阅读说明：本技术 一种基于液质联用技术快速精准分析茶鲜叶中茶氨酸的方法 (Method for rapidly and accurately analyzing theanine in fresh tea leaves based on liquid chromatography-mass spectrometry technology ) 是由 张丽 康法 刘腾飞 张季雨 于 2021-09-28 设计创作，主要内容包括：本发明提供了一种基于液质联用技术快速精准分析茶鲜叶中茶氨酸的方法,包括以下步骤：(1)茶鲜叶的前处理；(2)配制茶氨酸标准溶液；(3)获得最佳超高效液相色谱条件和质谱条件；(4)采用超高效液相色谱-质谱法,根据步骤(3)中的色谱/质谱条件对茶鲜叶中的茶氨酸进行定性确证,并进行定量分析。本发明以茶鲜叶代替茶叶干样作为样品,用纯水低温提取后,经一定比例的乙醇水溶液纯化除杂,利用超高效液相色谱-三重四级杆串联质谱检测,实现了快速精准测定茶鲜中茶氨酸成分的目的,为茶叶质量评价和等级评定提供了理想的分析手段。(The invention provides a method for quickly and accurately analyzing theanine in fresh tea leaves based on a liquid chromatography-mass spectrometry technology, which comprises the following steps of: (1) pretreating fresh tea leaves; (2) preparing a theanine standard solution; (3) obtaining the optimal ultra performance liquid chromatography condition and the optimal mass spectrum condition; (4) and (3) qualitatively confirming the theanine in the fresh tea leaves by adopting an ultra-performance liquid chromatography-mass spectrometry method according to the chromatographic/mass spectrometry condition in the step (3), and carrying out quantitative analysis. According to the method, fresh tea leaves are used as a sample to replace a tea dry sample, pure water is used for low-temperature extraction, the sample is purified by a certain proportion of ethanol water solution, and the ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry is used for detection, so that the purpose of rapidly and accurately determining the theanine component in the fresh tea leaves is realized, and an ideal analysis means is provided for tea quality evaluation and grade evaluation.)

1. A method for rapidly and accurately analyzing theanine in fresh tea leaves based on a liquid chromatography-mass spectrometry technology is characterized by comprising the following steps: the method comprises the following steps:

(1) precisely weighing a proper amount of theanine standard substance, placing the standard substance in a 25mL volumetric flask, adding ultrapure water for dissolving, and fixing the volume to scale to obtain the theanine concentration of 1mg/mL, and storing in a refrigerator at 4 ℃;

(2) preparing a theanine standard solution;

(3) obtaining ultra-high performance liquid chromatography conditions:

a chromatographic column: a Watertian velocity UPLC HSS T3 column;

column temperature: 40 ℃;

sample introduction volume: 1.0 μ L;

flow rate: 0.4 mL/min;

the mobile phase A is 2mmol/L ammonium formate solution, and the mobile phase B is methanol;

gradient elution: 0-0.5 min, 2% B; 0.5-6.0 min, 2% -90% B; 6.0-8.0 min, 90% B; 8.0-8.01 min, 2% B; 8.01-13.0 min, 2% B;

mass spectrum conditions:

electrospray ionization, positive ion scanning and multi-reaction monitoring modes;

capillary voltage: 0.5 kV;

ion source temperature: 150 ℃;

desolventizing gas temperature: 600 ℃;

desolventizing agent gas flow: 1000L/h;

collision gas: argon gas;

flow rate: 0.15 mL/min;

(4) pretreating fresh tea leaves;

(5) and (3) qualitatively confirming the tea leaves according to the chromatographic/mass spectrometric conditions in the step (3) by adopting an ultra-high performance liquid chromatography-mass spectrometric method, and carrying out quantitative analysis.

2. The method for rapidly and accurately analyzing theanine in fresh tea leaves based on the liquid chromatography-mass spectrometry technology as claimed in claim 1, wherein the method comprises the following steps: the mass concentrations of the theanine standard solution in the step (2) are respectively 1, 10, 50, 100, 200 and 400 mu g/L.

3. The method for rapidly and accurately analyzing theanine in fresh tea leaves based on the liquid chromatography-mass spectrometry technology as claimed in claim 1, wherein the method comprises the following steps: the chromatographic column in the step (3): 2.1 mm. times.100 mm,1.8 μm.

4. The method for rapidly and accurately analyzing theanine in fresh tea leaves based on the liquid chromatography-mass spectrometry technology as claimed in claim 1, wherein the method comprises the following steps: the mass spectrum parameters in the mass spectrum condition in the step (3) are as follows: parent ion: 175 m/z; and (3) quantifying ions: 84 m/z; and (3) qualitative ion: 158 m/z; taper hole voltage: 10V; collision energy: 20V and 15V.

5. The method for rapidly and accurately analyzing theanine in fresh tea leaves based on the liquid chromatography-mass spectrometry technology as claimed in claim 1, wherein the method comprises the following steps: the pretreatment of the fresh tea leaves in the step (4) comprises the following steps: taking fresh tea leaves, smashing and mixing uniformly, weighing 0.1g, adding 10mL of ultrapure water, vortex and mixing uniformly, extracting at 4 ℃ for 30min, centrifuging at 9000r/min for 2min, taking 4mL of upper layer extracting solution, adding an equivalent 80% ethanol aqueous solution, centrifuging at 4 ℃ at 9000r/min for 10min, taking supernatant, and passing through a 0.45 mu m water-phase filter membrane for detection.

Technical Field

The invention belongs to the field of food detection, relates to a method for detecting chemical components, and particularly relates to a method for quickly and accurately analyzing theanine in fresh tea leaves based on a liquid chromatography-mass spectrometry technology.

Background

The buds or leaves of the young shoots of the tea trees are rich in various amino acid substances, the composition and the content of the amino acid substances are closely related to the quality of the tea leaves, and the amino acid substances are one of important evaluation factors of the quality of the tea leaves. At present, the identifiable tea amino acids are more than 20, wherein theanine accounts for more than 40 percent of the total amount of free amino acids in the tea, is the special amino acid in the tea, has fresh and caramel fragrance similar to monosodium glutamate, can relieve the bitter taste of the tea and enhance the sweet taste of the tea, and is also the main source of the fresh and fresh taste of the tea. Therefore, the analysis of the theanine component in the tea has very important significance for the quality identification of the tea.

The fresh tea leaves are fresh and alive buds or leaves picked from the young shoots of tea trees and are used as raw materials for processing various finished tea products, and the quality of the fresh tea leaves directly influences the quality level of the tea products. At present, the research on the analysis method of amino acid in tea leaves is carried out in a dry sample. According to the regulation of the national standard GB/T8303-2013, a tea sample needs to be ground, then is heated in an electric heating constant-temperature drying oven, and is used for measuring the content of amino acid after water is removed to constant weight. The dry sample is prepared by high-temperature enzyme deactivation, drying and crushing, protein is denatured in a high-temperature process, the biological activity of protease is lost, the protein is easily degraded to increase the content of amino acid, and meanwhile, the steps of drying and the like can cause the conversion of various biochemical components in the tea leaves, so that the actual level of the amino acid in the tea leaves can not be truly reflected by test data, and detection errors are generated.

The analysis method of the amino acid components in the tea leaves is always searched. The ninhydrin colorimetric method is a conventional method for measuring the total amount of free amino acids at present, and is also a detection method adopted in national standard GB/T8314-2013 'determination of total amount of free amino acids in tea', but the method is only suitable for measuring the total amount of free amino acids in tea and cannot detect the content of each free amino acid component in the tea. The amino acid analyzer uses ninhydrin as a derivatization reagent for post-column derivatization determination, and has the problems of equipment price, instrument limitation and the like. The invention patent of China 'a rapid analysis method for theanine content in a fresh tea sample' (patent number ZL201710234515.1) reports a method for rapidly and quantitatively detecting theanine in the fresh tea sample by using 6-aminoquinoline-N-hydroxysuccinimide carbamate as a pre-column derivatization agent and combining a high performance liquid chromatography-fluorescence detector. However, the method needs complex pre-column derivatization, only depends on retention time for qualitative confirmation, and has the problems of complicated steps, high possibility of being interfered by impurities, low derivatization accuracy and detection accuracy and the like.

Disclosure of Invention

The technical problem to be solved is as follows: according to the method, the fresh tea leaves are used as the sample instead of the dry tea sample, and the dry tea sample is used as the sample to detect the theanine component in the traditional method, so that the detection error caused by the conversion among various biochemical components of the tea and the change of the content of theanine due to the steps of high temperature, drying and the like is avoided; after low-temperature extraction with pure water, purifying and removing impurities by using ethanol water solution in a certain proportion, and detecting by using ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry, the aim of quickly and accurately determining the theanine component in fresh tea is fulfilled, and an ideal analysis means is provided for quality evaluation and grade evaluation of tea.

The technical scheme is as follows: a method for rapidly and accurately analyzing theanine in fresh tea leaves based on a liquid chromatography-mass spectrometry technology comprises the following steps:

(1) precisely weighing a proper amount of theanine standard substance, placing the standard substance in a 25mL volumetric flask, adding ultrapure water for dissolving, and fixing the volume to scale to obtain the theanine concentration of 1mg/mL, and storing in a refrigerator at 4 ℃;

(2) preparing a theanine standard solution;

(3) obtaining ultra-high performance liquid chromatography conditions:

a chromatographic column: a Watertian velocity UPLC HSS T3 column;

column temperature: 40 ℃;

sample introduction volume: 1.0 μ L;

flow rate: 0.4 mL/min;

the mobile phase A is 2mmol/L ammonium formate solution, and the mobile phase B is methanol;

gradient elution: 0-0.5 min, 2% B; 0.5-6.0 min, 2% -90% B; 6.0-8.0 min, 90% B; 8.0-8.01 min, 2% B; 8.01-13.0 min, 2% B;

mass spectrum conditions:

electrospray ionization, positive ion scanning and multi-reaction monitoring modes;

capillary voltage: 0.5 kV;

ion source temperature: 150 ℃;

desolventizing gas temperature: 600 ℃;

desolventizing agent gas flow: 1000L/h;

collision gas: argon gas;

flow rate: 0.15 mL/min;

(4) pretreating tea leaves;

(5) and (4) qualitatively screening the tea leaves according to the chromatographic/mass spectrometric conditions in the step (3) by adopting an ultra-high performance liquid chromatography-mass spectrometric method, and carrying out quantitative analysis.

Further, the mass concentrations of the theanine standard solution in the step (2) are respectively 1, 10, 50, 100, 200 and 400 mug/L.

Further, the chromatographic column in the step (3): 2.1 mm. times.100 mm,1.8 μm.

Further, the mass spectrum parameters in the mass spectrum conditions in the step (3) are as follows: parent ion: 175 m/z; and (3) quantifying ions: 84 m/z; and (3) qualitative ion: 158 m/z; taper hole voltage: 10V; collision energy: 20V and 15V.

Further, the pretreatment of the tea leaves in the step (4): taking fresh tea leaves, smashing and mixing uniformly, weighing 0.1g, adding 10mL of ultrapure water, vortex and mixing uniformly, extracting at 4 ℃ for 30min, centrifuging at 9000r/min for 2min, taking 4mL of upper layer extracting solution, adding an equivalent 80% ethanol aqueous solution, centrifuging at 4 ℃ at 9000r/min for 10min, taking supernatant, and passing through a 0.45 mu m water-phase filter membrane for detection.

Has the advantages that:

1. the invention establishes an analysis method for rapidly and accurately analyzing theanine in fresh tea leaves by using a liquid chromatography-mass spectrometry technology for the first time, does not need derivatization treatment on a tea sample, simplifies the experimental steps and is simpler and more convenient to operate; the retention time and the characteristic ion pair are used for qualitative confirmation, so that false positive interference is effectively eliminated, and the detection result is more accurate.

2. According to the method, the fresh tea leaves are used as the samples, the traditional method that the dried tea leaves are used as the samples to detect the theanine components is replaced, and the detection errors caused by the conversion of various generated components of the fresh tea leaves and the change of the theanine content due to the steps of high temperature, drying and the like are avoided.

3. The method creatively utilizes the 80% ethanol water solution to purify the extracting solution, effectively removes the influence of protein impurities on the analysis of the theanine, ensures that the measured data is more accurate and reliable, and truly reflects the content level of the theanine in the fresh tea leaves.

Description of the drawings:

FIG. 1 is a total ion flux chromatogram of theanine.

FIG. 2 is a graph comparing the theanine recovery rates of example 1 and comparative example 1.

Detailed Description

Theanine standards (Sigma, usa); methanol (chromatographically pure, Merck, germany); formic acid, ethanol, ammonium formate (chromatographically pure, Sigma); the experimental water was ultrapure water (18.4 M.OMEGA.).

Precisely weighing a proper amount of theanine standard substance, placing the standard substance in a 25mL volumetric flask, adding ultrapure water for dissolving, and fixing the volume to a scale to obtain the theanine concentration of 1mg/mL, and storing the theanine in a refrigerator at 4 ℃.

Ultra-high performance liquid chromatography conditions:

watertian Acquity UPLC HSS T3 column (2.1 mm. times.100 mm,1.8 μm); column temperature: 40 ℃; sample introduction volume: 1.0 μ L; flow rate: 0.4 mL/min; mobile phase a is 2mmol/L ammonium formate solution, mobile phase B is methanol, gradient elution: 0-0.5 min, 2% B; 0.5-6.0 min, 2% -90% B; 6.0-8.0 min, 90% B; 8.0-8.01 min, 2% B; 8.01-13.0 min, 2% B.

Mass spectrum conditions:

electrospray ionization (ESI), positive ion (ESI)⁺) Scanning and a multi-reaction monitoring mode, wherein the capillary voltage is 0.5kV, the ion source temperature is 150 ℃, the desolvation gas temperature is 600 ℃, the desolvation gas flow is 1000L/h, the collision gas is argon, and the flow rate is 0.15 mL/min. The mass spectrum parameters of theanine are shown in table 1.

TABLE 1 analysis of spectral parameters of substances

The total ion current chromatogram of theanine is shown in FIG. 1.

1. Regression equation, correlation coefficient and detection limit of method

Preparing a theanine standard solution with the mass concentration of 1, 10, 50, 100, 200 and 400 mu g/L respectively, carrying out sample injection determination, drawing a standard working curve by taking the concentration (X) of the amino acid solution as a horizontal coordinate and the corresponding peak area (Y) as a vertical coordinate, carrying out linear regression analysis, and calculating a correlation coefficient. The result shows that the linear relation between the mass concentration of theanine and the peak area thereof is good within the range of 1-400 mu g/L, and the correlation coefficient is 0.9998. The detection limit of the method was calculated at 3 times the signal to noise ratio and the results are shown in table 2.

TABLE 2 Linear equation, correlation coefficient and detection limit

2. Method accuracy

The recovery of the process was determined by standard addition methods. Precisely weighing 5 parts of fresh tea leaf sample with 0.2g of known theanine content, adding a certain volume of theanine standard solution, measuring after sample preparation, quantifying by adopting an external standard method, and calculating the recovery rate and Relative Standard Deviation (RSD) of theanine, wherein the result shows that under two standard adding levels of 50 and 200 mu g/L, the average recovery rate of theanine is between 89.2 and 95.3 percent, and the relative standard deviation is between 6.84 and 9.21 percent, so that the method has higher accuracy.

TABLE 3 recovery of fresh tea leaves with standard addition

3. Precision of the method

Taking a proper amount of theanine standard solution, carrying out sample injection analysis according to the method, carrying out sample injection continuously for 5 times, calculating RSD by taking retention time and peak area of chromatographic peak as indexes, and inspecting precision. The retention time RSD of theanine is 0.1-0.55%, the peak area RSD is 2.02-3.21%, and the method is proved to have higher precision.

4. Method repeatability

5 parts of the same fresh tea leaf sample is taken, extracted and measured according to the method, the RSD is respectively calculated by taking the retention time and the peak area of the amino acid with the peak area of the chromatographic peak exceeding 1 percent as indexes, and the repeatability of the method is investigated. The retention time RSD of theanine in the fresh tea leaves is between 0.90 and 2.38 percent, the peak area RSD is between 3.28 and 4.88 percent, and the repeatability is good.

Example 1:

fresh tea leaf sample is obtained from Dongting mountain of Suzhou

Sample preparation

Taking fresh tea leaves, smashing and mixing uniformly, weighing 0.1g, adding 10mL of ultrapure water, vortex, mixing uniformly, extracting at 4 ℃ for 30min, centrifuging at 9000r/min for 2min, taking 4mL of upper layer extracting solution, adding an equivalent 80% ethanol water (volume ratio) solution, centrifuging at 4 ℃ at 9000r/min for 10min, taking supernatant, filtering with a 0.45 mu m water-phase filter membrane, and testing.

The results of the measurements carried out by the established method are shown in Table 4.

TABLE 4 determination of free amino acids in fresh leaves of Suzhou Dongtong tea

Example 2:

fresh tea leaf sample is obtained from West mountain of Dongting, Suzhou

Sample preparation

Taking fresh tea leaves, smashing and mixing uniformly, weighing 0.1g, adding 10mL of ultrapure water, vortex, mixing uniformly, extracting at 4 ℃ for 30min, centrifuging at 9000r/min for 2min, taking 4mL of upper layer extracting solution, adding an equivalent 80% ethanol water (volume ratio) solution, centrifuging at 4 ℃ at 9000r/min for 10min, taking supernatant, filtering with a 0.45 mu m water-phase filter membrane, and testing.

The results of the measurements carried out by the established method are shown in Table 5.

TABLE 5 determination of free amino acids in fresh tea leaves

Comparative example 1:

fresh tea leaf sample is obtained from Dongting mountain of Suzhou

Sample preparation

Taking fresh tea leaves, smashing and mixing uniformly, weighing 0.1g, adding 10mL of ultrapure water, vortex and mixing uniformly, extracting at 4 ℃ for 30min, centrifuging at 9000r/min for 2min, taking 4mL of upper layer extract, centrifuging at 9000r/min for 10min at 4 ℃, taking supernatant, filtering with a 0.45 mu m water-phase filter membrane, and testing.

The test recovery rate is shown in fig. 2, which is measured by the established method.