Method of isolating pyridine compound from tobacco ingredient-containing composition, refined tobacco ingredient-containing composition, and pyridine compound-containing composition

Abstract

To provide a method of easily and selectively isolating a pyridine compound form a tobacco ingredient-containing composition.SOLUTION: A method of isolating a pyridine compound from a tobacco ingredient-containing composition is provided, the method comprising a step of treating the tobacco ingredient-containing composition using a solid-phase phenylboronic acid.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for separating a pyridine compound from a tobacco component-containing composition, a purified tobacco component-containing composition, and a pyridine compound-containing composition. [Background technology]

[0002] Pyridine compounds are mainly produced in tobacco smoke during the biosynthesis and thermal decomposition of nicotine, and have a significant impact on the biological activity and aroma characteristics of tobacco smoke. For this reason, the pyridine compounds contained in tobacco smoke are analyzed, for example, by capturing tobacco components in tobacco smoke with a solvent and analyzing the resulting solution using multidimensional GC-MS or targeted analytical techniques (MS / MS or derivatization). However, because tobacco smoke contains more than 10,000 components, it is difficult to accurately detect trace amounts of pyridine compounds using these methods.

[0003] On the other hand, for example, Patent Document 1 and Non-Patent Documents 1 to 3 disclose the purification of non-glycated proteins and polyols using a phenylboronic acid solid phase (PBA-SPE). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2007-284425 A [Non-patent literature]

[0005] [Non-Patent Document 1] Keiji Gamoh et al., "Rapid and Selective Sample Preparation for the Chromatographic Determination of Brassinosteroids from Plant Material Using Solid-Phase Extraction Method," Analytical Sciences, volume 10, pages 913-917 (1994) [Non-Patent Document 2] Yoshiaki Fujii et al., “Simultaneous Determination of Aminoglycoside Residues in Livestock and Fishery Products by Phenylboronic Acid Solid-Phase Extraction and Liquid Chromatography-Tandem Mass Spectrometry”, Analytical Sciences, volume 35, Issue 9, pages 961-966 (2019) [Non-Patent Document 3] Jiaming Wang et al., “DETERMINATION OF GLYCIDOL IN E LIQUIDS AND EMISSIONS FROM E CIGARETTES”, 2020 Online CORESTA Congress, LABSTAT INTERNATIONAL INC. (https: / / www.coresta.org / sites / default / files / abstracts / 2020_ST05_Rodriguez-Lafuente.bak_.pdf) Summary of the Invention [Problem to be solved by the invention]

[0006] In order to accurately detect pyridine compounds in a tobacco component-containing composition, such as a solution containing tobacco components, it is desirable to selectively separate the pyridine compounds from the composition using a simple method.

[0007] The present invention aims to provide a method for easily and selectively separating pyridine compounds from a tobacco component-containing composition, a purified tobacco component-containing composition from which pyridine compounds have been selectively removed obtained by said method, and a pyridine compound-containing composition obtained by said method. [Means for solving the problem]

[0008] The present invention includes the following embodiments.

[0009] [1] A method for separating a pyridine compound from a tobacco component-containing composition, comprising a step of treating the tobacco component-containing composition with a solid phase of phenylboronic acid.

[0010] [2] The method according to [1], further comprising a step of washing the phenylboronic acid solid phase with a first washing agent prior to the step of treating the tobacco component-containing composition with the phenylboronic acid solid phase.

[0011] [3] The method according to [1] or [2], further comprising a step of washing the phenylboronic acid solid phase with a second washing agent after the step of treating the tobacco component-containing composition with the phenylboronic acid solid phase.

[0012] [4] The method according to [3], further comprising the step of treating the phenylboronic acid solid phase with an eluent after the step of washing the phenylboronic acid solid phase with the second detergent.

[0013] [5] The method according to any one of [1] to [4], wherein the tobacco component-containing composition contains a tobacco component and an aprotic solvent.

[0014] [6] The method according to any one of [2] to [4], wherein at least one of the first cleaning agent and the second cleaning agent is an aprotic solvent.

[0015] [7] The method according to [5] or [6], wherein the aprotic solvent does not contain a nitrogen atom.

[0016] [8] The method according to any one of [5] to [7], wherein the aprotic solvent is at least one selected from the group consisting of pentane, isopentane, hexane, cyclohexane, toluene, acetone, diethyl ether, methyl t-butyl ether, tetrahydrofuran, ethyl acetate, chloroform, dichloromethane, benzene, and xylene.

[0017] [9] The method according to [4], wherein the eluent is a protic solvent.

[0018]

[10] The method according to [9], wherein the protic solvent is at least one selected from the group consisting of methanol, ethanol, isopropanol, propanol, butanol, and isobutanol.

[0019]

[11] A purified tobacco component-containing composition obtained by treating the tobacco component-containing composition with the phenylboronic acid solid phase in the method according to any one of [1] to

[10] above, and selectively removing the pyridine compound contained in the tobacco component-containing composition.

[0020]

[12] A flavor inhaler comprising the refined tobacco component-containing composition described in

[11] .

[0021]

[13] A pyridine compound-containing composition obtained by treating the phenylboronic acid solid phase with the eluent in the method according to [4].

[0022]

[14] A flavoring agent comprising the pyridine compound-containing composition according to

[13] .

[0023]

[15] A method for analyzing pyridine compounds in a tobacco component-containing composition, comprising a step of treating the tobacco component-containing composition with a solid phase of phenylboronic acid. Effect of the Invention

[0024] According to the present invention, it is possible to provide a method for easily and selectively separating pyridine compounds from a tobacco component-containing composition, a purified tobacco component-containing composition from which pyridine compounds have been selectively removed obtained by the method, and a pyridine compound-containing composition obtained by the method. [Brief description of the drawings]

[0025] [Figure 1] 6 shows GC-MS spectra of the tobacco smoke extract, the MTBE washing solution, and the MeOH eluate in Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] [Method for separating pyridine compounds from tobacco component-containing compositions] The method for separating pyridine compounds from a tobacco component-containing composition according to this embodiment includes a step of treating the tobacco component-containing composition with a solid phase of phenylboronic acid (hereinafter also referred to as the "pyridine compound adsorption step").

[0027] The present inventors have found that when separating pyridine compounds from a tobacco component-containing composition, the tobacco component-containing composition is treated with a phenylboronic acid solid phase, whereby the pyridine compounds can be easily and selectively separated from the tobacco component-containing composition. The phenylboronic acid solid phase usually has a structure in which phenylboronic acid is bonded to a silanol group on the surface of a silica particle. When a tobacco component-containing composition is treated with a phenylboronic acid solid phase, an interaction accompanied by charge transfer occurs between the phenylboronic acid and the pyridine ring of the pyridine compound, so that the pyridine compound is adsorbed to the phenylboronic acid solid phase. This is presumed to allow the pyridine compound to be easily and selectively separated from the tobacco component-containing composition. In addition, as described below, after treating a tobacco component-containing composition with a phenylboronic acid solid phase, the phenylboronic acid solid phase is treated with an eluent, whereby the pyridine compound adsorbed to the phenylboronic acid solid phase can be easily released. Therefore, the pyridine compound in the tobacco component-containing composition can be accurately detected by analyzing the pyridine compound after release.

[0028] The method according to the present embodiment may include a step of washing the phenylboronic acid solid phase with a first washing agent (hereinafter also referred to as a "first washing step") before the pyridine compound adsorption step. The method according to the present embodiment may also include a step of washing the phenylboronic acid solid phase with a second washing agent (hereinafter also referred to as a "second washing step") after the pyridine compound adsorption step. The method according to the present embodiment may also include a step of treating the phenylboronic acid solid phase with an eluent (hereinafter also referred to as a "pyridine compound elution step") after the pyridine compound adsorption step or the second washing step. For example, the method according to the present embodiment may include a first washing step, a pyridine compound adsorption step, a second washing step, and a pyridine compound elution step in this order. Note that the method according to the present embodiment may include at least the pyridine compound adsorption step, and may further include one step of the first washing step, the second washing step, and the pyridine compound elution step in addition to the pyridine compound adsorption step, or may further include two or more steps.

[0029] (First cleaning step) In the method according to the present embodiment, it is preferable to carry out a step of washing the phenylboronic acid solid phase with a first washing agent prior to the pyridine compound adsorption step. By washing the phenylboronic acid solid phase with the first washing agent, unnecessary components such as impurities contained in the phenylboronic acid solid phase can be removed in advance.

[0030] The phenylboronic acid solid phase is not particularly limited and may be appropriately selected from known phenylboronic acid solid phases. For example, the phenylboronic acid solid phase may be used in a state where it is packed in a column, a syringe, or the like.

[0031] As the first cleaning agent, an aprotic solvent is preferable from the viewpoint of not hindering the adsorption of the pyridine compound to the phenylboronic acid solid phase in the pyridine compound adsorption step described later. Also, for the same reason, it is preferable that the aprotic solvent does not contain a nitrogen atom. Examples of such aprotic solvents include pentane, isopentane, hexane, cyclohexane, toluene, acetone, diethyl ether, methyl t-butyl ether (MTBE), tetrahydrofuran, ethyl acetate, chloroform, dichloromethane, benzene, xylene, etc. These may be used alone or in combination of two or more. Among these, the first cleaning agent is preferably methyl t-butyl ether or dichloromethane, and more preferably methyl t-butyl ether. In particular, it is preferable that the first cleaning agent is an aprotic solvent and does not contain a nitrogen atom. That is, it is preferable that the first cleaning agent does not contain any of water, alcohol, and nitrogen-containing solvent. Also, it is preferable that the first cleaning agent is the same as the solvent contained in the tobacco component-containing composition described later and the second cleaning agent.

[0032] The washing of the phenylboronic acid solid phase with the first detergent can be carried out by passing the first detergent through the phenylboronic acid solid phase. The amount of the first detergent passed through is not particularly limited, but for example, 200 to 1000% by volume of the first detergent can be passed through 100% by volume of the phenylboronic acid solid phase.

[0033] (Pyridine compound adsorption process) The method according to this embodiment includes a step of treating a tobacco component-containing composition with a phenylboronic acid solid phase. By treating the tobacco component-containing composition with the phenylboronic acid solid phase, pyridine compounds contained in the tobacco component-containing composition are selectively adsorbed to the phenylboronic acid solid phase. Therefore, the tobacco component-containing composition (refined tobacco component-containing composition) after treatment with the phenylboronic acid solid phase contains almost no pyridine compounds, and the pyridine compounds are selectively removed. The pyridine compounds adsorbed to the phenylboronic acid solid phase can be eluted by a pyridine compound elution step described below.

[0034] The tobacco component-containing composition is not particularly limited as long as it is a composition containing tobacco components at least including a pyridine compound, but may be, for example, a solution containing a tobacco component and a solvent. In this case, the treatment of the tobacco component-containing composition with a phenylboronic acid solid phase can be carried out by passing the tobacco component-containing composition through the phenylboronic acid solid phase. The tobacco component-containing composition preferably contains a tobacco component and an aprotic solvent. The aprotic solvent is used so as not to hinder the adsorption of the pyridine compound to the phenylboronic acid solid phase in the pyridine compound adsorption step. For the same reason, it is preferable that the aprotic solvent does not contain a nitrogen atom. Examples of such aprotic solvents include pentane, isopentane, hexane, cyclohexane, toluene, acetone, diethyl ether, methyl t-butyl ether, tetrahydrofuran, ethyl acetate, chloroform, dichloromethane, benzene, xylene, etc. These may be used alone or in combination of two or more. Among these, the solvent contained in the tobacco component-containing composition is preferably methyl t-butyl ether or dichloromethane, and more preferably methyl t-butyl ether. In particular, the solvent contained in the tobacco component-containing composition is preferably an aprotic solvent that does not contain a nitrogen atom. That is, the solvent preferably does not contain water, alcohol, or a nitrogen-containing solvent. In addition, the solvent is preferably the same as the first detergent and the second detergent described below.

[0035] Specifically, the tobacco component-containing composition can be a solution obtained by passing tobacco smoke through a solvent. In this case, the solvent through which the tobacco smoke passes corresponds to the solvent contained in the tobacco component-containing composition. The content of the pyridine compound contained in the tobacco component-containing composition is preferably 0.00000000001 to 10% by mass, more preferably 0.0000000001 to 1% by mass.

[0036] When the tobacco component-containing composition is a solution containing a tobacco component and a solvent, the preferred range of the amount of the tobacco component-containing composition passing through the phenylboronic acid solid phase varies depending on the content of the pyridine compound contained in the tobacco component-containing composition. However, for example, it is preferable to pass 0.0000000001 to 1 volume % of the tobacco component-containing composition through 100 volume % of the phenylboronic acid solid phase. The passing speed is not particularly limited, but can be, for example, 0.1 mL / min to 1 mL / min. In addition, in order to increase the passing speed, when passing the tobacco component-containing composition through the phenylboronic acid solid phase, the tobacco component-containing composition may be pressurized and forced through from the introduction side.

[0037] In this embodiment, the term "pyridine compound" refers to a compound having a pyridine ring. The pyridine compound is not particularly limited, but from the viewpoint of the adsorptivity to the phenylboronic acid solid phase, it is preferable that the pyridine compound does not have an electron-withdrawing group such as an acetyl group. In addition, from the viewpoint of the desorption from the phenylboronic acid solid phase in the pyridine compound elution step described later, it is preferable that the pyridine compound does not have an amino group.

[0038] (Second washing step) In the method according to the present embodiment, it is preferable to carry out a step of washing the phenylboronic acid solid phase with a second detergent after the pyridine compound adsorption step. By washing the phenylboronic acid solid phase with a second detergent after the pyridine compound adsorption step, unnecessary components other than the pyridine compound contained in the tobacco component-containing composition can be removed from the phenylboronic acid solid phase. Note that the pyridine compound is not released from the phenylboronic acid solid phase by washing with the second detergent.

[0039] As the second cleaning agent, an aprotic solvent is preferable from the viewpoint of preventing the detachment of the pyridine compound from the phenylboronic acid solid phase. Also, for the same reason, it is preferable that the aprotic solvent does not contain a nitrogen atom. Examples of such aprotic solvents include pentane, isopentane, hexane, cyclohexane, toluene, acetone, diethyl ether, methyl t-butyl ether, tetrahydrofuran, ethyl acetate, chloroform, dichloromethane, benzene, xylene, etc. These may be used alone or in combination of two or more. Among these, the second cleaning agent is preferably methyl t-butyl ether or dichloromethane, and more preferably methyl t-butyl ether. In particular, it is preferable that the second cleaning agent is an aprotic solvent and does not contain a nitrogen atom. That is, it is preferable that the second cleaning agent does not contain any of water, alcohol, and nitrogen-containing solvents. Also, it is preferable that the second cleaning agent is the same as the first cleaning agent and the solvent contained in the tobacco component-containing composition.

[0040] The washing of the phenylboronic acid solid phase with the second detergent can be carried out by passing the second detergent through the phenylboronic acid solid phase. The amount of the second detergent passed through is not particularly limited, but for example, 300 to 1000% by volume of the second detergent can be passed through 100% by volume of the phenylboronic acid solid phase.

[0041] (Pyridine compound elution step) In the method according to the present embodiment, it is preferable to carry out a step of treating the phenylboronic acid solid phase with an eluent after the pyridine compound adsorption step or the second washing step. By treating the phenylboronic acid solid phase with an eluent, the pyridine compounds adsorbed on the phenylboronic acid solid phase can be eluted. Therefore, by analyzing the pyridine compounds after elution, the pyridine compounds in the tobacco component-containing composition can be detected with high accuracy.

[0042] As the eluent, a protic solvent is preferred from the viewpoint of easily removing the pyridine compound from the phenylboronic acid solid phase. Examples of the protic solvent include methanol, ethanol, isopropanol, propanol, butanol, isobutanol, etc. These may be used alone or in combination of two or more. Among these, the eluent is preferably methanol or isopropanol, and more preferably methanol.

[0043] The treatment of the phenylboronic acid solid phase with an eluent can be carried out by passing the eluent through the phenylboronic acid solid phase. The amount of the eluent passed through is not particularly limited, but for example, 300 to 1000% by volume of the eluent can be passed through 100% by volume of the phenylboronic acid solid phase.

[0044] The quality and quantity of the eluted pyridine compound can be determined by analysis using GC-MS or LC-MS with the use of an internal standard substance.

[0045] [Composition containing refined tobacco components, flavor inhaler] The purified tobacco component-containing composition according to the present embodiment is obtained by treating the tobacco component-containing composition with a solid phase of phenylboronic acid in the above-described method for separating pyridine compounds from a tobacco component-containing composition according to the present embodiment, and selectively removing the pyridine compounds contained in the tobacco component-containing composition. Since pyridine compounds are selectively removed from the purified tobacco component-containing composition, components other than pyridine compounds can be analyzed with high accuracy. In addition, since pyridine compounds having a unique aroma are removed, a flavor inhaler having a desired flavor can be provided by using the purified tobacco component-containing composition as a tobacco raw material. The content of pyridine compounds contained in the purified tobacco component-containing composition according to the present embodiment is preferably 10% by mass or less, more preferably 0.0000000001 to 1% by mass.

[0046] In liquid-type electronic cigarettes, the refined tobacco component-containing composition according to the present embodiment can be used as is, or after removing the solvent and adding an aerosol source. When used in solid flavor sources such as tobacco sticks, the refined tobacco component-containing composition according to the present embodiment can be used by adding it by spraying, kneading it when preparing a tobacco sheet, or the like.

[0047] The flavor inhaler according to the present embodiment includes the refined tobacco component-containing composition according to the present embodiment. The flavor inhaler is not particularly limited, and can be, for example, a non-combustion heating type flavor inhaler, a combustion type flavor inhaler (cigarette), a non-heating type flavor inhaler (oral tobacco, etc.), etc. Since many pyridine compounds generate flavor when exposed to relatively high temperatures, the flavor inhaler according to the present embodiment is preferably a high-temperature heating type flavor inhaler or cigarette.

[0048] [Pyridine compound-containing composition, flavor imparting agent] The pyridine compound-containing composition according to the present embodiment is obtained by treating the phenylboronic acid solid phase with an eluent in the method for separating a pyridine compound from a tobacco component-containing composition according to the present embodiment described above. Since the pyridine compound-containing composition is concentrated with pyridine compounds, the pyridine compounds contained in the tobacco component-containing composition can be analyzed with high accuracy. In addition, by adding the pyridine compound-containing composition as a flavoring agent, a flavor inhaler having a desired flavor can be provided. The flavor inhaler is not particularly limited, and can be, for example, a non-combustion heating type flavor inhaler, a combustion type flavor inhaler (cigarette), a non-heating type flavor inhaler (oral tobacco, etc.), etc. The content of the pyridine compound contained in the pyridine compound-containing composition according to the present embodiment is preferably 0.00000000001% by mass or more, more preferably 0.0000000001 to 1% by mass.

[0049] [Method for analyzing pyridine compounds in tobacco component-containing compositions] The method for analyzing pyridine compounds in a tobacco component-containing composition according to this embodiment includes a step of treating the tobacco component-containing composition with a phenylboronic acid solid phase. The method for analyzing pyridine compounds in a tobacco component-containing composition according to this embodiment can be carried out in the same manner as the method for separating pyridine compounds from a tobacco component-containing composition according to this embodiment described above. According to the method for analyzing pyridine compounds in a tobacco component-containing composition according to this embodiment, the pyridine compounds can be easily and selectively separated from the tobacco component-containing composition by treatment with a phenylboronic acid solid phase, so that the pyridine compounds in the tobacco component-containing composition can be analyzed with high accuracy. The analysis (qualitative and quantitative) of pyridine compounds can be carried out, for example, by GC-MS analysis using an internal standard substance. EXAMPLES

[0050] The present embodiment will be described in detail below with reference to examples, but the present embodiment is not limited to these examples. The GC-MS analysis conditions in the examples are as follows.

[0051] [GC-MS analysis conditions] Qualitative and quantitative analysis was performed using an Agilent GC-MS (GC: Agilent 7890A, MS: 5975C inertXL, column: DB-heavyWAX (60 m × 0.25 mm id, 0.25 μm film thickness), injection volume: 1 μL, injection port conditions: 250 °C splitless mode, oven conditions: 40 °C (3 min. hold) to 280 °C at the rate of 5 °C / min., carrier gas: helium (flow rate 1 mL / min.), MS conditions: ionization energy 70 eV (EI), ion source temperature 30 °C, quadrupole temperature 150 °C, transfer line 280 °C).

[0052] [Example 1] (Preparation of model mixed solution) The following pyridine compounds and their analogues 1 to 20 (hereinafter also referred to as "model compounds") were prepared.

[0053] [ka]

[0054] The pyridine compound and its analogs 1 to 20 were mixed in each solvent (methyl t-butyl ether (MTBE), dichloromethane, acetone, isopropanol, methanol (MeOH), water (only compounds 1 and 14 were dissolved in water)) to a concentration of 50 mg / L to prepare a model mixed solution for each solvent. 2 mL of the model mixed solution was transferred to a 10 mL measuring flask, and 20 μL of 5 mg / mL pyridine-d5 MTBE solution was added as an internal standard substance, and then the solution was made up to the appropriate volume with each solvent (water was made up to the appropriate volume with MeOH). This was mixed well to prepare a standard solution for each solvent. The standard solution for each solvent was subjected to GC-MS analysis.

[0055] (Treatment of model mixed solution by PBA-SPE) PBA-SPE (Agilent, product name: Bond Elut PBA, syringe capacity 6 mL, resin capacity 500 mg, particle size 40 μm) was washed with 5 mL of each solvent used in the preparation of the model mixed solution. Then, 2 mL of the model mixed solution was passed through the PBA-SPE, and pressure was applied to a speed of about 2 drops / second. At that time, the solution after passing was collected in a 10 mL volumetric flask. Next, 5 mL of each solvent used in the preparation of the model mixed solution was passed through the PBA-SPE, and the solution after passing was collected in the 10 mL volumetric flask (a total of about 7 mL was collected in the volumetric flask). 20 μL of 5 mg / mL pyridine-d5 MTBE solution was added to the 10 mL volumetric flask as an internal standard substance, and then the volume was increased with each solvent. This was mixed well to prepare an eluate of each solvent. The eluate of each solvent was subjected to GC-MS analysis.

[0056] (Calculation of dissolution rate) The elution rate of each model compound in each solvent was calculated according to the following formula.

[0057]

number

[0058] In the above formula, A t indicates the area value of the characteristic ion of each model compound. IS indicates the area value of the characteristic ion (m / z = 84) of pyridine-d5 (ISTD). Table 1 shows the elution ratio of model compounds with each solvent.

[0059] [Table 1]

[0060] As shown in Table 1, when MTBE and dichloromethane were used as the solvent, almost all of the compounds except for compounds 8 and 10 to 13 were adsorbed to PBA-SPE and were not eluted. This shows that MTBE and dichloromethane (particularly MTBE) are useful as solvents contained in cleaning agents and tobacco component-containing compositions. On the other hand, when isopropanol and MeOH were used as the solvent, almost all of the compounds were not adsorbed to PBA-SPE, and most of them were eluted. This shows that isopropanol and MeOH (particularly MeOH) are useful as eluents. It is presumed that compound 8, which has an acetyl group that is an electron-withdrawing group, was difficult to adsorb to PBA-SPE even when MTBE was used. In addition, compounds 10 to 13 are non-pyridine compounds, so it is presumed that they were hardly adsorbed to PBA-SPE. Since compounds with pyridine-related structures such as these were not retained, high retention selectivity for pyridine-containing compounds was observed. In addition, since compounds 19 and 20 have free amino groups, they are presumably strongly adsorbed to PBA-SPE and are difficult to desorb even when MeOH is used.

[0061] [Example 2] (Treatment of model mixed solution by PBA-SPE) The same PBA-SPE as in Example 1 was washed with 5 mL of MTBE. Then, 2 mL of the model mixed solution prepared with MTBE was passed through the PBA-SPE, and pressure was applied to the PBA-SPE at a speed of about 2 drops / second. At that time, the solution after passing was collected in a 10 mL volumetric flask. Next, 5 mL of MTBE was passed through the PBA-SPE, and the solution after passing was collected in the 10 mL volumetric flask (a total of about 7 mL was collected in the volumetric flask). 20 μL of 5 mg / mL pyridine-d5 MTBE solution was added to the 10 mL volumetric flask as an internal standard substance, and then the volume was increased with MTBE. This was mixed well to prepare an MTBE washing solution. The MTBE washing solution was subjected to GC-MS analysis.

[0062] 5mL of MeOH was passed through the PBA-SPE after the MTBE washing, and the model compound held therein was eluted by applying pressure at a rate of about 2 drops / second. The solution after passing was then collected in a 10mL measuring flask. 20μL of 5mg / mL pyridine-d5 MTBE solution was added to the 10mL measuring flask as an internal standard, and then the flask was filled up with MTBE. This was mixed well to prepare a MeOH eluate. The MeOH eluate was subjected to GC-MS analysis.

[0063] (Calculation of removal rate by MTBE washing and recovery rate by MeOH elution) The removal rate of each model compound by MTBE washing and the recovery rate by MeOH elution were calculated using the following formula.

[0064]

number

[0065] In the above formula, A t indicates the area value of the characteristic ion of each model compound. ISindicates the area value of the characteristic ion (m / z = 84) of pyridine-d5 (ISTD). The removal rate by MTBE washing and the recovery rate by MeOH elution for each model compound are shown in Table 2. The removal rate by MTBE washing and the recovery rate by MeOH elution in Table 2 are the average values ​​when the same test was performed three times.

[0066] [Table 2]

[0067] As shown in Table 2, by using MTBE as a solvent for dissolving the model compounds and using MTBE as the first and second cleaning agents, it was found that the model compounds (except for compounds 8, 10 to 13) were hardly contained in the MTBE cleaning solution, and the model compounds were retained adsorbed on the PBA-SPE even after cleaning. On the other hand, by using MeOH as an eluent, it was found that most of the model compounds (except for compounds 19 and 20) were eluted in the MeOH eluent and could be recovered. From this, it was found that the method according to this embodiment can selectively separate pyridine compounds, which are representative of model compounds, in a simple manner.

[0068] [Example 3] (Preparation of model tobacco smoke extract) 1R6F, a standard cigarette for testing, was conditioned (temperature and humidity controlled) for more than 48 hours at a temperature of 22°C ± 1°C and a humidity of 60% ± 3% according to ISO3402. A 44mm Cambridge filter (hereinafter also referred to as "CF", manufactured by Borgwalt) and an impinger containing 20mL of MTBE cooled to -78°C were connected in series, and three 1R6F cigarettes were smoked using a smoking machine (manufactured by Cerulean, product name: Linear Smoking Machine SM450) with a specified inhalation method (inhalation volume: 55mL / inhalation, inhalation time: 2 seconds / inhalation, inhalation interval: 30 seconds). The smoking conditions were in accordance with ISO4387 and ISO20778. After smoking, the Cambridge filter and the MTBE in the impinger were added to a 50mL vial, and the mixture was shaken and extracted for 30 minutes using a shaking extractor. Then, insoluble matter was removed using a membrane filter (manufactured by Merck) to obtain a tobacco smoke extract.

[0069] Model compounds 1 to 20 were added to the tobacco smoke extract at 50 mg / L each to prepare a model tobacco smoke extract. 2 mL of the model tobacco smoke extract was transferred to a 10 mL measuring flask, and 20 μL of 5 mg / mL pyridine-d5 MTBE solution was added as an internal standard substance, and then the flask was filled up with MTBE. This was mixed well to prepare a model tobacco smoke standard solution. The model tobacco smoke standard solution was subjected to GC-MS analysis.

[0070] (Treatment of model cigarette smoke extracts using PBA-SPE) The same PBA-SPE as in Example 1 was washed with 5 mL of MTBE. Then, 2 mL of the model tobacco smoke extract was passed through the PBA-SPE, and pressure was applied to the PBA-SPE at a rate of about 2 drops / second. At that time, the solution after passing was collected in a 10 mL volumetric flask. Next, 5 mL of MTBE was passed through the PBA-SPE, and the solution after passing was collected in the 10 mL volumetric flask (a total of about 7 mL was collected in the volumetric flask). 20 μL of 5 mg / mL pyridine-d5 MTBE solution was added to the 10 mL volumetric flask as an internal standard substance, and then the volume was increased with MTBE. This was mixed well to prepare an MTBE washing solution of the model tobacco smoke extract. The MTBE washing solution of the model tobacco smoke extract was subjected to GC-MS analysis.

[0071] 5mL of MeOH was passed through the PBA-SPE after the MTBE washing, and the model compound held therein was eluted by applying pressure to a rate of about 2 drops / second. The solution after passing was then collected in a 10mL measuring flask. 20μL of 5mg / mL pyridine-d5 MTBE solution was added to the 10mL measuring flask as an internal standard, and then the flask was filled up with MTBE. This was mixed well to prepare a MeOH eluate of the model tobacco smoke extract. The MeOH eluate of the model tobacco smoke extract was subjected to GC-MS analysis.

[0072] (Calculation of removal rate by MTBE washing and recovery rate by MeOH elution) The removal rate of each model compound in the model cigarette smoke extract by MTBE washing and the recovery rate by MeOH elution were calculated using the following formula.

[0073]

number

[0074] In the above formula, A t indicates the area value of the characteristic ion of each model compound. ISindicates the area value of the characteristic ion (m / z = 84) of pyridine-d5 (ISTD). The removal rate by MTBE washing and the recovery rate by MeOH elution for each model compound are shown in Table 3. The removal rate by MTBE washing and the recovery rate by MeOH elution in Table 3 are the average values ​​when the same test was performed three times.

[0075] [Table 3]

[0076] As shown in Table 3, it was confirmed that the method according to this embodiment can similarly perform adsorption and desorption of pyridine compounds, which are representative of model compounds, even in a tobacco matrix.

[0077] [Example 4] (Preparation of tobacco smoke condensate) 1R6F, a standard cigarette for testing, was conditioned (temperature and humidity controlled) for more than 48 hours at a temperature of 22°C ± 1°C and a humidity of 60% ± 3% according to ISO3402. A 44mm Cambridge filter (hereinafter also referred to as "CF", manufactured by Borgwalt) and an impinger containing 20mL of MTBE cooled to -78°C were connected in series, and 15 cigarettes of the 1R6F were smoked using a smoking machine (manufactured by Cerulean, product name: Linear Smoking Machine SM450) with a specified inhalation method (inhalation volume: 55mL / inhalation, inhalation time: 2 seconds / inhalation, inhalation interval: 30 seconds). The smoking conditions were in accordance with ISO4387 and ISO20778. After smoking, the Cambridge filter and the MTBE in the impinger were added to a 50mL vial, and the mixture was shaken and extracted for 30 minutes using a shaking extractor. Then, insoluble matter was removed using a membrane filter (manufactured by Merck) to obtain a tobacco smoke condensate.

[0078] (Treatment of tobacco smoke condensate by PBA-SPE) The same PBA-SPE as in Example 1 was washed with 5 mL of MTBE. Then, 2 mL of the tobacco smoke condensate was passed through the PBA-SPE, and pressure was applied to the PBA-SPE at a speed of about 2 drops / second. At this time, the solution after passing was collected in a 10 mL volumetric flask. Next, 5 mL of MTBE was passed through the PBA-SPE, and the solution after passing was collected in the 10 mL volumetric flask (a total of about 7 mL was collected in the volumetric flask). The obtained MTBE washing solution was subjected to GC-MS analysis. Then, 5 mL of MeOH was passed through the PBA-SPE after the MTBE washing, and pressure was applied to the PBA-SPE at a speed of about 2 drops / second, thereby eluting the retained components. At this time, the solution after passing was collected in a 10 mL volumetric flask. The obtained MeOH eluate was subjected to GC-MS analysis.

[0079] (Qualitative characterization of each component) The qualitative analysis of each component was performed by deconvolution (deconvolution parameters: Absolute area filter>100000, RT window size 100, Extraction window left m / z delta 0.3, Extraction window right m / z delta 0.7, m / z delta units AMU) using Agilent software (unknown analysis), and comparing the detection time and mass spectrum of each component with the corresponding standard. The results are shown in Table 4 and Figure 1. Note that in order to make the results easier to see, Figure 1 shows an excerpt of the range of RT0 to 32.5 (the range in which compounds No. 1 to 14 in Table 4 can be detected), rather than the entire range analyzed.

[0080] [Table 4]

[0081] As shown in Table 4 and FIG. 1, by treating tobacco smoke condensate with PBA-SPE based on the method of this embodiment, it is possible to selectively recover pyridine compounds while removing many of the components contained in the tobacco smoke condensate, and these can be qualitatively and quantitatively analyzed with high accuracy.

[0082] The present embodiment includes the following aspects.

[0083] [1] A method for separating a pyridine compound from a tobacco component-containing composition, comprising a step of treating the tobacco component-containing composition with a solid phase of phenylboronic acid.

[0084] [2] The method according to [1], further comprising a step of washing the phenylboronic acid solid phase with a first washing agent prior to the step of treating the tobacco component-containing composition with the phenylboronic acid solid phase.

[0085] [3] The method according to [1] or [2], further comprising a step of washing the phenylboronic acid solid phase with a second washing agent after the step of treating the tobacco component-containing composition with the phenylboronic acid solid phase.

[0086] [4] The method according to [3], further comprising the step of treating the phenylboronic acid solid phase with an eluent after the step of washing the phenylboronic acid solid phase with the second detergent.

[0087] [5] The method according to any one of [1] to [4], wherein the tobacco component-containing composition contains a tobacco component and an aprotic solvent.

[0088] [6] The method according to any one of [2] to [4], wherein at least one of the first cleaning agent and the second cleaning agent is an aprotic solvent.

[0089] [7] The method according to [5] or [6], wherein the aprotic solvent does not contain a nitrogen atom.

[0090] [8] The method according to any one of [5] to [7], wherein the aprotic solvent is at least one selected from the group consisting of pentane, isopentane, hexane, cyclohexane, toluene, acetone, diethyl ether, methyl t-butyl ether, tetrahydrofuran, ethyl acetate, chloroform, dichloromethane, benzene, and xylene.

[0091] [9] The method according to [4], wherein the eluent is a protic solvent.

[0092]

[10] The method according to [9], wherein the protic solvent is at least one selected from the group consisting of methanol, ethanol, isopropanol, propanol, butanol, and isobutanol.

[0093]

[11] A purified tobacco component-containing composition obtained by treating the tobacco component-containing composition with the phenylboronic acid solid phase in the method according to any one of [1] to

[10] above, and selectively removing the pyridine compound contained in the tobacco component-containing composition.

[0094]

[12] A flavor inhaler comprising the refined tobacco component-containing composition described in

[11] .

[0095]

[13] A pyridine compound-containing composition obtained by treating the phenylboronic acid solid phase with the eluent in the method according to [4].

[0096]

[14] A flavoring agent comprising the pyridine compound-containing composition according to

[13] .

[0097]

[15] A method for analyzing pyridine compounds in a tobacco component-containing composition, comprising a step of treating the tobacco component-containing composition with a solid phase of phenylboronic acid.

Claims

1. A method for separating pyridine compounds from a tobacco component-containing composition, comprising the step of treating the tobacco component-containing composition with a phenylboronic acid solid phase.

2. The method according to claim 1, further comprising the step of washing the phenylboronic acid solid phase with a first cleaning agent before the step of treating the tobacco component-containing composition with the phenylboronic acid solid phase.

3. The method according to claim 1, further comprising the step of treating the tobacco component-containing composition with the phenylboronic acid solid phase, followed by the step of washing the phenylboronic acid solid phase with a second cleaning agent.

4. The method according to claim 3, further comprising the step of washing the phenylboronic acid solid phase with the second cleaning agent, followed by the step of treating the phenylboronic acid solid phase with an eluent.

5. The method according to claim 1, wherein the tobacco component-containing composition comprises a tobacco component and an aprotic solvent.

6. The method according to claim 2, wherein at least one of the first cleaning agent and the second cleaning agent is an aprotic solvent.

7. The method according to claim 5, wherein the aprotic solvent does not contain nitrogen atoms.

8. The method according to claim 5, wherein the aprotic solvent is at least one selected from the group consisting of pentane, isopentane, hexane, cyclohexane, toluene, acetone, diethyl ether, methyl t-butyl ether, tetrahydrofuran, ethyl acetate, chloroform, dichloromethane, benzene, and xylene.

9. The method according to claim 4, wherein the eluent is a protic solvent.

10. The method according to claim 9, wherein the protic solvent is at least one selected from the group consisting of methanol, ethanol, isopropanol, propanol, butanol, and isobutanol.

11. A purified tobacco component-containing composition obtained by treating the tobacco component-containing composition with the phenylboronic acid solid phase, thereby selectively removing the pyridine compound contained in the tobacco component-containing composition, according to the method of any one of claims 1 to 10.

12. A flavor inhaler comprising the refined tobacco component-containing composition described in claim 11.

13. A pyridine compound-containing composition obtained by treating the phenylboronic acid solid phase with the eluent, according to the method of claim 4.

14. A flavoring agent comprising the pyridine compound-containing composition described in claim 13.

15. A method for analyzing pyridine compounds in a tobacco component-containing composition, comprising the step of treating the tobacco component-containing composition with a phenylboronic acid solid phase.