High performance liquid chromatography determination method for tea catechin
The determination of amino acids and flavonoids in tea by high-performance liquid chromatography solves the problems that are difficult to analyze in existing technologies and achieves accurate assessment of tea quality and health value.
Patent Information
- Application Number
- CN202510773111.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-11
- Publication Date
- 2025-09-23
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Existing physical and chemical analysis methods for tea are difficult to effectively analyze amino acids and flavonoids.
High performance liquid chromatography (HPLC) was used to prepare mixed standard solutions and test solutions, combined with specific chromatographic detection conditions, to determine the amino acids and flavonoids in tea.
It achieves accurate measurement of amino acids and flavonoids in tea, providing a comprehensive assessment of tea quality and health value.
Smart Images
Figure CN120685804A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of tea detection, and in particular to a high performance liquid chromatography method for determining tea catechins. Background Art
[0002] Tea physical and chemical analysis uses the principles of chemistry and physics to examine the chemical composition and physical properties of tea. This method aims to fully understand the quality characteristics of tea and ensure its safety and nutritional value. Common methods for tea physical and chemical analysis include chromatography, spectroscopy, electrochemical analysis, and thermogravimetric analysis.
[0003] There are few existing physical and chemical analyses of tea that include analyses of amino acids and flavonoids. Summary of the Invention
[0004] The present invention hopes to provide a method for determining tea catechins by high performance liquid chromatography, the specific scheme is as follows: A high performance liquid chromatography method for determining tea catechins, including an HPLC method for determining amino acid components and an HPLC method for determining flavonoids; The HPLC determination method of flavonoids is as follows: (1) Preparation of mixed standard solution: Accurately weigh catechin standard, caffeine standard and flavonoid standard, add methanol solution to the volume respectively, and filter with 0.45 μm nylon organic membrane to obtain a single standard solution; take the standard solution into a centrifuge tube and make up the volume with methanol solution. The final mixed standard solution is stored in the refrigerator; (2) Preparation of the test solution: Weigh the sun-dried green tea sample, add methanol solution, and place it in a -4°C refrigerator for 24 hours. After centrifugation, extract the supernatant and filter it with a 0.45 μm nylon organic membrane. This is the test solution; (3) Chromatographic detection: The determination was performed using an Agilent 1200 chromatographic system from the United States; mobile phase A was 0.261% phosphoric acid and 5% acetonitrile aqueous solution; mobile phase B was 80% methanol.
[0005] The HPLC determination method of amino acid components is as follows: (1) Selection of amino acid standards; (2) Extraction of the sample: Accurately weigh the ground tea sample and place it in a conical flask. Add boiling water and extract it in a water bath at 80℃ for 1 hour. Shake it every 10 minutes. After cooling, filter it into a volumetric flask and make up the volume. After shaking, take the tea soup and add chloroform to extract it by shaking. Let it stand and then centrifuge it. The supernatant was extracted and filtered through a 0.45 μm nylon organic membrane and tested on an instrument; (3) Chromatographic detection: The determination was performed using an American Agilent 1200 chromatography system, with a Venusil AA column.
[0006] The elution gradient program in the chromatographic detection in step (3) of the HPLC determination method of flavonoid materials is as follows: 0-16 min, mobile phase B is linearly increased from 10% to 45%; 16-22 min, mobile phase B is linearly increased from 45% to 65%; 22-25.9 min, mobile phase B is linearly increased from 65% to 100%; 25.9-29 min, mobile phase B is kept unchanged at 100%; 29-30 min, mobile phase B is linearly decreased from 100% to 10%, the program is stopped at 30 min, and the column is equilibrated at 10% mobile phase B for 5 min; the flow rate is 1.0 mL / min, the column temperature is 40°C, and the detection wavelength is 280 nm.
[0007] The methanol solution in steps (1) and (2) of the HPLC determination method of flavonoid materials is a 70% methanol solution.
[0008] In step (3) of the amino acid component HPLC determination method, the flow rate is 1 mL / min; the column temperature is 40°C; an automatic sampler is used to perform OPA pre-column online derivatization, 5.0 μL of boric acid buffer solution and 0.5 μL of derivatization reagent are extracted, the needle is washed, 1.0 μL of standard solution or sample is extracted, and the mixture is mixed and then injected, with an injection volume of 10 μL.
[0009] The amino acid standards in step (1) of the amino acid component HPLC determination method include aspartic acid, glutamic acid, serine, histidine, glycine, threonine, arginine, alanine, tyrosine, cysteine, methionine, phenylalanine, isoleucine, leucine, lysine, proline, theanine and γ-aminobutyric acid.
[0010] The present invention provides an HPLC determination method capable of accurately measuring amino acid components and flavonoid materials in tea. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figure 1 This is a graph showing the detection content of flavonoid compounds in four regions; Figure 2 This is a chart showing the free amino acid content detected in four locations. DETAILED DESCRIPTION
[0012] .HPLC determination of amino acid components (1) The free amino acid components of tea leaves were determined by high performance liquid chromatography. The standards included aspartic acid (Asp), glutamic acid (Glu), serine (Ser), histidine (His), glycine (Gly), threonine (Thr), arginine (Arg), alanine (Ala), tyrosine (Tyr), cysteine (Cys), methionine (Met), phenylalanine (Phe), isoleucine (Ile), leucine (Leu), lysine (Lys), and proline (Pro). The concentration of theanine and γ-aminobutyric acid (GABA) standards was purchased from Agilent (1 nmol / μL).
[0013] (2) Extraction of the sample: Accurately weigh 1g of ground tea sample (accurate to 0.0001g) and place it in a 100mL conical flask, add 80mL of boiling water, and extract in an 80℃ water bath for 1h. Shake once every 10min. After cooling, filter and dilute to volume in a 100mL volumetric flask. After shaking, take 1mL of tea soup and add 200μL of chloroform for oscillation extraction. Let it stand for 10min, then centrifuge at 12000r for 10min. Draw out the supernatant and filter it with a 0.45μm nylon organic membrane, and then test it on the machine.
[0014] (3) Chromatographic detection method: The determination was performed using an Agilent 1200 high-speed liquid chromatography system from the United States. The chromatographic column was VenusilAA (4.6*250 mm, 5 μm, Agela, China); the flow rate was 1 mL / min; the column temperature was 40°C; an automatic injector was used for OPA pre-column online derivatization. 5.0 μL of boric acid buffer and 0.5 μL of derivatization reagent were extracted, the needle was washed, and 1.0 μL of standard solution or sample was extracted. The mixture was mixed and injected. The injection volume was 10 μL.
[0015] 2. HPLC determination of flavonoids (1) Preparation of mixed standard solution Accurately weigh 10 mg each of the catechin standard, caffeine standard, and flavonoid standard. Add 70% methanol solution to a volume of 10 mL (concentration: 1 mg / mL). Filter through a 0.45 μm nylon organic membrane to obtain a single standard solution. Transfer 1 mL of the standard solution to a 10 mL centrifuge tube and adjust the volume with methanol to obtain a 0.1 mg / mL mixed standard solution. Store at -80°C.
[0016] (2) Preparation of test solution Weigh 0.1g (accurate to 0.0001g) of sun-dried green tea as a sample, add 10mL of 70% methanol solution, and place it in a -4℃ refrigerator for 24h. After centrifugation at 4000r for 10min, extract the supernatant and filter it with a 0.45μm nylon organic membrane. This is the test solution.
[0017] (3) Chromatographic detection An Agilent 1200 high-speed liquid chromatography system was used for determination. Mobile phase A consisted of 0.261% phosphoric acid and 5% acetonitrile in water; mobile phase B consisted of 80% methanol. The elution gradient program was as follows: 0-16 min, linear gradient increase from 10% to 45% of mobile phase B; 16-22 min, linear gradient increase from 45% to 65% of mobile phase B; 22-25.9 min, linear gradient increase from 65% to 100% of mobile phase B; 25.9-29 min, mobile phase B remained constant at 100%; 29-30 min, linear gradient decrease from 100% to 10% of mobile phase B; the program was stopped at 30 min, and the mobile phase was equilibrated with 10% mobile phase B for 5 min. The flow rate was 1.0 mL / min, the column temperature was 40°C, and the detection wavelength was 280 nm.
[0018] Application examples: We used the above method to test the following regions:
[0019] The village is Figure 1-2 XC in the middle, Chang Liangzi is Figure 1-2 CLZ in Ganlongtan Figure 1-2 GLT and Banpo in Figure 1-2 The BP in was tested.
[0020] Flavonoids in tea are an important component of tea polyphenols, the main source of tea's astringency, and a key factor in its color. They are an important class of natural compounds with strong antioxidant properties that can effectively neutralize free radicals produced by ultraviolet rays and environmental pollutants. The flavonoid content in tea not only affects its health value, but also has a significant impact on the flavor and quality of the tea. Higher flavonoid content is generally associated with better antioxidant capacity and a richer taste. Figure 1 It can be seen that the flavonoid contents of the four tea gardens ranged from 6.9 to 10.36 mg / g, and there were significant differences among the four tea gardens. The flavonoid content was GLT>BP>XC>CLZ, with the highest flavonoid content in GLT being 10.36 mg / g and the lowest flavonoid content in CLZ being 6.9 mg / g.
[0021] The free amino acids in tea are the main source of the umami flavor of tea. Their types and content are important factors affecting the taste of tea soup. The content and ratio of many amino acids in tea directly affect the flavor characteristics of tea and are also key factors in evaluating the quality of tea. Figure 2 It can be seen that the amino acid content of the four tea gardens ranged from 3.09 to 5.63%, with significant differences. Among the four tea gardens, the amino acid content of GLT was significantly higher than that of XC, CLZ and BP.
[0022] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.
Claims
1. A method for determining tea catechins by high performance liquid chromatography, characterized in that: Including HPLC determination method of amino acid components and HPLC determination method of flavonoid materials; The HPLC determination method of flavonoids is as follows: Preparation of mixed standard solution: Accurately weigh catechin standard, caffeine standard, and flavonoid standard, add methanol solution to the volume, and filter through a 0.45μm nylon organic membrane to obtain a single standard solution; take the standard solution into a centrifuge tube and adjust the volume with methanol solution. The final mixed standard solution should be stored in the refrigerator; Preparation of the test solution: Weigh the sun-dried green tea sample, add methanol solution, and place it in a -4°C refrigerator for 24 hours. After centrifugation, extract the supernatant and filter it with a 0.45μm nylon organic membrane. This is the test solution; (3) Chromatographic detection: The determination was performed using an Agilent 1200 chromatographic system; mobile phase A was 0.261% phosphoric acid and 5% acetonitrile in water; mobile phase B was 80% methanol; The HPLC determination method of amino acid components is as follows: Selection of amino acid standards; (2) Extraction of the sample: Accurately weigh the ground tea sample and place it in a conical flask. Add boiling water and extract it in a water bath at 80℃ for 1 hour. Shake it every 10 minutes. After cooling, filter it into a volumetric flask and make up the volume. After shaking, take the tea soup and add chloroform to extract it by shaking. Let it stand and then centrifuge it. The supernatant was extracted and filtered through a 0.45 μm nylon organic membrane and tested on an instrument; (3) Chromatographic detection: The determination was performed using an American Agilent 1200 chromatography system, with a Venusil AA column.
2. The method for determining tea catechins by high performance liquid chromatography according to claim 1, wherein: The elution gradient program in the chromatographic detection in step (3) of the HPLC determination method of flavonoid materials is as follows: 0-16 min, mobile phase B is linearly increased from 10% to 45%; 16-22 min, mobile phase B is linearly increased from 45% to 65%; 22-25.9 min, mobile phase B is linearly increased from 65% to 100%; 25.9-29 min, mobile phase B is kept unchanged at 100%; 29-30 min, mobile phase B is linearly decreased from 100% to 10%, the program is stopped at 30 min, and the column is equilibrated at 10% mobile phase B for 5 min; the flow rate is 1.0 mL / min, the column temperature is 40°C, and the detection wavelength is 280 nm.
3. The method for determining tea catechins by high performance liquid chromatography according to claim 1, wherein: The methanol solution in steps (1) and (2) of the HPLC determination method of flavonoid materials is a 70% methanol solution.
4. The method for determining tea catechins by high performance liquid chromatography according to claim 1, wherein: In step (3) of the amino acid component HPLC determination method, the flow rate is 1 mL / min; the column temperature is 40°C; an automatic sampler is used to perform OPA pre-column online derivatization, 5.0 μL of boric acid buffer solution and 0.5 μL of derivatization reagent are extracted, the needle is washed, 1.0 μL of standard solution or sample is extracted, and the mixture is mixed and then injected, with an injection volume of 10 μL.
5. The method for determining tea catechins by high performance liquid chromatography according to claim 1, wherein: The amino acid standards in step (1) of the amino acid component HPLC determination method include aspartic acid, glutamic acid, serine, histidine, glycine, threonine, arginine, alanine, tyrosine, cysteine, methionine, phenylalanine, isoleucine, leucine, lysine, proline, theanine and γ-aminobutyric acid.