A method for constructing an HPLC characteristic chromatogram of stone lotus seed medicinal material, decoction piece, standard decoction and formula granules and application thereof

Abstract

The application provides a construction method and application of HPLC characteristic chromatogram of stone lotus seed medicinal materials, decoction pieces, standard decoction and formula granules, comprising the following steps: A) dissolving and extracting the test sample raw material with a solvent to obtain a test solution; B) determining the test solution by high performance liquid chromatography to obtain the HPLC characteristic chromatogram of stone lotus seed medicinal materials, decoction pieces, standard decoction and formula granules; the high performance liquid chromatography conditions are that the chromatographic column is a C 18 column; the mobile phase A is methanol, the mobile phase B is 0.1% phosphoric acid, and gradient elution is adopted. The application adopts high performance liquid chromatography, selects methanol-0.1% phosphoric acid as the mobile phase for gradient elution, takes protocatechuic acid as a reference, establishes the HPLC characteristic chromatogram of stone lotus seed medicinal materials, decoction pieces, standard decoction and formula granules, and has good repeatability and precision, and the method is stable and reliable, so that the quality of stone lotus seed medicinal materials, decoction pieces, standard decoction and formula granules can be controlled.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of detection, and particularly relates to a construction method of HPLC characteristic spectrum of stone lotus seed medicinal materials, decoction pieces, standard decoction and formula granules and application thereof. BACKGROUND

[0002] Stone lotus seed is also known as sweet stone lotus, shell lotus seed and skin lotus seed, which is a fruit with gray-black husk after frost of lotus house, and is also considered to be a lotus fruit sinking in water after frost. Stone lotus seed is oval or elliptical, slightly pointed at both ends, 1.5-2 cm long and 0.8-1.2 cm in diameter, with a gray-brown or gray-black surface. There is one seed, i.e. lotus meat, in the stone lotus seed, which tastes bitter and is cold in nature, enters heart and kidney channels, removes damp heat, stops vomiting and can promote appetite, and is used to treat lockjaw, diarrhea and loss of appetite.

[0003] In order to ensure the quality of stone lotus seed, scientific technical means need to be researched for identification. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a construction method of HPLC characteristic spectrum of stone lotus seed medicinal materials, decoction pieces, standard decoction and formula granules and application thereof. The HPLC characteristic spectrum of stone lotus seed medicinal materials, decoction pieces, standard decoction and formula granules constructed by the present application is stable and reliable, and can control the quality of stone lotus seed medicinal materials, decoction pieces, standard decoction and formula granules.

[0005] The present application provides a construction method of HPLC characteristic spectrum of stone lotus seed medicinal materials, decoction pieces, standard decoction and formula granules, which comprises the following steps:

[0006] A) dissolving and extracting the raw material of the test sample by using a solvent to obtain a test solution;

[0007] B) determining the test solution by using high performance liquid chromatography to obtain the HPLC characteristic spectrum of stone lotus seed medicinal materials, decoction pieces, standard decoction and formula granules;

[0008] The chromatography conditions of the high performance liquid chromatography are as follows: the chromatography column is a C18 column; the mobile phase A is methanol, and the mobile phase B is 0.1% phosphoric acid, and gradient elution is adopted.

[0009] The method provided by the present application further comprises a reference solution of a control sample:

[0010] The protocatechuic acid, 4-coumaric acid, isohispidin and nuciferine are respectively dissolved by using 30% methanol to prepare a solution of 25 μg / mL as the reference solution of the control sample.

[0011] The stone lotus seed control medicinal material is boiled with water, filtered, and the filtrate is evaporated to dryness. The residue is added with 30% methanol and ultrasonically treated, filtered, and the filtrate is taken as the reference solution of the control medicinal material.

[0012] The test sample raw material is dissolved by a solvent to obtain a test solution. In the present application, the solvent is 30% methanol; specifically, 30% methanol aqueous solution; the volume content of 30% methanol is 30%; the chromatographic peak shape and resolution of the test sample solution extracted by 30% methanol are better, so 30% methanol is used as the extraction solvent.

[0013] In the present application, the chromatographic peak information obtained by ultrasonic extraction of the test sample is large, and the peak shape is good, so the extraction method is ultrasonic treatment; the power of ultrasonic treatment is 580-620 W, and the frequency of ultrasonic treatment is 35-45 kHz.

[0014] If the test sample raw material is stone lily seed medicinal material and decoction piece, the volume mL of the solvent and the mass g of the test sample are in the ratio of (9-11):(0.95-1.05);

[0015] If the test sample raw material is stone lily seed standard decoction and formula granules, the volume mL of the solvent and the mass g of the test sample are in the ratio of (9-11):(0.18-0.22).

[0016] If the test sample raw material is stone lily seed medicinal material and decoction piece, the test sample raw material needs to be decocted with water, filtered, and the filtrate is evaporated to dryness; the residue is dissolved in 30% methanol, filtered, and the filtrate is obtained to obtain the test solution.

[0017] If the test sample raw material is stone lily seed extract or formula granules, the test sample raw material is directly mixed with 30% methanol, ultrasonically treated, cooled, shaken, filtered, and the filtrate is obtained to obtain the test solution.

[0018] The test solution is determined by high performance liquid chromatography to obtain the HPLC characteristic spectrum of stone lily seed medicinal material, decoction piece, standard decoction and formula granules.

[0019] When the injection amount is 3 μl, the chromatogram peak shape is good, and the resolution is moderate, therefore, 3 μl of the reference solution and the test sample solution (test solution) are precisely taken respectively and injected into the liquid chromatograph.

[0020] In the present application, octadecylsilane-bonded silica gel is used as the filler (the column length is 150 mm, the inner diameter is 2.1 mm, and the particle size is 1.8 μm); methanol is used as the mobile phase A, and 0.1% phosphoric acid aqueous solution (1 ml→1000 ml) is used as the mobile phase B for gradient elution; the flow rate is 0.3 ml / min, the column temperature is 30°C; the detection wavelength is 305 nm, and the theoretical plate number calculated according to protocatechuic acid should be not less than 5000.

[0021] In the present application, the gradient elution is specifically as follows:

[0022] In the present application, the gradient elution is specifically as follows:

[0023] 0~12min, A phase: 15%, B phase: 85%;

[0024] 12~20min, A phase: 15~22%, B phase: 85~78%;

[0025] 20~32min, A phase: 22~30%, B phase: 78~70%;

[0026] 32~40min, A phase: 30~36%, B phase: 70~64%;

[0027] 40~50min, A phase: 36~40%, B phase: 64~60%.

[0028] The application has good baseline separation under the elution gradient, and each peak has good separation degree and stable baseline.

[0029] The application adopts a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to evaluate the similarity of the characteristic spectrum of stone lotus seed, and obtains a stone lotus seed medicine, decoction piece, standard decoction and formula granule HPLC standard characteristic spectrum composed of 10 characteristic peaks, wherein peak 1 is protocatechuic acid; peak 6 is 4-coumaric acid; peak 9 is isohoutuyangoside; and peak 10 is nuciferine.

[0030] The application adopts a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to evaluate the similarity of the characteristic spectrum of stone lotus seed, and obtains a stone lotus seed medicine, decoction piece, standard decoction and formula granule HPLC standard characteristic spectrum composed of 10 characteristic peaks, wherein peak 1 is protocatechuic acid; peak 6 is 4-coumaric acid; peak 9 is isohoutuyangoside; and peak 10 is nuciferine.

[0031] The application adopts a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to evaluate the similarity of the characteristic spectrum of stone lotus seed, and obtains a stone lotus seed medicine, decoction piece, standard decoction and formula granule HPLC standard characteristic spectrum composed of 10 characteristic peaks, wherein peak 1 is protocatechuic acid; peak 6 is 4-coumaric acid; peak 9 is isohoutuyangoside; and peak 10 is nuciferine.

[0032] The application adopts a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to evaluate the similarity of the HPLC characteristic spectrum of Shilianzi formula granules, and obtains an HPLC standard characteristic spectrum of Shilianzi formula granules composed of 10 characteristic peaks, wherein the peak corresponding to the protocatechuic acid reference is an S peak; in the standard characteristic spectrum, the relative retention time of each characteristic peak to the S peak should be within ±10% of the specified value; the specified value is: 1.13 (peak 2), 1.41 (peak 3), 1.51 (peak 4), 3.40 (peak 5), 4.52 (peak 6), 6.10 (peak 7), 6.26 (peak 8), 6.56 (peak 9), and 7.55 (peak 10).

[0033] The method provided by the application can effectively monitor the quality of different batches of Shilianzi medicinal materials, decoction pieces, standard decoctions and formula granules, so that the quality is stable, and the method has the characteristics of high precision and good reproducibility, and is beneficial to the comprehensive monitoring of the quality of products.

[0034] The characteristic spectrum of Shilianzi medicinal materials, decoction pieces, standard decoctions and formula granules established by the application takes protocatechuic acid as a reference, focuses on the order of each characteristic peak and the correlation with medicinal materials and intermediate products, can comprehensively evaluate the overall quality characteristics of products, and the method is scientific and reliable.

[0035] The application also provides a method for identifying Shilianzi medicinal materials, decoction pieces, standard decoctions and formula granules, which adopts the method described in the above technical solution to detect and analyze the detection results.

[0036] The application provides a method for constructing an HPLC characteristic spectrum of Shilianzi medicinal materials, decoction pieces, standard decoctions and formula granules, which comprises the following steps: A) dissolving and extracting the raw material of the test sample with a solvent to obtain a test solution; B) determining the test solution by high performance liquid chromatography to obtain the HPLC characteristic spectrum of Shilianzi medicinal materials, decoction pieces, standard decoctions and formula granules; and the high performance liquid chromatography has the following chromatographic conditions: a C18 column is used as the chromatographic column; methanol is used as the mobile phase A, and 0.1% phosphoric acid is used as the mobile phase B, and gradient elution is performed. The application adopts high performance liquid chromatography, selects methanol-0.1% phosphoric acid as the mobile phase for gradient elution, takes protocatechuic acid as a reference, establishes the HPLC characteristic spectrum of Shilianzi medicinal materials, decoction pieces, standard decoctions and formula granules, and has good repeatability and precision, and the method is stable and reliable, so that the quality of Shilianzi medicinal materials, decoction pieces, standard decoctions and formula granules can be controlled. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 It is a UV absorption 3D spectrum of Shilianzi standard decoction;

[0038] Figure 2 It is a different wavelength chromatogram of Shilianzi standard decoction;

[0039] Figure 3 Chromatogram of standard decoction of Gynura japonica for column temperature investigation;

[0040] Figure 4 Chromatogram of standard decoction of Gynura japonica for flow rate investigation;

[0041] Figure 5 Chromatogram of standard decoction of Gynura japonica for different injection amount investigation;

[0042] Figure 6 Delay investigation of standard decoction of Gynura japonica;

[0043] Figure 7 Extraction method investigation of standard decoction of Gynura japonica;

[0044] Figure 8 Extraction solvent investigation of standard decoction of Gynura japonica;

[0045] Figure 9 Extraction time investigation of standard decoction of Gynura japonica;

[0046] Figure 10 Peak identification of standard decoction of Gynura japonica;

[0047] Figure 11 Chromatogram of standard decoction of Gynura japonica for different instruments investigation;

[0048] Figure 12 Durability investigation of chromatographic column for standard decoction of Gynura japonica;

[0049] Figure 13 Characteristic chromatogram of standard decoction of Gynura japonica;

[0050] Figure 14 Controlled characteristic chromatogram of standard decoction of Gynura japonica;

[0051] Figure 15 Extraction solvent investigation of Gynura japonica medicinal materials and decoction pieces;

[0052] Figure 16 Extraction method investigation of Gynura japonica medicinal materials and decoction pieces;

[0053] Figure 17 Extraction time investigation of Gynura japonica medicinal materials and decoction pieces;

[0054] Figure 18 Peak identification of Gynura japonica medicinal materials and decoction pieces;

[0055] Figure 19 Investigation of different instruments for Gynura japonica medicinal materials and decoction pieces;

[0056] Figure 20 Durability investigation of chromatographic column for Gynura japonica medicinal materials and decoction pieces;

[0057] Figure 21 Characteristic chromatogram of stone lily medicinal material

[0058] Figure 22 Reference characteristic chromatogram of stone lily medicinal material

[0059] Figure 23 UV absorption spectrum of stone lily formula granules

[0060] Figure 24 Different wavelength chromatogram of stone lily formula granules

[0061] Figure 25 Column temperature investigation chromatogram of stone lily formula granules

[0062] Figure 26 Flow rate investigation chromatogram of stone lily formula granules

[0063] Figure 27 Different injection amount investigation chromatogram of stone lily formula granules

[0064] Figure 28 Delay investigation of stone lily formula granules

[0065] Figure 29 Extraction solvent investigation chromatogram of stone lily formula granules

[0066] Figure 30 Extraction method investigation chromatogram of stone lily formula granules

[0067] Figure 31 Extraction time investigation of stone lily formula granules

[0068] Figure 32 Chromatographic peak identification of stone lily formula granules

[0069] Figure 33 Different chromatographic column investigation chromatogram of stone lily formula granules

[0070] Figure 34 Different chromatographic column investigation chromatogram of stone lily formula granules

[0071] Figure 35 Characteristic chromatogram verification of 3 batches of stone lily formula granules

[0072] Figure 36 Reference characteristic chromatogram of stone lily formula granules DETAILED DESCRIPTION

[0073] In order to further illustrate the present application, the construction method of the HPLC characteristic chromatogram of the stone lily medicinal material, decoction piece, standard decoction and formula granules provided by the present application is described in detail below in combination with examples, but they should not be understood as limiting the protection scope of the present application.

[0074] Establishment of standard decoction of Gastrodia elata Blume by HPLC

[0075] 1. Experimental instruments and materials

[0076] High performance liquid chromatograph 1; high performance liquid chromatograph 2; high performance liquid chromatograph 3

[0077] Cell type 1810A ultrapure water machine (Shanghai Moore Scientific Instrument Co., Ltd.);

[0078] KQ-600DB ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.);

[0079] Chromatographic column: chromatographic column 1; chromatographic column 2; chromatographic column 3

[0080] 2. Reagents and reagents

[0081] Methanol and phosphoric acid are chromatographically pure, water is ultrapure water, and the rest of the reagents are analytically pure. The mobile phase is laboratory-made ultrapure water, and the rest is laboratory-made pure water.

[0082] Protocatechuic acid reference substance (China Institute for Drug Control, batch number: 110809-201906),

[0083] 4-coumaric acid reference substance (China Institute for Drug Control, batch number: 112037-202102),

[0084] Isorhamnetin reference substance (China Institute for Drug Control, batch number: 111974-201401);

[0085] Nuciferine reference substance (China Institute for Drug Control, batch number: 111566-201706);

[0086] Gastrodia elata reference material (Shanghai Hongyong Biological Technology Co., Ltd., batch number: 230011-202201);

[0087] Standard decoction of Gastrodia elata Blume freeze-dried powder (Sichuan Xinglv Pharmaceutical Technology Development Co., Ltd.) Preparation: 21 batches of standard decoction freeze-dried powder.

[0088] 3. Proposed chromatographic conditions

[0089] Chromatographic conditions and system suitability test with octadecylsilane bonded silica gel as the filler (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol as mobile phase A, 0.1% phosphoric acid water as mobile phase B, gradient elution according to the following Table 1; flow rate: 0.3 ml / min, column temperature: 30℃; detection wavelength: 305 nm, theoretical plate number calculated by protocatechuic acid should not be less than 5000.

[0090] Table 1 Gradient elution procedure

[0091]

[0092]

[0093] Reference solution preparation: 0.5 g of the reference drug was added to 50 ml of water, decocted for 30 minutes, filtered, and the filtrate was evaporated to dryness. The residue was dissolved in 10 ml of 30% methanol, ultrasonically treated (power 600 W, frequency 40 kHz) for 30 minutes, allowed to cool, shaken, filtered, and the filtrate was used as the reference solution of the reference drug. An appropriate amount of protocatechuic acid was precisely weighed, dissolved in 30% methanol to prepare a solution containing 25 μg per 1 ml, and used as the reference solution of the reference substance.

[0094] Test solution preparation: an appropriate amount of the sample was taken, about 0.2 g was added to 10 ml of 30% methanol, ultrasonically treated (power 600 W, frequency 40 kHz) for 30 minutes, allowed to cool, shaken, centrifuged, the supernatant was filtered, and the filtrate was used as the test solution.

[0095] Determination: 3 μl of the reference solution and the test solution were precisely pipetted and injected into the liquid chromatograph, and determination was performed.

[0096] On the basis of the above proposed experimental conditions, the test solution of the Shilianzi Dispensing Granules was scanned by a diode array detector at full waveband, and the chromatograms of the test solution at 240 nm, 280 nm, 230 nm, 305 nm, 320 nm, and 360 nm were extracted, respectively. See Figures 1-2 . The results showed that the information amount of the chromatographic peaks was larger, and the baseline of the chromatogram was more stable when the detection wavelength was 305 nm, so the detection wavelength was determined to be 305 nm.

[0097] 3.1 Column temperature investigation

[0098] On the basis of the above proposed experimental conditions, the column temperature was investigated at 20 ℃, 25 ℃, 30 ℃, and 35 ℃, respectively. See Figure 3 , Table 2:

[0099] Table 2 Column temperature investigation - relative retention time

[0100]

[0101] The results of the column temperature investigation showed that when the column temperature was 30 ℃, the chromatographic peak shapes were relatively symmetrical, and the resolution was good, so 30 ℃ was finally determined as the column temperature of the characteristic chromatogram method of the Shilianzi Dispensing Granules.

[0102] 3.2 Flow rate investigation

[0103] On the basis of the above experimental conditions, the flow rate of 0.2 ml / min, 0.3 ml / min, 0.4 ml / min were investigated respectively. See Figure 4 Table 3: Flow rate investigation-relative retention time

[0104] Table 3: Flow rate investigation-relative retention time

[0105]

[0106]

[0107] The results showed that the flow rate was 0.3 ml / min, the chromatogram peak shape was better, and the separation degree was moderate. Therefore, the flow rate was determined to be 0.3 ml / min.

[0108] 3.3 Injection amount investigation

[0109] On the basis of the above experimental conditions, the flow rate of 2 μl, 3 μl, 4 μl were investigated respectively. See Figure 5 Table 4: Injection amount investigation-relative retention time

[0110] Table 4: Injection amount investigation-relative retention time

[0111]

[0112] The results showed that the injection amount was 3 μl, the chromatogram peak shape was better, and the separation degree was moderate. Therefore, the injection amount was determined to be 3 μl.

[0113] 3.4 Delay investigation

[0114] On the basis of the above experimental conditions, the analysis time was extended to 100 minutes, and whether there were chromatographic peaks after 50 minutes was observed. The results are shown in Figure 6 .

[0115] The results showed that there were basically no large chromatographic peaks after 50 minutes, and the analysis time of the characteristic chromatogram of Shilianzi formula granules was finally determined to be 50 minutes.

[0116] In summary, the chromatographic conditions and system suitability test of the characteristic chromatogram of Shilianzi formula granules were determined as follows: octadecylsilane bonded silica gel as the filler (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol as mobile phase A, 0.1% phosphoric acid water as mobile phase B, gradient elution according to the above Table 1; flow rate: 0.3 ml / min, column temperature: 30℃; detection wavelength: 305 nm, theoretical plate number should not be less than 5000 calculated by protocatechuic acid.

[0117] The subsequent standard decoction and medicinal decoction pieces were investigated under this chromatographic condition.

[0118] 4. Preparation of test solution

[0119] 4.1 Examination of Extraction Methods

[0120] Take about 0.2g of standard lotus seed decoction, place it in a stoppered conical flask, add 10ml of 30% methanol, sonicate (600W power, 40kHz frequency) and reflux for 30 minutes, cool, add 30% methanol to make up the weight, shake well, centrifuge, take the supernatant, filter, and take the filtrate to obtain the final product.

[0121] Inject and determine according to the above results. (See attached image) Figure 7 :from Figure 7 As can be seen, there is no significant difference in the final results between reflux extraction and ultrasonic extraction. Therefore, ultrasonic extraction, which is simpler to operate, was chosen as the extraction method for preparing the test solution in the determination of the characteristic spectrum of the lotus seed standard decoction.

[0122] 4.2 Investigation of Extraction Solvents

[0123] Take approximately 0.2g of the standard decoction of lotus seeds and place it in a stoppered conical flask. Add 10ml each of water, 30% methanol, 50% methanol, 70% methanol, methanol, 50% ethanol, and ethanol. Sonicate the mixture (600W, 40kHz) for 30 minutes, cool, shake well, centrifuge, collect the supernatant, filter, and collect the filtrate. Inject and determine the chromatographic results according to the above conditions. See the results below. Figure 8 The results showed that the peak shapes and resolutions of the chromatographic peaks in the test solutions extracted with water, 30% methanol, and 50% methanol were all good. In order to maintain consistency with the particles, 30% methanol was selected as the extraction solvent for the characteristic chromatogram of the standard decoction of lotus seeds.

[0124] 4.3 Examination of extraction time

[0125] Take approximately 0.2g of the standard decoction of lotus seeds and place it in a stoppered conical flask. Add 10ml of 30% methanol and sonicate (600W power, 40kHz frequency) for 20, 30, and 40 minutes respectively. After cooling, shake well, centrifuge, collect the supernatant, filter, and collect the filtrate. Inject and determine the chromatographic results according to the above conditions. The results are shown in the figure. Figure 9 The results showed that an extraction time of 20 minutes was sufficient, but in order to ensure sufficient extraction, the extraction time of the sample was tentatively set at 30 minutes.

[0126] In summary, the preparation method of the test solution for the characteristic chromatogram of the standard decoction of lotus seeds is determined as follows: Take about 0.2g of this product, place it in a stoppered conical flask, add 10ml of 30% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, centrifuge, take the supernatant, filter, and take the filtrate to obtain the product.

[0127] 5. Methodological Examination

[0128] 5.1 Chromatographic peak assignment

[0129] Preparation of test solution: The test solution of Asparagus cochinchinensis was prepared according to the experimental conditions above.

[0130] Preparation of reference solution: The reference solution was prepared by accurately weighing appropriate amounts of protocatechuic acid, 4-coumaric acid, isohispidin, and nuciferine, and dissolving them in 30% methanol to make a solution containing 25 μg per 1 ml. The results are shown in Table 2. Figure 10 Figure 10 It can be seen from Table 2 that four characteristic peaks were identified, in which the peak corresponding to peak 1 is the protocatechuic acid peak, the peak corresponding to peak 6 is the 4-coumaric acid peak, the peak corresponding to peak 9 is the isohispidin peak, and the peak corresponding to peak 10 is the nuciferine peak.

[0131] 5.2 Precision test

[0132] The test solution was prepared from the Asparagus cochinchinensis standard decoction, and 6 injections of 3 μl each were made successively according to the experimental method to calculate the retention time. The results are shown in Table 5.

[0133] Table 5 Precision test- retention time of characteristic peaks

[0134]

[0135] The results show that the RSD values of the retention time of each characteristic peak are between 0.06% and 0.67%, indicating that the precision of the instrument is good.

[0136] 5.3 Reproducibility test

[0137] Six portions of the Asparagus cochinchinensis standard decoction lyophilized powder were accurately weighed, and prepared and determined according to the experimental method. The results are shown in Table 6.

[0138]

[0139]

[0140] The results show that the RSD% values of the relative retention time of each characteristic peak are between 0.03% and 0.18%, indicating that the reproducibility of the method is good.

[0141] 5.4 Intermediate precision test

[0142] The Asparagus cochinchinensis standard decoction lyophilized powder was prepared and determined by different personnel (A, B) at different times (I, II) according to the experimental method, and the results were determined by instruments a and b, respectively. The results are shown in Table 7.

[0143] Table 7 Precision- relative retention time

[0144] ​

[0145] From Table 7, the RSD of the relative retention time of each characteristic peak was 0.15%-2.22% under different test solution preparation personnel and different test solution preparation time, indicating that the method was suitable.

[0146] 5.5 Stability Investigation

[0147] Based on the above experimental conditions, the same test solution was taken and measured at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h. See Table 8:

[0148] Table 8 Stability Investigation for 24 Hours-Characteristic Peak Retention Time

[0149]

[0150] The results showed that the RSD of the corresponding characteristic peak retention time was 0.07%-0.67%, and the sample solution was relatively stable within 24 hours.

[0151] 5.6 Durability Investigation

[0152] 5.6.1 Instrument Durability Investigation

[0153] Based on the above experimental conditions, a standard decoction of stone lotus was precisely weighed, and the test solution was prepared. The determination was performed on different brands of high-performance liquid chromatographs 1, 2, and 3. See Table 9, Figure 11 :

[0154] Table 9 Instrument Durability Investigation-Relative Retention Time Ratio

[0155]

[0156] The results showed that the RSD of the characteristic peak relative retention time was between 0.47%-4.57% when the sample was detected by the above three instruments, indicating that the method had good instrument column durability.

[0157] 5.6.2 Column Durability Investigation

[0158] Based on the above experimental conditions, column 1, column 2, and column 3 were investigated. See Figure 12 , Table 10:

[0159] Table 10 Column Durability Investigation-Characteristic Peak Relative Retention Time

[0160]

[0161] From the above table, the sample was detected by the above three kinds of chromatographic column, and it was found that each characteristic peak could be reproduced in different particle size chromatographic columns. From the above figure, it was found that in the investigation of different chromatographic columns, the separation effect of chromatographic column 2 for peak 1 and peak 2 was poor, and the separation effect of chromatographic column 3 for peak 7 and peak 8 was poor, and the separation effect of chromatographic column 1 for characteristic peaks was good. It is recommended to use chromatographic column 1 (octadecylsilane bonded silica gel as filler (column length is 150 mm, inner diameter is 2.1 mm, particle size is 1.8 μm)) for test. The chromatographic column 1 will be used for subsequent verification in this experiment, and a total of 10 characteristic peaks are included in the subsequent verification.

[0162] 5.7 Establishment of relative retention time specification limit

[0163] The results of each investigation item and verification of the methodology are shown in Table 11:

[0164] Table 11 Results of each item of the methodology RSD% summary standard-retention time, relative retention time

[0165]

[0166] From the above table, the chromatographic column has a greater influence on the peak time of each characteristic peak. After confirming the chromatographic column, in order to increase the reproducibility and applicability of the method, the relative retention time specification value of each peak is temporarily set as ±10%.

[0167] 6. Determination of characteristic peaks and establishment of control chromatogram

[0168] The proposed method was used to determine the characteristic chromatogram of 21 batches of samples, and the relative retention time was calculated. See Table 12, Figure 13 In which, peak 1 (S): protocatechuic acid; peak 6: 4-coumaric acid; peak 9: isoheteroside; peak 10: nuciferine (S22: control medicinal material, S1-S21: 21 batches of standard decoction):

[0169] Table 12 Relative retention time of 21 batches of standard decoction of Gynura japonica

[0170]

[0171] According to the principles of stable relative retention time and detectable in each batch of sample and relatively high peak, a total of 10 peaks with good reproducibility were selected as characteristic peaks. The results showed that the relative retention time RSD of 10 characteristic peaks of 21 batches of standard decoction of Gynura japonica was less than 2.0%.

[0172] The final provisions: the test sample chromatogram should present 10 characteristic peaks, and the retention time of the 10 characteristic peaks in the reference chromatogram of the control medicinal material should be corresponding, wherein the peak corresponding to the protocatechuic acid reference is S peak. The relative retention time of each characteristic peak to S peak should be within ±10% of the specified value. The specified value is: 1.13 (peak 2), 1.40 (peak 3), 1.51 (peak 4), 3.39 (peak 5), 4.52 (peak 6), 6.09 (peak 7), 6.25 (peak 8), 6.54 (peak 9), and 7.53 (peak 10).

[0173] The standard decoction of 21 batches of Gynura medica was synthesized by using Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 edition), and the control chromatogram of the standard decoction of Gynura medica was established, as shown in Figure 14 .

[0174] 7. Method for determining the standard decoction characteristic chromatogram of Gynura medica (Gynura divaricata)

[0175] The chromatographic conditions and system suitability test were as follows: octadecylsilane-bonded silica gel was used as the filler (the column length was 150 mm, the inner diameter was 2.1 mm, and the particle size was 1.8 μm); methanol was used as the mobile phase A, and 0.1% phosphoric acid was used as the mobile phase B, which was gradient eluted according to the above Table 1; the flow rate was 0.3 ml per minute, the column temperature was 30 °C; the detection wavelength was 305 nm, and the theoretical plate number calculated according to protocatechuic acid should not be less than 5000.

[0176] The reference solution was prepared as follows: 0.5 g of Gynura medica control medicinal material was added into 50 ml of water, decocted for 30 minutes, filtered, and the filtrate was evaporated to dryness. The residue was added into 10 ml of 30% methanol, ultrasonically treated (power 600 W, frequency 40 kHz) for 30 minutes, cooled, shaken uniformly, filtered, and the filtrate was taken as the control medicinal material reference solution. In addition, an appropriate amount of protocatechuic acid reference substance was precisely weighed and determined, and 30% methanol was added to prepare a solution containing 25 μg per 1 ml, which was taken as the reference substance reference solution.

[0177] The test sample solution was prepared as follows: an appropriate amount of the product was taken, about 0.2 g was added into 10 ml of 30% methanol, ultrasonically treated (power 600 W, frequency 40 kHz) for 30 minutes, cooled, shaken uniformly, centrifuged, the supernatant was taken, filtered, and the filtrate was taken as the test sample solution.

[0178] The determination method was as follows: 3 μl of each of the reference solution and the test sample solution was precisely taken and injected into the liquid chromatograph for determination.

[0179] Example 2: Construction of HPLC characteristic chromatogram of Gynura medica (Gynura divaricata) medicinal material and decoction pieces

[0180] 1. Reagents

[0181] Gynura medica medicinal material: 21 batches of Gynura medica medicinal material.

[0182] 2. Assumed chromatographic conditions

[0183] Take octadecylsilane bonded silica gel as filler (column length is 150 mm, inner diameter is 2.1 mm, particle size is 1.8 μm) with methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, gradient elution according to the provisions in Table 1 above; column temperature is 30°C; flow rate is 0.3 ml / min; detection wavelength is 305 nm.

[0184] Preparation of reference solution: take 0.5 g of Gynura japonica control medicinal material, add 50 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 10 ml of 30% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, take the subsequent filtrate as the control medicinal material reference solution. Take the appropriate amount of protocatechuic acid control, accurately weigh, add 30% methanol to prepare a solution containing 25 μg per 1 ml, which is used as the control reference solution.

[0185] Preparation of test solution: take 1.0 g of the product (pass through a No. 3 sieve), add 50 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue with 10 ml of 30% methanol, filter, take the subsequent filtrate as the test solution.

[0186] Determination: accurately pipette 3 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine, and then the determination is completed.

[0187] 3. Preparation of test solution for investigation

[0188] 3.1 Investigation of extraction solvent

[0189] Take about 1.0 g of the product, add 50 ml of water to a stoppered conical flask, decoct for 30 minutes, filter, evaporate to dryness, and then dissolve the residue with 10 ml of methanol, 70% methanol, 50% methanol, 30% methanol, water, ethanol, and 50% ethanol respectively, filter, and take the subsequent filtrate, and then the preparation is completed. See Table 2 below. Figure 15 The results show that when water and 30% methanol are used as the extraction solvent, the chromatographic peak information is large and the peak shape is good. Therefore, 30% methanol is selected as the extraction solvent for the test solution.

[0190] 3.2 Investigation of extraction method

[0191] Take two portions of the medicinal material of the product, about 1.0 g each, add 10 ml of 30% methanol to a stoppered conical flask, and investigate the extraction method by refluxing and ultrasonically treating respectively, the extraction time is 30 minutes, cool, shake well, filter, and take the subsequent filtrate. Take another portion of the medicinal material of the product, about 1.0 g, add 50 ml of water to a stoppered conical flask, decoct for 30 minutes, filter, evaporate to dryness, and then dissolve the residue with 10 ml of 30% methanol, filter, and take the subsequent filtrate, and then the preparation is completed.Figure 16 The results showed that the effect of ultrasonic extraction and reflux extraction on the test sample was consistent. The peak type of the medicinal material after water boiling was better, so the extraction method of the test sample was determined as water decoction for 30 min and dissolution with 30% methanol.

[0192] 3.3 Extraction time investigation

[0193] Take about 1.0 g of the medicinal material, accurately weigh, and put it in a conical flask with a plug. Add 50 ml of water, decoct for 30 min, filter, evaporate to dryness, add 10 ml of 30% methanol to the residue, and investigate the test sample under ultrasonic extraction times of 20 min, 30 min, and 40 min, respectively. Cool, shake well, filter, and take the filtrate, i.e. the test sample solution. Figure 17 The results showed that when the extraction time was 20 min, the components of the stone lily fruit were already extracted, so as to ensure sufficient extraction. Therefore, the extraction time of the test sample was determined as 30 min.

[0194] In summary, the preparation method of the stone lily fruit medicinal material characteristic chromatogram test sample solution was determined as follows: take about 1.0 g of the product, put it in a conical flask with a plug, add 50 ml of water, decoct for 30 min, filter, evaporate to dryness, add 10 ml of 30% methanol to the residue, dissolve, shake well, filter, and take the filtrate, i.e. the test sample solution.

[0195] 4. Methodology investigation

[0196] 4.1 Chromatographic peak identification

[0197] Preparation of the test sample solution: prepare the stone lily fruit medicinal material test sample solution according to the above proposed experimental conditions.

[0198] Preparation of the reference solution: accurately weigh appropriate amounts of protocatechuic acid, 4-coumaric acid, isohoutuyin, and nuciferine reference substances, and add 30% methanol to prepare a solution containing 25 μg per 1 ml as the reference substance solution.

[0199] Position the stone lily fruit medicinal material characteristic chromatogram peaks. See Table 14: Figure 18 .

[0200] 4.2 Precision test

[0201] Take the stone lily fruit medicinal material test sample solution, and continuously inject 6 times according to the proposed experimental method, 3 μl each time, and calculate the retention time of each characteristic peak. See Table 13:

[0202] Table 13 Precision investigation-retention time

[0203]

[0204] The results showed that the precision of each characteristic peak retention time RSD was 0.03%-0.18%, and the precision of the instrument was good.

[0205] 4.3 repeatability study

[0206] Precisely weigh 6 parts of Aspidium and prepare and determine according to the proposed experimental method. See Table 14:

[0207] Table 14 Repeatability study - relative retention time ratio

[0208]

[0209] The results show that the relative retention time RSD of each characteristic peak is 0.03%-0.15%, and the method has good repeatability.

[0210] 4.4 stability

[0211] On the basis of the above proposed experimental conditions, the same test solution was taken and determined at 0 h, 2 h, 4 h, 8 h, 12 h and 24 h. See Table 15:

[0212] Table 15 Stability study - retention time

[0213]

[0214] The results show that the RSD of the relative retention time of the corresponding characteristic peaks is 0.10-0.48%, and the sample solution is stable within 24 hours.

[0215] 4.5 intermediate precision study

[0216] 4.5.1 different instrument study

[0217] On the basis of the above proposed experimental conditions, three parts of Aspidium were precisely weighed, the test solution was prepared, and the determination was carried out on different brands of high performance liquid chromatograph 1; high performance liquid chromatograph 2; high performance liquid chromatograph 3. See Table 16, Figure 19 :

[0218] Table 16 Instrument durability study - relative retention time ratio

[0219]

[0220] The results show that when the test sample is detected by the above three instruments, the RSD of the relative retention time of each characteristic peak is 0.23%-4.1%.

[0221] 4.5.2 intermediate precision study

[0222] On the basis of the above proposed experimental conditions, (A, B) were prepared and determined at different times (I, II) according to the proposed experimental method, and the results were determined by instrument a and b, respectively. The results are shown in Table 17, Figure 20 :

[0223] Table 17 Personnel and time investigation - relative retention time ratio

[0224]

[0225] The results show that the RSD range of the relative retention time of each characteristic peak is 0.36%-1.6% when the same sample is detected by different personnel at different times, indicating that the method has good stability when the same sample is determined by different personnel at different times.

[0226] 4.6 Investigation of column durability

[0227] On the basis of the above proposed experimental conditions, the column was respectively investigated as follows: column 1; column 2; column 3. See Table 18, Figure 20 :

[0228] Table 18 Column durability investigation - relative retention time ratio

[0229]

[0230] From the above table, it can be seen that the detection of the sample by the above three columns shows that each characteristic peak can be reproduced in different particle size columns. From the above figure, it can be seen that in the investigation of different columns, it is found that column 2 has poor separation effect on peak 1 and peak 2, column 3 has poor separation effect on peak 7 and peak 8, and column 1 has good separation effect on characteristic peaks. It is suggested to use column 1 (octadecylsilane bonded silica gel as the filler (column length is 150 mm, inner diameter is 2.1 mm, and particle size is 1.8 μm)) for the test. Column 1 will be used for subsequent verification in this experiment, and a total of 10 characteristic peaks are included in the subsequent verification.

[0231] 4.7 Establishment of relative retention time limit

[0232] The results of each investigation item and verification of the method are shown in Table 19:

[0233] Table 19 Results of RSD% of each item of the method Standard - retention time, relative retention time

[0234]

[0235] From the above table, it can be seen that the column has a greater effect on the peak time of each characteristic peak. After confirming the column, in order to increase the reproducibility and applicability of the method, the relative retention time limit of each peak is temporarily set as ±10%.

[0236] 6. Verification of characteristic chromatogram of Gynostemma pentaphyllum medicinal materials

[0237] The 21 batches of Gynostemma pentaphyllum medicinal materials were determined by the preparation method of test solution of Gynostemma pentaphyllum medicinal materials characteristic chromatogram determined in the above "3. Preparation of test solution of test sample" and the characteristic chromatogram of 21 batches of Gynostemma pentaphyllum medicinal materials is shown in Figure 21 The relative retention time of characteristic peaks is shown in Table 20:

[0238] Table 20 Relative retention time of characteristic chromatogram of Gynostemma pentaphyllum medicinal materials

[0239]

[0240] According to the principle of stable relative retention time and detectable and relatively high peak of each batch of sample, a total of 10 peaks with good repeatability were selected as characteristic peaks. The results showed that the relative retention time RSD of 10 characteristic peaks of 21 batches of Gynostemma pentaphyllum medicinal materials was less than 2.0%. Finally, it was stipulated that 10 characteristic peaks should be present in the test sample chromatogram, and the relative retention time of the 10 characteristic peaks in the reference chromatogram of the control medicinal material should correspond, among which the peak corresponding to protocatechuic acid reference was S peak. The relative retention time of each characteristic peak to S peak should be within ±10% of the specified value. The specified value was: 1.13 (peak 2), 1.41 (peak 3), 1.51 (peak 4), 3.39 (peak 5), 4.51 (peak 6), 6.04 (peak 7), 6.20 (peak 8), 6.49 (peak 9), 7.46 (peak 10).

[0241] The 21 batches of Gynostemma pentaphyllum medicinal materials were synthesized by Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version) to establish the control characteristic chromatogram of Gynostemma pentaphyllum medicinal materials characteristic chromatogram. See Figure 22 Peak 1 (S): protocatechuic acid; peak 6: 4-coumaric acid; peak 9: isoheteroside; peak 10: nuciferine; the chromatographic column used was HSS T3 1.8 μm 150 x 2.1 mm.

[0242] 7. Determination of characteristic chromatogram method of Gynostemma pentaphyllum medicinal materials and decoction pieces

[0243] Octadecylsilane-bonded silica gel was used as the filler (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol was used as mobile phase A and 0.1% phosphoric acid solution was used as mobile phase B, which was gradient eluted according to the above Table 1; the detection wavelength was 305 nm; the flow rate was 0.3 ml per minute; the column temperature was 30°C, and the theoretical plate number calculated by protocatechuic acid should not be less than 5000.

[0244] Reference solution: 0.5 g of the reference material was added to 50 ml of water, boiled for 30 minutes, filtered, and the filtrate was evaporated to dryness. The residue was dissolved in 10 ml of 30% methanol, and ultrasonically treated (power 600 W, frequency 40 kHz) for 30 minutes. The solution was allowed to cool, mixed, filtered, and the filtrate was used as the reference solution. An appropriate amount of protocatechuic acid was precisely weighed, dissolved in 30% methanol to make a solution containing 25 μg per 1 ml, and used as the reference solution.

[0245] Test solution: 1.0 g of the sample was added to 50 ml of water, boiled for 30 minutes, filtered, and the filtrate was evaporated to dryness. The residue was dissolved in 10 ml of 30% methanol, filtered, and the filtrate was used as the test solution.

[0246] Determination: 3 μl of the reference solution and the test solution was precisely taken and injected into the liquid chromatograph for determination.

[0247] Example 3: HPLC characteristic chromatogram of Shilianzi dispensing granules

[0248] 1. Reagents

[0249] Shilianzi dispensing granules (Sichuan Xinlvgreen Pharmaceutical Science and Technology Development Co., Ltd.): three batches of dispensing granules were prepared.

[0250] 2. Proposed chromatographic conditions

[0251] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel was used as the filler (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol was used as the mobile phase A, and 0.1% phosphoric acid was used as the mobile phase B, which was gradient eluted according to the above Table 1; flow rate: 0.3 ml / min, column temperature: 30°C; detection wavelength: 305 nm, and the theoretical plate number calculated according to protocatechuic acid should not be less than 5000.

[0252] Reference solution: 0.5 g of the reference material was added to 50 ml of water, boiled for 30 minutes, filtered, and the filtrate was evaporated to dryness. The residue was dissolved in 10 ml of 30% methanol, and ultrasonically treated (power 600 W, frequency 40 kHz) for 30 minutes. The solution was allowed to cool, mixed, filtered, and the filtrate was used as the reference solution. An appropriate amount of protocatechuic acid was precisely weighed, dissolved in 30% methanol to make a solution containing 25 μg per 1 ml, and used as the reference solution.

[0253] Test solution: an appropriate amount of the sample was finely ground, about 0.2 g was added to 10 ml of 30% methanol, ultrasonically treated (power 600 W, frequency 40 kHz) for 30 minutes, allowed to cool, mixed, centrifuged, the supernatant was filtered, and the filtrate was used as the test solution.

[0254] The assay method was precisely pipetted 3 μl of the reference solution and the test solution, respectively, injected into the liquid chromatograph, and determined, and then obtained.

[0255] On the basis of the above experimental conditions, the diode array detector was used to scan the test solution of Shilianzi Dispensing Granules, and the chromatograms of the test solution at 240 nm, 280 nm, 230 nm, 305 nm, 320 nm, and 360 nm were extracted, respectively. See Figure 23 , Figure 24 The results showed that the information amount of the chromatographic peak was larger at the detection wavelength of 305 nm, and the baseline of the chromatogram was more stable, so the detection wavelength was determined to be 305 nm.

[0256] 2.1 Column temperature investigation

[0257] On the basis of the above experimental conditions, the column temperature at 20 ℃, 25 ℃, 30 ℃, and 35 ℃ was investigated, respectively. See Figure 25 , Table 21:

[0258] Table 21 Column temperature investigation-Relative retention time

[0259]

[0260] The results of the column temperature investigation showed that when the column temperature was 30 ℃, the chromatographic peak shape was relatively symmetrical, and the separation degree was good, so 30 ℃ was finally determined as the column temperature of the characteristic chromatogram method of Shilianzi Dispensing Granules.

[0261] 2.2 Flow rate investigation

[0262] On the basis of the above experimental conditions, the flow rate at 0.2 ml / min, 0.3 ml / min, and 0.4 ml / min was investigated, respectively. See Figure 26 , Table 22:

[0263] Table 22 Flow rate investigation-Relative retention time

[0264]

[0265] The results showed that when the flow rate was 0.3 ml / min, the chromatographic peak shape was good, and the separation degree was moderate. Therefore, the flow rate was determined to be 0.3 ml / min.

[0266] 2.3 Injection amount investigation

[0267] On the basis of the above experimental conditions, the injection amount at 2 μl, 3 μl, and 4 μl was investigated, respectively. See Figure 27 , Table 23:

[0268] Table 23 Injection amount investigation-Relative retention time

[0269]

[0270]

[0271] The results show that when the sample size is 3 μl, the chromatogram peak shape is good and the separation degree is moderate. Therefore, the sample size is determined to be 3 μl.

[0272] 2.4 Delayed investigation

[0273] On the basis of the above proposed experimental conditions, the analysis time is extended to 100 minutes, and whether there are chromatographic peaks after 50 minutes is observed. The results are shown in Figure 28 The results show that there are basically no large chromatographic peaks after 50 minutes of extension, and the analysis time of the characteristic spectrum of Shilianzi Dispensing Granules is finally determined to be 50 minutes.

[0274] In summary, the chromatographic conditions and system suitability test of the characteristic spectrum of Shilianzi Dispensing Granules are determined as follows: octadecylsilane-bonded silica gel is used as the filler (the column length is 150 mm, the inner diameter is 2.1 mm, and the particle size is 1.8 μm); methanol is used as the mobile phase A, and 0.1% phosphoric acid water is used as the mobile phase B, which is gradient eluted according to the above Table 1; the flow rate is 0.3 ml / min, the column temperature is 30°C; the detection wavelength is 305 nm, and the theoretical plate number calculated according to protocatechuic acid should not be less than 5000.

[0275] 3. Preparation of test solution

[0276] 3.1 Investigation of extraction solvent

[0277] Take an appropriate amount of the granules, grind them finely, and take about 0.2 g and put it in a conical flask with a plug. Add water, ethanol, 50% ethanol, methanol, 70% methanol, 50% methanol, and 30% methanol, each 10 ml, and ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes. Cool down, shake well, centrifuge and filter, and take the filtrate, which is obtained. See Figure 29 The results show that the chromatographic peak shape and separation degree of the test solution extracted by water and 30% methanol are good. In order to ensure the stability of the test solution, 30% methanol is selected as the extraction solvent for the characteristic spectrum of Shilianzi Standard Decoction.

[0278] 3.2 Investigation of extraction method

[0279] Take an appropriate amount of the granules, grind them finely, and take about 0.2 g and put it in a conical flask with a plug. Add 30% methanol 10 ml for extraction, and perform reflux and ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, respectively. Cool down, shake well, centrifuge and filter, and take the filtrate, which is obtained. See Figure 30 The results show that the chromatographic peak information is large when the test solution is extracted by ultrasonic extraction and reflux extraction, and the peak shape is better. Therefore, the extraction method of the test solution is determined to be ultrasonic extraction.

[0280] 3.3 Extraction time investigation

[0281] Take the granules of the product, grind finely, take about 0.2 g, put into a conical flask with a stopper, add 30% methanol 10 ml, tightly stopper, respectively investigate the test sample ultrasonic treatment (power 600 W, frequency 40 kHz) extraction time for 20 minutes, 30 minutes, 45 minutes, cool, shake well, centrifugal filter, take the subsequent filtrate, and obtain. See Figure 31 The results show that the extraction is completed at 20 minutes of extraction time, but in order to ensure more complete extraction, the test sample extraction time is determined to be 30 minutes.

[0282] 3.4 Determination of test sample preparation method

[0283] Take the product, grind finely, take about 0.2 g, accurately weigh, put into a conical flask with a stopper, accurately add 30% methanol 10 ml, tightly stopper, weigh, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, centrifugal filter, take the subsequent filtrate, and obtain.

[0284] 4. The characteristic chromatogram method is determined as:

[0285] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 edition four general rules 0512).

[0286] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as the filler (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol as mobile phase A, 0.1% phosphoric acid water as mobile phase B, gradient elution according to the provisions in the above table 1; flow rate: 0.3 ml / min, column temperature: 30℃; detection wavelength: 305 nm, theoretical plate number should not be less than 5000 calculated by protocatechuic acid.

[0287] Preparation of reference solution: take 0.5 g of stone lily seed control drug, add water 50 ml, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 30% methanol 10 ml to dissolve, filter, take the subsequent filtrate as the reference solution of the control drug; take the appropriate amount of protocatechuic acid control, accurately weigh, add 30% methanol to prepare a solution containing 25 μg per 1 ml as the reference solution of protocatechuic acid control.

[0288] Preparation of test sample solution: take the appropriate amount of the product, grind finely, take about 0.2 g, accurately weigh, put into a conical flask with a stopper, accurately add 30% methanol 10 ml, tightly stopper, weigh, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, centrifugal filter, take the subsequent filtrate, and obtain.

[0289] The determination method involves precisely pipetting 3 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0290] 5. Methodological Examination

[0291] 5.1 Chromatographic Peak Identification

[0292] Preparation of the test solution: Prepare the test solution of lotus seed formula granules according to the experimental conditions proposed above.

[0293] Preparation of reference solutions: Accurately weigh appropriate amounts of protocatechuic acid, isoharrenol, nuciferine, and 4-vanillic acid reference standards, and dissolve each in methanol to prepare solutions containing 25 μg per ml. These are used as reference solutions. See Figure 32 ;Depend on Figure 32 It can be seen that a total of 4 characteristic peaks were identified. Among them, the peak corresponding to peak 1 is the protocatechuic acid peak, the peak corresponding to peak 6 is the 4-coumaric acid peak, the peak corresponding to peak 9 is the isocarboxylic acid peak, and the peak corresponding to peak 10 is the nuciferine peak.

[0294] 5.2 Precision Test

[0295] Take the test solution of the lotus seed formula granules and inject it 6 times consecutively according to the proposed experimental method, 3 μl each time. Calculate the relative retention time of each characteristic peak. See Table 24:

[0296] Table 24 Precision Examination - Retention Time

[0297]

[0298] The results show that the retention time RSD values ​​of each characteristic peak are between 0.04% and 0.1%, indicating that the instrument has good precision.

[0299] 5.3 Repeatability Test

[0300] Six portions of the lotus seed formula granules were accurately weighed and prepared and measured according to the proposed experimental method. See Table 25:

[0301] Table 25 Repeatability Tests - Relative Retention Time Ratios

[0302]

[0303]

[0304] The results show that the relative retention time (RSD%) of each characteristic peak is between 0.02% and 0.13%, indicating that the method has good repeatability.

[0305] 5.4 Intermediate Precision Examination

[0306] Different personnel and time periods for inspection

[0307] On the basis of the above experimental conditions, the Shilianzi granules were prepared and determined by different personnel (A, B) at different times (I, II) according to the proposed experimental method. The results were determined by instruments a and b, respectively. The results are shown in Table 26:

[0308] Table 26 Personnel and time investigation-relative retention time ratio

[0309]

[0310] The results showed that the RSD% values of the relative retention time of each characteristic peak were between 0.29% and 1.56% when the same sample was determined by different personnel at different times, indicating that the method had good repeatability.

[0311] 5.5 Stability

[0312] On the basis of the above experimental conditions, the same test solution was measured at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h. See Table 27:

[0313] Table 27 Stability investigation-characteristic peak retention time

[0314]

[0315]

[0316] The results showed that the RSD of the corresponding characteristic peak retention time was 0.14-0.48%, indicating that the sample solution was stable within 24 hours.

[0317] 5.6 Investigation of column durability

[0318] On the basis of the above experimental conditions, the column was investigated as follows: column 1; column 2; column 3; column 4.

[0319] See Table 28, Figure 33

[0320] Table 28 Column durability investigation-relative retention time ratio

[0321]

[0322] From the above table, the sample was detected by the above four kinds of chromatographic column. From the above figure, in the investigation of different chromatographic columns, it was found that chromatographic column 1 lacked a peak, and the other three kinds of chromatographic columns for sample separation could reproduce each characteristic peak in different particle size chromatographic columns. The separation effect of chromatographic column 3 on peaks 1 and 2 was poor, and the separation effect of chromatographic column 4 on peaks 7 and 8 was poor. Chromatographic column 2 (octadecylsilane bonded silica gel as filler (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm)) had good separation effect on characteristic peaks. It was suggested to use chromatographic column for subsequent verification. A total of 10 characteristic peaks were included in the subsequent verification.

[0323] 5.7 Instrument durability investigation

[0324] Based on the above proposed experimental conditions, a portion of stone lotus seed formula granules was precisely weighed, and the test solution was prepared. The determination was carried out on different brands of high performance liquid chromatograph 1; high performance liquid chromatograph 2; high performance liquid chromatograph 3, respectively. See Table 29, Figure 34 .

[0325] Table 29 Instrument durability investigation-Relative retention time ratio

[0326]

[0327] The results showed that when the test sample was detected by the above three instruments, the RSD range of the relative retention time of each characteristic peak was 0.18% to 4.37%. It showed that the instrument durability of the method was good.

[0328] 6. Determination of characteristic peaks and establishment of control chromatogram

[0329] 6.1 Specification of relative retention time limit

[0330] The results of each investigation item and verification of the method are shown in Table 30:

[0331] Table 30 Results of each item of the method RSD% summary-Standard retention time-Relative retention time

[0332]

[0333] From the above table, the durability of the chromatographic column had a greater effect on the peak time of the sample. In order to increase the reproducibility and applicability of the method, the relative retention time limit of each peak was temporarily set as ±10%.

[0334] 6.2 Verification results of 3 batches of stone lotus seed formula granules

[0335] The proposed method was used to determine the characteristic chromatogram of 3 batches of samples, and the relative retention time was calculated. See Figure 35 , Table 31:

[0336] Table 3110 batches of stone lily formula granules relative retention time

[0337]

[0338] According to the relative retention time stability and each batch of sample can be detected and the peak relative is higher, a total of 10 peaks with good reproducibility were selected as characteristic peaks. The results showed that the relative retention time RSD of 10 characteristic peaks of 3 batches of stone lily formula granules was less than 2.0%. Finally, it was provided that:

[0339] The test sample chromatogram should present 10 characteristic peaks, which correspond to the retention time of the 10 characteristic peaks in the reference material chromatogram of the control medicinal material. Among them, the peak corresponding to protocatechuic acid reference is S peak. The relative retention time of each characteristic peak to S peak should be within ±10% of the specified value. The specified value is: 1.13 (peak 2), 1.41 (peak 3), 1.51 (peak 4), 3.40 (peak 5), 4.52 (peak 6), 6.10 (peak 7), 6.26 (peak 8), 6.56 (peak 9), 7.55 (peak 10)

[0340] The three batches of stone lily formula granules were synthesized by using Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version), and the control chromatogram of stone lily formula granule characteristic chromatogram was established. See Figure 36 ; Peak 1: protocatechuic acid (S); Peak 6: 4-coumaric acid; Peak 9: isohispidin; Peak 10: nuciferine; The chromatographic column used was HSS T3 1.8 μm 150×2.1 mm.

[0341] 7. Stone lily formula granule characteristic chromatogram method determination

[0342] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution according to the above Table 1; flow rate was 0.3 ml per minute; column temperature was 30℃; detection wavelength was 305 nm. The theoretical plate number calculated by protocatechuic acid peak should not be less than 5000.

[0343] Reference solution preparation: Take 0.5 g of stone lily control medicinal material, add 50 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 10 ml of 30% methanol to the residue, ultrasonic treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, take the filtrate as the control medicinal material reference solution. Take appropriate amount of protocatechuic acid reference substance, accurately weigh, add 30% methanol to prepare a solution containing 25 μg per 1 ml as the reference substance reference solution.

[0344] The test solution was prepared by taking the product, grinding it finely, taking about 0.2 g, adding 30% methanol 10 ml, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cooling, shaking, filtering, and taking the filtrate, to obtain the test solution.

[0345] The test solution was prepared by taking the product, grinding it finely, taking about 0.2 g, adding 30% methanol 10 ml, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cooling, shaking, filtering, and taking the filtrate, to obtain the test solution.

[0346] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for constructing an HPLC characteristic chromatogram of Aspidium, decoction pieces, standard decoction and formula granules of Aspidium, comprising the following steps: A) dissolving and extracting the raw material of the test sample with a solvent to obtain a test solution; the extraction method is ultrasonic treatment; B) determining the test solution by high performance liquid chromatography to obtain the HPLC characteristic chromatogram of Aspidium, decoction pieces, standard decoction and formula granules of Aspidium; The high performance liquid chromatography conditions are as follows: the chromatographic column is a C 18 column; the mobile phase A is methanol, the mobile phase B is 0.1% phosphoric acid, the detection wavelength is 305 nm, and gradient elution is used; The gradient elution is as follows: 0-12 min, A phase: 15%, B phase: 85%; 12-20 min, A phase: 15-22%, B phase: 85-78%; 20-32 min, A phase: 22-30%, B phase: 78-70%; 32-40 min, A phase: 30-36%, B phase: 70-64%; 40-50 min, A phase: 36-40%, B phase: 64-60%; It also includes a reference solution of control samples: Dissolve protocatechuic acid, 4-coumaric acid, isohispidin and nuciferine in 30% methanol respectively to prepare a solution of 25 μg / mL as a reference solution of control samples.

2. The construction method of claim 1, wherein, The similarity of the characteristic chromatogram of Aspidium is evaluated by a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to obtain an HPLC standard characteristic chromatogram of Aspidium, decoction pieces, standard decoction and formula granules of Aspidium composed of 10 characteristic peaks, wherein peak 1 is protocatechuic acid; peak 6 is 4-coumaric acid; peak 9 is isohispidin; and peak 10 is nuciferine.

3. The construction method of claim 1, wherein, The solvent is 30% methanol; The ultrasonic treatment power is 580-620 W, and the ultrasonic treatment frequency is 35-45 kHz.

4. The construction method of claim 1, wherein, The flow rate of the mobile phase is 0.3 ml / min, and the column temperature is 30°C; the theoretical plate number calculated based on protocatechuic acid should be not less than 5000.

5. The construction method of claim 1, wherein, The similarity of the HPLC characteristic chromatogram of the standard decoction of Aspidium is evaluated by a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to obtain an HPLC standard characteristic chromatogram of the standard decoction of Aspidium composed of 10 characteristic peaks, wherein the peak corresponding to the protocatechuic acid reference is the S peak; in the standard characteristic chromatogram, the relative retention times of the characteristic peaks and the S peak are calculated, which should be within ±10% of the specified value; the specified value is: 1.13 (peak 2), 1.40 (peak 3), 1.51 (peak 4), 3.39 (peak 5), 4.52 (peak 6), 6.09 (peak 7), 6.25 (peak 8), 6.54 (peak 9), and 7.53 (peak 10).

6. The construction method of claim 1, wherein, The similarity of HPLC characteristic spectrum of Gynostemma pentaphyllum medicinal materials and decoction pieces is evaluated by using traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and a standard HPLC characteristic spectrum of Gynostemma pentaphyllum medicinal materials and decoction pieces is obtained, which is composed of 10 characteristic peaks, wherein the peak corresponding to the reference substance of protocatechuic acid is S peak; in the standard characteristic spectrum, the relative retention time of each characteristic peak to S peak should be within ±10% of the specified value; the specified value is: 1.13 (peak 2), 1.41 (peak 3), 1.51 (peak 4), 3.39 (peak 5), 4.51 (peak 6), 6.04 (peak 7), 6.20 (peak 8), 6.49 (peak 9), and 7.46 (peak 10).

7. The construction method of claim 1, wherein, The similarity of HPLC characteristic spectrum of Gynostemma pentaphyllum formula granules is evaluated by using traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and a standard HPLC characteristic spectrum of Gynostemma pentaphyllum formula granules is obtained, which is composed of 10 characteristic peaks, wherein the peak corresponding to the reference substance of protocatechuic acid is S peak; in the standard characteristic spectrum, the relative retention time of each characteristic peak to S peak should be within ±10% of the specified value; the specified value is: 1.13 (peak 2), 1.41 (peak 3), 1.51 (peak 4), 3.40 (peak 5), 4.52 (peak 6), 6.10 (peak 7), 6.26 (peak 8), 6.56 (peak 9), and 7.55 (peak 10).

8. A method for identifying Asparagus cochinchinensis medicinal materials, decoction pieces, standard decoction and its formula granules, characterized in that, The detection is performed by using the method of any one of claims 1-7, and the detection results are analyzed.

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