© 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 Tests

As5

As3

DMA

MMA

5 Consecutive Injections of Apple Juice Diluted 2x

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?

Other Matrices: Rice

Run by Alternate Separation MethodHow Would the New Method Work? Better separation of other As peaks in brown rice?

Thai Rice

As5

As3

DMA

Brown Rice

As5

As3

DMA

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