Construction Method of HPLC Characteristic Chromatogram of Medicinal Materials, Sliced Medicinal Herbs, Standard Decoctions and Their Formula Granules of Luffa cylindrica (L.) Roem

Abstract

The present invention provides a method for constructing HPLC characteristic profiles of Cucurbitacin radiata medicinal materials, decoction pieces, standard decoctions, and formulated granules thereof. The method employs high-performance liquid chromatography (HPLC) with a mobile phase of acetonitrile-0.1% phosphoric acid in water for gradient elution. The method establishes HPLC characteristic profiles of Cucurbitacin radiata medicinal materials, decoction pieces, standard decoctions, and formulated granules thereof. The method demonstrates good repeatability and precision, and is stable and reliable, enabling quality control of Cucurbitacin radiata medicinal materials, decoction pieces, standard decoctions, and formulated granules thereof.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and detection, and in particular relates to a method for constructing HPLC characteristic maps of Cucurbita ulmoides medicinal materials, decoction pieces, standard decoctions and their formula particles. Background Art

[0002] Siceraria siceraria is the dried peel of the nearly mature fruit of Lagenaria siceraria (Molina) Standl. var. depressa (Ser.) Hara, a plant of the Cucurbitaceae family. It has diuretic, anti-inflammatory, and stranguria-relieving properties. Standard decoctions are freeze-dried powders made from the herbs after processing according to a fixed preparation process.

[0003] In order to ensure the uniformity and stability of the quality of gourd medicinal materials, decoction pieces and their standard decoctions, it is necessary to establish a characteristic spectrum method for gourd medicinal materials, decoction pieces and their standard decoctions 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 characteristic spectra of Cucurbitaceae medicinal materials, decoction pieces, standard decoctions and their formula granules. The HPLC characteristic spectra method of Cucurbitaceae 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 Cucurbitaceae medicinal materials, decoction pieces, standard decoctions and their formula granules.

[0005] The present invention provides a method for constructing an HPLC characteristic spectrum of a Chinese gourd herb, a decoction piece, a standard decoction, and a formula granule thereof, comprising the following steps:

[0006] A) treating the raw material containing the gourd with a solvent to obtain a test solution;

[0007] The raw material containing Gastrodia elata is selected from Gastrodia elata medicinal materials, decoction pieces, standard decoctions or their formula granules;

[0008] B) the need testing solution is measured by high performance liquid chromatography to obtain an HPLC characteristic spectrum of the raw material containing the gourd;

[0009] The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is a 0.1% phosphoric acid aqueous solution, and the elution is performed in a gradient manner.

[0010] Preferably, in the construction method, the preparation method of the test solution of the characteristic spectrum of the medicinal material of Cucurbitacin is: mixing the Cucurbitacin powder with water, heating and refluxing, filtering, evaporating the filtrate to dryness, dissolving the residue with methanol, filtering, and taking the filtrate to obtain the test solution of the characteristic spectrum of the medicinal material of Cucurbitacin;

[0011] The preparation method of the characteristic spectrum test solution of the Cucurbitacin decoction piece is as follows: mixing the Cucurbitacin decoction piece powder with water, heating and refluxing, filtering, evaporating the filtrate to dryness, dissolving the residue with methanol, filtering, and taking the filtrate to obtain the characteristic spectrum test solution of the Cucurbitacin decoction piece;

[0012] The preparation method of the characteristic spectrum test solution of the standard decoction of Cucurbitacin is as follows: taking the freeze-dried powder of the standard decoction of Cucurbitacin and mixing it with methanol, ultrasonically treating it, cooling it, filtering it, and taking the filtrate to obtain the characteristic spectrum test solution of the standard decoction of Cucurbitacin;

[0013] The preparation method of the characteristic spectrum test solution of the Chuhulu formula granules is as follows: taking the Chuhulu formula granules and mixing them with methanol, performing ultrasonic treatment, cooling, filtering, and taking the filtrate to obtain the characteristic spectrum test solution of the Chuhulu formula granules.

[0014] Preferably, the method further includes the preparation of a reference substance solution and a reference medicinal material solution:

[0015] Preparation of the reference substance solution: taking a vanillic acid reference substance, adding methanol to dissolve it to obtain a reference substance solution, wherein the concentration of vanillic acid in the reference substance solution is 20 μg / mL;

[0016] Preparation of the control medicinal material reference solution: taking the Cucurbitaceae control medicinal material, adding water, heating and refluxing extraction, filtering, evaporating the filtrate to dryness, adding methanol to dissolve the residue, filtering, and taking the filtrate as the control medicinal material reference solution;

[0017] The reference substance solution and the reference medicinal material reference solution are measured by high performance liquid chromatography to obtain chromatograms of the reference substance and the reference medicinal material reference; and the components of the HPLC characteristic spectra of the gourd medicinal material, decoction pieces, standard decoction and its formula granules are analyzed based on the chromatograms of the reference substance and the reference medicinal material reference.

[0018] Preferably, the gradient elution procedure is specifically as follows:

[0019] 0-25 min, phase A: 12%, phase B: 88%;

[0020] 25-28 min, phase A: 12%-14%, phase B 88%-86%;

[0021] 28-35 min, phase A: 14%, phase B 86%;

[0022] 35-40 min, phase A 14%-25%, phase B 86%-75%;

[0023] 40-60min, phase A 25%, phase B 75%.

[0024] Preferably, the chromatographic column is a C18 column, and the column temperature is 35°C.

[0025] Preferably, the flow rate of the mobile phase is 1.0 ml / min; the injection volume is 5 μL.

[0026] Preferably, the detection wavelength is 264 nm.

[0027] Preferably, in the HPLC characteristic spectrum of the standard decoction of Chu Hu Lu, the peak corresponding to the vanillic acid reference peak is taken as the S1 peak, and the relative retention times of peak 1, peak 2, peak 4, peak 5, peak 6 and peak 7 with respect to the S1 peak are calculated. The relative retention times should be within the range of ±10% of the specified values, and the specified values are 0.22 (peak 1), 0.80 (peak 2), 1.64 (peak 4), 2.34 (peak 5), 2.38 (peak 6), and 2.98 (peak 7), respectively;

[0028] In the HPLC characteristic spectrum of the medicinal material or slices of Cucurbitacin, the peak corresponding to the vanillic acid reference peak is taken as the S1 peak, and the relative retention times of peaks 1, 2, 4, 5, 6 and 7 to the S1 peak are calculated. The relative retention times should be within the range of ±10% of the specified values, which are 0.220 (peak 1), 0.802 (peak 2), 1.633 (peak 4), 2.392 (peak 5), 2.437 (peak 6) and 3.035 (peak 7), respectively.

[0029] In the HPLC characteristic spectrum of Chuhulu formula granules, the peak corresponding to the vanillic acid reference peak is taken as the S1 peak, and the relative retention times of peaks 1, 2, 4, 5, 6 and 7 relative to the S1 peak are calculated. The relative retention times should be within ±10% of the specified values, and the specified values are 0.22 (peak 1), 0.80 (peak 2), 1.64 (peak 4), 2.44 (peak 5), 2.49 (peak 6) and 3.09 (peak 7), respectively.

[0030] Compared with the prior art, the present invention provides a method for constructing an HPLC characteristic spectrum of the medicinal material, decoction pieces, standard decoctions, and formula granules thereof, comprising the following steps: A) treating a raw material containing the herb with a solvent to obtain a test solution; the raw material containing the herb is selected from the medicinal material, decoction pieces, standard decoctions, or formula granules thereof; B) subjecting the test solution to high performance liquid chromatography to obtain an HPLC characteristic spectrum of the raw material containing the herb; the chromatographic conditions of the high performance liquid chromatography are: a C18 column; mobile phase A is acetonitrile, and mobile phase B is a 0.1% phosphoric acid aqueous solution, with gradient elution. The present invention uses high performance liquid chromatography, selecting acetonitrile-0.1% phosphoric acid aqueous solution as the mobile phase for gradient elution, to establish an HPLC characteristic spectrum of the medicinal material, decoction pieces, standard decoctions, and formula granules thereof, with good repeatability and precision, and the method is stable and reliable, and can control the quality of the medicinal material, decoction pieces, standard decoctions, and formula granules thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The chromatograms of the test products at different wavelengths are shown;

[0032] Figure 2 is the column temperature investigation result;

[0033] Figure 3 is the flow velocity investigation result;

[0034] Figure 4 This is the result of delayed investigation;

[0035] Figure 5 The results of the investigation were obtained for the extraction method;

[0036] Figure 6 To investigate the results of the extraction solvent;

[0037] Figure 7 To extract the time inspection results;

[0038] Figure 8 The results of the investigation on the amount of solvent added for extraction;

[0039] Figure 9 To identify the chromatographic peaks of the characteristic spectrum of Chu Hu Lu Biao Tang;

[0040] Figure 10 is the UV absorption graph of vanillic acid;

[0041] Figure 11 To draw the results of different instruments for gourd standard soup;

[0042] Figure 12 To investigate the results of different chromatographic columns for extracting Hulu Biao Tang;

[0043] Figure 13 Characteristic spectrum of standard decoction of Chuhulu;

[0044] Figure 14 It is a comparison graph of the characteristic graph of the standard decoction of Chuhu Lu obtained in the present invention;

[0045] Figure 15 UV absorption spectrum of vanillic acid;

[0046] Figure 16 UV absorption spectrum of the test sample;

[0047] Figure 17 Chromatograms of different wavelengths of gourd medicinal materials;

[0048] Figure 18 To investigate the chromatogram for column temperature;

[0049] Figure 19 To investigate the chromatogram for flow rate;

[0050] Figure 20 This is a graph showing the delay of the finished product of the gourd;

[0051] Figure 21 The results of the investigation for the extraction method;

[0052] Figure 22 To investigate the results of the extraction solvent;

[0053] Figure 23 To extract the time inspection results;

[0054] Figure 24 The results of the investigation on the amount of solvent added;

[0055] Figure 25 To identify the chromatographic peaks of the characteristic spectrum of the gourd medicinal material;

[0056] Figure 26 Survey diagrams for different instruments;

[0057] Figure 27 To investigate the durability of the chromatographic column;

[0058] Figure 28 Verification diagram of the characteristic spectrum of 20 batches of gourd medicinal materials;

[0059] Figure 29 It is a comparative characteristic spectrum of the medicinal materials of gourd;

[0060] Figure 30 It is the characteristic spectrum of the gourd decoction piece;

[0061] Figure 31 It is the reference characteristic spectrum of the decoction pieces;

[0062] Figure 32 is the UV absorption spectrum of vanillic acid;

[0063] Figure 33 is the UV absorption spectrum of the test sample;

[0064] Figure 34 This is the chromatogram of different wavelengths of the gourd formula granules;

[0065] Figure 35 To investigate the chromatogram for column temperature;

[0066] Figure 36 To investigate the chromatogram for flow rate;

[0067] Figure 37 This is a graph showing the delay of the finished product of the gourd;

[0068] Figure 38 The results of the investigation for the extraction method;

[0069] Figure 39 To investigate the results of the extraction solvent;

[0070] Figure 40 To extract the time inspection results;

[0071] Figure 41 The results of the investigation on the amount of solvent added;

[0072] Figure 42 Identify the chromatographic peaks of the characteristic spectrum of the finished product of the gourd;

[0073] Figure 43 Survey results for different instruments;

[0074] Figure 44 This is the result of the durability investigation of the chromatographic column;

[0075] Figure 45 This is the verification diagram of the characteristic spectrum of three batches of gourd formula granules;

[0076] Figure 46 This is the reference characteristic spectrum of Chuhulu formula granules;

[0077] Figure 47 This is the chromatogram after changing the mobile phase;

[0078] Figure 48 This is the chromatogram after changing the elution program. DETAILED DESCRIPTION

[0079] The present invention provides a method for constructing an HPLC characteristic spectrum of a Chinese gourd herb, a decoction piece, a standard decoction, and a formula granule thereof, comprising the following steps:

[0080] A) treating the raw material containing the gourd with a solvent to obtain a test solution;

[0081] The raw material containing Gastrodia elata is selected from Gastrodia elata medicinal materials, decoction pieces, standard decoctions or their formula granules;

[0082] B) the need testing solution is measured by high performance liquid chromatography to obtain an HPLC characteristic spectrum of the raw material containing the gourd;

[0083] The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is a 0.1% phosphoric acid aqueous solution, and the elution is performed in a gradient manner.

[0084] The present invention first prepares the test solution. Depending on the type of raw materials, the test solution is prepared according to the following methods:

[0085] The preparation method of the characteristic spectrum of the medicinal material of Cucurbitacin is as follows: mixing the Cucurbitacin powder with water, heating and refluxing, filtering, evaporating the filtrate to dryness, dissolving the residue with methanol, filtering, and taking the filtrate to obtain the characteristic spectrum of the medicinal material of Cucurbitacin;

[0086] The mass volume ratio of the powder of the herbal medicine of the Chinese caltrop to water is 1g: (50-100)ml, preferably 1:50ml. In addition, using water as the extraction solvent can provide a large amount of information on the chromatographic peaks.

[0087] The heating reflux time is 30 to 60 minutes, preferably 30 minutes.

[0088] The preparation method of the characteristic spectrum test solution of the Cucurbitacin decoction piece is as follows: mixing the Cucurbitacin decoction piece powder with water, heating and refluxing, filtering, evaporating the filtrate to dryness, dissolving the residue with methanol, filtering, and taking the filtrate to obtain the characteristic spectrum test solution of the Cucurbitacin decoction piece;

[0089] The mass volume ratio of the powder of the herbal medicine piece of Chu Gourd to water is 1g: (50-100)ml, preferably 1:50ml. In addition, using water as the extraction solvent can provide a large amount of information on the chromatographic peak.

[0090] The heating reflux time is 30 to 60 minutes, preferably 30 minutes.

[0091] The preparation method of the characteristic spectrum test solution of the standard decoction of Chu Hu Lu is as follows: taking the freeze-dried powder of the standard decoction of Chu Hu Lu and mixing it with methanol, ultrasonically treating it, cooling it, filtering it, and taking the filtrate to obtain the characteristic spectrum test solution of the standard decoction of Chu Hu Lu.

[0092] The mass volume ratio of the freeze-dried powder of the standard decoction of Chu Hu Lu to methanol is 0.1 g: (10-45) ml, preferably 0.1 g: 20 ml. Under this ratio, the peak shape and separation of each chromatographic peak are good. In addition, when the extraction solvent is methanol, the peak shape and separation of the chromatogram are good.

[0093] The ultrasonic treatment conditions are preferably 600W power, 40kHz frequency, and 10 to 30 minutes, preferably 20 minutes. When the extraction time is 20 minutes, the chromatogram peak shape and separation are better.

[0094] The preparation method of the characteristic spectrum test solution of the Chuhulu formula granules is as follows: taking the Chuhulu formula granules and mixing them with methanol, performing ultrasonic treatment, cooling, filtering, and taking the filtrate to obtain the characteristic spectrum test solution of the Chuhulu formula granules.

[0095] Among them, the mass volume ratio of the gourd formula granules to methanol is 0.1g: (10~45)ml, preferably 0.1g: 20ml, and when the extraction solvent is methanol, the resulting chromatogram has a better peak shape, a flatter baseline, and a large amount of chromatographic peak information.

[0096] The ultrasonic treatment conditions are preferably 600W power, 40kHz frequency, and 10 to 30 minutes, preferably 20 minutes.

[0097] The chromatogram construction method provided by the present invention also includes the preparation of a reference substance solution and a reference medicinal material solution:

[0098] Preparation of the reference substance solution: taking a vanillic acid reference substance, adding methanol to dissolve it to obtain a reference substance solution, wherein the concentration of vanillic acid in the reference substance solution is 20 μg / mL;

[0099] Preparation of the control medicinal material reference solution: taking the Cucurbitaceae control medicinal material, adding water, heating and refluxing extraction, filtering, evaporating the filtrate to dryness, adding methanol to dissolve the residue, filtering, and taking the filtrate as the control medicinal material reference solution;

[0100] The reference substance solution and the reference medicinal material reference solution are measured by high performance liquid chromatography to obtain chromatograms of the reference substance and the reference medicinal material reference; and the components of the HPLC characteristic spectra of the gourd medicinal material, decoction pieces, standard decoction and its formula granules are analyzed based on the chromatograms of the reference substance and the reference medicinal material reference.

[0101] In the present invention, the mobile phase A is acetonitrile, the mobile phase B is 0.1% phosphoric acid aqueous solution, and the elution is performed by gradient elution.

[0102] The gradient elution procedure is specifically as follows:

[0103] 0-25 min, phase A: 12%, phase B: 88%;

[0104] 25-28 min, phase A: 12%-14%, phase B 88%-86%;

[0105] 28-35 min, phase A: 14%, phase B 86%;

[0106] 35-40 min, phase A 14%-25%, phase B 86%-75%;

[0107] 40-60min, phase A 25%, phase B 75%.

[0108] The present invention has good baseline separation under the above elution gradient, good separation of each peak and a stable baseline.

[0109] The chromatographic column is a C18 column, and the column temperature is 35°C. When the column temperature is 35°C, the peak shape of the chromatogram is relatively symmetrical, the separation is good, and the peak is more complete.

[0110] The flow rate of the mobile phase is 1.0 ml / min; when the flow rate is 1.0 ml / min, the chromatogram peak shape is good and the separation degree is moderate.

[0111] The injection volume was 5 μL.

[0112] The detection wavelength is 264 nm. When the detection wavelength is 264 nm, the chromatographic peak information volume is larger and the chromatogram baseline is more stable.

[0113] In the HPLC characteristic spectrum of the standard decoction of Chu Hu Lu, the peak corresponding to the vanillic acid reference peak is taken as the S1 peak, and the relative retention times of peaks 1, 2, 4, 5, 6, and 7 relative to the S1 peak are calculated. The relative retention times should be within the range of ±10% of the specified values, which are 0.22 (peak 1), 0.80 (peak 2), 1.64 (peak 4), 2.34 (peak 5), 2.38 (peak 6), and 2.98 (peak 7), respectively.

[0114] In the HPLC characteristic spectrum of the medicinal material or slices of Cucurbitacin, the peak corresponding to the vanillic acid reference peak is taken as the S1 peak, and the relative retention times of peaks 1, 2, 4, 5, 6 and 7 to the S1 peak are calculated. The relative retention times should be within the range of ±10% of the specified values, which are 0.220 (peak 1), 0.802 (peak 2), 1.633 (peak 4), 2.392 (peak 5), 2.437 (peak 6) and 3.035 (peak 7), respectively.

[0115] In the HPLC characteristic spectrum of Chuhulu formula granules, the peak corresponding to the vanillic acid reference peak is taken as the S1 peak, and the relative retention times of peaks 1, 2, 4, 5, 6 and 7 relative to the S1 peak are calculated. The relative retention times should be within ±10% of the specified values, and the specified values are 0.22 (peak 1), 0.80 (peak 2), 1.64 (peak 4), 2.44 (peak 5), 2.49 (peak 6) and 3.09 (peak 7), respectively.

[0116] The present invention establishes an HPLC characteristic spectrum method for Cucurbitacin medicinal materials, decoction pieces, standard decoctions and formula granules, providing a more scientific identification basis for identifying Cucurbitacin medicinal materials, decoction pieces, standard decoctions and formula granules.

[0117] The invention adopts a high performance liquid chromatography method, selects acetonitrile-0.1% phosphoric acid aqueous solution as a mobile phase for gradient elution, uses a vanillic acid reference substance as a reference substance, establishes HPLC characteristic spectra of the herbal medicine, herbal slices, standard decoction and formula granules thereof, has good repeatability and precision, and is stable and reliable, and can control the quality of the herbal medicine, herbal slices, standard decoction and formula granules thereof.

[0118] In order to further understand the present invention, the method for constructing the HPLC characteristic spectrum of the Cucurbitacin medicinal materials, decoction pieces, standard decoctions and formula granules provided by the present invention is described below in conjunction with the examples. The scope of protection of the present invention is not limited to the following examples.

[0119] Example 1

[0120] Determination of HPLC Characteristic Spectrum of Standard Decoction of Chuhulu

[0121] 1 Experimental instruments and materials

[0122] High performance liquid chromatograph: Agilent high performance liquid chromatograph, Shimadzu high performance liquid chromatograph, Waters high performance liquid chromatograph;

[0123] Electronic balances: ME-04E, ME204E, XPE26 (Mettler-Toledo Instruments Co., Ltd.);

[0124] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0125] Ultrasonic cleaner: KQ-600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0126] Electronic constant temperature water bath: DK-98-Ⅱ (Beijing Zhongxing Weiye Instrument Co., Ltd.)

[0127] Electronic constant temperature water bath: DZKW-4 (Beijing Zhongxing Weiye Instrument Co., Ltd.)

[0128] Chromatographic column: Shimadzu Shim-pack GIST C18.

[0129] Methanol (Norsch, chromatographic grade); phosphoric acid, acetonitrile (chromatographic grade); water was ultrapure water, and other reagents were of analytical grade.

[0130] Vanillic acid (China Food and Drug Inspection Institute, batch number: 110776-201503, content 99.8%);

[0131] Gourd control medicinal material (Shanghai Hongyong Biotechnology Co., Ltd., batch number: 230090-202301);

[0132] Standard decoction freeze-dried powder of Chuhulu (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch number: CHL-BT-230301, CHL-BT-230302, CHL-BT-230303, CHL-BT-230304, CHL-BT-230305, CHL-BT-230306, CHL-BT-230307, CHL-BT-230308, CHL-BT-230309) 9. CHL-BT-230310, CHL-BT-230311, CHL-BT-230312, CHL-BT-230313, CHL-BT-230314, CHL-BT -230315, CHL-BT-230316, CHL-BT-230318, CHL-BT-230319, CHL-BT-230320, CHL-BT-230321)

[0133] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the filler; acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B, gradient elution as specified in Table 1 below, detection wavelength at 264 nm, flow rate of 1.0 mL / min, and column temperature of 35°C. The mobile phase gradient is shown in Table 1 below.

[0134] Table 1

[0135]

[0136] Preparation of Reference Solution: Take 1 g of the reference medicinal material, Gastrodia elata, and add 50 ml of water. Heat and reflux for 30 minutes, filter, and evaporate the filtrate to dryness. Dissolve the residue in 5 ml of methanol, filter, and use the filtrate as the reference medicinal material solution. Separately, accurately weigh an appropriate amount of vanillic acid reference standard and add methanol to prepare a solution containing 20 μg of vanillic acid per 1 mL. This will serve as the reference solution.

[0137] Preparation of the test solution: Take about 0.1 g of the product powder, place it in a stoppered conical flask, add 20 ml of methanol, and ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes. Let it cool, shake well, filter, and take the filtrate to obtain the product.

[0138] Determination method: Accurately aspirate 5 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0139] 2 Chromatographic conditions and system suitability test

[0140] 2.1 Wavelength Selection

[0141] Based on the experimental conditions proposed above, the diode array detector was used to perform full-band scanning of the test solution, and the chromatograms of the test solution (batch number CHL-BT-230301) were extracted. Figure 1 , Figure 1 The chromatograms of the test samples at different wavelengths are shown.

[0142] The results showed that the chromatographic peak information content was larger and the chromatogram baseline was more stable when the detection wavelength was 264 nm, so the detection wavelength was determined to be 264 nm.

[0143] 2.2 Column temperature investigation

[0144] Based on the experimental conditions proposed above, the column temperatures of 25℃, 30℃ and 35℃ were investigated respectively. Figure 2 , Figure 2 Column temperature investigation results.

[0145] The results showed that when the column temperature was 35℃, the chromatogram peak shape was more symmetrical, the separation was better, and the peaks were more complete. Therefore, the column temperature was determined to be 35℃.

[0146] 2.3 Flow rate investigation

[0147] Based on the experimental conditions proposed above, the flow rates of 0.8ml / min, 1.0ml / min, and 1.2ml / min were investigated. Figure 3 , Figure 3 The flow rate investigation results.

[0148] The results showed that the flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were investigated. At a flow rate of 1.0 ml / min, the chromatogram peak shape was better and the separation was moderate. Therefore, the flow rate was determined to be 1.0 ml / min.

[0149] 2.4 Latency Investigation

[0150] Based on the experimental conditions proposed above, a delay test was conducted. The results are shown in Figure 4 , Figure 4 This is the result of delayed investigation.

[0151] The results showed that the sample had basically no useful chromatographic peak after 60 minutes, so the sample detection time was set at 60 minutes.

[0152] In summary, the chromatographic conditions and system suitability test for the characteristic spectrum of the standard decoction of Chuhulu were determined as follows: acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B, gradient elution according to the regulations in the following table, a flow rate of 1.0 ml per minute; column temperature of 35°C; detection wavelength of 264 nm.

[0153] 3 Preparation of test solution

[0154] 3.1 Extraction method

[0155] Take about 0.1g of this product (batch number: CHL-BT-230321), place it in a stoppered conical flask, add 20mL of methanol, and seal it tightly. The extraction method of the test product is reflux and ultrasound (power 600W, frequency 40kHz), respectively. The extraction time is 20 minutes. Let it cool, shake well, filter, and take the filtrate to obtain. Figure 5 , Figure 5 The results of the extraction method were investigated.

[0156] The results showed that ultrasonic extraction and reflux extraction had the same effect on the test sample. Because ultrasonic extraction is more convenient, ultrasonic extraction was determined to be the extraction method for the test sample.

[0157] 3.2 Extraction solvent investigation

[0158] Take about 0.1g of this product (batch number: CHL-BT-230321) and place it in a stoppered conical flask. Add 20mL each of water, 30% methanol, 70% methanol, methanol, and ethanol. Seal the flask tightly and sonicate (power 600W, frequency 40kHz) for 20 minutes. Let it cool, shake well, filter, and take the filtrate to obtain the product. Figure 6 , Figure 6 The results of the extraction solvent investigation are shown.

[0159] The results showed that the chromatogram peak shape and separation were better when methanol was used as the extraction solvent. Therefore, methanol was tentatively selected as the extraction solvent.

[0160] 3.3 Extraction time investigation

[0161] Take about 0.1g of this product (batch number: CHL-BT-230321), place it in a stoppered conical flask, add 20mL of methanol, seal it tightly, and treat it with ultrasound (power 600W, frequency 40kHz). Observe the extraction time of the test sample at 10 minutes, 20 minutes, and 30 minutes respectively. Let it cool, shake well, filter, and take the filtrate to obtain the product. Figure 7 , Figure 7 This is the result of the extraction time investigation.

[0162] The results showed that the chromatogram peak shape and separation were better when the extraction time was 20 minutes. Therefore, the extraction time was determined to be 20 minutes.

[0163] 3.4 Investigation of the amount of extraction solvent added

[0164] Take about 0.1g of this product (batch number: CHL-BT-230321) and place it in a stoppered conical flask. Add 10ml, 20ml and 45ml of methanol respectively. Seal the flask tightly and ultrasonically treat (power 600W, frequency 40kHz) for 20 minutes. Let it cool, shake well, filter and take the filtrate to obtain the product. Figure 8 , Figure 8 The results of the investigation on the amount of extraction solvent added.

[0165] The results showed that when the extraction solvent was 20 ml, the peak shape and separation of each chromatographic peak were better, so 20 ml of solvent was selected.

[0166] In summary, the preparation method of the test solution of the characteristic spectrum of the standard decoction of Chuhu Lu is determined as follows: take about 0.1 g of the powder of this product, place it in a conical flask, add 20 ml of methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, cool, shake well, filter, and take the filtrate.

[0167] 4 Methodological Investigation

[0168] 4.1 Chromatographic peak identification

[0169] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of standard decoction of Chuhulu.

[0170] Preparation of reference solution: Take 1 g of the reference herb, add 50 ml of water, heat under reflux for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5 ml of methanol, filter, and collect the filtrate. Separately, accurately weigh an appropriate amount of vanillic acid reference standard, and add methanol to prepare a solution containing 20 μg of vanillic acid per 1 mL. This will serve as the reference solution.

[0171] Preparation of negative control solution: According to the experimental conditions proposed above, a negative control solution of the standard decoction of gourd lacking tangerine peel was prepared, namely methanol (blank solution).

[0172] Position the characteristic peaks of the standard decoction of Chuhulu. Figure 9-10 , Figure 9 To identify the chromatographic peaks of the characteristic spectrum of Hulubiao Decoction. Figure 10 This is the UV absorption diagram of vanillic acid.

[0173] The results showed that peak 3 was vanillic acid. In the following methodological test, 7 characteristic peaks in the sample were investigated.

[0174] 4.2 Precision test

[0175] Accurately weigh 1 portion of the freeze-dried powder of the standard decoction of Chu Hu Lu (batch number: CHL-BT-230321), prepare the sample according to the proposed experimental method, and inject the sample 6 times continuously. See Table 2

[0176] Table 2 Precision investigation-retention time

[0177]

[0178] The results showed that the RSD values of the retention times of the peaks were 0.06% to 0.46%, indicating that the instrument had good precision.

[0179] 4.3 Repeatability study

[0180] Six portions of freeze-dried powder of the standard decoction of Gastrodia elata (Batch No. CHL-BT-230321) were accurately weighed and prepared and assayed according to the proposed experimental method (see Table 3).

[0181] Table 3 Repeatability study - characteristic peak relative retention time ratio

[0182]

[0183] The results showed that the RSDs of the relative retention times of the characteristic peaks were between 0.14% and 0.82%, indicating that the method had good repeatability.

[0184] 4.4 Intermediate precision study

[0185] 4.4.1 Investigation of different instruments

[0186] Based on the experimental conditions proposed above, two portions of freeze-dried powder of standard decoction of cucurbitacin (batch number: CHL-BT-230321) were accurately weighed to prepare the test solution, which was then measured on Agilent, Waters, and Shimadzu high performance liquid chromatographs. Figure 11 , Table 4. Figure 11 The results of the investigation of different instruments for drawing gourd standard soup.

[0187] Table 4. Relative retention time ratio of characteristic peaks in different instruments

[0188]

[0189] The results showed that when the three instruments were used to detect the test samples, the RSDs of the relative retention times of the characteristic peaks were between 0.73% and 4.63%.

[0190] 4.4.2 Inspection by different personnel and time

[0191] Based on the experimental conditions proposed above, two aliquots of lyophilized powder of the standard decoction of C. chulunii (Batch No.: CHL-BT-230321) were accurately weighed by different individuals (A and B) at different times (T1 and T2) to prepare test samples for determination (see Table 5).

[0192] Table 5. Relative retention time ratio of characteristic peaks for different personnel and time periods

[0193]

[0194] The results showed that under different sample preparation personnel and different sample preparation time conditions, the RSDs of the relative retention times of the characteristic peaks were between 0.12% and 0.29%.

[0195] 4.5 Durability inspection

[0196] 4.5.1 Column durability assessment

[0197] Based on the experimental conditions proposed above, different brands of chromatographic columns Shimadzu Shim-pack GISTC18, Agilent ZORBAXSB, Waters The results are shown in Figure 12 , Table 6. Figure 12 To investigate the results of different chromatographic columns for extracting Hulubiao Decoction.

[0198] Table 6 Column durability investigation - characteristic peak relative retention time ratio

[0199]

[0200] The results showed that the RSDs of the relative retention times of the characteristic peaks of different brands of chromatographic columns ranged from 1.9 to 17.07%.

[0201] 4.5.2 Stability investigation

[0202] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 4h, 8h, 12h, 16h, and 24h. See Table 7.

[0203] Table 7 Stability Study - Retention Time

[0204]

[0205]

[0206] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.06% and 0.32%.The sample solution was relatively stable within 24 hours.

[0207] In summary, the RSDs of the relative retention times of the characteristic peaks met the requirements in all the above investigations, indicating that the method was good. The above seven characteristic peaks were included in the subsequent investigations.

[0208] 5. Determination of characteristic peaks and establishment of reference maps

[0209] 5.1 Verification results of 20 batches of standard decoction of gourd

[0210] Using this method, characteristic spectrum analysis was performed on 20 batches of samples and relative retention times were calculated. Figure 13 , Table 8. Figure 13 Characteristic spectrum of standard decoction of Chuhulu. Figure 13 Among them, S1~S20 are: CHL-BT-230301, CHL-BT-230302, CHL-BT-230303, CHL-BT-230304, CHL-BT-2 30305, CHL-BT-230306, CHL-BT-230307, CHL-BT-230308, CHL-BT-230309, CHL-BT-230310, C HL-BT-230311, CHL-BT-230312, CHL-BT-230313, CHL-BT-230314, CHL-BT-230315, CHL-BT-230316, CHL-BT-230318, CHL-BT-230319, CHL-BT-230320, CHL-BT-230321; R is the reference atlas of the standard decoction of Chu Hu Lu.

[0211] Table 8 Relative retention time ratio of Chu Hu Lu Biao decoction

[0212]

[0213]

[0214] Based on the principle of stable relative retention times and the fact that peaks can be detected in all batches of samples at relatively high levels, a total of seven peaks with good reproducibility were selected as characteristic peaks. The RSDs of the relative retention times of peaks 1 to 7 were 0.04% to 0.17%.

[0215] 5.2 Establishment of relative retention time limit values

[0216] The summary of the methodology inspection items and verification results is shown in Table 9:

[0217] Table 9 Summary of RSD (%) results of each item of the methodology - relative retention time

[0218]

[0219] As can be seen from the table above, the relative retention time of each feature is stable and within the range of ±10% of the average value, so the relative retention time of each peak is temporarily set at ±10%.

[0220] Final regulations: The test sample chromatogram should show seven characteristic peaks, and their retention times should correspond to those of the seven characteristic peaks in the chromatogram of the reference medicinal material. Peak 3 should correspond to the retention time of the reference material. Calculate the relative retention time of each characteristic peak to the S peak, and the relative retention time should be within ±10% of the specified value. The specified values are 0.22 (peak 1), 0.80 (peak 2), 1.64 (peak 4), 2.34 (peak 5), 2.38 (peak 6), and 2.98 (peak 7).

[0221] The Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition) was used to synthesize 20 batches of standard decoctions of Chu Hu Lu, and a comparison spectrum of the characteristic spectrum of standard decoctions of Chu Hu Lu was established. Figure 14 , Figure 14 This is a comparison graph of the characteristic graph of the standard decoction of Chuhu Lu obtained in the present invention.

[0222] Determination of the characteristic spectrum of the standard decoction of 6-pulled gourd

[0223] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler; acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B, gradient elution as specified in the table below, and detection wavelength at 264 nm; the flow rate was 1.0 mL / min, and the column temperature was 35°C; the number of theoretical plates based on the vanillic acid peak was not less than 5000; the mobile phase gradient is shown in Table 1.

[0224] Preparation of Reference Solution: Take 1 g of the reference medicinal material, Gastrodia elata, and add 50 ml of water. Heat and reflux for 30 minutes, filter, and evaporate the filtrate to dryness. Dissolve the residue in 5 ml of methanol, filter, and use the filtrate as the reference medicinal material solution. Separately, accurately weigh an appropriate amount of vanillic acid reference standard and add methanol to prepare a solution containing 20 μg of vanillic acid per 1 mL. This will serve as the reference solution.

[0225] Preparation of the test solution: Take about 0.1 g of the product powder, place it in a stoppered conical flask, add 20 ml of methanol, and ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes. Let it cool, shake well, filter, and take the filtrate to obtain the product.

[0226] Determination method: Accurately aspirate 5 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0227] Example 2

[0228] HPLC characteristic spectrum of gourd medicinal materials and decoction pieces

[0229] 1 Experimental instruments and materials

[0230] High performance liquid chromatograph: Agilent high performance liquid chromatograph, Shimadzu high performance liquid chromatograph, Waters high performance liquid chromatograph;

[0231] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0232] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0233] Ultrasonic cleaner: KQ600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0234] Chromatographic columns: Shimadzu Shim-pack GIST C18, Agilent ZORBAX C18, Waters

[0235] 2 Reagents and test drugs

[0236] Acetonitrile (chromatographic grade, Sigma-Aldrich Shanghai Trading Co., Ltd.), phosphoric acid (chromatographic grade, Chengdu Norsch Technology Co., Ltd.), methanol (analytical grade, Chengdu Kelong Chemical Reagent Co., Ltd.), and the mobile phase water were laboratory-made ultrapure water, and the rest were laboratory-made pure water.

[0237] Gourd control medicinal material (Shanghai Hongyong Biotechnology Co., Ltd., batch number: 230090-202301);

[0238] Vanillic acid (China Food and Drug Inspection Institute, batch number: 110776-201503, content 99.8%);

[0239] Pumping gourd medicinal materials (batch number: XLS202109264, XLS202109265, XLS202109266, XLS202109267, XLS202109268, XLS202109269, XLS202109270, XLS202109271, XLS202109272, XLS2021092 73. XLS202109274, XLS202109275, XLS202109276, XLS202109277, XLS202109279, XLS202109281, XLS202109282, XLS202109283, 010606-2210001).

[0240] 3 Chromatographic conditions

[0241] Determined by high performance liquid chromatography (General Chapter 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia).

[0242] Chromatographic Conditions and System Suitability: Octadecylsilane bonded silica gel was used as the filler; acetonitrile was used as mobile phase A, and 0.1% aqueous phosphoric acid solution was used as mobile phase B. Gradient elution was performed as specified in the following table; the flow rate was 1.0 ml per minute; the column temperature was 30° C.; and the detection wavelength was 264 nm.

[0243] Table 10

[0244]

[0245] Preparation of reference solution: Take an appropriate amount of vanillic acid reference substance, weigh accurately, and add methanol to make a solution containing 20 μg per 1 ml.

[0246] Preparation of control medicinal material solution: Take 1 g of Cucurbitacin control medicinal material, add 50 ml of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 5 ml of methanol to dissolve the residue, filter, and take the filtrate as the control medicinal material reference solution.

[0247] Preparation of test solution: Take about 1.0 g of the product (passed through No. 3 sieve), place it in a stoppered conical flask, add 50 ml of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5 ml of methanol, filter, and take the filtrate to obtain.

[0248] Determination method: Accurately aspirate 5 μl of each of the above solutions, inject into liquid chromatograph, and determine.

[0249] 4 Chromatographic conditions and system suitability test

[0250] 4.1 Wavelength Selection

[0251] Based on the experimental conditions proposed above, the diode array detector was used to perform full-band scanning of vanillic acid and the test solution, and the chromatograms of the test solution at wavelengths of 254 nm, 264 nm, 280 nm, 300 nm, and 320 nm were extracted, respectively. Figures 15-17 . Figure 15 Vanillic acid UV absorption spectrum, Figure 16 UV absorption spectrum of the test sample, Figure 17 Chromatograms of different wavelengths of gourd medicinal materials.

[0252] The results showed that the chromatographic peak information content was larger and the chromatogram baseline was more stable when the detection wavelength was 264 nm, so the detection wavelength was determined to be 264 nm.

[0253] 4.2 Column temperature investigation

[0254] Based on the experimental conditions proposed above, the column temperatures of 25℃, 30℃ and 35℃ were investigated respectively. Figure 18 . Figure 18 This is a chromatogram for column temperature investigation.

[0255] The results showed that when the column temperature was 35℃, the chromatogram peaks were relatively symmetrical and the separation was good. Therefore, the column temperature for the characteristic spectrum of the medicinal material Cucurbita lappa was determined to be 35℃.

[0256] 4.3 Flow rate investigation

[0257] Based on the experimental conditions proposed above, the flow rates of 0.8ml / min, 1.0ml / min, and 1.2ml / min were investigated. Figure 19 , Figure 19 Examine the chromatogram for flow rate.

[0258] The results showed that when the flow rate was 1.0 ml / min, the peak shapes of the characteristic peaks were good and the separation was moderate. Therefore, the flow rate of 1.0 ml / min was determined to be the characteristic spectrum of the gourd herb.

[0259] 4.4 Delay test

[0260] Based on the experimental conditions proposed above, the chromatogram acquisition time was extended to 120 min. Figure 20 shown. Figure 20 This is a delay inspection diagram for the finished gourd product.

[0261] The results showed that the chromatogram peaks were completely collected at 60 minutes, so the chromatogram acquisition time was determined to be 60 minutes.

[0262] In summary, the chromatographic conditions and system suitability test for the characteristic spectrum of the medicinal material of Cucurbitacin were determined as follows: octadecylsilane bonded silica gel was used as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid aqueous solution was used as mobile phase B, the flow rate was 1.0 ml / min, the column temperature was 35°C, and the detection wavelength was 264 nm. Gradient elution was performed according to the requirements in Table 11 below;

[0263] Table 11

[0264]

[0265] 5 Preparation of test solution

[0266] 5.1 Investigation of extraction methods

[0267] Take about 1.0g of this product (batch number: 010303-2210001, passed through No. 3 sieve), place it in a stoppered conical flask, add 50ml of water, reflux and ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes respectively, let it cool, shake well, filter, and take the filtrate to obtain. Figure 21 , Figure 21 The results of the extraction method were investigated.

[0268] The results showed that heating reflux extraction was more effective when ultrasonic extraction and reflux extraction were performed on the sample. Therefore, heating reflux was determined to be the extraction method for the sample.

[0269] 5.2 Extraction solvent investigation

[0270] Take about 1.0g of this product (batch number: 010303-2210001, passed through No. 3 sieve), add 20ml each of water, 30% methanol, 70% methanol, methanol, and ethanol, heat and reflux for 30 minutes, cool, shake well, filter, and take the filtrate to obtain. Figure 22 , Figure 22 The results of the extraction solvent investigation are shown.

[0271] The results showed that water extraction had the best effect and the chromatographic peaks had a large amount of information, so water was determined to be the extraction solvent for the test sample.

[0272] 5.3 Extraction time investigation

[0273] Take about 1.0g of this product (batch number: 010303-2210001, passed through No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, add 50ml of water, heat and reflux for 30 minutes and 60 minutes respectively, filter, evaporate the filtrate to dryness, dissolve the residue in 5ml of methanol, filter, and take the filtrate to obtain. Figure 23 , Figure 23 This is the result of the extraction time investigation.

[0274] The results showed that the extraction time of 30 minutes and 60 minutes had the same effect. Considering all factors, 30 minutes was selected as the extraction time of the test sample.

[0275] 5.4 Investigation of solvent addition amount

[0276] Take about 1.0g of this product (batch number: 010303-2210001, passed through No. 3 sieve) and place it in a stoppered conical flask. Add 50ml and 100ml of water respectively. Heat and reflux for 30 minutes. Filter. Evaporate the filtrate to dryness. Dissolve the residue in 5ml of methanol. Filter. Take the filtrate to obtain the product. Figure 24 , Figure 24 The results are for the investigation of the amount of solvent added.

[0277] The results showed that the extraction effects were consistent when the extraction solvent was 50ml and 100ml. After comprehensive consideration, 50ml of solvent was chosen.

[0278] In summary, the preparation method of the test solution of the characteristic spectrum of the medicinal material of Cucurbitacin is determined as follows: take about 1.0 g of this product (passed through No. 3 sieve), place it in a stoppered conical flask, add 50 ml of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5 ml of methanol, filter, and take the filtrate to obtain.

[0279] 6 Methodological Investigation

[0280] 6.1 Chromatographic Peak Identification

[0281] Preparation of negative sample solution: According to the experimental conditions proposed above, the negative sample solution of the medicinal material of Cucurbita ulmoides is prepared, which is methanol (blank solution).

[0282] Preparation of reference solution: Take an appropriate amount of vanillic acid reference substance, weigh accurately, and add methanol to prepare a solution containing 20 μg per 1 ml.

[0283] Preparation of control medicinal materials: Take 1 g of Cucurbitacin control medicinal materials, add 50 ml of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 5 ml of methanol to dissolve the residue, filter, and take the filtrate as the control medicinal material reference solution.

[0284] Preparation of test solution: Take about 1.0 g of the product (passed through No. 3 sieve), place it in a stoppered conical flask, add 50 ml of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5 ml of methanol, filter, and take the filtrate to obtain.

[0285] Position the characteristic spectrum peaks of the medicinal material of Chu Hu Lu. Figure 25 , Figure 25 To identify the chromatographic peaks of the characteristic spectrum of the gourd medicinal material.

[0286] In the following methodological investigation, vanillic acid was set as the S peak, and the seven characteristic peaks in the sample spectrum were investigated.

[0287] 6.2 Precision test

[0288] Take approximately 1.0 g of this product (batch number: 010606-2210001, passed through a No. 3 sieve) and prepare the test solution of the herb Gastrodia elata according to "7.2.8.6". Inject 5 μl six times continuously according to the proposed test method, and calculate the retention time of each characteristic peak. See Table 12.

[0289] Table 12 Precision investigation - characteristic peak retention time

[0290]

[0291] The results showed that the RSD of the retention time of each characteristic peak in the precision investigation was between 0.02% and 0.13%, indicating a good precision.

[0292] 6.3 Repeatability Study

[0293] About 1.0 g of this product (batch number: 010606-2210001, passed through a No. 3 sieve) was prepared and assayed according to the proposed experimental method (see Table 13).

[0294] Table 13 Repeatability study - relative retention time of characteristic peaks

[0295]

[0296] The results showed that the RSD of the characteristic peak relative retention time was between 0.14% and 0.39%, with good repeatability.

[0297] 6.4 Intermediate precision study

[0298] 6.4.1 Inspection by different personnel and time

[0299] Based on the experimental conditions proposed above, two samples of approximately 1.0 g of this product (Batch No. 010606-2210001, passed through a No. 3 sieve) were weighed by different individuals (A and B) at different times (T1 and T2) to prepare test samples for measurement. See Table 14.

[0300] Table 14 Different personnel and time observation - characteristic peak relative retention time ratio

[0301]

[0302]

[0303] The results showed that under different sample preparation personnel and different sample preparation time conditions, the RSDs of the relative retention times of the characteristic peaks were between 0.14% and 0.39%.

[0304] 6.4.2 Investigation of different instruments

[0305] Take about 1.0g of this product (batch number: 010606-2210001, passed through No. 3 sieve) to prepare the test solution, and measure it on Agilent, Shimadzu, and Waters high performance liquid chromatographs respectively. See Table 15, Figure 26 , Figure 26 This is a diagram of an inspection of different instruments.

[0306] Table 15 Instrument investigation - relative retention time of characteristic peaks

[0307]

[0308] The results showed that the RSDs of the relative retention times of the characteristic peaks were between 0.57% and 1.26% when detected by the three instruments mentioned above.

[0309] 6.5 Durability inspection

[0310] 6.5.1 Column Durability Assessment

[0311] Based on the experimental conditions proposed above, the chromatographic columns were Shimadzu Shim-pack GIST C18, Agilent ZORBAX SB, Waters See Table 16. Figure 27 , Figure 27 To investigate the durability of the chromatographic column.

[0312] Table 16 Column durability investigation - relative retention time of characteristic peaks

[0313]

[0314] The results showed that the Shimadzu Shim-pack GIST C18 column had better peak shapes and resolution when used to detect samples using the three columns mentioned above. Therefore, the Shimadzu Shim-pack GIST C18 column is recommended for characteristic spectrum determination.

[0315] 6.5.2 Stability investigation

[0316] Based on the experimental conditions proposed above, the same test solution was measured at 0h, 2h, 4h, 8h, 12h, 18h, and 24h. See Table 17.

[0317] Table 17 24-hour stability study - characteristic peak retention time

[0318]

[0319]

[0320] The results showed that the RSDs of the retention times of the characteristic peaks were between 0.08% and 0.75%. The test solution was stable within 24 hours.

[0321] In summary, the RSDs of the relative retention times of the characteristic peaks met the requirements in all the above investigations, indicating that the method was good. The above seven characteristic peaks were included in the subsequent investigations.

[0322] 7 Determination of characteristic peaks and establishment of reference spectrum

[0323] 7.1 Verification results of 20 batches of bottle gourd medicinal materials

[0324] The proposed method was used to determine the characteristic spectra of 20 batches of samples of this product and calculate the relative retention time. Figure 28 , Table 18. Figure 28 This is the verification diagram of the characteristic spectrum of 20 batches of gourd medicinal materials. Figure 28In the example, S1 to S20 are: XLS202109264, XLS202109265, XLS202109266, XLS202109267, XLS202109268, XLS202109269, XLS202109270, XLS202109271, XLS202109272, XLS202109273, XLS202109274, XLS202109275, XLS202109276, XLS202109277, XLS202109278, XLS202109279, XLS202109280, XLS202109281, XLS202109282, XLS202109283, XLS202109284, XLS202109285, XLS202109286, XLS202109287, XLS202109288, XLS202109289, XLS202109290, XLS202109291, XLS202109292, XLS202109293, XLS202109294, XLS202109295, XLS202109296, XLS202109297, XLS202109298, XLS202109299, XLS202109290, XLS202109291, XLS202109292, XLS202109293, XLS202109294, XLS20210 LS202109274, XLS202109275, XLS202109276, XLS202109277, XLS202109278, XLS202109279, XLS202109281, XLS202109282, XLS202109283, 010606-2210001; R is the comparison chart of the medicinal material of Cucurbitaceae.

[0325] Table 18 Relative retention time of 20 batches of gourd medicinal materials

[0326]

[0327] According to the principle of stable relative retention time and the fact that all batches of samples can be detected with relatively high peaks, a total of 7 peaks with good reproducibility were selected as characteristic peaks.

[0328] 7.2 Establishment of relative retention time limits

[0329] The summary of the methodology inspection items and verification results is shown in Table 31:

[0330] Table 19 Summary of RSD% results for each item of the methodology - relative retention time (retention time)

[0331]

[0332] Therefore, the relative retention time of each peak is temporarily set at ±10%.

[0333] The test sample chromatogram should show seven characteristic peaks, and their retention times should correspond to those of the seven characteristic peaks in the chromatogram of the reference medicinal material. Peak 3 should correspond to the retention time of the vanillic acid reference material. The peak corresponding to Peak 3 is the S peak. Calculate the relative retention times of Peaks 1, 2, 4, 5, 6, and 7 relative to the S peak. These relative retention times should be within ±10% of the specified values. The specified values are: 0.220 (Peak 1), 0.802 (Peak 2), 1.633 (Peak 4), 2.392 (Peak 5), 2.437 (Peak 6), and 3.035 (Peak 7).

[0334] The characteristic spectra of 21 batches of Cucurbitacin were synthesized by using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a reference spectrum of the characteristic spectra of Cucurbitacin was established. Figure 29 , Figure 29 This is a comparative characteristic spectrum of gourd medicinal materials.

[0335] 7.3 Verification of the characteristic spectrum of gourd slices

[0336] According to the results of "5 Preparation of test solution", 20 batches of test solution of medicinal slices were prepared, and the samples were injected, measured and analyzed according to the results of "3 Chromatographic conditions and system suitability test", and the relative retention time was calculated. Figure 30 , Table 20. Figure 30 To draw the characteristic spectrum of gourd slices, Figure 30 In the figure, each atlas represents batch S1 to S20: CHL-230301, CHL-230302, CHL-230303, CHL-230304, CHL-230305, CHL-230306, CHL-230307, CHL-230308, CHL-230309, CHL-230310, CHL-230311, CHL-230312, CHL-230313, CHL-230314, CHL-230315, CHL-230316, CHL-230318, CHL-230319, CHL-230320, CHL-230321, and R is the reference atlas of the medicinal pieces.

[0337] Table 20 Relative retention time of characteristic peaks of gourd slices

[0338]

[0339]

[0340] Based on the principle of stable relative retention times and the ability to detect peaks at relatively high levels across all batches of samples, a total of seven peaks with good reproducibility were selected as characteristic peaks. The results showed that when Peak 3 was used as the S peak, the relative retention time RSDs of the seven characteristic peaks in 20 batches of gourd slices ranged from 0.22% to 3.05%.

[0341] Final regulations: The test sample's characteristic spectrum should show seven characteristic peaks, and their retention times should correspond to those of the seven characteristic peaks in the chromatogram of the reference medicinal material. Peak 3 should correspond to the retention time of the chromatogram peak of the vanillic acid reference. The peak corresponding to the vanillic acid reference is the S peak. The relative retention times of each characteristic peak and the S peak should be calculated and should be within ±10% of the specified values. The specified values are: 0.222 (peak 1), 0.799 (peak 2), 1.642 (peak 4), 2.410 (peak 5), 2.456 (peak 6), and 3.051 (peak 7).

[0342] The characteristic spectra of 21 batches of Chu Hu Lu decoction pieces were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a reference spectrum of the characteristic spectra of Chu Hu Lu decoction pieces was established. Figure 31 , Figure 31 It is a comparative characteristic spectrum of medicinal pieces.

[0343] 8. Determination of characteristic spectrum method of gourd medicinal materials and decoction pieces

[0344] Chromatographic Conditions and System Suitability: Octadecylsilane bonded silica gel was used as the filler; acetonitrile was used as mobile phase A, and 0.1% aqueous phosphoric acid was used as mobile phase B. Gradient elution was performed as specified in the table below. The flow rate was 1.0 ml / min; the column temperature was 30°C; and the detection wavelength was 264 nm. The number of theoretical plates based on the vanillic acid peak should be no less than 5000.

[0345] Preparation of reference solution: Take an appropriate amount of vanillic acid reference substance, weigh accurately, and add methanol to make a solution containing 20 μg per 1 ml.

[0346] Preparation of control medicinal material solution: Take 1 g of Cucurbitacin control medicinal material, add 50 ml of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 5 ml of methanol to dissolve the residue, filter, and take the filtrate as the control medicinal material reference solution.

[0347] Preparation of test solution: Take 1 g of the product (passed through No. 3 sieve), place it in a stoppered conical flask, add 50 ml of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5 ml of methanol, filter, and take the filtrate to obtain the product.

[0348] Determination method: Accurately aspirate 5 μl of each of the above solutions, inject into liquid chromatograph, and determine.

[0349] Example 3

[0350] HPLC characteristic spectrum of Chuhulu formula granules

[0351] 1 Experimental instruments and materials

[0352] High performance liquid chromatograph: Agilent high performance liquid chromatograph, Shimadzu high performance liquid chromatograph, Waters high performance liquid chromatograph;

[0353] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0354] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0355] Ultrasonic cleaner: KQ600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0356] Chromatographic columns: Shimadzu Shim-pack GIST C18, Agilent ZORBAX; Waters

[0357] 2. Reagents and test drugs

[0358] Acetonitrile (chromatographic grade, Sigma-Aldrich Shanghai Trading Co., Ltd.), phosphoric acid (chromatographic grade, Chengdu Norsch Technology Co., Ltd.), methanol (analytical grade, Chengdu Kelong Chemical Reagent Co., Ltd.), and the mobile phase water were laboratory-made ultrapure water, and the rest were laboratory-made pure water.

[0359] Gourd control medicinal material (Shanghai Hongyong Biotechnology Co., Ltd., batch number: 230090-202301);

[0360] Vanillic acid (China Food and Drug Inspection Institute, batch number: 110776-201503, content 99.8%);

[0361] Chuhulu formula granules (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: 2303215, 2303216, 2303217).

[0362] 3 Chromatographic conditions

[0363] According to the chromatographic conditions of the characteristic spectrum of the standard decoction of Chuhulu, the conditions for the Chuhulu formula granules were determined as follows: octadecylsilane bonded silica gel was used as the filler; acetonitrile was used as the mobile phase A, and 0.1% phosphoric acid aqueous solution was used as the mobile phase B, and gradient elution was performed as specified in Table 21 below; the flow rate was 1.0 ml per minute; the column temperature was 35°C; and the detection wavelength was 264 nm.

[0364] Table 21

[0365]

[0366]

[0367] Preparation of reference solution: Take an appropriate amount of vanillic acid reference substance, weigh accurately, and add methanol to make a solution containing 20 μg per 1 ml.

[0368] Preparation of control medicinal material solution: Take 1 g of Cucurbitacin control medicinal material, add 50 ml of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 5 ml of methanol to dissolve the residue, filter, and take the filtrate as the control medicinal material reference solution.

[0369] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take 1.0 g, place it in a stoppered conical flask, add 20 ml of methanol, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 20 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0370] Determination method: Accurately aspirate 5 μl of each of the above solutions, inject into liquid chromatograph, and determine.

[0371] 4 Chromatographic conditions and system suitability test

[0372] 4.1 Wavelength Selection

[0373] Based on the experimental conditions proposed above, the diode array detector was used to perform full-band scanning of vanillic acid and the test solution, and the chromatograms of the test solution at wavelengths of 254 nm, 264 nm, 280 nm, 300 nm, and 320 nm were extracted, respectively. Figures 32-34 . Figure 32 is the UV absorption spectrum of vanillic acid. Figure 33 is the UV absorption spectrum of the test sample, Figure 34 These are chromatograms of different wavelengths of gourd formula granules.

[0374] The results showed that the chromatographic peak information content was larger and the chromatogram baseline was more stable when the detection wavelength was 264 nm, so the detection wavelength was determined to be 264 nm.

[0375] 4.2 Column temperature investigation

[0376] Based on the experimental conditions proposed above, the column temperatures of 25℃, 30℃ and 35℃ were investigated respectively. Figure 35 . Figure 35 This is a chromatogram for column temperature investigation.

[0377] The results showed that when the column temperature was 35℃, the chromatogram peaks were relatively symmetrical and the separation was good, so the column temperature for the characteristic spectrum of gourd was determined to be 35℃.

[0378] 4.3 Flow rate investigation

[0379] Based on the experimental conditions proposed above, the flow rates of 0.8ml / min, 1.0ml / min, and 1.2ml / min were investigated. Figure 36 . Figure 36 Examine the chromatogram for flow rate.

[0380] The results showed that when the flow rate was 1.0 ml / min, the peak shapes of the characteristic peaks were good and the separation was moderate. Therefore, the flow rate of the gourd characteristic spectrum was determined to be 1.0 ml / min.

[0381] 4.4 Delay test

[0382] Based on the experimental conditions proposed above, the chromatogram acquisition time was extended to 120 min. Figure 37 shown. Figure 37 This is a delay inspection diagram for the finished gourd product.

[0383] The results showed that the chromatogram peaks were completely collected at 60 minutes, so the chromatogram acquisition time was determined to be 60 minutes.

[0384] In summary, the chromatographic conditions and system suitability test for the characteristic spectrum of Chuhulu formula granules were determined as follows: octadecylsilane bonded silica gel was used as the filler (column length was 250 mm, inner diameter was 4.6 mm, and particle size was 5 μm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid aqueous solution was used as mobile phase B; the flow rate was 1.0 ml per minute; the column temperature was 35°C; the detection wavelength was 264 nm; and gradient elution was performed according to the provisions in Table 22 below.

[0385] Table 22

[0386]

[0387] 5 Preparation of test solution

[0388] 5.1 Investigation of extraction methods

[0389] Take an appropriate amount of this product (batch number: 18080082), grind it into powder, about 1.0g, place it in a stoppered conical flask, add 20ml of methanol, reflux and ultrasonically treat (power 600W, frequency 40kHz) for 20 minutes respectively, let it cool, shake well, filter, and take the filtrate to obtain the product. Figure 38 , Figure 38 The results of the extraction method were investigated.

[0390] The results showed that ultrasonic extraction and reflux extraction had the same effect on the test sample. Because ultrasonic extraction is more convenient, ultrasonic extraction was determined to be the extraction method for the test sample.

[0391] 5.2 Extraction solvent investigation

[0392] Take an appropriate amount of this product (batch number: 18080082), grind it into powder, take 1.0g, add 20ml each of water, 30% methanol, 70% methanol, methanol, and ethanol, respectively, and ultrasonically treat (power 600W, frequency 40kHz) for 20 minutes, let it cool, shake well, filter, and take the filtrate to obtain the product. Figure 39 , Figure 39 The results of the extraction solvent investigation are shown.

[0393] The results showed that when the extraction solvent was methanol, the obtained chromatogram had a better peak shape, a relatively flat baseline, and a large amount of chromatographic peak information. Therefore, the extraction solvent of the test sample was determined to be methanol.

[0394] 5.3 Extraction time investigation

[0395] Take an appropriate amount of this product (batch number: 18080082), grind it into powder, take 1.0g, weigh it accurately, put it into a stoppered conical flask, add 20ml of methanol, and ultrasonically treat it (power 600W, frequency 40kHz) for 10 minutes, 20 minutes, and 30 minutes respectively. Let it cool, shake it well, filter it, and take the filtrate to obtain it. Figure 40 , Figure 40 This is the result of the extraction time investigation.

[0396] The results showed that the extraction time of 10 minutes, 20 minutes and 30 minutes had the same effect. Taking all factors into consideration, the extraction time of 20 minutes was selected.

[0397] 5.4 Investigation of solvent addition amount

[0398] Take an appropriate amount of this product (batch number: 18080082), grind it into powder, take 1.0g, place it in a stoppered conical flask, add 10ml, 20ml, and 45ml of methanol respectively, stopper it tightly, and ultrasonically treat it (power 600W, frequency 40kHz) for 20 minutes, let it cool, shake it well, filter it, and take the filtrate to obtain the product. Figure 41 , Figure 41 This is the result of investigating the amount of solvent added.

[0399] The results showed that when the extraction solvent was 20 ml, the size of each chromatographic peak was appropriate, so the amount of solvent added was selected to be 20 ml.

[0400] In summary, the preparation method of the test solution of the characteristic spectrum of the gourd formula granules is determined as follows: take an appropriate amount of the product, grind it into powder, take 1.0 g, put it in a stoppered conical flask, add 20 ml of methanol, ultrasonically treat it (power 600 W, frequency 40 kHz) for 20 minutes, cool it, shake it evenly, filter it, and take the filtrate to obtain it.

[0401] 6 Methodological Investigation

[0402] 6.1 Chromatographic Peak Identification

[0403] Preparation of negative sample solution: According to the experimental conditions proposed above, prepare the negative sample solution of Chuhulu granules, that is, methanol (blank solution).

[0404] Preparation of reference solution: Take an appropriate amount of vanillic acid reference substance, weigh accurately, and add methanol to prepare a solution containing 20 μg per 1 ml.

[0405] Preparation of control medicinal materials: Take 1 g of Cucurbitacin control medicinal materials, add 50 ml of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 5 ml of methanol to dissolve the residue, filter, and take the filtrate as the control medicinal material reference solution.

[0406] Preparation of test solution: Take an appropriate amount of this product, grind it into powder, take 1.0g, place it in a stoppered conical flask, add 20ml of methanol, ultrasonically treat (power 600W, frequency 40kHz) for 20 minutes, cool, shake well, filter, and take the filtrate.

[0407] Locate the characteristic spectrum peaks of Chuhulu formula granules. Figure 42 , Figure 42 Identification of the chromatographic peaks of the characteristic spectrum of the finished product of the gourd: Peak 3 (S): vanillic acid.

[0408] In the following methodological investigation, vanillic acid was set as the S peak, and the seven characteristic peaks in the sample spectrum were investigated.

[0409] 6.2 Precision test

[0410] Take an appropriate amount of this product (Batch No. 18080082), grind it finely, and take 1.0g. Prepare the test solution of Chuhulu Formula Granules according to "7.2.8.6". Inject 5μl of the solution six times in succession according to the proposed test method. Calculate the retention time of each characteristic peak. See Table 23.

[0411] Table 23 Precision investigation - characteristic peak retention time

[0412]

[0413] The results showed that the RSD of the retention time of each characteristic peak in the precision study was between 0.04% and 0.36%.

[0414] 6.3 Repeatability Study

[0415] Take an appropriate amount of this product (batch number: 18080082), grind it into powder, take 1.0g of it, and prepare and measure it according to the proposed experimental method. See Table 24.

[0416] Table 24 Repeatability study - relative retention time of characteristic peaks

[0417]

[0418] The results showed that the RSD of the characteristic peak relative retention time was between 0.02% and 0.08%, with good repeatability.

[0419] 6.4 Intermediate precision study

[0420] 6.4.1 Inspection by different personnel and time

[0421] Based on the experimental conditions proposed above, different individuals (A and B) weighed and ground 1.0g of Chuhulu Formula Granules (Batch No. 18080082) at different times (T1 and T2), collected two aliquots of each, and prepared the test samples for analysis (see Table 25).

[0422] Table 25 Different personnel and time observation - characteristic peak relative retention time ratio

[0423]

[0424] The results showed that under different sample preparation personnel and different sample preparation time conditions, the RSDs of the relative retention times of the characteristic peaks were between 0.09% and 0.26%.

[0425] 6.4.2 Investigation of different instruments

[0426] Take an appropriate amount of this product (batch number: 18080082), grind it into powder, take 1.0g, prepare the test solution, and measure it on Agilent 1260, Shimadzu LC-20AD, and Waters 2695 high performance liquid chromatographs respectively. See Table 26, Figure 43 . Figure 43 The results of investigations using different instruments.

[0427] Table 26 Instrument investigation - relative retention time of characteristic peaks

[0428]

[0429] The results showed that the RSDs of the relative retention times of the characteristic peaks were between 0.75% and 4.50% when the two instruments were used for detection, indicating that the different instruments had good durability.

[0430] 6.5 Durability inspection

[0431] 6.5.1 Column Durability Assessment

[0432] Based on the experimental conditions proposed above, the chromatographic columns were Shimadzu Shim-pack GIST C18, Agilent ZORBAX SB, Waters See Table 27. Figure 44 . Figure 44 This is the result of the chromatographic column durability investigation.

[0433] Table 27 Column durability investigation - relative retention time of characteristic peaks

[0434]

[0435] The results showed that the Shimadzu Shim-pack GIST C18 column had better peak shapes and resolution when used to detect samples using the three columns mentioned above. Therefore, the Shimadzu Shim-pack GIST C18 column is recommended for characteristic spectrum determination.

[0436] 6.5.2 Stability investigation

[0437] Based on the experimental conditions proposed above, take the same test solution and measure it at 0h, 2h, 4h, 8h, 12h, 18h, and 24h. See Table 28.

[0438] Table 28 Stability Study—Characteristic Peak Retention Time

[0439]

[0440] The results showed that the RSDs of the retention times of the characteristic peaks were between 0.05% and 0.51%. The test solution was stable within 24 hours.

[0441] In summary, the RSDs of the relative retention times of the characteristic peaks met the requirements in all the above investigations, indicating that the method was good. The above seven characteristic peaks were included in the subsequent investigations.

[0442] 7 Determination of characteristic peaks and establishment of reference maps

[0443] 7.1 Verification results of three batches of gourd formula granules

[0444] The proposed method was used to determine the characteristic spectra of three batches of samples of this product and calculate the relative retention time. Figure 45 , Table 29. Figure 45 This is the verification diagram of the characteristic spectrum of three batches of gourd formula granules. Figure 45 Among them, S1~S3 correspond to batch numbers 2303215, 2303216 and 2303217 of gourd formula granules respectively.

[0445] Table 29 Relative retention time of three batches of Chuhulu formula granules

[0446]

[0447]

[0448] According to the principle of stable relative retention time and the fact that all batches of samples can be detected with relatively high peaks, a total of 7 peaks with good reproducibility were selected as characteristic peaks.

[0449] 7.2 Establishment of relative retention time limits

[0450] The summary of the methodology inspection items and verification results is shown in Table 48:

[0451] Table 30 Summary of RSD% results for each item of the methodology - relative retention time

[0452]

[0453] Therefore, the relative retention time of each peak is temporarily set at ±10%.

[0454] The test sample chromatogram should show seven characteristic peaks, and their retention times should correspond to those of the seven characteristic peaks in the chromatogram of the reference medicinal material. Peak 3 should correspond to the retention time of the vanillic acid reference material. The peak corresponding to Peak 3 is the S peak. Calculate the relative retention times of Peaks 1, 2, 4, 5, 6, and 7 relative to the S peak. These relative retention times should be within ±10% of the specified values. The specified values are: 0.22 (Peak 1), 0.80 (Peak 2), 1.64 (Peak 4), 2.44 (Peak 5), 2.49 (Peak 6), and 3.09 (Peak 7).

[0455] The characteristic spectra of three batches of Chuhulu formula granules were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a reference spectrum of the characteristic spectra of Chuhulu formula granules was established. Figure 46 , Figure 46 This is the reference characteristic spectrum of Chu Hu Lu formula granules.

[0456] Determination of the characteristic spectrum method of 8-pulled gourd formula granules

[0457] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler; acetonitrile as mobile phase A, and 0.1% aqueous phosphoric acid as mobile phase B, with gradient elution as specified in the table below; the flow rate was 1.0 ml per minute; the column temperature was 35°C; the detection wavelength was 264 nm; the gradient elution was specified in Table 31 below, and the number of theoretical plates was not less than 5000 based on the vanillic acid peak.

[0458] Table 31

[0459]

[0460] Preparation of reference solution: Take an appropriate amount of vanillic acid reference substance, weigh accurately, and add methanol to make a solution containing 20 μg per 1 ml.

[0461] Preparation of control medicinal material solution: Take 1 g of Cucurbitacin control medicinal material, add 50 ml of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 5 ml of methanol to dissolve the residue, filter, and take the filtrate as the control medicinal material reference solution.

[0462] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take 1.0 g, place it in a stoppered conical flask, add 20 ml of methanol, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 20 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0463] Determination method: Accurately aspirate 5 μl of each of the above solutions, inject into liquid chromatograph, and determine.

[0464] Comparative Example 1

[0465] On the basis of Example 3, the mobile phase was changed to acetonitrile for mobile phase A and phosphoric acid for mobile phase B, and other conditions remained unchanged. The test results are shown in FIG. Figure 47 , Figure 47 The chromatograms after changing the mobile phase.

[0466] Comparative Example 2

[0467] On the basis of Example 3, the mobile phase was changed to acetonitrile for mobile phase A and formic acid for mobile phase B, and other conditions remained unchanged. The test results are shown in FIG. Figure 47 .

[0468] Depend on Figure 47 It can be seen that when the test solution uses acetonitrile-phosphoric acid as the mobile phase, the components can be better separated and the baseline is relatively flat.

[0469] Comparative Example 3

[0470] On the basis of Example 3, only the elution procedure was changed, see Table 32 for details, and other conditions remained unchanged. The test results are shown in Table 32. Figure 48 .

[0471] Table 32 Elution procedure of comparative example 3 (method 1)

[0472]

[0473] Comparative Example 4

[0474] On the basis of Example 3, only the elution procedure was changed, see Table 33 for details, and other conditions remained unchanged. The test results are shown in Table 33. Figure 48 , Figure 48 This is the chromatogram after changing the elution program.

[0475] Table 33 Elution procedure of comparative example 4 (method 2)

[0476]

[0477] Comparative Example 5

[0478] On the basis of Example 3, only the elution procedure was changed, see Table 34 for details, and other conditions remained unchanged. The test results are shown in Figure 48 .

[0479] Table 34 Elution procedure of comparative example 5 (method 3)

[0480]

[0481] in, Figure 48 , method 4 is the method described in Example 3.

[0482] Table 35 Elution procedure of Example 3 (Method 4)

[0483]

[0484]

[0485] Depend on Figure 48 It can be seen that the gradient elution in Method 4 is more effective in separating compounds, with a flatter baseline and appropriate sizes of the chromatographic peaks.

[0486] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for constructing HPLC characteristic patterns of Gastrodia elata medicinal materials, decoction pieces, standard decoctions and their formula granules, characterized in that: The following steps are involved: A) treating the raw material containing the gourd with a solvent to obtain a test solution; The raw material containing Gastrodia elata is selected from Gastrodia elata medicinal materials, decoction pieces, standard decoctions or their formula granules; The preparation method of the characteristic spectrum of the medicinal material of Cucurbitacin is as follows: mixing the Cucurbitacin powder with water, heating and refluxing, filtering, evaporating the filtrate to dryness, dissolving the residue with methanol, filtering, and taking the filtrate to obtain the characteristic spectrum of the medicinal material of Cucurbitacin; The preparation method of the characteristic spectrum test solution of the Cucurbitacin decoction piece is as follows: mixing the Cucurbitacin decoction piece powder with water, heating and refluxing, filtering, evaporating the filtrate to dryness, dissolving the residue with methanol, filtering, and taking the filtrate to obtain the characteristic spectrum test solution of the Cucurbitacin decoction piece; The preparation method of the characteristic spectrum test solution of the standard decoction of Cucurbitacin is as follows: taking the freeze-dried powder of the standard decoction of Cucurbitacin and mixing it with methanol, ultrasonically treating it, cooling it, filtering it, and taking the filtrate to obtain the characteristic spectrum test solution of the standard decoction of Cucurbitacin; The preparation method of the characteristic spectrum test solution of the Chuhulu formula granules is as follows: taking the Chuhulu formula granules and methanol, mixing them, performing ultrasonic treatment, cooling, filtering, and taking the filtrate to obtain the characteristic spectrum test solution of the Chuhulu formula granules; B) subjecting the test solution to high performance liquid chromatography to obtain a characteristic HPLC spectrum of the raw material containing the herb; The HPLC conditions are as follows: a C18 column, a detection wavelength of 264 nm; acetonitrile as mobile phase A, a 0.1% phosphoric acid aqueous solution as mobile phase B, and gradient elution. The gradient elution procedure is as follows: 0-25 min, phase A: 12%, phase B: 88%; 25-28 min, phase A: 12%-14%, phase B 88%-86%; 28-35 min, phase A: 14%, phase B 86%; 35-40 min, phase A 14%-25%, phase B 86%-75%; 40-60 min, phase A 25%, phase B 75%.

2. The construction method according to claim 1, characterized in that It also includes the preparation of reference substance solutions and reference substance solutions of reference medicinal materials: Preparation of the reference substance solution: taking a vanillic acid reference substance, adding methanol to dissolve it to obtain a reference substance solution, wherein the concentration of vanillic acid in the reference substance solution is 20 μg / mL; Preparation of the control medicinal material reference solution: taking the Cucurbitaceae control medicinal material, adding water, heating and refluxing extraction, filtering, evaporating the filtrate to dryness, adding methanol to dissolve the residue, filtering, and taking the filtrate as the control medicinal material reference solution; The reference substance solution and the reference medicinal material reference solution are measured by high performance liquid chromatography to obtain chromatograms of the reference substance and the reference medicinal material reference; and the components of the HPLC characteristic spectra of the gourd medicinal material, decoction pieces, standard decoction and its formula granules are analyzed based on the chromatograms of the reference substance and the reference medicinal material reference.

3. The method according to claim 1, characterized in that The chromatographic column is a C18 column, and the column temperature is 35°C.

4. The method according to claim 1, wherein The flow rate of the mobile phase was 1.0 ml / min; the injection volume was 5 μL.

5. The method according to claim 1, wherein In the HPLC characteristic spectrum of the standard decoction of Chu Hu Lu, the peak corresponding to the vanillic acid reference peak is taken as the S1 peak, and the relative retention times of peaks 1, 2, 4, 5, 6 and 7 to the S1 peak are calculated. The relative retention times should be within the range of ±10% of the specified values, which are 0.22 (peak 1), 0.80 (peak 2), 1.64 (peak 4), 2.34 (peak 5), 2.38 (peak 6), and 2.98 (peak 7), respectively. In the HPLC characteristic spectrum of the herbal medicine or slices of Cucurbitacin, the peak corresponding to the vanillic acid reference peak is taken as the S1 peak, and the relative retention times of peaks 1, 2, 4, 5, 6 and 7 to the S1 peak are calculated. The relative retention times should be within the range of ±10% of the specified values, which are 0.220 (peak 1), 0.802 (peak 2), 1.633 (peak 4), 2.392 (peak 5), 2.437 (peak 6) and 3.035 (peak 7), respectively. In the HPLC characteristic spectrum of Chuhulu formula granules, the peak corresponding to the vanillic acid reference peak is taken as S1 peak, and the relative retention times of peak 1, peak 2, peak 4, peak 5, peak 6 and peak 7 with respect to S1 peak are calculated. The relative retention times should be within the range of ±10% of the specified values, and the specified values are 0.22 (peak 1), 0.80 (peak 2), 1.64 (peak 4), 2.44 (peak 5), 2.49 (peak 6) and 3.09 (peak 7), respectively.

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