A method for quality detection or quality control of a polygonatum officinale formula granule

Abstract

The application discloses a quality detection or quality control method of polygonatum officinale formula granules, which comprises the following steps: preparation of a test sample solution, preparation of a control sample solution, liquid chromatography detection and data analysis, wherein the data analysis comprises calculating the content of (3R)-5,7-dihydroxy-6-methyl-3-(4'-hydroxybenzyl) chroman-4-one in the polygonatum officinale formula granule sample. The detection method is fast and accurate, has no interference of negative samples, is strong in specificity, has good reproducibility and stability, is low in detection cost and high in detection efficiency, and can provide a basis for internal quality monitoring, control and quality evaluation of the polygonatum officinale formula granules.

Description

TECHNICAL FIELD

[0001] The present application relates to a quality detection or quality control method of polygonatum formula granules, belonging to the technical field of drug detection. BACKGROUND

[0002] Polygonatum is the dry rhizome of Polygonatum odoratum (Mill.) Druce of Liliaceae, a commonly used traditional Chinese medicine, with mild cold nature and sweet taste, and has the effects of nourishing yin and moistening dryness, generating fluid and relieving thirst. It is used to treat lung and stomach yin damage, dry heat cough, dry throat and thirst, internal heat and polydipsia, and has a strong clinical application value. In addition to medicinal use, polygonatum has a wide market demand as health products, food, etc., and has export.

[0003] Polygonatum contains various chemical components, mainly including steroidal saponins, high isoflavones, volatile oils and polysaccharides. The flavonoids in polygonatum are one of the main components of its pharmacological effects. Flavonoids are a natural antioxidant with anti-aging and immune-enhancing effects. Some literature (Zhou X, Zhang Y, Zhao H, et al. Antioxidant homoisoflavonoids from Polygonatum odoratum [J]. Food Chemistry, 2015, 186: 63-68.) reported that three new homoisoflavones and eight homoisoflavones isolated from polygonatum showed strong antioxidant activity, and the compounds with dihydroxylated B ring showed stronger antioxidant activity than VC.

[0004] In recent years, the quality detection method of polygonatum mainly focuses on sugar components. The 2020 edition of Chinese Pharmacopoeia adopts colorimetric method to determine the polysaccharide content in polygonatum as a quality control method. However, this method is complicated and has poor accuracy. In addition, the addition of auxiliary materials in the production process of polygonatum formula granules will interfere with the colorimetric determination of polysaccharides, so the polysaccharide index is not suitable for the quality control of polygonatum formula granules. Some reports also measure monosaccharides such as fructose and glucose as the quality control method of polygonatum, but monosaccharides have poor specificity and exist in many traditional Chinese medicines. For flavonoid components, efficient and green extraction and separation technology and detection technology need to be further developed and improved, thereby providing a solid foundation for the further development and utilization of polygonatum and its quality control. SUMMARY

[0005] The technical problem solved by the present application is to provide a method for quickly and accurately detecting or controlling the quality of polygonatum officinale formula granules.

[0006] The technical scheme is that the present application provides a method for detecting the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one in polygonatum officinale formula granules, comprising the following steps:

[0007] (1) Take polygonatum officinale formula granules powder, dissolve it with an extraction solvent, weigh it, extract it, cool it, weigh it again, add methanol to make up for the weight loss, shake it, filter it, precisely take the filtrate, evaporate it, add 50% methanol to the residue to dissolve it, and obtain a test sample solution;

[0008] (2) Take (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one, add a solvent to prepare a control sample solution;

[0009] (3) Take the test sample solution and the control sample solution, respectively inject them into an ultra-high performance liquid chromatograph, compare the detection spectrum of the test sample solution with that of the control sample solution, and calculate the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one in the test sample according to the peak area normalization method.

[0010] In step (1), the extraction solvent includes 30%-100% methanol.

[0011] Preferably, in step (1), the extraction solvent includes 50%-100% methanol.

[0012] In step (1), the volume of the extraction solvent is 20-100ml.

[0013] Preferably, in step (1), the volume of the extraction solvent is 25-100ml.

[0014] Preferably, the volume percentage of alcohol in the aqueous alcohol solution is 100%.

[0015] The ratio of the use amount of the polygonatum officinale formula granule powder to the extraction solvent is 1g:20-100ml, preferably 1g:25ml.

[0016] In step (1), the extraction method includes ultrasonic extraction or heating reflux; the extraction time is 15-60 minutes.

[0017] Preferably, the extraction time in step (1) is 30-60 minutes.

[0018] Preferably, the extraction method is ultrasonic extraction.

[0019] Preferably, the extraction time is 15-30 minutes, more preferably 30 minutes.

[0020] The chromatographic conditions of the ultra-high performance liquid chromatography in step (3) are as follows: the mobile phase is acetonitrile:0.1% phosphoric acid = 45:55, acetonitrile:0.1% formic acid = 45:55 or acetonitrile:water = 40-60:40-60; the chromatographic column is ZORBAX SB-Aq, YMC-Triart-C18 or Shim-pack GIST C18-AQ; the elution mode is isocratic elution: the flow rate is 0.8-1.2 mL / min; the column temperature is 20-30℃; and the detection wavelength is 260nm-330nm.

[0021] Preferably, the volume ratio of the mobile phase is 45:55 of the acetonitrile-water binary system, the detection wavelength is 292nm, the flow rate is 1mL / min, and the column temperature is 25℃.

[0022] The linear relationship between the peak area of the chromatographic peak in step (3) and the injection amount of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one is Y=-0.0157+58.186X, R=0.99992, wherein X is the injection amount of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one, and Y is the peak area of the chromatographic peak; the injection amount of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one is 0.0100μg-0.0501μg.

[0023] The application also provides a method for recovering (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one in polygonatum officinale formula granules, comprising the following steps:

[0024] (1) Take polygonatum officinale formula granule powder, dissolve with an extraction solvent, weigh, extract, cool, weigh again, supplement the lost weight with methanol, shake well, filter, precisely pipette the filtrate, evaporate to dryness, add 50% methanol to dissolve, and obtain the test sample solution;

[0025] (2) Inject the test solution into the high performance liquid chromatograph, and (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one can be recovered.

[0026] The application further provides application of the method in quality detection and / or quality control of the polygonatum officinale formula granules.

[0027] The method provided by the application takes (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one as an index, adopts high performance liquid chromatography to detect the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one, and detects and monitors the internal quality of the polygonatum officinale formula granules.

[0028] Advantages: Compared with the prior art, the application has the following remarkable advantages: 1. The detection method of the polygonatum officinale formula granules provided by the application is fast and accurate in analysis, has no interference of negative samples, has strong specificity, good reproducibility and stability, has a smooth baseline, good separation degree and good chromatographic peak type of the obtained chromatogram, has low detection cost and high detection efficiency, and can accurately detect the content of isoflavone components in the polygonatum officinale sample; 2. The application provides a set of fast, efficient and feasible detection method for quality control of the polygonatum officinale formula granules; 3. The application can provide a basis for internal quality monitoring, control and quality evaluation of the polygonatum officinale formula granules. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 UV spectrum of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one reference substance under the condition of 190nm-400nm wavelength;

[0030] Figure 2 UPLC spectrum of the polygonatum officinale formula granule test sample under the condition of the mobile phase system and proportion;

[0031] Figure 3 UPLC spectrum of the polygonatum officinale formula granule test sample under the condition of different extraction solvents;

[0032] Figure 4 Linear relationship diagram of the reference substance;

[0033] Figure 5 Specificity test of the polygonatum officinale formula granule test sample;

[0034] Figure 6The delay test of the test sample of the Polygonatum multiflorum formula granules;

[0035] Figure 7 The UPLC chromatogram of the test sample of the Polygonatum multiflorum formula granules under different flow rate investigation conditions;

[0036] Figure 8 The UPLC chromatogram of the test sample of the Polygonatum multiflorum formula granules under different column temperature investigation conditions;

[0037] Figure 9 The UPLC chromatogram of the test sample of the Polygonatum multiflorum formula granules under different chromatographic column investigation conditions. DETAILED DESCRIPTION

[0038] The technical solutions of the present application are further described below in combination with the drawings.

[0039] 1. Instruments, reagents and samples

[0040] (1) Instruments: Thermo Vanquish high performance liquid chromatograph; chameleon 7.2 work station (Thermo Corporation), Agilent 1260 high performance liquid chromatograph, 1260TCC detector, 1260ALS automatic sampler, 1260Quat pump VL four-element pump, ChromeLeon chromatographic work station, KQ-250E ultrasonic cleaner (Kunshan ultrasonic instrument Co., Ltd.); ME204E / 02 type electronic analytical balance (Mettler-Toledo instrument (Shanghai) Co., Ltd.); METTLER TOLEDO XP6 millionth balance, GKC114 type temperature-controlled water bath (Nantong Huatai experimental instrument Co., Ltd.); HY-4 oscillator (Jintan City Ke Xing instrument factory); TGL-16C centrifuge (Shanghai Anting scientific instrument factory); Milli-Q water purification system (Millipore Corporation).

[0041] (2) Reagents: acetonitrile (chromatographically pure, Thermo Fisher Corporation); water is ultrapure water; other reagents are all analytical pure.

[0042] (3) Samples:

[0043] Reference substance: (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one (batch number: 114008) purchased from Shanghai Shidandeng Biotechnology Co., Ltd., for content determination, the content is 98%, and no treatment is required before use.

[0044] Preparation of reference solution: accurately weigh (3R)-5,7-Dihydroxy-6-methyl-3-(4'- hydroxybenzyl) chroman-4-one, add 50% methanol to make a solution with a concentration of 1 μg / mL, and the reference solution is obtained.

[0045] Test sample: Polygonatum multiflorum formula granules (batch numbers 19060139, 19060149, 19060159) were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0046] Determination of chromatographic conditions in Example 1

[0047] 1. Determination of detection wavelength

[0048] The UV absorption spectrum of the (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl) chroman-4-one reference substance was collected at 210-400 nm, and the results are shown in Figure 1 The (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl) chroman-4-one reference substance has a higher absorption between 260 nm and 330 nm in the UV absorption spectrum, which can be used as the detection wavelength. Considering that there is a maximum absorption around 292 nm, the detection wavelength is preferably 292 nm.

[0049] 2. Selection of mobile phase

[0050] Take the Polysaccharide of Polygonatum Officinale Formula Granules (batch number: 19060139), an appropriate amount, finely grind, take about 1 g, 3 groups in total, 2 replicates in each group, accurately weigh, place in a conical bottle with a stopper, accurately add 25 ml of methanol, tightly seal, weigh, ultrasonic treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, re-weigh, make up the weight loss with methanol, shake well, filter, accurately take 20 ml of the filtrate, evaporate to dryness, add 50% methanol to dissolve, transfer to a 5 ml volumetric flask, and the test sample solution is obtained. Accurately take 20 μl of each test solution and inject it into the high performance liquid chromatograph. The chromatographic conditions of this example are as follows: YMC-Triart-C18 chromatographic column (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); isocratic elution is used for elution; the flow rate is 1 mL / min; the column temperature is 30°C; and the detection wavelength is 292 nm. The theoretical plate number calculated according to the (3R)-5,7-dihydroxy-6-methyl-3-(4'-hydroxybenzyl) chroman-4-one peak should not be less than 3000. According to the existing experimental conditions, the ratio of the mobile phase is optimized (condition S1: acetonitrile: 0.1% phosphoric acid = 45:55; condition S2: acetonitrile: 0.1% formic acid = 45:55; condition S3: acetonitrile: water = 40:60; condition S4: acetonitrile: water = 45:55; condition S5: acetonitrile: water = 50:50; condition S6: acetonitrile: water = 55:45; condition S7: acetonitrile: water = 60:40), which is shown in Table 1. Figure 2 As can be seen from Figure 2 , under the above conditions, the target components can all obtain acceptable separation. The water phase has little difference with formic acid, phosphoric acid and pure water, and from the economic and environmental protection point of view, the acetonitrile-water mobile phase system is used. The acetonitrile proportion in condition S3 is low, and the detection time is long, so from the time cost point of view, the acetonitrile-water ratio is preferably conditions S4-S7, which can save about 10 minutes compared with condition S3. The following experiment uses acetonitrile-water with a volume ratio of 45:55 as the mobile phase.

[0051] Preparation of the test sample solution of Example 2

[0052] 1. Investigation of extraction solvent

[0053] Take Polysaccharide Peony Formula Granules (batch number: 19060139) appropriate amount, grind finely, take about 1 g, a total of 5 groups, 2 parallel each group, accurately weighed, placed in a conical flask with a stopper, respectively accurately add water, 30% methanol, 50% methanol, 70% methanol, methanol 25 ml, tightly sealed, weighed, ultrasonic treatment (power 600 W, frequency 40 kHz) 30 minutes, cool, reweigh, with the corresponding solvent to make up for the loss of weight, shake up, filter, accurately take 20 ml of the filtrate, evaporate to dryness, add 50% methanol to dissolve, transfer to a 5 ml volumetric flask, and get it. Respectively, accurately take 20 μl of each test solution, inject into high performance liquid chromatograph, determine according to the chromatographic conditions determined in Example 1, calculate the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl) chroman-4-one, the results are shown in Table 1, Figure 3 .

[0054] Table 1 Comparison of different extraction solvents

[0055]

[0056] Conclusion: From the results, the extraction rate is higher and the relative standard deviation is smaller, so methanol is selected as the extraction solvent.

[0057] 2. Extraction method investigation

[0058] Take the product Polysaccharide Peony Formula Granules (batch number: 19060139), appropriate amount, grind finely, take about 1 g, a total of 3 groups, 2 parallel each group, accurately weighed, placed in a conical flask with a stopper, accurately add methanol 25 ml, tightly sealed, weighed, respectively ultrasonic treatment (power 600 W, frequency 40 kHz), heating reflux, shaking extraction 30 minutes, cool, reweigh, with methanol to make up for the loss of weight, shake up, filter, accurately take 20 ml of the filtrate, evaporate to dryness, add 50% methanol to dissolve, transfer to a 5 ml volumetric flask, and get it. Respectively, accurately take 20 μl of each test solution, inject into high performance liquid chromatograph, determine according to the chromatographic conditions determined in Example 1, calculate the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl) chroman-4-one, the results are shown in Table 2.

[0059] Table 2 Comparison of different extraction methods

[0060]

[0061]

[0062] Conclusion: From the results, the efficiency of ultrasonic extraction and heating reflux extraction is similar, considering the ultrasonic treatment is easy to operate, therefore, ultrasonic treatment is selected as the extraction method.

[0063] 3. Extraction time investigation

[0064] Take YuZhu dispensing granules (batch number: 19060139), grind finely, take about 1 g, a total of 4 groups, 2 replicates in each group, accurately weigh, place in a conical flask with a plug, accurately add 25 ml of methanol, seal tightly, weigh, ultrasonic treatment (power 600 W, frequency 40 kHz) for 15 minutes, 45 minutes, 30 minutes, 60 minutes respectively, cool, weigh again, make up the weight loss with methanol, shake well, filter, accurately take 20 ml of the filtrate, evaporate to dryness, add 50% methanol to dissolve, transfer to a 5 ml volumetric flask, and obtain. Accurately take 20 μl of each test solution, inject into the high performance liquid chromatograph, determine the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl) chroman-4-one according to the chromatographic conditions determined in Example 1, and the results are shown in Table 3.

[0065] Table 3 Comparison of different extraction times

[0066]

[0067]

[0068] Conclusion: From the results, the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl) chroman-4-one measured at different extraction times is similar. In order to ensure sufficient extraction, the extraction time is determined to be 30 minutes.

[0069] 4. Extraction solvent volume investigation

[0070] Take Polysaccharide Peony Root Granules (batch number: 19060139) of the right amount, grind finely, take about 1 g, 4 groups in total, 2 parallel in each group, accurately weigh and place in a conical flask with a stopper, accurately add 20 ml, 25 ml, 50 ml and 100 ml of methanol respectively, seal tightly, weigh, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, re-weigh, make up the weight loss with methanol, shake well, filter, accurately take 20 ml of the filtrate, evaporate to dryness, add 50% methanol to dissolve, transfer to a 5 ml volumetric flask, and obtain. Accurately take 20 μl of each test solution, inject into the high performance liquid chromatograph, determine according to the chromatographic conditions determined in Example 1, calculate the content of (3R)-5,7-dihydroxy-6-methyl-3-(4'-hydroxybenzyl) chroman-4-one, and the results are shown in Table 4.

[0071] Table 4 Comparison of different extraction solvent volumes

[0072]

[0073] Conclusion: From the results, it can be seen that the extraction rates of different extraction volumes are similar, and the content of (3R)-5,7-dihydroxy-6-methyl-3-(4'-hydroxybenzyl) chroman-4-one is similar, indicating that the extraction solvent volume is 20 mL, which is basically completely extracted. In order to ensure sufficient extraction, the extraction solvent volume is selected as 25 mL.

[0074] Based on the above results, the chromatographic conditions used in the following Example 3 of the present application are as follows: YMC-Triart-C18 chromatographic column (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile-water (45:55) as the mobile phase; isocratic elution; flow rate 1 mL / min; column temperature 30℃; detection wavelength 292 nm. The theoretical plate number calculated according to the (3R)-5,7-dihydroxy-6-methyl-3-(4'-hydroxybenzyl) chroman-4-one peak should not be less than 5000.

[0075] The preparation method of the test solution is as follows: take the product of the right amount, grind finely, take about 1 g, accurately weigh and place in a conical flask with a stopper, accurately add 25 ml of methanol, weigh, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, take out, cool, re-weigh, make up the weight loss with methanol, shake well, filter, accurately take 20 ml of the filtrate, evaporate to dryness, add 50% methanol to dissolve and transfer to a 5 ml volumetric flask, add 50% methanol to the mark, shake well, filter, take the filtrate, and obtain.

[0076] Example 3: Methodology verification of the determination method of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one in Polygonatum sibiricum Preparations

[0077] 1. Linearity

[0078] Take 5.0 μl, 10.0 μl, 15.0 μl, 20.0 μl, 25.0 μl of the control solution ((3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one concentration is 2.00312 μg / mL) respectively, inject into the high performance liquid chromatograph, and determine according to the above chromatographic conditions, take the peak area as the vertical coordinate and the injection amount as the horizontal coordinate, draw the standard curve, and obtain the regression equation: Y=-0.0157+58.186X, R=0.99992, the results are shown in Table 5 and Figure 4 .

[0079] Table 5 Relationship between peak area integral value of control and injection amount

[0080]

[0081] The results show that the injection amount of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one is in the range of 0.0100 μg-0.0501 μg, and the injection amount and the peak area value show good linear relationship.

[0082] 2. Precision

[0083] 2.1 Instrument precision

[0084] Take Polygonatum sibiricum Preparations (batch number: 19060139) to prepare the test solution according to the test solution preparation method determined in Example 2, precisely take 20 μL, inject into the high performance liquid chromatograph, determine according to the chromatographic conditions determined in Example 1, continuously inject 6 times, record the peak area measurement value, calculate the relative standard deviation, and the results are shown in Table 6.

[0085] Table 6 Instrument precision test

[0086]

[0087] The results show that the instrument precision test is good.

[0088] 2.2 Intermediate precision

[0089] Take YuZhu formula granules (batch number: 19060139), respectively by two experimenters according to the preparation method of test solution in example 2, 3 test solution was prepared, 20 μL was injected at different time, in different instruments, according to the chromatographic conditions determined in example 1, the peak area value of (3R)-5, 7-dihydroxy-6-methyl-3-(4'-hydroxybenzyl) chroman-4-one was determined, and the content and RSD were calculated, and the results were shown in table 7.

[0090] Table 7 intermediate precision test

[0091]

[0092] Results: the RSD of (3R)-5, 7-dihydroxy-6-methyl-3-(4'-hydroxybenzyl) chroman-4-one content measured by intermediate precision test was 0.92%, and the results were good.

[0093] 2.3 repeatability

[0094] Take YuZhu formula granules (batch number: 19060139) appropriate amount, grind, take about 1g, accurately weigh, parallel 6 times, respectively according to the preparation method of test solution in example 2, test solution was prepared, 20 μL was injected, according to the chromatographic conditions determined in example 1, the peak area value of (3R)-5, 7-dihydroxy-6-methyl-3-(4'-hydroxybenzyl) chroman-4-one was determined, and the content and RSD were calculated, and the results were shown in table 8.

[0095] Table 8 repeatability test results of samples

[0096]

[0097] Results: the RSD of (3R)-5, 7-dihydroxy-6-methyl-3-(4'-hydroxybenzyl) chroman-4-one content measured by repeatability test was 0.87%, and the results were good.

[0098] 3. accuracy (spiked recovery test)

[0099] Take the known content of the sample (Yu zhu formula granules, (3R)-5, 7-dihydroxy-6-methyl-3- (4'-hydroxybenzyl) chroman-4-one content is: 0.00831 mg / g) 0.5 g, a total of 9 parts, precision weighing, respectively, add high, medium and low three concentrations of control solution (the amount of adding standard in table 9), according to the preparation method of the sample solution determined by example 2 to make sample recovery, according to the chromatographic conditions of example 1, respectively, 20 μL, according to the following formula to calculate the recovery rate and RSD, the results are shown in table 9.

[0100]

[0101] Table 9 recovery test results

[0102]

[0103]

[0104] Results: the content of (3R)-5, 7-dihydroxy-6-methyl-3-(4'-hydroxybenzyl) chroman-4-one is between 93.26% and 101.66%, and the accuracy test is good.

[0105] 4. Specificity

[0106] The added excipients in Yu zhu formula granules are malt dextrin, silicon dioxide and magnesium stearate. This test investigates the influence of negative samples without Yu zhu on the determination of the content of (3R)-5, 7-dihydroxy-6-methyl-3-(4'-hydroxybenzyl) chroman-4-one in Yu zhu formula granules. Take the negative sample (excipient) without Yu zhu and Yu zhu formula granules respectively according to the preparation method of the sample solution determined by example 2 and according to the chromatographic conditions determined by example 1 to detect the results Figure 5 .

[0107] Results: there is no chromatographic peak in the negative chromatogram at the corresponding retention time of the control sample, which shows that the excipients and solvents have no interference with the determination of (3R)-5, 7-dihydroxy-6-methyl-3-(4'-hydroxybenzyl) chroman-4-one. The determination of the content of (3R)-5, 7-dihydroxy-6-methyl-3-(4'-hydroxybenzyl) chroman-4-one in Yu zhu formula granules has specificity.

[0108] 5. Delay

[0109] Take the Yu Zhu standard formula granules (batch number: 19060139), according to the preparation method of the test sample solution in Example 2 to prepare the test sample solution, and analyze the sample under the chromatographic conditions obtained in Example 1. Under the same chromatographic conditions, the elution gradient with the highest proportion of acetonitrile is maintained, the elution time is doubled, and the chromatogram is recorded. The results are shown in Figure 6 .

[0110] Results: No obvious chromatographic peaks flow out after the original gradient elution, indicating that the chromatographic conditions basically meet the principle of maximum information.

[0111] 6. Durability

[0112] 6.1 Stability test

[0113] Take the Yu Zhu standard formula granules (batch number: 19060139) and prepare the test sample solution according to the preparation method of the test sample solution in Example 2. Determine at 0 h, 4 h, 8 h, 12 h, 16 h, and 24 h according to the chromatographic conditions in Example 1, and record the change of the peak area of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one. Calculate the RSD, and the results are shown in Table 10.

[0114] Table 10 Determination results of stability test

[0115]

[0116] Results: From the above data, the RSD value of the peak area of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one within 24 hours is 1.08%, which meets the requirements of the system applicability test.

[0117] 6.2 Different flow rate investigation

[0118] Take the Yu Zhu standard formula granules (batch number: 19060139) and prepare the test sample solution according to the preparation method of the test sample solution in Example 2. Investigate the chromatographic peak separation effect under different flow rates (0.8 mL / min, 1.0 mL / min, 1.2 mL / min) according to the chromatographic conditions in Example 1. The experimental results are shown in Table 11 and Figure 7 .

[0119] Table 11 Investigation results of different flow rates

[0120]

[0121] Results: At different flow rates, the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'- hydroxybenzyl) chroman-4-one was similar, and the peak separation was good, indicating that the durability was good when the flow rate fluctuated within the range of 0.8 mL / min to 1.2 mL / min.

[0122] 6.3 Investigation of different column temperatures

[0123] Take Yu Zhu standard formula granules (batch number: 19060139) to prepare the test solution according to the preparation method of the test solution in Example 2, and investigate the chromatographic peak separation effect at different column temperatures (20°C, 25°C, 30°C), with other chromatographic conditions being the same as in Example 2. The experimental results are shown in Table 12 and Figure 8 .

[0124] Table 12 Investigation results of different column temperatures

[0125]

[0126] Results: At different column temperatures, the chromatographic peak separation effect was good, and the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl) chroman-4-one was similar, indicating that the durability was good when the column temperature fluctuated within the range of 20°C to 30°C.

[0127] 6.4 Investigation of different chromatographic columns

[0128] Take Yu Zhu standard formula granules (batch number: 19060139) to prepare the test solution according to the preparation method of the test solution in Example 2, and investigate the chromatographic peak separation effect at different column temperatures (20°C, 25°C, 30°C), with other chromatographic conditions being the same as in Example 2. The experimental results are shown in Table 12 and Figure 9 .

[0129] Table 13 Investigation results of different chromatographic columns

[0130]

[0131] Results: The separation of the three chromatographic columns was good, and the retention time was moderate, indicating that the chromatographic column had little effect on the determination results of the sample, and this method had universal applicability.

[0132] Example 4: Determination of the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'- hydroxybenzyl)chroman-4-one in Polygonatum Formulation Granules

[0133] Three batches of Polygonatum Formulation Granules were taken, sample solutions were prepared according to the preparation method of the sample solution of the test product determined in Example 2, reference solution was prepared according to Example 2, sample was analyzed according to the chromatographic conditions determined in Example 2, 20 μL of sample was injected, the peak area was recorded, the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one was calculated, and the specific results are shown in Table 14.

[0134] Table 14 Determination of the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'- hydroxybenzyl)chroman-4-one in different batches of Polygonatum Formulation Granules

[0135]

[0136] The results show that the present application can be used for the determination of the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl) in Polygonatum Formulation Granules, and provides technical support for the quality control of Polygonatum Formulation Granules.

Claims

1. A method for detecting (3) in Polygonatum odoratum formula granules R The method for determining the content of 5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one is characterized by, Includes the following steps: (1) Take the Polygonatum odoratum formula granules powder, dissolve it with the extraction solvent, weigh it, extract it, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, accurately pipette the filtrate, evaporate it to dryness, add 50% methanol to dissolve the residue, and you will get the test solution; the extraction solvent includes 30%-100% methanol. (2) Take (3) R )-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one, add solvent to prepare a reference solution; (3) Inject the test solution and the reference solution into the high-performance liquid chromatograph, respectively. Compare the detection chromatogram of the test solution with that of the reference solution, and calculate the concentration of (3) in the test solution using the peak area normalization method. R The content of 5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one; the mobile phase of the high-performance liquid chromatograph is acetonitrile:0.1% phosphoric acid = 45:55, acetonitrile:0.1% formic acid = 45:55 or acetonitrile:water = 40-60:40-60; the chromatographic column is ZORBAX SB-Aq, YMC-Triart-C18 or Shim-pack GIST C18-AQ; the elution method is isocratic elution; the detection wavelength is 292 nm.

2. The method according to claim 1, characterized in that, The volume of the extraction solvent in step (1) is 20-100 ml.

3. The method according to claim 1, characterized in that, The extraction method described in step (1) includes ultrasonic extraction or heating reflux; the extraction time is 15-60 minutes.

4. The method according to claim 1, characterized in that, The flow rate of the high performance liquid chromatograph in step (3) is 0.8-1.2 mL / min; the column temperature is 20-30 ℃.

5. The method according to claim 1, characterized in that, The peak area of ​​the chromatographic peak described in step (3) is related to (3) R The linear relationship of the injection amount of )-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one is: Y = -0.0157 + 58.186X, R = 0.99992, where X is (3 R The injection volume of )-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one, where Y is the peak area of ​​the chromatographic peak; the (3 R The injection volume of 5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one was 0.0100 µg to 0.0501 µg.

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

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