Construction of HPLC characteristic chromatogram of Radix Morindae Officinalis and identification of its adulterant Radix Naucleae

Abstract

The application provides a construction method of HPLC characteristic atlas of Terminalia chebula medicinal material decoction pieces, standard decoction and formula granules, comprising: pretreating raw materials of a test sample to obtain a to-be-tested solution; the pretreatment method comprises water decoction, cooling, filtration, evaporation of the filtrate, and ultrasonic treatment of the residue with methanol; the to-be-tested solution is determined by HPLC to obtain the HPLC characteristic atlas of Terminalia chebula medicinal material decoction pieces, standard decoction and formula granules; preparation of reference solution: respectively dissolving gallic acid and 3,3'-O-dimethyl gallate with a solvent to obtain a reference solution of a control sample; the HPLC conditions are as follows: a C 18 column is used as the chromatographic column; mobile phase A is a methanol solution, mobile phase B is a 0.3% formic acid aqueous solution, and gradient elution is adopted; the application adopts high performance liquid chromatography, selects methanol-0.3% formic acid solution as the mobile phase for gradient elution, takes gallic acid as the reference, establishes the HPLC characteristic atlas method of Terminalia chebula medicinal material decoction pieces, standard decoction and formula granules, and can distinguish Terminalia chebula and its adulterants Terminalia chebula.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical and formulation analysis and detection technology, and in particular to a method for constructing HPLC characteristic chromatograms of Quisqualis indica decoction pieces, standard decoctions, and formulation granules, and a method for identifying the characteristic chromatograms of its adulterant product Terminalia chebula. Background Technology

[0002] Quisqualis indica L., the dried, ripe fruit of the plant (Combretaceae family), is used for its insecticidal and digestive properties. It is commonly used to treat ascariasis, pinworm infection, abdominal pain due to intestinal parasites, and infantile malnutrition. To ensure the uniformity and stability of the quality of Quisqualis indica raw materials, processed slices, standard decoctions, and formulated granules, this invention constructs a characteristic chromatogram detection method for Quisqualis indica to control its quality. Furthermore, in practical applications, Quisqualis indica is often difficult to distinguish from its adulterant, Terminalia chebula. Therefore, providing a method for effectively identifying Quisqualis indica and its adulterant Terminalia chebula is essential. Summary of the Invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing HPLC characteristic chromatograms of Quisqualis indica medicinal slices, standard decoctions, and formula granules. The method of the present invention is accurate and reliable, and can distinguish Quisqualis indica from its adulterant product Terminalia chebula.

[0004] A method for constructing HPLC characteristic chromatograms of Quisqualis indica medicinal slices, standard decoctions, and formulated granules, comprising:

[0005] The raw materials for testing are pretreated to obtain the test solution; the pretreatment method includes boiling in water, cooling, filtering, evaporating the filtrate to dryness, and ultrasonically treating the residue with methanol; the test solution is analyzed by HPLC to obtain the HPLC characteristic chromatograms of Quisqualis indica medicinal material, decoction pieces, standard decoction, and formula granules.

[0006] Preparation of reference solution: Ellagic acid and 3,3'-O-dimethylellagic acid were dissolved in solvent to obtain reference solution;

[0007] The HPLC conditions were as follows: C18 column; mobile phase A was methanol solution, mobile phase B was 0.3% formic acid aqueous solution, and gradient elution was used.

[0008] The gradient elution specifically refers to:

[0009] 0–10 min, Phase A: 20% → 30%, Phase B: 80% → 70%;

[0010] 10–30 min, Phase A: 30% → 40%, Phase B: 70% → 60%;

[0011] 30–45 min, Phase A: 40% → 52%, Phase B: 60% → 48%;

[0012] 45–50 min, Phase A: 52%, Phase B: 48%;

[0013] 50–55 min, Phase A: 52% → 55%, Phase B: 48% → 45%;

[0014] 55–75 min, Phase A: 55% → 70%, Phase B: 45% → 30%.

[0015] The raw materials for the test sample described in this invention are one or more of the following: Quisqualis indica medicinal slices, standard decoctions, and formula granules.

[0016] This invention provides a method for constructing HPLC characteristic chromatograms of Quisqualis indica medicinal slices, standard decoctions, and formulation granules. First, the raw materials for testing are pretreated. The pretreatment method includes boiling in water, cooling, filtering, evaporating the filtrate to dryness, and ultrasonically treating the residue with methanol.

[0017] Specifically, the test sample is placed in a stoppered conical flask, water is added, the mixture is boiled, cooled, filtered, the filtrate is evaporated to dryness, the residue is added to methanol, sonicated, filtered, and the filtrate is used as the test sample solution.

[0018] The decoction time described in this invention is 30 minutes; the power of the ultrasonic extraction device is set to 600W, the ultrasonic vibration frequency is 40kHz, and the duration of ultrasonic extraction is 30 minutes.

[0019] The present invention achieves complete extraction under the above-mentioned ultrasonic parameters. The chromatogram shows good peak shape and resolution.

[0020] The ratio of the mass (g) of the test sample raw material and the volume (mL) of water and methanol in this invention is 5g:100mL:10mL.

[0021] When using the extraction solvent and specific solvent addition amounts, the present invention achieves good peak shape and resolution, and moderate peak size for each chromatographic peak.

[0022] All the raw materials mentioned above can be subjected to quality control using the methods of this invention.

[0023] The present invention also includes the preparation of a reference solution: ellagic acid and 3,3'-O-dimethylellagic acid are dissolved in a solvent to obtain a reference solution;

[0024] The reference herb, Quisqualis indica, was decocted in water, cooled, filtered, and the filtrate was evaporated to dryness. The residue was treated with methanol by sonication, filtered, and the filtrate was used as the reference herb solution.

[0025] The reference standard solution and the reference medicinal material solution were injected into a high-performance liquid chromatograph for determination, and the chromatograms of the reference standard and the characteristic chromatograms of the reference medicinal material were obtained respectively. Based on the characteristic parameters of the chromatograms of the reference standard, the corresponding chromatograms in the HPLC characteristic chromatograms of the Quisqualis indica test sample, the standard decoction test sample, and the formula granule test sample were compared and identified.

[0026] The preferred concentration of the reference solution in this invention is 20 μg / mL of ellagic acid and 20 μg / mL of 3,3'-O-dimethylellagic acid.

[0027] The test solution was analyzed by high performance liquid chromatography to obtain the HPLC characteristic chromatograms of Quisqualis indica medicinal material, decoction pieces, standard decoction, and formula granules.

[0028] The chromatographic conditions for the high-performance liquid chromatography (HPLC) were as follows: the chromatographic column was a C18 column; the chromatographic column was a C18 column. 18 The specifications are 250×4.6mm5μm; this invention does not limit the specific chromatographic column model, any C18 chromatographic column that meets the above specifications is acceptable.

[0029] In some embodiments, the chromatographic column can be InertSustain TM C18 4.6×250mm 5μm, 5TC-C18(2)4.6×250mm 5μm, Kromasil 100-5-C18 4.6×250mm 5μm;

[0030] The column temperature of this invention is 25–35°C; preferably 30°C. At the above column temperature, the chromatographic peaks of this invention are symmetrical, the separation is good, and the peaks are eluted completely.

[0031] In this invention, mobile phase A is a methanol solution and mobile phase B is a 0.3% formic acid aqueous solution, with gradient elution.

[0032] Specifically, the gradient elution is as follows:

[0033] 0–10 min, Phase A: 20% → 30%, Phase B: 80% → 70%;

[0034] 10–30 min, Phase A: 30% → 40%, Phase B: 70% → 60%;

[0035] 30–45 min, Phase A: 40% → 52%, Phase B: 60% → 48%;

[0036] 45–50 min, Phase A: 52%, Phase B: 48%;

[0037] 50–55 min, Phase A: 52% → 55%, Phase B: 48% → 45%;

[0038] 55–75 min, Phase A: 55% → 70%, Phase B: 45% → 30%.

[0039] The theoretical plate number, calculated based on ellagic acid, should be no less than 5000.

[0040] The invention creatively discovers that, under the above-mentioned elution gradient, the baseline separation is good, the separation between each peak is good, the distribution is uniform, and the baseline is stable.

[0041] The flow rate of the mobile phase described in this invention is 1.0 mL / min.

[0042] The present invention found that the chromatographic peaks were well separated, with moderate resolution and complete elution at the above-mentioned flow rates, which is the optimal solution.

[0043] The injection volume was 10 μL.

[0044] The detection wavelength is 250 nm. The inventors have discovered that at 250 nm, the chromatographic peak information content is greater, the chromatographic baseline is more stable, and the peak areas are larger.

[0045] This invention is the first to establish a characteristic chromatographic detection method using ultra-high performance liquid chromatography (UHPLC) to distinguish between Quisqualis indica raw material, standard decoction, and formulated granules. This method is beneficial for comprehensively evaluating the scientific validity and rationality of the related processes of Quisqualis indica and its processed extracts and preparations, and can better control the intrinsic quality of Quisqualis indica and its processed extracts and preparations, thus ensuring the clinical efficacy of Quisqualis indica and its processed extracts and preparations.

[0046] This invention uses a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine to evaluate the similarity of HPLC characteristic chromatograms of Quisqualis indica raw materials, processed slices, standard decoctions, and formulated granules. A standard HPLC characteristic chromatogram consisting of 7 characteristic peaks was constructed, and the chemical composition information corresponding to each characteristic peak is as follows: Peak 3 (S): ellagic acid; Peak 6: 3,3'-O-dimethylellagic acid;

[0047] In the characteristic chromatograms of the Quisqualis indica medicinal material, processed slices, standard decoctions, and formulated granules, ellagic acid is used as the reference peak S. The relative retention time of each characteristic peak and the S peak is calculated. The allowable fluctuation range of the relative retention time of each characteristic peak is within ±10% of the specified value. The specified values ​​are: 0.40 (peak 1), 0.61 (peak 2), 1.09 (peak 4), 1.27 (peak 5), 1.44 (peak 6), and 1.71 (peak 7).

[0048] Quality judgment criteria: Take the extracts and preparations of Quisqualis indica and its processed products, and operate according to the above method to obtain the above characteristic chromatograms. Use the 2012 version of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" of the National Pharmacopoeia Commission to analyze the characteristic chromatograms of Quisqualis indica and its processed products and the sample characteristic chromatograms. The similarity is greater than 0.90.

[0049] This invention provides a method for identifying the characteristic spectral features of Quisqualis indica and its adulterant Terminalia chebula using the method described above, and analyzing the results.

[0050] The present invention has already clearly described the above technical solutions, and will not repeat them here.

[0051] Peak 4 was not detected at a relative retention time of 1.09, peak 6 was not detected at a relative retention time of 1.44, and peak 7 was not detected at a relative retention time of 1.71. Therefore, peaks 4, 6, and 7 are used as identification points for Quisqualis indica and its adulterant, Terminalia chebula.

[0052] This invention provides a method for constructing HPLC characteristic chromatograms of Quisqualis indica medicinal slices, standard decoctions, and formulated granules. The method includes: pretreatment of the test sample raw material to obtain a test solution; the pretreatment method includes decoction with water, cooling, filtration, evaporation of the filtrate to dryness, and ultrasonic treatment of the residue with methanol; determination of the test solution by HPLC to obtain HPLC characteristic chromatograms of Quisqualis indica medicinal slices, standard decoctions, and formulated granules; preparation of reference solution: dissolving ellagic acid and 3,3'-O-dimethylellagic acid in solvents to obtain a reference solution; the HPLC conditions are: a C18 column; mobile phase A is methanol solution, mobile phase B is 0.3% formic acid aqueous solution, gradient elution; this invention uses high-performance liquid chromatography, selecting methanol-0.3% formic acid solution as the mobile phase for gradient elution, and using ellagic acid as a reference, to establish a method for HPLC characteristic chromatograms of Quisqualis indica medicinal slices, standard decoctions, and formulated granules, while also distinguishing Quisqualis indica from its adulterant, Terminalia chebula.

[0053] This invention establishes a characteristic chromatographic method for identifying Quisqualis indica and its adulterants using high-performance liquid chromatography (HPLC). This method achieves effective separation within different wavelength, column temperature, and flow rate ranges. This method can effectively identify Quisqualis indica and its adulterant Terminalia chebula.

[0054] This invention establishes a characteristic chromatographic identification method for Quisqualis indica and its adulterant Terminalia chebula using high-performance liquid chromatography (HPLC). The method utilizes the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version) to synthesize characteristic chromatograms for 21 batches of Quisqualis indica raw materials, decoctions, and granules, establishing reference characteristic chromatograms for these materials. The test sample chromatogram should exhibit seven characteristic peaks, corresponding to the retention times of the seven characteristic peaks in the reference chromatogram of the control herb. Peak 3 should correspond to the retention time of the reference peak. The peak corresponding to the ellagic acid reference peak is designated as the S peak. The relative retention times of the remaining characteristic peaks and the S peak are calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are 0.41 (peak 1), 0.61 (peak 2), 1.09 (peak 4), 1.27 (peak 5), 1.44 (peak 6: 3,3'-O-dimethylellagic acid), and 1.71 (peak 7).

[0055] This invention establishes a characteristic chromatographic identification method for Quisqualis indica and its adulterant Terminalia chebula using high-performance liquid chromatography (HPLC). Peaks 4, 6, and 7 were identified as the identification points for Quisqualis indica and its adulterant Terminalia chebula. Peak 6 is identified as 3,3'-O-dimethylellagic acid.

[0056] This invention is characterized by its ease of operation, convenient analysis, and stability. It utilizes high-performance liquid chromatography (HPLC) fingerprinting technology to control the identification of Quisqualis indica and its adulterants, thereby effectively controlling the quality of the raw material. Attached Figure Description

[0057] Figure 1 Mobile phase selection;

[0058] Figure 2 Flow velocity study;

[0059] Figure 3 Column temperature investigation;

[0060] Figure 4 Injection volume investigation;

[0061] Figure 5 Delayed investigation;

[0062] Figure 6 Extraction solvent investigation;

[0063] Figure 7 Extraction time consideration;

[0064] Figure 8 Examining the extraction method;

[0065] Figure 9 Investigation of solvent addition amount;

[0066] Figure 10 Specificity assessment;

[0067] Figure 11 Chromatographic peak identification;

[0068] Figure 12 Ellagic acid spectrum - reference standard;

[0069] Figure 13 3,3'-O-dimethylellagic acid - Spectrum - Reference Standard;

[0070] Figure 14 Ellagic acid spectrum - medicinal materials;

[0071] Figure 15 3,3'-O-dimethylellagic acid - Spectrum - Medicinal material;

[0072] Figure 16 Different instruments;

[0073] Figure 17 Investigations using different chromatographic columns;

[0074] Figure 18 Superimposed image of the medicinal characteristics of Quisqualis indica;

[0075] Figure 19 Overlay of characteristic images of Quisqualis indica medicinal material.

[0076] Figure 20 Superimposed image of characteristic features of Quisqualis indica slices;

[0077] Figure 21 Superimposed image of characteristic features of Quisqualis indica slices;

[0078] Figure 22-a Comparative characteristic atlas of medicinal materials;

[0079] Figure 22-b Comparative characteristic atlas of medicinal slices;

[0080] Figure 23-a Mobile phase selection;

[0081] Figure 23-b Chromatograms of different wavelengths of Quisqualis indica decoction;

[0082] Figure 24 Column temperature investigation;

[0083] Figure 25 Flow velocity study;

[0084] Figure 26 Injection volume investigation;

[0085] Figure 27 Delayed investigation;

[0086] Figure 28 Extraction solvent investigation;

[0087] Figure 29 Extraction time consideration;

[0088] Figure 30 Sample quantity assessment;

[0089] Figure 31 Extraction methods were examined;

[0090] Figure 32 Chromatographic peak identification;

[0091] Figure 33 Ellagic acid spectrum - reference standard;

[0092] Figure 34 3,3'-O-dimethylellagic acid - Spectrum - Reference Standard;

[0093] Figure 35 Ellagic acid spectrum - standard soup;

[0094] Figure 36 3,3'-O-dimethylellagic acid - spectral diagram - standard soup;

[0095] Figure 37 Exploration using different instruments;

[0096] Figure 38 Investigations using different chromatographic columns;

[0097] Figure 39 Superimposed diagram of characteristic spectra of standard Quisqualis indica decoction;

[0098] Figure 40 Superimposed diagram of characteristic spectra of standard Quisqualis indica decoction;

[0099] Figure 41-a Superimposed diagram of characteristic spectra of standard Quisqualis indica decoction;

[0100] Figure 41-b A comparative chromatogram of the characteristic chromatograms of the standard decoction of Quisqualis indica.

[0101] Figure 42 Mobile phase selection;

[0102] Figure 43 Chromatograms of Quisqualis indica at different wavelengths;

[0103] Figure 44 Column temperature investigation;

[0104] Figure 45 Flow velocity study;

[0105] Figure 46 Injection volume investigation;

[0106] Figure 47 Delayed investigation;

[0107] Figure 48 Extraction solvent investigation;

[0108] Figure 49 Extraction time consideration;

[0109] Figure 50 Examining the extraction method;

[0110] Figure 51 Investigation of solvent addition amount;

[0111] Figure 52 Specificity assessment;

[0112] Figure 53 Chromatographic peak identification;

[0113] Figure 54 Ellagic acid spectrum - reference standard;

[0114] Figure 55 3,3'-O-dimethylellagic acid - Spectrum - Reference Standard;

[0115] Figure 56 Ellagic acid spectrum - particles;

[0116] Figure 57 3,3'-O-dimethylellagic acid - Spectrum - Particles

[0117] Figure 58 Different instruments;

[0118] Figure 59 Investigations using different chromatographic columns;

[0119] Figure 60 Overlay image of characteristic granules of Quisqualis indica formula;

[0120] Figure 61 Comparison of characteristic chromatograms of Quisqualis indica formula granules;

[0121] Figure 62 Comparative pictographic representations of Quisqualis indica and Terminalia chebula medicinal materials;

[0122] Figure 63 Comparison of characteristic chromatograms of Quisqualis indica and Terminalia chebula decoction;

[0123] Figure 64 Comparison of granule characteristic spectra of Quisqualis indica and Terminalia chebula;

[0124] Figure 65 To investigate different mobile phases. Detailed Implementation

[0125] This invention provides a method for constructing HPLC characteristic chromatograms of Quisqualis indica medicinal slices, standard decoctions, and formulated granules. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of protection of this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0126] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a method for constructing HPLC characteristic chromatograms of Quisqualis indica medicinal slices, standard decoctions, and formulation granules provided by the present invention.

[0127] Agilent 1260 HPLC system; Thermo Fisher HPLC system; Waters 2695 HPLC system;

[0128] Cellular 1810A Ultrapure Water System (Shanghai Moler Scientific Instruments Co., Ltd.);

[0129] KQ-600DB Ultrasonic Cleaner (Kunshan Ultrasonic Instrument Co., Ltd.);

[0130] Acetonitrile (chromatographic grade, Chengdu Kelong Chemical Reagent Co., Ltd.), methanol (chromatographic grade, Chengdu Kelong Chemical Reagent Co., Ltd.), phosphoric acid (chromatographic grade, Chengdu Kelong Chemical Reagent Co., Ltd.), formic acid (chromatographic grade, Chengdu Kelong Chemical Reagent Co., Ltd.), methanol (analytical grade, Chengdu Kelong Chemical Reagent Co., Ltd.), and water used for the mobile phase were laboratory-prepared ultrapure water; all other water was laboratory-prepared pure water.

[0131] Column: InertSustain TM C18 4.6×250mm 5μm, 5TC-C18(2)4.6×250mm5μm, Kromasil 100-5-C184.6×250mm 5μm Agilent ZORBAX Eclipse Plus C18 4.6×250mm 5μm, Agilent 5TC-C18 4.6×250mm 5μm, Agilent ZORBAX SB-C18 4.6×250mm 5μm;

[0132] Ellagic acid (China National Institute for Food and Drug Control, batch number: 111959-201903)

[0133] 3,3'-O-dimethylellagic acid (Chengdu DST Biotechnology Co., Ltd., batch number: DSTDE002001)

[0134] Quisqualis Fructus medicinal materials: SJZ-YC-0101, SJZ-YC-0102, SJZ-YC-0103, SJZ-YC-0104, SJZ-YC-0105, SJZ-YC-0106, SJZ-YC-0107, SJZ-YC-0108, SJZ-YC-0109, SJZ-YC-0110, SJZ-YC-0111, SJZ-YC-0112, SJZ-YC-0113, SJZ-YC-0114, SJZ-YC-0115, SJZ-YC-0116, SJZ-YC-0117, SJZ-YC-0118, SJZ-YC-0119, SJZ-YC-0120, SJZ-YC-0121, SJZ-YC-0122, SJZ-YC-0123, SJZ-YC-0124, SJZ-YC-0125, SJZ-YC-0126.

[0135] Quisqualis Fructus decoction pieces: SJZ-YP-0101, SJZ-YP-0102, SJZ-YP-0103, SJZ-YP-0104, SJZ-YP-0105, SJZ-YP-0106, SJZ-YP-0107, SJZ-YP-0108, SJZ-YP-0109, SJZ-YP-0110, SJZ-YP-0111, SJZ-YP-0112, SJZ-YP-0113, SJZ-YP-0114, SJZ-YP-0115, SJZ-YP-0116, SJZ-YP-0117, SJZ-YP-0118, SJZ-YP-0119, SJZ-YP-0120, SJZ-YP-0121, SJZ-YP-0122, SJZ-YP-0123, SJZ-YP-0124, SJZ-YP-0125, SJZ-YP-0126.

[0136] Standard decoction of Quisqualis indica: SJZ-BT-0101, SJZ-BT-0102, SJZ-BT-0103, SJZ-BT-0104, SJZ-BT-0105, SJZ-BT-0106, SJZ-BT-0107, SJZ-BT-0108, SJZ-BT-0109, SJZ-BT-0110, SJZ-BT-0111, SJZ-BT-0112, SJZ-BT-0 113. SJZ-BT-0114, SJZ-BT-0115, SJZ-BT-0116, SJZ-BT-0117, SJZ-BT-0118, SJZ-BT-0119, SJZ- BT-0120, SJZ-BT-0121, SJZ-BT-0122, SJZ-BT-0123, SJZ-BT-0124, SJZ-BT-0125, SJZ-BT-0126.

[0137] Quisqualis indica formula granules: SJZ-KL-0101, SJZ-KL-0102, SJZ-KL-0103.

[0138] Example 1: HPLC Characteristic Chromatography of Medicinal Materials and Slices

[0139] 2. Chromatographic conditions investigation

[0140] 2.1 Proposed chromatographic conditions

[0141] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol as mobile phase A and 0.3% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 360 nm. The theoretical plate number, calculated based on the ellagic acid peak, should be no less than 5000.

[0142]

[0143] 2.2 Selection of mobile phase

[0144] Based on the experimental conditions outlined above, the separation effects of three different mobile phases were investigated: methanol-0.3% formic acid, acetonitrile-0.3% formic acid, and methanol-0.3% phosphoric acid. (See attached figures.) Figure 1 . Figure 1 Mobile phase selection.

[0145] The results showed that the chromatographic peak baseline was relatively stable and there were many chromatographic peaks under the gradient elution condition of methanol-0.3% formic acid solution. Therefore, the gradient elution of methanol-0.3% formic acid solution was used as the mobile phase for the determination of the characteristic chromatogram of Quisqualis indica.

[0146] 2.3 Flow velocity investigation

[0147] Based on the above-specified experimental conditions, the flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were investigated respectively. (See attached figures.) Figure 2 Table 1-2. Figure 2 Flow velocity investigation

[0148] Table 1. Flow velocity assessment - retention time

[0149]

[0150] Table 2 Flow velocity study - relative retention time

[0151]

[0152] The results showed that the peak shapes were good under all three flow rate conditions, with the overall peak retention time being most suitable at a flow rate of 1.0 ml / min. Therefore, the flow rate was determined to be 1.0 ml / min.

[0153] 2.4 Column Temperature Investigation

[0154] Based on the above-specified experimental conditions, investigations were conducted at column temperatures of 25℃, 30℃, and 35℃, respectively. (See attached figures.) Figure 3 Table 3-4. Figure 3 Column temperature investigation.

[0155] Table 3 Column Temperature Study - Retention Time

[0156]

[0157] Table 4 Column Temperature Study - Relative Retention Time

[0158]

[0159] The results showed that the peak shapes were good at all three column temperatures, with the overall peak retention time being most suitable at a column temperature of 30℃. Taking all factors into consideration, the column temperature was determined to be 30℃.

[0160] 2.5 Injection Volume Investigation

[0161] Based on the above-established experimental conditions, the effects were investigated at injection volumes of 5 μl, 10 μl, and 15 μl. The results are shown below. Figure 4 Table 5-6. Figure 4 Injection volume investigation.

[0162] Table 5. Sample Volume and Retention Time

[0163]

[0164] Table 6. Injection Volume Analysis - Relative Retention Time

[0165]

[0166] The results showed that when the injection volume was 10 μl, the chromatogram peak shape was good and the resolution was moderate. Therefore, the injection volume was determined to be 10 μl.

[0167] 2.6 Delayed Examination

[0168] Based on the above-specified experimental conditions, a delay test was conducted. The results are shown below. Figure 5 . Figure 5 Delayed investigation.

[0169] The results showed that the sample had virtually no chromatographic peaks after 75 minutes, so the sample detection time was set at 75 minutes.

[0170] 2.7 Determination of chromatographic conditions

[0171] [Characteristic chromatogram] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0172] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol as mobile phase A and 0.3% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 360 nm. The theoretical plate number, calculated based on the ellagic acid peak, should be no less than 5000.

[0173]

[0174]

[0175] 3. Preparation of the test solution

[0176] 3.1 Investigation of extraction solvent

[0177] Take 5.0 g of the powder (passed through a No. 3 sieve, batch number: SJZ-YC-0101), place it in a stoppered conical flask, add 100 ml of water, decoct for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 10 ml each of methanol, 70% methanol, 50% methanol, 30% methanol, ethanol, and water to the residue, seal tightly, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the product. See Figure 6 . Figure 6 Investigation of extraction solvent.

[0178] The results showed that when methanol was used as the extraction solvent, the peak shapes of each characteristic peak were good and the separation was moderate. Taking all factors into consideration, methanol was tentatively chosen as the extraction solvent.

[0179] 3.2 Examination of extraction time

[0180] Take 5.0 g of the powder (passed through a No. 3 sieve, batch number: SJZ-YC-0101), place it in a stoppered conical flask, add 100 ml of water, decoct for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 10 ml of methanol to the residue, and sonicate (600 W power, 40 kHz frequency) for 20 minutes, 30 minutes, and 40 minutes respectively. Cool, shake well, filter, and collect the filtrate to obtain the product. See Figure 7 . Figure 7 Extraction time consideration;

[0181] The results showed that there were no significant differences in the chromatographic peaks at the three extraction times. Taking all factors into consideration, the extraction time was tentatively set at 30 minutes.

[0182] 3.3 Examination of Extraction Methods

[0183] Take 5.0 g of the powder (passed through a No. 3 sieve, batch number: SJZ-YC-0101), place it in a stoppered conical flask, add 10 ml of methanol, reflux and sonicate (600 W, 40 kHz) for 30 minutes respectively, cool, shake well, filter, and collect the filtrate. Separately, take another 5.0 g of the powder (passed through a No. 3 sieve, batch number: SJZ-YC-0101), place it in a stoppered conical flask, add 100 ml of water, decoct for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 10 ml of methanol to the residue, sonicate (600 W, 40 kHz) for 30 minutes to dissolve, filter, and collect the filtrate. See [link to product description]. Figure 8 . Figure 8 Examining the extraction method.

[0184] The results showed that the sample exhibited good peak shapes and moderate chromatographic peak resolution when extracted by water decoction, and the chromatographic baseline was relatively flat. Therefore, the proposed extraction method is to first decoct the sample in water and then treat it with ultrasound.

[0185] 3.4 Investigation of Solvent Addition Amount

[0186] Take 5g of the powder (passed through a No. 3 sieve, batch number: SJZ-YC-0101) and add 100ml of water. Boil for 30 minutes, cool, filter, and evaporate the filtrate to dryness. Add 10ml, 15ml, and 20ml of methanol to the residue respectively, and sonicate (600W power, 40kHz frequency) for 30 minutes. Filter and collect the filtrate to obtain the final product. See results below. Figure 9 . Figure 9 Investigation of the amount of solvent added.

[0187] The results showed that when the solvent volume was 10 ml, each chromatographic peak was slightly higher, therefore the solvent volume should be 10 ml.

[0188] 3.4 Determination of the preparation method of the test sample

[0189] In summary, the preparation method of the test solution of Quisqualis indica medicinal material characteristic spectrum is determined as follows: Take 5g of the powder (passed through a No. 3 sieve), place it in a stoppered conical flask, add 100ml of water, decoct for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 10ml of methanol to the residue, sonicate (power 600W, frequency 40kHz) for 30 minutes, shake well, filter, and take the filtrate to obtain the product.

[0190] 4. Methodological Investigation

[0191] 4.1 Specificity Examination

[0192] Preparation of the test solution: Prepare the test solution of Quisqualis indica medicinal material according to the experimental conditions proposed above.

[0193] Preparation of negative control solution: Prepare a negative control solution for Quisqualis indica without the above-mentioned experimental conditions.

[0194] See results Figure 10 . Figure 10 Specificity assessment.

[0195] The results showed that the negative control had no interference, indicating that the method has good specificity.

[0196] 4.2 Chromatographic Peak Identification

[0197] Preparation of the test solution: Prepare the test solution of Quisqualis indica medicinal material according to the experimental conditions proposed above.

[0198] Preparation of reference solution: Take 5g of Quisqualis indica reference material, place it in a stoppered conical flask, add 100ml of water, decoct for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 10ml of methanol to the residue, sonicate (power 600W, frequency 40kHz) for 30 minutes, filter, and take the filtrate as the reference solution of the reference material.

[0199] Separately, take appropriate amounts of ellagic acid and 3,3'-O-dimethylellagic acid reference standards, accurately weigh them, and add methanol to prepare a solution containing 20 μg per 1 ml, which will be used as the reference solution.

[0200] Preparation of negative control solution: Prepare a negative control solution for Quisqualis indica without the above-mentioned experimental conditions.

[0201] The characteristic spectral peaks of Quisqualis indica were located. (See...) Figure 11-15 . Figure 11 Chromatographic peak identification; Figure 12 Ellagic acid spectrum - reference standard;

[0202] Figure 13 3,3'-O-dimethylellagic acid - Spectrum - Reference Standard; Figure 14 Ellagic acid spectrum - medicinal materials; Figure 15 3,3'-O-dimethylellagic acid - Spectrum - Medicinal material;

[0203] The results showed that peak 3 was ellagic acid and peak 6 was 3,3'-O-dimethylellagic acid. In the following methodological investigation, seven characteristic peaks in the sample were examined.

[0204] 4.2 Precision Test

[0205] Take the test solution of Quisqualis indica (batch number: SJZ-YC-0101), inject it 6 times consecutively according to the proposed experimental method, 10 μL each time, and calculate the retention time of each characteristic peak. See Table 7.

[0206] Table 7 Precision Examination - Retention Time

[0207]

[0208] The results showed that the retention time RSD of each characteristic peak of the sample was 0.04% to 0.11%, and the peak area RSD was 0.62% to 4.60%, indicating that the instrument had good precision.

[0209] 4.3 Repeatability Test

[0210] Six samples of Quisqualis indica (batch number: SJZ-YC-0101) were prepared and tested according to the proposed experimental method. See Table 8.

[0211] Table 8 Repeatability Tests - Relative Retention Time

[0212]

[0213] The results showed that the relative retention time RSD of the six samples was 0.00%–0.23%, indicating that the method has good reproducibility.

[0214] 4.4 Intermediate Precision Examination

[0215] 4.4.1 Investigation with different instruments

[0216] Based on the above-established experimental conditions, the following samples of Quisqualis indica (batch number: SJZ-YC-0101) were weighed and tested: solutions were prepared and analyzed using Agilent, Thermo Fisher Scientific, and Waters high-performance liquid chromatographs, respectively. See Table 9. Figure 16 .

[0217] Table 9 Instrument Durability Test - Relative Retention Time

[0218]

[0219] Figure 16Different instruments were used; the results showed that when the test sample was detected using the above three instruments, the RSD of the relative retention time of each characteristic peak ranged from 0.00% to 2.75%, indicating that the instrument durability of this method is good.

[0220] 4.4.2 Investigations by different personnel and at different times

[0221] Based on the above-planned experimental conditions, different personnel (A, B) conducted measurements at different times (a, b) using different instruments (I, II). The Quisqualis indica medicinal material (batch number: SJZ-YC-0101) was weighed, the test sample was prepared, and the measurements were performed (see Table 10).

[0222] Table 10 Intermediate Precision - Relative Retention Time

[0223]

[0224] The results show that when different personnel measure the same sample at different times, the RSD of the relative retention time of each characteristic peak is 0.00% to 2.56%, indicating that the method has good applicability.

[0225] 4.5 Selection of Different Chromatographic Columns

[0226] Based on the above-planned experimental conditions, the InertSustain column was used for each test. TM C18 4.6×250mm 5μm, 5TC-C18(2) 4.6×250mm 5μm, Kromasil 100-5-C18 4.6×250mm 5μm; were analyzed and investigated, and the relative retention time and relative peak area of ​​each characteristic peak were calculated. The results are shown in Figure 17 Table 11. Figure 17 Investigation of different chromatographic columns.

[0227] Table 11 Column robustness study - relative retention time

[0228]

[0229] The results showed that when the samples were detected using the above three chromatographic columns, the RSD of the relative retention time of the characteristic peaks ranged from 0.00% to 5.52%, indicating that the columns had good robustness.

[0230] 4.6 Stability Test

[0231] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 4h, 8h, 16h, and 24h. See Table 12.

[0232] Table 12 Stability Study - Relative Retention Time

[0233]

[0234] The results showed that the RSD of the relative retention time of the characteristic peak was between 0.00% and 0.40%, and the sample solution was stable within 24 hours.

[0235] 5. Verification of Quisqualis indica medicinal materials and processed slices, and establishment of HPLC characteristic chromatographic methods.

[0236] 6.5.1 Verification results of 26 batches of Quisqualis indica medicinal materials, see... Figure 18-19 Table 13. Figure 18 Superimposed image of the medicinal characteristics of Quisqualis indica; Figure 19 Overlay of characteristic images of Quisqualis indica medicinal material.

[0237] Table 13 Relative retention time of Quisqualis indica medicinal material

[0238]

[0239]

[0240] Based on the principles of stable relative retention time, detectability in all batches of samples, and relatively high peak values, seven peaks with good robustness were selected as characteristic peaks. The results showed that the relative retention time RSD of the characteristic peaks in 26 batches of Quisqualis indica was less than 3.0%.

[0241] 5.2 Verification results of 26 batches of Quisqualis indica slices, see... Figure 20-21 Tables 21-22. Figure 20 Superimposed image of characteristic features of Quisqualis indica slices; Figure 21 Superimposed diagram of the characteristics of Quisqualis indica slices.

[0242] Table 14 Relative retention time of Quisqualis indica slices

[0243]

[0244]

[0245] 5.3 Establishment of Limits for Relative Retention Time

[0246] Table 23 summarizes the methodology, the examination items, and the verification results.

[0247] Table 15 Summary of RSD% for Methodological Results — Relative Retention Time / Retention Time

[0248]

[0249] The relative retention times of each characteristic peak were stable and within ±5% of the average value. Therefore, the specified range of relative retention times for each peak was tentatively set at ±10%. Since the chromatographic conditions for the characteristic chromatogram of Quisqualis indica were the same as those for the standard decoction, the specified values ​​for the characteristic chromatogram of the medicinal material were consistent with those for the standard decoction.

[0250] The final specification stipulates that the chromatogram of the test sample should show 7 characteristic peaks, and the retention times should correspond to the 7 characteristic peaks in the chromatogram of the reference medicinal material. Peak 3 should correspond to the retention time of the reference peak. The peak corresponding to the ellagic acid reference peak is designated as the S peak. The relative retention times of each characteristic peak and the S peak should be calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are 0.40 (peak 1), 0.61 (peak 2), 1.09 (peak 4), 1.28 (peak 5), 1.45 (peak 6), and 1.72 (peak 7).

[0251] The chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version) was used to synthesize fingerprints of 26 batches of Quisqualis indica raw materials and processed slices, establishing reference chromatograms for the characteristic chromatograms of Quisqualis indica raw materials and processed slices. Among them, Figure 22-a Characteristic chromatogram of medicinal material: Peak 3 (S): ellagic acid; Peak 6: 3,3'-O-dimethylellagic acid; Column: Kromasil 100-5-C18 4.6×250mm 5μm; Figure 22-b Characteristic chromatogram of medicinal slices; Peak 3 (S): Ellagic acid; Peak 6: 3,3'-O-dimethylellagic acid; Column: Kromasil 100-5-C18 4.6×250mm 5μm

[0252] Conclusion: The characteristic chromatographic determination method for Quisqualis indica raw materials and processed slices can effectively detect Quisqualis indica raw materials and processed slices, proving that the method is feasible.

[0253] 6. Determination of the characteristic chromatograms of Quisqualis indica medicinal materials and processed slices

[0254] [Characteristic chromatogram] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0255] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol as mobile phase A and 0.3% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 360 nm. The theoretical plate number, calculated based on the ellagic acid peak, should be no less than 5000.

[0256]

[0257] Preparation of the reference solution: Take 5.0 g of Quisqualis indica reference material, place it in a stoppered conical flask, add 100 ml of water, decoct for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 10 ml of methanol to the residue, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, filter, and take the filtrate as the reference solution. Separately, accurately weigh an appropriate amount of ellagic acid reference standard, add methanol to prepare a solution containing 20 μg per ml, as the reference solution.

[0258] Preparation of the test solution: Take 5.0 g of the powder of this product (passed through a No. 3 sieve) and prepare the test solution together with the reference solution of the reference medicinal material.

[0259] The assay involves precisely pipetting 10 μL each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0260] The chromatogram of the test sample should show 7 characteristic peaks, and the retention times should correspond to the 7 characteristic peaks in the chromatogram of the reference medicinal material. Peak 3 should correspond to the retention time of the reference peak. The peak corresponding to the ellagic acid reference peak is designated as the S peak. The relative retention times of each characteristic peak and the S peak should be calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are 0.40 (peak 1), 0.61 (peak 2), 1.09 (peak 4), 1.28 (peak 5), 1.45 (peak 6), and 1.72 (peak 7).

[0261] Example 2: HPLC Characteristic Chromatography of Standard Decoction

[0262] 2.1 Expected Chromatographic Conditions

[0263] The column was packed with octadecylsilane-bonded silica gel (250 mm column length, 4.6 mm inner diameter, 5 μm particle size); methanol was used as mobile phase A, and 0.3% formic acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the flow rate was 1.0 mL / min; the column temperature was 30 °C; and the detection wavelength was 360 nm. The theoretical plate number, calculated based on the ellagic acid peak, should be no less than 5000.

[0264]

[0265] 2.2 Selection of mobile phase

[0266] Based on the experimental conditions outlined above, the separation effects of three different mobile phases were investigated: methanol-0.3% formic acid, acetonitrile-0.3% formic acid, and methanol-0.3% phosphoric acid. (See attached figures.) Figure 23-a . Figure 23-a Mobile phase selection

[0267] The results showed that the chromatographic peak baseline was relatively stable and there were many chromatographic peaks under the gradient elution condition of methanol-0.3% formic acid solution. Therefore, the gradient elution of methanol-0.3% formic acid solution was used as the mobile phase for the determination of the characteristic chromatogram of the standard decoction of Quisqualis indica.

[0268] 2.3 Wavelength Selection

[0269] Based on the above-specified experimental conditions, a diode array detector was used to perform a full-band scan of the test solution, and chromatograms of the test solution were extracted at wavelengths of 220 nm, 250 nm, 280 nm, 300 nm, 330 nm, and 360 nm, respectively. The results are shown in the figure. Figure 23-b . Figure 23-b Chromatograms of different wavelengths of Shijunzi Biao Tang.

[0270] The results showed that the number of characteristic peaks did not differ significantly at different detection wavelengths, while the chromatogram baseline was more stable at a detection wavelength of 360 nm. Therefore, the detection wavelength was determined to be 360 ​​nm.

[0271] 2.4 Column Temperature Investigation

[0272] Based on the above-established experimental conditions, the results were investigated at column temperatures of 25℃, 30℃, and 35℃. See Tables 16 and 17. Figure 24 . Figure 24 Column temperature investigation.

[0273] Table 16 Column Temperature Study - Retention Time

[0274]

[0275] Table 17 Column Temperature Study - Relative Retention Time

[0276]

[0277] The results showed that the peak shapes of all chromatographic peaks were good and the resolution was moderate at different column temperatures. Taking all factors into consideration, the column temperature was determined to be 30℃.

[0278] 2.5 Flow velocity investigation

[0279] Based on the above-established experimental conditions, the flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were investigated. See Tables 26-27. Figure 25 . Figure 25 Flow velocity study.

[0280] Table 18 Flow velocity study - retention time

[0281]

[0282] Table 19 Flow velocity study - relative retention time

[0283]

[0284] The results showed that the peak shapes of all chromatograms were good and the resolution was moderate at different flow rates. Taking all factors into consideration, a flow rate of 1.0 ml / min was tentatively set.

[0285] 2.6 Sample Injection Volume Investigation

[0286] Based on the above-established experimental conditions, the effects were investigated at injection volumes of 5 μl, 10 μl, and 15 μl. (See...) Figure 26 Tables 20-21.

[0287] Figure 26 Injection volume investigation

[0288] Table 20 Injection Volume and Retention Time

[0289]

[0290] Table 21 Injection Volume Analysis - Relative Retention Time

[0291]

[0292]

[0293] The results showed that when the injection volume was 10 μl, the chromatogram peak shape was good and the resolution was moderate. Therefore, the injection volume was determined to be 10 μl.

[0294] 2.7 Delayedness Assessment

[0295] Based on the above-specified experimental conditions, a delay test was conducted. The results are shown below. Figure 27 . Figure 27 Delayed examination

[0296] The results showed that the sample had virtually no chromatographic peaks after 75 minutes, so the sample detection time was set at 75 minutes.

[0297] 2.8 Determination of chromatographic conditions

[0298] In summary, the chromatographic conditions and system suitability test were as follows: Octadecylsilane-bonded silica gel was used as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol was used as mobile phase A, and 0.3% formic acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the flow rate was 1.0 mL / min; the column temperature was 30 °C; and the detection wavelength was 360 nm. The theoretical plate number, calculated based on the ellagic acid peak, should not be less than 5000.

[0299]

[0300] The determination method involves precisely pipetting 10 μL each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0301] 3. Preparation of the test solution

[0302] 3.1 Investigation of extraction solvent

[0303] Take 2.0 g of this product powder (batch number: SJZ-BT-0101), place it in a stoppered conical flask, and add 15 ml each of methanol, 70% methanol, 50% methanol, 30% methanol, ethanol, and water. Sonicate the mixture (600 W, 40 kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate. See [link to product description]. Figure 28 . Figure 28 Extraction solvent investigation

[0304] The results showed that the peak shapes of the characteristic peaks were best and the resolution was moderate when methanol was used as the solvent. Considering all factors, methanol was tentatively chosen as the extraction solvent.

[0305] 3.2 Examination of extraction time

[0306] Take 2.0 g of this product powder (batch number: SJZ-BT-0101), place it in a stoppered conical flask, add 15 ml of methanol, and sonicate (600 W, 40 kHz) for 20, 30, and 40 minutes respectively. Cool, shake well, filter, and collect the filtrate. See [link to product description]. Figure 29 . Figure 29 Extraction time was investigated; the results showed that different extraction times resulted in better peak shape and resolution of the chromatograms. To ensure more complete extraction, the extraction time was determined to be 30 minutes.

[0307] 3.3 Investigation of Solvent Addition Amount

[0308] Take 2.0 g of this product powder (batch number: SJZ-BT-0101), place it in a stoppered conical flask, add 15 ml, 20 ml, and 25 ml of methanol respectively, and sonicate (600 W power, 40 kHz frequency) for 30 minutes. Cool, shake well, filter, and collect the filtrate to obtain the product. See [link to product description]. Figure 30 . Figure 30 The sample volume was investigated; the results showed that when the solvent volume was 15 ml, the peak shape and resolution of each chromatographic peak were better, so the solvent volume of 15 ml was selected.

[0309] 3.4 Examination of Extraction Methods

[0310] Take 2.0 g of this product powder (batch number: SJZ-BT-0101), place it in a stoppered conical flask, add 15 ml of methanol, stopper tightly, reflux and sonicate (power 600W, frequency 40kHz) for 30 minutes respectively, cool, shake well, filter, and collect the filtrate to obtain the product. See Figure 31.

[0311] Figure 31 Extraction methods were investigated; the results showed that reflux extraction was more effective, therefore reflux extraction was selected as the extraction method for the test sample.

[0312] 3.4 Determination of the preparation method of the test sample

[0313] In summary, the preparation method of the test solution for the characteristic chromatogram of Shijunzi decoction is determined as follows: Take 2.0g of the powder, add 15ml of methanol, reflux for 30 minutes, cool, filter, and collect the filtrate to obtain the product.

[0314] 4. Methodological Investigation

[0315] 4.1 Chromatographic Peak Identification

[0316] Preparation of the test solution: Prepare the test solution of the standard decoction of Quisqualis indica according to the experimental conditions proposed above.

[0317] Preparation of reference solution: Take 5.0g of Quisqualis indica reference material, place it in a stoppered conical flask, add 100ml of water, decoct for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 10ml of methanol to the residue, sonicate (power 600W, frequency 40kHz) for 30 minutes, filter, and take the filtrate as the reference solution of the reference material.

[0318] Preparation of reference solutions: Take appropriate amounts of ellagic acid and 3,3'-O-dimethylellagic acid reference standards, weigh them accurately, and add methanol to prepare a solution containing 20 μg of each per ml, which is used as the reference solution.

[0319] Preparation of negative control solution: Prepare a negative control solution for the standard decoction of Quisqualis indica without the experimental conditions proposed above.

[0320] The characteristic peaks of the standard decoction of Quisqualis indica were located. See [link / reference needed]. Figure 32-36 .

[0321] Figure 32 Chromatographic peak identification; Figure 33 Ellagic acid spectrum - reference standard; Figure 34 3,3'-O-dimethylellagic acid - Spectrum - Reference Standard; Figure 35 Ellagic acid spectrum - standard soup; Figure 36 3,3'-O-dimethylellagic acid - spectral diagram - standard soup.

[0322] The results showed that peak 3 was ellagic acid and peak 6 was 3,3'-O-dimethylellagic acid. In the following methodological investigation, seven characteristic peaks in the sample were examined.

[0323] 4.2 Precision Test

[0324] Take the test solution of the standard decoction of Quisqualis indica (batch number: SJZ-BT-0101), and inject it 6 times consecutively according to the proposed experimental method, 10 μl each time. Calculate the retention time and peak area of ​​each characteristic peak. See Table 22.

[0325] Table 22 Precision Examination - Retention Time

[0326]

[0327] The results showed that the retention time RSD of each characteristic peak was 0.04% to 0.16%, indicating that the instrument has good precision.

[0328] 4.3 Repeatability Test

[0329] Accurately weigh 6 portions of the standard decoction of Quisqualis indica (batch number: SJZ-BT-0101), prepare and measure it according to the proposed experimental method, and calculate the relative retention time and relative peak area of ​​each characteristic peak. See Table 23.

[0330] Table 23 Repeatability Tests - Relative Retention Time

[0331]

[0332] The results showed that the relative retention time RSD of the six samples was 0–0.37%, indicating that the method had good reproducibility.

[0333] 4.4 Intermediate Precision Examination

[0334] 4.4.1 Investigation with different instruments

[0335] Based on the above-established experimental conditions, three portions of the standard decoction of Quisqualis indica (batch number: SJZ-BT-0101) were accurately weighed to prepare test solutions. These solutions were then analyzed using Waters, Agilent, and Thermo Fisher high-performance liquid chromatographs, respectively. The relative retention times and relative peak areas of each characteristic peak were calculated. See Table 24. Figure 37 . Figure 37 Different instruments

[0336] Table 24 Instrument Durability Test - Relative Retention Time

[0337]

[0338]

[0339] The results showed that when the test samples were tested using the three different instruments, the RSD of the relative retention time of each characteristic peak was 1.38% to 3.68%, indicating that the instruments had good durability.

[0340] 4.4.2 Intermediate Precision Examination

[0341] Based on the experimental conditions outlined above, six portions of the standard decoction of Quisqualis indica (batch number: SJZ-BT-0101) were precisely weighed by different personnel (A and B) at different times (T1 and T2) to prepare test samples. The relative retention times and relative peak areas of each characteristic peak were then measured and calculated. See Table 25.

[0342] Table 25 Intermediate Precision - Relative Retention Time

[0343]

[0344] The results show that when different personnel measure the same sample at different times, the RSD of the relative retention time of each characteristic peak is 0.86% to 3.76%, indicating that the method has good applicability.

[0345] 4.5 Investigation of different chromatographic columns

[0346] Based on the above-planned experimental conditions, the InertSustain column was used for each test. TM C18 4.6×250mm 5μm, 5TC-C18(2) 4.6×250mm 5μm, Kromasil 100-5-C18 4.6×250mm 5μm; were analyzed and investigated, and the relative retention time and relative peak area of ​​each characteristic peak were calculated. The results are shown in Table 26. Figure 38 . Figure 38 Investigation of different chromatographic columns.

[0347] Table 26 Column Robustness Study - Relative Retention Time

[0348]

[0349] The results showed that when the samples were detected using the above three chromatographic columns, the RSD of the relative retention time of the characteristic peaks ranged from 0.96% to 4.27%, indicating that the columns had good robustness.

[0350] 4.6 Stability Test

[0351] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 4h, 8h, 16h, and 24h. See Table 27.

[0352] Table 27 Stability Study - Relative Retention Time

[0353]

[0354]

[0355] The results showed that the RSD of the characteristic peak relative retention time was 0–0.37%, the RSD of the characteristic peak relative peak area was 2.56–11.21%, and the sample solution was stable within 24 hours.

[0356] In summary, the RSD of the relative retention times of each characteristic peak meets the requirements in all the above tests, indicating that the method is effective. The above seven characteristic peaks will be included in subsequent investigations.

[0357] 5. Validation of the standard decoction of Quisqualis indica and establishment of HPLC characteristic chromatographic method.

[0358] 5.1 Characteristic spectral analysis was performed on 26 batches of samples, and the relative retention time and relative peak area were calculated. Results are shown below. Figure 39-4 1. Table 28. Figure 39 Superimposed diagram of characteristic spectra of standard Quisqualis indica decoction; Figure 40 Superimposed diagram of characteristic spectra of standard Quisqualis indica decoction; Figure 41-a Superimposed chromatogram of the characteristics of the standard decoction of Quisqualis indica.

[0359] Table 28 Relative retention time of standard decoction of Quisqualis indica

[0360]

[0361]

[0362] Based on the principles of stable relative retention time, detectability in all batches of samples, and relatively high peak values, seven peaks with good robustness were selected as characteristic peaks. The results showed that the relative retention time RSD of the characteristic peaks in 26 batches of the Quisqualis indica standard decoction was less than 2.0%.

[0363] 5.2 Establishment of Limits for Relative Retention Time

[0364] Table 29 summarizes the methodological examination items and validation results:

[0365] Table 29 Summary of RSD% for Methodological Results—Relative Retention Time / Retention Time

[0366]

[0367] The relative retention times of each characteristic peak are stable and within ±10% of the average value. Therefore, the specified range of relative retention times for each peak is tentatively set at ±10%.

[0368] The final specification stipulates that the chromatogram of the test sample should show 7 characteristic peaks, and the retention times should correspond to the 7 characteristic peaks in the chromatogram of the reference medicinal material. Peak 3 should correspond to the retention time of the reference peak. The peak corresponding to the ellagic acid reference peak is designated as peak S. The relative retention times of the remaining characteristic peaks and peak S should be calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are 0.41 (peak 1), 0.61 (peak 2), 1.09 (peak 4), 1.27 (peak 5), 1.44 (peak 6), and 1.71 (peak 7).

[0369] The similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine (2012 version) was used to synthesize 26 batches of Quisqualis indica standard decoction, and a system was established. Figure 41-b Comparative chromatogram of the characteristic chromatogram of Quisqualis indica standard decoction. Peak 3(S): Ellagic acid; Peak 6: 3,3'-O-dimethylellagic acid; Column: Kromasil 100-5-C18 4.6×250mm 5μm; Conclusion: The characteristic chromatogram determination method for Quisqualis indica standard decoction can effectively detect the product, proving the feasibility of this method.

[0370] 6. Determination of the characteristic chromatogram method for the standard decoction of Quisqualis indica

[0371] [Characteristic chromatogram] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0372] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol as mobile phase A and 0.3% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 360 nm. The theoretical plate number, calculated based on the ellagic acid peak, should be no less than 5000.

[0373]

[0374] Preparation of the reference solution: Take 5.0 g of Quisqualis indica reference material, place it in a stoppered conical flask, add 100 ml of water, decoct for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 10 ml of methanol to the residue, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, filter, and take the filtrate as the reference solution. Separately, accurately weigh an appropriate amount of ellagic acid reference standard, add methanol to prepare a solution containing 20 μg per ml, as the reference solution.

[0375] Preparation of the test solution: Take 2.0 g of the powder and place it in a stoppered conical flask. Add 15 ml of methanol, reflux for 30 minutes, cool, filter, and collect the filtrate to obtain the test solution.

[0376] The assay involves precisely pipetting 10 μL each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0377] The chromatogram of the test sample should show 7 characteristic peaks, and the retention times should correspond to the 7 characteristic peaks in the chromatogram of the reference medicinal material. Peak 3 should correspond to the retention time of the reference peak. The peak corresponding to the ellagic acid 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 0.41 (peak 1), 0.61 (peak 2), 1.09 (peak 4), 1.27 (peak 5), 1.44 (peak 6), and 1.71 (peak 7).

[0378] Example 3: HPLC Characteristic Chromatography of Formulation Particles

[0379] Chromatographic conditions investigation

[0380] 2.1 Proposed chromatographic conditions

[0381] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol as mobile phase A and 0.3% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 360 nm. The theoretical plate number, calculated based on the ellagic acid peak, should be no less than 5000.

[0382]

[0383] 2.2 Selection of mobile phase

[0384] Based on the experimental conditions outlined above, the separation effects of three different mobile phases were investigated: methanol-0.3% formic acid, acetonitrile-0.3% formic acid, and methanol-0.3% phosphoric acid. (See attached figures.) Figure 42 . Figure 42 Mobile phase selection

[0385] The results showed that the chromatographic peak baseline was relatively stable and there were many chromatographic peaks under the gradient elution condition of methanol-0.3% formic acid solution. Therefore, the gradient elution of methanol-0.3% formic acid solution was used as the mobile phase for the determination of the characteristic chromatogram of Quisqualis indica formula granules.

[0386] 2.3 Wavelength Selection

[0387] Based on the above-specified experimental conditions, a diode array detector was used to perform a full-band scan of the test solution, and chromatograms of the test solution were extracted at wavelengths of 220 nm, 250 nm, 280 nm, 300 nm, 330 nm, and 360 nm. See [link to chromatogram]. Figure 43 . Figure 43Chromatograms of Quisqualis indica at different wavelengths

[0388] The results showed that the chromatogram baseline was more stable at a detection wavelength of 360 nm, therefore the detection wavelength was determined to be 360 ​​nm.

[0389] 2.4 Column Temperature Investigation

[0390] Based on the above-specified experimental conditions, investigations were conducted at column temperatures of 25℃, 30℃, and 35℃, respectively. (See attached figures.) Figure 44 Tables 30-31. Figure 44 Column temperature investigation.

[0391] Table 30 Column Temperature Study - Retention Time

[0392]

[0393] Table 31 Column Temperature Study - Relative Retention Time

[0394]

[0395] The results showed that the peak shapes of the chromatograms were better and the resolution was moderate when the column temperature was 30℃. Taking all factors into consideration, the column temperature was determined to be 30℃.

[0396] 2.5 Flow velocity investigation

[0397] Based on the above-specified experimental conditions, the flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were investigated respectively. (See attached figures.) Figure 45 Tables 32-33. Figure 45 Flow velocity investigation

[0398] Table 32 Flow velocity study - retention time

[0399]

[0400]

[0401] Table 33 Flow velocity study - relative retention time

[0402]

[0403] The results showed that a flow rate of 1.0 ml / min resulted in good peak shape and moderate resolution. Therefore, the flow rate was determined to be 1.0 ml / min.

[0404] 2.6 Sample Injection Volume Investigation

[0405] Based on the above-established experimental conditions, the effects were investigated at injection volumes of 5 μl, 10 μl, and 15 μl. (See...) Figure 46 Tables 34-35.

[0406] Figure 46 Injection volume investigation

[0407] Table 34 Injection Volume and Retention Time

[0408]

[0409] Table 35 Injection Volume Analysis - Relative Retention Time

[0410]

[0411] The results showed that when the injection volume was 10 μl, the chromatogram peak shape was good and the resolution was moderate. Therefore, the injection volume was determined to be 10 μl.

[0412] 2.7 Delayedness Assessment

[0413] Based on the above-specified experimental conditions, a delay test was conducted. The results are shown below. Figure 47 . Figure 47 Delayed investigation;

[0414] The results showed that the sample had virtually no chromatographic peaks after 75 minutes, so the sample detection time was set at 75 minutes.

[0415] 2.8 Determination of chromatographic conditions

[0416] In summary, the method for determining the content of Quisqualis indica in medicinal materials is as follows: using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); using methanol as mobile phase A and 0.3% formic acid solution as mobile phase B, gradient elution is performed according to the specifications in the table below; the flow rate is 1.0 ml per minute; the column temperature is 30℃; and the detection wavelength is 360 nm. The theoretical plate number, calculated based on the ellagic acid peak, should not be less than 5000.

[0417]

[0418] 3. Preparation of the test solution

[0419] 3.1 Investigation of extraction solvent

[0420] Take an appropriate amount of this product (batch number: SJZ-KL-0101), grind it into a fine powder, take 2.0g, place it in a stoppered conical flask, add 25ml each of water, 30% methanol, 50% methanol, 70% methanol, methanol, and ethanol, seal tightly, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the product. See Figure 48 . Figure 48 Investigation of extraction solvents;

[0421] The results showed that the peak shapes of the test sample were best and the resolution was moderate when methanol was used as the solvent. Considering all factors, methanol was tentatively chosen as the extraction solvent.

[0422] 3.2 Examination of extraction time

[0423] Take an appropriate amount of this product (batch number: SJZ-KL-0101), grind it into a fine powder, take 2.0g, place it in a stoppered conical flask, add 25ml of methanol, and sonicate (power 600W, frequency 40kHz) for 20 minutes, 30 minutes, and 40 minutes respectively. Cool, shake well, filter, and collect the filtrate to obtain the product. See Figure 49 . Figure 49 Extraction time consideration;

[0424] The results showed that the chromatogram peak shape and resolution were good at different extraction times. In order to make the extraction more complete, the extraction time was determined to be 30 minutes.

[0425] 3.3 Examination of Extraction Methods

[0426] Take an appropriate amount of this product (batch number: SJZ-KL-0101), grind it into a fine powder, take 2.0g, place it in a stoppered conical flask, add 25ml of 70% methanol, reflux and sonicate (power 600W, frequency 40kHz) for 30 minutes respectively, cool, shake well, filter, and collect the filtrate to obtain the product. See Figure 50 . Figure 50 Extraction method investigation; the results showed that the sample had good peak shape and moderate separation under ultrasonic extraction, so ultrasonic extraction was tentatively selected as the extraction method.

[0427] 3.4 Investigation of Solvent Addition Amount

[0428] Take an appropriate amount of this product (batch number: SJZ-KL-0101), grind it into a fine powder, take 2.0g, add 15ml, 20ml, and 25ml of methanol respectively, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the final product. See [link to product description]. Figure 51 . Figure 51 The solvent addition amount was investigated; the results showed that when the solvent addition amount was 25 ml, the peak shape and resolution of each chromatographic peak were better, so the solvent addition amount of 25 ml was selected.

[0429] 3.5 Determination of Test Sample Method

[0430] In summary, the preparation method of the test solution of the characteristic chromatogram of Quisqualis indica formula granules is determined as follows: Take an appropriate amount of this product, grind it into a fine powder, take 2.0 g, place it in a stoppered conical flask, add 25 ml of methanol, seal tightly, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool, filter, and take the filtrate to obtain the product.

[0431] 4. Methodological Investigation

[0432] 4.1 Specificity Examination

[0433] Preparation of the test solution: Prepare the test solution of Quisqualis indica formula granules according to the experimental conditions proposed above.

[0434] Preparation of negative control solution: Prepare a negative control solution for the formula granules of Quisqualis indica without the experimental conditions proposed above.

[0435] See results Figure 52 . Figure 52 Specificity investigation showed that negative results did not interfere with the specificity of the granule characteristic spectrum method of Quisqualis indica formula.

[0436] 4.2 Chromatographic Peak Identification

[0437] Preparation of the test solution: Prepare the test solution of Quisqualis indica formula granules according to the experimental conditions proposed above.

[0438] Preparation of negative control solution: Prepare a negative control solution for the formula granules of Quisqualis indica without the experimental conditions proposed above.

[0439] Preparation of reference solution for control medicinal material: Take 5.0g of Quisqualis indica reference material, place it in a stoppered conical flask, add 100ml of water, decoct for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 10ml of methanol to the residue, sonicate (power 600W, frequency 40kHz) for 30 minutes, filter, and take the filtrate as the reference solution for control medicinal material.

[0440] Preparation of reference solutions: Take appropriate amounts of ellagic acid and 3,3'-O-dimethylellagic acid reference standards, accurately weigh them, and add methanol to prepare solutions containing 20 μg per ml, which are used as reference solutions.

[0441] The characteristic peaks of the Quisqualis indica formula granules were located. See [link / reference needed]. Figures 53-57 . Figure 53 Chromatographic peak identification; Figure 54 Ellagic acid spectrum - reference standard; Figure 55 3,3'-O-dimethylellagic acid - Spectrum - Reference Standard; Figure 56 Ellagic acid spectrum - particles; Figure 57 3,3'-O-dimethylellagic acid - Spectrum - Particles; The results showed that peak 3 was ellagic acid and peak 6 was 3,3'-O-dimethylellagic acid. In the following methodological investigation, seven characteristic peaks in the sample were examined.

[0442] 4.3 Precision Test

[0443] Take the test solution of Quisqualis indica granules (batch number: SJZ-KL-0101), and inject it 6 times consecutively according to the proposed experimental method, 10 μl each time. Calculate the retention time and peak area of ​​each characteristic peak. See Table 36.

[0444] Table 36 Precision Examination - Retention Time

[0445]

[0446]

[0447] The results showed that the retention time RSD of each characteristic peak of the sample was 0.04% to 0.13%, indicating that the instrument had good precision.

[0448] 4.4 Repeatability Test

[0449] Six portions of the Quisqualis indica formula granules (batch number: SJZ-KL-0101) were prepared and tested according to the proposed experimental method. See Table 37.

[0450] Table 37 Repeatability Tests - Relative Retention Time

[0451]

[0452] The results showed that the relative retention time RSD of the six samples was 0.00%–0.38%, indicating that the method has good reproducibility.

[0453] 4.5 Intermediate Precision Examination

[0454] 4.5.1 Investigation with different instruments

[0455] Based on the above-established experimental conditions, three portions of the Quisqualis indica formula granules (batch number: SJZ-KL-0101) were accurately weighed to prepare test solutions. These solutions were then analyzed using Waters, Agilent 1260, and Thermo Fisher high-performance liquid chromatographs, respectively. See Table 38. Figure 58 .

[0456] Table 38 Instrument Durability Test - Relative Retention Time

[0457]

[0458] Figure 58 Different instruments were used; the results showed that when the test sample was detected by the above three instruments, the RSD range of the relative retention time of each characteristic peak was 1.04%-3.95%, indicating that the instruments had good durability.

[0459] 4.5.2 Investigations by different personnel and at different times

[0460] Based on the experimental conditions outlined above, six portions of the Quisqualis indica formula granules (batch number: SJZ-KL-0101) were precisely weighed by different personnel (A and B) at different times (T1 and T2) to prepare test samples. The relative retention times and relative peak areas of each characteristic peak were then measured and calculated. See Table 39.

[0461] Table 39 Intermediate Precision - Relative Retention Time

[0462]

[0463]

[0464] The results show that when different personnel measure the same sample at different times, the RSD of the relative retention time of each characteristic peak is 0.86% to 3.81%, indicating that the method has good applicability.

[0465] 4.6 Investigation of different chromatographic columns

[0466] Based on the above-planned experimental conditions, the InertSustain column was used for each test. TM C18 4.6×250mm 5μm, 5TC-C18(2) 4.6×250mm 5μm, and Kromasil 100-5-C18 4.6×250mm 5μm were analyzed and investigated, and the relative retention time and relative peak area of ​​each characteristic peak were calculated. The results are shown in Table 40. Figure 59 . Figure 59 Investigation of different chromatographic columns.

[0467] Table 40 Column robustness study - relative retention time

[0468]

[0469] The results showed that when the samples were detected using the above three chromatographic columns, the RSD of the relative retention time of the characteristic peaks ranged from 0.91% to 4.27%, indicating that the columns had good robustness.

[0470] 4.7 Stability Test

[0471] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 4h, 8h, 12h, and 24h. See Table 41.

[0472] Table 41 Stability Study - Relative Retention Time

[0473]

[0474] The results showed that the RSD of the characteristic peak relative retention time was 0.00%–1.00%, the RSD of the characteristic peak relative peak area was 5.21%–8.57%, and the sample solution was stable within 24 hours.

[0475] In summary, the RSD of the relative retention times of each characteristic peak meets the requirements in all the above tests, indicating that the method is effective. The aforementioned seven characteristic peaks will be included in subsequent investigations.

[0476] 5. Validation of Quisqualis indica granules and establishment of HPLC characteristic chromatographic method

[0477] 5.1 Validation results of 3 batches of Quisqualis indica formula granules, see... Figure 60 Table 42. Figure 60 Superimposed image of the characteristic spectrum of Quisqualis indica granules

[0478] Table 42 Relative retention time of Quisqualis indica granules

[0479]

[0480]

[0481] Based on the principles of relatively stable retention time, detectability in all batches of samples, and relatively high peak values, seven robust peaks were selected as characteristic peaks. Based on the methodological investigation results and the particle validation results of three batches, the theoretical plate number, calculated using the ellagic acid peak, is tentatively set to be no less than 5000.

[0482] 5.2 Establishment of Limits for Relative Retention Time

[0483] Table 43 Summary of Methodological Examination Items and Validation Results

[0484]

[0485] The relative retention times of each characteristic peak were stable, with the RSD% of the relative retention time within the range of 10%. Therefore, the specified range of the relative retention time for each peak was tentatively set at ±10%. Since the chromatographic conditions of the characteristic chromatograms of the Quisqualis indica granules and the standard decoction were the same, the specified values ​​of the characteristic chromatograms of the Quisqualis indica granules were consistent with those of the standard decoction.

[0486] The final specification stipulates that the chromatogram of the test sample should show 7 characteristic peaks, and the retention times should correspond to the 7 characteristic peaks in the chromatogram of the reference medicinal material. Peak 3 should correspond to the retention time of the reference peak. The peak corresponding to the ellagic acid reference peak is designated as peak S. The relative retention times of the remaining characteristic peaks and peak S should be calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are 0.41 (peak 1), 0.61 (peak 2), 1.09 (peak 4), 1.27 (peak 5), 1.44 (peak 6), and 1.71 (peak 7).

[0487] The similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine (2012 version) was used to synthesize three batches of Quisqualis indica formula granules, and a similarity model was established. Figure 61 Comparative chromatograms of the characteristic chromatograms of Quisqualis indica formula granules. Peak 3(S): Ellagic acid; Peak 6: 3,3'-O-dimethylellagic acid; Column: Kromasil 100-5-C18 4.6×250mm 5μm; Conclusion: The characteristic chromatogram determination method for Quisqualis indica formula granules can effectively detect these granules, proving the feasibility of this method.

[0488] 7. Determination of the characteristic chromatogram method for Quisqualis indica formula granules

[0489] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol as mobile phase A and 0.3% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 360 nm. The theoretical plate number, calculated based on the ellagic acid peak, should be no less than 5000.

[0490]

[0491] Preparation of the reference solution: Take 5.0 g of Quisqualis indica reference material, place it in a stoppered conical flask, add 100 ml of water, decoct for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 10 ml of methanol to the residue, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, filter, and take the filtrate as the reference solution. Separately, accurately weigh an appropriate amount of ellagic acid reference standard, add methanol to prepare a solution containing 20 μg per ml, as the reference solution.

[0492] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take 2.0 g, place it in a stoppered conical flask, add 25 ml of methanol, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0493] The determination method involves precisely pipetting 10 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0494] Example 4: Comparison of HPLC characteristic chromatograms of Quisqualis indica and Terminalia chebula

[0495] 1. Experimental materials

[0496] Quisqualis indica medicinal materials: SJZ-YC-0101, SJZ-YC-0102, SJZ-YC-0103;

[0497] Quisqualis indica decoction: SJZ-BT-0101, SJZ-BT-0102, SJZ-BT-0103;

[0498] Quisqualis indica granules: SJZ-KL-0101, SJZ-KL-0102, SJZ-KL-0103;

[0499] Terminalia chebula medicinal materials: HZ-YC-0101, HZ-YC-0102, HZ-YC-0103;

[0500] Terminalia chebula decoction: HZ-BT-0101, HZ-BT-0102, HZ-BT-0103;

[0501] Terminalia chebula granules: HZ-KL-0101, HZ-KL-0102, HZ-KL-0103;

[0502] 2. Methods for determining the characteristic spectra of medicinal materials

[0503] 2.1 Methods for determining the characteristic spectra of medicinal materials

[0504] [Characteristic chromatogram] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0505] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol as mobile phase A and 0.3% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 360 nm. The theoretical plate number, calculated based on the ellagic acid peak, should be no less than 5000.

[0506]

[0507] Preparation of the test solution: Take 5.0 g each of the powdered herbs of Quisqualis indica and Terminalia chebula (passed through a No. 3 sieve), place them in stoppered conical flasks, add 100 ml of water, decoct for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 10 ml of methanol to the residue, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, filter, and take the filtrate as the test solution.

[0508] The assay involves precisely pipetting 10 μL each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0509] 2.2 Results of the determination of the characteristic spectra of medicinal materials; Figure 62 Comparison of the characteristic pictographs of Quisqualis indica and Terminalia chebula

[0510] The results showed that there were seven characteristic peaks in the Quisqualis indica herb. Taking peak 3 (the peak corresponding to the ellagic acid reference standard) as the S peak, the relative retention times of the remaining characteristic peaks and the S peak were calculated. The relative retention times of peak 1 and peak 2 were 0.41, peak 2 was 0.62, peak 4 was 1.09, peak 5 was 1.27, peak 6 was 1.44, and peak 7 was 1.71. However, peak 4 was not detected in Terminalia chebula at a relative retention time of 1.09, peak 6 was not detected at 1.44, and peak 7 was not detected at 1.71. Therefore, peaks 4, 6, and 7 can be used as identification points for Quisqualis indica herb and its adulterant Terminalia chebula herb.

[0511] Method for determining the characteristic spectrum of 3 standard decoction

[0512] 3.1 Method for determining the characteristic chromatogram of standard soup

[0513] [Characteristic chromatogram] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0514] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol as mobile phase A and 0.3% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 360 nm. The theoretical plate number, calculated based on the ellagic acid peak, should be no less than 5000.

[0515]

[0516] Preparation of the test solution: Take 2.0 g each of the standard decoction powder of Quisqualis indica and Terminalia chebula, place them in stoppered conical flasks, add 15 ml of methanol, reflux for 30 minutes, cool, filter, and take the filtrate to prepare the test solution.

[0517] The assay involves precisely pipetting 10 μL each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0518] 3.1. Results of the determination of the characteristic spectrum of the standard soup

[0519] Figure 63A comparison of the characteristic spectra of Quisqualis indica and Terminalia chebula decoction revealed that Quisqualis indica has seven characteristic peaks. Using peak 3 (the peak corresponding to the ellagic acid reference standard) as the S peak, the relative retention times of the remaining characteristic peaks and the S peak were calculated. The relative retention times of peak 1 were 0.41, peak 2 were 0.62, peak 4 were 1.09, peak 5 were 1.27, peak 6 were 1.44, and peak 7 were 1.71. Terminalia chebula showed no detectable peak 4 at a relative retention time of 1.09, no detectable peak 6 at 1.44, and no detectable peak 7 at 1.71. Therefore, peaks 4, 6, and 7 can be used as distinguishing features between Quisqualis indica decoction and its adulterant Terminalia chebula decoction.

[0520] 4. Methods for determining particle characteristic spectra

[0521] 4.1 Method for determining particle characteristic spectra

[0522] [Characteristic chromatogram] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0523] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol as mobile phase A and 0.3% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 360 nm. The theoretical plate number, calculated based on the ellagic acid peak, should be no less than 5000.

[0524]

[0525] Preparation of the test solution: Take appropriate amounts of Quisqualis indica and Terminalia chebula granules, grind them into a fine powder, take 2.0 g of each, place them in stoppered conical flasks, add 25 ml of methanol, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0526] The assay involves precisely pipetting 10 μL each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0527] 2. Results of Characteristic Spectrum Determination of Medicinal Materials

[0528] Figure 64A comparison of the characteristic spectra of Quisqualis indica and Terminalia chebula granules revealed that Quisqualis indica has seven characteristic peaks. Using peak 3 (the peak corresponding to the ellagic acid reference standard) as the S peak, the relative retention times of the remaining characteristic peaks and the S peak were calculated. The relative retention times of peak 1 were 0.41, peak 2 were 0.62, peak 4 were 1.09, peak 5 were 1.27, peak 6 were 1.44, and peak 7 were 1.71. Terminalia chebula showed no detectable peak 4 at a relative retention time of 1.09, no detectable peak 6 at 1.44, and no detectable peak 7 at 1.71. Therefore, peaks 4, 6, and 7 can be used as identification points for Quisqualis indica granules and their adulterant Terminalia chebula granules.

[0529] Comparative Example 1

[0530] 2.1 Comparison of different mobile phases, the only difference being: methanol-0.2% phosphoric acid aqueous solution and methanol-0.3% formic acid; gradient elution was performed according to the specifications in the table below; the flow rate was 1.0 ml / min; the column temperature was 30℃; and the detection wavelength was 360 nm. The theoretical plate number, calculated based on the ellagic acid peak, should not be less than 5000.

[0531]

[0532] 2.2 Preparation of the test solution: Take an appropriate amount of Quisqualis indica granules, grind them into a fine powder, take 2.0 g, place them in a stoppered conical flask, add 25 ml of methanol, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0533] The assay involves precisely pipetting 10 μL each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0534] 2.3 Test results, such as Figure 65 The above, Figure 65 The study investigated different mobile phases. The results showed that the resolution of peak 4 was poor when methanol-0.2% phosphoric acid aqueous solution was used as the mobile phase.

[0535] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for identifying the characteristic chromatograms of Quisqualis indica and its adulterant Terminalia chebula, comprising: The raw sample was pretreated to obtain the test solution; The pretreatment method includes boiling in water, cooling, filtering, evaporating the filtrate to dryness, and ultrasonically treating the residue with methanol. The test solution was analyzed by HPLC to obtain the characteristic HPLC spectra of Quisqualis indica raw material, processed slices, standard decoction, and formulated granules; the preparation of reference solutions of control medicinal materials was also included. The reference herb, Quisqualis indica, was decocted in water, cooled, filtered, and the filtrate was evaporated to dryness. The residue was treated with methanol by sonication, filtered, and the filtrate was used as the reference herb solution. Preparation of reference solution: Ellagic acid and 3,3'-O-dimethylellagic acid were dissolved in solvent to obtain reference solution; The HPLC conditions were as follows: C18 column; mobile phase A was methanol solution, mobile phase B was 0.3% formic acid aqueous solution, gradient elution; detection wavelength was 360 nm. The gradient elution specifically refers to: 0–10 min, Phase A: 20% → 30%, Phase B: 80% → 70%; 10–30 min, Phase A: 30% → 40%, Phase B: 70% → 60%; 30–45 min, Phase A: 40% → 52%, Phase B: 60% → 48%; 45–50 min, Phase A: 52%, Phase B: 48%; 50–55 min, Phase A: 52% → 55%, Phase B: 48% → 45%; 55–75 min, Phase A: 55% → 70%, Phase B: 45% → 30%; The reference standard solution and the reference medicinal material solution were injected into a high-performance liquid chromatograph for analysis, and the chromatograms of the reference standard and the characteristic chromatograms of the reference medicinal material were obtained respectively. Based on the characteristic parameters of the chromatograms of the reference standard, the corresponding chromatograms in the HPLC characteristic chromatograms of the Quisqualis indica test sample, the standard decoction test sample, and the formula granule test sample were compared and identified. Peak 4 was not detected at a relative retention time of 1.09, peak 6 was not detected at a relative retention time of 1.44, and peak 7 was not detected at a relative retention time of 1.

71. Therefore, peaks 4, 6, and 7 are used as identification points for Quisqualis indica and its adulterant, Terminalia chebula.

2. The method according to claim 1, characterized in that, The specific injection concentrations of the reference solution were: 20 μg / mL of ellagic acid and 20 μg / mL of 3,3'-O-dimethylellagic acid.

3. The method according to claim 1, characterized in that, The chromatographic column has the specifications of 250×4.6mm 5μm; the column temperature is 30℃; and the theoretical plate number, calculated based on ellagic acid, should not be less than 5000.

4. The method according to claim 3, characterized in that, The flow rate of the mobile phase was 1.0 mL / min; the injection volume was 10 μL.

5. The method according to claim 3, characterized in that, The decocting time in step A) is 30 minutes; the ultrasonic power is 600W, the frequency is 40kHz, and the extraction time is 30 minutes.

6. The method according to claim 1, characterized in that, The similarity of HPLC characteristic chromatograms of Quisqualis indica raw material, processed slices, standard decoction, and formula granules was evaluated using a chromatographic fingerprint similarity evaluation system for traditional Chinese medicine. A standard HPLC characteristic chromatogram consisting of 7 characteristic peaks was constructed, and the chemical composition information corresponding to each characteristic peak is as follows: Peak 3 (S): ellagic acid; Peak 6: 3,3'-O-dimethylellagic acid; In the characteristic chromatograms of the Quisqualis indica medicinal material, processed slices, standard decoctions, and formula granules, ellagic acid is used as the reference peak S. The relative retention time of each characteristic peak and the S peak is calculated. The allowable fluctuation range of the relative retention time of each characteristic peak is within ±10% of the specified value. The specified values ​​are: 0.40 (peak 1), 0.61 (peak 2), 1.09 (peak 4), 1.27 (peak 5), 1.44 (peak 6), and 1.71 (peak 7).

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