Method for constructing fingerprint spectrum and fingerprint spectrum of Xinshuning capsule

Abstract

To provide a method for establishing a fingerprint spectrum of a xinsuning capsule, and a fingerprint spectrum.SOLUTION: The present invention includes the steps of: collecting contents of xinsuning capsules with different batches, adding a methanol aqueous solution to perform ultrasonic extraction, and obtaining a test solution (S1); injecting the test solution into a rapid liquid chromatograph, performing gradient elution, performing chromatography analysis, and recording 0-140 min chromatogram (S2); introducing the chromatogram into the traditional Chinese medicine chromatographic Fingerprint similarity evaluation system for processing to obtain a fingerprint spectrum (S3); and performing mass spectrometry on the test solution and determining a chemical composition of chromatographic peaks in the fingerprint spectrum on the basis of mass spectrometry results (S4). A method for detecting a xinsuning capsule fingerprint spectrum is easy, stable, precise, and can be highly regenerated. Quality of xinsuning capsules can be evaluated comprehensively and scientifically and quality of a product and the effect of treatment can be guaranteed.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of traditional Chinese medicine detection, and in particular to the fingerprint spectrum construction method and fingerprint spectrum of Xinshuoning Capsules. [Background technology]

[0002] Xin Su Ning Capsules are prescribed to clear heat and eliminate phlegm, calm the heart, and stop palpitations. They conform to the theory of Lord, Minister, Assistant, and Secretary in traditional Chinese medicine. The principal drugs in the prescription are Coptis Rhizome and Meadowsweet, which clear heat and eliminate phlegm. The subordinate drugs are Sweet Artemisia Herb, Dichroa Febrifuga Lure, Clam Shell, and Plumera Nelumbinis, which help the principal drugs clear heat, clear phlegm, and detoxify, clear the mind, and calm the spirit. The auxiliary drug is Poria Cocos. , Aurantii Inmatulus Fructus, which regulates qi, exudes dampness and dispels phlegm, ginseng and dwarf lily turf tubers are also auxiliary medicines that nourish the heart and restore the pulse, licorice is an emissary that harmonizes the various medicines, and at the same time, Coptis Rhizome, Melilotus daisy and Dichroa febrifuga-rue release acid and reduce bitterness, which, together with Aurantii Inmatulus Fructus, eliminate stagnation of qi and regulate the pulse.

[0003] "Xin Suo Ning Capsules" is a compound medicine made from 11 kinds of herbal medicines: Coptis Rhizome, Meadowsweet, Poria Cocos, Aurantium Inmatullus Fructus, Dichroa Febrifuga Lure, Plumula Nelumbinis, Clam, Sweet Artemisia Herb, Ginseng, Dwarf Lily Turf Tubers, and Licorice. It is used to treat palpitations, chest tightness, heartburn, startleness, dry mouth, bitter mouth, insomnia, hypersomnia, dizziness, and arrhythmia (arrhythmia) caused by phlegm heat, and is also suitable for mild to moderate ventricular premature beats (premature ventricular contractions) caused by coronary heart disease and viral myocarditis.

[0004] The quality of traditional Chinese medicine is directly linked to its development. In recent years, traditional Chinese medicine has attracted increasing attention worldwide due to its reliable efficacy, few side effects, low toxicity, and low drug resistance. However, the complexity of the chemical composition of traditional Chinese medicines and the interactions between ingredients make quality control of traditional Chinese medicines increasingly difficult. Currently, the quality control of Xinshuoning Capsules is relatively simple, but the testing method is complicated. The current national standard for Xinshuoning Capsules only requires the content of berberine hydrochloride, which cannot reflect the overall composition of Xinshuoning Capsules and cannot provide a basis for quality control and evaluation. Summary of the Invention [Problem to be solved by the invention]

[0005] SUMMARY OF THE INVENTION In view of the technical shortcomings of existing detection methods, the present invention aims to provide a method for constructing a fingerprint spectrum of a cardiac speed monitoring capsule and a fingerprint spectrum thereof. [Means for solving the problem]

[0006] The present invention is achieved by the following technical solutions:

[0007] The fingerprint spectrum construction method of Xinshuning Capsule is S1: Collect the contents of different batches of Xinshuning capsules, add aqueous methanol solution, and ultrasonically extract to obtain test solutions; S2: Inject the test solution into a high performance liquid chromatograph, perform gradient elution, perform chromatographic analysis, and record the chromatogram from 0 to 140 min; S3, the chromatograms obtained in S2 are introduced into the traditional Chinese medicine chromatogram fingerprint spectrum similarity evaluation system, and the chromatogram peaks present in the chromatograms of different batches of Xinshuoning capsules are selected as common peaks, and the chromatograms of the test solutions are respectively subjected to data introduction, multi-point correction and data matching to obtain fingerprint spectra; S4, performing mass spectrometry on the test solution and determining the chemical composition of the chromatogram peak in the fingerprint spectrum based on the mass spectrometry results.

[0008] Preferably, in S1, the volume concentration of methanol in the aqueous methanol solution is 80%.

[0009] Preferably, in S1, the ultrasonic extraction time is 20 to 40 minutes.

[0010] Preferably, in S2, the detection wavelength used for the chromatographic analysis is 210 nm.

[0011] Preferably, in S2, the chromatographic column used in the high performance liquid chromatograph is a Hedera ODS-2-C18 (250 mm x 4.6 mm, 10 μm) chromatographic column.

[0012] Preferably, in S2, the mobile phase used for gradient elution is acetonitrile and 0.1% formic acid in water.

[0013] Preferably, in S2, the gradient elution procedure is as shown in the table below.

[0014] TIFF0007795819000001.tif42168

[0015] Preferably, in S2, the flow rate used for gradient elution is 0.8 mL / min.

[0016] Preferably, in S4, the chemical compositions of the chromatogram peaks in the fingerprint spectrum are determined to be: Peak 1 trifolirhizin, Peak 2 naringin, Peak 3 kaempferide, Peak 4 valine, Peak 5 palmatine, Peak 6 epiberberine, Peak 7 glycyrrhizin / isoglycyrrhizin, Peak 8 limonin, Peak 9 liquiritin, Peak 10 picrotoxin, Peak 11 methylopiogonanone A, Peak 12 rotacin, Peak 13 sophoridine / sophocarpidine, Peak 14 sophoranol / oxidized sophocarpidine, Peak 15 kurarinone, Peak 16 dehydropatiminic acid, Peak 17 polyporenic acid C, Peak 18 quercetin, and Peak 19 sophocarpine.

[0017] The present invention provides a fingerprint spectrum of a cardiac speed-up capsule constructed by the method according to any one of claims 1 to 9. Compared with existing technologies, the present invention has the following beneficial effects:

[0018] The present invention establishes a fingerprint spectrum detection method according to the structural characteristics of the active ingredients contained in Xinshuning Capsules. Through numerous experimental verifications, it has been found that the fingerprint spectrum detection method for Xinshuning Capsules provided by the present invention has better separation of each chromatogram peak, a smooth baseline, and good peak shapes, and can comprehensively respond to the type and amount of chemical ingredients contained in the capsules. Using the HPLC fingerprint spectrum of Xinshuning Capsules constructed by the present invention, 19 common peaks and standard fingerprint spectra were obtained and recognized, and the obtained chromatograms had high similarity. The relative retention time and relative peak area were used to calculate the stability, reproducibility, and accuracy. The RSD values ​​of the relative retention time and relative peak area were both less than 5.0%, indicating that the method provided by the present invention has good reproducibility and the fingerprint spectrum constructed by the method provided by the present invention is reliable. The fingerprint spectrum detection method for Xinshuning Capsules provided by the present invention has the advantages of being simple, stable, accurate, and reproducible, allowing for a more comprehensive and scientific evaluation of the quality of Xinshuning Capsules and ensuring the quality and therapeutic effect of the product. [Brief explanation of the drawings]

[0019] [Figure 1] 1 is a chromatogram obtained in Example 1 during the detection wavelength optimization process of the present invention. [Figure 2] 1 is a chromatogram obtained in Example 2 during the flow rate optimization process of the present invention. [Figure 3] 1 is a chromatogram obtained in Example 3 during the elution gradient optimization process of the present invention. [Figure 4] 1 is a chromatogram obtained in Example 4 during the elution gradient optimization process of the present invention. [Figure 5] 1 is a chromatogram obtained in Example 5 during the elution gradient optimization process of the present invention. [Figure 6] 1 is a chromatogram obtained in Example 6 during the elution gradient optimization process of the present invention. [Figure 7] 1 is a chromatogram obtained in Example 7 of the optimum method of the present invention. [Figure 8] 1 is a mass spectrum of trifolirhizin of the present invention. [Figure 9] 1 is a mass spectrum of naringin of the present invention. [Figure 10] 1 is a mass spectrum of kaempferide of the present invention. [Figure 11] 1 is a mass spectrum of valine of the present invention. [Figure 12] 1 is a mass spectrum of palmatine of the present invention. [Figure 13] 1 is a mass spectrum of epiberberine of the present invention. [Figure 14] 1 is a mass spectrum of glycyrrhizin / isoglycyrrhizin of the present invention. [Figure 15] 1 is a mass spectrum of limonin of the present invention. [Figure 16] 1 is a mass spectrum of liquiritin of the present invention. [Figure 17] 1 is a mass spectrum of picrotoxin of the present invention. [Figure 18]1 is a mass spectrum of methylopiogonanone A of the present invention. [Figure 19] 1 is a mass spectrum of rotacin of the present invention. [Figure 20] 1 is a mass spectrum of sophoridine / sophocarpidine of the present invention. [Figure 21] 1 is a mass spectrum of sophoranol / sophorpidine oxide of the present invention. [Figure 22] 1 is a mass spectrum of kurarinone of the present invention. [Figure 23] 1 is a mass spectrum of dehydropatimic acid of the present invention. [Figure 24] 1 is a mass spectrum of polyboronic acid C of the present invention. [Figure 25] 1 is a mass spectrum of quercetin of the present invention. [Figure 26] 1 is a mass spectrum of sophocarpine of the present invention. [Figure 27] 1 shows the fingerprint spectra of 15 batches of test samples based on the Xinshuning capsule of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0020] In order to further understand the present invention, the present invention will be described below in connection with examples. These examples are used to further interpret the features and advantages of the present invention, and are not intended to limit the scope of the claims of the present invention.

[0021] 1. The fingerprint spectrum construction method of Xinshuning Capsule includes the following steps: S1. Preparation of test solutions: 0.3-1.0 g of the contents of different batches of Xinsuo Ning capsules were precisely weighed and placed in a stoppered conical flask. 20-30 mL of aqueous methanol solution was added, and ultrasonic extraction was performed for 20-40 minutes. The resulting filtrate was filtered through a 0.45 μm microporous filter membrane to obtain the test solution. S2: 15-30 μL of the test solution prepared in S1 is precisely aspirated and injected into the high-performance liquid chromatograph. Gradient elution is performed, and the chromatogram from 0 to 140 min is recorded. The chromatograms obtained in S3 and S2 are extracted and introduced into the Chinese herbal medicine chromatogram fingerprint spectrum similarity evaluation system (2004 A version). The chromatogram peaks present in the chromatograms of different batches of Xinshuo Ning capsules are selected as common peaks. Data is input, multi-point correction and data matching are performed on the chromatograms of the test solutions, and the fingerprint spectrum is obtained and a similarity analysis is performed. The similarity is 0.90 or more. S4: Determine the chemical composition in the fingerprint spectrum, perform mass spectrometry on the above test solution to obtain a total ion flow diagram, import the detected data into Xcalibur software, enter it into the Qual Browser interface, and perform data analysis according to the chemical composition peak situation.

[0022] The preparation method of the test solution described in S1 above is as follows: 0.5 g of the contents of 15 batches of Xinsuo Ning capsules is precisely weighed and placed in a stoppered conical flask, 25 mL of 80% aqueous methanol solution is added, and ultrasonic extraction is performed for 30 minutes. The subsequent filtrate is filtered through a 0.45 μm microporous filter membrane to obtain the test solution.

[0023] The liquid-phase chromatographic conditions described in S2 above were as follows: chromatographic column: Hedera ODS-2-C18 (250 mm × 4.6 mm, 10 μm) chromatographic column; mobile phase: acetonitrile (A) and 0.1% formic acid aqueous solution (B); gradient elution; detection wavelength: 210 nm; column temperature: 30 °C; flow rate: 0.8 mL / min; sample injection volume: 20 μL; elution procedure: 0 min, 20% A; 0–35 min, 20%–27% A; 35–45 min, 27%–35% A; 45–85 min, 35–45% A; 85–90 min, 45–65% A; 90–140 min, 65–100% A.

[0024] 2. Optimization of fingerprint spectrum detection S1, Optimization of extraction method In this study, we conducted experimental comparisons using different extraction methods (ultrasonic 20 min, ultrasonic 30 min, reflux 60 min) and different extraction solvents (ultrapure water, 80% methanol aqueous solution, methanol). As a result, the spectral composition obtained after 30 min of ultrasonic extraction was more comprehensive and the degree of separation was better, so we chose the 30 min ultrasonic extraction method. In the examination of extraction solvents, the chromatogram information volume of the 80% methanol aqueous solution extract was the largest, and the composition content was the highest, so we chose 80% methanol aqueous solution for extraction.

[0025] S2, Optimization of chromatographic conditions In the present invention, the detection wavelengths (210 nm, 254 nm, 275 nm, 300 nm) were examined using an ultraviolet-visible light absorption detector. When the detection wavelength condition was 210 nm, the amount of information contained in the chromatogram was the most comprehensive and the baseline was the smoothest, so this method was selected as the detection wavelength condition.

[0026] In the present invention, the flow rates (0.6 mL / min, 0.8 mL / min, 1.0 mL / min) were screened, and it was found that a flow rate of 0.8 mL / min provided the best separation effect for each substance and the highest degree of separation, so a flow rate of 0.8 mL / min was adopted.

[0027] In the present invention, after screening temperatures (25°C, 30°C, 35°C), the temperature of 30°C was selected because the temperature had little effect on the peak time and number of peaks and had a good separation effect for each substance.

[0028] In this invention, the elution effects under different gradients of several different elution systems, including acetonitrile-water, methanol-water, acetonitrile-0.2% phosphoric acid aqueous solution, acetonitrile-0.1% formic acid, and acetonitrile-0.1% glacial acetic acid, are compared. As a result, when acetonitrile and 0.1% formic acid aqueous solution are used as the mobile phase, the peak conditions of each component in the Shinsokuning capsules are good and the separation effect is high, so acetonitrile and 0.1% formic acid aqueous solution are finally selected as the mobile phase.

[0029] After determining the optimal mobile phase composition, the present invention screened for the optimal gradient elution procedure through extensive experiments. The experimental results showed that using the following acetonitrile volume ratios: 20% at 0 min, 20% to 27% at 0-35 min, 27% to 35% at 35-45 min, 35% to 45% at 45-85 min, 45% to 65% at 85-90 min, and 65% to 100% at 90-140 min, achieved good resolution of each chromatographic peak in the fingerprint spectrum.

[0030] The equipment used in the present invention is shown in Table 1 below, the reagents used in the present invention are shown in Table 2 below, and the drugs used in the present invention are shown in Table 3 below.

[0031] [Table 1]

[0032] [Table 2]

[0033] [Table 3]

[0034] Hereinafter, embodiments of the present invention will be described in detail with reference to specific examples.

[0035] Example 1 The method for constructing a fingerprint spectrum of the Xinshuning capsule includes the following steps: S1. Preparation of test solutions: 0.5 g of the contents of the Shinsokuning capsule was weighed accurately and placed in a stoppered conical flask. 25 mL of aqueous methanol solution was added and subjected to ultrasonic extraction for 30 minutes. The resulting filtrate was filtered through a 0.45 μm microporous filter membrane to obtain the test solution. S2, chromatogram conditions Chromatography column: Hedera ODS-2-C18 (250 mm × 4.6 mm, 5 μm) chromatographic column, mobile phase: acetonitrile and 0.1% formic acid aqueous solution, gradient elution, detection wavelength: 210 nm / 254 nm / 270 nm / 300 nm, column temperature: 30 °C, flow rate: 0.8 mL / min, sample injection volume: 20 μL, elution procedure is shown in Table 4 below.

[0036] [Table 4]

[0037] The final experimental results are shown in Figure 1. As can be seen from the chromatograms, the resolution was poor overall under the four detection wavelengths, with few peaks between 0 and 255 min. However, the peak situation between 25 and 40 min was better when the detection wavelength was 210 nm, so 210 nm detection was used in the subsequent optimization process.

[0038] Example 2 The method for constructing a fingerprint spectrum of the Xinshuning capsule includes the following steps: S1. Preparation of test solutions: 0.5 g of the contents of the Shinsokuning capsule was weighed accurately and placed in a stoppered conical flask. 25 mL of aqueous methanol solution was added and subjected to ultrasonic extraction for 30 minutes. The resulting filtrate was filtered through a 0.45 μm microporous filter membrane to obtain the test solution. S2, chromatogram conditions Chromatography column: Hedera ODS-2-C18 (250 mm × 4.6 mm, 5 μm) chromatographic column, mobile phase: acetonitrile and 0.1% formic acid aqueous solution, gradient elution, detection wavelength: 210 nm, column temperature: 30 °C, flow rate: 0.6 mL / min, 0.8 mL / min, 1 mL / min, sample injection volume: 20 μL, elution procedure is shown in Table 5 below.

[0039] [Table 5]

[0040] The final experimental results are shown in Figure 2. As can be seen from the chromatogram, when the flow rate is 0.6 mL / min, there are many peaks, but the resolution between adjacent peaks is low, and the peaks after 125 minutes are incomplete. When the flow rate is 1 mL / min, there are few peaks, and there are no peaks between 75 and 100 minutes, which means the fingerprint spectrum requirements cannot be met. When the flow rate is 0.8 mL / min, the chromatogram peak conditions and resolution are good, so a flow rate of 0.8 mL / min is selected in subsequent examples.

[0041] Example 3 The method for constructing and detecting the Xinshuning capsule fingerprint spectrum includes the following steps: S1. Preparation of test solutions: 0.5 g of the contents of the Shinsokuning capsule was weighed accurately and placed in a stoppered conical flask. 25 mL of 80% aqueous methanol solution was added, and ultrasonic extraction was performed for 30 minutes. The resulting filtrate was filtered through a 0.45 μm microporous filter membrane to obtain the test solution. S2, chromatogram conditions Chromatography column: Hedera ODS-2-C18 (250 mm × 4.6 mm, 5 μm) chromatographic column, mobile phase: acetonitrile and 0.1% formic acid aqueous solution, gradient elution, detection wavelength: 210 nm, column temperature: 30 °C, flow rate: 0.8 mL / min, sample injection volume: 20 μL, elution procedure is shown in Table 6 below.

[0042] [Table 6]

[0043] The final experimental results are shown in Figure 3. As can be seen from the chromatogram, the overall resolution was poor, with few peaks and low content between 55 and 75 minutes, and many peaks between 30 and 45 minutes, but the peak density was high. During the optimization process, we focused on separating the peaks in this time period, taking into account the effect of the mobile phase organic phase concentration on the peak spacing, and continued to optimize the concentration gradient.

[0044] Example 4 The method for constructing a fingerprint spectrum of the Xinshuning capsule includes the following steps: S1. Preparation of test solutions: 0.5 g of the contents of the Shinsokuning capsule was weighed accurately and placed in a stoppered conical flask. 25 mL of 80% aqueous methanol solution was added, and ultrasonic extraction was performed for 30 minutes. The resulting filtrate was filtered through a 0.45 μm microporous filter membrane to obtain the test solution. S2, chromatogram conditions Chromatography column: Hedera ODS-2-C18 (250 mm × 4.6 mm, 5 μm) chromatographic column, mobile phase: acetonitrile and 0.1% formic acid aqueous solution, gradient elution, detection wavelength: 210 nm, column temperature: 30 °C, flow rate: 0.8 mL / min, sample injection volume: 20 μL, elution procedure is shown in Table 7 below.

[0045] [Table 7]

[0046] The final experimental results are shown in Figure 4. As can be seen from the chromatogram, the separation effect improved to some extent between 30 and 50 minutes, but the overall resolution did not meet the fingerprint spectrum requirements, so further optimization of the concentration gradient was required.

[0047] Example 5 The method for constructing a fingerprint spectrum of the Xinshuning capsule includes the following steps: S1. Preparation of test solutions: 0.5 g of the contents of the Shinsokuning capsule was weighed accurately and placed in a stoppered conical flask. 25 mL of 80% aqueous methanol solution was added, and ultrasonic extraction was performed for 30 minutes. The resulting filtrate was filtered through a 0.45 μm microporous filter membrane to obtain the test solution. S2, chromatogram conditions Chromatography column: Hedera ODS-2-C18 (250 mm × 4.6 mm, 5 μm) chromatographic column, mobile phase: acetonitrile and 0.1% formic acid aqueous solution, gradient elution, detection wavelength: 210 nm, column temperature: 30 °C, flow rate: 0.8 mL / min, sample injection volume: 20 μL, elution procedure is shown in Table 8 below.

[0048] [Table 8]

[0049] The final experimental results are shown in Figure 5. As can be seen from the chromatogram, the peak conditions are generally good, and the resolution of the chromatographic peaks is slightly improved. However, after 120 minutes, the peaks become incomplete, so we continued to optimize the concentration gradient and extend the elution time.

[0050] Example 6 The method for constructing a fingerprint spectrum of the Xinshuning capsule includes the following steps: S1. Preparation of test solutions: 0.5 g of the contents of the Shinsokuning capsule was weighed accurately and placed in a stoppered conical flask. 25 mL of 80% aqueous methanol solution was added, and ultrasonic extraction was performed for 30 minutes. The resulting filtrate was filtered through a 0.45 μm microporous filter membrane to obtain the test solution. S2, chromatogram conditions Chromatography column: Hedera ODS-2-C18 (250 mm × 4.6 mm, 5 μm) chromatographic column, mobile phase: acetonitrile and 0.1% formic acid aqueous solution, gradient elution, detection wavelength: 210 nm, column temperature: 30 °C, flow rate: 0.8 mL / min, sample injection volume: 20 μL, elution procedure is shown in Table 9 below.

[0051] [Table 9]

[0052] The final experimental results are shown in Figure 6. As can be seen from the chromatogram, the peak conditions after 100 minutes have improved somewhat, but the peaks are dense and the resolution is poor. Therefore, we will continue to optimize the concentration gradient, taking into account the effect of the mobile phase organic phase concentration on the peak spacing.

[0053] Example 7 The method for constructing a fingerprint spectrum of the Xinshuning capsule includes the following steps: S1. Preparation of test solutions 15 batches of Xinsuo Ning capsule contents (0.5g each) were precisely weighed and placed in a stoppered conical flask. 25mL of 80% methanol solution was added and ultrasonically extracted for 30 minutes. The resulting filtrate was filtered through a 0.45μm microporous filter membrane to obtain the test solution. S2, chromatogram conditions Chromatography column: Hedera ODS-2-C18 (250 mm × 4.6 mm, 5 μm) chromatographic column, mobile phase: acetonitrile and 0.1% formic acid aqueous solution, gradient elution, 210 nm, column temperature: 30 °C, flow rate: 0.8 mL / min, sample injection volume: 20 μL, elution procedure is shown in Table 10 below.

[0054] [Table 10]

[0055] The final chromatogram is shown in Figure 7. In this example, the chromatogram obtained by following the elution procedure has good peak conditions and good resolution, which meets the requirements for fingerprint spectrum construction. Therefore, this method was ultimately selected as the method for constructing the fingerprint spectrum of Xinshuning Capsules, and subsequent peak identification, similarity analysis, and methodological review were carried out.

[0056] The chromatograms of the Xinshuning Capsule test solution obtained in S3 and S2 were derived and introduced into the Chinese medicine chromatogram fingerprint spectrum similarity evaluation system (2004 A version). Data input, multi-point correction, and data matching were performed on the chromatogram of the test solution, respectively, to obtain the fingerprint spectrum (Figure 27), and similarity analysis was performed (Table 11).

[0057] [Table 11]

[0058] S4. To determine the chemical composition of the fingerprint spectrum, mass spectrometry was performed on the test solution. The instrument used was a Thermo Scientific Ultimate 3000 RSLC ultra-high performance liquid chromatograph - Thermo Scientific Q Exactive Focus benchtop high-resolution mass spectrometer. The mass spectrometry conditions were as follows: electrospray ionization (ESI), spray voltage 3800 V, sheath gas flow rate 45 arb, auxiliary gas flow rate 10 arb, capillary temperature 350°C, mass-to-charge ratio scan range m / z 100-1200, and full scan mode. A total ion flow diagram and mass spectrometry result diagrams of 19 chemical compositions were obtained (Figures 8-26). The detection data was imported into Xcalibur software, and the Qual Browser interface was used to perform data analysis according to the chemical composition peaks. Peak 1 is trifolirhizin at 14.73 min, peak 2 is naringin at 17.31 min, peak 3 is kaempferide at 22.85 min, peak 4 is valine at 24.98 min, peak 5 is palmatine at 26.89 min, peak 6 is epiberberine at 28.03 min, peak 7 is glycyrrhizin / isoglycyrrhizin at 32.76 min, peak 8 is limonin at 35.62 min, peak 9 is liquiritin at 37.68 min, peak 10 is picrotoxin at 44.75 min, and peak 11 is methylopiogonanone A. 50.83 min, peak 12 is rotacin 54.08 min, peak 13 is sophoridine / sophocarpidine 63.19 min, peak 14 is sophoranol / oxidized sophocarpidine 69.17 min, peak 15 is kurarinone 87.14 min, peak 16 is dehydropatiminic acid 96.23 min, peak 17 is polyporenic acid C 108.77 min, peak 18 is quercetin 113.61 min, and peak 19 is sophocarpine 118.39 min.

[0059] S5, Methodological Review

[0060] 1. Stability Experiment The test samples obtained in S1 were collected and analyzed according to the S2 chromatographic conditions at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h, respectively. Trifolirhizin, naringin, kaempferide, valine, palmatine, epiberberine, glycyrrhizin, isoglycyrrhizin, limonin, liquiritin, picrotoxin, methylopiogonanone, rotasin, sophoridine, sophocarpidine, sophoranol, oxidized sophocarpidine, kurarinone, dehydropatimic acid, porinic acid, quercetin, and sophocarpine were used as reference peaks. The peak areas and retention times of the common peaks in the HPLC fingerprint spectra of the samples were analyzed, and the RSD values ​​were calculated to record the chromatograms. The results are shown in Table 12. As can be seen from Table 12, the ratio of the relative retention time and the relative peak area of ​​each major chromatographic peak did not change significantly, with RSDs ranging from 0.32% to 3.07% and from 0.45% to 3.37%, respectively, with RSDs <5.0%, which means that the composition of the test solution was stable within 24 hours.

[0061] 2. Accuracy Experiment The test sample obtained in S1 was collected and measured six times in succession according to the chromatographic conditions of S2. Trifolirhizin, naringin, kaempferide, valine, palmatine, epiberberine, glycyrrhizin, isoglycyrrhizin, limonin, liquiritin, picrotoxin, methylopiogonanone, rotasin, sophoridine, sophocarpidine, sophoranol, oxidized sophocarpidine, kurarinone, dehydropatimic acid, porinic acid, quercetin, and sophocarpine were used as reference peaks. The peak areas and retention times of the common peaks in the HPLC fingerprint spectrum of the sample were analyzed and the RSD values ​​were calculated to record the chromatograms. The results are shown in Table 12. As can be seen, the ratio of the relative retention time of each major chromatographic peak to its relative peak area did not change significantly, and the RSDs were 0.55% to 3.40% and 0.27% to 3.22%, respectively, with RSDs <5.0%, which means that the precision of the instrument is good.

[0062] 3. Reproducibility Experiments Six test samples of Xinshuoning capsules were taken and analyzed according to the above chromatogram conditions. Trifolirhizin, naringin, kaempferide, valine, palmatine, epiberberine, glycyrrhizin, isoglycyrrhizin, limonin, liquiritin, picrotoxin, methylopiogonanone, rotasin, sophoridine, sophocarpidine, sophoranol, oxidized sophocarpidine, kurarinone, dehydropatimic acid, porinic acid, quercetin, and sophocarpine were used as reference peaks. The peak areas and retention times of the common peaks in the HPLC fingerprint spectra of the samples were analyzed to calculate the RSD values, and the chromatograms were recorded. The results are shown in Table 12. The ratios of the relative retention times of each major chromatographic peak and their relative peak areas did not change significantly, with RSDs ranging from 0.34% to 3.78% and from 0.41% to 3.91%, respectively, with RSDs <5.0%, indicating that the reproducibility of this experimental method was good.

[0063] [Table 12]

[0064] As can be seen from the above experimental results, the fingerprint spectrum construction method for Xinshuning Capsules provided by the present invention has the characteristics of good stability, high accuracy and good reproducibility, and can comprehensively and objectively evaluate the quality of Xinshuning Capsules and provide quality assurance of clinical efficacy.

[0065] The above embodiments are merely illustrative examples of the present invention and do not limit the present invention, and the protection scope of the present invention is limited by the claims. Those skilled in the art can make various modifications and equivalent substitutions to the present invention within the substantial protection scope of the present invention, and such modifications and equivalent substitutions shall also be deemed to be included in the protection scope of the present invention.

Claims

1. S1: Collect different batches of Xinxin capsule contents, add methanol aqueous solution, and ultrasonically extract to obtain test solution; S2: injecting the test solution into a high performance liquid chromatograph, performing gradient elution, and performing chromatographic analysis, and recording a chromatogram from 0 to 140 min; S3, the chromatogram obtained in S2 is introduced into the traditional Chinese medicine chromatogram fingerprint spectrum similarity evaluation system, and the chromatogram peaks present in all the chromatograms of the Xinshuoning capsules of different batches are selected as common peaks to obtain a fingerprint spectrum; S4. Mass spectrometry is performed on the test solution, and the chemical composition of the chromatogram peaks in the fingerprint spectrum is determined based on the mass spectrometry results. Trifolirhizin, naringin, kaempferide, valine, palmatine, epiberberine, glycyrrhizin / isoglycyrrhizin, limonin, liquiritin, picrotoxin, metylopiogonanone A, rotacin, sophoridine / sophocarpidine, sophoranol / oxidized sophocarpidine, kurarinone, dehydropatimic acid, polyporenic acid C, quercetin, and sophocarpine are determined as common peaks, and trifolirhizin, naringin, kaempferide, valine, palmatine, epiberberine, glycyrrhizin / isoglycyrrhizin, limonin, liquiritin, picrotoxin, metylopiogonanone A, rotacin, sophoridine / sophocarpidine, sophoranol / oxidized sophocarpidine, kurarinone, dehydropatimic acid, polyporenic acid C, quercetin, and sophocarpine are determined as common peaks. a step of selecting ruhidin, naringin, kaempferide, valine, palmatine, epiberberine, glycyrrhizin / isoglycyrrhizin, limonin, liquiritin, picrotoxin, metylopiogonanone A, rotasin, sophoridine / sophocarpidine, sophoranol / oxidized sophocarpidine, kurarinone, dehydropatimic acid, polyporenic acid C, quercetin, and sophocarpine as common peaks, and designating them as peaks 1 to 19 in order of retention time in high performance liquid chromatography.

2. The method for constructing a fingerprint spectrum of Xinshuning Capsule as described in claim 1, characterized in that in S1, the volume concentration of methanol in the methanol aqueous solution is 80%.

3. The method for constructing a fingerprint spectrum of Xinshuning Capsule as claimed in claim 1, characterized in that in S1, the ultrasonic extraction time is 20-40 minutes.

4. The method for constructing a fingerprint spectrum of Xinshuning Capsules according to claim 1, characterized in that in S2, the detection wavelength used for chromatographic analysis is 210 nm.

5. The fingerprint spectrum construction method for Xinshuning Capsules described in claim 1, characterized in that in S2, the chromatographic column used in the high-performance liquid chromatograph is a Hedera ODS-2-C18 (250 mm x 4.6 mm, 10 μm) chromatographic column.

6. The fingerprint spectrum construction method for Xinshuning Capsules according to claim 1, characterized in that in S2, the mobile phase used for gradient elution is acetonitrile and 0.1% formic acid aqueous solution.

7. In S2, the gradient elution procedure is shown in the table below: The method for constructing a fingerprint spectrum of a heart speeding capsule according to claim 6.

8. The method for constructing a fingerprint spectrum of Xinshuning Capsules according to claim 1, characterized in that in S2, the flow rate used for gradient elution is 0.8 mL / min.

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