Flavan-3-alcohol sulfonate detection method and application thereof

Abstract

The invention provides a detection method of flavan-3-alcohol sulfonate and application of the detection method. The method comprises the following specific steps: preparing and separating flavane-3-alcohol sulfonate, analyzing and screening molecular characteristic correlation and mass spectrum signal response, identifying the structure of flavane-3-alcohol sulfonate, drawing a standard curve of a flavane-3-alcohol sulfonate standard product, and detecting and analyzing flavane-3-alcohol sulfonate in wine. According to the invention, the epicatechin-(4beta)-sulfonate and the epigallocatechin-(4beta)-sulfonate are obtained by inserting sulfonyl into catechin compounds for preparation and separation, and the epicatechin-(4beta)-sulfonate and the epigallocatechin-(4beta)-sulfonate are used as standard substances for detecting the flavan-3-alcohol sulfonate in the grape wine and are applied to detection and analysis of the flavan-3-alcohol sulfonate in the grape wine. The method provided by the invention has the characteristics of simple and rapid compound synthesis route, easy control of operation conditions, high identification accuracy and sensitivity and strong specificity, provides a good method reference for detection of flavan-3-alcohol sulfonate in the wine aging process, and ensures the fragrant and mellow flavor and taste of wine.

Description

Technical Field

[0001] The present invention belongs to the technical field of food detection, and particularly relates to a method for detecting flavan-3-ol sulfonates and its application. Background Art

[0002] Tannin is an important polyphenolic compound, which mainly exists in the skin, seeds and stems of grapes. During the brewing process of wine, it dissolves into the wine through contact with grape juice, has a significant impact on the taste and structure of wine, and brings unique astringency and taste to wine. Tannin can not only provide a framework for wine, be extracted into the wine, react with salivary proteins, produce a sense of astringency, make the mouth feel dry and astringent, make its structure stable and plump, but also increase the complexity and aging potential of wine. At the same time, it has antioxidant effects, helps prevent wine from becoming sour due to oxidation, and thus maintains the best state of wine stored for a long time. During the aging process of wine, ethanol in the wine will gradually oxidize to form acetic acid, and ethanol and acetic acid will further react to form fragrant ester substances, adding rich aroma and flavor to the wine. And oxygen penetrates into the wine through the pores of the oak barrel or cork, reacts with the components in the wine, and can also endow the wine with more complex and rich oxidative aroma and flavor, such as the aroma of nuts, chocolate, coffee, etc. Moreover, tannin, as a natural preservative and antioxidant of wine, can enhance the aging potential of wine, make its taste more mellow, soft and smooth. At the same time, acidity helps to maintain the stability of wine and prevent the growth of microorganisms. However, under the aging conditions of wine, sulfonation of tannin is very easy to occur, releasing a large amount of flavan-3-ol sulfonates, such as sulfonated dimers, trimers and larger oligomers, and their content is greater than the unchanged tannin in wine. Therefore, it will greatly reduce the taste and astringency of wine. At present, there is no systematic, efficient, fast and sensitive method for monitoring the products of tannin sulfonation in wine to achieve the purpose of quantification. Only using conventional chromatographic analysis techniques, due to the high polarity of tannin sulfonation products, it is difficult to separate them from salts in typical chromatographic separations. Therefore, a method for separating, identifying and quantifying tannin sulfonation products in wine by using characteristic substances is invented, which can not only detect the changes of tannin in wine during the aging process in real time, but also broaden the application scope of characteristic substances in other products rich in tannin. Summary of the Invention

[0003] Technical problems to be solved: Aiming at the above technical problems, the purpose of the present invention is to prepare epicatechin-(4β)-sulfonate and epigallocatechin-(4β)-sulfonate as reference standards for detecting flavan-3-ol sulfonates in wine, and apply them to the detection and analysis of flavan-3-ol sulfonates during the aging process of grapes to ensure the fresh, mellow flavor and taste of wine.

[0004] Technical solution: A method for detecting flavan-3-ol sulfonate, comprising the following steps: S1. Add a 1.0 - 15.0 g / L sodium metabisulfite solution to the 1.0 - 5.0 g / L grape skin extract in a volume ratio of 0.5 - 2, adjust the pH to 1.0 - 5.0 with an HCl solution, and carry out a water bath overnight at 30 - 80°C under sealed conditions, and evaporate in a vacuum environment to obtain a sulfonated mixture; S2. Perform liquid chromatography separation on the sulfonated mixture, and concentrate the fractions by evaporating overnight under vacuum conditions to obtain sulfonate compounds with different molecular weights; S3. Analyze the sulfonate compounds with different molecular weights and the sulfonate compounds in aged wine by using liquid chromatography / quadrupole-time-of-flight mass spectrometry technology, and identify and screen 11 sulfonate compounds based on the correlation of molecular characteristics; S4. Perform mass spectrometry signal response analysis on the 11 sulfonate compounds and catechin, and screen 2 sulfonate compound monomers with high response values; S5. Based on the 2 sulfonate compound monomers with high response values, draw a standard curve of the sulfonate compound monomers with high response values; S6. Mix the wine and methanol evenly, perform ultrasonic extraction, centrifugation and filtration to obtain a 1.0 - 5.0 g / L wine extract; S7. According to the standard curve of the sulfonate compound monomers with high response values, quantitatively analyze the content of flavan-3-ol sulfonate in the wine extract by using liquid chromatography-mass spectrometry technology. Further, the concentration of the HCl solution in step S1 is 0.1 - 1 M. Further, the conditions for liquid chromatography separation in step S2 are: injection volume 100 μL, flow rate 3.0 mL / min, mobile phase A 0.1% formic acid, mobile phase B methanol, and the elution gradient is 0 - 2 min, 3.0% B; 2 - 22 min, 3.0% B; 22 - 24 min, 5.1% B; 24 min, 100% B. Further, the conditions for liquid chromatography / quadrupole-time-of-flight mass spectrometry technology in step S3 are: both the drying gas and the sheath gas are nitrogen, the drying gas flow rate is 10.0 L / min, the drying gas temperature is 325°C, the sheath gas temperature is 350°C, the sheath gas flow rate is 11.0 L / min, the atomization pressure is 35 psi, the capillary voltage is 3000 V, the fragmenter voltage is 130 V, the nozzle voltage is 500 V, and the mass-to-charge ratio is 100 - 2000 m / z. Further, the 11 sulfonate compounds in step S3 are composed of 3 flavan-3-ol monomer sulfonates and 8 flavan-3-ol dimer sulfonates. Further, the two sulfonate compound monomers in step S4 are epicatechin-(4β)-sulfonate and epigallocatechin-(4β)-sulfonate. Further, the volume ratio of wine to methanol in step S3 is 0.5 - 2. Use of the method according to any one of the above in detecting flavan-3-ol sulfonates during wine aging. Beneficial effects: 1. The present invention uses sulfonation of catechin compounds to generate sulfonate compounds, and then screens epicatechin-(4β)-sulfonate and epigallocatechin-(4β)-sulfonate according to liquid phase separation, the correlation of molecular characteristics, and the mass spectrometry signal response of catechins, as reference standards for detecting flavan-3-ol sulfonates in wine, so as to be applied to analyze the content of flavan-3-ol sulfonates in wine. 2. The synthesis conditions of epicatechin-(4β)-sulfonate and epigallocatechin-(4β)-sulfonate in the present invention are relatively simple and rapid. Its detection method has the characteristics of high specificity, high sensitivity, high precision, low detection limit, strong specificity, and short detection time, providing a rapid and accurate method for monitoring the sulfonation deterioration of tannins during wine aging, ensuring the quality of wine. It can not only broaden the application scope of characteristic substances in other products rich in tannins, but also provide research ideas for other new methods. Description of the drawings Figure 1 are the molecular structural formulas of epicatechin-(4β)-sulfonate (A) and epigallocatechin-(4β)-sulfonate (B) in Example 1; Figure 2 is the schematic diagram of the cleavage structure of the flavan-3-ol sulfonate monomer in Example 1; Figure 3 is the nuclear magnetic resonance hydrogen spectrum of epicatechin-sulfonate in Example 1; Figure 4 is the 1H-1H COSY nuclear magnetic resonance spectrum of epicatechin-sulfonate in Example 1; Figure 5 is the 1H-13C HSQC nuclear magnetic resonance spectrum of epicatechin-sulfonate in Example 1; Figure 6 is the nuclear magnetic resonance hydrogen spectrum of epigallocatechin-sulfonate in Example 1; Figure 7 is the 1H-1H COSY nuclear magnetic resonance spectrum of epigallocatechin-sulfonate in Example 1; Figure 8 is the 1H-13C HSQC nuclear magnetic resonance spectrum of epigallocatechin-sulfonate in Example 1; Figure 9: Figure 9 A is a comparison diagram of hydrogen chemical shifts of the standard substances epicatechin and epicatechin-(4β)-sulfonate in Example 1; Figure 9 B is a graph comparing the hydrogen chemical shifts of epigallocatechin and epigallocatechin-(4β)-sulfonate in Example 1. DETAILED DESCRIPTION The present invention will be further described below in conjunction with embodiments, which are explanations of the present invention and are not limited to the following embodiments: Example 1 A method for preparing a flavan-3-ol sulfonate standard product comprises the following steps: S1. The anhydrous methanol and the grape skin solution were mixed evenly, ultrasonically extracted, centrifuged at 5000 rpm for 15 min, and filtered to obtain a 1.0 g / L grape skin extract; S2. 10 g / L sodium metabisulfite solution was added to 1.0 g / L grape skin extract at a volume ratio of 1:1, the pH was adjusted to 2.0 with 0.1 M HCl solution, the mixture was placed in a water bath at 50° C. under sealed conditions overnight, and evaporated in a vacuum environment to obtain a sulfonated mixture; S3. The sulfonated mixture was separated by liquid chromatography, and the fractions were concentrated and evaporated overnight under vacuum conditions to obtain sulfonate compounds of different molecular weights; the chromatographic conditions were as follows: a chromatographic column LiChrospher 100RP-18 column, an injection volume of 100 μL, a flow rate of 3.0 mL / min, a mobile phase A of 0.1% formic acid, a mobile phase B of methanol, and an elution gradient of 0-2 min (3.0% B), 2-22 min (3.0% B), 22-24 min (5.1% B); 24 min (100% B); S4. Liquid chromatography / quadrupole-time of flight mass spectrometry (LC-QTOF) was used to analyze sulfonate compounds of different molecular weights and sulfonate compounds of aged wines, and 11 sulfonate compounds were identified and screened based on the correlation of molecular characteristics; (1) Liquid chromatography / quadrupole-time of flight mass spectrometry (LC-QTOF) analysis Liquid chromatography / quadrupole-time of flight mass spectrometry (LC-QTOF) technology was used to analyze the molecular features associated with sulfonate compounds of different molecular weights in aged wines, and a table of putative compounds was compiled based on the masses of monomeric and oligomeric flavan-3-ol sulfonates, as shown in Table 1 ; Specific test methods: ① The sample was run in the negative ion mode using an Agilent dual electrospray ionization (ESI) jet. Nitrogen was used for both the drying gas and the sheath gas, and the parameters were as follows: drying gas flow rate 10.0 L / min; drying gas temperature 325 °C; sheath gas temperature 350 °C; sheath gas flow rate 11.0 L / min; nebulizer pressure 35 psi; capillary voltage 3000 V; fragmentor voltage 130 V; nozzle 500 V. ② The sample was analyzed in the full MS scan mode of m / z 100 - 2000. The instrument was calibrated before each sequence run. During the entire run, reference mass ions were continuously introduced into the ion source at a rate of 3 μL / min to ensure accurate mass calibration. The reference ions were proton-extracted CF3 (m / z 68.9958), purine (m / z 119.0363), and HP-0921 (+ formate, m / z 966.0007). ③ The tandem mass spectrometry (MS / MS) spectra were obtained using the Auto MS / MS mode of the instrument. The source conditions were the same as those for single MS mode acquisition. Precursor ions were obtained with a narrow isolation width (-1.3 amu) to ensure ion selectivity, with a minimum threshold of 200 counts, a collision energy of 35 eV. MS data were obtained at a rate of 3 spectra per second in the range of m / z 10 - 1000, allowing a maximum of two precursors per cycle. The MS / MS acquisition range was m / z 50 - 1000 at a rate of 3 spectra per second. As shown in Table 1, based on the correlation of the molecular characteristics between sulfonate compounds in aged wines and sulfonate compounds with different molecular weights, 11 sulfonate compounds were identified and screened from more than 80% of aged wines. They consisted of 3 flavan-3-ol monomer sulfonates and 8 flavan-3-ol dimer sulfonates. The molecular formula was determined by appropriate isotope distribution and the expected accurate mass value (mass error of 2 ppm); the mass-to-charge ratio was 369.0281 m / z [M-H]. - The detection and isotope distribution of 15 C 14 H Figure 2 O9S of compound 1 (catechin-SO3H 1) and compound 2 (catechin-SO3H 2) indicated that they were catechin and / or epicatechin addition sulfonates. The mass spectrometry fragmentation data and structures of A1 (mass-to-charge ratio 216.9816 m / z), B1 (mass-to-charge ratio 241.9890 m / z), and C1 (mass-to-charge ratio 287.0567 m / z) are shown in Table 2 and Figure 2 as follows, indicating that the sulfonation occurred at the C-4 position. Table 1 Information of 11 sulfonate compounds Note: The compounds with the same molecular formula above were initially not distinguished by stereochemistry, the position of the sulfonic acid bond, or the order of the bond units. The incidence rate is the percentage of wines in which this signal was detected (in 10 tests). Table 2 Flavan-3-ol sulfonate monomers S5. Perform mass spectrometry signal response analysis on 11 sulfonate compounds and catechin, and screen to obtain 2 sulfonate compound monomers with high response values. (2) Mass spectrometry signal response analysis Use liquid chromatography / quadrupole-time-of-flight mass spectrometry to determine the MS signal responses of catechin and 11 sulfonate compounds in 10 different wines. The obtained mass spectrometry response signals are shown in Table 3; The specific test method is as follows: ① The sample is run in negative ion mode using an Agilent dual electrospray ionization (ESI) jet. Both the drying gas and the sheath gas use nitrogen, and the parameters are as follows: drying gas flow rate 10.0 L / min; drying gas temperature 325 °C; sheath gas temperature 350 °C; sheath gas flow rate 11.0 L / min; nebulization pressure 35 psi; capillary voltage 3000 V; fragmenter voltage 130 V; nozzle 500 V; ② The sample is analyzed in the full MS scan mode of m / z 100 - 2000. The instrument is calibrated before running each sequence; during the entire run, the reference mass ions are continuously run into the ion source at a rate of 3 μL / min to ensure accurate mass calibration; the reference ions are CF3 (m / z 68.9958) extracted by protons, purine (m / z 119.0363), and HP-0921 (+ formate, m / z 966.0007); catechin is used as a standard to determine the average relative response of the preliminarily identified flavan-3-ol sulfonates. All analyses are performed in triplicate. Table 3 Mass spectrometry signal responses between 11 sulfonate compounds and catechin Note: The relative response refers to the average relative MS response of potential compounds to catechin in 10 different wines. As can be seen from Table 3, based on the mass spectrometry signal response analysis of 11 sulfonate compounds and catechins, it was determined that the mass spectrometry signal response values of compound 1 (Catechin-SO3H 1, epicatechin-(4β)-sulfonate) and compound 3 (Gallocatechin-SO3H, epigallocatechin-(4β)-sulfonate) in flavan-3-ol monomer sulfonates were relatively strong. Therefore, both were selected as the reference standards for detecting flavan-3-ol sulfonates in wine. The following experiments analyzed the molecular structural formulas and purities of compound 1 (Catechin-SO3H 1, epicatechin-(4β)-sulfonate) and compound 3 (Gallocatechin-SO3H, epigallocatechin-(4β)-sulfonate). (2) Molecular formula and purity In Example 1, epicatechin-(4β)-sulfonate and epigallocatechin-(4β)-sulfonate were evaporated overnight under vacuum, collected using ChemStation software, and data were collected at 280 nm to analyze their molecular formulas and purities. As Figure 1 can be seen, the molecular formula of epicatechin-(4β)-sulfonate is C 15 H 13 O9S, the theoretical mass-to-charge ratio is 369.0280 m / z, and the measured data is 369.0281 m / z. Moreover, the molecular formula of epigallocatechin-(4β)-sulfonate is C 15 H 13 O 10 S, the theoretical mass-to-charge ratio is 385.0229 m / z, and the measured data is 385.0234 m / z, indicating that the theoretical mass-to-charge ratio and the measured data are approximately the same. At the same time, based on the total ion data, the purities of epicatechin-(4β)-sulfonate and epigallocatechin-(4β)-sulfonate are 95% and 97% respectively. (3) NMR analysis A Bruker Avance III 600 MHz nuclear magnetic resonance spectrometer equipped with a 5 mm CPTCIH / C / N / DZ gradient cryoprobe was used, and the temperature was adjusted to 298 K. Then the Bruker ZG30 pulse program was used to collect 1 H data, with an average of 32 scans and 4 dummy scans, a spectral width of 20 ppm, 1000 complex points, and then two-dimensional (2D) 1 H- 1 H correlation spectroscopy (COSY) data were collected. Each scan was performed 2 times, and 2048 and 128 points were obtained for F2 and F1 respectively, with a spectral width of 8.6 ppm and a recycle delay of 1 s; 2D 1 H- 13The acquisition of C heteronuclear single quantum correlation data uses the HSQCETGPSISP2.2 Bruker parameter set, with 4 or 8 scans per increment. 4096 and 256 points are obtained for F2 and F1 respectively, the spectral width is 16 ppm, the recycle delay is 1 s, and the nuclear magnetic resonance data are processed and analyzed in Mestrenova version 11. All chemical shifts ( 1 H and 13 C) are referenced to the methanol methyl peak ( 1 H and 13 C shifts of 3.31 ppm and 49.0 ppm respectively). Table 4 1H and 1 H and 13 13C nuclear magnetic resonance data of epicatechin, epigallocatechin, compound 1 and 3 Note: a indicates that the result of sulfonyl functionalization is a downfield shift at the C-4 position; b indicates a downfield shift of C-2 due to the γ-gauche effect after C-4 functionalization. As can be seen from Table 4, the structures of epicatechin-(4β)-sulfonate and epigallocatechin-(4β)-sulfonate are determined by 1 1H and 2D 1 H- 1 HCOSY and 1 H- 13 13C HSQC nuclear magnetic resonance data, and the corresponding spectra are as shown in Figures 3 - 8 Shown. From Figure 9 It can be seen that Figure 9 A and Figure 9 The arrows in B represent the relevant situations of the peaks in the spectra of epicatechin standard and epigallocatechin standard shifting after the introduction of the sulfonic acid group. The results show that the functionalization of epicatechin-(4β)-sulfonate and epigallocatechin-(4β)-sulfonate is at the C-4 position, because the non-functionalized CH2 protons H-4′ and H-4″ near 2.7 ppm disappear after sulfonation, generating a new CH proton downfield near 5.4 ppm, and due to the electron-withdrawing effect of the sulfonyl group, both C-4 and H-4 are significantly deshielded; the SO3H group is biaxial with H-2, and due to the γ-guche effect, both C-2 and H-2 at the C-4 position are significantly shifted forward after SO2 functionalization. In addition, the peak shapes and J coupling constants between adjacent protons H-2, H-3 and H-4 indicate a cis-trans relationship between the C-2, C-3 and C-4 substituents, and at the same time there are small J coupling values between adjacent protons, indicating that on the one hand, there are electronegative substituents at the C-2, C-3 and C-4 positions, tending to reduce 3 J HHCoupling values, on the other hand, the cis-trans relationship between adjacent protons results in a dihedral angle close to 90°, and according to the Karplus relationship, there is a small J value. Example 2 Determination of the limit of quantitation (LOQ) for flavan-3-ol sulfonates In Example 1, epicatechin-(4β)-sulfonate, epigallocatechin-(4β)-sulfonate, (+)-catechin, (-)-epicatechin, (+)-gallocatechin, (-)-epigallocatechin were quantified by LC-MS. The calibration curve ranges were 0.4 - 100 mg / L respectively. Sinapic acid (50 mg / L) was added as an internal standard to each single component. Method validation was determined by the spiked recovery rate of standard compounds, the precision of analysis, and the limit of quantitation (LOQ). Then, a low-concentration standard solution (0.4 mg / L) was injected 7 times to measure the LOQ. All analyses were performed in triplicate. Verification of the linear range and LOQ: The linearity was evaluated by the correlation coefficient (R 2 ) of the standard curve, where the calibration concentration was the independent variable (x), and the respective area ratio (compound area / respective internal standard area); the LOQ was defined as the dilution concentration when the signal-to-noise ratio (S / N) was 10. The data was obtained using weighted least-squares linear regression analysis, and the weighting factor was 1 / x; each substance showed good linearity within the corresponding range, the average recovery rate was between 85.83% and 99.37%, the detection limit was 0.07 mg / L, which was 10 times the standard deviation of the low-concentration standard (0.4 mg / L). The correlation coefficient (R 2 ) of all calibration curves was greater than 0.90. Table 5 Linear range and LOQ of each compound in Example 2 Table 6 Recovery rate and precision of each compound in Example 2 The linear range, LOQ, recovery rate, and precision of (+)-catechin, (-)-epicatechin, (+)-gallocatechin, and (-)-epigallocatechin were determined to ensure the accuracy and reliability of the detection results of the methodological parameters, and to reduce the influence of the detection results deviating from the actual values due to unqualified methodological parameters, thereby affecting the judgment of wine quality or process optimization. As can be seen from Table 5 and Table 6, the LOQ was 0.46 - 0.55 mg / L, indicating that this method has excellent detection ability for trace sulfonates; the R of the linear range 2All are greater than 0.99, which can ensure the quantitative accuracy of samples with different concentrations; the average recovery rate is 85.83 - 99.37%, which can verify the quantitative ability of the method for the target substance and ensure that the detection results are close to the true values; the relative standard deviation is calculated through multiple parallel experiments, RSD ≤ 6.11%, indicating that the method has good repeatability and ensures the consistency and stability of experimental operations; in summary, this method has the advantages of high accuracy, low detection limit and quantification limit, good linear correlation, and good precision. Example 3 A method for detecting flavan-3-ol sulfonates in Cabernet Sauvignon wine A (with a free SO2 content of 30 mg / L at bottling), comprising the following steps: S1. Mix anhydrous methanol and the wine extract evenly, perform ultrasonic extraction, centrifuge at 5000 rpm for 15 min, and filter to obtain a 1.0 g / L wine extract. S2. Use the epicatechin-(4β)-sulfonate and epigallocatechin-(4β)-sulfonate prepared in Example 1 as standards, draw the standard curves of epicatechin-(4β)-sulfonate and epigallocatechin-(4β)-sulfonate, and use liquid chromatography-mass spectrometry to detect and analyze the content of flavan-3-ol sulfonates in the 1.0 g / L wine extract. Example 4 A method for detecting flavan-3-ol sulfonates in Cabernet Sauvignon wine B (with a free SO2 content of 60 mg / L at bottling), comprising the following steps: S1. Mix anhydrous methanol and the wine extract evenly, perform ultrasonic extraction, centrifuge at 5000 rpm for 15 min, and filter to obtain a 1.0 g / L wine extract. S2. Use the epicatechin-(4β)-sulfonate and epigallocatechin-(4β)-sulfonate prepared in Example 1 as standards, draw the standard curves of epicatechin-(4β)-sulfonate and epigallocatechin-(4β)-sulfonate, and use liquid chromatography-mass spectrometry to detect and analyze the content of flavan-3-ol sulfonates in the 1.0 g / L wine extract. Example 5 A method for detecting flavan-3-ol sulfonates in Cabernet Sauvignon wine C (with a free SO2 content of 120 mg / L at bottling), comprising the following steps: S1. Mix anhydrous methanol and the wine extract evenly, perform ultrasonic extraction, centrifuge at 5000 rpm for 15 min, and filter to obtain a 1.0 g / L wine extract. S2. Using the epicatechin-(4β)-sulfonate and epigallocatechin-(4β)-sulfonate prepared in Example 1 as standard samples, draw the standard curves of epicatechin-(4β)-sulfonate and epigallocatechin-(4β)-sulfonate, and use liquid chromatography-mass spectrometry to detect and analyze the content of flavan-3-ol sulfonate in the 1.0 g / L wine extract. Index determination (1) Content of flavan-3-ol sulfonate Use liquid chromatography-mass spectrometry to detect the content of flavan-3-ol sulfonate in the wines of Examples 3-5, specifically as follows: ① Standard curve Drawing of the epicatechin-(4β)-sulfonate standard curve: Take a 1 mL centrifuge tube containing freeze-dried powder of epicatechin-(4β)-sulfonate, add 1 mL of 80% ethanol (v / v) for reconstitution, and sequentially dilute it with 80% ethanol (v / v) to prepare epicatechin-(4β)-sulfonate standard solutions with concentrations of 1, 5, 10, 20, 50, 75, and 100 mg / L. After passing through a 0.22 μm nylon organic filter, inject it into a brown sample bottle for LC-MS detection; Drawing of the epigallocatechin-(4β)-sulfonate standard curve: Take a 1 mL centrifuge tube containing freeze-dried powder of the epigallocatechin-(4β)-sulfonate standard curve, add 1 mL of 80% ethanol (v / v) for reconstitution, and sequentially dilute it with 80% ethanol (v / v) to prepare epigallocatechin-(4β)-sulfonate standard curve standard solutions with concentrations of 1, 5, 10, 20, 50, 75, and 100 mg / L. After passing through a 0.22 μm nylon organic filter, inject it into a brown sample bottle for LC-MS detection; The equation of the epicatechin-(4β)-sulfonate standard curve is y = 0.0985x + 0.0032 (R 2 = 0.9956), and the equation of the epigallocatechin-(4β)-sulfonate standard curve is y = 0.1024x + 0.0018 (R 2 = 0.9978). ② Sample detection: The wine samples were filtered through a 0.22 μm filter membrane and then subjected to LC-MS detection and analysis. The sample detection was repeated three times, and the external standard method based on peak area was used for quantification. The chromatographic conditions were as follows: Agilent 1290 ultra-high performance liquid chromatograph (UHPLC), C18 Phenomenex chromatographic column (100×4.60 mm, 2.6 μm), mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was 0.1% formic acid acetonitrile aqueous solution, the flow rate was 0.5 mL / min, and the column temperature was 25°C; the elution gradient was 0 min, 3% B; 2 min, 3% B; 10 min, 6% B; 25 min, 42% B; 30 min, 100% B; 32 min, 100% B; 33 min, 3% B; 36 min, 3% B; the samples in the auto-sampler tray were maintained at 10°C. Table 7 Content of flavan-3-ol sulfonates in the wine sample of Example 3 (mg / L) Compound Parallel 1 Parallel 2 Parallel 3 Mean Epicatechin-(4β)-sulfonate 18.76 18.49 18.03 18.47 Epigallocatechin-(4β)-sulfonate 10.73 10.96 10.21 10.63 Table 8 Content of flavan-3-ol sulfonates in the wine sample of Example 4 (mg / L) Compound Parallel 1 Parallel 2 Parallel 3 Mean Epicatechin-(4β)-sulfonate 23.95 23.25 23.14 23.45 Epigallocatechin-(4β)-sulfonate 18.16 17.99 18.66 18.27 Table 9 Content of flavan-3-ol sulfonates in the wine sample of Example 5 (mg / L) Compound Parallel 1 Parallel 2 Parallel 3 Mean Epicatechin-(4β)-sulfonate 45.61 44.83 45.17 45.20 Epigallocatechin-(4β)-sulfonate 34.57 34.14 35.76 34.82 As can be seen from Table 7, Table 8 and Table 9, this method can successfully quantify the flavan-3-ol sulfonates in the wines of Examples 3-5 and has good reproducibility. As described above, it is only a preferred embodiment of the present invention and does not impose any form of limitation on the present invention. Any person skilled in the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for detecting flavan-3-ol sulfonate, characterized in that: The following steps are involved: S1. Add 1.0-15.0 g / L sodium metabisulfite solution to 1.0-5.0 g / L grape skin extract at a volume ratio of 0.5-2, adjust the pH to 1.0-5.0 with HCl solution, place in a water bath at 30-80 ° C overnight under sealed conditions, and evaporate in a vacuum environment to obtain a sulfonated mixture; S2. The sulfonated mixture was separated by liquid chromatography, and the fractions were concentrated and evaporated overnight under vacuum conditions to obtain sulfonate compounds of different molecular weights; S3. Analyze sulfonate compounds of different molecular weights and sulfonate compounds of aged wine using liquid chromatography / quadrupole-time of flight mass spectrometry, and identify and screen 11 sulfonate compounds based on the correlation of molecular characteristics; S4. performing mass spectrometry signal response analysis on the 11 sulfonate compounds and catechins, and screening out 2 sulfonate compound monomers with high response values; S5. Based on two high response sulfonate compound monomers, a high response sulfonate compound monomer standard curve is drawn; S6. Evenly mix the wine and methanol, perform ultrasonic extraction, and centrifuge to obtain a 1.0-5.0 g / L wine extract; S7. Based on the high response value sulfonate compound monomer standard curve, liquid chromatography-mass spectrometry was used to quantitatively analyze the content of flavan-3-ol sulfonate in the wine extract.

2. The method for detecting flavan-3-ol sulfonate according to claim 1, characterized in that: The concentration of the HCl solution in step S1 is 0.1-1M.

3. The method for detecting flavan-3-ol sulfonate according to claim 1, characterized in that: The conditions for liquid chromatography separation in step S2 are: injection volume 100 μL, flow rate 3.0 mL / min, mobile phase A 0.1% formic acid, mobile phase B methanol, elution gradient 0-2 min, 3.0% B; 2-22 min, 3.0% B; 22-24 min, 5.1% B; 24 min, 100% B.

4. The method for detecting flavan-3-ol sulfonate according to claim 1, characterized in that: The conditions of the liquid chromatography / quadrupole-time-of-flight mass spectrometry technology in step S3 are: the drying gas and the sheath gas are both nitrogen, the drying gas flow rate is 10.0 L / min, the drying gas temperature is 325°C, the sheath gas temperature is 350°C, the sheath gas flow rate is 11.0 L / min, the nebulization pressure is 35 psi, the capillary voltage is 3000 V, the fragmentor voltage is 130 V, the nozzle voltage is 500 V, and the mass-to-charge ratio is 100-2000 m / z.

5. The method for detecting flavan-3-ol sulfonate according to claim 1, characterized in that: The 11 sulfonate compounds in step S3 are composed of 3 flavan-3-ol monomer sulfonates and 8 flavan-3-ol dimer sulfonates.

6. The method for detecting flavan-3-ol sulfonate according to claim 1, characterized in that: In the step S4, the two high response value sulfonate compound monomers are epicatechin-(4β)-sulfonate and epigallocatechin-(4β)-sulfonate.

7. The method for detecting flavan-3-ol sulfonate according to claim 1, characterized in that: In step S3, the volume ratio of wine to methanol is 0.5-2.

8. Use of the method according to any one of claims 1 to 7 in detecting flavan-3-ol sulfonates during wine aging.

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