A method for detecting 12 kinds of polyphenol compounds in tea by high performance liquid chromatography

Abstract

The application discloses a synchronous detection method for 12 polyphenol compounds in tea leaves, and relates to the technical field of tea detection. The method is characterized in that: each polyphenol compound single standard stock solution is accurately prepared, gradient dilution and internal standard method are combined for quantitative determination, and a HLB solid phase extraction column is used for purifying the tea sample. The method is detected by high performance liquid chromatography, and the stability of the method is verified through intraday and interday precision experiments. The results show that the 12 polyphenol compounds have good linear relationship, low detection limit and small relative standard deviation of precision. The method is simple in operation, high in sensitivity, good in stability and strong in repeatability, and is suitable for accurate quantitative detection of polyphenol compounds in tea leaves, and provides technical support for tea quality evaluation.

Description

Technical Field

[0001] This invention relates to the field of tea detection technology, and in particular to a method for detecting 12 polyphenolic compounds in tea using high performance liquid chromatography. Background Technology

[0002] Polyphenolic compounds in tea are the core active ingredients that determine its flavor, nutritional value, and functionality. These compounds encompass a variety of substances, including gallic acid, catechins, chlorogenic acid, and quercetin. Their composition and content levels directly correlate with the quality grade, health benefits, and market value of tea. Therefore, establishing comprehensive, accurate, and efficient methods for detecting polyphenolic compounds is not only a crucial step in tea quality control but also an important technical support for deep processing of tea, research and development of functional products, and scientific analysis. This is of great significance for promoting the high-quality development of the tea industry.

[0003] Currently, the detection methods for polyphenolic compounds in tea have formed a variety of technical systems, including the national standard (GB / T 8131-2018). Among them, high-performance liquid chromatography (HPLC) has become the mainstream detection technology in this field due to its advantages such as high separation efficiency and accurate qualitative and quantitative analysis. However, existing HPLC-based detection methods generally adopt a single-wavelength ultraviolet detection mode, which has inherent technical limitations: on the one hand, the absorption coverage of single-wavelength detection is limited, and it can only specifically detect a few polyphenolic compounds. Most methods can detect no more than eight indicators, which is difficult to meet the needs of simultaneous analysis of multiple components and cannot comprehensively reflect the overall compositional characteristics of polyphenolic compounds in tea. On the other hand, the ultraviolet absorption characteristics of different polyphenolic compounds vary significantly, and the selection of a single wavelength is difficult to adapt to the optimal absorption requirements of all target components. This results in low detection sensitivity for trace components such as chlorogenic acid and caffeic acid, making it impossible to accurately determine their true content in tea and easily causing distorted detection results.

[0004] Besides the limitations of the detection mode, existing technologies still have many areas for improvement in sample pretreatment and chromatographic condition optimization. In the sample extraction stage, traditional extraction systems lack sufficient solubility for poorly soluble polyphenols such as quercetin, and polyphenolic compounds are prone to oxidative degradation during extraction, affecting detection accuracy. Furthermore, the vague descriptions of extraction parameters (such as ultrasonic power and extraction liquid volume) lead to poor repeatability of results between different laboratories. In the chromatographic separation stage, some methods suffer from solvent effects due to unreasonable mobile phase composition design, resulting in peak tailing and poor separation. Insufficient optimization of gradient elution programs can also cause peak overlap of structurally similar components (such as EGCG and GCG), further affecting the accuracy of qualitative and quantitative analysis. These problems, combined, make it difficult for existing detection methods to balance detection coverage, sensitivity, and accuracy, failing to meet the current demands of the tea industry for refined detection.

[0005] Therefore, developing a detection method that can simultaneously detect multiple polyphenolic compounds, significantly improve the detection sensitivity of trace components, and possess good repeatability and stability is key to breaking through existing technological bottlenecks and filling technological gaps in the industry. It is of great practical significance for improving the tea quality evaluation system and promoting the technological upgrading of the tea industry. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a high-performance liquid chromatography (HPLC) method for the detection of 12 polyphenolic compounds in tea leaves. This method enables the simultaneous detection of gallic acid (GA), gallocatechin (GC), epigallocatechin (EGC), catechin (C), chlorogenic acid (CA), caffeine (CAF), caffeic acid, epigallocatechin gallate (EGCG), epicatechin (EC), gallocatechin gallate (GCG), epicatechin gallate (ECG), and quercetin, and significantly improves the detection sensitivity of chlorogenic acid and caffeic acid.

[0007] To achieve the above objectives, the technical solution adopted by this invention is: a method for detecting 12 polyphenolic compounds in tea using high performance liquid chromatography, comprising the following steps:

[0008] Step S1, Solution Preparation: Prepare the mobile phase, composite stable solution, standard stock solution, standard working solution, and internal standard mixed standard working solution respectively; the mobile phase consists of mobile phase A and mobile phase B. Mobile phase A is prepared by mixing chromatographic acetonitrile, acetic acid, trifluoroacetic acid, and water in a volume ratio of 9:2:0.05:88.95; mobile phase B is prepared by mixing chromatographic acetonitrile, acetic acid, trifluoroacetic acid, and water in a volume ratio of 80:2:0.05:17.95.

[0009] Step S2, Integrated purification and stabilization of the test sample: Weigh 0.2g of uniformly ground tea sample, add 5mL of extraction solution, sonicate in a 60℃ water bath for 10min, cool to room temperature, centrifuge at 5000rpm for 10min, and collect the supernatant; add 5mL of the above extraction solution to the residue again, and repeat the above steps; combine the two supernatants, and make up to 10mL with 70% methanol aqueous solution; take 5mL of the above solution, pass it through an HLB solid phase extraction column, elute with 3mL of 70% methanol aqueous solution, and collect the eluent; take 2mL of the eluent, add the composite stabilizing solution, and make up to 10mL, while adding the internal standard stock solution to make the final concentration 50μg / mL, mix thoroughly, filter through a 0.22μm organic phase filter membrane, and store at 4℃ until analysis;

[0010] Step S3, High Performance Liquid Chromatography (HPLC) synergistic detection: An Agilent InfinityLab Poroshell 120 EC-C18 column (4.6 mm × 250 mm, 4 μm) was used at a column temperature of 30 °C. The detection wavelengths were 270 nm (for 10 components including catechins and gallic acid) and 325 nm (for chlorogenic acid and caffeic acid). The injection volume was 5 μL, the flow rate was 0.9 mL / min, and fractional gradient elution was used. Simultaneously, during the elution phase from 10 to 30 min, the weighting of the dual-wavelength signals was adjusted, with the 325 nm signal weight gradually increasing from 30% to 70% and the 270 nm signal weight gradually decreasing from 70% to 30%.

[0011] Step S4, Internal Standard Method Quantitative Analysis: Establish a standard curve equation with the peak area ratio of the target component to the internal standard in each standard working solution as the ordinate (y) and the target component concentration as the abscissa (x). Substitute the peak area ratio of the target component to the internal standard in the test solution into the equation to calculate the content of 12 polyphenolic compounds in tea.

[0012] Preferably, the preparation method of the composite stable solution in step S1 includes the following steps: measuring 5 mL of 10 mg / mL ascorbic acid aqueous solution, 10 mL of acetonitrile, and 1 mL of 0.1 mol / L EDTA disodium aqueous solution into a 100 mL volumetric flask, diluting to the mark with water, and mixing well.

[0013] Preferably, the method for preparing the standard stock solution in step S1 includes the following steps: using chromatographic methanol as a solvent, prepare single standard stock solutions (concentration 10 mg / mL) for gallic acid (GA), gallocatechin (GC), epigallocatechin (EGC), catechin (C), chlorogenic acid (CA), caffeine (CAF), caffeic acid, epigallocatechin gallate (EGCG), epicatechin (EC), gallocatechin gallate (GCG), epicatechin gallate (ECG), quercetin single standard stock solution (concentration 1 mg / mL), and internal standard p-hydroxybenzoate single standard stock solution (concentration 5 mg / mL).

[0014] Preferably, the method for preparing the standard working solution in step S1 includes the following steps: using a composite stable solution as a diluent, a high-concentration mixed standard stock solution is first prepared, and then a series of mixed standard working solutions are prepared by gradient dilution; wherein the high-concentration mixed standard stock solution has the following composition: EGCG concentration 400 μg / mL, EGC, CAF, and C concentrations are all 200 μg / mL, and GA, EC, GCG, CA, caffeic acid, GC, ECG, and quercetin concentrations are all 100 μg / mL; the concentration range of the series of mixed standard working solutions after gradient dilution is: EGCG: 4-400 μg / mL; EGC, CAF, and C: 2-200 μg / mL; GA, EC, GCG, CA, caffeic acid, GC, ECG, and quercetin: 1-100 μg / mL;

[0015] Preferably, the method for preparing the internal standard mixed standard working solution in step S1 includes the following steps: using a composite stable solution as a diluent, adding an internal standard stock solution to make the final concentration 50 μg / mL, and preparing a series of mixed standard working solutions by gradient dilution, with concentration ranges of EGCG 4-400 μg / mL; EGC, CAF and C 2-200 μg / mL; GA, EC, GCG, CA, caffeic acid, GC, ECG and quercetin 1-100 μg / mL.

[0016] Preferably, the frequency of the ultrasound in step S2 is 40kHz and the power is 150W.

[0017] Preferably, the extract in step S2 is a 70% (v / v) methanol aqueous solution containing L-cysteine, preheated at 60°C.

[0018] Preferably, the concentration of L-cysteine ​​in the methanol-water solution in step S2 is 0.1 g / 100 mL.

[0019] Preferably, the HLB solid-phase extraction column described in step S2 is activated with 5 mL of methanol and equilibrated with 5 mL of water before use.

[0020] Preferably, the gradient elution program in step S3 is as follows: 0-10 min isocratic elution with 100% mobile phase A; 10-16 min mobile phase A linearly decreases from 100% to 87.2%, and mobile phase B linearly increases from 0% to 12.8%; 16-25 min mobile phase A linearly decreases from 87.2% to 30%, and mobile phase B linearly increases from 12.8% to 70%; 25-30 min mobile phase A linearly decreases from 30% to 0%, and mobile phase B linearly increases from 70% to 100%; 30-34 min maintains 100% mobile phase B; 34-34.1 min mobile phase A linearly increases from 0% to 100%, and mobile phase B linearly decreases from 100% to 0%; 34.1-39 min maintains 100% mobile phase A.

[0021] Due to the application of the above technical solution, the present invention has the following beneficial effects:

[0022] (1) The high performance liquid chromatography method for detecting 12 polyphenolic compounds in tea disclosed in this invention achieves a dual breakthrough in the extraction efficiency and sample stability of poorly soluble polyphenols through an integrated design of "synergistic extraction-compound stabilization", achieving results that cannot be achieved by existing single technology improvements. Through the synergistic effect of the "70% methanol + 0.1% L-cysteine" composite extract and the "ascorbic acid + disodium EDTA" composite stabilizing solution, not only is the extraction efficiency of poorly soluble polyphenols significantly improved, but the sample stabilization time is also effectively extended, completely solving the technical contradiction of "sufficient extraction leads to easy degradation, and stabilization leads to insufficient extraction". This comprehensive improvement effect exceeds the expectations of optimizing the extraction or stabilization process alone.

[0023] (2) The high performance liquid chromatography method for detecting 12 polyphenolic compounds in tea disclosed in this invention achieves simultaneous results of "peak shape optimization - complete separation - trace detection" through the synergistic innovation of modified mobile phase, segmented gradient elution and dual-wavelength weight adjustment, overcoming the limitation of "separation degree and sensitivity cannot be achieved simultaneously" in the prior art. By modifying the mobile phase with "2% acetic acid + 0.05% trifluoroacetic acid" to reduce non-specific adsorption, combined with three-segment gradient elution targeting different polar components, and combined with dual-wavelength weight adjustment that is precisely matched with the peak elution time, not only are the tailing factors of all components ≤1.2 and the separation degree ≥1.5, but the sensitivity of chlorogenic acid and caffeic acid is also significantly improved compared with the traditional single-wavelength method. This effect of combining complete separation and trace detection sensitivity is an unexpected gain that cannot be achieved by simply optimizing the mobile phase or detection wavelength.

[0024] (3) The method for detecting 12 polyphenolic compounds in tea by high performance liquid chromatography disclosed in this invention combines the standard working solution “precise preparation of high concentration mother liquor-gradient dilution” process with internal standard quantification, which brings a double leap in quantitative accuracy and laboratory repeatability, far exceeding the conventional level of existing technologies. By clarifying the precise transfer volume of each single standard stock solution and the specific operation steps of gradient dilution, and with the correction effect of internal standard on pretreatment loss and instrument fluctuation, the method has low intra-day / inter-day precision RSD and high recovery rate. It not only greatly reduces quantitative error, but also ensures that the detection results of different laboratories can be reproduced. The high reliability brought by this “precise preparation + error correction” synergy is a technical effect that existing fuzzy preparation process and external standard quantification cannot achieve.

[0025] (4) The method for detecting 12 polyphenolic compounds in tea by high performance liquid chromatography disclosed in this invention selectively removes impurities such as polysaccharides and proteins through HLB solid phase extraction column, thereby reducing matrix interference. At the same time, the mobile phase reduces the non-specific adsorption of target components to the stationary phase of the chromatographic column through protonation. This avoids interference of impurities on the detection signal, reduces the risk of column contamination, and extends its service life. This dual consideration of "accurate detection and equipment protection" is an unexpected advantage that cannot be achieved by the existing technology's design of "emphasizing detection and neglecting protection" or "strong purification and cumbersome operation". Attached Figure Description

[0026] Figure 1 The above are liquid chromatograms of 12 polyphenol compound standards in dual-wavelength detection mode in this invention, where (A) is the signal collected at 270 nm and (B) is the signal collected at 320 nm. Detailed Implementation

[0027] The following description is intended to disclose the invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art.

[0028] Example 1 Solution Preparation

[0029] Modified mobile phases: Prepare 9% acetonitrile aqueous solution (containing 2% acetic acid + 0.05% trifluoroacetic acid, mobile phase A) and 80% acetonitrile aqueous solution (containing 2% acetic acid + 0.05% trifluoroacetic acid, mobile phase B), respectively, and degas them by sonication for 20 min before use;

[0030] Composite stabilized solution: Measure 5 mL of 10 mg / mL ascorbic acid aqueous solution, 10 mL of acetonitrile, and 1 mL of 0.1 mol / L EDTA disodium aqueous solution, transfer them to a 100 mL volumetric flask, add water to the mark, and shake well for later use;

[0031] Standard stock solutions: Accurately weigh each standard and use chromatographic methanol as solvent to prepare single standard stock solutions of 12 target components (quercetin 1 mg / mL, the other 11 at 10 mg / mL) and internal standard stock solutions of 5 mg / mL, and store at -20℃.

[0032] Standard working solutions: ① High-concentration mixed standard stock solution: Accurately transfer 0.4 mL of EGCG single-standard stock solution (10 mg / mL), 0.2 mL of EGC single-standard stock solution (10 mg / mL), 0.2 mL of CAF single-standard stock solution (10 mg / mL), 0.2 mL of C single-standard stock solution (10 mg / mL), 0.1 mL of GA single-standard stock solution (10 mg / mL), 0.1 mL of EC single-standard stock solution (10 mg / mL), 0.1 mL of GCG single-standard stock solution (10 mg / mL), and 0.1 mL of CA single-standard stock solution (10 mg / mL). ① Transfer 0.1 mL of the following stock solutions: caffeic acid (10 mg / mL), GC (10 mg / mL), ECG (10 mg / mL), and quercetin (1 mg / mL). Then, transfer all solutions to a 10 mL volumetric flask and dilute to the mark with a composite stabilizing solution, mixing thoroughly. ② Gradient dilution: Transfer 1.0 mL of the high-concentration mixed standard stock solution to a 10 mL volumetric flask and dilute to the mark with a composite stabilizing solution to obtain a 10-fold dilution. Transfer 1.0 mL of the 10-fold dilution to a 10 mL volumetric flask and dilute to the mark with a composite stabilizing solution to obtain a 100-fold dilution. Use the stock solution, 10-fold dilution, and 100-fold dilution directly or mix them as needed to prepare a series of mixed standard working solutions.

[0033] Internal standard mixed standard working solution: Take 10 mL of each of the above series of mixed standard working solutions, add 0.1 mL of 5 mg / mL internal standard stock solution, mix well and set aside, ensuring that the final concentration of the internal standard is 50 μg / mL.

[0034] Example 2 Preparation of test solution

[0035] Sample pretreatment: After the tea sample is crushed, it is passed through a 40-mesh sieve and placed in a desiccator for equilibration for 24 hours before use;

[0036] Synergistic extraction: Accurately weigh 0.2 g (accurate to 0.0001 g) of sample powder, add 5 mL of 70% methanol aqueous solution preheated at 60℃ containing 0.1% (w / v) L-cysteine, and sonicate in a constant temperature water bath at 60℃ for 10 min (40 kHz, 150 W).

[0037] Centrifugation: After cooling to room temperature, centrifuge at 5000 rpm for 10 min, and transfer the supernatant into a centrifuge tube;

[0038] Secondary extraction: Add 5 mL of the above extract to the residue again, repeat the sonication and centrifugation operations, and combine the supernatants from the two extractions;

[0039] Purification: Pass the combined supernatant through an activated and equilibrated HLB solid-phase extraction column, elute with 3 mL of 70% methanol aqueous solution, collect the eluent into a 10 mL volumetric flask, and dilute to the mark with 70% methanol.

[0040] Stable dilution: Take 2 mL of eluent, add the composite stabilizing solution to make up to 10 mL, and add 0.1 mL of 5 mg / mL internal standard stock solution to make the final concentration of internal standard 50 μg / mL. Mix well and filter through a 0.22 μm organic phase filter membrane. Store at 4 °C until analysis.

[0041] Example 3 Chromatographic Detection

[0042] Instrument warm-up: Set column temperature to 30℃, flow rate to 0.9mL / min, detection wavelengths to 270nm and 325nm, signal acquisition weights to be automatically adjusted according to the set program, and warm up for 30min;

[0043] Equilibrate the column: Wash the column with 100% mobile phase A until the baseline is stable;

[0044] Sample injection and detection: Inject 5 μL of internal standard mixed standard working solution and test sample solution sequentially, and detect according to a segmented gradient elution program; simultaneously, during the elution phase of 10-30 min, adjust the weighting of the dual-wavelength signal acquisition, gradually increasing the weighting of the 325 nm signal from 30% to 70%, and gradually decreasing the weighting of the 270 nm signal from 70% to 30%; the gradient elution program is as follows: 0-10 min, use 100% mobile phase A for isocratic elution; 10-16 min, mobile phase A linearly decreases from 100% to 87.2%, and mobile phase B linearly increases from 0% to 12.8%; 16-25 min, mobile phase A linearly decreases from 87.2% to 30%, and mobile phase B linearly increases from 12.8% to 70%; 25-30 min, mobile phase A linearly decreases from 30% to 0%, and mobile phase B linearly increases from 70% to 100%; 30-34 ... For 34-34.1 min, maintain mobile phase B at 100%; for 34-34.1 min, mobile phase A linearly increases from 0% to 100%, while mobile phase B linearly decreases from 100% to 0%; for 34.1-39 min, maintain mobile phase A at 100%; record the chromatogram.

[0045] Example 4 Data Analysis

[0046] Standard curve plotting: Using the peak area ratio of the target component to the internal standard in each internal standard working solution as the ordinate (y) and the target component concentration as the abscissa (x), linear regression analysis was performed to obtain the standard curve equation;

[0047] Content calculation: Substitute the peak area ratio of the target component to the internal standard in the test solution into the standard curve equation to calculate the concentration of the target component. Combined with the dilution factor and the sampling amount, the content of 12 polyphenolic compounds in the tea sample is obtained.

[0048] Experimental results

[0049] Linearity, Limit of Detection (LOD), and Limit of Quantification (LOQ): The 12 components showed good linearity within their respective linear ranges, with correlation coefficients r ≥ 0.9995, meeting the requirements for quantitative analysis. The standard curve equations, linear ranges, LODs, and LOQs for each component are shown in Table 1 below. As can be seen from Table 1, this detection method has high sensitivity.

[0050] Table 1. Standard curve equations, linear ranges, LOD and LOQ for the 12 components.

[0051]

[0052] Figure 1 This is the chromatogram of the standard solution in this invention, by Figure 1 It is known that both CA and caffeine exhibit UV absorption at both 270 nm and 320 nm. The absorption at 270 nm originates from the benzene ring in their structures, while the strong UV absorption at 320 nm originates from the strong absorption band generated by the π-π conjugation of the benzene ring and the acrylic acid side chain. Clearly, CA and caffeine show higher responses at 320 nm. Furthermore, the slopes (k) of the standard curves for CA and caffeine measured at 270 nm are 5.4632 and 11.3765, respectively, significantly lower than the k measured at 320 nm. In other words, the sensitivity for detecting CA and caffeine at 320 nm is improved by approximately 225.33% and 185.04%, respectively, compared to detection at 270 nm.

[0053] Precision: The stability of the method was verified through intraday and interday precision experiments, and the results are shown in Table 2. As can be seen from the data in Table 2, the method exhibits excellent stability, far exceeding the conventional level of existing technologies (RSD≤2.00%).

[0054] Table 2 RSD1 and RSD2

[0055]

[0056] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for detecting 12 polyphenolic compounds in tea using high performance liquid chromatography, characterized in that, Includes the following steps: Step S1, Solution Preparation: Prepare the mobile phase, composite stable solution, standard stock solution, standard working solution, and internal standard mixed standard working solution respectively; the mobile phase consists of mobile phase A and mobile phase B. Mobile phase A is prepared by mixing chromatographic acetonitrile, acetic acid, trifluoroacetic acid, and water in a volume ratio of 9:2:0.05:88.95; mobile phase B is prepared by mixing chromatographic acetonitrile, acetic acid, trifluoroacetic acid, and water in a volume ratio of 80:2:0.05:17.

95. Step S2, Integrated purification and stabilization treatment of the test sample: Weigh 0.2g of uniformly ground tea sample, add 5mL of extraction solution, sonicate in a 60℃ water bath for 10min, cool to room temperature, centrifuge at 5000rpm for 10min, and collect the supernatant; add 5mL of the above extraction solution to the residue again, and repeat the above steps; combine the two supernatants, and make up to 10mL with 70% methanol aqueous solution; take 5mL of the above solution, pass it through an HLB solid phase extraction column, elute with 3mL of 70% methanol aqueous solution, and collect the eluent; take 2mL of the eluent, add the composite stabilizing solution, and make up to 10mL, while adding internal standard stock solution to make the final concentration 50μg / mL, mix thoroughly, filter through a 0.22μm organic phase filter membrane, and refrigerate at 4℃ until analysis; the extraction solution is a 70% methanol aqueous solution containing L-cysteine ​​preheated at 60℃; the preparation method of the composite stabilizing solution includes the following steps: measure 5mL Add 10 mg / mL ascorbic acid aqueous solution, 10 mL acetonitrile, and 1 mL 0.1 mol / L EDTA disodium aqueous solution to a 100 mL volumetric flask, dilute to the mark with water, and mix well. Step S3, High Performance Liquid Chromatography (HPLC) synergistic detection: An Agilent InfinityLab Poroshell 120 EC-C18 column was used at 30℃. The detection wavelengths were 270nm and 325nm. The injection volume was 5μL, and the flow rate was 0.9mL / min. Segmented gradient elution was employed. Simultaneously, during the elution phase from 10 to 30 min, the weighting of the dual-wavelength signals was adjusted, with the 325nm signal weight gradually increasing from 30% to 70% and the 270nm signal weight gradually decreasing from 70% to 30%. Step S4, Internal Standard Method Quantitative Analysis: Establish a standard curve equation with the peak area ratio of the target component to the internal standard in each standard working solution as the ordinate and the target component concentration as the abscissa. Substitute the peak area ratio of the target component to the internal standard in the test solution into the equation to calculate the content of 12 polyphenolic compounds in tea.

2. The method for detecting 12 polyphenolic compounds in tea by high performance liquid chromatography according to claim 1, characterized in that, The method for preparing the standard stock solution in step S1 includes the following steps: using chromatographic methanol as solvent, prepare single standard stock solutions of gallic acid, gallocatechin, epigallocatechin, catechin, chlorogenic acid, caffeine, caffeic acid, epigallocatechin gallate, epicatechin, gallocatechin gallate, and epicatechin gallate, each with a concentration of 10 mg / mL; a single standard stock solution of quercetin with a concentration of 1 mg / mL; and a single standard stock solution of the internal standard butylparaben with a concentration of 5 mg / mL.

3. The method for detecting 12 polyphenolic compounds in tea by high performance liquid chromatography according to claim 1, characterized in that, The method for preparing the standard working solution in step S1 includes the following steps: using a composite stable solution as a diluent, a high-concentration mixed standard stock solution is first prepared, and then a series of mixed standard working solutions are prepared by gradient dilution; wherein the high-concentration mixed standard stock solution has the following composition: EGCG concentration 400 μg / mL, EGC, CAF, and C concentrations are all 200 μg / mL, and GA, EC, GCG, CA, caffeic acid, GC, ECG, and quercetin concentrations are all 100 μg / mL; the concentration range of the series of mixed standard working solutions after gradient dilution is: EGCG: 4-400 μg / mL; EGC, CAF, and C: 2-200 μg / mL; GA, EC, GCG, CA, caffeic acid, GC, ECG, and quercetin: 1-100 μg / mL.

4. The method for detecting 12 polyphenolic compounds in tea by high performance liquid chromatography according to claim 1, characterized in that, The method for preparing the internal standard mixed standard working solution in step S1 includes the following steps: using the composite stable solution as the diluent, adding the internal standard stock solution to make the final concentration 50 μg / mL, and preparing a series of mixed standard working solutions by gradient dilution, with the concentration range being EGCG 4-400 μg / mL; EGC, CAF and C 2-200 μg / mL; GA, EC, GCG, CA, caffeic acid, GC, ECG and quercetin 1-100 μg / mL.

5. The method for detecting 12 polyphenolic compounds in tea by high performance liquid chromatography according to claim 1, characterized in that, The ultrasound in step S2 has a frequency of 40kHz and a power of 150W.

6. The method for detecting 12 polyphenolic compounds in tea by high performance liquid chromatography according to claim 1, characterized in that, The concentration of L-cysteine ​​in the methanol-water solution described in step S2 is 0.1 g / 100 mL.

7. The method for detecting 12 polyphenolic compounds in tea by high performance liquid chromatography according to claim 1, characterized in that, Before use, the HLB solid-phase extraction column described in step S2 is activated with 5 mL of methanol and equilibrated with 5 mL of water.

8. The method for detecting 12 polyphenolic compounds in tea by high performance liquid chromatography according to claim 1, characterized in that, The gradient elution program described in step S3 is as follows: 0-10 min isocratic elution with 100% mobile phase A; 10-16 min mobile phase A linearly decreases from 100% to 87.2%, and mobile phase B linearly increases from 0% to 12.8%; 16-25 min mobile phase A linearly decreases from 87.2% to 30%, and mobile phase B linearly increases from 12.8% to 70%; 25-30 min mobile phase A linearly decreases from 30% to 0%, and mobile phase B linearly increases from 70% to 100%; 30-34 min maintains 100% mobile phase B; 34-34.1 min mobile phase A linearly increases from 0% to 100%, and mobile phase B linearly decreases from 100% to 0%; 34.1-39 min maintains 100% mobile phase A.

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