Speciation analysis of trace elements– recent and future trends in food analysis

Jens J. SlothRikke V. Hedegaard, Kåre Julshamn and Erik H. Larsen

National Food Institute (DTU Food)Technical University of Denmark

Speciation - definition(IUPAC definition: Templeton et al., Pure and Appl. Chem., 2000, 72, 143)

Chemical species:specific form of an element defined as to isotopic composition, electronic or oxidation state, and/or complex or molecular structure.

Speciation of an element:distribution of an element amongst defined chemical species in a system.

Speciation analysis:analytical activities of identifying and/or measuring the quantities of one or more individual chemical species in a sample.

Speciation – some historical milestones

”Fish arsenic” (Chapman 1926)”…a different (and non-toxic) compound than As2O3”

Minamata, Japan (1950’ies)MeHg+ emissions from industry -> fish -> man

Archachon Bay, France (1980’ies)TBT from antifouling agents -> oysters

EU Tematic network ”speciation 21” (1997-2000)Participants from research, industry og authorities

EVISA (European Virtual Institute for Speciation Analysis)(2003)Information and technology transfer in the framework of speciation

As

www.speciation.net

See more info at the EVISA homepage: www.speciation.net

Selected examples

As - Arsenic Se - Selenium Sn - Organotin

HPLC (GC) ICPMS

Column

Chromatogram

The work-horse of speciation analysisHPLC-ICPMS

Arsenic compounds in the marine environment

As

O

OHOH OH

OH OH

OH

As As

O

OHOH

CH3As

O

CH3

OHCH3

As

O

CH3

CH3CH3

CH3

CH3

CH3CH3

As

CH3

CH3

CH2

CH3As CH2OH

CH3

CH3

CH2

CH3As COO As

O

CH3

CH2

CH3CH2OH

As

O

CH3

CH2

CH3CH2OH

+ +

+ -

As(V) As(III) MA DMA

TMAO TETRA AC

AB DMAA DMAE

O

CH3

CH2

CH3As COOH

TMAP

O OP

O OH

OOH

OH OH

O OSO3H

OH

COOH

NH

COOH

O

R

Dimethyl As-sugar 2

Dimethyl As-sugar 4

(7)

(8)

O OH

OHO O

PO OH

OOH

OH OH

O SO3H

OH

O OSO3H

OH

O RAs

OH OH

O

CH3

CH3

OMe

OAs

OH OH

CH3

CH3

CH3

R

H COOH

NH

COOH

O

AB2

Dimethylarsinoylriboside

Trimethylarsonioriboside

Dimethyl As-sugar 1 Dimethyl As-sugar 2

Dimethyl As-sugar 3 Dimethyl As-sugar 4

+

R = 1,2,3,4,5

(5) (6) (7)

(8)

More than 50 different arsenic species have been found in the marine environment –incl. lipid-soluble As compounds (arsenolipids).

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

55000

60000

65000

Time-->

( )

Sig

nal i

nten

sity

/ cps

- seven compounds identified by coelution with available standards- 16 non-identified peaks

Anions

DMA

DMAA

AB

AC

TETRATMAO TMAP

??

?

? ? ???

??

? ?? ?

Cation-exchange with gradient elution – extraction with aqueous methanolColumn: Chrompack Ionospher 5C; Mobile phase: Pyridine; pH = 2.7

Speciation analysis of arsenic of scallop kidney

Sloth et al, J.Anal.At.Spectrom., 2003, 18, 452-459

Food safety and arsenic – toxicity

WHO (1988) PTWI for inorganic arsenic: 15 µg/kg bw/week(Provisional Tolerable Weekly Intake)For a 70 kg person => 150 µg / day

Long term exposure => skin diseases• Keratosis, gangrene, melatosis• Skin cancer… and also• lung, kidney, liver, bladder cancers

Cancer slope factor: 1.5 (mg kg-1 day-1)-1 (for inorganic As) (US EPA 2005)

Focus on inorganic arsenic; As(III) and As(V)

No longer appropriate

EFSA opinion on arsenic in food (issued 23/10-09)

• DATA: >100.000 occurrence data; 98% on total arsenic• Conclusion: PTWI value not appropriate any more• NEW!: BMDL01 = 0.3 – 8 µg/kg bw per day for inorganic arsenic• => EU dietary exposures within this range• => Risk to some consumers cannot be excluded

• “…there is a need to produce speciation data for different food commodities to support dietary exposure assessment…”

• “There is a need for robust validated analytical methods for inorganic arsenic in a range of food items”

Analysis of inorganic arsenic in seafood samples

Subsample + oxidising extractant

Microwave treatment 20 min, 90°C

Determination of total inorganic arsenicas As(V) by anion-exchange HPLC-ICP-MS

I: Solubilisation of sample matrixII: Conversion of As(III) to As(V)

No conversion of other arsenic compounds to inorganic arsenic

Microwave assisted extraction

Sample identification Inorganic arsenic Total arsenic

Salmon (Salmo salar) < 0.0006 1.9 0.2

Cod (Gadus morhua) < 0.0006 17 2

Cod (Gadus morhua) < 0.0006 15 2

Wolffish (Anarhichas lupus) < 0.0006 4.1 0.5

Wolffish (Anarhichas lupus) < 0.0006 31 4

Anglerfish (Lophius piscatorius) < 0.0006 15 2

Anglerfish (Lophius piscatorius) < 0.0006 44 6

Atlantic halibut (Hippoglossus hippoglossus) < 0.0006 12 1

Mackerel (Scomber scombrus) < 0.0006 1.7 0.2

Mackerel (Scomber scombrus) < 0.0006 2.8 0.4

Herring (Clupea harengus) < 0.0006 1.5 0.2

Herring (Clupea harengus) < 0.0006 1.7 0.2

Herring (Clupea harengus) < 0.0006 1.7 0.2

Tuna fish (Thunnus alalunga) 0.008 0.001 0.9 0.1

Lobster, tail meat (Homarus gammarus) < 0.0006 14 2

Lobster, head and thorax meat (Homarus gammarus) 0.037 0.005 22 3

Crab, white meat (Cancer pagurus) 0.016 0.002 32 4

Crab, head and thorax meat (Cancer pagurus) 0.060 0.009 26 3

King crab, white meat (Paralithodes camschaticus) 0.005 0.001 26 3

Norway lobster (Nephrops norvegicus) 0.020 0.003 21 3

Shrimp (Pandalus borealis) < 0.0006 3.8 0.5

Shrimp (Pandalus borealis) < 0.0006 60 8

Shrimp (Pandalus borealis) < 0.0006 67 8

Horse mussel (Modilous modiolus) 0.0012 0.002 3.4 0.4

Scallop muscle (Pecten maximus) 0.008 0.001 3.1 0.3

Oyster (Ostrea edulis) 0.014 0.002 1.8 0.2

Mink whale (Balaenoptera Acutorostrata) < 0.0006 0.61 0.08

Harp seal (Pagophilus groenlandicus) < 0.0006 0.9 0.1

Hooded seal (Cystophora cristata) < 0.0006 0.22 0.03

Fish muscle

Marine mammals

Crustaceans& bivalves

Sloth et al., J.Agric.Food Chem., 2005, 53, 6011-6018

Inorganic arsenic in marine samples – in general

y = 0.51x - 1.51R2 = 0.9146

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14 16

Total As / mg kg-1

Inor

gani

c A

s / m

g kg

-1

Sloth and Julshamn , 2008, J. Agri.Food Chem., 56, 1269-1273

Data from 175 blue mussel (Mytilus edulis) samples collected along the Norwegian Coastline.

NORWAY

Total As = 13.8 mg/kgInorg As = 5.8 mg/kgFraction = 42 %

...but unusual high contents in some samples...

Arsenic in rice – an emerging health issue?

• 17 samples from supermarkets in Aberdeen• Total arsenic levels: 0.12 – 0.47 mg/kg• Inorganic arsenic: 0.06 – 0.16 mg/kg (33 – 69 % of tAs)• 35% above Chinese max level of 0.15 mg/kg iAs

• 19 rice milk samples from supermarkets• EU drinking water ML: 10 µg/L total As (100% of samples exceeded)• US drinking water ML: 10 µg/L iAs (80% of samples exceeded)

Present initiatives for standardised methods

• CEN (European Standardisation Organisation)- TC275 WG10 Foodstuffs (Elements and their species)

- method for inorganic arsenic in rice and seafood- method for methylmercury in seafood

• CONffIDENCE project- Focus on inorganic arsenic and methylmercury- Samples of marine origin

- Cytosensor methods (TUT, Finland)- SPE-HG-AAS and HPLC-ICPMS (DTU Food)

More about CONffIDENCE Friday L43 at 12:00 by Jacob de Jong

SPE-AAS – a speciation alternative…

µ-waveextraction

Separation by SPE

Detection by HG-AAS

Sequential elution for selective off-line separation of inorg As from organo As species by SPE

Selenium in commercial food supplements

Se speciation by HPLC-ICPMS

Se(IV)

Se(VI)Organic Se

Sig

nal I

nten

sity

/ co

unts

s-1

Time / sec

Supplement declared as organic bound Selenium (125 µg/tablet)

Larsen, unpublished data.

Larsen et al, Anal.Bioanal.Chem., 2006, 385, 1098

Se speciation in garlic by HPLC-ICPMS

+ Structural IDby ESI MS/MS

γ-Glu-MeSeCys – a proposed cancerpreventive Se compound

Hoch, A

ppl. Geochem

., 2001, 719-43

Seafood

Household commodities

Agriculture Antifoulings Industry

Sediment

Water

Seafood

Tap water pipes

Food, beverage

PVC-Materials

- Agriculture- Antifoulings- Industry

Used in

- PVC-Materials

TDI: 0.25 µg/kg bw/day∑TBT, DBT, TPhT and DOT

Legislation on OTCs in Food Contact Materials

Compounds Maximum level (µg Sn/kg foodstuff)

DBT, TBT, TPhT and DOT 40 (6)

MMT, DMT 180

MOT 1200

MDDT 12000 (50)

DDDT 24000 (50)

∑∑

- Max levels on organotin migrating from the packaging material- Testing by the use of food simulators (water, acid, oil, alcohol etc)- BUT no maximum levels on organotins in the foodstuff itself!!

Ref: EFSA (2005); proposed EFSA values in parenthesis

FCM → Foodstuff

migration→

Assumptions:

- 1 kg food per 6 dm2

- 100 mL in contact with 0.6 dm2

Organotin migration from Food Contact Materials II

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.000

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

8000

8500

Time-->

Abundance

TIC: 014SMPL.D

4.07

5.77

6.46

IS

Inorg SnDBT

6.306.326.346.366.386.406.426.446.466.486.506.526.546.566.586.600

500

1000

1500

2000

2500

3000

3500

4000

Time-->

Abundance

TIC: 005SMPL.DTIC: 014SMPL.D

DBT

DBT concentration: 9.9 µg/kgOverlaid standard and sample

> EFSA guideline value of 6 µg/kg

Output of DK survey:-33 samples-11 contained OT (mainly DBT)-3 exceeded EFSA guideline limit

Small scale survey on 33 FCMsBaking paper, PVC cling films, silicone baking forms, lids with PVC gasketsPUR-agglomerated cork wine stoppers

•PVC lid•3% acetic acid

Sloth et al, in prep

Speciation summary

speciation methods are more and more commonly used instrumentation is widely available legislation on species has started…and more is expected in the future!

standardised methods are not ready!…but the need is known by authorities legislation is still behind! Lack of CRMs (e.g. for iAs)

Acknowledgements + invitation

Coworkers:Rikke V. Hedegaard, Xenia T. Trier and Erik H. Larsen

Kåre Julshamn, Heidi Amlund, Siri Bargaard and A.K. Lundebye

Matti Karp and Anne Rantala

5th Nordic Conferenceon

Plasma SpectrochemistryJune 6-9, 2010, Loen, Norway

INVITATION