Construction method of HPLC (High Performance Liquid Chromatography) fingerprint spectrum of Jianer and digestion promoting oral liquid and multi-index component content

Abstract

The invention belongs to the technical field of food and medicine detection, and provides a construction method of an HPLC (High Performance Liquid Chromatography) fingerprint spectrum of Jianer and digestion promoting oral liquid and a multi-index component content determination method. According to the invention, the finger-print and multi-index component content determination and detection method for the Jianer digestion-promoting oral liquid is established, and the method is short in detection time, good in separation degree, accurate, reliable, simple to operate, high in precision, good in specificity, stability and repeatability, and provides a research basis for quality control of the preparation.

Description

Technical Field

[0001] The present invention belongs to the technical field of food and medicine detection, and specifically relates to a method for constructing an HPLC fingerprint of Jian'er Xiaoshi Koufuye and a method for determining the contents of multiple index components. Background Art

[0002] Jian'er Xiaoshi Koufuye is prepared from 7 herbs including Astragalus membranaceus, Atractylodes macrocephala, Ophiopogon japonicus, Citrus reticulata Blanco, Scutellaria baicalensis, Crataegus pinnatifida and Raphanus sativus, and has the effects of strengthening the spleen and stomach, regulating qi and promoting digestion. It is used for symptoms such as poor appetite, reduced food intake, feverish palms and soles, spontaneous sweating, fatigue, irregular stools, anorexia, and dislike of food caused by improper diet and spleen and stomach injury in children.

[0003] Currently, high performance liquid chromatography (HPLC) is mainly used to detect hesperidin and baicalin in Jian'er Xiaoshi Koufuye. However, the existing method has a long testing time and poor resolution, and cannot meet the requirements of chromatographic conditions. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for constructing an HPLC fingerprint of Jian'er Xiaoshi Koufuye and a method for determining the contents of multiple index components.

[0005] To achieve the purpose of the present invention, in the first aspect, the present invention provides a method for constructing an HPLC fingerprint of Jian'er Xiaoshi Koufuye and a method for determining the contents of multiple index components, which uses hesperidin, baicalin, sinapine thiocyanate, baicalein, wogonoside, and wogonin as active ingredients, and performs HPLC content determination and HPLC fingerprint construction on the active ingredients.

[0006] The chromatographic conditions for HPLC are as follows: using an Agilent ZORBAX Eclipse XDB-C18 chromatographic column with octadecylsilane chemically bonded silica as the filler, the chromatographic column specification is 4.6 mm × 250 mm, 5 µm; using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B; performing gradient elution according to Table 1: Table 1 Gradient Elution Program

[0007] The column temperature is 32°C, the flow rate is 1 mL / min, and the detection wavelength is 242 nm.

[0008] Further, the method includes the following steps: 1) Preparation of the reference solution: Take appropriate amounts of reference substances of hesperidin, baicalin, sinapine thiocyanate, baicalein, wogonoside, and wogonin, and prepare solutions containing 0.05 mg, 0.3 mg, 0.01 mg, 0.015 mg, 0.05 mg, and 0.015 mg per 1 ml with 50% methanol respectively, and that's it; 2) Preparation of the test solution: Take 5 ml of Jian'er Xiaoshi Koufuye, place it in a 50-ml volumetric flask, add 15 ml of water, shake well, then add 25 ml of methanol, shake well, perform ultrasonic treatment, let it cool, dilute to the mark with methanol, shake well, centrifuge, take the supernatant, filter, and take the consecutive filtrate to obtain the test solution; 3) Perform HPLC detection on the reference solution and the test solution respectively: 4) According to the HPLC detection results, construct an HPLC fingerprint with hesperidin, baicalin, sinapine thiocyanate, baicalein, wogonoside, and wogonin as the active ingredients, and use the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (version 2004) with baicalin as the reference peak to compare, calculate the similarity between the reference solution and the test solution, and calculate the content of each active ingredient through the chromatograms of the reference solution and the test solution.

[0009] Preferably, the conditions for ultrasonic treatment in step 2) are: power 250 W, frequency 50 kHz, ultrasonic treatment for 5 minutes.

[0010] Preferably, the conditions for centrifugation in step 2) are: 3000 revolutions per minute, centrifugation for 10 minutes.

[0011] Furthermore, hesperidin elutes at 55 - 65 min, baicalin elutes at 70 - 80 min, sinapine thiocyanate elutes at 15 - 25 min, baicalein elutes at 95 - 105 min, wogonoside elutes at 82 - 92 min, and wogonin elutes at 110 - 120 min.

[0012] In the second aspect, the present invention provides the application of the above method in the detection of commercially available Jian'er Xiaoshi Koufuye products.

[0013] In the third aspect, the present invention provides the application of the above method in the quality control of the production process of Jian'er Xiaoshi Koufuye.

[0014] By means of the above technical solutions, the present invention has at least the following advantages and beneficial effects: The present invention has established a fingerprint and a multi-index component content determination method for Jian'er Xiaoshi Koufuye. This method has a short detection time, good resolution, accuracy, reliability, simple operation, high precision, good specificity, stability, and repeatability.

[0015] The high-performance liquid chromatography method provided by the present invention simultaneously determines hesperidin, baicalin, and wogonoside in Jian'er Xiaoshi Koufuye, conducts linear relationship investigation, precision, repeatability and stability tests, recovery tests, and sample content determination, providing a research basis for the quality control of this preparation. Description of the Drawings

[0016] Figure 1 It is the chromatogram of the detection method of the prior art of the present invention.

[0017] Figure 2 Chromatogram of Comparative Example 1 of the present invention.

[0018] Figure 3 Chromatogram of Comparative Example 2 of the present invention.

[0019] Figure 4 Chromatogram of Comparative Example 3 of the present invention.

[0020] Figure 5 Chromatogram of the preferred embodiment of the present invention.

[0021] Figure 6 Comparison of the fingerprints of the finished products of each batch and the control fingerprint of the preferred embodiment of the present invention.

[0022] Figure 7 Control fingerprint in the preferred embodiment of the present invention, wherein, peak 3: Sinapine thiocyanate, peak 6: Hesperidin, peak 8: Baicalin, peak 9: Wogonoside, peak 10: Baicalein, peak 11: Wogonin.

[0023] Figure 8 Linear relationship diagram of hesperidin concentration - peak area in the preferred embodiment of the present invention.

[0024] Figure 9 Linear relationship diagram of wogonoside concentration - peak area in the preferred embodiment of the present invention. Detailed implementation mode

[0025] The existing HPLC detection method for Jian'er Xiaoshi Koufuye is as follows: Preparation of reference solution: Weigh appropriate amounts of Calycosin-7-O-β-D-glucoside, Hesperidin, and Baicalin reference substances accurately, and dissolve them in methanol to prepare a mixed reference solution 1 with mass concentrations of 0.0058, 0.1380, and 0.9500 mg / mL respectively; weigh appropriate amounts of Calycosin-7-O-β-D-glucoside, Hesperidin, Baicalin, Sinapine thiocyanate, Baicalein, and Wogonin reference substances accurately, and dissolve them in methanol to prepare a mixed reference solution 2 with mass concentrations of 0.014, 0.055, 0.380, 0.076, 0.050, and 0.190 mg / mL respectively.

[0026] Preparation of test solution: Precisely measure 5 mL of Jian'er Xiaoshi Koufuye, place it in a 100 mL conical flask, add 25 mL of methanol, ultrasonicate (power 300 W, frequency 50 kHz) for 20 min, centrifuge for 10 min, take all the supernatant and evaporate it to about 2 mL in a water bath, dilute it to 10 mL with methanol, and filter it through a 0.45 µm microporous membrane to obtain the test solution.

[0027] Chromatographic conditions: An Agilent ZORBAX SB C18 chromatographic column (150 mm × 4.6 mm, 3.5 µm) filled with octadecylsilane chemically bonded silica was used; acetonitrile was used as mobile phase A, and 1% acetic acid solution was used as mobile phase B; gradient elution was performed (Table 2), the column temperature was 30 °C, the flow rate was 1 mL / min, the detection wavelength was 242 nm, and the injection volume was 10 μL.

[0028] Table 2 Gradient elution program

[0029] Through experiments, it was found that the existing detection method takes a long time and has poor resolution ( Figure 1 ), and it cannot meet the requirements of the chromatographic conditions, so the chromatographic conditions need to be optimized.

[0030] In view of this, the present invention provides an improved method for constructing the HPLC fingerprint of Jian'er Xiaoshi Koufuye and a method for determining the contents of multiple index components, specifically as follows: Preparation of reference substance solution: Appropriate amounts of hesperidin, baicalin, sinapine thiocyanate, baicalein, wogonoside, and wogonin reference substances were accurately weighed and dissolved in 50% methanol to prepare solutions containing 0.05 mg, 0.3 mg, 0.01 mg, 0.015 mg, 0.05 mg, and 0.015 mg per 1 mL respectively, that is, obtained.

[0031] Preparation of test solution: 5 mL of this product was accurately measured and placed in a 50 mL volumetric flask, 15 mL of water was added, shaken well, then 25 mL of methanol was added, shaken well, sonicated (power 250 W, frequency 50 kHz) for 5 minutes, cooled, diluted to the mark with methanol, shaken well, centrifuged (rotation speed 3000 revolutions per minute) for 10 minutes, the supernatant was taken, filtered, and the subsequent filtrate was taken, that is, obtained.

[0032] Chromatographic conditions: An Agilent ZORBAX Eclipse XDB-C18 chromatographic column (4.6 mm × 250 mm, 5 µm) filled with octadecylsilane chemically bonded silica was used; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B; gradient elution was performed (Table 1), the column temperature was 32 °C, the flow rate was 1 mL / min, and the detection wavelength was 242 nm.

[0033] Determination method: 10 μL of the reference substance solution and the test solution were accurately pipetted and injected into the liquid chromatograph for determination, and the chromatogram was recorded, that is, obtained.

[0034] This method has a short detection time and good resolution of each active ingredient.

[0035] The following examples are used to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art, and the raw materials used are all commercially available products.

[0036] Comparative Example 1 Reference solution: Appropriate amounts of calycosin-7-O-β-D-glucoside, hesperidin, and baicalin reference substances were accurately weighed and dissolved in methanol to prepare a mixed reference solution 1 with mass concentrations of 0.005 8, 0.138 0, and 0.950 0 mg / mL, respectively; appropriate amounts of calycosin-7-O-β-D-glucoside, hesperidin, baicalin, sinapine thiocyanate, baicalein, and wogonin reference substances were accurately weighed and dissolved in methanol to prepare a mixed reference solution 2 with mass concentrations of 0.014, 0.055, 0.380, 0.076, 0.050, and 0.190 mg / mL, respectively. An appropriate amount of potassium sorbate was taken and dissolved in methanol to prepare a reference solution (auxiliary material reference) with a mass concentration of 1.501 mg / mL for standby.

[0037] Test solution: 5 mL of the test sample was accurately measured and placed in a 100 mL conical flask, 25 mL of methanol was added, sonicated (power 300 W, frequency 50 kHz) for 20 min, centrifuged for 10 min, the whole supernatant was evaporated to about 2 mL in a water bath, made up to 10 mL with methanol, and filtered through a 0.45 µm microporous membrane to obtain the test solution.

[0038] Chromatographic conditions: An Agilent ZORBAX SB C18 chromatographic column (150 mm×4.6 mm, 3.5 µm) filled with octadecylsilane-bonded silica gel was used; the mobile phase was acetonitrile (A)-1% acetic acid solution (B), the flow rate was 1.0 mL / min; the detection wavelength was 242 nm; the column temperature was 30 °C; the injection volume was 10 μL. The gradient elution program is shown in Table 3.

[0039] Table 3 Gradient elution program

[0040] After comparison, the elution gradient was optimized based on the existing detection method, and it was found that the resolution of hesperidin was not good ( Figure 2 ), and further optimization was still needed.

[0041] Comparative Example 2 Reference substance solution: Weigh appropriate amounts of calycosin-7-O-β-D-glucoside, hesperidin, and baicalin reference substances accurately, dissolve them in methanol to prepare a mixed reference substance solution 1 with mass concentrations of 0.005 8, 0.138 0, and 0.950 0 mg / mL respectively; weigh appropriate amounts of calycosin-7-O-β-D-glucoside, hesperidin, baicalin, sinapine thiocyanate, baicalein, and wogonin reference substances accurately, dissolve them in methanol to prepare a mixed reference substance solution 2 with mass concentrations of 0.014, 0.055, 0.380, 0.076, 0.050, and 0.190 mg / mL respectively; Weigh appropriate amount of potassium sorbate, dissolve it in methanol to prepare a reference substance solution (excipient reference substance) with a mass concentration of 1.501 mg / mL for standby.

[0042] Test solution: Precisely measure 5 mL of the test sample, transfer it to a 100 mL conical flask, add 25 mL of methanol, ultrasonicate (power 300 W, frequency 50 kHz) for 20 min, centrifuge for 10 min, take all the supernatant and evaporate it to about 2 mL in a water bath, dilute it to 10 mL with methanol, filter through a 0.45 µm microporous membrane to obtain the solution.

[0043] Chromatographic conditions: Use an Agilent ZORBAX SB C18 chromatographic column (150 mm×4.6 mm, 3.5 µm) filled with octadecylsilane chemically bonded silica gel; the mobile phase is acetonitrile (A)-1% acetic acid solution (B), the flow rate is 1.0 mL / min; the detection wavelength is 242 nm; the column temperature is 30 °C; the injection volume is 10 μL. The gradient elution program is shown in Table 4.

[0044] Table 4 Gradient elution program

[0045] After comparison, on the basis of the scheme of Comparative Example 1, the elution gradient was optimized. It was found that the resolution of hesperidin was not good ( Figure 3 ), and at the same time, the peak of calycosin glucoside was too small and was no longer treated as a common peak, and further optimization was still needed.

[0046] Comparative Example 3 Reference substance solution: Weigh appropriate amounts of hesperidin and wogonoside reference substances accurately, dissolve them in methanol to prepare a mixed reference substance solution 1 with mass concentrations of 0.05 and 0.05 mg / mL respectively; weigh appropriate amounts of hesperidin, baicalin, sinapine thiocyanate, baicalein, wogonoside, and wogonin reference substances accurately, dissolve them in methanol to prepare a mixed reference substance solution 2 with mass concentrations of 0.05, 0.3, 0.01, 0.015, 0.05, and 0.015 mg / mL respectively; Take an appropriate amount of potassium sorbate, dissolve it in methanol to prepare a reference solution (auxiliary reference) with a mass concentration of 1.501 mg / mL, and set aside.

[0047] Preparation of the test solution: Accurately measure 5 mL of this product, place it in a 50 mL volumetric flask, add 15 mL of water, shake well, then add 25 mL of methanol, shake well, ultrasonically treat (power 250 W, frequency 50 kHz) for 5 minutes, let it cool, dilute to the mark with methanol, shake well, centrifuge (rotation speed 3000 revolutions per minute) for 10 minutes, take the supernatant, filter, and take the continued filtrate to obtain the test solution.

[0048] Chromatographic conditions: Use an octadecylsilane chemically bonded silica gel as the filler, adopt an Agilent ZORBAX SB C18 chromatographic column (150 mm×4.6 mm, 3.5 µm); the mobile phase is acetonitrile (A) - 0.1% phosphoric acid solution (B), the flow rate is 1.0 mL / min; the detection wavelength is 242 nm; the column temperature is 32 °C; the injection volume is 10 μL. The gradient elution program is shown in Table 5.

[0049] Table 5 Gradient elution program

[0050] After comparison, based on the scheme of Comparative Example 2, the elution gradient was optimized. As a result, it was found that the resolution of hesperidin was improved, but the separation of wogonin was not good later ( Figure 4 ), and further optimization is still required.

[0051] Example 1 Reference solution: Take appropriate amounts of hesperidin and wogonoside reference substances, accurately weigh them, dissolve them in methanol to prepare a mixed reference solution 1 with mass concentrations of 0.05 and 0.05 mg / mL respectively; take appropriate amounts of hesperidin, baicalin, sinapine thiocyanate, baicalein, wogonoside, and wogonin reference substances, accurately weigh them, dissolve them in methanol to prepare a mixed reference solution 2 with mass concentrations of 0.05, 0.3, 0.01, 0.015, 0.05, and 0.015 mg / mL respectively; take an appropriate amount of potassium sorbate, dissolve it in methanol to prepare a reference solution with a mass concentration of 1.501 mg / mL, and set aside.

[0052] Preparation of the test solution: Accurately measure 5 mL of this product, place it in a 50 mL volumetric flask, add 15 mL of water, shake well, then add 25 mL of methanol, shake well, ultrasonically treat (power 250 W, frequency 50 kHz) for 5 minutes, let it cool, dilute to the mark with methanol, shake well, centrifuge (rotation speed 3000 revolutions per minute) for 10 minutes, take the supernatant, filter, and take the continued filtrate to obtain the test solution.

[0053] Chromatographic conditions: An octadecylsilyl silica gel bonded silica was used as the filler, and an Agilent ZORBAX SB C18 chromatographic column (150 mm×4.6 mm, 3.5 µm) was adopted; the mobile phase was acetonitrile (A) - 0.1% phosphoric acid solution (B), the flow rate was 1.0 mL / min; the detection wavelength was 242 nm; the column temperature was 32 °C; the injection volume was 10 μL. The gradient elution program is shown in Table 1.

[0054] After comparison, on the basis of the scheme of Comparative Example 3, the elution gradient was optimized, and the resolution of each component met the requirements ( Figure 5 ).

[0055] Example 2 The above reference substance, test sample preparation method and chromatographic conditions were used for the methodological verification of the fingerprint and multi-index component detection methods, as follows: 1. Methodological verification of fingerprint inspection 1.1 Precision Take 6 portions of this product (batch number of the test sample of Jian'er Xiaoshi Koufuye: 215260012), and examine the consistency of the retention time and chromatographic peak area of each main chromatographic peak. The determination results are shown in Table 6.

[0056] Table 6 Results of fingerprint precision investigation

[0057] The results showed that: the RSD of the retention time was between 0.00% and 0.08%, and the RSD of the chromatographic peak area was between 0.08% and 1.98%, indicating good precision.

[0058] 1.2 Repeatability investigation Take 6 portions of this product (batch number of the test sample of Jian'er Xiaoshi Koufuye: 215260012), and examine the consistency of the retention time and chromatographic peak area of each main chromatographic peak. The results are shown in Table 7.

[0059] Table 7 Results of fingerprint repeatability investigation

[0060] The results showed that: the RSD of the retention time was between 0.00% and 0.08%, and the RSD of the chromatographic peak area was between 0.08% and 1.98%, indicating good repeatability.

[0061] 1.3 Solution stability investigation Precisely pipette the test sample solution (batch number of the test sample of Jian'er Xiaoshi Koufuye: 215260012). At room temperature, inject 10 μl at 0, 2, 4, 6, 8, 10, 12, 24, 48, and 72 hours after preparation respectively, record the peak area of the sample, investigate the stability of the sample solution, calculate the relative standard deviation RSD (%), and the results are shown in Table 8.

[0062] Table 8 Results of the investigation on the stability of the fingerprint spectrum solution

[0063] The results show that the RSD of the retention time is between 0.02% and 0.10%, and the RSD of the peak area of the chromatographic peak is between 0.84% and 2.94%, indicating that the solution has good stability.

[0064] 1.5 Determination of the reference fingerprint spectrum and reference peaks In the reference spectrum ( Figure 7 ), a total of 11 common peaks appear. According to the selection requirements of the fingerprint reference peaks, the chromatographic peak of baicalin at peak No. 8 is stable, with good resolution and the largest peak area. Therefore, peak No. 8 is determined as the reference peak.

[0065] Among them, hesperidin elutes at 60 min, baicalin elutes at 72 min, sinapine thiocyanate elutes at 20 min, baicalein elutes at 99 min, wogonoside elutes at 87 min, and wogonin elutes at 114 min.

[0066] 2. Validation of the methodology for the determination of multiple indicator components 2.1 Specificity Table 9 Results of the specificity test

[0067] The results in Table 9 show that the retention times of the chromatographic peaks of hesperidin and baicalin in the test sample are consistent with those of the reference substances of hesperidin, baicalin, and wogonoside respectively. No corresponding peaks appear at the positions of the chromatographic peaks of hesperidin and baicalin reference substances in the chromatogram of the negative sample, indicating that the negative sample has no interference, and the method has good specificity.

[0068] 2.2 Linearity Table 10 Results of the linearity test of hesperidin Table 10 and Figure 8 The results show that in the range of 26.2926 μg / mL - 210.3408 μg / mL for hesperidin, the linear relationship between the concentration and the peak area is good. The linear equation of hesperidin is y = 6148.9x + 27387, R = 0.9992.

[0069] Table 11 Results of the linearity test for wogonoside Table 11 and Figure 9 The results showed that within the range of 25.4800 μg / mL - 203.8400 μg / mL, the linear relationship between the concentration and peak area of wogonoside was good. The linear equation of wogonoside was y = 11520x + 39900, with R = 0.9994.

[0070] 2.3 Accuracy 2.3.1 Recovery rate of hesperidin Table 12 Test for the recovery rate of sample addition

[0071] The results in Table 12 showed that the recovery rate of hesperidin was 97.57%, within the range of 85% - 110%; the RSD was 0.82%, less than 2.0%, meeting the verification requirements. This indicated that the accuracy of this content determination method was good.

[0072] 2.3.2 Recovery rate of wogonoside Table 13 Test for the recovery rate of sample addition

[0073] The results in Table 13 showed that the recovery rate of wogonoside was 96.36%, within the range of 85% - 110%; the RSD was 1.52%, less than 2.0%, meeting the verification requirements. This indicated that the accuracy of this content determination method was good.

[0074] 2.4 Precision 2.4.1 Investigation of intermediate precision Table 14 Results of the intermediate precision test for the content of hesperidin

[0075] The results in Table 14 showed that six test solution samples were prepared by two experimenters at different times, and the RSD values of the test results were 0.10% and 0.21% respectively; the RSD% of the intermediate precision was 1.72%, less than 8%, meeting the requirements. This indicated that the intermediate precision of the method for the content of hesperidin was good.

[0076] Table 15 Results of the intermediate precision test for wogonoside

[0077] The results showed that six test solution samples were prepared by two experimenters at different times, and the RSD values of the test results were 0.12% and 0.13% respectively; the RSD% of the intermediate precision was 1.02%, less than 8%, meeting the requirements. This indicated that the intermediate precision of the method for the content of wogonoside was good.

[0078] The results in Table 15 show that: Six test solutions were prepared by two experimenters at different times and with different instruments respectively. The RSD values of the hesperidin detection results were 0.03% and 0.08% respectively; the RSD values were both less than 2%, meeting the requirements, indicating good intermediate precision.

[0079] 2.5 Repeatability investigation Table 16 Repeatability investigation of hesperidin content

[0080] The experimental results in Table 16 show that: The RSD% of the repeatability of hesperidin content detection is 0.10, meeting the requirements.

[0081] Table 17 Repeatability investigation of wogonoside content

[0082] The experimental results in Table 17 show that: The RSD% of the repeatability of wogonoside content detection is 0.12, meeting the requirements.

[0083] 2.6 Solution stability investigation Table 18 Results of stability tests for hesperidin and wogonoside in the test solution

[0084] The results in Table 18 show that: Under the specified conditions, the RSD value of the hesperidin peak area obtained from the test solution within 72 hours is 1.37%, meeting the requirements, so the test solution of hesperidin is relatively stable; the RSD value of the wogonoside peak area obtained from the test solution within 72 hours is 1.51%, meeting the requirements, so the test solution of wogonoside is relatively stable.

[0085] 3. Sample inspection The finished products were inspected using the above-verified inspection method. The test results are shown in Table 19, and the fingerprint chromatogram is shown in Figure 6 : Table 19 Comparison of finished product similarity

[0086] The determination results of multiple index components are shown in Table 20: Table 20 Determination results of multiple index components

[0087] Although the present invention has been described in detail above with general descriptions and specific embodiments, on the basis of the present invention, some modifications or improvements can be made thereto, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection of the present invention.

Claims

1. A method for constructing the HPLC fingerprint of Jianer Xiaoshi oral liquid and a method for determining the content of multi-index components, characterized in that: Hesperidin, baicalin, sinapinic acid thiocyanate, baicalein, wogonin, and baicalein are used as active ingredients, and HPLC content determination and HPLC fingerprint construction are performed on the active ingredients; The chromatographic conditions of HPLC were as follows: using octadecylsilane bonded silica gel as filler and Agilent ZORBAX EclipseXDB-C18 column, column size 4.6 mm × 250 mm, 5 µm; using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B; gradient elution was performed according to Table 1: Table 1 Gradient elution program The column temperature was 32°C, the flow rate was 1 mL / min, and the detection wavelength was 242 nm.

2. The method according to claim 1, characterized in that The following steps are involved: 1) Preparation of reference solution: Take appropriate amount of hesperidin, baicalin, sinapinic acid thiocyanate, baicalin, wogonin, and baicalin reference substances, add 50% methanol to prepare reference solution containing 0.05 mg, 0.3 mg, 0.01 mg, 0.015 mg, 0.05 mg, and 0.015 mg per ml respectively; 2) Preparation of test solution: Take 5 ml of Jianer Xiaoshi oral solution, place it in a 50 ml volumetric flask, add 15 ml of water, shake well, then add 25 ml of methanol, shake well, ultrasonicate, cool, dilute to scale with methanol, shake well, centrifuge, take the supernatant, filter, and take the filtrate to obtain; 3) Perform HPLC test on reference solution and test solution respectively: 4) According to the HPLC test results, HPLC fingerprints with hesperidin, baicalin, sinapinic acid thiocyanate, baicalin, wogonin, and baicalein as active ingredients were constructed. Baicalin was used as the reference peak and the Chinese medicine chromatographic fingerprint similarity evaluation system was used to compare and calculate the similarity between the reference and the test samples. The content of each active ingredient was calculated through the chromatograms of the reference and test samples.

3. The method according to claim 2, characterized in that Step 2) The conditions for ultrasonic treatment are: power 250 W, frequency 50 kHz, and ultrasonication for 5 minutes.

4. The method according to claim 2, characterized in that: Step 2) The centrifugation conditions are: 3000 rpm, 10 minutes.

5. The method according to any one of claims 1 to 4, characterized in that: Hesperidin peaks at 55-65 min, baicalin peaks at 70-80 min, sinapinic thiocyanate peaks at 15-25 min, baicalein peaks at 95-105 min, wogonin peaks at 82-92 min, and wogonin peaks at 110-120 min.

6. Application of the method according to any one of claims 1 to 5 in the detection of commercially available Jianer Xiaoshi oral liquid products.

7. Application of the method according to any one of claims 1 to 5 in the quality control of the production process of Jianer Xiaoshi oral liquid.

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