A method for detecting enantiomers of furanyl linalool oxide glucoside in tea

Abstract

A method for detecting the enantiomers of furanyl linalool oxide glucoside in tea leaves belongs to the technical field of tea leaf detection and analysis. It comprises the following steps: preparing a furanyl linalool oxide glucoside racemic standard solution; performing chiral ultra-high performance liquid chromatography-mass spectrometry analysis on the furanyl linalool oxide glucoside standard solution to determine the qualitative parameters of the furanyl linalool oxide glucoside enantiomers, and drawing a standard working curve of the furanyl linalool oxide enantiomers; performing chiral analysis on the furanyl linalool oxide glucoside enantiomers in the tea soup under the same conditions; and calculating the content of the furanyl linalool oxide glucoside enantiomers in the tea leaves by referring to the qualitative parameters and the standard working curve in the standard product. The present invention can realize the accurate qualitative and quantitative detection of the furanyl linalool oxide glucoside enantiomers in tea leaves.

Description

Technical Field

[0001] The invention belongs to the technical field of tea detection and analysis, and particularly relates to a method for detecting furanyl linalool oxide glucoside enantiomers in tea. Background Art

[0002] Furan linalool oxide is a terpene alcohol compound with a five-membered furan ring as its structural skeleton. It was first detected in tea in 1964 and is widely present in nature. It is found in plants and processed foods such as tea, wine, coffee, honey, violets, carrots, and essential oils. Furan linalool oxide has a strong, sweet, woody, and penetrating aroma, with a floral, woody, and earthy undertone, and plays a pivotal role in the formation of the flavor quality of food or plants. According to the spatial position differences between the double bond carbon and other groups in its structure, furan linalool oxide can be divided into cis-furan linalool oxide and trans-furan linalool oxide. At the optical level, the chiral center in the furan ring structure gives it two pairs of enantiomers, so furan linalool oxide has four isomers in essence, including (2R,5R)-trans furan linalool oxide, (2S,5S)-trans furan linalool oxide, (2R,5S)-cis furan linalool oxide and (2S,5R)-cis furan linalool oxide. The optical isomers of these four linalool oxides have significant differences in aroma type and odor threshold. For example, (2R,5R)-trans furan linalool oxide and (2R,5S)-cis furan linalool oxide have green leaf and earthy aromas, and (2S,5S)-trans furan linalool oxide and (2S,5R)-cis furan linalool oxide have sweet, floral and creamy aromas. Whether in fresh tea leaves or finished tea, the content of furanyl linalool oxide is relatively high, which plays a vital role in the formation of tea aroma quality. The composition and content of the four enantiomers are closely related to the tea tree variety, origin, storage time, processing technology, etc., and have specific distribution characteristics in specific types of tea. Therefore, it has great potential in tea variety identification, origin traceability, and year determination.

[0003] The furanyl linalool oxide in tea mainly originates from the hydrolysis release of glycosidically bonded furanyl linalool oxide precursors, and some of them are converted by the oxidation of linalool. Furanyl linalool oxide glucoside is one of the most common glycoside structures in tea. It is stable and easy to store, and has broad application prospects in the field of aroma sustained release. This glycoside has four enantiomers corresponding to the structure of free furanyl linalool oxide, namely (2R, 5R)-trans furanyl linalool oxide glucoside, (2S, 5S)-trans furanyl linalool oxide glucoside, (2R, 5S)-cis furanyl linalool oxide glucoside and (2S, 5R)-cis furanyl linalool oxide glucoside. However, key scientific issues such as their corresponding transformation relationship with the free aglycones of the corresponding configuration, differences in aroma release characteristics and their specific contribution to the formation of tea aroma quality have not yet been clarified, so it has extremely high research significance and research value. However, since these four enantiomers are highly similar in terms of compound structure, physical and chemical properties, and lack conjugated groups in their chemical structures, have weak ultraviolet absorption, and cannot be detected in conventional liquid chromatography, there are great difficulties in the separation and characterization of the compounds. In recent years, although the mass spectrum information of furanyl linalool oxide glucoside can be accurately obtained by liquid chromatography-mass spectrometry to qualitatively characterize the compound, at most only cis- and trans-furanyl linalool oxide glucoside can be separated, and the deep optical configuration isomers cannot be directly separated, and there are few related research reports.

[0004] The classic analysis strategy for chiral compounds is to achieve separation by utilizing the difference in the strength of the interaction between the target enantiomer and the chiral environment (chiral stationary phase, mobile phase, etc.). However, since one type of chiral stationary phase (such as cyclodextrin structure) can be derived into chiral stationary phases with a variety of different groups, there are currently hundreds of chiral stationary phase structures reported and circulated on the market, and the price is also relatively expensive (an average of 10,000 yuan per root). Technicians need to rely on chemical foundations and previous analytical experience to determine the types of chiral stationary phases that may be effective, so as to achieve rapid separation and reduce economic costs. In the chiral separation of furanyl linalool oxide glucoside, early studies often used acid hydrolysis or enzymatic hydrolysis to decompose the glycoside to release the corresponding aglycone, and then chirally separated the aglycone to determine the configuration and content of the glycoside. However, under acidic conditions, glycosides are very likely to rearrange to form other terpene compounds with similar structures. The reaction efficiency and reaction conditions of enzymatic hydrolysis are unclear, and the hydrolysis results obtained may be inconsistent with the actual situation. The experimental accuracy cannot be guaranteed, and the experimental processing is also relatively complicated. Therefore, there are great limitations in the analytical method.

[0005] In addition, there is currently no intuitive analytical technology that can achieve the chiral separation of furanyl linalool oxide glucoside, which affects people's scientific understanding of the formation mechanism of tea aroma quality and also limits the practical application of such quality components in the tea industry. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention aims to design and provide a technical solution for a method for detecting the enantiomers of furanyl linalool oxide glucoside in tea. The method has simple pretreatment, easy operation, and accurate quantification, and can realize the detection of the enantiomers of furanyl linalool oxide glucoside in different types of tea.

[0007] The technical solution adopted by the present invention is as follows:

[0008] The method for detecting the enantiomers of furanyl linalool oxide glucoside in tea leaves comprises the following steps:

[0009] 1) preparing a furanyl linalool oxide glucoside racemate standard solution;

[0010] 2) performing chiral ultra performance liquid chromatography-mass spectrometry UPLC-QQQ-MS analysis on the standard solutions of furanyl linalool oxide glucoside with gradient concentrations to determine the qualitative parameters of the enantiomers of furanyl linalool oxide glucoside, and drawing a standard working curve of the enantiomers of furanyl linalool oxide;

[0011] 3) extracting the furanyl linalool oxide glucoside enantiomers in the tea soup, and performing chiral UPLC-QQQ-MS analysis on the furanyl linalool oxide glucoside enantiomers in the tea soup under the conditions of step 2);

[0012] 4) Calculate the enantiomer content of furanyl linalool oxide glucoside in tea by referring to the qualitative parameters and standard working curve in the standard.

[0013] Furthermore, in the step 1), the concentration of the racemized furanyl linalool oxide glucoside standard solution is 5 to 20 mg / L, preferably 8.0 mg / L.

[0014] Further, the gradient concentration of furanyl linalool oxide glucoside standard solution in step 2) is specifically as follows: accurately weigh the furanyl linalool oxide glucoside enantiomer standard, dissolve it in pure water to prepare a 1000.00 mg / L standard stock solution, and then use a pipette with a suitable range to respectively absorb 0.5 mL, 0.8 mL, and 1 mL of the standard stock solution, and gradually dilute it into a series of concentrations of standard curve working solutions.

[0015] Further, the chromatographic conditions in the UPLC-QQQ-MS analysis conditions in step 2) are:

[0016] Chromatographic column: Asec CYCLOBONDR I 2000HP-RSP, specification: 15 cm × 4.6 mm × 5 μm;

[0017] Mobile phase: Mobile phase A is pure aqueous solution, mobile phase B is acetonitrile;

[0018] Gradient elution conditions: 0.00-2.00min, 10-15% B; 2.00-4.00min, 15% B; 4.00-5.00min, 15-20% B; 5.00-7.00min, 20% B; 7.00-8.00min, 20-25% B; 8.00-11.00min, 25-40% B; 11.00-11.10min, 40-90% B; 11.10-14.00min, 90% B; 14.00-14.10min, 90-10% B; 14.10-19.00min, 10% B.

[0019] Further, the mass spectrometry conditions in the UPLC-QQQ-MS analysis conditions in step 2) are:

[0020] The ionization mode was positive ion mode ESI+, the scanning mode was selected ion monitoring, the capillary voltage was 3.0 kV, the desolvation gas N2 temperature was 500 °C, the collision gas was argon, the cone gas flow rate was 50 L / hr, the desolvation gas flow rate was 800 L / hr, the dwell time was 0.050 s, and the collision energy was 30 V.

[0021] Furthermore, the drawing of the standard curve in step 2) is specifically as follows: a linear equation is established with the concentration of the furanyl linalool oxide glucoside enantiomer standard as the abscissa and the peak area of ​​the furanyl linalool oxide glucoside enantiomer quantitative ion peak as the ordinate.

[0022] Furthermore, the preparation method of the tea extract in step 3) is:

[0023] Weigh 500.0 mg ± 0.5 mg of tea powder sample into a centrifuge tube, add 5 mL of boiling purified water, shake well, soak in a 70°C water bath for 30 minutes, and centrifuge at low temperature for 10 minutes; take the supernatant and filter it through a 0.22 μm membrane into a sample bottle for later use.

[0024] Furthermore, the hand chromatography column is Supelco Asec CYCLOBONDR I 2000HP-RSP; the injection volume is 2 μL, the flow rate is 0.4 mL / min, and the column temperature is 3°C.

[0025] The method provided by the present invention can detect the enantiomers of furanyl linalool oxide glucoside in tea samples, and the qualitative parameters are shown in Table 1.

[0026] Table 1 Qualitative parameters of furanyl linalool oxide glucoside enantiomers

[0027]

[0028] The method provided by the present invention shows a good linear relationship in a wide concentration range (0.0125-10.00 mg / L), with a correlation coefficient as high as 0.9933, and can achieve accurate qualitative and quantitative detection of the enantiomers of furanyl linalool oxide glucoside in tea.

[0029] The present invention uses high performance liquid chromatography-mass spectrometry combined with chiral chromatography technology to successfully separate the enantiomers of furanyl linalool oxide glucoside in tea for the first time. The method can achieve simultaneous and accurate quantitative detection of furanyl linalool oxide glucoside enantiomers, with a wide linear concentration range and a high correlation coefficient. The method uses less solvent, is simple in pretreatment, is easy to operate, and can greatly avoid the "peak overlap" phenomenon that is difficult to avoid in other detection methods. It has broad application prospects in the theoretical research of tea quality chemistry and the regulation of tea flavor quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the structural formula diagram of furanyl linalool oxide glucoside;

[0031] Figure 2 is the chromatogram of the enantiomer standard of furanyl linalool oxide glucoside;

[0032] Figure 3 This is the chromatogram of the enantiomers of furanyl linalool oxide glucoside detected in No. 4 Yunnan white tea;

[0033] Figure 4 This is the content distribution of furanyl linalool oxide glucoside enantiomers in Yunnan white tea. DETAILED DESCRIPTION

[0034] The present invention is further explained below with reference to examples and drawings. This example is only used to specifically illustrate the method, but not to limit the scope of the present invention. Any changes or improvements made according to the teachings of the present invention belong to the protection scope of the present invention.

[0035] Example 1: Analysis of enantiomers of furanyl linalool oxide glucoside by chromatographic and mass spectrometric conditions

[0036] Specific steps:

[0037] 1) Preparation of mixed standard solution of furanyl linalool oxide glucoside enantiomers

[0038] Unracemize the furanyl linalool oxide glucoside standard, add pure water to dissolve it, prepare a furanyl linalool oxide glucoside mixed racemate standard of appropriate concentration, and store it at -20℃ for later use.

[0039] 2) Optimization of chromatographic and mass spectrometric conditions for chiral UPLC-QQQ-MS analysis:

[0040] In order to obtain the best separation effect, this experiment first screened the chiral chromatographic columns, and then further optimized other chromatographic conditions on this basis. The specific optimization parameters included: type of mobile phase reagent (methanol / acetonitrile, pure water / formic acid water), elution gradient, column temperature, flow rate, etc., see Table 2 for details.

[0041] When optimizing the mass spectrometry conditions, the ionization mode and scanning mode were screened, and the relevant parameters were further optimized, including SIR target ions, MRM product ions, collision energy, dwell time, etc. (see Table 3 for details).

[0042] Table 2 Chromatographic conditions optimization information

[0043]

[0044] Table 3 Mass spectrometry condition optimization information

[0045]

[0046] The experimental results show that the separation effect of acetonitrile and pure water as mobile phases is better than that of methanol and formic acid water, so pure water is selected as mobile phase A and acetonitrile is selected as mobile phase B. The scanning mode used in this method is selected ion monitoring (SIR), and the quantitative parameter selects the ion (355.00) with the strongest response of furanyl linalool oxide glucoside enantiomer for qualitative and quantitative analysis of the target compound. The residence time is 0.050s, the collision energy is 20V, and the ion mode selects positive ions. The optimization results ensure the maximum signal intensity of the target compound. For specific conditions, refer to the content of the invention.

[0047] Example 2: Establishment of a quantitative analysis method for furanyl linalool oxide glucoside enantiomers in tea

[0048] Specific steps:

[0049] 1) Preparation of mixed standard solution of furanyl linalool oxide glucoside enantiomers

[0050] The racemic furanyl linalool oxide glucoside standard was removed and dissolved in pure water to prepare a mixed racemic furanyl linalool oxide glucoside standard with a concentration of 1000.00 mg / L as a mother solution, which was stored at -20°C for later use.

[0051] 2) Establishment of the standard curve of furanyl linalool oxide glucoside enantiomers by external standard method

[0052] Use a pipette with a suitable range to draw 0.5mL, 0.8mL, and 1.0mL of the mixed standard mother solution into a 15mL centrifuge tube, add 1.0mL, 1.5mL, 1.7mL or 4.5mL of pure water, prepare a series of concentrations (0.025-25mg / L) of furanyl linalool oxide glucoside mixed racemic standard solutions, transfer them to a 2.0mL liquid phase injection bottle containing a 200μL inner insert, and let stand for use. After UPLC-QQQ-MS analysis of each concentration of the standard, the concentration of the furanyl linalool oxide glucoside enantiomer is used as the abscissa and the peak area of ​​the corresponding quantitative ion peak is used as the ordinate to draw a standard curve to obtain the linear range and correlation coefficient.

[0053] Chiral UPLC-QQQ-MS analysis conditions: The chromatographic column used was Asec CYCLOBONDR I 2000HP-RSP (15 cm×4.6 mm, 5 μm, Supelco, Germany).

[0054] Chromatographic conditions: mobile phase A is pure aqueous solution, mobile phase B is acetonitrile; gradient elution conditions: 0.00-2.00min, 10-15%B; 2.00-4.00min, 15%B; 4.00-5.00min, 15-20%B; 5.00-7.00min, 20%B; 7.00-8.00min, 20-25%B; 8.00-11.00min, 25-40%B; 11.00-11.10min, 40-90%B; 11.10-14.00min, 90%B; 14.00-14.10min, 90-10%B; 14.10-19.00min, 10%B;; the injection volume is 2μL, the flow rate is 0.4mL / min, and the column temperature is 3°C.

[0055] Mass spectrometry conditions: ionization mode was positive ion mode ESI+, scanning mode was selected ion monitoring, capillary voltage was 3.0 kV, desolvation gas (N2) temperature was 500 °C, collision gas was argon, cone gas flow rate was 50 L / hr, desolvation gas flow rate was 800 L / hr, dwell time was 0.050 s, and collision energy was 30 V.

[0056] Finally, the linear range and correlation coefficient shown in Table 4 were obtained.

[0057] Table 4 Working curves of furanyl linalool oxide glucosides with different configurations

[0058]

[0059] Example 3: Separation of enantiomers of furanyl linalool oxide glucoside in Yunnan white tea

[0060] Tea sample preparation: 6 Yunnan white teas from different manufacturers were collected and numbered Yunnan white tea No. 1 to No. 6. The collected tea samples were ground with a grinder and passed through a 40-mesh sieve for later use.

[0061] The specific extraction steps of furanyl linalool oxide glucoside enantiomers in tea soup are as follows: weigh 500.0 mg (± 0.5 mg) of tea powder sample into a centrifuge tube, add 5 mL of boiling pure water, shake well, extract in a 70°C water bath for 30 minutes, and centrifuge at low temperature for 10 minutes; take the supernatant and filter it through a 0.22 μm membrane into a sample bottle for later use, and then perform chiral UPLC-QQQ-MS analysis. The chromatogram of furanyl linalool oxide glucoside enantiomers in No. 4 Yunnan white tea is shown in the figure. Figure 3 shown.

[0062] Chiral UPLC-QQQ-MS analysis conditions: The chromatographic column used was Asec CYCLOBONDR I 2000HP-RSP (15 cm×4.6 mm, 5 μm, Supelco, Germany). Chromatographic conditions: mobile phase A is pure aqueous solution, mobile phase B is acetonitrile; gradient elution conditions: 0.00-2.00min, 10-15%B; 2.00-4.00min, 15%B; 4.00-5.00min, 15-20%B; 5.00-7.00min, 20%B; 7.00-8.00min, 20-25%B; 8.00-11.00min, 25-40%B; 11.00-11.10min, 40-90%B; 11.10-14.00min, 90%B; 14.00-14.10min, 90-10%B; 14.10-19.00min, 10%B; the injection volume is 2μL, the flow rate is 0.4mL / min, the column is connected to an ice water bath, and the column temperature is 3°C. Mass spectrometry conditions: ionization mode was positive ion mode ESI+, scanning mode was selected ion monitoring, capillary voltage was 3.0 kV, desolvation gas (N2) temperature was 500 °C, collision gas was argon, cone gas flow rate was 50 L / hr, desolvation gas flow rate was 800 L / hr, dwell time was 0.050 s, and collision energy was 30 V.

[0063] Distribution of enantiomers of furanyl linalool oxide glucoside in Yunnan white tea Figure 4As shown, four furanyl linalool oxide glucosides with different configurations were detected in all six types of Yunnan white tea.

[0064] In summary, the method of the present invention uses less solvent, has simple pretreatment, and is easy to operate, so that the accurate qualitative and quantitative determination of the enantiomers of furanyl linalool oxide glucoside in tea can be achieved. In the examples of the present invention, only the content of the enantiomers of furanyl linalool oxide glucoside in some teas is determined. If more other types of teas or more types of glycosidically bonded terpenoid compound enantiomers need to be determined, the detection can be carried out according to the guidance of the present invention or after optimization by using appropriate methods. The above extended contents all belong to the protection scope of the present invention.

Claims

1. A method for detecting enantiomers of furanyl linalool oxide glucoside in tea, characterized in that The following steps are involved: 1) Prepare the racemic standard solution of furanyl linalool oxide glucoside; 2) Perform chiral ultra-performance liquid chromatography-mass spectrometry UPLC-QQQ-MS analysis on the standard solutions of furanyl linalool oxide glucoside with gradient concentrations to determine the qualitative parameters of the enantiomers of furanyl linalool oxide glucoside and draw the standard working curve of furanyl linalool oxide enantiomers; The chromatographic conditions in the UPLC-QQQ-MS analysis conditions are: Chromatographic column: Asec CYCLOBONDR I 2000 HP-RSP, size: 15 cm × 4.6 mm × 5 μm; Mobile phase: Mobile phase A is pure aqueous solution, mobile phase B is acetonitrile; Gradient elution conditions: 0.00-2.00 min, 10-15% B; 2.00-4.00min, 15%B; 4.00-5.00min, 15-20%B; 5.00-7.00min, 20%B; 7.00-8.00min, 20-25%B; 8.00-11.00min, 2 5-40%B; 11.00-11.10min, 40-90%B; 11.10-14.00min, 90%B; 14.00-14.10min, 90-10%B; 14.10-19.00min, 10%B; The mass spectrometry conditions in the UPLC-QQQ-MS analysis conditions are: The ionization mode was positive ion mode ESI+, the scanning mode was selected ion monitoring, the capillary voltage was 3.0 kV, the desolvation gas N2 temperature was 500 °C, the collision gas was argon, the cone gas flow rate was 50 L / hr, the desolvation gas flow rate was 800 L / hr, the dwell time was 0.050 s, and the collision energy was 30 V; 3) extracting the enantiomers of furanyl linalool oxide glucoside in the tea soup, and performing chiral UPLC-QQQ-MS analysis on the enantiomers of furanyl linalool oxide glucoside in the tea soup using the conditions of step 2); 4) Calculate the enantiomer content of furanyl linalool oxide glucoside in tea by referring to the qualitative parameters and standard working curve in the standard.

2. A method for detecting enantiomers of furanyl linalool oxide glucoside in tea leaves as claimed in claim 1, characterized in that In the step 1), the concentration of the racemic furanyl linalool oxide glucoside standard solution is 5-20 mg / L.

3. A method for detecting enantiomers of furanyl linalool oxide glucoside in tea leaves as claimed in claim 1, characterized in that The gradient concentration furanyl linalool oxide glucoside standard solution in step 2) is specifically as follows: accurately weigh the furanyl linalool oxide glucoside enantiomer standard, dissolve it in pure water to prepare a 1000.00 mg / L standard stock solution, and then use a pipette with a suitable range to respectively draw 0.5 mL, 0.8 mL, and 1 mL of the standard stock solution, and gradually dilute it into a series of concentrations of standard curve working solutions.

4. A method for detecting enantiomers of furanyl linalool oxide glucoside in tea leaves as claimed in claim 1, characterized in that The drawing of the standard curve in step 2) is specifically as follows: a linear equation is established with the concentration of the furanyl linalool oxide glucoside enantiomer standard as the abscissa and the peak area of ​​the furanyl linalool oxide glucoside enantiomer quantitative ion peak as the ordinate.

5. A method for detecting enantiomers of furanyl linalool oxide glucoside in tea leaves as claimed in claim 1, characterized in that The preparation method of the tea extract in step 3) is: Weigh 500.0 mg ± 0.5 mg of tea powder sample into a centrifuge tube, add 5 mL of boiling pure water, shake well, soak in a 70 ºC water bath for 30 minutes, and centrifuge at low temperature for 10 minutes; take the supernatant and filter it through a 0.22 μm membrane into a sample bottle for later use.

6. A method for detecting enantiomers of furanyl linalool oxide glucoside in tea leaves as claimed in claim 1, characterized in that The chromatographic column was Supelco Asec CYCLOBONDR I 2000 HP-RSP; the injection volume was 2 μL, the flow rate was 0.4 mL / min, and the column temperature was 3 ºC.

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