Method for determining content of astragaloside IV in astragalus membranaceus fine particles
By optimizing the high-performance liquid chromatography (HPLC) method for the determination of astragaloside A in Astragalus Extract Granules, and employing an ultraviolet detector and reflux extraction, the problems of solution emulsification and cumbersome preparation in the determination of astragaloside A content in Astragalus Extract Granules were solved, resulting in a simple and accurate detection method.
Patent Information
- Application Number
- CN202511579336.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-31
- Publication Date
- 2026-01-13
AI Technical Summary
The existing methods for determining the astragaloside A content in Astragalus extract granules have problems such as solution emulsification leading to large differences in detection results, and cumbersome and time-consuming preparation process.
High-performance liquid chromatography (HPLC) with a UV detector was employed. The sample preparation method was optimized by using heating reflux and water-saturated n-butanol extraction to simplify the operation process and improve detection sensitivity and efficiency.
This method achieves simplicity, accuracy, and stability in the determination of astragaloside A content in astragalus extract granules, reduces detection costs, and improves the repeatability and accuracy of detection results.
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Figure CN121324539A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to a method for determining the content of astragaloside A in astragalus extract granules. Background Technology
[0002] Astragalus Extract Granules are a traditional Chinese medicine. They are composed of Astragalus membranaceus. They have the effects of nourishing blood and qi, strengthening the body and stopping sweating. They are used for qi and blood deficiency, spontaneous sweating due to superficial deficiency, weakness of limbs, lack of energy, or weakness due to prolonged illness, and weak spleen and stomach.
[0003] Modern research has extracted and isolated various compounds from Astragalus membranaceus, including flavonoids, saponins, and alkaloids. Among them, astragaloside A is the most important active substance in Astragalus membranaceus.
[0004] Currently, the method for determining the content of astragaloside A in Astragalus Extract Granules mainly refers to the quality standard YBZ04082005-2012Z. It employs high-performance liquid chromatography (HPLC) with an evaporative light scattering detector. The preparation method of the test solution is as follows: Take an appropriate amount of the contents from the "Content Variation" section of this product, grind it finely, accurately weigh 4g, add 40mL of water to dissolve it, shake well, place it in a separatory funnel, add water-saturated n-butanol and shake to extract three times, 30mL each time (centrifuge if necessary), combine the n-butanol extracts, wash twice with 1% sodium hydroxide solution, 30mL each time, discard the sodium hydroxide solution, then wash with water saturated with n-butanol until neutral, discard the aqueous solution, evaporate the n-butanol to dryness on a water bath, dissolve the residue in methanol and transfer it to a 5mL volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate.
[0005] However, in actual testing, it was found that when the formulation was dispersed in water and then extracted with water-saturated n-butanol by shaking, the solution was prone to emulsification, making alcohol-water separation difficult. Direct centrifugation resulted in significant discrepancies in the final test results. Furthermore, the preparation process of this test sample was cumbersome and time-consuming, and the method needs optimization. Summary of the Invention
[0006] The purpose of this invention is to provide a method for determining the astragaloside A content in astragalus extract granules, based on existing technology. This method is a high-performance liquid chromatography (HPLC) method, employing the most commonly used ultraviolet detector in HPLC, which has higher sensitivity for detecting astragaloside A in low concentrations in the preparation. Simultaneously, the method for preparing the test sample is optimized, making the preparation process simpler, shortening the preparation time, and saving detection costs.
[0007] The technical solution of the present invention is as follows: A method for determining the content of astragaloside A in astragalus extract granules, comprising the following steps:
[0008] Step 1: Preparation of the reference solution:
[0009] Accurately weigh astragaloside A reference standard and add methanol to prepare a reference standard solution containing 0.2–0.8 mg per 1 mL;
[0010] Step 2, Preparation of the test solution:
[0011] Take a certain amount of Astragalus extract granules, place them in a stoppered conical flask, add 80% methanol solution containing 4% concentrated ammonia test solution (take 4 mL of concentrated ammonia test solution, add 80% methanol to 100 mL, shake well), stopper tightly, weigh, heat under reflux, cool, weigh again, make up the lost weight with solvent, shake well, filter, accurately measure the subsequent filtrate, evaporate to dryness, dissolve the residue in water, extract with water-saturated n-butanol, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in methanol, transfer to a volumetric flask, add methanol to the mark, shake well, filter, and take the subsequent filtrate to obtain the final product.
[0012] Step 3, Measurement Method:
[0013] Accurately pipette 10 μL each of the reference solution prepared in step 1 and the test solution prepared in step 2 into the liquid chromatograph. Calculate the content according to the following formula:
[0014]
[0015] In the above formula, m s The sample weight is the reference standard, P is the purity of the reference standard, and A is the reference standard purity. i V represents the peak area of the test sample. i V represents the dilution factor of the test solution. s A is the dilution factor of the reference solution. s The peak area of the reference solution is m. i Z represents the sample weight of the test sample, Z represents the fill weight of the preparation, and X represents the content of astragaloside A in each bag of preparation.
[0016] The chromatographic conditions for high performance liquid chromatography in step 3 are as follows: a column packed with octadecyl bonded silica gel; a UV detector with a detection wavelength of 200 nm; a mobile phase of acetonitrile and water in a volume ratio of 30–35:70–65; a column temperature of 25–35 °C; and a flow rate of 0.8–1.5 mL / min.
[0017] In some implementations, the ratio of the amount of Astragalus membranaceus granules to be tested to the amount of 80% methanol solution containing 4% concentrated ammonia solution in step 2 is 1g:5-20mL;
[0018] In some implementation schemes, the Astragalus extract granules to be tested in step 2 are ground into a fine powder and passed through a No. 3 sieve.
[0019] In some implementations, the heating reflux time in step 2 is 0.5 to 2 hours, preferably 1 to 2 hours.
[0020] In some implementations, the volume of the filtrate measured in step 2 is 20–50 mL.
[0021] In some implementations, in step 2, the residue is dissolved in 20 mL to 50 mL of water and extracted 2 to 6 times with 20 to 50 mL of water-saturated n-butanol each time.
[0022] In some implementations, the volume transferred to the volumetric flask in step 2 is 5–10 mL.
[0023] The beneficial effects of this invention are: the sample detection method established by this invention is simple to operate, has a short sample preparation time, strong specificity, and good repeatability, stability and accuracy. Attached Figure Description
[0024] Figure 1 The diagram shows the results of the specificity test, where A is the blank excipient sample, B is the astragaloside A reference standard, and C is the test sample.
[0025] Figure 2 HPLC chromatogram of Astragalus extract granules Detailed Implementation
[0026] The following embodiments are intended to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the invention to the scope of the embodiments described.
[0027] The instruments and reagents used in the examples are as follows:
[0028] Instruments: Shimadzu LC-2050C high performance liquid chromatograph; XSR105DU electronic analytical balance (Mettler Toledo); ME204E electronic analytical balance (Mettler Toledo); HH-6 digital display constant temperature water bath (Shanghai Lichen Instrument Technology Co., Ltd.)
[0029] Test drugs: Astragalus extract granules source is shown in Table 1; blank excipient samples were prepared according to the preparation method specified in the Astragalus extract granules quality standard YBZ04082005-2012Z; astragaloside A reference standard was purchased from the China National Institutes for Food and Drug Control; methanol (chromatographic grade), acetonitrile (chromatographic grade), concentrated ammonia (analytical grade); n-butanol (chromatographic grade); purified water.
[0030] Table 1. Sample Information of Astragalus Extract Granules
[0031]
[0032] Example 1: Determination of Astragaloside A Content in Astragalus Extract Granules
[0033] Step 1: Preparation of the reference solution:
[0034] Take an appropriate amount of astragaloside A reference standard, accurately weigh it, and add methanol to prepare a reference standard solution containing 0.4 mg per 1 mL.
[0035] Step 2, Preparation of the test solution:
[0036] Take the above three batches of Astragalus extract granules and blank excipients, grind them finely (pass through a No. 3 sieve), accurately weigh 4g of each of the Astragalus extract granules and blank excipient samples, place them in a stoppered conical flask, add 100ml of 80% methanol solution containing 4% concentrated ammonia test solution (take 4mL of concentrated ammonia test solution, add 80% methanol to 100mL, shake well), stopper tightly, weigh, heat under reflux for 1 hour, cool, weigh again, make up the lost weight with solvent, shake well, filter, accurately measure 50ml of the subsequent filtrate, evaporate to dryness, add 25ml of water to the residue, heat gently to dissolve, extract three times with water-saturated n-butanol, 25ml each time, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in methanol, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the subsequent filtrate to obtain the final product.
[0037] Step 3, Determination Method
[0038] Accurately pipette 10 μL each of the reference solution prepared in step 1 and the test solution prepared in step 2 into the liquid chromatograph and measure using a UV detector. Calculate the content based on peak area using the external standard method.
[0039] The liquid chromatography conditions were as follows: column packing material: octadecyl bonded silica gel; mobile phase: acetonitrile-water = 1:2; column temperature: 25℃; detection wavelength: 200nm; flow rate: 1.0ml / min.
[0040] The chromatogram of the test sample is as follows Figure 2 As shown in the figure, the results indicate that astragaloside A peaked at 12.053 min with a good peak shape.
[0041] According to the formula
[0042]
[0043] In the above formula, ms is the sample weight of the reference standard, P is the purity of the reference standard, Ai is the peak area of the test sample, Vi is the dilution factor of the test sample solution, Vs is the dilution factor of the reference standard solution, As is the peak area of the reference standard solution, mi is the sample weight of the test sample, Z is the dosage of the preparation, and X is the content of astragaloside A in each bag of preparation.
[0044] The astragaloside A content in samples S1 to S4 was calculated to be 4.4 mg / bag, 4.1 mg / bag, 4.1 mg / bag, and 4.2 mg / bag, respectively, showing little difference in content among the four batches. This indicates that the method of this invention can still effectively detect astragaloside A in granules with low astragaloside A content.
[0045] Example 2: Investigation of the preparation method of the test solution
[0046] 2.1 Investigation of extraction solvent
[0047] Preparation of reference solution: Take an appropriate amount of astragaloside A reference standard, accurately weigh it, and add methanol to prepare a reference solution containing 0.4 mg per 1 mL.
[0048] Preparation of test solution: Take the sample from the content difference section, grind it finely (pass through a No. 3 sieve), accurately weigh 4g, place it in a stoppered conical flask, add 100ml each of 80% methanol, 80% methanol solution containing 4% concentrated ammonia test solution (take 4mL of concentrated ammonia test solution, add 80% methanol to 100mL, shake well), and 80% methanol solution containing 1% sodium hydroxide, stopper tightly, weigh, heat under reflux for 1 hour, and ultrasonically extract for 1 hour respectively, cool, weigh again, make up the weight loss with solvent, shake well, filter, accurately measure 50ml of the subsequent filtrate, evaporate to dryness, add 25ml of water to the residue, heat gently to dissolve, extract 3 times with water-saturated n-butanol, 25ml each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue in methanol, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the subsequent filtrate to obtain the test solution.
[0049] Take the above-mentioned reference solution and test solution, and inject them for analysis.
[0050] Table 2 Results of the extraction solvent test
[0051] Extraction solvent Content (mg / bag) 80% methanol 2.9 80% methanol solution containing 4% concentrated ammonia test solution 4.4 80% methanol solution containing 1% sodium hydroxide 3.8
[0052] The results showed that using an 80% methanol solution containing 4% concentrated ammonia as the extraction solvent resulted in higher extraction and conversion efficiency of astragaloside A.
[0053] 2.2 Examination of Extraction Methods
[0054] Preparation of reference solution: Take an appropriate amount of astragaloside A reference standard, accurately weigh it, and add methanol to prepare a reference solution containing 0.4 mg per 1 mL.
[0055] Preparation of test solution: Take the sample from the content difference section, grind it finely (pass through a No. 3 sieve), accurately weigh 4g, place it in a stoppered conical flask, add 100ml of 80% methanol solution containing 4% concentrated ammonia test solution (take 4mL of concentrated ammonia test solution, add 80% methanol to 100mL, shake well), stopper tightly, weigh, heat under reflux for 1 hour, and ultrasonically extract for 1 hour, cool, weigh again, replenish the lost weight with solvent, shake well, filter, accurately measure 50ml of the subsequent filtrate, evaporate to dryness, add 25ml of water to the residue, heat gently to dissolve, extract 3 times with water-saturated n-butanol, 25ml each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue in methanol, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the subsequent filtrate to obtain the test solution.
[0056] Take the above-mentioned reference solution and test solution, and inject them for analysis.
[0057] Table 3 Results of the extraction method investigation experiment
[0058] Extraction method Content (mg / bag) Heating reflux 4.4 Ultrasonic extraction 3.5
[0059] The results showed that, under the same extraction time, the content of the sample solution extracted by heating and reflux was significantly higher than that extracted by ultrasonic extraction, thus the heating and reflux extraction method was chosen.
[0060] 2.3 Examination of extraction time
[0061] Preparation of reference solution: Take an appropriate amount of astragaloside A reference standard, accurately weigh it, and add methanol to prepare a reference solution containing 0.4 mg per 1 mL.
[0062] Preparation of test solution: Take the sample from the content difference section, grind it finely (pass through a No. 3 sieve), accurately weigh 4g, place it in a stoppered conical flask, add 100ml of 80% methanol solution containing 4% concentrated ammonia test solution (take 4mL of concentrated ammonia test solution, add 80% methanol to 100mL, shake well), stopper tightly, weigh, heat under reflux for 0.5 hours, 1 hour, and 2 hours respectively, cool, weigh again, make up the weight loss with solvent, shake well, filter, accurately measure 50ml of the subsequent filtrate, evaporate to dryness, add 25ml of water to the residue, heat gently to dissolve, extract 3 times with water-saturated n-butanol, 25ml each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue in methanol, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the subsequent filtrate to obtain the test solution.
[0063] Take the above-mentioned reference solution and test solution, and inject them for analysis.
[0064] Table 4 Results of the extraction time investigation experiment
[0065] Extraction time Content (mg / bag) Reflux for 0.5 hours 3.4 1 hour of reflux 4.4 2 hours of reflux 4.4
[0066] The results showed that sufficient extraction could be achieved by heating and refluxing for 1 hour, therefore the extraction time was 1 hour.
[0067] 2.4 Investigation of the amount of extraction solvent
[0068] Preparation of reference solution: Take an appropriate amount of astragaloside A reference standard, accurately weigh it, and add methanol to prepare a reference solution containing 0.4 mg per 1 mL.
[0069] Preparation of test solution: Take the sample from the content difference section, grind it finely (pass through a No. 3 sieve), accurately weigh 4g, place it in a stoppered conical flask, add 50ml, 100ml, and 150ml of 80% methanol solution containing 4% concentrated ammonia test solution (take 4mL of concentrated ammonia test solution, add 80% methanol to 100mL, and shake well), stopper tightly, weigh, heat under reflux for 1 hour, cool, weigh again, replenish the lost weight with solvent, shake well, filter, accurately measure 50ml of the subsequent filtrate, evaporate to dryness, add 25ml of water to the residue, heat gently to dissolve, extract 3 times with water-saturated n-butanol, 25ml each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue in methanol, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the subsequent filtrate to obtain the test solution.
[0070] Take the above-mentioned reference solution and test solution, and inject them for analysis.
[0071] Table 5 Results of the experiment on the amount of extraction solvent used
[0072] Extraction solvent volume Content (mg / bag) 50ml 3.3 100ml 4.4 150ml 4.0
[0073] The results showed that a solvent volume of 100 ml was sufficient for extraction, therefore the solvent volume was determined to be 100 ml.
[0074] 2.5 Investigation of the number of extractions with water-saturated n-butanol
[0075] Preparation of reference solution: Take an appropriate amount of astragaloside A reference standard, accurately weigh it, and add methanol to prepare a reference solution containing 0.4 mg per 1 mL.
[0076] Preparation of the test solution: Take the sample from the "Variation in Content" section, grind it finely (pass through a No. 3 sieve), accurately weigh 4g, place it in a stoppered conical flask, add 100ml of 80% methanol solution containing 4% concentrated ammonia test solution (take 4mL of concentrated ammonia test solution, add 80% methanol to 100mL, shake well), stopper tightly, weigh, heat under reflux for 1 hour, cool, weigh again, replenish the lost weight with solvent, shake well, filter, accurately measure 50ml of the subsequent filtrate, evaporate to dryness, add 25ml of water to the residue, heat gently to dissolve, extract with water-saturated n-butanol 2, 3, 4, and 5 times respectively, 25ml each time, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in methanol, transfer to a 5ml volumetric flask, add methanol to the mark, shake well, filter, and take the subsequent filtrate to obtain the test solution.
[0077] Take the above-mentioned reference solution and test solution, and inject them for analysis.
[0078] Table 6 Results of the n-Butanol Extraction Number Investigation Experiment
[0079] n-Butanol extraction times Content (mg / bag) 2 times 3.8 3 times 4.4 4 times 4.3 5 times 4.4
[0080] The results showed that extraction with water-saturated n-butanol was sufficient after three extractions, therefore the number of water-saturated n-butanol extractions was three.
[0081] Example 3 Specificity Test
[0082] The test solution and blank excipient solution were prepared according to the method in Example 1. The blank excipient solution (A), the reference solution (B), and the test solution (C) were respectively injected and analyzed under the detection conditions of Example 1. The results were as follows: the peak position of astragaloside A in the test solution (16.271 min) was consistent with that in the reference solution (16.262 min); at the corresponding retention time of astragaloside A, no corresponding chromatographic peak appeared in the blank excipient, indicating that the blank excipient did not interfere with the determination.
[0083] Example 4 Linear
[0084] Accurately pipette 10 μL of astragaloside A reference solution with concentrations of 0.170, 0.340, 0.679, 1.019, 1.358, and 2.038 mg / mL, respectively, and inject them into the liquid chromatograph. Linear regression was performed with peak area as the ordinate and injection concentration as the abscissa. The results showed that the linear equation was y = 629.5429x + 8.5170, R0. 2 =0.9999, indicating that astragaloside A showed good linearity in the concentration range of 0.170–2.038 mg / mL.
[0085] Example 5 System Precision
[0086] The astragaloside A reference solution from Example 1 was injected six times consecutively, and the RSD value of the peak area was calculated. The results showed that the RSD value of the peak area was 0.11%, indicating good system precision of the instrument.
[0087] Table 7 Results of System Precision Test
[0088]
[0089] Repeatability
[0090] Six S1 test solutions were prepared in parallel according to the method in Example 1, and analyzed to calculate the content of the test solution. The results showed that the RSD value of the astragaloside A content determination results of the six test solutions was 1.88%, indicating that the analytical method had good repeatability.
[0091] Table 8 Results of Repeatability Tests
[0092]
[0093] Example 6 Solution Stability
[0094] The S1 test solution was prepared in parallel according to the method in Example 1, and analyzed at 0, 2, 4, 8, 12, 16, and 24 hours. The RSD value of the peak area of the test solution was calculated. The results showed that the RSD value of the astragaloside peak area of the test solution was 1.94% within 24 hours, and the test solution had good stability at room temperature within 24 hours.
[0095] Table 9 Results of solution stability test
[0096]
[0097] Example 7 Accuracy
[0098] Nine 2g portions of sample S1 were accurately weighed, and 0.5ml, 1ml, and 2ml of a 1.6019mg / mL reference solution were accurately added to each portion. The sample was prepared according to the test sample preparation method, and the chromatographic conditions described above were used for determination. The recovery rate was calculated. The results showed that the average recovery rate at the three different concentrations was 87.65%, with an RSD of 2.23%, indicating good accuracy of the analytical method.
[0099] Table 10 Accuracy Test Results
[0100]
[0101] The above experimental results show that the method for determining the astragaloside A content in Astragalus Extract Granules provided by this invention has good repeatability, good solution stability, and high accuracy, and can be used to determine the astragaloside A content in Astragalus Extract Granules.
Claims
1. A method for determining the content of astragaloside A in astragalus extract granules, comprising the following steps: Step 1: Preparation of the reference solution: Accurately weigh astragaloside A reference standard and add methanol to prepare a reference standard solution containing 0.2–0.8 mg per 1 mL; Step 2, Preparation of the test solution: Take a certain amount of Astragalus extract granules, place them in a stoppered conical flask, add 80% methanol solution containing 4% concentrated ammonia test solution (take 4 mL of concentrated ammonia test solution, add 80% methanol to 100 mL, shake well), stopper tightly, weigh, heat under reflux, cool, weigh again, make up the lost weight with solvent, shake well, filter, accurately measure the subsequent filtrate, evaporate to dryness, dissolve the residue in water, extract with water-saturated n-butanol, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in methanol, transfer to a volumetric flask, add methanol to the mark, shake well, filter, and take the subsequent filtrate to obtain the product; Step 3, Measurement Method: Accurately pipette 10 μL each of the reference solution prepared in step 1 and the test solution prepared in step 2, and inject them into the liquid chromatograph. Calculate the content according to the following formula: In the formula m s The sample weight is the reference standard, P is the purity of the reference standard, and A is the reference standard purity. i V represents the peak area of the test sample. i V represents the dilution factor of the test solution. s A is the dilution factor of the reference solution. s The peak area of the reference solution is m. i Z represents the sample weight of the test sample, Z represents the fill weight of the preparation, and X represents the content of astragaloside A in each bag of preparation. The chromatographic conditions for high performance liquid chromatography in step 3 are as follows: a column packed with octadecyl bonded silica gel; a UV detector with a detection wavelength of 200 nm; a mobile phase of acetonitrile and water in a volume ratio of 30–35:70–65; a column temperature of 25–35 °C; and a flow rate of 0.8–1.5 mL / min.
2. The method for determining the astragaloside A content in Astragalus extract granules according to claim 1, characterized in that, In step 2, the mass-to-volume ratio of the Astragalus membranaceus granules to be tested to the 80% methanol solution containing 4% concentrated ammonia solution is 1:5-20.
3. The method for determining the astragaloside A content in Astragalus extract granules according to claim 1, characterized in that, Grind the Astragalus extract granules to be tested in step 2 into a fine powder and pass them through a No. 3 sieve.
4. The method for determining the astragaloside A content in Astragalus extract granules according to claim 1, characterized in that, In step 2, the heating and reflux time is 0.5 to 2 hours, preferably 1 to 2 hours.
5. The method for determining the astragaloside A content in Astragalus extract granules according to claim 1, characterized in that, The volume of the filtrate measured in step 2 is 20-50 mL.
6. The method for determining the astragaloside A content in Astragalus extract granules according to claim 1, characterized in that, In step 2, dissolve the residue in 20-50 mL of water, and extract with water-saturated n-butanol 2-6 times, 20-50 mL each time.
7. The method for determining the astragaloside A content in Astragalus extract granules according to claim 1, characterized in that, The volume transferred to the volumetric flask in step 2 is 5–10 mL.