A quantitative method for the derivatization of nitrogen nitroso compounds and its application

Abstract

This application provides a quantitative method for the derivatization of nitrogen nitrosamines and its application. Through simple derivatization, nitrogen nitrosamines with different structures can be determined. In the embodiments of this application, the sample derivatization method is studied, and optimal reaction conditions for nitrogen nitrosamines are proposed. This application establishes a simple and sensitive liquid chromatography quantitative analysis method, successfully establishing a method for the derivatization analysis of nitrogen nitrosamines with different structures. This method has high selectivity, sensitivity, and accuracy, and is suitable for the accurate determination of nitrogen nitrosamines in complex samples and complex structures.

Description

Technical Field

[0001] This application relates to the field of chemical detection and analysis, specifically to a quantitative method for the derivatization of nitrogen nitroso compounds. Background Technology

[0002] N-nitrosamines are considered highly carcinogenic due to their potent genotoxicity upon metabolic activation. The International Agency for Research on Cancer (IARC) of the World Health Organization classifies N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) as Group 2A carcinogens. The ICH M7 guidelines also explicitly state that these compounds have high carcinogenicity. The sources of N-nitrosamine impurities mainly include raw materials and processes. For example, in the synthesis of sartan drugs, impurities such as NDMA, NDEA, and N-nitroso-N-methyl-4-aminobutyric acid (NMBA) may be introduced to introduce a tetrazolium ring or remove excess raw materials. In the synthesis of metformin, ranitidine, and nizatidine, the generation of genotoxic N-nitrosamine impurities is related to the use of dimethylamine as a precursor.

[0003] As research progresses, more and more complex nitrogen-nitroso impurities have been discovered. Besides the aforementioned small molecules, there is also the possibility of nitrogen-nitroso functional groups directly binding to the active pharmaceutical ingredient (API). Taking nadroparin calcium as an example, the use of sodium nitrite during the degradation of heparin allows nitroso functional groups to directly bind to the branches of the repeating units of heparin. Similar cases of nitrogen-nitroso byproducts in APIs exhibit a wide molecular weight range, complex composition, and uncertain structure, significantly limiting the determination of nitrogen-nitroso content. Furthermore, drugs containing secondary amine structures also face the risk of nitroso formation during the formulation stage.

[0004] Existing methods for detecting such nitrogen-nitroso compounds with unknown structures and complex components rely on chemiluminescence immunoassay. This involves using di-n-propylamine nitrogen-nitroso as a standard, chemically cleaving the nitrogen-nitroso functional groups into nitric oxide using hydrobromic acid, and then quantitatively analyzing the light generated by the energy level transitions resulting from the reaction of nitric oxide with ozone. However, this method is limited by instrumentation and the chemical cleavage process, resulting in chromatograms with poor peak shape and severe tailing, failing to meet the requirements for detection sensitivity and quantitative analysis.

[0005] In impurity studies, small-molecule nitrogen nitrosyl impurities can be easily synthesized or their standards purchased. However, complex systems require the purchase of large quantities of standards, significantly increasing analytical costs. Furthermore, nitrogen nitrosyl standards with complex structures are often difficult to obtain, further limiting the research and development of analytical methods.

[0006] This study can transform the structure of nitrogen-containing nitroso functional groups with unknown structures and complex components into known structures through derivatization methods, enabling the detection of easily detectable target analytes. Then, existing methods can be used to perform quantitative analysis, thereby achieving specific quantitative detection of nitrogen-nitroso compounds. Summary of the Invention

[0007] To address the difficulties in detecting nitrogen nitrosamines in existing technologies, this application provides a detection method:

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

[0009] Step 1: Derivatization of the nitrogen nitroso compound sample to be tested. Dissolve 1,1-stilbene and the nitrogen nitroso compound sample in methanol, then add methanesulfonic acid, and irradiate 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 chromatography. TM Separation was performed using a C18 column (3.5 μm, 4.6 × 150 nm), with the column temperature controlled at 35 °C, the flow rate at 0.5 mL / min, the injection volume at 10 μL, and the UV detection wavelength at 290 nm. The mobile phase A consisted of water containing 0.1% acetic acid, and the mobile phase B consisted of acetonitrile containing 0.1% acetic acid. The ratio of mobile phase A to mobile phase B was (60%–40%): (60%–40%).

[0011] Step 3: Quantitatively calculate the content of nitrogen nitrosamines. After derivatization, nitrogen nitrosamines are converted into α-aminooxime derivatives. The content of nitrogen nitrosamines is quantitatively calculated by quantitative analysis of α-aminooxime derivatives.

[0012] This application also provides the application of the described quantitative method for the derivatization of nitrogen nitroso compounds in the field of detection.

[0013] Beneficial effects:

[0014] Currently, the detection of complex nitrogen nitrosamine compounds mostly involves separating and detecting them individually, which places high demands on analytical methods and instruments. The US FDA, in its latest "Control of Nitrosamine Impurities in Pharmaceuticals for Human Use" in September 2024, also stated that, provided a complete risk assessment methodology is established, for some drugs without special functional groups in their structure and without the risk of introducing nitrosamines during the manufacturing process, only the total nitrogen nitrosamine content needs to be controlled.

[0015] Currently, there is no good method for detecting total nitrogen nitrosamines. Only the European Pharmacopoeia EP 10.0 provides a chemiluminescence method based on a chemical reaction. The main principle is to convert nitrogen nitrosamines into NO, which then reacts with ozone to emit light, converting the light signal into an electrical signal for concentration detection. However, because the chemical reaction is not instantaneous, the peak signal often fails to meet analytical requirements. This application first captures the nitrogen nitrosamines and converts them into detectable α-aminooxime derivatives. In the embodiments of this application, sample pretreatment is studied, and optimal reaction conditions for nitrogen nitrosamine compounds are proposed, making the entire reaction process controllable, convenient, and reproducible, and obtaining a good detection signal in the liquid phase.

[0016] This application uses a single nitrogen nitrosamine standard for calibration and verifies its effectiveness with various other types of nitrogen nitrosamine compounds, demonstrating that the method maintains excellent detection accuracy. This indicates that the method can be used to detect multiple nitrogen nitrosamine compounds in complex compositions using the same approach. It significantly reduces analytical difficulties and the cost of purchasing different nitrogen nitrosamine reagents.

[0017] This application also demonstrates experimentally, using irbesartan, a drug with a relatively complex structure, that nitrogen nitrosyl groups in complex drug structures or components can be detected effectively, providing a new method for the detection of nitrogen nitrosyl groups in complex structures.

[0018] This application establishes a simple and sensitive liquid chromatography quantitative analysis method, successfully achieving the quantitative determination of nitrogen nitrosamines. This method exhibits high selectivity, sensitivity, and accuracy, making it suitable for the accurate determination of nitrogen nitrosamines in complex samples. This research provides a feasible means for further applying this method to fields such as environmental monitoring and food safety, especially for the quantitative analysis of total nitrogen nitrosamines in complex drug structures or components. Attached Figure Description

[0019] Figure 1 The effect of different molar amounts of methanesulfonic acid on derivatization is shown.

[0020] Figure 2 This shows the effect of different wavelengths of ultraviolet light irradiation on derivatization.

[0021] Figure 3 This chart shows the trend of benzophenone oxime formation over 8 hours.

[0022] Figure 4 This chart shows the trend of benzophenone oxime formation over 2 hours.

[0023] Figure 5 The characteristic spectra of each component in the gas chromatography are shown.

[0024] Figure 6The gas chromatogram of benzophenone oxime generated within 2 hours is shown, illustrating the issue of benzophenone oxime residue and demonstrating that gas chromatography cannot be used for quantification in this method.

[0025] Figure 7 Showing the specificity plots of each component in liquid chromatography

[0026] Figure 8 The linear fit is shown between the dosage of N-nitrosopyrrolidine and the response of the derivatized benzophenone oxime. Detailed Implementation

[0027] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

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

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] One embodiment of this application provides a quantitative method for the derivatization of nitrogen nitroso compounds, comprising the following steps:

[0031] Step 1: Derivatization of the nitrogen nitroso compound sample to be tested. Dissolve 1,1-stilbene and the nitrogen nitroso compound sample in methanol, then add methanesulfonic acid, and irradiate 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 chromatography. TM Separation was performed using a C18 column (3.5 μm, 4.6 × 150 nm), with the column temperature controlled at 35 °C, the flow rate at 0.5 mL / min, the injection volume at 10 μL, and the UV detection wavelength at 290 nm. The mobile phase A consisted of water containing 0.1% acetic acid, and the mobile phase B consisted of acetonitrile containing 0.1% acetic acid. The ratio of mobile phase A to mobile phase B was (60%–40%): (60%–40%).

[0033] Step 3: Quantitatively calculate the content of nitrogen nitrosamines. After derivatization, nitrogen nitrosamines are converted into α-aminooxime derivatives. The content of nitrogen nitrosamines is quantitatively calculated by quantitative analysis of α-aminooxime 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 nitrogen nitroso compound is (0.5-2):1.

[0036] In one embodiment, in step 1, the molar mass ratio of methanesulfonic acid and 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 illumination time under ultraviolet light is 0 to 8 hours.

[0040] In one embodiment, in step 1, the illumination 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 as follows:

[0042] 0 min–5 min, 40% B phase; 5 min–25 min, B phase increases from 40% to 100%; 25 min–27 min, 100% B phase; 27 min–28 min, B phase decreases from 100% to 40%; 28 min–35 min, 100% B phase.

[0043] In one embodiment, the method for quantitatively calculating nitrogen nitrosamines in step 3 is as follows: according to the detection methods in steps 1 and 2, the standard sample is detected and a standard curve is fitted, and the limit of quantification is confirmed. Then, according to the detection methods in steps 1 and 2, the sample to be tested is detected and the fitted curve is used to quantitatively calculate the content of nitrogen nitrosamines in the sample to be tested.

[0044] One embodiment of this application provides the application of the quantitative method for the 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 processes.

[0046] Example 1: Analytical Detection Method

[0047] 1 mmol of 1,1-stilbene and 0.5 mmol of N-nitrosopyrrolidine were dissolved in 2 mL of methanol, followed by the addition of 0.5 mmol of methanesulfonic acid. Irradiation under a 395 nm UV lamp for 2 hours converted the N-nitroso compound into a nitrosyl cation. During this process, N-nitrosylation and photoaddition reactions occurred, yielding the desired α-aminooxime derivative. Quantitative analysis of the α-aminooxime derivative indirectly quantifies the content of the N-nitroso compound.

[0048]

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

[0050] To ensure effective separation of the reaction solution, the injection sequence time was optimized. Isocratic elution was used during the main analytical time, but considering the need to empty the column to prevent interference from previous injection residues, the elution gradient time had to be adjusted. It was found that a 30-minute elution time still left 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, nor did the pressure stabilize. Therefore, a 35-minute analytical gradient time was ultimately selected.

[0051] The final gradients are shown in the table below.

[0052]

[0053] Example 2: Comparative Test of the Ratio of Methanesulfonic Acid and Nitrogen Nitrosides

[0054] The results of derivatization treatments with different ratios of methanesulfonic acid and nitrogen nitrosamines were compared. All conditions were the same as in Example 1, except for the ratio of methanesulfonic acid to nitrogen nitrosamines. The ratios of methanesulfonic acid to nitrogen nitrosamines were compared at 1:0.1, 1:0.25, 1:0.4, 1:0.5, and 1:0.1. The detection results are shown in [reference needed]. Figure 1

[0055] Experiments conducted at 395 nm wavelength on methanesulfonic acid of varying molar masses revealed that the highest yield was achieved when the molar ratio of methanesulfonic acid to N-nitrosopyrrolidine was 1:1. This indicates that a 1:1 molar ratio of methanesulfonic acid to N-nitrosopyrrolidine yields the best derivatization results, which is more conducive to subsequent quantitative detection.

[0056] Example 3: Excitation Light Wavelength Test Experiment

[0057] In photocatalytic radical reactions, a suitable excitation wavelength is crucial for experimental success. This example explores and optimizes the impact of the LED lamp's specified wavelength on the actual yield, taking into account the stability of the LED lamp and variations in experimental conditions. Except for the excitation wavelength, all other conditions were the same as in Example 1. Derivatization results at 365nm, 395nm, 405nm, and 455nm were compared. The detection results are shown in [link to relevant documentation]. Figure 2 .

[0058] Experimental results show that LEDs with wavelengths of 365nm, 395nm, and 405nm can effectively trigger the reaction, while LEDs with a wavelength of 455nm failed to generate the target product. In particular, the experimental yield was highest at a wavelength of 395nm, indicating that this is the optimal excitation wavelength.

[0059] Example 4: Derivatization Processing Time Test Experiment

[0060] In photocatalytic radical reactions, a relatively long reaction time is required to reach maximum yield. However, prolonged LED irradiation may cause the solvent temperature to rise; therefore, an optimal reaction time is needed to terminate the reaction at maximum yield.

[0061] Based on the results of previous embodiments, this embodiment selected an LED with a wavelength of 395nm and used a molar mass ratio of methanesulfonic acid and N-nitrosopyrrolidine of 1:1 for reaction monitoring.

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

[0063] 200 μL of the reaction solution was sampled at 0, 2, 4, 6, and 8 hours of reaction, diluted with 1800 μL of methanol, and then injected. The detection results are shown below. Figure 3 .

[0064] according to Figure 3 It was observed that the reaction almost reached equilibrium after 2 hours, and the subsequent fluctuations in the peak area of ​​the product likely indicated that the reaction had reached dynamic equilibrium. To further explore the reaction kinetics, it was decided to study the reaction process over 2 hours under the same experimental conditions. The detection results are shown below. Figure 4

[0065] according to Figure 4 The detection results showed that the reaction rapidly reached its peak within 2 hours, and the target product, benzophenone oxime, was rapidly generated within 0.5 hours. However, a reaction time of 0.5 hours may be too short for a photocatalytic free radical reaction. To further understand the changes in reactants and products within each 2-hour period...

[0066] Gas chromatography will be 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, using a splitless injection method. The injection port temperature was set to 220℃, and the injection volume was 1 μL. The thermal purge flow rate was 3 mL / min, and the purge time was 1 minute. The carrier gas flow rate was 3 mL / min. The temperature control mode was programmed, with a ramp rate of 10℃ / min, increasing the temperature from 150℃ to 230℃, and holding for 10 minutes. The ion source parameters were FID (Flame Ionization Detector), with a heater temperature of 240℃, an air flow rate of 300 mL / min, a hydrogen flow rate of 30 mL / min, a tail gas flow rate of 25 mL / min, and splitless injection.

[0068] The results of the gas chromatography standard localization experiment are as follows: Figure 5 As shown. The gas phase results of the reaction monitoring over two hours are as follows. Figure 6 According to the gas chromatography results, the peak of N-nitrosopyrrolidine was barely detectable after 2 hours, suggesting that the reaction reached the peak value of the major product within 2 hours. Therefore, 2 hours was chosen as the reaction termination time. While the gas chromatography conditions were simple and the peak shape was good, the results showed that the peak of the target product, benzophenone oxime, did not continuously increase within 2 hours, which contradicted the liquid chromatography results. Further analysis revealed the presence of benzophenone oxime residues in the gas chromatography, possibly due to its high boiling point, making it unsuitable for quantitative analysis using gas chromatography.

[0069] Example 5 Quantitative Detection Results - Specificity

[0070] The liquid chromatography retention times of standards 1,1-stilbene (CH), N-nitrosopyrrolidine (NPYR), benzophenone oxime (BO), and mixed standard samples were determined according to the conditions of Example 1. See the results below. Figure 7 The results showed that the target peaks were well separated and did not interfere with each other, indicating that the method has good specificity.

[0071] Example 6 Quantitative Detection Results - Linearity

[0072] Nitrosylpyrrolidine standard was used to prepare methanol solutions of 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 5 mg / mL, 8 mg / mL, and 10 mg / mL. Derivatization and quantitative analysis were performed according to the conditions of Example 1. A standard curve was plotted with the peak area of ​​benzophenone oxime as the ordinate and the concentration of added nitrosylpyrrolidine as the abscissa. The results are as follows. Figure 8As shown, benzophenone oxime exhibits linearity in the range of 0.5–10 mg / mL, with a linear curve of y = 4868.7864x - 9182.20027 and a correlation coefficient (R²). 2 The value is 0.99403.

[0073] Example 7 Quantitative Detection Results - Accuracy

[0074] Derivatization was performed on a 5 mg / mL NPYR solution according to the conditions of Example 1. The reaction solution was injected six times consecutively under the liquid chromatography conditions of Example 1. The peak area RSD of benzophenone oxime was calculated, and the results are shown in the table below.

[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 RSD was 0.08% after 6 repeated injections, indicating that the method has good accuracy.

[0077] Example 8 Quantitative Detection Results - Repeatability

[0078] Six parallel derivatizations of 5 mg / mL NPYR solution were performed according to the derivatization method described in Example 1. The reaction solution was injected under the liquid chromatography conditions described in Example 1, and the peak area RSD of benzophenone oxime was calculated. The results are shown in the table below.

[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 peak area RSD of the derivatized product was 4.8%, indicating that the method has good reproducibility.

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

[0082] A 10 mg / mL NPYR standard solution was prepared and serially diluted. After treating the derivatized sample as in Example 1, the sample was injected into the liquid chromatography system. The limit of quantitation (LOQ) was set at a signal-to-noise ratio (S / N) of 10, and the limit of detection (LOD) was set at a S / N of 3. The results showed that the LOQ was 0.1 mg / mL and the LOD was 0.05 mg / mL.

[0083] Example 10 Recovery rate

[0084] Take the N-nitrosopyrrolidine standard and prepare six parallel samples with different concentrations of 2 mg / mL, 5 mg / mL, 8 mg / mL, low, medium and high. Derivatize them in parallel according to Example 1. Inject the reaction solution according to the chromatographic conditions of Example 1. Analyze the samples at each spike level in parallel (n=6). Calculate the average recovery rate of the six spike levels. The results are shown in the table below.

[0085]

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

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

[0088] Standards of N-nitrosodipropylamine (NDPA), N-nitrosodimethylamine (NDMA), and N-methyl-N-nitrosoaniline (NMPA) were prepared in parallel at concentrations of 2 mg / mL, 5 mg / mL, 8 mg / mL, low, medium, and high. The samples were derivatized in parallel according to Example 1. The reaction solution was injected under the chromatographic conditions of Example 1. Three samples of each spiked level were measured in parallel (n=3). The average recovery rate of the three spiked levels was calculated. The results are shown in the table below.

[0089]

[0090] The recovery rates of three different concentrations of nitrogen nitrosamines ranged from 100.52% to 112.93%, indicating that the derivatization results basically meet the quantitative requirements for different types of nitrogen nitrosamines while maintaining good accuracy. This demonstrates that the total impurity content of nitrogen nitrosamines in drugs with complex components or structures can be detected.

[0091] Example Sample Detection in Example 12

[0092] This method can be used to detect total nitrogen nitrosamines in drugs with complex structures and components. This example uses irbesartan. When using this method for actual sample testing, the recovery rate needs to be verified first. The verification method and results are as follows:

[0093] Irbesartan raw material from the same batch was prepared with methanol to a concentration of 10 mg / mL as a sample stock solution. NPYR standard was prepared with methanol to a concentration of 20 mg / mL as a reference stock solution. 1 mL of the drug stock solution was added to 0.4 mL, 0.5 mL, and 0.8 mL of the reference stock solution, respectively, and diluted with methanol to 2 mL. This yielded three samples at different concentrations: 2 mg / mL, 5 mg / mL, and 8 mg / mL (low, medium, and high). Parallel derivatization was performed according to Example 1. The reaction solution was injected under the chromatographic conditions of Example 1. Three parallel determinations (n=3) were performed for each spiked level. The average recovery rate for the three spiked levels was calculated, and the results are shown in the table below.

[0094]

[0095] The recoveries of irbesartan at three levels were measured, and the recoveries ranged from 100.67% to 108.94%, indicating that the method has good accuracy in detecting the total nitrogen nitrosamine impurities in irbesartan.

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

[0097]

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

[0099] Ten batches of irbesartan active pharmaceutical ingredient were prepared at a concentration of 5 mg / mL and derivatized in parallel according to Example 1. The reaction solution was injected under the chromatographic conditions of Example 1, and the results are shown in the table below.

[0100]

[0101] This indicates that the results of this derivatization experiment can detect the total impurity content of nitrogen nitrosamines in drugs with complex components or structures, and can be applied to the detection and quantitative analysis of actual samples. Based on its precision and accuracy, it can be subsequently applied to detection fields including environmental monitoring, food testing, and the analysis of genotoxicity in pharmaceutical processes.

[0102] The above are merely preferred embodiments of the present invention. It should be noted that, for those skilled in the art, numerous improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for quantifying a derivative of a nitroso compound, characterized by, The method comprises the following steps: Step 1: Derivatization of the sample of the nitrogen nitroso compound to be tested, dissolving 1,1-diphenyl ethylene and the sample of the nitrogen nitroso compound in methanol, then adding methanesulfonic acid, and irradiating under the irradiation of a UV lamp to obtain the sample of the nitrogen nitroso compound to be tested after derivatization; Step 2: Liquid-phase analysis of the sample of the nitrogen nitroso compound after derivatization, separation is performed by using a Waters™ XBridge® C18 chromatographic column, 3.5 μm, 4.6*150 nm, column temperature control at 35°C, a flow rate of 0.5 mL / min, a sample injection amount of 10 μL, and a UV detection wavelength of 290 nm, wherein the mobile phase A is 0.1% acetic acid water, the mobile phase B is 0.1% acetic acid acetonitrile, and separation is performed by using the mobile phase A: mobile phase B = 60%~40%: 60%~40% in proportion; Step 3: Quantitative calculation of the content of the nitrogen nitroso compound, the sample of the nitrogen nitroso compound is converted into an α-amino oxime derivative after derivatization, the content of the nitrogen nitroso compound is quantitatively calculated by quantitative analysis of the α-amino oxime derivative; in the step 1, the molar mass ratio of the methanesulfonic acid to the nitrogen nitroso compound is (0.5~2): 1; In the step 1, the wavelength range of the UV lamp is 365 nm~405 nm; In the step 2, the analysis time and gradient of the liquid-phase detection method are as follows: 0 min~5 min, 40% B phase; 5 min~25 min, B phase increases from 40% to 100%; 25 min~27 min, 100% B phase; 27 min~28 min, B phase decreases from 100% to 40%; 28 min~35 min, 100% B phase.

2. The quantitative method for derivatization of nitrogen nitroso compounds according to claim 1, characterized in that, In the step 1, the wavelength of the UV lamp is 290 nm.

3. The quantitative method for derivatization of nitrogen nitroso compounds according to claim 1, characterized in that, In the step 1, the irradiation time under the irradiation of the UV lamp is 0 h~8 h.

4. The method of quantifying a diazeniumdiolate derivative according to claim 3, wherein In the step 1, the irradiation time under the irradiation of the UV lamp is 2 h.

5. The method of claim 1, wherein the diazeniumdiolate is administered in an amount of about 0.1 to about 1000 mg / kg. In the step 3, the method for quantitatively calculating the nitrogen nitroso compound is as follows: a standard sample is detected according to the detection methods of steps 1 and 2, a standard curve is fitted, a quantitative detection limit is confirmed, a sample to be tested is detected according to the detection methods of steps 1 and 2, and the content of the nitrogen nitroso compound in the sample to be tested is quantitatively calculated by substituting the sample to be tested into the fitted curve.

6. Application of the nitrogen nitroso compound derivatization quantitative method in the detection field according to any one of claims 1~5.

7. Use according to claim 6, characterized in that, The application includes environmental monitoring, food detection, and analysis of genotoxicity in pharmaceutical processes.

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