JP7263497B2 - Method for separating and analyzing positional isomers and/or enantiomers of triacylglycerols - Google Patents

Description

The present invention relates to a method for producing positional isomers and/or enantiomers of triacylglycerol, including a step of separating positional isomers and/or enantiomers of triacylglycerols by high performance liquid chromatography. More specifically, one or more positional isomers of triacylglycerols selected from the group consisting of AAB-type, ABA-type and BAA-type triacylglycerols consisting of two fatty acids A and B and glycerol, and / Or to a method for producing the positional isomer and / or the enantiomer, comprising the step of fractionating a raw material mixture of the enantiomer by high performance liquid chromatography using a specific column.

Among lipids, one of the three major nutrients, triacylglycerol (TAG) is the most abundant form of lipid in nature. TAG has a structure in which three fatty acids are ester-bonded at the sn-1 position (α position), sn-2 position (β position) and sn-3 position (α position) of glycerol, and is the main component of edible fats and oils. be. The physical properties and nutritional properties of edible fats and oils are affected by the types and bonding positions of fatty acids ester-bonded to glycerol.

When the carbon at the sn-2 position of a TAG composed of two fatty acids and glycerol is an asymmetric carbon, the TAG has enantiomers. For example, in a TAG having one eicosapentaenoic acid (E) and two caprylic acids (C) ester-bonded to glycerol, the eicosapentaenoic acid (E) is ester-bonded to the sn-3 position of glycerol and two TAG in which caprylic acid (C) is ester-bonded to the sn-1 and sn-2 positions of glycerol (hereinafter referred to as "sn-CCE"), and eicosapentaenoic acid (E) is glycerol's sn- There is a TAG having an ester bond at the 1-position and two caprylic acids (C) ester-bonded at the sn-2 and sn-3 positions of glycerol (hereinafter referred to as "sn-ECC"). In addition, in a TAG in which one docosahexaenoic acid (D) and two caprylic acids (C) are ester-bonded to glycerol, docosahexaenoic acid (D) is ester-bonded to the sn-3 position of glycerol and two caprylic acids (C) is an ester-bonded TAG at the sn-1 and sn-2 positions of glycerol (hereinafter referred to as "sn-CCD"), and docosahexaenoic acid (D) is at the sn-1 position of glycerol There is a TAG (hereinafter referred to as “sn-DCC”) in which two caprylic acids (C) are ester-bonded to the sn-2 and sn-3 positions of glycerol. sn-CCE and sn-ECC, sn-CCD and sn-DCC are each distinguished as enantiomers. Separation of sn-CCE and sn-ECC, and separation of sn-CCD and sn-DCC were each investigated by high performance liquid chromatography (HPLC) using a column filled with a separating agent in which a cellulose derivative was supported on silica gel. (See, for example, Non-Patent Document 1).

The present inventors have found that sn-PPO (palmitic acid (P), which is an enantiomer contained in edible fats and oils, is ester-bonded to the sn-1 and sn-2 positions of glycerol, and oleic acid (O) is glycerol. TAG attached to the sn-3 position) and sn-OPP (palmitic acid (P) is ester-bonded to the sn-2 and sn-3 positions of glycerol, and oleic acid (O) is attached to the sn-1 position of glycerol. TAG bound to) was examined by the above separation method, but since sn-PPO and sn-OPP could not be separated, a method for separating sn-PPO and sn-OPP by recycling HPLC was examined (for example, See Non-Patent Document 2). Recycle HPLC is HPLC that has the function of returning the UV detector outlet channel to the pump inlet to circulate the mobile phase.

In edible oils and fats, sn-POP (palmitic acid (P) is ester-bonded to the sn-1 and sn-3 positions of glycerol and oleic acid (O) is present as positional isomers of sn-PPO and sn-OPP) TAG attached to the sn-2 position of glycerol) is also included. Since it was impossible to separate these regio-enantiomers simultaneously using conventional chiral columns and recycling HPLC alone, amylose tris(3-chlorophenyl A column packed with a separating agent with a particle size of 3 μm in which amylose tris(3-chloro-4-methylphenylcarbamate) is supported on silica gel and a separating agent with a particle size of 5 μm in which amylose tris(3-chloro-4-methylphenylcarbamate) is supported on silica gel. A separation method by HPLC using each of these columns was investigated (see, for example, Non-Patent Document 3).

On the other hand, a sample containing TAG was injected into the flow channel to which the mobile phase containing the supercritical fluid was sent, and a separating agent having a particle size of 3 μm, in which amylose tris(3-chlorophenylcarbamate) was supported on silica gel, was filled. A method of separating the TAG enantiomers by passing the solution through a separate column was investigated (see, for example, Patent Document 1). However, simultaneous separation of TAG positional isomers and TAG enantiomers by the method for separating TAG enantiomers has not been investigated. Furthermore, chromatographic systems with supercritical fluids as mobile phases are significantly more expensive than conventional HPLC systems.

JP 2015-175747 A

Y. Iwasaki, M. Yasui, T. Ishikawa, R. Irimescu, K. Hata, T. Yamane. J. Chromatogr. A 905, 111-118 (2001) T. Nagai, H. Mizobe, I. Otake, K. Ichioka, K. Kojima, Y. Matsumoto, N. Gotoh, I. Kuroda, S. Wada. Enantiomeric separation of asymmetric triacylglycerol by recycle high-performance liquid chromatography with chiral column. J. Chromatogr. A 1218, 2880-2886(2011). Toshiharu Nagai and 7 others, Abstracts of the 54th Annual Meeting of the Japan Oil Chemists' Society, p.211, 2015

Enhanced separation of TAG regioisomers and enantiomers composed of two fatty acids and glycerol using conventional HPLC systems for the study of physical and nutritional properties of edible fats and oils. A method was expected, but no such separation method was provided. An object of the present invention is to provide a method for producing TAG regioisomers and/or enantiomers composed of two types of fatty acids and glycerol, including a step of separating these isomers by HPLC.

The present inventors have made intensive studies on a separation method with improved separation ability for TAG positional isomers and enantiomers using a conventional HPLC system that does not use a recycling system. found that the performance of HPLC using a specific column treated by passing the The present invention has been completed.

That is, the present invention relates to the following manufacturing method.
[1] Selected from the group consisting of AAB-, ABA-, and BAA-type triacylglycerols in which an arbitrary fatty acid A and an arbitrary fatty acid B different from fatty acid A, including the following steps A and B, are constituent fatty acids A method for producing one or more positional isomers and/or enantiomers of triacylglycerols.
A) Step A of preparing a raw material mixture containing positional isomers and/or enantiomers of two or more triacylglycerols selected from the group consisting of AAB-type, ABA-type and BAA-type triacylglycerols;
B) From the raw material mixture prepared in step A, high-performance liquid chromatography using a separation column packed with a stationary phase of amylose tris(3-chloro-4-methylphenylcarbamate) bound to silica gel with a particle size of 4 μm or less. A step of separating the positional isomer and/or enantiomer by B;
[2] The method for producing the positional isomer and/or enantiomer according to [1], further comprising the following step C.
C) A step of concentrating two or more regioisomers and/or enantiomers selected from the group consisting of AAB-type, ABA-type and BAA-type triacylglycerols from the raw material mixture by reversed-phase high-performance liquid chromatography. C.
[3] Before step B, at least the following 1) to 4 are added to a separation column packed with a stationary phase having a particle size of 4 μm or less, in which amylose tris(3-chloro-4-methylphenylcarbamate) is bound to silica gel. ), the method for producing the positional isomer and/or enantiomer according to [1] or [2], further comprising a step D of performing any one treatment.
1) After passing ethanol, pass N,N-dimethylformamide;
2) After passing ethanol, pass tetrahydrofuran;
3) pass isopropyl alcohol;
4) Passing a mixture of isopropyl alcohol and tetrahydrofuran;
[4] The positional isomer according to any one of [1] to [3], wherein the fatty acid is two selected from the group consisting of lauric acid, palmitic acid, stearic acid, oleic acid and linoleic acid, and / Or a method for preparing enantiomers.

In the production method of the present invention, the regioisomers and/or enantiomers of TAG can be simultaneously separated by chiral HPLC in a short time, and the separation performance is very high. Alternatively, according to the method for producing enantiomers, highly purified positional isomers and/or enantiomers of TAG can be produced in a short time.

Extracted ion chromatograms prepared by HPLC separation of TAG isomers obtained by reacting glycerol with palmitic acid, oleic acid and linoleic acid. Extracted ion chromatograms prepared by HPLC separation of TAG isomers obtained by reacting glycerol with palmitic acid, oleic acid, and stearic acid. Extracted ion chromatograms prepared by HPLC separation of TAG isomers obtained by reacting glycerol with linoleic acid, oleic acid, and stearic acid. Extracted ion chromatograms prepared by HPLC separation of TAG isomers obtained by reacting glycerol with palmitic acid, oleic acid and linoleic acid.

The TAG produced by the production method of the present invention is composed of two fatty acids A and B and glycerol. A TAG in which fatty acid A is ester-bonded to the sn-1 and sn-3 positions of glycerol constituting the TAG and fatty acid B is ester-bonded to the sn-2 position is called ABA type, the sn-1 position of the glycerol and A TAG in which fatty acid A is ester-bonded at the sn-2 position and fatty acid B is ester-bonded at the sn-3 position is AAB type, fatty acid B is ester-bonded at the sn-1 position of the glycerol, sn-2 position and A TAG in which fatty acid A is ester-bonded to the sn-3 position is referred to as a BAA type (hereinafter, a TAG composed of two types of fatty acids and glycerol is similarly referred to). AAB and BAA forms are enantiomers. These two TAGs (AAB and BAA) and ABA are positional isomers.

Fatty acids constituting the TAG produced by the production method of the present invention are not limited to specific fatty acids. The fatty acid may be two types selected from the group consisting of saturated fatty acids and unsaturated fatty acids that constitute glycerol esters contained in edible fats and oils. Specific examples of saturated fatty acids include lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, and lignoceric acid. Specific examples of unsaturated fatty acids are palmitoleic acid, oleic acid, linoleic acid, linolenic acid and the like. Preferred fatty acids are palmitic acid, stearic acid, oleic acid, linoleic acid.

In the production method of the present invention, two or more TAGs selected from the group consisting of AAB type, ABA type and BAA type TAGs, in which an arbitrary fatty acid A and an arbitrary fatty acid B different from fatty acid A are constituent fatty acids. It includes step A of providing a raw material mixture containing positional isomers and/or enantiomers. The method of preparing the mixture is not limited to a particular method. One specific example of the method for preparing the mixture is (a) an esterification reaction of two fatty acids A and B and glycerol, (b) a transesterification reaction of a triglyceride containing two fatty acids A and B, and (c) Esterification reaction of monoacylglycerol and/or diacylglycerol with fatty acid A and/or B as a constituent fatty acid and fatty acid A and/or B, or (d) methyl ester, ethyl ester, etc. of fatty acid A and/or B Another example of a method for preparing such mixtures is the transesterification of esters and triglycerides by (e) utilizing naturally occurring lipids containing positional isomers and/or enantiomers of the TAG; The use of mixtures of AAB-type and BAA-type TAGs that are commonly used.

In the production method of the present invention, the above regioisomeric and step B of separating the isomers and/or enantiomers. The particle size of the stationary phase is 4 μm or less, preferably the particle size is 3.5 μm or less, and more preferably the particle size is 3 μm or less. If the particle size is too large, the separation power of the column packed with the stationary phase will decrease, and the regioisomers and/or enantiomers of TAG composed of two fatty acids A and B and glycerol will not be separated. .

Separation columns packed with a stationary phase of amylose tris(3-chloro-4-methylphenylcarbamate) bound to silica gel with a particle size of less than 4 μm are commercially available. A specific example of the commercial product is CHIRALPAK IF-3 manufactured by Daicel Corporation.

Although the set temperature of the separation column varies somewhat depending on the constituent fatty acid to be separated, the preferred set temperature of the separation column used in the production method of the present invention is 15 to 40°C. A more preferable set temperature is 20 to 30°C, and a further preferable set temperature is 23 to 27°C. If the set temperature of the separation column is too low or too high, the separation performance of the separation column will decrease.

A preferred mobile phase used in the HPLC system employed in the production method of the present invention is composed of acetonitrile and methanol in a ratio ranging from 10:0 to 5:5 (volume ratio). A preferable composition ratio of acetonitrile and methanol is 10:0 to 8:2, and a more preferable composition ratio is 10:0 to 9:1. If the mobile phase consisting of acetonitrile and methanol contains too much methanol, the separation performance of the separation column is reduced.

A preferred linear velocity of the mobile phase used in the HPLC system employed in the production method of the present invention is 2-12 mL/min·cm ² . A more preferable linear velocity is 4 to 9 mL/min·cm ² , and a further preferable linear velocity is 5 to 7 mL/min·cm ² . If the linear velocity is too small, the separation performance of the separation column will decrease. The upper limit of the linear velocity is subject to technical restrictions.
The mixture of regioisomers and/or enantiomers of the TAG is separated into regioisomers and/or enantiomers of the TAG by passage through a separation column.
In the production method of the present invention, after step A, one or two selected from the group consisting of the above AAB type, ABA type and BAA type TAGs are subjected to reversed-phase high-performance liquid chromatography from the raw material mixture prepared in step A. A step C of concentrating more than one kind of TAG may be further provided. That is, the raw material mixture in which the TAG in the raw material mixture prepared in step A is concentrated can be subjected to step B.

Preferably, the production method of the present invention further includes, before step B, step D of subjecting the separation column to at least one of the following treatments 1) to 4).
1); After ethanol was passed through, N,N-dimethylformamide (DMF) was passed through. 2); After passing ethanol, tetrahydrofuran (THF) is passed.
3); Isopropyl alcohol (IPA) is passed through.
4); A mixture of IPA and THF is passed through.

Preferred linear velocities of the DMF in the treatment 1), the THF in the treatment 2), the IPA in the treatment 3), and the mixture of IPA and THF in the treatment 4) are 0.6 to 4.
2 mL/min·cm ² , and a more preferable linear velocity is 1.2 to 2.7 mL/min·c
m ² , and a more preferable linear velocity is 1.5 to 2.0 mL/min·cm ² .

DMF in the treatment 1) and THF in the treatment 2) are preferably passed for 3 hours to 3 days, more preferably 6 hours to 2 days, still more preferably 12 hours to 1.5 days. is. In the treatment 3), the IPA is preferably passed for 12 hours to 6 days, more preferably 1 to 5 days, and still more preferably 2 to 4 days. The liquid mixture of IPA and THF in the treatment 4) is preferably passed for 30 minutes to 5 hours, more preferably 1 hour to 4 hours, and still more preferably 1.5 hours to 3 hours. .

A preferred mixing volume ratio of the mixture of IPA and THF in the treatment 4) is IPA:THF = 1:20 to 20:1, a more preferred mixing volume ratio is IPA:THF = 1:15 to 15:1, and still more preferred. The mixing volume ratio is IPA:THF=1:10 to 10:1.

Using the positional isomer and/or enantiomer of TAG produced by the production method of the present invention as a standard product, using a mass chromatogram of the positional isomer and/or enantiomer by an atmospheric pressure ionization mass spectrometer, Quantitative analysis of triacylglycerol isomers contained in natural oils and fats and processed oil products can be performed.

Another aspect of the present invention is a method for separating positional isomers and/or enantiomers of triacylglycerols shown below.
[1] Positional isomers and/or enantiomers of two or more triacylglycerols selected from the group consisting of AAB-type, ABA-type and BAA-type triacylglycerols, consisting of two fatty acids A and B and glycerol A sample solution containing amylose tris(3-chloro-4-methylphenylcarbamate) bound to silica gel and passing through a column filled with a separating agent having a particle size of 4 μm or less, the above AAB type, ABA type and BAA-type triacylglycerols.
[2] The sample solution contains (a) esterification reaction of two fatty acids A and B and glycerol, (b) transesterification reaction of triglyceride containing two fatty acids A and B, (c) fatty acid A and / or Esterification reaction of monoacylglycerol and/or diacylglycerol with B as a constituent fatty acid and fatty acid A and/or B, or (d) ester such as methyl ester or ethyl ester of fatty acid A and/or B and triglyceride The separation method described in [1] obtained by an exchange reaction.
[3] The separation method described in [1], wherein the sample solution is obtained from natural lipids.
[4] The separation method described in [1], wherein the sample solution is a mixture of AAB-type and BAA-type triacylglycerols.
[5] The separation according to any one of [1] to [4], further comprising the step of subjecting the separation column to at least one of the following treatments 1) to 4) before passing the sample solution. Method.
1) After passing ethanol, pass N,N-dimethylformamide;
2) After passing ethanol, pass tetrahydrofuran;
3) pass isopropyl alcohol;
4) Passing a mixture of isopropyl alcohol and tetrahydrofuran;
[6] The separation method according to any one of [1] to [5], wherein the fatty acid is two selected from the group consisting of lauric acid, palmitic acid, stearic acid, oleic acid and linoleic acid.

Another aspect of the present invention includes a method for analyzing positional isomers and/or enantiomers of triacylglycerols shown below.
[7] Positional isomers and/or enantiomers of two or more triacylglycerols selected from the group consisting of AAB-type, ABA-type and BAA-type triacylglycerols, consisting of two fatty acids A and B and glycerol A sample solution containing amylose tris(3-chloro-4-methylphenylcarbamate) bound to silica gel and passing through a column filled with a separating agent having a particle size of 4 μm or less, the above AAB type, ABA type and a method for analyzing positional isomers and/or enantiomers of one or more triacylglycerols selected from the group consisting of BAA-type triacylglycerols by high-performance liquid chromatography.
The analytical method may comprise detecting the separated regioisomers and/or enantiomers. Mass spectrometry can be used as a detection method in the detection step.

EXAMPLES The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.
Example 1
Synthesis of TAG With respect to 1 molar equivalent of glycerol, 1 molar equivalent of palmitic acid (P), 1 molar equivalent of oleic acid (O) and 1 molar equivalent of linoleic acid (L) are mixed, and 1-(3- Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) 3 molar equivalents, N,N-dimethyl-4-aminopyridine (DMAP) 3 molar equivalents, N-ethyldiisopropylamine 3 molar equivalents as a catalyst, A TAG mixture was obtained by reacting in chloroform for 12 hours.

Separation of TAG Isomers by HPLC The above TAG mixture was diluted with acetonitrile (concentration 100 μg/mL) to obtain a TAG sample. 10 μL of the TAG sample was injected into an HPLC system equipped with a chiral column (Alliance e2695 manufactured by Waters) to separate and detect TAG positional isomers and enantiomers. The setting conditions of the above HPLC system were as follows.
Column: CHIRALPAK IF-3 manufactured by Daicel Corporation (4.6 × 250 mm, filled with a separating agent having a particle size of 3 µm, in which amylose tris (3-chloro-4-methylphenylcarbamate) is bound to silica gel)
Column temperature: 25°C
Flow rate: 1.0 mL/min
Mobile phase: acetonitrile (for LC/MS manufactured by Wako Pure Chemical Industries, Ltd.)

Detection of TAG isomers by electrospray ionization-mass spectrometry (ESI-MS) Method Half of the flow out of the HPLC system was discarded to the drain, and the remaining flow (0.5 mL/min) was filled with 100 mM ammonium formate. The methanol solutions were combined at a flow rate of 0.1 mL/min, the TAG regioisomers and enantiomers were ionized in positive mode, and peaks were detected with an ESI-MS system (Quattro micro API manufactured by Waters). The setting conditions of the ESI-MS system were as follows.
Source block temperature: 120°C
Desolvation temperature: 450°C
Desolvation gas: nitrogen (gas flow rate: 800 L/h)
Capillary voltage: 3 kV
Cone voltage: 40V
Detection mode: Full Scan m/z 200-1200

The results are shown in FIG. Extracted ion chromatograms corresponding to the regioisomers and enantiomers (LPL, LLP and PLL) of TAG with two linoleic acids and one palmitic acid esterified to glycerol from the total ion chromatogram (TIC). Once made, three peak components were observed (Fig. 1a). From the TIC, regioisomers and enantiomers of TAG with one linoleic acid and two palmitic acids esterified to glycerol (LPP, PPL and PLP; FIG. 1b), two oleic acids and one palmitic acid regioisomers and enantiomers of TAG esterified to glycerol (OPO, OOP and POO; FIG. 1c), regioisomers of TAG with one oleic acid and two palmitic acids esterified to glycerol and When extracted ion chromatograms corresponding to the enantiomers (OPP, PPO and POP; FIG. 1d) were generated, three peak components were observed for each. Therefore, from the TAG mixture containing the 12 types of positional isomers and enantiomers, the 12 types of positional isomers and enantiomers were separated by the HPLC system and detected by the ESI-MS system. was done.

Example 2
Instead of mixing 1 molar equivalent of palmitic acid (P), 1 molar equivalent of oleic acid (O) and 1 molar equivalent of linoleic acid (L) with respect to 1 molar equivalent of glycerol, On the other hand, the same operation as in Example 1 was performed except that 1 molar equivalent of palmitic acid (P), 1 molar equivalent of oleic acid (O) and 1 molar equivalent of stearic acid (S) were mixed. The results are shown in FIG.
The positional isomers and enantiomers of TAG (P+O+S), in which one molecule each of P, O and S are ester-bonded to one molecule of glycerol, were not well separated by the above HPLC system (Fig. 2a). On the other hand, from TIC, regioisomers and enantiomers of TAG with two oleates and one stearate esterified to glycerol (OSO, OOS and SOO; Fig. 2b), one oleate and two stearates When extracting ion chromatograms corresponding to the regioisomers and enantiomers of TAG (SSO, OSS and SOS; FIG. 2c), where the acid is esterified to glycerol, three peak components are observed, respectively. From the TAG mixture containing 6 types of positional isomers and enantiomers, it was confirmed that the 6 types of positional isomers and enantiomers were separated by the HPLC system and detected by the ESI-MS system.

Example 3
Instead of mixing 1 molar equivalent of palmitic acid (P), 1 molar equivalent of oleic acid (O) and 1 molar equivalent of linoleic acid (L) with respect to 1 molar equivalent of glycerol, On the other hand, the same operation as in Example 1 was performed except that 1 molar equivalent of linoleic acid (L), 1 molar equivalent of oleic acid (O) and 1 molar equivalent of stearic acid (S) were mixed. The results are shown in FIG. From TIC, regioisomers and enantiomers of TAG with two molecules of oleic acid and one molecule of stearic acid esterified to one molecule of glycerol (OSO, OOS and SOO; Fig. 3a), two molecules of linoleic acid and Positional isomers and enantiomers of TAG (LSS, SSL and SLS; Fig. 3a) in which one molecule of stearic acid is ester-bonded to one molecule of glycerol, two molecules of linoleic acid and one molecule of stearic acid are linked to one molecule of glycerol. When extracted ion chromatograms corresponding to the positional isomers and enantiomers (LSL, LLS and SLL; FIG. 3b) of TAG esterified to the It was confirmed that from the TAG mixture containing positional isomers and enantiomers, the 9 types of positional isomers and enantiomers were separated by the HPLC system and detected by the ESI-MS system.

Example 4
In the same manner as in Example 1, 1 molar equivalent of palmitic acid (P), 1 molar equivalent of oleic acid (O) and 1 molar equivalent of linoleic acid (L) were mixed with 1 molar equivalent of glycerol to form a TAG mixture. got The HPLC conditions were as follows.
Column: CHIRALPAK IF-3 manufactured by Daicel Corporation (2.0×250 mm, filled with a separating agent having a particle size of 3 μm, in which amylose tris (3-chloro-4-methylphenylcarbamate) is bound to silica gel)
Column temperature: 25°C
Flow rate: 0.2 mL/min
Mobile phase: acetonitrile (for LC/MS manufactured by Wako Pure Chemical Industries, Ltd.)
The results are shown in Figure 4a. Regioisomers and enantiomers of TAG in which two oleic acids and one palmitic acid are esterified to glycerol (peak at 26.40 is OPO, peak at 27.48 is OOP, peak at 28.68 is POO When the extracted ion chromatogram corresponding to FIG. 4 a) is generated, three peak components are observed, respectively, and from the TAG mixture containing the three positional isomers and enantiomers, the three positional isomers and It was confirmed that the enantiomers were separated by the HPLC system and detected by the ESI-MS system.

Example 5
Before separation of TAG isomers by HPLC, ethanol was passed through the column at a linear velocity of 2 mL/min·cm for ³ hours, and then DMF was passed at a linear velocity of 2 mL/min·cm for ¹ day. The same operations as in Example 1 were performed, except that pretreatment was performed. The results are shown in Figure 4b. From TIC, regioisomers and enantiomers of TAG with two oleic acids and one palmitic acid esterified to glycerol (peak at 25.37 OPO, peak at 26.18 OOP, peak at 27.44 The peak is POO; when the extracted ion chromatogram corresponding to FIG. It was confirmed that the isomers and enantiomers were separated by the HPLC system and detected by the ESI-MS system.

Example 6
Before separation of TAG isomers by HPLC, ethanol was passed through the column at a linear velocity of 2 mL/min·cm for ³ hours, and then THF was passed at a linear velocity of 2 mL/min·cm for ¹ day. The same operations as in Example 1 were performed, except that pretreatment was performed. The results are shown in Figure 4c. From TIC, regioisomers and enantiomers of TAG with two oleic acids and one palmitic acid esterified to glycerol (peak at 29.45 OPO, peak at 30.23 OOP, peak at 31.68 The peak is POO; when the extracted ion chromatogram corresponding to FIG. It was confirmed that the isomers and enantiomers were more clearly separated and detected by the ESI-MS system than the HPLC system using a column not pretreated with organic solvent in Example 4. . Furthermore, it was found that the pretreatment of passing THF after passing ethanol through the column contributes to the improvement of the separation ability of the column.

Example 7
The same procedure as in Example 1 was carried out, except that IPA was passed through the column at a linear velocity of 2 mL/min·cm ² for 1 day for pretreatment prior to separation of TAG isomers by HPLC. The results are shown in Figure 4d. From TIC, regioisomers and enantiomers of TAG with two oleic acids and one palmitic acid esterified to glycerol (peak at 29.61 OPO, peak at 30.70 OOP, peak at 32.22 The peak is POO; when the extracted ion chromatogram corresponding to FIG. It was confirmed that the isomers and enantiomers were more clearly separated and detected by the ESI-MS system than the HPLC system using a column not pretreated with organic solvent in Example 4. . Furthermore, it was found that the pretreatment of passing IPA through the column contributes to the improvement of the separation ability of the column.

Example 8
Prior to separation of TAG isomers by HPLC, a mixture of IPA and THF (mixed volume ratio IPA:THF = 9:1) was passed through the column for ² hours at a linear velocity of 2 mL/min cm for pretreatment. Except for this, the same operation as in Example 1 was performed. The results are shown in Figure 4e. From TIC, regioisomers and enantiomers of TAG with two oleic acids and one palmitic acid esterified to glycerol (peak at 29.96 OPO, peak at 31.14 OOP, peak at 32.69 The peak is POO; when the extracted ion chromatogram corresponding to FIG. It was confirmed that the isomers and enantiomers were more clearly separated and detected by the ESI-MS system than the HPLC system using a column not pretreated with organic solvent in Example 4. . Furthermore, it was found that pretreatment in which a mixed solution of IPA and THF (mixed volume ratio of 9:1) is passed through the column contributes to the improvement of the column's separation ability.

Example 9
Synthesis of TAG mixture To 1 molar equivalent of glycerol, 2 molar equivalents of palmitic acid (P) and 1 molar equivalent of oleic acid (O) are mixed, 3 molar equivalents of EDC, 3 molar equivalents of DMAP, N-ethyldiisopropyl Using 3 molar equivalents of amine as a catalyst, the reaction was carried out in dichloromethane for 12 hours to obtain a TAG mixture. The TAG mixture was fractionated by silica gel column chromatography using a mixture of hexane and ethyl acetate (volume ratio of 95:5) as an eluent to give tripalmitin, dipalmitoyl oleoylglycerol, dioleoyl palmitoylglycerol and trio 660 mg of TAG mixture containing rhein was obtained.

Fractionation of dipalmitoyl oleoylglycerol fraction by HPLC Dissolve 660 mg of the above TAG mixture in 13 mL of acetone to prepare a TAG sample, and inject the TAG sample in three portions into a preparative HPLC system to obtain POP, PPO and 200 mg of the dipalmitoyl oleoyl glycerol fraction containing OPP was fractionated. The setting conditions of the above HPLC system were as follows.
Column: Inertsil ODS-3 (50 × 250 mm) manufactured by GL Sciences Co., Ltd.
Column temperature: 40°C
Mobile phase flow rate: 120 mL/min
Mobile phase: acetone/acetonitrile (50/50, v/v)
Detector: UV205nm

Separation of Dipartoyloleoylglycerol Isomers by HPLC The above dipaltoyloleoylglycerol fraction was diluted with acetone/acetonitrile (50/50) (concentration 10 mg/mL) to obtain a dipalmitoyloleoylglycerol sample. 20 μL of the dipalmityl oleyl glycerol sample was injected into an HPLC system (Alliance e2695 manufactured by Waters) to separate POP, PPO and OPP. The setting conditions of the above HPLC system were as follows.
Column: CHIRALPAK IF-3 manufactured by Daicel Corporation (4.6 x 250 mm)
Column temperature: 25°C
Mobile phase flow rate: 1.0 mL/min
Mobile phase: acetonitrile (for LC/MS manufactured by Wako Pure Chemical Industries, Ltd.)
Detector: UV205nm

The operation of separating dipalmityloleylglycerol isomers by HPLC was repeated 100 times to obtain 4 mg each of POP, PPO and OPP.

In the production method of the present invention, the regioisomers and/or enantiomers of TAG isomers composed of two kinds of fatty acids and glycerol are separated with high performance. The improvement of the separation ability is expected to improve the accuracy of qualitative and quantitative analysis of the TAG isomers contained in edible fats and oils, and progress in research on the physical properties and nutritional properties of edible fats and oils is expected.

Claims (5)

A sample containing two or more positional isomers and/or enantiomers of triacylglycerols selected from the group consisting of AAB, ABA and BAA triacylglycerols consisting of two fatty acids A and B and glycerol AAB type, ABA type and BAA type, comprising a step of passing the solution through a column packed with a separating agent having a particle size of 4 μm or less, in which amylose tris(3-chloro-4-methylphenylcarbamate) is bound to silica gel. A method for separating positional isomers and/or enantiomers of one or more triacylglycerols selected from the group consisting of triacylglycerols by high performance liquid chromatography,
further comprising subjecting the column to at least any one of the following treatments 1) to 4) before passing the sample solution;
1) After passing ethanol, pass N,N-dimethylformamide;
2) After passing ethanol, pass tetrahydrofuran;
3) pass isopropyl alcohol;
4) passing a mixture of isopropyl alcohol and tetrahydrofuran; and
The sample solution contains (a) an esterification reaction of two fatty acids A and B and glycerol, (b) a transesterification reaction of a triglyceride containing two fatty acids A and B, and (c) fatty acids A and/or B. Those obtained by esterification reaction of monoacylglycerol and/or diacylglycerol as constituent fatty acids and fatty acids A and/or B, or (d) transesterification reaction of esters of fatty acids A and/or B and triglycerides. or (e) obtained from naturally occurring lipids. Claim 1, wherein the sample solution is a mixture of AAB-type and BAA-type triacylglycerols
Separation method described in. 3. The separation method according to claim 1 or 2 , wherein the fatty acid is two selected from lauric acid, palmitic acid, stearic acid, oleic acid and linoleic acid. A sample containing two or more positional isomers and/or enantiomers of triacylglycerols selected from the group consisting of AAB, ABA and BAA triacylglycerols consisting of two fatty acids A and B and glycerol The AAB type, ABA type and BAA type, comprising the step of passing the solution through a column filled with a separating agent having a particle size of 4 μm or less, in which amylose tris(3-chloro-4-methylphenylcarbamate) is bound to silica gel. A method for analyzing positional isomers and/or enantiomers of one or more triacylglycerols selected from the group consisting of triacylglycerols by high performance liquid chromatography,
further comprising subjecting the column to at least any one of the following treatments 1) to 4) before passing the sample solution;
1) After passing ethanol, pass N,N-dimethylformamide;
2) After passing ethanol, pass tetrahydrofuran;
3) pass isopropyl alcohol;
4) passing a mixture of isopropyl alcohol and tetrahydrofuran; and
The sample solution contains (a) an esterification reaction of two fatty acids A and B and glycerol, (b) a transesterification reaction of a triglyceride containing two fatty acids A and B, and (c) fatty acids A and/or B. Those obtained by esterification reaction of monoacylglycerol and/or diacylglycerol as constituent fatty acids and fatty acids A and/or B, or (d) transesterification reaction of esters of fatty acids A and/or B and triglycerides. or (e) obtained from naturally occurring lipids. 5. The analytical method according to claim 4 , further comprising the step of detecting positional isomers and/or enantiomers of triacylglycerols passed through the column by mass spectrometry.

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