Characteristic spectrum construction method for cyrtomium fortune, carbonized cyrtomium fortune decoction pieces, standard decoction and formula granules

Abstract

The present invention provides a characteristic spectrum construction method for cyrtomium fortune, carbonized cyrtomium fortune decoction pieces, a standard decoction and formula granules, comprising: A) using a solvent to dissolve a carbonized cyrtomium fortune raw material to obtain a solution to be tested; and B) using a high performance liquid chromatography to carry out measurement on said solution to obtain an UPLC characteristic spectrum of the carbonized cyrtomium fortune raw material, wherein the chromatographic conditions of the high performance liquid chromatography are: a chromatographic column is a C18 column, a mobile phase A is acetonitrile, a mobile phase B is 0.1% of formic acid, and gradient elution is carried out. The present invention uses the high performance liquid chromatography, selects acetonitrile and 0.1% of formic acid as mobile phases to carry out gradient elution, and uses protocatechuic acid as a reference object, so as to establish the UPLC characteristic spectrums of the carbonized cyrtomium fortune decoction pieces, the standard decoction and the formula granules, so that repeatability and precision are good, the method is stable and reliable, and the quality of the carbonized cyrtomium fortune decoction pieces, the standard decoction and the formula granules can be controlled.

Description

A method for constructing characteristic maps of Guanzhong and Guanzhong charcoal slices, standard decoctions, and formula granules

[0001] This application claims priority to a Chinese patent application filed with the Patent Office of China on September 7, 2023, with application number 202311154624.4 and invention name “A method for characterizing the characteristic spectrum of a decoction of Guanzhong and Guanzhong charcoal slices, standard decoctions, and formula granules and their identification”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to the technical field of analysis and detection, and in particular to a method for characterizing a standard decoction of Guanzhong and Guanzhong charcoal slices, a standard decoction, and formula granules and their identification. Background Art

[0003] Guanzhong (Guozhong) is the dried rhizome of Woodwardia unigemmata (Makino.) Nakai., a plant of the Pteridaceae family. Commonly known as "Guozhong Guanzhong," it is harvested in spring and autumn, with the petioles trimmed, fibrous roots and soil removed, washed, and sun-dried. Alternatively, it can be sliced ​​while still fresh and sun-dried. It has the effects of clearing heat and detoxifying, stopping bleeding, and healing sores and promoting tissue regeneration. Guanzhong standard decoction is a freeze-dried powder made from the processed Guanzhong herbal medicine according to a fixed preparation process. Guanzhong formula granules are formulated granules made from the processed Guanzhong herbal medicine according to the key quality standards of the standard decoction. Guanzhong is a commonly used Chinese herbal medicine, first recorded as a medicinal herb in the Shennong Bencao Jing (Shen Nong's Herbal Classic). It has the effects of clearing heat and detoxifying, as well as expelling parasites. Records of Guanzhong's morphology, habitat, and origin in historical compendiums of Materia Medica indicate a wide variety of native plant species. Documents indicate that as many as 58 species from 11 families, 18 genera, and 11 species of plants were used as Guanzhong in my country. Research by Ai Tiemin et al. indicates that in the 1980s, 27 plant species were used as the medicinal source of Guanzhong. The "Compendium of Chinese Materia Medica" lists seven Guanzhong plant species. Yang Chunyu et al., through research, have compiled a total of 38 Guanzhong plant species, 18 of which are widely used as Guanzhong medicinal plants. Even the Chinese Pharmacopoeia has undergone numerous changes in its definition of Guanzhong plant species. The 1977 edition included two species, Guanzhong scutellariae and Guanzhong scutellariae, but after the 1995 edition, only Guanzhong scutellariae was included. The 2010 edition added Guanzhong scutellariae. This demonstrates the immense complexity of Guanzhong medicinal materials.

[0004] Guanzhong Tan is made by stir-frying the Guanzhong herb according to the stir-frying method (General Rule 0213) until the outer surface is black and the interior is brown; this enhances its hemostatic properties. Guanzhong Tan standard decoction is a freeze-dried powder made from the herb after processing according to a fixed preparation process. Guanzhong Tan formula granules are formulated granules made from the Guanzhong herb after processing according to the key quality standards of the standard decoction. This ensures the uniformity and stability of the quality of Guanzhong Tan slices, standard decoction, and formula granules.

[0005] Therefore, a new characteristic spectrum method was established to control the quality of Guanzhong charcoal, and it can clearly distinguish Guanzhong charcoal from Guanzhong and its preparations.

[0006] Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing UPLC characteristic spectra of Guanzhong charcoal slices, standard decoctions, and formula granules. The UPLC characteristic spectra method of Guanzhong charcoal slices, standard decoctions, and formula granules constructed by the present invention is stable and reliable, and can control the quality of Guanzhong charcoal slices, standard decoctions, and formula granules.

[0008] A method for constructing UPLC characteristic profiles of Guanzhongchari slices, standard decoctions, and formula granules, comprising:

[0009] A) dissolving the carbon raw material with a solvent to obtain a solution to be tested;

[0010] B) the test solution is measured by high performance liquid chromatography to obtain a UPLC characteristic spectrum of the charcoal raw material;

[0011] 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 0.1% formic acid, and the elution is performed in a gradient manner.

[0012] The present invention provides a method for constructing a UPLC characteristic spectrum of a guanzhong charcoal slice, a standard decoction, or a formula granule. First, a guanzhong charcoal raw material is taken and dissolved in a solvent to obtain a test solution. The solvent is preferably 70% methanol.

[0013] The present invention adopts the above extraction solvent, which has large chromatographic peak information and good effect.

[0014] Specifically, the charcoal raw material is boiled in water, filtered, the filter residue is dissolved in a solvent, ultrasonically extracted, shaken, and filtered to obtain the product.

[0015] The extraction method of the present invention is ultrasonic extraction after boiling or direct ultrasonic extraction;

[0016] The specific parameters of ultrasound after boiling are as follows: the mass ratio of the smilax china charcoal to water is preferably 1:50; the boiling time is preferably 20 to 30 minutes; the ultrasonic power is preferably 600W, the frequency is preferably 40kHz; the extraction time is preferably 15 to 45 minutes, more preferably 15 to 30 minutes;

[0017] The specific parameters of direct ultrasonic extraction are: ultrasonication using 70% methanol for 10 to 15 minutes.

[0018] Medicinal materials, decoction pieces and control medicinal materials were extracted by ultrasonic method after boiling in water, while standard decoctions and formula granules were extracted by direct ultrasonic method.

[0019] The ratio of the mass g of the carbon raw material to the volume mL of the solvent is preferably (0.1-1): (10-50); more preferably 0.2:25.

[0020] The raw materials of the smilax china charcoal are smilax china medicinal materials, smilax china decoction pieces, standard decoctions, and formula granules. The present invention does not limit them, and the above raw materials can all be subjected to quality control and qualitative testing by the method of the present invention.

[0021] The present invention also includes preparing a reference solution: taking protocatechuic acid and 5-hydroxymethylfurfural respectively, adding water to boil, filtering, and dissolving the filter residue with 70% methanol to obtain a reference solution; the extraction method of the present invention is ultrasonic extraction after boiling or direct ultrasonic extraction; the mass ratio of the Guanzhong medicinal material to water is preferably 1:50; the boiling time is preferably 20 to 30 minutes; the ultrasonic power is preferably 600W, and the frequency is preferably 40kHz; the extraction time is preferably 15 to 45 minutes; more preferably 15 to 30 minutes. Wherein, the ratio of the mass g of the Guanzhong charcoal reference medicinal material to the volume mL of the solvent is preferably (0.1 to 1): (10 to 50); more preferably 0.2:25.

[0022] The reference solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the UPLC characteristic spectra of the Guanzhong carbonized slices, standard decoction, and formula granules are qualitatively measured based on the chromatogram of the reference.

[0023] Mobile phase A was acetonitrile, mobile phase B was 0.1% formic acid, and gradient elution was performed.

[0024] The gradient elution of the present invention is preferably specifically:

[0025] 0-8 min, phase A: 1%, phase B: 99%;

[0026] 8-18 min, phase A: 1%-15%, phase B: 99%-85%;

[0027] 18-35 min, phase A: 15%-25%, phase B: 85%-75%;

[0028] 35-50min, phase A: 25%-26%, phase B: 75%-74%.

[0029] The present invention has good baseline separation under the above elution gradient, good separation of each peak and a stable baseline.

[0030] 1.6μm, 2.1×150mm; column temperature 35℃.

[0031] The chromatographic column of the present invention has symmetrical chromatographic peaks and good separation under the above-mentioned 35°C condition.

[0032] The mobile phase flow rate is preferably 0.3 ml / min.

[0033] The present invention found that the chromatographic peaks were better separated and the peak shapes were more symmetrical at a flow rate of 0.3 ml / min, which is the most preferred solution.

[0034] The detection wavelength of the present invention is preferably 250 nm.

[0035] The present inventors have found that at the above wavelength, the chromatographic information is rich, each component has good absorption, the response value is moderate, the peak separation is good, and the baseline is stable.

[0036] The injection volume of the present invention is 1 μL.

[0037] The beneficial effect of the present invention is that under one liquid chromatography condition, the substance groups of the radix guanzhong charcoal slices, standard decoction, and formula granules are controlled by fingerprints, and the fingerprints are positioned by protocatechuic acid; the cost of detection can be greatly reduced, and qualitative detection can be achieved.

[0038] The similarity of the UPLC characteristic spectra of Guanzhongtan slices, standard decoction and formula granules was evaluated using the traditional Chinese medicine chromatographic fingerprint similarity evaluation system. The UPLC standard characteristic spectra of Guanzhongtan slices, standard decoction and formula granules were obtained, which consisted of 13 characteristic peaks. Peak 1 was hydroxymethylfurfural and peak 3 was protocatechuic acid.

[0039] In the characteristic spectrum of the standard decoction of Guanzhong Carbon, protocatechuic acid is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.77 (peak 1), 0.87 (peak 2), 1.51 (peak 4), 2.75 (peak 5), 2.81 (peak 6), 3.34 (peak 7), 3.43 (peak 8), 3.50 (peak 9), 3.67 (peak 10), 4.57 (peak 11), 4.72 (peak 12), and 4.77 (peak 13);

[0040] In the characteristic spectrum of the Guanzhong Charcoal slices, protocatechuic acid is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.77 (peak 1), 0.86 (peak 2), 1.48 (peak 4), 2.67 (peak 5), 2.72 (peak 6), 3.24 (peak 7), 3.32 (peak 8), 3.39 (peak 9), 3.55 (peak 10), 4.44 (peak 11), 4.57 (peak 12), and 4.61 (peak 13);

[0041] In the characteristic spectrum of the Guanzhong charcoal formula particles, protocatechuic acid is used as the reference peak S peak to calculate the relative retention time of each characteristic peak and the S peak, and the relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.77 (peak 1), 0.87 (peak 2), 1.50 (peak 4), 2.72 (peak 5), 2.77 (peak 6), 3.30 (peak 7), 3.39 (peak 8), 3.46 (peak 9), 3.62 (peak 10), 4.53 (peak 11), 4.65 (peak 12), and 4.70 (peak 13).

[0042] Through testing, the present invention shows that the cyperus rotundus and its preparation have only 11 peaks, which are less than the peak 1 and peak 2 of the cyperus rotundus charcoal, and can clearly identify the cyperus rotundus and the cyperus rotundus charcoal.

[0043] Quality judgment standard: Take samples of Guanzhong Charcoal slices, standard decoction, and formula granules, and operate according to the same method as above to obtain the characteristic spectra of Guanzhong Charcoal slices, standard decoction, and formula granules. Use the "Similarity Evaluation System of Chinese Medicine Chromatographic Fingerprints" (2012 edition) of the National Pharmacopoeia Committee to analyze the standard characteristic spectra of Guanzhong Charcoal slices, standard decoction, and formula granules and the sample characteristic spectra, and the similarity is greater than 0.90.

[0044] The method provided by the present invention can effectively monitor the quality of different batches of Guanzhong carbonized slices, standard decoctions, and formula granules, so as to stabilize their quality. The method has the characteristics of high precision and good reproducibility, and is conducive to comprehensive monitoring of product quality.

[0045] The characteristic spectra of Guanzhongchara medicinal slices, standard decoctions and formula granules established in the present invention use protocatechuic acid and 5-hydroxymethylfurfural as reference substances, focus on the order of each characteristic peak and the correlation with medicinal materials and intermediate products, can comprehensively evaluate the overall quality features of the products, and the method is scientific and reliable.

[0046] The newly developed characteristic spectrum method of the present invention can detect the components with higher polarity in Guanzhong and its standard decoction. In addition, the test sample preparation method is simple and easy to operate, and relatively more characteristic peaks can be identified. Accurate and reliable characteristic spectrum detection can be performed on Guanzhong and its preparations. The authenticity, quality consistency and stability of Guanzhong and its preparations can be effectively detected and controlled. It provides a basis for effectively controlling and comprehensively evaluating the quality of Guanzhong standard decoction. It ensures the uniformity and stability of the quality of Guanzhong and its standard decoction.

[0047] The present invention provides a method for constructing a UPLC characteristic spectrum of Guanzhong Charcoal slices, standard decoctions, and formula granules, comprising: A) dissolving Guanzhong Charcoal raw material in a solvent to obtain a test solution; B) determining the test solution by high-performance liquid chromatography to obtain a UPLC characteristic spectrum of the Guanzhong Charcoal raw material; the chromatographic conditions of the high-performance liquid chromatography are: a C18 column; mobile phase A is acetonitrile; mobile phase B is 0.1% formic acid, and gradient elution is performed. The present invention uses high-performance liquid chromatography, selects acetonitrile-0.1% formic acid as the mobile phase for gradient elution, and uses protocatechuic acid as a reference substance to establish a UPLC characteristic spectrum of the Guanzhong Charcoal slices, standard decoctions, and formula granules. The method has good repeatability and precision, is stable, and is reliable, and can be used to control the quality of the Guanzhong Charcoal slices, standard decoctions, and formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1-1 is a comparative characteristic spectrum of the Guanzhong Charcoal Slices disclosed in Example 1 of the present invention;

[0049] Figure 1-2 is a comparative characteristic spectrum of Guanzhong medicinal material under the same measurement conditions;

[0050] Figure 2 is a graph showing the results of an investigation into the extraction methods of the Guanzhong Carbonized Pieces;

[0051] Figure 3 is a chromatogram of extraction of Guanzhong carbonized slices with different solvents;

[0052] Figure 4 is a chromatogram of different extraction times of Guanzhong carbonized slices;

[0053] Figure 5 is a chromatogram of different extraction solvent amounts;

[0054] Figure 6 is a graph showing the chromatographic peak identification results;

[0055] Figure 7 shows the survey results of different instruments;

[0056] Figure 8 is a graph showing the results of investigations on different chromatographic columns;

[0057] Figure 9-1 is the characteristic spectrum of Guanzhongtan decoction pieces;

[0058] Figure 9-2 is the characteristic spectrum of Guanzhongtan decoction pieces;

[0059] Figure 9-3 is the characteristic spectrum of the medicinal material Guanzhong;

[0060] Figure 9-4 is a characteristic spectrum of the medicinal material Guanzhong;

[0061] Figure 9-5 is a comparative characteristic spectrum of Guanzhong medicinal materials;

[0062] Figure 10 is an investigation of the extraction methods of Guanzhong carbonized decoction;

[0063] Figure 11 is an investigation of the solvents used to extract the Guanzhong carbonized decoction;

[0064] Figure 12 Chromatograms at different extraction times;

[0065] Figure 13 chromatograms of different extraction solvent amounts;

[0066] Figure 14 Chromatographic peak identification;

[0067] Figure 15 Survey with different instruments;

[0068] Figure 16 Investigation of different chromatographic columns;

[0069] Figure 17 Characteristic spectrum of the standard decoction of Guanzhong carbon;

[0070] Figure 18 is a characteristic spectrum of the standard decoction of Guanzhong Carbon;

[0071] Figure 19 is a comparative characteristic spectrum of Guanzhong standard decoction;

[0072] Figure 20 Investigation of mobile phase types;

[0073] Figure 21 Column temperature investigation chromatogram;

[0074] Figure 22 Flow rate investigation;

[0075] Figure 23 Delay investigation;

[0076] Figure 24 Chromatograms of different extraction methods;

[0077] Figure 25 is a chromatogram of extraction with different solvents;

[0078] Figure 26 chromatograms at different extraction times;

[0079] Figure 27 chromatograms of different extraction solvent amounts;

[0080] Figure 28 Chromatographic peak identification;

[0081] Figure 29 Survey with different instruments;

[0082] Figure 30 Investigation of different chromatographic columns;

[0083] [Corrected 28.10.2024 according to Rule 91] Figure 31 is a verification diagram of the characteristic spectra of three batches of Guanzhong Formula Granules;

[0084] Figure 32 is a comparative characteristic spectrum of the Guanzhong carbon formula particles;

[0085] Figure 33 is a characteristic spectrum of the medicinal material Guanzhong;

[0086] Figure 34 is a characteristic spectrum of the medicinal material Guanzhong;

[0087] Figure 35 is a comparative characteristic spectrum of Guanzhong medicinal materials;

[0088] Figure 36 is a characteristic spectrum of Guanzhong standard decoction;

[0089] Figure 37 is a characteristic spectrum of Guanzhong standard decoction;

[0090] Figure 38 is a comparative characteristic spectrum of Guanzhong standard decoction;

[0091] Figure 39 is a characteristic spectrum of Guanzhong Formula Granules;

[0092] Figure 40 is a comparative characteristic spectrum of Guanzhong Formula Granules;

[0093] Figure 41 is a graph showing the results of different gradient conditions in comparative example 4. DETAILED DESCRIPTION

[0094] To further illustrate the present invention, the following describes in detail the method for constructing a UPLC characteristic spectrum of a Guanzhong charcoal slice, a standard decoction, and formula granules provided by the present invention in combination with the examples.

[0095] High performance liquid chromatograph: Agilent 1290; Shimadzu LC-40B X3; Thermo Fisher Vanquish Flex;

[0096] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0097] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0098] Ultrasonic cleaner: KQ600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0099] Chromatographic column: 2.1×150mm,1.6μm; Phenomenex Omega PS C18 100A 2.1×150mm, 1.6μm; ACQUITY BEH Shield RP18 2.1×150mm,1.7μm

[0100] Methanol and acetonitrile were of chromatographic grade, water was ultrapure water, and the remaining reagents were of analytical grade;

[0101] Protocatechuic acid reference substance (China Food and Drug Inspection Institute, batch number: 110809-201906),

[0102] 5-Hydroxymethylfurfural reference substance (China Food and Drug Inspection Institute, batch number: 111626-202215)

[0103] Rhizoma Cyperi (Sichuan Institute for Drug Control, batch number: SCZD003-201905);

[0104] Guanzhong Tan decoction pieces batch number (YP-01, YP-02, YP-03, YP-04, YP-05, YP-06, YP-07, YP-08, YP-09, YP -10, YP-11, YP-12, YP-13, YP-14, YP-15, YP-16, YP-17, YP-18, YP-19, YP-20);

[0105] Freeze-dried powder of standard decoction of Guanzhong carbon (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: BT-01, BT-02, BT-03, BT-04, BT-05, BT-06, BT-07, BT-08, BT-09, BT-10, BT-11, BT-12, BT-13, BT-14, BT-15, BT-16, BT-17, BT-18, BT-19, BT-20).

[0106] Guanzhong charcoal formula granules (batch numbers: KL-01, KL-001, KL-002, KL-003).

[0107] Example 1 Screening of Chromatographic Conditions for Medicinal Materials and Decoction Pieces of Guanzhong Carbon

[0108] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel as the filler (column length, 150 mm, inner diameter, 2.1 mm, particle size, 1.6 μm); acetonitrile as mobile phase A; 0.1% formic acid solution as mobile phase B; gradient elution as specified in the table below; column temperature, 35°C; flow rate, 0.3 ml / min; detection wavelength, 250 nm. The number of theoretical plates, calculated based on the protocatechuic acid peak, should be no less than 5000.

[0109] Preparation of reference solution: Take 1g of Guanzhong reference medicinal material, place it in a stoppered conical flask, add 50ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of 70% methanol to the residue, ultrasonicate (power 600W, frequency 40kHz) for 15 minutes, shake well, filter, and take the filtrate as the reference medicinal material solution. Separately, take an appropriate amount of protocatechuic acid reference substance, accurately weigh it, and accurately add methanol to make a solution containing 20μg of protocatechuic acid per 1ml, which is used as the reference substance solution.

[0110] Take about 1g of the powder of this product (passed through No. 3 sieve), place it in a stoppered conical flask, add 50ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of 70% methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 15 minutes, shake well, filter, and take the filtrate to obtain.

[0111] Determination method: Accurately aspirate 1 μl of the test solution and reference solution, inject them into the liquid chromatograph, and determine the result.

[0112] The test sample chromatogram should show 13 characteristic peaks. Except for peak 1 and peak 2, the retention times of all the peaks should correspond to the retention times of the 11 characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the protocatechuic acid reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.77 (peak 1), 0.86 (peak 2), 1.48 (peak 4), 2.67 (peak 5), 2.72 (peak 6), 3.24 (peak 7), 3.32 (peak 8), 3.39 (peak 9), 3.55 (peak 10), 4.44 (peak 11), 4.57 (peak 12), and 4.61 (peak 13). The results are shown in Figure 1. Figure 1-1 is a comparative characteristic spectrum of the Guanzhong charcoal slices disclosed in Example 1 of the present invention, and Figure 1-2 is a comparative characteristic spectrum of the Guanzhong medicinal material under the same measurement conditions. The method of the present invention can well distinguish between Guanzhong charcoal and Guanzhong medicinal materials.

[0113] Example 2: Investigation of extraction methods

[0114] Comparisons were made between ultrasonic extraction after water decoction, reflux extraction, and ultrasonic extraction. The chromatographic peak information from ultrasonic extraction after water decoction was consistent with that of the standard decoction of Guanzhong Charcoal. Ultimately, ultrasonic extraction after water decoction was chosen. See Figure 2, which shows the results of the investigation of extraction methods for Guanzhong Charcoal slices.

[0115] Example 3 Extraction solvent investigation:

[0116] Methanol, water, 50% methanol, and 70% methanol were investigated as extraction solvents. The results showed that 70% methanol provided a greater amount of information and better resolution of the chromatographic peaks. Therefore, 70% methanol was selected as the extraction solvent. See Figure 3, which shows the chromatograms of Guanzhong Charcoal slices extracted with different solvents.

[0117] Example 4 Extraction time investigation

[0118] Comparison of ultrasonic extractions for 15, 30, and 60 minutes revealed essentially identical chromatograms. Therefore, the extraction time was determined to be 15 minutes. See Figure 4, which shows chromatograms of Guanzhong Charcoal slices at different extraction times.

[0119] Example 5: Investigation of the amount of extraction solvent

[0120] Comparisons were made with extraction solvent additions of 25ml, 50ml, and 100ml. The chromatographic peak size was more appropriate when the extraction solvent volume was 25ml, so the extraction solvent volume was determined to be 25ml. See Figure 5, which shows chromatograms for different extraction solvent volumes.

[0121] Example 6 Methodological Investigation

[0122] 6.1 Chromatographic Peak Identification

[0123] Preparation of test solution: Prepare the Guanzhong herbal test solution according to the final test sample preparation method. Specifically, take approximately 1 g of powder (passed through a No. 3 sieve) and place it in a stoppered conical flask. Add 50 ml of water and boil for 30 minutes. Filter and evaporate the filtrate to dryness. Add 25 ml of 70% methanol to the residue and ultrasonically treat (power 600 W, frequency 40 kHz) for 15 minutes. Shake well, filter, and collect the filtrate.

[0124] Preparation of reference substance solution: Take an appropriate amount of protocatechuic acid reference substance, weigh accurately, and add methanol to make a reference substance solution containing 20 μg of protocatechuic acid per 1 ml.

[0125] Preparation of control medicinal material solution: Take 1 g of Guanzhong control medicinal material, place it in a stoppered conical flask, add 50 ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 25 ml of 70% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 15 minutes, shake well, filter, and take the filtrate as the control medicinal material reference solution.

[0126] Preparation of Negative Control Solution: Prepare a negative control solution lacking the Chinese cyperus rotundus medicinal material according to the experimental conditions proposed above. Locate the characteristic chromatographic peaks of the Chinese cyperus rotundus medicinal material. See Figure 6, which shows the chromatographic peak identification results.

[0127] 6.2 Precision test

[0128] Take the test solution of Guanzhong Charcoal Pieces (Batch No.: YP-01) and inject 1 μl of the sample six times according to the proposed experimental method. Calculate the retention time and peak area of ​​each characteristic peak. See Table 1-1.

[0129] Table 1-1 Precision Investigation-Retention Time

[0130] Table 1-2 Precision Investigation-Peak Area

[0131] The results showed that the RSD of each characteristic peak retention time was 0.02%-0.18%, and the RSD of peak area was 0.02%-0.18%. The instrument has good precision.

[0132] 6.3 Repeatability Study

[0133] Six portions of Guanzhong Charcoal Slices (Batch No.: YP-01) were prepared and assayed according to the proposed experimental method. See Tables 2-1 and 2-2.

[0134] Table 2-1 Repeatability Study - Relative Retention Time Ratio

[0135] Table 2-2 Repeatability study - relative peak area ratio

[0136] The results showed that the RSD of the relative retention time of each characteristic peak was 0.00%-0.54%, and the RSD of the relative peak area was 0.00%-9.08%. This method has good reproducibility.

[0137] 6.4 Intermediate precision study

[0138] 6.4.1 Different Persons, Different Times

[0139] Based on the experimental conditions proposed above, different personnel and different time periods were used to prepare the test solution of Guanzhong Charcoal Pieces (Batch No.: YP-01) and the test was performed. See Tables 3-1 and 3-2.

[0140] Table 3-1 Intermediate precision-relative retention time ratio

[0141] Table 3-2 Intermediate precision-relative peak area ratio

[0142] The results showed that the method had good precision when the same sample was measured by different personnel at different times.

[0143] 6.5 Different instruments

[0144] Based on the experimental conditions proposed above, Instrument 1, Instrument 2, and Instrument 3 were investigated separately. See Figure 7, which shows the results of the investigation of different instruments, and Tables 4-1 and 4-2.

[0145] Table 4-1 Different instruments investigated - relative retention time

[0146] Table 4-2 Different instruments investigated - relative peak areas

[0147] The results showed that when the three instruments were used for detection, the RSDs of the relative retention times of the characteristic peaks ranged from 0.67% to 17.16%; the RSDs of the relative peak areas of the characteristic peaks ranged from 0.00% to 43.84%, indicating that the durability of the different instruments was poor.

[0148] 6.6 Column Durability Assessment

[0149] Based on the experimental conditions proposed above, chromatographic columns 1, 2, and 3 were investigated respectively. See Figure 8, which shows the results of the investigation of different chromatographic columns, and Tables 5-1 and 5-2.

[0150] Table 5-1 Chromatographic column durability inspection - relative retention time

[0151] Table 5-2 Chromatographic column durability inspection - relative peak area

[0152] As shown in Figure 8, The chromatogram of 2.1×150mm, 1.6μm (column 1) is better, so it is recommended to use the column 2.1×150mm, 1.6μm.

[0153] 6.7 Stability Study

[0154] Based on the experimental conditions proposed above, take the same test solution and measure it at 0h, 3h, 6h, 9h, 12h, 18h, and 24h. See Tables 6-1 and 6-2.

[0155] Table 6-1 Stability Study - Retention Time

[0156] Table 6-2 Stability Study-Peak Area

[0157] The results showed that the RSDs for the retention times of the corresponding characteristic peaks ranged from 0.02% to 0.19%, and the RSDs for the peak areas ranged from 2.13% to 6.48%. The sample solution was relatively stable over a 24-hour period. In summary, the RSDs for the retention times of the characteristic peaks met the requirements in all of the above considerations, indicating that this method is robust. These 13 characteristic peaks will be included in subsequent investigations.

[0158] 6.8 Verification of the Characteristic Spectrum of Guanzhong Charcoal Pieces

[0159] Using this method, characteristic spectrum analysis was performed on 20 batches of Guanzhong carbonized slices samples, and the relative retention times and relative peak area ratios were calculated. The results are shown in Figures 9-1 and 9-2, and Tables 7-1 and 7-2.

[0160] Figure 9-1 is the characteristic spectrum of Guanzhongtan decoction pieces, and the batch numbers from bottom to top are: YP-01, YP-02, YP-03, YP-04, YP-05, YP-06, YP-07, YP-08, YP-09, YP-10;

[0161] Figure 9-2 is the characteristic spectrum of Guanzhong Charcoal Pieces. The batch numbers from bottom to top are: YP-11, YP-12, YP-13, YP-14, YP-15, YP-16, YP-17, YP-18, YP-19, and YP-20.

[0162] Table 7-1 Relative retention time of characteristic spectra of 20 batches of Guanzhong charcoal slices

[0163] Table 7-2 Relative peak areas of characteristic spectra of Guanzhong charcoal slices

[0164] Based on the principles of stable relative retention times, consistent detection across all batches of samples, and relatively high peaks, 13 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 characteristic peaks in 20 batches of Guanzhong Charcoal slices ranged from 0.00% to 0.26%, and the relative retention time RSDs of all 13 characteristic peaks in 20 batches of Guanzhong Charcoal slices were less than 10%.

[0165] Final regulations: The test sample chromatogram should show 13 characteristic peaks. Except for peaks 1 and 2, the retention times of all the peaks should correspond to the retention times of the 11 characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the protocatechuic acid reference peak is the S peak. The relative retention time of each characteristic peak and the S peak should be calculated. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.77 (peak 1), 0.86 (peak 2), 1.48 (peak 4), 2.67 (peak 5), 2.72 (peak 6), 3.24 (peak 7), 3.32 (peak 8), 3.39 (peak 9), 3.55 (peak 10), 4.44 (peak 11), 4.57 (peak 12), and 4.61 (peak 13).

[0166] 6.9 Result of the characteristic spectrum of the medicinal material Guanzhong:

[0167] The characteristic spectra of 20 batches of Guanzhong medicinal materials are shown in Figures 9-3 and 9-4, among which Figure 9-3 is the characteristic spectra of Guanzhong medicinal materials ((the batch numbers from bottom to top are: XLS202107277, XLS202107278, XLS202107279, XLS202107280, XLS202107281, XLS202107282, XLS202107283, XLS202107284, XLS202107285, XLS202107286, XLS202107287, XLS202107288, XLS202107289, XLS202107290, XLS202107291, XLS202107292, XLS202107293, XLS202107294, XLS202107295, XLS202107296, XLS202107297, XLS202107298, XLS202107299, XLS202107290, XLS202107291 ... S202107286); Figure 9-4 is the characteristic spectrum of Guanzhong medicinal materials (the batch numbers from bottom to top are: XLS202107287, XLS202107288, XLS202107289, XLS202107290, XLS202107291, XLS202107292, XLS202107293, XLS202107294, XLS202107295, 010176-2012001);

[0168] Figure 9-5 is a comparative characteristic spectrum of Guanzhong medicinal material; Peak 1 (S): protocatechuic acid; Note: Chromatographic column: 2.1×150mm, 1.6μm.

[0169] Example 7 Selection of Chromatographic Conditions for Standard Decoction of Guanzhong Carbon

[0170] The chromatographic conditions and system suitability test for the characteristic spectrum of Guanzhong standard decoction were determined as follows: octadecylsilane bonded silica gel was used as the filler (column length was 150 mm, inner diameter was 2.1 mm, and particle size was 1.6 μm); acetonitrile was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the column temperature was 35°C; the flow rate was 0.3 ml per minute, the detection wavelength was 250 nm, and the number of theoretical plates calculated based on the protocatechuic acid peak should be no less than 5000.

[0171] Preparation of reference solution

[0172] Take 1g of Guanzhong reference medicinal material, place it in a stoppered conical flask, add 50ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of 70% methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. Separately, take an appropriate amount of protocatechuic acid reference substance, accurately weigh it, and accurately add methanol to make a solution containing 20μg of protocatechuic acid per 1ml, which is used as the reference solution of the reference substance.

[0173] Preparation of test solution

[0174] Take 0.2 g of the standard decoction of Guanzhong Carbon, place it in a stoppered conical flask, add 50 ml of 70% methanol, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes. Take it out, let it cool, and take the filtrate to obtain the product.

[0175] Assay

[0176] Accurately pipette 1 μl of the test solution and reference solution, inject them into the liquid chromatograph, and measure to obtain the result.

[0177] Selection of wavelength, column temperature, and flow rate: The material composition of the standard soup and the finished particles is basically consistent, and the characteristic chromatographic conditions are consistent with the system adaptability and the characteristic chromatographic method of the particles.

[0178] 7.1 Investigation of extraction methods

[0179] A comparison of reflux extraction and ultrasonic extraction revealed similar chromatographic peak information, ultimately leading to the selection of ultrasonic extraction due to its ease of operation (see Figure 10). Figure 10 illustrates the investigation of extraction methods for Guanzhong Carbon Decoction.

[0180] 7.2 Extraction solvent investigation

[0181] Methanol, water, 50% methanol, and 70% methanol were investigated as extraction solvents. The results showed that 70% methanol produced a large amount of chromatographic peak information and good resolution, so 70% methanol was selected as the extraction solvent. (See Figure 11.) Figure 11 shows an investigation of extraction solvents for Guanzhong Charcoal Decoction.

[0182] 7.3 Extraction time investigation

[0183] The chromatograms of ultrasonic extractions at 15, 30, and 60 minutes were compared and were basically the same, so the extraction time was determined to be 15 minutes, as shown in Figure 12. Figure 12 Chromatograms of different extraction times.

[0184] 7.4 Investigation of the amount of extraction solvent

[0185] A comparison of 25ml, 50ml, and 100ml of extraction solvent was performed. The chromatographic peak size was more appropriate when the extraction solvent volume was 25ml, so the extraction solvent volume was determined to be 25ml. (See Figure 13.) Figure 13. Chromatograms of different extraction solvent volumes.

[0186] 7.5 Finalize the test sample preparation method

[0187] Take about 0.2 g, place it in a stoppered conical flask, add 25 ml of 70% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 15 minutes, shake well, filter, and take the filtrate to obtain the product.

[0188] Example 8 Methodological Investigation of Guanzhong Standard Decoction

[0189] 8.1 Chromatographic Peak Identification

[0190] Preparation of test solution: Prepare the test solution of Guanzhong Carbon standard decoction according to the final test sample preparation method.

[0191] Preparation of reference solution: Take an appropriate amount of protocatechuic acid reference substance, weigh it accurately, and add methanol to make a reference solution containing 20 μg of protocatechuic acid per 1 ml.

[0192] Preparation of control medicinal material solution: Take 1 g of Guanzhong control medicinal material, place it in a stoppered conical flask, add 50 ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 25 ml of 70% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 15 minutes, shake well, filter, and take the filtrate as the control medicinal material reference solution.

[0193] Preparation of Negative Control Solution: Prepare a negative control solution lacking the standard decoction of Guanzhong according to the experimental conditions outlined above. Locate the characteristic chromatographic peaks of the standard decoction of Guanzhong. See Figure 14. Figure 14. Chromatographic Peak Identification.

[0194] 8.2 Precision test

[0195] Take the test solution of Guanzhong Carbon Standard Decoction (Batch No.: BT-01) and inject 1 μl of the solution six times according to the proposed experimental method. Calculate the retention time and peak area of ​​each characteristic peak. See Tables 8 and 9.

[0196] Table 8 Precision Investigation-Retention Time

[0197] Table 9 Precision Investigation-Peak Area

[0198] The results showed that the RSD of each characteristic peak retention time was 0.02%-0.41%, and the RSD of peak area was 0.49%-2.18%. The instrument has good precision.

[0199] 8.3 Repeatability Study

[0200] Accurately weigh 6 portions of the standard decoction of Guanzhong Carbon (Batch No. BT-01) and prepare and assay according to the proposed experimental method. See Tables 10-1 and 10-2.

[0201] Table 10-1 Repeatability Study ~ Relative Retention Time Ratio

[0202] Table 10-2 Repeatability study - relative peak area ratio

[0203] The results showed that the repeatability of the method was good, with the relative retention time RSD of each characteristic peak ranging from 0.00% to 0.37% and the relative peak area RSD ranging from 0.00% to 15.65%.

[0204] 8.4 Intermediate Precision Examination

[0205] 8.4.1 Inspection by different personnel and at different times

[0206] Based on the experimental conditions proposed above, the standard decoction of Guanzhong (Batch No. BT-01) was accurately weighed, and different personnel and at different times prepared the test solution for determination. See Tables 11-1 and 11-2.

[0207] Table 11-1 Intermediate precision-relative retention time ratio

[0208] Table 11-2 Intermediate precision - relative peak area ratio

[0209] The results showed that the method had good precision when the same sample was measured by different personnel at different times.

[0210] 8.4.2 Inspection of different instruments

[0211] Based on the experimental conditions proposed above, Instrument 1, Instrument 2, and Instrument 3 were investigated separately. See Figure 15, Tables 12-1 and 12-2. Figure 15: Investigation of different instruments.

[0212] Table 12-1 Different Instruments - Relative Retention Time

[0213] Table 12-2 Different instruments investigated - relative peak areas

[0214] The results showed that when the three instruments were used for detection, the RSDs of the relative retention times of the characteristic peaks ranged from 9.21% to 16.74%; the RSDs of the relative peak areas of the characteristic peaks ranged from 0.00% to 68.79%, indicating that the durability of the different instruments was poor.

[0215] 8.5 Durability Assessment

[0216] 8.5.1 Column Durability Assessment

[0217] Based on the experimental conditions proposed above, chromatographic columns 1, 2, and 3 were investigated respectively. See Figure 16, Tables 13-1 and 13-2. Figure 16 Investigation of different chromatographic columns.

[0218] Table 13-1 Chromatographic column durability inspection - relative retention time

[0219] Table 13-2 Chromatographic column durability inspection - relative peak area

[0220] As shown in Figure 16, The chromatogram of 2.1×150mm, 1.6μm (column 1) is better, so it is recommended to use the column 2.1×150mm,1.6μm.

[0221] 8.5.2 Stability Study

[0222] Based on the experimental conditions proposed above, take the same test solution and measure it at 0h, 3h, 6h, 9h, 12h, 18h, and 24h. See Tables 14-1 and 14-2.

[0223] Table 14-1 Stability Study ~ Retention Time

[0224] Table 14-2 Stability Study - Peak Area

[0225] The results showed that the RSD of the corresponding characteristic peak retention time was 0.02% to 0.21%, and the RSD of the peak area was 0.27% to 5.41%. The sample solution was relatively stable within 24 hours.

[0226] 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 13 characteristic peaks were included in the subsequent investigation.

[0227] Example 9 Determination of characteristic peaks and establishment of reference spectrum

[0228] 9.1 Verification Results of 20 Batches of Guanzhong Charcoal Decoction: Using this method, characteristic chromatograms were performed on 20 batches of standard decoction samples, and relative retention times and relative peak area ratios were calculated. See Figures 17-18, Tables 15-1 and 15-2. Figure 17 shows the characteristic chromatogram of Guanzhong Charcoal Standard Decoction (from bottom to top, batch numbers are: BT-01, BT-02, BT-03, BT-04, BT-05, BT-06, BT-07, BT-08, BT-09, BT-10); Figure 18 shows the characteristic chromatogram of Guanzhong Charcoal Standard Decoction (from bottom to top, batch numbers are: BT-11, BT-12, BT-13, BT-14, BT-15, BT-16, BT-17, BT-18, BT-19, BT-20).

[0229] Table 15-1 Relative retention time of 20 batches of standard decoction of Guanzhong carbon

[0230] Table 15-2 Relative peak areas of 20 batches of standard decoction of Guanzhong carbon

[0231] 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 13 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 characteristic peaks in 20 batches of Guanzhongtan standard decoction ranged from 0.40% to 0.86%, and the relative retention time RSDs of the 13 characteristic peaks in 20 batches of Guanzhongtan standard decoction were all less than 10%.

[0232] Final regulations: There should be 13 characteristic peaks in the chromatogram of the test sample. Except for peak 1 and peak 2, the retention times of all the peaks should correspond to the retention times of the 11 characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the protocatechuic acid reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.77 (peak 1), 0.87 (peak 2), 1.51 (peak 4), 2.75 (peak 5), 2.81 (peak 6), 3.34 (peak 7), 3.43 (peak 8), 3.50 (peak 9), 3.67 (peak 10), 4.57 (peak 11), 4.72 (peak 12), 4.77 (peak 13). Figure 19 is the reference characteristic spectrum of Guanzhong standard decoction, Peak 1: 5-hydroxymethylfurfural; Peak 3 (S): protocatechuic acid; Note: Chromatographic column: 2.1×150mm,1.6μ.

[0233] Example 10 Chromatographic Conditions of the Formulated Granules of Guanzhong Carbon

[0234] 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: 1.6 μm); acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, and gradient elution as specified in the table below; the column temperature was 35°C; the flow rate was 0.3 ml / min, the detection wavelength was 250 nm, and the number of theoretical plates calculated based on the protocatechuic acid peak should be no less than 5000.

[0235] 10.1 Preparation of reference solution

[0236] Take 1g of Guanzhong reference medicinal material, place it in a stoppered conical flask, add 50ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of 70% methanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. Separately, take an appropriate amount of protocatechuic acid reference substance, accurately weigh it, and accurately add methanol to make a solution containing 20μg of protocatechuic acid per 1ml, which is used as the reference solution of the reference substance.

[0237] 10.2 Preparation of test solution

[0238] Take the Guanzhong carbon formula particles, grind them into powder, take about 0.2g, place it in a stoppered conical flask, add 50ml of 70% methanol, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, take it out, cool it, and take the filtrate to obtain it.

[0239] 10.3 Determination method

[0240] Accurately pipette 1 μl of reference solution and test solution respectively, inject into liquid chromatograph, and measure to obtain the result.

[0241] 10.4 Wavelength Selection

[0242] The wavelength is selected as 250nm.

[0243] 10.5 Investigation of Mobile Phase Types

[0244] Based on the experimental conditions proposed above, mobile phases of acetonitrile-water, acetonitrile-0.1% formic acid, and acetonitrile-0.1% phosphoric acid were investigated, as shown in Figure 20. Figure 20: Mobile phase investigation. The results showed that acetonitrile-0.1% formic acid provided excellent chromatographic peak resolution, so acetonitrile-0.1% formic acid was the preferred mobile phase.

[0245] 10.6 Column Temperature Investigation

[0246] Based on the experimental conditions proposed above, column temperatures of 25°C, 30°C, and 35°C were investigated. See Figure 21. Figure 21. Chromatogram of column temperature investigation.

[0247] The results showed that when the column temperature was 35℃, the chromatographic peak separation was good, so the column temperature was temporarily set at 35℃.

[0248] 10.7 Flow rate investigation

[0249] Based on the experimental conditions proposed above, flow rates of 0.25 ml / min, 0.3 ml / min, and 0.35 ml / min were investigated. See Figure 22. Figure 22 Flow rate investigation.

[0250] The results showed that the information content of each characteristic peak chromatogram was basically the same when the flow rate was 0.25ml / min, 0.3ml / min, and 0.35ml / min. At a flow rate of 0.3ml / min, the chromatographic peak separation was better, so the flow rate was determined to be 0.30ml / min.

[0251] 10.8 Delay Investigation

[0252] Based on the experimental conditions proposed above, the chromatogram acquisition time was set to double. The results are shown in Figure 23. Figure 23. Delayed performance test. The results show that no chromatographic peaks appear in the chromatogram after 50 minutes.

[0253] In summary, the chromatographic conditions and system suitability test for the characteristic spectrum of Guanzhong Formula Granules were determined as follows: octadecylsilane bonded silica gel as the filler (column length 150 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, gradient elution as specified in the table below; column temperature 35°C; flow rate 0.3 ml / min; detection wavelength 250 nm. The number of theoretical plates calculated based on the protocatechuic acid peak should be no less than 5000.

[0254] Example 11 Preferred Pretreatment Method of Guanzhong Carbon Formula Granules

[0255] 11.1 Investigation of extraction methods

[0256] A comparison of reflux extraction and ultrasonic extraction revealed similar chromatographic peak information, ultimately leading to the selection of ultrasonic extraction due to its ease of use (see Figure 24). Figure 24. Chromatograms of different extraction methods.

[0257] 11.2 Extraction Solvent Investigation

[0258] Methanol, water, 50% methanol, and 70% methanol were investigated as extraction solvents. The results showed that 70% methanol produced a greater amount of information and better resolution of the chromatographic peaks, so 70% methanol was selected as the extraction solvent. (See Figure 25.) Figure 25. Chromatograms of extractions using different solvents.

[0259] 11.3 Extraction time investigation

[0260] Comparison of ultrasonic extraction times of 15, 30, and 60 minutes revealed essentially identical chromatograms. Therefore, the extraction time was determined to be 15 minutes, as shown in Figure 26. Figure 26. Chromatograms of different extraction times.

[0261] 11.4 Investigation of the amount of extraction solvent

[0262] The addition amounts of extraction solvent (25 ml, 50 ml, and 100 ml) were compared. When the amount of extraction solvent was 25 ml, the chromatographic peak size was more appropriate, so the amount of extraction solvent was determined to be 25 ml, as shown in Figure 27. Figure 27 Chromatograms of different extraction solvent amounts.

[0263] 11.5 Finalize the test sample preparation method

[0264] Take an appropriate amount of this product, grind it into powder, take about 0.2g, place it in a stoppered conical flask, add 25ml of 70% methanol, ultrasonically treat (power 600W, frequency 40kHz) for 15 minutes, shake well, filter, and take the filtrate to obtain the product.

[0265] Example 12 Methodological Investigation of the Formulated Granules of Guanzhong Carbon

[0266] 12.1 Chromatographic Peak Identification

[0267] Preparation of test solution: Prepare the test solution of Guanzhong Carbon Formula Granules according to the final test sample preparation method.

[0268] Preparation of reference solution: Accurately weigh an appropriate amount of protocatechuic acid reference substance and add methanol to prepare a reference solution containing 20 μg of protocatechuic acid per 1 ml. Separately weigh an appropriate amount of 5-hydroxymethylfurfural reference substance and add methanol to prepare a reference solution containing 50 μg of 5-hydroxymethylfurfural per 1 ml.

[0269] Take 1 g of Guanzhong reference medicinal material, place it in a stoppered conical flask, add 50 ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 25 ml of 70% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 15 minutes, shake well, filter, and take the filtrate as the reference solution of the control medicinal material.

[0270] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of Quguanzhong formula granules.

[0271] The characteristic chromatographic peaks of Guanzhong Formula Granules were located, as shown in Figure 28. Figure 28 Chromatographic peak identification.

[0272] 12.2 Precision test

[0273] Take the test solution of Guanzhong Formula Granules (Batch No.: KL-01) and inject 1 μl of the sample six times according to the proposed experimental method. Calculate the retention time and peak area of ​​each characteristic peak. See Tables 16-1 and 16-2.

[0274] Table 16-1 Precision Investigation - Retention Time

[0275] Table 16-2 Precision Investigation-Peak Area

[0276] The results showed that the RSD of each characteristic peak retention time was 0.02%-0.11%, and the RSD of peak area was 0.23%-2.49%. The instrument has good precision.

[0277] 12.3 Repeatability Study

[0278] Accurately weigh 6 portions of Guanzhong Carbon Formula Granules (Batch No. KL-01) and prepare and measure according to the proposed experimental method. See Tables 17-1 and 17-2.

[0279] Table 17-1 Repeatability Study - Relative Retention Time Ratio

[0280] Table 17-2 Repeatability Study - Relative Peak Area Ratio

[0281] The results showed that the repeatability of the method was good, with the relative retention time RSD of each characteristic peak ranging from 0.00% to 0.28% and the relative peak area RSD ranging from 0.00% to 9.49%.

[0282] 12.4 Intermediate precision

[0283] 12.4.1 Different Persons, Different Times

[0284] Based on the experimental conditions proposed above, Guanzhong granules were accurately weighed, and test solutions were prepared by different personnel and at different times for determination. See Tables 18-1 and 18-2.

[0285] Table 18-1 Intermediate precision-relative retention time ratio

[0286] Table 18-2 Intermediate precision - relative peak area ratio

[0287] The results showed that the method had good precision when the same sample was measured by different personnel at different times.

[0288] 12.4.2 Instrument Durability Assessment

[0289] Based on the experimental conditions proposed above, Instrument 1, Instrument 2, and Instrument 3 were investigated separately. See Figure 29, Tables 19-1 and 19-2. Figure 29: Investigation of different instruments.

[0290] Table 19-1 Chromatographic column durability inspection - relative retention time

[0291] Table 19-2 Chromatographic column durability inspection - relative peak area

[0292] The results showed that when the three instruments were used for detection, the RSDs of the relative retention times of the characteristic peaks were between 0.67% and 17.08%, and the RSDs of the relative peak areas of the characteristic peaks were between 0.00% and 43.49%, indicating that the durability of the different instruments was poor.

[0293] 12.5 Durability Assessment

[0294] 12.5.1 Column Durability Assessment

[0295] Based on the experimental conditions proposed above, Column 1, Column 2, and Column 3 were investigated separately. See Figure 30, Tables 20-1 and 20-2. Figure 30: Investigation of different chromatographic columns.

[0296] Table 20-1 Chromatographic column durability inspection - relative retention time

[0297] Table 20-2 Chromatographic column durability inspection - relative peak area

[0298] As shown in Figure 30, The chromatogram of 2.1×150mm, 1.6μm (column 1) is better, so it is recommended to use the column 2.1×150mm,1.6μm.

[0299] 12.6 Stability Study

[0300] Based on the experimental conditions proposed above, take the same test solution and measure it at 0h, 3h, 6h, 9h, 12h, 18h, and 24h. See Tables 21-1 and 21-2.

[0301] Table 21-1 Stability Study ~ Retention Time

[0302] Table 21-2 Stability Study - Peak Area

[0303] The results showed that the RSD of the corresponding characteristic peak retention time was 0.02% to 0.24%, and the RSD of the peak area was 0.40% to 7.32%. The sample solution was relatively stable within 24 hours.

[0304] 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 13 characteristic peaks were included in the subsequent investigation.

[0305] Example 13 Determination of characteristic peaks and establishment of reference spectrum

[0306] 13.1 Batch Verification Results of Guanzhong Formula Granules

[0307] The proposed method was used to determine the characteristic spectra of three batches of this product, and the relative retention times and peak areas were calculated. See Figure 31, Tables 22-1 and 22-2. Figure 31: Verification of the characteristic spectra of three batches of Guanzhong Formula Granules.

[0308] Table 22-1 Relative retention time of three batches of guanyulu carbon formula granules

[0309] Table 22-2 Relative peak areas of 3 batches of Guanzhong carbon formula particles

[0310] 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 13 peaks with good repeatability 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 characteristic peaks of the three batches of Guanzhong carbon granules were between 0.00% and 0.37%, and the relative retention time RSDs of the 13 characteristic peaks of the three batches of Guanzhong carbon granules were all less than 10%.

[0311] Final regulations: The test sample chromatogram should show 13 characteristic peaks. Except for peaks 1 and 2, the retention times of all peaks should correspond to the retention times of the 11 characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the protocatechuic acid reference peak is the S peak. The relative retention time of each characteristic peak and the S peak should be calculated. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.77 (peak 1), 0.87 (peak 2), 1.50 (peak 4), 2.72 (peak 5), 2.77 (peak 6), 3.30 (peak 7), 3.39 (peak 8), 3.46 (peak 9), 3.62 (peak 10), 4.53 (peak 11), 4.65 (peak 12), and 4.70 (peak 13).

[0312] Three batches of Guanzhong Carbon Formula Granules were synthesized using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition), and a comparison of the characteristic spectra of Guanzhong Carbon Formula Granules was established. See Figure 32, which shows the comparison of the characteristic spectra of Guanzhong Carbon Formula Granules. Peak 1: 5-hydroxymethylfurfural; Peak 3 (S): protocatechuic acid; Chromatographic column: 2.1×150mm, 1.6μm.

[0313] Comparative Example 1

[0314] The same conditions as in Example 1 were used to determine the medicinal material Guanzhong. The results are shown in Figures 33 to 35, where Figure 33 is a characteristic spectrum of the medicinal material Guanzhong, Figure 34 is a characteristic spectrum of the medicinal material Guanzhong, and Figure 35 is a control characteristic spectrum of the medicinal material Guanzhong; Peak 1 (S): protocatechuic acid; Note: Chromatographic column: 2.1×150mm, 1.6μm. As can be seen from the above, the method of the present invention can well identify the decoction pieces of Guanzhong medicinal material and Guanzhong charcoal medicinal material decoction pieces.

[0315] Comparative Example 2

[0316] The same conditions as in Example 1 were used to determine the standard decoction of Guanzhong. The results are shown in Figures 36 to 38, wherein Figure 36 is a characteristic spectrum of the standard decoction of Guanzhong (the batch numbers from bottom to top are: BT-01, BT-02, BT-03, BT-04, BT-05, BT-06, BT-07, BT-08, BT-09, BT-10), Figure 37 is a characteristic spectrum of the standard decoction of Guanzhong (the batch numbers from bottom to top are: BT-11, BT-12, BT-13, BT-14, BT-15, BT-16, BT-17, BT-18, BT-19, BT-20), and Figure 38 is a control characteristic spectrum of the standard decoction of Guanzhong; Peak 1 (S): protocatechuic acid; Note: Chromatographic column: 2.1×150mm, 1.6μm. As can be seen from the above, the method of the present invention can well distinguish the standard decoction of Guanzhong and the standard decoction of Guanzhong charcoal.

[0317] Comparative Example 3

[0318] The same conditions as in Example 1 were used to determine the Guanzhong Formula Granules. The results are shown in Figures 39 and 40, where Figure 39 is a characteristic spectrum of the Guanzhong Formula Granules (from bottom to top: KL-001; KL-002; KL-003, and Figure 40 is a control characteristic spectrum of the Guanzhong Formula Granules; Peak 1 (S): protocatechuic acid; Note: Chromatographic column: 2.1×150mm, 1.6μm. As can be seen from the above, the method of the present invention can well distinguish the Guanzhong Guanzhong formula particles from the Guanzhong charcoal Guanzhong formula particles.

[0319] There are only 11 peaks for Guanzhong and its preparations, which are less than Peak 1 and Peak 2 for Guanzhong charcoal, and can clearly identify Guanzhong and Guanzhong charcoal.

[0320] Comparative Example 4

[0321] This comparative example uses different gradient conditions, and the rest of the results are the same as those in Example 1:

[0322] Mobile phase gradient:

[0323] Gradient 1:

[0324] The results are shown in Figure 41. It can be seen from the figure that peaks 1 and 2 are missing; peak 5 is separated, and peaks 12 and 13 have poor separation.

[0325] Gradient 2:

[0326] The results are shown in FIG41 . As can be seen from the figure, peaks 2 and 3 are not separated, and peaks 12 and 13 have poor separation.

[0327] Gradient 3:

[0328] The results are shown in FIG41 , from which it can be seen that peaks 11, 12, and 13 are missing.

[0329] It can be seen from the above that only the gradient conditions of the present invention can achieve a good peak separation effect.

[0330] 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 a characteristic spectrum of a standard decoction of Rhizoma Cynomorii and Rhizoma Cynomorii charcoal slices, a standard decoction, and a formula granule, comprising: A) dissolving the carbon raw material with a solvent to obtain a test solution; B) the test solution is measured by high performance liquid chromatography to obtain a UPLC characteristic spectrum of the charcoal raw material; 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 0.1% formic acid, and the elution is gradient.

2. The method according to claim 1, characterized in that The method also includes preparing a reference solution: taking protocatechuic acid and 5-hydroxymethylfurfural respectively, adding water to boil, filtering, and dissolving the filter residue with 70% methanol to obtain a reference solution; the concentration of the reference solution is 20 μg / mL; The reference solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the UPLC characteristic spectra of the charcoal slices, standard decoction, and formula granules are qualitatively measured based on the chromatogram of the reference.

3. The method according to claim 1, characterized in that The gradient elution is specifically: 0-8min, phase A: 1%, phase B: 99%; 8-18min, phase A: 1%-15%, phase B: 99%-85%; 18-35min, phase A: 15%-25%, phase B: 85%-75%; 35-50min, phase A: 25%-26%, phase B: 75%-74%.

4. The method according to claim 3, characterized in that The chromatographic column is a C18 column with specifications of 1.6 μm and 2.1×150 mm; the column temperature is 35° C.

5. The method according to claim 4, characterized in that The flow rate of the mobile phase was 0.3 mL / min; the detection wavelength was 250 nm; and the injection volume was 1 μL.

6. The method according to claim 1, characterized in that The similarity of the UPLC characteristic spectra of Guanzhongtan slices, standard decoction and formula granules was evaluated by the similarity evaluation system of traditional Chinese medicine chromatographic fingerprints, and the UPLC standard characteristic spectra of Guanzhongtan slices, standard decoction and formula granules were obtained, which consisted of 13 characteristic peaks, among which peak 1 was hydroxymethylfurfural and peak 3 was protocatechuic acid.

7. The method according to claim 6, characterized in that In the characteristic spectrum of the standard decoction of Guanzhong Carbon, protocatechuic acid is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.77 (peak 1), 0.87 (peak 2), 1.51 (peak 4), 2.75 (peak 5), 2.81 (peak 6), 3.34 (peak 7), 3.43 (peak 8), 3.50 (peak 9), 3.67 (peak 10), 4.57 (peak 11), 4.72 (peak 12), 4.77 (peak 13); In the characteristic spectrum of the Guanzhong Charcoal Slices, protocatechuic acid is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.77 (peak 1), 0.86 (peak 2), 1.48 (peak 4), 2.67 (peak 5), 2.72 (peak 6), 3.24 (peak 7), 3.32 (peak 8), 3.39 (peak 9), 3.55 (peak 10), 4.44 (peak 11), 4.57 (peak 12), 4.61 (peak 13); In the characteristic spectrum of the Guanzhong charcoal formula particles, protocatechuic acid is used as the reference peak S peak to calculate the relative retention time of each characteristic peak and the S peak, and the relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.77 (peak 1), 0.87 (peak 2), 1.50 (peak 4), 2.72 (peak 5), 2.77 (peak 6), 3.30 (peak 7), 3.39 (peak 8), 3.46 (peak 9), 3.62 (peak 10), 4.53 (peak 11), 4.65 (peak 12), and 4.70 (peak 13).

8. The method according to claim 1, characterized in that Step A) The solvent is 70% methanol; the extraction method is water boiling followed by ultrasonic extraction; the ultrasonic power is 600W and the frequency is 40kHz; the extraction time is 15 to 45 minutes.

9. The method according to claim 8, characterized in that In step A), the ratio of the mass g of the carbon raw material to the volume mL of the solvent is (0.1-1): (10-50).

10. The method according to claim 1, characterized in that The raw materials of the charcoal in step A) are charcoal slices, standard decoctions, and formula granules.

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