39.1.21
Determination
AOAC Official Method 973.31
Nitrites in Cured Meat
Colorimetric Method
First Action 1973
Final Action 1996
Codex-Adopted–AOAC Method*
A. Reagents and Apparatus
(a) NED reagent.—Dissolve 0.2 g N-(1-naphthyl)
ethylenediamine⋅2HCl in 150 mL 15% (v/v) CH3COOH. Filter, if
necessary, and store in glass-stoppered brown glass bottle.
(b) Sulfanilamide reagent.—Dissolve 0.5 g sulfanilamide in
150 mL 15% CH3COOH (v/v). Filter, if necessary, and store in
glass-stoppered brown glass bottle.
(c) Nitrite standard solutions.—(1) Stock solution.—1000 ppm
(µg/mL) NaNO2. Dissolve 1.000 g NaNO2 in H2O and dilute to 1 L.
(2) Intermediate solution.—100 µg/mL NaNO2. Dilute 100 mL
stock solution to 1 L with H2O. (3) Working solution.—1 µg/mL
NaNO2. Dilute 10 mL intermediate solution to 1 L with H2O.
(d) Filter paper.—Test for nitrite contamination by analyzing
3–4 sheets, at random, throughout box. Filter ca 40 mL H2O through
each sheet. Add 4 mL sulfanilamide reagent, mix, let stand 5 min,
add 4 mL NED reagent, mix, and wait 15 min. If any sheets are posi-
tive, discard entire box.
B.
Weigh 5 g finely comminuted and thoroughly mixed test sample
into 50 mL beaker. Add ca 40 mL H2O heated to 80°C. Mix thor-
oughly with glass rod, taking care to break up all lumps, and transfer
to 500 mL volumetric flask. Thoroughly wash beaker and rod with
successive portions of the hot H2O, adding all washings to flask. Add
enough hot H2O to bring volume to ca 300 mL, transfer flask to
steam bath, and let stand 2 h, shaking occasionally. Cool to room
temperature, dilute to volume with H2O, and remix. Filter. If turbid-
ity remains after filtration, centrifuging will usually clear the solu-
tion. Add 2.5 mL sulfanilamide reagent to aliquot containing
5–50 µg NaNO2 in 50 mL volumetric flask, and mix. After 5 min,
add 2.5 mL NED reagent, mix, dilute to volume, mix, and let color
develop 15 min. Transfer portion of solution to photometer cell and
determine A at 540 nm against blank of 45 mL H2O, 2.5 mL sulfanil-
amide reagent, and 2.5 mL NED reagent.
Determine nitrite present by comparison with standard curve
prepared as follows: Add 10, 20, 30, and 40 mL nitrite working
standard solution to 50 mL volumetric flasks, add 2.5 mL sulfa-
nilamide reagent, mix, and proceed as above, beginning “After
5 min,…”. Standard curve is straight line to 1µg/mL NaNO2 in fi-
nal solution.
References: JAOAC 56, 922(1973); 60, 594(1977).
Revised: March 1997
* Adopted as a Codex Reference Method (Type II) for nitrite and potassium
and/or sodium salts in canned corned beef and luncheon meat.
Adopted as a Codex Reference Method (Type II) for nitrite in cooked
cured chopped meat, cooked cured ham, and cooked cured pork shoulder.
© 2000 AOAC INTERNATIONAL