1. Zinc and Copper Analysis by Colourimetry By Carolyn Fahm TEQAS ~ Trace Elements External Quality Assessment Scheme EQ@ SEQ@ S: External Quality Assessment at Surrey EQ@ S University of Surrey Guildford

2. Why We Undertook this Study The majority of laboratories returning Zinc and/or Copper results to TEQAS analyse using Flame Atomic AbsorptionThe majority of laboratories returning Zinc and/or Copper results to TEQAS analyse using Flame Atomic Absorption But there are a growing number general chemistry laboratories measuring these analytes by colourimetry, since it does not require special equipment and can be easily integrated into the regular laboratory workload.But there are a growing number general chemistry laboratories measuring these analytes by colourimetry, since it does not require special equipment and can be easily integrated into the regular laboratory workload.

3. Why We Undertook this Study - 2 However over the course of time we observed that the number of times that colorimetric results had performance problems that exceeded those seen by labs running Zinc and Copper by FAA.However over the course of time we observed that the number of times that colorimetric results had performance problems that exceeded those seen by labs running Zinc and Copper by FAA. We decided to increase our understanding of what was going on by querying our Zinc and Copper participants about their current or past usage of colourimetric methods.We decided to increase our understanding of what was going on by querying our Zinc and Copper participants about their current or past usage of colourimetric methods.

4. Questions We asked each respondent to address: What is your current method? Have you ever used or evaluated a colourimetric method? If so ~ ~ Colourimetric Assay Questions Which Method? Method?Automated Or Manual? Type and Source of Calibrant Calibrant? TraceableStandard?EvaluationResults? ObservedPerformanceProblems?

5. Zinc Results 51 Labs Responded51 Labs Responded 19 Labs reported that they had evaluated colourimetry.19 Labs reported that they had evaluated colourimetry. Currently X labs report Zinc byCurrently X labs report Zinc by this method. this method. 9 used Wako 4 used Randox 1 used Boehringer Mannheim or Sentinel 2 used Audit

6. Detailed Zinc Information Analysers in use:Analysers in use: Deproteinisation used?Deproteinisation used? Aqueous or Serum Calibrant?Aqueous or Serum Calibrant? Olympus 600,640 Roche Modular, 917 Cobas Fara, Mira 14 6 14 yes; 6 no 163 16 Aqueous; 3 Serum

7. Detailed Zinc Information - 2 Source of Calibrant:Source of Calibrant: Is calibrant traceable to defined standard?Is calibrant traceable to defined standard? 14 14 from kit 3 3 Seronorm or other serum-based material 1 1 Merck standard solution 16 16 no or not known 4 4 yes

8. Zinc Comments “Precision and accuracy have improved since new analysers came into use in... We appear to over- estimate zinc by about 2  mol/L. Thus our accuracy looks worse at low Zinc levels…”“Precision and accuracy have improved since new analysers came into use in... We appear to over- estimate zinc by about 2  mol/L. Thus our accuracy looks worse at low Zinc levels…” “Few "flyers" when analyses moved from old to new analyser.” “Supplier of kit admitted that this lot of reagents we had been using was un- stable and replaced it.” “Occasional odd results in UK NEQAS Scheme.”

9. Copper Results 37 Labs Responded37 Labs Responded 6 Labs reported that they had used colourimetry, either currently or in the past6 Labs reported that they had used colourimetry, either currently or in the past Only 4 labs were currently using a colourimetric method.Only 4 labs were currently using a colourimetric method. 1 used Spinreact 1 used Roche 1used Randox 1 used Boehringer Mannheim

10. Detailed Copper Information Analysers in use:Analysers in use: Deproteinisation used?Deproteinisation used? Aqueous or Serum Calibrant?Aqueous or Serum Calibrant? Cobas Fara Roche Modular, 917 Cobas Fara, Mira 2 11 2 yes; 1 no; 1 only for plasma samples 31 3 Aqueous; 1 Serum

11. Detailed Copper Information - 2 Source of Calibrant:Source of Calibrant: Is calibrant traceable to defined standard?Is calibrant traceable to defined standard? 3 3 from kit 1 1 Serum-based reference material solution 2 2 no 2 2 did not state

12. Copper Comments “Haemolysed or lipemic samples give high results.”“Haemolysed or lipemic samples give high results.” “Occasional odd result in UK NEQAS Trace Elements Scheme.”“Occasional odd result in UK NEQAS Trace Elements Scheme.”

13. Zinc and Copper Precision Results Varies with Concentration ~ Several Labs gave two Values. Zinc Within Run Zinc Between Runs 0.4 – 5% Copper Within Run Copper Between Runs 5.2 – 7% 5.2 – 6.7%

14. The Next Step When this study was reported to the UK NEQAS Trace Elements Specialist Advisory Group, further analysis was suggested.When this study was reported to the UK NEQAS Trace Elements Specialist Advisory Group, further analysis was suggested. Since we have a database of TEQAS results going back for many years, a comparison of the TEQAS performance of colourimetric assays as opposed to other methods of analysis was undertaken.Since we have a database of TEQAS results going back for many years, a comparison of the TEQAS performance of colourimetric assays as opposed to other methods of analysis was undertaken.

15. Zinc Performance Profile Colourimetry typically yields higher reported concentrations across the concentration ranges.Colourimetry typically yields higher reported concentrations across the concentration ranges. However the statistics are not robust because of widely discrepant N’s (from 48 for FAA to 19 for colourimetry down to 4 for ICP-MS.) Hence the ALTM is most influenced by FAA and least influenced by ICP and Not Specified.However the statistics are not robust because of widely discrepant N’s (from 48 for FAA to 19 for colourimetry down to 4 for ICP-MS.) Hence the ALTM is most influenced by FAA and least influenced by ICP and Not Specified. CV’s (in increasing order) are: ICP, Flame AA, Colourimetry, and Not Specified.CV’s (in increasing order) are: ICP, Flame AA, Colourimetry, and Not Specified. Although there are just four in the ICP category (most precise), the least precise, not specified category has only 7!Although there are just four in the ICP category (most precise), the least precise, not specified category has only 7!

16. Zinc Concentration Chart

17. Zinc CV Chart

18. Zinc Concentration Chart - 2

19. Zinc CV Chart - 2

20. Zinc Concentration Chart -3

21. Zinc CV Chart - 3

22. Zinc Chart - 4

23. Zinc CV Chart - 4

24. Zinc Concentration Summary Slide: 21 Samples, over 7 Months

25. Zinc CV Summary Slide: 21 Samples, over 7 Months

26. Copper Performance Profile Colourimetry (for the two reporting participants) tended to be about the same (or slightly lower) than for other methods.Colourimetry (for the two reporting participants) tended to be about the same (or slightly lower) than for other methods. The CV’s were substantially lower – but again, with a mere two participants, few if any conclusions can be drawn.The CV’s were substantially lower – but again, with a mere two participants, few if any conclusions can be drawn. With the withdrawal of Roche’s copper reagents, it is unlikely that colourimetric copper analysis will play a significant role in the future.With the withdrawal of Roche’s copper reagents, it is unlikely that colourimetric copper analysis will play a significant role in the future.

27. Copper Concentration Chart

28. Copper CV Chart

29. Summary Zinc Conc. = Higher Common CV = Higher Copper Conc. =Same or Lower Extremely rare. CV = too few to assess

30. The entire study of TEQAS samples is available as an e-mail attachment.