Method for extracting, separating and detecting N-nitrosamine

Abstract

The invention relates to the technical field of chemical analysis, and discloses a method for extracting, separating and detecting N-nitrosamine, which comprises the following steps: obtaining a sample to be detected; a sample purification process; preparing an extraction solution; an extraction solution separation process; concentrating the extracted solution; performing chromatographic detection and quantitative analysis; the method comprises the following steps: crushing a to-be-detected sample, crushing the to-be-detected sample into 0.01 g of powder, and mixing the powder with sodium chloride, barium hydroxide and water. According to the invention, by carrying out step-by-step sample treatment procedures on the to-be-detected sample before the detection work, the interference factors of the to-be-detected sample can be effectively reduced, the influence on a detection instrument is reduced, so that the obtained detection result and detection data are more accurate, and in addition, when the method is used for detecting, the detection accuracy is improved. The method does not need to weigh a large number of samples like a traditional detection mode, the whole operation is more convenient, and the detection requirement of the N-nitrosamine in the smoked and cured food is met.

Description

Technical Field

[0001] The invention relates to the technical field of chemical analysis, in particular to a method for extracting, separating and detecting N-nitrosamines. Background Art

[0002] N-nitrosamine compounds are strong carcinogens, one of the important chemical carcinogens, and one of the four major food contaminants. Nitrosamines are contained in food, cosmetics, beer, and cigarettes, especially in smoked and cured foods, which contain large amounts of nitrosamines. In my country, smoked and cured foods have been around since ancient times and are consumed in huge quantities. This can easily lead to people consuming higher levels of nitrosamines while eating smoked and cured foods in normal times, thus causing the incidence of certain digestive system tumors such as esophageal cancer and the problem of dietary intake of nitrosamines.

[0003] At present, smoked and cured foods are prone to produce various N-nitrosamine compounds due to factors such as lack of oxygen, heat accumulation, microorganisms, and mildew during transportation, processing, and storage. As food safety is becoming more and more stringent, it is also receiving more and more attention. The traditional N-nitrosamine compound detection method discloses the use of steam distillation to separate and extract N-nitrosamine compounds according to my country's GB5009.26-2016 "Determination of N-nitrosamine Compounds in Foods", and then a large volume of dichloromethane is used for multiple liquid-liquid extraction to extract and purify N-nitrosamine compounds; however, the above-mentioned pretreatment method still has many disadvantages, and often requires weighing a large amount of samples. However, when the sample weighing amount is too large, there are many interfering factors in the smoked and cured foods themselves, such as fat, protein, pigments, and trace elements. After preliminary treatment, it still contains a certain amount of interfering substances, which affects the instrumental analysis test results, and easily leads to the problem of low readiness of the final test results and test data. Therefore, it is necessary to provide an extraction, separation, and detection method for N-nitrosamines to solve the above problems. Summary of the invention

[0004] 1. Technical issues to be solved

[0005] In view of the shortcomings of the prior art, the present invention provides a method for extracting, separating and detecting N-nitrosamines, which has the advantages of less sampling, reducing interfering substances to be detected, and improving data accuracy. It solves the problem that the detection of N-nitrosamine compound content in smoked foods is easily interfered with and the detection data is not accurate.

[0006] (II) Technical solution

[0007] To achieve the above object, the present invention provides the following technical solution: a method for extracting, separating and detecting N-nitrosamines, comprising the following steps:

[0008] S1, a step of obtaining a sample to be tested, taking 20 g of the sample from the sample to be tested, crushing it, and mixing the crushed sample to be tested with sodium chloride, barium hydroxide and water to obtain a sample raw material solution;

[0009] S2, sample purification step, the sample raw material liquid obtained in S1 is purified by steam distillation to obtain distillate, and the distillate is purified by GPC, wherein the GPC chromatographic conditions are: gel permeation chromatography column: 32cm×2.5cm; mobile phase: dichloromethane; flow rate: 5.0mL / min; injection volume: 5.0mL; ultraviolet detection wavelength: 254nm; discard the first 16min chromatographic effluent, collect 30min fractions, and rinse for 3min;

[0010] S3, a step of preparing an extraction solution, adding 40 ml of dichloromethane or chloroform to the solution collected in S2, mixing thoroughly to obtain a mixed solution, performing ultrasonic extraction for 10-30 min for separation, and separating and filtering the non-water-soluble solution as the extract, and re-diluting to 40 ml with dichloromethane or chloroform;

[0011] S4, extraction solution separation step, repeating step S3 of adding non-aqueous solvent to separate the remaining aqueous solution in S2, and combining all the extracts to obtain an N-nitrosamine extract;

[0012] S5, an extraction solution concentration step, subjecting the N-nitrosamine extract obtained in S4 to rotary evaporation and nitrogen blowing, removing most of the solvent and then concentrating to obtain an N-nitrosamine concentrated solution sample on the machine, wherein the N-nitrosamine concentrated solution includes N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosomethylethylamine, N-nitrosodipropylamine, N-nitrosodibutylamine, N-nitrosopyrrole, N-nitrosopiperidine, N-nitrosomorpholine, N-nitrosodiphenylamine, N-nitrosodibenzylamine, N-nitrosodiisopropylamine and N-nitrosoethylisopropylamine;

[0013] S6, chromatographic detection and quantitative analysis step, performing gas chromatography detection and mass spectrometry detection on the N-nitrosamine concentrated solution sample, the gas chromatography detection parameters include:

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

[0015] The injection port temperature was 230 °C;

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

[0017] The injection mode is splitless injection;

[0018] The injection volume is 1uL;

[0019] The heating program is as follows: initial temperature 40°C, hold for 3 min at 8°C / min to 170°C, then heat to 230°C at 20-30°C / min, hold for 3 min;

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

[0021] The temperature of the chromatographic and mass spectrometric interface was 230°C;

[0022] The ionization method is electron bombardment source;

[0023] The ionization energy is 70 eV;

[0024] Solvent delay 9.5min;

[0025] The ion source temperature was 230 °C;

[0026] The quadrupole temperature was 150°C.

[0027] Preferably, the sample to be tested is pulverized to a powder with a size of 0.01 g, and the powder is mixed with sodium chloride, barium hydroxide and water, wherein the ingredients include 40 ml of sodium chloride, 10 ml of barium hydroxide and 20 ml of water.

[0028] Preferably, a sealing test is required before starting the steam distillation method, and an N-nitrosamine extract is used to perform an equipment sealing test. 100 ml of distilled water is measured to replace 100 g of sample into a 1L ground-mouth sample storage flask, and an N-nitrosamine extract at the detection limit level is added. The pretreatment process refers to 2.1, and a distillation equipment sealing test is performed. The recovery rate is above 70%, and its sealing can meet the test requirements.

[0029] Preferably, during the steam distillation process, the temperature of the cooling water is ≤15° C., which can condense the distillate as quickly as possible to avoid loss, thereby facilitating improvement of the recovery rate of the distillation.

[0030] Preferably, the rotary evaporation and nitrogen blowing in S5 are to rotary evaporate the N-nitrosamine extract, concentrate it to 5-10 ml in a 40° C. water bath, then change to nitrogen blowing, and accurately determine the volume to 1.0 ml, shake well to obtain the N-nitrosamine concentrated solution sample to be loaded on the machine, and the N-nitrosamine concentrated solution sample to be loaded on the machine can be divided into samples of different concentrations.

[0031] Preferably, the quantitative analysis in S6 is a qualitative analysis by comparing the retention time and the abundance ratio of the corresponding compound daughter ions of the results obtained by the N-nitrosamine concentrate detected on the machine in S5, and by comparing and analyzing N-nitrosamine solutions of different concentrations, obtaining the corresponding standard curve, and obtaining the corresponding N-nitrosamine concentration in the sample injection solution to be tested and the N-nitrosamine concentration in the blank control, thereby obtaining the content of N-nitrosamines in the sample to be tested.

[0032] (III) Beneficial effects

[0033] Compared with the prior art, the present invention provides a method for extracting, separating and detecting N-nitrosamines, which has the following beneficial effects:

[0034] The method for extracting, separating and detecting N-nitrosamines can effectively reduce the interference factors of the samples to be tested and the impact on the detection instrument by performing a step-by-step sample processing procedure before the detection work is carried out, so that the test results and test data obtained are more accurate. In addition, when conducting the detection, the invention no longer needs to weigh a large number of samples as in the traditional detection method, and the overall operation is more convenient, which meets the detection needs of N-nitrosamines in smoked foods. DETAILED DESCRIPTION

[0035] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Embodiment 1:

[0037] A method for extracting, separating and detecting N-nitrosamines comprises the following steps:

[0038] S1, a step of obtaining a sample to be tested, taking 20 g of the sample from the sample to be tested and pulverizing it, wherein the sample to be tested is pulverized to a powder of 0.01 g, and the powder is mixed with sodium chloride, barium hydroxide and water to obtain a sample raw material solution, wherein the composition content is as follows: 40 ml of sodium chloride, 10 ml of barium hydroxide and 20 ml of water (the water is preferably deionized water);

[0039] S2, sample purification step, the sample raw material liquid obtained in S1 is purified by steam distillation to obtain distillate. Before starting the steam distillation, it is necessary to perform a sealing test, and use N-nitrosamine extract to perform the equipment sealing test. 100 ml of distilled water is measured to replace 100 g of sample into a 1L ground-mouth sample storage flask, and the detection limit level N-nitrosamine extract is added. The pretreatment process refers to 2.1, and the distillation equipment sealing test is performed. The recovery rate is above 70%, and its sealing can meet the test requirements. During the steam distillation process, the temperature of the cooling water is ≤15°C, which can condense the distillate as soon as possible to avoid loss, which is beneficial to improve the recovery rate of distillation; the distillate is subsequently subjected to GPC purification treatment, and the GPC purification treatment is used The GPC model is Freestyle (including full online solid phase extraction SPE / gel cleanup GPC / quantitative concentration EVA). GPC purification 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 will shorten the life of the analytical column and extend the maintenance time of the analytical equipment; GPC is a common and convenient sample post-processing purification technology for analyzing pesticide residues, PCBs, plasticizers, endocrine disruptors, semi-volatile organic compounds, pharmaceuticals, illegal dyes and other substances; and the Gilson GPC purification system meets the standards of various governments and independent agencies such as USEPA, USFDA, USGS, AOAC, International, AOCS, EN1528, EN12393, DFG S19, CFIA, among which the GPC chromatographic conditions are:

[0040] Gel permeation chromatography column: 32cmx2.5cm (gel filler Bio Beads S-X3, 200-400 mesh);

[0041] Mobile phase: dichloromethane;

[0042] Flow rate: 5.0 mL / min;

[0043] Injection volume: 5.0mL;

[0044] UV detection wavelength: 254nm;

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

[0046] S3, a step of preparing an extraction solution, adding 40 ml of dichloromethane or chloroform to the solution collected in S2, mixing thoroughly to obtain a mixed solution, performing ultrasonic extraction for 10-30 min for separation, and separating and filtering the non-water-soluble solution as the extract, and re-diluting to 40 ml with dichloromethane or chloroform;

[0047] S4, extraction solution separation step, repeating the non-aqueous solvent addition non-aqueous solution separation in step S3 with the remaining aqueous solution of S2, wherein an acid-modified activated carbon column is used to effectively adsorb N-nitrosodimethylamine and N-nitrosodiethylamine in the distillate, and then firstly rinsed with water to remove some water-soluble compounds, such as some small molecular amine compounds; then rinsed the acid-modified coconut shell activated carbon column with n-hexane to remove some fat-soluble impurities: then eluted N-nitrosodimethylamine and N-nitrosodiethylamine with dichloromethane, due to the strong adsorption of the acid-modified coconut shell activated carbon column to impurities, the impurities are not easily eluted by dichloromethane, so that the interfering impurities and N-nitrosodimethylamine and N-nitrosodiethylamine are effectively separated, and all extracts are combined to obtain N-nitrosodiamine extract;

[0048] S5, an extraction solution concentration step, rotary evaporating and nitrogen blowing the N-nitrosamine extract obtained in S4, removing most of the solvent and then concentrating to obtain an N-nitrosamine concentrated solution sample for use on the machine, wherein the N-nitrosamine concentrated solution includes N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosomethylethylamine, N-nitrosodipropylamine, N-nitrosodibutylamine, N-nitrosopyrrole, N-nitrosopiperidine, N-nitrosomorpholine, N-nitrosodiphenylamine, N-nitrosodibenzylamine, N-nitrosodiisopropylamine and N-nitrosoethylisopropylamine, and rotary evaporating and nitrogen blowing are to rotary evaporate the N-nitrosamine extract, concentrate it to 5-10 ml in a 40°C water bath, and then change to nitrogen blowing, and the temperature during nitrogen blowing is controlled at ≤25°C, which can effectively avoid the concentration of N-nitrosamine compounds by nitrogen blowing. The loss during the shrinking process is reduced, the recovery rate is improved, and the volume is accurately determined to 1.0 ml, and after shaking, the N-nitrosamine concentrated solution sample to be put on the machine is obtained. The N-nitrosamine concentrated solution sample to be put on the machine can be divided into samples of different concentrations, and the same standard concentration solutions of N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosomethylethylamine, N-nitrosodipropylamine, N-nitrosodibutylamine, N-nitrosopyrrole, N-nitrosopiperidine, N-nitrosomorpholine, N-nitrosodiphenylamine, N-nitrosodibenzylamine, N-nitrosodiisopropylamine and N-nitrosoethylisopropylamine are diluted step by step to dilute into 5ug / L, 10ug / L, 50ug / L, 100ug / L, 200ug / L, 500ug / L series standard solutions (the solvent is an aqueous solution containing 10% acetonitrile);

[0049] S6, chromatographic detection quantitative analysis step, performing gas chromatography detection and mass spectrometry detection on the N-nitrosamine concentrated solution sample, so as to obtain chromatographic information of N-nitrosamine compounds in the sample to be tested, and the parameters of the gas chromatography detection include:

[0050] The chromatographic column is a highly polar quartz capillary column, and the highly polar 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 was 230 °C;

[0052] The carrier gas is helium with a flow rate of 1 mL / min. The purity of the helium is preferably ≥ 99.999%;

[0053] The injection mode is splitless injection;

[0054] The injection volume is 1uL;

[0055] The heating program is as follows: initial temperature 40°C, hold for 3 min at 8°C / min to 170°C, then heat to 230°C at 20-30°C / min, hold for 3 min;

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

[0057] The temperature of the chromatographic and mass spectrometric interface was 230°C;

[0058] The ionization method is electron bombardment source;

[0059] The ionization energy is 70 eV;

[0060] Solvent delay 9.5min;

[0061] The ion source temperature was 230 °C;

[0062] The quadrupole temperature is 150°C;

[0063] The quantitative analysis was carried out by comparing the retention time and the abundance ratio of the corresponding compound daughter ions of the N-nitrosamine concentrates detected on the S5 machine. The results were compared with those of N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosomethylethylamine, N-nitrosodipropylamine, N-nitrosodibutylamine, N-nitrosopyrrole, N-nitrosopiperidine, N-nitrosomorpholine, N-nitrosodiphenylamine, N-nitrosodibenzylamine, N-nitrosodiisopropylamine and N-nitrosodimethylamine. Ethyl isopropylamine was diluted into a series of standard solutions of 5ug / L, 10ug / L, 50ug / L, 100ug / L, 200ug / L and 500ug / L (the solvent was an aqueous solution containing 10% acetonitrile), and the corresponding standard curve was obtained. The chromatographic conditions and mass spectrometry parameters provided in this embodiment were used for analysis to obtain the corresponding N-nitrosamine compound concentration in the sample injection solution to be tested, thereby obtaining the accurate content of N-nitrosamine compounds in the sample to be tested.

[0064] The beneficial effects of the present invention are as follows: the present invention can effectively reduce the interference factors of the samples to be tested themselves and the impact on the detection instrument by performing a step-by-step sample processing procedure before the detection work is carried out, so that the test results and test data obtained are more accurate. Moreover, when conducting the detection, the present invention no longer needs to weigh a large number of samples as in the traditional detection method, and the overall operation is more convenient, which meets the detection needs of N-nitrosamines in smoked foods.

[0065] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for extracting, separating and detecting N-nitrosamines, characterized in that: The following steps are involved: S1, a step of obtaining a sample to be tested, taking 20 g of the sample from the sample to be tested, crushing it, and mixing the crushed sample to be tested with sodium chloride, barium hydroxide and water to obtain a sample raw material solution; S2, sample purification step, the sample raw material liquid obtained in S1 is purified by steam distillation to obtain distillate, and the distillate is purified by GPC, wherein the GPC chromatographic conditions are: gel permeation chromatography column: 32cm×2.5cm; mobile phase: dichloromethane; flow rate: 5.0mL / min; injection volume: 5.0mL; ultraviolet detection wavelength: 254nm; discard the first 16min chromatographic effluent, collect 30min fractions, and rinse for 3min; S3, a step of preparing an extraction solution, adding 40 ml of dichloromethane or chloroform to the solution collected in S2, mixing thoroughly to obtain a mixed solution, performing ultrasonic extraction for 10-30 min for separation, and separating and filtering the non-water-soluble solution as the extract, and re-diluting to 40 ml with dichloromethane or chloroform; S4, extraction solution separation step, repeating step S3 of adding non-aqueous solvent to separate the remaining aqueous solution in S2, and combining all the extracts to obtain an N-nitrosamine extract; S5, an extraction solution concentration step, subjecting the N-nitrosamine extract obtained in S4 to rotary evaporation and nitrogen blowing, removing most of the solvent and then concentrating to obtain an N-nitrosamine concentrated solution sample on the machine, wherein the N-nitrosamine concentrated solution includes N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosomethylethylamine, N-nitrosodipropylamine, N-nitrosodibutylamine, N-nitrosopyrrole, N-nitrosopiperidine, N-nitrosomorpholine, N-nitrosodiphenylamine, N-nitrosodibenzylamine, N-nitrosodiisopropylamine and N-nitrosoethylisopropylamine; S6, chromatographic detection and quantitative analysis step, performing gas chromatography detection and mass spectrometry detection on the N-nitrosamine concentrated solution sample, the gas chromatography detection parameters include: The chromatographic column is a highly polar quartz capillary column; The injection port temperature was 230 °C; The carrier gas was helium with a flow rate of 1 mL min ; The injection mode is splitless injection; The injection volume is 1uL; The heating program is as follows: initial temperature 40°C, hold for 3 min at 8°C / min to 170°C, then heat to 230°C at 20-30°C / min, hold for 3 min; The parameters of the mass spectrometry detection include: The temperature of the chromatographic and mass spectrometric interface was 230°C; The ionization method is electron bombardment source; The ionization energy is 70 eV; Solvent delay 9.5min; The ion source temperature was 230 °C; The quadrupole temperature was 150°C.

2. The method for extracting, separating and detecting N-nitrosamines according to claim 1, characterized in that: The sample to be tested is pulverized into powder with an accuracy of 0.01 g, and the powder is mixed with sodium chloride, barium hydroxide and water, wherein the components include 40 ml of sodium chloride, 10 ml of barium hydroxide and 20 ml of water.

3. The method for extracting, separating and detecting N-nitrosamines according to claim 1, characterized in that: Before starting the steam distillation method, a sealing test is required. The equipment sealing test is carried out using N-nitrosamine extract. 100 ml of distilled water is measured to replace 100 g of sample into a 1L ground-mouth sample storage flask, and the detection limit level of N-nitrosamine extract is added. The pretreatment process refers to 2.1, and the distillation equipment sealing test is carried out. The recovery rate is above 70%, and its sealing can meet the test requirements.

4. The method for extracting, separating and detecting N-nitrosamines according to claim 1, characterized in that: During the steam distillation process, the temperature of the cooling water is ≤15° C., which can condense the distillate as quickly as possible to avoid loss, thereby facilitating an increase in the recovery rate of the distillation.

5. The method for extracting, separating and detecting N-nitrosamines according to claim 1, characterized in that: The rotary evaporation and nitrogen blowing in S5 are to perform rotary evaporation on the N-nitrosamine extract, concentrate it to 5-10 ml in a 40° C. water bath, then change to nitrogen blowing, and accurately determine the volume to 1.0 ml, shake well to obtain the N-nitrosamine concentrated solution sample to be loaded on the machine, and the N-nitrosamine concentrated solution sample to be loaded on the machine can be divided into samples of different concentrations.

6. The method for extracting, separating and detecting N-nitrosamines according to claim 1, characterized in that: The quantitative analysis in S6 is to perform qualitative analysis by comparing the retention time and the abundance ratio of the corresponding compound daughter ions of the N-nitrosamine concentrate obtained by the machine detection in S5, and to obtain the corresponding standard curve by comparing and analyzing N-nitrosamine solutions of different concentrations, and to obtain the corresponding N-nitrosamine concentration in the sample injection solution to be tested and the N-nitrosamine concentration in the blank control, thereby obtaining the content of N-nitrosamines in the sample to be tested.