Method for rapidly detecting chiral substances in white spirit and fermented grains and application

Abstract

The invention discloses a method for rapidly detecting chiral substances in white spirit and fermented grains and application, and belongs to the technical field of white spirit flavor analysis. The method specifically comprises the following steps: pre-treating a to-be-detected white spirit or fermented grain sample; the method comprises the following steps: constructing a multi-dimensional chromatographic detection system based on center cutting: adopting a polar column HP-INNOWAX with the specification of 60m * 0.32 mm * 0.5 mu m as a one-dimensional column, and connecting an FID (Flame Ionization Detector); the method comprises the following steps: adopting a low-heat capacity chiral chromatographic column LTM CP-Chirastil-Dex CB with the specification of 30m * 0.25 mm * 0.25 mu m as a two-dimensional column, and connecting the two-dimensional column with an MS (Murashige-Skoog) detector; and carrying out qualitative analysis on the chiral substances in the sample to be detected by adopting the constructed detection system. The technical scheme of the invention has the advantages of high accuracy and good reproducibility, provides a new technology for the quality control of the white spirit, and has important significance for improving the quality of the white spirit.

Description

Technical Field

[0001] The invention 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 central cutting technology, and specifically relates to a method and application for rapidly detecting chiral substances in liquor and fermented grains. Background Art

[0002] Liquor is a traditional distilled liquor in my country with a history of more than 2,000 years. There are many types of liquor in my country, with strong local characteristics, and each product has its own characteristics. The production process has its own characteristics and has good flavor and taste. There are many types of flavor compounds in liquor, and the content varies. These compounds determine the style and characteristics of liquor.

[0003] Chiral compounds play an important role in the fields of food, medicine, pesticides, etc. In the fields of spices and food, different configurations of chiral compounds may produce different flavor systems. Since biosynthesis is usually driven by enzymes, the chiral compounds of these reaction products usually exist in a specific enantiomeric ratio with a certain regularity, while the enantiomeric ratio of artificially synthesized chiral compounds is often quite different from this. Therefore, chiral analysis of liquor can be used for quality control, detecting whether samples are adulterated, determining whether there are exogenous additions, judging whether it is pure grain solid-state fermentation, etc. Clarifying the chiral compounds and enantiomeric composition in liquor is of great significance for determining the quality and authenticity of liquor.

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

[0005] The volatile chiral flavor substances detected in alcoholic beverages include esters, ketones, alcohols, thiols, organic acids and terpenes, among which esters, alcohols, organic acids and terpenes are more numerous. Ester compounds are an important indicator for the identification of quality in alcoholic beverages and are also the main aroma substances in liquor. The content of isomers of different configurations in alcoholic beverages varies at different brewing times and in different types of wines, and the flavor characteristics are also different. For example, in most wines, chiral ester compounds such as leucine ethyl ester, ethyl lactate, and 2-nonene-4-lactone mainly exist in the R configuration, and 2-methylbutyric acid ethyl ester mainly exists in the S configuration. In newly brewed wines, the content of 2-nonene-4-lactone with S configuration is greater than that with R configuration. In addition, 2-hexene-4-lactone with S configuration has a pleasant caramel and coconut smell, while its R configuration has a fat smell and an unpleasant smell of burning plastic. Alcohol compounds are a class of substances with a high content in alcoholic beverages. Almost all alcohols with a hydroxyl group at the 2nd position are chiral. There are also organic acid chiral compounds in alcoholic beverages, such as lactic acid, 2-methylbutyric acid, 3-hydroxybutyric acid, etc. Among them, lactic acid is an important organic acid in liquor and has a great influence on the taste and aftertaste of liquor. The study of chiral compounds of different configurations in liquor can be used as a judgment method.

[0006] It can be seen that detecting chiral substances in liquor and liquor production products is of great significance to improving the quality of liquor. However, the current analysis method of chiral substances in liquor often adopts the gas phase one-dimensional chiral column analysis method, and the analyzed substances are relatively small. There is currently no detection method that can quickly detect multiple chiral substances in liquor and liquor production products. Summary of the invention

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

[0008] The technical solution adopted by the present invention to solve its technical problem is:

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

[0010] S1 performs pre-treatment on the liquor or mash sample to be tested;

[0011] S2 constructs a multidimensional chromatography detection system based on heart cutting: a polar column HP-INNOWAX with a specification of 60m*0.32mm*0.5μm is used as a one-dimensional column, connected to an FID detector; a low thermal mass chiral column LTM CP-Chirasil-Dex CB with a specification of 30m*0.25mm*0.25μm is used as a two-dimensional column, connected to an MS detector;

[0012] S3 uses the detection system constructed in step S2, compares the retention time of the compound and the standard with the standard mass spectrum provided by the NIST14 database, and uses the matching degree, characteristic ions and retention time to perform qualitative analysis on the chiral substances in the white wine or mash sample to be tested.

[0013] In the above method, the chiral substances include R-2-methylbutyric acid ethyl ester and S-2-methylbutyric acid ethyl ester, D-lactate ethyl ester and L-lactate ethyl ester, R-leucine ethyl ester and S-leucine 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-methylbutyric acid and S-2-methylbutyric acid, R-2-methylvaleric acid and S-2-methylvaleric acid.

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

[0015] In step S1 of the above method, the headspace-solid phase microextraction is used to pre-treat the mash sample to be tested, specifically: after the mash is evenly mixed, 2 g of the mash sample to be tested is weighed into a 20 ml threaded headspace sample bottle, and the headspace bottle is sealed with a threaded cap equipped with a polytetrafluoroethylene septum, 8 ml of saturated NaCl solution is added, the extraction head is a 50 / 30 μm DVB / CAR / PDMS three-phase extraction head, preheated at 50°C for 15 min, extracted and adsorbed for 40 min, the extraction head is inserted into the gas chromatography inlet, and desorbed at 250°C GC for 3 min.

[0016] In step S3 of the above method, the gas chromatography conditions are: the injection port temperature is 240° C.; the carrier gas is helium with a purity of >99.999% and a flow rate of 1 mL / min; the injection volume is 1 μL, and the splitless mode is used;

[0017] The heart cutting conditions and the corresponding gas phase heating program can be selected from any of the following:

[0018] (1) Single-shot single-substance cutting mode: heart cutting and gas chromatography analysis of chiral substances respectively;

[0019] The temperature program of the first-dimensional column was as follows: 40 °C for 2 min, then heated to 240 °C at a rate of 5 °C / min and held for 15 min;

[0020] The temperature program of the 2D chromatographic column was as follows: 40 °C was maintained until the cutting was completed, then the temperature was increased to 180 °C at a rate of 5 °C / min and maintained for 20 min;

[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: esters and acids are divided into one group, alcohols into another group, and the chiral substances are divided into groups for heart cutting and gas chromatography analysis;

[0023] Esters and acids group:

[0024] The temperature program of the first-dimensional column was as follows: 40 °C for 3 min, then increased to 240 °C at a rate of 5 °C / min and maintained for 27 min;

[0025] The temperature program of the 2D column was as follows: 40 °C for 38 min, then heated to 180 °C at a rate of 5 °C / min and held for 4 min;

[0026] The common cutting times are: 23.2-23.8min, 31.8-32.4min, 37.0-37.7min, 39.5-40.5min, 42-43min;

[0027] Alcohol Group:

[0028] The temperature program of the first-dimensional column was as follows: 40 °C for 3 min, then heated to 240 °C at a rate of 5 °C / min and held for 20 min;

[0029] The temperature program of the 2D column was as follows: 40 °C for 29 min, then heated to 180 °C at a rate of 5 °C / min and held for 6 min;

[0030] The common cutting times are: 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 ionization source, electron energy 70 eV, ion source temperature 230°C, quadrupole temperature 150°C, interface temperature 250°C, full scan mode, and scan range 35.00-350.00 m / z.

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

[0033] In a second aspect, the present invention provides application of the above method in detecting flavor substances in liquor and its products.

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

[0035] The beneficial effects of the present invention are as follows: the present invention provides a method for rapidly detecting 8 pairs of chiral substances in liquor and its production products. By comparing the pretreatment efficiencies of liquid-liquid extraction and solid phase microextraction, the pretreatment method suitable for chiral analysis of liquor system is optimized; three chiral columns and different chromatographic column configuration methods are screened, and the one-dimensional column is determined to be the polar column HP-INNOWAX (60m*0.32mm*0.5μm) and the two-dimensional column is the low thermal capacity chiral chromatographic column module LTM CP-Chirasil-Dex CB (30m*0.25mm*0.25μm); by adding a single configuration standard product and performing a center cut on the samples before and after the addition, the configuration of the 8 chiral substances that may exist in the liquor system is judged; finally, the conditions are optimized, and the single center cut of a single substance is optimized to two times of multiple substance co-cutting, and the application of this method in liquor and other liquor products is realized.

[0036] The existing analysis methods of chiral substances in liquor often use gas phase one-dimensional chiral column analysis methods, and the substances that can be analyzed are relatively few. The present invention uses the center cutting method to analyze common chiral substances in liquor, and optimizes the experimental conditions, thus overcoming many defects in the previous liquor chiral analysis methods. The method of the present invention has the advantages of high accuracy and good reproducibility, and provides a new technology for the quality control of liquor, which is of great significance to improving the quality of liquor. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the FID chromatogram of the basic component (NBF) in the liquid-liquid extraction of a certain liquor sample in Example 1;

[0038] Figure 2 This is the FID chromatogram of the acidic component (AF) of the liquid-liquid extraction of a certain liquor sample in Example 1;

[0039] Figure 3 It is the solid phase microextraction FID chromatogram in Example 1;

[0040] Figure 4 This is the heart-cutting mass spectrum of the chiral standard product under chromatographic column configuration condition A in Example 2;

[0041] Figure 5 This is the heart-cutting mass spectrum of the chiral standard product under chromatographic column configuration condition B in Example 2;

[0042] Figure 6 This is the heart-cutting mass spectrum of the chiral standard product under chromatographic column configuration condition C in Example 2;

[0043] Figure 7 Add the front and back heart-cut mass spectra of the chiral substance standard in Example 3;

[0044] Figure 8 This is the center-cutting mass spectrum of three esters and two acids in a liquor sample in Example 4;

[0045] Fig. 9 This is the center-cutting mass spectrum of three alcohol chiral substances in a certain liquor sample in Example 4. DETAILED DESCRIPTION

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

[0047] A method for rapidly detecting chiral substances in liquor and fermented grains, wherein the chiral substances include R-2-methylbutyric acid ethyl ester and S-2-methylbutyric acid ethyl ester, D-lactate ethyl ester and L-lactate ethyl ester, R-leucine ethyl ester and S-leucine 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-methylbutyric acid and S-2-methylbutyric acid, R-2-methylvaleric acid and S-2-methylvaleric acid; the method specifically comprises the following steps:

[0048] 1. Pre-treat the liquor or mash samples to be tested:

[0049] (1) Liquor sample: 50 μl of liquor sample was pipetted into a 20 ml threaded headspace sample bottle and sealed with a threaded cap equipped with a polytetrafluoroethylene septum; 950 μl of saturated NaCl solution was added to dilute to 10%; the extraction head was a 50 / 30 μm DVB / CAR / PDMS three-phase extraction head, preheated at 50°C for 15 min, extracted and adsorbed for 40 min, and the extraction head was inserted into the gas chromatography injection port, desorbed at 250°C for 3 min, and used for GC-FID analysis.

[0050] (2) Grains and mash sample: After the grains and mash are mixed evenly, 2 g of the 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 septum; 8 ml of saturated NaCl solution is added, the extraction head is a 50 / 30 μm DVB / CAR / PDMS three-phase extraction head, preheated at 50 °C for 15 min, and the extraction adsorption is performed for 40 min. The extraction head is inserted into the gas chromatography injection port, and GC desorption is performed at 250 °C for 3 min for GC-FID analysis.

[0051] 2. Construction of a multidimensional chromatographic detection system based on heart cutting:

[0052] The present invention introduces multidimensional chromatography technology and solves the problem of co-peaks by adding a second chromatographic column. The working mode is heart cutting. During the sample operation, the flow control switch is used to selectively switch the effluent on the first chromatographic column to the second chromatographic column with a different stationary phase. The one-dimensional column is a polar column HP-INNOWAX (60m*0.32mm*0.5μm), connected to the 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 to the MS detector.

[0053] 3. Using the constructed detection system, the retention time of the compound and the standard is compared with the standard mass spectrum provided by the NIST14 database, and the chiral substances in the tested liquor or mash samples are qualitatively analyzed using the matching degree, characteristic ions and retention time.

[0054] The gas chromatography conditions were as follows: injection port temperature was 240°C; carrier gas was helium with a purity of >99.999% and a flow rate of 1 mL / min; injection volume was 1 μL, splitless mode;

[0055] Cutting conditions and corresponding heating program can be selected from any of the following:

[0056] (1) Single-shot single-substance cutting mode: heart cutting and gas chromatography analysis of chiral substances respectively;

[0057] The temperature program of the first-dimensional column was as follows: 40 °C for 2 min, then heated to 240 °C at a rate of 5 °C / min and held for 15 min;

[0058] The temperature program of the 2D chromatographic column was as follows: 40 °C was maintained until the cutting was completed, then the temperature was increased to 180 °C at a rate of 5 °C / min and maintained for 20 min;

[0059] 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;

[0060] (2) Single multi-substance co-cutting mode: esters and acids are divided into one group, alcohols into another group, and the chiral substances are divided into groups for heart cutting and gas chromatography analysis;

[0061] Esters and acids group:

[0062] The temperature program of the first-dimensional column was as follows: 40 °C for 3 min, then increased to 240 °C at a rate of 5 °C / min and maintained for 27 min;

[0063] The temperature program of the 2D column was as follows: 40 °C for 38 min, then heated to 180 °C at a rate of 5 °C / min and held for 4 min;

[0064] The common cutting times are: 23.2-23.8min, 31.8-32.4min, 37.0-37.7min, 39.5-40.5min, 42-43min;

[0065] Alcohol Group:

[0066] The temperature program of the first-dimensional column was as follows: 40 °C for 3 min, then heated to 240 °C at a rate of 5 °C / min and held for 20 min;

[0067] The temperature program of the 2D column was as follows: 40 °C for 29 min, then heated to 180 °C at a rate of 5 °C / min and held for 6 min;

[0068] The common cutting times are: 22.0-23.0min, 25.0-26.0min, 27.8-28.8min.

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

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

[0071] The technical solution and effects of the present invention are further illustrated below through practical examples.

[0072] Example

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

[0074] Example 1: Sample pretreatment method selection

[0075] This example first determines the pre-treatment method suitable for the liquor system, and the specific steps are as follows:

[0076] (1) Liquid-liquid extraction: Take 10 ml of wine sample and dilute it to 10% alcohol content with saturated sodium chloride solution. Extract with dichloromethane three times, use a separatory funnel oscillator, 30.0 ml each time, oscillate at a rate of 300 rpm, and take 8 min. Combine the organic phases. Concentrate to 50.0 ml with nitrogen blow, add saturated sodium bicarbonate solution to adjust the pH to 9.0, and separate to obtain organic phase solution A and aqueous phase solution B. Extract solution A twice with 20 ml of ultrapure water, combine the aqueous phase with solution B to obtain solution C, add anhydrous sodium sulfate to dry overnight, and finally concentrate to 500 microliters with nitrogen blow to obtain the medium alkaline 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 phases, add anhydrous sodium sulfate to dry overnight, and finally concentrate to 500 microliters with nitrogen blow to obtain the acidic component (NBF) for sample analysis.

[0077] (2) Solid phase microextraction: 50 μl of liquor sample was taken into a 20 ml threaded headspace sample bottle and sealed with a threaded cap equipped with a polytetrafluoroethylene septum. 950 μl of saturated NaCl solution was added to dilute to 10%. The extraction head was a 50 / 30 μm DVB / CAR / PDMS three-phase extraction head. The extraction head was preheated at 50°C for 15 min, and the extraction adsorption was performed for 40 min. The extraction head was inserted into the gas chromatography injection port and desorbed at 250°C for 3 min for GC-FID analysis.

[0078] Gas chromatography conditions: one-dimensional chromatographic column is HP-INNOWAX (60m*0.32mm*0.5μm); the injection port temperature is 240°C; the carrier gas is high-purity helium (He) (purity>99.999%), with a flow rate of 1mL / min; non-split mode; column heating program: maintain at 40°C for 2min, increase the temperature to 240°C at a rate of 5°C / min, and maintain for 15min.

[0079] FID parameters: detector temperature is 250°C, 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 was used to extract a certain liquor sample, where the FID chromatogram of the alkaline component (NBF) in liquid-liquid extraction is shown in Figure 1 As shown, the FID chromatogram of the liquid-liquid extraction acidic component (AF) is as follows Figure 2 As shown, the solid phase microextraction FID chromatogram is as follows Figure 3 As shown. Figures 1 to 3 It can be seen that both liquid-liquid extraction and solid phase microextraction have good performance in extracting flavor substances from liquor, but the pretreatment process of liquid-liquid extraction is relatively cumbersome. Considering the efficiency of subsequent sample measurement, the present invention selects solid phase microextraction as the pretreatment method for liquor and mash.

[0081] Example 2: Screening of chiral separation conditions

[0082] Gas chromatography plays a great role in the separation and analysis of volatile compounds. Most of the instruments used are one-dimensional chromatography, but when the sample is more complex, it is necessary to introduce multidimensional chromatography technology to solve the problem of co-elution peaks by adding a second chromatographic column. The working mode of the two-dimensional gas phase in the present invention is center cutting, that is, during the sample operation, the flow control switch is used to selectively switch the effluent on the first chromatographic column to the second chromatographic column with a different stationary phase, and the target analyte and the co-eluting substance can be fully separated. Chiral compounds have similar physical and chemical properties and cannot be separated using conventional media. They can only be separated by introducing a chiral environment. Preliminary experiments have found that the separation results of the same chiral column using different heating programs are not much different, so screening a suitable chiral column is crucial for chiral analysis.

[0083] This example determines the system configuration and chiral column suitable for separating chiral substances, and configures the following three conditions 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 (60 m*0.32 mm*0.5 μm), two-dimensional column: LTM CP-Chirasil-Dex CB (30 m*0.25 mm*0.25 μm).

[0087] The above three column configuration methods were used to perform heart cutting on 8 chiral standard products. The mass spectrum of the heart cutting of condition A chiral standard product is as follows: Figure 4 As shown, the central cutting mass spectrum of the chiral standard product under condition B is as follows Figure 5 As shown, the central cutting mass spectrum of the chiral standard product under condition C is as follows Figure 6 As shown. Figures 4 to 6It can be seen that under condition A, the two configurations of the chiral substances ethyl 2-methylbutyrate and ethyl lactate can be effectively distinguished, the separation effect on the two configurations of 2-methylvaleric acid, 2-butanol and 2-pentanol is poor, and there is no separation effect on the other substances; condition B can also effectively distinguish the two configurations of ethyl 2-methylbutyrate and ethyl lactate, the separation effect on the two configurations of 2-butanol and 2-pentanol is poor, and there is no separation effect on the configurations of ethyl leucine and 2-hexanol. The two acid substances 2-methylbutyric acid and 2-methylvaleric acid have late one-dimensional peaks, and the temperature of the one-dimensional column (LTM module) is high when the peaks appear, and the two-dimensional column (in the column box) has not yet heated up. At this time, the limiting column connecting the one-dimensional column and the FID detector is in the column box, and the temperature difference with the one-dimensional column is large, resulting in the deterioration of the "signal peaks" of 2-methylbutyric acid and 2-methylvaleric acid, so that they cannot be accurately cut to the two-dimensional column; condition C has better separation effects on the two configurations of 8 chiral substances than the above two conditions. Therefore, the present invention selects condition C as the chromatographic column configuration for rapid detection of 8 pairs of chiral substances in liquor and mash.

[0088] Example 3: Determination of the configuration of chiral substances

[0089] In the field of food, different configurations of chiral compounds may produce different flavor systems. Since biosynthesis is usually driven by enzymes, the chiral compounds of these reaction products usually exist in a specific enantiomeric ratio with a certain regularity, while the enantiomeric ratio of artificially synthesized chiral compounds is often quite different from this. Therefore, chiral analysis of liquor can be used for quality control, detecting whether samples are adulterated, determining whether there are exogenous additions, judging whether it is pure grain solid-state fermentation, etc. Clarifying the chiral compounds and enantiomeric composition in liquor is of great significance for determining the quality and authenticity of liquor. In this embodiment, by adding a standard product of a single configuration, the samples before and after the addition are centrally cut, and the configuration of chiral substances that may exist in the liquor is judged.

[0090] In this embodiment, the chiral substances used in the standard products of L-lactate, R-2-butanol, S-2-butanol, R-2-pentanol, S-2-pentanol, R-2-hexanol, S-2-hexanol, R-2-methylbutyric acid, S-2-methylbutyric acid, R-2-methylpentanoic acid and S-2-methylpentanoic acid are commercially available. D-lactate, R-leucine ethyl ester, S-leucine ethyl ester, R-2-methylbutyric acid ethyl ester and S-2-methylbutyric acid ethyl ester are synthesized by themselves; the specific synthesis method is: take the corresponding precursor acid and excess precursor alcohol, add 10% solid acid catalyst HND-580 to catalyze the esterification reaction, and detect the synthesis result by GC-MS.

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

[0092] Table 1 Cutting time

[0093]

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

[0095] FID parameters: detector temperature is 250°C, 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 heart-cut, and the peak area changes were analyzed by GC-MS to determine the configurations of the 8 chiral substances that may exist in the liquor. The results are as follows: Figure 7 As shown. On the chiral column CP-Chirasil-Dex CB, ethyl 2-methylbutyrate, ethyl leucine, 2-butanol, 2-pentanol, and 2-hexanol all have R configuration in front and S configuration in the back, ethyl lactate has D configuration in front and L configuration in the back, and 2-methylbutyric acid and 2-methylvaleric acid have S configuration in front and R configuration in the back.

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

[0098] For the analysis of 8 pairs of chiral flavor substances in liquor, the single pretreatment and gas chromatography analysis of a single substance takes about 1.5 hours on average. When the sample amount increases and parallel experiments are required in the later stage, it will take more time. In order to save time and other experimental costs, the cutting conditions are optimized. By comparing the retention time and qualifier ions, the 8 substances are divided into two groups, 3 ester substances and 2 acid substances in one group, and 3 alcohol substances in one group. The cutting time is adjusted to ensure that multiple substances can be cut at one time. This method not only ensures the separation of each chiral substance, but also greatly saves the analysis time of wine samples.

[0099] The sample pretreatment method uses solid phase microextraction, which is the same as in Example 1; the gas chromatography conditions, mass spectrometry conditions and FID parameters are the same as in Example 3; due to a slight adjustment in the cutting time, the gas chromatography heating program is adjusted to be suitable for single multi-substance cutting conditions:

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

[0101] When cutting 3 esters and 2 acids: one-dimensional column temperature program: 40°C for 3 min, increase the temperature to 240°C at a rate of 5°C / min, and maintain for 27 min; two-dimensional column temperature program: 40°C for 38 min, increase the temperature to 180°C at a rate of 5°C / min, and maintain for 4 min; the common cutting time was adjusted to: 23.2-23.8 min, 31.8-32.4 min, 37.0-37.7 min, 39.5-40.5 min, 42-43 min.

[0102] Under the above conditions, 8 chiral substances in a liquor sample were analyzed in a targeted manner: the retention time of the compound and the standard was compared with the standard mass spectrum provided by the NIST14 database, and qualitative analysis and identification were performed using matching degree, characteristic ions and retention time. The center-cut mass spectra of three esters and two acids in the liquor sample were as follows: Figure 8 As shown, the center-cutting mass spectra of three alcohol chiral substances are as follows Fig. 9 As shown. Figures 8-9 It can be seen that the grouped center-cutting mode also has a good separation effect on the two configurations of 8 pairs of chiral substances.

[0103] Example 5: Application of the method

[0104] In order to test the application scope of the method for rapid detection of chiral substances in liquor and mash of the present invention, liquor samples of different flavors were taken for chiral center cutting in this example; the gas chromatography conditions and heating program, mass spectrometry conditions and FID parameters were the same as those in Example 4; the percentages of R-configuration in the five liquor samples detected are shown in Table 2.

[0105] Table 2 The percentage of R-configuration in wine samples (%)

[0106] Luzhou-flavor wine sample Maotai-flavor liquor sample Rice-flavored wine sample Fengxiang wine sample Light aroma wine sample Ethyl 2-methylbutyrate 0.25 0.19 0.07 0.08 0.11 Ethyl lactate 0.85 0.77 0.68 0.67 0.39 Ethyl leucine 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-Methylbutyric acid 0.25 0.07 / 0.35 / 2-Methylvaleric acid / / / / /

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

Claims

1. A method for rapidly detecting chiral substances in liquor and fermented grains, characterized in that: The specific steps include: S1 performs pre-treatment on the liquor or mash sample to be tested; S2 constructs a multidimensional chromatography detection system based on heart cutting: a polar column HP-INNOWAX with a specification of 60m*0.32mm*0.5μm is used as a one-dimensional column, connected to an FID detector; a low thermal mass chiral column LTM CP-Chirasil-Dex CB with a specification of 30m*0.25mm*0.25μm is used as a two-dimensional column, connected to an MS detector; S3 uses the detection system constructed in step S2, compares the retention time of the compound and the standard with the standard mass spectrum provided by the NIST14 database, and uses the matching degree, characteristic ions and retention time to perform qualitative analysis on the chiral substances in the white wine or mash sample to be tested.

2. The method for rapid detection of chiral substances in liquor and fermented grains according to claim 1, characterized in that: The chiral substances include R-2-methylbutyric acid ethyl ester and S-2-methylbutyric acid ethyl ester, D-lactate ethyl ester and L-lactate, R-leucine ethyl ester and S-leucine 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-methylbutyric acid and S-2-methylbutyric acid, R-2-methylvaleric acid and S-2-methylvaleric acid.

3. The method for rapid detection of chiral substances in liquor and fermented grains according to claim 1, characterized in that: In step S1, headspace-solid phase microextraction is used to pre-treat the liquor sample to be tested, specifically: 50 μl of the liquor sample to be tested is aspirated into a 20 ml threaded headspace sample bottle, and the headspace bottle is sealed with a threaded cap equipped with a polytetrafluoroethylene septum, 950 μl of saturated NaCl solution is added to dilute it to 10%, the extraction head is a 50 / 30 μm DVB / CAR / PDMS three-phase extraction head, preheated at 50°C for 15 min, extracted and adsorbed for 40 min, the extraction head is inserted into the gas chromatography injection port, and desorbed at 250°C GC for 3 min.

4. The method for rapid detection of chiral substances in liquor and fermented grains according to claim 1, characterized in that: In step S1, the headspace-solid phase microextraction is used to pre-treat the mash sample to be tested, specifically: after the mash is evenly mixed, 2 g of the mash sample to be tested is weighed into a 20 ml threaded headspace sample bottle, and the headspace bottle is sealed with a threaded cap equipped with a polytetrafluoroethylene septum, 8 ml of saturated NaCl solution is added, the extraction head is a 50 / 30 μm DVB / CAR / PDMS three-phase extraction head, preheated at 50 ° C for 15 min, extracted and adsorbed for 40 min, the extraction head is inserted into the gas chromatography inlet, and desorbed at 250 ° C GC for 3 min.

5. The method for rapid detection of chiral substances in liquor and fermented grains according to claim 1, characterized in that: In step S3, the gas chromatography conditions are: the injection port temperature is 240°C; the carrier gas is helium, purity>99.999%, flow rate is 1mL / min; the injection volume is 1μL, non-split mode; the heart cutting condition and the corresponding gas phase heating program are selected from any of the following: (1) Single-shot single-substance cutting mode: heart cutting and gas chromatography analysis of chiral substances respectively; The temperature program of the first-dimensional column was as follows: 40 °C for 2 min, then heated to 240 °C at a rate of 5 °C / min and held for 15 min; The temperature program of the 2D chromatographic column was as follows: 40 °C was maintained until the cutting was completed, then the temperature was increased to 180 °C at a rate of 5 °C / min and maintained for 20 min; 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; (2) Single multi-substance co-cutting mode: esters and acids are divided into one group, alcohols into another group, and the chiral substances are divided into groups for heart cutting and gas chromatography analysis; Esters and acids group: The temperature program of the first-dimensional column was as follows: 40 °C for 3 min, then increased to 240 °C at a rate of 5 °C / min and maintained for 27 min; The temperature program of the 2D column was as follows: 40 °C for 38 min, then heated to 180 °C at a rate of 5 °C / min and held for 4 min; The common cutting times are: 23.2-23.8min, 31.8-32.4min, 37.0-37.7min, 39.5-40.5min, 42-43min; Alcohol Group: The temperature program of the first-dimensional column was as follows: 40 °C for 3 min, then heated to 240 °C at a rate of 5 °C / min and held for 20 min; The temperature program of the 2D column was as follows: 40 °C for 29 min, then heated to 180 °C at a rate of 5 °C / min and held for 6 min; The common cutting times are: 22.0-23.0min, 25.0-26.0min, 27.8-28.8min.

6. The method for rapid detection of chiral substances in liquor and fermented grains according to claim 1, characterized in that: In step S3, the mass spectrometry conditions are: EI ionization source, electron energy 70 eV, ion source temperature 230°C, quadrupole temperature 150°C, interface temperature 250°C, full scan mode, and scan range 35.00-350.00 m / z.

7. The method for rapid detection of chiral substances in liquor and fermented grains according to claim 1, characterized in that: In step S3, FID parameters are: detector temperature is 250°C, hydrogen flow rate is 30 ml / min, air flow rate is 300 ml / min, and nitrogen tail gas flow rate is 25 ml / min.

8. Application of the method according to any one of claims 1 to 6 in the detection of flavor substances in liquor and its products.

9. Application of the method according to any one of claims 1 to 6 in liquor quality identification.

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