Construction method and application of specific chromatogram of sandalwood volatile oil

Abstract

The invention relates to the technical field of traditional Chinese medicine identification and medicine analysis, in particular to a construction method and application of a specific chromatogram of sandalwood volatile oil. The construction method of the specific chromatogram comprises the following steps: carrying out high performance liquid chromatography detection on a test solution of sandalwood volatile oil under the detection conditions that a chromatographic column is a T3 chromatographic column and a filler is solid core-porous shell silica gel particles; acetonitrile is used as a mobile phase A, and water or an aqueous solution of phosphoric acid is used as a mobile phase B; the elution procedure is 0-25 minutes, and the mobile phase A is increased from 45v / v% to 50v / v%; the mobile phase A is increased to 65 v / v% from 50 v / v% in 25-40 min; the mobile phase A is increased to 85v / v% from 65v / v% in 40-50min. The specific chromatogram shows the key characteristic peaks of the sandalwood volatile oil, and can be visually used for identifying the quality and authenticity of the sandalwood volatile oil.

Description

Construction method and application of characteristic spectrum of sandalwood volatile oil Technical Field

[0001] This application relates to the field of identification and analysis of traditional Chinese medicine, and in particular to the method and application of constructing characteristic spectra of sandalwood volatile oil. Background Technology

[0002] Sandalwood medicinal material is the dried heartwood of the sandalwood tree (Santalum album L.), a plant in the Santalaceae family. It has a pungent taste and warm properties, and enters the spleen, stomach, heart, and lung meridians. It has the effects of regulating qi and warming the middle jiao, stimulating appetite and relieving pain. Clinically, it is used to treat symptoms such as cold stagnation of qi, chest discomfort, chest pain, abdominal pain, vomiting, and poor appetite. Modern research shows that sandalwood mainly contains volatile oils, phenolic acids, and flavonoids. Among them, the volatile oils have a central nervous system depressant effect and exhibit anti-inflammatory, analgesic, and nephroprotective pharmacological activities.

[0003] Due to the high price of sandalwood medicinal materials, and the long formation period and low proportion of the heartwood in the tree trunk, there are cases in the market where sandalwood sapwood is used to impersonate heartwood, or other tree trunks are used to impersonate sandalwood. Common sandalwood counterfeits include Dalbergia hupeana Hance (also known as sandalwood or white sandalwood), a deciduous tree of the Dalbergia genus in the Fabaceae family, and Platycladus orientalis (L.) Franco (also known as cypress or fragrant cypress), a tree of the Platycladus genus in the Cupressaceae family.

[0004] The 2025 edition of the Chinese Pharmacopoeia specifies methods for determining the volatile oil content and identifying it using thin-layer chromatography under the entry for sandalwood. However, existing research on sandalwood volatile oil mainly focuses on component analysis and activity evaluation; studies on its characteristic chromatograms using ultra-high performance liquid chromatography (UPLC) or high performance liquid chromatography (HPLC) have not been reported. To more comprehensively evaluate the quality of sandalwood medicinal materials and ensure its efficacy and safety, it is necessary to establish a detection method that can objectively and comprehensively reflect the intrinsic components of sandalwood volatile oil. This method would be used to distinguish sandalwood heartwood from sapwood, identify sandalwood from its common adulterants, and provide a basis for the quality control of sandalwood-containing preparations. Summary of the Invention

[0005] Based on this, one or more embodiments of this application provide a method for constructing and applying a characteristic spectrum of sandalwood volatile oil. The characteristic spectrum of sandalwood volatile oil in this application presents key characteristic peaks, which can more comprehensively and objectively present the internal components of sandalwood volatile oil, and can thus be used intuitively to identify sandalwood volatile oil from different sources, and provide an objective reference for the identification of sandalwood volatile oil and its counterfeits.

[0006] One or more embodiments of this application provide a method for constructing a characteristic spectrum of sandalwood volatile oil, including the following steps:

[0007] The sandalwood medicinal material is mixed with water and then steam distilled to collect the sandalwood volatile oil;

[0008] The sandalwood volatile oil was dissolved in an organic solvent to prepare a test solution;

[0009] The test solution was subjected to high performance liquid chromatography to obtain the characteristic spectrum of the sandalwood volatile oil;

[0010] The conditions for the high-performance liquid chromatography detection include:

[0011] The chromatographic column is a T3 column, and the packing material is core-shell silica particles, wherein the core-shell silica particles include a solid core and a porous shell layer located outside the solid core;

[0012] Acetonitrile was used as mobile phase A, and water or an aqueous solution of phosphoric acid was used as mobile phase B;

[0013] Gradient elution is used, and the gradient elution procedure includes:

[0014] From 0 to 25 minutes, the volume percentage of the mobile phase A increased from 45% to 50%.

[0015] Between 25 and 40 minutes, the volume percentage of mobile phase A increased from 50% to 65%.

[0016] Over 40 to 50 minutes, the volume percentage of the mobile phase A increased from 65% to 85%.

[0017] In some embodiments, the chromatographic column has a length of 150 mm and an inner diameter of 2.1 mm, and the packing material has a particle size of 1.6 μm to 1.9 μm.

[0018] In some embodiments, the conditions for high-performance liquid chromatography detection further include at least one of the following conditions:

[0019] (1) The volume concentration of the aqueous solution of phosphoric acid is 0.1%-0.2%;

[0020] (2) The flow rate is 0.2 mL / min - 0.4 mL / min;

[0021] (3) The column temperature is 30℃-40℃;

[0022] (4) The detection wavelength is 200nm-250nm; and

[0023] (5) The injection volume is 1μL-2μL.

[0024] In some embodiments, the characteristic spectrum of the sandalwood volatile oil exhibits the following characteristic peaks: peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, peak 10, and peak 11;

[0025] Among them, peaks 4 and 6 are characteristic peaks corresponding to santalol.

[0026] Furthermore, the characteristic peaks in the characteristic spectrum of the sandalwood volatile oil are as follows:

[0027] Taking peak 4 as peak S1, calculate the relative retention times of peaks 1-3 and peaks 5-11 with respect to peak S1. The relative retention times are within ±10% of a specified value, which is: peak 1: 0.71; peak 2: 0.84; peak 3: 0.96; peak 5: 1.10; peak 6: 1.14; peak 7: 1.22; peak 8: 1.54; peak 9: 1.64; peak 10: 1.78; peak 11: 1.82.

[0028] Taking peak 6 as the S2 peak, calculate the relative retention times of peaks 1-5 and peaks 7-11 with respect to peak S2. The relative retention times are within ±10% of the specified values, which are: peak 1: 0.62; peak 2: 0.73; peak 3: 0.85; peak 4: 0.88; peak 5: 0.96; peak 7: 1.07; peak 8: 1.36; peak 9: 1.44; peak 10: 1.56; peak 11: 1.60.

[0029] In some embodiments, the organic solvent includes one or more of methanol, ethanol, and acetonitrile; and / or,

[0030] The volume ratio of the sandalwood volatile oil to the organic solvent is (0.05-0.2):1.

[0031] In some embodiments, the conditions for steam distillation include: a feed-to-liquid ratio of 1g:(10mL-20mL) and a distillation time of 4h-6h.

[0032] In some embodiments, the method for constructing the feature map further includes the following steps:

[0033] The reference solution was subjected to high-performance liquid chromatography (HPLC) to obtain a chromatogram of the reference solution; the reference solution included santalol.

[0034] The chromatogram of the reference compound and the characteristic chromatogram of the sandalwood volatile oil are compared to identify the characteristic peaks in the characteristic chromatogram of the sandalwood volatile oil, wherein the high-performance liquid chromatography (HPLC) detection conditions are the same as those defined above; and / or,

[0035] The reference solution and the test solution were subjected to high performance liquid chromatography-mass spectrometry to obtain the total ion chromatogram of the reference solution and the total ion chromatogram of sandalwood volatile oil.

[0036] The total ion chromatogram of the reference material and the total ion chromatogram of the sandalwood volatile oil were compared to identify the characteristic peaks in the total ion chromatogram of the sandalwood volatile oil.

[0037] The high-performance liquid chromatography detection includes:

[0038] The chromatographic column is a T3 column, and the packing material is core-shell silica particles, wherein the core-shell silica particles include a solid core and a porous shell layer located outside the solid core;

[0039] Acetonitrile is used as mobile phase A, and water or an aqueous solution of formic acid is used as mobile phase B;

[0040] Gradient elution is used, and the gradient elution procedure includes:

[0041] From 0 to 25 minutes, the volume percentage of the mobile phase A increased from 45% to 50%.

[0042] Between 25 and 40 minutes, the volume percentage of mobile phase A increased from 50% to 65%.

[0043] Over 40 to 50 minutes, the volume percentage of the mobile phase A increased from 65% to 85%.

[0044] Furthermore, the conditions for the mass spectrometry detection include: using a heated electrospray ion source, positive ion mode detection, a scan range of m / z 90-500, a normalized collision energy of 35-45, and a peak-shaped mass spectrum.

[0045] One or more embodiments of this application also provide the application of the above-described method for constructing feature maps in the identification of sandalwood volatile oil.

[0046] The method for constructing the characteristic spectrum of sandalwood volatile oil in this application can more comprehensively and objectively present the intrinsic components of sandalwood volatile oil, with high separation, stable baseline, and good peak shape, including 11 characteristic peaks: peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, peak 10, and peak 11; among which, peak 4 and peak 6 are characteristic peaks corresponding to santalol. This characteristic spectrum can effectively distinguish the volatile oil of sandalwood heartwood and sapwood, as well as sandalwood and its common adulterants (such as rosewood and cypress), and can also be used to identify whether sandalwood volatile components have been added to related products, providing a basis for quality assessment.

[0047] The method for constructing the characteristic spectrum of this application can also be used to identify sandalwood volatile oil extracted from different sandalwood materials (sapwood and heartwood), as well as to identify common counterfeits such as arborvitae volatile oil and rosewood volatile oil. The presence or absence of characteristic peaks can be used to intuitively identify sandalwood volatile oil. Attached Figure Description

[0048] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0049] Figure 1 is a chromatogram of the specificity investigation in Example 1 of this application. As shown in the figure, from bottom to top, they are the chromatograms of blank solvent (methanol), santalol reference solution, and sandalwood volatile oil test solution.

[0050] Figure 2 is a superimposed image of the characteristic spectra of sandalwood volatile oil from 10 batches in Example 1 of this application.

[0051] Figure 3 is a comparative characteristic spectrum of sandalwood volatile oil generated in Example 1 of this application.

[0052] Figure 4 is a comparison of the total ion chromatograms of the sandalwood volatile oil test solution and the santalol reference solution in Example 1 of this application.

[0053] Figure 5 shows the ultraviolet absorption chromatograms of sandalwood volatile oil at different wavelengths in Example 1 of this application.

[0054] Figure 6 shows the results of the investigation on the extraction efficiency of sandalwood volatile oil by different organic solvents (methanol, acetonitrile, ethanol) in Example 2 of this application.

[0055] Figure 7 is a full wavelength scan of sandalwood volatile oil in the range of 190nm~400nm in Example 4 of this application.

[0056] Figure 8 shows the chromatograms of sandalwood volatile oil at different detection wavelengths in Example 4 of this application.

[0057] Figure 9 shows a comparison of chromatograms under different mobile phase conditions (acetonitrile-water, acetonitrile-0.1% phosphoric acid, acetonitrile-0.2% phosphoric acid) in Example 5 of this application.

[0058] Figure 10 shows a comparison of chromatograms at different column temperatures (30℃, 35℃, 40℃) in Example 6 of this application.

[0059] Figure 11 shows a comparison of chromatograms at different flow rates (0.20 mL / min, 0.25 mL / min, 0.30 mL / min) in Example 7 of this application.

[0060] Figure 12 shows a comparison of chromatograms under different mobile phase conditions (acetonitrile-water, methanol-water, acetonitrile-0.1% formic acid, acetonitrile-0.1% acetic acid) in Comparative Example 1 of this application.

[0061] Figure 13 shows a comparison of chromatograms of different elution gradients (gradient 1, gradient 2, and gradient 3) in Comparative Example 2 of this application.

[0062] Figure 14 shows a comparison of chromatograms of different types of chromatographic columns in Comparative Example 3 of this application.

[0063] Figure 15 is a comparison of the characteristic spectra of the volatile oils of sandalwood heartwood and sapwood in Example 8 of this application.

[0064] Figure 16 is a comparison of the characteristic spectra of the volatile oils of sandalwood and counterfeit products (Dalbergia odorifera and Platycladus orientalis) in Example 9 of this application.

[0065] Figure 17 is a chromatogram comparison of different sandalwood extracts (sandalwood volatile oil, degreased water extract, and alcohol extract) in Example 10 of this application. Detailed Implementation

[0066] The present application is further described below with reference to embodiments and examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Furthermore, it should be understood that after reading the teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the protection scope of the appended claims.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0068] The term "and / or" as used herein includes any one of two or more related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations encompass any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that, in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0069] In this document, "preferred" is only used to describe a better implementation method or embodiment, and should be understood as not constituting a limitation on the scope of protection of this application.

[0070] In this application, "further" is used to describe the purpose and indicate differences in content, but should not be construed as a limitation on the scope of protection of this application.

[0071] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0072] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, optional numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval points to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges may be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0073] In this application, weight can be a well-known unit of mass in the field of traditional Chinese medicine, such as μg, mg, g, or kg.

[0074] In this application, v / v% refers to volume percentage.

[0075] One or more embodiments of this application provide a method for constructing a characteristic spectrum of sandalwood volatile oil, including the following steps:

[0076] The sandalwood medicinal material is mixed with water and then steam distilled to collect the sandalwood volatile oil;

[0077] The volatile oil of sandalwood was dissolved in an organic solvent to prepare the test solution;

[0078] The test solution was subjected to high performance liquid chromatography to obtain the characteristic spectrum of sandalwood volatile oil;

[0079] The detection conditions for high performance liquid chromatography include:

[0080] The chromatographic column is a T3 column, and the packing material is core-shell silica particles, which include a solid core and a porous shell layer located outside the solid core.

[0081] Acetonitrile was used as mobile phase A, and water or an aqueous solution of phosphoric acid was used as mobile phase B;

[0082] Gradient elution is used, and the gradient elution procedure includes:

[0083] From 0 to 25 minutes, the volume percentage of mobile phase A increased from 45% to 50%.

[0084] Between 25 and 40 minutes, the volume percentage of mobile phase A increased from 50% to 65%.

[0085] Between 40 and 50 minutes, the volume percentage of mobile phase A increased from 65% to 85%.

[0086] In this article, "steam distillation" refers to passing steam into a mixture containing organic compounds that are insoluble or slightly soluble in water but have a certain degree of volatility, and heating it to boiling. This allows the organic compounds to be purified to be distilled out along with the steam at a temperature below 100°C, thereby achieving the purpose of separation and purification.

[0087] In some embodiments, the conditions for steam distillation include: a material-to-liquid ratio of 1g:(10mL-20mL), for example, 1g:10mL, 1g:15mL, 1g:20mL, etc.; and a distillation time of 4h-6h, for example, 4h, 5h, 6h, etc. In this embodiment, the material-to-liquid ratio is the mass-to-volume ratio of sandalwood and water.

[0088] In some embodiments, the preparation method of sandalwood volatile oil includes the following steps:

[0089] Place sandalwood in a still, add water and boiling stones, heat to boiling, and maintain a gentle boil. The volatile oil in the sandalwood will distill out with the steam. Cool the steam using a condenser, collect the condensed liquid, and collect the volatile oil that floats to the top. Optionally, the mass-to-volume ratio of sandalwood to water is 1g:(10mL-20mL), for example, 1g:10mL, 1g:15mL, 1g:20mL, etc.; maintain a gentle boil for 4h-6h, for example, 4h, 5h, 6h, etc.

[0090] In some embodiments, after collecting sandalwood volatile oil, the sandalwood volatile oil is washed with an organic solvent, and then dissolved in an organic solvent to obtain a test solution.

[0091] Alternatively, the organic solvent may include one or more of methanol, ethanol, and acetonitrile.

[0092] In some embodiments, the volume ratio of sandalwood volatile oil to organic solvent is (0.05-0.2):1, for example 0.05:1, 0.1:1, 0.15:1, 0.2:1, etc.

[0093] In some embodiments, acetonitrile is used as mobile phase A, and mobile phase B can be selected from water or an aqueous solution of phosphoric acid. Gradient elution using the above mobile phases can obtain a stable baseline and good resolution.

[0094] Preferably, the volume concentration of the aqueous phosphoric acid solution is 0.1%-0.2%, for example, 0.1%, 0.15%, 0.2%, etc. Using this volume concentration of the aqueous phosphoric acid solution as mobile phase B results in a more stable baseline and better resolution.

[0095] In some embodiments, the chromatographic column is a T3 column. Using a T3 column can achieve better peak resolution.

[0096] Furthermore, the chromatographic column has a length of 150 mm and an inner diameter of 2.1 mm, and the packing material has a particle size of 1.6 μm-1.9 μm.

[0097] In some embodiments, the chromatographic column can be selected from columns such as Waters CORTECS T3 (2.1mm × 150mm, 1.6μm), Waters ACQUITY UPLC BEH C18 (2.1mm × 150mm, 1.7μm), and ACQUITY UPLC HSS T3 (2.1mm × 150mm, 1.8μm). These columns provide good peak resolution. Further options include the Waters CORTECS T3 (2.1mm × 150mm, 1.6μm), which yields even better peak shape and resolution.

[0098] In some embodiments, the flow rate is 0.2 mL / min to 0.4 mL / min. Detection at these flow rates yields good characteristic peak separation.

[0099] Preferably, the flow rate is 0.25 mL / min-0.35 mL / min, which enables the elution time of the chromatographic peak to be evenly distributed in the middle of the entire detection time, resulting in more uniform elution and higher chromatographic peak resolution.

[0100] In some embodiments, the column temperature is 30℃-40℃, for example, 30℃, 35℃, 40℃, etc. Using the above column temperature for detection can ensure better response, resolution and peak shape of the chromatographic peak.

[0101] In some embodiments, the detection wavelength is 200nm-250nm, such as 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, etc. Using this detection wavelength can obtain rich chromatographic peak information, and the overall chromatographic peak response is relatively uniform.

[0102] Preferably, the detection wavelength is 210nm-220nm. Using this detection wavelength can obtain more uniform chromatographic peak information.

[0103] In some embodiments, the injection volume is 1 μL-2 μL, for example 1 μL, 1.5 μL, 2 μL, etc.

[0104] In some embodiments, the characteristic spectrum of sandalwood volatile oil exhibits the following characteristic peaks: peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, peak 10, and peak 11;

[0105] Among them, peaks 4 and 6 are characteristic peaks corresponding to santalol.

[0106] In some embodiments, the characteristic spectrum of sandalwood volatile oil includes:

[0107] Taking peak 4 as peak S1, calculate the relative retention times of peaks 1-3 and peaks 5-11 with respect to peak S1. The relative retention times are within ±10% of a specified value, which is: peak 1: 0.71; peak 2: 0.84; peak 3: 0.96; peak 5: 1.10; peak 6: 1.14; peak 7: 1.22; peak 8: 1.54; peak 9: 1.64; peak 10: 1.78; peak 11: 1.82.

[0108] Taking peak 6 as the S2 peak, calculate the relative retention times of peaks 1-5 and peaks 7-11 with respect to peak S2. The relative retention times are within ±10% of the specified values, which are: peak 1: 0.62; peak 2: 0.73; peak 3: 0.85; peak 4: 0.88; peak 5: 0.96; peak 7: 1.07; peak 8: 1.36; peak 9: 1.44; peak 10: 1.56; peak 11: 1.60.

[0109] In some embodiments, the method for constructing feature maps further includes the following steps:

[0110] The reference solution was analyzed by high performance liquid chromatography to obtain a chromatogram of the reference solution; the reference solution included santalol.

[0111] The chromatogram of the reference compound and the characteristic chromatogram of sandalwood volatile oil are compared to identify the characteristic peaks in the characteristic chromatogram of sandalwood volatile oil. Preferably, the high-performance liquid chromatography (HPLC) detection conditions are the same as those defined in any of the above technical solutions.

[0112] In some embodiments, the method for constructing feature maps further includes the following steps:

[0113] The reference solution and the test solution were subjected to high performance liquid chromatography-mass spectrometry to obtain the total ion chromatogram of the reference solution and the total ion chromatogram of sandalwood volatile oil.

[0114] The total ion chromatogram of the reference material was compared with that of sandalwood volatile oil, and the characteristic peaks in the total ion chromatogram of sandalwood volatile oil were identified.

[0115] In high-performance liquid chromatography-mass spectrometry (HPLC-MS) combined detection, considering the use of a volatile mobile phase in mass spectrometry detection, water or formic acid is used as mobile phase B. The conditions for HPLC detection include:

[0116] The chromatographic column is a T3 column, and the packing material is core-shell silica particles, wherein the core-shell silica particles include a solid core and a porous shell layer located outside the solid core;

[0117] Acetonitrile is used as mobile phase A, and water or an aqueous solution of formic acid is used as mobile phase B;

[0118] Gradient elution is used, and the gradient elution procedure includes:

[0119] From 0 to 25 minutes, the volume percentage of the mobile phase A increased from 45% to 50%.

[0120] Between 25 and 40 minutes, the volume percentage of mobile phase A increased from 50% to 65%.

[0121] Over 40 to 50 minutes, the volume percentage of the mobile phase A increased from 65% to 85%.

[0122] Optionally, the volume concentration of formic acid is 0.1%-0.2%, for example, it can be 0.1%, 0.15%, 0.2%, etc.

[0123] In some embodiments, the conditions for mass spectrometry detection include: using a heated electrospray ion source, positive ion mode detection, a scan range of m / z 90-500, a normalized collision energy of 35-45, and a peak-shaped mass spectrum.

[0124] In some embodiments, the conditions for mass spectrometry detection also include: sheath gas flow rate of 30-40 alb, auxiliary gas flow rate of 8-12 alb, spray voltage of 3.0-3.5 kV, S-lens voltage of 45-55 V, and ion transmission tube and heating temperature maintained at 345-355 °C.

[0125] The second aspect of this application provides the application of the aforementioned method for constructing characteristic maps in the identification of sandalwood volatile oil.

[0126] In some embodiments, the identification of sandalwood volatile oil includes identifying sandalwood volatile oil made from sandalwood heartwood and sapwood.

[0127] In some embodiments, the identification of sandalwood volatile oil includes distinguishing between volatile oils from raw materials that are sandalwood and those from counterfeit sandalwood. Common counterfeit sandalwood includes Dalbergia hupeana (also known as sandalwood or white sandalwood), a deciduous tree belonging to the genus Dalbergia in the family Leguminosae, and Platycladus orientalis (L.) Franco, a tree belonging to the genus Platycladus in the family Cupressaceae. The preparation method for the volatile oil of counterfeit sandalwood is the same as that for sandalwood volatile oil.

[0128] The following are some specific examples.

[0129] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines provided in this application, or consult experimental manuals or other known experimental methods in the field, or refer to the manufacturer's recommended experimental conditions. Raw materials and reagents not mentioned can be obtained commercially, or can be prepared by those skilled in the art using known methods.

[0130] The instruments, reagents, reagents, and samples used in the following examples or comparative examples are as follows:

[0131] Instruments: Thermo Ultra High Performance Liquid Chromatography (UHPLC) system (Vanquish, Thermo Fisher Scientific), Waters UHPLC system (H-Class, Waters Technologies), Thermo Vanquish Flex UHPLC-Thermo Fisher QE HHPLC system (Thermo Fisher Scientific), Waters CORTECS T3 column (2.1mm × 150mm, 1.6μm), Waters ACQUITY UPLC BEH C18 column (2.1mm × 150mm, 1.7μm), Waters ACQUITY UPLC HSS T3 column (2.1mm × 150mm, 1.8μm), YMC Triart C18 column (2.1mm × 150mm, 1.9μm), SHIMADZU Shim-pack Scepter C18-120 column (2.1mm × 150mm, 1.9μm), Agilent ZORBAX RRHD StableBond C18 chromatographic column (2.1mm×150mm, 1.8μm), 1 / 1000 electronic analytical balance (JJ600, Changshu Shuangjie Test Instrument Factory), 0.001% electronic analytical balance (ME204E, Mettler Toledo), 0.1% electronic analytical balance (XP26, Mettler Toledo), electric thermostatic water bath (HWS-28, Shanghai Yiheng Technology Co., Ltd.), CNC ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.), ultrapure water system (Milli-Q Direct, Merck AG).

[0132] Reagents: Ethanol and methanol (Xilong Technology Co., Ltd.) were analytical grade; phosphoric acid, acetic acid, formic acid (Tianjin Kemeio Chemical Reagent Co., Ltd.), and acetonitrile (Merck AG) were all chromatographic grade; formic acid (Merck AG) was liquid chromatography-mass spectrometry grade; water was ultrapure water (prepared in the laboratory).

[0133] Test reagents: santalol (α- and β-isomers, Merck AG); 10 batches of sandalwood were the dried heartwood of *Santalum album* L.; 3 batches of *Dalbergia hupeana* Hance were deciduous trees belonging to the genus *Dalbergia* of the family Fabaceae; 3 batches of *Platycladus orientalis* (L.) Franco were trees belonging to the genus *Platycladus* of the family Cupressaceae. See Table 1 for details of origin and serial numbers.

[0134] Table 1. Information on sandalwood and its counterfeit samples

[0135]

[0136] Example 1

[0137] This embodiment provides a method for constructing the characteristic spectrum of sandalwood volatile oil of this application, including the following steps:

[0138] 1. Preparation of reference solution

[0139] Take an appropriate amount of santalol reference standard and add methanol to prepare a solution containing 2 mg of santalol per 1 mL, which is used as the reference solution.

[0140] 2. Preparation of the test solution

[0141] Sandalwood material, after removing impurities and shavings (1 mm thick), is taken at approximately 30 g and placed in a flask. 300 mL–500 mL of water is added, along with a suitable amount of boiling chips. The mixture is thoroughly mixed. The volatile oil analyzer is connected to the reflux condenser. Water is added from the top of the condenser until it fills the graduated section of the analyzer and overflows into the flask. The flask is then placed in a heating mantle and slowly heated to boiling, maintaining a gentle boil for approximately 5 hours, until the oil level in the analyzer stops increasing. Heating is then stopped, and the mixture is allowed to stand for a short time. The volatile oil volume is read and collected in a 5 mL volumetric flask. The volatile oil is washed with methanol and diluted to the mark to obtain the volatile oil mother liquor. 1.0 mL of the volatile oil from the volumetric flask is accurately pipetted into a 10 mL volumetric flask, diluted to the mark with methanol, shaken well, filtered, and the filtrate is collected to prepare the test solution.

[0142] 3. Chromatographic detection

[0143] 3.1 Chromatographic detection conditions

[0144] A Waters CORTECS T3 column (2.1 mm × 150 mm, 1.6 μm) was used as the chromatographic column; acetonitrile was used as mobile phase A and water as mobile phase B, and gradient elution was performed according to the specifications in Table 2. The flow rate was 0.30 mL / min; the column temperature was 30 °C; and the detection wavelength was 215 nm. The injection volume was 2 μL.

[0145] Table 2 Gradient Elution Table

[0146]

[0147] 3.2 Determination Method

[0148] Accurately pipette 2 μL of the reference solution and the test solution into the liquid chromatograph for determination.

[0149] 4. Methodological Examination

[0150] 4.1 Specificity Examination

[0151] Prepare the reference solution according to the method in section "1"; take an appropriate amount of sandalwood (No.: SA-1) and prepare the test solution according to the method in section "2". Accurately pipette 2 μL each of the blank solvent (pure methanol), the reference solution, and the test solution, and inject them for analysis under the chromatographic conditions in section "3.1". The comparison of the chromatographic detection results is shown in Figure 1. The results show that the chromatogram of the test solution has the same chromatographic peak at the corresponding retention time as that of the reference solution, and the blank solvent does not interfere, indicating that the method has good specificity.

[0152] 4.2 Precision test

[0153] Take an appropriate amount of sandalwood (No.: SA-1) and prepare the test solution according to the method in section "2". Inject the sample 6 times repeatedly under the chromatographic conditions in section "3.1". Use the peaks (peak 4 and peak 6) of different configurations of santalol as reference peaks S1 and S2, respectively. Calculate the relative retention time and relative peak area of ​​each characteristic peak relative to peaks S1 and S2, and calculate the RSD value. The results are shown in Tables 3 and 4. The RSD values ​​of the relative retention times of each characteristic peak are all less than 1.0%, indicating good instrument precision.

[0154] Table 3. Results of precision study (retention time relative to peak 4)

[0155]

[0156] Table 4. Results of precision study (retention time relative to peak 6)

[0157]

[0158] 4.3 Stability Test

[0159] Take an appropriate amount of sandalwood (No.: SA-1) and prepare 6 test solutions according to the method in section "2". Inject the solutions at 0, 2, 4, 8, 12, 18, 24, 30, 36, and 45 hours according to the chromatographic conditions in section "3.1". Use the peaks of different configurations of santalol (peak 4 and peak 6) as reference peaks S1 and S1, respectively. Calculate the relative retention time and relative peak area of ​​each characteristic peak with respect to peaks S1 and S1, and calculate the RSD value. The results are shown in Tables 5 and 6. The RSD value of the relative retention time of each characteristic peak is less than 0.1%, indicating that the test solution has good stability within 45 hours.

[0160] Table 5. Results of stability study (relative retention time to peak 4)

[0161]

[0162] Table 6. Results of stability study (relative retention time to peak 6)

[0163]

[0164] 4.4 Repeatability Test

[0165] Take an appropriate amount of sandalwood (No.: SA-1) and prepare 6 test solutions according to the method in section "2". Inject and determine the chromatographic conditions in section "3.1". Use the peaks (peak 4 and peak 6) of different configurations of santalol as reference peaks S1 and S2, respectively. Calculate the relative retention time and relative peak area of ​​each characteristic peak relative to peaks S1 and S2, and calculate the RSD value. The results are shown in Tables 7 and 8. The RSD value of the relative retention time of each characteristic peak is less than 0.1%, indicating that the established method has good reproducibility.

[0166] Table 7 Results of reproducibility test (retention time relative to peak 4)

[0167]

[0168] Table 8 Results of reproducibility test (retention time relative to peak 6)

[0169]

[0170] 5. Establish a characteristic spectrum of sandalwood volatile oil.

[0171] 5.1 Determination of the characteristic spectrum of sandalwood volatile oil and establishment of a control characteristic spectrum

[0172] Ten batches of sandalwood medicinal materials from Table 1 were selected, and test solutions were prepared according to the method in section “2”. The samples were injected and determined according to the chromatographic conditions in section “3.1”. The peaks (peak 4 and peak 6) of different configurations of santalol were used as reference peaks S1 and S2, respectively. The relative retention time and relative peak area of ​​each characteristic peak and peaks S1 and S2 were calculated, and the RSD value was calculated. The results are shown in Tables 9 and 10.

[0173] Table 9. Characteristic spectral data of sandalwood volatile oil from 10 batches (relative retention time to peak 4)

[0174]

[0175] Table 10. Spectral characteristics of sandalwood volatile oil from 10 batches (retention time relative to peak 6)

[0176]

[0177] Chromatographic spectra of 10 batches of sandalwood volatile oil were recorded. The integrated characteristic spectra of the 10 batches were exported in CDF format and imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software (Version 2012.130723)" for peak matching and multi-point correction. The results showed that 11 common peaks were identified in the characteristic spectra of the 10 batches of sandalwood volatile oil, as shown in Figure 2. The characteristic spectrum of sandalwood volatile oil was generated using the mean method, as shown in Figure 3.

[0178] 5.2 Establishment of Spectral Standards for Sandalwood Volatile Oil

[0179] Based on the above research results, the chromatographic standard for sandalwood volatile oil is determined as follows: the test sample chromatogram should show 11 characteristic peaks. Peak 4 (α / β-santalol) is used as the reference peak S. The relative retention times of each characteristic peak and peak S are calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.71 (peak 1), 0.84 (peak 2), 0.96 (peak 3), 1.10 (peak 5), 1.14 (peak 6), 1.22 (peak 7), 1.54 (peak 8), and 1.64 (peak 9). 1.78 (peak 10), 1.82 (peak 11); Using peak 6 (α / β-santalol) as reference peak S, calculate the relative retention time of each characteristic peak and peak S. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.62 (peak 1), 0.73 (peak 2), 0.85 (peak 3), 0.88 (peak 4), 0.96 (peak 5), 1.07 (peak 7), 1.36 (peak 8), 1.44 (peak 9), 1.56 (peak 10), 1.60 (peak 11).

[0180] 6. Characteristic Peak Identification

[0181] Using the liquid chromatography conditions of the characteristic spectrum of sandalwood volatile oil, the compounds in the sandalwood volatile oil test solution were analyzed by mass spectrometry. The analysis was performed by matching the spectral data with the local mass spectrometry database for secondary fragment ions and by referring to relevant literature to identify unknown compounds.

[0182] 6.1 Liquid Chromatography-Mass Spectrometry Conditions

[0183] A Waters CORTECS T3 column (2.1 mm × 150 mm, 1.6 μm) was used; acetonitrile was used as mobile phase A and 0.1 v / v% formic acid was used as mobile phase B, and gradient elution was performed according to the specifications in Table 11; the flow rate was 0.30 mL per minute; the column temperature was 30 °C; the detection wavelength was 215 nm; and the injection volume was 1 μL.

[0184] Table 11 Gradient elution procedure

[0185]

[0186] Table 12 Mass Spectrometry Parameters

[0187]

[0188] 6.2 Compound Identification

[0189] The test solution was analyzed using the above-mentioned liquid chromatography and mass spectrometry conditions. Through comparison of the retention time of the reference standard in liquid chromatography and the precise molecular weight and fragment ion analysis in mass spectrometry, santalol in different configurations (α or β configurations) was identified as peaks 4 and 6 in the characteristic chromatogram of sandalwood volatile oil. The total ion chromatograms of the sandalwood volatile oil test solution and the santalol reference solution are shown in Figure 4, and the ultraviolet absorption chromatograms are shown in Figure 5.

[0190] Example 2

[0191] This embodiment investigates the effect of different organic solvents used in the preparation of the test sample on the chromatographic detection results. Six portions of the same batch of sandalwood (number: SA-1) were taken, divided into three parallel groups of two portions each. The test sample solution was prepared using a method essentially the same as that under "2. Preparation of Test Sample Solution" in Example 1, except that after collecting the volatile oil, it was washed with methanol, acetonitrile, and ethanol, respectively, and then diluted to the mark to obtain the volatile oil mother liquor. 1 mL of the volatile oil mother liquor was accurately pipetted into a 10 mL volumetric flask, diluted to the mark with the corresponding solvent, shaken well, filtered, and the filtrate was collected to obtain the volatile oil test sample solution.

[0192] Accurately pipette the above-mentioned test solution and inject it according to the method under "3.1 Chromatographic Detection Conditions" in Example 1. Record the peak areas of the 11 characteristic peaks and calculate "total peak area / volatile oil content". The results are shown in Table 13 and Figure 6.

[0193] Table 13 Results of Solvent Investigation of Sandalwood Volatile Oil

[0194]

[0195] The results showed that there were no significant differences in peak shape and resolution of the 11 characteristic peaks when different solvents were used. When methanol was selected as the solvent, the "total peak area / volatile oil content" value was the largest, indicating that methanol had the highest extraction efficiency for sandalwood volatile oil. Therefore, methanol was used as the solvent in subsequent studies.

[0196] Example 3

[0197] This embodiment investigates the effect of different material-to-liquid ratios (dilution factors) on chromatographic detection results during sample preparation. Six portions of the same batch of sandalwood (number: SA-1) were taken, divided into three parallel groups of two portions each. The volatile oil mother liquor was prepared according to the method described in "2. Preparation of Sample Solution" of Example 1. 0.5 mL, 1.0 mL, and 2.0 mL of the volatile oil mother liquor were accurately pipetted into 10 mL volumetric flasks, diluted to the mark with methanol, shaken well, filtered, and the filtrate was collected to obtain the volatile oil sample solutions at different dilution factors.

[0198] Accurately pipette the above-mentioned test solution and inject it according to the method under "3.1 Chromatographic Detection Conditions" in Example 1. Record the peak area of ​​each of the 11 characteristic peaks and calculate "total peak area / (volatile oil content × volume of mother liquor transferred)". The results are shown in Table 14.

[0199] Table 14 Results of the Dilution Factor Study of Sandalwood Volatile Oil

[0200]

[0201] The results showed that the value of "total peak area / (volatile oil content × volume of mother liquor transferred)" remained basically stable at different dilution factors, indicating that the response intensity of each characteristic peak had a good linear relationship with the injection volume within the range of dilution factors investigated. Among them, the calculated value was slightly higher when the volume of mother liquor transferred was 1.0 mL, so the volume of mother liquor transferred was 1.0 mL for subsequent studies.

[0202] Example 4

[0203] This embodiment examines the effect of different detection wavelengths on chromatographic detection results. Sandalwood material from the same batch (number: SA-1) was used, and a test solution was prepared according to the same method as under "2. Preparation of Test Solution" in Example 1. The sample was injected and analyzed using essentially the same method as under "3.1 Chromatographic Detection Conditions" in Example 1, except that the detection wavelength was switched to full-wavelength scanning. The absorption spectrum of the sandalwood volatile oil test solution in the range of 190 nm to 400 nm was recorded, and the results are shown in Figure 7. The results show that the characteristic peaks of sandalwood volatile oil are mainly concentrated between 200 nm and 250 nm, with virtually no ultraviolet absorption above 250 nm.

[0204] The chromatograms of sandalwood volatile oil at certain wavelengths are shown in Figure 8. The results show that there are abundant chromatographic peaks between 200 nm and 250 nm, with the peak information being most abundant and the overall response being more uniform at 215 nm. Therefore, 215 nm was selected as the detection wavelength for establishing the UPLC characteristic spectrum of sandalwood volatile oil.

[0205] Example 5

[0206] This embodiment examines the effect of different mobile phases on chromatographic detection results. Sandalwood material from the same batch (number: SA-1) was used, and a test solution was prepared according to the same method as under "2. Preparation of Test Solution" in Example 1. The solution was injected and analyzed according to essentially the same method as under "3.1 Chromatographic Detection Conditions" in Example 1, except that mobile phase A-mobile phase B was switched to acetonitrile-0.1% phosphoric acid and acetonitrile-0.2% phosphoric acid. The resulting chromatograms were compared with those obtained in Example 1 using acetonitrile-water as the mobile phase, and the comparison results are shown in Figure 9. The results show that when acetonitrile is used as mobile phase A and water or an aqueous solution of phosphoric acid is used as mobile phase B, the resolution and peak shape of each characteristic peak are better. For ease of operation, water is preferred as mobile phase B.

[0207] Example 6

[0208] This embodiment investigates the effect of different column temperatures on chromatographic detection results. Sandalwood material from the same batch (number: SA-1) was used, and a test solution was prepared according to the same method as under "2. Preparation of Test Solution" in Example 1. The solution was injected and analyzed using essentially the same method as under "3.1 Chromatographic Detection Conditions" in Example 1, except that the column temperature was switched to 35℃ and 40℃. The resulting chromatograms were compared with those obtained in Example 1 using a column temperature of 30℃. The comparison results are shown in Figure 10. The results show that a column temperature of 30℃~40℃ has little effect on the characteristic chromatogram of sandalwood volatile oil; the response, resolution, and peak shape of each characteristic peak are all excellent. To prolong the column life, a column temperature of 30℃ is preferred.

[0209] Example 7

[0210] This embodiment investigates the effect of different flow rates on chromatographic detection results. Sandalwood material from the same batch (number: SA-1) was used, and the test solution was prepared according to the same method as under "2. Preparation of Test Solution" in Example 1. The solution was injected and analyzed according to essentially the same method as under "3.1 Chromatographic Detection Conditions" in Example 1, except that the flow rate was switched to 0.20 mL / min and 0.25 mL / min. The resulting chromatograms were compared with those obtained in Example 1 using a flow rate of 0.30 mL / min. The comparison results are shown in Figure 11. The results show that at flow rates of 0.20 mL / min to 0.30 mL / min, except for retention time, the resolution, peak shape, and response of each characteristic peak are better. To concentrate the characteristic peaks in the middle region, a flow rate of 0.30 mL / min is preferred.

[0211] Comparative Example 1

[0212] This comparative study investigated the effect of different mobile phases on chromatographic detection results. Sandalwood material from the same batch (number: SA-1) was used, and a test solution was prepared according to the same method as under "2. Preparation of Test Solution" in Example 1. The solution was injected and analyzed according to essentially the same method as under "3.1 Chromatographic Detection Conditions" in Example 1, except that mobile phase A-mobile phase B was switched to methanol-water, acetonitrile-0.1% formic acid, and acetonitrile-0.1% acetic acid. The resulting chromatograms were compared with those obtained in Example 1 using acetonitrile-water as the mobile phase, and the comparison results are shown in Figure 12. The results showed that when methanol was used as mobile phase A, the resolution of each characteristic peak significantly deteriorated, and when an aqueous solution of formic acid or acetic acid was used as mobile phase B, the baseline of the chromatogram was unstable.

[0213] Comparative Example 2

[0214] This embodiment examines the effect of different elution gradients on chromatographic detection results. Sandalwood material from the same batch (number: SA-1) was used, and a test solution was prepared according to the same method as under "2. Preparation of Test Solution" in Example 1. The solution was injected and analyzed according to essentially the same method as under "3.1 Chromatographic Detection Conditions" in Example 1, except that the elution gradient was switched to either gradient 1 or gradient 2 as shown in Table 15. The resulting chromatograms were compared with those obtained in Example 1 using Table 2 as the elution gradient condition (gradient 1). The comparison results are shown in Figure 13. The results show that with gradient 1, the chromatographic peaks are mainly concentrated between 30 and 40 minutes, resulting in poor peak resolution. Although the optimized gradient 2 improved the resolution, some peaks still showed poor resolution.

[0215] Table 15 Preferred Gradient Elution Table

[0216]

[0217] Comparative Example 3

[0218] This embodiment examines the effect of different chromatographic columns on chromatographic detection results. Take sandalwood material from the same batch (number: SA-1), and prepare the test solution according to the same method as under "2. Preparation of test solution" in Example 1. Inject and determine the solution according to the basically the same method as under "3.1 Chromatographic Detection Conditions" in Example 1, except that the chromatographic column was changed to Waters ACQUITY UPLC BEH C18 (2.1mm × 150mm, 1.7μm), Waters ACQUITY UPLC HSS T3 (2.1mm × 150mm, 1.8μm), YMC Triart C18 (2.1mm × 150mm, 1.9μm), SHIMADZU Shim-pack Scepter C18-120 (2.1mm × 150mm, 1.9μm), or Agilent ZORBAX RRHD StableBond C18 (2.1mm × 150mm, 1.8μm). The obtained chromatogram is consistent with that obtained in Example 1 using Waters CORTECS... The chromatograms of columns using the T3 (2.1 mm × 150 mm, 1.6 μm) were compared, and the results are shown in Figure 14. The results show that when using columns other than the Waters CORTECS T3 (2.1 mm × 150 mm, 1.6 μm), the peak shapes and resolutions of the characteristic peaks are poor, which cannot meet the requirements for constructing characteristic chromatograms.

[0219] Example 8

[0220] This embodiment provides the application of the above-mentioned characteristic chromatogram construction method in the identification of the source of sandalwood volatile oil materials. Three batches of sandalwood trunks (numbered SA-1, SA-2, and SA-3) were taken according to Table 1. The heartwood and sapwood were separated. The test solution was prepared according to the same method as under "2. Preparation of Test Solution" in Example 1, and the chromatograms were injected and analyzed according to the same method as under "3.1 Chromatographic Detection Conditions" in Example 1. The superimposed chromatograms are shown in Figure 15. The results show that the characteristic chromatograms of the volatile oil from the three batches of sandalwood heartwood (SA-1, SA-2, and SA-3) exhibit the specified 11 characteristic peaks, while the corresponding sapwood volatile oil characteristic chromatograms showed almost no response. This indicates that the method can be used to distinguish between the heartwood and sapwood of sandalwood.

[0221] Example 9

[0222] This embodiment provides the application of the above-mentioned characteristic chromatogram construction method in the identification of genuine and counterfeit sandalwood volatile oil materials. Three batches of sandalwood (numbered SA-1, SA-2, SA-3), three batches of Dalbergia odorifera, and three batches of Platycladus orientalis were taken according to Table 1. Test solutions were prepared using the same method as under "2. Preparation of Test Solution" in Example 1, and the samples were injected and analyzed using the same method as under "3.1 Chromatographic Detection Conditions" in Example 1. The superimposed chromatograms are shown in Figure 16. The results show that the characteristic chromatograms of the volatile oils from the three batches of sandalwood heartwood (SA-1, SA-2, SA-3) exhibited the prescribed 11 characteristic peaks. The characteristic chromatograms of the volatile oils from the three batches of Dalbergia odorifera (DH-1, DH-2, DH-3) showed no obvious response. The volatile oils from the three batches of Platycladus orientalis (PO-1, PO-2, PO-3) exhibited four characteristic peaks different from those of sandalwood volatile oil. This indicates that the method can distinguish sandalwood volatile oil from its common counterfeit volatile oils.

[0223] Example 10

[0224] This embodiment provides the application of the above-mentioned characteristic spectrum construction method in the identification of sandalwood volatile oil and other sandalwood extracts. Three batches of sandalwood (numbered: SA-1, SA-2, SA-3) were taken according to Table 1, and each batch was divided into three parallel groups. Sandalwood decoction, sandalwood alcohol extract and sandalwood volatile oil were prepared respectively, and corresponding test solutions were prepared.

[0225] The specific preparation method is as follows:

[0226] Sandalwood volatile oil and its corresponding test solution: Remove impurities and 1mm thick sandalwood shavings from the raw material. Place approximately 30g in a flask, add 300mL-500mL of water, add an appropriate amount of boiling chips, mix well, connect the volatile oil analyzer to the reflux condenser, and add water from the top of the condenser until it fills the graduated section of the volatile oil analyzer and overflows into the flask. Heat slowly in a heating mantle to boiling, maintaining a gentle boil for about 5 hours until the oil level in the analyzer no longer increases. Stop heating, let stand for a moment, read the volatile oil volume, and collect the volatile oil in a 5mL volumetric flask. Wash with methanol and dilute to the mark to obtain the volatile oil mother liquor. Accurately pipette 1.0mL of the volatile oil from the volumetric flask into a 10mL volumetric flask, dilute to the mark with methanol, shake well, filter, and collect the filtrate to prepare the test solution.

[0227] Sandalwood oil decoction and its corresponding test solution: Sandalwood material, after removing impurities and sandalwood shavings (1 mm thick), about 30 g is placed in a flask, 300-500 mL of water is added, along with an appropriate amount of boiling chips, and mixed thoroughly. The volatile oil analyzer is connected to the reflux condenser. Water is added from the top of the condenser until it fills the graduated section of the volatile oil analyzer and overflows into the flask. The flask is placed in a heating mantle and slowly heated to boiling, maintaining a gentle boil for about 5 hours, until the oil volume in the analyzer no longer increases. Heating is then stopped, and the volatile oil volume is read and collected in a 5 mL volumetric flask. An appropriate amount of the decoction after extracting the volatile oil is taken, filtered, and the filtrate is collected to obtain the test solution.

[0228] Sandalwood alcohol extract and its corresponding test solution: Take 1.0 g of sandalwood heartwood powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 10 ml of methanol, weigh it, extract it by ultrasonication (300 W, 40 kHz) for 30 minutes, cool it, make up the weight with methanol, filter it, take the filtrate, and prepare the test solution.

[0229] The test solutions from the different extract sources were injected and analyzed using the same method as described in "3.1 Chromatographic Detection Conditions" of Example 1. The superimposed chromatograms are shown in Figure 17. The results showed that the characteristic chromatograms of the volatile oils of the three batches of sandalwood (SA-1, SA-2, SA-3) exhibited the 11 specified characteristic peaks. The chromatogram of the sandalwood decoction after removing the volatile oils showed no chromatographic peak response, indicating that the 11 characteristic peaks were all chemical components of the sandalwood volatile oil. This characteristic chromatographic method can be used to identify whether sandalwood products have added volatile sandalwood components. The characteristic chromatogram of the alcohol extract showed 5 characteristic peaks (peak 1, peak 4, peak 6, peak 7, peak 10), while the remaining 6 characteristic peaks had low or absent responses, indicating that alcohol extraction alone cannot fully represent the chemical components of the sandalwood volatile oil.

[0230] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0231] The embodiments described above merely illustrate several implementation methods of this application and should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Furthermore, it should be understood that after reading the above teachings, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the protection scope of the appended claims. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for constructing a characteristic spectrum of sandalwood volatile oil, characterized in that, The process includes the following steps: mixing sandalwood and water and then steam distilling the mixture to collect the sandalwood volatile oil; dissolving the sandalwood volatile oil in an organic solvent to obtain a test solution; The test solution was subjected to high-performance liquid chromatography (HPLC) to obtain the characteristic chromatogram of the sandalwood volatile oil. The HPLC conditions included: a T3 column, core-shell silica gel particles as packing material, the core-shell silica gel particles comprising a solid core and a porous shell outside the solid core; acetonitrile as mobile phase A, and an aqueous solution of water or phosphoric acid as mobile phase B; gradient elution, the gradient elution program including: 0-25 min, the volume percentage of mobile phase A increasing from 45% to 50%; 25 min-40 min, the volume percentage of mobile phase A increasing from 50% to 65%; 40 min-50 min, the volume percentage of mobile phase A increasing from 65% to 85%.

2. The method for constructing a feature map according to claim 1, characterized in that, The chromatographic column has a length of 150 mm and an inner diameter of 2.1 mm, and the packing material has a particle size of 1.6 μm-1.9 μm.

3. The method for constructing a feature map according to claim 1, characterized in that, The conditions for high performance liquid chromatography detection also include at least one of the following: (1) the volume concentration of the aqueous solution of phosphoric acid is 0.1%-0.2%; (2) the flow rate is 0.2 mL / min-0.4 mL / min; (3) the column temperature is 30℃-40℃; (4) the detection wavelength is 200 nm-250 nm; and (5) the injection volume is 1 μL-2 μL.

4. The method for constructing a feature map according to any one of claims 1-3, characterized in that, The characteristic spectrum of the sandalwood volatile oil shows the following characteristic peaks: peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, peak 10 and peak 11; among which, peak 4 and peak 6 are characteristic peaks corresponding to santalol.

5. The method for constructing a feature map according to claim 4, characterized in that, In the characteristic peaks of the characteristic spectrum of the sandalwood volatile oil: taking peak 4 as the S1 peak, the relative retention times of peaks 1-3 and peaks 5-11 with the S1 peak are calculated. The relative retention times are within ±10% of the specified values, which are: peak 1: 0.71; peak 2: 0.84; peak 3: 0.96; peak 5: 1.10; peak 6: 1.14; peak 7: 1.22; peak 8: 1.54; peak 9: 1.64; peak 10: 1.

78. Peak 11: 1.82; Taking peak 6 as the S2 peak, calculate the relative retention times of peaks 1-5, peaks 7-11 and the S2 peak. The relative retention times are within ±10% of the specified values, which are: peak 1: 0.62; peak 2: 0.73; peak 3: 0.85; peak 4: 0.88; peak 5: 0.96; peak 7: 1.07; peak 8: 1.36; peak 9: 1.44; peak 10: 1.56; peak 11: 1.

60.

6. The method for constructing a feature map according to any one of claims 1-3 and 5, characterized in that, The organic solvent includes one or more of methanol, ethanol, and acetonitrile; and / or, the volume ratio of the sandalwood volatile oil to the organic solvent is (0.05-0.2):

1.

7. The method for constructing a feature map according to any one of claims 1-3 and 5, characterized in that, The conditions for steam distillation include: a feed-to-liquid ratio of 1g:(10mL-20mL) and a distillation time of 4h-6h.

8. The method for constructing a feature map according to any one of claims 1-3 and 5, characterized in that, The method further includes the following steps: performing high-performance liquid chromatography (HPLC) on the reference solution to obtain a reference chromatogram; the reference solution includes santalol; comparing the reference chromatogram with the characteristic chromatogram of sandalwood volatile oil to identify the characteristic peaks in the characteristic chromatogram of sandalwood volatile oil, wherein the HPLC detection conditions are the same as those defined in any one of claims 1-3; and / or performing HPLC-MS / MS combined detection on the reference solution and the test solution to obtain a total ion chromatogram of the reference and a total ion chromatogram of sandalwood volatile oil; The total ion chromatogram of the reference material and the total ion chromatogram of the sandalwood volatile oil were compared to identify the characteristic peaks in the total ion chromatogram of the sandalwood volatile oil. The high-performance liquid chromatography (HPLC) detection conditions include: a T3 column, core-shell silica gel particles as packing material, the core-shell silica gel particles comprising a solid core and a porous shell outside the solid core; acetonitrile as mobile phase A, and an aqueous solution of water or formic acid as mobile phase B; gradient elution, the gradient elution program including: 0-25 min, the volume percentage of mobile phase A increasing from 45% to 50%; 25 min-40 min, the volume percentage of mobile phase A increasing from 50% to 65%; 40 min-50 min, the volume percentage of mobile phase A increasing from 65% to 85%.

9. The method for constructing a feature map according to claim 8, characterized in that, The conditions for mass spectrometry detection include: using a heated electrospray ion source, positive ion mode detection, a scan range of m / z 90-500, a normalized collision energy of 35-45, and a peak-shaped mass spectrum.

10. The application of the method for constructing the characteristic spectrum according to any one of claims 1-9 in the identification of sandalwood volatile oil.

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