Detection method for simultaneously detecting 27 pyrazine compounds in Maotai-flavor liquor

Abstract

The invention relates to the technical field of detection, and provides a detection method for simultaneously detecting 27 pyrazine compounds in Maotai-flavor liquor, which comprises the following steps: adjusting the pH value of a liquor sample, diluting the liquor sample, separating and extracting with a solvent, and finally detecting the sample by adopting a gas chromatography-tandem mass spectrometry dynamic multi-reaction detection mode. The pretreatment mode of the sample is screened and optimized, and the 27 pyrazine compounds in the Maotai-flavor liquor are measured at the same time. The method has the advantages that the pretreatment operation is simple, the solvent dosage is small, the detection cost is reduced, the environmental pollution is reduced, the advantages of high detection flux, high sensitivity, high accuracy and high resolution are achieved, and the 27 pyrazine compounds in the Maotai-flavor liquor can be measured at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of detection, and in particular to a detection method for simultaneously detecting 27 pyrazine compounds in Maotai-flavor Baijiu (Chinese liquor). Background Art

[0002] Pyrazine compounds are a very important class of trace components in Maotai-flavor Baijiu that possess both flavor and functional properties, and are mainly produced from brewing raw materials, microbial metabolism, and the Maillard reaction. Most pyrazine compounds have a pleasant aroma similar to roasted nuts and grilled meat, and have extremely low flavor thresholds, which can significantly enhance other flavor substances, making the aroma of Baijiu more abundant and contributing importantly to the flavor of Baijiu. At the same time, pyrazine compounds are also a very important class of functional active substances in Maotai-flavor Baijiu, with rich physiological activities and health-care functions. For example, tetramethylpyrazine can dilate blood vessels, inhibit platelet aggregation, improve microcirculation, and can also prevent cardiovascular diseases, ischemic stroke, cancer, diabetes, etc. Maotai-flavor Baijiu is mostly brewed with high-temperature Daqu (a type of starter culture) with a fermentation temperature exceeding 60 °C. Due to the rich variety of microorganisms and the Maillard reaction, the types and contents of pyrazine compounds in Maotai-flavor Baijiu are significantly higher than those in other types of Baijiu.

[0003] Currently, the detection methods for pyrazine compounds in Maotai-flavor Baijiu mainly focus on tetramethylpyrazine and cannot meet the need for synchronous analysis of multiple target substances. For example, Huang Jialing et al. used solid-phase extraction combined with GC-MS / MS to detect 12 pyrazines, but key flavor substances such as 2-isopropyl-3-methoxypyrazine were not covered; Sun Di et al. used LC-MS / MS to detect 8 pyrazines. Due to the low electrospray ionization efficiency (<30%), trace detection of chlorinated pyrazines (such as 3-chloro-2,5-dimethylpyrazine) could not be achieved. Moreover, the solid-phase extraction method requires multiple column activations and elutions, which not only takes a long time but also has a relatively high sample loss rate. At the same time, the detection limits of existing methods for trace pyrazines are generally higher than 1 μg / L, making it difficult to meet the accurate quantification requirements of ultra-trace components in actual Maotai-flavor Baijiu samples.

[0004] In addition, the types of pyrazine compounds in Maotai-flavor Baijiu are strongly related to the brewing process (for example, the high-temperature Daqu process is prone to generating tetramethylpyrazine isomers). Existing technologies cannot distinguish isomers such as 2,3-diethyl-5-methylpyrazine and 2-ethyl-3,5-dimethylpyrazine due to insufficient separation, resulting in a lack of data support for flavor tracing and process optimization. Therefore, there is an urgent need to develop a synchronous detection method for pyrazine compounds with wide coverage, high sensitivity, and simple pretreatment to solve the technical bottlenecks in the quality control and flavor analysis of Maotai-flavor Baijiu. Summary of the Invention

[0005] In view of this, the present invention proposes a detection method for simultaneously detecting 27 pyrazine compounds in Maotai-flavor Baijiu.

[0006] The technical solution of the present invention is implemented as follows: The present invention provides a detection method for simultaneously detecting 27 pyrazine compounds in Maotai-flavor liquor, which includes: concentrating and removing impurities from the sample by liquid-liquid microextraction, detecting by the dynamic multiple reaction monitoring mode of gas chromatography-tandem mass spectrometry, and quantifying by the internal standard standard curve method to calculate the content of pyrazine compounds.

[0007] In some embodiments, it specifically includes the following steps:

[0008] Step 1: Take the pyrazine compound to be measured, dissolve it with absolute ethanol, and prepare a single standard stock solution;

[0009] Step 2: Take 2-methoxy-3-methylpyrazine, dissolve it with dichloromethane, and prepare an internal standard working solution;

[0010] Step 3: Take the pyrazine compound standard stock solution in Step 1 and prepare a series of gradient standard mixed solutions with 53% vol ethanol aqueous solution;

[0011] Step 4: Take the liquor sample to be measured and the series of standard mixed solutions in Step 3, add the internal standard working solution in Step 2 and mix well, add sodium hydroxide solution to adjust the pH of the solution to 9-10, add ultrapure water to make the ethanol concentration in the to-be-detected standard mixed solution and the liquor sample 5-15%, and add an appropriate amount of sodium chloride to make the solution reach a saturated state;

[0012] Step 5: Add dichloromethane as an extraction agent to the solution in Step 4, mix well, shake, ultrasonicate, centrifuge, and refrigerate at 4°C and let stand overnight to separate the aqueous phase and the organic phase;

[0013] Step 6: Take the organic phase in Step 5, dehydrate it with anhydrous sodium sulfate, and then detect it by gas chromatography-tandem mass spectrometry to obtain the peak areas of pyrazine compounds and the internal standard;

[0014] Step 7: According to the detection results of the series of gradient standard mixed solutions in Step 6, take the ratio of the peak area of pyrazine compounds to the peak area of the internal standard as the ordinate, and the ratio of the content of pyrazine compounds to the content of the internal standard in the series of standard mixed solutions as the abscissa to draw a standard curve;

[0015] Step 8: Substitute the peak areas of pyrazine compounds and the internal standard in the liquor sample to be measured in Step 6 into the standard curve made in Step 7 to calculate the content of pyrazine compounds in the liquor sample to be measured.

[0016] By adjusting the liquor sample to pH 8-11, preferably 9-10, pyrazine compounds are more soluble in the organic phase in a weakly alkaline environment; adding sodium chloride, using the salting-out effect to reduce the water solubility of pyrazine compounds, and combining with the hydrophobicity of dichloromethane to achieve efficient extraction.

[0017] The wine sample is diluted to an ethanol concentration of 5-15% vol, preferably 10% vol, which can not only avoid the inhibition of extraction efficiency by high-concentration ethanol but also prevent the loss of target substances caused by excessive dilution.

[0018] In some embodiments, the target pyrazine compounds to be measured in step one include pyrazine, 2-methylpyrazine, 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, 2-ethylpyrazine, 2,3-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-5-methylpyrazine, trimethylpyrazine, 2-ethyl-3-methylpyrazine, 2-methyl-5-isopropylpyrazine, 2-methoxy-6-methylpyrazine, 2-isopropyl-3-methoxypyrazine, 2,6-diethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, 2,3-diethylpyrazine, 2,3-dimethyl-5-ethylpyrazine, tetramethylpyrazine, 2,3-diethyl-5-methylpyrazine, 2-vinyl-6-methylpyrazine, 2-sec-butyl-3-methoxypyrazine, 2-isobutyl-3-methylpyrazine, 3-chloro-2,5-dimethylpyrazine, methyl pyrazine-2-carboxylate, 2-isobutyl-3,5,6-trimethylpyrazine, 2-acetyl-3,5-dimethylpyrazine, pyrazinecarboxamide.

[0019] In some embodiments, the concentration of the internal standard working solution in step two is 100-500 mg / L.

[0020] In some embodiments, the addition amount of sodium chloride in step four is 30-40% w / v of the aqueous phase.

[0021] In some embodiments, the chromatographic conditions of gas chromatography-tandem mass spectrometry in step six are as follows: a polyethylene glycol capillary chromatographic column modified with nitroterephthalic acid (DB-FFAP: 60 m × 0.25 mm × 0.25 μm), the inlet temperature is 250 °C, the carrier gas is high-purity helium (purity ≥ 99.999%), injection is performed in splitless constant flow mode, the flow rate is 1 mL / min, and the injection volume is 1 μL.

[0022] The polyethylene glycol capillary chromatographic column modified with nitroterephthalic acid has strong polar characteristics, which can effectively separate pyrazine isomers.

[0023] In some embodiments, in step six, the temperature programming is as follows: the initial temperature is 40 °C, hold for 1 min, increase the temperature to 90 °C at a rate of 10 °C / min, hold for 2 min, increase the temperature to 140 °C at a rate of 3.5 °C / min, increase the temperature to 220 °C at a rate of 8 °C / min, hold for 4 min, increase the temperature to 250 °C at a rate of 15 °C / min, and hold for 2 min.

[0024] Through a multi-stage gradient temperature programming, 27 compounds can be completely eluted within 60 min without co-elution.

[0025] In some embodiments, the tandem mass spectrometry conditions are as follows: the ionization method is electron impact ionization source (EI), with an energy of 70 eV; the ion source temperature is 230 °C; the temperature of the first quadrupole is 150 °C; the temperature of the third quadrupole is 150 °C; the auxiliary channel heating temperature is 280 °C; the mass spectrometry scanning mode is the dynamic multiple reaction monitoring mode (dMRM); the mass spectrometry analysis uses the NIST20 spectral library.

[0026] For each pyrazine compound, ≥2 pairs of characteristic ion pairs are set. Combined with the optimization of the collision energy, the detection limit is low, and the sensitivity is improved compared with the existing LC-MS / MS method.

[0027] 2-Methoxy-3-methylpyrazine is selected as the internal standard. Its chemical structure is similar to that of the target compound but the retention time difference is significant, which can effectively offset the pretreatment error; the concentration range of the internal standard is 100 - 500 mg / L, and when the concentration is preferably 250 mg / L, the RSD of the quantitative result is lower.

[0028] In some embodiments, the calculation formula for pyrazine compounds in step eight is as follows:

[0029]

[0030] In the formula

[0031] Xi: the content of the i-th pyrazine compound in the sample to be measured, in micrograms per liter (μg / L);

[0032] Xf: the content of the internal standard 2-methoxy-3-methylpyrazine in the sample to be measured, in micrograms per liter (μg / L);

[0033] Ai: the chromatographic peak area of the i-th pyrazine compound in the sample to be measured;

[0034] Af: the chromatographic peak area of the internal standard 2-methoxy-3-methylpyrazine in the sample to be measured;

[0035] b: the intercept value of the standard curve;

[0036] k: the slope of the standard curve;

[0037] K: the dilution factor in step four.

[0038] The liquid-liquid microextraction method adopted in the present invention can reduce the influence of complex matrix in the sample on the detection result, and at the same time reduce the consumption of organic solvents. Only a small amount of organic solvents are required to efficiently extract pyrazine compounds in the wine sample, thereby effectively reducing the detection cost and significantly reducing the potential pollution to the environment. The pretreatment process is simple and fast, effectively improving the detection efficiency and accuracy.

[0039] The present invention adopts an internal standard method combined with the dynamic multiple reaction monitoring mode of gas chromatography - tandem mass spectrometry for dual correction, with a lower detection limit, and at the same time, the data accuracy and repeatability are significantly improved.

[0040] The present invention can simultaneously detect 27 pyrazine compounds in Maotai - flavor Baijiu, effectively improving the detection throughput, further expanding the research scope of pyrazine compounds, and having good detection repeatability, high accuracy, and reliable results.

[0041] The present invention has the following beneficial effects compared with the prior art:

[0042] The present invention first uses the liquid - liquid micro - extraction technology to extract and separate pyrazine compounds in Maotai - flavor Baijiu, reducing the interference of irrelevant matrices. And during the extraction process, only a small amount of organic solvent is used to effectively extract pyrazine compounds in the wine sample, greatly reducing the amount of organic solvent used, reducing the detection cost and environmental pollution. It uses the dynamic multiple reaction monitoring mode of gas chromatography - tandem mass spectrometry for detection, significantly improving the detection sensitivity and resolution, effectively reducing the interference of impurity peaks on the detection results, and greatly improving the detection accuracy. The internal standard curve method is used for quantification, eliminating the errors caused by changes in operating conditions during the sample pretreatment process, and can also effectively offset the result errors caused by sample matrix differences, making the quantitative results more accurate and reliable. The present invention can simultaneously detect 27 pyrazine compounds in Maotai - flavor Baijiu, with the advantages of high throughput, simple pretreatment, less interference, low detection limit, good repeatability, and high accuracy, and the detection limit is 0.03 μg / L - 1.10 μg / L. Description of the Drawings

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0044] Figure 1 It is the chromatogram of the mixed standard solution of 27 pyrazine compounds and the internal standard in Example 1 of the present invention;

[0045] Figure 2 It is the standard curve of pyrazine in Example 1 of the present invention;

[0046] Figure 3 It is the standard curve of 2 - methylpyrazine in Example 1 of the present invention;

[0047] Figure 4 It is the standard curve of 2,5 - dimethylpyrazine in Example 1 of the present invention;

[0048] Figure 5 It is the standard curve graph of 2,6-dimethylpyrazine in Example 1 of the present invention;

[0049] Figure 6 It is the standard curve graph of 2-ethylpyrazine in Example 1 of the present invention;

[0050] Figure 7 It is the standard curve graph of 2,3-dimethylpyrazine in Example 1 of the present invention;

[0051] Figure 8 It is the standard curve graph of 2-ethyl-6-methylpyrazine in Example 1 of the present invention;

[0052] Figure 9 It is the standard curve graph of 2-ethyl-5-methylpyrazine in Example 1 of the present invention;

[0053] Figure 10 It is the standard curve graph of trimethylpyrazine in Example 1 of the present invention;

[0054] Figure 11 It is the standard curve graph of 2-ethyl-3-methylpyrazine in Example 1 of the present invention;

[0055] Figure 12 It is the standard curve graph of 2-methyl-5-isopropylpyrazine in Example 1 of the present invention;

[0056] Figure 13 It is the standard curve graph of 2-methoxy-6-methylpyrazine in Example 1 of the present invention;

[0057] Figure 14 It is the standard curve graph of 2-isopropyl-3-methoxypyrazine in Example 1 of the present invention;

[0058] Figure 15 It is the standard curve graph of 2,6-diethylpyrazine in Example 1 of the present invention;

[0059] Figure 16 It is the standard curve graph of 2-ethyl-3,5-dimethylpyrazine in Example 1 of the present invention;

[0060] Figure 17 It is the standard curve graph of 2,3-diethylpyrazine in Example 1 of the present invention;

[0061] Figure 18 It is the standard curve graph of 2,3-dimethyl-5-ethylpyrazine in Example 1 of the present invention;

[0062] Figure 19 It is the standard curve graph of tetramethylpyrazine in Example 1 of the present invention;

[0063] Figure 20Standard curve graph of 2,3 - diethyl - 5 - methylpyrazine in Example 1 of the present invention;

[0064] Figure 21 Standard curve graph of 2 - vinyl - 6 - methylpyrazine in Example 1 of the present invention;

[0065] Figure 22 Standard curve graph of 2 - sec - butyl - 3 - methoxypyrazine in Example 1 of the present invention;

[0066] Figure 23 Standard curve graph of 2 - isobutyl - 3 - methylpyrazine in Example 1 of the present invention;

[0067] Figure 24 Standard curve graph of 3 - chloro - 2,5 - dimethylpyrazine in Example 1 of the present invention;

[0068] Figure 25 Standard curve graph of methyl pyrazine - 2 - carboxylate in Example 1 of the present invention;

[0069] Figure 26 Standard curve graph of 2 - isobutyl - 3,5,6 - trimethylpyrazine in Example 1 of the present invention;

[0070] Figure 27 Standard curve graph of 2 - acetyl - 3,5 - dimethylpyrazine in Example 1 of the present invention;

[0071] Figure 28 Standard curve graph of pyrazinecarboxamide in Example 1 of the present invention;

[0072] Figure 29 Chromatogram of the wine sample to be measured 1 in Example 1 of the present invention;

[0073] Figure 30 Chromatogram of the accuracy experiment of the wine sample to be measured in Example 1 of the present invention;

[0074] Figure 31 Chromatogram of the wine sample to be measured 2 in Example 2 of the present invention. Detailed implementation manners

[0075] Next, in combination with the implementation manners of the present invention, the technical solutions in the implementation manners of the present invention will be clearly and completely described. Obviously, the described implementation manners are only a part of the implementation manners of the present invention, rather than all of the implementation manners. Based on the implementation manners in the present invention, all other implementation manners obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments of the present invention belong. If the definitions stated in this section are contrary to or otherwise inconsistent with the definitions stated in the patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions listed in this section shall prevail over the definitions incorporated herein by reference.

[0077] Unless otherwise specified, the methods used in the following embodiments are all conventional methods. The materials, reagents, and instruments used, unless otherwise specified, are all conventional materials, reagents, and instruments in this field, and those skilled in the art can obtain them through commercial channels.

[0078] When an equivalent, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values and lower preferred values, it should be understood that all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value are specifically disclosed, regardless of whether the ranges are separately disclosed. For example, when the range "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its endpoint values and all integers and fractions within the range. In the specification and claims of this application, range limitations can be combined and / or interchanged, and if not otherwise stated, these ranges include all sub-ranges contained therein.

[0079] Example 1

[0080] This example provides a method for detecting the concentrations of 27 pyrazine compounds in Maotai-flavor Baijiu, specifically as follows:

[0081] 1. Reagents:

[0082] Absolute ethanol and dichloromethane are of chromatographic grade; water is ultrapure water; anhydrous sodium sulfate and sodium chloride are of superior grade pure.

[0083] 2. Samples to be tested and standards:

[0084] Sample to be tested 1: Shennongjia Maotai-flavor Baijiu, with an alcohol content of 53% vol, provided by Jinpai Co., Ltd.

[0085] Pyrazine, 2-methylpyrazine, 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, 2-ethylpyrazine, 2,3-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-5-methylpyrazine, trimethylpyrazine, 2-ethyl-3-methylpyrazine, 2-methyl-5-isopropylpyrazine, 2-methoxy-6-methylpyrazine, 2-isopropyl-3-methoxypyrazine, 2,6-diethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, 2,3-diethylpyrazine, 2,3-dimethyl-5-ethylpyrazine, tetramethylpyrazine, 2,3-diethyl-5-methylpyrazine, 2-vinyl-6-methylpyrazine, 2-sec-butyl-3-methoxypyrazine, 2-isobutyl-3-methylpyrazine, 3-chloro-2,5-dimethylpyrazine, methyl pyrazine-2-carboxylate, 2-isobutyl-3,5,6-trimethylpyrazine, 2-acetyl-3,5-dimethylpyrazine, pyrazinamide, 2-methoxy-3-methylpyrazine reference standards, with purity greater than 98.0%;

[0086] 3. Preparation of standard solution:

[0087] 3.1 Preparation of pyrazine compound standard stock solution:

[0088] Precisely weigh the above 27 pyrazine compound reference standards into a volumetric flask, add anhydrous ethanol to make the volume up to 10 mL to obtain 27 single-standard stock solutions of pyrazine compounds at one time. Then, respectively transfer appropriate amounts of the single-standard stock solutions into volumetric flasks, add anhydrous ethanol to make the volume up to 10 mL to obtain a mixed-standard stock solution of pyrazine compounds, and store it in the dark at -20 °C. The specific concentrations are shown in Table 1.

[0089] 3.2 Internal standard working solution:

[0090] Precisely weigh 2.5 mg of 2-methoxy-3-methylpyrazine reference standard into a 10 mL volumetric flask, dissolve it with anhydrous ethanol and make the volume up to the mark to obtain an internal standard working solution of 250 mg / L, and store it in the dark at -20 °C.

[0091] Table 1: Concentrations of single-standard and mixed-standard pyrazine compounds

[0092]

[0093]

[0094] 4. Sample pretreatment:

[0095] Wine sample extraction and concentration: Take 4 mL of wine sample (53% vol), add 20 μL of internal standard and shake. Add sodium hydroxide solution to adjust the pH of the solution to 9 - 10. Add 16 mL of ultrapure water to reduce the water - ethanol concentration to about 10% vol. Add 6.5 g of sodium chloride (the mass - volume ratio of sodium chloride to water is 0.36:1, that is, 0.36 g of sodium chloride is added to 1 mL of water) until the solution is saturated. Then add 2 mL of dichloromethane as the extraction solvent, shake for 5 min, sonicate for 15 min, centrifuge at 8000 rpm for 5 min, refrigerate at 4 °C and let stand overnight. Take the lower organic phase and dehydrate it with anhydrous Na2SO4. The obtained sample is detected by gas chromatography - tandem mass spectrometry.

[0096] 5. Sample detection

[0097] 5.1 Instrument conditions

[0098] Agilent 8890 - 7000D gas chromatography - tandem mass spectrometry;

[0099] Chromatographic conditions: Use a polyethylene glycol capillary column modified with nitroterephthalic acid (DB - FFAP: 60 m × 0.25 mm × 0.25 μm). The inlet temperature is 250 °C. The carrier gas is high - purity helium (purity ≥ 99.999%). Inject in splitless constant - flow mode with a flow rate of 1 mL / min and an injection volume of 1 μL. The temperature - programming procedure: The initial temperature is 40 °C, hold for 1 min, increase the temperature to 90 °C at a rate of 10 °C / min, hold for 2 min, increase the temperature to 140 °C at a rate of 3.5 °C / min, increase the temperature to 220 °C at a rate of 8 °C / min, hold for 4 min, increase the temperature to 250 °C at a rate of 15 °C / min, and hold for 2 min;

[0100] Tandem mass spectrometry conditions: The ionization method is electron impact ionization source (EI) with an energy of 70 eV; the ion source temperature is 230 °C; the temperature of the first quadrupole is 150 °C; the temperature of the third quadrupole is 150 °C; the auxiliary channel heating temperature is 280 °C; the mass spectrometry scanning mode is dynamic multiple reaction monitoring mode (dMRM); the mass spectrometry analysis uses the NIST20 spectral library.

[0101] Detection method: Dynamic multiple reaction monitoring; The monitored ions are shown in Table 2. First, perform qualitative analysis using the full - scan mode (SCAN) to optimize the instrument parameters, determine the retention times and characteristic ion pairs of each pyrazine compound and the internal standard, and then use the dynamic multiple reaction detection mode (dMRM), as shown in Table 2 specifically.

[0102] Table 2: Mass spectrometry parameters of 27 pyrazine compounds

[0103]

[0104]

[0105]

[0106]

[0107] 5.2 Preparation of Standard Curve

[0108] According to the concentration of the mixed standard compounds shown in "3.1", an appropriate amount of the standard solution was taken and diluted to a constant volume with an ethanol aqueous solution (53% vol) having the same alcohol content as the wine sample to be measured to obtain Standard Sample 1. Then, it was successively diluted 3.3 times to obtain a series of mixed standard solutions. 4 mL of each series of gradient standard mixed solutions was placed in a 50 mL centrifuge tube, and the pH of the solution was adjusted to 9 - 10 with sodium hydroxide solution. 16 mL of ultrapure water was added to reduce the ethanol concentration to about 10% vol. 6.5 g of sodium chloride was added to saturation, and then 2 mL of dichloromethane extractant was added. The mixture was shaken for 5 min, sonicated for 15 min, centrifuged at 8000 rpm for 5 min, refrigerated at 4 °C and allowed to stand overnight. The lower organic phase was taken and dehydrated with anhydrous Na2SO4. The obtained sample was detected by gas chromatography - tandem mass spectrometry. The ratio of the peak area of pyrazine compounds to the peak area of the internal standard was used as the ordinate, and the ratio of the content of pyrazine compounds to the content of the internal standard in the series of standard solutions was used as the abscissa to draw the standard curve. The standard curve parameters of 27 pyrazine compounds are shown in Table 3. The detection limit (LOD) was calculated as the sample content corresponding to the signal intensity / baseline noise (S / N) = 3:1; the quantification limit (LOQ) was calculated as the sample content corresponding to the signal intensity / baseline noise (S / N) = 10:1. The standard curves of 27 pyrazine compounds are respectively as Figures 2 - 28 shown Figure 1 In the figure, the chromatographic peaks numbered 1 - 29 are pyrazine, 2 - methylpyrazine, 2,5 - dimethylpyrazine, 2,6 - dimethylpyrazine, 2 - ethylpyrazine, 2,3 - dimethylpyrazine, 2 - methoxy - 3 - methylpyrazine (internal standard), 2 - ethyl - 6 - methylpyrazine, 2 - ethyl - 5 - methylpyrazine, trimethylpyrazine, 2 - ethyl - 3 - methylpyrazine, 2 - methyl - 5 - isopropylpyrazine, 2 - methoxy - 6 - methylpyrazine, 2 - isopropyl - 3 - methoxypyrazine, 2,6 - diethylpyrazine, 2 - ethyl - 3,5 - dimethylpyrazine, 2,3 - diethylpyrazine, 2,3 - dimethyl - 5 - ethylpyrazine, tetramethylpyrazine, 2,3 - diethyl - 5 - methylpyrazine, 2 - vinyl - 6 - methylpyrazine, 2 - sec - butyl - 3 - methoxypyrazine, 2 - isobutyl - 3 - methylpyrazine, 3 - chloro - 2,5 - dimethylpyrazine, 2 - isobutyl - 3,5,6 - trimethylpyrazine, 2 - acetyl - 3,5 - dimethylpyrazine I, 2 - acetyl - 3,5 - dimethylpyrazine II, methyl pyrazine - 2 - carboxylate, pyrazinecarboxamide respectively.

[0109] Table 3: Standard Curve Parameters of 27 Pyrazine Compounds

[0110]

[0111]

[0112]

[0113] 5.3 Sample Determination

[0114] The sample to be tested and the standard solution are tested under the same detection conditions. The peak areas A s of the 27 pyrazine compounds and the peak area A i of the internal standard are substituted into the standard curve to calculate the mass concentration of the corresponding pyrazine compound in the sample to be tested. The chromatogram of sample to be tested 1 is as shown in Figure 29 the figure.

[0115] 5.4 Calculation

[0116] The contents of the 27 pyrazine compounds in the sample to be tested are calculated according to the following formula:

[0117]

[0118] In the formula:

[0119] X i --- The content of the i-th pyrazine compound in the sample to be tested, in micrograms per liter (μg / L);

[0120] X f --- The content of the internal standard 2-methoxy-3-methylpyrazine in the sample to be tested, in micrograms per liter (μg / L);

[0121] A i --- The chromatographic peak area of the i-th pyrazine compound in the sample to be tested;

[0122] A f --- The chromatographic peak area of the internal standard 2-methoxy-3-methylpyrazine in the sample to be tested;

[0123] b --- The intercept value of the standard curve;

[0124] k --- The slope of the standard curve;

[0125] K --- The dilution factor.

[0126] The calculation results are expressed as the arithmetic mean of two independent determinations obtained under repeatability conditions, and are retained to 2 decimal places.

[0127] 6. Repeatability Experiment

[0128] Take a total of 6 samples to be tested, labeled 1 - 6, and process the samples according to the methods of "4. Sample Pretreatment" and "5. Sample Detection", determine the pyrazine compound content, and calculate the repeatability of the determination results of each pyrazine compound. The RSD values are all less than 5%, indicating that the method has good repeatability. The specific results are shown in Table 4.

[0129] Table 4: Results of the repeatability experiment data of 27 pyrazine compounds in the samples to be tested

[0130]

[0131]

[0132] Note: " / " indicates not detected.

[0133] 7. Accuracy experiment

[0134] Take 6 samples of the wine to be tested, divide them into 3 groups A, B, and C, with 2 samples in each group, and label them as Group A (Z1 - 1 and Z1 - 2), Group B (Z2 - 1 and Z2 - 2), and Group C (Z3 - 1 and Z3 - 2) respectively; take the mixed standard stock solution of 27 pyrazine compounds in "3.1", add 10 μL to Group A, 20 μL to Group B, and 30 μL to Group C respectively; add 20 μL of the internal standard working solution respectively, and process the samples according to the methods of "4. Sample Pretreatment" and "5. Sample Detection", determine the pyrazine compound content, and the chromatogram of the accuracy experiment of the wine sample to be tested is as Figure 30 shown.

[0135] Substitute the peak areas of the pyrazine compounds and the internal standard into the standard curve, calculate the content and recovery rate of each pyrazine compound. The recovery rates of the 27 pyrazine compounds are all between 90% and 105%, and the RSD values are all less than 10%, indicating that the accuracy of this method is good. The specific calculation results are shown in Table 6.

[0136] Table 6: Results of the accuracy experiment of 27 pyrazine compounds in the wine sample

[0137]

[0138] Example 2

[0139] This example provides a method for detecting the concentration of 27 pyrazine compounds in Maotai-flavor liquor, as follows: 1. Reagents: The same as in Example 1.

[0140] 2. Samples to be tested and standards:

[0141] Sample to be tested 2: Feitian Maotai, alcohol content 53% vol, purchased from the market as a Maotai-flavor liquor;

[0142] Standards: The same as in Example 1.

[0143] 3. Preparation of standard solution: The same as in Example 1.

[0144] 4. Pretreatment of sample: The same as in Example 1.

[0145] 5. Detection of sample: The same as in Example 1.

[0146] The determination conditions and method are the same as those in Example 1, and the chromatogram of the sample to be measured is as shown in Figure 31 The figure. The detection results of 27 pyrazine compounds in the sample to be measured in this example are shown in Table 7.

[0147] Table 7: Detection results of 27 pyrazine compounds in the sample

[0148]

[0149]

[0150] Note: " / " indicates not detected.

[0151] The above is only the preferred embodiment of the present invention, and it is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A detection method for simultaneously detecting 27 pyrazine compounds in Maotai-flavor Baijiu, characterized in that, It includes the following steps: Step 1: Take the pyrazine compound to be tested, dissolve it with absolute ethanol, and prepare a single standard stock solution; Step 2: Take 2-methoxy-3-methylpyrazine, dissolve it with dichloromethane, and prepare an internal standard working solution; Step 3: Take the pyrazine compound standard stock solution in Step 1, and prepare a series of gradient standard mixed solutions with 53% vol ethanol aqueous solution; Step 4: Take the wine sample to be tested and the series of standard mixed solutions in Step 3, add the internal standard working solution in Step 2 and mix well, add sodium hydroxide solution, adjust the pH of the solution to 9 - 10, add ultrapure water to make the ethanol concentration in the tested standard mixed solution and the wine sample 5 - 15%, and add an appropriate amount of sodium chloride to make the solution reach a saturated state; Step 5: Add dichloromethane as an extraction agent to the solution in Step 4, mix evenly, then shake, ultrasonicate, centrifuge, and refrigerate at 4°C and let stand overnight to separate the aqueous phase and the organic phase; Step 6: Take the organic phase in Step 5, dehydrate it with anhydrous sodium sulfate, and then detect it by gas chromatography - tandem mass spectrometry to obtain the peak areas of pyrazine compounds and the internal standard; Step 7: According to the detection results of the series of standard mixed solutions in Step 6, use the ratio of the peak area of pyrazine compounds to the peak area of the internal standard as the ordinate, and the ratio of the content of pyrazine compounds to the content of the internal standard in the series of standard mixed solutions as the abscissa to draw a standard curve; Step 8: Substitute the peak areas of pyrazine compounds and the internal standard in the wine sample to be tested in Step 6 into the standard curve made in Step 7, and calculate the content of pyrazine compounds in the wine sample to be tested.

2. The detection method for simultaneously detecting 27 pyrazine compounds in Maotai-flavor Baijiu as claimed in claim 1, wherein The target pyrazine compounds to be tested in Step 1 include pyrazine, 2 - methylpyrazine, 2,5 - dimethylpyrazine, 2,6 - dimethylpyrazine, 2 - ethylpyrazine, 2,3 - dimethylpyrazine, 2 - ethyl - 6 - methylpyrazine, 2 - ethyl - 5 - methylpyrazine, trimethylpyrazine, 2 - ethyl - 3 - methylpyrazine, 2 - methyl - 5 - isopropylpyrazine, 2 - methoxy - 6 - methylpyrazine, 2 - isopropyl - 3 - methoxypyrazine, 2,6 - diethylpyrazine, 2 - ethyl - 3,5 - dimethylpyrazine, 2,3 - diethylpyrazine, 2,3 - dimethyl - 5 - ethylpyrazine, tetramethylpyrazine, 2,3 - diethyl - 5 - methylpyrazine, 2 - vinyl - 6 - methylpyrazine, 2 - sec - butyl - 3 - methoxypyrazine, 2 - isobutyl - 3 - methylpyrazine, 3 - chloro - 2,5 - dimethylpyrazine, methyl pyrazine - 2 - carboxylate, 2 - isobutyl - 3,5,6 - trimethylpyrazine, 2 - acetyl - 3,5 - dimethylpyrazine, pyrazinecarboxamide.

3. The detection method for simultaneously detecting 27 pyrazine compounds in Maotai-flavor Baijiu as described in claim 1, characterized in that, The concentration of the internal standard working solution in Step 2 is 100 - 500 mg / L.

4. The detection method for simultaneously detecting 27 pyrazine compounds in Maotai-flavor Baijiu as described in claim 1, wherein The addition amount of sodium chloride in Step 4 is 30 - 40% w / v of the aqueous phase.

5. The detection method for simultaneously detecting 27 pyrazine compounds in Maotai-flavor Baijiu as claimed in claim 1, characterized in that, The chromatographic conditions of the gas chromatography - tandem mass spectrometry in Step 6 are as follows: use a polyethylene glycol capillary column modified with nitroterephthalic acid (DB - FFAP: 60 m×0.25 mm×0.25 μm), the inlet temperature is 250°C, the carrier gas is high - purity helium (purity ≥99.999%), inject in splitless constant - flow mode, the flow rate is 1 mL / min, and the injection volume is 1 μL.

6. The detection method for simultaneously detecting 27 pyrazine compounds in Maotai-flavor Baijiu according to claim 1, wherein, In Step 6, the temperature increase program: initial temperature 40°C, hold for 1 min, increase the temperature to 90°C at a rate of 10°C / min, hold for 2 min, increase to 140°C at a rate of 3.5°C / min, increase to 220°C at a rate of 8°C / min, hold for 4 min, increase to 250°C at a rate of 15°C / min, hold for 2 min.

7. The detection method for simultaneously detecting 27 pyrazine compounds in Maotai-flavor Baijiu as claimed in claim 1, wherein The conditions for tandem mass spectrometry are as follows: the ionization method is electron impact ionization source (EI), with an energy of 70 eV; the ion source temperature is 230°C; the temperature of the first quadrupole is 150°C; the temperature of the third quadrupole is 150°C; the temperature of the auxiliary channel heating is 280°C; the mass spectrometry scanning mode is dynamic multiple reaction monitoring mode (dMRM); the mass spectrometry analysis uses the NIST20 spectral library.

8. The detection method for simultaneously detecting 27 pyrazine compounds in Maotai-flavor Baijiu according to claim 1, wherein In Step 8, the calculation formula for pyrazine compounds is: In the formula Xi: the content of the i-th pyrazine compound in the sample to be measured, with the unit of micrograms per liter (μg / L); Xf: the content of the internal standard 2-methoxy-3-methylpyrazine in the sample to be measured, with the unit of micrograms per liter (μg / L); Ai: the chromatographic peak area of the i-th pyrazine compound in the sample to be measured; Af: the chromatographic peak area of the internal standard 2-methoxy-3-methylpyrazine in the sample to be measured; b: the intercept value of the standard curve; k: the slope of the standard curve; K: the dilution factor.

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