Method for determining contents of multi-index components of nerve-calming and mind-tranquilizing pills

Abstract

The invention discloses a method for determining the content of multi-index components of a nerve-soothing and mind-tranquilizing pill, and belongs to the technical field of traditional Chinese medicine component analysis. By optimizing a high performance liquid chromatography gradient elution procedure, strictly controlling an initial mobile phase ratio of the gradient elution procedure and matching with selection of a chromatographic column and a mobile phase, the method can be used for simultaneously, rapidly and comprehensively determining the content of the index components polygala # imgabs0 # ketone III, 3, 4-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5- The content of six index components, namely 6, 6 '-dimustard acyl sucrose, ginsenoside Rb1, beta-asarone, dehydrotumuloic acid and pachymic acid, is increased. The whole detection process is single-wavelength detection, the detection efficiency is high, the analysis time is short, the detection limits of the six components are all lower than 0.008 microgram / mL, and the method is high in sensitivity, good in specificity, accurate in result and good in reproducibility.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine component analysis, and particularly relates to a method for determining the content of multiple index components of Anshen Dingzhi Pills. Background Art

[0002] "Anshen Dingzhi Pills" is one of the classic prescriptions recorded in "Medical Mind". It is composed of Poria, Poria, Ginseng, Polygala, Acorus calamus, and Cinnabar. It has the effects of calming the mind and calming the spirit, invigorating qi and calming the heart. It is mainly used in the treatment of anxiety, depression, insomnia and other diseases in clinical practice. It is composed of Poria, Poria, Ginseng, Polygala, Acorus calamus, and Cinnabar. According to the basic theory of traditional Chinese medicine, the formula is analyzed. Poria and Poria in the formula are sweet, light, and flat, and they belong to the heart, lung, spleen, and kidney meridians, which have the effect of calming the mind and calming the mind; Ginseng greatly replenishes the vital energy; Acorus calamus is pungent, bitter, and warm, and belongs to the heart and stomach meridians, which has the effect of clearing phlegm and opening the mind, refreshing the mind and improving intelligence; Polygala is pungent, bitter, and warm, and belongs to the heart, kidney, and lung meridians, which can calm the mind and improve intelligence, eliminate phlegm, and reduce swelling. When used together with Acorus calamus, it can clear phlegm and open the mind, calm the mind and improve intelligence; Dragon tooth and Cinnabar calm the mind, and the whole formula has the effect of calming the mind and calming the mind. Modern research shows that Anshen Dingzhi Pills have multiple pharmacological activities, such as reducing the level of inflammatory factors, inhibiting inflammatory responses, alleviating neuronal damage in Alzheimer's rats, repairing the brain's learning and memory functions, and also has a good therapeutic effect on depression, anxiety, and primary insomnia.

[0003] Anshen Dingzhi Pills are a combination of various traditional Chinese medicines. The complex ingredients make it difficult to control the quality and conduct a comprehensive quality evaluation. Liquid chromatography is a commonly used method in the quality control of traditional Chinese medicines, but how to quickly and accurately determine the content of various index components in Anshen Dingzhi Pills in liquid chromatography has not been made public. Summary of the invention

[0004] In view of the above problems, according to the prescription composition of Anshen Dingzhi Pills, the present invention adopts high performance liquid chromatography, selects DAD detector, and simultaneously determines the content of Polygala tenuifolia in Anshen Dingzhi Pills within 90 minutes under the condition of 203nm. The contents of six indicator components, including ketone III, 3,6'-dienasinoylsucrose, ginsenoside Rb1, β-asarone, dehydrogenated turmeric acid and pachymic acid.

[0005] The present invention proposes a method for determining the content of multiple index components of Anshen Dingzhi Pills, comprising the following steps:

[0006] Prepare the Anshen Dingzhi Pills into a test solution;

[0007] Weigh Polygala Ketone III, 3,6'-dienasinoylsucrose, ginsenoside Rb1, β-asarone, dehydrogenated turmeric acid and pachymic acid to prepare a mixed reference solution;

[0008] The test solution and the mixed reference solution are respectively tested by high performance liquid chromatography to obtain a liquid chromatogram of the test solution and a liquid chromatogram of the mixed reference solution;

[0009] The liquid chromatogram of the test solution is compared with the liquid chromatogram of the mixed reference solution, and the characteristic peaks and contents of the multiple index components in Anshen Dingzhi Pills are determined according to the comparison results.

[0010] Further, preparing the Anshen Dingzhi Pills into a test solution comprises:

[0011] The Anshen Dingzhi Pills were ultrasonically treated and filtered in a water-saturated n-butanol solution. The filtrate was evaporated to dryness and then redissolved in methanol and passed through a microporous filter membrane to obtain a test solution of the Anshen Dingzhi Pills.

[0012] Furthermore, the ratio of the mass of Anshen Dingzhi Pills to the volume of water-saturated n-butanol solution is 1:15-20 g / mL, specifically 1:15 g / mL, 1:16 g / mL, 1:17 g / mL, 1:19 g / mL, 1:19 g / mL, 1:20 g / mL; the time of ultrasonic treatment is 20-40 min, and illustratively, it can be 20 min, 25 min, 28 min, 320 min, 32 min, 35 min, 40 min; the pore size of the microporous filter membrane is 0.22-0.45 μm, and illustratively, it can be 0.22 μm, 0.25 μm, 0.30 μm, 0.45 μm.

[0013] Further, preparing a mixed reference solution comprises:

[0014] Polygala tenuifolia The reference substances of ketone III, 3,6'-dienasinoylsucrose, ginsenoside Rb1, β-asarone, dehydrogenated turmeric acid and pachymic acid are accurately weighed, dissolved in methanol solution and fixed to volume, and diluted to prepare the reference substance solution.

[0015] Furthermore, the mixed reference solution contains The mass concentration of ketone III is 0.01 to 0.32 mg / mL, for example, 0.01 mg / mL, 0.07 mg / mL, 0.18 mg / mL, 0.20 mg / mL, 0.26 mg / mL, 0.32 mg / mL;

[0016] The mass concentration of 3,6'-dicerucyl sucrose is 0.03 to 1.00 mg / mL, for example, 0.03 mg / mL, 0.10 mg / mL, 0.18 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1.00 mg / mL;

[0017] The mass concentration of ginsenoside Rb1 is 0.02 to 0.60 mg / mL, for example, 0.02 mg / mL, 0.10 mg / mL, 0.17 mg / mL, 0.25 mg / mL, 0.35 mg / mL, 0.55 mg / mL, 0.60 mg / mL;

[0018] The mass concentration of β-asarone is 0.02 to 0.76 mg / mL, for example, 0.02 mg / mL, 0.10 mg / mL, 0.17 mg / mL, 0.25 mg / mL, 0.35 mg / mL, 0.60 mg / mL, 0.76 mg / mL;

[0019] The mass concentration of dehydrogenated tert-butyl ester is 0.01-0.16 mg / mL, for example, 0.01 mg / mL, 0.03 mg / mL, 0.10 mg / mL, 0.16 mg / mL;

[0020] The mass concentration of pachymic acid is 0.01 to 0.20 mg / mL, for example, 0.01 mg / mL, 0.03 mg / mL, 0.10 mg / mL, or 0.20 mg / mL.

[0021] Further, the test solution and the reference solution are detected by high performance liquid chromatography at 203 nm, comprising the following steps:

[0022] Determine HPLC test conditions and set up gradient elution run parameter programs;

[0023] Inject the test solution or reference solution into the injection valve;

[0024] The test solution or the reference solution is tested according to the operating parameter program.

[0025] Furthermore, the test conditions of the high performance liquid chromatography method include:

[0026] The mobile phase is acetonitrile-phosphoric acid aqueous solution, wherein phase A is acetonitrile and phase B is phosphoric acid aqueous solution, the volume percentage concentration of the phosphoric acid aqueous solution is 0.09-0.11%, for example, it can be 0.09%, 0.10%, 0.11%; the flow rate of the mobile phase is 0.95-1.05mL / min, for example, it can be 0.95mL / min, 1.0mL / min, 1.05mL / min.

[0027] Furthermore, the test conditions of the high performance liquid chromatography method include:

[0028] The detector was a DAD detector with a detection wavelength of 203 nm;

[0029] The chromatographic column is a C18 chromatographic column, and the column temperature is 33-37°C, for example, 33°C, 35°C, 36°C and 37°C;

[0030] The injection volume is 10-20 μL, for example, 10 μL, 12 μL, 15 μL, 20 μL; the analysis time is 90 min, and gradient elution is used.

[0031] Furthermore, the filler in the chromatographic column is octadecylsilane bonded silica gel.

[0032] Furthermore, the operating parameter program of the gradient elution is:

[0033] 0-3min, the volume percentage of phase B is 85-84%, and the rest is phase A;

[0034] 3-5min, the volume percentage of phase B is 84-78%, and the rest is phase A;

[0035] 5-15min, the volume percentage of phase B is 78%, and the rest is phase A;

[0036] 15-18min, the volume percentage of phase B is 78-75%, and the rest is phase A;

[0037] 18-23min, the volume percentage of phase B is 75-73%, and the rest is phase A;

[0038] 23-27min, the volume percentage of phase B is 73-67%, and the rest is phase A;

[0039] 27-40min, the volume percentage of phase B is 67-60%, and the rest is phase A;

[0040] 40-45min, the volume percentage of phase B is 60%, and the rest is phase A;

[0041] 45-54min, the volume percentage of phase B is 60-52%, and the rest is phase A;

[0042] 54-59min, the volume percentage of phase B is 52%, and the rest is phase A;

[0043] 59-65min, the volume percentage of phase B is 52-20%, and the rest is phase A;

[0044] 65-90min, the volume percentage of phase B is 20%, and the rest is phase A.

[0045] Beneficial effects of the present invention:

[0046] The chromatographic conditions and elution gradient explored in the present invention can simultaneously, quickly and comprehensively determine the six index components of Anshen Dingzhi Pills, Polygala tenuifolia The content of ketone III (representing ketones in Polygala), 3,6'-dienasoylsucrose (representing oligosaccharides in Polygala), ginsenoside Rb1 (representing ginsenosides in ginseng), β-asarone (representing volatile oil in Acorus calamus), dehydrogenated turmeric acid and pachymic acid (including triterpenoid components of Poria and Poria). The whole detection process is single wavelength detection, with high detection efficiency and short analysis time. The detection limits of the six components are all lower than 0.008μg / mL, with high sensitivity; the blank medium has no interference peaks and good specificity; and the linear relationship between the six index components is r 2 The RSDs of the peak areas of the six index components in each methodological investigation were all less than 2%, indicating good reproducibility.

[0047] The detection method of the multi-index components associated with the efficacy of Anshen Dingzhi Pills constructed by the present invention can accurately determine the content of the index components, comprehensively reflect the efficacy of the compound, and provide a basis for the quality control and evaluation of Anshen Dingzhi Pills. At the same time, it provides a reference for establishing a content determination method in a traditional Chinese medicine compound preparation containing Polygala, Ginseng, Acorus calamus, and Poria, and provides quality control standard support for the development of new products.

[0048] Other features and advantages of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0050] Figure 1 The HPLC chromatogram of the mixed reference substance solution of Anshen Dingzhi Pills at a wavelength of 203 nm is shown according to the detection method proposed in the embodiment of the present invention;

[0051] Figure 2 It is the HPLC chromatogram of the test solution of Anshen Dingzhi Pills at a wavelength of 203nm using the detection method proposed in the embodiment of the present invention;

[0052] Figure 3 The HPLC chromatogram of the blank medium at a wavelength of 203 nm is shown in the detection method proposed in the embodiment of the present invention;

[0053] Figure 4The HPLC chromatogram of the negative sample solution of Polygala tenuifolia under a wavelength of 203 nm according to the detection method proposed in the embodiment of the present invention is shown;

[0054] Figure 5 The HPLC chromatogram of the negative sample solution of ginseng deficiency at a wavelength of 203 nm according to the detection method proposed in the embodiment of the present invention;

[0055] Figure 6 The HPLC chromatogram of the negative sample solution of Acorus tatarinowii under a wavelength of 203 nm according to the detection method proposed in the embodiment of the present invention;

[0056] Figure 7 The HPLC chromatogram of the negative sample solution of Poria cocos deficiency at a wavelength of 203 nm according to the detection method proposed in the embodiment of the present invention;

[0057] Figure 8 This is the HPLC chromatogram of Anshen Dingzhi Pills at a wavelength of 203 nm using the detection method proposed in Comparative Example 1. DETAILED DESCRIPTION

[0058] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0059] Example 1

[0060] This embodiment proposes a method for detecting the content of ingredients in Anshen Dingzhi Pills, comprising the following process:

[0061] S1: Prepare the test solution of Anshen Dingzhi Pills;

[0062] Take 5 g of the reference sample of Anshen Dingzhi Pills, weigh it accurately, put it in a stoppered conical flask, accurately add 100 mL of water-saturated n-butanol solution, seal it for 30 minutes, extract it by ultrasonic for 30 minutes, filter it, take 25 mL of the filtrate, evaporate it to dryness in an evaporating dish, dissolve the residue in methanol and transfer it to a 5 mL volumetric flask, make up to the scale, shake it well, pass it through a 0.22 μm microporous filter membrane, take the filtrate, and obtain the test solution of Anshen Dingzhi Pills.

[0063] S2: preparing mixed reference solution of Anshen Dingzhi Pills;

[0064] Polygala tenuifolia The reference substances of ketone III, 3,6'-bisinacoyl sucrose, ginsenoside Rb1, β-asarone, dehydrogenated turmeric acid and pachymic acid were accurately weighed, dissolved in methanol solution and fixed to volume, and diluted to prepare mixed reference substance solutions of different concentrations. The mass concentration of ketone III was 0.07 mg / mL, the mass concentration of 3,6'-dicornoylsucrose was 0.25 mg / mL, the mass concentration of ginsenoside Rb1 was 0.10 mg / mL, the mass concentration of β-asarone was 0.10 mg / mL, the mass concentration of dehydrogenated tert-butyl ether was 0.03 mg / mL, and the mass concentration of pachymic acid was 0.04 mg / mL.

[0065] S3: Use high performance liquid chromatography to measure the test solution and the mixed reference solution respectively;

[0066] The test conditions of the high performance liquid chromatography are as follows: the mobile phase is acetonitrile-0.1% phosphoric acid water, wherein phase A is acetonitrile and phase B is 0.1% phosphoric acid water; the flow rate is 1.0 mL / min; the detector is a DAD detector; the detection wavelength is 203 nm; the chromatographic column is a C18 chromatographic column; the filler in the chromatographic column is octadecylsilane bonded silica gel; the column temperature is 35°C; the injection volume is 10 μL; the analysis time is 90 min; and the gradient elution is used.

[0067] The specific procedure of gradient elution is:

[0068] 0-3min, the volume percentage of phase B is 85-84%, and the rest is phase A;

[0069] 3-5min, the volume percentage of phase B is 84-78%, and the rest is phase A;

[0070] 5-15min, the volume percentage of phase B is 78%, and the rest is phase A;

[0071] 15-18min, the volume percentage of phase B is 78-75%, and the rest is phase A;

[0072] 18-23min, the volume percentage of phase B is 75-73%, and the rest is phase A;

[0073] 23-27min, the volume percentage of phase B is 73-67%, and the rest is phase A;

[0074] 27-40min, the volume percentage of phase B is 67-60%, and the rest is phase A;

[0075] 40-45min, the volume percentage of phase B is 60%, and the rest is phase A;

[0076] 45-54min, the volume percentage of phase B is 60-52%, and the rest is phase A;

[0077] At 54-59min, the volume percentage of phase B is 52%, and the rest is phase A.

[0078] 59-65min, the volume percentage of phase B is 52-20%, and the rest is phase A.

[0079] 65-90min, the volume percentage of phase B is 20%, and the rest is phase A.

[0080] The liquid chromatograms of the mixed reference solution and the test solution of Anshen Dingzhi Pills in this example are shown in Figure 2. Figure 1 and Figure 2 As shown, Figure 1 In the middle, peak 1 is Polygala tenuifolia The peaks are characterized by ketone III, peak 2 is 3,6'-dienasinoylsucrose, peak 3 is the characteristic peak of ginsenoside Rb1, peak 4 is the characteristic peak of β-asarone, peak 5 is the characteristic peak of dehydrogenated tert-butyl ester, and peak 6 is the characteristic peak of pachymic acid.

[0081] S4: Compare the liquid chromatogram of the test solution with the liquid chromatogram of the mixed reference solution, determine the characteristic peaks of the multiple index components in Anshen Dingzhi Pills according to the comparison results, and calculate the content of each component.

[0082] contrast Figure 1 and Figure 2 The peak time of each characteristic peak can be determined Figure 2 Polygala tenuifolia The peaks are characterized by ketone III, peak 2 is 3,6'-dienasinoylsucrose, peak 3 is the characteristic peak of ginsenoside Rb1, peak 4 is the characteristic peak of β-asarone, peak 5 is the characteristic peak of dehydrogenated tert-butyl ester, and peak 6 is the characteristic peak of pachymic acid.

[0083] Specifically, the mixed reference solution was sampled and measured using the external standard one-point method (the mixed reference solution and the test solution were sampled multiple times under the same conditions, the average value of the peak area was measured, and then the content of the component in the sample was determined by calculation); according to the chromatographic conditions in S3, sixteen batches of Anshen Dingzhi Pills benchmark samples were measured using a double-sample double-needle method, and the content determination results are shown in Table 1:

[0084] Table 1 Determination results of six components in sixteen batches of Anshen Dingzhi Pills reference samples

[0085]

[0086]

[0087] Example 2

[0088] Polygala tenuifolia L. in the Anshen Dingzhi Pills proposed in Example 1 The content detection methods of ketone III, 3,6'-dienasinoylsucrose, ginsenoside Rb1, β-asarone, dehydrogenated turmeric acid and pachymic acid were methodologically validated, including the specificity of the detection method, the linear relationship of the detection method, the stability, precision, accuracy and durability. The performance index results are as follows.

[0089] (1) Specificity testing of the detection method;

[0090] Accurately pipette 10 μL of each of the blank solvent methanol, mixed reference solution, negative reference solution, and test solution, inject them into the liquid chromatograph, and detect according to the determination conditions in Example 1S3, and record the liquid chromatogram. Figure 1-7 .

[0091] Determination of blank solvent methanol: The blank solvent methanol was tested according to the determination conditions of S3 in Example 1, and the obtained liquid chromatogram was as follows: Figure 3 As shown, it can be seen that the blank medium has no interfering peaks and has good specificity.

[0092] Determination of mixed reference solution: The mixed reference solution is prepared in S2 in Example 1, and the obtained liquid chromatogram is as follows: Figure 1 As shown; Peak 1 is Polygala tenuifolia The characteristic peaks of ketone III, peak 2 is 3,6'-dienasinoylsucrose, peak 3 is the characteristic peak of ginsenoside Rb1, peak 4 is the characteristic peak of β-asarone, peak 5 is the characteristic peak of dehydrogenated tert-butyl ester, and peak 6 is the characteristic peak of pachymic acid. It can be seen that the characteristic peaks of each reference solution are obvious and do not interfere with each other.

[0093] Determination of negative control solution: Remove any one of the medicinal materials from the Anshen Dingzhi Pills sample and prepare according to S1 in Example 1 to obtain negative control solution of Polygala tenuifolia, negative control solution of Ginseng tenuifolia, negative control solution of Acorus calamus, and negative control solution of Poria cocos. The liquid chromatogram of the negative control solution is as follows: Figure 4-7 shown. Figure 4 It is the liquid chromatogram of the negative sample solution of Polygala tenuifolia, wherein peak 1 is the characteristic peak of ginsenoside Rb1; peak 2 is the characteristic peak of β-asarone; peak 3 is the characteristic peak of dehydrogenated tert-butyl ether; and peak 4 is the characteristic peak of pachymic acid. Figure 5 This is the liquid chromatogram of the negative sample solution of ginseng deficiency, where peak 1 is Polygala tenuifolia The peak is the characteristic peak of ketone III; Peak 2 is the characteristic peak of 3,6'-diesinarosyl sucrose; Peak 3 is the characteristic peak of β-asarone; Peak 4 is the characteristic peak of dehydrogenated tert-butyl ester; Peak 5 is the characteristic peak of pachymic acid. Figure 6 This is the liquid chromatogram of the negative sample solution of Acorus tatarinowii, where peak 1 is Polygala tenuifolia. The peak is the characteristic peak of ketone III; Peak 2 is the characteristic peak of 3,6'-dienasinoylsucrose; Peak 3 is the characteristic peak of ginsenoside Rb1; Peak 4 is the characteristic peak of dehydrogenated tert-butyl ester; Peak 5 is the characteristic peak of pachymic acid. Figure 7 This is the liquid chromatogram of the negative sample solution of Poria cocos, where the middle peak 1 is Polygala tenuifolia The peak is the characteristic peak of ketone III; Peak 2 is the characteristic peak of 3,6'-diesinapoylsucrose; Peak 3 is the characteristic peak of ginsenoside Rb1; Peak 4 is the characteristic peak of β-asarone.

[0094] Determination of the test solution: The test solution was prepared in S1 in Example 1, and its liquid chromatogram was as follows: Figure 2 shown; contrast Figure 1 , Figure 4-7 Six characteristic peaks can be identified in the HPLC chromatogram of Anshen Dingzhi Pills, among which peak 1 is Polygala tenuifolia. The peaks are characterized by ketone III, peak 2 is the characteristic peak of 3,6'-dienasoylsucrose, peak 3 is the characteristic peak of ginsenoside Rb1, peak 4 is the characteristic peak of β-asarone, peak 5 is the characteristic peak of dehydrogenated tert-butyl ester, and peak 6 is the characteristic peak of pachymic acid. Figure 1-7 The retention times of the characteristic peaks are The characteristic peaks of ketone III (9.463-9.467), 3,6'-diesinapoylsucrose (15.387-15.423), ginsenoside Rb1 (35.413-35.433), β-asarone (55.617-55.623), dehydrogenated tert-butyl ester (68.343-68.347), and pachymic acid (77.533-77.547) showed that the negative test did not interfere with the content determination of this product, and this method had good specificity.

[0095] (2) Testing of the linear relationship of the detection method;

[0096] Weighing Polygala A suitable amount of reference substances of ketone III, 3,6'-bisinacoyl sucrose, ginsenoside Rb1, β-asarone, dehydrogenated turmeric acid and pachymic acid were added to the methanol solution according to S2 in Example 1 to prepare a series of reference substance solutions with different concentrations. According to the chromatographic conditions of S3 in Example 1, 10 μL of the mixed reference substance solution was accurately drawn and injected into the high performance liquid chromatograph, and a standard curve was drawn with the reference substance concentration as the ordinate and the peak area of ​​each index component as the abscissa. The specific data of the standard curve are shown in Table 2. At the same time, the polygala tenuifolia was calculated. The standard regression equations, correlation coefficients and linear ranges of ketone III, 3,6'-dienasinoylsucrose, ginsenoside Rb1, β-asarone, dehydrogenated turmeric acid and pachymic acid are shown in Table 2.

[0097] Table 2 Standard curve data of six index components

[0098]

[0099] According to Table 2, the six index components have good linear relationships within their respective injection mass concentration ranges, indicating that the method of the present invention has a wide linear range and high accuracy.

[0100] (3) Stability testing of the detection method;

[0101] Take the Anshen Dingzhi Pills sample with batch number ASDZW202405, prepare the test solution according to S1 in Example 1, and analyze the samples at 0h, 2h, 4h, 6h, 8h, 12h and 24h according to the chromatographic conditions of S3 in Example 1. The specific results are shown in Table 3.

[0102] Table 3 Stability test results

[0103]

[0104] The results showed that The RSDs of the chromatographic peak areas of ketone III, 3,6'-dienasinoylsucrose, ginsenoside Rb1, β-asarone, dehydrogenated turpentine acid and pachymic acid within 24 hours were all less than 2%, indicating that testing the test solution within 24 hours had no effect on the results and the detection method had good stability.

[0105] (4) Precision testing of the detection method;

[0106] 4.1) Intermediate precision test;

[0107] Different experimenters took samples of Anshen Dingzhi Pills with batch number ASDZW202405 from the same batch, prepared 3 test solutions using S1 in Example 1, and analyzed them on the same HPLC according to the chromatographic conditions of S3 in Example 1. The peak area results are shown in Table 4.

[0108] Table 4 Intermediate precision determination results

[0109]

[0110] The results showed that the RSDs of the peak areas of the six index components in Anshen Dingzhi Pills measured by different experimenters using the same instrument were all less than 2%, indicating that the instrument had good precision.

[0111] 4.2) Repeatability test;

[0112] Take the Anshen Dingzhi Pills sample with batch number ASDZW202405, prepare 6 test solutions in parallel according to S1 in Example 1, and measure each test solution twice according to the chromatographic conditions of S3 in Example 1. The specific results of peak area repeatability are shown in Table 5.

[0113] Table 5 Repeatability test results

[0114]

[0115]

[0116] The results showed that the peak area RSDs of the six index components were all less than 2%, which met the requirements of the 2020 edition of the "Chinese Pharmacopoeia", indicating that the method had good repeatability.

[0117] (5) Sample recovery test of the detection method;

[0118] Accurately weigh 2.5 g of 6 samples of Anshen Dingzhi Pills (batch number ASDZW202405) with known concentrations, and add 6 kinds of mixed reference solutions of index components at three levels of 50%, 100%, and 150%, respectively, with 3 parallels for each level. Prepare the test solution according to S1 in Example 1, and inject and analyze according to the chromatographic conditions of S3 in Example 1, and the results are shown in Table 6.

[0119] Table 6 Sample recovery determination results

[0120]

[0121]

[0122] It can be seen from Table 6 that the sample recovery rates of the six index components all meet the recovery rate limits specified in the 2020 edition of the "Chinese Pharmacopoeia" (Volume 4), indicating that the determination method has good accuracy.

[0123] (6) Durability testing of the testing method;

[0124] Take the Anshen Dingzhi Pills sample of the same batch with batch number ASDZW202405, use the test solution prepared in S1 in Example 1, and according to the chromatographic conditions of S3 in Example 1, measure under different types of chromatographic columns, different column temperatures, flow rates, different mobile phase ratios, and different sample extraction time conditions, and the peak area results are shown in Tables 7-11.

[0125] Table 7 Durability (Chromatographic column model)

[0126]

[0127]

[0128] Table 8 Durability (column temperature)

[0129]

[0130] Table 9 Durability (flow rate)

[0131]

[0132] Table 10 Durability (acid-water mobile phase ratio)

[0133]

[0134] Table 11 Durability (sample extraction time)

[0135]

[0136]

[0137] The results showed that after changing the corresponding conditions, the RSDs of the peak areas of the six index components were all less than 2%, indicating that the content determination method had good durability.

[0138] Comparative Example 1

[0139] According to the steps of Examples 1-3, the Anshen Dingzhi Pills were measured. The difference between this comparative example and Example 1 is that the data ratio of the AB phase of the elution procedure is:

[0140] 0-10min, the volume percentage of phase B is 95-85%, and the rest is phase A;

[0141] 10-20min, the volume percentage of phase B is 85-84%, and the rest is phase A;

[0142] 20-31min, the volume percentage of phase B is 84-82%, and the rest is phase A;

[0143] 31-33min, the volume percentage of phase B is 82-77%, and the rest is phase A;

[0144] 33-52min, the volume percentage of phase B is 77-60%, and the rest is phase A;

[0145] 52-70min, the volume percentage of phase B is 60-48%, and the rest is phase A;

[0146] 70-76min, the volume percentage of phase B is 48-27%, and the rest is phase A;

[0147] 76-93min, the volume percentage of phase B is 27-20%, and the rest is phase A;

[0148] 93-100min, the volume percentage of phase B is 20-5%, and the rest is phase A;

[0149] 100-102min, the volume percentage of phase B is 5%, and the rest is phase A.

[0150] The HPLC chromatogram obtained by the above chromatographic conditions is as follows: Figure 8 As shown, it can be seen that the detected characteristic peaks of Peak 1, Peak 5, and Peak 6 are not obvious, and are accompanied by the appearance of miscellaneous peaks, which interfere with the accuracy of the detection data.

[0151] The accuracy of the detection method of Comparative Example 1 was verified using the sample recovery rate, as follows:

[0152] Table 12 Sample recovery determination results

[0153]

[0154] The results show that compared with the detection data of comparative example 1, the detection method of the present invention has a short analysis time; the detection limits of the six components are all lower than 0.008 μg / mL, with high sensitivity; the blank medium has no interference peaks and good specificity; the linear relationship r of the six index components is 2 The results are all greater than 0.999, and the results are accurate; the peak area RSDs of the six index components in each methodological investigation are all less than 2%, and the reproducibility is good. The data of Comparative Example 1 show that after changing the AB phase data ratio of the elution program, the average sample recovery rate of the determination method is lower than the average sample recovery rate in Example 1, and the RSD value of Comparative Example 1 is greater than 2%, that is, the accuracy of Comparative Example 1 is lower than that of Example 1.

[0155] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for determining the content of multiple index components of Anshen Dingzhi Pills, characterized in that: The following steps are involved: Prepare the Anshen Dingzhi Pills into a test solution; Weigh Polygala Ketone III, 3,6'-dienasinoylsucrose, ginsenoside Rb1, β-asarone, dehydrogenated turmeric acid and pachymic acid to prepare a mixed reference solution; The test solution and the mixed reference solution are respectively tested by high performance liquid chromatography to obtain a liquid chromatogram of the test solution and a liquid chromatogram of the mixed reference solution; The liquid chromatogram of the test solution is compared with the liquid chromatogram of the mixed reference solution, and the characteristic peaks and contents of the multiple index components in Anshen Dingzhi Pills are determined according to the comparison results.

2. The method for determining the content of multiple index components of Anshen Dingzhi Pills according to claim 1, characterized in that: The Anshen Dingzhi Pills are prepared into a test solution comprising: The Anshen Dingzhi Pills were ultrasonically treated in a water-saturated n-butanol solution and then filtered. The filtrate was taken, evaporated to dryness, re-dissolved in methanol, and passed through a microporous filter membrane to obtain a test solution of the Anshen Dingzhi Pills.

3. The method for determining the content of multiple index components of Anshen Dingzhi Pills according to claim 2, characterized in that: The ratio of the mass of Anshen Dingzhi Pills to the volume of the water-saturated n-butanol solution is 1:15-20 g / mL; the time of ultrasonic treatment is 20-40 min; and the pore size of the microporous filter membrane is 0.22-0.45 μm.

4. The method for determining the content of multiple index components of Anshen Dingzhi Pills according to claim 1, characterized in that: The mixed reference solution is prepared by: Polygala tenuifolia The reference substances of ketone III, 3,6'-dienasinoylsucrose, ginsenoside Rb1, β-asarone, dehydrogenated turmeric acid and pachymic acid are accurately weighed, dissolved in methanol solution and fixed to volume, and diluted to prepare the reference substance solution.

5. The method for determining the content of multiple index components of Anshen Dingzhi Pills according to claim 1, characterized in that: Polygala tenuifolia The mass concentration of ketone III is 0.01-0.32 mg / mL, the mass concentration of 3,6'-dicornoylsucrose is 0.03-1.00 mg / mL, the mass concentration of ginsenoside Rb1 is 0.02-0.60 mg / mL, the mass concentration of β-asarone is 0.02-0.76 mg / mL, the mass concentration of dehydrogenated tert-butyl ether is 0.01-0.16 mg / mL, and the mass concentration of pachymic acid is 0.01-0.20 mg / mL.

6. The method for determining the content of multiple index components of Anshen Dingzhi Pills according to claim 1, characterized in that: The test solution and the reference solution are detected by high performance liquid chromatography at 203 nm, comprising the following steps: Determine HPLC test conditions and set up gradient elution run parameter programs; Inject the test solution or reference solution into the injection valve; The test solution or the reference solution is tested according to the operating parameter program.

7. The method for determining the content of multiple index components of Anshen Dingzhi Pills according to claim 6, characterized in that: The test conditions of the high performance liquid chromatography method include: The mobile phase is acetonitrile-phosphoric acid aqueous solution; wherein phase A is acetonitrile, phase B is phosphoric acid aqueous solution, and the volume percentage concentration of the phosphoric acid aqueous solution is 0.09-0.11%; The flow rate of the mobile phase was 0.95-1.05 mL / min.

8. The method for determining the content of multiple index components of Anshen Dingzhi Pills according to claim 6, characterized in that: The test conditions of the high performance liquid chromatography method include: The detector was a DAD detector with a detection wavelength of 203 nm; The chromatographic column is a C18 column, and the column temperature is 33-37°C; The injection volume was 10-20 μL; the analysis time was 90 min, and gradient elution was used.

9. The method for determining the content of multiple index components of Anshen Dingzhi Pills according to claim 8, characterized in that: The filler in the chromatographic column is octadecylsilane bonded silica gel.

10. The method for determining the content of multiple index components of Anshen Dingzhi Pills according to claim 8, characterized in that: The operating parameter program of the gradient elution is: 0-3min, the volume percentage of phase B is 85-84%, and the rest is phase A; 3-5min, the volume percentage of phase B is 84-78%, and the rest is phase A; 5-15min, the volume percentage of phase B is 78%, and the rest is phase A; 15-18min, the volume percentage of phase B is 78-75%, and the rest is phase A; 18-23min, the volume percentage of phase B is 75-73%, and the rest is phase A; 23-27min, the volume percentage of phase B is 73-67%, and the rest is phase A; 27-40min, the volume percentage of phase B is 67-60%, and the rest is phase A; 40-45min, the volume percentage of phase B is 60%, and the rest is phase A; 45-54min, the volume percentage of phase B is 60-52%, and the rest is phase A; 54-59min, the volume percentage of phase B is 52%, and the rest is phase A; 59-65min, the volume percentage of phase B is 52-20%, and the rest is phase A; 65-90min, the volume percentage of phase B is 20%, and the rest is phase A.

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