Fapas Food Chemistry Proficiency Test Report Sweeteners in Soft Drink. March-April Page 1 of 42

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1 Fapas Food Chemistry Proficiency Test Report Sweeteners in Soft Drink N O T C O N TR O LL ED W H EN PR IN TE D March-April 2017 Page 1 of 42

2 PARTICIPANT LABORATORY NUMBER Participants can log in to Fapas SecureWeb at any time to obtain their for this proficiency test. Laboratory numbers are displayed in SecureWeb next to the download link for this report. REPORT INTEGRITY Fapas reports are distributed as Adobe Certified Document Services (CDS) Adobe PDF documents [1]. The use of Adobe CDS allows the PDF files to certify that the author of the report is Fapas and that the document has not been altered in anyway. A blue ribbon and information bar indicates this validation when the document is opened using Adobe Reader v7 or later. Hard copies of Fapas reports can never incorporate this level of integrity and consequently when a Fapas report is printed a watermark, stating that printed copies are not controlled, appears on every page. End users of Fapas reports should ensure that either the opened PDF file displays a valid Fapas digital signature or that the content of any hard copy exactly matches the content of a PDF file that displays a valid Fapas digital signature. QUALITY SYSTEMS Fapas Food Chemistry is accredited by UKAS as complying with the requirements of ISO/IEC 17043:2010 [2]. Fera is an ISO 9001 certified organisation. Fera hereby excludes all liability for any claim, loss, demands or damages of any kind whatsoever (whether such claims, loss, demands or damages were foreseeable, known or otherwise) arising out of or in connection with the preparation of any technical or scientific report, including without limitation, indirect or consequential loss or damage; loss of actual or anticipated profits (including loss of profits on contracts); loss of revenue; loss of business; loss of opportunity; loss of anticipated savings; loss of goodwill; loss of reputation; loss or damage to or corruption of data; loss of use of money or otherwise, and whether or not advised of the possibility of such claim, loss demand or damages and whether arising in tort (including negligence), contract or otherwise. This statement does not affect your statutory rights. Nothing in this disclaimer excludes or limits Fera liability for: (a) death or personal injury caused by Fera negligence (or that of its employees, agents or directors); or (b) the tort of deceit; [or (c) any breach of the obligations implied by Sale of Goods Act 1979 or Supply of Goods and Services Act 1982 (including those relating to the title, fitness for purpose and satisfactory quality of goods);] or (d) any liability which may not be limited or excluded by law (e) fraud or fraudulent misrepresentation. The parties agree that any matters are governed by English law and irrevocably submit to the non-exclusive jurisdiction of the English courts. Copyright Fera Science Ltd (Fera) All rights reserved Page 2 of 42

3 SUMMARY 1. The test materials for Fapas Food Chemistry proficiency test were dispatched in March Each participant received two soft drink test materials. Test material A to be analysed for acesulfame-k, aspartame, cyclamate, and saccharin, and test material B to be analysed for total steviol glycosides, stevioside and rebaudioside A. Participants were asked to report any additional steviol glycosides found in the test material. 2. An assigned value (x a ) was determined for acesulfame-k, aspartame, cyclamate, saccharin and rebaudioside A and in conjunction with the standard deviation for proficiency (σ p ) was used to calculate a z-score for each result. However, those for rebaudioside A are given for information only. It was not possible to set an assigned value for total steviol glycosides and stevioside. 3. Results for this proficiency test are summarised as follows: analyte assigned value, xₐ mg/l number of scores, z 2 total number of scores % z 2 Acesulfame-K Aspartame Cyclamate Saccharin Rebaudioside A italics indicate for information only Page 3 of 42

4 CONTENTS 1. INTRODUCTION Proficiency Testing 5 2. TEST MATERIAL Preparation Homogeneity Dispatch 5 3. RESULTS 5 4. STATISTICAL EVALUATION OF RESULTS Calculation of the Assigned Value, x a Standard Deviation for Proficiency, σ p Individual z-scores 7 5. INTERPRETATION OF SCORES 7 6. REFERENCES 8 TABLES Table 1: Results and z-scores for Acesulfame-K, Aspartame, Cyclamate, Saccharin and Rebaudioside A 9 Table 2: Results for Total Steviol Glycosides (E960) and Stevioside 11 Table 3: Participants Comments 12 Table 4: Assigned Values and Standard Deviations for Proficiency 12 Table 5: Number and Percentage of z-scores where z 2 12 FIGURES Figure 1: z-scores for Acesulfame-K 13 Figure 2: z-scores for Aspartame 14 Figure 3: z-scores for Cyclamate 15 Figure 4: z-scores for Saccharin 16 Figure 5: z-scores for Rebaudioside A 17 Figure 6: Dot Plot for Total Steviol Glycosides (E960) and Stevioside 18 APPENDICES APPENDIX I: Analytical Methods Used by Participants 19 APPENDIX II: Fapas SecureWeb, Protocol and Contact Details 42 Page 4 of 42

5 1. INTRODUCTION 1.1. Proficiency Testing Proficiency testing aims to provide an independent assessment of the competence of participating laboratories. Together with the use of validated methods, proficiency testing is an essential element of laboratory quality assurance. Further details of the Fapas Food Chemistry proficiency testing scheme are available in our protocols [3, 4]. 2. TEST MATERIAL 2.1. Preparation Preparation of the samples for this proficiency test was sub-contracted to a laboratory meeting the quality requirements of the scheme s accreditation [2]. The test materials were purchased from a retail source. Acesulfame-K and aspartame were present in the test material A. Cyclamate and saccharin were spiked into the test material A. Steviol glycosides were present in test material B. Samples were stored at +4 C until dispatch Homogeneity To test for homogeneity, randomly selected test materials were analysed in duplicate. Testing was sub-contracted to a laboratory meeting the quality requirements of the scheme s accreditation [2]. These data showed sufficient homogeneity and were not included in the subsequent calculation of the assigned values Dispatch The start date was 02 March Test materials were sent to 60 participants. 3. RESULTS The instructions for reporting results were as follows: Test Material A Determine the level of acesulfame-k, aspartame, cyclamate and saccharin present in the test material in mg/l. PLEASE NOTE: Cyclamate should be expressed as the free acid and not the sodium salt. Saccharin should be expressed as the free imide and not the sodium salt. Test Material B Determine the level of stevioside and rebaudioside A present in the test material in mg/l, corrected for the purity of the standard. Page 5 of 42

6 Initially determine the level of total steviol glycosides (E960) in the test material in mg/l, corrected for the purity of the standard but before you submit these data you MUST express the results in steviol equivalents mg/l. The conversion factors for total steviol glycosides in mg/l to steviol equivalents mg/l are given in Table 1 on page 3 of this letter. The factors are taken from the specification, Ref: Commission regulation (EU) No 231/2012 of 09 March OJ L83/270. Please report any additional steviol glycosides found in the test material in mg/l and corrected for the purity of the standard. Results were submitted by 55 participants (92%) before the closing date for this test, 12 April Each participant was given a, assigned in order of receipt of results. The reported analyte concentrations are given in Table 1 and Table 2. Participants comments are given in Table 3. The analytical methods used by each participant are summarised in APPENDIX I. 4. STATISTICAL EVALUATION OF RESULTS The results submitted by participants were statistically analysed in order to provide an assigned value for acesulfame-k, aspartame, cyclamate, saccharin and rebaudioside A. The assigned values were then used in combination with the standard deviations for proficiency, σ p, to calculate a z-score [5] for each result. The procedure is detailed in the relevant protocols [3, 4]. Further background on the procedure followed can be found in the IUPAC International Harmonised Protocol for the Proficiency Testing of Analytical Chemistry Laboratories [6] Calculation of the Assigned Value, x a The assigned value, x a, for acesulfame-k, aspartame, cyclamate, saccharin and rebaudioside A was derived from the consensus of the results submitted by participants. The following results were excluded from the calculation of the assigned value: i) non numerical results i.e. qualitative or semi-quantitative results, ii) results reported as approximately 10, 100 or 1000 greater or smaller than the majority of submitted results (as these were considered to be reporting errors). For acesulfame-k, aspartame, cyclamate and saccharin, this procedure was straightforward and the robust mean was chosen as the assigned value. For rebaudioside A, the median was chosen as the assigned value because the uncertainty of the robust mean was too high due to the low number of submitted results. The assigned value and z-scores are given for information only due to unacceptably high uncertainty. For total steviol glycosides and stevioside an assigned value could not be set due to the low number of submitted results. The assigned values for acesulfame-k, aspartame, cyclamate, saccharin and rebaudioside A are shown in Table 4. Page 6 of 42

7 4.2. Standard Deviation for Proficiency, σ p The standard deviation for proficiency, σ p, was set at a value that reflects best practice for the analyses in question. For all analytes, σ p was derived from the appropriate form of the Horwitz equation [7]. The values for σ p used to calculate z-scores from the reported results of this test are given in Table Individual z-scores Participants z-scores were calculated as: ( x z = σ p x a ) where x = the participant s reported result, x a = the assigned value and σ p = the standard deviation for proficiency. Participants z-scores for acesulfame-k, aspartame, cyclamate, saccharin and rebaudioside A are given in Table 1 and shown as histograms in Figures 1 5. Those for rebaudioside A are given for information only. It is possible for the z-scores published in this report to differ slightly from the z-score that can be calculated using the formula given above. These differences arise from the necessary rounding of the actual assigned values and standard deviations for proficiency prior to their publication in Table 4. The number and percentage of z-scores in the range -2 z 2 for acesulfame-k, aspartame, cyclamate, saccharin and rebaudioside A are given in Table 5. Since no assigned value was calculated for total steviol glycosides and stevioside, it was not possible to derive z-scores for these analytes. The results reported by participants are given in Table 2 and shown as dot plots in Figure INTERPRETATION OF SCORES In normal circumstances, over time, about 95% of z-scores will lie in the range -2 z 2. Occasional scores in the range 2 < z <3 are to be expected, at a rate of 1 in 20. Whether or not such scores are of importance can only be decided by considering them in the context of the other scores obtained by that laboratory. Scores where z >3 are to be expected at a rate of about 1 in 300. Given this rarity, such z-scores very strongly indicate that the result is not fit-for-purpose and almost certainly requires investigation. The consideration of a set or sequence of z-scores over time provides more useful information than a single z-score. Examples of suitable methods of comparison are provided in the IUPAC International Harmonised Protocol for the Proficiency Testing of Analytical Chemistry Laboratories [6]. Page 7 of 42

8 6. REFERENCES 1 Adobe Certified Document Services, accessed 10/03/ ISO/IEC 17043:2010, Conformity assessment General requirements for proficiency testing. 3 Fapas, 2016, Protocol for Proficiency Testing Schemes, Part 1 Common Principles, Version 5, Issued September Fapas, 2016, Protocol for Proficiency Testing Schemes, Part 2 Fapas Food Chemistry Version 4, Issued September AMC Tech Brief No. 74, z-scores and other scores in chemical proficiency testing their meanings, and some common misconceptions, Anal. Methods, 2016, 8, Thompson, M., Ellison, S.L.R. and Wood, R., 2006, The International Harmonised Protocol for the Proficiency Testing of Analytical Chemistry Laboratories, Pure Appl. Chem., 78, No. 1, Thompson, M., 2000, Recent trends in inter-laboratory precision at ppb and sub-ppb concentrations in relation to fitness for purpose criteria in proficiency testing, Analyst, 125, Page 8 of 42

9 Table 1: Results and z-scores for Acesulfame-K, Aspartame, Cyclamate, Saccharin and Rebaudioside A laboratory number analyte Acesulfame-K Aspartame Cyclamate Saccharin Rebaudioside A assigned value: assigned value: assigned value: assigned value: assigned value: 39.9 mg/l 528 mg/l 89.7 mg/l 49.6 mg/l 66.5 mg/l result z-score result z-score result z-score result z-score result z-score z-scores outside z >2 are shown in bold, see Section 5 italics indicate for information only Page 9 of 42

10 Table 1 (continued): Results and z-scores for Acesulfame-K, Aspartame, Cyclamate, Saccharin and Rebaudioside A laboratory number analyte Acesulfame-K Aspartame Cyclamate Saccharin Rebaudioside A assigned value: assigned value: assigned value: assigned value: assigned value: 39.9 mg/l 528 mg/l 89.7 mg/l 49.6 mg/l 66.5 mg/l result z-score result z-score result z-score result z-score result z-score < LOQ (101 mg/l) < LOQ (40 mg/l) z-scores outside z >2 are shown in bold, see Section 5 italics indicate for information only Page 10 of 42

11 Table 2: Results for Total Steviol Glycosides (E960) and Stevioside laboratory number analyte Total Steviol Glycosides (E960) assigned value not set result Stevioside assigned value not set result < non-determinable Page 11 of 42

12 Table 3: Participants Comments comments 003 Limit of quantitation (LOQ) for stevioside 10 mg/l 014 detection limit = 2 mg/l 051 Stevioside was detected but non-determinable (< LOQ) 054 All results are corrected for recovery comments are as submitted by participants Table 4: Assigned Values and Standard Deviations for Proficiency analyte data points, n assigned value, xₐ, mg/l uncertainty, u standard deviation for proficiency, σ p, mg/l Acesulfame-K Horwitz [7] 3.67 Aspartame Horwitz [7] 32.9 Cyclamate Horwitz [7] 7.29 Saccharin Horwitz [7] 4.41 Rebaudioside A Horwitz [7] 5.66 italics indicate for information only Table 5: Number and Percentage of z-scores where z 2 analyte number of scores where z 2 total number of scores % z 2 Acesulfame-K Aspartame Cyclamate Saccharin Rebaudioside A italics indicate for information only Page 12 of 42

13 z-score Figure 1: z-scores for Acesulfame-K Laboratory Number 47.2 mg/l 39.9 mg/l 32.6 mg/l Page 13 of 42

14 mg/l z-score Figure 2: z-scores for Aspartame Laboratory Number 528 mg/l 462 mg/l Page 14 of 42

15 z-score Laboratory Number Figure 3: z-scores for Cyclamate mg/l 89.7 mg/l 75.1 mg/l Page 15 of 42

16 z-score Figure 4: z-scores for Saccharin Laboratory Number 58.4 mg/l 49.6 mg/l 40.8 mg/l Page 16 of 42

17 mg/l z-score Laboratory Number Figure 5: z-scores for Rebaudioside A 2 this histogram is given for information only mg/l 55.2 mg/l Page 17 of 42

18 Stevioside Total steviol glycosides Figure 6: Dot Plot for Total Steviol Glycosides (E960) and Stevioside Data Page 18 of 42

19 APPENDIX I: Analytical Methods Used by Participants Methods are tabulated according to the information supplied by participants, but some responses may have been combined or edited for clarity. Soft Drink (T03136A) Acesulfame-K Is The Method Used Accredited? yes no Reference AOAC Official Methods Vol 76 No BS EN BS EN BVL L CICC 027 CYS EN:12856: Deutsche Lebensmittelrundschau 1990 issue 11; 86th year EN 1999 UNI EN 12856: EN Food Additives and Contaminant (4) GB /T GB/T GB/T GB/T HPLC in Food Analysis In house method 048 Intense Sweetteners, Handbooks of Food Analysis ISO Page 19 of 42

20 Reference (continued) J. AOAC Int /5 024 J. AOAC Int J. Assoc. Off. Anal. Chem J. Chromatography A Korea Food Code LMBG Method L LMBG Method LMBG Method LMBG Method LMBG Method 2011 L National standard own method 030 own method 052 TFDA 039 TS Waters Application note EN AG-PDF ????? ????? Sample Amount Used for Analysis (g) < < < < < < < < Sample Treatment Prior to HPLC blend / homogenise with solvent centrifuge clarify 040 degas dilute dissolve in water evaporate 011 Page 20 of 42

21 Sample Treatment Prior to HPLC (continued) filter liquid/liquid extraction 046 ph adjustment 005 solid phase extraction (SPE) 011 solvent extraction 049 HPLC Injection Volume (µl) < < < < < HPLC Column Packing C C18 X-Terra type ACQUITY HSST3 054 HPLC Column Temperature ( C) ambient >ambient - < Mobile Phase Components acetate acetone 011 acetonitrile formic acid methanol phosphate Page 21 of 42

22 Mobile Phase Components (continued) water % Tetrapropyl ammonium hydroxide & MeOH 026 AA in water/ AA in Methanol 039 acetate buffer ph ammonium formate 030 KH2PO4+Acetonitrile 012 phosphate buffer/acetonitryle 028 potassium dihydrogen phosphate+ch3cn (90/10) 023 Isocratic Mobile Phase? yes no (gradient) Source of Standards Dr Ehrenstorfer Fluka Merck Sigma/Aldrich Supelco AppliChem GmbH, Darmstadt 019 national reference meterials centre of China 010 Riedel-de Haen 014 TCI 030 HPLC Detector Type Diode Array Detector MS 054 MS-MS PAD UV UV/Vis 028 PDA and QDA 030 Page 22 of 42

23 Wavelength (absorbance)(nm) 200/ nm nm Non HPLC Method Used ELSD 011 Aspartame Is The Method Used Accredited? yes no Reference BS EN BVL L CICC 027 CYS EN:12856: Page 23 of 42

24 Reference (continued) Deutsche Lebensmittelrundschau 1990 issue 11; 86th year EN 1999 UNI EN 12856: EN Food Additives and Contaminant (4) GB GB GB HPLC in Food Analysis In house method 048 Intense Sweetteners, Handbooks of Food Analysis internal Method L ISO J. AOAC Int. 049 J. AOAC Int /5 024 J. Assoc. Off. Anal. Chem J. Assoc. Off. Anal. Chem J. Chromatography 032 J. Chromatography A Journal Of Food Hygiene of Japan 1992 Vol Korea Food Code LMBG Method L LMBG Method LMBG Method Manufacturer's Instructions National standard NMKL own method 030 own method 052 TS Waters Application note EN AG-PDF Sample Amount Used for Analysis (g) < < < Page 24 of 42

25 Sample Amount Used for Analysis (g) (continued) 5 - < < < < < Sample Treatment Prior to HPLC blend / homogenise with solvent buffer 031 centrifuge clarify 040 degas dilute dissolve in water evaporate 011 filter ph adjustment 005 solid phase extraction (SPE) 011 HPLC Injection Volume (µl) < < < < < HPLC Column Packing C C18 X-Terra type 020 ACQUITY HSST3 054 Page 25 of 42

26 HPLC Column Temperature ( C) ambient >ambient - < Mobile Phase Components acetate ethanoic acid (acetic acid) 027 acetone 011 acetonitrile formic acid methanol phosphate water % Tetrapropyl ammonium hydroxide & MeOH 026 AA in water/ AA in Methanol 039 acetate buffer ph Acetic Acid 021 Acetonitrile + Phosphate 041 ammonium formate 030 KH2PO4+Acetonitrile 012 phosphate buffer/acetonitryle 028 potassium dihydrogen phosphate/methanol (72/28) Isocratic Mobile Phase? 023 yes no (gradient) Source of Standards Dr Ehrenstorfer Fluka Merck Sigma/Aldrich Page 26 of 42

27 Source of Standards (continued) Supelco AppliChem GmbH, Darmstadt 019 HPLC Detector Type Diode Array Detector MS 054 MS-MS PAD 033 UV UV/Vis 028 ELSD 042 PDA and QDA 030 Wavelength (absorbance)(nm) nm nm Non HPLC Method Used ELSD 011 Page 27 of 42

28 Cyclamate Is The Method Used Accredited? yes no Reference BS EN BVL L CICC 027 EN 1999 UNI EN 12857: EN Food Additives and Contaminant (4) GB GB GB GB/T In house method 048 Intense Sweetteners, Handbooks of Food Analysis ISO J. Assoc. Off. Anal. Chem / J. Chromatography LFGB 2006 L LMBG Method LMBG Method LMBG Method Manufacturer's Instructions National standard own method 052 SN/T TFDA 039 Waters Application note EN AG-PDF ????? ????? Page 28 of 42

29 Sample Amount Used for Analysis (g) < < < < < < < Sample Treatment Prior to HPLC blend / homogenise with solvent centrifuge 048 clarify 040 degas derivatisation dialysis 021 dilute dissolve in water evaporate 011 filter liquid/liquid extraction solid phase extraction (SPE) 011 solvent extraction 025 blend/homogenise with buffer 019 HPLC Injection Volume (µl) < < < < HPLC Column Packing C C18 X-Terra type 046 Ion Exchange 015 ACQUITY HSST3 054 Sactopore RP 003 Page 29 of 42

30 HPLC Column Temperature ( C) ambient >ambient - < Mobile Phase Components acetate ethanoic acid (acetic acid) 044 acetone acetonitrile formic acid methanol phosphate 046 water %methanol+20%water 028 AA in water/ AA in Methanol 039 ion pair buffer 043 o-phosphoric acid 009 Phosphoric Acid 027 Isocratic Mobile Phase? yes no (gradient) Source of Standards Dr Ehrenstorfer Merck Sigma/Aldrich Supelco national reference meterials centre of China 010 Roth 015 HPLC Detector Type conductivity 015 Diode Array Detector MS 054 Page 30 of 42

31 HPLC Detector Type (continued) MS-MS PAD 046 RI 027 UV UV/Vis ELSD Wavelength (absorbance)(nm) nm Non HPLC Method Used GC-FID 033 ELSD 011 GC-MS 032 Saccharin Is The Method Used Accredited? yes no Reference AOAC Official Methods Vol 76 No BS EN Page 31 of 42

32 Reference (continued) BVL L CICC 027 CYS EN:12856: Deutsche Lebensmittelrundschau 1990 issue 11; 86th year EN 1999 UNI EN 12856: EN Food Additives and Contaminant (4) GB /T GB GB GB HPLC in Food Analysis In house method 048 Intense Sweetteners, Handbooks of Food Analysis ISO J. AOAC Int /5 024 J. AOAC Int / J. AOAC Int J. Assoc. Off. Anal. Chem J. Chromatography J. Chromatography A Korea Food Code LFGB 2001 L LMBG Method LMBG Method LMBG Method LMBG Method 2001 L Manufacturer's Instructions National standard own method 030 own method 052 TFDA 039 TS Waters Application note EN AG-PDF ????? ????? Page 32 of 42

33 Sample Amount Used for Analysis (g) < < < < < < < < Sample Treatment Prior to HPLC blend / homogenise with solvent centrifuge clarify 040 degas dialysis 021 dilute dissolve in water evaporate 011 filter liquid/liquid extraction 046 ph adjustment 005 solid phase extraction (SPE) 011 solvent extraction 049 HPLC Injection Volume (µl) < < < < < Page 33 of 42

34 HPLC Column Packing C C18 X-Terra type ACQUITY HSST3 054 HPLC Column Temperature ( C) ambient >ambient - < Mobile Phase Components acetate acetone acetonitrile formic acid methanol phosphate water % Tetrapropyl ammonium hydroxide & MeOH 026 AA in water/ AA in Methanol 039 acetate buffer ph ammonium formate 030 KH2PO4+Acetonitrile 012 phosphate buffer/acetonitryle 028 Phosphoric Acid 027 potassium dihydrogen phosphate+ch3cn (90/10) 023 Isocratic Mobile Phase? yes no (gradient) Page 34 of 42

35 Source of Standards Acros 005 Dr Ehrenstorfer Fluka Merck Sigma/Aldrich Supelco AppliChem GmbH, Darmstadt 019 national reference meterials centre of China 010 HPLC Detector Type Diode Array Detector MS 054 MS-MS PAD RI 027 UV UV/Vis ELSD 042 PDA and QDA 030 Wavelength (absorbance)(nm) 200/ nm nm Page 35 of 42

36 Wavelength (absorbance)(nm) (continued) Non HPLC Method Used ELSD 011 Soft Drink (T03136B) Total Steviol Glycosides (E960) Is The Method Used Accredited? yes no Reference Analytical Chemistry FAO JECFA Monographs FAO JECFA Monographs In house method 036 own method 052 Waters Application note EN AG-PDF Sample Amount Used for Analysis (g) 054 < < < Sample Treatment Prior to HPLC centrifuge 048 degas dilute filter HPLC Injection Volume (µl) < < Page 36 of 42

37 HPLC Injection Volume (µl) (continued) 10 - < < HPLC Column Packing C endcapped ACQUITY HSST3 054 HPLC Column Temperature ( C) >ambient - < Mobile Phase Components acetate 052 acetonitrile formic acid phosphate water Isocratic Mobile Phase? yes 014 no (gradient) Source of Standards Dr Ehrenstorfer 054 Merck 052 Sigma/Aldrich LGC Standard/Chromadex 036 phytolab 003 HPLC Detector Type Diode Array Detector MS UV/Vis 003 Wavelength (absorbance)(nm) Page 37 of 42

38 Stevioside Is The Method Used Accredited? yes no Reference Analytical Chemistry FAO JECFA Monographs FAO JECFA Monographs own method 052 Waters Application note EN AG-PDF Sample Amount Used for Analysis (g) 054 < < < Sample Treatment Prior to HPLC centrifuge 048 degas dilute filter HPLC Injection Volume (µl) < < < < HPLC Column Packing C endcapped ACQUITY HSST3 054 HPLC Column Temperature ( C) >ambient - < Page 38 of 42

39 Mobile Phase Components acetate 052 acetonitrile formic acid 054 phosphate water Isocratic Mobile Phase? yes 014 no (gradient) Source of Standards Merck 052 Sigma/Aldrich Wako 014 HWI 054 LGC 051 phytolab 003 HPLC Detector Type Diode Array Detector MS 054 UV/Vis 003 Wavelength (absorbance)(nm) Rebaudioside A Is The Method Used Accredited? yes no Reference Analytical Chemistry FAO JECFA Monographs FAO JECFA Monographs Page 39 of 42

40 Reference (continued) FAO JECFA Monographs J. Chromatography A own method 052 Waters Application note EN AG-PDF Sample Amount Used for Analysis (g) < < < < Sample Treatment Prior to HPLC centrifuge 048 degas dilute dissolve in water 041 filter HPLC Injection Volume (µl) < < < < HPLC Column Packing C endcapped ACQUITY HSST3 054 HPLC Column Temperature ( C) >ambient - < Mobile Phase Components acetate 052 acetonitrile formic acid 054 phosphate water Page 40 of 42

41 Isocratic Mobile Phase? yes 014 no (gradient) Source of Standards Sigma/Aldrich Wako 014 Chromadex EXTRASYNTHESE 054 LGC 051 HPLC Detector Type Diode Array Detector MS 054 UV 041 UV/Vis 003 Wavelength (absorbance)(nm) Page 41 of 42

42 APPENDIX II: Fapas SecureWeb, Protocol and Contact Details 1. Fapas SECUREWEB Access to the secure area of our website is only available to participants in our proficiency tests. Please contact us if you require a UserID and Password. Fapas SecureWeb allows participants to: Obtain their s for the proficiency tests in which they have participated. View the results they submitted in past and current proficiency tests. Submit their results and methods for current tests. Review future tests they have ordered. Order proficiency tests, reference materials and quality control materials. Freely download copies of reports (PDF file), of proficiency tests in which they have participated. View charts of their z-scores obtained in previous Fapas Food Chemistry proficiency tests. 2. PROTOCOL The Protocols [3, 4] set out how Fapas Food Chemistry is organised. Copies can be downloaded from our website. 3. CONTACT DETAILS This report was prepared and authorised on behalf of Fapas by Izabele Cincyte (Round Coordinator). Participants with any comments or concerns about this proficiency test should contact: Fapas Fera Science Ltd (Fera) National Agri-Food Innovation Campus Sand Hutton York YO41 1LZ UK Tel: +44 (0) Fax: +44 (0) info@fapas.com Page 42 of 42