N-nitrosopyridine (NAB) purity standard, its preparation method, and its uses.

Abstract

This invention relates to a purity standard for N-nitrosopyridine (NAB), its preparation method, and its uses. The N-nitrosopyridine (NAB) purity standard of this invention is a pale yellow oily liquid with a density of 1.2 ± 0.1 g / cm³. 3 The sample is sealed in brown sample vials and has a purity of 99.18%. The purity standard material of this invention has high purity, accurate and traceable values, good homogeneity, and good stability.

Description

Technical Field

[0001] This invention relates to a purity standard of N-nitrosopseudoesequina (NAB), its preparation method, and its uses. Background Technology

[0002] N-nitrosopyridine (NAB), chemically named 1-nitroso-2-(3-pyridyl)piperidine, CAS number 37620-20-5, molecular formula C 10 H 13 Nitrogen oxide (N3O), with a relative molecular mass of 191.23, is a carcinogen produced during tobacco processing, smoking, and combustion. Reducing its content is a hot topic in international research on tobacco tar reduction and harm mitigation. Accurate determination of trace N-nitrosopyridine (NAB) content in the complex matrix of tobacco relies not only on standardized methods but also on the support of standard reference materials, ensuring the accuracy, reliability, comparability, and traceability of N-nitrosopyridine (NAB) analytical data within the industry.

[0003] However, a search revealed no purity standard for N-nitrosopyrhodoesequine (NAB), nor any single-solution standard for N-nitrosopyrhodoesequine (NAB). Only a few commercially available N-nitrosopyrhodoesequine (NAB) standards were found. Analysis and comparison of these standards revealed the following problems: First, some standards lack analytical certificates including accurate purity levels, affecting traceability. Second, N-nitrosopyrhodoesequine (NAB) is typically molten, with small individual packaging sizes, making accurate weighing difficult. Third, the standard materials used by different laboratories are purchased from different manufacturers, each with varying claimed purities, making it impossible to guarantee the consistency of test data.

[0004] Therefore, it is necessary to provide purity standard materials and single solution standard materials of N-nitrosopyridine (NAB). This will enable traceability of analytical test values, improve the consistency of comparative testing of harmful components of N-nitrosopyridine (NAB) in tobacco, tobacco products and their smoke, and serve as reference materials for assessing the ability of laboratories to participate in comparative experiments, debugging instruments and evaluating the proficiency of experimental staff. It will also further improve the industry standard material library and strongly support the industry's harm reduction efforts. Summary of the Invention

[0005] To overcome the above-mentioned shortcomings, this invention provides a purity standard of N-nitrosopseudoesequine (NAB), its preparation method, and its uses. The purity standard of this invention has high purity, accurate and traceable values, good homogeneity, and good stability.

[0006] The present invention is achieved by the following technical solution.

[0007] On the one hand, the present invention provides a purity standard for N-nitrosopseudoesequine (NAB), which is a pale yellow oily liquid with a density of 1.2 ± 0.1 g / cm³. 3 Brown bottles, such as brown sample bottles, are sealed and packaged with a purity of 99.18%.

[0008] The structural formula of the N-nitrosopseudoesequine (NAB) of the present invention is as follows:

[0009]

[0010] Preferably, the purity is the total purity (i.e., the purity before deducting impurities) minus the purity after deducting impurities, wherein the impurities are selected from anions, inorganic elements, solvent residues, xylene ketone, and water.

[0011] Preferably, the anion is selected from Cl. - NO2 - NO3 - and SO4 2- .

[0012] Preferably, the inorganic element is selected from Na, Mg, Al, K, Ca, Cr, Fe, Ni, Cu, Zn, As, Se, Cd, Sn, Sb, Te, Hg, and Pb.

[0013] Preferably, the solvent residue is selected from methanol, benzene, toluene, ethylbenzene, xylene, styrene, ethanol, isopropanol, n-propanol, n-butanol, acetone, butanone, cyclohexanone, ethyl acetate, n-propyl acetate, n-butyl acetate, isopropyl acetate, dichloromethane, tetrahydrofuran, propylene glycol methyl ether, ethylene glycol monoethyl ether, 4-methyl-2-pentanone, propylene glycol ethyl ether, m-p-xylene, ethylene glycol ethyl ether acetate, dimethyl succinate, dimethyl glutarate, and dimethyl adipate.

[0014] Preferably, the minimum packaging unit of the purity standard substance is 20 mg, and the packaging material is a 2 mL brown bottle.

[0015] Preferably, the relative standard uncertainty introduced by the purity is 0.30% as determined by the purity uncertainty assessment.

[0016] Preferably, the total purity is 99.51%.

[0017] Preferably, the anion content is 0.012%.

[0018] Preferably, the inorganic element content is 0.11%.

[0019] Preferably, the residual solvent is 0.06%.

[0020] Preferably, the benzophenone content is 0.005%.

[0021] Preferably, the moisture content is 0.037%.

[0022] On the other hand, the present invention provides a method for preparing the above-mentioned N-nitrosopyridine (NAB) purity standard material, the method comprising the following steps: (1) synthesis of N-nitrosopyridine (NAB); (2) purification of the N-nitrosopyridine (NAB) synthesized in step (1); (3) dispensing the N-nitrosopyridine (NAB) purified in step (2) to obtain the product.

[0023] Preferably, the synthesis of N-nitrosopyrhodoesequine (NAB) in step (1) includes the following steps:

[0024] (1-1) Starting with 3-aminomethylpyridine, it reacts with benzophenone via nucleophilic addition and dehydration to generate an imine compound 3;

[0025] (1-2) Compound 3 obtained in step (1-1) was reacted with 1,4-diiodobutane in the presence of LDA (diisopropylaminolithium) through a two-step nucleophilic substitution and cyclization reaction to generate compound 5;

[0026] (1-3) Compound 5 obtained in step (1-2) is subjected to a nitrosation reaction to generate N-nitrosopseudoesequina (NAB). The above reaction route is as follows: Figure 1 As shown.

[0027] Preferably, in step (1-1), the reaction temperature is 100-120°C, more preferably 115°C.

[0028] Preferably, in step (1-1), the reaction solvent is selected from toluene.

[0029] Preferably, in step (1-2), the reaction temperature is -80 to -60°C, more preferably -75 to -70°C.

[0030] Preferably, in step (1-2), the reaction solvent is selected from tetrahydrofuran.

[0031] Preferably, in steps (1-3), the reaction temperature is -5 to 10°C, more preferably 0 to 5°C.

[0032] Preferably, in steps (1-3), the reaction solvent is selected from deionized water.

[0033] Preferably, the synthesis of N-nitrosopyrhodoesequine (NAB) in step (1) further includes the steps of extracting the obtained N-nitrosopyrhodoesequine (NAB) in an organic solvent under alkaline conditions, drying, and concentrating under reduced pressure.

[0034] Preferably, the alkaline conditions refer to a pH value of 12 to 14.

[0035] Preferably, the alkaline conditions are obtained by adding sodium hydroxide, for example, 30% sodium hydroxide.

[0036] Preferably, the organic solvent is dichloromethane.

[0037] Preferably, the drying is achieved using anhydrous sodium sulfate.

[0038] Preferably, the purification in step (2) includes purification by column chromatography.

[0039] Preferably, the solvent used in the column chromatography is methanol and dichloromethane with a volume ratio of 1:40 or 1:50, more preferably 1:50.

[0040] Preferably, the N-nitrosopyridine (NAB) purified in step (2) is stored at -18°C.

[0041] Preferably, the packaging in step (3) includes the following steps:

[0042] (3-1) Clean the 2mL brown bottle with ultrapure water using ultrasonic cleaning and dry it in an oven at 100℃;

[0043] (3-2) Under a nitrogen atmosphere, use a pipette to take 20 mg of purified N-nitrosopyridine (NAB) and place it into a 2 mL brown bottle that was dried in step (3-1) to obtain the product.

[0044] Preferably, if the purified N-nitrosopyrhodoesequine (NAB) is stored at -18°C, it should be placed in the laboratory to return to room temperature before being dispensed.

[0045] Preferably, the present invention further includes determining the purity, evaluating the purity uncertainty, and testing the uniformity and stability of the N-nitrosopseudoesequina (NAB) after dispensing in step (3).

[0046] In one specific implementation, the packaging in step (3) includes:

[0047] The 2mL brown sample vial was ultrasonically cleaned with ultrapure water and dried in an oven at 100℃. The sample was removed from the -18℃ freezer and placed in a 25℃ laboratory to allow it to return to room temperature. The balance was then calibrated. The 2mL brown sample vial was then placed in the center of the balance. The sample was opened, and approximately 20mg of the sample was pipetted into the 2mL brown sample vial. After weighing, the cap was immediately screwed on and the vial was sealed with sealing film.

[0048] The reaction route of this invention uses inexpensive and readily available raw materials, involves fewer reaction steps, and produces a high-purity target product, reaching over 99%.

[0049] In another aspect, the present invention provides a N-nitrosopyrhodoesequine (NAB) solution standard, which is composed of the above-mentioned N-nitrosopyrhodoesequine (NAB) purity standard and methanol. The concentration of the solution standard is 0.099 mg / mL and the relative expanded uncertainty is 2% (expansion factor k = 2).

[0050] Preferably, the solution standard is dispensed into brown bottles, such as brown ampoules, with a packaging unit of 1.5 mL / bottle.

[0051] In another aspect, the present invention provides a method for preparing the above-mentioned solution standard substance, the method comprising (i) preparing the solution standard substance; and (ii) dispensing the solution standard substance prepared in step (i).

[0052] Preferably, the preparation of the solution standard in step (i) includes dissolving the above-mentioned N-nitrosoacetine (NAB) purity standard in methanol.

[0053] Preferably, the preparation of step (i) is completed in a Class 100,000 cleanroom.

[0054] Preferably, the solution standard prepared in step (i) is stored at -18°C.

[0055] Preferably, the packaging in step (ii) includes the following steps:

[0056] First, place the volumetric flask containing the standard solution prepared in step (i) in a -18°C freezer to cool it completely. Then, use a separator to add 1.5 mL of the standard solution to a clean brown 2 mL ampoule (under high-purity nitrogen protection). Quickly transfer the ampoule to a freezing water bath and store it at -18°C for about 20 minutes. Then, immediately remove it and seal it with a flame. Store each ampoule (1.5 mL) at -18°C.

[0057] In another aspect, the present invention provides the above-mentioned purity standard material of N-nitrosopyridine (NAB) and the above-mentioned solution standard material of N-nitrosopyridine (NAB) for the detection of tobacco-specific N-nitrosopyridine (NAB) content in tobacco and tobacco products, cigarette smoke and other tobacco-related products.

[0058] Compared with the prior art, the present invention has the following beneficial technical effects:

[0059] This invention provides for the first time a purity standard and a single solution standard for N-nitrosopseudoesequine (NAB).

[0060] The present invention verifies that the functional groups, structural formula, relative molecular mass and other information of the synthesized N-nitrosoanabasine (NAB) raw material are consistent with the relevant information of the corresponding target compound through high-resolution mass spectrometry combined with low-resolution mass spectrometry, nuclear magnetic resonance, infrared spectroscopy and ultraviolet spectroscopy.

[0061] The present invention adopts the liquid-phase area normalization method and purity analysis by multiple laboratories. After deducting impurities such as water, anions, inorganic elements and solvent residues, the purity of the N-nitrosoanabasine (NAB) purity reference material is 99.18%. Through the evaluation of purity uncertainty, the relative standard uncertainty introduced by the NAB purity is 0.30%.

[0062] The present invention conducts a homogeneity test (F-test) on the N-nitrosoanabasine (NAB) purity reference material. The results show that F-test < F-critical, indicating that the homogeneity of the N-nitrosoanabasine (NAB) purity reference material is good.

[0063] The present invention examines the long-term stability of the N-nitrosoanabasine (NAB) purity reference material. The results show that the slope of the fitting straight line equation for the long-term stability test is not significant. Therefore, the long-term stability (759 days) of the N-nitrosoanabasine (NAB) purity reference material is good.

[0064] The present invention uses the N-nitrosoanabasine (NAB) purity reference material as the raw material, chromatographic grade methanol as the solvent, and prepares a solution reference material by the weight-volume method. Under nitrogen protection, it is filled into brown bottles, and the packaging unit is 1.5 mL / bottle.

[0065] The present invention conducts a homogeneity test (F-test) on the solution reference material. The results show that for the "N-nitrosoanabasine (NAB) solution reference material in methanol (i.e., the solution reference material of the present invention)" after encapsulation, F-test < F-critical, indicating good homogeneity.

[0066] The present invention examines the short-term stability and long-term stability of the "N-nitrosoanabasine (NAB) solution reference material in methanol". The results show that the slopes of the fitting straight line equations for both the short-term stability and long-term stability tests are not significant. Therefore, the short-term stability (stored in the dark at 4 °C, 25 °C and 50 °C for 7 days) and long-term stability (12 months) of the "N-nitrosoanabasine (NAB) solution reference material in methanol" are good.

[0067] Through the research on certification and uncertainty evaluation, it is shown that the concentration value in the "N-nitrosoanabasine (NAB) solution reference material in methanol" is 0.099 mg / mL, and the relative expanded uncertainty is 2% (k = 2).

[0068] In the preparation of N-nitrosopyridine (NAB), 3-aminomethylpyridine is used as the starting material. It reacts with benzophenone via nucleophilic addition and dehydration to generate an imine compound 3. Subsequently, compound 3 reacts with 1,4-diiodobutane in the presence of lithium diisopropylaminoethyl (LDA) via a two-step nucleophilic substitution and cyclization reaction to generate compound 5. Finally, compound 5 undergoes nitrosation to generate the NAB candidate. This synthetic route uses inexpensive and readily available raw materials, involves few reaction steps, and yields a high-purity target product. After generating the target candidate N-nitrosopyridine (NAB), the product is further purified. The reaction process is monitored using TLC (thin-layer chromatography), and further purification is achieved by silica gel column chromatography to obtain a product with a purity >99%. In the process of determining the value of pure products, HPLC-DAD and GC-FID are used in combination, and the influence of impurities such as moisture, evaporation residue, and solvent residue in the pure products is fully considered, making the determination more accurate and traceable. The standard material of this invention can solve the problem that current testing methods cannot trace the value, improve the consistency of the test results of various units, and fill the gap in the development of N-nitrosamine standard materials at home and abroad.

[0069] The yield and purity of each step in this invention are high. Attached Figure Description

[0070] Figure 1 The synthetic route of NAB in this invention;

[0071] Figure 2 The liquid chromatogram is shown for the initial purity determination of NAB synthesized in Example 1.

[0072] Figure 3 The hydrogen spectrum of NAB synthesized in Example 1;

[0073] Figure 4 The carbon spectrum of NAB synthesized in Example 1;

[0074] Figure 5 High-resolution mass spectrometry data of NAB synthesized in Example 1;

[0075] Figure 6 This is a long-term stability graph of the NAB synthesized in Example 1.

[0076] Figure 7 The experimental results for the selection of developing solvents in the NAB purification stage are shown below. The developing solvents used in A, B, C, and D are as follows: A: methanol and dichloromethane with a volume ratio of 1:20; B: methanol and dichloromethane with a volume ratio of 1:40; C: methanol and dichloromethane with a volume ratio of 1:50; and D: methanol and dichloromethane with a volume ratio of 1:60. The samples on the left in A, B, C, and D are crude products, and the samples on the right are purified products. Detailed Implementation

[0077] The technical solution of the present invention will be further described below in conjunction with specific implementation methods.

[0078] Example 1: Preparation of a purity standard for N-nitrosopyridine (NAB)

[0079] (1) Synthesis of N-nitrosopseudoesequine (NAB)

[0080] The synthetic route of N-nitrosopseudoesequina (NAB) raw material is as follows: Figure 1 As shown. The specific synthesis steps are as follows:

[0081] (1-1) Preparation of compound 3

[0082] 32.3 g (1.0 eq) of 3-aminomethylpyridine, 57.2 g (1.05 eq) of benzophenone, 11.4 g (0.2 eq) of p-toluenesulfonic acid monohydrate, and 300 mL of toluene were added to a flask. The mixture was heated to reflux, and the water was separated. The mixture was stirred at this temperature for 3–4 h. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was poured into 100 mL of saturated sodium bicarbonate solution. The mixture was separated, and the aqueous phase was extracted with 100 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give 78.6 g of crude compound 3 (oily substance), with a crude product yield of 90.7%.

[0083] (1-2) Preparation of compound 5

[0084] Add 35.0 g (1.0 eq) of crude compound 3 and 100 mL of tetrahydrofuran to a flask, stir until dissolved, and cool to -75 to -70 °C in a dry ice-ethanol bath. Add 35 mL (1.2 eq) of 2.0 M LDA tetrahydrofuran solution dropwise, keeping the temperature below -70 °C. After the addition is complete, stir at -75 to -70 °C for 0.5 to 1 h. Then add 32.8 g (1.5 eq) of 1,4-diiodobutane dropwise, again keeping the temperature below -60 °C. After the addition is complete, stir at room temperature for 1 to 2 h. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was slowly poured into 200 mL of 3N hydrochloric acid solution and stirred for 20–30 min. 60 mL of ethyl acetate was added for extraction three times, and the aqueous phase was retained. The pH of the aqueous phase was adjusted to 10–12 with potassium carbonate and 40% potassium hydroxide, respectively, and then extracted three times with 100 mL of dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to column chromatography (methanol:dichloromethane = 1:12.5) to give 5.3 g of compound 4 (oil), with a yield of 24.4%.

[0085] (1-3) Synthesis of N-nitrosopseudoesequina

[0086] Add 5.3 g (1.0 eq) of compound 5 and 31 mL of 4N hydrochloric acid solution to a flask, and cool to 0–5 °C in an ice-water bath. Weigh 3.6 g (3.0 eq) of sodium nitrite and dissolve it in 24 mL of water to prepare a solution. Slowly add the sodium nitrite solution dropwise to the flask, controlling the temperature not to exceed 5 °C. After the addition is complete, stir at room temperature for 4–5 h. Monitor the reaction by TLC. After the reaction is complete, adjust the pH to 12–14 with 30% sodium hydroxide, extract three times with 100 mL of dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain crude N-nitrosopyrhodoesequine (NAB).

[0087] (2) Purification of N-nitrosopyridine (NAB)

[0088] The crude N-nitrosopyridine (NAB) obtained in step (1) was purified by column chromatography. The solvent used in the column chromatography was methanol and dichloromethane with a volume ratio of 1:50. 3.68 g of a yellow oily substance was obtained, with a yield of 65.7% and HPLC > 99%. The liquid chromatography conditions were as follows: elution program: 0-10 min: 60-90% B; 10-14 min: 90-90% B; 14-19 min: 90-10% B; 19-20 min: 10-60% B; 20-25 min: 60-60% B; injection volume: 10 μL; column temperature: 50℃; concentration: 1 mg / mL; column: Luna 3u PFP (4.6 × 150 mm × 3 μm); mobile phase: methanol and water. The initial liquid chromatogram of NAB purity is shown below. Figure 2 As shown.

[0089] High-resolution mass spectrometry, low-resolution mass spectrometry, and nuclear magnetic resonance spectroscopy were used (see...). Figures 3-5 The structure of N-nitrosopseudoesequina (NAB) obtained above was verified by means of infrared spectroscopy and ultraviolet spectroscopy. Its functional group information, structural formula and relative molecular mass information are consistent with the relevant information of the corresponding standard.

[0090] The proton NMR spectral data are as follows: δ: 8.59 (s, 1H-5, CH), 8.58 (d, J = 4.8 Hz, 1H-1, CH), 8.52 (d, J = 4.2 Hz, 1H-1, CH), 8.39 (s, 1H-5, CH), 7.55 (d, J = 7.8 Hz, 1H-3, CH), 7.32 (m, 1H-2, CH), 7.31 (m, 1H-3, CH), 7.27 (m, 1H-2, CH), 6.40 (d, J = 4.8 Hz, 1H-6, CH), 5.91 (t, J = 4.2 Hz, 1H-6, CH), 4.80 (dd, J = 14.7 Hz, J = 4.2 Hz, 1H-10, CH2). ),4.65(dt,J=13.8Hz,J=4.8Hz,1H-10,CH2),3.64(td,J=12.9Hz,J=4.2Hz,1H-10,CH2),2.89(td,J=12.9Hz,J=4.2Hz,1H-10,CH2),2.57(m,1H-7,C H2),2.41(d,J=13.8Hz,1H-7,CH2),2.16(m,1H-7,CH2),1.94(d,J=12.0Hz,1H-7,CH2),1.82(m,4H-9,CH2),1.69(m,2H-8,CH2),1.53(m,2H-8,CH2).

[0091] The carbon spectral data are as follows: δ: 148.97 (C-5), 148.53 (C-5), 148.34 (C-1), 147.98 (C-1), 134.75 (C-3), 134.41 (C-3), 133.55 (C-4), 132.11 (C-4), 123.53 (C-2), 123.51 (C-2), 59.86 (C-6), 47.90 (C-6), 47.58 (C-10), 37.48 (C-10), 29.14 (C-7), 26.97 (C-7), 25.90 (C-8), 24.55 (C-8), 20.41 (C-9), 19.55 (C-9).

[0092] The homogeneity and stability of the synthesized NAB raw material were tested, and the results showed that the NAB raw material was homogeneous and stable.

[0093] (3) Dispensing of purified N-nitrosopseudoesequina (NAB)

[0094] The purified N-nitrosopyridine (NAB) sample was aliquoted into 2mL brown sample vials. The vials were ultrasonically cleaned with ultrapure water and dried in an oven at 100°C. The samples were removed from a -18°C freezer and allowed to reach room temperature in a 25°C laboratory before aliquoting. Due to the sample characteristics, aliquoting required a low-moisture, oxygen-free environment; therefore, a glove box was used for sample aliquoting. The glove box was filled with high-purity nitrogen inert gas to control oxygen and moisture levels. Specific procedures are as follows:

[0095] Glove box operation preparation: Power on: Turn on the main power switch (red); open the working gas pressure reducing valve (main valve open to maximum, auxiliary valve open to 0.4-0.6 MPa); start the analyzer (oxygen index will start displaying when water index drops below 200 ppm); start circulation (ensure water and oxygen values ​​are both below 200 ppm before starting circulation, otherwise, purge to below 200 ppm before starting circulation); set the chamber pressure to +1-+6 mbar. Prepare high-purity nitrogen (99.999% purity), total pressure >10 bar, connect the gas line and set the pressure reducing valve pressure to 0.4 MPa. Check that the partial pressure is generally set between 4-6 bar, water and oxygen <0.1 ppm, and a balance (sensitivity 0.0001 g) is pre-installed in the chamber. Then turn on the vacuum pump and lighting, confirm that the transition chamber door is closed and the pressure gauge is at 0. After checking, prepare NAB samples, 2 mL brown sample vials (700 vials), sterile pipette tips, pipettes, and tweezers. Open the large transition chamber door, place the experimental equipment and samples inside, close the door, turn the transition chamber knob to the vacuum position, wait for the pressure gauge reading to reach the lowest value (-0.1 MPa), then turn the knob to the cleaning position, wait for the transition chamber pressure gauge to reach 0, and turn the knob to the closed position to complete one gas replacement cycle. Repeat this process three times, maintaining the vacuum for 5 minutes during the third cycle. After cleaning, turn the knob to the closed position. Slowly put on the operating box gloves, use the foot switch to reduce the pressure, and ensure that the pressure inside the chamber is within the normal range (3.2 mbar). Open the inner door of the transition chamber, remove the experimental equipment and samples from the large transition chamber, and then close the door.

[0096] Weighing procedure: First, calibrate the balance. Then, place a 2mL brown sample vial in the center of the balance. Open the sample vial and use a pipette to draw approximately 20mg of sample into the 2mL vial. After weighing, immediately screw on the cap and seal the vial opening with sealing film. Dispense a total of 150 vials. After dispensing, clean the work surface and confirm that the gas in the transition chamber is inert. Place the experimental materials and samples back into the large transition chamber, close the inner chamber door, slowly remove the gloves, and use the foot switch to ensure that the pressure inside the chamber is within the normal range. Remove the experimental materials, close the chamber door, and place the sealed samples in a desiccator and store them at a low temperature of -18℃.

[0097] Example 2: Purity determination, purity uncertainty assessment, homogeneity and stability testing of N-nitrosopyridine (NAB) I. Purity determination of N-nitrosopyridine (NAB)

[0098] The purity of the main component of N-nitrosopyrhodoesequine (NAB), the purified standard prepared in Example 1, was determined by liquid chromatography area normalization method. Simultaneously, impurities such as anions, inorganic elements, solvent residues, and moisture in the NAB raw material were determined by ion chromatography, inductively coupled plasma mass spectrometry, headspace gas chromatography, and gas chromatography, respectively. Specific test methods and results are as follows:

[0099] 1. Testing Method

[0100] 1.1 Liquid Chromatography Method

[0101] The determination was performed using liquid chromatography, and the liquid chromatography conditions were as follows:

[0102] Chromatographic column: Poroshell EC-C18 (4.6 x 250 mm x 4 μm); Mobile phase: Water (A) and methanol (B); Elution program: 0-5 min: 38-38% B; 5-8 min: 38-90% B; 8-13 min: 90-90% B; 13-15 min: 90-38% B; 15-20 min: 38-38% B; Column temperature: 40℃; Flow rate: 1.0 mL / min; Detection wavelength: 238 nm; Injection volume: 10 μL; Sample concentration: 1 mg / mL.

[0103] 1.2 Purity analysis by multiple laboratories

[0104] Taking into account the management capabilities, technical capabilities, and hardware facilities of each analytical unit, nine units were organized to conduct joint purity determination work, using the high-performance liquid chromatography (HPLC) area normalization method. The joint determination units and instrument models are shown in Table 1.

[0105] Table 1 Jointly set value units and instrument models

[0106]

[0107] Nine sets of fixed-value data were obtained from nine fixed-value units, from laboratory 01 to laboratory 09, as shown in Table 2 below.

[0108] Table 2. Collaborative NAB data from 9 laboratories (unit: %)

[0109]

[0110]

[0111] 1.3 Moisture Determination

[0112] The moisture content in NAB samples was determined according to YC / T 539-2016 "Determination of Moisture in Triacetin for Tobacco Products - Gas Chromatography". The internal standard method was used for quantification of moisture content. The specific instruments and equipment used are as follows:

[0113] The moisture content in the NAB candidate solution was determined using gas chromatography-thermal conductivity detector (GC-TCD, Agilent 6890N) and internal standard method (acetone (standard number: GBW06115)). The results are shown in Table 3. From the table, it can be calculated that the moisture content in the NAB candidate solution is 0.037%.

[0114] Table 3. Moisture content (mg / g) in NAB solution

[0115] Number of experiments 1 2 3 4 5 6 7 8 9 average value NAB 0.38 0.38 0.36 0.35 0.31 0.38 0.4 0.41 0.39 0.37

[0116] 1.4 Anion Determination

[0117] Referencing industry standard YC / T248-2008, "Determination of Inorganic Anions in Tobacco and Tobacco Products - Ion Chromatography Method". The anion detection results are shown in Table 4.

[0118] Table 4. Anion detection results (unit: μg / mL)

[0119]

[0120] 1.5 Determination of Inorganic Elements

[0121] 1.5.1 Measurement Process

[0122] The inorganic element content in NAB candidates was analyzed using inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7900). A 1 mg / mL candidate solution was prepared using chromatographic-grade methanol. Specifically, 10.07 mg of the candidate was weighed and placed in a 10 mL amber volumetric flask, then diluted to the mark with methanol to obtain a 1 mg / mL candidate solution. 1 mL of this solution was then transferred to each sample and diluted to 50 mL with 1% nitric acid. For analysis, a semi-quantitative analysis mode was first used. Elements with a CPS count exceeding 500 were accurately quantified using a standard curve. The full scan range covered elements from the periodic table.

[0123] 1.5.2 Instrumental Analysis Conditions

[0124] Under the selected instrument parameters, inductively coupled plasma mass spectrometry (ICP-MS) was performed with an internal standard added online. The element concentration in the sample solution was determined by the quantitative relationship between mass-to-charge ratio intensity and element concentration. A full-scan semi-quantitative analysis was first performed to accurately quantify elements with an instrument CPS count greater than 500 (Na, Mg, Al, K, Ca, Cr, Fe, Ni, Cu, Zn, As, Se, Cd, Sn, Sb, Te, Hg, Pb). The instrument operating parameters, selected isotopes, internal standard elements, and integration times are shown in Tables 5 and 6.

[0125] Table 5. Main parameters of ICP / MS

[0126]

[0127] Table 6 Measurement of isotopes, internal standard elements, and integration time.

[0128]

[0129] The results of inorganic elements in NAB are shown in Table 7 below. It was found that the types and contents of inorganic elements contained in the NAB candidates were not the same. They mainly contained trace amounts of Na, Sn, Sb and other elements, with a total amount of 22.57 mg / mL, accounting for 0.11%.

[0130] Table 7. Results of Inorganic Element Content Determination (Unit: mg / mL)

[0131]

[0132] Note: Italics in the table indicate not detected. The test results are expressed as detection limits and are included in the total element count.

[0133] 1.6 Determination of Volatile Impurities

[0134] Referring to the industry standard YC / T 207—2014 "Determination of Solvent Residues in Tobacco Paper by Headspace-Gas Chromatography / Mass Spectrometry", the solvent residues were determined by headspace-gas chromatography / mass spectrometry (HS-GC / MS).

[0135] Results Analysis: The content of volatile organic solvents in NAB was determined. Only dichloromethane was detected in all samples, with a peak time of 8.093 min. Other volatile organic solvents were not detected. The results are shown in Table 8.

[0136] Table 8. Dichloromethane content in NAB (unit: ng / mL)

[0137] Number of experiments 1 2 3 average value percentage dichloromethane 569 558 572 566 0.06％

[0138] 1.7 Determination of Benzophenone Solvent Residue

[0139] According to industry standard YQ / T 31-2013 "Determination of Photoinitiators in Cigarette Carton and Packaging Paper by Gas Chromatography-Mass Spectrometry", the content of xylene ketone in methanol and NAB candidates was analyzed using a gas chromatography-mass spectrometry system (Agilent 7890-5975C). Selected ion scanning was used. The results are shown in Table 9.

[0140] Table 9. Benzophenone content (μg / mL) in methanol solvent and NAB solution

[0141] Number of experiments 1 2 3 average value percentage methanol / / / / / NAB 0.05 0.05 0.05 0.05 0.005％

[0142] 2. Test Results

[0143] Using the liquid phase area normalization method, the purity values ​​from multiple laboratories were averaged, resulting in a purity of 99.52% (NAB) for the NAB candidates. The actual purity of the NAB candidates should be the result after deducting all impurities (anions, inorganic elements, residual solvent, benzophenone, and water), i.e., PB. 纯度 =(1-W) 阴离子 -W 无机元素 -W 溶剂残留 -W 二苯甲酮 -W 水 )×P 测定 The purity of NAB after deducting impurities is shown in Table 10 below.

[0144] Table 10. Purity test results (%) of liquid chromatography area normalization method

[0145]

[0146] II. Evaluation of the Purity Uncertainty of NAB

[0147] Referring to the technical specification JJF 1855-2022, the purity uncertainty of the NAB purity standard prepared in Example 1 after dispensing was determined. The uncertainty of the purity determination includes the uncertainty introduced by the instrument verification, raw material homogeneity, raw material stability and the determination of impurities (anions, moisture, inorganic elements and solvent residues).

[0148] The relative standard uncertainty introduced by NAB purity is 0.30% according to the purity uncertainty assessment.

[0149] III. Uniformity Test of NAB

[0150] According to the uniformity test sampling requirements in JJF 1343–2022 (when the total number of units is 100 < N ≤ 200, the number of units sampled shall not be less than 11), the NAB candidate samples prepared in Example 1 were coded according to the order of packaging, and then samples were taken according to a random number table, for a total of 11 bottles of samples (the sampling method is to code the packaging order and then take samples according to a random number table). Sample solution preparation steps: Take out 11 bottles of NAB raw material samples, take about 10.00 mg of sample from each bottle and place it in a 10 mL brown volumetric flask, and dilute to the mark with methanol to obtain a NAB test solution with a concentration of 1 mg / mL. A total of 11 solutions were prepared, and three samples were prepared from each bottle, for a total of 33 samples, and each sample was numbered. The method used was the liquid chromatography area normalization method.

[0151] The obtained purity data are subjected to an F-test. If the F-test value is less than the critical value, it indicates that the sample homogeneity is good; otherwise, it is not.

[0152] The results of the homogeneity test are shown in Table 11. According to JJF 1343-2022, the F-test was used for statistical analysis. Referring to the F-distribution table, at a confidence probability of 95%, F... 0.05 (10, 22) = 1.16. Since 1.16 calculated using F < F... 0.05 (10, 22) indicates that there is no significant difference between samples at the 95% confidence level, and the NAB candidate raw material samples after dispensing are homogeneous.

[0153] Table 11 Results of homogeneity test of NAB candidate raw materials

[0154]

[0155]

[0156] Note: In the table,

[0157] IV. Stability Testing of NAB

[0158] The sample used in this stability test was the purity standard material prepared in Example 1 after being dispensed.

[0159] The long-term stability of the samples needs to be assessed over a period of one year. Considering the characteristics of the raw materials, a 1 mg / mL NAB candidate solution needs to be prepared to test the stability of the raw materials. The preparation steps for the 1 mg / mL solution are as follows: Take 20.00 mg of the raw material sample from each of three vials and place it in a 20 mL amber volumetric flask. Dilute to the mark with methanol to obtain a 1 mg / mL NAB test solution. The method used is liquid chromatography with area normalization.

[0160] The results of the long-term stability test of NAB raw materials are shown in Table 12, and the long-term stability diagram of NAB raw materials is shown in [the table]. Figure 6 As shown.

[0161] Table 12 Long-term stability data of NAB feedstock

[0162]

[0163]

[0164] In accordance with the requirements of JJF 1343-2022, a linear model was selected as the empirical model for this standard material.

[0165] The long-term stability data in Table 12, with x representing time (days) and y representing NAB purity value, are fitted to a straight line y = kx + b, resulting in the equation y = -0.0000034x + 99.53. Therefore, the slope k = -0.0000034 and the intercept b = 99.53.

[0166] The standard deviation of each point on the straight line can be calculated using the following formula:

[0167]

[0168] The slope uncertainty is calculated using the following formula:

[0169]

[0170] The degrees of freedom are n-2 = 12-2 = 10 and p = 0.95 (95% confidence interval), and the t-value is 2.228 from the t-value table.

[0171] because Therefore, the slope is not significant, and thus the stability is good.

[0172] Example 3: Preparation of the NAB solution standard of the present invention

[0173] The NAB used in this embodiment is the purity standard substance prepared in Example 1 after being dispensed.

[0174] 1. Preparation of the solution standard substance of the present invention

[0175] Before weighing the samples, the weighing equipment (tweezers, 100μL microsyringe, calibrated Class A 1000mL volumetric flasks, and Class ① balance) was thoroughly cleaned with distilled water and dried at high temperature. The sample weighing and preparation process was completed in a Class 100,000 cleanroom. Gloves were worn throughout the weighing operation. The main steps were as follows: first, zero the balance; then, open the windproof cover door; next, use tweezers to place the container on the weighing pan; close the door and wait for the reading to stabilize before zeroing again; after adding the sample, close the door and wait for the balance reading to stabilize.

[0176] Since NAB is a liquid oil at room temperature, its density is 1.2 g / cm³, calculated using the conversion between density, volume, and mass. 3 100 mg corresponds to a volume of 83.34 μL. Using a 100 μL microsyringe, 84 μL was pipetted and added dropwise at a rate that was initially fast and then slowed down. When the balance reading approached 100 mg, the solution was added slowly dropwise, accurately weighing approximately 100 mg. Finally, the inner wall of the volumetric flask was rinsed with chromatographic grade methanol. When the volume was close to the mark, it was carefully added dropwise to the mark, bringing the volume to 1000 mL. The solution was shaken well and allowed to stand to obtain a 0.100 mg / mL NAB standard solution. The prepared NAB standard solution was then transferred to a -18°C freezer.

[0177] 2. Dispensing of the solution standard substance of the present invention

[0178] In a Class 100,000 cleanroom, volumetric flasks containing NAB standard solution were first placed in a -18°C freezer for thorough cooling. Then, using a dispenser, 1.5 mL of NAB standard solution was added to 2 mL clean brown ampoules (under high-purity nitrogen protection). The ampoules were then quickly transferred to a freezer bath and stored at -18°C for approximately 20 minutes. Afterward, they were immediately sealed with a flame. A total of 500 ampoules were dispensed, each containing 1.5 mL, and stored at -18°C.

[0179] Example 4: Evaluation of the homogeneity, stability and uncertainty of the NAB solution standard of the present invention. The NAB solution standard used in this example was prepared from Example 3.

[0180] I. Homogeneity test of the NAB solution standard substance of the present invention

[0181] 1. Uniformity test

[0182] According to the sampling quantity requirements of JJF 1343-2022 for homogeneity testing (when the total number of units is 200 < N ≤ 500, the number of units sampled shall not be less than 15), five bottles of NAB solution standard material were taken from each of the front, middle, and rear sections according to the packaging order (the sampling method was to use the packaging sequence code and then refer to a random number table for sampling), for a total of 15 bottles. Three samples were prepared from each bottle, resulting in a total of 45 samples, and each sample was numbered. The obtained data were converted to concentration using the single-point method, and the concentration values ​​were used for F-test. If the F-test value was less than the critical value, it indicated that the sample homogeneity was good; otherwise, it was not.

[0183] 2. Liquid Chromatography Conditions

[0184] The chromatographic conditions are as follows:

[0185] The NAB chromatographic conditions are as follows:

[0186] —Column: Poroshell EC-C18 column (4.6mm×250mm, 4μm);

[0187] —Flow rate: 1.0 mL / min;

[0188] —Column temperature: 40℃;

[0189] —Injection volume: 10 μL;

[0190] —Mobile phase A: water, mobile phase B: methanol;

[0191] —Full wavelength scanning mode (210-500nm), monitoring wavelength 238nm;

[0192] —The gradient elution conditions are shown in Table 13;

[0193] Table 13 High Performance Liquid Chromatography Gradient Elution Conditions (NAB)

[0194]

[0195] 3. Mathematical statistics methods

[0196] According to the JJF 1343-2022 technical specification, one-way ANOVA is adopted as the mathematical statistical method for homogeneity testing.

[0197] 4. Results

[0198] The test results showed that the NAB component of the "NAB solution standard in methanol" was well homogeneous after being dispensed.

[0199] II. Stability Testing of the NAB Solution Standard Material of the Present Invention

[0200] 1. Short-term stability

[0201] The samples were stored in brown ampoules protected from light. Therefore, the short-term stability was mainly assessed by examining the effect of temperature. The concentration changes of the reference material were examined on days 0, 1, 3, 5, and 7 at cold chain temperature (4℃), normal temperature (25℃), and extreme temperature (50℃) during cold chain transportation. Twelve ampoules of NAB solution reference material samples were placed in three temperatures (4℃ refrigerator, 25℃, and 50℃ artificial climate chamber). The sample on day 0 was measured after being placed at the corresponding temperature for 1 hour. Subsequently, three ampoules were removed from the refrigerator and artificial climate chamber at the corresponding temperatures on days 1, 3, 5, and 7, respectively, and the last three ampoules were removed for testing on day 7. Each sample was measured three times.

[0202] The liquid chromatography conditions are the same as those in Part 1 of this embodiment.

[0203] The concentration of the main component was converted using the single-point method, and the calibration solution for the single-point method was prepared in a Class 100,000 cleanroom.

[0204] The results of the short-term stability test of the NAB solution standard are shown in Table 14.

[0205] Table 14 Short-term stability data of NAB solution standard material

[0206]

[0207]

[0208] Conclusion: The short-term stability test results show that the concentration of the "NAB solution standard in methanol" did not change after short-term storage at 4℃, 25℃ and 50℃.

[0209] 2. Long-term stability of the sample

[0210] According to the metrological technical specification JJF 1343-2022, the long-term stability requirement for national secondary standard reference materials is 6 months or more. Considering the stability of NAB solution standard reference material samples under light-protected conditions at -18℃, the packaged NAB solution standard reference material samples were stored at -18℃ for a long period, following a principle of initial dense storage followed by sparse storage. Stability tests were conducted at 0, 1, 2, 4, 6, 9, 12, and 24 months. Three samples were randomly selected at each time point, and each sample was measured three times. The chromatographic conditions were the same as described in Part I of this embodiment.

[0211] The concentration of the main component was converted using the single-point method, and the calibration solution for the single-point method was prepared in a Class 100,000 cleanroom.

[0212] The measurement results are shown in Table 15 below.

[0213] Table 15 Long-term stability data of NAB solution standard material

[0214]

[0215] Conclusion: The long-term stability test results show that the t-value of the long-term stability test of the "NAB solution standard in methanol" is less than the critical value, indicating that the "NAB solution standard in methanol" sample has good long-term stability.

[0216] III. Uncertainty Assessment of the NAB Solution Standard Material of the Present Invention

[0217] The main sources of uncertainty in the NAB solution standard of this invention are: (1) solution preparation process (raw material purity analysis, volume and weighing); (2) solution homogeneity; (3) solution stability; and (4) filling loss. Uncertainty assessments were performed below:

[0218] 1. Uncertainty introduced by solution preparation

[0219] 1.1 Uncertainty introduced in the raw material purity analysis process

[0220] Referring to JJF 1855-2020 standard, the uncertainty in purity determination includes uncertainties introduced by instrument verification and the determination of impurities (anions, moisture, inorganic elements, and solvent residues). According to the results in Chapter 6, the uncertainty introduced by NAB purity is 0.30%.

[0221] 1.2 Uncertainty introduced by the balance

[0222] The electronic balance used in the experiment was calibrated to Class ① by Suzhou Langbo Calibration and Testing Co., Ltd. The standard uncertainty caused by balance calibration during the preparation of standard substance samples is as follows: Its relative uncertainty is:

[0223] 1.3 Uncertainty introduced by volume

[0224] (1) Type A Uncertainty. The Type A uncertainty caused by volume was examined by repeatedly making the volumetric flask to volume eight times and weighing it using a balance. The eight volumes were 999.96 mL, 999.93 mL, 999.87 mL, 1000.02 mL, 1000.01 mL, 999.94 mL, 1000.00 mL, and 1000.02 mL. Using Bessel's formula, the standard deviation is 0.9968 mL. Therefore, the uncertainty caused by volume in the preparation of the standard substance sample is 0.9968 mL, and the relative standard deviation expressed as a relative standard uncertainty is 0.1%.

[0225] (2) Type B uncertainty

[0226] Type B uncertainty arises from two sources: the calibration of the volumetric flask and the discrepancy between the calibration temperature and the experimental temperature. The volumetric flasks used in the experiment were calibrated to Class A by Suzhou Langbo Calibration and Testing Co., Ltd. The standard capacity tolerance of a Class A 1000mL volumetric flask at 20℃ is ±0.31mL. Therefore, its standard uncertainty is: Since the volumetric flask was calibrated at 20°C, while the laboratory temperature varied by 2°C, the uncertainty of the resulting volume change can be estimated using the temperature variation range and the coefficient of volume expansion. Because the volume expansion of methanol is significantly greater than that of the volumetric flask, only the former needs to be considered. The coefficient of volume expansion of methanol is 1.20 × 10⁻⁶. -3 ·℃ -1 Therefore, the resulting volume change is ±(1000×2×1.20×10). -3 If the volume is ±2.4 mL, then its standard uncertainty is: Type B combination uncertainty Relative standard uncertainty:

[0227] (3) Combined relative standard uncertainty

[0228] 2. Uncertainty introduced by solution homogeneity

[0229] According to JJF 1343-2022, and based on the homogeneity test results in this report, the relative standard uncertainty, expressed as the relative standard deviation of homogeneity, is: u c(H) =0.21%.

[0230] 3. Uncertainty introduced by stability

[0231] 3.1 Uncertainty introduced by short-term stability

[0232] According to JJF 1343-2022, and based on the short-term stability test results in this report, the uncertainty arising from short-term stability is evaluated using the following formula:

[0233] 3.2 Uncertainty introduced by long-term stability

[0234] According to JJF 1343-2022, and based on the long-term stability test results in this report, the uncertainty arising from long-term stability is evaluated using the following formula:

[0235] 4. Uncertainty introduced by filling loss

[0236] Since the solvents used in the NAB solution standard material, methanol, dichloromethane, and benzophenone, have relatively low boiling points, there may be some loss during the filling and heat sealing process. The concentration of NAB material in 25 filled samples was statistically analyzed, and its relative standard deviation was used as the relative standard uncertainty. The results are shown in Table 16.

[0237] Table 16 Uncertainty introduced by filling loss

[0238]

[0239]

[0240] 5. Overall combined relative standard uncertainty

[0241] Table 17 Relative Standard Uncertainty Components and Combination

[0242]

[0243] When the confidence probability is 95%, the expansion factor is taken as 2, and the expanded uncertainty is calculated according to u. rel =k×u crel The calculation yields the result: u rel =2 × 0.59% = 1.19% ≈ 2%.

[0244] Example 5: Selection of developing solvent for the purification stage of N-nitrosopyrheine (NAB)

[0245] The same method as in Example 1 was used, wherein the developing solvents used were as follows: A: methanol and dichloromethane in a volume ratio of 1:20, B: methanol and dichloromethane in a volume ratio of 1:40, C: methanol and dichloromethane in a volume ratio of 1:50, and D: methanol and dichloromethane in a volume ratio of 1:60.

[0246] The results are as follows Figure 7 As shown, when the methanol:dichloromethane ratio is 1:20, not only does NAB move upwards with the developing solvent in the crude NAB product, but impurity points also move upwards, increasing the difficulty of NAB purification during column chromatography and leading to a decrease in NAB purity. When the methanol:dichloromethane ratio is 1:60, the impurity points in the crude NAB product hardly move, but NAB tails during the plate climbing process. However, when the methanol:dichloromethane ratios are 1:40 and 1:50, the impurity points in the crude NAB product hardly shift, and there is no tailing of NAB. At these ratios, the R0 of NAB is significantly higher. f The values ​​were 0.43 and 0.42, respectively. Since dichloromethane has a lower boiling point than methanol, it is easier to remove during the concentration of the collected effluent. Therefore, the optimized developing solvent ratio used in this experiment was methanol:dichloromethane 1:50.

Claims

1. A purity standard for N-nitrosopyridine (NAB), a pale yellow oily liquid with a density of 1.2 ± 0.1 g / cm³. 3 It is packaged in a brown, sealed bottle and has a purity of 99.18%.

2. The purity standard substance according to claim 1, wherein, The purity is the total purity (i.e., the purity before deducting impurities) minus the purity after deducting impurities, wherein the impurities are selected from anions, inorganic elements, residual solvents, benzophenone and water. Preferably, the anion is selected from Cl. - NO2 - NO3 - and SO4 2- ; Preferably, the inorganic element is selected from Na, Mg, Al, K, Ca, Cr, Fe, Ni, Cu, Zn, As, Se, Cd, Sn, Sb, Te, Hg, and Pb; Preferably, the residual solvent is selected from methanol, benzene, toluene, ethylbenzene, xylene, styrene, ethanol, isopropanol, n-propanol, n-butanol, acetone, butanone, cyclohexanone, ethyl acetate, n-propyl acetate, n-butyl acetate, isopropyl acetate, dichloromethane, tetrahydrofuran, propylene glycol methyl ether, ethylene glycol monoethyl ether, 4-methyl-2-pentanone, propylene glycol ethyl ether, m-p-xylene, ethylene glycol ethyl ether acetate, dimethyl succinate, dimethyl glutarate, and dimethyl adipate.

3. The purity standard substance according to claim 1 or 2, wherein, The minimum packaging unit of the purity standard substance is 20 mg, and the packaging material is a 2 mL brown bottle; Preferably, the relative standard uncertainty introduced by the purity is 0.30% as determined by the purity uncertainty assessment. Preferably, the total purity is 99.51%; Preferably, the anion content is 0.012%; Preferably, the inorganic element content is 0.11%; Preferably, the residual solvent is 0.06%; Preferably, the benzophenone content is 0.005%; Preferably, the moisture content is 0.037%.

4. A method for preparing a purity standard of N-nitrosopyridine (NAB) according to any one of claims 1 to 3, the method comprising the following steps: (1) synthesis of N-nitrosopyridine (NAB); (2) purification of the N-nitrosopyridine (NAB) synthesized in step (1); (3) dispensing the N-nitrosopyridine (NAB) purified in step (2) to obtain the standard.

5. The preparation method according to claim 4, wherein, The synthesis of N-nitrosopyrhodoesequine (NAB) in step (1) includes the following steps: (1-1) Starting with 3-aminomethylpyridine, it reacts with benzophenone via nucleophilic addition and dehydration to generate an imine compound 3; (1-2) Compound 3 obtained in step (1-1) was reacted with 1,4-diiodobutane in the presence of LDA (diisopropylaminolithium) through a two-step nucleophilic substitution and cyclization reaction to generate compound 5; (1-3) The compound 5 obtained in step (1-2) is subjected to nitrosation reaction to generate N-nitrosopseudoesequina (NAB); Preferably, in step (1-1), the reaction temperature is 100-120°C, more preferably 115°C. Preferably, in step (1-1), the reaction solvent is selected from toluene; Preferably, in step (1-2), the reaction temperature is -80 to -60°C, more preferably -75 to -70°C; Preferably, in step (1-2), the reaction solvent is selected from tetrahydrofuran; Preferably, in steps (1-3), the reaction temperature is -5 to 10°C, more preferably 0 to 5°C; Preferably, in steps (1-3), the reaction solvent is selected from deionized water; preferably, the synthesis of N-nitrosopyrhodoesequine (NAB) in step (1) further includes the steps of extracting the obtained N-nitrosopyrhodoesequine (NAB) in an organic solvent under alkaline conditions, drying, and concentrating under reduced pressure. Preferably, the alkaline conditions refer to a pH value of 12 to 14; Preferably, the alkaline conditions are obtained by adding sodium hydroxide, for example, 30% sodium hydroxide; Preferably, the organic solvent is dichloromethane; Preferably, the drying is achieved using anhydrous sodium sulfate.

6. The preparation method according to claim 4 or 5, wherein, The purification in step (2) includes purification by column chromatography; Preferably, the solvent used in the column chromatography is methanol and dichloromethane with a volume ratio of 1:40 or 1:50, more preferably 1:

50. Preferably, the N-nitrosopyridine (NAB) purified in step (2) is stored at -18°C.

7. The preparation method according to any one of claims 4 to 6, wherein, Step (3) of the packaging includes the following steps: (3-1) Clean the 2mL brown bottle with ultrapure water using ultrasonic cleaning and dry it in an oven at 100℃; (3-2) Under a nitrogen atmosphere, use a pipette to take 20 mg of purified N-nitrosopyridine (NAB) and place it into a 2 mL brown bottle dried in step (3-1) to obtain the product; Preferably, if the purified N-nitrosopyrhodoesequine (NAB) is stored at -18°C, it should be placed in the laboratory to return to room temperature before being dispensed. Preferably, the present invention further includes determining the purity, evaluating the purity uncertainty, and testing the homogeneity and stability of the 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) after dispensing in step (3).

8. A solution standard of N-nitrosopyrhodoesequine (NAB), the solution standard being composed of the N-nitrosopyrhodoesequine (NAB) purity standard as described in any one of claims 1 to 3 and methanol, the concentration of the solution standard being 0.099 mg / mL and the relative expanded uncertainty being 2% (expansion factor k = 2); Preferably, the solution standard is dispensed into brown bottles, with each bottle containing 1.5 mL.

9. A method for preparing the solution standard substance according to claim 8, the method comprising (i) preparing the solution standard substance; and (ii) dispensing the solution standard substance prepared in step (i). Preferably, the preparation of the solution standard in step (i) includes dissolving the N-nitrosopyrhodoestiline (NAB) purity standard in methanol as described in any one of claims 1 to 3; Preferably, the preparation of step (i) is completed in a Class 100,000 cleanroom; Preferably, the solution standard prepared in step (i) is stored at -18°C; Preferably, the packaging in step (ii) includes the following steps: First, place the volumetric flask containing the standard solution prepared in step (i) in a -18°C freezer to cool it completely. Then, use a separator to add 1.5 mL of the standard solution to a clean brown 2 mL ampoule (under high-purity nitrogen protection). Quickly transfer the ampoule to a freezing water bath and store it at -18°C for about 20 minutes. Then, immediately remove it and seal it with a flame. Store each ampoule (1.5 mL) at -18°C.

10. The purity standard material of N-nitrosopyridine (NAB) according to any one of claims 1 to 3 and the N-nitrosopyridine (NAB) solution standard material according to claim 8 are used for the detection of tobacco-specific N-nitrosopyridine (NAB) content in tobacco and tobacco products, cigarette smoke and other tobacco-related products.

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