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This standard replaces GB/T 5009.26-2003 Determination of N-nitrosamines in Foods.
The following main changes have been made in this standard with respect to GB/T 5009.26-2003 (the previous edition):
—this standard is renamed as "National Standard for Food Safety - Determination of N-nitrosamine Compounds in Foods";
—"packed chromatographic column" in the previous method is revised as "adsorption capillary column";
—"high resolution gas chromatography - mass spectrometer" in the previous method is revised as "gas chromatograph-mass spectrometer".
National Food Safety Standard
Determination of N-nitrosamine Compounds in Foods
1 Scope
This standard specifies determination method of N-dimethyl nitrosamine content in foods.
This standard is applicable to the determination of N-dimethyl nitrosamine content in meat and meat products as well as aquatic animals and their products.
Method I Gas Chromatography - Mass Spectrometry
2 Theory
Subject the N-nitrosamine compounds in the specimen to steam distillation and organic solvent extraction, concentrate it to a certain volume and confirm and quantify with the gas chromatograph - mass spectrometer.
3 Reagents and Materials
Unless otherwise specified, analytically pure reagents and Class-II water (defined in GB/T 6682) are adopted for the purpose of this method.
3.1 Reagents
3.1.1 Methylene dichloride (CH2Cl2): chromatographically pure; 100mL of methylene dichloride shall be taken from each batch to be concentrated to 1mL with the rotary evaporator on a 40℃ water bath, after which it shall not induce positive response on the gas chromatograph - mass spectrometer; in case of positive response, the reagent shall be subjected to re-distillation with an all-glass device for another test until it gets negative response.
3.1.2 Anhydrous sodium sulfate (Na2SO4).
3.1.3 Sodium chloride (NaCl): guaranteed reagent.
3.1.4 Sulfuric acid (H2SO4).
3.1.5 Anhydrous ethanol (C2H5OH).
3.2 Preparation of reagents
Sulfuric acid solution (1+3): measure 30mL of sulfuric acid, and slowly pour it into 90mL of cold water, remove the heat thoroughly while stirring, and mix it carefully and uniformly after cooling down.
3.3 Standard product
N-nitrosamine standard product (C2H6N2O, CAS No.: 62-75-9): with purity ≥98.0%.
3.4 Preparation of n-nitrosamine standard solution
3.4.1 N-nitrosamine (C2H6N2O, CAS No.: 62-75-9) standard solution: prepare the methylene dichloride into solution of 1 mg/mL.
3.4.2 N-nitrosamine standard intermediate solution (C2H6N2O, CAS No.: 62-75-9) standard working solution: prepare the methylene dichloride into standard working solution of 1μg /mL.
4 Instruments and Apparatus
4.1 Gas chromatograph - mass spectrometer.
4.2 Rotary evaporator.
4.3 All-glass steam distillation device or equivalent full automatic steam distillation device (see Figure A.1).
4.4 Nitrogen blower.
4.5 Ice maker.
4.6 Electronic balance: with the sensibility of 0.01 g and 0.1 mg.
5 Analysis Steps
5.1 Preparation of specimen
5.1.1 Extraction
Distillation with water distilling apparatus: accurately weigh 200g (accurate to 0.01 g) of specimen, add it into 100mL of water and 50g of sodium chloride in the distillation tube, and then mix uniformly and sufficiently, after which check the air tightness. Add 100 mL of methylene chloride in a 500-mL flat-bottomed flask, together with a small amount of ice cubes to receive the condensate; insert the condensate tube outlet into the methylene dichloride liquid and put the flat-bottomed flask in an ice bath, and then turn on the distillation device for heating distillation; turn off the heating device and stop distillation after 400mL of condensate is collected.
5.1.2 Extractive purification
Add 20g of sodium chloride and 3mL of sulfuric acid (1+3) in a flat-bottomed flask containing distillate and stir to completely dissolve the sodium chloride. Then transfer the solution to a 500-mL separating funnel, oscillate for 5min, deflate if necessary; after keep it stand until layering , transfer the methylene chloride layer to another flat-bottomed flask, and then extract the aqueous layer with 150mL of methylene chloride by three times. Combine the methylene chloride extracts obtained by four times to get the total volume of about 250 mL.
5.1.3 Concentration
Dehydrate the dichloromethane extract with 10g of anhydrous sodium sulfate and subject it to rotary evaporation; concentrate it to 5mL ~ 10mL on a 40 °C water bath and change to nitrogen blowing, then scale the volume accurately to 1.0mL; shake well for determination.
5.2 Determination conditions for gas chromatography - mass spectrometry
5.2.1 Conditions for gas chromatography
Capillary gas chromatographic column: INNOWAX quartz capillary column (with column length of 30m, inner diameter of 0.25mm and film thickness of 0.25μm); injection port temperature: 220 ℃; programmed temperature rise conditions: with initial column temperature of 40 ℃, it is raised at a rate of 10 ℃/min to 80℃, then to 100℃ at a rate of 1 ℃/min, and then to 240℃ at a rate of 20 ℃/min, after which it is kept for 2 minutes; carrier gas: helium; flow rate: 1.0 mL/min; injection mode: splitless injection; injection volume: 1.0μL.
5.2.2 Conditions for mass spectrometry
Selected ion detection. Scan N-dimethyl nitrosamine from the 9.9th min for selected ions of 15.0, 42.0, 43.0, 44.0 and 74.0; voltage for electron impact ionization source (EI): 70 eV; ionization current: 300 μA; ion source temperature: 230 ℃; interface temperature: 230 ℃; ion source vacuum degree: 1.33 × 10-4 Pa.
5.3 Plotting of standard curve
Accurately pipet N-nitrosamine mixed standard stock solution (1μg / mL) respectively to prepare a series of mixed standard solution with concentrations of 0.01μg/mL, 0.02μg/mL, 0.05μg/mL, 0.1μg/mL, 0.2μg/mL, 0.5μg/mL for injection analysis, with the mass spectrum shown in Figure B.1. Perform linear regression of concentration with peak area to show that N-nitrosamine assumes linearity over a given concentration range; in the regression equation, y stands for the peak area and x for the concentration (μg / mL).
5.4 Determination of specimen solution
Inject the specimen solution into gas chromatograph - mass spectrometer to obtain the peak area of a certain specific monitoring ion, and calculate the concentration (μg/mL) of N-dimethyl nitrosamine in specimen solution according to the standard curve.
Foreword i
1 Scope
2 Theory
3 Reagents and Materials
4 Instruments and Apparatus
5 Analysis Steps
6 Expression of Analysis Results
7 Precision
8 Miscellaneous
9 Theory
10 Reagents and Materials
11 Instruments and Apparatus
12 Analysis Steps
13 Expression of Analysis Results
14 Precision
15 Miscellaneous
Annex A Drawing of the Steam Distillation Device
Annex B Gas Chromatography - Mass Spectrometry Diagram
Annex C Gas Chromatography - Thermal Energy Analyzer Spectrogram
