A method for simultaneously determining the contents of 16 tea components in instant black tea

Abstract

The invention discloses a method for simultaneously determining the contents of 16 kinds of tea components in instant black tea, mainly including the contents of catechins, purine bases, theaflavins and polyester catechins, and the specific components are: gallic acid, two alkaloids including caffeine and theobromine, polyester catechin A, eight kinds of catechins including gallic acid catechin, epigallic acid catechin gallate, epigallic acid catechin, epicatechin gallate, epicatechin, gallic acid catechin gallate, catechin gallate and catechin and four kinds of theaflavins monomers including theaflavins, theaflavins monogallate, theaflavins-3'-G gallate and theaflavins-3-3'-G digallate. The method utilizes high performance liquid chromatography, has good separation effect of each component, good precision, repeatability and stability, and provides a basis for the quality evaluation of instant black tea.

Description

Technical Field

[0001] The invention belongs to the technical field of multi-component detection of teas, and specifically relates to a method for simultaneously determining the contents of 16 components, including catechin components, purine base components, theaflavins components and polyester catechin components, in instant black tea. Background Art

[0002] Tea contains two major components, polyphenols and purine bases, accounting for 22.07% to 38.07% of the dry weight of tea. Tea polyphenols are a kind of polyphenol mixture, the main component of which is catechins, which can be used as antioxidants and scavengers of free radicals, and are widely used in many fields; clinically proven that catechins have antibacterial and anti-osteoporosis effects, can also reduce the three highs and prevent the occurrence of cardiovascular diseases, etc. Monomers such as epigallocatechin gallate and epigallocatechin gallate are fermented and oxidized to obtain dimer products theaflavins and polyester catechins. Polyester catechins have the effects of reducing toxicity, enhancing immunity, anti-tumor, and anti-cancer; theaflavins are important substances in black tea, and their content directly affects the color and aroma of black tea; purine bases are important flavor substances. The traditional high-performance liquid chromatography method for determining polyphenols and purine base components requires batch determination, which is time-consuming and labor-intensive; currently, the high-performance liquid chromatography method for detecting tea components has only been reported to detect 8 catechins and caffeine, and does not involve the detection of polyester catechin A and theaflavins, and the detection method has limitations. Therefore, a method for simultaneously determining catechin components, theaflavins components, and polyester catechin components in tea samples is developed to improve the traditional method that does not simultaneously detect theobromine, gallic acid, and caffeine components, and improve the existing method that theaflavins-3'-G gallate and theaflavins-3-3'-G digallate cannot be separated. It is of great significance to control the quality of instant black tea, guide the scientific production of instant black tea, the adaptability of various instant black teas, macroscopically control the changes in component content during the production of instant black tea, and guide the correct production process of instant black tea. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a method for simultaneously and rapidly and accurately determining the contents of 16 components, including catechin components, purine base components, theaflavins components and polyester catechin components, in an instant black tea sample by using high performance liquid chromatography. The 16 components are specifically: gallic acid (GA), two alkaloids: caffeine (CAF) and theobromine (TB), polyester-type catechin A (TSA), eight catechins: gallic acid catechin (GC), epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), epicatechin (EC), gallic acid catechin gallate (GCG), catechin gallate (CG) and catechin (C), four theaflavins monomers: theaflavins (TF), theaflavins monogallate (TF-3-G), theaflavins-3'-G gallate (TF-3'-G) and theaflavins-3-3'-G digallate (TF-3-3'-G).

[0004] The technical solution adopted to solve the above technical problems is:

[0005] 1. Prepare a mixed solution of standard substances of each component;

[0006] 2. Analyze the mixed solution of standard products in step 1 by high performance liquid chromatography. Chromatographic detection conditions: C 18 Chromatographic column, mobile phase A is a mixture of acetonitrile and tetrahydrofuran in a volume ratio of 7:1; mobile phase B is a 50mmol / L phosphoric acid aqueous solution; gradient elution program is: 0→39min: 4%~30% A, 96%~70% B; 39→50min: 30% A, 70% B; 50→54min: 30%~75% A, 70%~25% B; 54→55min: 75%~4% A, 26%~96% B; with the peak area value as the ordinate and the standard content as the abscissa, a standard curve of the peak area value of each standard as a inverse of the content is drawn;

[0007] 3. Prepare instant black tea test solution;

[0008] 4. Analyze the test solution using the method in step 2 and determine the contents of the 16 components in the test solution based on the standard curve.

[0009] Among the above determination methods, C 18 The chromatographic column was Cosmosil 5C18-AR-Ⅱ column (4.6mm×250mm, 5μm), the detection column temperature was 35°C, the detection wavelength was 280nm, and the mobile phase flow rate was 0.8mL / min.

[0010] The beneficial effects of the present invention are as follows:

[0011] The present invention uses a mixture of acetonitrile and tetrahydrofuran in a volume ratio of 7:1 and a 50mmol / L phosphoric acid aqueous solution as the mobile phase, and the mobile phase can optimize the peak shape of polyester catechins and theaflavins, so that there are more arched peaks and the substance peaks have no tailing. Therefore, the method of the present invention can quickly and accurately determine the contents of 16 components such as catechin components, purine base components, theaflavins components and polyester catechin components in instant black tea samples, which reduces the time for future tea sample detection and provides a reliable detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is the chromatogram of the instant black tea test solution (injection volume is 10 μL), among which (1) is GA, (2) is TB, (3) is GC, (4) is CAF, (5) is EGC, (6) is C, (7) is EC, (8) is EGCG, (9) is TSA, (10) is GCG, (11) is ECG, (12) is CG, (13) is TF, (14) is TF-3-G, (15) is TF-3'-G, and (16) is TF-3-3'G. DETAILED DESCRIPTION

[0013] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but the protection scope of the present invention is not limited to these embodiments.

[0014] Example 1

[0015] 1. Prepare standard solutions of each component

[0016] Accurately weigh gallic acid (GA), caffeine (CAF), polyester catechin A (TSA), gallic acid catechin (GC), epigallocatechin gallate (EGCG), epigallocatechin gallate (EGC), epicatechin (EC), catechin (C), theaflavin (TF), theaflavin monogallate (TF-3-G), theaflavin-3'-G gallate (TF-3'-G), theaflavin-3-3'-G digallate 0.002g (accurate to 0.0001g) of each (TF-3-3'-G) standard was placed in a 1mL centrifuge tube and diluted to 1mL with pure water; 0.002g of each of epicatechin gallate (ECG), gallocatechin gallate (GCG), and catechin gallate (CG) standards were accurately weighed in a 1mL centrifuge tube and diluted to 1mL with methanol; 0.001g of theobromine (TB) standard was accurately weighed in a 1mL centrifuge tube, dissolved with 50μL 2.0g / mL NaOH aqueous solution, and diluted to 1mL with methanol; the above 16 components of the standard stock solution were obtained. According to the proportion of the 16 components in the instant black tea, each standard stock solution was accurately aspirated, and the standard mixed solution of different contents was prepared by stepwise dilution method, and stored at 4°C for use.

[0017] 2. The standard mixed solution in step 1 was analyzed by high performance liquid chromatography (Agilent 1260 liquid chromatography). The chromatographic conditions were: Cosmosil 5C18-AR-Ⅱ chromatographic column (4.6 mm × 250 mm, 5 μm), mobile phase A was a mixture of acetonitrile and tetrahydrofuran in a volume ratio of 7:1; mobile phase B was a 50 mmol / L phosphoric acid aqueous solution; gradient elution program was: 0→39 min: 4% to 30% A, 96% to 70% B; 39→50 min: 30% A, 70% B; 50→54 min: 30% to 75% A, 70% to 25% B; 54→55 min: 75% to 4% A, 26% to 96% B; the detection column temperature was 35°C, the detection wavelength was 280nm, the mobile phase flow rate was 0.8mL / min, and the injection volume was 10μL; with the peak area value as the ordinate and the standard sample content as the abscissa, a standard curve of the peak area value (Y) of each standard sample versus the concentration (X) was plotted to obtain the linear regression equation shown in Table 1.

[0018] Table 1 Standard curves of 16 components in the standard mixed solution

[0019]

[0020]

[0021] 3. Prepare instant black tea test solution: weigh 0.2 g instant black tea powder and dissolve it in a 50 mL volumetric flask, add 5 mL acetonitrile and water to make up to volume, shake well, sample 5 mL and pass it through a 0.45 μm membrane to obtain the test solution.

[0022] 4. Analyze the test solution using the method in step 2. The results are shown in Figure 1 .Depend on Figure 1 It can be seen that the method of the present invention can effectively separate 16 test components in the instant black tea test sample. The contents of the 16 components in the test sample were determined in combination with the standard curve, and the results are shown in Table 2.

[0023] Table 2 Content of each component in instant black tea test sample

[0024]

[0025]

[0026] In order to prove the feasibility of the method of the present invention, a methodological investigation was carried out, and the specific experimental conditions are as follows:

[0027] (1) Repeatability test

[0028] Five samples of the same instant black tea sample solution were prepared at the same time, and the HPLC was used to detect five times continuously. After the detection, the RSD values ​​of the peak areas of the 16 components were calculated to examine the repeatability of the detection method. As shown in Table 3, the RSD of the contents of the 16 components in the sample was between 1.03% and 2.36%, and the RSD values ​​were all less than 5%. This shows that the method has good repeatability, the determination is repeatable, and it meets the standards of quantitative analysis.

[0029] Table 3 Sample repeatability results

[0030]

[0031] (2) Precision test

[0032] The 16 standard mixed solutions were injected 5 times continuously using high performance liquid chromatography, and then the RSD values ​​of the peak areas of the 16 components after detection were calculated to detect the accuracy of the method. The results in Table 4 show that the RSD values ​​of the 16 components are between 1.44% and 5.00%, of which the RSD values ​​of 15 components are less than 5%, indicating that the method has a high accuracy.

[0033] Table 4 Precision results of 16 standard mixed solutions

[0034]

[0035] (3) Stability test

[0036] Prepare 1 sample of instant black tea, place it in a refrigerator at 4℃ (0h, 4h, 8h, 12h, 24h), analyze it according to the method in step 2, and then calculate the RSD value of the peak area of ​​the 16 components after detection to detect the stability of the method. As shown in Table 5, the RSD values ​​of the contents of the 16 components are between 1.63% and 4.00%, and the RSD values ​​are all less than 5%. The results show that the 16 components of the test sample are stable within 24h.

[0037] Table 5 Sample stability results

[0038]

[0039]

[0040] (4) Spike recovery test

[0041] Prepare instant black tea samples, add reference solution according to 100% of the content of 16 components in instant black tea, repeat the test 3 times according to the method of step 2, and then calculate the spiked recovery and RSD value of 16 substances by standard curve according to the peak area. As can be seen from Table 6, the spiked recovery of each component ranges from 91.3% to 111.0%, the RSD value ranges from 0.37% to 4.03%, and the RSD value is less than 5%, indicating that the spiked recovery of 16 components meets the requirements, and the determination method of the present invention is stable and reliable.

[0042] Table 6 Results of the recovery test of 16 components

[0043]

[0044]

Claims

1. A method for simultaneously determining the contents of 16 tea components in instant black tea, characterized in that: The 16 tea components are gallic acid, caffeine, theobromine, polyester catechin A, gallic acid catechin, epigallic acid catechin gallate, epigallic acid catechin, epicatechin gallate, epicatechin, gallic acid catechin gallate, catechin gallate, catechin, theaflavins, theaflavins monogallate, theaflavins-3'-G gallate, theaflavins-3-3'-G digallate; the determination method is as follows: (1) Prepare mixed solutions of standard substances of each component; (2) Analyze the mixed solution of the standard substance in step (1) using high performance liquid chromatography. The chromatographic detection conditions are: C 18 Chromatographic column, mobile phase A is a mixture of acetonitrile and tetrahydrofuran in a volume ratio of 7:1; mobile phase B is a 50 mmol / L phosphoric acid aqueous solution; gradient elution program is: 0→39 min: 4%-30% A, 96%-70% B; 39→50 min: 30% A, 70% B; 50→54min: 30%-75% A, 70%-25% B; 54→55min: 75%-4% A, 26%-96% B; with the peak area value as the ordinate and the standard content as the abscissa, a standard curve of the peak area value of each standard as a inverse of the content was drawn; (3) Prepare instant black tea test solution: weigh 0.2 g instant black tea powder and dissolve it in a 50 mL volumetric flask, add 5 mL acetonitrile and water to make up to volume, shake well, take a 5 mL sample and pass it through a 0.45 μm membrane to obtain the test solution; (4) Analyze the test solution using the method in step (2) and determine the contents of the 16 components in the test solution based on the standard curve.

2. The method for simultaneously determining the contents of 16 tea components in instant black tea according to claim 1, characterized in that: The C18 chromatographic column is a Cosmosil 5C18-AR-Ⅱ column.

3. The method for simultaneously determining the contents of 16 tea components in instant black tea according to claim 1, characterized in that: The column temperature for chromatographic detection was 35°C.

4. The method for simultaneously determining the contents of 16 tea components in instant black tea according to claim 1, characterized in that: The wavelength of chromatographic detection was 280 nm.

5. The method for simultaneously determining the contents of 16 tea components in instant black tea according to claim 1, characterized in that: The mobile phase flow rate for chromatographic detection was 0.8 mL / min.

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