1 Proprietary & Confidential

The world leader in serving science

Mr. Kristan Bahten

Thermo Scientific, Sunnyvale, CA, USA

Dr. Shona McSheehy

Thermo Scientific, Bremen, Germany

Complete Inorganic Elemental Speciation Analysis Solutions for Food Safety

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What is Speciation Analysis?

• IUPAC Definition - the analytical activity of identifying and/or measuring the quantities of one or more individual chemical species in a sample

AsAs

As

As

AsAs

3

AsAs

As

As

AsAs

What is Speciation Analysis?

• IUPAC Definition - the analytical activity of identifying and/or measuring the quantities of one or more individual chemical species in a sample

4

AsAs

As

As

AsAs

What is Speciation Analysis?

• IUPAC Definition - the analytical activity of identifying and/or measuring the quantities of one or more individual chemical species in a sample

HO-As-OH|

OHO||

H3C-As-CH3

| OH

CH3

| H3C-As+-CH2CH2OH | CH3

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Different Types of Species

Redox Pairs

As (III), As (V)Cr (III), Cr (VI)Se (IV), Se (VI)

Alkylated Metals

MethylmercuryButyl- and phenyl-tins

Tetraethyllead

Biomolecules

Metal containing proteinsSe-containing amino acids

Arseno-sugars & -lipids

O

HO OH

As

O

CH3

H3COH

H3C - Hg+

Se O

NH2

OH

[Cr(H2O)6]3+

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Principles of Speciation Analysis

Sample containing various compounds

Individual detection of compounds

Separation

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Principles of Speciation Analysis

Sample containing various compounds

Individual detection of compounds

Separation

Total element concentration is the sum of all species↔Preservation of original species distribution

- Loss of species during sample preparation

- Transformation of species

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Principles of Speciation Analysis

Sample containing various compounds

Individual detection of compounds

Separation

Total element concentration is the sum of all species↔Preservation of original species distribution

- Loss of species during sample preparation

- Transformation of species

Different separation mechanisms ↔ Ability to tackle several analytes with same instrumentation Ion chromatography:Analyte retention is achieved by interaction of charges with stationary phase

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Principles of Speciation Analysis

Sample containing various compounds

Individual detection of compounds

Separation

Total element concentration is the sum of all species↔Preservation of original species distribution

- Loss of species during sample preparation

- Transformation of species

Different separation mechanisms ↔ Ability to tackle several analytes with same instrumentation Ion chromatography:Analyte retention is achieved by interaction of charges with stationary phase

ICP-MS can only detect the element enclosed in a species↔Accurate and reliable quantification of different compounds containing the same element

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SEM Image of 4 µm Supermacroporous Bead

What are the Advantages of Ion Chromatography?

• Metal-free PEEK systems• Powerful separation chemistries• Reagent-Free™ Ion

Chromatography (RFIC™)• Extensive IC product line for full

flexibility

• Smaller particles provide better performance

• Fast run times with higher flow rates using 150 mm columns

• High resolution with standard flow rates using 250 mm columns

New High Efficiency Thermo Scientific™ Dionex™ IonPac™ 4 µm HPIC™ Columns

Benefits:

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Dionex Ion Chromatography System Portfolio

Thermo Scientific Thermo Scientific Dionex ICS-1100

Basic Integrated Ion Chromatography

System

Thermo Scientific Thermo Scientific

Dionex ICS-900 Starter Line Ion

Chromatography System

Thermo Scientific Thermo Scientific Dionex ICS-1600

Standard Integrated Ion

Chromatography System

Thermo Scientific™ Thermo Scientific Dionex ICS-2100 Reagent-Free Ion Chromatography

(RFIC) System

Thermo Scientific Dionex ICS-5000+ HPIC Ion Chromatography

System

Thermo Scientific

Thermo Scientific Dionex ICS-4000

Capillary HPIC Ion Chromatography

System

RFIC

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What are the Advantages of IC for IC-ICP-MS?

• Dionex IonPac AS7 column• High efficiency hydrophobic anion-exchange column for separation of a

wide range of polyvalent anions

• Used for arsenic and chromium speciation

• Mixed anion-cation exchange capacity

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What are the Advantages of IC for IC-ICP-MS?

• Dionex IonPac CS5A column• Mixed mode anion and cation-exchange columns to separate transition

and lanthanide metals

• Available in 2 mm and 4 mm formats

• Used for Mercury speciation

Dual layer with both cation and anion exchange sites

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What are the Advantages of IC for IC-ICP-MS

• Dionex IonPac AS19 and Dionex IonPac AS19-4µm columns• Anion-exchange column optimized for the

separation of polarizable anions using hydroxide eluents

• Large separation of iodate and iodide regardless of the presence of high chloride matrices

• Ideal for biological and food/beverage matrices which can contain high chloride levels

• Available in 2 mm, 4 mm and capillary formats and 4 µm particle size versions

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Why Use ICP-MS for Speciation Analysis?

• It can measure almost the whole periodic table in just about everything

• Analysis of:• Elemental concentrations

• High precision isotope ratio determinations

• Species information when coupled to separation devices

•QCell technology• sub ppt detection limits•>9 orders dynamic range

Thermo Scientific™ iCAP™ Q ICP-MS

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Versatility of ICP-MS

Liquid Chromatography System

Field Flow Fractionation

Gas Exchange Device Laser Ablation

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Speciation with IC-ICP-MS

• Fully integrated hardware and software system:• Thermo Scientific™ Qtegra™ Intelligent

Scientific Data Solution (ISDS) drives the whole system and includes:

• Thermo Scientific™ Dionex™ Chromeleon™ Chromatography Data System (CDS) plug-in drivers to control IC or HPLC systems

• Simple hardware connection

• Powerful separation chemistries • Wide selection of columns

• Specialized applications

• Reagent-Free Ion Chromatography (RFIC)

• Thermo Scientific Dionex Ion Chromatography Systems (ICS) are entirely metal-free (PEEK)

IC-ICP-MS is the ideal choice for trace elemental speciation

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Data System

Inert tubing 0.125 mm i.d.

A Completely Integrated IC-ICP-MS System

Simple hardware connectionüEasy interchange between standard ICP-MS analysis and IC-ICP-MS

Single control software üOne software interface controls both ICS and ICP-MS

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Qtegra ISDS Advanced Instrument Control

Manual control features for AS and pump

Navigation tabs for each ICS module

Manual control features for AS and pump

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Qtegra ISDS LabBooks with Chromeleon CDS Drivers

Qtegra ISDS workflow

Chromeleon CDS workflow

ICS module parameters

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Principles of Speciation Analysis

Sample containing various compounds

Individual detection of compoundsSeparation

Total element concentration is the sum of all species↔Preservation of original species distribution

- Loss of species during sample preparation

- Transformation of species

Different separation mechanisms ↔ Ability to tackle several analytes with same instrumentation Ion chromatography:Analyte retention is achieved by interaction of charges with stationary phase

ICP-MS can only detect the element enclosed in a species↔Accurate and reliable quantification of different compounds containing the same element

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Analytical Workflow for Speciation Analysis

Total Elemental Determination

Acid Microwave Digestion

[M] – Total Analyte Concentration

Elemental SpeciesDetermination

[M] – Total Analyte Concentration

Soft Extraction Techniques

[M1,2...n] – Species Concentration

Difference = amount of element/elemental species extracted

Difference = amount of elemental species eluting from column

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Why Perform Arsenic Speciation?

• Toxicity of As depends on the species• Inorganic As compounds: As (III) and

As (V): highly toxic

• Organic As compounds: Arsenobetaine (AsB) or Arsenosugars: not toxic

• Determination of total As cannot allow us to evaluate potential hazards correctly

• Long term exposure to small amounts may lead to serious health effects

• Children particularly affected due to their lower body weight• Juice consumption

• Toddler formula

• Regulation: none until now, but under FDA scrutiny

http://www.consumerreports.org/cro/2012/01/arsenic-in-your-juice/index.htm

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Why Arsenic in Apple Juice?

• Dr. Oz Show

• Dr. Mehmet Oz, a cardiothoracic surgeon and teaching professor at Columbia University

• Reports high As content in apple juice (up to 36 ppb; 10 ppb is the FDA’s drinking water limit)

www.doctoroz.com

• No species information, only total As

• Subsequent investigations by FDA and manufacturers unable to confirm the result

• Analysis by the FDA showed concentrations in the range of 2-6 ppb

• http://www.fda.gov/Food/ResourcesForYou/Consumers/ucm271630.html

• http://www.fda.gove/Food/ResourcesForYou/Consumers/ucm271632.html

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Potential Sources of Arsenic in Fruit Juices

• Arsenic containing pesticides were outlawed in the 1970s, but residues can still be found today

• Wood treatment: also prohibited• Elevated concentrations from ground water• Country of origin: import of juice concentrate or fruit from

other countries

Source: http://eltern.t-online.de/-oeko-test-entdeckt-spuren-von-arsen-in-babysaeften/id_52749916/index

Source: www.salud.univision.com

Source: www.star365.cn/presscontent19232.html

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Arsenic Speciation using Gradient Elution

Column Dionex IonPac AS7 column (2 x 250 mm)

Mobile Phase Gradient elution: 20 to 200 mM ammonium carbonate

Flow rate 0.3 mL min-1

Injection volume 20 µL

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IC-ICP-MS for As Speciation

• 6 species detected• ~7000 cps / ppb• ~15 minute analysis• Narrow peak shape improves S/N

ratio (10 to 20s)• Reproducibility < 4% • Reduced solvent consumption

without comprising separation efficiency

Single species calibration – As (III)

Single species calibration – DMA

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Arsenic Speciation Analysis of Apple Juice

• Low concentration of the individual species after dilution

• Method detection limits (MDL): 0.001 ng g-1 As (V) to 0.011 ng g-1 (MMA)

DMA [ng g-1]

As (III) [ng g-1]

MMA [ng g-1]

As (V) [ng g-1]

Sum [ng g-1]

Total As [ng g-1]

Juice 3 - 0.5 ± 0.01 - 0.8 ± 0.01 1.3 1.7 ± 0.05

Juice 4 0.4 ± 0.05 0.3 ± 0.01 0.1 ± 0.05 0.7 ± 0.01 1.5 1.8 ± 0.05

- Not detected

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Organic Brown Rice Syrup (OBRS)

• Rice is commonly known to possibly contain high amounts of As• Inorganic As accumulates in the outer layer removed when polishing the

rice

• Organic brown rice syrup is commonly used as sweetener for toddler formulas and cereals• Healthier alternative to fructose containing corn syrups (obesity)

• Marketing directed to the organic foods market for customers especially aware of healthy living

• Also used in cereal bars or energy products for endurance athletes may contain OBRS as a major ingredient

Jackson, B. P. et al., Environmental Health Perspective, doi 10.1289/ehp.1104619

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Sample Preparation

• Three different samples were sourced in Germany

• Total As determination: closed vessel microwave digestion1

• 0.5 g sample + 4 mL of 50% HNO3, 10 minutes @180°C

• Dilution to 20 mL total volume

• Further dilution prior to measurement

• Species extraction: open vessel extraction2

• 1.5 g sample + 15mL of 1% HNO3, 90 minutes under reflux

• Centrifugation + filtration

• Dilution to 20 mL total volume

• Further dilution prior to measurement

1: Jackson, B. P. et al., Environmental Health Perspective, doi 10.1289/ehp.1104619 2: Huang et al., J. Anal. At. Spectrom. 25 (2010), 800-802

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Results

• All samples contained ≥ 100 µg kg-1 As• Sample 1: 118 ± 7 µg kg-1

• Sample 2: 136 ± 7 µg kg-1

• Sample 3: 107 ± 11 µg kg-1

• Other studies indicated even higher As levels (between 80-400 µg kg-1)

• Speciation analysis was conducted for all samples

Jackson, B. P. et al., Environmental Health Perspective, doi 10.1289/ehp.1104619

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Arsenic Speciation Analysis of OBRS

• Analysis was carried out using similar conditions

• Most abundant species is As (III)• Most abundant organic As species is DMA

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Arsenic Species Distribution

• Extraction efficiency was determined to be 74-78%

• Species distribution similar to previously reported values (80-90% inorganic As)

• Method detection limits (MDL): 0.001 – 0.01 ng g-1

Species distribution % DMA As (III) As (V) Sum

Sample 1 12% 82% 6% 100%12% 82% 6% 100%12% 81% 7% 100%

Sample 2 12% 83% 5% 100%12% 83% 5% 100%12% 82% 6% 100%

Sample 3 10% 84% 6% 100%10% 87% 3% 100%11% 86% 3% 100%

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Iodine Speciation in Milk and Milk Products

• Iodine is an essential element for the adequate function of thyroid hormones

• Speciation of iodine in milk and milk products can tell us if:• Enough iodine is present – to

avoid iodine deficiency

• Whether the iodine is in a bio available form

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What Information Can We Extract from Milk

• Samples:• 1 cow milk

• 1 baby formula,

• 1 breast milk

• CRM BCR-063

Procedure DescriptionTotal Elemental Concentration

Add 5 mL conc. HNO3 and 2 mL H2O2 to 0.5 - 1 g material and microwave digest at 1600 W for a total of 50 mins in closed PTFE vessels. Make up to 50 mL with 18.2 MΩ water.

Total Iodine extraction

Add 50 mL 0.5% ammonia to 0.25 - 0.35 g powder material or approx. 1 mL milk and shake. Leave overnight, then shake for 2 hrs. Centrifuge and analyze the same day.

Speciation Powder samples were diluted in deionized water water according to the manufacturer’s protocol and then all samples were diluted 25 fold in the mobile phase just prior to injection.

Iodine likely to be lost when using ‘typical’ acidic extraction media

Use basic extraction media for Iodine

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Total Elemental Concentrations

• Large concentration range of analytes• Blue = g/kg

• White = mg/kg

• Pink = µg/kg

• Good agreement with CRM concentrations

• In general, analyte concentrations found in cow and breast milk agree with literature

BCR-063Baby

Formula Cow milk Breast milk Conc % Conc % Conc Conc23Na 4.12 94 2.08 87 0.333 0.04724Mg 1.14 91 0.46 92 0.102 0.0331P 11.2 101 3.17 83 0.697 0.08939K 16.9 96 6.21 1.42 0.53144Ca 12.9 96 5.98 93 1.12 0.24556Fe 2.35 101 72.5 88 0.322 0.21565Cu 0.553 92 4.05 94 0.245 0.24466Zn 40.3 82 58.2 90 3.36 0.857127I 0.873 108 1.01 94 0.128 0.072

55Mn 221 466 82 17.1 2.2675As 3.04 2.12 0.19 0.378Se 107 79.1 66 13.6 9.26

111Cd 0.95 0.56 ND 0.07

120Sn 2.36 0.24 ND ND

202Hg 0.60 0.20 0.10 0.17

208Pb 20.7 112 3.75 0.88 0.59

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Iodine Speciation using IC-ICP-MS

Column Dionex IonPac AG19 column (2 x 50 mm) + Dionex IonPac AS19 column (2 x 250 mm)

Elution Gradient elution with RFICKOH, 10 to 100 mM in 14 min

Flow rate 0.3 mL min-1

Injection volume 20 µL

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Iodine Species in Powdered Milk and Baby Formula

Iodide only determined in extracts

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Iodine Species in Cow and Breast Milk

Unknown Iodine containing species found in Cow and Breast Milk

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Total Iodine vs Iodine Speciation in Milk

• Separation of iodate and iodide in 12 minutes on a Dionex IonPac AS19 anion-exchange column

• Two unknowns in cow milk and one unknown in breast milk that were not present in the baby formula or the reference material

• Iodine species not quantitatively extracted when compared to total iodine present

All conc. in µg/g Iodide Unknown 1 Unknown 2 Sum SpeciesBCR-063 1.007 1.007Baby Formula 0.452 0.452Cow Milk 0.131 0.002 0.018 0.151Breast Milk 0.043 0.004 0.047

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Mercury Speciation in Fish

• Why?• Hg (II) undergoes biomethylation to Methylmercury (MeHg)

• Species data required since Methylmercury is highly mobile, reactive and extremely toxic

• Who is at risk?• Pregnant, nursing women and young children due to the high sensitivity

of fetus and child brain in development • Provisional Tolerable Weekly Intakes

- 0.7 µg/kg body weight/week (NRC, 2000)- 1.6 µg/kg body weight/week (JECFA, 2003)

• Principal pathway for MeHg exposure is seafood consumption

• EPA/FDA & EFSA all have recommended consumption limits for certain fish/seafood

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Mercury Speciation: MeHg and Hg (II)

• Species have different positive charges:• MeHg: +1

• Hg (II): +2• Cationic exchange

• Both species are separated as cysteine complexes ([Hg(Cys)2]2+ and [MeHgCys]+) to reduce memory effects

• Short analysis time required to provide:• Fast turnaround to provide a rapid response

• High, automated sample throughput when processing large numbers of samples

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IC-ICP-MS: Instrumental Parameters for Hg Speciation• IC Parameters:

• Cation-exchange Dionex IonPac CS5A column

• Mobile Phase: 10mM NaClO4,10mM acetic acid, 10 mM cysteine

• Flow rate: 0.5 mL/min

• Injection volume: 20 µL

• ICP-MS Parameters:• Nebulizer gas: 1.01 L/min

• Mode: STD (no KED)

• Isotopes: 199Hg, 200Hg and 202Hg

• Dwell time: 100 ms

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IC-ICP-MS: MeHg and Hg (II) Calibration Curves

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IC-ICP-MS: Analysis of Certified Reference Material

• National Research Council of Canada, DORM-3 Fish protein• Total Hg: 0.382 mg kg-1

• MeHg: 0.355 ± 0.056 mg kg-1

• Extraction procedure1:• Approx. 0.1 g extracted with 2 g of 5 mol

L-1 HCl

• Ultrasonic bath for 30 minutes

• Centrifugation to separate solid material

• Neutralization with NaOH

• Filtration with 0.44 µm syringe filters

Vallant et al., J. Anal. At. Spectrom. 22 (2007), 322-325

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IC-ICP-MS: Hg Speciation of DORM-3 CRM

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IC-ICP-MS as a Tool for Speciation

• IC-ICP-MS is a fully integrated technique• One software, no triggering, simple hardware connection

• Large range of IC systems and columns• IC tailored for your application

• Flexibility and support for developing applications

• ICP-MS offers sensitive and interference free detection• Routine multi-elemental determination in almost any matrix

• Accurate and robust determination of species

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IC-ICP-MS as a Tool for Speciation

• The completely metal-free Dionex ICS product line eradicates possibility of contamination

• Dionex IC systems can run various chromatography types• The Dionex IC systems are not limited to running only ion

chromatography

• High-pressure systems can run HPLC and now HPIC

• For many elemental speciation applications, IC is the preferred chromatographic choice: • Mobile phase causes less interferences (lower background)

• Mobile phases are simpler and cleaner (lower background)

• Narrower bore columns (2 mm ID)• Give slimmer peak shapes (better S/N and thus lower LOD)

• Use a lower mobile phase consumption (less expensive, less waste)

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Elemental Speciation

• Speciation is giving a clearer picture of what is in our samples:• Chromium VI in drinking waters – what level is safe?

• Arsenic species in fruit juices and OBRS are principally inorganic – will legislation adapt to meet the needs for controlling these species in foods?

• Existing solutions for speciation applications• Conforming to legislation

• Flexibility to adapt to upcoming legislation

• Product quality and consumer safety

• Risk assessments

• How metals cycle in the environment

Speciation is needed for a healthier, safer, cleaner environment

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Helpful Resources

• www.thermoscientific.com/dramatic - iCAP Q Resource Center• www.thermoscientific.com/speciation - Speciation Resource Center• www.thermoscientific.com/HPIC - High-Pressure Ion Chromatography

For more information on speciation analysis, please visit EVISA’s website at: http://www.speciation.net

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Thank You!

WS71139_E 05/14S