HPLC (High Performance Liquid Chromatography) content determination construction method for pumpkin pedicel medicinal materials, decoction pieces, standard decoction and formula granules thereof

Abstract

The invention provides an HPLC (High Performance Liquid Chromatography) content determination construction method for a pumpkin pedicel medicinal material, a decoction piece, a standard decoction and formula granules thereof, which comprises the following steps: A) extracting a pumpkin pedicel raw material by adopting a solvent to obtain a to-be-determined solution; taking a vanillic acid reference substance, and dissolving the vanillic acid reference substance with 70% ethanol to obtain a reference substance solution; b) measuring the to-be-measured solution and the reference solution by adopting high performance liquid chromatography to respectively obtain chromatograms of the to-be-measured solution and the reference solution; according to the chromatogram of the reference substance, performing qualitative and quantitative determination on components for HPLC content determination of pumpkin pedicel medicinal materials, decoction pieces, standard decoction and formula granules thereof; chromatographic conditions of the high performance liquid chromatography are as follows: a chromatographic column is a C18 column; the mobile phase is methanol-0. 2% acetic acid. According to the method, high performance liquid chromatography is adopted, methanol-0. 2% acetic acid is selected as a mobile phase for elution, the content of vanillic acid in HPLC of pumpkin pedicel medicinal materials, decoction pieces, standard decoction and formula granules thereof is measured, repeatability and precision are good, and the method is stable and reliable.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a method for constructing HPLC content determination of pumpkin pedicle medicinal materials, decoction pieces, standard decoctions and formula particles thereof. Background Art

[0002] Pumpkin pedicles are the dried pedicles of Cucurbita moschata (Duch.ex Lam.) Duch.ex Poir. of the Cucurbitaceae family. The pedicles are picked after ripening and dried. They have the effects of harmonizing the middle, replenishing qi, calming the fetus, detoxifying, and healing sores. They are used for stomach deficiency, qi reversal, fetal movement disorder, and carbuncle. The standard decoction is a freeze-dried powder made from the medicinal materials after processing according to a fixed preparation process.

[0003] In order to ensure the uniformity and stability of the quality of pumpkin pedicle medicinal materials, herbal slices and their standard decoctions, a new content determination method was established in this paper 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 constructing HPLC content determination of pumpkin stalk medicinal materials, decoction pieces, standard decoctions and their formula granules. The HPLC content determination method for pumpkin stalk medicinal materials, decoction pieces, standard decoctions and their formula granules constructed by the present invention is stable and reliable, and can control the quality of vanillic acid in pumpkin stalk medicinal materials, decoction pieces, standard decoctions and their formula granules.

[0005] The present invention provides a method for constructing HPLC content determination of pumpkin pedicle medicinal materials, decoction pieces, standard decoctions and their formula particles, comprising:

[0006] A) extracting pumpkin pedicle raw material with solvent to obtain a test solution;

[0007] Take the vanillic acid reference substance and dissolve it in 70% ethanol to obtain a reference solution;

[0008] B) using high performance liquid chromatography to measure the test solution and the reference solution, respectively obtaining chromatograms of the test solution and the reference solution; and performing qualitative and quantitative determination of the components of pumpkin pedicle medicinal materials, decoction pieces, standard decoctions and their formula granules by HPLC content determination according to the chromatograms of the reference;

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

[0010] The invention provides a pumpkin stalk medicinal material, decoction pieces, standard decoction and formula granules thereof. The HPLC content determination construction method comprises first taking pumpkin stalk raw material, performing solvent extraction and obtaining a test solution.

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

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

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

[0014] The above extraction method and time can fully extract vanillic acid. At the same time, the inventors found that water as a solvent has a higher extraction effect than other organic solvents. Under the above ratio, the target component is extracted more completely. When the pumpkin stalk raw material is pumpkin stalk medicinal material or decoction piece, the preparation of the test solution specifically includes: mixing the pumpkin stalk medicinal material or decoction piece with water, heating the water flow, cooling, shaking, filtering, and obtaining.

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

[0016] The present invention adopts the above extraction method to achieve good chromatographic peak separation effect. Compared with ultrasonic extraction, the vanillic acid content of reflux extraction is higher.

[0017] The pumpkin pedicle raw material is pumpkin pedicle medicinal material, pumpkin pedicle decoction piece or decoction. The present invention does not limit it, and the above raw materials can all be subjected to quality control and qualitative detection by the method of the present invention.

[0018] Take the vanillic acid reference substance and dissolve it in 70% ethanol to obtain a reference solution.

[0019] When the injection concentration range of the vanillic acid reference substance of the present invention is 0.3514956-351.4956 μg / ml, the linear relationship is y=38549x+78.516, R 2 =1.

[0020] The minimum detection limit of the vanillic acid of the present invention is 0.3514956 ug / ml, and the quantitative limit is 0.3514956-351.4956 μg / ml.

[0021] The test solution and the reference solution are measured by high performance liquid chromatography to obtain chromatograms of the test solution and the reference solution, respectively; and the components of pumpkin pedicle medicinal materials, decoction pieces, standard decoctions and their formula granules are qualitatively and quantitatively determined according to the chromatograms of the reference;

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

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

[0024] The present invention has good baseline separation, large chromatographic peak information, good separation degree, moderate peak time and stable baseline under the above mobile phase.

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

[0026] The chromatographic column of the present invention is preferably: Agilent C18 4.6×250mm, 5μm, Shimadzu C18 4.6×250mm, 5μm, Waters C18 4.6×250mm, 5μm.

[0027] The chromatographic column of the present invention has symmetrical chromatographic peaks, good separation and moderate peak elution time under the above 30°C condition.

[0028] The mobile phase flow rate is preferably 1.0 ml / min.

[0029] The present invention found that at a flow rate of 1.0 ml / min, the chromatographic peaks were better separated, the peak shape was more symmetrical, and the separation degree was moderate. At the same time, based on the comprehensive theoretical plate number and the separation degree peak shape, a flow rate of 1.0 was adopted as the most preferred solution.

[0030] The detection wavelength of the present invention is preferably 260 nm.

[0031] The inventors found that at 260 nm, the chromatographic information is rich, 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 injection volume of the present invention is preferably 10 μL.

[0033] The present invention provides a method for constructing HPLC content determination of pumpkin stalk medicinal materials, decoction pieces, standard decoctions and their formula particles, comprising: A) extracting pumpkin stalk raw materials with solvents to obtain a test liquid; taking a vanillic acid reference substance and dissolving it with 70% ethanol to obtain a reference solution; B) determining the test liquid and the reference solution by high performance liquid chromatography to obtain chromatograms of the test liquid and the reference solution, respectively; and qualitatively and quantitatively determining the components of the HPLC content determination of pumpkin stalk medicinal materials, decoction pieces, standard decoctions and their formula particles according to the chromatograms of the reference; the chromatographic conditions of the high performance liquid chromatography are: the chromatographic column is a C18 column; the mobile phase is methanol-0.2% acetic acid. The present invention adopts high performance liquid chromatography, selects methanol-0.2% acetic acid as the mobile phase for elution, and determines the vanillic acid content in HPLC of pumpkin stalk medicinal materials, decoction pieces, standard decoctions and their formula particles, with good repeatability and precision, and the method is stable and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Investigation of different mobile phases;

[0035] Figure 2 UV absorption spectrum of vanillic acid;

[0036] Figure 3 Column temperature investigation results;

[0037] Figure 4 Flow rate survey results;

[0038] Figure 5 Exclusive inspection;

[0039] Figure 6 Standard curve of vanillic acid;

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

[0041] Figure 8 Example 2 full wavelength scanning results; Fig. 9 Example 2 Column temperature investigation results;

[0042] Fig.10 Example 2 Flow rate investigation results;

[0043] Fig.11 Embodiment 2 Specificity investigation;

[0044] Fig.12 Example 2 Vanillic acid standard curve diagram;

[0045] Fig.13 Example 2 Investigation of different instruments;

[0046] Fig.14 Example 2 Chromatographic column durability test results;

[0047] Fig.15 Example 3 Column temperature investigation results;

[0048] Fig.16 Example 3 Flow rate investigation results;

[0049] Fig.17 Embodiment 3 specificity investigation;

[0050] Fig.18 Example 3 Vanillic acid standard curve diagram;

[0051] Fig.19 Example 3 Investigation of different instruments;

[0052] Fig. 20 Example 3 Chromatographic column durability test results;

[0053] Fig.21 This is the result of the medicinal material test of pumpkin pedicles. The top is the overall picture, and the bottom is the enlarged picture.

[0054] Fig. 22 This is the test result of the standard decoction of pumpkin stem, the upper one is the whole picture, the lower one is the enlarged picture;

[0055] Fig.23 This is the test result of pumpkin stem formula particles, the upper one is the overall picture, and the lower one is the enlarged picture;

[0056] Fig.24 This is the chromatogram of vanillic acid standard. DETAILED DESCRIPTION

[0057] The present invention provides a method for constructing HPLC content determination of pumpkin pedicle medicinal materials, decoction pieces, standard decoctions and their formula particles. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they all belong to the scope of protection of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0058] To further illustrate the present invention, the following is a detailed description of the construction method for HPLC content determination of a pumpkin stalk medicinal material, decoction pieces, standard decoction and formula granules thereof provided by the present invention in combination with the examples.

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

[0060] 1. Experimental instruments and materials

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

[0062] Electronic balance: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instrument Co., Ltd.);

[0063] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0064] Ultrasonic cleaner: KQ-600DB (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 test drugs

[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 mobile phase water were laboratory-made ultrapure water, and the rest were laboratory-made pure water.

[0068] Vanillic acid (China Food and Drug Inspection Institute, batch number: 110776-201503, content is 99.8%)

[0069] Pumpkin pedicle standard decoction (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch number: 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. Propose test conditions

[0071] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol-0.2% acetic acid (15:85) was used as mobile phase; flow rate was 1.0 ml per minute; column temperature was 30°C; detection wavelength was 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 substance, weigh accurately, add 70% ethanol solution to make a solution containing 7ug of vanillic acid per 1ml.

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

[0074] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

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

[0076] 4.1 Mobile phase selection

[0077] Based on the experimental conditions proposed above, methanol-0.2% acetic acid, methanol-0.2% phosphoric acid, methanol-0.2% formic acid, acetonitrile-0.2% acetic acid, were investigated respectively. The results are shown in Figure 1 . Figure 1 Different mobile phases were investigated; the results showed that when the mobile phase was methanol-0.2% acetic acid, the chromatographic peak of vanillic acid had a larger amount of information, better separation, and a more moderate peak elution time. The mobile phase was tentatively determined to be methanol-0.2% acetic acid.

[0078] 4.2 Determination of detection wavelength

[0079] Based on the experimental conditions proposed above, a diode array detector was used to perform full-band scanning of vanillic acid. Figure 2 . Figure 2 Vanillic acid ultraviolet absorption spectrum; the results show that through the analysis of the spectrum, the optimal detection wavelength for the determination of vanillic acid in the standard decoction of pumpkin stalks is 260nm.

[0080] 4.3 Column temperature investigation

[0081] Based on the experimental conditions proposed above, column temperatures of 25°C, 30°C, and 35°C were investigated, with the separation degree of vanillic acid and the number of theoretical plates as evaluation indicators. Figure 3 , Table 1. Figure 3 Column temperature investigation results.

[0082] Table 1 Analysis results of different column temperatures

[0083]

[0084] The results showed that vanillic acid could be effectively detected at column temperatures of 25°C-35°C, and 30°C was proposed as the detection column temperature.

[0085] 4.4 Flow rate investigation

[0086] Based on the experimental conditions proposed above, the flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were investigated, and the separation degree of vanillic acid and the number of theoretical plates were used as evaluation indicators. Figure 4 , Table 2. Figure 4 Flow rate survey results.

[0087] Table 2 Analysis results of different flow rates

[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 ultimately determined to be 1.0 ml / min.

[0091] In summary, the chromatographic conditions and system suitability test for the content determination of pumpkin pedicle standard decoction are as follows: octadecylsilane bonded silica gel as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol-0.2% acetic acid (15:85) as mobile phase; flow rate 1.0 ml per minute; 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.

[0092] 5. Preparation of test solution

[0093] 5.1 Investigation of extraction methods

[0094] Take about 0.5g of pumpkin pedicle standard decoction freeze-dried powder (batch number: NGD--BT-230718), accurately weigh it, put it in a stoppered conical bottle, accurately add 20ml of 70% ethanol solution, weigh it, seal it, and conduct reflux and ultrasonic extraction respectively. The extraction time is 30 minutes, let it cool, weigh it again, make up the lost weight with 70% ethanol, shake it well, filter it, and take the filtrate. Analyze and calculate the vanillic acid content under different extraction methods. The results are shown in Table 36.

[0095] Table 3 Analysis results of different extraction methods

[0096]

[0097] The results showed that the contents of vanillic acid 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 much different from that of ultrasonic extraction, and ultrasonic extraction was more convenient. Ultrasonic extraction was determined to be the extraction method.

[0098] 5.2 Extraction time investigation

[0099] Take about 0.5g of the powder of this product (batch number: NGD--BT-230718), weigh it accurately, put it in a stoppered conical bottle, add 20ml of 70% ethanol solution accurately, stopper it, weigh it, extract it by ultrasonic for 20 minutes, 30 minutes, 60 minutes, let it cool, weigh it again, make up the lost weight with 70% ethanol, shake it well, filter it, and take the filtrate. Analyze and calculate the content of vanillic acid 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 when the extraction time was 30 minutes, sufficient extraction could be achieved. Therefore, the extraction time of the test sample was determined to be 30 minutes.

[0103] 5.3 Extraction solvent investigation

[0104] Take about 0.5g of the powder of this product (batch number: NGD--BT-230718), weigh it accurately, put it in a stoppered conical bottle, extract it with 30% ethanol, 50% ethanol, 70% ethanol, ethanol, methanol, and water, add 20ml accurately, stopper it, weigh it, ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, and take the filtrate to obtain it. Analyze and calculate the content of vanillic acid under different solvent extractions. The results are shown in Table 37.

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

[0106]

[0107]

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

[0109] 5.4 Investigation of solvent addition amount

[0110] Take about 0.5g of the powder of this product (batch number: NGD--BT-230718), weigh it accurately, put it in a stoppered conical bottle, add 15ml, 20ml, and 50ml of water accurately, stopper it, weigh it, treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with water, shake it well, filter it, and take the filtrate to obtain it. Analyze and calculate the content of vanillic acid under different solvent addition amounts. The results are shown in Table 39.

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

[0112]

[0113] The results show that when the sampling volume is 0.5g, the amount of solvent added is 20ml to ensure complete extraction of the target components, so the amount of solvent added is determined to be 20ml.

[0114] In summary, the preparation method of the test sample for the content determination of pumpkin stalk standard decoction is determined as follows: take about 0.5 g of the powder of the product, accurately add 20 ml of water, seal it, weigh it, ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with water, shake it well, and obtain it.

[0115] 5.5 Method for determination of content of pumpkin pedicle standard decoction

[0116] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol-0.2% acetic acid solution (15:85) was used as mobile phase; flow rate was 1.0 ml per minute; column temperature was 30°C; detection wavelength was 260 nm. The theoretical plate number calculated based on the vanillic acid peak should be no less than 4000.

[0117] Preparation of reference solution: Take an appropriate amount of vanillic acid reference substance, weigh accurately, and add 70% ethanol to make a solution containing 7 μg per 1 ml.

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

[0119] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0120] 6. Methodological investigation

[0121] 6.1 Specificity Experiment

[0122] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of pumpkin pedicle standard decoction.

[0123] Preparation of reference solution: Take an appropriate amount of vanillic acid reference, weigh accurately, and add 70% ethanol to make a solution containing 7 μg per 1 ml.

[0124] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of standard decoction lacking pumpkin pedicles. Figure 5 . Figure 5 Exclusive inspection.

[0125] The results showed that the chromatogram of negative solution had no interference with the determination of the peak to be tested, indicating that the method had good specificity.

[0126] 6.2 Precision investigation

[0127] The reference solution was sampled 6 times continuously, the peak area of ​​vanillic acid was recorded, and the RSD value was calculated. The results are shown in Table 7.

[0128] Table 7 Precision inspection results

[0129]

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

[0131] 6.3 Linear Relationship

[0132] Take an appropriate amount of vanillic acid, put it in a 10ml volumetric flask, dissolve it in 70% ethanol to make a mother solution of the reference substance containing 351.4956μg / ml (purity 99.8%) of vanillic acid, and then dilute it to a concentration of 21.089736μg / ml, 14.059824μg / ml, 7.029912μg / ml, 0.7029912μg / ml, and 0.3514956μg / ml. Accurately draw 10μl of each, inject it into the liquid chromatograph, obtain the peak area, and draw the response curve with the concentration of the vanillic acid reference substance (X, μg / ml) as the horizontal coordinate of the standard curve and the peak area (Y) as the vertical coordinate. The results are shown in Table 8. Figure 6 . Figure 6 Standard curve of vanillic acid.

[0133] Table 8 Vanillic acid standard curve analysis results

[0134]

[0135] The results showed that when the concentration of vanillic acid was in the range of 0.3514956 to 351.4956 μg / ml, the linear relationship was y=38549x+78.516, R2 = 1. This indicates that the concentration range is 0.3514956-351.4956 μg / ml, with a good linear relationship.

[0136] 6.4 Repeatability Experiment

[0137] About 0.5 g of the powder of this product (batch number: NGD--BT-230718) was taken and accurately weighed in 6 portions. The test solution was prepared by the same operator according to the determined method, the peak area was determined, and the vanillic acid content of the 6 test samples was calculated. The results are shown in Table 9.

[0138] Table 9 Repeatability test results

[0139]

[0140] The RSD value of the vanillic acid content obtained from 6 repeated tests was 2.0%, and this method had good repeatability.

[0141] 6.5 Stability test

[0142] The same test sample (batch number: NGD-BT-230718) solution was taken and the chromatographic peak area of ​​vanillic acid was determined 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 values ​​of the vanillic acid peak areas were all 1.4%, and the test solutions had good stability within 24 hours.

[0146] 6.6 Intermediate precision

[0147] 6.6.1 Investigation by different personnel and time

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

[0149] Table 11 Intermediate precision test results

[0150]

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

[0152] 6.7 Sample recovery rate

[0153] Take about 0.25g of the test sample with known content (batch number: NGD--BT-230718, vanillic acid content 0.29mg / g), a total of 6 portions, accurately weighed, accurately added 25ml of control solution containing vanillic acid (purity 98.0%), and prepared and measured the test sample solution according to the proposed method, and calculated the recovery rate. The results are shown in Table 12. The calculation formula is as follows:

[0154]

[0155]

[0156] Table 12 Vanillic acid sample recovery test results

[0157]

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

[0159] 6.8 Durability test

[0160] 6.8.1 Investigation with different instruments

[0161] Take about 0.5 g of the powder of this product (batch number: NGD-BT-230718), accurately weigh two portions, prepare the test solution according to the determined method, and examine it on Waters e2695, Agilent Inifity 1260, and Shimadzu high performance liquid chromatograph (the chromatographic columns are all Agilent C18 250mm×4.6mm, 5μm), calculate the vanillic acid content, and the results are shown in Table 13.

[0162] Table 13 Experimental results of different instruments

[0163]

[0164] The results showed that the RSD value of the results measured by Waters e2695, Agilent Inifity 1260, and Shimadzu HPLC was 1.9%, indicating that the instrument durability of this method was good.

[0165] 6.8.2 Investigation of different chromatographic columns

[0166] The same sample (batch number: NGD-BT-230718) was tested using different brands of C18 chromatographic columns: Agilent C18 4.6*250mm, 5μm, Shimadzu C18 4.6*250mm, 5μm, and Waters C184.6*250mm, 5μm. The results are shown in Table 14.

[0167] Table 14 Results of the investigation on the durability of the chromatographic column

[0168]

[0169]

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

[0171] 7. Sample content determination verification

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

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

[0174]

[0175] The results showed that the measured value of vanillic acid in the standard decoction of pumpkin pedicles ranged from 0.18mg / g to 0.60mg / g, the average total content was 0.32mg / g, and the SD value was 0.12. The limit of 70%-130% of the average value of the total content determination was 0.22-0.41mg / g of vanillic acid (C8H8O4), the limit range of the average value plus or minus 3 times the SD value was -0.06-0.69mg / g of vanillic acid (C8H8O4), and the limit range of the average value plus or minus 2 times the SD value was 0.069-0.57mg / g of vanillic acid (C8H8O4). In summary, the average value -2SD was rounded off to 0.07mg / g as the lower limit of the content, and the average value + 3SD, 0.69mg / g, was used as the upper limit of the content. It is tentatively estimated that the vanillic acid (C8H8O4) content of this product, calculated on the basis of dry product, ranges from 0.07mg / g to 0.69mg / g.

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

[0177] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol-0.2% acetic acid solution (15:85) was used as mobile phase; flow rate was 1.0 ml per minute; column temperature was 30°C; detection wavelength was 260 nm. The theoretical plate number calculated based on the vanillic acid peak should be no less than 4000.

[0178] Preparation of reference solution: Take an appropriate amount of vanillic acid reference substance, weigh accurately, and add 70% ethanol to make a solution containing 7 μg per 1 ml.

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

[0180] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0181] This product should contain 0.07mg~0.69mg of vanillic acid (C8H8O4) per 1g.

[0182] Example 2 Pumpkin pedicle medicinal materials, decoction pieces HPLC content determination

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

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

[0185] Pumpkin stem medicinal material batch number: 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 test: Octadecylsilane bonded silica gel was used as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol-0.2% acetic acid (15:85) was used as mobile phase; flow rate was 1.0 ml per minute; column temperature was 30°C; detection wavelength was 260 nm. The theoretical plate number calculated based on the vanillic acid peak should be no less than 4000.

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

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

[0191] The determination method is to accurately pipette 10ul of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

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

[0193] 4.1 Wavelength Selection

[0194] Based on the experimental conditions proposed above, the vanillic acid reference solution was scanned in the whole band using a diode array detector. The results are as follows: Figure 8 : Figure 8 Example 2 Full wavelength scanning results; The results show that when the detection wavelength is 260nm, the information content of the vanillic acid chromatographic peak is relatively large, so the detection wavelength is determined to be 260nm.

[0195] 4.2 Column temperature investigation

[0196] Based on the experimental conditions proposed above, column temperatures of 25°C, 30°C, and 35°C were investigated, with the separation degree of vanillic acid and the number of theoretical plates as evaluation indicators. Fig. 9 , Table 16. Fig. 9 Example 2 Column temperature investigation results.

[0197] Table 16 Analysis results of different column temperatures

[0198]

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

[0200] 4.3 Flow rate investigation

[0201] Based on the experimental conditions proposed above, the flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were investigated, and the separation degree of vanillic acid and the number of theoretical plates were used as evaluation indicators. Fig.10 , Table 17. Fig.10 Example 2 Flow rate investigation results.

[0202] Table 17 Analysis results of different flow rates

[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 temporarily set at 1.0 ml / min.

[0205] In summary, the chromatographic conditions and system suitability test for the determination of pumpkin pedicle particles are as follows: octadecylsilane bonded silica gel as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol-0.2% acetic acid (15:85) as mobile phase; flow rate 1.0 ml per minute; 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.

[0206] 5. Preparation of test solution

[0207] 5.1 Investigation of extraction methods

[0208] Take an appropriate amount of pumpkin stalk medicinal powder (batch number: XLS202109221), grind it into powder, take about 2.0g, weigh it accurately, put it in a stoppered conical flask, accurately add 20ml of water, stopper it, weigh it, and conduct ultrasonic (600W, frequency 40kHz) and reflux extraction methods respectively. The extraction time is 30 minutes. Cool it, weigh it again, make up the lost weight with water, shake it well, filter it, and take the filtrate to obtain it.

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

[0210] Table 18 Analysis results of different extraction methods

[0211]

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

[0213] 5.2 Investigation of extraction solvent

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

[0215] Table 19 Results of investigation on 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 Extraction time investigation

[0219] Take an appropriate amount of this product (batch number: XLS202109221), grind it into powder, take about 2.0g, weigh it accurately, put it in a stoppered conical flask, add 20ml of water accurately, stopper it, weigh it, reflux it for 20 minutes, 30 minutes, and 60 minutes respectively, let it cool, weigh it again, make up the lost weight with water, shake it well, filter it, and take the filtrate to obtain it. Analyze and calculate the content of vanillic acid at different extraction times. The results are shown in Table 20.

[0220] Table 20 Analysis results of different extraction times

[0221]

[0222]

[0223] The results showed that when the extraction time was 30 minutes, sufficient extraction could be achieved. Therefore, the extraction time of 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 powder, take about 0.5g, weigh it accurately, put it in a stoppered conical bottle, add 15ml, 20ml, and 50ml of water accurately, stopper it, weigh it, ultrasonically treat it (power 400W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with water, shake it well, filter it, take the filtrate, and examine the extraction efficiency of the amount of solvent added. Analyze and calculate the content of vanillic acid at different extraction times. The results are shown in Table 21.

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

[0227]

[0228] The results show that when the sampling volume is 0.5g, the amount of solvent added is 20ml to ensure complete extraction of the target components, so the amount of water added is determined to be 20ml.

[0229] In summary, the preparation method of the test sample for the content determination of pumpkin stalk particles is determined as follows: take an appropriate amount of the product, grind it into powder, take about 0.5 g, weigh it accurately, put it in a stoppered conical flask, accurately add 20 ml of water, weigh it, ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with water, shake it well, filter it, and take the filtrate to obtain it.

[0230] 5.5 Method for determination of medicinal material content in pumpkin pedicles

[0231] Chromatographic conditions and system suitability: Octadecylsilane bonded silica gel was used as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); methanol-0.2% acetic acid (15:85) was used as the mobile phase; the flow rate was 1.0 ml per minute; the column temperature was 30°C; 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 reference solution: Take an appropriate amount of vanillic acid reference substance, weigh accurately, and add 70% ethanol solution to make a solution containing 7 μg of vanillic acid per 1 ml.

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

[0234] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0235] 6. Methodological investigation

[0236] 6.1 Specificity Experiment

[0237] Preparation of test solution: According to the experimental conditions proposed above, prepare the pumpkin pedicle medicinal material test solution.

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

[0239] Preparation of negative control solution: According to the experimental conditions proposed above, a negative control solution lacking pumpkin pedicles was prepared. The results are shown in Fig.11 . Fig.11 Embodiment 2 specificity investigation.

[0240] The results showed that the chromatogram of negative solution had no interference with the determination of the peak to be tested, indicating that the method had good specificity.

[0241] 6.2 Precision investigation

[0242] The reference solution was sampled continuously 6 times, the peak area of ​​vanillic acid was recorded, and the RSD value was calculated. The results are shown in Table 22.

[0243] Table 22 Precision investigation results

[0244]

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

[0246] 6.3 Linear Relationship

[0247] Take an appropriate amount of vanillic acid, put it in a 10ml volumetric flask, dissolve it in 70% ethanol to make a mother solution of reference substance containing 351.4956μg / ml (purity 99.8%) of vanillic acid, and then dilute it to concentrations of 21.089736μg / ml, 14.059824μg / ml, 7.029912μg / ml, 0.7029912μg / ml, and 0.3514956μg / ml. Accurately draw 10μl of each, inject it into the liquid chromatograph, obtain the peak area, and draw the response curve with the concentration of the vanillic acid reference substance (X, μg / ml) as the horizontal coordinate of the standard curve and the peak area (Y) as the vertical coordinate. The results are shown in Table 23. Fig.12 . Fig.12 Example 2 Vanillic acid standard curve diagram.

[0248] Table 23 Vanillic acid standard curve analysis results

[0249]

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

[0251] 6.4 Repeatability Experiment

[0252] Take an appropriate amount of this product (batch number: XLS202109221), grind it into powder, take 2.0 g, 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 Repeatability test results

[0254]

[0255] The RSD value of the vanillic acid content obtained from 6 repeated tests was 2.9%, and this method had good repeatability.

[0256] 6.5 Stability test

[0257] Take the same test sample solution (batch number: XLS202109221) and determine the chromatographic 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 Investigation by different personnel and time

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

[0264] Table 26 Intermediate precision test results

[0265]

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

[0267] 6.7 Sample recovery rate

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

[0269]

[0270] Table 27 Vanillic acid sample recovery test results

[0271]

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

[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 into powder, take about 2.0g, weigh it accurately, and examine it on Waters, Shimadzu, and Agilent high performance liquid chromatographs (the chromatographic columns are all Agilent C18 250×4.6mm, 5μm), calculate the vanillic acid content, and the results are shown in Fig.13 , Table 28. Fig.13 Example 2 Investigation of different instruments.

[0276] Table 28 Experimental results of 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 instrument durability of this method was good.

[0279] 6.8.2 Investigation of different chromatographic columns

[0280] The same sample (batch number: 010634-2305001) was tested using C18 chromatographic columns of different brands: Agilent C18 4.6*250mm, 5μm, Shimadzu C18 4.6*250mm, 5μm, and Waters C184.6*250mm, 5μm. The results are shown in Table Fig.14 , Table 29. Fig.14 Example 2 Chromatographic column durability test results.

[0281] Table 29 Results of the chromatographic column durability test

[0282]

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

[0284] In summary, the method for determining the content of pumpkin pedicle medicinal materials is:

[0285] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel as filler; methanol-0.2% acetic acid (15:85) as mobile phase; flow rate of 1.0 ml per minute; column temperature of 30°C; 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 reference solution: Take an appropriate amount of vanillic acid reference substance, weigh it accurately, and add 70% ethanol solution to make a solution containing 7 ug of vanillic acid per 1 ml.

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

[0288] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0289] 7. Results of determination of medicinal material content in pumpkin pedicles

[0290] 21 batches of pumpkin pedicle medicinal materials were tested, and the results are shown in Table 30.

[0291] Table 30 Pumpkin pedicle medicinal material content determination results

[0292]

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

[0294] 8. Determination of the content determination method of pumpkin pedicle slices

[0295] The detection method of pumpkin pedicle slices refers to the method of [Content Determination] under the pumpkin pedicle medicinal material:

[0296] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol-0.2% acetic acid (15:85) was used as mobile phase; flow rate was 1.0 ml per minute; column temperature was 30°C; detection wavelength was 260 nm. The theoretical plate number calculated based on the vanillic acid peak should be no less than 4000.

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

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

[0299] The determination method is to accurately pipette 10ul of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0300] 9. Results of the content determination of pumpkin pedicle slices

[0301] The results are shown in Table 31.

[0302] Table 31 Decoction content determination results

[0303]

[0304]

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

[0306] Example 3 HPLC content determination of pumpkin pedicle formula granules

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

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

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

[0310] 3. Proposed Chromatographic Conditions

[0311] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol-0.2% acetic acid (15:85) was used as mobile phase; flow rate was 1.0 ml per minute; column temperature was 30°C; detection wavelength was 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 substance, weigh accurately, add 70% ethanol solution to make a solution containing 7ug of vanillic acid per 1ml.

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

[0314] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

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

[0316] 4.1 Column temperature investigation

[0317] Based on the experimental conditions proposed above, column temperatures of 25°C, 30°C, and 35°C were investigated, with the separation degree of vanillic acid and the number of theoretical plates as evaluation indicators. Fig.15 , Table 32. Fig.15 Example 3 Column temperature investigation results.

[0318] Table 32 Analysis results of different column temperatures

[0319]

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

[0321] 4.2 Flow rate investigation

[0322] Based on the experimental conditions proposed above, the flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were investigated, and the separation degree of vanillic acid and the number of theoretical plates were used as evaluation indicators. Fig.16 , Table 33. Fig.16 Example 3 flow rate investigation results.

[0323] Table 33 Analysis results of different flow rates

[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 temporarily set at 1.0 ml / min.

[0326] In summary, the chromatographic conditions and system suitability test for the determination of pumpkin pedicle particles are as follows: octadecylsilane bonded silica gel as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol-0.2% acetic acid (15:85) as mobile phase; flow rate 1.0 ml per minute; 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.

[0327] 5. Preparation of test solution

[0328] 5.1 Investigation of extraction methods

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

[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 ultrasonic and reflux extraction was 0.21 mg / g, and ultrasonic extraction was more convenient. Therefore, ultrasonic extraction was determined to be the extraction method.

[0334] 5.2 Investigation of extraction solvent

[0335] Take an appropriate amount of this product (batch number: 1904033), grind it into powder, take about 0.5g, weigh it accurately, put it in a stoppered conical bottle, add 20ml of 30% ethanol, 50% ethanol, 70% ethanol, ethanol, methanol, and water respectively, stopper it tightly, weigh it, treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, and take the filtrate to obtain it. Calculate the content of vanillic acid under different solvent extraction, the results are shown in Table 35.

[0336] Table 35 Results of investigation on 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 Extraction time investigation

[0340] Take an appropriate amount of this product (batch number: B2307007), grind it into powder, take about 0.5g, weigh it accurately, put it in a stoppered conical bottle, add 20ml of water accurately, stopper it, weigh it, and treat it with ultrasound (power 600W, frequency 40kHz) for 20 minutes, 30 minutes, and 60 minutes respectively, let it cool, weigh it again, make up the lost weight with water, shake it well, filter it, and take the filtrate to obtain it. Analyze and calculate the content of vanillic acid at different extraction times. The results are shown in Table 36.

[0341] Table 36 Analysis results of different extraction times

[0342]

[0343] The results showed that when the extraction time was 30 minutes, sufficient extraction could be achieved. Therefore, the extraction time of 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 powder, take about 0.5g, weigh it accurately, put it in a stoppered conical bottle, add 15ml, 20ml, and 50ml of water accurately, stopper it, weigh it, ultrasonically treat it (power 400W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with water, shake it well, filter it, take the filtrate, and examine the extraction efficiency of the solvent addition amount. Analyze and calculate the content of vanillic acid at different extraction times. The results are shown in Table 37.

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

[0347]

[0348] The results show that when the sampling volume is 0.5g, the amount of solvent added is 20ml to ensure complete extraction of the target components, so the amount of water added is determined to be 20ml.

[0349] In summary, the preparation method of the test sample for the content determination of pumpkin stalk particles is determined as follows: take an appropriate amount of the product, grind it into powder, take about 0.5 g, weigh it accurately, put it in a stoppered conical flask, accurately add 20 ml of water, weigh it, ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with water, shake it well, filter it, and take the filtrate to obtain it.

[0350] 5.5 Determination of pumpkin pedicle content

[0351] Chromatographic conditions and system suitability: Octadecylsilane bonded silica gel was used as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); methanol-0.2% acetic acid (15:85) was used as the mobile phase; the flow rate was 1.0 ml per minute; the column temperature was 30°C; 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 reference solution: Take an appropriate amount of vanillic acid reference substance, weigh accurately, and add 70% ethanol solution to make a solution containing 7 μg of vanillic acid per 1 ml.

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

[0354] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0355] 6. Methodological investigation

[0356] 6.1 Specificity Experiment

[0357] Preparation of test solution: According to the experimental conditions proposed above, prepare the pumpkin pedicle particle test solution.

[0358] Preparation of reference solution: According to the experimental conditions proposed above, prepare the pumpkin stalk granule reference solution.

[0359] Preparation of negative control solution: According to the experimental conditions proposed above, a negative control solution of pumpkin stalk-less granules was prepared. The results are shown in Fig.17 . Fig.17 Embodiment 3 specificity investigation.

[0360] The results showed that the chromatogram of negative solution had no interference with the determination of the peak to be tested, indicating that the method had good specificity.

[0361] 6.2 Precision investigation

[0362] The reference solution was sampled continuously for 6 times, the peak area of ​​vanillic acid was recorded, and the RSD value was calculated. The results are shown in Table 38.

[0363] Table 38 Precision inspection results

[0364]

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

[0366] 6.3 Linear Relationship

[0367] Take an appropriate amount of vanillic acid, put it in a 10ml volumetric flask, dissolve it in 70% ethanol to make a mother solution of the reference substance containing 351.4956μg / ml (purity 99.8%) of vanillic acid, and then dilute it to a concentration of 21.089736μg / ml, 14.059824μg / ml, 7.029912μg / ml, 0.7029912μg / ml, and 0.3514956μg / ml. Accurately draw 10μl of each, inject it into the liquid chromatograph, obtain the peak area, and draw the response curve with the concentration of the vanillic acid reference substance (X, μg / ml) as the horizontal coordinate of the standard curve and the peak area (Y) as the vertical coordinate. The results are shown in Table 39. Fig.18 . Fig.18 Example 3 Vanillic acid standard curve diagram.

[0368] Table 39 Vanillic acid standard curve analysis results

[0369]

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

[0371] 6.4 Repeatability Experiment

[0372] Take an appropriate amount of this product (batch number: B2307007), grind it into powder, take 0.5 g, 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 Repeatability test results

[0374]

[0375] The RSD value of the vanillic acid content obtained from 6 repeated tests was 0.9%, and this method had good repeatability.

[0376] 6.5 Stability test

[0377] Take the same test sample solution (batch number: B2307007) and determine the chromatographic 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 Investigation by different personnel and time

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

[0384] Table 42 Intermediate precision test results

[0385]

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

[0387] 6.7 Sample recovery rate

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

[0389]

[0390]

[0391] Table 43 Vanillic acid sample recovery experimental results

[0392]

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

[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 into powder, take about 0.5g, weigh it accurately, and examine it on Waters, Shimadzu, and Agilent high performance liquid chromatographs (the chromatographic columns are all Agilent C18250×4.6mm, 5μm), calculate the vanillic acid content, and the results are shown in Fig.19 , Table 44. Fig.19 Example 3 Investigation of different instruments.

[0397] Table 44 Experimental results of 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 instrument durability of this method was good.

[0400] 6.8.2 Investigation of different chromatographic columns

[0401] The same sample (batch number: B2307007) was tested using C18 chromatographic columns of different brands: Agilent C18 4.6*250mm, 5μm, Shimadzu C18 4.6*250mm, 5μm, and Waters C184.6*250mm, 5μm. The results are shown in Table Fig. 20 , Table 45. Fig. 20 Example 3 Chromatographic column durability test results.

[0402] Table 45 Results of the durability test of the chromatographic column

[0403]

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

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

[0406] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel as filler; methanol-0.2% acetic acid (15:85) as mobile phase; flow rate of 1.0 ml per minute; column temperature of 30°C; 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 reference solution: Take an appropriate amount of vanillic acid reference substance, weigh it accurately, and add 70% ethanol solution to make a solution containing 7 ug of vanillic acid per 1 ml.

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

[0409] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0410] 7. Verification

[0411] Using pumpkin stem formula granules as the test sample, three batches of process validation samples were tested using the established test method. The results are shown in Table 46.

[0412] Table 46 Test results of three batches of process verification formula granules

[0413]

[0414] Conclusion: The content determination method of pumpkin stalk formula granules can effectively detect pumpkin stalk formula granules, so this method is included in the text of pumpkin stalk formula granules.

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

[0416] Chromatographic conditions and system suitability: Octadecylsilane bonded silica gel was used as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); methanol-0.2% acetic acid (15:85) was used as the mobile phase; the flow rate was 1.0 ml per minute; the column temperature was 30°C; 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 reference solution: Take an appropriate amount of vanillic acid reference substance, weigh accurately, and add 70% ethanol solution to make a solution containing 7 μg of vanillic acid per 1 ml.

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

[0419] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0420] Comparative Example 1

[0421] Experimental instruments:

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

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

[0424] Electronic balance: ME204E, MS205DM, XPR10 (Mettler-Toledo Instrument Co., Ltd.);

[0425] Reference substance: vanillic acid; Source: China Food and Drug Inspection Institute; Batch number: 110776-201503, purity 99.8%.

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

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

[0428] The above method was used to test the medicinal materials, standard decoctions and formula granules of pumpkin pedicles.

[0429] The results are as follows Figure 21 to Figure 23 As shown, Fig.21 This is the result of the medicinal material test of pumpkin pedicles. The top is the overall picture, and the bottom is the enlarged picture. Fig. 22 This is the test result of the standard decoction of pumpkin stem, the upper one is the whole picture, the lower one is the enlarged picture; Fig.23 This is the test result of pumpkin stem formula particles, the upper one is the overall picture, and the lower one is the enlarged picture; Fig.24The chromatogram of vanillic acid standard. As can be seen from the above figure, at the vanillic acid peak time of about 15.622min, the medicinal materials, standard decoction, and formula granules all have serious peaks, so this method cannot effectively separate the vanillic acid in pumpkin pedicles.

[0430] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing HPLC content determination of pumpkin pedicle medicinal materials, decoction pieces, standard decoctions and their formula particles, comprising: A) extracting pumpkin pedicle raw material with solvent to obtain a test solution; Take the vanillic acid reference substance and dissolve it in 70% ethanol to obtain a reference solution; B) using high performance liquid chromatography to measure the test solution and the reference solution, respectively obtaining chromatograms of the test solution and the reference solution; and performing qualitative and quantitative determination of the components of pumpkin pedicle medicinal materials, decoction pieces, standard decoctions and their formula granules by HPLC content determination according to the chromatograms of the reference; The chromatographic conditions of the high performance liquid chromatography method are: the chromatographic column is a C18 column; the mobile phase is methanol-0.2% acetic acid.

2. The method according to claim 1, characterized in that The volume ratio of the methanol to 0.2% acetic acid is 15:

85.

3. The method according to claim 1, characterized in that The chromatographic column is a C18 column with specifications of 5 μm and 4.6×250 mm; the column temperature is 30° C.

4. The method according to claim 1, characterized in that: The flow rate of the mobile phase was 1.0 mL / min; the detection wavelength was 260 nm; and the injection volume was 10 μL.

5. The method according to claim 1, characterized in that When the pumpkin stalk raw material is pumpkin stalk standard decoction or formula granules, the preparation of the test solution specifically includes: mixing the pumpkin stalk standard decoction or formula granules with water, ultrasonic treatment, cooling, shaking, filtering, and obtaining the solution.

6. The method according to claim 5, characterized in that The mass ratio of the pumpkin pedicle raw material to water is 0.5 g:20 mL; the ultrasonic power is 600 W, the frequency is 40 kHz, and the ultrasonic time is 30 min.

7. The method according to claim 1, characterized in that When the pumpkin stalk raw material is pumpkin stalk medicinal material or decoction piece, the preparation of the test solution specifically includes: mixing the pumpkin stalk medicinal material or decoction piece with water, heating and refluxing, cooling, shaking, filtering, and obtaining the solution.

8. The method according to claim 7, characterized in that The mass ratio of the pumpkin stalk raw material to water is 2g:20mL; the heating reflux time is 30min.

9. The method according to claim 1, characterized in that: When the injection concentration range of the vanillic acid reference substance is 0.3514956-351.4956 μg / ml, the linear relationship is y=38549x+78.516, R 2 =1.

10. The method according to claim 1, characterized in that The minimum detection limit of the vanillic acid is 0.3514956ug / ml, and the quantitative limit is 0.3514956-351.4956μg / ml.

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