Method for determining six biogenic amines in Baijiu by silanization derivatization-gas chromatography-tandem mass spectrometry

Abstract

The invention discloses a method for determining six biogenic amines in Baijiu through silanization derivatization-gas chromatography-tandem mass spectrometry, and belongs to the technical field of detection. The method comprises the following steps: adding an isotope internal standard working solution into a to-be-detected sample solution, adding alkali to adjust the pH value to 11-13, adding a mixed solution of n-butyl alcohol and trichloromethane for extraction, adding acid into an extract, and removing a solvent; a derivatization reagent is added into the solid obtained after the solvent is removed for derivatization, normal hexane is added after derivatization for constant volume, a test solution is obtained, the pretreatment method is simple and effective, and the recovery rate is high; respectively detecting the mixed standard solution and the test solution by using a gas chromatography-tandem mass spectrometer, drawing a standard curve by using the mixed standard solution, quantifying by using a stable isotope internal standard method, and calculating the contents of the six biogenic amines in the test solution. The method disclosed by the invention is simple, rapid, environment-friendly and high in anti-interference capability, and the simultaneous determination of the six biogenic amines in the Baijiu is realized.

Description

Technical Field

[0001] The invention belongs to the field of detection technology, and specifically relates to a method for determining six biogenic amines in liquor by silylation derivatization-gas chromatography-tandem mass spectrometry. Background Art

[0002] Biogenic amines are a general term for a class of low molecular weight nitrogen-containing organic bases, which are mainly produced by the decarboxylation of free amino acids by microorganisms. Biogenic amines are usually found in food, especially alcoholic beverages that require a fermentation process, protein-rich meat and fish. Low concentrations of biogenic amines are not dangerous to consumers' health, but excessive intake can produce toxic reactions such as nausea, vomiting, diarrhea, respiratory failure and palpitations. The biogenic amines that change the most during the fermentation process are histamine and tyramine, of which histamine is the most toxic. Putrescine and cadaverine in biogenic amines can enhance the toxicity of histamine on the one hand, and on the other hand, they can react with nitrite to produce carcinogenic nitrosamines, which are harmful to human health. Studies have shown that ethanol in liquor can have a synergistic effect on histamine and phenylethylamine in biogenic amines, which can aggravate their toxicity. Some foreign countries have set limits on histamine in wine, among which the most stringent requirement is Germany, which stipulates that it should not exceed 2 mg / L. At present, there is no clear regulation on the limit of biogenic amines in alcoholic beverages in my country, but literature has reported that the detection value of biogenic amines in liquor is generally between 0.2201 and 13.027 mg / L. Therefore, monitoring of biogenic amine concentration is crucial to food quality and safety.

[0003] The main methods for detecting biogenic amines are currently spectrophotometry, high performance liquid chromatography, high performance liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. The sensitivity of spectrophotometry is too low to meet the requirements of low limit of quantification. High performance liquid chromatography requires derivatization with dansyl chloride to enhance its response on fluorescence or ultraviolet detectors. Various free amino acids in liquor will also react with the derivatization reagent, which will produce large matrix interference on the target. Liquid chromatography-mass spectrometry does not require sample derivatization, but the instrument is expensive and the detection cost is high, making it difficult to promote in ordinary laboratories. Gas chromatography-tandem mass spectrometry has the characteristics of simple principle, convenient operation, strong anti-interference ability, high accuracy and sensitivity, and is very suitable for the analysis and detection of trace compounds.

[0004] Biogenic amine compounds have strong polarity, low volatility and poor thermal stability. In gas chromatography, chemical derivatization reactions can be used to increase the volatility of samples and improve detection sensitivity. Common derivatization methods include chloroformate derivatization and dansyl chloride derivatization. Chloroformate reagents are highly toxic and have great harm to the human body and the environment; dansyl chloride derivatization pretreatment is cumbersome, the derivatization time is long, and the amount of extraction reagent is large. Therefore, it is particularly important to establish a green, environmentally friendly, rapid and efficient derivatization method. Silylation derivatization is a commonly used derivatization method in gas phase analysis. Its principle is to use trimethylsilyl to replace the active hydrogen atoms in the sample biogenic amine molecules, reduce hydrogen bond constraints, reduce the polarity of the compound, and the formed biogenic amine derivative products are more volatile. At the same time, due to the reduction in the number of reactive sites containing active hydrogen, the stability of the derivative products is also enhanced. Compared with the above two derivatization methods, silanization derivatization is simple and fast, and is more environmentally friendly. At present, there is no report on the detection method of combining silanization derivatization with gas chromatography-tandem mass spectrometry to analyze biogenic amines in liquor. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a method for determining six biogenic amines in liquor by silylation derivatization-gas chromatography-tandem mass spectrometry, establishes a rapid, efficient, green and environmentally friendly pretreatment method, uses stable isotope internal standards as a quantitative method, and combines the high sensitivity and high accuracy of gas chromatography-tandem mass spectrometry (GC-MS / MS) to accurately analyze phenylethylamine, putrescine, histamine, cadaverine, tyramine and tryptamine in liquor.

[0006] The present invention is achieved through the following technical solutions: A method for determining six biogenic amines in liquor by silylation derivatization-gas chromatography-tandem mass spectrometry comprises the following steps: (1) Take the standard samples of six biogenic amines and dissolve them in methanol to prepare a mixed standard solution; (2) Preparation of test solution: 1) Sample extraction: add isotope internal standard working solution to the sample solution to be tested, add alkali to adjust the pH to 11-13, add a mixed solution of n-butanol and chloroform for extraction, take the extract and add acid to remove the solvent; 2) Derivatization: After step 1) the solid after the solvent is removed is derivatized by adding a derivatization reagent, and after derivatization, n-hexane is added to the fixed volume to obtain a test solution; (3) Detection: The mixed standard solution and the test solution were detected by gas chromatography-tandem mass spectrometry, and a standard curve was drawn for the mixed standard solution. The stable isotope internal standard method was used for quantification to calculate the contents of the six biogenic amines in the test solution. The six biogenic amines are phenylethylamine, putrescine, histamine, cadaverine, tyramine and tryptamine.

[0007] Furthermore, the preparation method of the isotope internal standard working solution in step 1) is to take equal amounts of d4-histamine, d4-tryptamine and d4-phenylethylamine and dissolve them in methanol to prepare an isotope internal standard working solution with a concentration of 50-150 μg / mL.

[0008] Furthermore, in step 1), the volume ratio of the sample solution to be detected to the isotope internal standard working solution is 40-200:1.

[0009] Furthermore, in step 1), the volume ratio of n-butanol to chloroform is 1:1.

[0010] Furthermore, the derivatization reagent described in step 2) is a mixture of N-methyl-N-trimethylsilane heptafluorobutyramide and 1-methylimidazole; the derivatization temperature described in step 2) is 25~105°C, and the derivatization time is 10~30min.

[0011] Furthermore, the derivatization reagent described in step 2) is a mixture of N-methyl-N-trimethylsilane heptafluorobutyramide and 1-methylimidazole in a volume ratio of 95:5, the derivatization temperature described in step 2) is 75° C., and the derivatization time is 20 min.

[0012] Furthermore, the volume ratio of the extract to the derivatization reagent is 5:1.

[0013] Furthermore, the volume ratio of the extract in step 1) to the test solution in step 2) is 1:0.8~1.5. Further, the gas chromatography conditions were as follows: chromatographic column: HP-5MS capillary column, 30 m×0.25 mm, 0.25 μm; injection port temperature 250 °C; injection mode: splitless injection; carrier gas was helium; injection volume: 1 μL; programmed temperature: initial temperature 60 °C, maintained for 1 min, increased to 220 °C at 20 °C / min, maintained for 9 min, increased to 280 °C at 30 °C / min; post-operation temperature 310 °C, post-operation time: 6 min; Mass spectrometry conditions: electron bombardment ion source; ionization energy: 70 eV; ion source temperature: 300 ℃; transfer line temperature: 280 ℃; acquisition mode: multiple reaction monitoring (MRM); solvent delay was 5 min.

[0014] Furthermore, the base in step 1) is sodium hydroxide; the acid is concentrated hydrochloric acid, and the volume ratio of the extract to the concentrated hydrochloric acid is 100:1-4.

[0015] Beneficial Effects The method for determining six biogenic amines in liquor by silylation derivatization-gas chromatography-tandem mass spectrometry of the present invention has a linear range of 0.05-5.0 mg / L for the six biogenic amines and a correlation coefficient (r 2 ) was 0.9970-0.9991, the detection limit was 0.02-0.04 mg / L, the quantification limit was 0.05-0.12 mg / L, and when the addition levels were 0.25, 2.5, and 5.0 mg / L, the average recoveries of the method were 85.3%-107.8%, and the relative standard deviations (RSDs) were 1.8%-8.3%. The method of the invention is simple, rapid, environmentally friendly, and has strong anti-interference ability, and realizes the simultaneous determination of 6 biogenic amines in liquor. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The effect of the type of extractant on the extraction efficiency Figure 2 Effect of the amount of derivatization reagent on the extraction efficiency Figure 3 The total ion current chromatogram of six biogenic amine derivatives. DETAILED DESCRIPTION

[0017] The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0018] Materials and Methods 1. Materials and Reagents Phenylethylamine (purity 98.4%) (Guangzhou Jiatu Technology Co., Ltd.); cadaverine (purity 99.2%), histamine (purity 99.3%), tryptamine (purity 98.8%), tyramine (purity 99.7%), putrescine (purity 99.8%) (Manhag Detection Technology Co., Ltd.); d4-histamine (purity 98.9%), d4-tryptamine (purity 99.0%) (Manhag Detection Technology Co., Ltd.); d4-phenylethylamine (purity 98.7%) (Anolun (Beijing) Biological Technology Co., Ltd.); sodium hydroxide (NaOH), n-butanol, dichloromethane (CH2Cl2), chloroform (CHCl3), hydrochloric acid (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); acetonitrile (chromatographic grade, Merck, Germany); N-methyl-N-trimethylsilane heptafluorobutyramide (MSHFBA) (≥90%, Sigma), N-methyl-N-trimethylsilane trifluoroacetamide (MSTFA) (≥98.5%, Sigma); 1-methylimidazole (≥99%, Aladdin).

[0019] 2. Instruments and equipment Agilent 8890-7000D gas chromatograph-tandem mass spectrometer equipped with electron impact ion source (EI) (Agilent, USA); HP-5MS chromatographic column (30 m×0.25 mm, 0.25 μm, Agilent, USA); high-speed centrifuge (Sigma, Germany); SQP-electronic balance (Sartorius Scientific Instruments, USA); Milli-Q ultrapure water machine (Merck, USA); nitrogen blowdown apparatus (Organomation, USA).

[0020] Example 1 (1) Preparation of mixed standard solution: Accurately weigh 25 mg of each of the six biogenic amines (phenylethylamine, putrescine, histamine, cadaverine, tyramine and tryptamine) into the same 5 mL volumetric flask, dissolve with methanol and dilute to the mark to prepare a mixed standard stock solution with a mass concentration of 5 mg / mL. Further dilute with methanol to a mixed standard intermediate solution of 100 μg / mL; (2) Preparation of isotope internal standard working solution: Accurately weigh equal amounts of d4-phenylethylamine, d4-histamine, and d4-tryptamine into the same 5 mL volumetric flask, dilute to volume with methanol to prepare a mixed internal standard stock solution with a mass concentration of 2 mg / mL, and further dilute with methanol to a 100 μg / mL isotope internal standard working solution; (3) Preparation of test solution: 1) Sample extraction: Take 2 mL of the sample solution to be tested in a 50 mL centrifuge tube, add 25 μL of 100 μg / mL isotope internal standard working solution, let stand for 10 min, add 5 mol / L NaOH solution to adjust the pH to 12, add 5 mL of extractant (n-butanol / chloroform (1:1) mixed solution), vortex for 2 min, ultrasonicate for 10 min, centrifuge at 8000 r / min for 3 min, take 0.5 mL of the extract, add 10 μL of concentrated hydrochloric acid, mix well, and blow to dryness with nitrogen at 40 °C; 2) Derivatization: After removing the solvent from step 1), add 100 μL of derivatization reagent (a mixture of N-methyl-N-trimethylsilane heptafluorobutyramide (MSHFBA) and 1-methylimidazole (95:5, V / V)), vortex for 2 min, derivatize at 75 °C for 20 min, take out and cool to room temperature, add n-hexane to make up to 0.5 mL to obtain the test solution; (3) Detection: The mixed standard solution and the test solution were detected by gas chromatography-tandem mass spectrometry, and a standard curve was drawn for the mixed standard solution. The stable isotope internal standard method was used for quantification to calculate the contents of the six biogenic amines in the test solution. Gas chromatography (GC) conditions: chromatographic column: HP-5MS capillary column (30 m×0.25 mm, 0.25 μm); injection port temperature 250 ℃; injection mode: splitless injection; carrier gas: helium; injection volume: 1 μL; program temperature: initial temperature 60 ℃, hold for 1 min, increase to 220 ℃ at 20 ℃ / min, hold for 9 min, increase to 280 ℃ at 30 ℃ / min; post-operation temperature 310 ℃, post-operation time: 6 min; Mass spectrometry (MS / MS) conditions: electron impact (EI) ion source; ionization energy: 70 eV; ion source temperature: 300 °C; transfer line temperature: 280 °C; acquisition mode: multiple reaction monitoring (MRM); solvent delay of 5 min; MRM parameters of the six biogenic amine derivatives are shown in Table 1 below: Table 1 MRM parameters of 6 biogenic amine derivatives Example 2 Selection of extraction agent: Acetonitrile, ethyl acetate, and n-butanol-chloroform (1:1, V / V) were selected as the extractants, and the recovery rates of the target substances (six biogenic amines) were calculated according to the method of Example 1. The results are as follows: Figure 1 As shown by Figure 1 It can be seen that when n-butanol-chloroform (1:1, V / V) is used as the extractant, the recovery rates of all biogenic amines are higher than 85%; when ethyl acetate is used as the extractant, the recovery rate of putrescine is less than 30%, and the recovery rates of other biogenic amines are equivalent to those when n-butanol-chloroform (1:1) is used as the extractant; when acetonitrile is used as the extractant, the recovery rates of all target substances are no higher than 20%.

[0021] Example 3 1. Choice of derivatizing agent N-methyl-N-trimethylsilane trifluoroacetamide (MSTFA) and a mixture of N-methyl-N-trimethylsilane heptafluorobutyramide (MSHFBA) and 1-methylimidazole (95:5, V / V) were selected as derivatization reagents. When MSTFA was used as a derivatization reagent, the derivative products of histamine, tryptamine and putrescine were fully substituted compounds, while the derivative products of phenylethylamine and cadaverine were monosubstituted or disubstituted. The experimental conditions were more stringent, affecting the quantitative results. When MSHFBA and 1-methylimidazole were used as derivatization reagents, the derivative products of all biogenic amines were fully substituted, the experimental conditions were easy to control, and the response was higher. Therefore, MSHFBA and 1-methylimidazole (95:5, V / V) were selected as derivatization reagents.

[0022] 2. Selection of derivatization reagent volume The amount of derivatization reagent used will directly affect the accuracy and stability of the biogenic amine determination results. This study investigated the effect of different volumes of derivatization reagents (50, 75, 100, 150, 200 μL) on the derivatization of the target compound. Figure 2 As shown in the figure, when the volume of the derivatization reagent is less than 100 μL, the determination results of some biogenic amines have poor repeatability and low sensitivity. When the volume of the derivatization reagent is greater than 100 μL, the recovery rate of the biogenic amine derivatives is basically stable. When the volume of the derivatization reagent is too small, the biogenic amines containing multiple amine groups will be incompletely replaced, resulting in a decrease in the derivatization efficiency. In addition, the derivatization speeds of different biogenic amines are also different. When the derivatization volume is small, the target with a slow derivatization speed will not react completely and the sensitivity will also be reduced. Therefore, the volume of the derivatization reagent is 100 μL.

[0023] 3. Derivatization temperature and time Derivatization temperature and time will also affect the derivatization effect of the target. The derivatization efficiency of the target at different temperatures (room temperature, 50 ℃, 75 ℃, 105 ℃) of the derivatization reagent was studied. The results showed that when the derivatization temperature was lower than 75 ℃, the repeatability of the target determination results was poor, and the response increased with the increase of the derivatization temperature; when the derivatization temperature was higher than 75 ℃, the response of the target tended to be stable. Therefore, the derivatization reaction was selected to be carried out at 75 ℃. The derivatization efficiency of the target was investigated at different times (10, 15, 20, 30 min). The results showed that the response of all biogenic amines increased with the increase of reaction time within 10-20 min, and after 20 min, the response of all biogenic amines did not change significantly. Therefore, it was selected to derivatize at 75 ℃ for 20 min.

[0024] Example 4 Column selection The content of biogenic amines in the sample is low and the polarity is strong. The present invention compares three different polarity chromatographic columns, HP-5MS, TG-1701MS and HP-INNOWAX, to separate and determine biogenic amine derivatives. Through the optimization of precursor ions, fragment ions and collision energy, it is found that the weakly polar HP-5MS chromatographic column has a better separation effect on all biogenic amine derivatives, and the target peaks are baseline separated with high sensitivity. The polar groups of biogenic amines are replaced during the derivatization process, resulting in the polarity of the derivatives being lower than that of the precursor, which ensures that the chromatographic peaks on the weakly polar HP-5MS chromatographic column are sharp and symmetrical, without leading edges or tailing; the total ion flow chromatogram of biogenic amine derivatives is shown in the figure. Figure 3 shown.

[0025] Example 5 Evaluation of matrix effects In mass spectrometry analysis, the sample matrix may enhance or inhibit the ionization efficiency of biogenic amines, thereby causing deviations in the experimental results. Therefore, it is necessary to evaluate the matrix effects (ME) of the method of the present invention. The formula is ME = [slope of the matrix standard curve / slope of the pure solvent standard curve - 1] × 100%. |ME| = 0 means no matrix effect, |ME| ≤ 20% means weak matrix effect, |ME| > 50% means strong matrix effect, and 20% < |ME| ≤ 50% means moderate matrix effect.

[0026] According to the method of the test solution in Example 1, the biogenic amine standard sample was added and derivatized after nitrogen blowing to obtain a mixed blank matrix standard solution with a concentration range of 0.05~5.0 mg / L, and the derivative product after nitrogen blowing of the pure solvent standard curve of the same concentration directly prepared with methanol solution was compared and analyzed on the machine. The results showed that the ME (%) of phenylethylamine, putrescine, histamine, cadaverine, tyramine and tryptamine were -8.3%, 3.6%, 13.5%, -3.7%, 2.7% and -14.1%, respectively. It can be seen that the sample matrix has no significant effect on the quantitative results, so the present invention adopts the pure solvent standard solution for quantification.

[0027] Example 6 Linear range and sensitivity of the method The present invention adopts the stable isotope internal standard method for quantification. The six biogenic amine standard series solutions and the internal standard solution are measured according to the method of Example 1. The mass concentration of the biogenic amine derivatives is used as the abscissa and the peak area is used as the ordinate to draw the standard curve. An appropriate amount of the six biogenic amine standard solutions are added to the blank sample solution, and the six biogenic amine standard solutions are analyzed on the machine after derivatization. The detection limit (LOD) and quantification limit (LOQ) of the target object are determined by 3 times and 10 times the signal-to-noise ratio (S / N) of the target peak. The results are shown in Table 2: All biogenic amines have good linear relationships in the range of 0.05~5.0 mg / L, and the correlation coefficient (r 2) is 0.9970-0.9991, the detection limit is 0.02-0.04 mg / L, and the quantification limit is 0.05-0.12 mg / L. Therefore, the method established in the present invention has high sensitivity and is suitable for the simultaneous detection of 6 biogenic amines in liquor.

[0028] Table 2 Linear relationships, correlation coefficients, method detection limits and quantification limits of six biogenic amine derivatives Example 7 Method recovery and precision According to the method described in Example 1, six biogenic amines and internal standard solution were added to blank liquor samples for recovery experiments, with the addition levels being 0.25, 2.5, and 5.0 mg / L, respectively. Six parallel determinations were performed at each level to investigate the recovery and precision of the method, as shown in Table 3. As can be seen from Table 3, the average recovery of the six biogenic amines was 85.3% to 107.8%, and the relative standard deviation (RSD) was 1.8% to 8.3%. This indicates that the method established by the present invention has good accuracy and precision, and can meet the requirements for simultaneous detection of six biogenic amines in liquor. Table 3 Recovery and standard deviation of 6 biogenic amines in samples ( n =6) .

[0029] Example 8 Actual sample measurement The method described in Example 1 was used to test 20 batches of liquor of different brands and flavors randomly selected from local supermarkets (including 9 batches of Luzhou-flavor liquor, 2 batches of Light-flavor liquor, 6 batches of Maotai-flavor liquor, 3 batches of Sesame-flavor liquor, etc.). The analysis results showed that the detection value of tryptamine in 1 batch of Maotai-flavor liquor was 0.08 mg / L, and no biogenic amines were detected in other samples.

Claims

1. A method for determining six biogenic amines in liquor by silylation derivatization-gas chromatography-tandem mass spectrometry, characterized in that: The following steps are involved: (1) Take the standard samples of six biogenic amines and dissolve them in methanol to prepare a mixed standard solution; (2) Preparation of test solution: 1) Sample extraction: add isotope internal standard working solution to the sample solution to be tested, add alkali to adjust the pH to 11-13, add a mixed solution of n-butanol and chloroform for extraction, take the extract and add acid to remove the solvent; 2) Derivatization: After step 1) the solid after the solvent is removed is derivatized by adding a derivatization reagent, and after derivatization, n-hexane is added to the fixed volume to obtain a test solution; (3) Detection: The mixed standard solution and the test solution were detected by gas chromatography-tandem mass spectrometry, and a standard curve was drawn for the mixed standard solution. The stable isotope internal standard method was used for quantification to calculate the contents of the six biogenic amines in the test solution. The six biogenic amines are phenylethylamine, putrescine, histamine, cadaverine, tyramine and tryptamine.

2. The method for determining six biogenic amines in liquor by silylation derivatization-gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The preparation method of the isotope internal standard working solution described in step 1) is to take equal amounts of d4-histamine, d4-tryptamine and d4-phenylethylamine and dissolve them in methanol to prepare an isotope internal standard working solution with a concentration of 50-150 μg / mL.

3. The method for determining six biogenic amines in liquor by silylation derivatization-gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: In step 1), the volume ratio of the sample solution to be detected to the isotope internal standard working solution is 40-200:

1.

4. The method for determining six biogenic amines in liquor by silylation derivatization-gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The volume ratio of n-butanol to chloroform in step 1) is 1:0.8-1.

5.

5. The method for determining six biogenic amines in liquor by silylation derivatization-gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The derivatization reagent described in step 2) is a mixture of N-methyl-N-trimethylsilane heptafluorobutyramide and 1-methylimidazole; the derivatization temperature described in step 2) is 25~105°C, and the derivatization time is 10~30min.

6. The method for determining six biogenic amines in liquor by silylation derivatization-gas chromatography-tandem mass spectrometry according to claim 5, characterized in that: The derivatization reagent described in step 2) is a mixture of N-methyl-N-trimethylsilane heptafluorobutyramide and 1-methylimidazole in a volume ratio of 95:

5. The derivatization temperature described in step 2) is 75° C. and the derivatization time is 20 min.

7. The method for determining six biogenic amines in liquor by silylation derivatization-gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The volume ratio of the extract to the derivatization reagent was 5:

1.

8. The method for determining six biogenic amines in liquor by silylation derivatization-gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The volume ratio of the extract in step 1) to the test solution in step 2) is 1:

1.

9. The method for determining six biogenic amines in liquor by silylation derivatization-gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: Gas chromatography conditions: chromatographic column: HP-5MS capillary column, 30 m×0.25 mm, 0.25 μm; injection port temperature 250°C; injection mode: splitless injection; carrier gas: helium; injection volume: 1 μL; program temperature: initial temperature 60°C, hold for 1 min, increase to 220°C at 20°C / min, hold for 9 min, increase to 280°C at 30°C / min; post-operation temperature 310°C, post-operation time: 6 min; Mass spectrometry conditions: electron bombardment ion source; ionization energy: 70 eV; ion source temperature: 300 °C; transfer line temperature: 280 °C; acquisition mode: multiple reaction monitoring mode; solvent delay was 5 min.

10. The method for determining six biogenic amines in liquor by silylation derivatization-gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The base in step 1) is sodium hydroxide; the acid is concentrated hydrochloric acid, and the volume ratio of the extract to the concentrated hydrochloric acid is 100:1-4.

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