Method for detecting endogenous smoky substances in grape fruits

Abstract

The invention relates to the technical field of grape fruit detection, in particular to a method for detecting endogenous smoky substances in grape fruits. The method comprises the following steps: carrying out solid-phase extraction on a pretreated sample to respectively obtain a free-state eluent and a glycoside-state eluent, carrying out gas chromatography-mass spectrometry detection on the free-state eluent, and carrying out liquid chromatography-mass spectrometry detection on the glycoside-state eluent to obtain a detection result. The method can be used for simultaneously carrying out qualitative and quantitative detection on free eugenol, glycoside eugenol, free eugenol and glycoside eugenol, and a multi-reaction monitoring and scanning method for eugenol, eugenol and glycoside precursors thereof is established on the basis of gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. The detection steps are simple and efficient, the cost is low, and a technical support is provided for concentration evaluation of eugenol / eugenol and its glucoside state in grape fruits.

Description

Technical Field

[0001] The invention relates to the technical field of grape fruit detection, and in particular to a method for detecting endogenous smoky flavor substances in grape fruits. Background Art

[0002] Eugenol and eugenol are important aroma substances in wine. Both compounds can enhance the aroma characteristics of wine such as smoke, earth / dust / soil, medicinal taste, and wood. Their sensory threshold in wine is low, which can easily affect and modify the aroma style of wine. Eugenol and eugenol in wine mainly come from grape fruits and exist in glycoside and free forms. The existing methods can extract and detect the free and glycoside states in grape fruits respectively, but the operation procedures of the existing methods are relatively complicated. At the same time, the glycoside states of eugenol and eugenol in fruits exist in various forms, and these substances lack standard substances, which makes qualitative analysis difficult.

[0003] Liquid chromatography-mass spectrometry can only extract the glycoside form of eugenol / eugenol in the fruit, but cannot extract the free form, and there is a lack of detection methods for glycoside precursors of eugenol. The acid hydrolysis / enzyme hydrolysis method detects the content of eugenol / eugenol after the glycoside precursors are released in grape fruit, which requires enzymatic and acid hydrolysis of the glycoside substances, which is complicated and time-consuming; this method can only detect the total amount of eugenol / eugenol after release, and cannot measure its various glycoside forms separately, which cannot meet research needs. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for detecting endogenous smoky flavor substances in grape fruits, so as to solve the problem in the prior art that it is difficult to simultaneously detect free eugenol, glycoside eugenol, free eugenol and glycoside eugenol.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] The invention provides a method for detecting endogenous smoky flavor substances in grape fruits. The endogenous smoky flavor substances include free eugenol, glycoside eugenol, free eugenol and glycoside eugenol. The detection method comprises: performing solid phase extraction on a pre-treated sample to obtain a free eluent and a glycoside eluent respectively, performing gas chromatography-mass spectrometry detection on the free eluent, and performing liquid chromatography-mass spectrometry detection on the glycoside eluent to obtain a detection result.

[0007] Based on the above technical solution, the present invention can also be improved as follows.

[0008] Further, the following steps are included:

[0009] S1, mixing grape fruit, D-gluconolactone and cross-linked polyvinyl pyrrolidone, grinding, macerating and centrifuging to obtain a grape juice sample;

[0010] S2, adding an internal standard substance for gas chromatography-mass spectrometry detection and an internal standard substance for liquid chromatography-mass spectrometry detection to the grape juice sample to obtain a mixed sample;

[0011] S3, performing the solid phase extraction on the mixed sample, the solid phase extraction comprising the steps of activation, sample loading, elution and washing, wherein during the elution, dichloromethane is first used for washing to obtain the free eluent, and then methanol is used for washing to obtain the glycoside eluent; the volume ratio of the eluent to dichloromethane is 1:1-2, and the volume ratio of the eluent to methanol is 1:1-2;

[0012] S4, removing water and concentrating the free-state eluate, and then performing the gas chromatography-mass spectrometry detection, and calculating the contents of the free-state eugenol and the free-state eugenol according to the detection results;

[0013] S5. Concentrating, redissolving and filtering the glycoside eluate, and then performing the liquid chromatography-mass spectrometry detection, and calculating the contents of the glycoside eugenol and the glycoside eugenol according to the detection results.

[0014] Further, in step S1, the immersion condition is immersion for 3 to 5 hours in a closed environment at a temperature of 3.5°C to 4.5°C; the centrifugation condition is centrifugation at a speed of 7000 to 9000 r / min for 5 to 15 minutes in an environment at a temperature of 3.5°C to 4.5°C; after the centrifugation is completed, the supernatant is collected as the grape juice sample.

[0015] Further, in step S2, the internal standard substance detected by gas chromatography-mass spectrometry is 3,4-dimethylphenol solution, and the internal standard substance detected by liquid chromatography-mass spectrometry is glucose salicin solution; the concentration of the 3,4-dimethylphenol solution is 0.1-0.3 g / L, and the volume ratio of the 3,4-dimethylphenol solution to the grape juice sample is 1:1000; the concentration of the glucose salicin solution is 1.0-2.0 g / L, and the volume ratio of the glucose salicin solution to the grape juice sample is 1:1000.

[0016] Further, in step S3, the eluent is distilled water, the eluent is 5 mL, the dichloromethane is 10 mL, and the methanol is 10 mL.

[0017] Furthermore, in step S3, the solid phase extraction column is activated with methanol and deionized water, and the sample loading flow rate is 1 to 3 mL / min.

[0018] Furthermore, in step S4, anhydrous sodium sulfate is used for dehydration, and nitrogen purge is used for concentration.

[0019] Further, the gas chromatography-mass spectrometry detection is GC-QqQ-MS / MS detection, the chromatographic column is a capillary chromatographic column with a specification of 30m×250μm×0.25μm; the GC injection port temperature is 250°C, the injection volume is 1-2μL, and the injection mode is 2-5:1 split mode injection;

[0020] The carrier gas for GC-QqQ-MS / MS detection is helium with a volume percentage greater than 99.999% and a flow rate of 1.0 mL / min;

[0021] The temperature program of the GC-QqQ-MS / MS detection is as follows: the starting temperature is 40°C; the temperature is increased to 80°C at a rate of 5°C / min; the temperature is increased to 120°C at a rate of 2°C / min; the temperature is increased to 160°C at a rate of 5°C / min; the temperature is finally increased to 220°C at a rate of 30°C / min; the post-run is maintained at 280°C for 1 min;

[0022] The mass spectrometer uses an electron bombardment ion source, the ion source temperature is 230°C, the ionization energy is 70eV, the auxiliary heater temperature is 300°C, and the MS1 and MS2 quadrupole temperatures are both 150°C; the quenching gas is helium with a flow rate of 2.25mL / min; the collision gas is nitrogen with a flow rate of 1.5mL / min; the scanning method is multiple reaction monitoring mode, the characteristic ion pair of eugenol is 164149m / z, the collision energy is 10eV, and the retention time is 25.3min; the characteristic ion pair of eugenol is 154139m / z, the collision energy is 10eV, and the retention time is 25.0min; the characteristic ion pair of the internal standard detected by the gas chromatography-mass spectrometry is 122107m / z, the collision energy is 15eV, and the retention time is 16.7min.

[0023] Further, in step S5, the glycoside eluent is evaporated to dryness by rotary evaporation under vacuum conditions at a temperature of 30° C., and then re-dissolved by acetonitrile aqueous solution; in the acetonitrile aqueous solution, the volume ratio of acetonitrile to water is 5:100.

[0024] Further, the liquid chromatography-mass spectrometry detection is HPLC-QqQ-MS / MS detection, the chromatographic column is a C18 chromatographic column with specifications of 2.1×150 mm, 2.7 μm, and the column temperature is 50° C.;

[0025] Mobile phase A is a 0.1% by volume formic acid aqueous solution, and mobile phase B is a 0.1% by volume formic acid acetonitrile solution; the flow rate is 0.4 mL / min; the injection volume is 5 μL;

[0026] The elution procedure is: 0-12 min, the volume percentage of the mobile phase B is 5-10%; 12-14 min, the volume percentage of the mobile phase B is 10-40%; 14-16 min, the volume percentage of the mobile phase B is 40-100%;

[0027] The mass spectrometry parameters were as follows: AJS ESI ion source, negative ion mode, nebulizer pressure of 30 psi, drying gas flow rate of 7 L / min, and drying gas temperature of 250°C.

[0028] The beneficial effects of the present invention are:

[0029] (1) The method for detecting endogenous smoky flavor substances in grape fruits of the present invention can simultaneously perform qualitative and quantitative detection on free eugenol, glycoside eugenol, free eugenol and glycoside eugenol, so that the detection of endogenous smoky flavor substances is more comprehensive and the detection results are more accurate and objective;

[0030] (2) The method for detecting endogenous smoky flavor substances in grape fruits of the present invention can obtain free and glycoside substances at one time through solid phase extraction, and by optimizing the solid phase extraction process, the detection results of glycoside eugenol and glycoside eugenol are more accurate;

[0031] (3) The method for detecting endogenous smoky flavor substances in grape fruits of the present invention establishes multiple reaction monitoring (MRM) scanning methods for eugenol, eugenol and their glycoside precursors based on gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry, respectively, so that the detection steps are simple, efficient and low-cost;

[0032] (4) The method for detecting endogenous smoky flavor substances in grape fruits of the present invention can realize the convenient extraction and direct quantification of free and glycoside eugenol / eugenol in the fruit, greatly saving the experimental cost and providing technical support for the concentration assessment of eugenol / eugenol and its glycoside in grape fruits. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The chromatograms of the standard and sample of eugenol and eugenol obtained in Example 1 in the method for detecting endogenous smoky flavor substances in grape fruits of the present invention;

[0034] Figure 2 The chromatograms of the eugenol-β-D-rutin standard and sample obtained in Example 1 in the method for detecting endogenous smoky flavor substances in grape fruits of the present invention;

[0035] Figure 3 This is a comparison chart of the response ratios of different water washing liquid volumes and methanol elution volumes to glycoside eugenol in Example 2 in the method for detecting endogenous smoky flavor substances in grape fruits of the present invention; Figure 3 A is a comparison chart of the response ratios obtained when 10 mL of methanol is used for elution and 5 mL, 10 mL, and 15 mL of eluent are used, respectively. Figure 3 B is a comparison chart of the corresponding ratios obtained when 5 mL of eluent is used, and 5 mL, 10 mL, and 15 mL of methanol are used for elution respectively;

[0036] Figure 4 This is a comparison chart of the response ratios of different water washing liquid volumes and methanol elution volumes to glycoside eugenol in Example 2 in the detection method of endogenous smoky flavor substances in grape fruits of the present invention; Figure 4 A is a comparison chart of the corresponding ratios obtained when 10 mL of methanol is used for elution and 5 mL, 10 mL, and 15 mL of eluent are used, respectively. Figure 4 B is a comparison chart of the corresponding ratios obtained when 5 mL of eluent is used and 5 mL, 10 mL and 15 mL of methanol are used for elution. DETAILED DESCRIPTION

[0037] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0038] The invention discloses a method for detecting endogenous smoky flavor substances in grape fruits. The endogenous smoky flavor substances include free eugenol, glycoside eugenol, free eugenol and glycoside eugenol. The detection method includes: performing solid phase extraction on a pre-treated sample to obtain a free eluent and a glycoside eluent, respectively; performing gas chromatography-mass spectrometry detection on the free eluent; performing liquid chromatography-mass spectrometry detection on the glycoside eluent to obtain a detection result.

[0039] The detection method of endogenous smoky flavor substances in grape fruits of the present invention can simultaneously perform qualitative and quantitative detection on free eugenol, glycoside eugenol, free eugenol and glycoside eugenol, so that the detection of endogenous smoky flavor substances is more comprehensive and the detection results are more accurate and objective. The method can obtain free and glycoside substances at one time through solid phase extraction, and establish multiple reaction monitoring (MRM) scanning methods of eugenol, eugenol and their glycoside precursors based on gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry, so that the detection steps are simple, efficient and low in cost, and can realize the convenient extraction and direct quantification of free and glycoside eugenol / eugenol in fruits, greatly saving experimental costs, and providing technical support for the concentration assessment of eugenol / eugenol and its glycoside in grape fruits.

[0040] The glycoside eugenol in the present invention specifically includes eugenol-hexose (EU-H), eugenol-hexose-pentose (EU-HP), eugenol-hexose-deoxyhexose (EU-HDH), and eugenol-hexose-hexose (EU-HH); the glycoside syringol specifically includes syringol-hexose-pentose 1 (SY-HP 1), syringol-hexose-pentose 2 (SY-HP 2), syringol-hexose-deoxyhexose 1 (SY-HDH 1), syringol-hexose-deoxyhexose 2 (SY-HDH 2), and syringol-hexose-hexose (SY-HH).

[0041] The method for detecting endogenous smoky flavor substances in grape fruits of the present invention specifically comprises the following steps:

[0042] S1. Mix grape berries, D-gluconolactone and cross-linked polyvinyl pyrrolidone, grind, immerse and centrifuge to obtain a grape juice sample.

[0043] Among them, D-gluconolactone can inhibit the activity of endogenous glycosidase in grapes, and cross-linked polyvinyl pyrrolidone can prevent sample oxidation.

[0044] Preferably, the immersion condition is immersion in a closed environment at a temperature of 3.5°C to 4.5°C for 3 to 5 hours; the centrifugation condition is centrifugation at a speed of 7000 to 9000 r / min for 5 to 15 minutes in an environment at a temperature of 3.5°C to 4.5°C; after the centrifugation is completed, the supernatant is collected as the grape juice sample.

[0045] Soaking in a closed environment can prevent the loss of eugenol and eugenol in the grape fruit, effectively improving the accuracy of the test results.

[0046] Preferably, for grape fruits, the stalks and seeds can be removed first under the protection of liquid nitrogen to prevent the loss of eugenol and eugenol due to fruit rupture during the removal process, and to prevent impurities that may be contained in the stalks and seeds from contaminating the sample.

[0047] S2. Adding internal standard substances for gas chromatography-mass spectrometry detection and liquid chromatography-mass spectrometry detection to the grape juice sample to obtain a mixed sample; adding the internal standard substance can make subsequent quantitative detection simpler and the quantitative detection result more accurate and reliable.

[0048] Preferably, the internal standard substance for gas chromatography-mass spectrometry detection is 3,4-dimethylphenol solution, and the internal standard substance for liquid chromatography-mass spectrometry detection is glucose salicin solution.

[0049] Preferably, the concentration of the 3,4-dimethylphenol solution is 0.2 g / L, and the volume ratio of the 3,4-dimethylphenol solution to the grape juice sample is 1:1000; the concentration of the glucose salicin solution is 1.8 g / L, and the volume ratio of the glucose salicin solution to the grape juice sample is 1:1000.

[0050] S3. Performing solid phase extraction on the mixed sample, the solid phase extraction comprising the steps of activation, sample loading, rinsing and eluting, wherein during elution, dichloromethane is first used for elution to obtain a free eluent, and then methanol is used for elution to obtain the glycoside eluent.

[0051] Preferably, the solid phase extraction column is activated with methanol and deionized water; the sample loading flow rate is 2 mL / min; during elution, the volume ratio of dichloromethane to the grape juice sample is 1:1, and the volume ratio of methanol to the grape juice sample is 1:1.

[0052] Preferably, the eluent used during elution is distilled water.

[0053] S4. Dewatering and concentrating the free-state eluate, and then performing gas chromatography-mass spectrometry detection, and calculating the contents of free eugenol and free eugenol respectively according to the detection results.

[0054] Preferably, anhydrous sodium sulfate is used for dehydration, and nitrogen purge is used for concentration; in order to adapt to subsequent gas chromatography-mass spectrometry detection, the free eluent can be concentrated to 1 mL.

[0055] S5. Concentrate, redissolve and filter the glycoside eluate, and then perform liquid chromatography-mass spectrometry detection. Calculate the contents of glycoside eugenol and glycoside eugenol according to the detection results.

[0056] Preferably, the glycoside eluent is evaporated to dryness by rotary evaporation under vacuum conditions at a temperature of 30° C., and then re-dissolved by using a methanol-acetonitrile aqueous solution; in the methanol-acetonitrile aqueous solution, the volume ratio of methanol, acetonitrile and water is 50:5:100.

[0057] Preferably, in order to use subsequent liquid chromatography-mass spectrometry detection, the reconstituted solution is filtered through a 0.22 μm polyethersulfone filter membrane and then transferred into a 2 mL sample bottle for detection.

[0058] The method for detecting endogenous smoky flavor substances in grape fruits of the present invention organically combines the detection process of free eugenol and eugenol with the detection process of glycoside eugenol and eugenol, without the need to perform separate detections. Such a detection process is not only fast and simple, but also only requires one sample pretreatment, effectively reducing the loss of eugenol and eugenol-like substances, making the detection result more accurate.

[0059] The separate detection technology for free eugenol and free eugenol is relatively mature, but the technology for simultaneously detecting glycoside eugenol and glycoside eugenol on this basis is still blank. The detection method of the present invention achieves the above-mentioned purpose through a series of steps and parameter improvements, among which the most core improvement is mainly the eluent and methanol elution volume for glycoside eugenol and glycoside eugenol in solid phase extraction, which plays a decisive role in the elution effect. In addition, the method of the present invention also uses an internal standard substance, so that the detection results are more accurate when quantitative detection is subsequently performed using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry.

[0060] The detection method of the present invention has been verified by experiments. The detection limit for each test substance can reach 4.62μg / L, and the method quantitative limit is 4.72μg / L. The method accuracy is that the spike recovery rate is 101.0% to 105.3%. The method stability is evaluated by the intra-day and inter-day relative standard deviations, which are less than 11.27% and 14.71% respectively, both within 15%, indicating good stability.

[0061] The present invention is described in detail below through specific examples.

[0062] Example 1

[0063] The method of the present invention is used to extract and detect eugenol / eugenol and glycoside eugenol / eugenol in grape fruits. The specific steps of this embodiment are as follows:

[0064] (1) Pretreatment: Take 50 g of grape fruit, remove the stalks and seeds under the protection of liquid nitrogen. Add 0.5 g of D-gluconolactone and 1 g of PVPP, and crush into powder using a fruit grinder. Soak the powder in a sealed environment at 4°C for 4 h, then centrifuge at 4°C and 8000 r / min for 10 min, and collect the upper clarified grape juice.

[0065] (2) Solid phase extraction: The solid phase extraction column Cleanert PEP-SPE was activated by passing through 10 mL of methanol and 10 mL of deionized water in sequence.

[0066] To the 10 mL clarified grape juice sample obtained in step (1), add 0.2 g / L 3,4-dimethylphenol and 1.8 g / L glucose salicin, both in an amount of 10 μL. After mixing evenly, load the mixture onto the solid phase extraction column at a flow rate of 2 mL / min.

[0067] The solid phase extraction column was rinsed with 5 mL of distilled water. The solid phase extraction column was eluted with 10 mL of dichloromethane to obtain a free eluent. The free eluent contained free eugenol and free eugenol. The solid phase extraction column was eluted with 10 mL of methanol to obtain a glycoside eluent. The glycoside eluent contained glycoside eugenol and glycoside eugenol.

[0068] The free eluate was dehydrated with 1.5 g of anhydrous sodium sulfate and concentrated to 1 mL by nitrogen blowing for testing.

[0069] The glycoside eluate was rotary evaporated to dryness under vacuum conditions at 30°C, reconstituted with 2 mL (5% v / v acetonitrile water; 50% v / v methanol water), filtered through a 0.22 μm polyethersulfone filter membrane, and transferred into a 2 mL sample bottle for testing.

[0070] (3) Gas chromatography-mass spectrometry was used to qualitatively and quantitatively detect free eugenol and free eugenol.

[0071] Using eugenol and eugenol standards, a set of standard solutions with different concentration gradients of eugenol and eugenol were prepared based on simulated grape juice (glucose 200 g / L; tartaric acid 7 g / L; pH 3.3), and the same amount of internal standard substances as those in the samples were added to the standard solutions.

[0072] The specific concentration gradient of the standard is: eugenol: 0.25, 0.5, 1.0, 2.0, 5.0, 10, 20, 50, 100, 200 μg / L; eugenol: 0.6125, 1.225, 2.45, 4.9, 12.25, 24.5, 49, 122.5, 245, 490 μg / L.

[0073] The same solid phase extraction and gas chromatography-mass spectrometry were used to extract and detect eugenol and eugenol in the standard solution. The chromatograms obtained are shown in Figure 2. Figure 1 As shown in Table 1, a yx standard curve was established based on the ratio of the peak area of ​​eugenol and eugenol to the peak area of ​​the internal standard and the concentration of eugenol and eugenol.

[0074] Table 1 Standard curves of eugenol and eugenol

[0075]

[0076] The free eugenol and free eugenol substance peaks and the internal standard peak in the sample were integrated respectively, and the ratio of the free eugenol and free eugenol peak areas in the sample to the internal standard peak areas was substituted into the standard curve to calculate the concentrations of free eugenol and free eugenol.

[0077] In this embodiment, the gas chromatography mass spectrometry parameters are: Agilent Intuvo 9000-7010BGC-QqQ-MS / MS (Agilent Technologies, Santa Clara, CA) is used, and the chromatographic column is HP-5MS (30m×250μm×0.25μm). The GC injection port temperature is 250°C, the injection volume is 1μL, and the injection is in 4:1 split mode. The carrier gas is high-purity helium (>99.999%), and the flow rate is 1.0mL / min. The heating program is as follows: the starting temperature is 40°C; it is increased to 80°C at a rate of 5°C / min; it is increased to 120°C at a rate of 2°C / min; it is increased to 160°C at a rate of 5°C / min; and finally it is increased to 220°C at a rate of 30°C / min. The post-run is maintained at 280°C for 1min. The mass spectrometer used an electron impact ion source (EI), with an ion source temperature of 230°C, an ionization energy of 70 eV, an auxiliary heater temperature of 300°C, and quadrupole temperatures of MS1 and MS2 of 150°C. The quenching gas was helium with a flow rate of 2.25 mL / min; the collision gas was nitrogen with a flow rate of 1.5 mL / min. The scanning method was multiple reaction monitoring mode (MRM), and the characteristic ion pair of eugenol was 164149 m / z, collision energy of 10 eV, and retention time of 25.3 min; the characteristic ion pair of eugenol was 154139 m / z, collision energy of 10 eV, and retention time of 25.0 min; the characteristic ion pair of internal standard was 122107 m / z, collision energy of 15 eV, and retention time of 16.7 min.

[0078] (4) Liquid chromatography-mass spectrometry was used to qualitatively and quantitatively detect glycoside eugenol and glycoside eugenol.

[0079] A set of standard solutions with different concentration gradients were prepared using eugenol-β-D-rutinoside standard based on simulated grape juice (glucose 200 g / L; tartaric acid 7 g / L; pH 3.3), each of which contained the same amount of liquid phase internal standard substance as the sample. The specific concentration gradient was eugenol-β-D-rutinoside: 13, 26, 52, 104, 260, 520, 1040, 2600, 5200, 10400 μg / L.

[0080] The same solid phase extraction method and liquid chromatography-mass spectrometry were used to extract and detect eugenol-β-D-rutinoside in the standard solution. The chromatogram is shown in Figure 2 As shown, the yx standard curve was established based on the ratio of the peak area to the internal standard peak area and the concentration of eugenol-β-D-rutinoside, and the results are shown in Table 2.

[0081] Table 2 Eugenol-β-D-rutinoside standard curve

[0082]

[0083] The peaks of glycoside eugenol and glycoside eugenol in the sample were integrated respectively, and the ratios of the peak areas of glycoside eugenol and glycoside eugenol in the sample to the peak area of ​​the internal standard were substituted into the standard curve to calculate the concentrations of each glycoside eugenol and glycoside eugenol.

[0084] In this embodiment, liquid chromatography mass spectrometry parameters: HPLC-QqQ-MS / MS analysis was performed using an Agilent 1290-6470 triple quadrupole liquid chromatography-mass spectrometry device (Agilent Technologies, Santa Clara, CA, USA). The chromatographic column was InfinityLab Poroshell 120SB-C18 (2.1×150mm, 2.7μm), the column temperature was 50°C; the mobile phase A was 0.1% formic acid in water, and the mobile phase B was 0.1% formic acid in acetonitrile; the flow rate was 0.4mL / min. The injection volume was 5μL, and the elution program was: 0-12min, 5-10% B; 12-14min, 10-40% B; 14-16min, 40-100% B. Mass spectrometry parameters: AJS ESI ion source, negative ion mode, nebulizer pressure of 30psi, dry gas flow rate of 7L / min, dry gas temperature of 250°C. The scanning ion pairs and collision energies are shown in Table 3.

[0085] Table 3 MRM scanning ion pairs and collision energy information of glycoside eugenol and glycoside eugenol

[0086]

[0087]

[0088] Note: a EU, eugenol; SY, eugenol; H, hexose; P, pentose; DH, deoxyhexose.

[0089] After testing and calculation, in 50g of grape fruit of this example, the content of free eugenol was 4.4μg / L, and the total amount of glycoside eugenol was 232.9μg / L. Specifically, EU-H was 5.7μg / L, EU-HP was 166.5μg / L, EU-HDH was 50.1μg / L, and EU-HH was 10.6μg / L.

[0090] The content of free syringol was 7.3 μg / L, and the content of glycoside syringol was 262.0 μg / L. Specifically, SY-HP1 was 18.4 μg / L, SY-HP2 was 21.3 μg / L, SY-HDH1 was 5.4 μg / L, SY-HDH2 was 100.3 μg / L, and SY-HH was 116.6 μg / L.

[0091] Example 2

[0092] In order to obtain the maximum extraction efficiency of glycoside eugenol / eugenol, this example verifies the relationship between the volume of the eluent and the volume of methanol used for elution during solid phase extraction of glycoside eugenol and glycoside eugenol.

[0093] In the solid phase extraction method, the minimum volume of the eluent and methanol eluent used is generally 5 mL. When designing the experiment, due to the large differences in the responses of different compounds, the results need to be standardized for comparison and drawing. Therefore, the response after different volume treatments is compared with the response after 5 mL volume treatment to evaluate the results.

[0094] Specifically, the eluent volume of this embodiment was investigated at 5 mL, 10 mL, and 15 mL, and the methanol elution volume was also investigated at 5 mL, 10 mL, and 15 mL. The extraction and detection process of each group of experiments was the same as that of Example 1.

[0095] Figure 3 The results of different water washing volume and methanol elution volume on the extraction of glycoside eugenol. Figure 3 As can be seen in Figure A, when 10 mL of methanol was used for elution and 5 mL, 10 mL, and 15 mL of eluent were used respectively, the response ratios after 10 mL and 15 mL of elution were lower than those after 5 mL of eluent, indicating that the extraction efficiency was best when 5 mL of eluent was used. Figure 3 It can be seen from Figure B that when 5 mL of eluent is used, and 5 mL, 10 mL and 15 mL of methanol are used for elution respectively, the response ratios after 10 mL and 15 mL of elution are higher than that of 5 mL of methanol, and the elution effect of 10 mL of methanol is significantly higher than that of 15 mL of methanol, indicating that the extraction efficiency of elution with 10 mL of methanol is the best.

[0096] Figure 4 The results of different water washing volume and methanol elution volume on the extraction of glycoside syringol. Figure 4 As can be seen in Figure A, when 10 mL of methanol was used for elution and 5 mL, 10 mL, and 15 mL of eluent were used respectively, the response ratios after 10 mL and 15 mL of elution were lower than those after 5 mL of eluent, indicating that the extraction efficiency was best when 5 mL of eluent was used. Figure 4It can be seen from Figure B that when 5 mL of eluent is used, and 5 mL, 10 mL and 15 mL of methanol are used for elution respectively, the response ratios after 10 mL and 15 mL of elution are higher than that of 5 mL of methanol, and the elution effect of 10 mL of methanol is significantly higher than that of 15 mL of methanol, indicating that the extraction efficiency of elution with 10 mL of methanol is the best.

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting endogenous smoky flavor substances in grape fruits, characterized in that: The endogenous smoky flavor substances include free eugenol, glycoside eugenol, free eugenol and glycoside eugenol; the detection method includes performing solid phase extraction on the pre-treated sample to obtain free eluent and glycoside eluent respectively, performing gas chromatography-mass spectrometry detection on the free eluent, and performing liquid chromatography-mass spectrometry detection on the glycoside eluent to obtain the detection results.

2. The method for detecting endogenous smoky flavor substances in grape fruits according to claim 1, characterized in that: The following steps are involved: S1, mixing grape fruit, D-gluconolactone and cross-linked polyvinyl pyrrolidone, grinding, macerating and centrifuging to obtain a grape juice sample; S2, adding an internal standard substance for gas chromatography-mass spectrometry detection and an internal standard substance for liquid chromatography-mass spectrometry detection to the grape juice sample to obtain a mixed sample; S3, performing the solid phase extraction on the mixed sample, the solid phase extraction comprising the steps of activation, sample loading, elution and washing, wherein during the elution, dichloromethane is first used for washing to obtain the free eluent, and then methanol is used for washing to obtain the glycoside eluent; the volume ratio of the eluent to dichloromethane is 1:1-2, and the volume ratio of the eluent to methanol is 1:1-2; S4, removing water and concentrating the free-state eluate, and then performing the gas chromatography-mass spectrometry detection, and calculating the contents of the free-state eugenol and the free-state eugenol according to the detection results; S5. Concentrating, redissolving and filtering the glycoside eluate, and then performing the liquid chromatography-mass spectrometry detection, and calculating the contents of the glycoside eugenol and the glycoside eugenol according to the detection results.

3. The method for detecting endogenous smoky flavor substances in grape fruits according to claim 2, characterized in that: In step S1, the immersion condition is immersion for 3 to 5 hours in a closed environment at a temperature of 3.5°C to 4.5°C; the centrifugation condition is centrifugation at a speed of 7000 to 9000 r / min for 5 to 15 minutes in an environment at a temperature of 3.5°C to 4.5°C; after the centrifugation is completed, the supernatant is collected as the grape juice sample.

4. The method for detecting endogenous smoky flavor substances in grape fruits according to claim 2, characterized in that: In step S2, the internal standard substance detected by gas chromatography-mass spectrometry is 3,4-dimethylphenol solution, and the internal standard substance detected by liquid chromatography-mass spectrometry is glucose salicin solution; the concentration of the 3,4-dimethylphenol solution is 0.1-0.3 g / L, and the volume ratio of the 3,4-dimethylphenol solution to the grape juice sample is 1:1000; the concentration of the glucose salicin solution is 1.0-2.0 g / L, and the volume ratio of the glucose salicin solution to the grape juice sample is 1:1000.

5. The method for detecting endogenous smoky flavor substances in grape fruits according to claim 2, characterized in that: In step S3, the eluent is distilled water, the eluent is 5 mL, the dichloromethane is 10 mL, and the methanol is 10 mL.

6. The method for detecting endogenous smoky flavor substances in grape fruits according to claim 2, characterized in that: In step S3, the solid phase extraction column is activated with methanol and deionized water, and the sample loading flow rate is 1 to 3 mL / min.

7. The method for detecting endogenous smoky flavor substances in grape fruits according to any one of claims 1 to 6, characterized in that: In step S4, anhydrous sodium sulfate is used for dehydration, and nitrogen purge is used for concentration.

8. The method for detecting endogenous smoky flavor substances in grape fruits according to claim 7, characterized in that: The gas chromatography-mass spectrometry detection is GC-QqQ-MS / MS detection, the chromatographic column is a capillary chromatographic column with a specification of 30m×250μm×0.25μm; the GC injection port temperature is 250°C, the injection volume is 1-2μL, and the injection mode is 2-5:1 split mode injection; The carrier gas for GC-QqQ-MS / MS detection is helium with a volume percentage greater than 99.999% and a flow rate of 1.0 mL / min; The temperature program of the GC-QqQ-MS / MS detection is as follows: the starting temperature is 40°C; the temperature is increased to 80°C at a rate of 5°C / min; the temperature is increased to 120°C at a rate of 2°C / min; the temperature is increased to 160°C at a rate of 5°C / min; the temperature is finally increased to 220°C at a rate of 30°C / min; the post-run is maintained at 280°C for 1 min; The mass spectrometer adopts an electron bombardment ion source, the ion source temperature is 230°C, the ionization energy is 70eV, the auxiliary heater temperature is 300°C, and the MS1 and MS2 quadrupole temperatures are both 150°C; the quenching gas is helium with a flow rate of 2.25mL / min; the collision gas is nitrogen with a flow rate of 1.5mL / min; the scanning method is multiple reaction monitoring mode, the characteristic ion pair of eugenol is 164-149m / z, the collision energy is 10eV, and the retention time is 25.3min; the characteristic ion pair of eugenol is 154-139m / z, the collision energy is 10eV, and the retention time is 25.0min; the characteristic ion pair of the internal standard detected by the gas chromatography-mass spectrometry is 122-107m / z, the collision energy is 15eV, and the retention time is 16.7min.

9. The method for detecting endogenous smoky flavor substances in grape fruits according to any one of claims 1 to 6, characterized in that: In step S5, the glycoside eluate is evaporated to dryness by rotary evaporation under vacuum conditions at a temperature of 30° C., and then redissolved by acetonitrile aqueous solution; in the acetonitrile aqueous solution, the volume ratio of acetonitrile to water is 5:

100.

10. The method for detecting endogenous smoky flavor substances in grape fruits according to claim 9, characterized in that: The liquid chromatography-mass spectrometry detection is HPLC-QqQ-MS / MS detection, the chromatographic column is a C18 chromatographic column with a specification of 2.1×150 mm, 2.7 μm, and a column temperature of 50° C. Mobile phase A is a 0.1% by volume formic acid aqueous solution, and mobile phase B is a 0.1% by volume formic acid acetonitrile solution; the flow rate is 0.4 mL / min; the injection volume is 5 μL; The elution procedure is: 0-12 min, the volume percentage of the mobile phase B is 5-10%; 12-14 min, the volume percentage of the mobile phase B is 10-40%; 14-16 min, the volume percentage of the mobile phase B is 40-100%; The mass spectrometry parameters were as follows: AJS ESI ion source, negative ion mode, nebulizer pressure of 30 psi, drying gas flow rate of 7 L / min, and drying gas temperature of 250°C.

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