Evaluation Report proficiency test. DLA 43/ Free Amino Acids. in Infant Food. Waldemar-Bonsels-Weg Ahrensburg, Germany

Transcription

1 DLA Dienstleistung Lebensmittel Analytik GbR Evaluation Report proficiency test DLA 43/20 Free Amino Acids in Infant Food Dienstleistung Lebensmittel Analytik GbR Waldemar-Bonsels-Weg Ahrensburg, Germany Coordinator of this PT: Dr. Matthias Besler Page of 62

2 Inhalt / Content. Introduction Realisation Test material Homogeneity Test Submission of results Evaluation Assigned value Standard deviation Outliers Target standard deviation General model (Horwitz) Value by precision experiment Value by perception z'-score Quotient Standard uncertainty Results L-Alanine in g/00g L-Arginine in g/00g L-Aspartic Acid in g/00g L-Cyst(e)ine in g/00g L-Glutaminic Acid in g/00g Glycine in g/00g L-Histidine in g/00g L-Isoleucine in g/00g L-Leucine in g/00g L-Lysine in g/00g L-Methionine in g/00g L-Phenylalanine in g/00g L-Proline in g/00g L-Serine in g/00g L-Threonine in g/00g L-Tryptophan in g/00g L-Tyrosine in g/00g L-Valine in g/00g...4. Documentation Primary data Homogeneity Analytical Methods Index of participant laboratories Index of references...62 Page 2 of 62

3 . Introduction The participation in proficiency testing schemes is an essential element of the quality-management-system of every laboratory testing food and feed, cosmetics and food contact materials. The implementation of proficiency tests enables the participating laboratories to prove their own analytical competence under realistic conditions. At the same time they receive valuable data regarding the validity of the particular testing method. The purpose of DLA is to offer proficiency tests for selected parameters in concentrations with practical relevance. Realisation and evaluation of the present proficiency test follows the technical requirements of DIN EN ISO/IEC 7043 (200) and DIN ISO 328:2009 (6). 2. Realisation 2. Test material The test material is a common in commerce infant food formula based on free amino acids with < 20% protein equivalent content from a EU supplier with further addition of lactose. The powder was mixed and homogenized. Afterwards the samples were packaged lightproof in portions to approximately 0 g. The portions were numbered chronologically. The composition (list of ingredients) of the samples is given in table. Table : Composition of DLA-Samples Infant Food Milk Formula Ingredients: Dried glucose syrup, vegetable oils (coconut, sunflower, rape), L-Arginine-L-Aspartate, L-Leucine, L-Lysine acetate, Calcium diphosphate, Tripotassium citrate, L-Glutamine, L-Proline, Emulsifier E472c, L-Valine, Glycine, L-Isoleucine, L- Threonine, L-Phenylalanine, L-Tyrosine, L-Serine, L-Histidine, L-Alanine, Sodium chloride, L-Cystine, L-Tryptophane, Magnesium chloride, Cholin bitartrate, L-Methionine, arachidonic acid rich and docosahexaenic acid rich vegetable oils from protozoon, Magnesium-L-Aspartate, Tricalcium citrate, Inosit, L-Ascorbic acid, Taurine, Iron sulfate, Zinc sulfate, L-Carnitin, Uridin-'-Monophosphate sodium salt, Cytidin-'-Monophosphate, Nicotinamide, Inosin-'-Monophosphate disodium salt, Adenosin-'-Monophosphate, Calcium-D-Pantothenate, Guanosin-'-Monophosphate disodium salt, Antioxidants: E304, E307 and E306, DL-α-Tocopherylacetate, Copper sulfate, Thiamin hydrochloride, Pyridoxin hydrochloride, Riboflavin, Manganese sulfate, Retinylacetate, Potassium iodide, Pteroylmonoglutaminic acid, Chromium-(III)-chloride, Phyllochinone, Sodium selenite, Sodium molybdate, D-Biotin, Cholecalciferol, Cyanocobalamine further Additives: Lactose Nutrients per 00 g: protein 9,8 g, carbohydrates 64 g thereof sugar 28 g, fat 9 g Page 3 of 62

4 2.. Homogeneity Homogeneity of the test material was checked by fold determination of magnesium by ICP-OES. With a repeatability standard deviation of 4,8 % the homogeneity of the test material was considered acceptable. The results are given in the documentation. The calculation of the repeatability standard deviation of the participants was also used as an indicator of homogeneity. For L-methionine it was 0,4 % and for L-valine 6, % and in the range of common relative repeatability standard deviations of methods for amino acid determination (3-6). The repeatability standard deviation of the participants for both parameters is given in the documentation. Additionally in the documentation the portion numbers are graphically assigned to the results of L-alanine. There is no trend recognizable in the results which could suggest inhomogeneity. 2.2 Test Two portions of test material were sent to every participating laboratory in the 40 th week of 20. The testing method was optional. The tests should be finished at 3 th November Submission of results The participants submitted their results in provided standard forms by . The final concentration of the parameters as mean of both numbered samples (as far as available) was used for each statistical evaluation. Queried and documented were single results, recovery and the testing methods used. From participants of the PTs total amino acid composition (DLA 42/20) and free amino acids (DLA 43/20) 7 and 9 participants submitted results, respectively. One participant submitted no results. All other participants submitted their results in time. Page 4 of 62

5 3. Evaluation 3. Assigned value Because the analysed material was no certified reference material the robust mean of the submitted results was used as assigned value X (6). The distribution of submitted results showed no hint for bimodal distribution or other reasons for a higher variability. The statistical evaluation is performed when there are at least 7 results for a parameter. This was the case for all parameters except for cysteine/cystine, glutaminic acid/glutamine and tryptophan. Therefore for the parameters cysteine/cystine, glutaminic acid/glutamine and tryptophan no statistical evaluation was done. Single results giving values outside the measuring range of the participating laboratory were considered for statistical evaluation (e.g. results given as > 2 mg/kg and < 2, mg/kg, respectively) when a result indicating > is above and a result indicating < is below the target range. 3.2 Standard deviation For comparison to the target standard deviation a robust standard deviation (S x ) was calculated (6). 3.3 Outliers Statistical outliers were determined by Mandel s-h-statistic for 9% significance niveau (). Detected outliers were stated for information only, when z-score was < -2 or > Target standard deviation The target standard deviation of the assigned value is determined according to the following methods. In general the Horwitz target standard deviation is suitable for the statistical evaluation of interlaboratory tests where different analytical methods are applied. The standard deviation from precision experiments are derived from proficiency tests where a specific analytical method is mandatory. For information the z-scores from both models were given in the result tables, if available. For the valuation of the following parameters the target standard deviation according to the general model of horwitz was applied (3.4.): L- alanine, L-aspartic acid, glycine, L-isoleucine, L-leucine, L-lysine, L- methionine, L-tyrosine and L-valine. For the valuation of the following parameters the target standard devia- Page of 62

6 tion from precision experiments (s ) (German official ASU 64 method: 4) was applied: L-arginine, L-histidine, L-phenylalanine, L-proline, L-serine and L-threonine. Additionally the standard uncertainty was considered for glycine by evaluating with z'-score (see 3.6). For L-cysteine/L-cystine, L-glutaminic acid/glutamine and L-tryptophan there were < 7 quantitative results, thus no statistical evaluation could be done General model (Horwitz) The relative target standard deviation in % of the assigned value is derived from following equation (Horwitz) σ (%) = 2 (-0,logX) From the result the target standard deviation is calculated σ = X * σ (%) / Value by precision experiment Using the reproducibility standard deviation σ R and the repeatability standard deviation σ r of a precision experiment the between-laboratories standard deviation can be calculated σ L : L = R 2 r 2. And then, using the number of replicate measurements n, each participant is to perform, the target standard deviation for proficiency assessment is calculated : = L 2 r 2 /n. The target standard deviations given in table 2 were calculated from the precision data of the respective methods. Page 6 of 62

7 Table 2: Relative target standard deviations (RSD) according to precision experiments (4,, 6) Parameter Matrix RSD σ Method / Literature L-Alanine Dietetic food 3,7 % ASU 64 L (3) L-Arginine Dietetic food 3, % ASU 64 L (3) L-Aspartic acid Dietetic food, % ASU 64 L (3) L-Cyst(e)ine Dietetic food Protein concentrate 7, % 2, % ASU 64 L (3) VO 2/2009/EG (6) L-Glutaminic acid Dietetic food 6,6 % ASU 64 L (3) Glycine Dietetic food 4,9 % ASU 64 L (3) L-Histidine Dietetic food,9 % ASU 64 L (3) L-Isoleucine Dietetic food 4,4 % ASU 64 L (3) L-Methionine Dietetic food Protein concentrate 6,2 % 2,9 % ASU 64 L (3) VO 2/2009/EG (6) L-Leucine Dietetic food 4, % ASU 64 L (3) L-Lysine Dietetic food Protein concentrate 2,7 % 2,2 % ASU 64 L (3) VO 2/2009/EG (6) L-Phenylalanine Dietetic food,3 % ASU 64 L (3) L-Proline Dietetic food,8 % ASU 64 L (3) L-Serine Dietetic food 7,0 % ASU 64 L (3) L-Threonine L-Tryptophan Dietetic food Protein concentrate Dietetic food Dietetic food,7 % 3,0 % 9,4 % 6,8 % ASU 64 L (3) VO 2/2009/EG (6) ASU 64 L (3) ASU 64 L () L-Tyrosine Dietetic food 6,9 % ASU 64 L (3) L-Valine Dietetic food 4,7 % ASU 64 L (3) values given/applied in the evaluation (s. chapter 4) Page 7 of 62

8 3.4.3 Value by perception The target standard deviation for proficiency assessment can be set at a value that corresponds to the level of performance that the coordinator would wish laboratories to be able to achieve (6). For the present evaluation of results the target standard deviation according to Horwitz was applied for L-alanine, L-aspartic acid, glycine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-tyrosine and L-valine (3.4.) and the target standard deviation from precision experiments was applied for L-arginine, L-histidine, L-phenylalanine, L-proline, L-serine and L-threonine (3.4.2). Additionally the standard uncertainty was considered for glycine by valuating with z'-score (see 3.6). 3. To assess the results of the participants the z-score is used. It indicates about which multiple of the target standard deviation ( σ ) the result (x) of the participant is deviating from the assigned value (X) (6). Participants z-scores were derived as: z = (x X) / σ ; the requirements for the analytical performance are generally considered as fulfilled if -2 z z'-score The z'-score can be used for the valuation of the results of the participants, in cases the standard uncertainty has to be considered (s. 3.8). The z'-score represents the relation of the deviation of the result (x) of the participant from the respective assigned value (X) to the square root of quadrat sum of the target standard deviation ( σ ) and the standard uncertainty (Ux) (6). Participants z'-scores are derived as: In the following we define the denominator deviation σ '. as the target standard The requirements for the analytical performance are generally considered as fulfilled if Page 8 of 62

9 -2 z' 2. The z'-score was used for glycine by valuating with z'-score (see also 3.7 and 3.8). 3.7 Quotient S x / Following the Horrat-value the results of a proficiency-test (PT) can be considered convincing, if the quotient of robust standard deviation and target standard deviation does not exceed the value of 2. A value > 2 means an insufficient precision, i.e. the analytical method is too variable, or the variation between the test participants is higher than estimated. Thus the comparability of the results is not given (). In the present proficiency tests the quotients S x / σ were In the present proficiency test the quotient S x / σ of all parameters except glycine was below 2,0. For glycine the quotients S x / σ were clearly above 2,0. Therefore evaluation of these parameters was done according to 3.6 by z'-scores. The resulting quotients S x / σ ' were below 2,0 then. 3.8 Standard uncertainty The assigned value X has a standard uncertainty u X that depends on the analytical method, differences between the analytical methods used, the test material, the number of participant laboratories and perhaps on other factors. The standard uncertainty u X for this PT is calculated as follows (6). u X =,2 S x / p If u X 0,3 the standard uncertainty of the assigned value needs not to be included in the interpretation of the results of the PT (6). Values exceeding 0,3 imply, that the target standard deviation could be too low with respect to the standard uncertainty of the assigned value. In the present proficiency tests the quotients U x / σ were for all parameters except glycine between 0,23 and 0,79. For glycine the quotient was,3. At the same time the quotient S x / σ was > 2,0 for glycine. Therefore the target range for these parameters was extended according 3.6 z'-score considering the standard uncertainty. Page 9 of 62

10 4. Results All following tables are anonymized. With the delivering of the evaluation-report the participants are informed about their individual evaluation-number. In the first table the characteristics are listed: Statistic Data Number of results Number of outliers Mean Median Robust mean (X) Robust standard deviation (S x ) Target range: Target standard deviation σ or σ ' Target standard deviation for information lower limit of target range (X - 2 σ ) or (X - 2 σ ') * upper limit of target range (X + 2 σ ) or (X + 2 σ ') * Quotient S x / σ ' Standard uncertainty u X Quotient u X / ' Number of results in the target range * Target range is calculated with z-score or z'-score In the second table the individual results of the participating laboratories are listed: Auswertenummer Parameter Evaluation [Einheit / Unit] number Abweichung [Einheit / Unit] Deviation [Einheit / Unit] σ (Info) Hinweis Remark Page 0 of 62

11 4. L-Alanine in g/00g Vergleichsuntersuchung / Proficiency Test Statistic Data Number of results 8 Number of outliers 0 Mean 0,460 Median 0,46 Robust Mean (X) 0,460 Robust standard deviation (S x ) 0,0369 Target range: Target standard deviation σ 0,0207 Target standard deviation ASU (for Information) 0,068 lower limit of target range 0,49 upper limit of target range 0,0 Quotient S x / σ Standard uncertainty u x Quotient u x / σ,78 0,063 0,79 Results in the target range 7 Percent in the target range 88 0,80 Ergebnisse / Results 0,70 0,60 0,0 0,40 0,30 0,20 0,0 0, L-Alanin / L-Alanine obere Grenze upper limit rob. Mittelwert robust mean untere Grenze lower limit Abb. : Ergebnisse L-Alanin Fig. : Results L-Alanine Page of 62

12 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation number L-Alanin / L-Alanine Abweichung Deviation σ (Info) 0,423-0,0369 -,8-2,2 2 0,493 0,0329,6 2,0 3 0,4662 0,0062 0,3 0,4 4 0,422-0,038 -,8-2,3 6 0, 0,049 2,7 3,3 7 0,44-0,020 -,0 -,2 8 0,4-0,00-0,2-0,3 9 0,466 0,009 0,3 0,4 Hinweis Remark Abb. 2: Fig. 2: Z-Scores L-Alanin Z-Scores L-Alanine Page 2 of 62

13 4.2 L-Arginine in g/00g Vergleichsuntersuchung / Proficiency Test Statistic Data Number of results 7 Number of outliers 0 Mean 0,74 Median 0,776 Robust Mean (X) 0,749 Robust standard deviation (S x ) 0,066 Target range: Target standard deviation σ 0,0 Target standard deviation Horwitz (for Information) 0,033 lower limit of target range 0,47 upper limit of target range 0,9 Quotient S x / σ Standard uncertainty u x Quotient u x / σ 0,6 0,029 0,29 Results in the target range 7 Percent in the target range 00,20 Ergebnisse / Results,00 0,80 0,60 0,40 0,20 0, L-Arginin / L- Arginine obere Grenze upper limit rob. Mittelwert robust mean untere Grenze lower limit Abb. 3: Ergebnisse L-Arginin Fig. 3: Results L-Arginine Page 3 of 62

14 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation number L-Arginin / L-Arginine Abweichung Deviation σ (Info) 0,762 0,034 0, 0, ,7788 0,0267 0,3 0,9 4 0,63-0,8 -,2-3,8 6 0,68-0,064-0,6-2,0 7 0,78 0,0309 0,3,0 8 0,80 0 0,6,8 9 0,77 0 0,3 0,8 Hinweis Remark Abb. 4: Fig. 4: Z-Scores L-Arginin Z-Scores L-Arginine Page 4 of 62

15 4.3 L-Aspartic Acid in g/00g Vergleichsuntersuchung / Proficiency Test Statistic Data Number of results 8 Number of outliers Mean 0,686 Median 0,738 Robust Mean (X) 0,740 Robust standard deviation (S x ) 0,0332 Target range: Target standard deviation σ 0,030 Target standard deviation ASU (for Information) 0,0407 lower limit of target range 0,678 upper limit of target range 0,802 Quotient S x / σ Standard uncertainty u x Quotient u x / σ, 0,047 0,47 Results in the target range 7 Percent in the target range 88 Ergebnisse / Results,40,20,00 0,80 0,60 0,40 0,20 0, L-Asparaginsäure / L- Aspartic Acid obere Grenze upper limit rob. Mittelwert robust mean untere Grenze lower limit Abb. : Ergebnisse L-Asparaginsäure Fig. : Results L-Aspartic Acid Page of 62

16 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation number L-Asparaginsäure / L-Aspartic Acid Abweichung Deviation σ (Info) Hinweis Remark 0,29-0,480 -, -,8 Ausreisser / Outlier 2 0,78 0,04,3,0 3 0, ,0290 0,9 0,7 4 0,73-0,0269-0,9-0,7 6 0,73-0,0049-0,2-0, 7 0,74 0,000 0,0 0,0 8 0,7 0,0 0, 0,4 9 0,736-0,0039-0, -0, < Abb. 6: Fig. 6: Z-Scores L-Asparaginsäure Z-Scores L-Aspartic Acid Page 6 of 62

17 4.4 L-Cyst(e)ine in g/00g Vergleichsuntersuchung / Proficiency Test Due to the low number of results <7 no statistical evaluation was done. Below details to L-cystine: Statistic Data Number of results Number of outliers Mean 0,230 Median 0,290 Robust Mean (X) 0,23 Robust standard deviation (S x ) 0,0689 Target range: Target standard deviation σ Target standard deviation ASU (for Information) lower limit of target range upper limit of target range Quotient S x / σ Standard uncertainty u x Quotient u x / σ Results in the target range Percent in the target range 0,80 0,70 Ergebnisse / Results 0,60 0,0 0,40 0,30 0,20 0,0 0, L-Cystin / L- Cystine Median Abb. 7: Ergebnisse L-Cystin Fig. 7: Results L-Cystine Page 7 of 62

18 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation number L-Cystin / L-Cystine Abweichung Deviation 0,299 0, ,297 0, ,2283-0, ,29 0, ,04-0,208 σ (Info) Hinweis Remark For L-cysteine there were two additional results: Auswertenummer Evaluation number L-Cystein / L-Cysteine ,07 9 Abweichung Deviation σ (Info) Hinweis Remark Page 8 of 62

19 4. L-Glutaminic Acid in g/00g Vergleichsuntersuchung / Proficiency Test Due to the low number of results <7 no statistical evaluation was done. Below details to L-glutaminic acid: Statistic Data Number of results 4 Number of outliers Mean Median Robust Mean (X) Robust standard deviation (S x ) Target range: Target standard deviation σ Target standard deviation Horwitz (for Information) lower limit of target range upper limit of target range Quotient S x / σ Standard uncertainty u x Quotient u x / σ Results in the target range Percent in the target range Ergebnisse / Results 0,0 0,4 0,40 0,3 0,30 0,2 0,20 0, 0,0 0,0 0,00 < < < L-Glutamin-säure / L-Glutaminic Acid Abb. 8: Ergebnisse L-Glutaminsäure Fig. 8: Results L-Glutaminic Acid Page 9 of 62

20 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation number L-Glutaminsäure / L-Glutaminic Acid <0, 2 0, <0,0 9 <0, Abweichung Deviation σ (Info) Hinweis Remark For L-glutamine there was an additional result: Auswertenummer Evaluation number L-Glutamin / L-Glutamine Abweichung Deviation σ (Info) Hinweis Remark ,77 EG VO 2/ Page 20 of 62

21 4.6 Glycine in g/00g Vergleichsuntersuchung / Proficiency Test Statistic Data Number of results 8 Number of outliers Mean 0,680 Median 0,708 Robust Mean (X) 0,688 Robust standard deviation (S x ) 0,0964 Target range: Target standard deviation σ ' 0,06 Target standard deviation ASU (for Information) 0,0338 lower limit of target range 0,84 upper limit of target range 0,79 Quotient S x / σ ' Standard uncertainty u x Quotient u x / σ ',9 0,0426 0,83 Results in the target range 7 Percent in the target range 88,20 Ergebnisse / Results,00 0,80 0,60 0,40 0,20 0, Glycin / Glycine obere Grenze upper limit rob. Mittelwert robust mean untere Grenze lower limit Abb. 9: Ergebnisse Glycin Fig. 9: Results Glycine Page 2 of 62

22 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation number Glycin / Glycine Abweichung Deviation z'-score σ (Info) Hinweis Remark 0,823-0,03-2,0-3, 2 0,734 0,0464 0,9,4 3 0,4827-0,2048-4,0-6, Ausreisser / Outlier 4 0,69 0,0024 0,0 0, 6 0,68-0,0076-0, -0,2 7 0,79 0,024 2,0 3,0 8 0,7 0,3 2,0 9 0, ,7, 3 z'-score Abb. 0: Fig. 0: Z'-Scores Glycin Z'-Scores Glycine Page 22 of 62

23 4.7 L-Histidine in g/00g Vergleichsuntersuchung / Proficiency Test Statistic Data Number of results 7 Number of outliers 0 Mean 0,43 Median 0,420 Robust Mean (X) 0,43 Robust standard deviation (S x ) 0,034 Target range: Target standard deviation σ 0,069 Target standard deviation Horwitz (for Information) 0,097 lower limit of target range 0,296 upper limit of target range 0,74 Quotient S x / σ Standard uncertainty u x Quotient u x / σ 0, 0,067 0,24 Results in the target range 7 Percent in the target range 00 0,80 Ergebnisse / Results 0,70 0,60 0,0 0,40 0,30 0,20 0,0 0, L-Histidin / L- Histidine obere Grenze upper limit rob. Mittelwert robust mean untere Grenze lower limit Abb. : Ergebnisse L-Histidin Fig. : Results L-Histidine Page 23 of 62

24 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation number L-Histidin / L-Histidine Abweichung Deviation σ (Info) 0,429-0,027-0,3 -, 2 3 0,397-0,0376-0, -,9 4 0,47-0,076-0,3-0,9 6 0,42-0,046-0,2-0,7 7 0,4 0,04 0,2 0,8 8 0,48 0,004 0,7 2,6 9 0,460 0,029 0,4,3 Hinweis Remark Abb. 2: Fig. 2: Z-Scores L-Histidin Z-Scores L-Histidine Page 24 of 62

25 4.8 L-Isoleucine in g/00g Vergleichsuntersuchung / Proficiency Test Statistic Data Number of results 8 Number of outliers Mean 0,697 Median 0,687 Robust Mean (X) 0,686 Robust standard deviation (S x ) 0,0246 Target range: Target standard deviation σ 0,0290 Target standard deviation ASU (for Information) 0,030 lower limit of target range 0,628 upper limit of target range 0,744 Quotient S x / σ Standard uncertainty u x Quotient u x / σ 0,8 0,009 0,38 Results in the target range 7 Percent in the target range 88 Ergebnisse / Results,00 0,90 0,80 0,70 0,60 0,0 0,40 0,30 0,20 0,0 0, L-Isoleucin / L-Isoleucine obere Grenze upper limit rob. Mittelwert robust mean untere Grenze lower limit Abb. 3: Ergebnisse L-Isoleucin Fig. 3: Results L-Isoleucine Page 2 of 62

26 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation number L-Isoleucin / L- Isoleucine Abweichung Deviation σ (Info) Hinweis Remark 0,669-0,0238-0,8-0,8 2 0,8 0,29 4, 4,3 Ausreisser / Outlier 3 0,697-0,0260-0,9-0,9 4 0,693 0,0073 0,3 0,2 6 0,69 0,0043 0, 0, 7 0,67-0,07-0, -0, 8 0,70 0,093 0,7 0,6 9 0,683-0,0022-0, -0, 3 > Abb. 4: Fig. 4: Z-Scores L-Isoleucin Z-Scores L-Isoleucine Page 26 of 62

27 4.9 L-Leucine in g/00g Vergleichsuntersuchung / Proficiency Test Statistic Data Number of results 8 Number of outliers 0 Mean,7 Median,7 Robust Mean (X),7 Robust standard deviation (S x ) 0,0237 Target range: Target standard deviation σ 0,047 Target standard deviation ASU (for Information) 0,0483 lower limit of target range,08 upper limit of target range,26 Quotient S x / σ Standard uncertainty u x Quotient u x / σ 0,2 0,00 0,23 Results in the target range 8 Percent in the target range 00 Ergebnisse / Results,40,20,00 0,80 0,60 0,40 0,20 0, L-Leucin / L- Leucine obere Grenze upper limit rob. Mittelwert robust mean untere Grenze lower limit Abb. : Ergebnisse L-Leucin Fig. : Results L-Leucine Page 27 of 62

28 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation number L-Leucin / L-Leucine Abweichung Deviation σ (Info),89 0,0202 0,4 0,4 2,7 0,0007 0,0 0,0 3,463-0,0230-0, -0, 4,79 0,0097 0,2 0,2 6,6-0,0043-0, -0, 7, -0,093-0,4-0,4 8,24 0,077,7,6 9, -0,093-0,4-0,4 Hinweis Remark Abb. 6: Fig. 6: Z-Scores L-Leucin Z-Scores L-Leucine Page 28 of 62

29 4.0 L-Lysine in g/00g Vergleichsuntersuchung / Proficiency Test Statistic Data Number of results 8 Number of outliers 0 Mean 0,802 Median 0,800 Robust Mean (X) 0,797 Robust standard deviation (S x ) 0,0666 Target range: Target standard deviation σ 0,0 Target standard deviation Horwitz (for Information) 0,0330 lower limit of target range 0,9 upper limit of target range 0,999 Quotient S x / σ Standard uncertainty u x Quotient u x / σ 0,66 0,0294 0,29 Results in the target range 8 Percent in the target range 00,20 Ergebnisse / Results,00 0,80 0,60 0,40 0,20 0, L-Lysin / L- Lysine obere Grenze upper limit rob. Mittelwert robust mean untere Grenze lower limit Abb. 7: Ergebnisse L-Lysin Fig. 7: Results L-Lysine Page 29 of 62

30 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation number L-Lysin / L-Lysine Abweichung Deviation σ (Info) 0,760-0,0373-0,4 -, 2 0,809 0,06 0, 0,4 3 0,6677-0,30 -,3-3,9 4 0,767-0,0304-0,3-0,9 6 0,83 0,0376 0,4, 7 0,79-0,0074-0, -0,2 8 0,96 0,68,7, 9 0,823 0,026 0,3 0,8 Hinweis Remark Abb. 8: Fig. 8: Z-Scores L-Lysin Z-Scores L-Lysine Page 30 of 62

31 4. L-Methionine in g/00g Vergleichsuntersuchung / Proficiency Test Statistic Data Number of results 8 Number of outliers Mean 0,82 Median 0,78 Robust Mean (X) 0,79 Robust standard deviation (S x ) 0,064 Target range: Target standard deviation σ 0,00928 Target standard deviation ASU (for Information) 0,0 lower limit of target range 0,6 upper limit of target range 0,98 Quotient S x / σ Standard uncertainty u x Quotient u x / σ,8 0, ,78 Results in the target range 6 Percent in the target range 7 0,40 Ergebnisse / Results 0,3 0,30 0,2 0,20 0, 0,0 0,0 0, L-Methionin / L-Methionine obere Grenze upper limit rob. Mittelwert robust mean untere Grenze lower limit Abb. 9: Ergebnisse L-Methionin Fig. 9: Results L-Methionine Page 3 of 62

32 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation number L-Methionin / L-Methionine Abweichung Deviation σ (Info) Hinweis Remark 0,49-0,030-3,2-2,7 2 0,8 0,008 0,6 0, 3 0,2297 0,006,4 4,6 Ausreisser / Outlier 4 0,74-0,002-0,6-0, 6 0,7-0,0092 -,0-0,8 7 0,9 0,008,2,0 8 0,7-0,0042-0,4-0,4 9 0,8 0,008 0,2 0, > -4 Abb. 20: Fig. 20: Z-Scores L-Methionin Z-Scores L-Methionine Page 32 of 62

33 4.2 L-Phenylalanine in g/00g Vergleichsuntersuchung / Proficiency Test Statistic Data Number of results 8 Number of outliers Mean 0, Median 0,20 Robust Mean (X) 0, Robust standard deviation (S x ) 0,004 Target range: Target standard deviation σ 0,0273 Target standard deviation Horwitz (for Information) 0,0227 lower limit of target range 0,460 upper limit of target range 0,69 Quotient S x / σ Standard uncertainty u x Quotient u x / σ,8 0,0223 0,82 Results in the target range 6 Percent in the target range 7 0,80 0,70 0,60 0,0 0,40 0,30 0,20 0,0 0,00 Ergebnisse / Results > L-Phenylalanin / L- Phenylalanine obere Grenze upper limit rob. Mittelwert robust mean untere Grenze lower limit Abb. 2: Ergebnisse L-Phenylalanin Fig. 2: Results L-Phenylalanine Page 33 of 62

34 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation number L-Phenylalanin / L-Phenylalanine σ Hinweis Remark 0,308 0,063 0,6 0,7 2 0,73 0,08 2, 2,6 3 0,409-0,00-3,8-4,6 Ausreißer / Outlier 4 0,4 0,030,,3 3,9 Abweichung Deviation (Info) 6 0,47-0,044 -,6-2,0 7 0, -0,004-0,2-0,2 8 0, 0,3,6 9 0, ,6-0,7 Ergebnis ausgeschlossen / Result excluded Abb. 22: Fig. 22: Z-Scores L-Phenylalanin Z-Scores L-Phenylalanine Page 34 of 62

35 4.3 L-Proline in g/00g Vergleichsuntersuchung / Proficiency Test Statistic Data Number of results 8 Number of outliers Mean 0,80 Median 0,83 Robust Mean (X) 0,837 Robust standard deviation (S x ) 0,09 Target range: Target standard deviation σ 0,0483 Target standard deviation Horwitz (for Information) 0,0344 lower limit of target range 0,74 upper limit of target range 0,934 Quotient S x / σ Standard uncertainty u x Quotient u x / σ,9 0,040 0,84 Results in the target range 6 Percent in the target range 7 Ergebnisse / Results,40,20,00 0,80 0,60 0,40 0,20 0, L-Prolin / L- Proline obere Grenze upper limit rob. Mittelwert robust mean untere Grenze lower limit Abb. 23: Ergebnisse L-Prolin Fig. 23: Results L-Proline Page 3 of 62

36 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation number L-Prolin / L-Proline Abweichung Deviation σ (Info) Hinweis Remark,0844 0,247, 7,2 Ausreisser / Outlier 2 0,862 0,024 0, 0,7 3 0, ,47-3,0-4,3 4 0,83-0,024-0, -0,7 6 0,82-0,07-0,4-0, 7 0,78-0,07 -,2 -,7 8 0,9 0,062,3,8 9 0,8 0,02 0,3 0, > -4 Abb. 24: Fig. 24: Z-Scores L-Prolin Z-Scores L-Proline Page 36 of 62

37 4.4 L-Serine in g/00g Vergleichsuntersuchung / Proficiency Test Statistic Data Number of results 7 Number of outliers Mean 0,49 Median 0,20 Robust Mean (X) 0,00 Robust standard deviation (S x ) 0,094 Target range: Target standard deviation σ 0,0349 Target standard deviation Horwitz (for Information) 0,0222 lower limit of target range 0,430 upper limit of target range 0,70 Quotient S x / σ Standard uncertainty u x Quotient u x / σ,7 0,028 0,80 Results in the target range 6 Percent in the target range 86 0,80 Ergebnisse / Results 0,70 0,60 0,0 0,40 0,30 0,20 0,0 0, L-Serin / L- Serine obere Grenze upper limit rob. Mittelwert robust mean untere Grenze lower limit Abb. 2: Ergebnisse L-Serin Fig. 2: Results L-Serine Page 37 of 62

38 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation number L-Serin / L-Serine Abweichung Deviation σ (Info) Hinweis Remark 0,3769-0,230-3, -, Ausreisser / Outlier 2 3 0,4408-0,089 -,7-2,7 4 0,27 0,027 0,8,2 6 0,2 0,020 0,6 0,9 7 0, 0,00 0,3 0, 8 0,6 0,060,7 2,7 9 0,27 0,0276 0,8, Abb. 27: Fig. 27: Z-Scores L-Serin Z-Scores L-Serine Page 38 of 62

39 4. L-Threonine in g/00g Vergleichsuntersuchung / Proficiency Test Statistic Data Number of results 8 Number of outliers Mean 0,670 Median 0,96 Robust Mean (X) 0,608 Robust standard deviation (S x ) 0,0666 Target range: Target standard deviation σ 0,0348 Target standard deviation Horwitz (for Information) 0,0262 lower limit of target range 0,39 upper limit of target range 0,678 Quotient S x / σ Standard uncertainty u x Quotient u x / σ,9 0,0294 0,8 Results in the target range 6 Percent in the target range 7,40 Ergebnisse / Results,20,00 0,80 0,60 0,40 0,20 0, L-Threonin / L-Threonine obere Grenze upper limit rob. Mittelwert robust mean untere Grenze lower limit Abb. 27: Ergebnisse L-Threonin Fig. 27: Results L-Threonine Page 39 of 62

40 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation number L-Threonin / L-Threonine Abweichung Deviation σ (Info) Hinweis Remark 0,247-0,0834-2,4-3,2 2 0,9-0,08-0, -0,7 3 0,678 0,0634,8 2,4 4 0,602-0,006-0,2-0,2 6 0,6-0,048 -,4 -,8 7 0,62 0,09 0,3 0, 8,20 7,2 22,8 Ausreisser / Outlier 9 0,89 0-0,6-0,7 3 > Abb. 28: Fig. 28: Z-Scores L-Threonin Z-Scores L-Threonine Page 40 of 62

41 4.6 L-Tryptophan in g/00g Vergleichsuntersuchung / Proficiency Test Due to the low number of results <7 no statistical evaluation was done. Statistic Data Number of results 4 Number of outliers Mean 0,92 Median 0,26 Robust Mean (X) 0,92 Robust standard deviation (S x ) 0,062 Target range: Target standard deviation σ Target standard deviation ASU (for Information) lower limit of target range upper limit of target range Quotient S x / σ Standard uncertainty u x Quotient u x / σ Results in the target range Percent in the target range 0,80 0,70 Ergebnisse / Results 0,60 0,0 0,40 L-Tryptophan 0,30 0,20 0,0 Median 0, Abb. 29: Ergebnisse L-Tryptophan Fig. 29: Results L-Tryptophan Page 4 of 62

42 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation number L-Tryptophan Abweichung Deviation σ Hinweis Remark 2 3 0, ,0343 LC-MS/MS ,23 0,0376 VDLUFA 8 0,20 0, ,04-0,0879 (Info) keine Methode angegeben keine Methode angegeben Page 42 of 62

43 4.7 L-Tyrosine in g/00g Vergleichsuntersuchung / Proficiency Test Statistic Data Number of results 8 Number of outliers 0 Mean 0,24 Median 0,8 Robust Mean (X) 0,24 Robust standard deviation (S x ) 0,023 Target range: Target standard deviation σ 0,023 Target standard deviation ASU (for Information) 0,0362 lower limit of target range 0,477 upper limit of target range 0,70 Quotient S x / σ Standard uncertainty u x Quotient u x / σ 0,92 0, ,4 Results in the target range 8 Percent in the target range 00 0,80 Ergebnisse / Results 0,70 0,60 0,0 0,40 0,30 0,20 0,0 0, L-Tyrosin / L- Tyrosine obere Grenze upper limit rob. Mittelwert robust mean untere Grenze lower limit Abb. 30: Ergebnisse L-Tyrosin Fig. 30: Results L-Tyrosine Page 43 of 62

44 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation number L-Tyrosin / L- Tyrosine Abweichung Deviation σ (Info) 0,3-0,0084-0,4-0,2 2 0,2-0,0027-0, -0, 3 0,992 0,0362,6,0 4 0, -0,027-0, -0,3 6 0, 0,0278,2 0,8 7 0,0-0,037-0,6-0,4 8 0,2-0,0037-0,2-0, 9 0,06-0,077-0,8-0, Hinweis Remark Abb. 3: Fig. 3: Z-Scores L-Tyrosin Z-Scores L-Tyrosine Page 44 of 62

45 4.8 L-Valine in g/00g Vergleichsuntersuchung / Proficiency Test Statistic Data Number of results 8 Number of outliers 0 Mean 0,762 Median 0,79 Robust Mean (X) 0,762 Robust standard deviation (S x ) 0,0320 Target range: Target standard deviation σ 0,038 Target standard deviation ASU (for Information) 0,037 lower limit of target range 0,699 upper limit of target range 0,826 Quotient S x / σ Standard uncertainty u x Quotient u x / σ,0 0,04 0,44 Results in the target range 8 Percent in the target range 00,20 Ergebnisse / Results,00 0,80 0,60 0,40 0,20 0, L-Valin / L- Valine obere Grenze upper limit rob. Mittelwert robust mean untere Grenze lower limit Abb. 32: Ergebnisse L-Valin Fig. 32: Results L-Valine Page 4 of 62

46 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation number L-Valin / L-Valine Abweichung Deviation σ (Info) 0,7446-0,07-0,6-0, 2 0,72-0,037 -,2 -,0 3 0,76 0,0029 0, 0, 4 0,76 0,0029 0, 0, 6 0,8 0,038,2, 7 0,74-0,022-0,7-0,6 8 0,80 0,0429,3,2 9 0,72-0,0096-0,3-0,3 Hinweis Remark Abb. 33: Fig. 33: Z-Scores L-Valin Z-Scores L-Valine Page 46 of 62

47 . Documentation. Primary data Parameter Teilnehmer Einheit Proben-Nr. A Proben-Nr. B Ergebnis (Mittel) Ergebnis A Ergebnis B inkl. WF Wiederfindungsrate [%] Analyte Participant Unit Sample No. A Sample No. B Result (Mean) Result A Result B incl. RR Recovery rate [%] g/00g ,4232 0,4232 Not valuable no 2 g/00g ,493 0,446 0,4 no no L-Alanin / L-Alanine L-Arginin / L-Arginine L-Asparaginsäure / L-Aspartic Acid 3 g/00g ,4662 0, , yes see "further remarks" 4 g/00g ,422 0,383 0,46 no g/00g g/00g 7 4 0, 0, 0,2 7 g/00g ,44 0,4 0,43 no 8 g/00g 2 3 0,4 0,46 0,4 no 9 g/00g ,466 0,43 0,0 g/00g ,762 0,766 0,78 no 2 g/00g 6 24 ns ns ns no no 3 g/00g ,7787 0,8097 0,742 yes see "further remarks" 4 g/00g ,63 0,644 0,68 no g/00g g/00g 7 4 0,68 0,72 0,6 7 g/00g ,78 0,8 0,76 no 8 g/00g 2 3 0,80 0,82 0,79 no 9 g/00g ,77 0,79 0,792 g/00g ,29 0,2724 0,2466 no 2 g/00g ,78 0,774 0,788 no no 3 g/00g , ,797 0,7402 yes see "further remarks" 4 g/00g ,73 0,744 0,68 no g/00g g/00g 7 4 0,73 0,73 0,74 7 g/00g ,74 0,7 0,72 no 8 g/00g 2 3 0,7 0,76 0,7 no 9 g/00g ,736 0,722 0,7 Page 47 of 62

48 Parameter Teilnehmer Einheit Proben-Nr. A Proben-Nr. B Ergebnis (Mittel) Ergebnis A Ergebnis B inkl. WF Wiederfindungsrate [%] Analyte Participant Unit Sample No. A Sample No. B Result (Mean) Result A Result B incl. RR Recovery rate [%] g/00g ,299 0,2688 0,323 no 2 g/00g ,297 0,29 0,302 no no L-Cystin / L-Cystine L-Glutaminsäure / L-Glutaminic Acid 3 g/00g , , ,23 yes see "further remarks" 4 g/00g 2 44 g/00g g/00g g/00g ,29 0,3 0,27 no 8 g/00g g/00g ,04 0,04 0,048 g/00g 8 34 <0, <0, <0, no 2 g/00g ,02 0,02 0,02 no no 3 g/00g yes see "further remarks" 4 g/00g no g/00g g/00g g/00g ,77* 0,78* 0,7* no* 8 g/00g 2 3 <0,0 <0,0 <0,0 no 7 g/00g 9 36 <0, <0, <0, no g/00g ,823 0,6028 0,67 no 2 g/00g ,734 0,6 0,86 no no 3 g/00g ,4827 0, , yes see "further remarks" 4 g/00g ,69 0,68 0,699 no Glycin / Glycine g/00g g/00g 7 4 0,68 0,74 0,62 7 g/00g ,79 0,77 0,8 no 8 g/00g 2 3 0,7 0,74 0,77 no 9 g/00g ,726 0,704 0,748 * given / analysed as Glutamin Page 48 of 62

49 Parameter Teilnehmer Einheit Proben-Nr. A Proben-Nr. B Ergebnis (Mittel) Ergebnis A Ergebnis B inkl. WF Wiederfindungsrate [%] Analyte Participant Unit Sample No. A Sample No. B Result (Mean) Result A Result B incl. RR Recovery rate [%] g/00g ,429 0,4336 0,392 no 2 g/00g 6 24 ns ns ns no no L-Histidin / L-Histidine L-Isoleucin / L-Isoleucine L-Leucin / L-Leucine 3 g/00g ,397 0, , yes see "further remarks" 4 g/00g ,47 0,409 0,426 no g/00g g/00g 7 4 0,42 0,4 0,43 7 g/00g ,4 0,46 0,43 no 8 g/00g 2 3 0,48 0, 0,47 no 9 g/00g ,460 0,4 0,47 g/00g ,669 0,62 0,672 no 2 g/00g ,8 0,96 0,668 no no 3 g/00g ,697 0,6 0,669 yes see "further remarks" 4 g/00g ,693 0,707 0,679 no g/00g g/00g 7 4 0,69 0,7 0,68 7 g/00g ,67 0,7 0,64 no 8 g/00g 2 3 0,70 0,7 0,7 no 9 g/00g ,683 0,68 0,686 g/00g 8 34,89,788,200 no 2 g/00g 6 24,7,8,6 no no 3 g/00g 33 48, ,333333, yes see "further remarks" 4 g/00g 2 44,790,98,6 no g/00g g/00g 7 4,60,8, 7 g/00g 6 38,,9, no 8 g/00g 2 3,24,2,24 no 9 g/00g 9 36,,4,6 Page 49 of 62

50 Parameter Teilnehmer Einheit Proben-Nr. A Proben-Nr. B Ergebnis (Mittel) Ergebnis A Ergebnis B inkl. WF Wiederfindungsrate [%] Analyte Participant Unit Sample No. A Sample No. B Result (Mean) Result A Result B incl. RR Recovery rate [%] g/00g ,760 0,680 0,83 no 2 g/00g ,809 0,794 0,823 no no L-Lysin / L-Lysine L-Methionin / L-Methionine L-Phenylalanin / L-Phenylalanine 3 g/00g , , ,62000 yes see "further remarks" 4 g/00g ,767 0,77 0,763 no g/00g g/00g 7 4 0,83 0,88 0,79 7 g/00g ,79 0,8 0,77 no 8 g/00g 2 3 0,96,02 0,9 no 9 g/00g ,823 0,89 0,827 g/00g ,49 0,689 0,293 no 2 g/00g ,8 0,83 0,86 no no 3 g/00g ,2297 0, , yes see "further remarks" 4 g/00g ,74 0,82 0,67 no g/00g g/00g 7 4 0,7 0,6 0,8 7 g/00g ,9 0,2 0,8 no 8 g/00g 2 3 0,7 0,7 0,8 no 9 g/00g ,8 0,84 0,78 g/00g ,308 0,448 0,67 no 2 g/00g ,73 0,3 0,93 no no 3 g/00g ,409 0, , yes see "further remarks" 4 g/00g ,4 0,47 0,42 no g/00g ,9 3,7 4, NO 80 6 g/00g 7 4 0,47 0,49 0,4 7 g/00g , 0,3 0,49 no 8 g/00g 2 3 0, 0, 0, no 9 g/00g ,498 0, 0,497 Page 0 of 62

51 Parameter Teilnehmer Einheit Proben-Nr. A Proben-Nr. B Ergebnis (Mittel) Ergebnis A Ergebnis B inkl. WF Wiederfindungsrate [%] Analyte Participant Unit Sample No. A Sample No. B Result (Mean) Result A Result B incl. RR Recovery rate [%] g/00g 8 34,0844,082,0867 no 2 g/00g ,862 0,873 0,8 no no L-Prolin / L-Proline L-Serin / L-Serine L-Threonin / L-Threonine 3 g/00g , , ,67 yes see "further remarks" 4 g/00g ,83 0,827 0,8 no g/00g g/00g 7 4 0,82 0,84 0,8 7 g/00g ,78 0,79 0,77 no 8 g/00g 2 3 0,9 0,9 0,9 no 9 g/00g ,8 0,842 0,88 g/00g ,3769 0,3662 0,3876 no 2 g/00g 6 24 ns ns ns no no 3 g/00g , , , yes see "further remarks" 4 g/00g ,27 0,494 0,6 no g/00g g/00g 7 4 0,2 0,24 0,6 7 g/00g , 0,3 0,49 no 8 g/00g 2 3 0,6 0,9 0,3 no 9 g/00g ,27 0,3 0,42 g/00g ,247 0,38 0,09 no 2 g/00g ,9 0,82 0,97 no no 3 g/00g , ,647 0, yes see "further remarks" 4 g/00g ,602 0,63 0,92 no g/00g g/00g 7 4 0,6 0,7 0, 7 g/00g ,62 0,62 0,6 no 8 g/00g 2 3,20,22,9 no 9 g/00g ,89 0,87 0,9 Page of 62

52 Parameter Teilnehmer Einheit Proben-Nr. A Proben-Nr. B Ergebnis (Mittel) Ergebnis A Ergebnis B inkl. WF Analyte Participant Unit Sample No. A Sample No. B Result (Mean) Result A Result B incl. RR L-Tryptophan L-Tyrosin / L-Tyrosine L-Valin / L-Valine Wiederfindungsrate [%] Recovery rate [%] g/00g 8 34 nicht bestimmt nicht bestimmt nicht bestimmt no 2 g/00g 6 24 n/a n/a n/a n/a n/a 3 g/00g , , , yes see "further remarks" 4 g/00g 2 44 g/00g g/00g g/00g g/00g g/00g ,04 0,07 0,02 g/00g ,3 0,6 0,47 no 2 g/00g ,2 0,24 0,7 no no 3 g/00g , ,4 0, yes see "further remarks" 4 g/00g , 0,6 0,06 no g/00g g/00g 7 4 0, 0, 0,2 7 g/00g , 0, 0, no 8 g/00g 2 3 0,2 0,2 0,2 no 9 g/00g ,06 0,493 0,9 g/00g ,7446 0,723 0,767 no 2 g/00g ,72 0,739 0,7 no no 3 g/00g ,76 0,763 0,766 yes see "further remarks" 4 g/00g ,76 0,78 0,7 no g/00g g/00g 7 4 0,8 0,83 0,77 7 g/00g ,74 0,79 0,69 no 8 g/00g 2 3 0,80 0,8 0,8 no 9 g/00g ,72 0,72 0,73 Page 2 of 62

53 .2 Homogeneity.2. Homogeneity test before the PT Homogeneity test for magnesium by ICP-OES: Independant samples mg/00g 3,4 2 32,6 3 28,9 4 32,6 3,8 Mean Repeatability Standard Deviation 3,,2 4,8%.2.2 Repeatability standard deviation of duplicate tests of the participants The repeatability standard deviations were calculated with the data documented in.. It is 0,087 g/00g = 0,4 % of X for L-methionine. And it is 0,046 g/00g = 6, % of X for L-valine..2.3 Comparison of sample number / test results Homgenität / homogeneity L-Alanin / L-Alanine DLA-Nr. / No. Ergebnis *00 Page 3 of 62

54 .3 Analytical Methods Details by the participants L-Alanin / L-Alanine ASU 64 LFGB L / mod. 0,2g No- as 2 no Alanine (free) 0, received in same 3 sample (see LC-MS/MS "further no ~ 0 mg remarks") no 6 amino acid analyser g Ninhydrin one point yes Aufschluss / Extraktion Hydrolization / Extraction Derivatisation Reference material Yes Extraction with citrate buffer After column derivatisation with ninhydrin Yes Derivatisation with Waters AccQ tag individual standard dietary supplement powder Protein Total amino acids after acidic precipitation hydrolysis with amino acid 4 Mortar 0, - g with Ninhydrin Salami analyser (with after column sulfosalicylic derivatisation) acid Free amino acids (according to VO (EG) 2/2009) Appr. 3 g Feed (internal RM) Parameter Teilnehmer WF mit Homogenisierung gleicher Methodenbeschreibung Einwaage Matrix Analyte Participant Recovery Homogenization weight Sample with same Method description matrix Derivatisierung Referenzmaterial Kalibrierverfahren Calibration Internal standard Yes Methode ist akkreditiert Method accredited yes Yes 6-point external yes External standard, linear yes regression Internal std., - point-calibration yes yes yes Sonstige Hinweise Further remarks recovery corrected by stable isotope dilution assay Page 4 of 62

55 Parameter Teilnehmer WF mit Homogenisierung Extraktion rung terial fahren Aufschluss / Derivatisie- Referenzma- Kalibrierver- gleicher Methodenbeschreibung Einwaage Matrix Analyte Participant Recovery Homogenization weight / Extraction Derivatisation Reference Sample Hydrolization with same Method description material matrix Calibration 2 no Arginine (free) * 0, Yes Yes * Yes Yes 3 * 4 L-Arginin / L-Arginine yes L- Asparaginsäure / L-Aspartic Acid L-Cystin / L-Cystine 2 no Aspartic acid (free) * 0, Yes Yes * Yes Yes 3 * yes 2 no Cystine (free) * 0, Yes Yes * Yes Yes 3 * yes * siehe L-Alanin / see L-Alanine Methode ist akkreditiert Method accredited Sonstige Hinweise Further remarks Page of 62

56 Parameter Teilnehmer WF mit Methode Homogenisierung Extraktion rung terial fahren Aufschluss / Derivatisie- Referenzma- Kalibrierver- gleicher Methodenbeschreibung Einwaage ist akkreditiert Matrix Analyte Participant Recovery Homogenization weight / Extraction material accredited Sample Hydrolization Reference Method with same Method description Derivatisation Calibration matrix 2 no Glutaminc acid (free) * 0, Yes Yes * Yes Yes no 3 * L- 4 Glutaminsäure / L-Glutaminic Acid Glycin / Glycine L-Histidin / L-Histidine 2 no Glycine (free) * 0, Yes Yes * Yes Yes 3 * no Histidine (free) * 0, Yes Yes * Yes Yes 3 * * siehe L-Alanin / see L-Alanine Sonstige Hinweise Further remarks Page 6 of 62

57 Parameter Teilnehmer WF mit Homogenisierung Extraktion rung terial fahren Aufschluss / Derivatisie- Referenzma- Kalibrierver- gleicher Methodenbeschreibung Einwaage Matrix Analyte Participant Recovery Homogenization weight / Extraction Derivatisation Reference Sample Hydrolization with same Method description material matrix Calibration 2 no Isoleucine (free) * 0, Yes Yes * Yes Yes 3 * 4 L-Isoleucin / L-Isoleucine L-Leucin / L-Leucine L-Lysin / L-Lysine 2 no Leucine (free) * 0, Yes Yes * Yes Yes 3 * no Lysine (free) * 0, Yes Yes * Yes Yes 3 * * siehe L-Alanin / see L-Alanine Methode ist akkreditiert Method accredited Sonstige Hinweise Further remarks Page 7 of 62

58 Parameter Teilnehmer WF mit Homogenisierung Extraktion rung terial fahren Aufschluss / Derivatisie- Referenzma- Kalibrierver- gleicher Methodenbeschreibung Einwaage Matrix Analyte Participant Recovery Homogenization weight / Extraction material Sample Hydrolization Reference with same Method description Derivatisation matrix Calibration 2 no Methionine (free) * 0, Yes Yes * Yes Yes 3 * 4 L-Methionin / L-Methionine L- Phenylalanin / L-Phenylalanine L-Prolin / L-Proline 2 no Phenylalanine (free) No- as received 0, Yes Yes * Yes Yes 3 * no Proline (free) * 0, Yes Yes * Yes Yes 3 * * siehe L-Alanin / see L-Alanine Methode ist akkreditiert Method accredited Sonstige Hinweise Further remarks Page 8 of 62

59 Parameter Teilnehmer WF mit Homogenisierung Extraktion rung terial fahren Aufschluss / Derivatisie- Referenzma- Kalibrierver- gleicher Methodenbeschreibung Einwaage Matrix Analyte Participant Recovery Homogenization weight / Extraction Derivatisation Reference Sample Hydrolization with same Method description material matrix Calibration 2 no Serine (free) * 0, Yes Yes * Yes Yes 3 * 4 L-Serin / L-Serine L-Threonin / L-Threonine L-Tryptophan 2 no Threonine (free) No- as received 0, 3 * n/a n/a n/a n/a n/a n/a n/a n/a n/a 3 * * siehe L-Alanin / see L-Alanine HPLC (according to no VDLUFA methods III, 2. Amendment 988) Yes Yes individual standard Yes 3-pointcalibration Methode ist akkreditiert Method accredited Yes yes yes Sonstige Hinweise Further remarks Page 9 of 62

60 Parameter Teilnehmer WF mit Homogenisierung Extraktion rung terial fahren Aufschluss / Derivatisie- Referenzma- Kalibrierver- gleicher Methodenbeschreibung Einwaage Matrix Analyte Participant Recovery Homogenization weight / Extraction Derivatisation Reference Sample Hydrolization with same Method description material matrix Calibration 2 no Tyrosine ( (free) * 0, Yes Yes * Yes Yes 3 * 4 L-Tyrosin / L-Tyrosine L-Valin / L-Valine L-Glutamin / LGlutamine 2 no Valine ( (free) * 0, Yes Yes * Yes Yes 3 * free amino acics (according to 7 VO (EG) 2/2009) * siehe L-Alanin / see L-Alanine Methode ist akkreditiert Method accredited Sonstige Hinweise Further remarks Page 60 of 62

61 6. Index of participant laboratories in alphabetical order List of participants of PTs DLA and DLA 43-20: Teilnehmer / Participant Ort / Town Land / Country ENGLAND Germany ITALY DENMARK Germany Germany SPAIN Germany Germany NETHERLANDS Germany [Die Adressdaten der Teilnehmer wurden für die allgemeine Veröffentlichung des Auswerte-Berichts nicht angegeben.] [The address data of the participants were deleted for publication of the evaluation report.] Page 6 of 62

62 7. Index of references. DIN EN ISO/IEC 7043:200; Konformitätsbewertung Allgemeine Anforderungen an Eignungsprüfungen / Conformity assessment General requirements for proficiency testing 2. Verordnung / Regulation 882/2004/EU; Verordnung über amtliche Kontrollen / Regulation on official controls 3. DIN EN ISO/IEC 702:200; Allgemeine Anforderungen an die Kompetenz von Prüf- und Kalibrierlaboratorien / General requirements for the competence of testing and calibration laboratories 4. Richtlinie / Directive 993/99/EU; über zusätzliche Maßnahmen im Bereich der amtlichen Lebensmittelüberwachung / on additional measures concerning the official control of foodstuffs. ASU 64 LFGB: Planung und statistische Auswertung von Ringversuchen zur Methodenvalidierung 6. DIN ISO 328:2009; Statistische Verfahren für Eignungsprüfungen durch Ringversuche / Statistical methods for use in proficiency testing by interlaboratory comparisons 7. The International Harmonised Protocol for the Proficiency Testing of Ananlytical Laboratories ; J.AOAC Int., 76(4), (993) 8. The International Harmonised Protocol for the Proficiency Testing of Ananlytical Chemistry Laboratories ; Pure Appl Chem, 78, 4 96 (2006) 9. Evaluation of analytical methods used for regulation of food and drugs;w. Horwitz; Analytical Chemistry, 4, (982) 0.A Horwitz-like funktion describes precision in proficiency test; M. Thompson, P.J. Lowthian; Analyst, 20, (99).Protocol for the design, conduct and interpretation of method performance studies; W. Horwitz; Pure & Applied Chemistry, 67, (99) 2.Recent trends in inter-laboratory precision at ppb and sub-ppb concentrations in relation to fitness for purpose criteria in proficiency testing; M. Thompson; Analyst, 2, (2000) 3.ASU 64 LFGB: L49.07-; Bestimmung der Aminosäuren in Aminosäurengemischen. 4.ASU 64 LFGB: L ; Bestimmung der Aminosäuren in diätetischen Lebensmitteln auf Basis von Proteinhydrolysaten.ASU 64 LFGB: L ; Bestimmung des Tryptophangehaltes in diätetischen Lebensmitteln auf Basis von Proteinhydrolysaten 6.Verordnung 2/2009/EG zur Festlegung der Probenahmeverfahren und Analysemethoden für die amtliche Untersuchung von Futtermitteln / Regulation 2/2009/EC laying down the methods of sampling and analysis for the official control of feed Page 62 of 62