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Modification of AOAC method 973.31 for determination of nitrite in cured

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820 MOHAMED ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008
FOOD COMPOSITION AND ADDITIVES
Modification of AOAC Method 973.31 for Determination of
Nitrite in Cured Meats
ASHRAF A. MOHAMED
Ain Shams University, Faculty of Science, Department of Chemistry, Abbassia, Cairo-11566, Egypt
AHMED T. MUBARAK and KHALED F. FAWY
King Khalid University, Faculty of Science, Department of Chemistry, Abha-9033, Saudi Arabia
MOHAMED F. EL-SHAHAT
Ain Shams University, Faculty of Science, Department of Chemistry, Abbassia, Cairo-11566, Egypt
A modification of AOAC Method 973.31 is
proposed to improve the extraction efficiency of
nitrite from cured meat samples and its
subsequent quantification based on the
diazotization-coupling reaction of sulfanilamide
with N-(1-naphthyl)ethylenediamine
dihydrochloride (NED). The various experimental
parameters were thoroughly investigated. A 5 g
meat sample was mixed with 400 mL water; the pH
of the mixture was adjusted to 5.5 ± 0.3 and
allowed to stand for 2 h on a water bath at 80°C,
with occasional shaking for the complete
extraction of nitrite. After quantitative filtration, an
aliquot was mixed with chloroacetic–chloroacetate
buffer, pH 1.80 ± 0.05, sulfanilamide, and NED, and
the absorbance of the resulting azodye was
recorded at 540 nm against water as a reference.
Following the recommended procedure, a linear
calibration graph was obtained for up to 0.8 mg/mL
NO2–, with a correlation coefficient of 0.9996 and a
detection limit (based on the 3 Sb-criterion) of
5.6 ng/mL NO2–. The proposed method was
conveniently applied to various cured meat
samples and was validated by comparison with the
original AOAC method and by recovery
experiments that gave quantitative results
(94–98%) with convenient reproducibility.
Statistical analysis of the analytical data could not
detect any systematic error and revealed the high
accuracy and precision of the proposed method.
odium nitrite (20–120 mg/kg) is usually added during the
S curing of meat products because of its effect on color as
well as its antibotulinic activity. Nevertheless, excessive
concentrations of nitrites have potential influences on the health
of humans and aquatic organisms owing to its toxicity and its
important role as a precursor in the formation of N-nitrosamines,
Received December 27, 2007. Accepted by SG March 21, 2008.
Corresponding author’s e-mail: aamohamd@hotmail.com
many of which are well known as potent carcinogens (1).
Ascorbic acid (AA; 100–1000 mg/kg) is added to lower the
nitrite concentration and reduce the possible formation of
carcinogenic N-nitrosamines. Potassium sorbate (PS;
200–2600 mg/kg) is also added to lower the nitrite concentration
and maintain enhanced meat color and control Clostridium
botulinum growth and toxin production (1, 2). Other important
additives to cured meats include sodium chloride
(20 000–40 000 mg/kg), polyphosphates (4000–6000 mg/kg),
and ferulic acid (FA; 400–1000 mg/kg; 1, 2).
However, the nitrite content of cured meats is greatly
reduced and sometimes completely eliminated during curing,
cooking, aging, and/or sample treatment for extraction of
nitrite (1–9). Such depletion may be attributed to improper
sample treatment, reactions of nitrite with meat constituents
and food additives, and the conversion of nitrite to nitrate with
aging (1–9). For example, some nitrite may be bound to
reactive (sulfhydryl) groups in the insoluble proteins and
subsequently lost during the extraction of nitrite from meat
samples (7). Moreover, mixing of 4 mM NO2– with 3 mM
ascorbate, 30 mM cysteine, 50 mM histidine, and 4 mM
reduced nicotinamide adenine dinucleotide (NADH) resulted
in nitrite losses of 25–50, 30–50, 20–35, and 15–25%,
respectively (7). On the other hand, nitrite reacts with PS (3, 4)
and FA (5) at temperatures lower than those used during meat
curing processes to give well-known mutagens (3–5). Thus,
the actual levels of some food additives such as AA, cysteine,
histidine, NADH, PS, and FA in cured meats are more than
adequate to account for complete depletion of nitrite as a
result of improper processing and/or sample treatment prior to
the analysis step (1, 7, 8). However, a literature survey
revealed the absence of any quantitative examination of the
recovery of nitrite in the presence of the commonly used food
additives PS and FA during the extraction of nitrites and its
subsequent quantification in cured meats following the
AOAC recommendations (10). Therefore, a thorough
examination of the effects of reaction variables and the
possible interactions of nitrites with such commonly used
food additives seems to be necessary.
In the present study, we critically examined AOAC
Method 973.31 for the determination of nitrites in cured
 MOHAMED ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008 821
meats, and recommend several modifications. We have
optimized the experimental factors affecting the extraction
and quantification of nitrite in cured meats.
METHOD
Apparatus
Absorbance measurements were made on a Shimadzu
UV-1601 PC spectrophotometer (Kyoto, Japan), equipped
with a Shimadzu CPS 240A thermostatted multicell positioner
with a temperature stability of ±0.1°C, using 10 mm matched
cells. Eppendorf vary-pipets (10–100 and 100–1000 mL) were
used to deliver accurate volumes. pH measurements, with an
accuracy of ±0.01, were made on a calibrated Hanna pH meter
Model 211 equipped with a Hanna HI 1013B combination
glass electrode with a working temperature range of
–5–100°C (Hanna Instruments, Ltd, Leighton Buzzard, UK).
A thermostatted water bath, GFL 1003 type (GFL, Burgwedel,
Germany), with an accuracy of ±0.1°C, was also used.
Ultrapure water was prepared daily using a Barnstead
Nanopure diamond water purification system (Barnstead
International, Dubuque, IA) and was used throughout. All
glassware and storage bottles were soaked in 10% HNO3
overnight and thoroughly rinsed with water prior to use.
Reagents
Unless otherwise stated, all chemicals were of analytical
reagent grade and were purchased from Aldrich (Milwaukee,
WI), Fluka (Buchs, Switzerland), or Merck (Darmstadt,
Germany). Nitrite-free filter papers (10) and 0.45 mm
membrane filters were also used.
Stock standard aqueous solution of 1000 mg/mL NO2– was
prepared and further diluted as required (10). A working
buffer solution was prepared by dissolving 0.10 M
chloroacetic acid in ca 80 mL water, adjusting the pH to 1.80 ±
0.05 with NaOH and diluting to the mark in a 100 mL
measuring flask. Aqueous working solutions of 15.0 mM/L
sulfanilamide and 3.0 mM/L N-(1-naphthyl)ethylenediamine
dihydrochloride (NED) were prepared daily, wrapped with
aluminum foil, and kept at 4°C when not in use.
Preparation of Fresh Meat Samples
Aliquots of fresh cow meat samples were purchased from
the local market and homogenized twice in a blender. A 100 g
portion of homogenate was diluted in a 200 mL solution of
30 mg/mL NO2– and slurred for 1 h at room temperature. This
yielded 300 g slurry with a nitrite concentration of 20 mg/mL
NO2–. The homogenized mixture was stored in a screw-cap
glass container at room temperature until extraction [for <2 h
in order to prevent detectable changes in nitrite content (11)].
Recommended Procedure for Sample Treatment
Accurately weigh 5.00 g finely comminuted and
thoroughly mixed test sample and transfer to a 500 mL conical
flask. Add ca 400 mL H2O heated to 80°C, and mix
thoroughly with a glass rod, taking care to break up all lumps.
Add several drops of sodium hydroxide solution to adjust the
pH to 5.5 ± 0.3, using the pH meter (equipped with an
automatic temperature compensator), and further allow to
stand for 2 h on a steam bath with occasional shaking.
(Extracts of some samples, e.g., uncooked sausages,
underwent a pH change after a certain time and required
readjustment of pH by adding a little NaOH.) Cool to room
temperature, quantitatively transfer the contents and washings
to a 500 mL volumetric flask, dilute to volume with H2O,
remix, and filter through prewashed Whatman filter paper.
Samples of relatively high fat contents may show turbidity
after filtration; however, centrifugation and filtration through
filter paper and then through a 0.45 mm membrane filter will
usually clear the extract. Nevertheless, at pH values ³6.2,
samples of moderate to high fat contents gave turbid extracts
that cannot be easily clarified.
Recommended Procedure for Nitrite Analysis
Keep working solutions, unknown sample solutions, and
ultrapure water at 25°C in the thermostatted water bath for
10 min to attain the equilibrium temperature. Transfer
£2000 mL of a sample aliquot containing 0–2.4 mg NO2– to
one of the thermostatted spectrophotometric cells, dilute with
water to 2000 mL, and add 600 mL working buffer solution.
Add 200 mL working sulfanilamide solution, mix well using a
disposable Eppendorf tip, and let stand for 5 min for
diazotization. Add 200 mL working NED solution, mix well,
let stand for 15 min for complete color development, and
record the absorbance at 540 nm against water as a reference.
Calculate the nitrite content of the unknown sample from a
similarly prepared calibration graph.
Results and Discussion
The diazotization reaction of nitrite with sulfanilamide
followed by coupling with NED is the basis of AOAC Official
Method 973.31 for the quantification of nitrite in cured meat
samples (10) and was adopted as a Codex Reference Method
(Type II) for nitrite in canned corned beef, luncheon meat,
cooked cured chopped meat, cooked cured ham, and cooked
cured pork shoulder.
However, in the present work, during the application of the
AOAC method (10) to various cured meat samples, the
percent recoveries were frequently low and depended on the
type, weight, and dilution of the analyzed sample. Several
authors reported low nitrite recoveries, so that it was quite
possible to add widely different amounts of nitrite to
2 samples and, after processing and/or extraction, find the
same low amount of residual nitrite in both
samples (7, 11–14). Such capricious loss processes of nitrite
in meat samples were not satisfactorily accounted for
and/nor compensated for.
Briefly, in AOAC Method 973.31, 5 g finely comminuted
and thoroughly mixed sample is transferred to a 50 mL beaker,
mixed with about 40 mL H2O that is heated to 80°C. The
contents are mixed thoroughly with a glass rod, taking care to
break up all lumps, transferred to a 500 mL volumetric flask,
diluted to about 300 mL, and further allowed to stand for 2 h
822 MOHAMED ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008
Figure 1. Effects of food additive concentrations on
the percent recovery of nitrite from meat samples at
25°C (dotted lines) and 80°C (solid lines).
on a steam bath with occasional shaking. The flask is cooled to
room temperature, diluted to volume with H2O, remixed, and
filtered. An aliquot sample containing 5–50 mg NaNO2– is
transferred to a 50 mL volumetric flask, mixed with 2.5 mL
sulfanilamide reagent (0.375, w/v, in 15% acetic acid), and
allowed to stand for 5 min for diazotization. A 2.5 mL NED
coupling reagent (0.133, w/v, in 15% acetic acid) is then
added, and the reacting mixture is diluted to volume, mixed
well, and allowed to stand for another 15 min for complete
color development. Absorbance is measured at 540 nm
against a reagent blank, and the nitrite content is determined
from a similarly prepared calibration graph.
According to published analytical data, several apparent
influencing factors (AIFs) appeared a priori to be important in
the determination of nitrite content by the AOAC Method.
These AIFs fall into 2 categories: (A) factors affecting the
extraction of nitrite from cured meats, and (B) factors
affecting the color development procedure for the
quantification of nitrite. Basically, the AOAC
recommendations were followed during the study of these
AIFs in order to determine the optimum conditions that will
yield efficient extraction of nitrite.
(A) Optimization of the Extraction Procedure of
Nitrite
Effects of some food additives on the recovery of
nitrite.—In preliminary experiments, 5 and 10 g aliquots of
5 different canned meat samples were treated following the
AOAC procedure, while a standard nitrite solution was spiked
Figure 2. Effect of pH on the extraction of nitrite from
cured meats in the presence of some food additives.
into each sample aliquot in an initial dissolution volume of
40 mL. Average nitrite recoveries were 75 and 60% for the 5
and 10 g samples, respectively. When 300 mL initial
dissolution volumes were used, the average recoveries were
85 and 75% for the 5 and 10 g samples, respectively.
Moreover, analysis of 0.4 mg/mL NO2– in aqueous solutions in
the presence of 5 mg/mL AA, 20 mg/mL FA, or 40 mg/mL PS,
at room temperature, gave recoveries of 38, 43, or 65%,
respectively. Thus, the use of larger sample weights and/or
lower dissolution volumes resulted in increased difficulty in
sample treatment as the fat content increases, and poor nitrite
recovery due to the increased losses of nitrite via interactions
with the high local concentrations of some meat constituents
and/or additives that include AA, FA, and/or PS. Therefore, a
thorough examination of the effects of AA, FA, and PS on the
recovery of nitrite was undertaken.
In order to study the effects of various concentrations of
AA, FA, or PS on the recovery of nitrite from meat samples, a
series of 20 mL stoppered test tubes were placed in a water
bath at 80°C and were prepared to contain 13.85 mL aqueous
solutions of various concentrations of AA, PS, or FA. A
150 mL volume of 200 mg/mL NO2– was added to each test
tube (such volumes were selected to avoid the effect of
changing the initial dilution ratio). The reacting mixtures were
mixed well, kept in the water bath for 2 h, cooled under
running tap water, and diluted to the mark in 25 mL measuring
flasks. Sample aliquots were then analyzed following the
AOAC procedure for the quantification of nitrite. Another
series was similarly treated but without incubation in the water
 MOHAMED ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008 823
Figure 3. Effect of initial dilution ratio of meat
samples during extraction of nitrite.
bath. The data are given in Figure 1 and show that incubation
in the water bath at 80°C (solid lines) reduced the effects of
AA but enhanced the effects of both FA and PS on the
depletion of nitrite. However, quantitative recoveries of ³90%
for 0.4 mg/mL NO2– were obtained in the presence of 5, 6, and
7 mg/mL AA, FA, and PS at 80°C, respectively. Fortunately,
typical concentration levels of food additives in cured meats
are 20–120 mg/g sodium nitrite, 100–1000 mg/g AA,
400–1000 mg/g FA, and 200–2600 mg/g PS, respectively.
Therefore, the use of 500 mL as a final dissolution volume, as
recommended by the AOAC Method would give
concentration levels of AA, FA, and PS that are below their
tolerance levels for the quantitative extraction of nitrite from
cured meats.
The reactive nature of nitrite and its capricious loss
processes from meat products pose a considerable problem in
its detection and/or quantification. In general, neutral or
alkaline sample solutions are preferred because acidic
conditions tend to exacerbate losses through the generation of
nitrous acid that is prone to volatilization, decomposition, or
participation in electrophilic (nitrosation/nitration) reactions
with active organic functional groups present within samples,
especially upon digesting the sample suspension for 2 h on a
steam bath as recommended in the AOAC Method. Thus, the
recovery of 0.4 mg/mL NO2– was studied, following the
AOAC recommendations, in the presence of 1–10 mg/mL AA,
1–20 mg/mL FA, or 1–20 mg/mL PS at various pH values. In
the presence of 10 mg/mL AA, 20 mg/mL FA, or 20 mg/mL PS,
very poor recoveries were observed at pH <5.0; however, at
Figure 4. Effect of pH of diazotization and coupling
on color development.
pH ³6.0, recoveries of ³90% were observed with AA or PS
(and at pH ³8.0 with FA), respectively (Figure 2, dotted lines).
On the other hand, quantitative recoveries of ³95% nitrite
were observed in the presence of 5 mg/mL AA, 6 mg/mL FA,
or 7 mg/mL PS at pH values ³5.5 (Figure 2, solid lines).
Unfortunately, strong turbidity that cannot be easily clarified
was obtained at pH values ³6.2 with cured meat samples of
moderate to high fat content. Therefore, for practical
considerations and to avoid turbidity and emulsion formation
with extracts of samples of relatively high fat contents, a pH of
5.5 ± 0.3 was adopted in the recommended procedure.
Effect of sample weight.—The contents of a can of cured
meat were homogenized in a blender, and 5 replicates of 5, 10,
and 20 g aliquots were analyzed following the AOAC
recommendations, using 300 mL initial dissolution volume. In
another run, 5, 10, and 20 g aliquots were also analyzed after
spiking with 10.0 mg/g NO2–. The sample treatment and
filtration became more difficult with increasing sample
weight. However, the nitrite recovery was found to be 85, 75,
and 70 for the 5, 10, and 20 g aliquots, respectively. Such
decrease in percent nitrite recovery with increasing the sample
weight may be attributed to the increased loss of nitrite via its
interaction with high local concentrations of one (or more)
meat constituent and/or additive. Thus, the use of high weight
sample aliquots was avoided, and the use of 5 g sample
aliquots was adopted in the present work.
Effect of the initial dilution for the extraction of meat
samples.—The initial dilution of the meat sample in the
AOAC Method is 1:8 (5 g sample mixed with about 40 mL
824 MOHAMED ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008
Figure 5. Effect of sulfanilamide concentration on
color development.
water); the finial dilution is 1:99 (5 g sample in 500 mL
volumetric flask). However, we found that the initial dilution
is a critical factor that was not studied by AOAC Method
973.31 in the presence of high local concentrations of the
critically influencing food additives AA, PS, and/or FA.
Therefore, aqueous solutions equivalent to 20 mg NO2– were
added to AA, PS, or FA solutions at 2 different concentration
ratios in various initial dilutions in 20 mL stoppered test tubes.
The solutions were kept in the water bath at 80°C for 2 h,
cooled under running tap water, and diluted to the mark in
25 mL measuring flasks. Sample aliquots were then analyzed
following the AOAC procedure for the quantification of
nitrite. The data in Figure 3 (dotted lines) show that the
presence in the sample of 1:10 ratio of NO2–:AA, 1:100 ratio
of NO2–:FA, or 1:100 ratio of NO2–:PS gave poor recoveries
of nitrite, irrespective of the initial dilution, due to the
depletion of nitrite via its interactions with the high
concentrations of AA, PS, or FA used. However, the presence
in the sample of 1:5 ratio of NO2–:AA, 1:10 ratio of NO2–:FA,
or 1:10 ratio of NO2–:PS gave quantitative recoveries of nitrite
at initial dilutions of ³1:80 (Figure 3, solid lines). Therefore,
an initial dilution of 1:80 (5 g sample mixed with about
400 mL water) was adopted in the recommended procedure.
Deproteinization of the extract.—Many authors have used
various procedures to clean up the extracts prior to nitrite
quantification, using Carrez I (potassium ferricyanide) plus
Carrez II (zinc acetate), borax, or mercuric chloride solutions
to achieve efficient deproteinization; however, AOAC
Method 973.31 does not use any of these cleanup procedures,
as Carrez II did not act effectively on protein precipitation
Figure 6. Effect of NED concentration on color
development.
either on its own or when acting synergically with
Carrez I (10–12); the borax reagent not only failed to
efficiently eliminate the protein, but in some instances even
increased the level of protein present in the extract (10–12);
and the highly toxic mercuric chloride reagent always gives
low nitrite recoveries (13). Therefore, the proposed method
does not use any of these cleanup procedures.
Effects of extraction time and temperature.—The effects of
various extraction times from 15 to 240 min and extraction
temperatures of 40 to 90°C were studied on various cured
meats and on spiked fresh cow meats from the local market.
For the spiked fresh meat samples, extraction times of
30–180 min gave maximum nitrite recoveries at temperatures
of 60–90°C. However, extraction times of 90–150 min at
temperatures of 75–90°C gave maximum nitrite contents
and/or recoveries for the studied cured meat samples.
Therefore, an extraction time of 120 min at a temperature of
80°C on a water bath was adopted in the procedure as
recommended in the AOAC Method.
(B) Optimization of the Color Development
Procedure for Nitrite Quantification
Effects of pH and buffer type.—The effect of acidity (or pH)
on the diazotization and coupling steps was studied in the
analyses of nitrite. Five replicate analysis of 0.4 mg/mL NO2–,
following the AOAC recommendations, in the presence of
0.25 M acetic acid (pKa = 4.76, pH ca 2.70) gave an absorbance
of 0.318 ± 0.004, whereas in the presence of 0.25 M
 MOHAMED ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008 825

its low cost and high buffering capacity when compared to

Figure 7. Effect of standing time after addition of NED
on color development.
chloroacetic acid (pKa = 2.87, pH ca 1.74) or dichloroacetic
acid (pKa = 1.35, pH ca 1.0), the absorbance values were 0.430
± 0.003 or 0.437 ± 0.004, respectively. Thus, the use of
chloroacetic acid (or dichloroacetic acid) resulted in a 35% (or
37%) increase in sensitivity compared to the use of acetic acid.
However, meat samples contain various species that would alter
the pH of weak acids, and thus the use of an effective buffer
would be advantageous. Therefore, chloroacetic/chloroacetate
buffer was adopted in the recommended procedure because of
dichloroacetic/dichloroacetate buffer. Thus, a series of
solutions of 1.0 M chloroacetic acid were adjusted to various
pH values in the range of 1.60–3.60, and the effect of pH was
studied in the analysis of 0.4 mg/mL NO2– (Figure 4). However,
a pH of 1.80 ± 0.05 was adopted in the recommended
procedure because of its high sensitivity.
Effects of buffer concentration.—A 1.0 M
chloroacetic/chloroacetate buffer solution of pH 1.80 was
prepared, and the effect of 0.05–0.35 M buffer concentrations
was studied and found to have almost no effect on the
absorbance values. Therefore, a 0.20 M buffer concentration
was adopted in the recommended procedure to provide a
reasonable buffering action.
Effect of sulfanilamide concentration.—The effect of
0.08–2.0 mM sulfanilamide was studied and maximum
absorbance values were observed at concentrations ³0.6 mM
(Figure 5). A sulfanilamide concentration of 1.0 mM was
adopted in the recommended procedure.
Effect of NED concentration.—The effect of
0.005–0.500 mM concentrations of NED was studied and
maximum absorbance values were observed at concentrations
³0.10 mM (Figure 6). A NED concentration of 0.20 mM was
adopted in the recommended procedure.
Effects of added salts.—Cured meat samples are known to
contain salts such as sodium chloride (20 000–40 000 mg/kg)
and polyphosphates (4000–6000 mg/kg), among other
additives. It was found that, in the analysis of 0.4 mg/mL NO2–
following the recommended procedure, the presence of
0.005–0.20 M NaCl in the spectrophotometric cell had no
effect on the absorbance values. Also, the presence of
0.001–0.10 M sodium sulfate, 0.001–0.10 M boric acid, and
0.001–0.10 M phosphoric acid had almost no effect.
However, in the determination of 0.4 mg/mL NO2– following
the conditions of the original AOAC Method, the presence of
0.001, 0.005, 0.01, 0.05, and 0.1 M NaCl in the

Table 1. Some analytical characteristics of AOAC Method 973.31 and the proposed method

Characteristic AOAC Method 973.31 Proposed method

Calibration equation A = (0.008 ± 0.003) + (0.796 [NO2–] ± 0.003) A = (0.007 ± 0.002) + (1.076 [NO2–] ± .003)

LODa = 3Sb/slope, ng/mL 11.3 5.6

b
LOQ = 10Sb/slope, ng/mL 37.7 18.6
NED working solution, mM/L 5 (in 15% acetic acid) 3 (in water)
NED final concentration, mM/L 0.25 0.2
Sulfanilamide working solution, mM/L 20 (in 15% acetic acid) 15 (in water)
Sulfanilamide final concentration, mM/L 1 1
Type of acid or buffer Acetic acid (final concn = 1.5% or ca 0.25 M/L) Chloroacetic/chloroacetate buffer
(final concn = 0.2 M/L)
pH of diazotization and coupling ca 2.70c 1.80 ± 0.05

a
LOD = Limit of detection.
b
LOQ = Limit of quantitation.
c
Value is subject to marked changes, especially upon analyzing cured meat samples of variable compositions.
826 MOHAMED ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008

Table 2. Quantification of nitrite in some meat products

Nitrite concentration, mg/g

Found ± SD (n = 6)

Variance ratio
Sample Extract, pH Added AOAC Method Recovery, % Proposed method Recovery, % values, F6,6

Bologna 5.34 — 8.6 ± 0.3 — 11.2 ± 0.3 — 1.0

10 16.4 ± 0.5 78 20.9 ± 0.6 97 1.4
Sausage 4.96 — 3.7 ± 0.2 — 6.6 ± 0.3 — 2.3
10 11.9 ± 0.4 82 16.0 ± 0.5 94 1.6
Beef salami 4.87 — 5.8 ± 0.2 — 9.4 ± 0.3 — 2.3
10 14.0 ± 0.3 82 19.2 ± 0.5 98 2.8
Corned beef 5.65 — 4.3 ± 0.2 — 8.5 ± 0.3 — 2.3
10 12.8 ± 0.3 85 18.2 ± 0.4 97 1.8
Luncheon beef 5.12 — 6.8 ± 0.3 — 13.0 ± 0.3 — 1.0
10 14.9 ± 0.4 81 22.5 ± 0.5 95 1.6

spectrophotometric cell increased the nominal absorbance
value by 3, 10, 15, 21, and 26%, respectively.
Effect of color development–time.—Nitrite was allowed to
react with sulfanilamide in the presence of 0.2 M
chloroacetic/chloroacetate buffer of pH 1.8, for 1–15 min to
form a diazonium salt that is further allowed to react with
NED for 1–45 min (Figure 7). Maximum absorbance values
were obtained after standing times of ³2 and ³15 min for the
diazotization and coupling steps, respectively. Standing times
of 5 and 15 min, as recommended by the AOAC Method,
were adopted in the proposed procedure for the diazotization
and coupling steps, respectively.
Calibration graph, detection limit, and precision.—A linear
calibration graph (Amax vs concn) was obtained, following the
proposed procedure for up to 0.8 mg/mL NO2–, with a
correlation coefficient of 0.9996 and a limit of detection (LOD)
based on the 3Sb-criterion, 3 times the standard deviation (SD)
of the blank divided by the slope of the calibration graph (15) of
5.6 ng/mL NO2–. The linear regression equation is
A = (0.007 ± 0.002) + (1.076 [NO2–] ± .003) at 540 nm
where [NO2–] is the nitrite concentration expressed in mg/mL.
The small SDs of the slope and intercept indicate the high
accuracy and precision of the proposed method. Moreover,
the precision of the method was further assessed according to
the International Union of Pure and Applied Chemistry
recommendations (15) by analyzing 0.1, 0.3, 0.5, and
0.8 mg/mL NO2– in aqueous solutions that gave quantitative
recoveries ³99.7%, where the within- and between-day
relative standard deviation values were £1.2 and 1.8% (n = 6),
respectively; and the Student’s t-test values were £1.9,
showing that the t-test could not detect any systematic error
and revealing the high accuracy and precision of the proposed
method. The tabulated t-value for the 95% confidence level
and n = 6 is 2.45 (15). Table 1 compares some analytical
characteristics of the proposed method with the AOAC
Method, showing the advantageous sensitivity of the
proposed method.
Applications.—The proposed method was applied to
various cured meat products obtained from the local market
(Table 2). The proposed method was compared with the
original AOAC Method, and its reliability to analyze such
samples was further validated by recovery experiments that
gave quantitative recoveries (94–98%) with convenient
reproducibility. The variance ratio values F6,6 were £2.8,
where the threshold F6,6 value at the 95% confidence level is
4.28 (15), indicating the high accuracy and precision of the
proposed method.
Conclusions
To improve the extraction efficiency of nitrite from cured
meat samples followed by its quantification based on AOAC
Method 973.31, we proposed a modified procedure with
enhanced sensitivity, selectivity, and performance
characteristics. The proposed modifications included the use
of £5 g sample aliquots; the use of 400 mL water as an initial
extraction volume; adjusting the pH of aqueous sample
suspension to 5.5 ± 0.3 during the extraction step; the use of
aqueous reagent solutions; and the use of
chloroacetic–chloroacetate buffer of pH 1.8 ± 0.05 for the
diazotization and coupling steps.
Acknowledgments
This work was supported in part by a grant in aid (No. AT
22-80) from King Abdulaziz City for Science and
Technology. This support is gratefully acknowledged.
 MOHAMED ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 4, 2008 827
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