Quantitative nitroso compound derivatization method and application

Abstract

According to the nitroso-compound derivatization quantitative method and application, nitroso-compounds of different structures can be measured through simple derivatization treatment. In the embodiment of the invention, the sample derivatization method is researched, and the optimal reaction condition for the nitroso compound is provided. The invention establishes a simple, convenient and sensitive liquid chromatography quantitative analysis method, and successfully establishes a derivatization analysis method for nitroso compounds with different structures. The method has high selectivity, sensitivity and accuracy, and is suitable for accurate determination of nitroso compounds in complex samples and complex structures.

Description

Technical Field

[0001] The present application relates to the field of chemical detection and analysis, and in particular to a quantitative method for derivatization of nitrogen-nitroso compounds. Background Art

[0002] N-nitrosamine compounds are considered to be highly carcinogenic substances because of their strong genotoxicity after metabolic activation. In the list of carcinogens of the World Health Organization's International Agency for Research on Cancer (IARC), N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) are both listed as Class 2A carcinogens. The ICH M7 guideline also clearly states that this type of compound has a high carcinogenicity. The sources of N-nitroso impurities mainly include raw materials and processes. For example, in the synthesis process of sartan drugs, in order to introduce tetrazole rings or remove excess raw materials, impurities such as NDMA, NDEA and N-nitroso-N-methyl-4-aminobutyric acid (NMBA) may be introduced. In the synthesis process of metformin, ranitidine and nizatidine, the generation of N-nitroso genotoxic impurities is related to the use of dimethylamine as a precursor.

[0003] As research deepens, more and more nitrogen-nitroso impurities with complex structures are discovered. In addition to the above-mentioned small molecules, there is also the possibility that the nitrogen-nitroso functional group is directly bound to the API. Taking nadroparin calcium as an example, due to the use of sodium nitrite in the process of degrading heparin, the nitroso functional group is directly bound to the side chain of the repeating unit of heparin. The nitrogen-nitroso by-products of APIs similar to this situation have a large molecular weight span, complex components and uncertain structures, which greatly limits the determination of the nitrogen-nitroso content. In addition, drugs containing secondary amine structures also have the risk of binding to nitroso structures during the formulation stage.

[0004] The existing detection method for such nitrogen-nitroso compounds with unknown structures and complex components is chemiluminescence detection, that is, using nitrogen-nitroso di-n-propylamine as a standard sample, chemically cleaving the nitrogen-nitroso functional group into nitric oxide by hydrobromic acid, and performing quantitative analysis through the light generated by the energy level transition of the reaction between nitric oxide and ozone. This method is limited by the instrument and the chemical cleavage process, and the resulting chromatogram has poor peak shape and severe tailing, which cannot meet the requirements of detection sensitivity and quantitative analysis.

[0005] In impurity research, small-molecule nitrogen-nitroso impurities can be synthesized or purchased as standard products, but complex systems require the purchase of large quantities of standard products, greatly increasing the cost of analysis. Nitrogen-nitroso standards with complex structures are usually difficult to obtain, further limiting the research and development of analytical methods.

[0006] This study can transform the structure of nitrogen-nitroso functional groups of unknown structures and complex components into known structures through derivatization methods, detect easily detectable target analytes, and then use existing methods to perform quantitative analysis on them, thereby achieving specific quantitative detection of nitrogen-nitroso compounds. Summary of the invention

[0007] In order to solve the difficulties in detecting nitrogen nitroso compounds in the prior art, the present application provides a detection method:

[0008] A method for quantitative derivatization of nitrogen nitroso compounds comprises the following steps:

[0009] Step 1: Derivatizing the nitrogen-nitroso compound sample to be tested, dissolving 1,1-diphenylethylene and the nitrogen-nitroso compound sample in methanol, then adding methanesulfonic acid, and irradiating under ultraviolet light to obtain the derivatized nitrogen-nitroso compound sample to be tested;

[0010] Step 2: Liquid phase analysis of the derivatized nitrogen nitroso compound sample using Waters TM C18 chromatographic column (3.5 μm, 4.6×150 nm) was used for separation, the column temperature was controlled at 35° C., the flow rate was 0.5 mL / min, the injection volume was 10 μL, and the ultraviolet detection wavelength was 290 nm. The mobile phase A was water containing 0.1% acetic acid, and the mobile phase B was acetonitrile containing 0.1% acetic acid. The separation was performed in the ratio of mobile phase A:mobile phase B=(60%-40%):(60%-40%).

[0011] Step 3: quantitatively calculating the content of nitrogen-nitroso compounds. The nitrogen-nitroso compound samples are derivatized and converted into α-amino oxime derivatives. The content of nitrogen-nitroso compounds is quantitatively calculated by quantitative analysis of the α-amino oxime derivatives.

[0012] The present application also provides the application of the nitrogen-nitroso compound derivatization quantitative method in the detection field.

[0013] Beneficial effects:

[0014] At present, the detection of complex nitrogen-nitroso compounds is mostly done by separating them as much as possible and detecting them separately, which places high demands on analytical methods and instruments. The US FDA also stated in the latest "Control of Nitrosamine Impurities in Human Drugs" in September 2024 that if a complete risk assessment method is established, for some drugs that do not have special functional groups in their structure and do not have the risk of introducing nitrosamines in the pharmaceutical process, only the total nitrogen-nitrosamine content can be controlled.

[0015] However, there is currently no good method for detecting total nitrogen nitrosamines. Only the European Pharmacopoeia EP 10.0 provides a chemical reaction-chemiluminescence method. The main principle is to convert nitrogen nitroso into NO, emit light through reaction with ozone, and convert the light signal into an electrical signal for concentration detection. However, since the chemical reaction is not completed instantaneously, the peak signal often fails to meet the analytical detection requirements. The present application first captures the nitrogen nitroso and converts it into an α-amino oxime derivative that is detectable and easy to detect. In the embodiments of the present application, the sample pretreatment is studied, and the optimal reaction conditions for nitrogen nitroso compounds are proposed, so that the entire reaction process is controllable, convenient and repeatable, and a good detection signal can be obtained in the liquid phase.

[0016] The present application uses a nitrogen-nitroso standard for calibration and uses a variety of other types of nitrogen-nitroso compounds for verification, and still has good detection accuracy, indicating that the method can be used to detect a variety of nitrogen-nitroso compounds in complex components using the same method, greatly reducing the difficulty of analysis and the purchase cost of different nitrogen-nitroso reagents.

[0017] The present application also demonstrates through experiments using irbesartan, a relatively complex structure drug, that nitrogen nitroso groups in complex drug structures or components can also be well detected, providing a new method for the current detection of nitrogen nitroso groups in complex structures.

[0018] This application establishes a simple and sensitive liquid chromatography quantitative analysis method, which successfully realizes the quantitative determination of nitrogen nitroso compounds. The method has high selectivity, sensitivity and accuracy, and is suitable for the accurate determination of nitrogen nitroso compounds in complex samples. This study provides a feasible means for further application of this method in the fields of environmental monitoring, food safety, etc., especially for the quantitative analysis of total nitrogen nitrosamines in complex drug structures or complex components. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shows the effect of different molar amounts of methanesulfonic acid on the derivatization

[0020] Figure 2 Showing the effect of different wavelengths of UV light on derivatization

[0021] Figure 3 The trend of benzophenone oxime generated in 8 hours is shown in the figure

[0022] Figure 4 The trend graph of benzophenone oxime generated in 2 hours is shown

[0023] Figure 5 Shows the characteristic spectrum of each component of gas chromatography

[0024] Figure 6The gas chromatography spectrum of benzophenone oxime generated in 2 hours is shown, and the problem of benzophenone oxime residue is explained. Gas chromatography cannot be used for quantitative analysis in this method.

[0025] Figure 7 Spectrogram showing the specificity of each component in liquid chromatography

[0026] Figure 8 Linear fit showing the response of the nitrogen nitrosopyrrolidine dosage and the benzophenone oxime generated by derivatization DETAILED DESCRIPTION

[0027] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0029] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0030] An embodiment of the present application provides a method for quantifying the derivatization of nitrogen-nitroso compounds, comprising the following steps:

[0031] Step 1: Derivatizing the nitrogen-nitroso compound sample to be tested, dissolving 1,1-diphenylethylene and the nitrogen-nitroso compound sample in methanol, then adding methanesulfonic acid, and irradiating under ultraviolet light to obtain the derivatized nitrogen-nitroso compound sample to be tested;

[0032] Step 2: Liquid phase analysis of the derivatized nitrogen nitroso compound sample using Waters TM C18 chromatographic column (3.5 μm, 4.6×150 nm) was used for separation, the column temperature was controlled at 35° C., the flow rate was 0.5 mL / min, the injection volume was 10 μL, and the ultraviolet detection wavelength was 290 nm. The mobile phase A was water containing 0.1% acetic acid, and the mobile phase B was acetonitrile containing 0.1% acetic acid. The separation was performed in the ratio of mobile phase A:mobile phase B=(60%-40%):(60%-40%).

[0033] Step 3: quantitatively calculating the content of nitrogen-nitroso compounds. The nitrogen-nitroso compound samples are derivatized and converted into α-amino oxime derivatives. The content of nitrogen-nitroso compounds is quantitatively calculated by quantitative analysis of the α-amino oxime derivatives.

[0034] In one embodiment, the ratio of mobile phase A to mobile phase B is preferably 60%:40%.

[0035] In one embodiment, in step 1, the molar mass ratio of methanesulfonic acid to the nitrogen nitroso compound is (0.5-2):1.

[0036] In one embodiment, in step 1, the molar mass ratio of methanesulfonic acid to the nitrogen nitroso compound is preferably 1:1.

[0037] In one embodiment, in step 1, the wavelength range of the ultraviolet lamp is 365nm to 405nm.

[0038] In one embodiment, in step 1, the wavelength of the ultraviolet lamp is 290 nm.

[0039] In one embodiment, in step 1, the irradiation time under ultraviolet light is 0 hours to 8 hours.

[0040] In one embodiment, in step 1, the irradiation time under ultraviolet light is 2 hours.

[0041] In one embodiment, in step 2, the analysis time and gradient of the liquid phase detection method are:

[0042] 0min~5min, 40% B phase; 5min~25min, B phase increased from 40% to 100%; 25min~27min, 100% B phase; 27min~28min, B phase decreased from 100% to 40%; 28min~35min, 100% B phase.

[0043] In one embodiment, in step 3, the method for quantitatively calculating nitrogen-nitroso compounds is: according to the detection method of steps 1 and 2, the standard sample is detected and the standard curve is fitted, and the quantitative detection limit is detected and confirmed, and according to the detection method of steps 1 and 2, the sample to be tested is detected and brought into the fitting curve to quantitatively calculate the content of nitrogen-nitroso compounds in the sample to be tested.

[0044] An embodiment of the present application provides an application of the quantitative method for derivatization of nitrogen-nitroso compounds in the field of detection.

[0045] In one embodiment, the applications include environmental monitoring, food testing, and analysis of genotoxicity in pharmaceutical processing.

[0046] Example 1 Analysis and Detection Method

[0047] Dissolve 1 mmol of 1,1-stilbene and 0.5 mmol of nitrogen-nitrosopyrrolidine in 2 mL of methanol, and then add 0.5 mmol of methanesulfonic acid. Irradiate with 395 nm ultraviolet light for 2 hours, and the nitrogen-nitroso compound can be converted into a nitrosyl cation. In this process, N-nitrosylation and photoaddition reactions occur, thereby obtaining the desired α-amino oxime derivative. The content of nitrogen-nitroso compounds can be indirectly quantified by quantitative analysis of α-amino oxime derivatives.

[0048]

[0049] Waters TM C18 chromatographic column (3.5 μm, 4.6×150 nm) was used for separation, the column temperature was controlled at 35° C., the flow rate was 0.5 mL / min, the injection volume was 10 μL, and the UV detection wavelength was 290 nm.

[0050] In order to effectively separate the reaction solution, the injection sequence time was optimized. Isocratic elution was used during the main analysis time, but considering the need to drain the column to prevent the residue from the previous injection from interfering with the next injection, the elution gradient time had to be adjusted. It was found that a 30-minute elution time would still leave a residual peak within 2 minutes of the next injection, and the baseline did not return to the initial level in the last three minutes, and the pressure did not stabilize. Therefore, a 35-minute analytical gradient time was finally selected.

[0051] The final gradient is shown in the following table.

[0052]

[0053] Example 2 Comparative test of ratio of methanesulfonic acid and nitrogen nitroso compounds

[0054] The derivatization results with different ratios of methanesulfonic acid and nitrogen nitroso compounds were compared. Except for the ratio of methanesulfonic acid and nitrogen nitroso compounds, other conditions were the same as those in Example 1. The ratios of methanesulfonic acid and nitrogen nitroso compounds were 1:0.1, 1:0.25, 1:0.4, 1:0.5 and 1:01. The test results are shown in Figure 1

[0055] After experiments with methanesulfonic acid of different molar masses at a wavelength of 395 nm, it was found that the yield was highest when the molar mass ratio of methanesulfonic acid to nitrogen nitrosopyrrolidine was 1:1. That is, when the molar mass ratio of methanesulfonic acid to nitrogen nitrosopyrrolidine was 1:1, the derivatization treatment result was the best, which was more conducive to subsequent quantitative detection.

[0056] Example 3 Excitation light wavelength test experiment

[0057] In the photocatalytic free radical reaction, the appropriate excitation wavelength is crucial to the success of the experiment. In view of the differences in the stability of LED light and experimental conditions, this example explores and optimizes the effect of the LED light identification wavelength on the actual yield. Except for the different excitation light wavelengths, other conditions are the same as in Example 1. The derivatization results of 365nm, 395nm, 405nm and 455nm are compared respectively. The test results are shown in Figure 2 .

[0058] The experimental results showed that LED lights with wavelengths of 365nm, 395nm and 405nm were all able to effectively trigger the reaction, while LED lights with a wavelength of 455nm failed to generate the target product. In particular, the experimental yield was the highest at a wavelength of 395nm, indicating that this was the optimal excitation wavelength.

[0059] Example 4 Derivatization treatment time test experiment

[0060] In photocatalytic free radical reactions, a relatively long reaction time is required to achieve the maximum yield. However, long-term LED illumination may cause the solvent temperature to rise, so the optimal reaction time is to terminate the reaction at the maximum yield.

[0061] According to the results of the previous examples, this example selected an LED with a wavelength of 395 nm, and used a molar mass ratio of methanesulfonic acid to nitrogen nitrosopyrrolidine of 1:1 for reaction monitoring.

[0062] Except for the different derivatization treatment time, other conditions are the same as those in Example 1. The derivatization treatment results of 30 hours, 2 hours, 4 hours, 6 hours and 8 hours are compared respectively.

[0063] 200 μL of the reaction solution was sampled at 0, 2, 4, 6, and 8 hours of reaction, and diluted with 1800 μL of methanol before injection. Figure 3 .

[0064] according to Figure 3 It was observed that after 2 hours, the reaction almost reached equilibrium, and the subsequent fluctuations in the product peak area may indicate that the reaction has reached dynamic equilibrium. In order to further explore the kinetics of the reaction, it was decided to study the reaction process within 2 hours under the same experimental conditions. The test results are shown in Figure 4

[0065] according to Figure 4 The test results show that the reaction quickly reaches the peak within 2 hours, and the target product benzophenone oxime is quickly generated within 0.5 hours. However, the reaction time of 0.5 hours may be too short for the photocatalytic free radical reaction. In order to further understand the changes of each reactant and product within each 2 hours.

[0066] Gas chromatography is used to monitor the reaction process. The gas chromatography conditions are as follows:

[0067] The chromatographic column selected was a DB-624 medium polarity column, and a splitless injection method was used. The injection port temperature was set to 220°C, and the injection volume was 1μL. The adiabatic purge flow rate was 3mL / min, and the purge time was 1 minute. The carrier gas flow rate was 3mL / min. The heating mode was program controlled, the heating rate was 10°C / min, the temperature rose from 150°C to 230°C, and the holding time was 10 minutes. The ion source parameters were FID (hydrogen flame ionization detector), the heater temperature was 240°C, the air flow rate was 300mL / min, the hydrogen flow rate was 30mL / min, the tail gas flow rate was 25mL / min, and there was no split.

[0068] The results of the gas chromatography standard positioning experiment are as follows Figure 5 The gas phase results of the reaction monitoring within two hours are shown in Figure 6 According to the results of gas chromatography, nitrogen nitrosopyrrolidine was basically not detected by gas chromatography after 2 hours. It can be preliminarily considered that the reaction reached the peak of the main product within 2 hours, so 2 hours was selected as the reaction termination time. The gas chromatography conditions are simple and the peak shape is good. However, the gas chromatography results show that the peak of the target product benzophenone oxime does not continue to increase within 2 hours, which is inconsistent with the results of liquid chromatography. Further analysis found that there was a residue of benzophenone oxime in the gas chromatogram, which may be due to its high boiling point and is not suitable for quantitative analysis by gas chromatography.

[0069] Example 5 Quantitative Test Results - Specificity

[0070] According to the conditions of Example 1, the liquid phase retention time of the standard samples 1,1-diphenylethylene (CH), nitrogen nitrosopyrrolidine (NPYR), benzophenone oxime (BO) and the mixed standard samples was located. The results are shown in Figure 7 ,The results showed that the target peaks were well separated and had no interference with each other, ,which indicated that the method had good specificity.

[0071] Example 6 Quantitative Detection Results - Linearity

[0072] Take the nitrogen nitrosopyrrolidine standard, prepare 1.5mg / mL, 2mg / mL, 2.5mg / mL, 3mg / mL, 5mg / mL, 8mg / mL and 10mg / mL nitrogen nitrosopyrrolidine methanol solutions, and perform derivatization treatment and quantitative analysis according to the conditions of Example 1. Draw a standard curve with the peak area of ​​benzophenone oxime as the ordinate and the concentration of nitrogen nitrosopyrrolidine added as the abscissa. The results are as follows Figure 8As shown, benzophenone oxime is linear in the range of 0.5 to 10 mg / mL, and the linear curve is y=4868.7864x-9182.20027, with a correlation coefficient (R 2 ) is 0.99403.

[0073] Example 7 Quantitative Test Results - Accuracy

[0074] The derivatization treatment was carried out according to the conditions of Example 1 to derivatize a 5 mg / mL NPYR solution. The reaction solution was continuously injected 6 times according to the liquid phase conditions of Example 1. The peak area RSD value of benzophenone oxime was calculated. The results are shown in the following table:

[0075] Peak area Repeatability 1 Repeatability 2 Repeatability 3 Repeatability 4 Repeatability 5 Repeatability 6 RSD% NPYR 12172.511 12168.594 12161.045 12186.46 12163.631 12160.317 0.08

[0076] The sample was injected six times with an RSD of 0.08%, indicating that the method had good precision.

[0077] Example 8 Quantitative Test Results - Repeatability

[0078] The derivatization treatment method was carried out in parallel according to the conditions of Example 1 to derivatize 6 portions of 5 mg / mL NPYR solution. The reaction solution was injected according to the liquid phase conditions of Example 1, and the peak area RSD value of benzophenone oxime was calculated. The results are shown in the following table:

[0079] Peak area Repeatability 1 Repeatability 2 Repeatability 3 Repeatability 4 Repeatability 5 Repeatability 6 RSD% NPYR 13003.039 12439.430 12873.000 14038.444 13455.570 12387.613 4.8

[0080] The RSD value of the peak area of ​​the derivatized product was 4.8%, indicating that the method had good repeatability.

[0081] Example 9 Limit of Quantitation and Limit of Detection

[0082] A 10 mg / mL NPYR standard solution was prepared and diluted stepwise. The derivatized sample was treated according to Example 1 and then injected into the liquid phase. The signal-to-noise ratio S / N of 10 was taken as the quantification limit, and the signal-to-noise ratio S / N of 3 was taken as the detection limit. The results showed that the quantification limit was 0.1 mg / mL and the detection limit was 0.05 mg / mL.

[0083] Example 10 Recovery

[0084] Take nitrogen nitrosopyrrolidine standard, prepare 6 samples of different concentrations of 2 mg / mL, 5 mg / mL, 8 mg / mL, low, medium and high in parallel, and derivatize in parallel according to Example 1. The reaction solution is injected according to the chromatographic conditions of Example 1. Six samples of each spiked level are measured in parallel (n=6), and the average recovery of the 6 spiked levels is calculated. The results are shown in the following table.

[0085]

[0086] The recoveries of nitrogen nitrosopyrrolidine at three concentration levels ranged from 92.03% to 111.41%, indicating that the method had good accuracy.

[0087] Example 11 Recovery rates of different nitrogen nitroso compounds under this method - Example application

[0088] Take nitrogen-nitrosodipropylamine (NDPA), nitrogen-nitrosodimethylamine (NDMA), and N-methyl-N-nitrosoaniline (NMPA) standards, and prepare 3 samples of different concentrations of 2 mg / mL, 5 mg / mL, 8 mg / mL, low, medium, and high in parallel. Derivatize in parallel according to Example 1, inject the reaction solution according to the chromatographic conditions of Example 1, measure three samples of each spiked level in parallel (n=3), and calculate the average recoveries of the three spiked levels. The results are shown in the following table.

[0089]

[0090] The recovery rates of different types of nitrogen-nitroso compounds at three levels of concentration were between 100.52% and 112.93%, indicating that the results of the derivatization experiment basically meet the quantitative requirements of different types of nitrogen-nitroso and still have good accuracy. It shows that the total impurity content of nitrogen-nitroso in complex components or complex structure drugs can be detected.

[0091] Example 12 Example Sample Detection

[0092] This method can be used for the detection of total nitrogen nitroso groups in complex structures and complex component drugs. This example takes irbesartan as an example. When this method is used for actual sample detection, it is necessary to first perform sample recovery verification. The verification method and results are as follows:

[0093] Take the same batch of irbesartan API, and use methanol to prepare it to 10 mg / mL as sample stock solution; take NPYR standard, and use methanol to prepare it to 20 mg / mL as reference stock solution. Take 1 mL of drug stock solution, add 0.4 mL, 0.5 mL, and 0.8 mL of reference stock solution, respectively, and add methanol to dilute to 2 mL. You can get 2 mg / mL, 5 mg / mL, 8 mg / mL, 3 samples of low, medium, and high concentrations, and perform parallel derivatization according to Example 1. Inject the reaction solution according to the chromatographic conditions of Example 1. Each spiked level sample is measured in triplicate (n=3), and the average recovery of the 3 spiked levels is calculated. The results are shown in the following table.

[0094]

[0095] The recoveries of the samples added in three levels of irbesartan were detected, and the recoveries were between 100.67% and 108.94%, indicating that the method has good accuracy in detecting the total nitrogen nitroso impurity content in irbesartan.

[0096] Since the final determination result is the total nitrogen nitroso impurity content, this method uses NPYR as the standard sample, so it can be converted into the total nitrogen nitroso impurity concentration by the following formula.

[0097]

[0098] (c1: total nitrogen nitrite concentration, c2: NPYR concentration)

[0099] Ten batches of irbesartan bulk drug were prepared at 5 mg / mL and derivatized in parallel according to Example 1. The reaction solution was injected according to the chromatographic conditions of Example 1. The results are shown in the following table.

[0100]

[0101] The results of the derivatization experiment show that the total impurity content of nitrogen nitroso in complex components or complex structure drugs can be detected and applied to the detection and quantitative analysis of actual samples. According to the results of its precision and accuracy, it can be applied to the detection fields including environmental monitoring, food testing and analysis of genotoxicity in drug processing.

[0102] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for quantifying the derivatization of nitrogen nitroso compounds, characterized in that: The following steps are involved: Step 1: Derivatizing the nitrogen-nitroso compound sample to be tested, dissolving 1,1-diphenylethylene and the nitrogen-nitroso compound sample in methanol, then adding methanesulfonic acid, and irradiating under ultraviolet light to obtain the derivatized nitrogen-nitroso compound sample to be tested; Step 2: Liquid phase analysis of the derivatized nitrogen nitroso compound sample using Waters TM C18 chromatographic column (3.5 μm, 4.6×150 nm) was used for separation, the column temperature was controlled at 35° C., the flow rate was 0.5 mL / min, the injection volume was 10 μL, and the ultraviolet detection wavelength was 290 nm. The mobile phase A was water containing 0.1% acetic acid, and the mobile phase B was acetonitrile containing 0.1% acetic acid. The separation was performed in the ratio of mobile phase A:mobile phase B=(60%-40%):(60%-40%). Step 3: quantitatively calculating the content of nitrogen-nitroso compounds. The nitrogen-nitroso compound samples are derivatized and converted into α-amino oxime derivatives. The content of nitrogen-nitroso compounds is quantitatively calculated by quantitative analysis of the α-amino oxime derivatives.

2. The method for quantifying the derivatization of nitrogen nitroso compounds according to claim 1, characterized in that: In the step 1, the molar mass ratio of methanesulfonic acid to the nitrogen nitroso compound is (0.5-2):

1.

3. The method for quantifying the derivatization of nitrogen-nitroso compounds according to claim 1, characterized in that: In step 1, the wavelength range of the ultraviolet lamp is 365nm to 405nm.

4. The method for quantifying the derivatization of nitrogen nitroso compounds according to claim 3, characterized in that: In step 1, the wavelength of the ultraviolet lamp is 290nm.

5. The method for quantifying the derivatization of nitrogen-nitroso compounds according to claim 1, characterized in that: In the step 1, the illumination time under the irradiation of the ultraviolet lamp is 0 hours to 8 hours.

6. The method for quantifying the derivatization of nitrogen-nitroso compounds according to claim 5, characterized in that: In the step 1, the irradiation time under the ultraviolet lamp is 2 hours.

7. The method for quantifying the derivatization of nitrogen-nitroso compounds according to claim 1, characterized in that: In step 2, the analysis time and gradient of the liquid phase detection method are: 0min~5min, 40% phase B; 5min~25min, phase B increased from 40% to 100%; 25min~27min, 100% phase B; 27min~28min, phase B decreased from 100% to 40%; 28min~35min, 100% phase B.

8. The method for quantifying the derivatization of nitrogen-nitroso compounds according to claim 1, characterized in that: In the step 3, the method for quantitatively calculating the nitrogen-nitroso compounds is: according to the detection method of steps 1 and 2, the standard sample is detected and the standard curve is fitted, and the quantitative detection limit is detected and confirmed, and according to the detection method of steps 1 and 2, the sample to be tested is detected and substituted into the fitting curve to quantitatively calculate the content of the nitrogen-nitroso compounds in the sample to be tested.

9. Application of the nitrogen-nitroso compound derivatization quantitative method according to any one of claims 1 to 8 in the field of detection.

10. The use according to claim 9, characterized in that: The applications include environmental monitoring, food testing, and analysis of genotoxicity in pharmaceutical processing.

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