Method for Simultaneously Determining the Contents of Four Components in Shengxue Tiaoyuan Decoction
The separation and determination of various ingredients in Shengxuezhaoyuan Decoction by high performance liquid chromatography has solved the problem of incomplete quality evaluation in the prior art, and achieved more accurate ingredient content determination and quality control.
Patent Information
- Application Number
- CN202211230543.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-09-30
- Publication Date
- 2025-07-15
- Estimated Expiration
- 2042-09-30
AI Technical Summary
The prior art is difficult to comprehensively evaluate the quality of Shengxuezhaoyuan Decoction, and the lack of methods to determine the content of multiple ingredients simultaneously, resulting in insufficient comprehensive quality standards.
The contents of styrene glycoside, privetin glycoside, new North American saccharin and naringin were separated and measured by specific mobile phase, column temperature, gradient elution and detection wavelength in Shengxuezhaoyuan Decoction. A 50% methanol solution and a C18 chromatography column were used to improve the resolution and accuracy.
The accurate determination of various ingredients in Shengxuezhaoyuan Decoction is achieved, the reliability and accuracy of quality standards are improved, the interference between ingredients is reduced, and more comprehensive quality control means are provided.
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Figure CN116046917B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of component detection, and in particular to a method for simultaneously determining the contents of four components in Shengxue Tiaoyuan Decoction. Background Art
[0002] Shengxue Tiaoyuan Decoction is composed of 8 herbs such as Drynaria rhizome, Millettia reticulata Benth., Polygonum multiflorum Thunb., Astragalus membranaceus (Fisch.) Bunge, Codonopsis pilosula Nannf., etc., and has the effects of replenishing qi and nourishing blood, tonifying the kidney and strengthening the spleen, and is used for increasing peripheral blood white blood cells and weakness after leukopenia caused by other reasons. The current quality standard is included in the Chinese patent medicine formula preparations (Volume VII) in the Drug Standards of the Ministry of Health, standard number: WS3-B-1300-93, and there is no content determination item in the quality standard. Some studies have determined the contents of naringin in the main herb Drynaria rhizome and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (hereinafter referred to as "stilbene glucoside") in Polygonum multiflorum Thunb.
[0003] However, the complexity of the active ingredients in traditional Chinese medicine compound is relatively high. Evaluating the product quality with a single component is not comprehensive enough and difficult to meet the requirements for improving the quality standard of Shengxue Tiaoyuan Decoction. Therefore, there is an urgent need to develop a detection method that can simultaneously determine the contents of four components in Shengxue Tiaoyuan Decoction. Summary of the Invention
[0004] In order to simultaneously determine the contents of four components in Shengxue Tiaoyuan Decoction, the present application provides a method for simultaneously determining the contents of four components in Shengxue Tiaoyuan Decoction.
[0005] The method for simultaneously determining the contents of four components in Shengxue Tiaoyuan Decoction provided by the present application adopts the following technical solutions:
[0006] A method for simultaneously determining the contents of four components in Shengxue Tiaoyuan Decoction, comprising the following steps:
[0007] Step 1), mixing Shengxue Tiaoyuan Decoction with a solvent to obtain a test sample;
[0008] Step 2), adding the test sample into a chromatographic column, using a high performance liquid chromatography instrument to determine the chromatogram of the test sample, and calculating the contents of stilbene glucoside, acteoside, neoeriocitrin, and naringin according to the characteristic peak areas of stilbene glucoside, acteoside, neoeriocitrin, and naringin;
[0009] In the said step 2), the chromatographic conditions at least include:
[0010] Mobile phase A is acetonitrile, mobile phase B is phosphoric acid solution, and the concentration of phosphoric acid in the phosphoric acid solution is 0.2%;
[0011] The column temperature of the chromatographic column is 20-25°C;
[0012] Gradient elution is adopted;
[0013] The Shengxue Tiaoyuan Decoction is prepared by decocting Caulis Spatholobi, Drynaria rhizome, Polygonum multiflorum Thunb., Astragalus membranaceus, malt, Glossy Privet Fruit, Codonopsis pilosula, and Fructus Citri Sarcodactylis.
[0014] By adopting the above technical solution, through the combination of a specific mobile phase and a specific column temperature, the characteristic peaks of stilbene glucoside, specnuezhenide, neoeriocitrin, and naringin can appear simultaneously, with less interference between them, and the characteristic peaks of stilbene glucoside, specnuezhenide, neoeriocitrin, and naringin are less interfered by the characteristic peaks of other impurities, so that the characteristic peaks of stilbene glucoside, specnuezhenide, neoeriocitrin, and naringin are better resolved. When calculating the contents of stilbene glucoside, specnuezhenide, neoeriocitrin, and naringin through the peak area, the accuracy is higher. Moreover, by measuring four characteristic peaks at one time, the contents of stilbene glucoside, specnuezhenide, neoeriocitrin, and naringin in the complex component system of the Shengxue Tiaoyuan Decoction can be measured more accurately, and the detection result is more reliable, providing a feasible technical means for improving the quality standard of the Shengxue Tiaoyuan Decoction and helping to further improve the quality of the Shengxue Tiaoyuan Decoction.
[0015] Preferably, in the step 1), the solvent is a methanol solution with a concentration of 50%.
[0016] By adopting the above technical solution, by specifically selecting a methanol solution with a concentration of 50% as the solvent, the resolution of each characteristic peak can be better improved, further reducing interference and making the detection result more accurate.
[0017] Preferably, in the step 2), the chromatographic column is a C18 chromatographic column.
[0018] By adopting the above technical solution, by specifically selecting the chromatographic column, the detection result is more reliable, with less interference, and it is more conducive to obtaining a more accurate detection result.
[0019] Preferably, in the step 2), the chromatographic conditions further include: the flow rate is 1 - 1.2 mL / min.
[0020] By adopting the above technical solution, by selecting a special flow rate, the resolution of each characteristic peak is higher, and the result of calculating the content of each component is more accurate.
[0021] Preferably, in the step 2), the chromatographic conditions further include: the detection wavelength is 224 nm.
[0022] By adopting the above technical solution, by specifically selecting the detection wavelength, it is more helpful to improve the resolution of each characteristic peak, better reduce interference, the detection result is more accurate and reliable, and it is more helpful to improve the product quality.
[0023] Preferably, in the step 2), the chromatographic conditions further include: the injection volume is 10 μL.
[0024] By adopting the above technical solution, by specifically selecting the sample injection volume, the resolution of each characteristic peak can be better improved, the interference of impurity peaks can be reduced, and the calculation of the component content is more accurate and reliable.
[0025] Preferably, in the step 2), the time periods of gradient elution and the volume ratios of acetonitrile in the mobile phase are as follows: 0 - 4 min, acetonitrile is 12%; 4 - 4.5 min, acetonitrile rises from 12% to 18%; 4.5 - 18 min, acetonitrile is 18%; 18 - 19 min, acetonitrile rises from 18% to 30%; 19 - 28 min, acetonitrile is 30%; 28 - 30 min, acetonitrile drops from 30% to 12%.
[0026] By adopting the above technical solution, by adopting a special gradient elution process, the resolution of each characteristic peak can be better improved, the mutual interference and the interference of impurity peaks can be better reduced, and the contents of stilbene glucoside, specnuezhenide, neoeriocitrin, and naringin can be calculated more accurately.
[0027] Preferably, in the step 2), before or after the chromatogram of the test sample, the chromatogram of the reference substance is determined by high performance liquid chromatography;
[0028] By comparing the chromatogram of the reference substance with the chromatogram of the test sample, the content of each component in the test sample is calculated;
[0029] The preparation method of the reference substance is as follows:
[0030] Step 01), weigh neoeriocitrin, stilbene glucoside, specnuezhenide, and naringin;
[0031] Step 02), put neoeriocitrin, stilbene glucoside, specnuezhenide, and naringin into a methanol solution with a concentration of 40 - 60% and mix evenly to prepare the reference substance;
[0032] The process parameters for determining the chromatogram of the reference substance by high performance liquid chromatography are the same as those for determining the chromatogram of the test sample by high performance liquid chromatography.
[0033] By adopting the above technical solution, by measuring the chromatogram of the reference substance, knowing the concentration and peak area of the reference substance, and then comparing and calculating the area of the corresponding peak in the chromatogram of the test sample with the peak area of the reference substance, it is easier to calculate the contents of stilbene glucoside, specnuezhenide, neoeriocitrin, and naringin in the test sample, and the calculation result is more accurate.
[0034] In summary, the present application has the following beneficial effects:
[0035] 1. Since this application uses a specific mobile phase in combination with a specific column temperature, the characteristic peaks of stilbene glucoside, specnuezhenide, neoeriocitrin, and naringin can appear simultaneously, with less interference among them. Moreover, the characteristic peaks of stilbene glucoside, specnuezhenide, neoeriocitrin, and naringin are less interfered by the characteristic peaks of other impurities, enabling better resolution of the characteristic peaks of stilbene glucoside, specnuezhenide, neoeriocitrin, and naringin. When calculating the contents of stilbene glucoside, specnuezhenide, neoeriocitrin, and naringin through peak areas, the accuracy is relatively high. Additionally, measuring four characteristic peaks at once can more accurately determine the contents of stilbene glucoside, specnuezhenide, neoeriocitrin, and naringin in the complex component system of Shengxue Tiaoyuan Decoction, making the test results more reliable. This provides a feasible technical means for improving the quality standard of Shengxue Tiaoyuan Decoction and helps further enhance the quality of Shengxue Tiaoyuan Decoction.
[0036] 2. In this application, it is preferred to select a special flow rate to achieve higher resolution of each characteristic peak and more accurate results in calculating the contents of each component.
[0037] 3. In this application, it is preferred to adopt a special gradient elution process to better improve the resolution of each characteristic peak, better reduce mutual interference and the interference of impurity peaks, and more accurately calculate the contents of stilbene glucoside, specnuezhenide, neoeriocitrin, and naringin. Description of the Drawings
[0038] Figure 1 It is the chromatogram of the test sample in Example 1.
[0039] Figure 2 It is the chromatogram of the reference substance in Example 1.
[0040] Figure 3 It is the chromatogram of the test sample in Example 2.
[0041] Figure 4 It is the chromatogram of the test sample in Example 3.
[0042] Figure 5 It is the chromatogram of the test sample in Comparative Example 1.
[0043] Figure 6 It is the chromatogram of the test sample in Comparative Example 2.
[0044] Figure 7 It is the chromatogram of the test sample in Comparative Example 3.
[0045] Figure 8 It is the chromatogram of the test sample in Comparative Example 4.
[0046] Figure 9 It is the chromatogram of the test sample in Comparative Example 5.
[0047] Figure 10 It is the chromatogram of the sample to be tested in Comparative Example 6.
[0048] Figure 11 It is the chromatogram of the sample to be tested in Comparative Example 7.
[0049] Figure 12 It is the chromatogram of the sample to be tested in Comparative Example 8.
[0050] Figure 13 It is the chromatogram of the sample to be tested in Comparative Example 9.
[0051] Figure 14 It is the chromatogram of the sample to be tested in Comparative Example 10.
[0052] Figure 15 It is the chromatogram of the sample to be tested in Comparative Example 11.
[0053] Figure 16 It is the chromatogram of the sample to be tested in Comparative Example 12.
[0054] Figure 17 It is the chromatogram of the sample to be tested in Comparative Example 13.
[0055] Figure 18 It is the chromatogram of the sample to be tested lacking Drynaria rhizome measured by the method of Example 1 in Experiment 1.
[0056] Figure 19 It is the chromatogram of the sample to be tested lacking Drynaria rhizome measured by the method of Comparative Example 3 in Experiment 1. Detailed implementation manners
[0057] The following further elaborates on the present application in conjunction with the examples.
[0058] Example 1
[0059] A method for simultaneously determining the contents of four components in Shengxue Tiaoyuan Decoction includes the following steps:
[0060] Step 1), take 1 ml of Shengxue Tiaoyuan Decoction, place it in a 25-ml brown volumetric flask, add methanol with a concentration of 50% to the scale, shake well, filter with a 200-mesh filter cloth, take the filtrate, and prepare the sample to be tested;
[0061] Weigh 7.34 mg of neoeriocitrin, 1.822 mg of stilbene glucoside, 2.106 mg of specnuezhenide, and 9.108 mg of naringin, and place them all in a 10-ml brown volumetric flask. Add methanol with a concentration of 50% to dissolve and dilute to the scale, shake well, and prepare the reference substance. The mass concentrations of each component in the reference substance are respectively:
[0062] Neoeriocitrin 0.7333 mg / mL, stilbene glucoside 0.1727 mg / mL, specnuezhenide 0.1965 mg / mL, naringin 0.8489 mg / ml.
[0063] Step 2), add the test sample to the chromatographic column, and use a high-performance liquid chromatography instrument to measure the chromatogram of the test sample.
[0064] Add the reference substance to the chromatographic column, and use a high-performance liquid chromatography instrument to measure the chromatogram of the reference substance.
[0065] The chromatographic conditions for detecting the reference substance and the test sample are the same.
[0066] The chromatographic conditions are as follows:
[0067] The chromatographic column is Hypersil ODS C18 (4.6 mm × 150 mm, 5 μm); the flow rate is 1.0 mL / min; the detection wavelength is 224 nm; the column temperature is 25 °C; the injection volume is 10 μL; mobile phase A is acetonitrile, and mobile phase B is a phosphoric acid solution with a concentration of 0.2%, gradient elution.
[0068] The volume change of acetonitrile in the mobile phase at each time period of gradient elution is shown in Table 1.
[0069] Table 1
[0070] Time (min) A% (acetonitrile) 0 12％ 4 12％ 4.5 18％ 18 18％ 19 30％ 28 30％ 30 12％
[0071] By comparing and calculating the characteristic peak areas of stilbene glucoside, specnuezhenide, neoeriocitrin, and naringin in the test sample with those in the reference substance, the contents of stilbene glucoside, specnuezhenide, neoeriocitrin, and naringin in the test sample are obtained.
[0072] The calculation formula is as follows:
[0073]
[0074] W i is the content (mg / ml) of each specific substance;
[0075] C i对 is the concentration (mg / ml) of the reference substance solution of each specific substance;
[0076] A i对 is the peak area of the reference substance solution of each specific substance;
[0077] A i样 is the peak area of each specific substance;
[0078] The determination results of the contents of stilbene glycoside, ligustroside, neoeriocitrin, and naringin in the sample to be tested are as follows:
[0079] Content of neoeriocitrin: 1.082 mg / ml; content of stilbene glycoside: 0.2849 mg / ml; content of ligustroside: 0.2719 mg / ml; content of naringin: 1.240 mg / ml.
[0080] The preparation method of each batch of Shengxue Tiaoyuan Decoction determined in this example is as follows:
[0081] Weigh 450 g of Caulis Spatholobi, 900 g of Rhizoma Drynariae, 300 g of Radix Polygoni Multiflori, 300 g of Radix Astragali, 300 g of Fructus Hordei Germinatus, 150 g of Fructus Ligustri Lucidi, 150 g of Radix Codonopsis, and 150 g of Fructus Citri Sarcodactylis, add 8.1 kg of water, decoct at 100 °C for 1 h to obtain the first decoction, then add 5.4 kg of water, decoct at 100 °C for 1 h to obtain the second decoction. Combine the first decoction and the second decoction, filter through a 200-mesh filter cloth, take the filtrate, concentrate the filtrate into a thick paste under 0.01 MPa, add ethanol to make the alcohol content 65%, let it stand for 2 h, filter through a 200-mesh filter cloth, take the filtrate, recover the ethanol from the filtrate until there is no alcohol, add 300 g of sucrose, heat to boiling, add 6.75 g of benzoic acid (preservative), mix well, filter through a 200-mesh filter cloth, take the filtrate, add water to make the volume up to 1000 ml, stir well to obtain Shengxue Tiaoyuan Decoction.
[0082] The spectrogram of the sample to be tested measured in this example is shown in detail in Figure 1 .
[0083] The spectrogram of the reference substance measured in this example is shown in detail in Figure 2 .
[0084] The retention times and resolution of each characteristic peak in this example are shown in Table 2 in detail.
[0085] Table 2
[0086] Peak No. Peak component Retention time Resolution 1 Neoeriocitrin 11.440 2.25 2 Stilbene glucoside 12.256 2.30 3 Specnuezhenide 14.476 5.38 5 Benzoic acid 15.538 2.08 4 Naringin 16.924 2.44
[0087] According to Table 2, it can be obtained that the resolution of each peak in this example is greater than 2, and each peak is 100% separated. According to the results of Experiment 1, it is considered that there is no interference in Peak 1. When calculating the content of each component by the peak area, the accuracy is relatively high, which provides a feasible technical means for improving the quality standard of Shengxue Tiaoyuan Decoction and helps to further improve the quality of Shengxue Tiaoyuan Decoction.
[0088] Example 2
[0089] A method for simultaneously determining the contents of four components in Shengxue Tiaoyuan Decoction, compared with Example 1, the difference is only that:
[0090] In step 2), in the chromatographic conditions, the flow rate is 1.2 mL / min.
[0091] The spectrogram of the sample to be tested measured in this example is shown in detail in Figure 3 .
[0092] The retention time and resolution of each characteristic peak in this example are shown in detail in Table 3.
[0093] Table 3
[0094]
[0095]
[0096] According to Table 3, the resolution of each peak except benzoic acid in this example is greater than 2, and each peak is 100% separated. Benzoic acid is a preservative and does not participate in the calculation of the content of each component, and does not affect the test results.
[0097] Example 3
[0098] A method for simultaneously determining the contents of four components in Shengxue Tiaoyuan Decoction. Compared with Example 1, the difference is only that:
[0099] In step 2), in the chromatographic conditions, the column temperature is 20 °C.
[0100] The spectrogram of the sample to be tested measured in this example is shown in detail in Figure 4 .
[0101] The retention time and resolution of each characteristic peak in this example are shown in detail in Table 4.
[0102] Table 4
[0103] Peak No. Peak component Retention time Resolution 1 Neoeriocitrin 12.076 2.17 2 Stilbene glucoside 13.257 2.95 3 Specnuezhenide 15.165 4.14 5 Benzoic acid 16.562 2.48 4 Naringin 18.284 2.69
[0104] According to Table 4, the resolution of each peak in this example is greater than 2, and each peak is 100% separated. When calculating the content of each component by the peak area, the accuracy is relatively high.
[0105] Comparative Example 1
[0106] A method for simultaneously determining the contents of four components in Shengxue Tiaoyuan Decoction. Compared with Example 1, the difference is only that:
[0107] In step 2), when using a high performance liquid chromatography instrument to measure the chromatogram of the sample to be tested, the gradient elution in the chromatographic conditions is replaced by isocratic elution, the mobile phase is acetonitrile - phosphoric acid solution, the concentration of the phosphoric acid solution is 0.2%, the volume of acetonitrile is 18%, and the volume of the phosphoric acid solution is 82%.
[0108] The spectrogram of the sample to be tested measured in this comparative example is shown in detail in Figure 5 .
[0109] The retention time and resolution of each characteristic peak in this comparative example are shown in detail in Table 5.
[0110] Table 5
[0111] Peak No. Peak component Retention time Resolution 1 Neoeriocitrin 7.233 1.45 2 Stilbene glucoside 7.801 1.44 3 Specnuezhenide 9.482 3.99 4 Naringin 12.281 6.15 5 Benzoic acid 12.598 0.55
[0112] As can be seen from Table 5, the resolution of Peak 1 and Peak 2 is less than 2, and 100% separation cannot be achieved, resulting in mutual influence. When calculating the content of each component by peak area, the accuracy is relatively low.
[0113] Comparative Example 2
[0114] A method for simultaneously determining the contents of four components in Shengxue Tiaoyuan Decoction, compared with Example 1, the difference is only that:
[0115] In step 2), when using a high-performance liquid chromatography instrument to measure the chromatogram of the test sample, the gradient elution in the chromatographic conditions is replaced by isocratic elution, the mobile phase is acetonitrile-phosphoric acid solution, the concentration of the phosphoric acid solution is 0.2%, the volume of acetonitrile is 17%, and the volume of the phosphoric acid solution is 83%.
[0116] The chromatogram of the test sample measured in this comparative example is shown in detail in Figure 6 .
[0117] The retention times and resolutions of the characteristic peaks in this comparative example are shown in detail in Table 6.
[0118] Table 6
[0119] Peak No. Peak component Retention time Resolution 1 Neoeriocitrin 8.516 1.35 2 Stilbene glucoside 9.646 2.45 3 Specnuezhenide 12.836 5.85 5 Benzoic acid 14.091 2.01 4 Naringin 16.011 2.78
[0120] As can be seen from Table 6, the resolution of Peak 1 is less than 2, and 100% separation cannot be achieved, resulting in mutual influence. When calculating the content of each component by peak area, the accuracy is relatively low.
[0121] Comparative Example 3
[0122] A method for simultaneously determining the contents of four components in Shengxue Tiaoyuan Decoction, compared with Example 1, the difference is only that:
[0123] In step 2), when using a high-performance liquid chromatography instrument to measure the chromatogram of the test sample, the gradient elution in the chromatographic conditions is replaced by isocratic elution, the mobile phase is acetonitrile-phosphoric acid solution, the concentration of the phosphoric acid solution is 0.2%, the volume of acetonitrile is 17%, and the volume of the phosphoric acid solution is 83%.
[0124] In the chromatographic conditions, the column temperature is 20°C.
[0125] The chromatogram of the test sample measured in this comparative example is shown in detail in Figure 7 .
[0126] The retention times and resolutions of the characteristic peaks in this comparative example are shown in detail in Table 7.
[0127] Table 7
[0128] Peak No. Peak component Retention time Resolution 1 Neoeriocitrin 9.326 2.40 2 Stilbene glucoside 10.870 2.99 3 Specnuezhenide 13.818 4.84 5 Benzoic acid 15.231 2.05 4 Naringin 17.831 3.37
[0129] According to Table 7, the resolution of each peak is greater than 2, achieving 100% separation. However, according to the results of Experiment 1, due to interference in Peak 1, when calculating the content of each component by peak area, the accuracy is lower compared to Example 1.
[0130] Comparative Example 4
[0131] A method for simultaneously determining the contents of four components in Shengxue Tiaoyuan Decoction, compared with Example 1, the difference is only that:
[0132] In step 2), when using a high-performance liquid chromatography instrument to measure the chromatogram of the test sample, in the chromatographic conditions, gradient elution is replaced by isocratic elution, the mobile phase is acetonitrile-phosphoric acid solution, the concentration of the phosphoric acid solution is 0.2%, the volume of acetonitrile is 16.5%, and the volume of the phosphoric acid solution is 83.5%.
[0133] The chromatogram of the test sample measured in this comparative example is shown in detail in Figure 8 .
[0134] The retention time and resolution of each characteristic peak in this comparative example are shown in detail in Table 8.
[0135] Table 8
[0136] Peak No. Peak component Retention time Resolution 1 Neoeriocitrin 9.387 1.94 2 Stilbene glucoside 10.485 2.23 Overlapping peaks of 3 and 5 Specnuezhenide and benzoic acid 14.750 6.98 4 Naringin 17.909 4.20
[0137] According to Table 8, the resolution of Peak 1 is less than 2, and Peaks 3 and 5 overlap and cannot be separated. When calculating the content of each component by peak area, the accuracy is lower.
[0138] Comparative Example 5
[0139] A method for simultaneously determining the contents of four components in Shengxue Tiaoyuan Decoction, compared with Example 1, the difference is only that:
[0140] In step 2), when using a high-performance liquid chromatography instrument to measure the chromatogram of the test sample, in the chromatographic conditions, gradient elution is replaced by isocratic elution, the mobile phase is acetonitrile-phosphoric acid solution, the concentration of the phosphoric acid solution is 0.2%, the volume of acetonitrile is 15%, and the volume of the phosphoric acid solution is 85%.
[0141] The chromatogram of the test sample measured in this comparative example is shown in detail in Figure 9 .
[0142] The retention time and resolution of each characteristic peak in this comparative example are shown in detail in Table 9.
[0143] Table 9
[0144]
[0145]
[0146] According to Table 9, the resolution of Peak 2 and Peak 3 is less than 2, and 100% separation cannot be achieved. When calculating the content of each component by peak area, the accuracy is relatively low.
[0147] Comparative Example 6
[0148] A method for simultaneously determining the contents of four components in Shengxue Tiaoyuan Decoction, compared with Example 1, the difference is only that:
[0149] In step 2), when using a high performance liquid chromatography instrument to measure the chromatogram of the test sample, the changes in the mobile phase during gradient elution in the chromatographic conditions are shown in detail in Table 10.
[0150] Table 10
[0151] Time (min) A% (acetonitrile) 0 10 25 25 30 10
[0152] The chromatogram of the test sample measured in this comparative example is shown in detail in Figure 10 .
[0153] According to Figure 10 it can be obtained that Peak 1 and Peak 2 overlap and cannot be separated. When calculating the content of each component by peak area, the accuracy is relatively low.
[0154] Comparative Example 7
[0155] A method for simultaneously determining the contents of four components in Shengxue Tiaoyuan Decoction, compared with Example 1, the difference is only that:
[0156] In step 2), when using a high performance liquid chromatography instrument to measure the chromatogram of the test sample, the changes in the mobile phase during gradient elution in the chromatographic conditions are shown in detail in Table 11.
[0157] Table 11
[0158] Time (min) A% (acetonitrile) 0 16 20 19 30 16
[0159] The chromatogram of the test sample measured in this comparative example is shown in detail in Figure 11 .
[0160] According to Figure 11 it can be obtained that Peak 3 and Peak 5 overlap and cannot be separated. When calculating the content of each component by peak area, the accuracy is relatively low.
[0161] Comparative Example 8
[0162] A method for simultaneously determining the contents of four components in Shengxue Tiaoyuan Decoction, compared with Example 1, the difference is only that:
[0163] In step 2), when using a high performance liquid chromatography instrument to measure the chromatogram of the test sample, the changes in the mobile phase during gradient elution in the chromatographic conditions are shown in detail in Table 12.
[0164] Table 12
[0165] Time (min) A% (acetonitrile) 0 8 10 18 25 18 30 8
[0166] The spectrogram of the test sample measured in this comparative example is shown in detail in Figure 12 。
[0167] According to Figure 12 it can be obtained that the 3rd peak and the 5th peak overlap and cannot be separated. When calculating the content of each component by the peak area, the accuracy is relatively low.
[0168] Comparative Example 9
[0169] A method for simultaneously determining the contents of four components in Shengxue Tiaoyuan Decoction. Compared with Example 1, the difference is only that:
[0170] In step 2), when measuring the chromatogram of the test sample by a high performance liquid chromatography instrument, the changes in the mobile phase during gradient elution in the chromatographic conditions are shown in detail in Table 13.
[0171] Table 13
[0172] Time (min) A% (acetonitrile) 0 10 15 18 25 18 30 10
[0173] The spectrogram of the test sample measured in this comparative example is shown in detail in Figure 13 。
[0174] According to Figure 13 it can be obtained that the 1st peak and the 2nd peak are in contact and the separation is poor. When calculating the content of each component by the peak area, the accuracy is relatively low.
[0175] Comparative Example 10
[0176] A method for simultaneously determining the contents of four components in Shengxue Tiaoyuan Decoction. Compared with Example 1, the difference is only that:
[0177] In step 2), when measuring the chromatogram of the test sample by a high performance liquid chromatography instrument, the changes in the mobile phase during gradient elution in the chromatographic conditions are shown in detail in Table 14.
[0178] Table 14
[0179]
[0180]
[0181] The spectrogram of the test sample measured in this comparative example is shown in detail in Figure 14 。
[0182] From Figure 14 it can be obtained that the separation of each peak is relatively good, but the 1st peak is close to the small peak in front of it and is difficult to separate, which will cause interference. When calculating the content of each component by the peak area, the accuracy is relatively low.
[0183] Comparative Example 11
[0184] A method for simultaneously determining the contents of four components in Shengxie Tiaoyuan Decoction. Compared with Example 1, the only difference is that:
[0185] In step 2), when using a high performance liquid chromatography instrument to measure the chromatogram of the test sample, the column temperature in the chromatographic conditions is 35°C.
[0186] The chromatogram of the test sample measured in this comparative example is shown in detail in Figure 15 .
[0187] The retention times and resolutions of the characteristic peaks in this comparative example are shown in detail in Table 15.
[0188] Table 15
[0189] Peak No. Peak component Retention time Resolution 1 Neoeriocitrin 10.476 2.13 2 Stilbene glucoside 10.838 1.24 3 Specnuezhenide 13.321 6.73 5 Benzoic acid 13.945 1.39 4 Naringin 14.858 1.97
[0190] According to Table 15, the resolution of peak 2 and peak 4 is less than 2, and 100% separation cannot be achieved. When calculating the content of each component through the peak area, the accuracy is relatively low.
[0191] Comparative Example 12
[0192] A method for simultaneously determining the contents of four components in Shengxie Tiaoyuan Decoction. Compared with Example 1, the only difference is that:
[0193] In step 2), when using a high performance liquid chromatography instrument to measure the chromatogram of the test sample, the column temperature in the chromatographic conditions is 30°C.
[0194] The chromatogram of the test sample measured in this comparative example is shown in detail in Figure 16 .
[0195] The retention times and resolutions of the characteristic peaks in this comparative example are shown in detail in Table 16.
[0196] Table 16
[0197] Peak No. Peak component Retention time Resolution 1 Neoeriocitrin 10.828 2.10 2 Stilbene glucoside 11.376 1.77 3 Specnuezhenide 13.733 6.40 5 Benzoic acid 14.597 1.86 4 Naringin 15.663 2.09
[0198] According to Table 16, the resolution of peak 2 is less than 2, and 100% separation cannot be achieved. When calculating the content of each component through the peak area, the accuracy is relatively low.
[0199] Comparative Example 13
[0200] A method for simultaneously determining the contents of four components in Shengxie Tiaoyuan Decoction. Compared with Example 1, the only difference is that:
[0201] In step 2), when using a high performance liquid chromatography instrument to measure the chromatogram of the test sample, the flow rate in the chromatographic conditions is 0.8 ml / min.
[0202] The spectrogram of the test sample measured in this comparative example is shown in detail in Figure 17 .
[0203] The retention times and resolution of the characteristic peaks in this comparative example are shown in detail in Table 17.
[0204] Table 17
[0205]
[0206]
[0207] According to Table 17, the resolution of Peak 1 and Peak 4 is less than 2, and 100% separation cannot be achieved. When calculating the content of each component by peak area, the accuracy is relatively low.
[0208] Experiment 1
[0209] Negative control experiment
[0210] 1. Prepare a negative sample solution:
[0211] Weigh 450 g of Millettia reticulata, 300 g of Polygonum multiflorum, 300 g of Astragalus membranaceus, 300 g of Hordeum vulgare, 150 g of Ligustrum lucidum, 150 g of Codonopsis pilosula, and 150 g of Citrus medica var. sarcodactylis. Add 8.1 kg of water and decoct at 100 °C for 1 h to obtain the first decoction. Then add 5.4 kg of water and decoct at 100 °C for 1 h to obtain the second decoction. Combine the first and second decoctions, filter through a 200-mesh filter cloth, take the filtrate, concentrate the filtrate to a thick paste under 0.01 MPa, add ethanol to make the alcohol content 65%, let stand for 2 h, filter through a 200-mesh filter cloth, take the filtrate, recover the ethanol from the filtrate until there is no alcohol, add 300 g of sucrose, heat to boiling, add 6.75 g of benzoic acid (preservative), mix well, filter through a 200-mesh filter cloth, take the filtrate, add water to make the volume up to 1000 ml, stir well to obtain the Shengxue Tiaoyuan Decoction without Drynaria rhizome.
[0212] Measure the Shengxue Tiaoyuan Decoction without Drynaria rhizome by the method for simultaneously determining the contents of four components in the Shengxue Tiaoyuan Decoction in Example 1.
[0213] The spectrogram of the test sample of the Shengxue Tiaoyuan Decoction without Drynaria rhizome is shown in detail in Figure 18 .
[0214] Measure the Shengxue Tiaoyuan Decoction without Drynaria rhizome by the method for simultaneously determining the contents of four components in the Shengxue Tiaoyuan Decoction in Comparative Example 3.
[0215] The spectrogram of the test sample of the Shengxue Tiaoyuan Decoction without Drynaria rhizome is shown in detail in Figure 19 .
[0216] According to Figure 18 and Figure 1 comparison, it can be obtained that Figure 18The negative image of Drynaria fortunei (Kunze) J. Sm. has a small peak interference at peak 1, with a peak area of 16.6. Figure 1 The area of peak 1 in it is 1358. The negative interference peak accounts for 1.2% of the main peak area, and the interference peak accounts for less than 2% of the main peak area, so it is considered that there is no interference.
[0217] According to Figure 19 and Figure 7 By comparison, Figure 19 The negative image of Drynaria fortunei (Kunze) J. Sm. has 2 small peak interferences at peak 1, and the total area of the interference peaks is 74.2. Figure 7 The area of peak 1 in it is 1422. The negative interference peak accounts for 5.2% of the main peak area, and the interference peak accounts for more than 2% of the main peak area, so it is considered that there is obvious interference.
[0218] Experiment 2
[0219] Investigation of linear relationship
[0220] Precisely pipette 3 mL of the reference substance prepared in Example 1 into a 10 mL brown volumetric flask, dilute it to the mark with 50% methanol, and shake well to obtain the initial concentration mixed reference substance solution. Precisely pipette 5 mL of this solution into a 10 mL brown volumetric flask, dilute it to the mark with 50% methanol, and shake well to obtain the next half-concentration reference substance solution. Dilute it by half according to this method to obtain a series of mixed reference substance solutions. Inject the samples for determination. Using the mass concentration (X, μg·mL -1 ) of each reference substance solution as the abscissa and the peak area (Y) as the ordinate, perform linear regression, calculate the regression equation, and the results are shown in Table 18.
[0221] Table 18 Regression equations and linear ranges of 4 components to be determined
[0222] Component to be measured Regression equation Correlation coefficient r <![CDATA[Linear range (μg·mL -1 ) <!-- 11 -->]]> Neoeriocitrin y = 23.2865x + 7.9860 1.0000 6.874～220.0 Stilbene glucoside y = 26.1477x + 2.7664 1.0000 1.619～51.82 Specnuezhenide y = 12.3121x + 3.2239 1.0000 1.842～58.95 Naringin y = 29.8381x + 13.0537 1.0000 7.958～254.7
[0223] Experiment 3
[0224] Precision test
[0225] Precisely pipette the test sample prepared in Example 1 for injection and determination. Continuously determine 6 times, record the chromatographic peak areas of each component to be determined, and calculate the RSDs of the peak areas to be 0.12%, 0.75%, 1.33% and 0.16% respectively. The results show that the precision of the instrument is good.
[0226] Experiment 4
[0227] Stability test
[0228] The test samples prepared in Example 1 were injected for determination at 0 hour, 4 hours, 8 hours, 12 hours, 16 hours, 24 hours, and 36 hours respectively. The chromatographic peak areas of each component to be measured were recorded, and the RSDs of the peak areas were calculated to be 0.42%, 0.61%, 1.11%, and 0.43% respectively. The results showed that the test samples were stable within 36 hours.
[0229] Experiment 5
[0230] Repeatability test
[0231] Six test samples prepared in Example 1 were taken for injection determination. The chromatographic peak areas of each component to be measured were recorded and their contents were calculated. The RSDs of the contents of each component were 0.55%, 0.53%, 1.34%, and 0.56% respectively. The results showed that the repeatability of the method was good.
[0232] Experiment 6
[0233] Spiked recovery test
[0234] 1 ml of Shengxue Tiaoyuan Decoction prepared in Example 1 and 1 mL of the reference substance prepared in Example 1 were placed in a 25-ml brown volumetric flask, methanol with a concentration of 50% was added to the scale, shaken well, filtered through a 200-mesh filter cloth, and the filtrate was taken to prepare the test sample for the spiked recovery test. A total of 6 test samples for the spiked recovery test were prepared, injected for determination, and the spiked recoveries of 4 components to be measured were calculated. The results are shown in Table 19.
[0235] Table 19 Results of the spiked recovery test (n = 6)
[0236]
[0237]
[0238] This specific embodiment is only an explanation of the present application and does not limit the present application. Those skilled in the art can make modifications to this embodiment without creative contributions according to needs after reading this specification, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.
Claims
1. A method for simultaneously determining the contents of four components in Shengxue Tiaoyuan Decoction, characterized in that: It includes the following steps: Step 1), Mix the Shengxue Tiaoyuan Decoction with a solvent to obtain a test sample; Step 2), Add the test sample into a chromatographic column, use a high-performance liquid chromatography instrument to measure the chromatogram of the test sample, and calculate the content of stilbene glucoside or tenuifoliside or neoisoschaftoside or naringin according to the characteristic peak area of stilbene glucoside or tenuifoliside or neoisoschaftoside or naringin; In the said Step 2), the chromatographic conditions at least include: Mobile phase A is acetonitrile, mobile phase B is a phosphoric acid solution, and the concentration of phosphoric acid in the phosphoric acid solution is 0.2%; The column temperature of the chromatographic column is 20 - 25 °C; Gradient elution is adopted; The Shengxue Tiaoyuan Decoction is decocted from Caulis Spatholobi, Rhizoma Drynariae, Radix Polygoni Multiflori, Radix Astragali, Fructus Hordei Germinatus, Fructus Ligustri Lucidi, Radix Codonopsis, and Fructus Citri Sarcodactylis; In the said Step 1), the solvent is a methanol solution with a concentration of 50%; In the said Step 2), the chromatographic column is a C18 chromatographic column; In the said Step 2), the chromatographic conditions also include: the detection wavelength is 224 nm; In the said Step 2), the time periods of gradient elution and the volume ratio of acetonitrile in the mobile phase are respectively: 0 - 4 min, acetonitrile is 12%; 4 - 4.5 min, acetonitrile rises from 12% to 18%; 4.5 - 18 min, acetonitrile is 18%; 18 - 19 min, acetonitrile rises from 18% to 30%; 19 - 28 min, acetonitrile is 30%; 28 - 30 min, acetonitrile drops from 30% to 12%.
2. A method for simultaneously determining the contents of four components in Shengxue Tiaoyuan Decoction according to claim 1, characterized in that: In the said Step 2), the chromatographic conditions also include: the flow rate is 1 - 1.2 mL / min.
3. A method for simultaneously determining the contents of four components in Shengxue Tiaoyuan Decoction according to claim 1, characterized in that: In the said Step 2), the chromatographic conditions also include: the injection volume is 10 μL.
4. A method for simultaneously determining the contents of four components in Shengxue Tiaoyuan Decoction according to any one of claims 1-3, characterized in that: In the said Step 2), before or after the chromatogram of the test sample, use high-performance liquid chromatography to measure the chromatogram of the reference substance; By comparing the chromatogram of the reference substance with the chromatogram of the test sample, calculate the content of each component in the test sample; The preparation method of the said reference substance is as follows: Step 01), Weigh neoisoschaftoside, stilbene glucoside, tenuifoliside, and naringin; Step 02), Put neoisoschaftoside, stilbene glucoside, tenuifoliside, and naringin into a methanol solution with a concentration of 40 - 60% and mix evenly to prepare the reference substance; The process parameters when using high-performance liquid chromatography to measure the chromatogram of the reference substance are the same as those when using high-performance liquid chromatography to measure the chromatogram of the test sample.