Method for detecting content of effective components in Qingyin tea

Abstract

The invention belongs to the field of medicine detection, and particularly relates to a method for detecting the content of effective components in Qingyin tea. The invention relates to a method for detecting the content of citroobtusin, chlorogenic acid, luteoloside, 3, 5-O-dicaffeoylquinic acid and 4, 5-O-dicaffeoylquinic acid, which comprises the following steps of: measuring the content of citroobtusin, chlorogenic acid, luteoloside, 3, 5-O-dicaffeoylquinic acid and 4, 5-O-dicaffeoylquinic acid by adopting a triple quadrupole multiple reaction monitoring (MRM) quantitative mode through an ultra-high And establishing a standard curve of the five effective components, and calculating the content of the effective components through the standard curve and the sample peak area. The method can effectively separate five effective components, can perform simultaneous quantitative analysis, is quick to operate, good in repeatability and accurate and reliable in result, and can provide a basis for comprehensive quality control of the Qingyin tea.

Description

Technical Field

[0001] The present invention belongs to the field of drug detection, and particularly relates to a method for detecting the content of active ingredients in Qingyin Tea. Background Art

[0002] Qingyin Tea is a special traditional Chinese medicine preparation made by the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine. It contains Sterculia lychnophora, Lonicera japonica, Canarium album, Ophiopogon japonicus with the effects of relieving sore throat and moistening the lungs, Cassia obtusifolia with the effects of clearing heat and improving eyesight, and promoting bowel movement, and green tea or white tea powder with the effects of anti-inflammatory, antibacterial and antioxidant. The prescription components of Qingyin Tea are simple, with excellent effects and good taste. Cassia obtusifolia significantly enhances the effect of clearing the voice and moistening the lungs; all raw materials are health food raw materials that can be used both as food and medicine, with high safety. After stir-frying Cassia obtusifolia and Lonicera japonica, the cold nature is greatly reduced and the medicinal properties are alleviated, making it suitable for most people. After treating Sterculia lychnophora by enzymatic hydrolysis, the extraction of active ingredients is greatly improved, and the effects of relieving sore throat, hoarseness and dry itching of the throat are greatly enhanced.

[0003] The Qingyin Pills in the 2020 Edition of the Chinese Pharmacopoeia (Part I) are composed of eight traditional Chinese medicines, namely Fructus Chebulae, Bulbus Fritillariae Cirrhosae, Myrobalani Pulverata, Pulpa Mume, Pueraria lobata, Poria cocos, Glycyrrhiza uralensis, and Trichosanthis Radix. When determining the content, it is determined by high performance liquid chromatography (General Rule 0512), using octadecylsilane chemically bonded silica gel as the filler; using methanol - 0.5% acetic acid solution (20:80) as the mobile phase; the detection wavelength is 250 nm; only the content of puerarin is measured; Jinsang Qingyin Capsules are composed of sixteen traditional Chinese medicines, namely Scrophularia ningpoensis, Rehmannia glutinosa, Ophiopogon japonicus, Scutellaria baicalensis, Paeonia suffruticosa, Paeonia lactiflora, Bulbus Fritillariae Cirrhosae, Alisma orientale, Coix lacryma-jobi var. ma-yuen, Dendrobium officinale, Bombyx batryticatus, Mentha haplocalyx, Sterculia lychnophora, Cryptotympana pustulata, Inula britannica, and Glycyrrhiza uralensis. When determining the content, it is determined by high performance liquid chromatography (General Rule 0512), using octadecylsilane chemically bonded silica gel as the filler; using acetonitrile - 0.4% phosphoric acid solution (20:80) as the mobile phase; the detection wavelength is 280 nm; the content of puerarin and baicalin is measured; using octadecylsilane chemically bonded silica gel as the filler; using methanol - water (42:58) as the mobile phase; the detection wavelength is 274 nm; the content of paeonol is measured. For a preparation composed of eight or sixteen traditional Chinese medicines, it is difficult to comprehensively control the quality of the preparation by only measuring one or two of its components.

[0004] Due to the complex components of traditional Chinese medicine compounds, it is difficult to quantitatively study the main components that exert the medicinal effects and comprehensively reflect the overall quality of traditional Chinese medicine preparations. Currently, there are few quality detection methods for Qingyin Tea. The present invention uses ultra-high performance liquid chromatography - triple quadrupole mass spectrometry to detect the content of multiple active ingredients in Qingyin Tea at one time, which is of great significance for the component identification, quality evaluation and quality standard formulation of Qingyin Tea. Summary of the Invention

[0005] The object of the present invention is to solve the deficiencies of the prior art and provide a method for detecting the content of active ingredients in Qingyin tea. This detection method can objectively, comprehensively, and accurately evaluate the quality of Qingyin tea, which is of great significance for controlling the quality of Qingyin tea and ensuring clinical efficacy.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A method for detecting the content of active ingredients in Qingyin tea, comprising the following steps:

[0008] Step 1, preparation of the test solution:

[0009] Precisely weigh each traditional Chinese medicine sample of Qingyin tea, add 10 - 15 times the amount of water and decoct for extraction 1 - 3 times, each time for 1 - 3 hours. Combine the decoction solutions, filter, transfer to a volumetric flask, add water to make up the volume, and pass through a 0.22 μm microporous filter membrane to obtain the test solution.

[0010] Preferably, when decocting with water, add 12 times the amount of water for extraction 2 times.

[0011] Preferably, when decocting with water, after each decoction reaches boiling, boil strongly for 30 minutes and then turn to gentle heat for 2 hours.

[0012] Step 2, preparation of the reference solution:

[0013] Precisely take an appropriate amount of the standard stock solution, and make up the volume with 50% methanol - water to obtain a mixed reference solution of aurantio - obtusin, chlorogenic acid, luteoloside, 3,5 - O - dicaffeoylquinic acid, and 4,5 - O - dicaffeoylquinic acid. The above solution is stored at 4°C.

[0014] Preferably, the concentration of aurantio - obtusin is 600 ng / mL; the concentration of chlorogenic acid is 2000 ng / mL; the concentration of luteoloside is 400 ng / mL; the concentration of 3,5 - O - dicaffeoylquinic acid is 600 ng / mL; the concentration of 4,5 - O - dicaffeoylquinic acid is 800 ng / mL.

[0015] Step 3, detection conditions:

[0016] Chromatographic conditions: Waters ACQUITY UPLC BEH C18 column; mobile phase A is 0.1% formic acid - water, mobile phase B is acetonitrile, gradient elution; volume flow rate: 0.1 - 0.5 ml / min; injection volume: 2 - 8 μl; column temperature: 32 - 37°C.

[0017] Preferably, the specification of the chromatographic column is 2.1×100 μm, and the particle size is 1.7 μm.

[0018] Preferably, the volume flow rate is 0.3 ml / min, the injection volume is 5 μl, and the column temperature is 35°C.

[0019] Preferably, the conditions of the gradient elution are as follows:

[0020] Table 1 Gradient elution conditions

[0021]

[0022] Mass spectrometry conditions: electrospray ESI ion source, negative ion scanning mode; mass spectrometry parameters are as follows: ion spray voltage: 2.5 kV, ion source heating temperature: 150°C, desolvation temperature: 450°C, and detection in multiple reaction monitoring mode.

[0023] Table 2 Mass spectrometry parameter conditions

[0024]

[0025] Advantages of the present invention: Experimental results show that the system adaptability of the five active ingredients measured in Qingyin tea is good, and simultaneous quantitative analysis can be performed. The operation is fast, the repeatability is good, and the results are accurate and reliable, which can provide a basis for comprehensive quality control of Qingyin tea. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : MRM mass spectra of five components in the mixed reference solution, including 1. aurantin; 2. chlorogenic acid; 3. luteolin; 4. 3,5-O-dicaffeoylquinic acid; 5. 4,5-O-dicaffeoylquinic acid.

[0027] Figure 2 : MRM mass spectra of the five components in the test solution, including 1. aurantin; 2. chlorogenic acid; 3. luteolin; 4. 3,5-O-dicaffeoylquinic acid; 5. 4,5-O-dicaffeoylquinic acid. DETAILED DESCRIPTION

[0028] Example 1

[0029] A method for detecting the content of five active ingredients in Qingyin tea, the specific steps are as follows:

[0030] Step 1, preparation of test solution:

[0031] Precisely weigh each traditional Chinese medicine sample of Qingyin Tea according to the prescription amount (Cassia obtusifolia 45.47 g, Sterculia lychnophora 9.57 g, Canarium album 7.50 g, Lonicera japonica 22.32 g, Ophiopogon japonicus 18.81 g), place them in a decocting pot, add 12 times the amount of water and decoct for extraction twice. After each decoction reaches boiling, boil vigorously for 30 min and then turn to slow fire for 2 h. Combine the decoction liquids, filter, precisely measure 1 ml of the filtrate into a 50 ml volumetric flask, and pass through a 0.22 μm microporous filter membrane to obtain the test solution.

[0032] Step 2, Preparation of reference solution:

[0033] Precisely weigh the reference standards of aurantio-obtusin, chlorogenic acid, luteoloside, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid respectively, dissolve and make up the volume with 50% methanol aqueous solution, shake well, and a mixed reference solution can be prepared. In the mixed reference solution, the concentrations of aurantio-obtusin, chlorogenic acid, luteoloside, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid are 600 ng / mL, 2000 ng / mL, 400 ng / mL, 600 ng / mL, and 800 ng / mL respectively.

[0034] Step 3, Detection conditions:

[0035] Chromatographic conditions: Waters ACQUITY UPLC BEH C18 column; the specification is 2.1×100 μm, and the particle size is 1.7 μm; mobile phase A is 1‰ formic acid-water, mobile phase B is acetonitrile, gradient elution; volume flow rate: 0.3 ml / min; injection volume: 5 μl; column temperature: 35 °C.

[0036] The conditions of gradient elution are as follows:

[0037] Table 1 Gradient elution conditions

[0038]

[0039] Mass spectrometry conditions: Electrospray ESI ion source, negative ion scanning mode; the parameters of mass spectrometry are as follows: ion spray voltage: 2.5 kV, curtain gas pressure: 15 psi, nebulizing gas pressure: 50 psi, auxiliary heating gas pressure: 50 psi, ion source heating temperature: 150 °C, desolvation gas temperature: 450 °C, and detected by multiple reaction monitoring mode.

[0040] Table 2 Mass spectrometry parameter conditions

[0041]

[0042] Example 2 Methodology investigation

[0043] 1. Drawing of standard curve

[0044] Precisely pipette the mixed reference solution obtained in Step 2 of Example 1, and dilute it by half to obtain a series of standard solutions. Detect according to the conditions in Step 3 of Example 1. Use the peak area (Y) as the ordinate and the reference substance concentration (X, ng / mL) as the abscissa for linear regression to obtain the linear equation of the standard curve for each compound.

[0045] Table 3 Linear equations, correlation coefficients and linear ranges of active ingredients

[0046]

[0047] 2. Precision test

[0048] Take the mixed reference solution and inject it continuously 6 times according to the conditions in Step 3. Calculate the RSD (n = 6) of the peak areas of the quantitative ions of the 5 active ingredients. The final results show that the RSDs of the peak areas of aurantio-obtusin, chlorogenic acid, luteoloside, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid are 0.13%, 0.37%, 0.11%, 0.54%, and 0.26% respectively, indicating that the precision of the instrument is good.

[0049] 3. Repeatability test

[0050] Take samples from the same batch, prepare 6 test solutions according to the method in Step 1 of Example 1, and then detect according to the conditions in Step 3 of Example 1. The experimental results show that the RSDs of the contents of aurantio-obtusin, chlorogenic acid, luteoloside, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid are 1.21%, 0.87%, 1.54%, 1.08%, and 0.99% respectively, indicating that the repeatability of this method is good.

[0051] 4. Stability test

[0052] Take samples from the same batch, prepare test solutions according to the method in Step 1 of Example 1, inject and determine at 2h, 4h, 6h, 8h, 12h, and 24h respectively. The injection volume is 5 μL, and determine according to the conditions in Step 3 of Example 1. The RSDs of the stabilities of aurantio-obtusin, chlorogenic acid, luteoloside, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid are 1.56%, 0.87%, 1.98%, 1.28%, and 1.59% respectively, indicating that the stability of the samples is good.

[0053] 5. Recovery test

[0054] Take samples from the same batch and prepare the test solution according to the method in Step 1 of Example 1. Divide it equally into 6 portions, with each portion being 10 ml placed in a 50-ml volumetric flask. Add 5 ml of the reference substance mixed solution (containing aurantio-obtusin, chlorogenic acid, luteoloside, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid at 600 ng / mL, 2000 ng / mL, 400 ng / mL, 600 ng / mL, and 800 ng / mL respectively), and make up the volume to the mark with 50% methanol aqueous solution and mix well. Determine under the conditions in Step 3 of Example 1, and calculate the recovery rate and RSD according to the following formula. The average recovery rate range of the 5 components is between 99.25% and 101.58%, and the RSD range is between 0.82% and 1.71%, indicating that the recovery rate of the method is good. The specific results are shown in the table.

[0055] Recovery rate = (measured amount (ng) - content in the medicinal material (ng)) / added amount of reference substance (ng) × 100%

[0056] Table 4 Recovery rates of active ingredients

[0057]

[0058] 6. Sample determination

[0059] Take 3 batches of Qingyin Tea, prepare the test solution according to the method in Step 1 of Example 1, and determine under the chromatographic conditions in Step 3 of Example 1. Record the peak areas, and calculate the contents of each test component by the external standard method. The results are shown in Table 5. The deviation of the determination results of different batches of samples is small, indicating good homogeneity of the samples.

[0060] Table 5 Determination results of the contents of 5 active ingredients in three batches of Qingyin Tea

[0061]

[0062]

[0063] The determination of multi-component contents can better reflect the overall quality of traditional Chinese patent medicines. The determination method in the present invention can simultaneously determine the contents of 5 characteristic components such as aurantio-obtusin, chlorogenic acid, luteoloside, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid in Qingyin Tea. The operation is simple, with strong specificity and high resolution. The contents of the 5 test components in the 3 batches of samples are basically the same, indicating that the quality of Qingyin Tea samples is relatively consistent and the process is stable.

Claims

1. A method for detecting the content of active ingredients in Qingyin tea, characterized in that, It includes the following steps: (1) Preparation of the test solution: Weigh each traditional Chinese medicine sample of Qingyin Tea precisely, add 10 - 15 times the amount of water and decoct for extraction 1 - 3 times, each time for 1 - 3 h. Combine the decoction solutions, filter, transfer to a volumetric flask, add water to make up the volume, and pass through a 0.22 μm microporous filter membrane to obtain the test solution; (2) Preparation of the reference solution: Precisely take an appropriate amount of the standard stock solution and make up the volume with 50% methanol - water to obtain a mixed reference solution of aurantio - obtusin, chlorogenic acid, luteoloside, 3,5 - O - dicaffeoylquinic acid, and 4,5 - O - dicaffeoylquinic acid. Store the above solution at 4°C; (3) Detection conditions: Chromatographic conditions: The chromatographic column is a C18 column. The mobile phase A is formic acid - water, and the mobile phase B is acetonitrile. Gradient elution is carried out. The volume flow rate is 0.1 - 0.5 ml / min, the injection volume is 2 - 8 μl, and the column temperature is 32 - 37°C; Mass spectrometry conditions: Electrospray ESI ion source, negative ion scanning mode; Ion spray voltage: 2.5 kV, ion source heating temperature: 150°C, desolvent gas temperature: 450°C. Detection is carried out using the multiple reaction monitoring mode.

2. The method according to claim 1, wherein The chromatographic conditions in step (3) are: The chromatographic column is a Waters ACQUITY UPLC BEH C18 column with a specification of 2.1×100 μm and a particle size of 1.7 μm.

3. The method according to claim 1, wherein The chromatographic conditions in step (3) are: The volume flow rate is 0.3 ml / min, the injection volume is 5 μl; The column temperature is 35°C.

4. The method according to claim 1, characterized in that, The chromatographic conditions in step (3) are: Gradient elution conditions 5. The method according to claim 1, wherein The mass spectrometry conditions in (3) are: Mass spectrometry parameter conditions 6. The method according to claim 1, wherein In the above step (2), the concentration of aurantio - obtusin in the mixed reference solution is 600 ng / mL; the concentration of chlorogenic acid is 2000 ng / mL; the concentration of luteoloside is 400 ng / mL; the concentration of 3,5 - O - dicaffeoylquinic acid is 600 ng / mL; the concentration of 4,5 - O - dicaffeoylquinic acid is 800 ng / mL.

7. The method according to claim 1, wherein The specific steps for the preparation of the test solution in step (1) are: Weigh each traditional Chinese medicine sample of Qingyin Tea precisely according to the prescription amount, place it in a decocting pot, add 12 times the amount of water and decoct for extraction 2 times. After each decoction starts to boil, boil strongly for 30 min and then turn to slow fire for 2 h. Combine the decoction solutions, filter, precisely measure 1 ml of the filtrate into a 50 ml volumetric flask, add water to make up the volume to the scale, and pass through a 0.22 μm microporous filter membrane to obtain the test solution.

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