BELLOMONTE ET AL.: J. ASSOC. OFF. ANAL. CHEM. (VOL. 70, NO.
2, 1987) 227
VITAMINS
Comparison of Modified Automatic Dumas Method and the Traditional Kjeldahl Method for
Nitrogen Determination in Infant Food
GUIDO BELLOMONTE, ANNA COSTANTINI, and STEFANIA GIAMMARIOLI
Istituto Superiore Sanitd, Laboratorio Alimenti, Viale Regina Elena 299, Rome, Italy
This study compares 2 methods for determining nitrogen and protein
in various types of infant food: the Kjeldahl method, developed in
1883, which is time consuming and labor intensive, and a newer,
automatic method, based on the Dumas method. In each category of
infant food considered, the results obtained from both methods are
shown to be comparable; however, the modified Dumas method is
quicker, easier, and does not pollute the laboratory environment.
The standard national and international methods for nitro-
gen and protein determination are based on the original
method introduced by Kjeldahl (1) in 1883, which has been
modified through the years (2-7). To improve some parts of
the method (e.g., long digestion time, difficulties associated
with the quantitative conversion of organic nitrogen to am-
monia (6, 8), and contribution to environmental pollution),
several studies have been conducted, some of them promoted
by AOAC, in which a comparison is made between the of-
ficial method and the automated procedure based on the
principle of Kjeldahl (6, 9, 10), or the alternative fast method
proposed by Dumas. In the latter method, the pure sample
is combusted and the quantity of nitrogen formed is deter-
mined by volumetric techniques (11-14).
This paper presents a comparison of the results obtained
from the analysis of infant food by the Kjeldahl procedure,
which is the basis for the official Italian methods (15-19),
and a new automated nitrogen analyzer N A 1500, which uses
sample combustion (as originally proposed by Dumas) rather
than digestion. The only feature that this new analyzer has
in common with the Dumas method is the use of combustion
to obtain elemental nitrogen, and even in this the NA 1500
differs because it uses tin sample containers and an oxygen-
enriched atmosphere to achieve a combustion temperature
of around 1800°C. This high temperature assures complete
combustion of the entire sample. Nitrogen is separated from
the other combustion products by means of a chromato-
graphic column, and its concentration is determined by a
thermal conductivity detector. This separation and detection
system is much quicker (results every 3 min) than the method
used by Dumas (nitrometer) and also distinguishes the NA
1500 from other commercially available analyzers using the
combustion method. A plot of the chromatogram delivered
with the data print-out permits the operator to see eventual
interference.
We analyzed samples belonging to the main categories of
infant food: infant formula; food for weaning such as cereal-
based products and dietetic biscuits; lyophilized and ho-
mogenized products. Within each category, the samples were
selected in a manner to take into consideration the most
varied types of food.
Experimental
Reagents and Standards
Reagents are analytical grade.
(a) Atropine. —4.84% N (Carlo Erba Strumentazione, Milan,
Italy).
Received May 24, 1985. Accepted May 23, 1986.
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(b) Acetanilide. —10.36% N (Carlo Erba Strumentazione).
Apparatus
(a) Kjeldahl universal Model K3/E electric BICASA.
(b) Nitrogen analyzer. —Automatic nitrogen analyzer Model
1500 (NA 1500) (Carlo Erba Strumentazione) (20, 21).
Sample Preparation
Due to the small amount of sample used for the automatic
nitrogen analyzer in comparison to that used for the Kjeldahl
method, particular attention must be given to sample prep-
aration to ensure sufficient homogeneity for reliable analysis.
Cereal-based products and biscuits were ground to fine pow-
der by using a laboratory mill. Lyophilized products were
analyzed after samples had been dried to constant weight in
thermostated oven at 105°C. To avoid erroneous results and
to minimize differences between 2 methods, samples were
weighed as soon as possible after food containers were opened
and after homogenization, and analyses were run in parallel
by both methods. This applied to hygroscopic samples (infant
formula, biscuits, cereal-based products), to lyophilized
products, and to those products which tend to lose humidity
rapidly, such as homogenized products.
Analytical Method
Nitrogen determination with the Kjeldahl method was done
using the official method of the European Economic Com-
munity (18), which was the most appropriate for the samples
being analyzed. This method, which is similar to the official
AOAC method, specifies the mineralization of 1 g sample
with concentrated sulfuric acid in the presence of potassium
sulfate. We selected copper sulfate as the catalyst instead of
mercury oxide to avoid problems of ambient contamination
in the laboratory.
Determinations with the NA 1500 were done according to
instructions of the manufacturer, using 20-25 mg sample,
weighed accurately. Reference standards were selected as a
function of the nitrogen content of the sample: atropine (4.84%
N) for infant formula, cereal-based products, biscuits, and
homogenized products; acetanilide (10.36% N) for lyophi-
lized products.
Kjeldahl analyses were done in duplicate because this
method is accurately standardized in our laboratory. Anal-
yses done with the NA 1500 were done in triplicate to assure
the accuracy of the results.
To calculate the sample protein content from the nitrogen
data obtained, we used the conversion factors reported by
the manufacturer on the labels of the product. These factors,
which are shown at the bottom of the tables relative to the
products analyzed, correspond to those used by the United
Nations Food and Agriculture Organization (FAO) (22).
Results and Discussion
The reproducibility of the analysis performed with the NA
1500 was verified statistically from the data of a significant
number of analyses using standards and 3 samples belonging
to 3 categories of infant food (infant formula, cereal flour,
228 BELLOMONTE ET AL.: J. ASSOC. OFF. ANAL. CHEM. (VOL. 70, NO. 2, 1987)

Table 1. Reproducibility of analysis performed with NA 1500 on various food products

Homogenized beef and

Statistic Acetanilide Atropine Infant formula Cereal flour ham
Number of tests 13 15 14 15 9
Average* 10.39 4.85 2.74 2.66 1.67
Maximum value* 10.49 4.91 2.78 2.70 1.69
Minimum value* 10.21 4.78 2.69 2.63 1.65
Std dev. ±0.082 ±0.046 ±0.026 ±0.021 ±0.018
CV, % ±0.79 ±0.95 ±0.95 ±0.79 ±1.08

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* Value taken as g nitrogen per 100 g sample.

and homogenized products). The standard deviation and the
variation coefficient, shown in Table 1, are more than sat-
isfactory, which is in contrast with an earlier report of similar
studies conducted on a different automated instrument (12).
Thereafter, we analyzed several products belonging to 5
categories of infant food. Table 2 shows the nitrogen and
protein content of these products as determined by both
methods. The differences between the NA 1500 and the Kjei-
dahl methods are: 1-5% for infant formula, 2-4% for cereal-
based products, 2-3% for biscuits, about 2% for lyophilized
products, and 1-4% for homogenized products. The values
of samples obtained with the NA 1500 are higher than those
obtained using Kjeidahl, as was also observed for recovery
from the standard acetanilide (10.39 g N with NA 1500 vs
10.27 g with Kjeidahl). In both cases the difference between
the methods is around 2-3%. This slight but consistent dif-
ference is attributed to the principles involved (digestion vs
combustion) in the sample treatment, because different forms
of nitrogen may not be converted equally to ammonia or
nitrogen gas. It appears that the modified Dumas method is
slightly more vigorous in this regard, yielding the positive
differences compared to the Kjeidahl method.
Table 3 shows the differences between the protein content
declared by the manufacturers of the products and the values
obtained by the 2 nitrogen determination methods.
In conclusion, the values obtained with these 2 methods
are comparable. Nevertheless, the NA 1500 dynamic com-
bustion is of prime interest from the analytical viewpoint,
not only due to the speed of the analysis and the resulting
increase in the number of samples which can be analyzed

Table 2. Nitrogen and protein content of some infant food determined with NA 1500 and Kjeidahl method*

Nitrogen, % Protein, %

Sample NA 1500 Kjeidahl NA 1500 Kjeidahl Difference

Formula (% protein = % N x 6.38)

Formula 1 2.52 2.48 16.08 15.82 + 1.6

Formula 2 2.92 2.85 18.63 18.18 +2.5
Formula 3 2.62 2.50 16.72 15.95 +4.8
Formula 4 1.92 1.83 12.25 11.68 +4.9
Formula 5 2.20 2.16 14.04 13.78 + 1.9
Formula 6 2.08 2.00 13.27 12.76 +4.0
Formula 7 2.59 2.56 16.52 16.33 + 1.2

Cereal-based products (% protein = % N >: 6.25)

Creme of rice6 1.67 1.64 9.52 9.35 + 1.8

Semolina with honey" 1.32 1.27 7.52 7.24 +3.9
Creme of cereal 1.69 1.64 10.56 10.25 +3.0
Wheat flour with milk and oat 2.08 2.05 13.00 12.81 + 1.5
Milk soup with cereal and fruit 2.23 2.18 13.94 13.63 +2.3
Milk soup with cereal and apples 1.21 1.19 7.56 7.44 + 1.6

Baby biscuits"

Biscuits 1 1.87 1.81 10.66 10.32 +3.3

Biscuits 2 1.39 1.36 8.69 8.50 +2.2

Lyophilized products (% protein =• % N x 6.25)

Veal 9.46 9.26 59.13 57.88 +2.2

Ham and eggs 7.57 7.42 47.31 46.38 +2.0

Homogenized products (% protein = % N >

t 6.25)

Beef 1.59 1.56 9.94 9.75 + 1.9

Beef and ham 1.67" 1.65 10.44 10.31 + 1.2
Chicken 1.75 1.77 10.94 11.06 -1.1
Veal and brain 1.32 1.32 8.25 8.25 —
Turkey 1.99 1.92 12.44 12.00 +3.7
Chicken, carrots, and potatoes 1.03 1.01 6.44 6.31 +2.1

* Difference between NA 1500 and Kjeidahl results, expressed as percent relative to Kjeidahl.
6
% Protein = %N x 5.70.
e
% Protein = N x 5.70 for biscuits 1, x6.25 for biscuits 2.
" See reproducibility on Table 1.
 BELLOMONTE ET AL.: J. ASSOC. OFF. ANAL. CHEM. (VOL. 70, NO. 2, 1987) 229

Table 3. Comparison of the protein content of some infant foods determined by the NA 1500 and the Kjeldahl method with values
supplied by the manufacturer
Protein, % Difference from declared value, %
Sample Declared NA 1500 Kjeldahl NA1500 Kjeldahl

Infant formula
Milk 1 16.0 16.1 15.8 +0.6 -1.2
Milk 2 18.0 18.6 18.2 +3.3 + 1.1
Milk 3 15.8 16.7 16.0 +5.7 + 1.3
Milk 4 12.5 12.3 11.7 -1.6 -6.4

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Milk 5 13.8 14.0 13.8 + 1.4 —
Milk 6 13.6 13.3 12.8 -2.2 -5.9
Milk 7 16.1 16.5 16.3 +2.5 + 1.2

Cereal-based products
Creme of rice 9.0 9.5 9.4 +5.6 +4.4
Semolina with honey 7.0 7.5 7.2 +7.1 +2.9
Creme of cereal 10.8 10.6 10.3 -1.9 -4.6
Wheat flour with milk and oat 13.5 13.0 12.8 -3.7 -5.2
Milk soup with cereals and fruit 13.5 13.9 13.6 +3.0 +0.7
Milk soup with cereals and apples 7.9 7.6 7.4 -3.8 -6.3

Biscuits
Biscuits 1 10.5 10.7 10.3 + 1.9 -1.9
Biscuits 2 8.5 18.7 8.5 +2.4 —
Lyophilized products
Veal 59.2 59.1 57.9 -0.2 -2.2
Ham and eggs 49.2 47.3 46.4 -3.9 -5.7

Homogenized products
Beef 10.0 9.9 9.8 -1.0 -2.0
Beef and ham 10.5 10.4 10.3 -1.0 -1.9
Chicken 10.0 10.9 11.1 +9.0 + 11.0
Chicken, carrots, and potatoes 6.2 6.4 6.3 +3.2 + 1.6
Veal and brain 8.5 8.3 8.2 -2.4 -3.5
Turkey 11.9 12.4 12.0 +4.2 + 0.8

but above all because of the lack of interference by partic-
ularly resistant molecular structures and, finally, for not con-
taminating the laboratory environment with acid fumes or
other products of digestion.
Acknowledgment
The authors thank Carlo Erba Strumentazione for provid-
ing the apparatus and technical support and Roberto Hen-
riquez for his translation of the paper into English.
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