Analysis of PAH’s in food - AOAC Europe Section1).pdf · PAH profile analysis in food Member of NOE ECNIS AOAC Europe Section, International workshop, November 6-7, 2006, Limassol,

Albrecht Seidel

Biochemical Institute for Environmental CarcinogensProf. Dr. Gernot Grimmer-Foundation

Lurup 4D-22927 Grosshansdorf

[email protected]

Analysis of PAH’s in food

Member Member of NOE ECNISof NOE ECNIS

AOAC Europe Section, International workshop, November 6-7, 2006,Limassol, Cyprus

International Workshop‘Foods to Dye for –Contaminants-sampling, analysis,

legal limits’

PAH profile analysis in food


AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus

• Introduction

• Sample preparation / clean up

• Instrumental analysis

• HPLC-UV, HPLC-FD

• GC-MS(SIM) mode

Ambient Air

smoking Food

PAH-Exposure


Work place


PAH in human diet


CerealsOils and fats

Vegetables, grilled meat, fruits, smoked fish, milk and milk products

5

5-17

3

3

United Kingdom

(Dennis et al. 1983)

Netherlands

(de Vos et al. 1995)

Italy

(Lodovici et al. 1995)

New Zealand

(Thompson et al. 1996)

Dietary intakeof PAH(µg/day)

Country


Priority Polycyclic Aromatic Hydrocarbons (PAH)according to US-EPA

Benzo[a]pyrene

Phenanthrene

Chrysene

Naphthalene Acenaphthene Acenaphthylene Fluorene

Benz[a]anthraceneAnthracene PyreneFluoranthene

Benzo[b]fluoranthene

Dibenz[a,h]anthracene

Benzo[k]fluoranthene

Benzo[ghi]peryleneIndeno[1,2,3-cd]pyrene


Priority PAH according to EFSA and JECFA (FAO/WHO) to be includedin monitoring programs - EU Recommendation 256/2005

Cyclopenta[cd]pyrene

Benzo[a]pyrene

Dibenzo[a,l]pyrene

CH3

5-Methylchrysen

Dibenzo[a,i]pyrene

Chrysene

Dibenzo[a,h]pyrene

Dibenzo[a,e]pyrene

Benz[a]anthracene



Benzo[k]fluoranthene Benzo[j]fluoranthene

Benzo[ghi]peryleneIndeno[1,2,3-cd]pyrene

Benzo[c]fluorene



-

-

10

10

10

1

0,1

0,1

TEF

Benzo[c]fluorene

5-Methylchrysene

Dibenzo[a,l]pyrene

Dibenzo[a,h]pyrene

Dibenzo[a,i]pyrene

Dibenzo[a,e]pyrene

Benzo[j]fluoranthene

Cyclopenta[cd]pyrene

PAH - EFSA

Naphthaline

Acenaphthylene

Acenaphthene

Fluorene

Phenanthrene

Pyrene

Fluoranthene

Anthracene

PAH - EPA

0,1

1

0,1

0,1

0,01

0,01

0,1

1

Benz[a]anthracene

Benzo[a]pyren


Benzo[k]fluoranthene

Benzo[ghi]perylene

Chrysene

Indeno[1,2,3-cd]pyrene


0,001

0,001

0,001

0,001

0,001

0,001

0,001

0,01

TEFPAH - EPA/EFSATEF

Toxic Equivalence Factors (TEF) for PAH

a Collins, J.F. et al. (1998) Regul Toxicol Pharmacol, 28, 45-54; b Jacob, J. and Greim, H. (Ed‘s) Polycyclischearomatische Kohlenwasserstoffe (PAH) Forschungsberichte (DFG), Wiley-VCH, Weinheim (2004).


PAH in foodstuffs - EU Regulation 208/2005(Apply 1st April 2005)


1.0Dietary food for special medical purposesintended specifically for infants

7.1.9

1.0Infant milk and follow-on milk7.1.8

1.0Baby foods for infants and young children -processed cereal based food

7.1.710.0Bivalve molluscs7.1.6

5.0Crustaceans, cephalopods, other thansmoked

7.1.52.0Meat of fish, other than smoked fish7.1.45.0Smoked fish and smoked fish products7.1.35.0Smoked meat and smoked meat products7.1.2

2.0Oils and fats (excluding cocoa butter)7.1.1Benzo[a]pyrene (lead compound)7.1

Maximum levels(µg/kg fresh weight)


Sample dissolved in cyclohexaneor

Saponification with CH3OH/KOH +extraction with cyclohexane

Liquid-liquid partition ofcyclohexane extract with

DMF/H2O (9:1)

Liquid-liquid partition of DMFphase with H2O / cyclohexane

Cyclohexane phase washwith H2O

Concentration of cyclohexanephase

Concentration of cyclohexane phase andsolvent change to toluene

Silica gel,6% H2O

cyclohexane

GC / MSD


Extraction of the combined DMFphases with cyclohexane

Sample clean up - fats and oils(Grimmer & Böhnke (1975) Journal of the AOAC, 58, 725-733)


Sample clean up II


Caffeine complexation

Reextraction

GPC

Accelerated Solvent Extraction (ASE)

Gel PermeationChromatography (GPC)

Column Chromatographywith Silicagel

SPE with CN/SiOH

GC/MS

smoked meat, liquid smoke

GC/MS

beta-carotene, tocopherols,

fats, oil Sample, drying Material, ISn-hexane, 100°C100 bar

Bio-Bead S-X3,5 mL/minCyclohexane/Ethylacetate (50:50, v/v)

15% water,Elution with n-hexane/DCM (80:20, v/v)

Elution withACN/toluene (75:25, v/v)

EUROFINS

Jira (2004) Eur Food Res Technol,218, 208-212

2g sample dissolved, ISin 20mL cyclohexane,2 x 10 mL coffeine/formic acid

10% NaCl,cyclohexane

Automated GPC system(TACSTM),Ethylacetate/cyclohexane(1:1, v/v)50 g Bio-Beads S-X3



PAH profile (EFSA) analysis by HPLC

• Separation, identification, and quantification

Different columns in use:

Vydac 201 TP, Bakerbond PAH-16 plus, Nucleosil 5 C18 PAH, Cosmosil 5u PYE

etc.


PAH profile (EFSA) analysis by HPLCColumn: Knaur Eurospher-100 C18


1 Benzo[c]fluorene2 Cyclopenta[cd]pyrene3 Benz[a]anthracene4 Chrysene5 5-Methylchrysene6 Benzo[j]fluoranthene7 Benzo[b]fluoranthene8 Benzo[k]fluoranthene9 Benzo[a]pyrene10 Dibenzo[a,l]pyrene11 Dibenz[a,h]anthracene12 Benzo[ghi]perylene13 Indeno[1,2,3-cd]pyrene14 Dibenzo[a,e]pyrene15 Dibenzo[a,i]pyrene16 Dibenzo[a,h]pyrene

Column: KnaurEurospher-100 C18

100050

100030

80202

80200

CH3CNH2Ot(min.)

Flow: 1,0 ml/min.Detection: DAD 254 nm

1

2+3+4

5

6

7

8

9

11

13

14

10 12

15 16


PAH profile (EFSA) analysis by HPLC Column: Nacalai Tesque Cosmosil 5u PYE



Column: Nacalai TesqueCosmosil 5u PYE

1

2+3

4

5+6

8

79

11

13

12

1014

1615

100050

100030

60402

60400

CH3CNH2Ot(min.)



PAH profile (EFSA) analysis by HPLC Column: GRACE VYDAC 201TP5415


Column: GRACEVYDAC 201TP5415


100055

100020

70302

70300

CH3CNH2Ot(min.)


12

3

4 5

6

7

8

9

10

11

12

13

14

15 16


PAH Profile (EFSA) Analysis in Liquid Smoke using HPLC with UV and Fluorescence Detection

(Pinacle II RP column, Restek)


Simon et al., 2006IRMM,unpublished

UV at 375 nm

FluorescenceLambdaex = 270 nm

Lambdaem =370 nm420 nm470 nm500 nm

ACN/H2O gradient


Detection of Cyclopenta[cd]pyrene with UV-DAD within a PAH profile analysis using HPLC


CPP at 375 nm

Simon et al., 2006IRMM,unpublished


PAH profile (EFSA) analysis by GC-MS


• Isotope dilution methodology - addition of internal standards 2H-, 13C-labelled PAH

Separation, identification, and quantification

• GC capillary: FactorFour VF-5ms (30m x 0.25 mm x 0.25 µm) VF-17ms (60m x 0.25 mm x 0.25 µm) (Varian) DB5, (95% Dimethyl-5% Diphenyl-Polysiloxan) (J&W) etc.

recent review: D-L. Poster et al. (2006) Analysis of polycyclic aromatic hydrocarbons (PAHs) in environmental samples: a critical review of gas chromatography (GC) methods. Anal. Bioanal. Chem. 386, 859-881.


Separation of EFSA PAH by GC-MS(SIM)


Chrysene / Triphenylene - separation differs by type of capillary

Benzo[b]fluorantheneBenzo[j]fluoranthene - separation differs by type of capillaryBenzo[k]fluoranthene

5-Methylchrysene - (mono CH3-Chrysenes = 6 Isomers)

Benzo[c]fluorene - separation differs by type of capillary (B[a]F, B[b]F, B[c]F)

Dibenzo[a,l]pyrene (LOQ = 0.1 ppb; mw 302 specificity??)Dibenzo[a,e]pyrene 20 identified isomers with mw 302;Dibenzo[a,i]pyrene naphthopyrenes,Dibenzo[a,h]pyrene naphthofluoranthenes, dibenzofluoranthenes



Analysis of EFSA PAH in smoked pork belly by GC-MS(SIM) - Isotope dilution methodology I

(Jira et al., BFEL personal communication)

VF-17ms



Analysis of EFSA PAH in smoked pork belly by GC-MS(SIM) - Isotope dilution methodology II


VF-17ms



Analysis of EFSA PAH in smoked pork belly by GC-MS(SIM) - Isotope dilution methodology IV


VF-17ms



Analysis of EFSA PAH in smoked pork belly by GC-MS(SIM) - Isotope dilution methodology V


VF-17ms



Analysis of EFSA PAH in smoked pork belly by GC-MS(SIM) - Isotope dilution methodology VI


VF-17ms


Analysis of EFSA PAH in smoked pork belly by GC-MS(SIM) - Isotope dilution methodology VII



VF-17ms


PAH profile (EFSA) analysis in Cacoa Butterby GC-MS(SIM) - Isotope dilution methodology I


25.00 26.00 27.00 28.00 29.00 30.00 31.00 32.00 33.00 34.000

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Time-->

Abundance

Ion 216.00 (215.70 to 216.70): KBU0602.DIon 226.00 (225.70 to 226.70): KBU0602.DIon 228.00 (227.70 to 228.70): KBU0602.DIon 242.00 (241.70 to 242.70): KBU0602.D

BcF

BaA

CYC

CHR

TRI

5-CH3-CHR


PAH profile (EFSA) analysis in Cacoa Butterby GC-MS(SIM) - Isotope dilution methodology II


35.50 36.00 36.50 37.00 37.50 38.00 38.50 39.00 39.50 40.00 40.50 41.000

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

Time-->

Abundance

Ion 252.00 (251.70 to 252.70): KBU0602.D

BbFBkF

BjFBeP BaP

PER

Kakaobutter


PAH profile (EFSA) analysis in Cacoa Butterby GC-MS(SIM) - Isotope dilution methodology III


45.00 46.00 47.00 48.00 49.00 50.00 51.00 52.00 53.00 54.00 55.00 56.00

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

Time-->

Abundance

Ion 276.00 (275.70 to 276.70): KBU0602.DIon 278.00 (277.70 to 278.70): KBU0602.DIon 302.00 (301.70 to 302.70): KBU0602.D

INP

DBahA

BghiP

ANTHDBalP

DBaePDBaiP


HPLC-FD appears to be an acceptable method for determination of EFSAPAH profile provided that cyclopenta[cd]pyrene can be separated fromaccompanying PAHs to allow its UV-quantification under optimalconditions.

GC-MS(SIM) including isotope dilution methodology appears to be themethod of choice; resolution of benzofluoranthenes and the separation ofchrysene by the capillary is required.

Further validation of the two methods are needed for specific foodmatrices; the isomeric specificity of the two methods for analysis of5-methylchrysene and the dibenzopyrenes should be demonstrated.

In inter-laboratory trials native oils spiked with EFSA PAH arerecommended instead of material „blanked“ with active carbon and thanspiked with PAHs.

ConclusionsMember Member of NOE ECNISof NOE ECNIS


Thank you for

your attention

Analysis of PAH’s in food - AOAC Europe Section1).pdf · PAH profile analysis in food Member of NOE ECNIS AOAC Europe Section, International workshop, November 6-7, 2006, Limassol,

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