Journal of Cereal Science 58 (2013) 31e36

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Journal of Cereal Science

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Cereal protein analysis via Dumas method: Standardization of a

micro-method using the EuroVector Elemental Analyser
S. Serrano a, *, F. Rincón a, J. García-Olmo b
a
Department of Food Science and Technology, Charles Darwin Building Annex, Campus of Rabanales, University of Córdoba, E-14014 Córdoba, Spain
b
Central Service for Research Support (SCAI), Campus of Rabanales, University of Córdoba, E-14014 Córdoba, Spain

a r t i c l e i n f o a b s t r a c t

Article history: The Dumas method (DM) of protein analysis in foods was standardized using two cereal references in
Received 13 November 2012 order to identify the main sources of error during analysis; specifically, the study sought to establish
Received in revised form which of the apparent critical factors (ACFs) were really significant influencing factors (IF) for the
27 March 2013
application of the DM to protein analysis in cereals.
Accepted 8 April 2013
Using a non-geometric Plackett-Burman experimental design augmented to resolution IV via fold-over
by increasing to twenty-four runs, the following eleven ACFs were investigated: sample weight (WS),
Keywords:
capsule sealing process (CW), carrier gas flow (PC), purge gas flow (FHe), oxygen volume (O2), oxygen
Dumas method
Protein analysis
pressure (DO2), sample delay time (SD), sample run time (SR), front furnace temperature (FFT), rear
Kjeldahl method furnace temperature (RFT), and GC oven temperatures (GCT).
PlacketteBurman experimental design Two IFs were identified for each of the factors RFT and GCT, so an optimization process was carried out,
based on a central composite design.
Optimized conditions, ensuring enhanced accuracy and repeatability when determining protein
content in cereals using the DM, are reported.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction has it slowly begun to replace the traditional KM as the method of

The Dumas method (DM) for the quantitative determination of
nitrogen is based on the Dumas method originally employed to
determine the molecular weight of an unknown gas; it is used as an
alternative to the classical Kjeldahl method (KM) for analyzing the
protein content of foods (Jiménez and Lahda, 1993).
The two methods are based upon very different principles. The
DM consists in combusting a sample of known mass at high tem-
perature in the presence of oxygen; the gases produced are then
reduced by copper and dried while the CO2 is trapped, and finally
nitrogen is quantified using a universal detector. In the KM, by
contrast, the sample is wet digested to convert organic nitrogen
into ammonia nitrogen by boiling sulfuric acid; the ammonia
liberated is distilled, collected and titrated and a value for crude
protein is calculated. Although the DM was introduced by Jean-
Baptiste Dumas in 1831, and thus predates the KM by over 50
years (Kjeldahl, 1883), only over the last ten years e thanks to
improvements in dry combustion nitrogen analyzer technology e
* Corresponding author. Tel.: þ34 9 57 212654; fax: þ34 9 57 212000.
E-mail address: bt2sejis@uco.es (S. Serrano).
choice for protein analysis.
Certain difficulties have been reported with the quantitative
conversion of organic nitrogen to ammonia (Ma and Rittner, 1979).
Some forms of nitrogen are difficult, if not impossible, to convert to
ammonia (Sebranek and Cassens, 1973) and the KM achieves low
recoveries of nitrogen from refractory compounds with NeO or Ne
N bonds such as nitrite, nitrate, oximes azo-, nitro- and nitroso-
compounds (Kubota et al., 2011); as a result, values obtained using
the DM tend to be slightly higher than those provided by the KM. In
the case of cereal products, that difference is reported to range from
2% to 4% (Bellomonte et al., 1987) with a mean value of 1.7%
(Thompson et al., 2002). Most authors agree that DM gives a more
reliable measure of organic nitrogen than KM and offers greater
precision in the determination of protein content in cereals and
oilseeds (Beljkas et al., 2010). In addition, DM has been shown to
have certain major advantages: it is faster, more economical and
less polluting than KM (Chiacchierini et al., 2003) and probably
safer, according to our self experience.
The next step must therefore be to devise a suitable protocol for
each type of sample, with a goal of optimizing the analytical process
using the DM. Some authors have attempted to optimize the DM
using a 2k experimental design (Saint-Denis and Goupy, 2004).

0733-5210/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jcs.2013.04.006
32 S. Serrano et al. / Journal of Cereal Science 58 (2013) 31e36

However, this experimental way has proved that it is not the best 2.2. Reference materials
way of optimizing a process because two consecutive steps are
required. First, we need to determine which of the apparent critical The material used to calibrate analytical instruments for DM
factors (ACFs) are actually of major importance to the analytical protein analysis is generally selected on the basis of sample nitro-
procedure (step one, or screening phase). To address step two (or gen content, although there is usually a marked difference in matrix
optimization phase), experimental designs with three- to five-levels characteristics between calibration material and actual samples.
per factor are necessary (Myers and Montgomery, 1995), because For example, atropine (4.84% N) or acetanilide (10.36%) have been
when a variable is measured at three or more values, a quadratic used in infant-foods analysis (Bellomonte et al., 1987) and NaN3
response surface can be estimated by least-squares regression and solutions for milk (Jakob et al., 1995). Here, the reference standard
the predicted optimal value (i.e. the stationary point of the system) used for instrument calibration was 2, 5-bis (5-tert-butyl-benzox-
can be found from the estimated surface (SAS, 1999). azol-2-yl) thiophene (BBOT).
Previous obtained results unpublished on lycopene extraction Two cereal reference materials, BCR-381 rye (Secale cereale L.)
from tomato (Solanum lycopersicum L.) products highlighted the flour, 1.249  0.1 %N) and BCR-382 wheat (Triticum aestivum) flour,
differences between maximization (using a 2-levels- per-factor 2.12  0.1% N) were used for screening the factors affecting the DM
design, such as that employed by Saint-Denis and Goupy, 2004) analytical process.
and optimization (using a 5-levels-per-factor design, as reported
here). Marked quantitative differences were reported between the 2.3. Experimental design
two processes and between the end results, confirming that
maximization and optimization are clearly very different concepts. 2.3.1. Screening phase
Since the design to be used depends on the research stage An n  n matrix H ¼ hij is a Hadamard matrix of order n if the
involved e in the early stages, the goal is to detect or confirm the entries of H are either þ1 or 1, and such that HHt ¼ nI, where Ht is
importance of the variables or factors included in the experimental the transpose of H and I is the order n identity matrix. In other
design e and since screening designs are the most commonly-used words, a (þ1, 1) matrix is a Hadamard matrix if the inner product
factorial designs for determining the robustness of an analytical of two distinct rows is 0 and the inner product of a row with itself is
process (Youden and Steiner, 1975), the aim of this study was to n. Taking Hadamard matrices as their starting-point, Plackett and
analyze the effect of certain ACFs on the DM analytical process, Burman (1946) developed a screening-experiment design (PB)
using a PlacketteBurman experimental design to identify IFs and enabling estimation of main effects, assuming e on the basis of the
later to optimize the IF combination. effect sparsity principle e that all interactions are negligible when
compared with the few important main effects. Later, Box and
Hunter (1961) demonstrated that resolution IV designs can be ob-
2. Material and Methods
tained by folding over the PB experimental design.
Although other authors (e.g. Saint-Denis and Goupy, 2004)
2.1. Analytical instrument
considered only three factors for DM optimization, a review of the
instrument manual, a survey of the literature going back 10 years
The Dumas method is based on combustion of the whole sample
and a brainstorming session led to the identification of eleven
in an oxygen-enriched atmosphere at a high temperature in order
apparent critical factors (ACFs); a non-geometric PB design was
to ensure complete combustion. A EuroVector Elemental Analyser
therefore selected, because the number of experiments was a mul-
EA3000 equipped with Callidus software (EuroVector SpA, Milan,
tiple of four but not a power of two, so twelve runs were required
Italy) was used. The analyzer was generally operated in accordance
(k ¼ n  1). The ACFs examined and their experimental ranges are
with manufacturer’s instructions, but also bearing in mind the
shown in Table 1. Finally, to obtain resolution IV, the number of ex-
experimental design used for the screening phase (Table 1).
periments included in the experimental design was increased to
The present study included two samples obtained from the
twenty four runs via fold over. The experimental design is shown in
Community Bureau of Reference (BCR). The role of certified refer-
Tables 2 and 3 for CRM-381 and CRM-382, respectively.
ence materials (CRMs) in analytical chemistry is now well estab-
lished, particularly for calibration and for verifying the accuracy of
2.3.2. Optimization phase
analytical methods (Belliardo and Wagstaffe, 1988). The general
The simplicity of the screening phase lies in the assumption of a
point of using CRMs is to improve accuracy in an optimization
linear relationship between the settings of the factors and the re-
process such as that reported in this paper.
sponses. However, the optimization phase requires a more complex
Table 1 experimental design because only when each variable is measured
Eleven apparent critical factors (ACF) investigated, and experimental ranges tested at 3 or more levels, can a quadratic response surface be estimated
for the PlacketteBurman experimental design at resolution IV level. by least-squares regression and the optimal values (or stationary
ACF Symbol Levels point of the system) be found from the estimated surface (SAS,
1999). A two-level experimental design such as that used by
Code Actual 1 þ1
Saint-Denis and Goupy (2004) can thus never be used to optimize a
Sample weight, mg X1 WS 2 4 process. This is a fundamental consideration, since two points can
Capsule sealing processa X2 CW A B
Carrier gas flow, ml min1 X3 PC 80 120
only serve to estimate a linear model, but never a quadratic model.
Purge gas flow, ml min1 X4 FHe 60 100 Hence, at least a three-level design, e.g. a full factorial or Boxe
Oxygen volume, ml X5 O2 7 14 Behnken experimental design, must be used for optimization
Oxygen pressure, kPa X6 DO2 20 40 purposes.
Sample delay time, seg X7 SD 10 20
After the screening process, as indicated later under Results and
Sample run time, seg X8 SR 150 250
Front furnace temperature,  C X9 FFT 800 1000 Discussion, the number of key factors to be optimized turned out to
Rear furnace temperature,  C X10 RFT 500 750 be just 2. It was therefore possible to use a central composite design
GC oven temperature,  C X11 GCT 75 95 (CCD) (Box and Wilson, 1951) which is a sum of a two-level factorial
a
A ¼ capsule sealed with pliers to obtain the shape of a small bag. B ¼ capsule design and a star design, to obtain 5 levels per factor, as shown in
sealed with the help of tweezers to obtain a spherical shape. Table 4.
 S. Serrano et al. / Journal of Cereal Science 58 (2013) 31e36 33

Table 2
PlacketteBurman experimental design and results obtained for rye flour CRM-381 (R1 and R2) and wheat flour CRM-382 (R3 and R4). The responses considered were R1 and
R3 ¼ mean difference between detected and certified values of four replicates; R2 and R4 ¼ SD of replicate determinations.

Trial Run X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 R1 R2 R3 R4

1 14 1 1 1 1 1 1 1 1 1 1 1 1.831 2.094 0.711 0.844

2 10 1 1 1 1 1 1 1 1 1 1 1 0.013 0.046 0.020 0.075
3 17 1 1 1 1 1 1 1 1 1 1 1 0.120 0.102 0.129 0.069
4 15 1 1 1 1 1 1 1 1 1 1 1 0.438 0.463 0.479 0.551
5 20 1 1 1 1 1 1 1 1 1 1 1 6.532 5.686 22.135 13.946
6 22 1 1 1 1 1 1 1 1 1 1 1 0.153 0.034 0.325 0.022
7 19 1 1 1 1 1 1 1 1 1 1 1 0.181 0.144 0.267 0.024
8 13 1 1 1 1 1 1 1 1 1 1 1 0.120 0.030 0.200 0.032
9 9 1 1 1 1 1 1 1 1 1 1 1 0.005 0.015 0.001 0.013
10 2 1 1 1 1 1 1 1 1 1 1 1 0.101 0.014 0.175 0.032
11 18 1 1 1 1 1 1 1 1 1 1 1 0.459 0.354 0.423 0.227
12 6 1 1 1 1 1 1 1 1 1 1 1 0.467 0.089 0.349 0.141
13 4 1 1 1 1 1 1 1 1 1 1 1 0.202 0.184 1.078 0.428
14 1 1 1 1 1 1 1 1 1 1 1 1 5.911 2.269 7.886 2.764
15 5 1 1 1 1 1 1 1 1 1 1 1 0.501 0.030 0.550 0.062
16 7 1 1 1 1 1 1 1 1 1 1 1 0.022 0.025 0.022 0.007
17 3 1 1 1 1 1 1 1 1 1 1 1 0.029 0.022 0.007 0.018
18 12 1 1 1 1 1 1 1 1 1 1 1 0.009 0.010 0.017 0.037
19 23 1 1 1 1 1 1 1 1 1 1 1 0.748 0.039 1.469 0.253
20 21 1 1 1 1 1 1 1 1 1 1 1 0.033 0.029 0.028 0.015
21 24 1 1 1 1 1 1 1 1 1 1 1 0.688 0.040 1.106 0.098
22 11 1 1 1 1 1 1 1 1 1 1 1 0.141 0.018 0.285 0.140
23 16 1 1 1 1 1 1 1 1 1 1 1 0.690 0.214 1.989 0.445
24 8 1 1 1 1 1 1 1 1 1 1 1 0.312 0.246 0.833 0.541

Accuracy is the degree of concordance between the detected
value and the certified reference value, so the R1 response measures
the difference between detected (mean of four replicates) and
certified values. Precision, by contrast, is the variability of the re-
sults obtained when analyzing the same sample using the same
analytical method for validation purposes. The R2 response mea-
sures the SD of four replicate measurements.
Because accuracy is the concordance between the detected value
obtained and the certified reference value, the R1 (CRM-381) and R3
(CRM-382) responses will measure the difference between both
detected (mean of four replicates) and certified values (Table 2).
Precision is the variability of results obtained by analyzing the same
sample using the same method of analysis to validation, so R2 (CRM-
381) and R4 (CRM-382) responses were used to measure the SD of
four replicates determinations. In summary, factors affecting both
accuracy and precision of DM were detected and later optimized.
For both screening and optimization phases, the Design-Expert
(Stat-Ease, Inc. Minneapolis) and Statistical (StatSoft, Inc, Tulsa)
software packages were used to generate designs, fit the response
surface model to the experimental data, and draw response sur-
faces figures at optimization phase. In addition, runs were per-
formed in random order (trial order) because randomization helps
protect the experimenter, again reaching erroneous conclusions
due to extraneous sources of variability (Joglekar and May, 1987).
Effects identified as IF (p  0.25) for each of the responses
considered are shown in Table 4. Certain IFs were identified
amongst the apparent critical factors (ACF) included in the exper-
imental screening design (Table 1). On the basis of the effects
detected, a number of factors (X1 to X4 and also X6) appeared not to
be critical for the implementation of the DM, i.e. differences
between 1 and þ1 levels were not significant at the p  0.25
confidence level. Thus, over the experimental range considered
(Table 1), sample weight, capsule sealing process, carrier gas flow,
purge gas flow and oxygen pressure, appeared not to be critical in
the case of cereal samples. A smaller difference between detected
and certified nitrogen content (R1 and R3 responses, Table 4) and a
lower SD (R2 and R4 responses, Table 4), therefore needed to be
considered during the stage in optimization, as did the low levels
recorded for X1, X2, X4 and the high levels recorded for X3 and X6.
Similar results for the influence of sample weight were reported by
Saint-Denis and Goupy (2004), who concluded that DM, though
faster than KM, was less precise for milk samples. By contrast,
Kubota et al. (2011) reported that the DM provided excellent
Table 4
Effects in terms of coded factors on each response considered for strong effects. A
conventional criterion was considered to define a strong effect such as p  0.25 and
so to identify the factor as an influencing factor (IF).
Responsesa

Rye flour Wheat flour

3. Results and Discussion
Factors R1 R2 R3 R4
The results obtained for each of the trials included in the X1
experimental design for both rye and wheat CRM samples are X2
X3
shown in Table 2.
X4
Table 3 X5 0.84 2.67 1.36
Influencing factors to be optimized in a central composite experimental design, with X6
experimental ranges expressed in coded and actual units. X7 1.13 0.81 2.35 1.37
X8 0.89 0.52 2.57 1.36
Symbol Levels X9 0.52 1.38
X10 1.10 0.75 2.38
Code Actual 1.41 1 0 1 1.41
X11 1.11 0.87 2.36 1.50
Rear furnace temperature,  C X10 RFT 609 650 750 850 891 a
R1 and R3 ¼ difference between detected and certified values; R2 and R4 ¼ SD of
GC oven temperature,  C X11 GCT 53.8 60 75 90 96.2
replicate determinations.
34 S. Serrano et al. / Journal of Cereal Science 58 (2013) 31e36
precision for fish samples, while Daun and DeClercq (1994) found
that DM yielded more accurate measurements than KM in oilseed
samples and Nozawa et al. (2007) reported that DM provided
excellent precision and was at least as accurate as KM for soy
(Glycine max L.) sauce samples. It must therefore be concluded that
certain factors in DM implementation are critical for ensuring
adequate precision. The poorer precision reported by Saint-Denis
and Goupy (2004) may be ascribed to use of an experimental
range (700e1300 mg) much larger than that used here (Table 1)
and also greater than the sample size recommended by the
manufacturer (150e200 mg) for a similar instrument of the same
brand (Beljkas et al., 2010).
With regard to the IFs detected (Table 4), some preliminary
comments are warranted. First, similar IFs were detected for rye and
wheat samples: X5 and X7 to X11. These factors (oxygen volume,
sample delay time, sample run time, front and rear furnace tem-
perature and GC oven temperature) therefore needed, a priori, to be
optimized in a subsequent step. Second, although nitrogen content
in wheat samples was 1.7 times that of rye samples, the effects of
factors identified as IF in both sample types displayed a similar trend
(Table 4), reflecting the similar composition of the two cereal sam-
ples. However, this difference in nitrogen concentration prompted
a difference in uncertainty, a parameter e associated with the result
of a measurement e that characterizes the dispersion of the values
that could reasonably be attributed to the measurand, according to
Horwitz’s criteria (Horwitz, 2003), uncertainty for rye and wheat
samples was 4.8  104 and 7.5  104, respectively.
X5 (oxygen volume, Table 1) exerted a positive effect on R1 and R3
in both samples, the effect being particularly marked for wheat
(Table 4, R3). The higher the value, the further it lay from the real
value. Specifically, values rose from 0.4 to 1.24 units for rye
(effect ¼ 1.24  0.4 ¼ 0.84, Table 4, R1) and from 0.35 to 3.02 units for
wheat (effect ¼ 3.02  0.35 ¼ 2.67, Table 4, R3). In other words, as
oxygen volume increases, measurement becomes less accurate,
leading to a higher-than-actual result for N content. Moreover, this
loss in accuracy is accompanied by a sharp increase in R2 and R4 (2.6
for rye and 8 for wheat), thus prompting a major loss in precision.
Briefly, oxygen volume (Table 1, X5) displayed a positive linear
effect both for R1 (rye sample) and for R3 and R4 (wheat sample),
indicating that as the value of this parameter increases, both the
accuracy (Table 4, R1 and R3) and the precision (Table 4, R2 and R4)
of the method decline. Consequently, the value to be used for this
factor should lie at the lower end of the experimental range tested
(Table 1). Clearly, during the optimization phase, the value of X5
should be kept at the 1 level (Table 1, 7 ml), so that factor X5
becomes a constant in the optimization phase. The presence of an
excessive volume of O2 during sample combustion leads to (i)
greater oxidation of the reactor reducing agent (Cu) and a short-
ening of its useful life, (ii) the appearance of partially-overlapping
peaks, preventing their adequate integration, with the result that
the measured amount of N is higher than the real amount, and (iii)
an increase in background noise and thus a loss of precision.
Saint-Denis and Goupy (2004) failed to consider O2 volume as a
possible critical factor e since it was not included as a factor in the
experiment design used e so that use of an excessive oxygen vol-
ume (not shown by the authors) might have contributed to the poor
precision they reported in comparison to the KM. Despite the
critical role of oxygen volume in the acceptability of the analytical
result obtained, a role highlighted in the present study, many au-
thors fail to optimize this parameter in accordance with the sample
analyzed (Daun and DeClercq, 1994; Nozawa et al., 2007; Beljkas
et al., 2010; Kubota et al., 2011) opting simply to follow manufac-
turers’ instructions for any sample type. The results obtained here
underline the fact that the generic instructions provided by the
manufacturer are not the most suitable for all sample types; rather,
every sample type requires its own specific conditions in order to
ensure the best possible analytical performance.
A similar analysis can be carried out for X7 (sample delay time,
Table 1) leading to the same conclusion. A longer waiting-time
before the sample is placed in the oven-reactor leads to a larger
amount of air in the sample container, which would readily account
for the effects observed (Table 4).
X8 (sample run time, Table 1) exerted a negative effect on ac-
curacy in both samples, the effects were especially marked in the
wheat sample (Table 4, R1 and R3). The mean detected value at
level 1 was 2.16% N, compared with a level þ1 value of 1.27% N in
the rye sample (certified value 1.249% N), and with 4.49% N and
2.09% N, respectively, in the wheat sample (certified value 2.12% N).
In other words, as sample run time increased, so did measurement
accuracy. At the same time, the effect on both R2 and R4 was also
negative, i.e. SD was smaller and therefore measurement precision
was greater.
Briefly, X8 displayed a negative linear effect on all the responses
studied (Table 4), indicating that, in the optimization phase, the
value to be used should lie at the higher end of the experimental
range tested (Table 1, X8). The effects recorded for this factor can
readily be accounted for, given that a decrease in chromatogram
duration leads to the appearance of chromatographic peaks that fail
to reach the baseline, thus prompting errors in peak integration due
to poorer definition of baseline peaks. Saint-Denis and Goupy
(2004) obtained outliers when integration time was short, while
Williams et al. (1998) note that instrument malfunction, i.e. the use
of non-optimized instrument conditions, should be included
among the most important sources of error in Dumas testing,
which is why the equipment operating manual should specify that
optimum conditions need to be established for each sample type.
Regarding X9 (front furnace temperature, Table 1), either of the
tested values for this parameter (800  C and 1000  C, Table 1) were
sufficient to ensure complete sample combustion. Hence, there was
no difference between detected % N and certified % N for these
samples (Table 4). For example, a furnace temperature of 870  C, i.e.
lower than the maximum included in the experimental range used
here (Table 1), has been used successfully for other types of food
including soy sauce (Nozawa et al., 2007). However, this factor did
display a marked effect on the precision of the method (Table 4, R2
and R4). Precision was considerably improved at 1000  C, and this
temperature should therefore be used in the optimization phase.
What was a factor in the screening phase thus becomes a constant in
the optimization phase. At 800 C, the combustion of N-steam
generating compounds was ensured, higher temperatures enabled
greater and better combustion of other substances whose presence in
the combustion chamber and subsequent presence in the reactor
leads to considerable background noise and hence to loss of preci-
sion. For larger sample sizes (700e1300 mg), and thus for larger
amounts of interfering gaseous substances reported in the literature
(Saint-Denis and Goupy, 2004), it has even been suggested that
temperatures of up to 1150  C be used. When using a nitrogen
analyzer based on the DM, the intensive steam production during
injection may result in poor nitrogen recoveries for volatile nitrogen
compounds such as ethylene diamine (Jakob et al., 1995). By contrast,
heavy samples and low furnace temperatures are associated with a
very high number of low-end outliers (Saint-Denis and Goupy, 2004).
Similar temperatures to those recommended in this paper (1000  C)
have been used by other authors, including 950  C (AOAC, 1990;
Krotz et al., 2008; Beljkas et al., 2010) and, in some cases, 870 C
(Horwitz, 2003) or even 850  C (Daun and DeClercq, 1994).
Slightly different results were obtained for rear furnace tem-
perature (X10) and GC oven temperature (X11) (Table 1). The in-
crease in values for X10 (Table 4) was accompanied by a decrease of
the difference with respect to certified values from 1.37 to 0.27 for
 S. Serrano et al. / Journal of Cereal Science 58 (2013) 31e36 35

rye and from 2.88 to 0.5 for wheat. In other words, greater accuracy 1,41
is obtained at the þ1 level. Since the oven-reactor temperature 0,1 0,00 A
should not be kept constantly at 1000  C, two stand-by tempera- 1,00
tures were tested (500  C and 750  C; Table 1). It was found that 0,02
when a stand-by temperature of 500  C was used, the values ob-
0,04
tained for the first sample were lower than real values, probably
due to insufficient combustion of that sample. As a result, the mean 0,2 0,06
value for the four replicates decreased, prompting a loss of accu-

11
racy. A few minutes should therefore be allowed to elapse before 0,00

X
the sample is inserted, even though the instrument may signal that 0,08 0,1
the initial set temperature has been reached. A stand-by temper-
ature of 600  C has been used by other authors (Nozawa et al.,
2007) with satisfactory results in other types of sample (soy sauce). 0,0
Similarly, better results were obtained for R2 (Table 4) at -1,00 -0,1
level þ1; indeed, precision was improved 6.8-fold for rye and 7.7-
fold for wheat with respect to level 1. The effects of factor X11 were -0,2
-1,41
the reverse of those recorded for X10 (Table 4). The smallest dif- -1,41 -1,00 0,00 1,00 1,41
ference with respect to certified values was obtained at level 1 X10
(Table 4, R1 and R3), and a lower value for SD (Table 4, R2 and R4)
was also obtained at this level. Gases leaving the oven-reactor pass 1,41
into a chromatography column, whose temperature has a crucial
impact on the shape and position of the peaks obtained. Inadequate 1,00 0,00
B
integration and possible overlapping of these peaks might account
for the effects obtained. 0,2
0,02
The difference between factors X10 and X11 and the other factors 0,1
examined earlier is that their effect on detected nitrogen levels in 0,0
the sample appears ostensibly to be a quadratic rather than a linear
11

0,00
effect, as shown in Fig. 1. The presence of expected levels (certified 0,04
X

values) amongst the levels obtained points to the existence of -0,1

quadratic effects for both factors, which cannot be reliably detected
using a PlacketteBurman experimental design. Both these factors
should therefore be carried forward into the optimization phase,
using a central composite design, as outlined under Material and
Methods and detailed in Table 3. The results of this experimental
design enabled the optimization of conditions for parameters X10
and X11 in the case of rye (Fig. 2A) at 870  C and 92  C for X10 and X11,
respectively, and in the case of wheat (Fig. 2B) at as 720  C and
95  C, for X10 and X11, respectively. Beljkas et al. (2010) used a rear
furnace temperature of 850  C for N determination in wheat sam-
ples. Although they did not implement a fully-fledged optimization
process, simply following manufacturer’s instructions, this tem-
perature recommendation would agree with the results obtained
here for rye, but not with those obtained for wheat. This highlights
the need to optimize the analytical process for each different
sample type. Various combinations of these factors have been used
by other authors, including 840  C and 50  C for X11 and X12,
respectively (Krotz et al., 2008), and 850  C (Beljkas et al., 2010) or
600 C for X11 (Nozawa et al., 2007).
Rye Wheat
= certified value: 1.249 % N = certified value: 2.12 % N
-1 +1 -1
-0,2
0,06
-1,00
-1,41
-1,41 -1,00 0,00 1,00 1,41
X10
Fig. 2. Optimization graphics. Quadratic effect detected for rye (A) and wheat (B), and
identification of system stationary point.
In order to validate the conditions suggested here for the use of
the DM for protein measurement in cereals, five measurements
were performed for wheat (2.1067, 2.1253, 2.1254, 2.1365 and
2.1162, mean 2.122 e real values 2.12%- and SD 0.011) and rye
(1.2534, 1.2728, 1.2655, 1.2685 and 1.2533, mean 1.262 ereal value
1.249%- and SD 0.009).
For the measurement of crude protein in grains and oilseeds using
the combustion method, standard deviations for repeatability and
reproducibility are reported to range from 0.10 to 0.37 and from 0.25
to 0.54, respectively, while relative standard deviations for repeat-
ability and reproducibility range from 0.77 to 2.57% and from 1.24 to
3.15%, respectively (Bicsak, 1993). In a more recent review, Moore
et al. (2010) reported that intra- and interlaboratory relative stan-
+1
dard deviations for different matrices range from 0.30% to 8.3% and

X10 0.62e8.2%, respectively. In general, a significantly lower analytical

precision has been reported (Sweeny and Rexroad, 1987) for the
2.40 N level detected 1.03 4.75 N level detected 1.84
combustion method (0.013e0.052%) as compared with KM (0.006e
0.035%). Similar results are noted by Schmitter and Rihs (1989). The
-1 +1 -1 +1
results obtained in the present study suggest that the poorer preci-

X11 sion of DM compared to KM reported in the literature may be

attributable to the use of non-optimized instrumental conditions.
1.10 N level detected 2.33 1.94 N level detected 4.64
Beljkas et al. (2010) concluded that since the DM procedure is
Fig. 1. N levels detected as a function of X10 and X11 factor levels, and relationship with very simple and almost completely automated, there are relatively
certified value for rye and wheat samples (BCR-381 and BCR-382, respectively). few sources of uncertainty compared to the KM. The present results
36 S. Serrano et al. / Journal of Cereal Science 58 (2013) 31e36
show that edespite fewer sources of uncertainty than in the KM e
the failure to use instrumental conditions perfectly optimized for
each sample type may constitute a major source of error when
implementing the DM.
This study confirmed that measurement of nitrogen content
using the DM requires the prior optimization of analytical condi-
tions for each sample type, rather than simple reliance on the
general recommendations provided by the equipment manufac-
turer. Use of non-optimized conditions may be a significant cause of
error in analytical measurements.
In this study, application of a non-geometric PlacketteBurman
experimental design enabled the identification of two critical fac-
tors showing quadratic effects: RFT (rear furnace temperature) and
GCT (GC oven temperatures). Accordingly, an optimization process
was performed, based on a central composite design in order to
obtain perfectly-optimized conditions for the implementation of
the DM.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.jcs.2013.04.006.
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