Method for determining stable isotope ratio of caffeine hydrogen in tea leaves

Abstract

The invention belongs to the technical field of stable isotope analysis, and discloses a method for determining the stable isotope ratio of caffeine hydrogen in tea leaves. According to the method, caffeine in the tea leaves is extracted through the processes of water extraction, liquid-liquid extraction and the like, a large number of impurity components are effectively removed, the GC-IRMS with an online high-temperature cracking reaction device is used for carrying out caffeine separation and hydrogen stable isotope ratio determination on the caffeine extract, and the method has the advantages of being easy and convenient to operate, high in efficiency and good in repeatability; technical support and scientific basis are provided for tea producing area traceability by using a monomer isotope technology.

Description

Technical Field

[0001] This invention belongs to the field of stable isotope analysis technology, and specifically relates to a method for determining the stable isotope ratio of caffeine hydrogen in tea. Background Technology

[0002] Stable hydrogen isotope ratio (δ) 2 Caffeine (C8H) is an important indicator for geographical traceability and authenticity verification, widely used in the field of food authenticity identification. Hydrogen isotopes undergo fractionation in natural processes, and their compositional characteristics are closely related to regional climate, precipitation, and environmental factors, thus effectively reflecting the origin information of plants and animals. As a globally consumed beverage, tea's authenticity verification has significant economic and market value. 10 N4O2 is a characteristic component in tea with stable content and highly consistent molecular structure. All its hydrogen atoms exist in the form of CH bonds, which are non-exchangeable hydrogens. It is not easy to exchange hydrogen with water molecules in the environment, and can reliably retain the original isotopic signal related to the origin. Therefore, it is an ideal biomarker for tracing the origin of tea.

[0003] Currently, there are two main technical approaches for determining the stable hydrogen isotope ratios in organic compounds: one is to determine the overall δ¹⁸O values ​​of the purified sample using elemental analysis-stable isotope ratio mass spectrometry (EA-IRMS). 2 The method involves two main approaches: first, the extraction and high-purity purification of caffeine from a tea matrix; and second, the online separation and determination of specific compound monomers using gas chromatography-stable isotope ratio mass spectrometry (GC-IRMS). The EA-IRMS method requires the initial extraction and high-purity purification of caffeine from a tea matrix. This pretreatment process is complex, and tea often contains co-extractants such as pigments, polyphenols, and sugars, which are difficult to completely remove. These impurities are burned along with the caffeine in EA-IRMS, introducing hydrogen from non-caffeine sources, thus leading to δ-values. 2 The H measurement value showed a significant deviation, affecting the accuracy of the traceability results.

[0004] In contrast, GC-IRMS offers advantages in online separation and quantitative analysis, allowing direct δ²H determination of caffeine monomers in complex mixtures and effectively avoiding interference from co-extraction impurities. However, successfully applying this technology to the tea-caffeine system still faces a series of challenges: First, the polarity and thermal stability of caffeine require optimized chromatographic separation conditions to avoid interference from co-elution peaks; second, in GC-IRMS analysis, the chromatographic separation process, injection port discrimination effect, and high-temperature pyrolysis reactions may introduce additional hydrogen isotope fractionation, which must be controlled and corrected through rigorous calibration and standardization procedures; furthermore, existing literature has not yet established a GC-IRMS analytical method system specifically for tea matrix that can guarantee analytical accuracy and reproducibility.

[0005] Therefore, there is an urgent need in this field to develop a method for analyzing the hydrogen stable isotope ratio of tea caffeine based on GC-IRMS technology, to achieve efficient separation of caffeine, to have a reliable correction strategy to overcome instrument-related effects, and ultimately to provide accurate and stable isotope data support for tea origin traceability. Summary of the Invention

[0006] In view of this, the present invention provides a method for determining the stable isotope ratio of caffeine monomers in tea. By extracting and semi-purifying caffeine from tea through processes such as water extraction combined with liquid-liquid extraction, caffeine can be effectively extracted from tea while reducing impurity content, thus meeting the requirements for determining the stable hydrogen isotope ratio of caffeine in tea using GC-IRMS.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for determining the hydrogen stable isotope ratio of caffeine in tea involves extracting caffeine from tea using hot water extraction and a dichloromethane / water solution system, separating the caffeine from other impurities using gas chromatography, and analyzing the hydrogen stable isotope ratio δ of caffeine in tea using isotope mass spectrometry. 2 H, and then based on the δ in the hydrogen stable isotope standard material 2 H value was measured, and the δ value of caffeine in tea was calibrated and calculated. 2 H value.

[0008] Preferably, the specific steps include: (1) Caffeine extraction: The tea leaves were crushed and ground using a grinder. The tea powder sample was weighed into a centrifuge tube, pure water was added, and caffeine was extracted in a constant temperature water bath. Solid impurities were removed by filtration. The filtrate was extracted with a non-polar solvent. After standing and separating the layers, the lower layer solution was filtered through anhydrous sodium sulfate to remove water, and then rotary evaporated and dried to constant weight to obtain crude caffeine extract, which was then transferred to acetone solvent for analysis. (2) Caffeine separation: Caffeine was separated from other impurities using a gas chromatograph with a capillary gas chromatography column; (3) Hydrogen stable isotope determination: The H in caffeine was converted into H2 using an online high-temperature pyrolysis reaction tube, and the δ of H2 was determined by isotope mass spectrometry. 2 H; (4) Result correction: The measurement results were corrected using stable isotope standard substances to obtain the δ of caffeine in tea. 2 H value.

[0009] Preferably, the mass ratio of the tea powder sample to pure water in step (1) is 2:25.

[0010] Preferably, the temperature of the water bath in step (1) is 95 °C and the extraction time is 30 min.

[0011] The beneficial effects of the above technical solution are as follows: pure water extraction can reduce the interference of fat-soluble components such as fatty acids and fat-soluble pigments; under hot water conditions, it helps to increase the solubility of caffeine in water and improve the extraction rate of caffeine. Filtration can remove solid impurities such as cellulose, insoluble salts, and some proteins.

[0012] Preferably, the nonpolar solvent in step (1) is dichloromethane or trichloromethane, and the volume ratio of pure water to the nonpolar solvent is 1:1.

[0013] The beneficial effects of the above technical solution are: the filtrate is extracted using a non-polar solvent, which can effectively reduce the interference of components such as tea polyphenols.

[0014] Preferably, the drying temperature in step (1) is 100-105℃ and the time is 30min.

[0015] The beneficial effect of the above technical solution is that it can effectively remove the moisture bound to caffeine at this drying temperature.

[0016] Preferably, the specific operation of caffeine separation in step (2) includes: the injection volume is 1 μL, the injection port temperature is 250℃, the initial temperature is 80℃, the retention time is 1 min, the temperature is increased to 250℃ at 20℃ / min, the retention time is 2 min, the temperature is increased to 280℃ at 20℃ / min, and the retention time is 2 min; the carrier gas flow rate is 1.4 mL / min, the splitless mode is used, and the chromatographic column is a weakly polar chromatographic column.

[0017] Preferably, the high-temperature pyrolysis reaction tube in step (3) is made of ceramic and the temperature is 1380-1420 ℃.

[0018] Preferably, in step (4), one stable isotope standard material is first used to correct the signal effect of the measurement results, and then three stable isotope standard materials are used to establish a calibration curve to correct the signal effect correction results.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention extracts caffeine from tea leaves through processes such as water extraction and liquid-liquid extraction, effectively removing a large number of impurities. The caffeine extract is then separated and its hydrogen stable isotope ratio is determined using a GC-IRMS device equipped with an online high-temperature pyrolysis reaction device. This method has the advantages of being simple to operate, highly efficient, and reproducible, providing technical support and scientific basis for tracing the origin of tea leaves using monomeric isotope technology. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. The drawings in this description are merely embodiments of the present invention.

[0021] Figure 1 This is the hydrogen stable isotope ion flow map of caffeine in Example 1. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1 1. Instruments and reagents Gas chromatography / high-temperature pyrolysis-stable isotope mass spectrometry (Trace GC 1310 gas chromatograph, GC IsoLink II combustion apparatus, MAT 253 stable isotope ratio mass spectrometer, Thermo Fisher Scientific, USA); XP6 microbalance (sensitivity: 0.001 mg, Mettler Toledo, Switzerland); high-purity helium (99.999%, carrier gas); high-purity hydrogen (99.999%, reference gas); stable isotope standard USGS62 (caffeine, δ¹²) 2 H = -156.1‰); acetone (chromatographic grade, ≥99.8%, Beijing Tongguang Fine Chemical Co., Ltd.).

[0024] 2. Sample Preparation Standard solutions of different concentrations were prepared using acetone as solvent and USGS62: 62.5 mg / L, 125 mg / L, 250 mg / L, 500 mg / L, 1000 mg / L and 2000 mg / L.

[0025] 3. Instrumental analysis conditions (1) Gas chromatography conditions Chromatographic column: TG-5MS capillary column (30 m × 0.25 mm × 0.25 μm); Injector temperature: 250℃; Carrier gas: Helium (purity >99.999%); Carrier gas flow rate: 1.4 mL / min; Injection volume: 1 μL; Split mode: Splitless; Temperature program: Initial temperature 80℃, hold for 1 min, increase to 250℃ at 20℃ / min, hold for 2 min, then increase to 280℃ at 20℃ / min, hold for 2 min; (2) Conditions for pyrolysis tube The pyrolysis tube is a ceramic tube, and the reaction temperature is 1420 ℃; (3) Isotope mass spectrum conditions The ion source vacuum is 1.6 × 10⁻⁶. -6 mBar, voltage 9.5 kV, current 1.50 mA.

[0026] 4. Results Analysis After the hydrogen atoms in the caffeine molecule are converted into hydrogen gas through the pyrolysis tube of the GC-IRMS, they enter the isotope mass spectrometer, producing two isotope ions, m / z 2 and m / z 3. The isotope ion chromatogram is shown below. Figure 1 As shown. GC-IRMS analysis was performed on caffeine samples of different concentrations to investigate δ. 2 The stability of the H value is shown in Table 1. When the caffeine concentration is 62.5 mg / L, i.e., the H content is 6.6 ng, δ 2 The STDEV value of H reached 20.47‰, far exceeding the 3‰ precision requirement accepted in the literature. When the caffeine concentration was 125 mg / L, i.e., the H content was 13.3 ng, δ 2 The accuracy of H measurement is less than 3‰. As shown in Table 1, δ... 2 There is a linear relationship between the H value and the signal value; that is, the higher the signal value, the higher the δ value. 2 The lower the H value, the more necessary signal effect correction is.

[0027] Table 1

[0028] Example 2 1. Instruments and reagents Gas chromatography / high-temperature pyrolysis-stable isotope mass spectrometry (Trace GC 1310 gas chromatograph, GC IsoLink II combustion device, MAT 253 stable isotope ratio mass spectrometer, Thermo Fisher Scientific, USA); XP6 microbalance (sensitivity: 0.001 mg, Mettler Toledo, Switzerland); high-speed grinder (A11, IKA, UK); digital display constant temperature water bath (HH-2, Changzhou Guohua Electric Co., Ltd.); rotary evaporator (R210, BUCHI, Switzerland); high-purity helium (99.999%, carrier gas); high-purity hydrogen (99.999%, reference gas); stable isotope standard USGS76 (methyl heptadecanoate, δ¹⁸O). 2 H = -210.8‰); stable isotope standard USGS61 (caffeine, δ¹⁰) 2 H = +96.9‰); stable isotope standard USGS62 (caffeine, δ¹⁰) 2 H = -156.1‰); stable isotope standard USGS63 (caffeine, δ¹⁵) 2H = +174.5‰); dichloromethane (analytical grade, ≥99.5%, Changshu Hongsheng Fine Chemical Co., Ltd.); acetone (chromatographic grade, ≥99.8%, Beijing Tongguang Fine Chemical Co., Ltd.); anhydrous sodium sulfate (superior grade, Tianjin Kemeio Chemical Reagent Co., Ltd.); ultrapure water (prepared by Milli-Q Integral ultrapure water system).

[0029] 2. Sample pretreatment (1) Water extraction: After crushing the tea sample containing caffeine, weigh 2 g of the tea powder sample into a 50 mL centrifuge tube, add 25 mL of pure water, tighten the cap, and place it in a 95℃ constant temperature water bath for 30 min. (2) Organic solvent extraction: After removing solid impurities by filtration, the filtrate is transferred to a 100 mL separatory funnel and 25 mL of dichloromethane is added for extraction. After gentle shaking, the mixture is allowed to stand and separate into layers. (3) Concentration and drying: After filtering the lower layer solution through anhydrous sodium sulfate to remove water, it is transferred to a flask, and dichloromethane is removed by rotary evaporation. Then, it is dried in an oven at 105°C for 30 min to constant weight to obtain crude caffeine extract. Weigh an appropriate amount of crude caffeine extract, dissolve it in acetone, and then test it.

[0030] 3. Instrumental analysis conditions (1) Gas chromatography conditions Chromatographic column: TG-5MS capillary column (30 m × 0.25 mm × 0.25 μm); Injector temperature: 250℃; Carrier gas: Helium (purity >99.999%); Carrier gas flow rate: 1.4 mL / min; Injection volume: 1 μL; Split mode: Splitless; Temperature program: Initial temperature 80℃, hold for 1 min, increase to 250℃ at 20℃ / min, hold for 2 min, then increase to 280℃ at 20℃ / min, hold for 2 min; (2) Conditions for pyrolysis tube The pyrolysis tube is a ceramic tube, and the reaction temperature is 1420 ℃; (3) Isotope mass spectrum conditions The ion source vacuum is 1.6 × 10⁻⁶. -6 mBar, voltage 9.5 kV, current 1.50 mA.

[0031] 4.Isotope correction First, the signal value is calibrated using the USGS62, then the delta values ​​of the USGS76, USGS61, and USGS63 are used. 2 H measurement value is the x-axis, and δ of USGS76, USGS61 and USGS63 are used as the y-axis. 2 Using the actual value of H as the ordinate, an isotope ratio correction curve is established to correct the δ of the sample under test.2 H value.

[0032] The analytical results are shown in Table 2. The standard deviation of the measured samples was 1.8‰, indicating that the method has good reproducibility.

[0033] Table 2

[0034] Example 3 1. Instruments and reagents Gas chromatography / high-temperature pyrolysis-stable isotope mass spectrometry (Trace GC 1310 gas chromatograph, GC IsoLink II combustion device, MAT 253 stable isotope ratio mass spectrometer, Thermo Fisher Scientific, USA); XP6 microbalance (sensitivity: 0.001 mg, Mettler Toledo, Switzerland); high-speed grinder (A11, IKA, UK); digital display constant temperature water bath (HH-2, Changzhou Guohua Electric Co., Ltd.); rotary evaporator (R210, BUCHI, Switzerland); high-purity helium (99.999%, carrier gas); high-purity hydrogen (99.999%, reference gas); stable isotope standard USGS62 (caffeine, δ¹²) 2 H = -156.1‰); Stable isotope standard USGS76 (methyl heptadecanoate, δ¹⁵ ppm) 2 H = -210.8‰); stable isotope standard USGS61 (caffeine, δ¹⁰) 2 H = +96.9‰); stable isotope standard USGS63 (caffeine, δ¹⁸O) 2 H = +174.5‰); dichloromethane (analytical grade, ≥99.5%, Changshu Hongsheng Fine Chemical Co., Ltd.); acetone (chromatographic grade, ≥99.8%, Beijing Tongguang Fine Chemical Co., Ltd.); anhydrous sodium sulfate (superior grade, Tianjin Kemeio Chemical Reagent Co., Ltd.); ultrapure water (prepared by Milli-Q Integral ultrapure water system).

[0035] 2. Sample pretreatment (1) Addition of standard product: After pulverizing decaffeinated tea leaves, add a certain amount of USGS62; (2) Water extraction: Weigh 2 g of tea powder sample into a 50 mL centrifuge tube, add 25 mL of pure water, tighten the cap, and place in a 95 ℃ constant temperature water bath for 30 min; (3) Organic solvent extraction: After removing solid impurities by filtration, the filtrate is transferred to a 100 mL separatory funnel, and 25 mL of dichloromethane is added. After gentle shaking, the mixture is allowed to stand for separation. (4) Concentration and drying: After filtering the lower layer solution through anhydrous sodium sulfate to remove water, transfer it to a flask, remove dichloromethane by rotary evaporation, and dry it in an oven at 105 °C for 30 min to constant weight to obtain crude caffeine extract. Weigh an appropriate amount of crude caffeine extract, dissolve it in acetone, and then test it.

[0036] 3. Instrumental analysis conditions (1) Gas chromatography conditions Chromatographic column: TG-5MS capillary column (30 m × 0.25 mm × 0.25 μm); Injector temperature: 250 ℃; Carrier gas: Helium (purity >99.999%); Carrier gas flow rate: 1.4 mL / min; Injection volume: 1 μL; Split mode: Splitless; Temperature program: Initial temperature 80 ℃, hold for 1 min, increase to 250 ℃ at 20 ℃ / min, hold for 2 min, then increase to 280 ℃ at 20 ℃ / min, hold for 2 min; (2) Conditions for pyrolysis tube The pyrolysis tube is a ceramic tube, and the reaction temperature is 1420 ℃; (3) Isotope mass spectrum conditions The ion source vacuum is 1.6 × 10⁻⁶. -6 mBar, voltage 9.5 kV, current 1.50 mA; 4.Isotope correction First, the signal value is calibrated using the USGS62, then the delta values ​​of the USGS76, USGS61, and USGS63 are used. 2 H measurement value is the x-axis, and δ of USGS76, USGS61 and USGS63 are used as the y-axis. 2 Using the actual value of H as the ordinate, an isotope ratio correction curve is established to correct the δ of the sample under test. 2 H value.

[0037] The analytical results are shown in Table 3. The δ values ​​of the spiked samples measured according to the method of this invention are... 2 The H value deviates from the true value by 1.7‰, indicating that the method of the present invention will not cause significant fractionation of caffeine hydrogen isotopes.

[0038] Table 3

[0039] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for determining the stable hydrogen isotope ratio of caffeine in tea, characterized in that, Caffeine was extracted from tea leaves using hot water extraction and a dichloromethane / water solution system. Gas chromatography was then used to separate the caffeine from other impurities. The hydrogen stable isotope ratio δ of the caffeine in the tea leaves was analyzed using isotope mass spectrometry. 2 H, and then based on the δ in the hydrogen stable isotope standard material 2 H value was measured, and the δ value of caffeine in tea was calibrated and calculated. 2 H value.

2. The method for determining the stable hydrogen isotope ratio of caffeine in tea according to claim 1, characterized in that, Specifically, the steps include the following: (1) Caffeine extraction: The tea leaves were crushed and ground using a grinder. The tea powder sample was weighed into a centrifuge tube, pure water was added, and caffeine was extracted in a constant temperature water bath. Solid impurities were removed by filtration. The filtrate was extracted with a non-polar solvent. After standing and separating the layers, the lower layer solution was filtered through anhydrous sodium sulfate to remove water, and then rotary evaporated and dried to constant weight to obtain crude caffeine extract, which was then transferred to acetone solvent for analysis. (2) Caffeine separation: Caffeine was separated from other impurities using a gas chromatograph with a capillary gas chromatography column; (3) Hydrogen stable isotope determination: The H in caffeine was converted into H2 using an online high-temperature pyrolysis reaction tube, and the δ of H2 was determined by isotope mass spectrometry. 2 H; (4) Result correction: The measurement results were corrected using stable isotope standard substances to obtain the δ of caffeine in tea. 2 H value.

3. The method for determining the stable hydrogen isotope ratio of caffeine in tea according to claim 2, characterized in that, The mass ratio of the tea powder sample to pure water in step (1) is 2:

25.

4. The method for determining the stable hydrogen isotope ratio of caffeine in tea according to claim 2, characterized in that, The temperature of the water bath in step (1) is 95 ℃ and the extraction time is 30 min.

5. The method for determining the stable hydrogen isotope ratio of caffeine in tea according to claim 2, characterized in that, The non-polar solvent mentioned in step (1) is dichloromethane or trichloromethane, and the volume ratio of pure water to non-polar solvent is 1:

1.

6. The method for determining the stable hydrogen isotope ratio of caffeine in tea according to claim 2, characterized in that, The drying temperature in step (1) is 100-105℃.

7. The method for determining the stable hydrogen isotope ratio of caffeine in tea according to claim 2, characterized in that, The specific operation of caffeine separation in step (2) includes: injection volume of 1 μL, injection port temperature of 250℃, initial temperature of 80℃, retention for 1 min, increasing to 250℃ at 20℃ / min, retention for 2 min, increasing to 280℃ at 20℃ / min, retention for 2 min; carrier gas flow rate of 1.4 mL / min, splitless mode, and weakly polar column.

8. The method for determining the stable hydrogen isotope ratio of caffeine in tea according to claim 2, characterized in that, The high-temperature pyrolysis reaction tube mentioned in step (3) is made of ceramic and the temperature is 1380-1420℃.

9. The method for determining the stable hydrogen isotope ratio of caffeine in tea according to claim 2, characterized in that, In step (4), one stable isotope standard material is first used to correct the signal effect of the measurement results, and then three stable isotope standard materials are used to establish a calibration curve to correct the signal effect correction results.

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