A method for constructing UPLC characteristic spectrum of Qinglongyi medicinal material, standard decoction and formula granules

Abstract

The application provides a construction method of UPLC characteristic spectrum of Qinglongyi medicinal material, standard decoction and formula granules, comprising the following steps: dissolving and extracting raw materials of the test sample by using a solvent to obtain a test solution; determining the test solution by using high performance liquid chromatography to obtain the UPLC characteristic spectrum of Qinglongyi medicinal material, standard decoction and formula granules; and the high performance liquid chromatography conditions are as follows: a C 18 column is used as the chromatographic column; acetonitrile solution is used as mobile phase A, and 0.1% formic acid aqueous solution is used as mobile phase B, and gradient elution is adopted. The application adopts the high performance liquid chromatography, selects acetonitrile-0.1% formic acid solution as the mobile phase for gradient elution, takes protocatechuic acid, vanillic acid, syringic acid, ellagic acid, rutin and quercitrin as the reference substances, establishes the UPLC characteristic spectrum method of Qinglongyi medicinal material, standard decoction and formula granules, and provides more scientific technical means for controlling the medicinal quality of Qinglongyi medicinal material, standard decoction and formula granules.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical and preparation analysis and detection, and particularly relates to a method for constructing UPLC characteristic fingerprints of Qinglongyi medicinal materials, standard decoctions, and formula granules. Background Art

[0002] The Qinglongyi medicinal material is the dried pericarp of the Juglans regia L. plant of the Juglandaceae family; the Qinglongyi standard decoction is a freeze-dried powder prepared from the medicinal material by a fixed preparation process after processing; the Qinglongyi formula granule is a formula granule prepared from the medicinal material by processing according to the main quality indexes of the standard decoction. In order to ensure the uniformity and stability of the quality of the medicinal material, standard decoction, and formula granule, the present invention establishes a new characteristic fingerprint method to control its quality; and can significantly identify Qinglongyi and its preparations. [[ID= nine]] 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 UPLC characteristic fingerprints of Qinglongyi medicinal materials, standard decoctions, and formula granules, and the method of the present invention is accurate and reliable.

[0004] The present invention provides a method for detecting UPLC characteristic fingerprints of Qinglongyi medicinal materials, standard decoctions, and formula granules, including:

[0005] Taking the test sample raw material for pretreatment to obtain a test solution; the pretreatment method includes dissolving and extracting with a solvent; measuring the test solution by UPLC to obtain the UPLC characteristic fingerprints of Qinglongyi medicinal materials, standard decoctions, and formula granules;

[0006] Reference substance solution: Respectively taking protocatechuic acid, vanillic acid, syringic acid, ellagic acid, rutin, and quercitrin, and dissolving them with a solvent to obtain a reference substance solution of the reference substance;

[0007] The UPLC conditions are as follows: the chromatographic column is a C18 column; mobile phase A is an acetonitrile solution, and mobile phase B is a 0.1% formic acid aqueous solution, with gradient elution;

[0008] The specific gradient elution is as follows:

[0009] 0 - 6 min, phase A: 2%, phase B: 98%;

[0010] 6 - 35 min, phase A: 2% - 18%, phase B: 98% - 82%;

[0011] 35 - 40 min, phase A: 18% - 22%, phase B: 82% - 78%.

[0012] The test sample raw material described in the present invention is one or more of Qinglongyi medicinal materials, Qinglongyi standard decoctions, or Qinglongyi formula granules.

[0013] This invention provides a method for constructing UPLC characteristic spectra of Qinglongyi medicinal material, standard decoction, and formula granules. First, the raw materials for testing are pretreated. The pretreatment method includes solvent dissolution and extraction to obtain the test solution.

[0014] Specifically, the sample raw material is dissolved in a solvent, extracted, cooled, shaken, and filtered to obtain the final product.

[0015] The extraction method described in this invention is either heating and reflux extraction or ultrasonic extraction; ultrasonic extraction is preferred.

[0016] The extraction step is achieved using ultrasound-assisted extraction, wherein the power of the ultrasound extraction device is set to 600W, the ultrasound vibration frequency is 40kHz, and the duration of ultrasound extraction is 30 minutes.

[0017] The present invention achieves complete extraction under the above-mentioned ultrasonic parameters. The chromatogram shows good peak shape and resolution.

[0018] The ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent in this invention is 0.2 to 1:25.

[0019] In some specific embodiments, the ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent in this invention is 1:25.

[0020] 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.

[0021] Specifically, the test solution is prepared by taking the Qinglongyi herbal material, accurately weighing it, placing it in a stoppered conical flask, accurately adding methanol, sealing it tightly, weighing it, sonicating it for 30 minutes, taking it out, cooling it, weighing it again, replenishing the lost weight with methanol, shaking it well, filtering it, and taking the filtrate.

[0022] In some embodiments, the ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent is 0.2:25.

[0023] Specifically, to prepare the test solution, take the standard decoction and formula granules of Qinglongyi, weigh them accurately, place them in a stoppered conical flask, add methanol accurately, seal tightly, weigh, sonicate for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0024] All the raw materials mentioned above can be subjected to quality control using the methods of this invention.

[0025] The present invention also includes the preparation of a reference solution: protocatechuic acid, vanillic acid, syringic acid, ellagic acid, rutin, and quercetin are dissolved in 50% methanol to obtain a reference solution;

[0026] The reference herb, *Cynanchum paniculatum*, was dissolved in 50% methanol to obtain a reference herb solution.

[0027] The reference standard solution and the reference medicinal material solution were injected into a high-performance liquid chromatograph for analysis, and the chromatographic peak chromatograms of the reference standard and the characteristic chromatograms of the reference medicinal material were obtained respectively. Based on the characteristic parameters such as the retention time or peak area of ​​the chromatographic peaks of the reference standard, the corresponding chromatographic peaks in the UPLC characteristic chromatograms of the Qinglongyi medicinal material test sample, the standard decoction test sample, and the formula granule test sample were compared and identified qualitatively.

[0028] The preferred concentrations of the reference solution in this invention are: protocatechuic acid 30 μg / mL, vanillic acid 30 μg / mL, syringic acid 30 μg / mL, ellagic acid 30 μg / mL, rutin 30 μg / mL, and quercetin 30 μg / mL.

[0029] The test solution was analyzed by high performance liquid chromatography (HPLC) to obtain the UPLC characteristic spectra of Qinglongyi medicinal material, standard decoction, and formula granules.

[0030] 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 100×2.1mm and 1.6μm. This invention does not limit the specific chromatographic column model; any C18 chromatographic column that meets the above specifications is acceptable.

[0031] In some embodiments, the chromatographic column can be column 1 (Shim-pack GIST-HP C18-AQ 2.1*100mm, 1.6μm), column 2 (InfinityLab Poroshell 120HILIC-Z 2.1*100mm, 2.7μm), or column 3 (ZORBAX Eclipse Plus C18 2.1*100mm, 1.8μm).

[0032] The column temperature of this invention is 25–30°C; preferably 30°C. At the above-mentioned column temperature, the chromatographic peaks of this invention are symmetrical, the resolution is good, and the peaks are eluted completely.

[0033] In this invention, mobile phase A is an acetonitrile solution, and mobile phase B is a 0.1% formic acid aqueous solution, with gradient elution.

[0034] Specifically, the gradient elution is as follows:

[0035] 0–6 min, Phase A: 2%, Phase B: 98%;

[0036] 6–35 min, Phase A: 2%–18%, Phase B: 98%–82%;

[0037] 35 - 40 min, Phase A: 18% - 22%, Phase B: 82% - 78%.

[0038] The theoretical plate number calculated by syringic acid should be not less than 5000.

[0039] The creative discovery of the present invention is that under the above elution gradient, the baseline separation is good, the resolution between each peak is good, the distribution is uniform, and the baseline is stable.

[0040] The flow rate of the mobile phase in the present invention is 0.3 mL / min.

[0041] The present invention discovers that under the above flow rate, the separation of each chromatographic peak is good, the resolution is moderate, and the peaks come out completely, which is the most preferred scheme.

[0042] The injection volume is 2 μL.

[0043] The detection wavelength is 250 nm. The inventor of the present invention discovers that at 250 nm, the chromatographic peak information is larger, the baseline of the chromatogram is more stable, and the peak area of each peak is larger.

[0044] The present invention first adopts ultra - high performance liquid chromatography to establish a characteristic chromatogram detection method for differentiating Qinglongyi medicinal materials, standard decoctions, and formula granules. This method is beneficial to comprehensively evaluating the scientificity and rationality of the extraction process and its preparations of Qinglongyi and its processed products, and can better comprehensively control the internal quality of the extracts and their preparations of Qinglongyi and its processed products, ensuring the clinical efficacy of the extracts and their preparations of Qinglongyi and its processed products.

[0045] The present invention uses a similarity evaluation system for traditional Chinese medicine chromatographic fingerprints to evaluate the similarity of the UPLC characteristic chromatograms of Qinglongyi medicinal materials, standard decoctions, and formula granules, and obtains a UPLC standard characteristic chromatogram composed of 9 characteristic peaks, where Peak 1: protocatechuic acid; Peak 3: vanillic acid; Peak 4(S): syringic acid; Peak 7: ellagic acid; Peak 8: rutin; Peak 9: quercitrin;

[0046] In the characteristic chromatograms of the above - mentioned Qinglongyi medicinal materials, standard decoctions, and formula granules, taking syringic acid as the reference peak S peak, calculate the relative retention time of each characteristic peak to the S peak. The relative retention time is within ±10% of the specified value, and the specified values are respectively: 0.25 (Peak 1), 0.29 (Peak 2), 0.78 (Peak 3), 1.20 (Peak 5), 1.38 (Peak 6), 1.73 (Peak 7), 1.85 (Peak 8), 2.08 (Peak 9).

[0047] Quality judgment criteria: Take the extracts and preparations of Qinglongyi and its processed products, and operate in the same way as above to obtain the above characteristic chromatograms. Use the 2012 version of the "Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine" of the National Pharmacopoeia Commission to analyze the characteristic chromatograms of the extracts and preparations of Qinglongyi and its processed products and the sample characteristic chromatograms. The similarity is greater than 0.90.

[0048] This invention provides a method for constructing UPLC characteristic spectra of *Clerodendrum trichotomum* medicinal material, standard decoction, and formulated granules, comprising: A) dissolving and extracting the test sample raw material using a solvent to obtain a test solution; B) determining the test solution using high-performance liquid chromatography (HPLC) to obtain UPLC characteristic spectra of *Clerodendrum trichotomum* medicinal material, standard decoction, and formulated granules; the HPLC chromatographic conditions are as follows: a C18 column; mobile phase A is acetonitrile solution, mobile phase B is 0.1% formic acid aqueous solution, and gradient elution is performed; specifically, the gradient elution is as follows: 0–6 min, A phase: 2%, B phase: 98%; 6–35 min, A phase: 2%–18%, B phase: 98%–82%; 35–40 min, A phase: 18%–22%, B phase: 82%–78%. This invention employs high-performance liquid chromatography (HPLC) with acetonitrile-0.1% formic acid solution as the mobile phase for gradient elution. Using protocatechuic acid, vanillic acid, syringic acid, ellagic acid, rutin, and quercetin as references, a UPLC characteristic chromatographic method for Qinglongyi medicinal materials, standard decoctions, and formulated granules was established. This provides more scientific technical means for controlling the medicinal quality of Qinglongyi medicinal materials, standard decoctions, and formulated granules. Attached Figure Description

[0049] Figure 1 Extraction methods were examined;

[0050] Figure 2 Extraction solvent investigation;

[0051] Figure 3 Investigation of solvent addition amount;

[0052] Figure 4 Extraction time consideration;

[0053] Figure 5 Chromatographic peak identification;

[0054] Figure 6 Investigations using different chromatographic columns;

[0055] Figure 7 Exploration using different instruments;

[0056] Figure 8 Overlay of characteristic images of 14 batches of Qinglongyi medicinal materials;

[0057] Figure 9 Comparative characteristic atlas of Qinglongyi medicinal materials;

[0058] Figure 10 Chromatographic peak identification;

[0059] Figure 11 Investigation of different chromatographic columns;

[0060] Figure 12 Investigation of different instruments;

[0061] Figure 13 Overlay chromatograms of characteristic chromatograms of 14 batches of Qinglongyi standard decoctions;

[0062] Figure 14 Reference characteristic chromatogram of Qinglongyi standard decoction;

[0063] Figure 15 Chromatograms of different wavelengths of Qinglongyi formula granules;

[0064] Figure 16 Selection of mobile phase;

[0065] Figure 17 Investigation of column temperature;

[0066] Figure 18 Investigation of flow rate;

[0067] Figure 19 Investigation of delay;

[0068] Figure 20 Investigation of sample preparation method;

[0069] Figure 21 Investigation of extraction solvent;

[0070] Figure 22 Investigation of solvent addition amount;

[0071] Figure 23 Investigation of extraction time;

[0072] Figure 24 Chromatographic peak identification;

[0073] Figure 25 Investigation of different chromatographic columns;

[0074] Figure 26 Investigation of different instruments;

[0075] Figure 27 Overlay chromatograms of characteristic chromatograms of 3 batches of Qinglongyi formula granules;

[0076] Figure 28 Reference characteristic chromatogram of Qinglongyi formula granules;

[0077] Figure 29 Chromatogram of Qinglongyi medicinal materials under the chromatographic conditions in Comparative Example 1 adopted by this laboratory;

[0078] Figure 30The chromatogram of *Clerodendrum trichotomum* under the chromatographic conditions in Comparative Example 2 is shown below.

[0079] Figure 31 Chromatogram of Qinglongyi medicinal material under chromatographic conditions in Comparative Example 3. Detailed Implementation

[0080] This invention provides a method for constructing UPLC characteristic maps of *Gynostemma pentaphyllum* medicinal materials, 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.

[0081] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a method for constructing UPLC characteristic spectra of Qinglongyi medicinal material, standard decoction, and formula granules provided by the present invention.

[0082] High-performance liquid chromatographs: Instrument 1 (Agilent 1290), Instrument 2 (Thermo VF);

[0083] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Ltd.);

[0084] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0085] Ultrasonic cleaner: KQ600DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0086] Chromatographic columns: Column 1 (Shim-pack GIST-HP C18-AQ 2.1*100mm, 1.6μm), Column 2 (InfinityLab Poroshell120HILIC-Z 2.1*100mm, 2.7μm), Column 3 (ZORBAX Eclipse PlusC18 2.1*100mm, 1.8μm);

[0087] Acetonitrile and formic acid were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0088] Protocatechuic acid (China National Institutes for Food and Drug Control, batch number: 110809-202207, purity: 97.5%);

[0089] Vanillic acid (China National Institutes for Food and Drug Control, batch number: 110776-201503, purity: 99.8%);

[0090] Syringic acid (Chengdu Pusi Biotechnology Co., Ltd., batch number: PS010564, purity: 99.41%);

[0091] Ellagic acid (China National Institutes for Food and Drug Control, batch number: 111959-201903, purity: 88.8%);

[0092] Rutin (China National Institutes for Food and Drug Control, batch number: 100080-202012, purity: 92.2%);

[0093] Quercetin (China National Institutes for Food and Drug Control, batch number: 111538-202007, purity: 93.5%);

[0094] Reference material for Qinglongyi (360064-202409, Shanghai Hongyong Biotechnology Co., Ltd.);

[0095] Qinglongyi medicinal materials (batch numbers: QLY01, QLY02, QLY03, QLY04, QLY05, QLY06, QLY07, QLY08, QLY09, QLY10, QLY11, QLY12, QLY13, QLY14);

[0096] Qinglongyi Standard Decoction (prepared by Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch numbers: QLY-BT01, QLY-BT02, QLY-BT03, QLY-BT04, QLY-BT05, QLY-BT06, QLY-BT07, QLY-BT08, QLY-BT09, QLY-BT10, QLY-BT11, QLY-BT12, QLY-BT13, QLY-BT14).

[0097] Qinglongyi Formula Granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., Batch Nos.: QLY-KL01, QLY-KL02, QLY-KL03)

[0098] Example 1: UPLC Characteristic Map of Qinglongyi Herbal Material

[0099] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size...).

[0100] 1.6 μm); using acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, gradient elution was performed according to the specifications in the table below; the flow rate was 0.3 mL per minute; the column temperature was 30 °C; and the detection wavelength was 250 nm. The theoretical plate number, calculated based on the syringic acid peak, should not be less than 5000.

[0101]

[0102] Preparation of the reference solution: Take 1g of *Clerodendrum trichotomum* reference material, place it in a stoppered conical flask, add 25ml of 50% methanol, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate. Separately, accurately weigh appropriate amounts of protocatechuic acid, vanillic acid, syringic acid, ellagic acid, rutin, and quercetin reference standards, and dissolve them in 50% methanol to prepare a solution containing 30μg of each per ml, as the reference solution.

[0103] Preparation of the test solution: Take 1.0 g of Qinglongyi medicinal material powder (passed through No. 3 sieve), place it in a stoppered conical flask, add 25 ml of 50% methanol, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0104] The determination method involves precisely pipetting 2 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0105] 2.2.1.2 Chromatographic conditions and system suitability study

[0106] The characteristic chromatographic conditions and system adaptability, as well as the characteristic chromatographic methods for particles, are consistent with those for standard decoctions.

[0107] The chromatographic conditions and system suitability test for the characteristic chromatogram of *Clerodendrum trichotomum* medicinal material were determined as follows: Octadecylsilane-bonded silica gel was used as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B, with gradient elution as specified in the table below; the flow rate was 0.3 ml / min; the column temperature was 30℃; and the detection wavelength was 250 nm. The theoretical plate number, calculated based on the syringic acid peak, should not be less than 5000.

[0108]

[0109] 2.2.1.3 Investigation into the preparation of the test solution

[0110] 2.2.1.3.1 Examination of Sample Preparation Methods

[0111] Take 1g of *Clerodendrum trichotomum* powder (passed through a No. 3 sieve, batch number: QLY01), place it in a stoppered conical flask, add 25ml of 50% methanol, seal tightly, heat under reflux and sonicate (600W, 40kHz) for 30 minutes respectively, cool, shake well, filter, and collect the filtrate. Separately, take 1g of *Clerodendrum trichotomum* powder, place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 25ml of 50% methanol to the residue, sonicate (600W, 40kHz) for 30 minutes, shake well, filter, and collect the filtrate as the test solution. Figure 1 . Figure 1 Extraction methods were examined.

[0112] The results showed that the peaks were not significantly different when the test sample was extracted by boiling, reflux, and ultrasound. Ultrasonic extraction was simpler, so ultrasound was chosen as the extraction method for the test sample.

[0113] 2.2.1.3.2 Investigation of Extraction Solvents

[0114] Take 1g of *Clerodendrum trichotomum* powder (passed through a No. 3 sieve, batch number: QLY01), place it in a stoppered conical flask, add water, 30% methanol, 50% methanol, 70% methanol, and 25ml of methanol respectively, seal tightly, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the final product. Figure 2 . Figure 2 Investigation of extraction solvent.

[0115] The results showed that when the extraction solvent was 50% methanol, the peak shapes of each characteristic peak were good and the separation was moderate, so the extraction solvent was set at 50% methanol.

[0116] 2.2.1.3.3 Investigation of Solvent Addition Amount

[0117] Take 1g of *Clerodendrum trichotomum* powder (passed through a No. 3 sieve, batch number: QLY01), place it in a stoppered conical flask, and add 15ml, 25ml, and 50ml of 50% methanol respectively. Seal the flasks tightly, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate. See [link to flask description]. Figure 3 . Figure 3 Investigation of the amount of solvent added.

[0118] The results showed that when the extraction solvent volume was 25 ml, the peak shape and resolution of each chromatographic peak were good, and the peak size was appropriate. Therefore, the solvent volume was selected as 25 ml.

[0119] 2.2.1.3.4 Examination of extraction time

[0120] Take 1g of *Clerodendrum trichotomum* powder (passed through a No. 3 sieve, batch number: QLY01), place it in a stoppered conical flask, add 25ml of 50% methanol, seal tightly, and sonicate (600W power, 40kHz frequency) for 15 minutes, 30 minutes, and 60 minutes respectively. Cool, shake well, filter, and collect the filtrate. See [link to flask description]. Figure 4 . Figure 4 Extraction time was examined.

[0121] The results showed that extraction was complete at 30 minutes, with good peak shape and resolution in the chromatogram. Therefore, the extraction time was determined to be 30 minutes.

[0122] In summary, the preparation method of the test solution for the characteristic chromatogram of Qinglongyi medicinal material is determined as follows: Take 1g of Qinglongyi medicinal material powder (passed through a No. 3 sieve), place it in a stoppered conical flask, add 25ml of 50% methanol, seal tightly, and sonicate (power 600W, frequency 40kHz) for 130 minutes. After cooling, shake well, filter, and collect the filtrate to obtain the solution.

[0123] 2.2.1.4 Methodological Examination

[0124] 2.2.1.4.1 Chromatographic Peak Identification

[0125] Preparation of the test solution: Prepare the test solution of Qinglongyi medicinal material according to the experimental conditions proposed above.

[0126] Preparation of reference solutions: Take 1g of *Clerodendrum trichotomum* reference material, place it in a stoppered conical flask, add 25ml of 50% methanol, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, accurately weigh appropriate amounts of protocatechuic acid, vanillic acid, syringic acid, ellagic acid, rutin, and quercetin reference standards, and dissolve them in 50% methanol to prepare solutions containing 30μg of each per ml, as reference solutions.

[0127] Preparation of negative control solution: Prepare negative control solution for herbs lacking codonopsis pilosula according to the experimental conditions proposed above.

[0128] The characteristic spectral peaks of *Clerodendrum trichotomum* were located. (See...) Figure 5 . Figure 5 Chromatographic peak identification. The results showed that peak 1 was protocatechuic acid, peak 3 was vanillic acid, peak 4 (S) was syringic acid, peak 7 was ellagic acid, peak 8 was rutin, and peak 9 was quercetin. The following methodological investigation investigated the nine characteristic peaks in the sample.

[0129] 2.2.1.4.2 Precision Test

[0130] Take the test solution of *Clerodendrum trichotomum* (batch number: QLY01), and inject it 6 times consecutively according to the proposed experimental method, 2 μl each time. Calculate the retention time of each characteristic peak. See Table 1.

[0131] Table 1 Precision test - retention time

[0132]

[0133] The results showed that the retention time RSD of each characteristic peak of the sample ranged from 0.04% to 0.31%, indicating that the instrument had good precision.

[0134] 2.2.1.4.3 Repeatability Test

[0135] Six portions of Qinglongyi medicinal material (batch number: QLY01) were accurately weighed and prepared and measured according to the proposed experimental method. The relative retention times of each characteristic peak were calculated. See Table 2.

[0136] Table 2 Repeatability Tests - Relative Retention Time

[0137]

[0138] The results showed that the relative retention time (RSD) of the six samples ranged from 0.00% to 2.01%, indicating that the method had good reproducibility.

[0139] 2.2.1.4.4 Investigations by different personnel and at different times

[0140] Based on the experimental conditions outlined above, different personnel (A and B) precisely weighed one sample of Qinglongyi medicinal material (batch number: QLY01) at different times (T1 and T2) to prepare test samples for determination. See Table 3.

[0141] Table 3. Personnel and Time Assessment - Relative Retention Time

[0142]

[0143]

[0144] The results showed that when different personnel measured the same sample at different times, the RSD of the relative retention time of each characteristic peak was 0.00% to 0.43%, indicating good method stability.

[0145] 2.2.1.4.5 Column robustness test

[0146] Based on the above-planned experimental conditions, analyses were conducted using chromatographic columns 1 (Shim-pack GIST-HP C18-AQ 2.1*100mm, 1.6μm), 2 (InfinityLab Poroshell 120HILIC-Z 2.1*100mm, 2.7μm), and 3 (ZORBAX Eclipse Plus C18 2.1*100mm, 1.8μm), respectively. The results are shown in the table below. Figure 6 Table 4. Figure 6 Investigation of different chromatographic columns.

[0147] Table 4. Column robustness study - relative retention time

[0148]

[0149] 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 1.71% to 12.44%. The column robustness of this method was poor, while column 1 had better resolution. Therefore, column 1 is recommended.

[0150] 2.2.1.4.6 Instrument Durability Test

[0151] Based on the above-established experimental conditions, samples of *Qinglongyi* medicinal material (batch number: QLY01) were weighed and prepared into test solutions. These solutions were then analyzed on instrument 1 (Agilent 1290) and instrument 2 (Thermo VF), respectively. Results are shown below. Figure 7 Table 5.

[0152] Figure 7 Different instruments were used for the investigation.

[0153] Table 5 Instrument Durability Test - Relative Retention Time

[0154]

[0155] The results show that when the test sample is detected using the above two instruments, the RSD of the relative retention time of each characteristic peak is 0.00% to 2.32%, indicating that the instrument has good durability.

[0156] 2.2.1.4.7 Stability Test

[0157] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 3h, 6h, 9h, 15h, and 24h. See Table 6.

[0158] Table 6 Stability Study - Retention Time

[0159]

[0160] The results showed that the RSD of the retention time of the characteristic peak was between 0.20% and 1.60%, and the sample solution was stable within 24 hours.

[0161] 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 10 characteristic peaks will be included in subsequent investigations.

[0162] 2.2.1.4.8 Determination of characteristic peaks and establishment of reference spectra

[0163] Using this method, characteristic spectral analysis was performed on 14 batches of Qinglongyi medicinal material samples, and the relative retention times were calculated. (See attached image.) Figure 8 Table 13-14. Figure 8 The image shows a superimposed characteristic spectrum of 14 batches of Qinglongyi medicinal materials; among them: peak 1: protocatechuic acid; peak 3: vanillic acid; peak 4(S): syringic acid; peak 7: ellagic acid; peak 8: rutin; peak 9: quercetin.

[0164] (From bottom to top, S1-S14 are: QLY01, QLY02, QLY03, QLY04, QLY05, QLY06, QLY07, QLY08, QLY09, QLY10, QLY11, QLY12, QLY13, QLY14).

[0165] Table 7 shows the relative retention times of the characteristic chromatograms of 14 batches of medicinal materials.

[0166]

[0167]

[0168] Based on the principles of relatively stable retention time, detectability in all batches of samples, and relatively high peak values, nine peaks with good robustness were selected as characteristic peaks. According to the methodological investigation results and the validation results of 14 batches of medicinal materials, the theoretical plate number, calculated based on the syringic acid peak, is tentatively set to be no less than 5000.

[0169] The final specification stipulates that the chromatogram of the test sample should show 9 characteristic peaks, and the retention times should correspond to the 9 characteristic peaks in the chromatogram of the reference medicinal material. Peak 4 should correspond to the retention time of the reference peak, and the peak corresponding to the syringic acid reference peak is designated as peak S. The relative retention times of each characteristic peak and peak S should be calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.25 (peak 1), 0.29 (peak 2), 0.78 (peak 3), 1.20 (peak 5), 1.38 (peak 6), 1.73 (peak 7), 1.85 (peak 8), and 2.08 (peak 9).

[0170] The chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version) was used to synthesize chromatograms of 14 batches of *Clerodendrum trichotomum* (Qinglongyi) medicinal materials, and a reference chromatogram of the characteristic chromatograms of *Clerodendrum trichotomum* was established. See [link / reference]. Figure 9 . Figure 9 Characteristic spectrum of Qinglongyi medicinal material; Peak 1: protocatechuic acid; Peak 3: vanillic acid; Peak 4 (S): syringic acid; Peak 7: ellagic acid; Peak 8: rutin; Peak 9: quercetin.

[0171] In summary, the feature map method is as follows:

[0172] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 0.3 mL / min; column temperature 30 °C; detection wavelength 250 nm. The theoretical plate number, calculated based on the eugenol peak, should be no less than 5000.

[0173]

[0174] Preparation of the reference solution: Take 1g of *Clerodendrum trichotomum* reference material, place it in a stoppered conical flask, add 25ml of 50% methanol, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate. Separately, accurately weigh appropriate amounts of protocatechuic acid, vanillic acid, syringic acid, ellagic acid, rutin, and quercetin reference standards, and dissolve them in 50% methanol to prepare a solution containing 30μg of each per ml, as the reference solution.

[0175] Preparation of the test solution: Take 1.0 g of the powder (passed through a No. 3 sieve), place it in a stoppered conical flask, add 25 ml of 50% methanol, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0176] The determination method involves precisely pipetting 2 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0177] The chromatogram of the test sample should show 9 characteristic peaks, and the retention times should correspond to the 9 characteristic peaks in the chromatogram of the reference medicinal material. Peak 4 should correspond to the retention time of the reference peak, and the peak corresponding to the syringic acid reference peak is designated as peak S. The relative retention times of each characteristic peak and peak S should be calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.25 (peak 1), 0.29 (peak 2), 0.78 (peak 3), 1.20 (peak 5), 1.38 (peak 6), 1.73 (peak 7), 1.85 (peak 8), and 2.08 (peak 9).

[0178] Example 2: UPLC Characteristic Chromatography of Qinglongyi Standard Decoction

[0179] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 0.3 mL / min; column temperature 30 °C; detection wavelength 250 nm. The theoretical plate number, calculated based on the eugenol peak, should be no less than 5000.

[0180]

[0181]

[0182] Preparation of reference solutions: Take 1g of *Clerodendrum trichotomum* reference material, place it in a stoppered conical flask, add 25ml of 50% methanol, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, accurately weigh appropriate amounts of protocatechuic acid, vanillic acid, syringic acid, ellagic acid, rutin, and quercetin reference standards, and dissolve them in 50% methanol to prepare solutions containing 30μg of each per ml, as reference solutions.

[0183] Preparation of the test solution: Take 0.2g of Qinglongyi standard decoction, place it in a stoppered conical flask, add 25ml of 50% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0184] The determination method involves precisely pipetting 2 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0185] 2.2.2.2 Chromatographic conditions and system suitability test

[0186] The standard decoction and the finished granules have the same basic material composition, and the characteristic chromatographic conditions, system adaptability, and granule characteristic chromatographic methods are consistent.

[0187] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile was used as mobile phase A, and 0.1% 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 0.3 mL / min; the column temperature was 30 °C; and the detection wavelength was 250 nm. The theoretical plate number, calculated based on the eugenol peak, should not be less than 5000.

[0188]

[0189] 2.2.2.3 Examination of Sample Preparation Methods

[0190] The preparation of the test solution is the same as that of the Qinglongyi formula granules, specifically: take 0.2g of this product (batch number: QLY-BT01), place it in a stoppered conical flask, add 25ml of 50% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0191] 2.2.2.4 Methodological Examination

[0192] 2.2.2.4.1 Chromatographic Peak Identification

[0193] Preparation of the test solution: Prepare the test solution of the Qinglongyi standard decoction according to the experimental conditions proposed above.

[0194] Preparation of reference solutions: Take 1g of *Clerodendrum trichotomum* reference material, place it in a stoppered conical flask, add 25ml of 50% methanol, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, accurately weigh appropriate amounts of protocatechuic acid, vanillic acid, syringic acid, ellagic acid, rutin, and quercetin reference standards, and dissolve them in 50% methanol to prepare solutions containing 30μg of each per ml, as reference solutions.

[0195] Preparation of negative control solution: Prepare negative control solution of standard decoction lacking coix seed according to the experimental conditions proposed above.

[0196] The characteristic peaks of the standard decoction of *Clerodendrum trichotomum* were located. (See...) Figure 10 . Figure 10 Chromatographic peak identification.

[0197] The results showed that peak 1 was protocatechuic acid, peak 3 was vanillic acid, peak 4 (S) was syringic acid, peak 8 was ellagic acid, peak 9 was rutin, and peak 10 was quercetin. The following methodological investigations were conducted on the 10 characteristic peaks in the sample.

[0198] 2.2.2.4.2 Precision Test

[0199] Take the test solution of the standard decoction of *Clerodendrum trichotomum* (batch number: QLY-BT01), and inject it 6 times consecutively according to the proposed experimental method, 2 μl each time, and calculate the retention time of each characteristic peak. See Table 8.

[0200] Table 8 Precision Examination - Retention Time

[0201]

[0202] The results showed that the retention time RSD of each characteristic peak was 0.20% to 0.79%, indicating that the instrument has good precision.

[0203] 2.2.2.4.3 Repeatability Test

[0204] Accurately weigh 6 portions of the Qinglongyi standard decoction (batch number: QLY-BT01), prepare and determine it according to the proposed experimental method, and calculate the relative retention time of each characteristic peak. See Table 9.

[0205] Table 9 Repeatability Tests - Relative Retention Time

[0206]

[0207] The results showed that the relative retention time RSD of the six samples was 0.00%–1.88%, indicating that the method had good reproducibility.

[0208] 2.2.2.4.4 Investigations by different personnel and at different times

[0209] Based on the experimental conditions outlined above, different personnel (A and B) precisely weighed the Qinglongyi standard decoction (batch number: QLY-BT01) at different times (T1 and T2) to prepare test samples, and then measured and calculated the relative retention time and relative peak area of ​​each characteristic peak. See Table 10.

[0210] Table 10 Personnel and Time Assessment - Relative Retention Time

[0211]

[0212] The results showed that when different personnel measured the same sample at different times, the RSD of the relative retention time of each characteristic peak was 0.00% to 2.64%, indicating good method stability.

[0213] 2.2.2.4.5 Column robustness test

[0214] Based on the above-planned experimental conditions, chromatographic columns were used respectively: column 1 (Shim-pack GIST-HP C18-AQ 2.1*100mm, 1.6μm) and column 2 (InfinityLabPoroshell 120HILIC-Z).

[0215] The analysis was conducted using a chromatographic column (2.1*100mm, 2.7μm) and column 3 (ZORBAX Eclipse Plus C18 2.1*100mm, 1.8μm). The results are shown in the figure. Figure 11 Tables 21-22. Figure 11 Investigation of different chromatographic columns.

[0216] Table 11 Column Analysis - Relative Retention Times

[0217]

[0218] 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 1.71% to 12.44%. The column robustness of this method was poor, while column 1 had better resolution; column 1 is recommended.

[0219] 2.2.2.4.6 Instrument Durability Test

[0220] Based on the above-planned experimental conditions, analysis and investigation were conducted using instrument 1 (Agilent 1290) and instrument 2 (Thermo VF), respectively. The results are shown in [Figure 1]. Figure 12 Table 12. Figure 12 Different instruments were used for the investigation.

[0221] Table 12 Column robustness study - relative retention time

[0222]

[0223] The results show that when the test sample is detected using the above two instruments, the RSD of the relative retention time of each characteristic peak is 0.00% to 2.32%, indicating that the instrument has good durability.

[0224] 2.2.2.4.7 Stability Assessment

[0225] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 3h, 6h, 9h, 15h, and 24h. See Table 13.

[0226] Table 13 Stability Study - Retention Time

[0227]

[0228] The results showed that the RSD of the retention time of the characteristic peaks ranged from 0.20% to 1.58%, and the sample solution was stable within 24 hours. In summary, the RSD of the relative retention time of each characteristic peak met the requirements in the above investigations, indicating that the method is effective. The above nine characteristic peaks will be included in subsequent investigations.

[0229] 2.2.2.4.8 Determination of characteristic peaks and establishment of reference spectra

[0230] Using this method, characteristic spectral analysis was performed on 14 batches of samples, and the relative retention times were calculated. See... Figure 13 Table 14. Figure 13Superimposed characteristic chromatograms of 14 batches of Qinglongyi standard decoction; Peak 1: protocatechuic acid; Peak 3: vanillic acid; Peak 4 (S): syringic acid; Peak 7: ellagic acid; Peak 8: rutin; Peak 9: quercetin; from bottom to top, S1-S14 are: QLY-BT01, QLY-BT02, QLY-BT03, QLY-BT04, QLY-BT05, QLY-BT06, QLY-BT07, QLY-BT08, QLY-BT09, QLY-BT10, QLY-BT11, QLY-BT12, QLY-BT13, QLY-BT14).

[0231] Table 14 shows the relative retention times of 14 batches of Qinglongyi standard decoction.

[0232]

[0233] Based on the principles of relatively stable retention time, detectability in all batches of samples, and relatively high peak values, nine robust peaks were selected as characteristic peaks. Based on the methodological investigation results and validation results from 14 batches of standard solutions, the theoretical plate number, calculated using the syringic acid peak, is tentatively set at no less than 5000.

[0234] The final specification stipulates that the chromatogram of the test sample should show 9 characteristic peaks, and the retention times should correspond to the 9 characteristic peaks in the chromatogram of the reference medicinal material. Peak 4 should correspond to the retention time of the reference peak, and the peak corresponding to the syringic acid reference peak is designated as peak S. The relative retention times of each characteristic peak and peak S should be calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.25 (peak 1), 0.29 (peak 2), 0.78 (peak 3), 1.20 (peak 5), 1.38 (peak 6), 1.73 (peak 7), 1.85 (peak 8), and 2.08 (peak 9).

[0235] The chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version) was used to synthesize chromatograms of 14 batches of Qinglongyi standard decoction, and a reference chromatogram of the characteristic chromatograms of Qinglongyi standard decoction was established. See [link / reference]. Figure 14 . Figure 14 Characteristic spectrum of Qinglongyi standard decoction; Peak 1: protocatechuic acid; Peak 3: vanillic acid; Peak 4 (S): syringic acid; Peak 7: ellagic acid; Peak 8: rutin; Peak 9: quercetin.

[0236] In summary, the feature map method is as follows:

[0237] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 0.3 mL / min; column temperature 30 °C; detection wavelength 250 nm. The theoretical plate number, calculated based on the eugenol peak, should be no less than 5000.

[0238]

[0239]

[0240] Preparation of reference solutions: Take 1g of *Clerodendrum trichotomum* reference material, place it in a stoppered conical flask, add 25ml of 50% methanol, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, accurately weigh appropriate amounts of protocatechuic acid, vanillic acid, syringic acid, ellagic acid, rutin, and quercetin reference standards, and dissolve them in 50% methanol to prepare solutions containing 30μg of each per ml, as reference solutions.

[0241] Preparation of the test solution: Take 0.2 g of this product, place it in a stoppered conical flask, add 25 ml of 50% methanol, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0242] The determination method involves precisely pipetting 2 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0243] The chromatogram of the test sample should show 9 characteristic peaks, and the retention times should correspond to the 9 characteristic peaks in the chromatogram of the reference medicinal material. Peak 4 should correspond to the retention time of the reference peak, and the peak corresponding to the syringic acid reference peak is designated as peak S. The relative retention times of each characteristic peak and peak S should be calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.25 (peak 1), 0.29 (peak 2), 0.78 (peak 3), 1.20 (peak 5), 1.38 (peak 6), 1.73 (peak 7), 1.85 (peak 8), and 2.08 (peak 9).

[0244] Example 3: UPLC Characteristic Spectrum of Qinglongyi Formula Granules

[0245] 2.1 Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile was used as mobile phase A, and 0.1% 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 0.3 mL / min; the column temperature was 30 °C; and the detection wavelength was 250 nm. The theoretical plate number, calculated based on the eugenol peak, should not be less than 5000.

[0246]

[0247] Preparation of reference solutions: Take 1g of *Clerodendrum trichotomum* reference material, place it in a stoppered conical flask, add 25ml of 50% methanol, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, accurately weigh appropriate amounts of protocatechuic acid, vanillic acid, syringic acid, ellagic acid, rutin, and quercetin reference standards, and dissolve them in 50% methanol to prepare solutions containing 30μg of each per ml, as reference solutions.

[0248] Preparation of the test solution: Take 0.2g of Qinglongyi formula granules, place them in a stoppered conical flask, add 25ml of 50% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0249] The determination method involves precisely pipetting 2 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0250] 2.2.3.2 Wavelength, Column Temperature, Flow Rate, Mobile Phase Selection and Delay Test

[0251] 2.2.3.2.1 Wavelength Selection

[0252] Based on the experimental conditions (2.1) outlined above, 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 190 nm, 210 nm, 230 nm, 250 nm, 270 nm, 290 nm, 300 nm, and 330 nm. See [link to chromatogram]. Figure 15 . Figure 15 Chromatograms of Qinglongyi formula granules at different wavelengths.

[0253] The results showed that the chromatographic peak information was greater and the chromatographic baseline was more stable when the detection wavelength was 250 nm, so the detection wavelength was determined to be 250 nm.

[0254] 2.2.3.2.2 Mobile Phase Selection

[0255] Based on the experimental conditions (2.1) proposed above, the separation effects of three different mobile phases were investigated: acetonitrile-0.1% formic acid, acetonitrile-0.1% phosphoric acid, and acetonitrile-water. (See attached diagram.) Figure 16 . Figure 16 Mobile phase selection.

[0256] The results showed that the chromatogram baseline was relatively stable and uniformly distributed under the gradient elution condition of acetonitrile-0.1% formic acid solution. Therefore, gradient elution of acetonitrile-0.1% formic acid solution was used as the mobile phase for the determination of the characteristic chromatogram of Qinglongyi formulation particles.

[0257] 2.2.3.2.3 Column Temperature Investigation

[0258] Based on the experimental conditions (2.1) proposed above, the results were investigated at column temperatures of 25℃, 30℃, and 35℃. The results are shown below. Figure 17 Tables 15-16. Figure 17 Column temperature investigation.

[0259] Table 15 Column Temperature Study - Retention Time

[0260]

[0261] Table 16 Column Temperature Study - Relative Retention Time

[0262]

[0263]

[0264] The results showed that at a column temperature of 30℃, the chromatogram peaks were more symmetrical, the resolution was better, and the peaks were more complete. Therefore, the column temperature was determined to be 30℃.

[0265] 2.2.3.2.4 Flow velocity investigation

[0266] Based on the experimental conditions (2.1) proposed above, flow rates of 0.25 ml / min, 0.30 ml / min, and 0.35 ml / min were investigated. The results are shown below. Figure 18 Table 17-18. Figure 18 Flow velocity study.

[0267] Table 17 Flow velocity study - retention time

[0268]

[0269] Table 18 Flow velocity study - relative retention time

[0270]

[0271] The results showed that when the flow rate was 0.3 ml / min, the chromatogram had a good peak shape, moderate resolution, and complete peak elution. Therefore, the flow rate was determined to be 0.3 ml / min. 2.2.3.2.5 Investigation of Delay

[0272] Based on the above-determined experimental conditions (2.1), a delay test was conducted. The results are shown in Figure 19 . Figure 19 Investigation of Delay

[0273] The results showed that there were basically no chromatographic peaks after 40 minutes for the sample. Therefore, the sample detection time was set at 40 minutes.

[0274] To sum up, the chromatographic conditions and system suitability test for the characteristic chromatogram of Qinglongyi formula granules were determined as follows: Octadecylsilane-bonded silica gel was used as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B, and gradient elution was carried out according to the regulations in the following table; the flow rate was 0.3 ml per minute; the column temperature was 30 °C; the detection wavelength was 250 nm. The theoretical plate number calculated based on the syringic acid peak should be not less than 5000.

[0275]

[0276] 2.2.4.3 Investigation of the Preparation of Test Solution

[0277] 2.2.3.3.1 Investigation of Extraction Method

[0278] Take 0.2 g of Qinglongyi formula granules (batch number: QLY-KL01), grind them finely, place them in a stoppered conical flask, add 25 ml of 50% methanol, tightly stopper, heat under reflux and perform ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes respectively, cool, shake well, filter, and take the subsequent filtrate to obtain the solution, as shown in Figure 20 . Figure 20 Investigation of Sample Preparation Method

[0279] The results showed that there was little difference in the extraction effects of reflux and ultrasonic extraction for the test solution, and the ultrasonic method was fast and simple. Therefore, the extraction method for the test solution was determined to be ultrasonic extraction.

[0280] 2.2.3.3.2 Investigation of Extraction Solvent

[0281] Take 0.2 g of Qinglongyi formula granules (batch number: QLY-KL01), grind them finely, place them in a stoppered conical flask, and add 25 ml of water, 30% methanol, 50% methanol, 70% methanol, and methanol respectively, tightly stopper, perform ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the subsequent filtrate to obtain the solution. As shown in Figure 21 . Figure 21 Investigation of Extraction Solvent

[0282] The results showed that using 50% methanol as the extraction solvent yielded chromatographic peaks with a large amount of information, good peak shapes, and moderate resolution. The extraction solvent was tentatively set as 50% methanol.

[0283] 2.2.3.3.3 Investigation of Solvent Addition Amount

[0284] Take 0.2g of the Qinglongyi formula granules (batch number: QLY-KL01), grind them finely, and place them in stoppered conical flasks. Add 15ml, 25ml, and 50ml of 50% methanol respectively, seal tightly, and sonicate (600W power, 40kHz frequency) for 30 minutes. Cool, shake well, filter, and collect the filtrate to obtain the final product. See [link to product description] Figure 22 . Figure 22 Investigation of the amount of solvent added.

[0285] The results showed that the chromatographic peak size was more suitable when the extraction solvent volume was 25 ml, so the solvent volume was selected as 25 ml.

[0286] 2.2.3.3.4 Examination of extraction time

[0287] Take 0.2g of the Qinglongyi formula granules (batch number: QLY-KL01), grind them finely, place them in a stoppered conical flask, add 25ml of 50% methanol, seal tightly, and sonicate (600W power, 40kHz frequency) for 15 minutes, 30 minutes, and 60 minutes respectively. Cool, shake well, filter, and collect the filtrate to obtain the final product. (See...) Figure 23 . Figure 23 Extraction time was examined.

[0288] The results showed that extraction was complete at a time of 30 minutes. Therefore, the extraction time was determined to be 30 minutes.

[0289] In summary, the preparation method of the test solution of the Qinglongyi formula granule spectrum is determined as follows: Take 0.2g of this product, grind it into a fine powder, place it in a stoppered conical flask, add 25ml of 50% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0290] 2.2.3.4 Methodological Examination

[0291] 2.2.3.4.1 Chromatographic Peak Identification

[0292] Preparation of test solution: Prepare test solution of Qinglongyi formula granules according to the experimental conditions (2.1) above.

[0293] Preparation of reference solution: Take 1 g of the control crude drug of Qinglongyi, put it into a stoppered conical flask, add 25 ml of 50% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, take the continuous filtrate as the reference solution of the control crude drug. Separately weigh a proper amount of syringic acid reference substance accurately, dissolve it in 50% methanol to prepare a solution containing 15 μg per 1 ml as the reference solution of the reference substance. Separately weigh proper amounts of protocatechuic acid, vanillic acid, syringic acid, ellagic acid, rutin, and quercitrin reference substances accurately, dissolve them in 50% methanol to prepare a solution containing 30 μg per 1 ml each, and that's it.

[0294] Preparation of negative control solution: According to the above-mentioned determined experimental conditions, prepare the negative control solution of the Qinglongyi formula granules without Qinglongyi.

[0295] Locate the characteristic peaks of the Qinglongyi formula granules. See Figure 24 . Figure 24 Chromatographic peak identification;

[0296] The results show that peak 1 is protocatechuic acid, peak 3 is vanillic acid, peak 4 (S) is syringic acid, peak 8 is ellagic acid, peak 9 is rutin, and peak 10 is quercitrin. In the following methodological investigations, 10 characteristic peaks in the samples were investigated.

[0297] 2.2.3.4.2 Precision test

[0298] Take the test solution of the Qinglongyi formula granules (batch number: QLY-KL01), inject 2 μl each time continuously for 6 times according to the determined experimental method, and calculate the retention time of each characteristic peak. See Table 19.

[0299] Table 19 Precision investigation - retention time

[0300]

[0301] The results show that the RSD of the retention time of each characteristic peak is 0.04% - 0.19%, indicating that the precision of this instrument is good.

[0302] 2.2.3.4.3 Repeatability investigation

[0303] Accurately weigh 6 portions of the Qinglongyi formula granules (batch number: QLY-KL01), prepare and determine them according to the determined experimental method. See Tables 39 - 40.

[0304] Table 20 Repeatability investigation - relative retention time

[0305]

[0306] The results show that the RSD of the relative retention time of 6 samples is 0.00% - 1.53%, indicating that the repeatability of this method is good.

[0307] 2.2.3.4.4 Investigations by different personnel and at different times

[0308] Based on the experimental conditions outlined above, different personnel (A and B) precisely weighed one portion of the Qinglongyi formula granules (batch number: QLY-KL01) at different times (T1 and T2) to prepare test samples for determination. See Table 21.

[0309] Table 21 Personnel and Time Assessment - Relative Retention Time

[0310]

[0311]

[0312] The results showed that when different personnel measured the same sample at different times, the RSD of the relative retention time of each characteristic peak was 0.00% to 2.85%, indicating good method stability.

[0313] 2.2.3.4.5 Column robustness test

[0314] Based on the above-planned experimental conditions, analyses were conducted using chromatographic columns 1 (Shim-pack GIST-HP C18-AQ 2.1*100mm, 1.6μm), 2 (InfinityLab Poroshell 120HILIC-Z 2.1*100mm, 2.7μm), and 3 (ZORBAX Eclipse Plus C18 2.1*100mm, 1.8μm), respectively. The results are shown in the table below. Figure 25 Table 22. Figure 25 Investigation of different chromatographic columns.

[0315] Table 22 Column robustness study - relative retention time

[0316]

[0317] The results showed that when using the above three chromatographic columns to detect samples, the RSD of the relative retention time of the characteristic peaks ranged from 1.96% to 12.44%. The column robustness of this method was poor, while column 1 had better resolution; column 1 is recommended.

[0318] 2.2.3.4.6 Instrument Durability Test

[0319] Based on the above-planned experimental conditions, analysis and investigation were conducted using instrument 1 (Agilent 1290) and instrument 2 (Thermo VF), respectively. The results are shown in [Figure 1]. Figure 26 Table 23. Figure 26 Different instruments were used for the investigation.

[0320] Table 23 Column robustness study - relative retention time

[0321]

[0322] The results show that when the test sample is detected using the above two instruments, the RSD of the relative retention time of each characteristic peak is 0.00% to 0.92%, indicating that the instrument has good durability.

[0323] 2.2.3.4.7 Stability Test

[0324] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 3h, 6h, 9h, 15h, and 24h. See Table 24.

[0325] Table 24 Stability Study - Retention Time

[0326]

[0327] The results showed that the RSD of the retention time of the characteristic peak was between 0.21% and 1.49%, and the sample solution was stable within 24 hours.

[0328] 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 10 characteristic peaks will be included in subsequent investigations.

[0329] 2.2.3.4.8 Determination of characteristic peaks and establishment of reference spectra

[0330] Using this method, characteristic spectral analysis was performed on three batches of samples, and the relative retention times were calculated. See [link / reference]. Figure 27 Table 25. Figure 27 Superimposed characteristic chromatograms of three batches of Qinglongyi formula granules; Peak 1: protocatechuic acid; Peak 3: vanillic acid; Peak 4 (S): syringic acid; Peak 7: ellagic acid; Peak 8: rutin; Peak 9: quercetin.

[0331] Table 25 shows the relative retention times of the three batches of Qinglongyi formula granules.

[0332]

[0333]

[0334] Based on the principles of relatively stable retention time, detectability in all batches of samples, and relatively high peak values, a total of 9 peaks were selected as characteristic peaks. According to the methodological investigation results and the particle validation results of 3 batches, the theoretical plate number, calculated based on the syringic acid peak, is tentatively set to be no less than 5000.

[0335] Final Specification: Nine characteristic peaks should be presented in the chromatogram of the test sample, and their retention times should correspond to those of the nine characteristic peaks in the reference chromatogram of the reference crude drug. Among them, the retention time of Peak 4 should correspond to that of the reference peak of the reference substance, and the peak corresponding to the reference peak of syringic acid is the S peak. Calculate the relative retention times of each characteristic peak to the S peak, and the relative retention times should be within the range of ±10% of the specified values. The specified values are: 0.25 (Peak 1), 0.29 (Peak 2), 0.78 (Peak 3), 1.20 (Peak 5), 1.38 (Peak 6), 1.73 (Peak 7), 1.85 (Peak 8), 2.08 (Peak 9).

[0336] Using the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition), the fingerprints of 3 batches of Qinglongyi formula granules were synthesized to establish a reference chromatogram of the characteristic chromatogram of Qinglongyi formula granules. See Figure 28 . Figure 28 Reference Characteristic Chromatogram of Qinglongyi Formula Granules; Peak 1: Protocatechuic Acid; Peak 3: Vanillic Acid; Peak 4 (S): Syringic Acid; Peak 7: Ellagic Acid; Peak 8: Rutin; Peak 9: Quercitrin.

[0337] In summary, the method for the characteristic chromatogram is as follows:

[0338] Chromatographic Conditions and System Suitability Test Using octadecylsilane chemically bonded silica as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, and gradient elution was carried out according to the regulations in the following table; the flow rate was 0.3 ml per minute; the column temperature was 30 °C; the detection wavelength was 250 nm. The number of theoretical plates calculated based on the syringic acid peak should be not less than 5000.

[0339]

[0340] Preparation of Reference Solution Weigh 1 g of the reference crude drug of Qinglongyi, place it in a stoppered conical flask, add 25 ml of 50% methanol, ultrasonically treat it (power 600 W, frequency 4 kHz) for 30 minutes, cool it, shake it well, filter it, and take the subsequent filtrate as the reference crude drug reference solution. Separately weigh appropriate amounts of reference substances of protocatechuic acid, vanillic acid, syringic acid, ellagic acid, rutin, and quercitrin, accurately weigh them, and dissolve them in 50% methanol to prepare a solution containing 30 μg of each per 1 ml as the reference substance reference solution.

[0341] Preparation of Test Solution Weigh 0.2 g of this product, place it in a stoppered conical flask, add 25 ml of 50% methanol, ultrasonically treat it (power 600 W, frequency 4 kHz) for 30 minutes, cool it, shake it well, filter it, and take the subsequent filtrate, which is the test solution.

[0342] Determination Method Precisely pipette 2 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine, thus obtaining the results.

[0343] The chromatogram of the test sample should show 9 characteristic peaks, and the retention times should correspond to the 9 characteristic peaks in the chromatogram of the reference medicinal material. Peak 4 should correspond to the retention time of the reference peak, and the peak corresponding to the syringic acid reference peak is designated as peak S. The relative retention times of each characteristic peak and peak S should be calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.25 (peak 1), 0.29 (peak 2), 0.78 (peak 3), 1.20 (peak 5), 1.38 (peak 6), 1.73 (peak 7), 1.85 (peak 8), and 2.08 (peak 9).

[0344] Comparative Example 1

[0345] The pretreatment is the same as in Embodiment 1 of this invention.

[0346] The chromatographic column was an Agilent Poroshell 120SB-C18 (2.1 mm × 100 mm, 2.7 μm). Mobile phase A was 0.1% formic acid in water, and mobile phase B was acetonitrile. Gradient elution was used (0–5 min, 95%–87% A; 5–15 min, 87%–84% A; 15–25 min, 84%–78% A; 25–40 min, 78%–70% A; 40–42 min, 70%–0% A; 42–47 min, 0% A). The flow rate was 0.3 mL·min⁻¹. The detection wavelength for fingerprint chromatogram was 260 nm, and the detection wavelength for content determination was 325 nm. The column temperature was 30 °C, and the injection volume was 5 μL.

[0347] Figure 29 This is the chromatogram of *Clerodendrum trichotomum* medicinal material produced under the chromatographic conditions of Comparative Example 1 in our laboratory. Figure 29 The results show that the chromatographic method in Comparative Example 1, when used to detect the characteristic chromatograms of *Clerodendrum trichotomum*, has poor separation and indistinct characteristic peaks, making it impossible to effectively identify *Clerodendrum trichotomum* and indicating poor durability.

[0348] Comparative Example 2

[0349] Weigh 1g of the pulverized and sieved *Cynanchum paniculatum* sample powder accurately. Using a stoppered conical flask, add 50ml of 80% methanol, weigh accurately, and extract ultrasonically for 30min. Cool, replenish the lost weight with 80% methanol, and filter through a 0.45μm microporous membrane. UPLC was used with an Agilent TC-18 column (4.6mm × 250mm, 5μm); A-acetonitrile, B-0.1% phosphoric acid aqueous solution, binary linear gradient elution; flow rate: 1ml / min; column temperature: 30℃; detection wavelength: 274nm; injection volume: 10μl.

[0350]

[0351] Figure 30 The chromatogram of the Qinglongyi herb under the chromatographic conditions in Comparative Example 2 is shown.

[0352] The results show that most of the chromatographic peaks in Comparative Example 2 have poor resolution and symmetry factor. When the chromatographic method in Comparative Example 2 is used to detect the characteristic chromatographic spectrum of Qinglongyi medicinal material, the chromatographic peaks are densely concentrated in the middle section of the collection time, and the chromatographic peak resolution is poor, indicating that the robustness of this chromatographic method is not good.

[0353] Comparative Example 3

[0354] The pretreatment method is as follows: Step a: The dried *Cynanchum paniculatum* herb is pulverized into powder, then soaked in ethanol three times and filtered. Each soaking time is 48 hours. The filtrates are combined and the solvent is recovered by vacuum distillation to obtain *Cynanchum paniculatum* extract. Step b: Approximately 4g of *Cynanchum paniculatum* extract is added to 5mL of water, stirred thoroughly, allowed to stand for 30 minutes, filtered, and the filtrate is adsorbed onto an AB-8 type macroporous resin column. Then, it is eluted with distilled water until the effluent is colorless. The eluent obtained by distillation is discarded, and then eluted with a 30% (v / v) ethanol solution until the effluent is colorless. The effluent is collected and eluted with a 30% (v / v) ethanol solution. The eluent obtained from the liquid elution was subjected to vacuum distillation to recover the solvent, and dried to obtain dry solid A; Step c: The dry solid A obtained in step b was dissolved in distilled water, and then the pH was adjusted to 2 with a 1% hydrochloric acid solution. It was then extracted three times with ethyl acetate, the volume of ethyl acetate being equal to the volume of distilled water used to dissolve the dry solid A. The extracts were then combined, and the solvent was recovered by vacuum distillation. After drying, dry solid B was obtained; Step d: The dry solid B obtained in step c was accurately weighed, placed in a volumetric flask, dissolved in methanol to prepare a solution with a concentration of 400 μg / mL, filtered, and the filtrate was used as the test solution.

[0355] The chromatographic column was a YMC-Pack ODS AC18 (5 μm, 250 × 4.6 mm); the mobile phase was 0.2% phosphoric acid water (A).

[0356] Methanol-0.2% phosphoric acid aqueous solution (9:1) (B); Detection wavelength: 259nm; Column temperature: 35℃.

[0357] The gradient elution program was as follows: 0 min~35 min→35 min~60 min→60 min~100 min→100 min~130 min. From 0 min to 35 min, the volume concentration of solution B in the mixed mobile phase was increased uniformly from 15% to 20%. From 35 min to 60 min, the volume concentration of solution B in the mixed mobile phase was increased uniformly from 20% to 30%. From 60 min to 100 min, the volume concentration of solution B in the mixed mobile phase was increased uniformly from 30% to 40%. During the time period from 100 min to 130 min, the volume concentration of solution B in the mixed mobile phase was maintained at 40%.

[0358] The chromatographic conditions and sample preparation method of Comparative Example 3 were used. Figure 31 Chromatogram of Qinglongyi medicinal material under chromatographic conditions in Comparative Example 3.

[0359] The results show that the chromatographic peaks were poorly separated and the characteristic peaks were not clearly distinguishable within the first 20 minutes of the collection time. After 20 minutes of the collection time, the chromatographic peaks were not formed. Therefore, the results could not effectively identify the blue dragon skin and the durability was poor.

[0360] 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 detecting the UPLC characteristic spectra of Qinglongyi medicinal material, standard decoction, and formulated granules, comprising: The sample raw material is pretreated to obtain the test solution; the pretreatment includes solvent dissolution and extraction; The test solution was analyzed by UPLC to obtain the UPLC characteristic spectra of the Qinglongyi medicinal material, standard decoction, and formula granules; the solvent was 50% methanol; Preparation of reference solutions: Protocatechuic acid, vanillic acid, syringic acid, ellagic acid, rutin, and quercetin were dissolved in solvents to obtain reference solutions. The UPLC conditions were as follows: the column was a C18 column; mobile phase A was acetonitrile solution, mobile phase B was 0.1% formic acid aqueous solution, and gradient elution was used. The gradient elution specifically refers to: 0–6 min, Phase A: 2%, Phase B: 98%; 6–35 min, Phase A: 2%–18%, Phase B: 98%–82%; 35–40 min, Phase A: 18%–22%, Phase B: 82%–78%.

2. The method according to claim 1, characterized in that, This also includes the preparation of reference solutions of control medicinal materials: The reference herb *Clerodendrum trichotomum* was dissolved in 50% methanol to obtain a reference herb solution. Both the reference herb solution and the reference herb solution were injected into a high-performance liquid chromatograph (HPLC) for analysis, and chromatograms of the reference herb peaks and characteristic chromatograms of the reference herb were obtained. Based on the characteristic parameters of the chromatogram peaks of the reference herb, the corresponding chromatograms in the UPLC characteristic chromatograms of the *Clerodendrum trichotomum* sample, the standard decoction sample, and the formulated granule sample were compared and qualitatively analyzed.

3. The method according to claim 2, characterized in that, The specific injection concentrations of the reference solutions are as follows: protocatechuic acid 30 μg / mL, vanillic acid 30 μg / mL, syringic acid 30 μg / mL, ellagic acid 30 μg / mL, rutin 30 μg / mL, and quercetin 30 μg / mL.

4. The method according to claim 1, characterized in that, The chromatographic column has the specifications of 100×2.1mm 1.6μm; the column temperature is 25~30℃; the theoretical plate number calculated based on syringic acid should not be less than 5000.

5. The method according to claim 4, characterized in that, The detection wavelength is 250nm.

6. The method according to claim 4, characterized in that, The flow rate of the mobile phase was 0.3 mL / min; the injection volume was 2 μL.

7. The method according to claim 4, characterized in that, The extraction was achieved using an ultrasonic-assisted extraction method, wherein the power of the ultrasonic extraction device was set to 600W, the ultrasonic vibration frequency was 40kHz, and the duration of ultrasonic extraction was 30 minutes.

8. The method according to claim 1, characterized in that, The ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent is 0.2~1:25; The raw materials for the test sample are one or more of the following: Qinglongyi medicinal material, Qinglongyi standard decoction, or Qinglongyi formula granules.

9. The method according to claim 1, characterized in that, The similarity of UPLC characteristic spectra of Qinglongyi herbal material, standard decoction, and formula granules was evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system. A standard UPLC characteristic spectrum consisting of 9 characteristic peaks was constructed, and the chemical composition information corresponding to each characteristic peak is as follows: Peak 1: protocatechuic acid; Peak 3: vanillic acid; Peak 4: syringic acid; Peak 7: ellagic acid; Peak 8: rutin; Peak 9: quercetin. In the characteristic chromatograms of the Qinglongyi medicinal material, standard decoction, and formula granules, eugenol 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: 0.25 (peak 1), 0.29 (peak 2), 0.78 (peak 3), 1.20 (peak 5), 1.38 (peak 6), 1.73 (peak 7), 1.85 (peak 8), and 2.08 (peak 9).

10. A method for identifying the characteristic chromatograms of *Clerodendrum trichotomum* medicinal material, standard decoction, and formulated granules, characterized in that... The detection is performed using the method described in any one of claims 1 to 9, and the detection results are analyzed.

Images