Construction method and application of fingerprint spectrum of damp toxin clearing preparation

Abstract

The invention relates to a construction method and application of a fingerprint spectrum of a Rheuyanqing preparation, and the construction method comprises the following steps: extracting the Rheuyanqing preparation by using an extraction solvent to prepare a test solution; performing high performance liquid chromatography detection on the test solution, and constructing a fingerprint spectrum of the Huidoqing preparation; the conditions of the high performance liquid chromatography detection are as follows: a mobile phase A is a phosphoric acid aqueous solution, a mobile phase B is acetonitrile, and gradient elution is adopted. The construction method of the fingerprint spectrum of the damp toxin clearing preparation is simple, convenient, reliable, high in objectivity and good in precision, repeatability, specificity and stability. Furthermore, each characteristic peak of the fingerprint spectrum of the Rhodoqing preparation is relatively good in separation degree, contains 16 common peaks, and identifies four characteristic peaks including ferulic acid, astilbin, baicalin, salvianolic acid B and the like, so that the characteristics of main chemical components of the Rhodoqing preparation can be relatively comprehensively reflected, and an overall quality control basis can be provided for the quality standard of the Rhodoqing preparation.

Description

Technical Field

[0001] The present application relates to the technical field of detection of traditional Chinese medicine preparations, and in particular to a method for constructing and applying a fingerprint spectrum of a Shiduqing preparation. Background Art

[0002] Shiduqing preparation is a pure Chinese medicine preparation made by selecting a variety of Chinese medicines under the guidance of traditional Chinese medicine theory. It is a Chinese medicine prescription with mature concepts of dermatological diseases and a first-line medicine for skin allergies in traditional Chinese medicine. The prescription of Shiduqing preparation includes nine medicinal materials such as Rehmannia root, Angelica sinensis, Salvia miltiorrhiza, Sophora flavescens, Periostracum cicadae, Scutellaria baicalensis, Dictamni root, Smilax glabra, and Licorice. It has the effects of nourishing blood and moistening dryness, removing dampness and detoxifying, and dispelling wind and relieving itching. It is mainly used for skin pruritus caused by blood deficiency and dampness accumulation. The existing statutory quality control methods for Shiduqing preparation are mainly microscopic identification, thin layer identification and single content determination method of matrine, which have limitations on comprehensive product quality control.

[0003] Traditional Chinese medicine fingerprint is a comprehensive and quantifiable identification method based on the systematic study of chemical components of traditional Chinese medicine. It is mainly used to evaluate the authenticity, quality and stability of Chinese medicinal materials and semi-finished products of traditional Chinese medicine preparations. Traditional Chinese medicine is a complex multi-component system, so the evaluation of its quality should adopt a detection method that is suitable for it and can provide rich identification information. Traditional Chinese medicine fingerprint can more comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine, and can provide an overall description and evaluation of the quality of the medicine. It further reflects the quality of traditional Chinese medicine and the overall efficacy of traditional Chinese medicine. As a traditional Chinese medicine preparation, the fingerprint of Shiduqing preparation can reflect its unique chemical composition and pharmacodynamic characteristics, and has a wide range of application value in the quality control of traditional Chinese medicine. Summary of the invention

[0004] Based on this, the purpose of this application includes providing a method for constructing and applying a fingerprint spectrum of a Shiduqing preparation.

[0005] The technical solution of this application is as follows:

[0006] In one aspect of the present invention, a method for constructing a fingerprint of a Shiduqing preparation is provided, comprising the following steps:

[0007] Extract the wet-to-clear preparation using an extraction solvent to prepare a test solution;

[0008] Performing high performance liquid chromatography on the test solution to construct a fingerprint of the Shiduqing preparation;

[0009] The conditions for the high performance liquid chromatography detection include: mobile phase A is phosphoric acid aqueous solution, mobile phase B is acetonitrile, and gradient elution is adopted.

[0010] In one embodiment, the gradient elution procedure comprises:

[0011] 0-15min, the volume percentage of the mobile phase B increased from 5% to 20%;

[0012] 15min-25min, the volume percentage of the mobile phase B increased from 20% to 21%;

[0013] 25min-35min, the volume percentage of the mobile phase B increased from 21% to 28%;

[0014] 35min-40min, the volume percentage of the mobile phase B increased from 28% to 30%;

[0015] 40min-50min, the volume percentage of the mobile phase B increased from 30% to 48%;

[0016] 50min-60min, the volume percentage of the mobile phase B increased from 48% to 55%;

[0017] From 60 min to 65 min, the volume percentage of the mobile phase B was maintained at 55%.

[0018] In one embodiment, the volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.05%-0.2%.

[0019] In one embodiment, the high performance liquid chromatography detection satisfies at least one of the following conditions:

[0020] (1) The detection wavelength is 200nm-300nm;

[0021] (2) Flow rate: 0.5 mL / min-2 mL / min;

[0022] (3) Column temperature is 25°C-35°C;

[0023] (4) The injection volume is 1μL-10μL.

[0024] In one embodiment, the extracting step satisfies at least one of the following conditions:

[0025] (1) The extraction method is ultrasonic extraction or reflux extraction;

[0026] (2) The extraction time is 15min-60min;

[0027] (3) The extraction solvent includes methanol or methanol aqueous solution; optionally, the volume percentage of methanol in the methanol aqueous solution is 0.1%-100% methanol aqueous solution.

[0028] In one embodiment, the method for constructing the fingerprint of the wet detoxification preparation further comprises the following steps:

[0029] Dissolving the reference substance using a dissolving reagent to prepare a reference substance mixed solution;

[0030] Performing high performance liquid chromatography on the reference substance mixed solution to obtain a reference spectrum;

[0031] According to the reference spectrum, the chromatographic peaks of the fingerprint spectrum of the Shiduqing preparation are identified as common peaks;

[0032] Wherein, the reference substance includes one or more of ferulic acid, astilbin, baicalin, salvianolic acid B, matrine, oxymatrine, tanshinone IIA, liquiritin, ammonium glycyrrhizinate, verbascoside and rehmannoside D.

[0033] In one embodiment, the Shiduqing preparation includes Shiduqing tablets, Shiduqing capsules or Shiduqing granules.

[0034] In one embodiment, the dissolving agent includes methanol or a methanol-water solution; optionally, in the methanol-water solution, the volume percentage of methanol is 0.1%-100%.

[0035] In one embodiment, the fingerprint of the Shiduqing preparation contains 16 common peaks, among which the 4 identified characteristic peaks include: Peak 6 is ferulic acid, Peak 7 is astilbin, Peak 10 is baicalin, and Peak 11 is salvianolic acid B.

[0036] Another aspect of the present invention provides the application of the method for constructing the fingerprint of the Shiduqing preparation as described above in the quality control of the Shiduqing preparation.

[0037] Compared with the prior art, this application has the following beneficial effects:

[0038] The present application constructs a fingerprint spectrum of the Shiduqing preparation containing multiple characteristic peaks by using specific high performance liquid chromatography conditions to detect and analyze the Shiduqing preparation. The method for constructing the fingerprint spectrum of the Shiduqing preparation is simple, reliable, and objective, and has good precision, repeatability, specificity, and stability.

[0039] Furthermore, the fingerprint of the Shiduqing preparation has good separation of the characteristic peaks, contains 16 common peaks, and identifies four characteristic peaks, namely ferulic acid, astilbin, baicalin, and salvianolic acid B, which can more comprehensively reflect the main chemical composition characteristics of the Shiduqing preparation; in addition, the present application also investigates the medicinal material attribution of each characteristic peak, and the detection results of different batches of Shiduqing tablets have high similarity. The generated control fingerprint can be used as an overall similarity evaluation of the material basis of the Shiduqing tablets, and can provide an overall quality control basis for the quality standards of the Shiduqing preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0041] Figure 1 It is the HPLC superimposed fingerprint of Shiduqing Tablets in Example 1 and the control fingerprint R.

[0042] Figure 2 This is the HPLC detection chart of the mixed reference solution in Example 1.

[0043] Figure 3 This is the HPLC detection diagram of the medicinal materials attribution of the characteristic peaks of the fingerprint of Shiduqing Tablets in Example 1, wherein A: Shiduqing Tablets; B: Rehmannia root; C: Scutellaria baicalensis; D: Salvia miltiorrhiza; E: Licorice; F: Angelica sinensis; G: Sophora flavescens; H: Periostracum cicadae; I: Smilax glabra.

[0044] Figure 4 It is the HPLC detection diagram of the Shiduqing tablets, mixed reference substance and blank test solution in Example 1, wherein A: Shiduqing tablets; B: mixed reference substance; C: blank solvent.

[0045] Figure 5 This is the HPLC detection diagram of the intermediate precision of the fingerprint instrument of the Shiduqing Tablets in Example 1, wherein A: liquid phase equipment J-20-C-008; B: liquid phase equipment J-20-C-093.

[0046] Figure 6 These are the HPLC detection graphs of the intermediate precision of the chromatographic columns of the fingerprint of the Shiduqing Tablets in Example 1, A: chromatographic column JZ-SY-C002; B: chromatographic column QC-C18-24-0401; C: chromatographic column QC-C18-22-0604. DETAILED DESCRIPTION

[0047] The present application will be further described in detail below in conjunction with specific embodiments. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thoroughly understood.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0049] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0050] As used herein, "one or more" refers to any one, any two, or any two or more of the listed items.

[0051] Herein, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0052] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0053] Only some numerical ranges are specifically disclosed herein. However, any lower limit can be combined with any upper limit to form an undefined range; and any lower limit can be combined with other lower limits to form an undefined range, and any upper limit can be combined with any other upper limit to form an undefined range. In addition, each separately disclosed point or single value itself can be combined with any other point or single value as a lower limit or upper limit or with other lower limits or upper limits to form an undefined range. The use of numerical ranges represented by endpoints includes all numbers within the range and any range within the range, for example, 1 to 5 includes 1, 1.1, 1.3, 1.5, 2, 2.75, 3, 3.80, 4 and 5, etc.

[0054] The percentage contents involved in this application, unless otherwise specified, refer to mass percentage for solid-liquid mixing and solid-solid mixing, and refer to volume percentage for liquid-liquid mixing.

[0055] The percentage concentrations mentioned in this application, unless otherwise specified, refer to the final concentration, which refers to the percentage of the added component in the system after the addition of the component.

[0056] The temperature parameters in this application, unless otherwise specified, allow both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±2°C, ±1°C, ±0.5°C, ±0.4°C, ±0.3°C, ±0.2°C, and ±0.1°C are allowed. Normal temperature or room temperature in this application refers to no temperature control operation, generally 4°C~35°C, preferably 20±5°C.

[0057] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0058] Traditional Chinese medicine fingerprint is a comprehensive and quantifiable identification method based on the systematic study of chemical components of traditional Chinese medicine. It is mainly used to evaluate the authenticity, quality and stability of Chinese medicinal materials and semi-finished products of traditional Chinese medicine preparations. Traditional Chinese medicine is a complex multi-component system, so the evaluation of its quality should adopt a detection method that is suitable for it and can provide rich identification information. Traditional Chinese medicine fingerprint can more comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine, and can provide an overall description and evaluation of the quality of the medicine. It further reflects the quality of traditional Chinese medicine and the overall efficacy of traditional Chinese medicine. As a traditional Chinese medicine preparation, the fingerprint of Shiduqing preparation can reflect its unique chemical composition and pharmacodynamic characteristics, and has a wide range of application value in the quality control of traditional Chinese medicine.

[0059] Based on this, one aspect of the present invention provides a method for constructing a fingerprint of a wet detoxification preparation, comprising the following steps:

[0060] Extract the wet-to-clear preparation using an extraction solvent to prepare a test solution;

[0061] Performing high performance liquid chromatography on the test solution to construct a fingerprint of the Shiduqing preparation;

[0062] The conditions for the high performance liquid chromatography detection include: mobile phase A is phosphoric acid aqueous solution, mobile phase B is acetonitrile, and gradient elution is adopted.

[0063] In some examples, the gradient elution procedure includes:

[0064] 0-15min, the volume percentage of the mobile phase B increased from 5% to 20%;

[0065] 15min-25min, the volume percentage of the mobile phase B increased from 20% to 21%;

[0066] 25min-35min, the volume percentage of the mobile phase B increased from 21% to 28%;

[0067] 35min-40min, the volume percentage of the mobile phase B increased from 28% to 30%;

[0068] 40min-50min, the volume percentage of the mobile phase B increased from 30% to 48%;

[0069] 50min-60min, the volume percentage of the mobile phase B increased from 48% to 55%;

[0070] From 60 min to 65 min, the volume percentage of the mobile phase B was maintained at 55%.

[0071] In some examples, the volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.05%-0.2%. It can be understood that the volume percentage of phosphoric acid in the phosphoric acid aqueous solution includes but is not limited to 0.05%, 0.1%, 0.15%, and 0.2%.

[0072] In some examples, the conditions for the HPLC detection include: a detection wavelength of 200nm-300nm. It is understandable that the detection wavelength of the HPLC includes but is not limited to 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, and 300nm.

[0073] In some of the examples, the conditions for the HPLC detection include: a flow rate of 0.5 mL / min-2 mL / min. It is understandable that the flow rate for the HPLC detection includes but is not limited to 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1 mL / min, 1.1 mL / min, 1.2 mL / min, 1.3 mL / min, 1.4 mL / min, 1.5 mL / min, 1.6 mL / min, 1.7 mL / min, 1.8 mL / min, 1.9 mL / min, 2 mL / min.

[0074] In some examples, the conditions for the HPLC detection include: a column temperature of 25° C.-35° C. It is understandable that the column temperature for the HPLC detection includes but is not limited to 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., and 35° C.

[0075] In some examples, the conditions for the HPLC detection include: the injection volume is 1 μL-10 μL. It is understandable that the injection volume for the HPLC detection includes but is not limited to 1 μL, 3 μL, 5 μL, 7 μL, 9 μL, and 10 μL.

[0076] In some of the examples, the conditions of the extraction step include: the extraction method is ultrasonic extraction or reflux extraction.

[0077] In some examples, the conditions of the extraction step include: the extraction time is 15 min-60 min. It can be understood that the extraction time includes but is not limited to 15 min, 20 min, 30 min, 40 min, 45 min, 50 min, 60 min.

[0078] In some examples, the conditions of the extraction step include: the extraction solvent includes methanol or a methanol-water solution. Optionally, the volume percentage of methanol in the methanol-water solution is 0.1%-100%.

[0079] In some of the examples, the method for constructing the fingerprint of the wet detoxification preparation further includes the following steps:

[0080] Dissolving the reference substance using a dissolving reagent to prepare a reference substance mixed solution;

[0081] Performing high performance liquid chromatography on the reference substance mixed solution to obtain a reference spectrum;

[0082] According to the reference spectrum, the chromatographic peaks of the fingerprint spectrum of the Shiduqing preparation are identified as common peaks;

[0083] Wherein, the reference substance includes one or more of ferulic acid, astilbin, baicalin, salvianolic acid B, matrine, oxymatrine, tanshinone IIA, liquiritin, ammonium glycyrrhizinate, verbascoside and rehmannoside D.

[0084] In some examples, the Shiduqing preparation includes Shiduqing tablets, Shiduqing capsules or Shiduqing granules. Further, the Shiduqing preparation is Shiduqing tablets.

[0085] In one embodiment, the dissolving agent includes methanol or a methanol-water solution; optionally, in the methanol-water solution, the volume percentage of methanol is 0.1%-100%.

[0086] In some of the examples, the fingerprint of the Shiduqing preparation contains 16 common peaks, among which the 4 identified characteristic peaks include: Peak 6 is ferulic acid, Peak 7 is astilbin, Peak 10 is baicalin, and Peak 11 is salvianolic acid B.

[0087] Another aspect of the present invention provides the application of the method for constructing the fingerprint of the Shiduqing preparation as described above in the quality control of the Shiduqing preparation.

[0088] The following is further described in conjunction with specific embodiments. The raw materials involved in the following specific embodiments, unless otherwise specified, can all be commercially available; the instruments used, unless otherwise specified, can all be commercially available; the processes involved, unless otherwise specified, are all routinely selected by those skilled in the art.

[0089] Example 1

[0090] 1. Materials and Methods

[0091] Some of the instruments and materials used in the examples are as follows:

[0092] Testing instruments: electronic analytical balance, high performance liquid chromatograph, ultrasonic cleaner.

[0093] Test sample: The 19 batches of Shiduqing tablets used in the examples were sourced from Guangzhou Nuojin Pharmaceutical Co., Ltd., with batch numbers 325B004, 3259007, 3254005, 3254003, 3259001, 4252003, 4252002, 3257006, 325A010, 3257009, 325A010, 3257009, 325A004, 325B005, 3259008, 4251010, 325C002, 4252005, and 325A003, respectively.

[0094] The preparation method of the test solution is as follows: accurately weigh and grind the wet toxicity clearing tablets, take 2g, add 40mL of methanol, and ultrasonicate for 45min. Cool to room temperature, weigh again, make up the lost mass with methanol, shake well, filter with a 0.45μm microporous membrane, and collect the filtrate to obtain the solution.

[0095] Reference substances included ferulic acid, astilbin, baicalin, salvianolic acid B, matrine, oxymatrine, tanshinone IIA, liquiritin, ammonium glycyrrhizinate, verbascoside, and rehmannoside D.

[0096] The preparation method of the mixed reference solution is as follows: take appropriate amounts of ferulic acid reference, astilbin reference, baicalin reference and salvianolic acid B reference, accurately weigh them, and add methanol to prepare a mixed solution containing 100 μg of ferulic acid, 100 μg of astilbin, 100 μg of baicalin and 100 μg of salvianolic acid B per 1 mL.

[0097] The medicinal materials include: Rehmannia root, Scutellaria baicalensis, Salvia miltiorrhiza, Licorice root, Angelica sinensis, Sophora flavescens, Periostracum cicadae and Smilax glabra;

[0098] The preparation method of the medicinal material solution is as follows: according to the standard prescription proportion, take 2.2g of Rehmannia root, 0.4g of Scutellaria baicalensis, 1.0g of Salvia miltiorrhiza, 0.7g of Licorice, 1.7g of Angelica sinensis, 1.7g of Sophora flavescens, 0.7g of Cicadae Periostracum, and 0.4g of Smilax glabra, add 40mL of methanol respectively, and ultrasonicate for 45 minutes. Cool to room temperature, weigh again, make up the lost mass with methanol, shake well, filter with a 0.45μm microporous filter membrane, and collect the filtrate to obtain the solution.

[0099] The chromatographic conditions are shown in Table 1 below:

[0100] Table 1

[0101]

[0102] 2. Establishment of fingerprint and identification of common peaks

[0103] 19 batches of Wet Du Qing Tablets were used to prepare test solution according to the test solution preparation method described above. The test solution, medicinal material solution and mixed reference solution were injected and analyzed according to the above chromatographic conditions. The results are as follows: Figure 1 and Figure 2 As shown in the figure, the fingerprints of 19 batches of Shiduqing Tablets were analyzed to find their common characteristic peaks. The chromatograms of the 19 batches of Shiduqing Tablets were imported into the "Chinese Medicine Fingerprint Similarity Evaluation System (2012 Edition)", and the chromatogram of sample S11 was used as a reference spectrum. The superimposed fingerprint spectrum was automatically matched in the form of full spectrum peaks, and the reference fingerprint spectrum R was generated by the average method. The common peaks were identified by comparing with the spectrum of the mixed reference solution.

[0104] The results showed that through fingerprint analysis of Shiduqing Tablets and its negative samples, 16 common peaks were matched in the chromatograms of 19 batches of Shiduqing Tablets. By comparing with the spectrum of mixed reference solution, 4 characteristic peaks were identified, namely ferulic acid (peak 6), astilbin (peak 7), baicalin (peak 10), and salvianolic acid B (peak 11).

[0105] 3. Similarity analysis of fingerprints

[0106] By comparing the chromatograms, 16 characteristic peaks with good separation and stable peaks in each batch of fingerprints were determined. The 2012 version of the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" was used to generate a reference fingerprint based on the 16 peaks and perform similarity analysis. The results are shown in Table 2.

[0107] Table 2 Similarity evaluation results of 19 batches of Shiduqing tablets

[0108]

[0109] The results showed that the similarity of the batch with the lowest similarity was 0.998, and the others were all above 0.999. This indicated that the similarity of each batch of Shiduqing Tablets was high, and the generated control fingerprints could be used as an overall similarity evaluation of the material basis of Shiduqing Tablets.

[0110] 4. The single medicinal material attribution of each characteristic peak

[0111] The fingerprint analysis of Shiduqing Tablets and each single herbal medicine was carried out according to the above chromatographic conditions to investigate the herbal medicine attribution of each characteristic peak of Shiduqing Tablets. The results are as follows: Figure 3 And as shown in Table 3.

[0112] Table 3 The identification of the characteristic peaks of the fingerprints of 19 batches of Shiduqing tablets

[0113]

[0114] V. Methodological Validation

[0115] 5.1 Specificity inspection

[0116] Accurately pipette 5 μL of the test solution, mixed reference solution and blank solvent (0-100% methanol aqueous solution) of the Wet Du Qing Tablets, inject them into the liquid chromatograph, and measure according to the chromatographic conditions described in Table 1 to check whether there is interference with the extraction solvent. The results are as follows: Figure 4 shown.

[0117] The results showed that the blank solvent had almost no absorption under the chromatographic conditions and had no interference with the chromatographic analysis of the test samples, indicating that the established method had good specificity.

[0118] 5.2 Repeatability study

[0119] Weigh 6 portions of the same batch of Wet Du Qing Tablets to prepare the test solution, and inject and analyze under the optimal chromatographic conditions. Taking peak 10 as the reference peak, calculate the RSD value of the relative peak area and the RSD value of the relative retention time of each common peak. The results are shown in Tables 4 and 5.

[0120] Table 4 Relative peak area of ​​repeatability of fingerprint spectrum of Shiduqing tablets

[0121]

[0122] Table 5 Relative retention time of repeatability study of fingerprint spectrum of Shiduqing tablets

[0123]

[0124] The results showed that the RSD of the relative peak areas of the common peaks were all less than 3.48% (n=6), and the RSD of the relative retention time were all less than 0.03% (n=6), indicating that the method had good repeatability.

[0125] 5.3 Precision investigation

[0126] Take the test sample of Shiduqing tablets, inject 6 injections continuously, measure under the optimal chromatographic conditions, take peak No. 10 as the reference peak, calculate the RSD value of the relative peak area of ​​each common peak, and the results are shown in Tables 6 and 7.

[0127] Table 6 Relative peak area of ​​precision study of fingerprint of Shiduqing tablets

[0128]

[0129] Table 7 Relative retention time of precision study of fingerprint spectrum of Shiduqing tablets

[0130]

[0131] The results showed that the RSD of the relative peak areas of the common peaks were all less than 2.99% (n=6), and the RSD of the relative retention time were all less than 0.05% (n=6), indicating that the instrument had good precision.

[0132] 5.4 Stability investigation

[0133] The test sample of Shiduqing tablets was measured under the optimal chromatographic conditions at 0, 6h, 12h, 18h, 24h, 30h and 36h after preparation. Peak 10 was used as the reference peak to calculate the RSD value of the peak area. The results are shown in Tables 8 and 9.

[0134] Table 8 Relative peak areas of stability study of fingerprints of Shiduqing tablets

[0135]

[0136] Table 9 Relative retention time of stability study of fingerprint spectrum of Shiduqing tablets

[0137]

[0138] The results showed that the RSD of the relative peak areas of the common peaks were all less than 3.58% (n=7), and the RSD of the relative retention time were all less than 0.10% (n=7), indicating that the method had good stability.

[0139] 5.5 Intermediate precision study

[0140] 5.5.1 Liquid Phase Intermediate Precision Study

[0141] At present, the laboratory liquid chromatography is mainly Thermo Fisher U3000 (equipment number: J-20-C-093, J-20-C-008). One sample of Shiduqing Tablets was prepared and fingerprinted on different liquid chromatography, and similarity analysis was performed using the 2012 version of the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System". The results are shown in Table 10 and Figure 5 .

[0142] Table 10 Evaluation of the similarity of the intermediate precision of the fingerprint of Shiduqing Tablets

[0143]

[0144] The results showed that the similarity of the fingerprint spectra of Shiduqing tablets measured in liquid phases with different numbers reached 0.999 and above, indicating that the intermediate precision of the liquid phase was good.

[0145] 5.5.2 Column intermediate precision

[0146] One sample of Shiduqing Tablets was prepared and its fingerprint was determined using three ODS C18 (4.6×250mm, 5μm) chromatographic columns (department numbers: JZ-SY-C002, QC-C18-24-0401, QC-C18-22-0604), and similarity analysis was performed using the 2012 version of the “Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines”. The results are shown in Tables 11 and Figure 6 .

[0147] Table 11 Evaluation of similarity of intermediate precision of instrument for fingerprint of Shiduqing Tablets

[0148]

[0149] The results showed that the similarity of the fingerprint spectra of Shiduqing tablets measured in liquid phases with different numbers reached 0.999 and above, indicating that the intermediate precision of the instrument was good.

[0150] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0151] The above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be construed as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.

Claims

1. A method for constructing a fingerprint of a Shiduqing preparation, characterized in that: The following steps are involved: Extract the wet-to-clear preparation using an extraction solvent to prepare a test solution; Performing high performance liquid chromatography on the test solution to construct a fingerprint of the Shiduqing preparation; The conditions for the high performance liquid chromatography detection include: mobile phase A is phosphoric acid aqueous solution, mobile phase B is acetonitrile, and gradient elution is used; the procedure of the gradient elution includes: 0-15min, the volume percentage of the mobile phase B increased from 5% to 20%; 15min-25min, the volume percentage of the mobile phase B increased from 20% to 21%; 25min-35min, the volume percentage of the mobile phase B increased from 21% to 28%; 35min-40min, the volume percentage of the mobile phase B increased from 28% to 30%; 40min-50min, the volume percentage of the mobile phase B increased from 30% to 48%; 50min-60min, the volume percentage of the mobile phase B increased from 48% to 55%; From 60 min to 65 min, the volume percentage of the mobile phase B was maintained at 55%.

2. The method for constructing the fingerprint of the Shiduqing preparation according to claim 1, characterized in that: In the phosphoric acid aqueous solution, the volume percentage of phosphoric acid is 0.05%-0.2%.

3. The method for constructing the fingerprint of the Shiduqing preparation according to claim 1, characterized in that: The high performance liquid chromatography detection satisfies at least one of the following conditions: (1) The detection wavelength is 200nm-300nm; (2) Flow rate: 0.5 mL / min-2 mL / min; (3) Column temperature is 25°C-35°C; (4) The injection volume is 1μL-10μL.

4. The method for constructing the fingerprint of the Shiduqing preparation according to claim 1, characterized in that: The extraction step satisfies at least one of the following conditions: (1) The extraction method is ultrasonic extraction or reflux extraction; (2) The extraction time is 15min-60min; (3) The extraction solvent includes methanol or a methanol-water solution.

5. The method for constructing the fingerprint of the Shiduqing preparation according to claim 4, characterized in that: In the extraction step, the volume percentage of methanol in the methanol aqueous solution is 0.1%-100%.

6. The method for constructing the fingerprint of the Shiduqing preparation according to claim 1, characterized in that: The method for constructing the fingerprint spectrum of the wet detoxification preparation also includes the following steps: Dissolving the reference substance using a dissolving reagent to prepare a reference substance mixed solution; Performing high performance liquid chromatography on the reference substance mixed solution to obtain a reference spectrum; According to the reference spectrum, the chromatographic peaks of the fingerprint spectrum of the Shiduqing preparation are identified as common peaks; Wherein, the reference substance includes one or more of ferulic acid, astilbin, baicalin, salvianolic acid B, matrine, oxymatrine, tanshinone IIA, liquiritin, ammonium glycyrrhizinate, verbascoside and rehmannoside D.

7. The method for constructing the fingerprint of the Shiduqing preparation according to any one of claims 1 to 6, characterized in that: The Shiduqing preparation includes Shiduqing tablets, Shiduqing capsules or Shiduqing granules.

8. The method for constructing the fingerprint of the Shiduqing preparation according to claim 6, characterized in that: The dissolving agent includes methanol or a methanol aqueous solution.

9. The method for constructing the fingerprint of the Shiduqing preparation according to any one of claims 1 to 6, characterized in that: The fingerprint of the Shiduqing preparation contains 16 common peaks, among which the 4 identified characteristic peaks include: peak 6 is ferulic acid, peak 7 is astilbin, peak 10 is baicalin, and peak 11 is salvianolic acid B.

10. Application of the method for constructing the fingerprint of the Shiduqing preparation according to claims 1-9 in the quality control of the Shiduqing preparation.

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