Deuterium substitution rate analysis method and deuterium substitution rate prediction method

Abstract

The present invention relates to a method for analyzing and predicting the deuterium substitution rate of a deuterium-substituted sample, using the mass spectrometry spectrum of the deuterium-substituted sample.

Description

[Technical Field]

[0001] The present invention relates to a method for analyzing and predicting the deuterium substitution rate of a deuterium-substituted sample, using the mass spectrometry spectrum of the deuterium-substituted sample. [Background technology]

[0002] Hydrogen is the most abundant element in the universe and is found in many compounds on Earth, including water.

[0003] 1-Hydrogen( 1 H) and deuterium ( 2 H) is an isotope of hydrogen. 1- The nucleus of hydrogen consists of only one proton, whereas the nucleus of deuterium consists of one proton and one neutron.

[0004] Due to this difference in the number of neutrons, the atomic weight of monohydrogen and deuterium shows a difference in weight of 1 Dalton.Similarly, in a compound molecule in which monohydrogen is replaced with deuterium, the molecular weight of the compound will show a difference in weight of 1 Dalton depending on the number of substitutions.

[0005] This difference in atomic weight can lead to differences in physical and chemical properties, such as higher boiling and melting points for deuterium compared to hydrogen. Compounds with deuterium substituted for hydrogen positions in a compound also have the same properties as common hydrogen ( 1 H) are similar in chemical and physical properties to compounds to which they are bonded, but may behave differently due to the effect of the isotope.

[0006] Taking advantage of these chemical and physical characteristics, deuterium can be used to trace the presence or absence, reaction, and pathway of a substance through deuterium labeling (D-labling), which replaces hydrogen atoms in a molecule with deuterium atoms. Therefore, deuterium is widely used as an analytical tracer for functional substances in various fields such as agriculture, medicine, and natural sciences.

[0007] In particular, in organic electroluminescent (organic light-emitting diode) materials, which have recently become widely used, when the 1-hydrogen at a specific position is replaced with deuterium, it shows superior effects in terms of luminescence efficiency and lifespan compared to the 1-hydrogen isotope isomer, so research is underway to replace the 1-hydrogen site of luminescent materials with deuterium.

[0008] However, as mentioned above, compounds containing only one hydrogen atom (hereinafter referred to as "monohydrogen compounds") and compounds in which the hydrogen atom is replaced with deuterium (hereinafter referred to as "deuterated compounds") have very similar physicochemical properties other than molecular weight. Therefore, when monohydrogen compounds and deuterated compounds in which the hydrogen atom is replaced with deuterium are mixed, it is very difficult to determine the substitution rate through analysis.

[0009] Therefore, research is needed on methods that can accurately confirm the substitution rate in samples containing deuterated compounds. Summary of the Invention [Problem to be solved by the invention]

[0010] The present disclosure relates to an analytical method capable of accurately determining the deuterium substitution rate, etc., in a sample containing a deuterated compound.

[0011] The present disclosure also relates to a method for predicting the deuterium substitution rate of a product during the production of a deuterated compound. [Means for solving the problem]

[0012] The present specification provides a method for analyzing the deuterium substitution rate of a sample substance, comprising: a first step of obtaining a mass spectrum of a sample of an analyte containing one or more compounds selected from a set of chemical species represented by {C(0,n), C(1,n), C(2,n), C(3,n), ..., C(i,n), C(i+1,n), ..., C(n-1,n), C(n,n)}; a second step of expressing the mass spectrum of the sample substance as a linear combination of the mass spectra of each elemental compound belonging to the set; and a third step of calculating the relative abundance of each elemental compound belonging to the set from the coefficients of the linear combination.

[0013] In the compound C(i,n) as each element constituting the set, C means that the compounds as each element constituting the set are identical to each other except for whether or not they are substituted with hydrogen or deuterium, n means the total number of hydrogen and deuterium bonding sites in the compound C molecule, i means the number of deuterium substitutions in the elemental chemical species included in the set, and C(i,n) means a compound in which i out of the total n hydrogen and deuterium bonding sites are substituted with deuterium.

[0014] The set {C(0,n), C(1,n), C(2,n), C(3,n), ..., C(i,n), C(i+1,n), ..., C(n-1,n), C(n,n)} can also be expressed in other ways as follows: {C(i,n)|C(i,n) is a compound where i = 0 to n}; in the compound C(i,n) as each element constituting the set, C means that the compounds as each element constituting the set are identical except for the presence or absence of hydrogen or deuterium substitution, n means the total number of hydrogen and deuterium bonding sites in the compound C molecule, i means the number of deuterium substitutions in the elemental chemical species contained in the set, and C(i,n) means a compound in which i of the total n hydrogen and deuterium bonding sites are substituted with deuterium.

[0015] In some cases, the sample substance to be analyzed may contain only pure substances having the same number and position of deuterium substitutions, or may be in a mixture state in which compounds having different numbers and positions of deuterium substitutions are mixed together.

[0016] For example, the first step may be a step of obtaining a mass spectrum by MALDI-TOF MS measurement using an inorganic oxide matrix.

[0017] According to one example, the inorganic oxide matrix may be an inorganic oxide matrix containing one or more elements selected from the group consisting of silicon, zirconium, aluminum, cerium, and the like.

[0018] In one example, the second step can eliminate isotopic effects by utilizing the natural abundance of isotopes.

[0019] According to one example, in the second step, the mass spectrum of the sample material can be expressed as a linear combination of the mass spectra of each of the elemental chemical species belonging to the set using least squares approximation.

[0020] According to an example, the method for analyzing the deuterium substitution rate of the sample material may further include a fourth step of calculating an average deuterium substitution rate of the sample material from the relative content values ​​of each elemental species belonging to the set.

[0021] According to one example, in the fourth step, the average deuterium substitution rate of the sample material can be calculated using the following Equation 1: [Formula 1] [ka]

[0022] In Equation 1, SD (%) is the average deuterium substitution rate of the sample material to be analyzed, n is the total number of hydrogen and deuterium bonding sites in the compound C molecule, and Pi is the relative amount (%) of compound C(i,n) in which i of the total n hydrogen and deuterium bonding sites are substituted with deuterium.

[0023] According to one example, Pi can be calculated by the following Equation 2. [Formula 2] [ka]

[0024] In Equation 2, Pi is the relative amount (%) of a compound C(i,n) in which i of the total n hydrogen and deuterium bonding sites are substituted with deuterium, and ai is the weight of the C(i,n) molecule.

[0025] The present specification also provides a method for predicting a deuterium substitution rate of a reaction product, the method including the steps of: measuring an average deuterium substitution rate of a first reactant by the above-described method; measuring an average deuterium substitution rate of a second reactant by the above-described method; and predicting a deuterium substitution rate of a reaction product obtained by the reaction of the first reactant and the second reactant using the average deuterium substitution rate of the first reactant and the average deuterium substitution rate of the second reactant.

[0026] In the present invention, terms such as first and second are used to describe various components, and the terms are used only to distinguish one component from another.

[0027] Furthermore, the terms used in this specification are merely used to describe exemplary embodiments and are not intended to limit the present invention.

[0028] The singular expression includes the plural expression unless the context clearly indicates otherwise.

[0029] In this specification, the terms "comprises," "includes," "comprises," or "having" are intended to describe embodied features, numbers, steps, components, or combinations thereof, and do not exclude the possibility of one or more other features, numbers, steps, components, combinations, or additional features.

[0030] While the present invention can be modified in various ways and can have various forms, specific examples are exemplified and described in detail below, but it should be understood that the present invention is not limited to the specific disclosed forms, and includes all modifications, equivalents, and alternatives within the spirit and technical scope of the present invention.

[0031] The present invention will be described in detail below.

[0032] According to one aspect of the present invention, there is provided a method for analyzing a deuterium substitution rate of a sample substance, the method comprising: a first step of obtaining a mass spectrum of a sample of an analyte substance containing one or more compounds selected from a set of chemical species represented by {C(0,n), C(1,n), C(2,n), C(3,n), ..., C(i,n), C(i+1,n), ..., C(n-1,n), C(n,n)}; a second step of expressing the mass spectrum of the sample substance as a linear combination of the mass spectra of each of the elemental chemical species belonging to the set; and a third step of calculating the relative content value of each of the elemental chemical species belonging to the set from the coefficients of the linear combination.

[0033] In the compound C(i,n) as each element constituting the set, C means that the compounds as each element constituting the set are identical to each other except for whether or not they are substituted with hydrogen or deuterium, n means the total number of hydrogen and deuterium bonding sites in the compound C molecule, i means the number of deuterium substitutions in the elemental chemical species included in the set, and C(i,n) means a compound in which i out of the total n hydrogen and deuterium bonding sites are substituted with deuterium.

[0034] The set {C(0,n), C(1,n), C(2,n), C(3,n), ..., C(i,n), C(i+1,n), ..., C(n-1,n), C(n,n)} can also be expressed in other ways as follows: {C(i,n)|C(i,n) is a compound where i = 0 to n}; in the compound C(i,n) as each element constituting the set, C means that the compounds as each element constituting the set are identical except for the presence or absence of hydrogen or deuterium substitution, n means the total number of hydrogen and deuterium bonding sites in the compound C molecule, i means the number of deuterium substitutions in the elemental chemical species contained in the set, and C(i,n) means a compound in which i of the total n hydrogen and deuterium bonding sites are substituted with deuterium.

[0035] In a compound C whose atomic structure and interatomic bond relationships are the same except for the presence or absence of hydrogen or deuterium substitution, when n sites bonded with hydrogen or deuterium are present, a compound with no deuterium substitutions, i.e., a 1-hydrogen compound with a deuterium substitution number of 0, can be represented as C(0, n). A compound with one of n substitution sites substituted with deuterium instead of hydrogen, i.e., a deuterated compound with a deuterium substitution number of 1, can be represented as C(1, n). A compound with i of n substitution sites substituted with deuterium instead of hydrogen, i.e., a deuterated compound with a deuterium substitution number of i, can be represented as C(i, n). A compound with all n substitution sites substituted with deuterium instead of hydrogen, i.e., a deuterated compound with a deuterium substitution number of n, can be represented as C(n, n).

[0036] Here, the sample substance to be analyzed may contain one or more chemical species selected from a set of chemical species represented by {C(0,n), C(1,n), C(2,n), C(3,n), ..., C(i,n), C(i+1,n), ..., C(n-1,n), C(n,n)}.

[0037] In other words, the sample substance to be analyzed may contain only pure substances that are completely identical in terms of the number and position of deuterium substitutions, or it may be a mixture in which compounds with different numbers and positions of deuterium substitutions are mixed together.

[0038] This specification provides a method for determining the average deuterium substitution rate of a sample substance to be analyzed, which may be a pure substance or a mixture, i.e., the abundance ratio of deuterium to the total number of hydrogen or deuterium present in the sample substance to be analyzed, through interpretation of a mass spectrometry spectrum.

[0039] First, a mass spectrum is obtained for a sample of an analyte containing one or more compounds selected from a set of chemical species represented by {C(0,n), C(1,n), C(2,n), C(3,n), ..., C(i,n), C(i+1,n), ..., C(n-1,n), C(n,n)}.

[0040] In one example, the mass analysis can be obtained by MALDI-TOF MS measurement using a spectral inorganic oxide matrix.

[0041] The inorganic oxide matrix may be an inorganic oxide matrix containing one or more elements selected from the group consisting of silicon, zirconium, aluminum, cerium, etc. More specifically, the inorganic oxide may be silica, zirconia, alumina, ceria, or an inorganic oxide in which other silicon, zirconium, aluminum, and / or cerium atoms are mixed into the crystal lattice of these, but the present invention is not necessarily limited thereto.

[0042] When the number of deuterium substitutions increases by one, the mass number of the molecule also increases by 1. The inorganic oxide matrix described above does not generate a signal from the substance itself in MALDI-TOF MS analysis, and can generate a single ion species for the sample substance being analyzed. This eliminates the effect of an increase in mass number of one that occurs when another hydrogen atom is bonded to a molecule during MS analysis, and allows only the effect of an increase in mass number of one due to deuterium substitution or the effect of an increase in mass number due to an isotope of an element other than deuterium that makes up the compound (e.g., carbon) to appear.

[0043] 1 and 2 are examples of typical mass spectrometry spectra obtained by MALDI-TOF MS analysis.

[0044] Assume that FIG. 1 is a spectrum obtained by MALDI-TOF MS analysis of a sample containing the aforementioned compound C(i,n).

[0045] In Figure 1, the highest peak in the center is an isotope of an element other than deuterium (e.g., 13 C. 14 The parent peak of C(i, n) does not include C(i, n), and the molecular weight at this point is defined as M0. However, the parent peak M0 in Figure 1 is not a peak that appears due to a single chemical species, but rather appears as a combination of peaks from many chemical species that have the same mass number and are identically charged.

[0046] For example, the parent peak corresponding to the M0 peak is determined by the sum of the contributions described below. i) C(i,n), the radical ion formed when compound C(i,n) loses one electron +· Portion contributed by; ii) C(i-1,n), a radical ion formed by losing one electron from a compound C(i-1,n) (mass number: M0-1) which has one less deuterium substitution than the compound C(i,n). +· (Mass number: M0-1), and the isotopes of the elements contained therein (for example 13 C) The portion contributed by the compound present in (mass number: M0); iii) C(i-1,n), a radical ion formed by losing one electron from a compound C(i-1,n) (mass number: M0-1) which has one less deuterium substitution than the compound C(i,n). +· Compound C(i-1, n)+H with one additional hydrogen atom bonded to + (Mass number: M0) contribution; iv) C(i-2,n), a radical ion formed by losing one electron from compound C(i-2,n) (mass number: M0-2), which has two fewer deuterium substitutions than compound C(i,n). +· One hydrogen atom is further bonded to it (mass number: M0-1), and one of the elements contained in it is an isotope with a mass number of +1 (for example, one of the carbon atoms is 13 C) The portion contributed by the compound present in (mass number: M0); v) C(i-2,n), a radical ion formed by losing one electron from compound C(i-2,n) (mass number: M0-2), which has two fewer deuterium substitutions than compound C(i,n) +· (Mass number: M0-2), and one of the elements contained in it is an isotope with a mass number of +2 (for example, one carbon atom 14 C) The portion contributed by the compound present in (mass number: M0); vi) C(i-2,n), a radical ion formed by losing one electron from compound C(i-2,n) (mass number: M0-2), which has two fewer deuterium substitutions than compound C(i,n). +· (Mass number: M0-2), and one of the elements contained in it has two isotopes with a mass number of +1 (for example, two carbon atoms 13 C) The portion contributed by the compound (mass number: M0) present in...

[0047] According to the above logic, the number of deuterium substitutions, the number of additional hydrogen atoms bonded during the MS measurement process, and the number of isotopes of the elements contained in the molecule (e.g., 13 C. 14 The effect of C) can increase or decrease the mass number, and among these, species with the same charge-to-mass ratio are detected in one peak, as well as in other peaks in addition to the parent peak.

[0048] However, for example, when MALDI-TOF MS measurement is performed using an inorganic oxide matrix, it is possible to eliminate cases in which additional hydrogen is bonded and the mass number increases by 1, and only the effects of deuterium substitution and isotopes of elements other than deuterium (e.g., carbon) can be observed.

[0049] FIG. 2 is assumed to be a spectrum obtained when a sample containing the aforementioned compound C(i,n) is analyzed by MALDI-TOF MS using an inorganic oxide matrix.

[0050] In this case, the parent peak in FIG. 2 appears as the sum of the contributing parts, as explained below. i) C(i,n), the radical ion formed when compound C(i,n) loses one electron +· Portion contributed by; ii) C(i-1,n), a radical ion formed by losing one electron from a compound C(i-1,n) (mass number: M0-1) which has one less deuterium substitution than the compound C(i,n). +· (Mass number: M0-1), and one of the elements contained in it is an isotope with a mass number of +1 (for example, one of the carbon atoms is 13 C) The portion contributed by the compound present in (mass number: M0); iii) C(i-2,n), a radical ion formed by losing one electron from compound C(i-2,n) (mass number: M0-2), which has two fewer deuterium substitutions than compound C(i,n). +· (Mass number: M0-2), and two of the elements contained in it are isotopes with a mass number of +1 (for example, two of the carbon atoms are 13 C) The portion contributed by the compound present in (mass number: M0); iv) C(i-2,n), a radical ion formed by losing one electron from compound C(i-2,n) (mass number: M0-2), which has two fewer deuterium substitutions than compound C(i,n). +·(Mass number: M0-2), and one of the elements contained in it is an isotope with a mass number of +2 (for example, one of the carbon atoms is 14 C) The portion contributed by the compound (mass number: M0) present in...

[0051] By the above logic, the number of deuterium substitutions and the isotopes of elements present in the molecule (for example, isotopes of carbon atoms) 13 C. 14 The effect of the electrons (e.g., C) can increase or decrease the mass by one, and among these, species with the same charge-to-mass ratio will be detected as a single peak, as well as other peaks other than the parent peak.

[0052] Therefore, if the effect of isotopes (for example, carbon isotopes) can be eliminated, only the effect of the number of deuterium substitutions can be left.

[0053] According to one example, in the second step, the mass increase effect caused by isotopes other than deuterium can be eliminated by utilizing the natural abundance ratio of isotopes, specifically, elements (e.g., carbon) constituting the compound. In other words, the sample substance to be analyzed by the present invention is a compound in which some or all of the 1-hydrogen atoms in the compound have been intentionally replaced with deuterium, and the deuterium abundance ratio in the sample substance to be analyzed may differ from the natural abundance ratio of deuterium.

[0054] However, isotopes of other constituent elements excluding deuterium in the sample substance to be analyzed in the present invention, specifically elements such as carbon, oxygen, and nitrogen, will be present in their natural abundance ratios unless there are special circumstances.

[0055] For example, carbon ( 12 The natural abundance of carbon (C) is approximately 98.9%; 13 The natural abundance of carbon (C) is about 1.1%; 14The natural abundance of carbon (C) is known to be approximately 0.0000000001%, so this natural abundance can be used to eliminate carbon isotope effects in the mass spectrometry spectrum obtained.

[0056] Another example is oxygen ( 16 The natural abundance of oxygen (O) is approximately 99.757%; 17 The natural abundance of O is approximately 3.8 × 10 -4 ;Oxygen with mass number 18 ( 18 The natural abundance of O is approximately 2.05 × 10 -3 Therefore, by utilizing this natural abundance ratio, the effect of oxygen isotopes can be eliminated from the mass spectrum obtained.

[0057] Another example is nitrogen ( 14 The natural abundance of nitrogen (N) is approximately 99.636%; 15 The natural abundance of N is known to be approximately 0.364%, so this natural abundance can be used to eliminate nitrogen isotope effects in the mass spectra obtained.

[0058] In addition to the above elements, if an isotope is present in an element contained in a compound, the natural abundance ratio of the isotope can be used to eliminate the effect of the isotope, i.e., the mass number change effect.

[0059] According to one example, in the second step, the mass spectrum of the sample material can be expressed as a linear combination of the mass spectra of each of the elemental chemical species belonging to the set using least squares approximation.

[0060] In other words, the peaks that appear in the mass spectrum obtained by analyzing the entire sample material are separated by a linear combination of the mass spectra of each elemental chemical species contained in the sample material, which can be seen as a type of deconvolution.

[0061] FIG. 3 shows a schematic diagram of the deconvolution concept according to an example of the present invention.

[0062] In FIG. 3, 100 represents each peak that appears in the mass spectrum obtained by analyzing the entire sample material, and 200 represents peaks that may appear in a theoretical mass spectrum depending on the number of deuterium substitutions of a material having a specific chemical formula.

[0063] For convenience of explanation, 200 will first explain the peaks that may appear in a theoretical mass spectrometry spectrum depending on the number of deuterium substitutions of a substance having a specific chemical formula.

[0064] Assume that the molecular weight of C(i,n) is M0.

[0065] If 210 in FIG. 3 is a theoretical peak that appears due to C(i, n) (mass number: M0), then since the number of deuterium substitutions in C(i, n) is fixed at i, each peak in 210 is due to the effect of only the isotope (e.g., carbon isotope), and can be explained as follows. M0:i.0)C(i,n) +· Peak due to radical ions. M0+1:i.1)C(i,n) +· An isotope with a mass number of 1+1 (e.g., 13 C) Peak. M0+2:i.2.1)C(i,n) +· One isotope with a mass number of +2 (e.g., 14 C) Peak by; i.2.2) C(i,n) +· isotopes with a mass number of 2 + 1 (e.g., two 13 C) Peak. M0+3:i.3.1)C(i,n) +· One isotope with a mass number of +2 (e.g., 14 C) and one isotope with a mass number of +1 (e.g., 13 C) Peaks due to; i.3.2) C(i,n) +· isotopes with a mass number of 3 + 1 (e.g., 3 13 C) Peak. M0+4:i.4.1)C(i,n) +· isotopes with a mass number of 2 + 2 (e.g., two 14 C) peak, i.4.2) C(i,n) +· One isotope with a mass number of +2 (e.g., 14 C) and two isotopes with a mass number of +1 (e.g., two 13 C) peak, i.4.2) C(i,n) +· isotopes with a mass number of 4 + 1 (e.g., 4 13 C) Peak.

[0066] If 220 in Figure 3 is a theoretical peak that appears due to C(i+1, n) (mass number: M0+1), then since the number of deuterium substitutions in C(i+1, n) is fixed at i+1, only the effect of the carbon isotope remains for each peak of 220, which can be explained as follows. M0: Doesn't show up. M0+1:i+1.0)C(i+1,n) +· Peak due to radical ions. M0+2:i+1.1)C(i+1,n) +· An isotope with a mass number of 1+1 (e.g., 13 C) Peak. M0+3:i+1.2.1)C(i+1,n) +· One isotope with a mass number of +2 (e.g., 14 C) Peak by i+1.2.2) C(i+1,n) +· isotopes with a mass number of 2 + 1 (e.g., two 13 C) Peak. M0+4:i+1.3.1)C(i+1,n) +·One isotope with a mass number of +2 (e.g., 14 C) and one isotope with a mass number of +1 (e.g., 13 C) Peak by i+1.3.2) C(i+1,n) +· isotopes with a mass number of 3 + 1 (e.g., 3 13 C) Peak.

[0067] If 230 in Figure 3 is a theoretical peak that appears due to C(i+2, n) (mass number: M0+2), then since the number of deuterium substitutions in C(i+2, n) is fixed at i+2, only the effect of the carbon isotope remains for each peak in 230, which can be explained as follows. M0: Doesn't show up. M0+1: Does not appear. M0+2:i+2.0)C(i+2,n) +· Peak due to radical ions. M0+3:i+2.1)C(i+2,n) +· An isotope with a mass number of 1+1 (e.g., 13 C) Peak. M0+4:i+2.2.1)C(i+2,n) +· One isotope with a mass number of +2 (e.g., 14 C) Peak by i+2.2.2) C(i+2,n) +· isotopes with a mass number of 2 + 1 (e.g., two 13 C) Peak.

[0068] With this in mind, the reference numeral 100 in FIG. 3 will be explained again as follows. If we assume that the mass number excluding isotope effects (e.g., carbon isotopes) in C(i, n) is M0, each peak in 100 in Figure 3 appears as a sum of the following elements: M0:i.0)C(i,n) +· Contribution from radical ions. M0+1:i.1)C(i,n) +· An isotope with a mass number of 1+1 (e.g., 13 C), and the contribution by i+1.0)C(i+1, n) +·Contribution from radical ions. M0+2:i.2.1)C(i,n) +· One isotope with a mass number of +2 (e.g., 14 C) contribution by i.2.2) C(i,n) +· isotopes with a mass number of 2 + 1 (e.g., two 13 C) contribution by i+1.1)C(i+1,n) +· An isotope with a mass number of 1+1 (e.g., 13 C), and i+2.0)C(i+2,n) +· Contribution from radical ions. M0+3:i.3.1)C(i,n) +· One isotope with a mass number of +2 (e.g., 14 C) and one isotope with a mass number of +1 (e.g., 13 C) contribution by i.3.2) C(i,n) +· isotopes with a mass number of 3 + 1 (e.g., 3 13 C) contribution by i+1.2.1)C(i+1,n) +· One isotope with a mass number of +2 (e.g., 14 C) contribution by i+1.2.2)C(i+1,n) +· isotopes with a mass number of 2 + 1 (e.g., two 13 C) and i+2.1)C(i+2,n) +· An isotope with a mass number of 1+1 (e.g., 13 C) contribution by. M0+4:i.4.1)C(i,n) +· isotopes with a mass number of 2 + 2 (e.g., two 14 C) contribution, i.4.2) C(i, n) +· One isotope with a mass number of +2 (e.g., 14 C) and two isotopes with a mass number of +1 (e.g., two 13 C) contribution, i.4.2) C(i, n) +· isotopes with a mass number of 4 + 1 (e.g., 4 13 C) contribution by i+1.3.1)C(i+1,n) +·One isotope with a mass number of +2 (e.g., 14 C) and one isotope with a mass number of +1 (e.g., 13 C) contribution by i+1.3.2)C(i+1,n) +· isotopes with a mass number of 3 + 1 (e.g., 3 13 C) contribution by i+2.2.1)C(i+2,n) +· One isotope with a mass number of +2 (e.g., 14 C) and i+2.2.2)C(i+2,n) +· isotopes with a mass number of 2 + 1 (e.g., two 13 C) contribution by.

[0069] However, as mentioned above, carbon ( 12 The natural abundance of carbon (C) is approximately 98.9%; 13 The natural abundance of carbon (C) is about 1.1%; 14 Since the natural abundance of 1C is known to be approximately 0.0000000001%, even if we ignore the contribution of 14C or two or more 13C atoms, the final analytical result may not change significantly in arithmetic calculations, which can simplify calculations.

[0070] Furthermore, by the same principle, there may not be much difference even if peaks with mass numbers of M0+5 or more are ignored, based on the chemical species C(i, n) with mass number M0.

[0071] In other words, each peak appearing in the mass spectrum obtained by analyzing the entire sample material is a linear combination of the peaks in the mass spectrum that appear due to each chemical species contained in the sample material. Therefore, the reverse process can be performed to separate each peak appearing in the mass spectrum obtained by analyzing the entire sample material using a linear combination of the peaks in the mass spectrum that appear due to each chemical species contained in the sample material.

[0072] An example of such a method is the least squares method. In the case of the least squares method, the coefficient values ​​used in the linear combination of each peak in the mass spectrum appearing due to each chemical species, that is, the a of 200 in FIG. i , a i+1 , a i+2 , ... can be seen as the ratio of each chemical species.

[0073] According to one embodiment of the present invention, by analyzing the mass spectrum using the above method, the ratios a0, a1, a2, a3, ..., a4 of each set of elements belonging to each chemical species contained in the sample of the analyte, i.e., {C(0, n), C(1, n), C(2, n), C(3, n), ..., C(i, n), C(i+1, n), ..., C(n-1, n), C(n, n)}, are calculated. i , …, a n-1 , a n By rearranging the set to include the ratio of each chemical species, it can be expressed as follows: {C(0,n)×a0, C(1,n)×a1, C(2,n)×a2, C(3,n)×a3, ..., C(i,n)×a i , C(i+1,n)×a i+1 , …, C(n-1, n)×a n-1 , C(n, n)×a n}

[0074] According to an example, the method for analyzing the deuterium substitution rate of the sample material may further include a fourth step of calculating an average deuterium substitution rate of the sample material from the relative content values ​​of each elemental species belonging to the set.

[0075] According to one example, in the fourth step, the average deuterium substitution rate of the sample material can be calculated using the following Equation 1. [Formula 1] [ka]

[0076] In Equation 1, SD (%) is the average deuterium substitution rate of the sample material to be analyzed, n is the total number of hydrogen and deuterium bonding sites in the compound C molecule, and Pi is the relative amount (%) of compound C(i,n) in which i of the total n hydrogen and deuterium bonding sites are substituted with deuterium.

[0077] According to one example, Pi can be calculated by the following Equation 2. [Formula 2] [ka]

[0078] In Equation 2, Pi is the relative amount (%) of a compound C(i,n) in which i of the total n hydrogen and deuterium bonding sites are substituted with deuterium, and ai is the weight of the C(i,n) molecule.

[0079] That is, the above formula is obtained by the set {C(0,n)×a0, C(1,n)×a1, C(2,n)×a2, C(3,n)×a3, ..., C(i,n)×a i , C(i+1,n)×a i+1 , …, C(n-1, n)×a n-1 , C(n, n)×a n} is merely a formula that arithmetically arranges the relative proportions of each chemical species in the total amount of all chemical species contained in the sample of the substance to be analyzed, and the present invention is not necessarily limited to this.

[0080] Meanwhile, the present specification provides a method for predicting a deuterium substitution rate of a reaction product, the method including the steps of measuring an average deuterium substitution rate of a first reactant by the above-described method, measuring an average deuterium substitution rate of a second reactant by the above-described method, and predicting a deuterium substitution rate of a reaction product obtained by the reaction of the first reactant and the second reactant using the average deuterium substitution rate of the first reactant and the average deuterium substitution rate of the second reactant.

[0081] That is, according to the method, in a chemical reaction in which a specific deuterium-substituted reactant reacts to produce a reaction product, the relative amount by number of deuterium substitutions and the average substitution rate can be obtained through a deuterium substitution rate analysis, and the relative amount by number of substitutions of each chemical species present in the reaction product and the average deuterium substitution rate for all chemical species can be predicted without a separate analysis of the reaction product.

[0082] The following will be explained using an example of a reaction in which two molecules react to form one molecule.

[0083] Reactions in which two molecules with deuterium substituted at specific positions react to form one molecule can be classified into three cases: i) Case 1, in which when one deuterium-substituted compound (hereinafter referred to as Compound 1-1) reacts with one non-deuterium-substituted compound (hereinafter referred to as Compound 1-2) to form a product (hereinafter referred to as Compound 1), a functional group at a specific position of Compound 1-1 is substituted by Compound 1-2; ii) In the second case, when one deuterium-substituted compound (hereinafter referred to as Compound 2-1) reacts with one non-deuterium-substituted compound (hereinafter referred to as Compound 2-2) to form a product (hereinafter referred to as Compound 2), the deuterium substitution position of Compound 2-1 is maintained as it is; iii) A third case in which one deuterium-substituted compound (hereinafter, Compound 3-1) reacts with another deuterium-substituted compound (hereinafter, Compound 3-2) to form a product (hereinafter, Compound 3).

[0084] The first case will be explained first.

[0085] FIG. 4 is a conceptual diagram for explaining the change in deuterium substitution characteristics in an example reaction.

[0086] In FIG. 4, compound 1-1 is a deuterium compound substituted with deuterium, and compound 1-2 is a molecule in which deuterium is not substituted. Compound 1-2 is substituted at a specific substitution position X of compound 1-1 to form compound 1.

[0087] In this case, the probability that the X position of the 1-1 compound is 1-hydrogen is defined as a. The value of a can be calculated by using the "average substitution rate" value of the deuterium-substituted reactants obtained by mass spectrometry as described above, or by using other methods, such as NMR analysis.

[0088] In this case, the relative amount P() of each deuterium substitution number in the first compound, which is the product, can be expressed as the following Equation 3-1. [Formula 3-1] P i (first compound)={P i+1 (1-1 compound)×(1-a)}+{P i (Compound 1-1)×a}

[0089] In the above formula, P() is the relative amount of molecules with deuterium substituted at position i, and a is the probability that the original hydrogen is present at substitution position X in compound 1-1.

[0090] In this case, the average deuterium substitution rate (SD1) of the product can be expressed as the following Equation 3-2. [Formula 3-2] [ka]

[0091] Next, the second case will be described.

[0092] FIG. 5 is a conceptual diagram for explaining the change in deuterium substitution characteristics in an example reaction.

[0093] In FIG. 5, compound 2-1 is a deuterium-substituted compound, compound 2-2 is a molecule in which deuterium is not substituted, and the substitution position of compound 2-1 to which the original substituent X was attached is substituted with compound 2-2 to form compound 2.

[0094] In this case, the relative amount P() of the deuterium substitution number in the product compound 2 is the same as the relative amount P() of the deuterium substitution number in the reactant compound 2-1, and can be expressed as the following Equation 4-1. [Formula 4-1] P i (Second compound)=P i (Compound 2-1)

[0095] In this case, the average deuterium substitution rate (SD2) of the product can be expressed as the following Equation 4-2: [Formula 4-2] [ka]

[0096] Next, the third case will be described.

[0097] FIG. 6 is a conceptual diagram for explaining the change in deuterium substitution characteristics in an example reaction.

[0098] In FIG. 6, Compound 3-1 and Compound 3-2 are deuterium compounds substituted with deuterium, and the substitution position of Compound 3-1 to which the original substituent X was linked is substituted with Compound 3-2 to form Compound 3.

[0099] In this case, the relative amount P() of each deuterium substitution in the third compound, which is the product, can be expressed as the following Equation 5-1. [Formula 5-1] Pi(3rd compound)=sigma(m=0 to i){Pm(3rd-1st compound)×Pi-m(3rd-2nd compound)}

[0100] In this case, the average deuterium substitution rate (SD3) of the product can be expressed as the following Equation 5-2: [Formula 5-2] [ka]

[0101] Using the above method, when a deuterium compound reacts to produce another deuterium compound, the relative amount P() of each deuterium substitution in the product and the average deuterium substitution rate of the product can be predicted from the deuterium substitution analysis results of the reactant without performing a separate deuterium substitution analysis on the product. [Effects of the Invention]

[0102] According to one example of the present invention, the relative amount of deuterium substitution by number and the average deuterium substitution rate in an analysis sample can be accurately determined through MS analysis, and the relative amount of deuterium substitution by number of deuterium substitutions P() in a product and the average deuterium substitution rate in a product can be predicted from the results of the deuterium substitution analysis of a reactant without performing a separate deuterium substitution analysis on the product. [Brief explanation of the drawings]

[0103] [Figure 1] This is an example of a typical mass spectrum obtained by MALDI-TOF MS analysis. [Figure 2] This is an example of a typical mass spectrum obtained by MALDI-TOF MS analysis. [Figure 3] This is a schematic illustration of the concept of deconvolution according to one example of the present invention. [Figure 4] FIG. 1 is a conceptual diagram for explaining a change in deuterium substitution characteristics in an example reaction. [Figure 5] FIG. 1 is a conceptual diagram for explaining a change in deuterium substitution characteristics in an example reaction. [Figure 6] FIG. 1 is a conceptual diagram for explaining a change in deuterium substitution characteristics in an example reaction. [Figure 7] 1 is a MALDI-TOF mass spectrometry spectrum for an example compound. [Figure 8] 1 is a MALDI-TOF mass spectrometry spectrum for an example compound. [Figure 9] 1 shows the NMR analysis results for an example compound. [Figure 10]1 is a MALDI-TOF mass spectrometry spectrum for an example compound. DETAILED DESCRIPTION OF THE INVENTION

[0104] The functions and effects of the present invention will be described in more detail below through specific examples of the present invention. However, these examples are presented only as examples of the present invention and do not define the scope of the invention.

[0105] <Example> In the reaction of the compound shown below, the deuterium substitution rates of the precursor and intermediate (reactant) were analyzed, while the deuterium substitution rates of the product produced in the reaction of the first intermediate and the second intermediate were predicted. First Precursor: [ka] First intermediate: [ka] Second Precursor: [ka] Second intermediate: [ka]

[0106] Reaction schematic: First precursor → first intermediate; second precursor → second intermediate; first intermediate + second intermediate → product: [ka]

[0107] First, preliminary experiments were performed to select an appropriate matrix for analysis.

[0108] Preparation of analytical samples (preparation example) An Al2O3 matrix solution was prepared by adding 1 mg of Al2O3 powder to 300 μL of tetrahydrofuran (THF).

[0109] Separately, 1 mg of each of the substances to be analyzed (the H-form of the second intermediate) was dissolved in 1 mL of THF to prepare a solution of the substance to be analyzed.

[0110] The Al2O3 matrix solution was shaken to disperse the Al2O3, and 10 μL of the solution was taken and mixed with 10 μL of the target substance solution to prepare a mixed solution.

[0111] 1 μL of the mixed solution was placed on a MALDI sample dish and allowed to air dry to prepare a specimen.

[0112] Preparation of analytical samples (example of sample preparation)

[0113] An organic matrix solution was prepared by adding 1 mg of dihydroxybenzoic acid to 100 μL of tetrahydrofuran (THF).

[0114] Separately, 1 mg of each of the substances to be analyzed (the H-form of the second intermediate) was dissolved in 1 mL of THF to prepare a solution of the substance to be analyzed.

[0115] 10 μL of the organic matrix solution was taken and mixed with 10 μL of the analyte solution to prepare a mixed solution.

[0116] 1 μL of the mixed solution was placed on a MALDI sample dish and allowed to air dry to prepare a specimen.

[0117] Mass spectrometry spectrum acquisition and analysis

[0118] MALDI-TOF MS experimental conditions: Sample rate: 2.5GS / s or higher; Laser repetition rate: 100 or 200Hz; Laser condition: Shot at raster spot (50), Limit diameter (500μm); Mass spectrometer: positive mode

[0119] FIG. 7 shows the MALDI-TOF mass spectrum of the second intermediate (H-form).

[0120] Referring to FIG. 7, it can be seen that the peak intensity at 331 was higher in the reference preparation using an organic matrix than in the preparation using an inorganic oxide matrix.

[0121] This is thought to be due to an increase in mass number caused by the addition of another hydrogen atom during analysis. Depending on the type of matrix, chemical species with additional hydrogen atoms attached to the molecule may be generated, and it is difficult to eliminate the resulting change in mass number. In other words, when analyzing a compound substituted with deuterium, the accuracy of the analysis may be reduced due to the increase in mass number caused by hydrogenation, in addition to the increase in mass number of one due to deuterium substitution and the increase in mass number caused by isotopes of elements other than deuterium that make up the compound.

[0122] Therefore, in the following experiments of this example, an inorganic oxide matrix was used.

[0123] Preparation of analytical samples An Al2O3 matrix solution was prepared by adding 1 mg of Al2O3 powder to 300 μL of tetrahydrofuran (THF).

[0124] Separately, 1 mg of each analyte was dissolved in 1 mL of THF to prepare an analyte solution.

[0125] The Al2O3 matrix solution was shaken to disperse the Al2O3, and 10 μL of the solution was taken and mixed with 10 μL of the target substance solution to prepare a mixed solution.

[0126] 1 μL of the mixed solution was placed on a MALDI sample dish and allowed to air dry to prepare a specimen.

[0127] Mass spectrometry spectrum acquisition and analysis

[0128] MALDI-TOF MS experimental conditions: Sample rate: 2.5GS / s or higher; Laser repetition rate: 100 or 200Hz; Laser condition: Shot at raster spot (50), Limit diameter (500μm); Mass spectrometer: positive mode

[0129] Analysis of the average deuterium substitution rate of the first precursor

[0130] (Mass spectrometry results)

[0131] FIG. 8 is a MALDI-TOF mass spectrum of the first precursor.

[0132] The first precursor compound has a total of 16 deuterium substitution sites and 24 carbon atoms, and therefore each compound contained in the first precursor compound can be represented as C(i, 16), where i = 0 to 16.

[0133] This analysis result shows that the aforementioned hydrogen ( 1 Therefore, if the effect of the isotope can be eliminated, only the effect of the number of deuterium substitutions can be left. Since the first precursor compound contains only carbon in addition to hydrogen or deuterium, if the effect of the carbon isotope can be eliminated from the analysis results, the effect of the number of deuterium substitutions can be confirmed.

[0134] carbon( 12 The natural abundance of carbon (C) is about 98.9%; 13 The natural abundance of carbon (C) is about 1.1%; 14 The natural abundance of C) is approximately 0.0000000001%, and the effect of carbon isotopes was eliminated from the mass spectrum of Figure 8. This was then separated by linear combination of the peaks in the mass spectrum that appeared for each chemical species, and each coefficient value was calculated. The values ​​were rounded to one decimal place and summarized in Table 1.

[0135] {C(0, 16)×a0, C(1, 16)×a1, C(2, 16)×a2, C(3, 16)×a3, ..., C(i, 16)×a i , C(i+1, 16)×a i+1 , …, (15, 16) × a 15 , C(16, 16)×a 16}

[0136] [Table 1]

[0137] The average deuterium substitution rate was calculated using Equation 1 and was found to be approximately 91.2%. [Formula 1] [ka] n=16

[0138] FIG. 9 shows the NMR analysis results for the first precursor.

[0139] From the NMR analysis results of FIG. 9, the probability a of 1-hydrogen at the A-1 position of the ring of the first precursor was found to be approximately 6.8%.

[0140] Prediction of the average deuterium substitution rate of the first intermediate [ka]

[0141] In the above reaction, the reactive site of the first precursor is a site where deuterium substitution is possible, and this site is substituted with Br in the first intermediate. Since this corresponds to the first case described above, the average substitution rate of the first intermediate can be calculated using Equation 3-1 and Equation 3-2.

[0142] In Equation 3-1 below, the probability a that the A-1 position of the ring of the first precursor is 1-hydrogen may be the "average substitution rate" value of the deuterium-substituted reactant obtained by mass spectrometry, or may be the substitution rate value obtained by NMR analysis, etc. [Formula 3-1] P i (first intermediate)={P i+1 (first precursor) × (1-a)} + {P i (first precursor) × a}

[0143] The average deuterium substitution rate (SD1) of the first intermediate can be obtained by the following formula 3-2. [Formula 3-2] [ka] n=15

[0144] Table 1a below summarizes the values ​​measured by actual experiments for the first intermediate and the values ​​predicted by the above method.

[0145] [Table 1a] *When calculating the predicted value, the average substitution rate obtained through mass spectrometry is used as the a value in Equation 3-1. **When calculating the predicted value, the value of a in Equation 3-1 is the deuterium substitution rate at the functional group substitution position (X) obtained through NMR analysis.

[0146] From Table 1a, it can be seen that the ratio trends for each substitution number are the same between the values ​​measured in actual experiments and the predicted values, and it can be seen that there is almost no error in the obtained average substitution rate values.

[0147] Analysis of the average deuterium substitution rate of the second precursor

[0148] FIG. 10 is a MALDI-TOF mass spectrum of the second precursor.

[0149] The second precursor compound has a total of 11 deuterium substitution sites and 16 carbon atoms, and therefore each compound contained in the first precursor compound can be represented as C(i, 11), where i = 0 to 11.

[0150] This analysis result shows that the aforementioned hydrogen ( 1 Therefore, if the effect of the isotope can be eliminated, only the effect of the number of deuterium substitutions can be left. Since the second precursor compound contains carbon and chlorine in addition to hydrogen or deuterium, if the effect of the carbon and chlorine isotopes can be eliminated from the analysis results, the effect of the number of deuterium substitutions can be confirmed.

[0151] 12 The natural abundance of C is approximately 98.9%; 13 The natural abundance of C is about 1.1%; 14 The natural abundance of C is approximately 0.0000000001%; ​​and 35 The natural abundance of Cl is about 75.8%; 37 Using the natural abundance of Cl, which is approximately 24.2%, the effects of carbon and chlorine isotopes were eliminated from the mass spectrum in Figure 10, and the mass spectrum peaks appearing for each chemical species were separated using a linear combination to calculate the coefficient values. The values ​​were rounded to one decimal place and summarized in Table 2.

[0152] {C(0, 11)×a0, C(1, 11)×a1, C(2, 11)×a2, C(3, 11)×a3, ..., C(i, 11)×ai , C(i+1, 11)×a i+1 , …, C(10, 11)×a 10 , C(11, 11)×a 11}

[0153] [Table 2]

[0154] The average deuterium substitution rate was calculated using Equation 1 and was found to be approximately 81.7%. [Formula 1] [ka] n=11

[0155] Prediction of the average deuterium substitution rate of the second intermediate [ka]

[0156] In the above reaction, the reactive site of the second precursor is the site where Cl was located, and this site is replaced by a tetramethyl-1,3,2-dioxaborolan compound in the second intermediate, which corresponds to the second case described above.

[0157] Therefore, the deuterium substitution type and ratio of the second intermediate can be predicted using the results of Table 2, as shown in Table 2a below.

[0158] Table 2a below summarizes both the actual experimental results and the predicted values.

[0159] [Table 2a]

[0160] From Table 2a, it can be seen that the ratio trends for each substitution number are the same between the actual experimental values ​​and the predicted values, and there is also little error in the average substitution rate values.

[0161] Prediction of the average deuterium substitution rate of the product [ka]

[0162] In the reaction, a product is formed by reaction of a first intermediate with a second intermediate.

[0163] The reactive site of the first intermediate is the site where Br was located, and the reactive site of the second intermediate is the site replaced by the tetramethyl-1,3,2-dioxaborolan compound, so this corresponds to the third case mentioned above.

[0164] Therefore, the deuterium substitution type and ratio of the product can be predicted using the results of Tables 1a and 2a, as shown in Table 3 below.

[0165] Table 3 below summarizes the actual experimental results and the predicted values.

[0166] [Table 3]

[0167] From Table 3, it can be seen that the ratio trends for each substitution number are the same between the actual experimental values ​​and the predicted values, and there is almost no error in the average substitution rate values.

Claims

1. A first step of obtaining a mass spectrum for a sample of an analyte containing one or more compounds selected from a set of chemical species represented by {C(0,n), C(1,n), C(2,n), C(3,n), ..., C(i,n), C(i+1,n), ..., C(n-1,n), C(n,n)}; a second step of expressing the mass spectrum of the sample substance as a linear combination of the mass spectra of each of the elemental compounds belonging to the set; A third step of calculating the relative content values ​​of each element compound belonging to the set from the coefficients of the linear combination. Deuterium substitution rate analysis method for sample material: Compound C(i, n) as each element constituting the set, C means that the compounds as the elements constituting the group are the same except for whether or not they are substituted with hydrogen or deuterium, n represents the total number of hydrogen and deuterium bonding sites in the compound C molecule; i means the number of deuterium substitutions in the element compounds included in the set; C(i,n) means a compound in which i out of a total of n hydrogen and deuterium bonding sites are substituted with deuterium.

2. 2. The method of claim 1, wherein the first step involves obtaining a mass spectrum by MALDI-TOF MS measurement using an inorganic oxide matrix.

3. 2. The method for analyzing a deuterium substitution rate of a sample substance according to claim 1, wherein the inorganic oxide matrix is ​​an inorganic oxide matrix containing one or more elements selected from the group consisting of silicon, zirconium, aluminum, and cerium.

4. 2. The method of claim 1, wherein the second step further comprises the step of eliminating isotope effects by utilizing a natural abundance ratio of the isotopes.

5. 2. The method of claim 1, wherein the second step represents the mass spectrum of the sample material as a linear combination of mass spectra of each element compound belonging to the set using least squares approximation.

6. 2. The method for analyzing a deuterium substitution rate of a sample material according to claim 1, further comprising a fourth step of calculating an average deuterium substitution rate of the sample material from the relative content values ​​of each element compound belonging to the set.

7. 7. The method for analyzing a deuterium substitution rate of a sample material according to claim 6, wherein the fourth step calculates an average deuterium substitution rate of the sample material using the following Equation 1: [Formula 1] 【Chemistry 1】 In the above formula 1, SD (%) is the average deuterium substitution rate of the sample material to be analyzed, n represents the total number of hydrogen and deuterium bonding sites in the compound C molecule; Pi is the relative amount (%) of compound C(i,n) in which i out of the total n hydrogen and deuterium bonding sites are substituted with deuterium.

8. The method for analyzing a deuterium substitution rate of a sample material according to claim 7, wherein Pi is calculated by the following Equation 2: [Formula 2] 【Chemistry 2】 In the above formula 2, P is the relative amount (%) of compound C(i,n) in which i out of n total hydrogen and deuterium bonding sites are substituted with deuterium; ai is the weight of the C(i,n) numerator.

9. measuring the average deuterium substitution rate of the first reactant by the method of claim 1; measuring the average deuterium substitution rate of the second reactant by the method of claim 1; and predicting a deuterium substitution rate of a reaction product obtained by the reaction of the first reactant and the second reactant using the average deuterium substitution rate of the first reactant and the average deuterium substitution rate of the second reactant; A method for predicting the deuterium substitution rate of reaction products.

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