Quantitative Detection Method for Sulfur-Containing Compounds Suitable for Baijiu System

Abstract

The present invention discloses a method for quantitative detection of sulfur-containing compounds suitable for the Baijiu system, belonging to the technical field of Baijiu flavor analysis. In order to overcome the problem of inaccurate quantification caused by competitive adsorption in the existing quantification methods, the present invention provides a method for quantitative detection of sulfur-containing compounds suitable for the Baijiu system. This method selects corresponding specific internal standards for different sulfur-containing compounds and provides a standard curve background solution system for different flavor types of Baijiu, avoiding the problem of inaccurate quantification caused by different competitive adsorptions due to differences in the content of skeleton substances, etc. The present invention can quickly and accurately quantify 12 volatile sulfur-containing compounds in different flavor types of Baijiu. The quantification limit of each compound is less than 14 μg / L, the linear correlation coefficient R² is greater than 0.997, the relative standard deviation is less than 5%, and the recovery rate is between 83% - 117%. It provides a new technology for Baijiu quality control and has important significance for improving the quality of Baijiu.

Description

Technical Field

[0001] The present invention belongs to the technical field of flavor analysis of Chinese liquor, and particularly relates to a method for quantitatively detecting sulfur-containing compounds such as dimethyl sulfide, which is applicable to three Chinese liquor systems of strong flavor, light flavor and sauce flavor simultaneously. Background Art

[0002] Sulfur-containing compounds are a special type of flavor compounds in Chinese liquor, mainly presenting special aromas such as cooked potatoes, rotten cabbages, cabbages, onions, garlic, etc. Their characteristic is that the threshold value is extremely low (most are in the range of ng / L to μg / L level). Therefore, even the presence of extremely low concentrations or extremely small content changes of sulfur-containing compounds in Chinese liquor will affect the overall aroma of Chinese liquor. Therefore, the accurate quantification of sulfur-containing compounds in Chinese liquor is very important for the flavor research of Chinese liquor.

[0003] In the currently reported studies, there are relatively few quantitative analysis techniques for sulfur-containing compounds in Chinese liquor, mainly including direct injection, liquid-liquid extraction and solid-phase microextraction.

[0004] The direct injection technique for determining sulfur-containing compounds in Chinese liquor was proposed in the past two years. For example, in the Chinese patent with the authorization number CN111024872B and the patent name "A rapid detection method for 3-(methylthio)propan-1-ol in sesame-flavor Chinese liquor", 3-(methylthio)propan-1-ol in Chinese liquor was determined by the direct injection technique combined with gas chromatography-mass spectrometry (GC-MS), and the selected ion monitoring (SIM) mode was used to improve the sensitivity. This method is simple and fast, and can accurately reflect the specific content of 3-(methylthio)propan-1-ol in sesame-flavor Chinese liquor. However, since the content of most sulfur-containing compounds in Chinese liquor is extremely low and the response in the mass spectrometry is extremely low, it is difficult to extend this method to the determination of most sulfur-containing compounds in all flavors of Chinese liquor.

[0005] In addition, liquid-liquid extraction is also a commonly used technique for enriching sulfur-containing compounds. This technique extracts and concentrates volatile compounds in Baijiu to a certain extent through solvent extraction, and then combines different detectors for analysis. For example, in the Chinese patent application No. 201710515247.0, with the patent name "A Method for Economically and Rapidly Quantitatively Detecting Volatile Sulfur Compounds in Baijiu", volatile compounds in Baijiu are enriched by liquid-liquid microextraction method, and then detected by combining with a gas chromatography-flame photometric detector (GC-FPD) with specific response to sulfur, so as to realize the qualitative and quantitative analysis of sulfur-containing compounds in Baijiu. This method has established a quantitative method for 11 common sulfur-containing compounds in Baijiu and applied it to actual Baijiu. In addition, in the journal article "Quantitative Analysis of 3-Methylthiopropanol in Two Types of Liquors by GC-MS / SIM Internal Standard Method" in the Journal of Chinese Institute of Food Science and Technology, the authors established a method for determining the content of 3-methylthiopropanol in Baijiu by dichloromethane extraction combined with GC-MS. This method improves the accuracy of quantification by using the internal standard method. However, the liquid-liquid extraction method has problems such as cumbersome operation, long time consumption, large consumption of organic reagents, and certain material loss during the extraction process.

[0006] Headspace-solid phase microextraction is a commonly used method for quantifying sulfur-containing compounds in Baijiu. Due to its high selectivity, high sensitivity, simplicity, rapidity, and high degree of automation, it is widely used in the research of sulfur-containing compounds. For example, in the Chinese patent authorization No. CN108181390B, with the patent name "A Method for Quantitative Detection of Sulfur-Containing Compounds in Baijiu", a quantitative method for sulfur-containing compounds in Baijiu is established by combining headspace-solid phase microextraction technology with comprehensive two-dimensional gas chromatography-sulfur chemiluminescence detector (GC×GC-SCD). This method uses the external standard method for quantification, and the background of the standard curve uses a 10% ethanol aqueous solution with the pH adjusted to 3.7 with hydrochloric acid. In addition, in the journal article "Quantitative Detection of Two Off-odor Substances in Baijiu by Solid Phase Microextraction-GC-MS" in Food Science, a quantitative method for dimethyl disulfide and dimethyl trisulfide is established. This technique uses the external standard method for quantification and uses a 60% vol ethanol aqueous solution as the background of the standard curve. However, headspace solid phase microextraction has the disadvantage of competitive adsorption, resulting in its quantification limitations. Due to the limited adsorption capacity of the solid phase microextraction fiber head, the content of volatile compounds in Baijiu is rich, and the substances in different Baijiu vary greatly. Therefore, the change of the skeleton substances in Baijiu will seriously affect the adsorption amount of sulfur-containing compounds. Therefore, the results of simply using the external standard method for quantification or using one internal standard to quantify all sulfur-containing compounds are inaccurate, and the standard curve background solution containing only ethanol cannot avoid the influence of the skeleton substances in Baijiu on the quantification of sulfur-containing compounds.

[0007] Thus, optimizing the headspace solid phase microextraction technology and establishing a method for rapidly and accurately quantifying sulfur-containing compounds is of great significance for the flavor analysis and quality improvement of Baijiu. Summary of the Invention

[0008] The object of the present invention is to establish a method for quickly, simply and accurately simultaneously quantitatively detecting the concentrations of 12 common volatile sulfur-containing compounds in different fragrance-type baijiu, so as to solve the problem in the prior art that due to the complex volatile components in baijiu, different degrees of competitive adsorption are formed on the solid-phase extraction fiber head, resulting in inaccurate quantification.

[0009] To achieve the above object, the present invention provides a method for quantitatively detecting sulfur-containing compounds suitable for the baijiu system. It adopts headspace-solid phase microextraction-gas chromatography-sulfur chemiluminescence detector technology. Using at least one of ethyl methyl sulfide, diethyl sulfide, thiophene, and isopropyl disulfide as the internal standard, with the response ratio of the sulfur-containing compound standard to the corresponding internal standard as the ordinate and the concentration ratio of the sulfur-containing compound standard to the corresponding internal standard as the abscissa, a standard curve is plotted to quantitatively detect the content of at least one of dimethyl sulfide, allyl methyl sulfide, dimethyl disulfide, ethyl thioacetate, 3-methylthiophene, propyl thioacetate, ethyl disulfide, 2-methyl-3-(methylthio)furan, dimethyl trisulfide, methyl thioacetate, furfuryl methyl sulfide, or ethyl 3-(methylthio)propionate in baijiu; wherein, the corresponding internal standard for dimethyl sulfide and allyl methyl sulfide is ethyl methyl sulfide, the corresponding internal standard for dimethyl disulfide, ethyl thioacetate, propyl thioacetate, and methyl thioacetate is diethyl sulfide, the corresponding internal standard for 3-methylthiophene is thiophene, and the corresponding internal standard for ethyl disulfide, 2-methyl-3-(methylthio)furan, dimethyl trisulfide, furfuryl methyl sulfide, and ethyl 3-(methylthio)propionate is isopropyl disulfide. According to the sulfur-containing compounds determined in the baijiu, the corresponding internal standard (or internal standard mixture) is determined.

[0010] Among them, in the above method for quantitatively detecting sulfur-containing compounds suitable for the baijiu system, the following steps are specifically included:

[0011] A. Pretreatment:

[0012] Prepare standard samples to be measured: Mix brine, a simulated baijiu sample, a series of dosage standards of at least one of 12 sulfur-containing compounds (dimethyl sulfide, allyl methyl sulfide, dimethyl disulfide, ethyl thioacetate, 3-methylthiophene, propyl thioacetate, ethyl disulfide, 2-methyl-3-(methylthio)furan, dimethyl trisulfide, methyl thioacetate, furfuryl methyl sulfide, or ethyl 3-(methylthio)propionate) and the corresponding internal standard (the corresponding internal standard is determined according to the sulfur-containing compound to be detected) to obtain a series of concentration standard samples to be measured of at least one of the 12 sulfur-containing compounds;

[0013] Prepare a baijiu sample to be measured: Mix brine, a baijiu sample and the corresponding internal standard to obtain a baijiu sample to be measured;

[0014] B. Sample injection: The standard sample to be measured and the Baijiu sample to be measured are extracted and injected by headspace-solid phase microextraction;

[0015] C. Detection: The standard sample to be measured is tested by gas chromatography-sulfur chemiluminescence detector. A standard curve is plotted with the response ratio of the sulfur-containing compound to be measured to the corresponding internal standard as the ordinate and the concentration ratio of the sulfur-containing compound to be measured to the corresponding internal standard as the abscissa;

[0016] The Baijiu sample to be measured is tested under the same conditions to obtain the response ratio of the sulfur-containing compound in the Baijiu sample to its corresponding internal standard. According to the standard curve, the content of the corresponding sulfur-containing compound in the Baijiu is calculated.

[0017] The present invention provides a standard curve background solution system for strong, light, and sauce-flavored Baijiu respectively, avoiding the problem of inaccurate quantification caused by different competitive adsorptions on the solid phase microextraction fiber head due to different contents of skeleton substances. Therefore, in some embodiments of the present invention, according to the flavor type of the Baijiu sample to be measured, a standard sample is configured by selecting a simulated Baijiu sample of the corresponding flavor type. The flavor types include strong flavor type, sauce flavor type, and light flavor type. The simulated Baijiu samples of different flavor types are 30-65% vol aqueous alcohol solutions configured according to the following substances and their concentrations:

[0018]

[0019]

[0020] Among them, in the above method for quantitative detection of sulfur-containing compounds suitable for the Baijiu system, in step A, the dosage of the brine is controlled so that the ethanol concentration in the standard sample to be measured or the Baijiu sample to be measured is 3-15% vol.

[0021] Preferably, in the above method for quantitative detection of sulfur-containing compounds suitable for the Baijiu system, in step A, the dosage of the brine is controlled so that the ethanol concentration in the standard sample to be measured or the Baijiu sample to be measured is 5-10% vol.

[0022] Among them, in the above method for quantitative detection of sulfur-containing compounds suitable for the Baijiu system, in step A, the concentrations of the internal standards ethyl methyl sulfide, diethyl sulfide, thiophene, and isopropyl disulfide in the series of concentration standard samples to be measured are 0.1-200 ppb respectively.

[0023] Preferably, in the above method for quantitative detection of sulfur-containing compounds suitable for the Baijiu system, in step A, the concentrations of the internal standards ethyl methyl sulfide, diethyl sulfide, thiophene, and isopropyl disulfide in the series of concentration standard samples to be measured are 0.5-20 ppb respectively.

[0024] Among them, in the above-mentioned method for quantitative detection of sulfur-containing compounds suitable for the Baijiu system, in step A, the concentrations of the internal standards ethyl methyl sulfide, diethyl sulfide, thiophene, and isopropyl disulfide in the Baijiu sample to be measured are 0.1 - 200 ppb respectively.

[0025] Preferably, in the above-mentioned method for quantitative detection of sulfur-containing compounds suitable for the Baijiu system, in step A, the concentrations of the internal standards ethyl methyl sulfide, diethyl sulfide, thiophene, and isopropyl disulfide in the Baijiu sample to be measured are 0.5 - 20 ppb respectively.

[0026] The present invention can quantitatively analyze individually or simultaneously detect the concentrations of 12 common volatile sulfur-containing compounds in different types of Baijiu. For different types of Baijiu, the standard curves of the 12 sulfur-containing compounds contained therein need to be obtained. Therefore, in step C of the above-mentioned method for quantitative detection of sulfur-containing compounds suitable for the Baijiu system, the linear ranges of the standard curves of the 12 sulfur-containing compounds are as follows according to different types of Baijiu:

[0027]

[0028]

[0029] Those skilled in the art can know according to the description of the present invention that in step A, the "Baijiu simulated wine sample" + "series of dosage standards of at least one of the 12 sulfur-containing compounds" when preparing the standard sample to be measured is equivalent to the "Baijiu wine sample" when preparing the Baijiu sample to be measured; for the convenience of calculating the concentration of sulfur-containing compounds in the Baijiu sample to be measured according to the standard curve, the "concentration" in the above-mentioned "linear range" is the concentration of sulfur-containing compounds after mixing the "Baijiu simulated wine sample" and the "series of dosage standards of at least one of the 12 sulfur-containing compounds", and thus the concentration of sulfur-containing compounds in the Baijiu sample to be measured is calculated accordingly. Those skilled in the art can take several points (generally not less than 5) within the above linear range based on common analytical knowledge for drawing the standard curve.

[0030] Among them, in the above-mentioned method for quantitative detection of sulfur-containing compounds suitable for the Baijiu system, in step B, the solid-phase extraction fiber head is selected from any one of CAR, DVB, PDMS, DVB / PDMS, CAR / PDMS, and DVB / CAR / PDMS.

[0031] Preferably, in the above-mentioned method for quantitative detection of sulfur-containing compounds suitable for the Baijiu system, in step B, the solid-phase extraction fiber head is selected from any one of PDMS, CAR / PDMS, and DVB / CAR / PDMS.

[0032] Among them, in the above-mentioned method for quantitative detection of sulfur-containing compounds suitable for the Baijiu system, in step B, the solid-phase extraction fiber head is aged at 250 - 320 °C for 1 - 10 min in the aging station before extraction and after injection respectively.

[0033] Among them, in the quantitative detection method of sulfur-containing compounds suitable for the white liquor system, in step B, the extraction conditions are to balance for 3 - 20 min at 20 - 50°C under a rotation speed of 200 - 600 rpm, extract for 10 - 80 min, desorb at the injection port at 200 - 300°C for 1 - 8 min, and inject without splitting.

[0034] Among them, in the quantitative detection method of sulfur-containing compounds suitable for the white liquor system, in step C, the gas chromatography conditions are as follows: the carrier gas is He (99.999%), the flow rate is 1 - 3.5 mL / min; the chromatographic column is 30 m * 0.32 mm * 1.0 μm DB - FFAP; the injection port temperature is 200 - 300°C; inject without splitting; the column oven temperature programming is to hold at 40 - 60°C for 2 - 6 min, then rise to 220 - 240°C at a rate of 4 - 6°C / min and hold for 2 - 15 min.

[0035] Among them, in the quantitative detection method of sulfur-containing compounds suitable for the white liquor system, in step C, the sulfur chemiluminescence detector conditions are as follows: the base temperature is 250°C, the burner temperature is 800°C, the upper hydrogen flow rate is 40 mL / min, the lower hydrogen flow rate is 10 mL / min, and the oxidation gas flow rate is 50 mL / min.

[0036] Advantages of the present invention:

[0037] The method established by the present invention can quickly and accurately quantify 12 volatile sulfur-containing compounds in different flavor types of white liquors. These 12 compounds use the corresponding substances as internal standards, and the quantitative limits of each compound are all less than 14 μg / L, and the linear correlation coefficient R 2 is all greater than 0.997, the relative standard deviation is all less than 5%, and the recovery rate is between 83% - 117%.

[0038] The present invention adopts the headspace-solid phase microextraction-gas chromatography-sulfur chemiluminescence detector technology and establishes a specific internal standard system and different simulated systems containing the backbone components of white liquor for quantitative analysis. This technology is simple, fast, and accurate. The headspace-solid phase microextraction technology has high selectivity for the adsorption of volatile compounds, high sensitivity, and has good advantages for the quantification of sulfur-containing compounds. By using a specific internal standard system and different simulated systems containing the backbone components of white liquor, the problem of inaccurate quantification caused by competitive adsorption in the previous quantitative methods of sulfur-containing compounds in white liquor is solved, providing a new technology for the quality control of white liquor and having important significance for the improvement of the quality of white liquor. Description of the Drawings

[0039] Figure 1 It is the chromatogram of Luzhou-flavor white liquor 1 in Example 1.

[0040] Figure 2 It is the chromatogram of the Luzhou-flavor liquor 2 in Example 1.

[0041] Figure 3 It is the chromatogram of the Qingxiang-flavor liquor 1 in Example 1.

[0042] Figure 4 It is the chromatogram of the Qingxiang-flavor liquor 2 in Example 1.

[0043] Figure 5 It is the chromatogram of the Maotai-flavor liquor in Example 1.

[0044] Figure 6 It is the influence deviation (CAR / PDMS) of the change in the content of skeletal substances in liquor on the quantification of sulfur-containing compounds.

[0045] Figure 7 It is the influence deviation (DVB / CAR / PDMS) of the change in the content of skeletal substances in liquor on the quantification of sulfur-containing compounds. Detailed implementation manners

[0046] Specifically, for the method for quantitatively detecting sulfur-containing compounds suitable for the liquor system, it adopts the headspace-solid phase microextraction-gas chromatography-sulfur chemiluminescence detector technology, uses at least one of ethyl methyl sulfide, diethyl sulfide, thiophene, and isopropyl disulfide as the internal standard substance, takes the response ratio of the sulfur-containing compound standard substance to the corresponding internal standard substance as the ordinate, and the concentration ratio of the sulfur-containing compound standard substance to the corresponding internal standard substance as the abscissa to draw a standard curve, and quantitatively detects the content of at least one of 12 sulfur-containing compounds including dimethyl sulfide, allyl methyl sulfide, dimethyl disulfide, ethyl thioacetate, 3-methylthiophene, propyl thioacetate, ethyl disulfide, 2-methyl-3-(methylthio)furan, dimethyl trisulfide, methyl thioacetate, furfuryl methyl sulfide, or ethyl 3-(methylthio)propionate in liquor; wherein, the corresponding internal standard substances for dimethyl sulfide and allyl methyl sulfide are ethyl methyl sulfide, the corresponding internal standard substances for dimethyl disulfide, ethyl thioacetate, propyl thioacetate, and methyl thioacetate are diethyl sulfide, the corresponding internal standard substance for 3-methylthiophene is thiophene, and the corresponding internal standard substances for ethyl disulfide, 2-methyl-3-(methylthio)furan, dimethyl trisulfide, furfuryl methyl sulfide, and ethyl 3-(methylthio)propionate are isopropyl disulfide.

[0047] Based on the above analysis principle, the method for quantitatively detecting sulfur-containing compounds suitable for the liquor system provided by the present invention specifically includes the following steps:

[0048] A. Pretreatment:

[0049] Preparation of standard samples to be measured: Mix saline solution, simulated liquor samples of white liquor, series of standard samples with at least one of 12 sulfur-containing compounds (dimethyl sulfide, allyl methyl sulfide, dimethyl disulfide, ethyl thioacetate, 3-methylthiophene, propyl thioacetate, ethyl disulfide, 2-methyl-3-(methylthio)furan, dimethyl trisulfide, methyl thiomethyl acetate, furfuryl methyl sulfide or ethyl 3-(methylthio)propionate) and corresponding internal standards (the corresponding internal standards are determined according to the sulfur-containing compounds to be detected) to obtain series of concentration standard samples to be measured with at least one of 12 sulfur-containing compounds;

[0050] Preparation of white liquor samples to be measured: Mix saline solution, white liquor samples and corresponding internal standards to obtain white liquor samples to be measured;

[0051] B. Injection: Extract and inject the standard samples to be measured and white liquor samples to be measured by headspace-solid phase microextraction;

[0052] C. Detection: Use gas chromatography-sulfur chemiluminescence detector to test the standard samples to be measured. Draw a standard curve with the response ratio of the sulfur-containing compound to be measured to the corresponding internal standard as the ordinate and the concentration ratio of the sulfur-containing compound to be measured to the corresponding internal standard as the abscissa;

[0053] Test the white liquor samples to be measured under the same conditions to obtain the response ratio of the sulfur-containing compounds in the white liquor samples to their corresponding internal standards. Calculate the content of the corresponding sulfur-containing compounds in the white liquor according to the standard curve.

[0054] As in the embodiments of the present application, when preparing the standard samples to be measured and white liquor samples to be measured, for the convenience of operation and weighing, generally, the standard samples of 12 volatile sulfur-containing compounds are prepared into a stock solution with a certain concentration using chromatographic grade ethanol, and then the stock solution is diluted into mixed standard sample solutions with different concentration gradients. The selected corresponding internal standard system is respectively prepared into a stock solution with a certain concentration (such as about 10000 mg / L) using chromatographic grade methanol (sulfides will be oxidized when stored in ethanol for a long time, so all standard sample mother liquors in the present invention are prepared with methanol and stored below -20°C, with a validity period of 3 months), and then the internal standard solutions are mixed and diluted to the corresponding concentration for use as mixed internal standards. In step A, when preparing the samples, saturated saline solution is more conducive to the volatilization of substances, so the dilution reagent used in the HS-SPME analysis of white liquor flavor is generally saturated saline solution.

[0055] The research of the present invention finds that different skeletal substances in liquor have different quantitative effects on sulfur-containing compounds. At the same time, the types and contents of skeletal substances in different flavor liquors are different, which also affect the quantitative results of sulfur-containing compounds. Therefore, based on the differences in the quantitative effects of different flavor types and different skeletal substances on sulfur-containing compounds, the present invention designs standard samples by configuring simulated liquor samples of three flavor types, namely strong flavor, sauce flavor and light flavor, for drawing standard curves, thus avoiding the problem of inaccurate quantification caused by different competitive adsorptions of different skeletal substance contents on the solid-phase microextraction fiber head, and improving the accuracy of quantitative detection of sulfur-containing compounds in liquor samples.

[0056] The simulated liquor samples of different flavor types are 30-65% vol aqueous alcohol solutions configured according to the following substances and their concentrations;

[0057]

[0058] The research of the present invention finds that the ethanol concentration of the sample to be measured has a great influence on the quantification of sulfur-containing compounds. On the one hand, high-concentration ethanol will cause quantitative errors by competitively adsorbing with other volatile compounds on the solid-phase microextraction fiber head. On the other hand, ethanol as a solvent may wash off the adsorbed volatile compounds, thus forming a new equilibrium. Therefore, in order to avoid the influence of ethanol on the quantification of sulfur-containing compounds in liquor, the present invention controls the ethanol concentration in the standard sample to be measured or the liquor sample to be measured at 3-15% vol, preferably controlled at 5-10% vol; at the same time, at this concentration, the present invention studies the quantitative deviation of each internal standard in each internal standard system for each corresponding sulfur-containing compound when the ethanol concentration changes, so as to screen out a batch of corresponding specific internal standards for the quantification of sulfur-containing compounds.

[0059] Through comprehensive research, the present invention determines the quantitative deviation effects of a variety of internal standards on 12 sulfur-containing compounds under different liquor flavor types (including major component skeletal substances), ethanol concentrations, solid-phase microextraction fiber heads and other conditions, so as to determine a specific internal standard system corresponding to a specific sulfur-containing compound, and solves the problem of inaccurate quantification caused by competitive adsorption in the previous quantitative methods of sulfur-containing compounds in liquor. In step A of the present invention, the concentrations of the internal standards ethyl methyl sulfide, diethyl sulfide, thiophene and isopropyl disulfide in the series concentration standard samples to be measured are controlled at 0.1-200 ppb respectively; preferably, the concentrations of the internal standards ethyl methyl sulfide, diethyl sulfide, thiophene and isopropyl disulfide in the liquor sample to be measured are 0.1-200 ppb respectively.

[0060] The present invention needs to simultaneously quantitatively detect the concentrations of 12 common volatile sulfur-containing compounds in baijiu of different flavors. For different flavors, it is also necessary to obtain the standard curves of the 12 sulfur-containing compounds contained therein. Therefore, in step C of the above-mentioned method for quantitatively detecting sulfur-containing compounds suitable for the baijiu system, the linear ranges of the standard curves of the 12 sulfur-containing compounds are as follows according to different flavors:

[0061]

[0062] Those skilled in the art can take several points (for example, gradient concentration points not less than 5 or 6) from the above ranges for drawing the standard curve according to the common sense of analytical detection.

[0063] In step B of the present invention, the solid-phase extraction fiber head is selected from any one of CAR, DVB, PDMS, DVB / PDMS, CAR / PDMS, and DVB / CAR / PDMS; preferably, the solid-phase extraction fiber head is selected from any one of PDMS, CAR / PDMS, and DVB / CAR / PDMS.

[0064] In step B of the present invention, the solid-phase extraction fiber head is aged at 250-320 °C for 1-10 min in the aging station before extraction and after injection. In step B of the present invention, the extraction conditions are to balance at 20-50 °C for 3-20 min at a rotation speed of 200-600 rpm, extract for 10-80 min, and desorb at 200-300 °C at the injection port for 1-8 min, with splitless injection.

[0065] In step C of the present invention, the gas chromatography conditions are as follows: the carrier gas is He (99.999%), and the flow rate is 1-3.5 mL / min; the chromatographic column is 30 m * 0.32 mm * 1.0 μm DB-FFAP; the injection port temperature is 200-300 °C; splitless injection; the column oven temperature program is to hold at 40-60 °C for 2-6 min, and then rise to 220-240 °C at a rate of 4-6 °C / min and hold for 2-15 min. In step C of the present invention, the sulfur chemiluminescence detector conditions are as follows: the base temperature is 250 °C, the burner temperature is 800 °C, the upper hydrogen flow rate is 40 mL / min, the lower hydrogen flow rate is 10 mL / min, and the oxidation gas flow rate is 50 mL / min.

[0066] It should be noted that the present invention can achieve the individual quantitative detection of a certain sulfur-containing compound among the 12 volatile sulfur-containing compounds in baijiu, and can also simultaneously quantitatively detect several or all of the 12 sulfur-containing compounds.

[0067] The solution of the present invention will be explained below in conjunction with test examples and embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those without specific techniques or conditions noted in the embodiments, the techniques or conditions described in the literature in the art or according to the product specifications are followed. For reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through commercial purchases.

[0068] Example 1: Determination of the content of sulfur-containing compounds in finished baijiu of different fragrance types

[0069] (1) Pretreatment of baijiu

[0070] Boil ultrapure water for 5 min, cool it to room temperature, and then add NaCl until saturated to make saturated saline. Pipette 4.5 mL of saturated saline into a 20 mL headspace vial, purge with nitrogen to remove oxygen, and then accurately pipette 0.5 mL of the baijiu sample to be tested (a total of 5 baijiu products as shown in Table 9) into the headspace vial. Then add 10 μL of the mixed internal standard solution (the types of internal standards and their final concentrations in the sample to be tested are shown in Table 1) to obtain the baijiu sample to be tested. Tighten the bottle cap (the septum is PTFE / silicone) and place it at 4 °C for later measurement.

[0071] (2) Preparation of the standard curve

[0072] Boil ultrapure water for 5 min, cool it to room temperature, and then add NaCl until saturated to make saturated saline. Pipette 4.5 mL of saturated saline into a 20 mL headspace vial, purge with nitrogen to remove oxygen, and then accurately pipette 0.5 mL of the simulated baijiu sample systems of strong, light, and sauce fragrance types (as shown in Table 2) into the headspace vial. Add 20 μL of the sulfur-containing compound mixed standard solution at each concentration gradient (the concentrations of each sulfur-containing compound in the sulfur-containing compound mixed standard solution are shown in Tables 3 - 5), and then add 10 μL of the mixed internal standard solution (the types of internal standards and their final concentrations in the sample to be tested are shown in Table 1) to obtain the standard sample to be tested. Tighten the bottle cap (the septum is PTFE / silicone) and place it at 4 °C for later measurement.

[0073] Table 1 Internal standards and their final concentrations

[0074]

[0075]

[0076] For the above simulated baijiu sample systems of strong, light, and sauce fragrance types, their preparation schemes are shown in Table 2. The skeletal substances in baijiu are configured into a 52% vol aqueous ethanol solution according to the concentrations in Table 2. Among them, the reference standards and absolute ethanol involved in Table 2 are purchased from Sigma-Aldrich in the United States, TCI in Japan, or J&K in Northern Europe.

[0077] Table 2 Concentrations of various substances in the simulated wine samples of strong, light, and sauce-flavored Baijiu

[0078]

[0079]

[0080] Table 3 Mixed standard solution of sulfur-containing compounds (for drawing the standard curve of strong-flavored Baijiu system)

[0081]

[0082] Table 4 Mixed standard solution of sulfur-containing compounds (for drawing the standard curve of light-flavored Baijiu)

[0083]

[0084]

[0085] Table 5 Mixed standard solution of sulfur-containing compounds (for drawing the standard curve of sauce-flavored Baijiu)

[0086]

[0087] (3), Detection method

[0088] Headspace-solid phase microextraction operation: Extraction was carried out using a multi-functional autosampler with a headspace-solid phase microextraction module; the extraction conditions were equilibration at 45°C for 5 min at a rotation speed of 250 rpm, extraction for 40 min, desorption at 270°C in the injection port for 5 min, and splitless injection. The solid-phase extraction fiber head was CAR / PDMS (1 cm), and it was aged at 300°C for 2 min in the aging station before extraction and after injection respectively.

[0089] Gas chromatography conditions: The carrier gas was He (99.999%), the flow rate was 2.5 mL / min; the chromatographic column was DB-FFAP (30 m * 0.32 mm * 1.0 μm); the injection port temperature was 270°C; splitless injection; the column oven temperature program was to hold at 40°C for 5 min, then rise to 230°C at a rate of 4°C / min and hold for 5 min.

[0090] Sulfur chemiluminescence detector conditions: The base temperature was 250°C, the burner temperature was 800°C, and the flow rates of the upper hydrogen, lower hydrogen, and oxidation gas (air) were 40, 10, and 50 mL / min respectively.

[0091] Under the above instrument conditions, standard test samples of different flavors containing sulfur-containing compounds with different concentration gradients were tested. Using the response ratio of the standard product to the corresponding internal standard as the ordinate and the actual concentration ratio of the standard product to the corresponding internal standard as the abscissa, a standard curve was drawn, and the relevant methodological parameters are shown in Tables 6 - 8.

[0092] Table 6 Methodological parameters of the quantitative method for sulfur-containing compounds in Luzhou-flavor Baijiu

[0093]

[0094] Note: IS1: Ethyl methyl sulfide; IS2: Diethyl sulfide; IS3: Thiophene; IS4: Isopropyl disulfide; IS5: 4-Methylthiazole.

[0095] Table 7 Methodological parameters of the quantitative method for sulfur-containing compounds in Qingxiang-flavor Baijiu

[0096]

[0097]

[0098] Note: IS1: Ethyl methyl sulfide; IS2: Diethyl sulfide; IS3: Thiophene; IS4: Isopropyl disulfide; IS5: 4-Methylthiazole.

[0099] Table 8 Methodological parameters of the quantitative method for sulfur-containing compounds in Maotai-flavor Baijiu

[0100]

[0101] Note: IS1: Ethyl methyl sulfide; IS2: Diethyl sulfide; IS3: Thiophene; IS4: Isopropyl disulfide; IS5: 4-Methylthiazole.

[0102] Under the same instrument conditions, the test samples of Baijiu with different flavors were tested, and the response values of 12 common volatile sulfur-containing compounds in Baijiu were obtained respectively. According to the response ratio of the compounds in the sample to the corresponding internal standard, the contents of volatile sulfur-containing compounds in 12 Baijiu with different flavors were calculated by referring to the standard curves of the corresponding flavors. The results are shown in Table 9, and the chromatograms of representative Baijiu samples with different flavors are shown in Figures 1 - 3 .

[0103] Table 9 Contents of sulfur-containing compounds in finished Baijiu with different flavors

[0104]

[0105] Note: <ql. indicates less than the quantitation limit.

[0106] Example 2: Determination of recovery rate

[0107] To verify the reliability of the results, the recovery rate was determined.

[0108] Determination of recovery rate: Quantify the content of volatile sulfur compounds in finished baijiu of each flavor type (repeatedly determined 3 times). Add standard mixed solutions of sulfur compounds with two different concentrations into a 5 mL sample injection system (in the sample injection vial, the final concentrations of 12 sulfur compounds are two concentration gradient points of S5 and S6 in Tables 3 - 5), and determine the sulfur compounds in the two spiked samples (repeated 3 times). The recovery rate is calculated by the following formula (spiked recovery rate = (measured value of spiked sample - measured value of sample) ÷ spiked amount × 100%), and the average recovery rates of the two concentration spiked samples are shown in Table 10.

[0109] Table 10 Recovery Rates of the Quantitative Method for Sulfur Compounds in Baijiu

[0110]

[0111]

[0112] Note: IS1: Ethyl methyl sulfide; IS2: Diethyl sulfide; IS3: Thiophene; IS4: Isopropyl disulfide; IS5: 4 - Methylthiazole.

[0113] It can be seen from the results that the method of the present invention has good recovery rates in different baijiu, which proves that when using HS - SPME to quantify sulfur compounds in baijiu, this system can, to a certain extent, avoid the problem of inaccurate quantification caused by competitive adsorption of ethanol and skeletal substances on the solid - phase microextraction fiber head.

[0114] In summary, this method not only has the advantages of high selectivity, high sensitivity, simplicity, rapidity, and high automation, but also can, through a specific internal standard system and different simulated systems containing baijiu skeletal components, solve the problem of inaccurate quantification caused by competitive adsorption in the previous quantitative methods for sulfur compounds in baijiu, providing a new technology for the quality control of baijiu and having important significance for improving the quality of baijiu.

[0115] Test Example 1: Influence of Ethanol Concentration on the Quantification of Sulfur Compounds in Baijiu

[0116] (1) Preparation of Samples with Different Ethanol Concentrations

[0117] Boil ultrapure water for 5 min, add NaCl to saturation after cooling to room temperature to prepare saturated brine. Use this saturated brine and ethanol to prepare ethanol aqueous solutions with concentrations of 3, 5, 7, 9, 11, 13, and 15% vol respectively. Pipette 5 mL of ethanol aqueous solutions with different concentrations into 20 mL headspace vials, purge with nitrogen to remove oxygen, add 20 μL of sulfur compound mixed solution (the concentrations of each sulfur compound in the sulfur compound mixed solution are shown in Table 11), then add 10 μL of mixed internal standard solution (the types of internal standards and their final concentrations in the samples to be measured are shown in Table 1), to obtain the samples to be measured, tighten the vial caps (the septum is PTFE / silicone), and place them at 4 °C for later measurement.

[0118] Table 11 Concentrations of 12 sulfur compounds in the sulfur compound mixed solution

[0119]

[0120]

[0121] (2) Detection method

[0122] Headspace-solid phase microextraction operation: Use a multi-functional autosampler with a headspace-solid phase microextraction module for extraction; the extraction conditions are equilibration at 45 °C for 5 min at a rotation speed of 250 rpm, extraction for 40 min, and desorption at 270 °C at the injection port for 5 min, with splitless injection. The solid phase extraction fiber head is selected from any one of CAR / PDMS (1 cm) and DVB / CAR / PDMS (2 cm), and is aged at 300 and 270 °C for 2 min at the aging station before extraction and after injection respectively.

[0123] Gas chromatography conditions: The carrier gas is He (99.999%), the flow rate is 2.5 mL / min; the chromatographic column is DB-FFAP (30 m * 0.32 mm * 1.0 μm); the injection port temperature is 270 °C; splitless injection; the column oven temperature program is hold at 40 °C for 5 min, then increase to 230 °C at a rate of 4 °C / min and hold for 5 min.

[0124] Sulfur chemiluminescence detector conditions: The base temperature is 250 °C, the burner temperature is 800 °C, and the flow rates of the upper hydrogen, lower hydrogen, and oxidizing gas (air) are 40, 10, and 50 mL / min respectively.

[0125] Under the above instrument conditions, measure the responses of sulfur compounds and each internal standard at different ethanol concentrations. The quantitative deviation is calculated by the following formula (quantitative deviation = standard deviation of response ratio / average value of response ratio × 100%). Tables 12 and 13 list the quantitative deviation results on two different solid phase microextraction fiber heads. A series of corresponding internal standards are selected based on the deviation of the response ratio of sulfur compounds to internal standards at different ethanol concentrations.

[0126] Table 12 Quantitative deviation of sulfur-containing compounds caused by changes in ethanol content (CAR / PDMS)

[0127]

[0128]

[0129] Note: IS1: Ethyl methyl sulfide; IS2: Diethyl sulfide; IS3: Thiophene; IS4: Isopropyl disulfide; IS5: 4-Methylthiazole.

[0130] Table 13 Quantitative deviation of sulfur-containing compounds caused by changes in ethanol content (DVB / CAR / PDMS)

[0131]

[0132] Note: IS1: Ethyl methyl sulfide; IS2: Diethyl sulfide; IS3: Thiophene; IS4: Isopropyl disulfide; IS5: 4-Methylthiazole.

[0133] As can be seen from Table 12 and Table 13, the ethanol concentration during the sample introduction of Chinese liquor has a great influence on its quantification. On the one hand, high-concentration ethanol will competitively adsorb with other volatile compounds on the solid-phase microextraction fiber head, resulting in quantification errors. On the other hand, ethanol as a solvent may wash off the adsorbed volatile compounds, thus forming a new equilibrium. In addition, regardless of which fiber head is used, the change in ethanol concentration has an impact on the quantification of sulfur-containing compounds. Therefore, in the early research of the present invention, most Chinese liquors were diluted to 5% or 10% for sample introduction. At the same time, in order to avoid the influence of ethanol on the quantification of sulfur-containing compounds in Chinese liquor, the present invention studied the quantitative deviation of all internal standards in this internal standard system for each sulfur-containing compound when the ethanol concentration changes, so as to screen out a batch of corresponding internal standards for the quantification of sulfur-containing compounds.

[0134] Experimental Example 2: Influence of the alcohol concentration of the major component skeleton substances in Chinese liquor on the quantification of sulfur-containing compounds in Chinese liquor

[0135] (1) Preparation of samples with different concentrations of major component skeleton substances

[0136] Boil ultrapure water for 5 min, cool it to room temperature, and add NaCl until saturation to prepare saturated brine. Accurately pipette 5 mL of saturated brine into a 20 mL headspace vial, purge with nitrogen to remove oxygen, add 20 μL of the sulfur compound mixed solution (the concentrations of each sulfur compound in the sulfur compound mixed solution are shown in Table 8), 20 μL of the ethanol solution containing a certain concentration of the skeleton substance (the final concentration is shown in Table 14, with reference to its concentration in strong, light, and sauce-flavored Baijiu), and then add 10 μL of the mixed internal standard solution (the types of internal standards and their final concentrations in the sample to be measured are shown in Table 1) to obtain the sample to be measured. Tighten the bottle cap (the septum is PTFE / silicone), and place it at 4 °C for later measurement.

[0137] Table 14 Final concentrations of each skeleton substance in the sample to be measured

[0138]

[0139] (2) Detection method

[0140] Headspace-solid phase microextraction operation: Use a multi-functional autosampler with a headspace-solid phase microextraction module for extraction; the extraction conditions are to equilibrate at 45 °C for 5 min at a rotation speed of 250 rpm, extract for 40 min, desorb at 270 °C at the injection port for 5 min, and inject without splitting. The solid phase extraction fiber head is selected from any one of CAR / PDMS (1 cm) and DVB / CAR / PDMS (2 cm), and is aged at 300 and 270 °C for 2 min at the aging station before extraction and after injection, respectively.

[0141] Gas chromatography conditions: The carrier gas is He (99.999%), the flow rate is 2.5 mL / min; the chromatographic column is DB-FFAP (30 m * 0.32 mm * 1.0 μm); the injection port temperature is 270 °C; inject without splitting; the column oven temperature program is to hold at 40 °C for 5 min, then increase to 230 °C at a rate of 4 °C / min and hold for 5 min.

[0142] Sulfur chemiluminescence detector conditions: The base temperature is 250 °C, the burner temperature is 800 °C, and the flow rates of the upper hydrogen, lower hydrogen, and oxidizing gas (air) are 40, 10, and 50 mL / min, respectively.

[0143] Under the above instrument conditions, measure the responses of sulfur compounds and each internal standard at different concentrations of the skeleton substance. The quantitative deviation is calculated by the following formula (quantitative deviation = standard deviation of response ratio / average value of response ratio × 100%).

[0144] Figure 6 、 Figure 7The quantitative deviation results of two different solid-phase microextraction fiber heads are listed. The results show that for both CAR / PDMS and DVB / CAR / PDMS solid-phase microextraction fiber heads, the behavior of low-molecular-weight sulfur-containing compounds is basically consistent with the selected internal standards and is hardly affected by the change in the concentration of the backbone substances in Chinese liquor. However, the consistency of high-molecular-weight sulfur-containing compounds with these internal standards is poor. Therefore, when using CAR / PDMS, new internal standards should be selected for these sulfur-containing compounds. For individual backbone substances, from the perspective of the influence of backbone substances, the change in the concentration of ethyl hexanoate has the greatest impact on the quantification of sulfur-containing compounds, followed by ethyl butyrate, hexanoic acid, and 3-methylbutanol. The changes in the concentrations of 1-propanol and acetic acid have less impact on the quantification of sulfur-containing compounds. In order to avoid the influence of backbone substances on the quantification of sulfur-containing compounds in the Chinese liquor system, the present invention studies the quantitative deviation of all internal standards in the internal standard system screened in Comparative Example 1 for each sulfur-containing compound when the concentration of backbone substances changes, so as to screen out a batch of corresponding internal standards for the quantification of sulfur-containing compounds.

[0145] By comprehensively studying the quantitative deviation of a variety of internal standards and 12 sulfur-containing compounds during the processes of changes in the concentration of backbone substances, ethanol concentration, solid-phase microextraction fiber head, etc., and through comprehensive comparison, in order to simultaneously meet the condition that the quantitative deviation is not greater than 20% under various conditions, the present invention determines that the corresponding internal standard of dimethyl sulfide and allyl methyl sulfide is ethyl methyl sulfide, the corresponding internal standards of dimethyl disulfide, ethyl thioacetate, propyl thioacetate, and methyl methylthioacetate are diethyl sulfide, the corresponding internal standard of 3-methylthiophene is thiophene, and the corresponding internal standards of ethyl disulfide, 2-methyl-3-(methylthio)furan, dimethyl trisulfide, furfuryl methyl sulfide, and ethyl 3-(methylthio)propionate are isopropyl disulfide.

Claims

1. A method for quantitative detection of sulfur-containing compounds suitable for the Baijiu system, characterized in that: Using headspace-solid phase microextraction-gas chromatography-sulfur chemiluminescence detector technology, with ethyl methyl sulfide, diethyl sulfide, thiophene, and isopropyl disulfide as internal standards, taking the response ratio of sulfur-containing compound standards to the corresponding internal standards as the ordinate and the concentration ratio of sulfur-containing compound standards to the corresponding internal standards as the abscissa, a standard curve is plotted to quantitatively detect the contents of 12 sulfur-containing compounds in Baijiu, namely dimethyl sulfide, allyl methyl sulfide, dimethyl disulfide, ethyl thioacetate, 3-methylthiophene, propyl thioacetate, ethyl disulfide, 2-methyl-3-(methylthio)furan, dimethyl trisulfide, methyl thiolacetate, furfuryl methyl sulfide, and ethyl 3-(methylthio)propionate; among them, the corresponding internal standards for dimethyl sulfide and allyl methyl sulfide are ethyl methyl sulfide, the corresponding internal standards for dimethyl disulfide, ethyl thioacetate, propyl thioacetate, and methyl thiolacetate are diethyl sulfide, the corresponding internal standard for 3-methylthiophene is thiophene, and the corresponding internal standards for ethyl disulfide, 2-methyl-3-(methylthio)furan, dimethyl trisulfide, furfuryl methyl sulfide, and ethyl 3-(methylthio)propionate are isopropyl disulfide; Including the following steps: A. Pretreatment: Configure standard samples to be measured: Mix brine, Baijiu simulated wine samples, a series of dosage standards of 12 sulfur-containing compounds, and the corresponding internal standards to obtain a series of concentration standard samples to be measured for 12 sulfur-containing compounds; Configure Baijiu samples to be measured: Mix brine, Baijiu samples, and the corresponding internal standards to obtain Baijiu samples to be measured; B. Injection: Extract and inject the standard samples to be measured and Baijiu samples to be measured through headspace-solid phase microextraction; In step A, the dosage of the brine is based on controlling the ethanol concentration in the standard samples to be measured or Baijiu samples to be measured to be 3-15% vol; In step B, the solid-phase extraction fiber head is CAR / PDMS; Gas chromatography conditions: The carrier gas is He, with a flow rate of 1-3.5 mL / min; the chromatographic column is a 30 m × 0.32 mm × 1.0 μm DB-FFAP; the injection port temperature is 200-300 °C; splitless injection; the column oven temperature program is to hold at 40-60 °C for 2-6 min, and then rise to 220-240 °C at a rate of 4-6 °C / min and hold for 2-15 min.

2. The quantitative detection method of sulfur-containing compounds suitable for the baijiu system according to claim 1, characterized in that: It also includes the following steps: C. Detection: Use gas chromatography-sulfur chemiluminescence detector in combination to test the standard samples to be measured, taking the response ratio of the sulfur-containing compound to be measured to the corresponding internal standard as the ordinate and the concentration ratio of the sulfur-containing compound to be measured to the corresponding internal standard as the abscissa, and plot a standard curve; Test the Baijiu samples to be measured under the same conditions to obtain the response ratio of the sulfur-containing compounds in the Baijiu samples to their corresponding internal standards, and calculate the content of the corresponding sulfur-containing compounds in the Baijiu samples according to the standard curve.

3. The quantitative detection method of sulfur-containing compounds suitable for the Baijiu system according to claim 2, characterized in that: In step A, the Baijiu simulated wine samples are determined according to the fragrance type of the Baijiu samples, including strong fragrance type, sauce fragrance type, and light fragrance type. The Baijiu simulated wine samples of different fragrance types are 30-65% vol aqueous alcohol solutions configured according to the following substances and their concentrations; 4. The quantitative detection method of sulfur-containing compounds suitable for the baijiu system according to claim 2, characterized in that: In step A, the dosage of the brine is based on controlling the ethanol concentration in the standard samples to be measured or Baijiu samples to be measured to be 5-10% vol.

5. The quantitative detection method of sulfur-containing compounds suitable for the Baijiu system according to claim 2, wherein: In step A, the concentrations of internal standards ethyl methyl sulfide, diethyl sulfide, thiophene, and isopropyl disulfide in the series of standard samples to be measured with varying concentrations are 0.1 - 200 ppb respectively.

6. The method for quantitative detection of sulfur-containing compounds suitable for the Baijiu system according to claim 5, characterized in that: In step A, the concentrations of internal standards ethyl methyl sulfide, diethyl sulfide, thiophene, and isopropyl disulfide in the series of standard samples to be measured with varying concentrations are 0.5 - 20 ppb respectively.

7. The quantitative detection method of sulfur-containing compounds suitable for the baijiu system according to claim 2, characterized in that: In step A, the concentrations of internal standards ethyl methyl sulfide, diethyl sulfide, thiophene, and isopropyl disulfide in the liquor sample to be measured are 0.1 - 200 ppb respectively.

8. The method for quantitative detection of sulfur-containing compounds suitable for the Baijiu system according to claim 7, characterized in that: In step A, the concentrations of internal standards ethyl methyl sulfide, diethyl sulfide, thiophene, and isopropyl disulfide in the liquor sample to be measured are 0.5 - 20 ppb respectively.

9. The method for quantitative detection of sulfur-containing compounds suitable for the Baijiu system according to claim 2, wherein: In step C, the linear ranges of the standard curves of 12 sulfur-containing compounds are as follows according to different fragrance types of liquor:

10. The method for quantitative detection of sulfur-containing compounds suitable for the baijiu system according to claim 2, wherein: In step B, the solid-phase extraction fiber head is aged at 250 - 320 °C for 1 - 10 min at the aging station before extraction and after injection respectively.

11. The method for quantitative detection of sulfur-containing compounds suitable for the baijiu system according to claim 2, wherein: In step B, the extraction conditions are: equilibration at 20 - 50 °C for 3 - 20 min at a rotation speed of 200 - 600 rpm, extraction for 10 - 80 min, desorption at 200 - 300 °C at the injection port for 1 - 8 min, and splitless injection.

12. The method for quantitative detection of sulfur-containing compounds suitable for the Baijiu system according to any one of claims 2 to 11, characterized in that: In step C, the conditions of the sulfur chemiluminescence detector are: base temperature 250 °C, burner temperature 800 °C, upper hydrogen flow rate 40 mL / min, lower hydrogen flow rate 10 mL / min, and oxidation gas flow rate 50 mL / min.

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