Evaluation Report proficiency test. DLA 32/2015 Food Supplements I. Vitamin B1, Vitamin B2, Vitamin B6, Vitamin B12 and Folic Acid

Transcription

1 DLA Dienstleistung Lebensmittel Analytik GbR Evaluation Report proficiency test 32/2015 Food Supplement I: Vitamin B1, Vitamin B2, Vitamin B6, Vitamin B12 and Folic Acid in Multivitamin Drink Powder Dienstleistung Lebensmittel Analytik GbR Waldemar-Bonsels-Weg Ahrensburg, Germany Coordinator of this PT: Dr. Matthias Besler Page 1 of 33

2 Content 1. 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 z'-score Quotient Standard uncertainty Results Vitamin B1 (as Thiamine-Cation in mg / 100 g) Vitamin B2 (as Riboflavin in mg / 100 g) Vitamin B6 (as Pyridoxin in mg / 100 g) Vitamin B12 (as Cyanocobalamin in µg / 100 g) Folic Acid (as Pteroylmonoglutaminic acid in µg/100 g) Documentation Primary data Vitamin B Vitamin B Vitamin B Vitamin B Folic Acid Homogeneity Repeatability standard deviation of duplicate tests of the participants Comparison of sample / test result Analytical Methods Vitamin B Vitamin B Vitamin B Vitamin B Folic Acid Index of participant laboratories Index of references...33 Page 2 of 33

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 (2010) and DIN ISO 13528:2009 (6). 2. Realisation 2.1 Test material The test material is a common in commerce food supplement "multi vitamin drink powder" from an EU supplier with additional glucose and lactose. The materials were mixed and homogenized. Afterwards the samples were packaged lightproof in portions to approximately 25 g. The portions were ed chronologically. The composition (list of ingredients) and the amounts of vitamins were calculated according to the labeled values as given in table 1 and table 2 respectively. Table 1: Composition of DLA-Samples Multi vitamin drink powder (strawberry flavour) Ingredients: Maltodextrin, Starch, Vitamin C, Flavours, s: Sodium Cyclamate, Sodium Saccharin, Vitamin E, Niacin, Calcium-D-Pantothenate, Riboflavin, Vitamin B6, Vitamin B2, Vitamin B1, Folic acid, Biotin, Vitamin B12 Glucose, Acidifying agent: Citric acid, Calcium carbonate, Acid regulator: Sodium hydrogencarbonate, Lactose, Maltodextrin, Beetroot powder, Vitamin C, Flavour, Humectant: Sorbitol, Sweetener: Aspartam, Niacin, Vitamin E, Pantothenic Acid, Vitamin B2, Vitamin B6, Vitamin B1, Folic Acid, Biotin, Vitamin B12 Page 3 of 33

4 Table 2: Calculated amounts according to labeled values of vitamins Vitamin Content per 100 g Vitamin B1 Vitamin B2 VitaminB6 Vitamin B12 Folic Acid 2,7 mg 3,5 mg 3,5 mg 6,1 µg 500 µg Homogeneity Homogeneity of the test material was checked by 5fold determination of vitamin B6 by HPLC. With a repeatability standard deviation of 4,2 % 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 vitamin B6 it was 7,5 % and for folic acid 5,2 % and in the range of common relative repeatability standard deviations of methods for water-soluble vitamins (13-16). The repeatability standard deviation of the participants for both vitamins is given in the documentation. Additionally in the documentation the portion s are graphically assigned to the results of folic acid. 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 9 th week of The testing method was optional. The tests should be finished at 10 th April Submission of results The participants submitted their results in standard forms, which have been handed out with the samples. The finally calculated concentration of each vitamin as an average of a duplicate determination of both ed samples was used for each statistical evaluation. Queried and documented were single results, recovery and the testing methods used. On participant submitted no results and one participant submitted the results delayed. All other participants submitted their results in time. Page 4 of 33

5 3. Evaluation 3.1 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). Except for vitamin B2 the distribution of submitted results showed no hint for bimodal distribution or other reasons for a higher variability (for vitamin B2 see result chapter). 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 95% significance niveau (5). 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. For evaluation of all analytes the target standard deviation according to the general model (Horwitz) was applied General model (Horwitz) The relative target standard deviation in % of the assigned value is derived from following equation (Horwitz) σ (%) = 2 (1-0,5logX) From the result the target standard deviation is calculated σ = X * σ (%) / 100. Page 5 of 33

6 3.4.2 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 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 3 were calculated from the precision data of the respective methods. Table 3: Target standard deviations from precision experiments (ASU) Method Parameter Matrix relative Target standard deviation σ Literature HPLC Vitamin B1 Food Additive Mixture 13,3 % 1 L HPLC Vitamin B2 Milk powder 6,8 % 1 L HPLC Vitamin B6 Multivitamin Drink 7,5 % 1 L Microbiological Folic acid Milk powder 15,9 % 1 L values given in the evaluation 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 evaluation in the present proficiency testing the model according to Horwitz was applied. Page 6 of 33

7 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 -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 square 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 -2 z' 2. The z'-score was used for the analytes vitamin B1 and vitamin B2 (see also 3.7 and 3.8). Page 7 of 33

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 (11). In the present proficiency tests the quotients S x / σ were for vitamin B1 (2,1) and for vitamin B2 (3,2) above 2,0. Therefore evaluation was done according to 3.6 by z'-scores. The resulting quotients S x / σ were < 2,0 then. For vitamin B6, vitamin B12 and folic acid the quotients S x / σ were < 2, 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 of participant laboratories and perhaps on other factors. The standard uncertainty u X for this PT is calculated as follows (6). u X =1,25 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 between 0,31 and 0,57 for vitamin B6, vitamin B12 and folic acid, while the quotients S x / σ were < 2,0. For vitamin B1 and vitamin B2 the quotients U x / σ were 0,64 and 1,2. Because the quotients S x / σ were > 2,0 at the same time the target range was extended according 3.6 z'-score for vitamin B1 and vitamin B2. Page 8 of 33

9 4. Results All following tables are anonymized. With the delivering of the evaluation-report the participants are informed about their individual evaluation-. In the upper 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 for information Target standard deviation σ ' 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 lower table -laboratories- the individual results of the participating laboratories are listed: Evaluation Test result Deviation from assigned value Z-Score Horwitz Z'-Score Horwitz Remarks Page 9 of 33

10 4.1 Vitamin B1 (as Thiamine-Cation in mg / 100 g) Test Statistic Data Number of results 16 Number of outliers 1 Mean 3,12 Median 3,10 Robust mean (X) 3,11 Robust standard deviation (S x ) 0,606 Target range: Target standard deviation for information (ASU 64) 0,413 Target standard deviation σ ' (Horwitz) 0,352 lower limit of target range (X - 2 σ ') 2,40 upper limit of target range (X + 2 σ ') 3,81 Quotient S x / σ ' 1,7 Standard uncertainty u X 0,189 Quotient u X / ' 0,54 Number of results in the target range 13 (81%) 5,0 Ergebnisse / results Vitamin B1 4,0 mg / 100 g 3,0 2,0 1,0 0, Auswertenummer / evaluation Fig. 1: Results Vitamin B1 (red line = robust mean, yellow lines = target range) Page 10 of 33

11 Results of participants Evaluation Result Deviation Z'-Score Z-Score Remarks σ (Info) [mg/100g] X rob. Mean Horwitz ASU 1 4,20 1,09 3,1 2,6 2 2,57-0,54-1,5-1,3 result converted* 3 3,1-0,01 0,0 0,0 4 3,1-0,01 0,0 0,0 5 2,88-0,23-0,6-0,6 result converted* 7 3,21 0,10 0,3 0,2 8 3,42 0,31 0,9 0,8 result converted* 9 3,4 0,29 0,8 0,7 10 3,88 0,77 2,2 1,9 11 2,200-0,91-2,6-2,2 result converted* 13 2,72-0,39-1,1-0,9 14 3,52 0,41 1,2 1,0 15 2,4-0,71-2,0-1,7 16 3,77 0,66 1,9 1,6 17 2,68-0,43-1,2-1,0 outlier, result converted* 18 2,86-0,25-0,7-0,6 result converted* * conversion factor 0,787 (Thiamine-Hydrochloride to Thiamine-Cation) 4 z'-scores Vitamin B Auswertenummer / evaluation Fig. 2: Z'-Scores Vitamin B1 Page 11 of 33

12 4.2 Vitamin B2 (as Riboflavin in mg / 100 g) Test Statistic Data Number of results 11 * Number of outliers 1 Mean 2,87 Median 2,90 Robust mean (X) 2,89 Robust standard deviation (S x ) 0,890 Target range: Target standard deviation for information (ASU 64) 0,195 Target standard deviation σ ' (Horwitz) 0,436 lower limit of target range (X - 2 σ ') 2,02 upper limit of target range (X + 2 σ ') 3,77 Quotient S x / σ ' 2,0 Standard uncertainty u X 0,336 Quotient u X / ' 0,77 Number of results in the target range 7 (64%) * without results of participants 2, 5, 7 and 9 5,0 4,5 4,0 3,5 Ergebnisse / results Vitamin B2 mg / 100 g 3,0 2,5 2,0 1,5 1,0 0,5 0, Auswertenummer / evaluation Fig. 3: Results Vitamin B2 (red line = robust mean, yellow lines = target range) Page 12 of 33

13 Results of participants* Evaluation Result Deviation Z'-Score Z-Score Remarks σ (Info) [mg/100g] X rob. Mean Horwitz ASU 1 1,29-1,60-3,7-8,2 outlier 3 2,9 0,01 0,0 0,0 8 1,64-1,25-2,9-6,4 10 3,51 0,62 1,4 3,2 11 2,427-0,47-1,1-2,4 13 3,15 0,26 0,6 1,3 14 3,84 0,95 2,2 4,8 15 2,9 0,01 0,0 0,0 16 3,9 1,01 2,3 5,2 17 3,33 0,43 1,0 2,2 18 2,67-0,22-0,5-1,1 * without results of participants 2, 5, 7 and 9 4 z'-scores Vitamin B Auswertenummer / evaluation Fig. 4: Z'-Scores Vitamin B2 Page 13 of 33

14 Results of Participants 2, 5, 7 and 9 The results for vitamin B2 of all participants showed a bimodal distribution. The results no. 2, 5, 7 and 9 were approximately 10 times below the other results. The evaluation was therefore separated. The test samples contained riboflavin-5-phosphate. The final results should be given as riboflavin. It is not clear whether participants 2, 5, 7 and 9 tested for and/or included riboflavin-5-phosphate in their results. Evaluation Result Deviation Z-Score Z'-Score Remarks [mg/100g] X rob. Mean Horwitz Horwitz 2 0,22-0,05 5 0,11-0,16 7 0,57 0,29 9 0,2-0,07 Statistic Data Number of results 4 Mean 0,274 Median 0,210 Robust mean (X) 0,274 Robust standard deviation (S x ) 0,227 1,0 Ergebnisse / results Vitamin B2 0,8 mg / 100 g 0,6 0,4 0,2 0, Auswertenummer / evaluation Fig. 5: Results Vitamin B2 (red line = robust mean) Page 14 of 33

15 4.3 Vitamin B6 (as Pyridoxin in mg / 100 g) Test Statistic Data Number of results 14 Number of outliers 0 Mean 3,85 Median 3,78 Robust mean (X) 3,86 Robust standard deviation (S x ) 0,329 Target range: Target standard deviation for information (ASU 64) 0,289 Target standard deviation σ (Horwitz) 0,356 lower limit of target range (X - 2 σ ) 3,14 upper limit of target range (X + 2 σ ) 4,57 Quotient S x / σ 0,93 Standard uncertainty u X 0,110 Quotient u X / 0,31 Number of results in the target range 14 (100%) 5,0 Ergebnisse / results Vitamin B6 4,0 mg / 100 g 3,0 2,0 1,0 0, Auswertenummer / evaluation Fig. 6: Results Vitamin B6 (red line = robust mean, yellow lines = target range) Page 15 of 33

16 Results of participants Evaluation Result Deviation Z-Score Z-Score Remarks (Info) [mg/100g] X rob. Mean Horwitz ASU 2 4,06 0,21 0,6 0,7 3 4,2 0,35 1,0 1,2 5 3,45-0,40-1,1-1,4 result converted* 7 3,64-0,21-0,6-0,7 8 4,13 0,28 0,8 1,0 9 3,3-0,55-1,6-1,9 10 3,76-0,09-0,3-0,3 11 3,754-0,10-0,3-0,3 13 4,22 0,37 1,0 1,3 14 4,01 0,16 0,4 0,5 15 3,6-0,25-0,7-0,9 16 3,74-0,11-0,3-0,4 17 3,802-0,05-0,1-0,2 18 4,24 0,39 1,1 1,3 result converted* * conversion factor 0,823 (Pyridoxin-Hydrochloride to Pyridoxin) σ 3 z-scores Vitamin B Auswertenummer / evaluation Fig. 7: Z-Scores Vitamin B6 Page 16 of 33

17 4.4 Vitamin B12 (as Cyanocobalamin in µg / 100 g) Test Statistic Data Number of results 13 Number of outliers 1 Mean 10,2 Median 7,62 Robust mean (X) 7,90 Robust standard deviation (S x ) 2,66 Target range: Target standard deviation σ (Horwitz) 1,85 lower limit of target range (X - 2 σ ) 4,20 upper limit of target range (X + 2 σ ) 11,60 Quotient S x / σ 1,4 Standard uncertainty u X 0,923 Quotient u X / 0,50 Number of results in the target range 10 (77%) 30 Ergebnisse / results Vitamin B12 > 25 µg / 100 g Auswertenummer / evaluation Fig. 8: Results Vitamin B12 (red line = robust mean, yellow lines = target range) Page 17 of 33

18 Results of participants Evaluation Result Deviation Z-Score Remarks [µg/100g] X rob. Mean Horwitz 1 7,98 0,08 0,0 2 4,8-3,10-1,7 3 6,4-1,50-0,8 6 7,75-0,15-0,1 7 7,46-0,44-0,2 9 2,44-5,46-2, ,27 2,37 1,3 11 7,324-0,58-0,3 12 7,62-0,28-0,2 13 7,45-0,45-0, ,10 14,6 outlier 16 20,22 12,32 6,7 17 7,96 0,05 0,0 σ z-scores Vitamin B12 > > Auswertenummer / evaluation Fig. 9: Z-Scores Vitamin B12 Page 18 of 33

19 4.5 Folic Acid (as Pteroylmonoglutaminic acid in µg / 100 g) Test Statistic Data Number of results 15 Number of outliers 1 Mean 714 Median 693 Robust mean (X) 710 Robust standard deviation (S x ) 148 Target range: Target standard deviation for information (ASU 64) 113 Target standard deviation σ (Horwitz) 84,5 lower limit of target range (X - 2 σ ) 540 upper limit of target range (X + 2 σ ) 879 Quotient S x / σ 1,8 Standard uncertainty u X 47,8 Quotient u X / 0,57 Number of results in the target range 13 (87%) Ergebnisse Folsäure / results Folic Acid µg / 100 g Auswertenummer / evaluation Fig. 10: Results Folic Acid (red line = robust mean, yellow lines = target range) Page 19 of 33

20 Results of participants Evaluation Result Deviation Z-Score Z-Score Remark σ (Info) [µg/100g] X rob. MW Horwitz ASU ,6-1,9-1, ,6-1,5-1, ,4 0,7 0, ,4 2,0 1, ,6-1,7-1, ,05-212,5-2,5-1, ,5 110,9 1,3 1, ,4 1,7 1, ,6-0,2-0, ,4 0,0 0, ,7-31,9-0,4-0, ,4 1,0 0, ,6-0,8-0, ,77 302,2 3,6 2,7 outlier ,37-33,2-0,4-0,3 4 z-scores Folsäure / Folic Acid Auswertenummer / evaluation Fig. 11: Z-Scores Folic Acid Page 20 of 33

21 5. Documentation 5.1 Primary data Vitamin B1 Evaluation Result Sample-No. Sample-No. Result A Result B A B [mg/100g] [mg/100g] [mg/100g] % 1 5,34 * ,42 5,27 2 3,26 * ,37 3,14 N/A 3 3, , , ,13 3, * not determined 60 9 not determined not determined Recovery rate 7 3, ,13 3,29 91,5 8 4,35 * 16 ; ; 58 5,79 2,9 9 3,4 20 ; ; , ,88 11 ; ; 43 3,9 3,86 107,5 11 2,796 * 30 ; ; 59 2,777 2,816 (3,295) (3,233) 85,9/87, , ,71 2, , ,49 3, , , , ,71 2, ,64 * 24 ; ; 33 3,56 3,72 * as Thiamine-Hydrochloride Page 21 of 33

22 5.1.2 Vitamin B2 Evaluation Result Sample-No. Sample-No. Result A Result B A B [mg/100g] [mg/100g] [mg/100g] % 1 1, ,34 1,24 2 0, ,19 0,25 N/A 3 2, , , not determined 60 9 not determined not determined Recovery rate 7 0, ,55 0,58 99,5 8 1,64 16 ; ; 58 1,53 1,74 9 0,2 20 ; ; 36 0,19 0, ,51 11 ; ; 43 3,52 3,5 73,2 11 2, ; ; 59 2,492 2,361 (3,162) (2,728) 80,3/86, , ,11 3, , ,81 3, ,9 15 2, , , ,29 3, ,67 24 ; ; 33 2,41 2, Page 22 of 33

23 5.1.3 Vitamin B6 Evaluation Result Sample-No. A Sample-No. B [m g/100g] [m g/100g] [m g/100g] % , ,88 4,23 N/A 3 4, , * not determined 60 9 Result A Result B Recovery rate not determined not determined 7 3, ,61 3,67 98,9 8 4,13 16 ; ; 58 4,27 3,99 9 3,3 20 ; ; 36 3,6 3, ,76 11 ; ; 43 3,52 3,99 92,8 / 84,2 11 3, ; ; 59 3,817 3,828 (3,903) (3,605) 102/94, , ,26 4, , ,04 3, , , , ,969 3, ,15 * 24 ; ; 33 5,22 5,07 96,6 * as Pyridoxine-Hydrochloride Page 23 of 33

24 5.1.4 Vitamin B12 Evaluation Result Sample-No. A Sample-No. B Result A Result B Recovery rate [µg/100g] [µg/100g] [µg/100g] % 1 7, ,05 7,9 2 4, ,6 N/A 3 6, ,3 6, , , ,29 7,63 68, ; ; ,44 20 ; ; 36 2,41 2, ,27 11 ; ; 43 10,06 10, , ; ; 59 7,304 7,344 (7,866) (7,540) 92,9/97,4 12 7, ,53 7, , ,35 7, , , ,83 7, ; ; 33 Page 24 of 33

25 5.1.5 Folic Acid Evaluation Result Sample-No. A Sample-No. B Result A Result B Recovery rate [µg/100g] [µg/100g] [µg/100g] % N/A , , , ; ; ; ; ; ; , ; ; (678) 730 (691) 103/ , ,76 686, ; 31 5 ; , , ,03 651, ; ; 33 Page 25 of 33

26 5.2 Homogeneity Homogeneity test before the PT Homogeneity test for vitamin B6 by HPLC/fluorescence. Independant samples mg/100g 1 3,63 2 3,31 3 3,55 4 3,63 5 3,38 Mean Repeatability Standard Deviation 3,50 0,147 4,2% Repeatability standard deviation of duplicate tests of the participants The repeatability standard deviation was calculated with the data documented in for vitamin B6 acid. It is 0,288 mg/100g = 7,5 % of X. The repeatability standard deviation was calculated with the data documented in for folic acid. It is 36,9 µg/100g = 5,2 % of X Comparison of sample / test result (Folic Acid) Homgenität / homogeneity DLA-Nr. / No. Ergebnis / result :10 Page 26 of 33

27 5.3 Analytical Methods Details by the participants Vitamin B1 Evaluation Method description and further remarks Recovery w ith same matrix Accredited Rem arks 1 LUFA SP no microbiological Method (Thiamine), Standard curve with Thiamine-HCl 2 Vitamin B1 (as Thiamine Hydrochloride) no N/A 3 HPLC-DAD no no Thiamine 4 5 HPLC mit Fluorescence-Detection HPLC-UV - Thiamine-cation as thiamine HCl 6 7 PV 2.019/ Thiamin-Kation 8 LC-MS/MS no as Thiamine chloride Hydrochloride 9 LC/MS/MS no no Thiamine-cation 10 HPLC-FLD as Thiamine-cation 11 HPLC-Fluorescence Detection B1-HCl HPLC according to VDLUFA Method book III No LFGB, L result: as Thiamine-cation calculated as Thiamine-cation calculated 15 microbiologal Method, VitaFast-System by r-biopharm 16 HPLC-DAD 17 USP Acidic hydrolysis, release of Thiamine 18 phosphate, Oxidation of Thiamine into Thiochrom, determination by HPLC-FLD Determination of total vitamin contents (Thiamine, Thiamine hydrochloride and Thiamine-mono-nitrate) Thiamine Thiamine- cation no recovery determined. Vitamin B1 as Thiamine chloride Hydrochloride, according to Swiss Legislation Page 27 of 33

28 5.3.2 Vitamin B2 Evaluation Method description and further remarks Recovery w ith same matrix Accredited Rem arks 1 LUFA SP no microbiological Method (Riboflavin) 2 Vitamin B2 (Riboflavin) no Not as Riboflavin-5phosphate 3 HPLC-DAD no no Riboflavin 4 5 HPLC-UV - as riboflavin 6 7 PV 2.019/ Riboflavin 8 HPLC no given as Riboflavin 9 LC/MS/MS no no Riboflavin 10 HPLC-FLD 11 HPLC-Fluorescence Detection Riboflavin HPLC according to VDLUFA Method book III No result: as Riboflavin calculated (measured as Riboflavin-5-Phosphate, converted to Riboflavin; modification of gradient, because of bad peakt separation with standard conditions) in-house method HPLC-FLD calculated as Riboflavin microbiologal Method, VitaFast-System by r-biopharm nucleotide)) 16 HPLC-DAD Riboflavin 17 USP Riboflavin 18 according to SLMB-Method 1544 and EN 14152, respectively Determination of total vitamin contents (Riboflavin, Riboflavin-5-Sodium phosphate, FAD (Flavin-Adenin- Dinucleotide) and FMN (Flavin-mono not recovery corrected, Vitamin B2 given as Riboflavin Page 28 of 33

29 5.2.3 Vitamin B6 Evaluation Method description and further remarks Recovery w ith same matrix Accredited Remarks 1 2 Vitamin B6 (as Pyridoxine) NO N/A 3 HPLC-DAD no no Pyridoxin 4 5 HPLC-UV - Yes as pyridoxine HCl 6 7 PV 2.019/ Pyridoxin 8 9 according to SLMB :2007, microbiological LC/MS/MS no no no given as Pyridoxin Pyridoxin 10 HPLC-FLD as Pyridoxin 11 HPLC-Fluorescence Detection PN HPLC-DAD 17 USP 18 HPLC according to VDLUFA Method book III No result as Pyridoxin calculated; modification of gradient, because of bad peakt separation with standard conditions in-house method HPLC-FLD calculated as Pyridoxin microbiologal Method, VitaFast-System by r-biopharm according to 64-Method and EN 14663, respectively determination of total vitamin contents (Pyridoxin, Pyridoxin-5-Phosphate, Pyridoxal, Pyridoxal-5-Phosphate, Pyridoxamine and Pyridoxamine-5- Phosphate) Pyridoxin Pyridoxin not recovery corrected. Vitamin B6 as Pyridoxin hydrochloride according to Swiss Legislation Page 29 of 33

30 5.2.4 Vitamin B12 Evaluation Method description and further remarks Recovery w ith same matrix Accredited Rem arks 1 LUFA SP no microbiological Method (Cyanocobalamin) 2 Vitamin B12 (as cyanocobalamin) no N/A 3 HPLC-DAD no no 4 5 HPLC-UV - No result given Micro-Titer-Test (VitaFast by r-biopharm) LC/MS/MS after concentration with immuno affinity columns HPLC-DAD no no no no no Cyanocobalamin Cyanocobalamin Microtiter plate test vitafast r-biopharm microbiological microtiter plate test Vita Fast Vitamin B12 (RBiopharm) 15 determination of total vitamin contents microbiological method, VitaFast-System (Cyanocobalamin (Cobalamin) and by r-biopharm Hydroxycobalamin) 16 HPLC-DAD Cobalamin 17 USP 18 Page 30 of 33

31 5.2.5 Folic Acid Evaluation Method description and further remarks Recovery w ith sam e m atrix Accredited Rem arks 1 LUFA SP no microbiological Method 2 Free Folic Acid no N/A 3 HPLC-DAD no no 4 5 microbiological microtiter plate test HPLC-UV - 6 HPLC no 7 Micro-Titer-Test (VitaFast by r-biopharm) no no LC/MS/MS 10 HPLC-DAD HPLC-DAD 17 USP 18 no no folic acid Mikrotiterplattentest vitafast r-biopharm microbiological microtiter plate test Vita Fast Folic Acid (R-Biopharm) microbiological method, VitaFast-System by r-biopharm determination of total vitamin content folic acid-b9 Page 31 of 33

32 6. Index of participant laboratories Teilnehmer / Participant Ort / Town Land / Country UNITED KINGDOM GERMANY GERMANY GERMANY GERMANY DENMARK GERMANY GERMANY SWITZERLAND GERMANY GERMANY SWITZERLAND GERMANY GERMANY GERMANY GERMANY GERMANY GERMANY SWEDEN [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 32 of 33

33 7. Index of references 1. DIN EN ISO/IEC 17043:2010; 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 17025:2005; 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 13528:2009; 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), (1993) 8. The International Harmonised Protocol for the Proficiency Testing of Ananlytical Chemistry Laboratories ; Pure Appl Chem, 78, (2006) 9. Evaluation of analytical methods used for regulation of food and drugs;w. Horwitz; Analytical Chemistry, 54, (1982) 10.A Horwitz-like funktion describes precision in proficiency test; M. Thompson, P.J. Lowthian; Analyst, 120, (1995) 11.Protocol for the design, conduct and interpretation of method performance studies; W. Horwitz; Pure & Applied Chemistry, 67, (1995) 12.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, 125, (2000) 13.ASU 64 LFGB : L Bestimmung von Vitamin B1 mit HPLC, Juli ASU 64 LFGB : L Bestimmung von Vitamin B2 mit HPLC, Juli ASU 64 LFGB : L Bestimmung von Vitamin B6 (einschlieslich glucosidisch gebundener Verbindungen) in Lebensmitteln HPLC-Verfahren, Dezember ASU 64 LFGB : L Mikrobiologische Bestimmung von Folat, Juli Ministry of Health and Welfare, JSM, Japan Blake CJ (2007), Analytical procedures for water-soluble vitamins in foods and dietary supplements: a review. Anal Bioanal Chem 389(1): Blake CJ (2007) Status of methodology for the determination of fat-soluble vitamins in foods, dietary supplements, and vitamin premixes. J AOAC Int 90(4): Page 33 of 33