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Albrecht Seidel
Biochemical Institute for Environmental CarcinogensProf. Dr. Gernot Grimmer-Foundation
Lurup 4D-22927 Grosshansdorf
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
Member Member of NOE ECNISof NOE ECNIS
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
Member Member of NOE ECNISof NOE ECNIS
Work place
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
PAH in human diet
Member Member of NOE ECNISof NOE ECNIS
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
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
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
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
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[b]fluoranthene
Dibenz[a,h]anthracene
Benzo[k]fluoranthene Benzo[j]fluoranthene
Benzo[ghi]peryleneIndeno[1,2,3-cd]pyrene
Benzo[c]fluorene
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
Member Member of NOE ECNISof NOE ECNIS
-
-
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[b]fluoranthene
Benzo[k]fluoranthene
Benzo[ghi]perylene
Chrysene
Indeno[1,2,3-cd]pyrene
Dibenz[a,h]anthracene
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).
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
PAH in foodstuffs - EU Regulation 208/2005(Apply 1st April 2005)
Member Member of NOE ECNISof NOE ECNIS
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)
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
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
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Extraction of the combined DMFphases with cyclohexane
Sample clean up - fats and oils(Grimmer & Böhnke (1975) Journal of the AOAC, 58, 725-733)
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
Sample clean up II
Member Member of NOE ECNISof NOE ECNIS
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
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
Member Member of NOE ECNISof NOE ECNIS
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.
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
PAH profile (EFSA) analysis by HPLCColumn: Knaur Eurospher-100 C18
Member Member of NOE ECNISof NOE ECNIS
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
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
PAH profile (EFSA) analysis by HPLC Column: Nacalai Tesque Cosmosil 5u PYE
Member Member of NOE ECNISof NOE ECNIS
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: Nacalai TesqueCosmosil 5u PYE
1
2+3
4
5+6
8
79
11
13
12
1014
1615
100050
100030
60402
60400
CH3CNH2Ot(min.)
Flow: 1,0 ml/min.Detection: DAD 254 nm
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
PAH profile (EFSA) analysis by HPLC Column: GRACE VYDAC 201TP5415
Member Member of NOE ECNISof NOE ECNIS
Column: GRACEVYDAC 201TP5415
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
100055
100020
70302
70300
CH3CNH2Ot(min.)
Flow: 1,0 ml/min.Detection: DAD 254 nm
12
3
4 5
6
7
8
9
10
11
12
13
14
15 16
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
PAH Profile (EFSA) Analysis in Liquid Smoke using HPLC with UV and Fluorescence Detection
(Pinacle II RP column, Restek)
Member Member of NOE ECNISof NOE ECNIS
Simon et al., 2006IRMM,unpublished
UV at 375 nm
FluorescenceLambdaex = 270 nm
Lambdaem =370 nm420 nm470 nm500 nm
ACN/H2O gradient
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
Detection of Cyclopenta[cd]pyrene with UV-DAD within a PAH profile analysis using HPLC
Member Member of NOE ECNISof NOE ECNIS
CPP at 375 nm
Simon et al., 2006IRMM,unpublished
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
PAH profile (EFSA) analysis by GC-MS
Member Member of NOE ECNISof 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.
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
Separation of EFSA PAH by GC-MS(SIM)
Member Member of NOE ECNISof NOE ECNIS
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
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
Member Member of NOE ECNISof NOE ECNIS
Analysis of EFSA PAH in smoked pork belly by GC-MS(SIM) - Isotope dilution methodology I
(Jira et al., BFEL personal communication)
VF-17ms
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
Member Member of NOE ECNISof NOE ECNIS
Analysis of EFSA PAH in smoked pork belly by GC-MS(SIM) - Isotope dilution methodology II
(Jira et al., BFEL personal communication)
VF-17ms
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
Member Member of NOE ECNISof NOE ECNIS
Analysis of EFSA PAH in smoked pork belly by GC-MS(SIM) - Isotope dilution methodology IV
(Jira et al., BFEL personal communication)
VF-17ms
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
Member Member of NOE ECNISof NOE ECNIS
Analysis of EFSA PAH in smoked pork belly by GC-MS(SIM) - Isotope dilution methodology V
(Jira et al., BFEL personal communication)
VF-17ms
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
Member Member of NOE ECNISof NOE ECNIS
Analysis of EFSA PAH in smoked pork belly by GC-MS(SIM) - Isotope dilution methodology VI
(Jira et al., BFEL personal communication)
VF-17ms
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
Analysis of EFSA PAH in smoked pork belly by GC-MS(SIM) - Isotope dilution methodology VII
(Jira et al., BFEL personal communication)
Member Member of NOE ECNISof NOE ECNIS
VF-17ms
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
PAH profile (EFSA) analysis in Cacoa Butterby GC-MS(SIM) - Isotope dilution methodology I
Member Member of NOE ECNISof NOE ECNIS
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
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
PAH profile (EFSA) analysis in Cacoa Butterby GC-MS(SIM) - Isotope dilution methodology II
Member Member of NOE ECNISof NOE ECNIS
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
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
PAH profile (EFSA) analysis in Cacoa Butterby GC-MS(SIM) - Isotope dilution methodology III
Member Member of NOE ECNISof NOE ECNIS
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
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
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
AOAC Europe Section, International workshop, November 6-7, 2006, Limassol, Cyprus
Thank you for
your attention