A quantitative detection method for Ganweikang tablets and its application

Abstract

The present invention belongs to the technical field of drug detection, and discloses a quantitative detection method and application of Ganweikang tablets. The quantitative detection method comprises the following steps: preparing a reference solution and a test solution; then injecting into a high performance liquid chromatograph for detection, and calculating the content of each component in the test sample according to the peak area of ​​the chromatogram; in the detection, a mobile phase is used for gradient elution, and the mobile phase includes 0.05% to 0.2% formic acid aqueous solution and acetonitrile. The quantitative detection method of Ganweikang tablets provided by the present invention can simultaneously quantitatively determine the content of representative active ingredients in Ganweikang tablets, such as verbena glycosides, cimicifuga glycosides, 5-O-methylvisaminol glycosides, forsythiaside A, forsythiaside, liquiritin, ammonium glycyrrhizinate and calycosin. The quantitative detection method is simple to operate, has strong specificity, good repeatability, and accurate results. It can comprehensively evaluate the quality of Ganweikang tablets and is applied to the quality control and drug development of Ganweikang tablets.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and specifically relates to a quantitative detection method for Ganweikang tablets and application thereof. Background Art

[0002] Ganweikang Tablets are composed of Astragalus, Bupleurum, Atractylodes, Patchouli, Verbena, Saposhnikovia, Forsythia, Licorice, etc., which have the effects of soothing the liver and strengthening the spleen, clearing away heat and removing dampness. It is used for flank pain caused by liver depression and spleen deficiency, damp-heat internal obstruction, with symptoms of abdominal distension, loss of appetite, flank pain, fatigue, nausea, aversion to greasy food, uncomfortable bowel movements, red tongue with greasy coating; chronic hepatitis B with the above symptoms.

[0003] There are many active ingredients in Ganweikang Tablets, and most of them have the effect of protecting the liver and playing an important role in Ganweikang Tablets. However, the current quality standard of Ganweikang Tablets only uses thin-layer scanning to determine the content of astragaloside IV in Astragalus. This determination method not only has a cumbersome pretreatment process and low detection sensitivity, but also only detects astragaloside IV. The content determination of a single component cannot reflect the overall quality of Ganweikang Tablets.

[0004] Therefore, providing a method that can comprehensively evaluate and control the quality of Ganweikan tablets is of great significance to the quality control and drug development of Ganweikan tablets. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a quantitative detection method for Ganweikang tablets and its application. The quantitative detection method provided by the present invention can comprehensively evaluate the quality of Ganweikang tablets and is applied to the quality control and drug development of Ganweikang tablets.

[0006] The invention provides a quantitative detection method for Ganweikang tablets.

[0007] Specifically, a quantitative detection method of Ganweikang tablets comprises the following steps:

[0008] Weigh verbena glycoside, forsythiaside A, forsythiaside, cimicifuga glycoside, liquiritigenin, ammonium glycyrrhizinate, 5-O-methylvisaminol glycoside and calycosin isoflavone, and dissolve them in a solvent to prepare a reference solution;

[0009] Weigh Ganweikang tablets and dissolve them in solvent to prepare the test solution;

[0010] The reference solution and the test solution are injected into a high performance liquid chromatograph for detection to obtain chromatograms of the reference and the test sample; and the content of each component in the test sample is calculated based on the peak area of ​​the chromatogram;

[0011] In the detection process, a mobile phase is used for gradient elution, and the mobile phase includes a 0.05% to 0.2% formic acid aqueous solution (A) and acetonitrile (B); the process of the gradient elution is:

[0012] 0-25min, the volume percentage of acetonitrile in the mobile phase is 3%-8%;

[0013] 25-35 min, the volume percentage of acetonitrile in the mobile phase increases from 3%-8% to 32%-38%;

[0014] 35-40 min, the volume percentage of acetonitrile in the mobile phase increases from 32%-38% to 48%-55%.

[0015] Preferably, the mobile phase comprises a 0.08% to 0.15% formic acid aqueous solution; further preferably, the mobile phase comprises a 0.08% to 0.12% formic acid aqueous solution. It is understood that the volume percentage of formic acid in the 0.05% to 0.2% formic acid aqueous solution is 0.05% to 0.2%; the volume percentage of formic acid in the 0.08% to 0.15% formic acid aqueous solution is 0.08% to 0.15%; and the volume percentage of formic acid in the 0.08% to 0.12% formic acid aqueous solution is 0.08% to 0.12%.

[0016] Preferably, the process of gradient elution is:

[0017] 0-25min, the volume percentage of acetonitrile in the mobile phase is 4%-6%;

[0018] 25-35 min, the volume percentage of acetonitrile in the mobile phase increases from 4%-6% to 33%-36%;

[0019] 35-40 min, the volume percentage of acetonitrile in the mobile phase increases from 33%-36% to 48%-52%.

[0020] Further preferably, the process of gradient elution is:

[0021] 0-25min, the volume percentage of acetonitrile in the mobile phase is 4%-6%;

[0022] 25-35 min, the volume percentage of acetonitrile in the mobile phase increases from 4%-6% to 33%-36%;

[0023] 35-40 min, the volume percentage of acetonitrile in the mobile phase increases from 33%-36% to 48%-52%;

[0024] 40-42 min, the volume percentage of acetonitrile in the mobile phase is 100%;

[0025] At 45 to 47 minutes, the volume percentage of acetonitrile in the mobile phase is reduced from 100% to 4% to 6%.

[0026] Preferably, the solvent is selected from at least one of methanol, ethanol or acetonitrile. Further preferably, the solvent is methanol. Methanol has low toxicity and excellent solubility, and can better dissolve Ganweikang tablets and their main active ingredients.

[0027] Preferably, during the detection process, the detection wavelength is 230-250nm, 265-285nm and 300-320nm. Wherein, when the detection wavelength is 230-250nm, verbena glycoside, cimicifuga glycoside, 5-O-methylvisaminol glycoside, calycosin and ammonium glycyrrhizinate are detected; when the detection wavelength is 265-285nm, liquiritin and forsythiaside are detected; when the detection wavelength is 300-320nm, forsythiaside A is detected.

[0028] Further preferably, during the detection process, the detection wavelength is 235-245 nm, 270-280 nm and 305-315 nm; such as the detection wavelength is 240 nm, 275 nm and 310 nm.

[0029] Preferably, in the detection process, an Agilent ZOBAX SB-C18 column (250×4.6 mm, 5 μm) is used in combination with a ZOBAX SB-C18 pre-column (12.5×4.6 mm, 5 μm) with the same stationary phase for HPLC analysis.

[0030] Preferably, during the detection process, the flow rate is 0.8-1.2 mL / min, the column temperature is 25-35°C, and the injection volume is 5-15 μL. For example, during the detection process, the flow rate is 1.0 mL / min, the column temperature is 30°C, and the injection volume is 10 μL.

[0031] Preferably, the preparation process of the test solution is as follows: a certain amount of Ganweikan tablets is ground into uniform Ganweikan tablet powder, the Ganweikan tablet powder is weighed and added to a solvent, and after ultrasonication, the supernatant is filtered through a 0.22 μm filter membrane, and the filtrate is taken as the test solution.

[0032] Preferably, in the reference solution, the concentration of verbena glycoside is 62.5-312.50 μg / mL; the concentration of cimicifuga glycoside is 1.25-62.50 μg / mL; the concentration of forsythiaside A is 62.5-312.50 μg / mL; the concentration of liquiritin is 6.25-250.00 μg / mL; the concentration of 5-O-methylvisaminol glycoside is 3.13-125.00 μg / mL; the concentration of forsythia glycoside is 31.25-312.50 μg / mL; the concentration of calycosin is 1.25-125.00 μg / mL; and the concentration of ammonium glycyrrhizinate is 12.5-150.00 μg / mL.

[0033] The present invention also provides an application of the quantitative detection method of the Ganweikang tablets.

[0034] Specifically, the above-mentioned quantitative detection method of Ganweikan tablets is used in evaluating and controlling the quality of Ganweikan tablets.

[0035] Ganweikan tablets are composed of astragalus, bupleurum, atractylodes, patchouli, verbena, siler, forsythia, licorice, etc. Studies have found that verbena glycoside is an important active ingredient in verbena, which has multiple biological activities, such as liver protection, anti-hepatitis C virus, sleep promotion, and myocardial protection. The chromone compounds cimicifuga and 5-O-methylvisaminol glycoside in Saposhnikovia divaricata are important active ingredients in Saposhnikovia divaricata medicinal materials, and are the measurement standards for evaluating the quality of Saposhnikovia divaricata medicinal materials. Both the water extract and the alcohol extract of Saposhnikovia divaricata can significantly reduce the activities of ALT and AST in mouse serum, reduce the content of MDA in liver homogenate, and increase the activity of SOD, showing a good liver protection effect. Forsythiaside A and forsythiaside are the main active ingredients of Forsythia suspensa, a plant of the genus Forsythia. Forsythiaside A and forsythiaside have anti-inflammatory, antioxidant, and liver fibrosis improvement effects. Licorice glycoside, as a monomer active substance, is the main flavonoid compound in licorice, with multiple pharmacological activities such as anti-depression, protection of nerves, heart, and liver. Ammonium glycyrrhizinate is also one of the active ingredients in licorice and has a liver protection effect. As a representative of flavonoid components in Astragalus, calycosin has antioxidant, liver and kidney protection, and anti-tumor effects. Therefore, the present invention uses verbena glycoside, cimicifuga glycoside, 5-O-methylvisaminol glycoside, forsythiaside A, forsythiaside, liquiritigenin, ammonium glycyrrhizinate and verbascosin in Ganweikan tablets as representative active ingredients, and establishes a high performance liquid chromatography method to simultaneously and quantitatively detect the contents of the representative active ingredients. The quantitative detection method can comprehensively evaluate and control the quality of Ganweikan tablets, which is of great significance to the quality control and drug research and development of Ganweikan tablets.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) The quantitative detection method of Ganweikang Tablets provided by the present invention can simultaneously quantitatively determine the contents of representative active ingredients in Ganweikang Tablets, including verbena glycoside, cimicifuga glycoside, 5-O-methylvisaminol glycoside, forsythiaside A, forsythiaside, liquiritigenin, ammonium glycyrrhizinate and calycosin. The quantitative detection method can comprehensively evaluate and control the quality of Ganweikang Tablets.

[0038] (2) The quantitative detection method of Ganweikan Tablets provided by the present invention adopts a high-sensitivity high-performance liquid chromatograph to develop an analytical method capable of simultaneously determining 8 representative active ingredients. The method is simple to operate, has strong specificity, good repeatability, and accurate results, and can provide a technical reference for the quality control and quality standard improvement of Ganweikan Tablets. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is the linear relationship diagram of verbenaside, cimicifuga glycoside, 5-O-methylvisaminol glycoside and forsythiaside;

[0040] Figure 2 It is the linear relationship diagram of forsythiaside A, liquiritin, calycosin isoflavone and ammonium glycyrrhizinate;

[0041] Figure 3 It is the HPLC chromatogram of the reference substance mixed solution and the Ganweikang Tablets test solution at a detection wavelength of 240 nm;

[0042] Figure 4 It is the HPLC chromatogram of the reference substance mixed solution and the Ganweikang Tablets test solution at a detection wavelength of 275 nm;

[0043] Figure 5 It is the HPLC chromatogram of the reference mixed solution and the Ganweikang Tablets test solution at a detection wavelength of 310 nm. DETAILED DESCRIPTION

[0044] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0045] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0046] The embodiment of the present invention provides a quantitative detection method of Ganweikang tablets, which is as follows:

[0047] 1. Instruments and test drugs

[0048] 1.1 Instrument

[0049] Agilent 1260 Infinity high performance liquid chromatograph (Agilent Technologies Co., Ltd.), ultrasonic cleaning machine (Jiangsu Kunshan Jielimei Ultrasonic Instrument Co., Ltd.).

[0050] 1.2 Test drugs and reagents

[0051] Verbena glycoside (≥98%), cimicifuga glycoside (≥98%), forsythiaside A, 5-O-methylvisaminol glycoside and ammonium glycyrrhizinate (≥98%) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd., liquiritigenin (≥98%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., forsythia glycoside (95%) was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., and calycosin (≥98%) was purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.; acetonitrile (chromatographic grade) and formic acid (analytical grade) were from Chengdu Kelon Chemical Reagent Factory; Ganweikang tablets (batch numbers 210202, 210201, 200102, 200801 and 201201) were purchased from Henan Fusen Pharmaceutical Co., Ltd.

[0052] 2. Methods and Results

[0053] 2.1 Preparation of reference solution

[0054] Accurately weigh 2.5 mg of verbenaside, forsythiaside A and forsythiaside and place them in a centrifuge tube and add 1 mL of methanol respectively, 0.5 mg of cimicifuga glycoside and place them in a centrifuge tube and add 1 mL of methanol respectively, 2 mg of liquiritigenin and ammonium glycyrrhizinate and place them in a centrifuge tube and add 1 mL of methanol respectively, 1 mg of 5-O-methylvisaminol and 1 mg of calycosin are placed in a centrifuge tube and add 1 mL of methanol respectively and dissolve them by ultrasonication to prepare a reference solution.

[0055] 2.2 Preparation of test solution

[0056] Grind a certain amount of Ganweikang tablets into uniform powder, then weigh 0.5 g and add 5 mL of methanol for ultrasonication for 10 min. Pass the supernatant through a 0.22 μm filter membrane and take the filtrate as the test solution.

[0057] 2.3 Chromatographic conditions

[0058] HPLC analysis was performed using an Agilent ZOBAX SB-C18 column (250×4.6 mm, 5 μm) with a ZOBAX SB-C18 pre-column (12.5×4.6 mm, 5 μm) of the same stationary phase. Mobile phase A was 0.1% formic acid in water, and mobile phase B was acetonitrile. Gradient elution program: 0-25 min, 5% B; 25-35 min, 5%-35% B; 35-40 min, 35%-50% B; 40-42 min, 100% B; 45-47 min, 100%-5% B. The flow rate was 1.0 mL / min, and the detection wavelengths were 240 nm (verbenfoside, cimicifugoside, 5-O-methylvisaminol glycoside, calycosin and ammonium glycyrrhizinate), 275 nm (liquiritin and forsythiaside) and 310 nm (forsythiaside A), and the column temperature was 30°C. The injection volume for all samples was 10 μL.

[0059] 2.4 Investigation of linear relationship

[0060] Accurately measure the reference solution of verbena glycoside, cimicifuga glycoside, forsythiaside A, liquiritin, 5-O-methylvisaminol glycoside, forsythia glycoside, calycosin and ammonium glycyrrhizinate, dilute to a specific concentration with methanol solvent, filter with a 0.22 μm filter membrane and perform HPLC analysis. Perform linear regression on the concentration using the peak area. The corresponding linear relationship diagram is shown in Figure 1 to Figure 2 As shown, Figure 1 A, B, C, and D are the linear relationship diagrams of verbenaside, cimicifuga glycoside, 5-O-methylvisaminol glycoside, and forsythin, respectively; Figure 2 A, B, C, and D are the linear relationship diagrams of forsythiaside A, liquiritin, calycosin and ammonium glycyrrhizinate, respectively. The regression equation, the minimum detection limit (LOD, S / N=3) and the minimum quantification limit (LOQ, S / N=10) are shown in Table 1.

[0061] Table 1 Linear range, linear equation and correlation coefficient, LOD and LOQ data of eight compound contents

[0062]

[0063]

[0064] 2.5 Precision experiment

[0065] Take the same reference mixed solution, measure it 3 times on the same day, calculate the RSD of the peak area of ​​each component, and investigate the intra-day precision; inject 3 times a day, measure it for 3 consecutive days, calculate the RSD of the peak area of ​​each component, and investigate the inter-day precision. Results The intra-day precision RSD (n=3) of verbena glycoside, cimicifuga glycoside, forsythiaside A, liquiritin, 5-O-methylvisaminol glycoside, forsythiaside, calycosin and ammonium glycyrrhizinate were 1.3, 2.0, 0.8, 1.1, 1.0, 0.7, 1.5 and 2.4%, respectively, and the inter-day precision RSD (n=3) were 0.9, 1.0, 1.5, 0.7, 2.1, 2.0, 1.3 and 2.5%, respectively. The results show that the precision of the detection method provided by the present invention is good.

[0066] 2.6 Repeatability Experiment

[0067] Weigh 3 portions of the same Ganweikang Tablet powder (batch number: 210202) accurately, prepare the test solution according to the method under "2.2", and measure the contents of verbena glycoside, cimicifuga glycoside, forsythiaside A, liquiritin, 5-O-methylvisaminol glycoside, forsythia glycoside, calycosin isoflavone and ammonium glycyrrhizinate, respectively, and the RSD value is less than 2.1%. The results show that the detection method provided by the present invention has good repeatability.

[0068] 2.7 Recovery rate experiment

[0069] The standard addition method was used to carry out the recovery experiment, adding eight compound reference solutions of high, medium and low concentrations respectively, and then determining the contents. As shown in Table 2, the contents of verbena glycoside, cimicifuga glycoside, forsythiaside A, liquiritigenin, 5-O-methylvisaminol glycoside, forsythia glycoside, calycosus isoflavones and ammonium glycyrrhizinate in Ganweikang tablets (diluted 2 times) were measured to be 364.7 μg / g (36.47 μg / mL), 14.80 μg / g (1.48 μg / mL), 642.21 μg / g (64.59 μg / mL), 154.32 μg / g (15.61 μg / mL), 36.21 μg / g (3.62 μg / mL), 269.07 μg / g (27.00 μg / mL), 18.40 μg / g (1.84 μg / mL) and 78.12 μg / g (7.8 μg / mL), respectively. The recovery rate of the sample addition was between 97.5% and 104.8%, and the RSD value was less than 3.8%. The corresponding HPLC chromatogram is shown in Figures 3 to 5 As shown, Figure 3 A and B are HPLC chromatograms of the reference substance mixed solution and the Ganweikang Tablets test solution at a detection wavelength of 240 nm; Figure 4C and D in the figure are HPLC chromatograms of the reference substance mixed solution and the Ganweikang Tablets test solution at a detection wavelength of 275 nm; Figure 5 Figures E and F are HPLC chromatograms of the reference substance mixed solution and the Ganweikang Tablets test solution at a detection wavelength of 310 nm. Figures 3 to 5 In the figure, from left to right are verbena glycoside (1), cimicifuga glycoside (2), forsythiaside A (3), liquiritigenin (4), 5-O-methylvisaminol glycoside (5), forsythia glycoside (6), calycosin (7) and ammonium glycyrrhizinate (8).

[0070] Table 2 Determination results of the recovery of eight compounds

[0071]

[0072]

[0073] 2.8 Sample content determination

[0074] Five batches of Ganweikang tablets samples were taken, and the test solutions were prepared according to the above method. The contents of eight compounds therein were determined by the established HPLC method. The results are shown in Table 3. The content ranges of the eight compounds in the five different batches of Ganweikang tablets samples were slightly different, namely, verbena glycyrrhizin 727.60-729.92 μg / g, cimicifugo glycoside 29.16-29.80 μg / g, forsythiaside A 1283.10-1284.59 μg / g, liquiritin 307.05-308.73 μg / g, 5-O-methylvisamin glycoside 72.32-73.38 μg / g, forsythiaside 538.14-539.94 μg / g, calycosin 36.66-37.32 μg / g and ammonium glycyrrhizinate 155.86-158.52 μg / g. This indicates that the quality of the product is relatively stable, and the detection method provided by the present invention can be used as a quality control method for Ganweikan tablets.

[0075] Table 3 Contents of eight compounds in different batches of Ganweikang tablets (μg / g) (n=3)

[0076]

[0077] The quantitative detection method provided by the embodiment of the present invention has high linear fitting degree, high precision, good repeatability, and the sample recovery rate meets the requirements. It can be used to determine the content of representative active ingredients in Ganweikan tablets and is applied to the quality control and drug development of Ganweikan tablets.

[0078] The study found that in the above-mentioned quantitative detection method, if the conditions for controlling the gradient elution are: 0-25 min, the volume percentage of acetonitrile in the mobile phase is 4%-6%; 25-35 min, the volume percentage of acetonitrile in the mobile phase increases from 4%-6% to 33%-36%; 35-40 min, the volume percentage of acetonitrile in the mobile phase increases from 33%-36% to 48%-52%, the quantitative detection effect is similar to that of the above-mentioned embodiment, and has good linear fit, precision and repeatability.

[0079] The above-mentioned embodiment only expresses one implementation mode of the present invention, and its description is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A quantitative detection method for Ganweikang tablets, characterized in that: The following steps are involved: Weigh verbena glycoside, forsythia glycoside, cimicifuga glycoside, ammonium glycyrrhizinate and calycosin isoflavone, and dissolve them in a solvent to prepare a reference solution; Weigh Ganweikang tablets and dissolve them in methanol to prepare the test solution; Injecting the reference solution and the test solution into a high performance liquid chromatograph for detection to obtain chromatograms of the reference and test products; And calculating the content of each component in the test sample according to the peak area of ​​the chromatogram; In the detection process, a mobile phase is used for gradient elution, and the mobile phase includes 0.08% to 0.15% formic acid aqueous solution and acetonitrile; the process of the gradient elution is: 0-25min, the volume percentage of acetonitrile in the mobile phase is 5%; 25-35 min, the volume percentage of acetonitrile in the mobile phase increases from 5% to 35%; 35-40 min, the volume percentage of acetonitrile in the mobile phase increases from 35% to 50%; During the detection process, the detection wavelength for verbena glycoside, cimicifuga glycoside, calycosin and ammonium glycyrrhizinate was 240 nm; the detection wavelength for forsythiaside was 275 nm; In the detection process, an Agilent ZOBAX SB-C18 column, 250×4.6 mm, 5 μm, and a ZOBAX SB-C18 pre-column with the same stationary phase were used for HPLC analysis.

2. The quantitative detection method according to claim 1, characterized in that: The mobile phase includes 0.08% to 0.12% formic acid aqueous solution.

3. The quantitative detection method according to claim 1 or 2, characterized in that: The solvent is selected from at least one of methanol, ethanol or acetonitrile.

4. The quantitative detection method according to claim 1 or 2, characterized in that: During the detection process, the flow rate is 0.8-1.2 mL / min, the column temperature is 25-35° C., and the injection volume is 5-15 μL.

5. Use of the quantitative detection method for Ganweikan tablets according to any one of claims 1 to 4 in evaluating and controlling the quality of Ganweikan tablets.

Images