A method for detecting the fingerprint spectrum of Yangchao granules

Abstract

The application discloses a kind of Yangchao granules fingerprint detection methods, first the preparation method of Yangchao granules sample solution to be tested and chromatographic conditions are optimized, respectively precision draws different batches of sample solution to be tested and injects high performance liquid chromatograph, records chromatogram, then imports traditional Chinese medicine chromatographic fingerprint similarity evaluation system, by multiple point correction, with average method calculation generates Yangchao granules control fingerprint.The fingerprint of the sample to be tested Yangchao granules is compared with the control fingerprint of Yangchao granules, and the similarity is calculated.The Yangchao granules fingerprint detection method disclosed in the application can more comprehensively and effectively evaluate and control the quality of Yangchao granules, and the method is simple, stable and reproducible.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fingerprint spectrum analysis, and particularly relates to a detection method of a Yangchao granule fingerprint spectrum. BACKGROUND

[0002] The Yangchao granule is made of eight Chinese medicines, such as cuscuta, raspberry, pueraria, radix asparagi, cistanche, angelica, radix paeoniae alba and platycladus orientalis seed, and has the effects of benefiting kidney and filling essence and regulating yin and yang. The Yangchao granule is used for treating the kidney deficiency type of ovarian reserve function decline, and symptoms such as early or late menstruation, less menstruation, soreness of waist and knees, decreased libido, cold limbs, fatigue, frequent nocturia, dry mouth and throat, dizziness and tinnitus, hot flashes, insomnia and dreaminess, pale or red tongue, and slow or fine pulse.

[0003] In the current quality standard of the Yangchao granule, thin layer chromatography is used to identify three medicinal materials and main components, and high performance liquid chromatography is used to determine the content of puerarin. The complexity of multiple components of traditional Chinese medicine determines that a single component cannot accurately express the internal quality of traditional Chinese medicine. The fingerprint spectrum can comprehensively reflect the components contained in traditional Chinese medicine, and can represent the quality of traditional Chinese medicine products as a whole.

[0004] Therefore, in order to make the detection means of the preparation more rigorous and scientific, the detection method of the Yangchao granule fingerprint spectrum is provided, which can more comprehensively and effectively control the quality of the Yangchao granule, and provide a scientific basis for further exploring and researching the quality of the preparation. SUMMARY

[0005] The purpose of the application is to provide a detection method of a Yangchao granule fingerprint spectrum aiming at the deficiencies of the prior art.

[0006] The purpose of the application is achieved by the following technical solutions.

[0007] A detection method of a Yangchao granule fingerprint spectrum, specifically comprising the following steps:

[0008] (1) preparing a to-be-detected product solution, and the preparation process of the to-be-detected product solution is as follows: taking the Yangchao granule sample, grinding, accurately weighing 0.5 g, placing in a conical flask with a plug, accurately adding 25 mL of 50vol% methanol, weighing, ultrasonic or heating reflux treatment for 30 min, cooling, supplementing the weight loss with 50vol% methanol, shaking, centrifuging, and taking the supernatant, to obtain the to-be-detected product solution;

[0009] (2) preparing a reference solution: the reference solution is 50vol% methanol containing 80ug of paeoniflorin per 1mL;

[0010] (3) using high performance liquid chromatography, with concentration 0.1vol% phosphoric acid solution A, acetonitrile B as mobile phase, respectively, obtain the high performance liquid fingerprint of the product solution and the reference solution; the chromatographic conditions are as follows: using Agilent ZORBAX SB-C 18 The linear gradient elution procedure is as follows: 0-30 min, the volume fraction of A in the mobile phase is 96%-85%, and the volume fraction of B is 4%-15%; 30-50 min, the volume fraction of A in the mobile phase is 85%-80%, and the volume fraction of B is 15%-20%; 50-60 min, the volume fraction of A in the mobile phase is 80%-55%, and the volume fraction of B is 20%-45%; 60-65 min, the volume fraction of A in the mobile phase is 55%, and the volume fraction of B is 45%; the flow rate of the mobile phase is 1.0 mL / min; the column temperature is 30 DEG C; the injection amount is 10 mu L; and the detection wavelength is 240 nm; the obtained fingerprint of the reference solution and the product solution are introduced into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, Mark peak matching is carried out after multi-point correction, the fingerprint of the product solution is obtained, and the fingerprint includes 13 common peaks; through comparison with the control product, 5 index components are identified, and the 5 index components are as follows: No. 5 peak is puerarin, No. 9 peak is paeoniflorin, No. 11 peak is trilobatin, No. 12 peak is ellagic acid, and No. 13 peak is verbascoside.

[0011] A quality detection method for Yangchao granules, specifically comprising the following steps:

[0012] The fingerprint of the product solution is obtained by using the Yangchao granule fingerprint detection method, the high performance liquid chromatography fingerprint of the product is introduced into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system together with a control fingerprint, similarity is calculated, and samples with similarity greater than or equal to 0.95 are evaluated as products with stable quality; wherein the control fingerprint is generated by introducing product solutions of multiple batches into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, carrying out Mark peak matching after multi-point correction, and using an average method.

[0013] Further, the time window used by the average method is 0.1 min.

[0014] The method provides a simple and effective method for internal detection of the Yangchao granules, and the effectiveness, stability and uniformity of the quality of the Yangchao granules can be scientifically evaluated. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The chromatograms of Yangchao granule samples under different wavelengths;

[0016] Figure 2Chromatograms of the Nourishing the Nests Granules sample under different mobile phase conditions;

[0017] Figure 3 Chromatograms of the Nourishing the Nests Granules sample under different gradient elution procedure conditions;

[0018] Figure 4 Chromatograms of the Nourishing the Nests Granules sample under different extraction methods;

[0019] Figure 5 Chromatograms of the Nourishing the Nests Granules sample under different extraction solvent conditions;

[0020] Figure 6 Chromatograms of the Nourishing the Nests Granules sample under different extraction time conditions;

[0021] Figure 7 Fingerprint chromatograms of the same sample solution obtained by continuous sampling 6 times;

[0022] Figure 8 Fingerprint chromatograms of the same batch of sample solution obtained by repeating 6 times;

[0023] Figure 9 Fingerprint chromatograms of the same sample solution after being placed for different time;

[0024] Figure 10 Fingerprint chromatograms of 11 batches of the Nourishing the Nests Granules measured by the present application;

[0025] Figure 11 Control fingerprint chromatogram of the Nourishing the Nests Granules measured by the present application. DETAILED DESCRIPTION

[0026] The present application provides a fingerprint chromatographic detection method of the Nourishing the Nests Granules, specifically comprising the following steps:

[0027] (1) Prepare 10 or more batches of the sample solution, and the preparation process of each batch of the sample solution is as follows: take the Nourishing the Nests Granules sample, grind it finely, take about 0.5 g, accurately weigh it, place it in a conical flask with a plug, accurately add 25 mL of 50vol% methanol, weigh it, ultrasonically treat it for 30 min, cool it, add 50vol% methanol to make up the weight loss, shake it uniformly, centrifuge it, and take the supernatant, to obtain the sample solution of the batch.

[0028] (2) Preparation of the reference solution: take the appropriate amount of paeoniflorin reference substance, add 50vol% methanol to prepare a solution containing 80 μg of paeoniflorin per 1 mL, to obtain the reference solution.

[0029] (3) High performance liquid chromatography was used to obtain the high performance liquid fingerprint of the solution of all batches of the product to be tested and the reference solution, with 0.1 vol% phosphoric acid solution A and acetonitrile B as the mobile phase; the chromatographic conditions were as follows: an Agilent ZORBAX SB-C 18 chromatographic column, the length of the chromatographic column was 250 mm, the inner diameter of the chromatographic column was 4.6 mm, and the particle size of the filler was 5 μm; the linear gradient elution program was as follows: within 0-30 min, the volume fraction of A in the mobile phase was 96%-85%, and the volume fraction of B was 4%-15%; within 30-50 min, the volume fraction of A in the mobile phase was 85%-80%, and the volume fraction of B was 15%-20%; within 50-60 min, the volume fraction of A in the mobile phase was 80%-55%, and the volume fraction of B was 20%-45%; within 60-65 min, the volume fraction of A in the mobile phase was 55%, and the volume fraction of B was 45%; the flow rate of the mobile phase was 1.0 mL / min; the column temperature was 30°C; the injection volume was 10 μL; and the detection wavelength was 240 nm.

[0030] Mass stability evaluation: the above-obtained fingerprint of the reference solution and the fingerprint of all batches of the product to be tested were introduced into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, Mark peak matching was performed after multi-point correction, an average method was used to generate a control fingerprint, the obtained fingerprint of the product to be tested and the control fingerprint were introduced into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, Mark peak matching was performed after multi-point correction, the fingerprint of the product to be tested was obtained, the similarity was calculated, and the sample with a similarity ≥0.95 was evaluated as a product with stable quality. In the average method, the time window was 0.1 min.

[0031] The application will be further described below in combination with specific examples and the accompanying drawings. The reagents and instruments used in the following examples are as follows, but are not limited thereto:

[0032] 1. Reagents

[0033] Methanol, acetonitrile (chromatographic pure, Tedia, USA), water was ultrapure water, methanol, ethanol (analytically pure, Chengdu Kolon Chemical Co., Ltd.).

[0034] Paeoniflorin (batch number 110736-202145) was obtained from the China Institute for Food and Drug Control.

[0035] Eleven batches of Yangchao granules were provided by Hangzhou Hospital of Traditional Chinese Medicine, and the batch numbers were 20210120, 20210121A, 20210121B, 20210401, 20210403, 202103241, 202103242, 202103243, 220531, 220601, 220602.

[0036] 2. Instrument

[0037] METTLER-TOLEDO ME204E electronic balance (METTLER, Switzerland); Agilent 1260 high performance liquid chromatograph (Agilent, USA).

[0038] Example 1: Selection of detection wavelength

[0039] A method for detecting the fingerprint of Yangchao granules, specifically comprising the following steps:

[0040] (1) Preparation of the test product solution, the preparation process of the test product solution is as follows: take Yangchao granule sample, grind finely, take about 0.5 g, accurately weigh, place in a conical flask with a plug, accurately add 50 vol% methanol 25 mL, weigh, ultrasonic treat for 30 min, cool, make up the weight loss with 50 vol% methanol, shake well, centrifuge, take the supernatant, and the test product solution is obtained.

[0041] (2) Preparation of reference solution: take appropriate amount of paeoniflorin reference substance, add 50 vol% methanol to prepare a solution containing 80 μg paeoniflorin per 1 mL, and the reference solution is obtained.

[0042] (3) High performance liquid chromatography is used to obtain the high performance liquid fingerprint of the test product solution and the reference solution, with 0.1 vol% phosphoric acid solution A and acetonitrile B as the mobile phase; the chromatographic conditions are as follows: Agilent ZORBAX SB-C 18 chromatographic column is used, the length of the chromatographic column is 250 mm, the inner diameter of the chromatographic column is 4.6 mm, and the particle size of the filler is 5 μm; the linear gradient elution program is as follows: within 0-30 min, the volume fraction of A in the mobile phase is 96%-85%, and the volume fraction of B is 4%-15%; within 30-50 min, the volume fraction of A in the mobile phase is 85%-80%, and the volume fraction of B is 15%-20%; within 50-60 min, the volume fraction of A in the mobile phase is 80%-55%, and the volume fraction of B is 20%-45%; within 60-65 min, the volume fraction of A in the mobile phase is 55%, and the volume fraction of B is 45%; the flow rate of the mobile phase is 1.0 mL / min; the column temperature is 30℃; and the injection volume is 10 μL;

[0043] Considering that Yangchao granules are complex compound preparations, the selection of the detection wavelength should take into account various chemical components, and the chromatograms of Yangchao granule samples at 190, 240, 280, 320, and 360 nm wavelengths are investigated through multi-wavelength scanning and comparative analysis, and the results are as follows Figure 1As shown in the chromatogram, the apparent abundance of the chromatographic peak at 190 nm and 240 nm wavelengths is high, but the baseline drifts at 190 nm wavelength, and the impurity peak interferes more, affecting the resolution. Therefore, 240 nm is selected as the detection wavelength of Yangchao Granules high performance liquid chromatography fingerprint.

[0044] Example 2: Selection of mobile phase system

[0045] This example compares methanol-water, methanol-0.1vol% phosphoric acid, acetonitrile-0.1vol% phosphoric acid solution three mobile phase systems, other detection steps and conditions are the same as in Example 1, the obtained chromatogram is shown in Figure 2 As shown in the chromatogram, the apparent abundance of the chromatographic peak at 190 nm and 240 nm wavelengths is high, but the baseline drifts at 190 nm wavelength, and the impurity peak interferes more, affecting the resolution. Therefore, 240 nm is selected as the detection wavelength of Yangchao Granules high performance liquid chromatography fingerprint.

[0046] Example 3: Selection of mobile phase gradient

[0047] This example compares multiple gradient elution conditions, other detection steps and conditions are the same as in Example 1, wherein Agilent ZORBAX SB-C 18 (250mm×4.6mm, 5μm) chromatographic column; flow rate: 1.0mL / min; detection wavelength: 240nm; column temperature: 30℃; injection volume: 10μL; four linear gradients (gradient 1- gradient 4) conditions are as follows:

[0048] Gradient 1-Gradient 4 with 0.1vol% phosphoric acid solution A, acetonitrile B as mobile phase;

[0049] Gradient 1:

[0050] 0-20min: the volume fraction of A in the mobile phase is 95%-88%;

[0051] 20-40min: the volume fraction of A in the mobile phase is 88%-85%;

[0052] 40-60min: the volume fraction of A in the mobile phase is 85%-75%;

[0053] 60-70min: the volume fraction of A in the mobile phase is 75%-40%;

[0054] Gradient 2:

[0055] 0-20min: the volume fraction of A in the mobile phase is 95%-88%;

[0056] 20-40min: the volume fraction of A in the mobile phase is 88%-82%;

[0057] 40-50min: the volume fraction of A in the mobile phase is 82%-80%;

[0058] 50-65 min: 80-60% volume fraction of A in the mobile phase;

[0059] 65-70 min: 60% volume fraction of A in the mobile phase;

[0060] Gradient 3:

[0061] 0-30 min: 94-85% volume fraction of A in the mobile phase;

[0062] 30-50 min: 85-80% volume fraction of A in the mobile phase;

[0063] 50-60 min: 80-55% volume fraction of A in the mobile phase;

[0064] 60-65 min: 55% volume fraction of A in the mobile phase;

[0065] Gradient 4:

[0066] 0-30 min: 96-85% volume fraction of A in the mobile phase;

[0067] 30-55 min: 85-80% volume fraction of A in the mobile phase;

[0068] 55-60 min: 80-55% volume fraction of A in the mobile phase;

[0069] 60-65 min: 55% volume fraction of A in the mobile phase;

[0070] The chromatograms obtained under the conditions of Gradients 1-4 are shown in Figure 3 It can be seen from the chromatograms that the peaks are better separated and more spectral peaks are obtained under Gradient 4, which can provide more information. Therefore, Gradient 4 is selected as the chromatographic condition for the fingerprint analysis of Yangchao granules.

[0071] Example 5: Selection of extraction method

[0072] The same batch of Yangchao granule samples were extracted by the following methods:

[0073] Method (1): The Yangchao granule sample was finely ground, about 0.5 g was accurately weighed and placed in a conical flask with a stopper, 25 mL of 50 vol% methanol was accurately added, weighed, and heated to reflux extraction for 30 minutes.

[0074] Method (2): The Yangchao granule sample was finely ground, about 0.5 g was accurately weighed and placed in a conical flask with a stopper, 25 mL of 50 vol% methanol was accurately added, weighed, and ultrasonically extracted for 30 minutes.

[0075] The sample solution was cooled, made up to the weight loss with 50 vol% methanol, shaken, centrifuged, and the supernatant was injected into the high performance liquid chromatograph. Other detection steps and conditions were the same as in Example 1, and the chromatogram obtained is shown in Figure 4 As a result, there was no significant difference between the two extraction methods of heating reflux for 30 minutes and ultrasonic extraction for 30 minutes, and the ultrasonic extraction method was finally selected due to its simple operation.

[0076] Example 6: Selection of extraction solvent

[0077] The same batch of Yangchao granules was taken and extracted by the following method:

[0078] Method (1): The Yangchao granule sample was finely ground, about 0.5 g was accurately weighed and placed in a conical flask with a stopper, 25 mL of methanol was accurately added, weighed, ultrasonic treated for 30 minutes, cooled, made up to the weight loss with methanol, shaken, centrifuged, and the supernatant was obtained;

[0079] Method (2): The Yangchao granule sample was finely ground, about 0.5 g was accurately weighed and placed in a conical flask with a stopper, 25 mL of ethanol was accurately added, weighed, ultrasonic treated for 30 minutes, cooled, made up to the weight loss with ethanol, shaken, centrifuged, and the supernatant was obtained;

[0080] Method (3): The Yangchao granule sample was finely ground, about 0.5 g was accurately weighed and placed in a conical flask with a stopper, 25 mL of 50 vol% methanol aqueous solution was accurately added, weighed, ultrasonic treated for 30 minutes, cooled, made up to the weight loss with 50 vol% methanol, shaken, centrifuged, and the supernatant was obtained;

[0081] Method (4): The Yangchao granule sample was finely ground, about 0.5 g was accurately weighed and placed in a conical flask with a stopper, 50 mL of water was accurately added, weighed, ultrasonic treated for 30 minutes, cooled, made up to the weight loss with water, shaken, centrifuged, and the supernatant was obtained;

[0082] The sample solution was injected into the high performance liquid chromatograph, other detection steps and conditions were the same as in Example 1, and the chromatogram obtained is shown in Figure 5 As a result, the extraction effect and peak shape were better when 50 vol% methanol was used as the extraction solvent, and 50 vol% methanol was finally selected as the extraction solvent.

[0083] Example 7: Selection of extraction time

[0084] The same batch of Yangchao granules was taken and extracted by the following method:

[0085] Method (1): Take the Nangchao granules sample, grind finely, take about 0.5 g, accurately weigh, place in a conical flask with a stopper, accurately add 50 vol% methanol 25 mL, weigh, ultrasonic treat for 15 min;

[0086] Method (2): Take the Nangchao granules sample, grind finely, take about 0.5 g, accurately weigh, place in a conical flask with a stopper, accurately add 50 vol% methanol 25 mL, weigh, ultrasonic treat for 30 min;

[0087] Method (3): Take the Nangchao granules sample, grind finely, take about 0.5 g, accurately weigh, place in a conical flask with a stopper, accurately add 50 vol% methanol 25 mL, weigh, ultrasonic treat for 45 min;

[0088] The sample solution is cooled, the lost weight is made up with 50 vol% methanol, shaken, centrifuged, the supernatant is taken, injected into a high performance liquid chromatograph, other detection steps and conditions are the same as in Example 1, the chromatogram is recorded, and the obtained spectrum is shown in Figure 6 There is no obvious difference in the results under different extraction times, and the extraction time of 30 min is finally selected.

[0089] Example 8: Fingerprint spectrum methodological investigation

[0090] 1. Precision test

[0091] (1) Take the Nangchao granules sample, grind finely, take about 0.5 g, accurately weigh, place in a conical flask with a stopper, accurately add 50 vol% methanol 25 mL, weigh, ultrasonic treat for 30 min, cool, make up the lost weight with 50 vol% methanol, shake, centrifuge, take the supernatant, and the product solution to be tested is obtained.

[0092] (2) Preparation of reference solution: take the appropriate paeoniflorin reference substance, add 50 vol% methanol to prepare a solution containing 80 μg per 1 mL, and the reference solution is obtained.

[0093] (3) Repeat 6 times to obtain the fingerprint spectrum of the above solution, and obtain 6 fingerprint spectra. The method for obtaining the fingerprint spectrum is as follows: take 0.1 vol% phosphoric acid solution A and acetonitrile B as the mobile phase, and obtain the high performance liquid fingerprint spectrum of all batches of product solution to be tested; the chromatographic conditions are as follows: use Agilent ZORBAX SB-C 18Chromatographic column, column length 250 mm, column inner diameter 4.6 mm, packing particle size 5 μm; linear gradient elution procedure: 0-30 min, volume fraction of A in mobile phase 96%-85%, volume fraction of B 4%-15%; 30-50 min, volume fraction of A in mobile phase 85%-80%, volume fraction of B 15%-20%; 50-60 min, volume fraction of A in mobile phase 80%-55%, volume fraction of B 20%-45%; 60-65 min, volume fraction of A in mobile phase 55%, volume fraction of B 45%; flow rate of mobile phase 1.0 mL / min; column temperature 30 °C; injection volume 10 μL; detection wavelength 240 nm.

[0094] (4) Quality stability evaluation: six fingerprints obtained in step 3, results see Figure 7 , and the relevant data of the chromatogram see Table 1-1 and Table 1-2; taking peak 9 (paeoniflorin) as reference, the relative retention time and relative peak area of each common peak were calculated, the relative retention time RSD was less than 0.15%, and the relative peak area RSD was less than 4.0%. It showed that the precision of the method was good.

[0095] Table 1-1 Precision test results (relative retention time)

[0096] Common peak number 1 2 3 4 5 6 RSD (%) 1 0.555 0.555 0.555 0.555 0.555 0.555 0.06 2 0.658 0.657 0.657 0.657 0.657 0.658 0.07 3 0.686 0.685 0.685 0.684 0.684 0.685 0.12 4 0.784 0.784 0.784 0.784 0.784 0.785 0.07 5 0.853 0.852 0.852 0.851 0.851 0.853 0.07 6 0.893 0.893 0.892 0.892 0.891 0.893 0.06 7 0.908 0.908 0.908 0.908 0.907 0.908 0.05 8 0.932 0.931 0.931 0.932 0.931 0.932 0.05 9 1.000 1.000 1.000 1.000 1.000 1.000 0.00 10 1.034 1.034 1.034 1.034 1.034 1.034 0.03 11 1.095 1.094 1.095 1.095 1.096 1.095 0.06 12 1.275 1.272 1.273 1.273 1.273 1.273 0.08 13 1.407 1.405 1.408 1.408 1.409 1.410 0.13

[0097] Table 1-2 Precision test results (relative peak area)

[0098]

[0099]

[0100] 2. Reproducibility test

[0101] (1) Preparation of 6 Nourishing Granules test sample solutions from the same batch: the preparation process of each test sample solution was as follows: taking Nourishing Granules sample, finely ground, about 0.5 g was accurately weighed, placed in a conical flask with a stopper, 25 mL of 50 vol% methanol was accurately added, weighed, ultrasonically treated for 30 min, cooled, and the lost weight was made up with 50 vol% methanol, shaken well, centrifuged, and the supernatant was taken, to obtain 6 product solutions to be tested.

[0102] (2) Preparation of reference solution: an appropriate amount of paeoniflorin reference substance was added to 50 vol% methanol to prepare a solution containing 80 μg per 1 mL, and then the reference solution was obtained.

[0103] (3) The above test sample solution and reference solution were injected into the liquid chromatograph, and the chromatographic conditions were the same as those in the precision test, and the fingerprints of the above 6 product solutions to be tested were obtained, and the results are shown in Figure 8, the relevant data of the atlas are shown in Table 2-1 and Table 2-2; taking the 9th chromatographic peak (paeoniflorin) as the reference, the relative retention time and relative peak area of each common peak were calculated, the relative retention time RSD was less than 0.15%, and the relative peak area RSD was less than 5.0%. It showed that the method had good repeatability.

[0104] Table 2-1 repeatability test results (relative retention time)

[0105]

[0106]

[0107] Table 2-2 repeatability test results (relative peak area)

[0108] Common peak number 1 2 3 4 5 6 RSD (%) 1 0.57 0.56 0.56 0.55 0.56 0.56 1.16 2 0.38 0.38 0.38 0.38 0.38 0.39 0.66 3 1.78 1.84 1.84 1.83 1.84 1.84 1.30 4 0.19 0.20 0.20 0.20 0.20 0.20 3.00 5 15.60 16.08 16.13 16.02 16.07 16.11 1.26 6 0.82 0.84 0.84 0.87 0.84 0.84 2.20 7 2.33 2.39 2.39 2.40 2.38 2.39 1.01 8 2.98 3.05 3.06 3.04 3.04 3.06 0.94 9 1.00 1.00 1.00 1.00 1.00 1.00 0.00 10 2.56 2.64 2.65 2.63 2.64 2.66 1.37 11 0.24 0.26 0.26 0.25 0.26 0.25 2.12 12 0.63 0.65 0.67 0.67 0.64 0.65 2.39 13 0.08 0.09 0.09 0.08 0.08 0.09 4.09

[0109] 3. Stability test

[0110] (1) Take the Yangchao granules sample, grind it, take about 0.5 g, accurately weigh, put it in a conical flask with a plug, accurately add 50 vol% methanol 2 mL, weigh, ultrasonic treat for 30 min, cool, make up the weight lost with 50 vol% methanol, shake well, centrifuge, take the supernatant, and the product solution to be tested is obtained.

[0111] (2) Preparation of reference solution: take the paeoniflorin reference substance, add 50 vol% methanol to prepare a solution containing 80 μg per 1 mL, and the reference solution is obtained.

[0112] (3) The product solution to be tested was sampled and analyzed after being placed at room temperature for 0 h, 4 h, 7 h, 12 h, 20 h, and 28 h, respectively, and the chromatographic analysis method was the same as that in the precision test. Nine fingerprint maps were obtained, and the results are shown in Figure 9 , and the relevant data are shown in Table 3-1 and Table 3-2. Taking the 9th chromatographic peak (paeoniflorin) as the reference, the relative retention time and relative peak area of each common peak were calculated, the relative retention time RSD was less than 0.50%, and the relative peak area RSD was less than 5.0%. It showed that the Yangchao granules test solution extracted by the method had good sample stability at room temperature within 28 h.

[0113] Table 3-1 stability test results (relative retention time)

[0114]

[0115]

[0116] Table 3-2 stability test results (relative peak area)

[0117] Common peak number 0h 3h 7h 12h 20h 28h RSD (%) 1 0.57 0.58 0.58 0.55 0.58 0.58 1.85 2 0.38 0.39 0.39 0.38 0.39 0.38 0.65 3 1.78 1.83 1.84 1.84 1.80 1.81 1.38 4 0.19 0.20 0.20 0.20 0.19 0.19 3.04 5 15.60 16.03 16.12 16.10 15.70 15.88 1.36 6 0.82 0.84 0.84 0.84 0.82 0.83 1.14 7 2.33 2.37 2.39 2.38 2.35 2.35 0.90 8 2.98 3.04 3.06 3.05 3.00 3.03 0.99 9 1.00 1.00 1.00 1.00 1.00 1.00 0.00 10 2.56 2.63 2.65 2.65 2.61 2.64 1.32 11 0.24 0.26 0.26 0.26 0.24 0.25 2.49 12 0.63 0.63 0.64 0.64 0.62 0.64 1.21 13 0.08 0.08 0.09 0.09 0.08 0.08 4.58

[0118] Application Example 1: Stability Evaluation of Nourishing Granules

[0119] (1) Take 11 batches of Nourishing Granules samples (Zhejiang Province Traditional Chinese Medicine Research Co., Ltd. / Hangzhou Hospital of Traditional Chinese Medicine), and prepare 11 batches of test product solutions according to the following method: take Nourishing Granules samples, grind finely, take about 0.5 g, accurately weigh, place in a conical flask with a stopper, accurately add 50 vol% methanol 25 mL, weigh, ultrasonic treat for 30 min, cool, make up the weight lost with 50 vol% methanol, shake well, centrifuge, take the supernatant, and the test product solution is obtained.

[0120] (2) Preparation of reference solution: take appropriate amount of paeoniflorin reference substance, add 50 vol% methanol to prepare a solution containing 80 μg per 1 mL, and the reference solution is obtained.

[0121] (3) Obtain the high performance liquid chromatography fingerprints of all batches of test product solutions with 0.1 vol% phosphoric acid solution A and acetonitrile B as the mobile phase; the chromatographic conditions are as follows: use Agilent ZORBAX SB-C18 chromatographic column, the length of the chromatographic column is 250 mm, the inner diameter of the chromatographic column is 4.6 mm, and the particle size of the filler is 5 μm; the linear gradient elution program is as follows: within 0-30 min, the volume fraction of A in the mobile phase is 96%-85%, and the volume fraction of B is 4%-15%; within 30-50 min, the volume fraction of A in the mobile phase is 85%-80%, and the volume fraction of B is 15%-20%; within 50-60 min, the volume fraction of A in the mobile phase is 80%-55%, and the volume fraction of B is 20%-45%; within 60-65 min, the volume fraction of A in the mobile phase is 55%, and the volume fraction of B is 45%; the flow rate of the mobile phase is 1.0 mL / min; the column temperature is 30°C; the injection volume is 10 μL; and the detection wavelength is 240 nm.

[0122] (4) Quality stability evaluation: import the fingerprints of the 11 batches of test product solutions obtained in step (3) (such as Figure 10 ) into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2.0 version)", perform Mark peak matching after multi-point correction, and generate the control fingerprint (such as Figure 11), and the similarity was calculated. The fingerprint chromatogram included 13 common peaks. Peak 5 was puerarin, peak 9 was paeoniflorin, peak 11 was scopharin, peak 12 was ellagic acid, and peak 13 was verbascoside. The similarity of the fingerprint chromatogram of each batch of Yangchao granules was 1.000, 1.000, 1.000, 0.999, 1.000, 1.000, 1.000, 1.000, 0.998, 0.999, and 0.997, respectively. The similarity was greater than 0.95, indicating that the quality of each batch of Yangchao granules was uniform and stable.

Claims

1. A method for detecting the fingerprint of a Niche-keeping Granule, characterized in that, Specifically comprising the following steps: (1) preparing the product solution to be tested, the preparation process of the product solution to be tested is as follows: taking the granula utriculorum sample, grinding, precisely weighing 0.5 g, precisely adding 25 mL of 50vol% methanol, weighing, treating by ultrasonic or heating reflux for 30 min, cooling, supplementing the lost weight with 50vol% methanol, shaking, centrifuging, taking the supernatant, and thus obtaining the product solution to be tested; (2) preparing the reference solution: the reference solution is a 50vol% methanol solution containing 80 μg paeoniflorin per 1 mL; (3) using high performance liquid chromatography, with a concentration of 0.1 vol% phosphoric acid solution A, acetonitrile B as mobile phase, respectively, to obtain the high performance liquid chromatography fingerprint of the product solution and the reference solution; the chromatographic conditions are as follows: using Agilent ZORBAX SB-C 18 chromatographic column, the length of the chromatographic column is 250 mm, the inner diameter of the chromatographic column is 4.6 mm, and the particle size of the filler is 5 μm; the linear gradient elution program is as follows: within 0-30 min, the volume fraction of A in the mobile phase is 96%-85%, and the volume fraction of B is 4%-15%; within 30-50 min, the volume fraction of A in the mobile phase is 85%-80%, and the volume fraction of B is 15%-20%; within 50-60 min, the volume fraction of A in the mobile phase is 80%-55%, and the volume fraction of B is 20%-45%; within 60-65 min, the volume fraction of A in the mobile phase is 55%, and the volume fraction of B is 45%; the flow rate of the mobile phase is 1.0 mL / min; the column temperature is 30 °C; the injection amount is 10 μL; the detection wavelength is 240 nm; the obtained fingerprint of the reference solution and the product solution is introduced into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and after multi-point correction, Mark peak matching is performed to obtain the fingerprint of the product solution, which includes 13 common peaks; through comparison with the control, 5 index components are identified, which are as follows: No. 5 peak is puerarin, No. 9 peak is paeoniflorin, No. 11 peak is trilobatin, No. 12 peak is ellagic acid, and No. 13 peak is verbascoside.

2. A method for detecting the quality of a nest granule, characterized by, Specifically comprising the following steps: The fingerprint spectrum of the product solution to be tested is obtained by using the granula utriculorum fingerprint spectrum detection method of claim 1, the high performance liquid chromatography fingerprint spectrum of the product to be tested is introduced into the traditional Chinese medicine chromatography fingerprint spectrum similarity evaluation system together with the control fingerprint spectrum, the similarity is calculated, and the sample with a similarity ≥0.95 is evaluated as a product with stable quality; wherein the control fingerprint spectrum is obtained by introducing multiple batches of product solution to be tested into the traditional Chinese medicine chromatography fingerprint spectrum similarity evaluation system, performing multi-point correction, performing Mark peak matching, and using the average method.

3. The method of claim 2, wherein, The time window used by the average method is 0.1 min.

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