Method for simultaneously detecting four alkaloids and 10 catechin components in tea leaves

Abstract

The invention belongs to the technical field of tea leaf component detection methods, and particularly relates to a method for simultaneously detecting 4 alkaloids and 10 catechin components in tea leaves, which utilizes high performance liquid chromatography to simultaneously detect 4 alkaloids and 10 catechin components in tea leaf samples. And obtaining the contents of the 14 characteristic components in the tea sample according to the respective standard curves of the four alkaloids and the 10 catechin components. According to the detection method provided by the invention, 14 compounds in the tea leaves can be simultaneously detected and separated, and the problem that the separation effect is poor when multiple compounds in the tea leaves are simultaneously analyzed in the prior art is solved, so that the high-standard requirement of tea leaf component analysis is met; a more efficient, accurate and reliable analysis tool is provided for tea manufacturers, detection mechanisms and scientific researchers, and powerful support is provided for the fields of tea quality control, scientific research, deep processing and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of tea component detection methods, and specifically relates to a method for simultaneously detecting four alkaloids and ten catechin components in tea. Background Art

[0002] Tea is a widely consumed beverage, and the various bioactive ingredients it contains have important effects on human health. Among them, theophylline, theobromine, caffeine and theophylline are common alkaloids in tea, while catechins are a class of polyphenol compounds with a wide range of biological activities and are the most important physiologically active substances in tea. They are mainly composed of monomers such as epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG) and epicatechin (EC). Many physiological and pharmacological functions of catechins have been confirmed, such as antioxidant, anti-mutagenic, anti-cancer, anti-atherosclerotic and antibacterial. Accurate detection and separation of these compounds are of great significance for tea quality control, scientific research and deep processing.

[0003] In the existing technology, such as the "Test Method for Tea Polyphenols and Catechins in Tea" (GB / T8313-2018), although it provides a method for testing the content of tea polyphenols and catechins in tea, it is obviously insufficient in simultaneously detecting and separating 4 alkaloids and 10 catechins in tea. Using this method, many compounds cannot be effectively separated, resulting in inaccurate analysis results, which cannot meet the needs of tea quality control and scientific research. This limits the accuracy and reliability of tea chemical composition analysis, affecting the further development of the tea industry and the in-depth scientific research. Summary of the invention

[0004] The object of the present invention is to provide a method for simultaneously detecting four alkaloids and ten catechin components in tea leaves, so as to solve the above-mentioned problems.

[0005] According to one aspect of the present invention, a method for simultaneously detecting four alkaloids and ten catechin components in tea leaves is provided, comprising the following steps:

[0006] High performance liquid chromatography (HPLC) was used to simultaneously detect 4 alkaloids and 10 catechin components in tea samples, and the contents of the 14 characteristic components in the tea samples were obtained according to the respective standard curves of the 4 alkaloids and 10 catechin components; the chromatographic conditions adopted were as follows: the chromatographic column was Agilent Zorbax Eclipse Plus C18, the column temperature was 30-40°C, the mobile phase C was 100% acetonitrile, the mobile phase D was 0.05% phosphoric acid aqueous solution, the flow rate was 0.9-1.1 mL / min, the detection wavelength was 275-285 nm, the external standard method was used for quantification according to the peak area, and the elution gradient program was as follows.

[0007] Time (min) Mobile phase C (%) Mobile phase D (%) 0.01 6.5 93.5 30 16 84 34 20 80 39 20 80 40 35 65 45 35 65 50 6.5 93.5 60 Finish Finish

[0008] In some embodiments, the four alkaloids are theophylline, theobromine, caffeine, and theophylline; the ten catechins are epicatechin (EC), catechin (C), epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), catechin gallate (CG), gallocatechin gallate (GCG), gallocatechin (GC), trimethyl EGCG, and theaflavins.

[0009] In some embodiments, the standard curve is obtained by the following method: accurately weigh each standard sample of catechin and alkaloid, place them in a volumetric flask, dissolve and dilute them with methanol solution, prepare standard stock solutions with a content of each standard substance of 1 mg / mL, and then dilute the standard stock solutions with methanol solution to prepare standard solutions with a series of gradient concentrations of 0.0012, 0.002, 0.006, 0.01, 0.03, 0.05, and 0.2 mg / mL, respectively, detect according to the above chromatographic conditions to obtain the corresponding spectrum, and draw the standard curve with concentration as the horizontal axis and peak area as the vertical axis.

[0010] In some embodiments, the 0.05% phosphoric acid aqueous solution is prepared by the following method: pipette 0.50 mL of phosphoric acid, add water to a 1000 mL volumetric flask, mix well, and the pH value is 1.8-2.0.

[0011] In some embodiments, the flow rate is 1 mL / min.

[0012] In some embodiments, the column temperature is 35°C.

[0013] In some embodiments, the detection wavelength is 280 nm.

[0014] In some embodiments, the chromatography column has a size of 4.6×250 mm, 5 μm.

[0015] In some embodiments, the injection volume is 10 μL.

[0016] In some embodiments, the method for processing tea samples is as follows: after the tea leaves are ground, they are added to a 70% methanol solution preheated in a water bath, mixed thoroughly, and immediately transferred to a water bath for leaching. After leaching, they are cooled to room temperature, centrifuged, and the supernatant is taken; the residue after centrifugation is extracted once with a 70% methanol aqueous solution, the above operation is repeated, the extracts are combined to a fixed volume, shaken well, and filtered.

[0017] In some embodiments, the method for processing tea samples is as follows: weigh 0.2g (accurate to 0.0001g) of uniformly ground tea samples into a 10mL centrifuge tube, add 5mL of 70% methanol solution preheated in a 60-80℃ water bath, mix thoroughly, immediately transfer to a 60-80℃ water bath for immersion for 8-12min and shake every 3-6min, cool to room temperature after immersion, transfer to centrifuge at 3000-4000r / min for 8-12min, transfer the supernatant to a 10mL volumetric flask; extract the residue once with 5mL of 70% methanol aqueous solution, repeat the above operation, combine the extracts to make the volume 10mL, shake well, and filter with a 0.45μm filter membrane.

[0018] In some embodiments, the processing method of the tea sample is as follows: weigh 0.2g (accurate to 0.0001g) of uniformly ground tea sample into a 10mL centrifuge tube, add 5mL of 70% methanol solution preheated in a 70°C water bath, mix thoroughly, immediately transfer to a 70°C water bath for immersion for 10min and shake every 5min, cool to room temperature after immersion, transfer to centrifuge at 3500r / min for 10min, transfer the supernatant to a 10mL volumetric flask; extract the residue once with 5mL of 70% methanol aqueous solution, repeat the above operation, combine the extracts to make the volume 10mL, shake well, and filter with a 0.45μm filter membrane.

[0019] The detection method provided by the present invention can simultaneously detect and separate 4 alkaloids and 10 catechins, which solves the problem of poor separation effect when simultaneously analyzing multiple compounds in tea in the prior art, so as to meet the high standard requirements for tea component analysis, provide a more efficient, accurate and reliable analysis tool for tea producers, testing institutions and scientific researchers, and provide strong support for the fields of tea quality control, scientific research and deep processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The following are the chromatograms of the standard products of 4 alkaloids and 10 catechins.

[0021] Figure 2 This is the chromatogram of 4 alkaloids and 10 catechins in black tea.

[0022] Figure 3 This is the chromatogram of 4 alkaloids and 10 catechins in yellow tea.

[0023] Figure 4 This is a chromatogram of four alkaloids and ten catechins in green tea.

[0024] Figure 5 This is the chromatogram of 4 alkaloids and 10 catechins in dark tea.

[0025] Figure 6 This is the chromatogram of 4 alkaloids and 10 catechins in white tea.

[0026] Figure 7 This is the chromatogram of 4 alkaloids and 10 catechins in oolong tea.

[0027] Figure 8 This is the chromatogram of 4 alkaloids and 10 catechins in fresh leaves.

[0028] Figures 9 to 22 These are standard curves for different substances. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with a specific embodiment. In the embodiment, the reagents used and their sources are as follows: phosphoric acid, Tianjin Fuyu Fine Chemical Co., Ltd.; standard substances epicatechin (EC), catechin (C), epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), catechin gallate (CG), gallocatechin gallate (Gallocatechin Gallate, GCG), Gallocatechin (GC), Theophylline, Epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3"Me), Caffeine, Theobromine, Theophylline, Theaflavin, purity ≥ 98%, from Shanghai Aladdin Biochemical Technology Co., Ltd.; All liquid mobile phase organic solvents were domestically produced chromatographic grade.

[0030] This implementation method is mainly carried out according to the following steps:

[0031] Treatment of tea leaves: weigh 0.2g (accurate to 0.000 1g) of evenly ground tea leaves into a 10mL centrifuge tube, add 5mL of 70% methanol solution preheated in a 70℃ water bath, mix thoroughly, immediately transfer to a 70℃ water bath for 10min and shake once every 5min), soak in a 10mL volumetric flask. Extract the residue after centrifugation once with 5mL of 70% methanol aqueous solution, repeat the above operation, combine the extracts, dilute to 10mL in a volumetric flask, and shake well. The extracts are stored in a -20℃ refrigerator and used for the determination of catechins and alkaloid components after passing through a 0.45μm filter membrane.

[0032] Draw the standard curve: accurately weigh 10 mg of each standard sample of catechins and alkaloids, place them in a volumetric flask, dissolve them in methanol solution and dilute to 10 mL to prepare a 1 mg / mL standard stock solution, and then dilute the mother solution with methanol solution to a series of gradient concentration standard solutions of 0.0012, 0.002, 0.006, 0.01, 0.03, 0.05, and 0.2 mg / mL to draw the standard curve. The standard curve is used to quantify each compound. The standard curve results are shown in Table 1 and Figure 9-22 shown.

[0033] Then, the standard solution and the extract obtained after tea treatment were detected by HPLC, and the contents of 14 compounds were calculated.

[0034] Table 1 Standard curve formulas for 14 compounds

[0035] Peak Compound Standard curve calculation formula R-squared value 1 Theobromine y=0.0000007891x-0.0166831324 <![CDATA[R 2 =0.8292]]> 2 GC y=0.0000004017x+0.0010517384 <![CDATA[R 2 =0.9982]]> 3 Theophylline y=0.0000000375x+0.0033199745 <![CDATA[R 2 =0.9952]]> 4 EGC y=0.0000005741x-0.0051135036 <![CDATA[R 2 =0.9956 <!-- 3 -->]]> 5 Theophylline y=0.0000000566x-0.0000166743 <![CDATA[R 2 =0.9996]]> 6 C y=0.0000001059x-0.0004573687 <![CDATA[R 2 =0.9998]]> 7 Caffeine y=0.0000000301x+0.0008798432 <![CDATA[R 2 =0.9989]]> 8 EC y=0.0000000952x-0.0000938551 <![CDATA[R 2 =0.9996]]> 9 EGCG y=0.0000000524x-0.0007481208 <![CDATA[R 2 =0.9999]]> 10 GCG y=0.0000000491x-0.0000211166 <![CDATA[R 2 =0.9996]]> 11 EGCG3"Me y=0.0000000386x+0.0155558812 <![CDATA[R 2 =0.9444]]> 12 ECG y=0.0000000398x-0.0010347395 <![CDATA[R 2 =0.9999]]> 13 CG y=0.0000000357x+0.0003487674 <![CDATA[R 2 =0.9994]]> 14 Theaflavins y=0.0000000627x-0.0016788250 <![CDATA[R 2 =0.9997]]>

[0036] Example 1

[0037] A method for simultaneously detecting four alkaloids and ten catechin components in tea leaves comprises the following steps:

[0038] Accurately weigh 10 mg of each standard sample of catechins and alkaloids, place them in a volumetric flask, dissolve them with methanol solution and make up to 10 mL to prepare a 1 mg / mL standard stock solution, then dilute the standard stock solution with methanol solution to a series of standard solutions with gradient concentrations of 0.0012 mg / mL, 0.002 mg / mL, 0.006 mg / mL, 0.01 mg / mL, 0.03 mg / mL, 0.05 mg / mL, and 0.2 mg / mL, draw a standard curve, and use the standard curve to quantify each metabolite;

[0039] The content was determined by high performance liquid chromatography, and the chromatographic conditions used were as follows: the chromatographic column was Agilent Zorbax Eclipse Plus C18 (4.6×250mm, 5μm), the column temperature was 35°C, the mobile phase C was 100% acetonitrile, the mobile phase D was 0.05% phosphoric acid aqueous solution, the flow rate was 1mL / min, the detection wavelength was 280nm, the injection volume was 10μL, and the external standard method was used for quantification according to the peak area; the elution gradient program is shown in Table 1. Among them, the preparation method of 0.05% phosphoric acid aqueous solution is as follows: 0.50mL phosphoric acid is transferred, water is added to a 1000mL volumetric flask, mixed evenly, and the pH value is 1.9.

[0040] Table 1 Liquid chromatography elution program

[0041] Time (min) Mobile phase C (%) Mobile phase D (%) 0.01 6.5 93.5 30 16 84 34 20 80 39 20 80 40 35 65 45 35 65 50 6.5 93.5 60 Finish Finish

[0042] The test results of 14 kinds of compound standard samples are as follows Figure 1 And as shown in Table 2.

[0043] Table 2 Liquid chromatography information of 4 alkaloids and 10 catechin standards

[0044]

[0045]

[0046] The repeatability, precision and stability of the above method were tested.

[0047] Repeatability test: Take three copies of the same sample, analyze them separately, record the peak areas of the four alkaloids and 10 catechins, and calculate the contents of the four alkaloids and 10 catechins in the sample using the standard curve method;

[0048] Precision test: 10 μL of sample solution was drawn and injected 4 times in succession to determine the peak areas of 4 alkaloids and 10 catechins;

[0049] Stability test: The sample solution was taken and analyzed at 0h, 2h, 4h, 8h, 16h and 24h after preparation, and the peak areas of 4 alkaloids and 10 catechins were recorded to test the stability of the samples.

[0050] The results are shown in Table 3. The results show that the relative standard deviation (RSD) values ​​of each component are between 0.508% and 2.603%, which indicates that under the optimal gradient elution conditions, the method has excellent repeatability. The results of the precision experiment show that the RSD values ​​of each component are in the range of 0.581% to 3.190%, which indicates that the precision of this method is good. The results of the stability experiment show that the RSD values ​​of each component are between 0.544% and 2.659%, indicating that the method has good stability.

[0051] Table 3 Results of repeatability, precision and stability experiments of 4 alkaloids and 10 catechins

[0052]

[0053]

[0054] The above method was used to detect 14 compounds in black tea. The specific steps are as follows:

[0055] Weigh 0.2g (accurate to 0.0001g) of uniformly ground black tea sample into a 10mL centrifuge tube, add 5mL of 70% methanol solution preheated in a 70℃ water bath, mix thoroughly, immediately transfer to a 70℃ water bath for 10min and shake once every 5min, cool to room temperature after leaching, transfer to a centrifuge at 3500r / min for 10min, and transfer the supernatant to a 10mL volumetric flask. Extract the residue once with 5mL of 70% methanol aqueous solution, repeat the above operation, combine the extracts to make up to 10mL, shake well, filter with a 0.45μm filter membrane and use for liquid chromatography analysis. The results are as follows: Figure 2 and Table 4.

[0056] Table 4 Chromatographic information of 4 alkaloids and 10 catechins in black tea

[0057] Peak Compound Retention time (min) area Peak height Concentration (mg / mL) 1 Theobromine 6.572 65884 2625 1.080 2 GC 7.042 212144 15053 4.313 3 Theophylline 9.805 93421 5922 0.341 4 EGC 12.454 182954 6431 4.996 5 Theophylline 13.697 178528 9809 0.504 6 C 14.697 433079 21520 2.270 7 Caffeine 15.436 24603117 1079343 37.072 8 EC 21.303 858201 36945 4.080 9 EGCG 22.301 2017726 81555 5.249 10 GCG 26.075 259660 9587 0.636 11 EGCG3"Me 30.835 343137 12391 12.840 12 ECG 34.217 7418915 283375 14.712 13 CG 36.261 8247 939 0.032 14 Theaflavins 44.675 151601 34302 0.391

[0058] Example 2

[0059] The difference between Example 2 and Example 1 is that Example 2 detects yellow tea.

[0060] Weigh 0.2g (accurate to 0.0001g) of uniformly ground yellow tea sample into a 10mL centrifuge tube, add 5mL of 70% methanol solution preheated in a 70℃ water bath, mix thoroughly, immediately transfer to a 70℃ water bath for 10min and shake every 5min, cool to room temperature after leaching, transfer to a centrifuge at 3500r / min for 10min, transfer the supernatant to a 10mL volumetric flask. Extract the residue once with 5mL of 70% methanol aqueous solution, repeat the above operation, combine the extracts to make up to 10mL, shake well, filter with a 0.45μm filter membrane and use for liquid chromatography analysis. The results are as follows: Figure 3 and Table 5.

[0061] Table 5 Chromatographic information of 4 alkaloids and 10 catechins in yellow tea

[0062] Peak Compound Retention time (min) area Peak height Concentration (mg / mL) 1 Theobromine 7.142 252363 24748 4.298 2 GC 7.568 88360 7398 1.827 3 Theophylline 9.933 5368 403 0.176 4 EGC 13.87 13883 1556 0.143 5 Theophylline 14.173 290174 19103 0.820 6 C 14.845 486333 31900 2.552 7 Caffeine 15.621 9442840 619130 14.255 8 EC 21.428 1375821 75774 6.544 9 EGCG 22.415 7640311 384564 19.980 10 GCG 26.04 584865 30817 1.435 11 EGCG3"Me 30.95 1324000 67917 49.677 12 ECG 34.326 8770003 431036 17.401 13 CG 35.953 239282 15220 0.445 14 Theaflavins 44.551 31823 4276 0.016

[0063] Example 2

[0064] The difference between Example 2 and Example 1 is that Example 2 detects green tea.

[0065] Weigh 0.2g (accurate to 0.0001g) of uniformly ground green tea sample into a 10mL centrifuge tube, add 5mL of 70% methanol solution preheated in a 70℃ water bath, mix thoroughly, immediately transfer to a 70℃ water bath for 10min and shake once every 5min, cool to room temperature after leaching, transfer to centrifuge at 3500r / min for 10min, transfer the supernatant to a 10mL volumetric flask. Extract the residue once with 5mL of 70% methanol aqueous solution, repeat the above operation, combine the extracts to make up to 10mL, shake well, filter with a 0.45μm filter membrane and use for liquid chromatography analysis. The results are as follows: Figure 4 and Table 6.

[0066] Table 6 Chromatographic information of 4 alkaloids and 10 catechins in green tea

[0067] Peak Compound Retention time (min) Peak area Peak height Concentration (mg / mL) 1 Theobromine 7.019 593623 47939 10.188 2 GC 7.423 245290 14653 4.979 3 Theophylline 9.748 21836 1662 0.207 4 EGC 12.592 1224226 49205 34.886 5 Theophylline 13.476 53230 4604 0.150 6 C 14.672 818426 44157 4.311 7 Caffeine 15.415 28290086 1166753 42.621 8 EC 21.217 4083386 169637 19.432 9 EGCG 22.182 21854112 737539 57.220 10 GCG 25.904 772148 41731 1.895 11 EGCG3"Me 30.778 4470452 166504 167.842 12 ECG 34.082 26156035 804371 51.999 13 CG 35.808 200209 13529 0.375 14 Theaflavins 44.496 92933 14985 0.207

[0068] Example 3

[0069] The difference between Example 3 and Example 1 is that Example 3 detects dark tea.

[0070] Weigh 0.2g (accurate to 0.0001g) of uniformly ground black tea sample into a 10mL centrifuge tube, add 5mL of 70% methanol solution preheated in a 70℃ water bath, mix thoroughly, immediately transfer to a 70℃ water bath for 10min and shake once every 5min, cool to room temperature after leaching, transfer to a centrifuge at 3500r / min for 10min, transfer the supernatant to a 10mL volumetric flask. Extract the residue once with 5mL of 70% methanol aqueous solution, repeat the above operation, combine the extracts to make up to 10mL, shake well, filter with a 0.45μm filter membrane and use for liquid chromatography analysis. The results are as follows: Figure 5 and Table 7.

[0071] Table 7 Chromatographic information of 4 alkaloids and 10 catechins in dark tea

[0072]

[0073]

[0074] Example 4

[0075] The difference between Example 4 and Example 1 is that Example 4 detects white tea.

[0076] Weigh 0.2g (accurate to 0.0001g) of evenly ground white tea sample into a 10mL centrifuge tube, add 5mL of 70% methanol solution preheated in a 70℃ water bath, mix thoroughly, immediately transfer to a 70℃ water bath for 10min and shake once every 5min, cool to room temperature after leaching, transfer to a centrifuge at 3500r / min for 10min, and transfer the supernatant to a 10mL volumetric flask. Extract the residue once with 5mL of 70% methanol aqueous solution, repeat the above operation, combine the extracts to make up to 10mL, shake well, filter with a 0.45μm filter membrane and use for liquid chromatography analysis. The results are as follows: Figure 6 and Table 8.

[0077] Table 8 Chromatographic information of 4 alkaloids and 10 catechins in white tea

[0078]

[0079]

[0080] Example 5

[0081] The difference between Example 5 and Example 1 is that Example 5 detects oolong tea.

[0082] Weigh 0.2g (accurate to 0.0001g) of evenly ground oolong tea sample into a 10mL centrifuge tube, add 5mL of 70% methanol solution preheated in a 70℃ water bath, mix thoroughly, immediately transfer to a 70℃ water bath for 10min and shake every 5min, cool to room temperature after leaching, transfer to a centrifuge at 3500r / min for 10min, and transfer the supernatant to a 10mL volumetric flask. Extract the residue once with 5mL of 70% methanol aqueous solution, repeat the above operation, combine the extracts to make up to 10mL, shake well, filter with a 0.45μm filter membrane and use for liquid chromatography analysis. The results are as follows: Figure 7 and Table 9.

[0083] Table 9 Chromatographic information of four alkaloids and ten catechins in oolong tea

[0084] Peak Compound Retention time (min) Peak area Peak height Concentration (mg / mL) 1 Theobromine 6.754 214417 22726 3.643 2 GC 6.896 121984 16408 2.503 3 Theophylline 9.198 33006 2555 0.228 4 EGC 11.771 609312 49141 17.235 5 Theophylline 12.385 183052 13235 0.517 6 C 13.347 177228 15500 0.916 7 Caffeine 14.594 12608520 1077325 19.020 8 EC 19.891 664254 51107 3.157 9 EGCG 20.626 15916378 1038856 41.664 10 GCG 24.586 1291560 99349 3.170 11 EGCG3"Me 28.992 685617 48491 25.702 12 ECG 32.139 4811265 315960 9.523 13 CG 34.347 182905 11994 0.344 14 Theaflavins 44.182 181762 39346 0.486

[0085] Example 6

[0086] The difference between Example 6 and Example 1 is that Example 6 detects fresh leaves.

[0087] Weigh 0.2g (accurate to 0.0001g) of evenly ground fresh leaf sample into a 10mL centrifuge tube, add 5mL of 70% methanol solution preheated in a 70℃ water bath, mix thoroughly, immediately transfer to a 70℃ water bath for 10min and shake every 5min, cool to room temperature after leaching, transfer to centrifuge at 3500r / min for 10min, transfer the supernatant to a 10mL volumetric flask. Extract the residue once with 5mL of 70% methanol aqueous solution, repeat the above operation, combine the extracts to make up to 10mL, shake well, filter with a 0.45μm filter membrane and use for liquid chromatography analysis. The results are as follows: Figure 8 and Table 10.

[0088] Table 10 Chromatographic information of 4 alkaloids and 10 catechins in fresh leaves

[0089] Peak Compound Retention time (min) Peak area Peak height Concentration (mg / mL) 1 Theobromine 6.79 3263902 356763 56.277 2 GC 7.831 79845 3294 1.656 3 Theophylline 8.804 221860 21938 0.582 4 EGC 11.916 627968 42718 17.770 5 Theophylline 12.651 499055 23232 1.411 6 C 13.452 639177 43204 3.362 7 Caffeine 14.636 16558312 1361844 24.964 8 EC 19.976 786047 64299 3.737 9 EGCG 20.613 35469217 1913660 92.892 10 GCG 24.731 714589 57039 1.753 11 EGCG3"Me 29.25 153049 8515 5.701 12 ECG 32.272 9195351 586300 18.247 13 CG 34.413 1165866 78963 2.099 14 Theaflavins 44.558 514445 102708 1.529

[0090] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for simultaneously detecting four alkaloids and ten catechin components in tea, characterized in that: The steps include: The 4 alkaloids and 10 catechin components in the tea sample were simultaneously detected by high performance liquid chromatography, and the contents of the 14 characteristic components in the tea sample were obtained according to the respective standard curves of the 4 alkaloids and the 10 catechin components; the chromatographic conditions adopted were as follows: the chromatographic column was Agilent Zorbax Eclipse Plus C18, the column temperature was 30-40°C, the mobile phase C was 100% acetonitrile, the mobile phase D was 0.05% phosphoric acid aqueous solution, the flow rate was 0.9-1.1 mL / min, the detection wavelength was 275-285 nm, the external standard method was used for quantification according to the peak area, and the elution gradient program was as follows: Among them, the four alkaloids are theophylline, theobromine, caffeine, and theophylline, and the ten catechins are epicatechin, catechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, catechin gallate, gallocatechin gallate, gallocatechin, trimethyl EGCG, and theaflavins.

2. The method for simultaneously detecting four alkaloids and ten catechin components in tea according to claim 1, characterized in that: The standard curve is obtained by the following method: weighing standard samples of catechins and alkaloids respectively, dissolving and fixing the standard samples with methanol solution to prepare standard stock solutions with a content of 1 mg / mL for each standard substance, and then diluting the standard stock solutions with methanol solution to prepare standard solutions with a series of gradient concentrations of 0.0012, 0.002, 0.006, 0.01, 0.03, 0.05, and 0.2 mg / mL, respectively, performing detection according to the chromatographic conditions to obtain corresponding spectra, and drawing a standard curve with concentration as the horizontal axis and peak area as the vertical axis.

3. The method for simultaneously detecting four alkaloids and ten catechin components in tea according to claim 1 or 2, characterized in that: The tea sample is processed according to the following method: after the tea leaves are ground, they are added into a 70% methanol solution preheated in a water bath, fully mixed, and immediately transferred into a water bath for leaching. After leaching, they are cooled to room temperature, centrifuged, and the supernatant is taken; the residue after centrifugation is extracted once with a 70% methanol aqueous solution, the above operation is repeated, the extracts are combined to a fixed volume, shaken, and filtered.

4. The method for simultaneously detecting four alkaloids and ten catechin components in tea according to claim 3, characterized in that: The tea sample is processed according to the following method: weigh 0.2g of ground tea leaves into a 10mL centrifuge tube, add 5mL of 70% methanol solution preheated in a 60-80°C water bath, mix thoroughly, immediately transfer to a 60-80°C water bath for immersion for 8-12min and shake once every 3-6min, cool to room temperature after immersion, transfer to centrifuge at a speed of 3000-4000r / min for 8-12min, transfer the supernatant to a 10mL volumetric flask; extract the residue once with 5mL of 70% methanol aqueous solution, repeat the above operation, combine the extracts to make the volume 10mL, and filter with a 0.45μm filter membrane.

5. The method for simultaneously detecting four alkaloids and ten catechin components in tea according to claim 3, characterized in that: The tea sample was processed according to the following method: 0.2 g of ground tea leaves were weighed into a 10 mL centrifuge tube, 5 mL of 70% methanol solution preheated in a 70° C. water bath was added, and after mixing, the mixture was immediately transferred into a 70° C. water bath for immersion for 10 min and shaken every 5 min. After immersion, the mixture was cooled to room temperature, centrifuged at 3500 r / min for 10 min, and the supernatant was transferred to a 10 mL volumetric flask; the residue was extracted once with 5 mL of 70% methanol aqueous solution, the above operation was repeated, the extracts were combined to make up to 10 mL, and after mixing, the mixture was filtered with a 0.45 μm filter membrane.

6. The method for simultaneously detecting four alkaloids and ten catechin components in tea according to claim 1, characterized in that: The 0.05% phosphoric acid aqueous solution is prepared by the following method: 0.50 mL of phosphoric acid is pipetted, water is added to a 1000 mL volumetric flask, and the mixture is mixed, and the pH value is 1.8-2.

0.

7. The method for simultaneously detecting four alkaloids and ten catechin components in tea according to claim 1, characterized in that: The flow rate was 1 mL / min.

8. The method for simultaneously detecting four alkaloids and ten catechin components in tea according to claim 1, characterized in that: The column temperature was 35°C.

9. The method for simultaneously detecting four alkaloids and ten catechin components in tea leaves according to claim 1, characterized in that: The detection wavelength is 280 nm.

10. The method for simultaneously detecting four alkaloids and ten catechin components in tea leaves according to claim 1, characterized in that: The injection volume was 10 μL.

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