Method for separating and preparing megastigmatrienone isomeride

Abstract

The invention discloses a method for separating and preparing megastigmatrienone isomers, and belongs to the technical field of isomer separation and preparation. The technical problem to be solved is that in the prior art, mg-level separation preparation of a megastigmatrienone single-configuration isomer cannot be rapidly realized. The key point of the technical scheme is as follows: the method comprises the following steps: (1) mixing megastigmatrienone with methanol to obtain a mixed solution; (2) separating the mixed solution by adopting a reversed-phase preparative chromatography to obtain an isomer of megastigmatrienone; wherein the chromatographic conditions in the reversed-phase preparative chromatography in the step (2) are as follows: a chromatographic column is a reversed-phase semi-preparative chromatographic column, the sample size is 100-800 [mu] L, the flow velocity is 0.1-0.8 mL / min, the detection wavelength is 308 nm, an eluent is methanol-THF-H2O in a ratio of 37.5: 7.5: 55, and isocratic elution is performed.

Description

Technical Field

[0001] This invention belongs to the field of isomer separation and preparation technology, and provides a method for separating and preparing megatiterone isomers. Background Technology

[0002] For understanding the technical content of this invention:

[0003] Megastigrinone, also known as "flue-cured tobacco ketone," is naturally found in tobacco leaves such as Burley tobacco. Its chemical structure was isolated and identified from Burley and aromatic tobacco in 1972. It is primarily a mixture of four isomers, and is a colorless to pale yellow oily liquid with a strong and persistent tobacco-like, sweet, warm, and dry aroma, along with a spicy undertone. As an endogenous component of tobacco, megastigrinone's aroma is naturally harmonious with tobacco and is considered an indispensable key flavoring in cigarette manufacturing. Furthermore, its unique aroma characteristics have led to its widespread use in perfumes, cosmetics, beverages, and food flavorings. Currently, megastigmatrienones are typically mixtures of four isomers. The roles of megastigmatrienones with different structures in tobacco are unclear, and there is currently no effective method for separating these four isomers. In order to effectively separate these four isomers and further clarify the effects of megastigmatrienones with different structures on the aroma and quality of tobacco, it is essential to develop a reversed-phase preparative chromatography method for separating and preparing the four isomers.

[0004] Relevant patent documents retrieved: Chinese invention patent application CN120025241A (published on May 23, 2025) discloses a method for separating isomers of stigmatatrienone. The method involves mixing stigmatatrienone with methanol to obtain a mixture, filtering the mixture using an organic phase filter membrane, and then separating the filtered mixture using reversed-phase preparative chromatography to obtain the isomers of stigmatatrienone. This separation method can efficiently separate different isomers of stigmatatrienone, and it is also green, environmentally friendly, simple to operate, and easy to implement.

[0005] Relevant non-patent literature retrieved: The paper "Isolation and Identification of 3-oxo-α-ionol-β-D-glucopyranoside from Tobacco and Analysis of its Pyrolysis Products [J]" (Chen Zhenling, Zhang Haobo, Zhou Wanhong, et al. Tobacco Science and Technology, 2008, (07): 28-31) discloses the extraction of 3-oxo-α-ionol-β-D-glucopyranoside from Zimbabwean flue-cured tobacco leaves by methanol extraction and preparative chromatography at normal, medium and high pressures, with a yield of 0.0034%. The extract was identified by mass spectrometry, ultraviolet, infrared and nuclear magnetic resonance, and analyzed by pyrolysis-GC / MS in helium. The results showed that the extract was the target compound, and its pyrolysis products were mainly four isomers of megastigmatrienone and a small amount of 3-oxo-α-ionol.

[0006] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: The aforementioned separation methods cannot achieve mg-level separation and preparation of single-configuration mesostearne isomers or shorten the elution time of mesostearne isomers. Therefore, there is an urgent need to provide a method for separating and preparing mesostearne isomers, achieving mg-level separation and preparation of single-configuration mesostearne isomers and shortening the elution time of mesostearne isomers. Summary of the Invention

[0007] The purpose of this invention is to provide: To address the technical problems in the prior art, this invention provides a method for separating and preparing megastigmatrienone isomers, and related technologies, to solve technical problems such as the inability to achieve mg-level separation and preparation of megastigmatrienone with a single configuration and long peak elution time, or combinations thereof.

[0008] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0009] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0010] Definitions of standard chemical terms can be found in the reference book "High Performance Liquid Chromatography Methods and Applications" (Chemical Industry Press, Yu Shilin, published in January 2019).

[0011] Unless otherwise stated, conventional methods within the scope of the art, such as mixing methods, shall be used.

[0012] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0013] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0014] The term THF used in this article refers to tetrahydrofuran.

[0015] The term H2O used in this article refers to water.

[0016] In a first aspect, the present invention provides: a method for separating and preparing megastigmatrienone isomers, the method comprising the following steps: (1) Mix megastigmatrienone with methanol to obtain a mixture; (2) The mixture was separated by reversed-phase preparative chromatography to obtain the isomers of megastigmatrienone; The chromatographic conditions in the reversed-phase preparative chromatography method described in step (2) are as follows: the chromatographic column is a reversed-phase semi-preparative chromatographic column, the injection volume is 100-800 μL, the flow rate is 0.1-0.8 mL / min, the detection wavelength is 308 nm, and the eluent is methanol-THF-H2O = 37.5:7.5:55 isocratic elution.

[0017] In one embodiment of the present invention, the concentration of megastigmatrienone in the mixture of step (1) is 1-3 mg / mL.

[0018] In one embodiment of the present invention, the concentration of megastigmatrienone in the mixture of step (1) is preferably 1 mg / mL.

[0019] In one embodiment of the present invention, the concentration of megalotrienone in the mixture of step (1) is preferably 2 mg / mL.

[0020] In one embodiment of the present invention, the concentration of megalotrienone in the mixture of step (1) is preferably 3 mg / mL.

[0021] In one embodiment of the present invention, the reversed-phase semi-preparative chromatographic column is a PFP chromatographic column.

[0022] In one embodiment of the present invention, the reverse preparation chromatography column is preferably an XSelect HSS PFP chromatography column.

[0023] In one embodiment of the present invention, the reverse preparative chromatography column is further preferably an XSelect HSSPFP 5μm OBD 10×250 mm.

[0024] In one embodiment of the present invention, the column temperature of the reversed-phase semi-preparative chromatographic column is 25°C. In one embodiment of the present invention, the eluent is selected from at least two of methanol, THF, and H2O.

[0025] In one embodiment of the present invention, the eluent is preferably in the ratio of methanol-THF-H2O = 37.5:7.5:55.

[0026] In one embodiment of the present invention, the injection volume is selected from 100-800 μL.

[0027] As one embodiment of the present invention, the injection volume is preferably 100μL, 200μL, 300μL, 400μL, 500μL, 600μL, 700μL or 800μL.

[0028] In one embodiment of the present invention, the injection volume is selected from 1-2.5 mg.

[0029] In one embodiment of the present invention, the injection volume is preferably 1-2.4 mg.

[0030] In one embodiment of the present invention, the flow rate is 0.1-0.8 mL / min.

[0031] In one embodiment of the present invention, the flow rate is 0.1 mL / min, 0.2 mL / min, 0.3 mL / min, 0.4 mL / min, 0.5 mL / min, 0.6 mL / min, 0.7 mL / min or 0.8 mL / min.

[0032] In one embodiment of the present invention, the detection wavelength is selected from 308nm.

[0033] As one embodiment of the present invention, the isomers of stigmatrienone obtained in step (2) include four types, with structural formulas as shown in A, B, C and D: .

[0034] The beneficial effects of this invention are as follows: Compared with the prior art, this invention has better technical effects in terms of peak elution time and injection volume. According to experimental tests, this invention shortens the peak elution time of the four isomers of stigmatatrienone from more than 63-92 min in the prior art to 42-55 min, which is a significant reduction in peak elution time. At the same time, it achieves high-purity mg-level separation and preparation of the four isomers of stigmatatrienone. Attached Figure Description

[0035] Figure 1 The image shows the chromatogram of Experiment Example 1, where A is the full chromatogram and B is a magnified view of a portion of the chromatogram. Figure 2 The chromatograms for the water-methanol gradient separation of the mobile phase in Experiment Example 2 are shown, where A is the full chromatogram and B is a magnified view of a portion of the chromatogram. Figure 3 The chromatograms for isocratic separation of water and methanol as the mobile phase in Experiment Example 2 are shown, where A is the full chromatogram and B is a magnified partial chromatogram. Figure 4 The chromatogram of Experiment Example 4; Figure 5 This is the chromatogram of isocratic elution with 55% methanol / water in Experiment Example 5; Figure 6 This is the chromatogram of isocratic elution with 50% methanol / water in Experiment Example 5; Figure 7 The chromatogram is of methanol and acetonitrile mixed in a volume ratio of 50:5 as the organic phase in Experiment Example 6, and isocratically eluted with 55% organic phase / water. Figure 8 The chromatogram is of methanol and acetonitrile mixed in a volume ratio of 50:5 as the organic phase in Experiment Example 6, and isocratically eluted with 50% organic phase / water. Figure 9 The chromatogram is of methanol and acetonitrile mixed in a volume ratio of 50:5 as the organic phase in Experiment Example 6, and isocratically eluted with 45% organic phase / water. Figure 10 The chromatogram is of methanol and acetonitrile mixed at a volume ratio of 50:10 as the organic phase in Experimental Example 6, with 55% organic phase / water isocratic elution. Figure 11 The chromatogram is of methanol and acetonitrile mixed at a volume ratio of 50:10 as the organic phase in Experiment Example 6, with 50% organic phase / water isocratic elution. Figure 12 The chromatogram is of methanol and acetonitrile mixed at a volume ratio of 50:10 as the organic phase in Experiment Example 6, and isocratically eluted with 45% organic phase / water. Figure 13 Chromatogram of methanol and THF mixed at a volume ratio of 50:10 as the organic phase, eluted isocratically with 40% organic phase / water. Figure 14 Chromatogram of methanol and THF mixed at a volume ratio of 50:10 as the organic phase, eluted isocratically with 45% organic phase / water. Figure 15 Chromatogram of methanol and THF mixed at a volume ratio of 50:15 as the organic phase, with 43% organic phase / water isocratic elution. Figure 16 Chromatogram of methanol and THF mixed at a volume ratio of 50:15 as the organic phase, eluted isocratically with 45% organic phase / water. Figure 17 Chromatogram of methanol and THF mixed at a volume ratio of 50:20 as the organic phase, eluted isocratically with 45% organic phase / water. Figure 18 Chromatogram of methanol and THF mixed at a volume ratio of 50:30 as the organic phase, eluted isocratically with 40% organic phase / water. Figure 19 This is a chromatogram of a preparation example of the present invention. Detailed Implementation

[0036] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0037] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0038] The purified megastigmatrienone (purity > 90%) was prepared in-house and its purity was determined by testing with an Agilent GCMS8890-5977C and then based on the relative area.

[0039] Screening Experiment 1: Reverse Phase Column Screening Experimental Example 1 Megastigmatrienone was diluted with methanol to obtain a concentration of 3 mg / mL, and the injection volume was 0.1 mL. Chromatographic column: Venusil ABS C18 column, inner diameter 5 μm, 30 mm × 200 mm; column temperature 25℃, detection wavelength 308 nm; mobile phase: water-acetonitrile gradient separation, elution program as shown in Table 1 below.

[0040] Table 1: Elution program for Venusil ABS C18 column

[0041] Chromatogram as shown Figure 1 As shown, according to Figure 1 The results show that the separation effect of this column is very poor, and this bonded phase is not suitable for isomer separation and purification.

[0042] Experiment Example 2 Megastigmatrienone was diluted with methanol to obtain a concentration of 3 mg / mL, with an injection volume of 0.1 mL. A Shimadzu reversed-phase preparative column (Shim Pick GIS C18 (HSS)) with an inner diameter of 5 μm and dimensions of 20 mm × 250 mm was used; the column temperature was 25℃; and the detection wavelength was 308 nm. Two different mobile phases were used for elution, as detailed below: (1) Water-methanol gradient separation The program is shown in the table below.

[0043] Table 2: Gradient elution program for Shim Pick GIS C18 column

[0044] Chromatography Figure 2 As shown, the sample did not achieve baseline separation after 60 min, the peak shape was relatively wide, and a flat-topped peak appeared.

[0045] (2) Water-methanol isocratic separation The eluent was a methanol-water mixture of 75%:25% with isocratic elution at a flow rate of 3 mL / min. The chromatogram is shown below. Figure 3 As shown, the samples were not separated, the elution time was significantly advanced, and a flat-topped peak appeared. Therefore, it is necessary to reduce the proportion of organic phase to prolong the elution time and reduce the injection volume.

[0046] Experimental Example 3 Megastigmatrienone was diluted with methanol to obtain a concentration of 3 mg / mL, with an injection volume of 0.1 mL. The chromatographic column was an Elite Hypersil BDS C18, with an inner diameter of 5 μm and dimensions of 4.6 mm × 250 mm; the column temperature was 25℃; the detection wavelength was 308 nm; the flow rate was 1 mL / min; and the eluent was water-acetonitrile = 50%:50%.

[0047] The sample did not produce a peak under these conditions, indicating that this column is not suitable for the separation and purification of megastigmatrienone isomers.

[0048] Experiment Example 4 Megastigmatrienone was diluted with methanol to obtain a concentration of 1 mg / mL, and the injection volume was 1 μL. Chromatographic column: ACQUITY UPLC HSS PFP, 1.8 μm inner diameter, 2.1 mm × 100 mm analytical column; eluent: 55% methanol-45% water isocratic elution; flow rate: 0.3 mL / min; column temperature: 25℃; detection wavelength: 308 nm.

[0049] Chromatogram as shown Figure 4 As shown, the peak shape of the analytical column was not ideal; two substances achieved baseline separation, while the other two did not. However, the overall resolution was better than that of the C18 reversed column.

[0050] In summary, the C18 reversed-phase column is not effective in separating megastigmatrienone isomers, therefore the PFP reversed-phase column was chosen for the development of a separation method.

[0051] Screening Experiment 2: Optimization of Elution Conditions Experimental Example 5: Optimization of a Single Elution Phase Isocratic elution was performed using 55% methanol / water, 50% methanol / water, 45% methanol / water, 40% methanol / water, and 35% methanol / water, respectively; flow rate: 0.3 mL / min; injection volume: 1 μL; column temperature: 25℃; detection wavelength: 308 nm.

[0052] (1) The eluent is 55% methanol / water isocratic elution. Chromatogram as shown Figure 5 As shown, the column peak shape was poor; two substances achieved baseline separation, another did not, and a third was not separated at all. Therefore, reducing the organic phase ratio and extending the elution time was chosen to observe the degree of separation.

[0053] (2) Eluent: 50% methanol / water isocratic elution Chromatogram as shown Figure 6 As shown in the chromatogram, the peak shapes of the three substances are good, and the elution time is prolonged. The other substance was not completely separated, but the separation effect was better than 55% of the organic phase. Therefore, the ratio of the organic phase should be further optimized.

[0054] (3) Eluent: 45%, 40%, 35% methanol / water isocratic elution Elution with less than 50% methanol results in poor peak shape, unstable baseline, and poor separation. Furthermore, reducing the amount of organic solvent increases the separation time. However, the desired method is to achieve good separation and control the separation time to around 50 minutes.

[0055] Since the separation effect of a single mobile phase of methanol and acetonitrile cannot completely separate the four substances, a mixed elution phase was selected for further optimization.

[0056] Experimental Example 6: Optimization of Mixed Elution Phase Different eluents were selected. When methanol and acetonitrile were used as the organic phase, the flow rate was 0.3 mL / min. When methanol and THF were used as the organic phase, the flow rate was 0.1 mL / min. The injection volume was 1 μL. The column temperature was 25℃. The detection wavelength was 308 nm.

[0057] The eluents are as follows.

[0058] (1) Eluent: Methanol and acetonitrile are mixed in a volume ratio of 50:5 as the organic phase, and elution is carried out with 55% organic phase / water isocratic. Chromatogram as shown Figure 7 As shown, the peak shape of the analytical column is relatively good, showing better stereoscopicity than that of a single mobile phase peak. Two substances achieve baseline separation, while the other substance is not completely separated, although a peak tip is visible. However, the elution time is relatively fast, so the proportion of the organic phase was reduced to prolong the elution time, and the separation effect was observed.

[0059] (2) Eluent: Methanol and acetonitrile are mixed in a volume ratio of 50:5 as the organic phase, and elution is carried out by isocratic elution with 50% organic phase / water. Chromatogram as shown Figure 8 As shown in the figure, the column peak shape is good; three substances are baseline separated, while the third substance is not completely separated but a distinct peak tip is visible. The separation effect is better than that of 55% organic phase.

[0060] (3) Eluent: Methanol and acetonitrile are mixed in a volume ratio of 50:5 as the organic phase, and elution is carried out with 45% organic phase / water isocratic. Chromatogram as shown Figure 9 As shown in the figure. Analyzing the column peak shapes, three substances achieved baseline separation, while the third substance did not reach baseline separation but its separation effect was better than 50% of the organic phase. The elution time was relatively long, indicating incomplete separation; therefore, further optimization is needed.

[0061] (4) Eluent: Methanol and acetonitrile are mixed at a volume ratio of 50:10 as the organic phase, and elution is carried out with 55% organic phase / water isocratic. Chromatogram as shown Figure 10 As shown, the peak shape of the analytical column is not as good as that of 50 / 5 methanol acetonitrile. Three substances can achieve baseline separation, while the other substance cannot, so further optimization is needed.

[0062] (5) Eluent: Methanol and acetonitrile are mixed at a volume ratio of 50:10 as the organic phase, and elution is carried out by isocratic elution with 50% organic phase / water. Chromatogram as shown Figure 11 As shown, the peak shape of the analytical column is not as good as that of 50 / 5 methanol acetonitrile. Three substances can achieve baseline separation, while the other substance cannot, so further optimization is needed.

[0063] (6) Eluent: Methanol and acetonitrile are mixed at a volume ratio of 50:10 as the organic phase, and elution is carried out at 45% organic phase / water. Chromatogram as shown Figure 12As shown. The peak shape of the analytical column is not as good as that of 50 / 5 methanol-acetonitrile. The separation of the three substances is good, but the peak shape is not good. The other substance did not reach baseline separation, so further optimization is needed. Moreover, the baseline is unstable, so the type of eluent should be changed. If it is necessary to enhance the elution ability of weakly polar compounds, a small amount of tetrahydrofuran can be added.

[0064] (7) Eluent: Methanol and THF are mixed at a volume ratio of 50:10 as the organic phase, and elution is carried out at 40% organic phase / water. Chromatogram as shown Figure 13 As shown, the column peak shape was good and the separation of the four substances was good, but the separation time was long. Therefore, the proportion of organic phase was increased to shorten the peak elution time.

[0065] (8) Eluent: Methanol and THF are mixed at a volume ratio of 50:10 as the organic phase, and elution is carried out at 45% organic phase / water. Chromatogram as shown Figure 14 As shown. The analytical column peak shape was better, and the proportion of organic solvent was reduced. Although the time cost was reduced, the separation degree of the four substances was not as good as that of the organic phase with 40% separation.

[0066] (9) Eluent: Methanol and THF are mixed at a volume ratio of 50:15 as the organic phase, and elution is carried out with 43% organic phase / water isocratic. Chromatogram as shown Figure 15 As shown, the column peak shape is good, but there is not much difference compared with the isocratic elution conditions of methanol / THF=50:10, 45% methanol / THF mixture / water. Therefore, the eluent conditions need to be further optimized.

[0067] (10) Eluent: Methanol and THF are mixed at a volume ratio of 50:15 as the organic phase, and elution is carried out at 45% organic phase / water. Chromatogram as shown Figure 16 As shown, the column peak shape is good, but there is not much difference compared with the isocratic elution conditions of methanol / THF=50:10, 45% methanol / THF mixture / water. Therefore, the eluent conditions need to be further optimized.

[0068] (11) Eluent: Methanol and THF are mixed at a volume ratio of 50:20 as the organic phase, and elution is carried out at 45% organic phase / water. Chromatogram as shown Figure 17 As shown, the column peak shape is good, and the four substances can achieve baseline separation. However, the separation effect is not as good as that of 50 / 10 methanol tetrahydrofuran.

[0069] (12) Eluent: Methanol and THF are mixed at a volume ratio of 50:30 as the organic phase, and elution is carried out at 40% organic phase / water. Chromatogram as shown Figure 18As shown, the column peak shape is good, and the four substances can achieve baseline separation, but the separation effect is not as good as that of 50 / 10 methanol tetrahydrofuran.

[0070] After optimization, the eluent conditions were fixed as follows: methanol / THF = 50:10 as the organic phase, and 45% organic phase / water isocratic elution. Under these conditions, the separation of the four substances was good, and the peak shape was good.

[0071] Screening Experiment 3: Flow Rate Optimization A higher flow rate can shorten the time and narrow the peak shape, but the separation is generally poor, so the flow rate is not optimized.

[0072] Screening Experiment 4: Optimization of Sample Concentration Samples with concentrations of 2 mg / mL, 2.5 mg / mL, 3 mg / mL, and 5 mg / mL were prepared and the experiment was conducted according to the chromatographic conditions of Experiment Example X. The results showed that when the sample concentration reached 5 mg / mL and the injection volume was 500 μL, baseline separation could not be achieved, and the separation was poor.

[0073] Preparation example: Megastigmatrienone was diluted with methanol to prepare a concentration of 3 mg / mL, with an injection volume of 0.8 mL. The chromatographic column was transferred from the analytical column to a similar XSelect HSS PFP 5μm OBD 10×250 mm col semi-preparative column with increased column length. The column temperature was 25℃; the detection wavelength was 308 nm; the flow rate was 0.8 mL / min; and the mobile phase was methanol / THF / H2O = 37.5:7.5:55 with isocratic elution. Results are as follows... Figure 19 As shown.

[0074] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for separating and preparing megastigmatrienone isomers, characterized in that, Includes the following steps: (1) Mix megastigmatrienone with methanol to obtain a mixture; (2) The mixture was separated by reversed-phase preparative chromatography to obtain the isomers of megastigmatrienone; The chromatographic conditions in the reversed-phase preparative chromatography method described in step (2) are as follows: the chromatographic column is a reversed-phase semi-preparative chromatographic column, the injection volume is 100-800 μL, the flow rate is 0.1-0.8 mL / min, the detection wavelength is 308 nm, and the eluent is methanol-THF-H2O = 37.5:7.5:55 isocratic elution.

2. The method according to claim 1, characterized in that, The concentration of the megastigmatrienone in the mixture in step (1) is 1-3 mg / mL.

3. The method according to claim 1, characterized in that, The reversed-phase semi-preparative chromatographic column mentioned in step (2) is an XSelect HSS PFP 5μm OBD 10×250 mm column with a column temperature of 25℃.

4. The method according to claim 1, characterized in that, When separating the mixture using reversed-phase preparative chromatography in step (2), the single injection volume of the sample is 1-2.5 mg.

5. The method according to claim 1, characterized in that, The isomers of stigmatatrienone obtained in step (2) include four types, with structural formulas as shown in A, B, C, and D: 。

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