Method for identifying mantis egg-case related sample

Abstract

The invention discloses a mantis egg-case related sample identification method, which uses an ultra-high performance liquid chromatograph and takes acetonitrile and a formic acid-water solution as mobile phases to separate and identify acetyldopamine components in a sample. According to the invention, a rapid identification method which takes acetyldopamine as an index and is based on ultra-high performance liquid chromatography is established for the first time, and the method is suitable for detection of various mantis egg-case samples, including mantis egg-case medicinal materials, decoction pieces, extracts, standard decoction, formula granules, formula reference samples containing mantis egg-cases, intermediate products of preparations and finished products. The method is easy to operate, methodology verifies that the specificity is high, reproducibility and stability are good, the recovery rate is reliable, the detection cost is low, the detection efficiency is high, the method has good guiding significance in the aspects of mantis egg-case medicinal material selection, preparation processing process control and product quality stability, and a powerful guarantee is provided for industrialization and quality control of mantis egg-case medicinal materials.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine, and in particular to a method for identifying samples related to mantis egg case. Background Technology

[0002] Mantis egg case (Sangpiaoxiao) is an insect belonging to the Mantis family, specifically the Chinese mantis (Sinosaurus spp.). Tenodera sinensis Saussure, Brown Spotted Mantis Statilia maculata (Thunberg) or Broad Axe Mantis Hierodula patellifera The dried egg case of (Serville) is from multiple species, with the most common source being the Chinese mantis. It is sweet and salty in taste, neutral in nature, and enters the liver and kidney meridians. It has the effects of consolidating essence and reducing urination, tonifying the kidney and assisting yang. Clinically, it is often used to treat diseases such as urethral syndrome.

[0003] The main chemical components of mantis egg case include various proteins, fats, amino acids, vitamins, and trace elements. Modern pharmacological studies have shown that it has antioxidant, antibacterial, anti-fatigue, hypoglycemic, and immune-enhancing activities. With the modernization of traditional Chinese medicine, mantis egg case formula granules have gradually become more popular, but quality control is still relatively traditional. At present, the quality control standards for mantis egg case in the pharmacopoeia mainly focus on morphological identification, microscopic identification, thin-layer chromatography identification, and content determination. In particular, in local standards, the content determination item often uses amino acids such as tyrosine and tryptophan as indicators.

[0004] Existing analytical methods for mantis egg case samples include: Jia Kunjing et al. used the Coomassie brilliant blue method to determine the protein extraction rate of raw mantis egg case, the sulfuric acid phenol method and the weight loss method to determine the content of polysaccharides and total lipids in raw mantis egg case, respectively, and the ascorbic acid-molybdenum blue spectrophotometric method to determine the phospholipid content; Yang Huiquan et al. used the 835-50 high-speed automatic amino acid analyzer with the protein hydrolysis analysis standard program to determine the amino acid content of mantis egg case; Ye Yulan et al. used the JY-38 inductively coupled plasma atomic emission spectrometer and other instruments to determine the trace elements in different varieties of mantis egg case.

[0005] However, the specific components targeted by the above methods, although pharmacologically active, are widely present in various animal-derived traditional Chinese medicines, lacking specificity. This can easily lead to adulteration or the use of substitutes going undetected, affecting efficacy and safety. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to provide a method for identifying mantis egg case-related samples based on acetyldopamine.

[0007] Technical solution: The method for identifying mantis egg case related samples according to the present invention includes the following steps: (1) Preparation of test solutions for mantis egg case and related samples; (2) Setting chromatographic parameters on the ultra-high performance liquid chromatograph: (21) Chromatographic column, a chromatographic column with octadecylsilane-bonded silica gel filler as stationary phase; (22) Eluent, including mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile, and mobile phase B is 0.03% formic acid-water solution; (23) Elution mode, the flow rate of mobile phase is 0.25-0.35 mL / min, and gradient elution is performed: 0 min, the volume ratio of mobile phase A is 0%, and the volume ratio of mobile phase B is 100%, 4 min, the volume ratio of mobile phase A is 3%, and the volume ratio of mobile phase B is 97%, 14 min, the volume ratio of mobile phase A is 3%, and the volume ratio of mobile phase B is 97%, 15 min, the volume ratio of mobile phase A is 40%, and the volume ratio of mobile phase B is 60%; (24) Elution column temperature, 43-47℃; (3) Injecting the sample solution to be tested into the ultra-high performance liquid chromatograph, analyzing and recording the chromatogram, comparing with the chromatogram of acetyl dopamine standard, and identifying; The sample related to the egg of the snail Pupilla muscorum is any one of the medicinal material, decoction piece, extract, standard decoction, formula granule of the egg of the snail Pupilla muscorum, or the reference sample of a prescription containing the egg of the snail Pupilla muscorum, intermediate product and finished product of a preparation.

[0008] Preferably, the step 1 comprises: adding a methanol-water solution to the sample related to the egg of the snail Pupilla muscorum, extracting, filtering, and obtaining a sample solution.

[0009] Preferably, the concentration of methanol in the methanol-water solution is not higher than 70%.

[0010] Preferably, the amount of the methanol-water solution is 30-100 mL per gram of sample.

[0011] Preferably, the extraction is any one of ultrasonic treatment, heating reflux, and shaking extraction, and the extraction time is 15-60 min; further preferably, the extraction is ultrasonic extraction at a power of 200-300 W and a frequency of 35-45 kHz for 15-60 min.

[0012] Preferably, after the extraction, the method further comprises: evaporating to dryness and redissolving with a methanol-water solution.

[0013] Preferably, the octadecylsilane-bonded silica gel filler in step 21 is a chromatographic column with a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.5-2.0 µm; further preferably, the chromatographic column is selected from any one of ZORBAX Eclipse Plus C18 RRHD, specification 2.1×100 mm, 1.8 μm; Waters ACQUITY UPLC HSS T3, specification 2.1×100 mm, particle size 1.8 μm; Poroshell EC-C18, specification 2.1×100 mm, 1.9 μm.

[0014] Preferably, in step 3, the sample volume injected into the high-performance liquid chromatograph is 1-2 μL.

[0015] Preferably, the ultra-high-performance liquid chromatograph is an ultra-high-performance liquid chromatograph equipped with an ultraviolet detector; further preferably, the ultra-high-performance liquid chromatograph is selected from any one of Waters H-Class ultra-high-performance liquid chromatograph or Thermo VANQUISH ultra-high-performance liquid chromatograph.

[0016] Preferably, the detection wavelength parameter of the ultra-high-performance liquid chromatograph equipped with an ultraviolet detector is set to 275-285 nm.

[0017] Beneficial effects: Compared with the prior art, the present application has the following remarkable advantages: 1. The method uses acetyldopamine as an index for rapid determination by ultra-high-performance liquid chromatography, and is suitable for various kinds of Phascolosoma esculenta samples, including Phascolosoma esculenta medicinal materials, decoction pieces, extracts, standard decoctions, formula granules, Phascolosoma esculenta-containing formula reference samples, and intermediate products and finished products of preparations; 2. The method is simple to operate, has high specificity, good reproducibility and stability, reliable recovery rate, low detection cost, and high detection efficiency, and has good guiding significance for the selection of Phascolosoma esculenta medicinal materials, process control of preparation processing, and product quality stability, thereby providing strong support for industrialization and quality control. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 A representative ultra-high-performance liquid chromatography analysis spectrum of Phascolosoma esculenta formula granules; Figure 2 A representative ultra-high-performance liquid chromatography analysis spectrum of Phascolosoma esculenta medicinal materials; Figure 3 A representative ultra-high-performance liquid chromatography analysis spectrum of Phascolosoma esculenta decoction pieces; Figure 4 A representative ultra-high-performance liquid chromatography analysis spectrum of Phascolosoma esculenta standard decoction; Figure 5 Ultra-high-performance liquid chromatography analysis spectra of Phascolosoma esculenta formula granules under different extraction solvents; Figure 6 The UPLC analysis spectrum of the formula granules of the snail shell under different extraction methods; Figure 7 The UPLC analysis spectrum of the formula granules of the snail shell under different extraction times; Figure 8 The UPLC analysis spectrum of the formula granules of the snail shell under different extraction volumes; Figure 9 The injection amount-peak area linear relationship result graph of the UPLC analysis of acetyl dopamine; Figure 10 The UPLC analysis spectrum of the formula granules of the snail shell in the specificity test; Figure 11 The UPLC analysis spectrum of the formula granules of the snail shell in the integrity test; Figure 12 The UPLC analysis spectrum of the formula granules of the snail shell under different flow rates; Figure 13 The UPLC analysis spectrum of the formula granules of the snail shell under different column temperatures; Figure 14 The UPLC analysis spectrum of the formula granules of the snail shell under different chromatographic columns. DETAILED DESCRIPTION

[0019] The technical solutions of the present application are described below.

[0020] Example 1: Preparation of samples and standard samples The specific species sources and purchase places of the snail shell medicinal materials are shown in Table 1 below: Table 1 Information of specific species sources and purchase places of snail shell medicinal materials

[0021] Formula granules of Sangpaejiao, batch number: 2025060019, 2025060029, 2025060039; standard decoction of Sangpaejiao, batch number: 2018110401DG, 2018110402DG, 2018110403DG, 2018110404DG, 2018110405DG, 2018110406DG, 2021070307DG, 2021070308DG, 2021070309DG, 2021070310DG, 2021070311DG, 2024090212DG, 2024090213DG; pieces of Sangpaejiao, batch number: 2018110401YP, 2018110402YP, 2018110403YP, 2018110404YP, 2018110405YP, 2018110406YP, 2021070307YP, 2021070308YP, 2021070309YP, 2021070310YP, 2021070311YP, 2024090212YP, 2024090213YP; all prepared by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0022] Acetyldopamine, CAS number 2494-12-4, purchased from Shanghai Shidide Standard Technology Service Co., Ltd., item number STG5420120, RSG5421020, batch number 16082, 20532-P250401, purity 98.0%, 91.4% respectively, no need to be treated before use.

[0023] Example 2: Determination of the content of acetyldopamine in Sangpaejiao process preparation (1) Take the Sangpaejiao formula granules, Sangpaejiao medicinal materials, Sangpaejiao pieces, and Sangpaejiao standard decoction obtained in Example 1 respectively to prepare test solution; take acetyldopamine as reference substance to prepare reference substance solution; Preparation of formula granule test solution: Take Sangpaejiao formula granules, grind them, and take 0.5 g respectively, add 70% methanol 20 mL, tightly seal, weigh, ultrasonic treatment for 30 min at power 250 W, frequency 40 kHz, cool to room temperature and weigh again, make up the weight loss with 70% methanol, shake well, pass through 0.22 μm microporous filter membrane, take the filtrate, and Sangpaejiao formula granule test solution is obtained; Preparation of the test solution of the medicinal material: 0.5 g of the medicinal material was taken, 25 mL of 50% methanol was added, and the mixture was sealed and weighed. The mixture was heated and refluxed for 30 min, and then cooled to room temperature. The weight was determined again, and the lost weight was made up with 50% methanol. After shaking and filtering, 15 mL of the filtrate was precisely taken, evaporated, dissolved with 50% methanol, and diluted to 2 mL. The solution was filtered through a 0.22 μm microporous filter, and the filtrate was obtained, which was the test solution of the medicinal material of the snail egg; Preparation of the test solution of the medicinal material: 0.5 g of the medicinal material was taken, 25 mL of 50% methanol was added, and the mixture was sealed and weighed. The mixture was heated and refluxed for 30 min, and then cooled to room temperature. The weight was determined again, and the lost weight was made up with 50% methanol. After shaking and filtering, 15 mL of the filtrate was precisely taken, evaporated, dissolved with 50% methanol, and diluted to 2 mL. The solution was filtered through a 0.22 μm microporous filter, and the filtrate was obtained, which was the test solution of the medicinal material of the snail egg; Preparation of the test solution of the medicinal material: 0.5 g of the medicinal material was taken, 25 mL of 50% methanol was added, and the mixture was sealed and weighed. The mixture was heated and refluxed for 30 min, and then cooled to room temperature. The weight was determined again, and the lost weight was made up with 50% methanol. After shaking and filtering, 15 mL of the filtrate was precisely taken, evaporated, dissolved with 50% methanol, and diluted to 2 mL. The solution was filtered through a 0.22 μm microporous filter, and the filtrate was obtained, which was the test solution of the medicinal material of the snail egg; Acetyldopa was weighed and dissolved in 70% methanol-water solution to prepare a solution with a concentration of 90 μg / mL, which was used as the control solution for the determination of acetyldopa content in the snail egg formula granules and medicinal material.

[0024] Acetyldopa was weighed and dissolved in methanol to prepare a solution with a concentration of 230 μg / mL, which was used as the control solution for the determination of acetyldopa content in the snail egg standard decoction powder.

[0025] (2) Set the parameters of the chromatographic conditions on the Waters ultra-high performance liquid chromatography system: The chromatographic column was ACQUITY UPLC HSS T3 with a size of 2.1*100 mm and a particle size of 1.8 μm; The eluent included mobile phase A and mobile phase B, wherein the mobile phase A was acetonitrile and the mobile phase B was 0.03% formic acid-water solution; The elution mode was gradient elution, and the flow rate of the mobile phase was 0.3 mL / min. The specific elution gradient is shown in Table 2: Table 2 Elution gradient

[0026] The column temperature for elution was 45°C, and the detection wavelength was 280 nm. The test sample solution and the control sample solution were injected into the Waters H-Class ultra-high performance liquid chromatograph, wherein the sample injection amount of the test sample solution of the Arca granula was 2 μL, and the sample injection amount of the test sample solution of the Arca granula preparation and the standard decoction was 1 μL. The chromatogram and the peak area were analyzed and recorded, acetyl dopamine was used as the content determination index, and the quantitative analysis of the samples was performed.

[0027] The results are shown in Tables 3-6, and the representative chromatograms of each sample are shown in Figures 1-4 Table 3 Content of acetyl dopamine in Arca granula preparation

[0028] Table 4 Content of acetyl dopamine in Arca medicinal material

[0029] Table 5 Content of acetyl dopamine in Arca decoction pieces

[0030] Table 6 Content of acetyl dopamine in Arca standard decoction

[0031] From the above test results, it can be seen that the Arca granula preparation, Arca medicinal material, Arca decoction pieces and Arca standard decoction all contain acetyl dopamine, which can be used as a content index.

[0032] Example 3: Establishment of Arca identification method and methodological study 1. Preparation method of test sample solution 1.1 Selection of different concentrations of extraction solvents The Arca granula preparation (batch number: 2025060019) was finely ground, and 0.5 g was taken in parallel in 5 groups, 2 portions in each group. Water, 10% methanol, 30% methanol, 50% methanol, 70% methanol or methanol 20 mL was added, the weight was determined, and the weight was determined again after cooling to room temperature. The weight loss was made up with the corresponding concentration of methanol, shaken uniformly, filtered through a 0.22 μm microporous filter membrane, and the filtrate was obtained, which was the test sample solution of the Arca granula preparation extracted by different concentrations of methanol.

[0033] Each test solution 1 μL was injected into the liquid chromatograph, and the content of acetyl dopamine was calculated according to the chromatographic conditions of Example 2. The results are shown in Figure 5 Table 7, and the content of acetyl dopamine extracted by 70% methanol was relatively high. Therefore, 70% methanol was selected as the extraction solvent for subsequent experiments. ​

[0034] Table 7 Test results of different extraction solvents

[0035] 1.2, Selection of different extraction methods The Sangpiaoxiao formula granules (batch number: 2025060019) were ground and 0.5 g was taken in parallel in 3 groups, 2 portions in each group, 70% methanol 20 mL was added, tightly capped, the weight was determined, and a) power 250 W, frequency 40 kHz ultrasonic treatment for 30 min, or b) heating reflux for 30 min, or c) oscillation for 30 min was carried out, respectively. After all the treatments were cooled to room temperature, the weight was determined again, the lost weight was made up with 70% methanol, shaken well, and filtered through a 0.22 μm microporous filter membrane. The filtrate was taken, and the Sangpiaoxiao formula granule test sample solution of different extraction methods was obtained.

[0036] 1 μL of each test solution was injected into the liquid chromatograph, and the content of acetyl dopamine was calculated according to the chromatographic conditions of Example 2. The results are shown in Table 8. There was no significant difference in the content of acetyl dopamine obtained by the three extraction methods. The ultrasonic extraction method was selected subsequently. Figure 6

[0037] Table 8 Test results of different extraction methods

[0038] 1.3, Selection of different extraction time The Sangpiaoxiao formula granules (batch number: 2025060019) were ground and 0.5 g was taken in parallel in 4 groups, 2 portions in each group, 70% methanol 20 mL was added, tightly capped, the weight was determined, and ultrasonic treatment with power 250 W, frequency 40 kHz for 15, 30, 45 or 60 min was carried out, respectively. After all the treatments were cooled to room temperature, the weight was determined again, the lost weight was made up with 70% methanol, shaken well, and filtered through a 0.22 μm microporous filter membrane. The filtrate was taken, and the Sangpiaoxiao formula granule test sample solution of different extraction methods was obtained.

[0039] 1 μL of each test solution was injected into the liquid chromatograph, and the content of acetyl dopamine was calculated according to the chromatographic conditions of Example 2. The results are shown in Table 9. There was no significant difference in the content of acetyl dopamine obtained by different extraction time. The extraction time of 30 min was selected subsequently. Figure 7

[0040] Table 9 Test results of different extraction time

[0041] 1.4, Selection of different extraction volumes ​​Take 0.5 g of Sangpiaoji Dispensing Granules (Batch No. 2025060019), grind into powder through No. 2 sieve, add 70% methanol 15, 20 or 50 mL, seal tightly, weigh, treat with ultrasonic wave at a power of 250 W and a frequency of 40 kHz for 30 min, weigh again after cooling to room temperature, make up the weight loss with 70% methanol, shake well, pass through a 0.22 μm microporous filter membrane, and take the filtrate to obtain Sangpiaoji Dispensing Granules test sample solutions of different extraction methods.

[0042] Inject 1 μL of each test solution into the liquid chromatograph, determine the content of acetyl dopamine according to the chromatographic conditions of Example 2, and the results are shown in Figure 8 Table 10.

[0043] Table 10. Detection results of different extraction volumes

[0044] According to the above results, the test sample preparation method for determining the content of Sangpiaoji Granules is finally determined as follows: Weigh 0.5 g of the sample to be tested, add 70% methanol 20 mL, weigh, treat with ultrasonic wave at a power of 250 W and a frequency of 40 kHz for 30 min, weigh again after cooling to room temperature, make up the weight loss with 70% methanol, shake well, pass through a 0.22 μm microporous filter membrane, and take the filtrate to obtain the test sample.

[0045] 2. Methodology verification 2.1. Linearity verification Inject 0.2, 0.5, 0.8, 1.0, 2.0, 2.5 or 3.0 μL of acetyl dopamine control solution with a concentration of 88.1096 μg / mL into the liquid chromatograph, determine according to the chromatographic conditions of Example 2, take the peak area as the vertical coordinate and the injection amount (μg) as the horizontal coordinate to draw a standard curve, and obtain the regression equation of acetyl dopamine, as shown in Table 11. Figure 9 The injection amount of acetyl dopamine within the range of 0.0176-0.2643 μg has a good linear relationship with the peak area value, the regression equation is y = 2780339.4402x-15026.9942, R 2 = 1.0000, where x is the mass of acetyl dopamine (μg) and y is the peak area value.

[0046] Table 11. Relationship between injection amount of acetyl dopamine control and peak area

[0047] 2.2. Precision verification 2.2.1, Instrument precision test The Sangpaejiao Dispensing Granules (Batch No. 2025060019) were ground and 0.5 g was added to 20 mL of 70% methanol. The weight was determined, and then the mixture was treated with ultrasonic waves at a power of 250 W and a frequency of 40 kHz for 30 min. After cooling to room temperature, the weight was determined again, and the lost weight was made up with 70% methanol. The mixture was shaken and filtered through a 0.22 μm microporous filter. The filtrate was obtained, which was the test sample.

[0048] 1 μL of the test solution was injected into the liquid chromatograph, and the determination was performed according to the chromatographic conditions of Example 2. The peak area measurement value of acetyl dopamine was recorded, and the RSD was calculated. The results are shown in Table 12, and the peak area RSD was 0.29%, indicating that the instrument precision test was good.

[0049] Table 12 Instrument precision test

[0050] 2.2.2, Reproducibility test The Sangpaejiao Dispensing Granules (Batch No. 2025060019) were ground and 0.5 g was added to 20 mL of 70% methanol. The weight was determined, and then the mixture was treated with ultrasonic waves at a power of 250 W and a frequency of 40 kHz for 30 min. After cooling to room temperature, the weight was determined again, and the lost weight was made up with 70% methanol. The mixture was shaken and filtered through a 0.22 μm microporous filter. The filtrate was obtained, which was the test sample 1-6.

[0051] 1 μL of the test solution was injected into the liquid chromatograph, and the determination was performed according to the chromatographic conditions of Example 2. The peak area measurement value of acetyl dopamine was recorded, and the RSD was calculated. The results are shown in Table 12, and the peak area RSD was 0.29%, indicating that the instrument precision test was good.

[0052] Table 13 Reproducibility test results of samples

[0053] 2.2.3 Intermediate precision test The Sangpaejiao Dispensing Granules (Batch No. 2025060019) were ground by the experimenter A, and 0.5 g was added to 20 mL of 70% methanol. The weight was determined, and then the mixture was treated with ultrasonic waves at a power of 250 W and a frequency of 40 kHz for 30 min. After cooling to room temperature, the weight was determined again, and the lost weight was made up with 70% methanol. The mixture was shaken and filtered through a 0.22 μm microporous filter. The filtrate was obtained, which was the test sample A.

[0054] The Sangpaejiao Dispensing Granules (Batch No. 2025060019) were ground by the experimenter B, and the test sample B was prepared in the same way.

[0055] At different times, the tester A injects 1 μL of the test sample A into the Waters H-Class ultra-high performance liquid chromatograph; the tester B injects 1 μL of the test sample B into the Thermo VANQUISH ultra-high performance liquid chromatograph, and the determination is performed according to the chromatographic conditions in Example 2. The measurement of the peak area of acetyl dopamine is recorded, and the RSD is calculated. The results are shown in Table 14. The RSD of the intermediate precision of the sample is 2.54%, indicating that the intermediate precision is good.

[0056] Table 14. Intermediate precision test

[0057] 2.3. Accuracy test 0.25 g of the sample with an acetyl dopamine content of 3.16 mg / g is divided into 3 groups, each group has 3 parts, and a total of 9 parts. Each group is added with 1, 2 or 3 mL of acetyl dopamine solution with a concentration of 365.2344 μg / mL. Then 20 mL of 70% methanol is added, and after the weight is determined, the ultrasonic treatment is performed at a power of 250 W and a frequency of 40 kHz for 30 min. After cooling to room temperature, the weight is determined again, the lost weight is made up with 70% methanol, and the mixture is shaken and filtered through a 0.22 μm microporous filter. The filtrate is taken to obtain 3 groups of test sample solutions: 50%, 100% and 150%, each group has 3 parts.

[0058] Each part is injected into the liquid chromatograph, and the determination is performed according to the chromatographic conditions in Example 2. The recovery rate is calculated according to the following formula, and the RSD is calculated.

[0059] Table 15. Accuracy results

[0060] The results are shown in Table 15. The recovery rate of acetyl dopamine content is between 89.82-97.07%, and the accuracy test is good.

[0061] 2.4. Specificity test The auxiliary materials added in the Cantharis formula granules are malt dextrin, silicon dioxide and magnesium stearate. This experiment investigates the influence of the negative sample without Cantharis on the characteristic spectrum of the Cantharis formula granules.

[0062] The negative sample without Cantharis, i.e. the mixture of malt dextrin, silicon dioxide and magnesium stearate, is finely ground. 0.5 g of the mixture is taken, 20 mL of 70% methanol is added, and after the weight is determined, the ultrasonic treatment is performed at a power of 250 W and a frequency of 40 kHz for 30 min. After cooling to room temperature, the weight is determined again, the lost weight is made up with 70% methanol, and the mixture is shaken and filtered through a 0.22 μm microporous filter. The filtrate is taken to obtain the negative test sample (negative solution).

[0063] Sangpiaoxie Dispensing Granules (Batch Number: 2025060019) were ground and 0.5 g was added to 20 mL of 70% methanol. After weighing, the mixture was treated with ultrasonic waves at a power of 250 W and a frequency of 40 kHz for 30 min. After cooling to room temperature, the weight was re-weighed, and the lost weight was made up with 70% methanol. After shaking, the mixture was filtered through a 0.22 μm microporous filter, and the filtrate was obtained as the test sample.

[0064] 1 μL of the negative solution, the test sample and the acetyldopa solution (concentration: 88.1096 μg / mL) were injected into the liquid chromatograph, respectively, and determined according to the chromatographic conditions of Example 2.

[0065] The results are shown in Table 15. Figure 10 The solvent and excipient do not interfere with the determination of the characteristic chromatogram of Sangpiaoxie Dispensing Granules, and the method is specific.

[0066] 2.5, Integrity test Sangpiaoxie Dispensing Granules (Batch Number: 2025060019) were ground and 0.5 g was added to 20 mL of 70% methanol. After weighing, the mixture was treated with ultrasonic waves at a power of 250 W and a frequency of 40 kHz for 30 min. After cooling to room temperature, the weight was re-weighed, and the lost weight was made up with 70% methanol. After shaking, the mixture was filtered through a 0.22 μm microporous filter, and the filtrate was obtained as the test sample.

[0067] 1 μL of the test sample solution was injected into the liquid chromatograph, and determined according to the chromatographic conditions of Example 2, but the elution time of the elution gradient with the highest proportion of acetonitrile was doubled. The chromatogram was recorded, and the results are shown in Table 15. Figure 11 Doubling the analysis time does not interfere with the determination of the acetyldopa content.

[0068] 2.6, Durability test 2.6.1, Stability test Sangpiaoxie Dispensing Granules (Batch Number: 2025060019) were ground and 0.5 g was added to 20 mL of 70% methanol. After weighing, the mixture was treated with ultrasonic waves at a power of 250 W and a frequency of 40 kHz for 30 min. After cooling to room temperature, the weight was re-weighed, and the lost weight was made up with 70% methanol. After shaking, the mixture was filtered through a 0.22 μm microporous filter, and the filtrate was obtained as the test sample.

[0069] 1 μL of the test sample solution was injected into the liquid chromatograph, and determined according to the chromatographic conditions of Example 2, but the elution time of the elution gradient with the highest proportion of acetonitrile was doubled. The chromatogram was recorded, and the results are shown in Table 15.

[0070] Table 16 Determination results of stability test

[0071] 2.6.2, Test of different flow rates The formula granules of Sangpiaoxiao (batch number: 2025060019) were ground, 0.5 g of which was added into 20 mL of 70% methanol, and the weight was determined. Then, the mixture was treated with ultrasonic at a power of 250 W and a frequency of 40 kHz for 30 min. After cooling to room temperature, the weight was determined again. Then, the lost weight was made up with 70% methanol, and the mixture was shaken and filtered through a 0.22 μm microporous filter. The filtrate was obtained as the test sample.

[0072] The test sample was injected at 1 µL, and the flow rate of the mobile phase was set to 0.25, 0.30 or 0.35 mL / min. The other chromatographic conditions were the same as in Example 2. The peak area values were recorded and the RSD was calculated. The results are shown in Table 17 below. Within the range of 0.25-0.35 mL / min, the variation of flow rate had no significant effect on the determination of the content of the sample, and the flow rate was good in durability. Figure 12 Table 17 Investigation of different flow rates

[0073] 2.6.3, Test of different column temperatures

[0074] The formula granules of Sangpiaoxiao (batch number: 2025060019) were ground, 0.5 g of which was added into 20 mL of 70% methanol, and the weight was determined. Then, the mixture was treated with ultrasonic at a power of 250 W and a frequency of 40 kHz for 30 min. After cooling to room temperature, the weight was determined again. Then, the lost weight was made up with 70% methanol, and the mixture was shaken and filtered through a 0.22 μm microporous filter. The filtrate was obtained as the test sample. The test sample was injected at 1 µL, and the column temperature was set to 43, 45 or 47 ℃. The other chromatographic conditions were the same as in Example 2. The peak area values were recorded and the RSD was calculated. The results are shown in Table 18 below. Within the range of 43-47 ℃, the variation of column temperature had no significant effect on the determination of the content of the sample, and the column temperature was good in durability.

[0075] Figure 13 Table 18 Investigation of different column temperatures

[0076] 2.6.4, Test of different chromatographic columns

[0077] ​​The mulberry snail formula granules (batch number: 2025060019) were finely ground, 0.5 g was taken, 20 mL of 70% methanol was added, the weight was determined, then ultrasonic treatment was performed at a power of 250 W and a frequency of 40 kHz for 30 min, after cooling to room temperature, the weight was determined again, the lost weight was made up with 70% methanol, shaken well, filtered through a 0.22 μm microporous filter membrane, and the filtrate was taken as the test sample.

[0078] The test sample was injected at 1 µL, the chromatographic column was ZORBAX Eclipse Plus C18 RRHD, 2.1×100 mm, 1.8 μm, or ACQUITY UPLC HSS T3, 2.1 mm×100 mm, 1.8 μm, or Poroshell EC-C18, 2.1×100 mm, 1.9 μm, respectively, and the rest of the chromatographic conditions were the same as in Example 2, the peak area value was recorded and the RSD was calculated, and the results are shown in Table 19 below. Figure 14 , different chromatographic columns had no significant effect on the determination of the content of the sample, and the chromatographic column was suitable.

[0079] Table 19 Investigation of different chromatographic columns

Claims

1. A method for identifying samples related to mantis egg case, characterized in that the steps include... include: (1) Preparation of test solutions for mantis egg case and related samples; (2) Setting chromatographic parameters on the ultra-high performance liquid chromatograph: (21) Chromatographic column: a chromatographic column with octadecylsilane-bonded silica gel as the stationary phase; (22) The eluent includes mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile and mobile phase B is 0.03% formic acid-water solution; (23) Elution method: mobile phase flow rate is 0.25-0.35 mL / min, gradient elution: At 0 min, the volume percentage of mobile phase A was 0%, and the volume percentage of mobile phase B was 100%. After 4 minutes, the volume ratio of mobile phase A was 3%, and the volume ratio of mobile phase B was 97%. After 14 minutes, the volume percentage of mobile phase A was 3%, and the volume percentage of mobile phase B was 97%. After 15 minutes, the volume ratio of mobile phase A was 40%, and the volume ratio of mobile phase B was 60%. (24) Elution column temperature: 43-47℃; (3) Inject the sample solution to be tested into an ultra-high performance liquid chromatograph, analyze and record the chromatogram, and compare it with the chromatogram of acetyldopamine standard for identification; The samples related to mantis egg case include mantis egg case medicinal materials, processed slices, extracts, standard decoctions, formula granules, or any of the following: reference samples, intermediate products, and finished products containing mantis egg case.

2. The method for identifying mantis egg case related samples according to claim 1, characterized in that, Step 1 includes: adding methanol-water solution to the mantis egg case sample, extracting, filtering, and obtaining the test solution.

3. The method for identifying mantis egg case related samples according to claim 2, characterized in that, The methanol concentration in the methanol-water solution is not higher than 70%.

4. The method for identifying mantis egg case related samples according to claim 2, characterized in that, The amount of methanol-water solution used is 30-100 mL per gram of sample.

5. The method for identifying mantis egg case related samples according to claim 2, characterized in that, The extraction is performed by any one of ultrasonic treatment, heating and reflux, or shaking extraction, with an extraction time of 15-60 minutes.

6. The method for identifying mantis egg case related samples according to claim 2, characterized in that, The extraction process also includes: evaporation to dryness and reconstitution with methanol-water solution.

7. The method for identifying mantis egg case related samples according to claim 1, characterized in that, The chromatographic column with octadecylsilane-bonded silica gel as the stationary phase in step 21 has a length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.5-2.0 µm.

8. The method for identifying mantis egg case related samples according to claim 1, characterized in that, In step 3, the volume of sample injected into the high-performance liquid chromatograph is 1-2 μL.

9. The method for identifying mantis egg case related samples according to claim 1, characterized in that, The ultra-high performance liquid chromatograph is an ultra-high performance liquid chromatograph equipped with an ultraviolet detector.

10. The method for identifying mantis egg case related samples according to claim 9, characterized in that, The detection wavelength parameter of the ultra-high performance liquid chromatograph equipped with an ultraviolet detector is set to 275-285nm.

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