Method for detecting nitrosamine with high sensitivity

Abstract

A novel micro-extraction method is provided, and nitrite in food is extracted and measured by using a spectrophotometer. Three switchable hydrophilic solvents are prepared by mixing N, N-dimethyl cyclohexylamine, triethylamine and N, N-dimethyl benzylamine, and the molar ratio of the hydrophilic solvents to water is 1: 1 as an extraction solvent of nitrite. The protonation of the process is accomplished by dissolving it in water using dry ice, adding NaOH to convert it back to its original non-ionic state for phase separation and subsequent nitrite extraction. Ultrasonic waves are applied to improve the extraction capacity of nitrite in a sample. And various analysis parameters are optimized. The innovativeness of the method provided by the invention proves the availability of the method in the aspects of selective determination and nitrite extraction through multivariate statistical analysis. Furthermore, the method does not require heating and centrifugation steps, which is advantageous in cost and time compared to other similar methods.

Description

Technical Field

[0001] The present invention relates to a highly sensitive detection method for nitrosamines, in particular to a detection method for nitrite, a highly carcinogenic substance, in pickled meat foods. Background Art

[0002] Nitrite (NO2-) is a compound widely used in food production, as a preservative, fertilizer, and decomposition inhibitor in the environment. However, it has been studied that inhalation of low concentrations of nitrite can cause severe poisoning, and long-term ingestion can cause cancer. However, high concentrations of nitrite accelerate the conversion of regular hemoglobin to methemoglobin, which may cause hemoglobin to lose its oxygen-carrying capacity. In addition, nitrite may react with amides and amines to become carcinogenic N-nitrosamines, which may cause gastric cancer. Nitrite may damage cells and aid acidification and hydrolysis, and is toxic to many microorganisms. Therefore, it is of great significance to develop practical and effective analytical techniques to determine nitrite in food samples.

[0003] The World Health Organization (WHO) guideline for nitrite in drinking water is 3.0 mg / L. The maximum allowable amount of nitrite in meat products is 150 mg / kg. At the same time, the Joint Expert Committee of the Food and Agriculture Organization of the United Nations / World Health Organization (WHO) proposed an acceptable daily intake of 0.00 to 0.06 mg nitrite / Kg body weight.

[0004] The electrochemical detection of nitrite has a linear range of 0.5–105 µM and a detection limit of 0.22 µM. The electrochemical sensor was developed for the detection of nitrite with a linear range of 5–1000 μM and a detection limit of 0.094 μM. The spectrophotometric method described in this technology for the determination of nitrite has a linear range of 0.091–1.47 μg / mL and a detection limit of 0.053 μg / mL.

[0005] A variety of analytical techniques, including chromatography, spectrophotometry, fluorescence, Raman spectroscopy, chemiluminescence, colorimetry, and electrochemical methods. Several extraction methods, such as turbidity extraction, solid phase extraction, deep eutectic solvent extraction, liquid phase extraction, and microwave-assisted extraction, are used to extract nitrite from different samples. During the extraction process, the selection of switchable hydrophilicity solvents has the advantages of being green, inexpensive, having a high enhancement factor, and being sensitive and selective in analyte extraction. When a switchable hydrophilicity solvent is used as the extraction solvent, an ultrasonic bath and a centrifugation step after the ultrasonic treatment step are not required.

[0006] In the present method, the aim was to use sensitive switchable hydrophilic solvents in liquid-liquid microextraction and selective determination of nitrite in thermally processed foods and to use multivariate statistical analysis in method optimization. In this context, a new, simple and sustainable switchable hydrophilic solvent-based ultrasound-assisted liquid-liquid microextraction / spectrophotometry method was proposed for the extraction and determination of trace nitrite in samples. Several parameters were also optimized and examined for their effects on nitrite extraction recovery. Multivariate statistical analysis was performed, such as the use of factorial design to investigate the significant levels of factors and their effects on nitrite extraction recovery in samples. Overall, hydrophilic solvent ultrasound-assisted liquid-liquid microextraction provided a simple, selective and rapid method for the determination of nitrite in thermally processed foods. In addition, the proposed method does not require heating and centrifugation steps, which is advantageous in terms of cost and time compared to other similar methods. Summary of the invention

[0007] This method uses a hydrophilic solvent ultrasonic assisted liquid-liquid microextraction method to detect nitrosamines. Compared with the existing technology, it can significantly improve the extraction efficiency of nitrosamines, thereby greatly improving the detection efficiency of highly carcinogenic nitrosamines in food. The steps involved in this method include 1. Configuring a switchable hydrophilic solvent and an auxiliary solvent, and its beneficial effect is to significantly improve the extraction efficiency of various types of nitrites. 2. Pretreatment of the test sample, this step is to crush the sample as much as possible so that the internal nitrite is fully extracted. 3. Hydrophilic solvent ultrasonic assisted liquid-liquid microextraction, this step is to combine the effects of ultrasound and auxiliary liquid to fully extract the nitrite in the test sample, and the auxiliary liquid here refers to the hydrophilic solvent. Compared with the existing technology, the significant creativity of this method lies in the development of a group of hydrophilic solvents that can efficiently extract trace nitrites, and combined with ultrasonic microextraction technology, a method for determining the content of nitrosamines by spectrophotometry.

[0008] Instruments and equipment: First, the determination of nitrite was achieved using a spectrophotometer. An ultrasonic bath, using an ultrapure water system to obtain ultrapure water, was used to pretreat the collected samples and to disperse the extraction solvent into the sample solution. The pH control of the extraction medium was achieved using a digital pH meter, and an analytical balance was used in the weighing step.

[0009] Configuration of switchable hydrophilic solvents and auxiliary solvents: All chemicals were of at least analytical grade. A nitrite reference solution (1000 mg / L) was prepared by dissolving an appropriate amount of analytical grade sodium nitrite in ultrapure water. A drop of chloroform was added to preserve it. A working reference of nitrite was prepared by serially diluting this solution before the experiment. Phthalate, tris, borate, and phosphate buffer solutions were used to adjust the pH. A 500 µmol / L Fe(II) solution was prepared from FeCl2 (≥99.0%) solid in water. A 5 mol / L NaOH solution was prepared by dissolving an appropriate amount of solid NaOH (≥97.0%) in water. N,N-dimethylcyclohexylamine (99%, DMCA), triethylamine (≥99.5%, TEA), and N,N-dimethylbenzylamine (≥99%, DMBA) were used to prepare switchable hydrophilicity solvents. Amines of either DMCA, TEA, or DMBA were then added to three glass bottles with caps. An equimolar amount of ultrapure water was then added to the container, at which stage a two-phase system of a switchable hydrophilic solvent in a hydrophobic form was formed. Dry ice (10.0 g) was then slowly added to the resulting mixture to avoid excessive pressure. At this stage, turbidity appeared, indicating that it formed a protonated switchable hydrophilic solvent that was moderately dissolved by CO2 reaction. The mixture was then vortexed to dissolve the carbon dioxide and form a single phase. In this study, switchable hydrophilic solvent-1, switchable hydrophilic solvent-2, and switchable hydrophilic solvent-3 were obtained by mixing DMCA, TEA, and DMBA with water in a molar ratio of 1:1, respectively.

[0010] Pretreatment of test samples: In this method, the goal is to selectively and accurately analyze nitrite in heat-treated foods. In this case, all samples were from markets and restaurants. They included meat products such as roasted chicken, grilled meat, Chinese sausage, pickled food, mustard tuber, sauerkraut, kimchi, and meatballs.

[0011] The samples were pretreated by first grinding them into powders and increasing their homogeneity using a laboratory mixer. Then, 10 g of the powdered sample was weighed and transferred to a beaker. Then, 5 mL of 5% borax solution and 150 mL of hot distilled water were added to the beaker, respectively. The beaker was ultrasonically treated in an ultrasonic bath at 80 ° C for 30 minutes. After ultrasonic treatment, 2 mL of hexacyanoferrate, 0.25 mol / L potassium solution, and 2 mL of 0.25 mol / L Zn(CH3CO2)2 solution were added to the beaker, respectively, and manually shaken for 5 minutes. Finally, the obtained sample solution was filtered using a membrane filter.

[0012] Detection process of hydrophilic solvent ultrasonic assisted liquid-liquid microextraction First, 5 mL of the sample solution was added to a test tube containing 200 µg / L nitrite. In the second step, the pH of the resulting mixture was adjusted to 7.0 with a borate buffer solution. In the third step, a 50 µmol / L Fe(II) solution was added to the resulting mixture to complex the nitrite in the sample solution. In the fourth step, 800 µL of the prepared switchable hydrophilicity solvent (prepared from one of DMCA, TEA, and DMBA) was added to the resulting mixture in the presence of carbon dioxide and a solution containing water and TEA in a molar ratio of 1:1 (as an extraction solvent), followed by the addition of 450 µL of 50 µM NaOH, resulting in turbidity of the solution. In the fifth step, the obtained mixture was placed in an ultrasonic bath and sonicated for 12 minutes to increase the extraction of nitrite by the switchable hydrophilicity solvent. The sonication step achieved phase separation, resulting in a supernatant layer that encapsulated the switchable hydrophilicity solvent without the need for a centrifugation step. In the seventh step, the aqueous portion was removed by decantation, and the remaining solution was placed in a cuvette and analyzed spectrophotometrically at 406 nm. The blank sample was used as a reference solution for spectrophotometric colorimetry. Implementation

[0013] Example 1 First, a nitrite reference solution (1000 mg / L) was prepared by dissolving an appropriate amount of analytical grade sodium nitrite in ultrapure water. A drop of chloroform was added to preserve it. A 500 µmol / L Fe(II) solution was prepared from FeCl2 (≥99.0%) solid in water. An appropriate amount of its solid NaOH (≥97.0%) was dissolved in water to prepare a 5 mol / L NaOH solution. Then, amine-containing N,N-dimethylcyclohexylamine (99%, DMCA) was added to a glass bottle with a lid. Then, an equimolar amount of ultrapure water was added to the container, and a two-phase system of a switchable hydrophilic solvent in a hydrophobic form was formed at this stage. Then, dry ice (10.0 g) was slowly added to the resulting mixture to avoid excessive pressure. At this stage, turbidity appeared, indicating that it formed a protonation switchable hydrophilic solvent that was moderately soluble by CO2 reaction. Then, the mixture was vortexed to dissolve carbon dioxide and form a single phase. Several sauerkraut samples were extracted and ground into powder, and 10 g of the powder sample was weighed and transferred to a beaker. Then, 5 mL of 5% borax solution and 150 mL of hot distilled water were added to the beaker, respectively. The beaker was sonicated in an ultrasonic bath at 80°C for 30 minutes. After sonication, 2 mL of hexacyanoferrate, 0.25 mol / L potassium solution, and 2 mL of 0.25 mol / L Zn(CH3CO2)2 solution were added to the beaker, respectively, and manually shaken for 5 minutes. Finally, the obtained sample solution was filtered using a membrane filter. 5 mL of the sample solution was added to a test tube containing 200 µg / L nitrite. The pH of the obtained mixture was adjusted to 7.0 with a borate buffer solution. 50 µmol / L of Fe(II) solution was added to the obtained mixture to complex the nitrite in the sample solution. 800 µL of the prepared switchable protonation switchable hydrophilicity solvent and a solution containing water and DMCA in a molar ratio of 1:1 (as an extraction solvent) in the presence of carbon dioxide were added to the resulting mixture, followed by the addition of 450 µL of 50 µM NaOH, resulting in turbidity of the solution. The obtained mixture was then placed in an ultrasonic bath and sonicated for 12 minutes to increase the extraction of nitrite by the switchable hydrophilicity solvent. The sonication step achieved phase separation, resulting in a supernatant layer that encapsulated the switchable hydrophilicity solvent without the need for a centrifugation step. In the seventh step, the aqueous portion was removed by decantation, and the remaining solution was placed in a cuvette and analyzed spectrophotometrically at 406 nm. The blank sample was used as a reference solution for spectrophotometric colorimetry.

Claims

1. A method for highly sensitive detection of nitrosamines, characterized in that The method includes:

1. configuring a switchable hydrophilic solvent and an auxiliary solvent, which has the beneficial effect of significantly improving the extraction efficiency of various types of nitrites; 2. pretreatment of the test sample, which is a step of crushing the sample as much as possible so that the nitrite inside can be fully extracted; 3. hydrophilic solvent ultrasonic assisted liquid-liquid microextraction, which is a step of combining the effects of ultrasound and auxiliary liquid to fully extract the nitrite in the test sample.

2. The method according to claim 1, characterized in that The steps of configuring the switchable hydrophilic solvent and auxiliary solvent include: preparing a nitrite reference solution of 1000 mg / L by dissolving an appropriate amount of analytical grade sodium nitrite in ultrapure water, adding a drop of chloroform to preserve it, and preparing a working reference of nitrite by serially diluting the solution before the experiment, using phthalate, tris(hydroxymethyl)aminomethane, borate and phosphate buffer solutions to adjust the pH; 500µmol / LFe2+ solution is prepared by FeCl2≥99.0% solid in water, taking an appropriate amount of its solid NaOH≥97.0% and dissolving it in water to prepare a 5mol / LNaOH solution; N,N-dimethylcyclohexylamine 99%, DMCA, triethylamine ≥ 99.5%, TEA and N,N-dimethylbenzylamine ≥ 99%, DMBA are used to prepare switchable hydrophilic solvents, and then one of the amine-containing DMCA, TEA or DMBA is added to three glass bottles with lids, and then equimolar ultrapure water is added to the container. At this stage, a two-phase system of a switchable hydrophilic solvent in a hydrophobic form is formed. Then, 10.0 g of dry ice is slowly added to the resulting mixture to avoid excessive pressure. At this stage, turbidity appears, indicating that it forms a protonated switchable hydrophilic solvent that is moderately dissolved by CO2 reaction. Then, the mixture is vortexed to dissolve carbon dioxide and form a single phase. In this technology, switchable hydrophilic solvent-1, switchable hydrophilic solvent-2 and switchable hydrophilic solvent-3 are obtained by mixing DMCA, TEA and DMBA with water in a molar ratio of 1:1, respectively.

3. The method according to claim 1, characterized in that The pretreatment steps of the test samples include first grinding the samples into powders with a laboratory mixer and increasing their homogeneity, then weighing 10 g of the powdered sample and transferring it to a beaker, then adding 5 mL of a 5% borax solution and 150 mL of hot distilled water to the beaker, respectively, and sonicating the beaker in an ultrasonic bath at 80 °C for 30 min. After sonication, 2 mL of hexacyanoferrate, 0.25 mol / L potassium solution, and 2 mL of 0.25 mol / L Zn(CH3CO2)2 solution were added to the beaker, respectively, and manually shaken for 5 min. Finally, the obtained sample solution was filtered using a membrane filter.

4. The method according to claim 1, characterized in that The hydrophilic solvent ultrasound-assisted liquid-liquid microextraction steps include first, adding 5 mL of sample solution into a test tube containing 200 µg / L nitrite; in the second step, adjusting the pH of the resulting mixture to 7.0 with a borate buffer solution; in the third step, adding 50 µmol / L of Fe2+ solution to the resulting mixture to complex the nitrite in the sample solution; in the fourth step, adding 800 µL of a prepared switchable hydrophilic solvent, prepared from one of DMCA, TEA, and DMBA, in the presence of carbon dioxide, and a solution containing water and TEA in a molar ratio of 1:1 as an extraction solvent to the resulting mixture, followed by adding 450 µL of 50 µM NaOH, resulting in turbidity of the solution; in the fifth step, the obtained mixture is placed in an ultrasonic bath and ultrasonically treated for 12 minutes to increase the extraction of nitrite by the switchable hydrophilicity solvent, and the ultrasonic treatment step achieves phase separation, thereby producing a supernatant layer that seals the switchable hydrophilicity solvent without the need for a centrifugation step; in the seventh step, the aqueous portion is removed by decantation, and the remaining solution is placed in a colorimetric cup and analyzed spectrophotometrically at 406 nm, and the blank sample is used as a reference solution for spectrophotometric colorimetry.