CN115266981A - Method for detecting substance deuteration rate by GC-MS - Google Patents
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Abstract
The invention relates to a method for detecting substance deuteration rate by GC-MS, preparing a certain amount of sample before deuteration and sample solution after deuteration to be detected, and detecting by GC-MS; after blank is deducted, integrating main peaks of the samples before and after deuteration, and extracting ions, response values and abundance percentages of the main peaks; and calculating each deuteration rate of the sample according to the main peak ions before and after the extraction of deuteration, the response value and the abundance percentage data thereof. Compared with the traditional nuclear magnetic method for evaluating the deuteration rate, the method has low requirements on instruments and equipment, can separate impurities by adjusting analysis parameters of a GC end, has no special requirements on the purity of an analysis sample, is simple in sample pretreatment, can evaluate the quantity ratio of deuterium contained in the compound, and enables the analysis result to be more accurate and reliable by using the modes of correcting a detection instrument by a sample before deuteration, deducting isotope interference and the like.
Description
Technical Field
The invention belongs to the field of mass analysis, and particularly relates to a method for detecting substance deuteration rate by GC-MS.
Background
Deuterium (D), also called deuterium, is a stable isotope of hydrogen (H), deuterium carries neutrons lacking hydrogen, the mass is 2 times of that of hydrogen, and the deuterium has chemical properties similar to that of hydrogen, but a carbon-deuterium bond is shorter than a carbon-hydrogen bond, and the low vibrational stretching frequency of the carbon-deuterium bond makes the ground state energy lower than that of the carbon-hydrogen bond, so that the structure is more stable.
The organic electroluminescent (OLED) display technology with the advantages of high resolution, fast response, wide viewing angle, low power consumption, multiple colors, flexible screen, wide requirement on environmental temperature and the like has the advantages of increasing the application proportion in the fields of electronic and electrical products, medical treatment, traffic, commerce and the like year by year, but has the defects of poor material stability, short service life and the like, limits the application of organic light emitting diodes, and is a feasible method by carrying out deuteration on organic light emitting diode materials in order to improve the stability of OLED materials. For the analysis of the deuteration rate, most of the literature is about the analysis of some deuteration solvents such as heavy water, and the analysis of the deuteration rate of organic compounds is only reported, and patent CN112946092A also discloses an evaluation method of the deuteration purity of the deuteration organic luminescent material, which combines three methods of nuclear magnetism, mass spectrum and ultraviolet; the disclosed data shows that the deuteration rate basically uses nuclear magnetism or even quantitative nuclear magnetism, the capital investment on instruments and equipment is large, the deuteration rate is evaluated by the nuclear magnetism, the purity requirement on the sample is high for reducing impurity interference analysis, expensive deuteration reagents and the like are needed, in addition, the possible deuterium numbers of the sample in the deuteration process are not different according to the desired number of deuterium, and meanwhile, the deuteration samples have different numbers of deuterium, such as only one deuterium is used for some molecules, two or three deuterium is used for some molecules, even one deuterium is not used for some molecules, and how to evaluate the number of the previous deuterium in the sample, the number of the two deuterium is used for the previous deuterium, and the detection method is not reported.
Disclosure of Invention
Aiming at the requirements in the prior art, the invention provides a method for detecting the deuteration rate of a substance by using GC-MS (gas chromatography-mass spectrometry).
The invention is realized by the following technical scheme:
a method for detecting substance deuteration rate by GC-MS comprises the following steps:
1) Weighing a certain amount of pre-deuterated samples and post-deuterated samples to be detected, adding a proper amount of solvent to completely dissolve the samples, and preparing a blank sample;
2) Taking a proper amount of the solution obtained in the step 1, filtering the solution with an organic filter membrane, taking the solution as an upper machine sample solution, and bottling for later use;
3) Detecting the sample solution in the step 2 by using GC-MS;
4) Integrating the main peaks of the samples before and after deuteration after deducting blank from the result obtained by GC-MS detection in the step 3, and extracting ions, response values and abundance percentages of the main peaks;
5) And 4, calculating the deuteration rate of the sample by using the main peak ions before and after deuteration, the response value and the abundance percentage data thereof extracted in the step 4.
The structure of the pre-deuterated sample is shown in the formula I, and the molecular formula is C22H26Theoretical m/z is 290.20,291.21 and 292.21 respectively; the abundance percentages are respectively 100.0%,24.1% and 2.8%;
the method in the step 1) comprises the steps of weighing about 10mg of a pre-deuterated sample and a to-be-tested post-deuterated sample respectively, adding a corresponding volume of solvent to dissolve the pre-deuterated sample and the to-be-tested post-deuterated sample, and preparing an upper machine solution of 1 mg/mL; the sample before deuteration is a sample left before the product is subjected to deuteration; the blank sample is a blank solution prepared in the same way as the sample preparation except that no sample is added.
The solvent is dichloromethane and tetrahydrofuran, the solution is bottled in a 20mL brown glass bottle, and the dissolution is shaking or ultrasonic wave till the sample is completely dissolved.
The solvent is dichloromethane.
The organic filter membrane is bottled by selecting a 0.45 mu m nylon needle filter, and the bottle is a 2mL brown sample bottle for chromatographic detection.
The GC-MS detection method in the step 3) has the following conditions: temperature of the gasification chamber: 320 ℃; column flow rate: 1.2ml/min; column temperature: keeping the temperature at 100 ℃ for 1min initially, heating to 250 ℃ at the speed of 10 ℃/min and keeping the temperature for 1min; heating to 320 deg.C at 20 deg.C/min and maintaining for 9.5min; the split ratio is 30:1; and (3) chromatographic column: agilent HP-5MS UI 30m 250 μm 0.25 μm; MS interface temperature: at 330 ℃; intuvo flow path: 320 ℃; the sample injection amount is as follows: 1 microliter. An ion source: EI; electron energy: 70eV, MS ion Source 230 ℃; 150 ℃ for an MS quadrupole rod; 3min, respectively cleaning 5 times by using solvents A (dichloromethane) and B (methanol) before and after sample injection, cleaning 3 times by using a sample to be injected before sample injection, and then pumping 4 times to remove bubbles of a sample injection needle and carry gas: high purity helium, ion scan range: 35-350.
The retention time of the main peak in the step 4) is 19.5 +/-0.5 min.
The result of the ion base peak of the main peak molecule before deuteration in the step 4) is recorded as M, and the result of the isotopic abundance percentage of the main peak molecule ion before deuteration is recorded as 100C.
The method for calculating the deuteration rate of the sample comprises the following 4 steps:
(1) According to the molecular ion radical peak M of the sample before deuteration, the obtained non-deuteration molecular ion radical peak M/z is as follows: m +0; the ion radical peak m/z of the 1 deuterated (upper 1 deuterium) molecule is as follows: m +1; the ion radical peak m/z of the 2 deuterated (upper 2 deuterated) molecule is as follows: m +2; the peak m/z of the ionic group of the 3-deuterated (upper 3-deuterated) molecule is as follows: m +3; the ion radical peak m/z of the 4-deutero (the upper 4 deutero) molecule is: m +4; the ion radical peak m/z of the 5 deuterated (upper 5 deuterated) molecule is as follows: m +5; 823060, 8230;
(2) Calculating the possible existence of the deuterium in the sampleThe peak response value of each deuteron is calculated by the formula: response of 0 deuterated peak M is A0(ii) a 1 deuterated Peak m/z response A1Response value-A of = M +10* B/100;2 deuterated peak m/z response A2= M +2 response-a0*C/100-A1* B/100;3 deuterated peak m/z response A3= M +3 response-a1*C/100-A2* B/100;4 deuterated peak m/z response A4= M +4 response-a2*C/100-A3* B/100;5 deuterated Peak m/z response A5= M +5 response-a3*C/100-A4* B/100\8230, 8230; until the result appears negative;
(3) The deuterated nonnegative base peak responses are then summed, i.e., A0+A1+A2+A3+A4+A5+……＝K；
(4) Calculating each deuteration ratio of the sample after deuteration: deuterated = a0100% K, 1 deuteration = a1100% of/K, 2 deuteration = a2100% of/K, 3 deuteration = a3100% of/K, 4 deuteration = a4100% K, 5 deuteration = a5/K*100％……。
Compared with the traditional nuclear magnetic method for evaluating the deuteration rate, the method has low requirement on instruments and equipment, can separate impurities by adjusting the analysis parameters of a GC end, has no special requirement on the purity of an analysis sample, has simple sample pretreatment, can evaluate the amount ratio of deuterium in the compound, and enables the analysis result to be more accurate and reliable by using the modes of correcting a detection instrument by the sample before deuteration, deducting isotope interference and the like.
Drawings
FIG. 1 is the main peak molecular ion of the pre-deuterated sample under the method of 1.3.1;
FIG. 2 is the main peak molecular ion of sample 1 after deuteration under 1.3.1 approach;
FIG. 3 is the main peak molecular ion of sample 2 after deuteration under 1.3.1 method;
FIG. 4 is a graph comparing HNMR before and after deuteration of compound 2;
figure 5 is a structural change before and after deuteration of the compound with 2;
FIG. 6 is a TIC mass spectrum of 2-deuterated samples under the 1.3.1 procedure.
Detailed Description
The present invention will be described in further detail with reference to examples.
Example 1
The invention provides a method for detecting substance deuteration rate by GC-MS, which is completed by the following specific steps:
the sample has a structure before deuteration, a theoretical m/z and abundance percentage shown as the following formula I, and the molecular formula is C22H26The theoretical m/z and abundance percentages are 290.20 (100.0%), 291.21 (24.1%), 292.21 (2.8%).
1.1 laboratory instruments and reagents:
intuvo 9000-5977B of Agilent gas chromatograph-mass spectrometer; a mertlettolidone in ten thousandth analytical balance; 0.45 μm organic filter membrane; HPLC grade methanol, dichloromethane; 2mL sample bottles and other common laboratory glassware.
1.2 sample pretreatment:
respectively weighing 10mg of each of a sample 1 and a sample 2 before and after deuteration by different methods in a 20mL brown glass bottle, adding 10mL of dichloromethane, slightly shaking or carrying out ultrasound till complete dissolution, taking a proper amount of organic filter membrane as a sample solution, bottling and testing, wherein the sample concentration is 1mg/mL, and simultaneously preparing a sample blank.
1.3GC-MS method confirmation
1.3.1 the sample solution and blank obtained in step 1.2 are detected by the following conditions, wherein the GC-MS method comprises the following steps: temperature of the gasification chamber: 320 ℃; column flow rate: 1.2ml/min; column temperature: keeping the temperature at 100 ℃ for 1min initially, heating to 250 ℃ at the speed of 10 ℃/min and keeping the temperature for 1min; heating to 320 deg.C at 20 deg.C/min and maintaining for 9.5min; the split ratio is 30:1; a chromatographic column: agilent HP-5MS UI 30m 250 μm 0.25 μm; MS interface temperature: at 330 ℃; intuvo flow path: 320 ℃; the sample injection amount is as follows: 1 microliter. An ion source: EI; electron energy: 70eV, MS ion source 230 ℃; 150 ℃ for an MS quadrupole rod; 3min, respectively cleaning 5 times by using solvents A (dichloromethane) and B (methanol) before and after sample injection, cleaning 3 times by using a sample to be injected before sample injection, and then pumping 4 times to remove bubbles of a sample injection needle and carry gas: high purity helium, ion scan range: 35-350, the total analysis time is 30.0min, and the main peak retention time is about 19.5 +/-0.5 min; the results of the main peak ion before deuteration after blank deduction are shown in figure 1, the results of the main peak ion of sample 1 after deuteration are shown in figure 2, and the results of the main peak ion of sample 2 after deuteration are shown in figure 3.
1.3.2 As can be seen from FIG. 1, the peak M of the ion group of the pre-deuterated molecule is 290.2.
1.3.3 as can be seen in fig. 1, the percent abundance of pre-deuterated molecular ions results in 100 b.
1.3.4 peaks M of molecular ionic groups of the pre-deuterated sample according to the attached FIG. 1: 290.2, the ion group peak m/z of the non-deuterated molecule possibly existing in the deuterated sample is calculated as follows: 290.2+0=290.2; the ion radical peak m/z of 1 deutero (upper 1 deuterium) molecule is: 290.2+1=291.2; the ion radical peak m/z of the 2-deutero (upper 2 deutero) molecule is: 290.2+2=292.2; the peak m/z of the ionic group of the 3-deuterated (upper 3-deuterated) molecule is as follows: 290.2+3=293.2; the ion radical peak m/z of the 4-deutero (the upper 4 deutero) molecule is: 290.2+4=294.2; the ion radical peak m/z of the 5 deuterated (upper 5 deuterated) molecule is as follows: 290.2+5=295.2; \8230; \8230, from the results of fig. 2, the samples may have non-deuterated, 1-deuterated, 2-deuterated, 3-deuterated, 4-deuterated and 5-deuterated molecules.
1.3.5 based on the results shown in fig. 2, the peak response value of each deuteration group possibly existing in sample 1 after deuteration was calculated according to the following steps:
1.3.5.1 The m/z response value of 0 deuterated base peak is A0＝5177.13；
1.3.5.2 1 deuterated (upper 1 deuterium) base peak m/z response value A1Response value of =291.2
-A0*B/100＝10577.73-5177.13*25.12/100＝9277.24；
1.3.5.3 2 deuterated (2 deuterium above) radical peak m/z response A2Response =292.2
-A0*C/100-A1*B/100＝1953723.62-5177.13*2.82/100-9277.24*25.12/100＝1951247.18；
1.3.5.4 Peak m/z response value A of 3 deuterated (upper 3 deuterated) radical3Response =293.2
-A1*C/100-A2*B/100＝492863.69-9277.24*2.82/100-1951247.18*25.12/100＝2448.78；
1.3.5.5 Peak m/z response value A of 4 deuterated (upper 4 deuterated) radical4Response of =294.2
-A2*C/100-A3*B/100＝55068.71-1951247.18*2.82/100-2448.78*25.12/100＝-11.594；
1.3.5.6 5 deuterated (upper 5 deuterium) based peak m/z response A5=295.2 response
-A3*C/100-A4*B/100＝98.26-2448.78*2.82/100＝29.20；
1.3.5.6 results A0＝5177.13；A1＝9277.24；A2＝1951247.18；A3＝2448.78；A4＝-11.594；
A5＝29.20。
1.3.5.7 discussion of results: a. The4The calculated value shows a negative number, which proves that no 4-deuteration exists, and the GC-MS result shows that the peak 294.2 of the 4-deuteration group is the isotope response of 2-deuteration and 3-deuteration; a. The5The value of (a) is small, the percentage abundance is only 0.01%, and it can be concluded that 295.2 of the 5-deuteroradical peak is an isotopic response of 3 deuteroradicals.
1.3.5.8 the individual non-negative base peak response values are then summed, i.e.
K＝A0+A1+A2+A3＝5177.13+9277.24+1951247.18+2448.78＝1968150.33。
1.3.5.9 the ratio of each deuteration in sample 1 after deuteration was calculated:
1.3.5.10 non-deuterated = a0/K*100％＝5177.13/1968150.33*100％＝0.26％；
1.3.5.11 1 deuteration (last 1 deuterium) = a0/K*100％＝9277.24/1968150.33*100％＝0.47％；
1.3.5.12 2 deuteration (upper 2 deuterions) = a0/K*100％＝1951247.18/1968150.33*100％＝99.14％；
1.3.5.13 3 deuteration (upper 3 deuterions) = a0/K*100％＝2448.78/1968150.33*100％＝0.12％；
That is, the ratio of deuterium not added to the target compound in sample 1 after deuteration: 0.26%, 0.47% of deuterium in the upper 1, 99.14% of deuterium in the upper 2, and 0.12% of deuterium in the upper 3.
1.3.6 based on the results shown in FIG. 3, the peak response value of each deuteration group possibly existing in sample 2 after deuteration was calculated according to the following steps:
1.3.6.1 The m/z response value of the 0 deutero-peak is A0＝192091.78；
1.3.6.2 1 deuterated (upper 1 deuterium) base peak m/z response value A1Response value of =291.2
-A0*B/100＝65962.43-192091.78*25.12/100＝17708.97；
1.3.6.3 2 deuterated (2 deuterium above) radical peak m/z response A2Response of =292.2
-A0*C/100-A1*B/100＝1600078.88-192091.78*2.82/100-17708.97*25.12/100＝1590213.40；
1.3.6.4 3 deuterated (upper 3 deuterium) based peak m/z response A3Response =293.2
-A1*C/100-A2*B/100＝399935.88-17708.97*2.82/100-1590213.40*25.12/100＝-25.12；
1.3.6.5 4 deuterated (upper 4 deuterium) based peak m/z response A4Response =294.2
-A2*C/100-A3*B/100＝44794.23-2448.78*25.12/100＝-43.48；
1.3.6.6 results A0＝192091.78；A1＝17708.97；A2＝1590213.40；A3＝-25.12；A4＝-43.48。
1.3.6.7 discussion: a. The3The calculated values are negative, demonstrating no 3 deuterations (the last 3 deuterations), the GC-MS result is suspected to be that the peak 293.2 of 3 deuterations is an isotopic response of 1 deuteration and 2 deuterations; a. The4The calculated value shows negative number, and no 4 deuterations (upper 4 deuterations) are proved, and the GC-MS result showsThe question is that the 4-deuterated peak 294.2 is the isotopic response of 2-deuterated.
1.3.6.8 then sum the individual non-negative base peak response values, i.e., K = a0+A1+A2
＝192091.78+17708.97+1590213.40＝1800014.15。
1.3.6.9 calculate the percentage of deuterated samples 1:
1.3.6.10 non-deuterated = a0/K*100％＝192091.78/1800014.15*100％＝10.67％；
1.3.6.11 1 deuteration = a0/K*100％＝17708.97/1800014.15*100％＝0.89％；
1.3.6.12 2 deuteration = a0/K*100％＝1590213.40/1800014.15*100％＝88.34％；
That is, the ratio of deuterium not present in sample 2 after deuteration: 10.67%, the proportion of deuterium in the upper 1 is 0.89%, and the proportion of deuterium in the upper 2 is 88.34%.
1.4 methodological validation
1.4.1 Instrument stability verification
The deuterated pre-sample prepared in step 1.2 is continuously introduced into 3 pins by the analytical method of 1.3.1:
table 1: summary of instrument stability verification results
As a result, three needles are continuously injected into the same bottle of sample solution, the RSD of the main peak base peak response value and the main peak isotope peak abundance percentage are both less than 1%, and the instrument stability is good.
1.4.2 solution stability verification
The deuterated pre-sample solution prepared in the step 1.2 is placed in a laboratory environment (the temperature is 15-30 ℃, and the humidity is 20-80%), and is detected by using an analysis method of 1.3.1 at 0,2, 4 and 8 hours, and a solution stability experiment is carried out for 8 hours, and the result is shown in the following table 2:
table 2: summary of sample solution stability verification results
As a result, the same bottle of sample solution is placed in a laboratory environment (the temperature is 15-30 ℃, and the humidity is 20% -80%), the RSD of the main peak base peak response value and the main peak isotope peak abundance percentage in 8 hours is less than 1%, and the sample solution is proved to have good stability in 8 hours in the laboratory environment.
1.4.3 method stability verification
The same deuterated sample was analyzed using chromatographic conditions of 1.3.1 on the first, second and third days, respectively, in triplicate each day, and the results of the deuteration rates determined are given in table 3:
table 3: summary of method stability verification results
As a result, the relative standard deviation of the results of the deuteration rate measured in three days is less than 2.0%, and the method has good reproducibility.
1.4.4 method accuracy verification
1.4.4.1 the sample after being deuterated is confirmed by HNMR, and the HNMR chart shows that H on two cyclopentyl groups connected with benzene rings in the compound is replaced by deuterium, which is shown in figure 4, and H at other positions is unchanged, namely the compound is a 2-deuterated (upper 2-deuterium) compound, which is shown in figure 5.
1.4.4.2 the 2-deuterated (2-deuterium) samples described above were tested using chromatographic conditions of 1.3.1 and were found to be essentially free of other impurities from the total ion flux, see FIG. 6.
1.4.4.3 the above 2 deuterated samples (the upper 2 deuterium) were added to non-deuterated samples, the addition amounts are shown in the following table, two additions per group were made, the detection was performed under the chromatographic conditions of 1.3.1, the 2-deuteration ratio was calculated by the formula, and the recovery rate was calculated, and the results are shown in the following table.
Table 4: method accuracy verification result summary
As a result: the recovery rate verified by adding 2 deuterated (last 2 deuterium) samples from low to high in the non-deuterated samples is between 94.35 and 99.56 percent, the recovery rate is good, and the method is high in accuracy.
In conclusion, the method for detecting the substance deuteration rate by using the GC-MS has low requirements on a detection instrument, can evaluate the specific number of deuterium, and has important guiding significance on production.
Claims (10)
1. A method for detecting substance deuteration rate by GC-MS comprises the following steps:
1) Weighing a certain amount of pre-deuterated samples and post-deuterated samples to be detected, adding a proper amount of solvent to completely dissolve the samples, and preparing a blank sample;
2) Taking a proper amount of the solution obtained in the step 1), filtering the solution with an organic filter membrane, taking the solution as an upper computer sample solution, and bottling for later detection;
3) Detecting the sample solution obtained in the step 2) by using GC-MS;
4) Integrating the main peaks of the samples before and after deuteration after blank is deducted from the result obtained by GC-MS detection in the step 3), and extracting ions, response values and abundance percentages of the main peaks;
5) And 4) calculating the deuteration rate of the sample by using the main peak ions before and after deuteration, the response value and the abundance percentage data thereof extracted in the step 4).
2. The method of claim 1, wherein the pre-deuterated sample has the structure of formula I and the formula C22H26Theoretical m/z is 290.20,291.21 and 292.21 respectively; the abundance percentages are respectively 100.0%,24.1% and 2.8%;
3. the method of claim 2, wherein the step 1) comprises weighing the pre-deuterated sample and the pre-deuterated sample about 10mg respectively, adding a corresponding volume of solvent, dissolving, and making into 1mg/mL solution; the sample before deuteration is a sample left before the product is subjected to deuteration; the blank sample is a blank solution prepared in the same way as the sample preparation except that no sample is added.
4. The method for detecting substance deuteration rate by GC-MS in accordance with claim 3, wherein the solvent is dichloromethane or tetrahydrofuran, and the solution is bottled in a 20mL brown glass bottle, and the dissolution is shaking or ultrasonic wave until the sample is completely dissolved.
5. The method of claim 4, wherein the solvent is dichloromethane.
6. The method for detecting the deuteration rate of a substance by GC-MS as claimed in claim 5, wherein the organic membrane is bottled in a brown sample bottle for chromatographic detection with a 0.45 μm nylon needle filter and a 2mL bottle.
7. The method for detecting substance deuteration rate by GC-MS as claimed in claim 6, wherein the GC-MS detection conditions in step 3) are as follows: temperature of the gasification chamber: 320 ℃; column flow rate: 1.2ml/min; column temperature: keeping the temperature at 100 ℃ for 1min initially, heating to 250 ℃ at the speed of 10 ℃/min and keeping the temperature for 1min; heating to 320 deg.C at 20 deg.C/min and maintaining for 9.5min; the split ratio is 30:1; and (3) chromatographic column: agilent HP-5MS UI 30m 250 μm 0.25 μm; MS interface temperature: at 330 ℃; intuvo flow path: 320 ℃; the sample injection amount is as follows: 1 microliter. An ion source: EI; electron energy: 70eV, MS ion source 230 ℃; 150 ℃ for an MS quadrupole rod; 3min, respectively cleaning 5 times by using solvents A (dichloromethane) and B (methanol) before and after sample injection, cleaning 3 times by using a sample to be injected before sample injection, and then pumping 4 times to remove bubbles of a sample injection needle and carry gas: high purity helium, ion scan range: 35-350.
8. The method of claim 2, wherein the retention time of the main peak in step 4) is 19.5 ± 0.5min.
9. The method for detecting the deuteration rate of a substance by GC-MS as claimed in claim 2, wherein the results of the molecular ion base peak of the main pre-deuteration peak in step 4) are recorded as M and the results of the percentage isotopic abundance of the molecular ion of the main pre-deuteration peak are recorded as 100B.
10. The method for detecting the deuteration rate of a substance by GC-MS as claimed in claim 9, wherein the method for calculating the deuteration rate of a sample comprises the following 4 steps:
(1) According to the molecular ion radical peak M of the sample before deuteration, the obtained non-deuterated molecular ion radical peak M/z is as follows: m +0; the ion radical peak m/z of 1 deutero (upper 1 deuterium) molecule is: m +1; the ion radical peak m/z of the 2-deutero (upper 2 deutero) molecule is: m +2; the ion radical peak m/z of 3 deuterated (upper 3 deuterated) molecules is as follows: m +3; the ion radical peak m/z of the 4-deutero (the upper 4 deutero) molecule is: m +4; the 5-deuteration (upper 5 deuterons) molecular ion radical peak m/z is: m +5; 823060, 8230;
(2) Calculating the peak response value of each deuteration group possibly existing in the sample after deuteration by the following formula: response of 0 deuterated peak M is A0(ii) a 1 deuterated Peak m/z response A1Response value-A of = M +10* B/100;2 deuterated peak m/z response A2= M +2 response-a0*C/100-A1* B/100;3 deuterated Peak m/z response A3= M +3 response-a1*C/100-A2* B/100;4 deuterated peak m/z response A4= M +4 response-a2*C/100-A3* B/100;5 deuterated peak m/z response A5= M +5 response-a3*C/100-A4* B/100, 823060, 8230; until a negative value is obtained;
(3) The deuterated nonnegative base peak responses are then summed, i.e., A0+A1+A2+A3+A4+A5+……＝K；
(4) Calculating each deuteration ratio of the sample after deuteration: deuterated = a0100% K, 1 deuteration = a1100% K, 2 deuteration = a2100% of/K, 3 deuteration = a3100% K, 4 deuteration = a4100% of/K, 5 deuteration = a5/K*100％……。
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