Ultra-high performance liquid chromatography for detecting Buxue Yimu Pills, and preparation method and application thereof

Abstract

The present invention discloses an ultra-high performance liquid chromatography method for detecting Buxue Yimu Pills, and its preparation method and application; the preparation method comprises the following steps: the powder of Buxue Yimu Pills is extracted with ethanol with a volume percentage of 70-80% to obtain extract 1, impurities are removed, and the solvent is removed to obtain precipitate 1; the precipitate 1 is extracted with an aqueous solution to obtain extract 2, and the solvent is removed to obtain precipitate 2; precipitate 2 is fully dissolved with ethanol with a volume percentage of 70-80% to obtain a test sample. The present invention constructs a UPLC characteristic fingerprint of Buxue Yimu Pills and a corresponding detection method, which can simultaneously identify 8 components of 4 traditional Chinese medicines in Buxue Yimu Pills, with good precision, good repeatability, short time consumption, high efficiency, objective and reliable results, strengthening the specific identification of Buxue Yimu Pills, providing a new reference standard for the component identification and quality control of Buxue Yimu Pills, and helping to improve the overall quality control level of the industry.

Description

Technical Field

[0001] The present invention relates to the field of analytical detection, and specifically, to an ultra-high performance liquid chromatography method for detecting Buxue Yimu Pills, and its preparation method and application. Background Art

[0002] The main components of Buxue Yimu Pills are Angelica sinensis, Astragalus membranaceus, Colla Corii Asini, Leonurus japonicus Houtt., and Citrus reticulata Blanco, which have the effects of tonifying qi and blood, removing stasis and promoting new growth, and are commonly used clinically to treat postpartum abdominal pain with qi and blood deficiency and blood stasis syndrome.

[0003] Currently, high performance liquid chromatography technology has been used in the prior art to establish the fingerprint and corresponding quality detection method of Buxue Yimu Pills (CN113960184A), but at most only 5 characteristic components can be detected, and the detection method is complex, time-consuming, and costly.

[0004] Therefore, there is still a lack of a comprehensive, simple, and efficient quality detection technology for Buxue Yimu Pills. Summary of the Invention

[0005] In order to solve the problem that it is difficult to reflect the quality of finished medicines due to the qualitative identification and quantitative analysis of simple index components in the prior art, the present invention provides an ultra-high performance liquid chromatography method for detecting Buxue Yimu Pills, and its preparation method and application.

[0006] The first object of the present invention is to provide a preparation method of a test sample for detecting Buxue Yimu Pills by ultra-high performance liquid chromatography.

[0007] The second object of the present invention is to provide the application of the preparation method in constructing the UPLC characteristic fingerprint of Buxue Yimu Pills.

[0008] The third object of the present invention is to provide a test sample prepared by the preparation method.

[0009] The fourth object of the present invention is to provide the application of the test sample in constructing the UPLC characteristic fingerprint of Buxue Yimu Pills.

[0010] The fifth object of the present invention is to provide a method for constructing the UPLC characteristic fingerprint of Buxue Yimu Pills.

[0011] The sixth object of the present invention is to provide the application of the method in the component analysis and / or quality detection of Buxue Yimu Pills.

[0012] The seventh object of the present invention is to provide a quality detection method for Buxue Yimu Pills.

[0013] In order to achieve the above objects, the present invention is realized by the following solutions:

[0014] The present invention uses ultra - high performance liquid chromatography technology to establish the characteristic fingerprint of Buxue Yimu Pills and the corresponding quality detection method. 8 characteristic components are identified by using a reverse - phase column, which can simultaneously identify 4 traditional Chinese medicines, namely Angelica sinensis, Leonurus japonicus, Astragalus membranaceus and Citrus reticulata Blanco in Buxue Yimu Pills, enabling rapid detection and comprehensive component analysis.

[0015] A preparation method of a test sample for detecting Buxue Yimu Pills by ultra - high performance liquid chromatography includes the following steps:

[0016] The powder of Buxue Yimu Pills is extracted with ethanol with a volume percentage of 70 - 80% to obtain Extract 1. After impurity removal, the solvent of Extract 1 is removed to obtain Precipitate 1; the aqueous solution of Precipitate 1 is extracted with a n - butanol solution saturated with water to obtain Extract 2, and the solvent of Extract 2 is removed to obtain Precipitate 2; Precipitate 2 is fully dissolved with ethanol with a volume percentage of 70 - 80% to obtain the test sample.

[0017] Preferably, the water is distilled water.

[0018] Preferably, the powder of Buxue Yimu Pills is extracted with ethanol with a volume percentage of 75%.

[0019] Preferably, the powder of Buxue Yimu Pills is extracted by heating under reflux with ethanol with a volume percentage of 70 - 80%.

[0020] More preferably, the temperature of the heating under reflux extraction is 70 - 100 °C and the time is 2 - 3 hours.

[0021] Further preferably, the temperature of the heating under reflux extraction is 85 °C and the time is 2.5 hours.

[0022] Most preferably, the powder of Buxue Yimu Pills is extracted by heating under reflux with 75% ethanol at 85 °C for 2.5 hours.

[0023] Preferably, the method for impurity removal is centrifugation.

[0024] More preferably, the centrifugation is carried out at 8000 - 15000 revolutions per minute for 2 - 10 minutes.

[0025] Further preferably, the centrifugation is carried out at 12000 revolutions per minute for 5 minutes.

[0026] Preferably, the method for removing the solvent of Extract 1 is evaporation to dryness.

[0027] Preferably, in the aqueous solution of Precipitate 1, the concentration of Precipitate 1 is 0.03 - 0.1 g / mL.

[0028] More preferably, in the aqueous solution of Precipitate 1, the concentration of Precipitate 1 is 0.05 g / mL.

[0029] Preferably, the precipitate 1 is cooled to 20-25 °C and then made into an aqueous solution.

[0030] Preferably, the aqueous solution of precipitate 1 is shaken and extracted with a water-saturated n-butanol solution, and the number of shaking times is 1-3 times.

[0031] More preferably, the aqueous solution of precipitate 1 is shaken and extracted with a water-saturated n-butanol solution, and the number of shaking times is 3 times.

[0032] Even more preferably, the aqueous solution of precipitate 1 is shaken and extracted with a water-saturated n-butanol solution, and the number of shaking times is 1-3 times. The volume ratio of the water-saturated n-butanol solution to the aqueous solution of precipitate 1 for each shaking is (0.8-1.2):(0.8-1.2); the n-butanol layers obtained from 1-3 times of shaking extraction are combined to obtain the extract 2.

[0033] Most preferably, the aqueous solution of precipitate 1 is shaken and extracted with a water-saturated n-butanol solution, and the number of shaking times is 3 times. The volume ratio of the water-saturated n-butanol solution to the aqueous solution of precipitate 1 for each shaking is 1:1; the n-butanol layers obtained from 3 times of shaking extraction are combined to obtain the extract 2.

[0034] Preferably, the method for removing the solvent of the extract 2 is to evaporate to dryness.

[0035] Preferably, the test sample is obtained by fully dissolving the precipitate 2 with ethanol having a volume percentage of 75%.

[0036] Preferably, after fully dissolving the precipitate 2 with ethanol having a volume percentage of 70-80%, impurity removal is also carried out to obtain the test sample.

[0037] More preferably, the method for impurity removal is filtration.

[0038] Even more preferably, the filtration is carried out with a 0.22 μm microporous filter membrane.

[0039] Preferably, the content of ferulic acid in the methanol solution of the ferulic acid reference substance is 0.8-1.2 mg / mL.

[0040] More preferably, the content of ferulic acid in the methanol solution of the ferulic acid reference substance is 1 mg / mL.

[0041] The test sample prepared by the above preparation method should also be within the protection scope of the present invention.

[0042] The application of the above preparation method and / or the above test sample in constructing the UPLC characteristic fingerprint of Buxue Yimu Pills should also be within the protection scope of the present invention, and the test sample prepared by using the above preparation method is used to construct the UPLC characteristic fingerprint of Buxue Yimu Pills.

[0043] A method for constructing the UPLC characteristic fingerprint of Buxue Yimu Pills, comprising the following steps:

[0044] S1. Prepare the test sample using the above preparation method; use the methanol solution of ferulic acid reference substance as the reference substance;

[0045] S2. Perform ultra-high performance liquid chromatography detection on the test sample and the reference substance obtained in the previous step, and then obtain the UPLC characteristic fingerprint of Buxue Yimu Pills.

[0046] Preferably, in step S1, the water is distilled water.

[0047] Preferably, in step S1, extract with ethanol with a volume percentage of 75%.

[0048] Preferably, in step S1, perform heating reflux extraction with ethanol with a volume percentage of 70-80%.

[0049] More preferably, in step S1, the temperature of the heating reflux extraction is 70-100 °C, and the time is 2-3 hours.

[0050] Further preferably, in step S1, the temperature of the heating reflux extraction is 85 °C, and the time is 2.5 hours.

[0051] Most preferably, in step S1, perform heating reflux extraction with 75% ethanol at 85 °C for 2.5 hours.

[0052] Preferably, in step S1, the method for removing impurities is centrifugation.

[0053] More preferably, in step S1, the centrifugation is at 8000-15000 revolutions per minute for 2-8 minutes.

[0054] Further preferably, in step S1, the centrifugation is at 12000 revolutions per minute for 5 minutes.

[0055] Preferably, in step S1, the method for removing the solvent of the extraction solution 1 is evaporation to dryness.

[0056] Preferably, in the aqueous solution of the precipitate 1 in step S1, the concentration of the precipitate 1 is 0.003-0.1 g / mL.

[0057] More preferably, in the aqueous solution of the precipitate 1 in step S1, the concentration of the precipitate 1 is 0.005 g / mL.

[0058] Preferably, in step S1, the precipitate 1 is cooled to 20-25 °C and then made into an aqueous solution.

[0059] Preferably, in step S1, an aqueous solution of precipitate 1 is shaken and extracted with a water-saturated n-butanol solution, and the number of shaking times is 1 to 3 times.

[0060] More preferably, in step S1, an aqueous solution of precipitate 1 is shaken and extracted with a water-saturated n-butanol solution, and the number of shaking times is 3 times.

[0061] Further preferably, in step S1, an aqueous solution of precipitate 1 is shaken and extracted with a water-saturated n-butanol solution, and the number of shaking times is 1 to 3 times. The volume ratio of the water-saturated n-butanol solution to the aqueous solution of precipitate 1 for each shaking is (0.8 - 1.2):(0.8 - 1.2); the n-butanol layers obtained from 1 to 3 times of shaking extraction are combined to obtain extract 2.

[0062] Most preferably, in step S1, an aqueous solution of precipitate 1 is shaken and extracted with a water-saturated n-butanol solution, and the number of shaking times is 3 times. The volume ratio of the water-saturated n-butanol solution to the aqueous solution of precipitate 1 for each shaking is 1:1; the n-butanol layers obtained from 3 times of shaking extraction are combined to obtain extract 2.

[0063] Preferably, in step S1, the method for removing the solvent of extract 2 is to evaporate it to dryness.

[0064] Preferably, in step S1, the test sample is obtained by fully dissolving precipitate 2 with ethanol having a volume percentage of 75%.

[0065] Preferably, in step S1, after fully dissolving precipitate 2 with ethanol having a volume percentage of 70 - 80%, impurity removal is also performed to obtain the test sample.

[0066] More preferably, in step S1, the method for impurity removal is filtration.

[0067] Further preferably, in step S1, the filtration is performed using a 0.22 μm microporous membrane filter.

[0068] Preferably, in step S1, the content of ferulic acid in the methanol solution of the ferulic acid reference substance is 0.8 - 1.2 mg / mL.

[0069] More preferably, in step S1, the content of ferulic acid in the methanol solution of the ferulic acid reference substance is 1 mg / mL.

[0070] Preferably, in step S2, the detection conditions of the ultra-high performance liquid chromatography are as follows:

[0071] Use a T3 chromatographic column; the detection wavelength is 270 - 290 nm; the flow rate is 0.2 - 0.4 mL / min; the injection volume is 1 - 3 μL; an aqueous formic acid solution with a volume percentage of 0.4 - 0.8% is used as mobile phase A, and acetonitrile is used as mobile phase B. In the gradient elution program, the volume percentage change of mobile phase A is as follows: 0 - 2 min, mobile phase A is 92%; 2 - 5 min, mobile phase A decreases from 92% to 85%; 5 - 12 min, mobile phase A decreases from 85% to 84%; 12 - 13 min, mobile phase A decreases from 84% to 83%; 13 - 14 min, mobile phase A decreases from 83% to 81%; 14 - 17 min, mobile phase A decreases from 81% to 68%; 17 - 19 min, mobile phase A decreases from 68% to 60%; 19 - 25 min, mobile phase A decreases from 60% to 40%; 25 - 28 min, mobile phase A decreases from 40% to 20%

[0072] More preferably, in step S2, the T3 chromatographic column is ACQUITY UPLC HSS T3, with an inner diameter of 2.1 mm, a column length of 100 mm, and a particle size of 1.8 μm.

[0073] More preferably, in step S2, the detection wavelength is 280 nm.

[0074] More preferably, in step S2, the flow rate is 0.3 mL / min.

[0075] More preferably, in step S2, the injection volume is 2 μL.

[0076] More preferably, in step S2, acetonitrile is used as mobile phase A.

[0077] More preferably, in step S2, the column temperature of the T3 chromatographic column is 30 - 35 °C.

[0078] Even more preferably, in step S2, the column temperature of the T3 chromatographic column is 30 °C.

[0079] The application of any of the above methods in the component analysis and / or quality detection of Buxue Yimu Pills should also be within the protection scope of the present invention.

[0080] The UPLC characteristic chromatogram of Buxue Yimu Pills constructed by any of the above methods should also be within the protection scope of the present invention.

[0081] The application of the UPLC characteristic chromatogram of Buxue Yimu Pills constructed by any of the above methods in the component analysis and / or quality detection of Buxue Yimu Pills should also be within the protection scope of the present invention.

[0082] A quality inspection method for Buxue Yimu Pills, comprising the following steps: obtaining the UPLC chromatogram of the sample to be tested according to any of the above methods as the sample chromatogram; constructing the UPLC characteristic chromatogram of Buxue Yimu Pills according to any of the above methods as the control chromatogram; and comparing the sample chromatogram with the control chromatogram.

[0083] Preferably, calculate the similarity between the sample chromatogram and the control chromatogram according to the comparison result. If the similarity is greater than or equal to 0.90, the quality of the sample to be tested is qualified; otherwise, the quality is unqualified.

[0084] Compared with the prior art, the present invention has the following beneficial effects:

[0085] The present invention constructs the UPLC characteristic chromatogram of Buxue Yimu Pills and the corresponding detection method, which can simultaneously identify 8 components of 4 traditional Chinese medicines in Buxue Yimu Pills. It has good precision, excellent repeatability, short time consumption, high efficiency, objective and reliable results, strengthens the specific identification of Buxue Yimu Pills, provides a new reference standard for the component identification and quality control of Buxue Yimu Pills, and helps to improve the overall quality control level of the industry. Description of the Drawings

[0086] Figure 1 The UPLC detection results using different chromatographic columns, with black being the chromatographic column ACQUITY UPLC HSS T3 and blue being the chromatographic column ACQUITY UPLC BEH C18.

[0087] Figure 2 The UPLC detection results using different wavelengths.

[0088] Figure 3 The UPLC detection results of the test sample prepared by Method 1.

[0089] Figure 4 The UPLC detection results of the test sample prepared by Method 2.

[0090] Figure 5 The UPLC detection results of the test sample prepared by Method 3.

[0091] Figure 6 The UPLC detection results of the test sample prepared by Method 4.

[0092] Figure 7 The UPLC detection results of the test sample, the negative sample solution without Angelica sinensis, the negative sample solution without Astragalus membranaceus, the negative sample solution without Citrus reticulata Blanco, and the negative sample without Leonurus japonicus Houtt.

[0093] Figure 8 The UPLC comparison chromatogram for the confirmation of related components of the UPLC characteristic chromatogram of Buxue Yimu Pills.

[0094] Figure 9 It is a control characteristic chromatogram.

[0095] Figure 10 It is the UPLC characteristic chromatogram of 14 batches of Buxue Yimu Pills with different batch numbers; S1 is the Buxue Yimu Pills with batch number 20201217; S2 is the Buxue Yimu Pills with batch number 20210517; S3 is the Buxue Yimu Pills with batch number 20210607; S4 is the Buxue Yimu Pills with batch number 20210805; S5 is the Buxue Yimu Pills with batch number 20210916; S6 is the Buxue Yimu Pills with batch number 20211008; S7 is the Buxue Yimu Pills with batch number 20211014; S8 is the Buxue Yimu Pills with batch number 20220617; S9 is the Buxue Yimu Pills with batch number 20220618; S10 is the Buxue Yimu Pills with batch number 20220619; S11 is the Buxue Yimu Pills with batch number 20220621; S12 is the Buxue Yimu Pills with batch number 20220622; S13 is the Buxue Yimu Pills with batch number 20220623; S14 is the Buxue Yimu Pills with batch number 20220624; S15 is the control chromatogram; the "8" in the brackets indicates the number of mark peaks. Specific embodiments

[0096] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are, unless otherwise specified, reagents and materials that can be obtained from commercial channels.

[0097] Example 1 Chromatographic conditions for constructing the UPLC characteristic chromatogram of Buxue Yimu Pills

[0098] 1. Mixed reference substances

[0099] The main components of Buxue Yimu Pills are Angelica sinensis, Astragalus membranaceus, Colla Corii Asini, Leonurus japonicus, and Citrus reticulata Blanco.

[0100] Reference substances of the active ingredient ligustilide of Angelica sinensis, reference substances of the active ingredients senkyunolide I and calycosin-7-O-β-D-glucoside of Astragalus membranaceus, reference substance of the active ingredient leonurine hydrochloride of Leonurus japonicus, reference substance of the active ingredient hesperidin of Citrus reticulata Blanco, and reference substances of the common components ferulic acid and rutin of the four herbs of Angelica sinensis, Astragalus membranaceus, Leonurus japonicus, and Citrus reticulata Blanco. Due to the differences in the nature of Colla Corii Asini itself, the components in Colla Corii Asini were not detected in this example.

[0101] The reference substances of the above different herbs were dissolved in methanol to prepare a mixed reference substance. The specific preparation method is as follows:

[0102] Accurately weigh 20.45 mg of leonurine hydrochloride, 50.32 mg of ferulic acid, 19.98 mg of calycosin-7-O-β-D-glucoside, 99.80 mg of rutin, 20.63 mg of senkyunolide I, 20.04 mg of hesperidin, and 19.52 μL of ligustilide reference substance, and place them in 7 volumetric flasks respectively. Dissolve and make up the volume with methanol to obtain a leonurine hydrochloride reference stock solution with a concentration of 0.964 mg / mL, a ferulic acid reference stock solution with a concentration of 2.500 mg / mL, a calycosin-7-O-β-D-glucoside reference stock solution with a concentration of 0.967 mg / mL, a rutin reference stock solution with a concentration of 4.580 mg / mL, a senkyunolide I reference stock solution with a concentration of 1.020 mg / mL, a hesperidin reference stock solution with a concentration of 0.953 mg / mL, and a ligustilide reference stock solution with a concentration of 0.976 mg / mL. Then mix each reference stock solution in methanol to obtain a mixed reference substance.

[0103] The concentrations of leonurine hydrochloride, ferulic acid, calycosin-7-O-β-D-glucoside, rutin, senkyunolide I, hesperidin, and ligustilide in the mixed reference substance are 9.64 μg / mL, 15.80 μg / mL, 9.67 μg / mL, 1.33 μg / mL, 20.40 μg / mL, 9.53 μg / mL, and 97.60 μg / mL in sequence.

[0104] 2. Determination of chromatographic column

[0105] On a Waters ultra-high performance liquid chromatograph (Waters Acquity H-Class), using a Waters ACQUITY UPLC HSS T3 chromatographic column (inner diameter 2.1 mm, column length 100 mm, particle size 1.8 μm), the detection wavelength is 280 nm, the flow rate is 0.3 mL / min, the column temperature is 30 °C, the injection volume is 2 μL, 0.6% v / v formic acid aqueous solution is used as mobile phase A, and acetonitrile is used as mobile phase B. Injection and detection are carried out according to the mobile phase elution gradient shown in Table 1.

[0106] Table 1 Mobile phase elution gradient

[0107]

[0108] As Figure 1 shown, using two chromatographic columns, ACQUITY UPLC BEH C18 and ACQUITY UPLC HSS T3, for injection analysis of the mixed reference substance. Compared with the chromatographic column ACQUITY UPLC BEH C18, the characteristic chromatogram obtained by the chromatographic column ACQUITY UPLC HSS T3 has better separation, a stable baseline, and good peak shapes. Therefore, ACQUITY UPLC HSS T3 is selected as the optimal chromatographic column.

[0109] 3. Determination of Detection Wavelength

[0110] Take an appropriate amount of Buxue Yimu Pills, pulverize them, accurately weigh 1 g of the powder of Buxue Yimu Pills, add 20 mL of 75% v / v ethanol, heat under reflux at 85 °C for 2.5 hours, and collect the distillate; centrifuge the obtained distillate at a speed of 12,000 revolutions per minute for 5 minutes; take the supernatant, evaporate the solvent to dryness, and after the remaining residue is cooled to room temperature (20 - 25 °C), dissolve 1 g of the residue thoroughly with 20 mL of water, then shake and extract with a n-butanol solution saturated with 20 mL of water for 3 times, and combine the n-butanol layers to obtain a combined solution; evaporate the obtained combined solution to dryness, and after the remaining residue is cooled to room temperature (20 - 25 °C), dissolve all the residue thoroughly with 2 mL of 75% v / v ethanol, and filter through a 0.22 μm microporous membrane to obtain the test sample.

[0111] Using a diode array detector, set the detection wavelength of the ultra-high performance liquid chromatograph to 200 - 400 nm, scan the test sample solution, and select the spectra at wavelengths of 220 m, 240 nm, 260 nm, 280 nm, 300 nm, 320 nm, 340 nm, and 360 nm for analysis.

[0112] As Figure 2 shown, compared with other wavelengths, the number of absorption peaks is the largest at 280 nm, each absorption peak has a relatively high absorption, the absorption of each peak is the best, the amount of information is relatively large and the resolution is good; while at 260 nm close to 280 nm, the absorption of the ferulic acid characteristic peak is relatively small, and at 300 nm, the absorptions of the senkyunolide I and hesperidin characteristic peaks are relatively small. Therefore, 280 nm is selected as the detection wavelength.

[0113] 4. Determination of Chromatographic Conditions

[0114] According to the above determined conditions, the chromatographic conditions finally determined for constructing the UPLC characteristic chromatogram of Buxue Yimu Pills are as follows: The chromatographic column is Waters ACQUITY UPLC HSS T3 (inner diameter 2.1 mm, column length 100 mm, particle size 1.8 μm); the detection wavelength is 280 nm; the flow rate is 0.3 mL / min; the column temperature is 30 °C; the injection volume is 2 μL; the mobile phase is 0.6% v / v formic acid aqueous solution (A) - acetonitrile (B); in the gradient elution program, the volume percentage change of mobile phase A is: 0 - 2 min, mobile phase A is 92%; 2 - 5 min, mobile phase A drops from 92% to 85%; 5 - 12 min, mobile phase A drops from 85% to 84%; 12 - 13 min, mobile phase A drops from 84% to 83%; 13 - 14 min, mobile phase A drops from 83% to 81%; 14 - 17 min, mobile phase A drops from 81% to 68%; 17 - 19 min, mobile phase A drops from 68% to 60%; 19 - 25 min, mobile phase A drops from 60% to 40%; 25 - 28 min, mobile phase A drops from 40% to 20%.

[0115] Example 2 Preparation method of test sample for constructing UPLC characteristic chromatogram of Buxue Yimu Pills

[0116] 1. Experimental method

[0117] (1) Prepare test samples using different methods

[0118] Method 1:

[0119] Take an appropriate amount of Buxue Yimu Pills, pulverize them, and precisely weigh 1 g of the Buxue Yimu Pills powder. Add 20 mL of 75% v / v ethanol, heat under reflux at 85 °C for 2.5 hours, and collect the distillate; centrifuge the obtained distillate at a speed of 12,000 revolutions per minute for 5 minutes; take the supernatant, evaporate the solvent to dryness, and after the remaining residue cools to room temperature (20 - 25 °C), dissolve 1 g of the residue thoroughly with 20 mL of water, then shake and extract with 20 mL of water-saturated n-butanol solution for 3 times, and combine the n-butanol layers to obtain a combined solution; evaporate the obtained combined solution to dryness, and after the remaining residue cools to room temperature (20 - 25 °C), dissolve all the residue thoroughly with 2 mL of 75% v / v ethanol, and filter through a 0.22 μm microporous membrane to obtain the test sample.

[0120] Method 2:

[0121] Take an appropriate amount of Buxue Yimu Pills, pulverize them, accurately weigh 1 g of the pulverized Buxue Yimu Pills and 0.005 g of cellulase, add 10 mL of distilled water, and stir at 45 °C for 1.5 hours under a digital high-speed disperser; add 20 mL of 75% v / v ethanol, heat under reflux at 85 °C for 2.5 hours, and collect the distillate; centrifuge the obtained distillate at a speed of 12,000 revolutions per minute for 5 minutes; take the supernatant, evaporate the solvent to dryness, and after the remaining residue is cooled to room temperature (20 - 25 °C), dissolve it thoroughly with 20 mL of water, then shake and extract 3 times with a water-saturated n-butanol solution, 20 mL each time, combine the n-butanol layers to obtain a combined solution, evaporate the combined solution to dryness, and after the remaining residue is cooled to room temperature (20 - 25 °C), dissolve it thoroughly with 2 mL of 75% v / v ethanol, and then continue to make up the volume to 10 mL with 75% v / v ethanol, and filter through a 0.22 μm microporous membrane to obtain the test sample.

[0122] Method 3:

[0123] Take an appropriate amount of Buxue Yimu Pills, pulverize them, accurately weigh 4 g of the pulverized Buxue Yimu Pills, add 25 mL of water, adjust the pH to 10 with solid NaOH, stir evenly, and let it stand for extraction at room temperature (20 - 25 °C) for 3 hours; adjust the pH to 4 with HCL solution, and let it stand for 30 minutes; then centrifuge at a speed of 12,000 revolutions per minute for 5 minutes; take the supernatant, evaporate the solvent to dryness, and after the remaining residue is cooled to room temperature (20 - 25 °C), dissolve all the residue thoroughly with 2 mL of water, and filter through a 0.22 μm microporous membrane to obtain the test sample.

[0124] Method 4:

[0125] Take an appropriate amount of Buxue Yimu Pills, pulverize them, accurately weigh 4 g of the pulverized Buxue Yimu Pills, add 25 mL of water, adjust the pH to 4 with HCL solution, add 0.12 g of cellulase, and carry out enzymatic hydrolysis in a water bath at 35 °C with constant temperature oscillation for 4 hours; after the enzymatic hydrolysis is completed, adjust the pH to 10 with solid NaOH, and let it stand for 30 minutes; then centrifuge at a speed of 12,000 revolutions per minute for 5 minutes; take the supernatant, evaporate the solvent to dryness, and after the remaining residue is cooled to room temperature (20 - 25 °C), dissolve it thoroughly with 2 mL of water, and filter through a 0.22 μm microporous membrane to obtain the test sample.

[0126] (2) Detection and analysis

[0127] For the test samples prepared by the above 4 methods, detect and analyze them using the chromatographic conditions for finally determining the construction of the UPLC characteristic chromatogram of Buxue Yimu Pills in Example 1.

[0128] 2. Experimental results

[0129] As Figure 3As shown, the chromatogram of the test sample prepared by Method 1 has complete information, uniform distribution of characteristic peaks, moderate proportions, good resolution, and good integrity.

[0130] As Figure 4 shown, the chromatogram of the test sample prepared by Method 2 lacks the characteristic peak of rutin, and the peak shapes of the characteristic peaks of leonurine hydrochloride and calycosin-7-O-β-D-glucoside are not good, indicating that this method has poor extraction effects on rutin, leonurine hydrochloride, and calycosin-7-O-β-D-glucoside.

[0131] As Figure 5 shown, the chromatogram of the test sample prepared by Method 3 lacks the characteristic peaks of rutin and senkyunolide I, indicating that this method has poor extraction effects on rutin and senkyunolide I.

[0132] As Figure 6 shown, the chromatogram of the test sample prepared by Method 4 has only a small characteristic peak of ligustilide, indicating that this method has low extraction efficiency for each component of Buxue Yimu Pills.

[0133] In summary, Method 1 of this example is finally determined as the preparation method of the test sample for constructing the UPLC characteristic fingerprint of Buxue Yimu Pills.

[0134] Example 3 Construction of UPLC Characteristic Fingerprint of Buxue Yimu Pills

[0135] 1. Attribution of Characteristic Peaks of UPLC Characteristic Fingerprint of Buxue Yimu Pills

[0136] (1) Preparation of Test Sample Solution

[0137] The test sample solution was prepared according to Method 1 of Example 2.

[0138] (2) Preparation of Negative Sample Solution

[0139] Negative sample solution without Angelica sinensis:

[0140] Weigh 416.7 g of Astragalus membranaceus and 625 g of Leonurus japonicus, decoct them twice with water. For the first time, add 6 times the amount of water and decoct for 2 hours. For the second time, add 5 times the amount of water and decoct for 1.5 hours. Filter the decoction liquid, and concentrate the filtrate to a relative density of 1.10 - 1.15 (60 - 70 °C) to obtain the clear extract of Astragalus membranaceus and Leonurus japonicus. Filter it, and dissolve 125 g of donkey-hide gelatin in the above-mentioned clear extract of Astragalus membranaceus and Leonurus japonicus; crush 12.5 g of Citrus reticulata Blanco into coarse powder, and according to the percolation method under the item of "flowing extract and extract" in the Chinese Pharmacopoeia (2020 Edition), use 70% v / v ethanol as the solvent, soak for 48 hours, and then percolate at a flow rate of 5 mL / (min·kg) to collect about 1750 mL of percolate. Recover ethanol under reduced pressure and concentrate it to a clear extract with a relative density of 1.10 - 1.15 (60 - 70 °C) to obtain the clear extract of Citrus reticulata Blanco; take half of the mass of the clear extract of Citrus reticulata Blanco and mix it with the above-mentioned clear extract of Astragalus membranaceus and Leonurus japonicus, concentrate it to a relative density of 1.15 - 1.25 (80 °C), vacuum dry (0.08 - 0.10 MPa, 70 °C) for 60 - 70 minutes, crush it into fine powder, add 340 g of lactose, 90 g of microcrystalline cellulose and 60 g of sodium carboxymethyl starch, mix well, use the remaining clear extract of Citrus reticulata Blanco as the binder to make pills, and dry at 60 °C to make 1000 g, thus obtaining the negative sample without Angelica sinensis; according to Method 1 of Example 2, use the negative sample without Angelica sinensis to replace the Bushen Yimu Pills to prepare the negative sample solution without Angelica sinensis.

[0141] Negative sample solution without Astragalus membranaceus:

[0142] Weigh 625 g of Leonurus japonicus, decoct it twice with water. For the first time, add 6 times the amount of water and decoct for 2 hours. For the second time, add 5 times the amount of water and decoct for 1.5 hours. Filter the decoction liquid, and concentrate the filtrate to a relative density of 1.10 - 1.15 (60 - 70 °C) to obtain the clear extract of Leonurus japonicus. Filter it, and dissolve 125 g of donkey-hide gelatin in the above-mentioned clear extract of Leonurus japonicus; crush 416.7 g of Angelica sinensis and the 12.5 g of Citrus reticulata Blanco into coarse powder, and according to the percolation method under the item of "flowing extract and extract" in the Chinese Pharmacopoeia (2020 Edition), use 70% v / v ethanol as the solvent, soak for 48 hours, and then percolate at a flow rate of 5 mL / min·kg to collect about 1750 mL of percolate. Recover ethanol under reduced pressure and concentrate it to a clear extract with a relative density of 1.10 - 1.15 (60 - 70 °C) to obtain the clear extract of Angelica sinensis and Citrus reticulata Blanco; take half of the mass of the clear extract of Angelica sinensis and Citrus reticulata Blanco and mix it with the above-mentioned clear extract of Leonurus japonicus, concentrate it to a relative density of 1.15 - 1.25 (80 °C), vacuum dry (0.08 - 0.10 MPa, 70 °C) for 60 - 70 minutes, crush it into fine powder, add 340 g of lactose, 90 g of microcrystalline cellulose and 60 g of sodium carboxymethyl starch, mix well, use the remaining clear extract of Angelica sinensis and Citrus reticulata Blanco as the binder to make pills, and dry at 60 °C to make 1000 g, thus obtaining the negative sample without Astragalus membranaceus; according to Method 1 of Example 2, use the negative sample without Astragalus membranaceus to replace the Bushen Yimu Pills to prepare the negative sample solution without Astragalus membranaceus.

[0143] Negative sample solution lacking tangerine peel:

[0144] Weigh 416.7 g of Astragalus and 625 g of Leonurus japonicus, decoct them twice with water. Add 6 times the amount of water for the first time and decoct them for 2 hours. Add 5 times the amount of water for the second time and decoct them for 1.5 hours. The decoction is filtered and the filtrate is concentrated to a relative density of 1.10-1.15 (60-70 ° C). The Astragalus and Leonurus japonicus paste is obtained. Filter and dissolve 125 g of donkey-hide gelatin in the Astragalus and Leonurus japonicus paste. 416.7 g of Angelica sinensis is crushed into coarse powder. According to the percolation method under the "Fluid Extract and Extract Item" of the "Chinese Pharmacopoeia" (2020 edition), 70% v / v ethanol is used as the solvent. After soaking for 48 hours, percolation is carried out at an outflow rate of 5 mL / (min·kg). About 1750 mL of the percolate is collected, and the ethanol is recovered under reduced pressure and concentrated to The relative density is 1.10~1.15 (60~70 ℃) of the clear paste, namely obtains Angelica sinensis clear paste; Get 1 / 2 mass of Angelica sinensis clear paste and mix with above-mentioned Astragalus and Leonurus herba clear paste, be concentrated to relative density is 1.15~1.25 (80 ℃), vacuum drying (0.08~0.10MPa, 70 ℃) 60~70 minutes, grind into fine powder, add lactose 340g, microcrystalline cellulose 90g and sodium carboxymethyl starch 60g, mix, use the Angelica sinensis clear paste of the remaining amount as binder pilling, 60 ℃ drying, make 1000g, namely obtain lack of tangerine peel negative sample; According to the method one of Example 2, replace Buxue Yimu Wan with lack of tangerine peel negative sample, prepare lack of tangerine peel negative sample solution.

[0145] Negative sample solution lacking Motherwort:

[0146] Weigh 416.7 g of Astragali Radix, decoct it twice with water. For the first time, add 6 times the amount of water and decoct for 2 hours. For the second time, add 5 times the amount of water and decoct for 1.5 hours. Filter the decoction liquid, concentrate the filtrate to a relative density of 1.10 - 1.15 (60 - 70 °C) to obtain the clear extract of Astragali Radix, filter it, and dissolve 125 g of Colla Corii Asini in the above-mentioned clear extract of Astragali Radix; pulverize 416.7 g of Angelicae Sinensis Radix and 12.5 g of Citri Reticulatae Pericarpium into coarse powder, and according to the percolation method under the item of "Fluid Extracts and Extracts" in the Chinese Pharmacopoeia (2020 Edition), use 70% v / v ethanol as the solvent, soak for 48 hours, and then carry out percolation at a flow rate of 5 mL / (min·kg). Collect about 1750 mL of the percolate, recover ethanol under reduced pressure and concentrate it to a clear extract with a relative density of 1.10 - 1.15 (60 - 70 °C) to obtain the clear extract of Angelicae Sinensis Radix and Citri Reticulatae Pericarpium; take one-half of the mass of the clear extract of Angelicae Sinensis Radix and Citri Reticulatae Pericarpium and mix it evenly with the above-mentioned clear extract of Astragali Radix, concentrate it to a relative density of 1.15 - 1.25 (80 °C), carry out vacuum drying (0.08 - 0.10 MPa, 70 °C) for 60 - 70 minutes, pulverize it into fine powder, add 340 g of lactose, 90 g of microcrystalline cellulose and 60 g of sodium carboxymethyl starch, mix evenly, use the remaining amount of the clear extract of Angelicae Sinensis Radix and Citri Reticulatae Pericarpium as the binder to make pills, and dry at 60 °C to make 1000 g, thus obtaining the negative sample without Leonuri Herba; according to Method 1 of Example 2, use the negative sample without Leonuri Herba to replace the Buxue Yimu Pills, and prepare the negative sample solution without Leonuri Herba.

[0147] (2) Detection and analysis

[0148] For the above-mentioned test solution, negative sample solution without Angelicae Sinensis Radix, negative sample solution without Astragali Radix, negative sample solution without Citri Reticulatae Pericarpium, and negative sample solution without Leonuri Herba, conduct detection and analysis under the chromatographic conditions of the final determined UPLC characteristic chromatogram method of Buxue Yimu Pills in Example 1.

[0149] As Figure 7 shown, compared with the chromatogram of the test sample, the chromatogram of the negative sample without Angelicae Sinensis Radix has no characteristic peaks 6 and 8, so characteristic peaks 6 and 8 are attributed to Angelicae Sinensis Radix; the chromatogram of the negative sample without Astragali Radix has no characteristic peak 4, so characteristic peak 4 is attributed to Astragali Radix; the chromatogram of the negative sample without Citri Reticulatae Pericarpium has no characteristic peak 7, so characteristic peak 7 is attributed to Citri Reticulatae Pericarpium; the chromatogram of the negative sample without Leonuri Herba has no characteristic peaks 1 and 2, so characteristic peaks 1 and 2 are attributed to Leonuri Herba. Characteristic peaks 3 and 5 are the common peaks in the chromatograms of the test sample, negative sample without Angelicae Sinensis Radix, negative sample without Astragali Radix, negative sample without Citri Reticulatae Pericarpium, and negative sample without Leonuri Herba, so characteristic peaks 3 and 5 are attributed to Angelicae Sinensis Radix, Astragali Radix, Leonuri Herba, and Citri Reticulatae Pericarpium.

[0150] 2. Identification of characteristic peaks of the UPLC characteristic chromatogram of Buxue Yimu Pills

[0151] (1) Preparation of negative sample solution

[0152] The negative sample solutions lacking Angelica sinensis, Astragalus membranaceus, Leonurus japonicus, and Citrus reticulata Blanco were prepared according to the method of Example 3.

[0153] (2) Detection and analysis

[0154] The above-mentioned negative sample solutions were detected and analyzed under the chromatographic conditions of the final established UPLC characteristic chromatogram method of Buxue Yimu Pills in Example 1 to determine the attribution of each characteristic peak in the test solution chromatogram.

[0155] Table 2 Attribution of characteristic components of Buxue Yimu Pills

[0156]

[0157]

[0158] As Figure 8 shown in and Table 2, characteristic peak 1 is a common peak of each medicinal material, attributed to Angelica sinensis, Astragalus membranaceus, Leonurus japonicus, and Citrus reticulata Blanco; characteristic peak 2 is leonurine hydrochloride, attributed to Leonurus japonicus; characteristic peak 3 is ferulic acid, attributed to Angelica sinensis, Astragalus membranaceus, Leonurus japonicus, and Citrus reticulata Blanco; characteristic peak 4 is calycosin-7-O-β-D-glucoside, attributed to Astragalus membranaceus; characteristic peak 5 is rutin, attributed to Angelica sinensis, Astragalus membranaceus, Leonurus japonicus, and Citrus reticulata Blanco; characteristic peak 6 is senkyunolide I, attributed to Angelica sinensis; characteristic peak 7 is hesperidin, attributed to Citrus reticulata Blanco; peak 8 is ligustilide, attributed to Angelica sinensis.

[0159] In summary, the present invention successfully constructed the UPLC characteristic chromatogram of Buxue Yimu Pills as Figure 9 shown, with a total of 8 characteristic peaks, corresponding to 8 components of 4 medicinal materials in Buxue Yimu Pills, which can be used for the component identification and quality detection of Buxue Yimu Pills.

[0160] Example 4 Methodology verification of the UPLC characteristic chromatogram of Buxue Yimu Pills

[0161] 1. Usage method of the UPLC characteristic chromatogram of Buxue Yimu Pills

[0162] (1) Preparation of the test solution

[0163] The test solution was prepared according to Method 1 in Example 2.

[0164] (2) Preparation of the reference solution

[0165] Precisely weigh the ferulic acid reference substance and prepare a ferulic acid reference solution with a concentration of 1 mg / mL in methanol.

[0166] (3) Determination

[0167] Precisely pipette 2 μL each of the reference substance solution and the test solution, inject them into an ultra-high performance liquid chromatograph, and perform detection under the chromatographic conditions finally determined in Step 4 of Example 1. The number of theoretical plates calculated based on the ferulic acid peak should be not less than 8,000, and record the chromatogram.

[0168] 2. Precision test

[0169] Take the Buxue Yimu Pills (batch number: 20210606, Zhuzhou Qianjin Pharmaceutical Co., Ltd.), prepare the test solution according to Step 1 of this example, prepare the reference substance solution according to Step 1 of this example and conduct the determination.

[0170] Repeat the determination 6 times, use the software of "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints" (version 2012.130723) to obtain the test results, and generate the control characteristic chromatogram. Using ferulic acid (characteristic peak 3) as the reference peak (S peak), calculate the relative retention time and relative peak area of the characteristic peaks in the chromatogram, as well as their average values and relative standard deviations (RSD).

[0171] Table 3 Results of precision test - relative retention time of characteristic peaks

[0172]

[0173] Table 4 Results of precision test - relative peak area of characteristic peaks

[0174]

[0175]

[0176] The results are shown in Table 3 and Table 4. The RSD of the relative retention time of all 8 characteristic peaks is 0.00 - 0.59%, and the RSD of the relative peak area is 0.00 - 40.50%. Among them, the content of peak 5 (rutin) is relatively small, and the shaking extraction step during sample extraction is prone to batch-to-batch non-uniformity, resulting in large fluctuations in the results. After removing the rutin component, the RSD of the relative peak area of the remaining 7 characteristic peaks is 0.00 - 2.47%, indicating good instrument precision.

[0177] 3. Repeatability test

[0178] Take the Buxue Yimu Pills (batch number: 20210606, Zhuzhou Qianjin Pharmaceutical Co., Ltd.), prepare 6 test solutions according to Step 1 of this example, prepare the reference substance solution according to Step 1 of this example and conduct the determination.

[0179] Using ferulic acid (characteristic peak 3) as the reference peak (S peak), calculate the relative retention time and relative peak area of the characteristic peaks in the chromatogram.

[0180] Table 5 Results of Repeatability Test - Relative Retention Time of Characteristic Peaks

[0181]

[0182] Table 6 Results of Repeatability Test - Relative Peak Area of Characteristic Peaks

[0183]

[0184] The results are shown in Table 5 and Table 6. The RSD of the relative retention time of all 8 characteristic peaks is 0.00 - 0.22%, and the RSD of the relative peak area is 0.00 - 2.85%, indicating that the method has good repeatability.

[0185] 4. Stability Test

[0186] Take Buxue Yimu Pills (batch number: 20210606, Zhuzhou Qianjin Pharmaceutical Co., Ltd.), prepare the test solution according to the steps of Step 1 of this example, prepare the reference solution according to the steps of Step 1 of this example, and inject samples for determination at 0h, 1h, 2h, 4h, 8h, 12h, and 24h after the preparation of the test sample.

[0187] Using ferulic acid (characteristic peak 3) as the reference peak (S peak), calculate the relative retention time and relative peak area of the characteristic peaks in the chromatogram.

[0188] Table 7 Results of Stability Test - Relative Retention Time of Characteristic Peaks

[0189]

[0190] Table 8 Results of Stability Test - Relative Peak Area of Characteristic Peaks

[0191]

[0192] The results are shown in Table 7 and Table 8. The RSD of the relative retention time of all 8 characteristic peaks is 0.00 - 0.83%, and the RSD of the relative peak area of each common peak is 0.00 - 41.25%. Among them, the content of peak 5 (rutin) is relatively small, and the shaking extraction step during sample extraction is prone to batch - to - batch non - uniformity, resulting in large fluctuations in the results. After removing the rutin component, the RSD of the relative peak area of all 7 characteristic peaks is 0.00 - 6.06%. At 12h, the retention time and peak area of each component begin to change significantly, indicating that this method is stable within 8h after the preparation of the test sample.

[0193] 5. Investigation of Multi - batch Samples

[0194] Take 14 batches of Buxue Yimu Pills with different batch numbers (batch numbers: 20201217, 20210517, 20210607, 20210805, 20210916, 20211008, 20211014, 20220617, 20220618, 20220619, 20220621, 20220622, 20220623, and 20220624, Zhuzhou Qianjin Pharmaceutical Co., Ltd.), prepare the test solution according to the steps 1 of this example, prepare the reference solution according to the steps 1 of this example and conduct the determination.

[0195] Using the software of "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints" (2012 version), obtain the determination results of 14 batches of Buxue Yimu Pills. Set the chromatogram (S1) of the Buxue Yimu Pills with batch number 20201217 as the reference chromatogram, adopt the average method, with a time window width of 0.1, conduct multi-point calibration and characteristic peak matching, generate the control chromatogram (R), and generate the chromatogram as shown in Figure 10 the figure. Use this software to calculate the relative retention time of the characteristic peaks in the chromatogram and the similarity of 14 batches of samples.

[0196] Table 9 Relative retention time of characteristic chromatograms of 14 batches of Buxue Yimu Pills with different batch numbers

[0197]

[0198] As shown in Table 9, in the characteristic chromatogram of Buxue Yimu Pills, taking the peak corresponding to the ferulic acid reference peak as the S peak, its relative retention time RSD < 5%. According to the average value of the relative retention time of the characteristic peaks of 14 batches of samples, the specified values of the relative retention time of each characteristic peak are determined as: 0.66 (characteristic peak 1), 0.93 (characteristic peak 2), 1.11 (characteristic peak 4), 1.24 (characteristic peak 5), 1.68 (characteristic peak 6), 1.71 (characteristic peak 7), and 2.61 (characteristic peak 8).

[0199] Table 10 Similarity of 14 batches of Buxue Yimu Pills with different batch numbers

[0200]

[0201] As shown in Table 10, the similarity of 14 batches of samples is 0.958 - 0.999, all above 0.90, indicating that all 14 batches of samples are Buxue Yimu Pills with qualified quality.

[0202] In summary, the precision, stability, repeatability, etc. of the UPLC characteristic chromatogram and detection method of the Buxue Yimu Pills provided by the present invention all meet the regulations.

[0203] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description and ideas. It is not necessary and impossible to list all implementation manners here. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A method for constructing the UPLC characteristic fingerprint of Buxue Yimu Pills, which is characterized in that, It includes the following steps: S1. Prepare the test sample; methanol solutions of ferulic acid reference substance, leonurine hydrochloride reference substance, calycosin-7-O-β-D-glucoside reference substance, rutin reference substance, senkyunolide I reference substance, hesperidin reference substance and ligustilide reference substance are used as reference substances. The preparation method of the test sample includes the following steps: The powder of Buxue Yimu Pills is extracted by heating under reflux with ethanol with a volume percentage of 70-80% to obtain Extract 1. After removing impurities, the solvent of Extract 1 is removed to obtain Precipitate 1. The aqueous solution of Precipitate 1 is extracted with a water-saturated n-butanol solution to obtain Extract 2. The solvent of Extract 2 is removed to obtain Precipitate 2. Precipitate 2 is fully dissolved with ethanol with a volume percentage of 70-80% to obtain the test sample. S2. Perform ultra-high performance liquid chromatography detection on the test sample and reference substances obtained in the previous step to obtain the UPLC characteristic fingerprint of Buxue Yimu Pills. The characteristic peaks of the UPLC characteristic fingerprint of Buxue Yimu Pills include leonurine hydrochloride, ferulic acid, calycosin-7-O-β-D-glucoside, rutin, senkyunolide I, hesperidin and ligustilide. The detection conditions of the ultra-high performance liquid chromatography are as follows: Use a T3 chromatographic column; the detection wavelength is 270-290 nm; an aqueous solution of formic acid with a volume percentage of 0.4-0.8% is used as mobile phase A, and acetonitrile is used as mobile phase B. In the gradient elution program, the volume percentage change of mobile phase A is as follows: 0-2 min, mobile phase A is 92%; 2-5 min, mobile phase A drops from 92% to 85%; 5-12 min, mobile phase A drops from 85% to 84%; 12-13 min, mobile phase A drops from 84% to 83%; 13-14 min, mobile phase A drops from 83% to 81%; 14-17 min, mobile phase A drops from 81% to 68%; 17-19 min, mobile phase A drops from 68% to 60%; 19-25 min, mobile phase A drops from 60% to 40%; 25-28 min, mobile phase A drops from 40% to 20%.

2. The method according to claim 1, wherein In step S1, in the aqueous solution of Precipitate 1, the concentration of Precipitate 1 is 0.03-0.1 g / mL.

3. The method according to claim 1, characterized in that, In step S1, the temperature of the heating under reflux extraction is 70-100 °C, and the time is 2-3 hours.

4. The method according to claim 3, characterized in that, In step S1, the temperature of the heating under reflux extraction is 85 °C, and the time is 2.5 hours.

5. The method according to claim 1, characterized in that, In step S1, the aqueous solution of Precipitate 1 is shaken and extracted with a water-saturated n-butanol solution, and the number of shaking times is 1-3 times.

6. The method according to claim 5, wherein In step S1, the aqueous solution of Precipitate 1 is shaken and extracted with a water-saturated n-butanol solution, and the number of shaking times is 3 times.

7. The method according to claim 1, wherein In step S2, the detection conditions of the ultra-high performance liquid chromatography further include: the flow rate is 0.2-0.4 mL / min.

8. The method according to claim 1, wherein In step S2, the detection conditions of the ultra-high performance liquid chromatography further include: the injection volume is 1-3 μL.

9. The method according to claim 1, wherein In step S2, the detection conditions of the ultra-high performance liquid chromatography further include: the column temperature of the T3 chromatographic column is 30-35 °C.

10. Use of the method according to any one of claims 1 to 9 in the component analysis and / or quality inspection of Buxue Yimu Pills.

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