Characteristic chromatogram detection method and quality control method of winter melon peel medicinal preparation

Abstract

The present application relates to the field of traditional Chinese medicine detection, in particular to a characteristic spectrum detection method and quality control method of a drug preparation of winter melon peel. The detection method (1) provided by the present application can select S-peak vitexin-2"-O-rhamnose glycoside as the internal reference peak in the fingerprint spectrum, and can determine five common characteristic peaks of the winter melon peel formula granules, and calculate the relative retention time of each common characteristic peak according to S-peak vitexin-2"-O-rhamnose glycoside. The detection method (2) selects S-peak uridine as the internal reference peak in the fingerprint spectrum, and can determine six common characteristic peaks of the winter melon peel formula granules, and calculates the relative retention time of each common characteristic peak according to S-peak uridine. The detection method (1) and / or the detection method (2) are beneficial to the comprehensive quality detection and overall quality control of the winter melon peel drug preparation, thereby helping to improve the safety and stability uniformity of the drug use.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine testing, specifically to a method for detecting and controlling the quality of a medicinal preparation made from winter melon peel using characteristic chromatograms. Background Technology

[0002] Winter melon peel granules are obtained through extraction, concentration, and granulation of the traditional Chinese medicine winter melon peel. Winter melon peel is the dried outer pericarp of the plant *Benincasa hispida* (Thunb.) Cogn., belonging to the Cucurbitaceae family. It mainly contains flavonoids, nucleosides, organic acids, and other components. Its main functions are diuresis and reducing swelling, and it is used for edema, abdominal distension, difficulty urinating, thirst due to summer heat, and scanty dark urine.

[0003] The 2020 edition of the Chinese Pharmacopoeia includes quality control measures for winter melon peel, covering aspects such as the original plant variety, processing of the medicinal slices, characteristics of the slices, and physicochemical identification. Literature also describes the chemical components in winter melon peel, including flavonoids, nucleosides, and organic acids. However, on the one hand, identifying winter melon peel granules based on these simple characteristics is insufficient for comprehensive quality control. On the other hand, establishing multi-component fingerprint studies can provide better quality evaluation and control of the production process of winter melon peel granules and can be widely applied in production practice. However, current quality control measures for winter melon peel focus on the medicinal slices, not on pharmaceutical preparations made from winter melon peel. Existing detection methods for medicinal slices are not applicable to pharmaceutical preparations made from winter melon peel, and cannot provide a comprehensive and rapid detection of such preparations. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a characteristic spectrum detection method and quality control method for winter melon peel pharmaceutical preparations, which can comprehensively and rapidly detect winter melon peel pharmaceutical preparations, and is of great significance for their comprehensive quality detection and overall quality control.

[0005] Therefore, the present invention provides the following technical solution:

[0006] A method for detecting the characteristic chromatograms of a winter melon peel pharmaceutical preparation, comprising detection method (1) and / or detection method (2):

[0007] The detection method (1) includes: detection by high performance liquid chromatography (HPLC). The chromatographic conditions are as follows: using octadecylsilane-bonded silica gel as the packing material, acetonitrile as mobile phase A, and 0.1 v / v% formic acid aqueous solution as mobile phase B, the gradient elution is performed according to the following program: 0-13 min, mobile phase A: mobile phase B volume ratio is 8%:92% → 12%:88%; 13-50 min, mobile phase A: mobile phase B volume ratio is 12%:88% → 20%:80%; 50-55 min, mobile phase A: mobile phase B volume ratio is 20:80; 55-56 min, mobile phase A: mobile phase B volume ratio is 20%:80% → 8%:92%; 56-60 min, mobile phase A: mobile phase B volume ratio is 8%:92%.

[0008] The detection method (2) includes: detection by high performance liquid chromatography (HPLC). The chromatographic conditions are as follows: using octadecylsilane-bonded silica gel as the packing material, methanol as mobile phase A, and water as mobile phase B, the gradient elution is performed according to the following program: 0-10 min, mobile phase A: mobile phase B volume ratio is 0%:100%; 10-30 min, mobile phase A: mobile phase B volume ratio is 0%:100% → 5%:95%; 30-42 min, mobile phase A: mobile phase B volume ratio is 5%:95% → 10%:90%.

[0009] Optionally, the chromatographic conditions in the detection method (1) include at least one of the following conditions:

[0010] 1) The detection wavelength is 270nm;

[0011] 2) Column temperature is 33–37℃;

[0012] 3) The chromatographic column has a size of 4.6 mm × 250 mm and a diameter of 5 μm; optionally, the chromatographic column is selected from Dikma Platisil ODS C18.

[0013] 4) The chromatograph is selected from Agilent 1260, Waters H-Class, or Thermo U3000;

[0014] 5) The flow rate is 0.9–1.1 mL / min;

[0015] 6) The injection volume is 8–12 μL, preferably 10 μL;

[0016] And / or, the chromatographic conditions in the detection method (2) include at least one of the following conditions:

[0017] 1) The detection wavelength is 254nm;

[0018] 2) Column temperature is 23–27℃;

[0019] 3) The flow rate is 0.9–1.1 ml / min;

[0020] 4) The column specifications are 250mm × 4.6mm, 5μm; optionally, the column can be selected from Dikma Platisil ODS or Waters. T3 or Shimadzu Inertsil ODS;

[0021] 5) The chromatograph is selected from Agilent 1260, Waters e2695 or Thermo U3000;

[0022] 5) The injection volume is 8 to 12 μL, preferably 10 μL.

[0023] Optionally, the preparation of the test solution may also be included:

[0024] The preparation of the test solution in the detection method (1) includes: taking the test sample, adding solvent to extract, filtering, and taking the filtrate;

[0025] Optionally, take 0.5–3 parts by weight of the test sample, add 10–25 volumes of 20 v / v%–100 v / v% methanol solution, sonicate for 15–45 min, filter, and collect the filtrate; alternatively, take 2 parts by weight of the test sample, add 10 volumes of 50 v / v% methanol solution, sonicate for 30 min, filter, and collect the filtrate; the ratio of parts by weight to parts by volume is g / ml.

[0026] And / or, the preparation of the test solution in the detection method (2) includes: taking the test sample, adding solvent to extract, filtering, and taking the filtrate;

[0027] Optionally, take 0.8-1.2 parts by weight of the test sample and 15-25 parts by volume of 20 v / v% methanol solution, sonicate, filter, and collect the filtrate; or, take 1 g parts by weight of the test sample and 20 parts by volume of 20 v / v% methanol solution, sonicate, filter, and collect the filtrate; the ratio of parts by weight to parts by volume is g / ml.

[0028] Optionally, the preparation of a reference solution may also be included:

[0029] The preparation of the reference solution in the detection method (1) includes: taking 2-4 parts by weight of winter melon peel reference material, adding 30-80 parts by volume of water, heating under reflux for 30-60 minutes, filtering, evaporating the filtrate to dryness, adding 5 parts by volume of 50v / v% methanol to the residue, ultrasonically treating for 30-60 minutes, filtering, and taking the filtrate as the reference solution for the reference material; optionally, taking 3 parts by weight of winter melon peel reference material, adding 50 parts by volume of water, heating under reflux for 45 minutes, filtering, evaporating the filtrate to dryness, adding 5 parts by volume of 50v / v% methanol to the residue, ultrasonically treating for 45 minutes, filtering, and taking the filtrate as the reference solution for the reference material; the ratio of parts by weight to parts by volume is g / ml;

[0030] Take an appropriate amount of isovitexin-2”-O-rhamnoside reference standard and add 50 v / v% methanol to prepare solutions containing 30-80 μg per 1 ml as reference solutions; preferably, take an appropriate amount of isovitexin-2”-O-rhamnoside reference standard and add 50 v / v% methanol to prepare solutions containing 50 μg per 1 ml as reference solutions.

[0031] And / or, the preparation of the reference solution in the detection method (2) includes: reference solution of reference medicinal material: take 1-3 parts by weight of winter melon peel reference medicinal material, add 50-150 parts by volume of water, heat and reflux for 30-60 min, filter, evaporate the filtrate to dryness, add 10 parts by volume of 20v / v% methanol to the residue and sonicate for 30-60 min, take out, filter, and take the filtrate; take 2 parts by weight of winter melon peel reference medicinal material, add 100 parts by volume of water, heat and reflux for 45 min, filter, evaporate the filtrate to dryness, add 10 parts by volume of 20v / v% methanol to the residue and sonicate for 30 min, take out, filter, and take the filtrate; the ratio of the parts by weight to the parts by volume is g / ml;

[0032] Reference solution: Take an appropriate amount of uridine reference standard, accurately weigh it, and add 20% methanol to prepare a solution containing 30-50 μg per 1 mL; alternatively, take an appropriate amount of uridine reference standard, accurately weigh it, and add 20% methanol to prepare a solution containing 40 μg per 1 mL.

[0033] A method for determining the content of a medicinal preparation made from winter melon peel, comprising:

[0034] Take the test solution and the reference solution, and perform high performance liquid chromatography (HPLC) according to the detection method of the characteristic spectrum of the winter melon peel pharmaceutical preparation as described in any one of claims 1-4.

[0035] The use of the detection method of the characteristic spectrum of the winter melon peel drug preparation or the content determination method of the winter melon peel drug preparation in the quality testing of the winter melon peel drug preparation.

[0036] A quality testing method for a winter melon peel pharmaceutical preparation includes:

[0037] The method includes obtaining the characteristic spectrum of the test sample according to the detection method of the characteristic spectrum of the winter melon peel drug preparation, and comparing the characteristic spectrum with the control characteristic spectrum.

[0038] The control feature spectrum is obtained by fitting the feature spectrum obtained by using at least one batch of standard samples of winter melon peel drug preparations according to the detection method of the feature spectrum of winter melon peel drug preparations.

[0039] Optional, including:

[0040] In the detection method (1), the reference characteristic spectrum includes 5 characteristic peaks, of which peak 5 corresponds to isovitexin-2”-O-rhamnoside. Peak 5 is used as reference peak S. The relative retention times of peaks 1 to 4 with peak S should be within ±10% of the specified values. The specified value for peak 1 is 0.40, the specified value for peak 2 is 0.50, the specified value for peak 3 is 0.52, and the specified value for peak 4 is 0.97.

[0041] And / or, in the detection method (2), the reference characteristic spectrum includes 6 characteristic peaks, wherein peak 4 corresponds to uridine, peak 4 is used as reference peak S, and the relative retention times of peaks 1 to 3, peaks 5 to 6 and peak S should be within ±10% of the specified values, wherein the specified values ​​for peak 1 are 0.39, peak 2 are 0.73, peak 3 are 0.88, peak 5 are 1.46 and peak 6 are 1.84.

[0042] Optionally, in the detection method (1), peak 1 is 4-hydroxybenzoic acid; peak 4 is vitexin rhamnoside; peak 5 is isovitexin-2”-O-rhamnoside;

[0043] Optionally, in the detection method (2), peak 2 is guanine; peak 3 is xanthine; peak 4 is uridine; peak 5 is adenine; and peak 6 is guanosine.

[0044] A method for identifying medicinal preparations made from winter melon peel and watermelon peel includes obtaining a characteristic spectrum of the test sample according to the detection method (1) for the characteristic spectrum of the medicinal preparation made from winter melon peel, and comparing the characteristic spectrum with a control characteristic spectrum.

[0045] Optionally, if the characteristic chromatogram of the test sample has the same characteristic peaks as the control characteristic chromatogram, then the test sample is a winter melon peel drug preparation.

[0046] Optionally, if the characteristic chromatogram of the test sample differs from that of the control sample only in that it lacks characteristic peak 5, then the test sample is a watermelon rind drug preparation.

[0047] The method for detecting the characteristic chromatogram of the winter melon peel drug preparation, the method for determining the content of the winter melon peel drug preparation, or the method for quality testing of the winter melon peel drug preparation, is characterized in that the winter melon peel drug preparation includes winter melon peel formula granules and winter melon peel formula granule extract.

[0048] The technical solution of this invention has the following advantages:

[0049] 1. The present invention provides a method for detecting the characteristic chromatogram of a winter melon peel pharmaceutical preparation, comprising detection method (1) and / or detection method (2): the detection method (1) includes: detection by high performance liquid chromatography, the chromatographic conditions being: using octadecylsilane-bonded silica gel as the packing material, acetonitrile as mobile phase A, and 0.1 v / v% formic acid aqueous solution as mobile phase B, and performing gradient elution according to the following procedure: 0-13 min, mobile phase A: mobile phase B volume ratio of 8%:92% → 12%:88%; 13-50 min, mobile phase A: mobile phase B volume ratio of 12%:88% → 20%:80%; 55-56 min, mobile phase A: mobile phase B volume ratio of 20%:80% → 8%:92%; 56-60 min, mobile phase A: mobile phase B volume ratio of 20%:80% → 8%:92%; The volume ratio of mobile phase B is 8%:92%; the detection method (2) includes: detection by high performance liquid chromatography, the chromatographic conditions are: using octadecylsilane bonded silica gel as the packing material, using methanol as mobile phase A and water as mobile phase B, and performing gradient elution according to the following procedure: 0-10 min, the volume ratio of mobile phase A: mobile phase B is 0%:100%; 10-30 min, the volume ratio of mobile phase A: mobile phase B is 0%:100% → 5%:95%; 30-42 min, the volume ratio of mobile phase A: mobile phase B is 5%:95% → 10%:90%; the above detection method (1) and / or detection method (2) are beneficial to the comprehensive quality detection and overall quality control of winter melon peel drug preparations, thereby helping to improve the safety and stability of the drug.

[0050] Furthermore, the above-mentioned detection methods (1) and / or detection methods (2) can also determine the content of 4-hydroxybenzoic acid, vitexin rhamnoside, isovitexin-2”-O-rhamnoside, guanine, xanthine, uridine, adenine and guanosine in winter melon peel drug preparations, which can comprehensively and quickly detect the effective components and content of winter melon peel drug preparations.

[0051] Furthermore, the above-mentioned detection method (1) can also distinguish between medicinal preparations made from winter melon peel and medicinal preparations made from watermelon peel. Attached Figure Description

[0052] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0053] Figure 1 These are the characteristic chromatograms of 15 batches of winter melon peel formula granules in the detection method (1) of Embodiment 1 of the present invention;

[0054] Figure 2 It is the reference feature spectrum in the detection method (1) of Embodiment 1 of the present invention;

[0055] Figure 3 This is the reference standard localization map in the detection method (1) of Example 1 of the present invention; S1 is the reference characteristic map, S2: isovitexin-2"-O-rhamnoside, S3: 4-hydroxybenzoic acid, S4: vitexin rhamnoside;

[0056] Figure 4 This is the characteristic spectrum of the reference medicinal material in the detection method (1) of Embodiment 1 of the present invention;

[0057] Figure 5 These are the characteristic spectra of the reference medicinal materials for winter melon peel and watermelon peel in the identification method of Embodiment 1 of the present invention; S1: winter melon peel; S2: watermelon peel;

[0058] Figure 6 These are the characteristic spectra of winter melon peel formula particles and watermelon peel formula particles in the detection method (1) of Embodiment 1 of the present invention; S1: winter melon peel formula particles; S2: watermelon peel formula particles;

[0059] Figure 7 These are the characteristic chromatograms of 15 batches of winter melon peel formula granules obtained by the detection method (2) in Embodiment 2 of the present invention;

[0060] Figure 8 This is a comparative feature spectrum of the detection method (2) in Embodiment 2 of the present invention;

[0061] Figure 9 This is the reference standard positioning diagram of the detection method (2) in Example 2 of the present invention; S1 is the color spectrum of the formulation particles, S2: guanosine, S3: uridine, S4: guanine, S5: xanthine, S6: adenine;

[0062] Figure 10 This is a reference medicinal material characteristic spectrum of the detection method (2) in Example 2 of the present invention;

[0063] Figure 11 This is a characteristic chromatogram of the sample solution extracted with 20% methanol as the solvent in Example 4 of the present invention;

[0064] Figure 12 This is a characteristic chromatogram of the sample solution extracted with 50% methanol as the solvent in Example 4 of the present invention;

[0065] Figure 13 This is a characteristic chromatogram of methanol as the solvent extracted from the test sample solution in Example 4 of the present invention;

[0066] Figure 14 This is a characteristic chromatogram of the sample solution extracted for 15 min in Example 5 of the present invention;

[0067] Figure 15 This is a characteristic chromatogram of the sample solution extracted for 30 min in Example 5 of the present invention;

[0068] Figure 16 This is a characteristic chromatogram of the sample solution extracted for 45 min in Example 5 of the present invention;

[0069] Figure 17 The characteristic chromatogram is that of the sample solution with a sample amount of 0.5g in Example 6 of the present invention;

[0070] Figure 18 This is a characteristic chromatogram of a 1g sample taken from the test solution in Example 6 of the present invention;

[0071] Figure 19 This is a characteristic chromatogram of a 2g sample taken from the test solution in Example 6 of the present invention;

[0072] Figure 20 This is a characteristic chromatogram of a 3g sample taken from the test solution in Example 6 of the present invention;

[0073] Figure 21 The characteristic spectrum is that the flow rate is set to 0.9 mL / min in Example 7 of the present invention;

[0074] Figure 22 The characteristic spectrum is that the flow rate is set to 1.0 mL / min in Example 7 of the present invention;

[0075] Figure 23 The characteristic spectrum is that of Example 7 of the present invention, where the flow rate is set to 1.1 mL / min;

[0076] Figure 24 This is a characteristic spectrum of the column temperature set to 33°C in Embodiment 8 of the present invention;

[0077] Figure 25 This is a characteristic spectrum of the column temperature set to 35°C in Embodiment 8 of the present invention;

[0078] Figure 26 This is a characteristic spectrum of the column temperature set to 37°C in Embodiment 8 of the present invention;

[0079] Figure 27 This is a characteristic chromatogram of column 1 in Example 9 of the present invention;

[0080] Figure 28 This is a characteristic chromatogram of column 2 in Example 9 of the present invention;

[0081] Figure 29 This is a characteristic chromatogram of column 3 in Example 9 of the present invention;

[0082] Figure 30 These are characteristic chromatograms of the Agilent 1260 chromatograph used in Embodiment 10 of the present invention;

[0083] Figure 31 This is a characteristic chromatogram of the Waters H-Class chromatograph used in Embodiment 10 of the present invention;

[0084] Figure 32 These are characteristic chromatograms of the Thermo U3000 chromatograph used in Embodiment 10 of this invention;

[0085] Figure 33 This is a characteristic spectrum of the winter melon peel formula granules in "4. Specificity and integrity test" in Example 11 of this invention;

[0086] Figure 34 This is the characteristic spectrum of the winter melon peel reference medicinal material in "4. Specificity and integrity test" in Example 11 of this invention;

[0087] Figure 35 This is the characteristic chromatogram of the negative blank control in "4. Specificity and integrity test" in Example 11 of this invention;

[0088] Figure 36 This is the characteristic spectrum of the flow rate of 0.9 mL / min in Example 12 of the present invention;

[0089] Figure 37 This is a characteristic spectrum of the flow rate of 1.0 mL / min in Example 12 of the present invention;

[0090] Figure 38 This is the characteristic spectrum of the flow rate of 1.1 mL / min in Example 12 of the present invention;

[0091] Figure 39 This is a characteristic spectrum of the column temperature of 23°C in Embodiment 13 of the present invention;

[0092] Figure 40 This is a characteristic spectrum of the column temperature of 25°C in Embodiment 13 of the present invention;

[0093] Figure 41 This is a characteristic spectrum of the column temperature of 27°C in Embodiment 13 of the present invention;

[0094] Figure 42 In Example 14 of this invention, the chromatographic column is a Waters column. Characteristic map of T3;

[0095] Figure 43 This is a characteristic chromatogram of the Dikma Platisil ODS column used in Example 14 of this invention;

[0096] Figure 44 This is a characteristic chromatogram of the chromatographic column Shimadzu Inertsil ODS in Example 14 of the present invention;

[0097] Figure 45 This is a characteristic chromatogram of column 1 in Example 15 of the present invention;

[0098] Figure 46 This is a characteristic chromatogram of column 2 in embodiment 15 of the present invention;

[0099] Figure 47 This is a characteristic chromatogram of column 3 in Example 15 of the present invention;

[0100] Figure 48 These are characteristic chromatograms of the Agilent 1260 chromatograph used in Embodiment 16 of the present invention;

[0101] Figure 49 These are characteristic chromatograms of the Waters e2695 chromatograph used in Embodiment 16 of this invention;

[0102] Figure 50 These are characteristic chromatograms of the Thermo U3000 chromatograph used in Embodiment 16 of this invention;

[0103] Figure 51 This is the characteristic chromatogram of the winter melon peel formula granules in "4. Specificity Test" of Example 17 of the present invention;

[0104] Figure 52 This is the chromatogram of the winter melon peel reference medicinal material in "4. Specificity Test" of Example 18 of the present invention;

[0105] Figure 53 This is the characteristic spectrum of the negative blank control in "4. Specificity test" in Example 18 of the present invention. Detailed Implementation

[0106] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0107] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0108] Chromatograph 1: High Performance Liquid Chromatograph: Waters E2695 chromatography system, including a quaternary gradient pump (Alliance 2695 model), a 120-position high-performance autosampler, an original imported column oven, a Waters 2489 UV detector, and an Empower chromatography workstation.

[0109] Chromatograph 2: Agilent 1260 Infinity II, including G711B quaternary pump, G7129A autosampler, G7114A VWD detector, G7116A column oven, and OpenLab CDS chromatography workstation.

[0110] Chromatograph 3: Thermo Ultimate 3000, including Pump: LPG-3400SD; Columb Compartment: TCC-3000RS; Autosumpler: WPS-3000SL; Photometer: DAD-3000.

[0111] Electronic analytical balances: METTLERTOLEDO (Switzerland) XS-205, XS-204, XSE-205, MS36S.

[0112] Ultrasonic instrument: KQ-400KDB high-power CNC ultrasonic instrument (Kunshan Ultrasonic Instrument Co., Ltd.)

[0113] Water bath: HH-S6 digital display constant temperature water bath (Jiangsu Jinyi Instrument Technology Co., Ltd.)

[0114] Chromatographic column: (1) Dikma Platisil ODS (column length 250mm, inner diameter 4.6mm, particle size 5μm);

[0115] (2) Warwes T3 (column length 250mm, inner diameter 4.6mm, particle size 5μm);

[0116] (3) Shimadzu Inertsil ODS (column length 250mm, inner diameter 4.6mm, particle size 5μm).

[0117] Reagents: Acetonitrile was of chromatographic grade, methanol was of chromatographic grade, and water was ultrapure water; all other reagents were of analytical grade.

[0118] Drug trials:

[0119] 4-Hydroxybenzoic acid reference standard (China National Institutes for Food and Drug Control, batch number 101149-201903, purity 100%).

[0120] Vitexin rhamnoside (China National Institutes for Food and Drug Control, batch number 111668-200602, purity 100%).

[0121] Isovitilione-2”-O-rhamnoside reference standard (purchased from Shanghai Hongyong Biotechnology Co., Ltd., batch number 557142-202111, purity: 98.45%).

[0122] Winter melon peel reference material (purchased from Shanghai Hongyong Biotechnology Co., Ltd., batch number 240022-202006);

[0123] Guanine reference standard (China National Institute for Food and Drug Control, batch number: 140631-202008, purity: 98.9%).

[0124] Xanthine reference standard (China National Institute for Food and Drug Control, batch number: 140662-200802, purity: 100.0%).

[0125] Uric acid reference standard (China National Institute for Food and Drug Control, batch number: 110887-202305, purity: 99.6%).

[0126] Guanosine reference standard (China National Institute for Food and Drug Control, batch number: 111977-202202, purity: 88.6%).

[0127] The medicinal preparation made from winter melon peel in this invention is prepared by the following method:

[0128] Take winter melon peel, heat and reflux extract at least once, adding 6-12 times its weight of water each time, extracting for at least 0.5 hours. Filter, combine the filtrates, concentrate the filtrate to a relative density of 1.05-1.10 g / mL at 60℃, add conventional excipients, and follow conventional processes to prepare clinically acceptable tablets, capsules, pills, granules, honey-refined pills, sustained-release preparations, immediate-release preparations, controlled-release preparations, oral liquid preparations, or injectable preparations. The pharmaceutically acceptable excipients include: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, and matrix, etc. Fillers include: starch, pregelatinized starch, lactose, mannitol, chitosan, microcrystalline cellulose, sucrose, etc.; disintegrants include: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, croscarmellose, low-substituted hydroxypropyl cellulose, croscarmellose sodium, etc.; lubricants include: magnesium stearate, sodium lauryl sulfate, talc, silica, etc.; suspending agents include: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include: starch paste, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.; sweeteners include: sodium saccharin, aspartame, sucrose, cyclamate, glycyrrhetinic acid, etc.; flavoring agents include: sweeteners and various flavorings; preservatives include: parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chlorethidium acetate, eucalyptus oil, etc.; matrix includes: PEG6000, PEG4000, insect wax, etc.

[0129] The specific preparation method of the winter melon peel formula granules in this invention is as follows: take winter melon peel, heat and reflux extract twice. For the first extraction, add 10 times the weight of water and soak for 30 minutes, heat and reflux extract for 30 minutes, filter, add 8 times the weight of water and extract for 25 minutes, filter, combine the filtrates, concentrate the filtrate to a relative density of 1.05 g / mL at 60℃, spray dry, add maltodextrin as an excipient to the dry powder, mix evenly, and then dry granulate to make granules.

[0130] The preparation method of watermelon rind granules is the same as that of winter melon rind granules.

[0131] The above method yielded 15 batches of winter melon peel granules with the following batch numbers: 2005002S, 2007001W, 1909001W, 1911002W, 2002004S, 2001002W, 2003001W, 2104001W, 2011004S, 2102003S, 2106002S, 2109002W, 2111001S, 2111002S, and 2102002W.

[0132] Example 1

[0133] This embodiment provides a method for detecting the characteristic spectral features of a winter melon peel pharmaceutical preparation, using detection method (1):

[0134] High-performance liquid chromatography (HPLC) was used for detection. The chromatographic conditions were as follows: octadecylsilane-bonded silica gel (DikmaPlatisil ODS, column length 250 mm, inner diameter 4.6 mm, particle size 5 μm) was used as the stationary phase; acetonitrile was used as mobile phase A, and 0.1 v / v% formic acid aqueous solution was used as mobile phase B, with gradient elution as specified in the table below; the column temperature was 35℃, the flow rate was 1.0 mL / min, and the detection wavelength was 270 nm. The theoretical plate number, calculated based on the isovitexin-2”-O-rhamnoside peak, should be no less than 5000.

[0135] Table 1. Gradient elution program

[0136] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～13 8→12 92→88 13～50 12→20 88→80 50～55 20 80 55～56 20→8 80→92 56～60 8 92

[0137] Preparation of reference solution: Take 3g of winter melon peel reference material, place it in a stoppered conical flask, add 50ml of water, heat under reflux for 45 minutes, filter, evaporate the filtrate to dryness, add 5ml of 50% methanol to the residue and sonicate (power 250W, frequency 40kHz) for 30 minutes, remove, filter, and take the filtrate as the reference solution of the reference material; take an appropriate amount of isovitexin-2”-O-rhamnoside reference standard, add 50% methanol to prepare solutions containing 50μg per ml, and obtain the reference solution.

[0138] Preparation of the test solution: Take about 2g of the medicinal preparation powder of winter melon peel, place it in a stoppered conical flask, add 10ml of 50v / v% methanol, sonicate (power 250W, frequency 40kHz) for 30 minutes, shake well, filter, and take the filtrate to obtain the test solution.

[0139] Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0140] Fifteen batches of winter melon peel granule samples were taken as test samples, and the characteristic chromatograms of the winter melon peel granule were obtained according to the above method. Figure 1 ).like Figure 1 As shown in the table below, the Figure 1 In the batch number S1 to S15, the batch numbers are as follows: S1: 2005002S, S2: 2007001W, S3: 1909001W, S4: 1911002W, S5: 2002004S, S6: 2001002W, S7: 2003001W, S8: 2104001W, S9: 2011004S, S10: 2102003S, S11: 2106002S, S12: 2109002W, S13: 2111001S, S14: 2111002S, and S15: 2102002W.

[0141] Using the fingerprint chromatogram similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission, a reference characteristic chromatogram was generated, such as... Figure 2 As shown in the figure and the table below.

[0142] Table 2. Spectral characteristics of 15 batches of winter melon peel formulation granules.

[0143]

[0144]

[0145] Table 3. Relative peak area results of 15 batches of winter melon peel formulation granules

[0146]

[0147]

[0148] Table 4. Similarity results of characteristic fingerprint spectra of 15 batches of winter melon peel formulation granules

[0149] name Similarity R 1 2005002S 0.928 2007001W 0.985 1909001W 0.975 1911002W 0.982 2002004S 0.962 2001002W 0.985 2003001W 0.987 2104001W 0.974 2011004S 0.975 2102003S 0.972 2106002S 0.975 2109002W 0.973 2111001S 0.965 2111002S 0.971 2102002W 0.964

[0150] Table 5. Relative retention time of common patterns in the fingerprint spectrum of winter melon peel formulation granules

[0151] name Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 (S) Retention time 16.668 20.502 21.451 40.097 41.387 Relative retention time 0.40 0.50 0.52 0.97 1 Specified value -10% 0.36 0.45 0.468 0.873 0.9 Specified value + 10% 0.44 0.55 0.572 1.067 1.1

[0152] Table 6. Common Pattern Matching Data for Winter Melon Peel Granule Formula

[0153]

[0154] By identifying and locating the aforementioned common characteristic peaks, isovitexin-2”-O-rhamnoside was selected as a reference standard for localization. Literature review revealed that the main chemical components of winter melon peel are flavonoids, triterpenoids, polysaccharides, waxes, resins, and amino acids. Since winter melon peel formulation granules use water as a solvent, components with poor water solubility and low content cannot be represented in the characteristic chromatogram. Flavonoids have good water solubility; therefore, this characteristic chromatogram was further developed using flavonoids. The detection results of fingerprint chromatograms from multiple batches of winter melon peel samples were analyzed. By comparing winter melon peel medicinal materials, processed winter melon peel slices, and winter melon peel formulation granules, three common peaks were identified. The peaks were analyzed using UPLC-Q-TOF. By collecting the compounds represented by each characteristic peak and comparing them under HPLC chromatographic conditions, the components and retention times of the characteristic peaks were reconfirmed. The three characteristic peaks were identified through reference standard identification and MS analysis. Figure 3 As shown in the figure and the table below, peak 1 is identified as 4-hydroxybenzoic acid, peak 4 as vitexin rhamnoside, and peak 5 as isovitexin-2”-O-rhamnoside.

[0155] Table 7. LC / MS / MS Analysis Results of Winter Melon Peel Granules

[0156]

[0157] The results are shown in the table below. Figure 2 and Figure 4 The following criteria were established: The chromatogram of the winter melon peel pharmaceutical preparation should show five characteristic peaks, corresponding to the retention times of the five characteristic peaks in the chromatogram of the reference medicinal material. The retention time of peak 5 should correspond to the retention time of the reference peak of isovitexin-2”-O-rhamnoside. The peak corresponding to the isovitexin-2”-O-rhamnoside reference peak is designated as peak S. The relative retention times of each characteristic peak and peak S should be calculated and should be within ±10% of the specified values. The specified values ​​are 0.40 (peak 1), 0.50 (peak 2), 0.52 (peak 3), and 0.97 (peak 4). Three of the peaks (peaks 1, 4, and 5 are known components): Peak 1: 4-hydroxybenzoic acid; Peak 4: vitexin rhamnoside; Peak 5 (S): isovitexin-2”-O-rhamnoside.

[0158] Table 8. Relative retention time of granulated formulation of winter melon peel.

[0159] name Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 (S) Retention time 16.668 20.502 21.451 40.097 41.387 Relative retention time 0.40 0.50 0.52 0.97 1.00

[0160] Table 9. Relative Peak Areas of Winter Melon Peel Granule Formula Comparison Spectra

[0161] name Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 (S) Peak area 635.366 479.388 593.761 501.666 1242.827 relative peak area 0.51 0.39 0.48 0.40 1.00

[0162] Table 10. Relative retention times of characteristic chromatograms of winter melon peel as a control medicinal material

[0163] name Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 (S) Retention time 16.669 20.52 21.458 40.139 41.407 Relative retention time 0.40 0.50 0.52 0.97 1.00

[0164] Table 11. Relative peak areas of characteristic spectra of winter melon peel and other medicinal materials

[0165] name Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 (S) Peak area 499.795 642.168 174.147 257.742 589.994 relative peak area 0.85 1.09 0.30 0.44 1.00

[0166] Furthermore, when using the detection method of this embodiment to perform quality control on pharmaceutical preparations for winter melon peel, the sample to be tested is tested according to the above detection method, and the obtained characteristic spectrum and Figure 2Compare with the control characteristic chromatogram shown. If at least 5 characteristic peaks identical to those in the control characteristic chromatogram appear in the characteristic chromatogram of the test sample, the peak corresponding to the vitexin-2”-O-rhamnoside reference substance peak is the S peak. Calculate the relative retention times of each characteristic peak and the S peak, and the relative retention times should be within the range of ±10% of the specified values. The specified values are respectively: the specified values are: 0.40 (peak 1), 0.50 (peak 2), 0.52 (peak 3), 0.97 (peak 4). Then the quality of the medicinal preparation of wax gourd peel is qualified; otherwise, it is unqualified.

[0167] Furthermore, the detection method of this example is used to identify the medicinal preparations of wax gourd peel and watermelon peel. The method is as follows: Detect the test sample according to the above detection method, and the obtained characteristic chromatogram and Figure 2 Compare with the control characteristic chromatogram shown. When the characteristic chromatogram of the test sample has the same characteristic peaks as the control characteristic chromatogram, the test sample is the medicinal preparation of wax gourd peel; when the characteristic chromatogram of the test sample only lacks characteristic peak 5 compared with the control characteristic chromatogram, the test sample is the medicinal preparation of watermelon peel; The reference substance characteristic chromatograms of the control medicinal materials and the characteristic chromatograms of the formula granules of wax gourd peel and watermelon peel are as shown in Figure 5 and Figure 6 , it can be seen that characteristic peak 5 (vitexin-2”-O-rhamnoside) is missing in the characteristic chromatograms of watermelon peel medicinal materials and watermelon peel formula granules. Therefore, the presence or absence of the specified characteristic peak 5 can be used to distinguish the medicinal preparation of wax gourd peel from the medicinal preparation of watermelon peel.

[0168] Example 2

[0169] This example provides a characteristic chromatogram detection method for the medicinal preparation of wax gourd peel, using detection method (2): Perform high performance liquid chromatography detection. The chromatographic conditions are as follows: Use octadecylsilane chemically bonded silica gel as the filler (Waters T3, column length is 250 mm, column inner diameter is 4.6 mm, particle size is 5 μm), use methanol as mobile phase A, use water as mobile phase B, and perform gradient elution according to the regulations in the following table; The column temperature is 25 °C, the flow rate is 1.0 ml per minute, and the detection wavelength is 254 nm. The theoretical plate number calculated based on the uridine peak should be not less than 5000.

[0170] Table 12. Gradient elution program

[0171]

[0172] Preparation of reference solution: Take 2g of winter melon peel reference material, place it in a stoppered conical flask, add 100ml of water, heat under reflux for 45 minutes, filter, evaporate the filtrate to dryness, add 10ml of 20v / v% methanol to the residue and sonicate (power 250W, frequency 40kHz) for 30 minutes, remove, filter, and take the filtrate as the reference solution. Separately, take an appropriate amount of uridine reference standard, accurately weigh it, and add 20% methanol to prepare a solution containing 40μg per 1mL, as the reference solution.

[0173] Preparation of the test solution: Take an appropriate amount of winter melon peel preparation, grind it into a fine powder (about 1.0 g), place it in a stoppered conical flask, add 20 ml of 20 v / v% methanol, sonicate (power 250 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the filtrate to obtain the test solution.

[0174] Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0175] In this embodiment, 15 batches of winter melon peel formula granules were used as test solutions, and the fingerprint spectrum of the winter melon peel formula granules was obtained by detection according to the above method. Figure 7 ).like Figure 7 And as shown in the table below, the Figure 7 In the batch number S1 to S15, the batch numbers are as follows: S1: 2005002S, S2: 2007001W, S3: 1909001W, S4: 1911002W, S5: 2002004S, S6: 2001002W, S7: 2003001W, S8: 2104001W, S9: 2011004S, S10: 2102003S, S11: 2106002S, S12: 2109002W, S13: 2111001S, S14: 2111002S, and S15: 2102002W.

[0176] Using the fingerprint similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission, a control characteristic fingerprint spectrum was generated, such as... Figure 8 As shown.

[0177] Table 13. Results of Characteristic Spectrum Determination of 15 Batches of Winter Melon Peel Granules

[0178]

[0179]

[0180] Table 14 and 15 show the relative peak area results of winter melon peel formulation granules.

[0181]

[0182]

[0183] Table 15. Similarity results of characteristic fingerprint spectra of 15 batches of winter melon peel formulation granules

[0184] name Similarity R 1 2005002S 0.968 2007001W 0.985 1909001W 0.971 1911002W 0.953 2002004S 0.962 2001002W 0.984 2003001W 0.985 2104001W 0.940 2011004S 0.975 2102003S 0.974 2106002S 0.971 2109002W 0.959 2111001S 0.945 2111002S 0.977 2102002W 0.959

[0185] Table 16. Relative retention times of common patterns in the fingerprint spectrum of winter melon peel formula granules

[0186]

[0187] Table 17. Common Pattern Matching Data for Winter Melon Peel Granule Formula

[0188]

[0189]

[0190] The fingerprint chromatograms of multiple batches of winter melon peel samples were analyzed. By comparing winter melon peel medicinal materials, processed winter melon peel slices, and winter melon peel granules, six common peaks were identified. These peaks were analyzed using HPLC-Q-TOF. The compounds represented by each characteristic peak were collected and compared under HPLC chromatographic conditions to further confirm the composition and retention time of the characteristic peaks. The six characteristic peaks were identified using reference standards and analyzed by MS, as shown in the table below. Figure 9 As shown, peak 2 was identified as guanine, peak 3 as xanthine, peak 4 as uridine, peak 5 as adenine, and peak 6 as guanosine.

[0191] Table 18. LC / MS / MS Analysis Results of Winter Melon Peel Granules

[0192]

[0193] In summary, see Figure 8 and Figure 10 Based on the table below, it is determined that the chromatogram of the winter melon peel formula granules test sample shows 6 characteristic peaks, which should correspond to the retention times of the 6 characteristic peaks in the chromatogram of the reference medicinal material. The retention time of peak 4 should correspond to the retention time of the uridine reference peak. The peak corresponding to the uridine reference peak is the S peak. Calculate the relative retention times of each characteristic peak and the S peak; the relative retention times should be within ±10% of the specified values. The specified values ​​are 0.39 (peak 1), 0.73 (peak 2), 0.88 (peak 3), 1.46 (peak 5), and 1.84 (peak 6). Specifically, peak 2: guanine; peak 3: xanthine; peak 4 (S): uridine; peak 5: adenine; peak 6: guanosine.

[0194] Table 19. Relative retention time of winter melon peel granule formulation comparison chart

[0195]

[0196] Table 20. Relative peak areas of the chromatograms of winter melon peel granules.

[0197]

[0198] Table 21. Relative retention times of characteristic chromatograms of winter melon peel and other medicinal materials

[0199]

[0200] Table 22. Relative peak areas of characteristic spectra of winter melon peel as a reference medicinal material

[0201]

[0202] Furthermore, when using the detection method of this embodiment to perform quality control on pharmaceutical preparations for winter melon peel, the sample to be tested is tested according to the above detection method, and the obtained characteristic spectrum and Figure 8 Compare the sample's characteristic spectrum with the control spectrum shown. If the characteristic spectrum of the sample to be tested shows at least 6 characteristic peaks identical to those in the control spectrum, the peak corresponding to the uridine reference peak is designated as the S peak. Calculate the relative retention time of each characteristic peak and the S peak. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.39 (peak 1), 0.73 (peak 2), 0.88 (peak 3), 1.46 (peak 5), and 1.84 (peak 6). If these values ​​are not met, the medicinal preparation for winter melon peel is qualified; otherwise, it is unqualified.

[0203] Example 3

[0204] This embodiment provides a method for detecting the characteristic spectrum of a winter melon peel pharmaceutical preparation, using detection method (1) and detection method (2), and the methods are carried out according to Example 1 and Example 2.

[0205] Furthermore, when using detection methods (1) and (2) to conduct quality control on pharmaceutical preparations made from winter melon peel, it is necessary to simultaneously meet the following requirements: the quality testing of pharmaceutical preparations made from winter melon peel is more comprehensive.

[0206] The sample to be tested was tested according to the above detection method (1), and the obtained characteristic spectrum and Figure 2 Compare the sample's characteristic spectrum with the control spectrum shown. If the characteristic spectrum of the sample to be tested shows at least 5 characteristic peaks identical to those in the control spectrum, the peak corresponding to the reference peak of isovitexin-2”-O-rhamnoside is designated as the S peak. Calculate the relative retention time of each characteristic peak and the S peak. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.40 (peak 1), 0.50 (peak 2), 0.52 (peak 3), and 0.97 (peak 4). If these values ​​are not met, the quality of the medicinal preparation for winter melon peel is qualified; otherwise, it is unqualified.

[0207] The sample to be tested was tested according to the above detection method (2), and the resulting characteristic spectrum and Figure 8 Compare the sample's characteristic spectrum with the control spectrum shown. If the characteristic spectrum of the sample to be tested shows at least 6 characteristic peaks identical to those in the control spectrum, the peak corresponding to the uridine reference peak is designated as the S peak. Calculate the relative retention time of each characteristic peak and the S peak. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.39 (peak 1), 0.73 (peak 2), 0.88 (peak 3), 1.46 (peak 5), and 1.84 (peak 6). If these values ​​are not met, the medicinal preparation for winter melon peel is qualified; otherwise, it is unqualified.

[0208] Example 4

[0209] The difference between this embodiment and Example 1 is that the same winter melon peel granules (batch number: 2005002S) were used as the test sample, and the extraction solvents used in the test sample solutions were 20 v / v% methanol, 50 v / v% methanol, and 100 v / v% methanol, respectively. The results are shown in the table below. Figures 11-13 Based on the comprehensive system adaptability parameters, 20% methanol is the optimal extraction solvent.

[0210] Table 23. Parameters for Adaptability of Chromatographic Peak System to Extraction Solvent Methanol Concentration

[0211]

[0212] Example 5

[0213] The difference between this embodiment and Example 1 is that the same winter melon peel granule formula (batch number: 2005002S) was used as the test sample, and the extraction time of the test sample solution was set to 15 minutes, 30 minutes, and 45 minutes, respectively. The results are shown in the table below. Figures 14-16 The results showed that the system adaptability parameters of the test sample were relatively better when the extraction time was 30 minutes, so 30 minutes was selected as the optimal extraction time.

[0214] Table 24. Extraction time parameters for evaluating the adaptability of the chromatographic peak system

[0215]

[0216] Example 6

[0217] The difference between this embodiment and Example 1 is that the same winter melon peel granules (batch number: 2005002S) were used as the test sample, and the sampling amounts in the test sample solution were set to 0.5g, 1g, 2g, and 3g, respectively. The results are shown in the table below. Figures 17-20 As shown, the system adaptability parameters of the test sample are relatively better when the sampling amount is 2g, so 2g is selected as the optimal sampling amount.

[0218] Table 25. Sample Size Test Parameters for Adaptability of Chromatographic Peak System

[0219]

[0220] Example 7

[0221] The difference between this embodiment and Example 1 is that the same winter melon peel granules (batch number: 2005002S) were used as the test sample, and the flow rates in the chromatographic conditions were set to 0.9 mL / min, 1.0 mL / min, and 1.1 mL / min, respectively. The results are shown in the table below. Figures 21-23 The results show that small fluctuations in flow velocity have a relatively small impact on the specified values ​​of the relative retention time of each characteristic peak, indicating that the method has good robustness to small fluctuations in flow velocity.

[0222] Table 26. Results of relative retention times under different flow velocities

[0223]

[0224]

[0225] Table 27. Results of relative peak area under different flow velocities

[0226]

[0227] Example 8

[0228] The difference between this embodiment and Example 1 is that the same winter melon peel granules (batch number: 2005002S) were used as the test sample, and the column temperatures in the chromatographic conditions were set to 33℃, 35℃, and 37℃, respectively. The results are shown in the table below. Figures 24-26 The results show that small fluctuations in column temperature have little impact on the specified values ​​of the relative retention times of each characteristic peak, indicating that the method has good robustness to small temperature fluctuations.

[0229] Table 28. Results of relative retention times under different column temperatures

[0230]

[0231] Table 29. Results of relative peak area under different column temperatures

[0232]

[0233]

[0234] Example 9

[0235] The difference between this embodiment and Example 1 is that the same winter melon peel granules (batch number: 2005002S) were used as the test sample, and the chromatographic columns used in the chromatographic conditions were different batches of the same model of chromatographic column produced by the same manufacturer (brand and model: Dikma Platisil ODS C18 (250mm×4.6mm, 5.0μm; column 1: S / N: 2767062; column 2: S / N: 8068019; column 3: S / N: 4911512). The results are shown in the table below. Figure 27-29 This indicates that the specified values ​​for the relative retention times of each characteristic peak of the chromatographic column from this manufacturer vary little across different batches, and that this method has good durability for different batches of the same model of chromatographic column.

[0236] Table 30. Results of relative retention times for different chromatographic columns

[0237]

[0238] Table 31. Results of relative peak areas investigated using different chromatographic columns

[0239]

[0240] Example 10

[0241] The difference between this embodiment and Example 1 is that the same winter melon peel granules (batch number: 2005002S) were used as the test sample, and different brands of chromatograms were selected for the chromatographic conditions (manufacturers: (1) Agilent 1260; (2) Waters H-Class; (3) Thermo U3000). The results are shown in the table below. Figures 30-32 The results showed that the separation effect of the three major brands of chromatographs was good, and the relative retention time difference between different brands of chromatographs was small, indicating that the method has good durability for different brands of liquid chromatography.

[0242] Table 32. Results of Relative Retention Time for Different Brands

[0243]

[0244] Table 33. Results of relative peak area for different brands

[0245]

[0246] Example 11 Methodological Validation of Detection Method (1)

[0247] 1. Instrument precision test

[0248] Take the same sample solution (batch number: 2005002S), and test it according to the method in Example 1. Repeat the injection 6 times, record the chromatogram, and determine the relative retention time and relative peak area of ​​the 5 characteristic peaks. Analyze the results, as shown in the table below. The results indicate that the RSD of the relative retention time of each characteristic peak and the reference peak S (peak 5) is all within 2%, indicating that the instrument has good precision.

[0249] Table 34. Results of Instrument Precision Relative Retention Time Test

[0250]

[0251]

[0252] Table 35. Results of Instrument Precision Relative Peak Area Test

[0253]

[0254] 2. Method repeatability

[0255] Six samples of the same test sample (batch number: 2005002S) were tested according to the method in Example 1. Chromatograms were recorded, and the relative retention times and relative peak areas of the five characteristic peaks were determined and analyzed. The results are shown in the table below. The RSD of the relative retention times of each characteristic peak and the reference peak S (peak 5) is less than 2%, indicating that the method has good repeatability.

[0256] Table 36. Results of the relative retention time test for method repeatability (n=6)

[0257]

[0258] Table 37. Results of relative peak area test for method repeatability (n=6)

[0259]

[0260] 3. Intermediate precision (for different operators)

[0261] Three inspectors, at different times, used the same equipment to measure the relative retention time and relative peak area of ​​the same batch of winter melon peel formula granules (batch number: 2005002S) according to the method in Example 1. The results are shown in the table below. The RSD of the relative retention time of each characteristic peak and the reference peak S (peak 5) is less than 2%, indicating that the intermediate precision of this method (by different inspectors) is good.

[0262] Table 38. Results of Intermediate Precision Relative Retention Time Tests (Different Operators)

[0263]

[0264] Table 39. Results of Intermediate Precision Relative Peak Area Tests (Different Operators)

[0265]

[0266]

[0267] 4. Specificity and integrity test

[0268] Following the preparation and determination methods of the test sample in Example 1, the test sample solution and blank solvent were injected separately, and the chromatograms were recorded. The effect of the blank solvent was investigated, and the results are shown below. Figures 33-35 Experimental results show that negative samples exhibit no interference and good integrity.

[0269] 5. Stability test

[0270] The same test sample (batch number: 2005002S) was tested according to the method in Example 1. The sample was injected at 0, 4, 8, 12 and 24 hours after preparation. The relative retention time and relative peak area of ​​the five common peaks were measured and analyzed to determine the stability of the test sample solution. The results showed that the RSD of the relative retention time of each characteristic peak and the reference S peak was less than 2%, indicating that the test sample solution was stable within 24 hours and met the test requirements.

[0271] Table 40. Results of the relative retention time test for stability

[0272]

[0273] Table 41. Results of the relative peak area test for stability

[0274]

[0275]

[0276] Example 12

[0277] The difference between this embodiment and Example 2 is that the same winter melon peel granules (batch number: 2005002S) were used as the test sample, and the flow rates in the chromatographic conditions were set to 0.9 mL / min, 1.0 mL / min, and 1.1 mL / min, respectively. The results are shown in the table below. Figures 36-38 The results show that small fluctuations in flow velocity have a relatively small impact on the specified values ​​of the relative retention time of each characteristic peak, indicating that the method has good robustness to small fluctuations in flow velocity.

[0278] Table 42. Results of relative retention time under different flow velocities

[0279]

[0280] Table 43. Results of relative peak area under different flow velocities

[0281]

[0282] Example 13

[0283] The difference between this embodiment and Embodiment 2 is that the same winter melon peel granules (batch number: 2005002S) were used as the test sample, and the column temperatures in the chromatographic conditions were set to 23℃, 25℃, and 27℃, respectively. The results are shown in the table below. Figures 39-41 The results show that small fluctuations in column temperature have little impact on the specified values ​​of the relative retention times of each characteristic peak, indicating that the method has good robustness to small temperature fluctuations.

[0284] Table 44. Results of relative retention times under different column temperatures

[0285]

[0286]

[0287] Table 45. Results of relative peak area under different column temperatures

[0288]

[0289] Example 14

[0290] The difference between this embodiment and Embodiment 2 is that the same winter melon peel granules (batch number: 2005002S) were used as the test sample, and the chromatographic columns selected were Dikma Platisil ODS (250mm×4.6mm, 5μm) and Waters, respectively. T3 (250mm × 4.6mm, 5μm) and Shimadzu Inertsil ODS (250mm × 4.6mm, 5μm). Results are shown in the table below. Figures 42-44 The results showed that different brands of chromatographic columns had a significant impact on the separation of chromatographic peaks. It is recommended to use the Waters Atlantis RT3 (250 mm × 4.6 mm, 5 μm) as the chromatographic column for determining the characteristic chromatogram of winter melon peel formulation granules.

[0291] Table 46. Results of relative retention times for different brands of chromatographic columns

[0292]

[0293] Table 47. Results of relative peak areas for different brands of chromatographic columns

[0294]

[0295]

[0296] Example 15

[0297] The difference between this embodiment and Embodiment 2 is that the same winter melon peel granules (batch number: 2005002S) were used as the test sample, and the chromatographic columns used in this embodiment were different batches from the same manufacturer and of the same model (brand: Waters). T3 (250mm × 4.6mm, 5.0μm; Column 1: S / N: 01973134014020; Column 2: S / N: 01953130512488; Column 3: S / N: 01913125614073). Results are shown in the table below. Figures 45-47 This indicates that the specified values ​​for the relative retention times of each characteristic peak of the chromatographic column from this manufacturer vary little across different batches, and that this method has good durability for different batches of the same model of chromatographic column.

[0298] Table 48. Results of relative retention times for different batches of chromatographic columns

[0299]

[0300] Table 49. Results of relative peak areas for different batches of chromatographic columns

[0301]

[0302] Example 16

[0303] The difference between this embodiment and Example 2 is that the same winter melon peel granules (batch number: 2005002S) were used as the test sample, and different brands of liquid chromatographs were selected for the chromatographic conditions (manufacturers: (1) Agilent 1260; (2) Waterseed 2695; (3) Thermo U3000). The results are shown in the table below. Figures 48-50 The results indicate that the separation effects of the three major brands of chromatographs are all good, and the relative retention time differences between different brands of chromatographs are small, indicating that the method has good durability for different brands of liquid chromatography.

[0304] Table 50. Results of relative retention times in different liquid phases

[0305]

[0306] Table 51. Results of relative peak areas under different liquid phases

[0307]

[0308] Methodological Validation of Detection Method (2) in Example 17

[0309] 1. Instrument precision test

[0310] The same sample solution (batch number: 2005002S) was tested according to the method in Example 2. The injection was repeated six times, and the relative retention times and relative peak areas of the six characteristic peaks were measured and analyzed. The results showed that the RSD of the relative retention times of each characteristic peak and the reference peak S (peak 4) were all within 2%, indicating good instrument precision.

[0311] Table 52. Results of Instrument Precision Relative Retention Time Test

[0312]

[0313]

[0314] Table 53. Results of Instrument Precision Relative Peak Area Test

[0315]

[0316] 2. Method repeatability test

[0317] Six samples of the same batch (batch number: 2005002S) were taken, and the relative retention time and relative peak area of ​​the six common peaks were determined according to the method in Example 2. The results showed that the RSD of the relative retention time of each characteristic peak and the reference S peak (peak No. 4) was less than 2%, indicating that the method had good repeatability.

[0318] Table 54. Results of the method repeatability relative retention time test (n=6)

[0319]

[0320]

[0321] Table 55. Results of relative peak area test for method repeatability (n=6)

[0322]

[0323] 3. Intermediate precision (for different operators)

[0324] Three inspectors, at different times and using the same equipment, measured the relative retention time and relative peak area of ​​the same batch of winter melon peel granules (batch number: 2005002S) according to the method in Example 2. The results showed that the RSD of the relative retention time of each characteristic peak and the reference peak S (peak 4) was less than 2%, indicating that the intermediate precision of this method (by different inspectors) was good.

[0325] Table 56. Results of Intermediate Precision Relative Retention Time Tests (Different Operators)

[0326]

[0327] Table 57. Results of Intermediate Precision Relative Peak Area Tests (Different Operators)

[0328]

[0329]

[0330] 4. Specificity test

[0331] The test sample was taken and the procedure of Example 2 was followed. The test sample solution and blank solvent were injected separately, and the chromatograms were recorded. The effect of the blank solvent was investigated, and the results are as follows: Figures 51-53 Experimental results show that negative samples do not cause interference.

[0332] 5. Stability test

[0333] The same test sample (batch number: 2005002S) was prepared and tested according to the procedure in Example 2, under the preparation and determination method of the test sample solution. The sample was injected at 0, 4, 8, 12, and 24 hours after preparation. The relative retention time and relative peak area of ​​the six common peaks were measured and analyzed to determine the stability of the test sample solution. The results showed that the RSD of the relative retention time of each characteristic peak and the reference S peak was less than 2%, indicating that the test sample solution was stable within 24 hours and met the determination requirements.

[0334] Table 58. Results of the relative retention time test for stability

[0335]

[0336] Table 59. Results of Stability Relative Peak Area Test

[0337]

[0338]

[0339] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for detecting the characteristic chromatograms of a medicinal preparation made from winter melon peel, characterized in that, The winter melon peel pharmaceutical preparation includes winter melon peel formula granules or winter melon peel formula granule extract; Including detection method (1) and / or detection method (2): The detection method (1) includes: detection by high performance liquid chromatography (HPLC). The chromatographic conditions are as follows: using octadecylsilane-bonded silica gel as the packing material, acetonitrile as mobile phase A, and 0.1 v / v% formic acid aqueous solution as mobile phase B, the gradient elution is performed according to the following program: 0-13 min, mobile phase A: mobile phase B volume ratio is 8%:92%→12%:88%; 13-50 min, mobile phase A: mobile phase B volume ratio is 12%:88%→20%:80%; 50-55 min, mobile phase A: mobile phase B volume ratio is 20:80; 55-56 min, mobile phase A: mobile phase B volume ratio is 20%:80%→8%:92%; 56-60 min, mobile phase A: mobile phase B volume ratio is 8%:92%; the detection wavelength is 270 nm. The preparation of the test solution includes: taking the test sample, adding solvent for extraction, filtering, and taking the filtrate, wherein the solvent is a 20 v / v %~50 v / v % methanol solution; The reference standards include: 4-hydroxybenzoic acid, vitexin rhamnoside, and isovitexin-2”-O-rhamnoside were prepared into reference standard solutions, respectively; The detection method (2) includes: detection by high performance liquid chromatography, with the following chromatographic conditions: using octadecylsilane-bonded silica gel as the packing material, methanol as mobile phase A and water as mobile phase B, and performing gradient elution according to the following procedure: 0-10 min, mobile phase A: mobile phase B volume ratio of 0%:100%; 10-30 min, mobile phase A: mobile phase B volume ratio of 0%:100%→5%:95%; 30-42 min, mobile phase A: mobile phase B volume ratio of 5%:95%→10%:90%; detection wavelength of 254 nm; The preparation of the test solution includes: taking the test sample, adding solvent for extraction, filtering, and taking the filtrate, wherein the solvent is a 20 v / v% methanol solution; The reference standards include: guanine, xanthine, uridine, adenine, and guanosine prepared into reference standard solutions respectively.

2. The method for detecting the characteristic spectral features of winter melon peel pharmaceutical preparations according to claim 1, characterized in that, The chromatographic conditions in the detection method (1) include at least one of the following conditions: 1) Column temperature is 33~37℃; 2) The chromatographic column specifications are 4.6 mm × 250 mm, 5 μm; 3) The chromatograph is selected from Agilent 1260, Waters H-Class, or Thermo U3000; 4) The flow rate is 0.9~1.1 mL / min; 5) The injection volume is 8~12μL; And / or, the chromatographic conditions in the detection method (2) include at least one of the following conditions: 1) Column temperature is 23~27℃; 2) The flow rate is 0.9~1.1 ml / min; 3) The chromatographic column specifications are 250mm × 4.6mm, 5µm; 4) The chromatograph is selected from Agilent 1260, Waters e2695 or Thermo U3000; 5) The injection volume is 8~12μL.

3. The method for detecting the characteristic spectral features of winter melon peel pharmaceutical preparations according to claim 2, characterized in that, In the detection method (1), the injection volume is 10 μL; or, the chromatographic column is selected from Dikma Platisil ODS C18; And / or, in the detection method (2), the injection volume is 10 μL; or, the chromatographic column is selected from Dikma Platisil ODS, Waters Atlantis T3 or Shimadzu Inertsil ODS.

4. The method for detecting the characteristic spectral features of a winter melon peel pharmaceutical preparation according to claim 1 or 2, characterized in that, It also includes the preparation of the test solution: The preparation of the test solution in the detection method (1) involves taking 0.5 to 3 parts by weight of the test sample, adding 10 to 25 parts by volume of 20 v / v % to 50 v / v % methanol solution, sonicating for 15 to 45 minutes, filtering, and taking the filtrate. And / or, in the preparation of the test solution in the detection method (2), take 0.8-1.2 parts by weight of the test sample and 15-25 parts by volume of 20v / v% methanol solution, sonicate, filter, and take the filtrate.

5. The method for detecting the characteristic spectral features of a winter melon peel pharmaceutical preparation according to any one of claims 1-3, characterized in that, This also includes the preparation of the reference solution: The preparation of the reference solution in the detection method (1) includes: taking 2-4 parts by weight of winter melon peel reference material, adding 30-80 parts by volume of water, heating under reflux for 30-60 minutes, filtering, evaporating the filtrate to dryness, adding 5 parts by volume of 50v / v% methanol to the residue, ultrasonically treating for 30-60 minutes, filtering, and taking the filtrate as the reference solution for the reference material; the ratio of parts by weight to parts by volume is g / ml; Take an appropriate amount of isovitexin-2”-O-rhamnoside reference standard and add 50 v / v % methanol to prepare solutions containing 30-80 μg per 1 ml, which are used as reference solutions. And / or, the preparation of the reference solution in the detection method (2) includes: reference solution of reference medicinal material: take 1-3 parts by weight of winter melon peel reference medicinal material, add 50-150 parts by volume of water, heat and reflux to extract for 30-60 min, filter, evaporate the filtrate to dryness, add 10 parts by volume of 20v / v% methanol to the residue and sonicate for 30-60 min, take it out, filter, and take the filtrate; the ratio of the parts by weight to the parts by volume is g / ml; Reference solution: Take an appropriate amount of uridine reference standard, accurately weigh it, and add 20% methanol to prepare a solution containing 30-50 μg per mL.

6. A method for determining the content of a medicinal preparation made from winter melon peel, characterized in that, include: Take the test solution and the reference solution, and perform high performance liquid chromatography (HPLC) according to the detection method of the characteristic spectrum of the winter melon peel pharmaceutical preparation as described in any one of claims 1-5.

7. The method for detecting the characteristic chromatogram of the winter melon peel pharmaceutical preparation according to any one of claims 1-5 or the method for determining the content of the winter melon peel pharmaceutical preparation according to claim 6, is used in the quality testing of the winter melon peel pharmaceutical preparation.

8. A method for quality testing of a winter melon peel pharmaceutical preparation, characterized in that, include: The method includes the steps of obtaining the characteristic spectrum of the test sample by the detection method of the characteristic spectrum of the winter melon peel pharmaceutical preparation according to any one of claims 1-5, and comparing the characteristic spectrum with the control characteristic spectrum. The control feature spectrum is obtained by fitting the feature spectrum obtained by using at least one batch of standard samples of winter melon peel drug preparation according to the detection method of feature spectrum of winter melon peel drug preparation as described in any one of claims 1-5.

9. The quality inspection method according to claim 8, characterized in that, include: In the detection method (1), the reference characteristic spectrum includes 5 characteristic peaks, of which peak 5 corresponds to isovitexin-2”-O-rhamnoside. Peak 5 is used as reference peak S. The relative retention times of peaks 1 to 4 with peak S should be within ±10% of the specified values. The specified value for peak 1 is 0.40, the specified value for peak 2 is 0.50, the specified value for peak 3 is 0.52, and the specified value for peak 4 is 0.

97. And / or, in the detection method (2), the reference characteristic spectrum includes 6 characteristic peaks, wherein peak 4 corresponds to uridine, peak 4 is used as reference peak S, and the relative retention times of peaks 1 to 3, peaks 5 to 6 and peak S should be within ±10% of the specified values, with the specified values ​​for peak 1 being 0.39, peak 2 being 0.73, peak 3 being 0.88, peak 5 being 1.46, and peak 6 being 1.

84.

10. The quality inspection method according to claim 9, characterized in that, include: In the detection method (1), peak 1 is 4-hydroxybenzoic acid; peak 4 is vitexin rhamnoside; peak 5 is isovitexin-2”-O-rhamnoside; In the detection method (2), peak 2 is guanine; peak 3 is xanthine; peak 4 is uridine; peak 5 is adenine; and peak 6 is guanosine.

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