Method for detecting content of volatile substances in aquilaria sinensis

Abstract

According to the method for detecting the content of the volatile substances in the aquilaria sinensis, provided by the invention, through pretreatment steps such as extraction of an extraction solvent, heating reflux and filtration, a sample is selectively and preliminarily purified, and the matrix effect of subsequent detection and analysis is effectively reduced; an inert capillary column is used for separation, high-purity helium is used as carrier gas, and an optimized heating program and an MRM detection mode are combined; through the synergistic effect of the two components, comprehensive, accurate and high-repeatability qualitative and quantitative analysis of various volatile substances in the aquilaria sinensis, especially nookatone which is easy to leak in a conventional method, is realized, and a reliable technical means is provided for accurate identification and quality evaluation of the aquilaria sinensis.

Description

Technical Field

[0001] This invention relates to the field of analytical testing technology, specifically to a method for detecting the content of volatile ketones in agarwood. Background Technology

[0002] Agarwood from Dongguan, a precious resource unique to China, is characterized by its sweet and refreshing aroma with rich layers of flavor. The core of this unique sensory quality stems directly from its complex composition of volatile organic compounds (VOCs), specifically sesquiterpenes, aromatic compounds, and aliphatic derivatives. Accurate analysis of its components is an indispensable core support in key scenarios such as quality evaluation, origin identification, and product certification of Dongguan agarwood.

[0003] Currently, mass spectrometry, especially gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) coupled with chromatography, has become the mainstream method for analyzing the components of agarwood. For example, although the Chinese Pharmacopoeia includes agarwood testing standards, it only targets the content determination of linalool, which cannot cover the complex VOCs system of agarwood. Patent CN115575550A, a method for detecting the quality of living agarwood trees with minimal damage, provides an HPLC-MS coupled method that can simultaneously determine the content of linalool and 8-chloro-2-(2-phenylethyl)-5,6,7-tri-5,6,7,8-tetrahydroone, and uses living minimal damage sampling technology, which solves the need for living and minimal damage sampling of agarwood trees in artificial agarwood formation technology. However, its detection targets are limited to chromone components and do not involve the characteristic volatile components of agarwood. CN118777464A describes a method for identifying agarwood based on headspace gas chromatography-mass spectrometry. The method involves placing an agarwood sample in a headspace vial and heating it, then analyzing the gas using gas chromatography-mass spectrometry to differentiate it from other types of agarwood, including Qinan agarwood. While this method detects volatile components of agarwood, the headspace method improves sensitivity and allows for the collection of more semi-volatile components. However, prolonged purging may lead to the loss of volatile components, and water vapor interference is significant. For complex samples like agarwood that may contain moisture, subsequent processing is cumbersome.

[0004] Given the shortcomings of existing technologies, there is an urgent need to develop a gas chromatography-mass spectrometry method that can achieve systematic and simultaneous detection of complex volatile substances in agarwood. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention aims to provide a method for detecting volatile substances in agarwood. The method involves pretreatment steps such as solvent extraction, heating and reflux, and filtration. Based on gas chromatography-mass spectrometry (GC-MS), separation is performed using an inert capillary column with high-purity helium as the carrier gas. Combined with an optimized temperature program and MRM detection mode, the method achieves efficient separation and accurate quantification of the target components.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is: a method for detecting volatile substances in agarwood, comprising the following steps:

[0007] S1: Preparation of the sample to be tested: Take the agarwood sample, add the extraction solvent, weigh, let stand for extraction, then heat under reflux, cool and make up the weight loss with the extraction solvent, filter, and the sample to be tested is obtained.

[0008] S2: Preparation of mixed reference standard: Take the reference standard, dilute to the mark with the extraction solvent, shake well, and prepare a mixed reference standard solution;

[0009] S3: Gas chromatography-mass spectrometry was used to detect the test sample and mixed reference standard, determine the peak area, and calculate the content.

[0010] In some embodiments, in step S1, the agarwood sample is in powder form.

[0011] In some embodiments, in step S1, the amount of agarwood sample used is 0.05~0.15g; preferably 0.1g.

[0012] In some embodiments, in step S1, the amount of extraction solvent used is 8-15 mL; preferably 10 mL.

[0013] In some embodiments, in step S1, the extraction solvent is allowed to stand for 0.5 h to 2 h, and the heating and reflux time is 0.5 h to 2 h.

[0014] In some embodiments, in steps S1 and S2, the extraction solvent is ethyl acetate.

[0015] This invention uses ethyl acetate as the extraction solvent, combined with a heating reflux process, to efficiently and selectively extract target substances from agarwood. Ethyl acetate has good solubility for volatile substances in agarwood, especially 4-phenyl-2-butanone, 4-methoxyphenyl-2-butanone, and nocaketone; however, it has weak extraction effects on highly polar impurities (such as sugars and some proteins) and water-soluble components present in the agarwood matrix. This selective extraction characteristic helps reduce co-extraction impurities in the pretreatment stage, thereby reducing matrix interference in subsequent gas chromatography-mass spectrometry analysis and improving the specificity and sensitivity of the detection.

[0016] The optimized heating reflux pretreatment method of this invention can achieve efficient extraction within a set temperature range. Within this temperature range, on the one hand, it ensures the effective release and dissolution of volatile substances from the dense resin or bound precursor of agarwood; on the other hand, this temperature is far below the boiling point and thermal decomposition threshold of volatile substances in agarwood, thereby avoiding direct volatilization or degradation due to heating during the extraction process, and ensuring the stable retention of volatile substances in the ethyl acetate extract.

[0017] Compared with headspace analysis and other techniques that only collect free volatile components, the heating reflux process of this invention can more effectively break through physical encapsulation and extract ketones and their precursors that may exist in resin-bound or conjugate form, thereby obtaining more comprehensive and representative information on the volatile substances of agarwood, laying a more reliable material basis for subsequent accurate analysis and identification.

[0018] In some embodiments, in step S2, the reference standard includes 4-phenyl-2-butanone, 4-methoxyphenyl-2-butanone, and nocaconone.

[0019] In some embodiments, in step S2, the concentrations of the mixed reference solution are 0.14–11.58 μg / mL for 4-phenyl-2-butanone, 0.27–21.56 μg / mL for 4-methoxyphenyl-2-butanone, and 0.65–52.14 μg / mL for nocacolone.

[0020] In some embodiments, in step S3, the gas chromatography conditions are as follows: an inert capillary column is used, and the temperature is programmed as follows: initial temperature 80~95℃, held for 0.5~1.5 min; temperature increased to 130~150℃ at a rate of 4~6℃ / min, held for 4~6 min; temperature increased to 160~180℃ at a rate of 1~2℃ / min, held for 4~6 min; temperature increased to 260~300℃ at a rate of 8~12℃ / min, held for 12~16 min; linear velocity control mode, linear velocity is 35~40 cm / sec; injection port temperature is 220~240℃; 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.

[0021] 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.

[0022] More preferably, the linear velocity is 37 cm / sec; the injection port temperature is 230°C; the carrier gas flow rate is 1 mL / min; and the injection volume is 1 μL.

[0023] 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.

[0024] The gas chromatography conditions employed in this invention effectively separate volatile substances from agarwood, particularly nocaketones, which are difficult to detect using conventional analytical methods. The programmed temperature gradient in this invention ensures efficient vaporization and transport of nocaketones within the capillary column, avoiding condensation and adsorption losses. Simultaneously, it prevents severe overlap or co-eluting of nocaketones with other structurally similar sesquiterpenes and oxygen-containing derivatives present in the agarwood matrix, resulting in sharp, symmetrical chromatographic peaks and thus achieving accurate qualitative and quantitative analysis.

[0025] In some embodiments, in step S3, the 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 content determination is performed in MRM mode with a voltage of 0.25~0.35kV.

[0026] More preferably, the ionization energy is 70 eV; the ion source temperature is 230°C; the interface temperature is 250°C; the solvent delay time is 3 min; and the voltage is 0.3 kV.

[0027] This invention employs the MRM mode, which provides high sensitivity and selectivity for monitoring characteristic ion pairs, significantly improving the signal-to-noise ratio and anti-interference capability of the detection method, thereby ensuring excellent accuracy and reproducibility of the quantitative results.

[0028] In some embodiments, the detection limits for 4-phenyl-2-butanone, 4-methoxyphenyl-2-butanone, and nocaconone are 0.2, 1, and 25 μg / g, respectively.

[0029] In some embodiments, the limits of quantitation for 4-phenyl-2-butanone, 4-methoxyphenyl-2-butanone, and nocacolone in the detection method are 0.5, 3, and 65 μg / g, respectively.

[0030] Compared with the prior art, one of the above technical solutions has the following advantages or beneficial effects:

[0031] This invention establishes a gas chromatography-mass spectrometry (GC-MS) method for the simultaneous and accurate quantification of three key volatile biomarkers in agarwood—4-phenyl-2-butanone, 4-methoxyphenyl-2-butanone, and nocacolone—by organically combining the ethyl acetate reflux extraction step with an optimized GC-MS detection step. The pretreatment method ensures efficient dissolution of volatile substances in agarwood while utilizing solvent selectivity to pre-purify the sample, effectively reducing matrix effects in subsequent analysis. Optimized chromatographic and mass spectrometric conditions for subsequent analysis effectively avoid problems such as co-elution, poor peak shape, or insufficient detection sensitivity caused by the high boiling points and unique chromatographic retention behaviors of key substances like nocacolone. The synergistic effect of these two methods ultimately achieves comprehensive, accurate, and highly repeatable qualitative and quantitative analysis of multiple volatile substances in agarwood, especially nocacolone, which is easily missed by conventional methods, providing a reliable technical means for the accurate identification and quality evaluation of agarwood.

[0032] The sample pretreatment steps used in this invention are simple and the conditions are mild, ensuring good reproducibility and robustness among different personnel and batches.

[0033] The detection method provided by this invention has been examined for specificity, linearity and range, limit of detection, limit of quantitation, precision, repeatability, and recovery rate. All validation results meet the acceptance criteria, and the detection method can achieve accurate quantification. The obtained quantitative data can provide objective and scientific decision-making basis for the standardized cultivation, authenticity identification, quality grading, and formulation of relevant quality standards for agarwood medicinal materials, and has important practical significance and application prospects. Attached Figure Description

[0034] Figure 1 TIC chromatograms of each solution in Example 1: A - blank solvent, B - mixed reference solution, C - test sample solution.

[0035] Figure 2 MIC diagrams of the target analytes in the sample solution in Example 1: A-4-phenyl-2-butanone, B-4-methoxyphenyl-2-butanone, C-nocarone.

[0036] Terminology Explanation

[0037] 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.

[0038] 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.

[0039] 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 to which this invention pertains. All patents and publications related to this invention are incorporated herein by reference in their entirety.

[0040] 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.

[0041] 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 numbers with values ​​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

[0042] 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.

[0043] All reagents used in this invention can be purchased commercially or prepared by the methods described in this invention.

[0044] Example 1

[0045] A method for detecting volatile substances in agarwood includes the following steps:

[0046] S1: Preparation of the sample to be tested: Take 0.1g of agarwood sample powder and pass it through a No. 3 sieve. Add 10mL of ethyl acetate, weigh it, let it stand for 1h for extraction, heat it under reflux for 1h, cool it, and then make up the weight loss with ethyl acetate. Filter it to obtain the sample solution to be tested.

[0047] S2: Preparation of mixed reference standards: Take 4-phenyl-2-butanone, 4-methoxyphenyl-2-butanone, and nocacolone, dilute to the mark with ethyl acetate, shake well, and prepare a series of mixed reference standard solutions with concentrations of 0.14–11.58 μg / mL for 4-phenyl-2-butanone, 0.27–21.56 μg / mL for 4-methoxyphenyl-2-butanone, and 0.65–52.14 μg / mL for nocacolone.

[0048] S3: Gas chromatography-mass spectrometry was used to detect the test sample and mixed reference standard, determine the peak area, and calculate the content.

[0049] In step S3,

[0050] The gas chromatography conditions were as follows: An Agilent HP-5MS inert capillary column (30 m × 0.25 mm × 0.25 μm) was used; the temperature program was as follows: initial temperature 90 °C, hold for 1 min; increase to 140 °C at a rate of 5 °C / min, hold for 5 min; increase to 170 °C at a rate of 1 °C / min, hold for 5 min; increase to 280 °C at a rate of 10 °C / min, hold for 15 min. Linear velocity control mode was used, with a linear velocity of 37 cm / sec; the injection port temperature was 230 °C; high-purity helium was used as the carrier gas; the carrier gas flow rate was 1.0 mL / min; splitless injection was used; and the injection volume was 1 μL.

[0051] The mass spectrometry conditions were as follows: an electron impact (EI) ion source was used; the ionization energy was 70 eV; the ion source temperature was 230 °C; the interface temperature was 250 °C; the solvent delay time was 3 min; and the content determination was performed in MRM mode with a voltage of 0.3 kV.

[0052] The following are the relevant validation experiments on the methodological approach for detecting volatile substances in agarwood:

[0053] 1. Exclusivity

[0054] Take blank solvent (ethyl acetate), mixed reference solution and test sample solution, and inject them according to the above chromatographic and mass spectrometric conditions, and record the chromatograms.

[0055] The results showed that no interfering peaks appeared in the blank solvent chromatogram at the retention times corresponding to the target components (see [link]). Figure 1 (A-Blank solvent TIC chromatogram); In the mixed reference solution and the test sample solution, the chromatographic peaks of the three volatile substances in agarwood (4-phenyl-2-butanone, 4-methoxyphenyl-2-butanone, and nocaconone) were well separated, with sharp peak shapes, and the retention times of the target peaks in the test sample solution were consistent with those in the reference solution (see...). Figure 1 (B and C are TIC plots of the mixed reference solution and the test sample solution, respectively). The above results indicate that the method has good specificity, no blank interference, and can specifically identify and determine the analyte.

[0056] 2. Linear relationship

[0057] To verify the detection capability of the method, a series of mixed reference solutions of different concentrations were precisely measured and sequentially injected and analyzed under the chromatographic and mass spectrometric conditions described above. Linear regression fitting was performed with concentration as the x-axis and peak area as the y-axis. The linear regression equations for each component were as follows: 4-phenyl-2-butanone: Y = 478416 * X - 63148, r = 0.9995; 4-methoxyphenyl-2-butanone: Y = 600480 * X - 284238, r = 0.9990; Nocaketone: Y = 92778 * X - 94986, r = 0.9991. The results indicate that each component exhibits good linearity within its respective concentration range.

[0058] 3. Limit of Detection and Limit of Quantification

[0059] To verify the detection capability of the method, a series of mixed reference solutions of different concentrations were precisely measured and injected sequentially under the chromatographic and mass spectrometric conditions described above. Chromatograms were recorded, and the signal-to-noise ratio (S / N) of each component was calculated. The limits of detection (LOD) and limits of quantitation (LOQ) for each target component were defined as the concentrations corresponding to S / N ratios of approximately 3 and 10, respectively. The LOD and LOD results for each volatile substance are shown in Table 1.

[0060] Table 1. Limits of detection and limits of quantitation (μg / g) for each component

[0061] Volatile substances 4-Phenylon-2-Butanone 4-Methoxyphenyl-2-butanone Nocaketone Detection limit 0.2 1 25 Limit of Quantification 0.5 3 65

[0062] 4. Precision

[0063] To verify the precision of the instrument system, a mixed reference solution of 4-phenyl-2-butanone, 4-methoxyphenyl-2-butanone, and nocacolone with concentrations of 0.14 μg / mL, 0.27 μg / mL, and 0.65 μg / mL, respectively, was injected and measured six times consecutively. The peak areas of each target component were recorded, and their relative standard deviations (RSDs) were calculated. The calculated RSDs for 4-phenyl-2-butanone, 4-methoxyphenyl-2-butanone, and nocacolone were 4.1%, 3.9%, and 2.4%, respectively; these results indicate that the instrument has good precision.

[0064] 5. Recovery rate

[0065] To verify the accuracy of this analytical method, a spiked recovery experiment was conducted, as detailed below:

[0066] Nine portions of the same agarwood sample powder with known content were accurately weighed, each 0.1g, and divided into three groups on average.

[0067] Different volumes of mixed standard solution (containing 34.74 μg·mL⁻¹ of 4-phenyl-2-butanone, 64.68 μg·mL⁻¹ of 4-methoxyphenyl-2-butanone, and 208.56 μg·mL⁻¹ of nocacolone) were precisely added to each group of samples. The spiking volumes were 0.05 mL, 0.1 mL, and 0.2 mL, respectively, and three parallel samples were set up for each spiking level.

[0068] The spiked sample was treated in the same manner as described in the preparation method of the "test solution" of this invention, and then subjected to gas chromatography-mass spectrometry (GC-MS) analysis. The recovery rate of each component was calculated based on the analysis results.

[0069] Experimental results show that the average recoveries of the three target components are within a good range, specifically: 82%–98% for 4-phenyl-2-butanone, 80%–99% for 4-methoxyphenyl-2-butanone, and 75%–102% for nocacolone. These results meet the accuracy requirements for analytical method validation, fully demonstrating the reliability of the method of this invention.

[0070] 6. Stability

[0071] To investigate the stability of the test sample solution under the test conditions, the same sample solution prepared according to the method in Example 1 was taken and left at room temperature for 0, 1, 10, 24, and 48 hours, respectively. Then, it was injected and analyzed sequentially according to the gas chromatography and mass spectrometry conditions in Example 1. The peak areas of each target component were recorded, and their relative standard deviations (RSDs) were calculated. The results are as follows: the RSDs of 4-phenyl-2-butanone, 4-methoxyphenyl-2-butanone, and nocaketone were 2.9%, 1.5%, and 3.7%, respectively. The RSD values ​​of each component within 48 hours were all less than 4%, indicating that the test sample solution has good stability under the conditions specified in this method and can meet the requirements of analytical testing.

[0072] Example 2

[0073] A method for detecting volatile substances in agarwood differs from Example 1 in that the temperature program in the gas chromatography conditions is different; specifically: the initial temperature is 95°C, held for 1.5 min; the temperature is increased to 140°C at a rate of 4°C / min, held for 5 min; the temperature is increased to 170°C at a rate of 1°C / min, held for 4 min; and the temperature is increased to 280°C at a rate of 8°C / min, held for 16 min.

[0074] Specificity validation results showed that no interfering peaks appeared at the retention times corresponding to the target components in the blank solvent chromatogram. In both the mixed reference solution and the test sample solution, the chromatographic peaks of the three volatile substances from agarwood (4-phenyl-2-butanone, 4-methoxyphenyl-2-butanone, and nocaketone) were well separated, with sharp peak shapes, and the retention times of the target peaks in the test sample solution were consistent with those in the reference solution. This method demonstrates good specificity, with no interference from the blank, and can specifically identify and determine the analytes.

[0075] Comparative Example 1

[0076] A method for detecting volatile substances in agarwood differs from Example 1 in that the preparation of the test sample is different; specifically, 0.1g of agarwood sample powder is passed through a No. 3 sieve, 10mL of 95.0% ethanol is added, the weight is determined, the sample is allowed to stand for 1h for extraction, heated under reflux for 1h, cooled, and the weight loss is made up with ethyl acetate. After filtration, the test sample solution is obtained.

[0077] Specificity verification results showed that nocacaone was not detected.

[0078] Comparative Example 2

[0079] A method for detecting volatile substances in agarwood differs from Example 1 in that the sample preparation is different, and headspace sampling is used to collect volatile substances in agarwood. Specifically, 0.1 g of agarwood powder is placed in a 20 mL headspace sample vial, sealed, and then the temperature of the sample heating chamber is raised to 140 °C and heated for 5 min. The gas in the headspace sample vial is used as the injection gas, and the gas is analyzed under the same gas chromatography-mass spectrometry conditions as in Example 1.

[0080] Specificity verification results showed that nocacaone was not detected.

[0081] Comparative Example 3

[0082] A method for detecting volatile substances in agarwood differs from Example 1 in that the temperature program in the gas chromatography conditions is different; specifically: the initial temperature is 40°C, held for 2 min; then the temperature is increased to 240°C at a rate of 5°C / min, and held for 20 min.

[0083] Specificity verification results showed that nocacaone was not detected.

[0084] As can be seen from the above embodiments and comparative examples, the detection method provided by the present invention, while ensuring the efficient dissolution of volatile substances in agarwood, uses solvent selectivity to pre-purify the sample, effectively reducing the matrix effect in subsequent detection and analysis; it optimizes the gas chromatography and mass spectrometry conditions for subsequent analysis and detection, and successfully solves the problems of co-elution, poor peak shape, or insufficient detection sensitivity caused by the high boiling point and special chromatographic retention behavior of key substances such as nocarbamate.

[0085] 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 detecting volatile substances in agarwood, comprising the following steps: S1: Preparation of the sample to be tested: Take the agarwood sample, add the extraction solvent, weigh, let stand for extraction, then heat under reflux, cool and make up the weight loss with the extraction solvent, filter, and the sample to be tested is obtained. S2: Preparation of mixed reference standard: Take the reference standard, dilute to the mark with the extraction solvent, shake well, and prepare a mixed reference standard solution; S3: Gas chromatography-mass spectrometry was used to detect the test sample and mixed reference standard, determine the peak area, and calculate the content.

2. The method according to claim 1, characterized in that, In step S1, the amount of agarwood sample used is 0.05~0.15g; the amount of extraction solvent used is 8~15mL.

3. The method according to claim 1, characterized in that, In step S1, the extraction solvent is allowed to stand for 0.5 h to 2 h, and the heating and reflux time is 0.5 h to 2 h.

4. The method according to claim 1, characterized in that, In steps S1 and S2, the extraction solvent is ethyl acetate.

5. The method according to claim 1, characterized in that, In step S2, the reference standard includes 4-phenyl-2-butanone, 4-methoxyphenyl-2-butanone, and nocacolone.

6. The method according to claim 5, characterized in that, In step S2, the concentrations of the mixed reference solution are 0.14–11.58 μg / mL for 4-phenyl-2-butanone, 0.27–21.56 μg / mL for 4-methoxyphenyl-2-butanone, and 0.65–52.14 μg / mL for nocacolone.

7. The method according to claim 1, characterized in that, In step S3, the gas chromatography conditions are as follows: an inert capillary column is used, and the temperature is programmed 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 220~240℃; 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.

8. The method according to claim 1, characterized in that, In step S3, the 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 content determination is performed in MRM mode with a voltage of 0.25~0.35kV.

9. The method according to any one of claims 1-8, characterized in that, The detection limits for 4-phenyl-2-butanone, 4-methoxyphenyl-2-butanone, and nocacolone are 0.2, 1, and 25 μg / g, respectively; the quantitation limits for 4-phenyl-2-butanone, 4-methoxyphenyl-2-butanone, and nocacolone are 0.5, 3, and 65 μg / g, respectively.

10. The method according to claim 1, characterized in that, Includes the following steps: S1: Preparation of the sample to be tested: Take 0.1g of agarwood sample powder, add 10mL of ethyl acetate, weigh, let stand for 1h for extraction, heat to reflux for 1h, cool and make up the weight loss with ethyl acetate, filter, and the sample solution to be tested is obtained. S2: Preparation of mixed reference standards: Take 4-phenyl-2-butanone, 4-methoxyphenyl-2-butanone, and nocacolone, dilute to the mark with ethyl acetate, shake well, and prepare a series of mixed reference standard solutions with 4-phenyl-2-butanone concentrations of 0.14–11.58 μg / mL, 4-methoxyphenyl-2-butanone concentrations of 0.27–21.56 μg / mL, and nocacolone concentrations of 0.65–52.14 μg / mL; S3: Gas chromatography-mass spectrometry was used to detect the test sample and mixed reference standard, determine the peak area, and calculate the content.

11. Among them, In step S3, The gas chromatography conditions were as follows: an inert capillary column, Agilent HP-5MS (30m × 0.25mm × 0.25μm), was used; the temperature program was as follows: initial temperature 90℃, held for 1 min; temperature ramped to 140℃ at a rate of 5℃ / min, held for 5 min; temperature ramped to 170℃ at a rate of 1℃ / min, held for 5 min; temperature ramped to 280℃ at a rate of 10℃ / min, held for 15 min. Linear velocity control mode was used, with a linear velocity of 37 cm / sec; the injection port temperature was 230℃; high-purity helium was used as the carrier gas; the carrier gas flow rate was 1.0 mL / min; splitless injection was used; and the injection volume was 1 μL. The mass spectrometry conditions were as follows: an electron impact (EI) ion source was used; the ionization energy was 70 eV; the ion source temperature was 230 °C; the interface temperature was 250 °C; the solvent delay time was 3 min; and the content determination was performed in MRM mode with a voltage of 0.3 kV.

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