A method for detecting multiple main components in Baima oral liquid and its application

Abstract

The present invention belongs to the technical field of effective ingredient detection in veterinary drugs, and specifically relates to a method for detecting multiple main components in Baima oral liquid and its application. The method comprises: pre-treating a sample to be tested to obtain a test solution, and detecting the test solution based on UPLC-PDA. The present invention is based on ultra-high performance liquid chromatography to simultaneously detect five alkalis (matrine, oxymatrine, ephedrine hydrochloride, pseudoephedrine hydrochloride, berberine hydrochloride) and amygdalin in Baima oral liquid. The method is fast, sensitive, accurate, and highly specific. Through one-time sample processing, the above six main components are simultaneously detected and analyzed by liquid chromatography, which not only effectively saves reagents and time, but also improves work efficiency, which is beneficial to the quality monitoring of Baima oral liquid, and therefore has good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of effective ingredient detection in veterinary drugs, and specifically relates to a method for detecting multiple main ingredients in Baima oral liquid and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Bai Ma Oral Liquid is a Chinese veterinary medicine formula, included in the 2017 edition of "Quality Standards for Veterinary Drugs" (Chinese Medicine Volume), P200:

[0004] Prescription: 100g of Phellodendron, 50g of Ephedra, 75g of Bitter Apricot, 100g of Sophora flavescens, and 50g of Isatis indigotica. Add water to the above five flavors and boil them twice, let stand, filter, combine the filtrate, concentrate to 375ml, cool, fill, sterilize, and you are done. It can clear away heat and relieve asthma, dry dampness and stop diarrhea. It is used as an auxiliary treatment for infectious bronchitis in chickens; it is effective in treating tracheal and bronchial obstruction in chickens. In the original method, three thin layer chromatography methods are mainly used to identify berberine hydrochloride, ephedrine hydrochloride and Isatis indigotica, as shown below:

[0005] (1) Thin layer chromatography:

[0006] Operation: Take 50ml of this product, concentrate to 30ml, add diatomaceous earth and grind evenly, dry at 80℃, add appropriate amount of methanol, heat and reflux in a water bath for 15 minutes, filter, and concentrate the filtrate to 5ml as the test solution. Take another berberine hydrochloride reference substance, add methanol to make a solution containing 0.5mg per 1ml as the reference substance solution. According to the thin layer chromatography test, take 5μl of each of the above two solutions, and spot them on the same silica gel G thin layer plate, use n-butanol-glacial acetic acid-water (7:1:3) as the developing agent, develop, take out, dry, and examine under ultraviolet light (365nm). In the chromatogram of the test product, at the corresponding position of the chromatogram of the reference substance, a fluorescent spot of the same color appears.

[0007] (2) Thin layer chromatography:

[0008] Operation: Take 50ml of this product, add 1ml of concentrated ammonia solution to make it alkaline, extract twice with ether-ethanol (8:2) mixed solution, 10ml each time, combine the extracts, dehydrate with anhydrous sodium sulfate, filter, evaporate the ether from the filtrate, add 1ml of methanol to dissolve the residue, and use it as the test solution. Take another ephedrine hydrochloride reference substance, add methanol to make a solution containing 5mg per 1ml as the reference drug solution. According to the thin layer chromatography test, take 5μl of each of the above two solutions, spot them on the same silica gel G thin layer plate, use n-butanol-glacial acetic acid-water (8:2:1) as the developing agent, develop, take out, dry, spray with 0.5% ninhydrin acetone solution, and bake at 105℃ until the spots are clearly colored. In the chromatogram of the test product, spots of the same color appear at the corresponding position of the chromatogram of the reference medicinal material.

[0009] (3) Thin layer chromatography:

[0010] Operation: Take 50ml of this product, concentrate to dryness, add 20ml of ethanol to the residue to dissolve, filter, and concentrate the filtrate to 1ml as the test solution. Take 1g of the reference medicinal material of Isatis indigofera, add 20ml of ether, heat and reflux for 1 hour, filter, discard the filtrate, add 20ml of 70% ethanol to the residue, heat and reflux for 1 hour, filter, evaporate the filtrate, and add 1ml of ethanol to the residue to dissolve as the reference medicinal material solution. According to the thin layer chromatography test, take 5μl of each of the above two solutions, spot them on the same silica gel G thin layer plate, use n-butanol-anhydrous ethanol-concentrated ammonia test solution-water (8:8:1:3) as the developing agent, develop, take out, dry, spray with ninhydrin test solution, and bake at 105℃ until the spots are clearly colored. In the chromatogram of the test product, spots of the same color appear at the corresponding position of the chromatogram of the reference product. However, the developing agents such as n-butanol, concentrated ammonia solution, and ether are highly toxic and can easily cause harm to experimenters. Summary of the invention

[0011] In view of the deficiencies of the prior art, the present invention provides a method for detecting multiple main components in Baima oral liquid and its application. Specifically, the present invention simultaneously detects five alkalis (matrine, oxymatrine, ephedrine hydrochloride, pseudoephedrine hydrochloride, berberine hydrochloride) and amygdalin in Baima oral liquid based on ultra-high performance liquid chromatography, which not only effectively saves reagents and time, but also improves work efficiency. Based on the above research results, the present invention is completed.

[0012] In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0013] The first aspect of the present invention provides a method for detecting multiple main components in Baima oral liquid, wherein the main components include matrine, oxymatrine, ephedrine hydrochloride, pseudoephedrine hydrochloride, berberine hydrochloride and amygdalin, and the method comprises: pretreating a sample to be tested to obtain a test solution, and detecting the test solution based on UPLC-PDA; wherein, in the liquid chromatography method, mobile phase A is an acetonitrile solution containing phosphoric acid, and mobile phase B is an aqueous solution containing phosphoric acid.

[0014] The above detection method can realize qualitative and / or quantitative detection of the six main components in the Baima oral liquid, especially quantitative detection. The detection limit of the present invention is very low, which greatly improves the detection sensitivity and inspection effect, and is very beneficial to the quality monitoring of the Baima oral liquid.

[0015] Therefore, the second aspect of the present invention provides the application of the above-mentioned method for detecting multiple main components in Baima Oral Liquid in the quality monitoring of Baima Oral Liquid.

[0016] Compared with the prior art, the above one or more technical solutions have the following beneficial technical effects:

[0017] (1) The above technical solution detects the five alkaloids and amygdalin in the Baima oral liquid at the same time by ultra-high performance liquid chromatography. The method is simple and rapid, has good separation effect, high detection accuracy, good result reproducibility, is easy to observe, has strong specificity, and has a good chromatographic peak shape, and also saves time and reagents.

[0018] (2) The above technical solution is simple to process samples, saves reagents and time. The original standard of Bai Ma oral liquid used a thin layer method to identify ephedra, which has complex pre-treatment, high toxicity, and the spots are sometimes unclear and cannot be measured.

[0019] (3) The reagents used in the pretreatment and mobile phase of the above technical solution are less toxic: anhydrous ethanol, methanol, and acetonitrile are relatively less toxic; the various reagents used in the developing agent and color developer of the original method: n-butanol, ether, acetone, etc., are more toxic. The method of the present invention is more environmentally friendly and safe, and therefore has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0021] Figure 1 The spectrum of the reference substance is shown in Figure 1. The compounds from top to bottom are matrine at 1.89 min, oxymatrine at 3.07 min, ephedrine hydrochloride at 3.76 min, pseudoephedrine hydrochloride at 4.05 min, berberine hydrochloride at 14.31 min, and amygdalin at 15.91 min.

[0022] Figure 2 The chromatogram of the mixed reference substance at 208 nm; injection volume 0.5 μl; the compounds from left to right are 1.91 min matrine; 3.09 min oxymatrine; 3.78 min ephedrine hydrochloride; 4.07 min pseudoephedrine hydrochloride; 14.31 min berberine hydrochloride; 15.91 min amygdalin;

[0023] Figure 3 This is the 346nm chromatogram of the mixed reference substance; injection volume 0.5μl; the compound is only berberine hydrochloride at 14.31min;

[0024] Figure 4 This is the 251nm chromatogram of the mixed reference substance; the injection volume is 0.5μl; the compound is only berberine hydrochloride at 14.31min; amygdalin at 15.91min;

[0025] Figure 5 This is the 230nm chromatogram of the mixed reference substance; injection volume 0.5μl; the compound is only berberine hydrochloride at 14.31min;

[0026] Figure 6 Chromatogram of Yunhu sample at 208 nm treated by method A; injection volume 1 μl;

[0027] Figure 7 Chromatogram of Yunhu sample at 208 nm processed by method B; injection volume 1 μl;

[0028] Figure 8 Chromatogram of Yunhu sample at 208 nm processed by method D; injection volume 1 μl;

[0029] Fig. 9 Chromatogram of Yunhu sample at 208 nm treated by method C; injection volume 1 μl;

[0030] Fig.10 Chromatogram of Ruigu sample at 208nm processed by method B; injection volume 1μl. DETAILED DESCRIPTION

[0031] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0032] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention.

[0033] In a typical specific embodiment of the present invention, a method for detecting multiple main components in Baima oral liquid is provided, wherein the main components include matrine, oxymatrine, ephedrine hydrochloride, pseudoephedrine hydrochloride, berberine hydrochloride and amygdalin, and the method comprises: pretreating the sample to be tested to obtain a test solution, and detecting the test solution based on UPLC-PDA; wherein, in the liquid chromatography method, mobile phase A is an acetonitrile solution containing phosphoric acid, and mobile phase B is an aqueous solution containing phosphoric acid.

[0034] In another specific embodiment of the present invention, the pretreatment method may be any one of the following:

[0035] The pretreatment method A is: adding ethanol to the Baima oral liquid, and obtaining the test solution after ultrasonic, centrifugal and filtration treatment. This method has the lowest reagent toxicity, is the fastest, saves the most reagents, and is the safest;

[0036] Wherein, the volume ratio of the Baima oral liquid to ethanol is 1:2-5 (preferably 1:4), ultrasonic treatment is performed for 10-30 min (preferably 20 min), and centrifugation is performed for 5-20 min (preferably 10 min).

[0037] The pretreatment method B is: adding a mixed solution of methanol and hydrochloric acid to the Baima oral liquid, and obtaining the test solution after ultrasonic, centrifugal and filtration treatment. The reagent toxicity of this method is greater than that of ethanol, but it is relatively quick;

[0038] The volume ratio of the Baima oral liquid to the mixed solution of methanol and hydrochloric acid is 1:8-10 (preferably 1:9), the ultrasonic treatment is carried out for 10-30 minutes (preferably 20 minutes), and the centrifugation is carried out for 5-20 minutes (preferably 10 minutes). The volume ratio of methanol to hydrochloric acid in the mixed solution of methanol and hydrochloric acid is 100:1-10, preferably 100:5.

[0039] The pretreatment method C is: adding a mixed solution of ethanol and 3% phosphoric acid aqueous solution to the Baima oral liquid, and then subjecting it to ultrasonic, centrifugal and filtration treatment to obtain the test solution. This method has a better peak shape and better symmetry.

[0040] The volume ratio of the Baima oral liquid to the mixed solution of ethanol and 3% phosphoric acid aqueous solution is 1:2-5, preferably 1:4, ultrasonic treatment is performed for 10-30 minutes (preferably 20 minutes), and centrifugation is performed for 5-20 minutes (preferably 10 minutes). The volume ratio of ethanol to 3% phosphoric acid aqueous solution in the mixed solution of ethanol and 3% phosphoric acid aqueous solution is 1:0.5-2, preferably 1:1.

[0041] The pretreatment method D is a concentration method, specifically: take the Baima oral liquid and concentrate it to 1 / 5-4 / 5 (preferably 3 / 5) of the original volume, add diatomaceous earth and grind it evenly, add methanol for ultrasonication after drying, and filter and concentrate it as the test solution. This method is intended to improve the detection limit by concentration, but after actual detection, the peak area is relatively low. It is speculated that the diatomaceous earth adsorbs part of the drug, resulting in a low response, but the impurities are relatively small.

[0042] In another specific embodiment of the present invention, the volume ratio of the Baima oral liquid to methanol is 1:0.5-2, preferably 1:1; and the ultrasonic treatment is performed for 10-30 min (preferably 20 min).

[0043] In another specific embodiment of the present invention, the method also includes the preparation of a reference solution, the preparation method comprising: selecting matrine, oxymatrine, ephedrine hydrochloride, pseudoephedrine hydrochloride, berberine hydrochloride and amygdalin reference substances, adding anhydrous ethanol or methanol to prepare a reference solution as a reference stock solution; during detection, the above six reference stock solutions are mixed and sampled as a mixed reference solution for detection.

[0044] The specific condition parameters of the UPLC-PDA include:

[0045] The injection volume is 0.2-5 μL, the mobile phase A is a 0.2% phosphoric acid in acetonitrile solution, the mobile phase B is a 0.2% phosphoric acid in water solution, the flow rate is controlled at 0.1-0.5 ml / min (preferably 0.35 ml / min), and gradient elution is performed; PDA detector, scanning range: 190-400 nm, the detection wavelength is preferably 208 nm; the column temperature is 30-40° C. (preferably 35° C.).

[0046] The chromatographic column can be Waters CORTECS C 18 Chromatographic columns, 1.6μm,2.1mm*100mm; CORTECS C 18The columns are general-purpose, ultra-high-efficiency columns based on solid-core particles that provide balanced retention of acids, bases, and neutrals in the low to mid pH range.

[0047] The specific procedure of the gradient elution includes: 0-2 min, 3% A; 2-5 min, 3% A-5% A; 5-8 min, 5% A-5% A; 8-10 min, 5% A-15% A; 10-12 min, 15% A-15% A; 12-15 min, 15% A-45% A; 15-21 min, 45% A-45% A; 21-22 min, 45% A-3% A; 22-24 min, 3% A.

[0048] The above detection method can realize qualitative and / or quantitative detection of the six main components in the Baima oral liquid, especially quantitative detection. The detection limit of the present invention is very low, which greatly improves the detection sensitivity and inspection effect, and is very beneficial to the quality monitoring of the Baima oral liquid.

[0049] Therefore, in another specific embodiment of the present invention, there is provided application of the above-mentioned method for detecting multiple main components in Baima oral liquid in quality monitoring of Baima oral liquid.

[0050] The present invention is further explained by the following examples, but it does not constitute a limitation of the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The test samples used herein: Sample No. 1 was produced by Shandong Yunhu Biopharmaceutical Co., Ltd., and Sample No. 2 was produced by Yantai Ruigu Biotechnology Co., Ltd. All samples were produced in strict accordance with the national standard prescriptions for veterinary drugs and in accordance with the GMP management specifications for veterinary drugs.

[0051] Example 1: A method for simultaneously detecting six main components in Baima oral liquid

[0052] Here are the steps:

[0053] Step 1: Preparation of test solution:

[0054] Method A: Take 2.0 mL of Baima oral liquid into a 50 mL centrifuge tube, add 8 mL of ethanol, sonicate for 20 min, centrifuge for 10 min, and filter to use as the test solution.

[0055] Method B: Take 1ml or 2ml of Baima oral liquid into a 50ml centrifuge tube, add (methanol: hydrochloric acid = 100:5) solution to 10ml, ultrasonicate for 20min, centrifuge for 10min, filter, and use it as the test solution.

[0056] Method C: Take 2 ml of Baima oral liquid into a 50 ml centrifuge tube, add 8 ml of (ethanol: 3% phosphoric acid = 1:1) solution, ultrasonicate for 30 min, centrifuge for 10 min, filter, and use this as the test solution.

[0057] Step 2: Preparation of reference solution: Accurately weigh appropriate amount of matrine, oxymatrine, ephedrine hydrochloride, pseudoephedrine hydrochloride, berberine hydrochloride and amygdalin reference substances, add anhydrous ethanol or methanol and other solvents to make a solution of appropriate concentration as the reference substance stock solution; then take appropriate amount of 6 kinds of stock solutions, mix them evenly, and use them as mixed reference solution. Step 3: Liquid chromatography: injection volume: 0.5-2μL, item A: acetonitrile (0.2% phosphoric acid): item B water (0.2% phosphoric acid), gradient elution, specific gradient elution program see Table 1; PDA detector 3D scanning 190-400nm, detection wavelength 208nm; 35℃ column temperature; on-machine detection.

[0058] Table 1 Mobile phase gradient elution program

[0059] Flow rate Time / min Item A B curve 0.35ml / min initial 3 97 6 0.35ml / min 2 3 97 6 0.35ml / min 5 5 95 6 0.35ml / min 8 5 95 6 0.35ml / min 10 15 85 6 0.35ml / min 12 15 85 6 0.35ml / min 15 45 55 6 0.35ml / min 21 45 55 6 0.35ml / min 22 3 97 6 0.35ml / min 24 3 97 6

[0060] Comparison of results: After comparing the detection wavelengths, the response values ​​of the first 6 substances at 208nm were higher, and the wavelength of 208nm was finally selected, with appropriate response values ​​and less interference.

[0061] Result determination: According to the chromatogram, in the chromatogram of the positive sample of the test product, the same chromatographic peak appears at the corresponding position of the chromatogram of the six reference products, with good peak shape and qualified separation, indicating that the corresponding medicinal materials can be detected: Sophora flavescens (matrine, oxymatrine), Ephedra (ephedrine hydrochloride, pseudoephedrine hydrochloride), Phellodendron chinense (berberine hydrochloride), and Amygdalin (amygdalin). The results are shown in Tables 3-1 to 3-6.

[0062] Drugs and reagents:

[0063] Anhydrous ethanol was of analytical grade, phosphoric acid, acetonitrile, and methanol were of chromatographic grade, and reference substances were purchased from the China Food and Drug Inspection Institutes. The preparation information is shown in Table 2.

[0064] Table 2 Preparation information of reference substances

[0065]

[0066] The instruments used were: BP211D analytical balance (Sartorius, Germany); Waters ultra-high performance liquid chromatograph, PDA detector;

[0067] The chromatographic column is CORTECS C from Waters 18 Column, 1.6μm,2.1mm*100mm,1 / pk. CORTECS C 18 The columns are general-purpose, ultra-high-efficiency columns based on solid-core particles that provide balanced retention of acids, bases, and neutrals in the low to mid pH range.

[0068] Example 2 (Comparative Example): A method for simultaneously detecting six main components in Baima oral liquid

[0069] The steps are as follows: Method D

[0070] Step 1: Preparation of test solution: Take 50 ml of the product, concentrate to 30 ml, add diatomaceous earth and grind evenly, dry at 80°C, add 50 ml of methanol, ultrasonicate for 20 min, filter, and concentrate the filtrate to 5 ml as the test solution.

[0071] The second and third steps and the result determination are the same as those in Example 1. The results are shown in Tables 3-1 to 3-6. Comparison of the results of different samples and different extraction methods (injection volume 0.5 μl-1 μl)

[0072] Table 3-1 Comparison of results of matrine reference substance and samples treated by different methods

[0073]

[0074]

[0075] Table 3-2 Comparison of results of different treatment methods for oxymatrine reference and samples

[0076]

[0077] Table 3-3 Comparison of results of different treatment methods for ephedrine hydrochloride reference substance and samples

[0078]

[0079]

[0080] Table 3-4 Comparison of results of different treatment methods for pseudoephedrine hydrochloride reference substance and samples

[0081]

[0082] Table 3-5 Comparison of results of different treatment methods for berberine hydrochloride reference substance and samples

[0083]

[0084] Table 3-6 Comparison of the results of different treatment methods for amygdalin reference and samples

[0085]

[0086]

[0087] Spectrum is attached Figure 1The spectrum can assist in the qualitative analysis of the compounds. Due to the influence of different pretreatment methods, the response values ​​of the target compounds also vary to a certain extent. The chromatogram is attached. Figure 2-10 , the compounds can be further identified by retention time.

[0088] If the content of the compound in the market sample is relatively low, we can choose Example 1, and we can increase the detection sensitivity by increasing the sampling volume. In terms of time, we prefer Example 1, which saves the most time. The chromatographic column has the highest response value to the compound. The response values ​​of the three methods ABC are all acceptable, with method A having the lowest response value, method B having the highest response value, and method C having the second highest response value; in terms of safety, methods A and C are preferred; in terms of sample background, we prefer Example 2. Although the compound response value is not that high, the sample matrix background is small, which can meet the monitoring requirements and is better for the chromatographic column, but the sample pretreatment is slightly more complicated.

[0089] We found that the peak heights and peak areas of the six components vary due to different processing methods and different manufacturers, and are calculated and quantified by the ratio of the chromatographic peak areas of the reference and sample. The content varies (calculated by the 208nm channel data results), as shown in Table 4, which is closely related to the quality of the original medicinal materials used by the company and the production and extraction processes of each company. This method uses an ultra-high performance liquid chromatograph to present the results in the form of a chromatogram. The general detection limit can be inferred from the signal-to-noise ratio. Its detection limit is very low, as shown in Table 5 below, which greatly improves the detection sensitivity and inspection effect.

[0090] Table 4 Comparison of content of different samples and different treatment methods (content μg / ml)

[0091]

[0092]

[0093] Table 5 Comparison of detection limits of different methods

[0094]

[0095] If the response value is low when the sample is actually tested, the injection volume can be appropriately increased to 5-10 μl, and the chromatographic peaks of various components in the chromatogram can be clearly seen. Under the existing 2017 edition of the national veterinary drug quality standard, thin layer identification is difficult to control the quality of Baima oral liquid. The present invention can be used for accurate and rapid testing, laying a solid foundation for the formulation of the 2025 veterinary drug quality standard.

[0096] Advantages: 1. The solvent used in the present invention is less toxic and less harmful to humans and the environment; 2. According to the present invention, the detection of a sample from sample processing to machine detection and result determination can be completed in about 2 hours; while in the existing national standards, sample pretreatment is very complicated and requires a heating reflux device, thin layer running, drying, color spraying and other steps, and one sample takes about 8 hours, which greatly shortens the detection time and improves the inspection efficiency; 3. The present invention uses liquid chromatograms, the results are intuitive, easy to judge, and have a low detection limit; the national standard method uses thin layer chromatography, and the spots are greatly affected by the environmental temperature and humidity and human operation, the spots are often unclear, and the results are difficult to judge.

[0097] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for detecting multiple main components in Baima oral liquid, wherein the main components include matrine, oxymatrine, ephedrine hydrochloride, pseudoephedrine hydrochloride, berberine hydrochloride and amygdalin, characterized in that: The method comprises: pre-treating a sample to be tested to obtain a test solution, and detecting the test solution based on UPLC-PDA; wherein, in the liquid chromatography method, mobile phase A is an acetonitrile solution containing phosphoric acid, and mobile phase B is an aqueous solution containing phosphoric acid.

2. The method according to claim 1, characterized in that The pretreatment method is any one of the following: The pretreatment method A is as follows: ethanol is added to the Baima oral liquid, and the test solution is obtained after ultrasonic, centrifugal and filtration treatment; The pretreatment method B is: adding a mixed solution of methanol and hydrochloric acid to the Baima oral liquid, and obtaining a test solution after ultrasonic, centrifugal and filtration treatment; The pretreatment method C is: adding a mixed solution of ethanol and 3% phosphoric acid aqueous solution to the Baima oral liquid, and obtaining the test solution after ultrasonic, centrifugal and filtration treatment; The pretreatment method D is a concentration method, specifically: taking the Baima oral liquid and concentrating it to 1 / 5-4 / 5 of the original volume, adding diatomaceous earth to grind it evenly, adding methanol to perform ultrasound after drying, filtering and concentrating it as the test solution.

3. The method according to claim 2, characterized in that In the pretreatment method A, the volume ratio of the baima oral liquid to ethanol is 1:2-5, the ultrasonic treatment is performed for 10-30 minutes, and the centrifugation is performed for 5-20 minutes; In the pretreatment method B, the volume ratio of the Baima oral liquid to the mixed solution of methanol and hydrochloric acid is 1:8-10, the ultrasonic treatment is carried out for 10-30 minutes, and the centrifugation is carried out for 5-20 minutes; the volume ratio of methanol to hydrochloric acid in the mixed solution of methanol and hydrochloric acid is 100:1-10; In the pretreatment method C, the volume ratio of the baima oral liquid to the mixed solution of ethanol and 3% phosphoric acid aqueous solution is 1:2-5, ultrasonic treatment is performed for 10-30 minutes, and centrifugation is performed for 5-20 minutes; the volume ratio of ethanol to 3% phosphoric acid aqueous solution in the mixed solution of ethanol and 3% phosphoric acid aqueous solution is 1:0.5-2; In the pretreatment method D, the volume ratio of the Baima oral liquid to methanol is 1:0.5-2; and the ultrasonic treatment is performed for 10-30 minutes.

4. The method according to claim 1, characterized in that The method also includes the preparation of a reference solution, which includes: selecting matrine, oxymatrine, ephedrine hydrochloride, pseudoephedrine hydrochloride, berberine hydrochloride and amygdalin reference substances, adding anhydrous ethanol or methanol to prepare a reference solution as a reference stock solution.

5. The method according to claim 4, characterized in that During the test, six reference substance stock solutions were mixed and used as the mixed reference substance solution for sample loading test.

6. The method according to claim 1, characterized in that The specific condition parameters of the UPLC-PDA include: The injection volume is 0.2-5 μL, the mobile phase A is a 0.2% phosphoric acid in acetonitrile solution, the mobile phase B is a 0.2% phosphoric acid in water solution, the flow rate is controlled at 0.1-0.5 ml / min, and gradient elution is performed; the PDA detector has a scanning range of 190-400 nm and a detection wavelength of 208 nm; the column temperature is 30-40°C.

7. The method according to claim 5, characterized in that The chromatographic column is a Waters CORTECS C18 column. 1.6μm,2.1mm*100mm.

8. The method according to claim 5, characterized in that The specific procedure of the gradient elution includes: 0-2 min, 3% A; 2-5 min, 3% A-5% A; 5-8 min, 5% A-5% A; 8-10 min, 5% A-15% A; 10-12 min, 15% A-15% A; 12-15 min, 15% A-45% A; 15-21 min, 45% A-45% A; 21-22 min, 45% A-3% A; 22-24 min, 3% A.

9. The method according to any one of claims 1 to 8, characterized in that: The method is for qualitative and / or quantitative detection of six main components in Baima oral liquid.

10. Use of the method for detecting multiple main components in Baima oral liquid according to any one of claims 1 to 9 in the quality monitoring of Baima oral liquid.

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