A method for constructing a characteristic chromatogram of commelina communis and preparations thereof

Abstract

The application belongs to the field of traditional Chinese medicine quality detection, and specifically discloses a method for constructing a characteristic chromatogram of tradescantia fluminensis and preparations thereof, which comprises the following steps: (1) preparing a test sample solution; and (2) detecting the test sample solution by high performance liquid chromatography, using octadecylsilane bonded silica gel as a filler, using methanol as a mobile phase A, using water or an acid-containing aqueous solution as a mobile phase B, and through an optimized gradient elution program; finally, a plurality of common characteristic peaks are obtained, effective separation of the common characteristic peaks is realized, the characteristic chromatogram obtained has good peak shape and a stable baseline, the detection time is short, and the peak height or peak area is uniform.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine detection technology, specifically relating to a method for constructing characteristic spectra of Commelina communis and its preparations. Background Technology

[0002] The raw material for Commelina communis formula granules is the dried aerial parts of Commelina communis L., a plant in the Commelinaceae family. Commelina communis is sweet, bland, and cold in nature. It enters the lung, stomach, and small intestine meridians. It clears heat and drains fire, detoxifies, and promotes diuresis and reduces swelling. It is used for colds with fever, thirst due to febrile diseases, sore throat, edema with scanty urine, painful urination due to heat, and carbuncles and boils.

[0003] The main chemical components of Commelina communis are flavonoids and phenolic acids. Commelina communis is a traditional Chinese medicine listed in the current pharmacopoeia. Literature also describes the chemical components of Commelina communis; however, on the one hand, determining or identifying the content of a single component in Commelina communis and its preparations (e.g., granules) cannot comprehensively detect and control its quality; on the other hand, determining the content of a single component in conjunction with the identification of other components in Commelina communis and its preparations is time-consuming and labor-intensive, making it difficult to widely apply in production practice. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide a characteristic spectrum of Commelina communis and its preparations and a method for establishing it. This method establishes a characteristic spectrum of the variety based on the characteristics of the preparations, and achieves effective separation of each characteristic peak, providing a scientific basis for comprehensively establishing quality control standards for Commelina communis and its preparations (e.g., formulation granules).

[0005] Specifically, this invention discloses a method for constructing a characteristic spectrum of Commelina communis and its preparations, comprising the following steps:

[0006] (1) Preparation of the test solution;

[0007] (2) The test solution was analyzed by high performance liquid chromatography, with octadecylsilane bonded silica gel as the stationary phase, methanol as the mobile phase A, and water or acid-containing aqueous solution as the mobile phase B. The gradient elution program is as follows:

[0008] For 0-5 min, the volume ratio of mobile phase A to mobile phase B is 5%:95%;

[0009] From 5 to 30 minutes, the volume ratio of mobile phase A to mobile phase B changed from 5%:95% to 27%:73%.

[0010] Over 30-38 minutes, the volume ratio of mobile phase A to mobile phase B changed from 27%:73% to 30%:70%.

[0011] The time was 38-55 min, and the volume ratio of mobile phase A to mobile phase B was 30%:70%.

[0012] In some preferred embodiments, step (2) further satisfies at least one of the following 1)-4):

[0013] 1) The detection wavelength is 230-360nm, the flow rate is 0.25-0.35mL / min, and the column temperature is 25-35℃;

[0014] 2) The acid in the acid-containing aqueous solution is selected from one or more of formic acid, phosphoric acid, and acetic acid;

[0015] 3) The acid concentration in the acid-containing aqueous solution is 0.05%-0.2%;

[0016] 4) A chromatographic column with a length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.8-1.9 μm is used in the detection process.

[0017] In this invention, Commelina communis and its preparations can refer to Commelina communis or preparations made from Commelina communis, such as powders, granules, tablets, etc.

[0018] In some preferred embodiments, step (1) includes weighing the Commelina communis test sample, adding solvent to extract it, obtaining the extract, separating the solid and liquid, and taking the liquid, which is the test sample solution;

[0019] In some preferred embodiments, step (1) further satisfies any one or more of the following AEs:

[0020] A. The mass ratio of the test sample of Commelina communis to the volume of the solvent is 0.1-0.3:20-40; the mass-volume relationship is g / mL.

[0021] B. The extraction method is either reflux extraction or ultrasonic extraction;

[0022] C. The extraction time is ≥20 min, preferably 30-60 min;

[0023] D. The solid-liquid separation is selected from centrifugation or membrane filtration;

[0024] E. The solvent is selected from a methanol aqueous solution with a volume percentage of 40-80%.

[0025] In some preferred embodiments, the construction method further includes the step of preparing a reference solution by adding a solvent to at least one of vanillic acid reference standard, scutellarin reference standard, isoscutellarin reference standard, vitexin reference standard and rutin reference standard, and the step of detecting the reference solution by high performance liquid chromatography according to any of the construction methods of the present invention to obtain a reference chromatogram.

[0026] Preferably, each 1 mL of the reference solution contains 5-50 μg of each reference standard; and / or, the solvent used in the preparation of the reference solution is selected from methanol or an aqueous methanol solution.

[0027] In certain preferred embodiments, the characteristic chromatogram of Commelina communis and / or its preparations has nine common characteristic peaks, wherein peak 7 corresponds to the retention time of the isoharmonic acid reference standard peak; the peak corresponding to the isoharmonic acid reference standard peak is peak S, and the relative retention times of the remaining characteristic peaks and peak S are within ±10% of a specified value; the specified values ​​are: 0.32 (peak 1), 0.51 (peak 2), 0.60 (peak 3), 0.67 (peak 4), 0.91 (peak 5), 0.95 (peak 6), 1.04 (peak 8), and 1.23 (peak 9).

[0028] In some preferred embodiments, the method further includes constructing a reference characteristic chromatogram of Commelina communis formula granules. This involves generating a reference characteristic chromatogram of Commelina communis formula granules using a similarity evaluation system for chromatographic characteristic chromatograms of traditional Chinese medicine, based on the characteristic chromatograms obtained from multiple batches of Commelina communis formula granules. At least two batches of Commelina communis formula granules are used, for example, four, seven, eight, or fifteen batches.

[0029] In some preferred embodiments, after generating the control characteristic spectrum of the Commelina communis formula granules using the software for evaluating the similarity of chromatographic characteristic spectra of traditional Chinese medicine, the method further includes the step of marking common characteristic peaks.

[0030] The similarity of the characteristic chromatograms of the Commelina communis formula granules is greater than 0.90.

[0031] This invention also provides a method for determining the content of active ingredients in Commelina communis and its preparations, comprising the following steps:

[0032] (1) Preparation of test solution and reference solution; the method for preparing the reference solution includes: preparing the reference solution by adding a solvent to at least one of vanillic acid reference standard, scutellarin reference standard, isoscutellarin reference standard, vitexin reference standard and rutin reference standard;

[0033] (2) The test solution and the reference solution were detected by high performance liquid chromatography. Octadecylsilane bonded silica gel was used as the stationary phase, methanol was used as the mobile phase A, and water or acidic aqueous solution was used as the mobile phase B. The gradient elution program was as follows.

[0034] For 0-5 min, the volume ratio of mobile phase A to mobile phase B is 5%:95%;

[0035] 5-30 min, the volume ratio of mobile phase A to mobile phase B is 5%:95% → 27%:73%;

[0036] Over 30-38 minutes, the volume ratio of mobile phase A to mobile phase B changed from 27%:73% to 30%:70%.

[0037] 38-55 min, the volume ratio of mobile phase A to mobile phase B is 30%:70%;

[0038] (3) The content of reference standard contained in the test solution was calculated using the external standard method.

[0039] In some preferred embodiments, step (2) further satisfies at least one of the following 1)-4):

[0040] 1) The detection wavelength is 230-360nm, the flow rate is 0.25-0.35mL / min, and the column temperature is 25-35℃;

[0041] 2) The acid in the acid-containing aqueous solution is selected from one or more of formic acid, phosphoric acid, and acetic acid;

[0042] 3) The acid concentration in the acid-containing aqueous solution is 0.05%-0.2%;

[0043] 4) A chromatographic column with a length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.8-1.9 μm is used in the detection process.

[0044] In some preferred embodiments, step (1) includes weighing the *Commelina communis* sample, extracting it with a solvent to obtain an extract, separating the solid and liquid components, and taking the liquid as the sample solution; preferably, step (1) also satisfies any one or more of the following A:

[0045] A. The mass ratio of the test sample of Commelina communis to the volume of the solvent is 0.1-0.3:20-40; the mass-volume relationship is g / mL.

[0046] B. The extraction method is either reflux extraction or ultrasonic extraction;

[0047] C. The extraction time is ≥20 min, preferably 30-60 min;

[0048] D. The solid-liquid separation is selected from centrifugation or membrane filtration;

[0049] E. The solvent is selected from a methanol aqueous solution with a volume percentage of 40-80%.

[0050] In some preferred embodiments, each 1 mL of the reference solution contains 5-50 μg of each reference standard; and / or, the solvent used in the preparation of the reference solution is selected from methanol or an aqueous methanol solution.

[0051] The present invention also provides a quality detection method for Commelina communis and its preparations, including a method for constructing characteristic spectra of Commelina communis and its preparations as described above and / or a method for determining the content of active ingredients in Commelina communis and its preparations as described above.

[0052] Specifically, the characteristic spectrum of the Commelina communis product to be tested is constructed according to any of the above-described methods for constructing characteristic spectra of Commelina communis and its preparations, and / or the content of effective components in the Commelina communis product is determined according to any of the above-described methods for determining the content of effective components in Commelina communis and its preparations.

[0053] The product to be tested can be dayflower or a preparation made from dayflower, such as powder, granules, tablets, etc.

[0054] For example, when the product is a Commelina communis formula granule, the quality of the Commelina communis formula granule product is evaluated by chromatographic comparison. When the characteristic chromatogram of the Commelina communis formula granule product has at least 9 common peaks, peak 7 should correspond to the retention time of the isoharmonic acid reference peak; the peak corresponding to the isoharmonic acid reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.32 (peak 1), 0.51 (peak 2), 0.60 (peak 3), 0.67 (peak 4), 0.91 (peak 5), 0.95 (peak 6), 1.04 (peak 8), and 1.23 (peak 9), indicating that the quality is qualified; otherwise, the quality is unqualified.

[0055] The quality of the tested Commelina communis formula granules is evaluated using similarity. If the similarity between the characteristic chromatogram of the tested Commelina communis formula granules and the control characteristic chromatogram of Commelina communis formula granules is not lower than 0.90-1.00 (e.g., 0.9), the quality is qualified; if it is lower than 0.90-1.00 (e.g., 0.9), it is unqualified. Specifically, the similarity is obtained using software for evaluating the similarity of chromatographic characteristic chromatograms of traditional Chinese medicine.

[0056] In this invention, the terms "commelina communis and its preparations" or "commelina communis and / or its preparations" refer to one or more of the following: Commelina communis and its preparations. Commelina communis preparations are preparations made from Commelina communis using conventional processes, especially powders, granules, tablets, etc., obtained from Commelina communis water extract using conventional processes.

[0057] In this invention, vt% represents volume percentage. vt% methanol or % methanol both refer to the volume percentage of methanol in a methanol-water solution, and vt% ethanol or % ethanol refer to the volume percentage of ethanol in an ethanol-water solution. % formic acid is the volume percentage of formic acid in a formic acid-water solution; % acetic acid is the volume percentage of acetic acid in an acetic acid-water solution.

[0058] The technical solution of this invention has the following advantages:

[0059] 1. The method for constructing characteristic spectra of Commelina communis and its preparations according to the present invention uses octadecylsilane-bonded silica gel as a filler, methanol as mobile phase A, and water or an acidic aqueous solution as mobile phase B, through a preferred gradient elution procedure; finally, multiple common characteristic peaks are obtained, and the effective separation of multiple common characteristic peaks is achieved. Moreover, the obtained characteristic spectra have good peak shapes, stable baselines, short detection times, and uniform peak heights or peak areas, providing a scientific basis for comprehensively establishing quality control standards for Commelina communis formula granules. Furthermore, it can accurately locate the peak positions of vanillic acid, arbutin, isoarbutin, vitexin, and rutin, fully reflecting the integrity and characteristics of Commelina communis and its preparations (such as formula granules).

[0060] The detection method provided by this invention has good separation and can simultaneously determine the content of isopropargyl glycoside. Moreover, the detection method has high precision, good stability and repeatability. Therefore, it can comprehensively and quickly detect the active ingredients and their content in Commelina communis formula granules.

[0061] The S-peak isoharonin can be selected as the internal reference peak in the characteristic spectrum, and nine common characteristic peaks of Commelina communis formula granules can be identified. The relative retention time of each common characteristic peak can be calculated based on the S-peak isoharonin, which is beneficial for comprehensive quality testing and overall quality control of Commelina communis formula granules, thereby helping to improve the safety and stability of the drug.

[0062] 2. The method for constructing the characteristic chromatograms of Commelina communis and its preparations described in this invention involves optimizing chromatographic conditions, the type and amount of extraction solvent, extraction time, and other extraction conditions to determine the optimal extraction process and chromatographic conditions. This results in higher peak areas, better separation effects, and more comprehensive quality monitoring of Commelina communis formula granules. Attached Figure Description

[0063] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0064] Figure 1 This is the chromatogram of the mixed reference standards in Example 1;

[0065] Figure 2 The characteristic spectrum of the Commelina communis formula granules in Example 1;

[0066] Figure 3This is the chromatogram under gradient condition 1 in Experiment Example 1;

[0067] Figure 4 This is the chromatogram under gradient condition 2 in Experiment Example 1;

[0068] Figure 5 This is the chromatogram under gradient condition 3 in Experiment Example 1;

[0069] Figure 6 This is the chromatogram under gradient condition 4 in Experiment Example 1;

[0070] Figure 7 This is the chromatogram under gradient condition 5 in Experiment Example 1;

[0071] Figure 8 This is the chromatogram under gradient condition 6 in Experiment Example 1;

[0072] Figure 9 The chromatogram is for the flow rate of 0.25 mL / min in Experiment Example 1;

[0073] Figure 10 The chromatogram is for the flow rate of 0.3 mL / min in Experiment Example 1;

[0074] Figure 11 The chromatogram is for the flow rate of 0.35 mL / min in Experiment Example 1;

[0075] Figure 12 This is the chromatogram of the column temperature at 25℃ in Experiment Example 1;

[0076] Figure 13 This is the chromatogram of the column temperature at 30℃ in Experiment Example 1;

[0077] Figure 14 This is the chromatogram of the column temperature at 35℃ in Experiment Example 1;

[0078] Figure 15 The characteristic chromatograms of 15 batches of Commelina communis formula granules in Experiment Example 2;

[0079] Figure 16 The characteristic spectrum of the *Commelina communis* control herb in Experiment Example 2.

[0080] Figure 17 The characteristic spectrum of the Commelina communis formula granules in Experiment Example 2 is shown below; Peak 2: vanillic acid; Peak 6: arbutin; Peak 7 (S peak): isoarbutin; Peak 8: vitexin; Peak 9: rutin;

[0081] Figure 18 This is a comparison chart of the characteristic spectra of the mixed control and the control in Experiment Example 2.

[0082] Figure 19 Characteristic spectrum of Commelina communis formula granules;

[0083] Figure 20 This is a chromatogram of a negative blank solution. Detailed Implementation

[0084] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described herein. They do not constitute a limitation on the content and scope of protection of the present invention. Any product identical or similar to the present invention derived by anyone under the guidance of the present invention or by combining the features of the present invention with other prior art falls within the scope of protection of the present invention. Where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in the art. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products. The Commelina communis preparation described in this invention is prepared by the following method:

[0085] Take *Commelina communis* and extract it under reflux at least once, adding 8-14 times its weight of water each time for at least 0.5 hours. Filter, combine the filtrates, and concentrate them to a relative density of 1.05-1.10 g / mL at 60°C. Add conventional excipients and follow conventional processes to prepare clinically acceptable tablets, capsules, pills, granules, honey-processed pills, sustained-release preparations, immediate-release preparations, controlled-release preparations, oral liquid preparations, or injectable preparations. The pharmaceutically acceptable excipients include: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, and matrix, etc. Fillers include: starch, pregelatinized starch, lactose, mannitol, chitosan, microcrystalline cellulose, sucrose, etc.; disintegrants include: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, croscarmellose, low-substituted hydroxypropyl cellulose, croscarmellose sodium, etc.; lubricants include: magnesium stearate, sodium lauryl sulfate, talc, silica, etc.; suspending agents include: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include: starch paste, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.; sweeteners include: sodium saccharin, aspartame, sucrose, cyclamate, glycyrrhetinic acid, etc.; flavoring agents include: sweeteners and various flavorings; preservatives include: parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chlorethidium acetate, eucalyptus oil, etc.; matrix includes: PEG6000, PEG4000, insect wax, etc.

[0086] The test sample used in the following examples and experimental cases is Commelina communis formula granules. The specific preparation method of the Commelina communis formula granules is as follows: Take Commelina communis and extract it twice by heating and reflux. For the first extraction, add 12 times the weight of Commelina communis to water and soak for 30 minutes, then heat and reflux for 0.5 hours and filter. For the second extraction, add 10 times the weight of Commelina communis to water and heat and reflux for 0.5 hours and filter. Combine the filtrates and concentrate them to a relative density of 1.05 g / mL at 60°C. Spray dry the filtrate. Add 0.02 times the weight of Commelina communis to the dry powder as an excipient, mix evenly, and then dry granulate to make granules.

[0087] Example 1

[0088] This embodiment provides a method for determining the content of Commelina communis and its preparations and constructing characteristic chromatograms, including:

[0089] (1) Preparation of test solution: Using Commelina communis formula granules as test sample, take about 0.2g of test sample powder, accurately weigh it, place it in a stoppered conical flask, accurately add 20mL of 60% methanol, stopper tightly, weigh it, sonicate (power 250W, frequency 40kHz) for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with 60% methanol, shake well, filter, and take the filtrate to obtain the solution;

[0090] Preparation of mixed reference solution: Take appropriate amounts of vanillic acid reference standard, scutellarin reference standard, isoscutellarin reference standard, vitexin reference standard and rutin reference standard, accurately weigh them, add methanol to prepare a solution containing 50 μg of each reference standard per 1 mL, as the mixed reference solution.

[0091] (2) High-performance liquid chromatography (HPLC) detection: The test solution and mixed reference solution were analyzed under the following chromatographic conditions: Octadecylsilane-bonded silica gel was used as the packing material (column: Waters ACQUITY UPLC HSS T3; column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol was used as mobile phase A, and 0.1% formic acid was used as mobile phase B, with gradient elution as specified in Table 1; the flow rate was 0.3 mL / min, the column temperature was 30℃, and the detection wavelength was 270 nm. The theoretical plate number, calculated based on the isopropanol peak, should not be less than 10,000.

[0092] Table 1 Gradient Elution Table

[0093]

[0094] The results are shown in the table below. Figure 1-2 As shown, peak 2: vanillic acid; peak 6: arvense glycoside; peak 7 (S peak): isoarvense glycoside; peak 8: vitexin; peak 9: rutin. From Figure 2It was found that the characteristic chromatogram of Commelina communis formula granules had 9 characteristic peaks. Each peak had a good shape, stable baseline, short detection time, and uniform peak height or area. The retention times of peaks 2, 6, 7, 8, and 9 corresponded to the retention times of the reference peaks for vanillic acid, arbutin, isoarbutin, vitexin, and rutin, respectively. The peak corresponding to the isoarbutin reference peak was peak S. The relative retention times of the remaining characteristic peaks to peak S were: 0.32 (peak 1), 0.52 (peak 2), 0.61 (peak 3), 0.67 (peak 4), 0.91 (peak 5), 0.95 (peak 6), 1.04 (peak 8), and 1.23 (peak 9). The content of isoarbutin in Commelina communis formula granules was calculated to be 0.33 mg / g using the external standard method.

[0095] Table 2 System Adaptability Parameters

[0096]

[0097] Experimental Example 1

[0098] 1. Instruments and reagents

[0099] 1.1 Instruments

[0100] Chromatograph 1: Ultra-high performance liquid chromatograph: Thermo Fisher Vanquish TM The Flex UHPLC system includes a binary gradient pump F (VF-P10-A), an autosampler Split Sampler FT (VF-A10-A), a column compaction H (VF-C10-A), a diode array detector HL (VH-D10-A), and a Chromeleon 7 chromatographic data system.

[0101] Chromatograph 2: Ultra-high performance liquid chromatograph: Shimadzu LC-40X3 chromatography system, including binary gradient pump (LC-40BX3), autosampler (SIL-40C X3), column oven (CTO-40S), diode array detector (SPD-M40), and Shimadzu Labsolutions chromatography workstation.

[0102] Chromatograph 3: Ultra-high performance liquid chromatograph: Agilent 1290 chromatography system, including binary gradient pump (G732A), autosampler (G737A), column oven (G716B), diode array detector (G7117A), and Openlab chromatography workstation.

[0103] Electronic analytical balances: Sartorius SQP SECURA225D-1CN 1 / 100,000 (Sartorius Scientific Instruments (Beijing) Co., Ltd.), Shimadzu AY 1 / 200,000 (Shimadzu Corporation, Japan).

[0104] Ultrasonic instrument: KQ-400KDB high-power CNC ultrasonic instrument (Kunshan Ultrasonic Instrument Co., Ltd.)

[0105] Water Bath: HH-S6 Digital Display Thermostatic Water Bath (Jiangsu Jinyi Instrument Technology Co., Ltd.)

[0106] Chromatographic column:

[0107] (1) Waters ACQUITY UPLC HSS T3 (column length 100mm, inner diameter 2.1mm, particle size 1.8μm)

[0108] (2) SHIMADZU Shim-pack GISS (column length 100mm, inner diameter 2.1mm, particle size 1.9μm)

[0109] (3) HEXI-TECH Morphling AQ-C18 (column length 100mm, inner diameter 2.1mm, particle size 1.8μm)

[0110] 1.2 Reagents

[0111] Methanol was of chromatographic grade, and water was Watson's distilled water; all other reagents were of analytical grade.

[0112] 1.3 Drug Test

[0113] Isorhizonidine reference standard (purchased from the National Institutes for Food and Drug Control, batch number 111974-201401, purity ≥94%); Orhizonidine reference standard (purchased from the National Institutes for Food and Drug Control, batch number 111777-202003, purity ≥98%); Rutin reference standard (purchased from the National Institutes for Food and Drug Control, batch number 10080-202012, purity ≥98%); Vitexin reference standard (purchased from Chengdu Mansite Biotechnology Co., Ltd., DST210405-034, purity ≥98%); Vanillic acid reference standard (purchased from Chengdu Mansite Biotechnology Co., Ltd., DSTDX008801, purity ≥98%).

[0114] Dayflower reference material (purchased from the National Institutes for Food and Drug Control, batch number 121663-201602).

[0115] 2. Establishment of chromatographic conditions

[0116] Through exploration and optimization of the literature conditions, the liquid chromatography conditions for the characteristic chromatogram of Commelina communis formula granules were finally determined. These chromatographic conditions have good system adaptability and are suitable for the determination of the characteristic chromatogram of Commelina communis formula granules.

[0117] Based on relevant studies on the characteristic spectrum of Commelina communis and related literature, the reproducibility of the study was investigated using the sample processing methods and chromatographic conditions described in the literature. The results are as follows.

[0118] 2.1 Selection of chromatographic conditions

[0119] 2.1.1 Exploration and Results of Chromatographic Conditions

[0120] The test solution was prepared by taking the Commelina communis formula granules according to the method of Example 1. The same test solution was tested under the following gradient elution conditions, and the other chromatographic conditions were the same as in Example 1.

[0121] Table 3 Gradient Condition 1

[0122]

[0123] Table 4 Gradient Condition 2

[0124]

[0125] Table 5 Gradient Condition 3

[0126]

[0127]

[0128] Table 6 Gradient Condition 4

[0129]

[0130] Table 7 Gradient Condition 5

[0131]

[0132] Table 8 Gradient Conditions 6

[0133]

[0134] Table 9. System suitability parameters for each gradient chromatographic peak.

[0135]

[0136]

[0137]

[0138] By comparing the chromatograms of samples determined under different elution procedures ( Figure 3-8From the selected gradients, the one with the richest chromatographic information, best separation of major chromatographic peaks, most stable baseline, and most reasonable analysis time was chosen. The specific gradient settings are shown in the table above. The results showed that the chromatographic peak separation improved with increasing elution time. Gradient condition 3 exhibited better separation and richer chromatographic information than the previous two gradients. Referring to gradient condition 4 and combining it with gradient condition 5, gradient condition 6 was obtained. The resulting chromatogram after elution showed richer chromatographic information, more major chromatographic peaks, better separation, more stable baseline, and shorter detection time. Therefore, gradient condition 6 was selected as the mobile phase gradient for subsequent condition selection to achieve even better separation results.

[0139] 2.1.2 Selection of detection wavelength

[0140] The test solution of *Commelina communis* granules prepared according to the method of Example 1 was injected into a liquid chromatograph under the gradient conditions determined above, and a full-wavelength scan was performed. Based on the amount of chromatographic information, the chromatograms at 10 different absorption wavelengths (230 nm, 250 nm, 270 nm, 290 nm, 310 nm, 330 nm, 350 nm, and 360 nm) were compared. The remaining chromatographic conditions were the same as in Example 1. The number of chromatographic peaks, response value, and retention time were used as evaluation criteria to screen the detection wavelengths.

[0141] Table 10 System adaptability parameters for chromatographic peaks at different absorption wavelengths

[0142]

[0143]

[0144]

[0145] The main active ingredients in Commelina communis are flavonoids, with an effective detection wavelength range of 230–360 nm. Results showed that the chromatograms exhibited more peaks and greater information content, with a stable baseline, at wavelengths of 250–290 nm, with 270 nm being the optimal wavelength. Therefore, 270 nm, with its relatively rich chromatographic peak information and relatively superior system adaptability parameters, was selected as the detection wavelength for the characteristic chromatogram of Commelina communis formula granules for further investigation.

[0146] 2.1.3 Investigation of mobile phase composition

[0147] (1) Investigation of acid types

[0148] The test solution of *Commelina communis* formulation granules prepared according to the method of Example 1 was used. Based on the methanol-0.1% formic acid mobile phase composition used above, the effect of different types of added acid on the separation effect of characteristic chromatograms of *Commelina communis* formulation granules was investigated. All other chromatographic conditions were the same as in Example 1. The pure water system, the 0.1% formic acid solution system, the 0.1% acetic acid solution system, and the 0.1% phosphoric acid solvent system were compared respectively. The results are shown in the table below.

[0149] Table 11 Parameters for evaluating the applicability of the system for different types of acid addition

[0150]

[0151]

[0152] The results showed that compared with using 0.1% phosphoric acid as mobile phase B, the separation effect of characteristic peaks in the characteristic spectra obtained by using water, 0.1% formic acid and 0.1% acetic acid was significantly improved, especially for 0.1% formic acid, which had a larger peak area. Therefore, 0.1% formic acid solution was preferred for subsequent condition screening.

[0153] (2) Investigation of acid concentration

[0154] The test solution of *Commelina communis* formulation granules prepared according to the method of Example 1 was used. Based on the methanol-0.1% formic acid mobile phase composition determined above, the effect of different formic acid concentrations on the separation effect of characteristic chromatograms of *Commelina communis* formulation granules was investigated. The 0.05% formic acid solution system, the 0.1% formic acid system, and the 0.2% formic acid system were compared respectively, with other chromatographic conditions the same as in Example 1. The results are shown in the table below.

[0155] Table 12 Parameters for testing the applicability of the system at different formic acid concentrations

[0156]

[0157]

[0158] The results showed that different formic acid concentrations had a certain impact on each chromatographic peak. The chromatograms eluted with 0.1% formic acid solution showed better peak shapes and resolution of the main chromatographic peaks. Therefore, 0.1% formic acid solution was preferred for subsequent condition screening.

[0159] 2.1.4 Investigation of different flow velocities

[0160] The test solution of *Commelina communis* formula granules prepared according to the method of Example 1 was analyzed at flow rates of 0.25 mL / min, 0.30 mL / min, and 0.35 mL / min, respectively. Other chromatographic conditions were the same as in Example 1. The effect of different flow rates on the separation effect of the characteristic chromatograms of *Commelina communis* formula granules was investigated. The results are shown below. Figure 9-11 .

[0161] The results showed that different flow rates had a significant impact on the retention time of some chromatographic peaks, and also on the peak resolution and asymmetry. At a flow rate of 0.25 mL / min, the elution time was generally delayed, and the last peak was not completely separated. A flow rate of 0.3 mL / min showed relatively better peak separation; therefore, 0.3 mL / min was selected as the subsequent screening condition.

[0162] 2.1.5 Investigation at different column temperatures

[0163] The sample solution of *Commelina communis* granules prepared according to the method in Example 1 was used to investigate the effect of different column temperatures on the separation effect of the characteristic chromatograms of *Commelina communis* granules. The column temperatures were investigated at 25℃, 30℃, and 35℃, with other chromatographic conditions the same as in Example 1. The results are shown below. Figure 12-14 .

[0164] The results showed that different column temperatures had a significant impact on the retention time of chromatographic peaks. At a lower column temperature of 25℃, the elution time was prolonged and the peak shape was wider. At a column temperature of 35℃, the elution time was shortened and the peak separation effect was poor. At 30℃, the system adaptability parameters of each peak were relatively better. Taking all factors into consideration, a column temperature of 30℃ was selected for subsequent condition screening and investigation.

[0165] 2.1.6 Investigation of Chromatographic Columns of Different Brands

[0166] The test solution of *Commelina communis* formula granules prepared according to the method of Example 1 was used to investigate the effect of different brands of chromatographic columns on the characteristic chromatogram of *Commelina communis* formula granules. The different brands of chromatographic columns investigated were: (1) Waters ACQUITY UPLC HSST3 (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); (2) SHIMADZU Shim-pack GISS (column length 100 mm, inner diameter 2.1 mm, particle size 1.9 μm); (3) HEXI-TECH Morphling AQ-C18 (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm). The other chromatographic conditions were the same as in Example 1.

[0167] Table 13 Waters ACQUITY UPLC HSS T3 column test system suitability parameters

[0168]

[0169] Table 14. Suitability parameters for SHIMADZU Shim-pack GISS chromatographic columns.

[0170]

[0171]

[0172] Table 15. HEXI-TECH Morphling AQ-C18 Column System Suitability Parameters

[0173]

[0174] The results showed that different brands of chromatographic columns had a significant impact on the retention times of each chromatographic peak. The Waters ACQUITY UPLC HSS T3 column showed the best separation effect for each chromatographic peak, while the other two brands of columns performed slightly worse in terms of resolution, symmetry factor, and other parameters. Therefore, the Waters ACQUITY UPLC HSS T3 column (100 mm length, 2.1 mm inner diameter, 1.8 μm particle size) was selected as the chromatographic column.

[0175] 3. Preparation of test solution

[0176] (1) Investigation of methanol concentration in extraction solvent

[0177] The effects of different extraction solvents (40% methanol, 60% methanol, and 80% methanol) on the characteristic chromatograms of *Commelina communis* formula granules were investigated. Approximately 0.2 g of *Commelina communis* formula granules were accurately weighed and placed in stoppered conical flasks. 20 mL of each different extraction solvent was accurately added, and the flasks were sealed tightly. The flasks were then sonicated (250 W, 40 kHz) for 45 minutes, cooled, shaken well, filtered, and the filtrate was collected to obtain the test solution. The chromatographic conditions of Example 1 were used for detection, and the results are shown in the table below.

[0178] Table 16 shows the chromatographic peak system adaptability parameters for evaluating the methanol concentration of the extraction solvent.

[0179]

[0180]

[0181] Based on the chromatographic parameters of each peak, the results showed that the total peak area of ​​the characteristic chromatographic peaks obtained by extraction with different methanol concentrations was different. The chromatographic peaks obtained by extraction with 60-80% methanol had larger peak areas, higher separation of each peak, and better peak shapes. Considering all factors, the preferred extraction solvent was determined to be 60-80% methanol.

[0182] (2) Examination of extraction time

[0183] The extraction effects of different extraction times (30, 45, and 60 minutes) on the main characteristic peaks of the chromatogram of *Commelina communis* formula granules were investigated. Approximately 0.2 g of *Commelina communis* formula granules were accurately weighed and placed in stoppered conical flasks. 20 mL of 60% methanol was accurately added, and the flasks were tightly sealed. The flasks were then sonicated (250 W, 40 kHz) for 30, 45, and 60 minutes, respectively. After cooling, the flasks were shaken well, filtered, and the filtrate was collected to obtain the test solution. The chromatographic conditions of Example 1 were used for detection, and the results are shown in the table below.

[0184] Table 17 Extraction time parameters for chromatographic peak system adaptability.

[0185]

[0186] Results analysis: Comparing the results of the three different extraction times, the different extraction times did not affect the retention time of each peak; the peak areas showed some differences when ultrasonically treated for 30 minutes, 45 minutes and 60 minutes. Extraction was complete when ultrasonically treated for 30 minutes. Considering the need to save operation time, the preferred extraction time was 30 minutes.

[0187] (3) Extraction power investigation

[0188] The extraction effects of different extraction powers (150, 250, and 350 W) on the main characteristic peaks of the chromatogram of *Commelina communis* formula granules were investigated. Approximately 0.2 g of *Commelina communis* formula granules were accurately weighed and placed in stoppered conical flasks. 20 mL of 80% methanol was accurately added, and the flasks were tightly sealed. The flasks were then sonicated (powers of 150, 250, and 350 W, frequency 40 kHz) for 30 minutes each, cooled, shaken well, filtered, and the filtrate was collected to obtain the test solution. The chromatographic conditions of Example 1 were used for detection, and the results are shown in the table below.

[0189] Table 18 Extraction power evaluation parameters for chromatographic peak system suitability.

[0190]

[0191] Comparing the results of the three different extraction powers, it was found that different extraction powers did not affect the retention time of each peak. After ultrasonic treatment with powers of 150W, 250W, and 350W, the peak areas showed some differences. When ultrasonic treatment with 250W power for 30 minutes was performed, the extraction was complete, and the separation, asymmetry, and theoretical plate number of each peak were better. Therefore, the preferred extraction power was 250W.

[0192] (4) Sampling size investigation

[0193] The effect of different sampling amounts on the characteristic chromatogram of Commelina communis formula granules was investigated. Approximately 0.1 g, 0.2 g, and 0.3 g of Commelina communis formula granules were accurately weighed and placed in stoppered conical flasks. 20 mL of 60% methanol was accurately added, the flasks were sealed tightly, and the mixture was sonicated (250 W, 40 kHz) for 30 minutes. After cooling, the mixture was shaken well, filtered, and the filtrate was collected to obtain the test solution. The chromatographic conditions of Example 1 were used for detection, and the results are shown in the table below.

[0194] Table 19 Sampling quantity parameters for chromatographic peak system adaptability assessment

[0195]

[0196]

[0197] Experimental results show that different sample amounts have little effect on the retention time of each characteristic peak. The separation effect is better when the sample amount is 0.1-0.3g, and the peak areas of the characteristic peaks in the chromatogram of the Commelina communis formula granules are relatively moderate, especially when the sample amount is 0.2g. Therefore, 0.2g is the preferred sample amount for this method.

[0198] (5) Investigation of different solvent volumes

[0199] The effect of different solvent volumes (10 mL, 20 mL, and 40 mL) on the characteristic chromatogram of *Commelina communis* formulation granules was investigated. Approximately 0.2 g of *Commelina communis* formulation granules were accurately weighed and placed in stoppered conical flasks. 10 mL, 20 mL, and 40 mL of 60% methanol were accurately added respectively. The flasks were sealed tightly, sonicated (250 W, 40 kHz) for 30 minutes, cooled, shaken well, filtered, and the filtrate was collected to obtain the test solution. The chromatographic conditions of Example 1 were used for detection, and the results are shown in the table below.

[0200] Table 20 Solvent Volume Assessment: Chromatographic Peak System Suitability Parameters

[0201]

[0202]

[0203] Based on the information content and separation effect of the chromatographic peaks as the main evaluation indicators, the experimental results show that different solvent volumes have little impact on the chromatogram, so 20-40 mL can be used as the extraction solvent volume.

[0204] (6) Confirmation of the method for preparing the test solution

[0205] Based on the above results, the preparation method of the test solution of Commelina communis formula granules is determined as follows: Take about 0.2g of Commelina communis formula granules, place them in a stoppered conical flask, add 20mL of 60% methanol, seal tightly, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the solution.

[0206] Experimental Example 2: Construction and content determination of characteristic chromatograms of 15 batches of Commelina communis formula granules

[0207] (1) Construction of feature maps

[0208] Fifteen batches of Commelina communis formula granules were taken as test samples, and test sample solutions were prepared according to the method in Example 1. 1 g of Commelina communis reference material was accurately weighed, placed in a stoppered conical flask, 40 mL of water was added, and the mixture was heated under reflux for 45 minutes. The mixture was filtered, the filtrate was evaporated to dryness, and 10 mL of 60% methanol was added to the residue. The mixture was ultrasonically treated (power 250 W, frequency 40 kHz) for 30 minutes, filtered, and the filtrate was used as the reference solution for the reference material.

[0209] The above solution was analyzed using the high-performance liquid chromatography method described in Example 1. Characteristic chromatograms of 15 batches of Commelina communis formula granules and control medicinal materials were obtained. The results are shown in Tables 21-27. Figure 15-16 As shown in the figure. Figure 15 In the sample, S1–S15: 2002002W, 2004002S, 2001001S, 2003001W, 2005001W, 2010002S, 2012001W, 2104001S, 2111002W, 2205001S, 2208002S, 2212002S, 2303001W, 2309003S, 2310001S. The characteristic chromatograms of 15 batches of Commelina communis formula granules were analyzed. The fingerprint chromatogram similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission was used to generate control characteristic chromatograms, such as... Figure 17 As shown. According to Figure 17 The reference characteristic chromatograms shown can be used to analyze and compare the detection results of the characteristic chromatograms of formulated granules, and are used for the quality control of formulated granules. The specific method for quality control using reference characteristic chromatograms is as follows:

[0210] The characteristic fingerprint chromatogram of Commelina communis formula granules contains 9 chromatographic peaks. Peak 7 should correspond to the retention time of the isoharonium chloride reference peak. The peak corresponding to the isoharonium chloride reference peak is the S peak. The relative retention times of each characteristic peak and the S peak should be calculated, and the relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.32 (peak 1), 0.51 (peak 2), 0.60 (peak 3), 0.67 (peak 4), 0.91 (peak 5), 0.95 (peak 6), 1.04 (peak 8), and 1.23 (peak 9).

[0211] Table 21. Characteristic patterns and relative retention times of Commelina communis formula granules.

[0212]

[0213] Table 22 Common pattern matching data for Commelina communis formulation granules

[0214]

[0215] Table 23. Relative retention time results of characteristic chromatogram determination of 15 batches of Commelina communis formula granules.

[0216]

[0217] Table 24. Comparison chart of Commelina communis formula granules: relative retention time

[0218]

[0219] Table 25. Comparison of relative peak areas in the formula granules of Commelina communis.

[0220]

[0221] Table 26. Relative retention time of characteristic chromatograms of Commelina communis (compared medicinal materials)

[0222]

[0223] Table 27. Relative peak areas of characteristic spectra of Commelina communis (compared medicinal materials)

[0224]

[0225] (2) Characteristic peak identification

[0226] The chromatograms of the mixed reference standard obtained in Example 1 were compared with the above-mentioned characteristic reference chromatograms, and the results are shown in the figure. Figure 18 As shown, the retention times of peaks 2, 6, 7, 8, and 9 should correspond to the retention times of the reference peaks of vanillic acid, styracin, isostyracin, vitexin, and rutin, respectively.

[0227] (3) Similarity

[0228] The similarity of characteristic fingerprint spectra of 15 batches of Commelina communis formula granules was calculated using the 2012 version of the fingerprint spectra similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" compiled by the Pharmacopoeia Commission. The results are shown in the table below. The results show that the similarity of characteristic fingerprint spectra of all 15 batches of Commelina communis formula granules is greater than 0.90.

[0229] Table 28. Similarity results of characteristic fingerprint spectra of 15 batches of Commelina communis formula granules

[0230]

[0231] (4) Content determination

[0232] The content of isopropargyl glycosides was determined by taking 15 batches of Commelina communis formula granule test solutions according to the method in Example 1. The results are shown in the table below.

[0233] Table 29 Results of isopropargyl glycoside content in 15 batches of Commelina communis formula granules

[0234]

[0235]

[0236] The detection method described in Example 1 effectively obtains fingerprint spectra with good separation of various characteristic peaks, and can simultaneously determine the content of isoharmonin. Furthermore, by selecting the S peak of isoharmonin as the internal reference peak in the fingerprint spectrum, the relative retention times of the common characteristic peaks 1-6 and 8-9 of the Commelina communis formula granules can be determined. Therefore, it enables comprehensive and rapid detection of Commelina communis formula granules, which is beneficial for comprehensive quality testing and overall quality control of Commelina communis formula granules, thereby helping to improve the safety and stability of the drug.

[0237] Example 3: Analytical Method Verification

[0238] (1) Instrument precision test

[0239] Take the same sample solution of Commelina communis formula granules prepared according to the method of Example 1, and inject it 6 times under the chromatographic conditions of Example 1. Record the chromatogram, determine the relative retention time and relative peak area of ​​9 characteristic peaks, and perform analysis.

[0240] The results showed that the RSDs of the relative retention times of each characteristic peak and the reference peak S (peak 7) were 0.06%, 0.04%, 0.02%, 0.01%, 0.01%, 0.01%, 0%, 0.01%, and 0.04%, respectively, and the RSDs of the relative peak areas of each characteristic peak and the reference peak S (peak 7) were 6.04%, 3.57%, 4.49%, 4.46%, 3.48%, 3.87%, 0%, 2.50%, and 3.50%, respectively. This indicates that the instrument has good precision.

[0241] (2) Method repeatability test

[0242] Take the same batch of Commelina communis formula granules and prepare 6 test samples in parallel according to the method of Example 1. Determine the relative retention time and relative peak area of ​​the 9 common peaks according to the method of Example 1 and perform analysis.

[0243] The results show that the RSDs of the relative retention times of each characteristic peak and the reference peak S (peak 7) are 0.03%, 0.06%, 0.02%, 0.02%, 0.01%, 0.01%, 0%, 0.02%, and 0.01%, respectively, and the RSDs of the relative peak areas of each characteristic peak and the reference peak S (peak 7) are 2.92%, 2.82%, 3.53%, 3.89%, 2.97%, 3.58%, 0%, 1.80%, and 2.06%, respectively, indicating that the method has good repeatability.

[0244] (3) Intermediate precision (different operators)

[0245] Three inspectors, at different times, used the same equipment and the chromatographic conditions of Example 1 to determine the relative retention time and relative peak area of ​​the nine common peaks of the same batch of Commelina communis formula granules, and then analyzed them.

[0246] The results show that the RSDs of the relative retention times of each characteristic peak and the reference peak S (peak 7) are 0.02%, 0.01%, 0.01%, 0.02%, 0.01%, 0.01%, 0%, 0.01%, and 0.03%, respectively; and the RSDs of the relative peak areas of each characteristic peak and the reference peak S (peak 7) are 1.36%, 1.50%, 0.49%, 1.11%, 0.69%, 0.81%, 0%, 0.51%, and 0.30%, respectively, indicating that the intermediate precision (by different personnel) of this method is good.

[0247] (4) Specificity test

[0248] The test sample was prepared and analyzed according to the method described in Example 1. The test sample solution and negative blank solution (0.2 g of negative granules prepared by dry granulation of maltodextrin, prepared according to the method for preparing the test sample solution) were injected separately. Chromatograms were recorded, and the effect of the negative blank solution was investigated. The results are shown in [Figure 1]. Figures 19-20 Specificity test results showed that negative samples did not cause interference.

[0249] (5) Durability

[0250] ①24-hour stability test

[0251] Take the same sample solution prepared according to the method of Example 1, and determine the stability of the sample solution under the chromatographic conditions of Example 1. Inject the sample at 0, 2, 4, 8, 12, 18 and 24 hours after preparation, respectively, and determine the relative retention time and relative peak area of ​​the 9 common peaks.

[0252] The results showed that the RSDs of the relative retention times of each characteristic peak and the reference peak S were 0.06%, 0.06%, 0.02%, 0.02%, 0.01%, 0.01%, 0%, 0.01%, and 0.04%, respectively, and the RSDs of the relative peak areas of each characteristic peak and the reference peak S were 3.14%, 4.00%, 4.21%, 4.21%, 4.05%, 4.02%, 0%, 2.50%, and 3.22%, respectively. This indicates that the test solution was stable within 24 hours and met the determination requirements.

[0253] ② Investigation of different flow velocities

[0254] Take the same sample solution prepared according to the method of Example 1, and determine it according to the chromatographic conditions of Example 1. The flow rates are 0.28 mL / min, 0.30 mL / min and 0.32 mL / min, respectively. The relative retention time and relative peak area of ​​each characteristic peak of the Commelina communis formula granules and the reference S peak (peak 7) when the flow rate changes slightly are investigated and analyzed.

[0255] The relative retention times and relative peak areas of each characteristic peak and the reference peak S (peak 7) were examined when the flow rate changed slightly. The results showed that the relative retention time RSD% was 3.21%, 1.97%, 1.26%, 0.64%, 0.30%, 0.05%, 0%, 0.10%, and 0.80%, respectively, and the relative peak area RSD was 3.11%, 9.96%, 23.21%, 13.48%, 13.62%, 7.45%, 0%, 3.06%, and 0.76%, respectively. The results indicate that the relative retention time of the characteristic peaks varies considerably (but still within ±10%), so the flow rate was set at 0.3 mL per minute.

[0256] ③ Investigation at different column temperatures

[0257] Take the same sample solution prepared according to the method of Example 1, and determine it according to the chromatographic conditions of Example 1. Set the column temperature to 28℃, 30℃ and 32℃ respectively, and inject the sample for determination. Record the chromatogram and analyze the relative retention time and relative peak area of ​​the characteristic peaks.

[0258] The relative retention times and relative peak areas of each characteristic peak and the reference peak S (peak 7) were examined when the column temperature changed. The results showed that the RSD% of the relative retention times were 1.23%, 0.43%, 0.09%, 0.11%, 0.38%, 0.15%, 0%, 0.05%, and 1.26%, respectively, and the RSDs of the relative peak areas were 12.13%, 4.35%, 15.65%, 23.69%, 9.80%, 4.16%, 0%, 9.95%, and 3.71%, respectively. This indicates that the relative retention times of the characteristic spectra are robust to different column temperatures.

[0259] ④ Investigation of different chromatographic columns

[0260] Take the same sample solution prepared according to the method in Example 1, and determine it according to the chromatographic conditions in Example 1. Select the following chromatographic column brands for injection and determination: (1) Waters ACQUITY UPLC HSS T3 (column length 100mm, inner diameter 2.1mm, particle size 1.8μm); (2) SHIMADZU Shim-pack GISS (column length 100mm, inner diameter 2.1mm, particle size 1.9μm); (3) HEXI-TECH Morphling AQ-C18 (column length 100mm, inner diameter 2.1mm, particle size 1.8μm). Record the chromatogram and analyze the relative retention time and relative peak area of ​​the characteristic peaks.

[0261] The relative retention times and relative peak areas of each characteristic peak and the reference peak S were investigated using different brands of chromatographic columns. The results showed that the RSD% of the relative retention times were 3.34%, 2.93%, 5.95%, 4.29%, 0.28%, 0.40%, 0%, 1.45%, and 0.44%, respectively, and the RSD% of the relative peak areas were 23.30%, 9.77%, 38.18%, 28.18%, 1.60%, 24.41%, 0%, 14.27%, and 9.07%, respectively. This indicates that different brands of chromatographic columns have a significant impact on the relative retention times and relative peak areas of the characteristic chromatograms. It is recommended to consistently use the Waters ACQUITY UPLC HSS T3 column (100 mm length, 2.1 mm inner diameter, 1.8 μm particle size) for analysis.

[0262] Based on the above methodological investigation results, among the nine characteristic peaks of the established characteristic chromatogram of Commelina communis formula granules, each chromatographic peak is affected to a certain extent by different liquid chromatography instruments, chromatographic columns, column temperatures and flow rates. The relative retention time values ​​are in the range of -10% to 10%. In order to adapt to its robustness, the relative retention time of each peak and the S peak is controlled within ±10% of the specified value.

[0263] The characteristic chromatogram of the Commelina communis formula granules should show 9 characteristic peaks, which should correspond to the 9 characteristic peaks in the chromatogram of the reference medicinal material. Among them, the retention time of peak 7 should correspond to the retention time of the peak of the isoharonium glycoside reference standard.

[0264] The examples provided are not intended to limit the implementation of the invention. Those skilled in the art will recognize that various variations and modifications can be made based on the foregoing description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations and modifications derived therefrom remain within the scope of this invention.

Claims

1. A method for constructing a characteristic spectrum of Commelina communis and its preparations, characterized in that, Includes the following steps, (1) Preparation of test solution; When the test sample is a Commelina communis preparation, weigh the Commelina communis preparation test sample, add solvent to extract, obtain the extract, separate the solid and liquid, and take the liquid, which is the test solution; The solvent is selected from a methanol aqueous solution with a volume percentage of 40-80%; When the test sample is Commelina communis, take 1g of Commelina communis reference material, accurately weigh it, place it in a stoppered conical flask, add 40mL of water, heat under reflux for 45 minutes, filter, evaporate the filtrate to dryness, add 10mL of 60% methanol to the residue, sonicate for 30 minutes, filter, and take the filtrate as the test sample solution. The construction method further includes the step of preparing a reference solution by adding solvent to vanillic acid reference standard, scutellarin reference standard, isoscutellarin reference standard, vitexin reference standard and rutin reference standard; (2) The test solution and the reference solution were detected by high performance liquid chromatography, with methanol as mobile phase A and water or acid-containing aqueous solution as mobile phase B. The gradient elution program is as follows; For 0-5 min, the volume ratio of mobile phase A to mobile phase B is 5%:95%; From 5 to 30 minutes, the volume ratio of mobile phase A to mobile phase B changed from 5%:95% to 27%:73%. Over 30-38 minutes, the volume ratio of mobile phase A to mobile phase B changed from 27%:73% to 30%:70%. 38-55 min, the volume ratio of mobile phase A to mobile phase B is 30%:70%; the detection wavelength is 270 nm, the flow rate is 0.3-0.35 mL / min, and the chromatographic column is one of the following (1)-(3): (1) Waters ACQUITY UPLC HSS T3, specifications: column length 100 mm, inner diameter 2.1 mm, particle size 1.8 µm; (2) SHIMADZU Shim-pack GISS, specifications: column length 100 mm, inner diameter 2.1 mm, particle size 1.9 µm; (3) HEXI-TECH Morphling AQ-C18, specifications: column length 100 mm, inner diameter 2.1 mm, particle size 1.8 µm.

2. The method for constructing the characteristic spectrum of Commelina communis and its preparations according to claim 1, characterized in that, Step (2) also satisfies at least one of the following 1)-3): 1) Column temperature is 25-35℃; 2) The acid in the acid-containing aqueous solution is selected from one or more of formic acid, phosphoric acid, and acetic acid; 3) The acid concentration in the acid-containing aqueous solution is 0.05%-0.2%.

3. The method for constructing the characteristic spectrum of Commelina communis and its preparations according to claim 1, characterized in that, Step (1) also satisfies any one or more of the following AD: A. The mass ratio of the test sample to the volume of the solvent in the *Commelina communis* preparation is 0.1-0.3:20-40; the mass-volume relationship is expressed in g / mL. B. The extraction method is either reflux extraction or ultrasonic extraction; C. Extraction time is ≥20 min; D. The solid-liquid separation is selected from centrifugation or membrane filtration.

4. The method for constructing the characteristic spectrum of Commelina communis and its preparations according to claim 1, characterized in that, In step (1), the extraction time is 30-60 minutes.

5. The method for constructing the characteristic spectrum of Commelina communis and its preparations according to claim 1, characterized in that, Each 1 mL of the reference solution contains 5-50 µg of each reference standard.

6. The method for constructing the characteristic spectrum of Commelina communis and its preparations according to claim 5, characterized in that, The solvent used in the preparation of the reference solution is selected from methanol or an aqueous methanol solution.

7. The method for constructing the characteristic spectrum of Commelina communis and its preparations according to any one of claims 1-6, characterized in that, The characteristic chromatogram of Commelina communis and / or its preparations has 9 common characteristic peaks, among which peak 7 corresponds to the retention time of the isoharmonic acid reference standard peak; the peak corresponding to the isoharmonic acid reference standard peak is peak S, and the relative retention times of peaks 1-6, peaks 8-9 and peak S are within ±10% of the specified values; the specified values ​​are: 0.32, 0.51, 0.60, 0.67, 0.91, 0.95, 1.04, and 1.

23.

8. A method for determining the content of active ingredients in Commelina communis and its preparations, characterized in that, Includes the following steps: (1) Preparation of test solution and reference solution; the method for preparing the reference solution includes: preparing the reference solution by adding solvent to vanillic acid reference, scutellarin reference, isoscutellarin reference, vitexin reference and rutin reference; step (1) includes, when the test sample is a Commelina communis preparation, weighing the Commelina communis preparation test sample, adding solvent to extract, obtaining the extract, separating the solid and liquid, and taking the liquid, which is the test solution; the solvent is selected from a methanol aqueous solution with a volume percentage of 40-80%; When the test sample is Commelina communis, take 1g of Commelina communis reference material, accurately weigh it, place it in a stoppered conical flask, add 40mL of water, heat under reflux for 45 minutes, filter, evaporate the filtrate to dryness, add 10mL of 60% methanol to the residue, sonicate for 30 minutes, filter, and take the filtrate as the test sample solution. (2) The test solution and the reference solution were detected by high performance liquid chromatography, with methanol as mobile phase A and water or acid-containing aqueous solution as mobile phase B. The gradient elution program is as follows; For 0-5 min, the volume ratio of mobile phase A to mobile phase B is 5%:95%; From 5 to 30 minutes, the volume ratio of mobile phase A to mobile phase B changed from 5%:95% to 27%:73%. Over 30-38 minutes, the volume ratio of mobile phase A to mobile phase B changed from 27%:73% to 30%:70%. 38-55 min, the volume ratio of mobile phase A to mobile phase B is 30%:70%; the detection wavelength is 270 nm, the flow rate is 0.3-0.35 mL / min, and the chromatographic column is one of the following (1)-(3): (1) Waters ACQUITY UPLC HSS T3, specifications: column length 100 mm, inner diameter 2.1 mm, particle size 1.8 µm; (2) SHIMADZU Shim-pack GISS, specifications: column length 100 mm, inner diameter 2.1 mm, particle size 1.9 µm; (3) HEXI-TECH Morphling AQ-C18, specifications: column length 100 mm, inner diameter 2.1 mm, particle size 1.8 µm; (3) The content of reference standard contained in the test solution was calculated using the external standard method.

9. The method for determining the content of active ingredients in Commelina communis and its preparations according to claim 8, characterized in that, Step (2) also satisfies at least one of the following 1)-3): 1) Column temperature is 25-35℃; 2) The acid in the acid-containing aqueous solution is selected from one or more of formic acid, phosphoric acid, and acetic acid; 3) The acid concentration in the acid-containing aqueous solution is 0.05%-0.2%.

10. The method for determining the content of active ingredients in Commelina communis and its preparations according to claim 8, characterized in that, Step (1) also satisfies any one or more of the following AD: A. The mass ratio of the test sample to the volume of the solvent in the *Commelina communis* preparation is 0.1-0.3:20-40; the mass-volume relationship is expressed in g / mL. B. The extraction method is either reflux extraction or ultrasonic extraction; C. Extraction time is ≥20 min; D. The solid-liquid separation is selected from centrifugation or membrane filtration.

11. The method for determining the content of active ingredients in Commelina communis and its preparations according to claim 10, characterized in that, In step (1), the extraction time is 30-60 minutes.

12. A method for quality testing of Commelina communis and its preparations, characterized in that, The method includes the method for constructing the characteristic spectrum of Commelina communis and its preparations as described in any one of claims 1-7 and / or the method for determining the content of active ingredients in Commelina communis and its preparations as described in any one of claims 8-11.

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