A method for detecting the content of amide components in Polygonatum odoratum formula granules and its application

Abstract

This invention discloses a method for detecting the content of amide components in Polygonatum odoratum formula granules and its application, comprising the following steps: preparation of a reference solution, preparation of a test solution, detection by liquid chromatography, and calculation of the content of amide components in the Polygonatum odoratum formula granules; wherein the amide components are N-trans-p-coumaryl eutodamine and / or N-trans-feruloyl eutodamine. This invention provides a new analytical method for the quality detection of Polygonatum odoratum formula granules, and the results are not affected by excipients, 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] This invention relates to a method for detecting the content of amide components in Polygonatum odoratum formula granules and its application, belonging to the field of drug detection technology. Background Technology

[0002] Polygonatum odoratum (Mill.) Druce, a plant in the Liliaceae family, is a dried rhizome. It is a commonly used traditional Chinese medicine, slightly cold in nature and sweet in taste. It nourishes yin and moistens dryness, promotes body fluid production and quenches thirst. It is used to treat lung and stomach yin deficiency, dry cough due to heat, dry throat and thirst, and internal heat with thirst. Modern pharmacological studies have shown that Polygonatum odoratum has cardiotonic, blood pressure-lowering, blood sugar-lowering, blood lipid-lowering, immune-enhancing, anti-aging, anti-fatigue, and antioxidant effects, making it highly valuable in clinical applications. Besides its medicinal uses, Polygonatum odoratum is also widely available in health products and food, with broad market demand and export potential.

[0003] In recent years, quality control methods for Polygonatum odoratum have mainly focused on carbohydrate components. The 2020 edition of the Chinese Pharmacopoeia uses colorimetric methods to determine the polysaccharide content as a quality control measure. However, this method is cumbersome and inaccurate. Furthermore, the addition of excipients during the production of Polygonatum odoratum granules can interfere with the colorimetric determination of polysaccharides. Therefore, polysaccharide indicators are unsuitable for the quality control of Polygonatum odoratum granules. There are also reports on using the determination of monosaccharides such as fructose and glucose as quality control methods for Polygonatum odoratum, but monosaccharides have poor specificity and are present in many traditional Chinese medicines. The determination of monosaccharides often requires derivatization methods, which involve cumbersome pretreatment. There is an urgent need to develop a quality control method suitable for Polygonatum odoratum granules, thus providing an efficient and feasible technical means for the quality control of these granules.

[0004] Polygonatum odoratum contains various chemical components, mainly including steroidal saponins, flavonoids, amides, volatile oils, and polysaccharides. Among them, amides have multiple effects, including antioxidant, anti-inflammatory, immunomodulatory, lipid-regulating, and therapeutic effects on nervous system diseases. Multiple studies have confirmed that amides have strong antioxidant, anti-inflammatory, and lipid-regulating effects (Chen Dongjie, Guo Sheng, Yi Yanling, et al. Research progress on amide components and their bioactivity in Lycium spp. [J]. Chinese Traditional and Herbal Drugs, 2023). Therefore, amides are an important material basis for the antioxidant activity of Polygonatum odoratum, and the detection of amides is of great significance for the quality control of Polygonatum odoratum formulation granules. Summary of the Invention

[0005] The problem this invention aims to solve is the relative scarcity of quality evaluation indicators for Polygonatum odoratum formula granules and the corresponding lack of quality testing methods for these granules. To enrich the quality control methods for Polygonatum odoratum formula granules, this invention proposes the use of amide components for quality testing and establishes a method for determining the content of N-trans-coumaroyl eugenolamine and / or N-trans-feruloyl eugenolamine in Polygonatum odoratum formula granules, providing a new analytical means for the intrinsic quality control of Polygonatum odoratum formula granules.

[0006] Technical solution to the problem: To solve the above-mentioned 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 solutions: Take N-trans-coumaroyl eutodamine reference standard and N-trans-feruloyl eutodamine, add solvents respectively to prepare reference solutions;

[0008] (2) Preparation of test solution: Take the coarse powder of Polygonatum odoratum formula 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 test solution.

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

[0010] (4) The regression equation for the injection amount and peak area of ​​coumaroyl methoxymethylamine using the N-trans method was Y = 13011931.2709X - 12672.5495, R 2 =1.0000, calculate the content of N-trans-p-coumaroyl eugenol in the Polygonatum odoratum formulation granules; use the regression equation Y = 11739481.8188X - 38047.2301 for the injection amount and peak area of ​​N-trans-feruloyl eugenol, R 2 =1.0000, calculate the content of N-trans-feruloyl eugenolamine in the Polygonatum odoratum formula granules; the content of the amide component is the sum of the content of N-trans-p-coumaroyl eugenolamine and the content of N-trans-feruloyl eugenolamine.

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

[0012] The extraction methods described in step (2) include ultrasound, shaking, or heating and reflux.

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

[0014] In step (2), the volume of the 70%–100% methanol is 25–100 mL.

[0015] 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℃; 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] This invention also provides a method for recovering amide components from Polygonatum odoratum formula granules, comprising the following steps:

[0017] (1) Take the Polygonatum odoratum formula 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, make up to volume, and the test solution is obtained.

[0018] (3) The amide components can be recovered by injecting the test solution into an ultra-high performance liquid chromatograph; the amide components are N-trans-coumaroyl eugenolamine or N-trans-feruloyl eugenolamine.

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

[0020] In step (1), the volume of the 70%–100% methanol is 25–100 mL.

[0021] 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℃; 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] This invention also provides a method for detecting amide components in Polygonatum odoratum formula granules, comprising the following steps:

[0023] (1) Preparation of reference solutions: Take N-trans-coumaroyl eutodamine reference standard and N-trans-feruloyl eutodamine, add solvents respectively to prepare reference solutions;

[0024] (2) Preparation of test solution: Take the Polygonatum odoratum formula granules powder, add 70% to 100% methanol, weigh, extract, cool, weigh again, replenish 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.

[0025] (3) Inject the reference solution and the test solution into the ultra-high performance liquid chromatograph. When the test solution shows the same characteristic peak as the reference solution, it indicates that the Polygonatum odoratum formula granules contain amide components. The amide components are N-trans-coumaroyl eugenolamine and / or N-trans-feruloyl eugenolamine.

[0026] The chromatographic conditions of the ultra-high performance liquid chromatograph 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-ACQUITY UPLC 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.

[0027] The extraction methods described in step (2) include ultrasound, shaking, or heating and reflux.

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

[0029] In step (3), the volume of the 70%–100% methanol is 25–100 mL.

[0030] The present invention also provides the application of the method in the 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 existing technologies, this invention has the following significant advantages: It establishes a method for determining the content of amide components in Polygonatum odoratum formula granules, providing a new quality control method for these granules. The method of this invention is simple to operate, the detection results are not affected by excipients, have good reproducibility and stability, reliable recovery rate, low detection cost, and high detection efficiency, enabling more reasonable and efficient quality control and evaluation of Polygonatum odoratum formula granules. Attached Figure Description

[0033] Figure 1 The UV spectrum of N-trans-coumaroyl dimethoxylamine reference standard in the wavelength range of 210 nm to 400 nm;

[0034] Figure 2The UV spectrum of N-trans-feruloyl eugenolamine reference standard in the wavelength range of 210 nm to 400 nm;

[0035] Figure 3 UPLC chromatograms of Polygonatum odoratum granules under different flow rate conditions;

[0036] Figure 4 UPLC chromatograms of Polygonatum odoratum granule test sample under different column temperature conditions;

[0037] Figure 5 UPLC chromatograms of Polygonatum odoratum formula granules under different chromatographic column conditions;

[0038] Figure 6 Linearity graph of N-trans-coumaroyl ditocobalamin reference standard;

[0039] Figure 7 The relationship between peak area and injection volume of N-trans-feruloyl eugenolamine reference standard;

[0040] Figure 8 Specificity test for Polygonatum odoratum formula granules;

[0041] Figure 9 Delayedness test for Polygonatum odoratum formula granules. Detailed Implementation

[0042] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0043] Unless otherwise stated, the term “test sample” as used herein refers to an experimental sample used for testing or identification.

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

[0045] Unless otherwise stated, the terms “precise weighing” as used herein mean that the weight should be accurate to one-thousandth of the weight taken, the term “weighing” means that the weight should be accurate to one-hundredth of the weight taken, the term “precise measurement” means that the volume should be measured accurately to one-thousandth of the volume taken, and the term “precise aspiration” means the operation of accurately measuring the sample using a microsyringe.

[0046] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[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 Scientific); phosphoric acid (chromatographic grade, Aladdin Scientific); water was ultrapure water; all other reagents were of analytical grade.

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

[0051] N-transferoyl eugenolamine (batch number: PS013026) was purchased from Chengdu Pusi Biotechnology Co., Ltd. for content determination. The content is 99.63%. No treatment is required before use.

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

[0053] Preparation of reference solution: Take appropriate amounts of N-trans-coumaroyl euroxamine reference standard and N-trans-feruloyl euroxamine, accurately weigh them, and add 50% methanol to prepare solutions containing 10 μg of N-trans-coumaroyl euroxamine and 40 μg of N-trans-feruloyl euroxamine per 1 ml, which are the reference solution.

[0054] Example 1: Determination of chromatographic conditions

[0055] 1. Determination of detection wavelength

[0056] Accurately pipette 1 μl each of N-trans-coumaroyl metohematidine and N-trans-feruloyl metohematidine reference solutions and inject them separately into the ultra-high performance liquid chromatograph. Collect the ultraviolet absorption spectra at wavelengths of 210 nm to 400 nm. The results are shown in the figure. Figure 1 , Figure 2 As shown, in the ultraviolet absorption spectrum, N-trans-coumaroyl dimethoxylamine reference standard exhibits high absorption at 223 nm and 306 nm, while N-trans-feruloyl dimethoxylamine reference standard exhibits high absorption at 220 nm and 318 nm. 280 nm to 330 nm can be used as the detection wavelength. In this experiment, the preferred detection wavelength is 315 nm.

[0057] 2. Investigation of different flow velocities

[0058] Take an appropriate amount of Polygonatum odoratum formula granules (batch number: 19060159), grind them into a fine powder, take about 1g, make 5 groups in total, with 2 parallel portions for each group, accurately weigh them, place them in a stoppered conical flask, accurately add 25ml of 80% methanol, seal tightly, weigh, sonicate (power 600W, frequency 40kHz) for 30 minutes, remove, 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 it to a 5ml volumetric flask to obtain the test solution. Accurately pipette 3 μl of each test solution and inject it into the ultra-high performance liquid chromatograph. The chromatographic conditions are as follows: ZORBAX Eclipse Plus C18 (Agilent, 2.1×100mm, 1.8μm), with acetonitrile-0.1% phosphoric acid (13:87) as the mobile phase, isocratic elution, column temperature 25℃, and detection wavelength 315nm.

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

[0060] Table 1. Results of the investigation at different flow velocities

[0061]

[0062]

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

[0064] 3. Investigation at different column temperatures

[0065] Take an appropriate amount of Polygonatum odoratum formula granules (batch number: 19060159), grind them into a fine powder, take about 1g, make 5 groups in total, with 2 parallel portions for each group, accurately weigh them, place them in a stoppered conical flask, accurately add 25ml of 80% methanol, seal tightly, weigh, sonicate (power 600W, frequency 40kHz) for 30 minutes, remove, 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 it to a 5ml volumetric flask to obtain the test solution. Accurately pipette 3 μl of each test solution and inject it into the ultra-high performance liquid chromatograph. The chromatographic conditions are as follows: ZORBAX Eclipse Plus C18 (Agilent, 2.1×100mm, 1.8μm), with 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 separation effect of chromatographic peaks at different column temperatures (20℃, 25℃, and 30℃) was investigated. The experimental results are shown in Table 2. Figure 4 .

[0067] Table 2. Results of the investigation at different column temperatures

[0068]

[0069] Conclusion: The chromatographic peak separation was good at different column temperatures, and the measured contents were similar, indicating that the column has good robustness when the column temperature fluctuates within the range of 20℃ to 30℃.

[0070] 4. Investigation of different chromatographic columns

[0071] Take an appropriate amount of Polygonatum odoratum formula granules (batch number: 19060159), grind them into a fine powder, take about 1g, make 5 groups, with 2 parallel portions for each group, accurately weigh them, place them in a stoppered conical flask, accurately add 25ml of 80% methanol, seal tightly, weigh, sonicate (power 600W, frequency 40kHz) for 30 minutes, remove, cool, weigh again, make up the weight loss with 80% methanol, shake well, filter, accurately measure 20ml of the filtrate, evaporate to dryness, dissolve the residue in 50% methanol and transfer to a 5ml volumetric flask to obtain the test solution. Accurately pipette 3μl of each test solution and inject it into an 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 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 the Polygonatum odoratum formula were investigated. The experimental results are shown in Table 3. Figure 5 .

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

[0074]

[0075] Results: The separation of the three chromatographic columns was good and the retention time was moderate, indicating that the chromatographic column has little impact on the sample determination results and that this method has universal applicability.

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

[0077] Example 2: Preparation of the test solution

[0078] 1. Investigation of extraction solvent

[0079] Take an appropriate amount of Polygonatum odoratum formula granules (batch number: 19060159), grind them into a fine powder, take about 1g, make 5 groups, each group in duplicate, accurately weigh, place in a stoppered conical flask, accurately add 25ml each of water, 30% methanol, 50% methanol, 70% methanol, 80% methanol, and methanol, stopper tightly, weigh, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the lost weight with the appropriate solvent, shake well, filter, accurately pipette 20ml of the filtrate, evaporate to dryness, dissolve the residue in 50% methanol and transfer to a 5ml volumetric flask, thus obtaining the test solution. Accurately pipette 3μl of each test solution and inject it into an ultra-high performance liquid chromatograph, determine the content according to the chromatographic conditions determined in Example 1, and calculate the content as shown in Table 4.

[0080] Table 4. Comparison of different extraction solvents

[0081]

[0082]

[0083] Conclusion: High extraction rates were achieved using 70% to 100% methanol as the extraction solvent. The following experiments used 80% methanol as the extraction solvent.

[0084] 2. Examination of extraction methods

[0085] Take an appropriate amount of Polygonatum odoratum formula granules (batch number: 19060159), grind them into a fine powder, take about 1g, make 3 groups, 2 parallel portions for each group, accurately weigh them, place them in a stoppered conical flask, accurately add 25ml of 80% methanol, seal tightly, weigh, and sonicate (power 600W, frequency 40kHz), shake extract, heat under reflux for 30 minutes, cool, weigh again, make up the weight loss with 80% methanol, shake well, filter, accurately pipette 20ml of the filtrate, evaporate to dryness, dissolve the residue in 50% methanol, transfer to a 5ml volumetric flask, and obtain the final product. Accurately pipette 3μl of each test solution, inject it into an ultra-high performance liquid chromatograph, and determine the content 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 high 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 Polygonatum odoratum formula granules (batch number: 19060159), grind them into a fine powder, take about 1g, make 3 groups, each group in duplicate, accurately weigh, place in a stoppered conical flask, accurately add 25ml, 50ml and 100ml of 80% methanol respectively, stopper tightly, weigh, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the weight loss with 80% methanol, shake well, filter, accurately pipette 20ml, 40ml and 75ml of the filtrate respectively, evaporate to dryness, dissolve the residue in 50% methanol, transfer to a 5ml volumetric flask, and obtain the test solution. Accurately pipette 3μl of each test solution and inject it into an ultra-high performance liquid chromatograph, determine the content according to the above chromatographic conditions, and calculate the content as shown in Table 6.

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

[0093]

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

[0095] 4. Examination of extraction time

[0096] Take an appropriate amount of Polygonatum odoratum formula granules (batch number: 19060159), grind them into a fine powder, take about 1g, make 4 groups, with 2 parallel portions for each group, accurately weigh them, place them in a stoppered conical flask, accurately add 50mL of 80% methanol, seal tightly, weigh, and sonicate (power 600W, frequency 40kHz) for 15 minutes, 45 minutes, 30 minutes, and 60 minutes respectively, cool, weigh again, make up the weight loss with 80% methanol, shake well, filter, accurately pipette 20mL of the filtrate, evaporate to dryness, dissolve the residue in 50% methanol, transfer to a 5mL volumetric flask, and obtain the final product. Accurately pipette 3μl of each test solution and inject it into an ultra-high performance liquid chromatograph, determine the content according to the chromatographic conditions determined in Example 1, and calculate the content as shown in Table 7.

[0097] Table 7. Comparison of different extraction times

[0098]

[0099] Conclusion: The content was similar at different ultrasonic extraction times. The extraction time of 30 minutes was selected for the following experiments.

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

[0101] The preparation method of the test solution is as follows: Take an appropriate amount of Polygonatum odoratum formula granules, grind them into a fine powder, take about 1g, accurately weigh it, place it in a stoppered conical flask, accurately add 50mL of 80% methanol, seal tightly, weigh it, sonicate (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 well, filter, accurately measure 20ml of the filtrate, evaporate to dryness, add 50% methanol to dissolve the residue and transfer it to a 5ml volumetric flask to obtain the test solution.

[0102] Example 3: Methodological validation of the method for detecting the content of amide components in Polygonatum odoratum formulation 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-coumaroyl diotoluamide reference solution (21.4323 μg / ml) and N-trans-feruloyl diotoluamide reference solution (82.334232 μg / ml) respectively, and inject them into the ultra-high performance liquid chromatograph. Determine the chromatographic conditions as specified in Example 2. Plot a standard curve with peak area as the ordinate and injection volume as the abscissa, and obtain the regression equation. The results are shown in Tables 8 and 9. Figures 6-7 .

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

[0106]

[0107] Table 9. Regression Equation of Standard Curve for Standard Products

[0108]

[0109] Conclusions: For N-trans-coumaroyl dimethoxylamine, a good linear relationship was observed between the injection amount and peak area within the range of 0.00429 μg to 0.06430 μg. Similarly, for N-trans-feruloyl dimethoxylamine, a good linear relationship was observed between the injection amount and peak area within the range of 0.01647 μg to 0.24700 μg.

[0110] 2. Precision

[0111] 2.1 Instrument Precision

[0112] Take the Polygonatum odoratum formula granules (batch number: 19060159) and prepare the test solution according to the test solution preparation method in Example 2. Accurately pipette 3 μL and inject it into the ultra-high performance liquid chromatograph. Measure it according to the chromatographic conditions in Example 1. Inject the sample 6 times consecutively, 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 instrument's precision test was satisfactory.

[0117] 2.2 Intermediate Precision

[0118] Three portions of Polygonatum odoratum formula granules (batch number: 19060159) were taken and prepared by two experimenters according to the test solution preparation method in Example 2. 3 μl of each sample was injected at different times on different instruments, the peak area was measured, 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 six parallel portions of Polygonatum odoratum formula granules (batch number: 19060159), and prepare test solutions according to the test solution preparation method in Example 2. Inject 3 μl of each solution, measure its area value, calculate its content and RSD. The results are shown in Table 12.

[0124] Table 12. Results of repeatability tests on samples

[0125]

[0126]

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

[0128] 3. Accuracy (spiking recovery test)

[0129] Nine portions of 0.5 g of Polygonatum odoratum granules with known content (batch number: 19060159, N-trans-p-coumaryl ditocopherol amine content 0.01952 mg / g, N-trans-feruloyl ditocopherol amine content 0.09045 mg / g) were accurately weighed. Reference solutions of corresponding concentrations (high, medium, and low) were added to each portion. The sample recovery solution was prepared according to the method described in Example 2. 3 μL of each solution was injected under the chromatographic conditions described in Example 1. The recovery rate and RSD were calculated using the following formulas. The results are shown in Tables 13-14.

[0130]

[0131] Table 13. Results of the recovery test of N-trans-p-coumaroyl methylamine

[0132]

[0133]

[0134] Table 14. Results of the N-trans-feruloyl eugenol recovery test

[0135]

[0136] Conclusion: The recoveries of N-trans-coumaroyl eutodextrin and N-trans-feruloyl eutodextrin met the requirements (the recovery rate should be 80%–115% when the content of the analyte is below 10 mg / g and 85%–110% when the content of the analyte is above 0.1 mg / g), and the accuracy test was good.

[0137] 4. Exclusivity

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

[0139] Conclusion: The absence of chromatographic peaks at the corresponding retention times of the reference standard in the negative chromatogram indicates that the solvent does not interfere with the determination of amide components in Polygonatum odoratum granules, and this method is specific for determining the content of amide components in Polygonatum odoratum granules.

[0140] 5. Delay

[0141] Take the Polygonatum odoratum formula granules and prepare the test solution according to the preparation method of the test solution determined in Example 2. Inject and analyze the solution under the chromatographic conditions obtained in Example 1. Under the same chromatographic conditions, maintain the elution gradient with the highest acetonitrile ratio, and double the elution time. Record the chromatogram. The results are shown in [Figure 1]. Figure 9 .

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

[0143] 6. Durability

[0144] 6.1 Stability Test

[0145] The test solution was prepared by taking the Polygonatum odoratum formula granules and preparing the test solution according to the preparation method of the test solution in Example 2. The test solution was measured at 0h, 4h, 8h, 12h, 16h and 24h respectively according to the method of Example 1. The changes in peak area were recorded and the RSD was calculated. The results are shown in Table 15.

[0146] Table 15. Results of Stability Tests

[0147]

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

[0149] Example 4: Determination of amide content in Polygonatum odoratum formulation granules

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

[0151] Preparation of reference solution: Take appropriate amounts of N-trans-coumaroyl eurotoxin reference standard and N-trans-feruloyl eurotoxin, accurately weigh them, and add 50% methanol to prepare solutions containing 5 μg of N-trans-coumaroyl eurotoxin and 50 μg of N-trans-feruloyl eurotoxin per 1 ml.

[0152] Preparation of the test solution: Take 10 batches of Polygonatum odoratum formula granules, crush them, take about 1g of powder, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of 80% methanol, weigh it, sonicate (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 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, and the test solution is obtained.

[0153] Determination method: Accurately pipette 1 μl of the reference solution and 3 μl of the test solution into the ultra-high performance liquid chromatograph and determine the content of amide components in the Polygonatum odoratum formula granules as the total amount of N-trans-coumaroyl eugenolamine and N-trans-feruloyl eugenolamine.

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

[0155]

[0156] The average content of amide components in 10 batches of Polygonatum odoratum formula granules was 0.103 mg / g. Calculated by lowering the average by 30%, this is 0.07 mg / g. When the content of amide components in Polygonatum odoratum formula granules is higher than 0.07 mg / g, it indicates that the product quality is good. Using this as an example, this method was applied to the quality testing and quality control of different batches of Polygonatum odoratum formula granules.

Claims

1. A method for detecting the content of amide components in Polygonatum odoratum formula granules, characterized in that, Includes the following steps: (1) Preparation of reference solution: Take N-trans-coumaroyl eutodamine reference standard and N-trans-feruloyl eutodamine, add solvents respectively to prepare reference solution; (2) Preparation of test solution: Take the powder of Polygonatum odoratum formula, add 70%~100% methanol, weigh, 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, make up to volume, and the test solution is obtained. (3) Inject the reference solution and the test solution into the ultra-high performance liquid chromatograph respectively; the chromatographic column of the ultra-high performance liquid chromatograph is ZORBAX Eclipse Plus C18, ACQUITY UPLC HSS T3 or ZORBAX SB-C18; the mobile phase is acetonitrile-0.1% phosphoric acid with a volume ratio of 13:87, the elution method is isocratic elution, and the detection wavelength is 280-330nm; (4) The regression equation for the injection amount and peak area of ​​coumaroyl eugenolamine using the N-trans method is Y = 13011931.2709X - 12672.5495, R 2 =1.0000, calculate the content of N-trans-p-coumaroyl eugenol in the Polygonatum odoratum formulation granules; use the regression equation Y=11739481.8188X-38047.2301 for the injection amount and peak area of ​​N-trans-feruloyl eugenol, R 2 =1.0000, calculate the content of N-trans-feruloyl eugenolamine in the Polygonatum odoratum formula granules; the content of the amide component is the sum of the content of N-trans-coumaroyl eugenolamine and the content of N-trans-feruloyl eugenolamine.

2. The method according to claim 1, characterized in that, The extraction methods described in step (2) include ultrasound, shaking, or heating and reflux.

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

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

5. The method according to claim 1, characterized in that, The flow rate of the ultra-high performance liquid chromatograph mentioned in step (3) is 0.25~0.35mL / min; the column temperature is 20~30°C.

6. The application of the method according to any one of claims 1 to 5 in the quality testing of Polygonatum odoratum formula granules.

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