CN115598248A - Novel method for determining volatile N-nitrosamine in food - Google Patents

Novel method for determining volatile N-nitrosamine in food Download PDF

Info

Publication number
CN115598248A
CN115598248A CN202211226753.5A CN202211226753A CN115598248A CN 115598248 A CN115598248 A CN 115598248A CN 202211226753 A CN202211226753 A CN 202211226753A CN 115598248 A CN115598248 A CN 115598248A
Authority
CN
China
Prior art keywords
filler
nitrosamine
food
psa
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202211226753.5A
Other languages
Chinese (zh)
Inventor
闫薪竹
张雪丽
强维
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Anpu Experimental Technology Co ltd
Original Assignee
Shanghai Anpu Experimental Technology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Anpu Experimental Technology Co ltd filed Critical Shanghai Anpu Experimental Technology Co ltd
Priority to CN202211226753.5A priority Critical patent/CN115598248A/en
Publication of CN115598248A publication Critical patent/CN115598248A/en
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/025—Gas chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/062—Preparation extracting sample from raw material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G01N2030/143—Preparation by elimination of some components selective absorption

Landscapes

  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

The invention belongs to the technical field of chemical detection and analysis, and relates to a novel method for determining volatile N-nitrosamine in food. In the invention, a proper QuEChERS purification technology is applied, and then a detection scheme of the upper and lower streams is combined, a matrix dispersion extraction filler is used for extracting a target object from a matrix sample by using a small amount of organic reagent, and further impurities are removed by the purification filler, so that the effects of low matrix interference and high recovery rate are obtained. The method ensures low interference and high recovery, shortens the processing time and saves the time and labor cost. Meanwhile, the dosage of the organic solvent can be greatly reduced for the quantitative sample to be detected, and the method is an environment-friendly scheme.

Description

Novel method for determining volatile N-nitrosamine in food
Technical Field
The invention belongs to the technical field of chemical detection and analysis; more specifically, the invention relates to a new method for determining volatile N-nitrosamines in food products, said method being based on the QuEChERS purification technique.
Background
Nitrosamines, being potent carcinogens, are among the most important chemical carcinogens, not only causing carcinogenesis in animals or humans in small doses over a long period of time, but also causing carcinogenesis in a single "shot" of higher doses. N-nitrosamines have attracted considerable attention, and in particular how to prevent and control them is a matter of considerable concern.
In a carcinogen list issued by world health organization in 2017, multiple N-nitrosamine compounds are listed as first class carcinogenic substances and second class carcinogenic substances, and the nitrosamine compounds are the most important class of N-nitroso compounds, and are more than 200 in total, so that the nitrosamine compounds are one of the most important chemical carcinogens recognized in the field. In 1987, nitrosamines were classified as strongly carcinogenic substances by the international agency for research on cancer (IARC). Among them, N-Nitrosodimethylamine (NDMA) and N-Nitrosodiethylamine (NDEA) are listed as class 2A carcinogens (there is sufficient evidence of animal experiments to prove their potential carcinogenicity). Wherein the N-dimethyl nitrosamine has irritation effect on eyes and skin, and even a small dosage of N-dimethyl nitrosamine can increase the risk of cancer of human body by long-term contact.
In view of the toxicity and distribution of nitrosamines, there has been a push to establish standards and guidelines for nitrosamines in food products in different countries and regions of the world. The limit control of the national standard GB2762-2017 on the N-dimethyl nitrosamine is meat products (except canned meat), cooked dried meat products are 3.0 mu g/kg, aquatic products (except canned aquatic products) and dried aquatic products are 4.0 mu g/kg. N-dimethyl nitrosamine has no limit control requirement in CAC, european Union, australian New and other countries. Due to the large difference between the structure and the property of the N-nitrosamine compounds and the different content distribution of the N-nitrosamine compounds in different foods, an effective analysis method needs to be developed for the N-nitrosamine compounds with different contents in different food systems.
The second method gas chromatography-thermal energy analyzer method for detecting N-nitrosamine compounds in food in the current GB5009.26-2016 standard is only limited to the analysis of N-nitrosamine compounds due to the high price, narrow application range and low adaptability of the thermal energy analyzer, and common laboratories do not have the equipment, and the daily maintenance of the detector is troublesome, so that the wide application of the detector is limited. The first method of GB5009.26-2016 adopts water vapor distillation-gas chromatography-mass spectrometry/mass spectrometry, and the pretreatment adopts distillation extraction by a water distillation device, multiple extraction by dichloromethane and evaporation concentration to purify and enrich N-nitrosamine. However, distillation conditions are not easy to control, partial impurities can be extracted from dichloromethane in the extraction process, measurement is interfered, the method has more operation steps, the recovery rate is low and is only 40-70%, time and labor are wasted, reagent and reagent costs are wasted, batch detection is not facilitated, the detection target object is single, and the like (the national standard method only detects N-dimethyl nitrosamine), and the technical requirements of people on nitrosamine detection cannot be continuously met.
In some patent applications (CN 112730663A, CN110389188A, CN210626153U, CN 110208444B) mention is made of methods and devices which can effectively detect volatile N-nitrosamines in foods. However, these methods have problems of low recovery rate, poor purification effect, complicated apparatus, complicated operation, and limited application range.
Therefore, an experimental method which can effectively remove impurities, optimize a pretreatment process, reduce reagent loss and shorten treatment time under the condition of ensuring high recovery rate and can be simultaneously applied to treatment and detection of various N-nitrosamine compounds and matched is needed to be found.
Disclosure of Invention
The invention aims to provide a novel method for determining volatile N-nitrosamine in food, which is based on a QuEChERS purification technology.
In a first aspect of the present invention, there is provided a method for simultaneously detecting the content of a plurality (two or more) of N-nitrosamines in a foodstuff, the method comprising: (1) Extracting an extract containing (or potentially containing) N-nitrosamines from a food product; (2) Purifying the extract of (1) by QuEChERS purification method to obtain purified extract; the purification tubes used present a combination of the following fillers: c18 or C8 filler, PSA filler and Si (Silica) filler, optionally also GCB filler; (3) Detecting the purified extract by gas chromatography-mass spectrometry to determine N-nitrosamine content.
In one or more embodiments, the N-nitrosamine is a volatile N-nitrosamine; preferably, the plurality of N-nitrosamines is selected from: n-dimethylnitrosamine (NDMA), N-methylethylnitrosamine, N-diethylnitrosamine, N-dipropylnitrosamine, N-dibutylnitrosamine, N-nitrosopiperidine, N-nitrosopyrrolidine, N-nitrosomorpholine (NmorPh), N-diphenylnitrosamine (NDPhA).
In one or more embodiments, in step (2), the ratio by weight: the proportion of the C18 filler, the PSA filler and the Si filler is (2-10) to (1-5).
In one or more embodiments, the weight ratio of: the proportion of the C18 filler, the PSA filler and the Si filler is (4-8) to (2-4); more preferably 2.
In one or more embodiments, in step (2), the weight ratio of: the proportion of the C8 filler, the PSA filler and the Si filler is (6-30) to (4-20) to (1-5).
In one or more embodiments, the ratio of C8 filler, PSA filler, and Si filler is (12-24): (8-16): (2-4); more preferably 6.
In one or more embodiments, in step (2), the ratio by weight: the proportion of the C18 filler, the PSA filler, the Si filler and the GCB filler is (6-30): (6-30): 1-5): 0.2-1.
In one or more embodiments, the ratio of C18 filler, PSA filler, si filler, and GCB filler is (12-24): (2-4): (0.4-0.8); more preferably 30.
In one or more embodiments, step (1) comprises: extracting food with organic solvent to obtain extract containing N-nitrosamine, and taking the extract as sample to be detected; preferably, the organic solvent is acetonitrile; more preferably, the acetonitrile is 5 mL-50 mL per sample.
In one or more embodiments, the addition of the organic solvent may be accompanied by the addition of anhydrous magnesium sulfate and sodium chloride.
In one or more embodiments, the anhydrous magnesium sulfate is 50mg to 2g per sample;
in one or more embodiments, the sodium chloride is 50mg to 2g per sample.
In one or more embodiments, in step (1): before extraction, the method also comprises a step of crushing the food.
In one or more embodiments, in step (1): after extraction, the method also comprises the step of centrifuging to obtain a supernatant.
In one or more embodiments, upon the step (3) gas chromatography-mass spectrometry detection: adopting a gas chromatography-mass spectrometer; preferably, the column is CD-BASEWAX.
In one or more embodiments, when step (3) is performed by gc-mass spectrometry, the gas conditions are: a chromatographic column: CD-BASEWAX, length: 30m, inner diameter: 0.25mm film thickness: maximum 0.25 μm column temperature: temperature program at 240 ℃: maintaining the temperature at 40 ℃ for 0.5min, increasing the temperature to 190 ℃ at a rate of 15 ℃/min, increasing the temperature to 230 ℃ at a rate of 40 ℃/min, and maintaining the temperature of the injection port liner tube for 10 min: no split flow, injection port temperature: 220 ℃, carrier gas: helium, constant flow mode (1 ml/min).
In one or more embodiments, the mass spectrometry conditions at the time of step (3) gas chromatography-mass spectrometry detection are: solvent retardation: 5.5min ionization mode: EI ion source temperature: interface temperature 230 ℃:220 ℃ detection mode: MRM gain factor: 60 quantitative method: internal standard method.
In one or more embodiments, the method further comprises: performing qualitative and/or quantitative analysis by using a standard curve method; preferably, before the detection in step (3), a substrate labeling solution is prepared: taking a blank sample solution to be detected without N-nitrosamine, and then adding an N-nitrosamine standard solution to prepare substrate standard sample (standard) solutions with different concentrations; preferably, after the detection in step (3), the matrix labeling sample solutions with different concentrations are measured by using the detection conditions in step (3) to obtain a matrix labeling curve, and qualitative and/or quantitative analysis is realized by using a matrix external standard method.
In one or more embodiments, the standard curve is a matrix-spiking curve. Preferably, matrix labeling sample solutions with different concentrations are adopted to obtain a matrix labeling curve, and a matrix external labeling method is utilized to simultaneously realize qualitative and quantitative analysis.
In one or more embodiments, the formulation matrix spiking solution comprises: taking a blank sample solution to be detected without N-nitrosamine, and then adding an N-nitrosamine standard solution to prepare matrix standard solutions with different concentrations.
In a second aspect of the invention, there is provided a purge tube suitable for use in a QuEChERS purge method, the purge tube being used in the presence of a combination of packing materials: c18 or C8 filler, PSA filler and Si filler, optionally also GCB filler; preferably, the weight ratio of: the proportion of the C18 filler, the PSA filler and the Si filler is (2-10) to (1-5); or the proportion of the C8 filler, the PSA filler and the Si filler is (6-30) to (4-20) to (1-5); or the proportion of the C18 filler, the PSA filler, the Si filler and the GCB filler is (6-30) to (1-5) to (0.2-1).
In a third aspect of the invention, the use of said purification tube is provided for purifying a food extract as a purification tube based on the QuEChERS purification method.
In a fourth aspect of the present invention, there is provided a kit for detecting the content of N-nitrosamines in a food, the kit comprising:
(a) An organic agent for extracting an extract containing (or potentially containing) N-nitrosamines from a food product; preferably, the organic solvent is acetonitrile; preferably, anhydrous magnesium sulfate and sodium chloride are also included;
(b) A purge tube suitable for use in a QuEChERS purification method, the combination of packing present in the purge tube: c18 or C8 filler, PSA filler and Si filler, optionally also GCB filler;
in one or more embodiments, the kit further comprises: a standard of N-nitrosamine; and (5) a reference substance.
In one or more embodiments, the kit further comprises: instructions for use of the method for detecting N-nitrosamine content in a food product.
Detailed Description
The invention provides a method for detecting N-nitrosamine in a food matrix based on a novel QuEChERS purification technology combined with a gas chromatography-mass spectrometry. By applying a proper QuEChERS purification technology and combining the detection schemes of the upper and lower streams, the matrix dispersed extraction filler is used for extracting the target object from the matrix sample by using a small amount of organic reagent, and the impurities are further removed by the purification filler, so that the effects of low matrix interference and high recovery rate are obtained. The method has the advantages of ensuring low interference and high recovery, shortening the processing time and saving time and labor cost. Meanwhile, for a quantitative sample to be detected, the consumption of an organic solvent (such as acetonitrile) which exceeds 500mL in the conventional detection standard can be reduced to be less than 50mL, so that the method is an environment-friendly technical scheme and conforms to the currently advocated green environmental protection concept.
Term(s) for
As used herein, the "N-nitrosamines" are volatile N-nitrosamines; preferably, the plurality of N-nitrosamines is selected from: n-dimethylnitrosamines (including NDMA), N-methylethylnitrosamines, N-diethylnitrosamines, N-dipropylnitrosamines, N-dibutylnitrosamines, N-nitrosopiperidines, N-nitrosopyrrolidines, N-nitrosomorpholines (NmorPh), N-diphenylnitrosamines (NDPhA).
As used herein, "matrix" refers to a sample to be tested (including target analytes as well as other substances and impurities), since substances other than target analytes often interfere significantly with the analysis process of the analytes and affect the accuracy of the analysis result, these effects and interferences are called matrix effects (matrix effects).
As used herein, "target analyte" is also referred to as "target object", "target detector" or "detector"; unless otherwise indicated, the "target analyte" refers to "N-nitrosamine.
As used herein, the term "food (sample)" includes natural food samples, artificially processed food samples, or food waste.
As used herein, the amount of sample in "/sample" or "/sample" can refer to a sample having a weight of about 5 to 10 grams; the fluctuation in weight may be varied depending on the kind of the actual sample, such as. + -. 50%,. + -. 40%,. + -. 30%,. + -. 20%,. + -. 10%,. + -. 5%,. + -. 3%,. + -. 2%, and. + -. 1%.
Detection of N-nitrosamines in food samples
One of the keys to reducing the matrix effect is the pretreatment optimization of gas chromatography-mass spectrometry analysis. The sample pretreatment steps mainly comprise extraction and purification, and a common sample pretreatment method comprises the following steps: solid Phase Extraction (SPE), magnetic Solid Phase Extraction (MSPE), dispersion liquid-liquid microextraction (DLLME), quEChERS (Quick, easy, cheap, effective, rugged and Safe), immunoaffinity column (IAC) concentration, multifunctional purification column (MFC) purification, and the like. The prior art has been directed to the detection of N-nitrosamines in food products, although some criteria are already in operation, but still faces more aspects that need to be optimised. The invention provides an optimized detection method.
The method for detecting N-nitrosamine in food samples comprises the following steps: (1) Extracting an extract containing (or potentially containing) N-nitrosamines from a food product; (2) Purifying the extract of (1) by QuEChERS purification method to obtain purified extract; and (3) detecting the purified extract by gas chromatography-mass spectrometry to determine the content of N-nitrosamine.
Pretreatment of
The method of the invention comprises a pre-treatment step comprising the extraction of as close to the total amount of N-nitrosamines as possible from a food sample, thereby providing support for accurate subsequent testing.
Some organic solvents may be used in the present invention, such as methanol, ethanol, acetonitrile, and the like. In a preferred embodiment of the present invention, the food sample is extracted with acetonitrile to obtain an extract containing N-nitrosamine. The inventors have observed that the matrix effect of acetonitrile and ethanol extracts is weaker than that of other organic solvent extracts.
The pretreatment step may be preceded by a step of pulverizing the food sample, and this step may be performed by a mechanical treatment, which is not particularly limited in the present invention.
In a preferred embodiment of the present invention, the method further comprises the step of extracting the food sample with acetonitrile to obtain an extract, and then centrifuging the extract to obtain a supernatant.
In a preferred embodiment of the present invention, when food samples are extracted with acetonitrile, 5 to 50mL of acetonitrile is used per food sample. In particular embodiments, the inventors have optimized the amount of extractant used with the sample, and the recovery is relatively high.
From the pretreatment scheme, the method can solve the problems of low recovery rate, time and labor waste in a pretreatment method (GB 5009.26-2016 (steam distillation) -gas chromatography-mass spectrometry/mass spectrometry) by adopting steam distillation. The inventor finds that the low recovery rate of the pretreatment method of steam distillation is mainly caused by the fact that the distillation condition is difficult to control, partial impurities can be extracted from dichloromethane in the multiple extraction processes to interfere with the measurement, and the method has more operation steps and finally causes the low recovery rate; on the basis of this finding, the present inventors have created a motivation for improvement, and thus have achieved the technical solution as the present invention.
Extracting food with organic solvent to obtain extract containing N-nitrosamine, and taking the extract as sample to be detected; preferably, the organic solvent is acetonitrile; more preferably, the acetonitrile is 5-50 mL per sample.
As a preferable mode of the invention, the method also comprises the steps of adding anhydrous magnesium sulfate and sodium chloride while adding the organic solvent; preferably, the anhydrous magnesium sulfate is 50 mg-2 g per sample; preferably, the sodium chloride is 50mg to 2g per sample.
The addition of anhydrous magnesium sulfate and sodium chloride is suitable. The anhydrous magnesium sulfate can play a role in removing water; in cooperation with this, sodium chloride can reduce the solubility of the target substance in water, and increase the extraction efficiency of the organic solvent. In addition, other agents which have a water-removing effect, such as anhydrous sodium sulfate, have a problem of being easily agglomerated when they are exposed to water, and the present inventors have found that they cause a problem that the substrate cannot be sufficiently dispersed in a solution. In addition, other water scavengers or reagents for reducing the solubility in water are expensive and do not have the same effect as the anhydrous magnesium sulfate and sodium chloride selected by the invention.
QuEChERS decontamination
After the pretreatment step, the extract containing N-nitrosamine is obtained, and the extract is purified by a QuEChERS purification method, so that the purified extract which is beneficial to subsequent efficient and accurate detection is obtained.
To obtain good purification results, the inventors optimized the type of packing in the purification tubes used for QuEChERS purification, including the use of packing C18 or C8, PSA, si and optionally GCB as the main packing. Furthermore, the inventor optimizes the dosage proportion of the filler, and the proportion of the C18 filler, the PSA filler and the Si filler in the filler is (2-10) to (1-5) according to the weight ratio; or the proportion of the C8 filler, the PSA filler and the Si filler is (6-30) to (4-20) to (1-5); or the proportion of the C18 filler, the PSA filler, the Si filler and the GCB filler is (6-30) to (1-5) to (0.2-1).
The purifying recovery rate obtained by applying the mixed filler of the invention is obviously higher than that of a commercialized purifying column and that of the existing national standard, and the effect is excellent.
Detection by gas chromatography-mass spectrometry
The purified extract obtained by the purification step can be used for qualitative and/or quantitative detection of the target substance by gas chromatography-mass spectrometry.
After the measurement conditions are determined, the target substance can be qualitatively and quantitatively determined by a known qualitative and quantitative calculation method. As a preferred mode of the present invention, qualitative and/or quantitative analysis is performed by using a standard curve method. Preferably, a matrix labeling curve can be obtained by measuring matrix labeling sample solutions with different concentrations, and qualitative and quantitative analysis can be simultaneously realized by utilizing a matrix external standard method.
As a preferred mode of the present invention, qualitative and/or quantitative analysis is performed by using a standard curve method; preferably, the labeling solution can be prepared by: taking a blank sample solution to be detected without N-nitrosamine, and then adding an N-nitrosamine standard solution to prepare substrate standard sample (standard) solutions with different concentrations; preferably, the matrix labeled sample solution with different concentrations is measured by using the detection conditions detected by the gas chromatography-mass spectrometry to obtain a matrix labeled curve, and qualitative and/or quantitative analysis is realized by using a matrix external standard method.
The invention can solve the problems of complex pretreatment device and high experimental cost of methods involved in a gas chromatography-thermal energy analyzer method (GB 5009.26-2016 gas chromatography-thermal energy analyzer method) and patents (CN 112730663A, CN110389188A, CN210626153U and CN 110208444B). By adopting a simple and feasible experimental method, combining with a proper filler in a special QuEChERS method bag for N-nitrosamine and adopting low-cost general consumables, impurities in different matrixes can be effectively removed, and the detection of 9 nitrosamine compounds is realized.
The invention can solve the applicability problem in patents CN106053634B, CN111707769A and the like, has wide applicability, can be applied to traditional aquatic product substrates, seasoning substrates and complex substrates containing more grease and additives, such as: the spicy small fish larvae and the like can also be suitable, and can be applied to food substrates and different substrates which need to be subjected to nitrosamine substance detection, the adopted experimental scheme is unified, the time and the labor cost are greatly saved, and the operation difficulty of personnel is reduced.
Compared with the national standard method, the method of the invention greatly reduces the used base mass, and the national standard needs 200g, which is 5 g-10 g.
Application and kit
In the technical scheme of the invention, a simple and efficient determination method for determining N-nitrosamine in food is established by combining the optimized QuEChERs pretreatment technology of the invention. Can be applied to the determination of various foods/food products/processed products.
The method for detecting N-nitrosamine in food matrix based on the novel QuEChERS purification technology combined with the gas chromatography-mass spectrometry, which is established by the invention, enables the extraction solution to be purified by the optimized purification tube, so that the pretreatment work is simple and quick, and an ideal extract to be detected is obtained. Preferably the food sample is extracted with acetonitrile by shaking, preferably by chromatography using a CD-BASEWAX column. The method has good purification effect on the food matrix, high analysis speed, high sensitivity and good reproducibility, and provides a new detection method for evaluating the content of N-nitrosamine in the food. The method has the characteristics of rapidness, simplicity, low price, effectiveness, stability, safety and the like.
Based on the new findings of the present inventors, the present invention also provides a kit for detecting N-nitrosamines in a food sample, comprising: an organic agent for extracting an extract containing (or potentially containing) N-nitrosamines from a food sample; and a purge tube suitable for use in a QuEChERS purge process, the purge tube comprising the following packing: c18 or C8, PSA, si, GCB.
In a preferred embodiment of the present invention, the kit further comprises: a standard of N-nitrosamine; a control; eluting the reagent; and/or instructions for use that specify a method for detecting N-nitrosamines in a food sample.
The invention has the following beneficial effects:
(1) The detection method and the matched QuEChERS method package special for N-nitrosamine can greatly improve the recovery rate of N-nitrosamine, and the experimental method can improve the recovery rate of N-nitrosamine in a complex matrix to more than 85% by matching with the QuEChERS method package special for N-nitrosamine, and is accurate and efficient, and the special method package has excellent performance.
(2) Compared with the national standard (GB 5009.26-2016), the method has the advantages that the operation process is simple, nitrogen blowing is not needed in the whole process, the time can be saved (the processing period of a batch of samples is about 40-60 min), the cost for purchasing instruments such as a nitrogen blowing instrument and nitrogen is saved, and the condition that the parallelism is poor and the test result is influenced due to the fact that the nitrogen blowing degree is not good and the time control is controlled is eliminated. The method can avoid a series of influences caused by nitrogen blowing, and is simple and strong in operability. In addition, the method reduces more than 500mL of dichloromethane used in the standard to 50mL of acetonitrile, greatly reduces the reagent dosage, and belongs to an environment-friendly product and a method.
(3) The experiment method and the matched QuEChERS method bag special for the N-nitrosamine have wide application range, can be applied to various substrates, including relatively complex substrates such as fresh fish, grilled fish fillet, spicy small fish and the like, have unified experiment methods, do not need to select different fillers and methods according to different substrates, greatly facilitate experimenters, and save time and labor cost.
(4) The measured target compound is single), the method has simple and quick pretreatment; the compounds can be detected in various types, the method covers 9 kinds of volatile N-nitrosamines such as NDMA, NMEA, NDEA, NPYR, NMorPh, NDPA, NPIP, NDBA, NDPhA and the like, and the method can meet the requirement of rapid and safe screening of most compounds at present.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described below in conjunction with specific embodiments of the present invention. It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The retention time and MRM ion pair information of the N-nitrosamine target and the internal standard are shown in the table 1; in the table, N-dimethylnitrosamine-d 6 is an internal standard.
TABLE 1
Figure BDA0003880117960000101
Figure BDA0003880117960000111
Example 1 detection of fresh Fish meat as matrix
In the embodiment, fresh fish meat is used as a matrix, and various volatile N-nitrosamines in food are measured by a QuEChERS purification technology.
1. Detection process
The specific operation steps are as follows:
(1) Sample extraction
Weighing 5g of homogenized fresh fish meat in a centrifuge tube, and adding 15mL of acetonitrile corresponding to 5g of sample; 2g of anhydrous magnesium sulfate and 2g of sodium chloride were added. Mixing, ultrasonic treating, centrifuging, collecting supernatant, and purifying.
(2) Sample purification
A portion of the supernatant was transferred to a purge tube containing solid phase extraction packing as shown in table 2.
TABLE 2
Figure BDA0003880117960000112
Mixing, centrifuging, collecting supernatant, filtering, and measuring.
(3) Gas chromatography-mass spectrometer detection
Detecting by using a gas chromatography-mass spectrometer under the following detection conditions:
gas phase conditions: a chromatographic column: CD-BASEWAX, length: 30m, inner diameter: 0.25mm film thickness: maximum temperature of 0.25 μm column: temperature program at 240 ℃: keeping the temperature at 40 ℃ for 0.5min, increasing the temperature to 190 ℃ at a speed of 15 ℃/min, increasing the temperature to 230 ℃ at a speed of 40 ℃/min, and keeping the injection port liner tube for 10 min: no split flow, injection port temperature: 220 ℃, carrier gas: helium, constant flow mode (1 ml/min).
Mass spectrum conditions: solvent retardation: 5.5min ionization mode: EI ion source temperature: interface temperature 230 ℃:220 ℃ detection mode: MRM gain factor: 60, adding a solvent to the mixture; the quantitative method comprises the following steps: internal standard method.
The retention times and MRM ion pair information for the N-nitrosamine targets and internal standards are shown in Table 1.
2. The result of the detection
The recovery data of various N-nitrosamine compounds obtained by testing fresh fish meat as a substrate are shown in Table 3.
TABLE 3
Figure BDA0003880117960000121
As shown in Table 3, in the fresh fish meat matrix, the recovery rate of 9N-nitrosamines can reach more than 90 percent, and the recovery rate is extremely high; and most values tend to be close to 100%, and the parallelism is good.
The result shows that the QuEChERS method special for N-nitrosamine has excellent performance, and the experimental method can effectively collect 9N-nitrosamine targets with good compatibility.
Analytical comparisons showed that the filler of table 2 and its formulation were very much used, better than its filler selection, providing good purified products.
The time for the pretreatment part of the method is only about 30 minutes from the statistics of the treatment time.
Example 2 detection based on grilled Fish fillet
In the embodiment, roasted fish fillets (bulk products purchased from supermarkets) are used as a matrix, and various volatile N-nitrosamines in food are measured by a QuEChERS purification technology.
1. Detection process
The specific operation steps are as follows:
(1) Sample extraction
Weighing 5g of homogenized grilled fish fillet into a centrifuge tube, and adding 5mL of water and 15mL of acetonitrile corresponding to 5g of sample; 2g of anhydrous magnesium sulfate and 2g of sodium chloride were added. Mixing, ultrasonic treating, centrifuging, collecting supernatant, and purifying.
(2) Sample purification
A portion of the supernatant was transferred to a purge tube containing solid phase extraction packing as in table 4.
TABLE 4
Figure BDA0003880117960000131
And (4) uniformly mixing, centrifuging, taking supernatant, filtering by using a filter membrane, and measuring on a machine.
(3) And detecting conditions by using a gas chromatography-mass spectrometer.
The detection was carried out by gas chromatography-mass spectrometer under the same conditions as in example 1.
2. The result of the detection
The recovery data of various N-nitrosamine compounds obtained by detection using the grilled fish fillet as a substrate are shown in Table 5.
TABLE 5
Figure BDA0003880117960000132
Compared with the fresh fish meat of example 1, the grilled fish fillet of the present example as the substrate has relatively complex ingredients due to the processing process, additives, seasonings and the like. In the roasted fish slice matrix, the recovery rate of 9N-nitrosamines can reach more than 85 percent, and the recovery rate is higher; and most values tend to be close to 100%, with better parallelism.
The result shows that the QuEChERS method special for N-nitrosamine has excellent performance, and the experimental method can effectively collect 9N-nitrosamine targets and has good compatibility.
Analytical comparisons showed that the filler of table 4 and its formulation were very much used, better than its filler selection, providing good purified products.
The time taken for this pretreatment part is only about 30 minutes, counted from the treatment time.
Example 3 detection Using spicy little Fish as substrate
In the embodiment, spicy and hot small fish fries (products purchased in supermarkets) are used as a matrix, and various volatile N-nitrosamines in foods are measured by a QuEChERS purification technology.
1. Detection process
The specific operation steps are as follows:
(1) Sample extraction
5g of homogenized small fish is weighed into a centrifuge tube, 5mL of water and 15mL of acetonitrile are added corresponding to 5g of sample, and 2g of anhydrous magnesium sulfate and 2g of sodium chloride are added. Mixing, ultrasonic treating, centrifuging, collecting supernatant, and purifying.
(2) Sample purification
A portion of the supernatant was transferred to a purge tube containing solid phase extraction packing as in table 6.
TABLE 6
Figure BDA0003880117960000141
Mixing, centrifuging, collecting supernatant, filtering, and measuring.
(3) Detection condition of gas chromatography-mass spectrometer
The detection was carried out by gas chromatography-mass spectrometer under the same conditions as in example 1.
2. The result of the detection
The recovery rate data of various N-nitrosamine compounds obtained by detection with spicy small fish roe as matrix are shown in Table 7.
TABLE 7
Figure BDA0003880117960000151
Compared with the fresh fish meat in the embodiment 1, the spicy small fish meat of the embodiment as the substrate has more complex components due to the processing process and the additives, seasonings and the like; compared with the grilled fish fillet in the example 2, the grilled fish fillet has more additives and seasonings and is more complex. In the complex matrix of the spicy small fish fries, the recovery rate of 9N-nitrosamines can reach more than 90 percent, and the recovery rate is very high; and most values tend to be close to 100%, with better parallelism.
The result shows that the QuEChERS method special for N-nitrosamine has excellent performance, and the experimental method can effectively collect 9N-nitrosamine targets and has good compatibility.
Analytical comparisons showed that the filler of table 6 and its formulation were very useful, superior to its filler selection, providing good purified products.
The time taken for this pretreatment part is only about 30 minutes, counted from the treatment time.
Various changes and modifications may be suggested to one skilled in the art based on the teachings herein and are within the scope of the appended claims.

Claims (10)

1. A method for simultaneously detecting the contents of multiple N-nitrosamines in food, which is characterized by comprising the following steps:
(1) Extracting an extract containing N-nitrosamines from a food product;
(2) Purifying the extract of (1) by QuEChERS purification method to obtain purified extract; the purification tubes used present a combination of the following fillers: c18 or C8 filler, PSA filler and Si filler, optionally also GCB filler;
(3) Detecting the purified extract by gas chromatography-mass spectrometry to determine N-nitrosamine content.
2. The method of claim 1, wherein said N-nitrosamine is a volatile N-nitrosamine; preferably, the plurality of N-nitrosamines is selected from: n-dimethyl nitrosamine, N-methylethylnitrosamine, N-diethyl nitrosamine, N-dipropyl nitrosamine, N-dibutyl nitrosamine, N-nitrosopiperidine, N-nitrosopyrrolidine, N-nitrosomorpholine, N-diphenyl nitrosamine.
3. The method of claim 1, wherein in step (2), the weight ratio of:
the proportion of the C18 filler, the PSA filler and the Si filler is (2-10) to (1-5); or
The proportion of the C8 filler, the PSA filler and the Si filler is (6-30) to (4-20) to (1-5); or
The proportion of the C18 filler, the PSA filler, the Si filler and the GCB filler is (6-30): (6-30): 1-5): 0.2-1.
4. The method of claim 1, wherein step (1) comprises: extracting food with organic solvent to obtain extract containing N-nitrosamine, and taking the extract as sample to be detected; preferably, the organic solvent is acetonitrile; more preferably, the acetonitrile is 5-50 mL/sample.
5. The method of claim 4, wherein the addition of the organic solvent further comprises the addition of anhydrous magnesium sulfate and sodium chloride;
preferably, the anhydrous magnesium sulfate is 50 mg-2 g/sample;
preferably, the sodium chloride is 50 mg-2 g/sample.
6. The method of claim 1, wherein in step (1):
before extraction, the method also comprises a step of crushing the food; or
After extraction, a step of centrifuging to obtain a supernatant is also included.
7. The method of claim 1, wherein, in the step (3) of gas chromatography-mass spectrometry detection: adopting a gas chromatography-mass spectrometer; preferably, the column is CD-BASEWAX.
8. A purification tube suitable for use in the QuEChERS purification method, the purification tube used having a combination of packing materials: c18 or C8 filler, PSA filler and Si filler, optionally also GCB filler; preferably, the weight ratio of:
the proportion of the C18 filler, the PSA filler and the Si filler is (2-10) to (1-5); or
The proportion of the C8 filler, the PSA filler and the Si filler is (6-30) to (4-20) to (1-5); or
The proportion of the C18 filler, the PSA filler, the Si filler and the GCB filler is (6-30): (6-30): 1-5): 0.2-1.
9. Use of the purification tube of claim 8 as a purification tube for purifying a food extract based on the QuEChERS purification method.
10. A kit for detecting the content of N-nitrosamine in food, which is characterized by comprising:
(a) An organic agent for extracting an extract containing N-nitrosamine from a food; preferably, the organic solvent is acetonitrile; preferably, anhydrous magnesium sulfate and sodium chloride are also included;
(b) A purge tube suitable for use in a QuEChERS purification method, the combination of packing present in the purge tube: c18 or C8 filler, PSA filler and Si filler, optionally also GCB filler;
preferably, the kit further comprises: a standard of N-nitrosamine; and (5) a reference substance.
Preferably, the kit further comprises: instructions for use of the method for detecting N-nitrosamine content in a food product.
CN202211226753.5A 2022-10-09 2022-10-09 Novel method for determining volatile N-nitrosamine in food Pending CN115598248A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202211226753.5A CN115598248A (en) 2022-10-09 2022-10-09 Novel method for determining volatile N-nitrosamine in food

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202211226753.5A CN115598248A (en) 2022-10-09 2022-10-09 Novel method for determining volatile N-nitrosamine in food

Publications (1)

Publication Number Publication Date
CN115598248A true CN115598248A (en) 2023-01-13

Family

ID=84847352

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202211226753.5A Pending CN115598248A (en) 2022-10-09 2022-10-09 Novel method for determining volatile N-nitrosamine in food

Country Status (1)

Country Link
CN (1) CN115598248A (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103823001A (en) * 2013-12-19 2014-05-28 上海市食品药品检验所 Novel dispersion solid phase extractive purification tube as well as preparation method and application thereof
CN110261517A (en) * 2019-08-06 2019-09-20 南通市产品质量监督检验所 Utilize the method for N- nitrosamine compound in GPC-GC/MS/MS measurement meat products
CN110286163A (en) * 2019-04-30 2019-09-27 国家地质实验测试中心 The analysis method of 9 kinds or more nitrosamine compounds in water
CN114646706A (en) * 2022-03-24 2022-06-21 沈阳农业大学 Detection method for simultaneously detecting heterocyclic amine, acrylamide and nitrosamine in meat product

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103823001A (en) * 2013-12-19 2014-05-28 上海市食品药品检验所 Novel dispersion solid phase extractive purification tube as well as preparation method and application thereof
CN110286163A (en) * 2019-04-30 2019-09-27 国家地质实验测试中心 The analysis method of 9 kinds or more nitrosamine compounds in water
CN110261517A (en) * 2019-08-06 2019-09-20 南通市产品质量监督检验所 Utilize the method for N- nitrosamine compound in GPC-GC/MS/MS measurement meat products
CN114646706A (en) * 2022-03-24 2022-06-21 沈阳农业大学 Detection method for simultaneously detecting heterocyclic amine, acrylamide and nitrosamine in meat product

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
QIXIAN ZHANG 等: "Analysis of 7 volatile N-nitrosamines in Chinese Sichuan salted vegetables by gas chromatography-tandem mass spectrometry coupled to modified QuEChERS extraction", FOOD CONTROL, vol. 98, 30 April 2019 (2019-04-30), pages 349 - 347 *
蒋林蓉 等: "基于QuEChERS净化的液相色谱-串联质谱法分析食品中的6种生物胺", 中国调味品, vol. 42, no. 08, 31 August 2017 (2017-08-31), pages 127 - 132 *
谢亚兴: "气-质联用法测定食品中N-二甲基亚硝胺方法的改进", 现代食品, vol. 28, no. 10, 28 May 2022 (2022-05-28), pages 187 - 190 *
赵博 等: "分散固相萃取-气相色谱-串联质谱法测定动物源性食品中9种N-亚硝胺类化合物", 色谱, vol. 34, no. 10, 31 October 2016 (2016-10-31), pages 998 - 1004 *

Similar Documents

Publication Publication Date Title
Lehotay et al. Automated mini-column solid-phase extraction cleanup for high-throughput analysis of chemical contaminants in foods by low-pressure gas chromatography—tandem mass spectrometry
HonorÚáHansen Arsenic speciation in seafood samples with emphasis on minor constituents: an investigation using high-performance liquid chromatography with detection by inductively coupled plasma mass spectrometry
Li et al. Simultaneous determination of nine types of phthalate residues in commercial milk products using HPLC-ESI-MS-MS
Zeng et al. Application of QuEChERS-based purification coupled with isotope dilution gas chromatography-mass spectrometry method for the determination of N-nitrosamines in soy sauce
Fan et al. Screening of multi-mycotoxins in fruits by ultra-performance liquid chromatography coupled to ion mobility quadrupole time-of-flight mass spectrometry
Liang et al. Fast determination of 14 mycotoxins in chestnut by dispersive solid‐phase extraction coupled with ultra high performance liquid chromatography‐tandem mass spectrometry
CN106018624A (en) HPLC detection method for nitrosamines in food
Qi et al. Qualitative screening and quantitative determination of multiclass water-soluble synthetic dyes in foodstuffs by liquid chromatography coupled to quadrupole Orbitrap mass spectrometry
Amelin et al. Combination of microwave heating extraction and dispersive liquid-liquid microextraction for the determination of nitrosoamines in foods using gas-liquid chromatography with a mass-spectrometric detector
Chen et al. Development of a high-performance liquid chromatography method for the simultaneous quantification of four organoarsenic compounds in the feeds of swine and chicken
CN106053634B (en) N- N-nitrosodimethylamine detection method in a kind of cooked meat product
Amelin et al. Rapid identification and determination of N-nitrosamines in food products by ultra-high-performance liquid chromatography–high resolution quadrupole-time-of-flight mass spectrometry by exact masses of protonated molecules
Komarova et al. Determination of volatile N-nitrosamines in food by high-performance liquid chromatography with fluorescence detection
CN109633028A (en) The detection method of spice additive in a kind of special medicine purposes formula food
CN112285263A (en) Method for quantitatively analyzing non-volatile organic acid in tobacco essence and feed liquid
CN109633015B (en) Method for detecting additive in entolipide
CN115598248A (en) Novel method for determining volatile N-nitrosamine in food
Xing et al. Determination of nitrophenolate sodium in aquatic products by HPLC–MS/MS with atmospheric pressure chemical ionization
CN107102078B (en) A kind of method of aflatoxin B1 in measurement Gardenia Yellow
Shi et al. Determination of seven phenolic acids in honey by counter flow salting-out homogeneous liquid–liquid extraction coupled with ultra-high performance liquid chromatography-tandem mass spectrometry
Wang et al. A novel and sensitive screening method for β-agonists in porcine urine by using atmospheric solid analysis probe source coupled tandem mass spectrometry
Jiang et al. Determination of 2 Phthalic Acid Esters in Fermented Grains by GC–MS/MS Coupled with Modified QuEChERS
Sin et al. Application of pentafluorophenyl hydrazine on detecting 2-dodecylcyclobutanone and 2-tetradecylcyclobutanone in irradiated chicken, pork and mangoes by gas chromatography-mass spectrometry
Li et al. Evaluation of various commercial lipid removal sorbents for the detection of 18 neonicotinoid insecticides and their metabolites in meat by liquid chromatography tandem mass spectrometry
Polyakova et al. Improved sample preparation and GC–MS analysis of priority organic pollutants

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination