JP4999994B1 - Analysis method - Google Patents

Abstract

A method for accurately estimating the number of carbon atoms of a fragment ion of an organic compound having a large nitrogen atom content.
The first organic compound has a mass-to-charge ratio that is 1 larger than an ion obtained by ionizing a second organic compound obtained by changing the number of carbon atoms of the first organic compound. The first fragment ion and the first fragment ion obtained by mass analyzing the product ion generated from the second precursor ion and mass analyzing the product ion generated from the first precursor ion. A first fragment ion obtained by mass-analyzing a product ion generated from the second precursor ion, and a step of obtaining a peak height ratio of the second fragment ion having a mass-to-charge ratio smaller by 1 than Of the fragment ion corresponding to the corresponding fragment ion and the second fragment ion A step of obtaining a ratio of the height of the chromatography click.
[Selection] Figure 1

Description

  The present invention relates to an analysis method.

  Conventionally, various methods are known as methods for identifying fragment ions when performing mass analysis using a mass spectrometer.

In Non-Patent Document 1, using a tandem mass spectrometer, a precursor ion ([M + 1 + H] having a mass-to-charge ratio (m / z) 1 larger than an ion ([M + H] ⁺ ) obtained by adding a proton to 3-ethylascorbic acid. ) ⁺ ) Is mass-analyzed, and a method for estimating the number of carbon atoms of the fragment ion from the ratio of the fragment ion to the peak height of the fragment ion whose m / z is 1 smaller than the fragment ion is disclosed. ing.

  However, there is a problem that the number of carbon atoms of a fragment ion of an organic compound having a large nitrogen atom content such as arginine cannot be accurately estimated.

Summary of Mass Spectrometry Discussion Meeting, 2009, 57th, 506-507

  An object of the present invention is to provide an analysis method capable of accurately estimating the number of carbon atoms of a fragment ion of an organic compound having a large nitrogen atom content in view of the problems of the above-described conventional technology.

  The analysis method of the present invention uses a tandem mass spectrometer to mass-analyze product ions generated from a first precursor ion having a mass-to-charge ratio 1 larger than ions obtained by ionizing a first organic compound, Using a tandem mass spectrometer, the second organic compound obtained by changing the number of carbon atoms of the first organic compound is generated from a second precursor ion having a mass-to-charge ratio larger than that of an ion obtained by ionizing the second organic compound. Mass ion of the product ion to be obtained, and the mass fragment-to-charge ratio smaller than the first fragment ion and the first fragment ion obtained by mass analysis of the product ion generated from the first precursor ion A step of determining a peak height ratio of the second fragment ion, and the second precursor Obtaining a ratio of peak heights of fragment ions corresponding to the first fragment ions and fragment ions corresponding to the second fragment ions obtained by mass spectrometry of product ions generated from the ion .

  The analysis method of the present invention uses a tandem mass spectrometer to mass-analyze product ions generated from a first precursor ion having a mass-to-charge ratio 1 larger than ions obtained by ionizing a first organic compound, Using a tandem mass spectrometer, the second organic compound obtained by changing the number of carbon atoms of the first organic compound is generated from a second precursor ion having a mass-to-charge ratio larger than that of an ion obtained by ionizing the second organic compound. Mass ion of the product ion to be obtained, and the mass fragment-to-charge ratio smaller than the first fragment ion and the first fragment ion obtained by mass analysis of the product ion generated from the first precursor ion A step of determining a ratio of peak areas of the second fragment ions, and the second precursor Obtained by the product ion mass spectrometry to generate a down, comprising the step of determining the ratio of the area of the peak corresponding fragment ions to fragment ions and the second fragment ions corresponding to the first fragment ions.

  According to the present invention, it is possible to provide an analysis method capable of accurately estimating the number of carbon atoms of a fragment ion of an organic compound having a large nitrogen atom content.

It is a figure which shows the mass spectrometry result of pindolol. It is a figure which shows the mass spectrometry result (the 1) of arginine and arginine methyl ester. It is a figure which shows the mass spectrometry result (the 2) of arginine and arginine methyl ester. It is a figure which shows the mass spectrometry result (the 3) of arginine and arginine methyl ester. It is a figure which shows typically the peak of the fragment ion whose mass to charge ratio is 60 and 61 in FIG. 4 (a) and (b).

  Next, the form for implementing this invention is demonstrated with drawing.

  The analysis method of the present invention uses a tandem mass spectrometer to mass-produce product ions generated from a first precursor ion having a mass-to-charge ratio (m / z) 1 larger than that of an ion obtained by ionizing a first organic compound. A step of analyzing. Thereby, the ratio of the peak height of the first fragment ion and the second fragment ion whose m / z is 1 smaller than that of the first fragment ion is obtained.

  Note that the peak heights of the first fragment ion and the second fragment ion may be measured using analysis software of a tandem mass spectrometer, or may be measured using a ruler.

  At this time, an average value of peak heights obtained by performing mass spectrometry a plurality of times may be used as the peak height.

  The tandem mass spectrometer is not particularly limited, and examples thereof include a quadrupole ion trap mass spectrometer, a triple quadrupole mass spectrometer, and a quadrupole-time-of-flight hybrid mass spectrometer.

  A method for ionizing the first organic compound is not particularly limited, but an electron ionization (EI) method, a chemical ionization (CI) method, a field desorption (FD) method, a fast atom collision (FAB) method, a matrix-assisted laser. Examples include a desorption ionization (MALDI) method, an electrospray ionization (ESI) method, and an atmospheric pressure chemical ionization (APCI) method.

The ion obtained by ionizing the first organic compound is not particularly limited, but is a molecular ion (M ⁺ ), a proton added ion ([M + H] ⁺ ), a sodium ion added ion ([M + Na] ⁺ ), Ion added with ammonium ion ([M + NH ₄ ] ⁺ ), ion released from hydride ([M−H] ⁺ ), ion added with electron (M ⁻ ), ion added with halogen ion ([M + X] ⁻ ), Monovalent ions such as ions from which protons have been eliminated ([M−H] ⁻ ); ions to which protons have been added ([M + nH] ^{n +} ), ions from which protons have been eliminated ([M−nH] ⁿ⁻ ) Molecular weight related ions such as divalent or higher ions such as; fragment ions of molecular weight related ions and the like.

Examples of the fragment ion of the molecular weight related ion in the ion obtained by ionizing the first organic compound include an ion in which water is desorbed from an ion in which a proton is added to arginine ([M + H] ⁺ ).

  The first organic compound is not particularly limited as long as the number of carbon atoms can be changed.

  Here, in order to contrast with the analysis method of the present invention, the method described in Non-Patent Document 1 is used.

で表される分子量が２４８．３２のピンドロール（Ｃ_１４Ｈ_２０Ｎ_２Ｏ_２）を分析する場合について説明する。

A case of analyzing pindolol (C ₁₄ H ₂₀ N ₂ O ₂ ) having a molecular weight of 248.32 expressed by the following formula will be described.

  Mass spectrometry of pindolol was performed using a linear ion trap-Fourier transform type hybrid mass spectrometer LTQ-Orbitrap (manufactured by Thermo Scientific Co., Ltd.) connected with a high performance liquid chromatograph NANOSPACE SI-2 (manufactured by Shiseido Co., Ltd.) in FIG. The results are shown. Here, (a) is an MS spectrum, and (b) and (c) are precursor ions, which are ions obtained by ionizing pindolol and first ions whose m / z is 1 larger than ions obtained by ionizing pindolol, respectively. Is the MS / MS product ion spectrum.

From FIG. 1 (a), the ion obtained by ionizing pindolol is an ion ([M + H] ⁺ ) in which a proton is added to pindolol, and the first ion is m having a proton added to pindolol. It can be seen that / z is 250 ions ([M + 1 + H] ⁺ ).

FIG. 1B shows that fragment ions having m / z of 116, 146, and 172 exist as fragment ions of [M + H] ⁺ .

From FIG. 1C, as fragment ions of [M + 1 + H] ⁺ , fragment ions of m / z of 117, 147 and 173, and fragment ions of m / z 1 smaller than these fragment ions, m / z of 116 It can be seen that there are 146 and 172 fragment ions. This is because neutral species are desorbed from [M + 1 + H] ⁺ and fragment ions are generated, and isotopes having a mass number of 1 included in [M + 1 + H] ⁺ are included in neutral species desorbed and fragment ions. It is because it may be included in.

Here, the ratio of ¹³ C isotope abundance to ¹² C isotope abundance is 1.108 / 98.892, and the ratio of ² H isotope abundance to ¹ H isotope abundance is 0.015. /99.985, the ratio of the isotopic abundance of ¹⁵ N to the isotopic abundance of ¹⁴ N is 0.365 / 99.635, and the ratio of the isotopic abundance of ¹⁷ O to the isotopic abundance of ¹⁶ O is 0. 0.037 / 99.759.

The ratio of the ¹³ C isotope abundance to the ¹² C isotope abundance, the ratio of the ² H isotope abundance to the ¹ H isotope abundance, and the ¹⁵ N isotope abundance to the ¹⁴ N isotope abundance Various data exist for the ratio of the abundance of the body and the ratio of the isotope abundance of ¹⁷ O to the isotope abundance of ¹⁶ O.

^Since the isotope abundance of ² H, ¹⁵ N, and ¹⁷ O is sufficiently smaller than the isotope abundance of ¹³ C, the method described in Non-Patent Document 1 uses ¹³ The number of carbon atoms is estimated assuming only C is present. Specifically, since the ratio of the peak heights of the fragment ions with m / z of 173 and 172 is about 11: 3, the number of carbon atoms of the fragment ions with m / z of 172 and 173 is 11. It is estimated to be.

Note that fragment ions having m / z of 172 and 173 are generated by desorption of 2-aminopropane (C ₃ H ₉ N) and water (H ₂ O) from [M + 1 + H] ^+.

で表されるイオン（Ｃ_１１Ｈ_１０ＮＯ^＋）である。

(C ₁₁ H ₁₀ NO ⁺ )

At this time, in the case where m / z is contained fragment ions _{(C 11 H} 10 NO) to ¹³ C of 173, the ratio of the case contained in the ¹³ C is 2-amino-propane _{(C 3 H} 9 N), respectively 11: 3 because it is proportional to the number of carbon atoms.

  Similarly, since the ratio of the peak heights of the fragment ions with m / z of 147 and 146 is about 9: 5, the number of carbon atoms of the fragment ions with m / z of 146 and 147 is 9 Presumed. Further, since the ratio of the peak heights of the fragment ions having m / z of 117 and 116 is about 6: 8, it is estimated that the number of carbon atoms of the fragment ions having m / z of 116 and 117 is 6. The

However, since a compound having a high nitrogen atom content has a large contribution of ¹⁵ N in the peak of the fragment ion, the number of carbon atoms cannot be accurately estimated using the method described in Non-Patent Document 1. .

  Therefore, in the analysis method of the present invention, m / z is 1 more than ions obtained by ionizing the second organic compound obtained by changing the number of carbon atoms of the first organic compound using a tandem mass spectrometer. The method further includes the step of mass-analyzing product ions generated from the large second precursor ions. Thereby, the ratio of the peak height of the fragment ion corresponding to the first fragment ion and the fragment ion corresponding to the second fragment ion whose m / z is 1 smaller than that of the first fragment ion is obtained. As a result, it is possible to accurately estimate the number of carbon atoms of the fragment ion of the organic compound having a large nitrogen atom content.

  A method for ionizing the second organic compound is not particularly limited, but an electron ionization (EI) method, a chemical ionization (CI) method, a field desorption (FD) method, a fast atom collision (FAB) method, a matrix-assisted laser. Examples include a desorption ionization (MALDI) method, an electrospray ionization (ESI) method, and an atmospheric pressure chemical ionization (APCI) method.

The ion obtained by ionizing the second organic compound is not particularly limited, but a molecular ion (M ⁺ ), a proton added ion ([M + H] ⁺ ), a sodium ion added ion ([M + Na] ⁺ ), Ion added with ammonium ion ([M + NH ₄ ] ⁺ ), ion released from hydride ([M−H] ⁺ ), ion added with electron (M ⁻ ), ion added with halogen ion ([M + X] ⁻ ), Monovalent ions such as ions from which protons have been eliminated ([M−H] ⁻ ); ions to which protons have been added ([M + nH] ^{n +} ), ions from which protons have been eliminated ([M−nH] ⁿ⁻ ) Molecular weight related ions such as divalent or higher ions such as; fragment ions of molecular weight related ions and the like.

Examples of the fragment ion of the molecular weight related ion in the ion obtained by ionizing the second organic compound include an ion in which water is desorbed from an ion ([M + H] ⁺ ) obtained by adding a proton to arginine methyl ester.

  The method for changing the number of carbon atoms of the first organic compound is not particularly limited, and examples thereof include known reactions such as substitution reaction, addition reaction, elimination reaction, hydrolysis reaction, condensation reaction, and enzyme reaction.

  The second organic compound may be synthesized by changing the carbon number of the first organic compound, or a commercially available product of the second organic compound synthesized in advance may be used.

  As an example, the chemical formula

で表される分子量が１７４．２０のアルギニン（Ｃ_６Ｈ_１４Ｎ_４Ｏ_２）を質量分析する場合について説明する。このとき、アルギニンの炭素原子数を１増加させることにより得られる化合物として、アルギニンとメタノールを脱水縮合させることにより得られるアルギニンメチルエステルを用いる。

A case where arginine (C ₆ H ₁₄ N ₄ O ₂ ) having a molecular weight of 174.20 is represented by mass spectrometry will be described. At this time, as a compound obtained by increasing the number of carbon atoms of arginine by 1, arginine methyl ester obtained by dehydrating condensation of arginine and methanol is used.

  FIG. 2 shows arginine and arginine methyl using a linear ion trap-Fourier transform type hybrid mass spectrometer LTQ-Orbitrap (manufactured by Thermo Scientific) connected to a high performance liquid chromatograph NANOSPACE SI-2 (manufactured by Shiseido). The result (the 1) which carried out mass spectrometry of ester is shown. (A) and (b) are the MS spectra of arginine and arginine methyl ester, respectively.

From FIG. 2A, it can be seen that the ion obtained by ionizing arginine is an ion ([M ₁ + H] ⁺ ) having a m / z of 175 in which a proton is added to arginine. Further, it can be seen that the first ion whose m / z is 1 larger than the ion obtained by ionizing arginine is an ion ([M ₁ + 1 + H] ⁺ ) in which a proton is added to arginine and 176 is m / z.

From FIG. 2 (b), it can be seen that the ion obtained by ionizing arginine methyl ester is an ion ([M ₂ + H] ⁺ ) whose m / z is 189 in which a proton is added to arginine methyl ester. Further, it can be seen that the second ion whose m / z is 1 larger than the ion obtained by ionizing arginine methyl ester is an ion having m / z of 190 ([M ₂ + 1 + H] ⁺ ).

FIG. 3 shows the results (part 2) of mass spectrometry of arginine and arginine methyl ester. (A) and (b) are MS / MS product ion spectra when [M ₁ + H] ⁺ and [M ₂ + H] ⁺ are used as precursor ions, respectively.

FIG. 3A shows that fragment ions having m / z of 60, 70, 112, 116, and 140 exist as [M ₁ + H] ⁺ fragment ions.

FIG. 3B shows that fragment ions having m / z of 60, 70, 112, 130, and 140 exist as fragment ions of [M ₂ + H] ⁺ .

  At this time, it can be seen that even if the number of carbon atoms of arginine is changed, the m / z of fragment ions having m / z of 60, 70, 112 and 140 does not change. On the other hand, by changing the number of carbon atoms of arginine, it can be seen that the fragment ion having 116 m / z in FIG. 3A changed to the fragment ion having 130 m / z in FIG. .

FIG. 4 shows the results (part 3) of mass spectrometry of arginine and arginine methyl ester. (A) and (b) are MS / MS product ion spectra when [M ₁ + 1 + H] ⁺ and [M ₂ + 1 + H] ⁺ are used as precursor ions, respectively.

From FIG. 4 (a), as fragment ions of [M ₁ + 1 + H] ⁺ , m / z is 61, 71, 113, 117, 131 and 141, and m / z is more than m / z than fragment ions of [M ₁ + 1 + H] ^+. It can be seen that fragment ions having m / z of 60, 70, 112, 116, 130, and 140 exist as fragment ions having a small z.

From FIG. 4 (b), as fragment ions of [M ₂ + 1 + H] ⁺ , m / z is 61, 71, 113, 131 and 141, and m / z is higher than fragment ions of [M ₂ + 1 + H] ^+. It can be seen that fragment ions having m / z of 60, 70, 112, 130 and 140 exist as one small fragment ion.

  FIG. 5 schematically shows the peaks of fragment ions having m / z of 60 and 61 in FIGS. 4 (a) and 4 (b). 4A is a fragment ion peak with m / z of 60 and 61 in FIG. 4A, and FIG. 4B is a fragment ion peak with m / z of 60 and 61 in FIG. 4B. It is a peak. At this time, (a) and (b) are adjusted so that the peak heights of fragment ions having m / z of 61 are the same.

From FIG. 5, the peak height difference Δh between arginine methyl ester and arginine of m / z of 60 fragment ions corresponds to 1 carbon atom, and [M ₂ + 1 + H] ⁺ and [M ₁ + 1 + H] ⁺ The number of carbon atoms of the neutral species desorbed from is estimated to be 6 and 5, respectively. In addition, since the carbon atoms of arginine methyl ester and arginine are 7 and 6, respectively, it is estimated that the fragment ions having m / z of 60 and 61 have 1 carbon atom.

It should be noted that the fragment ions having m / z of 60 and 61 are 2-aminopentanoic acid (C ₅ H ₁₁ NO ₂ ) (or a neutral species from [M ₁ + 1 + H] ⁺ (or [M ₂ + 1 + H] ⁺ ) (or Chemical formula produced by elimination of methyl 2-aminopentanoate (C ₆ H ₁₃ NO ₂ ))

で表されるイオン（ＣＨ_６Ｎ_３ ^＋）である。

(CH ₆ N ₃ ⁺ ) represented by

On the other hand, the difference Δh ′ between the peak height of the fragment ion with m / z of 61 and the carbon number of the fragment ion with m / z of 61 and Δh, that is, Δh, is the mass number other than ¹³ C is 1. Presumed to be a large isotope contribution.

^Here, since the isotopic abundance of ² H and ¹⁷ O is sufficiently smaller than the isotopic abundance of ¹³ C and ¹⁵ N, the mass number one greater ^isotope, when only the ¹³ C and ¹⁵ N are present Assuming that the ratio of ¹³ C isotope abundance to ¹² C isotope abundance is 1.108 / 98.892, and the ratio of ¹⁵ N isotope abundance to ¹⁴ N isotope abundance is 0.365. /99.635, the number of nitrogen atoms of the fragment ions having m / z of 60 and 61 is given by the formula (1.108 / 98.892) / (0.365 / 99.635) × (Δh ′ / Δh)
Can be estimated from

At this time, since arginine has substantially the same Δh ′ as Δh, that is, the contribution of ¹⁵ N in the peak of the fragment ion having m / z of 61 is large, using the method described in Non-Patent Document 1, Even if the number of carbon atoms of arginine is estimated from the ratio of peak heights of fragment ions having m / z of 60 and 61, it cannot be estimated with high accuracy.

  In the same manner as above, fragment ions with m / z of 70 and 71, fragment ions with m / z of 112 and 113, fragment ions with m / z of 116 and 117, and m / z in FIG. Can estimate the number of carbon atoms of the fragment ions 140 and 141.

  On the other hand, in FIG. 4B, fragment ions with m / z of 70 and 71, fragment ions with m / z of 112 and 113, fragment ions with m / z of 130 and 131, and fragments of m / z of 140 and 141 The number of carbon atoms of the ion can be estimated.

  This shows that instead of increasing the number of carbon atoms in the first organic compound, it may be decreased.

  The difference in the number of carbon atoms between the second organic compound and the first organic compound obtained by changing the number of carbon atoms in the first organic compound may be other than 1, and usually 1 to 10 It is.

  Moreover, you may use 2 or more types of organic compounds as a 2nd organic compound. Thereby, the overlap of the peak of a fragment ion is detectable.

  Furthermore, when estimating the number of carbon atoms of a fragment ion, the ratio of peak areas may be used instead of the ratio of peak heights.

Incidentally, when the number of carbon atoms of the first organic compound is unknown, a mass number one greater isotope, ¹³ assuming that only ^C is present, ^{13 C} isotopic abundance of relative isotopic abundance of ^{12 C} Since the ratio of the peak of [M ₁ + H] ⁺ and [M ₁ + 1 + H] ⁺ is h ₁ and h ₂ respectively, the ratio of the first organic compound is 1.108 / 98.892. The number of carbon atoms is given by the formula (h ₂ / h ₁ ) / (1.108 / 98.892)
Can be estimated from

  The case where the analysis method of the present invention is applied to LC / MS / MS has been described above. However, the analysis method of the present invention includes GC / MS / MS, direct analysis MS / MS, direct sample introduction MS / MS, and MS / MS. It can be applied to MS / MS and the like.

Claims (4)

Using a tandem mass spectrometer to mass-analyze product ions generated from a first precursor ion having a mass-to-charge ratio that is 1 greater than the ion obtained by ionizing the first organic compound;
Using a tandem mass spectrometer, the second organic compound obtained by changing the number of carbon atoms of the first organic compound is generated from a second precursor ion having a mass-to-charge ratio larger than that of an ion obtained by ionizing the second organic compound. Mass spectrometry of product ions to be performed,
Peak heights of the first fragment ions and the second fragment ions having a mass-to-charge ratio smaller than that of the first fragment ions, obtained by mass spectrometry of product ions generated from the first precursor ions The process of determining the ratio of
The ratio of the peak height of the fragment ion corresponding to the first fragment ion and the fragment ion corresponding to the second fragment ion obtained by mass spectrometry of the product ion generated from the second precursor ion The analysis method characterized by having the process of calculating | requiring. Using a tandem mass spectrometer to mass-analyze product ions generated from a first precursor ion having a mass-to-charge ratio that is 1 greater than the ion obtained by ionizing the first organic compound;
Using a tandem mass spectrometer, the second organic compound obtained by changing the number of carbon atoms of the first organic compound is generated from a second precursor ion having a mass-to-charge ratio larger than that of an ion obtained by ionizing the second organic compound. Mass spectrometry of product ions to be performed,
The area of the peak of the first fragment ion and the second fragment ion having a mass-to-charge ratio smaller by 1 than that of the first fragment ion, obtained by mass analysis of the product ion generated from the first precursor ion. Determining the ratio;
The ratio of the peak areas of the fragment ions corresponding to the first fragment ions and the fragment ions corresponding to the second fragment ions obtained by mass spectrometry of the product ions generated from the second precursor ions The analysis method characterized by having the process to obtain | require.   The analysis method according to claim 1, further comprising a step of synthesizing the second organic compound by changing the number of carbon atoms of the first organic compound.   The analysis method according to any one of claims 1 to 3, wherein the tandem mass spectrometer is connected to a liquid chromatograph.

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