Construction method of aquilaria sinensis GC-MS fingerprint spectrum, fingerprint spectrum and application

Abstract

The invention provides an aquilaria sinensis GC-MS (gas chromatography-mass spectrometry) fingerprint spectrum construction method which comprises the following steps: extracting volatile substances in aquilaria sinensis by adopting a steam distillation method in combination with liquid-liquid extraction, and analyzing the extracted substances by adopting GC-MS; determining common components according to GC-MS data in combination with retention index calculation and NIST standard mass spectrum library retrieval; and constructing the aquilaria sinensis GC-MS fingerprint spectrum. The aquilaria sinensis GC-MS fingerprint spectrum constructed by the method can be used for quickly identifying coarse, inferior and counterfeit products obtained by soaking common wood in essence, can be used for effectively distinguishing the aquilaria sinensis from different producing areas, and fills the blank that the market is lack of exclusive identification standards.

Description

Technical Field

[0001] This invention relates to the field of analytical testing technology, specifically to a method for constructing a GC-MS fingerprint of agarwood, the fingerprint spectrum, and its applications. Background Technology

[0002] Agarwood, a precious spice and traditional Chinese medicine unique to China, specifically refers to a solid concretion rich in aromatic resin formed in the xylem of the native Aquilaria sinensis tree (Aquilaria agallocha), a member of the Thymelaeaceae family, after natural or human damage, through long-term infection by specific fungi and complex biochemical reactions. Agarwood is not only a core element of traditional incense culture due to its elegant, sweet, and layered aroma with unique floral and honey notes, but it is also listed in the Chinese Pharmacopoeia as a valuable medicinal material with effects such as promoting qi circulation, relieving pain, and warming the stomach to stop vomiting. However, its extremely high economic value has led to rampant market irregularities, such as using resin-free Aquilaria sinensis wood to imitate it, impregnating and processing other tree species, or mixing low-priced agarwood from other producing areas with high-priced Agarwood. Traditional identification methods rely heavily on the personal sensory experience of the appraiser, are highly subjective, have poor repeatability, and struggle to establish objective and unified judgment standards, failing to meet the urgent needs of market supervision, fair trade, and judicial appraisal.

[0003] Against this backdrop, gas chromatography-mass spectrometry (GC-MS) has become the mainstream technique for constructing "chemical fingerprints" of complex volatile components for substance identification, due to its combination of the high separation efficiency of GC and the powerful structural resolution capabilities of mass spectrometry. Although some studies have applied GC-MS fingerprinting to the analysis of agarwood, most studies aim to establish universal identification models covering agarwood from various producing regions, failing to focus on and extract the unique chemical characteristic patterns that distinguish Dongguan agarwood from other producing regions. This results in weak identification capabilities of the models against highly counterfeit products (such as agarwood from other producing regions).

[0004] For example, patent CN106053645A discloses a method for identifying the authenticity of agarwood. It involves constructing a GC / MS fingerprint of the agarwood sample to be tested. If the GC / MS fingerprint of the agarwood sample is substantially consistent with that of natural agarwood and contains the six characteristic components: ① benzylacetone, ② p-methoxybenzylacetone, ③ agarwood furan, ④ agarwood spirulinolate, ⑤ atractylol, and ⑥ agarwood aldehyde, it is determined to be natural agarwood. If the GC / MS fingerprint of the agarwood sample differs significantly from that of natural agarwood, containing only some of the six characteristic components or none of them, it is determined to be fake agarwood.

[0005] In summary, there is an urgent need for a GC-MS fingerprinting method that is highly targeted, capable of stably extracting specific chemical features from complex matrices, and capable of in-depth data analysis, in order to achieve scientific and accurate identification of the authenticity and quality of agarwood. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention aims to provide a method for constructing a GC-MS fingerprint spectrum of agarwood. The method involves analyzing the extracted substances using GC-MS; identifying common components based on the GC-MS data, combined with retention index calculation and NIST standard mass spectrometry library search; and constructing a GC-MS fingerprint spectrum of agarwood. This method is specifically designed to address the chemical characteristics of agarwood and can be used to distinguish its authenticity.

[0007] Firstly, a method for constructing a GC-MS fingerprint of agarwood is provided, comprising the following steps:

[0008] S1: Weigh multiple batches of agarwood sample powder, and use steam distillation combined with liquid-liquid extraction to obtain the sample solution;

[0009] S2: Inject the sample solution into GC-MS for detection and analysis;

[0010] S3: By analyzing GC-MS data from multiple batches of agarwood samples, combined with retention index calculation and NIST standard mass spectrometry library search, common components were identified;

[0011] S4: Import the data files corresponding to each batch of test solution into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software for data processing and establish the GC-MS fingerprint spectrum of agarwood.

[0012] In some embodiments, in step S1, the steam distillation method combined with liquid-liquid extraction is specifically as follows: water and glass beads are added to the agarwood sample powder, a volatile oil detector is connected, water is added from the top of the detector until it overflows into the flask, then an extraction solvent is added, a reflux condenser is connected, the mixture is heated to boiling and then gently boiled for extraction, the extraction solvent layer is collected, purified, and the sample solution is obtained.

[0013] This invention employs a combination of steam distillation and liquid-liquid extraction for the pretreatment of agarwood powder. This method comprehensively and efficiently extracts volatile / semi-volatile components from the sample, including characteristic substances with high boiling points and low volatility (such as high-boiling-point sesquiterpenes). These components are then physically separated from the abundant non-volatile resins, pigments, sugars, and lignin in the agarwood matrix, thereby reducing matrix interference in subsequent gas chromatography-mass spectrometry analysis and improving the specificity and sensitivity of the detection. Furthermore, the equipment used is simple and inexpensive, requiring no complex traps or other specialized equipment, making the method easier to standardize and popularize.

[0014] In some embodiments, in step S1, the solid-liquid ratio of the agarwood powder to the water is 1.5 to 2 g / L.

[0015] In some embodiments, in step S1, the extraction solvent is ethyl acetate.

[0016] In some embodiments, in step S1, the amount of the extraction solvent used is 1.5~5 mL.

[0017] In some embodiments, in step S1, the micro-boiling extraction takes 4-6 hours.

[0018] In some embodiments, in step S1, purification refers to adding anhydrous sodium sulfate to the collected extraction solvent layer, allowing it to stand for 0.5 to 2 hours, and then collecting the supernatant. Anhydrous sodium sulfate can further remove residual aqueous solution from the extraction solvent layer, resulting in less interference in subsequent analysis and detection of the prepared sample solution.

[0019] In some embodiments, in step S2, the gas chromatography conditions of the GC-MS are as follows: using an inert capillary column, with programmed temperature ramping: initial temperature 80~95℃, hold for 0.5~1.5 min; ramp to 130~150℃ at a rate of 4~6℃ / min, hold for 4~6 min; ramp to 160~180℃ at a rate of 1~2℃ / min, hold for 4~6 min; ramp to 260~300℃ at a rate of 8~12℃ / min, hold for 12~16 min; linear velocity control mode, with a linear velocity of 35~40 cm / sec; injection port temperature of 180~220℃; carrier gas is high-purity helium; carrier gas flow rate is 0.5~2.0 mL / min; splitless injection; injection volume of 0.5~2 μL.

[0020] More preferably, the programmed temperature rise is as follows: starting temperature 90°C, held for 1 min; rising to 140°C at a rate of 5°C / min, held for 5 min; rising to 170°C at a rate of 1°C / min, held for 5 min; rising to 280°C at a rate of 10°C / min, held for 15 min.

[0021] More preferably, the linear velocity is 37 cm / sec; the injection port temperature is 200°C; the carrier gas flow rate is 1 mL / min; and the injection volume is 1 μL.

[0022] In some embodiments, the inert capillary column is an Agilent HP-5MS, and the inert capillary column has dimensions of 30m × 0.25mm × 0.25μm.

[0023] The gas chromatography conditions employed in this invention effectively separate the volatile substances of agarwood. The programmed temperature gradient setting in this invention ensures efficient vaporization and transport of volatile substances within the capillary column, avoiding condensation and adsorption losses. Simultaneously, it prevents severe overlap or co-eluting of volatile substances with other structurally similar sesquiterpenes and oxygen-containing derivatives present in the agarwood matrix, resulting in sharp, symmetrical chromatographic peaks and thus achieving accurate characterization.

[0024] In some embodiments, in step S2, the GC-MS mass spectrometry conditions are as follows: an electron impact (EI) ion source is used; the ionization energy is 65~75eV; the ion source temperature is 220~240℃; the interface temperature is 240~260℃; the solvent delay time is 2~4min; the SCAN mode is used; and the mass spectrometry scanning range is 50-500m / z.

[0025] More preferably, the ionization energy is 70 eV; the ion source temperature is 230°C; the interface temperature is 250°C; and the solvent delay time is 3 min.

[0026] In some embodiments, the determination of the common components in step S3 specifically involves: taking a standard solution of n-alkanes (C8-C40), and collecting GC-MS data under the same gas chromatography-mass spectrometry conditions as the sample solution; using the automatic retention time adjustment (AART) function of the Shimadzu GC-MS solution workstation to calculate the retention indices (RI) of each chromatographic peak, and qualitatively analyzing the extracted mass spectrometry information in the NIST 17 standard spectral library in conjunction with the retention indices RI, with the substance having the highest matching degree between the mass spectrum and the RI value being the best identification result, and thus identifying the common components.

[0027] In some embodiments, step S3, the identification of the common components, further includes comparing the GC-MS data of the sample solution with the GC-MS data of the standard solution to achieve identification.

[0028] In some embodiments, the reference solution is an ethyl acetate solution of 1 mg / mL for 4-phenyl-2-butanone, 4-methoxyphenyl-2-butanone, and nocaketone.

[0029] In some embodiments, the common components include 4-phenyl-2-butanone, 4-methoxyphenyl-2-butanone, nootkaton-11,12-epoxide, and 3,5,11-Eudesmatriene. Using these common components as identifying characteristic compounds is more robust than relying on a large number of unstable common peak patterns; even if the minor components of the sample fluctuate due to differences in origin and grade, as long as the core marker group is present and the proportion is within a reasonable range, a positive result can be obtained.

[0030] In some embodiments, step S4, establishing the fingerprint spectrum, specifically involves: importing the total ion chromatogram data of multiple batches of samples obtained in step S2 into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System"; cutting the chromatograms from 5 to 70 minutes, and performing multi-point correction and automatic matching with a median and time width of 0.2 to automatically generate a reference fingerprint spectrum for agarwood. This invention uses the median method to generate the reference fingerprint spectrum, which, compared to the mean method, is better able to eliminate interference from abnormal samples, and the established "standard fingerprint" is more representative of the population.

[0031] Secondly, the present invention provides a GC-MS fingerprint spectrum of agarwood, which is constructed by the method described in the first aspect.

[0032] Thirdly, the present invention provides the application of the GC-MS fingerprint spectrum of agarwood described in the second aspect, including the following steps: importing the GC-MS spectrum of the sample to be tested into the similarity evaluation system of chromatographic fingerprint spectrum of traditional Chinese medicine, performing peak matching with the GC-MS fingerprint spectrum of agarwood, and if the similarity is above 0.90, the sample to be tested is identified as agarwood.

[0033] Compared with the prior art, one of the above technical solutions has the following advantages or beneficial effects:

[0034] This invention provides a method for constructing a GC-MS fingerprint of agarwood. The method employs steam distillation combined with liquid-liquid extraction for pretreatment of agarwood. This method can comprehensively and efficiently extract volatile / semi-volatile components from the sample and physically separate them from the large amounts of non-volatile resins, pigments, sugars, and lignin in the agarwood matrix. This reduces matrix interference in subsequent gas chromatography-mass spectrometry analysis, improving the specificity and sensitivity of the detection. The method also optimizes the chromatographic and mass spectrometric conditions for GC-MS detection and analysis, effectively avoiding problems such as co-elution, poor peak shape, or insufficient detection sensitivity caused by the high boiling point and special chromatographic retention behavior of key substances like nocaketone.

[0035] This invention provides a method for constructing a fingerprint spectrum of agarwood using GC-MS. When constructing the fingerprint spectrum, a retention index is introduced for dual qualitative analysis, which greatly enhances the specificity and reliability of feature peak identification and reduces the risk of misjudgment. At the same time, the fingerprint spectrum generated by the median method is more representative of the population.

[0036] The GC-MS fingerprint spectrum of agarwood provided by this invention can quickly identify inferior counterfeit products made by soaking ordinary wood in fragrance, and can also effectively distinguish agarwood from different origins, filling the gap in the market where there is a lack of exclusive identification standards. Attached Figure Description

[0037] Figure 1Example 1 provides the GC-MS fingerprint spectrum of agarwood. Terminology Explanation

[0038] Certain embodiments of the invention will now be described in detail, examples of which are illustrated by the accompanying structural and chemical formulas. The invention is intended to cover all alternatives, modifications, and equivalents, all of which are included within the scope of the invention as defined in the claims. Those skilled in the art will recognize that many similar or equivalent methods and materials can be used to practice the invention. The invention is by no means limited to the methods and materials described herein. In the event that one or more of the incorporated documents, patents, and similar materials differ from or contradict this application (including, but not limited to, defined terminology, application of terminology, described techniques, etc.), this application shall prevail.

[0039] It should be further appreciated that certain features of the invention, for clarity, have been described in multiple independent embodiments, but may also be provided in combination in a single embodiment. Conversely, various features of the invention, for brevity, have been described in a single embodiment, but may also be provided individually or in any suitable sub-combination.

[0040] Unless otherwise stated, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. All patents and publications related to this invention are incorporated herein by reference in their entirety.

[0041] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0042] In the following content, all numbers disclosed herein, whether or not they use words such as "approximately" or "about," are approximate values. The value of each number may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20%. Whenever a number with a value of N is disclosed, any number having a value of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15%, or N+ / -20% will be explicitly disclosed, where "+ / -" indicates addition or subtraction. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0044] All reagents used in this invention can be purchased commercially or prepared by the methods described in this invention.

[0045] Example 1

[0046] A method for constructing a GC-MS fingerprint of agarwood includes the following steps:

[0047] S1: Take 0.5g of each of 25 batches of agarwood sample powder (passed through a No. 3 sieve), place them in a 500mL flask, add 300mL of water and a few glass beads, shake to mix, connect the volatile oil analyzer, add water until it fills the graduated part of the volatile oil analyzer and overflows into the flask, add 2mL of ethyl acetate from the top of the volatile oil analyzer, connect the reflux condenser, heat slowly to boiling, and maintain a gentle boil for about 5 hours. After cooling, take the ethyl acetate layer, place it in a small bottle containing anhydrous sodium sulfate, let it stand for 1 hour, take the supernatant, filter it, and the sample solution is obtained.

[0048] S2: Inject the sample solution into the GC-MS for detection and analysis.

[0049] Gas chromatography conditions: An inert capillary column, Agilent HP-5MS, 30m × 0.25mm × 0.25μm, was used; the temperature program was as follows: initial temperature 90℃, hold for 1 min; increase to 140℃ at a rate of 5℃ / min, hold for 5 min; increase to 170℃ at a rate of 1℃ / min, hold for 5 min; increase to 280℃ at a rate of 10℃ / min, hold for 15 min; linear velocity control mode, the linear velocity was 37cm / sec; the injection port temperature was 220℃; the carrier gas was high-purity helium, the carrier gas flow rate was 1.0mL / min; splitless injection, injection volume was 1μL.

[0050] The mass spectrometry conditions were as follows: electron impact (EI) ion source; ionization energy of 70 eV; ion source temperature of 230 °C; interface temperature of 250 °C; solvent delay time of 3 min; SCAN mode; mass spectrometry scanning range of 50-500 m / z.

[0051] S3: Common components were identified. A standard solution of n-alkanes (C8-C40) was taken and GC-MS data was acquired under the same gas chromatography-mass spectrometry conditions as the sample solution. The obtained data were analyzed using the Automatic Retention Time Adjustment (AART) function of the Shimadzu GC-MS solution workstation to calculate the retention indices (RI) of each chromatographic peak. The extracted mass spectrometry information was used in the NIST 17 standard spectral library in conjunction with the retention indices (RI) for qualitative analysis. The substance with the highest match between the mass spectrum and the RI value was taken as the best identification result, and the common components were identified.

[0052] S4: Import the total ion chromatogram data of multiple batches of samples obtained in step S2 into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System"; cut the chromatograms from 5 to 70 minutes, and perform multi-point correction and automatic matching with a median and time width of 0.2 to automatically generate a reference fingerprint chromatogram of agarwood. This invention uses the median method to generate the reference fingerprint chromatogram, which can better eliminate the interference of abnormal samples compared with the mean method, and the established "standard fingerprint" is more representative of the population.

[0053] Of the 25 batches of agarwood samples mentioned above, 5 common components were identified, as detailed in Table 1; the generated agarwood GC-MS reference fingerprint is detailed in [reference needed]. Figure 1 .

[0054] Table 1. Peak information among 25 batches of agarwood samples

[0055] Component Name retention time min Retention Index 1 4-Phenylon-2-Butanone 8.241 1250 2 4-Methoxyphenyl-2-butanone 15.206 1503 3 Nocaketone 32.604 1796 4 Nootkaton-11,12-epoxide 34.834 1825 5 3,5,11-Eudesmatriene 53.531 2078

[0056] Example 2

[0057] Application of GC-MS fingerprinting of agarwood

[0058] Weigh out the agarwood powder soaked in fragrance from ordinary wood, and process and analyze it according to steps S1 and S2 in Example 1 to obtain the GC-MS spectrum of the sample to be tested; import the GC-MS spectrum of the sample to be tested into the similarity evaluation system of chromatographic fingerprint spectrum of traditional Chinese medicine, and perform peak matching with the GC-MS fingerprint spectrum of agarwood; the results show that the similarity is 0.77, and the sample to be tested is a counterfeit agarwood.

[0059] Example 3

[0060] Application of GC-MS fingerprinting of agarwood

[0061] Weigh out agarwood powder and process and analyze it according to steps S1 and S2 in Example 1 to obtain the GC-MS spectrum of the sample to be tested. Import the GC-MS spectrum of the sample to be tested into the similarity evaluation system of chromatographic fingerprint spectrum of traditional Chinese medicine and perform peak matching with the GC-MS fingerprint spectrum of agarwood. The results show that the similarity is 0.96, and the sample to be tested is agarwood.

[0062] Comparative Example 1

[0063] A method for constructing a GC-MS fingerprint of agarwood differs from Example 1 in that the sample solution preparation is different. Specifically, 0.2 g of agarwood powder is accurately weighed and placed in a 50 ml stoppered conical flask. 10 ml of 95.0% ethanol is added and the mixture is soaked at room temperature for half an hour. The weight is then measured, followed by ultrasonic extraction at room temperature for 40 min (300 W, 53 kHz). After cooling, the weight loss is made up, the solution is removed by rotary evaporation, and the solution is dissolved in 2 ml of anhydrous ethanol and filtered through a 0.22 μm microporous membrane to obtain the sample solution.

[0064] Nootkaton, Nootkaton-11,12-epoxide, and 3,5,11-Eudesmatriene were not detected by GC-MS.

[0065] Comparative Example 2

[0066] A method for constructing a GC-MS fingerprint of agarwood differs from Example 1 in that the temperature program in the gas chromatography conditions is different; specifically: the initial temperature is 40℃, held for 2 min; then the temperature is increased to 240℃ at a rate of 5℃ / min, and held for 20 min.

[0067] Nootkaton, Nootkaton-11,12-epoxide, and 3,5,11-Eudesmatriene were not detected by GC-MS.

[0068] As can be seen from the above embodiments and comparative examples, the method for constructing a GC-MS fingerprint spectrum of agarwood provided by the present invention uses steam distillation combined with liquid-liquid extraction to comprehensively and efficiently extract volatile / semi-volatile components inside agarwood samples. It optimizes the chromatographic and mass spectrometric conditions for GC-MS detection and analysis, and can effectively detect the volatile substances unique to agarwood, thereby identifying the authenticity of agarwood.

[0069] The method of this invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and context of this invention to implement and apply the technology of this invention. Those skilled in the art can refer to the content herein to appropriately improve process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention.

Claims

1. A method for constructing a GC-MS fingerprint of agarwood, comprising the following steps: S1: Weigh multiple batches of agarwood sample powder, and use steam distillation combined with liquid-liquid extraction to obtain the sample solution; S2: Inject the sample solution into GC-MS for detection and analysis; S3: By analyzing GC-MS data from multiple batches of agarwood samples, combined with retention index calculation and NIST standard mass spectrometry library search, common components were identified; S4: Import the data files corresponding to each batch of test solution into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software for data processing and establish the GC-MS fingerprint spectrum of agarwood.

2. The method according to claim 1, characterized in that, In step S1, the steam distillation method combined with liquid-liquid extraction is specifically as follows: water and glass beads are added to the agarwood sample powder, a volatile oil analyzer is connected, water is added from the top of the analyzer until it overflows into the flask, then an extraction solvent is added, a reflux condenser is connected, the mixture is heated to boiling and then gently boiled for extraction, the extraction solvent layer is collected, purified, and the sample solution is obtained.

3. The method according to claim 2, characterized in that, The solid-liquid ratio of the agarwood powder to the water is 1.5–2 g / L; the extraction solvent is ethyl acetate; the amount of the extraction solvent used is 1.5–5 mL; the extraction is carried out by gentle boiling for 4–6 hours.

4. The method according to claim 1, characterized in that, In step S2, the GC-MS gas chromatography conditions are as follows: an inert capillary column is used, and the temperature program is as follows: initial temperature 80~95℃, hold for 0.5~1.5 min; increase to 130~150℃ at a rate of 4~6℃ / min, hold for 4~6 min; increase to 160~180℃ at a rate of 1~2℃ / min, hold for 4~6 min; increase to 260~300℃ at a rate of 8~12℃ / min, hold for 12~16 min; linear velocity control mode, linear velocity is 35~40 cm / sec; injection port temperature is 180~220℃; carrier gas is high-purity helium; carrier gas flow rate is 0.5~2.0 mL / min; splitless injection; injection volume is 0.5~2 μL.

5. The method according to claim 1, characterized in that, In step S2, the GC-MS mass spectrometry conditions are as follows: an electron impact (EI) ion source is used; the ionization energy is 65~75eV; the ion source temperature is 220~240℃; the interface temperature is 240~260℃; the solvent delay time is 2~4min; the SCAN mode is used; and the mass spectrometry scanning range is 50-500m / z.

6. The method according to claim 1, characterized in that, In step S3, the determination of the common components specifically involves: taking a standard solution of n-alkanes (C8-C40), and collecting GC-MS data under the same gas chromatography-mass spectrometry conditions as the sample solution; correcting the retention time and calculating the retention index; using the extracted mass spectrometry information in the NIST 17 standard library in conjunction with the retention index for qualitative analysis; and identifying the substance with the highest match between the mass spectrum and the retention index as the best identification result for the common components.

7. The method according to claim 6, characterized in that, The common components include 4-phenyl-2-butanone, 4-methoxyphenyl-2-butanone, nootkaton-11,12-epoxide, and 3,5,11-Eudesmatriene.

8. The method according to claim 1, characterized in that, In step S4, the establishment of the fingerprint spectrum specifically involves: importing the total ion chromatogram data of multiple batches of samples obtained in step S2 into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System"; performing multi-point correction and automatic matching with a median and time width of 0.2 to automatically generate the fingerprint spectrum of agarwood.

9. A GC-MS fingerprint spectrum of agarwood, characterized in that, It is constructed by the method described in any one of claims 1-8.

10. The application of the GC-MS fingerprint spectrum of agarwood as described in claim 9, characterized in that, Includes the following steps: The GC-MS chromatogram of the sample to be tested is imported into the chromatographic fingerprint chromatogram similarity evaluation system for traditional Chinese medicine, and peak matching is performed with the GC-MS fingerprint chromatogram of agarwood. If the similarity is above 0.90, the sample to be tested is identified as agarwood.

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