Analysis method for determining multiple components of white spirit by high performance liquid chromatography-tandem mass spectrometry

Abstract

The invention discloses an analysis method for determining multiple components of white spirit by high performance liquid chromatography-tandem mass spectrometry, and relates to the technical field of white spirit analysis and detection, and the analysis method comprises the following steps: firstly, preparing an ethyl carbamate precursor substance standard stock solution and a mixed standard intermediate solution, and diluting the mixed standard intermediate solution step by step to obtain a mixed standard working solution; then determining chromatographic and mass spectrometry conditions, wherein BEH Ami chromatographic column is adopted as the chromatographic condition, and 0.1% formic acid water-acetonitrile is adopted as a mobile phase for gradient elution; a mass spectrum condition adopts a multi-reaction monitoring mode under an electrospray positive ion mode; drawing a quantitative calibration curve; finally, preparing a to-be-detected sample for content determination. According to the analysis method for determining the multiple components of the Baijiu through the high performance liquid chromatography-tandem mass spectrometry, through chromatographic condition optimization and mass spectrometry parameter innovation, the detection efficiency is improved, peak area data of urea, citrulline and arginine can be synchronously obtained through single sample injection, the analysis time is short, and quantification is accurate.

Description

Technical Field

[0001] The present invention relates to the technical field of liquor analysis and detection, and in particular to a method for analyzing multi-components of liquor using high performance liquid chromatography tandem mass spectrometry. Background Art

[0002] Ethyl carbamate (EC) is produced during the fermentation and storage of baijiu (white spirits). It is a class of chemical pollutants with carcinogenic effects (Class 2A). The presence of EC precursors can significantly affect the style and taste of baijiu. Baijiu flavor is a key characteristic that determines its quality. Studying the content of EC precursors in baijiu is crucial for improving its quality and controlling its EC content. Urea, citrulline, and arginine are common EC precursors. Their content is regulated by microbial metabolism during baijiu fermentation and can influence the aroma structure of baijiu. Excessive residues can lead to EC formation. Dynamically monitoring EC precursor levels during baijiu brewing and reducing EC formation by controlling fermentation and distillation temperatures are important methods for effectively reducing EC content in baijiu and improving product safety.

[0003] Common methods for determining urea in liquor include colorimetric-UV spectrophotometry, urease-titration, and high-performance liquid chromatography. Methods for determining amino acids in liquor primarily include PITC pre-column derivatization HPLC, reversed-phase HPLC, anion chromatography-pulsed amperometric detection, ninhydrin colorimetry, and amino acid analyzers. Consequently, existing methods are unable to simultaneously detect urea, arginine, and citrulline, requiring the development of separate detection systems. This results in doubled sample consumption, extended testing cycles, and increased testing costs. In addition, the current pre-treatment for urea detection is complex and requires derivatization. Derivatization reagents (such as 9-hydroxyxanthanol) are cytotoxic, and the exposure risk index for laboratory personnel reaches Class B hazard level, and the cost of waste liquid treatment is high; arginine / citrulline detection requires PITC pre-column derivatization, and the derivatization efficiency is easily affected by pH fluctuations, and the derivatization process can easily lead to loss of the analyte; the detection limit of urea under existing high-performance liquid chromatography methods is only at the ppm level, while the actual content of EC precursors in white wine is mostly at the ppb level, and traditional methods have low sensitivity.

[0004] Therefore, there is an urgent need to invent a high performance liquid chromatography tandem mass spectrometry method (HPLC-MS / MS) to achieve the simultaneous detection of urea, arginine and citrulline in liquor. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for multi-component analysis of liquor by high performance liquid chromatography tandem mass spectrometry to overcome the shortcomings of the prior art in that the simultaneous detection of urea, arginine and citrulline in liquor cannot be achieved, derivatization reagents need to be used, and the detection sensitivity is low.

[0006] In order to achieve the above object, the present invention provides the following technical solution: a method for multi-component analysis of liquor by high performance liquid chromatography tandem mass spectrometry, specifically comprising the following steps:

[0007] S1. Prepare a mixed standard working solution of three urethane precursor substances: weigh the three precursor substance standards and place them in a beaker. Add solvent and ultrasonically dissolve them. Transfer them to volumetric flask 1 and dilute to the mark to obtain the standard stock solution of the required concentration.

[0008] S2. Prepare mixed standard working solution: pipette the above standard stock solution into volumetric flask 2, add solvent to dilute and mix evenly to prepare mixed standard intermediate solution, and then dilute the mixed standard intermediate solution step by step to obtain mixed standard working solution;

[0009] S3. Determine the chromatographic and mass spectrometric conditions: The chromatographic conditions are: an Amide hydrophilic column, mobile phase A is 0.1% formic acid in water, mobile phase B is acetonitrile, and gradient elution; the mass spectrometric conditions are: an electrospray positive ion source, an interface voltage of 2 kV, and a multiple reaction monitoring scan mode;

[0010] S4. Drawing a quantitative calibration curve: Determine the mixed standard working solution in S2 by high performance liquid chromatography tandem mass spectrometry to draw a quantitative calibration curve for the three ethyl carbamate precursor substances;

[0011] S5. Prepare the sample to be tested: Take the liquor sample and filter the membrane to obtain the sample to be tested;

[0012] S6. Content determination: Analyze the sample to be tested in S5 by high performance liquid chromatography tandem mass spectrometry to obtain the peak area of ​​the sample to be tested, and calculate the content of the three ethyl carbamate precursor substances in the sample to be tested according to the quantitative calibration curve in S4.

[0013] Furthermore, the ethyl carbamate precursor in S1 is urea, citrulline, and arginine.

[0014] Furthermore, the solvent in S1 is at least one of 60% ethanol-water, 50% methanol-water, and pure water; and the concentration of the standard stock solution in S1 is 1000 mg / L.

[0015] Furthermore, the concentration of the mixed standard intermediate solution in S2 is 1000 μg / L; the concentration of the mixed standard working solution in S1 is 10 to 1000 μg / L.

[0016] Furthermore, the chromatographic conditions in S3 also include: column temperature of 30° C.; injection volume of 1.0 μL; flow rate of 0.3 mL / min; and delay column of BEH Amide Pre-Column.

[0017] Furthermore, the gradient elution method in S3 is:

[0018] 0-2 min, 2% A, 98% B;

[0019] 2-3 min, 10% A, 90% B;

[0020] 3-9 min, 25% A, 75% B;

[0021] 9~9.1min, 2%A, 98%B;

[0022] 9.1~15min, 2%A, 98%B.

[0023] Furthermore, the mass spectrometry conditions in S3 also include: nitrogen gas is used as nebulizing gas with a flow rate of 3.0 L / min; the heating gas flow rate is 10.0 L / min, and the ion source interface temperature is 300°C; the DL temperature is 250°C; the heating block temperature is 400°C; and the drying gas flow rate is 10.0 L / min.

[0024] Furthermore, the sample to be tested in S5 is at least one of the following types of liquor: strong-flavor liquor, sauce-flavor liquor, light-flavor liquor, rice-flavor liquor, phoenix-flavor liquor, Dong-flavor liquor, fermented soy-flavor liquor, sesame-flavor liquor, special-flavor liquor, mixed-flavor liquor, Laobaigan-flavor liquor, and rich-flavor liquor.

[0025] Furthermore, the filter membrane in S5 is a 0.22 μm to 0.45 μm water filter membrane.

[0026] Compared with the prior art, the high performance liquid chromatography tandem mass spectrometry method for multi-component analysis of liquor provided by the present invention has the following beneficial effects:

[0027] (1) High detection efficiency: A single injection can simultaneously obtain the peak area data of three substances: urea, citrulline, and arginine, achieving simultaneous detection, short analysis time, and high efficiency.

[0028] (2) Strong anti-interference ability: Using ethanol gradient dilution correction technology, in a 60% ethanol matrix, ion suppression and matrix effects are reduced by using a delay column to minimize background interference.

[0029] (3) High sensitivity: The detection limits of urea, citrulline, and arginine are reduced from the ppm level to the ppb level, and the sensitivity is improved, which can accurately monitor the dynamic changes of EC precursors during the aging process of liquor. Among them, the detection limit of urea is 10 μg / L, the detection limit of citrulline is 5 μg / L, and the detection limit of arginine is 2 μg / L.

[0030] (4) Green detection process: adopting a derivatization-free ultra-fast pre-treatment process, without using carcinogens such as 9-hydroxyxanthanol that are necessary for the three types of derivatization steps, thus reducing the amount of hazardous waste generated.

[0031] (5) Reliable data: The electrospray positive ionization mode (ESI+) was established, and the multiple reaction monitoring (MRM) of triple quadrupole mass spectrometry was used to greatly reduce the quantitative errors caused by incomplete and unstable derivatization, which is particularly suitable for the accurate determination of trace substances in complex liquor matrices. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments described in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0033] Figure 1 TIC diagrams of urea, citrulline, and arginine provided in Example 1 of the present invention;

[0034] Figure 2 The urea standard curve provided in Example 1 of the present invention;

[0035] Figure 3 This is a standard curve diagram of citrulline provided in Example 1 of the present invention;

[0036] Figure 4 This is a standard curve diagram of arginine provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0038] Example 1:

[0039] See also Figure 1-4 The method for determining the multi-component analysis of liquor by high performance liquid chromatography tandem mass spectrometry specifically comprises the following steps:

[0040] S1. Prepare a mixed standard working solution of three ethyl carbamate precursor substances: weigh the three precursor standard substances and place them in a beaker. Add solvent and ultrasonically dissolve them. Transfer them to volumetric flask 1 and adjust the volume to the scale line to obtain a standard stock solution of the required concentration; the EC precursor substances are urea, citrulline, and arginine; the solvent is at least one of 60% ethanol-water, 50% methanol-water, and pure water; the concentration of the standard stock solution is 1000 mg / L.

[0041] The specific implementation method is as follows: 0.0100 g of urea, citrulline, and arginine standards with a purity of more than 99% are accurately weighed and placed in a 10 mL beaker respectively, 50% methanol-water is added, and after ultrasonic dissolution, they are transferred to a 10 mL volumetric flask respectively. After constant volume, 1000 mg / L urea standard stock solution, 1000 mg / L citrulline standard stock solution, and 1000 mg / L arginine standard stock solution are obtained respectively.

[0042] S2. Prepare mixed standard working solution: pipette the above standard stock solution into volumetric flask 2, add solvent to dilute and mix evenly to prepare mixed standard intermediate solution, then dilute the mixed standard intermediate solution step by step to obtain mixed standard working solution; the concentration of the mixed standard intermediate solution is 1000 μg / L; the concentration of the mixed standard working solution is 10-1000 μg / L.

[0043] The specific implementation method is to accurately pipette 0.1 mL of 1000 mg / L urea, citrulline, and arginine standard stock solutions into a 100 mL volumetric flask, dilute to the scale with 60% ethanol-water to obtain a 1000 μg / L mixed standard intermediate solution; and dilute the mixed standard intermediate solution stepwise with 60% ethanol-water to obtain 10 μg / L, 20 μg / L, 50 μg / L, 100 μg / L, 200 μg / L, 500 μg / L, and 1000 μg / L mixed standard working solutions, respectively.

[0044] Comparing the effects of 50% methanol-water, 60% ethanol-water, and pure water as solvents on the responses of urea, citrulline, and arginine revealed that 50% methanol-water resulted in stronger background interference and lower sensitivity for urea using pure water. However, 60% ethanol-water reduced background interference and exhibited higher sensitivity for urea, citrulline, and arginine, indicating that different solvents have varying effects on the ionization of these three substances. Therefore, to ensure accurate measurements, 60% ethanol-water, which is closer to baijiu, was selected as the dilution solvent for the standard working solution.

[0045] S3. Determine the chromatographic and mass spectrometric conditions: The chromatographic conditions are to use an Amide hydrophilic column, mobile phase A is 0.1% formic acid water, mobile phase B is acetonitrile, and gradient elution; the mass spectrometric conditions are to use an electrospray positive ion source, an interface voltage of 2kV, and a multiple reaction monitoring scan mode; the chromatographic conditions also include: column temperature of 30°C; injection volume of 1.0μL; flow rate of 0.3mL / min; delay column is BEH Amide Pre-Column; gradient elution mode: 0-2 min, 2% A, 98% B; 2-3 min, 10% A, 90% B; 3-9 min, 25% A, 75% B; 9-9.1 min, 2% A, 98% B; 9.1-15 min, 2% A, 98% B; mass spectrometry conditions also include: nebulizing gas is nitrogen with a flow rate of 3.0 L / min; heating gas flow rate is 10.0 L / min, and the ion source interface temperature is 300°C; DL temperature is 250°C; heating block temperature is 400°C; and drying gas flow rate is 10.0 L / min.

[0046] The specific embodiment is as follows: the chromatographic conditions are as follows: a BEH Amide hydrophilic column with a particle size of 1.7 μm, a column length of 100 mm, and an inner diameter of 2.1 mm; mobile phase A is 0.1% formic acid, and mobile phase B is acetonitrile; the column temperature is 30°C; the injection volume is 1.0 μL; the flow rate is 0.3 mL / min; the detection time is 15 minutes; and the gradient elution mode is: 0-2 minutes, 2% A, 98% B; 2-3 minutes, 10% A, 90% B; 3-9 minutes, 25% A, 75% B; 9-9.1 minutes, 2% A, 98% B; 9.1-15 minutes, 2% A, 98% B. Comparison of isocratic and gradient elution modes revealed that urea was poorly retained under the isocratic elution mode, while the above gradient elution conditions were able to achieve good separation of the three substances.

[0047] The mass spectrometry conditions were as follows: electrospray positive ionization mode (ES I+) with an interface voltage of 2 kV and multiple reaction monitoring (MRM) scanning mode, wherein urea was detected using m / z 61.0→43.9, arginine m / z 175.1→70.1, and citrulline m / z 176.1→70.0; nitrogen was used as the nebulizer gas at a flow rate of 3.0 L / min; the heating gas flow rate was 10.0 L / min, and the ion source interface temperature was 300°C; the DL temperature was 250°C; the heating block temperature was 400°C; and the drying gas flow rate was 10.0 L / min.

[0048] Comparison of 0.1% formic acid + 0.2 mmol ammonium acetate and 0.1% formic acid in water revealed that ionization of arginine and citrulline was significantly suppressed when using 0.1% formic acid + 0.2 mmol ammonium acetate as mobile phase A, resulting in significantly lower responses. In contrast, using 0.1% formic acid in water as mobile phase A resulted in strong responses for urea, citrulline, and arginine, with sufficient sensitivity to meet quantitative requirements, enabling simultaneous and accurate quantification.

[0049] A delay column is added after the mixer to reduce background interference. The delay column is a BEH Amide Pre-Column (2.1 x 5 mm). By extending the residence time of the mobile phase in the chromatographic system, the solvent system can partially evaporate or achieve a more stable mixing state with sample components before entering the mass spectrometer, thereby reducing ion suppression. The delay column promotes thorough mixing of the sample and mobile phase, reduces competition between matrix components and target compounds in the ion source, and improves analytical accuracy and reproducibility.

[0050] Under the above chromatographic and mass spectrometric conditions, the retention time of urea was approximately 3.418 min, the retention time of citrulline was approximately 6.414 min, and the retention time of arginine was 6.170 min, with good separation. The compound information and multiple reaction monitoring (MRM) parameters of urea, citrulline, and arginine are shown in Table 1. The total ion current (TIC) of the three substances is shown in Table 1. Figure 1 shown.

[0051] Table 1 Compound information and MRM parameters of urea, citrulline, and arginine

[0052]

[0053] “*” is the quantitative ion of the compound.

[0054] S4. Drawing a quantitative calibration curve: Determine the mixed standard working solution in S2 by high performance liquid chromatography tandem mass spectrometry to draw quantitative calibration curves of the three ethyl carbamate precursor substances.

[0055] The specific implementation method is to perform high performance liquid chromatography tandem mass spectrometry on 10 μg / L, 20 μg / L, 50 μg / L, 100 μg / L, 200 μg / L, 500 μg / L, and 1000 μg / L mixed standard working solutions in order from low to high concentration, and measure the peak areas of urea, citrulline, and arginine in the mixed standard working solutions of different concentrations, respectively. The measured peak areas are used as the vertical coordinates and the corresponding standard concentrations are used as the horizontal coordinates to draw standard curves of urea, citrulline, and arginine, respectively.

[0056] like Figure 2-Figure 4 As shown in the figure, the linear ranges of the standard curves of urea, citrulline and arginine all meet the analytical requirements (r 2≥0.99), wherein, the urea standard curve is Y=1039.94X+24495.3, R 2 =0.994, the detection limit was 10 μg / L, and the quantification limit was 20 μg / L; the standard curve of citrulline was Y=11412.8X-36617.1, R 2 =0.998, the detection limit is 5μg / L, the quantification limit is 10μg / L; the arginine standard curve is Y=18568.8X-203399, R 2 =0.997, the detection limit was 2 μg / L, and the quantification limit was 5 μg / L.

[0057] S5. Prepare the sample to be tested: take a liquor sample and filter it through a membrane to obtain the sample to be tested; the sample to be tested should be at least one of the following types of liquor: Luzhou-flavor, Maotai-flavor, Qing-flavor, Rice-flavor, Feng-flavor, Dong-flavor, Chi-flavor, Sesame-flavor, Special-flavor, Mixed-flavor, Laobaigan-flavor, and Fuyu-flavor; the filter membrane should be a 0.22μm to 0.45μm water-based filter membrane.

[0058] S6. Content determination: Analyze the sample to be tested by high performance liquid chromatography tandem mass spectrometry to obtain the peak area of ​​the sample to be tested, and calculate the content of urea, citrulline, and arginine in the sample to be tested according to the quantitative calibration curve in S4.

[0059] The specific implementation method is to analyze 12 samples of different flavor liquors by high performance liquid chromatography tandem mass spectrometry, obtain the peak areas of the 12 different flavor liquor samples, and calculate the content of urea, citrulline, and arginine in the samples to be tested based on the quantitative calibration curves of urea, citrulline, and arginine in S4. The sample pretreatment specifically includes the following steps:

[0060] (1) Prepare 12 types of liquor samples, including strong-flavor, sauce-flavor, light-flavor, rice-flavor, phoenix-flavor, Dong-flavor, fermented soybean-flavor, sesame-flavor, special-flavor, mixed-flavor, Laobaigan-flavor, and rich-flavor liquor.

[0061] (2) 1 mL of each flavor liquor sample was taken and filtered through a 0.22 μm filter membrane to obtain 12 liquor samples to be tested, which were numbered 1-12, corresponding to strong-flavor liquor, sauce-flavor liquor, light-flavor liquor, rice-flavor liquor, phoenix-flavor liquor, Dong-flavor liquor, fermented soybean-flavor liquor, sesame-flavor liquor, special-flavor liquor, mixed-flavor liquor, Laobaigan-flavor liquor, and rich-flavor liquor.

[0062] (3) The 12 samples of liquor with different flavors were tested using the chromatographic and mass spectrometric conditions determined in S3 to obtain the peak areas of the 12 liquor samples to be tested. The peak areas of the corresponding substances in the 12 liquor samples were substituted into the quantitative calibration curves of urea, citrulline, and arginine obtained in S4 according to the retention times to obtain the contents of urea, citrulline, and arginine in the 12 liquor samples with different flavors. The test results are shown in Table 2.

[0063] Table 2 Urea, citrulline and arginine contents in 12 samples of different flavor liquors

[0064]

[0065] According to the test results of urea, citrulline and arginine content in 12 samples of different flavor liquors in Table 2:

[0066] Urea content testing revealed significant differences among the 12 different flavor types. Sesame-flavored liquor had the highest urea content, reaching 445.579 μg / L, while rich-flavored liquor had the lowest, at only 0.995 μg / L. The urea content of the other flavor types fell between these two, showing a more dispersed distribution.

[0067] Citrulline content testing revealed relatively small differences among different flavor types of liquor, compared to urea content. Sesame-flavored liquor had the highest citrulline content, at 26.738 μg / L. Light-flavored and mixed-flavor liquors had relatively low citrulline levels, at 12.474 μg / L and 12.600 μg / L, respectively. Overall, citrulline content fluctuated within a relatively narrow range among different flavor types of liquor.

[0068] Arginine content also showed significant differences among flavor types. Sesame-flavored white wine had a high arginine content of 366.185 μg / L, significantly higher than other flavor types. Soybean-flavored white wine had an intermediate arginine content of 26.972 μg / L. Phoenix-flavored white wine had a relatively low arginine content of 19.730 μg / L. The wide range of arginine content across different flavor types indicates that flavor significantly influences arginine content.

[0069] Based on the content of urea, citrulline and arginine in different types of liquor, it can be seen that this method can accurately detect the content differences of these three amino acids in different types of liquor, indicating that this method can be used for the accurate quantification of urea, citrulline and arginine in different types of liquor.

[0070] Example 2:

[0071] This example verifies the accuracy of the detection method of Example 1. In order to verify the accuracy of the detection method of Example 1, a sample recovery test was conducted.

[0072] Sample pretreatment specifically includes the following steps:

[0073] Take 0.02mL, 0.05mL, and 0.10mL of 1000μg / L mixed standard working solution and add them to three liquor samples 1 respectively, and make the volume to 1mL, so that the final concentration of the mixed standard solution in the three liquor samples 1 is 20μg / L, 50μg / L, and 100μg / L respectively. Set up three parallel samples for each concentration, filter with a 0.22μm filter membrane and directly detect on the machine.

[0074] Under the same chromatographic and mass spectrometric conditions as in Example 1, the concentrations of urea, citrulline, and arginine were measured in a liquor sample 1 without the addition of the mixed standard working solution and in liquor samples 1 with different concentrations of the mixed standard working solution added, and the recoveries were calculated. The recovery test results are shown in Table 3.

[0075] Table 3 Recovery results of urea, citrulline and arginine in liquor samples

[0076]

[0077]

[0078] According to the test results in Table 2, the recoveries of urea spiked into liquor sample 1 at concentrations of 20 μg / L, 50 μg / L, and 100 μg / L of the mixed standard working solution were 101%, 105%, and 109%, respectively; the recoveries of citrulline spiked were 104%, 102%, and 94%, respectively; and the recoveries of arginine spiked were 93%, 93%, and 97%, respectively. These results indicate that the recoveries of urea, citrulline, and arginine were all between 90% and 110%, with RSDs less than 7%. This demonstrates that this method has good recoveries for urea, citrulline, and arginine, meeting the requirements for quantitative analysis. It also demonstrates that this method has high accuracy and can accurately determine the content of EC precursors urea, citrulline, and arginine in liquor samples. It can be used for the rapid detection of these EC precursors in liquor, thereby ensuring the quality and safety of liquor products.

[0079] The above description is merely illustrative of certain exemplary embodiments of the present invention. It goes without saying that those skilled in the art will be able to modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and description are illustrative in nature and should not be construed as limiting the scope of protection of the claims.

Claims

1. A method for analyzing multi-components of liquor by high performance liquid chromatography tandem mass spectrometry, characterized in that: The specific steps include: S1. Prepare a mixed standard working solution of three urethane precursor substances: weigh the three precursor substance standards and place them in a beaker. Add solvent and ultrasonically dissolve them. Transfer them to volumetric flask 1 and dilute to the mark to obtain the standard stock solution of the required concentration. S2. Prepare mixed standard working solution: pipette the above standard stock solution into volumetric flask 2, add solvent to dilute and mix evenly to prepare mixed standard intermediate solution, and then dilute the mixed standard intermediate solution step by step to obtain mixed standard working solution; S3. Determine the chromatographic and mass spectrometric conditions: The chromatographic conditions are: an Amide hydrophilic column, mobile phase A is 0.1% formic acid in water, mobile phase B is acetonitrile, and gradient elution; the mass spectrometric conditions are: an electrospray positive ion source, an interface voltage of 2 kV, and a multiple reaction monitoring scan mode; S4. Drawing a quantitative calibration curve: Determine the mixed standard working solution in S2 by high performance liquid chromatography tandem mass spectrometry to draw a quantitative calibration curve for the three ethyl carbamate precursor substances; S5. Prepare the sample to be tested: Take the liquor sample and filter the membrane to obtain the sample to be tested; S6. Content determination: Analyze the sample to be tested in S5 by high performance liquid chromatography tandem mass spectrometry to obtain the peak area of ​​the sample to be tested, and calculate the content of the three ethyl carbamate precursor substances in the sample to be tested according to the quantitative calibration curve in S4.

2. The method for multi-component analysis of liquor by high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The ethyl carbamate precursor substances in S1 are urea, citrulline and arginine.

3. The method for multi-component analysis of liquor by high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The solvent in S1 is at least one of 60% ethanol-water, 50% methanol-water, and pure water; the concentration of the standard stock solution in S1 is 1000 mg / L.

4. The method for multi-component analysis of liquor by high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The concentration of the mixed standard intermediate solution in S2 is 1000 μg / L; the concentration of the mixed standard working solution in S1 is 10-1000 μg / L.

5. The method for multi-component analysis of liquor by high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The chromatographic conditions in S3 also include: column temperature of 30° C.; injection volume of 1.0 μL; flow rate of 0.3 mL / min; and delay column of BEH Amide Pre-Column.

6. The method for multi-component analysis of liquor by high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The gradient elution method in S3 is: 0-2 min, 2% A, 98% B; 2-3 min, 10% A, 90% B; 3-9 min, 25% A, 75% B; 9~9.1min, 2%A, 98%B; 9.1~15min, 2%A, 98%B.

7. The method for multi-component analysis of liquor by high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The mass spectrometry conditions in S3 also include: nitrogen as the nebulizing gas with a flow rate of 3.0 L / min; a heating gas flow rate of 10.0 L / min, an ion source interface temperature of 300°C; a DL temperature of 250°C; a heating block temperature of 400°C; and a drying gas flow rate of 10.0 L / min.

8. The method for multi-component analysis of liquor by high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The sample to be tested in S5 is at least one of the following types of liquor: strong-flavor liquor, sauce-flavor liquor, light-flavor liquor, rice-flavor liquor, phoenix-flavor liquor, Dong-flavor liquor, fermented soy-flavor liquor, sesame-flavor liquor, special-flavor liquor, mixed-flavor liquor, Laobaigan-flavor liquor, and rich-flavor liquor.

9. The method for multi-component analysis of liquor by high performance liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The filter membrane in the S5 is a 0.22 μm to 0.45 μm water filter membrane.

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