simplified and rapid determination of pcbs, pbdes, and ...€¦ · lc-fld pbde + hbcd pcb pah...
Post on 20-Jul-2020
5 Views
Preview:
TRANSCRIPT
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.
top related