Method for constructing UPLC characteristic profiles of Guanzhong medicinal materials, decoction pieces, standard decoctions, and formula granules

Abstract

The present invention provides a method for constructing a UPLC characteristic spectrum of a Chinese cyperus rotundus medicinal material, a decoction piece, a standard decoction, and a formula granule, comprising: A) dissolving the Chinese cyperus rotundus raw material in a solvent to obtain a test liquid; B) determining the test liquid by high-performance liquid chromatography to obtain a UPLC characteristic spectrum of the Chinese cyperus rotundus raw material; the chromatographic conditions of the high-performance liquid chromatography are: a chromatographic column is a C18 column; mobile phase A is methanol, mobile phase B is water, and gradient elution is performed. The present invention adopts high-performance liquid chromatography, selects methanol-water as the mobile phase for gradient elution, and uses uridine, guanosine, tryptophan, and adenosine as reference substances to establish a UPLC characteristic spectrum of the Chinese cyperus rotundus medicinal material, a decoction piece, a standard decoction, and a formula granule. The method has good repeatability and precision, is stable, and is reliable, and can be used to control the quality of the Chinese cyperus rotundus medicinal material, a decoction piece, a standard decoction, and a formula granule.

Description

Technical Field

[0001] The present invention relates to the field of analysis and detection technology, and in particular to a method for constructing UPLC characteristic maps of Guanzhong medicinal materials, decoction pieces, standard decoctions, and formula granules. Background Art

[0002] 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.

[0003] Lin Yongqiang et al. used UPLC-MS / MS to detect the presence of adulterated Cyperus rotundus in anti-cold granules using octadecylsilane bonded silica gel as the filler, and a mobile phase consisting of 0.02% formic acid in acetonitrile-0.02% formic acid solution with gradient elution. Mass spectrometry was performed in electrospray positive ionization mode (ESI+) and multiple reaction monitoring (MRM) for quantitative analysis. This method can be used to detect the presence of Cyperus rotundus in anti-cold granules. Ma Bingzhi et al. used UPLC fingerprinting to determine the presence of Cyperus rotundus in nine samples of Cyperus rotundus from different origins using an acetonitrile-water gradient elution method. UPLC fingerprints of Cyperus rotundus from different origins were established, and similarity comparisons were performed. Furthermore, the kaempferol-3-0-L-rhamnosyl group was identified.

[0004] Fingerprinting is an important method for macro-control of medicinal material quality. Currently, no relevant reports have been found for the fingerprint of Guanzhong. Fingerprint quality control research can not only identify the various types of compounds in the fingerprint, but also enable the identification of Guanzhong and its adulterants. Therefore, in order to comprehensively control the quality of Guanzhong, it is necessary to conduct fingerprint research.

[0005] Therefore, in order to better control the quality of Guanzhong medicinal materials, decoction pieces, standard decoctions, and formula granules, a characteristic spectrum method for Guanzhong was constructed to achieve the purpose of scientifically identifying Guanzhong and its preparations. Summary of the Invention

[0006] 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 medicinal materials, decoction pieces, standard decoctions, and formula granules. The UPLC characteristic spectra method of Guanzhong medicinal materials, decoction pieces, standard decoctions, and formula granules constructed by the present invention is stable and reliable, and can control the quality of Guanzhong medicinal materials, decoction pieces, standard decoctions, and formula granules.

[0007] A method for constructing UPLC characteristic profiles of Guanzhong medicinal materials, decoction pieces, standard decoctions, and formula granules, comprising:

[0008] A) dissolving the raw material of Rhizoma Cyperi in a solvent to obtain a solution to be tested;

[0009] B) the liquid to be tested is measured by high performance liquid chromatography to obtain the UPLC characteristic spectrum of the Rhizoma Cyperi raw material;

[0010] 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% phosphoric acid, and the elution is performed in a gradient manner.

[0011] The present invention provides a method for constructing a UPLC characteristic spectrum of a Chinese rhizoma cyrthospermi medicinal material, a decoction piece, a standard decoction, or a formula granule. First, the Chinese rhizoma cyrthospermi raw material is taken and dissolved in a solvent to obtain a test solution. The solvent is preferably 70% methanol.

[0012] The present invention adopts the above extraction solvent, which has large chromatographic peak information and good effect.

[0013] Specifically, the raw materials of Guanzhong are dissolved in a solvent, extracted, cooled, shaken, and filtered to obtain the product.

[0014] The extraction method of the present invention is ultrasonic extraction or heating reflux extraction; preferably ultrasonic extraction; 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.

[0015] Among them, the ratio of the mass g of the cyperus rotundus raw material to the volume mL of the solvent is preferably (0.1-0.5): (50-100); more preferably 0.1:50.

[0016] The raw materials of Rhizoma Cyperi are Rhizoma Cyperi medicinal materials, Rhizoma Cyperi 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.

[0017] The present invention further comprises preparing a reference solution: taking protocatechuic acid respectively and dissolving it in 70% methanol to obtain a reference solution;

[0018] 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 medicinal materials, decoction pieces, standard decoctions, and formula granules are qualitatively measured based on the chromatogram of the reference.

[0019] Mobile phase A was acetonitrile, mobile phase B was 0.1% phosphoric acid, and gradient elution was performed.

[0020] The gradient elution of the present invention is preferably specifically:

[0021] 0-5 min, phase A: 6%, phase B: 94%;

[0022] 5-10 min, phase A: 6%-15%, phase B: 94%-85%;

[0023] 10-25 min, phase A: 15%-25%, phase B: 85%-75%;

[0024] 25-40min, phase A: 25%-26%, phase B: 75%-74%.

[0025] The present invention has good baseline separation under the above elution gradient, good separation of each peak and a stable baseline.

[0026] 1.6μm, 2.1×150mm; column temperature 35℃.

[0027] The chromatographic column of the present invention has symmetrical chromatographic peaks and good separation under the above-mentioned 35°C condition.

[0028] The mobile phase flow rate is preferably 0.3 ml / min.

[0029] 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.

[0030] The detection wavelength of the present invention is preferably 250 nm or 260 nm.

[0031] The detection wavelength of Guanzhong medicinal material slices is 250nm, the detection wavelength of standard decoction is 260nm, and the detection wavelength of formula granules is 260nm.

[0032] 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.

[0033] The injection volume of the present invention is 1 μL.

[0034] The beneficial effect of the present invention is that under one liquid chromatography condition, the substance groups of Guanzhong medicinal materials, decoction pieces, standard decoctions, and formula granules are controlled by fingerprints, and protocatechuic acid is used to locate the fingerprints; the cost of detection can be greatly reduced and qualitative detection can be achieved.

[0035] The similarity of the UPLC characteristic spectra of Guanzhong medicinal materials, decoction pieces, standard decoctions, and formula granules was evaluated using the traditional Chinese medicine chromatographic fingerprint similarity evaluation system. The UPLC standard characteristic spectra of Guanzhong medicinal materials, decoction pieces, standard decoctions, and formula granules were obtained, which consisted of 11 characteristic peaks, among which peak 1 was protocatechuic acid.

[0036] In the characteristic spectrum of the Guanzhong standard decoction, 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: 2.09 (peak 2), 4.40 (peak 3), 4.50 (peak 4), 5.68 (peak 5), 5.84 (peak 6), 5.99 (peak 7), 6.34 (peak 8), 8.36 (peak 9), 8.66 (peak 10), and 8.79 (peak 11);

[0037] In the characteristic spectra of the Guanzhong medicinal materials and the decoction pieces, the relative retention time of each characteristic peak and the S peak is calculated with protocatechuic acid as the reference peak S peak, and the relative retention time is within ±10% of the specified value, and the specified values ​​are: 2.09 (peak 2), 4.39 (peak 3), 4.49 (peak 4), 5.67 (peak 5), 5.82 (peak 6), 5.97 (peak 7), 6.32 (peak 8), 8.33 (peak 9), 8.63 (peak 10), and 8.76 (peak 11);

[0038] In the characteristic spectrum of the Guanzhong formula granules, the relative retention time of each characteristic peak and the S peak is calculated with protocatechuic acid as the reference peak S peak. The relative retention time is within ±10% of the specified value, and the specified values ​​are:

[0039] 2.10 (peak 2), 4.41 (peak 3), 4.51 (peak 4), 5.70 (peak 5), 5.86 (peak 6), 6.01 (peak 7), 6.36 (peak 8), 8.39 (peak 9), 8.68 (peak 10), 8.81 (peak 11).

[0040] Quality judgment standard: Take samples of Guanzhong medicinal materials, decoction pieces, standard decoctions, and formula granules, and operate according to the same method as above to obtain the characteristic spectra of Guanzhong medicinal materials, decoction pieces, standard decoctions, 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 medicinal materials, decoction pieces, standard decoctions, and formula granules and the sample characteristic spectra, and the similarity is greater than 0.90.

[0041] The method provided by the present invention can effectively monitor the quality of different batches of Guanzhong medicinal materials, decoction pieces, standard decoctions, and formula granules to ensure stable quality. The method has the characteristics of high precision and good reproducibility, which is conducive to comprehensive monitoring of product quality.

[0042] The characteristic spectra of Guanzhong medicinal materials, decoction pieces, standard decoctions and formula granules established in the present invention use uridine, guanosine, tryptophan and adenosine as reference substances, focus on the order of each characteristic peak and the correlation with the medicinal materials and intermediate products, and can comprehensively evaluate the overall quality characteristics of the products. The method is scientific and reliable.

[0043] The newly developed characteristic spectrum method of the present invention can detect components with high 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 Gansui, Guanzhong and their 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.

[0044] The present invention provides a method for constructing a UPLC characteristic spectrum of a Chinese cyperus rotundus medicinal material, a decoction piece, a standard decoction, and a formula granule, comprising: A) dissolving the Chinese cyperus rotundus raw material in a solvent to obtain a test solution; B) measuring the test solution by high-performance liquid chromatography to obtain a UPLC characteristic spectrum of the Chinese cyperus rotundus 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% phosphoric acid, and gradient elution is performed. The present invention uses high-performance liquid chromatography, selects acetonitrile-0.1% phosphoric acid as the mobile phase for gradient elution, and uses protocatechuic acid as a reference substance to establish a UPLC characteristic spectrum of the Chinese cyperus rotundus medicinal material, a decoction piece, a standard decoction, and a formula granule. The method has good repeatability and precision, is stable, and is reliable, and can be used to control the quality of the Chinese cyperus rotundus medicinal material, the decoction piece, the standard decoction, and the formula granule. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a comparative characteristic spectrum of the Chinese medicinal material Guanzhong disclosed in Example 1 of the present invention;

[0046] Figure 2 This is the result diagram of the investigation of extraction method;

[0047] Figure 3 Extract chromatograms for different solvents;

[0048] Figure 4 Chromatograms at different extraction times;

[0049] Figure 5 The chromatograms are for different extraction solvent amounts;

[0050] Figure 6This is the result diagram of chromatographic peak identification;

[0051] Figure 7 The following are the results of investigations with different instruments;

[0052] Figure 8 The results of the investigation of different chromatographic columns are shown;

[0053] Figure 9-1 This is the characteristic spectrum of the medicinal material Guanzhong;

[0054] Figure 9-2 This is the characteristic spectrum of the medicinal material Guanzhong;

[0055] Figure 10 Characteristic spectrum of medicinal pieces;

[0056] Figure 11 Characteristic spectrum of medicinal pieces;

[0057] Figure 12 It is the reference characteristic spectrum of the characteristic spectrum of Guanzhong decoction pieces;

[0058] Figure 13 To investigate the extraction method;

[0059] Figure 14 Investigation of extraction solvents;

[0060] Figure 15 Chromatograms at different extraction times;

[0061] Figure 16 Chromatograms of different extraction solvent amounts;

[0062] Figure 17 Chromatographic peak identification;

[0063] Figure 18 Surveys using different instruments;

[0064] Figure 19 Investigation of different chromatographic columns;

[0065] Figure 20 Characteristic spectrum of Guanzhong standard decoction;

[0066] Figure 21 Characteristic spectrum of Guanzhong standard decoction;

[0067] Figure 22 This is the reference characteristic spectrum of the standard decoction of Guanzhong;

[0068] Figure 23 UV absorption spectrum of protocatechuic acid;

[0069] Figure 24 Chromatograms of Guanzhong Formula Granules at different wavelengths;

[0070] Figure 25 Investigation of mobile phase types;

[0071] Figure 26 Column temperature inspection chromatogram;

[0072] Figure 27 Flow rate investigation;

[0073] Figure 28 Delayed inspection;

[0074] Figure 29 Chromatograms of different extraction methods;

[0075] Figure 30 Chromatograms of extraction with different solvents;

[0076] Figure 31 Chromatograms at different extraction times;

[0077] Figure 32 Chromatograms of different extraction solvent amounts;

[0078] Figure 33 Chromatographic peak identification;

[0079] Figure 34 Surveys using different instruments;

[0080] Figure 35 Investigation of different chromatographic columns;

[0081] Figure 36 Verification diagram of characteristic spectra of 3 batches of Guanzhong formula granules;

[0082] Figure 37 Comparative characteristic spectrum of Guanzhong formula granules;

[0083] Figure 38 This is the result diagram of different mobile phase gradients in Comparative Example 1. DETAILED DESCRIPTION

[0084] To further illustrate the present invention, the following describes in detail a method for constructing a UPLC characteristic spectrum of a Guanzhong medicinal material, decoction pieces, standard decoction, and formula granules provided by the present invention in combination with examples.

[0085] High performance liquid chromatography: Instrument 1; Instrument 2; Instrument 3;

[0086] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0087] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0088] Ultrasonic cleaner: KQ600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0089] Chromatographic columns: Column 1; Column 2; Column 3

[0090] Methanol and acetonitrile were of chromatographic grade, water was ultrapure water, and the remaining reagents were of analytical grade;

[0091] Protocatechuic acid reference substance (China Food and Drug Inspection Institute, batch number: 110809-201906),

[0092] Guanzhong reference medicinal material (Sichuan Institute of Drug Control, batch number: NO.SCZD003-201905),

[0093] Guanzhong medicinal materials batch numbers: YC-01, YC-02, YC-03, YC-04, YC-05, YC-06, YC-07, YC-08, YC-09, YC -10, YC-11, YC-12, YC-13, YC-14, YC-15, YC-16, YC-17, YC-18, YC-19, YC-20

[0094] Lyophilized powder of standard decoction of Guanzhong (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).

[0095] Guanzhong formula granules (batch numbers: KL-001, KL-01, KL-02, KL-03).

[0096] Example 1 Screening of Chromatographic Conditions for Medicinal Materials and Decoction Pieces

[0097] Chromatographic conditions and system suitability testing were performed using 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.

[0098]

[0099]

[0100] Preparation of Reference Solution: Take 0.5 g of the Guanzhong reference medicinal material and place it in a conical flask. Add 50 ml of 70% methanol and sonicate (power 600 W, frequency 40 kHz) for 30 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 add methanol to make a solution containing 20 μg per 1 ml. This is the reference substance solution.

[0101] Preparation of test solution: Take about 0.5 g of medicinal material powder (passed through No. 3 sieve), place it in a stoppered conical flask, add 50 ml of 70% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the filtrate to obtain the product.

[0102] Determination method: Accurately aspirate 1 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0103] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize two batches of Guanzhong medicinal materials, and a reference characteristic spectrum of Guanzhong medicinal materials was established. Figure 1 As shown, Figure 1 This is a comparative characteristic spectrum of the Guanzhong medicinal material disclosed in Example 1 of the present invention.

[0104] Comparison of characteristic patterns.

[0105] The test sample's characteristic spectrum should show seven characteristic peaks (peak 3 is generated after vinegar treatment), and their retention times should correspond to the six characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the adenosine reference peak is the S peak. Calculate the relative retention time of each characteristic peak to the S peak. The relative retention time should be within ±10% of the specified value. The specified values ​​are 0.21 (peak 1), 0.40 (peak 2), 0.50 (peak 3), 0.64 (peak 4), 0.71 (peak 5), and 0.89 (peak 6).

[0106] Example 2: Investigation of extraction methods

[0107] Comparison of ultrasonic, reflux extraction and ultrasonic extraction after water decoction showed that the chromatographic peak information was roughly the same, and ultrasonic extraction was finally selected as it was easy to operate. Figure 2 , Figure 2 This is the result diagram of the extraction method investigation.

[0108] Example 3 Extraction solvent investigation:

[0109] Methanol, water, 30% methanol, 50% methanol, and 70% methanol were investigated as extraction solvents. The results showed that the chromatographic peaks obtained with 70% methanol as the extraction solvent had a large amount of information and good separation, so 70% methanol was selected as the extraction solvent. Figure 3 , Figure 3 Extract chromatograms for different solvents.

[0110] Example 4 Extraction time investigation

[0111] After comparing the ultrasonic extraction time of 15 minutes, 30 minutes and 45 minutes, the sample was completely extracted at 30 minutes. Therefore, the extraction time was determined to be 30 minutes. Figure 4 , Figure 4 Chromatograms at different extraction times.

[0112] Example 5: Investigation of the amount of extraction solvent

[0113] The addition of 25ml, 50ml and 100ml of extraction solvent was compared. When the amount of extraction solvent was 50ml, the size of the chromatographic peak was more appropriate, so the amount of extraction solvent was determined to be 50ml. Figure 5 , Figure 5 The chromatograms are obtained with different amounts of extraction solvent.

[0114] Example 6 Methodological Investigation

[0115] 6.1 Chromatographic Peak Identification

[0116] Preparation of test sample solution: Prepare the test sample solution of Guanzhong medicinal material according to the final test sample preparation method.

[0117] Preparation of reference substance solution: Take an appropriate amount of protocatechuic acid reference substance, accurately weigh it, and add methanol to make a reference substance solution containing 20 μg of protocatechuic acid per 1 ml.

[0118] Preparation of control medicinal material solution: Take 0.5 g of Guanzhong control medicinal material, place it in a stoppered conical flask, add 50 ml of 70% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the filtrate as the control medicinal material reference solution.

[0119] Preparation of negative control solution: According to the experimental conditions proposed above, prepare a negative control solution lacking the Chinese medicine Guanzhong. Locate the characteristic spectrum peak of the Chinese medicine Guanzhong. Figure 6 , Figure 6 This is the result diagram of chromatographic peak identification.

[0120] 6.2 Precision test

[0121] Take the test solution of Guanzhong herb (Batch No.: YC-20) 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.

[0122] Table 1-1 Precision Investigation-Retention Time

[0123]

[0124] Table 1-2 Precision Investigation-Peak Area

[0125]

[0126] The results showed that the RSD of each characteristic peak retention time was 0.03%-0.15%, and the RSD of peak area was 0.48%-8.95%. The instrument has good precision.

[0127] 6.3 Repeatability Study

[0128] Accurately weigh 6 portions of Cyperus rotundus (Batch No.: YC-20) and prepare and assay according to the proposed experimental method. See Tables 2-1 and 2-2.

[0129] Table 2-1 Repeatability Study - Relative Retention Time Ratio

[0130]

[0131] Table 2-2 Repeatability study - relative peak area ratio

[0132]

[0133]

[0134] The results showed that the RSD of the relative retention time of each characteristic peak was 0.26%-0.65%, and the RSD of the relative peak area was 1.90%-14.92%. This method has good reproducibility.

[0135] 6.4 Intermediate precision study

[0136] 6.4.1 Different Persons, Different Times

[0137] Based on the experimental conditions proposed above, the Chinese medicinal material Guanzhong (Batch No.: YC-20) was accurately weighed, and different personnel and at different times prepared the test solution for determination. See Tables 3-1 and 3-2.

[0138] Table 3-1 Intermediate precision-relative retention time ratio

[0139]

[0140] Table 3-2 Intermediate precision-relative peak area ratio

[0141]

[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 respectively. Figure 7 , Figure 7 Tables 4-1 and 4-2 show the results of investigations using different instruments.

[0145] Table 4-1 Chromatographic column durability inspection - relative retention time

[0146]

[0147] Table 4-2 Chromatographic column durability inspection - relative peak area

[0148]

[0149] 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 5.76% and 18.80%, and the RSDs of the relative peak areas of the characteristic peaks were between 2.19% and 10.46%, indicating that the durability of the different instruments was poor.

[0150] 6.6 Column Durability Assessment

[0151] Based on the experimental conditions proposed above, the chromatographic columns 1, 2, and 3 were investigated respectively. Figure 8 , Figure 8 The results of the investigation of different chromatographic columns are shown in Tables 5-1 and 5-2.

[0152] Table 5-1 Chromatographic column durability inspection - relative retention time

[0153]

[0154] Table 5-2 Chromatographic column durability inspection - relative peak area

[0155]

[0156] Depend on Figure 8 It can be seen that the chromatogram of column 1 has better separation, so column 1 is recommended.

[0157] 6.7 Stability Study

[0158] Based on the experimental conditions proposed above, take the same test solution and measure it at 0h, 2h, 4h, 8h, 12h, and 24h. See Tables 6-1 and 6-2.

[0159] Table 6-1 Stability Study - Retention Time

[0160]

[0161] Table 6-2 Stability Study-Peak Area

[0162]

[0163] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.06% and 0.57%, and the sample solution was relatively stable within 24 hours.

[0164] 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 11 characteristic peaks were included in the subsequent investigation.

[0165] 6.8 Verification of the Characteristic Spectrum of Guanzhong Medicinal Material

[0166] The Guanzhong medicinal material was tested and the ratios of relative retention time and relative peak area were calculated. The results are shown in Figures 9-10, Tables 7-1 and 7-2.

[0167] in Figure 9-1 This is the characteristic spectrum of Guanzhong medicinal materials. The batch numbers from bottom to top are: Guanzhong control medicinal materials, YC-01, YC-02, YC-03, YC-04, YC-05, YC-06, YC-07, YC-08, YC-09, and YC-10; Figure 9-2 The characteristic spectrum of Guanzhong medicinal materials, from bottom to top, the batch numbers are: Guanzhong control medicinal materials, YC-11, YC-12, YC-13, YC-14, YC-15, YC-16, YC-17, YC-18, YC-19, Y C-20 .

[0168] Table 7-1 Relative retention time of characteristic spectra of Guanzhong medicinal material

[0169]

[0170] Table 7-2 Relative peak areas of characteristic spectra of Guanzhong medicinal materials

[0171]

[0172]

[0173] Based on the principles of stable relative retention times, consistent detection across all batches, and relatively high peak heights, 11 peaks with good reproducibility were selected as characteristic peaks. Results showed that the relative peak areas of the characteristic peaks varied significantly, making it impossible to specify relative peak areas. Therefore, these peaks were not included in the quality standard. The final specification stipulates that the test sample's characteristic chromatogram should contain 11 characteristic peaks, which should correspond to the retention times of the 11 characteristic peaks in the chromatogram of the reference herbal medicine. The peak corresponding to the protocatechuic acid reference is the S peak. The relative retention time of each characteristic peak relative to the S peak should be calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 2.10 (peak 2), 4.30 (peak 3), 4.49 (peak 4), 5.67 (peak 5), 5.83 (peak 6), 5.97 (peak 7), 6.32 (peak 8), 8.34 (peak 9), 8.64 (peak 10), and 8.76 (peak 11).

[0174] 6.9 Verification of Characteristic Spectra of 20 Batches of Guanzhong Pieces

[0175] 20 batches of Guanzhong decoction pieces were tested, and the results are shown in Figure 10-11 , Tables 8-1 and 8-2. Figure 10 Characteristic spectrum of medicinal pieces (the batch numbers from bottom to top are: Guanzhong reference medicinal material, YP-01, YP-02, YP-03, YP-04, YP-05, YP-06, YP-07, YP-08, YP-09, YP-10); Figure 11 Characteristic spectrum of medicinal pieces (batch numbers from bottom to top are: Guanzhong control medicinal material, YP-11, YP-12, YP-13, YP-14, YP-15, YP-16, YP-17, YP-18, YP-19, YP-20).

[0176] Table 8-1 Relative retention time of characteristic spectra of medicinal pieces

[0177]

[0178]

[0179] Table 8-2 Relative peak areas of characteristic spectra of medicinal pieces

[0180]

[0181] Based on the principles of stable relative retention times, consistent detection across all batches, and relatively high peak heights, 11 peaks with good reproducibility were selected as characteristic peaks. Results showed that the relative peak areas of the characteristic peaks varied significantly, making it impossible to specify relative peak areas. Therefore, these peaks were not included in the quality standard. The final specification stipulates that the test sample's characteristic chromatogram should contain 11 characteristic peaks, which should correspond to the retention times of the 11 characteristic peaks in the chromatogram of the reference herbal medicine. The peak corresponding to the protocatechuic acid reference is the S peak. The relative retention time of each characteristic peak relative to the S peak should be calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 2.09 (peak 2), 4.39 (peak 3), 4.49 (peak 4), 5.67 (peak 5), 5.82 (peak 6), 5.97 (peak 7), 6.32 (peak 8), 8.33 (peak 9), 8.63 (peak 10), and 8.76 (peak 11).

[0182] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 20 batches of Guanzhong medicinal materials, and a reference characteristic spectrum of Guanzhong decoction pieces was established. The results are as follows Figure 12 , Figure 12 It is a comparison characteristic spectrum of the characteristic spectrum of Guanzhong decoction pieces.

[0183] Example 7 Selection of Chromatographic Conditions for Standardized Decoction of Guanzhong

[0184] 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.

[0185]

[0186] Preparation of reference solution

[0187] Take 0.5g of Guanzhong reference medicinal material and place it in a stoppered conical flask. Add 50ml of 70% methanol and ultrasonically treat (power 600W, frequency 40kHz) for 30 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 add methanol to make a solution containing 20μg of protocatechuic acid per 1ml. This is used as the reference substance solution.

[0188] Preparation of test solution

[0189] Take about 0.2 g of Guanzhong standard decoction and prepare a test solution using the same preparation method as in Example 1 "Control medicinal material reference solution".

[0190] Assay

[0191] Accurately pipette 1 μl of the test solution and reference solution, inject them into the liquid chromatograph, and measure to obtain the result.

[0192] 7.1 Investigation of extraction methods

[0193] Comparing reflux extraction and ultrasonic extraction, the chromatographic peak information was roughly the same, and ultrasonic extraction was finally selected due to its ease of operation. Figure 13 . Figure 13 To investigate the extraction method.

[0194] 7.2 Extraction solvent investigation

[0195] Methanol, water, 30% methanol, 50% methanol, and 70% methanol were investigated as extraction solvents. The results showed that the chromatographic peaks obtained with 70% methanol as the extraction solvent had a large amount of information and good separation, so 70% methanol was selected as the extraction solvent. Figure 14 . Figure 14 Investigation of extraction solvents.

[0196] 7.3 Extraction time investigation

[0197] The chromatograms of the ultrasonic extractions at 15, 30, and 45 minutes were basically the same, so the extraction time was determined to be 15 minutes. Figure 15 . Figure 15 Chromatograms at different extraction times.

[0198] 7.4 Investigation of the amount of extraction solvent

[0199] The addition of 25ml, 50ml and 100ml of extraction solvent was compared. When the amount of extraction solvent was 50ml, the size of the chromatographic peak was more appropriate, so the amount of extraction solvent was determined to be 50ml. Figure 16 . Figure 16 Chromatograms of different extraction solvent amounts.

[0200] 7.5 Finalize the test sample preparation method

[0201] Take about 0.2 g, place it in a stoppered conical flask, add 50 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.

[0202] Example 8 Methodological Investigation of Guanzhong Standard Decoction

[0203] 8.1 Chromatographic Peak Identification

[0204] Preparation of test sample solution: Prepare the test sample solution of Guanzhong standard decoction according to the final test sample preparation method.

[0205] 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.

[0206] Preparation of control medicinal material solution: Take 0.5 g of Guanzhong control medicinal material, place it in a conical flask, add 50 ml of 70% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the filtrate as the control medicinal material reference solution.

[0207] Preparation of negative control solution: According to the experimental conditions proposed above, prepare negative control solution lacking the standard decoction of Guanzhong. Locate the characteristic peaks of the standard decoction of Guanzhong. Figure 17 . Figure 17 Chromatographic peak identification.

[0208] 8.2 Precision test

[0209] Take the test solution of Guanzhong Standard Decoction (Batch No. BT-20) 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 9-1 and 9-2.

[0210] Table 9-1 Precision Investigation-Retention Time

[0211]

[0212] Table 9-2 Precision Investigation-Peak Area

[0213]

[0214] The results showed that the RSD of the retention time of each characteristic peak was 0.02%-0.17%, and the RSD of the peak area was 0.31%-4.48%. The instrument has good precision.

[0215] 8.3 Repeatability Study

[0216] Accurately weigh 6 portions of Guanzhong Standard Decoction (Batch No. BT-20) and prepare and assay according to the proposed experimental method. See Tables 10-1 and 10-2.

[0217] Table 10-1 Repeatability Study ~ Relative Retention Time Ratio

[0218]

[0219]

[0220] Table 10-2 Repeatability study - relative peak area ratio

[0221]

[0222] The results showed that the repeatability of the method was good, with the relative retention time RSD of each characteristic peak ranging from 0.49% to 0.62% and the relative peak area RSD ranging from 1.39% to 7.73%.

[0223] 8.4 Intermediate Precision Examination

[0224] 8.4.1 Inspection by different personnel and at different times

[0225] Based on the experimental conditions proposed above, the standard decoction of Guanzhong (Batch No. BT-20) was accurately weighed, and different personnel and at different times prepared the test solution for determination. See Tables 11-1 and 11-2.

[0226] Table 11-1 Intermediate precision-relative retention time ratio

[0227]

[0228] Table 11-2 Intermediate precision - relative peak area ratio

[0229]

[0230] The results showed that the method had good precision when the same sample was measured by different personnel at different times.

[0231] 8.4.2 Inspection of different instruments

[0232] Based on the experimental conditions proposed above, Instrument 1, Instrument 2, and Instrument 3 were investigated respectively. Figure 18 , Tables 12-1 and 12-2. Figure 18 Survey with different instruments.

[0233] Table 12-1 Different Instruments - Relative Retention Time

[0234]

[0235] Table 12-2 Different instruments investigated - relative peak areas

[0236]

[0237] 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 4.88% and 17.70%, and the RSDs of the relative peak areas of the characteristic peaks were between 0.86% and 22.77%, indicating that the durability of the different instruments was poor.

[0238] 8.5 Durability Assessment

[0239] 8.5.1 Column Durability Assessment

[0240] Based on the experimental conditions proposed above, the chromatographic columns 1, 2, and 3 were investigated respectively. Figure 19 , Tables 13-1 and 13-2. Figure 19 Investigation of different chromatographic columns.

[0241] Table 13-1 Chromatographic column durability inspection - relative retention time

[0242]

[0243] Table 13-2 Chromatographic column durability inspection - relative peak area

[0244]

[0245] Depend on Figure 19 It can be seen that the chromatogram of column 1 has better separation, so column 1 is recommended.

[0246] 8.5.2 Stability Study

[0247] Based on the experimental conditions proposed above, take the same test solution and measure it at 0h, 2h, 4h, 8h, 12h, and 24h. See Tables 14-1 and 14-2.

[0248] Table 14-1 Stability Study ~ Retention Time

[0249]

[0250]

[0251] Table 14-2 Stability Study - Peak Area

[0252]

[0253] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.03% and 0.57%, and the sample solution was relatively stable within 24 hours.

[0254] 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 11 characteristic peaks were included in the subsequent investigation.

[0255] Example 9 Determination of characteristic peaks and establishment of reference spectrum

[0256] 9.1 Verification results of 20 batches of Guanzhong decoction standard samples Using this method, characteristic spectrum analysis was performed on 20 batches of standard decoction samples to calculate relative retention time and relative peak area ratio. Figure 20-21 , Tables 15-1 and 15-2. Figure 20Characteristic spectrum of Guanzhong standard decoction (lot numbers from bottom to top are: Guanzhong reference medicinal material, BT-01, BT-02, BT-03, BT-04, BT-05, BT-06, BT-07, BT-08, BT-09, BT-10); Figure 21 Characteristic spectrum of Guanzhong standard decoction (batch numbers from bottom to top are: Guanzhong control medicinal material, BT-11, BT-12, BT-13, BT-14, BT-15, BT-16, BT-17, BT-18, BT-19, BT-20).

[0257] Table 15-1 Relative retention time of 20 batches of standard decoction of Guanzhong

[0258]

[0259]

[0260] Table 15-2 Relative peak areas of 20 batches of Guanzhong standard decoction

[0261]

[0262] Based on the principles of stable relative retention times, detectable peaks across all batches of samples, and relatively high peaks, 11 peaks with good reproducibility were selected as characteristic peaks. The results showed that when Peak 1 was used as the S peak, the relative retention time RSDs of the characteristic peaks for the three batches of Guanzhong Formula Granules ranged from 0.00% to 0.31%, and the relative retention time RSDs of the 11 characteristic peaks for the three batches of Guanzhong Formula Granules were all less than 5%.

[0263] Final regulations: The test sample chromatogram should show 11 characteristic peaks, and the retention times should correspond to 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: 2.09 (peak 2), 4.40 (peak 3), 4.50 (peak 4), 5.68 (peak 5), 5.84 (peak 6), 5.99 (peak 7), 6.34 (peak 8), 8.36 (peak 9), 8.66 (peak 10), and 8.79 (peak 11). Figure 22 This is the reference characteristic spectrum of Guanzhong standard decoction, Peak 1 (S): Protocatechuic acid Note: Chromatographic column: T3 2.1×150mm,1.6μ.

[0264] Example 10 Chromatographic Conditions of Guanzhong Formula Granules

[0265] 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.

[0266]

[0267] 10.1 Preparation of reference solution

[0268] Take 0.5g of Guanzhong reference medicinal material and place it in a stoppered conical flask. Add 50ml of 70% methanol and 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. This is used as the reference solution of the reference substance.

[0269] 10.2 Preparation of test solution

[0270] Take an appropriate amount of Guanzhong formula granules, grind them into powder, take 0.2g, and prepare the test solution using the same preparation method as in Example 1 "Control medicinal material reference solution".

[0271] 10.3 Determination method

[0272] Accurately pipette 1 μl of reference solution and test solution respectively, inject into liquid chromatograph, and measure to obtain the result.

[0273] 10.4 Wavelength Selection

[0274] Based on the experimental conditions proposed above, the diode array detector was used to perform full-band scanning of the protocatechuic acid reference substance and the test solution, and the chromatograms of each solution at wavelengths of 210nm, 230nm, 250nm, 270nm, 290nm, and 310nm were obtained. Figures 23-24 . Figure 23 UV absorption spectrum of protocatechuic acid; Figure 24 Chromatograms of Guanzhong formula granules at different wavelengths.

[0275] The results showed that the chromatographic peak had a larger amount of information when the detection wavelength was 250nm, so the detection wavelength was determined to be 250nm.

[0276] 10.5 Investigation of Mobile Phase Types

[0277] Based on the experimental conditions proposed above, the mobile phases of acetonitrile-water, acetonitrile-0.1% formic acid, and acetonitrile-0.1% phosphoric acid were investigated. Figure 25. Figure 25 The results showed that acetonitrile-0.1% formic acid was the best mobile phase for chromatographic peak separation, so acetonitrile-0.1% formic acid was the preferred mobile phase.

[0278] 10.6 Column Temperature Investigation

[0279] Based on the experimental conditions proposed above, the column temperatures of 25℃, 30℃ and 35℃ were investigated respectively. Figure 26 . Figure 26 Column temperature inspection chromatogram.

[0280] 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℃.

[0281] 10.7 Flow rate investigation

[0282] Based on the experimental conditions proposed above, the flow rates of 0.25ml / min, 0.3ml / min, and 0.35ml / min were investigated. Figure 27 . Figure 27 Flow rate investigation.

[0283] 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.

[0284] 10.8 Delay Investigation

[0285] Based on the experimental conditions proposed above, the chromatogram acquisition time was set to 2 times. Figure 28 . Figure 28 Delayed investigation: The results showed that no chromatographic peak appeared in the chromatogram after 40 minutes.

[0286] 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 260 nm. The number of theoretical plates calculated based on the protocatechuic acid peak should be no less than 5000.

[0287]

[0288] Example 11 Preparation of the test solution of Guanzhong formula granules

[0289] 11.1 Investigation of extraction methods

[0290] Comparing reflux extraction and ultrasonic extraction, the chromatographic peak information was roughly the same, and ultrasonic extraction was finally selected due to its ease of operation. Figure 29 . Figure 29 Chromatograms of different extraction methods.

[0291] 11.2 Extraction Solvent Investigation

[0292] Methanol, water, 30% methanol, 50% methanol, and 70% methanol were investigated as extraction solvents. The results showed that the chromatographic peaks obtained with 70% methanol as the extraction solvent had a large amount of information and good separation, so 70% methanol was selected as the extraction solvent. Figure 30 . Figure 30 Chromatograms of extractions with different solvents.

[0293] 11.3 Extraction time investigation

[0294] The chromatograms of ultrasonic extractions for 15 minutes, 30 minutes, and 45 minutes were basically the same. Therefore, the extraction time was determined to be 15 minutes. Figure 31 . Figure 31 Chromatograms at different extraction times.

[0295] 11.4 Investigation of the amount of extraction solvent

[0296] The extraction solvent addition amounts of 25ml, 50ml and 100ml were compared. When the extraction solvent amount was 50ml, the chromatographic peak size was more appropriate, so the extraction solvent amount was determined to be 50ml. Figure 32 . Figure 32 Chromatograms of different extraction solvent amounts.

[0297] 11.5 Finalize the test sample preparation method

[0298] Take an appropriate amount of this product, grind it 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 15 minutes, shake well, filter, and take the filtrate to obtain the product.

[0299] Example 12 Methodological Investigation

[0300] 12.1 Chromatographic Peak Identification

[0301] Preparation of test sample solution: Prepare the test sample solution of Guanzhong formula granules according to the final test sample preparation method.

[0302] Preparation of reference solution: Take an appropriate amount of protocatechuic acid reference substance, accurately weigh it, and accurately add methanol to make a reference solution containing 20 μg of protocatechuic acid per 1 ml.

[0303] Preparation of control medicinal material solution: Take 0.5 g of Guanzhong control medicinal material, place it in a conical flask, add 50 ml of 70% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the filtrate as the control medicinal material reference solution.

[0304] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of Quguanzhong formula granules.

[0305] Position the characteristic spectrum peaks of Guanzhong Formula Granules, see Figure 33 . Figure 33 Chromatographic peak identification.

[0306] 12.2 Precision test

[0307] Take the test solution of Guanzhong Formula Granules (Batch No.: KL-001) 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.

[0308] Table 16-1 Precision Investigation - Retention Time

[0309]

[0310] Table 16-2 Precision Investigation-Peak Area

[0311]

[0312] The results showed that the RSD of each characteristic peak retention time was 0.03%-0.15%, and the RSD of peak area was 0.40%-1.45%. The instrument has good precision.

[0313] 12.3 Repeatability Study

[0314] Accurately weigh 6 portions of Guanzhong Formula Granules (Batch No. KL-001) and prepare and measure according to the proposed experimental method. See Tables 17-1 and 17-2.

[0315] Table 17-1 Repeatability Study - Relative Retention Time Ratio

[0316]

[0317] Table 17-2 Repeatability Study - Relative Peak Area Ratio

[0318]

[0319]

[0320] The results showed that the repeatability of the method was good, with the relative retention time RSD of each characteristic peak ranging from 0.23% to 0.51% and the relative peak area RSD ranging from 0.00% to 5.95%.

[0321] 12.4 Intermediate precision

[0322] 12.4.1 Different Persons, Different Times

[0323] 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.

[0324] Table 18-1 Intermediate precision-relative retention time ratio

[0325]

[0326] Table 18-2 Intermediate precision - relative peak area ratio

[0327]

[0328] The results showed that the method had good precision when the same sample was measured by different personnel at different times.

[0329] 12.4.2 Instrument Durability Assessment

[0330] Based on the experimental conditions proposed above, Instrument 1, Instrument 2, and Instrument 3 were investigated respectively. Figure 34 , Tables 19-1 and 19-2. Figure 34 Survey with different instruments.

[0331] Table 19-1 Chromatographic column durability inspection - relative retention time

[0332]

[0333] Table 19-2 Chromatographic column durability inspection - relative peak area

[0334]

[0335]

[0336] 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 5.39% and 17.01%, and the RSDs of the relative peak areas of the characteristic peaks were between 0.00% and 9.49%, indicating that the durability of the different instruments was poor.

[0337] 12.5 Durability Assessment

[0338] 12.5.1 Column Durability Assessment

[0339] Based on the experimental conditions proposed above, the chromatographic columns 1, 2, and 3 were investigated respectively. Figure 35 , Tables 14-15. Figure 35 Investigation of different chromatographic columns.

[0340] Table 20-1 Chromatographic column durability inspection - relative retention time

[0341]

[0342] Table 20-2 Chromatographic column durability inspection - relative peak area

[0343]

[0344] Depend on Figure 35 It can be seen that the chromatogram of column 1 has better separation, so column 1 is recommended.

[0345] 12.6 Stability Study

[0346] Based on the experimental conditions proposed above, take the same test solution and measure it at 0h, 2h, 4h, 8h, 12h, and 24h. See Tables 21-1 and 21-2.

[0347] Table 21-1 Stability Study ~ Retention Time

[0348]

[0349] Table 21-2 Stability Study - Peak Area

[0350]

[0351]

[0352] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.06% and 0.48%, and the sample solution was relatively stable within 24 hours.

[0353] 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 11 characteristic peaks were included in the subsequent investigation.

[0354] Example 13 Determination of characteristic peaks and establishment of reference spectrum

[0355] 13.1 Batch Verification Results of Guanzhong Formula Granules

[0356] The proposed method was used to determine the characteristic spectra of three batches of samples of this product, and the relative retention time and relative peak area were calculated. Figure 36 , Tables 18-19. Figure 36Verification diagram of characteristic spectra of 3 batches of Guanzhong formula granules.

[0357] Table 22-1 Three batches of Guanzhong formula granules - relative retention time

[0358]

[0359] Table 22-2 Three batches of Guanzhong formula granules - relative peak areas

[0360]

[0361] Based on the principles of stable relative retention times, detectable peaks across all batches of samples, and relatively high peaks, 11 peaks with good reproducibility were selected as characteristic peaks. The results showed that when Peak 1 was used as the S peak, the relative retention time RSDs of the characteristic peaks for the three batches of Guanzhong Formula Granules ranged from 0.00% to 0.28%, and the relative retention time RSDs of the 11 characteristic peaks for the three batches of Guanzhong Formula Granules were all less than 5%.

[0362] Final regulations: The test sample chromatogram should show 11 characteristic peaks, and the retention times should correspond to 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: 2.10 (peak 2), 4.41 (peak 3), 4.51 (peak 4), 5.70 (peak 5), 5.86 (peak 6), 6.01 (peak 7), 6.36 (peak 8), 8.39 (peak 9), 8.68 (peak 10), and 8.81 (peak 11).

[0363] Three batches of Guanzhong Formula Granules were synthesized using the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition), and a comparison of the characteristic spectra of Guanzhong Formula Granules was established. Figure 37 . Figure 37 Reference characteristic spectrum of Guanzhong Formula Granules; Peak 1 (S): protocatechuic acid.

[0364] Comparative Example 1 Different mobile phase gradients:

[0365] Gradient 1:

[0366]

[0367] Gradient 2:

[0368]

[0369] Gradient 3:

[0370]

[0371] Gradient 4: (Last selected gradient)

[0372]

[0373] The above results are as follows Figure 38 As described, it can be seen from 38 that: under the gradient 1 condition, the baseline is uneven and the number of peaks is small; under the gradient 2 condition, peak 1 is missing; under the gradient 3 condition, peaks 9, 10, and 11 are missing; under the gradient 4 condition, the number of peaks is large, the separation is good, and the peaks are evenly distributed.

[0374] 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 UPLC characteristic profiles of Guanzhong medicinal materials, decoction pieces, standard decoctions, and formula granules, comprising: A) Extracting the Rhizoma Cyperi with a solvent to obtain a test solution; the solvent is 70% methanol; The extraction method is ultrasonic extraction or heating reflux extraction; the raw material of the cyperus rotundus is from Cibotium barometz; B) measuring the test solution by high performance liquid chromatography to obtain a UPLC characteristic spectrum of the Rhizoma Cyrthospermi raw material; The HPLC conditions are as follows: a C18 column; mobile phase A is acetonitrile; mobile phase B is 0.1% phosphoric acid; gradient elution; detection wavelength is 250 nm or 260 nm; the gradient elution is specifically as follows: 0-5 min, phase A: 6%, phase B: 94%; 5-10 min, phase A: 6%-15%, phase B: 94%-85%; 10-25 min, phase A: 15%-25%, phase B: 85%-75%; 25-40min, phase A: 25%-26%, phase B: 75%-74%.

2. The method according to claim 1, characterized in that The method also includes preparing a reference solution: taking protocatechuic acid and dissolving it in 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 Guanzhong medicinal materials, decoction pieces, standard decoctions, and formula granules are qualitatively measured based on the chromatogram of the reference.

3. The method according to claim 1, characterized in that The chromatographic column is a C18 column with a specification of 1.6 μm and 2.1×150 mm; the column temperature is 35° C.

4. The method according to claim 3, characterized in that The flow rate of the mobile phase was 0.3 mL / min; the injection volume was 1 μL.

5. The method according to claim 1, wherein The similarity of the UPLC characteristic spectra of Guanzhong medicinal materials, decoction pieces, standard decoctions, and formula granules was evaluated using the traditional Chinese medicine chromatographic fingerprint similarity evaluation system. The UPLC standard characteristic spectra of Guanzhong medicinal materials, decoction pieces, standard decoctions, and formula granules were obtained, which consisted of 11 characteristic peaks, among which peak 1 was protocatechuic acid.

6. The method according to claim 5, characterized in that In the characteristic spectrum of the Guanzhong standard decoction, 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: 2.09 (peak 2), 4.40 (peak 3), 4.50 (peak 4), 5.68 (peak 5), 5.84 (peak 6), 5.99 (peak 7), 6.34 (peak 8), 8.36 (peak 9), 8.66 (peak 10), and 8.79 (peak 11); In the characteristic spectra of the Guanzhong medicinal material and the decoction pieces, the relative retention time of each characteristic peak and the S peak is calculated with protocatechuic acid as the reference peak S, and the relative retention time is within ±10% of the specified value, and the specified values ​​are: 2.09 (peak 2), 4.39 (peak 3), 4.49 (peak 4), 5.67 (peak 5), 5.82 (peak 6), 5.97 (peak 7), 6.32 (peak 8), 8.33 (peak 9), 8.63 (peak 10), and 8.76 (peak 11); In the characteristic spectrum of the Guanzhong formula granules, the relative retention time of each characteristic peak and the S peak is calculated with protocatechuic acid as the reference peak S peak. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 2.10 (peak 2), 4.41 (peak 3), 4.51 (peak 4), 5.70 (peak 5), 5.86 (peak 6), 6.01 (peak 7), 6.36 (peak 8), 8.39 (peak 9), 8.68 (peak 10), 8.81 (peak 11).

7. The method according to claim 1, characterized in that Step A) The ultrasonic power is 600W and the frequency is 40kHz; the extraction time is 15-45 minutes.

8. The method according to claim 1, characterized in that In step A), the ratio of the mass g of the cyperus rotundus raw material to the volume mL of the solvent is (0.1-0.5): (50-100).

9. The method according to claim 1, characterized in that Step A): the Rhizoma Cyperi raw material is Rhizoma Cyperi medicinal material, Rhizoma Cyperi decoction pieces, formula granules or decoction.

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