Separation method of megastigmatrienone isomer

Abstract

The invention belongs to the technical field of isomer separation, and provides a separation method of megastigmatrienone isomers. The method comprises the following steps: mixing megastigmatrienone with methanol to obtain a mixed solution, and filtering the mixed solution by adopting an organic phase filter membrane; and then separating the filtered mixed solution by adopting a reversed-phase preparative chromatography to obtain the isomer of megastigmatrienone. According to the separation method, different isomers of megastigmatrienone can be obtained through efficient separation, and the separation method is environmentally friendly, easy to operate and easy to implement.

Description

Technical Field

[0001] The invention relates to the technical field of isomer separation, and in particular to a method for separating megastigmatrienone isomers. Background Art

[0002] Megastigmatrienoside is an important neutral aroma component in tobacco, which has the effect of improving tobacco aroma and masking off-flavors. Currently obtained megastigmatrienoside is usually a mixture of four isomers. The role of megastigmatrienosides with different structures in tobacco is not clear, and there is currently no separation method that can well separate these four isomers. In order to effectively separate these four isomers and further clarify the effects of megastigmatrienosides with different structures on tobacco aroma and quality, it is very necessary to develop a method for separating and preparing the four isomers by reverse phase preparative chromatography. Summary of the invention

[0003] In view of this, the present invention provides a method for separating isomers of megastigmatrienone to solve the problem that different isomers of megastigmatrienone are difficult to separate.

[0004] In order to achieve the above object, the present invention adopts the following technical solution:

[0005] The present invention provides a method for separating megastigmatrienone isomers, comprising the following steps:

[0006] 1) mixing megastigmatrienone with methanol to obtain a mixed solution, and then filtering the mixed solution through an organic phase filter membrane;

[0007] 2) using reverse phase preparative chromatography to separate the filtered mixed solution to obtain isomers of megastigmatrienone;

[0008] Wherein, the chromatographic conditions in the reverse phase preparative chromatography described in step 2) are: chromatographic column: EICOEE C18-PFP: 5 μm, 20 mm×150 mm, column temperature: 23° C., injection volume: 100 μL, flow rate: 10 mL / min, detection wavelength: 308 nm, mobile phase A: methanol, mobile phase B: water; elution procedure is isocratic elution or gradient elution;

[0009] When the elution program is isocratic elution, the isocratic elution program is: 50% A, 50% B, and the total elution time is 120 min;

[0010] When the elution program is gradient elution, the gradient elution program is: 0-40 min, 35-55% A, 65-45% B; 40-120 min, 55% A, 45% B.

[0011] Preferably, in step 1), the mass volume ratio of megastigmatrienone to methanol is 100 mg:900 μL; and the mixing time is 30 s.

[0012] Preferably, the pore size of the organic phase filter membrane in step 1) is 0.22 μm.

[0013] Preferably, the isomers of megastigmatrienone obtained in step 2) include 4 kinds, and the structural formulas of the 4 kinds of megastigmatrienone isomers are shown in Formula A, Formula B, Formula C and Formula D:

[0014]

[0015] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0016] The separation method of the present invention can realize the efficient separation of different isomers of megastigmatrienone in tobacco leaves, and the separation method is green, environmentally friendly, simple to operate, and easy to implement. The purity of the different isomers of megastigmatrienone separated is all above 95%, and they have different functional effects, and can contribute their respective roles in the process of cigarette flavoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0018] Figure 1 The separation analysis diagram of megastigmatrienone isomers in Example 1 (the abscissa is the method running time (min), and the ordinate is the absorbance (mAU));

[0019] Figure 2 The separation analysis and collection threshold diagram of megastigmatrienone isomers in Example 1 (the abscissa is the method running time (min), and the ordinate is the absorbance (mAU));

[0020] Figure 3 The separation analysis diagram of megastigmatrienone isomers in Example 2 (the abscissa is the method running time (min), and the ordinate is the absorbance (mAU));

[0021] Figure 4 The separation analysis diagram of megastigmatrienone isomers in Comparative Example 1 (the abscissa is the method running time (min), and the ordinate is the absorbance (mAU));

[0022] Figure 5The separation analysis diagram of megastigmatrienone isomers in Comparative Example 2 (the abscissa is the method running time (min), and the ordinate is the absorbance (mAU));

[0023] Figure 6 The separation analysis diagram of megastigmatrienone isomers in Comparative Example 3 (the abscissa is the method running time (min), and the ordinate is the absorbance (mAU));

[0024] Figure 7 The separation analysis diagram of megastigmatrienone isomers in Comparative Example 4 (the abscissa is the method running time (min), and the ordinate is the absorbance (mAU));

[0025] Figure 8 This is a separation analysis diagram of megastigmatrienone isomers in Comparative Example 5 (the abscissa is the method running time (min), and the ordinate is the absorbance (mAU));

[0026] Fig. 9 This is a separation analysis diagram of megastigmatrienone isomers in Comparative Example 6 (the abscissa is the method running time (min), and the ordinate is the absorbance (mAU)). DETAILED DESCRIPTION

[0027] The present invention provides a method for separating megastigmatrienone isomers, comprising the following steps:

[0028] 1) mixing megastigmatrienone with methanol to obtain a mixed solution, and then filtering the mixed solution through an organic phase filter membrane;

[0029] 2) using reverse phase preparative chromatography to separate the filtered mixed solution to obtain isomers of megastigmatrienone;

[0030] Wherein, the chromatographic conditions in the reverse phase preparative chromatography described in step 2) are: chromatographic column: EICOEE C18-PFP: 5 μm, 20 mm×150 mm, column temperature: 23° C., injection volume: 100 μL, flow rate: 10 mL / min, detection wavelength: 308 nm, mobile phase A: methanol, mobile phase B: water; elution procedure is isocratic elution or gradient elution;

[0031] When the elution program is isocratic elution, the isocratic elution program is: 50% A, 50% B, and the total elution time is 120 min;

[0032] When the elution program is gradient elution, the gradient elution program is: 0-40 min, 35-55% A, 65-45% B; 40-120 min, 55% A, 45% B.

[0033] In the present invention, the liquid chromatograph in the reverse phase preparative chromatography is preferably Waters Prep150; the mobile phase water is preferably provided by a Milli-Q ultrapure water machine.

[0034] In the present invention, the mass volume ratio of megastigmatrienone to methanol in step 1) is 100 mg:900 μL; and the mixing time is 30 s.

[0035] In the present invention, the pore size of the organic phase filter membrane in step 1) is 0.22 μm.

[0036] In the present invention, the isomers of megastigmatrienone obtained in step 2) include 4 kinds, and the structural formulas of the 4 kinds of megastigmatrienone isomers are shown in Formula A, Formula B, Formula C and Formula D:

[0037]

[0038] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0039] Example 1

[0040] 1) 100 mg of megastigmatrienone (purity of 4 isomers by peak area normalization method is 50%) was mixed with 900 μL of methanol (purity is 99.90%), vortexed for 30 seconds to obtain a mixed solution, and then the mixed solution was filtered through an organic phase filter membrane with a pore size of 0.22 μm;

[0041] 2) The filtered mixed liquid was separated by liquid chromatography (Waters Prep150), and the chromatographic conditions were set as follows: chromatographic column: EICOEE C18-PFP: 5 μm, 20 mm×150 mm, column temperature: 23°C, injection volume: 100 μL, flow rate: 10 mL / min, detection wavelength: 308 nm, mobile phase A: methanol, mobile phase B: water; the isocratic elution program was 50% A, 50% B, and the total elution time was 120 min. Four isomers of megastigmatrienone were obtained, among which the retention time of the first peak was 66.20 min, the retention time of the second peak was 73.24 min, the retention time of the third peak was 82.22 min, and the retention time of the fourth peak was 88.32 min.

[0042] The separation and analysis of megastigmatrienone isomers in Example 1 is shown in FIG. Figure 1 As shown, through analysis, it can be seen that the separation degree of the isomers of megastigmatrienone in Example 1 is large from the baseline, and the separation effect of each isomer is good. In addition, the separation analysis and collection threshold value diagram of the isomers of megastigmatrienone in this Example 1 are as shown in FIG. Figure 2As shown, it can be seen from the collection threshold that by setting an appropriate threshold (the threshold for the first and third peaks is set to 20 mAU, and the threshold for the second and fourth peaks is set to 60 mAU), four isomers of megastigmatrienone with relatively high purity can be separated.

[0043] The purity of the four megastigmatrienone isomers obtained in this example is all above 95%, and the recovery rate is about 45%.

[0044] Example 2

[0045] The difference between this embodiment and embodiment 1 is that the elution procedure is gradient elution, and the gradient elution procedure is: 0-40 min, 35-55% A, 65-45% B; 40-120 min, 55% A, 45% B.

[0046] The separation and analysis of megastigmatrienone isomers in Example 2 is shown in FIG. Figure 3 As shown, through analysis, it can be seen that in Example 2, the elution rate of the megastigmatrienone isomers is fast, the baseline separation is good, and the separation effect of each isomer is good.

[0047] Comparative Example 1

[0048] The difference between Comparative Example 1 and Example 1 is only that the ratio of the mobile phase is changed, and the isocratic elution program is 60% A and 40% B.

[0049] The separation and analysis of megastigmatrienone isomers in this comparative example is shown in FIG. Figure 4 As shown, through Figure 1 It can be seen from the comparison that the retention time is advanced, the separation from the baseline is poor, and the retention time separation between isomers is poor, which is not conducive to collection.

[0050] Comparative Example 2

[0051] The difference between Comparative Example 2 and Example 1 is only that the ratio of the mobile phase is changed, and the isocratic elution program is 40% A and 60% B.

[0052] The separation and analysis of megastigmatrienone isomers in this comparative example is shown in FIG. Figure 5 As shown, analysis of the chromatogram shows that no megastigmatrienone isomers were eluted within the programmed time.

[0053] Comparative Example 3

[0054] The difference between Comparative Example 3 and Example 2 is that the proportion of the mobile phase is changed, and the gradient elution program is: 0-40 min, 30-60% A, 70-40% B; 40-120 min, 60% A, 40% B.

[0055] The separation and analysis of megastigmatrienone isomers in this comparative example is shown in FIG. Figure 6As shown, through Figure 3 By comparison, it can be seen that the retention time of megastigmatrienone isomers is advanced, the separation from the baseline is poor, and the retention times of the isomers are too close, making collection difficult.

[0056] Comparative Example 4

[0057] The difference between this comparative example 4 and example 1 is that the mobile phase A is ethanol and the mobile phase B is water.

[0058] The separation and analysis of megastigmatrienone isomers in this comparative example is shown in FIG. Figure 7 As shown, by comparing with the separation analysis diagram of megastigmatrienoside isomers in Example 1, it can be seen that under isocratic elution conditions, different mobile phases have a great influence on the retention time and peak shape of the separation of megastigmatrienoside isomers. The four isomers in this comparative example were not successfully separated. When the mobile phase was methanol, the chromatographic peak separation effect of megastigmatrienoside was better.

[0059] Comparative Example 5

[0060] The difference between Comparative Example 5 and Comparative Example 4 is that the mobile phase ratio is changed, and the isocratic elution program is 40% A and 60% B.

[0061] The separation and analysis of megastigmatrienone isomers in Comparative Example 5 is shown in FIG. Figure 8 As shown, by comparing with the separation analysis diagram of megastigmatrienon isomers in Comparative Example 4, it can be seen that when isocratic elution occurs, the proportion of the organic phase decreases, which delays the retention time of the megastigmatrienon isomers and makes the separation between the isomers more thorough, but the four isomers are not successfully separated.

[0062] Comparative Example 6

[0063] The only difference between Comparative Example 6 and Example 2 is that the mobile phase A is ethanol.

[0064] The separation and analysis of megastigmatrienone isomers in Comparative Example 6 is shown in FIG. Fig. 9 As shown, by comparing with the separation analysis diagram of megastigmatrienoside isomers in Example 2, it can be seen that under the conditions of gradient elution, different mobile phases have a great influence on the retention time and peak shape of the separation of megastigmatrienoside isomers, and the chromatographic peak separation effect of megastigmatrienoside is better when the mobile phase is methanol.

[0065] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for separating megastigmatrienone isomers, characterized in that: The steps include: 1) mixing megastigmatrienone with methanol to obtain a mixed solution, and then filtering the mixed solution through an organic phase filter membrane; 2) using reverse phase preparative chromatography to separate the filtered mixed solution to obtain isomers of megastigmatrienone; Wherein, the chromatographic conditions in the reverse phase preparative chromatography described in step 2) are: chromatographic column: EICOEE C18-PFP: 5 μm, 20 mm×150 mm, column temperature: 23° C., injection volume: 100 μL, flow rate: 10 mL / min, detection wavelength: 308 nm, mobile phase A: methanol, mobile phase B: water; elution procedure is isocratic elution or gradient elution; When the elution program is isocratic elution, the isocratic elution program is: 50% A, 50% B, and the total elution time is 120 min; When the elution program is gradient elution, the gradient elution program is: 0-40 min, 35-55% A, 65-45% B; 40-120 min, 55% A, 45% B.

2. The method for separating megastigmatrienone isomers according to claim 1, characterized in that: The mass volume ratio of megastigmatrienone to methanol in step 1) is 100 mg:900 μL; The mixing time is 30s.

3. The method for separating megastigmatrienone isomers according to claim 2, characterized in that: The pore size of the organic phase filter membrane in step 1) is 0.22 μm.

4. The method for separating megastigmatrienone isomers according to any one of claims 1 to 3, characterized in that: The isomers of megastigmatrienone obtained in step 2) include 4 kinds, and the structural formulas of the 4 kinds of megastigmatrienone isomers are shown in Formula A, Formula B, Formula C and Formula D:

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