A method for determining the content of ferulic acid in a standard decoction of *Cinnamomum camphora* and a method for constructing its characteristic chromatogram.

Abstract

This invention relates to the field of pharmaceutical analysis technology, and in particular to a method for determining the content of ferulic acid in a standard decoction of *Cephalotaxus fortunei* and a method for constructing a characteristic chromatogram. The determination is performed using high-performance liquid chromatography (HPLC). The HPLC conditions include: a column packed with octadecylsilane-bonded silica gel; acetonitrile as mobile phase A; and a 0.1–0.3% (v / v) aqueous solution of phosphoric acid as mobile phase B, with gradient elution. This method exhibits high specificity, good repeatability and robustness, and high stability and accuracy. The constructed characteristic chromatogram and detection method can rapidly and comprehensively achieve the joint control of multiple components in the standard decoction of *Cephalotaxus fortunei*, realizing quality control of *Cephalotaxus fortunei* from raw material to preparation, which is beneficial to ensuring the effectiveness of clinical medication.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a method for determining the content of ferulic acid in a standard decoction of *Cinnamomum camphora* and a method for constructing a characteristic chromatogram. Background Technology

[0002] The original plant of *Jasminum lanceolaria* Roxburgh, belonging to the Oleaceae family, is the dried stem. The entire plant can be used medicinally, and the medicinal name is Qingxiangteng (Pogufeng). The Hunan Provincial Standard for Traditional Chinese Medicine and the Chongqing Municipal Standard for Traditional Chinese Medicine list the dried stem as the medicinal part, while the Guangxi Standard lists the whole dried plant. It is widely distributed in mountain slopes, thickets, and dense forests in valleys of Anhui, Taiwan, Fujian, Jiangxi, Hubei, Hunan, Hainan, Guangdong, Guangxi, Guizhou, Sichuan, and Yunnan provinces, at altitudes below 2200 meters. It has the effects of dispelling wind and dampness, cooling blood, and detoxifying. It is commonly used for rheumatic pain, traumatic injuries, unidentified boils, and gynecological inflammation; in folk medicine, it is also used to treat fever, rheumatic pain, inflammation, and eye pain.

[0003] The literature "Determination of Ferulic Acid Content in Chinese Vine by HPLC" discloses a method for determining the ferulic acid content in Chinese Vine by high performance liquid chromatography, using a VP-ODS-C18 column (4.6 mm × 150 mm, 5 μm) and acetonitrile-0.085% phosphoric acid aqueous solution (17:83) as the mobile phase.

[0004] The research subjects of the above literature are *Cephalotaxus fortunei* medicinal materials. However, the standard decoction of *Cephalotaxus fortunei* is a freeze-dried powder obtained from *Cephalotaxus fortunei* slices through water extraction, concentration, and drying. Compared with the raw medicinal materials and slices, it has lost its inherent form, and the corresponding active ingredients have also changed accordingly. Currently, only the Hunan Provincial Standard for Traditional Chinese Medicine (2009 edition), the Chongqing Municipal Standard for Traditional Chinese Medicine, and the Guangxi Standard for Traditional Chinese Medicine have implemented quality control for the microscopic and thin-layer chromatography identification of *Cephalotaxus fortunei* medicinal materials, which is insufficient to fully reflect its intrinsic quality. Furthermore, there are very few literature reports on the qualitative and quantitative detection techniques of high-performance liquid chromatography (HPLC) for *Cephalotaxus fortunei*. Therefore, it is necessary to establish an HPLC method for the determination of the content and characteristic chromatograms of the standard decoction of *Cephalotaxus fortunei*, providing a basis for effectively controlling and comprehensively evaluating the quality of the standard decoction. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes a method for determining the content of ferulic acid in a standard decoction of *Cephalotaxus fortunei* and a method for constructing a characteristic chromatogram, thereby providing assurance for the quality and consistency evaluation of the standard decoction of *Cephalotaxus fortunei*.

[0006] In a first aspect, the present invention provides a method for determining the content of ferulic acid in a standard decoction of *Clematis chinensis*, wherein the determination is performed by high-performance liquid chromatography (HPLC), and the conditions for the HPLC method include:

[0007] The chromatographic column was packed with octadecylsilane-bonded silica gel.

[0008] Acetonitrile was used as mobile phase A, and a 0.1% (v / v) aqueous solution of phosphoric acid was used as mobile phase B for gradient elution.

[0009] The conditions for gradient elution are as follows:

[0010]

[0011] The percentage sign in the table means volume percentage.

[0012] According to some embodiments of the present invention, the mobile phase B is an aqueous solution of phosphoric acid with a volume percentage of 0.1%.

[0013] According to some embodiments of the present invention, the chromatographic column is an ACQUITY UPLC HSS T3 C18.

[0014] According to some embodiments of the present invention, the chromatographic column has dimensions of 2.1 mm × 100 mm and 1.8 μm.

[0015] According to some embodiments of the present invention, the conditions for the high performance liquid chromatography method further include: the flow rate of the mobile phase is 0.28 to 0.32 mL / min; preferably 0.3 mL / min.

[0016] According to some embodiments of the present invention, the conditions for the high performance liquid chromatography method further include: the column temperature of the chromatographic column is 28-32°C; preferably 30°C.

[0017] According to some embodiments of the present invention, the conditions for the high performance liquid chromatography method further include: an injection volume of 1 μL.

[0018] According to some embodiments of the present invention, the conditions of the high performance liquid chromatography method further include: a detection wavelength of 320 nm.

[0019] According to some embodiments of the present invention, the detector of the high-performance liquid chromatography is a diode array detector.

[0020] According to some embodiments of the present invention, the theoretical plate number of the high-performance liquid chromatography, calculated based on the ferulic acid peak, is not less than 2000.

[0021] According to some embodiments of the present invention, the content determination method further includes the preparation of a test solution; the preparation step includes: mixing the standard decoction of *Cinnamomum camphora* with a solvent and sonicating to obtain the solution.

[0022] According to some embodiments of the present invention, the mass concentration of the test solution is 3 to 6 mg / mL.

[0023] Furthermore, the solvent of the test solution is water or an alcohol-water mixture.

[0024] Furthermore, the solvent is a methanol aqueous solution with a volume percentage of 50-70%, an ethanol aqueous solution with a volume percentage of 50-70%, or water; preferably, the solvent is an ethanol aqueous solution with a volume percentage of 50-70%; more preferably, the solvent is an ethanol aqueous solution with a volume percentage of 50%.

[0025] According to some embodiments of the present invention, the ultrasonic conditions described herein include at least one of the following i-iii:

[0026] i. Power: 400~600W;

[0027] ii. Frequency: 30–50 kHz;

[0028] iii. Time: 20–40 min.

[0029] According to some embodiments of the present invention, the content determination method further includes the preparation of a reference solution, wherein the preparation step includes: mixing the reference solution with a solvent to obtain the solution.

[0030] According to some embodiments of the present invention, the reference standard is ferulic acid.

[0031] According to some embodiments of the present invention, the mass concentration of the reference solution is 10–50 μg / mL.

[0032] Furthermore, the solvent of the reference solution is water or an alcohol-water mixture.

[0033] Furthermore, the solvent is a methanol aqueous solution with a volume percentage of 50-70%, an ethanol aqueous solution with a volume percentage of 50-70%, or water; preferably, the solvent is a methanol aqueous solution with a volume percentage of 50-60%; more preferably, the solvent is a methanol aqueous solution with a volume percentage of 50%.

[0034] In a second aspect, the present invention provides a method for constructing a characteristic chromatogram of a standard decoction of *Cinnamomum camphora*, wherein a reference solution, a reference herb solution, and a test solution are respectively determined by high performance liquid chromatography (HPLC) to identify common peaks, thereby obtaining the chromatogram; the HPLC method employs the conditions described above.

[0035] According to some embodiments of the present invention, the characteristic spectrum includes six characteristic peaks, wherein peak 3 (ferulic acid) is used as a reference peak, and the relative retention times of the remaining characteristic peaks and the reference peak are calculated. The relative retention times should be within ±10% of a specified value, which is: peak 1: 0.77, peak 2: 0.89, peak 4: 1.33, peak 5: 1.39, and peak 6: 1.95.

[0036] According to some embodiments of the present invention, the preparation steps of the reference medicinal material solution include: mixing the reference medicinal material with a solvent and sonicating.

[0037] According to some embodiments of the present invention, the mass concentration of the control medicinal material solution is 3 to 6 mg / mL.

[0038] According to some embodiments of the present invention, the solvent is water or an alcohol-water mixture;

[0039] According to some preferred embodiments of the present invention, the solvent is a methanol aqueous solution with a volume percentage of 50-70%, an ethanol aqueous solution with a volume percentage of 50-70%, or water. Preferably, the solvent is an ethanol aqueous solution with a volume percentage of 50-70%; more preferably, the solvent is an ethanol aqueous solution with a volume percentage of 50%.

[0040] According to some embodiments of the present invention, the ultrasound conditions include at least one of the following i-iii:

[0041] i. Power: 400~600W;

[0042] ii. Frequency: 30–50 kHz;

[0043] iii. Time: 20–40 min.

[0044] A third aspect of the present invention provides the application of the above-described content determination method or construction method in detecting the quality of the standard decoction of *Tetrapanax papyriferus*.

[0045] Beneficial effects:

[0046] This invention provides a characteristic chromatogram and content determination method for a standard decoction of *Cinnamomum camphora*, which identifies six common peaks, including ferulic acid. The constructed HPLC characteristic chromatogram shows good separation of chromatographic peaks, rich characteristic chromatographic information, and good peak shape, which can comprehensively reflect the main chemical components of the standard decoction of *Cinnamomum camphora*. The detection method has good stability and repeatability, and is easy to promote and apply.

[0047] This invention constructs and detects the HPLC characteristic chromatograms of the *Cinnamomum camphora* standard decoction using high-performance liquid chromatography (HPLC). This allows for the overall control of the characteristic components in the *Cinnamomum camphora* standard decoction, effectively ensuring the overall quality stability of the decoction and making the quality control technology for the decoction more complete and scientific. Furthermore, this method is simple to operate and has advantages such as high precision, good stability, good repeatability, and high accuracy, providing an effective basis for the quality identification of traditional Chinese medicinal materials.

[0048] definition:

[0049] The fragrant vine mentioned in this invention refers to the dried stem of the fragrant vine.

[0050] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0051] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0052] Figure 1 The chromatogram (210 nm) is the result of the investigation of the optimal absorption wavelength in Example 1.

[0053] Figure 2 The chromatogram (254 nm) is the result of the investigation of the optimal absorption wavelength in Example 1.

[0054] Figure 3 The chromatogram (320 nm) is the result of the investigation of the optimal absorption wavelength in Example 1.

[0055] Figure 4 The chromatogram is the result of gradient elution investigation in Example 1;

[0056] Figure 5 The chromatogram is the column temperature investigation result from Example 1;

[0057] Figure 6 The chromatogram shows the flow rate results from Example 1.

[0058] Figure 7 This is a chromatogram of the double retention time results in Example 1;

[0059] Figure 8 The chromatogram shows the results of the extraction method investigation in Example 1;

[0060] Figure 9 The chromatogram shows the extraction time results of Example 1;

[0061] Figure 10 The chromatogram shows the results of the solvent dosage investigation in Example 1;

[0062] Figure 11 The chromatogram shows the results of solvent extraction investigation in Example 1;

[0063] Figure 12 This is the chromatogram used in Example 1 to investigate the specificity of the characteristic chromatograms.

[0064] Figure 13 The characteristic chromatogram of ferulic acid reference standard in Example 1;

[0065] Figure 14 This is a characteristic chromatogram of the *Clematis chinensis* reference medicinal material in Example 1;

[0066] Figure 15 This is a superimposed image of the characteristic spectra of 18 batches of Qingxiangteng standard decoction in Example 1;

[0067] Figure 16 This is a characteristic chromatogram of the standard decoction of *Clematis chinensis* in Example 1;

[0068] Figure 17 This is the standard curve for ferulic acid reference standard in Example 2;

[0069] Figure 18 The chromatograms of different gradients of *Cinnamomum camphora* in Comparative Example 1 are shown. Detailed Implementation

[0070] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0071] The freeze-dried powder of the standard decoction of *Cinnamomum camphora* described in this invention is prepared as follows: *Cinnamomum camphora* medicinal material is taken, impurities are removed, it is slightly washed, moistened, cut into thick slices, and dried to obtain *Cinnamomum camphora* slices. 100g of *Cinnamomum camphora* slices are placed in an electric ceramic kettle and decocted twice with water: For the first decoction, 12 times the amount of water (by mass) is added and soaked for 30 minutes. After boiling over high heat, the decoction is simmered for 30 minutes. The decoction is then filtered through a 200-mesh sieve while hot and set aside. For the second decoction, 10 times the amount of water (by mass) is added, and the decoction is boiled over high heat and simmered for 20 minutes. The decoction is then filtered through a 200-mesh sieve while hot. The two decoctions are combined and concentrated under reduced pressure at 65°C to approximately 120g of extract. This extract is then dispensed into 6mL–14mL containers, with each bottle containing 2mL–3mL. After dispensing, the extract is freeze-dried, removed, and capped with an aluminum cap to obtain the final product. Properties of the freeze-dried powder of *Clematis chinensis* standard decoction: Yellow-brown to brownish-yellow powder; faint odor, bland taste. The preparation process complies with the relevant provisions of the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula Granules".

[0072] Example 1: Establishment of UPLC Characteristic Spectrum Method for Standard Decoction of *Clematis chinensis*

[0073] 1. Instruments and reagents

[0074] 1.1 Instruments

[0075] Shimadzu LC-30A High Performance Liquid Chromatograph (Shimadzu Corporation);

[0076] Agilent 1260 high-performance liquid chromatograph (Agilent Technologies, Inc., USA);

[0077] KM-5000DB CNC Ultrasonic Cleaner (Kunshan Ultrasonic Instrument Co., Ltd.);

[0078] Mettler Toledo Instruments (Shanghai) Co., Ltd. (ME104E, MS1050U);

[0079] MWO-ABS20-DW Ultrapure Water System (Mingwo Technology);

[0080] Column: ACQUITY UPLC HSS T3 C18; (2.1 mm × 100 mm, 1.8 μm).

[0081] 1.2 Reagents

[0082] Methanol was analytical grade (batch number: 20220824, Sinopharm Group Co., Ltd.), ethanol was analytical grade (batch number: 20221124, Sinopharm Group Co., Ltd.), methanol was chromatographic grade (batch number: 220852283, Tiandi Inc., USA), acetonitrile was chromatographic grade (batch number: 22115202, Tiandi Inc., USA), phosphoric acid was chromatographic grade (Tianjin Kemeio Chemical Reagent Co., Ltd.), and water was purified water (in-house prepared).

[0083] 1.3 Drug Test

[0084] Ferulic acid (purity 99.4%, China National Institutes for Food and Drug Control, batch number: 110773-201915), *Cinnamomum camphora* reference material (Zhuhai Anzhe Biotechnology Co., Ltd., batch number: 23041); *Cinnamomum camphora* standard decoction (lyophilized powder), batch numbers: QXT-BT230255~QXT-BT230267, QXT-BT230284~QXT-BT230288 (of which, QXT-BT230255~QXT-BT230257, QXT-BT230261~QXT-BT230263 are from Yueyang County, Hunan Province; QXT-BT230258~QXT-BT230260, QXT-BT230264~QXT-BT230267 are from Pingjiang County, Hunan Province; and QXT-BT230284~QXT-BT230288 are from Xiangyin County, Hunan Province).

[0085] 2. Chromatographic conditions and system suitability test

[0086] 2.1 Proposed chromatographic conditions

[0087] The column was packed with octadecylsilane-bonded silica gel (100 mm in length, 2.1 mm in inner diameter, and 1.8 μm in particle size); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid aqueous solution was used as mobile phase B. Gradient elution was performed according to the specifications in the table below; the flow rate was 0.3 mL / min; the column temperature was 30 °C; the detection wavelength was 336 nm; and the theoretical plate number, calculated based on the ferulic acid peak, should not be less than 2000.

[0088]

[0089] The percentage sign in the table means volume percentage.

[0090] 2.2 Solution Preparation

[0091] (1) Preparation of reference solution: Take an appropriate amount of ferulic acid reference standard, accurately weigh it, and add 50% methanol aqueous solution to prepare a solution containing 10 μg per 1 mL.

[0092] (2) Preparation of reference medicinal material solution: Take 0.1g of Qingxiangteng reference medicinal material, place it in a stoppered conical flask, add 25ml of 50% ethanol, sonicate (500W, frequency 40kHz) for 30 minutes, take it out, cool it, shake it well, filter it, and take the filtrate as the reference medicinal material solution.

[0093] (3) Preparation of test solution: Take about 0.1g of the powder of this product, accurately weigh it, put it in a stoppered conical flask, accurately add 25ml of 50% ethanol, weigh it, extract it by sonication for 30min, cool it, weigh it again, make up the weight loss with 50% ethanol, shake it well, filter it, and the test solution is obtained.

[0094] 2.3 Determination Method

[0095] Take 1 μL each of the obtained reference solution, reference medicinal material solution and test solution, inject them into a high-performance liquid chromatograph, and determine the result.

[0096] 3. Investigation of chromatographic conditions

[0097] 3.1 Investigation of Optimal Absorption Wavelength

[0098] Based on the established experimental conditions, a diode array detector was used to perform a full-wavelength scan of the *Pteris vittata* standard decoction test solution, and chromatograms of the test solution at wavelengths of 210 nm, 254 nm, and 320 nm were extracted. The results are as follows: Figures 1-3 As shown.

[0099] The results showed that the chromatographic peak information was greater, the chromatographic baseline was more stable, and the response values ​​of each characteristic peak were larger when the detection wavelength was 320 nm. Therefore, the detection wavelength was determined to be 320 nm.

[0100] 3.2 Elution gradient investigation

[0101] The chromatograms of *Phyllostachys edulis* under three different gradients were investigated. The gradient elution is shown in the table below. Other chromatographic conditions were the same as those described in section "2.1". The results are as follows: Figure 4 As shown.

[0102] Gradient elution 1

[0103]

[0104] Gradient elution 2

[0105]

[0106] Gradient elution 3

[0107]

[0108] The results showed that gradient elution 3 resulted in better separation, and gradient elution 3 was ultimately chosen.

[0109] 3.3 Column Temperature Investigation

[0110] The column temperatures were set to 28℃, 30℃, and 32℃, respectively, and the remaining chromatographic conditions were the same as those described in section "2.1". The results are shown in Table 1. Figure 5 As shown.

[0111] Table 1. Relative retention times at different column temperatures.

[0112]

[0113] The results showed that when the column temperature was (30±2)℃, the relative retention times (RSD) of the six characteristic peaks were relatively similar, and the column temperature robustness results met the requirements for method validation. Therefore, the column temperature was set at 28–32℃.

[0114] 3.4 Flow velocity investigation

[0115] The flow rates were set to 0.28 mL / min, 0.30 mL / min, and 0.32 mL / min, respectively, and the remaining chromatographic conditions were the same as those in section "2.1". The results are shown in Table 2. Figure 6 As shown.

[0116] Table 2. Results of relative retention time for different flow velocities

[0117]

[0118] The results showed that when the mobile phase flow rate varied at (0.3±0.02) ml / min, the relative retention time (RSD) of the six characteristic peaks was less than 3%, and the robustness results at different flow rates met the requirements for method validation; therefore, the proposed flow rate was 0.28–0.32 mL / min.

[0119] 3.5 times retention time

[0120] Take one portion of the above test solution and detect the characteristic chromatogram at twice the retention time. The results show that no other chromatographic peaks appeared after 30 minutes, indicating that the method has good overall reliability. The results are as follows... Figure 7 As shown.

[0121] In summary, the chromatographic conditions and system suitability test for the characteristic chromatogram of the *Phyllostachys edulis* standard decoction 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 1.8 μm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid aqueous solution was used as mobile phase B, with gradient elution as specified in the table below; the detection wavelength was 320 nm, the flow rate was 0.3 ml / min, the column temperature was 30 °C, and the theoretical plate number calculated based on the ferulic acid peak should not be less than 2000.

[0122] Prescribed gradient elution procedure:

[0123]

[0124] 4. Investigation of the preparation method of the test solution

[0125] 4.1 Examination of Extraction Methods

[0126] Compare two extraction methods: heating reflux and ultrasonic extraction. Take an appropriate amount of *Cinnamomum camphora* sample, weigh two portions of powder, approximately 0.1 g each, accurately weigh them, and place them in a stoppered conical flask. Accurately add 25 ml of 50% ethanol, weigh again, and reflux one portion for 30 minutes. Sonicate the other portion (250 W, 40 kHz) for 30 minutes. Cool, weigh again, and replenish the lost weight with 50% ethanol. Shake well, filter, and obtain the final product. Determine the chromatographic conditions as described above, and select a suitable extraction method based on the parameters of each characteristic peak (peak shape, separation degree, symmetry, total peak area, etc.).

[0127] The chromatographic conditions under section "2.1" were followed for injection and determination. The "total peak area / sample weight" of the six characteristic peaks was calculated to compare the effects of different extraction methods on the characteristic chromatogram of the *Pteris vittata* standard decoction, and the optimal extraction solvent was selected. The results are shown in Table 3. Figure 8 .

[0128] Table 3. Characteristic maps of *Pteris vittata* samples - different extraction methods

[0129]

[0130] The results showed that the total peak area of ​​the characteristic spectrum obtained by reflux extraction was not significantly different from that obtained by ultrasonic extraction. Taking all factors into consideration, ultrasonic extraction was selected as the extraction method for preparing the characteristic spectrum of the *Cinnamomum camphora* sample.

[0131] 4.2 Investigation of Extraction Solvents

[0132] The extraction solvents for preparing the characteristic chromatograms of *Cinnamomum camphora* samples were investigated. The extraction efficiency of the characteristic chromatograms of *Cinnamomum camphora* samples was investigated when methanol, 50% ethanol, 30% methanol, 50% methanol, and water were used as extraction solvents.

[0133] Take an appropriate amount of *Cinnamomum camphora* sample, weigh five portions of powder, each approximately 0.1 g, accurately weigh them, place them in a stoppered conical flask, accurately add 25 ml of 50% ethanol, weigh them, extract ultrasonically for 30 min, cool, weigh them again, replenish the lost weight with 50% ethanol, shake well, filter, and obtain the final product. Determine the chromatographic conditions as described above, and select a suitable extraction solvent based on the parameters of each characteristic peak (peak shape, separation degree, symmetry, total peak area, etc.).

[0134] The chromatographic conditions under section "2.1" were followed for injection and determination. The "total peak area / sample weight" of the six characteristic peaks was calculated to compare the effects of different extraction solvents on the characteristic chromatogram of the *Tetrapanax papyriferus* standard decoction, and the optimal extraction method was selected. The results are shown in Table 4. Figure 9 .

[0135] Table 4. Characteristic maps of *Phyllostachys edulis* samples - Applicability parameters for different extraction solvent systems

[0136]

[0137] The results showed that the extraction efficiencies of 50% methanol, 50% ethanol and 30% methanol were basically the same. Taking all factors into consideration, in order to ensure sufficient extraction and that the organic solvent is easy to preserve and conforms to the content determination, 50% ethanol was selected as the extraction solvent for the sample characteristic spectrum of *Cinnamomum camphora*.

[0138] 4.3 Examination of extraction time

[0139] Different extraction times affect the extraction efficiency of each characteristic peak. The extraction efficiency of six proposed characteristic peaks in the chromatogram of the *Pteris vittata* sample was compared at different extraction times, and a suitable time was determined based on the parameters of each characteristic peak. An appropriate amount of *Pteris vittata* sample was taken, and three portions of powder, approximately 0.1 g each, were accurately weighed and placed in a stoppered conical flask. 25 ml of 50% ethanol was accurately added, and the weight was measured. The samples were ultrasonically extracted for 20, 30, and 40 minutes respectively, cooled, and weighed again. The weight loss was replenished with 50% ethanol, shaken well, and filtered. The sample was then analyzed under the determined chromatographic conditions. The changes in chromatographic peaks at different extraction times (based on peak shape, separation effect, symmetry, total peak area, etc.) were compared to select a suitable extraction time.

[0140] The chromatographic conditions under section "2.1" were followed for injection and determination. The "total peak area / sample weight" of the six characteristic peaks was calculated to compare the effects of different extraction times on the characteristic chromatogram of the *Tetrapanax papyriferus* standard decoction, and the optimal extraction time was selected. The results are shown in Table 5. Figure 10 .

[0141] Table 5. Characteristic maps of *Phyllostachys edulis* samples - System applicability parameters for different extraction times.

[0142]

[0143] The results showed that, after extraction at 20, 30, and 40 minutes, there were no significant differences in the peak area of ​​each characteristic peak and the total peak area after conversion based on the sample size. The results indicated that the ultrasonic time had little effect on the spectrum. To ensure complete dissolution of the lyophilized powder, an ultrasonic time of 30 minutes was ultimately selected.

[0144] 4.4 Investigation of solvent dosage

[0145] Take an appropriate amount of *Phyllostachys edulis* sample, weigh three portions of powder (approximately 15 ml, 25 ml, and 35 ml respectively), accurately weigh them, place them in stoppered conical flasks, accurately add 25 ml of 50% ethanol, weigh them, and ultrasonically extract each portion for 30 minutes. After cooling, weigh them again, replenish the lost weight with 50% ethanol, shake well, and filter to obtain the final product. Perform chromatographic analysis according to the conditions determined above. Compare the changes in chromatographic peaks at different extraction times (based on peak shape, separation effect, symmetry, total peak area, etc.) to select an appropriate solvent volume.

[0146] The samples were injected and analyzed according to the chromatographic conditions in section "2.1". The "total peak area / sample weight" of the six characteristic peaks was calculated to compare the effect of different solvent volumes on the characteristic chromatogram of the *Tetrapanax papyriferus* standard decoction, and the optimal extraction method was selected. The results are shown in Table 6. Figure 11 .

[0147] Table 6. Characteristic chromatograms of *Phyllanthus praecox* samples - System suitability parameters for different solvent volumes.

[0148]

[0149] The results showed that the total peak area of ​​the test samples with solvent volumes of 15ml, 25ml, and 35ml was not significantly different after conversion of the sample volume. The extraction efficiency of the proposed characteristic peaks in the test samples with solvent volumes of 15ml, 25ml, and 35ml was basically the same, indicating that the above-determined extraction parameters can stably extract 0.1g of each characteristic peak substance. Taking all factors into consideration, 25ml was determined as the solvent volume for the characteristic spectrum of the *Phyllostachys edulis* sample.

[0150] In summary, the main parameters for the preparation method of the test solution are as follows: Take about 0.1g of the powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 50% ethanol, weigh it, extract it by sonication (power 500W, frequency 40kHz) for 30min, cool it, weigh it again, make up the lost weight with 50% ethanol, shake it well, filter it, and the solution is obtained.

[0151] 5. Validation of the Feature Mapping Methodology

[0152] 5.1 Specificity Examination

[0153] Take appropriate amounts of the above-mentioned reference herb solution, reference standard solution, test sample solution, and blank solvent (50% ethanol aqueous solution), and inject them according to the chromatographic conditions under section "2.1" for determination, and record the chromatograms. The results show that the baselines of the chromatograms of the *Clematis chinensis* reference herb, reference standard, and test sample are all stable, with good peak resolution and symmetry. The retention times of each chromatographic peak in the test sample are basically the same as the corresponding chromatographic peaks in the reference standard and reference herb, and there is no interference from the blank solvent. See the results below. Figure 12 .

[0154] 5.2 Precision Test

[0155] Weigh approximately 0.1 g of freeze-dried *Cinnamomum camphora* standard decoction powder (batch number QXT-BT230255), prepare according to the test sample method, inject continuously 6 times and record the chromatograms. Using peak 3 as the reference peak, calculate the relative retention time and relative peak area of ​​each characteristic peak. The results are shown in the table below. The relative retention time of each characteristic peak is less than 1%, and the RSD of the relative peak area is less than 5%. The experiment shows that the precision of this method is good. The results are shown in Tables 7 and 8.

[0156] Table 7. Precision test results of the standard decoction of *Clematis chinensis* (relative retention time)

[0157]

[0158] Table 8. Precision test results of the standard decoction of *Clematis chinensis* (relative peak area)

[0159]

[0160] 5.3 Repeatability Test

[0161] Weigh approximately 0.1 g of freeze-dried powder of the standard decoction of *Cymbidium faberi* (batch number BT230255), and prepare 6 parallel samples according to the test sample method. Inject each sample separately, record the chromatogram and integrate the results. Using peak 3 as the reference peak, calculate the relative retention time and relative peak area of ​​each characteristic peak. The results are shown in the table below. The relative retention time of each characteristic peak is less than 1%, and the RSD of the relative peak area is less than 5%. The experiment shows that the repeatability of this method is good. The results are shown in Tables 9 and 10.

[0162] Table 9. Repeatability test results of the standard decoction of *Clematis chinensis* (relative retention time)

[0163]

[0164] Table 10. Repeatability test results of the standard decoction of *Clematis chinensis* (relative peak area)

[0165]

[0166] 5.4 Stability Test

[0167] Weigh approximately 0.1 g of the freeze-dried powder of the *Cinnamomum camphora* standard decoction (batch number QXT-BT230255), prepare it according to the test sample method, and inject it at different time points within 24 hours. Record the chromatogram and integrate it. Using peak 3 as the reference peak, calculate the relative retention time and relative peak area of ​​each characteristic peak. The results are shown in the table below. The relative retention time of each characteristic peak is less than 1%, and the RSD of the relative peak area is less than 5%. The experiment shows that the test sample solution has good stability within 24 hours. The results are shown in Tables 11 and 12.

[0168] Table 11. Stability test results (relative retention time) of the standard decoction of *Clematis chinensis*.

[0169]

[0170] Table 12. Stability test results of the standard decoction of *Clematis chinensis* (relative peak area)

[0171]

[0172] 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, and the above six characteristic peaks will be included in subsequent tests.

[0173] 4.5 Establishment of characteristic spectra and identification of common peaks

[0174] The HPLC fingerprint chromatograms of 18 batches of *Citrus aurantium* standard decoction were imported into the "Traditional Chinese Medicine Chromatographic Characteristic Chromatographic Similarity Evaluation System Software (2012 Edition)" to generate overlay chromatograms and reference characteristic chromatograms. Six common peaks with good resolution, large peak area, and good peak shape were selected as characteristic peaks. Through comparison with reference standards, peak 3 was identified as ferulic acid. Ferulic acid is the main active ingredient of *Citrus aurantium* and has stable chemical properties. Its chromatographic peak showed good resolution and a large peak area among all chromatographic peaks; therefore, peak 3 (ferulic acid) was selected as the reference peak for the characteristic chromatogram. The relative retention times of peaks 1, 2, 4, 5, and 6 were calculated, and the results are shown in Table 13. The characteristic chromatogram of the ferulic acid reference standard is shown in Table 13. Figure 13 See the characteristic atlas of the reference medicinal materials. Figure 14 , 18 The superimposed characteristic chromatogram of the standard decoction of *Clematis chinensis* is shown in Figure 15; the comparative characteristic chromatogram of the standard decoction of *Clematis chinensis* is shown in Figure 15. Figure 16 .

[0175] Table 13 Relative retention times of 18 batches of freeze-dried powder samples of Qingxiangteng standard decoction

[0176]

[0177] 5.6 Establishment of Feature Map Standards

[0178] In summary, the criteria for determining the characteristic chromatograms are as follows: the chromatogram of the test sample should show 6 characteristic peaks, and the retention times should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material. The retention time of peak 3 should correspond to the retention time of the reference solution. The peak corresponding to the ferulic acid 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, which are 0.77 (peak 1), 0.89 (peak 2), 1.33 (peak 4), 1.39 (peak 5), and 1.95 (peak 6).

[0179] Example 2: Establishment of UPLC method for determining the content of *Clematis chinensis* in standard decoction

[0180] Based on the method for constructing the UPLC characteristic spectrum of the *Phyllostachys edulis* standard decoction in Example 1, Example 2 also conducted the following methodological investigation, establishing a UPLC content determination method for the *Phyllostachys edulis* standard decoction, specifically including:

[0181] 1. Examination of linear relationships

[0182] In this validation, reference solutions with ferulic acid concentrations of 1.0799E-07 mg / ml, 2.6999E-06 mg / ml, 6.7497E-05 mg / ml, 0.001687414 mg / ml, 0.04218536 mg / ml, and 0.2109268 mg / ml were prepared. A standard curve was plotted with the concentration of the reference solution on the x-axis and the corresponding peak area on the y-axis. The results showed that the ferulic acid concentration was in the range of 1.0799E-07 mg / ml to 0.2109268 mg / ml, i.e., the ferulic acid content of the test sample was in the range of 1.0799E-07 mg / ml to 0.2109268 mg / ml. The regression equation was y = 9,853,192.9747x + 19,122.9834, R² = 99991. The results are shown in Table 14, and the standard curve is shown in [Table 14]. Figure 15 .

[0183] Table 14 Results of linearity investigation of ferulic acid

[0184]

[0185] The results showed that, under the above chromatographic conditions, the injection concentration of ferulic acid in the range of 1.0799E-07 mg / ml to 0.2109268 mg / ml had a good linear relationship with the peak area.

[0186] 2 Repeatability Test

[0187] Six parallel test solutions were prepared from the same batch of *Cephalotaxus fortunei* powder according to the method described in "Preparation of Test Solution". The solutions were then injected and analyzed under determined chromatographic conditions. The data showed that the mean ferulic acid content in the *Cephalotaxus fortunei* powder was 0.715 mg / g, with an RSD of less than 4% (General Chapter 9101, Chinese Pharmacopoeia 2020), indicating good repeatability. The experimental results are shown in Table 15.

[0188] Table 15 Method validation for the determination of ferulic acid content in *Cinnamomum camphora* dried powder - Repeatability test data of the method for *Cinnamomum camphora* dried powder.

[0189]

[0190] The results showed that the average RSD of ferulic acid content in the standard decoction of *Citrus aurantium* was less than 3% in 6 measurements, indicating that the method had good repeatability.

[0191] 3. Precision test

[0192] This method was used to evaluate the repeatability of the response values ​​of a chromatographic system during continuous injection. The same reference solution was injected six times consecutively under determined chromatographic conditions. The data showed that the RSD value of the instrument's repeatable measurements was less than 2%, indicating good repeatability. The experimental results are shown in Table 16.

[0193] Table 16. Method validation and precision test data for the determination of ferulic acid content in dried *Cinnamomum camphora* powder.

[0194]

[0195] 4. Stability Test

[0196] Take the same batch of dried *Cinnamomum camphora* powder, prepare the test sample according to the method of "preparation of test sample solution", determine the chromatographic conditions, inject the sample for detection within 24 hours, record the chromatogram, and investigate the effect of different storage time on the test results of ferulic acid content determination. The experimental results are shown in Table 17.

[0197] Table 17 Effect of test solution standing time on test results - Ferulic acid

[0198]

[0199] The above results indicate that when the sample is injected and tested by personnel within 24 hours, the retention time RSD is less than 2%, and the peak area RSD is less than 2%, thus demonstrating good solution stability and meeting the method validation requirements.

[0200] 5. Intermediate Precision Test

[0201] For the same batch of samples, test samples were prepared according to the "Preparation of Test Sample Solution" method, and analyzed by injection into a Shimadzu LC-30A high-performance liquid chromatograph and an Agilent 1260 high-performance liquid chromatograph under determined chromatographic conditions. The experimental results are shown in Table 18.

[0202] Table 18 Methodological Validation for Determination of Ferulic Acid Content in Dry Powder of *Cinnamomum camphora* - Intermediate Precision Experimental Data for Different Instruments

[0203]

[0204]

[0205] The results showed that the RSD value of the decomposed powder of *Cinnamomum camphora* was less than 4% when determined by Shimadzu LC-30A HPLC and Agilent 1290 HPLC, respectively, indicating that the method has good intermediate precision among different instruments.

[0206] 6. Recovery Test

[0207] Nine samples of dried *Cinnamomum camphora* powder with known content were weighed. The ferulic acid content was 0.715 mg / g, and each sample was approximately 0.05 g. The ferulic acid reference standard was added to each sample, and the sample was prepared according to the test solution preparation method. The sample was then determined according to the method, and the chromatograms were recorded and integrated. The results are shown in Table 19. The recovery rate RSD was less than 5%. The experiment showed that the spiked recovery rate of this method was good.

[0208] Table 19 Results of the sample recovery test

[0209]

[0210] 7. Sample content determination

[0211] Eighteen batches of freeze-dried *Cymbidium goeringii* standard decoction powder were taken and tested according to the above method. The content of ferulic acid was calculated using the external standard method. The results are shown in Table 20. The content of ferulic acid ranged from 0.47 to 2.75 mg / g, with a mean of 1.26 mg / g. The mean ± 3SD range was -0.69 to 3.21. Considering the distribution of data from the 18 batches of standard decoction powder, the deviation of the 18 batches of samples was relatively large. The SD algorithm can be used, with the lower limit being the lowest measured value. The limit range for ferulic acid content is: per 1g of freeze-dried *Cymbidium goeringii* standard decoction powder, the content of ferulic acid (C...) is... 10 H 10 O4) should be 0.47mg to 3.21mg.

[0212] Table 20. Results of ferulic acid content determination in 18 batches of freeze-dried powder of Qingxiangteng standard decoction.

[0213]

[0214]

[0215] Comparative Example 1: Comparison of different gradients of *Phyllanthus praecox*

[0216] Chromatographic conditions:

[0217] The column was packed with octadecylsilane-bonded silica gel (100 mm column length, 2.1 mm inner diameter, and 1.8 μm particle size); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid aqueous solution was used as mobile phase B. Gradient elution was performed according to the specifications in the table below; the detection wavelength was 320 nm, the flow rate was 0.3 ml / min, the column temperature was 30 °C, and the theoretical plate number calculated based on the ferulic acid peak should not be less than 2000.

[0218] Elution procedure of Comparative Example 1:

[0219]

[0220] Example 1: Gradient elution procedure:

[0221]

[0222] Based on the above results, it can be seen that the elution program in Example 1 is gradient elution, while Comparative Example 1 uses isocratic elution. Gradient elution has the advantages of shortening the analysis cycle, improving separation ability, and improving peak shape with less tailing. In gradient elution, the peaks of the samples, regardless of whether the components elute first or later, generally maintain sharp and symmetrical peak shapes. In contrast, with isocratic elution, the chromatographic peaks of later eluting components become wider, resulting in poorer separation. Therefore, the results show that Example 1 has high peak separation, obvious peaks, stable baseline, and can achieve complete separation of the measured components, obtaining characteristic peaks with better shapes. The test results are shown in [Figure number missing]. Figure 18 In the figure, S1: Fragrant Vine (Comparative Example 1), S2: Fragrant Vine (Example 1), S3: Control (Comparative Example 1), S4: Control (Example 1).

[0223] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for constructing a characteristic chromatogram of a standard decoction of *Clematis chinensis*, characterized in that a reference solution, a reference herb solution, and a test solution are respectively analyzed by high-performance liquid chromatography (HPLC) to determine the common peak, thereby obtaining the chromatogram; the conditions for the HPLC method include: The chromatographic column was packed with octadecylsilane-bonded silica gel. Acetonitrile was used as mobile phase A, and 0.1% (v / v) aqueous phosphoric acid solution was used as mobile phase B, with gradient elution; flow rate: 0.28~0.32 mL / min; column temperature: 28~32℃; injection volume: 1 μl; detection wavelength: 320 nm; The conditions for gradient elution are as follows: The characteristic spectrum includes six characteristic peaks. Peak 3 (ferulic acid) is used as a reference peak. The relative retention times of the remaining characteristic peaks and the reference peak are calculated. These relative retention times should be within ±10% of a specified value, which is: Peak 1: 0.77, Peak 2: 0.89, Peak 4: 0.89, Peak 2: 0.89, Peak 3: 0.89, Peak 4: 0.89, Peak 2: 0.89, Peak 3: 0.89, Peak 4: 0.89, Peak 3 ... 1.33, Peak 5: 1.39, Peak 6: 1.95; Preparation of the reference solution: Take an appropriate amount of ferulic acid reference standard, accurately weigh it, and add 50% methanol aqueous solution to prepare a solution containing 10µg per 1mL. Preparation of the reference herb solution: Take 0.1 g of *Cinnamomum camphora* reference herb, place it in a stoppered conical flask, add 25 ml of 50% ethanol, sonicate at 500 W, frequency 40 kHz for 30 minutes, remove, cool, shake well, filter, and take the filtrate as the reference herb solution. Preparation of the test solution: Take about 0.1g of the powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 50% ethanol, weigh it, extract it by sonication for 30min, cool it, weigh it again, make up the weight loss with 50% ethanol, shake it well, filter it, and the test solution is obtained.

2. The method for constructing the characteristic spectrum of the standard decoction of *Clematis chinensis* according to claim 1, characterized in that, The theoretical plate number, calculated based on the ferulic acid peak, should be no less than 2000.

3. The application of the construction method as described in claim 1 or 2 in detecting the quality of the standard decoction of *Tetrapanax papyriferus*.

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