Method for simultaneously detecting 4 alkaloids and 10 catechin components in tea leaves

Abstract

The present application belongs to the technical field of tea component detection method, and particularly relates to a method for simultaneously detecting four kinds of alkaloids and ten kinds of catechin components in tea, which simultaneously detects four kinds of alkaloids and ten kinds of catechin components in tea samples by using high performance liquid chromatography, and obtains the content of the 14 characteristic components in the tea sample according to the standard curve of each of the four kinds of alkaloids and the ten kinds of catechin components. The detection method provided by the present application can simultaneously detect and separate 14 kinds of compounds in tea, solves the problem of poor separation effect in the prior art when simultaneously analyzing multiple compounds in tea, meets the high standard requirement of tea component analysis, provides a more efficient, accurate and reliable analysis tool for tea producers, detection agencies and scientific researchers, and provides strong support for the fields of tea quality control, scientific research and deep processing.

Description

Technical Field

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

[0002] Tea, as a widely consumed beverage, contains a variety of bioactive components that have a significant impact on human health. Among these, theophylline, theobromine, caffeine, and thalassemia are common alkaloids found in tea, while catechins are a class of polyphenolic compounds with broad biological activity. These are the most important physiologically active substances in tea, primarily 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, antimutagenic, anticancer, anti-atherosclerotic, and antibacterial effects. Accurate detection and separation of these compounds are of great significance for tea quality control, scientific research, and deep processing.

[0003] Existing technologies, such as the "Determination Method of Tea Polyphenols and Catechins in Tea" (GB / T8313-2018), while providing methods for detecting the content of tea polyphenols and catechins in tea, have significant shortcomings in simultaneously detecting and separating four alkaloids and ten catechins in tea. Using this method, many compounds cannot be effectively separated, leading to inaccurate analytical results and failing to meet the needs of tea quality control and scientific research. This limits the accuracy and reliability of tea chemical composition analysis, hindering the further development of the tea industry and in-depth scientific research. Summary of the Invention

[0004] The purpose of this invention is to provide a method for simultaneously detecting four alkaloids and ten catechin components in tea, in order to solve the aforementioned problems.

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

[0006] High-performance liquid chromatography (HPLC) was used to simultaneously detect four alkaloids and ten catechins in tea samples. The contents of these 14 characteristic components in the tea samples were obtained based on the standard curves of the four alkaloids and ten catechins. The chromatographic conditions used were as follows: Agilent Zorbax Eclipse Plus C18 column, column temperature 30-40℃, mobile phase C 100% acetonitrile, mobile phase D 0.05% phosphoric acid aqueous solution, flow rate 0.9-1.1 mL / min, detection wavelength 275-285 nm. External standard method was used for quantification based on peak area. 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 thallium catechin; the ten catechins are epicatechin (EC), catechin (C), epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), catechin gallate (CG), gallocatechin gallate (GCG), gallocatechin (GC), trimethylEGCG, 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 with methanol solution to prepare a standard stock solution with a content of 1 mg / mL for each standard substance, then dilute the standard stock solution with methanol solution to prepare a series of standard solutions with gradient concentrations of 0.0012, 0.002, 0.006, 0.01, 0.03, 0.05, and 0.2 mg / mL, respectively, and detect them under the above chromatographic conditions to obtain the corresponding spectra, and plot the standard curve with concentration as the abscissa and peak area as the ordinate.

[0010] In some embodiments, a 0.05% phosphoric acid aqueous solution is prepared by transferring 0.50 mL of phosphoric acid, adding water to make up to a volumetric flask of 1000 mL, mixing thoroughly, and setting the pH value to 1.8-2.0.

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

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

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

[0014] In some implementations, the chromatographic column has dimensions of 4.6 × 250 mm and a diameter of 5 μm.

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

[0016] In some embodiments, the tea sample is processed as follows: the tea leaves are ground and added to a 70% methanol solution preheated in a water bath, mixed thoroughly, and immediately transferred to a water bath for extraction. After extraction, the mixture is cooled to room temperature, centrifuged, and the supernatant is collected. The residue after centrifugation is extracted once with a 70% methanol aqueous solution. The above operation is repeated, the extracts are combined, the volume is adjusted, shaken well, and filtered.

[0017] In some embodiments, the tea sample is processed 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 60-80℃ water bath, mix thoroughly, and immediately transfer to a 60-80℃ water bath for extraction for 8-12min, shaking once every 3-6min. After extraction, cool to room temperature, centrifuge at 3000-4000r / min for 8-12min, 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 and make up to 10mL, shake well, and filter through a 0.45μm filter membrane.

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

[0019] The detection method provided by this invention can simultaneously detect and separate four alkaloids and ten catechins, which solves the problem of poor separation effect when analyzing multiple compounds in tea at the same time in the prior art. It meets the high standard requirements for tea component analysis and provides a more efficient, accurate and reliable analytical tool for tea producers, testing institutions and researchers, and provides strong support for tea quality control, scientific research and deep processing. Attached Figure Description

[0020] Figure 1 Chromatograms of standards for 4 alkaloids and 10 catechins are shown.

[0021] Figure 2 Chromatograms of four alkaloids and ten catechins in black tea.

[0022] Figure 3 Chromatograms of four alkaloids and ten catechins in yellow tea.

[0023] Figure 4 Chromatograms of four alkaloids and ten catechins in green tea.

[0024] Figure 5 Chromatograms of four alkaloids and ten catechins in dark tea.

[0025] Figure 6 Chromatograms of four alkaloids and ten catechins in white tea.

[0026] Figure 7 Chromatograms of four alkaloids and ten catechins in oolong tea.

[0027] Figure 8 Chromatograms of four alkaloids and ten catechins in fresh leaves.

[0028] Figures 9 to 22 Standard curves for different substances. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to specific embodiments. The reagents used in the embodiments and their sources are as follows: Phosphoric acid, Tianjin Fuyu Fine Chemical Co., Ltd.; Standards: Epicatechin (EC), Catechin (C), Epigallocatechin (EGC), Epicatechin gallate (ECG), Epigallocatechin gallate (EGCG), Catechingallate (CG), Gallatechin gallate (Gallocatechin... Gallate (GCG), gallatechin (GC), theophylline, epigallocatechin-3-O-(3-O-methyl)gallate (EGCG3"Me), caffeine, theobromine, theophylline, and theaflavins, with a purity ≥98%, were sourced from Shanghai Aladdin Biochemical Technology Co., Ltd.; all organic solvents in the liquid mobile phase were domestically produced chromatographic grade.

[0030] This implementation method mainly follows the steps below:

[0031] Tea processing: Weigh 0.2g (accurate to 0.0001g) of uniformly ground tea leaves into a 10mL centrifuge tube, add 5mL of 70% methanol solution preheated in a 70℃ water bath, mix thoroughly, and immediately transfer to a 70℃ water bath for extraction (shaking once every 5 minutes). Transfer the extract to a 10mL volumetric flask. Extract the residue once with 5mL of 70% methanol aqueous solution. Repeat the above operation, combine the extracts, and dilute to 10mL in a volumetric flask. Store the extract at -20℃. After filtering through a 0.45μm filter membrane, it can be used for the determination of catechins and alkaloids.

[0032] Standard curve preparation: Accurately weigh 10 mg of each catechin and alkaloid standard sample, place them in volumetric flasks, dissolve them in methanol solution, and dilute to 10 mL to prepare a 1 mg / mL standard stock solution. Then, dilute the stock solution with methanol solution to prepare a series of standard solutions with gradient concentrations of 0.0012, 0.002, 0.006, 0.01, 0.03, 0.05, and 0.2 mg / mL. Plot a standard curve. Quantify each compound using the standard curve. The results are shown in Table 1 and [Table data would be inserted here]. Figure 9-22 As shown.

[0033] Then, HPLC was used to detect the standard solution and the extract obtained after tea treatment, and the contents of 14 compounds were calculated.

[0034] Table 1. Formulas for standard curves of 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 includes the following steps:

[0038] Accurately weigh 10 mg of each catechin and alkaloid standard sample, place them in volumetric flasks, dissolve them in methanol solution and dilute to 10 mL to prepare a 1 mg / mL standard stock solution. Then dilute the standard stock solution with methanol solution to prepare 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. Plot a standard curve and use the standard curve to quantify each metabolite.

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

[0040] Table 1. Liquid Chromatography Elution Procedure

[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 compound standard samples are as follows Figure 1 As shown in Table 2.

[0043] Table 2. Liquid Chromatography Information for 4 Alkaloids and 10 Catechins Standards

[0044]

[0045]

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

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

[0048] Precision test: Take 10 μL of sample solution and inject the sample 4 times consecutively to determine the peak area of ​​4 alkaloids and 10 catechins.

[0049] Stability test: Sample solutions were injected and analyzed at 0h, 2h, 4h, 8h, 16h and 24h after preparation. The peak areas of the four alkaloids and ten 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 ranged from 0.508% to 2.603%, indicating that the method has excellent repeatability under optimal gradient elution conditions. Precision experiments showed that the RSD values ​​of each component ranged from 0.581% to 3.190%, indicating good precision of the method. Stability experiments showed that the RSD values ​​of each component ranged from 0.544% to 2.659%, indicating good stability of the method.

[0051] Table 3. Results of repeatability, precision, and stability experiments for 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 0.2 g (accurate to 0.0001 g) of uniformly ground black tea sample into a 10 mL centrifuge tube, add 5 mL of 70% methanol solution preheated in a 70℃ water bath, mix thoroughly, and immediately transfer to a 70℃ water bath for extraction for 10 min, shaking once every 5 min. After extraction, cool to room temperature, centrifuge at 3500 r / min for 10 min, and transfer the supernatant to a 10 mL volumetric flask. Extract the residue once with 5 mL of 70% methanol aqueous solution, repeat the above operation, combine the extracts, and dilute to 10 mL, shake well, filter through a 0.45 μm 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 tested yellow tea.

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

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

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

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

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

[0083] Table 9. Chromatographic Information of 4 Alkaloids and 10 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 tested fresh leaves.

[0087] Weigh 0.2 g (accurate to 0.0001 g) of uniformly ground fresh leaf sample into a 10 mL centrifuge tube, add 5 mL of 70% methanol solution preheated in a 70℃ water bath, mix thoroughly, and immediately transfer to a 70℃ water bath for extraction for 10 min, shaking once every 5 min. After extraction, cool to room temperature, centrifuge at 3500 r / min for 10 min, and transfer the supernatant to a 10 mL volumetric flask. Extract the residue once with 5 mL of 70% methanol aqueous solution, repeat the above operation, combine the extracts, and dilute to 10 mL, shake well, filter through a 0.45 μm filter membrane, and use for liquid chromatography analysis. The results are as follows. Figure 8 See 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 descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A method for simultaneous detection of 4 alkaloids and 10 catechin components in tea leaves, characterized in that, Comprising the following steps: Simultaneous detection of 4 alkaloids and 10 catechins in tea samples by high performance liquid chromatography, and obtaining the content of the 14 characteristic components in the tea sample according to the standard curve of each of the 4 alkaloids and 10 catechins; the chromatographic conditions are as follows: the chromatographic column is Agilent Zorbax Eclipse Plus C18, the column temperature is 30-40 DEG C, the mobile phase C is 100% acetonitrile, the mobile phase D is 0.05% phosphoric acid aqueous solution, the flow rate is 0.9-1.1 mL / min, the detection wavelength is 275-285 nm, the external standard method is used for quantification according to the peak area, and the elution gradient program is as follows: The 4 alkaloids are theophylline, theobromine, caffeine and theacrine, and the 10 catechins are epicatechin, catechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, catechin gallate, gallocatechin gallate, gallocatechin, epigallocatechin-3-O-(3-O-methyl) gallate and theaflavin.

2. The method for simultaneous detection of 4 alkaloids and 10 catechin components in tea leaves according to claim 1, characterized in that, The standard curve is obtained by the following method: respectively taking catechin and alkaloid standard samples, dissolving and constant volume with methanol solution, preparing 1 mg / mL standard stock solution of each standard substance, diluting the standard stock solution with methanol solution, respectively preparing 0.0012, 0.002, 0.006, 0.01, 0.03, 0.05, 0.2 mg / mL series gradient concentration standard solution, detecting according to the chromatographic conditions to obtain the corresponding spectrum, and drawing the standard curve with concentration as abscissa and peak area as ordinate.

3. The method for simultaneous detection of 4 alkaloids and 10 catechin components in tea leaves according to claim 1 or 2, characterized in that, The tea sample is treated according to the following method: after the tea is ground, 70% methanol solution preheated in a water bath is added, mixed thoroughly, immediately transferred into the water bath for extraction, cooled to room temperature after extraction, centrifuged, and the supernatant is taken; the residue after centrifugation is extracted once with 70% methanol solution, and the above operation is repeated, the extract is combined, constant volume, shaken, and filtered.

4. The method for simultaneous detection of 4 alkaloids and 10 catechin components in tea leaves according to claim 3, characterized in that, The tea sample is treated according to the following method: 0.2 g of ground tea is weighed in a 10 mL centrifuge tube, 5 mL of 70% methanol solution preheated in a 60-80 DEG C water bath is added, mixed thoroughly, immediately transferred into the 60-80 DEG C water bath for 8-12 min and shaken every 3-6 min, cooled to room temperature after extraction, centrifuged at 3000-4000 r / min for 8-12 min, and the supernatant is transferred to a 10 mL volumetric flask; the residue is extracted once with 5 mL of 70% methanol solution, and the above operation is repeated, the extract is combined, constant volume to 10 mL, filtered with a 0.45 mu m filter membrane, and then used.

5. The method for simultaneous detection of four alkaloids and ten catechin components in tea leaves according to claim 3, characterized in that, The tea sample is treated according to the following method: 0.2 g of ground tea is weighed in a 10 mL centrifuge tube, 5 mL of 70% methanol solution preheated in a 70 ℃ water bath is added, after mixing, it is immediately transferred into a 70 ℃ water bath for extraction for 10 min and shaken every 5 min, after extraction, it is cooled to room temperature, centrifuged at a speed of 3500 r / min for 10 min, the supernatant is transferred into a 10 mL volumetric flask; the residue is extracted once with 5 mL of 70% methanol solution, the above operation is repeated, the extract is combined and diluted to 10 mL, after mixing, it is filtered with a 0.45 μm filter membrane and then used.

6. The method for simultaneous detection of 4 alkaloids and 10 catechin components in tea leaves 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 taken, water is added to a 1000 mL volumetric flask, mixed, and the pH value is 1.8-2.

0.

7. The method for simultaneous detection of 4 alkaloids and 10 catechin components in tea leaves according to claim 1, characterized in that, The flow rate is 1 mL / min.

8. The method for simultaneous detection of 4 alkaloids and 10 catechin components in tea leaves according to claim 1, characterized in that, The column temperature is 35 ℃.

9. The method for simultaneous detection of 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 simultaneous detection of 4 alkaloids and 10 catechin components in tea leaves according to claim 1, characterized in that, The injection volume is 10 μL.

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