Fapas Food Chemistry Proficiency Test Report Mycotoxins in Maize. November-December Page 1 of 48

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1 Fapas Food Chemistry Proficiency Test Report Mycotoxins in Maize N O T C O N TR O LL ED W H EN PR IN TE D November-December 2017 Page 1 of 48

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 digitally signed Adobe PDF files. When these files are opened with Adobe Reader v9 or later, a blue ribbon and information bar indicates that the certificate has been validated. This confirms that the author of the report is Fapas and that the document has not been altered since it was signed [1, 2]. The integrity of hard copies of Fapas reports cannot be assured in this way, i.e. printed copies are not controlled. A watermark stating this appears on every page when a Fapas report is printed. End users of Fapas reports should ensure that either the PDF file they are viewing displays a valid Fapas digital signature or that the content of their 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 [3]. 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 48

3 SUMMARY 1. The test materials for Fapas Food Chemistry proficiency test were dispatched in November Each participant received a maize test material to be analysed for aflatoxin B 1, deoxynivalenol, zearalenone and ochratoxin A. 2. An assigned value (xa) was determined for aflatoxin B 1, deoxynivalenol, zearalenone and ochratoxin A and in conjunction with the standard deviation for proficiency (σp) was used to calculate a z-score for each result. Results for this proficiency test are summarised as follows: analyte assigned value, xₐ µg/kg number of scores, z 2 total number of scores % z 2 Aflatoxin B Deoxynivalenol (DON) Zearalenone (ZON) Ochratoxin A Page 3 of 48

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 Aflatoxin B 1 9 Table 2: Results and z-scores for Deoxynivalenol 12 Table 3: Results and z-scores for Zearalenone 15 Table 4: Results and z-scores for Ochratoxin A 18 Table 5: Participants Comments 21 Table 6: Assigned Values and Standard Deviations for Proficiency 21 Table 7: Number and Percentage of z-scores where z 2 21 FIGURES Figure 1: z-scores for Aflatoxin B 1 22 Figure 2: z-scores for Deoxynivalenol (DON) 23 Figure 3: z-scores for Zearalenone (ZON) 24 Figure 4: z-scores for Ochratoxin A 25 APPENDICES APPENDIX I: Analytical Methods Used by Participants 26 APPENDIX II: Fapas SecureWeb, Protocol and Contact Details 48 Page 4 of 48

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 [4, 5]. 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 [3]. The test materials were prepared from maize. Aflatoxin B 1, deoxynivalenol, zearalenone and ochratoxin A were spiked into the test material. Samples were stored at -20 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 [3]. These data showed sufficient homogeneity and were not included in the subsequent calculation of the assigned values Dispatch The start date was 9 November Test materials were sent to 75 participants. 3. RESULTS The instructions for reporting results were as follows: Determine the level of aflatoxin B 1, deoxynivalenol, zearalenone and ochratoxin A present in the test material, in μg/kg, as received, corrected for recovery. PLEASE NOTE: Please state your % recovery when submitting your results. If a different correction factor to a % recovery is used, please make a note of this in the comments section when submitting your results. Results were submitted by 69 participants (92%) before the closing date for this test, 19 December Each participant was given a, assigned in order of receipt of results. The reported analyte concentrations are given in Tables 1-4. Participants comments are given in Table 5. Page 5 of 48

6 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 aflatoxin B 1, deoxynivalenol, zearalenone and ochratoxin A. The assigned values were then used in combination with the standard deviation for proficiency, σp, to calculate a z-score [6] for each result. The procedure is detailed in the relevant protocols [4, 5]. Further background on the procedure followed can be found in the IUPAC International Harmonised Protocol for the Proficiency Testing of Analytical Chemistry Laboratories [7] Calculation of the Assigned Value, xa The assigned value, xa, for aflatoxin B 1, deoxynivalenol, zearalenone and ochratoxin 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) iii) 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), results uncorrected for recovery. For aflatoxin B 1, deoxynivalenol, zearalenone and ochratoxin A, this procedure was straightforward and the robust mean was chosen as the assigned value. The assigned values for aflatoxin B 1, deoxynivalenol, zearalenone and ochratoxin A are shown in Table Standard Deviation for Proficiency, σp The standard deviation for proficiency, σp, was set at a value that reflects best practice for the analyses in question. For aflatoxin B 1, deoxynivalenol, zearalenone and ochratoxin A, σp was derived from the appropriate form of the Horwitz equation [8]. The values for σp used to calculate z-scores from the reported results of this test are given in Table 6. Page 6 of 48

7 4.3. Individual z-scores Participants z-scores were calculated as: ( x z = σ p x a ) where x = the participant s reported result, xa = the assigned value and σp = the standard deviation for proficiency. Participants z-scores for aflatoxin B 1, deoxynivalenol, zearalenone and ochratoxin A are given in Tables 1-4 and shown as histograms in Figures 1 4. 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 6. The number and percentage of z-scores in the range -2 z 2 for aflatoxin B 1, deoxynivalenol, zearalenone and ochratoxin A are given in Table 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 [7]. Page 7 of 48

8 6. REFERENCES 1 Adobe Approved Trust List, accessed 01/06/ GlobalSign PDF Signing Tool, accessed 01/06/ ISO/IEC 17043:2010, Conformity assessment General requirements for proficiency testing. 4 Fapas, 2017, Protocol for Proficiency Testing Schemes, Version 6, April 2017, Part 1 Common Principles. 5 Fapas, 2017, Protocol for Proficiency Testing Schemes, Version 5, April 2017, Part 2 Fapas Food Chemistry scheme (FAPAS). 6 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 48

9 Table 1: Results and z-scores for Aflatoxin B1 analyte Aflatoxin B1 assigned value: 14.1 µg/kg result Recovery, % Limit of detection (µg/kg) z-score , Uncorreted 2, z-scores outside z >2 are shown in bold, see Section 5 Page 9 of 48

10 Table 1 (continued): Results and z-scores for Aflatoxin B1 analyte Aflatoxin B1 assigned value: 14.1 µg/kg result Recovery, % Limit of detection (µg/kg) z-score % % % z-scores outside z >2 are shown in bold, see Section 5 Page 10 of 48

11 Table 1 (continued): Results and z-scores for Aflatoxin B1 analyte Aflatoxin B1 assigned value: 14.1 µg/kg result Recovery, % Limit of detection (µg/kg) z-score % N/A z-scores outside z >2 are shown in bold, see Section 5 Page 11 of 48

12 Table 2: Results and z-scores for Deoxynivalenol analyte Deoxynivalenol (DON) assigned value: 1786 µg/kg result Recovery, % Limit of detection (µg/kg) z-score Uncorreted ppm ppm z-scores outside z >2 are shown in bold, see Section 5 Page 12 of 48

13 Table 2 (continued): Results and z-scores for Deoxynivalenol analyte Deoxynivalenol (DON) assigned value: 1786 µg/kg result Recovery, % Limit of detection (µg/kg) z-score < z-scores outside z >2 are shown in bold, see Section 5 Page 13 of 48

14 Table 2 (continued): Results and z-scores for Deoxynivalenol analyte Deoxynivalenol (DON) assigned value: 1786 µg/kg result Recovery, % Limit of detection (µg/kg) z-score % N/A z-scores outside z >2 are shown in bold, see Section 5 Page 14 of 48

15 Table 3: Results and z-scores for Zearalenone analyte Zearalenone (ZON) assigned value: 389 µg/kg result Recovery, % Limit of detection (µg/kg) z-score Uncorreted ppb z-scores outside z >2 are shown in bold, see Section 5 Page 15 of 48

16 Table 3 (continued): Results and z-scores for Zearalenone analyte Zearalenone (ZON) assigned value: 389 µg/kg result Recovery, % Limit of detection (µg/kg) z-score z-scores outside z >2 are shown in bold, see Section 5 Page 16 of 48

17 Table 3 (continued): Results and z-scores for Zearalenone analyte Zearalenone (ZON) assigned value: 389 µg/kg result Recovery, % Limit of detection (µg/kg) z-score % N/A z-scores outside z >2 are shown in bold, see Section 5 Page 17 of 48

18 Table 4: Results and z-scores for Ochratoxin A analyte Ochratoxin A assigned value: 5.90 µg/kg result Recovery, % Limit of detection (µg/kg) z-score , Uncorreted ppb z-scores outside z >2 are shown in bold, see Section 5 Page 18 of 48

19 Table 4 (continued): Results and z-scores for Ochratoxin A analyte Ochratoxin A assigned value: 5.90 µg/kg result Recovery, % Limit of detection (µg/kg) z-score z-scores outside z >2 are shown in bold, see Section 5 Page 19 of 48

20 Table 4 (continued): Results and z-scores for Ochratoxin A analyte Ochratoxin A assigned value: 5.90 µg/kg result Recovery, % Limit of detection (µg/kg) z-score % N/A z-scores outside z >2 are shown in bold, see Section 5 Page 20 of 48

21 Table 5: Participants Comments comments 032 Is the method you used to determine the result accredited to ISO 17025? YES 033 the current results are before calculation 054 GB The method used was submitted to accreditation in ISO comments are as submitted by participants Table 6: analyte Assigned Values and Standard Deviations for Proficiency data points, n assigned value, xₐ µg/kg uncertainty, u standard deviation for proficiency, σ p Aflatoxin B Horwitz [8] 3.11 Deoxynivalenol (DON) Horwitz [8] Zearalenone (ZON) Horwitz [8] 71.8 Ochratoxin A Horwitz [8] 1.30 Table 7: analyte Number and Percentage of z-scores where z 2 number of scores where z 2 total number of scores % z 2 Aflatoxin B Deoxynivalenol (DON) Zearalenone (ZON) Ochratoxin A Page 21 of 48

22 z-score Figure 1: z-scores for Aflatoxin B1 Laboratory Number 20.3 µg/kg 14.1 µg/kg 7.91 µg/kg Page 22 of 48

23 z-score Figure 2: z-scores for Deoxynivalenol (DON) Laboratory Number 2309 µg/kg 1786 µg/kg 1262 µg/kg Page 23 of 48

24 µg/kg z-score Figure 3: z-scores for Zearalenone (ZON) Laboratory Number 389 µg/kg 246 µg/kg Page 24 of 48

25 z-score Figure 4: z-scores for Ochratoxin A Laboratory Number 8.50 µg/kg 5.90 µg/kg 3.30 µg/kg Page 25 of 48

26 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. Aflatoxin B1 Is the Method Used Accredited? yes no What is Your Method Based On? International Standard National Standard Manufacturer/Kit Instructions/Technical Note In house method Sample Weight (g) 2 - < < < < Page 26 of 48

27 Extraction Solvent Components acetonitrile ethanoic acid (acetic acid) methanol phosphate buffer water acetic acid, acetonitrile and water 057 ethanol 012 methanol/water 062 methanol/water(7:3) 026 see kit instructions 018 Extraction Procedure add NaCl blend / homogenise with solvent shake with solvent shaking sonicate/ultrasonic bath Ultra Turrax vortex mix see kit instructions 018 Extraction Type single multiple Page 27 of 48

28 Sample Work Up centrifuge defatted with hexane dilute evaporate filter none 054 ph adjustment 062 Ultra Turrax 069 see kit instructions 018 Sample Clean-up by Immunoaffinity Column (Brand) R-Biopharm Rhone VICAM Micotox 007 AflaClean - LCTech 050 AflaStar Coring Systems 053 gl-science 034 HORIBA 035 huaanmaike 046 LC-Tech 030 Romer Labs 062 Sample Clean-up by SPE Cation exchange showadenkou 043 Page 28 of 48

29 Mycotoxin Determination ELISA fluorometric 033 HPLC HPLC/MS 026 Immunochromatography 012 UPLC 037 UPLC-MSMS 069 HPLC Injection Volume (μl) < < < < < < HPLC Column Packing C non-endcapped 024 HPLC Column Temperature ( C) ambient >ambient - < Page 29 of 48

30 Mobile Phase Components acetate ethanoic acid (acetic acid) acetonitrile formic acid (methanoic acid) HNO3 & KBr (for Kobra Cell) methanol water Ammonium formate 037 Ammoniumformiate 007 Mobile Phase Flow Rate (ml/min) < < < Post Column Mobile Phase Flow Rate (ml/min) < < HPLC Pre Column Derivatisation TFA none HPLC Post Column Derivatisation Kobra cell pyridinium bromide perbromide (C5H6Br3N) 032 none Page 30 of 48

31 Source of Standards Acros 069 R-Biopharm Rhone Romer Labs Sigma/Aldrich Supelco Wako Biopure 050 Fermentek 038 LGC 058 Trilogy 061 HPLC Detector Type fluorescence MS 026 MS-MS FLD 054 Page 31 of 48

32 Deoxynivalenol (DON) Is the Method Used Accredited? yes no What is Your Method Based On? International Standard National Standard Paper Published In An International Journal Manufacturer/Kit Instructions/Technical Note In house method Sample Weight (g) 1 - < < < < < Extraction Solvent Components acetone 035 acetonitrile ethanoic acid (acetic acid) ethyl acetate 031 hexane 031 methanol water acetic acid, acetonitrile and water 057 Formic acid 004 PEG 061 Page 32 of 48

33 Extraction Procedure activated charcoal 031 add filter aid 031 add NaCl 037 blend / homogenise with solvent shake with solvent shaking sonicate/ultrasonic bath 038 Ultra Turrax 069 vortex mix Extraction Type single multiple Extraction Time 1 hour min hours 011 1h and 30 min min min minutes min min 061 see kit instructions 018 Page 33 of 48

34 Sample Work Up centrifuge defatted with hexane 037 dilute evaporate filter none Ultra Turrax 030 see kit instructions 018 Sample Clean-up by Immunoaffinity Column (Brand) R-Biopharm Rhone VICAM gl-science 034 hua an maike 046 ROMER 024 Romer Labs 062 Sample Clean-up by SPE Cation exchange Activated Charcoal / Celite 031 Autoprep MF-T MF-T Multifunctional 044 showadenkou 043 Page 34 of 48

35 Mycotoxin Determination ELISA HPLC TLC/HPTLC 031 UPLC 037 UPLC-MSMS 069 HPLC Pre Column Derivatisation none HPLC Injection Volume (μl) < < < < < < HPLC Column Packing C non-endcapped 024 HPLC Column Temperature ( C) ambient >ambient - < Page 35 of 48

36 Mobile Phase Components acetate ethanoic acid (acetic acid) acetonitrile formic acid (methanoic acid) methanol water % acetonitrile 051 ammonium acetate Ammonium formate 037 NH4HCO2 015 Mobile Phase Flow Rate (ml/min) < < < Post Column Mobile Phase Flow Rate (ml/min) < < HPLC Detector Type Diode Array Detector fluorescence 051 MS 042 MS-MS UV/Vis 058 Diode Array and MS 014 Page 36 of 48

37 Isothermal Column Temp? yes GC Flow Rate < Source of Standards Dr Ehrenstorfer 046 R-Biopharm Rhone Romer Labs Sigma/Aldrich Supelco 051 Wako Biopure 015 Trilogy Page 37 of 48

38 Zearalenone (ZON) Is the Method Used Accredited? yes no What is Your Method Based On? International Standard National Standard Paper Published In An International Journal 058 Manufacturer/Kit Instructions/Technical Note In house method Sample Weight (g) 2 - < < < < Extraction Solvent Components acetonitrile ethanoic acid (acetic acid) methanol phosphate buffer 011 water acetic acid, acetonitrile and water 057 Formic acid 004 Page 38 of 48

39 Extraction Procedure add NaCl blend / homogenise with solvent shake with solvent shaking sonicate/ultrasonic bath 038 Ultra Turrax 069 vortex mix 057 Extraction Type single multiple Extraction Time 1 h min h and 30 min 037 2min min min min min min 061 Page 39 of 48

40 Sample Work Up centrifuge defatted with hexane 037 dilute evaporate filter none 038 ph adjustment 062 Ultra Turrax 030 Sample Clean-up by Immunoaffinity Column (Brand) R-Biopharm Rhone VICAM gl-science 034 Romer Labs 062 Sample Clean-up by SPE Cation exchange # Multifunctional 044 showadenkou 043 VRA Mycotoxin Determination ELISA fluorometric 033 HPLC UPLC 037 UPLC-MSMS 069 Page 40 of 48

41 HPLC Pre Column Derivatisation none HPLC Injection Volume (μl) < < < < < < HPLC Column Packing C non-endcapped 024 HPLC Column Temperature ( C) ambient >ambient - < Mobile Phase Components acetate ethanoic acid (acetic acid) acetonitrile carbonate buffer 015 formic acid (methanoic acid) methanol water ammonium acetate 035 Ammonium formate 037 Page 41 of 48

42 Mobile Phase Flow Rate (ml/min) < < < < Post Column Mobile Phase Flow Rate (ml/min) < < HPLC Detector Type fluorescence MS-MS UV/Vis 058 Isothermal Column Temp? yes Source of Standards Dr Ehrenstorfer 046 R-Biopharm Rhone 060 Romer Labs Sigma/Aldrich Supelco 033 Wako Biopure 015 Trilogy Page 42 of 48

43 Ochratoxin A Is the Method Used Accredited? yes no What is Your Method Based On? International Standard National Standard Manufacturer/Kit Instructions/Technical Note In house method Sample Weight (g) 2 - < < < < Extraction Solvent Components acetonitrile ethanoic acid (acetic acid) methanol phosphate buffer water acetic acid, acetonitrile and water 057 Formic acid 004 Sodium bi carbonate 011 Sodium bicarbonate 006 Page 43 of 48

44 Extraction Procedure add Na2CO3 046 add NaCl blend / homogenise with solvent shake with solvent shaking sonicate/ultrasonic bath Ultra Turrax 030 vortex mix wash with solvent 037 Extraction Type single multiple Sample Work Up centrifuge defatted with hexane dilute evaporate filter none 038 ph adjustment Page 44 of 48

45 Sample Clean-up by Immunoaffinity Column (Brand) R-Biopharm Rhone VICAM Micotox 007 HORIBA 034 hua an mai ke 046 Neogen 024 Ochra star R 058 OTAClean - LCTech 050 Romer Labs 062 Sample Clean-up by SPE Cation exchange 035 Multifunctional 044 OchraKing 034 Mycotoxin Determination ELISA fluorometric 033 HPLC UPLC 037 HPLC Injection Volume (μl) < < < < < Page 45 of 48

46 HPLC Column Packing C non-endcapped 024 PFP 012 HPLC Column Temperature ( C) ambient >ambient - < Isocratic Mobile Phase? yes no (gradient) Mobile Phase Components acetate ethanoic acid (acetic acid) acetone 034 acetonitrile formic acid (methanoic acid) methanol water ammonium acetate 035 Ammonium formate 037 Mobile Phase Flow Rate (ml/min) < < < Page 46 of 48

47 Post Column Mobile Phase Flow Rate (ml/min) < HPLC Post Column Derivatisation none HPLC Detector Type fluorescence MS-MS Source of Standards Dr Ehrenstorfer 046 Fluka 050 R-Biopharm Rhone Romer Labs Sigma/Aldrich Supelco 033 Wako Fermentek 038 LGC 058 Trilogy 061 Page 47 of 48

48 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 [4, 5] 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 Jacob Darlow (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 48 of 48