Detection method for measuring isotope abundance of 15N or 18O labeled nitrate sample by using organic mass spectrum

Abstract

The invention relates to a detection method for determining isotope abundance of a 15N or 18O labeled nitrate sample by using an organic mass spectrum. The detection method comprises the following steps: (1) dissolving the 15N or 18O labeled nitrate sample in a methanol aqueous solution; (2) taking the supernatant, filtering with a filter membrane, and diluting with a methanol aqueous solution to obtain a solution to be detected; and (3) detecting the solution to be detected by using a high-resolution liquid chromatograph-mass spectrometer, selecting a mass spectrum in a peak appearance time window, recording relative mass spectrum signal intensities with mass-to-charge ratios of 61.988 and 62.985, and calculating to obtain the 15N isotope abundance. Or recording the relative mass spectrum signal intensities with the mass-to-charge ratios of 61.988, 63.993, 65.997 and 68.001, and calculating to obtain the abundance of the < 18 > O isotope. Compared with the prior art, the method has the advantages that the sample pretreatment time is greatly shortened, the economic cost is low, and the test result is accurate.

Description

Technical Field

[0001] The present invention relates to the technical field of stable isotope detection, in particular to a method for determining the presence of an organic mass spectrometer. 15 N or 18 Method for detecting isotopic abundance of O-labeled nitrate samples. Background Art

[0002] With the continuous development of isotope labeling technology and the iterative update of high-precision instruments such as mass spectrometry, stable isotope labeled nitrate reagents have shown their indispensable importance in many scientific research fields such as environment and agriculture. For example, by studying the migration and transformation of nitrate in the environment, water quality and soil pollution can be evaluated; studying the absorption, utilization and transformation process of nitrate by plants is of great significance for guiding the exploration of plant physiological and biochemical characteristics and the dynamic analysis of soil and plant nutrition. Therefore, the relevant nitrate 15 N. 18 The accuracy of O isotope abundance evaluation is crucial.

[0003] The main methods for detecting the abundance of stable isotopes are nuclear magnetic resonance spectroscopy and mass spectrometry. 1 H spectrum and 13 C spectrum is the most widely used. Currently, nuclear magnetic resonance is mainly used for the determination of organic compounds. 13 C and deuterium isotope abundance detection, invention patent CN112305007 A discloses a method for determining the deuterium isotope abundance of organic compounds using nuclear magnetic hydrogen spectrum or deuterium spectrum, using the internal standard method to measure the content of various deuterium-labeled compounds through the spectrum peak area, thereby obtaining the isotope abundance, and also obtaining the information of the deuterium labeling site. The method is simple to operate and the detection data is accurate. Invention patent CN 116879341 A discloses a method for determining the deuterium isotope abundance of organic compounds using nuclear magnetic carbon spectrum. 13 The isotope abundance of C-labeled compounds is determined by deconvoluting the carbon spectrum and integrating the peak area. 13 C isotope abundance. This method has the advantages of wide application range, simple operation process and high accuracy.

[0004] Mass spectrometry mainly includes gas isotope mass spectrometry, "mass cluster" method and high-resolution mass spectrometry. Among them, "mass cluster" method and high-resolution mass spectrometry are mainly suitable for isotope abundance detection of organic compounds. "Mass cluster" method uses the acquired target mass spectrum data to analyze the mass spectrum data through the classification calculation method of "mass cluster" to achieve D, 13 C or 15 Isotope abundance detection of N-labeled organic compounds. Invention patent CN 104122339 A discloses a method for isotope abundance detection based on high performance liquid chromatography-mass spectrometry and the "mass cluster" method. Invention patent CN 104330515 A discloses a method for determining13 The method for determining the isotopic abundance and chemical purity of C-labeled linear fatty acids uses the ratio of isotope peak clusters to calculate the isotopic abundance value of the labeling reagent, which can be achieved 13 Accurate calculation of isotope abundance and chemical purity of C-labeled fatty acids. High-resolution mass spectrometry has ultra-high resolution and high sensitivity, and can directly detect organic compounds in different isotope-labeled states. Therefore, it has great advantages in isotope abundance detection. Invention patent CN 112630345 A discloses a method for detecting the isotope distribution and abundance of deuterium-labeled organic compounds based on a high-resolution mass spectrometer. The method is for deuterium-labeled organic compounds. Its sampling method is flow injection sampling, and multiple mass spectra are collected to calculate the deuterium isotope abundance according to the formula. Invention patent CN116482256 A discloses a method for measuring deuterium isotope abundance using a high-resolution liquid chromatography-mass spectrometer. 13 C. 15 The method for determining the isotopic abundance of N-labeled organic compounds can achieve the simultaneous determination of the abundance of two isotopes with a single injection. The method is simple to operate, requires a small amount of sample, and has high accuracy and stability.

[0005] However, the above methods are all applicable to the detection of isotope abundance of organic compounds. For the detection of isotope abundance of inorganic compounds, the commonly used method is gas isotope mass spectrometry, which converts the sample into gas through a complex pre-treatment process before detection. Invention patent CN 111983007 A discloses a method for determining nitric acid or nitrate 15 The method or device for determining the N isotope abundance comprises adding a sample, an oxide, and a reducing agent into a reaction tube, evacuating the tube, sealing the tube, heating the tube to generate nitrogen gas, and then introducing the gas isotope mass spectrometer to calculate the N isotope abundance based on the relative intensity of the peaks. 15 N isotope abundance; Invention patent CN 110763535A discloses a method for determining nitrite 15 The sample preparation method for N isotope abundance is to mix the sample with a reducing agent to generate 15 NO gas is detected by gas isotope mass spectrometry.

[0006] Existing nitrate 15 The only method for detecting N isotope abundance is gas isotope mass spectrometry. Pretreatment usually requires the use of a complex reaction device. After vacuuming, the sample 15 The N-labeled sample is mixed with the oxide and the reducing agent in sequence to generate 15 N-labeled nitrogen is then introduced into a gas isotope mass spectrometer for detection. This method consumes a lot of sample, is costly, requires specialized pre-treatment equipment, has complex and time-consuming steps, and can only measure the abundance of a single isotope. Therefore, it has certain defects in detection applicability. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for determining the 15 N or 18 A method for detecting the isotopic abundance of O-labeled nitrate samples was developed, and organic mass spectrometry was applied to the detection of inorganic compounds.

[0008] The purpose of the present invention can be achieved by the following technical solutions: a method for determining the 15 N or 18 The method for detecting the isotopic abundance of an O-labeled nitrate sample comprises the following steps:

[0009] (1) 15 N or 18 O-labeled nitrate samples were dissolved in methanol-water solution;

[0010] (2) taking the supernatant, filtering it with a filter membrane, and diluting it with methanol-water solution to obtain the test solution;

[0011] (3) Use high-resolution liquid chromatography-mass spectrometry to detect the test solution, select the mass spectrum within the peak time window, record the relative mass spectrum signal intensity of the mass-to-charge ratio of 61.988 and 62.985, and calculate 15 N isotope abundance; alternatively, record the relative mass spectrometry signal intensities at mass-to-charge ratios of 61.988, 63.993, 65.997, and 68.001 and calculate 18 O isotope abundance.

[0012] Preferably, the 15 N or 18 O-labeled nitrate samples were 15 N or 18 O-labeled nitrate reagents or complex matrix samples such as water, soil, and cells.

[0013] Preferably, the 15 N or 18 O-labeled nitrate samples were 15 N-labeled potassium nitrate, 15 N-labeled sodium nitrate or 18 O marks potassium nitrate.

[0014] Preferably, in the methanol-water solution of step (1), the volume ratio of methanol to water is (1-3):(1-2).

[0015] In the present invention, the ratio of methanol-water solution used in step (1) is V:V=(1-3):(1-2).

[0016] Preferably, step (1) specifically includes the following steps:

[0017] Take 0.001-0.1 mg solid or 2.5-10 μL liquid 15 N or18 Place the O-labeled nitrate sample in a centrifuge tube, add 0.5-1 mL of methanol-water solution, and vortex for 1-3 minutes to fully dissolve it.

[0018] Preferably, the filter membrane in step (2) is a hydrophilic filter membrane.

[0019] Preferably, the pore size of the filter membrane in step (2) is 0.20-0.24 μm.

[0020] Preferably, in the methanol-water solution of step (2), the volume ratio of methanol to water is 2:(1-2).

[0021] In the present invention, the ratio of methanol-water solution used in step (2) is V:V=2:(1-2).

[0022] Preferably, in step (2), the supernatant after membrane filtration is diluted 10-1000 times using methanol aqueous solution.

[0023] Preferably, the method comprises the following steps: 15 N or 18 The method for detecting the isotopic abundance of an O-labeled nitrate sample comprises the following steps:

[0024] (1) Take 0.001-0.1 mg solid or 2.5-10 μL liquid 15 N or 18 Place the O-labeled nitrate sample in a centrifuge tube, add 0.5-1 mL of methanol-water solution, and vortex for 2 minutes to fully dissolve it;

[0025] (2) taking the supernatant, filtering it with a 0.22 μm filter membrane, and then diluting it 10-1000 times with a methanol-water solution to obtain a test solution;

[0026] (3) Using high-resolution liquid chromatography-mass spectrometry, the mass spectrometer was selected within the peak time window, and the relative mass spectrometry signal intensities of the mass-to-charge ratios of 61.988 and 62.985 were recorded. The calculated 15 N isotope abundance; record the relative mass spectrometry signal intensities of mass-to-charge ratios of 61.988, 63.993, 65.997, and 68.001, and calculate 18 O isotope abundance.

[0027] Preferably, the high-resolution mass spectrometer in the high-resolution liquid chromatography-mass spectrometry instrument in step (3) is calibrated before detection, and the mass deviation is less than 5 ppm.

[0028] Preferably, the mass spectrum selection range in step (3) is 10-50 superimposed mass spectra within the nitrate peak time range.

[0029] Preferably, when the test solution is detected by high-resolution liquid chromatography-mass spectrometry in step (3), the chromatographic conditions are as follows:

[0030] The chromatographic column is a C18 column, the mobile phase A is water, the mobile phase B is methanol, the elution conditions are 90% B isocratic elution for 1-3 min, the flow rate is 0.2-0.3 mL / min, the column temperature is 30-40°C, and the injection volume is 1 μL.

[0031] Preferably, when the test solution is detected by high-resolution liquid chromatography-mass spectrometry in step (3), the mass spectrometry conditions are as follows:

[0032] The ion source is ESI; the electrospray voltage is 2000-2800 V; the nebulizing gas is 2-6 L / min; the auxiliary gas is 0-10 L / min; the ion transfer tube temperature is 300-350°C; the scanning mode is negative ion scanning in full scan mode; the resolution is 60000-120000; and the scanning mass-to-charge ratio range is 40-500 m / z.

[0033] Preferably, in step (3), 15 The calculation formula for N isotope abundance is:

[0034]

[0035] The unit is atom% 15 N, where I 61.998 is the relative mass spectrum signal intensity with a mass-to-charge ratio of 61.988, I 62.985 is the relative mass spectrum signal intensity with a mass-to-charge ratio of 62.985, in %.

[0036] Preferably, in step (3), 18 The formula for calculating O isotope abundance is:

[0037]

[0038] The unit is atom% 18 O, where I 61.998 is the relative mass spectrum signal intensity with a mass-to-charge ratio of 61.988, I 63.993 is the relative mass spectrum signal intensity with a mass-to-charge ratio of 63.993, I 65.997 is the relative mass spectrum signal intensity with a mass-to-charge ratio of 65.997, I 68.001 is the relative mass spectrum signal intensity with a mass-to-charge ratio of 68.001, in %.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] 1. The present invention provides a method for preparing nitrate in an inorganic reagent. 15 N or 18O isotope abundance detection method, using organic mass spectrometry 15 N or 18 O-labeled nitrate sample isotope abundance can directly detect nitrate in the sample 5 N or 18 O-labeled state, compared with traditional gas isotope mass spectrometry, greatly shortens sample pre-treatment time, has low economic cost and accurate test results;

[0041] 2. Compared with gas isotope mass spectrometry, this method greatly shortens sample pretreatment time and does not require complex pretreatment steps such as vacuuming and reaction conversion. The detection time for a single sample is less than 10 minutes, which improves detection efficiency and realizes high-throughput detection.

[0042] 3. This invention expands the detection range of high-resolution liquid chromatography-mass spectrometry, applies organic mass spectrometry to the detection of inorganic compounds, and combines the high resolution and high sensitivity of high-resolution mass spectrometry to achieve 15 N or 18 O isotope abundance determination, with higher accuracy and precision of test data;

[0043] 4. The sample amount used in the experimental process of the present invention is small, and no oxides and reducing agents are used in the pre-treatment reaction. Compared with gas isotope mass spectrometry, it is more economical and practical. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is embodiment 1 of the present invention 15 High-resolution mass spectrum of N-potassium nitrate sample;

[0045] Figure 2 This is embodiment 2 of the present invention 15 High-resolution mass spectrum of N-sodium nitrate sample;

[0046] Figure 3 This is embodiment 3 of the present invention 18 High-resolution mass spectrum of O-potassium nitrate sample. DETAILED DESCRIPTION

[0047] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0048] Unless otherwise specified, the reagents, methods, instruments and equipment used in the present invention are conventional reagents, methods, instruments and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0049] Example 1

[0050] 15N-labeled potassium nitrate reagent isotope abundance detection (product of CIL, USA, NLM-765-1, 99.0 atom% 15 N)

[0051] Take 0.01mg 15 N-potassium nitrate reagent was placed in a 1.5 mL centrifuge tube, 1 mL of 50% methanol aqueous solution was added to fully dissolve it, filtered using a 0.22 μm filter membrane, and then diluted 1000 times with 50% methanol aqueous solution to obtain a test solution.

[0052] High-resolution liquid spectrometry was used for detection, and calibration was performed before detection. The mass detection deviation was less than 5ppm.

[0053] Chromatographic conditions were as follows: column: BEH C18 column (100 mm×2.1 mm, 1.9 μm); mobile phase: mobile phase A was pure water, mobile phase B was methanol; elution conditions: 90% B isocratic elution for 2 min; flow rate: 0.25 mL / min; injection volume: 1 μL.

[0054] The mass spectrometry conditions were as follows: ion source: ESI; electrospray voltage (Ion Sray Voltage): 2500 V; sheath gas (Sheath Gas): 5.34 L / min; auxiliary gas (Aux Gas): 9.35 L / min; ion transfer tube temperature: 320°C; scan mode: negative ion scan in full scan mode; resolution: 120,000; scanning mass-to-charge ratio range: 40-500 m / z.

[0055] The obtained high-resolution mass spectrum is as follows Figure 1 shown.

[0056] Extract 20 mass spectra within the nitrate peak time window and overlay them. Record the relative mass spectrometry signal intensities of mass-to-charge ratios of 61.988 and 62.985, which are 0.08% and 99.92% respectively. Substitute them into the formula:

[0057]

[0058] The calculated abundance is 99.92atom% 15 N, the results are basically consistent with the reagent identification value.

[0059] Example 2

[0060] 15 N-labeled sodium nitrate reagent isotope abundance detection (product of CIL, USA, NLM-157-1, 99.0 atom% 15 N)

[0061] Take 0.01mg 15N-potassium nitrate reagent was placed in a 1.5 mL centrifuge tube, 1 mL of 50% methanol aqueous solution was added to fully dissolve it, filtered using a 0.22 μm filter membrane, and then diluted 1000 times with 50% methanol aqueous solution to obtain a test solution.

[0062] High-resolution liquid spectrometry was used for detection, and calibration was performed before detection. The mass detection deviation was less than 5ppm.

[0063] Chromatographic conditions were as follows: column: BEH C18 column (100 mm×2.1 mm, 1.9 μm); mobile phase: mobile phase A was pure water, mobile phase B was methanol; elution conditions: 90% B isocratic elution for 2 min; flow rate: 0.25 mL / min; injection volume: 1 μL.

[0064] The mass spectrometry conditions were as follows: ion source: ESI; electrospray voltage (Ion Sray Voltage): 2400 V; sheath gas (Sheath Gas): 5.34 L / min; auxiliary gas (Aux Gas): 9.35 L / min; ion transfer tube temperature: 320°C; scan mode: negative ion scan in full scan mode; resolution: 120,000; scanning mass-to-charge ratio range: 40-500 m / z.

[0065] The obtained high-resolution mass spectrum is as follows Figure 2 shown.

[0066] Extract 20 mass spectra within the nitrate peak time window and overlay them. Record the relative mass spectrometry signal intensities of mass-to-charge ratios of 61.988 and 62.985, which are 0.11% and 99.89% respectively. Substitute them into the formula:

[0067]

[0068] The calculated abundance is 99.89atom% 15 N, the results are basically consistent with the reagent identification value.

[0069] Example 3

[0070] 18 Isotopic abundance detection of O-labeled potassium nitrate samples

[0071] Take 5 μL 18 The O-potassium nitrate sample solution was placed in a 1.5 mL centrifuge tube, 1 mL of 50% methanol aqueous solution was added to fully dissolve it, the solution was filtered using a 0.22 μm filter membrane, and then diluted 100 times with 50% methanol aqueous solution to obtain the test solution.

[0072] High-resolution liquid spectrometry was used for detection, and calibration was performed before detection. The mass detection deviation was less than 5ppm.

[0073] Chromatographic conditions were as follows: column: BEH C18 column (100 mm×2.1 mm, 1.9 μm); mobile phase: mobile phase A was pure water, mobile phase B was methanol; elution conditions: 90% B isocratic elution for 3 min; flow rate: 0.25 mL / min; injection volume: 1 μL.

[0074] The mass spectrometry conditions were as follows: ion source: ESI; electrospray voltage (Ion Sray Voltage): 2400 V; sheath gas (Sheath Gas): 5.34 L / min; auxiliary gas (Aux Gas): 9.35 L / min; ion transfer tube temperature: 320°C; scan mode: negative ion scan in full scan mode; resolution: 120,000; scanning mass-to-charge ratio range: 40-500 m / z.

[0075] The obtained high-resolution mass spectrum is as follows Figure 3 shown.

[0076] Extract 20 mass spectra within the nitrate peak time window and overlay them. Record the relative mass spectrometry signal intensities of mass-to-charge ratios of 61.988, 63.993, 65.997, and 68.001, which are 7.43%, 26.20%, 41.95%, and 24.42%, respectively. Substitute them into the formula:

[0077]

[0078] The calculated abundance is 61.12 atom% 18 O, which is consistent with the results of gas isotope mass spectrometry.

[0079] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A method for determination using organic mass spectrometry 15 N or 18 The method for detecting the isotopic abundance of an O-labeled nitrate sample is characterized in that: The following steps are involved: (1) 15 N or 18 O-labeled nitrate samples were dissolved in methanol-water solution; (2) taking the supernatant, filtering it with a filter membrane, and diluting it with methanol-water solution to obtain the test solution; (3) Use high-resolution liquid chromatography-mass spectrometry to detect the test solution, select the mass spectrum within the peak time window, record the relative mass spectrum signal intensity of the mass-to-charge ratio of 61.988 and 62.985, and calculate 15 N isotope abundance; alternatively, record the relative mass spectrometry signal intensities at mass-to-charge ratios of 61.988, 63.993, 65.997, and 68.001 and calculate 18 O isotope abundance.

2. The method of claim 1 wherein the organic mass spectrometry method is used to determine the 15 N or 18 The method for detecting the isotopic abundance of an O-labeled nitrate sample is characterized in that: described 15 N or 18 O-labeled nitrate samples were 15 N-labeled potassium nitrate, 15 N-labeled sodium nitrate or 18 O marks potassium nitrate.

3. The method of claim 1 wherein the organic mass spectrometry method is used to determine the 15 N or 18 The method for detecting the isotopic abundance of an O-labeled nitrate sample is characterized in that: In the methanol-water solution of step (1), the volume ratio of methanol to water is (1-3):(1-2).

4. The method of claim 1 wherein the organic mass spectrometry method is used to determine the 15 N or 18 The method for detecting the isotopic abundance of an O-labeled nitrate sample is characterized in that: Step (1) specifically includes the following steps: Take 0.001-0.1 mg solid or 2.5-10 μL liquid 15 N or 18 Place the O-labeled nitrate sample in a centrifuge tube, add 0.5-1 mL of methanol-water solution, and vortex for 1-3 minutes to fully dissolve it.

5. The method of claim 1 wherein the organic mass spectrometry method is used to determine the 15 N or 18 The method for detecting the isotopic abundance of an O-labeled nitrate sample is characterized in that: The filter membrane in step (2) is a hydrophilic filter membrane with a pore size of 0.20-0.24 μm.

6. The method of claim 1 wherein the organic mass spectrometry method is used to determine the 15 N or 18 The method for detecting the isotopic abundance of an O-labeled nitrate sample is characterized in that: In the methanol aqueous solution of step (2), the volume ratio of methanol to water is 2:(1-2), and the supernatant after filtering with the filter membrane is diluted 10-1000 times using the methanol aqueous solution.

7. The method of claim 1 wherein the organic mass spectrometry method is used to determine the 15 N or 18 The method for detecting the isotopic abundance of an O-labeled nitrate sample is characterized in that: The high-resolution mass spectrometer in the high-resolution liquid chromatography-mass spectrometry instrument of step (3) is calibrated before detection, and the mass deviation is less than 5 ppm; The mass spectrum selection range in step (3) is 10-50 superimposed mass spectra within the nitrate peak time range.

8. The method of claim 1 wherein the organic mass spectrometry method is used to determine the 15 N or 18 The method for detecting the isotopic abundance of an O-labeled nitrate sample is characterized in that: When the test solution is detected by high-resolution liquid chromatography-mass spectrometry in step (3), the chromatographic conditions are as follows: The chromatographic column was a C18 column, mobile phase A was water, mobile phase B was methanol, the elution conditions were 90% B isocratic elution for 1-3 min, flow rate was 0.2-0.3 mL / min, column temperature was 30-40 °C, and the injection volume was 1 μL; Mass spectrometry conditions are as follows: The ion source is ESI; the electrospray voltage is 2000-2800V; the nebulizing gas is 2-6L / min; the auxiliary gas is 0-10L / min; the ion transfer tube temperature is 300-350℃; the scanning mode is negative ion scanning in full scan mode; the resolution is 60000-120000; and the scanning mass-to-charge ratio range is 40-500m / z.

9. The method of claim 1 wherein the organic mass spectrometry method is used to determine the 15 N or 18 The method for detecting the isotopic abundance of an O-labeled nitrate sample is characterized in that: In step (3), 15 The calculation formula for N isotope abundance is: The unit is atom% 15 N, where I 61.998 is the relative mass spectrum signal intensity with a mass-to-charge ratio of 61.988, I 62.985 is the relative mass spectrum signal intensity with a mass-to-charge ratio of 62.985, in %.

10. The method of claim 1 wherein the organic mass spectrometry method is used to determine the 15 N or 18 The method for detecting the isotopic abundance of an O-labeled nitrate sample is characterized in that: In step (3), 18 The formula for calculating O isotope abundance is: The unit is atom% 18 O, where I 61.998 is the relative mass spectrum signal intensity with a mass-to-charge ratio of 61.988, I 63.993 is the relative mass spectrum signal intensity with a mass-to-charge ratio of 63.993, I 65.997 is the relative mass spectrum signal intensity with a mass-to-charge ratio of 65.997, I 68.001 is the relative mass spectrum signal intensity with a mass-to-charge ratio of 68.001, in %.

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