganic acid and methanol is 0.45 mg:1 mL, the dosage ratio of mangiferin and methanol is 0.65 mg:1 mL, the dosage ratio of paeonolactone and methanol is 0.60 mg:1 mL, the dosage ratio of paeoniflorin and methanol is 0.62 mg:1 mL, the dosage ratio of vanillin and methanol is 0.50 mg:1 mL, the dosage ratio of p-coumaric acid and methanol is 0.60 mg:1 mL, the dosage ratio of ferulic acid and methanol is 0.63 mg:1 mL, the dosage ratio of isochlorogenic acid B and methanol is 0.51 mg:1 mL, the dosage ratio of epimedin B and methanol is 0.46 mg:1 mL, the dosage ratio of epimedin C and methanol is 0.45 mg:1 mL, and the dosage ratio of icariin and methanol is 0.46 mg:1 mL, and the dosage ratio of baohuoside I and methanol is 0.52 mg:1 mL.

[0016] Preferably, the high performance liquid chromatography conditions are as follows: a chromatographic column: Shim-pack VP-ODS, column length 250 mm, inner diameter 4.6 mm, and filler particle size 5 μm; a detector: a diode array detector, detection wavelength 210 nm; volume flow rate: 1.0 mL / min; column temperature: 30 °C; sample injection amount: 20 μL; and mobile phase: phase A is acetonitrile, and phase B is 0.1% (volume fraction) phosphoric acid aqueous solution, gradient elution is performed, and the elution program is as follows: 0 min-10 min, 95%-88% B phase; 10 min-26 min, 88%-79% B phase; 26 min-32 min, 79%-76% B phase; 32 min-36 min, 76%-74% B phase; 36 min-40 min, 74%-72% B phase; 40 min-45 min, 72%-70% B phase; 45 min-51 min, 70%-66% B phase; and 51 min-77 min, 66%-50% B phase.

[0017] Preferably, similarity analysis is performed on the chromatograms of different batches of test solution, and the specific method is as follows: the chromatograms of different batches of test solution are introduced into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system; chromatographic peaks existing in the chromatograms of different batches of test solution are selected as common peaks, an average value calculation method is used to generate a control fingerprint, similarity analysis is performed on the chromatograms of different batches of test solution and the generated control fingerprint, a similarity result table between the chromatograms of different batches of test solution and the control fingerprint is obtained, and the table is exported; and the reliability of the result is confirmed according to the similarity result table and the chromatogram of the test solution.

[0018] Preferably, the high resolution mass spectrometry analysis conditions are: electrospray ionization, spray voltage 3500V, sheath gas flow rate 40arb, auxiliary gas flow rate 10arb, capillary temperature 300 DEG C, auxiliary gas temperature 300 DEG C, scan mode is full scan mode, mass-to-charge ratio scan range m / z is 100~1500.

[0019] The application provides a Gushenbao tablet fingerprint spectrum, which is obtained by the construction method.

[0020] By comparing with the chromatograms of 15 reference substance solutions of Danggui-shaonain B and Danggui-shaonain C, the chemical components corresponding to the 15 characteristic peaks in the Gushenbao tablet fingerprint spectrum are analyzed, and the chemical components are identified as follows: gallic acid is derived from red peony root, protocatechuic acid is derived from rhizoma sparganii, catechin is derived from red peony root, strychnine glycoside acid is derived from dogwood, mangiferin is derived from anemarrhena asphodeloides, peony lactone glycoside is derived from red peony root, paeoniflorin is derived from red peony root, vanillin is derived from rhizoma sparganii, p-coumaric acid is derived from rhizoma sparganii, asafeotoic acid is derived from chuanxiong, Danggui-shaonain B is derived from herba epimedii, Danggui-shaonain C is derived from herba epimedii, icariin is derived from herba epimedii, and baohuoglycoside I is derived from herba epimedii.

[0021] The application provides application of the Gushenbao tablet fingerprint spectrum in quality detection, quality evaluation or quality control of the Gushenbao tablet and same formula medicines.

[0022] Compared with the prior art, the application has the following beneficial effects:

[0023] The present application adopts high performance liquid chromatography and mass spectrometry technology, namely HPLC-MS combined technology, to detect Gushangbao tablets, takes gallic acid, protocatechuic acid, catechin, strychnine acid, mangiferin, paeonolactone glycoside, paeoniflorin, vanillin, p-coumaric acid, ferulic acid, isochlorogenic acid B, damianol B, damianol C, icariin and baohuoglycoside I as reference substances, and can identify and analyze 39 common peaks and identify 15 characteristic components in the common peaks through systematic and reasonable control of high performance liquid chromatography conditions, so as to represent 6 medicinal materials, i.e., Herba Epimedii, Dipsacus asper, Radix Paeoniae Rubra, Rhizoma Chuanxiong, Rhizoma Anemarrhenae and Rhizoma Sparganii, in Gushangbao tablets. The established fingerprint spectrum covers main effective components in Gushangbao tablets, greatly improves the detection precision of chemical components in Gushangbao tablets, and comprehensively reflects the types and quantities of chemical components, which is beneficial to comprehensively control product quality and help to ensure the quality consistency of each batch of products.

[0024] The construction method of the fingerprint spectrum of the present application has significant advantages in precision, reproducibility and accuracy. Through optimization of chromatography conditions, the detection process is not only efficient but also has strong reproducibility, which guarantees the reliability of each experiment, provides accurate basis for quality control of Gushangbao tablets, can effectively monitor the quality consistency of different batches of products, and ensures the stability of efficacy and safety. Compared with traditional detection methods, the HPLC-MS combined technology of the present application greatly improves the sensitivity and resolution of detection, can identify trace components, avoids the problems of component loss and inaccurate quantification in traditional methods. The method can quickly and comprehensively analyze multiple characteristic components, improves the quality control level of key medicinal materials such as Herba Epimedii, Dipsacus asper and Radix Paeoniae Rubra, and thus ensures the stability of raw material quality of Gushangbao tablets, providing protection for the efficacy and safety of drugs. In addition, the detection method of the present application is simple in operation, good in reproducibility, and accurate and reliable. At least 15 components are confirmed at the same chromatography condition at one time, which not only saves time but also greatly reduces the testing cost and significantly improves the detection efficiency. Therefore, the present application provides a scientific, fast and efficient solution, and provides strong technical support for traditional Chinese medicine quality management and product stability. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The fingerprint spectrum of Gushangbao tablets constructed by the present application.

[0026] Figure 2 The chromatogram obtained by optimizing the extraction method in the preparation process of the test solution of the present application.

[0027] Figure 3 The chromatogram obtained by optimizing the extraction solvent in the preparation process of the test solution of the present application.

[0028] Figure 4 The chromatogram obtained by optimizing the extraction time in the preparation process of the test solution of the present application.

[0029] Figure 5 Full wavelength 3D scan for the test product of the invention - diode array detector.

[0030] Figure 6 Chromatogram obtained by optimizing the detection wavelength in the chromatographic conditions of the invention.

[0031] Figure 7 Chromatogram obtained by optimizing the column temperature in the chromatographic conditions of the invention.

[0032] Figure 8 Chromatogram obtained by optimizing the flow rate in the chromatographic conditions of the invention.

[0033] Figure 9 Chromatogram obtained by optimizing the injection volume in the chromatographic conditions of the invention.

[0034] Figure 10 Chromatogram obtained by optimizing the composition of the mobile phase in the chromatographic conditions of the invention.

[0035] Figure 11 Chromatogram obtained by optimizing the elution program in the chromatographic conditions of the invention.

[0036] Figure 12 Chromatogram of gallic acid of the invention.

[0037] Figure 13 UV spectrum of gallic acid of the invention.

[0038] Figure 14 Full wavelength 3D scan of gallic acid of the invention.

[0039] Figure 15 Chromatogram of protocatechuic acid of the invention.

[0040] Figure 16 UV spectrum of protocatechuic acid of the invention.

[0041] Figure 17 Full wavelength 3D scan of protocatechuic acid of the invention.

[0042] Figure 18 Chromatogram of catechin of the invention.

[0043] Figure 19 UV spectrum of catechin of the invention.

[0044] Figure 20 Full wavelength 3D scan of catechin of the invention.

[0045] Figure 21 Chromatogram of loganic acid of the invention.

[0046] Figure 22The UV spectrum of the aristofuranic acid of the present application.

[0047] Figure 23 The full wavelength 3D scan of the aristofuranic acid of the present application.

[0048] Figure 24 The chromatogram of the mangiferin of the present application.

[0049] Figure 25 The UV spectrum of the mangiferin of the present application.

[0050] Figure 26 The full wavelength 3D scan of the mangiferin of the present application.

[0051] Figure 27 The chromatogram of the paeonolactone of the present application.

[0052] Figure 28 The UV spectrum of the paeonolactone of the present application.

[0053] Figure 29 The full wavelength 3D scan of the paeonolactone of the present application.

[0054] Figure 30 The chromatogram of the paeonoside of the present application.

[0055] Figure 31 The UV spectrum of the paeonoside of the present application.

[0056] Figure 32 The full wavelength 3D scan of the paeonoside of the present application.

[0057] Figure 33 The chromatogram of the vanillin of the present application.

[0058] Figure 34 The UV spectrum of the vanillin of the present application.

[0059] Figure 35 The full wavelength 3D scan of the vanillin of the present application.

[0060] Figure 36 The chromatogram of the p-coumaric acid of the present application.

[0061] Figure 37 The UV spectrum of the p-coumaric acid of the present application.

[0062] Figure 38 The full wavelength 3D scan of the p-coumaric acid of the present application.

[0063] Figure 39 The chromatogram of the ferulic acid of the present application.

[0064] Figure 40 The UV spectrum of the ferulic acid of the present application.

[0065] Figure 41 Full wavelength 3D scanning diagram of ferulic acid of the present application.

[0066] Figure 42 Chromatogram of iso- chlorogenic acid B of the present application.

[0067] Figure 43 UV spectrum diagram of iso- chlorogenic acid B of the present application.

[0068] Figure 44 Full wavelength 3D scanning diagram of iso- chlorogenic acid B of the present application.

[0069] Figure 45 Chromatogram of Daurisamine B of the present application.

[0070] Figure 46 UV spectrum diagram of Daurisamine B of the present application.

[0071] Figure 47 Full wavelength 3D scanning diagram of Daurisamine B of the present application.

[0072] Figure 48 Chromatogram of Daurisamine C of the present application.

[0073] Figure 49 UV spectrum diagram of Daurisamine C of the present application.

[0074] Figure 50 Full wavelength 3D scanning diagram of Daurisamine C of the present application.

[0075] Figure 51 Chromatogram of Icariin of the present application.

[0076] Figure 52 UV spectrum diagram of Icariin of the present application.

[0077] Figure 53 Full wavelength 3D scanning diagram of Icariin of the present application.

[0078] Figure 54 Chromatogram of Baohuoside I of the present application.

[0079] Figure 55 UV spectrum diagram of Baohuoside I of the present application.

[0080] Figure 56 Full wavelength 3D scanning diagram of Baohuoside I of the present application.

[0081] Figure 57 Mass spectrum diagram of Gushangbao tablet in positive ion mode of the present application.

[0082] Figure 58 Mass spectrum diagram of Gushangbao tablet in negative ion mode of the present application.

[0083] Figure 59Mass spectrum of epimedin B of the present application.

[0084] Figure 60 Mass spectrum of icariin of the present application.

[0085] Figure 61 Mass spectrum of baohuoside I of the present application.

[0086] Figure 62 Mass spectrum of loganic acid of the present application.

[0087] Figure 63 Mass spectrum of paeonol glycoside of the present application.

[0088] Figure 64 Mass spectrum of mangiferin of the present application.

[0089] Figure 65 Mass spectrum of chlorogenic acid of the present application.

[0090] Figure 66 Mass spectrum of paeonol glycoside of the present application.

[0091] Figure 67 Mass spectrum of senkyunolide I of the present application.

[0092] Figure 68 Mass spectrum of coniferyl ferulate of the present application.

[0093] Figure 69 Mass spectrum of timosaponin BII of the present application.

[0094] Figure 70 Mass spectrum of timosaponin A3 of the present application.

[0095] Figure 71 Mass spectrum of 5-hydroxymethylfurfural of the present application.

[0096] Figure 72 Mass spectrum of rehderin D of the present application.

[0097] Figure 73 Mass spectrum of verbascoside of the present application.

[0098] Figure 74 Mass spectrum of pterospanoside of the present application.

[0099] Figure 2 Chromatogram of 15 batches of GuSongBao tablets and the generated control fingerprint. DETAILED DESCRIPTION

[0100] In order to enable the technical personnel in the art to better understand the technical solutions of the present application and to implement them, the present application will be further described below in conjunction with specific embodiments and drawings, but the embodiments are not limiting to the present application. The experimental methods and detection methods described in the following embodiments are all conventional methods, unless otherwise specified; the reagents and materials described, unless otherwise specified, can be purchased on the market.

[0101] A method for constructing a fingerprint spectrum of Gushenbao tablets, comprising the following steps:

[0102] S1, Preparation of test sample solution: Remove the coating from different batches of Gushenbao tablets, grind into powder, take an appropriate amount of powder, place it in a conical flask with a stopper, add methanol, ultrasonic extraction, filter, take the filtrate, and obtain test sample solutions of different batches.

[0103] S2, Preparation of control sample solution: Take appropriate amounts of gallic acid, protocatechuic acid, catechin, strychnine acid, mangiferin, paeonol glycoside, paeoniflorin, vanillin, p-coumaric acid, ferulic acid, isochlorogenic acid B, damianol B, damianol C, icariin, and baohuoglycoside I control samples, respectively, and dissolve them in methanol to prepare corresponding single component control sample solutions.

[0104] S3, The test sample solutions of different batches prepared in S1 and the control sample solutions prepared in S2 are injected into a high performance liquid chromatograph, respectively, and analyzed under the same high performance liquid chromatography conditions, and the chromatograms of the test sample solutions of different batches and the chromatograms of the control sample solutions are recorded.

[0105] S4, Similarity evaluation of the chromatograms of the test sample solutions of different batches is performed to confirm the reliability of the results.

[0106] The chromatograms of the test sample solutions of different batches obtained in S3 are imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System, version 2012 A; the chromatographic peaks that exist in the chromatograms of the test sample solutions of different batches are selected as common peaks, and the average value calculation method is used to generate a control fingerprint spectrum; similarity analysis is performed on the chromatograms of the test sample solutions of different batches and the generated control fingerprint spectrum to obtain a similarity result table between the chromatograms of the test sample solutions of different batches and the control fingerprint spectrum, and the table is exported; the reliability of the results is confirmed according to the similarity result table and the chromatogram of the test sample solution.

[0107] S5, High resolution mass spectrometry analysis is performed on one of the test sample solutions prepared in S1 to obtain a total ion flow chart; the total ion flow chart is imported into Xcalibur software, data analysis is performed according to the peak conditions of the chromatogram of the test sample solution, and mass spectrum result charts of each chemical component are obtained.

[0108] S6, according to the total ion current chromatogram and the mass spectrum result chromatogram of each chemical component, and in combination with the chromatogram of each reference solution, the chemical components of each peak in the chromatogram of the test solution are determined, and a fingerprint spectrum is obtained.

[0109] With peak 28 as the reference peak of doriden B, it is identified that in the chromatogram of the test solution, peak 2 is gallic acid with a retention time of 7.830 min; peak 6 is protocatechuic acid with a retention time of 12.610 min; peak 8 is catechin with a retention time of 17.877 min; peak 10 is strychnine acid with a retention time of 20.703 min; peak 11 is mangiferin with a retention time of 21.600 min; peak 12 is paeonolactone glycoside with a retention time of 22.863 min; peak 14 is paeoniflorin with a retention time of 24.373 min; peak 16 is vanillin with a retention time of 25.963 min; peak 17 is p-coumaric acid with a retention time of 27.723 min; peak 18 is ferulic acid with a retention time of 29.820 min; peak 21 is isochlorogenic acid B with a retention time of 31.120 min; peak 29 is doriden C with a retention time of 48.193 min; peak 31 is icariin with a retention time of 49.893 min; and peak 39 is baohuoglycoside I with a retention time of 73.690 min, and the fingerprint spectrum of Gushangbao tablets is obtained.

[0110] In S1, the preparation method of the test solution is as follows: 15 batches of Gushangbao tablets are removed from the coating, ground into powder, 1.5 g of the powder is taken and placed in a conical flask with a plug, 20 mL of methanol is added, the volume fraction of methanol is ≥ 99.5%, which is pure methanol, ultrasonic extraction is performed for 30 min, the ultrasonic power is 250 W, the frequency is 40 kHz, filtration is performed, the filtrate is taken, and 0.45 µm microporous filter membrane is used for filtration, and 15 batches of test solutions are prepared.

[0111] In S2, the preparation method of the reference solution is as follows: gallic acid, protocatechuic acid, catechin, strychnine acid, mangiferin, paeonolactone glycoside, paeoniflorin, vanillin, p-coumaric acid, ferulic acid, isochlorogenic acid B, doriden B, doriden C, icariin and baohuoglycoside I are precisely weighed, and the above components are dissolved in methanol, the volume fraction of methanol is ≥ 99.5%, which is pure methanol, and the reference solutions of corresponding single components are prepared, specifically: gallic acid reference solution, protocatechuic acid reference solution, catechin reference solution, strychnine acid reference solution, mangiferin reference solution, paeonolactone glycoside reference solution, paeoniflorin reference solution, vanillin reference solution, p-coumaric acid reference solution, ferulic acid reference solution, isochlorogenic acid B reference solution, doriden B reference solution, doriden C reference solution, icariin reference solution, and baohuoglycoside I reference solution.

[0112] The amount ratio of gallic acid and methanol is 0.57 mg:1 mL, the amount ratio of protocatechuic acid and methanol is 0.63 mg:1 mL, the amount ratio of catechin and methanol is 0.47 mg:1 mL, the amount ratio of loganic acid and methanol is 0.45 mg:1 mL, the amount ratio of mangiferin and methanol is 0.65 mg:1 mL, the amount ratio of paeonolactone and methanol is 0.60 mg:1 mL, the amount ratio of paeoniflorin and methanol is 0.62 mg:1 mL, the amount ratio of vanillin and methanol is 0.50 mg:1 mL, the amount ratio of p-coumaric acid and methanol is 0.60 mg:1 mL, the amount ratio of ferulic acid and methanol is 0.63 mg:1 mL, the amount ratio of isochlorogenic acid B and methanol is 0.51 mg:1 mL, the amount ratio of epimedin B and methanol is 0.46 mg:1 mL, the amount ratio of epimedin C and methanol is 0.45 mg:1 mL, the amount ratio of icariin and methanol is 0.46 mg:1 mL, and the amount ratio of baohuoside I and methanol is 0.52 mg:1 mL.

[0113] In S3, the high performance liquid chromatography conditions are as follows: a chromatographic column is Shim-pack VP-ODS, the column length is 250 mm, the inner diameter is 4.6 mm, and the filler particle size is 5 μm; a detector is a diode array detector, the detection wavelength is 210 nm; the flow rate is 1.0 mL / min; the injection amount is 20 μL; and the mobile phase is that the A phase is acetonitrile, the B phase is 0.1% (by volume) phosphoric acid aqueous solution, and gradient elution is carried out, and the elution program is as follows: 0 min-10 min, 95%-88% B phase; 10 min-26 min, 88%-79% B phase; 26 min-32 min, 79%-76% B phase; 32 min-36 min, 76%-74% B phase; 36 min-40 min, 74%-72% B phase; 40 min-45 min, 72%-70% B phase; 45 min-51 min, 70%-66% B phase; and 51 min-77 min, 66%-50% B phase.

[0114] In S5, the high resolution mass spectrometry analysis conditions are as follows: electrospray ionization, a spray voltage of 3500 V, a sheath gas flow rate of 40 arb, an auxiliary gas flow rate of 10 arb, a capillary temperature of 300 DEG C, an auxiliary gas temperature of 300 DEG C, a scanning mode of full scanning mode, and a mass-to-charge ratio scanning range m / z of 100-1500.

[0115] The optimization process of the fingerprint detection is specifically as follows.

[0116] 1. Optimization in the preparation of the test sample solution:

[0117] The present application investigates different extraction methods such as ultrasonic extraction, reflux extraction and immersion extraction. Figure 3As shown, it is found through analysis that the ultrasonic extraction has less difference with the reflux extraction and the immersion extraction in the chromatogram. Considering the operation simplicity and the extraction effect, the ultrasonic extraction method is more efficient than the other two extraction methods, so the ultrasonic extraction method is adopted.

[0118] The present application compares the extraction effects of different extraction solvents such as methanol with a volume fraction of 99.5%, 50% methanol aqueous solution, water and ethanol, as shown in the following table: Figure 4 As shown, it is found that when the methanol with a volume fraction of 99.5% is used as the extraction solvent, the obtained chromatogram has a relatively stable baseline, a smaller signal noise, the most information of the extract chromatogram, the most abundant types of extracted compounds and the highest content of each component, so the methanol with a volume fraction of 99.5% is selected for extraction.

[0119] The present application compares the extraction effects of different extraction times of 20min, 30min and 45min, as shown in the following table: Figure 5 As shown, it is found that the ultrasonic extraction effects of 30min and 45min are similar, and the obtained chromatogram has little difference in component content and peak shape, but compared with the ultrasonic extraction of 20min, the component content is obviously higher and the peak shape is more symmetrical, showing good resolution and stability. Meanwhile, considering the extraction efficiency and the overall feasibility of the experiment, the ultrasonic extraction time of 30min is selected.

[0120] 2. Optimization of high performance liquid chromatography conditions:

[0121] The present application uses a diode array detector to investigate the detection wavelength, and performs full wavelength scanning of 190nm-800nm on the sample, extracts the chromatogram at 210nm, 230nm, 254nm and 270nm, and the results are shown in the following table: Figure 6 and Figure 7 As shown, when the detection wavelength condition is 210nm, the chromatogram contains the most comprehensive information and has a stable baseline, so 210nm is selected as the detection wavelength condition.

[0122] The present application screens the column temperature of 25℃, 30℃ and 35℃, and the results are shown in the following table: Figure 8 As shown, when the column temperature is 35℃, the content of each component in the chromatogram is lower, and the number of components is less, which cannot effectively separate each component in the sample, and the separation effect is not ideal. On the contrary, when the column temperature is kept at 30℃ and 25℃, the peak shape symmetry in the chromatogram is better, which can effectively separate multiple components in the sample, and has a higher separation degree. However, when the column temperature is 25℃, an abnormal large peak appears in the last few minutes of the chromatogram, which may affect the separation of subsequent components. Considering the separation effect and the peak shape symmetry, the column temperature of 30℃ is finally selected as the best condition.

[0123] The present application screens different flow rates of 0.6 mL / min, 0.8 mL / min and 1.0 mL / min, as shown in Figure 9 The results show that the peak components at different flow rates are not much different, but the content of each component at a flow rate of 0.6 mL / min is slightly higher than that at a flow rate of 1.0 mL / min. However, considering the high flow rate can improve the analysis efficiency, shorten the analysis time, and provide a relatively stable separation effect while ensuring good separation degree and peak shape symmetry, the flow rate of 1.0 mL / min is finally selected.

[0124] The present application screens injection amounts of 10 μL, 15 μL and 20 μL, as shown in Figure 10 The results show that the injection amount of 20 μL can effectively improve the analysis signal intensity, and the peak area and peak height in the chromatogram are relatively obvious, which helps to improve the accuracy of quantitative analysis. At this injection amount, the peak shape is still symmetrical and no obvious tailing or broadening phenomenon occurs, ensuring good separation degree and stable analysis effect, so the injection amount of 20 μL is finally selected.

[0125] The present application compares various elution systems, including acetonitrile-0.1% formic acid aqueous solution, methanol-0.1% formic acid aqueous solution, acetonitrile-water, methanol-water, acetonitrile-0.1% phosphoric acid aqueous solution, methanol-0.1% phosphoric acid aqueous solution, etc., all under the same gradient conditions for elution, to evaluate the effect of the elution system on the separation of each component in Gushengbao tablets. The concentration unit of the aqueous solution in the above elution system is volume percent. As shown in Figure 11 The results show that the acetonitrile-0.1% phosphoric acid aqueous solution as the mobile phase shows the best separation effect. This mobile phase composition can effectively separate each component in the sample, and the peak shape is symmetrical and the separation degree is good, which can accurately reflect the content and properties of each component in the sample. In contrast, when methanol is used as the organic phase, although elution can be performed, the peak components are fewer and the separation effect is not as good as that of the acetonitrile-0.1% phosphoric acid aqueous solution mobile phase, which cannot effectively separate each component in the sample. When 0.1% formic acid or water is used as the aqueous phase, there is a significant baseline drift phenomenon, and the eluted components are fewer, which affects the accuracy and stability of the analysis results, so the acetonitrile-0.1% phosphoric acid aqueous solution is finally selected as the mobile phase.

[0126] After determining the optimal mobile phase composition, the present application screens the best gradient elution program through a large number of experiments, and some elution programs are shown as follows.

[0127] Table 1 Elution program 1

[0128]

[0129] Table 2 Elution program 2

[0130]

[0131] Table 3 Elution procedure 3

[0132]

[0133] Table 4 Elution procedure 4

[0134]

[0135] Table 5 Elution procedure 5

[0136]

[0137] Table 6 Elution procedure 6

[0138]

[0139] The detection results are shown in Table 7. Figure 12~Figure 56 As shown in Table 7, the elution procedure 6 has good separation degree, high peak height, stable baseline and complete chromatographic information, and thus is selected as the optimal elution procedure.

[0140] The embodiments of the present application will be described in detail below with reference to the examples, and the specific conditions not specified in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments not specified by the manufacturers are all conventional products that can be obtained by purchase.

[0141] 1. The instruments used in the present application are shown in Table 7.

[0142] Table 7 Instruments used in the present application

[0143]

[0144] 2. Medicines and reagents

[0145] The different batches of bone marrow pills used in the present application are all purchased from the market and are produced by Shaanxi Panlong Pharmaceutical Group Co., Ltd.

[0146] The reagents used in the present application are shown in Table 8.

[0147] Table 8 Reagents used in the present application

[0148]

[0149] Reference substance: Gallic acid reference substance, batch number: 110831-202408, purity: 96.5%; protocatechuic acid reference substance, batch number: 110809-202207, purity: 97.5%; catechin reference substance, batch number: 110877-202306, purity: 96.6%; loganin acid reference substance, batch number: 111865-202406, purity: 98.6%; mangiferin reference substance, batch number: 111607-202406, purity: 98.5%; paeoniflorin reference substance, batch number: 110736-202447, purity: 98.1%; vanillin reference substance, batch number: 100491-202203, purity: 99.3%; p-coumaric acid reference substance, batch number: 112037-202102, purity: 99.7%; ferulic acid reference substance, batch number: 110773-202316, purity: 99.3%; icariin reference substance, batch number: 110737-202418, purity: 98.4%; baohuoside I reference substance, batch number: 111852-202104, purity: 99.2%, all purchased from China Institute for Drug Control; paeonilactoside reference substance, batch number: 22040205, purity: 99.7%, purchased from Chengdu Pufide Biotechnology Co., Ltd.; isochlorogenic acid B reference substance, batch number: 104028-240901, purity: 98.5%, purchased from Jiangsu Yongjian Pharmaceutical Technology Co., Ltd.; desmosdumortiin B reference substance, batch number: PS010935, purity: 98.96%, purchased from Chengdu Pus Bio-technology Co., Ltd.; desmosdumortiin C reference substance, batch number: AZBG1212, purity: 99.34%, purchased from Chengdu Efa Biological Technology Co., Ltd.

[0150] Example 1

[0151] A method for constructing a fingerprint spectrum of Gushenbao tablets, comprising the following steps:

[0152] S1, preparation of Gushenbao tablet test solution:

[0153] Take 15 batches of Gushenbao tablets, remove the coating, grind into powder, take 1.5 g of powder, place in a conical flask with a stopper, add 20 mL of pure methanol, ultrasonic extraction for 30 min, ultrasonic power 250 W, frequency 40 kHz, filter, take the filtrate, pass through a 0.45 µm microporous filter membrane to obtain 15 batches of test solution.

[0154] S2, preparation of reference substance solution:

[0155] Gallic acid, protocatechuic acid, catechin, loganin acid, mangiferin, paeonolactone, paeoniflorin, vanillin, p-coumaric acid, ferulic acid, isochlorogenic acid B, epimedin B, epimedin C, icariin, and baohuoside I were precisely weighed, respectively, and dissolved in pure methanol to prepare a corresponding single component reference solution containing 0.57 mg of gallic acid, 0.63 mg of protocatechuic acid, 0.47 mg of catechin, 0.45 mg of loganin acid, 0.65 mg of mangiferin, 0.60 mg of paeonolactone, 0.62 mg of paeoniflorin, 0.50 mg of vanillin, 0.60 mg of p-coumaric acid, 0.63 mg of ferulic acid, 0.51 mg of isochlorogenic acid B, 0.46 mg of epimedin B, 0.45 mg of epimedin C, 0.46 mg of icariin, or 0.52 mg of baohuoside I per 1 mL of pure methanol.

[0156] S3, the 15 batches of test sample solutions in S1 and the respective reference sample solutions in S2 were injected into a high performance liquid chromatograph for chromatographic analysis, detection was performed under the same conditions, and the corresponding chromatograms were recorded. The high performance liquid chromatography conditions were as follows: a chromatographic column: Shim-pack VP-ODS, column length 250 mm, inner diameter 4.6 mm, and filler particle size 5 μm; a detector: a diode array detector, detection wavelength 210 nm; volume flow rate: 1.0 mL / min; sample injection amount: 20 μL; and mobile phase: phase A was acetonitrile, phase B was 0.1% (volume fraction) phosphoric acid aqueous solution, gradient elution, and the elution program was the above-mentioned elution program 6. Figure 75 The respective reference sample corresponding chromatograms are shown in

[0157] S4, the chromatograms of the 15 batches of test sample solutions obtained in S3 were imported into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, version 2012 A; the chromatographic peaks present in the chromatograms of the 15 batches of test sample solutions were selected as common peaks, the average value calculation method was used to generate a control fingerprint of Gushenbao tablets, similarity analysis was performed on the chromatograms of the 15 batches of test sample solutions and the generated control fingerprint, the chromatograms of the test sample solutions of different batches were as shown in S1-S15, the control fingerprint was as shown in R, a similarity result table between the chromatograms of the 15 batches of test sample solutions and the control fingerprint was obtained, and was exported; the reliability of the results was confirmed according to the similarity result table and the chromatograms of the test sample solutions. Figure 75 Figure 57 S4, the chromatograms of the 15 batches of test sample solutions obtained in S3 were imported into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, version 2012 A; the chromatographic peaks present in the chromatograms of the 15 batches of test sample solutions were selected as common peaks, the average value calculation method was used to generate a control fingerprint of Gushenbao tablets, similarity analysis was performed on the chromatograms of the 15 batches of test sample solutions and the generated control fingerprint, the chromatograms of the test sample solutions of different batches were as shown in S1-S15, the control fingerprint was as shown in R, a similarity result table between the chromatograms of the 15 batches of test sample solutions and the control fingerprint was obtained, and was exported; the reliability of the results was confirmed according to the similarity result table and the chromatograms of the test sample solutions.

[0158] S5, one batch of test sample solution prepared in S1 was subjected to high resolution mass spectrometry analysis, and total ion chromatograms were obtained, as shown in Figure 58 and Figure 59~Figure 74 ​The total ion current chromatogram was imported into Xcalibur software, and data analysis was performed according to the peak situation of the chromatogram of the test solution, and the mass spectrum result of each chemical component was obtained. Through high-resolution mass spectrometric analysis of the GuSongBao tablet substance reference, a total of 16 chemical components were resolved, as shown in Figure 1 The results showed that, as shown in the table, the 15 characteristic components were identified, which were: No. 28 peak of damian B as the reference peak, No. 2 peak of gallic acid in the chromatogram, the retention time was 7.830 min; No. 6 peak of protocatechuic acid, the retention time was 12.610 min; No. 8 peak of catechin, the retention time was 17.877 min; No. 10 peak of strychnine acid, the retention time was 20.703 min; No. 11 peak of mango glycoside, the retention time was 21.600 min; No. 12 peak of peony lactone glycoside, the retention time was 22.863 min; No. 14 peak of paeoniflorin, the retention time was 24.373 min; No. 16 peak of vanillin, the retention time was 25.963 min; No. 17 peak of p-coumaric acid, the retention time was 27.723 min; No. 18 peak of ferulic acid, the retention time was 29.820 min; No. 21 peak of isochlorogenic acid B, the retention time was 31.120 min; No. 29 peak of damian C, the retention time was 48.193 min; No. 31 peak of icariin, the retention time was 49.893 min; No. 39 peak of baohuoglycoside I, the retention time was 73.690 min, and the fingerprint of GuSongBao tablet was obtained.

[0159] The high-resolution mass spectrometry detection conditions were: electrospray ionization, spray voltage 3500V, sheath gas flow rate 40arb, auxiliary gas flow rate 10arb, capillary temperature 300℃, auxiliary gas temperature 300℃, scanning mode was full scan mode, mass-to-charge ratio scanning range m / z was 100~1500.

[0160] S6, according to the total ion current chromatogram and the mass spectrum result of each chemical component, and combined with the chromatogram of each reference solution, the chemical components of each peak in the chromatogram of the test solution were determined, and the fingerprint was obtained. As shown in ​ The results showed that, as shown in the table, the 15 characteristic components were identified, which were: No. 28 peak of damian B as the reference peak, No. 2 peak of gallic acid in the chromatogram, the retention time was 7.830 min; No. 6 peak of protocatechuic acid, the retention time was 12.610 min; No. 8 peak of catechin, the retention time was 17.877 min; No. 10 peak of strychnine acid, the retention time was 20.703 min; No. 11 peak of mango glycoside, the retention time was 21.600 min; No. 12 peak of peony lactone glycoside, the retention time was 22.863 min; No. 14 peak of paeoniflorin, the retention time was 24.373 min; No. 16 peak of vanillin, the retention time was 25.963 min; No. 17 peak of p-coumaric acid, the retention time was 27.723 min; No. 18 peak of ferulic acid, the retention time was 29.820 min; No. 21 peak of isochlorogenic acid B, the retention time was 31.120 min; No. 29 peak of damian C, the retention time was 48.193 min; No. 31 peak of icariin, the retention time was 49.893 min; No. 39 peak of baohuoglycoside I, the retention time was 73.690 min, and the fingerprint of GuSongBao tablet was obtained.

[0161] By comparing with the chromatograms of 15 control product solutions such as epimedin B, epimedin C and the like, the chemical components corresponding to the 15 characteristic peaks in the Gushenbao tablet fingerprint are analyzed and attributed, wherein gallic acid is derived from red peony root, protocatechuic acid is derived from sparganium stoloniferum, catechin is derived from red peony root, strychnine glycoside acid is derived from rhizoma dangshen, mangiferin is derived from anemarrhena asphodeloides, peony lactone glycoside is derived from red peony root, paeoniflorin is derived from red peony root, vanillin is derived from sparganium stoloniferum, p-coumaric acid is derived from sparganium stoloniferum, ferulic acid is derived from cnidium officinalis, isochlorogenic acid B is derived from rhizoma dangshen, epimedin B is derived from epimedium, epimedin C is derived from epimedium, icariin is derived from epimedium, and baohuoglycoside I is derived from epimedium.

[0162] The present application uses the automatically generated control fingerprint R to generate the common chromatographic peak mode, and the common chromatographic peaks of the 15 batches of Gushenbao tablet test products have relatively good similarity, which shows that the Gushenbao tablet fingerprint established by the method can well detect the quality of Gushenbao tablet and the 15 batches of Gushenbao tablet, and the results are shown in Table 9.

[0163] Table 9 Similarity evaluation results of chromatograms of 15 batches of test product solutions

[0164]

[0165] Example 2

[0166] Methodological study of the fingerprint detection method:

[0167] S1, precision study

[0168] The test product solution prepared by the method of Example 1 is analyzed according to the detection method of Example 1, and 6 parallel injections are performed, with an injection amount of 20 μL, and epimedin B is used as the reference peak. By analyzing the relative peak area and the relative retention time and calculating the relative standard deviation RSD value, the results show that the RSD of the relative retention time is less than 0.45%, and the RSD of the relative peak area is less than 0.52%, as shown in Tables 10 and 11, which indicates that the parallel injection precision of the equipment is good.

[0169] Table 10 Peak area and retention time of precision study

[0170]

[0171] Table 11 Continuation of Table 10

[0172]

[0173] S2, stability study

[0174] The sample solution prepared according to the method of Example 1 was analyzed according to the detection method of Example 1, and the sample was injected at different times of 0 h, 2 h, 6 h, 12 h, 18 h, 24 h, the injection amount was 20 μL, and the peak area and retention time of the common peaks in the sample HPLC fingerprint were analyzed and the RSD value was calculated, and the results showed that the RSD of the relative retention time was less than 0.222%, and the RSD of the relative peak area was less than 0.622%, as shown in Tables 12 and 13, indicating that the chromatographic peaks of the GuSongBao tablet sample solution had little change within 24 h, and the stability was good.

[0175] Table 12 Peak area and retention time of stability study

[0176]

[0177] Table 13 Continuation of Table 12

[0178]

[0179] S3, repeatability study

[0180] Six batches of sample solutions were prepared according to the sample solution method in Example 1, and the sample was injected in an amount of 20 μL according to the chromatographic conditions of Example 1, and the peak area and retention time of the common peaks in the sample HPLC fingerprint were analyzed and the RSD value was calculated, and the results showed that the RSD of the relative retention time was less than 0.514%, and the RSD of the relative peak area was less than 0.445%, as shown in Table 14, indicating that the sample chromatographic peaks had good reproducibility, and the repeatability of the method was good.

[0181] Table 14 Peak area and retention time of repeatability study

[0182]

[0183] The above experimental results show that the fingerprint spectrum construction method of GuSongBao tablets and its application in quality standards have the characteristics of good stability, high precision and good repeatability, and can comprehensively and objectively evaluate the quality of GuSongBao tablets, and provide quality assurance for clinical efficacy.

[0184] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, these modifications and variations are also intended to be included in the present application.

Claims

1. A method for constructing the fingerprint spectrum of Gusongbao tablets, characterized in that, Includes the following steps: Different batches of bone-strengthening tablets were decoated, ground into powder, and the powder was added to methanol for ultrasonic extraction. The mixture was then filtered, and the filtrate was collected to prepare test solutions for different batches. Gallic acid, protocatechuic acid, catechin, loganic acid, mangiferin, paeoniflorin, paeoniflorin, vanillin, p-coumaric acid, ferulic acid, isochlorogenic acid B, chomprolin B, chomprolin C, icariin and baicalin I were used as reference standards and dissolved in methanol to prepare reference solutions for the corresponding single components. Different batches of test solutions and each reference solution were injected into a high-performance liquid chromatograph and analyzed under the same high-performance liquid chromatographic conditions. The chromatograms of different batches of test solutions and each reference solution were recorded. Similarity analysis was performed on the chromatograms of different batches of test solutions to confirm the reliability of the results; One batch of test solution was subjected to high-resolution mass spectrometry analysis to obtain a total ion chromatogram. The total ion chromatogram was then imported into Xcalibur software. Data analysis was performed based on the peak elution of the chromatogram of the test solution to obtain the mass spectrometry results of each chemical component. Based on the total ion chromatogram and the mass spectrometry results of each chemical component, and in conjunction with the chromatograms of each reference solution, the chemical components of each peak in the chromatogram of the test solution are determined, and the fingerprint spectrum is obtained. The high performance liquid chromatography conditions were as follows: column: Shim-pack VP-ODS, column length 250 mm, inner diameter 4.6 mm, packing particle size 5 μm; Detector: Diode array detector, detection wavelength 210 nm; Volumetric flow rate: 1.0 mL / min; Column temperature: 30℃; injection volume: 20μL; mobile phase: phase A is acetonitrile, phase B is 0.1% (v / v) aqueous phosphoric acid solution, gradient elution, elution program by volume percentage: 0 min~10 min, 95%~88% phase B; 10 min~26 min, 88%~79% phase B; 26 min~32 min, 79%~76% phase B; 32 min~36 min, 76%~74% phase B; 36 min~40 min, 74%~72% phase B; 40 min~45 min, 72%~70% phase B; 45 min~51 min, 70%~66% phase B; 51 min~77 min, 66%~50% phase B.

2. The method for constructing the fingerprint spectrum of Gusongbao tablets according to claim 1, characterized in that, In the test solution, the ratio of powder to methanol is 1.5g:20mL, the volume fraction of methanol is ≥99.5%, the ultrasonic power is 250W, the frequency is 40kHz, the ultrasonic extraction time is 30min, and the filtrate is filtered through a 0.45µm microporous membrane. When preparing reference solutions for each single component, the volume fraction of methanol should be ≥99.5%, the volume ratio of gallic acid to methanol should be 0.57 mg:1 mL, the volume ratio of protocatechuic acid to methanol should be 0.63 mg:1 mL, the volume ratio of catechin to methanol should be 0.47 mg:1 mL, the volume ratio of loganic acid to methanol should be 0.45 mg:1 mL, the volume ratio of mangiferin to methanol should be 0.65 mg:1 mL, the volume ratio of paeoniflorin to methanol should be 0.60 mg:1 mL, and the volume ratio of paeoniflorin to methanol should be 0.62 mg:1 mL. The dosage ratios of vanillin to methanol were 0.50 mg:1 mL, p-coumaric acid to methanol were 0.60 mg:1 mL, ferulic acid to methanol were 0.63 mg:1 mL, isochlorogenic acid B to methanol were 0.51 mg:1 mL, ascorbic acid B to methanol were 0.46 mg:1 mL, ascorbic acid C to methanol were 0.45 mg:1 mL, icariin to methanol were 0.46 mg:1 mL, and cymosin I to methanol were 0.52 mg:1 mL.

3. The method for constructing the fingerprint spectrum of Bone-Suppressing Tablets according to claim 1, characterized in that, Similarity analysis was performed on the chromatograms of different batches of test solutions. The specific method was as follows: chromatograms of different batches of test solutions were imported into a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine; chromatographic peaks present in all batches of test solutions were selected as common peaks, and a reference fingerprint was generated using the average value calculation method; similarity analysis was performed on the chromatograms of different batches of test solutions and the generated reference fingerprint, and a similarity result table between the chromatograms of different batches of test solutions and the reference fingerprint was obtained and exported; the reliability of the results was confirmed based on the similarity result table and the chromatograms of the test solutions.

4. The method for constructing the fingerprint spectrum of Gusongbao tablets according to claim 1, characterized in that, The conditions for high-resolution mass spectrometry analysis were: electrospray ionization, spray voltage 3500V, sheath gas flow rate 40arb, auxiliary gas flow rate 10arb, capillary temperature 300℃, auxiliary gas temperature 300℃, full scan mode, and mass-to-charge ratio scan range m / z of 100~1500.

5. The application of the method for constructing the fingerprint spectrum of Gusongbao tablets according to any one of claims 1 to 4 in the quality testing of Gusongbao tablets and its related drugs.

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