JP7019281B2 - Analysis method of triacylglycerol - Google Patents

Description

The present disclosure relates to a method for analyzing triacylglycerol, particularly a method for analyzing the triacylglycerol composition of fats and oils.

Triacylglycerol, which is a component of fats and oils, is a triester of glycerol and fatty acids. The composition of triacylglycerol is considered to affect the physical characteristics and nutrition of fats and oils. Therefore, knowing the triacylglycerol composition of fats and oils is useful for the development, manufacture and quality control of foods containing fats and oils. Generally, gas chromatography or liquid chromatography has been used for composition analysis of triacylglycerol. However, in the conventional gas chromatography or liquid chromatography method, it is difficult to separate a plurality of triacylglycerols composed of fatty acids having the same total carbon number but different fatty acids from each other.

In addition, supercritical fluid chromatography is characterized by the low viscosity and high diffusivity of supercritical fluids, which enables separation (clearly separated from peaks of other substances) and detection (high sensitivity is obtained by sharp peaks). The ability is said to be better than conventional high performance liquid chromatography. For example, Japanese Patent Publication No. 6-500783 discloses an analysis of polyol polyester using supercritical fluid chromatography.

Special Table 6-500783 Gazette

However, even if supercritical fluid chromatography is applied, although the treatment time is short and the accuracy is improved, it is still not possible to effectively separate multiple triacylglycerols having the same total carbon number and different constituent fatty acids from each other. rice field.

The present disclosure has been made in view of the above circumstances, and an object of the present disclosure is to provide a method for analyzing a plurality of triacylglycerol compositions composed of different fatty acids having the same total carbon number of constituent fatty acids. It is to be. Another object of the present disclosure is to provide a method for analyzing the triacylglycerol composition of fats and oils.

The present disclosers have made diligent studies to solve the above problems. As a result, the mass of the first step of decomposing each triacylglycerol of a composition containing two or more different triacylglycerols having the same total number of fatty acid residues (total carbon number) into diacylglycerol and fatty acid. It has been found that the composition of triacylglycerol can be analyzed by combining the analysis and the mass analysis of the second step of decomposing each diacylglycerol obtained in the first step into fatty acid. That is, the present disclosure may be in the following aspects.

[1] A method for analyzing the triacylglycerol composition of a composition containing two or more different triacylglycerols having the same total number of fatty acid residues (total carbon number).
A method comprising the following steps (a) and (b).
(A) By mass spectrometry, each triacylglycerol is decomposed into corresponding diacylglycerol and fatty acid fragments.
(B) By mass spectrometry, each diacylglycerol detected in step (a) is decomposed into the corresponding fatty acid fragments.
[2] By the steps (a) and (b), the triacylglycerol to which each fatty acid fragment of the step (b) belongs is determined, and two or more different triacyls in which the total number of the fatty acid residues is the same as each other are determined. The method of [1], wherein the triacylglycerol composition of the composition containing glycerol is calculated based on the detection intensity of the fatty acid fragment belonging to each triacylglycerol.
[3] Compositions of two or more different triacylglycerols having the same total number of fatty acid residues are obtained by chromatographic separation of fats and oils based on total carbon number, [1] or [ 2] method.
[4] A method for analyzing the triacylglycerol composition of fats and oils.
The method according to [1] or [2], wherein the method is applied to each of two or more peaks obtained by separating fats and oils based on the total number of carbon atoms by chromatography.
[5] Among all the fatty acids constituting the fat and oil, the chain length difference (difference in carbon number) between the fatty acid having the largest carbon atom number and the fatty acid having the smallest carbon atom number is 6 or more. The method of [3] or [4].
[6] The method of [3] or [4], wherein the content of the fatty acid having 14 or less carbon atoms in the total amount of the fatty acids constituting the fat and oil is 10% by mass or more.
[7] From processed fats and oils containing lauric-based fats and oils, milk-derived fats and oils, lauric-based fats and oils as raw material fats and / or milk-derived fats and oils (separated fats and oils, transesterified fats and oils, hydrogenated fats and oils, and combinations of fats and oils). The method of [3] or [4], which comprises one or more selected fats and oils.

According to the present disclosure, it is possible to provide a composition analysis method for a plurality of triacylglycerol compositions composed of different fatty acids having the same total carbon number of the constituent fatty acids, depending on the type of the constituent fatty acids. Further, by applying this, it is possible to provide a method for analyzing the composition of triacylglycerols constituting fats and oils depending on the type of constituent fatty acids.

It is a mass spectrum chart of TAG-CN36. It is explanatory drawing which shows the relationship between TAG-CN36 and DG and FA precursor ion generated by a mass spectrum. It is explanatory drawing which shows the relationship between TAG-CN36 and DG and FA precursor ion generated by a mass spectrum. It is explanatory drawing which shows the relationship between TAG-CN36 and DG and FA precursor ion generated by a mass spectrum. It is explanatory drawing which shows the relationship between TAG-CN36 and DG and FA precursor ion generated by a mass spectrum. It is a schematic diagram of the TAG composition calculation procedure of a random transesterified fat and oil. It is an analysis flow of the TAG composition analysis method of fats and oils. It is a chromatographic chart of the palm kernel oil and fat with random transesterification by GC-FID. It is explanatory drawing of the matching of GC-FID and the theoretical value. It is a figure which shows the corresponding m / z value of each FA, DG precursor ion in the extracted ion chromatograph. It is an overlay diagram of the chromatograph of each FA precursor ion generated from each TAG-CN peak by mass spectrometry. It is an overlay diagram of the chromatograph of each DG precursor ion generated from each TAG-CN peak by mass spectrometry.

Hereinafter, the present disclosure will be described in detail.
"Triacylglycerol (TAG)" is a triester of glycerol and fatty acids. The fatty acid may be a linear or branched fatty acid having 4 to 24 carbon atoms. Further, either saturated fatty acid or unsaturated fatty acid may be used. Preferred are linear saturated fatty acids and / or linear unsaturated fatty acids having 6 to 22 carbon atoms. Examples of these fatty acids include butyric acid (FA4), caproic acid (FA6), caprylic acid (FA8), capric acid (FA10), lauric acid (FA12), myristic acid (FA14), palmitoleic acid (FA16), and stear. Acid (FA18), araquinic acid (FA20), behenic acid (FA22), palmitoleic acid (FA16: 1), oleic acid (FA18: 1), linoleic acid (FA18: 2), linolenic acid (FA18: 3), erucic acid Acids (FA22: 1), arachidonic acid (FA20: 4), EPA (FA20: 5), DHA (FA22: 6), and the like can be mentioned.

The present disclosure is a method for analyzing the triacylglycerol composition (constituent ratio) of a composition containing two or more different triacylglycerols having the same total number of fatty acid residues (total carbon number). For two or more different triacylglycerols having the same total number of fatty acid residues (total carbon number), triacylglycerol having a total carbon number of 36 in the constituent fatty acids is taken as an example (hereinafter, triacylglycerols are referred to as triacylglycerols). Depending on the total carbon number of the constituent fatty acids, it may be expressed as TAG-CN36 (TAG with a total carbon number of 36). For example, TAG12 / 12/12, TAG10 / 12/14, TAG10/10/16, TAG8 / 14/14, TAG8 / 12/16, TAG8 / 10/18, TAG6 / 14/16, TAG6 / 12/18, Triglycerides, which are a combination of TAG8 / 10/18: 1, TAG6 / 12/18: 1, TAG8 / 10/18: 2, TAG6 / 12/18: 2, etc., have 36 fatty acid residues in each other. Corresponds to triacylglycerol (TAG-CN36), which is the same in and has different constituent fatty acids. Here, for example, TAG10 / 12/14 represents triacylglycerol in which the constituent fatty acids are capric acid (FA10), lauric acid (FA12) and myristic acid (FA14), and of capric acid, lauric acid and myristic acid. , The binding position on the glycerol skeleton is indistinguishable. That is, according to the method of the present disclosure, no distinction is made between the positional isomer and the optical isomer of triacylglycerol having the same constituent fatty acids. In addition, TAG 10/12/14 may be expressed as 10/12/14.

According to the methods of the present disclosure, compositions comprising two or more different triacylglycerols having the same total number of fatty acid residues (total carbon number) will be subjected to mass spectrometry to each triacylglycerol, the corresponding diacyl. Decomposes into fragments (fragments, precursor ions) of glycerol (DG) and fatty acids (FA) (step (a)). According to the principle of mass spectrometry, the m / z values obtained by dividing the relative mass (m) of the ions of the decomposed diacylglycerol and the fatty acid by the charge number (z) of the ions are in ascending order of molecular weight, and the relative strength (relative strength (m). Peak area) is proportional to the number of molecules x the size of the molecular weight. For example, in the case of capric acid (FA10), lauric acid (FA12), and myristic acid (FA14), the m / z values are in the order of FA10 (155) <FA12 (183) <FA14 (211), and the measurement conditions are as follows. Since the unique value (value in parentheses) is shown by, the fatty acid can be identified. Similarly, in the case of diacylglycerols of FA10 + 12, FA12 + 12, FA10 + 14, FA12 + 14, for example, the m / z value is FA10 + 12 (411) <FA12 + 12 = FA10 + 14 Since (439) <FA12 + 14 (467), and the unique value (value in ()) is shown depending on the measurement conditions, diacylglycerol can be identified. Here, for example, FA10 + 14 represents diacylglycerol in which the constituent fatty acids are capric acid (FA10) and myristic acid (FA14), and the binding positions of capric acid and myristic acid on the glycerol skeleton are indistinguishable. Further, hereinafter, diacylglycerol (DG) may be expressed as DG24 (DG having a total carbon number of 24) depending on the total carbon number of the constituent fatty acids. For example, FA10 + FA14 are included in DG24.

According to the methods of the present disclosure, for example, compositions containing two or more different triacylglycerols having the same total number of fatty acid residues (total carbon number) are TAG12 / 12/12 and TAG10 / 12/14. In the case of a mixture of TAG-CN36 (1 example of TAG-CN36, TAG-CN36-1), in step (a), TAG12 / 12/12 is decomposed and ionized into FA12 + 12 and FA12 by mass spectrometry. Detected. Further, TAG10 / 12/14 is detected by being decomposed and ionized into any of FA10 + 12 and FA14, FA10 + 14 and FA12, and FA12 + 14 and FA10 by mass spectrometry. That is, as diacylglycerol, peaks were detected at the three m / z values (values in parentheses) of FA10 + 12 (411), FA12 + 12 = FA10 + 14 (439), and FA12 + 14 (467). , Peaks are detected in the three m / z values (values in parentheses) of FA10 (155), FA12 (183), and FA14 (211) as fatty acids (see FIG. 1). Here, the peak with an m / z value of 439 contains two types of diacylglycerols having different constituent fatty acids of FA12 + 12 and FA10 + 14 (see FIG. 2).

Next, in step (b), each diacylglycerol (FA10 + 12 (411), FA12 + 12 = FA10 + 14 (439), FA12 + 14 (467)) detected in step (a) is divided into two stages. It is detected by being decomposed and ionized into FA by mass spectrometry of the eye. FA12 + 12 (439) is the only DG derived from TAG12 / 12/12, and FA12 + 12 contained in the m / z value 439 is detected by being decomposed and ionized into FA12 (183). On the other hand, FA10 + 14 contained in m / z value 439 is decomposed and ionized into FA10 (155) and FA14 (211) and detected, so DG with m / z value 439 is decomposed and ionized and detected. Only the peak intensity (area) of FA12 (183) represents the intensity of the TAG12 / 12/12 content in TAG-CN36-1. Regarding FA10 + 12 (411), FA10 + 14 (439) and FA12 + 14 (467) derived from TAG10 / 12/14, FA10 + 12 (411) is FA10 (155) and FA12 (183). FA10 + 14 (439) is decomposed into FA10 (155) and FA14 (211), FA12 + 14 (467) is decomposed into FA12 (183) and FA14 (211), and is detected. Therefore, the peak intensities of m / z values 155 and 183 detected by decomposing and ionizing the DG of m / z value 411, and the m / z values 155 and 211 detected by decomposing and ionizing the DG of m / z value 439. The sum of the peak intensity of m / z value 183 and the peak intensity of 211 detected by decomposition ionization of DG with m / z value 467 is the TAG10 / 12/14 content in TAG-CN36-1. Represents strength. Then, from the ratio of the total peak intensities of FA fragments derived from TAG12 / 12/12 and TAG10 / 12/14, the content of TAG12 / 12/12 and TAG10 / 12/14, which is TAG-CN36-1, is It can be calculated (see FIGS. 2 to 5). A triple quadrupole mass spectrometer capable of continuously processing the above steps (a) and (b) is suitable for the method of the present disclosure.

According to the method of the present disclosure, as described above, in the composition of triacylglycerol having the same total carbon number of the constituent fatty acids (for example, the TAG-CN36 composition), the average molecular weight (≈mass) of the constituent fatty acids is the same. Therefore (for example, in the case of TAG contained in the TAG-CN36 composition, the average molecular weight of the constituent fatty acids is equal to the molecular weight of FA12), triacylglycerols having the same total carbon number of the constituent fatty acids are decomposed by mass analysis and have a probability. It can be assumed that diacylglycerols having the same average molecular weight are produced. Based on this assumption, the sum of the FA peak intensities from one type of triacylglycerol (eg, FA10-12-14) derived from that triacylglycerol, which is identified through two-step mass spectrometry (eg, m /). Peak intensities of m / z values 155 and 183 detected by decomposition ionization of DG with z value 411, peak intensities of m / z values 155 and 211 detected by decomposition ionization of DG with m / z value 439, The sum of the peak intensities of m / z values 183 and 211 detected by decomposition ionization of DG with m / z value 467) is the intensity of the content of the triacylglycerol in the TAG-CN36 composition. Can be said to represent. As described above, the method of the present disclosure is premised on assumptions based on the principle of mass spectrometry. Since the method of the present disclosure does not require a standard substance required for quantification in mass spectrometry, TAG composition analysis can be performed very easily. The TAG composition analysis of fats and oils, which is one aspect of the present disclosure, will be described below, and a series of analyzes and analyzes can be performed in about 2 hours.

According to one aspect of the present disclosure, the reliability of the method of the present disclosure is based on the TAG composition of a triacylglycerol composition or fat or oil in which constituent fatty acids whose TAG composition can be specified by calculation from the fatty acid composition are randomly distributed. The TAG composition obtained by the above method can be verified by the degree of dissociation from the reference value. Methods for calculating the TAG composition of triacylglycerol compositions or fats and oils in which constituent fatty acids are randomly distributed are known, for example, a review of RJVANDER WAL (Journal of American Oil Chemists' Society 40, 242-247 (1963)). Can be referred to. The calculation procedure is shown in FIG.

According to one aspect of the present disclosure, the above method is a triacylglycerol composition of a triacylglycerol peak (triacylglycerol composition) obtained by chromatographically separating fats and oils based on the total carbon number of the constituent fatty acids of the acylglycerol. Can be applied to the analysis of. Further, according to one aspect of the present disclosure, the above method can be applied to two or more peaks in which fats and oils are separated based on the total carbon number of constituent fatty acids. Further, by applying the above method to all peaks whose TAG composition is unknown, the triacylglycerol composition of fats and oils can be analyzed. That is, according to one aspect of the present disclosure, a method for analyzing the triacylglycerol composition of fats and oils is provided. The fat and oil is not particularly limited as long as the main component is acylglycerol (for example, the content of triacylglycerol is 70% by mass or more). It may be any of natural fats and oils, synthetic fats and oils, vegetable fats and oils, animal fats and oils, and microorganism-derived fats and oils.

According to one aspect of the present disclosure, in the method of separating fats and oils based on the total carbon number of the constituent fatty acids of acylglycerol, triacylglycerols are separated according to the total carbon number of the constituent fatty acids, and the mass thereof is equal to the separated peak. Any separation method may be used as long as the analysis is applicable. However, the separation method is preferably gas chromatography or liquid chromatography, more preferably gas chromatography.

According to one aspect of the present disclosure, first, by gas chromatography (GC-FID) equipped with a FID as a detector, fats and oils are separated into triacylglycerol compositions (each peak) having the same total carbon number of constituent fatty acids. Obtain the ratio of each peak intensity to the whole. In gas chromatography in which the detector is FID, there is almost no difference in sensitivity depending on the type of triacylglycerol, so that the peak intensity (peak area) contains a triacylglycerol composition having a specific total carbon number in fats and oils. It is proportional to the amount. Further, by using the separated phase (column) to which the polarity is imparted, the triacylglycerol composition having the same total carbon number of the constituent fatty acids can be further separated into a plurality of peaks. In recent years, the type of triacylglycerol belonging to a peak in which a triacylglycerol composition having the same total carbon number of the constituent fatty acids is further separated is being identified. Basically, if the total carbon number of the constituent fatty acids is the same triacylglycerol, the larger the difference in the chain length of the constituent fatty acids and the larger the number of double bonds contained in the constituent fatty acids, the more the corresponding tri. The retention time of acylglycerol becomes longer. Utilizing such information makes it easier to analyze the TAG composition contained in one peak by applying the method of the present disclosure.

In parallel, the above method comprising step (a) and step (b) is applied to each peak consisting of a triacylglycerol composition having the same total carbon number of constituent fatty acids separated by gas chromatography. That is, the composition ratio of TAG contained in each peak is analyzed by applying a series of flows (GC-MSMS) of gas chromatography (GC) → mass spectrometry (MS) → mass spectrometry (MS). That is, in the TAG composition analysis method of the present disclosure, the ratio (% by mass) of each peak area of the triacylglycerol composition having the same total carbon number of the constituent fatty acids obtained by separating the fat and oil by GC-FID to the total fat and oil (area). This is a method for obtaining the TAG composition of the whole fat and oil by applying the composition ratio of the TAG contained in each peak obtained by GC-MSMS (see FIG. 7). A triple quadrupole mass spectrometer linked with gas chromatography can be applied to the above-mentioned series of GC-MSMS flows. Examples of such an analyzer include a GCMS-TQ8050NX manufactured by Shimadzu Corporation.

According to one aspect of the present disclosure, the TAG composition analysis of the fat and oil can be analyzed very simply as compared with the conventional method when the chain length difference (difference in the number of carbon atoms) of the fatty acids constituting the fat and oil is large. That is, in the TAG composition analysis of the above fats and oils, the chain length difference (difference in carbon number) between the fatty acid having the largest number of carbon atoms and the fatty acid having the smallest number of carbon atoms among the fatty acids constituting the fats and oils is preferably 6 or more. It can be suitably applied to the analysis of fats and oils, which are more preferably 8 to 14, and even more preferably 10 to 12.

According to one aspect of the present disclosure, in the TAG composition analysis of the fats and oils, the content of fatty acids having 14 or less carbon atoms in the total amount of fatty acids constituting the fats and oils is preferably 10% by mass or more, more preferably. When it is 10 to 95% by mass, more preferably 20 to 90% by mass, and even more preferably 30 to 85% by mass, it can be analyzed very simply as compared with the conventional method.

According to one aspect of the present disclosure, in the TAG composition analysis of the above fats and oils, the fats and oils are processed fats and oils containing lauric acid-based fats and oils, milk-derived fats and oils, lauric-based fats and oils as raw material fats and / or milk-derived fats and oils (separated fats and oils, transesterification). When one or more fats and oils selected from fats and oils, hydrogenated fats and oils, and combinations of fats and oils processed thereof are contained, the analysis can be performed very easily as compared with the conventional method. Here, the lauric-based fat and oil is a fat and oil in which the content of lauric acid in the total amount of the constituent fatty acids is 30% by mass or more. The milk-derived fats and oils are fats and oils obtained from milk (human milk, milk, goat milk, mare milk, etc.). Examples of lauric oils and fats include coconut oil, palm kernel oil, and babassu oil.

According to one aspect of the present disclosure, the TAG composition analysis of fats and oils can be applied to quality control of fats and oils. For example, it can be applied to manufacturing process control and shipping standard control of transesterified fats and oils and separated fats and oils. Further, according to one aspect of the present disclosure, the TAG composition analysis of the above-mentioned fats and oils can be utilized for designing TAG compositions for development such as breast milk substitute fats and oils, milk fat substitute fats and oils, and lauric chocolate fats and oils (CBS). Further, according to one aspect of the present disclosure, the TAG composition analysis of the above-mentioned fats and oils can be utilized for analysis of fats and oils physical characteristics (solid fat content, viscosity, emulsion stability, etc.). Further, according to one aspect of the present disclosure, the TAG composition analysis of the above-mentioned fats and oils can be applied to the analysis of nutritional properties such as medium-chain fatty acid triglyceride and milk fat.

Hereinafter, the present disclosure will be described in more detail by way of examples, but the present disclosure is not limited thereto.

<Creating a standard sample>
Random transesterified palm kernel oil (PKO-CIE) was prepared as a standard sample. That is, RBD palm kernel oil (manufactured by Intercontinental Specialty fats, Malaysia) was randomly transesterified using sodium methylate as a catalyst according to a conventional method.
Next, the fatty acid composition (based on mass%) of the random transesterified palm kernel oil was analyzed according to AOCS Ce 1h-05. The obtained fatty acid composition (based on mass%) was converted to a molar composition based on mol% based on the carbon number or molecular weight of the fatty acid. Based on the obtained fatty acid composition (mol% basis), the triacylglycerol composition when the constituent fatty acids of palm kernel oil were randomly rearranged on the glycerol skeleton was calculated. For example, triacylglycerol ABC (TGABC), which consists of fatty acids A (a mol%), B (b mol%), and C (c mol%) and can be located at any position on the glycerol skeleton of A, B, C, is ABC. , ACB, BAC, BCA, CAB, CBA exist, and it can be calculated that a × b × c / 10000 × 6 (mol%) is probabilistically produced by random transesterification. Nine constituent fatty acids (caproic acid (FA6), capric acid (FA8), caproic acid (FA10), lauric acid (FA12), myristic acid (FA14), palmitic acid (FA16), stearic acid (FA18), oleic acid In the case of palm kernel oil having (FA18: 1), linoleic acid (FA18: 2)), the type of triacylglycerol after random ester exchange must be distinguished by the binding position of the constituent fatty acid on the glycerol skeleton. Then, ₉ C ₁ + ₉ C ₂ × 2 + ₉ C ₃ = 165, and the content (mol%) of each triacylglycerol can be calculated. Then, the TAG composition (mass% base) of the random transesterified palm kernel oil is obtained by converting the mol%-based TAG composition obtained by calculation into a mass base based on the molecular weight of each TAG. This was used as a standard sample for TAG composition analysis. The TG composition of the standard sample obtained by calculation is shown in Table 1. In Table 1, RPKO_CIE is random transesterified palm kernel oil, CN is the total carbon number of the constituent fatty acids of triacylglycerol, TAG is triacylglycerol, and FA1 to FA3 are fatty acids constituting triacylglycerol (however, glycerol). The binding position of the skeleton is indistinguishable), Simulation represents the content of each triacylglycerol obtained by the above calculation.

<Gas chromatographic analysis of standard sample>
The composition of the PKO-CIE prepared above was analyzed by gas chromatography (GC-FID) equipped with a FID (hydrogen flame ionization detector) using a strong polar column based on the total carbon number of the constituent fatty acids. .. The analysis conditions are as follows (the analysis conditions may be fine-tuned as appropriate according to the characteristics of the equipment).

Column: Rtx (R) -65TG (manufactured by GL Sciences Co., Ltd.) Length 15m, Inner diameter 0.25mm, Film thickness 0.1μm
GC condition: Injection side split specification, temperature 365 ℃ FID detector side temperature 365 ℃
Carrier gas helium (flow rate 1 ml / min)
Temperature rise conditions 230 ℃ (hold for 3 minutes) → 3 ℃ / min → 365 ℃ (hold for 4 minutes)

The chart obtained by the above gas chromatography is shown in FIG. As shown in the GC chart, in RPKO-CIE, triacylglycerol is separated by the total carbon number of the constituent fatty acids, and even in the triacylglycerol having the same total carbon number of the constituent fatty acids, the triacylglycerol has a small amount. Due to the difference in polarity, it is separated into a plurality of peaks (TAG compositions). The tendency of separation in triacylglycerols having the same total number of carbon atoms of the constituent fatty acids has been confirmed. The smaller the length difference, the faster the elution. Next, the TAG having a double bond in the constituent fatty acid is eluted, and as the number of double bonds contained in the constituent fatty acid increases, the elution of the corresponding TAG becomes slower (retention time becomes longer). For each peak on the GC chart, the main types of TAG included in the peak are entered based on the conventional knowledge. However, in the peak containing a plurality of TAGs (TAG composition), the content ratio of each TAG is unknown from the GC analysis. In the following, for the sake of explanation, a plurality of peaks (peak groups) in which the total carbon number of the constituent fatty acids is 36 will be taken up. FIG. 9 is an enlarged view of a peak group in which the total carbon number of the constituent fatty acids is 36 extracted from the GC-FID chart of FIG. The TAG having a total carbon number of 36 in the constituent fatty acids is separated into five peaks, and from the conventional knowledge and the above theoretical calculation values, for example, the first peak 1 to be eluted is TAG12 / 12/12 and TAG10 / 12 /. It is assumed that 14 types of TAGs are included, but the content ratio (ratio to the peak area) of each TAG is unknown from the GC chart. In the table in FIG. 9, for reference, the content of each TAG in the calculated RPKO-CIE (total TAG amount) is entered.

<TAG composition analysis of each peak (TAG composition) by mass spectrometry>
(Specification of fragments (fragments, precursor ions) obtained from each peak (TAG composition))
The fraction corresponding to each peak (TAG composition having the same total carbon number of constituent fatty acids) obtained by the above GC-FID analysis is subjected to GC-MSMS analysis in advance by time-of-flight mass spectrometry (GC-TOFMS). Fragments (precursor ions) obtained from the above were identified. FIG. 10 shows the m / z value (value obtained by dividing the relative mass (m) of the ion by the charge number (z) of the ion) corresponding to each FA and each DG of the precursor ion generated from the TAG by mass spectrometry. ).
Next, the precursor ions generated from each peak (TAG composition having the same total carbon number of constituent fatty acids) are specified. FIG. 11 shows fragments (precursor ions) of constituent fatty acids detected from each peak. For example, FA8, FA10, FA12, FA14, and FA16 were detected as fatty acid fragments from the peak (TAG-CN36 composition) in which the total number of carbon atoms of the constituent fatty acids was 36, and FA6, FA18, and FA18: 1 were slightly present. Detected. That is, it can be seen that the TAG-CN36 composition contains FA6, FA8, FA10, FA12, FA14, FA16, FA18, and FA18: 1 as constituent fatty acids. And FIG. 12 shows a fragment (precursor ion) of diacylglycerol produced from each peak (TAG composition having the same total carbon number of constituent fatty acids). For example, DG18, DG20, DG22, and DG24 are detected as DG fragments, and DG26 and DG28 are slightly detected from the peak (TAG-CN36 composition) in which the total carbon number of the constituent fatty acids is 36. That is, the TAG constituting the TAG-CN36 composition is DG18, DG20, DG22, DG24, DG26, DG28, and further fatty acids (any of FA6, FA8, FA10, FA12, FA14, FA16, FA18, FA18: 1). It can be seen that is a structure in which one is combined. The m / z values corresponding to each FA and each DG fragment are specified (confirmed) by GC-TOFMS, and the DG and FA fragments generated from each peak (TAG composition having the same total carbon number of constituent fatty acids). The specification is an arbitrary step, and if it has already been specified, it can be omitted.

Before being subjected to mass spectrometry, it is necessary to separate fats and oils into each TAG composition (each peak) having the same total carbon number of constituent fatty acids on a GC column. Therefore, the following conditions were adopted (analytical conditions may be fine-tuned as appropriate according to the characteristics of the equipment). However, the elution pattern of each peak does not change from the above GC-FID.

Column: Rtx (R) -65TG (manufactured by GL Sciences Co., Ltd.) Length 15m, Inner diameter 0.25mm, Film thickness 0.1μm
GC conditions: Injection side split specification, temperature 370 ℃
Carrier gas helium (flow rate 2.4 ml / min)
Temperature rise conditions 150 ℃ (hold for 1 minute) → 5 ℃ / min → 350 ℃ (hold for 20 minutes)

The conditions of the mass spectrometry (MS analysis) device were set as follows.
Interface temperature: 350 ℃
Ion source:
--Temperature: 330 ℃
--Electron energy: 70 eV
--Collision energy (MRM mode): 10 eV

(Detailed analysis of TAG composition (each peak) by GC-MSMS)
Next, the mass spectrum of the triple quadrupole mass spectrometer obtained from each peak separated by the GC column was analyzed by GC-MSMS. Here, the first peak (TAG-CN36_1st) of the five peak groups of triacylglycerol (TAG-CN36 composition) having a total carbon number of 36 of the constituent fatty acids separated by the GC column is taken as an example. .. It has already been identified by conventional knowledge that TAG-CN36_1st contains two types of triacylglycerols, TAG12 / 12/12 and TAG10 / 12/14 (however, positional isomers and optical isomers are not distinguished). There is. However, its content ratio is unknown. Then, the ratio of TAG12 / 12/12 and TAG10 / 12/14 contained in TAG-CN36_1st is subjected to two-step mass spectrometry using a triple quadrupole mass spectrometer (step (a) and step (b) of the present disclosure. The method for analyzing from the mass spectrum obtained by)) has already been described in detail in paragraphs 0013 to 0016 above for TAG-CN36-1.
As a result of the analysis, the composition ratio (mass ratio) of TAG12 / 12/12 and TAG10 / 12/14 obtained from the GC-MSMS analysis of the present disclosure is TAG12 / 12/12: TAG10 / 12/14 = 85.2: 14.8. The composition ratio (mass ratio) of TAG12 / 12/12 and TAG10 / 12/14 of the standard sample calculated from the fatty acid composition in paragraph 0027 above is TAG12 / 12/12: TAG10 / 12/14 = 87.9: 12. Since it is .1, it can be said that the accuracy is practically usable. In addition, the content of TAG-CN36_1st obtained by GC-FID (area content of TAG-CN36_1st with respect to the total area of the peak) is 13.65% by mass, and the composition ratio of TAG12 / 12/12 and TAG10 / 12/14 (mass). Ratio) By applying 85.2: 14.8, 11.6% by mass of TAG12 / 12/12 content and 2.0% by mass of TAG10 / 12/14 content in fats and oils can be obtained.
Table 2 shows the results of analyzing the mass spectra obtained by two-step mass spectrometry for all the peaks separated by the GC column. In Table 2, RPKO_CIE is random transesterified palm kernel oil, CN is the total carbon number of the constituent fatty acids of triacylglycerol, TAG is triacylglycerol, and FA1 to FA3 are fatty acids constituting triacylglycerol (however, glycerol). The binding position of the skeleton is indistinguishable), Analysis represents the content of each triacylglycerol obtained by multiplying the GC-FID analysis and the GC-MSMS analysis of the present disclosure. Even when compared with the results in Table 1, which are standard calculated values, almost the same results are obtained.

Palm kernel oil obtained in the same manner as above (the chain length difference between the constituent fatty acid having the largest number of carbon atoms and the constituent fatty acid having the smallest carbon atom number is 12 (= 18-6), and the number of carbon atoms is 14 or less. Fatty acid content 70.6% by mass) and coconut oil (chain length difference between constituent fatty acid with the largest number of carbon atoms and constituent fatty acid with the smallest carbon atom is 12 (= 18-6)), carbon number 14 or less The analysis results of the constituent fatty acid content of 83.7% by mass) are shown in Tables 3 and 4. However, in Table 3, RPKO is palm kernel oil, CN is the total carbon number of the constituent fatty acids of triacylglycerol, TAG is triacylglycerol, and FA1 to FA3 are fatty acids constituting triacylglycerol (however, the binding of the glycerol skeleton). (Position is indistinguishable), Analysis represents the content of each triacylglycerol obtained by multiplying the GC-FID analysis and GC-MSMS analysis of the present disclosure, and in Table 4, CNO is coconut oil, CN. Is the total carbon number of the constituent fatty acids of triacylglycerol, TAG is triacylglycerol, FA1 to FA3 are fatty acids constituting triacylglycerol (however, the binding position of the glycerol skeleton is not distinguished), and Analysis is GC- of the present disclosure. Represents the content of each triacylglycerol obtained by multiplying FID analysis and GC-MSMS analysis.

Claims (7)

A method for analyzing the triacylglycerol composition of a composition comprising two or more different triacylglycerols having the same total number of fatty acid residues (total carbon number).
A method comprising the following steps (a) and (b).
(A) By mass spectrometry, each triacylglycerol is decomposed into corresponding diacylglycerol and fatty acid fragments.
(B) By mass spectrometry, each diacylglycerol detected in step (a) is decomposed into the corresponding fatty acid fragments. The steps (a) and (b) determine the triacylglycerol to which each fatty acid fragment of step (b) belongs and contain two or more different triacylglycerols having the same total number of fatty acid residues. The method of claim 1, wherein the triacylglycerol composition of the composition is calculated based on the detection intensity of a fatty acid fragment belonging to each triacylglycerol. Claim 1 or 2, wherein two or more different triacylglycerol compositions having the same total number of fatty acid residues are obtained by chromatographic separation of fats and oils based on total carbon number. Method. A method for analyzing the triacylglycerol composition of fats and oils .
Two or more peaks containing two or more different triacylglycerols having the same total number of fatty acid residues (total carbon number) as each other, obtained by separating fats and oils based on total carbon number by chromatography. A method of applying the following steps (a), (b) and (c) to each .
(A) By mass spectrometry, each triacylglycerol contained in the peak is decomposed into corresponding diacylglycerol and fatty acid fragments.
(B) By mass spectrometry, each diacylglycerol detected in step (a) is decomposed into the corresponding fatty acid fragments.
(C) By the steps (a) and (b), the triacylglycerol to which each fatty acid fragment of the step (b) belongs is determined, and two or more different triacyls in which the total number of the fatty acid residues is the same as each other are determined. The triacylglycerol composition of peaks containing glycerol is calculated based on the detection intensity of fatty acid fragments belonging to each triacylglycerol. The fourth aspect of claim 4 , wherein the chain length difference (difference in carbon number) between the fatty acid having the largest carbon atom number and the fatty acid having the smallest carbon atom number among all the fatty acids constituting the fat and oil is 6 or more. Method. The method according to claim 4 , wherein the content of the fatty acid having 14 or less carbon atoms in the total amount of the fatty acids constituting the fat and oil is 10% by mass or more. The fat is selected from lauric-based fats and oils, milk-derived fats and oils, processed fats and oils containing lauric-based fats and oils and / or milk-derived fats and oils as raw material fats and oils (separated fats and oils, transesterified fats and oils, hydrogenated fats and oils, and combinations of fats and oils thereof). The method according to claim 4 , which comprises one or more fats and oils.

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