N-nitrosamine mixed solution standard reference and its preparation method

Abstract

This invention relates to a mixed solution standard of N-nitrosamines and its preparation method. The standard is composed of N-nitrosonornicotinic acid (NNN) purity standard, 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) purity standard, N-nitrosoneonicotinic acid (NAT) purity standard, N-nitrosoacetine (NAB) purity standard, and methanol. The standard of this invention exhibits high purity, accurate and traceable values, good homogeneity, and good stability.

Description

Technical Field

[0001] This invention relates to a mixed solution standard of N-nitrosamines and its preparation method. Background Technology

[0002] Nitrosamines (TSNAs) are a class of carcinogenic substances produced during tobacco processing and smoking. They mainly include four substances: N-nitrosonornicotinic acid (NNN), 4-(N-methylnitroso)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoneonicotinic acid (NAT), and N-nitrosopseudoesequine (NAB). Accurate determination of TSNA content or release levels is crucial for precisely assessing the harmfulness of tobacco products and promoting harm reduction efforts.

[0003] Currently, only a few TSNA standard products are available on the market; see Table 1 for details.

[0004] Table 1. Structure, physicochemical properties, and commercially available candidate information of four nitrosamines

[0005]

[0006]

[0007] Therefore, it is necessary to provide industry quality inspection agencies and cigarette manufacturing enterprises with a complete set of TSNAs solution standard material products. Summary of the Invention

[0008] To overcome the above-mentioned shortcomings, this invention provides an N-nitrosamine mixed solution standard and its preparation method. The solution standard of this invention has high purity, accurate and traceable values, good homogeneity, and good stability.

[0009] The present invention is achieved by the following technical solution.

[0010] On one hand, the present invention provides a mixed solution standard material of N-nitrosamine, which is composed of N-nitrosonornicotinine (NNN) purity standard material, 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) purity standard material, N-nitrosoneonicotinine (NAT) purity standard material, N-nitrosopseudoestipine (NAB) purity standard material and methanol.

[0011] Preferably, the minimum packaging unit of the solution standard is 1.5 mL, and the packaging material is a 2 mL brown bottle, preferably a brown ampoule bottle.

[0012] Preferably, in the mixed solution standard material, the concentration standard values ​​of N-nitrosonornicotinine (NNN), 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoneonicotinine (NAT), and N-nitrosopseudoestipine (NAB) are 0.050, 0.050, 0.050, and 0.010 mg / mL, respectively, with relative expanded uncertainties of 3%, 3%, 3%, and 4% (k=2), respectively. This constitutes a high-concentration mixed solution standard material.

[0013] Preferably, in the mixed solution standard material, the concentration standard values ​​of N-nitrosonornicotinine (NNN), 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoneonicotinine (NAT), and N-nitrosopseudoestipine (NAB) are 0.010, 0.010, 0.010, and 0.005 mg / mL, respectively, with relative expanded uncertainties of 4%, 4%, 4%, and 5% (k=2), respectively. This constitutes a low-concentration mixed solution standard material.

[0014] The structural formulas of N-nitrosonornicotinine (NNN), 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoneonicotinine (NAT), and N-nitrosopseudoestipine (NAB) of the present invention are as follows:

[0015]

[0016] Preferably, the purities of N-nitrosonornicotinine (NNN), 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoneonicotinine (NAT), and N-nitrosopseudoestiline (NAB) are 99.35%, 99.38%, 99.45%, and 99.18%, respectively.

[0017] 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.

[0018] Preferably, the anion is selected from Cl. - NO2 - NO3 - and SO4 2- .

[0019] 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.

[0020] 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.

[0021] Preferably, through purity uncertainty evaluation, the relative standard uncertainties introduced by the purity of N-nitrosonornicotinine (NNN), 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoneonicotinine (NAT), and N-nitrosoestipine (NAB) are 0.24%, 0.67%, 0.25%, and 0.30%, respectively.

[0022] Preferably, the total purities of the N-nitrosonornicotinine (NNN), 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoneonicotinine (NAT), and N-nitrosopseudoestiline (NAB) purity standards are 99.76%, 99.67%, 99.78%, and 99.51%, respectively.

[0023] Preferably, the anions in the purity standards of N-nitrosonornicotinine (NNN), 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoneonicotinine (NAT), and N-nitrosopseudoestipine (NAB) are 0.077%, 0.068%, 0.079%, and 0.012%, respectively.

[0024] Preferably, the inorganic elements in the purity standards of N-nitrosonornicotinine (NNN), 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoneonicotinine (NAT), and N-nitrosophagusine (NAB) are 0.18%, 0.16%, 0.11%, and 0.11%, respectively.

[0025] Preferably, the residual solvents in the N-nitrosonornicotinine (NNN), 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoneonicotinine (NAT), and N-nitrosopseudoestiline (NAB) purity standard substances are 0.09%, 0%, 0.05%, and 0.06%, respectively.

[0026] Preferably, the benzophenone content in the N-nitrosonornicotinine (NNN) purity standard, the 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) purity standard, the N-nitrosoneonicotinine (NAT) purity standard, and the N-nitrosopseudoestiphenine (NAB) purity standard is 0.008%, 0%, 0.025%, and 0.005%, respectively.

[0027] Preferably, the water content in the N-nitrosonornicotinine (NNN), 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoneonicotinine (NAT), and N-nitrosopseudoestiline (NAB) purity standard substances is 0.055%, 0.063%, 0.062%, and 0.037%, respectively.

[0028] Preferably, the preparation method of the N-nitrosonornicotinic acid (NNN) purity standard material includes the following steps: (N1) synthesis of N-nitrosonornicotinic acid (NNN); (N2) purification of the N-nitrosonornicotinic acid (NNN) synthesized in step (N1); (N3) dispensing the N-nitrosonornicotinic acid (NNN) purified in step (N2) to obtain the product.

[0029] Preferably, the synthesis of N-nitrosonornicotinic acid (NNN) in step (N1) includes the following steps:

[0030] (N1-1) Starting with 3-aminomethylpyridine, it reacts with benzophenone via nucleophilic addition and dehydration to generate an imine compound 3;

[0031] (N1-2) Compound 3 obtained in step (N1-1) is reacted with 1,3-diiodopropane in the presence of LDA (diisopropylaminolithium) through a two-step nucleophilic substitution and cyclization reaction to generate compound 6;

[0032] (N1-3) Compound 6 obtained in step (N1-2) is subjected to a nitrosation reaction to generate N-nitrosonornicotinic acid (NNN). The above synthetic route is as follows: Figure 1 As shown.

[0033] Preferably, in step (N1-1), the reaction temperature is 100-120°C, more preferably 115°C.

[0034] Preferably, in step (N1-1), the reaction reagent is selected from toluene.

[0035] Preferably, in step (N1-2), the reaction temperature is -80 to -70°C, and more preferably -78°C.

[0036] Preferably, in step (N1-2), the reaction reagent is selected from tetrahydrofuran.

[0037] Preferably, in step (N1-3), the reaction temperature is -5 to 10°C, and more preferably 0-5°C.

[0038] Preferably, in step (N1-3), the reaction reagent is selected from deionized water.

[0039] Preferably, the synthesis of N-nitrosonornicotinine (NNN) in step (N1) further includes the steps of extracting the obtained N-nitrosonornicotinine (NNN) in an organic solvent under alkaline conditions, drying, and concentrating under reduced pressure.

[0040] Preferably, the alkaline conditions refer to a pH value of 12 to 14.

[0041] Preferably, the alkaline conditions are obtained by adding sodium hydroxide, for example, 30% sodium hydroxide.

[0042] Preferably, the organic solvent is dichloromethane.

[0043] Preferably, the drying is achieved using anhydrous sodium sulfate.

[0044] Preferably, the purification in step (N2) includes purification by column chromatography.

[0045] Preferably, the solvent used in the column chromatography is methanol and dichloromethane in a volume ratio of 1:33.

[0046] Preferably, the N-nitrosonornicotinic acid (NNN) purified in step (N2) is stored at -18°C.

[0047] Preferably, the packaging in step (N3) includes the following steps:

[0048] (N3-1) 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.

[0049] (N3-2) Under a nitrogen atmosphere, use a pipette to take 20 mg of purified N-nitrosonornicotinic acid (NNN) and place it into a 2 mL brown bottle that was dried in step (N3-1) to obtain the product.

[0050] Preferably, if the purified N-nitrosonornicotinamide (NNN) is stored at -18°C, it should be placed in the laboratory to return to room temperature before being dispensed.

[0051] In this application, purified N-nitrosonornicotinamide (NNN) can be directly aliquoted. If purified N-nitrosonornicotinamide (NNN) is not directly aliquoted, it can be stored at -18°C. When aliquoting, it needs to be placed in the laboratory to return to room temperature before aliquoting.

[0052] Preferably, the present invention further includes determining the purity, evaluating the purity uncertainty, and testing the uniformity and stability of the N-nitrosonornicotinic acid (NNN) after dispensing in step (N3).

[0053] In one specific implementation, the packaging in step (N3) includes:

[0054] 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.

[0055] Preferably, the preparation method of the purity standard of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) includes the following steps: (K1) synthesis of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK); (K2) purification of the 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) synthesized in step (K1); (K3) dispensing the 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) purified in step (K2) to obtain the product.

[0056] Preferably, the synthesis of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) in step (K1) comprises the following steps:

[0057] (K1-1) starts with ethyl nicotinate and reacts with NMP (N-methylpyrrolidone) via a nucleophilic substitution reaction under the action of LDA (diisopropylaminolithium) to generate compound 1;

[0058] (K1-2) Compound 1 obtained in step (K1-1) is heated under acidic conditions and refluxed to undergo hydrolysis and decarboxylation reaction to generate compound 2;

[0059] (K1-3) Compound 2 obtained in step (K1-2) is subjected to nitrosation to generate 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK). The above synthetic route is as follows: Figure 3 As shown.

[0060] Preferably, in step (K1-1), the reaction temperature is -80 to -70°C, and more preferably -78°C.

[0061] Preferably, in step (K1-1), the reaction solvent is selected from tetrahydrofuran.

[0062] Preferably, in step (K1-2), the reaction temperature is 95-110°C, more preferably 105°C.

[0063] Preferably, in step (K1-2), the acidic condition is a 6 mol / L hydrochloric acid aqueous solution.

[0064] Preferably, in step (K1-3), the reaction temperature is -5 to 10°C, more preferably 0 to 5°C.

[0065] Preferably, in steps (K1-3), the reaction solvent is selected from deionized water.

[0066] Preferably, the synthesis of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) in step (K1) 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.

[0067] Preferably, the alkaline condition refers to a pH value of 12 to 13.

[0068] Preferably, the alkaline conditions are obtained by adding sodium hydroxide, for example, 30% sodium hydroxide.

[0069] Preferably, the organic solvent is dichloromethane.

[0070] Preferably, the drying is achieved using anhydrous sodium sulfate.

[0071] Preferably, the purification in step (K2) includes purification using column chromatography.

[0072] 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.

[0073] Preferably, the 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) purified in step (K2) is stored at -18°C.

[0074] Preferably, the packaging in step (K3) includes the following steps:

[0075] (K3-1) 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.

[0076] (K3-2) 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 (K3-1) to obtain the product.

[0077] 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.

[0078] 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 (K3).

[0079] In one specific implementation, the packaging step (K3) includes:

[0080] 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.

[0081] Preferably, the preparation method of the above-mentioned N-nitrosonicotinic acid (NAT) purity standard material includes the following steps: (T1) synthesis of N-nitrosonicotinic acid (NAT); (T2) purification of the N-nitrosonicotinic acid (NAT) synthesized in step (T1); (T3) dispensing the N-nitrosonicotinic acid (NAT) purified in step (T2) to obtain the product.

[0082] Preferably, the synthesis of N-nitrosoneonicazone (NAT) in step (T1) includes the following steps:

[0083] (T1-1) Starting with 3-aminomethylpyridine, it reacts with benzophenone via nucleophilic addition and dehydration to generate an imine compound 3;

[0084] (T1-2) Compound 3 obtained in step (T1-1) is reacted with 1,4-dichloro-2-butene in the presence of LDA (diisopropylaminolithium) through a two-step nucleophilic substitution and cyclization reaction to generate compound 4.

[0085] (T1-3) Compound 4 obtained in step (T1-2) is subjected to a nitrosation reaction to generate N-nitrosoneonicotinic acid (NAT). The above synthetic route is as follows: Figure 5 As shown.

[0086] Preferably, in step (T1-1), the reaction temperature is 100-120°C, more preferably 115°C.

[0087] Preferably, in step (T1-1), the reaction solvent is selected from toluene.

[0088] Preferably, in step (T1-2), the reaction temperature is -80 to -60°C, more preferably -75 to -70°C.

[0089] Preferably, in step (T1-2), the reaction solvent is selected from tetrahydrofuran.

[0090] Preferably, in step (T1-3), the reaction temperature is -5 to 10°C, more preferably 0 to 5°C.

[0091] Preferably, in step (T1-3), the reaction solvent is selected from deionized water.

[0092] Preferably, the synthesis of N-nitrosonicotinic acid (NAT) in step (T1) further includes the steps of extracting the obtained N-nitrosonicotinic acid (NAT) in an organic solvent under alkaline conditions, drying, and concentrating under reduced pressure.

[0093] Preferably, the alkaline condition refers to a pH value of 13 to 14.

[0094] Preferably, the alkaline conditions are obtained by adding sodium hydroxide, for example, 30% sodium hydroxide.

[0095] Preferably, the organic solvent is dichloromethane.

[0096] Preferably, the drying is achieved using anhydrous sodium sulfate.

[0097] Preferably, the purification in step (T2) includes purification by column chromatography.

[0098] 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.

[0099] Preferably, the N-nitrosonicotinic acid (NAT) purified in step (T2) is stored at -18°C.

[0100] Preferably, the packaging in step (T3) includes the following steps:

[0101] (T3-1) Use ultrapure water to ultrasonically clean a 2mL brown bottle, for example, brown sample, and dry it in an oven at 100℃.

[0102] (T3-2) Under a nitrogen atmosphere, use a pipette to take 20 mg of purified N-nitrosonicotinic acid (NAT) and place it into a 2 mL brown bottle that has been dried in step (T3-1) to obtain the product.

[0103] Preferably, if the purified N-nitrosonicotinic acid (NAT) is stored at -18°C, it should be placed in the laboratory to reach room temperature before being dispensed.

[0104] Preferably, the method further includes determining the purity, evaluating the purity uncertainty, and testing the homogeneity and stability of the N-nitrosonicotinic acid (NAT) after dispensing in step (T3).

[0105] In one specific implementation, the packaging in step (T3) includes:

[0106] 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.

[0107] Preferably, the preparation method of the above-mentioned N-nitrosopyridine (NAB) purity standard material includes the following steps: (B1) synthesis of N-nitrosopyridine (NAB); (B2) purification of the N-nitrosopyridine (NAB) synthesized in step (1); (B3) dispensing the N-nitrosopyridine (NAB) purified in step (B2) to obtain the product.

[0108] Preferably, the synthesis of N-nitrosopyrhodoesequine (NAB) in step (B1) includes the following steps:

[0109] (B1-1) Starting with 3-aminomethylpyridine, it reacts with benzophenone via nucleophilic addition and dehydration to generate an imine compound 3;

[0110] (B1-2) Compound 3 obtained in step (B1-1) is reacted with 1,4-diiodobutane in the presence of LDA (diisopropylaminolithium) through a two-step nucleophilic substitution and cyclization reaction to generate compound 5;

[0111] (B1-3) Compound 5 obtained in step (B1-2) is subjected to a nitrosation reaction to generate N-nitrosopseudoesequine (NAB). The above synthetic route is as follows: Figure 7 As shown.

[0112] Preferably, in step (B1-1), the reaction temperature is 100-120°C, more preferably 115°C.

[0113] Preferably, in step (B1-1), the reaction solvent is selected from toluene.

[0114] Preferably, in step (B1-2), the reaction temperature is -80 to -60°C, more preferably -75 to -70°C.

[0115] Preferably, in step (B1-2), the reaction solvent is selected from tetrahydrofuran.

[0116] Preferably, in step (B1-3), the reaction temperature is -5 to 10°C, more preferably 0 to 5°C.

[0117] Preferably, in step (B1-3), the reaction solvent is selected from deionized water.

[0118] Preferably, the synthesis of N-nitrosopyrhodoesequine (NAB) in step (B1) further includes the steps of extracting the obtained N-nitrosopyrhodoesequine (NAB) in an organic solvent under alkaline conditions, drying, and concentrating under reduced pressure.

[0119] Preferably, the alkaline conditions refer to a pH value of 12 to 14.

[0120] Preferably, the alkaline conditions are obtained by adding sodium hydroxide, for example, 30% sodium hydroxide.

[0121] Preferably, the organic solvent is dichloromethane.

[0122] Preferably, the drying is achieved using anhydrous sodium sulfate.

[0123] Preferably, the purification in step (B2) includes purification by column chromatography.

[0124] 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.

[0125] Preferably, the N-nitrosopyridine (NAB) purified in step (B2) is stored at -18°C.

[0126] Preferably, the packaging in step (B3) includes the following steps:

[0127] (B3-1) Clean the 2mL brown bottle with ultrapure water using ultrasonic cleaning and dry it in an oven at 100℃.

[0128] (B3-2) 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 has been dried in step (B3-1) to obtain the product.

[0129] 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.

[0130] Preferably, the present invention further includes determining the purity, evaluating the purity uncertainty, and testing the homogeneity and stability of the N-nitrosopseudoesequina (NAB) after dispensing in step (B3).

[0131] In one specific implementation, the packaging in step (B3) includes:

[0132] 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.

[0133] 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%.

[0134] On the other hand, 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).

[0135] Preferably, the preparation of the solution standard in step (i) includes dissolving the N-nitrosonornicotinine (NNN) purity standard, 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) purity standard, N-nitrosoneonicotinine (NAT) purity standard, and N-nitrosopseudoestipine (NAB) purity standard in methanol.

[0136] Preferably, the purity standards for N-nitrosonicotinic acid (NNN), 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonicotinic acid (NAT), and N-nitrosopseudoestiline (NAB) are prepared using the methods described above.

[0137] Preferably, the preparation of step (i) is completed in a Class 100,000 cleanroom.

[0138] Preferably, the solution standard prepared in step (i) is stored at -18°C.

[0139] Preferably, the packaging in step (ii) includes the following steps:

[0140] First, place the volumetric flask containing the solution reference material prepared in step (i) in a refrigerator at -18°C for sufficient cooling. Then, use a dispenser to add 1.5 mL of the said reference material solution into a clean brown ampoule bottle with a specification of 2 mL (under the protection of high-purity nitrogen). Quickly transfer the ampoule bottle to a freezing water bath, store it at -18°C for about 20 min, then take it out and immediately seal it with a flame. Each bottle contains 1.5 mL and is stored in a refrigerator at -18°C.

[0141] Compared with the prior art, the present invention has the following beneficial technical effects:

[0142] The present invention uses HPLC method to test the purity of chemically synthesized NNN, NNK, NAT, and NAB candidates, and the purity > 99%.

[0143] Through high-resolution mass spectrometry combined with low-resolution mass spectrometry, nuclear magnetic resonance, infrared spectroscopy, and ultraviolet spectroscopy analysis, it is verified that the functional group information, structural formula, relative molecular mass, etc. of the highly pure NNN, NNK, NAT, and NAB candidates obtained by chemical synthesis are consistent with the relevant information of the corresponding target compounds.

[0144] Using the liquid phase area normalization method and the purity定值 of multiple laboratories, the actual purity of the 4 candidates should be the result after deducting all impurities (moisture, inorganic elements, and solvent residues). The purities are 99.35% (NNN), 99.38% (NNK), 99.45% (NAT), and 99.18% (NAB) respectively. Through the evaluation of purity uncertainty, the relative standard uncertainties introduced by the purities of NNN, NNK, NAT, and NAB are 0.24%, 0.67%, 0.25%, and 0.30% respectively.

[0145] Perform a homogeneity test (F test) on 11 bottles of samples of each of the 4 candidate raw materials extracted. The results show that for the encapsulated NNN, NNK, NAT, and NAB candidates, F test < F critical, which indicates that the 4 raw material samples have good homogeneity.

[0146] The long-term stability of the 4 candidate raw materials was investigated. 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 4 candidate raw materials is good.

[0147] Use "4 kinds of N-nitrosamines (NNN, NNK, NAT, NAB)" as candidate raw materials, use chromatographic grade methanol as the solvent, and prepare a solution reference material by the weight - volume method. Under the protection of nitrogen, it is filled into brown bottles, and the packaging unit is 1.5 mL / bottle.

[0148] The homogeneity test (F - test) was carried out on the 15 extracted samples. The results showed that for each component in the "Standard Substance of 4 - N - nitrosamines Mixed Solution in Methanol" after encapsulation, F - test < F - critical, indicating good homogeneity.

[0149] The short - term stability and long - term stability of the "Standard Substance of 4 - N - nitrosamines Mixed Solution in Methanol" were investigated. The results showed that the fitting straight - line equations for both the short - term stability and long - term stability tests satisfied The slope was 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 of the "Standard Substance of 4 - N - nitrosamines Mixed Solution in Methanol" were good.

[0150] Through the study of value assignment and uncertainty evaluation, it was shown that for the "Standard Substance of 4 - N - nitrosamines Mixed Solution in Methanol (High - concentration Solution Standard Substance)", the standard values of NNN, NNK, and NAT were 0.050 mg / mL (relative expanded uncertainties were 3%, 3%, 3% respectively; k = 2), and the standard value of NAB was 0.010 mg / mL (relative expanded uncertainty was 4%; k = 2).

[0151] Through the study of value assignment and uncertainty evaluation, it was shown that for the "Standard Substance of 4 - N - nitrosamines Mixed Solution in Methanol (Low - concentration Solution Standard Substance)", the standard values of NNN, NNK, and NAT were 0.010 mg / mL (relative expanded uncertainties were 4%; 4%; 4% respectively; k = 2), and the standard value of NAB was 0.005 mg / mL (relative expanded uncertainty was 5%; k = 2). Description of the Drawings

[0152] Figure 1 is the synthesis route of NNN of the present invention;

[0153] Figure 2 is the high - resolution mass spectrum of NNN prepared by the present invention;

[0154] Figure 3 is the synthesis route of NNK of the present invention;

[0155] Figure 4 is the high - resolution mass spectrum of NNK prepared by the present invention;

[0156] Figure 5 is the synthesis route of NAT of the present invention;

[0157] Figure 6 is the high - resolution mass spectrum of NAT prepared by the present invention;

[0158] Figure 7 is the synthesis route of NAB of the present invention;

[0159] Figure 8This is a high-resolution mass spectrometer of NAB prepared according to the present invention;

[0160] Figure 9 shows the short-term stability of NNN in the "mixed solution standard material", where A represents 4℃, B represents 25℃, and C represents 50℃.

[0161] Figure 10 shows the short-term stability of NNK in the "mixed solution standard material", where A represents 4℃, B represents 25℃, and C represents 50℃.

[0162] Figure 11 shows the short-term stability of NAT in the "mixed solution standard material", where A represents 4℃, B represents 25℃, and C represents 50℃.

[0163] Figure 12 shows the short-term stability of NAB in the "mixed solution standard material", where A represents 4℃, B represents 25℃, and C represents 50℃. Detailed Implementation

[0164] The technical solution of the present invention will be further described below in conjunction with specific implementation methods.

[0165] Example 1: Synthesis of NNN

[0166] The synthesis route of NNN is as follows Figure 1 As shown.

[0167] 1. Preparation of Compound 3

[0168] 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%.

[0169] 2. Preparation of Compound 6

[0170] Add 24 g (1.0 eq) of crude compound 3 and 120 mL of tetrahydrofuran to a flask, stir until dissolved, and cool to -75 to -70 °C in a dry ice-ethanol bath. Add 50 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 29.6 g (1.2 eq) of 1,3-diiodopropane dropwise, again keeping the temperature below -60 °C. After the addition is complete, stir at room temperature for 1 to 2 h. Monitor the reaction by TLC. After the reaction is complete, slowly pour the reaction solution into a 200 mL container. The mixture was stirred in 3N hydrochloric acid solution for 20–30 min, then extracted three times with 60 mL of ethyl acetate. The aqueous phase was retained and adjusted to pH 10–12 with potassium carbonate and 40% potassium hydroxide, respectively. The mixture was 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 = 1:10) to give 5 g of compound 6 (oily substance), with a yield of 36.5%.

[0171] 3. Preparation of N-nitrosonornicotinamide (N-nitroso-3-(2-pyrrolidinyl)pyridine)

[0172] 5 g (1.0 eq) of compound 6 and 50 mL of 4N hydrochloric acid solution were added to a flask and cooled to 0–5 °C in an ice-water bath. 6.2 g (3.0 eq) of sodium nitrite was dissolved in 40 mL of water to prepare a solution. The sodium nitrite solution was slowly added dropwise to the flask, controlling the temperature not to exceed 5 °C. After the addition was complete, the mixture was stirred at room temperature for 4–5 h. The reaction was monitored by TLC. After the reaction was completed, the pH was adjusted to 12–14 with 30% sodium hydroxide. The mixture was 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 = 1:33) to obtain 3.44 g of a yellow oily substance, with a yield of 64.5% and HPLC >99%.

[0173] High-resolution mass spectrometry (HS-MS) was used Figure 2 The structure of N-nitrosopseudoesequina (NAB) obtained above was verified by means of low-resolution mass spectrometry, nuclear magnetic resonance spectroscopy, 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.

[0174] Example 2: Synthesis and Preparation of NNK

[0175] NNK's synthesis route is as follows Figure 3 As shown.

[0176] 1. Preparation of Compound 1

[0177] 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%.

[0178] 2. Preparation of Compound 2

[0179] 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%.

[0180] 3. Preparation of 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone

[0181] 8.0 g (1.0 eq) of crude compound 2 and 80 mL of 4N hydrochloric acid solution were added to a flask and cooled to 0–5 °C in an ice-water bath. 8.4 g (3.0 eq) of sodium nitrite was dissolved in 64 mL of water to prepare a solution. The sodium nitrite solution was slowly added dropwise to the flask, controlling the temperature not to exceed 5 °C. After the addition was complete, the mixture was stirred overnight at room temperature (16–17 h). The reaction was monitored by TLC. After the reaction was complete, the pH was adjusted to 12–13 with 30% sodium hydroxide. The mixture was extracted twice 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 = 1:50) to obtain 3.1 g of solid, yield 10.8%, HPLC >99%.

[0182] High-resolution mass spectrometry (HS-MS) was used Figure 4 The structure of N-nitrosopseudoesequina (NAB) obtained above was verified by means of low-resolution mass spectrometry, nuclear magnetic resonance spectroscopy, 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.

[0183] Example 3: Synthesis and Preparation of NAT

[0184] NNK's synthesis route is as follows Figure 5 As shown.

[0185] 1. Preparation of Compound 3

[0186] 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%.

[0187] 2. Preparation of Compound 4

[0188] Add 15 g (1.0 eq) of crude compound 3 and 90 mL of tetrahydrofuran to a flask, stir until dissolved, and cool to -75 to -70 °C in a dry ice-ethanol bath. Add 31 mL (1.2 eq) of LDA tetrahydrofuran solution (2.0 M) dropwise, controlling the temperature not to exceed -70 °C. After the addition is complete, stir at -75 to -70 °C for 0.5 to 1 h. Then add 9.7 g (1.5 eq) of cis-1,4-dichloro-2-butene dropwise, again controlling the temperature not to exceed -60 °C. After the addition is complete, stir at room temperature for 0 h. The reaction was monitored by TLC for 5–1 h. 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, 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 column chromatography (methanol:dichloromethane = 1:20) was performed to give 5 g of compound 4 (oil), with a yield of 54.3%.

[0189] 3. Preparation of N-nitrosonephrine (1-nitroso-1,2,3,6-tetrahydro-2,3'-bipyridine)

[0190] 5.0 g (1.0 eq) of compound 4 and 50 mL of 4N hydrochloric acid solution were added to a flask and cooled to 0–5 °C in an ice-water bath. 5.8 g (3.0 eq) of sodium nitrite was dissolved in 40 mL of water to prepare a solution. The sodium nitrite solution was slowly added dropwise to the flask, controlling the temperature not to exceed 5 °C. After the addition was complete, the mixture was stirred overnight at room temperature (16–17 h). The reaction was monitored by TLC. After the reaction was completed, the pH was adjusted to 13–14 with 30% sodium hydroxide. The mixture was extracted twice 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 = 1:50) to obtain 3.77 g of a yellow oily substance, with a yield of 47.7% and HPLC >99%.

[0191] High-resolution mass spectrometry (HS-MS) was used Figure 6 The structure of N-nitrosopseudoesequina (NAB) obtained above was verified by means of low-resolution mass spectrometry, nuclear magnetic resonance spectroscopy, 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.

[0192] Example 4: Synthesis and Preparation of NAB

[0193] NNK's synthesis route is as follows Figure 7 As shown.

[0194] 1. Preparation of Compound 3

[0195] 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%.

[0196] 2. Preparation of Compound 5

[0197] Add 20.5 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. Monitor the reaction by TLC. After the reaction is complete, slowly pour the reaction solution into a 200 mL container. The mixture was stirred in 3N hydrochloric acid solution for 20–30 min, then extracted three times with 60 mL of ethyl acetate. The aqueous phase was retained and adjusted to pH 10–12 with potassium carbonate and 40% potassium hydroxide, respectively. The mixture was 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 3.1 g of compound 4 (oil), with a yield of 24.4%.

[0198] 3. Preparation of N-nitrosopseudoesequine (N-nitroso-3-(2-piperidinyl)pyridine)

[0199] 3.1 g (1.0 eq) of compound 5 and 31 mL of 4N hydrochloric acid solution were added to a flask and cooled to 0–5 °C in an ice-water bath. 3.6 g (3.0 eq) of sodium nitrite was dissolved in 24 mL of water to prepare a solution. The sodium nitrite solution was slowly added dropwise to the flask, controlling the temperature not to exceed 5 °C. After the addition was complete, the mixture was stirred at room temperature for 4–5 h. The reaction was monitored by TLC. After the reaction was completed, the pH was adjusted to 12–14 with 30% sodium hydroxide. The mixture was 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 = 1:50) to obtain 3.68 g of a yellow oily substance, with a yield of 65.7% and HPLC >99%.

[0200] High-resolution mass spectrometry (HS-MS) was used Figure 8 The structure of N-nitrosopseudoesequina (NAB) obtained above was verified by means of low-resolution mass spectrometry, nuclear magnetic resonance spectroscopy, 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.

[0201] Example 5: Purity Analysis of NNN, NNK, NAT, and NAB

[0202] I. Raw material packaging and preparation

[0203] The four candidate samples (NNN, NNK, NAT, and NAB) synthesized in Examples 1-4 were aliquoted into 2mL brown vials. Each 2mL vial was 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 aliquoting. The glove box was filled with high-purity nitrogen inert gas to control oxygen and moisture levels.

[0204] Preparation for glove box operation: Power on: Turn on the main power switch (red); open the working gas pressure reducing valve (main valve fully open, 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. After connecting the gas line, 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, confirming that the transition chamber door is closed and the pressure gauge is at 0. After checking, prepare the sample, 700 2mL brown sample vials, sterile pipette tips, pipettes, tweezers, and 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.

[0205] Weighing Procedure: First, calibrate the balance. Then, place a 2mL brown sample vial in the center of the balance. Open the sample vial. For oily samples (NNN, NAT, NAB), use a pipette to take approximately 2mg of sample and place it in a 2mL vial. After weighing, immediately cap the vial and seal it with sealing film. Dispense 150 vials for each candidate sample. For solid samples (NNK), use a weighing spoon to take approximately 20mg of sample and place it in a 2mL vial. After weighing, immediately cap the vial and seal it 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. Return the experimental materials and samples to the large transition chamber, close the inner door, slowly remove the gloves, and use the foot switch to ensure the pressure inside the chamber is within the normal range. Remove the experimental materials, close the door, and place the sealed samples in a desiccator and store them at -18℃.

[0206] II. Purity Analysis of NNN, NNK, NAT, and NAB

[0207] The purity of the main components of the four raw materials (NNN, NNK, NAT, and NAB) after repackaging was determined by liquid chromatography area normalization. Anions, inorganic elements, residual solvents, and moisture in the candidate raw materials 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:

[0208] 1. Principal component separation method

[0209] The determination was performed using liquid chromatography, and the liquid chromatography conditions were as follows:

[0210] Column: Poroshell EC-C18 (4.6 x 250 mm x 4 μm); Mobile phase: Water (A) and methanol (B); Elution program: 0-2 min: 25-55% B; 2-2.1 min: 55-40% B; 2.1-8 min: 40-60% B; 8-8.1 min: 60-90% B; 8.1-10 min: 90-90%; 10-10.1 min: 90-25%; 10.1-15 min: 25-25%; Column temperature: 50℃; Flow rate: 1.0 mL / min; Injection volume: 10 μL; Detection wavelength: individual standard wavelengths.

[0211] 2. Purity analysis by multiple laboratories

[0212] 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 2.

[0213] Table 2 Jointly set value units and instrument models

[0214]

[0215]

[0216] Nine sets of fixed-value data were obtained from nine fixed-value units, from laboratory 01 to laboratory 09, as shown in Table 3-6 below.

[0217] Table 3. Collaborative NNN data from 9 laboratories (unit: %)

[0218]

[0219] Table 4. NNK data from collaborative assessments by 9 laboratories (unit: %)

[0220]

[0221] Table 5. NAT data from collaborative assessments by 9 laboratories (unit: %)

[0222]

[0223]

[0224] Table 6. Collaborative NAB data from 9 laboratories (unit: %)

[0225]

[0226] 3. Moisture content determination

[0227] 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. The moisture content in the solutions of four candidate compounds (NNN, NNK, NAT, and NAB) 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 7. From the table, it can be calculated that the moisture content in the NNN candidate is 0.055%; in the NNK candidate is 0.063%; in the NAT candidate is 0.062%; and in the NAB candidate is 0.037%.

[0228] Table 7. Moisture content (mg / g) in four types of nitrosamine solutions

[0229]

[0230]

[0231] 4. Anion determination

[0232] Referencing industry standard YC / T248-2008, "Determination of Inorganic Anions in Tobacco and Tobacco Products - Ion Chromatography," the anion detection results are shown in Table 8.

[0233] Table 8. Anion detection results (unit: μg / mL)

[0234]

[0235] 5. Inorganic element determination

[0236] 5.1 Measurement Process

[0237] The inorganic element content of four candidate compounds (NNN, NNK, NAT, and NAB) was analyzed using inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7900). 1 mg / mL solutions of each candidate compound were prepared using chromatographic-grade methanol. Specifically, 10.07 mg of NNN was weighed and placed in a 10 mL amber volumetric flask, then diluted to the mark with methanol to obtain a 1 mg / mL NNN solution. Similarly, 1 mg / mL solutions of NNK, NAT, and NAB were prepared. 1 mL of each solution was then transferred 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 all elements in the periodic table.

[0238] 5.2 Instrumental Analysis Conditions

[0239] 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 9 and 10.

[0240] Table 9 Main Parameters of ICP / MS

[0241]

[0242] Table 10 Measurement of isotopes, internal standard elements, and integration time.

[0243]

[0244] The results of inorganic elements in the four candidates are shown in Table 11 below. It was found that the types and contents of inorganic elements contained in the four candidates were not the same. They mainly contained trace amounts of Na, Sn, Te and other elements, with a total amount of 21.04-35.65 mg / mL, accounting for 0.11%-0.18%.

[0245] Table 11 Results of Inorganic Element Content Determination (Unit: mg / mL)

[0246]

[0247]

[0248] Note: Italics in the table indicate not detected. The test results are expressed as detection limits and are included in the total element count.

[0249] 6. Determination of volatile impurities

[0250] 6.1 Measurement Process

[0251] For the solvents used in the synthesis of four candidate raw materials (NNN, NNK, NAT, and NAB), and considering common solvent impurities, 28 substances were selected as target analytes for solvent residue determination. Referring to the industry standard YC / T 207—2014 "Determination of Solvent Residues in Tobacco Paper by Headspace-Gas Chromatography / Mass Spectrometry", headspace-gas chromatography / mass spectrometry (HS-GC / MS) was used to determine the solvent residues.

[0252] Sample preparation: Triacetylglycerol was used to prepare 1 mg / mL candidate solutions. The specific steps were as follows: 10 mg of candidate was weighed and placed in a 10 mL brown volumetric flask, and the volume was adjusted to the mark with triacetylglycerol to obtain a candidate solution with a concentration of 1 mg / mL.

[0253] The instruments and conditions were as follows: static headspace analyzer; headspace vial: 20 mL to 25 mL; sample loop: 3.0 mL; sample equilibration temperature: 80 °C; sample loop temperature: 160 °C; transfer line temperature: 180 °C; sample equilibration time: 45.0 min; sample vial pressurization pressure: 138 kPa; pressurization time: 0.20 min; gas filling time: 0.20 min; sample loop equilibration time: 0.05 min; sample injection time: 1.0 min.

[0254] Gas chromatograph (GC): VOC-specific capillary column (VOCOL column); 60m × 0.32mm × 1.8μm; carrier gas: He; injection port temperature: 180℃; constant flow mode; column flow rate 2.0mL / min; split ratio 20:1; temperature program: 40℃; hold for 2 min; increase to 200℃ at a rate of 4℃ / min; hold for 10 min.

[0255] Mass spectrometer (MS): Auxiliary interface temperature: 220℃; Ionization method: Electron impact source (EI); Ion source temperature: 230℃; Ionization energy: 70eV; Quadrupole temperature: 150℃; Full scan monitoring mode, scan range: 29 amu~350 amu; Selected ion monitoring mode, ion selection parameter principle: Among the mass spectrometric ion fragments of each solvent residue, select the ion with the highest relative abundance as the quantitative ion. If there is interference, select the ion with the second highest relative abundance as the quantitative ion; select 1-2 other fragment ions as auxiliary quantitative ions.

[0256] 6.2 Results Analysis

[0257] The content of volatile organic solvents in the TSNAs solution was determined. 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 12.

[0258] Table 12. Content of dichloromethane in four types of N-nitrosamines (unit: ng / mL)

[0259] Number of experiments NNN NNK NAT NAB 1 892 / 515 569 2 890 / 520 558 3 898 / 531 572 average value 893 / 522 566 percentage 0.09％ / 0.05％ 0.06％

[0260] 7. Determination of Benzophenone Solvent Residue

[0261] According to industry standard YQ / T 31-2013 "Determination of Photoinitiators in Cigarette Packaging Paper by Gas Chromatography-Mass Spectrometry", benzophenone is a substance used in large quantities during synthesis, and due to its high boiling point, it cannot be detected using solvent residue methods. Therefore, gas chromatography-mass spectrometry (Agilent 7890-5975C) was used to analyze the benzophenone content in four candidate compounds: methanol, NNN, NNK, NAT, and NAB. Selected ion scanning was used. The results are shown in Table 13.

[0262] Table 13. Benzophenone content (μg / mL) in methanol solvent and nitrosamine solution

[0263] Number of experiments methanol NNN NNK NAT NAB 1 / 0.08 / 0.27 0.05 2 / 0.09 / 0.25 0.05 3 / 0.07 / 0.22 0.05 average value / 0.08 / 0.25 0.05 percentage / 0.008％ / 0.025％ 0.005％

[0264] 8. Test Results

[0265] Using the liquid phase area normalization method, the average purity values ​​from multiple laboratories were averaged. The resulting purities for the four candidates were 99.76% (NNN), 99.67% (NNK), 99.78% (NAT), and 99.51% (NAB). The actual purity of the four candidates 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 The purity of the four candidates after deducting inorganic elements, solvent residues, and benzophenone is shown in Table 14 below.

[0266] Table 14. Purity test results (%) of liquid chromatography area normalization method

[0267]

[0268] Example 6: Packaging and Uniformity / Stability Testing of NNN, NNK, NAT, and NAB

[0269] 1. Raw material packaging and preparation

[0270] The four candidate samples (NNN, NNK, NAT, and NAB) synthesized in Examples 1-4 were aliquoted into 2mL brown vials. Each 2mL vial was 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 aliquoting. The glove box was filled with high-purity nitrogen inert gas to control oxygen and moisture levels.

[0271] Preparation for glove box operation: Power on: Turn on the main power switch (red); open the working gas pressure reducing valve (main valve fully open, 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. After connecting the gas line, 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, confirming that the transition chamber door is closed and the pressure gauge is at 0. After checking, prepare the sample, 700 2mL brown sample vials, sterile pipette tips, pipettes, tweezers, and 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.

[0272] Weighing Procedure: First, calibrate the balance. Then, place a 2mL brown sample vial in the center of the balance. Open the sample vial. For oily samples (NNN, NAT, NAB), use a pipette to take approximately 2mg of sample and place it in a 2mL vial. After weighing, immediately cap the vial and seal it with sealing film. Dispense 150 vials for each candidate sample. For solid samples (NNK), use a weighing spoon to take approximately 20mg of sample and place it in a 2mL vial. After weighing, immediately cap the vial and seal it 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. Return the experimental materials and samples to the large transition chamber, close the inner door, slowly remove the gloves, and use the foot switch to ensure the pressure inside the chamber is within the normal range. Remove the experimental materials, close the door, and place the sealed samples in a desiccator and store them at -18℃.

[0273] 2. Raw material uniformity inspection

[0274] According to the sampling quantity requirements for homogeneity testing 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 four candidate samples 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 10.00 mg of the raw material sample from each of the 11 bottles and place it in a 10 mL brown volumetric flask. Dilute to the mark with methanol to obtain a test solution with a concentration of 1 mg / mL. A total of 11 solutions were prepared for each candidate, with three samples prepared from each bottle, for a total of 33 samples. Each sample was then numbered. The method used was the area normalization method in liquid chromatography.

[0275] 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.

[0276] 2.1 Liquid Chromatography Conditions

[0277] The liquid chromatography conditions are as follows:

[0278] —Column: Poroshell EC-C18 column (4.6mm×250mm, 4μm);

[0279] —Flow rate: 1.0 mL / min;

[0280] —Column temperature: 40℃;

[0281] —Injection volume: 10 μL;

[0282] —Mobile phase A: water, mobile phase B: methanol;

[0283] —Full wavelength scanning mode (210-500nm), monitoring wavelengths NNN (235nm), NNK (230nm), NAT (236nm), NAB (238nm);

[0284] —The gradient elution conditions are shown in Table 15;

[0285] Table 15 Gradient elution conditions for high performance liquid chromatography

[0286]

[0287] 2.2 Uniformity Results

[0288] The results of the homogeneity test are shown in Tables 16 to 19. 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) = 2.30. Since the calculated NNN(1.18), NNK(1.25), NAT(1.22), and NAB(1.16) are less than F... 0.05 (10, 22) = 2.30, indicating that there is no significant difference between the samples at the 95% confidence level, and the candidate raw material samples after dispensing are homogeneous.

[0289] Table 16. Results of Homogeneity Test of Raw Materials for NNN Candidates

[0290]

[0291] Note: In the table, The same applies below.

[0292] Table 17 Results of Homogeneity Test of NNK Candidate Raw Materials

[0293]

[0294]

[0295] Table 18. Results of Homogeneity Test of Raw Materials for NAT Candidates

[0296]

[0297] Table 19 Results of homogeneity test of NAB candidate raw materials

[0298]

[0299]

[0300] 3. Raw material stability testing

[0301] 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 single 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 the three vials and place it in a 20 mL brown volumetric flask. Dilute to the mark with methanol to obtain a test solution with a concentration of 1 mg / mL. The method used is the liquid chromatography area normalization method.

[0302] The results of the long-term stability test of the four raw materials are shown in Table 20-23.

[0303] Table 20 Long-term stability data of NNN feedstock

[0304]

[0305] In accordance with the requirements of JJF 1343-2022, a linear model was selected as the empirical model for this standard material.

[0306] The long-term stability data in Table 20, with x representing time (days) and y representing the NNN purity value, are fitted to a straight line y = kx + b, resulting in the equation y = 0.000013x + 99.74. Therefore, the slope k = 0.000013 and the intercept b = 99.74.

[0307] The standard deviation of each point on the straight line can be calculated using the following formula:

[0308]

[0309] The slope uncertainty is calculated using the following formula:

[0310]

[0311] 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.

[0312] because Therefore, the slope is not significant, and thus the stability is good.

[0313] Table 21 Long-term stability data of NNK raw materials

[0314]

[0315] In accordance with the requirements of JJF 1343-2022, a linear model was selected as the empirical model for this standard material.

[0316] The long-term stability data in Table 21, with x representing time (days) and y representing the NNK purity value, are fitted to a straight line y = kx + b, resulting in the equation y = 0.000022x + 99.68. Therefore, the slope k = 0.000022 and the intercept b = 99.68.

[0317] The standard deviation of each point on the straight line can be calculated using the following formula:

[0318]

[0319] The slope uncertainty is calculated using the following formula:

[0320]

[0321] 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.

[0322] because Therefore, the slope is not significant, and thus the stability is good.

[0323] Table 22 Long-term stability data of NAT raw materials

[0324]

[0325] In accordance with the requirements of JJF 1343-2022, a linear model was selected as the empirical model for this standard material.

[0326] The long-term stability data in Table 22, with x representing time (days) and y representing NAT purity value, are fitted to a straight line y = kx + b, resulting in the equation y = 0.0000080x + 99.78. The slope k = 0.0000090 and the intercept b = 99.78.

[0327] The standard deviation of each point on the straight line can be calculated using the following formula:

[0328]

[0329] The slope uncertainty is calculated using the following formula:

[0330]

[0331] 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.

[0332] because Therefore, the slope is not significant, and thus the stability is good.

[0333] Table 23 Long-term stability data of NAB raw materials

[0334]

[0335]

[0336] In accordance with the requirements of JJF 1343-2022, a linear model was selected as the empirical model for this standard material.

[0337] The long-term stability data in Table 23, 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.

[0338] The standard deviation of each point on the straight line can be calculated using the following formula:

[0339]

[0340] The slope uncertainty is calculated using the following formula:

[0341]

[0342] 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.

[0343] because Therefore, the slope is not significant, and thus the stability is good.

[0344] Example 7: Evaluation of Purity Uncertainty for NNN, NNK, NAT, and NAB

[0345] The NNN, NNK, NAT, and NAB used in this embodiment are the products after being packaged in Embodiment 6.

[0346] 1. Sources of uncertainty

[0347] The main sources of uncertainty in the purity of the four raw materials 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).

[0348] 2. Uncertainty assessment of purity analysis

[0349] Referring to the JJF 1855-2020 standard, the uncertainty of purity determination includes the uncertainty introduced by instrument verification, the determination process of impurities (anions, moisture, inorganic elements and solvent residues), raw material homogeneity and raw material stability.

[0350] 3. Results

[0351] Based on the purity uncertainty assessment, the relative standard uncertainties introduced by the purity of NNN, NNK, NAT, and NAB are 0.24%, 0.67%, 0.25%, and 0.30%, respectively.

[0352] Example 8: Preparation of solution standard (high concentration)

[0353] The NNN, NNK, NAT, and NAB used in this embodiment are the products after being packaged in Embodiment 6.

[0354] 1. Preparation of standard solutions

[0355] Before weighing the samples, the weighing instruments (tweezers, weighing spoon, weighing boat, microsyringe, 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 door; next, use tweezers to place the container or weighing boat 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.

[0356] Based on the relationship between density, volume, and mass, taking NNN as an example, since it is an oily substance at room temperature, its density is 1.3 g / cm³. 3 The volume corresponding to 50 mg is 38.46 μL. Using a 50 μL microsyringe, 39 μL was drawn up and added dropwise at a rate that was initially fast and then slowed down. When the balance reading was close to 50 mg, the remaining liquid was added slowly dropwise, and 50 mg was accurately weighed. Similarly, NAT (density 1.2 g / cm³) was accurately weighed sequentially. 3 50mg, NAB (density 1.2g / cm³) 3 Add 10 mg of NNK to the same volumetric flask. Finally, accurately weigh and add NNK. Since NNK is a solid powder, a weighing boat is used to assist in weighing. Place the weighing boat in the center of the balance and zero it. Use a weighing spoon to accurately place approximately 50 mg onto the weighing boat. Then, rinse the weighing boat several times with chromatographic grade methanol. Finally, rinse the inner wall of the volumetric flask neck with methanol. When near the mark, carefully add the methanol dropwise to the mark, making up to 1000 mL. Shake well and let stand to obtain mixed standard solutions with concentrations of 0.01, 0.05, 0.05, and 0.05 mg / mL (NAB, NAT, NNK, NNN). Transfer the prepared mixed standard solutions to a -18°C freezer.

[0357] 2. Dispensing of standard solutions

[0358] The specific aliquoting procedure is as follows: First, place the volumetric flask containing the mixed standard solution in a -18°C freezer to cool completely. Then, using a dispenser, add 1.5 mL of the mixed standard solution to a clean 2 mL brown ampoule (performed under nitrogen protection in a biosafety cabinet). Quickly transfer the ampoule to a freezer bath and store at -18°C for approximately 20 minutes. Then, immediately remove the ampoule and seal it with a flame. A total of 500 ampoules were dispensed, each containing 1.5 mL, and stored at -18°C.

[0359] 3. Constant value

[0360] 3.1 Basis for setting values

[0361] According to JJF 1343-2022 "General Principles and Statistical Principles for the Determination of Standard Reference Materials", one of the following methods may be used to determine the value of a standard reference material: ① determination by a single laboratory using a single reference method; ② determination by a laboratory using two or more independent reference methods with different principles; ③ determination by multiple laboratories in cooperation using one or more methods with proven accuracy; ④ determination using a specific method; ⑤ determination by comparison using primary standard reference materials.

[0362] This example uses ① a single laboratory using a single reference method to determine the value, that is, using a balance to weigh a certain mass of standard substance and dilute it to a certain volume of volumetric flask with methanol whose purity has been verified.

[0363] Using the area normalization method, the purity of the four solution standards (NNN, NNK, NAT, and NAB) after removing anions, inorganic elements, solvent residues, and water is determined to be 99.35%, 99.38%, 99.45%, and 99.18%, respectively.

[0364] 3.2 Constant Value Formula

[0365] The content of each component in the "Standard Reference Material for Mixed Solution of Four N-nitrosamines in Methanol" can be traced back to the standard reference material and the balance and volumetric flask used for metrological verification.

[0366] The standard value c of the main component concentration in the prepared mixed solution standard substance is calculated according to the following formula:

[0367]

[0368] Where: c—the standard value of the concentration of the main component of the prepared solution; m—the mass of the purity standard substance weighed; v—the volume of the solution prepared; ρ—the purity of the standard substance.

[0369] 3.3 Characteristic values ​​of the main component in the mixed solution standard substance

[0370] Based on the purity and weight of the four standard substances and the above formula, the standard values ​​of each component in the "mixed standard solution of four N-nitrosamines in methanol" were calculated, as shown in Table 24.

[0371] Table 24 Standard values ​​of each component in the mixed solution standard substance

[0372]

[0373] Example 9: Homogeneity test of mixed solution standard substance

[0374] The mixed sample used in this embodiment is the sample prepared in Example 8.

[0375] 1. Uniformity test

[0376] According to the sampling quantity requirements for homogeneity testing in JJF 1343-2022 (when the total number of units is 200 < N ≤ 500, the number of units sampled shall not be less than 15), five bottles of samples were taken from each of the pre-packaged mixed solution standard substance solutions, following the order of packaging (sampling method: sampling was performed by coding the packaging sequence and then referring to a random number table), for a total of 15 bottles of samples. 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.

[0377] 2. Liquid Chromatography Conditions

[0378] Liquid chromatography conditions:

[0379] —Column: Poroshell EC-C18 column (4.6mm×250mm, 4μm);

[0380] —Flow rate: 1.0 mL / min;

[0381] —Column temperature: 40℃;

[0382] —Injection volume: 10 μL;

[0383] —Mobile phase A: water, mobile phase B: methanol;

[0384] —Full wavelength scanning mode (210-500nm), with simultaneous setting of monitoring wavelengths NNK (232nm), NNN (235nm), NAT (236nm), and NAB (238nm).

[0385] —The gradient elution conditions are shown in Table 25.

[0386] Table 25 Gradient elution conditions for high performance liquid chromatography

[0387]

[0388] 3. Mathematical statistics methods

[0389] According to the JJF 1343-2022 technical specification, one-way ANOVA was adopted as the mathematical statistical method for testing the uniformity of this project.

[0390] 4. Statistical analysis of principal components in samples and interpretation of results

[0391] The concentration of the principal component was converted using a single-point method. The calibration solutions for the single-point method were prepared in a Class 100,000 cleanroom, yielding mixed calibration solutions with concentrations of 0.01, 0.05, 0.05, and 0.05 mg / mL. The conversion formula is as follows:

[0392]

[0393] In the formula:

[0394] c1 -- Concentration of the standard substance solution being filled, mg / mL;

[0395] S1 -- Peak area of ​​the main component in the liquid chromatography of the standard substance solution;

[0396] S2 -- Peak area of ​​the main component in the standard substance calibration solution of the liquid chromatography;

[0397] C 2-- Concentration of the standard substance calibration solution, mg / mL;

[0398] The results of the homogeneity test are shown in Tables 26 to 29. According to JJF 1343-2022, one-way ANOVA (F-test) was used for statistical analysis. Referring to the F-distribution table, at a confidence probability of 95%, F... 0.05 (14, 30) = 2.04. Since the calculated NNN (1.67), NNK (1.39), NAT (1.60), and NAB (1.15) values ​​for F are less than F... 0.05 (14, 30) indicates that there is no significant difference between the samples at the 95% confidence level, and the aliquoted "mixed solution standard material" samples are homogeneous.

[0399] Table 26 Results of NNN homogeneity test in “Mixed Solution Standard Material” samples

[0400]

[0401] Note: In the table, The same applies below.

[0402] Table 27 Results of NNK homogeneity test in "Mixed Solution Standard Material" samples

[0403]

[0404]

[0405] Table 28 Results of NAT homogeneity test in "Mixed Solution Standard Material" samples

[0406]

[0407] Table 29 Results of NAB homogeneity test in “Mixed Solution Standard Material” samples

[0408]

[0409] The test results above show that the components of the "mixed solution standard substance in methanol" are well homogeneous after repackaging.

[0410] Example 10: Stability Test

[0411] The sample used in this embodiment is the sample from Example 8.

[0412] 1. Short-term stability of the sample

[0413] The samples were stored in brown ampoules protected from light; therefore, short-term stability was primarily assessed by examining the effect of temperature. The concentration changes of the standard substance during cold chain transportation under normal weather (4°C), normal temperature (25°C), and extreme conditions (50°C) on days 0, 1, 3, 5, and 7 were examined. Twelve ampoules of the mixed solution standard substance sample were placed in three temperatures (4°C refrigerator, 25°C, and 50°C artificial climate chamber). The sample from day 0 was measured after being placed at the corresponding temperature for one hour. Subsequently, three ampoules were removed from the refrigerator and artificial climate chamber at each of the three temperatures on days 1, 3, 5, and 7, respectively, and the last three ampoules were measured on day 7. Each sample was measured three times. Chromatographic conditions were used for stability testing, referring to the homogeneity chromatographic conditions in Example 9.

[0414] The results of the short-term stability test of the "mixed solution standard material" samples are shown in Tables 30 to 33.

[0415] Table 30 shows the short-term stability data of NNN in the "Mixed Solution Standard Material" table.

[0416]

[0417]

[0418] Conclusion: Short-term stability test results show that the concentration of NNN in the "mixed solution standard of four N-nitrosamines in methanol" did not change after short-term storage at 4℃, 25℃, and 50℃, indicating good sample stability. The uncertainty arising from short-term stability is assessed by the uncertainty of the slope. (T = 7 days, S) (k) (Taking the maximum value among the experimental results under the three conditions), therefore the short-term uncertainty of NNN is 0.70%.

[0419] Table 31 shows the short-term stability data of NNK in the "Mixed Solution Standard Material" table.

[0420]

[0421] Conclusion: Short-term stability test results show that the concentration of NNK in the "mixed solution standard of four N-nitrosamines in methanol" did not change after short-term storage at 4℃, 25℃, and 50℃, indicating good sample stability. The uncertainty arising from short-term stability is assessed by the uncertainty of the slope. (T = 7 days, S) (k) (Taking the maximum value among the experimental results under the three conditions), therefore the short-term uncertainty of NNK is 0.61%.

[0422] Table 32 shows the short-term stability data of NAT in "Mixed Solution Standard Materials".

[0423]

[0424]

[0425] Conclusion: Short-term stability test results show that the NAT concentration in the "mixed solution standard of four N-nitrosamines in methanol" did not change after short-term storage at 4℃, 25℃, and 50℃, indicating good sample stability. The uncertainty arising from short-term stability was assessed by the uncertainty of the slope. (T = 7 days, S) (k) Taking the maximum value among the experimental results under the three conditions, the short-term uncertainty of NAT is 0.68%.

[0426] Table 33 shows the short-term stability data of NAB in "Mixed Solution Standards".

[0427]

[0428] Conclusion: Short-term stability test results show that the NAB concentration in the "mixed solution standard of four N-nitrosamines in methanol" did not change after short-term storage at 4℃, 25℃, and 50℃, indicating good sample stability. The uncertainty arising from short-term stability was assessed by the uncertainty of the slope. (T = 7 days, S) (k) (Taking the maximum value among the experimental results under the three conditions), therefore the short-term uncertainty of NAB is 0.87%.

[0429] 4. Long-term stability of the sample

[0430] 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 the "mixed solution standard reference material of four N-nitrosamines in methanol" sample under light-protected conditions at -18℃, the packaged sample was stored at -18℃ in the dark for a long period of time. Following a principle of initial dense storage followed by sparse storage, stability tests were performed at 0, 1, 2, 4, 6, 9, 12, and 24 months. Three samples were randomly selected at each time point, and each sample was tested three times.

[0431] The measurement results are shown in Tables 34 to 37 below.

[0432] Table 34 shows the long-term stability data of NNN in the "Mixed Solution Standard Material" table.

[0433]

[0434] Conclusion: The results of the long-term stability test show that the t-value of NNN in the "mixed solution standard of four N-nitrosamines in methanol" is less than the critical value, which indicates that the NNN sample in the "mixed solution standard of four N-nitrosamines in methanol" has good long-term stability.

[0435] Table 35 shows the long-term stability data of NNK in "Mixed Solution Standard Materials".

[0436]

[0437] Conclusion: The results of the long-term stability test show that the t-value of NNK in the "mixed solution standard of four N-nitrosamines in methanol" is less than the critical value, which indicates that the NNK sample in the "mixed solution standard of four N-nitrosamines in methanol" has good long-term stability.

[0438] Table 36 "Long-term stability data of NAT in mixed solution standard materials"

[0439]

[0440]

[0441] Conclusion: The results of the long-term stability test show that the t-value of NAT in the "mixed solution standard of four N-nitrosamines in methanol" is less than the critical value, which indicates that the NAT sample in the "mixed solution standard of four N-nitrosamines in methanol" has good long-term stability.

[0442] Table 37 shows the long-term stability data of NAB in "Mixed Solution Standard Materials".

[0443]

[0444] Conclusion: The results of the long-term stability test show that the t-value of NAB in the "mixed solution standard of four N-nitrosamines in methanol" is less than the critical value, which indicates that the NAB sample in the "mixed solution standard of four N-nitrosamines in methanol" has good long-term stability.

[0445] Example 11: Uncertainty Assessment of Solution Standards

[0446] The solution standard used in this embodiment was prepared as in Example 8.

[0447] 1. Sources of uncertainty

[0448] The main sources of uncertainty in the determination of mixed solution standard substances are: (1) solution preparation process (purity analysis of raw materials, volume and weighing); (2) solution homogeneity; (3) solution stability; and (4) filling loss.

[0449] 2. Uncertainty assessment

[0450] 2.1 Uncertainty introduced by solution preparation

[0451] 2.1.1 Uncertainty introduced in the raw material purity analysis process

[0452] Referring to JJF 1855-2020 standard, the uncertainty in purity analysis includes uncertainties introduced by instrument verification, the determination process of impurities (anions, moisture, inorganic elements, and solvent residues), raw material homogeneity, and raw material stability. According to the results in Chapter 6, the uncertainties introduced by the purity of NNN, NNK, NAT, and NAB are 0.24%, 0.60%, 0.25%, and 0.30%, respectively.

[0453] 2.1.2 Uncertainty introduced by the balance

[0454] 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:

[0455] The relative uncertainties of its NNN, NNK, and NAT are: The same evaluation process yields a relative uncertainty of 0.98% for NAB.

[0456] 2.1.3 Uncertainty introduced by volume

[0457] (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 998.01 mL, 999.93 mL, 998.87 mL, 1001.02 mL, 1000.01 mL, 999.94 mL, 1000.00 mL, and 1001.02 mL. Using Bessel's formula, the standard deviation is 1.007 mL. Therefore, the uncertainty caused by volume in the preparation of the standard substance sample is 1.007 mL, and the relative standard uncertainty expressed by the relative standard deviation is 0.100%.

[0458] (2) Type B uncertainty

[0459] Type B uncertainty. 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., and the metrology certificate is shown in Appendix E. 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:

[0460] (3) Combined relative standard uncertainty:

[0461] 2.2 Uncertainty introduced by homogeneity

[0462] According to JJF 1343-2022, and based on the homogeneity test results in this report, the relative standard uncertainty u is expressed as the relative standard deviation of homogeneity. c(H) That is: NNN = 0.35%, NNK = 0.25%, NAT = 0.34%, NAB = 0.34%.

[0463] 2.3 Uncertainty introduced by short-term stability

[0464] 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: That is: NNN = 0.72%, NNK = 0.61%, NAT = 0.68%, NAB = 0.87%.

[0465] 2.4 Uncertainty introduced by long-term stability

[0466] 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: That is: NNN = 1.10%, NNK = 1.04%, NAT = 0.94%, NAB = 1.07%.

[0467] 2.5 Uncertainty introduced by filling loss

[0468] Because the solvents methanol, dichloromethane, and benzophenone used in the mixed solution standard substances all have relatively low boiling points, some loss may occur during the filling and heat-sealing process. Therefore, the relative standard deviations of the concentration changes of each substance in the 25 filled samples were statistically analyzed, and these relative standard deviations were used as the relative standard uncertainties. The uncertainties for NNN, NNK, NAT, and NAB were 0.0023%, 0.0023%, 0.0023%, and 0.0023%, respectively.

[0469] 2.6 Overall Combined Relative Standard Uncertainty

[0470] Table 38 Relative Standard Uncertainty Components and Combination

[0471]

[0472] 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 calculations are shown in Table 39 below.

[0473] Table 39 Expanded Uncertainty of Each Sample

[0474]

[0475] Example 12: Preparation of solution standard (low concentration)

[0476] The NNN, NNK, NAT, and NAB used in this embodiment were prepared in Examples 1-4. This embodiment uses the same method as Example 8 for preparation, dispensing, and determination. In the prepared mixed solution standard, the concentrations of NNN, NNK, NAT, and NAB are 0.010 mg / mL, 0.010 mg / mL, 0.010 mg / mL, and 0.005 mg / mL, respectively.

[0477] The standard values ​​of the content of each component of the mixed solution standard substance are shown in Table 40.

[0478] Table 40

[0479]

[0480]

[0481] Example 13: Homogeneity test of mixed solution standard substance

[0482] The mixed sample used in this embodiment was the sample prepared in Example 12. This embodiment uses the same method as Example 9. The results are shown in Tables 41-44.

[0483] Table 41 shows the uniformity results of NNN.

[0484]

[0485] Note: In the table, The same applies below.

[0486] Table 42 shows the uniformity results of NNK.

[0487]

[0488]

[0489] Table 43 shows the uniformity results of NAT.

[0490]

[0491] Table 44 shows the uniformity results of NAB.

[0492]

[0493] It can be seen that the components of the "mixed solution standard substance in methanol" after being dispensed have good homogeneity.

[0494] Example 14: Stability Test

[0495] The mixed sample used in this embodiment is the sample prepared in Example 12. This embodiment uses the same method as in Example 10. The short-term stability diagrams of the "mixed solution standard substance" sample are shown in Figures 9(AC) to 12(AC), where A represents 4°C, B represents 25°C, and C represents 50°C.

[0496] Conclusion: The short-term stability test results show that the NAB concentration in the "mixed solution standard of four N-nitrosamines in methanol" did not change after short-term storage at 4℃, 25℃ and 50℃, thus the sample has good stability.

[0497] The long-term stability plots of the “mixed solution standard material” samples are similar to those in Figures 9(AC) to 12(AC).

[0498] Conclusion: The results of the long-term stability test show that the t-value of NAB in the "mixed solution standard of four N-nitrosamines in methanol" is less than the critical value, which indicates that the NAB sample in the "mixed solution standard of four N-nitrosamines in methanol" has good long-term stability.

[0499] The test results above show that the components of the "mixed solution standard substance in methanol" are well homogeneous after repackaging.

[0500] Example 15: Uncertainty Assessment of Solution Standards

[0501] The solution standard used in this embodiment was prepared as in Example 12. The experimental procedure was as described in Example 11. The relative standard uncertainty components, the synthesis, and the expanded uncertainty of each sample are shown in Tables 45-46.

[0502] Table 45 Relative Standard Uncertainty Components and Combination

[0503]

[0504] 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 calculations are shown in Table 46 below.

[0505] Table 46 Expanded Uncertainty for Each Sample

[0506]

Claims

1. A mixed solution standard of N-nitrosamine, which is composed of N-nitrosonornicotinine (NNN) purity standard, 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) purity standard, N-nitrosoneonicotinine (NAT) purity standard, N-nitrosopseudoestipine (NAB) purity standard and methanol.

2. The solution standard substance according to claim 1, wherein, In the mixed solution standard material, the concentration standard values ​​of N-nitrosonornicotinine (NNN), 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoneonicotinine (NAT), and N-nitrosopseudoestipine (NAB) are 0.050, 0.050, 0.050, and 0.010 mg / mL, respectively, with relative expanded uncertainties of 3%, 3%, 3%, and 4% (k=2), respectively. Preferably, in the mixed solution standard material, the concentration standard values ​​of N-nitrosonornicotinine (NNN), 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoneonicotinine (NAT), and N-nitrosopseudoestipine (NAB) are 0.010, 0.010, 0.010, and 0.005 mg / mL, respectively, with relative expanded uncertainties of 4%, 4%, 4%, and 5% (k=2), respectively. Preferably, the minimum packaging unit of the solution standard is 1.5 mL, and the packaging material is a 2 mL brown bottle, preferably a brown ampoule bottle.

3. The solution standard substance according to claim 1 or 2, wherein, The purities of N-nitrosonornicotinine (NNN), 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoneonicotinine (NAT), and N-nitrosophagusine (NAB) are 99.35%, 99.38%, 99.45%, and 99.18%, respectively.

4. The solution standard substance according to any one of claims 1 to 3, 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, solvent residues, xylene ketones 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 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.

5. The solution standard substance according to any one of claims 1 to 4, wherein, Based on purity uncertainty assessment, the relative standard uncertainties introduced by the purity of N-nitrosonornicotinine (NNN), 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoneonicotinine (NAT), and N-nitrosophagusine (NAB) are 0.24%, 0.67%, 0.25%, and 0.30%, respectively.

6. The solution standard substance according to any one of claims 1 to 5, wherein, The total purities of the N-nitrosonornicotinine (NNN), 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoneonicotinine (NAT), and N-nitrosopseudoesequine (NAB) purity standards were 99.76%, 99.67%, 99.78%, and 99.51%, respectively. Preferably, the anions in the purity standards of N-nitrosonornicotinine (NNN), 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoneonicotinine (NAT), and N-nitrosopseudoestipine (NAB) are 0.077%, 0.068%, 0.079%, and 0.012%, respectively.

7. The solution standard substance according to any one of claims 1 to 6, wherein, The inorganic elements in the purity standards of N-nitrosonicotinic acid (NNN), 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonicotinic acid (NAT), and N-nitrosophagusine (NAB) are 0.18%, 0.16%, 0.11%, and 0.11%, respectively. Preferably, the residual solvents in the N-nitrosonornicotinine (NNN), 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoneonicotinine (NAT), and N-nitrosopseudoestiline (NAB) purity standard substances are 0.09%, 0%, 0.05%, and 0.06%, respectively.

8. The solution standard substance according to any one of claims 1 to 7, wherein, The benzophenone content in the purity standards of N-nitrosonicotinic acid (NNN), 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonicotinic acid (NAT), and N-nitrosopseudoestiline (NAB) is 0.008%, 0%, 0.025%, and 0.005%, respectively. Preferably, the water content in the N-nitrosonornicotinine (NNN), 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoneonicotinine (NAT), and N-nitrosopseudoestiline (NAB) purity standard substances is 0.055%, 0.063%, 0.062%, and 0.037%, respectively.

9. A method for preparing a solution standard substance according to any one of claims 1 to 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-nitrosonornicotinine (NNN) purity standard, 4-(N-methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) purity standard, N-nitrosoneonicotinine (NAT) purity standard, and N-nitrosopseudoestiline (NAB) purity standard 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.

10. The solution standard substance according to claim 9, wherein, 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.

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