Method for detecting content of amide components in polygonatum odoratum formula granules and application of method

Abstract

The invention discloses a method for detecting the content of amide components in polygonatum odoratum formula granules and application of the method. The method comprises the following steps: preparation of a reference solution, preparation of a test solution, liquid chromatography detection and calculation of the content of the amide components in the polygonatum odoratum formula granules. The amide component is N-trans-p-coumaroyl true octoamine and / or N-trans-feruloyl true octoamine. The invention further discloses a preparation method of the compound. The method provides a new analysis means for quality detection of the polygonatum odoratum formula granules, the result is not interfered by auxiliary materials, the operation is simple, the reproducibility is good, the stability is good, the recovery rate is reliable, and the detection efficiency is high.

Description

Technical Field

[0001] The invention relates to a method for detecting the content of amide components in polygonatum odoratum formula granules and application thereof, belonging to the technical field of drug detection. Background Art

[0002] Polygonatum odoratum is the dried rhizome of Polygonatum odoratum (Mill.) Druce, a plant of the Liliaceae family. It is a commonly used Chinese medicine with a slightly cold nature and sweet taste. It has the effects of nourishing yin and moistening dryness, promoting body fluid and quenching thirst. It is used to treat lung and stomach yin damage, dryness and cough, dry throat and thirst, and internal heat and thirst. Modern pharmacological studies have shown that Polygonatum odoratum has the effects of strengthening the heart, lowering blood pressure, lowering blood sugar, lowering blood lipids, enhancing immune function, anti-aging, anti-fatigue, and anti-oxidation, and has a strong clinical application value. In addition to medicinal uses, Polygonatum odoratum is used as a health product, food, etc., with a broad market demand and export.

[0003] In recent years, the quality control methods for Polygonatum odoratum have mainly focused on sugar components, including the 2020 edition of the Chinese Pharmacopoeia, which uses colorimetry to determine the polysaccharide content in Polygonatum odoratum as a quality control method. However, this method is cumbersome to operate and has poor accuracy. In addition, the addition of excipients during the production process of Polygonatum odoratum formula granules will interfere with the colorimetric determination of polysaccharides. Therefore, polysaccharide indicators are not suitable for the quality control of Polygonatum odoratum formula granules. There are also reports on the determination of monosaccharides such as fructose and glucose as a quality control method for Polygonatum odoratum, but the specificity of monosaccharides is poor and they are present in many traditional Chinese medicines. The determination of monosaccharides often requires the use of derivatization methods, and the pretreatment is cumbersome. It is urgent to develop a quality control method suitable for Polygonatum odoratum formula granules, so as to provide an efficient and feasible technical means for the quality control of Polygonatum odoratum formula granules.

[0004] Polygonatum odoratum contains a variety of chemical components, which mainly include steroidal saponins, flavonoids, amides, volatile oils and polysaccharides. Among them, amide components have multiple effects such as antioxidant, anti-inflammatory, immunomodulatory, lipid regulation, and treatment of nervous system diseases. Many papers have confirmed that amide components have strong antioxidant, anti-inflammatory and lipid regulation effects (Chen Dongjie, Guo Sheng, Yi Yanling, et al. Research progress on amide components and their biological activities in Lycium barbarum plants [J]. Chinese Herbal Medicine, 2023.). Therefore, amide components are an important material basis for the antioxidant activity of Polygonatum odoratum, and the detection of amide components is of great significance for the quality control of Polygonatum odoratum formula granules. Summary of the invention

[0005] Problem to be solved by the invention: In view of the relatively few quality evaluation indicators in the yuzhu formula granules and the corresponding lack of quality detection methods for yuzhu formula granules samples, in order to enrich the quality control means of yuzhu formula granules, the present invention proposes to use amide components for quality detection of yuzhu formula granules, and establishes a method for determining the content of N-trans-p-coumaroyl octopamide and / or N-trans-feruloyl octopamide in yuzhu formula granules, providing a new analytical means for the intrinsic quality control of yuzhu formula granules.

[0006] Technical solution for solving the problem: In order to solve the above technical problem, the present invention provides a method for detecting the content of amide components in Polygonatum odoratum formula granules, comprising the following steps:

[0007] (1) Preparation of reference solution: Take N-trans-p-coumaryl octopamide reference substance and N-trans-feruloyl octopamide, add solvents respectively, and prepare reference solution;

[0008] (2) Preparation of test solution: Take coarse powder of Polygonatum odoratum formula granules, add 70% to 100% methanol, weigh to determine the weight, extract, cool, weigh again, make up the lost weight with corresponding solvent, shake well, filter, accurately pipette the filtrate, evaporate to dryness, dissolve the residue, and make up to volume to obtain the test solution;

[0009] (3) Injecting the reference solution and the test solution into the ultra-high performance liquid chromatograph respectively;

[0010] (4) Using the regression equation of the injection volume and peak area of ​​N-trans-p-coumaryl octopamide, Y=13011931.2709X-12672.5495, R 2 =1.0000, calculate the content of N-trans-p-coumaroyl octopamide in the Yuzhu formula granules; use the regression equation of the injection volume and peak area of ​​N-trans-feruloyl octopamide Y=11739481.8188X-38047.2301, R 2 =1.0000, calculate the content of N-trans feruloyl octopamide in the Polygonatum odoratum formula granules; the content of the amide components is the sum of the content of N-trans p-coumaryl octopamide and the content of N-trans feruloyl octopamide.

[0011] Among them, "isocratic elution" refers to an elution method in which the composition, proportion and flow rate of the mobile phase remain constant during the chromatographic analysis cycle of the sample.

[0012] Wherein, the extraction method in step (2) includes ultrasound, shaking or heating reflux.

[0013] Wherein, the extraction time in step (2) is 30 to 60 minutes.

[0014] Wherein, the volume of the 70% to 100% methanol in step (2) is 25 to 100 mL.

[0015] Wherein, the chromatographic conditions of the ultra-high performance liquid chromatograph in step (3) are as follows: flow rate of 0.25-0.35 mL / min; column temperature of 20-30° C.; chromatographic column of 1-ZORBAX Eclipse Plus C18, 2-ACQUITYUPLC HSS T3 or 3-ZORBAX SB-C18; mobile phase of acetonitrile-0.1% phosphoric acid, elution mode of gradient elution or isocratic elution, and detection wavelength of 280-330 nm.

[0016] The present invention also provides a method for recovering amide components in the granules of Polygonatum odoratum, comprising the following steps:

[0017] (1) Take the powder of Polygonatum odoratum formula granules, add 70% to 100% methanol, weigh the weight, extract, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, accurately pipette the filtrate, evaporate to dryness, dissolve the residue, and make up to volume to obtain the test solution;

[0018] (3) The test solution is injected into an ultra-high performance liquid chromatograph to recover the amide components; the amide components are N-trans-p-coumaryl octopamide or N-trans-feruloyl octopamide.

[0019] Wherein, the extraction method in step (1) includes ultrasound, shaking or heating reflux; and the extraction time is 30 to 60 minutes.

[0020] Wherein, the volume of the 70% to 100% methanol in step (1) is 25 to 100 mL.

[0021] Wherein, the chromatographic conditions of the ultra-high performance liquid chromatograph in step (2) are as follows: flow rate of 0.25-0.35 mL / min; column temperature of 20-30° C.; chromatographic column of 1-ZORBAX Eclipse Plus C18, 2-ACQUITYUPLC HSS T3 or 3-ZORBAX SB-C18; mobile phase of acetonitrile-0.1% phosphoric acid, elution mode of gradient elution or isocratic elution, and detection wavelength of 280-330 nm.

[0022] The present invention also provides a method for detecting amide components in the granules of Polygonatum odoratum formula, comprising the following steps:

[0023] (1) Preparation of reference solution: Take N-trans-p-coumaryl octopamide reference substance and N-trans-feruloyl octopamide, add solvents respectively, and prepare reference solution;

[0024] (2) Preparation of test solution: Take the powder of Polygonatum odoratum granules, add 70% to 100% methanol, weigh it, extract, cool, weigh it again, make up the lost weight with the corresponding solvent, shake well, filter, accurately pipette the filtrate, evaporate to dryness, dissolve the residue, and make up to volume to obtain the solution;

[0025] (3) The reference solution and the test solution are respectively injected into an ultra-high performance liquid chromatograph. When the same characteristic peaks as those of the reference solution appear in the spectrum of the test solution, it indicates that the Polygonatum odoratum formula granules contain amide components; the amide components are N-trans-p-coumaroyl octopamide and / or N-trans-feruloyl octopamide.

[0026] The chromatographic conditions of the ultra-high performance liquid chromatograph are as follows: a flow rate of 0.25 to 0.35 mL / min; a column temperature of 20 to 30° C.; a chromatographic column of 1-ZORBAX Eclipse Plus C18, 2-ACQUITY UPLC HSS T3 or 3-ZORBAX SB-C18; a mobile phase of acetonitrile-0.1% phosphoric acid, an elution mode of gradient elution or isocratic elution, and a detection wavelength of 280 to 330 nm.

[0027] Wherein, the extraction method in step (2) includes ultrasound, shaking or heating reflux.

[0028] Wherein, the extraction time in step (2) is 30 to 60 minutes.

[0029] Wherein, the volume of 70% to 100% methanol in step (3) is 25 to 100 mL.

[0030] The invention also provides application of the method in quality detection or quality control of polygonatum odoratum formula granules.

[0031] Among them, when the content of amide components in Polygonatum odoratum is higher than 0.07 mg / g, it indicates that the quality of Polygonatum odoratum is high.

[0032] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: a method for determining the content of amide components in the yuzhu formula granules is established, providing a new quality control method for the yuzhu formula granules. The method of the present invention is simple to operate, the test results are not interfered by auxiliary materials, have good reproducibility, good stability, reliable recovery rate, low test cost, high test efficiency, and can more reasonably and efficiently perform quality control and quality evaluation of the yuzhu formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the UV spectrum of N-trans-p-coumaryl octopamide reference substance at a wavelength of 210nm to 400nm;

[0034] Figure 2It is the UV spectrum of N-trans-feruloyl octopamide reference substance at a wavelength of 210nm to 400nm;

[0035] Figure 3 This is the UPLC spectrum of the test product of Yuzhu Formula Granule under different flow rate conditions;

[0036] Figure 4 This is the UPLC spectrum of the test product of Yuzhu Formula Granule under different column temperature conditions;

[0037] Figure 5 This is the UPLC spectrum of the test product of Yuzhu Formula Granule under different chromatographic column conditions;

[0038] Figure 6 This is the linear relationship diagram of the N-trans-p-coumaryl octopamide reference substance;

[0039] Figure 7 is the relationship between the peak area of ​​N-trans-feruloyl octopamine reference substance and the injection volume;

[0040] Figure 8 This is a specificity test for the Polygonatum odoratum formula granules;

[0041] Fig. 9 This is a delayed test of the test sample of Polygonatum odoratum formula granules. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0043] Unless otherwise specified, the term "test article" as used herein refers to an experimental sample used for detection or identification.

[0044] Unless otherwise specified, the term "reference substance" as used herein refers to a standard substance used for identification, inspection, content determination and calibration of instrument performance.

[0045] Unless otherwise specified, the term "precision weighing" used in this article means that the weight should be weighed accurately to one thousandth of the weight taken, the term "weighing" means that the weight should be weighed accurately to one hundredth of the weight taken, the term "precision measuring" means that the volume should be measured accurately to one thousandth of the volume taken, and the term "precision pipetting" refers to the operating method of accurately measuring samples through a microinjector.

[0046] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0047] Instruments, reagents and samples:

[0048] Instruments: Waters-ACQUIYT-UPLC-H-Class ultra-high performance liquid chromatography system; Waters-Quaternary-Solvent-Manager-quaternary pump; Sample-manager-FTN autosampler; Waters-UPLC-PDA detector; Empower-3 chromatography workstation.

[0049] Reagents: acetonitrile (chromatographic grade, Thermo Fisher); phosphoric acid (chromatographic grade, Aladdin); ultrapure water; other reagents were of analytical grade.

[0050] Standard: N-trans-p-coumaryl octopamide (batch number: PS000082) was purchased from Chengdu Pusi Biotechnology Co., Ltd. for content determination. The content was calculated as 99.5% and no treatment was required before use.

[0051] N-trans-feruloyl octopamide (batch number: PS013026) was purchased from Chengdu Pusi Biotechnology Co., Ltd. for content determination. The content was 99.63% and no treatment was required before use.

[0052] Samples: Polygonatum odoratum formula granules (batch numbers: 19060139, 19060149, 19060159, 23050221, 23050231, 23050241, 23050271, 18070261, 18070271, 18070281) were obtained from Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0053] Preparation of reference solution: Take appropriate amount of N-trans-p-coumaryl octopamide reference substance and N-trans-feruloyl octopamide, accurately weigh them, and add 50% methanol to make solutions containing 10 μg of N-trans-p-coumaryl octopamide and 40 μg of N-trans-feruloyl octopamide per 1 ml to obtain reference solution.

[0054] Example 1: Determination of chromatographic conditions

[0055] 1. Determination of detection wavelength

[0056] Accurately pipette 1 μl of N-trans-p-coumaryl octopamide and N-trans-ferulyl octopamide reference solution, respectively, and inject them into the ultra-high performance liquid chromatograph to collect the ultraviolet absorption spectrum at a wavelength of 210 nm to 400 nm. The results are shown in Figure 1 , Figure 2 As shown, in the ultraviolet absorption spectrum, the N-trans-p-coumaroyl octopamide reference substance has higher absorption at 223nm and 306nm, and the N-trans-feruloyl octopamide reference substance has higher absorption at 220nm and 318nm. 280nm-330nm can be used as the detection wavelength. The detection wavelength of this experiment is preferably 315nm.

[0057] 2. Investigation of different flow rates

[0058] Take an appropriate amount of Polygonatum odoratum formula granules (batch number: 19060159), grind it into powder, take about 1g, and make 5 groups in total, with 2 parallel portions in each group. Accurately weigh, place in a stoppered conical flask, accurately add 25ml of 80% methanol, seal, weigh, and ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes. Take out, cool, weigh again, make up the lost weight with 80% methanol, shake well, filter, accurately measure 20ml of the filtrate, evaporate to dryness, add 50% methanol to dissolve the residue and transfer to a 5ml volumetric flask to obtain the test solution. Accurately pipette 3 μl of each test solution and inject it into ultra-high performance liquid chromatography. The chromatographic conditions are as follows: ZORBAX Eclipse Plus C18 (Agilent, 2.1×100 mm, 1.8 μm), acetonitrile-0.1% phosphoric acid (13:87) as the mobile phase, isocratic elution, column temperature 25°C; detection wavelength is 315 nm.

[0059] The chromatographic peak separation effect at different flow rates (0.25mL / min, 0.30mL / min, 0.35mL / min) was investigated. The experimental results are shown in Table 1. Figure 3 .

[0060] Table 1. Investigation results of different flow rates

[0061]

[0062]

[0063] Conclusion: At different flow rates, the measured contents were similar and the chromatographic peak separation was good, indicating that the durability was good when the flow rate fluctuated within the range of 0.25 mL / min to 0.35 mL / min.

[0064] 3. Investigation of different column temperatures

[0065] Take an appropriate amount of Polygonatum odoratum formula granules (batch number: 19060159), grind it into powder, take about 1g, and make 5 groups in total, with 2 parallel portions in each group. Accurately weigh, place in a stoppered conical flask, accurately add 25ml of 80% methanol, seal, weigh, and ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes. Take out, cool, weigh again, make up the lost weight with 80% methanol, shake well, filter, accurately measure 20ml of the filtrate, evaporate to dryness, add 50% methanol to dissolve the residue and transfer to a 5ml volumetric flask to obtain the test solution. Accurately pipette 3 μl of each test solution and inject it into ultra-high performance liquid chromatography. The chromatographic conditions are as follows: ZORBAX Eclipse Plus C18 (Agilent, 2.1×100 mm, 1.8 μm), acetonitrile-0.1% phosphoric acid (13:87) as the mobile phase, isocratic elution, flow rate 0.3 mL / min; detection wavelength is 315 nm.

[0066] The chromatographic peak separation effect at different column temperatures (20℃, 25℃, 30℃) was investigated. The experimental results are shown in Table 2. Figure 4 .

[0067] Table 2. Investigation results of different column temperatures

[0068]

[0069] Conclusion: Under different column temperatures, the chromatographic peak separation effect is good and the measured contents are similar, indicating that the column has good durability when the temperature fluctuates within the range of 20℃~30℃.

[0070] 4. Investigation of different chromatographic columns

[0071] Take an appropriate amount of Yuzhu Formula Granules (batch number: 19060159), grind it into powder, take about 1g, and make 5 groups in total, with 2 parallel copies in each group, accurately weigh it, put it in a stoppered conical bottle, accurately add 25ml of 80% methanol, seal it, weigh it, ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with 80% methanol, shake it well, filter it, accurately measure 20ml of the filtrate, evaporate it to dryness, add 50% methanol to dissolve the residue and transfer it to a 5ml volumetric bottle to obtain the test solution. Accurately take 3μl of each test solution and inject it into the ultra-high performance liquid chromatograph. The chromatographic conditions are: acetonitrile-0.1% phosphoric acid (13:87) as the mobile phase, isocratic elution, flow rate 0.3mL / min, column temperature 25℃; detection wavelength is 315nm.

[0072] The separation effects of different chromatographic columns 1-ZORBAX Eclipse Plus C18 (Agilent, 2.1×100mm, 1.8μm), 2-ACQUITY UPLC HSS T3 (Waters, 2.1×100mm, 1.8μm), and 3-ZORBAX SB-C18 (Agilent, 2.1×100mm, 1.8μm) on the granules of Polygonatum odoratum were investigated. The experimental results are shown in Table 3. Figure 5 .

[0073] Table 3. Results of investigation on different chromatographic columns

[0074]

[0075] Results: The separation of the three chromatographic columns was good and the retention time was moderate, which showed that the chromatographic columns had little influence on the determination results of the samples and this method had universal adaptability.

[0076] The chromatographic conditions used in the following experiments are as follows: 1-ZORBAX Eclipse Plus C18 (Agilent, 2.1×100mm, 1.8μm) as the chromatographic column; acetonitrile-0.1% phosphoric acid (13:87) as the mobile phase, isocratic elution, flow rate 0.3mL / min, column temperature 25°C; detection wavelength is 315nm, and the theoretical plate number calculated based on the N-trans-p-coumaryl octopamide peak must not be less than 3000.

[0077] Example 2: Preparation of test solution

[0078] 1. Investigation of extraction solvent

[0079] Take an appropriate amount of Yuzhu Formula Granules (batch number: 19060159), grind it into powder, take about 1g, and add 5 groups in total, 2 parts in parallel for each group, accurately weigh it, put it in a stoppered conical bottle, accurately add 25ml of water, 30% methanol, 50% methanol, 70% methanol, 80% methanol, and methanol respectively, plug it tightly, weigh it, ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, accurately take 20ml of the filtrate, evaporate it to dryness, add 50% methanol to dissolve the residue and transfer it to a 5ml volumetric bottle, and obtain it. Accurately take 3μl of each test solution, inject it into the ultra-high performance liquid chromatograph, and determine it according to the chromatographic conditions determined in Example 1. The calculated content is shown in Table 4.

[0080] Table 4. Comparison of different extraction solvents

[0081]

[0082]

[0083] Conclusion: Using 70% methanol to 100% methanol as the extraction solvent, the extraction rate is relatively high. The following experiments use 80% methanol as the extraction solvent.

[0084] 2. Investigation of extraction methods

[0085] Take an appropriate amount of Yuzhu Formula Granules (batch number: 19060159), grind it into powder, take about 1g, and make 3 groups in total, with 2 parts in each group, accurately weigh it, put it in a stoppered conical bottle, accurately add 25ml of 80% methanol, plug it tightly, weigh it, ultrasonically treat it (power 600W, frequency 40kHz), shake it to extract, heat it under reflux for 30 minutes, let it cool, weigh it again, make up the lost weight with 80% methanol, shake it well, filter it, accurately take 20ml of the filtrate, evaporate it to dryness, add 50% methanol to dissolve the residue, transfer it to a 5ml volumetric bottle, and get it. Accurately take 3μl of each test solution, inject it into the ultra-high performance liquid chromatograph, and determine it according to the chromatographic conditions determined in Example 1. The calculated content is shown in Table 5.

[0086] Table 5. Comparison of different extraction methods

[0087]

[0088]

[0089] Conclusion: Ultrasonic extraction and heating reflux extraction have higher efficiency. Ultrasonic treatment was selected as the extraction method in the following experiments.

[0090] 3. Investigation of different extraction volumes

[0091] Take an appropriate amount of Yuzhu Formula Granules (batch number: 19060159), grind it into powder, take about 1g, and make 3 groups in total, with 2 parallel portions in each group, accurately weigh it, put it in a stoppered conical bottle, accurately add 80% methanol 25ml, 50ml, 100ml respectively, plug it tightly, weigh it, ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 80% methanol, shake it well, filter it, accurately take 20ml, 40ml, 75ml of the filtrate respectively, evaporate it to dryness, add 50% methanol to dissolve the residue, transfer it to a 5ml volumetric bottle, and obtain it. Accurately take 3μl of each test solution, inject it into the ultra-high performance liquid chromatograph, and determine it according to the above chromatographic conditions. The calculated content is shown in Table 6.

[0092] Table 6. Comparison of different extraction solvent volumes

[0093]

[0094] Conclusion: The extraction rates of different extraction volumes are similar, and the measured contents are similar, indicating that the extraction is basically complete when the extraction solvent volume is 25mL. To ensure sufficient extraction, the extraction solvent volume is selected as 50mL in the following experiments.

[0095] 4. Extraction time investigation

[0096] Take an appropriate amount of Yuzhu Formula Granules (batch number: 19060159), grind it into powder, take about 1g, and make 4 groups in total, with 2 parallel portions in each group, accurately weigh it, put it in a stoppered conical bottle, accurately add 50mL of 80% methanol, plug it tightly, weigh it, and ultrasonically treat it (power 600W, frequency 40kHz) for 15 minutes, 45 minutes, 30 minutes, and 60 minutes respectively, let it cool, weigh it again, make up the lost weight with 80% methanol, shake it well, filter it, accurately take 20mL of the filtrate, evaporate it to dryness, add 50% methanol to dissolve the residue, and transfer it to a 5mL volumetric bottle to obtain it. Accurately take 3μl of each test solution, inject it into the ultra-high performance liquid chromatograph, and determine it according to the chromatographic conditions determined in Example 1. The calculated content is shown in Table 7.

[0097] Table 7. Comparison of different extraction times

[0098]

[0099] Conclusion: The contents of different ultrasonic extraction times are similar. The extraction time of 30 minutes was selected in the following experiments.

[0100] Based on the above results, the chromatographic conditions used in the following examples of the present invention are: ZORBAX EclipsePlus C18 (Agilent, 2.1×100 mm, 1.8 μm) as the chromatographic column; acetonitrile-0.1% phosphoric acid (13:87) as the mobile phase, the flow rate is 0.3 mL / min, the column temperature is 25° C.; the detection wavelength is 315 nm, and the theoretical plate number calculated based on the N-trans-p-coumaryl octopamide peak shall not be less than 3000.

[0101] The preparation method of the test solution is as follows: take an appropriate amount of Polygonatum odoratum granules, grind them into powder, take about 1g, accurately weigh it, put it in a stoppered conical flask, accurately add 50mL of 80% methanol, stopper it, weigh it, ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes, take it out, cool it, weigh it again, make up the lost weight with 80% methanol, shake it well, filter it, accurately measure 20ml of the filtrate, evaporate it to dryness, add 50% methanol to dissolve the residue and transfer it to a 5ml volumetric flask to obtain the solution.

[0102] Example 3: Methodological verification of the content of amide components in Polygonatum odoratum formula granules

[0103] 1. Linear

[0104] Accurately pipette 0.2 μl, 0.4 μl, 0.6 μl, 1.0 μl, 1.5 μl, 2.0 μl, and 3.0 μl of N-trans-p-coumaryl octopamide reference solution (21.4323 μg / ml) and N-trans-feruloyl octopamide reference solution (82.334232 μg / ml), respectively, and inject into ultra-high performance liquid chromatograph, and measure according to the chromatographic conditions determined in Example 2, plot the standard curve with the peak area as the ordinate and the injection volume as the abscissa, and obtain the regression equation. The results are shown in Tables 8 to 9. Figure 6-7 .

[0105] Table 8. Relationship between peak area integral value of reference substance and injection volume

[0106]

[0107] Table 9. Regression equation of standard curve

[0108]

[0109] Conclusion: The injection amount of N-trans-p-coumaroyl octopamide is in the range of 0.00429μg to 0.06430μg, and the injection amount and peak area value show a good linear relationship. The injection amount of N-trans-feruloyl octopamide is in the range of 0.01647μg to 0.24700μg, and the injection amount and peak area value show a good linear relationship.

[0110] 2. Precision

[0111] 2.1 Instrument precision

[0112] Take Yuzhu Formula Granules (Batch No.: 19060159) and prepare a test solution according to the test solution preparation method of Example 2. Accurately pipette 3 μL and inject it into the ultra-high performance liquid chromatograph. Determine according to the chromatographic conditions of Example 1. Inject 6 times continuously, record the peak area measurement value, and calculate the relative standard deviation. The results are shown in Table 10.

[0113] Table 10. Instrument precision test

[0114]

[0115]

[0116] Conclusion: The precision test of the instrument is good.

[0117] 2.2 Intermediate precision

[0118] Take 3 portions of Polygonatum odoratum formula granules (batch number: 19060159), and two experimenters prepared the test solution according to the test solution preparation method of Example 2. 3 μl was injected on different instruments at different times, the peak area value was determined, the content was calculated, and the RSD was calculated. The results are shown in Table 11.

[0119] Table 11. Intermediate precision test

[0120]

[0121] Conclusion: The intermediate precision (RSD% = 0.80) is good.

[0122] 2.3 Repeatability

[0123] Take 6 portions of Polygonatum odoratum formula granules (batch number: 19060159), prepare the test solution according to the test solution preparation method in Example 2, inject 3 μl of each, measure the area value, calculate the content and RSD, and the results are shown in Table 12.

[0124] Table 12. Repeatability test results of samples

[0125]

[0126]

[0127] Conclusion: The repeatability test (RSD% = 0.34%) is good.

[0128] 3. Accuracy (sample recovery test)

[0129] Take 0.5 g of Polygonatum odoratum formula granules with known content (batch number: 19060159, N-trans-p-coumaryl octopamide content 0.01952 mg / g, N-trans-feruloyl octopamide content 0.09045 mg / g), a total of 9 portions, accurately weighed, and added with high, medium and low corresponding concentrations of reference solution, respectively, and prepared according to the test solution preparation method of Example 2 to prepare the sample recovery test solution, according to the chromatographic conditions of Example 1, respectively inject 3 μL, and calculate the recovery rate and RSD according to the following formula. The results are shown in Tables 13 to 14.

[0130]

[0131] Table 13. N-trans-p-coumaryl octopamide recovery test results

[0132]

[0133]

[0134] Table 14. N-trans-feruloyl octopamide recovery test results

[0135]

[0136] Conclusion: The recovery rates of N-trans-p-coumaryl octopamide and N-trans-feruloyl octopamide meet the requirements (when the content of the test sample component is lower than 10 mg / g, the sample recovery requirement is 80% to 115%; when the content of the test component is higher than 0.1 mg / g, the sample recovery requirement is 85% to 110%), and the accuracy test is good.

[0137] 4. Exclusivity

[0138] The yuzhu formula granules (batch number: 19060159), negative samples (excipients added to the yuzhu formula granules: maltodextrin, silicon dioxide and magnesium stearate) and N-trans-p-coumaroyl octopamide and N-trans-feruloyl octopamide mixed standard reference substances were tested according to the method of Example 2. The results are shown in Figure 8 .

[0139] Conclusion: There is no chromatographic peak in the negative chromatogram at the retention time corresponding to the reference substance, indicating that the solvent has no interference with the determination of amide components in the Yuzhu Formula Granules. This method is specific for determining the content of amide components in the Yuzhu Formula Granules.

[0140] 5. Latency

[0141] Take the yuzhu formula granules, prepare the test solution according to the preparation method of the test solution determined in Example 2, and analyze the sample according to the chromatographic conditions obtained in Example 1. Under the same chromatographic conditions, keep the elution gradient when the acetonitrile ratio is the highest, double the elution time, record the chromatogram, and the results are shown in Fig. 9 .

[0142] Conclusion: No obvious chromatographic peaks were eluted after the original gradient elution, indicating that the chromatographic conditions basically met the principle of maximum information content and had no effect on the analysis of subsequent samples.

[0143] 6. Durability

[0144] 6.1 Stability test

[0145] Take the Polygonatum odoratum formula granules and prepare the test solution according to the preparation method of the test solution in Example 2. The test was carried out according to the method of Example 1 after 0h, 4h, 8h, 12h, 16h and 24h, and the changes in the peak area were recorded. The RSD was calculated. The results are shown in Table 15.

[0146] Table 15. Stability test results

[0147]

[0148] Conclusion: The test solution has good stability within 24 hours.

[0149] Example 4: Determination of the content of amide components in Polygonatum odoratum formula granules

[0150] Chromatographic conditions: the chromatographic column is 1-ZORBAX Eclipse Plus C18 (column length is 100 mm, inner diameter is 2.1 mm, particle size is 1.8 μm); acetonitrile-0.1% phosphoric acid (13:87) is used as the mobile phase; the flow rate is 0.3 ml / min; the column temperature is 25°C, and the elution is isocratic; the detection wavelength is 315 nm, and the theoretical plate number calculated based on the N-trans-p-coumaryl octopamide peak shall not be less than 3000.

[0151] Preparation of reference solution: Take appropriate amount of N-trans-p-coumaryl octopamide reference substance and N-trans-feruloyl octopamide, accurately weigh them, and add 50% methanol to make solutions containing 5 μg of N-trans-p-coumaryl octopamide and 50 μg of N-trans-feruloyl octopamide per 1 ml.

[0152] Preparation of test solution: Take 10 batches of Polygonatum odoratum formula granules, crush them, take about 1g of powder, weigh it accurately, put it in a stoppered conical flask, add 50ml of 80% methanol accurately, weigh the weight, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, take it out, cool, weigh it again, make up the lost weight with 80% methanol, shake well, filter, accurately pipette 20ml of the filtrate, evaporate to dryness, dissolve the residue in 50% methanol and transfer it to a 5ml volumetric flask.

[0153] Determination method: Accurately aspirate 1 μl of the reference solution and 3 μl of the test solution, respectively, and inject them into the ultra-high performance liquid chromatography for determination. The content of amide components in the Polygonatum odoratum formula granules is calculated as the total amount of N-trans-p-coumaroyl octopamide and N-trans-feruloyl octopamide.

[0154] Table 16. Determination results of the content of amide components in different batches of Polygonatum odoratum formula granules

[0155]

[0156] The average content of amide components in 10 batches of yuzhu formula granules is 0.103 mg / g, which is 0.07 mg / g when the average is reduced by 30%. When the content of amide components in yuzhu formula granules is higher than 0.07 mg / g, it indicates that the product quality is good. Taking this as an example, this method is used for quality detection and quality control of different batches of yuzhu formula granules.

Claims

1. A method for detecting the content of amide components in Polygonatum odoratum formula granules, characterized in that: The following steps are involved: (1) Preparation of reference solution: Take N-trans-p-coumaryl octopamide reference substance and N-trans-feruloyl octopamide, add solvents respectively, and prepare reference solution; (2) Preparation of test solution: Take the powder of Polygonatum odoratum granules, add 70% to 100% methanol, weigh it, extract, cool, weigh it again, make up the lost weight with the corresponding solvent, shake well, filter, accurately pipette the filtrate, evaporate to dryness, dissolve the residue, and make up to volume to obtain the solution; (3) Injecting the reference solution and the test solution into the ultra-high performance liquid chromatograph respectively; (4) Using the regression equation of the injection volume and peak area of ​​N-trans-p-coumaryl octopamide, Y=13011931.2709X-12672.5495, R 2 =1.0000, calculate the content of N-trans-p-coumaroyl octopamide in the Yuzhu formula granules; use the regression equation of the injection volume and peak area of ​​N-trans-feruloyl octopamide Y=11739481.8188X-38047.2301, R 2 =1.0000, calculate the content of N-trans feruloyl octopamide in the Polygonatum odoratum formula granules; the content of the amide components is the sum of the content of N-trans p-coumaryl octopamide and the content of N-trans feruloyl octopamide.

2. The method according to claim 1, characterized in that: The extraction method in step (2) includes ultrasound, shaking or heating reflux.

3. The method according to claim 1, characterized in that: The extraction time in step (2) is 30 to 60 minutes.

4. The method according to claim 1, characterized in that: The volume of the 70% to 100% methanol in step (2) is 25 to 100 mL.

5. The method according to claim 1, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatograph in step (3) are as follows: a flow rate of 0.25 to 0.35 mL / min; a column temperature of 20 to 30° C.; a chromatographic column of 1-ZORBAX Eclipse Plus C18, 2-ACQUITYUPLC HSS T3 or 3-ZORBAX SB-C18; a mobile phase of acetonitrile-0.1% phosphoric acid, an elution mode of gradient elution or isocratic elution, and a detection wavelength of 280 to 330 nm.

6. A method for recovering amide components in Polygonatum odoratum formula granules, characterized in that: The following steps are involved: (1) Take the powder of Polygonatum odoratum formula granules, add 70% to 100% methanol, weigh the weight, extract, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, accurately pipette the filtrate, evaporate to dryness, dissolve the residue, and make up to volume to obtain the test solution; (3) The test solution is injected into an ultra-high performance liquid chromatograph to recover the amide components; the amide components are N-trans-p-coumaryl octopamide or N-trans-feruloyl octopamide.

7. The method according to claim 6, characterized in that: The extraction method in step (1) includes ultrasound, shaking or heating reflux; the extraction time is 30 to 60 minutes.

8. The method according to claim 6, characterized in that: The volume of the 70% to 100% methanol in step (1) is 25 to 100 mL.

9. The method according to claim 6, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatograph in step (2) are as follows: a flow rate of 0.25 to 0.35 mL / min; a column temperature of 20 to 30° C.; a chromatographic column of 1-ZORBAX Eclipse Plus C18, 2-ACQUITYUPLC HSS T3 or 3-ZORBAX SB-C18; a mobile phase of acetonitrile-0.1% phosphoric acid, an elution mode of gradient elution or isocratic elution, and a detection wavelength of 280 to 330 nm.

10. Use of the method according to any one of claims 1 to 9 in quality detection or quality control of Polygonatum odoratum formula granules.

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