Method for determining contents of multiple components in a preparation for treating static blood obstruction arthralgia and constructing fingerprint spectrum

Abstract

The present invention relates to the field of traditional Chinese medicine analysis, and particularly to a method for determining the contents of multiple components in a Yuxuebi preparation and constructing a fingerprint. The present invention uses 5-hydroxymethylfurfural, danshensu, chlorogenic acid, ferulic acid, isochlorogenic acid B, rosmarinic acid, salvianolic acid B, salvianolic acid A, dihydrotanshinone I, cryptotanshinone, tanshinone IIA, and 3-acetyl-11-keto-β-boswellic acid as index components, and adopts ultra-high performance liquid chromatography for content determination and fingerprint construction. The method provided by the present invention can simply, rapidly, and accurately determine the contents of the above 12 components in the Yuxuebi preparation, with good accuracy, high sensitivity, and good stability. It can be used for more comprehensive quality control of the Yuxuebi preparation and for constructing the fingerprint of the Yuxuebi preparation. Based on this method, the present invention constructs the fingerprint of the Yuxuebi capsule, provides a basis for the quality control of the Yuxuebi capsule, and further improves the quality consistency and clinical medication effectiveness of this drug.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine analysis, and particularly to a method for determining the contents of multiple components in a Yuxuebi preparation and constructing a fingerprint. Background Art

[0002] Commercially available Yuxuebi preparations include Yuxuebi tablets, Yuxuebi granules, and Yuxuebi capsules, etc., which are made from 11 Chinese medicinal materials including frankincense, clematis root, safflower, salvia miltiorrhiza, myrrh, cyathula root, chuanxiong rhizome, angelica sinensis, turmeric, processed lindera root, and roasted astragalus membranaceus, and have the effects of promoting blood circulation to remove blood stasis and dredging collaterals to relieve pain, and are used for arthralgia syndrome caused by blood stasis blocking collaterals, with symptoms such as severe pain in muscles and joints, pain at the affected area being refused to be pressed, fixed without moving, and there may be hard nodules or ecchymosis.

[0003] The clinical efficacy of Yuxuebi is remarkable, but the quality detection method is not yet perfect. The current standards for Yuxuebi capsules and Yuxuebi granules are included in the Chinese Pharmacopoeia (2020 Edition). The inspection items for Yuxuebi capsules include microscopic identification of cyathula root and astragalus membranaceus, and thin-layer chromatography identification of clematis root, chuanxiong rhizome, angelica sinensis, and astragalus membranaceus; determination of the content of salvianolic acid B, and it is required that the content of salvianolic acid B contained in each Yuxuebi capsule shall not be less than 2.8 mg. The inspection items for Yuxuebi granules include thin-layer chromatography identification of cyathula root, angelica sinensis, chuanxiong rhizome, and astragalus membranaceus; determination of the content of salvianolic acid B, and it is required that each bag contains not less than 25.0 mg of salvia miltiorrhiza calculated as salvianolic acid B.

[0004] The chemical components of Yuxuebi preparations are complex. A single index component cannot reflect the situation of other components in Yuxuebi preparations, and it is impossible to effectively control the quality of the preparations. The types and contents of chemical components in the preparations directly affect the product quality and further affect its clinical efficacy. Existing analysis and detection methods have certain limitations in the quantitative analysis of index components, and it is difficult to simultaneously detect the contents of multiple components in Yuxuebi preparations. There is an urgent need to construct a multi-component quantitative analysis method and a characteristic fingerprint evaluation method.

[0005] Therefore, in order to better control the quality of Yuxuebi preparations and effectively guarantee their clinical efficacy, it is particularly necessary to establish a method for detecting multiple index components and analyzing the characteristic fingerprint. Summary of the Invention

[0006] Aiming at the problem that the prior art cannot simultaneously detect multiple components in Yuxuebi preparations, the present invention provides a method for determining the contents of multiple components in a Yuxuebi preparation and constructing a fingerprint. This content determination method can simultaneously detect more than ten active components in Yuxuebi preparations, with good accuracy, high sensitivity, and good stability, which helps to comprehensively reflect its internal quality. On this basis, the fingerprint of Yuxuebi preparation constructed can provide a basis for the quality control of Yuxuebi preparation.

[0007] To achieve the above invention purpose, the present invention provides the following technical solutions:

[0008] A method for determining the contents of multiple components in a Yuxuebi preparation comprises extracting the Yuxuebi preparation with a methanol aqueous solution, and determining the contents of the target components by ultra-high performance liquid chromatography (UPLC), wherein the target components include 5-hydroxymethylfurfural, danshensu, chlorogenic acid, ferulic acid, isochlorogenic acid B, rosmarinic acid, salvianolic acid B, salvianolic acid A, dihydrotanshinone I, cryptotanshinone, tanshinone IIA and 3-acetyl-11-keto-β-boswellic acid; and the chromatographic conditions of the ultra-high performance liquid chromatography include:

[0009] Chromatographic column: C18 bonded silica gel column;

[0010] Mobile phase: Mobile phase A is 0.19% to 0.21% formic acid aqueous solution (v / v), mobile phase B is acetonitrile, and the mobile phase A and mobile phase B are used for gradient elution. The procedure of the gradient elution is as follows:

[0011]

[0012] The detection wavelengths are as follows:

[0013]

[0014]

[0015] The above-mentioned components to be tested are all active ingredients in the Yuxuebi preparation and are related to the clinical efficacy of the Yuxuebi preparation. For example, 5-hydroxymethylfurfural has antioxidant and anti-inflammatory effects, chlorogenic acid, isochlorogenic acid B, danshensu, salvianolic acid A, salvianolic acid B, rosmarinic acid, cryptotanshinone, and tanshinone IIA have antioxidant, anti-inflammatory, antibacterial, and antiplatelet aggregation effects, ferulic acid has anti-inflammatory, antithrombotic, and lipid-lowering effects, and dihydrotanshinone I and 3-acetyl-11-keto-β-boswellic acid have anti-inflammatory effects.

[0016] The content determination method provided by the present invention has good accuracy, high sensitivity and good stability, and can simply, quickly and accurately determine the contents of the above 12 components in the Yuxuebi preparation, can be used for more comprehensive quality inspection and control of the Yuxuebi preparation, and can be used for the construction of the fingerprint of the Yuxuebi preparation.

[0017] The gradient elution procedure specifically represents: from 0 to 2 min, the volume percentage of mobile phase A decreases uniformly from 97% to 90%, from 2 to 3 min, the volume percentage of mobile phase A decreases uniformly from 90% to 80%, from 3 to 7 min, the volume percentage of mobile phase A decreases uniformly from 80% to 72%, from 7 to 7.5 min, the volume percentage of mobile phase A decreases uniformly from 72% to 55%, and from 7.5 to 20 min, the volume percentage of mobile phase A decreases uniformly from 55% to 10%.

[0018] In combination with the first aspect, mobile phase A is preferably an aqueous solution of 0.2% formic acid (v / v).

[0019] In combination with the first aspect, the chromatographic conditions further include a column temperature of 35 - 45 °C; preferably 40 °C.

[0020] In combination with the first aspect, the chromatographic conditions further include a flow rate of 0.25 - 0.35 mL / min.

[0021] In combination with the first aspect, the C18-bonded silica gel chromatographic column is ACQUITY BEH C18 (2.1×100 mm, 1.7 μm).

[0022] In combination with the first aspect, the specific operation of extracting the Yuxue Bi preparation with an aqueous methanol solution is as follows: performing ultrasonic extraction with a 25% - 100% aqueous methanol solution (v / v). Further preferably, it is methanol, that is, 100% methanol.

[0023] Preferably, the temperature of the ultrasonic extraction is 55 - 65 °C; the time of the ultrasonic extraction is 20 - 40 min.

[0024] Optionally, the temperature of the ultrasonic extraction is 60 °C; the time of the ultrasonic extraction is 30 min.

[0025] In combination with the first aspect, the mass-volume ratio of the Yuxue Bi preparation to the aqueous methanol solution is 1:62.5 - 250 (g:mL), and further preferably 1:125 (g:mL). When the Yuxue Bi preparation is in capsule form, the calculation of the mass-volume ratio is based on the mass of the capsule content.

[0026] In combination with the first aspect, the content determination method includes the following steps:

[0027] Step a: Extract the Yuxue Bi preparation with an aqueous methanol solution to prepare a test solution.

[0028] Step b: Prepare a reference solution of the target index component to be measured.

[0029] Step c: Use the ultra-high performance liquid chromatography method to measure the reference solution and the test solution, and calculate the content of the target index component in the test sample by the external standard method.

[0030] Preferably, in step a, the aqueous methanol solution is methanol (i.e., 100% methanol), and the obtained extract is diluted with water or methanol to prepare a test solution.

[0031] Water-soluble components such as 5-hydroxymethylfurfural, danshensu, and chlorogenic acid will be affected by the solvent effect of methanol, which will affect their peak shapes. Therefore, when detecting 5-hydroxymethylfurfural, danshensu, and chlorogenic acid, water dilution can be selected. After dilution, filter, and use the obtained test solution for the detection of 5-hydroxymethylfurfural, danshensu, and chlorogenic acid.

[0032] When diluted with water, the methanol concentration in the solution is too low, and the detection of lipophilic components is incomplete. Therefore, when detecting other compounds, methanol dilution can be selected. After dilution, the obtained test solution is used for the detection of ferulic acid, isochlorogenic acid B, rosmarinic acid, salvianolic acid B, salvianolic acid A, dihydrotanshinone I, cryptotanshinone, tanshinone IIA, and 3-acetyl-11-keto-β-boswellic acid.

[0033] Second, the present invention also provides a method for constructing a fingerprint of a Yuxuebi preparation, including the following steps:

[0034] S1. Prepare a reference solution of the index components; the index components include ferulic acid, isochlorogenic acid B, rosmarinic acid, salvianolic acid B, salvianolic acid A, dihydrotanshinone I, cryptotanshinone, tanshinone IIA, and 3-acetyl-11-keto-β-boswellic acid;

[0035] S2. Take different batches of Yuxuebi preparations, extract the preparations with methanol respectively to obtain test solutions of different batches of Yuxuebi preparations, use ultra-high performance liquid chromatography in the above content determination method to measure the reference solution and the test solutions, record the fingerprint, import all the fingerprints into the similarity evaluation system software for traditional Chinese medicine chromatographic fingerprints, select the common characteristic chromatographic peaks in the fingerprints of different batches of Yuxuebi capsules as characteristic fingerprint peaks, generate a standard reference fingerprint, and calculate the relative retention time and relative peak area of each common characteristic fingerprint peak.

[0036] The fingerprint constructed by the method for constructing a fingerprint of a Yuxuebi preparation provided by the present invention can obtain the fingerprint information of nine active ingredients, so that common peaks can be determined therefrom and a characteristic fingerprint can be constructed, which is used for a comprehensive and rapid quality evaluation of the Yuxuebi preparation, provides a basis for the quality control of the Yuxuebi preparation, and further improves the quality consistency and clinical medication effectiveness of the drug.

[0037] Third, the present invention also provides the application of the above content detection method and fingerprint construction method in the quality control of Yuxuebi preparations.

[0038] Using the above content detection method of Yuxuebi preparations provided by the present invention and the fingerprint obtained by using the above fingerprint construction method can accurately, quickly, and comprehensively reflect the content of various active ingredients in the Yuxuebi preparation, thus facilitating its quality control.

[0039] Fourthly, the present invention also provides a fingerprint of Yuxuebi Capsules, and the fingerprint of Yuxuebi Capsules contains 9 characteristic fingerprint peaks: ferulic acid, isochlorogenic acid B, rosmarinic acid, salvianolic acid B, salvianolic acid A, dihydrotanshinone I, cryptotanshinone, tanshinone IIA, and 3-acetyl-11-keto-β-boswellic acid in sequence.

[0040] Combined with the fourth aspect, taking salvianolic acid B as the reference peak (Z4), the relative retention times of each common peak and the reference peak are within the range of ±5% of the specified value; the specified values are: peak Z1 is 0.7728, peak Z2 is 0.8058, peak Z3 is 0.9172, peak Z4 is 1.000, peak Z5 is 1.106, peak Z6 is 1.701, peak Z7 is 1.928, peak Z8 is 2.190, and peak Z9 is 2.811.

[0041] The beneficial effects of the present invention are as follows:

[0042] Based on the UPLC-PDA timing switching wavelength technology, the content determination method for multiple components in the Yuxuebi preparation established by the present invention can simultaneously determine 12 index components related to its clinical efficacy in the Yuxuebi preparation. After methodological verification, the precision, repeatability, stability, and sample addition recovery rate of this detection method are all good and meet the methodological requirements. Therefore, the data obtained by this content detection method are accurate and reliable and can be used for the content determination of the above 12 index components in the Yuxuebi preparation. The present invention also establishes a fingerprint construction method for the Yuxuebi preparation and obtains 9 characteristic fingerprint peaks of the commercially available Yuxuebi Capsules. The similarity values of the fingerprints of 20 batches of Yuxuebi Capsules and the control fingerprint are all greater than 0.9. The present invention selects salvianolic acid B as the reference peak and determines the relative retention times of the common peaks in the fingerprint of the Yuxuebi preparation. The above content determination method, fingerprint construction method, and the fingerprint obtained therefrom provided by the present invention can comprehensively, effectively, and efficiently reflect the situation of multiple active components in the Yuxuebi preparation, which is beneficial to the comprehensive quality detection and overall quality control of the Yuxuebi preparation, thereby helping to improve the quality stability and clinical medication effectiveness of this drug. Description of the Drawings

[0043] Figure 1 It is the ultra-high performance liquid chromatogram of the mixed reference substance solution in Example 1;

[0044] Figure 2 It is the ultra-high performance liquid chromatogram of the test solution diluted with water in Example 1;

[0045] Figure 3It is the ultra - performance liquid chromatography (UPLC) chromatogram of the test sample solution diluted with methanol in Example 1; in the figure, peak 1 is 5 - hydroxymethylfurfural, peak 2 is danshensu, peak 3 is chlorogenic acid, peak 4 (Z1) is ferulic acid, peak 5 (Z2) is isochlorogenic acid B, peak 6 (Z3) is rosmarinic acid, peak 7 (Z4) is salvianolic acid B, peak 8 (Z5) is salvianolic acid A, peak 9 (Z6) is dihydrotanshinone I, peak 10 (Z7) is cryptotanshinone, peak 11 (Z8) is tanshinone IIA, and peak 12 (Z9) is 3 - acetyl - 11 - keto - β - boswellic acid;

[0046] Figure 4 It is the ultra - performance liquid chromatography fingerprint (Z1 - Z9) of 20 batches of Yuxuebi Capsules in Example 2;

[0047] Figure 5 It is the characteristic fingerprint of Yuxuebi Capsules in Example 2. Detailed implementation manners

[0048] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0049] Currently, the quality control indexes of Yuxuebi preparations are all too single and cannot reflect the content of multiple components in Yuxuebi preparations at the same time. For traditional Chinese medicine preparations, the content of active ingredients has a direct impact on their clinical efficacy. In addition, fingerprint technology can comprehensively reflect the chemical characteristics of multiple index components in traditional Chinese medicine preparations, provide rich drug component information, and is a supplement and improvement to the quality control of traditional Chinese medicine. Therefore, a method for accurately, efficiently and rapidly detecting the content of multiple active ingredients in Yuxuebi preparations and a method for constructing fingerprint maps are of great significance for comprehensively reflecting its internal quality and ensuring clinical efficacy.

[0050] The present invention will be further described below with multiple embodiments.

[0051] The Yuxuebi Capsules used in the following examples are 20 batches of drugs provided by Liaoning Shangyao Haohushi Pharmaceutical Co., Ltd.

[0052] Example 1

[0053] The embodiment of the present invention provides a method for determining the content of multiple components in Yuxuebi Capsules, including the following steps:

[0054] Step a. Preparation of the test sample solution

[0055] Accurately weigh 0.2 g of the contents of Yuxuebi Capsules, place it in a 50 mL stoppered conical flask, accurately add 25 mL of methanol with a pipette, weigh it, ultrasonically extract at 60 °C for 30 min, after allowing to stand at room temperature, weigh it again, make up the lost weight with methanol, shake well, take the supernatant and centrifuge for 10 min, take 1 mL of the supernatant, add 1 mL of water, mix well, and use it for the determination of 5-hydroxymethylfurfural, danshensu, and chlorogenic acid; take 1 mL of the supernatant, add 1 mL of methanol, mix well, and use it for the determination of ferulic acid, isochlorogenic acid B, rosmarinic acid, salvianolic acid B, salvianolic acid A, dihydrotanshinone I, cryptotanshinone, tanshinone IIA, and 3-acetyl-11-keto-β-boswellic acid.

[0056] Step b. Preparation of the reference substance solution

[0057] Weigh an appropriate amount of 12 reference substances accurately. The reference substances of 5-hydroxymethylfurfural, danshensu, chlorogenic acid, ferulic acid, isochlorogenic acid B, rosmarinic acid, and salvianolic acid B are dissolved in 50% methanol aqueous solution (v / v), and the reference substances of salvianolic acid A, dihydrotanshinone I, cryptotanshinone, tanshinone IIA, and 3-acetyl-11-keto-β-boswellic acid are dissolved in methanol, and respectively prepare reference substance stock solutions with concentrations of 0.3216 mg / mL, 0.5020 mg / mL, 0.1006 mg / mL, 0.05020 mg / mL, 0.05020 mg / mL, 0.2510 mg / mL, 2.004 mg / mL, 0.2008 mg / mL, 0.04980 mg / mL, 0.1992 mg / mL, 0.2000 mg / mL, and 0.4980 mg / mL.

[0058] Accurately measure an appropriate amount of each reference substance stock solution, place it in a 10 mL volumetric flask, add water to the scale, shake well, and obtain a mixed reference substance solution. The mixed reference substance solution contains 5-hydroxymethylfurfural 15.76 μg / mL, danshensu 33.63 μg / mL, chlorogenic acid 5.835 μg / mL, ferulic acid 3.112 μg / mL, isochlorogenic acid B 5.823 μg / mL, rosmarinic acid 21.59 μg / mL, salvianolic acid B 356.7 μg / mL, salvianolic acid A 20.08 μg / mL, dihydrotanshinone I 3.486 μg / mL, cryptotanshinone 10.96 μg / mL, tanshinone IIA 10.00 μg / mL, and 3-acetyl-11-keto-β-boswellic acid 40.34 μg / mL; use 50% methanol aqueous solution (v / v) to gradually dilute it into a series of mixed reference substance solutions with different concentrations

[0059] Step c. Use a Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph to determine the above-mentioned mixed reference substance solution and the test solution. The chromatographic conditions are as follows:

[0060] Chromatographic column: ACQUITY BEH C18 (2.1×100 mm, 1.7 μm);

[0061] Column temperature: 40 °C;

[0062] Sample injection volume: 2 μL;

[0063] Flow rate: 0.3 mL / min;

[0064] Detection wavelengths are as follows:

[0065]

[0066] Mobile phase: Phase A is 0.2% formic acid aqueous solution (v / v), and Phase B is acetonitrile;

[0067] Gradient elution conditions are:

[0068]

[0069]

[0070] The ultra-high performance liquid chromatography (UHPLC) chromatogram of the mixed reference substance solution is as Figure 1 shown;

[0071] The UHPLC chromatogram of the test solution diluted with water is as Figure 2 shown; The UHPLC chromatogram of the test solution diluted with methanol is as Figure 3 shown.

[0072] The external standard method is used to calculate the contents of 12 components in the sample, and the formula is as follows:

[0073]

[0074] Among them, w is the content of the target component to be measured in the content of the Yuxuebi Capsules to be measured, with the unit of μg / g;

[0075] Cs is the concentration of the component in the reference substance solution, with the unit of μg / mL;

[0076] An is the peak area of the target component to be measured in the test solution;

[0077] As is the peak area of the component in the reference substance solution;

[0078] W is the actual weighed amount of the content of the Yuxuebi Capsules to be measured, with the unit of g.

[0079] Test Example 1

[0080] This test example of the present invention provides a methodological investigation of the method for determining the contents of multiple components in the Yuxuebi Capsules in Example 1, where:

[0081] (1) Study on linear relationship, detection limit and quantitation limit

[0082] Prepare a mixed reference solution according to the method in step b of Example 1, and determine the peak area by ultra-high performance liquid chromatography. The chromatographic conditions are the same as those in step c of Example 1. Inject samples in parallel 2 times. Take the concentration of each compound reference as the abscissa x (μg / mL), and the corresponding reference peak area as the ordinate y to plot the regression equation; take the signal-to-noise ratio S / N = 3 as the detection limit (LOD) and S / N = 10 as the quantitation limit (LOQ) to determine the detection limit and quantitation limit of 5-hydroxymethylfurfural, danshensu, chlorogenic acid, ferulic acid, isochlorogenic acid B, rosmarinic acid, salvianolic acid B, salvianolic acid A, dihydrotanshinone I, cryptotanshinone, tanshinone IIA, 3-acetyl-11-keto-β-boswellic acid. The results are shown in Table 1.

[0083] Table 1 Linear regression equations, linear ranges, correlation coefficients, detection limits and quantitation limits of 12 index components

[0084]

[0085] As can be seen from Table 1, each component has a good linear relationship in its respective concentration range (r 2 ≥0.999). The concentration range of the detection limit is 0.02279 - 0.6303 μg / mL, and the concentration range of the quantitation limit is 0.04558 - 1.261 μg / mL, indicating that the content determination method for multiple components in the Yuxuebi Capsules provided by the present invention has relatively high sensitivity.

[0086] (2) Precision test

[0087] (a) Intra-day precision test

[0088] Prepare a test solution of Yuxuebi Capsules according to the method in step a of Example 1. Precisely pipette the same portion of the above test solution and inject samples repeatedly 6 times. Determine the peak area by ultra-high performance liquid chromatography. The chromatographic conditions are the same as those in step c of Example 1. Calculate the RSD values based on the peak areas of 5-hydroxymethylfurfural, danshensu, chlorogenic acid, ferulic acid, isochlorogenic acid B, rosmarinic acid, salvianolic acid B, salvianolic acid A, dihydrotanshinone I, cryptotanshinone, tanshinone IIA, 3-acetyl-11-keto-β-boswellic acid respectively. The results are shown in Table 2 (where the data of 5-hydroxymethylfurfural, danshensu, and chlorogenic acid are from the test solution diluted with water, and the data of the remaining compounds are from the test solution diluted with methanol).

[0089] Table 2 Results of intra-day precision test for multi-component determination of Yuxuebi Capsules (n = 6)

[0090]

[0091] As can be seen from Table 2, the intra-day precision RSD of the determination methods for multiple components in the Yuxue Bi Capsules provided by the present invention is < 2.9%.

[0092] (b) Inter-day precision test

[0093] Prepare the test solution of Yuxue Bi Capsules according to the method in step a of Example 1. Continuously for 3 days, precisely pipette the same portion of the above test solution every day, inject samples repeatedly 6 times, and determine the peak area by ultra-high performance liquid chromatography. The chromatographic conditions are the same as those in step c of Example 1. Calculate the RSD value based on the average peak areas of 5-hydroxymethylfurfural, danshensu, chlorogenic acid, ferulic acid, isochlorogenic acid B, rosmarinic acid, salvianolic acid B, salvianolic acid A, dihydrotanshinone I, cryptotanshinone, tanshinone IIA, and 3-acetyl-11-keto-β-boswellic acid. The results are shown in Table 3 (where the data of 5-hydroxymethylfurfural, danshensu, and chlorogenic acid are from the test solution diluted with water, and the data of the remaining compounds are from the test solution diluted with methanol).

[0094] Table 3 Results of the inter-day precision test for the determination of multiple components in Yuxue Bi Capsules (n = 3)

[0095]

[0096] As can be seen from Table 3, the inter-day precision RSD of the determination methods for multiple components in the Yuxue Bi Capsules provided by the present invention is < 2.7%.

[0097] As can be seen from Table 2 and Table 3, the precision of the determination methods for multiple components in the Yuxue Bi Capsules provided by the present invention is good.

[0098] (3) Repeatability test

[0099] Parallelly prepare 6 test solutions of Yuxue Bi Capsules according to the method in step a of Example 1. Determine the above test solutions by ultra-high performance liquid chromatography, calculate the contents of each compound, and the chromatographic conditions are the same as those in step c of Example 1. Calculate the contents of 5-hydroxymethylfurfural, danshensu, chlorogenic acid, ferulic acid, isochlorogenic acid B, rosmarinic acid, salvianolic acid B, salvianolic acid A, dihydrotanshinone I, cryptotanshinone, tanshinone IIA, and 3-acetyl-11-keto-β-boswellic acid by the external standard method and calculate the RSD value. The results are shown in Table 4 (where the data of 5-hydroxymethylfurfural, danshensu, and chlorogenic acid are from the test solution diluted with water, and the data of the remaining compounds are from the test solution diluted with methanol).

[0100]

[0101] As can be seen from Table 4, in this example, the content of 5-hydroxymethylfurfural is 511.6 μg / g, the content of danshensu is 1070 μg / g, the content of chlorogenic acid is 194.5 μg / g, the content of ferulic acid is 102.9 μg / g, the content of isochlorogenic acid B is 181.1 μg / g, the content of rosmarinic acid is 649.4 μg / g, the content of salvianolic acid B is 10.34 mg / g, the content of salvianolic acid A is 687.7 μg / g, the content of dihydrotanshinone I is 89.16 μg / g, the content of cryptotanshinone is 377.0 μg / g, the content of tanshinone IIA is 327.6 μg / g, and the content of 3-acetyl-11-keto-β-boswellic acid is 1543 μg / g. The RSD of each compound is less than 2.7%, indicating that the repeatability of this detection method is good.

[0102] (4) Stability test

[0103] Prepare the test solution of Yuxue Bi Capsule according to the method of step a in Example 1, store it at room temperature, and accurately absorb the same portion of the above test solution at 0, 2, 4, 6, 8, 10, and 12 h respectively. Determine the above test solution by ultra-high performance liquid chromatography, and the chromatographic conditions are the same as those in step c of Example 1. Calculate the RSD value based on the peak areas of 5-hydroxymethylfurfural, danshensu, chlorogenic acid, ferulic acid, isochlorogenic acid B, rosmarinic acid, salvianolic acid B, salvianolic acid A, dihydrotanshinone I, cryptotanshinone, tanshinone IIA, and 3-acetyl-11-keto-β-boswellic acid. The results are shown in Table 5 (where the data of 5-hydroxymethylfurfural, danshensu, and chlorogenic acid are from the test solution diluted with water, and the data of the remaining compounds are from the test solution diluted with methanol).

[0104]

[0105] As can be seen from Table 5, the RSD of the stability test of the method for determining multiple components of Yuxue Bi Capsule provided by the present invention is <2.3%, indicating that the test solution is stable within 12 h under normal temperature conditions.

[0106] (5) Recovery test

[0107] Take about 0.1 g of the content of Yuxuebi Capsules, accurately weigh it, and accurately add 5 mL of a mixed reference substance solution equivalent to the mass of each component in the sample (5-hydroxymethylfurfural 10.29 μg / mL, danshensu 21.08 μg / mL, chlorogenic acid 4.024 μg / mL, ferulic acid 2.008 μg / mL, isochlorogenic acid B 3.614 μg / mL, rosmarinic acid 13.05 μg / mL, salvianolic acid B 0.2044 mg / mL, salvianolic acid A 13.65 μg / mL, dihydrotanshinone I 1.793 μg / mL, cryptotanshinone 7.570 μg / mL, tanshinone IIA 6.800 μg / mL, 3-acetyl-11-keto-β-boswellic acid 30.88 μg / mL).

[0108] Prepare 6 test solution samples of Yuxuebi Capsules according to the method in step a of Example 1. Each sample is determined in parallel 2 times. The above test solution samples are determined by ultra-high performance liquid chromatography, and the chromatographic conditions are the same as those in step c of Example 1. Calculate the recovery rate and RSD value of 5-hydroxymethylfurfural, danshensu, chlorogenic acid, ferulic acid, isochlorogenic acid B, rosmarinic acid, salvianolic acid B, salvianolic acid A, dihydrotanshinone I, cryptotanshinone, tanshinone IIA, and 3-acetyl-11-keto-β-boswellic acid according to the spiked recovery rate formula to investigate the accuracy of this method.

[0109] The spiked recovery rate formula is as follows:

[0110]

[0111] The results are shown in Table 6 (where the data of 5-hydroxymethylfurfural, danshensu, and chlorogenic acid are from the test solution samples diluted with water, and the data of the remaining compounds are from the test solution samples diluted with methanol).

[0112] Table 6 Results of the spiked recovery rate test for the determination of multi-component contents in Yuxuebi Capsules (n = 6)

[0113]

[0114]

[0115]

[0116] As can be seen from Table 6, the average recovery rate of the method for the determination of multi-component contents in the Yuxuebi Capsules provided by the present invention is between 89.56% - 109.6% (except for 5-hydroxymethylfurfural which is 83.70% - 88.96%), and the RSD values are all less than 2.9%. This indicates that the spiked recovery rates of each compound basically meet the requirements, and this method is accurate and feasible.

[0117] As can be seen from Tables 1 to 6, the content determination method for multiple components in the Yuxuebi Capsules provided by the present invention meets the requirements of content determination methodology research and can be used for the quantitative analysis of Yuxuebi preparations. The content determination results of multiple components in different batches of Yuxuebi Capsules are shown in Table 7.

[0118]

[0119]

[0120] As can be seen from the results in Table 7, among the 20 batches of Yuxuebi Capsule samples, the content of 5-hydroxymethylfurfural is 52.42 - 1286 μg / g; the content of danshensu is 720.6 - 1236 μg / g; the content of chlorogenic acid is 111.3 - 233.8 μg / g; the content of ferulic acid is 18.85 - 158.8 μg / g; the content of isochlorogenic acid B is 161.8 - 262.5 μg / g; the content of rosmarinic acid is 493.5 - 716.3 μg / g; the content of salvianolic acid B is 8.104 - 10.94 mg / g; the content of salvianolic acid A is 542.9 - 838.8 μg / g; the content of dihydrotanshinone I is 57.16 - 156.5 μg / g; the content of cryptotanshinone is 255.9 - 450.5 μg / g; the content of tanshinone IIA is 223.0 - 409.8 μg / g; the content of 3-acetyl-11-keto-β-boswellic acid is 822.6 - 1271 μg / g.

[0121] Example 2

[0122] The embodiment of the present invention provides a method for constructing a characteristic fingerprint of Yuxuebi Capsules, which includes the following steps:

[0123] S1. Preparation of reference substance solutions: Weigh an appropriate amount of 9 reference substances accurately. The reference substances of ferulic acid, isochlorogenic acid B, rosmarinic acid, and salvianolic acid B are dissolved in 50% methanol aqueous solution (v / v), and the reference substances of salvianolic acid A, dihydrotanshinone I, cryptotanshinone, tanshinone IIA, and 3-acetyl-11-keto-β-boswellic acid are dissolved in methanol, and reference substance stock solutions with concentrations of 0.05020 mg / mL, 0.05020 mg / mL, 0.2510 mg / mL, 2.004 mg / mL, 0.2008 mg / mL, 0.04980 mg / mL, 0.1992 mg / mL, 0.2000 mg / mL, and 0.4980 mg / mL are prepared respectively.

[0124] Precisely measure appropriate amounts of each reference solution, transfer them into a 10 mL volumetric flask, add water to the mark, shake well to obtain a mixed reference solution. The mixed reference solution contains ferulic acid at 3.112 μg / mL, isochlorogenic acid B at 5.823 μg / mL, rosmarinic acid at 21.59 μg / mL, salvianolic acid B at 356.7 μg / mL, salvianolic acid A at 20.08 μg / mL, dihydrotanshinone I at 3.486 μg / mL, cryptotanshinone at 10.96 μg / mL, tanshinone IIA at 10.00 μg / mL, and 3-acetyl-11-keto-β-boswellic acid at 40.34 μg / mL; gradually dilute it with 50% methanol aqueous solution into a series of mixed reference solutions with different concentrations.

[0125] S2. Precisely weigh 0.2 g of the contents of Yuxuebi Capsules, transfer it into a 50 mL stoppered conical flask, accurately add 25 mL of methanol solution with a pipette, weigh it, ultrasonically extract for 30 min at 60 °C, let it stand at room temperature, weigh it again, make up the lost weight with methanol, shake well, take the supernatant and centrifuge for 10 min, take 1 mL and add 1 mL of methanol, mix well.

[0126] Use a Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph to determine the above-mentioned mixed reference solution and test solution, record the fingerprint, import all the fingerprints into the similarity evaluation system software for traditional Chinese medicine chromatographic fingerprints, select the common chromatographic peaks in the fingerprints of different batches of Yuxuebi Capsules as characteristic fingerprint peaks, and generate fingerprints using the timed wavelength switching technique according to the different absorption characteristics of each characteristic fingerprint peak to obtain a standard control fingerprint, and calculate the relative retention time and relative peak area of each characteristic fingerprint peak; the chromatographic conditions are as follows:

[0127] Chromatographic column: BEH C18 (2.1×100 mm, 1.7 μm);

[0128] Column temperature: 40 °C;

[0129] Injection volume: 2 μL;

[0130] Flow rate: 0.3 mL / min;

[0131] Detection wavelengths are as follows:

[0132]

[0133]

[0134] Mobile phase: Phase A is 0.2% formic acid aqueous solution (v / v), and phase B is acetonitrile;

[0135] Gradient elution conditions are as follows:

[0136]

[0137] According to the determination results of the relative retention times of each compound in the fingerprints of 20 batches of Yuxuebi Capsules, 9 common chromatographic peaks with larger peak areas, better peak shapes and resolutions were selected as characteristic fingerprint peaks. By comparing with the reference substance chromatogram, each characteristic fingerprint peak was identified as ferulic acid (Z1), isochlorogenic acid B (Z2), rosmarinic acid (Z3), salvianolic acid B (Z4), salvianolic acid A (Z5), dihydrotanshinone I (Z6), cryptotanshinone (Z7), tanshinone IIA (Z8), 3-acetyl-11-keto-β-boswellic acid (Z9). The standard reference fingerprint was generated. Taking the Z4 peak as the reference peak, the relative retention times and relative peak areas of the 9 common chromatographic peaks are shown in Tables 8 and 9;

[0138]

[0139]

[0140]

[0141] Test Example 2

[0142] The test example of the present invention provides a methodological investigation of the method for constructing the characteristic fingerprint of Yuxuebi Capsules in Example 2:

[0143] (1) Precision test

[0144] Prepare the test solution of Yuxuebi Capsules according to the method in step S2 of Example 2. Precisely absorb the same portion of the above test solution and inject it repeatedly for 6 times, and record the fingerprint. The relative retention time RSD of each common chromatographic peak < 0.2%, and the relative peak area RSD of the main chromatographic peaks < 2.9%.

[0145] (2) Reproducibility test

[0146] Prepare 6 parallel test solutions of Yuxuebi Capsules according to the method in step S2 of Example 2. Determine the above test solutions by ultra-high performance liquid chromatography, and the chromatographic conditions are the same as those in step S2 of Example 2, and record the fingerprint. The relative retention time RSD of each main chromatographic peak < 0.5%, and the relative peak area RSD of the main common chromatographic peaks < 2.9%, indicating that the detection method has good reproducibility.

[0147] (3) Stability test

[0148] Prepare the test solution of Yuxuebi Capsules according to the method in step S2 of Example 2, and store it at room temperature. Precisely pipette the same portion of the above test solution at 0, 2, 4, 6, 8, 10, and 12 h respectively, and record the fingerprint chromatogram. The relative retention time RSD of each main chromatographic peak < 0.3%, and the relative peak area RSD of the common chromatographic peaks < 2.8%, indicating that the test solution has good stability within 12 h under normal temperature conditions.

[0149] (4) Calculation of the fingerprint chromatogram similarity of Yuxuebi Capsules

[0150] Take 20 batches (S1 - S20) of Yuxuebi Capsules and prepare the test solutions according to the operation in step S2 respectively. Perform ultra-high performance liquid chromatography determination successively under the chromatographic conditions in step S2, record the fingerprint chromatograms, and import all the fingerprint chromatograms into the software of the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version) to calculate the similarity. The results show that the similarities of the 20 batches of Yuxuebi Capsules (S1 - S20) are 0.999, 0.999, 0.999, 0.999, 0.999, 0.999, 0.999, 0.999, 0.998, 1.000, 0.941, 0.999, 1.000, 0.997, 0.999, 0.999, 0.998, 1.000, 0.999, 0.997, 0.999 respectively. The similarity results show that the similarities of the 20 batches of Yuxuebi Capsules are all above 0.9.

[0151] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for determining the contents of multiple components in a preparation for treating static blood obstruction syndrome, characterized in that, Extract the Yuxuebi preparation with aqueous methanol solution or pure methanol, and determine the content of the target components to be measured by ultra-high performance liquid chromatography. The target components to be measured include 5-hydroxymethylfurfural, danshensu, chlorogenic acid, ferulic acid, isochlorogenic acid B, rosmarinic acid, salvianolic acid B, salvianolic acid A, dihydrotanshinone I, cryptotanshinone, tanshinone IIA and 3-acetyl-11-oxo- β -boswellic acid; The chromatographic conditions of the ultra-high performance liquid chromatography include: Chromatographic column: C18 bonded silica gel chromatographic column; Mobile phase: Mobile phase A is an aqueous solution of formic acid with a concentration of 0.19% - 0.21%, and mobile phase B is acetonitrile. Gradient elution is performed using mobile phase A and mobile phase B. The gradient elution program is as follows: v / v ​ The detection wavelength is as follows: 。 2. The content determination method according to claim 1, characterized in that, Mobile phase A is 0.2% v / v aqueous formic acid solution; and / or The chromatographic conditions further include that the column temperature is 35 - 45 °C; and / or The chromatographic conditions further include that the flow rate is 0.25 - 0.35 mL / min; and / or The C18 bonded silica gel chromatographic column is ACQUITY UPLC ® BEH C18, with a specification of 2.1×100 mm, 1.7 μm.

3. The assay method according to claim 1, characterized in that, The specific operation of extracting the Yuxue Bi preparation with an aqueous methanol solution or pure methanol is as follows: using an aqueous methanol solution with a concentration greater than or equal to 25% v / v , less than 100% v / v for ultrasonic extraction, or using pure methanol for ultrasonic extraction.

4. The content determination method according to claim 3, wherein, The temperature of the ultrasonic extraction is 55 - 65 °C; the time of the ultrasonic extraction is 20 - 40 min; and / or The mass-to-volume ratio of the Yuxuebi preparation to the methanol aqueous solution is 1:62.5 - 250, g:mL.

5. The content determination method according to any one of claims 1 to 4, characterized in that, It includes the following steps: Step a: Extract the Yuxuebi preparation with a methanol aqueous solution or pure methanol to prepare a test solution; Step b: Prepare a reference solution of the component to be measured; Step c: Measure the reference solution and the test solution by the ultra-high performance liquid chromatography, and calculate the content of the component to be measured in the test solution by the external standard method.

6. The content determination method according to claim 5, characterized in that, In step a, the Yuxuebi preparation is extracted with pure methanol, and the obtained extract is diluted with water or methanol to prepare a test solution.

7. A method for constructing a fingerprint of a Yuxue Bi preparation, characterized in that, It includes the following steps: S1. Prepare a reference substance solution of the target components; the target components include ferulic acid, isochlorogenic acid B, rosmarinic acid, salvianolic acid B, salvianolic acid A, dihydrotanshinone I, cryptotanshinone, tanshinone IIA, and 3-acetyl-11-keto- β -boswellic acid; S2. Take different batches of Yuxuebi preparations, extract the preparations with methanol respectively to obtain test solutions of different batches of Yuxuebi preparations, measure the reference solution and the test solution by the ultra-high performance liquid chromatography in the content determination method according to claims 1 - 6, record the fingerprint, import all the fingerprints into the software of the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints, select the common characteristic chromatographic peaks in the fingerprints of different batches of Yuxuebi capsules as characteristic fingerprint peaks, generate a standard reference fingerprint, and calculate the relative retention time and relative peak area of each common characteristic fingerprint peak.

8. The application of the content determination method according to claims 1 - 6 or the fingerprint construction method according to claim 7 in the quality control of Yuxuebi preparations.

9. The application according to claim 8, characterized in that, The fingerprint spectrum contains 9 characteristic fingerprint peaks: ferulic acid, isochlorogenic acid B, rosmarinic acid, salvianolic acid B, salvianolic acid A, dihydrotanshinone I, cryptotanshinone, tanshinone IIA, 3-acetyl-11-oxo- β -Boswellic acid.

10. The application according to claim 9, characterized in that, Taking salvianolic acid B as the reference peak, the relative retention times of each common peak and the reference peak are within the range of ±5% of the specified value; the specified values are: peak Z1 is 0.7728, peak Z2 is 0.8058, peak Z3 is 0.9172, peak Z4 is 1.000, peak Z5 is 1.106, peak Z6 is 1.701, peak Z7 is 1.928, peak Z8 is 2.190, peak Z9 is 2.811.

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