Analytical Methods for Compound Concentration and Uniformity in Liquor

Abstract

This invention discloses a method for analyzing the concentration and uniformity of compounds in liquor, belonging to the technical field of liquor detection. By systematically studying the relationship between pretreatment processes and the quantitative accuracy of target compounds, the method combines target compounds with specific pretreatment processes and GC-MS conditions, thereby ensuring accurate quantitative detection of the concentrations of up to 108 compounds in liquor. The concentrations of each compound are then used to calculate the uniformity index of the compounds in the liquor sample, facilitating the identification of liquor aging time.

Description

Technical Field

[0001] The invention belongs to the technical field of liquor detection, and particularly relates to a method for analyzing the concentration and uniformity of compounds in Luzhou-flavor liquor. Background Art

[0002] Baijiu (Chinese liquor), one of the world's six major distilled spirits, is composed of approximately 98% ethanol and water. The remaining 2% comprises a diverse range of trace components, with over 2,000 compounds identified. These compounds are crucial components of baijiu's flavor and contribute significantly to its quality. Gas chromatography-mass spectrometry (GC-MS) is one of the most commonly used techniques for qualitative and quantitative analysis of compounds in baijiu. Analyzing compound variations in aged baijiu using GC-MS can improve the universality and scalability of research techniques. While GC-MS has explored numerous sample pretreatment methods, there is still room for parameter optimization for each pretreatment technique, and their scope of application remains under-researched.

[0003] Therefore, it is necessary to select appropriate pretreatment and detection conditions according to the characteristics of the compounds contained in the liquor, so as to systematically ensure the accuracy of the detection of each compound in the liquor, and then use it to calculate the uniformity. Summary of the Invention

[0004] In order to accurately identify numerous compounds in liquor and provide a basis for identifying the aging time of liquor, the present invention first provides a method for analyzing the concentration of compounds in Luzhou-flavor liquor, which comprises the following steps:

[0005] A. Pretreatment: Liquor samples were pretreated using headspace solid phase microextraction, liquid-liquid microextraction, and liquid-liquid microextraction-BSTFA derivatization;

[0006] B. GC-MS determination: GC-MS was used to detect the pretreated samples to obtain the concentration of compounds in the liquor;

[0007] The compounds in the liquor are isobutanol, n-pentanol, furfuryl alcohol, n-hexanol, phenylethyl alcohol, ethyl acetate, ethyl caproate, ethyl lactate, ethyl butyrate, ethyl heptanoate, ethyl octanoate, ethyl nonanoate, ethyl caprate, ethyl palmitate, ethyl linoleate, ethyl oleate, ethyl stearate, ethyl laurate, ethyl myristate, acetic acid, butyric acid, hexanoic acid, heptanoic acid, isopentanol, ethyl propionate, ethyl isobutyrate, ethyl valerate, ethyl isovalerate, DL-leucine ethyl ester, amyl caproate, ethyl 2-methylbutyrate, hexanoic acid, ethyl ethyl Methyl phthalate, ethyl phenylpropionate, phenol, hexyl hexanoate, dibutyl phthalate, diisobutyl phthalate, palmitic acid, lauric acid, caprylic acid, nonanoic acid, 2,3,5-trimethylpyrazine, capric acid, lauric acid, myristic acid, linoleic acid, oleic acid, stearic acid, hexanal, 3-methylbutanal, furfural, 2-methylbutanal, benzaldehyde, nonanal, 3-hydroxy-2-butanone, 2-pentanone, isophorone, 4-ethylphenol, 2,3,5,6-tetramethylpyrazine, longifolene, butyl butyrate, isopropyl lactate amyl ester, isoamyl butyrate, dimethyl phthalate, ethyl 4-methylvalerate, ethyl phenylacetate, propionic acid, isoamyl hexanoate, 4-methylvaleric acid, methyl palmitate, n-valeric acid, isovaleric acid, hexyl acetate, 2-butanol, 2-pentanol, 2-octanol, isobutyl hexanoate, ethyl acrylate, ethyl crotonate, methyl (S)-2-hydroxy-3-methylbutyrate, ethyl 2-furoate, ethyl 5-hexenoate, ethyl 2-hexenoate, diethyl succinate, propyl hexanoate, hexyl butyrate, diethyl azelaic acid, Ethyl 9-hexadecenoate, dioctyl phthalate, methyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate, isobutyric acid, butenoic acid, benzoic acid, phenylacetic acid, 2-hydroxy-4-methylvaleric acid, 3-phenyllactic acid, 2-ketovaleric acid, myristic aldehyde, stearalkanal, p-cresol, 2,4-di-tert-butylphenol, 2,6-di-tert-butyl-4-methylphenol, oleamide, methyl stearate, 2-ethylhexylvalerate, ethylfurfuryl ether, butyl hexanoate, and 4-methyl-2-pentanol;

[0008] The target detection compounds are hexyl butyrate, ethyl phenylacetate, ethyl propionate, ethyl 3-phenylpropionate, 2,3,5-trimethylpyrazine, ethyl butyrate, benzaldehyde, butyl butyrate, isovaleraldehyde, ethyl isovalerate, ethyl isobutyrate, isobutanol, DL-2-methylbutyrate, methyl hexanoate, octyl formate, ethyl acetate, ethyl isocaproate, phenol, furfural, 2,3,5,6-tetramethylpyrazine, ethyl hexanoate, ethyl lactate, isopentyl valerate, ethyl valerate, isophorone, n-hexanol, n-pentanol, Headspace solid phase microextraction was used to pretreat the liquor samples for isopentanol, amyl hexanoate, ethyl heptanoate, hexyl acetate, isopentanol, longifolene, heptanoic acid, hexanoic acid, acetic acid, ethyl decanoate, hexyl hexanoate, dimethyl phthalate, ethyl octanoate, butyl hexanoate, octanoic acid, propyl hexanoate, isopentyl hexanoate, ethyl nonanoate, myristic aldehyde, ethyl laurate, isovaleric acid, valeric acid, butyl valerate, nonanal, butyric acid, n-hexanal, β-phenylethanol, DL-leucine ethyl ester, sec-octanol, and 2-methylbutanal;

[0009] The target detection compounds are ethyl isohexanoate, ethyl decanoate, n-hexanol, hexyl acetate, ethyl valerate, ethyl hexanoate, phenylacetaldehyde, ethyl butyrate, isoamyl alcohol, diisobutyl phthalate, butyl valerate, ethyl propionate, butyric acid, nonanal, ethyl heptanoate, ethyl phenylacetate, 2-pentanone, lauric acid, ethyl nonanoate, ethyl linoleate, hexyl hexanoate, ethyl isovalerate, propionic acid, isobutyric acid, longifolene, DL-2-methylbutyrate, ethyl oleate, furfuryl alcohol, isoamyl lactate, acetic acid, isophorone, ethyl octanoate, furfural, ethyl isobutyrate, dimethyl phthalate, isoamyl valerate, hexanoic acid, methyl palmitate, butyl hexanoate, methyl hexanoate, 3- When ethyl phenylpropionate, dibutyl phthalate, 4-ethylphenol, 4-methylvaleric acid, diethyl phthalate, 2,3,5,6-tetramethylpyrazine, heptanoic acid, ethyl palmitate, isobutyl alcohol, isopropyl palmitate, benzaldehyde, 2-methylbutanal, ethyl acetate, valeric acid, isopentyl hexanoate, phenol, ethyl lactate, ethyl stearate, diacetyl, isovaleraldehyde, isovaleric acid, octanoic acid, butyl butyrate, palmitic acid, isopentyl butyrate, octyl formate, β-phenylethanol, acetoin (3-hydroxy-2-butanone), myristic acid, 2,3,5-trimethylpyrazine, hexanal, n-pentanol and nonanoic acid were used for pretreatment of liquor samples by liquid-liquid microextraction;

[0010] When the target detection compounds were ethyl linoleate, ethyl oleate, oleic acid, ethyl laurate, β-phenylethanol, myristic acid, ethyl myristic acid, diethyl phthalate, lauric acid, linoleic acid, isopropyl palmitate, caprylic acid, heptanoic acid, nonanoic acid, capric acid, lactic acid, methyl palmitate, palmitic acid and ethyl stearate, liquid-liquid microextraction-BSTFA derivatization was used to pretreat the liquor samples.

[0011] Among them, in the above-mentioned method for analyzing the concentration of compounds in liquor, in step A,

[0012] The headspace solid phase microextraction operation is as follows: diluting the liquor sample to a final ethanol content of 5-15 vol%, adding NaCl, using 2-methylhexanoic acid as an internal standard for ethyl lactate and compounds with a retention time greater than acetic acid, using tert-amyl alcohol as an internal standard for alcohols with a retention time less than acetic acid, and using amyl acetate as an internal standard for other compounds, using an SPME fiber to extract volatile compounds from the sample headspace, pre-equilibrating the sample at 45-60° C. for 0-10 min, then extracting at 45-60° C. for 30-60 min, and then performing subsequent GC-MS analysis;

[0013] The liquid-liquid microextraction process is as follows: three internal standards, namely amyl acetate, 2-methylhexanoic acid and tert-amyl alcohol, and a saturated sodium chloride solution (wherein the volume ratio of anhydrous ether to pentane is 1:1) are added to the liquor sample, followed by a mixed solution of anhydrous ether and pentane, which is stirred thoroughly for more than 3 minutes. After static stratification, the upper organic phase is collected, and the organic phase is concentrated by nitrogen purge at room temperature before subsequent GC-MS analysis.

[0014] The operation of liquid-liquid microextraction-BSTFA derivatization is as follows: add heptadecanoic acid internal standard to the liquor sample, then add saturated sodium chloride solution and dilute to an ethanol concentration of about 10%, add a mixed solution of anhydrous ether and pentane (wherein the volume ratio of anhydrous ether: pentane = 1:1) to the diluted system for extraction, vortex for more than 3 minutes, let stand for more than 20 minutes, collect the organic layer, and extract the remaining aqueous phase with acetonitrile and dichloromethane in turn. Combine the organic phases, add pyridine containing v / v 2.5% hydroxylamine hydrochloride to the organic phase, add BSTFA containing 1% TMCs, vortex for more than 5 seconds, incubate in a metal bath at 45-85°C for 1-5 hours, centrifuge at 6000-12000 rpm for more than 3 minutes, take the supernatant, and perform subsequent GC-MS analysis.

[0015] Among them, in the above-mentioned method for analyzing the concentration of compounds in liquor, in step B,

[0016] When using headspace solid-phase microextraction, the GC-MS conditions were as follows: DB-WAX column; carrier gas: 99.999% pure helium at 0.8–1.4 mL / min; electron impact mode: 70 eV; transfer line temperature at the connection between the column and the ion source set at 200–230°C; scan mode, m / z 33–350; inlet temperature: 250–300°C; desorption time: 5–15 min; split ratio: 2–20:1; temperature program: starting from 50°C and increasing in stages to 235°C, maintained for at least 2 min;

[0017] When using liquid-liquid microextraction, the GC-MS conditions were as follows: DB-WAX column; carrier gas 0.8-1.4 mL / min of 99.999% pure helium; electron impact mode: 70 eV; the transfer line temperature at the connection between the column and the ion source was set to 200-230°C; scan mode, scanning range m / z 33-350; sample volume 1 μL, injection port temperature 250-300°C, desorption time 5-15 minutes, split ratio 2-20:1; temperature program: starting from 35°C and gradually increasing to 235°C and holding for more than 10 minutes;

[0018] When using liquid-liquid microextraction-BSTFA derivatization, the GC-MS conditions were as follows: HP-5MS column; carrier gas: 99.999% pure helium at 0.8-1.4 mL / min; electron impact mode: 70 eV; the transfer line temperature at the connection between the chromatographic column and the ion source was set to 200-230°C; scan mode, the scan range was m / z 33-350; the sample volume was 1 μL, the injection port temperature was 200-250°C, and the split ratio was 2-20:1; the heating program was: 65°C gradually increased to 280°C, and maintained for more than 2 minutes.

[0019] In the above-mentioned method for analyzing the concentration of compounds in liquor, in step B, 73 compounds were quantitatively determined by establishing a standard curve using a mixed standard, and the remaining 35 compounds were quantitatively determined by relative internal standards:

[0020] The target compounds for GC-MS detection were isobutanol, n-pentanol, furfuryl alcohol, n-hexanol, phenylethyl alcohol, ethyl acetate, ethyl hexanoate, ethyl lactate, ethyl butyrate, ethyl heptanoate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, ethyl palmitate, ethyl linoleate, ethyl oleate, ethyl stearate, ethyl laurate, ethyl myristate, acetic acid, butyric acid, hexanoic acid, heptanoic acid, isopentanol, ethyl propionate, ethyl isobutyrate, ethyl valerate, ethyl isovalerate, DL-leucine ethyl ester, amyl hexanoate, ethyl 2-methylbutyrate, methyl hexanoate, ethyl phenylpropionate, phenol, hexyl hexanoate, dibutyl phthalate, diisobutyl phthalate, palmitic acid, lauric acid, octanoic acid, nonanoic acid, 2,3,5-trimethylpyrazine, decanoic acid, lauric acid, myristic acid, linoleic acid, oil When the following test samples are tested, an internal standard calibration curve is established: when the following test samples are tested, an internal standard calibration curve is established: the abscissa is the peak area ratio of the target compound to the corresponding internal standard substance, and the ordinate is the mass concentration ratio of the target compound to the corresponding internal standard substance. The calibration curve is established, and the concentration of the target compound is obtained through a linear equation;

[0021] The target compounds for GC-MS detection were 2-butanol, 2-pentanol, 2-octanol, isobutyl hexanoate, ethyl acrylate, ethyl butenoate, (S)-2-hydroxy-3-methylbutanoate, ethyl 2-furoate, ethyl 5-hexenoate, ethyl 2-hexenoate, diethyl succinate, propyl hexanoate, hexyl butanoate, diethyl azelaic acid, ethyl 9-hexadecenoate, dioctyl phthalate, methyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate, isobutyric acid, For butenoic acid, benzoic acid, phenylacetic acid, 2-hydroxy-4-methylvaleric acid, 3-phenyllactic acid, 2-ketovaleric acid, myristic aldehyde, stearalkanal, p-cresol, 2,4-di-tert-butylphenol, 2,6-di-tert-butyl-4-methylphenol, oleamide, methyl stearate, 2-ethylhexylvalerate, ethylfurfuryl ether, butyl hexanoate, and 4-methyl-2-pentanol, amyl acetate, 2-methylhexanoic acid, tert-amyl alcohol, or heptadecanoic acid was used as the internal standard to obtain the relative concentrations of the target compounds.

[0022] Among them, in the above-mentioned method for analyzing the concentration of compounds in white wine, in step B, the compounds in the sample are first qualitatively analyzed, and then the target compounds are quantitatively analyzed.

[0023] Based on the above method, the present invention also provides a method for analyzing the uniformity of compound concentration in liquor, which, in addition to steps A and B, further comprises the following steps:

[0024] C. Calculate the evenness index of the compounds in the liquor sample by chemometrics. The calculation formula is: P i =N i / N, where i is 1, 2, ..., S, S is the total number of compounds in the liquor sample in step A, N is the sum of the contents of all compounds in the liquor sample, and N i is the content of a certain compound i in the liquor sample.

[0025] Among them, in the above two methods, in step A, the liquor sample is a Luzhou-flavor liquor.

[0026] Preferably, in the above two methods, in step A, the aging time of the Luzhou-flavor liquor is 0 to 11 years.

[0027] It should be noted that the method of the present invention can ensure accurate quantitative detection of the concentrations of the aforementioned 108 compounds by combining the target compounds with specific pretreatment processes and GC-MS conditions. However, some wine samples may not contain all 108 compounds, that is, they may not contain a small amount of compounds. In this case, the method of the present invention is also applicable. Figure 4 It can be seen that although the number of compounds detected in liquors with different aging times is different, the difference is not large, and the method of the present invention is also applicable to these liquor samples.

[0028] In addition, the target compounds targeted by the three pretreatment processes of the present invention overlap, and in this case, one of them can be selected according to needs.

[0029] Beneficial effects of the present invention:

[0030] The present invention systematically studies the relationship between the pretreatment process and the quantitative accuracy of the target compounds. By coordinating the target compounds with specific pretreatment processes and GC-MS conditions, it is possible to ensure accurate quantitative detection of the concentrations of up to 108 compounds in liquor. On this basis, the concentration of each compound can be used to calculate the evenness index of the compounds in the liquor sample, which is conducive to the identification of the aging time of the liquor. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The figure shows the heat map drawn after normalization of 54 wine samples and the HCA cluster analysis results based on Spearman similarity.

[0032] Figure 2This is a diagram of abnormal results that occurred during the HS-SPME-GC-MS standard curve determination process.

[0033] Figure 3 This is the compound uniformity index diagram of aged liquor.

[0034] Figure 4 Figure 2 is a graph showing the number of compounds detected in liquors with different aging times. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below through examples, but the protection scope of the present invention is not limited to the scope of the examples.

[0036] Materials and samples:

[0037] Sodium chloride, anhydrous ether, n-pentane, acetonitrile, anhydrous ethanol, and dichloromethane used for salting out or extraction were all from Shanghai Sinopharm Chemical Reagent Co., Ltd., China. 40 Retention indices (RIs) of the compounds were calculated using a mixture of n-alkanes (Sigma-Aldrich, Shanghai, China). Four internal standards (amyl acetate (IS1), 2-methylhexanoic acid (IS2), tert-amyl alcohol (IS3), and heptadecanoic acid (IS4), as well as 98 standards (see Tables 1 and S2) were purchased from Sigma-Aldrich or Aladdin Reagent Co., Ltd. (Shanghai, China) with a purity greater than 98%.

[0038] Authentic bottled aged liquor samples were provided by the preservation center of Sichuan Luzhou Laojiao Co., Ltd. All samples were premium grade liquor (Guojiao 1573) with the same alcohol content (52% by volume). Samples of varying vintages (0-11 years) were packaged and stored at room temperature.

[0039] Example 1: HS-SPME is applicable to the quantification of low-volatility compounds in a 91-compound system

[0040] Materials: 90 standard products (hexyl butyrate, ethyl phenylacetate, ethyl propionate, ethyl 3-phenylpropionate, 2,3,5-trimethylpyrazine, ethyl butyrate, benzaldehyde, butyl butyrate, heptanoic acid, isovaleraldehyde, ethyl isovalerate, ethyl isobutyrate, isobutanol, DL-2-methylbutyrate, methyl hexanoate, octyl formate, ethyl acetate, ethyl isohexanoate, phenol, hexanoic acid, furfural, acetic acid, 2,3,5,6- Tetramethylpyrazine, ethyl hexanoate, ethyl lactate, isopentyl valerate, ethyl decanoate, ethyl valerate, hexyl hexanoate, isophorone, n-hexanol, n-pentanol, isopentyl alcohol, amyl hexanoate, ethyl heptanoate, hexyl acetate, isopentyl butyrate, dimethyl phthalate, ethyl octanoate, butyl hexanoate, octanoic acid, propyl hexanoate, isopentyl hexanoate, ethyl nonanoate, longifolene, myristic aldehyde, ethyl laurate, isovaleric acid, amyl hexanoate The following substances (acid, butyl valerate, nonanal, butyric acid, n-hexanal, β-phenylethanol, DL-leucine ethyl ester, sec-octanol, 2-methylbutyraldehyde, furfuryl alcohol, 4-methylvaleric acid, 3-hydroxy-2-butanone, lactic acid, ethyl myristate, ethyl palmitate, ethyl stearate, ethyl linoleate, ethyl oleate, methyl palmitate, isopropyl palmitate, propionic acid, isobutyric acid, capric acid, nonanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, oleic acid, diacetyl, 2-pentanone, p-cresol, phenylacetaldehyde, 4-ethylphenol, diethyl phthalate, isoamyl lactate, n-heptanol, dibutyl phthalate, 2,4-di-tert-butylphenol, diisobutyl phthalate, and stearalkanal) were dissolved in 52% ethanol aqueous solution in proportion to prepare a mixed standard stock solution system 1, and the calibration curve was obtained by gradient dilution to determine the samples.

[0041] HS-SPME-GC-MS method parameters:

[0042] The standard mix for each gradient was diluted to a final ethanol content of 8% (6 mL) and placed in a 20 mL glass vial. 2.0 g of NaCl was added. 2-Methylhexanoic acid (10 μL, 14.00 g / L) was used as the internal standard for ethyl lactate and compounds with retention times greater than acetic acid. Tert-amyl alcohol (10 μL, 8.05 g / L) was used as the internal standard for alcohols with retention times less than acetic acid, while amyl acetate (10 μL, 10.33 g / L) was used for other compounds. Volatile compounds were extracted from the sample headspace using a PAL3 autosampler (Sterus Analytical Instruments, Switzerland) and an SPME fiber (80 μm thick, 10 mm long, DVB / C-WR / PDMS) (Agilent Technologies, USA). Samples were equilibrated at 60°C for 5 min, followed by extraction at 60°C for 40 min.

[0043] GC-MS analysis was performed using a 7890B gas chromatograph (GC), a 5977B mass spectrometer (MS) (Agilent Technologies, USA) and a DB-WAX (30 m × 0.25 mm × 0.25 μm) column. The carrier gas was helium (99.999% purity) at a flow rate of 1.2 mL / min. The MS was operated in electron impact (EI) mode (70 eV). The transfer line connecting the column and the ion source required auxiliary heating, and the temperature was set to 230°C. Detection was performed in scan mode over the range of m / z 33–350. The inlet temperature was 300°C, the desorption time was 15 min, and the split ratio was 4:1. The temperature program started at 50°C, held for 2 min, then increased to 145°C at 3°C / min, and then increased to 235°C at 15°C / min and held for 8 min.

[0044] The results are shown in Table 1.

[0045] Table 1 Standard curve of mixed standard system 1 determined by HS-SPME-GC-MS

[0046]

[0047]

[0048]

[0049] After testing, the standard samples of the 57 compounds in Table 1 above are suitable for determination by HS-SPME, and the test results are accurate. However, compounds such as furfuryl alcohol, 4-methylvaleric acid, 3-hydroxy-2-butanone, lactic acid, ethyl myristate, ethyl palmitate, ethyl stearate, ethyl linoleate, ethyl oleate, methyl palmitate, isopropyl palmitate, propionic acid, isobutyric acid, capric acid, nonanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, oleic acid, diacetyl, 2-pentanone, p-cresol, phenylacetaldehyde, 4-ethylphenol, diethyl phthalate, isoamyl lactate, n-heptanol, dibutyl phthalate, 2,4-di-tert-butylphenol, diisobutyl phthalate, and stearalkanal are difficult to determine by HS-SPME (e.g. Figure 2 ).

[0050] Example 2: HS-SPME is not suitable for the quantification of medium-chain acids and long-chain esters in a 17-compound system

[0051] Materials: 17 standards (acetic acid, butyric acid, caproic acid, lactic acid, ethyl acetate, ethyl butyrate, ethyl caproate, ethyl lactate, ethyl heptanoate, ethyl caprate, ethyl laurate, ethyl phenylacetate, ethyl palmitate, ethyl stearate, ethyl oleate, ethyl linoleate, and isopropyl palmitate) were dissolved in 52% ethanol aqueous solution in proportion to prepare a mixed standard stock solution system 2. Calibration curves were obtained by gradient dilution to determine the samples.

[0052] HS-SPME-GC-MS method parameters:

[0053] The standard mix for each gradient was diluted to a final ethanol content of 8% (6 mL) and placed in a 20 mL glass vial. 2.0 g of NaCl was added. 2-Methylhexanoic acid (10 μL, 14.00 g / L) was used as the internal standard for ethyl lactate and compounds with retention times greater than acetic acid. Tert-amyl alcohol (10 μL, 8.05 g / L) was used as the internal standard for alcohols with retention times less than acetic acid, while amyl acetate (10 μL, 10.33 g / L) was used for other compounds. Volatile compounds were extracted from the sample headspace using a PAL3 autosampler (Sterus Analytical Instruments, Switzerland) and an SPME fiber (80 μm thick, 10 mm long, DVB / C-WR / PDMS) (Agilent Technologies, USA). Samples were equilibrated at 60°C for 5 min, followed by extraction at 60°C for 40 min.

[0054] GC-MS was performed using a 7890B gas chromatograph (GC), a 5977B mass spectrometer (MS) (Agilent Technologies, Inc., USA), and a DB-WAX (30 m × 0.25 mm × 0.25 μm) column. The carrier gas was helium (99.999% purity) at a flow rate of 1.2 mL / min. The MS was operated in electron impact (EI) mode (70 eV). The transfer line connecting the column and the ion source required auxiliary heating, and the temperature was set to 230°C. Detection was performed in scan mode over the m / z 33-350 range. The inlet temperature was 250°C, the desorption time was 5 min, and the split ratio was 4:1. The temperature program started at 50°C, held for 2 min, then increased to 145°C at 3°C / min, and then increased to 235°C at 15°C / min and held for 8 min.

[0055] The results are shown in Table 2.

[0056] Table 2 Standard curve of mixed standard system 2 determined by HS-SPME-GC-MS

[0057]

[0058]

[0059] Table 2 shows that HS-SPME-GC-MS cannot quantify compounds such as lactic acid. When quantifying short-chain, low-volatile acids in liquor using HS-SPME-GC-MS, the detection limit for hexanoic acid exceeds 330 mg / L. When the concentration of substances with carbon chain lengths greater than 12C ranges from 8 to 80 mg / L, HS-SPME-GC-MS quantification is inaccurate, with the linear fit of the calibration curve falling below 0.43 and the slope even exhibiting a negative value, resulting in abnormal results. This may be due to the high boiling points of long-chain fatty acid esters. The inlet temperature of 250°C and a desorption time of 5 minutes cannot completely desorb the esters on the extraction head, resulting in residual residues. This results in cross-contamination between samples, and the mass spectrometer response area does not truly reflect the actual content.

[0060] Example 3: HS-SPME is not suitable for the quantification of medium-chain acids and long-chain esters in a 33-compound system

[0061] Materials: 35 standards (acetic acid, butyric acid, hexanoic acid, lactic acid, ethyl acetate, ethyl butyrate, ethyl hexanoate, ethyl lactate, valeric acid, heptanoic acid, octanoic acid, decanoic acid, 4-methylvaleric acid, palmitic acid, stearic acid, linoleic acid, ethyl phenylacetate, ethyl heptanoate, ethyl nonanoate, ethyl decanoate, ethyl palmitate, isopropyl palmitate, ethyl linoleate, ethyl 3-phenylpropionate, lauric acid, myristic acid, oleic acid, elaidic acid, methyl palmitate, ethyl stearate, ethyl myristate, ethyl valerate, ethyl octanoate, ethyl laurate, nonanoic acid) were dissolved in 52% ethanol aqueous solution in proportion to prepare mixed standard stock solution system three. Calibration curve was obtained by gradient dilution to measure samples.

[0062] HS-SPME-GC-MS method parameters:

[0063] The standard mix for each gradient was diluted to a final ethanol content of 8% (6 mL) and placed in a 20 mL glass vial. 2.0 g of NaCl was added. 2-Methylhexanoic acid (10 μL, 14.00 g / L) was used as the internal standard for ethyl lactate and compounds with retention times greater than acetic acid. Tert-amyl alcohol (10 μL, 8.05 g / L) was used as the internal standard for alcohols with retention times less than acetic acid, while amyl acetate (10 μL, 10.33 g / L) was used for other compounds. Volatile compounds were extracted from the sample headspace using a PAL3 autosampler (Sterus Analytical Instruments, Switzerland) and an SPME fiber (80 μm thick, 10 mm long, DVB / C-WR / PDMS) (Agilent Technologies, USA). Samples were equilibrated at 60°C for 5 min, followed by extraction at 60°C for 40 min.

[0064] GC-MS analysis was performed using a 7890B gas chromatograph (GC), a 5977B mass spectrometer (MS) (Agilent Technologies, USA) and a DB-WAX (30 m × 0.25 mm × 0.25 μm) column. The carrier gas was helium (99.999% purity) at a flow rate of 1.2 mL / min. The MS was operated in electron impact (EI) mode (70 eV). The transfer line connecting the column and the ion source required auxiliary heating, and the temperature was set to 230°C. Detection was performed in scan mode over the range of m / z 33–350. The inlet temperature was 300°C, the desorption time was 15 min, and the split ratio was 4:1. The temperature program started at 50°C, held for 2 min, then increased to 145°C at 3°C / min, and then increased to 235°C at 15°C / min and held for 8 min.

[0065] The results are shown in Table 3.

[0066] Table 3 Standard curves of abnormal compounds when mixed standard system 3 was determined using HS-SPME-GC-MS

[0067] Compound <![CDATA[Mixed standard R 2 > Marking the song Concentration range mg / L lactic acid ND ND 100-1000 Ethyl laurate ND ND 0.06-0.6 Myristic acid ND ND 0.25-2.5 Palmitic acid ND ND 1.01-10.1 stearic acid ND ND 1-10 Linoleic acid ND ND 0.24-2.4 Oleic acid ND ND 0.236-2.36 Elaidic acid ND ND 0.19-1.9 Heptanoic acid 0.8466 y=0.6169x-0.0063 0.596-5.96 4-Methylvaleric acid 0.5669 y=0.108x+0.0045 0.406-4.06 Lauric acid 0.0816 y=0.3639x+0.3496 5-20 Ethyl stearate 0.000004 y=0.0007x+0.0019 0.048-0.48 Ethyl linoleate 0.5315 y=-0.1906x+0.016 0.306-3.06 Ethyl myristate 0.5188 y=-3.8173x+0.0718 0.052-0.52

[0068] Table 3 shows that HS-SPME-GC-MS quantification of acidic compounds in liquor showed poor linearity for heptanoic acid, while the detection limits for most acids, including lactic acid, stearic acid, linoleic acid, and oleic acid, were high or non-detectable, likely due to their low volatility. In a complex system containing 35 compounds, HS-SPME-GC-MS quantification was inaccurate when the concentration of esters with carbon chain lengths greater than 12C ranged from 0.048 to 20 mg / L. The linear fit of the calibration curve was less than 0.53, and the slope even exhibited a negative value, indicating anomalous results. This may be due to interactions between the various compounds in the complex system, resulting in non-linear increases and decreases in the volatility coefficients of the compounds at different concentrations.

[0069] Example 4: HS-SPME is not suitable for the quantification of medium-chain acids and long-chain esters in a 41-compound system

[0070] Materials: 41 standards (acetic acid, butyric acid, hexanoic acid, lactic acid, ethyl acetate, ethyl butyrate, ethyl hexanoate, ethyl lactate, valeric acid, heptanoic acid, octanoic acid, decanoic acid, 4-methylvaleric acid, palmitic acid, stearic acid, linoleic acid, ethyl phenylacetate, ethyl heptanoate, ethyl nonanoate, ethyl decanoate, ethyl palmitate, isopropyl palmitate, ethyl linoleate, ethyl 3-phenylpropionate, lauric acid, myristic acid, oleic acid, elaidic acid, methyl palmitate, ethyl stearate, ethyl myristate, ethyl valerate, n-hexanol, n-heptanol, ethyl octanoate, propionic acid, isobutyric acid, diethyl succinate, ethyl laurate, β-phenylethanol, nonanoic acid) were dissolved in 52% ethanol aqueous solution in proportion to prepare a mixed standard stock solution system IV. Calibration curves were obtained by gradient dilution.

[0071] HS-SPME-GC-MS method parameters:

[0072] The standard mix for each gradient was diluted to a final ethanol content of 8% (6 mL) and placed in a 20 mL glass vial. 2.0 g of NaCl was added. 2-Methylhexanoic acid (10 μL, 14.00 g / L) was used as the internal standard for ethyl lactate and compounds with retention times greater than acetic acid. Tert-amyl alcohol (10 μL, 8.05 g / L) was used as the internal standard for alcohols with retention times less than acetic acid, while amyl acetate (10 μL, 10.33 g / L) was used for other compounds. Volatile compounds were extracted from the sample headspace using a PAL3 autosampler (Sterus Analytical Instruments, Switzerland) and an SPME fiber (80 μm thick, 10 mm long, DVB / C-WR / PDMS) (Agilent Technologies, USA). Samples were equilibrated at 60°C for 5 min, followed by extraction at 60°C for 40 min.

[0073] GC-MS analysis was performed using a 7890B gas chromatograph (GC), a 5977B mass spectrometer (MS) (Agilent Technologies, USA) and a DB-WAX (30 m × 0.25 mm × 0.25 μm) column. The carrier gas was helium (99.999% purity) at a flow rate of 1.2 mL / min. The MS was operated in electron impact (EI) mode (70 eV). The transfer line connecting the column and the ion source required auxiliary heating, and the temperature was set to 230°C. Detection was performed in scan mode over the range of m / z 33–350. The inlet temperature was 300°C, the desorption time was 15 min, and the split ratio was 4:1. The temperature program started at 50°C, held for 2 min, then increased to 145°C at 3°C / min, and then increased to 235°C at 15°C / min and held for 8 min.

[0074] The results are shown in Table 4.

[0075] Table 4 Standard curves of compounds with abnormalities when the mixed standard system was determined using HS-SPME-GC-MS

[0076]

[0077]

[0078] Table 4 shows that HS-SPME-GC-MS quantification of acidic compounds in liquor showed poor linearity for octanoic and nonanoic acids. The detection limits for most acids, including lactic, stearic, linoleic, and oleic acids, were high or non-detectable, likely due to their low volatility. In a complex system containing 41 compounds, HS-SPME-GC-MS quantification was inaccurate when the concentration of esters with carbon chain lengths greater than 12C ranged from 0.08 to 25.4 mg / L. The linear fit of the calibration curve was less than 0.43, and the slope even exhibited a negative value, indicating anomalous results. This may be due to interactions between the various compounds in the complex system, resulting in non-linear increases and decreases in the volatility coefficients of the compounds at different concentrations.

[0079] Example 5: LLME has a wide range of applications, but it still cannot quantify all non-volatile acids.

[0080] 5.1 Materials: 90 standard products (hexyl butyrate, ethyl phenylacetate, ethyl propionate, ethyl 3-phenylpropionate, 2,3,5-trimethylpyrazine, ethyl butyrate, benzaldehyde, butyl butyrate, heptanoic acid, isovaleraldehyde, ethyl isovalerate, ethyl isobutyrate, isobutanol, DL-2-methylbutyrate, methyl hexanoate, octyl formate, ethyl acetate, ethyl isohexanoate, phenol, hexanoic acid, furfural, acetic acid, 2,3,5 ,6-Tetramethylpyrazine, Ethyl hexanoate, Ethyl lactate, Isoamyl valerate, Ethyl decanoate, Ethyl valerate, Hexyl hexanoate, Isophorone, Hexanol, Pentyl alcohol, Isoamyl alcohol, Pentyl hexanoate, Ethyl heptanoate, Hexyl acetate, Isoamyl butyrate, Dimethyl phthalate, Ethyl octanoate, Butyl hexanoate, Octanoic acid, Propyl hexanoate, Isoamyl hexanoate, Ethyl nonanoate, Longifolene, Myristic aldehyde, Ethyl laurate, Isovaleric acid , valeric acid, butyl valerate, nonanal, butyric acid, n-hexanal, β-phenylethanol, DL-leucine ethyl ester, sec-octanol, 2-methylbutanal, furfuryl alcohol, 4-methylvaleric acid, 3-hydroxy-2-butanone, lactic acid, ethyl myristate, ethyl palmitate, ethyl stearate, ethyl linoleate, ethyl oleate, methyl palmitate, isopropyl palmitate, propionic acid, isobutyric acid, capric acid, nonanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, oleic acid, diacetyl, 2-pentanone, p-cresol, phenylacetaldehyde, 4-ethylphenol, diethyl phthalate, isoamyl lactate, n-heptanol, dibutyl phthalate, 2,4-di-tert-butylphenol, diisobutyl phthalate, stearalkanal) were dissolved in 52% ethanol aqueous solution in proportion to prepare a mixed standard stock solution system 1, and the calibration curve was obtained by gradient dilution to determine the samples.

[0081] LLME-GC-MS method parameters:

[0082] Transfer 4 mL of each gradient standard mixture to a 30 mL glass centrifuge tube, add 10 μL of each of the three internal standards (10.33 g / L amyl acetate, 14.00 g / L 2-methylhexanoic acid, and 10.22 g / L tert-amyl alcohol) and 14 mL of saturated sodium chloride solution. Then, add 1.5 mL of Solution A (anhydrous ether:pentane = 1:1, v / v), stir thoroughly for at least 3 minutes, and allow static separation to collect the upper organic phase. The organic phase was concentrated to 250 μL at room temperature using nitrogen purge and analyzed using an Agilent 7890B-5977B GC-MS.

[0083] GC-MS analysis was performed using a 7890B gas chromatograph (GC) and a 5977B mass spectrometer (MS) (Agilent Technologies, Inc., USA) using a DB-WAX (30 m × 0.25 mm × 0.25 μm) column. Helium (99.999% purity) was used as the carrier gas at a flow rate of 1.4 mL / min. The MS was operated in electron impact (EI) mode (70 eV). The transfer line connecting the column and ion source required auxiliary heating and was set to 230°C. Detection was performed in scan mode over the m / z range of 33–350. 1 μL of the concentrated organic phase was injected with a split ratio of 4:1. The GC temperature program was as follows: 35°C for 0.5 min, then 10°C / min to 50°C for 4 min, then 3°C / min to 100°C for 3 min, and finally 3°C / min to 230°C for 23 min.

[0084] The results are shown in Table 5.

[0085] Table 5 Standard curve of compounds in mixed standard system 1 determined by LLME-GC-MS

[0086]

[0087]

[0088]

[0089] As shown in Table 5, the standards of the above 73 compounds are relatively suitable for determination by LLME. However, compounds such as lactic acid, amyl hexanoate, oleic acid, capric acid, stearic acid, DL-leucine ethyl ester, linoleic acid, hexyl butyrate, n-heptanol, ethyl laurate, ethyl myristate, sec-octanol, propyl hexanoate, 2,4-di-tert-butylphenol, p-cresol, myristic aldehyde, and stearic aldehyde are difficult to accurately quantify using LLME.

[0090] Example 6: LLME-BSTFA is suitable for the quantification of C3-C20 low-volatile acids and esters in liquor

[0091] Material:

[0092] 90 standard products (hexyl butyrate, ethyl phenylacetate, ethyl propionate, ethyl 3-phenylpropionate, 2,3,5-trimethylpyrazine, ethyl butyrate, benzaldehyde, butyl butyrate, heptanoic acid, isovaleraldehyde, ethyl isovalerate, ethyl isobutyrate, isobutanol, DL-2-methylbutyrate, methyl hexanoate, octyl formate, ethyl acetate, ethyl isohexanoate, phenol, hexanoic acid, furfural, acetic acid, 2,3,5,6-tetramethyl Pyrazine, ethyl hexanoate, ethyl lactate, isopentyl valerate, ethyl decanoate, ethyl valerate, hexyl hexanoate, isophorone, n-hexanol, n-pentanol, isopentyl alcohol, amyl hexanoate, ethyl heptanoate, hexyl acetate, isopentyl butyrate, dimethyl phthalate, ethyl octanoate, butyl hexanoate, octanoic acid, propyl hexanoate, isopentyl hexanoate, ethyl nonanoate, longifolene, myristic aldehyde, ethyl laurate, isovaleric acid, valeric acid , butyl valerate, nonanal, butyric acid, n-hexanal, β-phenylethanol, DL-leucine ethyl ester, sec-octanol, 2-methylbutanal, furfuryl alcohol, 4-methylvaleric acid, 3-hydroxy-2-butanone, lactic acid, ethyl myristate, ethyl palmitate, ethyl stearate, ethyl linoleate, ethyl oleate, methyl palmitate, isopropyl palmitate, propionic acid, isobutyric acid, capric acid, nonanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, oleic acid, diacetyl, 2-pentanone, p-cresol, phenylacetaldehyde, 4-ethylphenol, diethyl phthalate, isoamyl lactate, n-heptanol, dibutyl phthalate, 2,4-di-tert-butylphenol, diisobutyl phthalate, and stearalkanal) were dissolved in 52% ethanol aqueous solution in proportion to prepare a mixed standard stock solution system 1, and the calibration curve was obtained by gradient dilution to determine the samples.

[0093] LLME-GC-MS method parameters:

[0094] Transfer 4 mL of each gradient standard mixture to a 30 mL glass vial and add 100 μL of internal standard (0.0308 g / L heptadecanoic acid). Then, add 14 mL of saturated sodium chloride solution to dilute the liquor to approximately 10%. Extract the diluted sample with 1.5 mL of mixed solution A, vortex for 3 minutes, and let it sit for at least 20 minutes. Transfer the organic layer (containing the acid) to a new test tube. Extract the remaining aqueous phase with 1.5 mL of acetonitrile and then 1.5 mL of dichloromethane. Combine the organic layer with the previous layers, purge to dryness with nitrogen, and then derivatize. Add 50 μL of pyridine (containing 2.5% hydroxylamine hydrochloride, v / v) to dissolve the less volatile compounds. Add 200 μL of BSTFA (Bis(trimethylsilyl)trifluoroacetamide, containing 1% TMCs), cap tightly, and vortex for 5 seconds. The mixture was incubated in a metal bath at 55°C for 5 h. After the reaction, the mixture was centrifuged at 12,000 rpm for 3 min, and the supernatant was transferred to a sample bottle for GC-MS analysis.

[0095] GC-MS was performed using a 7890B gas chromatograph (GC) and a 5977B mass spectrometer (MS) (Agilent Technologies, Inc., USA) using an HP-5MS column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies, Inc., USA). The carrier gas was helium (99.999% purity) at 1.0 mL / min. The sample volume was 1 μL, the inlet temperature was 250°C, and the split ratio was 5:1. The GC temperature program was: 65°C for 2 min, then 6°C / min to 280°C, where it was held for 8 min. The MS was operated in electron impact (EI) mode (70 eV). The transfer line connecting the column and ion source required auxiliary heating and was set to 230°C. Detection was performed in scan mode over the m / z range of 33–350.

[0096] The results are shown in Table 6.

[0097] Table 6 Standard curve of compounds in mixed standard system 1 determined by LLME-BSTFA-GC-MS

[0098] substance <![CDATA[Mixed standard R 2 > Slope intercept LODmg / L LQDmg / L Ethyl linoleate 0.9999 2.1259 -0.0788 0.2202 0.7341 Ethyl oleate 0.9996 1.1044 -0.1219 0.1382 0.4605 Oleic acid 0.9992 0.8066 0.0682 0.2005 0.6682 Ethyl laurate 0.9984 1.2919 0.0615 0.2679 0.8931 β-phenylethanol 0.9971 2.2956 0.7317 0.4562 1.5208 Myristic acid 0.9959 0.8386 -0.1722 0.0348 0.1161 Ethyl myristate 0.9959 1.2508 -0.0165 0.1847 0.6155 Diethyl phthalate 0.9929 1.2349 -1.4262 0.2569 0.8563 Lauric acid 0.9926 0.8183 0.0781 0.1816 0.6052 Linoleic acid 0.992 1.4517 0.0319 0.2068 0.6892 Isopropyl palmitate 0.9917 1.251 0.0354 0.1858 0.6192 bitter 0.9908 1.007 0.3881 0.1302 0.4340 Heptanoic acid 0.9903 1.421 2.1879 0.3733 1.2442 Nonanoic acid 0.9899 0.9684 -0.0667 0.0703 0.2342 Decanoic acid 0.9886 0.6799 -0.1475 0.0639 0.2130 lactic acid 0.9876 42.781 -44.677 1.3393 4.4643 Methyl palmitate 0.9876 1.1651 -1.1957 0.0101 0.0337 Palmitic acid 0.9863 0.8458 -24.782 0.0100 0.0334 Ethyl stearate 0.9836 1.2604 0.0348 0.2100 0.7000

[0099] As shown in Table 6, the standards of the above 19 compounds are relatively suitable for quantification using LLME-BSTFA. However, volatile compounds and compounds that cannot be derivatized with BSTFA cannot be quantified using LLME-BSTFA.

[0100] Example 7

[0101] In order to better and objectively understand the similarity of compounds in samples of different ages and reduce the computational complexity of year identification, this example analyzed 54 samples (the aging time of each sample is shown in Figure 2). Figure 1 The data on the horizontal axis are all clustered using the unsupervised hierarchical clustering algorithm (HCA), and the results are as follows Figure 2 The PCA analysis results of the compound content were highly consistent with the clustering results. Therefore, in this example, the samples were divided into four groups based on the 108 compounds. Group I consisted of samples aged for [0, 1) years, while Groups II, III, and IV comprised samples aged for [1, 5), [5, 9), and [9, 11] years, respectively.

[0102] 1. Pretreatment and GC-MS detection methods:

[0103] (1) GC-MS combined with headspace solid phase microextraction (HS-SPME):

[0104] Liquor samples were diluted to a final ethanol content of 8% (6 mL) and placed in a 20 mL glass vial. 2.0 g of NaCl was added. Ethyl lactate and compounds with retention times greater than acetic acid were internally standardized with 2-methylhexanoic acid (10 μL, 14.00 g / L). Alcohols with retention times less than acetic acid were internally standardized with tert-amyl alcohol (10 μL, 8.05 g / L), while other compounds were internally standardized with amyl acetate (10 μL, 10.33 g / L). Volatile compounds were extracted from the sample headspace using a PAL 3 autosampler (Sterus Analytical Instruments, Switzerland) and an SPME fiber (80 μm thick, 10 mm long, DVB / C-WR / PDMS) (Agilent Technologies, USA). The sample was equilibrated at 60°C for 5 min, followed by extraction at 60°C for 40 min.

[0105] GC-MS: Analyses were performed using a 7890B gas chromatograph (GC) and a 5977B mass spectrometer (MS) (Agilent Technologies, Inc., USA) using a DB-WAX (30 m × 0.25 mm × 0.25 μm) column. The carrier gas was helium (99.999% purity) at a flow rate of 1.2 mL / min. The MS was operated in electron impact (EI) mode (70 eV). The transfer line connecting the column and ion source required auxiliary heating, set to 230°C. Detection was performed in scan mode over the m / z 33–350 range. The inlet temperature was 300°C, the desorption time was 15 min, and the split ratio was 4:1. The temperature program started at 50°C, held for 2 min, then increased at 3°C / min to 145°C, and then increased to 235°C at 15°C / min, where it was held for 8 min.

[0106] (2) GC-MS combined with liquid-liquid microextraction (LLME):

[0107] Transfer 4 mL of liquor sample to a 30 mL glass centrifuge tube, add 10 μL of each of three internal standards (10.33 g / L amyl acetate, 14.00 g / L 2-methylhexanoic acid, and 10.22 g / L tert-amyl alcohol) and 14 mL of saturated sodium chloride solution. Then, add 1.5 mL of Solution A (anhydrous ether:pentane = 1:1, v / v) and stir thoroughly for at least 3 minutes. After static separation, collect the upper organic phase. Concentrate the organic phase to 250 μL at room temperature using nitrogen purge.

[0108] GC-MS: An Agilent 7890B-5977B GC-MS was used for the determination. The column was DB-WAX (30 m × 0.25 mm × 0.25 μm, Agilent Technologies). 1 μL of the concentrated organic phase was injected, with a split ratio of 4:1. The GC temperature program was as follows: 35°C for 0.5 min, then 10°C / min to 50°C for 4 min, then 3°C / min to 100°C for 3 min, and finally 3°C / min to 240°C for 23 min. The carrier gas was helium (99.999% purity) at 1.4 mL / min. Other parameters were the same as in 1.(1).

[0109] (3) GC-MS combined with liquid-liquid microextraction (LLME)-BSTFA derivatization:

[0110] Transfer 4 mL of the liquor sample to a 30 mL glass vial and add 100 μL of internal standard (0.0308 g / L heptadecanoic acid). Then, add 14 mL of saturated sodium chloride solution to dilute the liquor to approximately 10%. Extract the diluted sample with 1.5 mL of Solution A, vortex for 3 minutes, and let it sit for at least 20 minutes. Transfer the organic layer (containing the acid) to a new test tube. Extract the remaining aqueous phase with 1.5 mL of acetonitrile and then 1.5 mL of dichloromethane. Combine the organic layer with the previous layers, purge to absolute dryness with nitrogen, and then derivatize. Add 50 μL of pyridine (containing 2.5% hydroxylamine hydrochloride, v / v) to dissolve the less volatile compounds. Add 200 μL of BSTFA (Bis(trimethylsilyl)trifluoroacetamide, containing 1% TMCs), cap tightly, and vortex for 5 seconds. The mixture was incubated in a metal bath at 55°C for 5 h. After the reaction, the mixture was centrifuged at 12,000 rpm for 3 min, and the supernatant was transferred to a sample bottle for GC-MS analysis.

[0111] GC-MS: The chromatographic column was an HP-5MS (30 m × 0.25 mm × 0.25 μm, Agilent Technologies, Inc., USA). The sample volume was 1 μL, the inlet temperature was 250°C, and the split ratio was 5:1. The GC temperature program was: 65°C for 2 min, then 6°C / min to 280°C, and then held for 8 min. The carrier gas was helium (99.999% purity) at 1.0 mL / min. Other parameters were the same as in 1.(1).

[0112] 2. Detection limit, quantification limit and precision:

[0113] The limit of detection (LOD) and limit of quantification (LOQ) were the concentrations corresponding to signal-to-noise ratios of 3 and 10, respectively. Intra-day precision was calculated based on the results of a mixed standard assay that was closest to the actual concentration of liquor. The assay was repeated three times within a day, and inter-day precision was calculated after repeated assays on three different days. Precision was calculated using the mean, standard deviation, and relative standard deviation (%) of the measured values.

[0114] 3. Qualitative analysis of compounds:

[0115] The identification of volatile compounds is based on two or three qualitative methods. One is to compare with the NIST17 database (MS), and the similarity should be greater than 80. One is to compare the retention index (RI) of the compound to be identified with the retention index (RIs) of the standard, and the other is to compare the RI with the retention index (RIs) of the compound in the reference literature. lit ) for comparison, the difference in retention index should be less than 30.

[0116] Specific operation: The samples were measured using a variety of extraction and pretreatment methods such as HS-SPME, LLME, and LLME-BSTFA derivatization. Then, the compounds were qualitatively identified based on their NIST library matching and RI similarity. The RI values ​​of 98 compounds were referenced to the standard, and the other compounds were referenced to the literature, and 234 compounds were preliminarily identified. Next, the modified "50% rule" was used for data processing. When the detection rate of a compound in all samples was less than 50%, the compound was removed from the data set. Therefore, a total of 108 compound data sets were obtained, such as Figure 2 shown.

[0117] 4. Quantitative analysis of compounds:

[0118] A mixed standard solution was prepared by dissolving 91 standard compounds in a 52% ethanol solution of chromatographic-grade ethanol and ultrapure water. Six concentrations of mixed standard stock solutions were then serially diluted. The proportions of each compound should approximate those in the liquor sample. The standard curve was assayed using the same method as the sample. An internal standard calibration curve was constructed, with the abscissa and ordinate representing the peak area ratio and mass concentration ratio of the target compound to the corresponding internal standard, respectively.

[0119] Since the aged liquor samples analyzed in this example are rare, LLME with a minimum sample volume (4 mL) was used. In addition, the LLME-BSTFA derivatization method was used to determine the non-volatile acids in the liquor. Water can affect the derivatization efficiency of BSTFA. In this case, LLME combined with nitrogen drying was used to provide anhydrous conditions for BSTFA derivatization, which saves more time than rotary evaporation to remove water. The LLME-BSTFA-GC-MS method can accurately quantify ethyl laurate, ethyl myristate, ethyl hexadecanoate, ethyl stearate, ethyl oleate, lauric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, etc.

[0120] The standard curves of 73 compounds were established using mixed standards (see Table 7 ), and the relative quantification of the other 35 compounds using internal standards (see Table 8 ).

[0121] Table 7 Quantitative standard curves of three pretreatment methods

[0122]

[0123]

[0124]

[0125]

[0126] Note: a indicates that amyl acetate was used as the internal standard, b indicates that 2-methylhexanoic acid was used as the internal standard, c indicates that tert-amyl alcohol was used as the internal standard, d indicates that heptadecanoic acid was used as the internal standard, e indicates that HS-SPME was used for pretreatment, f indicates that LLME was used for pretreatment, g indicates that LLME-BSTFA was used for pretreatment, and h indicates that the RSD% was calculated based on the gradient of the standard closest to the actual concentration of liquor.

[0127] Table 8 Corresponding internal standards and qualitative methods for relative quantification of compounds

[0128]

[0129]

[0130] Note: a: IS1 is amyl acetate, IS2 is 2-methylhexanoic acid, IS3 is tert-amyl alcohol, and IS4 is heptadecanoic acid. b: MS, identified by comparison with the NIST spectral library; RI, identified by calculating the compound's retention index (reference values ​​can be found at https: / / webbook.nist.gov / chemistry / ); S, identified by standard sample.

[0131] Example 8: Compound Uniformity Index of Aged Liquor

[0132] Based on Examples 1 to 6, tests were conducted on test compounds suitable for different pretreatment processes to ensure the accuracy of the concentration test results of each compound in the liquor. The concentration results of 108 compounds tested in Example 7 were accurate and could be used to calculate the evenness index of the compounds in the liquor sample.

[0133] Based on the results obtained in Example 7, the evenness index of the compounds in each liquor sample was calculated by chemometrics using the following formula: P i =N i / N, where i is 1, 2, ..., S, S is the total number of compounds in a certain liquor sample in step A, N is the sum of the contents of all compounds in the liquor sample, N i is the content of a certain compound i in the liquor sample. Figure 3 and Table 9.

[0134] Table 9 Uniformity index of compounds in liquor samples of different years

[0135] Age (months) Age (years) Uniformity Index Age (months) Age (years) Uniformity Index 4.5 0.38 0.555880 57.5 4.79 0.557221 7.0 0.58 0.550408 62.0 5.17 0.569318 10.0 0.83 0.552593 67.5 5.63 0.570448 20.0 1.67 0.558795 81.0 6.75 0.566001 20.5 1.71 0.557142 98.0 8.17 0.567247 32.0 2.67 0.572681 109.0 9.08 0.567247 35.5 2.96 0.566869 115.0 9.58 0.584498 43.5 3.63 0.570026 118.0 9.83 0.588906 45.0 3.75 0.577795 128.0 10.67 0.585145

[0136] The results showed that the uniformity index of compounds in liquor was linearly positively correlated with aging time (R 2 =0.7012), increasing from 0.55 to 0.59. The uniformity of the compounds tends to increase with aging. Therefore, the uniformity index was used as an important input feature for vintage identification in subsequent analyses.

Claims

1. A method for analyzing the concentration of compounds in liquor, characterized by: The following steps are involved: A. Pretreatment: Liquor samples were pretreated using headspace solid phase microextraction, liquid-liquid microextraction, and liquid-liquid microextraction-BSTFA derivatization; B. GC-MS determination: GC-MS was used to detect the pretreated samples to obtain the concentration of compounds in the liquor; The target compounds were hexyl butyrate, ethyl phenylacetate, ethyl propionate, ethyl 3-phenylpropionate, 2,3,5-trimethylpyrazine, ethyl butyrate, benzaldehyde, butyl butyrate, isovaleraldehyde, ethyl isovalerate, ethyl isobutyrate, isobutanol, DL-2-methylbutyrate, methyl hexanoate, octyl formate, ethyl acetate, ethyl isocaproate, phenol, furfural, 2,3,5,6-tetramethylpyrazine, ethyl hexanoate, ethyl lactate, isopentyl valerate, ethyl valerate, isophorone, n-hexanol, n-pentanol, isopentanol, hexane Headspace solid phase microextraction was used to pretreat the liquor samples, including amyl heptanoate, ethyl heptanoate, hexyl acetate, isoamyl butyrate, longifolene, heptanoic acid, hexanoic acid, acetic acid, ethyl decanoate, hexyl hexanoate, dimethyl phthalate, ethyl octanoate, butyl hexanoate, octanoic acid, propyl hexanoate, isoamyl hexanoate, ethyl nonanoate, myristic aldehyde, ethyl laurate, isovaleric acid, valeric acid, butyl valerate, nonanal, butyric acid, n-hexanal, β-phenylethanol, DL-leucine ethyl ester, sec-octanol, 2-methylbutanal, isobutyl hexanoate and p-cresol. The target detection compounds are ethyl isohexanoate, ethyl decanoate, n-hexanol, hexyl acetate, ethyl valerate, ethyl hexanoate, phenylacetaldehyde, ethyl butyrate, isopentanol, diisobutyl phthalate, butyl valerate, ethyl propionate, butyric acid, nonanal, ethyl heptanoate, ethyl phenylacetate, 2-pentanone, lauric acid, ethyl nonanoate, ethyl linoleate, hexyl hexanoate, ethyl isovalerate, propionic acid, isobutyric acid, longifolene, ethyl DL-2-methylbutyrate, ethyl oleate, furfuryl alcohol, Isoamyl lactate, acetic acid, isophorone, ethyl octanoate, furfural, ethyl isobutyrate, dimethyl phthalate, isoamyl valerate, hexanoic acid, methyl palmitate, butyl hexanoate, methyl hexanoate, ethyl 3-phenylpropionate, dibutyl phthalate, 4-ethylphenol, 4-methylvaleric acid, diethyl phthalate, 2,3,5,6-tetramethylpyrazine, heptanoic acid, ethyl palmitate, isobutyl alcohol, isopropyl palmitate, benzaldehyde, 2-methylbutanal, ethyl Ethyl acetate, valeric acid, isopentyl hexanoate, phenol, ethyl lactate, ethyl stearate, diacetyl, isovaleraldehyde, isovaleric acid, octanoic acid, butyl butyrate, palmitic acid, isopentyl butyrate, octyl formate, β-phenylethanol, acetoin (3-hydroxy-2-butanone), myristic acid, 2,3,5-trimethylpyrazine, hexanal, n-pentanol, nonanoic acid, 2-butanol, 2-pentanol, 2-octanol, ethyl acrylate, ethyl crotonate, (S)-2-hydroxy-3- When methyl methylbutyrate, ethyl 2-furoate, ethyl 5-hexenoate, ethyl 2-hexenoate, diethyl succinate, propyl hexanoate, hexyl butyrate, diethyl azelaic acid, ethyl 9-hexadecenoate, methyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate, myristic aldehyde, stearic aldehyde, methyl stearate, 2-ethylhexylvalerate, ethyl furfuryl ether and 4-methyl-2-pentanol were used, the liquor samples were pretreated by liquid-liquid microextraction; When the target compounds were ethyl linoleate, ethyl oleate, oleic acid, ethyl laurate, β-phenylethanol, myristic acid, ethyl myristate, diethyl phthalate, lauric acid, linoleic acid, isopropyl palmitate, octanoic acid, heptanoic acid, nonanoic acid, decanoic acid, lactic acid, methyl palmitate, palmitic acid, ethyl stearate, dioctyl phthalate, crotonic acid, benzoic acid, phenylacetic acid, 2-hydroxy-4-methylvaleric acid, 3-phenyllactic acid, 2-ketovaleric acid, 2,4-di-tert-butylphenol, 2,6-di-tert-butyl-4-methylphenol, and oleamide, the liquor samples were pretreated by liquid-liquid microextraction-BSTFA derivatization. In step A, The headspace solid phase microextraction operation is as follows: the liquor sample is diluted to a final ethanol content of 5-15 vol%, NaCl is added, ethyl lactate and compounds with a retention time greater than acetic acid are all treated with 2-methylhexanoic acid as internal standards, alcohols with a retention time less than acetic acid are treated with tert-amyl alcohol as internal standards, and other compounds are treated with amyl acetate as internal standards, volatile compounds are extracted from the sample headspace using an SPME fiber, the sample is pre-equilibrated at 45-60°C for 0-10 minutes, then extracted at 45-60°C for 30-60 minutes, and subsequently subjected to GC-MS analysis; The liquid-liquid microextraction process is as follows: three internal standards, namely amyl acetate, 2-methylhexanoic acid and tert-amyl alcohol, and a saturated sodium chloride solution are added to the liquor sample, followed by a mixed solution of anhydrous ether and pentane, and the mixture is stirred for more than 3 minutes. After standing and stratification, the upper organic phase is collected, and the organic phase is concentrated by nitrogen purge at room temperature before subsequent GC-MS analysis. The liquid-liquid microextraction-BSTFA derivatization operation is as follows: add heptadecanoic acid as an internal standard to the liquor sample, then add saturated sodium chloride solution and dilute to an ethanol concentration of 10%. Add a mixed solution of anhydrous ether and pentane to the diluted system for extraction, vortex for more than 3 minutes, let it stand for more than 20 minutes, collect the organic layer, and extract the remaining aqueous phase with acetonitrile and dichloromethane in sequence. Combine the organic phases, add pyridine containing 2.5% v / v hydroxylamine hydrochloride to the organic phase, add BSTFA containing 1% TMCs, vortex for more than 5 seconds, incubate in a metal bath at 45-85°C for 1-5 hours, centrifuge at 6000-12000 rpm for more than 3 minutes, and collect the supernatant for subsequent GC-MS analysis. In step B, When using headspace solid-phase microextraction, the GC-MS conditions were as follows: DB-WAX column; carrier gas: 99.999% pure helium at 0.8–1.4 mL / min; electron impact mode: 70 eV; transfer line temperature at the connection between the column and the ion source set at 200–230°C; scan mode, scanning range: m / z 33–350; inlet temperature: 250–300°C; desorption time: 5–15 min; split ratio: 2–20:1; temperature program: starting from 50°C and increasing in stages to 235°C, maintained for more than 2 min; When using liquid-liquid microextraction, the GC-MS conditions were as follows: DB-WAX column; carrier gas 0.8-1.4 mL / min of 99.999% pure helium; electron impact mode: 70 eV; transfer line temperature at the connection between the column and the ion source set to 200-230°C; scan mode, scanning range m / z 33-350; sample volume 1 μL, injection port temperature 250-300°C, split ratio 2-20:1; temperature program: starting from 35°C and increasing in stages to 235°C and holding for more than 10 minutes; When using liquid-liquid microextraction-BSTFA derivatization, the GC-MS conditions were as follows: HP-5MS column; carrier gas: 99.999% pure helium at 0.8-1.4 mL / min; electron impact mode: 70 eV; the transfer line temperature at the connection between the chromatographic column and the ion source was set to 200-230°C; scan mode, scanning range: m / z 33-350; sample volume: 1 μL, injection port temperature: 200-250°C, split ratio: 2-20:1; heating program: gradually increase the temperature from 65°C to 280°C and maintain for more than 2 minutes.

2. The method for analyzing compound concentrations in liquor according to claim 1, wherein: In step B, 73 compounds were quantified using a mixed standard to establish a standard curve, and the remaining 35 compounds were quantified using an internal standard: The target compounds for GC-MS detection were isobutanol, n-pentanol, furfuryl alcohol, n-hexanol, β-phenylethanol, ethyl acetate, ethyl hexanoate, ethyl lactate, ethyl butyrate, ethyl heptanoate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, ethyl palmitate, ethyl linoleate, ethyl oleate, ethyl stearate, ethyl laurate, ethyl myristate, acetic acid, butyric acid, hexanoic acid, heptanoic acid, isopentanol, ethyl propionate, ethyl isobutyrate, ethyl valerate, ethyl isovalerate, DL-leucine ethyl ester, amyl hexanoate, DL-2-methylbutyrate, methyl hexanoate, ethyl 3-phenylpropionate, phenol, hexyl hexanoate, dibutyl phthalate, diisobutyl phthalate, palmitic acid, lactic acid, octanoic acid, nonanoic acid, 2,3,5-trimethylpyrazine, decanoic acid, lauric acid, myristic acid, and linoleic acid. , oleic acid, stearic acid, n-hexanal, 3-methylbutyraldehyde, furfural, 2-methylbutyraldehyde, benzaldehyde, nonanal, 3-hydroxy-2-butanone, 2-pentanone, isophorone, 4-ethylphenol, 2,3,5,6-tetramethylpyrazine, longifolene, butyl butyrate, isopentyl lactate, isopentyl butyrate, dimethyl phthalate, ethyl 4-methylvalerate, ethyl phenylacetate, propionic acid, isopentyl hexanoate, 4-methylvaleric acid, methyl palmitate, n-valeric acid, isovaleric acid and hexyl acetate, establish an internal standard calibration curve, the abscissa and ordinate are respectively the peak area ratio of the target compound to be measured and the corresponding internal standard substance, and the ordinate is the mass concentration ratio of the target compound to be measured and the corresponding internal standard substance, establish a calibration curve, and obtain the concentration of the target compound through a linear equation; The target compounds for GC-MS detection were 2-butanol, 2-pentanol, 2-octanol, isobutyl hexanoate, ethyl acrylate, ethyl butenoate, (S)-2-hydroxy-3-methylbutanoate, ethyl 2-furoate, ethyl 5-hexenoate, ethyl 2-hexenoate, diethyl succinate, propyl hexanoate, hexyl butanoate, diethyl azelaic acid, ethyl 9-hexadecenoate, dioctyl phthalate, methyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate, isobutyric acid, For butenoic acid, benzoic acid, phenylacetic acid, 2-hydroxy-4-methylvaleric acid, 3-phenyllactic acid, 2-ketovaleric acid, myristic aldehyde, stearalkanal, p-cresol, 2,4-di-tert-butylphenol, 2,6-di-tert-butyl-4-methylphenol, oleamide, methyl stearate, 2-ethylhexylvalerate, ethylfurfuryl ether, butyl hexanoate, and 4-methyl-2-pentanol, amyl acetate, 2-methylhexanoic acid, tert-amyl alcohol, or heptadecanoic acid was used as the internal standard to obtain the relative concentrations of the target compounds.

3. The method for analyzing compound concentrations in liquor according to claim 1 or 2, characterized in that: In step B, the compounds in the sample are first qualitatively analyzed, and then the target compounds are quantitatively analyzed.

4. A method for analyzing the uniformity of compound concentration in liquor, characterized by: On the basis of any one of claims 1 to 3, the method further comprises the following steps: C. Calculate the evenness index of the compounds in the liquor sample by chemometrics. The calculation formula is: Evenness index = , , where i is 1, 2, ... S, S is the total number of compounds in the liquor sample in step A, N is the sum of the contents of all compounds in the liquor sample, N i is the content of a certain compound i in the liquor sample.

5. The method for analyzing compound concentration in liquor according to claim 1 or the method for analyzing compound concentration uniformity in liquor according to claim 4, characterized in that: In step A, the liquor sample is a Luzhou-flavor liquor.

6. The method according to claim 5, characterized in that: In step A, the aging time of the Luzhou-flavor liquor is 0 to 11 years.

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