4-(methyl nitrosyl amino)-1-(3-pyridyl)-1-butanone (NNK) purity standard substance as well as preparation method and application thereof

Abstract

The invention relates to a 4-(methyl nitrosyl amino)-1-(3-pyridyl)-1-butanone (NNK) purity standard substance as well as a preparation method and application thereof. The 4-(methyl nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) purity standard substance is white powder, has the melting point of 63-65 DEG C, the boiling point of 346 DEG C and the density of 1.2 g / cm < 3 >, is hermetically packaged with a brown bottle, and has the purity of 99.38%. The purity standard substance disclosed by the invention is high in purity, accurate and traceable in quantity value, good in uniformity and good in stability.

Description

Technical Field

[0001] This invention relates to the field of purity standard material technology, specifically to the purity standard material of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), its preparation method, and its uses. Background Technology

[0002] With increasing public concern about smoking and health issues, the content of nitrosamines in tobacco, tobacco products (including cigarettes and smokeless tobacco products), and cigarette smoke has received much attention. 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the aforementioned nitrosamines, and its content detection has also received much attention.

[0003] Accurate determination of the content of 4-(methylnitroso)-1-(3-pyridyl)-1-butanone (NNK) relies not only on standardized methods but also on the support of standard substances. However, there are currently no purity standards for 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), nor are there any single-solution standards for 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK). Only a few commercially available 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) standards exist, such as: 1) Brand: Dr. Ehrenstorfer; Catalog No.: DRE-C15606300; Specification: 25mg; Purity: 99.61% ± 0.49%, Shelf life: 4 years; 2) Brand: TRC; Catalog No.: CDDM-M325750; Specification: 100mg; 3) Brand: STEEMA; Catalog No.: SN181114554; Specification: 10mg / 20mg / 30mg / 50mg. However, some of these standards lack accurate purity certificates, affecting traceability; moreover, the small size of individual packages makes precise weighing difficult. Furthermore, the standards used in different laboratories come 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 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) to improve the comparative consistency of the testing of harmful components of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) in tobacco, tobacco products and their smoke. Summary of the Invention

[0005] To overcome the above-mentioned deficiencies, this invention provides a purity standard of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), its preparation method, and its uses. The purity standard of this invention exhibits high purity, good homogeneity, good stability, and accurate and traceable metrological values.

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

[0007] On one hand, the present invention provides a purity standard for 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), which is a white powder with a melting point of 63-65℃, a boiling point of 346℃, and a density of 1.2 g / cm³. 3 It is packaged in a brown, sealed bottle and has a purity of 99.38%.

[0008] The structural formula of the 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) 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, 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 minimum packaging unit of the purity standard substance is 20 mg, and the packaging material is a 2 mL brown bottle, such as a brown sample bottle or a brown ampoule.

[0014] Preferably, the relative standard uncertainty introduced by the purity is 0.67% as determined by the purity uncertainty assessment.

[0015] Preferably, the total purity is 99.67%.

[0016] Preferably, the anion content is 0.068%.

[0017] Preferably, the inorganic element content is 0.16%.

[0018] Preferably, the moisture content is 0.063%.

[0019] On the other hand, the present invention provides a method for preparing the purity standard of the above-mentioned 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), the method comprising the following steps: (1) synthesis of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK); (2) purification of the 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) synthesized in step (1); (3) dispensing the 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) purified in step (2) to obtain the product.

[0020] Preferably, the synthesis of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) in step (1) comprises the following steps:

[0021] (1-1) Starting with ethyl nicotinate, it reacts with NMP (N-methylpyrrolidone) via a nucleophilic substitution reaction under the action of LDA (diisopropylaminolithium) to generate compound 1;

[0022] (1-2) Compound 1 obtained in step (1-1) is heated under acidic conditions and refluxed to undergo hydrolysis and decarboxylation reaction to generate compound 2;

[0023] (1-3) Compound 2 obtained in step (1-2) is subjected to a nitrosation reaction to generate 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK). The above reaction route is as follows: Figure 1 As shown.

[0024] Preferably, in step (1-1), the reaction temperature is -80 to -70°C, and more preferably -78°C.

[0025] Preferably, in step (1-1), the reaction solvent is selected from tetrahydrofuran.

[0026] Preferably, in step (1-2), the reaction temperature is 95-110°C, more preferably 105°C.

[0027] Preferably, in step (1-2), the acidic condition is a 6 mol / L hydrochloric acid aqueous solution.

[0028] Preferably, in steps (1-3), the reaction temperature is -5 to 10°C, more preferably 0 to 5°C.

[0029] Preferably, in steps (1-3), the reaction solvent is selected from deionized water.

[0030] Preferably, the synthesis of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) in step (1) further includes the steps of extracting the obtained 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) in an organic solvent under alkaline conditions, drying, and concentrating under reduced pressure.

[0031] Preferably, the alkaline condition refers to a pH value of 12 to 13.

[0032] Preferably, the alkaline conditions are obtained by adding sodium hydroxide, for example, 30% sodium hydroxide.

[0033] Preferably, the organic solvent is dichloromethane.

[0034] Preferably, the drying is achieved using anhydrous sodium sulfate.

[0035] Preferably, the purification in step (2) includes purification by column chromatography.

[0036] 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.

[0037] Preferably, the 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) purified in step (2) is stored at -18°C.

[0038] Preferably, the packaging in step (3) includes the following steps:

[0039] (3-1) Use ultrapure water to ultrasonically clean a 2mL brown bottle, such as a brown sample vial or brown ampoule, and dry it in an oven at 100°C.

[0040] (3-2) Under a nitrogen atmosphere, weigh 20 mg of purified 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) and place it in a 2 mL brown bottle dried in step (3-1) to obtain the product.

[0041] Preferably, if the purified 4-(methylnitroso)-1-(3-pyridyl)-1-butanone (NNK) is stored at -18°C, it should be placed in the laboratory to return to room temperature before being dispensed.

[0042] 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).

[0043] In one specific implementation, the packaging in step (3) includes:

[0044] Clean the 2mL brown sample vial ultrasonically with ultrapure water and dry it in an oven at 100℃. Remove the sample from the -18℃ freezer and place it in a 25℃ laboratory to allow it to return to room temperature. Perform balance calibration, then place the 2mL brown sample vial in the center of the balance. Open the sample vial and use a weighing spoon to weigh approximately 20mg of the sample into the 2mL brown sample vial. After weighing, immediately screw on the cap and seal the vial opening with sealing film.

[0045] 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%.

[0046] In another aspect, the present invention provides a 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) solution standard, which is composed of the above-mentioned 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) 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).

[0047] Preferably, the solution standard is dispensed into brown bottles, such as brown ampoules, with a packaging unit of 1.5 mL / bottle.

[0048] 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).

[0049] Preferably, the preparation of the solution standard in step (i) includes dissolving the above-mentioned 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) purity standard in methanol.

[0050] Preferably, the preparation of step (i) is completed in a Class 100,000 cleanroom.

[0051] Preferably, the solution standard prepared in step (i) is stored at -18°C.

[0052] Preferably, the packaging in step (ii) includes the following steps:

[0053] 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.

[0054] On the other hand, the present invention provides the use of the above-mentioned 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) purity reference material and the above-mentioned 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) solution reference material for the determination of the content of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in tobacco and tobacco products, cigarette smoke and other tobacco-related products.

[0055] Compared with the prior art, the present invention has the following beneficial technical effects:

[0056] The present invention first provides a purity reference material and a single solution reference material for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

[0057] The present invention verifies that the functional groups, structural formula and relative molecular mass of the synthesized 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) raw material are consistent with the relevant information of the corresponding target compound by combining high-resolution mass spectrometry with low-resolution mass spectrometry, nuclear magnetic resonance, infrared spectroscopy and ultraviolet spectroscopy.

[0058] The present invention uses the liquid phase area normalization method and the purity analysis of multiple laboratories. After deducting impurities such as water, anions and inorganic elements, the purity of the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) purity reference material is 99.38%. Through the evaluation of the purity uncertainty, the relative standard uncertainty introduced by the NNK purity is 0.67%.

[0059] The present invention conducts a homogeneity test (F test) on the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) purity reference material. The results show that F test < F critical, indicating that the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) purity reference material has good homogeneity.

[0060] The present invention examines the long-term stability of the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) purity reference material. The results show that the slope of the fitting straight line equation of the long-term stability test is not significant. Therefore, the long-term stability (759 days) of the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) purity reference material is good.

[0061] The present invention uses the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) purity reference material as the raw material, prepares a solution reference material by the weight-volume method with chromatographic grade methanol as the solvent, and fills it into brown bottles under nitrogen protection. The packaging unit is 1.5 mL / bottle.

[0062] The present invention conducts a homogeneity test (F-test) on the solution reference material. The results show that for the "4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) solution reference material in methanol (i.e., the solution reference material of the present invention)" after encapsulation, F test < F critical, indicating good homogeneity.

[0063] The present invention examines the short-term stability and long-term stability of the "4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) solution reference material in methanol". The results show that the fitting straight-line equations for both the short-term stability and long-term stability tests are satisfied with a non-significant slope. Therefore, the short-term stability (stored in the dark at 4 °C, 25 °C, and 50 °C for 7 days) and long-term stability of the "4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) solution reference material in methanol" are good.

[0064] Through the study of value assignment and uncertainty evaluation, it is shown that the concentration value in the "4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) solution reference material in methanol" is 0.099 mg / mL, and the relative expanded uncertainty is 2% (k = 2).

[0065] When synthesizing 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the present invention, especially when synthesizing compound 1, the strong base lithium diisopropylamide (LDA) is used. As a sterically hindered base, LDA performs excellently in ortho-directed reactions. Therefore, the yield of the crude product of the reaction in the present invention is relatively high, up to 97.8%.

[0066] The reaction yields of each step in the synthesis route of the present invention are relatively high. Brief Description of the Drawings

[0067] Figure 1 is the synthesis route of NNK of the present invention;

[0068] Figure 2 is the preliminary liquid chromatography purity test chromatogram of NNK synthesized in Example 1;

[0069] Figure 3 is the hydrogen spectrum of NNK synthesized in Example 1;

[0070] Figure 4 is the carbon spectrum of NNK synthesized in Example 1;

[0071] Figure 5 is the high-resolution mass spectrometry data of NNK synthesized in Example 1;

[0072] Figure 6 is the long-term stability graph of NNK synthesized in Example 1.

[0073] Figure 7 The experimental results for the selection of developing solvent in the purification stage of 4-(methylnitroso)-1-(3-pyridyl)-1-butanone (NNK) are shown. The developing solvents used in A, B, C, and D are as follows: A: methanol and dichloromethane at a volume ratio of 1:20; B: methanol and dichloromethane at a volume ratio of 1:40; C: methanol and dichloromethane at a volume ratio of 1:50; and D: methanol and dichloromethane at 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

[0074] The technical solution of the present invention will be further described below in conjunction with specific implementation methods.

[0075] Example 1: Preparation of a purity standard for 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK)

[0076] (1) Synthesis of 4-(methylnitroso)-1-(3-pyridyl)-1-butanone (NNK)

[0077] The synthetic route for the starting material 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) is as follows: Figure 1 As shown. The specific synthesis steps are as follows:

[0078] (1-1) Preparation of Compound 1

[0079] 6.6 g (1.0 eq) of N-methylpyrrolidone (NMP) and 100 mL of tetrahydrofuran were added to a flask. The mixture was cooled to -75 to -70 °C in a dry ice-ethanol bath. 40 mL (1.2 eq) of LDA tetrahydrofuran solution (2.0 M) was added dropwise, keeping the temperature below -70 °C. After the addition was complete, the mixture was stirred at -75 to -70 °C for 0.5 to 1 h. Then, 10 g (1.0 eq) of ethyl nicotinate was added dropwise, again keeping the temperature below -70 °C. After the addition was complete, the mixture was stirred at room temperature for 1 to 2 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was slowly poured into 100 mL of 2N hydrochloric acid solution to quench the reaction. The mixture was extracted twice with 100 mL of ethyl acetate, and the aqueous phase was retained. The pH of the aqueous phase was adjusted to 7 with 30% sodium hydroxide, and the mixture was extracted three times with 100 mL of dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 13.2 g of crude compound 1 (oily substance), with a crude product yield of 97.8%.

[0080] (1-2) Preparation of Compound 2

[0081] 18.4 g of crude compound 1 and 150 mL of 6N hydrochloric acid solution were added to a flask, heated to reflux, and the reaction was maintained at this temperature for 7–8 h. The reaction was monitored by TLC. After the reaction was completed, the mixture was cooled in an ice bath, the pH was adjusted to 12–14 with 30% sodium hydroxide, and the mixture was extracted three times with 100 mL of dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 8 g of crude compound 2 (oily substance). The yield of the crude product was 45.2%.

[0082] Synthesis of (1-3) 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone

[0083] Add 8.0 g (1.0 eq) of crude compound 2 and 80 mL of 4N hydrochloric acid solution to a flask, and cool to 0–5 °C in an ice-water bath. Weigh 8.4 g (3.0 eq) of sodium nitrite and dissolve it in 64 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 overnight at room temperature (16–17 h). Monitor the reaction by TLC. After the reaction is complete, adjust the pH to 12–13 with 30% sodium hydroxide, extract twice with 100 mL of dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain crude 4-(methylnitroso)-1-(3-pyridyl)-1-butanone (NNK).

[0084] (2) Purification of 4-(methylnitroso)-1-(3-pyridyl)-1-butanone (NNK)

[0085] The crude 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) obtained in step (1) was purified by column chromatography. The solvent used in the column chromatography was methanol and dichloromethane in a volume ratio of 1:50. 3.1 g of solid was obtained, with a yield of 35.9% and HPLC > 99%.

[0086] The liquid chromatography conditions were as follows: Specific 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 for NNK purity determination is shown below. Figure 2 As shown.

[0087] High-resolution mass spectrometry (HS-MS) was used Figure 5 ) and low-resolution mass spectrometry, nuclear magnetic resonance spectroscopy (see Figures 3-4The structure of the obtained 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) was verified by 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.

[0088] The homogeneity and stability of the synthesized NNK raw material were tested, and the results showed that the NNK raw material was homogeneous and stable.

[0089] (3) Dispensing of purified 4-(methylnitroso)-1-(3-pyridyl)-1-butanone (NNK)

[0090] The purified 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) sample was aliquoted into 2mL brown sample vials. The 2mL vials were ultrasonically cleaned with ultrapure water and dried in an oven at 100℃. The samples were removed from a -18℃ freezer and allowed to reach room temperature in a 25℃ 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:

[0091] Preparation for glove box operation: 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. A balance (sensitivity 0.0001 g) should be 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 NNK samples, 700 2 mL brown sample vials, tweezers, and a weighing spoon. 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.

[0092] 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 weighing spoon to weigh approximately 20mg of the sample, placing it into the 2mL sample vial. After weighing, immediately screw on the cap and seal the vial opening with sealing film. A total of 150 vials are dispensed. 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℃.

[0093] Example 2: Purity determination, purity uncertainty assessment, homogeneity and stability testing of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK)

[0094] The sample tested in this embodiment is the purified 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) purity standard prepared in Example 1.

[0095] I. Purity determination of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK)

[0096] The purity of NNK was determined using a mass balance method to identify the standard reference material. The purity of the main component of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) after repackaging in Example 1 was determined using liquid chromatography with area normalization. Simultaneously, ion chromatography, inductively coupled plasma mass spectrometry, headspace gas chromatography, and gas chromatography were used to determine impurities such as anions, inorganic elements, residual solvents, and moisture in the NNK raw material. Specific test methods and results are as follows:

[0097] 1. Testing Method

[0098] 1.1 Liquid Chromatography Method

[0099] The determination was performed using liquid chromatography, and the liquid chromatography conditions were as follows:

[0100] Column: Poroshell EC-C18 (4.6 x 250 mm x 4 μm); Mobile phase: Water (A) and methanol (B); Elution program: 0-2 min: 25-25% B; 2-5 min: 25-70% B; 5-8 min: 70-70% B; 8-10 min: 70-90% B; 10-15 min: 90-90% B; 15-17 min: 90-25% B; 17-22 min: 25-25% B; Column temperature: 40℃; Flow rate: 1.0 mL / min; Detection wavelength: 230 nm; Injection volume: 10 μL; Concentration: 1 mg / mL.

[0101] 1.2 Purity analysis by multiple laboratories

[0102] 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.

[0103] Table 1 Jointly set value units and instrument models

[0104]

[0105] 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.

[0106] Table 2. NNK data from collaborative assessments by 9 laboratories (unit: %)

[0107]

[0108]

[0109] 1.3 Moisture Determination

[0110] The moisture content in NNK 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. The moisture content in the NNK candidate solution was detected using a gas chromatograph with a thermal conductivity detector (GC-TCD, Agilent 6890N) and the 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 NNK candidate is 0.063%.

[0111] Table 3. Moisture content (mg / g) in methanol solvent and nitrosamine solution

[0112] Number of experiments 1 2 3 4 5 6 7 8 9 average value NNK 0.64 0.63 0.61 0.70 0.60 0.65 0.62 0.64 0.60 0.63

[0113] 1.4 Anion Determination

[0114] 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.

[0115] Table 4. Anion detection results (unit: μg / mL)

[0116]

[0117] 1.5 Determination of Inorganic Elements

[0118] 1.5.1 Measurement Process

[0119] The inorganic element content in NNK 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, 10.13 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.

[0120] 1.5.2 Instrumental Analysis Conditions

[0121] 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.

[0122] Table 5 Main parameters of ICP / MS

[0123]

[0124] Table 6 Measurement of isotopes, internal standard elements, and integration time.

[0125]

[0126]

[0127] The results of inorganic elements in NNK are shown in Table 7 below. It was found that the types and contents of inorganic elements contained in the NNK candidates were not the same. They mainly contained trace amounts of Na, Sn, Te and other elements, with a total amount of 31.38 mg / mL, accounting for 0.16%.

[0128] Table 7. Results of Inorganic Element Content Determination (Unit: mg / mL)

[0129]

[0130]

[0131] Note: Italics in the table indicate not detected. The test results are expressed as detection limits and are included in the total element count.

[0132] 1.6 Determination of Volatile Impurities

[0133] 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).

[0134] Results analysis: The content of volatile organic solvents in the NNK candidate solution was determined, and none were detected.

[0135] 1.7 Determination of Benzophenone Solvent Residue

[0136] Since benzophenone was not used in the synthesis of the NNK candidate, it was not detected.

[0137] 2. Test Results

[0138] Using the liquid phase area normalization method, the purity values ​​from multiple laboratories were averaged, and the resulting average purity for the NNK candidate was 99.67% (NNK). The actual purity of the NNK candidate should be the result after deducting all impurities (anions, inorganic elements, residual solvent, benzophenone, and water), i.e., P 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 It can be seen that the purity of the NNK candidate after deducting impurities is 99.38%, as shown in Table 8.

[0139] Table 8. Purity test results (%) of liquid chromatography area normalization method

[0140]

[0141] II. Evaluation of the Purity Uncertainty of NNK

[0142] Referring to the technical specification JJF 1855-2022, the purity uncertainty of NNK after being dispensed in Example 1 was determined. The main sources of purity analysis uncertainty are: (1) instrument verification (measurement repeatability, liquid chromatograph response factor difference and detection limit), (2) raw material homogeneity, (3) raw material stability, and (4) impurity determination (anions, moisture, inorganic elements, solvent residue and benzophenone).

[0143] The relative standard uncertainty introduced by the purity assessment of NNK is 0.67%.

[0144] III. Uniformity Test of NNK

[0145] 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 NNK candidate samples packaged 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 NNK raw material samples, take 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 an NNK 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 area normalization method of liquid chromatography.

[0146] 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.

[0147] The results of the homogeneity test are shown in Table 9. 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) = 1.25. Since the calculated value of F is 1.25 < F... 0.05 (10, 22) indicates that there is no significant difference between the samples at the 95% confidence level, and the NNK raw material samples after dispensing are homogeneous.

[0148] Table 9. Results of homogeneity test of NNK candidate raw materials

[0149]

[0150]

[0151] Note: In the table,

[0152] IV. Stability Test of NNK

[0153] The sample used in this stability test was the purity standard material prepared in Example 1 after being dispensed.

[0154] 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 NNK 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 NNK test solution. The method used is the liquid chromatography area normalization method.

[0155] The long-term stability test results of NNK raw materials are shown in Table 10, and the long-term stability diagram of NNK raw materials is shown in [the table]. Figure 6 As shown.

[0156] Table 10. Long-term stability data of NNK raw materials

[0157]

[0158]

[0159] In accordance with the requirements of JJF 1343-2022, a linear model was selected as the empirical model for this standard material.

[0160] The long-term stability data in Table 10 are used, with x representing time (days) and y representing the NNK purity value. A straight line y = kx + b is fitted, and the equation of the line is: y = 0.000022x + 99.68. Then the slope k = 0.000022 and the intercept b = 99.68.

[0161] The standard deviation of each point on the straight line can be calculated using the following formula:

[0162]

[0163] The slope uncertainty is calculated using the following formula:

[0164]

[0165] 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.

[0166] because Therefore, the slope is not significant, and thus the stability is good.

[0167] Example 3: Preparation of the NNK solution standard of the present invention

[0168] The NNK used in this embodiment is the purity standard material prepared in Example 1 after being dispensed.

[0169] 1. Preparation of the solution standard substance of the present invention

[0170] Before weighing the samples, the weighing instruments (tweezers, weighing spoon, weighing boat, calibrated Class A 1000mL volumetric flasks, and Class ① balance) were 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.

[0171] Since NNK is a solid powder, a weighing boat was used to assist in weighing. The weighing boat was placed in the center of the balance and zeroed. Using a weighing spoon, approximately 100 mg was accurately transferred onto the weighing boat, starting with a larger amount and gradually decreasing it. The weighing boat was then rinsed several times with chromatographic grade methanol. Finally, the inner wall of the volumetric flask neck was rinsed with methanol. Near the final volume mark, a dropper was used to carefully add the methanol 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 NNK standard solution. The prepared NNK standard solution was then transferred to a -18°C freezer.

[0172] 2. Dispensing of the solution standard substance of the present invention

[0173] In a Class 100,000 cleanroom, volumetric flasks containing NNK standard solution were first placed in a -18°C freezer for thorough cooling. Then, using a dispenser, 1.5 mL of NNK standard solution was added to 2 mL clean brown ampoules (under nitrogen protection in a biosafety cabinet). 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.

[0174] Example 4: Evaluation of the homogeneity, stability and uncertainty of the NNK solution standard material of the present invention. The NNK solution standard material used in this example was prepared from Example 3.

[0175] I. Homogeneity test of the NNK solution standard substance of the present invention

[0176] 1. Uniformity test

[0177] 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 NNK 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 coding 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.

[0178] 2. Liquid Chromatography Conditions

[0179] The NNK chromatographic conditions are as follows:

[0180] —Column: Poroshell EC-C18 column (4.6mm×250mm, 4μm);

[0181] —Flow rate: 1.0 mL / min;

[0182] —Column temperature: 40℃;

[0183] —Injection volume: 10 μL;

[0184] —Mobile phase A: water, mobile phase B: methanol;

[0185] —Full wavelength scanning mode (210-500nm), monitoring wavelength 230nm;

[0186] —The gradient elution conditions are shown in Table 11;

[0187] Table 11 Gradient elution conditions for high performance liquid chromatography (NNK)

[0188]

[0189] 3. Mathematical statistics methods

[0190] According to the JJF 1343-2022 technical specification, one-way ANOVA is adopted as the mathematical statistical method for homogeneity testing.

[0191] 4. Results

[0192] The test results show that the NNK component of the "NNK solution standard in methanol" has good homogeneity after being dispensed.

[0193] II. Stability testing of the NNK solution standard material of the present invention

[0194] 1. Short-term stability

[0195] 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 standard substance during cold chain transportation were examined at cold chain temperature (4℃), normal temperature (25℃), and extreme temperature (50℃) on days 0, 1, 3, 5, and 7. Twelve bottles of NNK solution standard substance 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 bottles were removed from the refrigerator and artificial climate chamber at the two temperatures on days 1, 3, 5, and 7, respectively, and the last three bottles were removed for testing on day 7. Each sample was measured three times.

[0196] The liquid chromatography conditions are the same as those in Part 1 of this embodiment.

[0197] 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.

[0198] The results of the short-term stability test of NNK solution standard are shown in Table 12.

[0199] Table 12 Short-term stability data of NNK solution standard material

[0200]

[0201] Conclusion: The short-term stability test results show that the concentration of the "NNK solution standard in methanol" did not change after short-term storage at 4℃, 25℃ and 50℃.

[0202] 2. Long-term stability of the sample

[0203] 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 NNK solution standard reference material samples under light-protected conditions at -18℃, the packaged NNK 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.

[0204] 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.

[0205] The measurement results are shown in Table 13 below.

[0206] Table 13 Long-term stability data of NNK solution standard material

[0207]

[0208] Conclusion: The long-term stability test results show that the t-value of the long-term stability test of the "NNK solution standard in methanol" is less than the critical value, which indicates that the "NNK solution standard in methanol" sample has good long-term stability.

[0209] III. Uncertainty Assessment of the NNK Solution Standard Material of the Present Invention

[0210] The main sources of uncertainty in the NNK 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:

[0211] 1. Uncertainty introduced by solution preparation

[0212] 1.1 Uncertainty introduced in the raw material purity analysis process

[0213] 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 NNK purity is 0.67%.

[0214] 1.2 Uncertainty introduced by the balance

[0215] 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:

[0216] 1.3 Uncertainty introduced by volume

[0217] (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 1001.03 mL, 998.94 mL, 999.86 mL, 1001.02 mL, 1000.01 mL, 999.93 mL, 1001.04 mL, and 998.87 mL. Using Bessel's formula, the standard deviation is 0.8902 mL. Therefore, the uncertainty caused by volume in the preparation of the standard substance sample is 0.8902 mL, and the relative standard uncertainty expressed by the relative standard deviation is 0.09%.

[0218] (2) Type B uncertainty

[0219] Type B uncertainty. Type B uncertainty arises from two aspects: volumetric flask calibration 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., and the metrology certificate is attached as Appendix A. 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:

[0220] (3) Combined relative standard uncertainty:

[0221] 2. Uncertainty introduced by solution homogeneity

[0222] 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) =0.16%.

[0223] 3. Uncertainty introduced by stability

[0224] 3.1 Uncertainty introduced by short-term stability

[0225] 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:

[0226] 3.2 Uncertainty introduced by long-term stability

[0227] 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:

[0228] 4. Uncertainty introduced by filling loss

[0229] Since the solvents used in the NNK solution standard substance, such as methanol, have low boiling points, there may be some loss during the filling and heat sealing process. The concentration of NNK substance 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 14.

[0230] Table 14 Uncertainty introduced by filling loss

[0231]

[0232]

[0233] 5. Overall combined relative standard uncertainty

[0234] Table 15 Relative Standard Uncertainty Components and Combination

[0235]

[0236] 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 × 0.79% = 1.59% ≈ 2%.

[0237] Example 4: Selection of developing solvent for the purification stage of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK)

[0238] 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.

[0239] The results are as follows Figure 7 As shown, under all developing solvent conditions, only NNK in the crude NNK product moves upward, and the rate of NNK movement slows down with increasing dichloromethane concentration, while impurity points hardly move. However, when the methanol:dichloromethane ratio is 1:20, the R of NNK... f A value of 0.55 indicates that NNK is eluted at a relatively fast rate during column chromatography, increasing the risk of introducing impurities; while when methanol:dichloromethane is 1:60, the R of NNK is... f The value is only 0.15 and there is a slight tailing phenomenon; when the ratio of methanol to dichloromethane is 1:40 and 1:50, the Rf values ​​of NNK are 0.40 and 0.32 respectively, which are more suitable. Since the boiling point of dichloromethane is lower than that of methanol, it is easier to remove it when the collected effluent is concentrated. Therefore, the optimized developing solvent ratio used in this experiment is methanol:dichloromethane 1:50.

Claims

1. A purity standard for 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), a white powder with a melting point of 63-65℃, a boiling point of 346℃, and a density of 1.2 g / cm³. 3 It is packaged in a brown, sealed bottle and has a purity of 99.38%.

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 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.

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 0.67% as determined by the purity uncertainty assessment. Preferably, the total purity is 99.67%; Preferably, the anion content is 0.068%; Preferably, the inorganic element content is 0.16%; Preferably, the moisture content is 0.063%.

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

5. The preparation method according to claim 4, wherein, The synthesis of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) in step (1) includes the following steps: (1-1) Starting with ethyl nicotinate, it reacts with NMP (N-methylpyrrolidone) via a nucleophilic substitution reaction under the action of LDA (diisopropylaminolithium) to generate compound 1; (1-2) Compound 1 obtained in step (1-1) is heated under acidic conditions and refluxed to undergo hydrolysis and decarboxylation reaction to generate compound 2; (1-3) The compound 2 obtained in step (1-2) was subjected to nitrosation to generate 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK); Preferably, in step (1-1), the reaction temperature is -80 to -70°C, more preferably -78°C; Preferably, in step (1-1), the reaction solvent is selected from tetrahydrofuran; Preferably, in step (1-2), the reaction temperature is 95-110°C, more preferably 105°C; Preferably, in step (1-2), the acidic condition is a 6 mol / L hydrochloric acid aqueous solution; 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 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) in step (1) further includes the steps of extracting the obtained 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) in an organic solvent under alkaline conditions, drying, and concentrating under reduced pressure; Preferably, the alkaline condition refers to a pH value of 12 to 13; 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 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) 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, weigh 20 mg of purified 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) and place it in a 2 mL brown bottle dried in step (3-1) to obtain the product; Preferably, if the purified 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) 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 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) solution standard, wherein the solution standard is composed of the 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) purity standard as described in any one of claims 1 to 3 and methanol, wherein the concentration of the solution standard is 0.099 mg / mL and the relative expanded uncertainty is 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 purity standard of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) as described in any one of claims 1 to 3 in methanol; 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 of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) according to any one of claims 1 to 3 and the solution standard of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) according to claim 8 are used for the detection of the content of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) in tobacco and tobacco products, cigarette smoke and other tobacco-related products.

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