A method for constructing a feature map of purple dragon gold flakes and its application

Abstract

This invention relates to the field of traditional Chinese medicine analysis technology, and in particular to a method for constructing a characteristic chromatogram of Zilongjin tablets and its application. The method includes the following steps: preparation of a reference solution; preparation of a test solution; determination conditions; and generation of a control characteristic chromatogram. This invention can comprehensively present the overall chemical composition information of Zilongjin tablets through a single detection, exhibiting 14 characteristic peaks, with minimal variation in the relative retention time of each peak (RDS < 1.0%). The sample extraction method is simple and has good reproducibility.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine analysis technology, and in particular to a method for constructing a characteristic spectrum of Zilongjin tablets and its application. Background Technology

[0002] The quality standard for Zilongjin tablets is included in the 2020 edition of the Chinese Pharmacopoeia, Volume I. The pharmacopoeia standard only includes three identification criteria and one content determination criterion, lacking a characteristic chromatographic item that can comprehensively control the quality of Zilongjin tablets. Furthermore, there are few studies in the literature concerning the characteristic chromatographic item of Zilongjin tablets, and only one patent study has investigated the fingerprint spectrum of Zilongjin tablets. For example, CN113640434A, "Construction Method and Application of High Performance Liquid Chromatography Fingerprint Spectrum of Zilongjin Tablets," identifies the characteristic peaks of different medicinal materials contained in Zilongjin tablets by constructing a reference fingerprint spectrum and a test sample solution fingerprint spectrum, and verifies the characteristic chromatographic method. However, the fingerprint spectrum in this study has relatively few chromatographic peaks, and the only specifically identified chromatographic peaks are tanshinone IIA and salvianolic acid B, both of which are contained in tanshinone. The components contained in other medicinal materials were not studied.

[0003] In addition, the construction of the feature map of Zilong Gold Flakes faces the following challenges:

[0004] (1) The complexity of the sample makes it difficult to prepare and purify the effective components and remove the interference of impurities: complex samples such as traditional Chinese medicine contain a variety of components with different polarities, molecular weights and chemical properties, which increases the difficulty of separation and detection.

[0005] (2) Difficulty in selecting high-precision detection methods: Selecting the appropriate analytical method is the key to preparing characteristic chromatograms. Commonly used analytical methods include high performance liquid chromatography (HPLC) and ultra-high performance liquid chromatography (UPLC); however, different methods have different sample processing and detection capabilities, and the most suitable method needs to be selected according to the characteristics of the sample in order to improve the accuracy of analysis and detection.

[0006] (3) Standardization issues: To ensure the comparability and reproducibility of characteristic chromatograms, standardized preparation and analysis procedures need to be established; this includes sample pretreatment, selection of chromatographic conditions, and data processing methods. A lack of standardization may lead to inconsistencies in results between different laboratories.

[0007] (4) Identification and assignment of characteristic peaks: In characteristic spectra, it is necessary to accurately identify and assign characteristic peaks; this involves a deep understanding of the chemical properties and chromatographic behavior of each component in the sample. Incorrect identification and assignment of characteristic peaks may lead to inaccurate characteristic spectra. Summary of the Invention

[0008] To overcome the shortcomings of the prior art, this invention provides a method for constructing a feature map of purple dragon gold flakes and its application.

[0009] This invention is achieved through the following technical solution:

[0010] In a first aspect, the present invention provides a method for constructing a feature map of purple dragon gold flakes, comprising the following steps:

[0011] (1) Preparation of reference solution: Take the reference standard, weigh it accurately, add methanol to prepare a solution containing 30ug per ml, shake well, and use it as the reference solution; the reference standards are: salvianolic acid B, ferulic acid, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid;

[0012] (2) Preparation of the test solution: Take Zilongjin tablets, grind them finely, accurately weigh 0.5g, add 30mL of petroleum ether with a boiling range of 60~90℃, extract with ultrasonic assistance at 40℃ and 200W for 15 minutes, centrifuge at 8000 rpm for 10 minutes, discard the petroleum ether layer, and air-dry the residue; add 30mL of anhydrous ethanol:phosphoric acid:water mixed solvent with a volume ratio of 70:0.1:29.9 to the defatted residue after centrifugation, then reflux extract at 80℃ for 30 minutes, centrifuge to collect the supernatant, and concentrate the supernatant to 5mL under reduced pressure at 40℃; use 200mg / 3 A C18 solid-phase extraction column was activated sequentially with 3 mL of pure methanol and 3 mL of water. The concentrate obtained by vacuum concentration at 40 °C was loaded onto the activated column at a flow rate of 1 mL / min. Impurities were eluted with 3 mL of water, and the target component was eluted with 3 mL of 70% methanol containing 0.1% formic acid. The eluent was collected and diluted to 5 mL with 50% methanol solution. The eluent was mixed and filtered through a 0.22 μm filter membrane to obtain the test solution.

[0013] (3) Determination: The reference solution and the test solution were injected into the high performance liquid chromatograph under the following chromatographic conditions, and the chromatograms were recorded;

[0014] The chromatographic conditions were as follows: octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile-tetrahydrofuran (35:13 v / v) was used as mobile phase A, and 0.1 mol / L ammonium acetate solution (0.5 ml of glacial acetic acid per 1000 ml) was used as mobile phase B, eluted according to a specified gradient; the column temperature was 30°C; the flow rate was 1 ml per minute; and the detection wavelength was 300–340 nm.

[0015] The elution gradient program is as follows:

[0016] From 0 to 20 minutes, the volume ratio of mobile phase A to phase B changed from 8:92 to 16:84.

[0017] 20-27 min, the volume ratio of mobile phase A: phase B changes from 16:84 to 19:81;

[0018] 27~40min, the volume ratio of mobile phase A: phase B changes from 19:81 to 23:77;

[0019] For 40-60 minutes, the volume ratio of mobile phase A to phase B changes from 23:77 to 55:45.

[0020] 60~65min, the volume ratio of mobile phase A: phase B changes from 55:45 to 8:92;

[0021] 65~75 min, mobile phase A: phase B, volume ratio 8:92;

[0022] (4) Generate a reference characteristic chromatogram: Using the "Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine" formulated by the National Pharmacopoeia Commission, the chromatographic peaks that are present in the chromatograms of different batches of Zilongjin tablets are selected as common peaks, and the reference characteristic chromatogram of Zilongjin tablets is generated by the average value calculation method.

[0023] Preferably, the chromatographic column in step (3) is: InertSustain C18 4.6x250mm, 5um.

[0024] Preferably, the peak corresponding to the reference peak of salvianolic acid B is designated as peak S, and the relative retention times of each characteristic peak and peak S are within ±10% of the specified values. 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), and 0.98 (peak 13), 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.

[0025] Secondly, this invention provides the application of the characteristic spectrum of Zilong Gold Flakes in the quality control of characteristic components of Zilong Gold Flakes.

[0026] Thirdly, this invention provides the application of the characteristic spectrum of Zilong gold flakes in the quality control of Zilong gold flake production.

[0027] Compared with the prior art, the advantages of the present invention are as follows:

[0028] This invention establishes an HPLC characteristic chromatogram of Zilongjin tablets, identifying 14 characteristic peaks, which fully demonstrates the chemical characteristics of Zilongjin tablets.

[0029] This invention investigated the extraction solvent and extraction method of the sample. Within the scope of investigation, the preparation method, chromatographic conditions and elution gradient of the test solution were optimized. The characteristic chromatograms obtained by the optimized method showed 14 characteristic peaks, and the peak shape and resolution of each characteristic peak were good.

[0030] The methodological evaluation of the constructed characteristic spectral method for Zilongjin tablets was conducted, including instrument precision experiments, method repeatability experiments, and sample stability experiments. In all experimental results, the RSD values ​​of the relative retention times of each peak were ≤1.0%, and the RSD values ​​of the relative peak areas were ≤5.0%. This indicates that the method is effective and can accurately reflect the major chemical components in Zilongjin tablets. It allows for more efficient and rapid control of the overall quality of Zilongjin tablets. Attached Figure Description

[0031] The invention will now be further described with reference to the accompanying drawings.

[0032] Figure 1 The chromatogram of the extraction solvent of the test sample was examined; where S1: an anhydrous ethanol: phosphoric acid: water mixed solvent in a ratio of 30:0.1:29.9; S2: an anhydrous ethanol: phosphoric acid: water mixed solvent in a ratio of 50:0.1:29.9; S3: an anhydrous ethanol: phosphoric acid: water mixed solvent in a ratio of 70:0.1:29.9.

[0033] Figure 2 The chromatograms of the extraction methods for the test sample were examined; where S1: ultrasonic extraction; S2: reflux extraction;

[0034] Figure 3 The wavelength was examined in the chromatogram; where S1: 300nm; S2: 320nm; S3: 340nm;

[0035] Figure 4 Specific chromatogram; where 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 solution;

[0036] Figure 5 Chromatograms of precision test; where S1~S6: precision 1~6;

[0037] Figure 6 Chromatograms of repeatability tests; where S1~S6: repeatability 1~6;

[0038] Figure 7 Chromatograms of stability test; where S1~S6: stability 1~6;

[0039] Figure 8Chromatograms of eight batches of Zilongjin tablets; S1: 350114; S2: 350151; S3: 350142; S4: 350232; S5: 350243; S6: 350231; S7: 350252; S8: 350262;

[0040] Figure 9 Purple Dragon Gold Flakes Comparison Characteristic Atlas. Detailed Implementation

[0041] To facilitate understanding by those skilled in the art, the present invention will be further described below in conjunction with embodiments and accompanying drawings.

[0042] Example 1

[0043] 1. Instruments and reagents

[0044] 1.1 Instruments and Equipment

[0045] .

[0046] 1.2 Test Materials

[0047] .

[0048] 1.3 Reference Standard Information

[0049] .

[0050] 1.4 Sample Information

[0051] .

[0052] Example 2: Investigation of the method for constructing the feature map of Zilong Gold Flakes

[0053] 2.1 Investigation of the preparation method of the test sample

[0054] 2.1.1 Investigation of extraction solvent for test sample

[0055] Chromatographic conditions

[0056] Chromatographic column: InertSustain C18 (4.6 x 250 mm, 5 μm); acetonitrile-tetrahydrofuran (35:13) as mobile phase A, and 0.1 mol / L ammonium acetate solution (0.5 ml of glacial acetic acid per 1000 ml) as mobile phase B, with gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 320 nm. The theoretical plate number, calculated based on the Danshensu B peak, should be no less than 5000.

[0057] .

[0058] Preparation of reference solution

[0059] Take appropriate amounts of salvianolic acid B, ferulic acid, chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid reference standards, accurately weigh them, add methanol to prepare solutions containing 30 μg per ml, shake well, and use them as reference solutions.

[0060] Preparation of test solution

[0061] Take an appropriate amount of Zilongjin tablets (batch number: 350252), grind them into a fine powder, accurately weigh 0.5g, add 30mL of petroleum ether with a boiling range of 60~90℃, extract with ultrasonic assistance at 40℃ and 200W 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 in the ratio of 30:0.1:29.9, 50:0.1:29.9, and 70:0.1:29.9 to the defatted residue, reflux extract at 80℃ for 30 minutes, centrifuge to collect the supernatant, and concentrate to 5 mL under reduced pressure at 40℃; use a 200 mg / 3 mL C18 solid phase extraction column, activate with 3 mL methanol and 3 mL water sequentially; load the concentrate into the activation column at a flow rate of 1 mL / min; wash away impurities with 3 mL water, 3 The target component was eluted with 70% methanol containing 0.1% formic acid in mL, and the eluent was collected. The eluent was then diluted to 5 mL with 50% methanol solution, mixed well, and filtered through a 0.22 μm filter membrane to obtain the test solution.

[0062] Determination method

[0063] Accurately pipette 10 μl each of the reference solution and the test solution and inject them into the liquid chromatograph, then record the chromatograms.

[0064] The RSDs of the relative retention times for all solvent-based extraction results were less than 1.0%, indicating that the extraction solvent of 40%–80% methanol had little impact on the relative retention times of the peaks in the chromatogram. The results are shown in the appendix. Figure 1 .

[0065] Table 1. Results of relative retention times for solvent testing

[0066] .

[0067] 2.1.2 Investigation of the sample extraction method

[0068] Preparation of test solution

[0069] Take an appropriate amount of Zilongjin tablets (batch number: 350252), grind them into a fine powder, accurately weigh 0.5g, add 30mL of petroleum ether with a boiling range of 60~90℃, and extract with ultrasonic assistance at 40℃ and 200W for 15 minutes. Centrifuge at 8000 rpm for 10 minutes, discard the petroleum ether layer, and air-dry the residue. Add 30mL of anhydrous ethanol:phosphoric acid:water mixed solvent with a ratio of 30:0.1:29.9, 50:0.1:29.9, and 70:0.1:29.9 to the defatted residue, and extract with ultrasonic extraction (250W, 40Hz) or reflux at 80℃ for 30 minutes, respectively. Centrifuge to collect the supernatant, and concentrate it to 5mL under reduced pressure at 40℃. Use a 200 mg / 3 mL C18 solid-phase extraction column, and activate it with 3 mL of methanol and 3 mL of water sequentially. Load the concentrated solution into the activation column at a flow rate of 1. mL / min; wash away impurities with 3 mL of water, and elute the target component with 3 mL of 70% methanol containing 0.1% formic acid, and collect the eluent; dilute the eluent to 5 mL with 50% methanol solution, mix well, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0070] The chromatographic conditions are the same as those in section “2.1.1”; the reference solution is the same as that in section “2.1.1”.

[0071] Determination method

[0072] Accurately pipette 10 μl each of the reference solution and the test solution and inject them into the liquid chromatograph, then record the chromatograms.

[0073] The RSDs of the relative retention times for all extraction methods were less than 1.0%, indicating that ultrasonic extraction or reflux extraction had minimal impact on the relative retention times of the peaks in the chromatogram. (See attached figure for results.) Figure 2 .

[0074] Table 2 Results of relative retention time for extraction methods

[0075] .

[0076] 2.2.1 Wavelength Investigation

[0077] Chromatographic conditions

[0078] Chromatographic column: InertSustain C18 (4.6 x 250 mm, 5 μm); acetonitrile-tetrahydrofuran (35:13) was used as mobile phase A, and 0.1 mol / L ammonium acetate solution (0.5 ml of glacial acetic acid per 1000 ml) was used as mobile phase B, with gradient elution as specified in the table below; column temperature was 30°C; detection wavelengths were selected at 300 nm, 320 nm, and 340 nm. The theoretical plate number, calculated based on the B peak of salvianolic acid, should be no less than 5000.

[0079] .

[0080] The reference solution used is the reference solution described in section “2.1.1”.

[0081] Preparation of test solution

[0082] Take Zilongjin tablets, grind them into a fine powder, accurately weigh 0.5g, add 30mL of petroleum ether with a boiling range of 60~90℃, extract with ultrasonic assistance at 40℃ 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 solvent with a ratio of 70:0.1:29.9 to the defatted residue, reflux extract at 80℃ for 30 minutes, centrifuge to collect the supernatant, and concentrate to 5 mL under reduced pressure at 40℃; 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 into the activated column at a flow rate of 1 mL / min; wash away impurities with 3 mL of water, and wash away the target component with 3 mL of 70% methanol containing 0.1% formic acid, collect the eluent; make up the volume of the eluent to 5 mL with 50% methanol solution, mix well, filter through a 0.22μm filter membrane to obtain the test solution.

[0083] Determination method

[0084] Accurately pipette 10 μl each of the reference solution and the test solution and inject them into the liquid chromatograph, then record the chromatograms.

[0085] The RSDs of the relative retention times for all wavelength-dependent peaks were less than 1.0%, indicating that wavelengths in the 300 nm–340 nm range have minimal impact on the relative retention times of the peaks in the chromatogram. (See attached figure for results.) Figure 3 .

[0086] Table 3. Results of relative retention time for wavelength studies

[0087] .

[0088] 2.2.2 Investigation of Mobile Phase Types

[0089] Chromatographic conditions 1: Column: InertSustain C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-tetrahydrofuran (35:13); mobile phase B: 0.1 mol / L ammonium acetate solution (0.5 ml of glacial acetic acid per 1000 ml); gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 320 nm. The theoretical plate number, calculated based on the Danshensu B peak, should be no less than 5000.

[0090] .

[0091] Chromatographic conditions 2: Column: InertSustain C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-tetrahydrofuran (35:13); mobile phase B: 0.1 mol / L ammonium dihydrogen phosphate solution (0.5 mL phosphoric acid per 1000 mL); gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 320 nm. The theoretical plate number, calculated based on the Danshensu B peak, should be no less than 5000.

[0092] .

[0093] Chromatographic conditions 3: Column: InertSustain C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-tetrahydrofuran (35:13); mobile phase B: 0.1 mol / L ammonium acetate solution; gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 320 nm. The theoretical plate number, calculated based on the Danshensu B peak, should be no less than 5000.

[0094] .

[0095] The reference solution used is the reference solution described in section “2.1.1”.

[0096] Preparation of test solution

[0097] Take Zilongjin tablets, grind them into a fine powder, accurately weigh 0.5g, add 30mL of petroleum ether with a boiling range of 60~90℃, extract with ultrasonic assistance at 40℃ 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 solvent with a ratio of 70:0.1:29.9 to the defatted residue, reflux extract at 80℃ for 30 minutes, centrifuge to collect the supernatant, and concentrate to 5 mL under reduced pressure at 40℃; 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 into the activated column at a flow rate of 1 mL / min; wash away impurities with 3 mL of water, and wash away the target component with 3 mL of 70% methanol containing 0.1% formic acid, collect the eluent; make up the volume of the eluent to 5 mL with 50% methanol solution, mix well, filter through a 0.22μm filter membrane to obtain the test solution.

[0098] Determination method

[0099] Accurately pipette 10 μl each of the reference solution and the test solution and inject them into the liquid chromatograph, then record the chromatograms.

[0100] The results showed that when elution was performed using acetonitrile-tetrahydrofuran (35:13 v / v) as mobile phase A and 0.1 mol / L ammonium acetate solution (0.5 ml glacial acetic acid per 1000 ml) as mobile phase B, the chromatogram of the test sample showed 14 characteristic peaks with good peak resolution and superior peak shape. However, when elution was performed using acetonitrile-tetrahydrofuran (35:13) as mobile phase A and 0.1 mol / L ammonium dihydrogen phosphate solution (0.5 ml phosphoric acid per 1000 ml) as mobile phase B, or using acetonitrile-tetrahydrofuran (35:13) as mobile phase A and 0.1 mol / L ammonium acetate solution as mobile phase B, both resulted in poor peak shape and peak loss, failing to achieve the desired effect.

[0101] Table 4 Results of Relative Retention Time Based on Mobile Phase Type

[0102] .

[0103] 2.2.3 Investigation of Mobile Phase Ratio

[0104] Chromatographic conditions: Column: InertSustain C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-tetrahydrofuran (35:13); mobile phase B: 0.1 mol / L ammonium acetate solution (0.5 mL glacial acetic acid per 1000 mL); gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 320 nm. The theoretical plate number, calculated based on the Danshensu B peak, should be no less than 5000.

[0105] Mobile phase ratio 1:

[0106] .

[0107] Mobile phase ratio 2:

[0108] .

[0109] Mobile phase ratio 3:

[0110] .

[0111] The reference solution shall be the reference solution under section “2.2.2”.

[0112] Preparation of the test solution: Take Zilongjin tablets, grind them finely, accurately weigh 0.5g, add 30mL of petroleum ether (boiling range 60~90℃), extract with ultrasonic assistance at 40℃ and 200W for 15 minutes, centrifuge at 8000 rpm for 10 minutes, discard the petroleum ether layer, and air-dry the residue; add 30mL of anhydrous ethanol:phosphoric acid:water mixed solvent with a ratio of 70:0.1:29.9 to the defatted residue, reflux at 80℃ for 30 minutes, centrifuge to collect the supernatant, and concentrate to 5mL under reduced pressure at 40℃; use a 200 mg / 3 mL C18 solid phase extraction column, activate with 3 mL of methanol and 3 mL of water sequentially; load the concentrated solution into the activated column at a flow rate of 1 mL / min; elute impurities with 3 mL of water, and elute the target component with 3 mL of 70% methanol containing 0.1% formic acid, collect the eluent; dilute the eluent to 5 mL with 50% methanol solution. Mix mL, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0113] Determination method

[0114] Accurately pipette 10 μl each of the reference solution and the test solution and inject them into the liquid chromatograph, then record the chromatograms.

[0115] The results showed that when eluting with mobile phase ratio 1, the chromatogram of the test sample showed 14 characteristic peaks with good peak resolution and excellent peak shape. When eluting with mobile phase ratio 2 or mobile phase ratio 3, there were poor peak shapes and peak missing, and neither achieved the desired effect.

[0116] Table 5 Results of Relative Retention Time Based on Mobile Phase Acid Concentration

[0117] .

[0118] Example 3: Validation of the Methodology for Characteristic Mapping of Purple Dragon Gold Flakes

[0119] 3.1 Exclusivity

[0120] Chromatographic conditions

[0121] Chromatographic column: InertSustain C18 (4.6 x 250 mm, 5 μm); acetonitrile-tetrahydrofuran (35:13) as mobile phase A, and 0.1 mol / L ammonium acetate solution (0.5 ml of glacial acetic acid per 1000 ml) as mobile phase B, with gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 320 nm. The theoretical plate number, calculated based on the Danshensu B peak, should be no less than 5000.

[0122] .

[0123] Preparation of reference solution

[0124] Take appropriate amounts of salvianolic acid B, ferulic acid, chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid reference standards, accurately weigh them, add methanol to prepare solutions containing 30 μg per ml, shake well, and use them as reference solutions.

[0125] Preparation of test solution

[0126] Take Zilongjin tablets, grind them into a fine powder, accurately weigh 0.5g, add 30mL of petroleum ether with a boiling range of 60~90℃, extract with ultrasonic assistance at 40℃ 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 solvent with a ratio of 70:0.1:29.9 to the defatted residue, reflux extract at 80℃ for 30 minutes, centrifuge to collect the supernatant, and concentrate to 5 mL under reduced pressure at 40℃; 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 into the activated column at a flow rate of 1 mL / min; wash away impurities with 3 mL of water, and wash away the target component with 3 mL of 70% methanol containing 0.1% formic acid, collect the eluent; make up the volume of the eluent to 5 mL with 50% methanol solution, mix well, filter through a 0.22μm filter membrane to obtain the test solution.

[0127] Preparation of negative solution

[0128] Take an appropriate amount of excipients, grind them finely, take 0.5g, add 30mL of petroleum ether with a boiling range of 60~90℃, extract with ultrasonic assistance at 40℃ and 200W for 15 minutes, centrifuge at 8000 rpm for 10 minutes, discard the petroleum ether layer, and air-dry the residue; add 30mL of anhydrous ethanol:phosphoric acid:water mixed solvent with a ratio of 70:0.1:29.9 to the defatted residue, reflux extract at 80℃ for 30 minutes, centrifuge to collect the supernatant, and concentrate to 5mL under reduced pressure at 40℃; 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 into the activated column at a flow rate of 1 mL / min; wash away impurities with 3 mL of water, and wash away the target component with 3 mL of 70% methanol containing 0.1% formic acid, collect the eluent; make up the volume of the eluent to 5 mL with 50% methanol solution, mix well, and filter through a 0.22μm filter membrane to obtain the final product.

[0129] Determination method

[0130] Accurately pipette 10 μl each of the reference solution and the test solution and inject them into the liquid chromatograph, then record the chromatograms.

[0131] The results showed that the negative solution had no interference and exhibited good specificity. See attached results. Figure 4 .

[0132] 3.2 Precision

[0133] Grind 0.5g of Zilongjin tablets (batch number: 350252) into a fine powder. Prepare the test solution according to the test solution preparation method in section "3.1". Inject the sample 6 times under the chromatographic conditions in section "3.1", record the chromatogram, determine the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0134] Table 6. Results of Precision Relative Retention Time

[0135] .

[0136] Table 7 Precision Relative Peak Area Results

[0137] .

[0138] 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 3.0%, indicating good precision. The chromatograms are attached. Figure 5 .

[0139] 3.3 Repeatability

[0140] Grind Zilongjin tablets (batch number: 350252) into a fine powder, take 0.5g, make 6 portions, prepare the test solution according to the test solution preparation method under section "3.1", inject the sample according to the chromatographic conditions under section "3.1", record the chromatogram, determine the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0141] Table 8. Repeatability Relative Retention Time Results

[0142] .

[0143] Table 9. Repeatability Relative Peak Area Results

[0144] .

[0145] 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 repeatability. The chromatograms are attached. Figure 6 .

[0146] 3.4 Stability

[0147] Grind 0.5g of Zilongjin tablets (batch number: 350252) into a fine powder. Prepare a test solution according to the test solution preparation method in section "3.1". Inject the sample at 0h, 2h, 4h, 8h, 12h and 24h according to the chromatographic conditions in section "3.1". Record the chromatograms, determine the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0148] Table 10 Results of relative retention time for stability

[0149] .

[0150] Table 11. Stability relative peak area results

[0151] .

[0152] 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 attached. Figure 7 .

[0153] In summary, this method has demonstrated good specificity, with no interference from the negative solution in the specificity test. In the precision test, the RSD of the relative retention time of each chromatographic peak was less than 1.0%, and the RSD of the relative peak area was less than 3.0%, indicating good precision. In the repeatability test, the RSD of the relative retention time of each chromatographic peak was less than 1.0%, and the RSD of the relative peak area was less than 5.0%, indicating good repeatability. In the stability test, the RSD of the relative retention time of each chromatographic peak was less than 1.0%, and the RSD of the relative peak area was less than 5.0%, indicating good solution stability. This method has been validated and the results are reliable and accurate.

[0154] Example 4: Construction of the characteristic map of Zilongjin tablets

[0155] Chromatographic conditions

[0156] Chromatographic column: InertSustain C18 (4.6 x 250 mm, 5 μm); acetonitrile-tetrahydrofuran (35:13) as mobile phase A, and 0.1 mol / L ammonium acetate solution (0.5 ml of glacial acetic acid per 1000 ml) as mobile phase B, with gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 320 nm. The theoretical plate number, calculated based on the Danshensu B peak, should be no less than 5000.

[0157]

[0158] Preparation of reference solution

[0159] Take appropriate amounts of salvianolic acid B, ferulic acid, chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid reference standards, accurately weigh them, add methanol to prepare solutions containing 30 μg per ml, shake well, and use them as reference solutions.

[0160] Preparation of test solution

[0161] Add 30 mL of petroleum ether (boiling range 60-90℃), extract with ultrasound at 40℃ and 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 in a ratio of 70:0.1:29.9 to the defatted residue, reflux at 80℃ for 30 minutes, centrifuge to collect the supernatant, and concentrate to 5 mL under reduced pressure at 40℃; 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 into the activated column at a flow rate of 1 mL / min; elute impurities with 3 mL of water, and elute the target component with 3 mL of 70% methanol containing 0.1% formic acid, collect the eluent; make up the volume of the eluent to 5 mL with 50% methanol solution, mix well, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0162] Determination method

[0163] Accurately pipette 10 μl each of the reference solution and the test solution and inject them into the liquid chromatograph, then record the chromatograms.

[0164] Chromatograms from eight batches of Zilongjin tablets were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System." Chromatographic peaks present in all batches of Zilongjin tablets were selected as common peaks. A reference characteristic chromatogram was generated using the average value method, and the relative retention time and relative peak area of ​​each common peak were calculated. (See attached image) Figure 8 , 9 .

[0165] Table 12 Relative retention time results of eight batches of Zilongjin flakes samples

[0166] .

[0167] Table 13 Relative peak area results of eight batches of Zilongjin tablets

[0168] .

[0169] The chromatograms of all 8 batches of Zilongjin tablets showed 14 common peaks. The RSD of the relative retention time of the 14 common peaks was less than 1.0%, and the RSD of the relative peak area was less than 10.0%.

[0170] The chromatogram of the test sample shows 14 characteristic peaks. Peaks 1, 3, 4, 8, and 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 is the S peak. Calculate the relative retention times of each characteristic peak and the S peak. The relative retention times should all be within ±10% of the specified values. 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), and 0.98 (peak 13).

Claims

1. A method for constructing a feature map of purple dragon gold flakes, characterized in that: Includes the following steps: (1) Preparation of reference solution: Take the reference standard, weigh it accurately, add methanol to prepare a solution containing 30ug per ml, shake well, and use it as the reference solution; the reference standards are: salvianolic acid B, ferulic acid, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid; (2) Preparation of test solution: Take Zilongjin tablets, grind them into a fine powder, weigh them accurately, add petroleum ether with a boiling range of 60~90℃, extract with ultrasonic assistance at 40℃ and 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 anhydrous ethanol:phosphoric acid:water mixed solvent to the defatted residue after centrifugation, then reflux at 80℃ for 30 minutes, collect the supernatant by centrifugation, 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 sequence; load the concentrated solution obtained by concentrated solution under reduced pressure at 40℃ onto the activated column at a flow rate of 1 mL / min; Impurities were washed away with water, and the target component was eluted with formic acid-methanol solution. The eluent was collected. The eluent was diluted to 5 mL with 50% methanol solution, mixed well, and filtered through a 0.22 μm filter membrane to obtain the test solution. The volume ratio of the anhydrous ethanol:phosphoric acid:water mixed solvent was 70:0.1:29.9, and the formic acid-methanol solution was a 70% methanol solution containing 0.1% formic acid. (3) Determination: The reference solution and the test solution were injected into the high performance liquid chromatograph under the following chromatographic conditions, and the chromatograms were recorded; The chromatographic conditions were as follows: octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile-tetrahydrofuran (35:13 v / v) was used as mobile phase A, and 0.1 mol / L ammonium acetate solution was used as mobile phase B, with elution performed according to a specified gradient; the column temperature was 30°C; the flow rate was 1 ml per minute; and the UV detector wavelength was 300–340 nm. The elution gradient program is as follows: From 0 to 20 minutes, the volume ratio of mobile phase A to phase B changed from 8:92 to 16:

84. 20-27 min, the volume ratio of mobile phase A: phase B changes from 16:84 to 19:81; 27~40min, the volume ratio of mobile phase A: phase B changes from 19:81 to 23:77; For 40-60 minutes, the volume ratio of mobile phase A to phase B changes from 23:77 to 55:

45. 60~65min, the volume ratio of mobile phase A: phase B changes from 55:45 to 8:92; 65~75 min, mobile phase A: phase B, volume ratio 8:92; (4) Generate a reference characteristic chromatogram: Select the chromatographic peaks that are present in the chromatograms of different batches of Zilongjin tablets as common peaks, and generate a reference characteristic chromatogram of Zilongjin tablets by calculating the average value; take the peak corresponding to the reference peak of salvianolic acid B as the S peak, and the relative retention time of each characteristic peak and the S peak is within ±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.

2. The method according to claim 1, characterized in that, The chromatographic column in step (3) is: InertSustain C184.6x250mm, 5um.

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