A method for ultra-rapid and specific detection of active ingredients in tea

Abstract

The present invention discloses a method for ultra-rapid and specific detection of active ingredients in tea leaves. Supercritical extraction is used to obtain an extract from a tea sample, and then the content of the active ingredients in the tea sample is determined by an internal standard method based on a supercritical fluid chromatography-mass spectrometry system. The active ingredients include 17 types of sugars, amino acids, and flavonoids. The supercritical extraction agent is supercritical CO2 and a modifier containing formic acid, ammonium formate, water, and methanol. The present invention can accurately detect 17 active ingredients in tea samples simultaneously, and the total time taken from extraction to analysis of each sample is about 20 minutes. After supercritical fluid extraction, the tea sample can be directly sampled and analyzed without the need for additional purification treatment, which significantly saves analysis time and cost. Moreover, the present invention effectively reduces sample matrix interference, improves detection sensitivity, ensures the reliability of analysis results, and can provide comprehensive and efficient information support for the research and quality control of tea active ingredients.

Description

Technical Field

[0001] The invention belongs to the technical field of food analysis and detection, and particularly relates to a method for ultra-fast and specific detection of active ingredients in tea. Background Art

[0002] As the birthplace of tea culture, China has a long history of cultivated tea trees and tea drinking. Tea is rich in a variety of bioactive ingredients, including sugars, alkaloids, amino acids, and flavonoids. These ingredients not only contribute to tea's unique flavor and quality attributes, but also possess multiple physiological benefits, including antioxidant, anti-inflammatory, and immune-modulating properties. As living standards improve, consumers' demand for healthier diets is growing, prompting a continuous improvement in tea quality standards.

[0003] Currently, tea quality testing primarily relies on sensory evaluation and instrumental analysis. Traditional sensory evaluation relies on subjective judgment, resulting in complex procedures and large errors, making it difficult to meet the demand for rapid and accurate tea quality testing. Methods based on modern analytical instruments have become the mainstream for tea quality analysis, including near-infrared spectroscopy (NIRS), ultraviolet-visible spectrophotometry (UV-Vis), gas chromatography (GC), and high-performance liquid chromatography (HPLC). However, these analytical methods still have certain limitations in tea quality testing.

[0004] NIRS and UV-Vis as rapid, nondestructive detection methods offer the advantage of high throughput, but due to their lack of effective separation capabilities, they typically require integration with other analytical techniques (such as chemometric modeling) to accurately quantify target components. GC, limited by compound polarity and thermal stability, is primarily used for the analysis of volatile components in tea. HPLC, with its superior separation efficiency and broad solvent compatibility, has become the mainstream method for the analysis of multiple active components in tea. However, it still has several limitations: Firstly, tea samples require complex pretreatment procedures such as solid-phase extraction to eliminate matrix interferences before injection; secondly, a single injection cannot simultaneously detect compounds with significantly different polarities (such as sugars and flavonoids). Supercritical fluid chromatography (SFC), an emerging separation technology, uses supercritical fluids such as carbon dioxide as the mobile phase, offering approximately 30-50% improvements in separation speed and resolution compared to HPLC. However, due to mobile phase compatibility and detector limitations, the sensitivity for highly polar components such as amino acids and soluble sugars is poor.

[0005] The above technical bottlenecks indicate that the existing analytical methods still have unmet technical needs in terms of balancing detection throughput, separation efficiency and scope of application. It is urgent to develop new combined technologies or improve existing methods to achieve efficient and accurate analysis of multi-component active substances in the complex matrix of tea. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for ultra-fast and specific detection of active ingredients in tea leaves in response to the deficiencies of the above-mentioned existing technologies. The method can simultaneously determine the content of sugars, amino acids and flavonoids in tea leaves, and has the advantages of high detection sensitivity, strong specificity and short analysis cycle.

[0007] The technical solution adopted by the present invention to solve the above-mentioned problems is:

[0008] A method for ultra-rapid and specific detection of active ingredients in tea leaves comprises the following steps:

[0009] (1) After mixing the tea sample to be tested with the dispersant, the internal standard solution is added dropwise, and then filled into the SFE extraction tank, covered with ash-free filter paper and sealed to obtain the filled tea sample to be tested;

[0010] (2) Quantitatively pipette the standard solution of the target analyte, add the dispersant and internal standard solution, and then fill it into the SFE extraction tank. Cover it with ash-free filter paper and seal it to obtain the filled standard sample;

[0011] (3) The samples loaded in step (1) and step (2) are subjected to supercritical fluid extraction, and the extracts are collected in injection vials; the injection vials containing the extracts are placed in a supercritical fluid chromatography-mass spectrometry system for analysis, and after supercritical fluid chromatography separation, data is collected in the multiple reaction monitoring (MRM) mode of the mass spectrometer to obtain an MRM chromatogram;

[0012] (4) Based on the MRM chromatogram of the standard sample, the standard curve is established with the peak area ratio of the target analyte to the internal standard as the ordinate and the concentration of the standard sample as the abscissa. The content of the target analyte in the tea sample to be tested is then calculated using the internal standard method.

[0013] According to the above scheme, the active ingredients in tea include 17 kinds of sugars, amino acids, and flavonoids; among them, sugars include: maltose, arabinogalactan, mannose, arabinose, fructose, rhamnose, and sucrose; amino acids include: theanine, tryptophan, allothreonine, homoserine, glutamine, and homocitrulline; flavonoids include: myricetin, naringin, naringin, and vitexin.

[0014] According to the above scheme, in step (1), the sample to be tested can be directly loaded into the extraction tank without using a dispersion medium. The present invention preferably uses polyacrylamide powder as the dispersion medium, with an amount of about 1 gram, to absorb moisture, support, and uniformly disperse the tea sample to be tested. The mass ratio of the tea sample to the dispersant is 1:(4-6). The dispersant is preferably polyacrylamide powder with a molecular weight range of 2 million to 10 million.

[0015] According to the above scheme, the internal standard solution is a methanol solution containing dihydrocarbamidoacetate and ligustilide. The concentrations of dihydrocarbamidoacetate and ligustilide are 3~5 μg / mL and 5~7 μg / mL, respectively. The volume of the internal standard solution added to the tea samples to be tested and the standard samples is equal.

[0016] According to the above scheme, in step (2), the standard solution of the target analyte is a stock solution containing multiple target analytes (i.e., the active ingredients in tea to be detected), divided into two groups A and B, which need to be added simultaneously in equal volumes when used; the solvent of group A is pure water, and the solvent of group B is methanol. Group A includes maltose, arabinogalactan, mannose, arabinose, fructose, rhamnose, sucrose, theanine, allothreonine, homoserine, and glutamine; Group B includes myricetin, naringin, tryptophan, galangin, vitexin, and homocitrulline.

[0017] According to the above scheme, the ratio range between the tea sample to be tested, the dispersant and the internal standard solution is preferably as follows: the mass ratio of the tea sample to be tested to the dispersant is 1: (4~6); the mass volume ratio of the tea sample to be tested to the internal standard solution is 200 mg: (90~110) μL.

[0018] According to the above scheme, the ratio range between the target analyte standard solution, dispersant and internal standard solution is preferably as follows: the volume mass ratio of the target analyte standard solution to the dispersant is (90~110) μL:1 g; the volume ratio of the target analyte standard solution to the internal standard solution is preferably 1:1.

[0019] According to the above scheme, dihydrocarbamidoacetate was used as the internal standard when detecting maltose, arabinogalactan, mannose, arabinose, fructose, sucrose, theanine, and tryptophan; ligustilide was used as the internal standard when detecting rhamnose, allothreonine, homoserine, glutamine, homocitrulline, myricetin, naringin, naringin, and vitexin.

[0020] According to the above scheme, the supercritical extraction conditions in step (3) are as follows:

[0021] a. Extractant composition: by volume percentage, comprising 60 to 70% supercritical CO2 and 30 to 40% modifier; wherein the composition of the modifier is shown in Table 1;

[0022] Table 1

[0023]

[0024] b. Extraction flow rate: 4-7 mL / min;

[0025] c. Extraction process (single sample): Static extraction for 2–4 minutes followed by dynamic extraction for 2–4 minutes. Collect 0.9–1.1 mL of extract in a vial. Rinse and equilibration time is 5–9 minutes, for a total run time of 9–17 minutes.

[0026] d. Extraction temperature: 35-40°C;

[0027] e. Back pressure: 10~20 MPa.

[0028] According to the above scheme, the analysis conditions of step (3) are as follows:

[0029] a. Mobile phase A: supercritical CO2; mobile phase B: modifier, the modifier composition is shown in Table 1;

[0030] b. Analytical procedure: Isocratic elution using 65-75% A + 35-25% B by volume, flow rate: 2-4 mL / min, analysis time: 8-10 min;

[0031] c. Chromatographic column: Shim-pack GIS RP-Shield (150 mm × 4.6 mm × 5 μm), or other equivalent columns;

[0032] d. Column temperature: 35-40°C;

[0033] e. Back pressure: 10~20 MPa;

[0034] f. Post-column compensation: 50%-100% water + 0%-50% methanol by volume; flow rate: 0.1-0.2 mL / min;

[0035] g. The mass spectrometry MRM parameters are shown in Table 2. The internal standard was analyzed in positive ion mode, and the target analytes were analyzed in negative ion mode.

[0036] Table 2

[0037]

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] (1) The present invention can simultaneously and accurately detect multiple active substances such as sugars, amino acids and flavonoids in tea samples, and the content indicators of these components can be obtained in one analysis; and the total time taken from extraction to completion of analysis for each sample is about 20 minutes, which significantly saves analysis time and cost, and provides comprehensive and efficient information support for the research and quality control of tea active ingredients.

[0040] (2) The present invention uses a triple quadrupole mass spectrometer detector, which can accurately identify active substances such as sugars, amino acids, and flavonoids in tea. On the one hand, the present invention uses chromatographic retention time and mass spectrometry MRM ion pair information for qualitative identification. After supercritical fluid extraction (SFE), the sample can be directly sampled and analyzed without additional purification. On the other hand, the present invention effectively reduces sample matrix interference, improves detection sensitivity, and ensures the reliability of the analysis results. In addition, the present invention has the ability to detect multiple active substances simultaneously, and multiple concentration indicators can be obtained in one analysis, which significantly saves analysis time and cost, and provides more comprehensive information support for the research and quality control of tea active ingredients.

[0041] (3) The present invention achieves the following improvements by optimizing supercritical fluid extraction and analysis conditions:

[0042] a. The use of specific modifiers significantly improves the extraction and analysis capabilities of supercritical fluids for polar substances.

[0043] b. Adding polyacrylamide as a dispersant guides the extraction solvent to diffuse evenly throughout the extraction tank, thereby improving the extraction efficiency and reproducibility of the target components. At the same time, the dispersant can effectively inhibit water seepage and prevent equipment malfunctions such as blockage.

[0044] c. A high water phase is introduced as a compensation liquid at the back end of the back pressure controller to prevent the loss of polar components after the supercritical fluid is depressurized, thereby significantly enhancing the signal intensity of polar substances such as sugars and amino acids.

[0045] In summary, the present invention takes the active ingredients in tea as the research object and establishes a rapid, accurate and highly sensitive quantitative analysis method, which can provide technical support for the quality inspection and origin identification of tea. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the standard curve of the representative target analytes in Example 1.

[0047] Figure 2 The chromatogram is a chromatogram of the quantitative limit concentrations of 17 active ingredients in the standard sample of Example 1.

[0048] Figure 3 The chromatogram is a chromatogram of the quantitative limit concentrations of the two internal standards in the standard sample of Example 1.

[0049] Figure 4 The chromatogram is of 17 active ingredients of the sample to be tested in Example 1.

[0050] Figure 5 The chromatograms of two internal standards in the sample to be tested in Example 1 are shown.

[0051] Figure 6 The chromatograms obtained by using different modifiers in Comparative Example 1 are shown.

[0052] Figure 7 The chromatograms obtained in Comparative Example 2 using different chromatographic columns are shown.

[0053] Figure 8 This is the total ion map of Comparative Example 3 when the post-column aqueous phase compensation measures were not implemented and when the post-column aqueous phase compensation measures were implemented.

[0054] Figure 9 These are the first extraction total ion map and the second extraction total ion map obtained in Comparative Example 4, respectively, without using a dispersant and with using a dispersant. DETAILED DESCRIPTION

[0055] In order to better understand the present invention, the content of the present invention is further illustrated below in conjunction with examples, but the present invention is not limited to the following examples.

[0056] In the following examples, the dispersant polyacrylamide (molecular weight 5 million) and the ash-free filter paper consumables were purchased from Shimadzu (Shanghai) Laboratory Equipment Co., Ltd.

[0057] In the following examples, the supercritical fluid chromatography-triple quadrupole mass spectrometry system used is model SFE-30A-SFC-30A-LCMS-8060NX, manufactured by Shimadzu, Japan.

[0058] Example 1

[0059] A method for ultra-rapid and specific detection of sugars, amino acids, and flavonoids in tea leaves comprises the following steps:

[0060] (1) Sample loading

[0061] Filling of the sample to be tested: Weigh about 200 mg of Pu'er tea leaves (after crushing) from a certain origin into a weighing boat, add dispersant (1 g), add 100 μL of internal standard solution, mix, fill into the SFE extraction tank, cover with ash-free filter paper and seal.

[0062] Loading of standard samples: Prepare mixed standard solutions of different concentrations according to Table 3 and divide them into two groups, A and B. Prepare one mixed standard solution for the compounds in Group A and another mixed standard solution for the compounds in Group B. The solvent for Group A is pure water, and the solvent for Group B is methanol. Drop 100 μL of the mixed standard solution (Group A or Group B) into the dispersant (1 g), add 100 μL of the internal standard solution, mix, and load into the SFE extraction tank. Cover with ash-free filter paper and seal.

[0063] The gradient concentration intervals of the two mixed standard solutions and the corresponding concentration ranges of the standard curves are detailed in Table 3. Because the standard sample contained 100 μL of the mixed standard solution and the subsequent online extract collection was 1 mL, the linear range of the standard curve was one-tenth of the concentration of the mixed standard solution. The linear range can be adjusted based on the actual sample conditions to ensure that the linear range covers the target detection value.

[0064] Table 3

[0065]

[0066] Preparation of internal standard solution: Dihydroanthenoyl angelate and ligustilide standard powders were diluted with methanol to obtain stock solutions, and internal standard solutions containing 4 μg / mL of dihydroanthenoyl angelate and 6 μg / mL of ligustilide were prepared using methanol.

[0067] (2) Sample extraction

[0068] The SFE extraction tanks filled with the test samples or standard samples in (1) were placed in the SFE equipment for supercritical fluid extraction, and 1 mL of the extract was collected online in the injection vial. The processing time for a single sample was 11.4 minutes.

[0069] The SFE extraction conditions are as follows:

[0070] a. SFE extractant: 65% supercritical CO2 + 35% modifier (methanol solution containing 10% water, 5mM ammonium formate and 0.2% formic acid);

[0071] b. Extraction flow rate: 5 mL / min;

[0072] c. Extraction mode: Static extraction for 3 minutes followed by dynamic extraction for 3 minutes. The extract was collected in a vial. The total SFE analysis time was 11.4 minutes.

[0073] d. Extraction temperature: 36°C;

[0074] e. Back pressure: 10 MPa;

[0075] (3) Sample collection

[0076] The sample injection vials containing the sample to be tested or the standard sample extract in (2) are respectively sent to supercritical fluid chromatography and mass spectrometry (SFC-MS) for analysis. After separation by supercritical fluid chromatography, data is collected in the MRM mode of mass spectrometry to obtain an MRM chromatogram; wherein, the SFC-MS analysis conditions are as follows:

[0077] a. Mobile phase A: supercritical CO2; mobile phase B: modifier (methanol solution containing 10% water, 5 mM ammonium formate, and 0.2% formic acid);

[0078] b. Gradient program: 70% A + 30% B isocratic elution, flow rate: 3 mL / min, single sample analysis time: 9 min;

[0079] c. Chromatographic column: Shim-pack GIS RP-Shield (150 mm × 4.6 mm × 5 μm) (Shimadzu Laboratory Instruments (Shanghai) Co., Ltd., catalog number: 227-30589-06);

[0080] d. Column temperature: 35°C;

[0081] e. Back pressure: 10 MPa;

[0082] f. Post-column compensation: pure water (0.1 mL / min);

[0083] g. Mass spectrometry ion source parameters are shown in Table 4:

[0084] Table 4

[0085]

[0086] h. MRM parameters are shown in Table 5:

[0087] Table 5

[0088]

[0089] (4) Based on the MRM chromatogram of the standard sample, a standard curve is established with the peak area ratio of the target analyte to the internal standard as the ordinate and the concentration of the target analyte in the extract as the abscissa. Then, based on the peak area ratio of the target analyte to the internal standard in the MRM chromatogram of the tea sample to be tested, the content of the target analyte (i.e., active ingredient) in the tea sample to be tested is calculated in combination with the standard curve.

[0090] The standard curves of the 17 target analyses are shown in Table 6. The linear correlation coefficients were all higher than 0.995, indicating a good linear relationship. Figure 1 Figure 4 is the standard curve of three representative target analytes.

[0091] Table 6

[0092]

[0093] Note: In Table 6, x is the concentration of the corresponding target analyte in the extract of the standard sample, and y is the ratio of the peak area of ​​the corresponding target analyte to the peak area of ​​the internal standard.

[0094] like Figure 2 and Figure 3 In the quantitative limit concentration chromatogram shown, the chromatographic peaks of each target analyte and internal standard are symmetrical, and the signal-to-noise ratio is greater than 5, which meets the requirements of quantitative analysis.

[0095] like Figure 4 As shown in the chromatogram of the sample to be tested, the chromatographic peaks of each target analyte are symmetrical, and no interference from matrix impurities is observed, indicating that the detection method has good specificity.

[0096] According to income Figure 4 and Figure 5 , substitute the peak area ratio of the target analyte to the internal standard into the standard curve to obtain the concentration value a of each target compound in the sample extract to be tested. Then, the content information of the target analyte (i.e., the active ingredient to be tested) in the tea leaves is obtained according to the following formula. The calculation results are shown in Table 7:

[0097] (mg / kg)

[0098] Y: target substance content in tea, unit: mg / kg

[0099] a: SFC-MS measured concentration (i.e., concentration in the extract), unit: μg / mL

[0100] b: Extraction volume, where b=1, unit: mL

[0101] c: SFE extraction mass (mg).

[0102] Table 7

[0103]

[0104] Note: “ND” means not detected.

[0105] Example 2

[0106] In order to investigate the accuracy of the method for detecting the content of active ingredients in tea provided by the present invention, a spike recovery experiment was conducted. The specific steps are as follows: a tea sample was selected, a standard sample solution was added, and then the sample was pretreated according to steps (1)-(2) of Example 1. Then, the measurement and calculation were performed according to steps (3)-(4). The measurement was repeated three times, and the average measurement results are shown in Table 8.

[0107] As shown in Table 8, the spiked recoveries of the 17 active ingredients ranged from 83.5% to 109.1%, meeting the spiked recoveries requirement of 80% to 120%, indicating that the method had good accuracy.

[0108] Table 8

[0109]

[0110] Example 3

[0111] To investigate the precision of the method provided by the present invention for detecting the active ingredient content in tea leaves, repeated analysis experiments were conducted. The specific steps were as follows: low-concentration and high-concentration standard sample solutions were used, and sample pretreatment and analysis were performed according to the above steps (1)-(2)-(3)-(4). The determination was repeated five times. The precision RSD results are shown in Table 9 below. As shown in Table 9, the repeatability test RSDs at different spiked concentrations were all <20%, indicating that the method had good precision.

[0112] Table 9

[0113]

[0114] Comparative Example 1

[0115] The composition of the modifier used in the SFE extraction and SFC analysis of the present invention plays a vital role in the accuracy of the test results. If only a single alcohol substance is selected as a modifier, it will make it difficult to effectively extract sugars and amino acids. This comparative example uses D-mannitol, rhamnose, sucrose and DL-homoserine as representative target analytes, and uses the same mixed standard solution to perform measurements according to steps (1), (2) and (3) of Example 1, confirming the important role of the modifier of the present invention. Except for the different modifiers, other operations are the same. Figure 6 As shown, when pure methanol is used as a modifier without the modifier of the present invention, the response of sugars and amino acids is weak and the detection efficiency of the present invention cannot be obtained.

[0116] Comparative Example 2

[0117] In the present invention, the preferred chromatographic column type (hydrophilic modified silica-based ODS column) plays an important role in ensuring the accuracy of the test results. The use of chromatographic columns with other filler specifications (non-hydrophilic modified silica-based ODS column) may cause abnormal chromatographic peaks of some analytes. In this comparative example, D-mannitol, myricetin, sucrose, and DL-homoserine were used as representative target analytes. The same mixed standard solution was used to perform the determination according to steps (1), (2), and (3) of Example 1, and the determination results of different chromatographic columns were compared. Except for the different chromatographic columns, all other operations were the same.

[0118] Test results such as Figure 7As shown, when a UC-Basic column was used instead of the preferred GISRP-Shield column, the sensitivity for detecting sugars and amino acids was low, and the chromatographic peaks of flavonoids exhibited severe tailing. In contrast, the preferred column significantly improved the chromatographic peak shape and effectively increased detection sensitivity, providing a strong guarantee for accurate detection.

[0119] Comparative Example 3

[0120] In the present invention, the post-column aqueous phase compensation method is used to prevent the loss of polar components after the supercritical fluid is depressurized, which plays an important role in improving the detection sensitivity. The same mixed standard solution is used to perform the determination according to steps (1), (2), and (3) of Example 1 to prove the effect of the post-column aqueous phase compensation measure. Apart from this, the other operations are the same. Figure 8 As shown in the figure, without post-column aqueous phase compensation, the overall sensitivity of the target analyte is significantly reduced. In contrast, the aqueous phase compensation scheme adopted by the present invention successfully increased the detection sensitivity by approximately 4 times, which provides a strong guarantee for achieving accurate detection.

[0121] Comparative Example 4

[0122] In the present invention, polyacrylamide powder is used as a dispersant for the sample to guide the extraction solvent to diffuse evenly throughout the extraction tank, thereby significantly improving the extraction efficiency and reproducibility of the target component. At the same time, the dispersant effectively inhibits water seepage and prevents the occurrence of equipment malfunctions such as blockage. This comparative example uses the same mixed standard solution to perform measurements according to steps (1), (2), and (3) of Example 1 to demonstrate the effect of the dispersant. Except for the different dispersants, all other operations are the same.

[0123] like Figure 9 As shown, using ashless filter paper as the dispersion medium results in a low single-extraction recovery of the target analyte, with the maximum ion strength of the secondary extraction being approximately 34% of that of the primary extraction. However, using the dispersant described in this invention significantly improves the single-extraction recovery, with the maximum ion strength of the secondary extraction being only 4% or less of that of the primary extraction. This improvement provides a strong guarantee for accurate detection.

[0124] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this field, several improvements and changes can be made without departing from the creative concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. A method for ultra-rapid and specific detection of active ingredients in tea, characterized in that: Supercritical extraction is used to obtain an extract from a tea sample, and then the content of the active ingredient in the tea sample is determined by an internal standard method based on a supercritical fluid chromatography-mass spectrometry system; The active ingredients include sugars, amino acids, and flavonoids; The extractant used in the supercritical extraction comprises, by volume percentage, 60-70% supercritical CO2 and 30-40% modifier; wherein the modifier is a mixed solvent of methanol and water, with water accounting for 5-15% of the total volume of the mixed solvent, and the solutes are formic acid and ammonium formate, with the volume concentration of formic acid being 0.1%-0.3% and the concentration of ammonium formate being 1-10 mM; The analysis conditions of the supercritical fluid chromatography include: a. Mobile phase A: supercritical CO2; mobile phase B: modifier; the modifier is a mixed solvent of methanol and water, with water accounting for 5-15% of the total volume of the mixed solvent; the solutes are formic acid and ammonium formate, with a formic acid concentration of 0.1%-0.3% and an ammonium formate concentration of 1-10 mM. b. Analytical procedure: Isocratic elution using 65-75% mobile phase A + 35-25% mobile phase B, by volume, flow rate: 2-4 mL / min, analysis time: 8-10 min; c. Chromatographic column: GIS RP-Shield column; Supercritical fluid chromatography sets a post-column compensation solution, which includes 50% to 100% water and 0% to 50% methanol by volume; A dispersant and an internal standard were pre-added to the tea sample for supercritical extraction; the dispersant was polyacrylamide, and the internal standard was dihydrocarbendazim angelate and ligustilide.

2. The method for ultra-rapid specific detection of active ingredients in tea according to claim 1, characterized in that: The active ingredients include 17 kinds, namely maltose, arabinogalactan, mannose, arabinose, fructose, rhamnose, sucrose, theanine, tryptophan, allo-threonine, homoserine, glutamine, homocitrulline, myricetin, naringin, galangin, and vitexin.

3. A method for ultra-rapid specific detection of active ingredients in tea according to any one of claims 1 to 2, characterized in that: The steps include: (1) After mixing the tea sample to be tested with the dispersant, the internal standard solution is added dropwise, and then filled into the SFE extraction tank, covered with ash-free filter paper and sealed to obtain the filled tea sample to be tested; (2) Quantitatively pipette the standard solution of the target analyte, add the dispersant and internal standard solution, and then fill it into the SFE extraction tank. Cover it with ash-free filter paper and seal it to obtain the filled standard sample; (3) The samples loaded in step (1) and step (2) are subjected to supercritical fluid extraction respectively, and the extracts are collected in injection vials; the injection vials containing the extracts are placed in a supercritical fluid chromatography-mass spectrometry system for analysis, and after supercritical fluid chromatography separation, data is collected in the multiple reaction monitoring mode of the mass spectrometry to obtain an MRM chromatogram; (4) Based on the MRM chromatogram of the standard sample, a standard curve is established with the peak area ratio of the target analyte to the internal standard as the ordinate and the concentration of the standard sample as the abscissa. The content of the target analyte in the tea sample to be tested, that is, the content of the active ingredient, is then calculated using the internal standard method.

4. The method for ultra-rapid specific detection of active ingredients in tea according to claim 3, characterized in that: The mass ratio of the tea sample to be tested to the dispersant is 1:(4-6); the dispersant is polyacrylamide powder with a molecular weight of 2 million to 10 million; The internal standard solution is a methanol solution containing dihydrocarbamidoacetate and ligustilide. The concentrations of dihydrocarbamidoacetate and ligustilide are 3-5 μg / mL and 5-7 μg / mL, respectively. The volume of the internal standard solution added to the tea samples to be tested and the standard samples is equal.

5. The method for ultra-rapid specific detection of active ingredients in tea according to claim 3, characterized in that: In step (2), the standard solution of the target analyte is a stock solution containing multiple target analyte standards, which is divided into two groups, A and B, and equal volumes are added simultaneously when used; the solvent of group A is pure water, and the solvent of group B is methanol; wherein group A includes maltose, arabinogalactan, mannose, arabinose, fructose, rhamnose, sucrose, theanine, allothreonine, homoserine, and glutamine; and group B includes myricetin, naringin, tryptophan, galangin, vitexin, and homocitrulline.

6. The method for ultra-rapid and specific detection of active ingredients in tea leaves according to claim 3, characterized in that: The ratio ranges of the tea sample to be tested, the dispersant, and the internal standard solution are as follows: the mass ratio of the tea sample to be tested to the dispersant is 1:(4-6); the mass volume ratio of the tea sample to be tested to the internal standard solution is 200 mg:(90-110) μL; The ratio ranges among the standard solution of the target analyte, dispersant, and internal standard solution were as follows: the volume mass ratio of the standard solution of the target analyte to the dispersant was (90-110) μL:1 g; the volume ratio of the standard solution to the internal standard solution was 1:(0.9-1.1).

7. The method for ultra-rapid and specific detection of active ingredients in tea according to claim 3, characterized in that: The conditions for supercritical extraction in step (3) include: a. Extractant composition: 60-70% supercritical CO2 and 30-40% modifier, by volume; wherein the modifier is a mixed solvent of methanol and water, with water accounting for 5-15% of the total volume of the mixed solvent; the solutes are formic acid and ammonium formate, with the volume concentration of formic acid being 0.1%-0.3% and the concentration of ammonium formate being 1-10 mM; b. Extraction flow rate: 4-7 mL / min; c. Single sample extraction process: static extraction for 2–4 minutes followed by dynamic extraction for 2–4 minutes. Collect 0.9–1.1 mL of extract into a vial. Wash and equilibrate for 5–9 minutes, for a total run time of 9–17 minutes. d. Extraction temperature: 35-40°C; e. Back pressure: 10~20 MPa.

8. The method for ultra-rapid and specific detection of active ingredients in tea leaves according to claim 3, characterized in that: The analysis conditions of supercritical fluid chromatography in step (3) also include: d. Column temperature: 35-40°C; e. Back pressure: 10~20 MPa; f. Post-column compensation: 50%-100% water + 0%-50% methanol by volume; flow rate: 0.1-0.2 mL / min.

9. The method for ultra-rapid and specific detection of active ingredients in tea leaves according to claim 3, characterized in that: During the mass spectrometry analysis in step (3), the internal standard is analyzed in positive ion mode, and the target analyte is analyzed in negative ion mode.

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