GB 5009.33-2016 PDF in English
GB 5009.33-2016 (GB5009.33-2016) PDF English
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National food safety standard -- Determination of nitrite and nitrate in foods
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Standards related to: GB 5009.33-2016
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GB 5009.33-2016: PDF in English GB 5009.33-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Determination of Nitrite and Nitrate in Foods
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;
State Food and Drug Administration of the People’s Republic
of China.
Table of Contents
Foreword ... 3
1 Scope ... 4
Method I Ion Chromatography ... 4
2 Principle ... 4
3 Reagents and Materials ... 4
4 Instruments and Equipment ... 5
5 Analytical Procedures ... 6
6 Expression of Analysis Results ... 8
7 Precision ... 9
8 Others ... 9
Method II Spectrophotometry ... 9
9 Principle ... 9
10 Reagents and Materials... 10
11 Instruments and Equipment ... 12
12 Analytical Procedures ... 15
13 Expression of Analysis Results ... 17
14 Precision ... 18
15 Others ... 18
Method III Determination of Nitrate in Vegetables and Fruits - UV
Spectrophotometry ... 18
16 Principle ... 18
17 Reagents and Materials... 19
18 Instruments and Equipment ... 20
19 Analytical Procedures ... 20
20 Calculation Result ... 21
21 Precision ... 21
22 Others ... 21
Appendix A Chromatogram of Nitrite and Nitrate ... 22
National Food Safety Standard -
Determination of Nitrite and Nitrate in Foods
1 Scope
This Standard specifies the method of determining nitrite and nitrate in foods.
This Standard is applicable to the determination of nitrite and nitrate in foods.
Method I -- Ion Chromatography
2 Principle
Precipitate protein and remove fat from the sample; adopt the corresponding method
to extract and purify; take potassium hydroxide solution as the eluent; adopt anion
exchange column for separation; take conductance detector or UV detector for
detection. Determine the nature with the retention time and quantify with the external
standard method.
3 Reagents and Materials
Unless otherwise indicated, the reagents adopted under this method are of analytical
purity. The water is first-grade water as specified in GB/T 6682.
3.1 Reagents
3.1.1 Acetic acid (CH3COOH).
3.1.2 Potassium hydroxide (KOH).
3.2 Preparation of Reagents
3.2.1 Acetic acid solution (3%). weigh-take 3 mL of acetic acid, place it in 100 mL
volumetric flask. Add water to dilute to the constant volume; mix it up.
3.2.2 Potassium hydroxide solution (1 mol/L). weigh-take 6 g of potassium hydroxide;
dissolve it in cold water that’s newly boiled; dilute to 100 mL; mix it up.
3.3 Standards
volumetric flask; add water to dilute to the constant volume, mix it up. Use filter paper
to filter the solution; take part of the solution, then, centrifuge for 15 min under 10,000
r/min. Reserve the supernatant for later usage.
5.2.2 Meat, egg, fish and other products. weigh-take 5 g (accurate to 0.001 g) of
sample homogenate, place it in 150 mL conical flask with a plug; add 80 mL of water.
Start ultrasonic extraction for 30 min; shake once every 5 min, then, maintain the
completely dispersed solid phase. Place it in water bath at 75 °C for 5 min; take it out
and wait till it reaches the room temperature. Quantitatively transfer it into 100 mL
volumetric flask; add water to dilute to the constant volume, mix it up. Use filter paper
to filter the solution; take part of the solution, then, centrifuge for 15 min under 10,000
r/min. Reserve the supernatant for later usage.
5.2.3 Marinated fish, marinated meat and other marinated products. weigh-take 2 g
(accurate to 0.001 g) of sample homogenate, place it in 150 mL conical flask with a
plug; add 80 mL of water. Start ultrasonic extraction for 30 min; shake once every 5
min, then, maintain the completely dispersed solid phase. Place it in water bath at
75 °C for 5 min; take it out and wait till it reaches the room temperature. Quantitatively
transfer it into 100 mL volumetric flask; add water to dilute to the constant volume, mix
it up. Use filter paper to filter the solution; take part of the solution, then, centrifuge for
15 min under 10,000 r/min. Reserve the supernatant for later usage.
5.2.4 Dairy products. weigh-take 10 g (accurate to 0.01 g) of sample, place it in 100
mL conical flask with a plug; add 80 mL of water, mix it up. Start ultrasonic extraction
for 30 min. Add 2 mL of 3% acetic acid solution, place it under 4 °C for 20 min; take it
out and wait till it reaches the room temperature. Add water to dilute to the constant
volume. Use filter paper to filter the solution; reserve the filtrate for later usage.
5.2.5 Milk powder and cottage cheese. weigh-take 2.5 g (accurate to 0.01 g) of
sample, place it in 100 mL conical flask with a plug; add 80 mL of water, mix it up. Start
ultrasonic extraction for 30 min. Take it out and wait till it reaches the room
temperature. Quantitatively transfer it into 100 mL volumetric flask; add 2 mL of 3%
acetic acid solution. Add water to dilute to the constant volume. Place it under 4 °C for
20 min; take it out and wait till it reaches the room temperature. Use filter paper to filter
the solution; reserve the filtrate for later usage.
5.2.6 Take approximately 15 mL of the above-mentioned reserved solution; adopt
0.22 μm water filter needle filter and C18 column to remove 3 mL of supernatant (if
chloride is >100 mg/L, adopt needle filter, C18 column, Ag column and Na column to
remove 7 mL of supernatant); gather the remaining eluent for detection.
Before the usage of solid phase extraction column, it needs to be activated. The
activation of C18 column (1.0 mL), Ag column (1.0 mL) and Na column (1.0 mL).
before the usage of C18 column (1.0 mL), respectively inject 10 mL of methanol and 15
mL of water; start static activation for 30min. Inject 10 mL of water to Ag column (1.0
mL) and Na column (1.0 mL); start static activation for 30 min.
add 100 mL of water; mix it up. Add 65 mL of ammonia; add water to dilute to 1,000
mL; mix it up. Adjust pH to 9.6~9.7.
10.2.5 Diluent of ammonia buffer solution. weigh-take 50 mL of pH 9.6~9.7 ammonia
buffer solution; add water to dilute to 500 mL; mix it up.
10.2.6 Hydrochloric acid (0.1 mol/L). weigh-take 8.3 mL of hydrochloric acid; add
water to dilute to 1,000 mL.
10.2.7 Hydrochloric acid (2 mol/L). weigh-take 167 mL of hydrochloric acid; add water
to dilute to 1,000 mL.
10.2.8 Hydrochloric acid (20%). weigh-take 20 mL of hydrochloric acid; add water to
dilute to 100 mL.
10.2.9 Sulfanilic acid solution (4 g/L). weigh-take 0.4 g of sulfanilic acid; dissolve it in
100 mL of 20% hydrochloric acid; mix it up. Place it in brown bottle; keep away from
light.
10.2.10 Naphthalene ethylenediamine hydrochloride solution (2 g/L). weigh-take 0.2 g
of naphthalene ethylenediamine hydrochloride; dissolve it in 100 mL of water; mix it
up. Place it in brown bottle; keep away from light.
10.2.11 Copper sulfate solution (20 g/L). weigh-take 20 g of copper sulfate; add water
to dilute it to 1,000 mL.
10.2.12 Cadmium sulfate solution (40 g/L). weigh-take 40 g of cadmium sulfate; add
water to dilute it to 1,000 mL.
10.2.13 Acetic acid solution (3%). weigh-take 3 mL of glacial acetic acid; place it in
100 mL volumetric flask. Add water to dilute to the constant volume; mix it up.
10.3 Standards
10.3.1 Sodium nitrite (NaNO2, CAS No.. 7632-00-0). reference reagents, or standard
nitrite solution supported by Reference Material Certificate.
3.3.2 Sodium nitrate (NaNO3, CAS No.. 7631-99-4). reference reagents, or standard
nitrate solution supported by Reference Material Certificate.
10.4 Preparation of Standard Solutions
10.4.1 Standard solution of sodium nitrite (200 μg/mL, calculated by sodium nitrite).
accurately weigh-take 0.1000 g of sodium nitrite that’s dried to constant weight under
110 °C~120 °C. Add water to dissolve it; transfer it to 500 mL volumetric flask. Add
water to dilute to the constant volume; mix it up.
10.4.2 Standard solution of sodium nitrate (200 μg/mL, calculated by sodium nitrate).
Keys.
1 Liquid funnel, internal diameter. 35 mm, external diameter. 37 mm;
2 Inlet capillary, internal diameter. 0.4 mm, external diameter. 6 mm;
3 Rubber stopper;
4 Cadmium column glass tube, internal diameter. 12 mm, external diameter. 16 mm;
5,7 Glass wool;
6 Spongy cadmium;
8 Outlet capillary, internal diameter. 2 mm, external diameter. 8 mm.
Figure 1 -- Cadmium Column Sketch
11.6.4 After each usage of cadmium column, use 25 mL of hydrochloric acid (0.1
mol/L) to wash it, then, use water to wash it for 2 times (25 mL of water for each time).
In the end, cover the cadmium column with water.
11.6.5 Determination of cadmium column reduction efficiency. extract 20 mL of
standard sodium nitrate working fluid, add 5 mL of diluent of ammonia buffer; mix it up
and inject it into the liquid funnel and pass the cadmium column for reduction. Use a
100 mL volumetric flask to gather the eluent. The flow rate of the eluent shall not
exceed 6 mL/min. When the liquid storage cup is about to be drained, use
approximately 15 mL of water to rinse the cup. After the water is drained, use extra 15
mL of water to rinse it repeatedly. After the water is drained for the second time, fill the
cup up with water; let it pass the cadmium column at the largest flow rate. When the
eluent in the volumetric flask is approaching 100 mL, take out the volumetric flask
from the bottom of the column. Add water to the constant volume; mix it up. Take 10.0
mL of restored solution (equivalent to 10 μg of sodium nitrite) and place it in 50 mL
colorimetric tube. Follow the steps described in 12.3 from “extract 0.00 mL, 0.20 mL,
0.40 mL, 0.60 mL, 0.80 mL, 1.00 mL...”. Calculate the result in accordance with the
standard curve line. If it is consistent with the added amount and the reduction
efficiency is > 95%, then, it can satisfy the requirements.
11.6.6 The reduction efficiency shall be calculated in accordance with Formula (2).
Where.
X - Reduction efficiency, expressed in %;
m1 - The content of sodium nitrite, expressed in (μg);
10 - The content of sodium nitrite in the solution to be determined, expressed in (μg).
to cool it down; wait till it reaches the room temperature. Quantitatively transfer it into
200 mL volumetric flask; add 5 mL of 106 g/L potassium ferrocyanide solution; mix it
up. Add 5 mL of 220 g/L zinc acetate solution to precipitate protein. Add water to the
constant volume, mix it up; place it evenly for 30 min. Remove the supernatant fat;
use filter paper to filter the supernatant; get rid of 30 mL of initial filtrate, and reserve
the filtrate for later usage.
12.3 Determination of Nitrite
Extract 40.0 mL of the above-mentioned filtrate and place it in 50 mL colorimetric tube
with a plug. Extract 0.00 mL, 0.20 mL, 0.40 mL, 0.60 mL, 0.80 mL, 1.00 mL, 1.50 mL,
2.00 mL and 2.50 mL of standard working solution of sodium nitrite (equivalent to 0.0
μg, 1.0 μg, 2.0 μg, 3.0 μg, 4.0 μg, 5.0 μg, 7.5 μg, 10.0 μg and 12.5 μg of sodium
nitrite). Place it respectively in 50 mL colorimetric tube with a plug. Add 2 mL of 4 g/L
sulfanilic acid solution respectively to the standard tube and sample tube, mix it up;
place it evenly for 3 min~5 min. Respectively add 1 mL of 2 g/L naphthalene
ethylenediamine hydrochloride solution; add water to the constant volume, mix it up.
Place it evenly for 15 min. Adopt 1 cm colorimetric cup; adjust the zero point through
the zero tube; measure absorbance at the wavelength of 538 nm. Draw a standard
curve for comparison. Simultaneously, prepare blank reagent.
12.4 Determination of Nitrate
12.4.1 Cadmium Column Reduction
12.4.1.1 Adopt 25 mL of ammonia buffer solution to rinse the cadmium column;
control the flow rate at 3 mL/min~5 mL/min (2 mL/min~3 mL/min if it is replaced by
burette).
12.4.1.2 Extract 20 mL of filtrate and place it in 50 mL beaker. Add 5 mL of pH 9.6~9.7
ammonia buffer solution, mix it up; inject it into liquid funnel and pass the cadmium
column for reduction. After the sample solution is drained in the cup, add 15 mL of
water to rinse the beaker; pour into the cup again. After the water is drained, use 15
15 mL of water to rinse again. When the water is about to be drained for the second
time, fill up the cup with water and pass the column at the largest flow rate. When the
eluent in the volumetric flask is approaching 100 mL, take out the volumetric flask;
add water to the constant volume, and mix it up.
12.4.2 Determination of Total Sodium Nitrite
Extract 10 mL~20 mL of restored sample solution, place it in 50 mL colorimetric tube.
Follow the steps described in 12.3 from “extract 0.00 mL, 0.20 mL, 0.40 mL, 0.60 mL,
0.80 mL, 1.00 mL...”.
mg/L, 4.0 mg/L, 6.0 mg/L, 8.0 mg/L, 10.0 mg/L and 12.0 mg/L respectively.
18 Instruments and Equipment
18.1 UV spectrophotometer.
18.2 Analytical balance. division value. 0.01 g and 0.0001 g.
18.3 Tissue shredder.
18.4 Adjustable oscillator.
18.5 pH meter. accuracy. 0.01.
19 Analytical Procedures
19.1 Pre-processing of Samples
Select a certain quantity of representative samples. Use tap water to rinse it; use
water to clean it; dry the superficial moisture. Adopt quartile method to take the
sample, chop it, thoroughly mix it up. Homogenize it with tissue shredder (equivalent
amount of water can be added under a certain ratio to some juiceless samples); add a
drop of N-octanol to the homogenate to remove bubbles.
19.2 Extraction
Weigh-take 10 g (accurate to 0.01 g) of homogenate sample (convert in accordance
with the amount of added water if any water is added during the preparation); place it
in 250 mL conical flask. Add 100 mL of water; add 5 mL of ammonia buffer solution
(pH=9.6~9.7), and 2 g of powdered activated carbon. Shake it (at the reciprocating
speed of 200 times/min) for 30 min. Quantitatively transfer it into 200 mL volumetric
flask; add 2 mL of 150 g/L potassium ferrocyanide solution and 2 mL of 300 g/L zinc
sulfate solution; mix it up. Add water to the constant volume, mix it up; place it evenly
for 5 min. Use quantitative filter paper to filter the supernatant; reserve the filtrate for
later usage. Simultaneously, conduct blank test.
19.3 Determination
In accordance with the content of nitrate in the sample, extract 2 mL~10 mL of the
above-mentioned filtrate; place it in 50 mL volumetric flask. Add water to dilute to the
constant volume; mix it up. Adopt 1 cm quartz colorimetric vessel to determine the
absorbance at 219 nm.
19.4 Draw a Standard Curve Line
Adopt 1 cm quartz colorimetric vessel to determine the absorbance of the standard
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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