Determination of Rehymn D in Baihe Gugan Tablets and Its Application

Abstract

This application relates to the field of traditional Chinese medicine analysis technology, and in particular to a method for determining the content of rehmannia glycoside D in Baihe Gujin tablets and its application. The method includes: taking rehmannia glycoside D and adding a solvent to prepare a reference solution with a known concentration; taking the test Baihe Gujin tablets and adding an extraction solvent to extract the sample to obtain a test solution; performing liquid chromatography analysis on the reference solution and the test solution with the known concentration, and obtaining the peak area corresponding to rehmannia glycoside D respectively; and calculating the concentration of rehmannia glycoside D in the test solution using the external standard method. The determination method of this application has strong specificity, good accuracy and robustness, and is more conducive to the comprehensive and effective control of the quality of Baihe Gujin tablets.

Description

Technical Field

[0001] This application relates to the field of traditional Chinese medicine detection technology, and in particular to a method for determining the content of rehmannia glycoside D in Baihe Gujin tablets and its application. Background Technology

[0002] Baihe Gujin Tablets are made from ten medicinal herbs: lily bulb, rehmannia root, prepared rehmannia root, ophiopogon root, angelica root, scrophularia root, fritillaria bulb, platycodon root, licorice root, and white peony root. They have the effects of nourishing yin and moistening the lungs, resolving phlegm and relieving cough. They are used for symptoms such as lung and kidney yin deficiency, dry cough with little phlegm, dry throat and sore throat.

[0003] The existing standard for Baihe Gujin tablets in the 2020 edition of the Chinese Pharmacopoeia specifies the identification of Scrophularia, Angelica sinensis, Paeonia lactiflora, Platycodon grandiflorus, Glycyrrhiza uralensis, and Fritillaria cirrhosa under the identification section, and the determination of paeoniflorin under the content determination section. To more comprehensively assess the quality of Baihe Gujin tablets, it is necessary to develop new detection methods to determine the key indicator components. Summary of the Invention

[0004] Based on this, one or more embodiments of this application provide a method for determining the content of rehmannia glycoside D in Baihe Gujin tablets and its application. This method can be used to accurately and rapidly determine the content of rehmannia glycoside D in Baihe Gujin tablets as a basis for its quality evaluation.

[0005] The technical solution of this application includes the following:

[0006] A method for determining the content of rehmannia glycoside D in Baihe Gujin tablets includes the following steps:

[0007] Take rehmannia glycoside D and prepare a reference solution of known concentration using a solvent;

[0008] Take the tested Lily Stemona Tablets, add extraction solvent to extract, and obtain the test solution;

[0009] The reference solution and the test solution with known concentrations were analyzed by liquid chromatography, and the peak areas corresponding to rehmannia glycoside D were obtained. The concentration of rehmannia glycoside D in the test solution was calculated using the external standard method.

[0010] The conditions for the liquid chromatography analysis include:

[0011] (1) Gradient elution is adopted, wherein the mobile phase A of the gradient elution is acetonitrile, and the mobile phase B is an aqueous solution of phosphoric acid with a mass concentration of 0.08%~0.12%;

[0012] (2) The gradient elution procedure includes:

[0013] From 0 min to 8 min, the volume percentage of mobile phase A is maintained at 1%;

[0014] Over 8 to 9 minutes, the volume percentage of mobile phase A increased from 1% to 4%.

[0015] From 9 min to 50 min, the volume percentage of mobile phase A remained at 4%.

[0016] From 50 min to 50.01 min, the volume percentage of mobile phase A decreased from 4% to 1%;

[0017] From 50.01 min to 55 min, the volume percentage of mobile phase A was maintained at 1%.

[0018] Furthermore, the conditions for the liquid chromatography analysis also include at least one of the following:

[0019] (1) The chromatographic column was packed with octadecylsilane-bonded silica gel;

[0020] (2) The detection wavelength is 200nm~210nm;

[0021] (3) The injection volume is 5 μL to 15 μL.

[0022] Furthermore, the extraction solvent is an aqueous solution of methanol with a volume concentration of 20% to 30%.

[0023] Furthermore, the mass-to-volume ratio of the tested lily-based tablets to the extraction solvent is (2~3) g: 25 mL.

[0024] Further, the tested lily and gold tablets were extracted with an extraction solvent to obtain an extract. The extract was then processed and loaded onto a macroporous resin column. The macroporous resin column was then eluted with water, and the eluent was collected to obtain the test solution.

[0025] Furthermore, the macroporous resin column is made of polyvinylbenzene; and / or

[0026] The macroporous resin column has a length of 16cm~17cm and an inner diameter of 1.4cm~1.6cm; and / or

[0027] The mass-to-volume ratio of the tested lily-derived gold tablets to the water used for washing was (2~3) g: 100 mL.

[0028] One or more embodiments of this application also provide an application of the determination method described in any of the above technical solutions in the quality control of Baihe Gujin tablets, wherein the Baihe Gujin tablets to be tested are tested using the determination method described in any of the above technical solutions to obtain the content of rehmannia glycoside D in the Baihe Gujin tablets to be tested.

[0029] The quality of the Lily Bulb Gujin Tablets to be tested is determined based on whether the content of rehmannia glycoside D in the tablets is within a preset range.

[0030] Furthermore, the preset range is obtained by testing different batches of Baihe Gujin tablets using the determination method described in any of the above technical solutions to obtain the content of Baihe Gujin tablets in different batches, and the content range of rehmannia glycoside D in Baihe Gujin tablets is set according to the principle of statistical analysis.

[0031] Furthermore, based on the principles of statistical analysis, the content range of rehmannia glycoside D in Baihe Gujin tablets is set to 80% of the median value of the content of rehmannia glycoside D in different batches of Baihe Gujin tablets.

[0032] Furthermore, the preset range is that the amount of Rehmannia glutinosa and processed Rehmannia glutinosa contained in Baihe Gujin tablets, calculated as rehmannia glycoside D, is not less than 13.5 μg / tablet.

[0033] The method for determining the content of rehmannia glycoside D in Baihe Gujin tablets disclosed in this application can accurately determine the content of rehmannia glycoside D in Baihe Gujin tablets by adopting appropriate extraction conditions and liquid phase analysis conditions. This method has strong specificity, good accuracy and robustness, and is more conducive to the comprehensive and effective control of the quality of Baihe Gujin tablets. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0035] Figure 1 The results of liquid chromatography detection of the negative control solution under "2.1 Specificity Examination" in Example 1 of this application;

[0036] Figure 2 The results of liquid chromatography detection of the positive control solution under "2.1 Specificity Examination" in Example 1 of this application;

[0037] Figure 3 The results of liquid chromatography detection of the test sample reference solution under "2.1 Specificity Examination" in Example 1 of this application;

[0038] Figure 4 The graph showing the linear relationship between rehmannia glycoside D concentration and peak area established under "2.4 Linearity Examination" in Example 1 of this application is shown. The horizontal axis represents concentration in μg / mL, and the vertical axis represents peak area in mV×S. Detailed Implementation

[0039] The present application is further described below with reference to embodiments and examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Furthermore, it should be understood that after reading the teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the protection scope of the appended claims.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0041] the term

[0042] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0043] In this document, terms such as "preferred" are merely descriptions of better implementation methods or embodiments, and should be understood as not constituting a limitation on the scope of protection of this application.

[0044] In this application, terms such as "further" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0045] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0046] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, optional numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0047] In this application, weight can be a well-known unit of mass in the chemical industry, such as μg, mg, g, or kg.

[0048] One aspect of this application provides a method for determining the content of rehmannia glycoside D in Baihe Gujin tablets, which can be used to accurately determine the content of rehmannia glycoside D in Baihe Gujin tablets as a basis for quality evaluation of Baihe Gujin tablets.

[0049] In one embodiment, the method for determining the content of rehmannia glycoside D in Baihe Gujin tablets includes the following steps:

[0050] Take rehmannia glycoside D and prepare a reference solution of known concentration using a solvent;

[0051] Take the tested Lily Stemona Tablets, add extraction solvent to extract, and obtain the test solution;

[0052] The reference solution and the test solution with known concentrations were analyzed by liquid chromatography, and the peak areas corresponding to rehmannia glycoside D were obtained. The concentration of rehmannia glycoside D in the test solution was calculated using the external standard method.

[0053] The conditions for liquid chromatography analysis include:

[0054] (1) Gradient elution is used, with acetonitrile as the mobile phase A and phosphoric acid as the mobile phase B with a mass concentration of 0.08%~0.12%;

[0055] (2) The gradient elution procedure includes:

[0056] From 0 min to 8 min, the volume percentage of mobile phase A is maintained at 1%;

[0057] Over 8 to 9 minutes, the volume percentage of mobile phase A increased from 1% to 4%.

[0058] From 9 min to 50 min, the volume percentage of mobile phase A remained at 4%.

[0059] From 50 min to 50.01 min, the volume percentage of mobile phase A decreased from 4% to 1%;

[0060] From 50.01 min to 55 min, the volume percentage of mobile phase A is maintained at 1%. In one embodiment, the column is packed with octadecylsilane-bonded silica gel.

[0061] Since the concentration of rehmannia glycoside D in the reference solution is known, under the same chromatographic detection conditions, the concentration of rehmannia glycoside D and the intensity of the response signal should have a one-to-one linear relationship. Therefore, the concentration of rehmannia glycoside D in the test solution can be calculated by mathematical conversion based on the ratio of the response signal (peak area) of rehmannia glycoside D and the specific concentration of rehmannia glycoside D in the reference solution.

[0062] In one embodiment, the detection wavelength is 200nm~210nm.

[0063] In one embodiment, the injection volume is 5 μL to 15 μL.

[0064] In one embodiment, the extraction solvent is an aqueous solution of methanol with a volume concentration of 20% to 30%.

[0065] In one embodiment, the mass-to-volume ratio of the tested lily extract tablets to the extraction solvent was (2~3) g: 25 mL.

[0066] In one embodiment, the extraction method is oscillation.

[0067] In one embodiment, the tested lily tablets were extracted with an extraction solvent to obtain an extract. The extract was then processed and loaded onto a macroporous resin column. The macroporous resin column was then eluted with water, and the eluent was collected to obtain the test solution.

[0068] In one embodiment, the extract was evaporated to dryness, dissolved in water, and then loaded onto a macroporous resin column.

[0069] In one embodiment, the macroporous resin column is made of polyvinylbenzene.

[0070] In one embodiment, the macroporous resin column is of type DA-201.

[0071] In one embodiment, the macroporous resin column has a length of 16cm to 17cm and an inner diameter of 1.4cm to 1.6cm.

[0072] In one embodiment, the mass-to-volume ratio of the tested lily-based tablets to the water used for washing was (2-3) g: 100 mL.

[0073] One or more embodiments of this application also provide an application of the determination method of any of the above technical solutions in the quality control of Baihe Gujin tablets, wherein the Baihe Gujin tablets to be tested are tested using the determination method of any of the above technical solutions to obtain the content of rehmannia glycoside D in the Baihe Gujin tablets to be tested.

[0074] The quality of the tested Baihe Gujin tablets is determined by whether the content of rehmannia glycoside D in the tablets is within a preset range.

[0075] In one embodiment, the preset range is determined by testing different batches of Baihe Gujin tablets using any of the above-mentioned technical solutions to obtain the content of different batches of Baihe Gujin tablets, and the content range of rehmannia glycoside D in Baihe Gujin tablets is set according to the principle of statistical analysis.

[0076] In one embodiment, based on the principle of statistical analysis, the content range of rehmannia glycoside D in Baihe Gujin tablets is set to be limited to 80% of the median value of the content of rehmannia glycoside D in different batches of Baihe Gujin tablets.

[0077] In one embodiment, the preset range is that the amount of Rehmannia glutinosa and processed Rehmannia glutinosa contained in Baihe Gujin tablets, calculated as rehmannia glycoside D, is not less than 13.5 μg / tablet.

[0078] The following are some specific examples.

[0079] The raw materials and reagents involved in the following specific embodiments can be obtained commercially or prepared by those skilled in the art using known methods.

[0080] Example 1

[0081] 1.1 Preparation of reference solution

[0082] Take an appropriate amount of rehmannia glycoside D reference standard, accurately weigh it, and add 25% methanol to prepare a solution containing 70 μg of rehmannia glycoside D per 1 mL to obtain the positive control solution;

[0083] Take samples of Lily Bulb Stemona Tablets (containing Rehmannia glutinosa and Rehmannia glutinosa in preparation), grind them finely, weigh them accurately, place them in a stoppered conical flask, accurately add 25 mL of 25% methanol, seal tightly, shake for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 5 mL of water, pass it through a DA-201 macroporous adsorption resin (inner diameter 1.5 cm, length 16.5 cm), elute with 100 mL of water, collect the eluent, take 50 mL of the eluent, evaporate to dryness, dissolve in 25% methanol and transfer to a 10 mL volumetric flask, add 25% methanol to the mark, shake well, and obtain the negative control solution.

[0084] Take a standard sample of Lilium oxyphylla tablets, grind it finely, weigh it accurately, place it in a stoppered conical flask, accurately add 25 mL of 25% methanol, seal tightly, shake for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 5 mL of water, pass it through a DA-201 macroporous adsorption resin (inner diameter 1.5 cm, length 16.5 cm), elute with 100 mL of water, collect the eluent, take 50 mL of the eluent, evaporate it to dryness, dissolve it in 25% methanol and transfer it to a 10 mL volumetric flask, add 25% methanol to the mark, shake well to obtain the test solution.

[0085] 1.2 Preparation of the test solution

[0086] The proposed method for preparing the test solution is as follows: Take lily root extract tablets, grind them into a fine powder, weigh them accurately, place them in a stoppered conical flask, accurately add 25 mL of 25% methanol, stopper tightly, shake for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 5 mL of water, pass it through a DA-201 macroporous adsorption resin (inner diameter 1.5 cm, length 16.5 cm), elute with 100 mL of water, collect the eluent, take 50 mL of the eluent, evaporate it to dryness, dissolve it in 25% methanol and transfer it to a 10 mL volumetric flask, add 25% methanol to the mark, shake well, and the test solution is obtained.

[0087] 1.2.1 Analysis of eluent volume

[0088] Take 2.5g of Baihe Gujin tablets, grind them finely, weigh them accurately, place them in a stoppered conical flask, accurately add 25mL of 25% methanol, seal tightly, sonicate for 1 hour (power 400w, frequency 40kHz), filter, evaporate the filtrate to dryness, dissolve the residue in 5mL of water, and pass it through a DA-201 macroporous adsorption resin (inner diameter 1.5cm, length 16.5cm), elute with 50mL, 80mL, and 100mL of water, collect the eluent, take 25mL, 40mL, and 50mL of the eluent respectively, evaporate to dryness, dissolve in 25% methanol and transfer to a 10mL volumetric flask, add 25% methanol to the mark, shake well, and the test solution is obtained. Perform the detection according to the provisions under "1.3 Liquid Chromatography Detection", and the results are shown in Table 1.

[0089] According to Table 1, the content of rehmannia glycosides was highest when the sample solution was collected after elution with 100 mL of elution buffer. Therefore, the elution volume was selected as 100 mL.

[0090] Table 1 Results of Elution Volume Study

[0091]

[0092] 1.2.2 Examination of Extraction Methods

[0093] Take 2.5g of Baihe Gujin tablets, grind them finely, weigh them accurately, place them in a stoppered conical flask, accurately add 25mL of 25% methanol, seal tightly, shake, sonicate (400W power, 40kHz frequency), and reflux for 1 hour, cool, filter, evaporate the filtrate to dryness, dissolve the residue in 5mL of water, pass through DA-201 macroporous adsorption resin (inner diameter 1.5cm, length 16.5cm), elute with 100mL of water, collect the eluent, take 50mL of the eluent, evaporate to dryness, dissolve in 25% methanol and transfer to a 10mL volumetric flask, add 25% methanol to the mark, shake well, and the test solution is obtained. The content determination was performed according to the above method, and the results are shown in Table 2.

[0094] According to Table 2, the sample with the highest content was obtained by shaking extraction, which may be because the content of rehmannia glycoside D decreased due to the increase in temperature. Therefore, shaking was chosen as the extraction method.

[0095] Table 2 Results of the extraction method evaluation

[0096]

[0097] 1.2.3 Investigation of Extraction Solvents

[0098] Take 2.5g of Baihe Gujin tablets, grind them finely, weigh them accurately, and place them in a stoppered conical flask. Accurately add 25mL each of methanol, 50% methanol, and 25% methanol, respectively. Seal the flask tightly, shake for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 5mL of water, and pass it through a DA-201 macroporous adsorption resin (inner diameter 1.5cm, length 16.5cm). Elute with 100mL of water, collect the eluent, evaporate 50mL of the eluent to dryness, dissolve it in 25% methanol, transfer it to a 10mL volumetric flask, add 25% methanol to the mark, and shake well to obtain the test solution. Determine the content according to the above method, and the results are shown in Table 3. According to Table 3, the sample content was highest when extracted with 25% methanol; therefore, 25% methanol was chosen as the extraction solvent.

[0099] Table 3 Results of the investigation of extraction solvents

[0100]

[0101] 1.3 Conditions for Liquid Chromatography Detection

[0102] Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine.

[0103] The proposed liquid chromatography detection conditions include: using octadecylsilane-bonded silica gel as the stationary phase; using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B; gradient elution as specified in Table 4; and a detection wavelength of 203 nm. The theoretical plate number, calculated based on the rehmannia glycoside D peak, should be no less than 5000.

[0104] Table 4

[0105]

[0106] 2. Validation of the content determination method

[0107] 2.1 Specificity Examination

[0108] Take the negative control solution, positive control solution, and test sample control solution separately for liquid chromatography analysis, using the same method as specified in section "1.3 Liquid Chromatography Detection". The detection results are as follows: Figures 1-3 .according to Figures 1-3 It can be seen that when the determination method of this application is used to detect Baihe Gujin tablets, there are no other peaks interfering with the absorption peak around the retention time of rehmannia glycoside D, indicating that the method has strong specificity.

[0109] 2.2 Accuracy

[0110] Accuracy was determined using the spiking recovery test method, which included the following steps:

[0111] (1) Accurately weigh 7.5 mg of rehmannia glycoside D reference standard, dissolve it in 25% methanol and dilute to 500 mL to obtain the rehmannia glycoside D reference standard solution.

[0112] (2) Take the known content of Lily Stem Gold Tablets, grind them into a fine powder, take about 1.25g, make 6 portions, weigh them accurately, put them in a stoppered conical flask, accurately add 25mL of the rehmannia glycoside D reference solution from step (1) above, stopper tightly, shake for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 5mL of water, pass it through DA-201 macroporous adsorption resin (inner diameter 1.5cm, length 16.5cm), elute with 100mL of water, collect the eluent, take 50mL of the eluent to dryness, dissolve it in 25% methanol and transfer it to a 10mL volumetric flask, add 25% methanol to the mark, and shake well.

[0113] (3) The solution obtained after shaking in step (2) was subjected to liquid chromatography analysis, using the same method as specified in section "1.3 Liquid Chromatography Detection". The peak area was recorded, and the RSD value of the peak area of ​​rehmannia glycoside D was calculated. The results of the spiking recovery test are shown in Table 5. According to Table 5, the RSD of the spiking recovery rate of rehmannia glycoside D in Baihe Gujin tablets was 2.11%, which is less than 3%, and the average recovery rate was 87.59%, which is within the recovery limit range of 85% to 110%. This indicates that the method used to determine the content of rehmannia glycoside D in Baihe Gujin tablets is accurate and reliable.

[0114] Table 5 Results of the recovery test of rehmannia glutinosa D

[0115]

[0116] 2.3 Precision

[0117] Precision refers to the degree of similarity between results obtained from multiple sampling and measurement of the same homogeneous sample under specified conditions. It is expressed as deviation (d), standard deviation (SD), and relative standard deviation (RSD) (coefficient of variation, CV). The precision of results obtained under the same conditions by the same analyst is called repeatability; the precision between results obtained under changing conditions within the same laboratory, such as at different times, by different analysts, or using different equipment, is called intermediate precision; the precision between results obtained from different laboratories is called reproducibility.

[0118] 2.3.1 Precision Test

[0119] Take samples of Baihe Gujin tablets and prepare the test solution according to the provisions under "1.2 Preparation of Test Solution". Perform liquid chromatography analysis on the test solution according to the provisions under "1.3 Liquid Chromatography Detection". Repeat the injection 6 times, record the peak area each time, and calculate the RSD value of the rehmannia glycoside D peak area. The precision results are shown in Table 6. According to Table 6, it can be shown that the determination method of this application for the content of rehmannia glycoside D in Baihe Gujin tablets has good precision.

[0120] Table 6. Precision test results of rehmannia glycoside D

[0121]

[0122] 2.3.2 Repeatability Test

[0123] The same analyst took six samples of the same Baihe Gujin tablets and prepared test solutions according to the provisions under "1.2 Preparation of test solution". The test solutions were analyzed by liquid chromatography according to the provisions under "1.3 Liquid Chromatography Detection". The peak area was recorded and the RSD value of the peak area of ​​rehmannia glycoside D was calculated. The results of the repeatability test are shown in Table 7. According to Table 7, the RSD of the rehmannia glycoside D content is 3.46%, which is less than 4% and meets the requirements.

[0124] Table 7 Results of repeatability test of rehmannia glutinosa D

[0125]

[0126] 2.3.3 Intermediate Precision Test

[0127] The same Baihe Gujin tablet sample was analyzed using three different instruments. The test solution was prepared according to the specifications in section "1.2 Preparation of Test Solution," and the test solution was analyzed by liquid chromatography according to the specifications in section "1.3 Liquid Chromatography Detection." Peak areas were recorded, and the RSD value of the rehmannia glycoside D peak area was calculated. The results of the intermediate precision test are shown in Table 8. According to Table 8, the RSD of rehmannia glycoside D in the same batch of Baihe Gujin tablets determined by different high-performance liquid chromatographs was 2.70%, less than 3%, indicating that the rehmannia glycoside D content in Baihe Gujin tablets can be accurately quantified using different instruments.

[0128] Table 8 Results of intermediate precision test

[0129]

[0130] 2.4 Examination of Linear Relationships

[0131] Accurately weigh 2.577 mg of rehmannia glycoside D reference standard and place it in a 20 mL volumetric flask. Add 25% methanol solution to dissolve it completely and dilute to volume to obtain the reference standard stock solution. Accurately transfer four portions of the above stock solution (1.25 mL, 2.5 mL, 2.5 mL, and 5 mL) to 25% methanol solution and dilute to volume to 20 mL, 20 mL, 10 mL, and 10 mL respectively to obtain four concentration gradients of rehmannia glycoside D reference standard solutions.

[0132] The above five concentrations of reference solutions were analyzed by high-performance liquid chromatography (HPLC) according to the method specified in "1.3 HPLC Detection". The peak areas were recorded, and the regression equation for rehmannia glycoside D (n=5) was obtained as y=5782.6x+1089.9 (R0). 2 =1), such as Figure 4 As shown, this method exhibits good linearity in the determination of rehmannia glycoside D between 7.506 and 120.1 μg / mL.

[0133] 2.5 Durability

[0134] 2.5.1 Stability Test

[0135] Prepare the test solution according to the provisions under "1.2 Preparation of test solution". Take the same test solution and perform liquid chromatography analysis at 0h, 4h, 8h, 12h, 18h and 24h according to the above method. The method is the same as that under "1.3 Liquid chromatography detection". Record the peak area and calculate the RSD value. The stability test results are shown in Table 9. According to Table 9, the Baihe Gujin tablet test solution has good stability within 24h.

[0136] Table 9 Results of stability test of rehmannia glycoside D

[0137]

[0138] 3. Setting limits

[0139] Ten batches of Baihe Gujin tablets were taken, and test solutions were prepared according to the provisions under "1.2 Preparation of test solution". The test solutions were analyzed by liquid chromatography according to the provisions under "1.3 Liquid Chromatography Detection". The peak areas were recorded and the content of rehmannia glycoside D was calculated. The content determination results of nine batches of samples are shown in Table 10 below.

[0140] Table 10. Results of content determination in 9 batches of samples

[0141]

[0142] Based on the results of the above 10 batches of samples, the content of rehmannia glutinosa D in Baihe Gujin tablets is 0.0421~0.2100mg / g, and each tablet weighs 0.4g, which is 16.84~78.84μg / tablet. Therefore, the limit is 80% of the lowest content value. It is tentatively determined that the amount of rehmannia glutinosa and prepared rehmannia glutinosa contained in each tablet of this product, calculated as rehmannia glutinosa D, shall not be less than 13.5μg.

[0143] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0144] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0145] The embodiments described above merely illustrate several implementation methods of this application and should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Furthermore, it should be understood that after reading the above teachings, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the protection scope of the appended claims. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for determining the content of rehmannia glycoside D in Baihe Gujin tablets, characterized in that, Includes the following steps: Take rehmannia glycoside D and prepare a reference solution of known concentration using a solvent; Take the tested Lily Stemona Tablets, add an extraction solvent to extract the sample, and then load the extract onto a macroporous resin column. Elute the macroporous resin column with water and collect the eluent to obtain the test solution. The extraction solvent is an aqueous solution of methanol with a volume concentration of 20% to 30%. The macroporous resin column is made of polyvinylbenzene. The reference solution and the test solution with known concentrations were analyzed by liquid chromatography, and the peak areas corresponding to rehmannia glycoside D were obtained. The concentration of rehmannia glycoside D in the test solution was calculated using the external standard method. The conditions for the liquid chromatography analysis include: (1) Gradient elution is adopted, wherein the mobile phase A of the gradient elution is acetonitrile, and the mobile phase B is an aqueous solution of phosphoric acid with a mass concentration of 0.08%~0.12%; (2) The gradient elution procedure includes: From 0 min to 8 min, the volume percentage of mobile phase A is maintained at 1%; Over 8 to 9 minutes, the volume percentage of mobile phase A increased from 1% to 4%. From 9 min to 50 min, the volume percentage of mobile phase A remained at 4%. From 50 min to 50.01 min, the volume percentage of mobile phase A decreased from 4% to 1%; From 50.01 min to 55 min, the volume percentage of mobile phase A remained at 1%. (3) The chromatographic column is packed with octadecylsilane-bonded silica gel; (4) The detection wavelength is 200nm~210nm.

2. The determination method according to claim 1, characterized in that, The conditions for the liquid chromatography analysis also include: an injection volume of 5 μL to 15 μL.

3. The determination method according to claim 1 or 2, characterized in that, The mass-to-volume ratio of the tested lily-based extract tablets to the extraction solvent was (2~3) g: 25 mL.

4. The determination method as described in claim 1 or 2, characterized in that, The macroporous resin column has a length of 16cm to 17cm and an inner diameter of 1.4cm to 1.6cm.

5. The determination method as described in claim 1 or 2, characterized in that, The mass-to-volume ratio of the tested lily-based tablets to the eluted water was (2~3) g: 100 mL.

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