A method for simultaneously detecting multiple medicinal components in Yuhuang oral liquid using UPLC-PDA

Abstract

The present invention belongs to the technical field of veterinary drug detection, and specifically relates to a method for simultaneously detecting multiple medicinal components in Yuhuang oral liquid by UPLC-PDA. The present invention can simultaneously determine the contents of 10 components in Yuhuang oral liquid, including berberine hydrochloride, bamipine hydrochloride, baicalin, gardenia glycoside, gallic acid, paeoniflorin, rhein, rhein, physophanol, and chrysophanol, thereby simultaneously detecting 7 medicinal components, including coptis root, phellodendron, scutellaria baicalensis, gardenia jasminoides, terminalia chebula, white peony root, and rhubarb. The present invention uses spectrum and chromatography for qualitative and quantitative determination, which is rapid, sensitive, accurate, and highly specific. Through one sample processing, the contents of 10 components can be simultaneously determined, which not only saves reagents and time, but also improves work efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary drug detection, and particularly relates to a method for simultaneously detecting multiple medicinal components in Yuhuang oral liquid by using UPLC-PDA. 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] Yuhuang oral liquid is a Chinese veterinary medicine formula, which is included in the 2017 edition of the Chinese medicine volume of the "National Standard for Veterinary Drugs" p184. Its prescription includes: 250g of Curcuma, 220g of Terminalia chebula, 50g of Gardenia, 50g of Scutellaria baicalensis, 50g of Rhubarb, 30g of White Peony, 50g of Phellodendron, and 50g of Coptis chinensis. The standard uses thin layer chromatography to detect Gardenia (gardena glycoside), Terminalia chebula (gallic acid), Coptis chinensis (berberine hydrochloride), and Rhubarb in Yuhuang oral liquid, and uses high performance liquid chromatography to detect the content of baicalin. It takes about 2-3 days to complete the detection of a sample using the detection method in the standard.

[0004] When using thin layer chromatography, the spots are greatly affected by the environmental temperature and humidity and human operation. The spots are often unclear, the results are difficult to judge, and it is time-consuming and reagent-consuming. In actual inspection work, it is found that the developing agents used for chromatography are highly toxic, such as chloroform, ethyl acetate, methanol, acetone, formic acid, benzene, isopropanol, ether, petroleum ether (30-60°C), ethyl formate and other reagents, which are relatively harmful to the experimenters. In addition, the operation is cumbersome and time-consuming, and it is impossible to detect multiple effective ingredients at the same time.

[0005] In addition, the preliminary preparation of thin layer chromatography is complicated, requiring multiple steps such as pre-saturation, development, drying, and inspection of results. It is time-consuming and labor-intensive, and is greatly affected by factors such as environmental temperature and humidity, edge effects, etc. Thin layer spots are sometimes difficult to identify and have poor reproducibility. Summary of the invention

[0006] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a method for simultaneously detecting multiple medicinal components in Yuhuang oral liquid by UPLC-PDA. The present invention can simultaneously determine the contents of berberine hydrochloride, bamipine hydrochloride, baicalin, gardenia glycoside, gallic acid, paeoniflorin, rhein, rhein, physophanol, and chrysophanol in Yuhuang oral liquid, thereby simultaneously detecting seven medicinal components, namely, coptis chinensis, phellodendron, scutellaria baicalensis, gardenia jasminoides, terminalia chebula, white peony root, and rhubarb. The present invention uses spectrum and chromatography for qualitative and quantitative determination, which is rapid, sensitive, accurate, and highly specific. Through one sample processing, the contents of 10 components can be simultaneously determined, which not only saves reagents and time, but also improves work efficiency.

[0007] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0008] In a first aspect, the present invention provides a method for simultaneously detecting multiple medicinal components in Yuhuang oral liquid using UPLC-PDA, comprising the following steps:

[0009] A. Take berberine hydrochloride, palmatine hydrochloride, baicalin, gardenia glycoside, gallic acid, paeoniflorin, rhein, rhein, physophanol methyl ether, and chrysophanol reference substances, dissolve them in solvent, prepare reference stock solutions respectively, mix the reference stock solutions, and obtain a mixed reference stock solution;

[0010] B. Take the test sample of Yuhuang oral liquid, dissolve it with a solvent, perform ultrasonic extraction, centrifuge, dilute, and filter to obtain the sample solution to be tested;

[0011] C. Inject the mixed control stock solution into the UPLC-PDA chromatograph, perform gradient elution and detection, and qualitatively determine the quality by retention time. Draw a standard curve based on the corresponding relationship between the peak area measured by the injection volume and the injection volume.

[0012] D. Inject the sample solution to be tested into the UPLC-PDA chromatograph, perform gradient elution and detection under the same conditions as step C, measure the peak area of ​​each target, qualitatively determine the retention time, and quantitatively determine the content of the active ingredient in the Yuhuang oral liquid test sample based on the standard curve prepared in step C.

[0013] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0014] 1. The present invention adopts UPLC-PDA method for determination, using waters ACQUITY UPLC HSS T 3 The chromatographic column is a preferred chromatographic column, acetonitrile (0.05% phosphoric acid)-0.05% phosphoric acid aqueous solution is used for gradient elution, and 3D scanning (190nm-400nm) is used for detection with detection wavelengths of 232nm, 238nm, 257nm, 278nm and 289nm, so that 10 main components in the Yuhuang oral liquid can be determined at one time. The method is simple and rapid, has good separation effect, high determination accuracy, good result reproducibility, is easy to observe, has strong specificity, and has a good chromatographic peak shape, which not only meets the requirements of the Chinese Pharmacopoeia of Veterinary Medicine, but also saves time and reagents.

[0015] 2. The determination method of the present invention uses (V 50% methanol: V 0.05% phosphoric acid aqueous solution = 1:1) as the extraction solvent to extract the test sample. Compared with chloroform ether and the like used in the thin layer, the toxicity is less, the peak area response value of the compound is moderate, the sample treatment is simple, reagents are saved, and time is saved. The solvent used is less toxic, the operation is safe and environmentally friendly, and the harm to humans and the environment is small. The original standard 4 thin layer determinations take a long time to process, develop, and dry, and use a variety of reagents: benzene, acetone, ethyl acetate, isopropanol, methyl acetate, etc., and benzene belongs to a class of carcinogens. The reagents used are highly toxic, the spots are sometimes unclear, and are greatly affected by the ambient temperature and humidity and personnel operation, and sometimes the spot results are difficult to judge.

[0016] 3. The determination method of the present invention uses a high performance liquid chromatograph to present the results in the form of chromatograms and spectrograms, which is convenient and fast, the results are intuitive, easy to judge, and have a low detection limit, which greatly shortens the test time and improves the test efficiency, detection sensitivity and test effect.

[0017] 4. The method of the present invention can complete the detection of a sample in about 2 hours from sample processing to machine detection and product determination results; while in the existing 2017 edition of the National Standard for Veterinary Drugs, the sample pretreatment during thin layer detection is relatively complicated, some require heating and reflux, some require water bath evaporation, and some require shaking extraction. It is necessary to prepare a developing agent, pre-saturation, development, drying and other steps, and one sample takes about 2-3 days. The present invention greatly shortens the detection time and improves the inspection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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.

[0019] Figure 1 The spectra are berberine hydrochloride, palmatine hydrochloride, baicalin, gardenia glycoside, gallic acid, paeoniflorin, rhein, physophanol, physophanol methyl ether, and chrysophanol;

[0020] Figure 2 The chromatogram of the mixed control sample in Example 1 when the detection wavelength is 232 nm;

[0021] Figure 3 The chromatogram of the mixed control sample in Example 1 when the detection wavelength is 238 nm;

[0022] Figure 4 The chromatogram of the mixed control sample in Example 1 at a detection wavelength of 257 nm;

[0023] Figure 5 The chromatogram of the mixed control sample in Example 1 at a detection wavelength of 278 nm;

[0024] Figure 6 The chromatogram of the mixed control sample in Example 1 at a detection wavelength of 289 nm;

[0025] Figure 7 The chromatogram of sample 0037 in Example 1 when the detection wavelength is 232 nm;

[0026] Figure 8 The chromatogram of sample 0037 in Example 1 when the detection wavelength is 238 nm;

[0027] Fig. 9 The chromatogram of sample 0037 in Example 1 at a detection wavelength of 257 nm;

[0028] Fig.10 The chromatogram of sample 0037 in Example 1 at a detection wavelength of 278 nm;

[0029] Fig.11 The chromatogram of sample 0037 in Example 1 at a detection wavelength of 289 nm;

[0030] Fig.12 The chromatogram of the 1751 sample in Example 2 when the detection wavelength is 232 nm;

[0031] Fig.13 The chromatogram of the 1751 sample in Example 2 when the detection wavelength is 238 nm;

[0032] Fig.14 The chromatogram of the sample 1751 in Example 2 at a detection wavelength of 257 nm;

[0033] Fig.15 The chromatogram of the 1751 sample in Example 2 at a detection wavelength of 278 nm;

[0034] Fig.16 This is the chromatogram of sample 1751 in Example 2 at a detection wavelength of 289 nm. DETAILED DESCRIPTION

[0035] It should be noted that the following detailed descriptions are exemplary and are 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.

[0036] 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, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0037] The first typical embodiment of the present invention is a method for simultaneously detecting multiple medicinal components in Yuhuang oral liquid using UPLC-PDA, comprising the following steps:

[0038] A. Take berberine hydrochloride, palmatine hydrochloride, baicalin, gardenia glycoside, gallic acid, paeoniflorin, rhein, rhein, physophanol methyl ether, and chrysophanol reference substances, dissolve them in solvent, prepare reference stock solutions respectively, mix the reference stock solutions, and obtain a mixed reference stock solution;

[0039] B. Take the test sample of Yuhuang oral liquid, dissolve it with a solvent, perform ultrasonic extraction, centrifuge, dilute, and filter to obtain the sample solution to be tested;

[0040] C. Inject the mixed control stock solution into the UPLC-PDA chromatograph, perform gradient elution and detection, and qualitatively determine the quality by retention time. Draw a standard curve based on the corresponding relationship between the peak area measured by the injection volume and the injection volume.

[0041] D. Inject the sample solution to be tested into the UPLC-PDA chromatograph, perform gradient elution and detection under the same conditions as step C, measure the peak area of ​​each target, qualitatively determine the retention time, and quantitatively determine the content of the active ingredient in the Yuhuang oral liquid test sample based on the standard curve prepared in step C.

[0042] In one or more examples of this embodiment, the solvent in step A is one or more of anhydrous ethanol, methanol, and chloroform.

[0043] In one or more examples of this embodiment, the solvent in step B is one or more of methanol, anhydrous ethanol, 50% methanol aqueous solution, 50% ethanol aqueous solution, and a mixed solution of methanol: 0.05% phosphoric acid = 50:50.

[0044] The concentrations in the 50% methanol aqueous solution, the 50% ethanol aqueous solution, and the mixed solution of methanol:0.05% phosphoric acid = 50:50 are all volume concentrations.

[0045] In one or more examples of this embodiment, in step B, the ultrasonic extraction time is 15-30 min, the centrifugal speed is 10000 r / min, and the centrifugal time is 5-20 min.

[0046] In one or more examples of this embodiment, in step C, the injection volume is 0.5-5 μL, the mobile phase flow rate is 0.34-0.36 mL / min, and the column temperature is 39-41°C.

[0047] In one or more examples of this embodiment, the mobile phase in step C comprises mobile phase A and mobile phase B, mobile phase A is a phosphoric acid acetonitrile solution with a volume concentration of 0.05%, and mobile phase B is a phosphoric acid aqueous solution with a volume concentration of 0.05%.

[0048] In one or more examples of this embodiment, the gradient elution in step C includes the following stages:

[0049] 0 min, 8% mobile phase A, 92% mobile phase B, initial curve;

[0050] 0.5 min, 8% mobile phase A, 92% mobile phase B, curve 6;

[0051] 3 min, 20% mobile phase A, 80% mobile phase B, curve 6;

[0052] 4.5 min, 25% mobile phase A, 75% mobile phase B, curve 6;

[0053] 5.5 min, 28% mobile phase A, 72% mobile phase B, curve 6;

[0054] 8.5 min, 28% mobile phase A, 72% mobile phase B, curve 6;

[0055] 10 min, 50% mobile phase A, 50% mobile phase B, curve 6;

[0056] 11 min, 50% mobile phase A, 50% mobile phase B, curve 6;

[0057] 11.5 min, 70% mobile phase A, 30% mobile phase B, curve 6;

[0058] 14 min, 70% mobile phase A, 30% mobile phase B, curve 6;

[0059] 14.5 min, 8% mobile phase A, 92% mobile phase B, curve 6;

[0060] 15 min, 8% mobile phase A, 92% mobile phase B, curve 6.

[0061] In one or more examples of this embodiment, 3D scanning (190-400 nm) is used in step C, and the detection wavelength is one or more of 232 nm, 238 nm, 257 nm, 278 nm, and 289 nm.

[0062] In one or more examples of this embodiment, in step B, 8% mobile phase A and 92% mobile phase B are used as diluents, which are mixed and diluted with the supernatant obtained by centrifugation in a ratio of 1:1.

[0063] In one or more embodiments of this implementation mode, the contents of berberine hydrochloride, bamipine hydrochloride, baicalin, gardenia glycoside, gallic acid, paeoniflorin, rhein, rhein, physophanol and chrysophanol in the Yuhuang oral liquid sample can be detected simultaneously through chromatograms and spectrograms, and the contents of coptis chinensis, phellodendron, scutellaria baicalensis, gardenia jasminoides, terminalia chebula, white peony root and rhubarb in the Yuhuang oral liquid sample can be detected simultaneously.

[0064] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments and comparative examples.

[0065] In the embodiment of the present invention:

[0066] Source of reference substances: purchased from China Veterinary Drug Administration, China Food and Drug Inspection Institute, China National Institute for the Control of Biological Products and other institutions.

[0067] Source of test samples: Huangyu oral solution: Sample 1571 is from Jinan Guangshengyuan Biotechnology Co., Ltd.; Sample 0037 is produced by Shandong Paison Pharmaceutical Co., Ltd. These are samples produced by various companies in strict accordance with the pharmacopoeia prescriptions.

[0068] Instruments: BP211D analytical balance (Sartorius, Germany); Waters AcquityTM ultra-high performance liquid chromatograph (Waters, USA), PDA detector, chromatographic column: Waters ACQUITY UPLC HSS T3 chromatographic column (2.1 mm×100 mm, 1.8 μm).

[0069] Example 1

[0070] 0037 Sample Testing

[0071] Take berberine hydrochloride, palmatine hydrochloride, baicalin, gardenia glycoside, gallic acid, paeoniflorin, rhein, rhein, physophanol, and chrysophanol reference substances, dissolve them in appropriate solvents and make up to volume, and prepare their own reference stock solutions; mix the reference stock solutions according to Table 1 to obtain a mixed reference stock solution. Weigh an appropriate amount of benzoic acid reference substance, dissolve it in ethanol alone and prepare a benzoic acid stock solution, add 10 μL of benzoic acid stock solution to 10 ml of the mixed reference substance solution, and the total volume is almost negligible.

[0072] Table 1 Preparation table of 10 mixed comparisons of Yuhuang oral liquid

[0073]

[0074] Accurately measure 2.0 mL of the 0037 sample, place it in a 10 mL volumetric flask, dissolve it with a mixed solution of methanol: (0.05% phosphoric acid) = 50:50 and make up to the scale, weigh it, perform ultrasonic extraction for 20 minutes, cool to room temperature, weigh it, make up the weight loss with a suitable solvent, take out 5 mL, centrifuge it at 10000 rpm / min for 10 minutes, take the supernatant and dilute it 1:1 with a mixed solution consisting of 8% volume concentration of 0.05% phosphoric acid acetonitrile solution and 92% volume concentration of 0.05% phosphoric acid aqueous solution, filter it, and obtain the 0037 sample solution for liquid chromatography determination.

[0075] The UPLC detection instrument is an ultra-high performance liquid chromatograph, the PDA detector is a photodiode array detector, and the chromatographic column is a Waters ACQUITY UPLC HSS T 3 The column temperature was 40°C, the mobile phase contained mobile phase A and mobile phase B, mobile phase A was a phosphoric acid acetonitrile solution with a volume concentration of 0.05%, mobile phase B was a phosphoric acid aqueous solution with a volume concentration of 0.05%, the flow rate was 0.35 mL / min, and the elution program was shown in Table 2. PDA selected 3D scanning (190-400 nm), and multi-wavelength detection selected 232 nm, 238 nm, 257 nm, 278 nm, and 289 nm channels.

[0076] Table 2 Gradient elution program

[0077] Flow rate A％ B％ curve initial 0.35 8 92 initial 0.5 0.35 8 92 6 3 0.35 20 80 6 4.5 0.35 25 75 6 5.5 0.35 28 72 6 8.5 0.35 28 72 6 10 0.35 50 50 6 11 0.35 50 50 6 11.5 0.35 70 30 6 14 0.35 70 30 6 14.5 0.35 8 92 6 15 0.35 8 92 6

[0078] The mixed control stock solution was injected into the UPLC-PDA chromatograph, and the gradient was qualitatively determined by retention time. A standard curve was drawn based on the corresponding relationship between the peak area measured by the injection volume and its injection volume. The test results of the mixed control stock solution are shown in Table 3.

[0079] Table 3 Chromatographic data of mixed control stock solution

[0080]

[0081]

[0082] The 0037 sample solution was injected into the UPLC-PDA chromatograph for gradient elution and detection, and the peak area of ​​each target was measured. The retention time was used for qualitative analysis, and the standard curve was used for quantitative analysis to calculate the content of the active ingredient in the Yuhuang oral liquid test sample. The chromatographic data of the 0037 sample solution are shown in Table 4.

[0083] Table 4 Chromatographic data of 0037 sample solution

[0084]

[0085]

[0086] like Figure 1 As shown, the spectra of multiple compounds have the following characteristics: wavelengths at the peak of gallic acid: 215.8nm, 272.0nm;

[0087] The wavelength of the peak of Gardenia glycoside is 240.2nm;

[0088] The wavelength of the peak of paeoniflorin: 232.3nm;

[0089] The wavelengths at the peaks of baicalin are: 277.5nm, 316.3nm;

[0090] The wavelengths of the peaks of palmatine hydrochloride are: 226.2nm, 273.9nm, and 345.5nm;

[0091] The wavelengths at the peaks of berberine hydrochloride are: 229.2nm, 264.7nm, and 347.9nm;

[0092] Wavelengths at the peaks of chrysophanol: 225.2nm, 257.9nm, 288.0nm;

[0093] The wavelengths of the peaks of rheum officinale are: 222.5nm, 288.6nm;

[0094] Wavelengths at the peaks of rhein: 231.0nm, 258.5nm;

[0095] The peak wavelengths of physcion are: 223.1nm, 266.5nm, and 286.1nm.

[0096] Multiple compounds were qualitatively identified by chromatographic retention time and spectral characteristics.

[0097] like Figure 2-6 As shown, the chromatograms of the mixed control stock solution obtained under PDA channels with different wavelengths indicate that: at 278nm and 289nm, gardenia glycoside and paeoniflorin have almost no response; at 257nm, there is no response to paeoniflorin; at 238nm and 232nm, all substances have response values; and the response of rhein at 238nm is much lower than that at 232nm. Figure 7-11 The chromatograms of the 0037 sample solution obtained under the PDA channels with different wavelengths are shown in Table 5. By comparing the chromatograms, the response of each compound at 232nm is relatively better, which is the optimized wavelength. Therefore, the chromatogram data of the 0037 sample solution obtained by the 232nm channel is used to calculate the content of the active ingredient, and the results are shown in Table 5.

[0098] Table 5 Chromatographic data of 0037 sample solution at 232nm channel and calculation results of active ingredient content

[0099]

[0100]

[0101] Nine compounds were detected in sample 0037, among which baicalin had the highest content of 10.8mg / ml, followed by gallic acid 3.1mg / ml, gardenia glycoside 2.8mg / ml and paeoniflorin 267.3μg / ml, rhein 158.862μg / ml, rhein 82.33μg / ml, berberine hydrochloride 78.326μg / ml, bamipine hydrochloride 27.788μg / ml, chrysophanol 18.612μg / ml, and physcion methyl ether was not detected. In addition, benzoic acid, a preservative, was detected in the sample, but it was not quantified. The preservative was well separated from other compounds, meeting the requirements.

[0102] Example 2

[0103] Testing of 1751 samples

[0104] The difference from Example 1 is that the test sample is replaced by sample 1751 from sample 0037.

[0105] The chromatogram of the 1751 sample solution at 232nm, 238nm, 257nm, 278nm, and 289nm channels is as follows: Figure 12-16 The data are shown in Table 6. Similar to Example 1, at 278nm and 289nm, gardenia glycoside and paeoniflorin almost have no response; at 257nm, paeoniflorin has no response; at 238nm and 232nm, all substances have response values; the response of rhein at 238nm is much lower than that at 232nm. By comparing the chromatograms, the response of each compound at 232nm is relatively better, which is the optimized wavelength.

[0106] Table 6 Chromatographic data of 1751 sample solution

[0107]

[0108]

[0109] Therefore, the chromatographic data of 1571 sample solutions obtained by the 232nm channel were used to calculate the content of the effective ingredients, and the results are shown in Table 7. Among the 1751 samples, 7 compounds were detected, among which baicalin had the highest content of 2.2mg / ml, followed by gallic acid 468.276μg / ml, gardenia glycoside 279.765μg / ml and paeoniflorin 336.440μg / ml, and rhein, berberine hydrochloride, and palmatine hydrochloride were lower in the range of 30-50μg / ml. Rhein, chrysophanol and rhein methyl ether were not detected. Benzoic acid, a preservative in the sample, was detected but not quantified. The preservative was well separated from other compounds, meeting the requirements.

[0110] Table 7 Chromatographic data of 1571 sample solution at 232nm channel and calculation results of active ingredient content

[0111]

[0112]

[0113] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may be modified and varied in various ways. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention, such as using different brands of liquid phases, different brands of chromatographic columns, different mobile phase compositions and elution methods, shall be included in the protection scope of the present invention.

Claims

1. A method for simultaneously detecting multiple medicinal components in Yuhuang oral liquid using UPLC-PDA, characterized in that: The following steps are involved: A. Take berberine hydrochloride, palmatine hydrochloride, baicalin, gardenia glycoside, gallic acid, paeoniflorin, rhein, rhein, physophanol methyl ether, and chrysophanol reference substances, dissolve them in solvent, prepare reference stock solutions respectively, mix the reference stock solutions, and obtain a mixed reference stock solution; In step A, the solvent is one or more of anhydrous ethanol, methanol, and chloroform; B. Take the test sample of Yuhuang oral liquid, dissolve it with a solvent, perform ultrasonic extraction, centrifuge, dilute, and filter to obtain the sample solution to be tested; In step B, the solvent is one or more of methanol, anhydrous ethanol, 50% methanol aqueous solution, 50% ethanol aqueous solution, and a mixed solution of methanol: 0.05% phosphoric acid = 50:50; C. Inject the mixed control stock solution into the UPLC-PDA chromatograph, perform gradient elution and detection, and qualitatively determine the quality by retention time. Draw a standard curve based on the corresponding relationship between the peak area measured by the injection volume and the injection volume. The mobile phase comprises mobile phase A and mobile phase B, mobile phase A is a phosphoric acid acetonitrile solution with a volume concentration of 0.05%, and mobile phase B is a phosphoric acid aqueous solution with a volume concentration of 0.05%; The gradient elution includes the following stages: 0 min, 8% mobile phase A, 92% mobile phase B; 0.5 min, 8% mobile phase A, 92% mobile phase B; 3 min, 20% mobile phase A, 80% mobile phase B; 4.5 min, 25% mobile phase A, 75% mobile phase B; 5.5 min, 28% mobile phase A, 72% mobile phase B; 8.5 min, 28% mobile phase A, 72% mobile phase B; 10 min, 50% mobile phase A, 50% mobile phase B; 11 min, 50% mobile phase A, 50% mobile phase B; 11.5 min, 70% mobile phase A, 30% mobile phase B; 14 min, 70% mobile phase A, 30% mobile phase B; 14.5 min, 8% mobile phase A, 92% mobile phase B; 15 min, 8% mobile phase A, 92% mobile phase B; 3D scanning using a wavelength of 190-400 nm, with a detection wavelength of 232 nm or 238 nm; The chromatographic column was a Waters ACQUITY UPLC HSS T3 column; D. Inject the sample solution to be tested into the UPLC-PDA chromatograph, perform gradient elution and detection under the same conditions as step C, measure the peak area of ​​each target, qualitatively determine the retention time, quantitatively determine the standard curve prepared in step C, and calculate the content of the active ingredient in the Yuhuang oral liquid test sample; Multiple compounds were qualitatively identified by chromatographic retention time and spectral characteristics.

2. The method for simultaneously detecting multiple medicinal components in Yuhuang oral liquid by using UPLC-PDA as claimed in claim 1, characterized in that: In step B, the ultrasonic extraction time is 15-30 min, the centrifugal speed is 10000 r / min, and the centrifugal time is 5-20 min.

3. The method for simultaneously detecting multiple medicinal components in Yuhuang oral liquid by using UPLC-PDA as claimed in claim 1, characterized in that: In step C, the injection volume is 0.5-5 μL, the mobile phase flow rate is 0.34-0.36 mL / min, and the column temperature is 39-41 °C.

4. The method for simultaneously detecting multiple medicinal components in Yuhuang oral liquid by UPLC-PDA according to claim 1, characterized in that: In step B, 8% mobile phase A and 92% mobile phase B were used as diluents and mixed with the supernatant obtained by centrifugation in a 1:1 ratio.

5. The method for simultaneously detecting multiple medicinal components in Yuhuang oral liquid by using UPLC-PDA according to any one of claims 1 to 4, characterized in that: The chromatogram and spectrum can be used to simultaneously detect the contents of berberine hydrochloride, bamipine hydrochloride, baicalin, gardenia glycoside, gallic acid, paeoniflorin, rhein, rhein, rhein methyl ether and chrysophanol in the Yuhuang oral liquid test sample, and then the contents of coptis chinensis, phellodendron, scutellaria baicalensis, gardenia jasminoides, terminalia chebula, white peony root and rhubarb in the Yuhuang oral liquid test sample can be simultaneously detected.

Images