Quality detection method of cinobufagin capsule

Abstract

The invention discloses a quality detection method of cinobufagin capsules, which is characterized in that according to active ingredients with different properties in the cinobufagin capsules, analysis conditions such as optimal mobile phase composition, gradient elution mode and mass spectrum condition are screened out through a large number of experiments. According to the UPLC-Q / TOF-MS qualitative analysis method established by the invention, 51 compounds are identified in total. Besides, the fingerprint spectrum of the cinobufagin capsule is established, the content of 11 functional components in the cinobufagin capsule is measured, the HPLC method is good in precision, repeatability and stability, the characteristics of the main components in the cinobufagin capsule are reflected more comprehensively, and reference is provided for improvement of the quality standard and comprehensive development and application of the cinobufagin capsule. The method also has important significance for controlling the quality of cinobufagin capsules and ensuring the clinical curative effect.

Description

Technical Field

[0001] The present invention relates to a quality control method for Chinese patent medicine, and specifically to a quality detection method for Cinobufacini Capsules. Background Art

[0002] Cinobufacini Capsules are a model for the modern application of traditional Chinese medicine. They are a traditional Chinese medicine preparation with the effects of clearing heat and detoxifying, reducing swelling and alleviating pain, which is made from the water-soluble extract of the dried epidermis of the toad Bufo bufo gargarizans Cantor of the Bufonidae family. Cinobufacini Capsules are widely used in clinical practice, showing significant anti-tumor activity, especially outstanding performance in the treatment of advanced cancers. It exerts its effects through multiple pathways, significantly delaying the progression of cancer and improving the quality of life of patients. In addition, Cinobufacini Capsules also have positive effects in the treatment of cancer pain and immune function regulation.

[0003] Nowadays, the component identification mainly focuses on the identification of related components in toad skins and toads, while the research on the component identification of Cinobufacini Capsules is relatively less. The chemical composition system of Cinobufacini Capsules is complex and has a multi-component synergistic effect. Clarifying the chemical composition of Cinobufacini Capsules is the key to deeply understanding its pharmacological action mechanism and improving the clinical treatment effect.

[0004] The UPLC-Q / TOF-MS technology has unique advantages in the co-detection of multiple components and is suitable for the multi-component analysis of complex samples.

[0005] HPLC-PDA has important applications in the analysis of traditional Chinese medicine components and quality control due to its high-efficiency separation and multi-wavelength detection characteristics. At the same time, the fingerprint of traditional Chinese medicine is an effective means for the identification and quality evaluation of medicinal materials, and the quality stability of medicinal materials can be evaluated by constructing the fingerprints of multiple batches of medicinal materials. Summary of the Invention

[0006] Object of the Invention: The object of the present invention is to solve the deficiencies of the prior art. Through a large number of experiments to screen chromatographic conditions and mass spectrometry conditions, UPLC-Q / TOF-MS is used to identify the chemical components in Cinobufacini Capsules. In addition, the present invention uses HPLC-PDA technology to quantitatively analyze the main active components of Cinobufacini Capsules, and establish fingerprints for the active components in different batches of drugs, so as to more comprehensively and objectively reflect the internal quality of the drugs, thereby providing a scientific basis for the overall quality control and evaluation of Cinobufacini Capsules.

[0007] Technical Solution: In order to achieve the above object, the technical solution adopted by the present invention is as follows: A quality detection method for Cinobufacini Capsules, including qualitative analysis and quantitative analysis; (1) The qualitative analysis includes the following steps: Step (1.1): Preparation of the test solution Accurately weigh the content of the Cinobufacini Capsules, place it in a stoppered conical flask, accurately add methanol, weigh, ultrasonically treat at room temperature, weigh again, make up the lost amount with methanol, shake well, and let stand; before analysis, dilute the sample, filter, and take the subsequent filtrate to obtain the test solution; Step (1.2) Analyze the compounds in the Cinobufacini Capsules under the established UPLC-Q / TOF-MS conditions Inject the test solution from step (1) into the UPLC, analyze the components in the Cinobufacini Capsules under the established UPLC-Q / TOF-MS conditions, obtain the total ion current chromatograms of the Cinobufacini Capsules in positive and negative ion modes, and identify the chemical components in the Cinobufacini Capsules by comparing the accurate mass-to-charge ratio with the information in the database; (2) The quantitative analysis includes the following steps: (2.1) Preparation of the mixed reference solution Take appropriate amounts of reference standards of 5-hydroxytryptamine, bufothionine, resibufogenin, arenobufagin, deacetylresibufogenin, telocinobufagin, bufotalin, bufotenine, bufalin, cinobufagin, and esterscinobufagin, accurately weigh, dissolve in methanol to prepare single reference stock solutions; accurately measure appropriate amounts of the above reference stock solutions, mix, and dilute with methanol to prepare a mixed reference solution; (2.2) Preparation of the test solution Take the contents of different batches of Cinobufacini Capsules, add methanol, ultrasonically treat at room temperature, weigh again, make up the lost amount with methanol, shake well, let stand, take the supernatant and filter through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain it; (2.3) Investigation of the linear relationship Take the mixed reference solution from step (2.1), add methanol and dilute it step by step by 1 - 5 times, inject it into the HPLC-PDA for determination respectively, and construct a regression equation based on the mass concentration and peak area; (2.4) Establishment of the fingerprint Take the test solutions of different batches of Cinobufacini Capsules, inject them into the HPLC-PDA respectively, record their HPLC chromatograms respectively, import the HPLC chromatograms of different batches of Cinobufacini Capsule samples into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints", take the chromatogram of sample S6 as the reference chromatogram, select the median method as the generation method of the reference fingerprint, set the time window width to 0.1, use the multi-point calibration method for full-peak matching to generate the reference fingerprint (R), and conduct common peak comparison and similarity investigation; (2.5) Determination of the sample content Accurately weigh the test sample solutions of Cinobufacini capsules from different batches, inject them into HPLC-PDA to determine the test sample solutions of Cinobufacini capsules, and measure in parallel 3 times. Calculate the mass fractions of each component in the Cinobufacini capsules according to the linear equation in step (2.3).

[0008] As an optimized scheme, for the quality inspection method of the Cinobufacini capsules described above, step (1.1): The preparation method of the test sample solution is as follows: Accurately weigh 1.0 - 5.0 g of the content of Cinobufacini capsules, place it in a stoppered conical flask, accurately add 10 - 50 mL of methanol, weigh it, ultrasonically treat it at room temperature for 30 - 60 min, weigh it again, make up the lost amount with methanol, shake well, and let it stand; before analysis, dilute the sample to 1 mg / mL, take the supernatant and filter it through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain the test sample solution.

[0009] As an optimized scheme, for the quality inspection method of the Cinobufacini capsules described above, the chromatographic conditions in step (2) are Waters Acquity BEH C 18 chromatographic column, with specifications of 2.1 mm × 100 mm, 1.7 μm, mobile phase being 0.1% formic acid aqueous solution A - acetonitrile B; gradient elution program: 0 - 1 min, 95% A; 1 - 6.5 min, 95 - 53% A; 6.5 - 13 min, 53 - 5% A; 13 - 14 min, 5% A; 14 - 15 min, 5 - 95% A; 15 - 16 min, 95% A; injection volume is 1 μL, flow rate is 0.3 mL / min, column temperature is 35°C; The mass spectrometry conditions are as follows: Negative ion mode, source conditions: capillary voltage is 2.5 kV, sample cone temperature is 40°C, source temperature is 120°C, desolvation gas temperature is 280°C, cone gas flow rate is 50 L / h, desolvation gas N2 flow rate is 600 L / h; the data acquisition in this mode covers the mass spectrometry range of mass-to-charge ratio 50 - 1200 Da; In the positive ion mode, source conditions: capillary voltage is 3.0 kV, sample cone temperature is 55°C, source temperature is 100°C, desolvation gas temperature is 350°C, cone gas flow rate is 50 L / h, desolvation gas N2 flow rate is 800 L / h; the data acquisition in this mode covers the mass spectrometry range of mass-to-charge ratio 50 - 1200 Da; The control of data acquisition and analysis is carried out by using Waters MassLynx 4.2 software.

[0010] As an optimized scheme, for the quality inspection method of the Cinobufacini capsules described above, the preparation method of the mixed reference solution in step (2.1) is as follows: Take appropriate amounts of serotonin, bufotalin, resibufogenin, arenobufagin, deacetylresibufogenin, telocinobufagin, bufotalidin, bufotenine, bufalin, cinobufagin, and cinobufotalin reference substances, accurately weigh them, dissolve them in methanol to prepare a single reference substance stock solution; accurately measure an appropriate amount of the above reference substance stock solution, mix them, and dilute with methanol to prepare a mixed reference substance solution containing 0.16 mg of serotonin, 0.015 mg of bufotalin, 0.05 mg of resibufogenin, 0.1 mg of arenobufagin, 0.03 mg of deacetylresibufogenin, 0.03 mg of telocinobufagin, 0.16 mg of bufotalidin, 0.12 mg of bufotenine, 0.05 mg of bufalin, 0.1 mg of cinobufagin, and 0.12 mg of cinobufotalin per 1 mL.

[0011] As a preferred embodiment, for the quality inspection method of the cinobufacin capsules described above, the preparation method of the test solution in step (2.2) is as follows: Take the contents of cinobufacin capsules of different batches, accurately weigh about 1.5 g, place them in a stoppered conical flask, accurately add 6 mL of methanol, weigh, ultrasonically treat at a power of 500 W and a frequency of 40 kHz at room temperature for 30 min, weigh again, make up the lost amount with methanol, shake well, let stand, take the supernatant and filter it through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain the test solution.

[0012] As a preferred embodiment, for the quality inspection method of the cinobufacin capsules described above, the chromatographic elution conditions in steps (2.3) to (2.5) are as follows: Use Waters SunFire C 18 , with a specification of 4.6×250 mm and 5 µm chromatographic column; the mobile phase is acetonitrile A - 0.1% formic acid aqueous solution B, and the gradient elution conditions are: 0 - 6 min, 5 - 8% A; 6 - 10 min, 8 - 18% A; 10 - 30 min, 18 - 43% A; 30 - 32 min, 43 - 47% A; 32 - 40 min, 47 - 60% A; 40 - 42 min, 60 - 45% A; 42 - 44 min, 45% A; 44 - 46 min, 45 - 90% A; 46 - 48 min, 90 - 5% A; 48 - 50 min, 5% A.

[0013] As an optimal solution, for the quality inspection method of the Cinobufacini capsules described above, in step (2.4), the chromatogram of sample S6 is used as the reference chromatogram, and the test solution of 13 batches of Cinobufacini capsules is compared. Among them, 15 chromatographic peaks are common to the 13 batches of Cinobufacini capsule samples and are determined as common peaks. The similarity is calculated with reference to the control fingerprint chromatogram; 11 of the peaks are identified with reference substances, which are peak 1 5-hydroxytryptamine, peak 2 bufothionine, peak 3 gamabufotalin, peak 4 arenobufagin, peak 5 deacetylgamabufotalin, peak 6 telocinobufagin, peak 7 bufotalin, peak 8 bufotenine, peak 9 bufalin, peak 10 cinobufagin, and peak 11 esterscinobufagin peak.

[0014] As an optimal solution, for the quality inspection method of the Cinobufacini capsules described above, the linear regression equation in step (2.3) is: Ingredient Regression equation <![CDATA R 2 > <![CDATA[Linear range / μg·mL -1 > 5-Hydroxytryptamine y = 2E+07x + 37356 0.9993 3.12~100.00 Bufothionine y = 3E+06x + 19123 0.9999 50.00-1600.00 3alpha-hydroxy-5alpha,14alpha-Bufadienolide y = 1E+07x + 7887.2 0.9995 2.50-80.00 Cinobufotalin y = 1E+07x + 13850 0.9997 7.81-250.00 3-argininylsuccinoyl bufotalin y =1E+07x - 2680.9 0.9997 2.50-80.00 5beta-Pregn-11-ene-3,20-dione y = 2E+07x - 6434.7 0.9999 1.56-50.00 Bufalin y = 2E+07x - 12904 0.9993 1.56-50.00 Bufotenine y = 1E+07x + 9969.1 0.9993 3.90-125.00 Bufalin y = 2E+07x + 30601 0.9990 3.90-125.00 Cinobufagin y = 2E+07x + 7568.3 0.9993 1.95-62.50 Resibufogenin y = 1E+07x + 6919.7 0.9999 3.12-100.00 Beneficial effects: The quality inspection method of the Cinobufacini capsules provided by the present invention has the following advantages compared with the prior art: According to the active ingredients with different properties in the Cinobufacini capsules, the present invention screens out the best analysis conditions such as the composition of the mobile phase, the elution program mode, and the mass spectrometry conditions through a large number of experiments. The present invention uses UPLC-Q / TOF-MS to analyze the chemical components in the Cinobufacini capsules, and a total of 51 compounds are identified, including 44 bufadienolide compounds, 2 tryptamine compounds, 1 steroid compound, 1 heterocyclic compound, 1 polypeptide compound, 1 amino acid compound, and 1 dicarboxylic acid compound. This result highlights that the Cinobufacini capsules are mainly composed of bufadienolide compounds and have the structural characteristics of multiple types of active substances such as tryptamine compounds, steroid compounds, and polypeptide compounds, reflecting the chemical diversity of the raw medicinal material toad skin and the complexity of its processing process, laying a material foundation for subsequent clarification of the key pharmacodynamic components, quality control standards, and mechanism of action research of the Cinobufacini capsules.

[0015] The present invention uses HPLC-PDA to qualitatively and quantitatively analyze the main efficacy components of the Cinobufacini capsules. First, the contents of each component in the Cinobufacini capsules are determined by HPLC-PDA, and then the fingerprint chromatogram of 13 batches of Cinobufacini capsules is established to characterize the efficacy components. 13 common peaks are determined, and 11 components including 5-hydroxytryptamine, bufothionine, gamabufotalin, arenobufagin, deacetylgamabufotalin, telocinobufagin, bufotalin, bufotenine, bufalin, cinobufagin, and esterscinobufagin are identified with reference substances. Among them, 5-hydroxytryptamine, bufothionine, and bufotenine are indole alkaloid components, and the remaining 8 components are all bufadienolide components. At the same time, the similarity of different batches of Cinobufacini capsules is compared, and it can be seen that the similarity of the 13 batches of drugs is relatively high, and the quality of the medicinal materials is stable among different batches.

[0016] The present invention establishes the fingerprint of Cinobufacini capsules and determines the contents of 11 active ingredients in Cinobufacini capsules. The HPLC method has good precision, repeatability and stability, and can more comprehensively reflect the characteristics of the main components in Cinobufacini capsules, providing a reference for the improvement of the quality standard and the comprehensive development and application of Cinobufacini capsules. This method is also of great significance for controlling the quality of Cinobufacini capsules and ensuring clinical efficacy. Description of the Drawings

[0017] Figure 1 Total ion chromatograms of the Cinobufacini capsule sample in positive (A) and negative (B) modes.

[0018] Figure 2 Chromatograms of the Cinobufacini capsule sample (A), the mixed reference substance (B) solution, and the chemical structural formula diagram of the identified components (C).

[0019] Figure 3 Fingerprint of batch Cinobufacini capsules. Detailed Embodiments

[0020] The present invention will be further illustrated below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. After reading the present invention, various equivalent modifications of the present invention by those skilled in the art fall within the scope defined by the appended claims of this application.

[0021] Example 1 Qualitative Analysis of Chemical Components in Cinobufacini Capsules Based on UPLC-Q / TOF-MS Method 1 Instruments and Reagents Instruments and Reagents Manufacturing Company ACQUITYTM UPLC System Waters Corporation, USA SYNAPT G2-Si Q-TOF High Resolution Time-of-Flight Mass Spectrometer Waters Corporation, USA Waters Acquity BEH C18 Column (2.1 mm × 100 mm, 1.7 μm) Waters Corporation, USA Microfuge 20R Desktop High-Speed Refrigerated Centrifuge Beckman Coulter, Inc., USA Milli-Q Ultrapure Water System Millipore Corporation, USA Acetonitrile (Mass Spectrometry Grade) Honeywell Burdick&Jackson Company Formic Acid (Mass Spectrometry Grade) Merck KGaA, Darmstadt, Germany Formic Acid (Mass Spectrometry Grade) Merck KGaA, Darmstadt, Germany Cinobufagin Capsules (Lot No.: 2H13 03932) Shaanxi Dongtai Pharmaceutical Co., Ltd. 2 Experimental Methods 2.1 Chromatographic Conditions Use a Waters Acquity BEH C18 chromatographic column (2.1 mm × 100 mm, 1.7 μm), and the mobile phase is 0.1% formic acid aqueous solution (A) - acetonitrile (B). Gradient elution program: 0 - 1 min, 95% A; 1 - 6.5 min, 95 - 53% A; 6.5 - 13 min, 53 - 5% A; 13 - 14 min, 5% A; 14 - 15 min, 5 - 95% A; 15 - 16 min, 95% A. The injection volume is 1 μL, the flow rate is 0.3 mL / min, and the column temperature is 35°C.

[0022] 2.2 Mass Spectrometry Conditions Negative ion mode, source conditions: capillary voltage is 2.5 kV, sample cone temperature is 40 °C, source temperature is 120 °C, desolvation gas temperature is 280 °C, cone gas flow rate is 50 L / h, desolvation gas (N2) flow rate is 600 L / h. The data acquisition in this mode covers the mass spectrometry range of m / z 50 - 1200 Da.

[0023] In positive ion mode, source conditions: capillary voltage is 3.0 kV, sample cone temperature is 55 °C, source temperature is 100 °C, desolvation gas temperature is 350 °C, cone gas flow rate is 50 L / h, desolvation gas (N2) flow rate is 800 L / h. The data acquisition in this mode covers the mass spectrometry range of m / z 50 - 1200 Da. The control of data acquisition and analysis is carried out by using Waters MassLynx 4.2 software.

[0024] 2.3 Preparation of test samples Precisely weigh 1.0 g of the content of Cinobufacini capsules, place it in a stoppered conical flask, precisely add 10 mL of methanol, weigh it, ultrasonically treat it (power 500 W, frequency 40 kHz) at room temperature for 30 min, weigh it again, make up the lost amount with methanol, shake well, and let it stand. Before analysis, dilute the sample to 1 mg / mL, take the supernatant and filter it through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain the test sample solution.

[0025] 2.4 Data processing and result analysis Use MassLynx v4.2 software to process the data. According to the retention time and secondary mass spectrometry fragments of each chemical component in positive and negative ion modes, compare with the UNIFI database and relevant literature data to confirm the chemical components.

[0026] 3. Experimental results 3.1 Identification of chemical components in Cinobufacini capsules Analyze the components in Cinobufacini capsules under the established UPLC - Q / TOF - MS conditions to obtain the total ion current chromatograms of Cinobufacini capsules in positive and negative ion modes ( Figure 1 ). By searching the literature for comparison, a total of 46 compounds were identified, and the detailed information is shown in Tables 1 - 4.

[0027] Table 1 Serial Number English Name Molecular Formula Theoretical Value Actual Value Molecular Ion Peak Retention Time (min) Secondary Fragment Ion 1 Hexahydropyrrolo[1,2-A]pyrazine-1,4-dione <![CDATA[C7H 10 N2O2]]> 154.0742 193.0376 <![CDATA[[M+K] + > 0.94 115.0949 2 Bufothionine <![CDATA[C 12 H 14 N2O4S]]> 282.0674 281.0610 <![CDATA[[M-H] - > 3.94 189.858，141.868 3 3alpha-hydroxy-5alpha,14alpha-Bufadienolide <![CDATA[C 24 H 34 O3]]> 370.2508 388.2879 <![CDATA[[M+NH4] + > 3.95 191.1397 4 Cinobufotalin <![CDATA[C 26 H 34 O7]]> 459.2377 459.2373 <![CDATA[[M+H] + > 5.20 345.1859，417.2235 5 3alpha-hydroxy-5alpha,14alpha-Bufadienolide <![CDATA[C 24 H 34 O3]]> 370.2508 371.2576 <![CDATA[[M+H] + > 5.97 353.2501，165.1298 6 3-argininylsuccinoyl bufotalin <![CDATA[C 36 H 52 N4O 10 > 700.3683 718.4062 <![CDATA[[M+NH4] + > 6.15 547.3463 7 5beta-Pregn-11-ene-3,20-dione <![CDATA[C 21 H 30 O2]]> 314.2246 353.1843 <![CDATA[[M+K] + > 6.40 299.196 8 Suberic acid <![CDATA[C8H 14 O4]]> 174.0892 173.0777 <![CDATA[[M-H] - > 6.44 111.0819，127.0741 9 Marinobufagin-3-pimeloyl-L-arginine ester <![CDATA[C 37 H 54 N4O9]]> 698.3891 699.3975 <![CDATA[[M+H] + > 6.96 97.0199 10 7beta-Hydroxybufalin <![CDATA[C 24 H 34 O5]]> 402.2406 447.2389 <![CDATA[[M+HCOO] - > 7.56 405.2612，319.9258 11 Glaucorigenin <![CDATA[C 23 H 32 O6]]> 404.2199 463.2346 <![CDATA[[M+CH3COO] - > 7.99 373.2313 12 19-hydroxydesacetylcinobufagin <![CDATA[C 24 H 32 O6]]> 416.2199 461.2183 <![CDATA[[M+HCOO] - > 8.37 353.2141，397.1979 13 Cinobufaginol <![CDATA[C 26 H 34 O7]]> 458.54396 458.2305 <![CDATA[[M+HCOO] - > 10.13 440.2238，357.0602 14 3beta-Acetoxy-15alpha-Hydroxybufalin <![CDATA[C 26 H 36 O6]]> 444.2512 489.2500 <![CDATA[[M+HCOO] - > 10.34 329.1779 15 19-oxo-bufalin <![CDATA[C 24 H 32 O5]]> 400.2250 445.2244 <![CDATA[[M+HCOO] - > 10.55 121.0295，329.1808 16 Bufalin <![CDATA[C 24 H 34 O4]]> 386.2457 431.2446 <![CDATA[[M+HCOO] - > 10.64 425.2098，373.2820 Table 2 Serial Number English Name Molecular Formula Theoretical Value Actual Value Molecular Ion Peak Retention Time (min) Secondary Fragment Ion 17 19-hydroxybufalin-3-(16-hydroxy-palmitate) <![CDATA[C 40 H 64 O7]]> 656.4652 695.4265 <![CDATA[[M+K] + > 10.97 545.3885，462.4108，257.2426 18 Cinobufotoxin <![CDATA[C 40 H 58 N4O 10 > 754.4153 772.4465 <![CDATA[[M+NH 4 + > ​ 11.13 341.1352，596.3931，480.3225，339.2821，618.4009 19 Marinobufagin3-hemisuberate <![CDATA[C 32 H 46 O8]]> 556.3036 579.2875 <![CDATA[[M+Na] + > 11.53 437.2329，308.1636，298.1797，246.0879 20 Argentinogenin <![CDATA[C 24 H 30 O6]]> 414.2042 459.2022 <![CDATA[[M+HCOO] - > 11.82 387.258 21 Bufalin 3-sulfate <![CDATA[C 24 H 34 O7S]]> 466.2025 505.1679 <![CDATA[[M+K] + > 12.30 259.2071 22 3-O-(methylsuberoyl)-gamabufotalin <![CDATA[C 33 H 48 O8]]> 572.3349 611.2943 <![CDATA[[M+K] + > 12.46 365.2349，409.2542 23 Bufalone <![CDATA[C 24 H 32 O4]]> 384.2301 383.2228 <![CDATA[[M-H] - > 13.17 221.1182 24 2beta-acetoxy-3,5-di-O-acetylhellebrigenin <![CDATA[C 30 H 38 O 10 > 558.2465 581.2358 <![CDATA[[M+Na] + > 13.30 360.1567 25 Gamabufotalin-3-hemiadipat-methylester <![CDATA[C 31 H 44 O8]]> 544.3036 545.3127 <![CDATA[[M+H] + > 13.39 351.2919 26 Hellebrigenin-3-(14-hydroxy-6Z-tetradecenoate) <![CDATA[C 38 H 56 O8]]> 640.3975 658.4344 <![CDATA[[M+NH4] + > 13.46 183.1758 27 Scillaridin A <![CDATA[C 24 H 30 O3]]> 366.2195 367.2260 <![CDATA[[M+H] + > 13.53 324.2061 28 Resibufogenin <![CDATA[C 24 H 32 O4]]> 385.2379 385.2382 <![CDATA[[M+H] + > 13.59 351.2393 Table 3 Serial Number English Name Molecular Formula Theoretical Value Actual Value Molecular Ion Peak Retention Time (min) Secondary Fragment Ion 29 ResibufogeninAcetate <![CDATA[C 26 H 34 O5]]> 426.2406 427.2486 <![CDATA[[M+H] + > 13.60 385.2372 30 Bufalin 3-suberateBochydrazine <![CDATA[C 37 H 56 N2O8]]> 656.4037 674.4377 <![CDATA[[M+NH4] + > 13.61 385.2372，614.3923，359.2266，403.2469 31 GLY-LEU-LEU-GLY <![CDATA[C 16 H 30 N4O5]]> 358.2216 359.2264 <![CDATA[[M+H] + > 13.62 300.1915，243.0725，240.1842，241.1821 32 Bufalin <![CDATA[C 24 H 34 O4]]> 386.2457 409.2321 <![CDATA[[M+Na] + > 13.73 359.2261 33 3alpha-hydroxy-5alpha,14alpha-bufadienolide <![CDATA[C 24 H 34 O3]]> 370.2508 393.2409 <![CDATA[[M+Na] + > 14.22 315.1961 34 Acetylmarinobufogenin <![CDATA[C 26 H 34 O6]]> 442.2355 443.2441 <![CDATA[[M+H] + > 14.32 169.1224 35 16beta-hydroxy-pseudobufarenogin <![CDATA[C 24 H 32 O7]]> 432.2148 450.2522 <![CDATA[[M+NH4] + > 14.56 140.0486 36 3beta-Acetoxyresibufogenin <![CDATA[C 26 H 34 O5]]> 426.2406 449.2332 <![CDATA[[M+Na] + > 14.75 253.1989 37 Bufalin-3-hemisuberate <![CDATA[C 32 H 46 O7]]> 542.3244 543.3333 <![CDATA[[M+H] + > 14.78 365.3049 38 BufogargarizinA <![CDATA[C 25 H 30 O8]]> 458.1941 497.1541 <![CDATA[[M+K] + > 14.85 413.1218，178.1354，165.1298 39 Bufotenidine <![CDATA[C 13 H 18 N2O]]> 218.1419 241.1288 <![CDATA[[M+Na] + > 14.90 160.0827 40 Cinobufagin-3-succinoylargininester <![CDATA[C 36 H 50 N4O 10 > 698.3527 721.3409 <![CDATA[[M+Na] + > 15.01 279.1438，621.2945，666.3426，638.3106 41 Resibufagin <![CDATA[C 24 H 30 O5]]> 398.2093 437.1728 <![CDATA[[M+K] + > 15.24 152.1187 42 Scillaridin A <![CDATA[C 24 H 30 O3]]> 366.2195 384.2560 <![CDATA[[M+NH4] + > 15.29 215.1407 Table 4 Serial Number English Name Molecular Formula Theoretical Value Actual Value Molecular Ion Peak Retention Time (min) Secondary Fragment Ion 43 Leucine <![CDATA[C6H 13 NO2]]> 132.1025 132.1031 <![CDATA[[M+H] + > 15.30 113.0153 44 Acetylbufalin <![CDATA[C 26 H 36 O5]]> 428.2563 429.2666 <![CDATA[[M+H] + > 15.32 339.2312 45 19-Oxocinobufagin <![CDATA[C 26 H 32 O7]]> 456.2148 479.2008 <![CDATA[[M+Na] + > 15.33 140.0485，168.0464，407.1495，79.0559 46 Xanthine <![CDATA[C5H4N4O2]]> 152.0334 153.0407 <![CDATA[[M+H] + > 15.45 125.0179，138.0249 Example 2 Quantitative Analysis of the Main Active Components in Cinobufacini Capsules by HPLC-PDA Method 1 Experimental Materials 1.1 Reagents The purity of all reference substances determined by liquid chromatography was ≥ 98%. Acetonitrile (Honeywell International Inc., chromatographic grade); formic acid (Merck KGaA, chromatographic grade).

[0028] Reagent Manufacturing Company Batch Number 5-Hydroxytryptamine Nanjing Yuanzhi Biotechnology Co., Ltd. Yz0711222 Bufotenine Nanjing Yuanzhi Biotechnology Co., Ltd. Yz0626222 Gamabufotalin Nanjing Yuanzhi Biotechnology Co., Ltd. Yz0620222 Cinobufagin Nanjing Yuanzhi Biotechnology Co., Ltd. Yz0624222 Desacetylcinobufotalin Chengdu Biaoyang Biotechnology Co., Ltd. wkq23012909 Telocinobufagin Chengdu Biaoyang Biotechnology Co., Ltd. wkq22092913 Bufotalin Nanjing Yuanzhi Biotechnology Co., Ltd. Yz0621222 Cinobufotalin Nanjing Yuanzhi Biotechnology Co., Ltd. Yz0622222 Bufalin Nanjing Yuanzhi Biotechnology Co., Ltd. Yz0421222 Cinobufacine Nanjing Yuanzhi Biotechnology Co., Ltd. Yz081421 Resifugin Chengdu Standard Biotechnology Co., Ltd. wkq22060601 1.2 Instruments Instruments and reagents Production Company Waters 2695 HPLC Waters Corporation 2998 PDA Delector Photodiode Array Detector Waters Corporation KQ-500DE CNC Ultrasonic Cleaner Kunshan Ultrasonic Instrument Co., Ltd. Mili-Q IQ7000 Ultrapure Water System Merck Millipore Mettler MS105DU Electronic Analytical Balance Mettler-Toledo AG Mettler ME204 electronic balance Mettler-Toledo AG 1.3 Cinobufacini Capsules All cinobufacini capsules were purchased from Shaanxi Dongtai Pharmaceutical Co., Ltd. The batch information of 13 batches of cinobufacini capsules is shown in Table 5.

[0029] Table 5 Batch Numbers and Sources of 13 Batches of Cinobufacini Capsules Serial number batch source Serial number batch source S1 2C01 Shaanxi Dongtai Pharmaceutical Co., Ltd. S2 1E02 09601 Shaanxi Dongtai Pharmaceutical Co., Ltd. S3 2H13 03932 Shaanxi Dongtai Pharmaceutical Co., Ltd. S4 2H13 03968 Shaanxi Dongtai Pharmaceutical Co., Ltd. S5 1I05 13682 Shaanxi Dongtai Pharmaceutical Co., Ltd. S6 2F09 02504 Shaanxi Dongtai Pharmaceutical Co., Ltd. S7 2G04 03079 Shaanxi Dongtai Pharmaceutical Co., Ltd. S8 2G04 03074 Shaanxi Dongtai Pharmaceutical Co., Ltd. S9 2F11 02722 Shaanxi Dongtai Pharmaceutical Co., Ltd. S10 2F11 02695 Shaanxi Dongtai Pharmaceutical Co., Ltd. S11 2E11 01509 Shaanxi Dongtai Pharmaceutical Co., Ltd. S12 2F11 02729 Shaanxi Dongtai Pharmaceutical Co., Ltd. S13 2H05 03364 Shaanxi Dongtai Pharmaceutical Co., Ltd. 2 Experimental Methods 2.1 Chromatographic Elution Conditions A Waters SunFire C18 column (4.6×250 mm, 5 µm) was used; the mobile phase was acetonitrile (A) - 0.1% formic acid aqueous solution (B), and the gradient elution conditions were as follows: 0 - 6 min, 5 - 8% A; 6 - 10 min, 8 - 18% A; 10 - 30 min, 18 - 43% A; 30 - 32 min, 43 - 47% A; 32 - 40 min, 47 - 60% A; 40 - 42 min, 60 - 45% A; 42 - 44 min, 45% A; 44 - 46 min, 45 - 90% A; 46 - 48 min, 90 - 5% A; 48 - 50 min, 5% A.

[0030] 2.2 Preparation of Reference Substance Solution Appropriate amounts of reference substances of 5-hydroxytryptamine, bufothionine, resibufogenin, arenobufagin, deacetylresibufogenin, telocinobufagin, bufotalin, bufotenine, bufalin, cinobufagin, and estersbufagin were accurately weighed and dissolved in methanol to prepare single reference substance stock solutions; appropriate amounts of the above stock solutions were accurately measured, mixed, and diluted with methanol to finally prepare a mixed reference substance solution containing 0.16 mg of 5-hydroxytryptamine, 0.015 mg of bufothionine, 0.05 mg of resibufogenin, 0.1 mg of arenobufagin, 0.03 mg of deacetylresibufogenin, 0.03 mg of telocinobufagin, 0.16 mg of bufotalin, 0.12 mg of bufotenine, 0.05 mg of bufalin, 0.1 mg of cinobufagin, and 0.12 mg of estersbufagin per 1 mL.

[0031] 2.3 Preparation of Test Solution Take the content of Cinobufacini Capsules, accurately weigh about 1.5 g, place it in a stoppered conical flask, accurately add 6 mL of methanol, weigh it, ultrasonically treat it at room temperature (power 500 W, frequency 40 kHz) for 30 minutes, weigh it again, make up the lost amount with methanol, shake well, let it stand, take the supernatant and filter it through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain the solution.

[0032] 2.4 Methodology Investigation 2.4.1 Investigation of Linear Relationship Take the mixed reference solution, add methanol for stepwise dilution, and determine it according to the chromatographic conditions under item "2.1", and construct a regression equation based on the mass concentration and peak area.

[0033] 2.4.2 Precision Test Precisely pipette 10 μL of the mixed reference solution, prepare the reference solution according to the method under item "2.2", continuously inject samples 6 times according to the chromatographic conditions under item "2.1", record the peak area, and calculate the RSD.

[0034] 2.4.3 Repeatability Test Take the Cinobufacini Capsules of batch S1, prepare 6 portions of test solutions in parallel according to the method under item "2.3". Continuously inject samples according to the chromatographic conditions under item "2.1", record the peak area, and calculate the RSD.

[0035] 2.4.4 Stability Test Take the Cinobufacini Capsules of batch S1, prepare 6 portions of test solutions in parallel according to the method under item "2.3". According to the chromatographic conditions under item "2.1", inject samples and determine at 0, 2, 4, 8, 12, and 24 hours respectively, record the peak area, and calculate the RSD.

[0036] 2.4.5 Spiking Recovery Test Take 6 portions of the content of Cinobufacini Capsules (S1) with known content, each about 0.1 g, accurately weigh them, respectively add the reference substance equivalent to the content of the component to be measured in the sample, prepare the test solution according to the method under item "2.3", determine it according to the chromatographic conditions under item "2.1", record the peak area, and calculate the recovery rate.

[0037] 2.5 Fingerprint Study Prepare 13 batches of test solution of Cinobufacini capsules according to the method under item "2.3", inject samples according to the chromatographic conditions under item "2.1", and record their HPLC chromatograms respectively. Input the HPLC chromatograms of 13 batches of Cinobufacini capsules into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints" (version 2012.130723). Using the chromatogram of sample S6 as the reference chromatogram, select the median method as the method for generating the control fingerprint, set the time window width to 0.1, and use the multi-point calibration method for full-peak matching to generate the control fingerprint (R), and conduct comparison of common peaks and similarity investigation.

[0038] 2.5 Determination of sample content Precisely weigh the contents of 13 batches of Cinobufacini capsules, prepare the test solution according to the method under item "2.3", determine the test solution of Cinobufacini capsules according to the chromatographic conditions under item "2.1", and measure in parallel for 3 times. Calculate the mass fractions of each component in Cinobufacini capsules according to the linear equation.

[0039] 3 Experimental results 3.1 Methodology investigation 3.1.1 Investigation of linear relationship Perform regression with the mass concentration of each component as the abscissa (X) and the peak area as the ordinate (Y). The results are shown in Table 6, indicating that each component has a good linear relationship within its respective range.

[0040] Table 6 Linear relationships of each component Element Regression equation <![CDATA R 2 > <![CDATA[Linear range (μg·mL -1 )]]> Serotonin y = 2E+07x + 37356 0.9993 3.12~100.00 Toad Thien y = 3E+06x + 19123 0.9999 50.00-1600.00 Nitofatin y = 1E+07x + 7887.2 0.9995 2.50-80.00 Sand toad venom y = 1E+07x + 13850 0.9997 7.81-250.00 Deacetyl cinobufolin y =1E+07x - 2680.9 0.9997 2.50-80.00 Cinotoxin y = 2E+07x - 6434.7 0.9999 1.56-50.00 Bufalin y = 2E+07x - 12904 0.9993 1.56-50.00 Toad venom tryptamine y = 1E+07x + 9969.1 0.9993 3.90-125.00 Bufalin y = 2E+07x + 30601 0.9990 3.90-125.00 Cinobufacine y = 2E+07x + 7568.3 0.9993 1.95-62.50 Resifugin y = 1E+07x + 6919.7 0.9999 3.12-100.00 3.1.2 Precision test The results show that the RSDs of the peak areas of serotonin, bufothionine, gamabufotalin, arenobufagin, deacetylgamabufotalin, telocinobufagin, bufotalin, bufotenine, bufalin, cinobufagin, and esterscinobufagin are 0.86%, 1.67%, 2.88%, 1.01%, 3.11%, 1.54%, 1.62%, 1.18%, 0.93%, 0.88%, and 0.96% respectively, indicating good precision of the instrument.

[0041] 3.1.3 Repeatability experiment The results show that the RSDs of the peak areas of serotonin, bufothionine, gamabufotalin, arenobufagin, deacetylgamabufotalin, telocinobufagin, bufotalin, bufotenine, bufalin, cinobufagin, and esterscinobufagin are 2.65%, 2.63%, 2.45%, 3.18%, 1.61%, 1.97%, 2.74%, 3.63%, 2.48%, 2.28%, and 2.72% respectively, indicating good repeatability of this method.

[0042] 3.1.4 Stability test The results showed that the RSDs of the peak areas of serotonin, bufotalinine, gamabufotalin, arenobufagin, deacetylcinobufagin, telocinobufagin, cinobufotalin, bufotenine, bufalin, cinobufagin, and resibufogenin were 2.74%, 1.98%, 3.47%, 3.25%, 2.71%, 3.44%, 3.28%, 2.26%, 3.78%, 3.54%, and 3.56%, respectively, indicating that the solution had good stability within 24 h.

[0043] 3.1.5 Sampling Recovery Test As shown in Tables 7 and 8, the recoveries of serotonin, bufotalinine, gamabufotalin, arenobufagin, deacetylcinobufagin, telocinobufagin, cinobufotalin, bufotenine, bufalin, cinobufagin, and resibufogenin were 101.33%, 105.05%, 94.12%, 91.89%, 96.91%, 91.89%, 95.79%, 96.71%, 100.08%, 104.21%, and 94.53%, respectively, and the recoveries met the requirements.

[0044] Table 7 Sampling Recovery Results (n = 6)

[0045] Table 8 Sampling Recovery Results (n = 6)

[0046] 3.2 Fingerprint Taking the chromatogram of sample S6 as the reference chromatogram, the test solutions of 13 batches of cinobufacini capsules were compared. Among them, 15 chromatographic peaks were common to the 13 batches of cinobufacini capsule samples and were determined as common peaks. The similarity was calculated with reference to the control fingerprint. Eleven of the peaks were identified with reference substances, namely peak 1 serotonin, peak 2 bufotalinine, peak 3 gamabufotalin, peak 4 arenobufagin, peak 5 deacetylcinobufagin, peak 6 telocinobufagin, peak 7 cinobufotalin, peak 8 bufotenine, peak 9 bufalin, peak 10 cinobufagin, and peak 11 resibufogenin peak, as shown in Figure 3 ... The similarities of the 13 batches of samples were between 0.945 and 0.999, as shown in Table 9, indicating that the chemical compositions of each batch of cinobufacini capsules were relatively consistent and the quality stability between batches was good.

[0047] Table 9 Similarities of Cinobufacini Capsule Fingerprints serial number Similarity serial number Similarity S1 0.995 S2 0.945 S3 0.993 S4 0.994 S5 0.992 S6 0.996 S7 0.999 S8 0.998 S9 0.997 S10 0.998 S11 0.998 S12 0.977 S13 0.992 3.3 Determination of Sample Content The mass fraction obtained from three parallel determinations of the test solution was averaged to obtain the sample content value, and the results are shown in Tables 10 and 11.

[0048] Table 10

[0049] Table 11

[0050] In this study, the fingerprint of Cinobufacini Capsules was established, and the contents of 11 active ingredients in Cinobufacini Capsules were determined. The HPLC method has good precision, repeatability and stability, which can more comprehensively reflect the characteristics of the main components in Cinobufacini Capsules, providing a reference for the improvement of the quality standard and the comprehensive development and application of Cinobufacini Capsules.

Claims

1. A quality inspection method for cinobufacin capsules, characterized in that, It includes qualitative analysis and quantitative analysis; (1) The qualitative analysis mentioned above includes the following steps: Step (1.1): Preparation of the test solution Weigh precisely the content of Cinobufacini Capsules and place it in a stoppered conical flask. Precisely add methanol, weigh it, ultrasonically treat it at room temperature, weigh it again, make up the lost amount with methanol, shake well, and let it stand. Before analysis, dilute the sample, filter it, and take the subsequent filtrate to obtain the test solution; Step (1.2) Analyze the compounds in Cinobufacini Capsules under the established UPLC-Q / TOF-MS conditions Inject the test solution from step (1.1) into the UPLC and analyze the components in Cinobufacini Capsules under the established UPLC-Q / TOF-MS conditions to obtain the total ion current chromatograms of Cinobufacini Capsules in positive and negative ion modes. Identify the chemical components in Cinobufacini Capsules by comparing the accurate mass-to-charge ratio with the information in the database; (2) The quantitative analysis mentioned above includes the following steps: (2.1) Preparation of the mixed reference substance solution Take appropriate amounts of reference substances of serotonin, bufothionine, gamabufotalin, arenobufagin, deacetylgamabufotalin, telocinobufagin, bufotalin, bufotenine, bufalin, cinobufagin, and esterscinobufagin, weigh them precisely, dissolve them in methanol to prepare single reference substance stock solutions. Precisely measure appropriate amounts of the above reference substance stock solutions, mix them, and dilute with methanol to prepare the mixed reference substance solution; (2.2) Preparation of the test solution Take the contents of Cinobufacini Capsules of different batches, add methanol, ultrasonically treat it at room temperature, weigh it again, make up the lost amount with methanol, shake well, let it stand, take the supernatant and filter it through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain it; (2.3) Investigation of the linear relationship Take the mixed reference substance solution from step (2.1), add methanol and dilute it step by step by 1 - 5 times, inject it into the HPLC-PDA for determination respectively, and construct a regression equation based on the mass concentration and peak area; (2.4) Establishment of the fingerprint Take the test solutions of Cinobufacini Capsules of different batches from step (2.2), inject them into the HPLC-PDA respectively, record their HPLC chromatograms respectively, import the HPLC chromatograms of Cinobufacini Capsule samples of different batches into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints", take the chromatogram of sample S6 as the reference chromatogram, select the median method as the generation method of the control fingerprint, set the time window width to 0.1, use the multi-point correction method for full-peak matching to generate the control fingerprint (R), and conduct common peak comparison and similarity investigation; (2.5) Determination of the sample content Precisely weigh the test solutions of Cinobufacini Capsules of different batches, inject them into the HPLC-PDA to determine the test solutions of Cinobufacini Capsules, measure them in parallel 3 times, and calculate the mass fractions of each component in Cinobufacini Capsules according to the linear equation in step (2.3).

2. The quality inspection method of the cinobufacini capsule according to claim 1, wherein, Step (1.1): The preparation method of the test solution is as follows: Accurately weigh 1.0 - 5.0 g of the content of Cinobufacini Capsules, place it in a stoppered conical flask, accurately add 10 - 50 mL of methanol, weigh it, ultrasonically treat it at room temperature for 30 - 60 min, weigh it again, make up the lost amount with methanol, shake well, and let it stand; before analysis, dilute the sample to 1 mg / mL, take the supernatant and filter it through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain the test solution.

3. The quality inspection method of the cinobufacini capsule according to claim 1, characterized in that, The chromatographic conditions in step (2) are Waters Acquity BEH C 18 chromatographic column, with a specification of 2.1 mm × 100 mm, 1.7 μm, the mobile phase is 0.1% formic acid aqueous solution A - acetonitrile B; gradient elution program: 0 - 1 min, 95% A; 1 - 6.5 min, 95~53% A; 6.5 - 13 min, 53 - 5% A; 13 - 14 min, 5% A; 14 - 15 min, 5 - 95% A; 15 - 16, 95% A; the injection volume is 1 μL, the flow rate is 0.3 mL / min, and the column temperature is 35°C; The mass spectrometry conditions are as follows: Negative ion mode, source conditions: capillary voltage is 2.5 kV, sample cone temperature is 40°C, source temperature is 120°C, desolvation gas temperature is 280°C, cone gas flow rate is 50 L / h, desolvation gas N2 flow rate is 600 L / h; the data acquisition of this mode covers the mass spectrometry range of mass-to-charge ratio 50 - 1200 Da; In the positive ion mode, source conditions: capillary voltage is 3.0 kV, sample cone temperature is 55°C, source temperature is 100°C, desolvation gas temperature is 350°C, cone gas flow rate is 50 L / h, desolvation gas N2 flow rate is 800 L / h; the data acquisition of this mode covers the mass spectrometry range of mass-to-charge ratio 50 - 1200 Da; the control of data acquisition and analysis is carried out by using Waters MassLynx 4.2 software.

4. The quality inspection method of the cinobufagin capsule according to claim 1, characterized in that, Step (2.1) Preparation of the mixed reference solution Take appropriate amounts of reference substances of serotonin, bufothionine, gamabufotalin, arenobufagin, deacetylgamabufotalin, telocinobufagin, bufotalin, bufotenine, bufalin, cinobufagin, and resibufogenin, accurately weigh them, dissolve them in methanol to prepare single reference substance stock solutions; accurately measure appropriate amounts of the above reference substance stock solutions, mix them and dilute with methanol to prepare a mixed reference solution containing 0.16 mg of serotonin, 0.015 mg of bufothionine, 0.05 mg of gamabufotalin, 0.1 mg of arenobufagin, 0.03 mg of deacetylgamabufotalin, 0.03 mg of telocinobufagin, 0.16 mg of bufotalin, 0.12 mg of bufotenine, 0.05 mg of bufalin, 0.1 mg of cinobufagin, and 0.12 mg of resibufogenin per 1 mL.

5. The quality inspection method of cinobufacini capsules according to claim 1, characterized in that, Step (2.2) Preparation of the test solution: Take the content of Cinobufacini Capsules of different batches, accurately weigh about 1.5 g, place it in a stoppered conical flask, accurately add 6 mL of methanol, weigh it, ultrasonically treat it at room temperature for 30 min with a power of 500 W and a frequency of 40 kHz, weigh it again, make up the lost amount with methanol, shake well, let it stand, take the supernatant and filter it through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain it.

6. The quality inspection method of the cinobufotalin capsule according to claim 1, characterized in that, Chromatographic elution conditions for steps (2.3) - (2.5) Using Waters SunFire C 18 , with a specification of 4.6×250mm, 5µm chromatographic column; the mobile phase is acetonitrile A - 0.1% formic acid aqueous solution B, and the gradient elution conditions are: 0 - 6 min, 5 - 8% A; 6 - 10 min, 8 - 18% A; 10 - 30 min, 18~43% A; 30 - 32 min, 43~47% A; 32 - 40 min, 47~60% A; 40 - 42 min, 60~45% A; 42 - 44 min, 45% A; 44 - 46 min, 45~90% A; 46 - 48 min, 90~5% A; 48~50 min, 5% A.

7. The quality inspection method of the cinobufacini capsule according to claim 1, characterized in that Step (2.4): Using the chromatogram of sample S6 as the reference chromatogram, compare it with the test solution of 13 batches of Cinobufagin capsules. Among them, 15 chromatographic peaks are common to the 13 batches of Cinobufagin capsule samples and are determined as common peaks. Calculate the similarity with the reference fingerprint chromatogram; identify 11 of the peaks with reference substances, which are peak 1 5 - hydroxytryptamine, peak 2 bufothionine, peak 3 gamabufotalin, peak 4 arenobufagin, peak 5 deacetylgamabufotalin, peak 6 telocinobufagin, peak 7 bufotalin, peak 8 bufotenine, peak 9 cinobufagin, peak 10 cinobufagin, and peak 11 estersbufagin peak.

8. The quality inspection method of the cinobufacini capsule according to claim 1, wherein, The linear regression equation of step (2.3) is: 。

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