Construction method of high performance liquid chromatography for determination of content of herba potentillae discolor, medicinal slices, extract and preparation and its application

Abstract

This invention provides a method for determining the content of *Potentilla chinensis* medicinal materials, processed slices, extracts, and preparations using high-performance liquid chromatography (HPLC), and its application. This invention establishes a new indicator component by determining the content of *Potentilla chinensis* using HPLC. The method exhibits good stability, high precision, good reproducibility, convenience, and ease of mastery. It ensures the efficacy of the medicine and enables more standardized quality control of the medicinal materials and related preparations, thereby achieving comprehensive and effective quality control of *Potentilla chinensis* medicinal materials, processed slices, and related preparations.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, and in particular relates to a method for constructing and applying a high-performance liquid chromatography method for the determination of the content of Agrimonia pilosa medicinal materials, decoction pieces, extracts and preparations. Background Technology

[0002] Potentilla discolor Bge., a plant in the Rosaceae family, is a dried whole herb. It has a sweet and slightly bitter taste, is neutral in nature, and enters the liver, stomach, and large intestine meridians. It has the functions of clearing heat and detoxifying, stopping dysentery, and stopping bleeding. It is commonly used for damp-heat diarrhea, carbuncles and boils, hematemesis due to blood heat, hematochezia, and metrorrhagia. Modern research shows that Potentilla discolor has antibacterial, antidiarrheal, antitumor, immunosuppressive, and hypoglycemic effects. The main active ingredients in Potentilla discolor are flavonoids, terpenes, steroids, tannins, and organic acids, as well as phenolic acids such as protocatechuic acid, chlorogenic acid, and caffeic acid; saponins such as shampoosin and trigonelline; steroidal components such as β-sitosterol and carotene; and other components such as adenosine, naringenin, and polyterpenoids.

[0003] Quality control is the fundamental guarantee for the safe and effective clinical use of traditional Chinese medicine (TCM). The current basic model for TCM quality control mainly relies on qualitative and quantitative analysis of indicative components. The 2020 edition of the Chinese Pharmacopoeia does not specify the content determination of *Potentilla discolor*. Literature reports mainly use high-performance liquid chromatography (HPLC) to determine flavonoids and triterpenoids in *Potentilla discolor*, and ultraviolet spectrophotometry to determine total flavonoids and total triterpenoids. Kong Xiaoni et al. established an HPLC method to determine four flavonoids, including quercetin and rutin, in *Potentilla discolor*, and used ultraviolet-visible spectrophotometry to determine total flavonoids. This method is stable, precise, and reproducible, providing a scientific basis for better controlling the intrinsic quality of *Potentilla discolor*. Qu Yuanjun et al. purified *Potentilla discolor* using macroporous adsorption resin. The study investigated the triterpenoid components in *Potentilla chinensis* and evaluated their hypoglycemic effect using an in vivo mouse hypoglycemic experiment. The purification method was simple, feasible, and of controllable quality, demonstrating the hypoglycemic effect of *Potentilla chinensis* triterpenoid components. Chen Junhua et al. established a high-performance liquid chromatography (HPLC) method to separate and determine seven components in *Potentilla chinensis*, including chlorogenic acid, caffeic acid, hyperoside, and quercetin, providing experimental evidence for the quality control of the medicinal plant. Liu Siman et al. established a reversed-phase HPLC method to determine the contents of kaempferol and quercetin. Zhang Deke et al. established an HPLC method to determine the contents of oleanolic acid and total triterpenoids in *Potentilla chinensis*. Zhang Ying et al. established an HPLC method for determining the content of ursolic acid in *Potentilla chinensis*.

[0004] Potentilla chinensis granules are made from Potentilla chinensis slices through water extraction, vacuum concentration, and spray drying, and are intended for clinical use. Currently, there are few research reports on its extracts and granules. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a method for the construction and application of high performance liquid chromatography (HPLC) for the content determination of *Potentilla chinensis* herbal materials, processed slices, extracts and preparations. This method can effectively separate and determine the isoquercitrin content in *Potentilla chinensis* extracts and preparations. It can effectively reflect the quality level of *Potentilla chinensis* and its related preparations, and provide guidance for the quality control of *Potentilla chinensis* herbal materials, processed slices and related preparations.

[0006] This invention establishes a new indicator component by using high-performance liquid chromatography (HPLC) to determine the content of *Potentilla chinensis*. The method is stable, precise, reproducible, convenient, and easy to master. This ensures the efficacy of the medicine and allows for more standardized quality control of the medicinal materials and related preparations, thereby achieving comprehensive and effective quality control of *Potentilla chinensis* medicinal materials, processed slices, and related preparations.

[0007] This invention provides a method for determining the content of Agrimonia pilosa medicinal materials, processed slices, extracts, or preparations using high-performance liquid chromatography, comprising the following steps:

[0008] S1) Take the reference solution and detect it using high performance liquid chromatography. Establish a standard curve based on the concentration of the reference solution and the corresponding peak area.

[0009] S2) Take the test sample raw material, extract it with a solvent to obtain the test sample solution; determine the peak area of ​​the test sample solution by high performance liquid chromatography;

[0010] S3) Obtain the content of the target substance in the Agrimonia pilosa herb, decoction pieces, extract or preparation based on the peak area of ​​the standard curve and the test sample solution;

[0011] The reference solution includes isoquercitrin reference solution;

[0012] The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.05% phosphoric acid solution, and gradient elution is used.

[0013] In one specific embodiment of the present invention, the reference solution is prepared by the following method: mixing the reference standard with a solvent to obtain the reference solution; the reference standard is isoquercitrin; the solvent is preferably 60% ethanol.

[0014] In one specific embodiment of the present invention, reference solutions of different concentrations are provided, and high-performance liquid chromatography is used for detection. A standard curve is established based on the concentration of the reference solution and the corresponding peak area.

[0015] In one specific embodiment of the present invention, the raw material of the test sample is preferably Agrimonia pilosa herb, decoction pieces, extract or preparation; the preparation can be any preparation well known to those skilled in the art, and there are no special limitations. In the present invention, it includes, but is not limited to, standard decoction, formula granules, etc.

[0016] In a specific embodiment of the present invention, the test sample raw material is taken and extracted with a solvent to obtain a test sample solution; the solvent is preferably 60% ethanol; the mass ratio of the test sample to the solvent is preferably (0.2-1):(20-50) based on g / mL, more preferably (0.2-1):(20-30), and even more preferably (0.2-1):25.

[0017] In one specific embodiment of the present invention, the raw material of the test sample is taken, extracted with a solvent, preferably cooled, weighed, and the weight loss is replenished with a solvent. The sample is then shaken, filtered, and the filtrate is taken to obtain the test sample solution.

[0018] In one specific embodiment of the present invention, the extraction is reflux extraction or ultrasonic extraction; the extraction time is preferably 15 to 45 minutes, more preferably 30 minutes.

[0019] In one specific embodiment of the present invention, the power of the ultrasonic extraction is 600W; the frequency of the ultrasonic extraction is 40kHz.

[0020] In one specific embodiment of the present invention, the chromatographic column is preferably a C18 column; specifically, the chromatographic column may be a Waters C18, Agilent C18, or Shimadzu C18.

[0021] In one specific embodiment of the present invention, the theoretical plate number of the chromatographic column, calculated based on the isoquercitrin peak, is not less than 5000.

[0022] In a specific embodiment of the present invention, the preferred dimensions of the chromatographic column are 5 μm and 4.6 × 250 mm; the preferred column temperature is 30℃ to 40℃, specifically 30℃, 35℃ or 40℃.

[0023] In a specific embodiment of the present invention, the gradient elution, expressed as a volume percentage, specifically comprises:

[0024] 0 min, mobile phase A 18%, mobile phase B 82%;

[0025] From 0 to 12 min, mobile phase A changed from 18% to 19%, and mobile phase B changed from 82% to 81%.

[0026] 12–17 min, mobile phase A 19% → 21%, mobile phase B 81% → 79%;

[0027] Over 17–18 minutes, mobile phase A decreased from 21% to 18%, and mobile phase B decreased from 79% to 82%.

[0028] In this invention, unless otherwise specified, "→" indicates the transition from the previous percentage to the next percentage.

[0029] In one specific embodiment of the present invention, the flow rate of the mobile phase is 0.8 to 1.2 mL / min, specifically 0.8 mL / min, 1 mL / min or 1.2 mL / min.

[0030] In one specific embodiment of the present invention, the detection wavelength of the high-performance liquid chromatography is preferably 354 nm.

[0031] In one specific embodiment of the present invention, the injection volume of the high-performance liquid chromatography (HPLC) is 10 μL.

[0032] In one specific embodiment of the present invention, the isoquercitrin exhibits a linear relationship in the range of 4.99–398.91 μg / mL, with the linear relationship being y = 19860x - 16223, R. 2 =1.0000.

[0033] The content of the target substance in the herbal medicine, decoction pieces, extracts or preparations of Agrimonia pilosa can be obtained based on the standard curve of isoquercitrin and the peak area of ​​the test solution.

[0034] This invention establishes a method for determining the content of Agrimonia pilosa, identifies a new indicator component, and can control the intrinsic quality of Agrimonia pilosa and its related preparations from an overall and macroscopic perspective, ensuring the efficacy of the drugs and enabling more standardized quality control of the medicinal materials and their related preparations. Furthermore, the method provided by this invention has good stability, high precision, good reproducibility, and is convenient and easy to master.

[0035] The present invention also provides a method for quality evaluation of Agrimonia pilosa medicinal materials, decoction pieces, extracts or preparations, wherein the sample to be tested is taken and the above-mentioned construction method is used for detection to obtain the content of isoquercitrin in the sample to be tested;

[0036] The sample to be tested is a medicinal herb, sliced ​​herb, extract, or preparation of Agrimonia pilosa.

[0037] In one specific embodiment of the present invention, the preparation can be any preparation well known to those skilled in the art, and there are no special limitations. In the present invention, it includes, but is not limited to, standard decoctions, formula granules, etc.

[0038] In one specific embodiment of the present invention, if the content of isoquercitrin in the Agrimonia pilosa herb is not less than 0.060% when calculated on a dried basis, it is deemed qualified; if the content of isoquercitrin in the Agrimonia pilosa herb is less than 0.060%, it is deemed unqualified.

[0039] In one specific embodiment of the present invention, the content of isoquercitrin in the dried Agrimonia pilosa slices is not less than 0.060%, which is considered qualified; the content of isoquercitrin in the Agrimonia pilosa herb is less than 0.060%, which is considered unqualified.

[0040] In one specific embodiment of the present invention, the isoquercitrin content in the Agrimonia pilosa extract ranges from 0.80 mg to 5.70 mg per 1 g, calculated on a dried basis.

[0041] This invention employs HPLC to establish a content determination methodology using isoquercitrin as an indicator component, which can effectively control the quality of Agrimonia pilosa formula granules from raw materials, processed slices, extracts to finished products. Attached Figure Description

[0042] Figure 1 This is the UV absorption spectrum of isoquercitrin;

[0043] Figure 2 Chromatograms selected for different mobile phases;

[0044] Figure 3 The chromatogram shows the column temperature test results;

[0045] Figure 4 The chromatogram shows the results of the flow rate investigation.

[0046] Figure 5 To investigate the specificity of the chromatograms;

[0047] Figure 6 This is a quasi-curve diagram of isoquercitrin;

[0048] Figure 7 To examine chromatograms using different instruments;

[0049] Figure 8 Chromatograms were examined for different chromatographic columns;

[0050] Figure 9 Specific chromatogram for the determination of Agrimonia pilosa content;

[0051] Figure 10 This is a quasi-curve diagram of isoquercitrin;

[0052] Figure 11 To examine chromatograms using different instruments;

[0053] Figure 12Chromatograms were used to investigate the durability of different chromatographic columns. Detailed Implementation

[0054] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0055] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a method for constructing and applying a high-performance liquid chromatography method for determining the content of Agrimonia pilosa medicinal materials, decoction pieces, extracts and preparations.

[0056] All reagents used in the following examples are commercially available.

[0057] Instruments and reagents

[0058] High-performance liquid chromatographs: Waters e2695 high-performance liquid chromatograph, Shimadzu LC-20AD high-performance liquid chromatograph;

[0059] Electronic balances: ME204E / 02, XP26 (Mettler-Toledo Instruments Ltd.);

[0060] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0061] Ultrasonic cleaner: KQ600-DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0062] Chromatographic columns: Waters C18 4.6mm×250mm, 5μm; Agilent C18 4.6mm×250mm, 5μm; Shimadzu C18 4.6mm×250mm, 5μm;

[0063] Acetonitrile and phosphoric acid were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0064] Isoquercetin (China National Institutes for Food and Drug Control, batch number: 111809-202205, purity: 96.3%);

[0065] The extract of Agrimonia pilosa was prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: TQW-01, TQW-02, TQW-03, TQW-04, TQW-05, TQW-06, TQW-07, TQW-08, TQW-09, TQW-10, TQW-11, TQW-12, TQW-13, TQW-14, TQW-15, TQW-16;

[0066] The finished granules of Agrimonia pilosa were prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: KL-1, KL-2, KL-3;

[0067] The medicinal materials for *Potentilla discolor* come from Sichuan, Hubei, Henan and other places; the batch numbers are: YC-01, YC-02, YC-03, YC-04, YC-05, YC-06, YC-07, YC-08, YC-09, YC-10, YC-11, YC-12, YC-13, YC-14, YC-15, YC-16;

[0068] The processed slices of Agrimonia pilosa were prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., with batch numbers: YP-01, YP-02, YP-03, YP-04, YP-05, YP-06, YP-07, YP-08, YP-09, YP-10, YP-11, YP-12, YP-13, YP-14, YP-15, and YP-16.

[0069] Example 1: Establishment of a method for determining the content of extracts

[0070] 1.1 Solution Preparation

[0071] Preparation of the test solution: Weigh approximately 0.2 g of Agrimonia pilosa extract accurately, place it in a stoppered conical flask, add 25 mL of 60% ethanol accurately, stopper tightly, weigh, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, weigh again, replenish the lost weight with 60% ethanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0072] Preparation of reference solution: Weigh an appropriate amount of isoquercitrin reference standard accurately, add 60% ethanol to prepare a solution containing 80 μg per mL.

[0073] 1.2 Chromatographic conditions

[0074] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, and 0.05% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the detection wavelength was 354 nm. The theoretical plate number, calculated based on the isoquercitrin peak, should not be less than 5000.

[0075]

[0076]

[0077] Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0078] 1.3 Selection of chromatographic conditions and system suitability assessment

[0079] 1.3.1 Determination of detection wavelength

[0080] Based on the above-specified experimental conditions, a diode array detector was used to perform a full-band scan of isoquercitrin in the reference standard, Agrimonia pilosa herb, extract, and finished product. Except for the wavelength, all other conditions were as specified. The results are shown in [Figure number missing]. Figure 1 . Figure 1 This is the UV absorption spectrum of isoquercitrin.

[0081] The results showed that, through analysis of the spectra, the optimal detection wavelength for determining the content of isoquercitrin in Agrimonia pilosa extract was 354 nm.

[0082] 1.3.2 Selection of mobile phase

[0083] Based on the proposed experimental conditions, the separation effects of three different mobile phases were investigated: acetonitrile-0.05% acetic acid, acetonitrile-0.05% phosphoric acid, and acetonitrile-0.05% formic acid. Except for the mobile phase, all other conditions were the proposed conditions. The results are shown in [Figure number missing]. Figure 2 . Figure 2 Chromatograms for different mobile phases.

[0084] The results showed that the chromatographic peak baseline was relatively stable and there were many chromatographic peaks under the gradient elution condition of acetonitrile-0.05% phosphoric acid solution. Therefore, the gradient elution of acetonitrile-0.05% phosphoric acid solution was used as the mobile phase for the determination of the characteristic chromatogram of Agrimonia pilosa extract.

[0085] 1.3.3 Column Temperature Investigation

[0086] Based on the above-specified experimental conditions, column temperatures of 30℃, 35℃, and 40℃ were investigated. The peak area of ​​isoquercitrin, theoretical plate number, and resolution were used as evaluation indicators. Except for column temperature, all other conditions were the proposed conditions. The results are shown in [Figure number missing]. Figure 3 Table 1. Figure 3 The chromatogram shows the results of column temperature testing.

[0087] Table 1. Analytical results at different column temperatures

[0088]

[0089] The results showed that isoquercitrin could be effectively detected at different column temperatures, and 35℃ was selected as the detection column temperature.

[0090] 1.3.4 Flow velocity investigation

[0091] Based on the above-specified experimental conditions, flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min were investigated. The peak area of ​​isoquercitrin, resolution, and theoretical plate number were used as evaluation indicators. Except for the flow rate, all other conditions were the pre-specified conditions. The results are shown in [Figure number missing]. Figure 4 Table 2. Figure 4 The chromatogram shows the results of the flow rate investigation.

[0092] Table 2 Analysis results at different flow velocities

[0093]

[0094] The results showed that different flow rates could effectively detect isoquercitrin, with a tentative flow rate of 1.0 mL / min.

[0095] In summary, the chromatographic conditions and system suitability test for the determination of *Potentilla discolor* extract content were determined as follows: Octadecylsilane-bonded silica gel was used as the packing material; acetonitrile was used as mobile phase A, and 0.05% phosphoric acid solution was used as mobile phase B, with gradient elution as specified in the table below; the detection wavelength was 354 nm. The theoretical plate number, calculated based on the isoquercitrin peak, should not be less than 5000.

[0096]

[0097] 1.4 Investigation on the preparation of test solution

[0098] 1.4.1 Examination of Extraction Methods

[0099] Accurately weigh approximately 0.2 g of *Potentilla chinensis* extract (batch number: TQW-16), place it in a stoppered conical flask, accurately add 25 mL of 60% ethanol, seal tightly, and weigh. The extraction methods were investigated for reflux and ultrasonication, with an extraction time of 30 minutes. After cooling, the sample was weighed again, and the lost weight was replenished with 60% ethanol. The mixture was shaken well, filtered, and the filtrate was collected as the final product. The isoquercitrin content under different extraction methods was analyzed and calculated, and the results are shown in Table 3.

[0100] Table 3. Analysis results of different extraction methods

[0101]

[0102]

[0103] The results showed that the content of isoquercitrin obtained by reflux extraction and ultrasonic extraction was not significantly different. Ultrasonic extraction was simple to operate, and ultrasonic extraction was selected as the extraction method.

[0104] 1.4.2 Investigation of Extraction Solvents

[0105] Accurately weigh approximately 0.2 g of *Potentilla chinensis* extract (batch number: TQW-16) and place it in a stoppered conical flask. Add 25 mL each of ethanol, 60% ethanol, water, 80% methanol, 30% methanol, and methanol, respectively. Seal the flask tightly and weigh it. Sonicate the flask (600 W, 40 kHz) for 30 minutes, cool it, and weigh it again. Make up the weight loss with the appropriate solvent, shake well, filter, and collect the filtrate. The isoquercitrin content under different extraction solvents was analyzed and calculated, and the results are shown in Table 4.

[0106] Table 4 Results of the investigation of different extraction solvents

[0107]

[0108] The results showed that 60% ethanol had a higher extraction efficiency of isoquercitrin, and 60% ethanol was selected as the extraction solvent for Agrimonia pilosa extract.

[0109] 1.4.3 Examination of extraction time

[0110] Accurately weigh approximately 0.2 g of *Potentilla chinensis* extract (batch number: TQW-16), place it in a stoppered conical flask, accurately add 25 mL of 60% ethanol, seal tightly, weigh, and sonicate (600 W, 40 kHz) for 15, 30, and 45 minutes. After cooling, weigh again, replenish the lost weight with 60% ethanol, shake well, filter, and collect the filtrate. The isoquercitrin content at different extraction times was analyzed and calculated; the results are shown in Table 5.

[0111] Table 5. Analysis results of different extraction times

[0112]

[0113] The results showed that sufficient extraction was achieved when the extraction time was 30 minutes. Therefore, the extraction time for the test sample was determined to be 30 minutes.

[0114] 1.4.4 Investigation of Solvent Addition Amount

[0115] Accurately weigh approximately 0.2 g of Agrimonia pilosa extract (batch number: TQW-16) and place it in a stoppered conical flask. Accurately add 10 mL, 25 mL, and 50 mL of 60% ethanol, respectively. Seal the flask tightly and weigh the contents. Sonicate the flask (600 W, 40 kHz) for 30 minutes, cool, and weigh again. Make up the weight loss with 60% ethanol, shake well, filter, and collect the filtrate. The isoquercitrin content was analyzed and calculated for different solvent additions; the results are shown in Table 6.

[0116] Table 6 Results of the investigation of different solvent addition amounts

[0117]

[0118] The results showed that when the sample size was 0.2g and the solvent added was 25mL, the target component could be completely extracted. Therefore, the solvent added was determined to be 25mL.

[0119] In summary, the preparation method for the test sample for the determination of the content of Agrimonia pilosa extract is as follows: Take about 0.2g of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of 60% ethanol, seal tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, weigh it again, make up the lost weight with 60% ethanol, shake well, filter, and take the filtrate to obtain the product.

[0120] 1.5 Determination of content of Agrimonia pilosa extract

[0121] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the stationary phase; acetonitrile as mobile phase A and 0.05% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; the detection wavelength was 354 nm. The theoretical plate number, calculated based on the isoquercitrin peak, should not be less than 5000.

[0122]

[0123] Preparation of reference solution: Weigh an appropriate amount of isoquercitrin reference standard accurately, add 60% ethanol to prepare a solution containing 80 μg per mL.

[0124] Preparation of the test solution: Weigh approximately 0.2 g of this product accurately, place it in a stoppered conical flask, accurately add 25 mL of 60% ethanol, stopper tightly, weigh, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, weigh again, replenish the lost weight with 60% ethanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0125] Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0126] 1.6 Methodological Investigation of Extraction

[0127] 1.6.1 Specificity Experiment

[0128] Preparation of test solution: Prepare the test solution of Agrimonia pilosa extract according to the experimental conditions proposed above.

[0129] Preparation of reference solution: Weigh an appropriate amount of isoquercitrin reference standard accurately, add 60% ethanol to prepare a solution containing 80 μg per mL.

[0130] Preparation of negative control solution: A negative control solution lacking *Potentilla discolor* extract was prepared according to the experimental conditions outlined above. Results are shown below. Figure 5 . Figure 5The chromatogram was used to investigate specificity.

[0131] The results showed that the negative solution chromatogram did not interfere with the determination of the target peak, indicating that the method has good specificity.

[0132] 1.6.2 Precision Test

[0133] The reference solution was injected six times consecutively, and the peak area of ​​isoquercitrin was recorded. The RSD value was calculated, and the results are shown in Table 8.

[0134] Table 8 Precision test results

[0135]

[0136] The results showed that the peak area RSD of isoquercitrin was 1.0% in the precision study. The injection precision of this method is good.

[0137] 1.6.3 Linear Relationship

[0138] An appropriate amount of isoquercitrin was placed in a 25 mL volumetric flask and dissolved in 60% ethanol to prepare a solution containing 398.91 μg / mL isoquercitrin (purity 96.3%). This solution was then diluted to concentrations of 199.46 μg / mL, 99.73 μg / mL, 49.86 μg / mL, 19.95 μg / mL, and 4.99 μg / mL. 10 μL of each solution was accurately injected into the liquid chromatograph, and the peak area was obtained. A response curve was plotted with concentration (X, μg / mL) on the x-axis and peak area (Y) on the y-axis. The results are shown in Table 9. Figure 6 . Figure 6 This is a quasi-curve diagram of isoquercitrin.

[0139] Table 9. Results of the standard curve analysis of isoquercitrin

[0140]

[0141] The results showed that when the concentration of isoquercitrin was in the range of 4.99–398.91 μg / mL, the linear relationship was y = 19860x - 16223, R0 2 =1.0000. This indicates a good linear relationship within the concentration range of 4.99–398.91 μg / mL.

[0142] 1.6.4 Repeatability Experiment

[0143] Take about 0.2g of Agrimonia pilosa extract (batch number: TQW-16), accurately weigh 6 portions, and prepare test solutions by the same operator according to the established method. Measure the peak area and calculate the isoquercitrin content of the 6 test samples. The results are shown in Table 10.

[0144] Table 10 Results of Repeatability Experiments

[0145]

[0146]

[0147] The RSD value of isoquercitrin content obtained from 6 repeatability tests was 2.0%, indicating that the method has good repeatability.

[0148] 1.6.5 Stability Test

[0149] The peak area of ​​isoquercitrin was determined at 0h, 2h, 4h, 8h, 12h, 16h and 24h using the same Agrimonia pilosa extract test sample (batch number: TQW-16). The results are shown in Table 11.

[0150] Table 11 Stability test results

[0151]

[0152] The results showed that the RSD values ​​of the peak areas of isoquercitrin were all 2.0%, and the test solution had good stability within 24 hours.

[0153] 1.6.6 Intermediate Precision

[0154] 1.6.6.1 Investigations by different personnel and at different times

[0155] The same Agrimonia pilosa extract (batch number: TQW-16) was used as a test sample. Different personnel (A, B) prepared test sample solutions at different times (I, II) for analysis. The content of isoquercitrin in the test sample was calculated. The results are shown in Table 12.

[0156] Table 12 Results of intermediate precision experiments

[0157]

[0158] The results showed that the RSD value of isoquercitrin was 1.0%, indicating good intermediate precision of this method.

[0159] 1.6.7 Recovery rate

[0160] Six portions of a known concentration of *Potentilla chinensis* extract (batch number: TQW-16, isoquercitrin content 5.40 mg / g) were accurately weighed. A precise amount of isoquercitrin reference standard (purity: 96.3%) was added to each portion. The test solutions were prepared and determined according to the prescribed method. The recovery rate was calculated, and the results are shown in Table 13. The calculation formula is as follows:

[0161] Recovery rate (%) = (Measured amount - Content in sample) / Amount added × 100%

[0162] Table 13 Results of the isoquercitrin recovery experiment

[0163]

[0164] The results showed that the average recovery rate of isoquercitrin was 100.5%, and the RSD was 1.3%. The method had good accuracy.

[0165] 1.6.8 Durability Assessment

[0166] 1.6.8.1 Investigation with different instruments

[0167] Two portions of approximately 0.2 g of *Potentilla chinensis* extract powder (batch number: TQW-16) were accurately weighed and prepared according to the established method. The solutions were then analyzed using a Waters e2695 and a Shimadzu LC-20AD high-performance liquid chromatograph (both columns were Waters C18 4.6 mm × 250 mm, 5 μm). The isoquercitrin content was calculated, and the results were obtained. Figure 7 See Table 14. Figure 7 Chromatograms were examined using different instruments.

[0168] Table 14 Experimental Results with Different Instruments

[0169]

[0170] The results showed that the RSD value of the results measured by the Waters e2695 and Shimadzu LC-20AD high performance liquid chromatographs was 3.7%, indicating that the instruments used in this method have good durability.

[0171] 1.6.8.2 Investigation of different chromatographic columns

[0172] Different brands of C18 columns were used to detect the same *Potentilla discolor* extract (batch number: TQW-16). These columns included Waters C18 (4.6mm×250mm, 5μm), Agilent C18 (4.6mm×250mm, 5μm), and Shimadzu C18 (4.6mm×250mm, 5μm). The results are shown in the table below. Figure 8 Table 15. Figure 8 Chromatograms were examined for different chromatographic columns.

[0173] Table 15 Results of column robustness test

[0174]

[0175] The results showed that the analytical chromatographic parameters of different chromatographic columns were good.

[0176] 1.7 Validation of Extract Content Determination

[0177] Using Agrimonia pilosa extract as the test sample, the test sample solution was prepared and measured according to the proposed method. The peak area was recorded, and the content of isoquercitrin was calculated. The results are shown in Table 16.

[0178] Table 16. Isoquercitrin content in Agrimonia pilosa extract

[0179]

[0180]

[0181] The results showed that the measured content of isoquercitrin in *Potentilla chinensis* extract ranged from 2.02 mg / g to 4.21 mg / g, with an average content of 3.22 mg / g and a SD value of 0.82. The 70%–130% limit of the average content was 2.25 mg / g–4.19 mg / g, and the SD value (plus or minus three times the average) was 0.76 mg / g–5.68 mg / g. The range of ±30% of the average was relatively narrow; therefore, the range of ±3 SD of the average was temporarily used as the limit range for *Potentilla chinensis* extract, rounded to 0.80 mg / g–5.70 mg / g. That is, the limit range for isoquercitrin in *Potentilla chinensis* extract is "containing isoquercitrin (C... 21 H 20 O 12 The range is 0.80 mg / g to 5.70 mg / g.

[0182] 1.8 Verification of Finished Product Particle Content Determination

[0183] The results of testing 16 batches of finished Agrimonia pilosa granules are shown in Table 18.

[0184] Table 18 Results of Particle Content Determination in Finished Agrimonia pilosa Products

[0185]

[0186] The results showed that the method for determining the content of Agrimonia pilosa extract can effectively detect Agrimonia pilosa finished product granules.

[0187] Example 2: Establishment of a method for determining the content of medicinal materials

[0188] 2.1 Preparation of the test solution

[0189] Take about 1g of the powder of Potentilla chinensis (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of 60% ethanol, seal tightly, weigh it, reflux and extract for 30 minutes, filter, and collect the filtrate to obtain the product.

[0190] 2.2 Preparation of reference solution

[0191] Take an appropriate amount of isoquercitrin reference standard, accurately weigh it, and add 60% ethanol to prepare a solution containing 80 μg per 1 mL.

[0192] 2.3 Chromatographic conditions: Same as 1.3 Selection of chromatographic conditions for extracts and evaluation of system suitability.

[0193] 2.4 Investigation on the preparation of test solution

[0194] 2.4.1 Investigation of Extraction Solvents

[0195] Twelve samples of *Potentilla chinensis* herb (batch number: YC-16), each approximately 1.0 g, were accurately weighed and placed in stoppered conical flasks. Extraction was performed using methanol, 80% methanol, 30% methanol, water, 60% ethanol, and 95% ethanol, respectively. 25 mL of the corresponding solvent was accurately added to each flask, the flasks were sealed, and the samples were weighed. The flasks were then sonicated (600 W, 40 kHz) for 30 minutes, cooled, and weighed again. The weight loss was replenished with the corresponding solvent, the flasks were shaken well, filtered, and the filtrate was collected. 10 μL of each sample solution was accurately injected into the liquid chromatograph, and the isoquercitrin content under different solvent extraction conditions was calculated. The results are shown in Table 19.

[0196] Table 19 Analytical results of different extraction solvents

[0197]

[0198] The results showed that the highest content of isoquercitrin in the sample was obtained when 60% ethanol was used as the extraction solvent, and 60% ethanol was determined to be the extraction solvent.

[0199] 2.4.2 Examination of Extraction Methods

[0200] Four samples of *Potentilla chinensis* herb (batch number: YC-16), each approximately 1.0 g, were accurately weighed and placed in stoppered conical flasks. 25 mL of 60% ethanol was accurately added, the flasks were sealed, and the samples were weighed. Ultrasonic extraction (600 W, 40 kHz) and reflux extraction were performed separately for 30 minutes each. After cooling, the samples were weighed again, and the weight loss was replenished with 60% ethanol. The mixture was shaken well and filtered to obtain the final product. 10 μL of each solution was accurately injected into a liquid chromatograph to analyze and calculate the isoquercitrin content under different extraction methods. The results are shown in Table 20.

[0201] Table 20 Analysis results of different extraction methods

[0202]

[0203] The results showed that the isoquercitrin content obtained by reflux extraction was high, and reflux extraction was determined to be the optimal extraction method.

[0204] 2.4.3 Examination of extraction time

[0205] Six portions of *Potentilla chinensis* herbal powder (batch number: YC-16), each approximately 1.0 g, were accurately weighed and placed in stoppered conical flasks. 25 mL of 60% ethanol was accurately added, the flasks were sealed, and the contents were weighed. The flasks were then refluxed for 15 minutes, 30 minutes, and 45 minutes, respectively. After cooling, the contents were weighed again, and the lost weight was replenished with 60% ethanol. The flasks were shaken well, filtered, and the filtrate was collected. 10 μL of each sample solution was accurately injected into the liquid chromatograph, and the isoquercitrin content at different extraction times was analyzed and calculated. The results are shown in Table 21.

[0206] Table 21 Analysis results of different extraction times

[0207]

[0208] The results showed that isoquercitrin was basically completely extracted from the sample within 30 minutes, so the extraction time was chosen to be 30 minutes.

[0209] 2.4.4 Investigation of Solvent Addition Amount

[0210] Six portions of *Potentilla chinensis* herbal powder (batch number: YC-16), each approximately 1.0 g, were accurately weighed and placed in stoppered conical flasks. 10 mL, 25 mL, and 50 mL of 60% ethanol were accurately added to each flask, and the flasks were sealed. The flasks were weighed, and the mixture was refluxed for 30 minutes. After cooling, the flasks were weighed again. The lost weight was replenished with 60% ethanol, and the mixture was shaken well. The filtrate was then collected. 10 μL of each test solution was accurately injected into the liquid chromatograph. The isoquercitrin content was analyzed and calculated under different solvent addition amounts. The results are shown in Table 22.

[0211] Table 22 Analysis results for different solvent addition amounts

[0212]

[0213] The results showed that isoquercitrin could be completely extracted from the sample when the solvent volume was 25 mL. Considering factors such as the appropriate peak area, the optimal solvent volume was determined to be 25 mL.

[0214] 2.4.5 Determination of the preparation method for the test solution

[0215] Take about 1.0g of powdered Potentilla chinensis (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of 60% ethanol, seal tightly, weigh it, reflux and extract for 30 minutes, cool, weigh it again, replenish the lost weight with 60% ethanol, shake well, filter, and collect the filtrate to obtain the final product.

[0216] 2.5 Method for determining the content of Agrimonia pilosa in medicinal materials

[0217] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile was used as mobile phase A, and 0.05% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the detection wavelength was 354 nm. The theoretical plate number, calculated based on the isoquercitrin peak, should not be less than 5000.

[0218]

[0219] Preparation of reference solution: Weigh an appropriate amount of isoquercitrin reference standard accurately, add 60% ethanol to prepare a solution containing 80 μg per mL.

[0220] Preparation of the test solution: Weigh approximately 1.0 g of the powder (passed through a No. 3 sieve) accurately, place it in a stoppered conical flask, accurately add 25 mL of 60% ethanol, stopper tightly, weigh, reflux for 30 minutes, cool, weigh again, replenish the lost weight with 60% ethanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0221] Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0222] 2.6 Methodological Investigation of Medicinal Materials

[0223] 2.6.1 Specificity Experiment

[0224] The test solution, control solution, and negative control solution of Agrimonia pilosa were prepared according to the proposed method, and then tested. Results are shown below. Figure 9 . Figure 9 Specific chromatogram for the determination of Agrimonia pilosa content.

[0225] The results showed that the negative solution chromatogram did not interfere with the determination of the target peak, indicating that the method has good specificity.

[0226] 2.6.2 Precision Test

[0227] The reference solution was injected six times consecutively, and the peak area of ​​isoquercitrin was recorded. The RSD value was calculated, and the results are shown in Table 23.

[0228] Table 23 Precision test results

[0229]

[0230] The results showed that the peak area RSD of isoquercitrin was 1.0% in the precision study, indicating that the injection precision of this method was good.

[0231] 2.6.3 Linear Relationship

[0232] Take an appropriate amount of isoquercitrin and place it in a 25 mL volumetric flask. Dissolve it in 60% ethanol to prepare an isoquercitrin concentration of 398.91 μg / mL (purity 96.3%). Then dilute to concentrations of 199.46 μg / mL, 99.73 μg / mL, 49.86 μg / mL, 19.95 μg / mL, and 4.99 μg / mL. Accurately pipette 10 μL and inject it into the liquid chromatograph. Obtain the peak area and plot the response curve with concentration (X, μg / mL) on the x-axis and peak area (Y) on the y-axis. The results are shown in Table 24. Figure 10 . Figure 10 This is a quasi-curve diagram of isoquercitrin.

[0233] Table 24 Results of isoquercitrin standard curve analysis

[0234]

[0235] The results showed that when the concentration of isoquercitrin was in the range of 4.99–398.91 μg / mL, the linear relationship was y = 19860x - 16223, R0 2 =1.0000. This indicates a good linear relationship within the concentration range of 4.99–398.91 μg / mL.

[0236] 2.6.4 Stability Test

[0237] The same test solution of Agrimonia pilosa (batch number: YC-16) was used to determine the peak area of ​​isoquercitrin at 0, 2h, 4h, 8h, 12h, 16h and 24h. The results are shown in Table 25.

[0238] Table 25 Results of isoquercitrin stability test

[0239]

[0240] The results showed that under the experimental conditions, the peak area RSD of isoquercitrin was 1.0%, and the test solution had good stability within 24 hours.

[0241] 2.6.5 Repeatability Test

[0242] Take 1.0g of the same Agrimonia pilosa herb test sample (batch number: YC-16), accurately weigh 6 portions, and have the same operator prepare the test sample solution according to the proposed method. Calculate the content of the 6 test samples. The results are shown in Table 26.

[0243] Table 26 Results of Repeatability Tests

[0244]

[0245] The results showed that the RSD value of isoquercitrin was 1.0%, and the method had good reproducibility.

[0246] 2.6.6 Intermediate Precision

[0247] 2.6.6.1 Investigations by different personnel and at different times

[0248] The same *Potentilla discolor* herb (batch number: YC-16) was used as a test sample. Different personnel (A, B) prepared test sample solutions at different times (I, II) for analysis. The content of isoquercitrin in the test sample was calculated, and the results are shown in Table 27.

[0249] Table 27 Results of Time-Based Surveys of Different Personnel

[0250]

[0251]

[0252] The results showed that the RSD value of isoquercitrin content determination was 1.0%, indicating good intermediate precision of this method.

[0253] 2.6.7 Recovery rate

[0254] Take approximately 0.5 g of the *Potentilla chinensis* herb sample (batch number: YC-16, content: 0.147%), in six portions, accurately weigh them, and accurately add a certain amount of isoquercitrin reference standard (purity: 96.3%) to each sample. Prepare and determine the test solution according to the prescribed method, calculate the recovery rate, and the results are shown in Table 28. The calculation formula is as follows:

[0255] Recovery rate (%) = (Measured amount - Content in sample) / Amount added × 100%

[0256] Table 28 Results of the recovery experiment

[0257]

[0258] The results showed that the RSD of the recovery rate was 1.1%, indicating that the method had good accuracy.

[0259] 2.6.8 Durability Test

[0260] 2.6.8.1 Investigation with different instruments

[0261] According to the determined method, accurately weigh 1.0 g of Agrimonia pilosa powder (batch number: YC-16), and accurately determine two portions. Analyze each portion separately using a Waters or Shimadzu high-performance liquid chromatograph (both columns: Waters C18 4.6 mm × 250 mm, 5 μm). Calculate the isoquercitrin content. The results are shown in [Figure number missing]. Figure 11 Table 29. Figure 11 Chromatograms were examined using different instruments.

[0262] Table 29 Experimental Results with Different Instruments

[0263]

[0264]

[0265] The results show that the RSD value of the results measured by different instruments is 1.0%, indicating that the instruments used in this method have good durability.

[0266] 2.6.8.2 Investigation of different chromatographic columns

[0267] Different brands of chromatographic columns were used: Waters C18 4.6mm×250mm, 5μm; Agilent C18 4.6mm×250mm, 5μm; and Shimadzu C18 4.6mm×250mm, 5μm to detect the same *Potentilla discolor* herb sample (batch number: YC-16). The results are shown in the figure. Figure 12 Table 30. Figure 12 Chromatograms were used to investigate the durability of different chromatographic columns.

[0268] Table 30 Results of column durability study

[0269]

[0270] The results showed that the RSD value of the results measured by different chromatographic columns was 4.5%, indicating that the chromatographic columns of this method have good robustness.

[0271] 2.7 Verification of Medicinal Material Content Determination

[0272] The results of testing 16 batches of Agrimonia pilosa medicinal materials are shown in Table 31.

[0273] Table 31 Results of content determination of Agrimonia pilosa.

[0274]

[0275]

[0276] The results showed that the isoquercitrin content of the 16 batches of *Potentilla chinensis* herb tested, calculated on a dried basis, ranged from 0.101% to 0.168%, with an average of 0.129% and a SD value of 0.022. The range of 70% to 130% of the average was 0.090% to 0.168%, and the SD value (plus or minus three times the average) ranged from 0.063% to 0.195%. Taking the average value minus three SD values ​​as the minimum limit for isoquercitrin content, rounded to 0.060%, it is tentatively defined as "This product, calculated on a dried basis, contains isoquercitrin (C... 21 H 20 O 12 "(Not less than 0.060%)".

[0277] 2.8 Verification of the content determination of medicinal slices

[0278] The results of testing 16 batches of Agrimonia pilosa decoction pieces are shown in Table 32.

[0279] Table 32 Results of content determination of Agrimonia pilosa slices

[0280]

[0281]

[0282] The results showed that the measured value of isoquercitrin in Agrimonia pilosa slices was 0.093%–0.169%, with an average content of 0.126% and a SD of 0.021. Calculated on a dried basis, the limit of 70%–130% of the average content is 0.088%–0.164%; the limit range of the average content plus or minus 3 times the SD is 0.063%–0.189%. Taking the average value minus 3SD as the minimum limit for isoquercitrin content, rounded to 0.060%, it is tentatively defined as "This product, calculated on a dried basis, contains isoquercitrin (C... 21 H 20 O 12 "(Not less than 0.060%)".

[0283] 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 determining the content of Agrimonia pilosa medicinal materials, processed slices, extracts, and preparations by high-performance liquid chromatography, characterized in that, Includes the following steps: S1) Take the reference solution and detect it using high performance liquid chromatography. Establish a standard curve based on the concentration of the reference solution and the corresponding peak area. S2) Take the test sample raw material, extract it with a solvent to obtain the test sample solution; determine the peak area of ​​the test sample solution by high performance liquid chromatography; S3) Obtain the content of the target substance in the Agrimonia pilosa herb, decoction pieces, extract or preparation based on the peak area of ​​the standard curve and the test sample solution; The reference solution includes isoquercitrin reference solution; The chromatographic conditions for the high performance liquid chromatography are as follows: the column is a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.05% phosphoric acid solution, and gradient elution is used. The gradient elution, expressed as a volume percentage, specifically comprises: 0 min, mobile phase A 18%, mobile phase B 82%; From 0 to 12 min, mobile phase A changed from 18% to 19%, and mobile phase B changed from 82% to 81%. Over 12-17 minutes, mobile phase A decreased from 19% to 21%, and mobile phase B decreased from 81% to 79%. 17-18 min, mobile phase A 21%→18%, mobile phase B 79%→82%; The solvent is 60% ethanol; the extraction is reflux extraction or ultrasonic extraction.

2. The construction method according to claim 1, characterized in that, The extraction time is 15-45 minutes.

3. The construction method according to claim 1, characterized in that, The mass ratio of the test sample to the solvent, expressed in g / mL, is (0.2~1):(20~50).

4. The construction method according to claim 1, characterized in that, The chromatographic column is a C18 column with a size of 5 μm and 4.6 × 250 mm; the column temperature is 30℃~40℃.

5. The construction method according to claim 1, characterized in that, The mobile phase flow rate is 0.8~1.2 mL / min; the detection wavelength is 354 nm; and the injection volume is 10 μL.

6. The construction method according to claim 1, characterized in that, The theoretical plate number of the chromatographic column, calculated based on the isoquercitrin peak, shall not be less than 5000.

7. The construction method according to claim 1, characterized in that, The isoquercitrin showed a linear relationship in the range of 4.99~398.91 μg / mL, with the linear relationship being y=19860x-16223 and R²=1.0000.

8. A method for quality evaluation of Agrimonia pilosa medicinal materials, processed slices, extracts, or preparations, characterized in that, Take the sample to be tested and use the construction method described in any one of claims 1 to 7 to detect the content of isoquercitrin in the sample to be tested; The sample to be tested is a medicinal herb, sliced ​​herb, extract, or preparation of Agrimonia pilosa.

9. The quality evaluation method according to claim 8, characterized in that, If the content of isoquercitrin in the *Potentilla discolor* herb is not less than 0.060% when calculated on a dried basis, it is deemed qualified; if the content of isoquercitrin in the *Potentilla discolor* herb is less than 0.060%, it is deemed unqualified. Based on the dried product, the content of isoquercitrin in the prepared slices of Agrimonia pilosa is not less than 0.060%, which is considered qualified; the content of isoquercitrin in the herbal medicine of Agrimonia pilosa is less than 0.060%, which is considered unqualified. Based on dried product, the isoquercitrin content in the *Potentilla chinensis* extract ranges from 0.80 mg to 5.70 mg per 1 g.

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