Method for detecting primary metabolites and / or secondary metabolites in flavors and fragrances

Abstract

The invention relates to a method for detecting primary metabolites and / or secondary metabolites in flavors and fragrances, which comprises the following steps: (1) mixing flavors and fragrances, an internal standard substance and a solvent, extracting, taking clear liquid, and removing the solvent to obtain a dry product; (2) mixing the dry product, an oximation reagent and a solvent for an oximation reaction, and then mixing a reaction system and a silanization reagent for a silanization reaction to obtain a solution to be detected; the silanization reagent comprises a combination of N-methyl-N-(trimethylsilyl) trifluoroacetamide and N, O-bis (trimethylsilyl) trifluoroacetamide, and the silanization reagent comprises N-methyl-N-(trimethylsilyl) trifluoroacetamide and N, O-bis (trimethylsilyl) trifluoroacetamide; and (3) detecting the solution to be detected by adopting a gas chromatography-mass spectrometry method, and obtaining the content of primary metabolites and / or secondary metabolites in the flavors and fragrances by utilizing an internal standard method. According to the method provided by the invention, the flavors and fragrances are subjected to oximation and silanization treatment, and then the flavors and fragrances are detected by using the gas chromatography-mass spectrometry, so that the metabolites contained in the flavors and fragrances can be precisely and accurately detected.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, and in particular to a method for detecting primary metabolites and / or secondary metabolites in fragrances and flavorings. Background Technology

[0002] Flavors and fragrances are highly complex mixtures whose sensory characteristics and functional properties depend primarily on their primary and secondary metabolites. Primary metabolites are essential building blocks for life, mainly including small molecules such as sugars, amino acids, organic acids, and nucleotides. They directly participate in cellular metabolic processes, providing the basic flavor precursors for flavors and fragrances. Secondary metabolites, on the other hand, are specialized compounds produced by organisms under specific conditions. They possess more complex chemical structures and more specific biological activities, including alkaloids, flavonoids, terpenes, and phenols. These substances not only contribute to the characteristic aromas of flavors and fragrances but also significantly influence product stability and bioactivity.

[0003] The analysis of metabolites in fragrances and flavorings currently relies primarily on chromatography-mass spectrometry (GC-MS). Gas chromatography (GC) and its coupled techniques are effective at separating volatile components, but are not suitable for large, thermally unstable, or non-volatile secondary metabolites. High-performance liquid chromatography (HPLC), while capable of analyzing large molecules, is not suitable for analyzing complex fragrance and flavoring components. Traditional mass spectrometry techniques, such as single quadrupole mass spectrometry (SQMS) or triple quadrupole tandem mass spectrometry (TPMS), typically require pre-identification of target compounds and the establishment of specific monitoring ion pairs, limiting their applicability to targeted analysis. Furthermore, existing technologies suffer from limitations in detection precision.

[0004] Therefore, it is necessary to provide a precise and comprehensive method for detecting primary and / or secondary metabolites in fragrances and flavorings. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for detecting primary and / or secondary metabolites in fragrances and flavorings. The method provided by this invention, through oximation and silanization treatment of the fragrances and flavorings, followed by detection using gas chromatography-mass spectrometry, enables precise, accurate, and comprehensive detection of the metabolites contained therein.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] This invention provides a method for detecting primary metabolites and / or secondary metabolites in fragrances and flavorings, the method comprising the following steps:

[0008] (1) Mix the flavoring, internal standard and solvent to extract, remove the solvent from the clear liquid, and obtain the dried product;

[0009] (2) The dry product, oxime reagent and solvent are mixed to carry out oxime reaction, and then the reaction system is mixed with silanizing reagent to carry out silanization reaction to obtain the test solution; the silanizing reagent includes a combination of N-methyl-N-(trimethylsilyl)trifluoroacetamide and N,O-bis(trimethylsilyl)trifluoroacetamide;

[0010] (3) Gas chromatography-mass spectrometry was used to detect the test solution, and the content of primary metabolites and / or secondary metabolites in the fragrance and flavor was obtained by internal standard method.

[0011] This invention first oximates and silanizes the fragrance and flavoring. The silanizing agent is a combination of N-methyl-N-(trimethylsilyl)trifluoroacetamide and N,O-bis(trimethylsilyl)trifluoroacetamide, which work synergistically to further promote the silanization reaction. Then, gas chromatography-mass spectrometry is used for detection. The content of primary metabolites and / or secondary metabolites can be obtained by using the internal standard method, which effectively improves the precision and accuracy of the detection.

[0012] The flavorings and fragrances described in this invention are preferably tobacco flavorings and fragrances, including but not limited to tobacco leaf extract, fig extract, passion fruit extract, Indian gooseberry extract, jujube extract, Alpinia oxyphylla extract, dragon fruit extract, etc.

[0013] The primary metabolites of this invention include any one or a combination of at least two of sugars, amino acids, organic acids or nucleotides; the secondary metabolites include any one or a combination of at least two of alkaloids, flavonoids, terpenes or phenols.

[0014] Preferably, the internal standard in step (1) includes any one or a combination of at least two of n-heptadecane, ribitol, or phenethyl acetate.

[0015] In this invention, the composition of the substance being detected is complex. When heptadecane is used as an internal standard, its peaks do not interfere with those of the analyte, which can further improve the precision and accuracy of the detection.

[0016] Preferably, the solvent in step (1) includes any one or a combination of at least two of methanol, dichloromethane, ethanol or propylene glycol, and is preferably a combination of methanol and dichloromethane.

[0017] Preferably, the mass ratio of the internal standard and the flavoring in step (1) is 1:(0.5-2.0) (for example, it can be 1:0.5, 1:0.8, 1:1, 1:1.5, 1:2.0, etc.).

[0018] Preferably, the extraction method in step (1) includes centrifugation.

[0019] Preferably, the centrifugation speed is 4000-6000 rpm (e.g., 4000 rpm, 4500 rpm, 5000 rpm, 5500 rpm, 6000 rpm, etc.), and the time is 10-20 min (e.g., 10 min, 12 min, 15 min, 18 min, 20 min, etc.).

[0020] Preferably, the oxime reagent in step (2) includes methoxyamine hydrochloride.

[0021] Preferably, the solvent in step (2) includes any one or a combination of at least two of pyridine, ethanol, methanol or dichloromethane.

[0022] Preferably, the ratio of the dry product, oxime reagent and silanizing reagent used in step (2) is 1 mg:(0.4-2.0) mg:(20-40) μL.

[0023] The values ​​mentioned above (0.4-2.0) can be, for example, 0.4, 0.5, 0.6, 0.7, 0.8, 1, 2, etc.; (20-40) can be, for example, 20, 25, 30, 35, 40, etc.

[0024] Preferably, the volume ratio of N-methyl-N-(trimethylsilyl)trifluoroacetamide and N,O-bis(trimethylsilyl)trifluoroacetamide in step (2) is 1:(0.8-1.2) (for example, it can be 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, etc.).

[0025] Preferably, the temperature of the oxime reaction in step (2) is 30-40℃ (e.g., 30℃, 32℃, 35℃, 38℃, 40℃, etc.), and the time is 60-120min (e.g., 60min, 80min, 100min, 120min, etc.).

[0026] Preferably, the temperature of the silanization reaction in step (2) is 45-55℃ (e.g., 45℃, 48℃, 50℃, 52℃, 55℃, etc.), and the time is 60-120min (e.g., 60min, 80min, 100min, 120min, etc.).

[0027] Preferably, the stationary phase of the gas chromatography column in step (3) is a methyl polysiloxane containing 30-40% (e.g., 30%, 32%, 35%, 38%, 40%, etc.) phenyl.

[0028] Preferably, the gas chromatograph has a column length of 50-70m (e.g., 50m, 55m, 60m, 65m, 70m, etc.), a diameter of 0.2-0.3mm (e.g., 0.2mm, 0.22mm, 0.25mm, 0.28mm, 0.3mm, etc.), and a film thickness of 0.2-0.3μm (e.g., 0.2μm, 0.22μm, 0.25μm, 0.28μm, 0.3μm, etc.).

[0029] Preferably, the column temperature program for the gas chromatography in step (3) is as follows: 95-105℃ (e.g., 95℃, 98℃, 100℃, 102℃, 105℃, etc.) held for 0.5-1.5 min (e.g., 0.5 min, 0.8 min, 1 min, 1.2 min, 1.5 min, etc.), then increased to 180-185℃ (e.g., 180℃, 182℃, 184℃, 185℃, etc.) at a rate of 2.5-3.5℃ / min (e.g., 2.5℃ / min, 2.8℃ / min, 3℃ / min, 3.2℃ / min, 3.5℃ / min, etc.), and then increased at a rate of 0.4-0.6℃ / min (e.g., 0.4℃ / min, 0.45℃ / min, 0.5℃ / min, etc.). Increase the temperature at a rate of 0.55℃ / min, 0.6℃ / min, etc., to 186-195℃ (e.g., 186℃, 188℃, 190℃, 192℃, 195℃, etc.), hold for 0.5-1.5min (e.g., 0.5min, 0.8min, 1min, 1.2min, 1.5min, etc.), and finally increase the temperature at a rate of 12-18℃ / min (e.g., 12℃ / min, 14℃ / min, 16℃ / min, 18℃ / min, etc.) to 275-285℃ (e.g., 275℃, 278℃, 280℃, 282℃, 285℃, etc.), hold for 3-8min (e.g., 3min, 4min, 5min, 6min, 8min, etc.).

[0030] In this invention, the gas chromatography employs a three-stage temperature program, which can improve the separation of the analytes and achieve the technical effect of improving detection precision and accuracy.

[0031] Preferably, the injection port temperature of the gas chromatograph in step (3) is 240-260℃ (for example, it can be 240℃, 245℃, 250℃, 255℃, 260℃, etc.).

[0032] Preferably, the split ratio of the gas chromatograph in step (3) is (5-15):1 (for example, it can be 5:1, 8:1, 10:1, 12:1, 15:1, etc.).

[0033] Preferably, the transfer line temperature of the mass spectrometer in step (3) is 240-260℃ (e.g., 240℃, 245℃, 250℃, 255℃, 260℃, etc.).

[0034] Preferably, the ion source temperature of the mass spectrometer in step (3) is 200-300℃ (for example, it can be 200℃, 220℃, 250℃, 280℃, 300℃, etc.).

[0035] Preferably, the mass spectrometry scanning range in step (3) is 45-600 m / z.

[0036] Compared with the prior art, the present invention has at least the following beneficial effects:

[0037] This invention first oximates and silanizes the fragrance and flavoring. The silanizing agent is a combination of N-methyl-N-(trimethylsilyl)trifluoroacetamide and N,O-bis(trimethylsilyl)trifluoroacetamide, which work synergistically to further promote the silanization reaction. Then, gas chromatography-mass spectrometry is used for detection. The content of primary metabolites and / or secondary metabolites can be obtained by using the internal standard method, achieving precise, accurate and comprehensive detection results. Attached Figure Description

[0038] Figure 1 This is the GCMS total ion chromatogram of primary metabolites derived in Example 1. Detailed Implementation

[0039] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0040] Example 1

[0041] This embodiment provides a method for detecting primary metabolites and / or secondary metabolites in fragrances and flavorings, comprising the following steps:

[0042] (1) Prepare 300 μL of 0.1 mg / mL methanol solution of fragrance and flavoring, add 200 μL of 0.2 mg / mL n-heptadecane in dichloromethane solution, centrifuge at 25℃ and 5000 rpm for 15 min; pipette 0.1 mL of supernatant into a 2.0 mL sample bottle, and blow dry the liquid with a nitrogen blower to obtain the dry product;

[0043] (2) Take 5 mg of dried product, add 150 μL of pyridine solution of 20 mg / mL methoxyamine hydrochloride, and carry out oxime reaction at 37℃ for 90 min; then add 200 μL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) at a volume ratio of 1:1 and carry out silanization reaction at 50℃ for 90 min. After filtering the reaction solution with a 0.45 μm organic phase microporous filter membrane, the test solution is obtained and placed at 4℃ for gas chromatography-mass spectrometry detection.

[0044] (3) Gas chromatography-mass spectrometry (GC-MS) was used to detect the test solution. The total ion chromatogram was analyzed by GC-MS reanalysis software. The mass spectral fragment peaks of each metabolite were compared with the NIST05 and Wiley databases. Substances with similarity greater than 80% were qualitatively identified. Then, the corresponding mass spectral peaks were manually integrated. The internal standard method was used to perform relative quantitative analysis of each substance to obtain the content of primary metabolites and / or secondary metabolites in the fragrance and flavor.

[0045] The detection conditions for gas chromatography are as follows:

[0046] Chromatographic column: DB-35MS capillary column (60m×0.25mm×0.25μm); Injector temperature: 250℃; Carrier gas: High-purity nitrogen (99.999%), Carrier gas flow rate: 1.2mL / min; Split ratio: 10:1; Injection volume: 1μL; Temperature program: Hold at 100℃ for 1min, increase to 184℃ at 3℃ / min, then increase to 190℃ at 0.5℃ / min and hold for 1min, and finally increase to 280℃ at 15℃ and hold for 5min.

[0047] The detection conditions for mass spectrometry are as follows:

[0048] Ion source: 70eV; transfer line temperature: 250℃; ion source temperature: 230℃; scan range: 45-600m / z.

[0049] Example 2

[0050] This embodiment provides a method for detecting primary metabolites and / or secondary metabolites in fragrances and flavorings, comprising the following steps:

[0051] (1) Prepare 200 μL of 0.3 mg / mL methanol solution of fragrance and flavoring, add 300 μL of 0.2 mg / mL n-heptadecane in dichloromethane solution, and centrifuge at 25℃ and 4000 rpm for 20 min; pipette 0.1 mL of supernatant into a 2.0 mL sample bottle, and blow dry the liquid with a nitrogen blower to obtain the dry product;

[0052] (2) Take 5 mg of dried product, add 100 μL of pyridine solution of 20 mg / mL methoxyamine hydrochloride, and carry out oxime reaction at 30 °C for 120 min; then add 100 μL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) at a volume ratio of 1:0.8 and carry out silanization reaction at 45 °C for 120 min. After filtering the reaction solution with a 0.45 μm organic phase microporous filter membrane, the test solution is obtained and placed at 4 °C for gas chromatography-mass spectrometry detection.

[0053] (3) Gas chromatography-mass spectrometry (GC-MS) was used to detect the test solution. The total ion chromatogram was analyzed by GC-MS reanalysis software. The mass spectral fragment peaks of each metabolite were compared with the NIST05 and Wiley databases. Substances with similarity greater than 80% were qualitatively identified. Then, the corresponding mass spectral peaks were manually integrated. The internal standard method was used to perform relative quantitative analysis of each substance to obtain the content of primary metabolites and / or secondary metabolites in the fragrance and flavor.

[0054] The detection conditions for gas chromatography are as follows:

[0055] Chromatographic column: DB-35MS capillary column (60m×0.25mm×0.25μm); Injector temperature: 240℃; Carrier gas: High-purity nitrogen (99.999%), Carrier gas flow rate: 1.2mL / min; Split ratio: 5:1; Injection volume: 1μL; Temperature program: Hold at 105℃ for 0.5min, increase to 180℃ at 2.5℃ / min, then increase to 186℃ at 0.4℃ / min and hold for 1.5min, and finally increase to 275℃ at 12℃ and hold for 8min;

[0056] The detection conditions for mass spectrometry are as follows:

[0057] Ion source: 70eV; transfer line temperature: 240℃; ion source temperature: 240℃; scan range: 45-600m / z.

[0058] Example 3

[0059] This embodiment provides a method for detecting primary metabolites and / or secondary metabolites in fragrances and flavorings, comprising the following steps:

[0060] (1) Prepare 400 μL of 0.1 mg / mL methanol solution of fragrance and flavoring, add 300 μL of 0.2 mg / mL n-heptadecane in dichloromethane solution, centrifuge at 25℃ and 6000 rpm for 10 min; pipette 0.1 mL of supernatant into a 2.0 mL sample bottle, and blow dry the liquid with a nitrogen blower to obtain the dry product;

[0061] (2) Take 7 mg of dried product, add 250 μL of pyridine solution of 20 mg / mL methoxyamine hydrochloride, and carry out oxime reaction at 40 °C for 70 min; then add 250 μL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) at a volume ratio of 1:1.2 and carry out silanization reaction at 55 °C for 80 min. After filtering the reaction solution with a 0.45 μm organic phase microporous filter membrane, the test solution is obtained and placed at 4 °C for gas chromatography-mass spectrometry detection.

[0062] (3) Gas chromatography-mass spectrometry (GC-MS) was used to detect the test solution. The total ion chromatogram was analyzed by GC-MS reanalysis software. The mass spectral fragment peaks of each metabolite were compared with the NIST05 and Wiley databases. Substances with similarity greater than 80% were qualitatively identified. Then, the corresponding mass spectral peaks were manually integrated. The internal standard method was used to perform relative quantitative analysis of each substance to obtain the content of primary metabolites and / or secondary metabolites in the fragrance and flavor.

[0063] The detection conditions for gas chromatography are as follows:

[0064] Chromatographic column: DB-35MS capillary column (60m×0.25mm×0.25μm); Injector temperature: 260℃; Carrier gas: High-purity nitrogen (99.999%), Carrier gas flow rate: 1.2mL / min; Split ratio: 15:1; Injection volume: 1μL; Temperature program: Hold at 95℃ for 1.5min, increase to 185℃ at 3.5℃ / min, then increase to 195℃ at 0.6℃ / min and hold for 0.5min, and finally increase to 285℃ at 18℃ and hold for 3min.

[0065] The detection conditions for mass spectrometry are as follows:

[0066] Ion source: 70eV; transfer line temperature: 260℃; ion source temperature: 270℃; scan range: 45-600m / z.

[0067] Example 4

[0068] This embodiment provides a method for detecting primary metabolites and / or secondary metabolites in fragrances and flavorings. The only difference between this method and Example 1 is that in step (1), n-heptadecane is replaced with an equal amount of ribitol, and methanol is used as the solvent. Other steps are the same as in Example 1.

[0069] Example 5

[0070] This embodiment provides a method for detecting primary metabolites and / or secondary metabolites in fragrances and flavorings. The only difference between this method and Example 1 is that in step (1), n-heptadecane is replaced with an equal amount of deuterated tridecanoic acid, and ethanol is used as the solvent. Other steps are the same as in Example 1.

[0071] Example 6

[0072] This embodiment provides a method for detecting primary metabolites and / or secondary metabolites in fragrances and flavorings. The only difference between this method and Example 1 is that the heating program in step (3) is as follows: maintain 100°C for 1 min, increase the temperature to 184°C at 3°C / min, increase the temperature to 280°C at 15°C and maintain for 5 min, and the rest is the same as in Example 1.

[0073] Example 7

[0074] This embodiment provides a method for detecting primary metabolites and / or secondary metabolites in fragrances and flavorings. The only difference between this method and Example 1 is that the heating procedure in step (3) is as follows: maintain 100°C for 1 min, then increase the temperature to 280°C at 15°C and maintain for 5 min. Other steps are the same as in Example 1.

[0075] Comparative Example 1

[0076] This comparative example provides a method for detecting primary metabolites and / or secondary metabolites in fragrances and flavorings. The only difference between this method and Example 1 is that MSTFA is not added in the silanization reaction in step (2), and its amount is allocated to BSTFA. Other aspects are the same as in Example 1.

[0077] Comparative Example 2

[0078] This comparative example provides a method for detecting primary metabolites and / or secondary metabolites in fragrances and flavorings. The only difference between this method and Example 1 is that BSTFA is not added in the silanization reaction in step (2), and its amount is allocated to MSTFA. Other aspects are the same as in Example 1.

[0079] Test Example 1

[0080] Precision testing

[0081] The tobacco leaf extract was tested using the methods of Examples 1-7 and Comparative Examples 1-2. Three tests were performed, and the average value was taken. The detection results of some primary and secondary metabolites are shown in Table 1 (Examples 2-7 and Comparative Examples 1-2 present data representing some metabolites). For example, the total ion chromatogram (0-24 min) in Example 1 is shown below. Figure 1 As shown.

[0082] Table 1

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] Test results:

[0089] (1) As can be seen from Examples 1 to 7, the present invention can improve the precision of detection by first oximating and silanizing the fragrance and flavor and then using gas chromatography-mass spectrometry.

[0090] (2) By comparing Example 1 with Examples 4-5, it can be seen that the present invention can achieve a better technical effect of improving detection precision by further optimizing the internal standard, which does not interfere with the metabolite components.

[0091] A comparison of Examples 1 and 6-7 shows that the present invention employs a three-stage heating program, resulting in better separation of chromatographic peaks and further improving the precision of detection.

[0092] (3) As can be seen from the comparison between Example 1 and Comparative Examples 1-2, the present invention uses a compounded silanizing reagent, which can promote the silanization reaction and improve the precision of detection.

[0093] Test Example 2

[0094] Accuracy test

[0095] The recovery rate was calculated by taking the average value of three tests on a standard sample of tobacco extract containing 600 mg / L glucose. The results are shown in Table 2.

[0096] Table 2

[0097] Grouping Average recovery rate (%) RSD (%) Example 1 94.08 0.89 Example 2 93.46 1.41 Example 3 92.94 2.04 Example 4 92.46 1.69 Example 5 90.22 2.32 Example 6 89.63 2.85 Example 7 89.47 2.69 Comparative Example 1 85.08 1.32 Comparative Example 2 85.94 1.85

[0098] The test results show that the detection method provided by this invention has a high recovery rate, which can reach 85.08-94.08% within the preferred range, indicating that the method has high accuracy.

[0099] Test Example 3

[0100] The fig Maillard reactant was used as the flavoring and fragrance sample to be tested, and the method provided in Example 1 was used for testing. The test results are shown in Table 3.

[0101] Table 3

[0102]

[0103]

[0104] The test results show that the detection method provided by this invention can be applied to a variety of fragrances and flavorings, and has high detection accuracy and precision.

[0105] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for detecting primary metabolites and / or secondary metabolites in fragrances and flavorings, characterized in that, The method includes the following steps: (1) Mix the flavoring, internal standard and solvent to extract, remove the solvent from the clear liquid, and obtain the dried product; (2) The dry product, oxime reagent and solvent are mixed to carry out oxime reaction, and then the reaction system is mixed with silanizing reagent to carry out silanization reaction to obtain the test solution; the silanizing reagent includes a combination of N-methyl-N-(trimethylsilyl)trifluoroacetamide and N,O-bis(trimethylsilyl)trifluoroacetamide; (3) Gas chromatography-mass spectrometry was used to detect the test solution, and the content of primary metabolites and / or secondary metabolites in the fragrance and flavor was obtained by internal standard method.

2. The method according to claim 1, characterized in that, The internal standard in step (1) includes any one or a combination of at least two of n-heptadecane, ribitol, or phenethyl acetate; Preferably, the solvent in step (1) includes any one or a combination of at least two of methanol, dichloromethane, ethanol or propylene glycol, and is preferably a combination of methanol and dichloromethane; Preferably, the mass ratio of the internal standard and the flavoring in step (1) is 1:(0.5-2.0).

3. The method according to claim 1 or 2, characterized in that, The extraction method described in step (1) includes centrifugation; Preferably, the centrifugation speed is 4000-6000 rpm and the time is 10-20 min.

4. The method according to any one of claims 1-3, characterized in that, The oxime reagent in step (2) includes methoxyamine hydrochloride; Preferably, the solvent in step (2) includes any one or a combination of at least two of pyridine, ethanol, methanol or dichloromethane.

5. The method according to any one of claims 1-4, characterized in that, The ratio of the dry product, oxime reagent, and silanizing reagent used in step (2) is 1 mg:(0.4-2.0) mg:(20-40) μL; Preferably, the volume ratio of N-methyl-N-(trimethylsilyl)trifluoroacetamide and N,O-bis(trimethylsilyl)trifluoroacetamide in step (2) is 1:(0.8-1.2).

6. The method according to any one of claims 1-5, characterized in that, The oxime reaction in step (2) is carried out at a temperature of 30-40℃ for a time of 60-120 min; Preferably, the temperature of the silanization reaction in step (2) is 45-55°C and the time is 60-120 min.

7. The method according to any one of claims 1-6, characterized in that, The stationary phase of the gas chromatography column in step (3) is a methyl polysiloxane containing 30-40% phenyl groups; Preferably, the gas chromatograph has a column length of 50-70m, a diameter of 0.2-0.3mm, and a film thickness of 0.20-0.30μm.

8. The method according to any one of claims 1-7, characterized in that, The column temperature program for gas chromatography in step (3) is as follows: hold at 95-105℃ for 0.5-1.5 min, then increase the temperature to 180-185℃ at a rate of 2.5-3.5℃ / min, then increase the temperature to 186-195℃ at a rate of 0.4-0.6℃ / min, hold for 0.5-1.5 min, and finally increase the temperature to 275-285℃ at a rate of 12-18℃ / min, hold for 3-8 min.

9. The method according to any one of claims 1-8, characterized in that, The injection port temperature of the gas chromatograph in step (3) is 240-260℃; Preferably, the split ratio of the gas chromatography in step (3) is (5-15):

1.

10. The method according to any one of claims 1-9, characterized in that, The transfer line temperature of the mass spectrometer in step (3) is 240-260℃; Preferably, the ion source temperature of the mass spectrometer in step (3) is 200-300℃; Preferably, the mass spectrometry scanning range in step (3) is 45-600 m / z.

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