A method for determining quality control indicators of Erding oral liquid

Abstract

The invention belongs to the technical field of analysis and detection of compound Chinese medicine preparations, and discloses a method for determining the quality control index of dibutyl oral liquid, comprising the following steps: the dibutyl oral liquid is a reddish brown to brownish brown liquid, slightly bitter, and the relative density is not less than 1.01, and the pH value is 4.0-6.0, and thin layer chromatography is used to qualitatively identify Viola yedoensis and dandelion, Lobelia chinensis, and Radix Isatidis in the dibutyl oral liquid; a reference substance and different batches of test sample solutions are configured, and the content of the main active ingredient in the oral liquid is determined by high performance liquid chromatography; the main active ingredient control index of the dibutyl oral liquid is determined according to the results of high performance liquid chromatography: each 1mL of dibutyl oral liquid contains not less than 0.5mg of fraxin, not less than 50μg of caffeic acid, and not less than 10μg of (R,S)-gaoyichun. The present invention adopts a method combining qualitative and quantitative analysis, which lays a foundation for the establishment of the quality standard of dibutyl oral liquid.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection of compound Chinese medicine preparations, and relates to a method for determining quality control indicators of dibutyl oral liquid. Background Art

[0002] Erding oral liquid is composed of medicinal materials such as Viola yedoensis, dandelion, Lobelia and Radix Isatidis, and is mainly used to treat chickens with swollen head and face, conjunctivitis and other symptoms. Swollen head and face, conjunctivitis and other symptoms of chickens are more common in the clinical manifestations of chicken diseases, and are mostly caused by bacteria or viruses, such as avian pneumovirus, avian influenza virus, J subtype avian leukosis, infectious laryngotracheitis, Escherichia coli disease, infectious rhinitis and chronic respiratory diseases. In the prescription, Viola yedoensis and dandelion (Yellow Flower Yedoensis) are bitter and cold, clearing away heat and detoxifying, and eliminating carbuncle as the main medicine; Radix Isatidis detoxifies and relieves sore throat, and eliminates swelling as the minister medicine; Lobelia is sweet and mild, penetrating and beneficial as the assistant, which can make the heat go out with the body fluids, and also has the effect of eliminating swelling and detoxification. Modern pharmacological research shows that Viola yedoensis has antibacterial, anti-inflammatory, antiviral, and immune-regulating effects; dandelion has antibacterial, antiviral, anti-inflammatory, antioxidant, and immune-regulating effects; Lobelia has antibacterial, analgesic, anti-inflammatory, and antioxidant effects; Isatis root has antibacterial, anti-inflammatory, antiviral, anti-pathogenic microorganisms, and improves the body's immunity effects; the various medicinal materials in the prescription complement each other through compatibility, which can not only directly kill pathogens such as viruses or bacteria, but also support the body's positive energy and regulate the body's immune function. By strengthening the body's positive energy and eliminating evil, and taking into account both the symptoms and the root causes, it can treat chicken swollen head and face, conjunctivitis, etc. caused by various reasons.

[0003] In recent years, chromatographic fingerprints have become internationally recognized as the most effective means for identifying and controlling the quality of Chinese medicinal materials. It is an organic fusion of the outstanding achievements of modern analytical science and the overall comprehensive view of traditional Chinese medicine. It is the best method that can be achieved by modern scientific and technological means to express the characteristics of complex Chinese medicine systems that is recognized internationally so far. The invention patent with the authorization announcement number CN 109856262 B discloses a method that can be used for qualitative and quantitative analysis of the main components of dibutyl preparations at the same time. Only one chromatographic condition can be used to simultaneously determine the characteristic qualitative fingerprint of the preparation and the content of the five main active ingredients, but it is suitable for the detection of granules, capsules or tablets. At present, there is no quality control standard for dibutyl oral liquid preparations. Summary of the invention

[0004] The purpose of the present invention is to provide a method for determining the quality control index of Erding oral liquid, and finally determine the quality control index of the main active ingredient in Erding oral liquid through qualitative identification by property characteristics, inspection indexes, thin layer chromatography and content determination by high performance liquid chromatography.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for determining the quality control index of Erding oral liquid, comprising the following steps:

[0007] 1) Erding oral liquid is a reddish brown to brownish brown liquid with a slightly bitter taste. Its relative density is not less than 1.01 and its pH value is 4.0-6.0. Thin layer chromatography is used to qualitatively identify Viola yedoensis and dandelion, Lobelia chinensis, and Radix Isatidis in Erding oral liquid;

[0008] 2) Prepare reference substances and different batches of test solution, and determine the content of the main active ingredient in the oral solution by high performance liquid chromatography;

[0009] 3) Based on the results of HPLC, the control indicators of the main active ingredients of dibutyl oral solution are determined as follows: each 1mL of dibutyl oral solution contains no less than 0.5mg of quinidine, no less than 50μg of caffeic acid, and no less than 10μg of (R,S)-gaitrin.

[0010] In one technical solution, the above step 1) comprises the following specific steps:

[0011] a. Identification of Viola yedoensis and Taraxacum officinale: Take 1 mL of Erding oral solution, add 30 mL of methanol, ultrasonically treat for 30 min, filter, evaporate the filtrate to dryness, add 20 mL of hot water to dissolve the residue, cool, shake and extract twice with ethyl acetate, 20 mL each time, combine the ethyl acetate solution, evaporate to dryness, add 1 mL of methanol to dissolve the residue, and use it as the test solution; separately take the quinoline reference substance and the caffeic acid reference substance, add methanol to prepare a reference substance solution containing 0.5 mg per 1 mL; then take the negative samples of Viola yedoensis and Taraxacum officinale to prepare a negative control solution according to the preparation method of the test sample; take 5 μL of each of the above solutions, spot them on the same silica gel G thin layer plate, use a mixed solution of toluene-ethyl acetate-formic acid in a ratio of 5:3:1 as the developing solvent, develop, take out, dry, and fumigate in iodine vapor until the spots are clearly colored; spots of the same color appear in the chromatogram of the test sample at the corresponding position of the chromatogram of the reference substance, and there is no interference with the negative control;

[0012] b. Identification of Lobelia: Take 8 mL of Erding oral solution, adjust pH to 11 with ammonia test solution, shake and extract twice with chloroform, 10 mL each time, combine the chloroform solution, evaporate to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the test solution; then take 2 g of Lobelia control medicinal material, add 50 mL of water, boil for 30 minutes, centrifuge, concentrate the supernatant to 10 mL, adjust pH to 11 with ammonia test solution, shake and extract twice with chloroform, 10 mL each time, combine the chloroform solution , evaporate to dryness, add 1mL of methanol to the residue to dissolve it as the reference solution; take another negative sample of Lobelia chinensis and prepare a negative control solution according to the preparation method of the test sample; take 5μL of each of the above solutions and spot them on the same silica gel G thin layer plate, use the lower layer solution with a ratio of chloroform to formic acid of 10:1 as the developing agent, develop, take out, dry, and examine under a 365nm ultraviolet lamp; in the chromatogram of the test sample, at the corresponding position of the chromatogram of the reference medicinal material, a spot of the same color appears, and there is no interference with the negative control;

[0013] Step c, identification of Radix Isatidis: Take 10 mL of Erding oral liquid, shake and extract with chloroform 3 times, 10 mL each time, combine the chloroform solution, evaporate to dryness, add 0.5 mL of methanol to the residue to dissolve it as the test solution; then take 1 g of Radix Isatidis reference medicinal material, add 20 mL of water, ultrasonicate for 30 min, centrifuge, shake the supernatant with chloroform for 3 times, 20 mL each time, combine the chloroform solution, evaporate to dryness, add 0.5 mL of methanol to the residue to dissolve it as the reference solution; take (R, S)-gaoyichun reference, add methanol to make a reference solution containing 0.5 mg per 1 mL; take another Radix Isatidis negative sample and make a negative control solution according to the preparation method of the test sample; take 10 μL of each of the above solutions and spot them on the same silica gel GF 254 On the thin layer plate, a mixed solution of petroleum ether and ethyl acetate in a ratio of 1:1 was used as the developing agent. The plate was developed, taken out, dried, and examined under a 254nm ultraviolet lamp. Spots of the same color appeared in the chromatogram of the test sample at the corresponding positions of the chromatogram of the control medicinal material and the chromatogram of the reference sample, and there was no interference with the negative control.

[0014] In one technical solution, the above step 2) includes the following specific steps:

[0015] a. Preparation of reference solution: Take the reference substance of aesculetin, the reference substance of caffeic acid and the reference substance of (R,S)-gaotizone, and add methanol to prepare reference solution containing 0.2-0.3 mg of aesculetin, 15-20 μg of caffeic acid and 2-5 μg of (R,S)-gaotizone per 1 mL respectively;

[0016] b. Preparation of test solution: Measure 5 mL of dibutyl oral solution, place in a 150 mL conical flask, add 20 mL of methanol, weigh, ultrasonicate for 30 min, cool, make up the lost weight with methanol, shake well, filter, and take the filtrate;

[0017] c. Determination: 10 μL of reference solution and test solution are taken, and injected into liquid chromatograph. Chromatographic conditions: chromatographic column with octadecylsilane bonded silica as filler, column temperature 30°C, detection wavelength 240nm; mobile phase with acetonitrile as phase A and 0.1% phosphoric acid solution as phase B, elution program: 0-20min, 2%A; 20-40min, 2%-20%A; theoretical plate number is calculated according to each peak of esculetin, caffeic acid and (R,S)-goitrin and is not less than 3000, determination.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The method of the invention uses property characteristics, inspection indexes, thin-layer chromatography to perform qualitative identification and high-performance liquid chromatography to perform content determination. The method first uses thin-layer chromatography to perform qualitative analysis, which is rapid and simple, and then uses high-performance liquid chromatography to perform quantitative analysis, which is simple and easy to operate. The high-performance liquid chromatography used has passed methodology verification and has high precision and good reproducibility. The combination of qualitative and quantitative analysis lays a foundation for the establishment of the quality standard of Erding oral liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The figure is a thin layer chromatogram of aesculetin and caffeic acid. In the figure, 1 to 3 are three batches of test samples, 4 is aesculetin reference substance, 5 is a caffeic acid reference substance, and 6 is a negative sample lacking Viola yedoensis and Taraxacum officinale.

[0021] Figure 2 This is a thin layer chromatogram of Lobelia chinensis. In the figure, 1 to 3 are three batches of test samples, 4 is a Lobelia chinensis control medicinal material, and 5 is a negative sample lacking Lobelia chinensis.

[0022] Figure 3 This is a thin layer chromatogram of Radix Isatidis. In the figure, 1 is the (R, S)-gaoichun reference substance, 2 to 4 are three batches of test substances, 5 is the Radix Isatidis reference medicinal material, and 6 is a negative sample lacking Radix Isatidis.

[0023] Figure 4 The HPLC chromatogram of the reference solution in the specificity investigation of Example 1 is shown in FIG.

[0024] Figure 5 The HPLC chromatogram of the test solution in the specificity investigation of Example 1 is shown in FIG.

[0025] Figure 6 The HPLC chromatogram of the negative control solution in the specificity investigation of Example 1 is shown in FIG. DETAILED DESCRIPTION

[0026] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified.

[0027] Embodiment 1

[0028] 1. Materials and Reagents

[0029] Instruments: DZKW-4 water bath (Beijing Zhongxing Weiye Century Instrument Co., Ltd.); LC-1260 high performance liquid chromatograph (Agilent Technologies, Inc., USA); KQ5200B ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); 1 / 100,000 analytical balance (MS205DU, Mettler-Toledo); TD5Z low-speed centrifuge (Hunan Kaida Scientific Instrument Co., Ltd.).

[0030] Reagents: Fraxin (batch number: 110741-201708, purity: 99.9%), caffeic acid (110885-201703, purity: 99.7%), (R, S)-gaoyichun (111753-202007, purity: 100%) were purchased from the China Food and Drug Administration; dibutyl oral solution was three batches of pilot samples (batch numbers 210601, 210602, 210603, 210604, 210605) from Zhengzhou Bairui Animal Pharmaceutical Co., Ltd. 0603), the preparation process is as follows: take the crude powder of each raw material drug of Viola yedoensis, Taraxacum officinale, Lobelia chinensis and Isatis indigotica in equal proportions, add water for ultrasonic decoction, filter after decoction, separate by membrane to obtain clear liquid, further concentrate the clear liquid to 1g / mL concentrate, add suspending agent and preservative, adjust the pH of the concentrate to 4.0-6.0 with HCl or NaOH solution, make the volume with water to 1-2 times of the weight of the raw material drug, shake well, filter, fill and sterilize to obtain.

[0031] 2. Determination of quality control indicators

[0032] (1) Properties: This product is a reddish brown to brown liquid, slightly bitter;

[0033] (2) Inspection: relative density is 1.10, pH value is 4.82;

[0034] (3) Qualitative identification by thin layer chromatography:

[0035] Identification of Viola yedoensis and Taraxacum officinale: Take 1 mL of Erding oral solution, add 30 mL of methanol, ultrasonically treat for 30 min, filter, evaporate the filtrate to dryness, add 20 mL of hot water to dissolve the residue, cool, shake and extract twice with ethyl acetate, 20 mL each time, combine the ethyl acetate solution, evaporate to dryness, add 1 mL of methanol to dissolve the residue as the test solution; separately take the quinoline reference substance and the caffeic acid reference substance, add methanol to prepare a reference substance solution containing 0.5 mg per 1 mL; then take the negative samples of Viola yedoensis and Taraxacum officinale to prepare a negative control solution according to the preparation method of the test sample; according to the thin layer chromatography method (General Rule 0502), take 5 μL of each of the above solutions, spot them on the same silica gel G thin layer plate, use a mixed solution of toluene-ethyl acetate-formic acid (5:3:1) as the developing solvent, develop, take out, dry, and fumigate in iodine vapor until the spots are clearly colored; in the chromatogram of the test sample, spots of the same color appear at the corresponding position of the chromatogram of the reference substance, and there is no interference with the negative control. The results are as follows Figure 1 As shown;

[0036] Identification of Lobelia: Take 8 mL of Erding oral solution, adjust pH to 11 with ammonia test solution, shake and extract twice with chloroform, 10 mL each time, combine the chloroform solution, evaporate to dryness, add 1 mL of methanol to the residue to dissolve, and use it as the test solution; then take 2 g of Lobelia control medicinal material, add 50 mL of water, decoct for 30 minutes, centrifuge, concentrate the supernatant to 10 mL, adjust pH to 11 with ammonia test solution, shake and extract twice with chloroform, 10 mL each time, combine the chloroform solution, evaporate to dryness, and add 1 mL of methanol to the residue Dissolve it as the reference solution; take another negative sample of Lobelia chinensis and prepare a negative control solution according to the preparation method of the test sample; according to the thin layer chromatography method (Appendix 0502), take 5μL of each of the above solutions and spot them on the same silica gel G thin layer plate, use chloroform-formic acid (10:1) as the lower layer solution as the developing agent, develop, take out, dry, and examine under ultraviolet light (365nm); in the chromatogram of the test sample, at the corresponding position of the chromatogram of the control medicinal material, a spot of the same color appears, and there is no interference with the negative control. The results are as follows Figure 2 shown.

[0037] Identification of Radix Isatidis: Take 10mL of Erding oral solution, shake and extract with chloroform for 3 times, 10mL each time, combine the chloroform solution, evaporate to dryness, add 0.5mL of methanol to the residue to dissolve it, and use it as the test solution; then take 1g of Radix Isatidis reference medicinal material, add 20mL of water, ultrasonicate for 30min, centrifuge, shake and extract the supernatant with chloroform for 3 times, 20mL each time, combine the chloroform solution, evaporate to dryness, add 0.5mL of methanol to the residue to dissolve it, and use it as the control medicinal material solution; take (R,S)-gaoyichun reference substance, add methanol to make a solution containing 0.5mg per 1mL, and use it as the reference substance solution; take another Radix Isatidis negative sample and make a negative control solution according to the preparation method of the test sample; according to the thin layer chromatography method (Appendix 0502), take 10μL of each of the above solutions and spot them on the same silica gel GF 254 On the thin layer plate, a mixed solution of petroleum ether (60-90°C)-ethyl acetate (1:1) is used as the developing agent. Develop, take out, dry, and examine under ultraviolet light (254nm); spots of the same color appear in the chromatogram of the test sample at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference substance. There is no interference with the negative control. The results are as follows Figure 3 shown.

[0038] (4) Content determination:

[0039] Chromatographic conditions: Chromatographic column: ZORBAX SB-C 18 (4.6mm×250mm, 5μm); column temperature 30℃, detection wavelength 240nm; mobile phase with acetonitrile as phase A, 0.1% phosphoric acid solution as phase B, elution program: 0~20min, 2%A; 20~40min, 2%~20%A; theoretical plate number is calculated according to the peaks of esculetin, caffeic acid and (R,S)-goitrin and is not less than 3000;

[0040] Preparation of reference solution: Take appropriate amount of aesculetin reference substance, caffeic acid reference substance and (R,S)-gaoizolin reference substance, accurately weigh them, and add methanol to prepare reference solution containing 250 μg aesculetin, 20 μg caffeic acid and 5 μg (R,S)-gaoizolin per 1 mL respectively;

[0041] Preparation of test solution: accurately measure 5 mL of dibutyl oral solution, place in a 150 mL conical flask, accurately add 20 mL of methanol, weigh, ultrasonically treat (power 250 W, frequency 50 kHz) for 30 min, cool, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain;

[0042] Determination: Accurately pipette 10 μL of reference solution and test solution, inject into liquid chromatograph, and determine. The determination results of three batches of dibutyl oral solution are shown in Table 1.

[0043] Table 1 Content determination results of three batches of dibutyl oral liquid samples

[0044]

[0045]

[0046] As determined in Table 1, the control indicators of the main active ingredients in the dibutyl phthalate oral solution are: each 1mL contains no less than 0.5mg of quinidine (C9H6O4), no less than 50μg of caffeic acid, and no less than 10μg of (R,S)-gaitrin.

[0047] 3. Exclusive inspection

[0048] Accurately pipette 10 μL of each reference solution, test solution and negative control solution, inject and measure, the liquid chromatogram results are as follows Figures 4 to 6 The results showed that the retention time of the chromatographic peaks of the test solution and the reference solution was consistent, and no chromatographic peaks appeared in the negative control solution at the corresponding retention time of the reference solution, indicating that under this chromatographic condition, aesculetin, caffeic acid and (R, S)-gaitrin were well absorbed, and other components in the drug solution did not interfere with the determination of their content.

[0049] 4. Linear relationship investigation

[0050] Prepare a series of solutions of 53.09, 106.18, 159.27, 212.36, 265.45, 318.54, 371.63 μg / mL of esculetin reference substance, then prepare a series of solutions of 3.96, 7.92, 11.88, 15.84, 19.80, 23.76, 27.72 μg / mL of caffeic acid reference substance, and then prepare a series of solutions of 0.62, 1.24, 1.85, 2.47, 3.09, 3.71, 4.33 μg / mL of (R, S)-gaoyichun reference substance. Accurately pipette 10 μL of the above-mentioned reference substance series solutions of different concentrations, inject and measure, and record the peak area. Linear regression was performed on the peak area (Y) of each reference substance and its injection mass concentration (X), and regression equations were obtained respectively. The results are shown in Table 2. The results showed that the linear relationship was good in the range of mass concentration of esculetin 53.09-371.63 μg / mL, mass concentration of caffeic acid 3.96-27.72 μg / mL, and mass concentration of (R,S)-goitrin 0.62-4.33 μg / mL.

[0051] Table 2 Linear relationship table of reference substances

[0052]

[0053] 5. Precision test

[0054] Accurately pipette 10 μL of each of the reference solutions of aesculetin, caffeic acid and (R,S)-gaitrin, repeat the injection 6 times, record the peak area, and calculate the RSD values ​​of each reference solution to be 0.73%, 1.37% and 0.92%, respectively. The results show that the instrument has good precision.

[0055] 6. Stability test

[0056] 10 μL of the same test solution was accurately pipetted and injected at 0, 2, 4, 6, 8, 10 and 12 h respectively. The peak areas were recorded and the RSD values ​​of esculetin, caffeic acid and (R, S)-goitrin were calculated to be 0.68%, 1.54% and 1.92%, respectively. The results showed that the test solution had good stability within 12 h.

[0057] 7. Repeatability test

[0058] Take the same batch of drug solution to prepare 6 test solutions, accurately pipette 10 μL, inject and measure respectively, record the peak area, the results are shown in Table 3, indicating that the method has good repeatability.

[0059] Table 3 Repeatability test results

[0060]

[0061] 8. Sample recovery test

[0062] Accurately measure 6 samples of 2.5 mL of known content, accurately add 9 mL of esculetin reference solution (265.45 μg / mL), 10.5 mL of caffeic acid reference solution (19.80 μg / mL), and 3 mL of (R, S)-goitrin reference solution (12.36 μg / mL) to each sample, weigh the weight, ultrasonically treat (power 250 W, frequency 50 kHz) for 30 min, cool, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the sample recovery. Accurately draw 10 μL, sample and measure respectively, calculate the sample recovery rate, and the results are shown in Table 4. This shows that the method has good accuracy.

[0063] Table 4 Sample recovery test results (n = 6)

[0064]

[0065]

[0066] The present invention first uses thin layer chromatography to perform qualitative analysis on Erding oral liquid, and performs trace and rapid prediction. Thin layer chromatography identification test is performed on each medicinal material in the preparation. The results show that spots of the same color appear at corresponding positions in the chromatogram of the test sample and the chromatogram of the reference sample and the reference medicinal material, and there is no interference with the negative control. Therefore, the thin layer chromatography identification methods of the four medicinal materials are combined for the qualitative analysis of Erding oral liquid.

[0067] The invention adopts high performance liquid chromatography to carry out quantitative analysis, and simultaneously determines aesculetin, caffeic acid and (R, S)-coitrin in Erding oral liquid. Under the same chromatographic conditions, each peak has an appropriate size, does not interfere with each other, and is accurate, simple and fast. The high performance liquid chromatography adopted by the invention has passed the methodological verification, is simple and easy to operate, has high sensitivity and good reproducibility, combines qualitative identification with quantitative analysis, and can be used for the quality control of Erding oral liquid.

[0068] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, other implementation methods can certainly be easily made by replacement or modification based on the technical contents disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. A method for determining the quality control index of Erding oral liquid, characterized in that: The following steps are involved: 1) The properties of Erding oral liquid are reddish brown to brown liquid, slightly bitter, and the relative density is not less than 1.01, and the pH value is 4.0-6.

0. The thin layer chromatography method is used to qualitatively identify Viola yedoensis, dandelion, lobelia, and isatis root in Erding oral liquid; Step 1) The specific steps are as follows: a. Identification of Viola yedoensis and Taraxacum officinale: Take 1 mL of Erding oral solution, add 30 mL of methanol, ultrasonically treat for 30 min, filter, evaporate the filtrate to dryness, add 20 mL of hot water to dissolve the residue, cool, shake and extract with ethyl acetate twice, 20 mL each time, combine the ethyl acetate solution, evaporate to dryness, add 1 mL of methanol to dissolve the residue as the test solution; separately take the quinoline reference substance and the caffeic acid reference substance, add methanol to prepare a reference substance solution containing 0.5 mg per 1 mL; then take the negative samples of Viola yedoensis and Taraxacum officinale to prepare a negative control solution according to the preparation method of the test sample; take 5 μL of each of the above solutions, spot them on the same silica gel G thin layer plate, use a mixed solution of toluene-ethyl acetate-formic acid in a ratio of 5:3:1 as the developing agent, develop, take out, dry, and fumigate in iodine vapor until the spots are clearly colored; spots of the same color appear in the chromatogram of the test sample at the corresponding position of the chromatogram of the reference substance, and there is no interference with the negative control; b. Identification of Lobelia: Take 8 mL of dibutyl oral solution, adjust pH to 11 with ammonia test solution, shake and extract twice with chloroform, 10 mL each time, combine the chloroform solution, evaporate to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the test solution; then take 2 g of Lobelia control medicinal material, add 50 mL of water, boil for 30 minutes, centrifuge, concentrate the supernatant to 10 mL, adjust pH to 11 with ammonia test solution, shake and extract twice with chloroform, 10 mL each time, combine the chloroform solution , evaporate to dryness, add 1mL of methanol to the residue to dissolve it as the reference solution; take another negative sample of Lobelia chinensis and prepare a negative control solution according to the preparation method of the test sample; take 5μL of each of the above solutions and spot them on the same silica gel G thin layer plate, use the lower layer solution with a ratio of chloroform to formic acid of 10:1 as the developing agent, develop, take out, dry, and examine under a 365nm ultraviolet lamp; in the chromatogram of the test sample, at the corresponding position of the chromatogram of the reference medicinal material, a spot of the same color appears, and there is no interference with the negative control; Step c, identification of Radix Isatidis: Take 10 mL of Erding oral liquid, shake and extract with chloroform 3 times, 10 mL each time, combine the chloroform solution, evaporate to dryness, add 0.5 mL of methanol to the residue to dissolve it as the test solution; then take 1 g of Radix Isatidis reference medicinal material, add 20 mL of water, ultrasonicate for 30 min, centrifuge, shake the supernatant with chloroform for 3 times, 20 mL each time, combine the chloroform solution, evaporate to dryness, add 0.5 mL of methanol to the residue to dissolve it as the reference solution; take (R, S)-gaoyichun reference, add methanol to make a reference solution containing 0.5 mg per 1 mL; take another Radix Isatidis negative sample and make a negative control solution according to the preparation method of the test sample; take 10 μL of each of the above solutions and spot them on the same silica gel GF 254 On the thin layer plate, a mixed solution of petroleum ether and ethyl acetate in a ratio of 1:1 was used as the developing solvent, developed, taken out, dried, and examined under a 254nm ultraviolet lamp; spots of the same color appeared in the chromatogram of the test sample at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference substance, and there was no interference with the negative control; 2) Prepare reference substance and different batches of test solution, and determine the content of the main active ingredient in the oral solution by high performance liquid chromatography; Step 2) The specific steps are as follows: a. Preparation of reference solution: Take the reference substance of aesculetin, the reference substance of caffeic acid and the reference substance of (R,S)-gaotizone, and add methanol to prepare reference solution containing 0.2-0.3 mg of aesculetin, 15-20 μg of caffeic acid and 2-5 μg of (R,S)-gaotizone per 1 mL respectively; b. Preparation of test solution: Measure 5 mL of dibutyl oral solution, place in a 150 mL conical flask, add 20 mL of methanol, weigh, ultrasonicate for 30 min, cool, make up the lost weight with methanol, shake well, filter, and take the filtrate; c. Determination: 10 μL of reference solution and test solution were taken and injected into the liquid chromatograph. Chromatographic conditions: chromatographic column with octadecylsilane bonded silica as filler, column temperature 30°C, detection wavelength 240nm; mobile phase with acetonitrile as phase A and 0.1% phosphoric acid solution as phase B, elution program: 0-20min, 2%A; 20-40min, 2%-20%A; theoretical plate number was calculated according to the peaks of esculetin, caffeic acid and (R,S)-goitrin and was not less than 3000, determination; 3) Based on the results of HPLC, the control indicators of the main active ingredients of dibutyl oral solution are determined as follows: each 1mL of dibutyl oral solution contains no less than 0.5mg of quinidine, no less than 50μg of caffeic acid, and no less than 10μg of (R,S)-gaitrin.

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