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As Speciation in Apple Juice

As Speciation in Apple Juice. Charles Schneider, Presenter Kenneth Neubauer, Co-Author PerkinElmer.

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As Speciation in Apple Juice

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

  2. …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.

  3. 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

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

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

  6. 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

  7. 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

  8. 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

  9. 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

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

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

  12. Effect of Dilution Factor Black: Juice – 5x dilution Blue: Juice – 2x dilution As5 As3 DMA MMA • 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 • Benefit: Lower concentrations can be measured • How low is relevant?

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

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

  15. Effect of Injection Volume Black = 20 µL Blue = 40 µL 0.1 ppb Standard As5 As3 MMA DMA • 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

  16. Effect of Sampling Rate As5 2 points/sec As3 DMA MMA As5 4 points/sec As3 DMA MMA • 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?

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

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

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

  20. 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

  21. 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

  22. 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 2nd 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

  23. 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?

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

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