An extraction method and a detection method of N-nitrosamines in aquatic products

Abstract

The application belongs to the technical field of substance analysis and detection, and provides an extraction method and a detection method for N-nitrosamine compounds in aquatic products. The N-nitrosamine compounds in the aquatic products to be measured are extracted by steam distillation, and the extraction rate of the N-nitrosamine compounds is high. The obtained distillate is purified and enriched by using an acid-modified coconut shell activated carbon column, the acid-modified coconut shell activated carbon column can effectively adsorb N-dimethyl-nitrosamine and N-diethyl-nitrosamine in the distillate, the acid-modified coconut shell activated carbon column is first eluted with n-hexane to remove part of the fat-soluble impurities, then N-dimethyl-nitrosamine and N-diethyl-nitrosamine are eluted with dichloromethane, due to the strong adsorption of the acid-modified coconut shell activated carbon column to the impurities, the impurities are not easily eluted by dichloromethane, so that the impurities, N-dimethyl-nitrosamine and N-diethyl-nitrosamine are effectively separated, and the purpose of purification is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of substance analysis and detection, and particularly relates to a method for extracting and detecting N-nitrosamine compounds in aquatic products. BACKGROUND

[0002] N-nitrosamine compounds are a class of compounds containing N-N=O structure. Research has found that more than 90% of the more than 300 known N-nitrosamine compounds have toxicity and carcinogenicity, can cause tumors in offspring through mother-to-child transmission, and also have strong teratogenic and mutagenic effects. Common N-nitrosamine compounds in food include N-dimethyl-nitrosamine (NDMA), N-methyl-ethyl-nitrosamine (NMEA), N-nitrosopyrrolidine (NPyr), N-nitrosopiperidine (NPIP), N-dipropyl-nitrosamine (NDPA), N-diethyl-nitrosamine (NDEA), N-dibutyl-nitrosamine (NDBA) and N-nitrosomorpholine (NMOR), etc., among which NDMA and NDEA are the most toxic. The International Agency for Research on Cancer (IARC) has rated NDMA and NDEA as 2A carcinogens and NPyr, NMEA, NPIP and NDBA as B-class general carcinogens.

[0003] In recent years, the products with NDMA content exceeding the standard mainly include aquatic products, including squid, cod, lionfish and other fish processing foods, so it is very important to detect and monitor NDMA and NDEA in aquatic products.

[0004] N-nitrosamine compounds have the characteristics of small relative molecular weight and easy volatilization, and their content in food is generally in the μg / kg level, the response is low, and the detection is difficult. In addition, the fish slice sample is usually complex after marinating and heat processing, and other impurities are easy to interfere with the determination accuracy, so the requirements for pretreatment and instrument analysis technology are relatively high. The GB 5009.26-2016 'National Food Safety Standard Determination of N-nitrosamine Compounds in Food' discloses using water vapor distillation to separate and extract N-nitrosamine compounds, and then using large-volume dichloromethane for multiple liquid-liquid extraction to extract and purify N-nitrosamine compounds. However, the above pretreatment method still has many shortcomings, such as a large sample size of 200g, a large amount of organic reagent for multiple extraction and rotary evaporation concentration, large consumption of organic reagent, long pretreatment process, low processing efficiency, especially difficult blank control, and high protection requirements for experimental operators.

[0005] Especially for aquatic products with rich oil content, simple organic solvent extraction cannot effectively remove interfering substances and enrich the analyte, and it is difficult to meet the detection requirements, and a large amount of sample needs to be weighed; distillation extraction or alkali treatment is first carried out, and then solid phase extraction, matrix solid phase dispersion microextraction or solid phase microextraction and other steps are carried out on the extraction liquid or supernatant to remove impurities, and finally rotary evaporation or nitrogen blowing is used to achieve the purpose of concentration and enrichment. According to relevant literature research and pre-experiment, when the sample weight is large, due to the oil contained in the aquatic products and the edible oil added in the processing process, the sample after alkali treatment still contains a certain amount of oil and other interfering substances, which needs to be removed and concentrated in multiple steps, which not only consumes manpower and material resources, but also easily leads to the loss of the analyte, resulting in inaccurate detection results. SUMMARY

[0006] Therefore, the purpose of the present application is to provide an extraction method and a detection method for N-nitrosamine compounds in aquatic products. The extraction method provided by the present application is simple to operate, and the loss of the analyte is small. When the detection is carried out, the detection result is accurate.

[0007] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0008] The present application provides an extraction method for N-nitrosamine compounds in aquatic products, comprising the following steps:

[0009] Mixing the aquatic product to be tested, N-nitrosamine compound mixed internal standard, sodium chloride, barium hydroxide and water to obtain a raw material solution;

[0010] Carrying out water vapor distillation on the raw material solution to obtain a distillation liquid;

[0011] Passing the distillation liquid through an acid-modified coconut shell activated carbon column, and sequentially carrying out water leaching, n-hexane leaching and dichloromethane elution to obtain a dichloromethane eluate;

[0012] Sequentially concentrating and redissolving the dichloromethane eluate to obtain an on-machine sample;

[0013] The N-nitrosamine compounds include N-dimethyl-nitrosamine and N-diethyl-nitrosamine.

[0014] Preferably, the N-nitrosamine compound mixed internal standard includes N-dimethyl-nitrosamine-d6 and N-diethyl-nitrosamine-d4.

[0015] Preferably, based on the mass of the aquatic product to be tested, the addition amount of N-dimethyl-nitrosamine-d6 and N-diethyl-nitrosamine-d4 is 2.0-10.0 ng / g, respectively.

[0016] Preferably, the ratio of the amounts of the to-be-tested aquatic product, the N-nitrosamine compound mixed internal standard, sodium chloride, barium hydroxide and water is 5-20 g: 20-100 ng: 10-20 g: 0.5-2.0 g: 30-50 mL.

[0017] Preferably, the temperature of the cooling water is ≤15℃ during the water vapor distillation.

[0018] Preferably, before the distillate passes through the acid-modified coconut shell activated carbon column, the acid-modified coconut shell activated carbon column is activated, and the activation comprises sequentially performing dichloromethane activation, methanol activation and water activation.

[0019] Preferably, after the dichloromethane elution, the obtained eluate is dehydrated; the dehydration is performed by a desiccant dehydration, and the desiccant for the desiccant dehydration is anhydrous sodium sulfate.

[0020] Preferably, the concentration is performed by nitrogen blowing concentration; and the reagent for the redissolution comprises methanol.

[0021] Preferably, the temperature for the nitrogen blowing concentration is ≤25℃.

[0022] The application further provides a method for detecting N-nitrosamine compounds in aquatic products, comprising the following steps:

[0023] The method for extracting N-nitrosamine compounds in to-be-tested aquatic products is used to extract N-nitrosamine compounds in to-be-tested aquatic products to obtain an on-machine sample;

[0024] The on-machine sample is subjected to gas chromatography-mass spectrometry detection to obtain chromatographic information of N-nitrosamine compounds in the to-be-tested aquatic products;

[0025] The chromatographic information of N-nitrosamine compounds in the to-be-tested aquatic products is substituted into a preset standard curve to obtain the content of N-nitrosamine compounds in the to-be-tested aquatic products;

[0026] The N-nitrosamine compounds comprise N-dimethyl-nitrosamine and N-diethyl-nitrosamine;

[0027] The gas chromatography-mass spectrometry detection comprises gas chromatography detection and mass spectrometry detection;

[0028] The parameters of the gas chromatography detection comprise:

[0029] The chromatographic column is a strong-polarity quartz capillary column;

[0030] The temperature of the injection port is 230℃;

[0031] The carrier gas is helium, and the flow rate is 1 mL / min;

[0032] The injection mode is splitless injection.

[0033] The injection volume was 1 μL;

[0034] The temperature program was initial temperature 40 °C, hold 3 min, then increased to 170 °C at 8 °C / min, and then increased to 230 °C at 20-30 °C / min, hold 3 min;

[0035] The parameters of the mass spectrometry detection included:

[0036] The interface temperature of the chromatography and mass spectrometry was 230 °C;

[0037] The ionization mode was electron impact source;

[0038] The ionization energy was 70 eV;

[0039] The solvent delay was 9.5 min;

[0040] The ion source temperature was 230 °C;

[0041] The quadrupole rod temperature was 150 °C.

[0042] The monitoring mode was multiple reaction monitoring mode;

[0043] The monitoring conditions were shown in Table 1:

[0044] Table 1 Monitoring conditions

[0045]

[0046] The application provides an extraction method of N-nitrosamine compounds in aquatic products, comprising the following steps: mixing to-be-tested aquatic products, N-nitrosamine compound mixed internal standard, sodium chloride, barium hydroxide and water to obtain a raw material liquid; performing water vapor distillation on the raw material liquid to obtain a distillation liquid; passing the distillation liquid through an acid-modified coconut shell activated carbon column, and sequentially performing water leaching, n-hexane leaching and dichloromethane elution to obtain a dichloromethane elution liquid; and sequentially performing concentration and redissolution on the dichloromethane elution liquid to obtain an on-machine sample; the N-nitrosamine compounds include N-dimethyl-nitrosamine and N-diethyl-nitrosamine. The application extracts N-nitrosamine compounds in to-be-tested aquatic products by water vapor distillation, and has a high extraction rate of N-nitrosamine compounds. The obtained distillation liquid is purified and enriched by an acid-modified coconut shell activated carbon column, the acid-modified coconut shell activated carbon column can effectively adsorb N-dimethyl-nitrosamine and N-diethyl-nitrosamine in the distillation liquid, then the acid-modified coconut shell activated carbon column is leached with n-hexane to remove part of fat-soluble impurities, and then N-dimethyl-nitrosamine and N-diethyl-nitrosamine are eluted with dichloromethane, because of the strong adsorption of the acid-modified coconut shell activated carbon column to impurities, the impurities are not easily eluted with dichloromethane, so that the impurities, N-dimethyl-nitrosamine and N-diethyl-nitrosamine are effectively separated, and the purpose of purification is achieved. It can be seen that the acid-modified coconut shell activated carbon column effectively solves the problem of impurity interference, and achieves good purification effect; multiple impurity removal and concentration are avoided, the loss of to-be-tested N-nitrosamine compounds is reduced, and when detection is performed, the accuracy of the detection result of N-nitrosamine compounds in to-be-tested aquatic products is improved.

[0047] The application further provides a detection method of N-nitrosamines in aquatic products, comprising the following steps: extracting N-nitrosamines in the aquatic products to be detected according to the extraction method in the above technical solution, and obtaining a sample for machine; performing gas chromatography-mass spectrometry detection on the sample for machine, and obtaining chromatographic information of N-nitrosamines in the aquatic products to be detected; inputting the chromatographic information of N-nitrosamines in the aquatic products to be detected into a preset standard curve, and obtaining the content of N-nitrosamines in the aquatic products to be detected; the N-nitrosamines include N-dimethyl-nitrosamine and N-diethyl-nitrosamine; the gas chromatography-mass spectrometry detection comprises gas chromatography detection and mass spectrometry detection; parameters of the gas chromatography detection include that a chromatographic column is a strong polarity quartz capillary column, an injection port temperature is 230 DEG C, a carrier gas is helium with a flow rate of 1 mL / min, an injection mode is splitless injection, an injection amount is 1 mu L, and a temperature rising program is that an initial temperature is 40 DEG C, maintained for 3 min, then raised to 170 DEG C at a rate of 8 DEG C / min, then raised to 230 DEG C at a rate of 20-30 DEG C / min, and maintained for 3 min; parameters of the mass spectrometry detection include that a chromatographic and mass spectrometry interface temperature is 230 DEG C, an ionization mode is an electron impact source, an ionization energy is 70 eV, a solvent delay is 9.5 min, an ion source temperature is 230 DEG C, a quadrupole temperature is 150 DEG C, and a monitoring mode is a multiple reaction monitoring mode; and monitoring conditions are shown in Table 1. The application realizes rapid, environment-friendly, high-sensitivity and high-accuracy detection of N-dimethyl-nitrosamine and N-diethyl-nitrosamine in aquatic products by using gas chromatography-mass spectrometry detection and analysis. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 A structure schematic diagram of a steam distillation instrument used for the steam distillation of the application; wherein, 1-reflux condenser, 2-condensed liquid, 3-steam, 4-sample to be distilled, 5-receiving device, 6-ice bath cup;

[0049] Figure 2 N-dimethyl-nitrosamine and N-diethyl-nitrosamine standard and internal standard total ion chromatogram and MRM diagram (target: 20.0 mu g / L, internal standard: 50 mu g / L);

[0050] Figure 3 Total ion chromatogram and MRM diagram of baked cod fillet purified by coconut shell activated carbon column;

[0051] Figure 4 Total ion chromatogram and MRM diagram of baked cod fillet purified by acid modified coconut shell activated carbon column;

[0052] Figure 5 Total ion chromatogram and MRM diagram of a blank test;

[0053] Figure 6Determine the total ion chromatogram and MRM chart for the black fish slice with 1.0 μg / kg standard;

[0054] Figure 7 Determine the total ion chromatogram and MRM chart for the squid silk sample in Example 7;

[0055] Figure 8 Determine the total ion chromatogram and MRM chart for the shrimp sample in Example 8. DETAILED DESCRIPTION

[0056] The application provides an extraction method of N-nitrosamine compounds in aquatic products, comprising the following steps:

[0057] Mixing the to-be-tested aquatic product, N-nitrosamine compound mixed internal standard, sodium chloride, barium hydroxide and water to obtain a raw material solution;

[0058] Carrying out water vapor distillation on the raw material solution to obtain a distillation solution;

[0059] Passing the distillation solution through an acid-modified coconut shell activated carbon column, and sequentially performing water leaching, n-hexane leaching and dichloromethane elution to obtain a dichloromethane eluate;

[0060] Sequentially performing concentration and redissolution on the dichloromethane eluate to obtain an on-machine sample;

[0061] The N-nitrosamine compounds include N-dimethyl nitrosamine and N-diethyl nitrosamine.

[0062] The application mixes the to-be-tested aquatic product, N-nitrosamine compound mixed internal standard, sodium chloride, barium hydroxide and water to obtain a raw material solution.

[0063] In the application, the to-be-tested aquatic product preferably includes one or more of roasted fish slices, fresh fish slices, shrimp and squid silk; the roasted fish slices preferably include roasted cod slices; and the fresh fish slices preferably include black fish slices.

[0064] In the application, the to-be-tested aquatic product is preferably pretreated before extraction; and the pretreatment preferably includes mincing and homogenization, and is subjected to frozen preservation. In the application, the N-nitrosamine compounds include N-dimethyl nitrosamine and N-diethyl nitrosamine.

[0065] In the application, the N-nitrosamine compound mixed internal standard preferably includes N-dimethyl nitrosamine-d6 and N-diethyl nitrosamine-d4.

[0066] In the application, the water is preferably deionized water.

[0067] In the present application, the dosage ratio of the to-be-tested aquatic product, the N-nitrosamine compound mixed internal standard, sodium chloride, barium hydroxide and water is preferably 5-20 g: 20-100 ng: 10-20 g: 0.5-2.0 g: 30-50 mL.

[0068] In the present application, the addition amount of N-dimethyl-nitrosamine-d6 and N-diethyl-nitrosamine-d4 is preferably 2.0-10.0 ng / g based on the mass of the to-be-tested aquatic product.

[0069] After obtaining the raw material solution, the present application performs water vapor distillation on the raw material solution to obtain a distillate.

[0070] In the present application, the temperature of the cooling water during the water vapor distillation is preferably ≤15℃.

[0071] In the present application, the temperature of the cooling water during the water vapor distillation is controlled to be ≤15℃, which can make the distillate condense as soon as possible, avoid loss, and be conducive to improving the distillation recovery rate.

[0072] In the present application, the water vapor distillation is preferably performed on a water vapor distillation instrument, and a structural diagram of the water vapor distillation instrument is shown in Figure 1 , wherein 1 is a reflux condenser tube, 2 is a condensate, 3 is water vapor, 4 is a to-be-distilled sample, 5 is a receiving device, and 6 is an ice bath cup.

[0073] In the present application, the water vapor distillation instrument is preferably cleaned with distilled water before use.

[0074] After obtaining the distillate, the present application passes the distillate through an acid-modified coconut shell activated carbon column and sequentially performs water rinsing, n-hexane rinsing and dichloromethane elution to obtain a dichloromethane eluate.

[0075] In the present application, the acid-modified coconut shell activated carbon column is preferably purchased from Kangyuan Technology Co., Ltd.

[0076] In the present application, before the distillate passes through the acid-modified coconut shell activated carbon column, the acid-modified coconut shell activated carbon column is preferably activated; the activation preferably includes sequentially performing dichloromethane activation, methanol activation and water activation. The present application does not make specific limitations on the operation of the activation, and any activation method known to those skilled in the art can be adopted.

[0077] The present application does not make specific limitations on the operation of the water rinsing and n-hexane rinsing, and any rinsing method known to those skilled in the art can be adopted.

[0078] The present application does not make specific limitations on the dichloromethane elution method, and any elution method known to those skilled in the art can be adopted.

[0079] After the dichloromethane elution, the application preferably further comprises dehydrating the obtained eluent.

[0080] In the application, the acid-modified coconut shell activated carbon column can effectively adsorb N-dimethyl nitrosamine and N-diethyl nitrosamine in the distillate, then part of water-soluble compounds such as small-molecule amine compounds are removed by water elution, then part of fat-soluble impurities are removed by n-hexane elution, then N-dimethyl nitrosamine and N-diethyl nitrosamine are eluted by 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 interference impurities and N-dimethyl nitrosamine and N-diethyl nitrosamine are effectively separated, and the purpose of purification is achieved.

[0081] After obtaining the dichloromethane eluent, the application sequentially concentrates and redissolves the dichloromethane eluent to obtain an on-machine sample.

[0082] In the application, the concentration is preferably nitrogen blowing concentration, the nitrogen blowing concentration is preferably ≤25℃, and the nitrogen blowing concentration is preferably not dry.

[0083] In the application, controlling the nitrogen blowing concentration temperature ≤25℃ can effectively avoid the loss of N-nitrosamine compounds in the nitrogen blowing concentration process, and improve the recovery rate.

[0084] The application also provides a detection method of N-nitrosamine compounds in aquatic products, comprising the following steps:

[0085] According to the extraction method in the above technical solution, the N-nitrosamine compounds in the aquatic products to be tested are extracted to obtain an on-machine sample.

[0086] The on-machine sample is detected by gas chromatography-mass spectrometry to obtain the chromatographic information of the N-nitrosamine compounds in the aquatic products to be tested.

[0087] The chromatographic information of the N-nitrosamine compounds in the aquatic products to be tested is substituted into the preset standard curve to obtain the content of the N-nitrosamine compounds in the aquatic products to be tested.

[0088] According to the extraction method in the above technical solution, the N-nitrosamine compounds in the aquatic products to be tested are extracted to obtain an on-machine sample.

[0089] The parameters of the extraction are preferably consistent with the above technical solution, which will not be repeated here.

[0090] After obtaining the sample, the sample is detected by gas chromatography-mass spectrometry to obtain the chromatographic information of the N-nitrosamine compound in the water product to be detected.

[0091] In the present application, the gas chromatography-mass spectrometry detection includes gas chromatography detection and mass spectrometry detection.

[0092] In the present application, the parameters of the gas chromatography detection include:

[0093] The chromatographic column is a strong polar quartz capillary column, which is preferably an HP-INNOWAX column, and the size of the HP-INNOWAX column is preferably 30 m x 0.25 mm (inner diameter) x 0.25 μm (film thickness).

[0094] The inlet temperature is 230℃;

[0095] The carrier gas is helium with a flow rate of 1 mL / min, and the purity of the helium is preferably ≥ 99.999%;

[0096] The injection mode is splitless injection;

[0097] The injection volume is 1 μL;

[0098] The temperature program is an initial temperature of 40℃, holding for 3 min, increasing to 170℃ at a rate of 8℃ / min, then increasing to 230℃ at a rate of 20-30℃ / min, holding for 3 min.

[0099] In the present application, the parameters of the mass spectrometry detection include:

[0100] The chromatographic and mass spectrometry interface temperature is 230℃;

[0101] The ionization mode is electron impact source;

[0102] The ionization energy is 70 eV;

[0103] The solvent delay is 9.5 min;

[0104] The ion source temperature is 230℃;

[0105] The quadrupole rod temperature is 150℃.

[0106] The monitoring mode is multiple reaction monitoring mode;

[0107] The monitoring conditions are shown in Table 1.

[0108] After obtaining the chromatographic information of the N-nitrosamine compound in the water product to be detected, the chromatographic information of the N-nitrosamine compound in the water product to be detected is substituted into the preset standard curve to obtain the content of the N-nitrosamine compound in the water product to be detected.

[0109] In the present application, the abscissa of the preset standard curve is preferably the concentration of N-nitrosamines, and the ordinate is preferably the peak area ratio of N-nitrosamines and the corresponding deuterium isotope internal standard.

[0110] The method for establishing the preset standard curve is not specifically limited in the present application, and the numerical standard curve establishing method of those skilled in the art can be used.

[0111] The extraction method and detection method of N-nitrosamines in aquatic products provided by the present application will be described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0112] 1. Instruments and equipment

[0113] Gas chromatography-tandem mass spectrometer (GC-MS / MS), all-glass steam distillation apparatus, nitrogen blowing instrument, electronic balance with a sensitivity of 0.001 g, ice maker, condensate water machine, solid phase extraction device (with vacuum pump).

[0114] 2. Detection conditions of gas chromatography-mass spectrometry

[0115] Gas chromatography conditions

[0116] Chromatographic column: strong polarity quartz capillary column HP-INNOWAX column, 30 m x 0.25 mm (inner diameter) x 0.25 μm (film thickness), or equivalent;

[0117] Inlet temperature: 230°C;

[0118] Carrier gas: helium, purity ≥ 99.999%; flow rate 1 mL / min;

[0119] Injection mode: splitless injection;

[0120] Injection volume: 1 μL;

[0121] Temperature program: initial temperature 40°C, hold for 3 min, increase to 170°C at 8°C / min, then increase to 230°C at 20°C / min, hold for 3 min.

[0122] Mass spectrometry conditions

[0123] Chromatography and mass spectrometry interface temperature: 230°C;

[0124] Ionization mode: electron impact source (EI source);

[0125] Ionization energy: 70 eV;

[0126] Solvent delay: 9.5 min;

[0127] Ion source temperature: 230 °C; quadrupole temperature: 150 °C;

[0128] Monitoring mode: multiple reaction monitoring (MRM) mode;

[0129] The monitoring conditions are shown in Table 1.

[0130] 3. Reagents and materials

[0131] Unless otherwise specified, the reagents used in the present application are of analytical purity, and the water is first-grade water specified in GB / T 6682.

[0132] 3.1 Reagents

[0133] 3.1.1 Dichloromethane (CH2Cl2): chromatographically pure.

[0134] 3.1.2 Anhydrous sodium sulfate (Na2SO4), roasted at 400 °C for 3 hours, and bottled after cooling for storage in a desiccator.

[0135] 3.1.3 Sodium chloride (NaCl): extra pure.

[0136] 3.1.4 Barium hydroxide (Ba(OH)2).

[0137] 3.1.5 n-Hexane (C6H 14 ).

[0138] 3.1.6 Methanol (CH3OH): chromatographically pure.

[0139] 3.1.7 Acid-modified coconut shell activated carbon column and coconut shell activated carbon column: both with a specification of 1000 mg / 6 mL, purchased from Kangyuan Technology Co., Ltd.

[0140] 3.1.8 The aquatic products were all collected from supermarkets and stored at -18 °C.

[0141] 3.2 Standards

[0142] 3.2.1 N-nitrosamine mixed standard solution (containing N-dimethyl-nitrosamine, N-diethyl-nitrosamine): with a concentration of 1000 μg / mL, a solvent of methanol, and purchased from O2SI.

[0143] 3.2.2 N-dimethyl-nitrosamine-d6 internal standard solution (NDMA-d6): with a concentration of 100 μg / mL, a solvent of methanol, and purchased from BePure.

[0144] 3.2.3 N-diethyl-nitrosamine-d4 internal standard solution (NDEA-d4): with a concentration of 100 μg / mL, a solvent of methanol, and purchased from BePure.

[0145] 3.3 Preparation of standard solutions

[0146] 3.3.1 N-nitrosamine mixed standard stock solution (100 μg / mL): Accurately pipette 1 mL of N-nitrosamine mixed standard solution (3.2.1) into a 10 mL volumetric flask, dilute to the mark with methanol, and mix well. Transfer the solution to a brown glass container, and store at -18°C in the dark for 6 months.

[0147] 3.3.2 N-nitrosamine mixed standard application solution (1.0 μg / mL): Accurately pipette 1 mL of N-nitrosamine mixed standard stock solution (100 μg / mL) into a 100 mL volumetric flask, dilute to the mark with methanol, and mix well. Transfer the solution to a brown glass container, and store at -18°C in the dark for 3 months.

[0148] 3.3.3 N-nitrosamine mixed internal standard application solution (1.0 μg / mL): Accurately pipette 1 mL of N-dimethyl-nitrosamine-d6 internal standard solution (3.2.2) and 1 mL of N-diethyl-nitrosamine-d4 internal standard solution (3.2.3) into a 100 mL volumetric flask, dilute to the mark with methanol, and mix well. Transfer the solution to a brown glass container, and store at -18°C in the dark for 6 months.

[0149] 3.3.4 N-nitrosamine standard and internal standard mixed series working solution: Pipette 5.0 μL, 10.0 μL, 20.0 μL, 50.0 μL, 100.0 μL, and 200.0 μL of N-nitrosamine mixed standard application solution (1.0 μg / mL) and 50 μL of N-nitrosamine mixed internal standard application solution (1.0 μg / mL) into a 1 mL sample vial, and supplement with methanol to 1 mL. Mix well. The N-dimethyl-nitrosamine standard series working solution has concentrations of 5.0 μg / L, 10.0 μg / L, 20.0 μg / L, 50.0 μg / L, 100.0 μg / L, and 200.0 μg / L series mixed standard solution, and the internal standard has a concentration of 50.0 μg / L. Prepare immediately before use.

[0150] Example 1

[0151] Preparation of a preset standard curve

[0152] Inject the N-nitrosamine standard and internal standard mixed series working solution into a gas chromatograph-tandem mass spectrometer in order of decreasing concentration, and plot a standard curve with the concentration of N-nitrosamine compounds as the abscissa x and the peak area ratio of N-nitrosamine compounds and their corresponding deuterated isotopic internal standards as the ordinate y. The results are shown in Table 2.

[0153] Table 2 Linear range, regression equation, and fitting coefficient of N-nitrosamine compounds

[0154]

[0155] Figure 2 N-nitrosodimethylamine and N-nitrosodiethylamine standard and internal standard total ion chromatogram and MRM chromatogram (target: 20.0 μg / L, internal standard: 50 μg / L) from Figure 2 It can be seen that: N-nitrosodimethylamine and its internal standard N-nitrosodimethylamine-d6 standard solution at about 11.0 min, N-nitrosodiethylamine standard and its internal standard N-nitrosodiethylamine-d4 internal standard solution at about 12.3 min. The chromatographic peak is symmetrical and well separated, the GC-MS / MS sensitivity is high, and it can meet the experimental requirements of water product detection.

[0156] Example 2

[0157] Extraction method of N-nitrosamines in aquatic products

[0158] 2.1 Preparation of sample

[0159] The grilled cod fillets were minced and homogenized, and about 200 g of sample was taken into a sample bottle, labeled and stored at -18°C for future use. Before detection, the sample was thawed and mixed at 4°C, and then taken for detection.

[0160] 2.2 Extraction using Figure 1 a steam distillation apparatus as shown in the figure

[0161] 10.0 g of sample (accurate to 0.001 g) was weighed, 50 μL of N-nitrosamine mixed internal standard solution (1.0 μg / mL) was added, 40 mL of water, 20 g of sodium chloride and 1 g of barium hydroxide were added to the distillation flask, and the distillation device was fully mixed and the steam outlet was passed below the liquid surface. 50 mL of ice water was added to a 250 mL triangular flask, the outlet of the condenser tube (the cooling water temperature was controlled below 15°C) was inserted below the ice water surface, and the triangular flask was placed in an ice bath. The condenser water was turned on, and the steam distillation apparatus (see Figure 1 ) was turned on for heating and distillation. After collecting 150 mL of condensate, the heating device was turned off and the distillation was stopped, and the distillate was obtained.

[0162] 2.3 Purification

[0163] An acid-modified coconut shell activated carbon column (as a comparison, the acid-modified coconut shell activated carbon column was replaced with a coconut shell activated carbon column) was taken, the vacuum pump of the solid phase extraction device was turned on, and the acid-modified coconut shell activated carbon column was activated with 10 mL of dichloromethane, 10 mL of methanol and 10 mL of water in turn. Then 100 mL of distillate was passed through the modified coconut shell carbon column at a speed of 5 mL / min, the column was washed with 10 mL of water, vacuumed for 2 min to remove the water in the column, then washed with 10 mL of n-hexane, and finally eluted with 10 mL of dichloromethane. The eluate was dehydrated with a small amount of anhydrous sodium sulfate and collected in a 15 mL plastic centrifuge tube, 0.5 mL of methanol was added, and the dichloromethane eluate was obtained.

[0164] 2.4 Concentration

[0165] The dichloromethane eluate was concentrated under nitrogen at 25°C or below, and the nitrogen was blown off to 0.5 mL or less (not dried). Methanol was added to 1 mL, and the mixture was vortexed and then transferred to a sample vial for instrument detection.

[0166] 2.5 Detection

[0167] The instrument sample obtained in 2.4 was detected according to the parameters shown in 2.

[0168] Figure 3 The total ion chromatogram and MRM chromatogram of baked cod fillet purified by acid-modified coconut shell activated carbon column were determined; from Figure 3 It can be seen that: the acid-modified coconut shell activated carbon column for the concentration and purification of nitrosamine compounds in the distillation liquid of baked cod fillet, compared with

[0169] Figure 4 The total ion chromatogram and MRM chromatogram of baked cod fillet purified by acid-modified coconut shell activated carbon column were determined; from Figure 4 It can be seen that: the acid-modified coconut shell activated carbon column for the concentration and purification of nitrosamine compounds in the distillation liquid of baked cod fillet, compared with Figure 3 compared, the impurity interference before the N-dimethyl nitrosamine peak (about 10.8 min) is significantly reduced, reducing the qualitative and quantitative interference of N-dimethyl nitrosamine (10.97 min), greatly improving the sensitivity of the method, improving the accuracy and precision, sensitivity of the detection method, and it is speculated that the acid-modified coconut shell activated carbon column improves the adsorption capacity of impurities, so that the dichloromethane elution of N-nitrosamine compounds cannot elute the interfering substances, thereby separating the impurities and N-nitrosamine compounds.

[0170] 2.5.1 Qualitative determination

[0171] Compared with the standard series working solution, the mass chromatographic peak retention time of the sample should be consistent with the standard solution, and the allowed deviation is less than ± 2.5%, and in the sample mass spectrum after deducting the background, the selected ions all appear and the signal-to-noise ratio is ≥3, and the relative abundance of the qualitative ion pair (expressed by the intensity percentage relative to the strongest ion abundance) is equivalent to the relative abundance of the standard use solution, and the allowed deviation is not more than the range specified in Table 3, then it can be judged that the corresponding measured substance exists in the sample, and the specific requirements are shown in Table 3.

[0172] Table 3 Maximum allowed deviation of relative ion abundance in qualitative determination

[0173] Relative ion abundance >50％ 20％～50％ 10％～20％ ≤10％ Allowed maximum deviation ±20％ ±25％ ±30％ ±50％

[0174] 2.5.2 Quantitative determination

[0175] The content of N-nitrosamines in the aquatic product sample is calculated according to Formula 1:

[0176]

[0177] In Formula 1:

[0178] X - the content of N-nitrosamines in the sample, in units of micrograms per kilogram (μg / kg);

[0179] p - the peak area ratio of the chromatographic peak of N-nitrosamines in the sample to the chromatographic peak of the corresponding internal standard, the corresponding N-nitrosamine concentration obtained by the standard curve, in units of micrograms per liter (μg / L);

[0180] p0 - the peak area ratio of the chromatographic peak of N-nitrosamines in the blank test to the chromatographic peak of the corresponding internal standard, the corresponding N-nitrosamine concentration obtained by the standard curve, in units of micrograms per liter (μg / L);

[0181] V - the final constant volume of the test solution, in units of milliliters (mL);

[0182] m - the mass of the sample, in units of grams (g);

[0183] 1000 - conversion coefficient;

[0184] The calculation result is kept to two decimal places.

[0185] The ratio of the peak area of N-nitrosamines to the peak area of the corresponding isotopic internal standard is calculated, and the concentration of the measured compound in the sample solution is obtained according to the standard curve. The response values of the standard working solution and the sample solution of N-nitrosamines should be within the linear response range of the standard curve. If the content exceeds the range of the standard curve, the sample mass or the constant volume needs to be adjusted for re-detection.

[0186] Example 3

[0187] The same as Example 2, except that no aquatic product sample is added.

[0188] Figure 5 The total ion flow chromatogram and the MRM diagram are determined for the blank test, and the chromatographic peak of N-nitrosamines is not found. Figure 5It can be seen that: the blank test does not detect N-dimethyl nitrosamine and N-diethyl nitrosamine. N-dimethyl nitrosamine and N-diethyl nitrosamine have small molecular weight and large impurity interference, and the high blank value is caused by the distillation device, large amount of reagent used and the like. The extraction method provided in the application uses a Teflon tube instead of a plastic tube to avoid the dissolution of interfering substances from the plastic tube under high temperature, thereby reducing the high blank value. In addition, the extraction method provided in the application avoids large-volume liquid-liquid extraction as much as possible, reduces the amount of reagent (only 10 mL of dichloromethane), avoids large-volume concentration, and reduces the blank value.

[0189] Example 4 recovery experiment

[0190] The blank matrix of roasted cod fillets, black fish fillets, shrimp and squid filaments is taken, and 1.0 μg / kg, 5.0 μg / kg and 10.0 μg / kg of N-dimethyl nitrosamine and N-diethyl nitrosamine are added. The extraction method in Example 2 is used for extraction, and the detection conditions of the gas chromatography tandem mass spectrometer (GC-MS / MS) shown in 2 are used for parallel determination for 6 times, and the results are shown in Table 4.

[0191] Table 4 recovery rate of N-nitrosamine compounds in aquatic products (%)(n=6)

[0192]

[0193] It can be seen from Table 4 that the recovery rate of N-dimethyl nitrosamine is 74.3% to 107.8%, and the RSD is 3.2% to 12.3%; the recovery rate of N-diethyl nitrosamine is 83.5% to 115.5%, and the RSD is 1.4% to 9.6%; which meets the methodological requirements.

[0194] Figure 6 The black fish fillets are added with 1.0 μg / kg to determine the total ion chromatogram and MRM diagram; from Figure 6 It can be seen that: the blank matrix of roasted cod fillets, black fish fillets, shrimp and squid filaments is taken, and 1.0 μg / kg, 5.0 μg / kg and 10.0 μg / kg of N-dimethyl nitrosamine and N-diethyl nitrosamine are added. The extraction method in Example 2 is used for extraction, and the detection conditions of the gas chromatography tandem mass spectrometer (GC-MS / MS) shown in 2 are used for parallel determination for 6 times, and the results are shown in Table 4.

[0195] Example 5 precision

[0196] The blank matrix of roasted cod fillets, black fish fillets, shrimp and squid filaments is taken, and 1.0 μg / kg of N-dimethyl nitrosamine and N-diethyl nitrosamine is added. The extraction method in Example 2 is used for extraction, and the detection conditions of the gas chromatography-mass spectrometry shown in 2 are used for parallel determination for 6 times, and the precision results are obtained.

[0197] The results are: precision 7.5%~9.6%, which meets the methodological requirements.

[0198] Example 6 quantitative limit and detection limit

[0199] The blank matrix of baked cod fillets was taken, and N-dimethyl nitrosamine and N-diethyl nitrosamine were added at 1.0 μg / kg, respectively, and the determination was carried out according to Example 2. The average signal-to-noise ratio of the sample was 35.2 and 42.6, respectively, both of which were greater than 10. Therefore, the quantitative limit was determined to be 1.0 μg / kg. Then, according to the detection limit being 1 / 3 of the quantitative limit, the detection limit was determined to be 0.3 μg / kg.

[0200] Example 7

[0201] The difference from Example 2 is that the source of the squid threads is different.

[0202] Figure 7 The total ion flow chromatogram and MRM diagram of the squid thread sample were determined. Figure 7 It can be seen that the content of N-dimethyl nitrosamine and N-diethyl nitrosamine in the squid thread is 3.2 μg / kg and 0.7 μg / kg, respectively.

[0203] Example 8

[0204] The difference from Example 2 is that the source of the shrimp is different.

[0205] Figure 8 The total ion flow chromatogram and MRM diagram of the shrimp sample were determined. Figure 8 It can be seen that the content of N-dimethyl nitrosamine and N-diethyl nitrosamine in the shrimp is 24.3 μg / kg and 0.5 μg / kg, respectively.

[0206] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for extracting N-nitrosamine compounds from aquatic products, characterized in that, Includes the following steps: The aquatic product to be tested, N-nitrosamine compounds mixed internal standard, sodium chloride, barium hydroxide and water were mixed to obtain the raw material solution; The raw material liquid is subjected to steam distillation to obtain a distillate; The distillate was used to modify a coconut shell activated carbon column with acid, and then sequentially eluted with water, hexane, and dichloromethane to obtain a dichloromethane eluent. The dichloromethane eluent was concentrated and reconstituted sequentially to obtain the sample for use on the instrument; The N-nitrosamine compounds include N-dimethylnitrosamine and N-diethylnitrosamine; The N-nitrosamine compound mixture internal standard includes N-dimethylnitrosamine-d6 and N-diethylnitrosamine-d4; The ratio of the tested aquatic product, the mixed internal standard of N-nitrosamine compounds, sodium chloride, barium hydroxide and water is 5-20g: 20-100ng: 10-20g: 0.5-2.0g: 30-50mL; During the steam distillation process, the temperature of the cooling water is ≤15℃; The concentration method is nitrogen blowing concentration; the redissolution reagent includes methanol; The nitrogen blowing concentration temperature is ≤25℃.

2. The extraction method according to claim 1, characterized in that, Based on the quality of the aquatic product to be tested, the addition amounts of N-dimethylnitrosamine-d6 and N-diethylnitrosamine-d4 are 2.0 to 10.0 ng / g, respectively.

3. The extraction method according to claim 1, characterized in that, Before the distillate passes through the acid-modified coconut shell activated carbon column, the acid-modified coconut shell activated carbon column is further activated, which includes sequential activation with dichloromethane, methanol and water.

4. The extraction method according to claim 1, characterized in that, After elution with dichloromethane, the eluent is further dehydrated; the dehydration is performed by desiccant dehydration, and the desiccant used for dehydration is anhydrous sodium sulfate.

5. A method for detecting N-nitrosamine compounds in aquatic products, characterized in that, Includes the following steps: The N-nitrosamine compounds in the aquatic product to be tested are extracted according to the extraction method described in any one of claims 1 to 4 to obtain the sample for use in the instrument; The samples were subjected to gas chromatography-mass spectrometry to obtain chromatographic information of N-nitrosamine compounds in the aquatic products to be tested. The chromatographic information of N-nitrosamine compounds in the aquatic product to be tested is substituted into a preset standard curve to obtain the content of N-nitrosamine compounds in the aquatic product to be tested. The N-nitrosamine compounds include N-dimethylnitrosamine and N-diethylnitrosamine; The gas chromatography-mass spectrometry (GC-MS) detection includes both gas chromatography and mass spectrometry detection. The parameters detected by gas chromatography include: The chromatographic column is a highly polar quartz capillary column; The injection port temperature is 230℃; The carrier gas is helium, and the flow rate is 1 mL / min; The injection method is splitless injection; The injection volume was 1 μL; The heating program is as follows: initial temperature 40℃, hold for 3 min, increase to 170℃ at 8℃ / min, then increase to 230℃ at 20-30℃ / min, hold for 3 min; The parameters for mass spectrometry detection include: The interface temperature between chromatography and mass spectrometry is 230℃. The ionization method is electron bombardment source; The ionization energy is 70 eV; Solvent delay 9.5 min; The ion source temperature is 230℃; The quadrupole temperature is 150℃; The monitoring mode is a multi-response monitoring mode; The monitoring conditions are shown in Table 1: Table 1 Monitoring Conditions

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