Method for establishing the HPLC characteristic spectrum of Xiongdan Jiuxin Pill

Abstract

The present invention relates to a method for establishing a high-performance liquid phase characteristic spectrum of a Bear Gallbladder Heart-Saving Pill, and the method comprises the following steps: (1) preparation of a test solution; (2) preparation of a reference solution; (3) high-performance liquid chromatography determination: respectively taking appropriate amounts of a test solution and a reference solution and injecting them into a high-performance liquid chromatograph, and recording a chromatogram; analyzing the high-performance liquid chromatograms of multiple batches of test solutions to obtain a reference characteristic spectrum of the Bear Gallbladder Heart-Saving Pill, and calculating the relative retention time and relative peak area of ​​each common peak. The method has the advantages of high compound separation and good repeatability, and the characteristic compounds in the Bear Gallbladder Heart-Saving Pill are determined using the characteristic spectrum provided by the method, so that the quality of the Bear Gallbladder Heart-Saving Pill can be effectively characterized and comprehensively monitored.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for establishing a high-performance liquid phase characteristic spectrum of Xiongdan Jiuxin Pills. Background Art

[0002] Bear Bile Heart-Rescue Pills are composed of 9 Chinese medicinal materials: 0.2g bear bile powder, 1.67g toad venom, 2g borneol, 0.2g artificial musk, 6.7g ginseng, 3.4g pearl, 0.5g artificial bezoar, 1.5g pig bile powder, and 1.67g buffalo horn concentrated powder. It has the effects of strengthening the heart and replenishing qi, and aromatically opening the orifices. It is used for chest pain caused by insufficient heart qi, with symptoms such as chest tightness, heart pain, shortness of breath, palpitations, etc.

[0003] Bear Bile Heart-Rescue Pills have many flavors and complex chemical components, and there has been little research on quality control. The Bear Bile Heart-Rescue Pills recorded in the standard are only quality controlled by inspections under the properties and pill items. The quality standards are low and cannot fully reflect the intrinsic quality of the Bear Bile Heart-Rescue Pills, and cannot guarantee comprehensive control of the quality of the Bear Bile Heart-Rescue Pills. Summary of the invention

[0004] In order to make up for the deficiencies of the prior art, the present invention provides a method for establishing a high-performance liquid phase characteristic spectrum of the Bear Gallbladder Heart-Saving Pills, which is conducive to more comprehensive and effective control of the quality of the Bear Gallbladder Heart-Saving Pills.

[0005] The technical solution adopted by the present invention to solve the above technical problems is:

[0006] A method for establishing a high-performance liquid chromatography characteristic spectrum of Xiongdan Jiuxin Pills comprises the following steps: (1) preparing a test solution: taking a certain weight of Xiongdan Jiuxin Pills and placing them in a container, adding an appropriate amount of methanol for extraction, cooling, shaking, filtering, and taking the filtrate as the test solution; (2) preparing a reference solution: taking a certain weight of ginsenoside Rg1, bufotoxin, bufotoxin, ginsenoside Rb1, bufotoxin, taurohyodeoxycholic acid, cinobufolin, bufotoxin and Put the test solution and the reference solution in a volumetric flask, add an appropriate amount of methanol and perform ultrasonic treatment, cool, shake and filter to obtain a reference solution; wherein, the test solution and the reference solution are used as a reference; (3) high performance liquid chromatography determination: take an appropriate amount of the test solution and the reference solution and inject them into a high performance liquid chromatograph to record the chromatogram; analyze the high performance liquid chromatograms of multiple batches of the test solution to obtain the reference characteristic spectrum of the Xiongdan Jiuxin Pills, and calculate the relative retention time and relative peak area of ​​each common peak;

[0007] The chromatographic conditions used are as follows: select a C18 chromatographic column 250mm×4.6mm, 5μm; the column temperature is 29-31°C; the detection wavelength is 205nm; the flow rate is 0.9-1.1mL / min; the injection volume is 5μL; acetonitrile is used as mobile phase A, and a phosphoric acid aqueous solution with a volume concentration of 0.1% is used as mobile phase B for gradient elution, and the gradient elution conditions are as follows: 0-10min, the volume concentration of mobile phase A is maintained at 5%, and the volume concentration of mobile phase B is maintained at 95%; 10-15min, the volume concentration of mobile phase A is increased from 5% to 25%, and the volume concentration of mobile phase B is reduced from 95% to 75%; 15-50min, the volume concentration of mobile phase A is increased from 25% to 52%, and the volume concentration of mobile phase B is reduced from 75% to 48%.

[0008] Furthermore, the column temperature of the chromatographic column in the chromatographic conditions is 30° C.; and the flow rate is 1.0 mL / min.

[0009] Furthermore, the preparation method of the test solution is specifically as follows: take about 0.3 g of Xiongdan Jiuxin Pills powder, place it in a stoppered conical flask, add 25 mL of methanol, reflux for 1 hour, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0010] Furthermore, the control characteristic spectrum of the Bear Bile Heart-saving Pills includes 15 common peaks, peaks 5, 6, 9, 11, 13 and 15 are characteristic peaks of toad venom, peaks 4 and 8 are characteristic peaks of ginseng, and peak 10 is the characteristic peak of pig bile powder.

[0011] Furthermore, Peak 6 is toad venom, as a reference peak, Peak 4 is ginsenoside Rg1, Peak 5 is toad venom, Peak 8 is ginsenoside Rb1, Peak 9 is toad venom, Peak 10 is taurohyodeoxycholic acid, Peak 11 is cinobufal, Peak 13 is toad venom, and Peak 15 is cinobufal.

[0012] Furthermore, the relative standard deviations (RSDs) of the relative retention times of the 15 common peaks were all less than 2%. The similarities were calculated using the characteristic spectrum of the Bear Bile Heart-Saving Pills as a reference, and the similarities of the characteristic spectrum of the Bear Bile Heart-Saving Pills were all above 0.9.

[0013] The present invention adopts the above structure, and has the advantages of:

[0014] 1. The method for establishing the HPLC characteristic spectrum of Bear Gallbladder Heart-Saving Pills in the present application is based on the medicinal effects of the components contained in Bear Gallbladder Heart-Saving Pills, and takes the main components of medicinal materials such as toad venom, ginseng, pig bile powder, such as toad venom, ginsenoside Rg1, toad venom, ginsenoside Rb1, toad venom, taurohyodeoxycholic acid, cinobufalfa, toad venom and cinobufalfa as research indicators, to study the characteristic spectrum of Bear Gallbladder Heart-Saving Pills, and provides a method for establishing and determining the characteristic spectrum of Bear Gallbladder Heart-Saving Pills, which provides a reference and basis for the quality control of Bear Gallbladder Heart-Saving Pills; in addition, the method also has the advantages of simplicity, stability, high precision and good reproducibility.

[0015] 2. The characteristic spectrum of Bear Bile Rescue Heart Pills established according to the method for establishing the high-performance liquid chromatography characteristic spectrum of Bear Bile Rescue Heart Pills in this application has 15 characteristic peaks that are effectively separated. Therefore, the obtained characteristic spectrum can more effectively characterize the quality of Bear Bile Rescue Heart Pills, and thus facilitate the comprehensive monitoring of the quality of the drug.

[0016] 3. The method for establishing the high-performance liquid chromatography characteristic spectrum of Bear Bile Rescue Heart Pills of the present application was used to test 10 different batches of Bear Bile Rescue Heart Pills, and the RSDs of the relative retention times of the chromatographic peaks at the same position were all less than 2%. Therefore, the characteristic spectrum established by the method for establishing the high-performance liquid chromatography characteristic spectrum of Bear Bile Rescue Heart Pills provided in the present application has good reliability. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the control characteristic spectrum of the Bear Bile Heart-saving Pills generated using the average value calculation method.

[0018] Figure 2 The characteristic spectra of Xiongdan Jiuxin Pill sample solution and mixed reference substance;

[0019] Figure 3 This is the characteristic spectrum of the Xiongdan Jiuxin Pill sample solution at a wavelength of 194nm;

[0020] Figure 4 This is the characteristic spectrum of the Xiongdan Jiuxin Pill sample solution at a wavelength of 210nm;

[0021] Figure 5 This is the characteristic spectrum of the Xiongdan Jiuxin Pill sample solution at a wavelength of 220nm;

[0022] Figure 6 This is the characteristic spectrum of the Xiongdan Jiuxin Pill sample solution at a wavelength of 240nm;

[0023] Figure 7 This is the characteristic spectrum of the Xiongdan Jiuxin Pill sample solution at a wavelength of 260nm;

[0024] Figure 8 This is the characteristic spectrum of the Xiongdan Jiuxin Pill sample solution of elution procedure 1#;

[0025] Fig. 9 This is the characteristic spectrum of the Xiongdan Jiuxin Pill sample solution of elution procedure 2#;

[0026] Fig.10 This is the characteristic spectrum of the Xiongdan Jiuxin Pill sample solution of elution procedure 3#;

[0027] Fig.11 This is the characteristic spectrum of the sample solution of one batch of Xiongdan Jiuxin Pills;

[0028] Fig.12 These are the characteristic spectra of multiple batches of Xiongdan Jiuxin Pill sample solutions. DETAILED DESCRIPTION

[0029] In order to clearly illustrate the technical features of the present solution, the present invention is described in detail below through specific implementation methods and in conjunction with the accompanying drawings.

[0030] 1. Instruments and reagents

[0031] Agilent 1260 high performance liquid chromatograph; DAD detector; BSA224S-CW electronic analytical balance; KDM electric heating mantle; KQ-800VSM digitally controlled ultrasonic cleaner.

[0032] Among the reference substances, bufotoxin and cinobufolin were purchased from Chengdu Pufeide Biotechnology Co., Ltd., cinobufolin was purchased from Shanghai Shidande Standard Technology Service Co., Ltd., taurohyodeoxycholic acid, bufotoxin, bufotoxin, and bufotoxin were purchased from Shanghai Zerui Biotechnology Co., Ltd., and ginsenoside Rg1 and ginsenoside Rb1 were purchased from China Food and Drug Inspection Institute, with a purity of ≥98%. The Xiongdan Jiuxin Pills used as the test substance were from Hongjitang, and the selected batch numbers were 2204001, 230501, 230502, 230503, 230504, 230505, 230506, 230507, 230508, and 230509. Methanol and acetonitrile were chromatographically pure and purchased from Yuexu Technology (Shanghai) Co., Ltd. Water was Wahaha purified water, and the other reagents were analytically pure.

[0033] 2. Method for establishing the HPLC characteristic spectrum of Xiongdan Jiuxin Pills

[0034] The preparation method of the reference solution is as follows: accurately weigh 18.42 mg of toad venom, 3.57 mg of ginsenoside Rg1, 10.53 mg of bufotoxin, 20.59 mg of ginsenoside Rb1, 10.46 mg of bufotoxin, 10.27 mg of taurohyodeoxycholic acid and 9.51 mg of bufotoxin, and place them in 10 mL volumetric flasks respectively, and place 3.26 mg of cinobuccal venom and 5.65 mg of cinobuccal venom in 5 mL volumetric flasks respectively, add methanol for ultrasonic dissolution, cool, make up to volume, shake well, filter, and take the filtrate as the reference solution.

[0035] The preparation method of the test solution is as follows: take an appropriate amount of Xiongdan Jiuxin Pills, grind them, weigh about 0.3g and place them in a stoppered conical flask, then accurately add 25mL of methanol, reflux for 1h, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0036] The chromatographic conditions for high performance liquid chromatography determination of the test solution and the reference solution are as follows: select a C18 chromatographic column, the column temperature is 30°C; the detection wavelength is 205nm; the flow rate is 1.0mL / min; the injection volume is 5μL; acetonitrile is used as mobile phase A, and a phosphoric acid aqueous solution with a volume concentration of 0.1% is used as mobile phase B for gradient elution, and the gradient elution conditions are as follows: 0-10min, the volume concentration of mobile phase A is maintained at 5%, and the volume concentration of mobile phase B is maintained at 95%; 10-15min, the volume concentration of mobile phase A is increased from 5% to 25%, and the volume concentration of mobile phase B is reduced from 95% to 75%; 15-50min, the volume concentration of mobile phase A is increased from 25% to 52%, and the volume concentration of mobile phase B is reduced from 75% to 48%.

[0037] Through the HPLC determination of a single reference solution (chromatogram omitted), it can be seen that among the 9 compounds, the content of sabdariffa in Xiongdan Jiuxin Pills is relatively stable, and the retention time in the spectrum is moderate and the separation is good. Therefore, sabdariffa was selected as the reference substance, and the chromatographic peak of sabdariffa was used as the reference peak accordingly. The mixed reference solution and the test solution were determined by HPLC, and the results are shown in Figure 2 The main peaks in the chromatogram of the mixed reference solution and the test solution can be confirmed through the chromatogram of a single reference solution, among which peak 4 is ginsenoside Rg1, peak 5 is bufotoxin, peak 8 is ginsenoside Rb1, peak 9 is bufotoxin, peak 10 is taurohyodeoxycholic acid, peak 11 is cinobuccal toxin, peak 13 is bufotoxin, and peak 15 is cinobuccal toxin.

[0038] 3. Basis for selection of chromatographic conditions

[0039] 3.1 Selection of detection wavelength

[0040] The wavelength range of the diode array detector is 190-400nm. Taking the detection wavelength as a variable, high performance liquid chromatography detection is performed on different test samples according to the chromatographic detection steps of the test samples. By comparing the chromatograms of the samples at wavelengths of 194nm, 205nm, 210nm, 220nm, 240nm, and 260nm, the chromatogram at 205nm is shown in Fig.10 The rest of the chromatograms are shown in Figure 3-7 According to the number of chromatographic peaks, chromatographic peak response values, and whether the baseline is stable, 205nm was finally selected as the detection wavelength of the characteristic spectrum of the Bear Bile Heart-saving Pills.

[0041] 3.2 Investigation of elution gradient

[0042] Using the gradient elution conditions in Table 1, Table 2, and Table 3 as variables, accurately aspirate 5 μL of the test solution, inject it into the HPLC, and record the chromatogram for 50 minutes. The results are shown in Figure 8-Figure 10 .

[0043] Table 1 Solvent elution procedure 1#

[0044]

[0045]

[0046] Table 2 Solvent elution procedure 2#

[0047]

[0048] Table 3 Solvent elution procedure 3#

[0049]

[0050] pass Figure 8-Figure 10 It can be seen that based on factors such as the number of chromatographic peaks, chromatographic peak response values, and separation, elution program 3# was finally selected as the characteristic spectrum elution condition.

[0051] 4. Optimization experiment of test solution preparation conditions

[0052] 4.1 Selection of extraction method

[0053] Take an appropriate amount of Xiongdan Jiuxin Pills with batch number 2204001, grind it into powder, accurately weigh 2 portions, 0.2015g and 0.2008g respectively, place them in stoppered conical bottles, accurately add 25mL of methanol, observe reflux extraction for 1h and ultrasonic extraction (300W, 40KHz) for 1h, cool, filter, and take the filtrate as the test solution. Accurately weigh 5μL of each of the two test solutions, inject them into the high performance liquid chromatograph, record the chromatogram for 50min, and use the number of chromatographic peaks and the main peak area divided by the sampling amount as evaluation indicators. The results are shown in Table 4.

[0054] Table 4 Extraction method investigation results (peak area)

[0055]

[0056] From the results in Table 4, it can be seen that the main peak chromatographic peak area of ​​the reflux sample is basically larger than that of the ultrasonic sample, so the reflux extraction method is selected.

[0057] 4.2 Investigation of extraction solvent

[0058] Take the contents of the batch number 2204001 Xiongdan Jiuxin Pills, grind them finely, and accurately weigh 3 portions, 0.2012g, 0.2018g and 0.2008g respectively, and place them in stoppered conical bottles, and accurately add 50% methanol, 70% methanol and 25mL of methanol, reflux and extract for 1h, investigate different solvents, cool, filter, and take the filtrate as the test solution. Accurately weigh 5μL of each of the above 3 test solutions, inject them into the high performance liquid chromatograph, record the chromatogram for 50min, and analyze them with the number of chromatographic peaks and the main peak area divided by the sampling amount as evaluation indicators. The results are shown in Table 5.

[0059] Table 5 Extraction solvent investigation results (peak area)

[0060]

[0061] From the results in Table 5, it can be seen that the main peak area of ​​methanol as the extraction solvent is mostly larger than 50% methanol and 70% methanol, so methanol is selected as the extraction solvent.

[0062] 4.3 Selection of extraction time

[0063] Take the contents of Xiongdan Jiuxin Pills with batch number 2204001, grind them finely, weigh 2 portions accurately, 0.1998g, 0.2001g, 0.2012g and 0.2007g respectively, put them in stoppered conical bottles, accurately add 25mL of methanol, observe reflux extraction for 30min, reflux extraction for 45min, reflux extraction for 1h and reflux extraction for 1.5h, cool, filter, and take the filtrate as the test solution. Accurately weigh 5μL of each of the above 2 test solutions, inject them into the high performance liquid chromatograph, record the chromatogram for 50min, and analyze them with the number of chromatographic peaks and the peak area of ​​the main peak divided by the sampling amount as evaluation indicators. The results are shown in Table 6.

[0064] Table 6 Extraction time investigation results (peak area)

[0065]

[0066] From the results in Table 6, it can be seen that the extraction effects of reflux for 1 h and reflux for 1.5 h are equivalent, and the number of chromatographic peaks and the main peak area are greater than those of reflux for 30 min and reflux for 45 min. Considering that reflux for 1 h is time-consuming, the extraction time is selected as 1 h.

[0067] 4.4 Selection of sample weight

[0068] Take the contents of Xiongdan Jiuxin Pills with batch number 2204001, grind them finely, weigh 3 portions accurately, 0.1006, 0.2013, and 0.3003 g respectively, place them in stoppered conical bottles, accurately add 25 mL of methanol, reflux and extract for 1 hour, examine different sample amounts, cool, filter, and take the filtrate as the test solution. Accurately weigh 5 μL of each of the above 3 test solutions, inject them into the high performance liquid chromatograph, record the chromatogram for 50 minutes, and analyze them with the number of chromatographic peaks and the main peak area divided by the sampling amount as evaluation indicators. The results are shown in Table 7.

[0069] Table 7 Sample weight investigation results (peak area)

[0070]

[0071] From the results in Table 7, it can be seen that the extraction effects of sample weights of 0.1g, 0.2g and 0.3g are equivalent. Considering that the response of 0.3g sample weight is higher, 0.3g sample weight is selected.

[0072] Based on the above test results, the specific preparation method of the test solution of the characteristic spectrum of Bear Bile Rescue Heart Pills is determined as follows: take 0.3 g of Bear Bile Rescue Heart Pills powder, put it in a stoppered conical flask, add 25 mL of methanol, reflux for 1 hour, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0073] 5. Methodological validation of the characteristic profile establishment method

[0074] 5.1 Precision experiment

[0075] Take the contents of Xiongdan Jiuxin Pills with batch number 2204001, grind, accurately weigh 0.3016g, put it in a stoppered conical flask, add 25mL of methanol to the conical flask, heat and reflux for 1h, cool, shake, filter, and take the filtrate to obtain the test solution. The test solution was injected 6 times in parallel, and the relative retention time and relative peak area of ​​the main chromatographic peaks were calculated to examine the precision of the instrument. The results are shown in Tables 8 and 9.

[0076] Table 8 Precision investigation results (relative retention time)

[0077]

[0078] Table 9 Precision investigation results (relative peak area)

[0079]

[0080]

[0081] The results in Tables 8 and 9 show that the RSD of the relative retention time is significantly less than 1%, and the RSD of the relative peak area is less than 5%. Both RSDs are within ±10%, indicating that the instrument has good precision.

[0082] 5.2 Solution stability test

[0083] Take the Xiongdan Jiuxin Pills with batch number 2204001, grind, accurately weigh 0.3013g, put it in a stoppered conical flask, add 25mL of methanol to the conical flask, heat and reflux for 1h, cool, shake, filter, and take the filtrate to obtain the test solution. The test solution was injected at 0h, 2h, 4h, 12h, 18h, and 24h, and the fingerprint chromatogram was recorded. A total of 6 time points were measured, and the relative retention time and relative peak area of ​​the main chromatographic peaks were calculated. The results are shown in Tables 10 and 11.

[0084] Table 10 Stability test results (relative retention time)

[0085]

[0086]

[0087] Table 11 Stability investigation results (relative peak area)

[0088]

[0089]

[0090] The results in Tables 8 and 9 show that the RSD of the relative retention time is less than 2%, and the RSD of the relative peak area is within 5%, indicating that the test solution has good stability within 24 hours at room temperature.

[0091] 5.3 Solution repeatability test

[0092] Take the Xiongdan Jiuxin Pills with batch number 2204001, grind, accurately weigh 0.3009g, 0.3017g, 0.3016g, 0.2995g, 0.3008g, 0.3016g, place in a stoppered conical flask, add 25mL of methanol to the conical flask, heat and reflux for 1h, cool, shake, filter, and take the filtrate to obtain the test solution. Accurately aspirate the test solution and the reference peak solution respectively, inject into the high performance liquid chromatograph, perform gradient elution according to the chromatographic conditions provided, and record the chromatogram. Calculate the relative retention time and relative peak area of ​​the main chromatographic peaks, and the results are shown in Tables 12 and 13.

[0093] Table 12 Repeatability test results (relative retention time)

[0094]

[0095]

[0096] Table 13 Repeatability test results (relative peak area)

[0097]

[0098] It can be seen from Tables 12 and 13 that the RSD of the relative retention time is less than 1%, and the RSD of the relative peak area is less than 3%, indicating that the method has good repeatability.

[0099] 5.4 Durability test at different flow rates

[0100] Take the Xiongdan Jiuxin Pills with batch number 2204001, grind them, accurately weigh 0.3012g and place them in a stoppered conical flask, add 25mL of methanol to the conical flask, heat and reflux for 1h, cool, shake well, filter, and take the filtrate to obtain the test solution. Accurately aspirate the test solution and the reference peak solution respectively, inject them into the high performance liquid chromatograph, and the flow rates are 0.9mL / min, 1.0mL / min and 1.1mL / min respectively. The rest of the conditions are gradient eluted according to the chromatographic conditions provided, and the chromatogram is recorded. Calculate the relative retention time and relative peak area of ​​the main chromatographic peaks, and the results are shown in Tables 14 and 15.

[0101] Table 14 Durability test results at different flow rates (relative retention time)

[0102]

[0103] Table 15 Durability test results at different flow rates (relative peak area)

[0104]

[0105]

[0106] It can be seen from Tables 14 and 15 that the relative retention times at flow rates of 0.9 mL / min and 1.1 mL / min are within ±10% of the relative retention time at a flow rate of 1.0 mL / min, and the RSD of the relative peak area is less than 5%, indicating that the method has good durability at flow rates between 0.9 mL / min and 1.1 mL / min.

[0107] 5.5 Durability test at different column temperatures

[0108] Take the Xiongdan Jiuxin Pills with batch number 2204001, grind them, accurately weigh 0.3012g and place them in a stoppered conical flask, add 25mL of methanol to the conical flask, heat and reflux for 1h, cool, shake, filter, and take the filtrate to obtain the test solution. Accurately aspirate the test solution and the reference peak solution respectively, inject them into the high performance liquid chromatograph, the column temperatures are 29℃, 30℃ and 31℃ respectively, and the other conditions are gradient eluted according to the chromatographic conditions provided, and record the chromatogram. Calculate the relative retention time and relative peak area of ​​the main chromatographic peaks, and the results are shown in Tables 16 and 17.

[0109] Table 16 Results of durability study at different column temperatures (relative retention time)

[0110]

[0111] Table 17 Results of durability study at different column temperatures (relative peak area)

[0112]

[0113]

[0114] It can be seen from Tables 16 and 17 that the relative retention times at column temperatures of 29°C and 31°C are within ±10% of the relative retention time at a column temperature of 30°C, and the RSD of the relative peak area is less than 5%, indicating that the method has good durability when the column temperature is between 29°C and 31°C.

[0115] 5.7 Durability test of different chromatographic columns

[0116] Take the Xiongdan Jiuxin Pills with batch number 2204001, grind them, accurately weigh 0.3012g and put them in a stoppered conical flask, add 25mL of methanol to the conical flask, heat and reflux for 1h, cool, shake, filter, and take the filtrate to obtain the test solution. Accurately aspirate the test solution and the reference peak solution respectively, inject them into the high performance liquid chromatograph, and perform gradient elution according to the chromatographic conditions provided except for the chromatographic column conditions. The following chromatographic columns were selected for investigation: SIMADZU InertSustain C18 (250mm×4.6mm, 5μm), Thermo Acclaim TM 120 C18 (250mm×4.6mm, 5μm), Agilent 5TC-C18(2) (250mm×4.6mm, 5μm), Waters Atlantis TM T3 (250 mm × 4.6 mm, 5 μm), the above chromatographic columns are referred to as chromatographic column 1, chromatographic column 2, chromatographic column 3, and chromatographic column 4. The retention time of the main chromatographic peaks was calculated, and the results are shown in Table 18.

[0117] Table 18 Results of durability study of different chromatographic columns (relative retention time)

[0118]

[0119]

[0120] As can be seen from Table 18, the relative retention times of columns 2, 3, and 4 are within ±10% of the relative retention time of column 1, indicating that the method has good durability among different columns.

[0121] 6. Establishment of feature map

[0122] 6.1 Characteristic spectrum determination of Xiongdan Jiuxin Pills

[0123] Take the Bear Bile Rescue Heart Pills with batch numbers of 204001, 230501, 230502, 230503, 230504, 230505, 230506, 230507, 230508, and 230509 (the sample weights are: 0.3012g, 0.3007g, 0.2998g, 0.3012g, 0.3001g, 0.3006g, 0.3011g, 0.2999g, 0.3004g, and 0.3002g, respectively), and obtain the corresponding test solutions according to the preparation method of the test solution in 2.1. Accurately aspirate the test solution, inject it into the high performance liquid chromatograph, perform gradient elution according to the above chromatographic conditions, record the chromatogram, and the results are shown in Fig.11 The relative retention times of the main chromatographic peaks of the chromatograms of 10 batches of Xiongdan Jiuxin Pills were calculated, and the results are shown in Table 19.

[0124] Table 19 Characteristic spectra of 10 batches of Xiongdan Jiuxin Pills (relative retention time)

[0125]

[0126]

[0127] 6.2 Determination of common peaks and acquisition of reference characteristic spectra

[0128] The AIA format of the characteristic spectra of 10 batches of Xiongdan Jiuxin Pills was exported from the instrument and imported into the similarity evaluation system of Chinese medicine chromatographic characteristic spectra, see attached Fig.12 , select the chromatographic peaks present in the characteristic spectra of 10 batches of Xiongdan Jiuxin Pills as the common peaks. Use the average calculation method to generate the control characteristic spectrum of Xiongdan Jiuxin Pills, see the attached Figure 1 , calculate the similarity of each common peak, and the results are shown in Table 20.

[0129] Table 20 Similarity results of characteristic spectra of 10 batches of Xiongdan Jiuxin Pills

[0130]

[0131] The results in Table 20 show that the similarity of the characteristic spectra of the 10 batches of Bear Bile Heart-Saving Pills is above 0.9 when the similarity is calculated using the control characteristic spectrum as a reference. Therefore, the standard for the characteristic spectrum of Bear Bile Heart-Saving Pills is tentatively set as follows: the test sample spectrum should be consistent with the control characteristic spectrum, and according to the similarity evaluation system for traditional Chinese medicine chromatographic characteristic spectra, the similarity between the test sample characteristic spectrum and the control characteristic spectrum shall not be less than 0.90 after similarity calculation.

[0132] The above specific implementations cannot be used as limitations on the scope of protection of the present invention. For those skilled in the art, any replacement, improvement or change made to the implementations of the present invention falls within the scope of protection of the present invention. The parts not described in detail in the present invention are all known technologies to those skilled in the art.

Claims

1. A method for establishing a high performance liquid chromatography characteristic spectrum of Xiongdan Jiuxin Pills, characterized in that: The method comprises the following steps: (1) preparing a test solution: taking a certain weight of Xiongdan Jiuxin Pills and placing them in a container, adding an appropriate amount of methanol for extraction, cooling, shaking, filtering, and taking the filtrate as the test solution; (2) preparing a reference solution: taking a certain weight of ginsenoside Rg1, bufotoxin, ginsenoside Rb1, bufotoxin, taurohyodeoxycholic acid, cinobufolin, bufotoxin and cinobufolin, placing them in volumetric flasks respectively, adding an appropriate amount of methanol and performing ultrasonic treatment, cooling, shaking, filtering, and obtaining a reference solution; wherein cinobufolin is used as a reference; (3) high performance liquid chromatography determination: taking an appropriate amount of the test solution and the reference solution and injecting them into a high performance liquid chromatograph respectively, and recording the chromatogram; analyzing the high performance liquid chromatograms of multiple batches of the test solution to obtain a reference characteristic spectrum of Xiongdan Jiuxin Pills, and calculating the relative retention time and relative peak area of ​​each common peak; The chromatographic conditions used are as follows: select C18 chromatographic column 250mm×4.6mm, 5μm; column temperature 29-31℃; detection wavelength 205nm; flow rate 0.9-1.1mL / min; injection volume 5μL; acetonitrile is used as mobile phase A, and phosphoric acid aqueous solution with a volume concentration of 0.1% is used as mobile phase B for gradient elution. The gradient elution conditions are as follows: 0-10min, the volume concentration of mobile phase A is maintained at 5%, and the volume concentration of mobile phase B is maintained at 95%; 10-15min, the volume concentration of mobile phase A increases from 5% to 25%, and the volume concentration of mobile phase B decreases from 95% to 75%; 15-50min, the volume concentration of mobile phase A increases from 25% to 52%, and the volume concentration of mobile phase B decreases from 75% to 48%; The preparation method of the test solution is specifically as follows: take about 0.3 g of Xiongdan Jiuxin Pill powder, place it in a stoppered conical flask, add 25 mL of methanol, reflux for 1 hour, cool, shake well, filter, and take the filtrate to obtain the test solution.

2. The method for establishing the high-performance liquid phase characteristic spectrum of Xiongdan Jiuxin Pills according to claim 1 is characterized in that: The column temperature of the chromatographic conditions was 30°C and the flow rate was 1.0 mL / min.

3. The method for establishing the high-performance liquid phase characteristic spectrum of Xiongdan Jiuxin Pills according to claim 1 is characterized in that: The control characteristic spectrum of Xiongdan Jiuxin Pills includes 15 common peaks, peaks 5, 6, 9, 11, 13 and 15 are characteristic peaks of toad venom, peaks 4 and 8 are characteristic peaks of ginseng, and peak 10 is the characteristic peak of pig bile powder.

4. The method for establishing the high-performance liquid phase characteristic spectrum of Xiongdan Jiuxin Pills according to claim 3 is characterized in that: Peak No. 6 is toad venom, which is used as a reference peak. Peak No. 4 is ginsenoside Rg1, Peak No. 5 is toad venom, Peak No. 8 is ginsenoside Rb1, Peak No. 9 is toad venom, Peak No. 10 is taurohyodeoxycholic acid, Peak No. 11 is cinobuccal venom, Peak No. 13 is toad venom, and Peak No. 15 is cinobuccal venom.

5. The method for establishing the high-performance liquid phase characteristic spectrum of Xiongdan Jiuxin Pills according to claim 4 is characterized in that: The relative standard deviations (RSDs) of the relative retention times of the 15 common peaks were all less than 2%. The similarities were calculated using the characteristic spectrum of the Bear Bile Heart-Saving Pills as a reference, and the similarities of the characteristic spectrum of the Bear Bile Heart-Saving Pills were all above 0.9.

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