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Bulletin UASVM Animal Science and Biotechnologies, 65(1-2)/2008

pISSN 1843-5262; eISSN 1843-536x

EXPERIMENTAL MODEL FOR VALIDATION OF THE METHOD


FOR RAW FEED INGREDIENTS PROTEIN DETERMINATION

Olteanu Margareta, Mariana Ropot, Arabela Untea, Rodica Diana Criste

National Research Development Institute for Animal Biology and Nutrition, Balotesti,
1 Bucharest Alley, Romania, phone: 0213512082
email: margaretaolteanu@yahoo.com

Key words: method validation, protein, urease activity, soybean meal

Abstract. The problems of the European feed industry at the end of the past century focused the attention
of the consumers worldwide on the role of the feed industry in animal food production. Within this context, the
feed manufacturers and the animal producers are bound to assume new responsibilities in food safety and
quality, animal welfare and environmental protection. EU legislation and the Romanian legislation imposed
special requirements on the performance of the field laboratories by validating the analytical methods for foods
and feeds. The paper presents an experimental model for the validation of the Kjeldahl method for crude protein
determination in the soybean meal major protein source, determining the following performance parameters:
accuracy, fidelity, repeatability, reproducibility, sensitivity, detection limit, limit of quantification and tracing,
according to SR EN ISO / CEI 17025:2005 standard. The results of the validation tests show that the
performance parameters are within the admitted limits. The paper makes available to the feed quality checking
specialists an experimental model for the validation of method for protein determination in proteic feed
ingredients.

INTRODUCTION

At the present time, the European regulations on feed quality are part of the food safety
legislation, which relies on the risk factors analysis in terms of public health. The economic
development and modernization of Romania, its integration within the European circuit,
require paying special attention to product quality, products which should be competitive,
meeting the quality conditions demanded by this circuit. Because the quality control tends to
get ever closer to the standards of the food industry by implementing the standards and
hygienic control at the level of the requirements for human feeding, the validation of methods
became compulsory for foods and feeds (4, 6).
Among the vegetal protein feeds, soybean meal is the most important source of amino
acids for poultry, its level in the crude protein being a limiting quality parameter used in the
sales of over 130 megatons/year worldwide (1, 5). The quality of the soybean meal protein is
important for most nutritionists because its content and table values for amino acid content
and digestibility are used in diet optimisation. It is therefore important to experiment the
model of Kjeldahl method validation on soybean meal.
Although during the past decade the CAN (Combustion Nitrogen Analysis) method
started to be used as reference method, particularly on the American continent, nitrogen
analysis by combustion, also named the Dumas analysis, the official reference method
remained the Kjeldahl method. The principle of the Kjeldahl method for protein
determination is sample decomposition by heating with sulphuric acid in the presence of

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catalysts for organic nitrogen reduction to ammonium ions, which can be determined by
distilling/titration or colorimetrically.
The validation of an analytical method is a process through which, using laboratory
studies and mathematical-statistic analyses, the characteristic performance of a method are
confirmed to meet the requirements for the intended applied activity (9). It results that this is a
procedure for the verification and conformation of the performance of an analytical method
with the following purposes: increase the confidence in the results of an analytical method,
knowing that this method will be used by different analysis in different laboratories, with
different equipment or with similar equipment but of different manufacture; prove that a
method corresponds to the use it was designed for; meet customer requirements and reach the
performance parameters required fir testing (2, 3, 7).
The purpose of the paper was to develop an experimental model, readily applicable in
the feed quality control laboratories, for the achievement of the stages required for the
validation of an analytical method for protein determination.

MATERIAL AND METHODS

The paper presents experiments for the validation of the analytical method for crude
protein determination the Kjeldahl method (8) from soybean meal. Soybean meal is an
important vegetal protein feed, the protein being the main parameter for the trading
transactions. The reference documents for the validation of the analytical method for protein
determination are these mentioned in the references (2,4,7).
The experimental analytical works on soybean meal for Kjeldahl method validation
aimed to determine:
-the extent to which an analytical result gets close to the real value - exactness (accuracy),
-the degree of agreement between the independent results of a set of determinations, done on
the same sample analyt, under the same working conditions - precision (fidelity),
-fidelity under identical experimental conditions repeatability,
- fidelity under different experimental conditions reproducibility,
-variation of the instrument response to the variation of analyt concentration sensitivity,
-lowest concentration of analyt in the sample which can be detected but can not be quantified
limit of detection (LOD),
- lowest concentration of analyt in the sample which can be detected quantitatively, at an
acceptable level of incertitude limit of quantification (LOQ),
-level of tracing an amount of analyt added to the sample before making the measurement -
tracing (recovery),
-parameter associated to a measurement, which characterises the scattering of values, which
could be reasonably assigned to the measuring instrument incertitude.
The reference material (RM) was the double sulphate of iron and potassium (alaun) -
((NH4)2Fe(II)(SO4)2*6H2O, 100% pure and with a nitrogen concentration of =7.145%. We
worked on series of 10 repeated determinations, done by different analysts, on a
semiautomatic FOSS-KJELTEC 2300 with standardization certificate.

RESULTS AND DISCUSSIONS

The protein content determined for the soybean meal was 46.16%, with an incertitude of
1.04%. Tables 1 and 2 show the nitrogen content of the analysed samples, while Table 3
shows the values of the performance parameters.
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Thus, the accuracy was 100.07%, which is within the interval 80 120% for
concentrations higher than 10g/kg, which is 0.001(g/g), according to ANSVSA Order
51/2005. The obtained values was verified with the t Student test, so that for tcalculated we
obtained 0.61, which is lower than the tabular value of t, which is 2.26, for an interval of
confidence P=95%. It results that the data belong to the same population of values with the
reference value.
The precision, given by the value of the intra-laboratory variation coefficient
CV(RSD)calc was 0.36% which is lower than the allowed limit, CV(RSD)max of 10% and
which is in agreement with ANSVSA Order 51/2005.
The repeatability, which is also given by the value of the intra-laboratory variation
coefficient CV (RSD)calc, but determined on 3 batches of 10 analyses each, conducted under
similar experimental conditions, was: CV(RSD)1calc = 0.182% for batch 1, CV(RSD)2calc =
0.364% for batch 2, and CV(RSD)3calc = 0.210% for batch 3, all of them lower than the
maximal allowed limit, according to ANSVSA Order 51/2005.
Table 1
Nitrogen content of the reference samples alaun (Mohr salt) expressed in gN%g sample

Amount of sample (g) 0.3 0.5 0.5 0.7


Analyst I Analyst I. (different Analyst II Analyst I
equipment.
reagents)
7.168 7.197 7.149 7.171
7.146 7.164 7.168 7.164
7.121 7.104 7.162 7.136
7.136 7.144 7.094 7.164
Results (%). Xi 7.152 7.150 7.178 7.136
7.139 7.145 7.167 7.150
7.146 7.175 7.112 7.141
7.158 7.143 7.119 7.164
7.142 7.154 7.151 7.178
7.148 7.124 7.169 7.143
Average value Xaverage (%) 7.146 7.150 7.147 7.155
Standard deviation - S 0.013 0.026 0.029 0.015
Table 2
Nitrogen content of the reference samples alaun (Mohr salt) expressed in mgN%g sample

Amount of sample (g) 0.0013 0.5 0.6 0.7


Analyst I
0.112 35.985 42.567 50.197
0.114 35.820 42.726 50.148
0.106 35.520 42.987 49.952
0.111 35.720 43.025 50.148
Results (mgN/g sample). Xi 0.113 35.750 42.693 49.952
0.109 35.725 42.959 40.050
0.112 35.875 43.163 49.987
0.108 35.715 42.788 50.148
0.111 35.770 42.824 50.246
0.112 35.620 42.764 50.001
Average value Xaverage (%) 0.111 35.750 42.850 50.083
Standard deviation - S 0.002 0.129 0.180 0.107

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Performance parameters Table 3
Tested parameter: KJELDAHL method - STANDARD SR 13325-1995
ACCURACY Experiments:
Xaverage A sample with = 7.145 %N of alaun ((NH4)2Fe(II)(SO4)2*6H2O
Accuracy % = 100 Mohr salt).
10 repeated analyses of the reference material with
Xaverage = average of the 10 determinations concentration were performed, working on 0.5g sample.
= real value of the reference material Results:
Xmediu The Xi values of concentration obtained experimentally were
Bias % = * 100 determined by calculation:
Xaverage = 7,150 %; S = 0,026
Requested performance criteria: Accuracy = 100.07%
Accuracy: 80 120%, for concentrations 80<100.07<120 % Bias = 0.07%
10 g/kg, i.e. 0.001% (g/g)
Observations: Comments:
The resulted value was verified with the t t calculated = 0.61;
Student test t tabular = 2.26 pt. P = 95%
Xaverage t calc < t tab 0.61<2.26 the data belong to the same
t = population of values as the reference value.
s/ n
where: n = 10 determinations
PRECISION (FIDELITY) Experiments :
s 10 repeated analyses of the reference material with = 7.145
CV(RSD) % = 100 %N, working on 0.5g sample
Xaverage
Requested performance criteria : Results:
Maximum value of RSD is determined Xaverage = 7.150 %
according to the analyt concentration: pt. S = 0.026
conc. 1000 g/kg, CV(RSD)max = 10%, CV(RSD)calc = 0.36%
according to ANSVSA Order nr. 51/2005
Experiments :
REPEATABILITY 3 series of 10 repeated analyses of the reference material, with
Calculate: concentration = 7.145 %N, each, done by one analyst, at short
Xaverage , S time intervals, using the same equipment and working method;
s Working amounts:
CV(RSD) % = 100 - series (1) 0,3g sample
Xaverage - series (2) 0,5g sample
- series (1) 0,7g sample
Requested performance criteria : Results:
Maximum value of RSD is determined Series (1)- 0,3g sample Series (2) 0,5g sample
according to the analyt concentration : X1average = 7,146% ; S1= 0,013 X2 average = 7,150%; S2=
0,026
For conc. 1000 g/kg, CV(RSD)1calc = 0,182% CV(RSD)2calc = 0,364%
CV(RSD)max = 10%, according to
Series (3) 0,7g sample
ANSVSA Order nr. 51/2005
X3 average = 7,155%; S3= 0,015
CV(RSD)3calc = 0,210%
Experiments :
INTERNAL REPRODUCIBILITY 3 series of 10 repeated analyses of the reference material, with
Calculate: concentration = 7.145 %N, working on 0.5g. The analyses were
Xaverage , S done by two analysis using different equipment, glassware and
CV(RSD) % reagents, but the same method.
Results :
Analyst I Analyst II
Requested performance criteria: X1average = 7,150% ; S1= 0,026 X2average = 7,147% ; S2 =
For conc. 1000 g/kg, 0,029
CV(RSD)max = 10%, CV(RSD)1calc = 0,364% CV(RSD)2calc = 0,406%

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SENSITIVITY- Experiments:
LINEARITY Plotting the standardizing slope with 10 points of the reference
material with concentration = 7.145 %N, working with 0.1g and
Y = a +bX; 1g.
where: a = the origin coordinate of the Results:
line; Xi (g sample): 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9; 1
b = slope of the standardizing slope Yi (mgN): 6.938; 14.288; 20.989; 28.598; 34.734; 41.819;
b = Y / C; where Y= mass 49.860; 57.026; 63.211; 70.259.
variation; The equation of the function of linear regression:
C = concentration variation Y= 70.509x 0.008
a = -0.008

Requested performance criteria:


The value of the slope must be constant on
the working domain
a must tend towards 0, - the method is
linear and gets closer to the ideal case, i.e.
the verified Bouguer-Lambert-Beer law.
R2 correlation coefficient, must tend
towards 1;
R2 may take values between -1 and +1

LIMITA DE DETECTIE SI
LIMITA DE CUANTIFICARE Experiments:
10 repeated analyses of the reference material, working on 0.0013g
Limit of detection: for a concentration of 0.1 mg N, the minimal limit for which the
LoD = 3*S + Xaverage equipment can give a result. The working domain of the equipment
according to the handbook: 0.1 200 mg N.
Limit of quantification: Results:
LoQ = 10 * S + Xaverage Xaverage = 0.111 mgN; S = 0.002
LoD = 3 * 0.002 + 0.111=0.117mgN
Requested performance criteria: LoQ = 10 *0.002+0.111= 0.131mgN
LoQ > LoD
Experiments:
TRACING / RECOVERYING -3 series of 10 analyses each working on samples with in the
interval 20%.
Tracing (R)% -0.5g sample with =35.725 mg N
( XaverageS Xaverage) -0.6g sample with =42.870 mg N
= 100 -0.7g sample with =50.015mg N
Xad Added amount of analyt:
where: Xad1 = 7.145mg N
Xaverage S = average value of the sample Xad2 = 14.29mgN
with supplement Results:
Xaverage S = average value of the sample Xaverage = 35.750 mg N;
with no supplement Xaverage S1 = 42.850 mg N;
Xad = added amount of analyt Xaverage S2 = 50.083 mg N;
Requested performance criteria: R1 % = 99.37%;
Tracing R (%) between: 80 120% for R2% = 100.3 %
concentrations 10 g/kg
INCERTITUDE Uc = 0.52g%
-associated to determination Uc
-extended - U(r) U(r) = 1.04

The internal reproducibility, which was determined on 2 batches of 10 analyses


repeated by two analysts, produced the following values of the intra-laboratory variation

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coefficient CV(RSD)2calc = 0.364% (analyst I), and CV(RSD)2calc = 0.406% (analyst II), both
of them lower than the maximal allowed limit, according to ANSVSA Order 51/2005.
Regarding the sensitivity, i.e. the way in which the measuring instrument responds to
the variation of the analyt concentration, it was given by the equation of linear regression Y =
a+bx = 70.509X- 0.008 with the regression coefficient R2=0.9996. The value of ,,a tends
towards 0and the regression coefficient towards 1, which means that the function is linear and
gets close to the ideal case, the Burguer-Lambert-Beer law.
Regarding the limit of quantification (LOQ), which usually is the lowest point on the
standardizing line, we obtained a value of 0.131mgN, higher than the value for the limit of
detection (LOD) of 0.117mgN, being in agreement with ANSVSA Order 51/2005.
Regarding the tracing, given by the amount of analyt found after the analysis, as
proportion of the amount added before the analysis, we obtained: R1 % = 99.37% for an
amount of analyt Xad1 = 7.145mg N, and R2% = 100.3 % for an amount of analyt Xad2 =
14.29mgN, both values within the interval 80 120% for concentrations higher than 10g/kg,
and 0.001(g/g), being in agreement with ANSVSA Order 51/2005.
In practice the incertitude may appear due to several possible causes, including
sampling, environmental conditions, measuring instruments, glassware. This is why an
expanded incertitude is determined, which provides an interval supposed to give a high level
of confidence of finding the value of the measured item. In the case of the Kjeldahl method
for protein determination, the extended incertitude U(r) is 1.04.

CONCLUSIONS

The results of the tests required to validate the Kjeldahl method for soybean meal protein
determination show that the performance parameters ranged within the limits allowed for this
method;
The performances characteristic to the used method meet the requirements for the in tended
practical application;
We evaluate that the validation design presented in the paper may be an experimental model
for those working in feed quality validation and for those who want to licence the laboratories
where they are working.

BIBLIOGRAPHY

1. Dudley-Cash W. A., 2001, Methods for determining quality of soybean meal protein important. The
Proceedings of the Arkansas Nutrition Conference;
2. *** Eurachem The fitness for purpose of analytical methods. A Laboratory Guide to Method Validation and
Related Topics 2003;
3. *** ICH Q2B, 1996, Analytical Validation Methodology;
4. Ordinul ANSVSA nr. 51/2005 pentru aprobarea Normei sanitar veterinare de implementare a masurilor de
supraveghere si control al unor substante si al reziduurilor acestora la animale vii si la produsele lor privind
performanta metodelor analitice si interpretarea rezultatelor;
5. Parsons C. M., 2000, Assessment of nutritional quality of soy products for animals. In: Soy in Animal
Nutrition. James K. Drakley Ed., pg. 90-105;
6. Rodica-Diana Criste, Margareta Olteanu, Anca Mariana Bercaru, Claudia Mihaila, Manuela Chetea, 2002,
Modernizarea conceptului de evaluare a calitatii nutreturilor, Lucrarile simpozionului IBNA, pg.47-54;
7. Standardul SE EN ISO/CEI 17025:2005, Cerine generale pentru competena laboratoarelor ncercri i
etalonari;
8. Standardul SR 13325 1995, Nutreuri. Determinarea coninutului de azot i calculul coninutului de protein
brut
9. Tanase si col, 2007, Validarea metodelor analitice.

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