A method for extracting, separating and detecting N-nitrosamines

Abstract

The application relates to the technical field of chemical analysis, and discloses a method for extracting, separating and detecting N-nitrosamine, which comprises the following steps: a sample obtaining step; a sample purification step; a preparation and extraction solution step; an extraction solution separation step; an extraction solution concentration step; a chromatographic detection and quantitative analysis step; the sample to be detected is crushed to a size of 0.01g of powder, and the powder is mixed with sodium chloride, barium hydroxide and water. In the application, the sample to be detected is subjected to a step-by-step sample treatment process before detection, so that the interference factors of the sample to be detected can be effectively reduced, the influence on the detection instrument is reduced, the detection result and detection data are more accurate, and when the application is detected, a large amount of sample does not need to be weighed like in the traditional detection mode, the overall operation is more convenient, and the detection demand of N-nitrosamine in smoked food is met.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chemical analysis, in particular to a method for extracting, separating and detecting N-nitrosamine. BACKGROUND

[0002] N-nitrosamine compounds are strong carcinogens, one of the important chemical carcinogens, and one of the four major food contaminants. Food, cosmetics, beer and cigarettes all contain nitrosamines, especially in smoked bacon, which contains a large amount of nitrosamine compounds. Smoked bacon has been consumed in China since ancient times, and the consumption is huge, which can easily lead to the fact that people consume more nitrosamines while eating smoked bacon during ordinary times, thereby causing the incidence of some digestive system tumors such as esophageal cancer and the problem of dietary nitrosamine intake.

[0003] At present, due to the lack of oxygen, heat accumulation, microorganisms, mold and other factors during the transportation, processing and storage of smoked bacon, various N-nitrosamine compounds are easily produced. With the increasing strictness of food safety, more and more attention is paid to the detection of N-nitrosamine compounds. According to the GB5009.26-2016 "National Food Safety Standard Determination of N-nitrosamine Compounds in Food" published by China, a water vapor distillation method is used to separate and extract N-nitrosamine compounds, and then large-volume dichloromethane is used for liquid-liquid extraction to extract and purify N-nitrosamine compounds. However, the above pretreatment method still has many shortcomings, and a large amount of sample needs to be weighed. However, when the sample weight is too large, there are many interference factors in smoked bacon itself, such as fat, protein, pigment and trace elements. After preliminary treatment, there are still a certain amount of interfering substances, which affect the instrument analysis and detection results, and easily lead to the problem of low preparedness of the final detection results and detection data. Therefore, a method for extracting, separating and detecting N-nitrosamine is needed to solve the above problems. SUMMARY

[0004] (I) Technical problems to be solved

[0005] In view of the deficiencies in the prior art, the present application provides a method for extracting, separating and detecting N-nitrosamine, which has the advantages of less sampling, reducing the interference of the tested substances, improving the accuracy of the data, and solving the problem that the detection of N-nitrosamine compounds in smoked bacon is easily interfered and the accuracy of the detection data is not high.

[0006] (II) Technical solutions

[0007] To achieve the above purpose, the present application provides the following technical solutions: a method for extracting, separating and detecting N-nitrosamine, comprising the following steps:

[0008] S1, a sample acquisition process, 20g of a sample is taken from a sample to be measured, and the sample is crushed and mixed with sodium chloride, barium hydroxide and water to obtain a sample raw liquid;

[0009] S2, a sample purification process, the sample raw liquid obtained in S1 is purified by a water vapor distillation method to obtain a distillation liquid, and the distillation liquid is purified by GPC, wherein the GPC chromatographic conditions are: gel permeation chromatography column: 32 cm x 2.5 cm; mobile phase: dichloromethane; flow rate: 5.0 mL / min; sample size: 5.0 mL; UV detection wavelength: 254 nm; discard the first 16 min of chromatographic effluent, collect 30 min fractions, and elute for 3 min;

[0010] S3, a preparation of an extraction solution process, 40ml of dichloromethane or trichloromethane is added to the solution collected in S2, and the mixture is mixed to obtain a mixed solution, which is separated by ultrasonic extraction for 10-30min. The non-aqueous solution after filtration is the extraction solution, and the non-aqueous solution is reconstituted to 40ml with dichloromethane or trichloromethane;

[0011] S4, an extraction solution separation process, the remaining aqueous solution in S2 is repeatedly added with non-aqueous solvent in step S3 to separate the non-aqueous solution, and all the extraction solutions are combined to obtain an N-nitrosamine extraction solution;

[0012] S5, an extraction solution concentration process, the N-nitrosamine extraction solution obtained in S4 is rotary evaporated and nitrogen blown, and after most of the solvent is removed, it is concentrated to obtain an N-nitrosamine concentrated solution to be loaded onto a machine. The N-nitrosamine concentrated solution includes N-nitrosodi-methylamine, N-nitrosodi-ethylamine, N-nitrosomethyl-ethylamine, N-nitrosodi-propylamine, N-nitrosodi-butylamine, N-nitrosopyrrole, N-nitrosopiperidine, N-nitrosomorpholine, N-nitrosodiphenylamine, N-nitrosobenzylamine, N-nitrosodi-isopropylamine and N-nitrosodi-ethyl-isopropylamine;

[0013] S6, a chromatographic detection and quantitative analysis process, the N-nitrosamine concentrated solution is subjected to gas chromatography detection and mass spectrometry detection, and the parameters of the gas chromatography detection include:

[0014] The chromatographic column is a strong polar quartz capillary column;

[0015] The injection port temperature is 230℃;

[0016] The carrier gas is helium with a flow rate of 1mL / min;

[0017] The injection mode is splitless injection;

[0018] The sample size is 1uL;

[0019] The temperature rising procedure is: initial temperature 40℃, holding for 3 minutes, rising to 170℃ at 8℃ / min, then rising to 230℃ at 20-30℃ / min, holding for 3 minutes;

[0020] The parameters of the mass spectrum detection include:

[0021] The interface temperature of the chromatography and the mass spectrum is 230℃;

[0022] The ionization mode is electron impact source;

[0023] The ionization energy is 70eV;

[0024] The solvent delay is 9.5 minutes;

[0025] The ion source temperature is 230℃;

[0026] The quadrupole rod temperature is 150℃.

[0027] Preferably, the sample to be detected is crushed to a size of 0.01g powder, and the powder is mixed with sodium chloride, barium hydroxide and water, wherein the content of each component is 40ml of sodium chloride, 10ml of barium hydroxide and 20ml of water.

[0028] Preferably, the sealing test is required before the water vapor distillation method, and the N-nitrosamine extraction solution is used for the equipment sealing test. 100ml of distilled water is measured instead of 100g of sample into a 1L ground mouth sample storage flask, and the detection limit level N-nitrosamine extraction solution is added for the distillation equipment sealing test. The recovery rate is more than 70%, and the sealing property can meet the test requirements.

[0029] Preferably, during the water vapor distillation method, the temperature of the cooling water is ≤15℃, which can make the distillation liquid condense as soon as possible, avoid loss, and be beneficial to improve the recovery rate of distillation.

[0030] Preferably, in S5, the N-nitrosamine extraction solution is rotary evaporated, concentrated to 5-10ml on a 40℃ water bath, then changed to nitrogen blowing, and accurately measured to 1.0ml. After shaking, the N-nitrosamine concentrated solution to be detected is obtained, which can be divided into different concentration samples.

[0031] Preferably, in S6, the quantitative analysis is qualitative analysis by comparing the retention time and corresponding compound sub-ion abundance ratio of the N-nitrosamine concentrated solution to be detected in S5. By comparing and analyzing different concentrations of N-nitrosamine solution, the corresponding standard curve is obtained, and the corresponding N-nitrosamine concentration in the sample injection liquid and the N-nitrosamine concentration in the blank control are obtained, so as to obtain the content of N-nitrosamine in the sample to be detected.

[0032] (Three) beneficial effects

[0033] Compared with the prior art, the application provides a kind of N-nitrosamine extraction separation detection method, with the following beneficial effects:

[0034] The N-nitrosamine extraction separation detection method can effectively reduce the interference factors of the sample to be tested by performing the sample processing procedure step by step before detection, reduce the influence on the detection instrument, so that the detection results and detection data are more accurate, and the detection instrument is not required to take a large amount of sample as in the traditional detection method, the overall operation is more convenient, and the detection demand of N-nitrosamine in smoked food is met. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the application will be described below in conjunction with the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, not all. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the application.

[0036] Embodiment one:

[0037] A kind of N-nitrosamine extraction separation detection method, comprising the following steps:

[0038] S1, sample acquisition procedure, 20g sample is taken from the sample to be tested, and is crushed, wherein the sample to be tested is crushed to 0.01g size powder, and the powder is mixed with sodium chloride, barium hydroxide and water to obtain sample raw material liquid, wherein the component content is as follows: 40ml sodium chloride, 10ml barium hydroxide and 20ml water (water is preferably deionized water);

[0039] S2, sample purification and cleaning process, the sample raw material liquid obtained in S1 is purified by water vapor distillation method to obtain a distillation liquid. Before starting the water vapor distillation method, a sealing test needs to be performed. An N-nitrosamine extraction liquid is used to test the sealing of the equipment. 100 ml of distilled water is measured instead of 100 g of sample into a 1 L ground mouth sample storage flask. The detection limit level N-nitrosamine extraction liquid is added, and the distillation equipment sealing test is performed. The recovery rate is more than 70%, and the sealing can meet the test requirements. During the water vapor distillation process, the temperature of the cooling water is ≤15℃, which can make the distillation liquid condense as soon as possible, avoid loss, and be beneficial to improve the recovery rate of distillation. The distillation liquid is then subjected to GPC purification treatment. The GPC model used in the GPC purification treatment is Freestyle (including full online solid phase extraction SPE / gel purification GPC / quantitative concentration EVA). The GPC purification treatment can remove interfering substances in environmental samples, food and animal tissue samples before GC, GC / MS, HPLC or LC / MS analysis. GPC purification can selectively remove large molecular weight interfering substances such as lipids, pigments, proteins, humic acid, plasticizers, while ensuring the integrity of the sample. The presence of these interfering substances can shorten the service life of the analysis column and prolong the maintenance time of the analysis equipment. GPC is a commonly used and convenient sample post-treatment and purification technology for analyzing pesticide residues, PCBs, plasticizers, endocrine disruptors, semi-volatile organic compounds, pharmaceuticals, illegal dyes and other substances. Moreover, the Gilson GPC purification system meets the standards of various governments and independent institutions such as USEPA, USFDA, USGS, AOAC, International, AOCS, EN1528, EN12393, DFG S19, CFIA. The GPC chromatographic conditions are as follows:

[0040] Gel permeation chromatography column: 32 cm x 2.5 cm (gel packing Bio Beads S-X3, 200-400 mesh);

[0041] Mobile phase: dichloromethane;

[0042] Flow rate: 5.0 mL / min;

[0043] Injection volume: 5.0 mL;

[0044] UV detection wavelength: 254 nm;

[0045] Discard the first 16 min of chromatographic effluent, collect the 17-47 min fraction, and elute for 3 min;

[0046] S3, a preparation of extraction solution process, 40 ml of dichloromethane or chloroform is added to the solution collected in S2, and mixed to obtain a mixed solution, and separated by ultrasonic extraction for 10-30 min, and the non-aqueous solution after filtration is the extraction solution, and the non-aqueous solution is redissolved in 40 ml of dichloromethane or chloroform;

[0047] S4, an extraction solution separation process, the remaining aqueous solution in S2 is repeatedly subjected to the non-aqueous solvent addition and non-aqueous solution separation in step S3, wherein an acid-modified activated carbon column is used to effectively adsorb N-dimethyl nitrosamine and N-diethyl nitrosamine in the distillate, then water is used to elute to remove part of the water-soluble compounds, such as small-molecule amine compounds; then n-hexane is used to elute the acid-modified coconut activated carbon column to remove part of the fat-soluble impurities; then dichloromethane is used to elute N-dimethyl nitrosamine and N-diethyl nitrosamine, and due to the strong adsorption of the acid-modified coconut activated carbon column to impurities, the impurities are not easily eluted by dichloromethane, so that the interfering impurities and N-dimethyl nitrosamine and N-diethyl nitrosamine are effectively separated, and all the extraction solutions are combined to obtain an N-nitrosamine extraction solution;

[0048] S5, an extraction solution concentration process, the N-nitrosamine extraction solution obtained in S4 is subjected to rotary evaporation and nitrogen blowing, most of the solvent is removed, and then concentrated to obtain an N-nitrosamine concentrated solution sample for machine analysis, wherein the N-nitrosamine concentrated solution includes N-nitrosodi-methylamine, N-nitrosodi-ethylamine, N-nitrosomethyl-ethylamine, N-nitrosodi-propylamine, N-nitrosodi-butylamine, N-nitrosopyrrole, N-nitrosopiperidine, N-nitrosomorpholine, N-nitrosodiphenylamine, N-nitrosobenzylamine, N-nitrosodi-isopropylamine, and N-nitrosoethyl-isopropylamine, and the rotary evaporation and nitrogen blowing are rotary evaporation of the N-nitrosamine extraction solution, concentrated to 5-10 ml on a 40℃ water bath, and then nitrogen blowing, the temperature during nitrogen blowing is controlled to ≤25℃, which can effectively avoid the loss of N-nitrosamine compounds during nitrogen blowing concentration, improve the recovery rate, and accurately dissolve to 1.0 ml, and then shake to obtain the N-nitrosamine concentrated solution for machine analysis, which can be divided into samples of different concentrations, and the same standard concentration solutions of N-nitrosodi-methylamine, N-nitrosodi-ethylamine, N-nitrosomethyl-ethylamine, N-nitrosodi-propylamine, N-nitrosodi-butylamine, N-nitrosopyrrole, N-nitrosopiperidine, N-nitrosomorpholine, N-nitrosodiphenylamine, N-nitrosobenzylamine, N-nitrosodi-isopropylamine, and N-nitrosoethyl-isopropylamine are diluted step by step to obtain a series of standard solutions of 5ug / L, 10ug / L, 50ug / L, 100ug / L, 200ug / L, 500ug / L (the solvent is 10% acetonitrile aqueous solution);

[0049] S6, chromatographic detection and quantitative analysis process, the N-nitrosamine concentrated liquid sample is subjected to gas chromatographic detection and mass spectrometric detection, so as to obtain the chromatographic information of N-nitrosamine compounds in the sample to be detected, the parameters of the gas chromatographic detection include:

[0050] The chromatographic column is a strong polarity quartz capillary column, and the strong polarity quartz capillary column is preferably an HP-INNOWAX column, and the size of the HP-INNOWAX column is preferably 35mx0.35mm (inner diameter) x0.35um (film thickness);

[0051] The injection port temperature is 230℃;

[0052] The carrier gas is helium, and the flow rate is 1mLmin, and the purity of the helium is preferably ≥99.999%;

[0053] The injection mode is splitless injection;

[0054] The injection amount is 1uL;

[0055] The temperature rising program is that the initial temperature is 40℃, which is kept for 3min, then increased to 170℃ at a rate of 8℃ / min, then increased to 230℃ at a rate of 20-30℃ / min, and kept for 3min;

[0056] The parameters of the mass spectrometric detection include:

[0057] The chromatographic and mass spectrometric interface temperature is 230℃;

[0058] The ionization mode is electron impact source;

[0059] The ionization energy is 70eV;

[0060] The solvent delay is 9.5min;

[0061] The ion source temperature is 230℃;

[0062] The quadrupole rod temperature is 150℃;

[0063] And quantitative analysis is through comparing the retention time and corresponding compound sub-ion abundance ratio of the N-nitrosamine concentrated liquid in S5 to be detected by machine to carry out qualitative analysis, through comparative analysis of different concentrations of N-nitrosodi-methylamine, N-nitrosodi-ethylamine, N-nitrosomethyl-ethylamine, N-nitrosodi-propylamine, N-nitrosodi-butylamine, N-nitrosopyrrole, N-nitrosopiperidine, N-nitrosomorpholine, N-nitrosodiphenylamine, N-nitrosobenzylamine, N-nitrosodi-isopropylamine and N-nitrosodi-ethyl-isopropylamine diluted into 5ug / L, 10ug / L, 50ug / L, 100ug / L, 200ug / L, 500ug / L series standard solution (solvent is 10% acetonitrile aqueous solution), corresponding standard curve is obtained, the corresponding N-nitrosamine compound concentration in the sample injection liquid is obtained by using the chromatographic conditions and mass spectrometry parameters provided in the embodiment, so as to obtain the accurate content of N-nitrosamine compound in the sample to be detected.

[0064] The beneficial effects of the present application are: the present application can effectively reduce the interference factors of the sample to be detected by carrying out the sample processing procedure step by step before detection, reduce the influence on the detection instrument, so that the detection results and detection data are more accurate, and the present application does not need to take a large amount of sample as in the traditional detection method, the overall operation is more convenient, and meets the detection requirements of N-nitrosamine in smoked food.

[0065] Although the embodiments of the present application have been shown and described, it can be understood by those skilled in the art that various changes, modifications, replacements and variations can be made to the embodiments without departing from the principles and spirits of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for extracting, separating and detecting an N-nitrosamine, characterized by, The method comprises the following steps: S1, a sample acquisition process, 20g of a sample is taken from a sample to be tested, and the sample is crushed and treated, and the crushed sample is mixed with sodium chloride, barium hydroxide and water to obtain a sample raw material solution; S2, a sample purification process, the sample raw material solution obtained in S1 is purified by a water vapor distillation method to obtain a distillation solution, and the distillation solution is purified by GPC, wherein the GPC chromatographic conditions are as follows: a gel permeation chromatographic column: 32cm*2.5cm; a mobile phase: dichloromethane; a flow rate: 5.0mL / min; a sample injection amount: 5.0mL; an ultraviolet detection wavelength: 254nm; discarding the first 16min of chromatographic effluent, collecting 30min of flow fraction, and eluting for 3min; S3, a preparation of an extraction solution process, 40ml of dichloromethane or trichloromethane is added to the solution collected in S2, and the solution is mixed to obtain a mixed solution, and the mixed solution is separated by ultrasonic extraction for 10-30min, and the non-aqueous solution after filtration is an extraction solution, and the extraction solution is reconstituted to 40ml with dichloromethane or trichloromethane; S4, an extraction solution separation process, the aqueous solution remaining in S2 is repeatedly subjected to the non-aqueous solvent addition and non-aqueous solution separation in step S3, and all the extraction solutions are combined to obtain an N-nitrosamine extraction solution; S5, an extraction solution concentration process, the N-nitrosamine extraction solution obtained in S4 is subjected to rotary evaporation and nitrogen blowing, most of the solvent is removed, and then concentrated to obtain an N-nitrosamine concentrated solution sample, and the N-nitrosamine concentrated solution comprises N-nitrosodi-methylamine, N-nitrosodi-ethylamine, N-nitrosomethyl-ethylamine, N-nitrosodi-propylamine, N-nitrosodi-butylamine, N-nitrosopyrrole, N-nitrosopiperidine, N-nitrosomorpholine, N-nitrosodiphenylamine, N-nitrosobenzylideneamine, N-nitrosodi-isopropylamine and N-nitrosodi-ethyl-isopropylamine; S6, a chromatographic detection and quantitative analysis process, the N-nitrosamine concentrated solution sample is subjected to gas chromatography detection and mass spectrometry detection, and the parameters of the gas chromatography detection include: a strong polarity quartz capillary column is used as a chromatographic column; the temperature of a sample inlet is 230℃; helium is used as a carrier gas, and the flow rate is 1mL / min; the sample is injected in a splitless manner; the sample injection amount is 1uL; the temperature rising program is as follows: the initial temperature is 40℃, maintained for 3min, then increased to 170℃ at a rate of 8℃ / min, then increased to 230℃ at a rate of 20-30℃ / min, and maintained for 3min; the parameters of the mass spectrometry detection include: the temperature of a chromatographic and mass spectrometry interface is 230℃; an electron impact source is used as an ionization mode; the ionization energy is 70eV; the solvent delay is 9.5min; the ion source temperature is 230℃; the quadrupole rod temperature is 150℃.

2. The method according to claim 1, wherein The sample to be tested is crushed to a size of 0.01g of powder, and the powder is mixed with sodium chloride, barium hydroxide and water, wherein the content of each component is 40ml of sodium chloride, 10ml of barium hydroxide and 20ml of water.

3. The method according to claim 1, wherein the method is characterized by, The sealing test needs to be carried out before the start of the water vapor distillation method, the N-nitrosamine extraction solution is used for equipment sealing test, 100ml distilled water is measured instead of 100g sample into 1L ground mouth sample storage flask, the detection limit level N-nitrosamine extraction solution is added, the distillation equipment sealing test is carried out, and the recovery rate is more than 70%, so that the sealing can meet the test requirements.

4. The method according to claim 1, wherein the method is characterized by, In the process of the water vapor distillation method, the temperature of the cooling water is ≤15℃, which can make the distillation liquid condense as soon as possible, avoid loss, and be beneficial to improve the recovery rate of distillation.

5. The method according to claim 1, wherein the method is characterized by, In S5, rotary evaporation and nitrogen blowing are used to concentrate the N-nitrosamine extraction solution to 5-10ml on a 40℃ water bath, then nitrogen blowing is used, and accurate volume is 1.0ml, then shake well to obtain the N-nitrosamine concentrated solution for machine, and the N-nitrosamine concentrated solution for machine is divided into different concentration samples.

6. The method for extraction, separation, and detection of N-nitrosamines according to claim 1, characterized in that, In S6, quantitative analysis is carried out by comparing the retention time and corresponding compound sub-ion abundance ratio of the N-nitrosamine concentrated solution for machine detection in S5, comparing and analyzing different concentrations of N-nitrosamine solution, obtaining the corresponding standard curve, obtaining the corresponding N-nitrosamine concentration in the sample injection liquid of the sample to be measured and the N-nitrosamine concentration in the blank control, so as to obtain the content of N-nitrosamine in the sample to be measured.