A method for rapid detection of 30 compounds in tea

Abstract

The application discloses a method for rapidly detecting 30 compounds in tea, such as catechins, flavonoids, anthocyanins, phenolic acids and amino acids, wherein the 30 compounds are gallic acid, theobromine, GC, protocatechuic acid, theophylline, EGC, theanine, chlorogenic acid, caffeine, catechin, syringic acid, caffeic acid, vanillic acid, epicatechin, EGCG, delphinidin, salicylic acid, GCG, p-coumaric acid, cyanidin, ferulic acid, sinapic acid, rutin, ECG, pelargonidin, myricetin, theaflavin, quercetin, apigenin and kaempferol. The application can realize simultaneous rapid qualitative and quantitative detection of the 30 compounds, and provides technical support for development of active substances in tea.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to a method for rapidly detecting 30 compounds in tea, including catechins, flavonoids, anthocyanins, phenolic acids, and amino acids. Background Technology

[0002] Tea is considered one of the best natural health beverages for humans, and its pure, natural qualities meet modern people's demands for a high quality of life. Current research has found that the main organic compounds in tea include: carbohydrates (approximately 10%-20% cellulose) accounting for 25%-30%, protein for 20%-30%, tea polyphenols for 20%-30%, caffeine for 3%-5%, amino acids for 1%-4%, pigments, vitamins, and organic acids. Among these, tea polyphenols are a mixture of various phenols present in tea, the most important of which are flavanols, primarily catechins, accounting for 70%-80% of the total polyphenols. These are important components of secondary metabolism in tea plants and play a crucial role in the formation of the color, aroma, and flavor of tea. Phenolic acids are aromatic compounds with carboxyl and hydroxyl groups; research on phenolic acids in tea began as early as the mid-19th century. Gallic acid accounts for 0.5%-1.4% of the dry matter in tea. Alkaloids are important metabolic products of tea, mainly including caffeine, theobromine, and theophylline, which are present in the highest concentration in the dry matter of tea leaves. These nutrients have excellent health benefits, such as the antioxidant, cardiovascular protective, and anti-tumor effects of catechins; the stimulating and vasodilatory effects of caffeine; and the strong antioxidant properties of gallic acid, as well as its anti-tumor and anti-thrombotic pharmacological effects.

[0003] Currently, there are numerous reported methods for determining gallic acid, catechins, and caffeine in tea, mainly including thin-layer chromatography (TLC), liquid chromatography-UV detection (LC-UV), liquid chromatography-mass spectrometry (LC-MS), and capillary zone electrophoresis. High-performance liquid chromatography (HPLC) is the most widely used, but methods for simultaneously detecting multiple components are time-consuming. Ultra-high performance liquid chromatography (UPLC), based on small-particle packing materials, low system volume, rapid operation, and ultra-high pressure, has the advantages of high efficiency, high resolution, sharp peaks, low cost, environmental friendliness, and accurate results, and is gradually becoming an effective tool for food analysis. However, it has not yet been widely applied to the separation and detection of tea polyphenols and alkaloids in tea, and methods for simultaneously detecting catechins, phenolic acids, anthocyanins, amino acids, and flavonoids in tea have not been reported. Therefore, establishing a rapid, high-throughput, highly sensitive, and specific UPLC method for determining the content of catechins, phenolic acids, anthocyanins, amino acids, and flavonoids in tea is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a method for rapidly detecting 30 compounds in tea, including catechins, flavonoids, anthocyanins, phenolic acids, and amino acids.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for rapid detection of 30 compounds in tea leaves includes the following steps:

[0007] (1) Preparation of standard stock solution

[0008] Accurately weigh 5.00 mg of each of the 30 reference standards, dissolve them in chromatographic grade methanol, and dilute to 5.00 mL to prepare a 1.00 g / L stock solution for later use;

[0009] (2) Preparation of sample solution

[0010] Take 0.1g of tea leaves, add 3mL of 80vol% methanol solution and extract by sonication for 10min. After standing and separating the layers, collect the supernatant. Take the lower layer and extract it three times with 80vol% methanol solution. Combine the supernatants, add chromatographic grade methanol to make up to 10mL, redissolve by sonication, and filter through a 0.22 μm microporous membrane to obtain the sample solution.

[0011] (3) Plotting the standard curve:

[0012] A certain volume of standard stock solution was taken according to the gradient, diluted with chromatographic grade methanol to prepare a series of standard solutions of different concentrations, filtered through a 0.22 μm microporous membrane, and detected by ultra-high performance liquid chromatography. Then, a standard curve was plotted with the concentration of the standard solution as the abscissa and the peak area of ​​the standard solution as the ordinate.

[0013] (4) Sample testing:

[0014] The sample solution was detected using an ultra-high performance liquid chromatograph, and the content of 30 compounds in the sample was calculated based on the obtained sample peak area and the corresponding standard curve.

[0015] Furthermore, the 30 compounds specifically include gallic acid, theobromine, GC, protocatechuic acid, theophylline, EGC, theanine, chlorogenic acid, caffeine, catechin, syringic acid, caffeic acid, vanillic acid, epicatechin, EGCG, delphinidin, salicylic acid, GCG, p-coumaric acid, cyanidin, ferulic acid, sinapic acid, rutin, ECG, pelargonidin, myricetin, theaflavins, quercetin, apigenin, and kaempferol.

[0016] Furthermore, the ultra-high performance liquid chromatography (UHPLC) conditions were as follows: column: C18, 3 µm, 4.6 × 100 mm; mobile phase: a mixture of 0.01 vol% acetic acid solution and acetonitrile, using gradient elution; detector settings: DAD, wavelength scanning range 210–900 nm; FLD, excitation wavelength scanning range 250–400 nm, emission wavelength scanning range 340–800 nm; other settings: column temperature 30℃, flow rate 0.03 mL / min, injection volume 1.0 μL; qualitative judgment was performed by combining UV absorption spectrum scan, fluorescence emission spectrum scan, and chromatographic peak retention time; detection wavelengths: 280 nm, 330 nm, 530 nm, and quantification was performed using the external standard method.

[0017] Furthermore, the gradient elution procedure is as follows:

[0018]

[0019] The significant advantages of this invention are:

[0020] This invention primarily employs ultra-high performance liquid chromatography (UPLC) to simultaneously determine the content of 30 components in tea, including gallic acid (GA), theobromine, gallocatechin (GC), protocatechinic acid, theophylline, epigallocatechin (EGC), theanine, chlorogenic acid, caffeine, catechin (C), syringic acid, caffeic acid, vanillic acid, epicatechin (EC), epigallocatechin gallate (EGCG), delphinidin, salicylic acid, gallocatechin gallate (GCG), p-coumaric acid, cyanidin, ferulic acid, sinapic acid, rutin, epicatechin gallate (ECG), pelargonidin, myricetin, theaflavins, quercetin, apigenin, and kaempferol. This method is rapid and simple, meeting the need for simultaneous detection of multiple components in tea and providing technical support for the development of active substances in tea. Attached Figure Description

[0021] Figure 1 Liquid chromatograms of components eluted from acetic acid solutions of different concentrations as mobile phase components at a wavelength of 280 nm.

[0022] Figure 2 Liquid chromatograms of mixed standard solutions at different detection wavelengths;

[0023] Figure 3 The liquid chromatograms of tea samples at different detection wavelengths are shown.

[0024] Among them, 1-gallic acid, 2-theobromine, 3-GC, 4-protocatechuic acid, 5-theophylline, 6-EGC, 7-theanine, 8-chlorogenic acid, 9-caffeine, 10-catechin (C), 11-syringic acid, 12-caffeic acid, 13-vanillic acid, 14-epicatechin (EC), 15-EGCG, 16-deltaenoside, 17-salicylic acid, 18-GCG, 19-coumaric acid, 20-cyanidin, 21-ferulic acid, 22-sinapic acid, 23-rutin, 24-ECG, 25-pelargonidin, 26-myricetin, 27-theaflavins, 28-quercetin, 29-apigenin, and 30-kaempferol. Detailed Implementation

[0025] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto. Example

[0026] (1) Preparation of standard stock solution

[0027] Accurately weigh 5.00 mg each of the reference standards for gallic acid, theobromine, GC, protocatechuic acid, theophylline, EGC, theanine, chlorogenic acid, caffeine, catechin, syringic acid, caffeic acid, vanillic acid, epicatechin, EGCG, delphinidin, salicylic acid, GCG, p-coumaric acid, cyanidin, ferulic acid, sinapic acid, rutin, ECG, pelargonidin, myricetin, theaflavins, quercetin, apigenin, and kaempferol. Dissolve each in chromatographic grade methanol and dilute to 5.00 mL to prepare a 1.00 g / L standard stock solution for later use.

[0028] (2) Preparation of sample solution

[0029] Take 0.1g of fresh tea leaves, add 3mL of 80vol% methanol solution and shake to extract for 10min. After standing and separating the layers, collect the supernatant. Take the lower layer and repeat the ultrasonic extraction with 80vol% methanol solution 3 times. Combine the supernatants, add chromatographic grade methanol to the residue after rotary evaporation and make up to 10mL. Soluble and redissolve the residue by ultrasonication. Filter through a 0.22 μm microporous membrane to obtain the sample solution.

[0030] (3) Screening of the mobile phase:

[0031] Take 10 μL of each standard stock solution and dilute to 10 mL with chromatographic grade methanol to prepare standard solutions. Filter the solutions through a 0.22 μm microporous membrane. Use mixed solutions of different acids (acetic acid, formic acid, and phosphoric acid) with acetonitrile as the mobile phase. Detect the solutions using ultra-high performance liquid chromatography (UHPLC) to investigate the separation effect of different mobile phases. The results are shown in Table 1. Furthermore, the effect of different concentrations of the determined acid solutions on the separation effect was investigated, and the results are shown in Table 1. Figure 1 ;

[0032] The ultra-high performance liquid chromatography (UHPLC) conditions were as follows: column: C18, 3 µm, 4.6 × 100 mm; detector settings: DAD, wavelength scan range 210–900 nm; FLD, excitation wavelength scan range 250–400 nm, emission wavelength scan range 340–800 nm; other settings: column temperature 30℃, flow rate 0.03 mL / min, injection volume 1.0 μL; qualitative judgment was performed by combining the UV absorption spectrum scan, fluorescence emission spectrum scan, and chromatographic peak retention time; detection wavelengths: 280 nm, 330 nm, 530 nm, 210 nm; quantification was performed using the external standard method.

[0033] Table 1. Effect of different mobile phase compositions on separation degree

[0034]

[0035] Based on Table 2 Figure 1 It is evident that using 0.01 vol% acetic acid solution-acetonitrile as the mobile phase can achieve good separation results for 30 compounds.

[0036] (4) Plotting the standard curve:

[0037] Take 5 μL, 10 μL, 50 μL, 100 μL, 500 μL, 1000 μL, and 1500 μL of the standard stock solution respectively, and dilute to 10 mL with chromatographic grade methanol to prepare a series of standard solutions of different concentrations. Filter the solutions through a 0.22 μm microporous membrane and detect them using ultra-high performance liquid chromatography (UHPLC). The mobile phase used in UHPLC was a mixture of 0.01 vol% acetic acid solution and acetonitrile. The elution gradient is shown in Table 2, and other parameters are the same as in step 3. Then, a standard curve was plotted with the concentration of the standard solution as the abscissa and the peak area of ​​the standard solution as the ordinate. The results are shown in Table 3.

[0038] Table 2 Mobile phase elution gradient

[0039]

[0040] Table 3. Standard curve regression equations, correlation coefficients, linear ranges, detection limits, precision, and repeatability for 30 substances.

[0041]

[0042]

[0043] (5) Sample testing:

[0044] The sample solution was analyzed using ultra-high performance liquid chromatography (UHPLC). The contents of 30 compounds in the sample were then calculated based on the obtained peak areas and corresponding standard curves. The results are shown in Table 4. The white tea sample used was purchased from Fuding Dingcun Tea Co., Ltd.

[0045] Table 4. Content of characteristic substances (mg / g, DW) in white tea samples harvested in different seasons

[0046]

[0047] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A method for rapid detection of 30 compounds in tea, characterized in that: Includes the following steps: (1) Preparation of standard stock solution Accurately weigh 5.00 mg of each of the 30 reference standards, dissolve them in chromatographic grade methanol, and dilute to 5.00 mL to prepare a 1.00 g / L stock solution for later use; (2) Preparation of sample solution Take 0.1g of tea leaves, add 3mL of 80vol% methanol solution and extract by sonication for 10min. After standing and separating the layers, collect the supernatant. Take the lower layer and extract it three times with 80vol% methanol solution. Combine the supernatants, add chromatographic grade methanol to make up to 10mL, redissolve by sonication, and filter through a 0.22 μm microporous membrane to obtain the sample solution. (3) Plotting the standard curve: A certain volume of standard stock solution was taken according to the gradient, diluted with chromatographic grade methanol to prepare a series of standard solutions of different concentrations, filtered through a 0.22 μm microporous membrane, and detected by ultra-high performance liquid chromatography. Then, a standard curve was plotted with the concentration of the standard solution as the abscissa and the peak area of ​​the standard solution as the ordinate. (4) Sample testing: The sample solution was detected using an ultra-high performance liquid chromatograph, and the contents of 30 compounds in the sample were calculated based on the obtained sample peak area and the corresponding standard curve. The 30 compounds are specifically gallic acid, theobromine, GC, protocatechuic acid, theophylline, EGC, theanine, chlorogenic acid, caffeine, catechin, syringic acid, caffeic acid, vanillic acid, epicatechin, EGCG, delphinidin, salicylic acid, GCG, p-coumaric acid, cyanidin, ferulic acid, sinapic acid, rutin, ECG, pelargonidin, myricetin, theaflavins, quercetin, apigenin, and kaempferol. The ultra-high performance liquid chromatography (UHPLC) conditions used were as follows: column: C18, 3 µm, 4.6 × 100 mm; mobile phase: a mixture of 0.01 vol% acetic acid solution and acetonitrile, using gradient elution; detector settings: DAD, wavelength scan range 210–900 nm; FLD, excitation wavelength scan range 250–400 nm, emission wavelength scan range 340–800 nm; other settings: column temperature 30℃, flow rate 0.03 mL / min, injection volume 1.0 μL; qualitative analysis was performed by combining UV absorption spectrum, fluorescence emission spectrum, and chromatographic peak retention time; detection wavelengths: 280 nm, 330 nm, 530 nm; quantification was performed using the external standard method. The gradient elution program was as follows: 0-12 min, acetonitrile volume increased from 7% to 16%; 12-16 min, acetonitrile volume increased from 16% to 50%; 16-20 min, acetonitrile volume decreased from 50% to 7%.

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