Analysis of PAH's in Food

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

legal limits’
Analysis of PAH’s in food
Albrecht Seidel
Biochemical Institute for Environmental Carcinogens
Prof. Dr. Gernot Grimmer-Foundation
Lurup 4
D-22927 Grosshansdorf
Member of NOE ECNIS Germany
albrecht.seidel@biu-grimmer.de
AOAC Europe Section, International workshop, November 6-7, 2006,

Limassol, Cyprus
PAH profile analysis in food
Member of NOE ECNIS
• Introduction
• Sample preparation / clean up
• Instrumental analysis
• HPLC-UV, HPLC-FD
• GC-MS(SIM) mode
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
PAH-Exposure
Member of NOE ECNIS
Work place Ambient Air
smoking Food
PAH in human diet
Member of NOE ECNIS
Dietary intake
Country of PAH
(µg/day) Oils and fats Cereals
United Kingdom 5
(Dennis et al. 1983)
Netherlands 5-17
(de Vos et al. 1995)
Italy 3
(Lodovici et al. 1995)
New Zealand 3
(Thompson et al. 1996) Vegetables, grilled meat, fruits,
smoked fish, milk and milk products
Priority Polycyclic Aromatic Hydrocarbons (PAH)
according to US-EPA
Naphthalene Acenaphthene Acenaphthylene Phenanthrene Fluorene
Anthracene Fluoranthene Pyrene Benz[a]anthracene
Chrysene Benzo[k]fluoranthene Benzo[b]fluoranthene Benzo[a]pyrene
Dibenz[a,h]anthracene Indeno[1,2,3-cd]pyrene Benzo[ghi]perylene
Priority PAH according to EFSA and JECFA (FAO/WHO) to be included
in monitoring programs - EU Recommendation 256/2005
CH3
Chrysene 5-Methylchrysen Benz[a]anthracene Cyclopenta[cd]pyrene
Benzo[b]fluoranthene Benzo[k]fluoranthene Benzo[j]fluoranthene Benzo[a]pyrene
Dibenz[a,h]anthracene Indeno[1,2,3-cd]pyrene Benzo[ghi]perylene Dibenzo[a,e]pyrene
Benzo[c]fluorene Dibenzo[a,h]pyrene Dibenzo[a,l]pyrene Dibenzo[a,i]pyrene
Toxic Equivalence Factors (TEF) for PAH
Member of NOE ECNIS
PAH - EPA TEF PAH - EPA/EFSA TEF PAH - EFSA TEF
Naphthaline 0,001 Benz[a]anthracene 0,1 Benzo[c]fluorene -
Acenaphthylene 0,001 Benzo[a]pyren 1 5-Methylchrysene -
Acenaphthene 0,001 Benzo[b]fluoranthene 0,1 Dibenzo[a,l]pyrene 10
Fluorene 0,001 Benzo[k]fluoranthene 0,1 Dibenzo[a,h]pyrene 10
Phenanthrene 0,001 Benzo[ghi]perylene 0,01 Dibenzo[a,i]pyrene 10
Pyrene 0,001 Chrysene 0,01 Dibenzo[a,e]pyrene 1
Fluoranthene 0,001 Indeno[1,2,3-cd]pyrene 0,1 Benzo[j]fluoranthene 0,1
Anthracene 0,01 Dibenz[a,h]anthracene 1 Cyclopenta[cd]pyrene 0,1
a
Collins, J.F. et al. (1998) Regul Toxicol Pharmacol, 28, 45-54; b Jacob, J. and Greim, H. (Ed‘s) Polycyclische
aromatische Kohlenwasserstoffe (PAH) Forschungsberichte (DFG), Wiley-VCH, Weinheim (2004).
PAH in foodstuffs - EU Regulation 208/2005
(Apply 1st April 2005)
Member of NOE ECNIS
Maximum levels
(µg/kg fresh weight)
7.1 Benzo[a]pyrene (lead compound)
7.1.1 Oils and fats (excluding cocoa butter) 2.0
7.1.2 Smoked meat and smoked meat products 5.0
7.1.3 Smoked fish and smoked fish products 5.0
7.1.4 Meat of fish, other than smoked fish 2.0
7.1.5 Crustaceans, cephalopods, other than 5.0
smoked
7.1.6 Bivalve molluscs 10.0
7.1.7 Baby foods for infants and young children - 1.0
processed cereal based food
7.1.8 Infant milk and follow-on milk 1.0
7.1.9 Dietary food for special medical purposes 1.0
intended specifically for infants
Sample clean up - fats and oils
(Grimmer & Böhnke (1975) Journal of the AOAC, 58, 725-733)
Member of NOE ECNIS
Sample dissolved in cyclohexane Liquid-liquid partition of

or cyclohexane extract with
Saponification with CH3OH/KOH + DMF/H2O (9:1)
extraction with cyclohexane
Liquid-liquid partition of DMF Extraction of the combined DMF

phase with H2O / cyclohexane phases with cyclohexane
Cyclohexane phase wash Concentration of cyclohexane

with H2O phase
GC / MSD
Silica gel,
6% H2O
cyclohexane
Concentration of cyclohexane phase and

solvent change to toluene
Sample clean up II
Member of NOE ECNIS
beta-carotene, tocopherols,
smoked meat, liquid smoke EUROFINS
fats, oil
Sample, drying
Accelerated Solvent Material, IS 2g sample dissolved, IS
n-hexane, 100°C Caffeine complexation in 20mL cyclohexane,
Extraction (ASE)
100 bar 2 x 10 mL coffeine/formic acid
Bio-Bead S-X3,
Gel Permeation 5 mL/min
Chromatography (GPC) Cyclohexane/
Ethylacetate (50:50, v/v) 10% NaCl,
Reextraction cyclohexane
Column Chromatography 15% water,
with Silicagel Elution with n-hexane/
DCM (80:20, v/v) Automated GPC system
(TACSTM),
SPE with CN/SiOH Elution with GPC Ethylacetate/cyclohexane
ACN/toluene (75:25, v/v) (1:1, v/v)
50 g Bio-Beads S-X3
GC/MS
GC/MS
Jira (2004) Eur Food Res Technol,
218, 208-212
PAH profile (EFSA) analysis by HPLC
Member of NOE ECNIS
• Separation, identification, and quantification
Different columns in use:
Vydac 201 TP,

Bakerbond PAH-16 plus,
Nucleosil 5 C18 PAH,
Cosmosil 5u PYE
etc.
Column: Knaur Eurospher-100 C18
Member of NOE ECNIS
2+3+4 1 Benzo[c]fluorene
t(min.) H2O CH3CN
2 Cyclopenta[cd]pyrene
0 20 80 3 Benz[a]anthracene
4 Chrysene
2 20 80
5 5-Methylchrysene
30 0 100 6 Benzo[j]fluoranthene
Column: Knaur 7 Benzo[b]fluoranthene
50 0 100
Eurospher-100 C18 8 Benzo[k]fluoranthene
Flow: 1,0 ml/min. 9 Benzo[a]pyrene
Detection: DAD 254 nm 10 Dibenzo[a,l]pyrene
5 11 Dibenz[a,h]anthracene
12 Benzo[ghi]perylene
7 13 Indeno[1,2,3-cd]pyrene
14 Dibenzo[a,e]pyrene
9 15 Dibenzo[a,i]pyrene
16 Dibenzo[a,h]pyrene
8 13
6 10 12
1
14
11
15 16
Column: Nacalai Tesque Cosmosil 5u PYE
Member of NOE ECNIS
t(min.) H2O CH3CN

1 Benzo[c]fluorene
5+6
0 40 60
2 Cyclopenta[cd]pyrene
3 Benz[a]anthracene 2 40 60
4 Chrysene 4 30 0 100
5 5-Methylchrysene
6 Benzo[j]fluoranthene 50 0 100
7 Benzo[b]fluoranthene
8 Benzo[k]fluoranthene
2+3
7 Flow: 1,0 ml/min.
9 Benzo[a]pyrene Detection: DAD 254 nm
10 Dibenzo[a,l]pyrene
9
11 Dibenz[a,h]anthracene
8 13
13 Indeno[1,2,3-cd]pyrene
14 Dibenzo[a,e]pyrene 14
15 Dibenzo[a,i]pyrene
16 Dibenzo[a,h]pyrene
10
1
12
Column: Nacalai Tesque 11 16
Cosmosil 5u PYE 15
Column: GRACE VYDAC 201TP5415
Member of NOE ECNIS
4 5
t(min.) H2O CH3CN 1 Benzo[c]fluorene
0 30 70 2 Cyclopenta[cd]pyrene
3 Benz[a]anthracene
2 30 70 4 Chrysene
3
20 0 100 5 5-Methylchrysene
7 6 Benzo[j]fluoranthene
55 0 100
7 Benzo[b]fluoranthene
9 8 Benzo[k]fluoranthene
Flow: 1,0 ml/min.
Detection: DAD 254 nm 9 Benzo[a]pyrene
8 10 Dibenzo[a,l]pyrene
13 11 Dibenz[a,h]anthracene
10 Column: GRACE 13 Indeno[1,2,3-cd]pyrene
6 14 VYDAC 201TP5415 14 Dibenzo[a,e]pyrene
2
15 Dibenzo[a,i]pyrene
1 16 Dibenzo[a,h]pyrene
12
11
15 16
PAH Profile (EFSA) Analysis in Liquid Smoke using HPLC
with UV and Fluorescence Detection
(Pinacle II RP column, Restek)
Member of NOE ECNIS
ACN/H2O gradient
UV at 375 nm
Fluorescence
Lambdaex =
270 nm
Lambdaem =
370 nm
420 nm
470 nm
500 nm
Simon et al., 2006

IRMM,
unpublished
Detection of Cyclopenta[cd]pyrene with UV-DAD within a
PAH profile analysis using HPLC
Member of NOE ECNIS
CPP at 375 nm
Simon et al., 2006

IRMM,
unpublished
PAH profile (EFSA) analysis by GC-MS
Member of NOE ECNIS
• 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)
Member of NOE ECNIS
Chrysene / Triphenylene - separation differs by type of capillary
Benzo[b]fluoranthene
Benzo[j]fluoranthene - separation differs by type of capillary
Benzo[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
Member of NOE ECNIS
(Jira et al., BFEL personal communication)
VF-17ms
by GC-MS(SIM) - Isotope dilution methodology II
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VF-17ms
by GC-MS(SIM) - Isotope dilution methodology IV
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VF-17ms
by GC-MS(SIM) - Isotope dilution methodology V
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VF-17ms
by GC-MS(SIM) - Isotope dilution methodology VI
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VF-17ms
by GC-MS(SIM) - Isotope dilution methodology VII
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VF-17ms
PAH profile (EFSA) analysis in Cacoa Butter
by GC-MS(SIM) - Isotope dilution methodology I
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Abundance
Ion 216.00 (215.70 to 216.70): KBU0602.D
CHR
Ion 226.00 (225.70 to 226.70): KBU0602.D
5000
Ion 228.00 (227.70 to 228.70): KBU0602.D
Ion 242.00 (241.70 to 242.70): KBU0602.D
4500 BaA
4000
3500
3000
2500
BcF TRI
2000
1500 CYC
5-CH3-CHR
1000
500
0
25.00 26.00 27.00 28.00 29.00 30.00 31.00 32.00 33.00 34.00
Time-->
by GC-MS(SIM) - Isotope dilution methodology II
Member of NOE ECNIS
Abundance
2200
Ion 252.00 (251.70 to 252.70): KBU0602.D Kakaobutter
2000
1800
BbF
BkF BeP BaP
1600
1400 BjF
1200
1000
800
600
PER
400
200
0
35.50 36.00 36.50 37.00 37.50 38.00 38.50 39.00 39.50 40.00 40.50 41.00
Time-->
by GC-MS(SIM) - Isotope dilution methodology III
Member of NOE ECNIS
Abundance
INP Ion 276.00 (275.70 to 276.70): KBU0602.D

Ion 278.00 (277.70 to 278.70): KBU0602.D
750
Ion 302.00 (301.70 to 302.70): KBU0602.D
700
650
600
BghiP
550
500
450
400
350
DBalP
300
250
DBahA DBaiP
200
ANTH DBaeP
150
100
50
45.00 46.00 47.00 48.00 49.00 50.00 51.00 52.00 53.00 54.00 55.00 56.00
Time-->
Conclusions
Member of NOE ECNIS
HPLC-FD appears to be an acceptable method for determination of EFSA

PAH profile provided that cyclopenta[cd]pyrene can be separated from
accompanying PAHs to allow its UV-quantification under optimal
conditions.
GC-MS(SIM) including isotope dilution methodology appears to be the

method of choice; resolution of benzofluoranthenes and the separation of
chrysene by the capillary is required.
Further validation of the two methods are needed for specific food
matrices; the isomeric specificity of the two methods for analysis of
5-methylchrysene and the dibenzopyrenes should be demonstrated.
In inter-laboratory trials native oils spiked with EFSA PAH are

recommended instead of material „blanked“ with active carbon and than
spiked with PAHs.
Thank you
for
your
attention

Analysis of PAH's in Food

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Analysis of PAH's in Food

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International Workshop

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

Analysis of PAH’s in food

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

• Sample preparation / clean up

Work place Ambient Air

Naphthalene Acenaphthene Acenaphthylene Phenanthrene Fluorene

Anthracene Fluoranthene Pyrene Benz[a]anthracene

Chrysene Benzo[k]fluoranthene Benzo[b]fluoranthene Benzo[a]pyrene

Dibenz[a,h]anthracene Indeno[1,2,3-cd]pyrene Benzo[ghi]perylene

Benzo[b]fluoranthene Benzo[k]fluoranthene Benzo[j]fluoranthene Benzo[a]pyrene

Dibenz[a,h]anthracene Indeno[1,2,3-cd]pyrene Benzo[ghi]perylene Dibenzo[a,e]pyrene

Benzo[c]fluorene Dibenzo[a,h]pyrene Dibenzo[a,l]pyrene Dibenzo[a,i]pyrene

PAH - EPA TEF PAH - EPA/EFSA TEF PAH - EFSA TEF

Naphthaline 0,001 Benz[a]anthracene 0,1 Benzo[c]fluorene -

Acenaphthylene 0,001 Benzo[a]pyren 1 5-Methylchrysene -

Acenaphthene 0,001 Benzo[b]fluoranthene 0,1 Dibenzo[a,l]pyrene 10

Fluorene 0,001 Benzo[k]fluoranthene 0,1 Dibenzo[a,h]pyrene 10

Phenanthrene 0,001 Benzo[ghi]perylene 0,01 Dibenzo[a,i]pyrene 10

Pyrene 0,001 Chrysene 0,01 Dibenzo[a,e]pyrene 1

Fluoranthene 0,001 Indeno[1,2,3-cd]pyrene 0,1 Benzo[j]fluoranthene 0,1

Anthracene 0,01 Dibenz[a,h]anthracene 1 Cyclopenta[cd]pyrene 0,1

Member of NOE ECNIS

Sample dissolved in cyclohexane Liquid-liquid partition of

Liquid-liquid partition of DMF Extraction of the combined DMF

Cyclohexane phase wash Concentration of cyclohexane

Concentration of cyclohexane phase and

• Separation, identification, and quantification

Different columns in use:

Vydac 201 TP,

t(min.) H2O CH3CN

Simon et al., 2006

Simon et al., 2006

• Isotope dilution methodology -

Separation, identification, and quantification

Chrysene / Triphenylene - separation differs by type of capillary

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

Benzo[c]fluorene - separation differs by type of capillary

Dibenzo[a,l]pyrene (LOQ = 0.1 ppb; mw 302 specificity??)

Ion 216.00 (215.70 to 216.70): KBU0602.D

INP Ion 276.00 (275.70 to 276.70): KBU0602.D

HPLC-FD appears to be an acceptable method for determination of EFSA

GC-MS(SIM) including isotope dilution methodology appears to be the

In inter-laboratory trials native oils spiked with EFSA PAH are

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