GB 5009.26-2016 English PDF

GB 5009.26-2016: Determination of N-nitrosamines in foods
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GB 5009.26-2016
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard - Determination of N-
nitrosamine Compounds in Food
ISSUED ON: DECEMBER 23, 2016
IMPLEMENTED ON: JUNE 23, 2017
Issued by: National Health and Family Planning Commission of the
People's Republic of China;
China Food and Drug Administration.
Table of Contents
Foreword ... 3 
1 Scope ... 4 
Method One -- Gas chromatography - Mass spectrometry ... 4 
2 Principle ... 4 
3 Reagents and materials ... 4 
4 Apparatus and equipment ... 5 
5 Analysis steps ... 5 
6 Expression of analysis results ... 7 
7 Precision ... 7 
8 Other ... 7 
Method Two -- Gas chromatography - Thermal energy analyzer ... 8 
9 Principle ... 8 
10 Reagents and materials ... 8 
11 Apparatus and equipment ... 9 
12 Analysis steps ... 9 
13 Expression of analysis results ... 11 
14 Precision ... 12 
15 Other ... 12 
Annex A Steam distillation device diagram ... 13 
Annex B Gas chromatogram ... 14 
Annex C Gas chromatography - thermal energy analyzer spectrogram ... 15 
Foreword
This Standard replaces GB/T 5009.26-2003 “Determination of N-nitrosamines
in foods”.
Compared with GB/T 5009.26-2003, the main changes in this Standards are as
follows:
- modified standard’s name to “National Food Safety Standard -
Determination of N-nitrosamine Compounds in Food”;
- modified filling chromatographic column in original method to capillary
chromatographic column;
- modified gas chromatography high resolution mass spectrometer in original
method to gas chromatography mass spectrometer.
National Food Safety Standard - Determination of N-
nitrosamine Compounds in Food
1 Scope
This Standard specifies determination method of N-dimethylnitrosamine in food.
This Standard is applicable to determination of N-dimethylnitrosamine in meat
and meat products, aquatic animals and their products.
Method One -- Gas chromatography - Mass
spectrometry
2 Principle
After N-nitrosamine compounds in sample are distilled by water steam and are
extracted by organic solvent, they are concentrated to a certain volume. Use
gas chromatography mass spectrometry to confirm and quantify.
3 Reagents and materials
Unless otherwise stated, reagents used in this Method are analytically pure;
water is grade-two water specified in GB/T 6682.
3.1 Reagents
3.1.1 Dichloromethane (CH2Cl2): Chromatographically pure. Take 100mL from
each batch on 40°C water bath. Use rotary evaporator to concentrate to 1mL.
On gas chromatography - mass spectrometry, there shall be no positive
response. If there is positive response, it shall be re-evaporated by full-glass
device till it is negative.
3.1.2 Anhydrous sodium sulfate (Na2SO4).
3.1.3 Sodium chloride (NaCl): superb pure.
3.1.4 Sulfuric acid (H2SO4).
3.1.5 Anhydrous ethanol (C2H5OH).
3.2 Reagent preparation
Sulfuric acid solution (1+3): Weigh 30mL of sulfuric acid. Slowly pour into 90mL
of cold water. Keep stirring to make it fully dissipated. Carefully mix after cooling.
3.3 Standard product
N-nitrosamine standard (C2H6N2O, CAS number: 62-75-9): purity ≥98.0%.
3.4 Preparation of N-nitrosamine standard solution
3.4.1 N-nitrosamine (C2H6N2O, CAS No.: 62-75-9) standard solution: Use
dichloromethane to prepare into 1mg/mL solution.
3.4.2 N-nitrosamine standard intermediate solution (C2H6N2O, CAS No.: 62-75-
9) standard use solution: Use dichloromethane to prepare into 1μg/mL standard
use solution.
4 Apparatus and equipment
4.1 Gas chromatography - mass spectrometry.
4.2 Rotary evaporator.
4.3 Full-glass steam distillation device or equivalent fully-automatic steam
distillation device (see Figure A.1).
4.4 Nitrogen blower.
4.5 Ice maker.
4.6 Electronic balance: resolutions are 0.01g and 0.1mg.
5 Analysis steps
5.1 Sample preparation
5.1.1 Extraction
Distillation of water distillation device: Accurately weigh 200g (to the nearest of
0.01g) of sample. Add 100mL of water and 50g of sodium chloride into
distillation tube. Fully mix. Check air tightness. In 500mL flat bottom flask, add
into 100mL of dichloromethane and a small amount of ice to receive condensate.
Stick condenser outlet under methylene chloride level. Place flat bottom flask
into water bath. Start distillation device to heat and distill. Collect 400mL of
condensate then turn off heating device to stop distillation.
5.1.2 Extraction and purification
In flat bottom flask that is filled with distillate, add into 20g of sodium chloride
and 3mL of sulfuric acid (1+3). Stir to make sodium chloride fully dissolved.
Then move solution to 500mL separating funnel. Oscillate 5min. Deflate if
necessary. After standing layering, move methylene chloride layer to another
flat bottom flask. Then use 150mL of dichloromethane to extract water layer for
three times. Combine 4 times dichloromethane extract. Total volume is about
250mL.
5.1.3 Concentration
Use 10g of anhydrous sodium sulfate to dehydrate dichloromethane extract.
Perform rotary evaporation. On 40°C water batch, concentrate to 5mL~10mL
change nitrogen blowing. Accurately set volume to 1.0mL. Shake well for
determination.
5.2 Determination conditions for gas chromatography - mass
spectrometry
5.2.1 Gas chromatographic conditions
Capillary gas chromatographic column: INNOWAX quartz capillary column
(column length is 30m, inner diameter is 0.25mm, film thickness is 0.25μm).
Inlet temperature: 220°C. Program temperature rising conditions: Initial column
temperature is 40°C; Rise to 80°C at a speed rate of 10°C/min; Rise to 100°C
at a speed rate of 1°C/min; Rise to 240°C at a speed rate of 20°C/min; Remain
2min. Carrier gas: helium. Flow rate: 1.0mL/min. Injection method: split injection.
Inlet volume: 1.0μL.
5.2.2 Mass spectrometry conditions
Select ion detection. At 9.9min, start scanning N-dimethylnitrosamine. Selected
ions are 15.0, 42.0, 43.0, 44.0, 74.0. Electron bombardment ionization source
(EI), voltage: 70eV. Ionization current: 300μA. Ion source temperature: 230°C.
Interface temperature: 230°C. Ion source vacuum: 1.33×10-4 Pa.
5.3 Production of standard curve
Respectively pipette standard series mixed standard series solution that is
prepared by mixed standard stock solution of N-nitrosamine (1μg/mL), that
concentrations are 0.01μg/mL, 0.02μg/mL, 0.05μg/mL, 0.1μg/mL, 0.2μg/mL,
0.5μg/mL. Perform injection analysis. See Figure B.1 for mass spectrum. Use
peak area to concentration for linear regression to indicate that within given
concentration range, N-nitrosamine is linear. Regression equation is: y for peak
area and x for concentration (μg/mL).
5.4 Determination of sample solution
Inject sample solution into gas chromatography - mass spectrometer to obtain
peak area of a particular monitored ion. According to standard curve, calculate
N-dimethylnitrosamine (μg/mL) in sample solution.
6 Expression of analysis results
Calculate N-dimethylnitrosamine content in sample according to formula (1):
Where,
X - N-dimethylnitrosamine content in sample, in micrograms per kilogram or
micrograms per liter (μg/kg or μg/L);
h1 - peak area of some N-nitrosamine compound in concentrate;
h2 - standard peak area of N-nitrosamine;
ρ - concentration of N-nitrosamine compound in standard solution, in
micrograms per milliliter (μg/mL);
V - volume of test solution (concentrate), in milliliters (mL);
m - sample mass or volume, in grams or milliliters (g or mL);
1000 - conversion factor.
Results remain three significant digits.
7 Precision
The absolute difference between two independent determinations obtained
under repeatability conditions shall not exceed 15% of the arithmetic mean.
8 Other
When sampling amount is 200g, when concentrated volume is 1.0mL, detection
limit of this Method is 0.3μg/kg, the limit of quantification is 1.0μg/kg.
Method Two -- Gas chromatography - Thermal energy
analyzer
9 Principle
Sample is distilled by water steam. N-dimethylnitrosamine in sample, as steam,
is absorbed through dichloromethane. Then it is extracted, separated by
dichloromethane solution for determination of gas chromatography - thermal
energy analyzer (GC-TEA).
Principle of GC-TEA determination: N-dimethylnitrosamine that is separated by
gas chromatographic column in pyrolysis chamber, is split by specific catalysis
to generate nitric oxide (NO) group. The later one reacts with ozone to generate
excited-state NO*. When excited-state NO* returns to basic state, it emits near-
infrared light (600nm~2800nm) and tested by photomultiplier tube
(600nm~800nm). Due to specific catalytic cleavage, and impurities are
eliminated by CTR filter, thermal analyzer can only test NO group to become a
specific detector for N-nitrosamine compounds.
10 Reagents and materials
Unless otherwise stated, reagents used in this Method are analytically pure;
water is grade-two water specified in GB/T 6682.
10.1 Reagents
10.1.1 Dichloromethane (CH2Cl2): Chromatographically pure. Take 100mL from
each batch on 40°C water bath. Use rotary evaporator to concentrate to 1mL.
On gas chromatography - mass spectrometry, there shall be no positive
response. If there is positive response, it shall be re-evaporated by full-glass
device till it is negative.
10.1.2 Sodium chloride (NaCl).
10.1.3 Anhydrous sodium sulfate (Na2SO4).
10.1.4 Sulfuric acid (H2SO4).
10.1.5 Anhydrous ethanol (C2H5OH).
10.2 Reagent preparation
Sulfuric acid solution (1+3): Weigh 30mL of sulfuric acid. Slowly pour into 90mL
of cold water. Keep stirring to make it fully dissipated. Carefully mix after cooling.
10.3 Standard product
N-nitrosamine standard (C2H6N2O, CAS number: 62-75-9): purity ≥98.0%.
10.4 Preparation of N-nitrosamine standard solution
10.4.1 N-nitrosamine (C2H6N2O, CAS No.: 62-75-9) standard solution: Use
dichloromethane to prepare into 1mg/mL solution.
10.4.2 N-nitrosamine standard intermediate solution (C2H6N2O, CAS No.: 62-
75-9) standard use solution: Use dichloromethane to prepare into 1μg/mL
standard use solution.
11 Apparatus and equipment
11.1 Gas chromatograph (GC).
11.2 Thermal energy analyzer (TEA).
11.3 Full-glass steam distillation device or equivalent fully-automatic steam
distillation device (see Figure A.1).
11.4 Rotary evaporator.
11.5 Nitrogen blower.
11.6 Ice maker.
11.7 Electronic balance: resolutions are 0.01g and 0.1mg.
12 Analysis steps
12.1 Sample preparation
12.1.1 Extraction
Distillation of water distillation device: Accurately weigh 200g (to the nearest of
0.01g) of sample. Add 100mL of water and 50g of sodium chloride into
distillation tube. Fully mix. Check air tightness. In 500mL flat bottom flask, add
into 100mL of dichloromethane and a small amount of ice to receive condensate.
Stick condenser outlet under methylene chloride level. Place flat bottom flask
into water bath. Start distillation device to heat and distill. Collect 400mL of
condensate then turn off heating device to stop distillation.
12.1.2 Extraction and purification
In flat bottom flask that is filled with distillate, add into 20g of sodium chloride
and 3mL of sulfuric acid (1+3). Stir to make sodium chloride fully dissolved.
Then move solution to 500mL separating funnel. Oscillate 5min. Deflate if
necessary. After standing layering, move methylene chloride layer to another
flat bottom flask. Then use 150mL of dichloromethane to extract water layer for
three times. Combine 4 times dichloromethane extract. Total volume is about
250mL.
12.1.3 Concentration
Use 10g of anhydrous sodium sulfate to dehydrate dichloromethane extract.
Perform rotary evaporation. On 40°C water batch, concentrate to 5mL~10mL
change nitrogen blowing. Accurately set volume to 1.0mL. Shake well for
determination.
12.2 Determination conditions for gas chromatography - mass
spectrometry
12.2.1 Gas chromatographic conditions
Gas chromatographic conditions are listed as follows:
a) Chromatographic column: VF-WAX capillary column (column length is
30m, inner diameter is 0.32mm, film thickness is 0.25μm, fixing solution is
polyethylene glycol) or equivalent type column;
b) Inlet temperature: 120°C;
c) Program temperature rising conditions: Initial column temperature is 60°C;
remain 2min. Rise to 150°C at a speed rate of 6°C/min. Rise to 200°C at
a speed rate of 20°C/min; remain 5min;
d) Carrier gas: Nitrogen (≥99.999%);
e) Flow rate: 1.0mL/min;
f) Injection method: split injection;
g) Inlet volume: 2μL.
12.2.2 Thermal analyzer conditions
Thermal analyzer conditions are listed as follows:
a) Interface temperature: 250°C;
b) Pyrolysis chamber temperature: 500°C;
c) Vacuum degree: 59.85Pa~66.5Pa;
d) Oxygen pressure: 13.79kPa;
e) Ozone level: 244 (22.8V).
12.3 Production of standard curve
Respectively and accurately pipette 200μL, 400μL, 600μL, 800μL, 1000μL of
N-dimethylnitrosamine standard working solution (1μg/mL) in 1.0mL volumetric
flask. Use dichloromethane to set volume to 1.0mL. Their concentrations are
0.2μg/mL, 0.4μg/mL, 0.6μg/mL, 0.8μg/mL, 1.0μg/mL.
Inject the above concentration gradient N-dimethylnitrosamine standard series
working solutions into GC-TEA. Use retention time to determine nature.
Measure peak area of N-dimethylnitrosamine. See Figure C.1 for spectrum.
Use concentration of N-dimethylnitrosamine standard working solution as
abscissa (μg/mL), use peak area of N-dimethylnitrosamine as Y-axis to draw
standard curve.
12.4 Determination of sample solution
Inject sample solution into GC-TEA. Use retention time to determine nature.
Measure peak area of N-dimethylnitrosamine. According to standard curve
method, calculate concentration of N-dimethylnitrosamine in sample solution
(μg/mL).
13 Expression of analysis results
Calculate N-dimethylnitrosamine content in sample according to formula (2):
Where,
X - N-dimethylnitrosamine content in sample, in microgr...