Construction method and application of specific chromatogram of Zilong Jin tablets

Abstract

The invention relates to the technical field of traditional Chinese medicine analysis, in particular to a construction method and application of a specific chromatogram of a purple Longjin tablet. Comprising the following steps: preparing a reference substance solution; preparing a test solution; determining conditions; and generating a control characteristic spectrum. According to the method, the overall chemical component information of the purple Longjin tablet can be comprehensively presented through one-time detection, namely 14 characteristic peaks are presented, the relative retention time change of each peak is small, and RDS is smaller than 1.0%. The sample extraction method is simple and convenient and has good reproducibility.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine analysis, and particularly relates to a method for constructing a characteristic chromatogram of Zilongjin tablets and its application. Background Art

[0002] The quality standard of Zilongjin tablets is included in Part I of the Chinese Pharmacopoeia 2020 Edition. There are only three identifications and one content determination in the pharmacopoeia standard, lacking the characteristic chromatogram item that can comprehensively control the quality of Zilongjin tablets. Moreover, there are few studies on the characteristic chromatogram item of Zilongjin tablets in the literature research, and only 1 patent study has been conducted on the fingerprint chromatogram of Zilongjin tablets. For example, CN113640434A, a method for constructing and applying a high-performance liquid chromatography fingerprint chromatogram of Zilongjin tablets, determines the characteristic peaks of different medicinal materials contained in Zilongjin tablets by constructing a reference fingerprint chromatogram and a test solution fingerprint chromatogram, and conducts methodological verification of the characteristic chromatogram. However, the chromatographic peaks in the fingerprint chromatogram in this study are relatively few, and only tanshinone IIA and salvianolic acid B are specifically identified. These two components are both contained in the salvia miltiorrhiza medicinal material, and the components contained in the other medicinal materials have not been studied.

[0003] In addition, the construction of the characteristic chromatogram of Zilongjin tablets faces the following difficulties: (1) The complexity of the sample, resulting in difficulty in preparing and purifying the active ingredients and removing impurity interference: Complex samples such as traditional Chinese medicine contain multiple components with different polarities, molecular weights, and chemical properties, increasing the difficulty of separation and detection; (2) Difficulty in selecting a high-precision detection method: Selecting a suitable analysis method is the key to preparing the characteristic chromatogram. Commonly used analysis methods include high-performance liquid chromatography (HPLC), ultra-high performance liquid chromatography (UPLC), etc.; however, different methods have different capabilities for sample processing and detection, and the most suitable method needs to be selected according to the characteristics of the sample to improve the accuracy of analysis and detection; (3) Standardization issue: To ensure the comparability and repeatability of the characteristic chromatogram, a standardized preparation and analysis process need to be established; this includes sample pretreatment, selection of chromatographic conditions, data processing methods, etc. The lack of standardization may lead to inconsistent results between different laboratories; (4) Identification and attribution of characteristic peaks: In the characteristic chromatogram, accurate identification and attribution of characteristic peaks are required; this involves in-depth understanding of the chemical properties and chromatographic behaviors of each component in the sample. Incorrect identification and attribution of characteristic peaks may lead to inaccurate characteristic chromatograms. Summary of the Invention

[0004] In order to make up for the deficiencies of the prior art, the present invention provides a method for constructing a characteristic chromatogram of Zilongjin tablets and its application.

[0005] The present invention is achieved by the following technical solutions: In a first aspect, the present invention provides a method for constructing a characteristic chromatogram of Zilongjin tablets, comprising the following steps: (1) Preparation of the reference substance solution: Take the reference substances, accurately weigh them, and separately dissolve them in methanol to prepare solutions containing 30 μg per 1 ml, shake well, and use them as the reference substance solutions; the reference substances are: salvianolic acid B, ferulic acid, chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid. (2) Preparation of the test sample solution: Take Zilongjin tablets, grind them finely, accurately weigh 0.5 g, add 30 mL of petroleum ether with a boiling range of 60 - 90 °C, perform ultrasonic-assisted extraction at 40 °C with 200 W for 15 minutes, centrifuge at 8000 rpm for 10 min, discard the petroleum ether layer, and air-dry the residue; add 30 mL of a mixed solvent of anhydrous ethanol: phosphoric acid: water with a volume ratio of 70:0.1:29.9 to the defatted residue after centrifugation, then reflux and extract at 80 °C for 30 minutes, centrifuge and collect the supernatant, and concentrate the supernatant under reduced pressure at 40 °C to 5 mL; use a C18 solid-phase extraction column with 200 mg / 3 mL, activate the C18 solid-phase extraction column with 3 mL of pure methanol and 3 mL of water in sequence; load the concentrated solution obtained by concentration under reduced pressure at 40 °C onto the activated column at a flow rate of 1 mL / min; wash away impurities with 3 mL of water, elute the target components with 3 mL of 70% methanol containing 0.1% formic acid, and collect the eluate; dilute the eluate to 5 mL with a 50% methanol solution, mix well, and filter through a 0.22 μm filter membrane to obtain the test sample solution. (3) Determination: Inject the reference substance solution and the test sample solution into a high-performance liquid chromatograph under the following chromatographic conditions, and record the chromatogram. Chromatographic conditions: Use octadecylsilane-bonded silica gel as the filler; use acetonitrile - tetrahydrofuran (volume ratio 35∶13) as mobile phase A, and use 0.1 mol / L ammonium acetate solution (add 0.5 ml of glacial acetic acid per 1000 ml) as mobile phase B, and perform elution according to the specified gradient; the column temperature is 30 °C; the flow rate is 1 ml per minute; the detection wavelength is 300 - 340 nm. The elution gradient program is as follows: 0 - 20 min, the volume ratio of mobile phase A to B changes from 8:92 to 16:84; 20 - 27 min, the volume ratio of mobile phase A to B changes from 16:84 to 19:81; 27 - 40 min, the volume ratio of mobile phase A to B changes from 19:81 to 23:77; 40 - 60 min, the volume ratio of mobile phase A to B changes from 23:77 to 55:45; 60 - 65 min, the volume ratio of mobile phase A to B changes from 55:45 to 8:92; 65 - 75 min, mobile phase A: B, volume ratio 8:92; (4) Generating a control characteristic chromatogram: Using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" formulated by the Pharmacopoeia Commission of the People's Republic of China, select the chromatographic peaks that exist in the chromatograms of different batches of Zilongjin Tablets as common peaks, and generate the control characteristic chromatogram of Zilongjin Tablets by the average value calculation method.

[0006] Preferably, the chromatographic column in step (3) is: InertSustain C18 4.6x250mm, 5um.

[0007] Preferably, taking the peak corresponding to the reference substance of salvianolic acid B as the S peak, the relative retention times of each characteristic peak and the S peak are all within the range of ±10% of the specified value; the specified values are: 0.20 (peak 1), 0.27 (peak 2), 0.31 (peak 3), 0.32 (peak 4), 0.34 (peak 5), 0.38 (peak 6), 0.55 (peak 7), 0.62 (peak 8), 0.70 (peak 9), 0.72 (peak 10), 0.76 (peak 11), 0.94 (peak 12), 0.98 (peak 13), where peak 1 corresponds to neochlorogenic acid, peak 3 corresponds to chlorogenic acid, peak 4 corresponds to cryptochlorogenic acid, peak 8 corresponds to ferulic acid, and peak 14 corresponds to salvianolic acid B.

[0008] In the second aspect, the present invention provides the application of the characteristic chromatogram of Zilongjin Tablets in the quality control of the characteristic components of Zilongjin Tablets.

[0009] In the third aspect, the present invention provides the application of the characteristic chromatogram of Zilongjin Tablets in the production quality control of Zilongjin Tablets.

[0010] Compared with the prior art, the beneficial effects of the present invention are: The present invention has established an HPLC characteristic chromatogram for Zilongjin Tablets, identified 14 characteristic peaks, and relatively fully demonstrated the chemical characteristics of Zilongjin Tablets; The present invention has investigated the extraction solvent and extraction method of the sample. Within the investigated range, the preparation method, chromatographic conditions, and elution gradient of the test solution have been optimized. The characteristic chromatogram measured by the optimized method shows 14 characteristic peaks, and the peak shapes and resolutions of each characteristic peak are all good; Methodological investigations were carried out on the established characteristic chromatogram method of Zilongjin Tablets, including instrument precision experiments, method repeatability experiments, and sample stability experiments. In the results of each experiment, the RSD values of the relative retention times of each peak are all ≤1.0%, and the RSD values of the relative peak areas are all ≤5.0%. It shows that the method is good, and the major chemical components in Zilongjin Tablets can be reflected by this method. The overall quality of Zilongjin Tablets can be controlled more efficiently and quickly. Description of the Drawings

[0011] The present invention will be further described below in conjunction with the accompanying drawings.

[0012] Figure 1 Chromatogram for investigating the extraction solvent of the test sample; among them, S1: a mixed solvent of absolute ethanol: phosphoric acid: water with a ratio of 30:0.1:29.9; S2: a mixed solvent of absolute ethanol: phosphoric acid: water with a ratio of 50:0.1:29.9; S3: a mixed solvent of absolute ethanol: phosphoric acid: water with a ratio of 70:0.1:29.9; Figure 2 Chromatogram for investigating the extraction method of the test sample; among them, S1: ultrasonic extraction; S2: reflux extraction; Figure 3 Chromatogram for wavelength investigation; among them, S1: 300 nm; S2: 320 nm; S3: 340 nm; Figure 4 Specificity chromatogram; among them, S1: negative solution; S2: ferulic acid control; S3: salvianolic acid B control; S4: chlorogenic acid control; S5: neochlorogenic acid control; S6: cryptochlorogenic acid control; S7: test sample solution; Figure 5 Precision test chromatogram; among them, S1 - S6: precisions 1 - 6; Figure 6 Repeatability test chromatogram; among them, S1 - S6: repeatabilities 1 - 6; Figure 7 Stability test chromatogram; among them, S1 - S6: stabilities 1 - 6; Figure 8 Chromatograms of eight batches of Zilongjin tablets samples; among them, S1: 350114; S2: 350151; S3: 350142; S4: 350232; S5: 350243; S6: 350231; S7: 350252; S8: 350262; Figure 9 Characteristic reference chromatogram of Zilongjin tablets. Detailed implementation manners

[0013] For the convenience of understanding by those skilled in the art, the present invention will be further described below in conjunction with the embodiments and the accompanying drawings.

[0014] Example 1 1. Instruments and test drugs 1.1 Instrument equipment .

[0015] 1.2 Test materials .

[0016] 1.3 Control information .

[0017] 1.4 Sample Information .

[0018] Investigation on the Construction Method of the Characteristic Chromatogram of Zilongjin Tablets in Example 2 2.1 Investigation on the Preparation Method of Test Samples 2.1.1 Investigation on the Extraction Solvent of Test Samples Chromatographic Conditions Chromatographic column: InertSustain C18 (4.6x250mm, 5um); Acetonitrile - tetrahydrofuran (35:13) was used as mobile phase A, and 0.1 mol / L ammonium acetate solution (adding 0.5 ml glacial acetic acid per 1000 ml) was used as mobile phase B, and gradient elution was carried out according to the regulations in the following table; The column temperature was 30 °C; The detection wavelength was 320 nm. The number of theoretical plates calculated by the salvianolic acid B peak should be not less than 5000.

[0019] .

[0020] Preparation of Reference Substance Solution Appropriately weigh an appropriate amount of reference substances of salvianolic acid B, ferulic acid, chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid, and dissolve them in methanol respectively to prepare solutions containing 30 μg per 1 ml, shake well, and use them as reference substance solutions.

[0021] Preparation of Test Sample Solution Take an appropriate amount of Zilongjin Tablets (batch number: 350252), grind them finely, accurately weigh 0.5 g, add 30 mL of petroleum ether with a boiling range of 60 - 90 °C, perform ultrasonic - assisted extraction at 40 °C and 200 W for 15 minutes, centrifuge at 8000 rpm for 10 min, discard the petroleum ether layer, and air - dry the residue; Add 30 mL of anhydrous ethanol: phosphoric acid: water mixed solvents with ratios of 30:0.1:29.9, 50:0.1:29.9, and 70:0.1:29.9 respectively to the defatted residue, reflux and extract at 80 °C for 30 minutes, centrifuge and collect the supernatant, and concentrate it under reduced pressure to 5 mL at 40 °C; Use a 200 mg / 3 mL C18 solid - phase extraction column, and activate it successively with 3 mL of methanol and 3 mL of water; Load the concentrated solution onto the activated column at a flow rate of 1 mL / min; Wash impurities with 3 mL of water, elute the target components with 3 mL of 70% methanol containing 0.1% formic acid, and collect the eluate; Dilute the eluate to 5 mL with 50% methanol solution, mix well, and filter through a 0.22 μm filter membrane to obtain the test sample solution.

[0022] Determination Method Precisely pipette 10 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0023] The RSDs of the relative retention times in the solvent investigation results were all less than 1.0%, indicating that when the extraction solvent was 40% - 80% methanol, the influence on the relative retention times of each peak in the chromatogram was relatively small. The results are shown in the appendix Figure 1 .

[0024] Table 1 Results of Relative Retention Times in Solvent Investigation .

[0025] 2.1.2 Investigation of the Extraction Method of the Test Sample Preparation of the Test Solution Take an appropriate amount of Zilongjin Tablets (batch number: 350252), grind them finely, accurately weigh 0.5 g, add 30 mL of petroleum ether with a boiling range of 60 - 90 °C, perform ultrasonic-assisted extraction at 40 °C and 200 W for 15 minutes, centrifuge at 8000 rpm for 10 min, discard the petroleum ether layer, and air-dry the residue; add 30 mL of a mixed solvent of anhydrous ethanol: phosphoric acid: water with ratios of 30:0.1:29.9, 50:0.1:29.9, and 70:0.1:29.9 respectively to the defatted residue, perform ultrasonic extraction (250 W, 40 Hz) or heat reflux extraction at 80 °C for 30 minutes, centrifuge to collect the supernatant, and concentrate it under reduced pressure to 5 mL at 40 °C; use a 200 mg / 3 mL C18 solid-phase extraction column, activate it successively with 3 mL of methanol and 3 mL of water; load the concentrated solution onto the activated column at a flow rate of 1 mL / min; wash away impurities with 3 mL of water, elute the target components with 3 mL of 70% methanol containing 0.1% formic acid, and collect the eluate; dilute the eluate to 5 mL with a 50% methanol solution, mix well, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0026] The chromatographic conditions are the same as those in item "2.1.1"; the reference solution used is the reference solution in item "2.1.1".

[0027] Determination Method Precisely pipette 10 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0028] The RSDs of the relative retention times in the extraction method investigation results were all less than 1.0%, indicating that when the extraction method was ultrasonic extraction or heat reflux extraction, the influence on the relative retention times of each peak in the chromatogram was relatively small. The results are shown in the appendix Figure 2 .

[0029] Table 2 Results of Relative Retention Times in Extraction Method Investigation 。

[0030] 2.2.1 Wavelength Investigation Chromatographic Conditions Chromatographic column: InertSustain C18 (4.6x250mm, 5um); Acetonitrile - tetrahydrofuran (35∶13) was used as mobile phase A, and 0.1mol / L ammonium acetate solution (adding 0.5ml glacial acetic acid per 1000ml) was used as mobile phase B. Gradient elution was carried out according to the regulations in the following table; the column temperature was 30°C; the detection wavelengths were selected as 300nm, 320nm, and 340nm respectively. The number of theoretical plates calculated by salvianolic acid B peak should be not less than 5000.

[0031] 。

[0032] The reference solution was taken from the reference solution under "2.1.1".

[0033] Preparation of Test Solution Take Zilongjin Tablets, grind them finely, accurately weigh 0.5g, add 30 mL of petroleum ether with a boiling range of 60 - 90°C, perform ultrasonic - assisted extraction at 40°C with 200 W for 15 minutes, centrifuge at 8000 rpm for 10 min, discard the petroleum ether layer, and air - dry the residue; add 30 mL of a mixed solvent of anhydrous ethanol: phosphoric acid: water with a ratio of 70:0.1:29.9 to the defatted residue, reflux and extract at 80°C for 30 minutes, centrifuge and collect the supernatant, concentrate it under reduced pressure to 5 mL at 40°C; use a 200 mg / 3 mL C18 solid - phase extraction column, activate it successively with 3 mL of methanol and 3 mL of water; load the concentrated solution onto the activated column at a flow rate of 1 mL / min; wash off impurities with 3 mL of water, elute the target components with 3 mL of 70% methanol containing 0.1% formic acid, collect the eluate; dilute the eluate to 5 mL with 50% methanol solution, mix well, and filter through a 0.22μm filter membrane to obtain the test solution.

[0034] Determination Method Precisely pipette 10μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0035] The RSD of the relative retention time of the wavelength investigation results was less than 1.0%, indicating that when the wavelength was in the range of 300nm - 340nm, the influence on the relative retention time of each peak in the chromatogram was small. The results are shown in the appendix Figure 3 。

[0036] Table 3 Results of Relative Retention Time in Wavelength Investigation 。

[0037] 2.2.2 Investigation of Mobile Phase Types Chromatographic conditions 1: Chromatographic column: InertSustain C18 (4.6x250mm, 5um); acetonitrile - tetrahydrofuran (35:13) as mobile phase A, 0.1mol / L ammonium acetate solution (adding 0.5ml glacial acetic acid per 1000ml) as mobile phase B, gradient elution was carried out according to the regulations in the following table; column temperature was 30°C; detection wavelength was 320nm. The number of theoretical plates calculated by Salvianolic acid B peak should be not less than 5000.

[0038] 。

[0039] Chromatographic conditions 2: Chromatographic column: InertSustain C18 (4.6x250mm, 5um); acetonitrile - tetrahydrofuran (35:13) as mobile phase A, 0.1mol / L ammonium dihydrogen phosphate solution (adding 0.5ml phosphoric acid per 1000ml) as mobile phase B, gradient elution was carried out according to the regulations in the following table; column temperature was 30°C; detection wavelength was 320nm. The number of theoretical plates calculated by Salvianolic acid B peak should be not less than 5000.

[0040] 。

[0041] Chromatographic conditions 3: Chromatographic column: InertSustain C18 (4.6x250mm, 5um); acetonitrile - tetrahydrofuran (35:13) as mobile phase A, 0.1mol / L ammonium acetate solution as mobile phase B, gradient elution was carried out according to the regulations in the following table; column temperature was 30°C; detection wavelength was 320nm. The number of theoretical plates calculated by Salvianolic acid B peak should be not less than 5000.

[0042] 。

[0043] The reference substance solution was taken from the reference substance solution under item "2.1.1".

[0044] Preparation of the test solution Take Zilongjin tablets, grind them finely, accurately weigh 0.5 g, add 30 mL of petroleum ether with a boiling range of 60 - 90 °C, perform ultrasonic-assisted extraction at 40 °C and 200 W for 15 minutes, centrifuge at 8000 rpm for 10 min, discard the petroleum ether layer, and air-dry the residue; add 30 mL of a mixed solvent of anhydrous ethanol: phosphoric acid: water in a ratio of 70:0.1:29.9 to the defatted residue, reflux and extract at 80 °C for 30 minutes, centrifuge and collect the supernatant, concentrate it under reduced pressure to 5 mL at 40 °C; use a C18 solid-phase extraction column of 200 mg / 3 mL, activate it successively with 3 mL of methanol and 3 mL of water; load the concentrated solution onto the activated column at a flow rate of 1 mL / min; wash away impurities with 3 mL of water, elute the target component with 3 mL of 70% methanol containing 0.1% formic acid, and collect the eluate; dilute the eluate to 5 mL with a 50% methanol solution, mix well, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0045] Determination method Precisely pipette 10 μl each of the reference substance solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0046] The results showed that when acetonitrile - tetrahydrofuran (volume ratio 35∶13) was used as mobile phase A and 0.1 mol / L ammonium acetate solution (adding 0.5 ml of glacial acetic acid per 1000 ml) was used as mobile phase B for elution, a total of 14 characteristic peaks appeared in the chromatogram of the test sample, and the peak separation was good and the peak shape was excellent; when acetonitrile - tetrahydrofuran (35∶13) was used as mobile phase A and 0.1 mol / L ammonium dihydrogen phosphate solution (adding 0.5 ml of phosphoric acid per 1000 ml) was used as mobile phase B or when acetonitrile - tetrahydrofuran (35∶13) was used as mobile phase A and 0.1 mol / L ammonium acetate solution was used as mobile phase B for elution, there were situations of poor peak shape and peak missing, and the corresponding effects could not be achieved.

[0047] Table 4 Results of relative retention time for investigation of mobile phase types 。

[0048] 2.2.3 Investigation of mobile phase ratio Chromatographic conditions Column: InertSustain C18 (4.6x250mm, 5um); acetonitrile - tetrahydrofuran (35∶13) as mobile phase A, 0.1 mol / L ammonium acetate solution (adding 0.5 ml of glacial acetic acid per 1000 ml) as mobile phase B, perform gradient elution according to the regulations in the following table; column temperature is 30 °C; detection wavelength is 320 nm. The number of theoretical plates calculated based on the salvianolic acid B peak should be not less than 5000.

[0049] Mobile phase ratio 1: 。

[0050] Mobile phase ratio 2: 。

[0051] Mobile phase ratio 3: 。

[0052] The reference solution used is the reference solution under item "2.2.2".

[0053] Preparation of test solution: Take Zilongjin tablets, grind them finely, accurately weigh 0.5 g, add 30 mL of petroleum ether with a boiling range of 60 - 90 °C, perform ultrasonic-assisted extraction at 40 °C with 200 W for 15 minutes, centrifuge at 8000 rpm for 10 min, discard the petroleum ether layer, and air-dry the residue; add 30 mL of a mixed solvent of anhydrous ethanol: phosphoric acid: water in a ratio of 70:0.1:29.9 to the defatted residue, reflux and extract at 80 °C for 30 minutes, centrifuge to collect the supernatant, and concentrate it under reduced pressure to 5 mL at 40 °C; use a C18 solid-phase extraction column of 200 mg / 3 mL, activate it successively with 3 mL of methanol and 3 mL of water; load the concentrated solution onto the activated column at a flow rate of 1 mL / min; wash off impurities with 3 mL of water, elute the target components with 3 mL of 70% methanol containing 0.1% formic acid, collect the eluate; dilute the eluate to 5 mL with 50% methanol solution, mix well, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0054] Assay method Precisely pipette 10 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0055] The results show that when eluting with mobile phase ratio 1, a total of 14 characteristic peaks appear in the chromatogram of the test sample, and the peak resolution is good and the peak shape is excellent; when eluting with mobile phase ratio 2 or mobile phase ratio 3, there are situations of poor peak shape and peak missing, and the corresponding effects cannot be achieved.

[0056] Table 5 Results of relative retention time for investigation of mobile phase acid concentration 。

[0057] Example 3 Methodology verification of characteristic chromatogram of Zilongjin tablets 3.1 Specificity Chromatographic conditions Chromatographic column: InertSustain C18 (4.6x250mm, 5um); Acetonitrile - tetrahydrofuran (35∶13) is used as mobile phase A, and 0.1 mol / L ammonium acetate solution (adding 0.5 mL of glacial acetic acid per 1000 mL) is used as mobile phase B, and gradient elution is carried out according to the regulations in the following table; the column temperature is 30 °C; the detection wavelength is 320 nm. The number of theoretical plates calculated based on the salvianolic acid B peak should be not less than 5000.

[0058] 。

[0059] Preparation of reference solution Weigh appropriate amounts of reference substances of salvianolic acid B, ferulic acid, chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid accurately, and dissolve them in methanol respectively to prepare solutions containing 30 μg per 1 ml. Shake well to obtain the reference solution.

[0060] Preparation of test solution Take Zilongjin tablets, grind them into fine powder, accurately weigh 0.5 g, add 30 mL of petroleum ether with a boiling range of 60 - 90 °C, perform ultrasonic-assisted extraction at 40 °C with 200 W for 15 minutes, centrifuge at 8000 rpm for 10 min, discard the petroleum ether layer, and dry the residue; add 30 mL of a mixed solvent of anhydrous ethanol: phosphoric acid: water with a ratio of 70:0.1:29.9 to the defatted residue, reflux extract at 80 °C for 30 minutes, centrifuge to collect the supernatant, and concentrate it under reduced pressure to 5 mL at 40 °C; use a 200 mg / 3 mL C18 solid-phase extraction column, activate it successively with 3 mL of methanol and 3 mL of water; load the concentrated solution onto the activated column at a flow rate of 1 mL / min; elute impurities with 3 mL of water, elute the target components with 3 mL of 70% methanol containing 0.1% formic acid, and collect the eluate; dilute the eluate to 5 mL with 50% methanol solution, mix well, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0061] Preparation of negative solution Take appropriate amounts of excipients, grind them into fine powder, weigh 0.5 g, add 30 mL of petroleum ether with a boiling range of 60 - 90 °C, perform ultrasonic-assisted extraction at 40 °C with 200 W for 15 minutes, centrifuge at 8000 rpm for 10 min, discard the petroleum ether layer, and dry the residue; add 30 mL of a mixed solvent of anhydrous ethanol: phosphoric acid: water with a ratio of 70:0.1:29.9 to the defatted residue, reflux extract at 80 °C for 30 minutes, centrifuge to collect the supernatant, and concentrate it under reduced pressure to 5 mL at 40 °C; use a 200 mg / 3 mL C18 solid-phase extraction column, activate it successively with 3 mL of methanol and 3 mL of water; load the concentrated solution onto the activated column at a flow rate of 1 mL / min; elute impurities with 3 mL of water, elute the target components with 3 mL of 70% methanol containing 0.1% formic acid, and collect the eluate; dilute the eluate to 5 mL with 50% methanol solution, mix well, and filter through a 0.22 μm filter membrane to obtain the negative solution.

[0062] Assay method Precisely pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0063] The results show that the negative solution has no interference and good specificity. The results are shown in the appendix Figure 4 。

[0064] 3.2 Precision Take Zilongjin Tablets (batch number: 350252), grind them finely, take 0.5 g, prepare the test solution according to the preparation method of the test solution under item "3.1", inject 6 needles under the chromatographic conditions under item "3.1", record the chromatogram, measure the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD.

[0065] Table 6 Results of relative retention time for precision 。

[0066] Table 7 Results of relative peak area for precision 。

[0067] The results show that the RSD of the relative retention time of each chromatographic peak is less than 1.0%, and the RSD of the relative peak area is less than 3.0%, indicating good precision. The chromatogram is shown in the appendix Figure 5 。

[0068] 3.3 Repeatability Take Zilongjin Tablets (batch number: 350252), grind them finely, take 0.5 g, a total of 6 portions, prepare the test solution according to the preparation method of the test solution under item "3.1", inject under the chromatographic conditions under item "3.1", record the chromatogram, measure the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD.

[0069] Table 8 Results of relative retention time for repeatability 。

[0070] Table 9 Results of relative peak area for repeatability 。

[0071] The results show that the RSD of the relative retention time of each chromatographic peak is less than 1.0%, and the RSD of the relative peak area is less than 5.0%, indicating good repeatability. The chromatogram is shown in the appendix Figure 6 。

[0072] 3.4 Stability Take Zilongjin Tablets (batch number: 350252), grind them finely, take 0.5 g, prepare the test solution according to the preparation method of the test solution under item "3.1", inject under the chromatographic conditions under item "3.1" at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h respectively, record the chromatogram, measure the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD.

[0073] Table 10 Results of relative retention time for stability 。

[0074] Table 11 Results of relative peak area for stability 。

[0075] The results showed that the relative retention time RSD of each chromatographic peak was less than 1.0%, and the relative peak area RSD was less than 5.0%, indicating good solution stability. The chromatogram is shown in the appendix Figure 7 。

[0076] In summary, through the specificity test of this method, the negative solution showed no interference, indicating good specificity; in the precision test, the relative retention time RSD of each chromatographic peak was less than 1.0%, and the relative peak area RSD was less than 3.0%, indicating good precision; in the repeatability test, the relative retention time RSD of each chromatographic peak was less than 1.0%, and the relative peak area RSD was less than 5.0%, indicating good repeatability; in the stability test, the relative retention time RSD of each chromatographic peak was less than 1.0%, and the relative peak area RSD was less than 5.0%, indicating good solution stability. This method has been verified by methodology, and the results are reliable and accurate.

[0077] Construction of the reference characteristic chromatogram of Zilongjin Tablets in Example 4 Chromatographic conditions Chromatographic column: InertSustain C18 (4.6x250mm, 5um); acetonitrile - tetrahydrofuran (35:13) was used as mobile phase A, and 0.1mol / L ammonium acetate solution (adding 0.5ml glacial acetic acid per 1000ml) was used as mobile phase B, and gradient elution was carried out according to the regulations in the following table; the column temperature was 30°C; the detection wavelength was 320nm. The theoretical plate number calculated based on the salvianolic acid B peak should be not less than 5000.

[0078]

[0079] Preparation of the reference substance solution Appropriate amounts of reference substances of salvianolic acid B, ferulic acid, chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid were accurately weighed and dissolved in methanol to prepare solutions containing 30ug per 1ml respectively, and shaken well to obtain the reference substance solution.

[0080] Preparation of the test solution Add 30 mL of petroleum ether with a boiling range of 60 - 90°C, perform ultrasonic-assisted extraction at 40°C with 200 W for 15 minutes, centrifuge at 8000 rpm for 10 min, discard the petroleum ether layer, and air-dry the residue; add 30 mL of a mixed solvent of anhydrous ethanol: phosphoric acid: water with a ratio of 70:0.1:29.9 to the defatted residue, reflux and extract at 80°C for 30 minutes, centrifuge to collect the supernatant, and concentrate it under reduced pressure to 5 mL at 40°C; use a C18 solid-phase extraction column of 200 mg / 3 mL, activate it successively with 3 mL of methanol and 3 mL of water; load the concentrated solution onto the activated column at a flow rate of 1 mL / min; elute impurities with 3 mL of water, elute the target components with 3 mL of 70% methanol containing 0.1% formic acid, and collect the eluate; dilute the eluate to 5 mL with a 50% methanol solution, mix well, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0081] Assay Precisely pipette 10 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0082] Import the chromatograms of the test samples of 8 batches of Zilongjin Tablets into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints", select the chromatographic peaks that exist in the chromatograms of different batches of Zilongjin Tablets as common peaks; generate a reference characteristic fingerprint by the average value calculation method, and calculate the relative retention time and relative peak area of each common peak. See Appendix Figure 8 、 9 。

[0083] Table 12 Results of Relative Retention Time of Samples of 8 Batches of Zilongjin Tablets 。

[0084] Table 13 Results of Relative Peak Area of Samples of 8 Batches of Zilongjin Tablets 。

[0085] Fourteen common peaks are presented in the chromatograms of the samples of 8 batches of Zilongjin Tablets. The RSD of the relative retention time of the fourteen common peaks is less than 1.0%, and the RSD of the relative peak area is less than 10.0%.

[0086] Fourteen characteristic peaks were presented in the chromatogram of the test sample. Among them, peak 1, peak 3, peak 4, peak 8, and peak 14 should correspond to the reference peaks of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, ferulic acid, and salvianolic acid B respectively; the peak corresponding to the reference peak of salvianolic acid B was the S peak. The relative retention times of each characteristic peak and the S peak were calculated, and their relative retention times should all be within the range of ±10% of the specified values. The specified values were: 0.20 (peak 1), 0.27 (peak 2), 0.31 (peak 3), 0.32 (peak 4), 0.34 (peak 5), 0.38 (peak 6), 0.55 (peak 7), 0.62 (peak 8), 0.70 (peak 9), 0.72 (peak 10), 0.76 (peak 11), 0.94 (peak 12), 0.98 (peak 13).

Claims

1. A method for constructing a characteristic spectrum of Zilongjin tablets, characterized in that: The following steps are involved: (1) Preparation of reference solution: Take the reference substance, weigh it accurately, add methanol to make a solution containing 30ug per 1ml, shake well, and use it as the reference solution; the reference substances are: salvianolic acid B, ferulic acid, chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid; (2) Preparation of the test solution: Take the purple dragon gold flakes, grind them into powder, weigh them accurately, add petroleum ether with a boiling range of 60-90℃, perform ultrasonic assisted extraction at 40℃, 200 W for 15 minutes, centrifuge at 8000 rpm for 10 minutes, discard the petroleum ether layer, and air-dry the residue; add 30 mL of anhydrous ethanol: phosphoric acid: water mixed solvent to the defatted residue after centrifugation, then reflux extract at 80℃ for 30 minutes, collect the supernatant by centrifugation, and concentrate the supernatant under reduced pressure at 40℃; use a C18 solid phase extraction column, activate the C18 solid phase extraction column with pure methanol and water in turn; load the concentrate obtained by reduced pressure concentration at 40℃ to the activated column at a flow rate of 1 mL / min; The impurities were eluted with water, and the target components were eluted with formic acid-methanol solution, and the eluate was collected; the eluate was made up to 5 mL with 50% methanol solution, mixed, and filtered through a 0.22 μm filter membrane to obtain the test solution; (3) Determination: Inject the reference solution and the test solution into a high performance liquid chromatograph under the following chromatographic conditions and record the chromatogram; The chromatographic conditions were as follows: octadecylsilane bonded silica gel was used as filler; Acetonitrile-tetrahydrofuran with a volume ratio of 35:13 was used as mobile phase A, and 0.1 mol / L ammonium acetate solution was used as mobile phase B, and elution was performed according to the specified gradient; the column temperature was 30°C; the flow rate was 1 ml per minute; the detection wavelength of the ultraviolet detector was 300-340 nm; The elution gradient program was: 0-20min, mobile phase A:B, volume ratio changed from 8:92 to 16:84; From 20 to 27 min, the volume ratio of mobile phase A:phase B changed from 16:84 to 19:81; From 27 to 40 min, the volume ratio of mobile phase A:phase B changed from 19:81 to 23:77; From 40 to 60 min, the volume ratio of mobile phase A:phase B changed from 23:77 to 55:45; 60-65 min, the volume ratio of mobile phase A:phase B changed from 55:45 to 8:92; 65-75 min, mobile phase A:B, volume ratio 8:92; (4) Generate a reference characteristic spectrum: Select the chromatographic peaks that exist in the chromatograms of different batches of Zilongjin Tablets as common peaks, and use the average value calculation method to generate a reference characteristic spectrum of Zilongjin Tablets.

2. The method according to claim 1, characterized in that In step (2), the volume ratio of the mixed solvent of anhydrous ethanol: phosphoric acid: water is 70:0.1:29.

9.

3. The method according to claim 1, characterized in that In step (2), the formic acid-methanol solution is a 70% methanol solution containing 0.1% formic acid.

4. The method according to claim 1, characterized in that: The chromatographic column in step (3) is: InertSustain C18 4.6x250mm, 5um.

5. The method according to any one of claims 1 to 4, characterized in that: The peak corresponding to the reference peak of the salvianolic acid B reference substance is the S peak, and the relative retention time of each characteristic peak and the S peak is within the range of ±10% of the specified value; the specified values ​​are: peak 1 is 0.20, peak 2 is 0.27, peak 3 is 0.31, peak 4 is 0.32, peak 5 is 0.34, peak 6 is 0.38, peak 7 is 0.55, peak 8 is 0.62, peak 9 is 0.70, peak 10 is 0.72, peak 11 is 0.76, peak 12 is 0.94, and peak 13 is 0.98, among which peak 1 corresponds to neochlorogenic acid, peak 3 corresponds to chlorogenic acid, peak 4 corresponds to cryptochlorogenic acid, peak 8 corresponds to ferulic acid, and peak 14 corresponds to salvianolic acid B.

6. The application of the method for constructing a characteristic spectrum of Zilongjin tablets according to any one of claims 1 to 5, characterized in that: The application of the characteristic spectrum of Zilongjin tablets in the quality control of the characteristic components of Zilongjin tablets.

7. The application of the method for constructing a characteristic spectrum of Zilongjin tablets according to any one of claims 1 to 5, characterized in that: The application of the characteristic spectrum of Zilongjin tablets in the production quality control of Zilongjin tablets.

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