Determination of multiple components in senna leaf formula granules or standard decoction freeze-dried powder

Abstract

The invention discloses a method for determining the content of multiple components in senna leaf formula granules or standard decoction freeze-dried powder, and belongs to the technical field of Chinese medicine component detection, which includes: (1) preparing a test solution, a double-label mixed reference solution of a sennoside B reference solution and apigenin-6,8-di-C glucoside, and mixed reference solutions of different concentrations; (2) determining liquid chromatography conditions; (3) collecting chromatograms of the test solution and the mixed reference solution according to the chromatographic conditions, comparing and analyzing the two spectra, and calibrating characteristic peaks; (4) performing qualitative and quantitative analysis on the calibrated characteristic peaks. The present invention uses apigenin-6,8-di-C glucoside and sennoside B as double-label reference substances, adopts a double-label linear correction method to effectively predict and locate the chromatographic peaks of the other three components with two reference substances, and performs well in scientific accuracy and durability, and is relatively economical; at the same time, using sennoside B as an internal standard, combining relative correction factors to determine the contents of the other four components, the method has high accuracy and durability.

Description

Technical Field

[0001] The invention relates to a method for determining the contents of multiple components in senna leaf formula granules or standard decoction freeze-dried powder, and belongs to the technical field of traditional Chinese medicine component detection. Background Art

[0002] Senna is the dried leaflet of Cassia angustifolia Vahl or Cassiaacutifolia Delile, both of which belong to the Leguminosae family. Its main functions are to relieve heat and stagnation, relieve constipation, and promote diuresis. It is used for heat stagnation, constipation, abdominal pain, and edema. Modern research shows that senna mainly contains anthraquinone compounds, flavonoid compounds, polysaccharides and other ingredients, and has pharmacological effects such as laxative, antibacterial, hemostatic, muscle relaxant and antispasmodic. In terms of the content determination of active ingredients in senna, the content determination indicators under the senna item of the 2020 edition of the Chinese Pharmacopoeia are sennoside A and sennoside B. Cai Jinzhang et al. used high-performance liquid chromatography to determine the content of sennoside A in senna and its formula granules, but lacked overall control over the active ingredients of senna. Wu Qiuping et al. used HPLC to determine the contents of five main chemical components in senna leaves. This study adopted the external standard method and only used one chromatographic column to establish the method, which has the disadvantages of poor durability, complex operation, poor repeatability and high cost.

[0003] It should be noted that the above contents belong to the technical knowledge of the inventor and do not necessarily constitute prior art. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a method for determining the content of multiple components in senna leaf formula granules or standard decoction freeze-dried powder, wherein a double-standard linear correction method is used to perform qualitative analysis of the chromatogram and a relative correction factor is used to perform content determination, and the accuracy and durability of the method meet the experimental requirements.

[0005] The present invention achieves the above-mentioned purpose by adopting the following technical solutions:

[0006] A method for determining the contents of multiple components in senna leaf formula granules or standard decoction freeze-dried powder comprises the following steps:

[0007] (1) Prepare the test solution, the double-label mixed reference solution of apigenin-6,8-di-C glucoside and sennoside B, and mixed reference solutions of different concentrations of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside A and sennoside B;

[0008] (2) Determine the liquid chromatography conditions:

[0009] The chromatographic column was filled with octadecylsilane bonded silica gel; acetonitrile (A) and 0.1% trifluoroacetic acid (B) were used as mobile phases for gradient elution; detection wavelength: 270 nm; flow rate: 1.0 mL / min; column temperature: 40°C; injection volume: 10 μl;

[0010] (3) collecting chromatograms of the test solution and the mixed reference solution according to the chromatographic conditions in step (2), comparing and analyzing the two spectra, and calibrating the characteristic peaks of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C-glucoside, quercetin-3-O-gentiobioside, sennoside A, and sennoside B;

[0011] (4) Qualitative and quantitative analysis of the calibrated characteristic peaks:

[0012] (4.1) Determine the predicted retention time and relative correction factor:

[0013] Using multiple chromatographic columns of different brands and types, collecting chromatograms of the mixed reference solution and the test solution according to the chromatographic conditions in step (2), obtaining the standard retention times of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside B, and sennoside A; using apigenin-6,8-di-C glucoside and sennoside B as double-label reference substances, and using a double-label linear calibration method to obtain the predicted retention times of kaempferol-3,7-O-diglucoside, quercetin-3-O-gentiobioside, and sennoside A;

[0014] According to the chromatographic conditions in step (2), chromatograms of mixed reference solutions of different concentrations are collected, and relative correction factors of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C-glucoside, quercetin-3-O-gentiobioside, and sennoside A are calculated respectively using sennoside B as the internal standard;

[0015] (4.2) Determination:

[0016] According to the chromatographic conditions in step (2), the chromatograms of the double-label mixed reference solution of apigenin-6,8-di-C glucoside and sennoside B and the test solution are collected, and the chromatographic peaks of each component to be tested are located according to the predicted retention time determined in step (4.1). The concentration of the sennoside B reference substance and the peak area of ​​the chromatographic peak are used as controls, and the contents of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, and sennoside A are calculated according to the relative correction factors determined in step (4.1).

[0017] In a preferred embodiment, the gradient elution procedure in step (2) is:

[0018] 0~5min: 5%→10%A, 95%→90%B;

[0019] 5~25min: 10%→13%A, 90%→87%B;

[0020] 25~35min: 13%→15%A, 87%→85%B;

[0021] 35~65min: 15%→20%%A, 85%→80%B;

[0022] 65~70min: 20%→35%A, 80%→65%;

[0023] 70~75min: 35%→55%A, 65%→45%B;

[0024] 75~80min: 55%A, 45%B.

[0025] In a preferred embodiment, the preparation method of the test solution in step (1) is:

[0026] Take an appropriate amount of senna leaf formula granules or standard decoction freeze-dried powder, grind it into powder, take about 0.5g, accurately weigh it, put it in a stoppered conical flask, accurately add 25ml of 0.1% sodium bicarbonate in 50% methanol, weigh the weight, ultrasonically treat it at a power of 250W and a frequency of 40kHz for 30 minutes, cool it, make up the lost weight with 0.1% sodium bicarbonate in 50% methanol, shake it well, filter it, take the filtrate, and prepare the test solution.

[0027] In a preferred embodiment, the preparation method of the mixed reference solution in step (1) is:

[0028] Take appropriate amount of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside A and sennoside B reference substances respectively, weigh them accurately, and add 50% methanol to prepare a mixed reference substance solution.

[0029] Specifically, in step (4.1), the specific method for obtaining the predicted retention time using the double-standard linear calibration method is:

[0030] The linear fitting equation was obtained with the actual retention time of the chromatographic peaks of the double-labeled reference substances apigenin-6,8-di-C glucoside and sennoside B as the ordinate and the standard retention time as the abscissa; the standard retention times of kaempferol-3,7-O-diglucoside, quercetin-3-O-gentiobioside, and sennoside A were substituted into the equation to obtain the corresponding predicted retention times; wherein the standard retention time is the average retention time of the test sample collected on multiple chromatographic columns.

[0031] Specifically, the standard retention times of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside B, and sennoside A are 21.04 min, 23.76 min, 33.25 min, 49.25 min, and 62.33 min, respectively.

[0032] Specifically, in step (4.1), the relative correction factors of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, and sennoside A are 1.02, 0.77, 1.09, and 0.99, respectively.

[0033] The calculation formula of the relative correction factor is:

[0034]

[0035] In the formula, s is sennoside B, i is the component to be tested, and one of the four components, kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, and sennoside A, is selected in turn; C s is the concentration of sennoside B, C i is the concentration of the component i to be tested, A s is the peak area of ​​sennoside B, A i is the peak area of ​​the component to be tested; f s is the correction factor for sennoside B, f i is the correction factor for the component to be measured.

[0036] The beneficial effects of this application include but are not limited to:

[0037] The present invention establishes a method for determining the contents of five components, namely, kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside A and sennoside B in senna leaf (senna angustifolia) formula granules. Specifically, the present invention uses apigenin-6,8-di-C glucoside and sennoside B as double-label reference substances, adopts a double-label linear correction method to use two reference substances to effectively predict and locate the chromatographic peaks of the other three components, and based on multi-point linear regression correction, the chromatographic column applicability and prediction accuracy are significantly better than the relative retention time method currently commonly used for chromatographic peak qualitative analysis, greatly eliminating the problem of insufficient characteristic peak qualitative accuracy and chromatographic column applicability of the relative retention time method, and has good scientific accuracy and durability, and is more economical, thereby improving the simplicity of the experiment and reducing the cost; at the same time, sennoside B is used as an internal standard, and the contents of the other four components are determined in combination with a relative correction factor, and the method has high accuracy and durability, providing a reference for the quality control of senna leaf (senna angustifolia) formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The drawings described herein are used to provide further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute improper limitations on the present application.

[0039] Figure 1 The chromatograms of the mixed reference substance (A) and senna leaf (Cassia angustifolia) formula granules (B) (1 is kaempferol-3,7-O-diglucoside, 2 is apigenin-6,8-di-C glucoside, 3 is quercetin-3-O-gentiobioside, 4 is sennoside A, and 5 is sennoside B).

[0040] Figure 2 Chromatograms of mixed reference solution, dextrin negative control solution, and blank solvent. DETAILED DESCRIPTION

[0041] The present invention will be further described in detail in the following content. However, it should be pointed out that the following specific embodiments only provide specific operation examples of the present invention in an exemplary manner, but the protection scope of the present invention is not limited thereto. The protection scope of the present invention is limited only by the claims. It is obvious to those skilled in the art that various other improvements and replacements can be made to the embodiments of the present invention within the protection scope defined by the claims of the present invention, and the same technical effects can still be achieved, thereby achieving the ultimate technical purpose of the present invention.

[0042] Unless otherwise specified, all raw materials in this specification were purchased from commercial sources.

[0043] 1. Instruments and test drugs

[0044] 1.1 Instrument:

[0045] Agilent 1260 high performance liquid chromatograph; DRS Origin software (China Food and Drug Inspection Institute); TS8606 freeze dryer (Fiveco), BSA224S-CW 1 / 10,000 electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.), XS105 1 / 100,000 electronic balance (Mettler Toledo), PS-60AL ultrasonic cleaner (Shandong Dongyue International Economic and Trade Cooperation Co., Ltd.).

[0046] 1.2 Reference substances, reagents and samples:

[0047] Kaempferol-3,7-O-diglucoside (batch number: CFN91159, purity: 98%) reference substance was purchased from Suzhou Yani Biotechnology Co., Ltd., and apigenin-6,8-di-C-glucoside (batch number: C579772, purity: 99.43%), quercetin-3-O-gentiobioside (batch number: P209198, purity: 99.82%), sennoside B (batch number: X900213, purity: 99.08%), and sennoside A (batch number: F77097, purity: 99.25%) reference substances were purchased from Shandong Walderson Biotechnology Co., Ltd.;

[0048] Water was from Watsons, acetonitrile, phosphoric acid, and trifluoroacetic acid were of chromatographic grade, and other reagents were of analytical grade.

[0049] The senna leaf (Cassia angustifolia) formula granules are products of Shandong Hongjitang Pharmaceutical Group, with batch numbers 2202001, 2202002, and 2202003. The sources of the medicinal materials of 15 batches of senna leaves (Cassia angustifolia) are detailed in Table 1, and they were identified as dried leaflets of Cassia angustifolia Vahl, a plant of the Leguminosae family.

[0050] Table 1 Information on 15 batches of senna leaves (Senna angustifolia) medicinal materials / decoction pieces

[0051]

[0052] 2. Determination method

[0053] The present invention provides a method for determining the contents of multiple components in a senna leaf formula granule or a standard decoction freeze-dried powder, comprising the following steps:

[0054] (1) Prepare a double-label mixed reference solution of apigenin-6,8-di-C glucoside and sennoside B, mixed reference solutions of different concentrations of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside A and sennoside B, and test solution:

[0055] The preparation method of the double-label mixed reference solution of apigenin-6,8-di-C glucoside and sennoside B is as follows: take appropriate amounts of apigenin-6,8-di-C glucoside and sennoside B reference substances, accurately weigh them, and add 50% methanol to prepare a double-label mixed reference solution containing 50 μg of each substance per 1 ml.

[0056] The preparation method of the mixed reference solution is as follows:

[0057] Take appropriate amount of reference substances kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside A and sennoside B respectively, weigh accurately, add 50% methanol to make a mixed reference substance solution containing 50 μg of each substance per 1 ml as single standard stock solution.

[0058] The preparation method of mixed reference substance solutions of different concentrations is as follows: accurately transfer appropriate amounts of single-label stock solutions into volumetric flasks (the amount of each single-label stock solution transferred is in multiples), dilute them with 50% methanol, and prepare mixed reference substance solutions of different concentrations.

[0059] The preparation method of the test solution is:

[0060] Take an appropriate amount of senna leaf formula granules or standard decoction freeze-dried powder, grind it into powder, take about 0.5g, accurately weigh it, put it in a stoppered conical flask, accurately add 25ml of 0.1% sodium bicarbonate in 50% methanol, weigh it, ultrasonically treat it (power 250W, frequency 40kHz) for 30 minutes, cool it, make up the lost weight with 0.1% sodium bicarbonate in 50% methanol, shake it well, filter it, take the filtrate, and prepare the test solution.

[0061] Negative control solution preparation:

[0062] Take an appropriate amount of the excipient (dextrin) used in the senna leaf formula granules and prepare a negative control solution according to the preparation method of the test solution under "2. Determination method".

[0063] Among them, the preparation method of standard decoction freeze-dried powder is: take no less than 100g of senna leaf slices, put them in a casserole, soak for 30 minutes, add 14 times the amount of water of the slices for the first decoction, boil over high heat and then simmer for 30 minutes over low heat, add 12 times the amount of water of the slices for the second decoction, boil over high heat and then simmer for 20 minutes over low heat, filter while hot, combine the decoctions, concentrate under reduced pressure, and freeze-dry to prepare 15 batches of standard decoction freeze-dried powder samples (numbered S1 to S15).

[0064] (2) Determine the liquid chromatography conditions:

[0065] Chromatographic columns: Octadecylsilane bonded silica gel is used as filler. The 11 chromatographic columns are numbered COL1 to COL11, with the same specifications (4.6 mm × 250 mm, 5 μm). The specific brands and models are shown in Table 2.

[0066] Mobile phase: acetonitrile (A) and 0.1% trifluoroacetic acid (B) as mobile phase; gradient elution, elution program see Table 3;

[0067] Detection wavelength: 270nm; flow rate: 1.0mL / min; column temperature 40℃; injection volume 10μl.

[0068] Take the mixed reference solution mother solution (A) and the test solution (Senna (Senna angustifolia) formula granules) (B), and inject and measure according to the above chromatographic conditions. The chromatogram is shown in Figure 1 The results showed that under the above chromatographic conditions, the chromatographic peak separation effect was good and the peak shape was symmetrical.

[0069] Table 2 Column information

[0070]

[0071] Table 3 Elution program

[0072]

[0073] (3) collecting chromatograms of the test solution (senna (Cassia angustifolia) formula granules) and the mixed reference solution according to the chromatographic conditions in step (2), comparing and analyzing the two spectra, and calibrating the characteristic peaks of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside A, and sennoside B;

[0074] (4) Qualitative and quantitative analysis of the calibrated characteristic peaks:

[0075] (4.1) Determine the predicted retention time and relative correction factor:

[0076] Using multiple chromatographic columns of different brands and types, collecting chromatograms of the mixed reference solution and the test solution according to the chromatographic conditions in step (2), the standard retention times of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside B, and sennoside A are 21.04 min, 23.76 min, 33.25 min, 49.25 min, and 62.33 min, respectively; wherein the standard retention time is the average retention time of the test sample collected on multiple chromatographic columns;

[0077] The actual retention time of the double-labeled reference substance was collected on another chromatographic column, and the double-labeled linear correction method was used, with the actual retention time of the chromatographic peaks of the double-labeled reference substances apigenin-6,8-di-C glucoside and sennoside B as the ordinate and the standard retention time as the abscissa to obtain a linear fitting equation; the standard retention times of kaempferol-3,7-O-diglucoside, quercetin-3-O-gentiobioside, and sennoside A were substituted into the equation to obtain the corresponding predicted retention times;

[0078] According to the chromatographic conditions in step (2), chromatograms of mixed reference solution of different concentrations are collected, and the concentration of each reference solution is used as the abscissa and the corresponding chromatographic peak area is used as the ordinate to draw a standard curve to obtain regression equations of the five components; according to the regression equation, sennoside B is used as the internal standard to calculate the relative correction factors of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, and sennoside A respectively;

[0079] The calculation formula of relative correction factor is:

[0080]

[0081] In the formula, s is sennoside B, i is the component to be tested, and one of the four components, kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, and sennoside A, is selected in turn; C s is the concentration of sennoside B, C i is the concentration of the component i to be tested, A s is the peak area of ​​sennoside B, A i is the peak area of ​​the component to be tested; f s is the correction factor for sennoside B, f i is the correction factor of the component to be measured; the relative correction factors are shown in Table 4.

[0082] Table 4 Relative correction factors of the four ingredients in the senna (Senna angustifolia) formula granules

[0083]

[0084]

[0085] (4.2) Determination:

[0086] According to the chromatographic conditions in step (2), the chromatograms of the double-label mixed reference solution of apigenin-6,8-di-C glucoside and sennoside B and the test solution are collected, and the chromatographic peaks of each component to be tested are located according to the predicted retention time determined in step (4.1). The concentration of the sennoside B reference substance and the peak area of ​​the chromatographic peak are used as controls, and the contents of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, and sennoside A are calculated according to the relative correction factors determined in step (4.1).

[0087] The content determination formula of the component to be tested is:

[0088]

[0089] Where W iis the content of the component to be tested in the test solution, C i is the concentration of the component to be tested in the test solution, V is the volume of the test solution, m is the sample weight of the test solution, A i is the chromatographic peak area of ​​each component to be tested in the test solution, C s is the concentration of the internal standard, f s / i is the relative correction factor of each component to be measured, A s is the chromatographic peak area of ​​the internal standard.

[0090] 3. Methodological investigation

[0091] 3.1 Exclusiveness inspection:

[0092] Take the senna leaf formula granule test solution, kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside A, sennoside B mixed reference solution, dextrin negative control solution, and blank solvent and measure according to the chromatographic conditions under "2. Determination method" to obtain the HPLC chart, see Figure 2 The results showed that the dextrin negative control solution and blank solvent had no chromatographic peaks at the retention times of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside A, and sennoside B, indicating good method specificity.

[0093] 3.2 Linear investigation:

[0094] Accurately measure kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside A, and sennoside B reference substances to prepare 5 mixed reference substance solutions of different concentrations, and detect them according to the chromatographic conditions under "2. Determination method". With mass concentration as the abscissa and peak area as the ordinate, linear regression was performed and a standard curve was drawn to calculate the regression equation and correlation coefficient. The results showed that the linear relationship of each component was good within a certain concentration range, as shown in Table 5.

[0095] Table 5 Investigation of the linear relationship between the five components in the senna leaf (Senna angustifolia) granules

[0096]

[0097] 3.3 Repeatability study:

[0098] Six test solutions were prepared in parallel and injected into the liquid chromatograph for determination. The RSDs of the contents of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside A, and sennoside B were 0.60%, 1.20%, 0.55%, 0.37%, and 0.44%, respectively, with good repeatability, meeting the analysis requirements.

[0099] 3.4 Intermediate precision study:

[0100] Different analysts conducted repeatability tests using different liquid chromatographs at different times. The RSDs of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside A and sennoside B were 0.30%, 0.30%, 0.78%, 0.41% and 1.30%, respectively. The RSDs of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside A and sennoside B in senna leaf formula granules determined by different instruments were 0.49%, 0.80%, 0.65%, 0.61% and 1.14%, respectively, which met the analysis requirements.

[0101] 3.5 Precision inspection:

[0102] Take 1 portion of the test solution and inject 6 times continuously for measurement. The peak areas of the 5 components tested were counted, and the RSDs of the peak areas were 0.23%, 0.82%, 0.04%, 0.21% and 0.11%, respectively, indicating good precision.

[0103] 3.6 Stability study:

[0104] The contents of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside A and sennoside B in the same test solution were measured at 0h, 4h, 8h, 12h and 24h, and the RSD values ​​were calculated. The results showed that the RSDs of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside A and sennoside B were 0.39%, 0.75%, 0.06%, 0.25% and 0.24%, respectively. The test solution had good stability and met the analysis requirements.

[0105] 3.7 Sample recovery rate investigation:

[0106] Take about 0.25 g of senna leaf formula granule powder, weigh it accurately, add appropriate amount of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside A, and sennoside B reference substance solution, and perform the remaining operations with the test sample content determination method in 2.1 to determine the contents of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside A, and sennoside B, and calculate the recovery rate. The results showed that the recovery rates of kaempferol-3,7-O-diglucoside were between 96.26% and 98.04%, the recovery rates of apigenin-6,8-di-C-glucoside were between 93.45% and 96.88%, the recovery rates of quercetin-3-O-gentiobioside were between 92.25% and 98.56%, the recovery rates of sennoside A were between 92.93% and 95.87%, and the recovery rates of sennoside B were between 92.47% and 94.53%, which met the analysis requirements.

[0107] 4. Research on dual label multiple measurement method (TRSDMC)

[0108] 4.1 Qualitative Research:

[0109] Take the test solution and inject and measure it according to the chromatographic conditions under "2. Determination method". Use the 11 liquid chromatography columns in Table 1 to analyze the samples in turn, determine the positions of the 5 characteristic peaks, and record the chromatogram data.

[0110] The chromatogram data were imported into DRS Origin software, and the chromatographic column results with 5 chromatographic peaks to be tested reaching complete baseline separation were used as the calibration chromatogram, and the attribution of each chromatographic peak was input.

[0111] DRS Origin was used for regression analysis to calculate the retention time regression deviation, average chromatographic peak recognition rate, and chromatographic column compliance rate of the double-label peak combination of apigenin-6,8-di-C glucoside and sennoside B (see Table 6).

[0112] Table 6 Optimization results of double-standard linear calibration method

[0113]

[0114] The results showed that the prediction accuracy of apigenin-6,8-di-C glucoside to sennoside B was 100%, and the chromatographic column compliance rate was 98.18%. This combination was selected for double calibration, with a high accuracy and good durability.

[0115] 4.2 Comparison between the double-label linear calibration method (LCTRS) and the relative retention time method (ESM):

[0116] The double-label linear calibration method used apigenin-6,8-di-C glucoside~sennoside B as the double-label peak, and the relative retention time calibration method used sennoside B as the reference peak. The two different prediction methods were compared with the actual retention of the samples. The results are shown in Table 7.

[0117] Table 7 Comparison of two prediction methods: LCTRS and ESM

[0118]

[0119] The results showed that the retention time prediction value of the double-standard linear correction method had a lower absolute deviation and smaller fluctuation. This method had higher prediction accuracy and better applicability of the chromatographic column, and was superior to the relative retention time method in its application to this variety.

[0120] 4.3 Qualitative Verification:

[0121] After the measured retention times of all the components of 18 batches of samples, including 15 batches of senna leaf (Senna sennae) standard decoction (S1-S15) and 3 batches of senna leaf (Senna sennae) formula granules (S16-S18), were determined on the chromatographic column COL12 by the mixed reference external standard method, the double-standard linear correction method was used to predict the components in the samples and verify the applicability of the method. The chromatograms were imported into DRS Origin software, the double-standard retention times were extracted, and the retention times of other target components were predicted using the software. The results showed that the predicted retention times were compared with the measured retention times, and both achieved a relatively ideal prediction effect, with △TR <0.5min and relative deviation <1.3%. The method was feasible, and the results are shown in Table 8.

[0122] Table 8 Comparison data between the predicted retention time and the actual retention time by LCTRS method

[0123]

[0124]

[0125] 4.4 Quantitative Research

[0126] 4.4.1 Durability study of relative correction factor

[0127] The f values ​​of the five components in the senna leaf (Senna angustifolia) granules were investigated under different HPLC systems, chromatographic columns, flow rates, and column temperatures, as shown in Table 9. The results showed that each factor had no significant effect on the f of the tested components, and the RSD was between 0.0% and 5.1%, with good reproducibility.

[0128] Table 9 Relative correction factors of the five components measured under different instrument, chromatographic column, flow rate and column temperature conditions

[0129]

[0130]

[0131] 4.4.2 Comparison of the results between the double-label multiple-measurement method and the external standard method:

[0132] TRSDMC and ESM were used to calculate the contents of five components in 18 batches of samples, including 15 batches of senna (Cassia angustifolia) standard decoction and 3 batches of senna (Cassia angustifolia) formula granules. The results are shown in Tables 10-1 and 10-2.

[0133] Table 10-1 Results of determination by double standard multiple measurement method (TRSDMC) and external standard method (ESM) 1 (mg·g －1 )

[0134]

[0135]

[0136] Table 10-2 Results of determination by double standard multiple measurement method (TRSDMC) and external standard method (ESM) 2 (mg·g －1 )

[0137]

[0138]

[0139] The results showed that the RSD between the contents calculated by the two methods was less than 3.2%, indicating that the established TRSDMC method has good accuracy and can be used for the content determination of senna (Senna angustifolia) formula granules and standard decoctions.

[0140] The above specific implementation manner cannot be used as a limitation on the protection scope of the present invention. For those skilled in the art, any substitution, improvement or change made to the implementation manner of the present invention falls within the protection scope of the present invention.

[0141] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

Claims

1. A method for determining the content of multiple components in senna leaf formula granules or standard decoction freeze-dried powder, characterized in that: The steps include: (1) Prepare the test solution, the double-label mixed reference solution of apigenin-6,8-di-C glucoside and sennoside B, and mixed reference solutions of different concentrations of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside A and sennoside B; The preparation method of the test solution is: Take an appropriate amount of senna leaf formula granules or standard decoction freeze-dried powder, grind it into powder, take about 0.5g, weigh it accurately, put it in a stoppered conical bottle, accurately add 25ml of 0.1% sodium bicarbonate in 50% methanol, weigh it, treat it with ultrasound at a power of 250W and a frequency of 40kHz for 30 minutes, let it cool, make up the lost weight with 0.1% sodium bicarbonate in 50% methanol, shake it well, filter it, take the filtrate, and prepare the test solution; (2) Determine the liquid chromatography conditions: The chromatographic column was filled with octadecylsilane bonded silica gel; acetonitrile was used as mobile phase A and 0.1% trifluoroacetic acid was used as mobile phase B for gradient elution; detection wavelength: 270 nm; flow rate: 1.0 mL / min; column temperature: 40°C; injection volume: 10 μl; The gradient elution program was: 0~5min: 5%→10%A, 95%→90%B; 5~25min: 10%→13%A, 90%→87%B; 25~35min: 13%→15%A, 87%→85%B; 35~65min: 15%→20%A, 85%→80%B; 65~70min: 20%→35%A, 80%→65%; 70~75min: 35%→55%A, 65%→45%B; 75~80min: 55%A, 45%B; (3) collecting chromatograms of the test solution and the mixed reference solution according to the chromatographic conditions in step (2), comparing and analyzing the two spectra, and calibrating the characteristic peaks of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C-glucoside, quercetin-3-O-gentiobioside, sennoside A, and sennoside B; (4) Qualitative and quantitative analysis of the calibrated characteristic peaks: (4.1) Determine the predicted retention time and relative correction factor: Using multiple chromatographic columns of different brands and types, collecting chromatograms of the mixed reference solution and the test solution according to the chromatographic conditions in step (2), obtaining the standard retention times of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside B, and sennoside A; using apigenin-6,8-di-C glucoside and sennoside B as double-label reference substances, and using a double-label linear calibration method to obtain the predicted retention times of kaempferol-3,7-O-diglucoside, quercetin-3-O-gentiobioside, and sennoside A; According to the chromatographic conditions in step (2), chromatograms of mixed reference solutions of different concentrations are collected, and relative correction factors of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C-glucoside, quercetin-3-O-gentiobioside, and sennoside A are calculated respectively using sennoside B as the internal standard; (4.2) Determination: According to the chromatographic conditions in step (2), the chromatograms of the double-label mixed reference solution of apigenin-6,8-di-C glucoside and sennoside B and the test solution are collected, and the chromatographic peaks of each component to be tested are located according to the predicted retention time determined in step (4.1). The concentration of the sennoside B reference substance and the peak area of ​​the chromatographic peak are used as controls, and the contents of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, and sennoside A are calculated according to the relative correction factors determined in step (4.1).

2. The method for determining the contents of multiple components in the senna leaf formula granules or standard decoction freeze-dried powder according to claim 1, characterized in that: The preparation method of the mixed reference solution in step (1) is: Take appropriate amount of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside A and sennoside B reference substances respectively, weigh them accurately, and add 50% methanol to prepare a mixed reference substance solution.

3. The method for determining the contents of multiple components in the senna leaf formula granules or standard decoction freeze-dried powder according to claim 1, characterized in that: In step (4.1), the specific method for obtaining the predicted retention time using the double-standard linear calibration method is: The linear fitting equation was obtained with the actual retention time of the chromatographic peaks of the double-labeled reference substances apigenin-6,8-di-C glucoside and sennoside B as the ordinate and the standard retention time as the abscissa; the standard retention times of kaempferol-3,7-O-diglucoside, quercetin-3-O-gentiobioside, and sennoside A were substituted into the equation to obtain the corresponding predicted retention times; among which, the standard retention time was the average retention time of the test sample collected on multiple chromatographic columns.

4. The method for determining the contents of multiple components in the senna leaf formula granules or standard decoction freeze-dried powder according to claim 1, characterized in that: In step (4.1), the standard retention times of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, sennoside B, and sennoside A are 21.04 min, 23.76 min, 33.25 min, 49.25 min, and 62.33 min, respectively.

5. The method for determining the contents of multiple components in the senna leaf formula granules or standard decoction freeze-dried powder according to claim 1, characterized in that: In step (4.1), the relative correction factors of kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, and sennoside A are 1.02, 0.77, 1.09, and 0.99, respectively.

6. The method for determining the contents of multiple components in the senna leaf formula granules or standard decoction freeze-dried powder according to claim 1, characterized in that: In step (4.1), the calculation formula of the relative correction factor is: In the formula, s is sennoside B, i is the component to be tested, and one of the four components, kaempferol-3,7-O-diglucoside, apigenin-6,8-di-C glucoside, quercetin-3-O-gentiobioside, and sennoside A, is selected in turn; C s is the concentration of sennoside B, C i is the concentration of the component i to be tested, A s is the peak area of ​​sennoside B, A i is the peak area of ​​the component to be tested; f s is the correction factor for sennoside B, f i is the correction factor for the component to be measured.

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