JP2018205013A - Method for quantifying anthocyanin pigment - Google Patents

Abstract

To provide a technique capable of quantifying an exact total amount of natural anthocyanin pixels which are a set of compositions of analogue compounds which is the technique capable of even quantifying the anthocyanin compound which is the individual component having no reference sample.SOLUTION: A quantifying method of an anthocyanin compound includes the steps of: performing degradation treatment on a measurement sample including the anthocyanin compound with varied collision energies more than three times and acquiring a mass spectrum by the MS/MS analysis including accumulating fragmented compound ions (step 1); and quantifying the anthocyanin compound using a common structure contained in the anthocyanin compound as a target fragment ion serving as an index from the mass spectrum acquired at the step 1 (step 2).SELECTED DRAWING: Figure 2

Description

  The present invention relates to a technique that enables accurate quantification of anthocyanin dyes and is applicable to the quantification of natural anthocyanin dyes that are aggregate compositions of similar compounds.

Anthocyanin pigments are natural pigment compounds derived from plant materials that exhibit colors such as red, blue, purple, etc., and overlap with the recent safety orientation of consumers, and many foods, cosmetics, quasi drugs, pharmaceuticals, etc. It is used as an excellent pigment material in the field.
An anthocyanin pigment is a pigment composition that is an aggregate of a plurality of similar compounds, and the compound composition varies greatly depending on the type and variety of the source plant, the growing period, the growing environment, and the like. In addition to the combination of an aglycone and a sugar molecule, anthocyanin dyes have a large number of combinations of similar compounds depending on the presence or type of organic acids and various modifying groups, and each has different molecular properties relating to color tone and stability.

In the field of production of coloring agents and foods that use anthocyanin dyes, the accurate determination of anthocyanin dyes must be performed quickly and easily in terms of quality requirements such as color tone and stability in production lots, etc. Is required.
Here, as a method for quantifying the anthocyanin dye, a standard curve of a typical anthocyanin compound such as pelargonidin-3-glucoside was used to prepare a calibration curve by absorbance, and the total anthocyanin converted to the dye compound as the standard sample. The method of calculating as an amount is mentioned. This method is used in the field as a simple method. However, in this method, it is difficult to apply to a sample having a different absorbance from the dye compound of the standard sample, and it is an aggregate composition of similar compounds having different color tones. Natural anthocyanin pigments cannot be accurately quantified. Further, in this method, it is not possible in principle to quantify individual anthocyanin components only by estimating the total amount of anthocyanins.
Moreover, as a method for quantifying individual components contained in an anthocyanin dye, a method for quantifying individual components by HPLC, LC / MS, or the like can be mentioned. However, only a part of the compounds for which standard samples can be prepared can be quantified by these general techniques. In addition, when estimating the total anthocyanin amount, total aglycone amount, etc. using a conversion formula, etc., due to the circumstances of anthocyanin dyes, which are various similar compound aggregates with different molecular weights, chemical properties, etc., these estimates are accurate. Can't do it.

  The situation in the above general technology will be described with specific examples. For example, the red radish pigment, which is one of the anthocyanin pigments, contains a large number of anthocyanin compounds classified as pelargonidin-based anthocyanins, Only a very small portion of standard samples such as pelargonidin-3-glucoside can be easily obtained, and there is no standard sample for most natural compounds. In addition, the compound composition of the red radish pigment varies greatly depending on the type of raw radish. For this reason, the above-described conventional technique cannot accurately determine the total amount of anthocyanins and the total amount of pelargonidin contained in the red radish pigment. In addition, for individual anthocyanin compounds without a standard sample, individual quantification for each component cannot be performed.

Journal of Agricultural and Food Chemistry, 2004 Feb 25 52 (4) p688-691, Zhang Z. et al., Comparison of HPLC methods for determination of anthocyanins and anthocyanidins in bilberry extracts.

  The present invention has been made in view of the circumstances of the prior art described above, and the subject of the present invention is a technique that enables accurate quantification of the total amount of natural anthocyanin pigments that are aggregate compositions of similar compounds. It is an object of the present invention to provide a technique that enables quantification of anthocyanin compounds that are individual components without a sample.

  In the situation of the above-mentioned prior art, the present inventors conducted extensive research and found that, when the anthocyanin compound in the target sample was subjected to MS / MS analysis, the anthocyanin compound was decomposed into the compound ions decomposed and produced by the collision energy. We focused on the fact that aglycon ions, which are common structures, are included. However, only by measuring the amount of fragment compound ions obtained by performing normal MS / MS analysis, when the anthocyanin dye, which is an aggregate composition of similar compounds, is measured, the amount of aglycone decomposed is quantitative. I couldn't find it.

Therefore, the inventors of the present invention further studied and obtained a mass spectrum in an MS / MS analysis including an aglycon ion accumulated by performing a decomposition process with varying collision energy three times or more. The present inventors have found that the total amount of anthocyanin compounds containing the aglycone as a common structure can be quantified using the amount of aglycone ions in the acquired mass spectrum as an index.
Further, as a result of further investigations, the present inventors conducted at least one of the three or more decomposition processes in the MS / MS analysis using a low energy collision energy containing an undecomposed peak of the anthocyanin compound. , At least one more time with a high energy collision energy substantially free of unresolved peaks of the anthocyanin compound and including the peak of the aglycon ions of the common structure, and at least another one time with the two times As a result, the amount of aglycone ions that can be quantified can be stably generated even from natural anthocyanin dyes, which are aggregate compositions of similar compounds with various molecular weights and structures. I found it.

  Based on the above findings, the present inventors conducted a quantitative verification test using red radish pigment and purple potato pigment as measurement samples. It has been found that anthocyanin pigments can be quantified very accurately. The quantification could be performed simultaneously with the total amount of anthocyanin compounds having the desired aglycone, the total amount of all anthocyanin compounds, and individual quantification of the desired anthocyanin compounds. Further, in the quantification means, it is not necessary to prepare individual components in the measurement sample as standard compounds, and only by creating a calibration curve using a known compound containing the aglycone, the total amount or individual component amount described above can be obtained. Quantification was possible.

The above-described quantification principle of the anthocyanin dyes found by the present inventors is based on the principle of the AIF (All Ion Fragmentation) method, in which the accumulated target fragment ions (specifically, aglycone ions) are stable. The original AIF method is known to be used in a method for “qualitative analysis” of unknown compounds in a sample. There has been no known knowledge that quantification is imparted to the target fragment ions in the setting.
In this respect, the present invention was conceived in the presence of common technical knowledge that the conventional AIF analysis is an analysis method used only for “qualitative analysis”, and was accumulated in a plurality of settings of the above-mentioned collision energy. It is recognized that there was no technical common sense reminiscent of imparting “quantitativeness” to the target fragment ions.

Although not widely used as a general method, as a conventional technique for quantifying anthocyanin dyes other than those described above, an anthocyanin compound in a measurement sample is subjected to acid hydrolysis to liberate aglycone, and an organic solvent layer A method for extracting an aglycone and quantifying anthocyanin content by LC / MS or the like has been reported (Non-patent Document 1).
However, this method has a large variation in the amount of aglycone extracted obtained after the acid hydrolysis reaction and organic solvent extraction, and it is in principle difficult to perform accurate quantification. Furthermore, in this method, since it is essential to perform acid hydrolysis and organic solvent extraction as pretreatment of the measurement sample, the quantitative operation cannot be performed easily and quickly in terms of sample preparation and handling.
On the other hand, the above-described quantification method found by the present inventors enables precise quantification using MS / MS analysis, and does not require pretreatment of the measurement sample by chemicals or enzyme treatment. Therefore, the quantitative operation can be performed easily and quickly.

  Based on the above findings, the present inventors have completed the present invention. The present invention specifically relates to the invention described below.

[Claim 1]
The steps described below,
(Step 1) A measurement sample containing an anthocyanin compound is subjected to a decomposition process with varying collision energy three or more times to obtain a mass spectrum by MS / MS analysis including the integrated fragmented compound ions. And the process of
(Step 2) Quantifying the anthocyanin compound from the mass spectrum obtained in the (Step 1) using the common structure contained in the anthocyanin compound as a target fragment ion as an index,
A method for quantifying an anthocyanin compound, comprising:
[Section 2]
Three or more decomposition processes in the MS / MS analysis described in (Step 1)
At least once with low energy collision energy containing undegraded peaks of the anthocyanin compound,
At least another time is performed in a high-energy collision energy that does not substantially include the undecomposed peak of the anthocyanin compound and includes the peak of the fragment ion of the common structure,
At least one more time is performed with the energy between the two collision energy,
Item 2. The method according to Item 1.
[Section 3]
Said (Step 1)
During three or more decomposition processes with varying collision energy, the compound ions after each decomposition process are electrically supplemented and accumulated within a certain spatial range, and the total number of decomposition processes It is a step that is performed by integrating and detecting this at once after the end of the minute,
Item 3. The method according to Item 1 or 2.
[Claim 4]
Item 4. The method according to any one of Items 1 to 3, wherein the MS / MS analysis in (Step 1) is performed on a measurement sample obtained by LC / MS analysis.
[Section 5]
Item 5. The method according to any one of Items 1 to 4, wherein the device described below is used:
(Apparatus) MS / MS analysis can be performed with three or more decomposition processes with different collision energy, and compound ions after the decomposition process with collision energy are electrically captured and integrated to detect or detect Mass spectrometer that can be integrated.
[Claim 6]
Item 6. The method according to Item 5, wherein the mass spectrometer is a mass spectrometer based on an electric field type Fourier transform.
[Claim 7]
The common structure as the target fragment ion is
An anion of an anthocyanin compound or an ion of a similar compound derived from the aglycone,
Item 7. A method for quantifying an anthocyanin dye according to any one of Items 1 to 6.
[Section 8]
The quantification method comprises:
An anthocyanin pigment composition comprising a plurality of anthocyanin compounds is a method capable of performing the total amount of anthocyanin compounds having a desired aglycone, the total amount of all anthocyanin compounds, and / or individual quantification of the desired anthocyanin compounds.
Item 8. A method for quantifying an anthocyanin dye according to any one of Items 1 to 7.

The present invention is a technique that enables accurate quantification of the total amount of natural anthocyanin dyes, which are aggregate compositions of similar compounds, and also enables the quantification of anthocyanin compounds that are individual components without a standard sample. I will provide a.

It is the flowchart which showed the outline of the main process about the determination method of the anthocyanin pigment | dye concerning this invention.

It is the conceptual diagram which showed the principle of the main processes which comprise the fixed_quantity | assay method which concerns on this invention. FIG. 2A: Step of acquiring MS mass spectrum by MS analysis. FIG. 2B: A process of obtaining a mixture of three decomposition compound ions having different degrees of decomposition by performing three different decomposition processes of collision energy. FIG. 2C: a step of measuring the integrated value of fragment ions by MS / MS analysis. The symbol “M” in the figure indicates the compound ion (parent ion) before decomposition, which is the object of quantification. The symbol “F” in the figure indicates target fragment ions generated after the decomposition process.

It is the structural diagram illustrated using pelargonidin-3-caffeoylsophoroside-5-glucoside about the general molecular structure which comprises an anthocyanin compound.

It is the conceptual diagram which illustrated the decomposition product of the anthocyanin compound in the decomposition process by MS / MS analysis in the quantification method based on this invention.

In the decomposition | disassembly process which changed the collision energy which concerns on Example 1, it is a result figure which measured the peak area of the aglycon (peonidine) ion which is the anthocyanin compound ion before decomposition | disassembly and the produced | generated target fragment.

In the decomposition process by MS / MS analysis which changed the collision energy which concerns on Example 2, it is a result figure which measured the peak area of the aglycon (cyanidine) ion which is the anthocyanin compound ion before decomposition | disassembly and the produced | generated target fragment. .

In the decomposition treatment by MS / MS analysis with varying collision energy according to Example 3, pelargonidin-3-caffeoyl, feruloylsophoroside-5-malonylglucoside (m / It is a result figure showing a result of MS / MS mass spectrum about z: 1195.3102). FIG. 7A: Mass spectrum with HCD10. FIG. 7B: Mass spectrum with HCD40. FIG. 7C: Mass spectrum on HCD80.

It is the result figure which measured the peak area of the aglycon (pelargonidin) ion which is the produced | generated target fragment in the decomposition | disassembly process by MS / MS analysis which changed the collision energy which concerns on Example 3. FIG.

It is the result figure of the regression line obtained by performing the detection using the AIF analysis with respect to the dilution series sample of pelargonidin chloride by HCD conditions of Example 3, and plotting the peak area of the obtained pelargonidin ion.

It is the result figure of the regression curve obtained by performing the detection using AIF analysis with respect to the dilution series solution of a red radish pigment | dye according to HCD conditions of Example 4, and plotting the peak area of the obtained pelargonidin ion.

In LC / MS analysis with respect to the red radish pigment | dye which concerns on Example 6, it is a result figure which shows the obtained total ion chromatogram.

FIG. 6 is a diagram showing a structural formula of an anthocyanin compound contained in a red radish pigment whose structure is estimated in Example 6.

It is the result figure which measured the peak area of the aglycon (peonidine, cyanidin) ion which is the produced | generated target fragment in the decomposition | disassembly process by MS / MS analysis which changed the collision energy which concerns on Example 7. FIG.

It is the result figure of the regression line obtained by performing the detection using the AIF analysis with respect to the dilution series sample of pelargonidin chloride by HCD conditions of Example 7, and plotting the peak area of the obtained peonidin ion.

It is the result of the regression line obtained by performing the detection using the AIF analysis with respect to the dilution series sample of cyanidin chloride by HCD conditions of Example 7, and plotting the peak area of the obtained cyanidin ion.

Hereinafter, embodiments of the present invention will be described in detail. Moreover, the description regarding the principle, process, etc. of the determination method according to the present invention is exemplarily shown in FIGS.
In this specification, “Pn” is peonidin, “PnCl” is peonidin chloride, “Cy” is cyanidin, “CyCl” is cyanidin chloride, “Pg” is pelargonidin, and “PgCl” is pelargonidin chloride. May be used as an abbreviation for
It should be noted that the technical scope according to the present invention is not intended to exclude aspects including configurations other than those described below, unless they substantially hinder the operational effects of the technical features according to the present invention. Absent. Further, the technical scope according to the present invention is not limited to an embodiment including all of the following configurations.

1. Principle of Quantification The present invention relates to a technique that enables accurate quantification of anthocyanin dyes, and is applicable to the quantification of natural anthocyanin dyes that are aggregate compositions of similar compounds.

[Measurement sample]
The measurement sample in the method for quantifying an anthocyanin dye according to the present invention is a sample containing an anthocyanin compound.
Here, in the present specification, the “anthocyanin compound” is a natural pigment compound derived from a plant material exhibiting a color tone such as red, blue, purple, etc., and a sugar molecule (for example, glucose, galactose, rhamnose) is added to an anthocyanidin that is an aglycone. Etc.). In addition to sugar molecules, organic acids (for example, sinapinic acid, caffeic acid, succinic acid, malonic acid, etc.) may bind to form acylated anthocyanins. As for the structure of anthocyanin compounds, there are enormous types of molecular species having different molecular weights and structures depending on the combination of aglycone type, sugar molecule type and bonding position, presence / absence or type of organic acid, and bonding position (see FIG. 3). ). Anthocyanin compounds vary in color tone due to the difference in structure.

  Examples of the measurement sample of the present invention include those containing an anthocyanin compound as a single component, and specific examples include a dye composition containing two or more anthocyanin compounds which are similar compounds having different molecular weights. . In addition, the measurement sample according to the present invention can be suitably used as an measurement sample for an anthocyanin dye composition comprising two or more anthocyanin compounds having different types of aglycones.

As a measurement sample according to the present invention, an anthocyanin pigment derived from a natural raw material containing various types of anthocyanin compounds can be suitably used as a measurement sample.
Examples of natural anthocyanin pigments include red radish pigment, red cabbage pigment, purple potato pigment, grape juice pigment, grape skin pigment, colored potato pigment, purple corn pigment, butterfly pigment, black bean pigment, elder Berry dye, Cranberry dye, Gooseberry dye, Salmon berry dye, Strawberry dye, Cherry dye, Dark sweet cherry dye, Timbleberry dye, Duberry dye, Hibiscus dye, Hakulberry dye, Black currant dye, Blackberry dye, Red currant Dye, Loganberry dye, Plum dye, Blueberry dye, Boysenberry dye, Whiteberry dye, Mulberry dye, Morero cherry dye, Raspberry dye, Perilla dye, Red rice dye, Purple carrot dye, etc. It is possible. Moreover, the mixed pigment | dye composition which comprises 2 or more chosen from these can be mentioned.

The measurement samples according to the present invention include various forms of anthocyanin pigment compositions such as pigment preparations, pigment extracts, fruit juices, vegetable juices and the like.
The measurement sample according to the present invention includes a mixed dye composition in which other dye compounds are contained in addition to the anthocyanin compound. For example, natural pigments such as gardenia red pigment, gardenia yellow pigment, gardenia blue pigment, safflower pigment, red beech pigment, turmeric pigment, tamarind pigment, oyster pigment, caramel pigment, spirulina pigment, cucurian pigment, cochineal pigment, tomato pigment, etc. However, it is not limited to these. Also, mixed dye compositions such as various synthetic dyes and various fragrance compounds can be used as the measurement sample according to the present invention.

The measurement sample according to the present invention is preferably subjected to analysis in the form of a liquid sample. Specifically, it is desirable to use as an aqueous solution diluted with water.
The sample concentration of the measurement sample to be used in the analysis process according to the present invention can be determined as appropriate according to the performance of the apparatus used. In consideration of performing the acquisition, it is preferable to employ a sample concentration that contains a dye compound in an amount that ensures quantitativeness and is prepared with a small amount of ions of all compounds.
As an example of such a sample concentration, a sample solution containing an anthocyanin compound so as to have a color value of 0.001 to 1, preferably about 0.005 to 0.1 can be mentioned.
In the present specification, the term "color values" means "color value ^E _{10% 1 cm",} the "color value ^E _{10% 1 cm",} when prepared with 10 wt% of the dye composition containing a solution In FIG. 2, the value is calculated by measuring the absorbance (A: Absorbance) of the maximum absorption wavelength (λmax) in the visible light region using a measurement cell having an optical path length of 1 cm. Sometimes referred to as “color value (10% E)”. Specifically, it can be calculated according to the method described in the 8th edition Food Additives Official (Ministry of Health, Labor and Welfare).

[measuring device]
The method for quantifying anthocyanin dyes according to the present invention is capable of MS / MS analysis with three or more decomposition processes with different collision energy, and electrically supplements the compound ions after the decomposition process with the collision energy. Then, a mass spectrometer that can be integrated and detected or detected and integrated is used.
In view of the accuracy of the analysis and the great simplicity of the analytical operation, the apparatus is preferably: i) constant compound ions after each decomposition treatment during three or more decomposition treatments with varying collision energy. It is preferable that the mass spectrometer be capable of being added and accumulated in an electrically supplemented space range and integrated and detected after completion of the number of times of all decomposition processes. .
As the apparatus, it is also possible to adopt a mode in which ii) compound ions after the decomposition treatment are detected first, and this is repeated for the set amount of collision energy, and then the detection values are integrated afterwards. . This embodiment is not excluded from the scope of the present invention, however, in the present invention, it is preferable to adopt the embodiment i) described in the above paragraph from the viewpoint of both analysis accuracy and ease of analysis operation. It is.

As a device that can be used in the method according to the present invention, any known or unknown device or instrument can be used as long as it is a mass spectrometer capable of realizing the above analysis. As an embodiment of the apparatus, for example, a mass spectrometer based on the principle of an electric field type Fourier transform method, a magnetic field type Fourier transform method, an ion trap method, or the like can be used.
In the present invention, in particular, when a mass spectrometer based on the electric field type Fourier transform method is used, it is preferable from the viewpoint of precision in precision measurement. Here, as the mass spectrometer based on the electric field type Fourier transform method, at the time of the present application, an example mass spectrometer based on the orbitrap method manufactured by Thermo Fisher Scientific Co., Ltd. can be exemplified. However, the embodiment of the present invention is not limited to the embodiment using the apparatus.

Moreover, as an apparatus that can be used in the method according to the present invention, it is preferable that the apparatus is capable of LC / MS analysis as an analysis prior to the MS / MS analysis. That is, it is preferable that the apparatus that can be used in the method according to the present invention is an apparatus that uses the above principle and that can perform LC / MS / MS analysis.
As the device, in order to secure a large number of scan points for MS / MS analysis and to acquire high-resolution data, it is possible to perform the MS / MS analysis after selecting a compound in a desired molecular weight range. Is preferred. As this aspect, a device equipped with a quadrupole type MS analysis means or the like is preferably used as an analysis instrument in the previous stage of MS / MS analysis.

[Analysis process]
The quantification method according to the present invention can perform quantification using AIF analysis using a mass spectrometer using the above principle. In this specification, “AIF (All Ion Fragmentation) analysis” means performing MS / MS with multiple decomposition processes in which the collision energy of the target compound is changed, and integrating the fragmented compound ions. This refers to an analysis method for obtaining a mass spectrum including the mass spectrum (see FIGS. 1 to 4).
Specifically, in the method for quantifying anthocyanin dyes according to the present invention, a measurement sample containing an anthocyanin compound is subjected to a decomposition treatment with varying collision energy three or more times, and the fragmented compound ions are integrated. And a step of acquiring a mass spectrum in the MS / MS analysis including the method.

Acquisition of MS scan data MS / MS analysis in the AIF analysis step is generated by decomposing and generating energy (collision energy) for the peak ion of the mass spectrum acquired in the first MS analysis for decomposition processing. This is a step of detecting compound ions. The first MS analysis is a mass spectrometry step for obtaining a mass spectrum in which an undecomposed compound contained in a measurement sample is detected as it is. The MS / MS analysis is a second mass analysis step for fragmenting and detecting the peak ions of the first MS analysis.

Here, as the first MS analysis performed before the MS / MS analysis, in order to comprehensively quantify even a low content of anthocyanin compounds, it is desirable to obtain full MS scan data, In order to secure a large number of scan points for MS / MS analysis and acquire high-resolution data, it is possible to perform the MS / MS analysis after selecting compounds within a desired molecular weight range to some extent. Is preferred.
Therefore, in the quantification method according to the present invention, i) since the molecular weight of the anthocyanin compound to be measured can be predicted within a certain range, the target compound for the first MS analysis is m / z value of 150 to 2000, preferably It is preferable to obtain the first MS scan data by narrowing down to a molecular weight range of m / z value of about 400 to 1500.

In addition, as another mode of MS / MS analysis in the analysis step, ii) in the first MS analysis, full MS scan data for all compounds is obtained without limiting the molecular weight range, and the subsequent MS In the / MS analysis, it is also possible to adopt a mode in which the MS / MS analysis is performed by narrowing down to a signal peak equal to or higher than a certain threshold and / or a signal peak in a higher predetermined range. The quantitative method according to the present invention does not exclude the embodiment described in ii). However, in this embodiment, there is a risk that a compound having a small content in the sample leaks.
Therefore, in the present invention, from the viewpoint of satisfying both completeness and resolution, it is preferable to adopt the aspect i) described in the above paragraph as the first MS analysis.
The first MS analysis in the analysis step is preferably LC / MS analysis because the analysis target is an anthocyanin compound in a liquid sample.

Decomposition process by collision energy In the AIF analysis step of the quantification method according to the present invention, MS / MS analysis is performed by decomposing process by changing the collision energy for each of the peak ions constituting the first MS mass spectrum. The process to perform is included. The MS / MS analysis is comprehensively executed in the above-described aspect i), and is performed in a data-dependent manner in the above-mentioned aspect ii).
As an example of the embodiment, in the case of an apparatus based on the orbitrap method described in the examples, the decomposition process can be performed in a high energy collision divergence cell (HCD collision cell). In this specification, “HCD” is described as an abbreviation for higher energy collisional dissociation.

  In the quantification method according to the present invention, when a natural anthocyanin dye composition composed of an enormous number of similar compounds having different molecular weights and structures is used as a measurement sample, as a decomposition process three or more times in the MS / MS analysis, It is desirable that the energy application and the like in each decomposition process on the measurement sample is performed independently in three or more modes. Here, the term “independently three times or more” refers to a mode in which the target sample is separately decomposed three or more times independently. Here, the number of times of performing the decomposition treatment in the quantification method according to the present invention can be three times or more in order to cover the presence of similar compounds having different energy states required for decomposition in the sample. Specifically, it can be mentioned 3 to 40 times, preferably about 3 to 20 times.

Specifically, as the decomposition treatment of three or more times in the MS / MS analysis, (Condition 1) is performed at least once with a low energy collision energy containing an undecomposed peak of the anthocyanin compound. Is preferred. The collision energy of (Condition 1) is an energy condition for recovering the target fragment ions derived from the low molecular weight anthocyanin compound without excessive decomposition.
Here, the collision energy is preferably a collision energy which includes an undecomposed peak of the anthocyanin compound and also includes a peak of the fragment ion having the common structure.

In addition, as the decomposition treatment of three times or more in the MS / MS analysis, (Condition 2) At least another time is the peak of the fragment ion of the common structure substantially not including the undecomposed peak of the anthocyanin compound It is preferable to use a high energy collision energy containing The collision energy of (Condition 2) is an energy condition for collecting sufficient fragment ions derived from the polymer compound by giving sufficient energy to the decomposition of the polymer anthocyanin compound.
Here, “substantially free of the undecomposed peak of the anthocyanin compound” described in the (Condition 2) indicates a state in which the anthocyanin compound as a measurement sample is sufficiently decomposed. This shows a sufficient condition that the target fragment ions are decomposed and produced to the extent that the quantitative property of the above is ensured. Specifically, it can be determined that the condition is satisfied if the state is 10% or less, preferably 5% or less, more preferably 1% or less of the amount of an anthocyanin compound in an undegraded state before imparting collision energy. . Whether or not an anthocyanin compound has an undecomposed peak under the conditions can be determined using the anthocyanin compound having the highest molecular weight as an index among the anthocyanin compounds contained in the anthocyanin dye composition.
In addition, the “including fragment ion peak” described in (Condition 2) is preferably 1% or more, preferably 5% or more, more preferably 10%, relative to the highest peak area of fragment ions. If it is the above peak area, it can be judged that the said conditions are satisfy | filled clearly. When the peak area is too small, the target fragment ions may be excessively decomposed excessively, which is not preferable.

In addition, as the decomposition process of three times or more in the MS / MS analysis, it is preferable that (Condition 3) at least one more time is performed with energy between the two times of collision energy. The collision energy of the decomposition process described in (Condition 3) is preferably in an energy state that is different from the collision energy in the other decomposition processes. Further, when the total number of decomposition processes in the MS / MS analysis is four times or more, it is preferable that the decomposition process in the collision energy of (Condition 3) is performed a plurality of times.
Preferably, as the collision energy, in order to reliably recover the target fragment ions derived from the medium molecular weight anthocyanin compound, the energy value intervals between the decomposition treatments in (Conditions 1 to 3) are equal or substantially equal. It is preferred to employ a uniform collision energy. Here, “substantially equal in the conditions” means that the error is within 10%, preferably within 5%, more preferably within 1%, and the target fragment ions are sufficient to ensure the quantitative property of the present invention. The range that is decomposed and generated.

  As the decomposition process in the MS / MS analysis, it is preferable that the collision energies in all the decomposition processes are in different energy states, but the overlapping decomposition process is performed in the collision energy in the same energy state. The manner of performing is not excluded. Moreover, there is no restriction | limiting in particular about the order of the height of the energy state of the collision energy in multiple decomposition | disassembly processes, and it is also possible to perform a decomposition | disassembly process in what order.

In the method for quantifying anthocyanin dyes according to the present invention, an aglycone is a common structure for an anthocyanin dye composition, which is a collection of similar compounds, by performing a decomposition treatment in MS / MS analysis using such a collision energy. Quantification of the compound possessed as can be performed.
In particular, in the quantification method according to the present invention, in order to reliably produce aglycone from anthocyanin compounds of similar molecular weights contained in natural anthocyanin dye compositions, a wide and appropriate range of collision energy is set. It is preferable to set to. Here, when the setting range of the collision energy in the AIF analysis is not appropriate, it is not preferable because the anthocyanin compound in the measurement sample cannot be accurately quantified.

  In the method for quantifying anthocyanin dyes according to the present invention, when the setting range of the collision energy related to a predetermined sample is examined with a predetermined apparatus, the quantification related to the same type of sample is performed after that. By adopting the determined setting range of collision energy, it becomes possible to perform a quantitative operation quickly.

Accumulation of Compound Ions after Decomposition In the AIF analysis step, after performing the above decomposing process with varying collision energy three or more times, the mass in MS / MS analysis including integrating the fragmented compound ions is performed. Including a step of acquiring a spectrum.

Here, as a mode of integrating the compound ions after the decomposition treatment, it is preferable to perform i) a mode in which the compound ions after the decomposition treatment in the collision energy are electrically supplemented and integrated and detected. Specifically, in this aspect, during the sequential execution of three or more decomposition processes with varying collision energy, various fragmented compound ions (including undecomposed compound ions depending on the energy state) are constant. It is preferable to adopt a mode in which a mixture state is obtained that is electrically supplemented and accumulated in the spatial range of and is accumulated and detected after completion of the number of times of all decomposition processes. .
To further exemplify this aspect together with the decomposition treatment with collision energy, (first time) The compound ion obtained by performing the decomposition treatment of the compound in the sample with the first collision energy of the intensity, (Including undecomposed compound ions and fragmented fragment ions) are electrically supplemented and accumulated in another space (second time), and then at a collision energy with a strength different from that of the first time. Similarly, the compound in the sample is decomposed, and the compound ions obtained by the treatment are electrically supplemented and accumulated together with the compound ions in the previous stage in the space. ) Further, the compound in the sample is decomposed in the same manner with a collision energy having a strength different from that in the previous stage, and the compound ions obtained by the process are converted into the compound ions up to the previous stage in the space. And electrically to supplement accumulated together, it may be mentioned a mode to detect this at a time after the end of the number of times for all of the decomposition process.

The electrical supplementary state of the compound ions in this process is different from the site where the decomposition process is performed in the collision energy in the mass spectrometer based on the electric field type Fourier transform method, the magnetic field type Fourier transform method, the ion trap method, etc. It can be realized in a space where electrodes are arranged.
Further, as an aspect of “electrically supplementing and adding and accumulating in a fixed space” in the AIF analysis step, an electrode arrangement space capable of supplementary holding by rotating ions in an electric field around the electrode is provided. Separately, a collision energy decomposition reaction space is arranged, and compound ions treated in the collision energy decomposition reaction space are sequentially introduced into the electrode arrangement space, and the compound ions are generated in the electric field around the electrode. A mode in which are sequentially added and accumulated in an additive manner can be preferably mentioned. The preferred embodiment is particularly suitable for precise quantification, and can be realized in a mass spectrometer based on the principle of electric field type Fourier transform.
As an embodiment described in the examples, there can be mentioned an aspect in which an electric field generated around the spindle electrode of the orbitrap MS2 in an apparatus using the principle of the orbitrap method is used.

  In addition, as a step of obtaining a mass spectrum in MS / MS analysis including the integrated fragmented compound ions in the AIF analysis step, as an aspect of integrating the compound ions after the decomposition treatment, In addition to the mode of i), a mode is adopted in which the compound ion after the decomposition treatment is detected first, and this is repeated by the amount set for the collision energy, and then the data processing for integrating the detected values is performed afterwards. Is also possible. The embodiment of ii) is not excluded from the scope of the present invention. However, in the present invention, the embodiment of i) described in the above paragraph is adopted from the viewpoints of analysis accuracy and simplicity of analysis operation. Is preferred.

  In the AIF analysis step, as a value indicating the integrated compound ion, a value obtained by performing various arithmetic processes can be used. For example, a value obtained by dividing by the number of times the decomposition process is performed can be used for the determination.

Target fragment ions The mass spectrum obtained by MS / MS analysis in the AIF analysis step according to the present invention constituted undecomposed anthocyanin compound ions, intermediate product ions, and anthocyanins according to the progress of decomposition. Various compound ions such as common structure ions and excessively decomposed low-molecular compound ions are included as integrated peak values at various stages of the degree of decomposition.
In the present invention, among these, an anthocyanin having the common structure contained in the measurement sample is used by using the integrated peak of compound ions constituting the common structure of the molecular species of the anthocyanin compound in the measurement sample as an index. The total amount of the compound can be quantified.
That is, the AIF analysis step according to the present invention includes a step of quantifying the anthocyanin compound from the mass spectrum obtained by the MS / MS analysis, using the common structure contained in the anthocyanin compound as a target fragment ion as an index.

In detail, in this process, based on the accurate quantification of the anthocyanin dye, it is a common structure possessed by the molecular species of the anthocyanin compound in the measurement sample and is not a constituent structure of the compound in the measurement sample other than the anthocyanin compound. It is preferred to employ compound ions.
As the compound produced after the decomposition treatment satisfying such conditions, it is preferable to employ an aglycone of an anthocyanin compound contained in the measurement sample or a similar compound derived therefrom. That is, as the target fragment ion according to the present invention, it is preferable that an aglycone ion of the anthocyanin compound or a similar compound ion derived from the aglycone is a detection target. Here, examples of ions of similar compounds derived from aglycone include functional group-substituted compounds generated by changes in metabolism in a plant body, storage and processing of a measurement sample, and the like.

The aglycone decomposed and produced from the anthocyanin compound which is the target fragment ion according to the present invention specifically refers to anthocyanidin. The general structural formula of anthocyanidins is shown below (Formula 1). _R 1 to R ₇ in the following (formula 1) indicates either a hydrogen atom (-H), a hydroxyl group (-OH), a or methoxyl group (-OCH _3).

Anthocyanidins consist of three ring structures, A ring, B ring, and C ring, depending on the number of hydroxyl groups (—OH) and methoxyl groups (—OCH ₃ ) added to the B ring (the benzene ring on the right side of the formula (1)). Pelargonidin, cyanidin, delphinidin, peonidin, petunidin and malvidin are mainly classified. Other examples include aurantinidine, luteolinidine, europeinidine, and rosinidine. When these become constituent aglycones of anthocyanin compounds, their color tones vary.
The target fragment ion according to the present invention is not limited to these enumerated anthocyanidins, and is an aglycon of an anthocyanin compound in a measurement sample and a structural structure of a compound in the measurement sample other than the anthocyanin compound If it is a compound ion that is not, anthocyanidins other than those described above can be used as target fragment ions.

2. Specific Quantitative Technique In the present invention, the anthocyanin dye can be accurately quantified by utilizing the principle and knowledge described in the above paragraph.

[Concentration conversion]
In the quantification method according to the present invention, in order to calculate the actual concentration value from the detection data of the target fragment ion which is the common structure, one or more compounds containing the target fragment structure are prepared and diluted. A concentration value can be calculated from detection data of a measurement sample only by creating a calibration curve by creating a series.
For example, in an embodiment in which aglycone is used as a target fragment, a calibration curve is created by analyzing a known compound containing the aglycone under the same conditions as the AIF method according to the present invention (such as setting of collision energy). Thus, the concentration value and content value of the target anthocyanin compound in the measurement sample can be calculated from the detection data and molecular weight information of the measurement sample.

  Here, as a calibration curve for performing concentration conversion, it is not necessary to prepare a calibration curve by preparing individual components in a measurement sample as standard compounds.For example, if an aglycon is adopted as a target fragment, Any known low molecular anthocyanin compound containing the aglycon, chloride of the aglycon, and the like can be used as long as they are compounds capable of generating a common structural compound that is a target fragment.

[Various aspects]
Quantitative determination of all compounds containing a common structure Anthocyanin compounds have a huge variety of molecular species with different molecular weights and structures, and ordinary natural anthocyanin dyes exist as aggregate compositions of these similar compounds. Since the quantification method according to the above is a quantification method using the compound ion having a common structure utilizing the AIF analysis as a target fragment ion, quantification of all anthocyanin compounds including the common structure is possible (FIGS. 1 to 4). reference).
Therefore, in the quantification method according to the present invention, the total amount of anthocyanin compounds containing the aglycone as a “common structure” is quantified by detecting the aglycone constituting the anthocyanin compound contained in the measurement sample as a target fragment ion in AIF analysis. It becomes possible to do.
As a specific measurement example of this aspect, for example, when red radish dye is used as a measurement sample and pelargonidin ion is detected as a target fragment ion, the total amount of pelargonidin anthocyanins contained in red radish dye can be quantified. Become.

Quantification of total amount of all anthocyanin compounds In the quantification method according to the present invention, it is possible to quantify the “total amount of all anthocyanin compounds” contained in the measurement sample using the AIF analysis. Specifically, as the aspect, it is possible to quantify the total amount of all anthocyanin compounds by performing the quantification according to the present invention using all types of aglycones constituting the anthocyanin compound contained in the measurement sample as target fragment ions. Become.
As a specific measurement example of the embodiment, for example, when the total amount of all anthocyanins is quantified using a red radish pigment (containing only a pelargonidin-based anthocyanin compound as an anthocyanin compound) as a measurement sample, the above AIF using the pelargonidin ion as a target fragment ion The total amount of pelargonidin-based anthocyanins can be quantified by analysis, and this value can be used as the total amount of all anthocyanin compounds contained in the red radish pigment.
Moreover, as another aspect, it can be set as a measurement sample suitably also with respect to the anthocyanin pigment | dye composition containing the anthocyanin compound of 2 or more types from which the kind of aglycone differs. As a measurement example of this aspect, for example, when measuring the total amount of all anthocyanins using a purple potato dye (including a peonidin-based anthocyanin compound and a cyanidin-based anthocyanin compound as an anthocyanin compound) as a measurement sample, the peonidin ion and the cyanidin ion are the target fragment ions. As described above, the total amount of peonidin-based anthocyanins and the total amount of cyanidin-based anthocyanins can be quantified by the above AIF analysis, and the sum of these can be used as the total amount of all anthocyanin compounds contained in the purple potato pigment.

Quantification of individual components In the quantification method according to the present invention, it is possible to quantify the “individual component amount of each anthocyanin compound” contained in the measurement sample using the AIF analysis. Specifically, as the mode, the aglycone constituting the desired anthocyanin compound contained in the measurement sample is quantified by the AIF method according to the present invention using the target fragment ion, and the signal content derived from the desired individual component is measured. Thus, the individual content of the desired anthocyanin compound in the measurement sample can be quantified.
Here, when calculating the actual concentration value from the measurement data of the individual component, since the calibration curve using the above-mentioned common structure can be used uniformly, the individual component to be measured is prepared as a standard compound. Thus, it is not necessary to perform an operation for creating a calibration curve for each standard compound.

  In the quantification method according to the present invention, it is possible to determine the structure of the individual components of the desired anthocyanin compound contained in the measurement sample at the same time as quantification based on the common structure described above. In one embodiment of the quantification method according to the present invention, normal MS scan data is also obtained before the AIF analysis, and the mass spectrum of the MS scan data is fragmented by MS / MS analysis. The structure of the desired anthocyanin compound can be determined from the mass spectrum and the like by the function normally provided in the apparatus described above. In the structural analysis, it may be effective to perform multi-stage analysis by (MS) n-stage analysis as necessary.

[Main advantages of the quantitative method according to the present invention]
The quantification method according to the present invention is a method characterized by analyzing fragment ions having a common structure decomposed and generated by MS / MS analysis so as to have quantification. The natural anthocyanin pigment, which is an aggregate composition of similar compounds, can be accurately quantified.
In the quantification method according to the present invention, it is possible to simultaneously execute the total amount of anthocyanin compounds having a desired aglycone, the total amount of all anthocyanin compounds, the individual quantification of the desired anthocyanin compounds, and the like. It is also possible to perform these separately.
In the quantification method according to the present invention, it is not necessary to prepare individual components in a measurement sample as standard compounds, and if an aglycone is adopted as a target fragment, a calibration curve is created using a known compound containing the aglycone. It is possible to quantify the total amount and individual component amount as described above.
In the quantification method according to the present invention, because of the measurement principle, it is a technique that does not require pretreatment of the measurement sample by chemicals, enzyme treatment, etc., so that sample preparation and quantification operations can be performed easily and quickly. It becomes.

Here, differences from the conventional anthocyanin compound quantification method compared with the advantages of the quantification method according to the present invention will be described.
As a quantification widely used as a conventional technique, i) an absorbance method is used in which a standard curve of a representative anthocyanin compound is used to create a calibration curve based on absorbance, and is calculated as a total anthocyanin amount converted to the dye compound as the standard sample. Are known. This method is a method widely used in the field as a simple method. However, this technique is difficult to apply to a standard sample having a different absorbance from the dye compound, and the natural anthocyanin dye, which is an aggregate composition of similar compounds having different color tones, cannot be accurately determined.
Another conventional technology is ii) acid hydrolysis of the anthocyanin compound in the measurement sample to release aglycone, extract the aglycone in the organic solvent layer, and determine the anthocyanin content by LC / MS etc. How to do it has been reported. However, this method has a large variation in the amount of aglycone extracted obtained after the acid hydrolysis reaction and organic solvent extraction, and it is in principle difficult to perform accurate quantification. Furthermore, in this method, since it is essential to perform acid hydrolysis and organic solvent extraction as pretreatment of the measurement sample, the quantitative operation cannot be performed easily and quickly in terms of sample preparation and handling.
As another conventional technique, iii) a method for quantifying individual components contained in an anthocyanin dye includes a method for quantifying individual components by HPLC, LC / MS, or the like. However, this method only allows the quantification of anthocyanin dyes, which are aggregates of various similar compounds with different molecular weights and chemical properties, etc. It cannot be done accurately.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, the scope of the present invention is not limited by these.

[Example 1] “Quantification of anthocyanin compound as a single component”
Using an AIF analysis of LC / MS / MS based on the electric field type Fourier transform principle, it was examined whether the quantification was possible using the fragment ion of the aglycone constituting the anthocyanin dye as an index.

(1) “Measurement sample”
A measurement sample having a color value of 0.01 was prepared by diluting peonidin-3-glucoside (Pn-3-glc), which is an anthocyanin compound contained in the purple sweet potato pigment, with water. As the anthocyanin compound, a purified compound manufactured by Wako Pure Chemical Industries, Ltd. was used.

(2) “Examination of collision energy in MS / MS”
The measurement sample was subjected to LC / MS / MS analysis using an orbitrap mass spectrometer, and fragment ions showing aglycone formation when MS / MS collision energy was changed in the analysis were detected. Here, peonidin (m / z: 301.0698) was used as a fragment ion of detection ions. The orbitrap mass spectrometer and conditions used for the analysis are shown below. The solvent used was LC / MS grade. The value indicating the size of the collision energy was expressed as an HCD (higher energy collisional dissociation) value. The results of measuring the obtained ion peak area are shown in the following table and FIG.

[Orbitrap LC / MS / MS conditions]
Equipment: UPLC Ultimate 3000 (Thermo Fisher Scientific Inc.)
Q-Exactive HPLC system (Thermo Fisher Scientific Inc.)
Column: L-column ODS (column inner diameter φ2.1mm x column length 150mm, particle diameter 3μm)
Mobile phase: (A) 0.1% formic acid aqueous solution → (B) 0.1% formic acid-containing acetonitrile Gradient condition (B content): 5% (0 minutes) → 18% (15 minutes)
→ 70% (40-42 minutes)
Flow rate: 0.2 mL / min Column temperature: 40 ° C

Ionization method: ESI positive
Spray voltage: 3kV
Capillary temperature: 300 ° C
Quadrupole MS scan width: m / z 400-1500
Orbitrap MS2 scan width: m / z 150-1500
HCD conditions: 10-50
Detection ion: Peonidin (m / z: 301.0698)

  As a result, when the collision energy in MS / MS in the Orbitrap mass spectrometer is set to HCD10, the fragment ion peonidin is detected, but the parent ion peonidin-3-glucoside before decomposition also remains. Were detected at the same time. Here, when the collision energy was set to HCD20, it was shown that peonidin, which is a fragment ion, is sufficiently detected while peonidin-3-glucoside, which is a parent ion, disappears. Moreover, when collision energy was set to HCD40, it was shown that the fragment ion peonidin is decomposed | disassembled and further fragmentation advances.

  From the results, the conditions covering the collision energy in which fragment ions (m / z: 301.0698) are sufficiently detected when peonidin-3-glucoside (m / z: 463.1226) is subjected to analysis are as follows: It was judged to be in the range of HCD 10-30.

(3) “Measurement of integrated value of fragment ion peak area by AIF method”
Based on the above, an ionized product is detected simultaneously at three measurement points (Stepped Collision) using an Orbitrap mass spectrometer, and the amount of aglycone in the sample is determined by the integrated peak area of fragment ions indicating the resulting aglycone. We examined whether quantification was possible.
A measurement sample is prepared by diluting peonidin chloride (manufactured by EXTRASYNTHESE) with water, and the integrated value of the area of the peonidin ion peak (m / z: 301.0698) at three measurement points of HCD10, HCD20, and HCD30 is obtained. It was measured. The integrated value analysis was performed by simultaneously detecting ionized products at three measurement points using the AIF (All Ion Fragmentation) mode of the orbitrap mass spectrometer described in (2) above.

As a result, the integrated value of the detected peonidin ion peak area showed a correlation with the added amount of added peonidin chloride, and it was shown that a calibration curve capable of converting the concentration could be created. Moreover, since the contribution ratio (R ² ) of the correlation was a value very close to 1, it was shown that precise quantification with high accuracy is possible.

(4) “Summary”
From the above results, by measuring the integrated value of the peak areas of fragment ions obtained by performing simultaneous detection of ionization products at three measurement points using the AIF analysis according to the present invention, anthocyanins using aglycone as an index are measured. It was shown that the compound could be quantified. The quantitative accuracy was shown to be extremely high.

[Example 2] “Examination with anthocyanin compounds having other aglycones”
Regarding the quantification method using the AIF method according to the above example, the applicability to an anthocyanin compound containing cyanidin different from peonidin as an aglycone was examined.

(1) “Measurement sample”
A measurement sample having a color value of 0.01 was prepared by diluting cyanidin-3-glucoside (Cy-3-glc), which is an anthocyanin compound contained in the purple sweet potato pigment, with water. As the anthocyanin compound, a purified compound manufactured by Wako Pure Chemical Industries, Ltd. was used.

(2) “Examination of collision energy in MS / MS”
The measurement sample was subjected to LC / MS / MS analysis using an orbitrap mass spectrometer, and fragment ions showing aglycone formation when MS / MS collision energy was changed in the analysis were detected. The orbitrap mass spectrometer and conditions used for the analysis were the same as described in Example 1 except that cyanidin (m / z: 287.0537) was targeted as the fragment ion to be detected. It was. The results are shown in the following table and FIG.

      Detection ion: Cyanidin (m / z: 287.0537)

  As a result, a result almost similar to the detection result with peonidin-3-glucoside in Example 1 was obtained, and when the cyanidin-3-glucoside (m / z: 449.1065) was subjected to analysis, a fragment ion (m / Z: 287.0537) was determined to be in the range of HCD 10-30 to cover the collision energy sufficiently detected.

(3) “Measurement of integrated value of fragment ion peak area by AIF method”
Based on the above, an ionized product is detected simultaneously at three measurement points (Stepped Collision) using an Orbitrap mass spectrometer, and the amount of aglycone in the sample is determined by the integrated peak area of fragment ions indicating the resulting aglycone. We examined whether quantification was possible.
A measurement sample in which cyanidin chloride (manufactured by EXTRASYNTHESE) was diluted with water was prepared, and the integrated value of the area of cyanidin ion peak (m / z: 287.0537) at three measurement points of HCD10, HCD20, and HCD30 was measured. . The integrated value analysis was performed by simultaneously detecting ionized products at three measurement points using the AIF (All Ion Fragmentation) mode of the orbitrap mass spectrometer described in (2) above.

As a result, the integrated value of the detected cyanidin ion peak area showed a correlation with the added amount of added cyanidin chloride, and it was shown that a calibration curve capable of converting the concentration could be created. Moreover, since the contribution ratio (R ² ) of the correlation was a value very close to 1, it was shown that precise quantification with high accuracy is possible.

(4) “Summary”
From the above results, it was shown that the quantification method using the AIF method according to the present invention can quantitate anthocyanin compounds composed of aglycones other than peonidin.

[Example 3] "Study on natural anthocyanin dye composition"
The applicability to a natural anthocyanin dye composition, which is a collection of anthocyanin compounds of various molecular weights, was examined for a quantitative method using AIF analysis according to the above-described Examples.

(1) “Measurement sample”
A red radish pigment preparation (manufactured by Saneigen FFI Co., Ltd.) was diluted with water to prepare a measurement sample having a color value of 0.01. The red radish pigment is an aggregate composition comprising a large number of compounds classified as pelargonidin-based anthocyanins.

(2) “Examination of collision energy in MS / MS”
The measurement sample was subjected to LC / MS / MS analysis using an orbitrap mass spectrometer, and fragment ions showing aglycone formation when MS / MS collision energy was changed in the analysis were detected. The orbitrap mass spectrometer and conditions used for the analysis are the same as those described in Example 1 except that the following conditions were set for pelargonidin (m / z: 271.0599) as a fragment ion to be detected. The same was done. The results are shown in the following table and FIG. A part of the results of MS / MS mass spectrum is shown in FIG.

Gradient condition (B content): 10% (0 minutes) → 20% (40 to 44 minutes)
→ 90% (48-50 minutes)
HCD conditions: 10-100
Detection ion: pelargonidin (m / z: 271.0599)

As a result, when the collision energy in MS / MS in the orbitrap mass spectrometer is set to HCD10, the pelargonidin that is a fragment ion is not detected, and pelargonidin-3-one that is one of the macromolecular anthocyanin compounds before decomposition is detected. It was shown that a large amount of caffeoyl, feruloyl sophoroside-5-malonyl glucoside (m / z: 1195.3102) was detected (FIG. 7A, FIG. 8).
Here, when collision energy is set to HCD40-50, pelargonidin-3-caffeoyl and feruloyl sophoroside-5-malonyl glucoside disappear, and pelargonidin which is a fragment ion is sufficiently detected. (FIG. 7B, FIG. 8). Moreover, when collision energy was set to HCD90-100, it was shown that the pelargonidin which is a fragment ion is also decomposed | disassembled and decreased, and further fragmentation advances (FIG. 7C, FIG. 8).

From this result, when the red radish dye, which is an aggregate composition of similar compounds, is subjected to analysis, the polymer anthocyanin compound is decomposed to sufficiently detect fragment ions (m / z: 271.0599). It was determined that the collision energy conditions for covering HCD were in the range of HCD10-80.
Here, the reason why the HCD range covering the detection of fragment ions in this test is wide is considered to be because pelargonidin-based anthocyanins having various molecular structures having different molecular weights are contained in the measurement sample.
Specifically, it is recognized that pelargonidin produced by decomposition of a low molecular weight compound is detected among the pelargonidin-based anthocyanin dyes constituting the red radish dye composition under the low energy conditions in the vicinity of HCD 10 to 40 in this test. . Moreover, it is recognized that pelargonidin produced by decomposition of a polymer compound among pelargonidin-based anthocyanin dyes is detected under a high energy condition around HCD 50 to 80.

  Based on the above, it was recognized that by setting the collision energy in the AIF analysis in the range of HCD 10 to 80, pelargonidin, which is an aglycone generated by the decomposition of the anthocyanin compound in the red radish pigment, can be comprehensively detected.

(3) “Measurement of integrated value of fragment ion peak area by AIF method”
Based on the above, an ionized product is detected simultaneously at three measurement points (Stepped Collision) using an Orbitrap mass spectrometer, and the amount of aglycone in the sample is determined by the integrated peak area of fragment ions indicating the resulting aglycone. We examined whether quantification was possible.
A measurement sample in which pelargonidin chloride (manufactured by EXTRASYNTHESE) was diluted with water was prepared, and the integrated value of the area of the pelargonidin ion peak (m / z: 271.0606) at three measurement points with HCD10, HCD45, and HCD80 Was measured. The integrated value analysis was performed by simultaneously detecting ionized products at three measurement points using the AIF (All Ion Fragmentation) mode of the orbitrap mass spectrometer described in (2) above. The results are shown in the following table and FIG.

As a result, the integrated value of the detected pelargonidin ion peak area shows a correlation with the added amount of added pelargonidin chloride, and a calibration curve (y = 9.0 × 10 ⁷ X-8) capable of converting the concentration. 0.0 × 10 ⁷ ) can be created. Further, the correlation contribution ratio (R ² ) was 0.9996, which was a value very close to 1, indicating that precise quantification with high accuracy is possible.

(4) “Summary”
From the above results, the quantification method using the AIF method according to the present invention is a compound having an aglycone as a common structure with respect to a natural anthocyanin dye composition that is a collection of similar compounds (that is, pelargonidin-based anthocyanins in this example). It was shown that this technique is capable of quantitative determination. It was also shown that it is effective to set a wide range of collision energy in AIF analysis in order to reliably produce aglycone from anthocyanin compounds of various molecular weights contained in natural anthocyanin dye compositions. .

[Example 4] “Examination of color value of measurement sample”
In carrying out the quantification methods of the above-mentioned Examples, the color values of anthocyanin dyes when subjected to AIF analysis were examined.

(1) "Color value examination"
A red radish pigment preparation (manufactured by San-Ei Gen FFI Co., Ltd.) is diluted with water to prepare measurement samples having the color values described in the following table, and orbitrap mass spectrometry is performed in the same manner as described in Example 3. The integrated value of the area of the pelargonidin ion peak (m / z: 271.0606) at three measurement points with HCD10, HCD45, and HCD80 was measured by the AIF method using an apparatus. The results are shown in FIG.
As a result, in the analysis using the apparatus described in Example 1, in the sample prepared so that the color value was 1 or less, preferably 0.5 or less, It was shown that the peak area can be measured. In addition to the test, a low-concentration sample having a color value of 0.01 to 0.1 was used for analysis. However, even when such a thin sample was used for the test, it was dependent on the concentration. It was possible to measure the peak area of fragment ions.

(2) “Summary”
As described above, in the analysis using the orbitrap mass spectrometer described in Example 1, it is preferable to adjust the color value of the measurement sample to 1 or less, preferably to 0.5 or less. It was shown that.
Here, in the AIF analysis using the apparatus described in Example 1, it is preferable that the total amount of ions contained in the sample is smaller in order to secure as much scan point data as possible in the apparatus. Then, it was shown that stable quantification is possible even when an extremely thin sample having a color value of about 0.01 to 0.1 is used for analysis.

[Example 5] "Quantification of red radish pigment"
Using the knowledge and technique obtained in the above examples, the total amount of anthocyanin dye contained in the red radish dye, which is an assembly of anthocyanin compounds having various molecular weights, was quantified.

(1) “Quantification of total amount of anthocyanins”
A red radish pigment preparation (manufactured by San-Ei Gen FFI Co., Ltd.) is diluted with water to prepare a measurement sample having a color value of 0.01, and an orbitrap mass spectrometer is prepared in the same manner as described in Example 3. The integrated value of the area of the pelargonidin ion peak (m / z: 271.0606) in HCD10, HCD45, and HCD80 was measured by the AIF method used. About the integrated value of the obtained peak area, the pelargonidin content was calculated using the calibration curve described in Example 3, and the total anthocyanin content was calculated from the average molecular weight of the pelargonidin-based anthocyanins. The results are shown in the table below.

As a result, it was shown that the total amount of pelargonidin-based anthocyanins determined by the AIF method according to the present invention was 13.8% (w / w) in all compounds contained in the red radish pigment. Here, since the anthocyanin pigment constituting the red radish pigment used in this test is composed of pelargonidin-based anthocyanins, the total amount of pelargonidin-based anthocyanins can be regarded as the “total amount of all anthocyanins”. .
On the other hand, with respect to the measurement sample, the result of measuring the total amount of all anthocyanins by the absorbance of pelargonidin-3-5-diglucoside, which is a conventional absorbance method, is 12.7% (w / w). It was.
Here, in the AIF method according to the present invention, it has been confirmed that precise quantification is possible with the calibration curve of Example 3 above, whereas in the conventional absorbance method, natural anthocyanins have different colors. It is an approximate value calculated only by the absorbance of pelargonidin-3-5-diglucoside, which is a single compound, without taking into account the fact that it is an aggregate composition. In other words, the conventional absorbance method was recognized as a measurement method including an error by a numerical difference from the analysis method according to the present invention.

(2) “Summary”
From the above results, in the quantification method using the AIF method according to the present invention, the total amount of compounds having an aglycone as a common structure (that is, pelargonidin-based anthocyanins in this example) in the natural anthocyanin dye composition which is a collection of similar compounds. It was shown that it can calculate correctly. Thus, in the present invention, it was shown that the total amount of all anthocyanins contained in the natural anthocyanin pigment composition can be accurately quantified.

[Example 6] "Simultaneous determination of individual components contained in natural anthocyanin pigment composition"
With respect to natural anthocyanin dyes, which are a collection of similar compounds, it was examined whether the anthocyanin compounds as the respective constituent components can be individually quantified using the AIF method.

(1) “Quantification and structure estimation of individual components of anthocyanins”
A red radish pigment preparation (manufactured by San-Ei Gen FFI Co., Ltd.) is diluted with water to prepare a measurement sample having a color value of 0.01, and an orbitrap mass spectrometer is prepared in the same manner as described in Example 3. LC / MS / MS analysis used was performed. Compound A (m / z: 919.2487), compound B (m / z: 1019.2624), compound having different molecular weights from the total ion chromatogram (FIG. 11) obtained by the LC / MS analysis obtained in this analysis Focusing on C (m / z: 1195.3102), the integrated value of the area of the pelargonidin ion peak (m / z: 271.0606) on HCD10, HCD45, and HCD80 by AIF method for these compounds It was measured. About the integrated value of the obtained fragment ion peak area derived from each compound A to C, the pelargonidin content is calculated using the calibration curve described in Example 3, and the individual content of each compound is calculated from the molecular weight of each individual component. did. The orbitrap mass spectrometer and conditions used for the analysis were the same as the method described in Example 3.
The structures of the compounds A to C were estimated by a conventional method using the accurate mass obtained by the orbitrap mass spectrometer and the fragment profiling data by MS / MS. The quantification and identification results are shown in the table below. The estimated structural formula is shown in FIG.

  As a result, it was shown that Compound A was pelargonidin-3-caffeoylsophoroside-5-glucoside and contained 0.20% (w / w) in the red radish pigment as a measurement sample. Compound B is pelargonidin-3-feruloylsophoroside-5-malonylglucoside, and it was shown that 0.88% (w / w) was contained in the red radish pigment as a measurement sample. Compound C was pelargonidin-3-caffeoyl, feruloylsophoroside-5-malonylglucoside, and was found to be contained in the red radish pigment as a measurement sample in an amount of 0.26% (w / w). .

(2) “Summary”
From the above results, by using the quantification method by the AIF method according to the present invention, it is possible to perform “individual quantification” of the anthocyanin compound as a composition component with respect to the natural anthocyanin pigment as a collection of similar compounds. Indicated. Further, it was shown that the quantification method enables accurate quantification without using a standard sample of the individual component that is a measurement target.
Furthermore, it has been shown that the method can simultaneously estimate the structure of an unidentified anthocyanin compound.

[Example 7] “Examination with a dye composition containing two types of anthocyanin compounds having different types of aglycones”
The applicability of the quantification method using AIF analysis according to the above example to a dye composition containing two types of anthocyanin compounds, peonidin-type anthocyanin and cyanidin-type anthocyanin, as an aglycon was examined.

(1) “Measurement sample”
A purple potato pigment preparation (manufactured by Saneigen FFI Co., Ltd.) was diluted with water to prepare a measurement sample having a color value of 0.01. The purple potato dye is a dye composition comprising a large number of compounds classified into peonidin-based anthocyanins and a large number of anthocyanin compounds classified into cyanidin-based anthocyanins.

(2) “Examination of collision energy in MS / MS”
The measurement sample was subjected to LC / MS / MS analysis using an orbitrap mass spectrometer, and fragment ions showing aglycone formation when MS / MS collision energy was changed in the analysis were detected. The orbitrap mass spectrometer and conditions used for the analysis were as follows for peonidin (m / z: 301.0698) or cyanidin (m / z: 287.0537) as fragment ions to be detected. Except this, the procedure was the same as that described in Example 1. The results are shown in the table below and FIG.

HCD conditions: 10-100
Detection ion: Peonidin (m / z: 301.0698)
Cyanidin (m / z: 287.0537)

As a result of evaluating the measurement results in the same manner as the analysis with the red radish dye described in Example 3, when the purple potato dye was subjected to the analysis, peonidin (m / z: 301.0698) and cyanidin (m / Z: 287.0537) was determined to be in the range of HCD 20-60, covering the sufficiently detected collision energy.
In other words, by setting the collision energy in the AIF analysis within the range of HCD 20 to 60, it is possible to comprehensively detect peonidin and cyanidin, which are aglycones generated by decomposition of anthocyanin compounds in purple potato pigments, including high molecular anthocyanin compounds. It was recognized that it would be possible.

(3) “Measurement of integrated value of fragment ion peak area by AIF method”
Based on the above, an ionized product is detected simultaneously at three measurement points (Stepped Collision) using an Orbitrap mass spectrometer, and the amount of aglycone in the sample is determined by the integrated peak area of fragment ions indicating the resulting aglycone. We examined whether quantification was possible.

  Samples prepared by diluting each of peonidin chloride (manufactured by EXTRASYNTHESE) or cyanidin chloride (manufactured by EXTRASYNTHESE) with water were prepared, and peonidin ion peaks (target fragment 7-1) at three measurement points of HCD20, HCD40, and HCD60. , M / z: 301.0698) or the integrated value of the area of the cyanidin ion peak (target fragment 7-2, m / z: 287.0537). The integrated value analysis was performed by simultaneously detecting ionized products at three measurement points using the AIF (All Ion Fragmentation) mode of the orbitrap mass spectrometer described in (2) above. The results are shown in the following table, FIG. 14 and FIG.

As a result, the integrated value of the peak area of the detected peonidin ion (fragment 7-1) shows a correlation with the added amount of the added peonidin chloride, and a calibration curve (y = 9.0 ×) capable of converting the concentration. 10 ⁷ X-9.0 × 10 ⁷ ) can be produced. Moreover, since the contribution rate (R ² ) of the correlation was a value close to 1 of 0.9994, it was shown that precise quantification with high accuracy was possible (Table 8, FIG. 14). ).
Similarly, the integrated value of the peak area of the detected cyanidin ion (fragment 7-2) shows a correlation with the added amount of added cyanidin chloride, and a calibration curve (y = 1. 0 × 10 ⁸ X−1.0 × 10 ⁸ ) can be created. Moreover, since the contribution rate (R ² ) of the correlation was an extremely close value of 0.9990, it was shown that precise quantification with high accuracy was possible (Table 9, FIG. 15). ).

(4) “Summary”
From the above results, the quantification method using the AIF method according to the present invention is a compound quantification in which each aglycone has a common structure with respect to a natural anthocyanin dye composition containing two or more anthocyanin compounds having different types of aglycones. Has been shown to be a possible technology. In addition, in order to reliably produce aglycone from anthocyanin compounds of various molecular weights contained in the natural anthocyanin dye composition, it has been shown that it is effective to set a wide range of collision energy in the AIF analysis. It was.

[Example 8] "Quantification of purple potato pigment that is a natural anthocyanin pigment composition"
Using the knowledge and technique obtained in the above Examples, the total amount of anthocyanin compounds contained in the dye composition containing two systems of anthocyanin compounds was quantified.

(1) “Quantification of total amount of anthocyanins”
A purple potato pigment preparation (manufactured by San-Ei Gen FFI Co., Ltd.) is diluted with water to prepare a measurement sample having a color value of 0.5. The integrated value of the area of the peonidin ion peak (m / z: 301.0698) or cyanidin ion peak (m / z: 287.0537) at HCD20, HCD40, and HCD60 was measured by the AIF method used.
About the integrated value of the obtained peak area, the peonidin content and the cyanidin content were calculated using the calibration curve described in Example 7, and the total anthocyanin content was calculated from the average molecular weight of the peonidin-based anthocyanins and cyanidin-based anthocyanins. The results are shown in the table below.

As a result, it was shown that the total amount of peonidin-based anthocyanins quantified by the AIF method according to the present invention was 15.2% (w / w) in all compounds contained in the purple potato pigment. Moreover, it was shown that the total amount of cyanidin-based anthocyanins was 3.4% (w / w) in all compounds contained in the purple potato pigment.
Here, since the anthocyanin dye constituting the purple-io dye used in this test is composed of peonidin-based anthocyanin and cyanidin-based anthocyanin, the sum of these total amounts shall be the “total amount of all anthocyanins”. Is possible. That is, it was shown that the total amount of anthocyanin compounds contained in the purple potato pigment determined by the AIF method according to the present invention was 18.6 (w / w).

(2) “Summary”
From the above results, in the quantification method using the AIF method according to the present invention, in a natural anthocyanin dye composition containing two systems of anthocyanin compounds having different aglycones, each compound having a common structure for each aglycone (ie, in this example, It was shown that the total amount of peonidin-based anthocyanins and cyanidin-based anthocyanins) can be calculated accurately.
Thereby, in this invention, it was shown that the total amount of all the anthocyanins contained in the natural anthocyanin pigment | dye composition containing the anthocyanin compound of two systems from which an aglycon differs can be quantified correctly.

1. 1. Anthocyanin compound, compound before decomposition, parent ion Intermediate product compound 3. Target fragment ion, aglycone, aglycone structure part Overly decomposed fragment compound

M.M. Compound ion before decomposition, parent ion F. Target fragment ion

5. 5. Sugar molecule structure part 6. Organic acid molecular structure part Sugar molecule structure part

11. Pelargonidin-3-caffeoyl, feruloyl sophoroside-5-malonyl glucoside 31. Pelargonidin

A. Pelargonidin-3-caffeoylsophoroside-5-glucoside B. Pelargonidin-3-feruloylsophoroside-5-malonylglucoside C.I. Pelargonidin-3-caffeoyl, feruloylsophoroside-5-malonylglucoside

Claims (8)

The steps described below,
(Step 1) A measurement sample containing an anthocyanin compound is subjected to a decomposition process with varying collision energy three or more times to obtain a mass spectrum by MS / MS analysis including the integrated fragmented compound ions. And the process of
(Step 2) Quantifying the anthocyanin compound from the mass spectrum obtained in the (Step 1) using the common structure contained in the anthocyanin compound as a target fragment ion as an index,
A method for quantifying an anthocyanin compound, comprising: Three or more decomposition processes in the MS / MS analysis described in (Step 1)
At least once with low energy collision energy containing undegraded peaks of the anthocyanin compound,
At least another time is performed in a high-energy collision energy that does not substantially include the undecomposed peak of the anthocyanin compound and includes the peak of the fragment ion of the common structure,
At least one more time is performed with the energy between the two collision energy,
The method of claim 1. Said (Step 1)
During three or more decomposition processes with varying collision energy, the compound ions after each decomposition process are electrically supplemented and accumulated within a certain spatial range, and the total number of decomposition processes It is a step that is performed by integrating and detecting this at once after the end of the minute,
The method according to claim 1 or 2.   The method according to any one of claims 1 to 3, wherein the MS / MS analysis in (Step 1) is performed on a measurement sample obtained by LC / MS analysis. A method according to any of claims 1 to 4, characterized in that it uses an apparatus as described below:
(Apparatus) MS / MS analysis can be performed with three or more decomposition processes with different collision energy, and compound ions after the decomposition process with collision energy are electrically captured and integrated to detect or detect Mass spectrometer that can be integrated.   The method according to claim 5, wherein the mass spectrometer is a mass spectrometer based on an electric field type Fourier transform method. The common structure as the target fragment ion is
An anion of an anthocyanin compound or an ion of a similar compound derived from the aglycone,
The quantification method of the anthocyanin pigment | dye in any one of Claims 1-6. The quantification method comprises:
An anthocyanin pigment composition comprising a plurality of anthocyanin compounds is a method capable of performing the total amount of anthocyanin compounds having a desired aglycone, the total amount of all anthocyanin compounds, and / or individual quantification of the desired anthocyanin compounds.
The quantification method of the anthocyanin pigment | dye in any one of Claims 1-7.