Proficiency study 312. Acesulfame-K, aspartame and caffeine in energy drink. Report no. ND Author: Mariëlle van Vondel.

Transcription

1 Proficiency study 312 Acesulfame-K, aspartame and caffeine in energy drink Report no. ND Author: Mariëlle van Vondel 29 April 2005 Food and Consumer Product Safety Authority Region North Inspectorate for Health Protection and Veterinary Public Health (VWA/KvW) PO Box AL Groningen The Netherlands Phone Fax CHEK Proficiency Studies ISSN No: CHEK Proficiency Study 312; Acesulfame-K, aspartame and caffeine in energy drink

2 CONTENTS 1. Introduction 1 2. Time-table 1 3. Sample preparation 1 4. Statistical results Acesulfame-K in energy drink samples A and B split level Aspartame in energy drink samples A and B split level Caffeine in energy drink samples A and B split level 5 5. Methods of analysis 6 6. Remarks from participants List of invited participants Explanation of graphical presentations Tables and graphical presentations CHEK Proficiency Study 312; Acesulfame-K, aspartame and caffeine in energy drink

3 1. Introduction Proficiency study number 312 as directed by the working-group CHEK concerned the investigation of acesulfame-k, aspartame and caffeine in energy drink samples. To get an impression of the performance of the quantitative determination of acesulfame-k, aspartame and caffeine in energy drink 23 laboratories were invited to join this proficiency study. One laboratory was not able to do the analysis for acesulfame-k, two laboratories were not able to do the analysis for aspartame and four laboratories were not able to do the analysis for caffeine. 2. Time-table Distribution of samples 7 March 2005 Deadline for the production of results 8 April 2005 Final report 29 April Sample preparation Energy drink samples A and B split level 17.5 L Power Up energy drink sample containing aspartame (80 mg/l) and caffeine (150 mg/l according to label) was put in an ultrasonic bath for 2 minutes to remove CO2. After homogenisation of the bulk sample g acesulfame-k and g aspartame were added to 17.5 L energy drink sample and thoroughly minced using a planet mincer yielding 351 mg/l acesulfame-k, 601 mg/l aspartame and 150 mg/l caffeine. After homogenisation, the bulk sample was divided in two portions of approximately 5 and 12.5 L. Sample A 12.5 L of the bulk sample energy drink was divided in approximately 50 sub-samples of about 100 ml, prepared and packed in sterilised bottles of glass. Sample B 570 ml of water was added to 5 L of the bulk sample energy drink. The mixture was thoroughly minced using a planet mincer yielding 315 mg/l acesulfame-k, 539 mg/l aspartame and 135 mg/l caffeine L of sample B of energy drink was divided in approximately 50 sub-samples of about 100 ml, prepared and packed in sterilised bottles of glass. Samples were stored in a refrigerator until shipment. To each of the participants two samples coded A and B were sent. CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 1 of 29

4 4. Statistical results The results of the laboratories and the Z-scores are given in Table I to III. Figures I to XII give graphical presentations of the results. One-way analysis of variance (ANOVA), preceded by checking for normality and outlier checking of the results (Cochran/Grubbs) is the statistical procedure for obtaining the estimates of within laboratory and between laboratory variability. The performance of a determination is assessed as follows: satisfactory = 0-2 x maximum allowable RSDR questionable = >2-3 x maximum allowable RSDR unsatisfactory = >3 x maximum allowable RSDR The value of a certain parameter, resulting from a single analysis, above which a sample is regarded as rejectable with a probability of 95%, is expressed as xmax. CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 2 of 29

5 4.1. Acesulfame-K in energy drink samples A and B split level 22 labs : Results of all laboratories. 21 labs : Results of laboratory 1 are rejected by the Grubbs outlier test. 22 labs 21 labs Unit Average A mg/kg Average B mg/kg Repeatability standard deviation (sr') mg/kg Repeatability rel. standard deviation (RSDr') % Reproducibility standard deviation (sr') mg/kg Reproducibility rel. standard deviation (RSDR') % Horwitz acceptable value for (RSDR') % Repeatability r' mg/kg Reproducibility R' mg/kg xmax value at level 350 mg/kg mg/kg Summary acesulfame-k in energy drink samples A and B split level Prof. study Number of labs Average A-B mg/kg RSDR % Horwitz RSDR % xmax/standard mg/kg / / Conclusions acesulfame-k in energy drink samples A and B split level - The performance of the determination of acesulfame-k in energy drink is satisfactory (see 4). - The results of sample A and B are drawn from a normal distribution. - No Cochran outliers are found in the datasets of sample A and B. - The results of laboratory 1 are marked as outliers by the Grubbs test indicating systematic errors and are rejected. CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 3 of 29

6 4.2 Aspartame in energy drink samples A and B split level 21 labs : Results of all laboratories. 19 labs : Results of laboratories 5 and 15 are rejected by the Cochran outlier test. 21 labs 19 labs Unit Average A mg/kg Average B mg/kg Repeatability standard deviation (sr') mg/kg Repeatability rel. standard deviation (RSDr') % Reproducibility standard deviation (sr') mg/kg Reproducibility rel. standard deviation (RSDR') % Horwitz acceptable value for (RSDR') % Repeatability r' mg/kg Reproducibility R' mg/kg xmax value at level 600 mg/kg 675 mg/kg Summary aspartame in energy drink samples A and B split level Prof. study Number of labs Average A-B mg/kg RSDR % Horwitz RSDR % xmax/standard mg/kg / / Conclusions aspartame in energy drink samples A and B split level - The performance of the determination of aspartame in energy drink is satisfactory (see 4). - The results of sample A and B are drawn from a normal distribution. - The results of laboratories 5 and 15 are marked as outliers by the Cochran test indicating bad repeatability and are rejected. - No Grubbs outliers are found in the datasets of sample A and B. - The absolute Z-score for sample B of laboratory 15 is greater than 3 and suggest poor performance in terms of accuracy. CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 4 of 29

7 4.3. Caffeine in energy drink samples A and B split level 19 labs : Results of all laboratories. 15 labs : Results of laboratories 14, 16, 19 and 21 are rejected by the Grubbs outlier test. 19 labs 15 labs Unit Average A mg/kg Average B mg/kg Repeatability standard deviation (sr') mg/kg Repeatability rel. standard deviation (RSDr') % Reproducibility standard deviation (sr') mg/kg Reproducibility rel. standard deviation (RSDR') % Horwitz acceptable value for (RSDR') % Repeatability r' mg/kg Reproducibility R' mg/kg xmax value at level 350 mg/kg mg/kg Summary caffeine in energy drink samples A and B split level Prof. study Number of labs Average A-B mg/kg RSDR % Horwitz RSDR % xmax/standard mgkg / / Conclusions caffeine in energy drink samples A and B split level - The performance of the determination of caffeine in energy drink is satisfactory (see 4). - The results are drawn from a normal distribution. - No Cochran outliers are found in the datasets of sample A and B. - The results of laboratories 14 and 19, based on the differences, are marked as outliers by the Grubbs test indicating bad repeatability and are rejected. - The results of laboratories 16 and 21 are marked as outliers by the Grubbs test indicating systematic errors and are rejected. CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 5 of 29

8 5. Method of analysis Lab1 Acesulfame-K and aspartame Extraction of a test portion with mobile phase. Injection of an aliquot of the extract in a HPLC system using diode array detection. : RP-18. Mobile phase : KH2PO4 solution/acetonitrile (90:10), adjusted to ph 4.3. Detection : diode array. Lab 2 Acesulfame-K and aspartame Extraction of a test portion with mobile phase. Filtration and injection of an aliquot of the filtrate in a HPLC system using UV detection. : 150 x 4.6 mm Alltech C18, 5 µm. Mobile phase : 0.11 M phosphoric acid /acetonitrile (87:13). Detection : UV 220 nm. Caffeine Extraction of a test portion with water. Filtration and injection of an aliquot of the extract in a HPLC system using UV detection. : 150 x 4.6 mm Alltech C18, 5 µm. Mobile phase : 4% acetic acid solution/methanol (88:12). Detection : UV 280 nm. Lab 3 Acesulfame-K and caffeine Extraction of a test portion. Degassing and injection of an aliquot of the sample in a HPLC system using UV detection. : Nucleosil 100 C18, 5 µm with guard column. Mobile phase : 0.02 M KH2PO4 buffer, ph 4.3/acetonitrile (90:10). Detection : diode array 220 nm. Lab 4 Acesulfame-K and aspartame Extraction of a test portion with water. Clean up using Carrez reagents. Injection of an aliquot of the extract in a HPLC system using diode array detection. : C18. Mobile phase : phosphate buffer/acetonitrile. Detection : diode array 210 and 230 nm. Lab 5 Acesulfame-K and aspartame Extraction of a test portion with water. Clean up using Carrez reagents. Injection of an aliquot of the sample in a HPLC system using diode array detection. : 250 x 4 mm Nucleosil 100-5C 18AB. Mobile phase : 0.02 M KH2PO4/acetonitrile (90:10), ph 4.3. Detection : diode array 220 nm. CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 6 of 29

9 Caffeine Extraction of a test portion with water. Injection of an aliquot of the sample in a HPLC system using diode array detection. : 250 x 4 mm RP-18. Mobile phase : M KH2PO4 /methanol (70:30). Detection : diode array 272 nm. Lab 6 Acesulfame-K, aspartame and caffeine Extraction of a test portion with mobile phase. Injection of an aliquot of the extract in a HPLC system using diode array detection. : 250 x 3 mm Purosphere STAR RP-18, 5 µm with guard column. Mobile phase A : 0.02 M NaHPO4, ph 5. Mobile phase B : acetonitrile. Gradient: t0 : 85% A - 15% B, hold 2 min. t8 : 70% A - 30% B, hold 4 min. Detection : diode array 220 and 254 nm. Lab 7 Acesulfame-K, aspartame and caffeine Extraction of a test portion with dilution of water (1:5) after addition of theophylline as internal standard. Clean up using Carrez reagents. Injection of an aliquot of the extract in a HPLC system using diode array detection. : 250 x 4 mm Purosphere RP18, 5 µm with guard column. Mobile phase A : 0.02 M KH2PO4, ph Mobile phase B : acetonitrile. Gradient: t0 : 90% A - 10% B, hold 7 min. t7 : 70% A - 30% B. Detection : diode array 210 and 220 nm. Lab 8 Acesulfame-K Extraction of a test portion with water after addition of acesulfame-k as internal standard. Filtration and injection of an aliquot of the filtrate in a HPLC system using UV detection. : 250 x 4 mm Lichrocart RP-select B. Mobile phase : KH2PO4 buffer/acetonitrile (98:2). Detection : UV 220 nm. Aspartame Extraction of a test portion with water after addition of aspartame as internal standard. Filtration and injection of an aliquot of the filtrate in a HPLC system using UV detection. : 250 x 4 mm Lichrocart RP-select B. Mobile phase : KH2PO4 buffer/acetonitrile (90:10). Detection : UV 220 nm. CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 7 of 29

10 Aspartame Filtration and injection of an aliquot of the sample in a HPLC system using UV detection after addition of caffeine 99% as internal standard. : Lichrosphere 100 RP8. Mobile phase : water/acetonitrile (90:10). Detection : UV 220 nm. Lab 9 Acesulfame-K, aspartame and caffeine Extraction of a test portion with water after addition of 3-hydroxybenzoic acid as internal standard. Clean up using Carrez reagents. Filling up to mark, filtration and injection of an aliquot of the filtrate in a HPLC system using diode array detection. : 150 x 3.9 mm Novak C18, 4 µm with guard column. Mobile phase A : potassium phosphate buffer/water. Mobile phase B : methanol. Gradient: t0 : 93% A - 7% B. t14 : 60% A - 40% B, hold 2 min. Detection : diode array 220 nm. Lab 10 Acesulfame-K, aspartame and caffeine Extraction of a test portion with water. Clean up using Carrez reagents. Filtration and injection of an aliquot of the extract in a HPLC system using diode array detection. : Xterra C18. Mobile phase : M phosphate buffer ph 3/acetonitrile (93:7). Detection : diode array 235, 214 and 275 nm. Lab 11 Acesulfame-K and aspartame Extraction of a test portion with water. Clean-up using Carrez reagents. Injection of an aliquot of the extract in a HPLC system using diode array detection. : 250 x 4.6 mm Spherisorb ODS, 5 µm with guard column. Mobile phase A : 0.02 M K2H2PO4/methanol (98:2). Mobile phase B : acetonitrile. Gradient: t0 : 98% A - 2% B. t10 : 92% A - 8% B. t10.1 : 80% A - 20% B. t20 : 75% A - 25% B. Detection : diode array 210 and 225 nm. Caffeine Extraction of a test portion with water. Injection of an aliquot of the extract in a HPLC system using diode array detection. : 125 x 4.6 mm RP8, 5 µm with guard column. Mobile phase : water/acetonitrile (90:10). Detection : diode array 280 nm. CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 8 of 29

11 Lab 12 Acesulfame-K, aspartame and caffeine Direct injection of an aliquot of the sample in a HPLC system using diode array detection. : 150 x 4.6 mm Chromopack Inertsil ODS2, 5 µm. Mobile phase A : 0.02 M KH2PO4. Mobile phase B : acetonitrile. Gradient: t0 : 91% A - 9% B, hold 3 min. t12 : 85% A - 15% B. t20 : 75% A - 25% B. Detection : diode array 226.2, and nm. Lab 13 Acesulfame-K and aspartame Dilution of a test portion with mobile phase. Microfiltration and injection of an aliquot of the extract in a HPLC system using diode array detection. : 250 x 4 mm Nucleosil 100 C18 AB, 5 µm with guard column. Mobile phase : 0.02 M KH2PO4 solution ph 4.3/acetonitrile (90:10). Detection : diode array 217 and 227 nm. Caffeine Dilution of a test portion with mobile phase. Microfiltration and injection of an aliquot of the extract in a HPLC system using UV detection. : 250 x 4 mm Nucleosil 7C 18 with guard column. Mobile phase : water/acetonitrile (90:10). Detection : UV 276 nm. Lab 14 Acesulfame-K, aspartame and caffeine Extraction of a test portion with water. Injection of an aliquot of the extract in a HPLC system using UV detection. : 250 x 4.6 mm C18, 5 µm. Mobile phase : 0.02 M KH2PO4 solution ph 4.3/acetonitrile (86:14). Detection : diode array 220 nm. Lab 15 Acesulfame-K, aspartame and caffeine Extraction of a test portion with water. Injection of an aliquot of the extract in a HPLC system using UV detection. : Lichrocart-Nucleosil C18, 5 µm with guard column. Mobile phase : 0.02 M KH2PO4 buffer ph 4.3/acetonitrile (90:10). Detection : UV 220 and 265 nm. Lab 16 Acesulfame-K, aspartame and caffeine Extraction of a test portion with mobile phase. Injection of an aliquot of the extract in a HPLC system using diode array detection. : 250 x 4 mm Lichrospher 100 C18, 5 µm with guard column. Mobile phase : M KH2PO4 solution ph 3.5/acetonitrile (90:10). Detection : diode array 210, 220 and 270 nm. CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 9 of 29

12 Lab 17 Acesulfame-K, aspartame and caffeine Extraction of a test portion with water/methanol (75:25). Injection of an aliquot of the extract in a HPLC system using diode array detection. : 250 x 4.6 mm Lichrosorb 10 RP18 with guard column. Mobile phase : sodium acetate ph 5.4/methanol (75:25). Detection : diode array 210, 230 and 271 nm. Lab 18 Acesulfame-K and aspartame Extraction of a test portion with water. Clean-up using Carrez reagents. Injection of an aliquot of the extract in a HPLC system using diode array detection. : 300 x 3.9 mm Bondapak C18 with guard column. Mobile phase : phosphate buffer ph 3.5/acetonitrile (89:11). Detection : diode array 220 nm. Lab 19 Acesulfame-K and aspartame Extraction of a test portion with water. Clean up using Carrez reagents. Injection of an aliquot of the extract in a HPLC system using diode array detection. : 250 x 4 mm Nucleodur C18. Mobile phase A : buffer ph 4.2. Mobile phase B : methanol/buffer ph 4.2 (50:50). Gradient t0 : 90% A - 10% B. t8 : 10% A - 90% B, hold 2 min. t10.5 : 90% A - 10% B. Detection : diode array 220 and 230 nm. Caffeine Extraction of a test portion with mixture of 0.01 M ammonium acetate buffer/methanol (60:40). Clean up using Carrez reagents. Injection of an aliquot of the extract in a HPLC system using diode array detection. : 250 x 4 mm Nucleodur C18. Mobile phase : water/methanol (82:18). Detection : diode array 272 nm. Lab 20 Acesulfame-K and aspartame Direct injection of an aliquot of the sample in a HPLC system using diode array detection. : 250 x 4.6 mm ODS, 5 µm with guard column. Mobile phase : tetrabutyl ammonium hydrogen sulphate + sodium acetate solution/methanol (70:30). Detection : diode array 220 nm. Lab 21 Acesulfame-K and aspartame Direct injection of an aliquot of the sample in a HPLC system using UV detection. : C18 with guard column. Mobile phase : aqueous buffer ph 4.50/methanol (70:30). Detection : UV 220 nm. CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 10 of 29

13 Caffeine Direct injection of an aliquot of the sample in a HPLC system using UV detection. : C18 with guard column. Mobile phase : ammonium acetate ph 4.40/acetonitrile. Detection : UV 233 nm. Lab 22 Acesulfame-K and aspartame Extraction of a test portion with 12.5 mm phosphate buffer ph 5. Injection of an aliquot of the extract in a HPLC system using UV detection. : 150 x 3.9 mm Novapack C18 with guard column. Mobile phase A : acetonitrile. Mobile phase B : 12.5 mm phosphate buffer ph 5. Gradient t0 : 3% A - 97% B. t9 : 20% A - 80% B. Detection : UV 214 and 227 nm. Caffeine Extraction of a test portion with water. Injection of an aliquot of the extract in a HPLC system using UV detection. : 150 x 3.9 mm Novapack C18 with guard column. Mobile phase : phosphate buffer ph 5/methanol (72:28). Detection : UV 214 and 275 nm. Lab 23 Caffeine Direct injection of an aliquot of the sample in a HPLC system using UV detection. : Novapack RCM C18 with guard column. Mobile phase : water/methanol (80:20). Detection : UV 280 nm. CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 11 of 29

14 6. Remarks from participants Lab 8 At the day of arrival (9 March 2005) there were mould recognised in sample B by eye. Lab 21 An extra peak was deleted in both samples during the analysis for acesulfame-k and aspartame. This extra peak was very close in retention time to acesulfame-k (difference 0.4 min). 7. List of invited participants Austria AGES ILMU-Wien. Belgium Brussels Intercommunaal Laboratorium, Brussels. Denmark Fødevareregion København Og Nordøstsiaelland, København. Finland Finnish Customs, Espoo. France Laboratoire de la DGCCRF Bordeaux-Talence, Talence Cédex. Germany Chemisches- und Lebensmitteluntersuchungsamt, Aachen. Amt für Umwelt, Verbraucherschutz und Lokale Agenda, Bonn. Chemisches Untersuchungsamt der Stadt Hamm. Institut für Lebensmittelchemie, Koblenz. Stadt Köln, Institut für Lebensmitteluntersuchungen, Köln. Stadt Leverkusen Fachbereich Umwelt Chemisches Untersuchungsinstitut, Leverkusen. Landeslabor Schleswig-Holstein, Neumünster. Greece General Chemical State, Division of Thessaloniki. Ireland Southern Health Board, Public Analyst s, Cork. Public Analyst s, Dublin 2. Luxembourg Laboratoire National de Santé, Luxembourg. The Netherlands Silliker B.V., Ede. CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 12 of 29

15 Portugal Direcção Geral de Fiscalização e Controlo da Qualidade Alimentar, Lisboa. Réunion (France) Laboratoire Régional DGCCRF, Saint Denis Cedex. Slovenia Institute of Public Health of the Republic Slovenia, Department of sanitary chemistry, Ljubljana. Spain Health Department of Basque Government, Laboratorio Salud Publica Gipuzkoa, San Sebastian (Gipuzkoa). Food and Consumer Product Safety Authority, Inspectorate for Health Protection and Veterinary Public Health, The Netherlands: Location: Eindhoven. Location: Groningen. CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 13 of 29

16 8. Explanation of graphical presentations Z-score: As a criterion for evaluation of the performance of an individual laboratory a so-called Z-score is used. The Z-score is given by the following equation: Z = x µ s Where: x = an (average) laboratory result. µ = the average result of all laboratories (calculated exclusive outliers). s = an assigned precision standard or fixed target value. As a fixed target value the maximum allowable standard deviation is used at a given concentration-level. The Z-score for an individual laboratory can be compared with those of previous proficiency studies to determine whether the laboratory performance has improved. Because Z is standardised, it is comparable for all analytes, testmaterials and analytical methods. In general, an absolute value of Z greater than three suggests poor performance in terms of accuracy. When overall performance in a specific interlaboratory test is graded as "good", values of Z <1 would be very common and values of Z >3 would be very rare. It is possible to classify these scores: Satisfactory = Z 2 Questionable = 2< Z < 3 Unsatisfactory = Z 3 Saw-tooth plot: The results of the samples are presented in a so-called saw-tooth plot. In this figure individual results of two (nearly) identical individual results are plotted. The average of the sample, the 2s- and 3s-intervals of the target values and the 2s- and 3s-intervals after removal of outliers are also included. For split-level samples 2s- and 3s-intervals are presented for each sample. Youden-plot: Based on Youden statistics the calculated variance is split in a variance caused by systematic and random errors of the individual laboratories. It is necessary that the samples are similar. Generally the points form an elliptical pattern with the major axis of the ellipse running diagonally at an angle of 45 to the X-axis. The lengths of the perpendiculars drawn from the points to the 45 line are directly related to the random errors. Systematic errors will be presented along the 45 line. The perpendiculars intersect the 45 line at various distances from the point through which the 45 line was drawn. These distances are directly related to the systematic errors. CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 14 of 29

17 9. Tables and graphical presentations Table I; Proficiency study 312: Acesulfame-K in energy drink Units: mg/kg Lab Sample A Sample B Z-score A Z-score B Samples/year 1 (2) (1) = Cochran outlier (2) = Grubbs outlier (3) = Results removed by hand Calculations of the Z-scores is based on the average of sample A resp. sample B and the assigned standard deviation for sample A resp. sample B calculated from the Horwitz equation. CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 15 of 29

18 Table II; Proficiency study 312: Aspartame in energy drink Units: mg/kg Lab Sample A Sample B Z-score A Z-score B Samples/year (1) (#) (134) (126) 15 (1) (1) = Cochran outlier (2) = Grubbs outlier (3) = Results removed by hand (#) = Results are corrected after contact with participant. Results between brackets are initial results. Calculations of the Z-scores is based on the average of sample A resp. sample B and the assigned standard deviation for sample A resp. sample B calculated from the Horwitz equation. CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 16 of 29

19 Table III; Proficiency study 312: Caffeine in energy drink Units: mg/kg Lab Sample A Sample B Z-score A Z-score B Samples/year (#,2) (560) (501) (2) (2) (2) (1) = Cochran outlier (2) = Grubbs outlier (3) = Results removed by hand (#) = Results are corrected after contact with participant. Results between brackets are initial results. Calculations of the Z-scores is based on the average of sample A resp. sample B and the assigned standard deviation for sample A resp. sample B calculated from the Horwitz equation. CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 17 of 29

20 Graphical presentations acesulfame-k in energy drink Figure Ia; Saw-tooth plot acesulfame-k in energy drink sample A, calculated with target s. 450 Acesulfame-K in energy drink mg/kg, sample A sample A mean -2s/+2s -3s/+3s Figure Ib; Saw-tooth plot acesulfame-k in energy drink sample A, calculated with group s (exclusive laboratory 1). 450 Acesulfame-K in energy drink mg/kg, sample A sample A mean -2s/+2s -3s/+3s CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 18 of 29

21 Figure IIa; Saw-tooth plot acesulfame-k in energy drink sample B, calculated with target s. 400 Acesulfame-K in energy drink mg/kg, sample B sample B mean -2s/+2s -3s/+3s Figure IIb; Saw-tooth plot acesulfame-k in energy drink sample B, calculated with group s (exclusive laboratory 1). 400 Acesulfame-K in energy drink mg/kg, sample B sample B mean -2s/+2s -3s/+3s CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 19 of 29

22 Figure III; Youden plot acesulfame-k in energy drink. 400 mg/kg Acesulfame-K in energy drink, sample B mg/kg Acesulfame-K in energy drink, sample A CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 20 of 29

23 Figure IVa; Z-score acesulfame-k in energy drink sample A. 3 1 Z-score A Acesulfame-K in energy drink Figure IVb; Z-score acesulfame-k in energy drink sample B. 3 1 Z-score B Acesulfame-K in energy drink CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 21 of 29

24 Graphical presentations aspartame in energy drink Figure Va; Saw-tooth plot aspartame in energy drink sample A, calculated with target s. 800 Aspartame in energy drink mg/kg, sample A sample A mean -2s/+2s -3s/+3s Figure Vb; Saw-tooth plot aspartame in energy drink sample A, calculated with group s (exclusive laboratories 5 and 15). 800 Aspartame in energy drink mg/kg, sample A sample A mean -2s/+2s -3s/+3s CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 22 of 29

25 Figure VIa; Saw-tooth plot aspartame in energy drink sample B, calculated with target s. 700 Aspartame in energy drink mg/kg, sample B sample B mean -2s/+2s -3s/+3s Figure VIb; Saw-tooth plot aspartame in energy drink sample B, calculated with group s (exclusive laboratories 5 and 15). 700 Aspartame in energy drink mg/kg, sample B sample B mean -2s/+2s -3s/+3s CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 23 of 29

26 Figure VII; Youden plot aspartame in energy drink mg/kg Aspartame in energy drink, sample B mg/kg Aspartame in energy drink, sample A CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 24 of 29

27 Figure VIIIa; Z-score aspartame in energy drink sample A Z-score A Aspartame in energy drink Figure VIIIb; Z-score aspartame in energy drink sample B Z-score B Aspartame in energy drink CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 25 of 29

28 Graphical presentations caffeine in energy drink Figure IXa; Saw-tooth plot caffeine in energy drink sample A, calculated with target s. 180 Caffeine in energy drink mg/kg, sample A sample A mean -2s/+2s -3s/+3s Figure IXb; Saw-tooth plot caffeine in energy drink sample A, calculated with group s (exclusive laboratories 14, 16, 19 and 21). 180 Caffeine in energy drink mg/kg, sample A sample A mean -2s/+2s -3s/+3s CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 26 of 29

29 Figure Xa; Saw-tooth plot caffeine in energy drink sample B, calculated with target s. 180 Caffeine in energy drink mg/kg, sample B sample B mean -2s/+2s -3s/+3s Figure Xb; Saw-tooth plot caffeine in energy drink sample B, calculated with group s (exclusive laboratories 14, 16, 19 and 21). 180 Caffeine in energy drink mg/kg, sample B sample B mean -2s/+2s -3s/+3s CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 27 of 29

30 Figure XI; Youden plot caffeine in energy drink. 145 mg/kg Caffeine in energy drink, sample B mg/kg Caffeine in energy drink, sample A CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 28 of 29

31 Figure XIIa; Z-score caffeine in energy drink sample A. 3 2 Z-score A caffeine in energy drink Figure XIIb; Z-score caffeine energy drink sample B. 3 Z-score B caffeine in energy drink CHEK Proficiency study 312; Acesulfame-K, aspartame and caffeine in energy drink page 29 of 29