LC-MS / MS synchronous detection method for 14 active components in tea leaves

Abstract

The invention relates to an LC-MS / MS synchronous detection method for 14 active components in tea, and belongs to the technical field of food detection. Comprising the following steps: step S10, preparing a standard working solution of active components with multiple concentration gradients; step S20, analyzing the standard working solution of each group of active components under different concentration gradients by adopting an LC-MS / MS method, and drawing a peak area response value and a concentration making standard curve; step S30, pretreating a sample, mixing with ultrapure water, performing water bath extraction, cooling, filtering, performing high-speed centrifugation on filtrate, diluting supernate with the solution A, and filtering through a membrane to obtain a to-be-detected sample solution B; and step S40, establishing isomer exclusive ion pair detection parameters, and detecting the solution of the sample B to be detected by using an LC-MS / MS method: carrying out gradient periodic elution by using a mobile phase A and a mobile phase B. The problems that high-flux quantitative / qualitative detection cannot be carried out on the xyephedrine in the tea leaves through an HPLC method, and large-scale sample screening is difficult to support are solved; the accurate quantitative requirement of trace amount of the xyephedrine in the cultivated tea tree is difficult to meet.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food detection, and particularly relates to a LC-MS / MS synchronous detection method for 14 active components in tea leaves. BACKGROUND

[0002] Casuarinin is a hydrolytic gallotannin and a minor active component in tea plant species. It is abundant in wild tea plants but usually has a very low content in cultivated tea plants. Studies have shown that casuarinin has multiple biological activities such as antiviral, antibacterial, anti-obesity, laxative, anti-caries, anti-allergy, improving psoriasis, improving hypertension, improving diabetes, and anticancer, and is one of the key targets for functional component research of tea.

[0003] Currently, the detection methods for casuarinin in tea are all focused on high-performance liquid chromatography (HPLC) in the published literature, which has significant limitations.

[0004] Mari Maeda-Yamamoto's method takes 45 minutes per sample and only realizes the co-detection of casuarinin and a single catechin derivative, which is low in analysis efficiency. In the HPLC method for simultaneously detecting catechins, gallic acid, casuarinin and purine alkaloids in green tea, the limit of detection (LOD) of casuarinin is only 0.41 μg / mL, the limit of quantification (LOQ) is 1.24 μg / mL, which is difficult to meet the accurate quantification requirement of trace casuarinin in cultivated tea plants, and the analysis period is more than 60 min, which is difficult to support large-scale sample screening. The HPLC method is only suitable for qualitative analysis of casuarinin and does not provide quantitative verification parameters (such as recovery rate and precision).

[0005] The existing methods cannot simultaneously detect 14 related active components (such as chlorogenic acid and theophylline), and some cannot distinguish key isomers such as catechins, which leads to questionable data reliability. The existing HPLC technology is seriously restricted in the in-depth study of casuarinin and other active components due to insufficient sensitivity, low analysis efficiency, and limited coverage of multiple components.

[0006] Patent No. CN119901854B discloses a kit and a detection method for simultaneously detecting catecholamines and their metabolites. The kit contains a diluent, a solid-phase extraction reagent, an LC-MS / MS detection reagent, a solid-phase extraction device, and a solid-phase extraction filler. The solid-phase extraction filler creatively uses bipolar ion exchange microspheres containing both carboxyl and quaternary ammonium groups. Catecholamines and their metabolites are simultaneously detected by high-performance liquid chromatography tandem mass spectrometry.

[0007] The patent with the publication number CN104330482A discloses a method for simultaneously determining 17 characteristic components in tea by HPLC, comprising: simultaneously determining 17 characteristic components in tea by high performance liquid chromatography, obtaining the content of the 17 characteristic components in the tea sample according to the standard curve of each of the 17 characteristic components; the chromatographic conditions are: the column temperature of the chromatographic column is 31-33 DEG C, methanol and 1% formic acid solution are used as the mobile phase, gradient elution is adopted, and the detection wavelength is 280-285 nm.

[0008] The prior art has at least the following problems in use:

[0009] The HPLC method in the prior art cannot perform high-throughput quantitative / qualitative detection on casuarine in tea, and it is difficult to support large-scale sample screening; it is difficult to meet the accurate quantitative demand of trace casuarine in cultivated tea trees. SUMMARY

[0010] The present application provides a LC-MS / MS synchronous detection method for 14 active components in tea, which is used to solve the technical problems that the HPLC method in the prior art cannot perform high-throughput quantitative / qualitative detection on casuarine in tea, and it is difficult to support large-scale sample screening; it is difficult to meet the accurate quantitative demand of trace casuarine in cultivated tea trees.

[0011] In order to achieve the above purpose, the present application realizes the following technical scheme:

[0012] A LC-MS / MS synchronous detection method for 14 active components in tea, the active components including casuarine, catechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, epicatechin gallate, gallocatechin gallate, epigallocatechin gallate, gallic acid, chlorogenic acid, caffeine, theobromine and theophylline, characterized in that the synchronous detection method comprises the following steps:

[0013] Step S10, preparing a standard working solution of multiple concentration gradient active components; step S20, analyzing the standard working solution of each group of active components under different concentration gradients by LC-MS / MS method, and drawing a standard curve of peak area response value and concentration; step S30, sample pretreatment, weighing tea powder and mixing with 70 DEG C ultrapure water, extracting in a 70 DEG C water bath, cooling and filtering, filtering the filtrate by high-speed centrifugation, diluting the supernatant with A solution and filtering through a membrane to obtain a to-be-tested sample B solution; step S40, establishing isomer-specific ion pair detection parameters, synchronously detecting and outputting the detection results, and detecting the to-be-tested sample B solution by LC-MS / MS method: gradient elution is performed with mobile phase A-mobile phase B, a multiple reaction monitoring mode is adopted, qualitative and quantitative detection is realized through specific ion pairs of each active component, and the single sample analysis period is T.

[0014] Further, the method for preparing the standard working solution in step S10 comprises the following steps: step S11, accurately weighing 0.002 g of each active ingredient standard, dissolving in 70% methanol and diluting to volume, and dissolving the theobromine in 70°C water to prepare a single standard stock solution with a concentration of 200 μg / mL; step S12, mixing the respective single standard stock solutions, and diluting with 0.1% formic acid aqueous solution to form the standard working solution with multiple concentrations, with a concentration range of 0.048-176.923 μg / mL.

[0015] Further, the pretreatment in step S30 comprises the following steps: step S31, accurately weighing 0.5000 g of tea powder, adding 50 mL of 70°C ultrapure water, and placing in a 70°C constant temperature water bath for water bath for 30 min, stirring once every 5 min; step S32, after water bath, cooling to room temperature, and filtering with qualitative filter paper; step S33, transferring the filtrate to a centrifuge tube, centrifuging at 10000 rpm and 4°C for 10 min; step S34, the A solution is 0.1% formic acid aqueous solution, diluting the supernatant after centrifugation 100 times with the A solution, filtering through a 0.22 μm water filter membrane to obtain the sample B solution to be tested.

[0016] Further, the gradient elution of the LC-MS / MS method in step S40 comprises the following steps: step S41, at 0 min, the proportion of mobile phase A is 87% and the proportion of mobile phase B is 13%, with a flow rate of 0.3 mL / min; maintaining the above proportions for 1.6 min; step S42, from 1.6 to 2.5 min, the proportion of mobile phase A is reduced to 86% and the proportion of mobile phase B is increased to 14%, and maintaining the proportions from 2.5 to 3.5 min; step S43, from 3.5 to 4.5 min, the proportion of mobile phase A is reduced to 85% and the proportion of mobile phase B is increased to 15%, and maintaining the proportions from 4.5 to 6 min; step S44, from 6 to 6.5 min, the proportion of mobile phase A is reduced to 82% and the proportion of mobile phase B is increased to 18%, and maintaining the proportions from 6.5 to 9 min.

[0017] Further, the parameters of the exclusive ion pairs of the active ingredients in step S40 are as follows:

[0018] Casuarinin: parent ion m / z 633.1, qualitative daughter ion m / z 301, quantitative daughter ion m / z 275.2, collision energy 41 V / 49 V;

[0019] Catechin: parent ion m / z 289, qualitative daughter ion m / z 245.15, quantitative daughter ion m / z 203.05, collision energy 13 V / 17 V;

[0020] Epicatechin: parent ion m / z 289.2, qualitative daughter ion m / z 202.9, quantitative daughter ion m / z 123, collision energy 25 V / 25 V;

[0021] Gallocatechin: parent ion m / z 305.2, qualitative daughter ion m / z 125.1, quantitative daughter ion m / z 179, collision energy 21V / 13V;

[0022] Epigallocatechin: parent ion m / z 307.05 / 305.1, qualitative daughter ion m / z 139.05 / 219.1, quantitative daughter ion m / z 125, collision energy 17V / 17V / 21V;

[0023] Catechin gallate: parent ion m / z 441.1, qualitative daughter ion m / z 289.1, quantitative daughter ion m / z 169, collision energy 17V / 21V;

[0024] Epicatechin gallate: parent ion m / z 441.1, qualitative daughter ion m / z 289.1, quantitative daughter ion m / z 169, collision energy 17V / 21V;

[0025] Gallocatechin gallate: parent ion m / z 457.1, qualitative daughter ion m / z 169, quantitative daughter ion m / z 125.1, collision energy 17V / 49V;

[0026] Epigallocatechin gallate: parent ion m / z 457.1, qualitative daughter ion m / z 169, quantitative daughter ion m / z 125, collision energy 21V / 49V;

[0027] Gallic acid: parent ion m / z 169, qualitative daughter ion m / z 125.1, quantitative daughter ion m / z 78.95, collision energy 13V / 29V;

[0028] Chlorogenic acid: parent ion m / z 353, qualitative daughter ion m / z 191.15, quantitative daughter ion m / z 85, collision energy 13V / 45V;

[0029] Caffeine: parent ion m / z 195, qualitative daughter ion m / z 138.1, quantitative daughter ion m / z 110.15, collision energy 21V / 25V;

[0030] Theobromine: parent ion m / z 181, qualitative daughter ion m / z 138.05, quantitative daughter ion m / z 163.1, collision energy 17V / 21V;

[0031] Kurchine: parent ion m / z 225, qualitative daughter ion m / z 210.1, quantitative daughter ion m / z 153.2, collision energy 21V / 33V.

[0032] Further, the T of the single sample analysis cycle is 9 min.

[0033] The application provides a LC-MS / MS synchronous detection method for 14 active ingredients in tea leaves, and has the beneficial effects of:

[0034] Based on the specific ion pair of casuarine, the 14 active ingredients are separated in 9 minutes, high-throughput quantitative / qualitative detection of casuarine in tea is realized, large-scale sample screening is supported, the accurate quantitative demand of trace casuarine in cultivated tea trees is met, and 70 DEG C pure water extraction is used instead of organic solvent extraction, so that the use of toxic reagents such as acetonitrile / methanol is avoided, and the safety and accuracy are improved. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0036] Figure 1 The column performance comparison chart of three chromatographic columns provided by the embodiments of the present application under the condition of 70% mobile phase B is shown in the following table:

[0037] Figure 2(a) is a comparison chart of different mobile phases provided by the embodiments of the present application;

[0038] Figure 2(b) is a comparison chart of different mobile phases provided by the embodiments of the present application;

[0039] Figure 3 The TIC chart of 14 active ingredients such as casuarine provided by the embodiments of the present application is shown in the following table:

[0040] Figure 4 The flowchart of the LC-MS / MS synchronous detection method for 14 active ingredients in tea provided by the embodiments of the present application is shown in the following table: DETAILED DESCRIPTION

[0041] The embodiments of the present application will be described in detail below with reference to the drawings.

[0042] In the description of the present application, it should be understood that the terms "center", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, therefore it cannot be understood as a limitation to the present application.

[0043] The terms "first", "second", "third", etc. are used only for descriptive purposes and do not imply or suggest a relative importance or an implicit indication of the number of the technical features indicated. Thus, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. In the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specified.

[0044] In the description of the present application, it should be noted that, unless otherwise explicitly specified and limited, the terms "mounting", "connection", "connecting" should be understood broadly, for example, it can be welding, or bolted connection, or riveting; it can be fixed connection, or detachable connection, or integrally connected; it can be mechanical connection, or electrical connection; it can be directly connected, or indirectly connected through an intermediate medium, or the communication inside two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0045] Embodiment:

[0046] The embodiment provides a kind of synchronous detection method of 14 kinds of active ingredients in tea leaf by LC-MS / MS, and active ingredient includes Casuarine, catechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, epicatechin gallate, gallocatechin gallate, epigallocatechin gallate, gallic acid, chlorogenic acid, caffeine, theobromine and amarogentin, wherein, synchronous detection method includes the following steps:

[0047] Step S10, prepare multiple concentration gradient active ingredient standard working solution;Step S20, LC-MS / MS method is analyzed to different concentration gradient under each group of active ingredient standard working solution, and standard curve is drawn for peak area response value and concentration;Step S30, sample pretreatment, tea powder is weighed and mixed with 70 ℃ ultrapure water, after 70 ℃ water bath extraction, cooling filtration, filter liquor is centrifuged at high speed, supernatant is diluted with A solution and filtered, to obtain to be measured sample B solution;Step S40, establish isomer exclusive ion pair detection parameter, synchronous detection and output detection result, LC-MS / MS method is detected to to be measured sample B solution: with mobile phase A-mobile phase B gradient elution is carried out, using multiple reaction monitoring mode, by the exclusive ion pair of each active ingredient, qualitative and quantitative are realized, and single sample analysis cycle is T.

[0048] In the embodiment, Agilent 1290 Infinity II high performance liquid chromatography system and Agilent 6470 triple quadrupole mass spectrometer;Electronic analytical balance, water bath, centrifuge;

[0049] Chromatographic column: Agilent ZORBAX RRHD EclipsePlus C18 column (2.1 × 100 mm, 1.8 μm); flow rate: 0.3 mL / min; injection volume: 2 μL; column temperature: 35 °C;

[0050] Water is laboratory grade water specified in GB / T 6682; acetonitrile, methanol and formic acid are HPLC grade; see Table 1 for standard information.

[0051] Table 1 Standard information

[0052] The principle of the determination method of the present application is that after 14 kinds of active ingredients such as casuarine in tea are separated by high performance liquid chromatography, they enter the mass spectrometer detector for detection, and the existence of each substance is determined by the qualitative ion detected, and the content of the substance is determined by the quantitative characteristic ion.

[0053] The preparation method of the 14 kinds of active ingredient standard working solution is: accurately weigh 0.002 g of each compound, dissolve with 70% methanol and dilute to volume. Theobromine is dissolved in water at 70 °C to prepare a stock solution (200 μg / mL). Then, the single standard stock solution is mixed in a certain amount and diluted with a mobile phase, 0.1% formic acid aqueous solution, to a series of concentrations of mixed standard working solution, and the concentration range of each substance is from 0.048 to 176.923 μg / mL, to construct a standard solution curve.

[0054] The mass spectrometry conditions in the LC-MS / MS method are as follows:

[0055] Ion mode: electrospray ion source; ion source body: ESI electrospray ion source; scanning mode: positive / negative ion mode; detection mode: multiple reaction monitoring mode; ESI ion source parameters: dry gas temperature 300 °C, dry gas flow rate 8 L / min, atomizing gas pressure 45 psi, sheath gas temperature 250 °C, sheath gas flow rate 11 L / min, capillary voltage 3500 V (+) / 2500 V (-), nozzle voltage 500 V.

[0056] The method for pretreating the sample is as follows: accurately weigh 0.5000 g of tea powder and mix with 50 ml of ultrapure water (70 °C) in a conical flask, and water bath in a 70 °C water bath for 30 min, stirring every 5 min. After water bath, cool to room temperature, filter. The filtrate is centrifuged at 10000 rpm (9380g, 4 °C) for 10 min. The supernatant is diluted 100 times with 0.1% (by volume) formic acid aqueous solution, then filtered with a 0.22 μm water system membrane, and then subjected to LC-MS / MS analysis.

[0057] Further, the method for preparing the standard working solution in step S10 comprises the following steps: in step S11, 0.002 g of each active ingredient standard is accurately weighed, dissolved in 70% methanol and diluted to volume, and theobromine is dissolved in 70°C water to prepare a single standard stock solution with a concentration of 200 μg / mL; in step S12, the respective single standard stock solutions are mixed, diluted with 0.1% formic acid aqueous solution to form a standard working solution with multiple concentrations, and the concentration range is 0.048-176.923 μg / mL.

[0058] In this embodiment, the mobile phase A is 0.1% formic acid aqueous solution; the mobile phase B is acetonitrile solution; the method for preparing the standard working solution is as follows: 0.002 g of each compound is accurately weighed, dissolved in 70% methanol and diluted to volume (theobromine is dissolved in 70°C water), to prepare a stock solution (200 μg / mL). Then, the single standard stock solutions are mixed in a certain amount and diluted with the mobile phase (0.1% formic acid aqueous solution) to form a mixed standard working solution with a series of concentrations, and the concentration range of each substance is 0.048-176.923 μg / mL, to construct a standard solution curve.

[0059] Further, the pretreatment in step S30 comprises the following steps: in step S31, 0.5000 g of tea powder is accurately weighed, 50 mL of 70°C ultrapure water is added, and the mixture is placed in a 70°C constant temperature water bath for water bath for 30 min, and stirred every 5 min; in step S32, after water bath, the mixture is cooled to room temperature and filtered with qualitative filter paper; in step S33, the filtrate is transferred to a centrifuge tube, centrifuged at 10000 rpm and 4°C for 10 min; in step S34, the A solution is 0.1% formic acid aqueous solution, the supernatant after centrifugation is diluted 100 times with the A solution, filtered through a 0.22 μm water filter membrane, and the B solution of the sample to be tested is obtained.

[0060] Further, the gradient elution of the LC-MS / MS method in step S40 comprises the following steps: in step S41, at 0 min, the proportion of mobile phase A is 87% and the proportion of mobile phase B is 13%, and the flow rate is 0.3 mL / min; the above proportions are maintained for 1.6 min; in step S42, from 1.6 min to 2.5 min, the proportion of mobile phase A is reduced to 86% and the proportion of mobile phase B is increased to 14%, and the proportions are maintained from 2.5 min to 3.5 min; in step S43, from 3.5 min to 4.5 min, the proportion of mobile phase A is reduced to 85% and the proportion of mobile phase B is increased to 15%, and the proportions are maintained from 4.5 min to 6 min; in step S44, from 6 min to 6.5 min, the proportion of mobile phase A is reduced to 82% and the proportion of mobile phase B is increased to 18%, and the proportions are maintained from 6.5 min to 9 min.

[0061] In this embodiment, the chromatographic conditions in the LC-MS / MS method are as follows:

[0062] Mobile phase A: 0.1% formic acid in water; mobile phase B: acetonitrile; gradient elution. As a further preferred technical solution, the gradient elution program is shown in Table 2.

[0063] Table 2 Gradient elution program

[0064] wherein the ratio of mobile phase A to mobile phase B is in volume percentage.

[0065] Further, the parent ions, qualitative daughter ions, quantitative daughter ions, retention times, residence times, collision energies, ionization polarities, and the like of the 14 active ingredients are shown in Table 3.

[0066] Table 3 Exclusive ion pair parameter table of active ingredients

[0067] wherein, a MRM is the qualitative daughter ion, b MRM is the quantitative daughter ion; Frag (Q1) is the voltage of quadrupole 1 for selecting the parent ion; Cell Acc (Q3) is the voltage of quadrupole 3 for selecting the daughter ion.

[0068] Example 2:

[0069] Optimization of determination conditions, in the condition optimization process, the influence of different columns, mobile phases on the results was investigated respectively. In the column optimization, (a) Agilent EclipsePlus C18 RRHD (2.1 x 50 mm, 1.8 μm; Agilent Technologies, Santa Clara, CA, USA), (b) Agilent ZORBAX RRHD EclipsePlus C18 (2.1 x 100 mm, 1.8 μm; Agilent Technologies, Santa Clara, CA, USA), (c) Waters ACQUITY UPLC HSS T3 (2.1 x 100 mm, 1.8 μm; Waters Corporation, Milford, MA, USA) were investigated respectively. As shown in Figure 1 (b) Agilent ZORBAX RRHD EclipsePlus C18 column was used, the peak shape, separation degree, and response value of the 14 active ingredients were optimal.

[0070] In the mobile phase optimization process, first of all, the effect of acetonitrile-H2O, acetonitrile-0.1% formic acid water, acetonitrile-0.1% ammonium formate water, methanol-H2O, methanol-0.1% formic acid water, methanol-0.1% ammonium formate water was investigated respectively, as shown in Figure 2(a), the separation degree and response value of acetonitrile-0.1% formic acid water were optimal, and the time was shortest in the case of completing the separation of all compounds. Then in the case of acetonitrile as mobile phase A, the different proportions of formic acid water in mobile phase B were investigated respectively, and the results are shown in Figure 2(b), the effect of different proportions of formic acid water on the results was not significant. In summary, the selection of mobile phase is as follows: mobile phase A: 0.1% formic acid water solution by volume fraction, mobile phase B: acetonitrile solution. The gradient elution program is as shown above, and the ideal TIC graph of 14 active ingredients such as casuarinin is obtained, as shown in Figure 2(c). Figure 3

[0071] Example 3:

[0072] The standard curve was prepared by analyzing the standard solution with a concentration range of 0.048 to 176.923 μg / mL prepared in Example 1 in turn, and the standard curve was obtained; the detection limit and the quantitative limit of the method were calculated by formulas (1) and (2).

[0073]

[0074]

[0075] δ is the standard deviation of the signal and the blank sample (noise)

[0076] S is the slope of the standard curve (the relationship between the signal and the concentration)

[0077] The results are shown in Table 4, the correlation coefficient R2 of the linear equation of all compounds reached 0.999, which was linear and ideal, and met the actual use requirements. For casuarinin, the detection limit of the method was 0.013 μg / mL, and the quantitative limit was 0.042 μg / mL. It is proved that compared with the existing technology such as the HPLC method of Yuzo Mizukami et al. (2007) with a detection limit of 0.41 μg / mL and a quantitative limit of 1.24 μg / mL for casuarinin, the detection limit and the quantitative limit of the method of the present application are lower and more sensitive.

[0078] Table 4 Linear equation, LOD and LOQ, precision and recovery rate evaluation of 14 active ingredients such as casuarinin

[0079] Example 4:

[0080] ​Recovery and precision tests were added to evaluate the sensitivity and precision of the method established by the present application. The standard solution with a concentration range of 0.048 to 176.923 μg / mL was used for evaluation. The intra-day precision was determined by analyzing the mixed standard solution every 4 hours (6 times a day), and the inter-day precision was determined by analyzing 6 times a day for 3 consecutive days. The precision results were expressed as the percentage of relative standard deviation (%RSD). The accuracy was evaluated by adding standard substances at three levels (50%, 100% and 200% of the amount of the compound naturally present in the tea sample, respectively) to 0.5000 grams of tea sample, then treating the spiked sample according to the pretreatment method of the sample to be tested, and performing three experiments for each level. The recovery rate was calculated by comparing the actual concentration and the measured concentration.

[0081]

[0082] The results are shown in Table 4. The parallelism and recovery results of the 14 active ingredients including casuarine at three concentration points (low, medium and high) are ideal, the relative standard deviations of inter-day precision and intra-day precision are less than 2%, and the overall precision is good; the recovery rate is 80.417%~118.93%, which meets the requirement of the recovery rate in the range of 80%~120%, and meets the testing needs.

[0083] In summary, the present application realizes the rapid separation of 14 active ingredients within 9 minutes based on the specific ion pair of casuarine, and performs high-throughput quantitative / qualitative detection of casuarine in tea to support large-scale sample screening. It can meet the accurate quantitative demand of trace casuarine in cultivated tea trees. The 70℃ pure water extraction replaces the organic solvent extraction, avoids the use of toxic reagents such as acetonitrile / methanol, and improves safety and accuracy.

[0084] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for simultaneous detection of 14 active ingredients in tea leaves including casuarine, catechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, epicatechin gallate, gallocatechin gallate, epigallocatechin gallate, gallic acid, chlorogenic acid, caffeine, theobromine and theacrine, characterized in that, The synchronous detection method comprises the following steps: ​ Step S10, preparing a standard working solution of multiple concentration gradient active ingredients; Step S20, analyzing the standard working solution of each group of active ingredients under different concentration gradients by LC-MS / MS method, and drawing a standard curve of peak area response value and concentration; Step S30, sample pretreatment, weighing tea powder and mixing with 70 DEG C ultrapure water, extracting in a 70 DEG C water bath, cooling, filtering, filtering the filtrate by high-speed centrifugation, diluting the supernatant with A solution and filtering to obtain a to-be-tested sample B solution; Step S40, establishing isomer-specific ion pair detection parameters, synchronous detection and outputting detection results, and detecting the to-be-tested sample B solution by LC-MS / MS method: gradient elution is performed with mobile phase A-mobile phase B, a multiple reaction monitoring mode is adopted, qualitative and quantitative analysis is realized through specific ion pairs of each active ingredient, and the single sample analysis cycle is T. 2.The method according to claim 1, wherein, The preparation method of the standard working solution in step S10 comprises the following steps: Step S11, accurately weighing 0.002 g of each active ingredient standard, dissolving in 70% methanol and constant volume, dissolving theobromine in 70 DEG C water to prepare a single standard stock solution with a concentration of 200 μg / mL; Step S12, mixing the single standard stock solution, diluting with 0.1% formic acid solution to prepare a standard working solution with multiple concentrations, and the concentration range is 0.048-176.923 μg / mL. 3.The method according to claim 1, wherein, The pretreatment in step S30 comprises the following steps: Step S31, accurately weighing 0.5000 g of tea powder, adding 50 mL of 70 DEG C ultrapure water, placing in a 70 DEG C constant temperature water bath, water bathing for 30 min, and stirring every 5 min; Step S32, after water bathing, cooling to room temperature, and filtering with qualitative filter paper; Step S33, transferring the filtrate to a centrifuge tube, centrifuging at 10000 rpm and 4 DEG C for 10 min; Step S34, the A solution is 0.1% formic acid solution, the supernatant after centrifugation is diluted 100 times with the A solution, filtered through a 0.22 μm water filter membrane to obtain the to-be-tested sample B solution. 4.The method according to claim 1, wherein, The gradient elution of the LC-MS / MS method in step S40 comprises the following steps: Step S41, at 0 min, the proportion of mobile phase A is 87%, the proportion of mobile phase B is 13%, and the flow rate is 0.3 mL / min; the above proportions are maintained for 1.6 min; Step S42, 1.6-2.5 min, mobile phase A is reduced to 86%, mobile phase B is increased to 14%, and the proportions are maintained for 2.5-3.5 min; Step S43, 3.5-4.5 min, mobile phase A is reduced to 85%, mobile phase B is increased to 15%, and the proportions are maintained for 4.5-6 min; Step S44, 6-6.5 min, mobile phase A is reduced to 82%, mobile phase B is increased to 18%, and the proportions are maintained for 6.5-9 min. 5.The method according to claim 1, wherein, The specific ion pair parameters of the active ingredients in step S40 are as follows: Casuarine: parent ion m / z 633.1, qualitative daughter ion m / z 301, quantitative daughter ion m / z 275.2, and collision energy 41 V / 49 V; Catechin: parent ion m / z 289, qualifier m / z 245.15, quantifier m / z 203.05, collision energy 13V / 17V; Epicatechin: parent ion m / z 289.2, qualifier m / z 202.9, quantifier m / z 123, collision energy 25V / 25V; Gallocatechin: parent ion m / z 305.2, qualifier m / z 125.1, quantifier m / z 179, collision energy 21V / 13V; Epigallocatechin: parent ion m / z 307.05 / 305.1, qualifier m / z 139.05 / 219.1, quantifier m / z 125, collision energy 17V / 17V / 21V; Catechin gallate: parent ion m / z 441.1, qualifier m / z 289.1, quantifier m / z 169, collision energy 17V / 21V; Epicatechin gallate: parent ion m / z 441.1, qualifier m / z 289.1, quantifier m / z 169, collision energy 17V / 21V; Gallocatechin gallate: parent ion m / z 457.1, qualifier m / z 169, quantifier m / z 125.1, collision energy 17V / 49V; Epigallocatechin gallate: parent ion m / z 457.1, qualifier m / z 169, quantifier m / z 125, collision energy 21V / 49V; Gallic acid: parent ion m / z 169, qualifier m / z 125.1, quantifier m / z 78.95, collision energy 13V / 29V; Chlorogenic acid: parent ion m / z 353, qualifier m / z 191.15, quantifier m / z 85, collision energy 13V / 45V; Caffeine: parent ion m / z 195, qualifier m / z 138.1, quantifier m / z 110.15, collision energy 21V / 25V; Theobromine: parent ion m / z 181, qualifier m / z 138.05, quantifier m / z 163.1, collision energy 17V / 21V; Theacrine: parent ion m / z 225, qualifier m / z 210.1, quantifier m / z 153.2, collision energy 21V / 33V. 6.The method according to claim 1, wherein, The T for single sample analysis period is 9 min.

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