Evaluation Report proficiency test. DLA 39/ Diet Product I: Fat-soluble Vitamins. Meal Replacement

Transcription

1 DLA Dienstleistung Lebensmittel Analytik GbR Evaluation Report proficiency test DLA 39/015 Diet Product I: Fat-soluble Vitamins in Meal Replacement Dienstleistung Lebensmittel Analytik GbR Waldemar-Bonsels-Weg Ahrensburg, Germany Coordinator of this PT: Dr. Matthias Besler Page 1 of 30

2 Inhalt / Content 1. Introduction...3. Realisation Test material Homogeneity...4. 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 z'-score Quotient Standard uncertainty Results Vitamin A (as Retil in µg / 100 g) Vitamin D3 (as Cholecalciferol in µg / 100 g) Vitamin E (as D-alpha-Tocopherol in mg / 100 g) Vitamin K1 (as Phyllochine in µg / 100 g) β-carotene (in mg / 100 g) Documentation Primary data Homogeneity Analytical Methods Index of participant laboratories Index of references...30 Page of 30

3 1. 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 (010) and DIN ISO 1358:009 (6).. Realisation.1 Test material The test material is a mixture of common in commerce diet products (meal replacement, drink powder) with addition of food supplements from European Suppliers. The materials were ground, sieved, mixed and homogenized. Afterwards the samples were packaged lightproof in portions to approximately 50 g. The portions were numbered chrologically. The composition (summary list of ingredients) and the amounts of vitamins were calculated according to the labelled values as given in table 1 and table respectively. Table 1: Composition of DLA-Samples Meal Replacement Powder with added Food Supplements Ingredients: Soy protein isolate, skimmed milk yogurt powder, honey, skimmed milk powder, demineralized whey powder, yoghurt powder, vegetable oils (palm, rapeseed, soybean, sunflower), lactose, inulin, milk protein, starch, natural flavor, lutein, maltodextrin, L-tyrosine, taurine, L phenylalanine, L-tryptophan Minerals: tri-calcium phosphate, calcium carbonate, potassium citrate, di-potassium phosphate, potassium chloride, tri-magnesiumcitrat, tri-magnesium hydroxide, magnesium oxide, magnesium carbonate, sodium citrate, iron fumarate, iron (III) diphosphate, ferrous lactate, zinc oxide, zinc sulfate, manganese sulfate, copper gluconate, copper sulfate, copper carbonate, chromium (III) chloride, sodium molybdate, potassium iodide, potassium iodate, sodium selenite, sodium selenate Vitamins: L-ascorbic acid, D-alpha-tocopherol, DL-alpha-tocopherol, nicotinamide, calcium D-pantothenate, pyridoxine hydrochloride, thiamin monitrate, riboflavin, retinyl acetate, beta-carotene, folic acid, D-biotin, vitamin K1, cholecalciferol, cyacobalamin Further additives: E551 and E470b, E464, soya lecithin, E101, titanium dioxide, iron oxides and hydroxides, polyethylene glycols. Page 3 of 30

4 Table : Calculated amounts according to labelled values of vitamins Vitamin Vitamin A Vitamin D Vitamin E Vitamin K beta-carotene Content per 100 g 650 µg 5,6 µg 8 mg 30 µg 0,88 mg.1.1 Homogeneity Homogeneity of the test material was checked by 5fold determination of vitamin E by HPLC. With a repeatability standard deviation of 5,3 % the homogeneity of the test material was considered acceptable. The relative repeatability standard deviation of the official German ASU 64 method for the determination of vitamin E (α-tocopherol) in milk powder ( samples) by HPLC was 4,0 % and 3,0 %, respectively (14). 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 vitamin A it was 18,4 % and for vitamin K 10,8 %. The relative repeatability standard deviation of the official German ASU 64 method was,1 % for vitamin A, while the target standard deviation according to Horwitz is 11,4 % (15). For vitamin K the mean of the relative repeatability standard deviations of the official German ASU 64 method was 4,5 % in infant food, while the target standard deviation according to Horwitz is 18,3 %. The repeatability standard deviations of the participants' results are given in the documentation. Additionally in the documentation the portion numbers are graphically assigned to the results of vitamin E. There is trend recognizable in the results which could suggest inhomogeneity.. Test Two portions of test material were sent to every participating laboratory in the 4 nd week of 015. The testing method was optional. The tests should be finished at 8 th vember 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 was used for each statistical evaluation, in case at least 7 results were submitted. Queried and documented were single results, recovery and the testing methods used. Except three participants, who submitted results, all other participants submitted their results in time. Page 4 of 30

5 3. Evaluation 3.1 Assigned value Because the analysed material was certified reference material the robust mean of the submitted results was used as assigned value X (6). The distribution of submitted results showed 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. Single results giving values outside the measuring range of the participating laboratory were considered for statistical evaluation (e.g. results given as > 5 mg/kg and <,5 mg/kg, respectively) when a result indicating > is above and a result indicating < is below the target range. 3. 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 95% significance niveau (5). Detected outliers were stated for information only, when z-score was < - or >. 3.4 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 vitamin A, vitamin D, vitamin K and β-carotene the target standard deviation according to the model of Horwitz was applied (3.4.1). For vitamin A additionally the standard uncertainty was considered by valuating with z'-score (see 3.6). For vitamin E the target standard deviation according to a precision experiment was applied (3.4., official German ASU 64 method L (14)). Page 5 of 30

6 3.4.1 General model (Horwitz) The relative target standard deviation in % of the assigned value is derived from following equation (Horwitz) σ (%) = (1-0,5logX) 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 r. And then, using the number of replicate measurements n, each participant is to perform, the target standard deviation for proficiency assessment is calculated : = L r /n. The target standard deviations given in table 3 were calculated from the precision data of the respective methods. Tabelle 3: Relative target standard deviations (RSD) from precision experiments (13-17) Method Parameter Matrix Mean Relative Literature [per 100g] target (14) standarddeviation σ HPLC Vitamin A Milk powder 653 µg,9 % 1 L /1 HPLC Vitamin D3 Milk powders 14,3/9,95 µg 3, %/11,6 % 1 L HPLC Vitamin E Milk powders 9,89/10, mg 6,1 %/1,5 % 1 L HPLC Vitamin K1 6 Infant food on base of milk and soy protein µg 4,4 % 1 (Mean) L HPLC β-carotene Pudding powder 1,53 mg,9 % 1 L /1 1 given in evaluation (s. chapter 4) Page 6 of 30

7 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 vitamin A, vitamin D, vitamin K and β-carotene (3.4.1), for vitamin A considering the standard uncertainty by valuating with z'-score (see 3.6). For vitamin E the target standard deviation according to a precision experiment was applied (3.4., official German ASU 64 method L (14)). 3.5 z-score 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 - z. 3.6 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: If applied, we define the deminator deviation σ ' in following sections. as the target standard The requirements for the analytical performance are generally considered as fulfilled if - z'. Page 7 of 30

8 The z'-score was applied for vitamin A in the present evaluation. 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 t exceed the value of. A value > 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 t given (11). In the present proficiency tests the quotients S x / σ were In the present proficiency test for vitamin D, vitamin E, vitamin K and β-carotene satisfactory quotients S x / σ <,0 were obtained. For vitamin A the quotient S x / σ above,0. Therefore evaluation was done according to 3.6 by z'-score. The resulting quotient S x / σ ' was,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 =1,5 S x / p If u X 0,3 the standard uncertainty of the assigned value needs t 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 between 0,51 and 0,70 for the parameters. For vitamin A, for which the quotients U x / σ and S x / σ were > 0,3 and >,0 at the same time, the target range was extended considering the standard uncertainty according 3.6 z'-score. Page 8 of 30

9 4. Results All following tables are anymized. 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 - σ ) or (X - σ ') * upper limit of target range (X + σ ) or (X + 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] z-score σ z-score (Info) Hinweis Remark Page 9 of 30

10 4.1 Vitamin A (as Retil in µg / 100 g) Vergleichsuntersuchung / Proficiency Test Statistic Data Number of results 13 Number of outliers Mean 987 Median 80 Robust Mean (X) 939 Robust standard deviation (S x ) 301 Target range: Target standard deviation σ ' 150 Target standard deviation ASU (for Information) 7,0 lower limit of target range 640 upper limit of target range 140 Quotient S x / σ ' Standard uncertainty u x Quotient u x / σ ', ,70 Results in the target range 10 Percent in the target range 77 Ergebnisse / Results Auswertenummer / evaluation number Vitamin A [µg/100 g] Obergrenze upper limit robuster Mittelwert robust mean Untergrenze lower limit Abb. 1: Ergebnisse Vitamin A Fig. 1: Results vitamin A Page 10 of 30

11 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation Vitamin A [µg/100 g] Abweichung [µg/100 g] Deviation z'-score σ z-score (Info) Hinweis Remark number [µg/100 g] ,8 4, ,6-9, ,5 349,3 1, ,4-7, , ,3 9,3 Ausreisser / Outlier 6 754, , -6, ,5-8, ,8 6,9 Ausreisser / Outlier ,1-0, ,9-5, , ,9-10, ,9-5, ,7 66 0,4,4 6 z'-score Auswertenummer / evaluation number 8 5 Abb. : Fig. : Z'-Scores Vitamin A Z'-Scores vitamin A Page 11 of 30

12 4. Vitamin D3 (as Cholecalciferol in µg / 100 g) Vergleichsuntersuchung / Proficiency Test Statistic Data Number of results 10 Number of outliers 1 Mean 6,97 Median 6,66 Robust Mean (X) 7,06 Robust standard deviation (S x ),18 Target range: Target standard deviation σ 1,68 Target standard deviation ASU (for Information) 0,819 lower limit of target range 3,69 upper limit of target range 10,4 Quotient S x / σ Standard uncertainty u x Quotient u x / σ 1,3 0,863 0,51 Results in the target range 8 Percent in the target range 80 Ergebnisse / Results > 14 1 Vitamin D [µg/100 g] Obergrenze upper limit robuster Mittelwert robust mean Auswertenummer / evaluation number Untergrenze lower limit Abb. 3: Ergebnisse Vitamin D Fig. 3: Results vitamin D Page 1 of 30

13 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation Vitamin D [µg/100 g] z-score σ Hinweis Remark number [µg/100 g] 1 5,96-1,10-0,7-1,3 1,56-5,50-3,3-6,7 Ausreisser / Outlier 3 5,9-1,16-0,7-1,4 4 6,11-0,95-0,6-1, 5 77,5 Abweichung [µg/100 g] Deviation z-score (Info) 6 9,8,74 1,6 3, ,563-0,50-0,3-0, ,09 1,03 0,6 1,3 11 7,31 0,5 0,1 0,3 1 6,75-0,31-0, -0, ,7 4,64,8 5,7 Ergebnis ausgeschlossen / Result excluded 3 z-score Auswertenummer / evaluation number 6 14 Abb. 4: Fig. 4: Z-Scores Vitamin D Z-Scores vitamin D Page 13 of 30

14 4.3 Vitamin E (as D-alpha-Tocopherol in mg / 100 g) Vergleichsuntersuchung / Proficiency Test Statistic Data Number of results 1 Number of outliers 1 Mean 34,3 Median 34,0 Robust Mean (X) 34,0 Robust standard deviation (S x ) 6,03 Target range: Target standard deviation σ 4,6 Target standard deviation Horwitz (for Information),6 lower limit of target range 5,5 upper limit of target range 4,5 Quotient S x / σ Standard uncertainty u x Quotient u x / σ 1,4,18 0,51 Results in the target range 11 Percent in the target range 9 Ergebnisse / Results Auswertenummer / evaluation number Vitamin E [mg/100 g] Obergrenze upper limit robuster Mittelwert robust mean Untergrenze lower limit Abb. 5: Ergebnisse Vitamin E Fig. 5: Results vitamin E Page 14 of 30

15 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation number z-score σ Hinweis Remark 1 37,5 3,49 0,8 1,5 34,9 0,89 0, 0,4 3 5,7-8,31-1,9-3, ,01-0, -0,4 5 46,71 1,70 3,0 5,6 Ausreisser / Outlier 6 31,7 -,31-0,5-1,0 7 9,0-5,01-1, -, 8 Vitamin E [mg/100 g] Abweichung [mg/100 g] Deviation [mg/100 g] z-score (Info) 9 36,15,14 0,5 0, ,3 7,9 1,7 3, 11 7,94-6,07-1,4 -,7 1 37, 3,19 0,7 1, ,7-3,31-0,8-1,5 nicht nachweisbar / t detected 3 z-score Auswertenummer / evaluation number 10 5 Abb. 6: Fig. 6: Z-Scores Vitamin E Z-Scores vitamin E Page 15 of 30

16 4.4 Vitamin K1 (as Phyllochine in µg / 100 g) Vergleichsuntersuchung / Proficiency Test Statistic Data Number of results 7 Number of outliers 1 Mean 54,8 Median 40,5 Robust Mean (X) 41,6 Robust standard deviation (S x ) 10,4 Target range: Target standard deviation σ 7,60 Target standard deviation ASU (for Information) 1,84 lower limit of target range 6,4 upper limit of target range 56,8 Quotient S x / σ Standard uncertainty u x Quotient u x / σ 1,4 4,90 0,64 Results in the target range 6 Percent in the target range Ergebnisse / Results > > Auswertenummer / evaluation number Vitamin K [µg/100 g] Obergrenze upper limit robuster Mittelwert robust mean Untergrenze lower limit Abb. 7: Ergebnisse Vitamin K Fig. 7: Results vitamin K Page 16 of 30

17 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation Vitamin K [µg/100 g] z-score σ Hinweis Remark number [µg/100 g] 1 4 0,38 0,0 0, 9,7-11,9-1,6-6,5 3 48,8 7,18 0,9 3, ,3 107,61 14, 58,4 Ausreisser / Outlier 6 40,5-1,1-0,1-0,6 7 33,9-7,7-1,0-4, Abweichung [µg/100 g] Deviation z-score (Info) 9 39,3 -,3-0,3-1, Ergebnis ausgeschlossen / Result excluded z-score 3 > Auswertenummer / evaluation number 5 Abb. 8: Fig. 8: Z-Scores Vitamin K Z-Scores vitamin K Page 17 of 30

18 4.5 β-carotene (in mg / 100 g) Vergleichsuntersuchung / Proficiency Test Statistic Data Number of results 10 Number of outliers 1 Mean 0,847 Median 0,814 Robust Mean (X) 0,834 Robust standard deviation (S x ) 0,147 Target range: Target standard deviation σ 0,0970 Target standard deviation Horwitz (for Information) 0,066 lower limit of target range 0,640 upper limit of target range 1,03 Quotient S x / σ Standard uncertainty u x Quotient u x / σ 1,5 0,058 0,60 Results in the target range 8 Percent in the target range 80 Ergebnisse / Results > 1,40 1,0 1,00 0,80 0,60 0,40 0,0 0, Auswertenummer / evaluation number β-carotin / β-carotene [mg/100 g] Obergrenze upper limit robuster Mittelwert robust mean Untergrenze lower limit Abb. 9: Ergebnisse β-carotin Fig. 9: Results β-carotene Page 18 of 30

19 Ergebnisse der teilnehmenden Institute: Results of Participants: Auswertenummer Evaluation number β-carotin / β- Carotene [mg/100 g] z-score σ Hinweis Remark 1 0,591-0,43 -,5-3,7 0,793-0,041-0,4-0,6 3 1, 0,366 3,8 5,5 Ausreisser / Outlier ,47 Abweichung [mg/100 g] Deviation [mg/100 g] z-score (Info) 7 0,707-0,17-1,3-1, ,166 1,7,5 9 0,865 0,031 0,3 0,5 10 0,81-0,013-0,1-0, 11 0,90 0,068 0,7 1,0 1 0,78-0,054-0,6-0,8 13 0,806-0,08-0,3-0,4 14 Ergebnis ausgeschlossen / Result excluded 4 z-score Auswertenummer / evaluation number 3 Abb. 10: Fig. 10: Z-Scores β-carotin Z-Scores β-carotene Page 19 of 30

20 5. Documentation 5.1 Primary data 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 Vitamin A Wiederfindungsrate [%] Recovery rate [%] 1 µg/100 g µg/100 g µg/100 g , µg/100 g µg/100 g ,6 1917,8 1539,4-6 µg/100 g ,3 747,8 760,8-7 µg/100 g µg/100 g n/a 9 µg/100 g µg/100 g µg/100 g ,5 585,5 731,5 1 µg/100 g µg/100 g µg/100 g ,7 985, Page 0 of 30

21 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 [%] 1 µg/100 g ,96 6,08 5,84 - µg/100 g ,56 1,6 1, µg/100 g ,9 5, µg/100 g ,11 5,84 6,37 5 µg/100 g ,5 45,5 31,5-6 µg/100 g ,8 10,1 9,5 - Vitamin D3 7 µg/100 g µg/100 g , n/a 9 µg/100 g µg/100 g ,09 6,94 9,4 11 µg/100 g 45 7,31 7,3 7,9 1 µg/100 g ,75 7,74 5, µg/100 g µg/100 g ,7 13,1 10,4 79 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 [%] 1 mg/100 g ,5 38,4 36,6 - mg/100 g ,9 34,3 35,5 3 mg/100 g ,7 6, mg/100 g , mg/100 g ,71 43,39 50,03-6 mg/100 g ,7 31,8 31,5 - Vitamin E 7 mg/100 g mg/100 g 8 61 t detected t detected t detected n/a 9 mg/100 g 47 36,15 35,5 36,8 10 mg/100 g ,3 4,1 40,5 11 mg/100 g 45 7,94 6,51 9,36 1 mg/100 g , 37,83 36, mg/100 g mg/100 g ,7 30,6 30,9 93 Page 1 of 30

22 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 [%] 1 µg/100 g , 4,8 - µg/100 g ,7 30 9,4 3 µg/100 g ,8 48, µg/100 g µg/100 g ,3 143,5 154,95-6 µg/100 g ,5 39,1 41,9 - Vitamin K1 7 µg/100 g µg/100 g n/a 9 µg/100 g 47 39,3 39, 39,4 10 µg/100 g µg/100 g 45 1 µg/100 g µg/100 g µg/100 g 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 [%] 1 mg/100 g ,59 0,63 0,55 - mg/100 g ,79 0,79 0,79 3 mg/100 g , 1,13 1, mg/100 g mg/100 g mg/100 g ,47 743,99 736,95 - β-carotin / 7 mg/100 g β-carotene 8 mg/100 g n/a 9 mg/100 g 47 0,87 0,8 0,91 10 mg/100 g ,8 0,84 0,8 11 mg/100 g 45 0,9 0,9 0,89 1 mg/100 g ,78 0,78 0, mg/100 g ,81 0,84 0,78 14 mg/100 g Page of 30

23 5. Homogeneity 5..1 Homogeneity test before the PT Homogeneity test for vitamin E by HPLC: Independant samples mg/100 g 1 37,5 35,8 3 34,7 4 3,4 5 34,6 Mean Repeatability Standard Deviation 35,0 1,86 5,3% 5.. Repeatability standard deviation of duplicate tests of the participants The repeatability standard deviations were calculated with the data documented in 5.1. Vitamin A Einheit / Unit Vitamin K Einheit / Unit Wiederholstandardabweichung SD der Teilnehmer / Repeatability Standard Deviation SD of Participants 173 µg/100 g 4,51 µg/100 g robuster Mittelwert / robust Mean 939 µg/100 g 41,6 µg/100 g relative Wiederholstandardabweichung / relative SD 18,4 % 10,8 % 5..3 Comparison of sample number / test results Homgenität / homogeneity Vitamin E DLA-Nr. / No. Ergebnis / result Page 3 of 30

24 5.3 Analytical Methods Details by the participants Parameter Analyte Vitamin A Teilnehmer Methodenbeschreibung Wiederfindung mit Methode ist gleicher Matrix akkreditiert Sonstige Hinweise Participant Method description Recovery with same matrix Method accredited Further remarks 1 after saponification by LC-DAD 64 LFGB L /1ª 3 ASU L / LFGB L /1 5 HPLC-UV 6 ASU L /1 ne 7 Saponification followed by extraction with cyclohexane. HPLC with ami column and UV detection at 35 nm. 8 HPLC n/a 9 VO (EG) 15/009, IV, A 10 HPLC ne 11 ASU L : Determination of vitamin E in food by HPLC (according to EN 18) very little sample material saponification with methalic KOH, liquid-liquid-extraction, HPLC with 1 UV-Detection for feed according to Regulation EG 15/009 Annex IV/A; for food: basic rm EN L /1 poor separation of layers Page 4 of 30

25 Parameter Analyte Vitamin D3 Teilnehmer Methodenbeschreibung Wiederfindung mit Methode ist gleicher Matrix akkreditiert Sonstige Hinweise Participant Method description Recovery with same matrix Method accredited Further remarks 1 after saponification by LC-MS 64 LFGB L ª 3 ASU L LFGB L HPLC-UV 6 ASU L ne 7 8 HPLC n/a 9 t analysed 10 HPLC ne 11 ASU 64, L , Determination of Vitamin D 3 or Vitamin D in food, HPLC saponification, liquid-liquid-extraction, clean up by HPLC and fraction 1 collection, HPLC with PDA-Detection: food: basic rm EN 181, feed: VDLUFA Method L poor separation of layers Page 5 of 30

26 Parameter Analyte Vitamin E Teilnehmer Methodenbeschreibung Wiederfindung mit Methode ist gleicher Matrix akkreditiert Sonstige Hinweise Participant Method description Recovery with same matrix Method accredited Further remarks 1 after saponification by LC-DAD 64 LFGB L ª 3 ASU L LFGB L HPLC-UV 6 ASU L ne 7 Saponification followed by extraction with cyclohexane. HPLC with ami column and fluorescence detection (ex. 95 nm, em. 37 nm). Peak found in the sample does t 8 HPLC n/a correspond to d-a-tocopherol. Suspect is vit e acetate. 9 VO (EG) 15/009, IV, B 10 HPLC ne 11 ASU L : Determination of vitamin E in food by HPLC (according to EN 18) saponification with methalic KOH, liquid-liquid-extraction, HPLC with 1 fluorescence-detection for feed according to Regulation EG 15/009 Annex IV/B; for food: basic rm EN L For sythetic vitamin E the analytical values were corrector for the factor of 0,74 poor separation of layers Page 6 of 30

27 Parameter Analyte Vitamin K1 Teilnehmer Methodenbeschreibung Wiederfindung mit Methode ist gleicher Matrix akkreditiert Sonstige Hinweise Participant Method description Recovery with same matrix Method accredited Further remarks 1 after saponification by LC-FLD with post-column derivatisation 64 LFGB L ª 3 LC-MS/MS 4 5 HPLC-UV 6 ASU L ne 7 Extraction with n-heptane. HPLC with C-18 column and Zn-column, and fluorescence detection (ex. 48 nm, em. 418 nm). 8 HPLC n/a 9 in-house method t performed 11 1 t analysed Page 7 of 30

28 Parameter Analyte β-carotin / β-carotene Teilnehmer Methodenbeschreibung Wiederfindung mit Methode ist gleicher Matrix akkreditiert Sonstige Hinweise Participant Method description Recovery with same matrix Method accredited Further remarks 1 after saponification by LC-DAD 64 L ª 3 ASU L / ASU L / ne 7 8 Extraction with ethal and dichloromethane. HPLC with C-30 column and diode-array-detection (wavelength 451 nm used for calculation). HPLC n/a 9 10 DIN EN 183- mod. HPLC ne in-house method: determination of ß-carotene and lycopene in food (HPLC) saponification with methalic KOH, liquid-liquid-extraction, HPLC with UV-detection; basic rm EN 183- DIN EN 183- (HPLC/UV) 14 Page 8 of 30

29 6. Index of participant laboratories in alphabetical order Teilnehmer / Participant Ort / Town Land / Country AUSTRIA UNITED KINGDOM Germany Germany DENMARK IRELAND Germany Germany Germany Germany Germany SWEDEN Germany USA Germany 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 9 of 30

30 7. Index of references 1. DIN EN ISO/IEC 17043:010; Konformitätsbewertung Allgemeine Anforderungen an Eignungsprüfungen / Conformity assessment General requirements for proficiency testing. Verordnung / Regulation 88/004/EU; Verordnung über amtliche Kontrollen / Regulation on official controls 3. DIN EN ISO/IEC 1705:005; Allgemeine Anforderungen an die Kompetenz von Prüf- und Kalibrierlaboratorien / General requirements for the competence of testing and calibration laboratories 4. Richtlinie / Directive 1993/99/EU; über zusätzliche Maßnahmen im Bereich der amtlichen Lebensmittelüberwachung / on additional measures concerning the official control of foodstuffs 5. ASU 64 LFGB : Planung und statistische Auswertung von Ringversuchen zur Methodenvalidierung 6. DIN ISO 1358:009; 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), (1993) 8. The International Harmonised Protocol for the Proficiency Testing of Ananlytical Chemistry Laboratories ; Pure Appl Chem, 78, (006) 9. Evaluation of analytical methods used for regulation of food and drugs;w. Horwitz; Analytical Chemistry, 54, (198) 10.A Horwitz-like funktion describes precision in proficiency test; M. Thompson, P.J. Lowthian; Analyst, 10, 71-7 (1995) 11.Protocol for the design, conduct and interpretation of method performance studies; W. Horwitz; Pure & Applied Chemistry, 67, (1995) 1.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, 15, (000) 13.ASU 64 LFGB L Bestimmung von Vitamin D (Cholecalciferol (D 3 ) und Ergocalciferol (D )) in Lebensmitteln mittels HPLC / EN 181:009 Foodstuffs. Determination of vitamin D by high performance liquid chromatography. Measurement of cholecalciferol (D3) or ergocalciferol (D) 14.ASU 64 LFGB L Bestimmung von Vitamin E (α-, β-, γ- und δ- Tocopherol) in Lebensmitteln mittels HPLC / EN 18:014 Foodstuffs. Determination of vitamin E by high performance liquid chromatography. Measurement of α-, β-, γ- and δ-tocopherol 15.ASU 64 LFGB L /1 Bestimmung von Vitamin A in Lebensmitteln mittels HPLC, Teil 1: Bestimmung von all-trans-retil und 13-cis- Retil / EN 183-1:014 Foodstuffs. Determination of vitamin A by high performance liquid chromatography. Measurement of all-e-retil and 13-Zretil 16.ASU 64 LFGB L / Bestimmung von Vitamin A in Lebensmitteln mittels HPLC, Teil : Bestimmung von β-carotin / EN 183-:000 Foodstuffs. Determination of vitamin A by high performance liquid chromatography. Measurement of β-carotene 17.ASU 64 LFGB L Bestimmung von Vitamin K 1 mit HPLC / EN 14148:003 Foodstuffs. Determination of vitamin K1 by HPLC 18.Ministry of Health and Welfare, JSM, Japan Blake CJ (007), Analytical procedures for water-soluble vitamins in foods and dietary supplements: a review. Anal Bioanal Chem 389(1): Blake CJ (007) Status of methodology for the determination of fat-soluble vitamins in foods, dietary supplements, and vitamin premixes. J AOAC Int 90(4): Page 30 of 30