Chromatography: Complete Inorganic Elemental Speciation Analysis Solutions for Food Safety

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

2
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
As
As
As
As
As
As

3
As
As
As
As
As
As
species in a sample

4
As
As
As
As
As
As
species in a sample
HO-As-OH
|
OH
O
||
H3C-As-CH3
|
OH
CH3
|
H3C-As+-CH2CH2OH
|
CH3

5
Different Types of Species
Redox Pairs
As (III), As (V)
Cr (III), Cr (VI)
Se (IV), Se (VI)
Alkylated Metals
Methylmercury
Butyl- and phenyl-tins
Tetraethyllead
Biomolecules
Metal containing proteins
Se-containing amino acids
Arseno-sugars & -lipids
O
HO OH
As
O
CH3
H3C
OH
H3C - Hg+
Se O
NH2
OH
[Cr(H2O)6]3+

6
Principles of Speciation Analysis
Sample containing
various compounds
Individual detection
of compounds
Separation

7
Sample containing
various compounds
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

8
Sample containing
various compounds
of compounds
Separation
Total element
sum of all species
↔
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

9
Sample containing
various compounds
of compounds
Separation
Total element
sum of all species
↔
sample preparation
-Transformation of
species
mechanisms
↔
analytes with same
instrumentation
Ion chromatography:
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

10
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:

11
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

12
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

13
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

14
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

15
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

16
Versatility of ICP-MS
Liquid Chromatography
System
Field Flow Fractionation
Gas Exchange Device Laser Ablation

17
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

18
Data
System
Inert tubing
0.125 mm i.d.
A Completely Integrated IC-ICP-MS System
Simple hardware
connection
Easy interchange
between standard
ICP-MS analysis and
IC-ICP-MS
Single control
software
One software
interface controls both
ICS and ICP-MS

19
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

20
Qtegra ISDS LabBooks with Chromeleon CDS Drivers
Qtegra ISDS
workflow
Chromeleon
CDS workflow
ICS module
parameters

21
Sample containing
various compounds
of compoundsSeparation
Total element
sum of all species
↔
sample preparation
-Transformation of
species
mechanisms
↔
analytes with same
instrumentation
Ion chromatography:
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

22
Analytical Workflow for Speciation Analysis
Total Elemental
Determination
Acid Microwave
Digestion
[M] – Total Analyte
Concentration
Elemental Species
Determination
[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

23
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

24
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/ucm271
630.html
• http://www.fda.gove/Food/ResourcesForYou/Consumers/ucm27
1632.html

25
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/presscontent
19232.html

26
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

27
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

28
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

30
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

31
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

32
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

33
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%

34
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

35
What Information Can We Extract from Milk
• Samples:
• 1 cow milk
• 1 baby formula,
• 1 breast milk
• CRM BCR-063
Procedure Description
Total 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

36
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-063
Baby
Formula Cow milk Breast milk
Conc % Conc % Conc Conc
23Na 4.12 94 2.08 87 0.333 0.047
24Mg 1.14 91 0.46 92 0.102 0.03
31P 11.2 101 3.17 83 0.697 0.089
39K 16.9 96 6.21 1.42 0.531
44Ca 12.9 96 5.98 93 1.12 0.245
56Fe 2.35 101 72.5 88 0.322 0.215
65Cu 0.553 92 4.05 94 0.245 0.244
66Zn 40.3 82 58.2 90 3.36 0.857
127I 0.873 108 1.01 94 0.128 0.072
55Mn 221 466 82 17.1 2.26
75As 3.04 2.12 0.19 0.3
78Se 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

37
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 RFIC
KOH, 10 to 100 mM in 14 min
Flow rate 0.3 mL min-1
Injection volume 20 µL

38
Iodine Species in Powdered Milk and Baby Formula
Iodide only
determined in
extracts

39
Iodine Species in Cow and Breast Milk
Unknown Iodine
containing species
found in Cow and
Breast Milk

40
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 Species
BCR-063 1.007 1.007
Baby Formula 0.452 0.452
Cow Milk 0.131 0.002 0.018 0.151
Breast Milk 0.043 0.004 0.047

41
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

42
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

43
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

44
IC-ICP-MS: MeHg and Hg (II) Calibration Curves

45
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

46
IC-ICP-MS: Hg Speciation of DORM-3 CRM

47
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

48
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)

49
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

50
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

51
Thank You!
WS71139_E 05/14S

Chromatography: Complete Inorganic Elemental Speciation Analysis Solutions for Food Safety

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Chromatography: Complete Inorganic Elemental Speciation Analysis Solutions for Food Safety