Method for separating, identifying and quantifying 3-methylthiopropionaldehyde in Baijiu
Through PFBHA derivatization method and VSLLME-GC/MS technology, the problem of insufficient detection enrichment ability of 3-methylthiopropionaldehyde in rich-flavored liquor is solved, and efficient and accurate quantitative detection is achieved, which improves detection sensitivity and accuracy. sex.
Patent Information
- Application Number
- CN202411631699.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-15
- Publication Date
- 2025-02-21
AI Technical Summary
The prior art is inadequate when detecting 3-methylthiopropionaldehyde in rich-flavored liquor, and accurate analysis cannot be achieved.
The detection sensitivity and accuracy of 3-methylthiopropionaldehyde were improved by derivatization reaction and vortex extraction by combining PFBHA derivatization method with vortex liquid extraction-gas chromatography-mass spectrometry technology (VSLLME-GC/MS).
The problem of insufficient enrichment ability of 3-methylthiopropionaldehyde in traditional extraction analysis methods was successfully solved, and efficient and accurate quantity detection of the substance was achieved, which improved the detection sensitivity and accuracy.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of food detection, and in particular relates to a method for separating, identifying and quantifying 3-methylthiopropionaldehyde in liquor. Background Art
[0002] Luzhou-flavor liquor is an important representative of Chinese liquor. It is deeply favored by consumers for its unique "rich cellar aroma" flavor. As the main force in my country's liquor market, Luzhou-flavor liquor has the widest production and distribution in the country, accounting for about 60% of the liquor market. In-depth research on "cellar aroma" flavor substances is not only the key to revealing the characteristics of Luzhou-flavor liquor, but also an important means to enhance and optimize its flavor. "Cellar aroma" is a typical feature of Luzhou-flavor liquor, which is specifically manifested as a strong, complex aroma with certain aging characteristics. However, although this feature is widely recognized, there are still certain challenges in the analysis of its chemical composition. At present, studies have shown that 3-methylthiopropionaldehyde is the main contributor to "cellar aroma". By combining gas chromatography-mass spectrometry olfaction (GC-MS / O) and standard sensory evaluation methods, researchers have confirmed the key role of this compound in the formation of "cellar aroma". However, the quantitative detection method for this key "cellar aroma" substance is still insufficient, especially in the complex liquor matrix, and achieving efficient and accurate separation and identification remains an urgent problem to be solved.
[0003] In the prior art, 3-methylthiopropionaldehyde is difficult to enrich to a sufficient concentration by the existing simple extraction method due to its extremely low concentration in liquor and poor direct extraction effect, making it difficult to achieve accurate detection and quantification. Therefore, how to effectively separate, identify and quantify this key cellar aroma substance in liquor has become a major problem in the current research on the flavor of Luzhou-flavor liquor. When faced with a complex liquor matrix, the existing detection technology has insufficient enrichment capacity for low-concentration substances and cannot meet the needs of accurate analysis. Therefore, there is an urgent need to develop a new method to more effectively separate and detect 3-methylthiopropionaldehyde in liquor, so as to provide technical support for the flavor research and quality control of Luzhou-flavor liquor. Summary of the invention
[0004] The technical problem to be solved by the present invention is that when the prior art detects 3-methylthiopropionaldehyde, a key flavor substance that contributes to the cellar aroma in Luzhou-flavor liquor, the enrichment capacity is insufficient, resulting in an inability to accurately analyze.
[0005] The technical solution adopted by the present invention to solve its technical problem is:
[0006] The invention provides a method for separating, identifying and quantifying 3-methylthiopropionaldehyde in liquor. The method comprises the following steps: diluting a liquor sample to be tested to an alcohol content of 10% to 20%, adding NaCl to saturation, and then adding 3-methylthiobutyraldehyde and a PFBHA derivatization reagent to carry out a derivatization reaction; subsequently adding carbon tetrachloride to carry out vortex extraction and centrifugation, taking a clear liquid to carry out GC-MS analysis; and calculating the substance concentration of 3-methylthiopropionaldehyde in the liquor sample to be tested according to a standard curve established with a 3-methylthiopropionaldehyde standard product.
[0007] In the above method, the concentration of 3-methylthiobutyraldehyde is 3-6 mg / L, the solvent is ethanol; and the concentration of the PFBHA derivatization reagent is 15-25 g / L.
[0008] Furthermore, the concentration of 3-methylthiobutyraldehyde is 5 mg / L, the solvent is ethanol, and the concentration of the PFBHA derivatization reagent is 20 g / L.
[0009] In the above method, the volume ratio of the wine sample to be tested, 3-methylthiobutyraldehyde and PFBHA derivatization reagent is 90-110:3-5:1.
[0010] Furthermore, the volume ratio of the wine sample to be tested, 3-methylthiobutyraldehyde and PFBHA derivatization reagent is 100:4:1.
[0011] In the above method, the derivatization reaction conditions are 35-50° C. for 25-35 min.
[0012] Furthermore, the derivatization reaction was carried out at 45° C. for 30 min.
[0013] In the above method, the volume ratio of the wine sample to be tested to carbon tetrachloride is 8 to 12:1.
[0014] Furthermore, the volume ratio of the alcohol sample to be tested to carbon tetrachloride is 10:1.
[0015] In the above method, the vortex extraction condition is vortex extraction at a rotation speed of 650 to 750×g for 25 to 45 seconds.
[0016] Furthermore, the vortex extraction was performed at a speed of 701.2×g for 30 seconds.
[0017] In the above method, the centrifugal condition is 3500-4000×g for 10-15 min.
[0018] Furthermore, the centrifugation condition was 3778.8×g for 10 min.
[0019] In the above method, the gas chromatography conditions for GC-MS analysis are as follows: the chromatographic column adopts a DB-WAX capillary column; the carrier gas is He; the constant flow mode has a flow rate of 1.0 mL / min; the split mode is split injection, and the split ratio is 10:1; the injection volume is 1 μL; the injection port temperature is 250°C; the initial temperature is 40°C, increased to 205°C at 10°C / min, and maintained for 5 minutes; then increased to 245°C at 15°C / min, and maintained for 7 minutes.
[0020] In the above method, the mass spectrometry conditions of GC-MS analysis are: using an electron bombardment source; the electron energy is 70eV; in qualitative analysis, a full scan mode is used; the scanning mass range is 45 to 450amu; the temperatures of the ion source, quadrupole and transmission line are 230°C, 150°C and 250°C respectively; in quantitative analysis, a selected ion scan mode is used.
[0021] In the above method, the method for establishing a standard curve for 3-methylthiopropionaldehyde standard is: using the ratio of the mass concentration of the analyte to the corresponding internal standard in each gradient standard mixed solution as the horizontal coordinate (x), and using the ratio of the peak area of their respective quantitative ions as the vertical coordinate (y), to establish the standard curve linear regression equation y=ax+b.
[0022] The beneficial effects of the present invention are as follows: the present invention establishes an analytical method for "3-methylthiopropionaldehyde", a key flavor substance of "cellar aroma", quantitatively analyzes 3-methylthiopropionaldehyde by PFBHA derivatization method combined with VSLLME-GC / MS, and establishes a standard curve. The method successfully solves the problem that the 3-methylthiopropionaldehyde cannot reach the quantitative limit due to low content and low extraction enrichment rate in traditional extraction analysis methods. Accurately evaluating the contribution of different aroma compounds to the aroma characteristics of liquor is of great value for accurately analyzing the aroma characteristics and intensity of liquor samples, and for accurately separating and identifying aroma compounds, and has wide applicability. The method improves the detection sensitivity and accuracy, provides a reliable tool for the quantitative evaluation of flavor substances, and has important scientific research value and application prospects. It also provides theoretical support for the improvement of the flavor of liquor, especially provides technical guidance for the flavor analysis of the "cellar aroma" of Luzhou-flavor liquor, and promotes the practical technology of analyzing some flavor substances in food that are difficult to analyze. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 A is a flow chart of the derivatization reaction of 3-methylmercaptopropionaldehyde; B is the structural formula of 3-methylmercaptopropionaldehyde after derivatization and its characteristic ion fragments; C is the SIM image of 3-methylmercaptopropionaldehyde before and after derivatization. DETAILED DESCRIPTION
[0024] The technical solution of the present invention can be specifically implemented in the following manner.
[0025] Example
[0026] In this example, the PFBHA derivatization method combined with the VSLLME-GC / MS method was used to improve the accuracy of quantitative detection of 3-methylthiopropionaldehyde in wine samples. The concentration of 3-methylthiopropionaldehyde in 16 kinds of Luzhou-flavor base wines was determined by the PFBHA derivatization method combined with the VSLLME-GC / MS method. The specific detection method is as follows:
[0027] (1) Construction of standard curve
[0028] The linear regression equation of the standard curve y=ax+b was established by taking the mass concentration ratio of the analyte to the corresponding internal standard in each gradient standard mixed solution as the abscissa (x) and the peak area ratio of their respective quantitative ions as the ordinate (y).
[0029] (2)PFBHA-VSLLME-GC / MS
[0030] Take 5mL of wine sample diluted to 15% in a 10mL centrifuge tube and add 1.2g NaCl to saturation. Then add 200μL 3-methylthiobutyraldehyde (5mg / L, prepared in ethanol) and 50μL PFBHA derivatization reagent (20g / L), and mix by vortexing at room temperature. Derivatize the mixed sample at 45℃ for 30min, add 500μL carbon tetrachloride as the extraction solvent, and vortex extract for 30s at a speed of 701.2×g. Finally, centrifuge at a speed of 3778.8×g for 10min, and take the clear liquid for GC-MS analysis.
[0031] Gas chromatography conditions for GC-MS analysis: chromatographic column using DB-WAX capillary column (30m×0.25mm×0.25μm); carrier gas is He (99.999%); constant flow mode, flow rate is 1.0mL / min; split mode: split injection, split ratio is 10:1; injection volume is 1μL; injection port temperature is 250℃; initial temperature is 40℃, increased to 205℃ at 10℃ / min, maintained for 5min; then increased to 245℃ at 15℃ / min, maintained for 7min;
[0032] The mass spectrometry conditions for GC-MS analysis were as follows: electron impact (EI) source was used; electron energy was 70 eV; full scan mode was used for qualitative analysis; the scanning mass range (m / z) was 45-450 amu; the temperatures of the ion source, quadrupole and transmission line were 230°C, 150°C and 250°C, respectively; selected ion monitoring (SIM) mode was used for quantitative analysis.
[0033] (3) Results analysis
[0034] The reaction flow chart of the derivatization of 3-methylthiopropionaldehyde with PFBHA reagent is as follows: Figure 1 As shown in A.
[0035] The MS analysis results show that (see Figure 1 In B), the derivatization reaction of 3-methylthiopropionaldehyde was successful. Among them, the characteristic fragment ion at m / z 299 corresponds to the [M + ] ion; in addition, due to the introduction of -NH2- group on PFBHA, the oxygen atom on the carbonyl group of 3-methylthiopropionaldehyde is replaced by nitrogen atom, thus converting the characteristic ion fragment from m / z=104 to m / z=102; the characteristic ion fragments of m / z=75 and 61 are attributed to the drop of -C3H7S and -C2H5S functional groups, respectively. Finally, in this embodiment, m / z 102 is selected as the quantitative ion, and m / z 75 and 61 are selected as the qualitative ions, respectively.
[0036] like Figure 1 As shown in C, after derivatization, the peak area of 3-methylthiopropionaldehyde increased significantly by more than 100 times.
[0037] The concentrations of 3-methylthiopropionaldehyde in 16 liquors were detected according to the above method, and the results are shown in Table 1.
[0038] Table 1 Concentration of 3-methylthiopropionaldehyde in 16 liquors
[0039]
[0040] In summary, the method of the present invention can significantly reduce the detection limit of 3-methylmercaptopropionaldehyde, improve its sensitivity, and ensure the efficient and accurate detection of 3-methylmercaptopropionaldehyde in the wine sample to be tested.
[0041] Comparative Example
[0042] In this comparative example, the concentration of 3-methylthiopropionaldehyde in 16 kinds of Luzhou-flavor base wines was determined by a common pretreatment method combined with GC-MS. The specific detection method is as follows:
[0043] (1) Sample pretreatment
[0044] 1) Liquid-liquid extraction: Take 30.0 mL of each wine sample to be tested, dilute it with ultrapure water to 15.0% vol, add 37.0 g of baked sodium chloride and stir evenly to reach saturation, then add 45.0 mL of chromatographically pure dichloromethane for extraction, let it stand for 3 minutes after degassing, so that the organic phase and the aqueous phase are completely separated, repeat the above extraction operation 3 times, collect a total of 135.0 mL of organic phase, add anhydrous sodium sulfate after impurities removal to the organic phase until the solid sodium sulfate is in the form of quicksand, and refrigerate overnight. Adjust pH-liquid extraction Repeat the above steps, collect 135.0 mL of organic phase, extract the organic phase 3 times with 45.0 mL of sodium carbonate solution with pH 10, let it stand for stratification, and collect about 135.0 mL of organic phase, which is recorded as the neutral / basic fraction (NBF). Then combine the aqueous phases, adjust the aqueous phase to pH 2 with 4M hydrochloric acid solution, add sodium chloride until the solution is saturated, add 45.0mL of dichloromethane to extract, repeat the extraction operation 3 times, collect about 135.0mL of the organic phase, and record it as the acid phase (Acid Fraction, AF). The alkaline phase and the acid phase components are respectively added with anhydrous sodium sulfate after impurity removal for dehydration and placed in a refrigerator overnight. Filter to remove the anhydrous sodium sulfate in the organic phase, and then evaporation concentrate to about 2mL, transfer to an injection vial, and further concentrate to 500μL under a nitrogen flow (99.999%, 1mL / min) to obtain a concentrated sample. After sealing, store in a refrigerator at -20°C for GC-MS detection and analysis.
[0045] 2) Headspace solid phase microextraction: Insert the solid phase microextraction head into the gas chromatography injection port for aging, the aging time is 30 minutes, the aging temperature is set to 250 ° C, and the carrier gas flow rate is 3mL / min. Then, use ultrapure water to dilute the sample of Luzhou-flavor liquor to be tested to an ethanol volume fraction of 10%, take 8mL of the diluted sample, add 20μL of internal standard (diisopropyl disulfide, concentration 1.2656mg / L), and add NaCl to saturate the solution, shake well and transfer to a 20mL headspace bottle. Next, place the headspace bottle in an automatic microextraction device, insert the solid phase microextraction head into the headspace bottle to a depth of 22mm, the sample equilibration time is 1 minute, the extraction temperature is 50 ° C, and the extraction time is 45 minutes. Finally, perform injection port desorption, and the desorption time is 5 minutes.
[0046] (2) GC-MS analysis
[0047] Gas chromatography conditions: The chromatographic columns are DB-WAX capillary column (30m×0.25mm×0.25μm) and HP-5MS capillary column (30m×0.25mm×0.25μm); the injection port temperature is set to 250°C, the injection volume is 1μL, non-split injection, the carrier gas is He (99.999%), and the flow rate is 1.5mL / min. The temperature program of the column box is as follows: DB-WAX column: the initial temperature is 40°C, maintained for 2min, increased to 70°C at a heating rate of 15°C / min, and then increased to 180°C at a heating rate of 3°C / min, maintained for 5min, and finally increased to 245°C at a heating rate of 5°C / min, maintained for 2min. HP-5MS capillary column: the initial temperature was 40°C, which was increased to 120°C at a rate of 2°C / min and maintained for 3 min, then increased to 250°C at a rate of 15°C / min and maintained for 2 min, and finally increased to 325°C at a rate of 15°C / min and maintained for 2 min.
[0048] Mass spectrometry conditions: Analyzed in electron ionization (EI) mode at 70 eV, using full scan mode, with a range of (m / z) 45 to 450. When using DB-WAX, the ion source temperature was 230°C, and when using HP-5MS, the ion source temperature was 300°C.
[0049] (3) Results analysis
[0050] The concentration of 3-methylmercaptopropionaldehyde in 16 liquors was analyzed based on the above two pretreatment methods combined with GC-MS detection. The results showed that 3-methylmercaptopropionaldehyde only reached the detection limit but did not reach the quantification limit.
Claims
1. A method for separating, identifying and quantifying 3-methylthiopropionaldehyde in liquor, characterized in that: The wine sample to be tested is diluted to an alcohol content of 10% to 20%, NaCl is added to saturation, and then 3-methylthiobutyraldehyde and PFBHA derivatization reagent are added to carry out derivatization reaction; then carbon tetrachloride is added to carry out vortex extraction and centrifugation, and the clear liquid is taken for GC-MS analysis; The substance concentration of 3-methylmercaptopropionaldehyde in the wine sample to be tested was calculated based on the standard curve established with the 3-methylmercaptopropionaldehyde standard.
2. The method for separating, identifying and quantifying 3-methylthiopropionaldehyde in liquor according to claim 1, characterized in that: The concentration of 3-methylthiobutyraldehyde is 3-6 mg / L, and the solvent is ethanol; the concentration of the PFBHA derivatization reagent is 15-25 g / L.
3. The method for separating, identifying and quantifying 3-methylthiopropionaldehyde in liquor according to claim 1, characterized in that: The volume ratio of the wine sample to be tested, 3-methylthiobutyraldehyde and PFBHA derivatization reagent is 90-110:3-5:
1.
4. The method for separating, identifying and quantifying 3-methylthiopropionaldehyde in liquor according to claim 1, characterized in that: The conditions for the derivatization reaction are 35-50°C for 25-35 minutes.
5. The method for separating, identifying and quantifying 3-methylthiopropionaldehyde in liquor according to claim 1, characterized in that: The volume ratio of the wine sample to be tested to carbon tetrachloride is 8 to 12:
1.
6. The method for separating, identifying and quantifying 3-methylthiopropionaldehyde in liquor according to claim 1, characterized in that: The conditions for vortex extraction are vortex extraction at a rotation speed of 650 to 750×g for 25 to 45 seconds.
7. The method for separating, identifying and quantifying 3-methylthiopropionaldehyde in liquor according to claim 1, characterized in that: The centrifugation conditions are 3500-4000×g for 10-15 min.
8. The method for separating, identifying and quantifying 3-methylthiopropionaldehyde in liquor according to claim 1, characterized in that: The gas chromatography conditions for GC-MS analysis are as follows: the chromatographic columns are DB-WAX capillary columns; the carrier gas is He; the flow rate is 1.0 mL / min in constant flow mode; the split mode is split injection with a split ratio of 10:1; the injection volume is 1 μL; the injection port temperature is 250°C; the initial temperature is 40°C, which is increased to 205°C at 10°C / min and maintained for 5 min; then increased to 245°C at 15°C / min and maintained for 7 min.
9. The method for separating, identifying and quantifying 3-methylthiopropionaldehyde in liquor according to claim 1, characterized in that: The mass spectrometry conditions for GC-MS analysis are: using an electron bombardment source; the electron energy is 70 eV; for qualitative analysis, the full scan mode is used; the scanning mass range is 45-450 amu; the temperatures of the ion source, quadrupole and transmission line are 230°C, 150°C and 250°C respectively; for quantitative analysis, the selected ion scanning mode is used.
10. The method for separating, identifying and quantifying 3-methylthiopropionaldehyde in liquor according to claim 1, characterized in that: The method for establishing a standard curve for 3-methylthiopropionaldehyde standard is as follows: the ratio of the mass concentration of the analyte to the corresponding internal standard in each gradient standard mixed solution is used as the horizontal coordinate (x), and the ratio of the peak area of their respective quantitative ions is used as the vertical coordinate (y), and the linear regression equation of the standard curve y=ax+b is established.