Construction method of characteristic spectrum of euphorbia kansuensis medicine preparation and content determination method

Abstract

This invention belongs to the field of traditional Chinese medicine detection technology, specifically providing a method for constructing characteristic chromatograms and determining the content of Euphorbia pekinensis drug preparations. In the method for constructing characteristic chromatograms of Euphorbia pekinensis drug preparations, octylsilane-bonded silica gel is used as the packing material, and an acidic aqueous solution-acetonitrile is used as the mobile phase. By continuously optimizing the gradient elution program, combined with controlling the 250-260 nm wavelength, the flow rate at 0.8-1.2 mL / min, and the column temperature at 33-40℃, 9-10 common characteristic peaks are finally obtained, and the effective separation of these 9-10 common characteristic peaks is achieved. This provides a basis for the quality detection and control of Euphorbia pekinensis drug preparations and realizes the overall component characterization of Euphorbia pekinensis drug preparations.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine detection technology, specifically relating to a method for constructing characteristic spectra and determining the content of Euphorbia pekinensis drug preparations. Background Technology

[0002] Euphorbia pekinensis Rupr., a plant in the Euphorbiaceae family, is the dried root of the Chinese herb. Euphorbia pekinensis is bitter, cold in nature, and toxic. It enters the lung, spleen, and kidney meridians. It has the effects of draining water and eliminating phlegm, reducing swelling and dissipating nodules. It is used for edema, abdominal distension, ascites, phlegm accumulation, cough and asthma, difficulty in urination and defecation, carbuncles, boils, scrofula, and phlegm nodules. The main chemical components of Euphorbia pekinensis are phenolic acids and flavonoids. Euphorbia pekinensis granules are obtained through extraction, concentration, drying, and granulation of the Chinese herb. These granules can be used to produce other types of Euphorbia pekinensis pharmaceutical preparations, such as tablets and capsules.

[0003] Euphorbia pekinensis is a traditional Chinese medicine listed in the 2020 edition of the Chinese Pharmacopoeia. Literature also describes the chemical components of Euphorbia pekinensis. However, on the one hand, using existing technologies to determine or identify the content of a single component in Euphorbia pekinensis granules cannot comprehensively detect and control its quality; on the other hand, combining the determination of one component with the identification of other components in Euphorbia pekinensis granules is time-consuming and labor-intensive, making it difficult to widely apply in production practice. Summary of the Invention

[0004] Therefore, the purpose of this invention is to establish a method for comprehensively and rapidly detecting Euphorbia pekinensis formulation granules. Furthermore, the purpose of this invention is to provide a method for constructing characteristic spectra and determining the content of Euphorbia pekinensis drug preparations.

[0005] Specifically, this invention discloses a method for constructing the characteristic spectrum of Euphorbia pekinensis drug preparations, including the following steps:

[0006] (1) Prepare the test solution from Euphorbia pekinensis drug preparation;

[0007] (2) The test solution was analyzed by high performance liquid chromatography. Octylsilane bonded silica gel was used as the stationary phase. The detection wavelength was 250-260 nm, the flow rate was 0.8-1.2 mL / min, the column temperature was 33-40 °C, and an acidic aqueous solution-acetonitrile was used as the mobile phase for gradient elution. The gradient elution program included: 0 → 7 min → 10 min → 20 min → 33 min → 60 min. The volume percentage of acetonitrile in the mobile phase was 5% → 5% → 9% → 9% → 14% → 40%.

[0008] According to any one of the construction methods of the present invention, step (1) includes the steps of extracting the Euphorbia pekinensis drug preparation with a solvent, separating solid and liquid, and taking the filtrate.

[0009] According to any one of the construction methods of the present invention, step (1) satisfies any one or more of the following AE:

[0010] A. The mass ratio of the Euphorbia pekinensis drug preparation to the volume ratio of the organic solvent is (0.05-0.2):(50-100), and the ratio is g / mL;

[0011] B. The solvent is a methanol aqueous solution with a volume fraction of 20-80%;

[0012] C. The extraction method is heating reflux extraction or ultrasonic extraction, preferably with an extraction time of 15-45 min;

[0013] D. Solid-liquid separation is achieved by centrifugation or filtration;

[0014] E. Ultrasonic power is 150-350W.

[0015] Optionally, the preparation method of the test solution in step 1) includes the following steps: Take 0.05-0.2g of Euphorbia pekinensis powder, accurately weigh it, place it in a stoppered conical flask, accurately add 50-100mL of 20-80% methanol, stopper tightly, weigh it, sonicate for 15-45 minutes, take it out, let it cool, weigh it again, replenish the lost weight with 20-80% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0016] According to any one of the construction methods of the present invention, in step (2), the volume percentage of formic acid in the aqueous solution containing formic acid is 0.05-0.2%; and / or, the detection wavelength is 254 nm; and / or, the flow rate is 1.0 ml / min; and / or, the column temperature is 35 °C; and / or, during the high performance liquid chromatography detection, the injection volume is 1-20 μl; and / or, an Acclaim 120C8 column is used; and / or, the acid in the aqueous solution containing acid is selected from one or more of formic acid, acetic acid, and phosphoric acid.

[0017] According to any one of the construction methods of the present invention, the construction method further includes the steps of preparing a reference solution using at least one of gallic acid, ellagic acid, and kaempferol-3-O-galactoside as a reference standard, and replacing the test solution in step (2) with the reference solution to obtain a reference standard characteristic chromatogram; and / or, using the aqueous extract of Euphorbia pekinensis reference medicinal material instead of Euphorbia pekinensis drug preparation to prepare a reference medicinal material reference solution according to any one of the above construction methods and detecting it by high performance liquid chromatography to obtain a reference medicinal material reference chromatogram.

[0018] According to any one of the construction methods of the present invention, the solvent of the reference solution is selected from a methanol aqueous solution with a volume fraction of 20-80%; and / or, 1 mL of the reference solution contains 10-30 μg of each reference standard.

[0019] Optionally, the preparation method of the reference solution includes the following steps: take appropriate amounts of gallic acid, ellagic acid, and kaempferol-3-O-galactoside, accurately weigh them, add 20-80% methanol to prepare a solution containing 10-30 μg of reference standard per 1 ml, and use it as the reference solution.

[0020] According to any one of the construction methods of the present invention, the preparation method of the aqueous extract of Euphorbia pekinensis reference material includes the following steps: taking Euphorbia pekinensis reference material, adding water, heating under reflux, filtering, and evaporating the filtrate to dryness to obtain the extract. Preferably, the mass ratio of the Euphorbia pekinensis reference material to the volume ratio of water is (0.3-0.8):(20-100), with a ratio of g / mL. Preferably, the heating under reflux time is 0.5-2 hours.

[0021] According to any one of the construction methods of the present invention, the preparation method of the aqueous extract of Euphorbia pekinensis reference material includes the following steps: take 0.3-0.8g of Euphorbia pekinensis reference material, place it in a stoppered conical flask, add 20-100ml of water, heat under reflux for 0.5-2 hours, filter, and evaporate the filtrate to dryness to obtain the extract.

[0022] According to any one of the construction methods of the present invention, the preparation method of the reference solution of the control medicinal material includes the following steps: take 0.3-0.8g of Euphorbia pekinensis reference medicinal material, place it in a stoppered conical flask, add 20-100ml of water, heat under reflux for 0.5-2 hours, filter, evaporate the filtrate to dryness, add 50-100mL of 20-80% methanol to the residue and sonicate for 15-45 minutes, take it out, cool it, shake it well, filter it, and take the filtrate as the reference solution of the control medicinal material.

[0023] According to any one of the construction methods of the present invention, the pharmaceutical preparation may optionally be a tablet, capsule, pill, granule, honey-processed pill, sustained-release preparation, immediate-release preparation, controlled-release preparation, oral liquid preparation, or injectable preparation.

[0024] According to any one of the construction methods of the present invention, the characteristic chromatogram of the Euphorbia pekinensis drug preparation has 10 common characteristic peaks, peak 6 corresponds to the retention time of the ellagic acid reference peak; the peak corresponding to the ellagic acid reference peak is the S peak, and the relative retention time of each characteristic peak and the S peak is within ±10% of a specified value; the specified values ​​are: 0.16 (peak 1), 0.42 (peak 2), 0.51 (peak 3), 0.60 (peak 4), 0.73 (peak 5), 1.08 (peak 7), 1.13 (peak 8), 1.19 (peak 9), and 1.37 (peak 10).

[0025] This invention also provides a method for determining the content of active ingredients in Euphorbia pekinensis pharmaceutical preparations, comprising the following steps:

[0026] Prepare the test solution from the Euphorbia pekinensis drug preparation;

[0027] Prepare a reference solution by taking one or more of gallic acid, ellagic acid, and kaempferol-3-O-galactoside;

[0028] Test procedure: The test solution and reference solution were analyzed by high performance liquid chromatography (HPLC) using octylsilane-bonded silica gel as the stationary phase, with a detection wavelength of 250-260 nm, a flow rate of 0.8-1.2 mL / min, and a column temperature of 33-40 °C. Gradient elution was performed using an acidic aqueous solution-acetonitrile as the mobile phase. The gradient elution program included: 0 → 7 min → 10 min → 20 min → 33 min → 60 min. The volume percentage of acetonitrile in the mobile phase was 5% → 5% → 9% → 9% → 14% → 40%. The content of the reference standard in the Euphorbia pekinensis drug preparation was calculated.

[0029] According to any one of the content determination methods of the present invention, step (1) includes the steps of extracting the Euphorbia pekinensis drug preparation with a solvent, separating solid and liquid, and taking the filtrate;

[0030] Preferably, step (1) satisfies any one or more of the following AEs:

[0031] A. The mass ratio of the Euphorbia pekinensis drug preparation to the volume ratio of the organic solvent is (0.05-0.2):(50-100), and the ratio is g / mL;

[0032] B. The solvent is a methanol aqueous solution with a volume fraction of 20-80%;

[0033] C. The extraction method is heating reflux extraction or ultrasonic extraction, preferably with an extraction time of 15-45 min;

[0034] D. Solid-liquid separation is achieved by centrifugation or filtration;

[0035] E. Ultrasonic power is 150-350W.

[0036] Optionally, the preparation method of the test solution in step 1) includes the following steps: Take 0.05-0.2g of Euphorbia pekinensis powder, accurately weigh it, place it in a stoppered conical flask, accurately add 50-100mL of 20-80% methanol, stopper tightly, weigh it, sonicate for 15-45 minutes, take it out, let it cool, weigh it again, replenish the lost weight with 20-80% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0037] Optionally, the power of the ultrasonic treatment is 150–350W.

[0038] According to any one of the construction methods of the present invention, in step (2), the volume percentage of formic acid in the aqueous solution containing formic acid is 0.05-0.2%; and / or, the detection wavelength is 254 nm; and / or, the flow rate is 1.0 ml / min; and / or, the column temperature is 35 °C; and / or, during the high performance liquid chromatography detection, the injection volume is 1-20 μl; and / or, an Acclaim 120C8 column is used; and / or, the acid in the aqueous solution containing acid is selected from one or more of formic acid, acetic acid, and phosphoric acid.

[0039] According to any one of the construction methods of the present invention, the solvent of the reference solution is selected from a methanol aqueous solution with a volume fraction of 20-80%; and / or, 1 mL of the reference solution contains 10-30 μg of each reference standard.

[0040] Optionally, the preparation method of the reference solution includes the following steps: take appropriate amounts of gallic acid, ellagic acid, and kaempferol-3-O-galactoside, accurately weigh them, add 20-80% methanol to prepare a solution containing 10-30 μg of reference standard per 1 ml, and use it as the reference solution.

[0041] This invention also provides a quality testing method for a Euphorbia pekinensis drug preparation, comprising constructing a characteristic spectrum of the Euphorbia pekinensis drug preparation to be tested using any of the above-described construction methods; and / or, comprising determining the content of the active ingredient in the Euphorbia pekinensis drug preparation to be tested according to the above-described method for determining the content of the active ingredient in the Euphorbia pekinensis drug preparation.

[0042] In some implementations, a similarity evaluation software for chromatographic feature maps of traditional Chinese medicine is used to generate a reference feature map of the Euphorbia pekinensis drug preparation.

[0043] If the similarity between the characteristic chromatogram of the tested Euphorbia pekinensis product and the control characteristic chromatogram of the Euphorbia pekinensis drug preparation is not less than 0.90, the product is considered qualified; if it is less than 0.90, it is considered unqualified. Specifically, the similarity is obtained by software for evaluating the similarity of chromatographic characteristic chromatograms of traditional Chinese medicine.

[0044] In some preferred embodiments, after generating the control characteristic chromatogram of Euphorbia pekinensis drug preparation using the software for evaluating the similarity of chromatographic characteristic chromatograms of traditional Chinese medicine, the method further includes the step of marking common characteristic peaks.

[0045] % methanol indicates the volume percentage of methanol in an aqueous methanol solution.

[0046] The technical solution of this invention has the following advantages:

[0047] 1. The method for constructing the characteristic spectrum of Euphorbia pekinensis drug preparations provided by this invention uses octylsilane-bonded silica gel as the packing material and an acidic aqueous solution-acetonitrile as the mobile phase. Through continuous optimization of the gradient elution program, gradient elution is performed according to the following procedure: 0 → 7 min → 10 min → 20 min → 33 min → 60 min, with the volume percentage of acetonitrile in the mobile phase being 5% → 5% → 9% → 9% → 14% → 40%. Combined with controlled detection wavelength of 250-260 nm, flow rate of 0.8-1.2 mL / min, and column temperature of 33-40 °C, 9-10 common characteristic peaks are finally obtained, achieving effective separation of these 9-10 common characteristic peaks. Furthermore, the obtained characteristic spectrum has a stable baseline, good peak shape, high separation, and short detection time, providing a basis for the quality detection and control of Euphorbia pekinensis drug preparations and realizing the overall component characterization of Euphorbia pekinensis drug preparations. Furthermore, it can accurately locate the peak positions of ellagic acid, gallic acid, and kaempferol-3-O-galactoside, increasing the known information of characteristic peaks and fully reflecting the integrity and characteristics of Euphorbia pekinensis drug preparations (such as formulation granules).

[0048] This study established a characteristic chromatogram (high performance liquid chromatography) for *Euphorbia pekinensis* formula granules. The sample preparation method is simple, the chromatographic conditions are easy to achieve, and the analytical method validation results show that the method is simple to operate, accurate, and has good reproducibility. Ellagic acid peak S was selected as the internal reference peak in the characteristic chromatogram, and 9-10 common characteristic peaks of *Euphorbia pekinensis* formula granules could be identified. The relative retention times of each common characteristic peak were calculated based on ellagic acid peak S, which is beneficial for comprehensive quality detection and overall quality control of *Euphorbia pekinensis* formula granules, thereby helping to improve the safety and stability of this drug.

[0049] 2. This invention provides a method for determining the content of active ingredients in a *Euphorbia pekinensis* pharmaceutical preparation. Using octylsilane-bonded silica gel as the packing material and an acidic aqueous solution-acetonitrile as the mobile phase, a gradient elution program is continuously optimized and performed according to the following procedure: 0 → 7 min → 10 min → 20 min → 33 min → 60 min, with the volume percentage of acetonitrile in the mobile phase being 5% → 5% → 9% → 9% → 14% → 40%. Combined with controlled detection wavelength of 250-260 nm, flow rate of 0.8-1.2 mL / min, and column temperature of 33-40 °C, a method for determining the content of this pharmaceutical preparation is established. This method has high sensitivity and excellent separation, and can simultaneously detect the content of gallic acid, ellagic acid, and kaempferol-3-O-galactoside. Furthermore, this detection method has high precision, good stability, and good repeatability; therefore, it can comprehensively and rapidly detect the active ingredients and their content in *Euphorbia pekinensis* formulation granules.

[0050] 3. This invention provides a quality testing method for Euphorbia pekinensis pharmaceutical preparations, offering a comprehensive and scientific quality standard testing method, including the determination of relevant component content and characteristic chromatographic detection. The testing method uses precisely controlled parameters, fully reflecting the quality of Euphorbia pekinensis pharmaceutical preparations and serving as an evaluation basis for the consistency of efficacy in large-scale production of Euphorbia pekinensis pharmaceutical preparations, thus ensuring the safe, effective, and standardized production of Euphorbia pekinensis pharmaceutical preparations. Attached Figure Description

[0051] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0052] Figure 1 The characteristic spectrum of the Euphorbia pekinensis formulation granules obtained in Example 1; Figure 2 The characteristic spectra of 15 batches of Euphorbia pekinensis formula granules obtained in Example 1; Figure 3 This is the chromatogram of gallic acid reference standard in Example 1; Figure 4 The chromatogram of ellagic acid reference standard in Example 1 is shown below. Figure 5 The chromatogram of kaempferol-3-O-galactoside reference standard in Example 1 is shown below. Figure 6 The chromatogram of the reference herb *Euphorbia pekinensis* in Example 1 is shown below. Peak 1: gallic acid; Peak 6 (S): ellagic acid; Peak 8: kaempferol-3-O-galactoside; Column: Acclaim 120C 85μm 4.6*250mm. Figure 7 The reference characteristic spectrum of the Euphorbia pekinensis formula granules in Example 1; Peak 1: gallic acid, Peak 6 (S): ellagic acid, Peak 8: kaempferol-3-O galactoside; Figure 8 The above is a localization diagram of the reference standards in Example 1; (S1 is the reference characteristic chromatogram, S2 is gallic acid reference standard, S3 is ellagic acid reference standard, S4 is kaempferol-3-O galactoside reference standard); Figure 9 The chromatogram of gradient 1 in Experiment Example 2; Figure 10 The gradient 2 chromatogram in Experiment Example 2; Figure 11 The gradient 3 chromatogram in Experiment Example 2; Figure 12 The gradient 4 chromatogram in Experiment Example 2; Figure 13 The gradient 5 chromatogram from Experiment Example 2; Figure 14 The gradient 6 chromatogram in Experiment Example 2; Figure 15 The gradient 7 chromatogram from Experiment Example 2;

[0053] Figure 16 The gradient 8 chromatogram from Experiment Example 2; Figure 17 The chromatogram at a wavelength of 200 nm is shown in Experiment Example 2. Figure 18 The chromatogram at wavelength 220 nm is from Experiment Example 2;

[0054] Figure 19 The chromatogram at wavelength 240 nm is from Experiment Example 2; Figure 20 The chromatogram at wavelength 254 nm is from Experiment Example 2; Figure 21 The chromatogram at wavelength 280 nm is from Experiment Example 2; Figure 22 The chromatogram at a wavelength of 300 nm is shown in Experiment Example 2. Figure 23 The chromatogram at wavelength 320 nm is shown in Experiment Example 2. Figure 24 The chromatogram at wavelength 340 nm is shown in Experiment Example 2. Figure 25 The chromatogram at wavelength 360 nm is from Experiment Example 2; Figure 26 The chromatogram of the 0.1% phosphoric acid aqueous solvent system in Experimental Example 2 is shown below. Figure 27 The chromatogram of the 0.1% formic acid aqueous solvent system in Experimental Example 2 is shown below. Figure 28 The chromatogram of the 0.1% acetic acid aqueous solvent system in Experimental Example 2 is shown below. Figure 29 The chromatogram for the flow rate of 0.8 mL / min in Experiment Example 2 is shown below. Figure 30 The chromatogram for a flow rate of 1.0 mL / min in Experiment Example 2 is shown below. Figure 31 The chromatogram for the flow rate of 1.2 mL / min in Experiment Example 2 is shown. Figure 32 This is a control diagram for the column temperature of 30℃ in Experiment Example 2; Figure 33 This is the chromatogram of Experiment 2 at a column temperature of 35℃; Figure 34 This is the chromatogram of the column temperature at 40℃ in Experiment Example 2; Figure 35 The chromatogram of 20% methanol in Experiment Example 3 is shown. Figure 36 The chromatogram of 50% methanol in Experiment Example 3 is shown. Figure 37 The chromatogram of 80% methanol in Experiment Example 3 is shown. Figure 38 Extract the chromatogram for 15 minutes in Experiment Example 3; Figure 39 Extract the chromatogram for 30 minutes in Experiment Example 3; Figure 40 Extract the chromatogram for 45 minutes in Experiment Example 3; Figure 41 The chromatogram for the extraction power of 150W in Experiment Example 3 was examined. Figure 42 The chromatogram for the extraction power of 250W in Experiment Example 3 was examined. Figure 43 The chromatogram for the extraction power of 350W in Experiment Example 3 was examined. Figure 44 This is the chromatogram of the blank solvent in Experiment Example 1. Detailed Implementation

[0055] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0056] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0057] The instruments and reagents used in this invention are as follows:

[0058] 1. Instruments and reagents

[0059] 1.1 Instruments:

[0060] Chromatograph 1: High Performance Liquid Chromatograph: Shimadzu LC-2040 3D Chromatography System, including a quaternary gradient pump (LC-40B X3), an autosampler (SIL-40C X3), a column oven (CTO-40S), a dual ultraviolet detector (SPD-M40), and a Shimadzu Labsolutions chromatography workstation;

[0061] Chromatograph 2: Thermo SR-3000 chromatography system, including LPG-3400 quaternary pump, WPS-3000SL autosampler, VWD-3100 PDA diode array detector, TCC-3000SD column oven, and chromatography workstation.

[0062] Chromatograph 3: Waters e2695, PDA detector (2998).

[0063] Electronic balance with a ratio of 0.0001 (Shanghai Sunny Hengping Scientific Instruments Co., Ltd., FA1004); Electronic balance with a ratio of 0.0001 (Sartorius Scientific Instruments Co., Ltd., SQP); Ultrasonic cleaner: WB400US Shanghai Wangbiao Instruments Co., Ltd.

[0064] Chromatographic column:

[0065] (1) Acclaim 120C8 (column length 250mm, inner diameter 4.6mm, particle size 5μm)

[0066] 1.2 Reagents

[0067] Acetonitrile was of chromatographic grade, and water was Watson's distilled water; all other reagents were of analytical grade.

[0068] 1.3 Drug Testing

[0069] Euphorbia pekinensis reference material (purchased from the National Institutes for Food and Drug Control, batch number 121675-201201).

[0070] Ellagic acid reference standard (purchased from the National Institutes for Food and Drug Control, batch number 111959-201903, purity 88.8%).

[0071] Gallic acid reference standard (purchased from the National Institutes for Food and Drug Control, batch number 110831-202407, purity 90.8%).

[0072] Kaempferol-3-O-galactoside reference standard (purchased from Shanghai Hongyong Biotechnology Co., Ltd., batch number 190408-202409, purity 99.99%).

[0073] Example 1

[0074] A method for constructing a characteristic spectrum of a pharmaceutical preparation of Euphorbia pekinensis, comprising:

[0075] (1) Preparation of the test solution: Accurately weigh approximately 0.1 g of the powdered preparation of Euphorbia pekinensis, place it in a stoppered conical flask, accurately add 50 mL of 50% methanol, seal tightly, weigh, sonicate (power 250 W, frequency 40 kHz) for 30 minutes, remove, cool, weigh again, replenish the lost weight with 50% methanol, shake well, filter, and collect the filtrate to obtain the test solution. The Euphorbia pekinensis preparation described in this invention is prepared by the following method:

[0076] Take slices of Euphorbia pekinensis, and extract them by heating and reflux at least once, adding 6-14 times their weight of water each time for at least 0.5 hours. Filter, combine the filtrates, and concentrate the filtrate to a relative density of 1.05-1.10 g / mL at 60℃. Add conventional excipients and follow conventional processes to prepare clinically acceptable tablets, capsules, pills, granules, honey-refined pills, sustained-release preparations, immediate-release preparations, controlled-release preparations, oral liquid preparations, or injectable preparations. The pharmaceutically acceptable excipients include: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, and matrix, etc. Fillers include: starch, pregelatinized starch, lactose, mannitol, chitosan, microcrystalline cellulose, sucrose, etc.; disintegrants include: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, croscarmellose, low-substituted hydroxypropyl cellulose, croscarmellose sodium, etc.; lubricants include: magnesium stearate, sodium lauryl sulfate, talc, silica, etc.; suspending agents include: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include: starch paste, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.; sweeteners include: sodium saccharin, aspartame, sucrose, cyclamate, glycyrrhetinic acid, etc.; flavoring agents include: sweeteners and various flavorings; preservatives include: parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chlorethidium acetate, eucalyptus oil, etc.; matrix includes: PEG6000, PEG4000, insect wax, etc.

[0077] In this embodiment, the test sample used was Euphorbia pekinensis formula granules. The preparation method of Euphorbia pekinensis formula granules is as follows: Euphorbia pekinensis slices were taken and extracted twice by heating and reflux. For the first extraction, 9 times the weight of water was added and soaked for 30 minutes, and then heated and refluxed for 0.5 hours. After filtration, 7 times the weight of water was added and extracted for 0.5 hours. After filtration, the filtrates were combined and concentrated to a relative density of 1.05 g / mL at 60°C. The filtrates were then spray-dried. The dry powder was mixed with 0% to 4% of the total mass of the granules as excipient maltodextrin. After mixing evenly, the mixture was dry-granulated to produce granules.

[0078] (2) Detection by high performance liquid chromatography

[0079] Accurately pipette 5 μl of the test solution and inject it into the high-performance liquid chromatograph (HPLC). Perform the determination under the following chromatographic conditions: use octylsilane-bonded silica gel as the packing material, employ an Acclaim 120C8 column (250 mm length, 4.6 mm inner diameter, 5 μm particle size), with acetonitrile as mobile phase A, followed by 0.1% formic acid solution as mobile phase B, and perform gradient elution as specified in Table 1. The flow rate is 1.0 mL / min, the column temperature is 35 °C, and the detection wavelength is 254 nm. The theoretical plate number, calculated based on the ellagic acid peak, should be no less than 3000.

[0080] Table 1 Gradient Elution Table

[0081]

[0082] Table 2 System Adaptability Parameters

[0083]

[0084]

[0085] The results are shown in the table above. Figure 1 As shown, the baseline of the characteristic spectrum of the Euphorbia pekinensis formula granules is stable, with 10 common characteristic peaks. These 10 common characteristic peaks have good separation effect, good peak shape, and short detection time.

[0086] The common pattern of the characteristic spectrum of Euphorbia pekinensis formula granules was established using the method described above.

[0087] Fifteen batches of *Euphorbia pekinensis* formula granules were used as test samples to prepare test solutions. The fingerprint chromatograms of the *Euphorbia pekinensis* formula granules were obtained by high-performance liquid chromatography (HPLC). Figure 2 As shown in the figure, the Figure 2 In the series, S1 to S15 are: 1901002W, 1906002S, 1907001S, 1912001W, 2001001W, 2004002S, 2008001W, 2102001S, 2105002W, 2111001S, 2203002S, 2207002S, 2210001W, 2308003S, and 2311001S.

[0088] Preparation of reference solutions: Take 0.5g of Euphorbia pekinensis reference material, place it in a stoppered conical flask, add 50ml of water, heat under reflux for 1 hour, filter, evaporate the filtrate to dryness, add 50ml of 50% methanol to the residue, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, filter, and take the filtrate as the reference solution. Accurately weigh gallic acid reference standard, add 50% (v / v) methanol aqueous solution to prepare a solution containing 20μg of reference standard per 1mL, shake well, as reference solution A; accurately weigh ellagic acid reference standard, add 50% (v / v) methanol aqueous solution to prepare a solution containing 20μg of reference standard per 1mL, shake well, as reference solution B; accurately weigh kaempferol-3-O-galactoside reference standard, add 50% (v / v) methanol aqueous solution to prepare a solution containing 20μg of reference standard per 1mL, shake well, as reference solution C. The above reference solution was analyzed using the high-performance liquid chromatography method described above to obtain a reference chromatogram, see [link to reference chromatogram]. Figures 3-6 As shown.

[0089] Ten characteristic peaks were located and identified using reference standards. Peak 1 was determined to be gallic acid, peak 6 to be ellagic acid, and peak 8 to be kaempferol-3-O-galactoside.

[0090] The relative retention times of the characteristic chromatograms were determined based on the research results: the characteristic chromatogram of the Euphorbia pekinensis formula granules should show 10 characteristic peaks, which should correspond to the 10 characteristic peaks in the chromatogram of the reference medicinal materials. Among them, the retention times of peaks 1, 6, and 8 should correspond to the retention times of the reference peaks of gallic acid, ellagic acid, and kaempferol-3-O-galactoside.

[0091] Using the fingerprint chromatogram similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission, a reference characteristic chromatogram was generated, such as... Figure 7 As shown. According to Figure 7 The reference characteristic chromatograms shown can be used to analyze and compare the detection results of the characteristic chromatograms of formulated granules, and are used for the quality control of formulated granules. The specific method for quality control using reference characteristic chromatograms is as follows:

[0092] pass Figure 7 It is known that the HPLC characteristic chromatogram of the Euphorbia pekinensis formula granules has 10 chromatographic peaks, which should correspond to the retention times of the 10 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 6 should correspond to the retention time of the corresponding reference peak. The peak corresponding to the ellagic acid reference peak is the S peak. The relative retention times of each characteristic peak and the S peak are calculated, and the relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.16 (peak 1), 0.42 (peak 2), 0.51 (peak 3), 0.60 (peak 4), 0.73 (peak 5), 1.08 (peak 7), 1.13 (peak 8), 1.19 (peak 9), and 1.37 (peak 10). The results are shown in Tables 3-6.

[0093] The characteristic chromatograms of 15 batches of *Euphorbia pekinensis* granules were analyzed. The relative retention times of peaks 1-5, 7-10, and peak S were calculated. These relative retention times were within ±10% of the specified values, which were: 0.16 (peak 1), 0.42 (peak 2), 0.51 (peak 3), 0.60 (peak 4), 0.73 (peak 5), 1.08 (peak 7), 1.13 (peak 8), 1.19 (peak 9), and 1.37 (peak 10). The similarity of the characteristic chromatograms of the 15 batches of *Euphorbia pekinensis* granules was greater than 0.90. The results are shown in Tables 3-6. The reference standards for different characteristic peaks are located as follows. Figure 8 As shown.

[0094] Table 3. Relative retention times of common patterns in the characteristic chromatograms of Euphorbia pekinensis formulation granules.

[0095]

[0096]

[0097] Table 5. Common pattern matching data for Euphorbia pekinensis formulation granules

[0098]

[0099]

[0100] Table 6. Results of relative retention time determination of characteristic spectra of 15 batches of Euphorbia pekinensis formulation granules.

[0101]

[0102] Table 7. Similarity results of characteristic spectral data for 15 batches of Euphorbia pekinensis formulation granules.

[0103]

[0104] Example 2

[0105] A method for determining the content of a pharmaceutical preparation of Euphorbia pekinensis, comprising:

[0106] (1) Preparation of test solution: Same as step (1) of Example 1.

[0107] (2) Preparation of reference solutions: Accurately weigh gallic acid reference standard, add 50% (v / v) methanol aqueous solution to prepare a solution containing 20 μg of reference standard per 1 mL, shake well, and use as reference solution A; accurately weigh ellagic acid reference standard, add 50% (v / v) methanol aqueous solution to prepare a solution containing 20 μg of reference standard per 1 mL, shake well, and use as reference solution B; accurately weigh kaempferol-3-O-galactoside reference standard, add 50% (v / v) methanol aqueous solution to prepare a solution containing 20 μg of reference standard per 1 mL, shake well, and use as reference solution C.

[0108] (3) Determination method: Accurately pipette 1 μl of the reference solution and the test solution and inject them into the liquid chromatograph. The chromatographic conditions are the same as in Example 1. Determine the contents of gallic acid, ellagic acid and kaempferol-3-O-galactoside in the test solution by external standard method.

[0109] Fifteen batches of Euphorbia pekinensis granule samples were used to prepare test solutions, and the content of Euphorbia pekinensis granules was obtained by high performance liquid chromatography. The results are shown in the table below.

[0110] Table 8. Results of granule content in 15 batches of Euphorbia pekinensis formulation.

[0111]

[0112] The detection methods described in Examples 1 and 2 effectively obtain characteristic spectra with good separation of each characteristic peak. They also allow for the simultaneous determination of the contents of gallic acid, ellagic acid, and kaempferol-3-O-galactoside. Furthermore, by selecting the S peak (ellagic acid) as the internal reference peak in the characteristic spectrum, the relative retention times of the common characteristic peaks 1-5 and 7-10 of the Euphorbia pekinensis formula granules can be determined. Therefore, this comprehensive and rapid detection method is beneficial for the overall quality testing and control of Euphorbia pekinensis formula granules, thereby contributing to improved safety and stability of the drug.

[0113] Experimental Example 1

[0114] This experimental example provides the process and results of testing the precision, stability, and repeatability of the construction method described in Example 1 above, as follows:

[0115] 1 System Suitability Test

[0116] 1.1 Instrument precision test

[0117] The same sample solution (batch number: 1901002W of Euphorbia pekinensis formula granules) prepared by the method in Example 1 was injected 6 times under the chromatographic conditions in section (2) of Example 1. The relative retention times of 10 common peaks were determined. The RSD values ​​of the relative retention times of each peak were 0.27%, 0.22%, 0.42%, 0.23%, 0.26%, 0%, 0.08%, 0.11%, 0.14%, and 0.17%, respectively. The RSD of the relative retention times of each characteristic peak and the reference peak S were all less than 2%, indicating that the instrument precision was good.

[0118] 1.2 Method repeatability test

[0119] Six portions of the same batch of Euphorbia pekinensis granules were taken, and the relative retention times of 10 common peaks were determined according to the method in Example 1. The RSD values ​​of the relative retention times of each peak were 0.28%, 0.23%, 0.43%, 0.23%, 0.31%, 0%, 0.09%, 0.13%, 0.15%, and 0.15%, respectively. The RSD of the relative retention times of each characteristic peak and the reference peak S were all less than 2%, indicating that the method has good repeatability.

[0120] 1.3 Intermediate Precision (by different operators)

[0121] Three inspectors, at different times and using the same equipment, measured the relative retention times of the same batch of Euphorbia pekinensis granules according to the method in Example 1. The relative retention times of the 10 common peaks were measured and analyzed. The RSD values ​​of the relative retention times of the 10 common peaks were 0.31%, 0.29%, 0.54%, 0.31%, 0.04%, 0%, 0.08%, 0.11%, 0.14%, and 0.18%, respectively. The RSD of the relative retention times of each characteristic peak and the reference peak S were all less than 2%, indicating good intermediate precision (by different personnel) of this method.

[0122] 2. Specificity test

[0123] Take a blank solvent (50% methanol) and inject it under the chromatographic conditions described in section (2) of Example 1. Record the chromatogram and investigate the effect of the blank solvent. The results are shown in […]. Figure 44 As shown, the specificity test results indicate that the negative sample did not cause interference.

[0124] 3. Stability Experiment

[0125] The same batch of Euphorbia pekinensis granules was used as the test sample. The preparation method in Example 1 was followed, and the sample was injected at 0, 2, 4, 8, 12, and 24 hours after preparation. The relative retention times of 10 common peaks were measured. The RSD values ​​of the relative retention times of each peak were 0.23%, 0.23%, 0.41%, 0.23%, 0.26%, 0%, 0.08%, 0.11%, 0.14%, and 0.17%, respectively. The results showed that the RSD of the relative retention times of each characteristic peak and the reference peak S were all less than 2%, indicating that the test sample solution was stable within 24 hours and met the determination requirements.

[0126] Experiment Example 2

[0127] 1) Gradient selection: The same batch of *Euphorbia pekinensis* granules was used to prepare test solutions according to the method in Example 1. Except for the gradient elution program, all other conditions were the same as in Example 1. The gradient elution program was used as a variable. Different gradient elution programs are shown in Table 1, and the chromatograms of different gradient elution programs are shown in Table 1. Figures 9-16 As shown. By Figures 9-16 It can be seen that the chromatogram after elution with gradient 8 has rich chromatographic information, more major chromatographic peaks, better resolution, and a more stable baseline. Therefore, gradient 8 was determined as the mobile phase gradient for subsequent condition screening in order to achieve better separation results.

[0128] Table 9 Gradient elution procedures for gradients 1-3

[0129]

[0130] Table 10 Gradient elution procedures for gradients 4-6

[0131]

[0132] Table 11 Gradient elution procedures for gradients 7-8

[0133]

[0134]

[0135] Table 12 System Suitability Parameters for Each Gradient Chromatographic Peak

[0136]

[0137]

[0138] 2) Selection of detection wavelength: Test solutions were prepared from the same batch of *Euphorbia pekinensis* granules according to the method in Example 1. Except for the detection wavelength, all other conditions were the same as in Example 1. Using the detection wavelength as a variable, chromatograms at nine different absorption wavelengths (200 nm, 220 nm, 240 nm, 254 nm, 280 nm, 300 nm, 320 nm, 340 nm, 360 nm) were compared. The number of chromatographic peaks, response value, and retention time were used as evaluation criteria to screen detection wavelengths. The main active ingredients in *Euphorbia pekinensis* are triterpenoids and phenolic acids, and their effective detection wavelength range is 200–360 nm. The results are shown in Table 13 below. Figures 17-25 The results showed that the chromatogram at a wavelength of 254 nm had more chromatographic peaks, more information, and a stable baseline. Therefore, the absorption wavelength of 254 nm, which has relatively rich chromatographic peak information and relatively better system adaptability parameters, was selected as the detection wavelength for the characteristic chromatogram of Euphorbia pekinensis formulation granules for further investigation.

[0139] Table 13 System adaptability parameters for chromatographic peaks at different absorption wavelengths

[0140]

[0141]

[0142]

[0143] 3) Acid type investigation: Test solutions were prepared from the same batch of *Euphorbia pekinensis* granules according to the method in Example 1. Except for the type of acid in the mobile phase, all other conditions were the same as in Example 1. Chromatograms of 0.1% phosphoric acid aqueous solution, 0.1% formic acid aqueous solution, and 0.1% acetic acid aqueous solution were compared. Chromatograms at different flow rates are shown below. Figures 26-28 As shown. By Figures 26-28It can be seen that different acids have a certain impact on each chromatographic peak. The peak shape and resolution of the three solvent systems are better. Compared with the 0.1% phosphoric acid aqueous solvent system and the 0.1% acetic acid aqueous solvent system, the chromatogram presented by 0.1% formic acid solution elution has better peak shape and resolution of the main chromatographic peaks. Therefore, 0.1% formic acid solution is preferred for subsequent condition screening.

[0144] Table 14 Parameters for evaluating the applicability of the system to different types of acid addition

[0145]

[0146] 4) Investigation of different flow rates: The same batch of *Euphorbia pekinensis* granules was used to prepare test solutions according to the method in Example 1. Except for the flow rate, all other conditions were the same as in Example 1. Flow rate was used as the variable, and measurements were taken at flow rates of 0.8 mL / min, 1 mL / min, and 1.2 mL / min. The chromatograms for different flow rates are shown below. Figures 29-31 As shown. By Figures 29-31 It can be seen that the chromatographic effect is better when the flow rate is 0.8-1.2 mL / min, and the preferred flow rate is 1.0 mL / min.

[0147] Table 15 Parameters for assessing system suitability at a flow rate of 0.8 mL / min

[0148]

[0149] Table 16 Parameters for assessing system suitability at a flow rate of 1.0 mL / min

[0150]

[0151] Table 17 Parameters for assessing system suitability at a flow rate of 1.2 mL / min

[0152]

[0153] 5) Selection of different column temperatures: The same batch of *Euphorbia pekinensis* granules was used to prepare the test solution according to the method in Example 1. Except for the column temperature, all other conditions were the same as in Example 1. Using column temperature as a variable, the separation effect of different column temperatures (30℃, 35℃, and 40℃) on the product was investigated. The chromatograms for different column temperatures are shown below. Figures 32-34 As shown in the figure. The results indicate that different column temperatures have a certain impact on the retention time of chromatographic peaks. Compared with a column temperature of 30℃, the system adaptability parameters of each chromatographic peak are relatively better at column temperatures of 35℃ and 40℃, especially at 35℃. Therefore, it is recommended to select a column temperature of 35℃ for subsequent condition screening.

[0154] Table 18 Parameters for System Suitability Testing at 30℃ Column Temperature

[0155]

[0156]

[0157] Table 19 Parameters for System Suitability Testing at 35℃ Column Temperature

[0158]

[0159] Table 20 Parameters for System Suitability Testing at 40℃ Column Temperature

[0160]

[0161] Experimental Example 3

[0162] 1) Selection of extraction solvent: The same batch of *Euphorbia pekinensis* granules was used to prepare test solutions according to the method in Example 1. Except for the extraction solvent, all other conditions were the same as in Example 1. The extraction solvent was used as a variable, with 20% methanol, 50% methanol, and 80% methanol as the extraction solvents. The results are shown in […]. Figures 35-37 As shown in Table 21, the system adaptability parameters of the main characteristic peaks are as follows. The results show that the total peak area of ​​the characteristic chromatographic peaks obtained by extraction with different methanol concentrations varies to some extent. The peak area of ​​the chromatographic peaks obtained by extraction with 50% methanol is larger, and the peak shape of each peak is better. Considering all factors, 50% methanol is the preferred extraction solvent.

[0163] Table 21. Adaptability Parameters of Chromatographic Peak System for Extraction Solvent Methanol Concentration Evaluation

[0164]

[0165]

[0166] 2) Selection of extraction time: The same batch of *Euphorbia pekinensis* granules was used to prepare the test solution according to the method in Example 1. Except for the extraction time, all other conditions were the same as in Example 1. See the attached figures for the results. Figures 38-40 As shown in Table 22, with extraction time as the variable, the system adaptability parameters of the main characteristic peaks at extraction times of 15, 30, and 45 minutes are as follows. Results analysis: The chromatographic peaks obtained by ultrasonic treatment for 15-45 minutes showed good resolution, asymmetry, and theoretical plate number, with 30 minutes being the optimal value.

[0167] Table 22 Extraction time parameters for chromatographic peak system adaptability.

[0168]

[0169]

[0170] 3) Extraction power evaluation: The same batch of *Euphorbia pekinensis* granules was used to prepare test solutions according to the method in Example 1. Except for the different extraction powers of the samples, other conditions were the same as in Example 1. Extraction power was used as a variable to evaluate different extraction powers of the samples: 150, 250, and 350 W. The system adaptability parameters of the characteristic peaks were used as evaluation indicators. The system adaptability parameters of the main characteristic peaks for different sample amounts are shown in Table 23. See the attached figures for the results. Figures 41-43 As shown. Results analysis: The resolution, asymmetry, and theoretical plate number of each chromatographic peak obtained by ultrasonic treatment with power of 150-350W were all good, and the optimal extraction power was 250W.

[0171] Table 23 Extraction time parameters for chromatographic peak system adaptability.

[0172]

[0173] 4) Selection of Sample Amount: The same batch of *Euphorbia pekinensis* formula granules was used to prepare test solutions according to the method in Example 1. Except for the sample amount, other conditions were the same as in Example 1. The sample amount was used as a variable to investigate different extraction amounts: 0.05 g, 0.1 g, and 0.2 g. The system adaptability parameters of the characteristic peaks were used as the evaluation index. The system adaptability parameters of the main characteristic peaks at different sample amounts are shown in Table 24. The experimental results show that different sample amounts have no effect on the retention time of each characteristic peak, and the resolution, symmetry factor, and theoretical plate number have little impact. At a sample amount of 0.1 g, the peak area of ​​the characteristic peaks in the *Euphorbia pekinensis* formula granule characteristic spectrum is relatively moderate. Therefore, 0.1 g is the preferred sample amount for this method.

[0174] Table 24 Sampling quantity parameters for evaluating the adaptability of the chromatographic peak system

[0175]

[0176] 5) Investigation of different solvent volumes: The same batch of *Euphorbia pekinensis* granules was used to prepare test solutions according to the method in Example 1. Except for the solvent volume, other conditions were the same as in Example 1. Solvent volume was used as a variable to investigate different solvent volumes: 50 mL, 75 mL, and 100 mL. The system adaptability parameters of the characteristic peaks were used as the evaluation index. The system adaptability parameters of the main characteristic peaks at different sampling amounts are shown in Table 25. The results showed that different solvent volumes had little impact on the chromatogram; therefore, the minimum solvent volume of 50 mL was selected as the optimal extraction solvent volume.

[0177] Table 25 Solvent volume analysis of chromatographic peak system adaptability parameters

[0178]

[0179]

[0180] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a characteristic spectrum of Euphorbia pekinensis formula granules, characterized in that, Includes the following steps, (1) Preparation of the test solution: including the steps of extracting the Euphorbia pekinensis formula granules with a solvent, separating solid and liquid, and taking the filtrate; the solvent is a methanol aqueous solution with a volume fraction of 20-80%; the extraction method is heating reflux extraction or ultrasonic extraction; It also includes the preparation of reference solutions using gallic acid, ellagic acid, and kaempferol-3-O-galactoside as reference standards; (2) The test solution and the reference solution were detected by high performance liquid chromatography. Octylsilane bonded silica gel was used as the packing material. The detection wavelength was 250-260 nm, the flow rate was 0.8-1.2 mL / min, the column temperature was 33-40 °C, and the mobile phase was acid-containing aqueous solution-acetonitrile. Gradient elution was performed. The gradient elution program included: 0 → 7 min → 10 min → 20 min → 33 min → 60 min. The volume percentage of acetonitrile in the mobile phase was 5% → 5% → 9% → 9% → 14% → 40%. The acid in the acid-containing aqueous solution was selected from one or more of formic acid, acetic acid, and phosphoric acid.

2. The construction method according to claim 1, characterized in that, Step (1) satisfies any one or more of the following AD: A. The mass ratio of the Euphorbia pekinensis granules to the volume ratio of the organic solvent is (0.05-0.2):(50-100), and the ratio is g / mL; B. Extraction time is 15-45 minutes; C. Solid-liquid separation is achieved by centrifugation or filtration; D. Ultrasonic power is 150-350W.

3. The construction method according to claim 1 or 2, characterized in that, In step (2), the volume percentage of formic acid in the aqueous solution containing formic acid is 0.05-0.2%; and / or, the detection wavelength is 254 nm; and / or, the flow rate is 1.0 ml / min; and / or, the column temperature is 35 °C; and / or, during the high performance liquid chromatography detection, the injection volume is 1-20 μl; and / or, an Acclaim 120 C8 column is used.

4. The construction method according to claim 1 or 2, characterized in that, A reference herb solution was prepared by using the aqueous extract of Euphorbia pekinensis as a substitute for Euphorbia pekinensis granules according to the construction method described in claim 1 or 2, and the reference herb solution was detected by high performance liquid chromatography to obtain the reference herb chromatogram.

5. The construction method according to claim 1 or 2, characterized in that, The characteristic chromatogram of the *Euphorbia pekinensis* formula granules has 10 common characteristic peaks. Peak 6 corresponds to the retention time of the ellagic acid reference peak. The peak corresponding to the ellagic acid reference peak is peak S. The relative retention time of each characteristic peak and peak S is within ±10% of the specified value. The specified values ​​of peaks 1-5 and peaks 6-10 are 0.16, 0.42, 0.51, 0.60, 0.73, 1.08, 1.13, 1.19, and 1.37, respectively.

6. A method for determining the content of active ingredients in Euphorbia pekinensis formula granules, characterized in that, Includes the following steps: The sample solution was prepared by taking the granules of Euphorbia pekinensis; the granules were then extracted with solvent, followed by solid-liquid separation and collection of the filtrate; the extraction method was either reflux extraction or ultrasonic extraction. A reference solution was prepared by taking gallic acid, ellagic acid, and kaempferol-3-O-galactoside; Test Procedure: The test solution and reference solution were analyzed by high performance liquid chromatography (HPLC) using octylsilane-bonded silica gel as the packing material, with a detection wavelength of 250-260 nm, a flow rate of 0.8-1.2 mL / min, and a column temperature of 33-40 °C. Gradient elution was performed using an acidic aqueous solution-acetonitrile as the mobile phase. The gradient elution program included: 0 → 7 min → 10 min → 20 min → 33 min → 60 min. The volume percentage of acetonitrile in the mobile phase was 5% → 5% → 9% → 9% → 14% → 40%. The content of the reference standard in the Euphorbia pekinensis granules was calculated. The acid in the acidic aqueous solution was selected from one or more of formic acid, acetic acid, and phosphoric acid.

7. The content determination method according to claim 6, characterized in that, Step (1) satisfies any one or more of the following AEs: A. The mass ratio of the Euphorbia pekinensis granules to the volume ratio of the organic solvent is (0.05-0.2):(50-100), and the ratio is g / mL; B. The solvent is a methanol aqueous solution with a volume fraction of 20-80%; C. Extraction time is 15-45 minutes; D. Solid-liquid separation is achieved by centrifugation or filtration; E. Ultrasonic power is 150-350W.

8. The content determination method according to claim 6 or 7, characterized in that, In step (2), the volume percentage of formic acid in the aqueous solution containing formic acid is 0.05-0.2%; and / or, the detection wavelength is 254 nm; and / or, the flow rate is 1.0 ml / min; and / or, the column temperature is 35 °C; and / or, during the high performance liquid chromatography detection, the injection volume is 1-20 μl; and / or, an Acclaim 120 C8 column is used.

9. The content determination method according to claim 6 or 7, characterized in that, The solvent of the reference solution is selected from a methanol aqueous solution with a volume fraction of 20-80%; and / or, 1 mL of the reference solution contains 10-30 μg of each reference standard.

10. A method for quality testing of Euphorbia pekinensis granules, characterized in that, The method includes constructing a characteristic spectrum of the *Euphorbia pekinensis* formula granules to be tested using the construction method described in any one of claims 1-5; and / or, including the step of determining the content of the active ingredient in the *Euphorbia pekinensis* formula granules to be tested using the content determination method of the active ingredient in the *Euphorbia pekinensis* formula granules described in any one of claims 6-9.

Images