SIMPLIFIED AND RAPID

DETERMINATION OF PCBs,

PBDEs, AND PAHs IN FISH AND

SEAFOOD INTEGRATED INTO A

SINGLE METHODJana Hajslova, Kamila Kalachova, Lucie Drabova, Tom as Cajka, Vladimir Kocourek and Jana Pulkrabova

www.con ffidence.eu

Session on the World Aquaculture Society, conference 2011“Aquaculture for a Changing World”, Natal Convention Center (Natal, Brasil

Fish & fish feed

Dioxin-like polychlorinated biphenylsBrominated flame retardantsPolycyclic aromatic hydrocarbons

Fish & fish feed

Dioxin-like polychlorinated biphenylsBrominated flame retardantsPolycyclic aromatic hydrocarbons

DESIRED LOQ FOR FISH TISSUE:

� Dioxin-like PCB: 2 ng WHO-PCB-TEQ/kg

� BFR: ≤≤≤≤ 0.2 µg/kg� B[a]P: ≤≤≤≤ 2 µg/kg

DESIRED LOQ FOR FISH TISSUE:

� Dioxin-like PCB: 2 ng WHO-PCB-TEQ/kg

� BFR: ≤≤≤≤ 0.2 µg/kg� B[a]P: ≤≤≤≤ 2 µg/kg

CONffIDENCE – Project objectives , part I

To develop a simplified sample preparation strategy for simultaneous determination of wide range of contami nants in food and feed focused on fish and cereal based f ood.

To develop a simplified sample preparation strategy for simultaneous determination of wide range of contami nants in food and feed focused on fish and cereal based f ood.

Cereal-based products

Polycyclic aromatic hydrocarbons

Cereal-based products

Polycyclic aromatic hydrocarbons

DESIRED LOQ FOR CEREAL BASED FOOD: � B[a]P: ≤≤≤≤ 1 µg/kg

DESIRED LOQ FOR CEREAL BASED FOOD: � B[a]P: ≤≤≤≤ 1 µg/kg

Target analytes� POLYCHLORINATED BIPHENYLS (PCB)

� Non-ortho PCB # 77, 81, 126, 169� Mono-ortho PCB # 105, 114, 118, 123, 156, 157, 167, 189� Major PCB # 28, 52, 101, 138, 153, 180

� BROMINATED FLAME RETARDANTS (BFR)� Polybrominated diphenyl ethers (PBDE) # 28, 47, 99, 100, 153, 154, 183� Hexabromocyclododecane (HBCD)� Polybrominated biphenyls (PBB) # 153

� POLYCYCLIC AROMATIC HYDROCARBONS (PAH)� Benz(a)anthracene BaA� Benzo(a)pyrene BaP � Benzo(b)fluoranthene BbFA� Benzo(c)fluorene BcFL� Benzo(j)fluoranthene BjFA� Benzo(k)fluoranthene BkFA� Benzo(g,h,i)perylene BghiP� Chrysene CHR

B[a]AB[a]A� Cyklopenta(c,d)pyrene CPP � Dibenzo(a,h)anthracene DBahA� Dibenzo(a,e)pyrene DBaeP� Dibenzo(a,h)pyrene DBahP� Dibenzo(a,i)pyrene DBaiP� Dibenzo(a,l)pyrenee DBalP� Indeno(1,2,3-cd)pyrene IP � 5-Methylchrysene 5-MC

O

Br x Br y

PBDE

x + y = 1 až10

OO

Br x Br y

PBDE

x +

Clx

ClyPCB

y = 1 až 10

Clx

ClyPCB

x +

Baby foodFats / oils Fish / sea food

Mean concentrations of the sum of the eight PAHs in 4,065 samplessplit across 33 food categories/subcategories (EU market)

Chrysene

Benzo[a]pyrene

Benzo[b]fluoranthene

Benzo[a]anthracene

PAHs: Mean concentrations of carcinogenic / genotoxic species in 1375 food products at EU market (http://www.efsa.europa.eu)

����benzo(a)pyrene (BaP) not a good marker for the presence and toxicity of priority PAH present in food….

����benzo(a)pyrene (BaP) not a good marker for the presence and toxicity of priority PAH present in food….

--- PCBs

--- PBDEs

--- PAHs

SaponificationFreeze dryingDessication by salt

Soxhlet MAE Sonic. MSPD PLE SFE

GPC Adsorption chromatogr.

Concentrated sulphuric acid

SPE cartridge

SPE cartridge (acidic silicagel)

GC-MS (NCI) GC-MS (EI) LC-

FLDGC-ECD LC-UVLC-MS

(APPI)

A. SAMPLE PRETREATEMENT

B. EXTRACTION STRATEGY

C1. NON-DESTRUCTIVE CLEAN-UP

C2. DESTRUCTIVE CLEAN-UP

D. FINAL SEPARATION, IDENTI.& QUANTIFICATION

B + C1. INTEGRATED EXTRACTION & CLEAN-UP

PLE SFE

Conventional sample prep for POPs analysis

Identification &quantification1 h / 1 sample

Soxhlet extractionhexane – dichloromethane

(1:1, v/v)

Gel permeation chromatography

cyclohexane-ethylacetate

GC-MS (EI)GC-MS (NCI)GC-MS (EI)

LC-FLD

PBDE + HBCD PCB PAH Non-ortho PCB

Soxhlet extractionhexane – dichloromethane

(1:1, v/v)

GC-MS (NCI)

Clean up on silica column

Fractionation on pyrenyl column

Gel permeation chromatography

chlorophorm

Isolation7h / 10 samples

Clean up1 h / 1 sample

����LABORIOUS, TIME CONSUMING ����

BFR PCB PAH Non-ortho PCB

PLE (Pressurized liquid extraction)hexane-dichloromethane

Na2SO4, silica

Identification & quantification GC-TOFMS (EI)

25 min

Isolation & Clean -up25 min

Identification &quantification

1 h

� PCBs: 86 – 110%, RSD max 12%

� BFRs: 80 – 90%, RSD max 12%

� PAHs: 55 – 75%, RSD up to 28%

ASE300, Dionex

Agilent 6890NPegasus III, LECO Corp.

Integrated sample preparation: set-up I

����

Integrated sample preparation: set-up 2

Isolation10 min

Clean up30 min

Identification &quantification

1 h

BFR PCB PAH Non-ortho PCB

Extraction Shaking (H2O + ethylacetate)

Partition (transfer into organic phase)induced by MgSO4 + NaCl

Clean up Silica minicolumn

Identification & quantificationGC-TOFMS (EI)

GC GC-TOFMS (EI)

6 SAMPLES /

< 1 HOUR

SINGLE GC

INJECTION

VALIDATION STUDYProcedure blank

� With each batch of six samples, the procedure blank was prepa red.

Recovery (%) and repeatability RSD (%)

� Spiking level 1 and 2 = 1 and 5 µg/kg for PAHs, PCBs and PBDEs(5 and 25 µg/kg for major PCB 138, 153, 180 and for PBDE 47).

Procedure blank

� With each batch of six samples, the procedure blank was prepa red.

Recovery (%) and repeatability RSD (%)

� Spiking level 1 and 2 = 1 and 5 µg/kg for PAHs, PCBs and PBDEs(5 and 25 µg/kg for major PCB 138, 153, 180 and for PBDE 47).

PERFORMANCE CHARACTERISTICS - TROUTAnalytes Rec [%] RSD [%] LOQ [µg/kg]

PAHs 73-97 2-13 0.05-0.25

PCBs 74-113 4-18 0.1-0.5

PBDEs 82-107 5-9 0.5

PERFORMANCE CHARACTERISTICS - SHRIMPSAnalytes Rec [%] RSD [%] LOQ [µg/kg]

PAHs 73-109 2-15 0.05-0.25

PCBs 93-124 5-21 0.1-0.5

PBDEs 79-122 4-11 0.5

Trout

Shrimps

QA / QC – accuracy control (CRMs)

SRM 2977 – Mussel Tissue

Analytes Determinedvalue [µg/kg]

Certified value[µg/kg]

B[a]A 20.42 20.34 ± 0.78B[b]Fln 10.86 11.01 ± 0.28B[a]P 8.19 8.35 ± 0.72

B[ghi]P 9.22 9.53 ± 0.43I[1,2,3cd]P 4.19 4.84 ± 0.81DB[ah]A 1.29 1.41 ± 0.19

Chr 50.57 49 ± 2B[ j]Fln 4.48 4.6 ± 0.2B[ f]Fln 3.46 4 ± 1

SRM 1947 – Lake Michigan Fish Tissue

AnalytesDetermined

value [ µg/kg]Certified

value [ µg/kg]

Mon

o-or

tho

PC

Bs

PCB 105 53.2 ± 4.8 50.3 ± 3.7PCB 118 115.1 ± 10.4 112 ± 6PCB 156 15.0 ± 0.5 13.3 ± 0.9PCB 157 3.8 ± 0.3 4.08 ± 0.77

Maj

or

PC

Bs PCB 138 167.0 ± 13.4 162.0 ± 6.9PCB 153 204.6 ± 10.2 201 ± 3PCB 180 83.3 ± 9.2 80.8 ± 5.0

PB

DE

s

PBDE 47 70.7 ± 6.4 73.3 ± 2.9PBDE 99 18.4 ± 1.3 19.2 ± 0.8PBDE 100 17.7 ± 1.4 17.1 ± 0.6PBDE 153 3.8 ± 0.3 3.83 ± 0.04PBDE 154 6.3 ± 0.5 6.88 ± 0.52

11

CONffIDENCE – project objectives, part II

� To implement a GC GC/TOFMS for thesimultaneous determination of PCBs,PBDEs and PAHs in fish muscle tissue andcereal based food.

� To obtain the best chromatographic resolutionof all target analytes within a single run.

� To reach desired limits of quantification (LOQ).

� To chose and optimize suitableinjection technique.

� To chose the best capillarycolumn system to get the bestchromatographic resolution.

Agilent6890NPegasus III, LECO Corp.

SECOND COLUMN DIMENSION

More polar narrow-bore (0.5-2) m 0.1mm i.d. 0.1µm.

π –π interactions, hydrogen bonding etc.

GC×GC-TOFMS

J. Zrostlikova, J. Hajslova, T. Cajka, J. Chromatogr. A (2003), 1019, 173

FIRST COLUMN DIMENSION

Non-polar columns

(15-30) m (0.25-0.32) mm i.d. (0.1-1) µm.

Separation based on volatility.

High peak capacity.High chromatographic resolution needed for complex matrix / analytes mixture (separation of isomers). Focusing of peaks in 2 nd dimension → decrease of LOQs.Structured chromatograms.

High peak capacity.High chromatographic resolution needed for complex matrix / analytes mixture (separation of isomers). Focusing of peaks in 2 nd dimension → decrease of LOQs.Structured chromatograms.

Column systems tested:A. 1st column dimension: BPX 5 (30 m ×××× 0.25 mm ×××× 0.25 µm)

2nd column dimension: BPX 50 (1 m ×××× 0.1 mm ×××× 0.1 µm) B. 1st column dimension: BPX-5 (30 m ×××× 0.25 mm ×××× 0.25 µm)

2nd column dimension: Rt-LC35 (1 m ×××× 0.15 mm ×××× 0.1 µm)C. 1st column dimension: BPX 5 (30 m ×××× 0.25 mm ×××× 0.25 µm)

2nd column dimension: HT 8 (1 m ×××× 0.1 mm ×××× 0.1 µm)D. 1st column dimension: BPX 50 (30 m ×××× 0.25 mm ×××× 0.25 µm)

2nd column dimension: BPX 5 (1 m ×××× 0.1 mm ×××× 0.1 µm)

E. 1st column dimension: BPX 50 (30 m ×××× 0.25 mm ×××× 0.25 µm) 2nd column dimension: HT 8 (1 m ×××× 0.1 mm ×××× 0.1 µm)

GC GC-TOFMSHigh chromatographic resolution needed for complex matrix / analytes mixture (separation of isomers) Focusing of peaks in 2 nd

dimension – decrease of LODs (DL-PCBs, BFRs, PAHs)

Column BPX-5,

solvent standard

50 ng/ml

PCB 28

13.3333 15 16.6667 18.3333 20 21.6667 23.3333 25

20000

40000

60000

80000

100000

120000

Time (min)

256 290 326 360 394m/z

PCB

81

PCB

77

PCB

123

PCB

118

PCB 52

PCB 101

PCB

126

PCB

114 PCB

105PCB

153PCB

138

PCB

169

PCB

167

PCB

156

PCB

189

PCB

180

PCB

157

Halogenated POPs separation using 1D GC-TOFMS

23.3333 26.6667 30 33.3333 36.6667 40 43.3333

10000

20000

30000

40000

50000

60000

70000

80000

Time (min)

226

228

242

252

276

278

302

CP[cd]PB[a]A

Chr

5MeChr

B[b]F

B[k]F

B[j]F

B[a]P

B[ghi]P

D[ah]A

I[cd]P

DB[al]P

DB[ai]P

DB[ah]P

DB[ae]P

m/z

PAHs separation using 1D GC-TOFMS

Column BPX-5,

solvent standard

50 ng/ml

01138

11138

01140

11140

01142

11142

01144

0

500

1000

1500

2000

2500

3000

3500

4000

1st Time (s)2nd Time (s)

486

1130 1135 1140 1145 1150

0

100

200

300

400

500

600

700

800

900

Time (s)

486

1D-GC1D-GC GC××××GCGC××××GC

S/N: 127:1S/N: 923:1

7.3××××

GC×GC: Signal-to-noise enhancement

17

BD

E 4

7 (m

/z48

6)C

B 1

26 (

m/z

326)

900 905 910 915 920 925 930 935 940

0

10000

20000

30000

40000

50000

Time (s)

326

1910

0912

1912

0914

1914

0916

1916

0918

1918

0

20000

40000

60000

80000

100000

120000

1st Time (s)2nd Time (s)

326

S/N: 1,4:1S/N: 22 :1

16.7××××

PCB & PBDE - separation

Bream (Abramis brama)

Yellow – mono-ortho PCB

Green– non-ortho PCB

White – major PCB

Red - PBDE

2852

101

77

81138

105

114

153

126

167

156

157

180

169

189

118 +123

47

99100

153

154

28

◄Standard of PCB and PBDE

� Mono-ortho PCB – 105,118,123,156,157,167,189

� Non-ortho PCB – 77, 81, 126, 169

� Major PCB – 28, 52, 138, 153, 180

� PBDE – 28, 47, 99, 100, 153, 154, 183

PCB m/z 256, 290, 324, 358, 390

PBDE m/z 404, 484, 564, 644,724

28

52

101

105153

118

138167

156

157

47 100

77

PBDE

PCB

PAH

114

126

PCB m/z 256, 290, 324, 358, 390

PBDE m/z 404, 484, 564, 644,724

BPX 50 (30 m ×××× 0.25 mm ×××× 0.25 μm)

HT 8 (1 m ×××× 0.1 mm ×××× 0.1 µm)

Critical groups of PAHs - separation� 1st group: BaA, CPP and CHR� 2nd group: BjFA, BkFA and BbFA� 3rd group: DBahA, IP and BghiP

BaPBbFA

BjFA

BkFA

BPX-5 × BPX-50 BPX-5 × Rt-LC35 BPX-5 × HT-8

BkFA

BbFA

BjFA

BaP

BPX-50 × HT-8

BaPBbFA

BjFA

BkFA

BaPBbFA

BjFA

BkFA

BPX5 × BPX50

CP[cd]P

B[a]A

Chr

B[b]F

B[k]F

B[j]F

B[a]P

B[ghi]P

D[ah]A

I[cd]P

BPX50 × HT8 BPX5 × HT8

LOQs for all target analytes obtained by current analytical procedure for fish matrix

AnalytesFish

Desired in DoW Obtained*

BFRs

PBDE

≤ 0.2 µg/kg

0.05 µg/kg

HBCD 0.2 µg/kg

PBB 153 0.05 µg/kg

dl-PCB

2 ng WHO-PCB-

TEQ/kg

1.3 ng WHO-

PCB-TEQ/kg*

B[a]P ≤ 2 µg/kg 0.05 µg/kg

Oil Spill - Gulf Of Mexico 2010

Methods for Measurement of Polycyclic Aromatic Hydrocarbon (PAH) Compounds in Gulf of Mexico SeafoodAOAC INTERNATIONAL is inviting method developers to submit methods for consideration and possible evaluation through the AOAC Official MethodsSM

program. Prospective methods must be able to quantify polycyclic aromatic hydrocarbon (PAH) “seafood”. Acceptable methods must be able to demonstrate a Limit of Quantification of 1 ppb (ng/g) for benzo(a)pyrene in seafood. Currently accepted analytical methods require 96 to 120 hours to complete. Evaluation of analytical methods that significantly reduce the time-to-signal (including sample preparation and extraction) is a primary goal of this call for methods .

� Dibenz(a,h)anthracene� Fluorene� Fluoranthene� Ideno(1,2,3-cd)pyrene� Naphthalene� Pyrene

� Anthracene/phenanthrene� Benz(a)anthracene� Benzo(b)fluoranthene� Benzo(k)fluoranthene� Benzo(g,h,i)perylene� Chrysene

Call for Methods

16 EU PAHs EPA PAHs + methylated homologues

Benz[a]anthracene BaA 2-Methylanthracene 2-MA

Benzo[b]fluoranthene BbFA 1-Methylchrysene 1-MC

Benzo[k]fluoranthene BkFA 3-Methylchrysene 3-MC

Benzo[j]fluoranthene BjFA 1-Methylnaphthalene 1-MN

Benzo[c]fluorene BcFL 2-Methylnaphthalene 2-MN

Benzo[ghi]perylene BghiP 1-Methylphenanthrene 1-MPH

Benzo[a]pyrene BaP 1-Methylpyrene 1-MP

Chrysene CHR 1,7-Dimethylphenanthracene 1,7-DMP

Cyclopenta[cd]pyrene CPP 2,6-Dimethylnaphthalene 2,6-D MN

Dibenz[a,h]anthracene DBahA Acenaphthene AC

Dibenzo[a,e]pyrene DBaeP Acenaphthylene ACL

Dibenzo[a,h]pyrene DBahP Anthracene AN

Dibenzo[a,i]pyrene DBaiP Benzo[e]pyrene BeP

Dibenzo[a,l]pyrene DBalP Dibenzothiophene DBT

Indeno[1,2,3-cd]pyrene IP Fluoranthene FA

5-Methylchrysene 5-MC Fluorene FL

Naphthalene NA

Phenanthrene PHE

Pyrene PY

Target analytes – AOAC study

Method Performance Characteristics – PAHs and their homologues (GC-TOFMS)

750 1000 1250 1500 1750 2000 2250 2500

0

10000

20000

30000

40000

50000

60000

Time (s)

128 142 152 153 166 178 202 216 226 228 242 252 276 278

302

Na

ph

1-M

N

AC

LA

CFL

DB

TP

HE

AN

FA

PY

1-M

PH

2-M

A

Ba

AC

PP

CH

R

1-M

C

5-M

C

Bb

FA

Bk

FA

BjF

A

Ba

P

DB

ah

AIP

Bg

hiP

DB

alP

DB

ae

P

DB

aiP

DB

ah

P

2-M

N

3-M

C

Column: BPX-50 (30 x 0.25 mm x 0.25 µm)

Thank you for your attention.