Method for quantitatively detecting selenium form content in tea leaves
Through specific pretreatment of tea samples and high-performance liquid chromatography-inductively coupled plasma mass spectrometry combined detection, the problem of low extraction rate and doubtful accuracy of the five selenium morphological content detection in tea leaves was solved, and more efficient and accurate detection results were achieved.
Patent Information
- Application Number
- CN202411040125.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-30
- Publication Date
- 2025-03-21
AI Technical Summary
The existing detection methods have problems with low extraction rates and doubts about the accuracy of the detection results when detecting the five selenium morphological contents in tea.
High performance liquid chromatography and inductively coupled plasma mass spectrometer were used to conduct targeted pretreatment of tea samples, including cleaning, lyophilization, grinding, enzymatic decomposition and extraction, and quantitative analysis was performed in combination with normalization.
The detection accuracy and efficiency of the five selenium morphological contents in tea has been improved, experimental errors have been reduced, and detection methods have been optimized.
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Figure CN119667068A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a method for quantitatively detecting the contents of five selenium forms in tea leaves, and belongs to the field of analysis and testing. Background Art
[0002] At present, the only method standard for detecting the content of selenium forms is "High Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry for the Determination of Five Selenium Forms in Agricultural Products" (GH / T1429-2023) issued by the China National Federation of Supply and Marketing Cooperatives. This standard is mainly used for analysis and testing of agricultural products such as grains, fruits and vegetables. However, for tea, due to its unique cell structure, it is not suitable for this standard. When this standard is used for extraction and testing, the content of selenium forms extracted is low, and the extraction rate is low. Compared with the extraction rate of conventional grains and fruit and vegetable samples, there is a large gap, and the accuracy of the test results is questionable. . Summary of the invention
[0003] In view of this, the main purpose of the present invention is to provide a method for quantitatively detecting the content of selenium forms in tea, to perform targeted pretreatment on tea samples, to optimize the detection method, to reduce experimental errors, and to improve detection efficiency and accuracy.
[0004] The standard selenium form solution and tea sample enzymatic hydrolyzate were detected by high performance liquid chromatography and inductively coupled plasma mass spectrometry, and the tetravalent selenium (Se4+), hexavalent selenium (Se6+), selenomethionine (SeMet), methylselenocysteine (SeMeCys) and selenocystine (SeCys2) in the samples were quantitatively analyzed by normalization method. The contents of the five selenium forms in the samples were obtained by calculation, which includes the following steps:
[0005] (1) Sample preparation: cleaning, freeze-drying, grinding and sieving of tea samples;
[0006] (2) Sample treatment: Add cellulase solution and protease solution to the sample to be tested, shake and sonicate, enzymolyze overnight, extract and centrifuge, concentrate and re-dissolve,
[0007] (3) Instrument configuration: High performance liquid chromatography and inductively coupled plasma mass spectrometry were used to detect the selenium form of the sample.
[0008] (4) Preparation of mobile phase: ammonium salt solution and methanol are prepared into methanol-diammonium citrate buffer, and formic acid is used to adjust the pH. The mobile phase is filtered through a 0.22 μm filter membrane and ultrasonically degassed to obtain a mobile phase;
[0009] (5) Data processing: The normalization method was used to quantitatively analyze tetravalent selenium (Se4+), hexavalent selenium (Se6+), selenomethionine (SeMet), methylselenocysteine (SeMeCys) and selenocysteine (SeCys2) in the samples.
[0010] Furthermore, the freeze-drying in step (1) is performed by vacuum freeze-drying at -80°C, and the freeze-drying time is 12 to 24 hours, preferably 24 hours.
[0011] Furthermore, the grinding and pulverizing in step (1) is performed using a grinding pestle under liquid nitrogen conditions.
[0012] Furthermore, the sieving in step (1) is performed using a 60-100 mesh sieve, preferably a 100 mesh sieve.
[0013] Furthermore, the cellulase content in the cellulase solution in step (2) is 0.1% to 0.5%, preferably 0.4%, which is beneficial to the enzymatic hydrolysis of the tea sample.
[0014] Furthermore, the protease content in the protease solution in step (2) is 0.1% to 0.5%, preferably 0.4%. This optimization is beneficial to the enzymatic hydrolysis of selenium forms in the tea sample and the separation of the five selenium forms.
[0015] Furthermore, the oscillation ultrasound in step (2) is ultrasound at 37°C for 30 minutes, and then the ultrasound is followed by shaking overnight at 37°C and 200 rpm for 12 to 24 hours, preferably 12 hours, which is conducive to the accuracy of the test results.
[0016] Furthermore, the extraction in step (2) uses dichloromethane as the extraction liquid.
[0017] Furthermore, the normalization method in step (5) is mainly as follows: according to the positive proportional relationship between the content of each component in the sample and its peak area detected by the HPLC-ICP MS coupling device, all components in the sample can generate signals, corresponding chromatographic peaks can be obtained, and the corresponding morphology can be known from the retention time of each component, thereby the content of each component can be calculated using the normalization formula.
[0018] Compared with the existing detection technology, the present invention has the following beneficial effects:
[0019] The present invention optimizes and adjusts the pretreatment method of tea samples, and uses HPLC-ICP MS to detect the selenium forms of samples, so as to qualitatively determine the selenium forms in the samples; and quantitatively determine the selenium forms in the samples by normalization. The method is simple and fast, reduces experimental errors, and improves detection efficiency and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 It is a schematic diagram of the on-machine detection result of the present invention. DETAILED DESCRIPTION
[0021] The following is an explanation with reference to an actual implementation example.
[0022] Unless otherwise specified, the following materials or reagents are commercially available.
[0023] The embodiment of the present invention provides a method for quantitatively detecting the content of selenium forms in tea leaves, comprising the following steps:
[0024] (1) Sample preparation: The tea samples were washed with ultrapure water and then refrigerated in a -80°C refrigerator. After refrigeration for 24 hours, the samples were taken out and placed in a freeze dryer for freeze drying for 24 hours. The samples were then ground and pulverized under liquid nitrogen conditions, and the ground samples were sieved through a 100-mesh screen.
[0025] (2) Sample treatment: Weigh 0.5-1g of sample into a centrifuge tube, add 10-15mL of cellulase solution and 10-15mL of protease solution to the sample to be tested, and the enzyme content in the enzyme solution is 0.1%-0.5%. Ultrasonicate the centrifuge tube at 37℃ for 30min, and then shake at 37℃ and 200rpm for 12-24h. After shaking, add 10mL of dichloromethane to the sample, shake well, and store in a 4℃ refrigerator until the sample turns white and the upper aqueous phase is clear. Take out the sample, centrifuge the sample at 3000rpm for 10min, and transfer all the upper aqueous phase to a new 50mL polypropylene tube. Use 5mL of the liquid to detect the total selenium content, and place the remaining liquid in a vacuum refrigerated centrifugal concentrator, adjust the cold trap temperature to -105℃, and the concentrator temperature to -4℃, and concentrate until the remaining liquid is about 2mL.
[0026] (3) Preparation of mobile phase: Weigh ammonium citrate and add ultrapure water, stir evenly with a glass rod, add frozen chromatography methanol, and adjust the pH to 5.7 with 10w% formic acid; filter the mobile phase with a 0.22μm filter membrane, and then ultrasonically degas the prepared mobile phase for 30min.
[0027] (4) Data processing: The normalization method was used to quantitatively analyze tetravalent selenium (Se4+), hexavalent selenium (Se6+), selenomethionine (SeMet), methylselenocysteine (SeMeCys) and selenocysteine (SeCys2) in the samples.
[0028] Example 1
[0029] (1) Sample preparation: The tea samples were washed with ultrapure water, dried at 45°C for 24 hours, crushed and ground, and passed through a 100-mesh sieve.
[0030] (2) Sample treatment: Weigh 0.5g of sample into a centrifuge tube, add 10mL of cellulase solution and 15mL of protease solution to the sample to be tested, and the enzyme content in the enzyme solution is 0.5%. Ultrasonicate the centrifuge tube at 37℃ for 30min, and then shake at 37℃ and 200rpm for 12h. After shaking, add 10mL of dichloromethane to the sample, shake well, and store in a 4℃ refrigerator until the sample turns white and the upper aqueous phase is clear. Take out the sample, centrifuge the sample at 3000rpm for 10min, and transfer all the upper aqueous phase to a new 50mL polypropylene tube. Use 5mL of the liquid to detect the total selenium content, and place the remaining liquid in a vacuum refrigerated centrifugal concentrator, adjust the cold trap temperature to -105℃, and the concentrator temperature to -4℃, and concentrate until the remaining liquid is about 2mL.
[0031] (3) Preparation of mobile phase: Weigh ammonium citrate and add ultrapure water, stir evenly with a glass rod, add frozen chromatography methanol, and adjust the pH to 5.7 with 10w% formic acid; filter the mobile phase with a 0.22μm filter membrane, and then ultrasonically degas the prepared mobile phase for 30min.
[0032] (4) Test on the machine, the test results are shown in Figure 1 .
[0033] Example 2
[0034] (1) Sample preparation: The tea samples were washed with ultrapure water and then refrigerated in a -80°C refrigerator. After 24 hours, the samples were taken out and placed in a freeze dryer for freeze drying for 24 hours. The samples were then ground and pulverized under liquid nitrogen conditions, and the ground samples were sieved through a 100-mesh sieve.
[0035] (2) Sample treatment: Weigh 0.5g of sample into a centrifuge tube, add 10mL of cellulase solution and 15mL of protease solution to the sample to be tested, and the enzyme content in the enzyme solution is 0.1%. Ultrasonicate the centrifuge tube at 37℃ for 30min, and then shake at 37℃ and 200rpm for 12h. After shaking, add 10mL of dichloromethane to the sample, shake well, and store in a 4℃ refrigerator until the sample turns white and the upper aqueous phase is clear. Take out the sample, centrifuge the sample at 3000rpm for 10min, and transfer all the upper aqueous phase to a new 50mL polypropylene tube. Use 5mL of the liquid to detect the total selenium content, and place the remaining liquid in a vacuum refrigerated centrifugal concentrator, adjust the cold trap temperature to -105℃, and the concentrator temperature to -4℃, and concentrate until the remaining liquid is about 2mL.
[0036] (3) Preparation of mobile phase: Weigh ammonium citrate and add ultrapure water, stir evenly with a glass rod, add frozen chromatography methanol, and adjust the pH to 5.7 with 10w% formic acid; filter the mobile phase with a 0.22μm filter membrane, and then ultrasonically degas the prepared mobile phase for 30min.
[0037] (4) Test on the machine, the test results are shown in Figure 1 .
[0038] Example 3
[0039] (1) Sample preparation: The tea samples were washed with ultrapure water and then refrigerated in a -80°C refrigerator. After 24 hours, the samples were taken out and placed in a freeze dryer for freeze drying for 24 hours. The samples were then ground and pulverized under liquid nitrogen conditions, and the ground samples were sieved through a 100-mesh sieve.
[0040] (2) Sample treatment: Weigh 1g of sample into a centrifuge tube, add 10mL of cellulase solution and 15mL of protease solution to the sample to be tested, and the enzyme content in the enzyme solution is 0.5%. Ultrasonicate the centrifuge tube at 37℃ for 30min, and then shake at 37℃ and 200rpm for 12h. After shaking, add 10mL of dichloromethane to the sample, shake well, and store in a 4℃ refrigerator until the sample turns white and the upper aqueous phase is clear. Take out the sample, centrifuge the sample at 3000rpm for 10min, and transfer all the upper aqueous phase to a new 50mL polypropylene tube. Use 5mL of the liquid to detect the total selenium content, and place the remaining liquid in a vacuum refrigerated centrifugal concentrator, adjust the cold trap temperature to -105℃, and the concentrator temperature to -4℃, and concentrate until the remaining liquid is about 2mL.
[0041] (3) Preparation of mobile phase: Weigh ammonium citrate and add ultrapure water, stir evenly with a glass rod, add frozen chromatography methanol, and adjust the pH to 5.7 with 10w% formic acid; filter the mobile phase with a 0.22μm filter membrane, and then ultrasonically degas the prepared mobile phase for 30min.
[0042] (4) Test on the machine, the test results are shown in Figure 1 .
[0043] Example 4
[0044] (1) Sample preparation: The tea samples were washed with ultrapure water and then refrigerated in a -80°C refrigerator. After 24 hours, the samples were taken out and placed in a freeze dryer for freeze drying for 24 hours. The samples were then ground and pulverized under liquid nitrogen conditions, and the ground samples were sieved through a 100-mesh sieve.
[0045] (2) Sample treatment: Weigh 1g of sample into a centrifuge tube, add 15mL of cellulase solution and 10mL of protease solution to the sample to be tested, and the enzyme content in the enzyme solution is 0.1%. Ultrasonicate the centrifuge tube at 37℃ for 30min, and then shake at 37℃ and 200rpm for 12h. After shaking, add 10mL of dichloromethane to the sample, shake well, and store in a 4℃ refrigerator until the sample turns white and the upper aqueous phase is clear. Take out the sample, centrifuge the sample at 3000rpm for 10min, and transfer all the upper aqueous phase to a new 50mL polypropylene tube. Use 5mL of the liquid to detect the total selenium content, and place the remaining liquid in a vacuum refrigerated centrifugal concentrator, adjust the cold trap temperature to -105℃, and the concentrator temperature to -4℃, and concentrate until the remaining liquid is about 2mL.
[0046] (3) Preparation of mobile phase: Weigh ammonium citrate and add ultrapure water, stir evenly with a glass rod, add frozen chromatography methanol, and adjust the pH to 5.7 with 10w% formic acid; filter the mobile phase with a 0.22μm filter membrane, and then ultrasonically degas the prepared mobile phase for 30min.
[0047] (4) Test on the machine, the test results are shown in Figure 1 .
[0048] Example 5
[0049] (1) Sample preparation: The tea samples were washed with ultrapure water and then refrigerated in a -80°C refrigerator. After 24 hours, the samples were taken out and placed in a freeze dryer for freeze drying for 24 hours. The samples were then ground and pulverized under liquid nitrogen conditions, and the ground samples were sieved through a 100-mesh sieve.
[0050] (2) Sample treatment: Weigh 1g of sample into a centrifuge tube, add 15mL of cellulase solution and 15mL of protease solution to the sample to be tested, and the enzyme content in the enzyme solution is 0.5%. Ultrasonicate the centrifuge tube at 37℃ for 30min, and then shake at 37℃ and 200rpm for 12h. After shaking, add 10mL of dichloromethane to the sample, shake well, and store in a 4℃ refrigerator until the sample turns white and the upper aqueous phase is clear. Take out the sample, centrifuge the sample at 3000rpm for 10min, and transfer all the upper aqueous phase to a new 50mL polypropylene tube. Use 5mL of the liquid to detect the total selenium content, and place the remaining liquid in a vacuum refrigerated centrifugal concentrator, adjust the cold trap temperature to -105℃, and the concentrator temperature to -4℃, and concentrate until the remaining liquid is about 2mL.
[0051] (3) Preparation of mobile phase: Weigh ammonium citrate and add ultrapure water, stir evenly with a glass rod, add frozen chromatography methanol, and adjust the pH to 5.7 with 10w% formic acid; filter the mobile phase with a 0.22μm filter membrane, and then ultrasonically degas the prepared mobile phase for 30min.
[0052] (4) Test on the machine, the test results are shown in Figure 1 .
[0053] Depend on Figure 1 It can be seen that under different treatments, the content and extraction rate of different selenium forms in the same sample are different. By comparing the five groups of examples, it is found that under the treatment of Example 2, the content and extraction rate of different selenium forms are better, so the best treatment is Example 2.
[0054] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A method for quantitatively detecting the content of selenium forms in tea, characterized in that The standard selenium form solution and tea sample enzymatic hydrolysate were detected by high performance liquid chromatography and inductively coupled plasma mass spectrometry. The tetravalent selenium (Se 4+ ), hexavalent selenium (Se 6+ ), selenomethionine (SeMet), methylselenocysteine (SeMeCys) and selenocysteine (SeCys2), and the contents of the five selenium forms in the sample can be obtained by calculation.
2. A method for quantitatively detecting the content of selenium forms in tea according to claim 1, characterized in that The method comprises the following steps: (1) Sample preparation: cleaning, freeze-drying, grinding and sieving of tea samples; (2) Sample processing: Add cellulase solution and protease solution to the sample to be tested, shake and sonicate, enzymolyze overnight, extract and centrifuge, concentrate and re-dissolve, (3) Instrument configuration: High performance liquid chromatography and inductively coupled plasma mass spectrometry were used to detect the selenium form of the sample. (4) Preparation of mobile phase: ammonium salt solution and methanol are prepared into methanol-diammonium citrate buffer, and formic acid is used to adjust the pH. The mobile phase is filtered through a 0.22 μm filter membrane and ultrasonically degassed to obtain a mobile phase; (5) Data processing: The normalization method was used to analyze the tetravalent selenium (Se 4+ ), hexavalent selenium (Se 6+ ), selenomethionine (SeMet), methylselenocysteine (SeMeCys) and selenocysteine (SeCys2) were quantitatively analyzed.
3. Sample preparation and freeze drying: vacuum freeze drying is adopted at -80℃, and the freeze drying time is 12-24 hours.
4. Sample preparation Grinding and crushing: Grind with a pestle under liquid nitrogen conditions.
5. Sample preparation and screening: Use a 60-100 mesh sieve for screening.
6. Sample treatment Cellulase solution: The cellulase content is 0.1% to 0.5%.
7. Sample treatment protease solution: the protease content is 0.1% to 0.5%.
8. Sample treatment: Ultrasound shaking: ultrasonication at 37°C for 30 min, and then shaking at 37°C, 200 rpm for 12 h.
9. Sample processing and extraction: Use dichloromethane as the extraction liquid.
10. The data processing normalization method is mainly: according to the positive proportional relationship between the content of each component in the sample and its peak area detected by high performance liquid chromatography and inductively coupled plasma mass spectrometry, all components in the sample can generate signals and obtain corresponding chromatographic peaks. The corresponding morphology can be known from the retention time of each component, so the content of each component can be calculated by the normalization method.