Method for rapid detection of chiral substances in baijiu and daqu and application thereof

Abstract

The application discloses a kind of methods for rapidly detecting chiral substances in liquor and fermented grains and application, belong to liquor flavor analysis technical field.The method is specifically as follows: pretreatment is carried out to the liquor or fermented grains sample to be measured;Center-cutting-based multidimensional chromatographic detection system is constructed: the polarity column HP-INNOWAX with the specification of 60m*0.32mm*0.5μm is used as one-dimensional column, and is connected with FID detector;Low heat capacity chiral chromatographic column LTM CP-Chirasil-Dex CB with the specification of 30m*0.25mm*0.25μm is used as two-dimensional column, and is connected with MS detector;The chiral substances in the sample to be measured are qualitatively analyzed using the constructed detection system.The technical scheme of the application has the advantages of high accuracy and good reproducibility, provides a new technology for the quality control of liquor, and has important significance for improving the quality of liquor.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of liquor flavor analysis, and relates to a method for rapidly detecting the proportion of chiral substances in liquor and liquor production products by using gas chromatography combined with center cutting technology, in particular to a method for rapidly detecting chiral substances in liquor and fermented grains and application thereof. BACKGROUND

[0002] Liquor is a traditional distillate in China, and its history has been more than 2000 years. There are various types of Chinese liquor, and each type has its own characteristics and production process, and has good flavor and taste. There are various types of flavor compounds in liquor, and the contents of these compounds are different, which determine the style and characteristics of the liquor.

[0003] Chiral compounds play an important role in the fields of food, medicine and pesticide. In the field of spices and food, different configurations of chiral compounds may produce different flavor systems. Since biological synthesis is usually driven by enzymes, the chiral compounds of these reaction products usually exist in a specific enantiomeric ratio with certain regularity, while the enantiomeric ratio of artificially synthesized chiral compounds often differs greatly. Therefore, chiral analysis of liquor can be used for quality control, detection of sample adulteration, determination of exogenous addition, judgment of pure grain solid-state fermentation, etc. Therefore, it is of great significance to determine the chiral compounds and enantiomeric composition in liquor for determining the quality and authenticity of liquor.

[0004] At present, the common extraction methods of chiral flavor substances in beverage wine mainly include liquid-liquid extraction, headspace-solid phase microextraction, solvent-assisted flavor evaporation technology and direct injection. Common chiral substance analysis methods include chiral gas chromatography, high performance liquid chromatography, capillary electrophoresis, etc., and chiral gas chromatography and liquid chromatography are often used for analysis and determination.

[0005] The volatile chiral flavoring substances detected in beverage wine include esters, ketones, alcohols, mercaptans, organic acids and terpene compounds, wherein the types of esters, alcohols, organic acids and terpene compounds are more. The ester compounds are important indicators for identifying the quality of beverage wine and are also the main aroma substances in liquor. The content of isomers with different configurations in beverage wine is different in different brewing times and different wine types, and the flavor characteristics are also different. For example, in most grape wines, chiral ester compounds such as ethyl lactate, ethyl lactate and 2-nonen-4-lactone mainly exist in the R configuration, and 2-methyl butyric acid ethyl ester mainly exists in the S configuration. In newly brewed grape wine, the content of 2-nonen-4-lactone in the S configuration is greater than that in the R configuration. In addition, 2-hexene-4-lactone in the S configuration has a pleasant caramel and coconut smell, while 2-hexene-4-lactone in the R configuration has a fatty smell and a foul smell of burning plastic. Alcohols are a kind of substances with high content in beverage wine, and almost all alcohols with hydroxyl group at position 2 are chiral. There are also chiral organic acid compounds in beverage wine, such as lactic acid, 2-methyl butyric acid and 3-hydroxy butyric acid. Lactic acid is an important organic acid in liquor, which has a great influence on the taste and aftertaste of liquor. The research on chiral compounds with different configurations in liquor can be used as a kind of judgment method.

[0006] It can be seen that the detection of chiral substances in liquor and liquor production products is of great significance for improving the quality of liquor. However, the current analysis method of chiral substances in liquor often uses one-dimensional gas chromatography with chiral column, and the number of analyzed substances is small. There is no detection method for rapidly detecting multiple chiral substances in liquor and liquor production products. SUMMARY

[0007] In view of the above technical problems, the present application provides a method for rapidly detecting multiple chiral substances in liquor and liquor production products.

[0008] The technical scheme adopted by the present application to solve its technical problems is:

[0009] In a first aspect, the present application provides a method for rapidly detecting chiral substances in liquor and fermented grains, which specifically comprises the following steps:

[0010] S1, pretreating the liquor or fermented grain sample to be tested;

[0011] S2, constructing a multi-dimensional chromatography detection system based on center cutting: using a polar column HP-INNOWAX with a specification of 60m*0.32mm*0.5μm as a one-dimensional column, and connecting a FID detector; using a low heat capacity chiral chromatographic column LTM CP-Chirasil-Dex CB with a specification of 30m*0.25mm*0.25μm as a two-dimensional column, and connecting a MS detector;

[0012] S3 uses the detection system constructed in step S2, and through comparison of the retention time of the compound with the standard product with the standard mass spectrum provided by NIST14 database, the chiral substance in the to-be-tested liquor or distiller's grains sample is qualitatively analyzed by using matching degree, characteristic ion and retention time.

[0013] In the above method, the chiral substance includes R-2-methyl butyl acetate and S-2-methyl butyl acetate, D-ethyl lactate and L-ethyl lactate, R-ethyl leucine and S-ethyl leucine, R-2-butanol and S-2-butanol, R-2-pentanol and S-2-pentanol, R-2-hexanol and S-2-hexanol, R-2-methyl butyric acid and S-2-methyl butyric acid, and R-2-methyl valeric acid and S-2-methyl valeric acid.

[0014] In step S1 of the above method, headspace-solid phase microextraction is used for pretreatment of the to-be-tested liquor sample, specifically: 50 μl of the to-be-tested liquor sample is taken into a 20 ml threaded headspace sample bottle, and the headspace bottle is sealed with a threaded cap equipped with a polytetrafluoroethylene spacer, 950 μl of saturated NaCl solution is added for dilution to 10%, the extraction head is a 50 / 30 μm DVB / CAR / PDMS three-phase extraction head, preheating at 50°C for 15 min, extraction adsorption for 40 min, the extraction head is inserted into the gas chromatography sample inlet, and desorption at 250°C GC for 3 min.

[0015] In step S1 of the above method, headspace-solid phase microextraction is used for pretreatment of the to-be-tested distiller's grains sample, specifically: after the distiller's grains are mixed uniformly, 2 g of the to-be-tested distiller's grains sample is weighed into a 20 ml threaded headspace sample bottle, and the headspace bottle is sealed with a threaded cap equipped with a polytetrafluoroethylene spacer, 8 ml of saturated NaCl solution is added, the extraction head is a 50 / 30 μm DVB / CAR / PDMS three-phase extraction head, preheating at 50°C for 15 min, extraction adsorption for 40 min, the extraction head is inserted into the gas chromatography sample inlet, and desorption at 250°C GC for 3 min.

[0016] In step S3 of the above method, the gas chromatography conditions are: the sample inlet temperature is 240°C; the carrier gas is helium with a purity of >99.999%, and the flow rate is 1 mL / min; the sample size is 1 μL, and the split mode is not split;

[0017] The center cutting conditions and the corresponding gas phase temperature rising program are selected as follows:

[0018] (1) Single substance cutting mode: center cutting and gas chromatography analysis of chiral substances are performed respectively;

[0019] One-dimensional chromatographic column temperature rising program: 40°C for 2 min, rising at a rate of 5°C / min to 240°C, and maintaining for 15 min;

[0020] Two-dimensional chromatographic column temperature program: 40℃ was kept until cutting was completed, and then the temperature was raised to 180℃ at a rate of 5℃ / min, and kept for 20min;

[0021] Cutting time: 21.8-22.4min, 22.9-23.4min, 24.2-24.8min, 27.0-27.5min, 30.8-31.6min, 36.0-36.6min, 38.8-39.6min, 41.0-41.7min;

[0022] (2) Single multi-substance co-cutting mode: ester substances and acid substances were divided into one group, and alcohol substances were divided into another group, and the chiral substances were subjected to center cutting and gas chromatography analysis;

[0023] Ester substances and acid substances group:

[0024] One-dimensional chromatographic column temperature program: 40℃ was kept for 3min, and then the temperature was raised to 240℃ at a rate of 5℃ / min, and kept for 27min;

[0025] Two-dimensional chromatographic column temperature program: 40℃ was kept for 38min, and then the temperature was raised to 180℃ at a rate of 5℃ / min, and kept for 4min;

[0026] Co-cutting time: 23.2-23.8min, 31.8-32.4min, 37.0-37.7min, 39.5-40.5min, 42-43min;

[0027] Alcohol substance group:

[0028] One-dimensional chromatographic column temperature program: 40℃ was kept for 3min, and then the temperature was raised to 240℃ at a rate of 5℃ / min, and kept for 20min;

[0029] Two-dimensional chromatographic column temperature program: 40℃ was kept for 29min, and then the temperature was raised to 180℃ at a rate of 5℃ / min, and kept for 6min;

[0030] Co-cutting time: 22.0-23.0min, 25.0-26.0min, 27.8-28.8min.

[0031] In step S3 of the above method, the mass spectrometry conditions are: EI ion source, electron energy 70eV, ion source temperature 230℃, quadrupole temperature 150℃, interface temperature 250℃, full scan mode is used, and the scan range is 35.00-350.00m / z.

[0032] In step S3 of the above method, the FID parameters are: detector temperature 250℃, hydrogen flow rate 30ml / min, air flow rate 300ml / min, and nitrogen tail gas flow rate 25ml / min.

[0033] In a second aspect, the application provides application of the above method in detection of flavor substances in baijiu and its production products.

[0034] In a third aspect, the application provides application of the above method in quality identification of baijiu.

[0035] The application has the beneficial effect that the application provides a method for rapidly detecting 8 pairs of chiral substances in baijiu and its production products. By comparing the pretreatment efficiency of liquid-liquid extraction and solid-phase microextraction, the pretreatment method suitable for chiral analysis of baijiu system is optimized; three kinds of chiral columns and different chromatographic column configuration methods are screened to determine that the one-dimensional column is the polar column HP-INNOWAX (60m*0.32mm*0.5μm) and the two-dimensional column is the low heat capacity chiral chromatographic column module LTM CP-Chirasil-Dex CB (30m*0.25mm*0.25μm); by adding a single configuration standard, and performing center cutting on the samples before and after the addition, the configuration of 8 pairs of chiral substances possibly existing in baijiu system is determined; finally, the condition is optimized, the single center cutting of a single substance is optimized into twice cutting of multiple substances, and the application of the method in baijiu and other products of baijiu is realized.

[0036] The existing analysis method of chiral substances in baijiu often uses gas phase one-dimensional chiral column analysis method, and the number of analyzable substances is small. The application uses the center cutting method to analyze the common chiral substances in baijiu, and optimizes the experimental conditions, overcomes many defects in the previous baijiu chiral analysis method, and the method has the advantages of high accuracy and good reproducibility, provides a new technology for quality control of baijiu, and has important significance for improving the quality of baijiu. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 FID chromatogram of basic components (NBF) in liquid-liquid extraction of a baijiu sample in Example 1;

[0038] Figure 2 FID chromatogram of acid components (AF) in liquid-liquid extraction of a baijiu sample in Example 1;

[0039] Figure 3 FID chromatogram of solid-phase microextraction in Example 1;

[0040] Figure 4 Mass spectrum of center cutting of chiral standard in chromatographic column configuration condition A in Example 2;

[0041] Figure 5 The center-cut mass spectrum of the chiral marker for the chromatographic column configuration condition B in Example 2;

[0042] Figure 6 The center-cut mass spectrum of the chiral marker for the chromatographic column configuration condition C in Example 2;

[0043] Figure 7 The center-cut mass spectrum of the chiral marker before and after the addition of the chiral marker in Example 3;

[0044] Figure 8 The center-cut mass spectrum of three ester and two acid chiral markers in a liquor sample in Example 4;

[0045] Figure 9 The center-cut mass spectrum of three alcohol chiral markers in a liquor sample in Example 4. DETAILED DESCRIPTION

[0046] The technical solution of the present application can be implemented in the following manner.

[0047] The method for rapidly detecting chiral markers in liquor and fermented grains, wherein the chiral markers include R-2-methyl butyric acid ethyl ester and S-2-methyl butyric acid ethyl ester, D-lactic acid ethyl ester and L-lactic acid ethyl ester, R-alanine ethyl ester and S-alanine ethyl ester, R-2-butanol and S-2-butanol, R-2-pentanol and S-2-pentanol, R-2-hexanol and S-2-hexanol, R-2-methyl butyric acid and S-2-methyl butyric acid, and R-2-methyl pentanoic acid and S-2-methyl pentanoic acid, and the method specifically comprises the following steps:

[0048] 1. Pretreatment of the liquor or fermented grain sample to be tested:

[0049] (1) Liquor sample: 50 μl of the liquor sample is taken in a 20 ml screw thread headspace sample bottle, and the headspace bottle is sealed with a screw thread cap equipped with a polytetrafluoroethylene spacer; 950 μl of saturated NaCl solution is added for dilution to 10%, the extraction head is a 50 / 30 μm DVB / CAR / PDMS three-phase extraction head, preheating at 50°C for 15 min, extraction adsorption for 40 min, the extraction head is inserted into the gas chromatography inlet, and desorption is performed at 250°C for 3 min for GC-FID analysis.

[0050] (2) Fermented grain sample: after the fermented grains are mixed evenly, 2 g of the sample is taken in a 20 ml screw thread headspace sample bottle, and the headspace bottle is sealed with a screw thread cap equipped with a polytetrafluoroethylene spacer; 8 ml of saturated NaCl solution is added, the extraction head is a 50 / 30 μm DVB / CAR / PDMS three-phase extraction head, preheating at 50°C for 15 min, extraction adsorption for 40 min, the extraction head is inserted into the gas chromatography inlet, and desorption is performed at 250°C for 3 min for GC-FID analysis.

[0051] 2. Construct a multi-dimensional chromatography detection system based on center cutting:

[0052] The present application introduces multi-dimensional chromatography technology to solve the co-elution problem by adding a second chromatographic column. The working mode is center cutting. During the sample running process, the effluent on the first chromatographic column is selectively switched to the second chromatographic column with different stationary phase by using a flow control switch. The one-dimensional column is a polar column HP-INNOWAX (60m*0.32mm*0.5μm) connected with an FID detector, and the two-dimensional column is a low thermal capacity chiral chromatographic column module LTM CP-Chirasil-Dex CB (30m*0.25mm*0.25μm) connected with an MS detector.

[0053] 3. Using the constructed detection system, the chiral substances in the liquor or distiller's grains sample to be tested are qualitatively analyzed by comparing the retention time of the compound with the standard and the standard mass spectrum provided by the NIST14 database, using matching degree, characteristic ions, and retention time.

[0054] The gas chromatography conditions are as follows: the injection port temperature is 240℃; the carrier gas is helium with a purity of >99.999%, and the flow rate is 1mL / min; the injection amount is 1μL in splitless mode;

[0055] The cutting conditions and the corresponding temperature program selection are as follows:

[0056] (1) Single-substance cutting mode: center cutting and gas chromatography analysis of chiral substances are performed respectively;

[0057] The one-dimensional chromatographic column temperature program is as follows: 40℃ for 2min, then increased to 240℃ at a rate of 5℃ / min, and maintained for 15min;

[0058] The two-dimensional chromatographic column temperature program is as follows: 40℃ until the cutting is completed, then increased to 180℃ at a rate of 5℃ / min, and maintained for 20min;

[0059] The cutting time is as follows: 21.8-22.4min, 22.9-23.4min, 24.2-24.8min, 27.0-27.5min, 30.8-31.6min, 36.0-36.6min, 38.8-39.6min, and 41.0-41.7min;

[0060] (2) Multi-substance cutting mode: ester and acid substances are divided into one group, and alcohol substances are divided into another group, and center cutting and gas chromatography analysis of chiral substances are performed in groups;

[0061] Ester and acid substance group:

[0062] One-dimensional column temperature program: 40℃ for 3min, heated to 240℃ at a rate of 5℃ / min, and kept for 27min;

[0063] Two-dimensional column temperature program: 40℃ for 38min, heated to 180℃ at a rate of 5℃ / min, and kept for 4min;

[0064] Common cutting time: 23.2-23.8min, 31.8-32.4min, 37.0-37.7min, 39.5-40.5min, 42-43min;

[0065] Alcohol substance group:

[0066] One-dimensional column temperature program: 40℃ for 3min, heated to 240℃ at a rate of 5℃ / min, and kept for 20min;

[0067] Two-dimensional column temperature program: 40℃ for 29min, heated to 180℃ at a rate of 5℃ / min, and kept for 6min;

[0068] Common cutting time: 22.0-23.0min, 25.0-26.0min, 27.8-28.8min.

[0069] Mass spectrometry conditions: EI ion source, electron energy 70eV, ion source temperature 230℃, quadrupole temperature 150℃, interface temperature 250℃, full scan mode, scan range 35.00-350.00m / z.

[0070] FID parameters: detector temperature 250℃, hydrogen flow rate: 30ml / min, air flow rate: 300ml / min, nitrogen tail gas flow rate: 25ml / min.

[0071] The technical solutions and effects of the present application are further described below through actual examples.

[0072] Examples

[0073] The instruments used in this example are: Agilent 7890B-5977B gas chromatograph-mass spectrometer, equipped with microplate flow control technology Deans Switch, one-dimensional Agilent capillary column HP-INNOWax (60m x 0.32mm x 0.5μm), two-dimensional Agilent low heat capacity chiral chromatographic column module LTM CP-ChiraSil-DEX CB LTM (30m x 0.25mm x 0.25μm), SPME special straight liner, GERSTEL MPSpro multifunctional full-automatic sample pretreatment platform, hydrogen flame detector.

[0074] Example 1: Sample pretreatment method selection

[0075] This embodiment first determines the pretreatment method suitable for the liquor system, the specific steps are as follows:

[0076] (1) Liquid-liquid extraction: take 10 ml of liquor sample, dilute the alcohol content to 10% with saturated sodium chloride solution. Extract with dichloromethane three times, use a separatory funnel shaker, 30.0 ml each time, the shaking rate is 300 rpm, the time is 8 min, and the organic phase is combined. Concentrated to 50.0 ml by nitrogen blowing, add saturated sodium bicarbonate solution to adjust the pH to 9.0, separate to get organic phase solution A and aqueous solution B. Extract solution A with 20 ml of ultrapure water twice, combine the aqueous phase with solution B to get solution C, dry with anhydrous sodium sulfate overnight, and finally concentrate to 500 microliters by nitrogen blowing to obtain the intermediate basic component (NBF) for sample analysis. Add 1 mol / L sulfuric acid solution to solution C to adjust the pH to 2.0, add sodium chloride until saturated, extract with dichloromethane three times, combine the organic phase, dry with anhydrous sodium sulfate overnight, and finally concentrate to 500 microliters by nitrogen blowing to obtain the acidic component (NBF) for sample analysis.

[0077] (2) Solid phase microextraction: take 50 μl of liquor sample into a 20 ml screw thread headspace sample bottle, and seal the headspace bottle with a screw thread cap equipped with a polytetrafluoroethylene spacer. Dilute to 10% by adding 950 μl of saturated NaCl solution, the extraction head is a 50 / 30 μm DVB / CAR / PDMS three-phase extraction head, preheat for 15 min at 50℃, extract and adsorb for 40 min, insert the extraction head into the gas chromatograph injection port, and desorb at 250℃ for 3 min for GC-FID analysis.

[0078] Gas chromatography conditions: one-dimensional chromatographic column is HP-INNOWAX (60 m*0.32 mm*0.5 μm); the injection port temperature is 240℃; the carrier gas is high-purity helium (He) (purity > 99.999%), the flow rate is 1 mL / min; no split mode; the column temperature program: 40℃ for 2 min, increase the temperature to 240℃ at a rate of 5℃ / min, and maintain for 15 min.

[0079] FID parameters: detector temperature is 250℃, hydrogen flow rate: 30 ml / min, air flow rate: 300 ml / min, nitrogen tail gas flow rate: 25 ml / min.

[0080] The above pretreatment method is used for extraction of a liquor sample, wherein: the FID chromatogram of the liquid-liquid extraction basic component (NBF) is as shown in Figure 1 , the FID chromatogram of the liquid-liquid extraction acidic component (AF) is as shown in Figure 2 , and the FID chromatogram of the solid phase microextraction is as shown inFigure 3 As shown in Figures 1 to 3 It can be known that the liquid-liquid extraction and the solid phase microextraction have good performances on the extraction of liquor flavor substances, but the pretreatment process of the liquid-liquid extraction is more complicated, and considering the sample testing efficiency in the later stage, the solid phase microextraction is selected as the pretreatment mode of the liquor and the distiller's grains.

[0081] Example 2: Screening of chiral separation conditions

[0082] Gas chromatography plays a great role in the separation and analysis of volatile compounds, and most of the instruments used are one-dimensional chromatography, but when the sample is more complex, it is necessary to introduce multi-dimensional chromatography technology to solve the problem of co-elution by increasing the second chromatographic column. The working mode of two-dimensional gas phase in the application is center cutting, that is, during the sample running process, the effluent on the first chromatographic column is selectively switched to the second chromatographic column with different stationary phases by using a flow control switch, and the target analyte and co-eluted substances can be fully separated. Chiral compounds have similar physicochemical properties and cannot be separated by conventional media, and only by introducing a chiral environment can they be separated. Through the pre-experiment, it is found that there is no big difference in the separation results of the same chiral column using different temperature programs, so it is very important to select a suitable chiral column for chiral analysis.

[0083] This example determines the system configuration and chiral column suitable for separating chiral substances, and the following three conditions are configured on the gas chromatograph:

[0084] A. One-dimensional column: HP-INNOWAX (60m*0.32mm*0.5μm), two-dimensional column: LTM Cyclosil B (30m*0.25mm*0.25μm);

[0085] B. One-dimensional column: LTM DB-Wax (30m*0.25mm*0.25μm), two-dimensional column: Astec B-DM (50m*0.25mm*0.12μm);

[0086] C. One-dimensional column: HP-INNOWAX (60m*0.32mm*0.5μm), two-dimensional column: LTM CP-Chirasil-Dex CB (30m*0.25mm*0.25μm).

[0087] The above three chromatographic column configuration methods are used for center cutting of 8 pairs of chiral substance standards, and the mass spectrum of chiral standard center cutting under condition A is as shown in Figure 4 , the mass spectrum of chiral standard center cutting under condition B is as shown in Figure 5 , and the mass spectrum of chiral standard center cutting under condition C is as shown in Figure 6 . From Figures 4 to 6It can be known that the two configurations of chiral substance 2-methyl butyric acid ethyl ester and ethyl lactate can be effectively distinguished under condition A, the separation effect of the two configurations of 2-methyl pentanoic acid, 2-butanol and 2-pentanol is poor, and no separation effect is obtained for the remaining substances; the two configurations of 2-methyl butyric acid ethyl ester and ethyl lactate can also be effectively distinguished under condition B, the separation effect of the two configurations of 2-butanol and 2-pentanol is poor, and no separation effect is obtained for the configurations of ethyl leucine and 2-hexanol, the two acid substances 2-methyl butyric acid and 2-methyl pentanoic acid are synthesized by the inventors, and the specific synthesis method is as follows: taking the corresponding precursor acid and excess precursor alcohol, 10% solid acid catalyst HND-580 is added to catalyze the esterification reaction, and the synthesis result is detected by GC-MS.

[0088] Example 3: Chiral substance configuration determination

[0089] In the field of food, different configurations of chiral compounds can produce different flavor systems. Because biological synthesis is usually driven by enzymes, the chiral compounds of these reaction products usually exist in a specific enantiomeric ratio with certain regularity, while the enantiomeric ratio of artificially synthesized chiral compounds often differs greatly. Therefore, chiral analysis of liquor can be used for quality control, detection of whether the sample is adulterated, determination of whether there is exogenous addition, judgment of whether it is pure grain solid-state fermentation, etc., and the composition of chiral compounds and enantiomers in liquor is of great significance for determining the quality and authenticity of liquor. In this embodiment, single configuration markers are added, and the configurations of chiral substances possibly existing in the samples before and after the addition are determined by center cutting.

[0090] In this embodiment, L-ethyl lactate, R-2-butanol, S-2-butanol, R-2-pentanol, S-2-pentanol, R-2-hexanol, S-2-hexanol, R-2-methyl butyric acid, S-2-methyl butyric acid, R-2-methyl pentanoic acid and S-2-methyl pentanoic acid in the chiral substance markers used are commercially available. D-ethyl lactate, R-ethyl leucine, S-ethyl leucine, R-2-methyl butyric acid ethyl ester and S-2-methyl butyric acid ethyl ester are synthesized by the inventors; the specific synthesis method is as follows: taking the corresponding precursor acid and excess precursor alcohol, 10% solid acid catalyst HND-580 is added to catalyze the esterification reaction, and the synthesis result is detected by GC-MS.

[0091] Gas chromatography conditions: the injection port temperature was 240℃; the carrier gas was high-purity helium (He) (purity > 99.999%), the flow rate was 1 mL / min; the injection volume was 1 μL, in splitless mode; the one-dimensional chromatographic column temperature program was 40℃ for 2 min, then increased to 240℃ at a rate of 5℃ / min, and maintained for 15 min; the two-dimensional chromatographic column temperature program was 40℃ until the cutting was completed, then increased to 180℃ at a rate of 5℃ / min, and maintained for 20 min; the compound center cutting time is shown in Table 1.

[0092] Table 1 Cutting time

[0093]

[0094] Mass spectrometry conditions: EI ion source, electron energy 70 eV, ion source temperature 230℃, quadrupole temperature 150℃, interface temperature 250℃, full scan mode, scan range (m / z) 35.00-350.00.

[0095] FID parameters: detector temperature 250℃, hydrogen flow rate 30 ml / min, air flow rate 300 ml / min, nitrogen tail gas flow rate 25 ml / min.

[0096] By adding a single configuration standard, the samples before and after the addition were respectively subjected to center cutting, and the peak area changes were analyzed by GC-MS to determine the configurations of the 8 chiral substances possibly present in the liquor, and the results are shown in Table 1. Figure 7 On the chiral column CP-Chirasil-Dex CB, ethyl 2-methylbutanoate, ethyl leucine, 2-butanol, 2-pentanol and 2-hexanol were all R configuration in front and S configuration in back, ethyl lactate was D configuration in front and L configuration in back, and 2-methylbutanoic acid and 2-methylvaleric acid were S configuration in front and R configuration in back.

[0097] Example 4: Optimization of chiral center cutting conditions

[0098] For the analysis of 8 pairs of chiral flavor substances in liquor, the single pre-treatment and gas chromatography analysis of a single substance required about 1.5 h on average, and when the sample amount increased in the later stage and parallel experiments were required, the time used would be more. In order to save time and other experimental costs, the cutting conditions were optimized. By comparing the retention time and qualitative ions, the 8 substances were divided into two groups, 3 ester substances and 2 acid substances in one group, and 3 alcohol substances in another group. The cutting time was adjusted to ensure that multiple substances could be cut at one time. This method not only ensured the separation of each pair of chiral substances, but also greatly saved the analysis time of the liquor sample.

[0099] The sample pretreatment method uses solid-phase microextraction, the gas chromatography conditions, the mass spectrometry conditions, and the FID parameters are the same as in Example 1; due to slight adjustment of the cutting time, the gas chromatography temperature program is adjusted to be suitable for single multi-substance cutting conditions:

[0100] When cutting the three alcohol substances: the one-dimensional chromatographic column temperature program is 40°C for 3 min, and then the temperature is raised to 240°C at a rate of 5°C / min, and maintained for 20 min; the two-dimensional chromatographic column temperature program is 40°C for 29 min, and then the temperature is raised to 180°C at a rate of 5°C / min, and maintained for 6 min; the common cutting time is adjusted to be 22.0-23.0 min, 25.0-26.0 min, and 27.8-28.8 min.

[0101] When cutting the three ester and two acid substances: the one-dimensional chromatographic column temperature program is 40°C for 3 min, and then the temperature is raised to 240°C at a rate of 5°C / min, and maintained for 27 min; the two-dimensional chromatographic column temperature program is 40°C for 38 min, and then the temperature is raised to 180°C at a rate of 5°C / min, and maintained for 4 min; the common cutting time is adjusted to be 23.2-23.8 min, 31.8-32.4 min, 37.0-37.7 min, 39.5-40.5 min, and 42-43 min.

[0102] Under the above conditions, eight pairs of chiral substances in a certain liquor sample are subjected to directional analysis: through comparison of the retention time of the compounds with the standard substance and the standard mass spectrum provided by the NIST14 database, qualitative analysis and identification are performed by using the matching degree, characteristic ions, and retention time; the mass spectrum of the three ester and two acid chiral substances in the center of the cutting is as shown in Figure 8 , and the mass spectrum of the three alcohol chiral substances in the center of the cutting is as shown in Figure 9 . As can be seen from Figures 8 to 9 , the grouping center cutting mode also has good separation effect on the two configurations of the eight pairs of chiral substances.

[0103] Example 5: Application of the method

[0104] In order to test the application scope of the method for rapidly detecting chiral substances in liquor and fermented grains according to the application, liquor samples of different flavor types are taken for chiral center cutting; the gas chromatography conditions and the temperature program, the mass spectrometry conditions and the FID parameters are the same as in Example 4; the percentage of R-configuration in the five kinds of liquor samples is as shown in Table 2.

[0105] Table 2 Percentage of R-configuration in liquor samples (%)

[0106] Luzhou-flavor liquor sample Maotai-flavor liquor sample Rice-flavor liquor sample Feng-flavor liquor sample Feng-flavor liquor sample Ethyl 2-methylbutanoate 0.25 0.19 0.07 0.08 0.11 Ethyl lactate 0.85 0.77 0.68 0.67 0.39 Ethyl lactate 0.83 0.65 0.56 0.55 0.83 2-Butanol 0.42 0.65 0.66 0.56 0.73 2-Pentanol 0.66 0.24 0.24 0.49 0.15 2-Hexanol 0.43 0.24 / 0.46 / 2-Methylbutanoic acid 0.25 0.07 / 0.35 / 2-Methylpentanoic acid / / / / /

[0107] It can be seen from Table 2 that the method for detecting chiral substances can quickly and effectively detect 8 pairs of chiral substances in different flavor types of liquor and determine the proportion of different configurations of chiral substances in the liquor sample. Different flavor types of liquor samples have different systems. Based on the verification of the present embodiment, the method for quickly detecting chiral substances in liquor and fermented grains of the present application is suitable for the detection of chiral substances in different flavor types of liquor, and has high reproducibility and accuracy.

Claims

1. A method for rapidly detecting chiral substances in liquor and fermented grains, characterized in that, Specifically comprising the following steps: S1 pretreating the sample of the liquor or distiller's grains to be tested; S2 constructing a multi-dimensional chromatographic detection system based on center cutting: using a polar column HP-INNOWAX with a specification of 60m x 0.32mm x 0.5μm as a one-dimensional column, and connecting an FID detector; using a low-heat-capacity chiral chromatographic column LTM CP-Chirasil-Dex CB with a specification of 30m x 0.25mm x 0.25μm as a two-dimensional column, and connecting an MS detector; Wherein, the gas chromatography conditions are: the temperature of the sample inlet is 240℃; the carrier gas is helium with a purity of >99.999%, and the flow rate is 1 mL / min; the sample injection amount is 1μL, and the split mode is not divided; the mass spectrometry conditions are: EI ionization source, electron energy 70eV, ion source temperature 230℃, quadrupole temperature 150℃, interface temperature 250℃, full scan mode is adopted, and the scan range is 35.00-350.00 m / z; the FID parameters are: the detector temperature is 250℃, the hydrogen flow rate is 30 ml / min, the air flow rate is 300 ml / min, and the nitrogen tail gas flow rate is 25ml / min; S3 using the detection system constructed in step S2, and performing qualitative analysis on the chiral substances in the sample of the liquor or distiller's grains to be tested by comparing the retention time of the compounds with the standard substance with the standard mass spectrum provided by the NIST14 database, and using the matching degree, characteristic ions and retention time; the chiral substances are: R-2-methyl butyric acid ethyl ester and S-2-methyl butyric acid ethyl ester, D-ethyl lactate and L-ethyl lactate, R-ethyl leucine and S-ethyl leucine, R-2-butanol and S-2-butanol, R-2-pentanol and S-2-pentanol, R-2-hexanol and S-2-hexanol, R-2-methyl butyric acid and S-2-methyl butyric acid, and R-2-methyl valeric acid and S-2-methyl valeric acid.

2. The method for rapidly detecting chiral substances in Baijiu and Daqu according to claim 1, characterized in that: In step S1, the sample of the liquor to be tested is pretreated by headspace-solid phase microextraction, specifically: 50μl of the sample of the liquor to be tested is taken into a 20ml threaded headspace sample bottle, the headspace bottle is sealed with a threaded cap equipped with a polytetrafluoroethylene spacer, 950μl of saturated NaCl solution is added for dilution to 10%, the extraction head is a 50 / 30μm DVB / CAR / PDMS three-phase extraction head, preheating is performed at 50℃ for 15min, extraction adsorption is performed for 40min, the extraction head is inserted into the gas chromatography sample inlet, and GC desorption is performed at 250℃ for 3min.

3. The method for rapidly detecting chiral substances in Baijiu and Daqu according to claim 1, characterized in that: In step S1, the sample of the distiller's grains to be tested is pretreated by headspace-solid phase microextraction, specifically: after the distiller's grains are mixed uniformly, 2g of the sample of the distiller's grains to be tested is taken into a 20ml threaded headspace sample bottle, the headspace bottle is sealed with a threaded cap equipped with a polytetrafluoroethylene spacer, 8ml of saturated NaCl solution is added, the extraction head is a 50 / 30μm DVB / CAR / PDMS three-phase extraction head, preheating is performed at 50℃ for 15min, extraction adsorption is performed for 40min, the extraction head is inserted into the gas chromatography sample inlet, and GC desorption is performed at 250℃ for 3min.

4. The method for rapidly detecting chiral substances in Baijiu and Daqu according to claim 1, characterized in that: In step S2, the center cutting conditions and the corresponding gas temperature rising program are selected as any one of the following: (1) Single single-substance cutting mode: center cutting and gas chromatography analysis of chiral substances respectively; One-dimensional chromatographic column temperature program: 40℃ for 2 min, temperature rising to 240℃ at a rate of 5℃ / min, holding for 15 min; Two-dimensional chromatographic column temperature program: 40℃ for 2 min, temperature rising to 180℃ at a rate of 5℃ / min, holding for 20 min; Cutting time: 21.8-22.4 min, 22.9-23.4 min, 24.2-24.8 min, 27.0-27.5 min, 30.8-31.6 min, 36.0-36.6 min, 38.8-39.6 min, 41.0-41.7 min; (2) Single multi-substance cutting mode: ester and acid substances are divided into one group, and alcohol substances are divided into another group, and center cutting and gas chromatography analysis of chiral substances are carried out in groups; Ester and acid substance group: One-dimensional chromatographic column temperature program: 40℃ for 3 min, temperature rising to 240℃ at a rate of 5℃ / min, holding for 27 min; Two-dimensional chromatographic column temperature program: 40℃ for 38 min, temperature rising to 180℃ at a rate of 5℃ / min, holding for 4 min; Common cutting time: 23.2-23.8 min, 31.8-32.4 min, 37.0-37.7 min, 39.5-40.5 min, 42-43 min; Alcohol substance group: One-dimensional chromatographic column temperature program: 40℃ for 3 min, temperature rising to 240℃ at a rate of 5℃ / min, holding for 20 min; Two-dimensional chromatographic column temperature program: 40℃ for 29 min, temperature rising to 180℃ at a rate of 5℃ / min, holding for 6 min; Common cutting time: 22.0-23.0 min, 25.0-26.0 min, 27.8-28.8 min.

5. The method of any one of claims 1-4 for use in the detection of flavor substances in Baijiu and its production products.

6. The method of any one of claims 1-4 for use in the quality identification of Baijiu.

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