EXPERIMENTAL MODEL FOR VALIDATION OF THE METHOD FOR RAW FEED INGREDIENTS PROTEIN DETERMINATION

Size: px

Start display at page:

Download "EXPERIMENTAL MODEL FOR VALIDATION OF THE METHOD FOR RAW FEED INGREDIENTS PROTEIN DETERMINATION"

Tracey Malone
6 years ago
Views:

1 Bulletin UASVM Animal Science and Biotechnologies, 65(1-2)/2008 pissn ; eissn x EXPERIMENTAL MODEL FOR VALIDATION OF THE METHOD FOR RAW FEED INGREDIENTS PROTEIN DETERMINATION Olteanu Margareta, Mariana Ropotă, Arabela Untea, Rodica Diana Criste National Research Development Institute for Animal Biology and Nutrition, Balotesti, 1 Bucharest Alley, Romania, phone: 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 77

2 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) - ((NH 4 ) 2 Fe(II)(SO4) 2 *6H 2 O, 100% pure and with a nitrogen concentration of µ=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. 78

3 Thus, the accuracy was %, which is within the interval % for concentrations higher than 10µg/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 t calculated 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) Analyst I Analyst I. (different Analyst II Analyst I equipment. reagents) Results (%). X i Average value X average (%) Standard deviation - S Table 2 Nitrogen content of the reference samples alaun (Mohr salt) expressed in mgn%g sample Amount of sample (g) Analyst I Results (mgn/g sample). X i Average value X average (%) Standard deviation - S

4 Accuracy: %, for concentrations 10 µg/kg, i.e % (g/g) Observations: The resulted value was verified with the t Student test t Xaverage µ = s / n where: n = 10 determinations PRECISION (FIDELITY) Performance parameters Table 3 Tested parameter: KJELDAHL method - STANDARD SR ACCURACY Xaverage A sample with µ = %N of alaun ((NH 4 ) 2 Fe(II)(SO4) 2 *6H 2 O Accuracy % = 100 Mohr salt). µ 10 repeated analyses of the reference material with µ X average = average of the 10 determinations concentration were performed, working on 0.5g sample. µ = real value of the reference material Bias % = Xmediu µ The Xi values of concentration obtained experimentally were * 100 determined by calculation: µ X average = 7,150 %; S = 0,026 Accuracy = % 80<100.07<120 % Bias = 0.07% s Xaverage CV(RSD) % = 100 Requested performance criteria : Maximum value of RSD is determined according to the analyt concentration: pt. conc µg/kg, CV(RSD) max = 10%, according to ANSVSA Order nr. 51/2005 REPEATABILITY Calculate: X average, S s Xaverage CV(RSD) % = 100 Requested performance criteria : Maximum value of RSD is determined according to the analyt concentration : For conc µg/kg, CV(RSD) max = 10%, according to ANSVSA Order nr. 51/2005 INTERNAL REPRODUCIBILITY Calculate: X average, S CV(RSD) % For conc µg/kg, CV(RSD) max = 10%, Comments: t calculated = 0.61; t tabular = 2.26 pt. P = 95% t calc < t tab 0.61<2.26 the data belong to the same population of values as the reference value. Experiments : 10 repeated analyses of the reference material with µ = %N, working on 0.5g sample X average = % S = CV(RSD) calc = 0.36% Experiments : 3 series of 10 repeated analyses of the reference material, with concentration µ = %N, each, done by one analyst, at short time intervals, using the same equipment and working method; Working amounts: - series (1) 0,3g sample - series (2) 0,5g sample - series (1) 0,7g sample Series (1)- 0,3g sample Series (2) 0,5g sample X 1average = 7,146% ; S 1 = 0,013 X 2 average = 7,150%; S 2 = 0,026 CV(RSD) 1calc = 0,182% CV(RSD) 2calc = 0,364% Series (3) 0,7g sample X 3 average = 7,155%; S 3 = 0,015 CV(RSD) 3calc = 0,210% Experiments : 3 series of 10 repeated analyses of the reference material, with concentration µ = %N, working on 0.5g. The analyses were done by two analysis using different equipment, glassware and reagents, but the same method. Results : Analyst I Analyst II X 1average = 7,150% ; S 1 = 0,026 X 2average = 7,147% ; S 2 = 0,029 CV(RSD) 1calc = 0,364% CV(RSD) 2calc = 0,406% 80

5 SENSITIVITY- LINEARITY Y = a +bx; where: a = the origin coordinate of the line; b = slope of the standardizing slope b = Y / C; where Y= mass variation; C = concentration variation Plotting the standardizing slope with 10 points of the reference material with concentration µ = %N, working with 0.1g and 1g. X i (g sample): 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9; 1 Yi (mgn): 6.938; ; ; ; ; ; ; ; ; The equation of the function of linear regression: Y= x a = 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. R 2 correlation coefficient, must tend towards 1; R 2 may take values between -1 and +1 LIMITA DE DETECTIE SI LIMITA DE CUANTIFICARE Limit of detection: LoD = 3*S + X average Limit of quantification: LoQ = 10 * S + X average LoQ > LoD TRACING / RECOVERYING Tracing (R)% ( XaverageS Xaverage) = 100 Xad where: X average S = average value of the sample with supplement X average S = average value of the sample with no supplement X ad = added amount of analyt Tracing R (%) between: % for concentrations 10 µg/kg INCERTITUDE -associated to determination Uc -extended - U(r) 10 repeated analyses of the reference material, working on g for a concentration of 0.1 mg N, the minimal limit for which the equipment can give a result. The working domain of the equipment according to the handbook: mg N. X average = mgn; S = LoD = 3 * =0.117mgN LoQ = 10 * = 0.131mgN -3 series of 10 analyses each working on samples with µ in the interval ± 20%. -0.5g sample with µ= mg N -0.6g sample with µ= mg N -0.7g sample with µ=50.015mg N Added amount of analyt: X ad1 = 7.145mg N X ad2 = 14.29mgN X average = mg N; X average S1 = mg N; X average S2 = mg N; R 1 % = 99.37%; R 2 % = % Uc = 0.52g% 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 81

6 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 = X with the regression coefficient R 2 = 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: R 1 % = 99.37% for an amount of analyt X ad1 = 7.145mg N, and R 2 % = % for an amount of analyt X ad2 = 14.29mgN, both values within the interval % for concentrations higher than 10µg/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 ± 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 ; 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, CerinŃe generale pentru competenńa laboratoarelor încercări şi etalonari; 8. Standardul SR , NutreŃuri. Determinarea conńinutului de azot şi calculul conńinutului de proteină brută 9. Tanase si col, 2007, Validarea metodelor analitice. 82

ADAPTATION OF THE QUALITY CONTROL OF THE POLYUNSATURATED FATTY ACIDS AND VITAMIN E- ENRICHED FEEDS TO THE EUROPEAN STANDARDS

Lucrări ştiinńifice Zootehnie şi Biotehnologii, vol. 41(1) (2008), Timişoara ADAPTATION OF THE QUALITY CONTROL OF THE POLYUNSATURATED FATTY ACIDS AND VITAMIN E- ENRICHED FEEDS TO THE EUROPEAN STANDARDS