A method for determining the content of multiple components in Tongshukoushuang tablets

Abstract

This invention discloses a method for determining the content of multiple components in Tongshu Kou Shuang tablets. The specific steps are as follows: Step 1, prepare a reference solution; Step 2, perform HPLC on the reference solution according to preset HPLC chromatographic conditions; Step 3, prepare a test solution for the Tongshu Kou Shuang tablets; Step 4, determine the content of the test solution of the Tongshu Kou Shuang tablets according to the preset HPLC chromatographic conditions described in Step 2, and use the standard curves of each of the nine components to detect the content of each of the nine components in the test solution. This invention can simultaneously detect nine active substances in the two principal drugs and two assistant drugs of Tongshu Kou Shuang tablets, thereby enabling more comprehensive control and evaluation of the intrinsic quality of Tongshu Kou Shuang tablets. Furthermore, the simultaneous detection of the nine active substances effectively improves detection efficiency and reduces detection costs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical testing, and in particular to a method for determining the content of multiple components in Tongshu Kou Shuang tablets. Background Technology

[0002] Tongshu Kou Shuang tablets are composed of eight herbs: rhubarb, peony bark, angelica, immature bitter orange, gentian root, prunella vulgaris, artemisia capillaris, and horsetail. They have the effects of clearing heat and dampness, resolving turbidity, and promoting bowel movements. Clinically, they are used for halitosis, swollen and painful gums, and constipation caused by damp-heat in the large intestine, with significant efficacy. Traditional Chinese medicine compound preparations have complex components with synergistic and interactive effects. Quality and efficacy emphasize the holistic aspect; therefore, simultaneous determination of the content of multiple components is of great significance for the quality control of these preparations.

[0003] Currently, there are no literature reports on the content determination of Tongshu Kou Shuang tablets, and the existing quality standards only control the quality of rhein and chrysophanol in rhubarb, which is difficult to comprehensively control and evaluate the quality of this preparation. Rhubarb in the formula clears heat and detoxifies, promotes bowel movement, and its main active ingredients include anthraquinone components such as aloe-emodin, rhein, rhein, chrysophanol, and rhein methyl ether. Immature bitter orange breaks up qi stagnation and promotes bowel movement, and its main active ingredient is hesperidin. Gentiana macrophylla dispels wind and dampness and clears damp-heat, and its main active ingredients are loganic acid and gentianin. Moutan bark clears heat and cools blood, promotes blood circulation and removes blood stasis, and its main active ingredient is paeonol. Therefore, this invention uses HPLC to simultaneously determine the content of nine components in this preparation: loganic acid, gentianin, hesperidin, paeonol, aloe-emodin, rhein, rhein, chrysophanol, and rhein methyl ether, in order to more comprehensively control and evaluate the intrinsic quality of Tongshu Kou Shuang tablets, thereby ensuring the stability of clinical efficacy. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a method for determining the content of multiple components in Tongshu Kou Shuang tablets.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A method for determining the content of multiple components in Tongshu Kou Shuang tablets, wherein the multiple components of Tongshu Kou Shuang tablets include loganic acid, gentiopicrin, hesperidin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and emodin methyl ether, and the specific steps are as follows:

[0007] Step 1: Prepare the reference solution;

[0008] Step 2: Perform HPLC analysis on the reference solution under the preset HPLC chromatographic conditions to obtain the standard curves for each of the nine components. The preset HPLC chromatographic conditions are as follows: using Thermo... Acclaim-C18 column; mobile phase: 0.15% phosphoric acid-acetonitrile; gradient elution: 0–10 min, 8% B; 10–20 min, 8%→15% B; 20–35 min, 15%→25% B; 35–50 min, 25%→40% B; 50–65 min, 40%→70% B; 65–70 min, 70%→80% B; 75–80 min, 80% B; run for 6 min; flow rate: 1.0 mL·min⁻¹; column temperature: 30℃; injection volume: 10 μL; the detection wavelengths for loganic acid, gentiopicrin, hesperidin, and paeonol were 280 nm, and the detection wavelengths for aloe-emodin, rhein, emodin, chrysophanol, and emodin methyl ether were 254 nm.

[0009] Step 3: Prepare the test solution for the Tongshu Shuang tablets to be tested;

[0010] Step 4: The test solution of Tongshu Kou Shuang tablets is tested according to the preset HPLC chromatographic conditions described in Step 2. The content of each of the nine components in the test solution is determined by using the standard curves of the nine components.

[0011] In a further improvement, the reference solution is a mixed reference stock solution, wherein the concentrations of loganic acid, gentiopicrin, hesperidin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and emodin methyl ether in the mixed reference stock solution are 78.55 μg / mL, 224.1 μg / mL, 62.75 μg / mL, 42.36 μg / mL, 14.20 μg / mL, 19.32 μg / mL, 21.14 μg / mL, 24.17 μg / mL, and 11.19 μg / mL, respectively.

[0012] A further improvement is made to the preparation method of the mixed reference standard stock solution as follows:

[0013] Accurately weigh 10.07 mg, 8.07 mg, 10.61 mg, 6.02 mg, 6.88 mg, and 7.60 mg of loganic acid, hesperidin, paeonol, aloe-emodin, rhein, and chrysophanol reference standards into 25 mL volumetric flasks, respectively. Accurately weigh 6.08 mg and 5.64 mg of rhein and rhein methyl ether reference standards into 50 mL volumetric flasks, respectively. Dissolve in methanol and dilute to the mark to obtain stock solutions of each single reference standard. Accurately weigh 5.77 mg of gentiopicroside reference standard into a 25 mL volumetric flask, and then add the stock solutions of each single reference standard to the same volumetric flask: 5 mL of loganic acid, 5 mL of hesperidin, 2.5 mL of paeonol, 1.5 mL of aloe-emodin, 4 mL of rhein, 2 mL of rhein, 2 mL of chrysophanol, and 2.5 mL of rhein methyl ether. Dilute to the mark with methanol to obtain a mixed stock solution of reference standards.

[0014] Further improvements resulted in the following regression equations for the nine components:

[0015]

[0016] A further improvement is made to the preparation method of the test solution for the Tongshu Kou Shuang tablets as follows: Take a number of Tongshu Kou Shuang tablets, remove the film coating, crush them, weigh the powder, dissolve the powder in methanol solution, weigh to obtain the initial weight, heat under reflux for 60 minutes, cool, weigh again, add methanol to the initial weight, shake well, filter, and filter the filtrate through a 0.45μm microporous membrane to obtain the test solution.

[0017] In a further improvement, the volume-to-mass ratio of the powder to the methanol solution is 0.7:50 g / ml.

[0018] The beneficial effects of this invention are as follows:

[0019] This invention can simultaneously detect nine active substances in the two principal drugs and two assistant drugs of Tongshu Kou Shuang tablets, thereby enabling more comprehensive control and evaluation of the intrinsic quality of Tongshu Kou Shuang tablets. At the same time, the simultaneous detection of nine active substances effectively improves detection efficiency and reduces detection costs. Attached Figure Description

[0020] The invention will be further illustrated with reference to the accompanying drawings, but the contents of the drawings do not constitute any limitation on the invention.

[0021] Figure 1 The HPLC chromatogram is for the mixed reference standards.

[0022] Figure 2 The HPLC chromatogram of the test solution is shown below.

[0023] Figure 3 This is the HPLC chromatogram of a negative sample lacking Gentiana macrophylla;

[0024] Figure 4 The HPLC chromatogram is for a negative sample lacking Citrus aurantium.

[0025] Figure 5 The HPLC chromatogram is for a negative sample lacking peony bark.

[0026] Figure 6 This is the HPLC chromatogram of a negative sample lacking rhubarb.

[0027] In the diagram: 1. Loganic acid 2. Gentianoside 3. Hesperidin 4. Paeonol 5. Aloe-emodin 6. Emodin 7. Emodin 8. Rhein 9. Emodin methyl ether. Detailed Implementation

[0028] To make the purpose, technical solution, and advantages of the invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and examples.

[0029] 1. Reagents and Instruments

[0030] Agilent 1260 high performance liquid chromatograph (equipped with diode array detector, autosampler, quaternary pump, column oven, Agilent Technologies); XSE-205Du electronic balance (Mettler Toledo); electric thermostatic water bath (Beijing Yongguangming Medical Instrument Co., Ltd.); KQ-500VDE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0031] Reference standards, reagents, and samples: Reference standards include loganic acid (batch number 111865-20202005, content 97.5%), gentiopicrin (batch number 110770-201918, content 97.1%), hesperidin (batch number 110721-202220, content 97.2%), paeonol (batch number 110708-201908, content 99.8%), and aloe-emodin (batch number 110795-). The following substances were purchased from the National Institutes for Food and Drug Control: 201710 (98.3% purity), rhein (batch number 110757-201607, 99.3% purity), emodin (batch number 110756-201913, 96.0% purity), chrysophanol (batch number 110796-201922, 99.4% purity), and emodin methyl ether (batch number 110758-201817, 99.2% purity). Formic acid, acetic acid, and phosphoric acid (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); methanol and acetonitrile (chromatographic grade, Merck GmbH, Germany); and water was Watson's purified water.

[0032] Tongshu Kou Shuang Tablets (batch numbers: 20211001, 20211101, 20220101, 20220201, 20220401, 20220402, Jilin Junhong Pharmaceutical Co., Ltd.).

[0033] 2. Chromatographic conditions

[0034] Use a Thermo Acclaim-C18 (250 mm × 4.6 mm, 5 μm) chromatographic column; mobile phase: acetonitrile (A) - 0.15% phosphoric acid (B), gradient elution (0 - 10 min, 8% A; 10 - 20 min, 8% → 15% A; 20 - 35 min, 15% → 25% A; 35 - 50 min, 25% → 40% A; 50 - 65 min, 40% → 70% A; 65 - 70 min, 70% → 80% A; 75 - 80 min, 80% A; post-run for 6 min); flow rate: 1.0 mL·min-1; column temperature 30 °C; injection volume: 10 μL; detection wavelengths are 236 nm (for detecting loganinic acid), 280 nm (for detecting gentiopicroside, hesperidin, paeonol), and 254 nm (for detecting aloe-emodin, rhein, emodin, chrysophanol, and physcion).

[0035] 3. Preparation of solutions

[0036] Test solution: Take 20 Tongshu Kou爽 tablets, remove the film coating, grind them finely, accurately weigh 0.7 g of the powder, place it in a stoppered conical flask, accurately add 50 mL of methanol, weigh it, heat under reflux for 60 min, cool, weigh again, make up the lost weight with methanol, shake well, filter, and filter the filtrate through a 0.45 μm microporous filter membrane to obtain the test solution.

[0037] Negative sample solution: According to the proportion of each medicinal material in the prescription, take the other medicinal materials except gentiana macrophylla, aurantii fructus immaturus, moutan cortex, and rheum officinale, prepare each negative sample according to the process, and prepare the negative sample solutions lacking gentiana macrophylla, lacking aurantii fructus immaturus, lacking moutan cortex, and lacking rheum officinale according to the preparation method of the test solution.

[0038] Accurately weigh 10.07, 8.07, 10.61, 6.02, 6.88, and 7.60 mg of loganic acid, hesperidin, paeonol, aloe-emodin, rhein, and chrysophanol reference standards into 25 mL volumetric flasks, respectively. Accurately weigh 6.08 and 5.64 mg of rhein and rhein methyl ether reference standards into 50 mL volumetric flasks, respectively. Dissolve in methanol and dilute to volume to obtain stock solutions of each single reference standard. Accurately weigh 5.77 mg of gentiopicroside reference standard and place it in a 25 mL volumetric flask. Then, add the following single reference standard stock solutions to the same volumetric flask: 5 mL of loganic acid, 5 mL of hesperidin, 2.5 mL of paeonol, 1.5 mL of aloe-emodin, 4 mL of emodin, 2 mL of emodin, 2 mL of chrysophanol, and 2.5 mL of emodin methyl ether. Dilute to the mark with methanol to obtain a mixed reference standard stock solution (containing 78.55, 224.1, 62.75, 42.36, 14.20, 19.32, 21.14, 24.17, and 11.19 μg / mL of loganic acid, gentiopicroside, hesperidin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and emodin methyl ether, respectively).

[0039] 4. Specificity test

[0040] Take appropriate amounts of the test sample solution, four negative sample solutions, and the mixed reference solution, filter them through a 0.45 μm microporous membrane, and inject them separately into a high-performance liquid chromatograph (HPLC). Perform the chromatographic analysis under the conditions described above, and record the chromatograms. Results show that the retention times of the nine component peaks in the test sample chromatogram are consistent with those of the mixed reference solution, and the resolution with adjacent impurity peaks is greater than 1.9. No chromatographic peaks appeared at the corresponding retention time positions in the negative samples, indicating good method specificity.

[0041] 5. Linear relationship

[0042] Accurately measure 0.5, 1, 2, 3, 4, and 5 mL of the mixed reference standard stock solution into 5 mL volumetric flasks, and add methanol to the mark to obtain a series of standard curve working solutions. Take an appropriate amount, filter through a 0.45 μm microporous membrane, and inject and determine the peak area of ​​each analyte according to the chromatographic conditions in section "2.1". Perform linear regression on the peak area with the mass concentration (μg / mL) of each analyte to plot the standard curve for each component, and obtain the regression equation (see Table 1). The results show that the linear relationship of each component is good within the experimental range (r = 0.9991–0.9999).

[0043] Table 1 Results of linear relationship investigation of 9 components

[0044]

[0045] 6. Instrument precision test

[0046] The mixed reference standard stock solution was diluted 2-fold and filtered through a 0.45 μm microporous membrane. The peak areas of each component were determined by continuous injection six times under the chromatographic conditions described above. The RSDs of the peak areas of loganic acid, gentiopicrin, hesperidin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and emodin methyl ether were 0.50%, 0.15%, 0.61%, 0.19%, 0.23%, 0.20%, 0.42%, 0.17%, and 0.44%, respectively, indicating good instrument precision.

[0047] 7. Stability test

[0048] The same test solution (batch number: 20220101) was used, and the peak areas of loganic acid, gentiopicrin, hesperidin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and emodin methyl ether were determined at 0, 6, 12, 18, 24, and 36 h under the chromatographic conditions described above. The RSD values ​​were 0.57%, 0.55%, 0.73%, 0.66%, 0.58%, 0.93%, 1.67%, 0.55%, and 0.58%, respectively, indicating good stability of the test solution within 36 h.

[0049] 8. Repeatability test

[0050] Six portions of the same batch of samples (batch number: 20220101) were accurately weighed and prepared in parallel. The peak areas of loganic acid, gentiopicrin, hesperidin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and emodin methyl ether were then determined under the chromatographic conditions described above. The values ​​were substituted into the standard curve to calculate the content of the nine components. The average contents of the nine components were 3.555, 10.09, 2.929, 1.819, 0.5039, 0.8046, 0.6231, 1.111, and 0.4166 mg / g, respectively, with RSD values ​​of 0.94%, 1.04%, 1.68%, 1.97%, 1.94%, 1.90%, 1.16%, 1.84%, and 1.42%. The method showed good repeatability.

[0051] 9. Recovery test

[0052] Take 0.35g of Tongshu Kou Shuang tablets (batch number: 20220101) with known contents of loganic acid, gentiopicroside, hesperidin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and emodin methyl ether. Divide the sample into 6 portions and place each portion in a stoppered conical flask. Add the corresponding reference solution (0.4228mg / mL loganic acid 3mL, 0.3555mg / mL gentiopicroside 10mL, 0.2047mg / mL hesperidin 5mL, 0.4375mg / mL paeonol 1.5mL) to each portion at 100% of the content of each component in the sample. L, 0.1846 mg / mL aloe-emodin 1 mL, 0.1501 mg / mL rhein 2 mL, 0.2312 mg / mL rhein 1 mL, 0.2712 mg / mL chrysophanol 1.5 mL, 0.1192 mg / mL rhein methyl ether 1.5 mL), add methanol to make up to 50 mL, then inject according to the above chromatographic conditions to determine the peak areas of loganic acid, gentiopicrin, hesperidin, paeonol, aloe-emodin, rhein, rhein, chrysophanol, and rhein methyl ether. Substitute into the corresponding standard curves to calculate the recovery rates of the nine components. The results are shown in Table 2.

[0053] Table 2. Results of recovery tests for 9 components (n=6)

[0054]

[0055]

[0056] 10. Sample content determination

[0057] Six batches of Tongshu Kou Shuang tablets were sampled, and two portions of each batch were weighed. Test solutions were prepared separately, and the peak areas of loganic acid, gentiopicrin, hesperidin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and emodin methyl ether were determined by injection. The contents of each were calculated by substituting them into the standard curve (see Table 3).

[0058] Table 3. Results of the content of 9 components in the Tongshu Kou Shuang tablets sample (mg / g, n=2)

[0059]

[0060] This invention is the first to employ HPLC technology to establish a method for simultaneously determining the content of nine active ingredients in Tongshu Kou Shuang tablets. This invention provides a comprehensive, objective, and accurate evaluation of the quality of Tongshu Kou Shuang tablets, offering a basis for quality control and enabling more comprehensive control and evaluation of the intrinsic quality of this preparation, thus improving the quality control methods for this preparation.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for determining the content of multiple components in a soothing and refreshing tablet, characterized in that, The multiple components of the Tongshu Kou Shuang tablets include loganic acid, gentiopicrin, hesperidin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and emodin methyl ether. The specific steps are as follows: Step 1: Prepare the reference solution; Step 2: Perform HPLC analysis on the reference solution under the preset HPLC chromatographic conditions to obtain the standard curves for each of the nine components. The preset HPLC chromatographic conditions are as follows: using Thermo... Acclaim-C18 column; mobile phase: 0.15% phosphate A-acetonitrile B; gradient elution: 0–10 min, 8% B; 10–20 min, 8%→15% B; 20–35 min, 15%→25% B; 35–50 min, 25%→40% B; 50–65 min, 40%→70% B; 65–70 min, 70%→80% B; 75–80 min, 80% B; run for 6 min; flow rate: 1.0 mL·min⁻¹; column temperature: 30℃; injection volume: 10 μL; the detection wavelengths for loganic acid, gentiopicrin, hesperidin, and paeonol were 280 nm, and the detection wavelengths for aloe-emodin, rhein, emodin, chrysophanol, and emodin methyl ether were 254 nm. Step 3: Prepare the test solution for the Tongshu Shuang tablets to be tested; Step 4: The test solution of Tongshu Kou Shuang tablets is tested according to the preset HPLC chromatographic conditions described in Step 2. The content of each of the nine components in the test solution is obtained by using the standard curves of the nine components. The preparation method of the test solution for the Tongshushuang tablets to be tested is as follows: Take a number of Tongshu Kou Shuang tablets, remove the film coating, crush them, weigh the powder, dissolve the powder in methanol solution, weigh to obtain the initial weight, heat under reflux for 60 minutes, cool, weigh again, add methanol to the initial weight, shake well, filter, and filter the filtrate through a 0.45μm microporous membrane to obtain the test solution.

2. The method for determining the multi-component content of Tongshu Kou Shuang tablets as described in claim 1, characterized in that, The reference solution is a mixed reference stock solution, in which the concentrations of loganic acid, gentiopicrin, hesperidin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and emodin methyl ether are 78.55 μg / mL, 224.1 μg / mL, 62.75 μg / mL, 42.36 μg / mL, 14.20 μg / mL, 19.32 μg / mL, 21.14 μg / mL, 24.17 μg / mL, and 11.19 μg / mL, respectively.

3. The method for determining the multi-component content of Tongshu Kou Shuang tablets as described in claim 2, characterized in that, The preparation method of the mixed reference standard stock solution is as follows: Accurately weigh 10.07 mg, 8.07 mg, 10.61 mg, 6.02 mg, 6.88 mg, and 7.60 mg of loganic acid, hesperidin, paeonol, aloe-emodin, rhein, and chrysophanol reference standards into 25 mL volumetric flasks, respectively. Accurately weigh 6.08 mg and 5.64 mg of rhein and rhein methyl ether reference standards into 50 mL volumetric flasks, respectively. Dissolve in methanol and dilute to the mark to obtain stock solutions of each single reference standard. Accurately weigh 5.77 mg of gentiopicroside reference standard into a 25 mL volumetric flask, and then add the stock solutions of each single reference standard to the same volumetric flask: 5 mL of loganic acid, 5 mL of hesperidin, 2.5 mL of paeonol, 1.5 mL of aloe-emodin, 4 mL of rhein, 2 mL of rhein, 2 mL of chrysophanol, and 2.5 mL of rhein methyl ether. Dilute to the mark with methanol to obtain a mixed stock solution of reference standards.

4. The method for determining the multi-component content of Tongshu Kou Shuang tablets as described in claim 1, characterized in that, The regression equations for the nine components are shown below:

5. The method for determining the multi-component content of Tongshu Kou Shuang tablets as described in claim 1, characterized in that, The volume-to-mass ratio of the powder to the methanol solution is 0.7:50 g / ml.

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