© 2009 Perkin Elmer
As Speciation in Apple Juice
Charles Schneider, PresenterKenneth Neubauer, Co-AuthorPerkinElmer
…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.
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
As5
As3
MMA DMA
AsB
Improving the Separation
1 ppb Mixed As Standard
Faster Separation, Better Resolution Reversed-Phase Ion Pairing Chromatography
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
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
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
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
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
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
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
MMADMA
AsB
ICP-MS in Standard Mode No Interferences
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 dilutionBlue: Juice – 2x dilution
As5
As3
MMADMA
Benefit: Lower concentrations can be measured How low is relevant?
As5As3
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
Stability
20 Injections over 100 Minutes of Apple Juice Diluted 2x
Good Stability Still need to evaluate longer-term stability
As5
As3
MMADMA
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 µLBlue = 40 µL
As5
As3
MMA DMA
0.1 ppb Standard
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
Calibration
MMAAs5As3 DMA
As5
As3
DMA
MMA
Calibration StandardsBlank, 0.1, 0.25, 0.5, 1.0 ppb
(prepared in diluent)
Initial Quantitation Results: Various Apple Juices
All Apple Juice Diluted 2x and Run Against Aqueous External Calibration Curve
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
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
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
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?
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