A method for constructing HPLC content determination of pumpkin stem medicinal materials, decoction pieces, standard decoction and formula granules

Abstract

The application provides a kind of pumpkin stem medicinal materials, decoction piece, standard decoction and its formula granule HPLC content determination construction method, comprising: A) pumpkin stem raw material is extracted using solvent, to obtain the test solution;Vanillic acid control sample is dissolved using 70% ethanol, to obtain reference solution;B) the test solution and reference solution are determined using high performance liquid chromatography, to obtain the chromatogram of test solution and reference solution respectively;And according to the chromatogram of reference, the components of pumpkin stem medicinal materials, decoction piece, standard decoction and its formula granule HPLC content determination are qualitatively and quantitatively determined;The high performance liquid chromatography chromatographic conditions are: the chromatographic column is C18 column;The mobile phase is methanol-0.2% acetic acid.The application uses high performance liquid chromatography, selects methanol-0.2% acetic acid as mobile phase for elution, determines the content of vanillic acid in pumpkin stem medicinal materials, decoction piece, standard decoction and its formula granule HPLC, and has good repeatability, precision, stable and reliable method.

Description

Technical Field

[0001] This invention relates to the field of analytical testing technology, and in particular to a method for constructing an HPLC content determination method for pumpkin stem medicinal materials, decoction pieces, standard decoctions and their formulation granules. Background Technology

[0002] Pumpkin stem is the dried stem of *Cucurbita moschata* (Duch. ex Lam.) Duch. ex Poir., a plant in the Cucurbitaceae family. It is harvested after the pumpkin is ripe and then dried. It has the functions of harmonizing the stomach, replenishing qi, calming the fetus, detoxifying, and astringing sores. It is used for stomach deficiency with qi stagnation, threatened abortion, and carbuncles. The standard decoction is a freeze-dried powder made from the medicinal material according to a fixed preparation process.

[0003] To ensure the uniformity and stability of the quality of pumpkin stem medicinal materials, decoction pieces, and their standard decoctions, this paper establishes a new content determination method to control their quality. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for determining the content of pumpkin stem medicinal materials, decoction pieces, standard decoctions and their formulation granules by HPLC. The method for determining the content of pumpkin stem medicinal materials, decoction pieces, standard decoctions and their formulation granules constructed by the present invention is stable and reliable, and can control the quality of vanillic acid in pumpkin stem medicinal materials, decoction pieces, standard decoctions and their formulation granules.

[0005] This invention provides a method for constructing an HPLC content determination method for pumpkin stem medicinal materials, processed slices, standard decoctions, and their formulation granules, comprising:

[0006] A) Extract the pumpkin stem material with a solvent to obtain the test solution;

[0007] Vanillic acid reference standard was dissolved in 70% ethanol to obtain a reference solution;

[0008] B) The test solution and the reference solution were determined by high performance liquid chromatography, and the chromatograms of the test solution and the reference solution were obtained respectively; and the components of pumpkin stem medicinal material, decoction pieces, standard decoction and its formula granules were qualitatively and quantitatively determined by HPLC based on the chromatogram of the reference solution.

[0009] The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase is methanol-0.2% acetic acid.

[0010] The present invention provides a method for determining the content of pumpkin stem medicinal material, decoction pieces, standard decoction and its formula granules by HPLC. First, pumpkin stem raw material is taken, extracted with solvent to obtain the test solution.

[0011] According to the present invention, the raw material of pumpkin stem is pumpkin stem medicinal material, decoction pieces, standard decoction and its formula granules.

[0012] When the raw material of pumpkin stem is standard decoction or formula granules of pumpkin stem, the preparation of the test solution specifically includes: mixing the standard decoction or formula granules of pumpkin stem with water, ultrasonic treatment, cooling, shaking, and filtering to obtain the solution.

[0013] The mass ratio of pumpkin stem material to water was 0.5g:20mL; the ultrasonic power was 600W, the frequency was 40kHz, and the ultrasonic time was 30min.

[0014] The above extraction method and time can fully extract vanillic acid. Furthermore, the inventors discovered that water as a solvent has a higher extraction efficiency compared to other organic solvents. Under the above ratio, the target component is extracted more completely. When the pumpkin stem raw material is pumpkin stem medicinal material or processed slices, the preparation of the test solution specifically includes: mixing the pumpkin stem medicinal material or processed slices with water, heating the water flow, cooling, shaking well, and filtering to obtain the final product.

[0015] The mass ratio of the pumpkin stem raw material to water is 2g:20mL; the heating and reflux time is 30min.

[0016] The extraction method described above provides good chromatographic peak separation, and compared with ultrasonic extraction, reflux extraction yields a higher vanillic acid content.

[0017] The pumpkin stem raw material can be pumpkin stem medicinal material, pumpkin stem slices, or decoction. This invention does not limit its use; all of the above raw materials can be subjected to quality control and qualitative testing using the methods of this invention.

[0018] Vanillic acid reference standard was dissolved in 70% ethanol to obtain a reference solution.

[0019] When the injection concentration of the vanillic acid reference standard described in this invention is in the range of 0.3514956–351.4956 μg / ml, the linear relationship is y = 38549x + 78.516, R0. 2 =1.

[0020] The limit of detection for vanillic acid described in this invention is 0.3514956 μg / ml, and the limit of quantitation is 0.3514956–351.4956 μg / ml.

[0021] The test solution and the reference solution were determined by high performance liquid chromatography (HPLC), and chromatograms of the test solution and the reference solution were obtained respectively. Based on the chromatogram of the reference solution, the components of pumpkin stem medicinal material, decoction pieces, standard decoction and its formula granules were qualitatively and quantitatively determined by HPLC.

[0022] The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase is methanol-0.2% acetic acid.

[0023] The mobile phase of this invention is methanol-0.2% acetic acid. The volume ratio of methanol to 0.2% acetic acid is 15:85.

[0024] The present invention provides good baseline separation, abundant chromatographic peak information, good resolution, moderate elution time, and stable baseline under the above-mentioned mobile phase.

[0025] C18 column, 5μm, 4.6×250mm; column temperature 30℃.

[0026] The preferred chromatographic columns of this invention are: Agilent C18 4.6×250mm, 5μm, Shimadzu C18 4.6×250mm, 5μm, and Waters C18 4.6×250mm, 5μm.

[0027] The chromatographic column of this invention exhibits symmetrical peaks, good resolution, and moderate elution time under the above-mentioned 30℃ conditions.

[0028] The preferred flow rate of the mobile phase is 1.0 ml / min.

[0029] This invention found that at a flow rate of 1.0 ml / min, the chromatographic peaks were well separated, the peak shapes were relatively symmetrical, and the resolution was moderate. Based on the comprehensive theoretical plate number, resolution, and peak shape, a flow rate of 1.0 ml / min was selected as the optimal solution.

[0030] The preferred detection wavelength of this invention is 260 nm.

[0031] The inventors have discovered that the chromatographic information at 260 nm is abundant, the chromatographic peak information is large, vanillic acid has good absorption, the response value is moderate, the separation is good, and the baseline is stable.

[0032] The preferred injection volume in this invention is 10 μL.

[0033] This invention provides a method for determining the content of pumpkin stem medicinal materials, processed slices, standard decoctions, and their formulated granules by HPLC. The method includes: A) extracting the pumpkin stem raw material with a solvent to obtain a test solution; dissolving vanillic acid reference standard in 70% ethanol to obtain a reference solution; B) determining the content of the test solution and the reference solution by high-performance liquid chromatography (HPLC), obtaining chromatograms of the test solution and the reference solution respectively; and qualitatively and quantitatively determining the components in the pumpkin stem medicinal materials, processed slices, standard decoctions, and their formulated granules by HPLC based on the chromatograms of the reference solution. The HPLC conditions are: a C18 column; and a mobile phase of methanol-0.2% acetic acid. This invention uses HPLC with methanol-0.2% acetic acid as the mobile phase for elution to determine the vanillic acid content in pumpkin stem medicinal materials, processed slices, standard decoctions, and their formulated granules by HPLC. The method exhibits good repeatability and precision, and is stable and reliable. Attached Figure Description

[0034] Figure 1 Investigation of different mobile phases;

[0035] Figure 2 Vanillic acid UV absorption spectrum;

[0036] Figure 3 Column temperature investigation results;

[0037] Figure 4 Flow velocity study results;

[0038] Figure 5 Specificity assessment;

[0039] Figure 6 Vanillic acid standard curve;

[0040] Figure 7 Distribution diagram of vanillic acid content in standard pumpkin stem decoction;

[0041] Figure 8 Example 2: Full wavelength scan results; Figure 9 Example 2: Column temperature investigation results;

[0042] Figure 10 Example 2: Flow rate investigation results;

[0043] Figure 11 Example 2: Specificity investigation;

[0044] Figure 12 Example 2: Vanillic acid standard curve;

[0045] Figure 13 Example 2: Investigation using different instruments;

[0046] Figure 14 Example 2: Results of column robustness study;

[0047] Figure 15 Example 3: Column temperature investigation results;

[0048] Figure 16 Example 3: Flow rate investigation results;

[0049] Figure 17 Example 3: Specificity investigation;

[0050] Figure 18 Example 3: Vanillic acid standard curve;

[0051] Figure 19 Example 3: Investigation using different instruments;

[0052] Figure 20 Example 3: Results of column robustness test;

[0053] Figure 21 The images show the results of the medicinal material testing for pumpkin stems; the top image is an overall view, and the bottom image is a magnified view.

[0054] Figure 22 The image shows the test results of the standard decoction of pumpkin stems. The top image is the overall view, and the bottom image is a magnified view.

[0055] Figure 23 The images show the test results of the pumpkin stem formulation granules; the top image is the overall view, and the bottom image is a magnified view.

[0056] Figure 24 This is the chromatogram of vanillic acid standard. Detailed Implementation

[0057] This invention provides a method for determining the content of pumpkin stem medicinal materials, processed slices, standard decoctions, and their formulation granules using HPLC. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0058] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a method for constructing an HPLC content determination method for pumpkin stem medicinal material, decoction pieces, standard decoction and its formulation granules provided by the present invention.

[0059] Example 1: HPLC determination of pumpkin stem standard decoction content

[0060] 1. Experimental instruments and materials

[0061] High-performance liquid chromatographs: Shimadzu LC-20AD, Agilent 1260, Waters e2695 high-performance liquid chromatographs;

[0062] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Ltd.);

[0063] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0064] Ultrasonic cleaner: KQ-600DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0065] Chromatographic columns: Agilent 5C18 4.6×250mm, 5μm, Shimadzu C18 4.6×250mm, 5μm, Waters C18 4.6×250mm, 5μm.

[0066] 2. Reagents and reagents

[0067] Acetonitrile (chromatographic grade, Sigma-Aldrich Shanghai Trading Co., Ltd.), acetic acid (chromatographic grade, Chengdu Kelong Chemical Reagent Co., Ltd.), methanol (analytical grade, Chengdu Kelong Chemical Reagent Co., Ltd.), and water used as the mobile phase were laboratory-prepared ultrapure water; all other components were laboratory-prepared pure water.

[0068] Vanillic acid (China National Institutes for Food and Drug Control, batch number: 110776-201503, content calculated as 99.8%)

[0069] Pumpkin stem standard decoction (prepared by Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch numbers: NGD-BT-230701, NGD-BT-230702, NGD-BT-230703, NGD-BT-230704, NGD-BT-230705, NGD-BT-230706, NGD-BT-230707, NGD-BT-230708, NGD-BT-230709, NGD-BT- 230710, NGD-BT-230711, NGD-BT-230712, NGD-BT-230713, NGD-BT-230714, NGD-BT-230715, NGD- BT-230716, NGD-BT-230717, NGD-BT-230718, NGD-BT-230719, NGD-BT-230720, NGD-BT-230721).

[0070] 3. Determine the experimental conditions

[0071] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol-0.2% acetic acid (15:85) as the mobile phase; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 260 nm. The theoretical plate number, calculated based on the vanillic acid peak, should be no less than 4000.

[0072] Preparation of reference solution: Take an appropriate amount of vanillic acid reference standard, accurately weigh it, and add 70% ethanol solution to prepare a solution containing 7ug of vanillic acid per ml.

[0073] Preparation of the test solution: Take about 0.5g of freeze-dried pumpkin stem standard decoction powder (batch number: NGD--BT-230718), accurately weigh it, place it in a stoppered conical flask, accurately add 20ml of water, seal tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with water, shake well, filter it, and take the filtrate to obtain the test solution.

[0074] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0075] 4. Selection of chromatographic conditions and system suitability assessment

[0076] 4.1 Selection of mobile phase

[0077] Based on the above-established experimental conditions, the effects of methanol-0.2% acetic acid, methanol-0.2% phosphoric acid, methanol-0.2% formic acid, and acetonitrile-0.2% acetic acid were investigated respectively. The results are shown in the table below. Figure 1 . Figure 1 Different mobile phases were investigated; the results showed that when the mobile phase was methanol-0.2% acetic acid, the vanillic acid chromatographic peak had a large amount of information, good resolution, and a moderate elution time. The mobile phase was tentatively set as methanol-0.2% acetic acid.

[0078] 4.2 Determination of detection wavelength

[0079] Based on the above-specified experimental conditions, vanillic acid was scanned across the entire wavelength range using a diode array detector. (See...) Figure 2 . Figure 2 The ultraviolet absorption spectrum of vanillic acid was analyzed. The results showed that the optimal detection wavelength for determining the vanillic acid content in the standard decoction of pumpkin stem was 260 nm.

[0080] 4.3 Column Temperature Investigation

[0081] Based on the above-specified experimental conditions, column temperatures of 25℃, 30℃, and 35℃ were investigated, with vanillic acid separation degree and theoretical plate number used as evaluation indicators. Results are shown below. Figure 3 Table 1. Figure 3 Column temperature test results.

[0082] Table 1. Analytical results at different column temperatures

[0083]

[0084] The results showed that vanillic acid could be effectively detected at column temperatures ranging from 25℃ to 35℃, and 30℃ was selected as the detection column temperature.

[0085] 4.4 Flow velocity investigation

[0086] Based on the above-established experimental conditions, flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were investigated, with vanillic acid separation degree and theoretical plate number used as evaluation indicators. Results are shown below. Figure 4 Table 2. Figure 4 Results of flow velocity study.

[0087] Table 2 Analysis results at different flow velocities

[0088]

[0089]

[0090] The results showed that vanillic acid could be effectively detected at a flow rate of 1.0 ml / min, and the flow rate was finally determined to be 1.0 ml / min.

[0091] In summary, the chromatographic conditions and system suitability test for the determination of the content in the pumpkin stem standard decoction were determined as follows: Octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol-0.2% acetic acid (15:85) as the mobile phase; flow rate 1.0 ml / min; column temperature 30℃; detection wavelength 260 nm. The theoretical plate number, calculated based on the vanillic acid peak, should be no less than 4000.

[0092] 5. Investigation on the preparation of the test solution

[0093] 5.1 Examination of Extraction Methods

[0094] Accurately weigh approximately 0.5g of freeze-dried pumpkin stem standard decoction powder (batch number: NGD--BT-230718), place it in a stoppered conical flask, accurately add 20ml of 70% ethanol solution, weigh again, seal tightly, and investigate both reflux and ultrasonic extraction methods. Extraction time is 30 minutes for both. After cooling, weigh again, replenish the lost weight with 70% ethanol, shake well, filter, and collect the filtrate. The vanillic acid content under different extraction methods is analyzed and calculated; the results are shown in Table 36.

[0095] Table 3. Analysis results of different extraction methods

[0096]

[0097] The results showed that the vanillic acid contents obtained by reflux extraction and ultrasonic extraction were 0.27 mg / g and 0.26 mg / g, respectively. The efficiency of reflux extraction was not significantly different from that of ultrasonic extraction, and ultrasonic extraction was simpler. Therefore, ultrasonic extraction was selected as the extraction method.

[0098] 5.2 Examination of extraction time

[0099] Accurately weigh approximately 0.5 g of the powder (batch number: NGD--BT-230718), place it in a stoppered conical flask, accurately add 20 ml of 70% ethanol solution, seal tightly, and weigh. Extract using ultrasound for 20 minutes, 30 minutes, and 60 minutes respectively. After cooling, weigh again, replenish the lost weight with 70% ethanol, shake well, filter, and collect the filtrate. Analyze and calculate the vanillic acid content at different extraction times; the results are shown in Table 38.

[0100] Table 4. Analysis results of different extraction times

[0101]

[0102] The results showed that sufficient extraction was achieved when the extraction time was 30 minutes. Therefore, the extraction time for the test sample was determined to be 30 minutes.

[0103] 5.3 Investigation of Extraction Solvents

[0104] Accurately weigh approximately 0.5 g of the powder (batch number: NGD--BT-230718) and place it in a stoppered conical flask. Extract with 30% ethanol, 50% ethanol, 70% ethanol, ethanol, methanol, and water respectively. Accurately add 20 ml of each solvent, seal tightly, weigh, and sonicate (600 W, 40 kHz) for 30 minutes. Cool, weigh again, and replenish the lost weight with the corresponding solvent. Shake well, filter, and collect the filtrate. Analyze and calculate the vanillic acid content under different solvent extractions; the results are shown in Table 37.

[0105] Table 5 Results of the investigation of different extraction solvents

[0106]

[0107]

[0108] The results showed that water extraction efficiency was high, so aqueous solution was used as the extraction solvent.

[0109] 5.4 Investigation of Solvent Addition Amount

[0110] Accurately weigh approximately 0.5 g of the powder (batch number: NGD--BT-230718) and place it in a stoppered conical flask. Accurately add 15 ml, 20 ml, and 50 ml of water respectively, seal tightly, weigh, and sonicate (600 W, 40 kHz) for 30 minutes. Cool, weigh again, replenish the lost weight with water, shake well, filter, and collect the filtrate. Analyze and calculate the vanillic acid content under different solvent addition amounts; the results are shown in Table 39.

[0111] Table 6 Results of the investigation of different solvent addition amounts

[0112]

[0113] The results showed that when the sample size was 0.5g and the solvent volume was 20ml, the target component could be completely extracted. Therefore, the solvent volume was determined to be 20ml.

[0114] In summary, the preparation method for determining the content of pumpkin stem standard decoction is as follows: Take about 0.5g of the powder, accurately add 20ml of water, seal tightly, weigh, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, weigh again, replenish the lost weight with water, shake well, and the sample is ready.

[0115] 5.5 Method for determining the content of pumpkin stem in standard decoction

[0116] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol-0.2% acetic acid solution (15:85) as the mobile phase; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 260 nm. The theoretical plate number, calculated based on the vanillic acid peak, should be no less than 4000.

[0117] Preparation of the reference solution: Take an appropriate amount of vanillic acid reference standard, accurately weigh it, and add 70% ethanol to prepare a solution containing 7 μg per ml.

[0118] Preparation of the test solution: Take 0.5g of freeze-dried powder of pumpkin stem standard decoction, accurately weigh it, place it in a stoppered conical flask, accurately add 20ml of water, seal tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with water, shake well, filter it, and take the filtrate to obtain the test solution.

[0119] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0120] 6. Methodological Examination

[0121] 6.1 Specificity Experiment

[0122] Preparation of the test solution: Prepare the pumpkin stem standard decoction test solution according to the experimental conditions proposed above.

[0123] Preparation of reference solution: Take an appropriate amount of vanillic acid reference standard, accurately weigh it, and add 70% ethanol to prepare a solution containing 7 μg per ml.

[0124] Preparation of negative control solution: Prepare a negative control solution for the standard decoction lacking the pumpkin stem according to the experimental conditions outlined above. See the results below. Figure 5 . Figure 5 Specificity assessment.

[0125] The results showed that the negative solution chromatogram did not interfere with the determination of the target peak, indicating that the method has good specificity.

[0126] 6.2 Precision test

[0127] The reference solution was injected six times consecutively, and the peak area of ​​vanillic acid was recorded. The RSD value was calculated, and the results are shown in Table 7.

[0128] Table 7 Precision test results

[0129]

[0130] The results showed that the peak area RSD of vanillic acid in the precision study was 0.9%. The injection precision of this method is good.

[0131] 6.3 Linear Relationship

[0132] Take an appropriate amount of vanillic acid and place it in a 10 ml volumetric flask. Dissolve it in 70% ethanol to prepare a stock solution containing 351.4956 μg / ml vanillic acid (99.8% purity). Then dilute to concentrations of 21.089736 μg / ml, 14.059824 μg / ml, 7.029912 μg / ml, 0.7029912 μg / ml, and 0.3514956 μg / ml. Accurately pipette 10 μl of each solution into the liquid chromatograph and obtain the peak area. Plot the response curve with the concentration of vanillic acid (X, μg / ml) as the x-axis and the peak area (Y) as the y-axis. The results are shown in Table 8. Figure 6 . Figure 6 Vanillic acid standard curve.

[0133] Table 8. Results of vanillic acid standard curve analysis

[0134]

[0135] The results showed that when the vanillic acid concentration ranged from 0.3514956 to 351.4956 μg / ml, the linear relationship was y = 38549x + 78.516, R0.2 =1. This indicates a good linear relationship when the concentration range is 0.3514956 to 351.4956 μg / ml.

[0136] 6.4 Repeatability Experiment

[0137] Take about 0.5g of this product powder (batch number: NGD--BT-230718), accurately weigh 6 portions, and have the same operator prepare the test solution according to the established method. Measure the peak area and calculate the vanillic acid content of the 6 test samples. The results are shown in Table 9.

[0138] Table 9 Results of Repeatability Experiments

[0139]

[0140] The RSD value of vanillic acid content obtained from 6 repeatability tests was 2.0%, indicating that the method has good repeatability.

[0141] 6.5 Stability Test

[0142] Take the same test sample (batch number: NGD-BT-230718) solution and determine the peak area of ​​vanillic acid at 0h, 3h, 6h, 9h, 15h and 24h respectively. The results are shown in Table 10.

[0143] Table 10 Stability test results

[0144]

[0145] The results showed that the RSD value of the vanillic acid peak area was 1.4%, and the test solution had good stability within 24 hours.

[0146] 6.6 Intermediate Precision

[0147] 6.6.1 Investigations by different personnel and at different times

[0148] The same test sample (batch number: NGD--BT-230718) was used, and different personnel (A, B) prepared test sample solutions at different times (I, II) for analysis. The content of vanillic acid in the test sample was calculated, and the results are shown in Table 11.

[0149] Table 11 Results of intermediate precision experiments

[0150]

[0151] The results showed that the RSD value of vanillic acid was 0.3%, indicating good intermediate precision of the method.

[0152] 6.7 Recovery rate

[0153] Take approximately 0.25 g of the test sample (batch number: NGD--BT-230718, vanillic acid content 0.29 mg / g), a total of 6 portions, accurately weigh them, and accurately add 25 ml of a control solution containing vanillic acid (purity 98.0%) to each portion. Prepare and determine the test solution according to the prescribed method, calculate the recovery rate, and the results are shown in Table 12. The calculation formula is as follows:

[0154]

[0155]

[0156] Table 12 Results of Vanillic Acid Recovery Experiment

[0157]

[0158] The results showed that the average recovery rate of vanillic acid was 98.7%, and the method had good accuracy.

[0159] 6.8 Durability Test

[0160] 6.8.1 Investigation with different instruments

[0161] Take approximately 0.5 g of the powder (batch number: NGD-BT-230718), accurately weigh two portions, prepare test solutions according to the established method, and investigate on Waters e2695, Agilent Inifity 1260, and Shimadzu high performance liquid chromatographs (all columns were Agilent C18 250mm×4.6mm, 5μm). Calculate the vanillic acid content, and the results are shown in Table 13.

[0162] Table 13 Experimental results using different instruments

[0163]

[0164] The results showed that the RSD value of the results measured by the Waters e2695, Agilent Inifity 1260, and Shimadzu high performance liquid chromatographs was 1.9%, indicating that the instruments used in this method have good durability.

[0165] 6.8.2 Investigation of different chromatographic columns

[0166] Different brands of C18 columns, Agilent C18 4.6*250mm, 5μm, Shimadzu C18 4.6*250mm, 5μm and Waters C18 4.6*250mm, 5μm, were used to detect the same test sample (batch number: NGD-BT-230718). The results are shown in Table 14.

[0167] Table 14 Results of column durability study

[0168]

[0169]

[0170] The results showed that the analytical chromatographic parameters of different chromatographic columns were good.

[0171] 7. Sample content determination verification

[0172] Using the freeze-dried powder of pumpkin stem standard decoction as the test sample, the test sample solution was prepared and measured according to the proposed method. The peak area was recorded, and the vanillic acid content was calculated. The results are shown in Table 15. Figure 7 . Figure 7 Distribution of vanillic acid content in standard pumpkin stem decoction

[0173] Table 15 Vanillic acid content of 21 batches of pumpkin stem standard decoction

[0174]

[0175] The results showed that the measured vanillic acid content in the pumpkin stem standard decoction ranged from 0.18 mg / g to 0.60 mg / g, with an average total content of 0.32 mg / g and a SD value of 0.12. The 70%-130% limit of the total content was 0.22-0.41 mg / g of vanillic acid (C8H8O4). The limit range of the average plus or minus three times the SD value was -0.06-0.69 mg / g of vanillic acid (C8H8O4), and the limit range of the average plus or minus two times the SD value was 0.069-0.57 mg / g of vanillic acid (C8H8O4). Therefore, the lower limit was determined by rounding the average value to 2SD (0.07 mg / g), and the upper limit was determined by adding three times the SD value (0.69 mg / g). The vanillic acid (C8H8O4) content of this product is tentatively set at 0.07 mg / g-0.69 mg / g, calculated on a dried basis.

[0176] In summary, the content determination method is as follows:

[0177] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol-0.2% acetic acid solution (15:85) as the mobile phase; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 260 nm. The theoretical plate number, calculated based on the vanillic acid peak, should be no less than 4000.

[0178] Preparation of the reference solution: Take an appropriate amount of vanillic acid reference standard, accurately weigh it, and add 70% ethanol to prepare a solution containing 7 μg per ml.

[0179] Preparation of the test solution: Take about 0.5g of the powder of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 20ml of water, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the weight loss with water, shake well, filter it, and take the filtrate to obtain the test solution.

[0180] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0181] This product contains 0.07mg to 0.69mg of vanillic acid (C8H8O4) per 1g.

[0182] Example 2: HPLC content determination of pumpkin stem medicinal materials and processed slices

[0183] 1. Experimental instruments and materials: Same as in Example 1

[0184] 2. Reagents and reagents are the same as in Example 1.

[0185] Pumpkin stem medicinal material batch numbers: XLS202109204, XLS202109205, XLS202109206, XLS202109207, XLS202109208, XLS202109209, XLS202109210, XLS202109211, XLS202109212, XLS202109213, XLS 202109214, XLS202109215, XLS202109216, XLS202109217, XLS202109218, 9219, XLS202109220, XLS202109221, XLS202109222,

[0186] Pumpkin stem slices batch number: NGD-230701, NGD-230702, NGD-230703, NGD-230704, NGD-230705, NGD-230706, NGD-230707, NGD-230708, NGD-230709, NGD-2307010, N GD-230711, NGD-230712, NGD-230713, NGD-230714, NGD-230715, NGD-230716, NGD-230717, NGD-230718, NGD-230719, NGD-230720, NGD-230721.

[0187] 3. Proposed chromatographic conditions

[0188] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol-0.2% acetic acid (15:85) as the mobile phase; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 260 nm. The theoretical plate number, calculated based on the vanillic acid peak, should be no less than 4000.

[0189] Preparation of the reference solution: Take an appropriate amount of vanillic acid reference standard, accurately weigh it, and add 70% ethanol solution to prepare a solution containing 7 μg of vanillic acid per 1 ml.

[0190] Preparation of the test solution: Take about 2g of pumpkin stem powder (XLS202109221) (passed through No. 3 sieve), weigh it accurately, place it in a stoppered conical flask, add 20ml of water accurately, stopper tightly, weigh it, reflux for 30 minutes, cool it, weigh it again, make up the lost weight with water, shake well, and the test solution is ready.

[0191] The assay involves precisely pipetting 10 μL each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0192] 4. Selection of chromatographic conditions and system suitability assessment

[0193] 4.1 Wavelength Selection

[0194] Based on the above-specified experimental conditions, a full-band scan of the vanillic acid reference solution was performed using a diode array detector. The results are as follows. Figure 8 : Figure 8 Example 2: Full wavelength scan results; The results show that the vanillic acid chromatographic peak has a large amount of information at a detection wavelength of 260 nm, so the detection wavelength was determined to be 260 nm.

[0195] 4.2 Column Temperature Investigation

[0196] Based on the above-specified experimental conditions, column temperatures of 25℃, 30℃, and 35℃ were investigated, with vanillic acid separation degree and theoretical plate number used as evaluation indicators. Results are shown below. Figure 9 Table 16. Figure 9 Example 2: Column temperature investigation results.

[0197] Table 16 Analytical results at different column temperatures

[0198]

[0199] The results showed that vanillic acid could be effectively detected at column temperatures of 25℃ and 30℃, so the column temperature was tentatively set at 30℃.

[0200] 4.3 Flow velocity investigation

[0201] Based on the above-established experimental conditions, flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were investigated, with vanillic acid separation degree and theoretical plate number used as evaluation indicators. Results are shown below. Figure 10 Table 17. Figure 10 Flow rate investigation results in Example 2.

[0202] Table 17 Analysis results at different flow velocities

[0203]

[0204] The results showed that vanillic acid could be effectively detected at flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min, so the flow rate was tentatively set at 1.0 ml / min.

[0205] In summary, the chromatographic conditions and system suitability test for the determination of pumpkin stem particle content were determined as follows: Octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol-0.2% acetic acid (15:85) as the mobile phase; flow rate 1.0 mL / min; column temperature 30℃; detection wavelength 260 nm. The theoretical plate number, calculated based on the vanillic acid peak, should be no less than 4000.

[0206] 5. Investigation on the preparation of the test solution

[0207] 5.1 Examination of Extraction Methods

[0208] Take an appropriate amount of pumpkin stem powder (batch number: XLS202109221), grind it into a fine powder, take about 2.0g, weigh it accurately, place it in a stoppered conical flask, add 20ml of water accurately, seal tightly, weigh it, and conduct two extraction methods: ultrasonic (600W, frequency 40kHz) and reflux extraction. The extraction time is 30 minutes for both. After cooling, weigh it again, make up the weight loss with water, shake well, filter, and take the filtrate to obtain the final product.

[0209] The vanillic acid content under different extraction methods was analyzed and calculated, and the results are shown in Table 18.

[0210] Table 18 Analysis results of different extraction methods

[0211]

[0212] The results showed that the vanillic acid content obtained by reflux extraction was slightly higher than that obtained by ultrasound, so reflux extraction was determined as the extraction method.

[0213] 5.2 Investigation of Extraction Solvents

[0214] Take an appropriate amount of this product (batch number: XLS202109221), grind it into a fine powder, and accurately weigh about 2.0g. Place it in a stoppered conical flask, and accurately add 20ml each of 30% ethanol, 50% ethanol, 70% ethanol, ethanol, methanol, and water. Seal the flask tightly, weigh it, reflux for 30 minutes, cool it, weigh it again, and make up the weight loss with the corresponding solvent. Shake well, filter it, and collect the filtrate. Calculate the vanillic acid content extracted with different solvents; the results are shown in Table 19.

[0215] Table 19 Results of the investigation of different extraction solvents

[0216]

[0217] The results showed that water extraction efficiency was better, so aqueous solution was used as the extraction solvent.

[0218] 5.3 Examination of extraction time

[0219] Take an appropriate amount of this product (batch number: XLS202109221), grind it into a fine powder, accurately weigh about 2.0g, place it in a stoppered conical flask, accurately add 20ml of water, seal tightly, weigh, and reflux for 20 minutes, 30 minutes, and 60 minutes respectively. After cooling, weigh again, replenish the lost weight with water, shake well, filter, and collect the filtrate. The vanillic acid content at different extraction times was analyzed and calculated, and the results are shown in Table 20.

[0220] Table 20 Analysis results for different extraction times

[0221]

[0222]

[0223] The results showed that sufficient extraction was achieved when the extraction time was 30 minutes. Therefore, the extraction time for the test sample was determined to be 30 minutes.

[0224] 5.4 Investigation of Solvent Addition Amount

[0225] Take an appropriate amount of this product (batch number: XLS202109221), grind it into a fine powder, accurately weigh about 0.5g, place it in a stoppered conical flask, and accurately add 15ml, 20ml, and 50ml of water respectively. Seal the flask tightly, weigh it, and sonicate it (power 400W, frequency 40kHz) for 30 minutes. After cooling, weigh it again, replenish the lost weight with water, shake well, filter, and collect the filtrate. Examine the extraction efficiency of the solvent addition amount. Analyze and calculate the vanillic acid content at different extraction times. The results are shown in Table 21.

[0226] Table 21 Results of the investigation of different solvent addition amounts

[0227]

[0228] The results showed that when the sample size was 0.5g and the solvent added was 20ml, the target component could be completely extracted. Therefore, the amount of water added was determined to be 20ml.

[0229] In summary, the preparation method for determining the content of pumpkin stem granules is as follows: Take an appropriate amount of this product, grind it into a fine powder, take about 0.5g, accurately weigh it, place it in a stoppered conical flask, accurately add 20ml of water, weigh it, sonicate it (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with water, shake it well, filter it, and take the filtrate to obtain the product.

[0230] 5.5 Method for determining the medicinal content of pumpkin stem

[0231] Chromatographic conditions and system suitability: Octadecylsilane-bonded silica gel was used as the stationary phase (250 mm column length, 4.6 mm inner diameter, 5 μm particle size); methanol-0.2% acetic acid (15:85) was used as the mobile phase; the flow rate was 1.0 mL / min; the column temperature was 30 °C; and the detection wavelength was 260 nm. The theoretical plate number, calculated based on the vanillic acid peak, should be no less than 4000.

[0232] Preparation of the reference solution: Take an appropriate amount of vanillic acid reference standard, accurately weigh it, and add 70% ethanol solution to prepare a solution containing 7 μg of vanillic acid per ml.

[0233] Preparation of the test solution: Take about 2g of pumpkin stem powder (batch number: XLS202109221) (passed through No. 3 sieve), weigh accurately, place it in a stoppered conical flask, add 20ml of water accurately, stopper tightly, weigh, reflux for 30 minutes, cool, weigh again, replenish the lost weight with water, shake well, and the test solution is ready.

[0234] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0235] 6. Methodological Examination

[0236] 6.1 Specificity Experiment

[0237] Preparation of the test solution: Prepare the pumpkin stem test solution according to the experimental conditions proposed above.

[0238] Preparation of reference solution: Prepare vanillic acid reference solution according to the experimental conditions proposed above.

[0239] Preparation of negative control solution: A negative control solution for pumpkin stemless medicinal material was prepared according to the experimental conditions outlined above. The results are shown below. Figure 11 . Figure 11 Example 2: Specificity investigation.

[0240] The results showed that the negative solution chromatogram did not interfere with the determination of the target peak, indicating that the method has good specificity.

[0241] 6.2 Precision test

[0242] The reference solution was injected six times consecutively, and the peak area of ​​vanillic acid was recorded. The RSD value was calculated, and the results are shown in Table 22.

[0243] Table 22 Precision test results

[0244]

[0245] The results showed that the peak area RSD of vanillic acid in the precision study was 0.9%. The injection precision of this method is good.

[0246] 6.3 Linear Relationship

[0247] Take an appropriate amount of vanillic acid and place it in a 10 ml volumetric flask. Dissolve it in 70% ethanol to prepare a stock solution containing 351.4956 μg / ml vanillic acid (99.8% purity). Then dilute to concentrations of 21.089736 μg / ml, 14.059824 μg / ml, 7.029912 μg / ml, 0.7029912 μg / ml, and 0.3514956 μg / ml. Accurately pipette 10 μl of each solution and inject it into the liquid chromatograph. Obtain the peak area. Plot the response curve with the concentration of vanillic acid (X, μg / ml) as the x-axis and the peak area (Y) as the y-axis. The results are shown in Table 23. Figure 12 . Figure 12 Example 2: Vanillic acid standard curve.

[0248] Table 23. Results of vanillic acid standard curve analysis

[0249]

[0250] The results showed that when the vanillic acid concentration ranged from 0.3514956 to 351.4956 μg / ml, the linear relationship was y = 38549x + 78.516, R0. 2 =1. This indicates a good linear relationship when the concentration range is 0.3514956 to 351.4956 μg / ml.

[0251] 6.4 Repeatability Experiment

[0252] Take an appropriate amount of this product (batch number: XLS202109221), grind it into a fine powder, take 2.0g, accurately weigh 6 portions, and have the same operator prepare the test solution according to the above-mentioned method. Calculate the vanillic acid content of the 6 test samples. The results are shown in Table 24.

[0253] Table 24 Results of Repeatability Tests

[0254]

[0255] The RSD value of vanillic acid content obtained from 6 repeatability tests was 2.9%, indicating that the method has good repeatability.

[0256] 6.5 Stability Test

[0257] Take the same test solution (batch number: XLS202109221) and determine the peak area of ​​vanillic acid at 0h, 3h, 6h, 9h, 15h and 24h respectively. The results are shown in Table 25.

[0258] Table 25 Stability test results

[0259]

[0260] The results showed that the RSD value of the vanillic acid peak area was 1.0%, and the test solution had good stability within 24 hours.

[0261] 6.6 Intermediate Precision

[0262] 6.6.1 Investigations by different personnel and at different times

[0263] The same test sample (batch number: XLS202109221) was prepared into test solution by different personnel (A, B) at different times (Ⅰ, Ⅱ) for analysis. The content of vanillic acid in the test sample was calculated, and the results are shown in Table 26.

[0264] Table 26 Results of intermediate precision experiments

[0265]

[0266] The results showed that the RSD value of vanillic acid was 5.5%, indicating that the intermediate precision of this method was good.

[0267] 6.7 Recovery rate

[0268] Take approximately 1 g of the test sample (batch number: XLS202109221, vanillic acid content 0.0010%), totaling 6 portions, accurately weigh them, and accurately add 20 ml of a reference stock solution containing vanillic acid (purity 99.8%, concentration 0.04477 μg / ml) to each portion. Prepare and determine the test solution according to the prescribed method, calculate the recovery rate, and the results are shown in Table 27. The calculation formula is as follows:

[0269]

[0270] Table 27 Results of Vanillic Acid Recovery Experiment

[0271]

[0272] The results showed that the average recovery rate of vanillic acid was 97.8%, and the method had good accuracy.

[0273] 6.8 Durability Test

[0274] 6.8.1 Investigation with different instruments

[0275] Prepare the test solution according to the proposed method. Take an appropriate amount of this product (batch number: XLS202109221), grind it finely, and accurately weigh about 2.0 g. Analyze the sample using a Waters, Shimadzu, or Agilent high-performance liquid chromatograph (all columns are Agilent C18 250×4.6 mm, 5 μm). Calculate the vanillic acid content. The results are shown in the figure. Figure 13 Table 28. Figure 13 Example 2: Investigation using different instruments.

[0276] Table 28 Experimental Results with Different Instruments

[0277]

[0278] The results showed that the RSD value of the results measured by Agilent, Shimadzu, and Waters high performance liquid chromatographs was 5.4%, indicating that the instruments used in this method have good durability.

[0279] 6.8.2 Investigation of different chromatographic columns

[0280] Different brands of C18 chromatographic columns—Agilent C18 4.6*250mm, 5μm, Shimadzu C18 4.6*250mm, 5μm, and Waters C18 4.6*250mm, 5μm—were used to detect the same test sample (batch number: 010634-2305001). The results are shown in the table. Figure 14 Table 29. Figure 14 Example 2: Results of column durability study.

[0281] Table 29 Results of column robustness test

[0282]

[0283] The results showed that the RSD of vanillic acid content in different chromatographic columns was 4.2%, indicating good analytical performance.

[0284] In summary, the method for determining the medicinal content of pumpkin stem is as follows:

[0285] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the stationary phase; methanol-0.2% acetic acid (15:85) as the mobile phase; a flow rate of 1.0 mL / min; a column temperature of 30 °C; and a detection wavelength of 260 nm. The theoretical plate number, calculated based on the vanillic acid peak, should be no less than 4000.

[0286] Preparation of the reference solution: Take an appropriate amount of vanillic acid reference standard, accurately weigh it, and add 70% ethanol solution to prepare a solution containing 7 μg of vanillic acid per 1 ml.

[0287] Preparation of the test solution: Take about 2g of pumpkin stem powder (batch number: XLS202109221) (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 20ml of water, seal tightly, weigh it, heat under reflux for 30 minutes, cool it, weigh it again, replenish the lost weight with water, shake well, filter, and take the filtrate to obtain the test solution.

[0288] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0289] 7. Results of the determination of the medicinal content of pumpkin stem

[0290] The results of testing 21 batches of pumpkin stem medicinal materials are shown in Table 30.

[0291] Table 30 Results of determination of pumpkin stem medicinal material content

[0292]

[0293] The vanillic acid content of the 21 batches of pumpkin stem medicinal materials tested, calculated on a dried basis, ranged from 0.0010% to 0.0022%, with an average of 0.0015%. The range of the average ±30% was 0.0011% to 0.0020%, and the range of the average ±3SD was 0.0004% to 0.0026%. Therefore, the lower limit of the content was set at the average -3SD, i.e., 0.0004%. Thus, the standard for pumpkin stem medicinal materials is tentatively set as "the vanillic acid (C8H8O4) content of this product, calculated on a dried basis, shall not be less than 0.0004%".

[0294] 8. Determination of the method for determining the content of pumpkin stem slices

[0295] The detection method for pumpkin stem slices is the same as the method for [content determination] under the section on pumpkin stem medicinal materials:

[0296] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol-0.2% acetic acid (15:85) as the mobile phase; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 260 nm. The theoretical plate number, calculated based on the vanillic acid peak, should be no less than 4000.

[0297] Preparation of the reference solution: Take an appropriate amount of vanillic acid reference standard, accurately weigh it, and add 70% ethanol solution to prepare a solution containing 7 μg of vanillic acid per 1 ml.

[0298] Preparation of the test solution: Take about 2g of pumpkin stem powder (batch number: XLS202109221) through a No. 3 sieve, place it in a stoppered conical flask, add 20ml of water, stopper tightly, weigh, heat under reflux for 30 minutes, cool, weigh again, make up the lost weight with water, shake well, filter, and take the filtrate to obtain the test solution.

[0299] The assay involves precisely pipetting 10 μL each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0300] 9. Results of the determination of pumpkin stem content in medicinal slices

[0301] The results are shown in Table 31.

[0302] Table 31 Results of Determination of Content in Medicinal Herbs

[0303]

[0304]

[0305] The vanillic acid content of 21 batches of pumpkin stem slices ranged from 0.0009% to 0.0021%, with an average of 0.0014%. The average ±30% ranged from 0.0010% to 0.0018%, and the average ±3SD ranged from 0.0005% to 0.0023%. Therefore, this standard refers to the standard for pumpkin stem medicinal materials, and the standard for pumpkin stem slices is tentatively set as "the vanillic acid (C8H8O4) content of this product, calculated on a dried basis, shall not be less than 0.0004%".

[0306] Example 3: HPLC content determination of pumpkin stem formulation granules

[0307] 1. Experimental instruments and materials: Same as in Example 1

[0308] 2. Reagents and reagents: Same as in Example 1

[0309] Pumpkin stem formula granules batch numbers: 1904033, B2307007, B2307008, B2307009.

[0310] 3. Proposed chromatographic conditions

[0311] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol-0.2% acetic acid (15:85) as the mobile phase; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 260 nm. The theoretical plate number, calculated based on the vanillic acid peak, should be no less than 4000.

[0312] Preparation of reference solution: Take an appropriate amount of vanillic acid reference standard, accurately weigh it, and add 70% ethanol solution to prepare a solution containing 7ug of vanillic acid per ml.

[0313] Preparation of the test solution: Take an appropriate amount of pumpkin stem formula granules (batch number: B2307007), grind them into a fine powder, take about 0.5g, weigh accurately, place in a stoppered conical flask, accurately add 20ml of water, seal tightly, weigh, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, weigh again, replenish the lost weight with water, shake well, and the test solution is ready.

[0314] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0315] 4. Selection of chromatographic conditions and system suitability assessment

[0316] 4.1 Column Temperature Investigation

[0317] Based on the above-specified experimental conditions, column temperatures of 25℃, 30℃, and 35℃ were investigated, with vanillic acid separation degree and theoretical plate number used as evaluation indicators. Results are shown below. Figure 15 Table 32. Figure 15 Example 3: Column temperature investigation results.

[0318] Table 32 Analytical results at different column temperatures

[0319]

[0320] The results showed that vanillic acid could be effectively detected at column temperatures of 25℃ and 30℃, so the column temperature was tentatively set at 30℃.

[0321] 4.2 Flow velocity investigation

[0322] Based on the above-established experimental conditions, flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were investigated, with vanillic acid separation degree and theoretical plate number used as evaluation indicators. Results are shown below. Figure 16 Table 33. Figure 16 Example 3: Flow rate investigation results.

[0323] Table 33 Analysis results for different flow velocities

[0324]

[0325] The results showed that vanillic acid could be effectively detected at flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min, so the flow rate was tentatively set at 1.0 ml / min.

[0326] In summary, the chromatographic conditions and system suitability test for the determination of pumpkin stem particle content were determined as follows: Octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol-0.2% acetic acid (15:85) as the mobile phase; flow rate 1.0 mL / min; column temperature 30℃; detection wavelength 260 nm. The theoretical plate number, calculated based on the vanillic acid peak, should be no less than 4000.

[0327] 5. Investigation on the preparation of the test solution

[0328] 5.1 Examination of Extraction Methods

[0329] Take an appropriate amount of this product (batch number: B2307007), grind it into a fine powder, take about 0.5g, accurately weigh it, place it in a stoppered conical flask, accurately add 20ml of water, seal tightly, weigh it, and conduct two extraction methods: ultrasonic (600W, frequency 40kHz) and reflux extraction. The extraction time is 30 minutes for both. Cool it, weigh it again, make up the weight loss with water, shake well, filter, and take the filtrate to obtain the product.

[0330] The vanillic acid content under different extraction methods was analyzed and calculated, and the results are shown in Table 34.

[0331] Table 34 Analysis results of different extraction methods

[0332]

[0333] The results showed that the vanillic acid content obtained by both ultrasonic and reflux extraction was 0.21 mg / g, and ultrasonic extraction was simpler. Therefore, ultrasonic extraction was selected as the extraction method.

[0334] 5.2 Investigation of Extraction Solvents

[0335] Take an appropriate amount of this product (batch number: 1904033), grind it into a fine powder, accurately weigh about 0.5g, place it in a stoppered conical flask, and accurately add 20ml each of 30% ethanol, 50% ethanol, 70% ethanol, ethanol, methanol, and water. Seal the flask tightly, weigh it, and sonicate it (600W power, 40kHz frequency) for 30 minutes. After cooling, weigh it again, replenish the lost weight with the corresponding solvent, shake well, filter, and collect the filtrate. Calculate the vanillic acid content extracted with different solvents; the results are shown in Table 35.

[0336] Table 35 Results of the investigation of different extraction solvents

[0337]

[0338] The results showed that water extraction efficiency was better, so aqueous solution was used as the extraction solvent.

[0339] 5.3 Examination of extraction time

[0340] Take an appropriate amount of this product (batch number: B2307007), grind it into a fine powder, accurately weigh about 0.5g, place it in a stoppered conical flask, accurately add 20ml of water, seal tightly, weigh, and sonicate (power 600W, frequency 40kHz) for 20 minutes, 30 minutes, and 60 minutes respectively. After cooling, weigh again, replenish the lost weight with water, shake well, filter, and collect the filtrate. Analyze and calculate the vanillic acid content at different extraction times; the results are shown in Table 36.

[0341] Table 36 Analysis results for different extraction times

[0342]

[0343] The results showed that sufficient extraction was achieved when the extraction time was 30 minutes. Therefore, the extraction time for the test sample was determined to be 30 minutes.

[0344] 5.4 Investigation of Solvent Addition Amount

[0345] Take an appropriate amount of this product (batch number: B2307007), grind it into a fine powder, accurately weigh about 0.5g, place it in a stoppered conical flask, and accurately add 15ml, 20ml, and 50ml of water respectively. Seal the flasks tightly, weigh them, and sonicate them (power 400W, frequency 40kHz) for 30 minutes. After cooling, weigh them again, replenish the lost weight with water, shake well, filter, and collect the filtrate. Examine the extraction efficiency based on the amount of solvent added. Analyze and calculate the vanillic acid content at different extraction times. The results are shown in Table 37.

[0346] Table 37 Results of the investigation of different solvent addition amounts

[0347]

[0348] The results showed that when the sample size was 0.5g and the solvent added was 20ml, the target component could be completely extracted. Therefore, the amount of water added was determined to be 20ml.

[0349] In summary, the preparation method for determining the content of pumpkin stem granules is as follows: Take an appropriate amount of this product, grind it into a fine powder, take about 0.5g, accurately weigh it, place it in a stoppered conical flask, accurately add 20ml of water, weigh it, sonicate it (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with water, shake it well, filter it, and take the filtrate to obtain the product.

[0350] 5.5 Method for determining the content of pumpkin stem particles

[0351] Chromatographic conditions and system suitability: Octadecylsilane-bonded silica gel was used as the stationary phase (250 mm column length, 4.6 mm inner diameter, 5 μm particle size); methanol-0.2% acetic acid (15:85) was used as the mobile phase; the flow rate was 1.0 mL / min; the column temperature was 30 °C; and the detection wavelength was 260 nm. The theoretical plate number, calculated based on the vanillic acid peak, should be no less than 4000.

[0352] Preparation of the reference solution: Take an appropriate amount of vanillic acid reference standard, accurately weigh it, and add 70% ethanol solution to prepare a solution containing 7 μg of vanillic acid per ml.

[0353] Preparation of the test solution: Take an appropriate amount of pumpkin stem formula granules (batch number: B2307007), grind them into a fine powder, take about 0.5g, weigh it accurately, place it in a stoppered conical flask, accurately add 20ml of water, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with water, shake well, and the test solution is ready.

[0354] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0355] 6. Methodological Examination

[0356] 6.1 Specificity Experiment

[0357] Preparation of the test solution: Prepare the pumpkin stem granule test solution according to the experimental conditions proposed above.

[0358] Preparation of reference solution: Prepare a pumpkin stem granule reference solution according to the experimental conditions proposed above.

[0359] Preparation of negative control solution: A negative control solution lacking pumpkin stem particles was prepared according to the experimental conditions outlined above. The results are shown below. Figure 17 . Figure 17 Example 3: Specificity investigation.

[0360] The results showed that the negative solution chromatogram did not interfere with the determination of the target peak, indicating that the method has good specificity.

[0361] 6.2 Precision test

[0362] The reference solution was injected six times consecutively, and the peak area of ​​vanillic acid was recorded. The RSD value was calculated, and the results are shown in Table 38.

[0363] Table 38 Precision test results

[0364]

[0365] The results showed that the peak area RSD of vanillic acid in the precision study was 0.9%. The injection precision of this method is good.

[0366] 6.3 Linear Relationship

[0367] Take an appropriate amount of vanillic acid and place it in a 10 ml volumetric flask. Dissolve it in 70% ethanol to prepare a stock solution containing 351.4956 μg / ml vanillic acid (99.8% purity). Then dilute to concentrations of 21.089736 μg / ml, 14.059824 μg / ml, 7.029912 μg / ml, 0.7029912 μg / ml, and 0.3514956 μg / ml. Accurately pipette 10 μl of each solution and inject it into the liquid chromatograph. Obtain the peak area. Plot the response curve with the concentration of vanillic acid (X, μg / ml) as the x-axis and the peak area (Y) as the y-axis. The results are shown in Table 39. Figure 18 . Figure 18 Example 3: Vanillic acid standard curve.

[0368] Table 39. Results of Vanillic Acid Standard Curve Analysis

[0369]

[0370] The results showed that when the vanillic acid concentration ranged from 0.3514956 to 351.4956 μg / ml, the linear relationship was y = 38549x + 78.516, R0. 2 =1. This indicates a good linear relationship when the concentration range is 0.3514956 to 351.4956 μg / ml.

[0371] 6.4 Repeatability Experiment

[0372] Take an appropriate amount of this product (batch number: B2307007), grind it into a fine powder, take 0.5g, accurately weigh 6 portions, and have the same operator prepare the test solution according to the above-mentioned method. Calculate the vanillic acid content of the 6 test samples. The results are shown in Table 40.

[0373] Table 40 Results of Repeatability Experiments

[0374]

[0375] The RSD value of vanillic acid content obtained from 6 repeatability tests was 0.9%, indicating that the method has good repeatability.

[0376] 6.5 Stability Test

[0377] Take the same test solution (batch number: B2307007) and determine the peak area of ​​vanillic acid at 0h, 3h, 6h, 9h, 15h and 24h respectively. The results are shown in Table 41.

[0378] Table 41 Stability test results

[0379]

[0380] The results showed that the RSD value of the vanillic acid peak area was 1.8%, and the test solution had good stability within 24 hours.

[0381] 6.6 Intermediate Precision

[0382] 6.6.1 Investigations by different personnel and at different times

[0383] The same test sample (batch number: B2307007) was prepared into test solution by different personnel (A, B) at different times (I, II) for analysis. The content of vanillic acid in the test sample was calculated, and the results are shown in Table 42.

[0384] Table 42 Results of intermediate precision experiments

[0385]

[0386] The results showed that the RSD value of vanillic acid was 0.3%, indicating good intermediate precision of the method.

[0387] 6.7 Recovery rate

[0388] Take approximately 0.25 g of the test sample (batch number: B2307007, vanillic acid content 0.22 mg / g), a total of 6 portions, accurately weigh them, and accurately add 20 ml of a reference stock solution containing vanillic acid (purity 99.8%, concentration 2.45708 μg / ml) to each portion. Prepare and determine the test solution according to the prescribed method, calculate the recovery rate, and the results are shown in Table 43. The calculation formula is as follows:

[0389]

[0390]

[0391] Table 43 Results of Vanillic Acid Recovery Experiment

[0392]

[0393] The results showed that the average recovery rate of vanillic acid was 98.9%, and the method had good accuracy.

[0394] 6.8 Durability Test

[0395] 6.8.1 Investigation with different instruments

[0396] Prepare the test solution according to the proposed method. Take an appropriate amount of this product (batch number: B2307007), grind it finely, and accurately weigh about 0.5 g. Analyze the sample using a Waters, Shimadzu, or Agilent high-performance liquid chromatograph (all columns are Agilent C18250 × 4.6 mm, 5 μm). Calculate the vanillic acid content. The results are shown in [Figure number missing]. Figure 19 Table 44. Figure 19 Example 3: Investigation using different instruments.

[0397] Table 44 Experimental Results with Different Instruments

[0398]

[0399] The results showed that the RSD value of the results measured by Agilent, Shimadzu, and Waters high performance liquid chromatographs was 3.2%, indicating that the instruments used in this method have good durability.

[0400] 6.8.2 Investigation of different chromatographic columns

[0401] Different brands of C18 columns (Agilent C18 4.6*250mm, 5μm, Shimadzu C18 4.6*250mm, 5μm, and Waters C18 4.6*250mm, 5μm) were used to detect the same test sample (batch number: B2307007). The results are shown in the table. Figure 20 Table 45. Figure 20 Example 3: Results of column durability study.

[0402] Table 45 Results of column durability testing

[0403]

[0404] The results showed that the RSD of vanillic acid content in different chromatographic columns was 3.7%, indicating good analytical performance.

[0405] In summary, the content determination method is as follows:

[0406] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the stationary phase; methanol-0.2% acetic acid (15:85) as the mobile phase; a flow rate of 1.0 mL / min; a column temperature of 30 °C; and a detection wavelength of 260 nm. The theoretical plate number, calculated based on the vanillic acid peak, should be no less than 4000.

[0407] Preparation of the reference solution: Take an appropriate amount of vanillic acid reference standard, accurately weigh it, and add 70% ethanol solution to prepare a solution containing 7 μg of vanillic acid per 1 ml.

[0408] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 0.5g, weigh it accurately, place it in a stoppered conical flask, accurately add 20ml of water, seal it tightly, weigh it, sonicate it (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with water, shake it well, and the test solution is ready.

[0409] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0410] 7. Verification

[0411] Using pumpkin stem granules as the test sample, the established determination method was used to test three batches of process verification samples. The results are shown in Table 46.

[0412] Table 46. Test results of particle content in three batches of process validation formulations.

[0413]

[0414] Conclusion: The method for determining the content of pumpkin stem formulation granules can effectively detect the content of pumpkin stem formulation granules, therefore this method is included in the main text of pumpkin stem formulation granules.

[0415] 7. Determination of the content of pumpkin stem granules in the formula

[0416] Chromatographic conditions and system suitability: Octadecylsilane-bonded silica gel was used as the stationary phase (250 mm column length, 4.6 mm inner diameter, 5 μm particle size); methanol-0.2% acetic acid (15:85) was used as the mobile phase; the flow rate was 1.0 mL / min; the column temperature was 30 °C; and the detection wavelength was 260 nm. The theoretical plate number, calculated based on the vanillic acid peak, should be no less than 4000.

[0417] Preparation of the reference solution: Take an appropriate amount of vanillic acid reference standard, accurately weigh it, and add 70% ethanol solution to prepare a solution containing 7 μg of vanillic acid per ml.

[0418] Preparation of the test solution: Take an appropriate amount of pumpkin stem formula granules (batch number: B2307007), grind them into a fine powder, take about 0.5g, weigh it accurately, place it in a stoppered conical flask, accurately add 20ml of water, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with water, shake well, and the test solution is ready.

[0419] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0420] Comparative Example 1

[0421] Experimental apparatus:

[0422] High performance liquid chromatograph: Agilent 1260 high performance liquid chromatograph, serial number: XLS-HPLC-060;

[0423] Column: Agilent ZORBAX Eclipse Plus C18 4.6mm×150mm, 3.5μm;

[0424] Electronic balances: ME204E, MS205DM, XPR10 (Mettler-Toledo Instruments Ltd.);

[0425] Reference standard: vanillic acid; Source: China National Institutes for Food and Drug Control; Batch number: 110776-201503; Purity: 99.8%.

[0426] Detection wavelength: 270 nm; Mobile phase: 0.2% phosphoric acid solution as mobile phase A, methanol as mobile phase B; Gradient elution program: 0–5 min, 10%–15% B; 5–10 min, 15%–20% B; 10–30 min, 20%–40% B; 30–40 min, 40%–60% B; 40–50 min, 60%–70% B; 50–60 min, 70%–80% B. Column temperature: 30℃; Flow rate: 1.0 ml / min; Injection volume: 10 μl.

[0427] Preparation of the test sample: Take appropriate amounts of pumpkin stem medicinal material, standard decoction freeze-dried powder, and formula granule powder, grind them into a fine powder, take about 1.0g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of 70% ethanol, weigh it, sonicate (power 300W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the weight loss with 70% ethanol, shake it well, filter it, and take the filtrate to obtain the test sample.

[0428] The above methods were used to test the medicinal materials, standard decoctions, and formula granules of pumpkin stems.

[0429] The results are as follows Figures 21-23 As shown, where Figure 21 The images show the results of the medicinal material testing for pumpkin stems; the top image is an overall view, and the bottom image is a magnified view. Figure 22 The image shows the test results of the standard decoction of pumpkin stems. The top image is the overall view, and the bottom image is a magnified view. Figure 23 The images show the test results of the pumpkin stem formulation granules; the top image is the overall view, and the bottom image is a magnified view. Figure 24The above figure shows the chromatogram of vanillic acid standard. As can be seen from the figure, severe peak inclusion occurs in the medicinal material, standard decoction, and formula granules at approximately 15.622 min, indicating that this method cannot effectively separate vanillic acid from pumpkin stems.

[0430] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing HPLC content determination of Cucurbita L. fruit pedicel medicinal material, decoction piece, standard decoction and its formula granules, comprising: A) extracting Cucurbita L. fruit pedicel raw material with solvent to obtain a test solution; when the Cucurbita L. fruit pedicel raw material is a standard decoction or formula granules of Cucurbita L. fruit pedicel, the preparation of the test solution specifically comprises: mixing the standard decoction or formula granules of Cucurbita L. fruit pedicel with water, ultrasonic treatment, cooling, shaking, and filtering to obtain the test solution; when the Cucurbita L. fruit pedicel raw material is a medicinal material or decoction piece of Cucurbita L. fruit pedicel, the preparation of the test solution specifically comprises: mixing the medicinal material or decoction piece of Cucurbita L. fruit pedicel with water, heating reflux, cooling, shaking, and filtering to obtain the test solution; taking vanillic acid reference substance, dissolving with 70% ethanol to obtain a reference solution; B) determining the test solution and the reference solution by high performance liquid chromatography to obtain the chromatograms of the test solution and the reference solution, respectively; and qualitatively and quantitatively determining the components of the HPLC content determination of Cucurbita L. fruit pedicel medicinal material, decoction piece, standard decoction and its formula granules according to the chromatogram of the reference solution; the high performance liquid chromatography conditions are: the chromatographic column is a C18 column; the mobile phase is methanol-0.2% acetic acid; the volume ratio of the methanol and the 0.2% acetic acid is 15:85; and the detection wavelength is 260 nm.

2. The method of claim 1, wherein, the chromatographic column is a C18 column with a specification of 5μm, 4.6×250mm; and the column temperature is 30℃.

3. The method of claim 1, wherein, the flow rate of the mobile phase is 1.0mL / min; and the injection amount is 10μL.

4. The method of claim 1, wherein, the mass ratio of the Cucurbita L. fruit pedicel raw material to water is 0.5g:20mL; the ultrasonic power is 600W, the frequency is 40kHz, and the ultrasonic time is 30min.

5. The method of claim 1, wherein, the mass ratio of the Cucurbita L. fruit pedicel raw material to water is 2g:20mL; and the heating reflux time is 30min.

6. The method of claim 1, wherein, when the injection concentration of the vanillic acid reference substance ranges from 0.3514956μg / ml to 351.4956μg / ml, the linear relationship is y =38549x + 78.516, R² =1.

7. The method of claim 1, wherein, the lowest detection limit of the vanillic acid is 0.3514956μg / ml, and the quantitative limit is from 0.3514956μg / ml to 351.4956μg / ml.

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