A method for detecting oxidized condensed tannins in red wines

Abstract

This invention relates to the field of chemical analysis technology, specifically to a method for detecting oxidized condensed tannins in red wine. The test solution containing oxidized condensed tannins is divided into a treatment solution and a blank control solution. An acidic depolymerization reagent is used to depolymerize the treatment solution, yielding a depolymerized solution. Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS) is used to detect the depolymerized solution and the blank control solution separately, and the content of oxidized condensed tannins is calculated. The acidic depolymerization reagent includes menthol furan and hydrochloric acid, with the concentration of menthol furan being 15-25 mmol / L and the concentration of hydrochloric acid being 0.4-0.6 mol / L. This method is the first to achieve simultaneous qualitative and quantitative analysis of the constituent units of natural, quinone-oxidized, and acetaldehyde-oxidized condensed tannins. It has the advantages of being non-toxic and odorless, suitable for routine laboratory use, and provides an effective method for predicting the degree of oxidation and aging potential of wine.

Description

Technical Field

[0001] This invention relates to the field of chemical analysis technology, and more specifically, to a method for detecting oxidized condensed tannins in red wine. Background Technology

[0002] Condensed tannins are polymers composed of flavan-3-ol compounds such as epicatechin, epicatechin, and epicatechin gallate, linked by C4–C8 or C4–C6 bonds. They play a crucial role in the formation of astringency and structure in red wine.

[0003] However, naturally condensed tannins are prone to oxidation during wine fermentation and aging, primarily through quinone and acetaldehyde oxidation pathways. In the non-enzymatic oxidation process of wine, oxygen, mediated by metal ions, produces various reactive intermediates (such as Fe). 3+ Hydrogen peroxide radicals (HOO•, etc.) can oxidize the hydroxyl groups on flavan-3-ols or condensed tannins, generating highly reactive quinone structures. These quinone structures then polymerize with other flavan-3-ol units through the formation of new flavan bonds or intramolecular rearrangements, producing quinone oxidized condensed tannins. Some reactive intermediates (such as the hydroxyl radical HO•) can also oxidize ethanol in wine to acetaldehyde. Acetaldehyde can then undergo nucleophilic addition reactions with flavan-3-ols, mediating the polymerization between flavan-3-ol units through the formation of ethyl-bridged or vinyl-bridged structures, generating acetaldehyde oxidized condensed tannins. The formation of these two types of oxidized condensed tannins can significantly alter the composition, content, and structural characteristics of tannins in wine, thus affecting its astringency. Simultaneously, oxidized condensed tannins can also serve as key chemical markers for predicting the degree of oxidation and aging potential of wine.

[0004] Currently, the main analytical methods for detecting condensed tannins in wine are as follows: (1) Chemical methods such as DMACA method, butanol-hydrochloric acid method and methylcellulose precipitation method are used to detect the total amount of condensed tannins (including both natural and oxidized condensed tannins) with the aid of spectrophotometer or enzyme-linked immunosorbent assay (ELISA) reader. However, this method can only determine the total content of condensed tannins and cannot distinguish between condensed tannins with different degrees of polymerization and different structural compositions.

[0005] (2) The condensed tannin extract was directly detected by liquid chromatography-mass spectrometry. The condensed tannin molecules were analyzed by scanning and extracting specific ions with known structures. However, as the degree of polymerization of condensed tannins increases, the possible molecular structures and the number of isomers also increase sharply. Furthermore, the ionization efficiency of compounds with larger molecular weights in mass spectrometry will decrease significantly. Therefore, this method is only suitable for detecting oligomeric condensed tannins (degree of polymerization less than 4), and cannot be used to analyze the composition of high polymers.

[0006] (3) After chemically depolymerizing condensed tannins using nucleophilic reagents (phloroglucinol, mercaptoacetic acid, mercaptoethylamine, etc.) under acidic conditions, the depolymerization products (flavan-3-ol constituent units) of the condensed tannins are detected by liquid chromatography-mass spectrometry. However, existing analytical methods can only analyze natural condensed tannins, and there are currently no detection methods for quinone oxidized and acetaldehyde oxidized condensed tannins. In addition, commonly used nucleophilic reagents such as phloroglucinol, mercaptoacetic acid, and mercaptoethylamine have low cleavage efficiency for condensed tannins, and some nucleophilic reagents also have an irritating odor and are highly toxic and corrosive. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for detecting oxidized condensed tannins in red wine.

[0008] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: This invention provides a method for detecting oxidized condensed tannins. The test solution containing oxidized condensed tannins is divided into a treatment solution and a blank control solution. An acidic depolymerization reagent is used to depolymerize the treatment solution to obtain a depolymerized solution. Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry is used to detect the depolymerized solution and the blank control solution separately, and the content of oxidized condensed tannins is calculated. The acidic depolymerization reagent includes menthol furan and hydrochloric acid, wherein the concentration of menthol furan is 15-25 mmol / L and the concentration of hydrochloric acid is 0.4-0.6 mol / L.

[0009] Furthermore, the depolymerization temperature is 40-60℃, and the depolymerization time is 60-120 minutes.

[0010] Furthermore, the volume ratio of the acidic depolymerization reagent to the tannin extract is 1:1-1.5.

[0011] Furthermore, the oxidized condensed tannins include natural condensed tannins, quinone oxidized condensed tannins, and acetaldehyde oxidized condensed tannins.

[0012] Furthermore, the depolymerization solution includes condensed tannin terminal units not linked to menthol furan, and also includes condensed tannin extension units linked to the menthol furan; the blank control solution contains only free condensed tannin units.

[0013] Furthermore, in the ultra-high performance liquid chromatography-triple tandem quadrupole mass spectrometry (UHPLC-MS / MS) method, the mobile phase A of the UHPLC is an aqueous formic acid solution with a volume percentage of 0.1%, and the mobile phase B is a formic acid-acetonitrile solution with a volume percentage of 0.1%. The ultra-high performance liquid chromatography employed gradient elution. During elution, the column temperature was 40℃, the flow rate of each mobile phase was 0.4 mL / min, and the injection volume was 2 μL. With the total volume of mobile phase A and mobile phase B being 100%, the elution procedure is as follows:

[0014] Furthermore, the ultra-high performance liquid chromatography column is an InfinityLab Poroshell 120SB-C18 with dimensions of 2.1×150mm and 2.7μm.

[0015] Furthermore, in the ultra-high performance liquid chromatography-triple tandem quadrupole mass spectrometry (UHPLC-MS / MS) method, the triple tandem quadrupole mass spectrometry uses an AJS-ESI ion source, the ionization mode is negative ion mode, the data acquisition mode is multiple reaction monitoring (MRM), the nebulizer pressure is 35 psi, the drying gas flow rate is 8 L / min, and the drying gas temperature is 320℃. In mass spectrometry detection, the qualitative and quantitative ion pair information is as follows:

[0016] Furthermore, the step of calculating the content of oxidized condensed tannins is as follows: the peak area obtained by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry is substituted into the standard curve to calculate the concentration of oxidized condensed tannins; The standard curve is established using standard solutions of multiple concentration gradients, each of which includes a natural flavan-3-ol monomer standard and / or proanthocyanidin dimer B2 standard.

[0017] The present invention also provides a method for detecting oxidized condensed tannins in red wine, wherein tannins are extracted from red wine to obtain a tannin extract, and the above-described detection method is used for detection.

[0018] The beneficial effects of this invention are as follows: (1) The detection method of oxidized condensed tannins of the present invention is based on the principle of chemical depolymerization of nucleophilic reagents, and establishes a brand-new detection method for condensed tannins. For the first time, it realizes the simultaneous qualitative and quantitative analysis of the constituent units of natural, quinone oxidized and acetaldehyde oxidized condensed tannins, filling the gap in the existing technology. (2) The detection method of oxidized condensed tannins of the present invention uses menthol furan as a nucleophilic reagent for chemical depolymerization. The cleavage efficiency of oxidized condensed tannins is not only significantly better than that of traditional nucleophilic reagents, but also has the advantages of being non-toxic and odorless, making it suitable for routine laboratory use. (4) The method for detecting oxidized condensed tannins of the present invention determines the optimal concentrations of HCl and menthol furan, as well as the optimal reaction conditions. It has the advantages of efficient depolymerization, simple experimental operation, and is suitable for high-throughput analysis of wine samples. (5) The method for detecting oxidized condensed tannins of the present invention uses UHPLC-QqQ-MS technology to construct and optimize a comprehensive condensed tannin detection database. The method can achieve targeted detection of 8 natural and 30 oxidized condensed tannin components, and has the characteristics of high sensitivity and high stability. (6) The method for detecting oxidized condensed tannins in red wine of the present invention can effectively detect oxidized condensed tannins in red wine qualitatively and quantitatively, providing an effective method for predicting the degree of oxidation and aging potential of wine. Attached Figure Description

[0019] Figure 1 This is a comparison chart of the yields of quinone oxidized condensed tannin constituent units after chemical depolymerization using four different nucleophiles in Example 1 of the present invention. Figure 2 This is a comparison chart of the yields of acetaldehyde oxidized condensed tannin constituent units after chemical depolymerization using four different nucleophiles in Example 1 of the present invention. Figure 3 This is a graph showing the results of a single-factor analysis of the yield of oxidized tannin components after chemical depolymerization in Example 2 of the present invention. Figure 3 The single factor for A is the concentration of hydrochloric acid. Figure 3 The single factor for B was the concentration of menthol furan. Figure 3 The single factor for C is the reaction temperature. Figure 3 The single factor for D is reaction time. Detailed Implementation

[0020] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0021] The method for detecting oxidized condensed tannins of the present invention involves dividing the test solution containing oxidized condensed tannins into a treatment solution and a blank control solution. An acidic depolymerization reagent is used to depolymerize the treatment solution to obtain a depolymerized solution. Ultra-high performance liquid chromatography-triple tandem quadrupole mass spectrometry is used to detect the depolymerized solution and the blank control solution respectively, and the content of oxidized condensed tannins is calculated. The acidic depolymerization reagent includes menthol furan and hydrochloric acid, with the concentration of menthol furan being 15-25 mmol / L and the concentration of hydrochloric acid being 0.4-0.6 mol / L.

[0022] The detection method for oxidized condensed tannins of the present invention employs a menthol furan nucleophilic reagent to efficiently chemically depolymerize condensed tannins with different degrees of polymerization, thereby enabling a comprehensive analysis of the various constituent units of oxidized condensed tannins. The menthol furan reagent used is non-toxic, harmless, and odorless.

[0023] Meanwhile, this detection method establishes a highly sensitive targeted quantitative method for oxidized condensed tannins based on ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-QqQ-MS). The overall detection procedure is simple and rapid, suitable for high-throughput analysis of wine samples.

[0024] Preferably, the depolymerization temperature is 40-60℃ and the depolymerization time is 60-120 minutes.

[0025] Preferably, the concentration of hydrochloric acid is 0.5 mol / L, the concentration of menthol furan is 20 mmol / L, the depolymerization reaction temperature is 50℃, and the reaction time is 90 min. These conditions ensure the efficient and stable release of oxidized tannin components.

[0026] Preferably, the volume ratio of the acidic depolymerization reagent to the tannin extract is 1:1-1.5; the optimal volume ratio is 1:1.

[0027] The oxidized condensed tannins targeted by the detection method of the present invention, according to whether they are oxidized and the type of oxidized linkage, specifically include natural condensed tannins, quinone oxidized condensed tannins (including β / ε type and γ / δ type) and acetaldehyde oxidized condensed tannins (including ethyl-bridged type and vinyl-bridged type, i.e. e type and v type).

[0028] The oxidized condensed tannins detected in this invention can be divided into three constituent units according to their structure in the tannin molecule and their existence form after chemical depolymerization: free condensed tannin units that are not linked to mentholatum before depolymerization (present in the blank group), terminal units that are not linked to mentholatum after depolymerization, and extended condensed tannin units linked to mentholatum.

[0029] Based on the two classification methods described above, we can obtain 5 natural free / terminal units, 6 quinone oxidized free / terminal units, 9 acetaldehyde oxidized free / terminal units, 3 natural extended units, 10 quinone oxidized extended units, and 5 acetaldehyde oxidized extended units. Their specific names and chemical structures are as follows: Natural free / terminal units: EC, ECG, EGC, EGCG, PB2; Quinone oxidation free / terminal monomers: EC-β / ε-EC, EC-γ / δ-EC, ECG-β / ε-EC, ECG-γ / δ-EC, EGC-β / ε-EC, EGC-β / ε-EGC; Acetaldehyde oxidized free / terminal units: EC-e, EC-e-EC, EC-v-EC, ECG-e, ECG-e-EC, ECG-v-EC, EGC-e, EGC-e-EGC, EGC-v-EC; Natural extension units: EC-MF, ECG-MF, EGC-MF; Quinone oxidation type extension unit: EC-β / ε-EC-MF, EC-γ / δ-EC-MF, EC-β / ε-EC-2MF, ECG-β / ε-EC-MF, ECG-β / ε-ECG-M F, ECG-γ / δ-ECG-MF, ECG-β / ε-EC-2MF, EGC-γ / δ-EC-MF, EGC-β / ε-EC-2MF, EGC-γ / δ-EC-2MF; Acetaldehyde oxidative extension units: EC-e-MF, EC-e-EC-2MF, ECG-e-MF, EGC-e-MF, EGC-v-MF.

[0030] In this context, EC represents catechin and epicatechin; ECG represents epicatechin-3-O-gallate; EGC represents gallatechin and epigallatechin; EGCG represents epigallate-3-O-gallate; PB2 represents proanthocyanidin B2; and MF represents menthol furan adduct.

[0031] In the detection method of this invention, the mobile phase A used in UHPLC-QqQ-MS analysis is an aqueous solution of formic acid with a volume percentage of 0.1%, and the mobile phase B is a formic acid-acetonitrile solution with a volume percentage of 0.1%. The ultra-high performance liquid chromatography employs gradient elution. During elution, the column temperature is 40℃, the flow rate of each mobile phase is 0.4 mL / min, and the injection volume is 2 μL. Based on a total volume of 100% for mobile phases A and B, the elution program is as follows:

[0032] Preferably, the ultra-high performance liquid chromatography column is an InfinityLab Poroshell 120SB-C18 with dimensions of 2.1×150mm and 2.7μm.

[0033] Preferably, in the UHPLC-QqQ-MS analysis, the triple tandem quadrupole mass spectrometer uses an AJS-ESI ion source in negative ion mode, with an nebulizer pressure of 35 psi, a drying gas flow rate of 8 L / min, and a drying gas temperature of 320℃.

[0034] The present invention discloses a method for detecting oxidized condensed tannins in red wine, wherein tannins are extracted from red wine to obtain a tannin extract, and the extract is then detected using the method described above.

[0035] Preferably, tannins in red wine can be extracted using conventional methods, such as solid-phase extraction.

[0036] The specific detection process for oxidized condensed tannins in red wine is as follows: S1. Extraction of condensed tannins from wine: A solid-phase extraction column (Oasis HLB, 3 cc / 60 mg) was activated sequentially with 2 mL of methanol and 2 mL of distilled water. Then, 5 mL of wine sample was added (the wine samples used in Examples 2 and 3 were 2023 vintage Cabernet Sauvignon and Marselan wines from the Manas region of Xinjiang, aged for 12 months in two different oak barrels). The sample was first rinsed with 5 mL of distilled water, then eluted with 20 mL of acetone / water / acetic acid (80:19.5:0.5, v / v / v) solution, and the eluent was collected. The organic phase was removed by rotary evaporation, and the remaining aqueous phase was freeze-dried and redissolved in 1 mL of 50% methanol aqueous solution to obtain the tannin extract.

[0037] S2. Chemical depolymerization of condensed tannins: Place 100 μL of tannin extract in a centrifuge tube, add 100 μL of an acidic depolymerization reagent containing 0.5 mol / L hydrochloric acid and 20 mmol / L menthol furan, and vortex mix for 1 minute. Incubate the mixture in a 50°C metal bath for 90 minutes to carry out the depolymerization reaction. After the reaction is complete, immediately place the centrifuge tube in an ice-water bath to cool and terminate the reaction.

[0038] S3. Filter the depolymerized sample through a 0.22 μm polytetrafluoroethylene microporous membrane. The resulting filtrate is the test solution for subsequent UHPLC-QqQ-MS analysis.

[0039] S4, UHPLC-QqQ-MS analysis: The analysis was performed using an Agilent 1290-6470 ultra-high performance liquid chromatography-triple quadrupole mass spectrometry system.

[0040] Specifically, the chromatographic column was an InfinityLab Poroshell 120SB-C18 (2.1 × 150 mm, 2.7 μm). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. The column temperature was 40 ℃, the flow rate was 0.4 mL / min, and the injection volume was 2 μL. The elution program was: 0–16 min, 5–43% B; 16–17 min, 43–100% B; 17–22 min, 100% B.

[0041] The mass spectrometry parameters were as follows: AJS-ESI ion source, negative ion mode, nebulizer pressure 35 psi, drying gas flow rate 8 L / min, and drying gas temperature 320 ℃. The detection parameters for each compound were obtained in multiple reaction monitoring (MRM) scan mode, as shown in Table 1.

[0042] Table 1. UHPLC-QqQ-MS qualitative and quantitative detection parameters of free / terminal and extended units of natural, quinone-oxidized, and acetaldehyde-oxidized condensed tannins. Note: EC stands for catechin and epicatechin; ECG stands for epicatechin-3- O - Gallic acid esters; EGC, gallatechin and epigallocatechin; EGCG, epigallocatechin-3- O - Gallic acid ester; PB2, proanthocyanidin B2; MF, indicating menthol furan adduct.

[0043] Since there are no commercially available standards for the three classes (free, terminal, and extended) of quinone oxidized and acetaldehyde oxidized condensed tannins, as well as the first class (extended) of natural condensed tannins, only the free and terminal units of natural condensed tannins have commercially available standards, semi-quantitative analysis using standard curves of commercially available standards with the closest structural similarity is necessary for each compound without standards to ensure accuracy.

[0044] Therefore, for each natural free / terminal unit with reference standards, accurate quantification is performed using its corresponding reference standard calibrates; for the monomeric compounds EC-e, ECG-e, and EGC-e in the acetaldehyde oxidized free / terminal unit, semi-quantification is performed using the standard curves of epicatechin (EC), epicatechin-3-O-gallate (ECG), and epicatechin (EGC), respectively; the remaining acetaldehyde oxidized free / terminal units and quinone oxidized free / terminal units are all in dimer form, so semi-quantification is performed using the standard curve of proanthocyanidin B2.

[0045] Following acidic depolymerization of menthol furan, a menthol furan group was added to the structure of all extended units (natural, quinone oxidized, and acetaldehyde oxidized). Therefore, epicatechin-menthol furan adducts can be generated by depolymerizing proanthocyanidin dimer B2 standard solutions at multiple concentration gradients, thereby establishing a standard curve for semi-quantitative analysis of all extended units.

[0046] Specifically, after processing the standard solutions of six natural flavan-3-ol monomers and proanthocyanidin dimer B2 at multiple concentration gradients according to steps 1-3, they were detected by UHPLC-QqQ-MS under the same conditions to obtain the standard curves of each compound concentration versus peak area (Table 2).

[0047] The concentration gradients of catechin and epicatechin standard solutions were: 4, 10, 40, 100, 200, 400, 800, 1200, 1600, and 2000 μmol / L; the concentration gradients of epicatechin-3-O-gallate, gallatechin, epigallocatechin, and epigallocatechin-3-O-gallate were: 1, 2.5, 10, 25, 50, 100, 200, 300, 400, and 500 μmol / L; and the concentration gradients of proanthocyanidin dimer B2 standard solutions were: 1, 5, 10, 50, 250, 500, 1000, 1500, 3000, and 6000 μmol / L.

[0048] In addition, the standard solutions of proanthocyanidin dimer B2 with multiple concentration gradients were processed in the same way as the depolymerized samples in step 2, and the peak area of ​​the epicatechin-menthol furan adduct generated by the depolymerization was detected. A standard curve was established by comparing the peak area with the concentration of the proanthocyanidin dimer B2 standard solution (Table 2).

[0049] Table 2 Quantitative standard curves of UHPLC-QqQ-MS method The effects of the present invention will be illustrated below through specific embodiments.

[0050] Example 1: Verification of the chemical depolymerization efficiency of oxidized tannins by different nucleophiles This embodiment specifically compares the chemical depolymerization efficiency of menthol furan with that of common nucleophiles in the prior art (phloroglucinol, mercaptoacetic acid, mercaptoethylamine).

[0051] Specifically, in this embodiment, quinone oxidized and acetaldehyde-derived condensed tannins were prepared by laccase oxidation and acetaldehyde treatment of epicatechin (EC), epicatechin-3-O-gallate (ECG), epicardocatechin (EGC), and proanthocyanidin B2 in a simulated wine system. Using these two types of oxidized tannins as substrates, chemical depolymerization was performed under uniform conditions (10 mmol / L nucleophile, 0.3 mol / L HCl, 50℃, 30 min) using four nucleophiles: mercaptoethylamine, phloroglucinol, mercaptoacetic acid, and menthol furan. After filtration, the composition and content of oxidized units were determined using the UHPLC-QqQ-MS method established in this invention, thereby comparing the depolymerization efficiency of each reagent on the oxidized tannins.

[0052] The test results of this embodiment are as follows: Figure 1 and 2 As shown in the bar chart, different letters indicate significant differences between different nucleophiles (Duncan test, p<0.05).

[0053] It can be seen that among different nucleophiles, menthol furan exhibits the highest depolymerization efficiency for quinone oxidized ECG units. For acetaldehyde-derived structures, menthol furan also demonstrates the best depolymerization efficiency for both EC and ECG units, significantly outperforming phloroglucinol, mercaptoethylamine, and mercaptoacetic acid. Although phloroglucinol shows good depolymerization effect on EGC-type acetaldehyde-derived units, its overall abundance is low. In summary, menthol furan exhibits the highest overall depolymerization efficiency for both types of oxidized units.

[0054] Example 2 Optimization of chemical depolymerization reaction conditions for menthol furan To maximize the depolymerization efficiency of menthol furan on oxidative condensation tannins, this embodiment employs a systematic approach combining single-factor experiments and orthogonal experiments to comprehensively optimize the acid-catalyzed depolymerization reaction conditions.

[0055] This embodiment selected samples of Cabernet Sauvignon and Marselan wines (2023 vintage from Manas region, Xinjiang) aged for 12 months in two different oak barrels. Equal volumes of condensed tannin extracts from each sample were mixed to create a composite validation sample. Further, hydrochloric acid concentration (A), menthol furan concentration (B), reaction temperature (C), and reaction time (D) were identified as key factors. First, single-factor experiments were conducted to determine the reasonable level ranges for each factor. Based on this, an L9(3) model was designed. 4 Orthogonal experiment.

[0056] The results of the univariate analysis are as follows Figure 3 As shown, different letters on the bar chart indicate significant differences between different levels (Duncan test, p<0.05).

[0057] according to Figure 3 It can be seen that the highest amount of oxidized tannin components were generated when the hydrochloric acid concentration was 0.4, 0.5, and 0.6 mol / L, the menthol furan concentration was 15, 20, and 25 mmol / L, the reaction temperature was 40, 50, and 60 ℃, and the reaction time was 60, 90, and 120 min.

[0058] Further orthogonal experiments (Table 3) showed that the order of significance of the effects of each factor on the yield of the oxidized unit was: hydrochloric acid concentration (A) > reaction time (D) > menthol furan concentration (B) > reaction temperature (C). Through range and variance analysis, the optimal combination of depolymerization conditions was finally determined to be: hydrochloric acid concentration 0.5 mol / L, menthol furan concentration 20 mmol / L, reaction temperature 50℃, and reaction time 90 min. These conditions ensured the efficient and stable release of the oxidized tannin components.

[0059] Table 3. Conditions for the chemical depolymerization of menthol furan (L9 (3) 4 Orthogonal Experiment Data Analysis Table Note: Factor A is the HCl concentration, with three levels of 0.4, 0.5, and 0.6 mol / L; Factor B is the menthol furan concentration, with three levels of 15, 20, and 25 mmol / L; Factor C is the reaction temperature, with three levels of 40, 50, and 60 ℃; Factor D is the reaction time, with three levels of 60, 90, and 120 min.

[0060] Example 3 Methodological Validation To confirm the reliability of the detection method established in this invention, this embodiment systematically validated the optimized menthol furan depolymerization method combined with UHPLC-QqQ-MS / MS quantification, including linearity, sensitivity, and intra-day / inter-day stability. Since there is a lack of commercially available standards for the oxidized tannin unit, this embodiment specifically used mixed samples for validation.

[0061] This embodiment selected samples of Cabernet Sauvignon and Marselan wines (2023 vintage from Manas region of Xinjiang) aged for 12 months in two different oak barrels for the determination of intraday / interday stability. Specifically, equal volumes of condensed tannin extracts from each sample were mixed as a mixed validation sample, and then serially diluted with 50% methanol aqueous solution for the determination of linearity and sensitivity. The results are shown in Table 4.

[0062] Table 4. Linear regression equations, limits of detection (LOD), limits of quantitation (LOQ), intra-day stability, and inter-day stability for the detection methods of natural, quinone-oxidized, and acetaldehyde-oxidized condensed tannin constituent units. Note: EC stands for catechin and epicatechin; ECG stands for epicatechin-3- O - Gallic acid esters; EGC, gallatechin and epigallocatechin; EGCG, epigallocatechin-3- O - Gallic acid ester; PB2, proanthocyanidin B2; MF, indicating menthol furan adduct.

[0063] The above methodological validation results show that this method performs excellently in terms of linearity, sensitivity, and precision: the regression equations R for all target compounds are as follows: 2 >0.99, with most maintaining good linearity in dilution ranges of 50-fold and above; the method limit of detection (LOD) is 0.19–0.87 μmol / L, and the limit of quantitation (LOQ) is 0.62–2.90 μmol / L, with sensitivity significantly superior to traditional detection methods; intra-day and inter-day stability validation showed that, except for a few compounds, the relative standard deviation (RSD) of all targets was less than 15%, demonstrating that the method has good repeatability and stability.

[0064] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for detecting oxidized condensed tannins, characterized in that, The test solution containing oxidized condensed tannins was divided into a treatment solution and a blank control solution. The treatment solution was depolymerized using an acidic depolymerization reagent to obtain a depolymerized solution. The depolymerized solution and the blank control solution were detected by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry to calculate the content of oxidized condensed tannins. The acidic depolymerization reagent includes menthol furan and hydrochloric acid, wherein the concentration of menthol furan is 15-25 mmol / L and the concentration of hydrochloric acid is 0.4-0.6 mol / L.

2. The method for detecting oxidized condensed tannins according to claim 1, characterized in that, The depolymerization temperature is 40-60℃, and the depolymerization time is 60-120 minutes.

3. The method for detecting oxidized condensed tannins according to claim 1, characterized in that, The volume ratio of the acidic depolymerization reagent to the tannin extract is 1:1-1.

5.

4. The method for detecting oxidized condensed tannins according to claim 1, characterized in that, The oxidized condensed tannins include natural condensed tannins, quinone oxidized condensed tannins, and acetaldehyde oxidized condensed tannins.

5. A method for detecting oxidized condensed tannins according to any one of claims 1-4, characterized in that, The depolymerization solution includes condensed tannin terminal units not linked to menthol furan, and also includes condensed tannin extension units linked to the menthol furan; the blank control solution contains only free condensed tannin units.

6. The method for detecting oxidized condensed tannins according to claim 5, characterized in that, In the ultra-high performance liquid chromatography-triple tandem quadrupole mass spectrometry (UHPLC-MS / MS) method, mobile phase A of UHPLC is an aqueous formic acid solution with a volume percentage of 0.1%, and mobile phase B is a formic acid-acetonitrile solution with a volume percentage of 0.1%. The ultra-high performance liquid chromatography employed gradient elution. During elution, the column temperature was 40℃, the flow rate of each mobile phase was 0.4 mL / min, and the injection volume was 2 μL. With the total volume of mobile phase A and mobile phase B being 100%, the elution procedure is as follows: 。 7. The method for detecting oxidized condensed tannins according to claim 6, characterized in that, The ultra-high performance liquid chromatography column used was an InfinityLab Poroshell 120SB-C18 with dimensions of 2.1 × 150 mm and 2.7 μm.

8. The method for detecting oxidized condensed tannins according to claim 6, characterized in that, In the ultra-high performance liquid chromatography-triple tandem quadrupole mass spectrometry (UHPLC-MS / MS) method, the triple tandem quadrupole mass spectrometry uses an AJS-ESI ion source, the ionization mode is negative ion mode, the data acquisition mode is multiple reaction monitoring (MRM) mode, the nebulizer pressure is 35 psi, the drying gas flow rate is 8 L / min, and the drying gas temperature is 320℃. In mass spectrometry detection, the qualitative and quantitative ion pair information is as follows: 。 9. The method for detecting oxidized condensed tannins according to claim 8, characterized in that, The step of calculating the content of oxidized condensed tannins is as follows: the peak area obtained by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry is substituted into the standard curve to calculate the concentration of oxidized condensed tannins; The standard curve is established using standard solutions of multiple concentration gradients, each of which includes a natural flavan-3-ol monomer standard and / or proanthocyanidin dimer B2 standard.

10. A method for detecting oxidized condensed tannins in red wine, characterized in that, Tannins were extracted from red wine to obtain a tannin extract, which was then tested using the detection method described in any one of claims 1-9.

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