1. © 2009 Perkin Elmer As Speciation in Apple Juice Charles Schneider, Presenter Kenneth Neubauer, Co-Author PerkinElmer

3. … we explain that some scientific studies have shown that two forms of organic arsenic found in apple juice could also be harmful, and for that reason, the FDA counts these two forms of organic arsenic in with the overall content for inorganic arsenic.

5. Initial Goals Develop an Improved Method for As Speciation in Urine  As(III), As(V), MMA, DMA, AsB Increase the Speed of Analysis  Current methods require 10 minutes or longer  Some methods use of LC gradients  Shorter chromatograms but long sample-to-sample times  i.e. 8 minute chromatogram, but 20 minutes between samples Attain Baseline Resolution of All Species

6. As5 As3 MMA DMA AsB Improving the Separation 1 ppb Mixed As Standard Faster Separation, Better Resolution  Reversed-Phase Ion Pairing Chromatography

7. As5 As3 MMA DMA AsB Separation in Urine 2 Consecutive Injections of a Urine Sample Diluted 2x Urine Matrix Does Not Hinder the Separation  All 5 species are baseline resolved  Good Injection-to-Injection Stability

8. Other Matrices: Apple Juice Recent Media Focus on As in Apple Juice  What forms? Will the Newly-Developed Separation Work in Apple Juice?  Urine: High salt content  Juice: High sugar content FDA Method in Development for As Speciation in Fruit Juices

9. FDA Method Elemental Analysis Manuals: Section 4.10: HPLC-ICP-MS Determination of Four Arsenic Species in Fruit Juice  Version Draft 0.82 (August 2010)  Author: Sean D. Conklin Species:  As(III), As(V), MMA, DMA  Check for AsB interference Can this separation be improved?  Shorter time  Better resolution between peaks

10. Goals Focus on the Separation Chemistry of As Species in Apple Juice  Sample preparation  Chromatographic conditions  Affect of juice matrix  Variability among different apple juices Once the LC Conditions are Established, Focus on Quantitation  Optimal ICP-MS conditions  Calibration strategies Characterize Method

11. Following FDA LC Methodology Separation of 5 Species Slightly better separation and shorter chromatogram  Different version of ion-exchange column listed in the FDA Method

12. Following FDA LC Methodology Apple Juice – 5x Dilution Juice matrix does not affect retention times or separation/resolution of peaks Apple Juice - 5x Dilution + 5 ppb Spike Dilution in mobile phase

13. Applying Improved As in Urine LC Methodology Apple Juice +/- 1 ppb Spike Apple Juice Matrix Does Not Affect the Separation  No matrix-induced peak shift  Peak shape is unaffected  Dilution in mobile phase Black: Juice (5x dilution) Blue: Juice (5x) + 1ppb spike As5 As3 MMA DMA AsB ICP-MS in Standard Mode  No Interferences

14. Effect of Dilution Factor Lower Dilution Factor Doesn’t Affect the Chromatography  No change in retention time or peak shape  But some dilution is necessary  Matrix match mobile phase and sample Black: Juice – 5x dilution Blue: Juice – 2x dilution As5 As3 MMA DMA Benefit: Lower concentrations can be measured  How low is relevant?

15. As5 As3 MMA DMA AsB Effect of Dilution Factor Apple Juice Diluted 2x +/- 1 ppb Spike Lower Dilution Factor Does Not Affect the Chromatography  No matrix-induced peak shift  Peak shape is unaffected  No interferences Black: Juice (2x dilution) Blue: Juice (2x) + 1ppb spike

16. Stability 20 Injections over 100 Minutes of Apple Juice Diluted 2x Good Stability  Still need to evaluate longer-term stability As5 As3 MMA DMA

17. Effect of Injection Volume Benefit: Ability to See Lower Levels  How low is relevant? Drawbacks of Larger Injection Volumes  Overload the column for samples with high concentrations  Possible increased sample matrix effects on the chromatography Black = 20 µL Blue = 40 µL As5 As3 MMA DMA 0.1 ppb Standard

18. 2 points/sec 4 points/sec Effect of Sampling Rate Higher Sampling Rate Gives Noisier Peaks  More difficult to integrate low levels  Can’t sample too slow or else peak integration is less accurate  How low is relevant? As5 As3 MMA DMA As5 As3 MMA DMA

19. Calibration MMA As5 As3 DMA As5 As3 DMA MMA Calibration Standards Blank, 0.1, 0.25, 0.5, 1.0 ppb (prepared in diluent)

20. Initial Quantitation Tests As5 As3 DMA MMA 5 Consecutive Injections of Apple Juice Diluted 2x

21. Initial Quantitation Results: Various Apple Juices All Apple Juice Diluted 2x and Run Against Aqueous External Calibration Curve

22. Initial Quantitation Results: Various Apple Juices Comparison: Total As vs. Sum of Species Total As  Measured directly by ICP-MS (i.e. no LC) on a different instrument in another lab by a different chemist  Summed As Species  Sum of individual species from previous slide  MMA and DMA standards made to final concentration of total molecule  As is ≈ 54% of the total molecule  Summed species are adjusted

23. Summary – Initial Results Four As species present in apple juice can be separated and measured in 4 minutes Various aspects of the chromatography have been characterized  Effects of sample matrix, dilution factor, sampling rate, injection volume Method has been applied to several apple juice samples  Separation is not affected by the different juices tested Initial results show good stability  More stability tests need to be performed Accurate quantitative results are obtained using aqueous calibration standards  Sum of species agrees with total As measurements All measurements can be made at As75 in Standard mode  No interferences observed, so cell mode is not needed

24. Going Forward Initial results are encouraging and suggest the method will work Test Method Ruggedness  Day–to-day results  Run same samples over several days  Stability over long runs  8-12 hours  Variation from column-to-column  Repeat tests on a 2 nd column of the same type  Matrix Effects  Can this method be applied to other matrices? Once these criteria have been met, the details of the method will be published

25. Questions That Need Answering Other juices need testing? Which ones? What levels need to be measured?  How low is biologically relevant? How will the test be implemented?  Measure juices for total As and only those about a certain level subject to speciation analysis? Are there SRMS? What else needs to be shown for the method to gain acceptance?

26. Other Matrices: Rice Run by Alternate Separation Method How Would the New Method Work?  Better separation of other As peaks in brown rice? Thai Rice As5 As3 DMA Brown Rice As5 As3 DMA ? ? ?