A method for quality control of a sample of muira puama

Abstract

The application discloses a quality control method for a sample of Radix Tylophori, and comprises the following steps: (1) preparing reference solution and test solution; taking caffeic acid, caffeoyl malic acid, ferulic acid and rutin as reference substances, reference substance solutions are prepared respectively; (2) respectively taking the reference solution and the test solution to perform ultra-high performance liquid chromatography analysis, and constructing a characteristic spectrum of the sample to be measured, wherein four characteristic peaks in the characteristic spectrum correspond to the reference substance peaks of caffeic acid, caffeoyl malic acid, ferulic acid and rutin respectively; (3) performing qualitative analysis and quantitative analysis. The method is simple in operation, and is verified by a method, is good in reproducibility and stability, is reliable in recovery rate, is low in detection cost, is high in detection efficiency, and can realize qualitative and quantitative analysis of Radix Tylophori simultaneously by using one set of method.

Description

Technical Field

[0001] The present invention relates to a drug quality control method, in particular to a comprehensive quality control method for tianxianteng samples. Background Art

[0002] Tianxianteng is the dried aerial part of Aristolochia debilis Sieb.et Zucc. or Aristolochia contorta Bge., a plant of the Aristolochiaceae family (National Pharmacopoeia Committee, Chinese Pharmacopoeia, China Traditional Chinese Medicine Science and Technology Press, 2015: 55). Tianxianteng contains flavonoids, alkaloids, volatile oils, phenolic acids, and other ingredients (Xu Yuqiong, Shang Mingying, Ge Yuewei, et al. Study on the Chemical Constituents of Aristolochia [J]. Chinese Journal of Traditional Chinese Medicine, 2010, 35(21): 2862-2865). It has a bitter taste and warm nature; it enters the liver, spleen, and kidney meridians. It has anti-tumor, anti-inflammatory, diuretic, analgesic, antitussive, and antibacterial pharmacological activities; it can be used to treat rheumatic pain, abdominal pain, etc. (National Pharmacopoeia Committee, Chinese Pharmacopoeia, China Traditional Chinese Medicine Science and Technology Press, 2015: 55).

[0003] Modern pharmacological and clinical application studies have shown that Caulis Sinomenii has good effects in anti-tumor (Li C, Wang MH. Aristolochia debilis Sieb. et Zucc. induces apoptosis and reactive oxygen species in the HT-29 human colon cancer cell line [J]. Cancer Biother Radiopharm, 2013, 28(10): 717-724), anti-inflammation (SUN D, HAN YQ, WANG WY, et al. Screening and identification of Caulis Sinomenii bioactive ingredients with dual-target NF-κB inhibition and β2-AR agonizing activities [J]. Biomed Chromatogr, 2016, 30: 1843-1853), diuresis (Zhang L, Li J, Jiang Z, et al. Mechanisms of aristolochic acid I and aristolochic lactam I on renal tubular injury and their effects on the expression of aquaporin 1 in rats [J]. Chinese Journal of New Drugs and Clinical Remedies, 2011, 22(4): 359-364), analgesia (Sui Da yuan, Zhang Da xu. Study on analgesic effect of Caulis Aristolochiae Miltschukianae [J]. Journal of Baishouen Medical University, 1995, 21(5): 500-501), and relieving cough (Wu Zefang, Xiong Zhaomin. Comparative study on relieving cough, asthma and anti-inflammatory effects of Caulis Aristolochiae Miltschukianae and Lycium Fruit [J]. Chinese Journal of Pharmacology and Clinical Remedies, 1989(4): 34-36).

[0004] Traditional Chinese medicine formula granules are a new form of reform of single herb pieces, which are prepared by water extraction, concentration, drying and granulation. Compared with traditional decoction, formula granules are convenient to carry and easy to use, which can meet the requirements of modern fast-paced work and life. In recent years, traditional Chinese medicine formula granules have developed rapidly, and the national standards of formula granules of multiple traditional Chinese medicine varieties have been introduced one after another, which has made great contributions to standardizing the production of traditional Chinese medicine formula granules and promoting the modernization of traditional Chinese medicine. Caulis Sinomenii formula granules are prepared by modern technology extraction, concentration, drying and granulation of Caulis Sinomenii, which retains the clinical effect of Caulis Sinomenii and takes into account the portability of use.

[0005] There are some reports on the quality control methods of Aristolochiae Fructus. In the 2015 edition of Chinese Pharmacopoeia, the quality of Aristolochiae Fructus is controlled by appearance and thin layer chromatography identification (State Pharmacopoeia Commission, Chinese Pharmacopoeia, China Medical Science Press, 2015: 55). Xu YQ et al. established a high performance liquid chromatography method for the determination of aristolochic acid and aristolochic lactone components in Aristolochia fruit (Xu YQ, Li XW, Liu GX, et al. Comparative study of the contents of analogues of aristolochic acid in two kinds of Aristolochiae Fructus by high-performance liquid chromatography [J]. J Nat Med, 2013, 67(1): 113-122). Liu Jing et al. established a method for the determination of five aristolochic acid components in Aristolochia by UHPLC-MS / MS (Liu Jing, Wu Yingxue, Dai Zhong, et al. UHPLC-MS / MS determination of five aristolochic acid components in Aristolochia [J]. Chinese Journal of Pharmacy, 2022, 57(19): 1679-1684). Aristolochiae Fructus is not included in the 2020 edition of Chinese Pharmacopoeia. Although there are some studies on the quality control of Aristolochiae Fructus, there is no comprehensive, effective and systematic quality evaluation method and standard. For example, there is no study on the characteristic chromatogram of Aristolochiae Fructus, and the content determination is focused on the toxic components aristolochic acid. There is no quantitative analysis of other water-soluble active ingredients in Aristolochiae Fructus. SUMMARY

[0006] The purpose of the present application is to provide a quality control method for Aristolochiae Fructus samples with good reproducibility, stability, reliable recovery rate, low detection cost and high detection efficiency.

[0007] Technical scheme: The quality control method for Aristolochiae Fructus samples provided by the present application comprises the following steps:

[0008] (1) Select Aristolochiae Fructus samples and prepare test sample solutions; prepare reference sample solutions of Aristolochiae Fructus as control materials; prepare reference sample solutions of caffeic acid, caffeic acid, ferulic acid and rutin as control samples;

[0009] (2) Take the reference sample solution and the test sample solution for ultra-high performance liquid chromatography analysis respectively, and construct the characteristic chromatogram of the sample to be tested. The characteristic peaks include four characteristic peaks corresponding to the caffeic acid control sample peak, the caffeic acid control sample peak, the ferulic acid control sample peak and the rutin control sample peak.

[0010] (3) through the characteristic map obtained in step (2), qualitative analysis of the sample to be tested; with rutin as the content determination index, quantitative analysis of the sample to be tested.

[0011] Further, in step (1), the extraction solvent for preparing the reference solution or the test solution is water, methanol or a methanol aqueous solution with a volume concentration of 30% to 70%, and the corresponding extraction agent is used in an amount of 30 ml to 100 ml per gram of Stephania tetrandra, and the extraction time is 15 to 60 minutes.

[0012] Further, the Stephania tetrandra formula granules are finely ground, water, methanol or a methanol aqueous solution with a volume concentration of 30% to 70% is added for extraction, and the filtrate is taken to obtain the test solution.

[0013] Further, the Stephania tetrandra control medicinal materials are added with water, methanol or a methanol aqueous solution with a volume concentration of 30% to 70% for extraction, and the filtrate is taken as the reference solution of the control medicinal materials; and the coffee acid, caffeoyl malic acid, ferulic acid, and rutin control samples are added with methanol for dissolution as the reference solution of the control samples.

[0014] Further, in step (1), the concentration of the coffee acid control sample reference solution is 5 to 20 μg / ml; the concentration of the caffeoyl malic acid control sample reference solution is 5 to 20 μg / ml; the concentration of the ferulic acid control sample reference solution is 5 to 20 μg / ml; and the concentration of the rutin control sample reference solution is 20 to 450 μg / ml.

[0015] Further, 1 μl to 4 μl of the reference solution and the test solution are respectively taken for determination.

[0016] Further, the conditions for the ultra-high performance liquid chromatography analysis are as follows: the chromatographic column is filled with octadecylsilane-bonded silica gel; acetonitrile is used as the mobile phase A, and a phosphoric acid solution with a volume fraction of 0.02% to 0.1% is used as the mobile phase B, and gradient elution is adopted: 0 to 2 min, 8→12% A, 2 to 12 min, 12→15% A, 12 to 20 min, 15→18% A, 20 to 23 min, 18→43% A, 23 to 29 min, 43→60% A.

[0017] Further, the detection wavelength is 210 to 320 nm; the flow rate is 0.25 ml to 0.35 ml per minute; the column temperature is 15 to 25°C; and an ultraviolet detector is used.

[0018] Further, the ultra-high performance liquid chromatography characteristic map of the sample of Radix Sinomenii includes 8 characteristic peaks; and corresponds to the retention time of 8 characteristic peaks in the reference chromatogram of the control medicinal material; the peak corresponding to the rutin reference is S1 peak, the relative retention time of peak 1, peak 3 and S1 peak is calculated; the peak corresponding to the rutin reference is S2 peak, the relative retention time of peak 6, peak 8 and S2 peak is calculated; the relative retention time should be within ±10% of the specified value, and the specified value is: 0.77 (peak 1), 1.36 (peak 3), 0.93 (peak 6), 1.08 (peak 8).

[0019] Further, the sample of Radix Sinomenii includes Radix Sinomenii medicinal material, Radix Sinomenii decoction pieces, Radix Sinomenii preparation intermediates or Radix Sinomenii preparation finished products.

[0020] Further, the step (3) quantitative analysis meets: the rutin content of Radix Sinomenii formula granules is above 1.1 mg / g, and the rutin content of Radix Sinomenii medicinal material is above 0.07%, so as to ensure the effectiveness of Radix Sinomenii formula granules and medicinal material.

[0021] Beneficial effects: compared with the prior art, the present application has the following obvious advantages:

[0022] (1) The characteristic map meeting the overall characteristics of Radix Sinomenii is established for the first time. The characteristic map containing 8 characteristic peaks is established, 4 peaks are identified, and the consistency of the formula granules and the medicinal material is better embodied by using the control medicinal material as the reference;

[0023] (2) Rutin is used as the content index of Radix Sinomenii for the first time, and the flavonoid effective component in Radix Sinomenii is analyzed;

[0024] (3) The selection of the effectiveness index is more targeted. Rutin is used as the content index component, which has the effects of anti-inflammatory and antibacterial, and can fully represent the anti-inflammatory and antibacterial effects of Radix Sinomenii. The content of rutin is high, the component is stable, and it has good guiding significance for process control and product quality stability of Radix Sinomenii formula granules. The quality of Radix Sinomenii raw materials, intermediates and formula granules is provided with technical control means, which provides strong guarantee for industrialization and quality control;

[0025] (4) The ultra-high performance liquid chromatography method is adopted, which is more efficient and effective than the content control of high performance liquid chromatography (HPLC), and is more conducive to the industrialization application of Radix Sinomenii formula granules;

[0026] (5) A set of chromatographic methods is adopted to realize the qualitative and quantitative control of Radix Sinomenii. That is, the characteristic map of Radix Sinomenii and the content determination method of the effective component are established at the same time, which is more conducive to the overall quality control of Radix Sinomenii formula granules, and takes into account the effectiveness and overall quality evaluation of traditional Chinese medicine, and has high detection efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Characteristic spectrum of the recipe granules of Radix Sinomenii, peak 2 (S1): caffeic acid; peak 4: caffeoyl malic acid; peak 5: ferulic acid; peak 7 (S2): rutin;

[0028] Figure 2 Characteristic spectrum of the intermediate of Radix Sinomenii, peak 2 (S1): caffeic acid; peak 4: caffeoyl malic acid; peak 5: ferulic acid; peak 7 (S2): rutin;

[0029] Figure 3 Characteristic spectrum of the medicinal material / slice of Radix Sinomenii, peak 2 (S1): caffeic acid; peak 4: caffeoyl malic acid; peak 5: ferulic acid; peak 7 (S2): rutin;

[0030] Figure 4 Chromatogram of the reference substance, reference medicinal material, recipe granules of Radix Sinomenii;

[0031] Figure 5 UPLC chromatogram of the recipe granules of Radix Sinomenii under different detection wavelengths;

[0032] Figure 6 Full wavelength scanning diagram of rutin;

[0033] Figure 7 UPLC diagram of the investigation of different extraction solvents;

[0034] Figure 8 UPLC diagram of the investigation of different extraction methods;

[0035] Figure 9 UPLC diagram of the investigation of different extraction volumes;

[0036] Figure 10 UPLC diagram of the investigation of different extraction times;

[0037] Figure 11 Investigation diagram of the chromatographic column;

[0038] Figure 12 Investigation diagram of the column temperature;

[0039] Figure 13 Investigation diagram of the flow rate;

[0040] Figure 14 Linear relationship diagram of the rutin reference substance;

[0041] Figure 15 Specificity test;

[0042] Figure 16 Integrity test. DETAILED DESCRIPTION

[0043] The technical solutions of the present application will be further described below in combination with the drawings.

[0044] Example 1

[0045] Take 3 batches of Radix Aconiti Kusnezoffii Dispensing Granules and 10 batches of Radix Aconiti Kusnezoffii as Radix Aconiti Kusnezoffii dispensing samples, and respectively perform characteristic map and content determination according to the following steps:

[0046] (1) Select Radix Aconiti Kusnezoffii samples and prepare test sample solutions; take Radix Aconiti Kusnezoffii as a control drug, and prepare a reference solution of the control drug; take caffeic acid, caffeic acid malic acid, ferulic acid and rutin as reference substances, and respectively prepare reference substance solutions;

[0047] Preparation of the reference solution of the control drug: take 1 g of Radix Aconiti Kusnezoffii control drug, place it in a conical flask with a plug, add 25 ml of 70% methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, take the filtrate, and use it as the reference solution of the control drug.

[0048] Preparation of the reference substance solution: take appropriate amounts of caffeic acid reference substance, caffeic acid malic acid reference substance, ferulic acid reference substance and rutin reference substance, accurately weigh, add methanol to prepare a solution containing 5-20 μg of caffeic acid, 5-20 μg of caffeic acid malic acid, 5-20 μg of ferulic acid and 20-450 μg of rutin per 1 ml, and use it as the reference substance solution.

[0049] Preparation of the test sample solution of Radix Aconiti Kusnezoffii dispensing granules: take about 0.3 g of Radix Aconiti Kusnezoffii dispensing granules, accurately weigh, place it in a conical flask with a plug, accurately add 25 ml of 70% methanol, tightly plug, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the weight loss with 70% methanol, shake well, filter, take the filtrate, and obtain it.

[0050] Preparation of the test sample solution of Radix Aconiti Kusnezoffii: take about 1 g of Radix Aconiti Kusnezoffii powder, accurately weigh, place it in a conical flask with a plug, accurately add 25 ml of 70% methanol, tightly plug, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the weight loss with 70% methanol, shake well, filter, take the filtrate, and obtain it.

[0051] (2) Ultrasonically treat the test sample solution and the reference solution prepared in step (1) respectively, and obtain the corresponding ultrahigh performance liquid chromatogram; accurately take 2 μl of the reference solution and the test sample solution respectively, inject them into the liquid chromatograph, and determine, and obtain it.

[0052] Among them, the chromatographic condition parameters are as follows:

[0053] The octadecylsilane-bonded silica gel was used as the filler (the column length was 100 mm, the inner diameter was 2.1 mm, and the particle size was 2.2 μm); the acetonitrile was used as the mobile phase A, and the 0.05% phosphoric acid solution was used as the mobile phase B, which was gradient eluted according to the following table; the flow rate was 0.3 ml per minute; the column temperature was 20 °C; and the detection wavelength was 320 nm.

[0054]

[0055] As shown in Figure 4 , the characteristic chromatogram of the Formulation Granules of Tetrastigma hemsleyanum is shown in the following figure, and the test sample solution chromatogram presents 8 characteristic peaks, which correspond to the retention time of the 8 characteristic peaks in the reference chromatogram of the control medicinal material; among them, the peak 2, the peak 4, the peak 5, and the peak 7 should be consistent with the retention time of the corresponding control reference peaks. The peak corresponding to the caffeic acid reference is the S1 peak, and the relative retention time of the peak 1 and the peak 3 to the S1 peak is calculated; the peak corresponding to the rutin reference is the S2 peak, and the relative retention time of the peak 6 and the peak 8 to the S2 peak is calculated; the relative retention time should be within ±10% of the specified value, and the specified value is: 0.77 (peak 1), 1.36 (peak 3), 0.93 (peak 6), and 1.08 (peak 8).

[0056] The determination results of the test sample solution are shown in the following table.

[0057] Table 1 Determination results of the Formulation Granules of Tetrastigma hemsleyanum (relative retention time)

[0058]

[0059] Table 2 Determination results of 10 batches of Tetrastigma hemsleyanum medicinal material samples (relative retention time)

[0060]

[0061] (3) The ultra-high performance liquid chromatogram obtained in step (2) is compared with the characteristic chromatogram of the Formulation Granules (the characteristic chromatogram of the Formulation Granules of Tetrastigma hemsleyanum is shown in the following figure Figure 1 ), the intermediate (the characteristic chromatogram of the intermediate of Tetrastigma hemsleyanum is shown in the following figure Figure 2 ), or the medicinal material and decoction piece (the characteristic chromatogram of the Tetrastigma hemsleyanum medicinal material / decoction piece is shown in the following figure Figure 3 ), to perform qualitative analysis of the sample;

[0062] The rutin is used as the content determination index to perform quantitative analysis of the sample. The full wavelength scan chromatogram of the rutin is shown in the following figure. Figure 6

[0063] The content of the rutin in the sample is determined, and the experimental results are shown in Tables 3 and 4.

[0064] Table 3 Determination of the content of rutin in the Formulation Granules of Tetrastigma hemsleyanum ​

[0065]

[0066] Table 4 Determination of the content of rutin in the Radix Sinomenii

[0067]

[0068] From the above test results, from the qualitative analysis, the characteristic chromatograms of 3 batches of Sinomenium Formula Granules and 10 batches of Radix Sinomenii meet the requirements of the characteristic chromatogram in step (2). From the quantitative analysis, the content of rutin in 3 batches of Sinomenium Formula Granules and 10 batches of Radix Sinomenii is stable, which ensures the effectiveness of Sinomenium Formula Granules and Radix Sinomenii.

[0069] Example 2 Establishment of the characteristic chromatogram method of Sinomenium Formula Granules and its methodological study

[0070] This example is used to confirm the optimal scheme of the establishment of the characteristic chromatogram in the quality control method of the Radix Sinomenii sample, which specifically includes the comparison of the schemes of the chromatographic conditions, wavelength selection, and preparation of the test solution, and selects the optimal chromatographic conditions and test solution preparation scheme; and the optimal scheme is respectively investigated for specificity, integrity, precision, intermediate precision, stability, repeatability, and durability.

[0071] 1 Instruments and reagents

[0072] Instruments: Waters ultra-high performance liquid chromatograph (H-Class UPLC, Waters Corporation), Agilent ultra-high performance liquid chromatograph (1290 II, Agilent Corporation), one-hundredth balance (AP224W, Mettler-Toledo Corporation), one-millionth balance (XBR36, Mettler-Toledo Corporation), digital ultrasonic cleaner (2700TH, Shanghai Anpu Experimental Technology Co., Ltd.), electric heating constant temperature water bath (J-HH-6A, Shanghai Shengwei Electronic Technology Co., Ltd.), shaker (SHIPING SPH-310, Shanghai Shiping Experimental Equipment Co., Ltd.), ultrapure water system (Comfort 1, Germany Sartorius Corporation).

[0073] Reagents: methanol (AR, Tianjin Damao Chemical Reagent Co., Ltd.), phosphoric acid (HPLC, Tianjin Kemioke Science and Technology Co., Ltd.), acetonitrile (HPLC, CNW Corporation), water is ultrapure water (laboratory self-made).

[0074] Reagents: caffeic acid (batch number: 110885-201703, purity: 99.7%, China Institute for Drug Control); caffeic acid malate (batch number: PS230714-05, purity: 98%, Chengdu Pusai Biological Technology Co., Ltd.); ferulic acid (batch number: 110773-202316, purity: 99.3%, China Institute for Drug Control); rutin (batch number: 100080-202012, purity: 91.6%, China Institute for Drug Control).

[0075] Reference material of Sargentodoxa

[0076] Sargentodoxa capillata formula granules were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0077] 2Determination of detection wavelength

[0078] Take an appropriate amount of the product, grind it finely, take about 0.3g, put it in a conical flask with a plug, precisely add 25ml of 70% methanol, tightly plug, weigh, ultrasonic treatment (power 250W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the weight loss with 70% methanol, shake well, filter, take the filtrate, and get it. The chromatogram of the sample collected at 210nm, 254nm and 320nm is shown in Figure 5 .

[0079] The results show that the chromatogram detected at 320nm has good peak shape and stable baseline, and 320nm is finally selected as the detection wavelength.

[0080] 3Determination of chromatographic conditions

[0081] Octadecylsilane-bonded silica gel is used as the filler (column length 100mm, inner diameter 2.1mm, particle size 2.2μm); acetonitrile is used as mobile phase A, and 0.05% phosphoric acid solution is used as mobile phase B, gradient elution is carried out according to the following table; the flow rate is 0.30ml per minute; the column temperature is 20℃; the detection wavelength is 320nm. The theoretical plate number should not be less than 5000 calculated by the rutin peak.

[0082]

[0083] 4Preparation of reference solution

[0084] Take 1 g of Radix Sinomenii control material, place it in a conical flask with a stopper, add 25 ml of 70% methanol, tightly stopper, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, take the subsequent filtrate as the control material reference solution. Take the control solution under the item of "content determination" as the control reference solution. Take the appropriate amount of caffeic acid control, caffeic acid malic acid control, and ferulic acid control, precisely weigh, add methanol to make a mixed solution containing 10 μg per 1 ml, as the control reference solution.

[0085] 5. Preparation of test solution

[0086] 5.1 Investigation of different extraction solvents

[0087] This experiment investigates the influence of different extraction solvents on the characteristic chromatogram of Radix Sinomenii formula granules. Water, 30% methanol, 50% methanol, 70% methanol, and methanol are selected as extraction solvents. The peak area / sample weight of 8 chromatographic peaks and the chromatogram are used to compare the influence of different extraction solvents on the characteristic chromatogram of Radix Sinomenii formula granules.

[0088] Take an appropriate amount of the product, finely grind, take about 0.3 g, divide into 5 parts, precisely weigh, place in a conical flask with a stopper, precisely add 25 ml of water, 30% methanol, 50% methanol, 70% methanol, and methanol respectively, tightly stopper, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the weight loss with the corresponding solvent, shake well, filter, take the subsequent filtrate. Inject the sample according to the chromatographic conditions in the text, the results are shown in Table 5, Figure 7 .

[0089] Table 5 Comparison of extraction efficiency of different extraction solvents (peak area / sample weight)

[0090]

[0091] From the above table, it can be seen that when the extraction solvent is 70% methanol, the extraction efficiency is higher, therefore, 70% methanol is selected as the extraction solvent.

[0092] 5.2 Investigation of different extraction methods

[0093] This experiment investigates the influence of different extraction methods on the characteristic chromatogram of Radix Sinomenii formula granules. Ultrasonic treatment, heating reflux, and shaking extraction are selected as extraction methods. The peak area / sample weight of 8 chromatographic peaks and the chromatogram are used to compare the influence of different extraction methods on the characteristic chromatogram of Radix Sinomenii formula granules.

[0094] Take the product, grind, take about 0.3g, parallel 3, accurately weighed, placed in a conical flask with a plug, accurately add 70% methanol 25ml, tightly sealed, weighed, ultrasonic treatment (power 250W, frequency 40kHz), heating reflux, shaking extraction 30 minutes, cool, reweigh, make up the weight loss with 70% methanol, shake, filter, take the filtrate, sample by the chromatographic conditions in the text, the results are shown in Table 6, Figure 8 .

[0095] Table 6 Comparison of extraction efficiency of different extraction methods (peak area / weight)

[0096]

[0097] From the above table, it can be seen that the number of chromatographic peaks obtained by using three kinds of treatment methods is consistent, and the extraction efficiency of chromatographic peaks is similar. Therefore, the extraction method is selected as ultrasonic treatment.

[0098] 5.3 Investigation of different extraction volumes

[0099] This experiment investigated the influence of three different extraction solvent volumes, 15ml, 25ml and 50ml, on the characteristic chromatogram of the Stephania tetrandra formula granules. The influence of different extraction volumes on the characteristic chromatogram of the Stephania tetrandra formula granules was compared by the peak area / weight*extraction volume / 25 of 8 chromatographic peaks and the chromatogram.

[0100] Take the product, grind, take about 0.3g, parallel 3, accurately weighed, placed in a conical flask with a plug, respectively accurately add 70% methanol 15ml, 25ml and 50ml, tightly sealed, weighed, ultrasonic treatment (power 250W, frequency 40kHz) 30 minutes, cool, reweigh, make up the weight loss with 70% methanol, shake, filter, take the filtrate, sample by the chromatographic conditions in the text, the results are shown in Table 7, Figure 9 .

[0101] Table 7 Comparison of extraction efficiency of different extraction volumes (peak area / weight*extraction volume / 25)

[0102]

[0103] From the above table, it can be seen that the number of chromatographic peaks obtained by using three kinds of extraction solvent volumes is consistent, and there is no obvious difference in extraction efficiency. In order to ensure sufficient extraction and save solvent, the extraction volume is determined as 25ml.

[0104] 5.4 Investigation of different extraction times

[0105] The experiment investigated the effects of four different extraction times, 15 minutes, 30 minutes, 45 minutes and 60 minutes, on the characteristic chromatogram of Stephania tetrandra formula granules. The effects of different extraction times on the characteristic chromatogram of Stephania tetrandra formula granules were compared by the peak area / sample weight of 8 chromatographic peaks.

[0106] Take the product, grind finely, take about 0.3g, accurately weigh, place in a conical flask with a stopper, accurately add 70% methanol 25ml, tightly seal, weigh, ultrasonic treatment (power 250W, frequency 40kHz) for 15 minutes, 30 minutes, 45 minutes and 60 minutes, cool, re-weigh, make up the weight loss with 70% methanol, shake well, filter, take the filtrate, and then inject the sample according to the chromatographic conditions in the text, the results are shown in Table 8, Figure 10 .

[0107] Table 8 Comparison of extraction efficiency of different extraction times (peak area / sample weight)

[0108]

[0109] From the above table, it can be seen that the number of chromatographic peaks obtained by each extraction time is consistent. When ultrasonic treatment is 15 minutes, it is basically fully extracted, in order to ensure complete extraction, therefore the extraction time is selected as 30 minutes.

[0110] 5.5 Determination of test solution preparation method

[0111] According to the above research results, the test solution preparation method for the characteristic chromatogram of Stephania tetrandra formula granules is determined as follows:

[0112] Take the product, grind finely, take about 0.3g, accurately weigh, place in a conical flask with a stopper, accurately add 70% methanol 25ml, tightly seal, weigh, ultrasonic treatment (power 250W, frequency 40kHz) for 30 minutes, cool, re-weigh, make up the weight loss with 70% methanol, shake well, filter, take the filtrate, and then inject the sample according to the chromatographic conditions in the text, the results are shown in Table 8,

[0113] 6 Methodology study of characteristic chromatogram

[0114] 6.1 Investigation of specificity

[0115] The excipients added in Stephania tetrandra formula granules are malt dextrin, silicon dioxide and magnesium stearate. This experiment investigates the effects of negative samples without Stephania tetrandra on the detection of characteristic chromatogram.

[0116] The negative sample solution was prepared according to the test sample preparation method, and the sample was injected according to the chromatographic conditions in the text, and the results are shown below Figure 15 .

[0117] The results showed that the solvent and excipient had no interference with the determination of the characteristic chromatogram of the Formula Granules of Stephania tetrandra, and the specificity of the analysis method was good.

[0118] 6.2 Investigation of integrity

[0119] Under the same chromatographic conditions, the elution gradient with the highest proportion of organic phase was maintained, the elution time was extended by one time, the chromatogram was recorded, and the results are shown below Figure 16 .

[0120] From the experimental results, no obvious chromatographic peaks flowed out after the original gradient elution ended, indicating that the chromatographic conditions basically met the principle of maximum information.

[0121] 6.3 Investigation of precision

[0122] Take the Stephania tetrandra granules sample, grind it finely, take about 0.3g, prepare the test solution according to the preparation method of the test solution, continuously sample 6 times, calculate the relative retention time according to the requirements of the text, and the results are shown in Table 9.

[0123] Table 9 Precision test results (relative retention time)

[0124]

[0125] The results showed that the same sample was continuously sampled 6 times, and the relative retention time RSD value of each characteristic peak was less than 1.0%, indicating that the instrument precision of the method was good.

[0126] 6.4 Investigation of intermediate precision

[0127] Take the Stephania tetrandra granules sample, grind it finely, take about 0.3g, prepare the test solution according to the preparation method of the test solution, continuously sample 6 times, calculate the relative retention time according to the requirements of the text, and the results are shown in Table 9.

[0128] Table 10 Investigation of intermediate precision (relative retention time)

[0129]

[0130]

[0131] The results showed that the relative retention time RSD of each characteristic peak was less than 2%, and the intermediate precision test was good.

[0132] 6.5 Investigation of stability

[0133] Take the Stephania tetrandra granules sample, grind it finely, take about 0.3g, prepare the test solution according to the preparation method of the test solution, respectively at 0, 2, 4, 8, 12, 18, 24 hours time point sampling, calculate the relative retention time according to the requirements of the text, and the results are shown in Table 11.

[0134] Table 11 Stability Investigation (Relative Retention Time)

[0135]

[0136] The results show that the RSD of the relative retention time of each characteristic peak is less than 1%, and the test solution is stable within 24 hours.

[0137] 6.6 Reproducibility Investigation

[0138] Take an appropriate amount of Radix Aconiti Kusnezoffii granules, grind them finely, take about 0.3g, prepare 6 parallel samples according to the preparation method of the test solution, analyze the samples, and calculate the relative retention time according to the requirements of the text. The results are shown in Table 12.

[0139] Table 12 Reproducibility Test (Relative Retention Time)

[0140]

[0141] The results show that the RSD of the relative retention time of each characteristic peak is less than 1%, and the reproducibility test is good.

[0142] 6.7 Durability Investigation

[0143] (1) Chromatographic column investigation

[0144] Take the same batch of samples, prepare the test solution according to the preparation method of the test solution in the text, and use Acclaim RSLC 120C18 (2.1*100mm, 2.2μm); Agilent ZORBAX SB-C18 (2.1*100mm, 1.8μm); Waters CORTECS UPLC T3 (2.1*100mm, 1.6μm) three kinds of chromatographic columns to investigate their separation effect, and the results are shown in the figure.

[0145] Table 13 Chromatographic Column Investigation (Relative Retention Time)

[0146]

[0147] The results show that the Radix Aconiti Kusnezoffii sample can be well separated in the Acclaim RSLC 120C18 chromatographic column (100*2.1mm, 2.2μm), and the peak shape is good. In the remaining two chromatographic columns, the separation degree of some characteristic peaks is not good, and the relative retention time is within the specified range. The subsequent study uses the Acclaim RSLC 120C18 chromatographic column (100*2.1mm, 2.2μm).

[0148] (2) Investigation of different column temperatures

[0149] The same batch of samples was prepared according to the preparation method of the test solution in the text to investigate the separation effect of the samples at 15℃, 20℃ and 25℃, and the results are shown in Table 14. Figure 12

[0150] Table 14 Column temperature investigation (relative retention time)

[0151]

[0152] The results show that when the column temperature is 15℃-25℃, the relative retention time of each characteristic peak is within the specified range, and small changes in column temperature can meet the system durability requirements. The subsequent study uses a column temperature of 20℃.

[0153] (3) Investigation of different flow rates

[0154] The same batch of samples was prepared according to the preparation method of the test solution in the text to investigate the separation effect of the samples at 0.25ml / min, 0.30ml / min and 0.35ml / min, and the results are shown in Table 15. Figure 13

[0155] Table 15 Flow rate investigation (relative retention time)

[0156]

[0157] The results show that when the flow rate is 0.25ml / min-0.35ml / min, small changes in flow rate can meet the system applicability requirements. The subsequent study uses a flow rate of 0.30ml / min.

[0158] Establishment of content determination method of Radix Stephaniae Tetrandrae Dispensing Granules and its methodological study

[0159] 1 Instruments and reagents

[0160] Instruments: Waters ultra-high performance liquid chromatograph (H-Class UPLC, Waters Corporation), Agilent ultra-high performance liquid chromatograph (1290 II, Agilent Corporation), one-hundredth scale (AP224W, Mettler-Toledo Corporation), one-millionth scale (XBR36, Mettler-Toledo Corporation), digital ultrasonic cleaner (2700TH, Shanghai Anpu Experimental Technology Co., Ltd.), electric heating constant temperature water bath (J-HH-6A, Shanghai Shengwei Electronics Technology Co., Ltd.), shaker (SHIPING SPH-310, Shanghai Shiping Experimental Equipment Co., Ltd.), ultrapure water system (Comfort 1, Germany Sartorius Corporation).

[0161] ​​Reagents: methanol (AR, Tianjin Damao Chemical Reagent Co., Ltd.), phosphoric acid (HPLC, Tianjin Kemioke Technology Co., Ltd.), acetonitrile (HPLC, CNW company), water is ultrapure water (laboratory self-made).

[0162] Reagents: Methanol (AR, Tianjin Damao Chemical Reagent Co., Ltd.), phosphoric acid (HPLC, Tianjin Kemioke Technology Co., Ltd.), acetonitrile (HPLC, CNW company), water is ultrapure water (laboratory self-made).

[0163] 2. Control product source and purity inspection

[0164] 2. Control product source and purity inspection

[0165] The control product solution rutin control product solution was scanned at full wavelength, and its ultraviolet absorption graph was recorded, see Figure 6 .

[0166] The results showed that rutin had strong absorption at 254 nm and 352 nm. Combined with the characteristic chromatographic conditions, 320 nm was finally selected as the detection wavelength.

[0167] 4. Determination of chromatographic conditions

[0168] Referring to the determination method of the characteristic spectrum of sinomenine, the content determination method of rutin in sinomenine formula granules was established, and the chromatographic conditions were as follows:

[0169] Octadecylsilane bonded silica gel was used as the filler (column length was 100 mm, inner diameter was 2.1 mm, particle size was 2.2 μm); acetonitrile was used as mobile phase A, and 0.05% phosphoric acid solution was used as mobile phase B, which was gradient eluted according to the following table; the flow rate was 0.30 ml per minute; the column temperature was 20℃; the detection wavelength was 320 nm. The theoretical plate number should not be less than 5000 calculated by rutin peak.

[0170]

[0171] 5. Preparation of control solution

[0172] Take rutin control product, accurately weigh, add methanol to make 35 μg per 1 ml solution, shake well, and get it.

[0173] 6. Preparation of test solution

[0174] 6.1 Investigation of different extraction solvents

[0175] Take the product, grind, take about 0.3g, parallel 5 groups, each group in parallel 2 copies, precision weighing, placed in a conical flask with a plug, respectively, precision add water, 30% methanol, 50% methanol, 70% methanol and methanol, methanol 25ml, sealed, weighed, ultrasonic treatment (power 250W, frequency 40kHz) 30 minutes, cool, reweigh, with the corresponding solvent to make up for the weight loss, shake, filter, take the filter, get. Respectively, precision suction each test solution 2μl, injected into liquid chromatograph, according to the above chromatographic conditions, the content of rutin was calculated, the results were shown in table 16. Figure 7 , table 16.

[0176] Table 16 comparison of different extraction solvents

[0177]

[0178]

[0179] The results show that: when the extraction solvent is 70% methanol, the content of rutin is higher, and the final extraction solvent is 70% methanol.

[0180] 6.2 investigation of different extraction methods

[0181] Take the product, grind, take about 0.3g, parallel 3 groups, each group in parallel 2 copies, precision weighing, placed in a conical flask with a plug, respectively, precision add 70% methanol 25ml, sealed, weighed, ultrasonic treatment (power 250W, frequency 40kHz), shaking extraction, ultrasonic treatment (power 250W, frequency 40kHz) 30 minutes, cool, reweigh, with 70% methanol to make up for the weight loss, shake, filter, take the filter, get. Respectively, precision suction each test solution 2μl, injected into liquid chromatograph, according to the above chromatographic conditions, the content of rutin was calculated, the results were shown in table 17. Figure 8 , table 17.

[0182] Table 17 comparison of different extraction methods

[0183]

[0184] The results show that: when the extraction solvent is 70% methanol, the content of rutin is higher, and the final extraction solvent is 70% methanol.

[0185] 6.3 investigation of different extraction volumes

[0186] Take the product, grind, take about 0.3g, parallel 3 groups, each group in parallel 2 copies, precision weighing, placed in a conical flask with plug, respectively, precision 70% methanol 15ml, 25ml, 50ml, tight, weighing, ultrasonic treatment (power 250W, frequency 40kHz) 30 minutes, cool, reweigh, with 70% methanol to make up for the weight loss, shake, filter, take the filter, get. Precision 2μl of each test solution was injected into liquid chromatograph, and the content of rutin was calculated by the above chromatographic conditions. The results are shown in Table 18. Figure 9 , Table 18.

[0187] Table 18 Comparison of different extraction volumes

[0188]

[0189] The results show that when the extraction solvent is 25ml, the content of rutin is higher. In order to ensure sufficient extraction, the final extraction solvent is 25ml.

[0190] 6.4 Investigation of different extraction time

[0191] Take the product, grind, take about 0.3g, parallel 4 groups, each group in parallel 2 copies, precision weighing, placed in a conical flask with plug, respectively, precision 70% methanol 25ml, tight, weighing, ultrasonic treatment (power 250W, frequency 40kHz) 15 minutes, 30 minutes, 45 minutes and 60 minutes, cool, reweigh, with 70% methanol to make up for the weight loss, shake, filter, take the filter, get. Precision 2μl of each test solution was injected into liquid chromatograph, and the content of rutin was calculated by the above chromatographic conditions. The results are shown in Table 19. Figure 10 , Table 19.

[0192] Table 19 Comparison of different extraction time

[0193]

[0194]

[0195] The results show that when the extraction time is 30 minutes, the content of rutin is higher. The final extraction time is 30 minutes.

[0196] 6.5 Determination of test solution preparation method

[0197] According to the above research results, the test solution preparation method for determination of Radix Aristolochiae Granules is finally determined as follows:

[0198] Take the product, grind, take about 0.3g, accurately weighed, placed with plug conical flask, accurately add 70% methanol 25ml, tightly sealed, weighed, ultrasonic treatment (power 250W, frequency 40kHz) 30 minutes, cool, reweigh, with 70% methanol to make up for the loss of weight, shake, filter, take the filtrate, i.e.

[0199] 7Methodology validation

[0200] 7.1Linearity

[0201] Accurately weighed rutin reference substance 11.763mg, placed in 25ml volumetric flask, add methanol to the mark, i.e. the concentration of rutin reference substance stock solution is 432.4079μg / ml.

[0202] Respectively, accurately measure the above rutin reference substance stock solution 0.1ml, 0.3ml, 0.5ml, 1ml, 3ml, 5ml, respectively, placed in 10ml volumetric flask, add methanol to the mark, shake, respectively, make 4.3241μg, 12.9722μg, 21.6204μg, 43.2408μg, 129.7224μg, 216.2039μg rutin per 1ml of the control solution, respectively, accurately take the above control solution and control stock solution, in turn, 2μl, record the chromatographic peak area. With peak area as ordinate (y), control concentration as abscissa (x), draw the standard curve, get the regression equation, results Figure 14 , table 20 and 21.

[0203] Table 20 regression equation

[0204]

[0205] Table 21 control concentration and peak area

[0206]

[0207] The results show that: rutin sample concentration in the range of 4.3241~432.4079μg / ml, with peak area value showed a good linear relationship.

[0208] 7.2Precision test

[0209] 7.2.1Instrument precision test

[0210] Take the product, grind, take about 0.3g, accurately weighed, prepared into test solution according to the preparation method of test solution, injected into liquid chromatograph, according to the above chromatographic conditions, continuously sample 6 times, record the rutin peak area measurement value, calculate its RSD, the results are shown in the following table 22.

[0211] Table 22 instrument precision test

[0212]

[0213] The results show that the instrument precision test is good.

[0214] 7.2.2 repeatability test

[0215] Take the product, grind finely, take about 0.3g, accurately weigh, parallel 6 groups, prepare the test solution according to the method of test solution preparation, analyze the sample, determine the peak area value of rutin, calculate the content and RSD, the results are shown in Table 23.

[0216] Table 23 repeatability test results of samples

[0217]

[0218] The results show that the RSD of rutin content is 0.44%, and the repeatability test is good.

[0219] 7.2.3 intermediate precision test

[0220] Take the product, grind finely, take about 0.3g, accurately weigh, respectively by two experimenters A, B, respectively according to the requirements in the text, 3 test solution, respectively at different times in Agilent ultra high performance liquid chromatograph (1290 Infinity, Agilent company) and Waters ultra high performance liquid chromatograph (Waters UPLC, Waters company) respectively, determine the peak area value of rutin, calculate the content and RSD, the results are shown in Table 24.

[0221] Table 24 intermediate precision test

[0222]

[0223] The results show that the RSD of rutin content is 0.43%, and the intermediate precision is good.

[0224] 7.3 accuracy test

[0225] Take the sample with known content (rutin content: 2.72mg / g), take about 0.15g, parallel 3 groups, accurately weigh, respectively add 0.15g sample containing 50%, 100%, 150% of the control, prepare the sample recovery test solution according to the method of test solution preparation, according to the above chromatographic conditions, respectively, calculate the recovery rate and RSD by the following formula, the results are shown in Table 25.

[0226]

[0227] Table 25 accuracy results

[0228]

[0229]

[0230] The results show that the rutin recovery rate is between 95.70% and 100.70%, and the accuracy test is good.

[0231] 7.4 Specificity test

[0232] The excipients added in the Radix Mitrephorae Macrocarpae dispensing granules are malt dextrin, silicon dioxide and magnesium stearate. This experiment investigates the influence of negative samples without Radix Mitrephorae Macrocarpae on the content determination of Radix Mitrephorae Macrocarpae dispensing granules.

[0233] Take the negative sample without Radix Mitrephorae Macrocarpae, and prepare the negative sample solution according to the preparation method of the test sample.

[0234] Inject the test sample solution of Radix Mitrephorae Macrocarpae dispensing granules, the negative control solution and the rutin control solution into the liquid chromatograph.

[0235] Take 2 μl of each of the Radix Mitrephorae Macrocarpae dispensing granules, the negative control solution and the rutin control solution according to the text chromatography conditions, and the results are shown in Table 7. Figure 15

[0236] The experimental results show that the excipients and solvents have no interference with the determination of rutin content, and the method has strong specificity.

[0237] 7.5 Integrity test

[0238] Under the same chromatography conditions, maintain the elution gradient with the highest proportion of organic phase, extend the elution time by one time, record the chromatogram, and the results are shown in Table 8. Figure 16

[0239] From the experimental results, it can be seen that extending the analysis time by one time has no interference with the determination of rutin content.

[0240] 7.6 Durability test

[0241] 7.6.1 Stability test

[0242] Take an appropriate amount of the product, grind it finely, take about 0.3 g, accurately weigh it, prepare the test sample solution according to the preparation method of the test sample solution, and inject it at 0, 2, 4, 8, 12, 18 and 24 hours, respectively, measure the peak area value, calculate the RSD, and the results are shown in Table 26.

[0243] Table 26 Determination results of stability test

[0244]

[0245] The results show that the test sample solution is stable within 24 hours.

[0246] 7.6.2 Investigation of different flow rates​​

[0247] Take an appropriate amount of this product, grind it into powder, take about 0.3g, weigh it accurately, and prepare the test solution according to the above test solution preparation method. The rutin content at flow rates of 0.25ml / min, 0.30ml / min, and 0.35ml / min is examined respectively. The results are shown in the figure. Figure 13 , Table 27.

[0248] Table 27 Investigation of different flow rates

[0249]

[0250] The results show that when the flow rate is in the range of 0.25ml / min to 0.35ml / min, small changes in the flow rate have no significant effect on the content determination of the sample, and the durability is good.

[0251] 7.6.3 Investigation of different column temperatures

[0252] Take an appropriate amount of this product, grind it into powder, take about 0.3g, weigh it accurately, prepare the test solution according to the above test solution preparation method, and examine the rutin content at three temperatures of 15℃, 20℃, and 25℃ respectively. The results are shown in the figure. Figure 12 , Table 28.

[0253] Table 28 Investigation of different column temperatures

[0254]

[0255] The results showed that when the column temperature was within the range of 15℃~25℃, small changes in column temperature had no significant effect on the content determination of the sample, and the column had good durability.

[0256] 7.6.4 Chromatographic column inspection

[0257] Take an appropriate amount of this product, grind it into powder, take about 0.3g, weigh it accurately, and prepare the test solution according to the preparation method of the test solution above. The rutin content under three different chromatographic column conditions, Acclaim RSLC 120C18 column (2.1*100mm, 2.2μm); Agilent ZORBAX SB-C18 column (2.1*100mm, 1.8μm); Waters CORTECS UPLC T3 column (2.1*100mm, 1.6μm), was investigated. The results are shown in the table. Figure 11 , Table 29.

[0258] Table 29 Comparison of different chromatographic columns

[0259]

[0260] The results showed that the different types of chromatographic columns had good separation of target components, similar rutin contents, and good durability.

Claims

1. A quality control method for a Thunbergia oxyphylla sample, characterized in that: The following steps are involved: (1) Select a Thunb. chinensis sample and prepare a test solution; use Thunb. chinensis as a control medicinal material and prepare a reference solution of the control medicinal material; use caffeic acid, caffeoylmalic acid, ferulic acid and rutin as reference substances and prepare reference solutions respectively; (2) Take the reference solution and the test solution separately for ultra-performance liquid chromatography analysis to construct a characteristic spectrum of the sample to be tested. The characteristic spectrum of the Thunb. chinensis sample has 8 characteristic peaks, and 4 characteristic peaks are identified among the characteristic peaks, which correspond to the caffeic acid reference peak, the caffeoylmalic acid reference peak, the ferulic acid reference peak and the rutin reference peak respectively; (3) Performing qualitative analysis of the sample to be tested using the characteristic spectrum obtained in step (2); and performing quantitative analysis of the sample to be tested using rutin as the content determination indicator; The extractant of the test solution is water, methanol or a methanol-water solution with a volume concentration of 30% to 70%; the conditions of the ultra-performance liquid chromatography analysis are as follows: acetonitrile is used as the mobile phase A, and a phosphoric acid solution with a volume fraction of 0.02% to 0.1% is used as the mobile phase B, and gradient elution is adopted: 0-2 min, 8→12%A, 2-12 min, 12→15%A, 12-20 min, 15→18%A, 20-23 min, 18→43%A, 23-29 min, 43→60%A, the chromatographic column is an Acclaim RSLC 120 C18 chromatographic column, 100×2.1 mm, 2.2 μm, and the detection wavelength is 320 nm. The Tianxianteng sample is Tianxianteng medicinal material, Tianxianteng decoction slices or Tianxianteng formula granules.

2. The quality control method of the Tianxianteng sample according to claim 1, characterized in that: In step (1), the extractant for preparing the reference solution of the control medicinal material is water, methanol, or a methanol-water solution with a volume concentration of 30% to 70%.

3. The quality control method of the Tianxianteng sample according to claim 2, characterized in that: In the reference solution for preparing the control medicinal material, the amount of extractant used per gram of Tianxianteng is 50ml~150ml, and the extraction time is 15~60 minutes.

4. The quality control method of Tianxianteng Teng sample according to claim 1, characterized in that: In step (1), the concentration of the caffeic acid reference substance solution is 5-20 μg / ml, the concentration of the caffeoylmalic acid reference substance solution is 5-20 μg / ml, the concentration of the ferulic acid reference substance solution is 5-20 μg / ml, and the concentration of the rutin reference substance solution is 20-450 μg / ml.

5. The quality control method of Tianxianteng Teng sample according to claim 4, characterized in that: The flow rate was 0.25 ml to 0.35 ml per minute; the column temperature was 15°C to 25°C; and an ultraviolet detector was used.

6. The quality control method of Tianxianteng Teng sample according to claim 5, characterized in that: The characteristic peaks of the Tianxianteng sample correspond to the retention times of the eight characteristic peaks in the chromatogram of the reference medicinal material; the peak corresponding to the caffeic acid reference is peak S1, and the relative retention times of peaks 1 and 3 to peak S1 are calculated; the peak corresponding to the rutin reference is peak S2, and the relative retention times of peaks 6 and 8 to peak S2 are calculated; their relative retention times should be within the range of ±10% of the specified values. The specified values ​​of peaks 1, 3, 6, and 8 are: 0.77、1.36、0.93、1.08。

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