JP2020186953A - Mass analysis labeling composition, metabolite quantitative analysis metho, and metabolite kinetic analysis method - Google Patents

Abstract

To provide a mass analysis labeling composition, metabolite quantitative analysis method, and metabolite kinetic analysis method that can discriminate and evaluate a plurality of labeling compositions to be used in an analysis even when the plurality of labeling compositions undergo a metabolic conversion in a sample.SOLUTION: A mass analysis labeling composition includes labeling compounds of two or more kinds having a metabolite in vivo labelled, in which the labeling compounds of the two or more kinds are labeling compounds of two or more kinds having phosphate compounds of two or more kinds in a series of routes of a metabolic conversion labelled by a stable isotope of one or more kinds including 18O, and the labeling compounds of the two or more kinds are labelled so as to allow transition of a multiple reaction monitoring to mutually distinguish the labeling compounds of two or more kinds from a labeling compound generating by a metabolic conversion of the labeling compound of the two or more kinds. Further, the present disclosure relates to a metabolite quantitative analysis and kinetic analysis method that use the mass analysis labeling composition.SELECTED DRAWING: None

Description

The present invention relates to a labeling composition for mass spectrometry, a method for quantitative analysis of biotransforms, and a method for dynamic analysis of biotransforms.

In vivo phosphate compounds such as nucleic acids, coenzymes, sugar phosphates, phospholipids, and phosphorylated proteins play an important role in life activities. Therefore, phosphoric acid compound measurement technology and analysis technology such as comprehensive analysis of biotransformers are required not only for elucidation of biological phenomena but also in various fields such as medicine, pharmacy, and agriculture.

As a method for observing a phosphoric acid compound, there is a method using ³¹ P-NMR. In this method, it is possible to understand the detailed reaction mechanism such as phosphorylation and dephosphorylation by directly observing ³¹ P. However, when this method is applied to a biologically extracted sample, a large number of NMR signals derived from a huge variety of phosphate compounds contained in the living body are observed, and attribution is difficult. Not suitable for analysis.

As an analysis of protein phosphorylation, there is a method of analyzing a phosphorylated protein labeled with ³² P, which is a radioisotope, by radioactivity measurement, autoradiography, or the like. However, this method requires RI experimental equipment, and there is also the problem of exposure of the experimenter. In addition, the method using a radioisotope may analyze a state different from that in the living body because the object to be analyzed may be damaged by radiation.
With ^{2 H,} 13 ^{C, 18} phosphoric acid compounds labeled with stable isotopes such as ^O, it is also known methods for analyzing the reaction mechanism and the like in 31 P-NMR and the like (for example, Non-Patent Documents 1 and 2) .. The use of stable isotopes for labeling can prevent radiation from damaging the analyzed object.

In the comprehensive analysis of metabolites, it is extremely important to prepare and analyze a sample while maintaining the composition of the metabolite in vivo in order to ensure the reliability of the analysis result. Therefore, for example, when a phosphoric acid compound is extracted from a biological sample, these reactions are stopped so that the acid or alkali in the extract or the remaining phosphoric acid enzyme does not decompose or diffuse the phosphoric acid compound after extraction. I need to let you. Various studies have been conducted so far (Non-Patent Documents 3 and 4), and quantitative analysis of phosphoric acid compounds extracted from biological samples has also been performed (Non-Patent Document 5). However, there is no method for evaluating the decomposition or diffusion of the target compound in the process of preparing the analysis sample, and in the conventional method, it is necessary to know whether or not the biotransformation composition in the analysis sample matches the composition in the living body. It is not possible to grasp the status of metabolic conversion.

Blattler, W. et. Al. Stereochemical Course of Phosphokinases. The Use of Adenosine [Gamma-(S) -16O, 17O, 18O] triphosphate and the Mechanistic Consequences for the Reactions Catalyzed by Glycerol Kinase, Hexokinase, Pyruvate Kinase, and Acetate Kinase. Biochemistry 1979, 18 (18), 3927-3933. Mildred Cohn and Angela Hu. Isotopic l8O Shifts in 31P NMR of Adenine Nucleotides Synthesized with l80 in Various Positions, 1980, J. Am. Chem. Soc. 102, 913. E. G. Bligh, W. J. Dyer, A rapid method of total lipid extraction and purification. Can. J. Biochem., Physiol. 1959 Aug; 37 (8): 911-7. van Gulik WM. Fast sampling for quantitative microbial metabolomics. Curr Opin Biotechnol. 2010 Feb; 21 (1): 27-34. Kim H. et. Al. A fit-for-purpose LC-MS / MS method for the simultaneous quantitation of ATP and 2,3-DPG in human K2EDTA whole blood. J Chromatogr B Analyt Technol Biomed Life Sci. 2017 Sep 1; 1061-1062: 89-96.

The present invention provides a labeling composition for mass spectrometry, a quantitative analysis method for biotransforms, and a dynamic analysis method for biotransforms, which can distinguish and evaluate a plurality of labeled compounds used for analysis even if they are metabolically converted in the sample. The purpose is to provide.

The present invention has the following configurations.
[1] A labeling composition for mass spectrometry containing two or more kinds of labeling compounds.
The above-mentioned two or more kinds of labeled compounds are two or more kinds in which two or more kinds of phosphate compounds in a series of pathways of metabolic conversion in the living body are labeled with one or more kinds of stable isotopes including ¹⁸ O. It is a labeled compound and
The two or more labeled compounds are labeled so that the two or more labeled compounds and the labeled compound produced by the metabolic conversion of the two or more labeled compounds can be distinguished from each other by the transition of multiple reaction monitoring. Labeled compositions for mass analysis.
[2] The labeling composition for mass spectrometry according to [1], wherein the stable isotope is a combination of ¹⁸ O and one or more selected from the group consisting of ² H, ¹³ C, ¹⁵ N and ¹⁷ O. Stuff.
[3] The two or more labeled compounds are labeled compounds selected from the group consisting of nucleosides, nucleoside monophosphates, nucleoside diphosphates, and nucleoside triphosphates [1]. ] Or [2]. The labeling composition for mass analysis.
[4] The mass according to [1] or [2], wherein the two or more kinds of labeled compounds include a labeled compound labeled with nucleoside diphosphate and a labeled compound labeled with nucleoside triphosphate. Labeling composition for analysis.
[5] Using the labeling composition for mass spectrometry according to any one of [1] to [4], and using the above-mentioned two or more kinds of labeling compounds as internal standard substances, the target biotransformation is absolutely quantified. Quantitative analysis method.
[6] A method for analyzing the dynamics of a metabolite, which analyzes the dynamics of the target metabolite using the labeling composition for mass spectrometry according to any one of [1] to [4].

According to the present invention, even if a plurality of labeled compounds used for analysis are metabolically converted in a sample, a labeled composition for mass spectrometry, a quantitative analysis method for biotransforms, and a dynamic analysis of biotransforms can be evaluated separately. Can provide law.

It is a graph which showed the mass spectrometry result of ATP, ADP and AMP in Example 1. It is a graph which showed the ¹⁸ O enrichment calculated from the mass spectrometry result of ATP-β, γ- ¹⁸ O ₆ in Example 2. It is a graph which showed the ¹⁸ O enrichment calculated from the mass spectrometric result of ADP-α, β- ¹⁸ O ₆ in Example 2.

[Label composition for mass spectrometry]
The labeling composition for mass spectrometry of the present invention (hereinafter, also referred to as “the labeling composition”) contains two or more kinds of labeling compounds.
In the two or more labeled compounds in the present labeling composition, two or more phosphate compounds in a series of pathways for metabolic conversion in vivo are labeled with one or more stable isotopes containing ¹⁸ O. It is a thing. Further, in the two or more kinds of labeled compounds in the present labeling composition, the two or more kinds of labeled compounds and the labeled compound generated by the metabolic conversion of the two or more kinds of the labeled compounds are subjected to Multiple Reaction Monitoring (MRM). It is labeled so that it can be distinguished from each other by the transition of Monitoring). That is, two or more kinds of labeled compounds are labeled so that the two or more kinds of labeled compounds and the labeled compounds generated by a series of metabolic decomposition and resynthesis from the two or more kinds of labeled compounds can be distinguished from each other by MRM transition. Has been done.

The MRM is, for example, a triple quadrupole mass spectrometer (MS / MS) in which two quadrupole mass spectrometers (Q) are connected in series (QqQ) with a collision chamber (q) in between. ) Can be used. The mass spectrometer used for the MRM may be a GC-MS / MS linked with a gas chromatograph (GC) or an LC-MS / MS linked with a liquid chromatograph (LC).

Labeling two or more labeled compounds and labeled compounds produced by the metabolic conversion of the two or more labeled compounds so that they can be distinguished from each other by MRM transitions means that they are labeled as either one or both of precursor ions and product ions in MRM. Means to be labeled differently.

The labeling in the labeling compound may be labeled so that only the m / z value of the precursor ion is different, or may be labeled so that only the m / z value of the product ion is different, and the m / z of the precursor ion may be different. Both the value and the m / z value of the product ion may be labeled differently.

Examples of the phosphoric acid compound to be labeled include nucleoside, nucleoside monophosphate (NMP), nucleoside diphosphate (NDP), and nucleoside triphosphate (NTP) in a series of metabolic conversion pathways. The phosphoric acid compound to be labeled may be nucleic acid, coenzyme, sugar phosphate, phospholipid, phosphorylated protein or the like.

Examples of the nucleoside include ribonucleosides such as adenosine, guanosine, uridine, 5-methyluridine and cytidine, and deoxynucleosides such as deoxyadenosin, deoxyguanosine, deoxythymidine, deoxyuridine and deoxycytidine.

The NMP, adenosine monophosphate (AMP), guanosine monophosphate (GMP), uridine monophosphate (UMP), 5-methyl uridine monophosphate ^(m 5 UMP), cytidine monophosphate (CMP), deoxy Examples thereof include adenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP), and deoxycytidine monophosphate (dCMP).

The NDP, adenosine diphosphate (ADP), guanosine diphosphate (GDP), uridine diphosphate (UDP), 5-methyl uridine diphosphate ^(m 5 UDP), cytidine diphosphate (CDP), deoxy Examples thereof include adenosine diphosphate (dADP), deoxyguanosine diphosphate (dGDP), deoxythymidine diphosphate (dTDP), deoxyuridine diphosphate (dUDP), and deoxycitidine diphosphate (dCDP).

The NTP, adenosine triphosphate (ATP), guanosine triphosphate (GTP), uridine triphosphate (UTP), 5-methyl uridine triphosphates ^(m 5 UTP), cytidine triphosphate (CTP), deoxy Examples thereof include adenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP), deoxyuridine triphosphate (dUTP), and deoxythytidine triphosphate (dCTP).

As the two or more phosphate compounds in the series of metabolic conversion pathways to be labeled, two or more selected from the group consisting of nucleosides, NMPs, NDPs and NTPs are preferable.
The combination of ATP, ADP, AMP and adenosine is a phosphate compound in a series of metabolic transformation pathways. Similarly, combinations of GTP, GDP, GMP and guanosine, combinations of UTP, UDP, UMP and uridine, combinations of m ⁵ UTP, m ⁵ UDP, m ⁵ UMP and 5-methyluridine, CTP, CDP, CMP and cytidine. Combinations, dATP, dADP, dAMP and deoxyadenosin combinations, dGTP, dGDP, dGMP and deoxyguanosine combinations, dUTP, dUDP, dUMP and deoxyuridine combinations, dTTP, dTDP, dTPP and deoxythymidine combinations, dCTP, dCDP, The combination of dCMP and deoxycytidine is a phosphate compound in a series of pathways of metabolic conversion.

The two or more kinds of labeled compounds in the present labeling composition include an NDP-labeled labeled compound and an NTP-labeled labeled compound because they play an important role in intracellular metabolism and regulation. Is preferable. Further, it is more preferable that the two or more kinds of labeled compounds include an ADP-labeled labeled compound and an ATP-labeled labeled compound. This makes it possible to analyze the rate of decomposition of ATP into ADP, the rate of resynthesis of ATP, and the rate of other conjugated reactions.

Stable isotope labeling a phosphate compound may if it contains ^{18 O} may be only ^{18 O,} may be a combination of ^{18 O} and ^{non-18 O} stable isotope. Examples of stable isotopes other than ¹⁸ O include ² H, ¹³ C, ¹⁵ N, and ¹⁷ O.
^When a stable isotope other than ¹⁸ O is used, the stable isotope that labels the phosphate compound consists of ¹⁸ O and ² H, ¹³ C, ¹⁵ N and ¹⁷ O because of the high probability of existence in the biological compound. A combination with one or more selected from the group is preferable, and a combination of ¹⁸ O and one or both of ¹³ C and ¹⁵ N is more preferable.

The labeled compounds of the present labeling composition, from the analysis sample ^{16 O} and ^{18 O} exchange reaction hardly causes points, it is preferred that phosphoric acid moiety of the phosphoric acid compound is labeled with ^{18 O.}
In general, the carbon skeleton portion of a phosphoric acid compound contained in a living body is often difficult to synthesize by a chemical synthesis method such as a complicated sugar or lipid. Therefore, from the viewpoint of being cheaper and easier to obtain, the labeled compound in the present labeling composition is preferably a labeled compound in which the carbon skeleton portion of the phosphoric acid compound is an unlabeled compound and the phosphoric acid portion is labeled with ¹⁸ O.

In the present labeled composition, 2 to 20 kinds of labeled compounds labeled with 2 or more kinds of phosphoric acid compounds in a series of metabolic conversion pathways are preferable, and 2 to 5 kinds are more preferable.

Specific examples of two or more kinds of labeled compounds in the present labeling composition are, for example, labeled compounds represented by the following formula (A-1) (hereinafter, referred to as “labeled compound (A-1)”). The labeled compound represented by the formula and the unlabeled compound are also described in the same manner), and are selected from the group consisting of the labeled compound (A-2), the labeled compound (A-3) and the labeled compound (A-4). More than a species can be exemplified.

In a series of metabolic conversions of the labeled compound (A-1), the labeled compound (A-11), the labeled compound (A-12), the labeled compound (A-13), the labeled compound (A-14), the compound (A-). 15) occurs. In the electrospray ionization method (ESI method) generally used in LC-MS / MS, the labeled compound (A-1) (m / z = 528 → 424, 136) and the labeled compound (A-11) ( Precursor ions of m / z = 442 → 136, 119) and labeled compound (A-12) (m / z = 356 → 136, 71) generate deproton molecules in the negative ion detection mode, and the labeled compound (A-13) ) (M / z = 270 → 136), the precursor ions of the labeled compound (A-14) and the compound (A-15) generate protonized molecules in the positive ion detection mode. However, the m / z value in parentheses is the m / z value of the precursor ion and the product ion, and the same applies to the following.

In a series of metabolic conversions of the labeled compound (A-2), the labeled compound (A-21), the labeled compound (A-22), the labeled compound (A-23), the labeled compound (A-24), the compound (A-). 25) occurs. In the ESI method, labeled compound (A-2) (m / z = 431 → 136, 119), labeled compound (A-21) (m / z = 511 → 408, 136), labeled compound (A-22) (m). Precursor ions of / z = 353 → 136, 70) generate deproton molecules in the negative ion detection mode, and labeled compounds (A-23) (m / z = 267 → 136), labeled compounds (A-24), compounds. In (A-25), trihydrogen cations are generated in the positive ion detection mode.

In a series of metabolic conversions of the labeled compound (A-3), the labeled compound (A-31), the labeled compound (A-32), the labeled compound (A-33), the labeled compound (A-24), the compound (A-). 35) occurs. In the ESI method, labeled compound (A-3) (m / z = 355 → 136, 73), labeled compound (A-31) (m / z = 435 → 136, 125), labeled compound (A-32) (m). Precursor ions of / z = 515 → 417, 136) generate deprotonated molecules in the negative ion detection mode, and labeled compounds (A-33) (m / z = 275 → 136, 72) and labeled compounds (A-24). ), Compound (A-35) produces trihydrogen cations in the positive ion detection mode.

In a series of metabolic conversions of the labeled compound (A-4), the labeled compound (A-41), the labeled compound (A-42), the labeled compound (A-43), the labeled compound (A-44), the compound (A-) 45) occurs. In the ESI method, labeled compound (A-41) (m / z = 361 → 73, 146), labeled compound (A-42) (m / z = 441 → 127, 146), labeled compound (A-43) (m). Precursor ions of / z = 521 → 423, 146) generate deproton molecules in the negative ion detection mode, and labeled compounds (A-4) (m / z = 281 → 146), labeled compounds (A-44), compounds. In (A-45), trihydrogen cations are generated in the positive ion detection mode.

As described above, the labeled compounds (A-1) to (A-4) are a series of the labeled compounds (A-1) to (A-4) and the labeled compounds (A-1) to (A-4). Each labeled compound produced by metabolic conversion is labeled with ¹⁸ O, ¹³ C and ¹⁵ N so that they can be distinguished from each other by MRM transitions.

Specifically, the labeled compound (A-1) which is a labeled ATP, the labeled compound (A-21) produced from the labeled compound (A-2), and the labeled compound (A-) produced from the labeled compound (A-3). 32) and the labeled compound (A-43) resulting from the labeled compound (A-4) can be distinguished from each other by the MRM transition.

Labeled ADP labeled compound (A-2), labeled compound (A-11) derived from labeled compound (A-1), labeled compound (A-31) resulting from labeled compound (A-3), and labeled. Labeled compounds (A-42) resulting from compound (A-4) can be distinguished from each other by MRM transitions.

Labeled compound (A-3) which is a labeled AMP, labeled compound (A-12) derived from labeled compound (A-1), labeled compound (A-22) generated from labeled compound (A-2), and labeled. Labeled compounds (A-41) resulting from compound (A-4) can be distinguished from each other by MRM transitions.

Labeled compound (A-4) which is labeled adenosine, labeled compound (A-13) derived from labeled compound (A-1), labeled compound (A-23) derived from labeled compound (A-2), and labeled. Labeled compounds (A-33) resulting from compound (A-3) can be distinguished from each other by MRM transitions.

As the labeling compounds (A-1) to (A-4), any two of these may be selected and used, any three may be selected and used, or all of them may be used. Good.

Specific examples of the two or more labeled compounds labeled only with ¹⁸ O are selected from the group consisting of, for example, the labeled compound (B-1), the labeled compound (B-2) and the labeled compound (B-3). More than a species can be exemplified.

In a series of metabolic conversions of the labeled compound (B-1) (m / z = 350 → 136, 71), the labeled compound (B-11) (m / z = 430 → 119, 136), the labeled compound (B-12) ) (M / z = 510 → 400, 136). In the ESI method, precursor ions of the labeled compound (B-1), the labeled compound (B-11), and the labeled compound (B-12) generate deproton molecules in the negative ion detection mode.

In a series of metabolic conversions of the labeled compound (B-2) (m / z = 432 → 119, 136), the labeled compound (B-21) (m / z = 352 → 69, 136) and the labeled compound (B-22) ) (M / z = 512 → 404, 136). In the ESI method, deproton molecules are generated in the precursor ions of the labeled compound (B-2), the labeled compound (B-21), and the labeled compound (B-22) in the negative ion detection mode.

In a series of metabolic conversions of the labeled compound (B-3) (m / z = 520 → 406, 136), the labeled compound (B-31) (m / z = 434 → 119, 136), the labeled compound (B-32) ) (M / z = 348 → 69, 136). In the ESI method, precursor ions of the labeled compound (B-3), the labeled compound (B-31), and the labeled compound (B-32) generate deproton molecules in the negative ion detection mode.

As described above, the labeled compounds (B-1) to (B-3) are a series of the labeled compounds (B-1) to (B-3) and the labeled compounds (B-1) to (B-3). Each labeled compound produced by metabolic conversion is labeled with ¹⁸ O so that it can be distinguished from each other by MRM transitions.

Specifically, the labeled compound (B-1) which is a labeled AMP, the labeled compound (B-21) produced from the labeled compound (B-2), and the labeled compound (B) produced from the labeled compound (B-3). -32) can be distinguished from each other by MRM transitions.

The labeled compound (B-2) which is a labeled ADP, the labeled compound (B-11) produced from the labeled compound (B-1), and the labeled compound (B-31) produced from the labeled compound (B-3) are They can be distinguished from each other by MRM transitions.

The labeled compound (B-3) which is a labeled ATP, the labeled compound (B-12) produced from the labeled compound (B-1), and the labeled compound (B-22) produced from the labeled compound (B-2) are They can be distinguished from each other by MRM transitions.

As the labeling compounds (B-1) to (B-3), any two of these may be selected and used, or all of them may be used.

The labeled compound in the present labeling composition is not limited to the above-mentioned labeled compounds (A-1) to (A-4) and labeled compounds (B-1) to (B-3). For example, the labeling compound in the present labeling composition may be a combination of the labeling compound (D-1) and the labeling compound (D-2).
The method for synthesizing the labeled compound is not particularly limited, and a known method can be adopted.

With conventional methods, it is not possible to determine whether or not the labeled compound has been metabolized in the analytical sample. Further, when two or more kinds of conventional labeled compounds are used, when the labeled compound is metabolically converted in the analysis sample, the added labeled compound and the labeled compound generated by the metabolic conversion of the added labeled compound cannot be distinguished. , Multiple metabolites cannot be comprehensively analyzed.

On the other hand, in the two or more kinds of labeled compounds in the present labeling composition, the two or more kinds of labeled compounds and the labeled compound generated by the metabolic conversion of the two or more kinds of labeled compounds are distinguished from each other by the MRM transition. It is labeled with a stable isotope containing ¹⁸ O so that it can be. Therefore, even if the labeled compound is metabolized in the analysis sample, it is possible to identify which of the labeled compounds added to the analysis sample is the metabolic conversion of the labeled compound generated by the metabolic conversion in the analysis sample. it can. This makes it possible to recognize the presence or absence of metabolic conversion of the labeled compound in the analysis sample, and even if mutual conversion between ADP and ATP occurs in the analysis sample, for example, accurate absolute quantification that takes this into account. Dynamic analysis can be performed.

[Quantitative analysis method of metabolites]
The method for quantitative analysis of biotransforms of the present invention is a method for absolutely quantifying a target biotransform using the present labeled composition and using two or more kinds of labeled compounds as internal standard substances. Specifically, two or more kinds of labeled compounds in the present labeled composition are used as internal standard substances, and MRM measurement is performed by GC-MS / MS, LC-MS / MS or the like to absolutely quantify the target biotransforms.
The measurement conditions for MRM measurement other than using two or more labeled compounds in the labeling composition as an internal standard substance can be appropriately set.

By MRM measurement using two or more kinds of labeled compounds in this labeling composition as an internal standard substance, even if the labeled compound is metabolized and converted in the analysis sample, the target metabolite is comprehensively and absolutely quantified in consideration of it. can do.

[Biotransformation dynamics analysis method]
The method for analyzing the dynamics of a metabolite of the present invention is a method for analyzing the dynamics of a target metabolite using the labeled composition. Specifically, two or more kinds of labeled compounds in the present labeled composition are added to the analysis sample, held under predetermined conditions, and then MRM measurement is performed by GC-MS / MS, LC-MS / MS or the like. Analyze the dynamics of the target metabolite.
The measurement conditions for MRM measurement other than using two or more labeled compounds in the labeling composition can be appropriately set.

By MRM measurement using two or more kinds of labeled compounds in this labeling composition, those labeled compounds and the labeled compounds produced by the metabolic conversion of these labeled compounds can be distinguished from each other, so that the dynamics of the target metabolite is comprehensive. Can be analyzed.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to the following description.
[Example 1] Experiment on evaluation of oxidative decomposition status of ATP in cell extract HeLa cells cultured in a 10 cm dish were washed 3 times with phosphate buffered saline (PBS) and metabolized with cold methanol (1 mL). Later, the cell turbidity extract was recovered by scraping treatment. Two-phase fractionation was carried out by adding 10 nmol of the labeled compound (C-1) as an internal standard substance to the cell extract, and further adding chloroform and water. The upper phase water-methanol extract was recovered as a hydrophilic metabolite fraction to obtain an analytical sample. The labeled compound (C-1) in the analysis sample was subjected to MRM measurement by Nexera UHPLC-LCMS-8060 (Tsu Seisakusho Co., Ltd.) as LC-MS / MS.
The MRM transition of the labeled compound (C-1) is m / z = 526 → 422, 136. The MRM transition of the labeled compound (C-11), which is a decomposition product of the labeled compound (C-1), is m / z = 440 → 136, 119. The MRM transition of the labeled compound (C-12), which is a decomposition product of the labeled compound (C-11), is m / z = 354 → 69, 136.
The MRM measurement of the analytical sample to which the labeled compound (C-1) was added was performed twice (n = 2). In addition, MRM measurement was also performed on ATP (unlabeled ATP) contained in the cell extract and not labeled with ¹⁸ O. The results are shown in FIG.

As shown in FIG. 1, the degree of decomposition of the added labeled compound (C-1) was about the same as the degree of decomposition of unlabeled ATP. From this result, it was found that the degree of decomposition of ATP in the cell extract from HeLa cells can be evaluated by using the labeled compound (C-1).

[Example 2] Solution stability test of labeled compound The labeled compound (D-1) is 200 mass ppm in each of water and an aqueous solution of formic acid having a concentration of 0.1 mass%, 1 mass%, and 5 mass%. And stored at 4 ° C, room temperature (25 ° C), and 40 ° C. Immediately after preparation, 5 days and 20 days after each solution, mass spectrometry was performed using ACQUITY H class (manufactured by waters) and XEVO-G2XSQTOF (manufactured by waters) as LC-MS / MS under the following conditions, and ¹⁸ O concentration was performed. The degree was calculated. The results are shown in FIG.
(Mass spectrometry conditions)
Mobile phase A: 5 mM ammonium acetate aqueous solution,
Mobile phase B: 5 mM ammonium acetate / acetonitrile,
Separation column: CAPCELLLPAK ADME 2 μm 2.1 × 100 mm,
Ionization mode: Negative,
Capillary voltage: 2.0 kV,
Cone voltage: 40V,
Source temperature: 150 ° C,
Desolvent gas temperature: 350 ° C,
Desolvent gas flow rate: 800 L / h,
Cone gas flow rate 50 L / h.
In the case where the labeled compound (D-2) was used instead of the labeled compound (D-1), the ¹⁸ O enrichment was calculated in the same manner. The results are shown in FIG.

When the labeled compound (D-1) was stored in a 1% formic acid aqueous solution at 40 ° C. for 20 days, decomposition into AMP and ADP was observed, but as shown in FIG. 2, ¹⁸ O was observed in the solution. ^No exchange with ¹⁶ O was observed and the ¹⁸ O enrichment did not decrease. Also, under other conditions, no exchange of ¹⁸ O with ¹⁶ O was observed in the solution, and the ¹⁸ O enrichment was hardly reduced.
Further, as shown in FIG. 3, even in the labeled compound (D-2), the exchange of ¹⁸ O with ¹⁶ O was not observed in the solution, and the ¹⁸ O concentration was hardly reduced.

Claims (6)

A labeling composition for mass spectrometry containing two or more labeling compounds.
The above-mentioned two or more kinds of labeled compounds are two or more kinds in which two or more kinds of phosphate compounds in a series of pathways of metabolic conversion in the living body are labeled with one or more kinds of stable isotopes including ¹⁸ O. It is a labeled compound and
The two or more labeled compounds are labeled so that the two or more labeled compounds and the labeled compound produced by the metabolic conversion of the two or more labeled compounds can be distinguished from each other by the transition of multiple reaction monitoring. Labeled compositions for mass analysis. The labeling composition for mass spectrometry according to claim 1, wherein the stable isotope is a combination of ¹⁸ O and one or more selected from the group consisting of ² H, ¹³ C, ¹⁵ N and ¹⁷ O. Claim 1 or 2 wherein the two or more labeled compounds are labeled compounds selected from the group consisting of nucleosides, nucleoside monophosphates, nucleoside diphosphates, and nucleoside triphosphates. The labeling composition for mass analysis according to. The labeling composition for mass spectrometry according to claim 1 or 2, wherein the two or more kinds of labeling compounds include a labeling compound labeled with nucleoside diphosphate and a labeling compound labeled with nucleoside triphosphate. .. A method for quantitative analysis of biotransforms, which uses the labeling composition for mass spectrometry according to any one of claims 1 to 4 and absolutely quantifies the target biotransform using the above-mentioned two or more kinds of labeled compounds as internal standards. .. A method for analyzing the dynamics of a biotransformate, which analyzes the dynamics of the target biotransformer using the labeling composition for mass spectrometry according to any one of claims 1 to 4.

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