CN112305007B - Method for measuring deuterium isotope abundance of deuterium labeled compound by using nuclear magnetic hydrogen spectrum or deuterium spectrum - Google Patents
Method for measuring deuterium isotope abundance of deuterium labeled compound by using nuclear magnetic hydrogen spectrum or deuterium spectrum Download PDFInfo
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Abstract
The invention relates to a method for measuring deuterium isotope abundance of a deuterium labeled compound by utilizing a nuclear magnetic hydrogen spectrum or a deuterium spectrum, which comprises the following steps of: (1) Weighing a deuterium labeled compound, an internal standard substance and a deuterated solvent, and filling into a nuclear magnetic tube; (2) Then placing the nuclear magnetic tube into a sample injector of a nuclear magnetic instrument, and running and performing sample testing; (3) And (4) performing peak area integration on the tested nuclear magnetic spectrum, and calculating to obtain the deuterium isotope abundance of the deuterium labeled compound according to the integrated peak area. Compared with the prior art, the method has the advantages of simple operation process, wide application range and accurate test data.
Description
Technical Field
The invention belongs to the technical field of deuterium isotope abundance, and relates to a method for measuring deuterium isotope abundance of a deuterium labeled compound by using a nuclear magnetic hydrogen spectrum or a deuterium spectrum.
Background
The deuterium labeling reagent with stable isotope is an essential reagent in scientific research work, and besides being used in a large amount of nuclear magnetic solvents, deuterium labeling compounds are also widely applied to the fields of proteomics (deuterium labeling amino acids, used for SILAC quantitative detection and the like), newborn metabolic genetic disease screening (deuterium labeling compounds such as carnitine and the like), deuterium labeling medicines (2017 year deuterium labeling tetrabenazine (SD-809) obtains FDA approval for marketing), food safety (deuterium labeling internal standard reagent for detecting pesticide and veterinary medicine residues), molecular building blocks (deuterium labeling basic reagent is used for research and development of new medicines and customized reagents), modern agriculture (deuterium labeling endogenous plant hormones such as jasmonic acid and the like) and the like.
At present, methods for detecting the isotopic abundance of deuterium labeled compounds mainly comprise stable isotope ratio mass spectrometry, gas chromatography, infrared spectrometry, raman spectrometry, mass spectrometry and nuclear magnetic resonance spectrometry, wherein the former four methods have relatively narrow application ranges, and the mass spectrometry is the most common test means at present. However, because the polarity, boiling point and other properties of the compounds are different, some compounds cannot be accurately calculated by conventional mass spectrometry. Compared with mass spectrometry, nuclear magnetism can accurately reflect the chemical environment of deuterium atoms and the structural site information of deuterium atoms. This property provides technical support for the research and development of multi-site deuterium labeled compounds with site information.
The NMR spectroscopy mainly uses Quantitative NMR (qNMR) technique, wherein qNMR is to measure hydrogen in deuterium-labeled compound by integral area of spectral peak by internal standard method 1 H) The isotopic abundance of the deuterium labeled compound is further obtained, and simultaneously, the information of the deuterium labeled site can also be obtained.
Goldbolat M et al (Goldbolat M, jones W H. Detecting CONTENT OF THE same deuterorium CONTENT OF A same OF deuterorium OXIDE BY THE same METHODS [ J].Analytical Chemistry,1964,36 (2): 431-432.) was used in 1964 1 H NMR detects deuterium isotope abundance of the heavy water, only proves that nuclear magnetism can carry out deuterium isotope abundance test, and specific test conditions are not given. Ma X, deng et al [ Ma X, deng P, wang X, et al].Talanta,2012:450-455.]In 2012 use 1 H NMR or 2 H NMR also detected deuterium isotope abundance in heavy water, ranging from natural abundance (0.015tom% D) to 100%, with recovery rates of 95% -110%, enabling rapid detection of heavy water. The existing deuterium isotope abundance nuclear magnetic resonance detection method is found through retrieval and mainly develops around heavy water, a known deuterium isotope abundance value and a measured relative peak area are used for carrying out linear fitting to obtain a calibration curve, the relative peak area measured by a measured object is taken into the calibration curve to obtain the deuterium isotope abundance value, a relative quantification method is mainly adopted, meanwhile, the internal standard substance is single in selection, when the internal standard substance or the measured object is replaced, the detection method needs to be re-optimized, and the influence of different internal standard substances on a quantification result is not researched.
Disclosure of Invention
The invention aims to provide a method for measuring deuterium isotope abundance of a deuterium labeled compound by utilizing a nuclear magnetic hydrogen spectrum or a deuterium spectrum, so as to simplify the test process and improve the accuracy and the application range of test data.
The purpose of the invention can be realized by the following technical scheme:
a method for determining deuterium isotope abundance of a deuterium labeled compound using nuclear magnetic hydrogen spectroscopy or deuterium spectroscopy, comprising the steps of:
(1) Weighing a deuterium labeled compound, an internal standard substance and a deuterated solvent, and filling into a nuclear magnetic tube;
(2) Then placing the nuclear magnetic tube into a sample injector of a nuclear magnetic instrument, and running and performing sample testing;
(3) And performing peak area integration on the tested nuclear magnetic spectrum, and calculating to obtain the deuterium isotope abundance of the deuterium labeled compound according to the integrated peak area.
Further, the deuterium labeled compound is selected from the group consisting of deuterium oxide, deuterium-oxygenated water, deuterium methanol, deuterium ethanol, deuterium acetone, deuterium benzene, deuterium toluene, deuterium chloroform, deuterium dimethyl sulfoxide, deuterium N, N-dimethylformamide, deuterium iodomethane, deuterium iodobenzene, deuterium bromobenzene, deuterium chlorobenzene, dimethyl phthalate-D 4 One or more of deuterated acetonitrile, deuterated tetrahydrofuran, deuterated o-xylene or a compound containing a deuterium atom.
Further, the internal standard substance is one or more of N, N-dimethylformamide, maleic acid, fumaric acid, acetic acid, dimethyl sulfone, ethanol, acetone, 1, 2, 4-trimethoxybenzene, ethyl p-hydroxybenzoate, dimethyl phthalate, toluene, acetic acid, benzoic acid, deuterated methanol, deuterated ethanol, deuterated acetone, deuterated iodobenzene, deuterated N, N-dimethylformamide, heavy water, deuterated toluene, deuterated dimethyl sulfoxide, deuterated acetonitrile, deuterated tetrahydrofuran and deuterated o-xylene compounds.
Further, the deuterated solvent is one or more of deuterium oxide, deuterated methanol, deuterated ethanol, deuterated acetone, deuterated benzene, deuterated toluene, deuterated chloroform, deuterated dimethyl sulfoxide, deuterated N, N-dimethylformamide, deuterated iodomethane, deuterated iodobenzene, deuterated bromobenzene, deuterated chlorobenzene, deuterated acetonitrile or deuterated tetrahydrofuran.
Further, in the step (1), the sampling amount of the deuterium labeled compound is 1mg to 1000mg, correspondingly, the sampling amount of the internal standard substance is 0mg to 1000mg, and the sampling amount of the deuterated solvent is 0mg to 1000mg. When the sample amount of the internal standard substance or the deuterated solvent is 0, it indicates that the internal standard substance or the deuterated solvent is not required to be added at this time. Mixing a precisely weighed sample and an internal standard substance to prepare a solution, and calculating the deuterium content of the sample by comparing the peak area of a specified peak of the sample with the peak area of the internal standard substance, wherein the internal standard substance plays a role in quantification; the deuterated solvent is liquid, and is used for dissolving the sample and the internal standard substance into the liquid and simultaneously serving as nuclear magnetism calibration. An internal standard substance is not required to be added when a part of deuterium labeled compounds are tested, and the internal standard substance is required to be added when all deuterium labeled compounds are tested; when the tested sample is solid, the deuterated solvent is required to be added, and when the tested sample is solution, the deuterated solvent can be not added.
Further, in the step (2), the operating parameters of the nuclear magnetic instrument are as follows: the relaxation delay time is 0.1 s-10 h; the pulse angle is 30-90 ℃; the scanning times are 1 to 2000; detecting frequency: 400 MHz-1000 MHz; the spectrum width is as follows: -2.5ppm to 12.5ppm; collecting time: 0.5 s-10 s.
Further, the nuclear magnetic spectrum tested is nuclear magnetic hydrogen spectrum or nuclear magnetic deuterium spectrum.
Furthermore, the nuclear magnetic spectrum obtained by the test is a nuclear magnetic hydrogen spectrum, and is suitable for measuring a part of deuterium-labeled compounds, and the calculation formula corresponding to the deuterium isotope abundance value of the deuterium-labeled compounds is as follows:
wherein D is i Is the deuterium isotope abundance value of the deuterium-labelled compound, unit atom% D; n is a radical of x Specifying the number of hydrogen atoms of the peak for the deuterium-labeled compound; n is a radical of y Designating the number of hydrogen atoms of the peak for the non-labeled moiety; I.C. A x With specified peaks for deuterium labeling 1 H NMR peak area; I.C. A y Assigning peaks to non-labelled moieties 1 H NMR peak area.
For example: CH (CH) 3 CH 2 OD，N x Is 1; n when methyl is selected as the designated peak y Is 3, N when methylene is selected as the designated peak y Is 2.
Furthermore, the nuclear magnetic spectrum obtained by testing is a nuclear magnetic hydrogen spectrum, and is suitable for measuring all deuterium-labeled compounds, and the calculation formula corresponding to the deuterium isotope abundance value of the deuterium-labeled compound is as follows:
wherein D is i Is the deuterium isotope abundance value of the deuterium-labelled compound, in units of atom% D; p is the true content value of deuterium labeled compound (by convention of chromatography, density, DSC, etc.)Test means acquisition); h y Assigning peaks to non-labeled moieties 1 H NMR detected content value;
wherein H y Assigning peaks to non-labeled moieties 1 H NMR detected content value; m is Inner part Is the mass of the internal standard substance, unit mg; m is y Is the mass of the deuterium labeled compound, in mg; n is a radical of Inner part Designating the number of hydrogen atoms of the peak for the internal standard substance; n is a radical of y Designating the number of hydrogen atoms of the peak for the non-labeled moiety; m Inner part Is the molecular weight of the internal standard substance; m y Is the molecular weight of the deuterium-labeled compound not labeled with deuterium; i is Inner part With peaks assigned to internal standard substances 1 H NMR integrated area; i is y Assigning peaks to non-labelled moieties 1 H NMR integrated area; h Inner part Is the internal standard purity.
For example: the sample is iodobenzene-D 5 The internal standard substance is maleic acid; m is a unit of Inner part And m y The peak N is obtained by weighing with a balance and is specified by maleic acid Inner part 2, iodobenzene designates peak N y Is 5, maleic acid molecular weight M Inner part 116.07, iodobenzene molecular weight M y Is 198.21 and I Inner part And I y Is the nuclear magnetic peak area, H Inner part The purity of the maleic acid is 99.4 percent, and P is iodobenzene-D 5 And (4) purity.
Furthermore, the nuclear magnetic spectrum obtained by the test is a nuclear magnetic hydrogen spectrum, and the calculation formula of the deuterium isotope abundance value of the deuterium labeled compound is as follows:
wherein D is i Is the deuterium isotope abundance value of the deuterium-labelled compound, in units of atom% D; m is Inner part The mass of the deuterium labeled internal standard substance is in mg; m is D Mass in mg of deuterium labelled compound; n is a radical of Inner part Designating the number of deuterium atoms of a peak for deuterium labeling an internal standard substance; n is a radical of D Assigning a peak deuterium atom number to the deuterium-labelled moiety; m Inner part Labeling the molecular weight of the internal standard substance for deuterium; m D Is the molecular weight of the deuterium-labelled compound; i is Inner part Specifying peaks for deuterium-labelled internal standard substances 2 H NMR integrated area; I.C. A D With peaks assigned to deuterium-labelled moieties 2 H NMR integrated area.
For example: sample is dimethyl sulfoxide-D 6 The internal standard substance is deuterated ethanol, m Inner part And m D Obtaining a specified peak N of deuterated ethanol methyl by balance weighing Inner part Is 3, dimethyl sulfoxide-D 6 Assigned peak N D Is 6, molecular weight M of deuterated ethanol Inner part Is 52.11, dimethyl sulfoxide-D 6 Molecular weight M D Is 84.17, I Inner part And I D The area of the nuclear magnetic peak.
The invention adopts an absolute quantitative method to measure hydrogen (in) in a deuterium-labeled compound through the integral area of a spectrum peak 1 H) The deuterium isotope abundance value is indirectly obtained, and besides, the information of the deuterium labeling site can be obtained. Based on 2 The quantitative nuclear magnetic resonance technology of H NMR adopts an absolute quantitative method to measure the content of deuterium (D) in a deuterium-labeled compound through the integral area of a spectrum peak, and directly obtains the deuterium isotope abundance value. The invention does not need to re-optimize the detection method and linearly fit the calibration curve while replacing the internal standard substance, so the detection technology provided by the invention has wide application range.
Compared with the prior art, the invention has the following advantages:
(1) Compared with the conventional mass spectrometry, due to different properties such as polarity, boiling point and the like of the compounds, some compounds (deuterated benzene, deuterated methanol, deuterated ethanol and the like) cannot be accurately calculated by the conventional mass spectrometry. Compared with mass spectrometry, nuclear magnetism can accurately reflect the chemical environment of deuterium atoms and the structural site information of deuterium atoms.
(2) The existing nuclear magnetic detection method mainly comprises the steps of testing around heavy water, utilizing a relative method to establish a fitting standard curve to carry out quantitative analysis, and needing to optimize the detection method again when an internal standard substance or a detected deuterium labeled compound is replaced and to fit the standard curve again at the same time. The invention utilizes an absolute method, and does not need to re-optimize the detection method and linearly fit a calibration curve when replacing an internal standard substance or a detected deuterium-labeled compound, so the detection technology provided by the invention has wide application range.
Detailed Description
The present invention will be described in detail with reference to specific examples. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the scope of the present invention is not limited to the following embodiments.
In the following embodiments or examples, the nuclear magnetic instrument used, i.e., JEOL JNM-ECZ 500MHz superconducting nuclear magnetic resonance spectrometer, is a conventional commercial product or conventional commercial raw material in the art, unless otherwise specified.
A method for determining deuterium isotope abundance of a deuterium labeled compound using nuclear magnetic hydrogen spectroscopy or deuterium spectroscopy, comprising the steps of:
(1) Weighing a deuterium labeled compound, an internal standard substance and a deuterated solvent, and filling into a nuclear magnetic tube;
(2) Then placing the nuclear magnetic tube into a sample injector of a nuclear magnetic instrument, and running and performing sample testing;
(3) And performing peak area integration on the tested nuclear magnetic spectrum, and calculating to obtain the deuterium isotope abundance of the deuterium labeled compound according to the integrated peak area.
In a specific embodiment of the present invention, the deuterium labeled compound is selected from the group consisting of deuterium oxide, deuterium-oxygenated water, deuterated methanol, deuterated ethanol, deuterated acetone, deuterated benzene, deuterated toluene, deuterated chloroform, deuterated dimethyl sulfoxide, deuterated N, N-dimethylformamide, deuterated iodomethane, deuterated iodobenzene, deuterated bromobenzene, deuterated chlorobenzene, dimethyl phthalate-D 4 One or more of deuterated acetonitrile, deuterated tetrahydrofuran, deuterated o-xylene or a compound containing a deuterium atom.
In a specific embodiment of the present invention, the internal standard is one or more of N, N-dimethylformamide, maleic acid, fumaric acid, acetic acid, dimethyl sulfone, ethanol, acetone, 1, 2, 4-trimethoxybenzene, ethyl p-hydroxybenzoate, dimethyl phthalate, toluene, acetic acid, benzoic acid, deuterated methanol, deuterated ethanol, deuterated acetone, deuterated iodobenzene, deuterated N, N-dimethylformamide, heavy water, deuterated toluene, deuterated dimethyl sulfoxide, deuterated acetonitrile, deuterated tetrahydrofuran, and deuterated o-xylene compound.
In a specific embodiment of the present invention, the deuterated solvent is one or more of deuterium oxide, deuterated methanol, deuterated ethanol, deuterated acetone, deuterated benzene, deuterated toluene, deuterated chloroform, deuterated dimethyl sulfoxide, deuterated N, N-dimethylformamide, deuterated iodomethane, deuterated iodobenzene, deuterated bromobenzene, deuterated chlorobenzene, deuterated acetonitrile or deuterated tetrahydrofuran.
In a specific embodiment of the present invention, in step (1), the deuterium labeled compound is sampled at 1mg to 1000mg, and correspondingly, the internal standard is sampled at 0mg to 1000mg, and the deuterated solvent is sampled at 0mg to 1000mg. When the sample amount of the internal standard substance or the deuterated solvent is 0, it indicates that the internal standard substance or the deuterated solvent is not required to be added at this time.
Mixing a precisely weighed sample and an internal standard substance to prepare a solution, and calculating the deuterium content of the sample by comparing the peak area of a specified peak of the sample with the peak area of the internal standard substance, wherein the internal standard substance plays a role in quantification; the deuterated solvent is liquid, and is used for dissolving the sample and the internal standard substance into the liquid and simultaneously serving as nuclear magnetism calibration. An internal standard substance is not required to be added when a part of deuterium labeled compounds are tested, and the internal standard substance is required to be added when all deuterium labeled compounds are tested; when the tested sample is solid, the deuterated solvent is required to be added, and when the tested sample is solution, the deuterated solvent can be not added.
In a specific embodiment of the present invention, in step (2), the operating parameters of the nuclear magnetic instrument are: the relaxation delay time is 0.1 s-10 h; the pulse angle is 30-90 ℃; the scanning times are 1 to 2000; detecting frequency: 400 MHz-1000 MHz; spectrum width: -2.5ppm to 12.5ppm; collecting time: 0.5 s-10 s.
In a specific embodiment of the invention, the nuclear magnetic spectrum tested is nuclear magnetic hydrogen spectrum or nuclear magnetic deuterium spectrum.
In a more specific embodiment, the nuclear magnetic spectrum obtained by the test is a nuclear magnetic hydrogen spectrum, and is suitable for the determination of a part of deuterium-labeled compound, and the calculation formula corresponding to the deuterium isotope abundance value of the deuterium-labeled compound is as follows:
wherein D is i Is the deuterium isotope abundance value of the deuterium labeled compound, in units of atom% D; n is a radical of hydrogen x Specifying the number of hydrogen atoms of the peak for the deuterium-labeled compound; n is a radical of y Designating the number of hydrogen atoms of the peak for the non-labeled moiety; I.C. A x With specified peaks for deuterium labeling 1 H NMR peak area; I.C. A y Assigning peaks to non-labelled moieties 1 H NMR peak area.
For example: CH (CH) 3 CH 2 OD，N x Is 1; n when methyl is selected as the designated peak y Is 3, N when methylene is selected as the designated peak y Is 2.
In a more specific embodiment, the nuclear magnetic spectrum obtained by the test is a nuclear magnetic hydrogen spectrum, and is suitable for the determination of all deuterium-labeled compounds, and the calculation formula corresponding to the deuterium isotope abundance value of the deuterium-labeled compound is as follows:
wherein D is i Is the deuterium isotope abundance value of the deuterium-labelled compound, in units of atom% D; p is the true content value of the deuterium-labelled compound (obtained by conventional testing means such as chromatography, density, DSC, etc.); h y Assigning peaks to non-labeled moieties 1 H NMR detected content value;
wherein H y Assigning peaks to non-labeled moieties 1 Content by H NMRA value; m is Inner part Is the mass of the internal standard substance, unit mg; m is a unit of y Is the mass of the deuterium labeled compound, in mg; n is a radical of Inner part Designating the number of hydrogen atoms of the peak for the internal standard substance; n is a radical of y Designating the number of hydrogen atoms of the peak for the non-labeled moiety; m Inner part Is the molecular weight of the internal standard substance; m y Is the molecular weight of the deuterium-labeled compound not labeled with deuterium; i is Inner part Specifying peaks for internal standard substances 1 H NMR integrated area; i is y Assigning peaks to non-labelled moieties 1 H NMR integrated area; h Inner part Is the internal standard purity.
For example: sample was iodobenzene-D 5 The internal standard substance is maleic acid; m is Inner part And m y The specified peak N of the maleic acid is obtained by weighing with a balance Inner part 2, iodobenzene designates peak N y 5, molecular weight of maleic acid M Inner part 116.07, iodobenzene molecular weight M y 198.21 and I Inner part And I y Is the nuclear magnetic peak area, H Inner part The purity of maleic acid is 99.4 percent, and P is iodobenzene-D 5 And (4) purity.
In a more specific embodiment, the nuclear magnetic spectrum obtained by the test is a nuclear magnetic spectrum, and the calculation formula of the deuterium isotope abundance value of the deuterium-labeled compound is as follows:
wherein D is i Is the deuterium isotopic abundance value of the deuterium-labelled compound, in units of atom% D; m is Inner part The mass of the deuterium labeled internal standard substance is in mg; m is D Is the mass of the deuterium labeled compound, in mg; n is a radical of Inner part Designating the number of deuterium atoms of a peak for deuterium labeling an internal standard substance; n is a radical of hydrogen D Designating the number of deuterium atoms of the peak for the deuterium-labeled moiety; m Inner part Labeling the molecular weight of the internal standard substance for deuterium; m D Is the molecular weight of the deuterium-labelled compound; i is Inner part Specifying peaks for deuterium-labelled internal standard substances 2 H NMR integrated area; I.C. A D With peaks assigned to deuterium-labelled moieties 2 H NMR integrated area.
For example: sample is dimethyl sulfoxide-D 6 The internal standard substance is deuterated ethanol, m Inner part And m D Obtaining a specified peak N of deuterated ethanol methyl by balance weighing Inner part Is 3, dimethyl sulfoxide-D 6 Assigned peak N D Is 6, molecular weight M of deuterated ethanol Inner part Is 52.11, dimethyl sulfoxide-D 6 Molecular weight M D Is 84.17, I Inner part And I D The area of the NMR peak was determined.
The above embodiments may be implemented individually, or in any combination of two or more.
The above embodiments will be described in more detail with reference to specific examples.
Example 1:
quantitative partial deuterium-labeled compound hydrogen spectra-ethanol-D (commercially available deuterium labeling chemical, deuterium isotopic abundance value 99.5atom% D) isotopic abundance analysis, comprising the steps of:
(1) Weighing 500mg of ethanol-D and putting into a nuclear magnetic tube;
(2) Setting nuclear magnetic instrument parameters, wherein the relaxation delay time is 5s, the pulse angle is 30 ℃, the scanning times are 32 times, the detection frequency is 500MHz, and the spectrum width is as follows: -2.5-12.5 ppm, acquisition time: 2.1s, placing the nuclear magnetic tube into a sample injector, operating a nuclear magnetic instrument, and performing sample injection test;
(3) Performing peak area integration on the spectrogram of the tested parallel sample by utilizing nuclear magnetic software, wherein the first group is as follows: n is a radical of x Is 1, N y Is 3; I.C. A x Is 0.52, I y Is 300; second group: n is a radical of x Is 1, N y Is 3; i is x Is 0.48, I y Is 300;
substituting the integrated peak area into a formula
Calculating the deuterium isotope abundance of the parallel samples as 99.48atom% D, 99.52atom% D, the average value is 99.5atom% D, respectively.
The measured data are compared with the marked value of a commercially available reagent ethanol-D, and the result is basically consistent, so that the isotope abundance measuring method also has very high measuring accuracy.
Example 2:
quantitative determination of hydrogen spectra of partially deuterium labeled compounds-dimethyl phthalate-D 4 Isotopic abundance analysis, comprising the steps of:
(1) 20mg of dimethyl phthalate-D are weighed 4 500mg of deuterated ethanol is filled into a nuclear magnetic tube;
(2) Setting nuclear magnetic instrument parameters, wherein the relaxation delay time is 50s, the pulse angle is 60 ℃, the scanning times are 1600 times, the detection frequency is 500MHz, and the spectrum width is as follows: 0ppm to 15ppm, acquisition time: 3.8s, placing the nuclear magnetic tube into a sample injector, operating a nuclear magnetic instrument, and performing sample injection test;
(3) Performing peak area integration on the spectrogram of the tested parallel sample by utilizing nuclear magnetic software, wherein the first group is as follows: n is a radical of hydrogen x Is 4, N y Is 6; i is x Is 3.55, I y Is 600; second group: n is a radical of hydrogen x Is 4, N y Is 6; i is x Is 3.48, I y Is 600;
substituting the integrated peak area into a formula
The calculated deuterium isotopic abundance of the replicates was 99.11tom% and 99.13tom% respectively, with an average of 99.1tom% and d.
And for the same dimethyl phthalate-D 4 The results of parallel tests using the existing mass spectrometry were 99.08atom% D and 99.00atom% D, respectively, and the average value was 99.0atom% D, and the results of the measurements by the two methods were substantially identical, and it was found that the method for measuring isotopic abundance of the present invention also had very high measurement accuracy.
Example 3:
total deuterium labeled compound hydrogen spectra quantitation iodobenzene-D 5 Isotopic abundance analysis, comprising the steps of:
(1) Respectively weighing 300mg of iodobenzene-D 5 10mg of maleic acid and 300mg of deuterated acetone-methanol are filled into a nuclear magnetic tube;
(2) Setting nuclear magnetic instrument parameters, wherein the relaxation delay time is 6000s, the pulse angle is 30 ℃, the scanning times are 10 times, the detection frequency is 500MHz, and the spectrum width is as follows: 0 ppm-10 ppm, acquisition time: 6.6s, placing the nuclear magnetic tube into a sample injector, operating a nuclear magnetic instrument, and performing sample injection test;
(3) Performing peak area integration on the spectrogram of the tested parallel sample by utilizing nuclear magnetic software, wherein the first group is as follows: m is a unit of Inner part 10.1mg, m y 300.4mg, N Inner part Is 2, N y Is 5, M Inner part Is 116.07, M y 198.21 and I Inner part Is 2000, I y Is 336.9, H Inner part 99.4% of P and 99.2% of P; second group: m is Inner part 10.7mg, m y 300.9mg, N Inner part Is 2, N y Is 5, M Inner part Is 116.07, M y Is 198.21 and I Inner part Is 2000, I y Is 348.1 and H Inner part 99.4% of P and 99.2% of P; substituting the integrated peak area into a formula
And formula
Calculating the deuterium isotope abundance of the parallel samples as 99.61atom% D, 99.58atom% D, respectively, the average value as 99.6atom% D.
Likewise, p-iodobenzene-D 5 The results of the tests carried out by the existing mass spectrometry were 99.60tom% and 99.64tom% respectively, and the average 99.6tom% was 99.60tom% and 99.64tom% respectively, and the results of the measurements by the two methods were substantially identical, and it was found that the method for measuring isotopic abundance of the present invention also had very high measurement accuracy.
Example 4:
total deuterium labeled compound hydrogen spectra quantification-deuterated methanol-D 4 (commercial deuterium labeling chemical reagent, deuterium isotopic abundance value 99.8atom% d) isotopic abundance analysis comprising the steps of:
(1) 400mg of methanol-D are respectively weighed 4 200mg of N, N-dimethylformamide and 100mg of deuterated chloroform are filled into a nuclear magnetic tube;
(2) Setting nuclear magnetic instrument parameters, wherein the relaxation delay time is 600s, the pulse angle is 45 ℃, the scanning times are 32 times, the detection frequency is 600MHz, and the spectrum width is as follows: -2.5ppm to 10ppm, acquisition time: 5.2s, placing the nuclear magnetic tube into a sample injector, operating a nuclear magnetic instrument, and performing sample injection test;
(3) Performing peak area integration on the spectrogram of the tested parallel sample by utilizing nuclear magnetic software, wherein the first group is as follows: m is a unit of Inner part Is 201.4mg, m y 413.5mg, N Inner part Is 6, N y Is 3, M Inner part Is 73.09, M y Is 32.04 and I Inner part Is 6000, I y Is 56.15 and H Inner part 99.5% of P and 99.8% of P; second group: m is Inner part Is 217.9mg, m y 417.8mg, N Inner part Is 6, N y Is 3, M Inner part Is 73.09, M y Is 32.04 and I Inner part Is 6000, I y Is 52.19 and H Inner part 99.5% of P and 99.8% of P; substituting the integrated peak area into a formula
And formula
The calculated deuterium isotopic abundance of the parallel samples was 99.60tom% D, and the average value was 99.6 tom% D, respectively.
The above test data were compared with commercially available deuterated methanol-D 4 The labeled values of the reagents are compared, and the results are equivalent, so that the isotope abundance measuring method also has very high measuring accuracy.
Example 5:
all deuterium-labelled compound hydrogen spectra quantification-heavy water (commercial deuterium-labelled chemical reagent, deuterium isotope abundance value 99.8 atom%) isotope abundance analysis, comprising the steps of:
(1) Respectively weighing 400mg of heavy water and 5mg of dimethyl sulfoxide, and filling into a nuclear magnetic tube;
(2) Setting nuclear magnetic instrument parameters, wherein the relaxation delay time is 5s, the pulse angle is 90 ℃, the scanning times are 16 times, the detection frequency is 400MHz, and the spectrum width is as follows: 0ppm to 12.5ppm, acquisition time: 1s, placing the nuclear magnetic tube into a sample injector, operating a nuclear magnetic instrument, and performing sample injection test;
(3) After testing by nuclear magnetic softwarePeak area integration was performed on the spectra of the replicates of (a) first set: m is a unit of Inner part 5.1mg, m y 431.8mg, N Inner part Is 6, N y Is 2, M Inner part Is 78.13, M y Is 18.02, I Inner part Is 600, I y Is 83.15, H Inner part 99.9% of P and 99.5% of P; second group: m is a unit of Inner part Is 6.1mg, m y 424.7mg, N Inner part Is 6, N y Is 2, M Inner part Is 78.13, M y Is 18.02, I Inner part Is 600, I y Is 47.93, H Inner part 99.9% of P and 99.5% of P; substituting the integrated peak area into a formula
And formula
Calculating the deuterium isotope abundance of the parallel samples as 99.89atom% D, 99.92atom% D, respectively, the average value is 99.9atom% D.
Meanwhile, the above-mentioned heavy water raw materials were tested by the conventional nuclear magnetic method to obtain the results of parallel tests of 99.88atom% D and 99.86atom% D, respectively, and the average value of 99.9atom% D, respectively, and the results of the measurements by the two methods were substantially identical and also equivalent to the values indicated by commercially available reagents, and it was found that the method for measuring isotopic abundance of the present invention also had very high measurement accuracy.
Example 6:
deuterium spectrometry-benzene-D 6 (commercial deuterium labeling chemical reagent, deuterium isotope abundance value 99.5atom% d) isotopic abundance analysis comprising the steps of:
(1) 300mg of acetone-D was weighed out separately 6 300mg of deuterated toluene is put into a nuclear magnetic tube;
(2) Setting nuclear magnetic instrument parameters, wherein the relaxation delay time is 400s, the pulse angle is 30 ℃, the scanning times are 200 times, the detection frequency is 500MHz, and the spectrum width is as follows: 0ppm to 14.5ppm, acquisition time: 2.8s, placing the nuclear magnetic tube into a sample injector, operating a nuclear magnetic instrument, and performing sample injection test;
(3) Performing peak area on spectrogram of the tested parallel sample by utilizing nuclear magnetic softwareIntegration, first set: m is Inner part 315.8mg, m D 338.5mg, N Inner part Is 5, N D Is 6, M Inner part Is 100.19, M D Is 64.12, I Inner part Is 500, I D Is 1002; second group: m is a unit of Inner part 323.7mg, m D 339.8mg of N Inner part Is 5, N D Is 6, M Inner part Is 100.19, M D Is 64.12 and I Inner part Is 500, I D Is 978; substituting the integrated peak area into a formula
The calculated deuterium isotope abundances of the replicates were 99.61atom% D, 99.28atom% D, and the average was 99.4atom% D, respectively.
The above test data were compared with commercially available benzene-D 6 The comparison of the reagent mark values shows that the results are equivalent, and the method for measuring the isotopic abundance has very high measurement accuracy.
Example 7:
deuterium spectrometry-dimethyl sulfoxide-D 6 (commercial deuterium labeling chemistry, deuterium isotopic abundance value 99.9atom% d) isotopic abundance analysis comprising the steps of:
(1) 100mg of dimethyl sulfoxide-D were weighed out separately 6 600mg of deuterated ethanol is filled into a nuclear magnetic tube;
(2) Setting nuclear magnetic instrument parameters, wherein the relaxation delay time is 30s, the pulse angle is 30 ℃, the scanning times are 100 times, the detection frequency is 500MHz, and the spectrum width is as follows: -2.5-12.5 ppm, acquisition time: 4.1s, placing the nuclear magnetic tube into a sample injector, operating a nuclear magnetic instrument, and performing sample injection test;
(3) Performing peak area integration on the spectrogram of the tested parallel sample by utilizing nuclear magnetic software, wherein the first group is as follows: m is Inner part 95.4mg, m D 579.3mg, N Inner part Is 3, N D Is 6, M Inner part Is 52.11, M D Is 84.17, I Inner part Is 300, I D Is 2252; second group: m is Inner part 93.8mg, m D 592.1mg, N Inner part Is 3, N D Is 6, M Inner part Is 52.11, M D Is 84.17, I Inner part Is 300, I D Is 2343; substituting the integrated peak area into a formula
The deuterium isotopic abundance of the parallel samples was calculated as 99.84atom% D, 99.92atom% D, and the average value was 99.9atom% D, respectively.
The above test data were compared with commercially available dimethylsulfoxide-D 6 The results of comparison of the labeled values of the reagents are equivalent, and the method for measuring the isotopic abundance has very high measurement accuracy.
The embodiments described above are intended to facilitate a person of ordinary skill in the art in understanding and using the invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make modifications and alterations without departing from the scope of the present invention.
Claims (6)
1. A method for determining deuterium isotope abundance of a deuterium labeled compound by using nuclear magnetic hydrogen spectrum or deuterium spectrum, comprising the steps of:
(1) Weighing a deuterium labeled compound, an internal standard substance and a deuterated solvent, and filling into a nuclear magnetic tube;
(2) Then placing the nuclear magnetic tube into a sample injector of a nuclear magnetic instrument, and running and performing sample-in test;
(3) Performing peak area integration on the tested nuclear magnetic spectrum, and calculating to obtain deuterium isotope abundance of the deuterium labeled compound according to the integrated peak area;
the nuclear magnetic spectrum to be tested is nuclear magnetic hydrogen spectrum or nuclear magnetic deuterium spectrum;
the nuclear magnetic spectrum obtained by testing is a nuclear magnetic hydrogen spectrum and is suitable for measuring a part of deuterium-labeled compound, and the calculation formula corresponding to the deuterium isotope abundance value of the deuterium-labeled compound is as follows:
wherein D is i Is the deuterium isotope abundance value of the deuterium-labelled compound, unit atom% D; n is a radical of x Specifying the number of hydrogen atoms of the peak for the deuterium-labeled compound; n is a radical of y Designating the number of hydrogen atoms of the peak for the non-labeled moiety; i is x With specified peaks for deuterium labeling 1 H NMR peak area; I.C. A y Assigning peaks to non-labelled moieties 1 H NMR peak area;
or the nuclear magnetic spectrum obtained by testing is a nuclear magnetic hydrogen spectrum and is suitable for measuring all deuterium-labeled compounds, and the calculation formula corresponding to the deuterium isotope abundance value of the deuterium-labeled compounds is as follows:
wherein D is i Is the deuterium isotopic abundance value of the deuterium-labelled compound, in units of atom% D; p is the true content value of the deuterium labeled compound; h y Assigning peaks to non-labeled moieties 1 H NMR detected content value;
wherein H y Assigning peaks to non-labeled moieties 1 H NMR detected content value; m is a unit of Inner part The mass of the internal standard substance is in mg; m is a unit of y Is the mass of the deuterium labeled compound, in mg; n is a radical of Inner part Designating the number of hydrogen atoms of the peak for the internal standard substance; n is a radical of y Designating the number of hydrogen atoms of the peak for the non-labeled moiety; m is a group of Inner part Is the molecular weight of the internal standard substance; m y Is the molecular weight of the deuterium-labeled compound not labeled with deuterium; I.C. A Inner part With peaks assigned to internal standard substances 1 H NMR integrated area; I.C. A y Assigning peaks to non-labelled moieties 1 H NMR integrated area; h Inner part Is the internal standard purity;
or the nuclear magnetic spectrum obtained by testing is nuclear magnetic deuterium spectrum, the calculation formula of the deuterium isotope abundance value of the deuterium labeled compound is as follows:
wherein D is i Is the deuterium isotope abundance value of the deuterium-labelled compound, in units of atom% D; m is a unit of Inner part Labeling mass of internal standard substance for deuterium in mg; m is a unit of D Is the mass of the deuterium labeled compound, in mg; n is a radical of hydrogen Inner part Designating the number of deuterium atoms of a peak for deuterium labeling an internal standard substance; n is a radical of D Assigning a peak deuterium atom number to the deuterium-labelled moiety; m Inner part Labeling the molecular weight of the internal standard substance for deuterium; m is a group of D Is the molecular weight of the deuterium-labelled compound; i is Inner part Specifying peaks for deuterium-labelled internal standard substances 2 HNMR integrated area; I.C. A D With peaks assigned to deuterium-labelled moieties 2 H NMR integrated area.
2. The method of claim 1, wherein the deuterium labeled compound is selected from the group consisting of deuterium, deuterium-oxygenated water, deuterium-methanolic acid, deuterium-ethanolic acid, deuterium-acetone, deuterium-benzene, deuterium-toluene, deuterium-chloroform, deuterium-dimethyl sulfoxide, deuterium-N, N-dimethylformamide, deuterium-iodomethane, deuterium-iodobenzene, deuterium-bromobenzene, deuterium-chlorobenzene, dimethyl phthalate-D 4 One or more of deuterated acetonitrile, deuterated tetrahydrofuran, deuterated o-xylene or a compound containing a deuterium atom.
3. The method for determining deuterium isotope abundance of deuterium-labeled compound according to claim 1, wherein the internal standard is one or more of N, N-dimethylformamide, maleic acid, fumaric acid, acetic acid, dimethyl sulfone, ethanol, acetone, 1, 2, 4-trimethoxybenzene, ethyl p-hydroxybenzoate, dimethyl phthalate, toluene, acetic acid, benzoic acid, deuterated methanol, deuterated ethanol, deuterated acetone, deuterated iodobenzene, deuterated N, N-dimethylformamide, heavy water, deuterated toluene, deuterated dimethyl sulfoxide, deuterated acetonitrile, deuterated tetrahydrofuran, and deuterated o-xylene compound.
4. The method of claim 1, wherein the deuterated solvent is one or more of deuterium, deuterated methanol, deuterated ethanol, deuterated acetone, deuterated benzene, deuterated toluene, deuterated chloroform, deuterated dimethyl sulfoxide, deuterated N, N-dimethylformamide, deuterated iodomethane, deuterated iodobenzene, deuterated bromobenzene, deuterated chlorobenzene, deuterated acetonitrile or deuterated tetrahydrofuran.
5. The method for determining deuterium isotope abundance of deuterium-labeled compound according to claim 1, wherein in step (1), the sampling amount of deuterium-labeled compound is 1 mg-1000 mg, and correspondingly, the sampling amount of internal standard substance is 0 mg-1000 mg and the sampling amount of deuterated solvent is 0 mg-1000 mg.
6. The method for determining deuterium isotope abundance of deuterium labeled compound according to claim 1, wherein in step (2), the operating parameters of nuclear magnetic instrument are: the relaxation delay time is 0.1 s-10 h; the pulse angle is 30-90 degrees; the scanning times are 1 to 2000; detecting the frequency of 400 MHz-1000 MHz; the spectrum width is-2.5 ppm to 12.5ppm; the collection time is 0.5 s-10 s.
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