Method for determining multi-component content of Xintong oral liquid

Abstract

The application discloses a content determination method of Xintong oral liquid, which adopts UPLC method to determine the contents of puerarin, daidzin, 2,3,5,4 ’ tetrahydroxystilbene-2-O-beta-D-glucoside, naringin and icariin in the preparation. The quality detection method is stable and reliable, has strong specificity and good reproducibility, can comprehensively and effectively control the quality of the Xintong oral liquid, is favorable for stabilizing the product quality, ensures the safety and effectiveness of clinical medication, and thus better meets the needs of medical treatment and market.

Description

Technical Field

[0001] This invention relates to a quality testing method for Xintong oral liquid, and more particularly to a method for determining the content of multiple components of Xintong oral liquid based on UPLC, which belongs to the field of traditional Chinese medicine preparation analysis. Background Technology

[0002] Xintong Oral Liquid is included in the national ministerial standards. It is made from thirteen Chinese medicinal materials, including Astragalus membranaceus, Codonopsis pilosula, Ophiopogon japonicus, Polygonum multiflorum, Epimedium brevicornu, Pueraria lobata, Angelica sinensis, Salvia miltiorrhiza, Gleditsia sinensis thorns, Sargassum fusiforme, Laminaria japonica, Ostrea gigas, and Citrus aurantium. It has the effects of invigorating qi and blood, resolving phlegm and unblocking collaterals. It is used for chest pain caused by deficiency of both qi and yin and phlegm and blood stasis, such as coronary heart disease and angina pectoris, with symptoms such as chest pain, chest tightness, shortness of breath, nausea and vomiting, and poor appetite. It has significant clinical effects.

[0003] Currently, the national standard YBZ16392009-2017 for the inspection of Xintong preparations mainly includes thin-layer chromatography identification of Astragalus membranaceus, Polygonum multiflorum, Epimedium, and Salvia miltiorrhiza, and content determination of Pueraria lobata. Traditional Chinese medicine compound preparations have complex components. Simply using thin-layer chromatography for qualitative identification and ultra-high performance liquid chromatography for content determination of single active ingredients, such as puerarin, is insufficient to comprehensively reflect the quality of Xintong oral liquid.

[0004] Ultra-high performance liquid chromatography (UPLC), based on the principles of HPLC, incorporates novel technologies such as small-particle packing materials, very low system volume, and rapid detection methods, increasing analytical throughput and peak capacity. It is characterized by its simplicity, accuracy, high sensitivity, good repeatability, and versatility in detectors, making it a crucial method for determining the content of multiple components in pharmaceutical preparations. Currently, no patents or literature reports have documented studies using UPLC to determine the content of seven components in Xintong oral liquid. This invention discloses a UPLC method for the quantification of five components in Xintong oral liquid, enabling comprehensive evaluation and control of its quality, thereby ensuring product stability and the safety and efficacy of clinical use. Simultaneously, UPLC is used to determine multiple components in Xintong oral liquid, completing the detection of five components within 56 minutes. The experimental results demonstrate that UPLC has high separation efficiency, while also saving analytical costs, reducing solvent usage, and being more environmentally friendly. The separation method established in this experiment provides data reference for improving the quality standards of Xintong oral liquid. Summary of the Invention

[0005] The technical problem this invention aims to solve is to further optimize the formulation detection method based on the national ministerial standard YBZ16392009-2017, simplifying the operation method and improving the detection efficiency. Through long-term exploration and repeated experiments, the inventors have successfully detected puerarin, daidzein, and 2,3,5,4-diethylstilbestrol in formulations. ‘ The UPLC content determination of five components, including tetrahydroxystilbene-2-O-β-D-glucoside, naringin, and icariin, is combined into one, providing a new method for more comprehensive and accurate detection of Xintong oral liquid.

[0006] The purpose of this invention is to provide a quality testing method for Xintong oral liquid. Xintong oral liquid is formulated from thirteen traditional Chinese medicines: Astragalus membranaceus, Codonopsis pilosula, Ophiopogon japonicus, Polygonum multiflorum, Epimedium brevicornu, Pueraria lobata, Angelica sinensis, Salvia miltiorrhiza, Gleditsia sinensis thorns, Sargassum fusiforme, Laminaria japonica, Ostrea gigas, and Citrus aurantium, and is prepared through extraction and purification. Puerarin and daidzein are components of Pueraria lobata, 2,3,5,4... ‘ -Tetrahydroxystilbene-2-O-β-D-glucoside is a component of Polygonum multiflorum, naringin is a component of Citrus aurantium, and icariin is a component of Epimedium. This invention uses UPLC to simultaneously determine puerarin, daidzein, and 2,3,5,4-diethylstilbestrol in the formulation. ‘ The content of tetrahydroxystilbene-2-O-β-D-glucoside, naringin, and icariin is measured to achieve a comprehensive evaluation and control of the quality of Xintong oral liquid, thereby providing an accurate basis for identifying the authenticity and detecting the intrinsic quality of Xintong oral liquid. Specifically, the following steps are included:

[0007] A. Preparation of test solution: Accurately measure 2 ml of Xintong oral liquid, place it in a 25 ml volumetric flask, dilute to the mark with 50% methanol, shake well, filter, and use as the test solution;

[0008] B. Preparation of reference solution: Accurately weigh appropriate amounts of puerarin, daidzein, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, naringin, and icariin reference standards, place them in a 25ml volumetric flask, dissolve and dilute to the mark with 50% methanol, and shake well to obtain the solution.

[0009] C. UPLC chromatographic conditions: Gradient elution was performed using acetonitrile (A)-water (B) as the mobile phase; Detection wavelength: puerarin, daidzein, naringin, and icariin were detected at 283 nm, and stilbene glycosides were detected at 320 nm; Injection volume: 2 μL;

[0010] D. Determination method: Accurately pipette 2 μL of the reference solution and the test solution into the liquid chromatograph and determine the chromatographic conditions as described in step C.

[0011] Preferably, the changes in the mobile phase gradient content and flow rate in step C are as follows:

[0012] Time (min) Flow rate (ml / min) Mobile phase A (%) Mobile phase B (%) 0-18 0.5 7％→25％ 93％→75％ 18-25 0.5 25％→70％ 75％→30％ 25-28 0.5 70％→7％ 30％→93％

[0013] Compared with the prior art, the present invention achieves the following beneficial technical effects:

[0014] 1) This invention establishes an upgraded quality testing method for Xintong oral liquid, using UPLC to quantitatively determine puerarin, daidzein, and 2,3,5,4-diethylstilbestrol in the preparation. ‘ The content of five components, namely tetrahydroxystilbene-2-O-β-D-glucoside, naringin, and icariin, can be further and effectively controlled to achieve a comprehensive evaluation of the preparation quality.

[0015] 2) This invention enables simultaneous detection of five components in Xintong oral liquid through a single test and multiple evaluation, thereby simplifying the operation method, improving detection efficiency, saving consumables and labor costs, and providing a faster, more convenient, energy-saving and environmentally friendly detection method.

[0016] 3) This invention is the first to successfully combine the UPLC content determination of five components in the preparation, namely puerarin, daidzein, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, naringin and icariin, through a gradient elution procedure, providing a new method for more comprehensive and accurate detection and evaluation of the stability of Xintong oral liquid.

[0017] 4) Compared with existing quality testing methods for Xintong oral liquid, the method of the present invention has the advantages of being fast, sensitive, and reproducible. It can accurately determine the five components in Xintong oral liquid, with strong specificity and good reproducibility. It can comprehensively and effectively control the quality of Xintong oral liquid, which is conducive to stabilizing product quality, ensuring the safety and effectiveness of clinical medication, and better meeting the needs of medical care and the market. Attached Figure Description

[0018] Figure 1 This is the UPLC chromatogram of the mixed reference standard. The chromatographic peaks from left to right are puerarin, daidzein, and peaks 2, 3, 5, and 4. ‘ -Tetrahydroxystilbene-2-O-β-D-glucoside, naringin, icariin;

[0019] Figure 2 The UPLC chromatogram of the test sample at a wavelength of 283 nm is shown.

[0020] Figure 3 The UPLC chromatogram of the test sample at a wavelength of 320 nm is shown.

[0021] Figure 4 This is the UPLC chromatogram for a negative control lacking kudzu root;

[0022] Figure 5 The UPLC chromatogram for the negative control of Polygonum multiflorum is missing.

[0023] Figure 6 This is the UPLC chromatogram for the negative control of Citrus aurantium lacking.

[0024] Figure 7 This is the UPLC chromatogram for the epimedium negative control. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments.

[0026] Example 1: UPLC Methodology Study of Five Components in Xintong Oral Liquid

[0027] 1. Medicines and Reagents

[0028] 1.1 Drugs and Reagents: Puerarin reference standard (batch number: 110752-201816, purity 95.4%), daidzein reference standard (batch number: 111738-201904, purity 93.4%), 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (batch number: 110844-201814, purity 91.0%), naringin reference standard (batch number: 110722-201815, purity 91.7%), and icariin (batch number: 110737-201516, purity 94.2%) were all purchased from the National Institutes for Food and Drug Control; methanol and acetonitrile were of chromatographic grade, water was double-distilled water, and all other reagents were of analytical grade.

[0029] 1.2 Test Drugs

[0030] Xintong Oral Solution was provided by Lunan Houpu Pharmaceutical Co., Ltd., and the sample batch number is shown in Table 1.

[0031] Table 1. Sample batch numbers for Xintong oral liquid.

[0032]

[0033]

[0034] 2. Instrument: Waters Acquity Arc high-performance liquid chromatograph (USA): 2998PDA detector;

[0035] 3. Chromatographic conditions: Column: Waters C18 column (3.0x100mm, 2.7um); acetonitrile as mobile phase A, aqueous solution as mobile phase B; column temperature: 25℃; injection volume: 2μl;

[0036] Table 2. Changes in mobile phase gradient content and flow rate:

[0037] Time (min) Flow rate (ml / min) Mobile phase A (%) Mobile phase B (%) 0-18 0.5 7％→25％ 93％→75％ 18-25 0.5 25％→70％ 75％→50％ 25-28 0.5 70→7 30→93

[0038] 4. Methodological investigation of content determination

[0039] 4.1 Preparation of the test solution

[0040] Accurately measure 2 ml of Xintong oral liquid, place it in a 25 ml volumetric flask, dilute to the mark with 50% methanol, shake well, filter, and use as the test solution;

[0041] 4.2 Preparation of mixed reference solution

[0042] Accurately weigh puerarin, daidzein, and 2,3,5,4 ‘ A mixture of tetrahydroxystilbene-2-O-β-D-glucoside, naringin, icariin, and reference standard in appropriate amounts was prepared by adding methanol to form a mixed solution containing 174.78 μg, 42.67 μg, 21.6 μg, 33.56 μg, and 19.66 μg per mL, respectively.

[0043] 4.3 Preparation of negative control solution

[0044] Negative control solutions were prepared by taking samples without kudzu root, Polygonum multiflorum, Citrus aurantium, and Epimedium, respectively, following the same procedure as in the preparation of the test solution. The chromatograms showed the presence of puerarin, daidzein, and 2,3,5,4... ’ No interference was observed at the positions of -tetrahydroxystilbene-2-O-β-D-glucoside, naringin, and icariin. Figure 4 , Figure 5 , Figure 6 and Figure 7 .

[0045] 4.4 Examination of Linear Relationships

[0046] Accurately weigh an appropriate amount of the reference standard, add 50% methanol to prepare the following concentrations, and perform the determination under the above chromatographic conditions. Inject 2 μL of each sample and measure the peak area. The results are shown in Tables 3, 4, 5, 6, and 7.

[0047] Table 3 Results of the linear relationship investigation of puerarin

[0048]

[0049]

[0050] Table 4 Results of linear relationship investigation of daidzin

[0051]

[0052] Table 5. Results of linearity investigation of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside

[0053]

[0054] Table 6 Results of linear relationship investigation of naringin

[0055]

[0056] Table 7 Results of linear relationship investigation of icariin

[0057]

[0058]

[0059] For the determination of puerarin, a standard curve was plotted with the measured peak area as the ordinate and the concentration of the reference standard (μg / mL) as the abscissa. The regression equation was Y = 2960.1811X - 2360.8667, and R² = 0.9979, indicating that the puerarin concentration showed good linearity in the range of 87.39–524.34 μg / mL.

[0060] For the determination of daidzin, a standard curve was plotted with the measured peak area as the ordinate and the concentration of the reference standard (μg / mL) as the abscissa. The regression equation was Y = 3167.2913x - 313.1414, R² = 0.9992, indicating that the daidzin concentration showed good linearity in the range of 21.34–128.01 μg / mL.

[0061] The determination of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside was performed. A standard curve was plotted with the measured peak area as the ordinate and the concentration of the reference standard (μg / mL) as the abscissa. The regression equation was Y = 5571.0000x - 12,393.1333, R² = 0.9989, indicating that the concentration of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside showed good linearity in the range of 10.80–64.80 μg / mL.

[0062] For the determination of naringin, a standard curve was plotted with the measured peak area as the ordinate and the concentration of the reference standard (ug / mL) as the abscissa. The regression equation was Y = 4538.9654x + 779.8199, R² = 0.9995, indicating that the naringin concentration showed good linearity in the range of 16.78–100.67 μg / mL.

[0063] For the determination of icariin, a standard curve was plotted with the measured peak area as the ordinate and the concentration of the reference standard (μg / mL) as the abscissa. The regression equation was Y = 2176.6070x + 1261.9869, R² = 0.9996, indicating that the icariin concentration showed good linearity in the range of 9.83–58.95 μg / mL.

[0064] 5. Precision test

[0065] The reference solution was precisely pipetted and injected six times, 2 μL each time. The peak area RSD was less than 2%, indicating good instrument precision. The results are shown in Table 8.

[0066] Table 8 Precision Test Results

[0067]

[0068]

[0069] 6. Stability Test

[0070] The same sample solution was measured at 0, 2, 4, 6, 8, 12, 16, 20, and 24 hours after preparation. The results showed that the sample was stable within 24 hours. The results are shown in Table 9.

[0071] Table 9. Stability Test Results

[0072]

[0073] 7 Repeatability Test

[0074] Six samples from the same batch (217210481) were measured and their contents were calculated according to the sample preparation method under the "Sample Determination" section. The results are shown in Tables 10, 11, 12, 13, and 14.

[0075] Table 10 Results of repeatability tests of puerarin

[0076]

[0077] Table 11 Results of repeatability tests on daidzin

[0078]

[0079]

[0080] Table 12 Results of repeatability tests on 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside

[0081]

[0082] Table 13 Results of repeatability test of naringin

[0083]

[0084] Table 14 Results of repeatability tests of icariin

[0085]

[0086] 8 Recovery rate test

[0087] Take samples with known content, accurately measure 6 portions, 1 ml each, and place them in 25 ml volumetric flasks. Accurately add 6 ml of 0.8739 mg / ml puerarin reference solution, 2.5 ml of 0.4267 mg / ml daidzein reference solution, 1 ml of 0.2257 mg / ml 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference solution, 1 ml of 0.5243 mg / ml naringin reference solution, and 0.5 ml of 0.4090 mg / ml icariin to the respective solutions. Determine the results using the same method as in the "Preparation of Test Solution" section. The results are shown in Tables 15, 16, 17, 18, and 19.

[0088] Table 15 Results of Puerarin Recovery Rate Test

[0089]

[0090] Table 16 Results of the daidzein recovery test

[0091]

[0092] Table 17 Results of the recovery test of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside

[0093]

[0094] Table 18 Results of the naringin recovery test

[0095]

[0096]

[0097] Table 19 Results of the icariin recovery test

[0098]

[0099] The results of the content determination of 9 components are shown in Table 20 (5 components in 18 batches of Xintong oral liquid).

[0100] Table 20 Contents of 5 Components in 18 Batches of Xintong Oral Liquid

[0101]

[0102]

[0103] Example 2: UPLC determination of five components of Xintong oral liquid

[0104] A. Preparation of test solution: Accurately measure 2 ml of Xintong oral liquid, place it in a 25 ml volumetric flask, dilute to the mark with 40% methanol, shake well, filter, and use as the test solution;

[0105] B. Preparation of reference solution: Accurately weigh appropriate amounts of puerarin, daidzein, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, naringin, and icariin reference standards, place them in a 25ml volumetric flask, dissolve and dilute to the mark with 40% methanol, and shake well to obtain the solution.

[0106] C. UPLC chromatographic conditions: Gradient elution was performed using acetonitrile (A)-water (B) as the mobile phase; detection wavelengths: puerarin, daidzein, naringin, and icariin were detected at 283 nm, and stilbene glycosides were detected at 320 nm; injection volume was 2 μL; and elution conditions were as follows: [Details omitted for brevity].

[0107] Time (min) Flow rate (ml / min) Mobile phase (A %) Mobile phase (B %) 0-18 0.5 7％→25％ 93％→75％ 18-25 0.5 25％→70％ 75％→30％ 25-28 0.5 70％→7％ 30％→93％

[0108] D. Determination method: Accurately pipette 2 μL of the reference solution and the test solution into the liquid chromatograph and determine the chromatographic conditions as described in step C.

[0109] Example 3: UPLC determination of five components of Xintong oral liquid

[0110] A. Preparation of test solution: Accurately measure 2 ml of Xintong oral liquid, place it in a 25 ml volumetric flask, dilute to the mark with 60% methanol, shake well, filter, and use as the test solution;

[0111] B. Preparation of reference solution: Accurately weigh appropriate amounts of puerarin, daidzein, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, naringin, and icariin reference standards, place them in a 25ml volumetric flask, dissolve and dilute to the mark with 60% methanol, and shake well to obtain the solution.

[0112] C. UPLC chromatographic conditions: Gradient elution was performed using acetonitrile (A)-water (B) as the mobile phase; detection wavelengths: puerarin, daidzein, naringin, and icariin were detected at 283 nm, and stilbene glycosides were detected at 320 nm; injection volume was 2 μL; and elution conditions were as follows: [Details omitted for brevity].

[0113] Time (min) Flow rate (ml / min) Mobile phase (A %) Mobile phase (B %) 0-18 0.5 7％→25％ 93％→75％ 18-25 0.5 25％→70％ 75％→30％ 25-28 0.5 70％→7％ 30％→93％

[0114] D. Determination method: Accurately pipette 2 μL of the reference solution and the test solution into the liquid chromatograph and determine the chromatographic conditions as described in step C.

Claims

1. A quality testing method for Xintong oral liquid, characterized in that, This method uses ultra-high performance liquid chromatography (UHPLC) to determine puerarin, daidzein, and 2,3,5,4-dimethylglycine in the formulation. ‘ The content of tetrahydroxystilbene-2-O-β-D-glucoside, naringin, and icariin is determined by the following steps: A. Preparation of test solution: Accurately measure 2 ml of Xintong oral liquid, place it in a 25 ml volumetric flask, dilute to the mark with 40%-60% methanol, shake well, filter, and use as the test solution; B. Preparation of reference solution: Accurately weigh puerarin, daidzein, 2,3,5,4 ‘ Take appropriate amounts of tetrahydroxystilbene-2-O-β-D-glucoside, naringin, and icariin reference standards, place them in a 25 ml volumetric flask, dissolve and dilute to the mark with 40-60% methanol, and shake well to obtain the final product. C. UPLC chromatographic conditions: Gradient elution was performed using acetonitrile as mobile phase A and water as mobile phase B; Waters CORTECS ® C18 column, 3.0x100mm, 2.7µm; detection wavelength: 283nm for puerarin, daidzein, naringin, and icariin; 320nm for stilbene glycosides; injection volume 2µL; D. Determination method: Accurately pipette 2µL of the reference solution and the test solution into the liquid chromatograph and determine them according to the chromatographic conditions described in step C. The elution conditions for step C, namely, the gradient content and velocity gradient of the mobile phase, are as follows: 。 2. The method according to claim 1, characterized in that, In step A, add 50 ml of 50% methanol, shake well, and then dilute to the mark with 50% methanol, shaking well.

3. The method according to claim 1, characterized in that, Step B: Accurately weigh puerarin, daidzein, and 2,3,5,4... ‘ Take appropriate amounts of tetrahydroxystilbene-2-O-β-D-glucoside, naringin, and icariin reference standards, place them in a volumetric flask, add 50% methanol to dissolve and dilute to the mark, and shake well.

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