CN107202836B - A rapid analysis method for theanine content in fresh tea samples - Google Patents

Abstract

A method for rapidly analyzing the content of theanine in a fresh tea sample is characterized by comprising the following steps: crushing and uniformly mixing fresh tea leaves, extracting for 20min at 90 ℃ by using ultrapure water as an extraction solvent, performing centrifugal filtration, performing pre-column derivatization by using 6-aminoquinoline-N-hydroxysuccinimide carbamate as a derivatization agent, performing gradient elution by using an Xbridge C18 chromatographic column, and performing separation and detection by using a high performance liquid chromatography-fluorescence detector. The method has the advantages of rapid and simple sample preparation, low cost, good precision, accuracy, stability and linear relation, rapid and sensitive whole analysis process and good reproducibility, and is suitable for rapid analysis of theanine in fresh tea samples.

Description

A rapid analysis method for theanine content in fresh tea samples

technical field

The invention relates to the field of analytical chemistry, in particular to a method for rapid analysis of theanine content in fresh tea samples.

Background technique

Tea is one of the three major natural beverages in the world. It has unique aroma, rich nutrition and health benefits, and is deeply loved by consumers. Tea is rich in amino acids, which are important components that make up the taste of tea and directly affect the quality of tea. Among them, theanine is a unique amino acid in tea, accounting for more than 50% of the total amino acid in tea. Theanine is safe and non-toxic, and has various biological functions such as lowering blood pressure, anti-fatigue, and anti-tumor. With the discovery of theanine's physiological function and medical value, theanine is widely used in medical treatment, health care, food, beverages and fine chemicals. and other fields are widely used. The accurate quantitative analysis of theanine is of great significance for the evaluation of tea quality, the research and development of theanine's application and its functional metabolism.

At present, the dry samples are more and more mature in the research on theanine in tea. As stipulated in the national standard GB/T8303-2013, the tea samples need to be ground and heated in an electric heating constant temperature drying box to remove moisture to constant weight. And then used for the determination of amino acid content. Since the dry sample is usually made by high temperature fixing, drying and crushing, the protein is denatured during the high temperature process, and the biological activity of its protease is lost, which is easy to cause protein degradation and lead to an increase in amino acid content. At the same time, drying and other steps may cause various biochemical components of tea. Therefore, the measured data cannot reflect the true level of amino acids in tea leaves, resulting in detection errors.

The analysis methods of theanine in tea mainly include ninhydrin colorimetry, gas chromatography and mass spectrometry, capillary electrophoresis, and high performance liquid chromatography. The classical analytical method generally adopts an amino acid analyzer and uses ninhydrin as a derivatizing reagent for post-column derivatization determination. However, the amino acid analyzer is expensive, has a long analysis time and is highly specific, and can only be used to analyze free amino acids such as theanine, which limits its wide application. Compared with several other methods, pre-column derivatization-high performance liquid chromatography does not require special reaction equipment, and has the advantages of high instrument penetration, short analysis time, flexible and diverse methods, high sensitivity, and easy promotion. It has gradually become the method for theanine detection. conventional means. Chinese invention patent "method for detecting free amino acids in tea by reversed-phase high performance liquid chromatography" (patent number ZL201510109474.4) uses 6-aminoquinoline-N-hydroxysuccinimidyl carbamate as pre-column derivatizing agent , using a special analytical column for amino acids for gradient elution, combined with reversed-phase high performance liquid chromatography, to achieve the quantitative analysis of 19 amino acids such as theanine in tea. However, the invention uses a special chromatographic column for amino acid separation, which requires complex gradient separation, high cost of use, long operation period (about 1 h), and is very time-consuming in the process of analyzing a large number of samples, which is not conducive to the rapid analysis and promotion of theanine. universal. More importantly, the invention does not carry out necessary methodological tests on the accuracy, sensitivity and precision of the method, and the technical effect cannot be effectively guaranteed.

SUMMARY OF THE INVENTION

The invention provides a rapid analysis method for theanine content in fresh tea samples, which aims to solve the problems of slow speed, low efficiency, high cost, and difficulty in basic application promotion in the existing tea theanine analysis methods.

In order to achieve the above object, the technical scheme adopted in the present invention is: a rapid analysis method of theanine content in a fresh tea sample, comprising the following two parts:

The first part is to prepare a solution, and then use the high performance liquid chromatography-fluorescence detection method to establish a standard curve of the measured theanine with a known gradient concentration; the establishment of the standard curve consists of the following steps:

Step (1), prepare the theanine standard, and then prepare 0.14 mol/L acetate buffer, 0.4 mol/L borate buffer, 1 mg/mL AQC derivative solution, and 6 concentrations of tea. Amino acid standard working solution, the concentration of theanine standard working solution is 1 μmol/L, 5 μmol/L, 50 μmol/L, 500 μmol/L, 1000 μmol/L, 1250 μmol/L;

Step (2), derivatizing the theanine standard working solution;

Pipette 6 concentrations of the theanine standard working solution, take 10 μL of each concentration of theanine standard working solution, put it in an automatic sampling bottle, add 70 μL of the borate buffer solution, vortex Mix; respectively take 15 μL of the AQC derivative solution and 5 μL of acetonitrile, add them to the autosampler vial under vortex, mix by vortex, heat at 50~55°C for 8~15min after standing, take out and cool to room temperature for for analysis;

In step (3), the chromatographic peak retention time and chromatographic peak area of theanine in the derivatized theanine standard working solution are determined by high performance liquid chromatography-fluorescence detection method, and the chromatographic peak retention time is used for qualitative determination, and then the all The molar concentration of the theanine standard working solution is the abscissa, and the chromatographic peak area is the ordinate to draw the standard curve;

Among them, the instrument separation conditions are:

Chromatographic column: XBridge C18 column (3.9mm×15cm, 4μm); column temperature 37°C; flow rate 2.0mL/min;

Fluorescence detection: excitation wavelength 250nm, emission wavelength 395nm;

Mobile phase: A is acetate buffer, diluted with ultrapure water at a volume ratio of 1:10; B is 100% acetonitrile; C is 100% ultrapure water; gradient elution program: 0 min, 100% A; 0.5 min, 98%A+2.0B%; 0.5-9.0min, 96.5%A+3.5%B; 9.0-9.5min, 95.0%A+5.0%B; 9.5-11.5min, 91.5%A+8.5%B; 11.5- 13.0min, 83.0%A+17.0%B, hold for 4min; 17.0min, 60.0%B+40%C, hold for 2min; 19-23min, 100%A; injection volume 10μL;

The second part, measuring the theanine content in the first part in the fresh tea sample, including the following steps:

Step (1), sample preparation;

Take a fresh tea sample and mix it evenly, add boiling water to the fresh tea sample, the input ratio of the fresh tea sample to the boiling water is to put 40 mL of boiling water into every 1 g of fresh tea sample, and soak it at 88~92°C. Lift for 18~22min, cool to room temperature, centrifuge at 2500~3500r/min for 4~6min, add water to the supernatant to dilute to 10mL, and pass through a 0.45μm microporous membrane to obtain a fresh tea leaf sample solution;

Step (2), derivatizing the fresh tea leaf sample solution;

Pipette 10 μL of tea fresh leaf sample solution, put it into an automatic sampling bottle, add 70 μL of the borate buffer, and vortex to mix; respectively, take 15 μL of the AQC derivative solution and 5 μL of acetonitrile, and add the automatic In the sample bottle, vortex mixing, let stand and heat at 50~55℃ for 8~15min, take out and cool to room temperature for analysis;

Step (3), according to the chromatographic peak retention time of theanine in the theanine standard working solution, quantify the theanine in the fresh tea leaf sample solution, and use the external standard curve method to calculate the tea in the fresh tea leaf sample solution. The content of amino acid; wherein, the instrument separation conditions for measuring theanine in the fresh tea leaf sample solution are the same as the instrument separation conditions in step (3) of the first part.

The relevant contents in the above technical solutions are explained as follows:

1. In the above scheme, in step (1) of the first part, the preparation method of 0.14 mol/L acetate buffer is as follows: Weigh 19.0g of sodium acetate trihydrate and 1.72g of triethylamine and dissolve it in 1000mL Water, adjust the pH to 5.05 with phosphoric acid, add EDTA, and filter with a 0.45µm filter;

The preparation method of 0.4mol/L borate buffer is as follows: weigh 12.36g of boric acid, add 400mL of water to dissolve, adjust the pH to 8.8 with 400g/L of sodium hydroxide solution, and then add water to dilute to 500mL;

The preparation method of 1 mg/mL AQC derivative solution is as follows: add 1 mL of acetonitrile to 1 mg of AQC powder, mix by vortex, and heat to dissolve at 55°C. AQC refers to 6-aminoquinolinyl-N-hydroxysuccinimidylcarbamate, produced by Waters Company, and acetonitrile is chromatographically pure.

Preparation of theanine standard mother solution: Accurately weigh an appropriate amount of theanine standard product, place it in a 25mL volumetric flask, add ultrapure water to dissolve and dilute to the mark to obtain the theanine standard solution with a concentration of 12.5 μmol/L, Store in -20°C refrigerator. Then the theanine standard mother solution was used to prepare the theanine standard working solution of the above 6 concentrations.

The working principle and beneficial effects of the present invention are as follows: the present invention aims at the problems of slow speed, low efficiency, high cost, and difficulty in popularization existing in the existing theanine analysis technology. An accurate method for the separation and analysis of theanine in fresh tea samples. The fresh tea samples were crushed and mixed, and extracted with ultrapure water at 90 °C for 20 min. After centrifugal filtration, 6-aminoquinoline-N-hydroxysuccinimidyl carbamate was used as the extraction solvent. The derivatizing agent was derivatized before the column, and the gradient elution was carried out using an XBridge C18 chromatographic column, and the separation and detection were carried out with a high performance liquid chromatography-fluorescence detector. The traditional methods use dry tea samples as samples to detect theanine content. This is because fresh tea samples contain more water, pigment, water-soluble ash, tea polyphenols and other interfering substances than dry tea samples, which is easy to cause subsequent separation difficulties. , that is to say, the existing detection method for measuring theanine in dry tea samples is not suitable for the determination of fresh tea samples. In the present invention, fresh tea samples are used as samples instead of dried tea samples. In order to solve the difficult problem of separation, 6-aminoquinoline-N-hydroxysuccinimidyl carbamate is creatively and targetedly selected as the pre-column derivatizing agent, Combined use of XBridge C18 chromatographic column and high performance liquid chromatography-fluorescence detector for separation and analysis, it is found that theanine in fresh tea samples can be separated and analyzed quickly, efficiently and accurately. In particular, the high-efficiency XBridge C18 chromatographic column with a specification of 4 μm is used in combination with the AQC pre-column derivatization method, which can well separate theanine in fresh tea samples while ensuring faster analysis speed. Qualitative and quantitative analysis of theanine.

Compared with the existing tea theanine analysis technology, the present invention has the following beneficial effects:

① A rapid quantitative analysis method for theanine in fresh tea samples was established for the first time. The conversion between various biochemical components of tea may be caused by drying and other steps, and the detection error caused by the change of theanine content, the measured data is more accurate and reliable, and can truly reflect the content of theanine in tea leaves;

②The XBridge C18 chromatographic column was used instead of the special analysis column for amino acids to separate and analyze theanine. Through technical optimization, the entire analysis period was only 14 minutes, which greatly improved the analysis efficiency of theanine and could meet the needs of large-scale sample analysis. progress requirements. At the same time, the XBridge C18 chromatographic column is relatively cheap and not very specific, and can be used for the detection of other substances. On the premise of saving analysis costs, it makes up for the current inability to accurately analyze theanine in tea based on high performance liquid chromatography. It is especially suitable for promotion and use in the majority of grass-roots testing institutions and units.

In a word, the sample preparation of the present invention is fast, simple, low in cost, has good precision, accuracy, stability and linear relationship, and the whole analysis process is fast, sensitive and reproducible, and is suitable for the rapid analysis of theanine in fresh tea samples , provides an effective analytical method for the quality control of theanine in tea, and is suitable for popularization and application.

Description of drawings

Accompanying drawing 1 is the HPLC spectrum of the theanine standard working solution of the present invention.

Detailed ways

Below in conjunction with accompanying drawing and embodiment, the present invention is further described:

Example: A method for rapid analysis of theanine content in fresh tea samples

The rapid analysis method includes the following two parts:

The first part is to prepare a solution, and then use the high performance liquid chromatography-fluorescence detection method to establish a standard curve of the measured theanine with a known gradient concentration; the establishment of the standard curve consists of the following steps:

Step (1), prepare the theanine standard, and then prepare 0.14 mol/L acetate buffer, 0.4 mol/L borate buffer, 1 mg/mL AQC derivative solution, and 6 concentrations of tea. Amino acid standard working solution, the concentration of theanine standard working solution is 1 μmol/L, 5 μmol/L, 50 μmol/L, 500 μmol/L, 1000 μmol/L, 1250 μmol/L;

The preparation method of 0.14 mol/L acetate buffer is as follows: Weigh 19.0 g of sodium acetate trihydrate and 1.72 g of triethylamine and dissolve it in 1000 mL of water, adjust the pH to 5.05 with phosphoric acid, add EDTA, and filter with a 0.45 µm filter membrane. ;

The preparation method of 0.4mol/L borate buffer is as follows: weigh 12.36g of boric acid, add 400mL of water to dissolve, adjust the pH to 8.8 with 400g/L of sodium hydroxide solution, and then add water to dilute to 500mL;

The preparation method of 1 mg/mL AQC derivative solution is as follows: add 1 mL of acetonitrile to 1 mg of AQC powder, mix by vortex, and heat to dissolve at 55°C.

Preparation of theanine standard mother solution: Accurately weigh an appropriate amount of theanine standard product, place it in a 25mL volumetric flask, add ultrapure water to dissolve and dilute to the mark to obtain the theanine standard solution with a concentration of 12.5 μmol/L, Store in -20°C refrigerator. Then the theanine standard mother solution was used to prepare the theanine standard working solution of the above 6 concentrations.

Preparing instruments and equipment: 2695 high performance liquid chromatograph with 2475 fluorescence detector (Waters Company); TG16-WS desktop high-speed centrifuge (Hunan Xiangyi Experimental Instrument Company); K600 pulverizer (German Braun Company); LE-3000 Electrothermal constant temperature water bath (Shanghai Yuejin Medical Equipment Co., Ltd.); Direct-Q 5 UV ultrapure water machine (Millipore, USA).

Step (2), derivatizing the theanine standard working solution;

Pipette 6 concentrations of the theanine standard working solution, take 10 μL of each concentration of theanine standard working solution, put it in an automatic sampling bottle, add 70 μL of the borate buffer solution, vortex Mix; respectively take 15 μL of the AQC derivative solution and 5 μL of acetonitrile, add them to the autosampler vial under vortexing, mix by vortexing for 10-20 s, let stand for 1 min, heat at 55 °C for 10 min, take out and cool to room temperature for for analysis;

In step (3), the chromatographic peak retention time and chromatographic peak area of theanine in the derivatized theanine standard working solution are determined by high performance liquid chromatography-fluorescence detection method, and the chromatographic peak retention time is used for qualitative determination, and then the all The molar concentration of the theanine standard working solution is the abscissa, and the chromatographic peak area is the ordinate to draw the standard curve;

Among them, the instrument separation conditions are:

Chromatographic column: XBridge C18 chromatographic column (3.9mm×15cm, 4μm, Waters company); column temperature 37℃; flow rate 2.0mL/min;

Fluorescence detection: excitation wavelength 250nm, emission wavelength 395nm;

Mobile phase: A is acetate buffer, diluted with ultrapure water at a volume ratio of 1:10; B is 100% acetonitrile; C is 100% ultrapure water; gradient elution program: 0 min, 100% A; 0.5 min, 98%A+2.0B%; 0.5-9.0min, 96.5%A+3.5%B; 9.0-9.5min, 95.0%A+5.0%B; 9.5-11.5min, 91.5%A+8.5%B; 11.5- 13.0min, 83.0%A+17.0%B, hold for 4min; 17.0min, 60.0%B+40%C, hold for 2min; 19-23min, 100%A; injection volume 10μL;

The second part, measuring the theanine content in the first part in the fresh tea sample, including the following steps:

Step (1), sample preparation;

Take a fresh sample of tea leaves and mix them evenly. Weigh 0.25 g, add 10 mL of boiling water, and extract in a water bath at 90°C for 20 minutes. After cooling to room temperature, centrifuge at 3000 r/min for 5 minutes. Add water to the supernatant to dilute to 10 mL. 0.45μm microporous filter membrane was used to obtain fresh tea leaf sample solution;

Step (2), derivatizing the fresh tea leaf sample solution;

Pipette 10 μL of tea fresh leaf sample solution, put it into an automatic sampling bottle, add 70 μL of the borate buffer, and vortex to mix; respectively, take 15 μL of the AQC derivative solution and 5 μL of acetonitrile, and add the automatic In the injection bottle, vortex for 10-20s, let stand for 1min, heat at 55°C for 10min, take out and cool to room temperature for analysis;

Step (3), according to the chromatographic peak retention time of theanine in the theanine standard working solution, quantify the theanine in the fresh tea leaf sample solution, and use the external standard curve method to calculate the tea in the fresh tea leaf sample solution. The content of amino acid; wherein, the instrument separation conditions for measuring theanine in the fresh tea leaf sample solution are the same as the instrument separation conditions in step (3) of the first part.

The test results of this example:

1. Chromatographic separation of theanine standard working solution

As can be seen from Figure 1, the theanine standard working solution is determined after derivatization, the peak time is about 11min, and there is no other interference before and after the peak, indicating that this method can be used for the accurate qualitative and quantitative determination of theanine.

2. The regression equation, correlation coefficient and detection limit of the method

Prepare the theanine standard solution with concentrations of 1, 5, 50, 500, 1000, and 1250 μmol/L, respectively. After derivatization, the samples are injected and measured. The concentration of theanine solution ( X ) is the abscissa, and the corresponding peak area ( Y ) is On the ordinate, draw a standard working curve, and perform linear regression analysis to calculate the correlation coefficient (Table 1).

The results showed that the concentration of theanine had a good linear relationship with its peak area in the range of 5-250 μmol/L, and the correlation coefficient was 0.9991. The detection limit was 0.05 μmol/L calculated by 3 times the signal-to-noise ratio.

Table 1 Linear equation, correlation coefficient and detection limit of theanine

3. Method recovery rate

Accurately weigh 0.25 g of 5 fresh tea leaf samples with known theanine content, add a certain volume of standard solution to them, and the addition level is equivalent to 50 μmol/L. After sample preparation and derivatization, the determination is carried out, and the external standard method is used for quantification. , the average recovery rate of theanine was 87.91, and the RSD was 9.02. It shows that the method has high accuracy, good reproducibility and reliable method.

4. Method precision

An appropriate amount of the standard solution containing theanine was taken, derivatized and analyzed by the above method, and the samples were continuously injected for 5 times. The RSD was calculated with the retention time and peak area of the chromatographic peak as indicators, and its precision was investigated. The RSD of the theanine retention time was 0.49%, and the RSD of the peak area was 1.97%, indicating that the method has high precision.

5. Method repeatability

Take 5 samples of the same fresh tea leaves, extract, derivatize and measure according to the above method, calculate the RSD with the retention time and peak area of chromatographic peaks as indicators, and investigate the repeatability of the method. The RSD of peak retention time of theanine contained in fresh tea leaves was 1.68%, and the RSD of peak area was 2.55, with good repeatability.

6. Method stability

Take the same fresh tea leaf sample for test solution, derivatize it according to the above method, inject samples at 0, 4, 8, 12, 24 h respectively, calculate the RSD with the retention time and peak area of the chromatographic peak as indicators, and investigate the derivatized solution of fresh tea leaves. stability. The RSD of retention time of the theanine derivatized product was 1.50% (n=5), and the RSD of peak area was 2.54% (n=5). It shows that the amino acid derivatization solution can be stable for 24 h at room temperature.

7. Method application

The established method was used to determine the theanine in two fresh samples of Biluochun tea from Dongting East Mountain and Dongting West Mountain in Suzhou, respectively. The results are shown in Table 2.

Table 2 Detection results of theanine in fresh tea samples

The above-mentioned embodiments are only intended to illustrate the technical concept and characteristics of the present invention, and the purpose thereof is to enable those who are familiar with the art to understand the content of the present invention and implement them accordingly, and cannot limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be included within the protection scope of the present invention.

Claims (2)

1. A method for rapidly analyzing the content of theanine in a fresh tea sample is characterized by comprising the following steps: the rapid analysis method comprises the following two parts:

preparing a solution, and establishing a standard curve of the tested theanine with known gradient concentration by using a high performance liquid chromatography-fluorescence detection method; the establishment of the standard curve comprises the following steps:

step (1), preparing a theanine standard substance, and then respectively preparing 0.14 mol/L acetate buffer solution, 0.4mol/L borate buffer solution, 1mg/mL AQC derivative solution and 6 kinds of theanine standard working solutions with concentrations of 1 mu mol/L, 5 mu mol/L, 50 mu mol/L, 500 mu mol/L, 1000 mu mol/L and 1250 mu mol/L; the preparation method of 0.14 mol/L acetate buffer solution comprises the following steps: weighing 19.0g of sodium acetate trihydrate and 1.72g of triethylamine, dissolving the sodium acetate trihydrate and the 1.72g of triethylamine in 1000mL of water, adjusting the pH value to 5.05 by using phosphoric acid, adding EDTA, and filtering by using a 0.45 mu m filter membrane;

step (2), performing derivatization reaction on the theanine standard working solution;

transferring the theanine standard working solutions with 6 concentrations, putting 10 mu L of the theanine standard working solution with each concentration into an automatic sampling bottle, respectively adding 70 mu L of the borate buffer solution, and mixing in a vortex manner; respectively taking 15 mu L of AQC derivative liquid and 5 mu L of acetonitrile, adding the AQC derivative liquid and the acetonitrile into an automatic sample injection bottle in a vortex state, carrying out vortex mixing, standing, heating at 50-55 ℃ for 8-15 min, taking out, and cooling to room temperature for analysis;

step (3), determining the chromatographic peak retention time and the chromatographic peak area of theanine in the derivatized theanine standard working solution by using a high performance liquid chromatography-fluorescence detection method, determining the nature by using the chromatographic peak retention time, and then drawing the standard curve by using the molar concentration of the theanine standard working solution as a horizontal coordinate and using the chromatographic peak area as a vertical coordinate;

wherein, the separation conditions of the instrument are as follows:

a chromatographic column: an Xbridge C18 chromatographic column with the specification of 3.9mm multiplied by 15cm and 4 μm; the column temperature is 37 ℃; the flow rate is 2.0 mL/min;

fluorescence detection: excitation wavelength of 250nm and emission wavelength of 395 nm;

mobile phase: a is acetate buffer solution which is diluted by ultrapure water according to the volume ratio of l to 10; b is 100% acetonitrile; c is 100% ultrapure water; gradient elution procedure: 0min, 100% A; 0.5min, 98% A + 2.0B%; 0.5-9.0min, 96.5% A +3.5% B; 9.0-9.5min, 95.0% A +5.0% B; 9.5-11.5min, 91.5% A + 8.5% B; 11.5-13.0min, 83.0% A +17.0% B, keeping for 4 min; 17.0min, 60.0% B +40% C, keeping for 2 min; 19-23min, 100% A; the sample volume is 10 mu L;

the second part, the content of theanine in the first part in the fresh tea sample is measured, and the method comprises the following steps:

step (1), preparing a sample;

taking a fresh tea sample, crushing and uniformly mixing, adding boiling water into the fresh tea sample, wherein the adding ratio of the fresh tea sample to the boiling water is that 40mL of boiling water is added into each 1g of fresh tea sample, leaching for 18-22 min at 88-92 ℃, cooling to room temperature, centrifuging for 4-6 min at 2500-3500 r/min, adding water into supernatant to a constant volume of 10mL, and filtering through a 0.45-micrometer microporous filter membrane to obtain a fresh tea sample solution;

step (2), carrying out derivatization reaction on the fresh tea leaf sample solution;

transferring 10 mu L of fresh tea leaf sample solution, placing the fresh tea leaf sample solution in an automatic sample feeding bottle, adding 70 mu L of borate buffer solution, and mixing by vortex; respectively taking 15 mu L of AQC derivative liquid and 5 mu L of acetonitrile, adding the AQC derivative liquid and the acetonitrile into an automatic sample injection bottle in a vortex state, carrying out vortex mixing, standing, heating at 50-55 ℃ for 8-15 min, taking out, and cooling to room temperature for analysis;

step (3), determining the quality of the theanine in the fresh tea leaf sample solution according to the retention time of the chromatographic peak of the theanine in the standard working solution of the theanine, and calculating the content of the theanine in the fresh tea leaf sample solution by using an external standard curve method; wherein the separation conditions of the instrument for measuring the theanine in the fresh tea leaf sample solution are the same as those of the instrument in the step (3) of the first part.

2. The method for rapidly analyzing the content of theanine in the fresh tea sample according to claim 1, which is characterized in that: in the first step (1), 0.4mol/L borate buffer solution is prepared by the following method: weighing 12.36g of boric acid, adding 400mL of water for dissolving, adjusting the pH to 8.8 by using 400g/L of sodium hydroxide solution, and then adding water for diluting to 500 mL;

the preparation method of the 1mg/mL AQC derivative solution comprises the following steps: to 1mg of AQC powder, 1mL of acetonitrile was added, mixed by vortexing, and heated to dissolve at 55 ℃.

Images