Advances in the Analysis of Environmental
Toxicants
MAXXAM Science Summit
November 24, 2011
Co-Authors
Simon Zhou, Li Shen
Alina Muscalu
Eric Reiner, Adrienne Boden, Terry Kolic,
Karen MacPherson, Sheng Yang,
Karl Jobst, Paul Helm, Patrick Crozier,
Liad Haimovici, Jessica D’eon,
Satyendra Bhavsar, Vince Taguchi,
Tony Chen, Dave Poirier, Myrna Simpson
Frank Dorman, Jack Cochran,
Michelle Misselwitz
Nicole Riddell, Bob McCrindle, Brock Chittim
Ian Brindle, Chris Marvin
Jef Focant
2
2
Outline
•
•
•
•
Brief history / analytical challenges
Method attributes – the 4 Ss
Ways to reduce analysis time and increase capacity
Applications
•
•
•
•
•
3
Fast gas chromatography (Fast GC)
Parallel column analysis
Two-dimensional gas chromatography (GCxGC)
Liquid chromatography-tandem mass spectrometry (LC-MSMS)
Screening procedures (PCR, Metabolomics, Kendrick Plots)
3
Background
Muir & Howard, ES&T, 2006, 40, 7157-7166
US TSCA – United States Toxic Substances Control Act
4
4
Background
•
Industrial chemicals have been used for over 100 years
•
5
Lindane (1825), Polychlorinated Naphthalenes - PCN (1833) and
DDT (1873)
•
PCNs used as antifungal agents (gas masks) and flame
retardants (airplane cloth, WW I uniforms, electrical cabling)
•
Pesticide characteristics discovered for DDT and Lindane in
the late 1930’s and used extensively to control malaria,
typhus and typhoid.
•
Used extensively (PCB -106 tons, DDT - 2 x 106 tons)
•
Only a very few chemicals monitored in the environment
Stockholm Agreement Compounds
6
Original 12
Added 2009
Under Discussion
aldrin, chlordane, dieldrin, DDT, endrin, heptachlor, hexachlorobenzene (HCB), mirex, toxaphene, polychlorinated biphenyls (PCBs), polychlorinated dibenzodioxins (PCDD) and dibenzofurans (PCDF)
short chain chlorinated chlordecone, paraffin (SCCPs), α‐hexachlorocyclohexane, endosulfan, β‐hexachlorocyclohexane, hexabromobiphenyl, hexabromocyclododecane
(HBCD)
tetra ‐ to hepta ‐
bromodiphenylether,
lindane, (δ‐hexachlorocyclohexane), pentachlorobenzene, perfluoroctanesulfonic acid (PFOS), its salts and prefluoroctanesulfonyl fluoride
Method Attributes
•
•
Sensitivity
Selectivity
•
•
•
(Precision and Accuracy)
Speed
$ (Cost)
The method should be fit for purpose
7
7
Early Analytical Challenges
8
•
Early methods not sensitive or selective enough to
protect the environment or human health
•
They required extensive workup with hazardous
reagents
•
No way of separating toxic components from non-toxic
ones
•
Gas Chromatography (GC) developed (1952) to address
this issue
•
Early GC detectors not very sensitive or selective
BC (Before Chromatography)
9
Method
Gravimetric
Carius
Titration
Stepanow/Bacon
Photometric
Schechter
Date range
1800s
1900 to 1940s
1940s to 1950
Details
Boil samples in fuming
nitric acid. Add silver
nitrate and determine
weight of silver
chloride formed
Treat halide with
sodium in presence of
ethanol and determine
halide by Volhard
method
Boil samples in fuming
nitric acid. React with
sodium methylate –
methanol and measure
adsorption at 600 nm
Selectivity
Halogens
Halo organics
DDT and related
compounds
Sensitivity
% level
Milligrams (PPTh)
Micrograms (PPM)
1950s Pesticide Ad – DDT
10
10
1950s Pesticide Ad (Dieldrin)
11
11
DDT Container
12
It all began with…
Rachel Carson
(1907–1964)
– author of Silent
Spring, (1962)
13
The Result: Environmental Effects of DDT
14
16
Discovery of PCB in the Environment
•
PCBs were used in a number of applications including
dielectrics, heat transfer fluids, additives in sealants and
pesticides
•
Additional peaks were originally thought to be pesticide
degradation products. Too few peaks to be PCBs
•
Jensen analyzed eagle feathers dating back to 1888 and
unknown peaks were observed starting in the early 1930s
•
Used gas chromatography-mass spectrometry to confirm
presence
•
Took 7 years to confirm identity of PCBs
16
17
POPs in Environmental Samples:
the Analytical Challenge
Many congeners per analyte group
•
dioxins/furans: 210; PCBs: 209, toxaphene: >600
Separate and accurately quantify all toxic congeners
•
dioxins/furans: 17; PCBs: 12; toxaphene: 22
Toxicity can range up to 6 orders of magnitude
•
TCDD – NOEL = 3g/kg to LD50 = 1 ug/kg
Range of concentrations
•
fg/g (10-15g/g ) to %
Range of sample types, complexities
•
18
biota, air, water, soil, hazardous waste, other
TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin
NOEL: No-observable effect-level
LD50: Lethal dose (50% test population)
18
19
19
Dioxin Toxicity
NOEL = 3g / kg
20
LD50 = 1ug / kg
20
Chromatography
Phenomenon observed by D.T. Day (1903)
Reported as chromatography by M. Tswett (1906)
Martin & Synge (1941) published on liquid-liquid partitioning and stated that
the mobile phase could be a gas and it should be possible to perform very
refined separations of volatile compounds
Martin & James (1952) published first gas chromatogram
Peak Capacity
Packed: 5–6
Capillary: 50–100
GCxGC: >1000
nc =
21
(N)1/2
4Rs
nc = Peak Capacity
Rs= Resolution for separation = 1.5
Modern Approaches
•
•
•
•
22
Selection of appropriate method
Quantitative extraction of analytes from
matrix
Cleanup of sample extract to remove
interfering matrix and coextractable
compounds
Chromatographic separation – selective
detection
Dioxin-Like Toxicity
Most toxic are planar with 3Å x 10Å dimensions
23
Extract Fractionation
•
Multi-analyte groups are extracted together (Dioxins/furans, PCNs ,
PCBs, PBDEs, HFRs) but cannot be separated in a single GC column
analysis
•
Co-eluting / interfering compounds can be separated physically (into
fractions) or by using multiple GC column phases (e.g. PCB 77 and 110)
•
Sample extracts are separated using silica, carbon and alumina (forward
carbon fraction)
•
Dioxins/furans, PCNs and non-ortho PCBs in reverse carbon fraction
•
Ortho substituted PCBs, PBDEs and other HFRs in forward carbon
fraction
Combining these analyses can save significant time and costs
•
24
24
Classical
Open Column
Sample
Preparation
FMS PowerPrep
FMS = fluid management systems
26
34
4
CRM = certified reference material
13
,4
ct
C
-P
C
)
4)
)
N
(7
5
(6
N
(5
2
N
-P
C
N
aC
N
,5
,7
C
-o
,3
N
-P
C
100
13
C
-1
,2
7)
120
,7
C
140
,5
BDEs
,3
100
-P
C
N
(2
80
C
N
2)
-2
13 3'4'
5C
te
-3
tra
44
13
'5
-te pcb
C
13
-3
tra
(7
3'
C
PC 0)
44
-2
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'
B
te
44
13
tra
(8
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C
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PC 1)
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3'
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n
44
ta
13
(
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PC 77)
C
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en
34
(1
ta
13
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PC 23)
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pe
B
33
nt
(1
13
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a
4'
PC 18)
C
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en
13 3'4
(1
ta
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5PC 14)
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e
(1
13 44'5 nta
PC 05)
C
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33
ex
13
(1
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)
33
he CB
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(1
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7)
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13 33'
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6)
5'
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(1
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PC 57)
'-h
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ep
(1
ta
PC 69)
B
(1
89
)
13
C
120
,4
9
100
,3
-P
C
N
(4
20
0
C
N
D
E
140
13
C
-1
,2
B
3
4
18
3
15
15
99
47
20
,7
D
E
D
E
D
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BD
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B
B
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40
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28
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en
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PCDD/F
13
C
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-2
44
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4'
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e
-tr
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120
2'
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2'4
44
44
'
13
13 C-2
C 37
13 123 8-T
C
C 7
13 -23 8-P DF
C 47 eC
13 123 8-P DF
C 47 eC
13 123 8-H DF
C 67 x C
13 234 8-H DF
C 67 x C
13 -12 8- D
C 37 Hx F
13 123 89- CD
C 46 Hx F
-1
23 78- CD
4 7 Hp F
8 9 CD
-H F
pC
13
D
C
F
13 -2
C 37
13 -12 8C 37 TC
13 123 8-P DD
C 47 eC
13 -12 8- D
C 36 Hx D
-1
23 78- CD
46 Hx D
78 CD
-H D
13 pC
C DD
-O
C
D
D
140
13
C
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2
C
-2
C
-2
2'
C
-2
140
120
100
80
60
40
20
0
13
C
13
13
13
FMS – WMF-01 – 10 replicates
DLPCB
80
60
40
20
0
PCNs
80
60
40
20
0
27
Cape Sample Prep System
28
Fast GC – Method Attributes
Changes in parameters
• shorter & narrower columns
• thinner stationary phase films
• faster oven temperature programming rates
• higher pressures, faster carrier gas flow rates
Faster analyses  Increased sample
throughput
Narrower peak widths place new demands on
detection systems
29
Comparison of GC Columns
30
Column Length (m)
10
30
20
60
40
i.d. (mm)
0.1
0.25
0.1
0.25
0.18
Film Thickness (m)
0.1
0.25
0.1
0.25
0.18
Theoretical Plates/m
8,600
3,300
8,600
3,300
5,300
Total Theor. Plates
86,600
99,000
172,000
198,000
212,000
Height Equiv. Theor. Plate
(mm)
0.1
0.3
0.1
0.3
0.2
Rel. Col. Efficiency
0.93
1
1.32
1.41
1.46
30
Comparison of Different Column Dimensions
Handbook of GC/MS – H.J. Hubschmman
31
What is Fast GC ?
GC ramp rates >50ºC/min.
GC column diameters <0.18 mm
Stationary phase <0.18 µm
GC column head pressures >60 psi
Phase ratio:  = r / df
r = column diameter
df = film thickness
32
Fast GC Chromatogram
60m – 0.25μm,
0.25mm 5% phenyl
RT = 23.4
25% Valley
20m – 0.1 μm,
0.1mm
5% phenyl
25% Valley
33
RT = 9.00
RTA = 38%
Time of Flight Mass Spectrometry
p,p’-DDE
Dieldrin
Endrin
PCB-81
PCB-87
34
PCB-77
PCB-110
PCB-151
PCB-82
34
Sediment CRM
CRM WMF01 – Lake Ontario
Analysis Time: 7.3 minutes
35
PAHs, PCBs & Organochlorine
Pesticides by GC-TOF
Pyrene
a-Chlordane
g-Chlordane
Endosulphan-1
PCB-99
PCB-101
PCB-119
36
Sediment CRM
CRM WMF01 – Lake Ontario
3
1
5
7
4
2
37
6
8
1. Phenanthrene
2. Anthracene
3. Fluoranthene
4. Pyrene
5. Benzofluoranthenes
6. Benzo[e]pyrene
7. Benzo[a]pyrene
8. Perylene
Reductions in Analysis Times
Using Microbore Columns
38
60 m
40 m
30 m
20 m
10 m
(.25/.25)
(.18/.18)
(.25/.25)
(.10/.10)
(.10/.10)
Dioxins
[50]
28 (44%)
PCB
Congeners
[90]
14 (72%)
18 (80%)
PAH
[40]
22 (55%) 14 (65%)
OC
Pesticides
[55]
12* (78%)
Parallel Columns
Most compound classes cannot be uniquely separated on a
single chromatographic phase – e.g., PCBs, dioxins,
PAHs
Extracts must be separated or analyzed on 2 phases
Parallel columns can be used to analyze multiple fractions
simultaneously
•
e.g., dioxins/furans/coplanar PCBs and ortho PCBs
Extracts can also be analyzed separately
39
•
Analysis and confirmation in the same run
•
Both columns must be temperature compatible
39
GC Configuration
HP6890 + model GC
• 2 injection ports and 2 autosamplers
2 GC Columns
40
•
Dioxin/Furan & coplanar PCBs injected on
40M Rtx5, 0.18mm x 0.20µm
•
Mono-ortho PCBs
– 20M Rtx5, 0.1mm x 0.1µm
40
The Gas Chromatograph
41
41
42
4242
Parallel Column Advantages
Mono-ortho PCBs elute from 20m column well before
PCDD/Fs elute from 40m column
DPEs, and interfering PCBs in mono-ortho DLPCB sample
fraction
Avoid potential interferences:
1) Furan formation in ion source
2) Reduces co-elution of higher chlorinated PCBs with
coplanars (i.e. PCB 110 with PCB 77)
43
43
Fraction 2: 40M
Tetra - Octa PCDDs
12378-PCDD
123678-HxCDD
/
123789-HxCDD
123478123478-HxCDD /
HpCDD
\
/
OCDD
2378-TCDD
81
\
77
/
126
167
123 118
156
\ / 114
\
/
105
/
44
157
/
189
/
169
Fraction 2: 40M
Coplanar PCBs
Fraction 1: 20M
Mono Ortho PCBs
44
Dioxins and WHO PCBs in NIST 1944
T4CDDs
Tetra to Octa
Dioxins on a
40M column
100
P5CDD
s
H6CDD
s
H7CDDs OCDD
PCDFs and
coplanar PCBs
(77/81/126/169)
not shown
%
0
100
7.50
10.00
12.50
%
15.00
17.50
20.00
22.50
25.00
27.50
30.00
32.50
35.00
37.50
Time
40.00
H7CBs on 20 M column (PCB189)
0
100
H6CBs on 20 M column (PCB156/157/167)
%
0
100
P5CBs on 20 M column (PCB105/114/118/123)
%
0
7.50
45
10.00
12.50
15.00
17.50
20.00
22.50
25.00
27.50
30.00
32.50
35.00
37.50
Time
40.00
45
Two Dimensional GC (GCxGC)
46
•
Produces higher peak capacity (more chromatographic peaks per
space). Increases peak capacity to 50 x 20 = 1000 compounds
•
Eliminates the need for second column confirmation. Can do
multiple analyte groups in same run and may eliminate need for
extract fractionation
•
Fast analysis – requires fast detector – e.g., time-of-flight mass
spectrometer (TOFMS), ECD
•
Provides structured chromatograms for excellent selectivity
•
Provides much more information
•
Results in increased sensitivity
46
GC x GC Schematic Diagram
Injector
TOFMS
Modulator
1D
2D
PM
X
X+Y
Y
1t
PM = modulation time
D = dimension
2t
R
Retention Time (tR)
RX
2t Y
R
47
Classical GC – First Dimension
L
N =
H
30m
=
0.3mm
=
105 Plates
5)1/2
(N)1/2
(10
~300
nc =
=
=
=
~50
Peaks
4Rs
4 x 1.5
6
Classical GC
…
…
50 peaks
Injector
Detector
30m x 0.25mm x 0.25µm
Fast GC – (Second Dimension)
L
N =
H
=
(N)1/2
nc =
4Rs
2m
0.1mm
=
2x104 Plates
(2x104)1/2 140
= 4 x 1.5 =
= ~25 Peaks
6
Practical GCxGC
…
…
1D
= 30-60m
(50-100 peaks)
2D
= 0.1-5m
(2-25 peaks)
GCxGC (2 columns)
…
…
1D
= 30 m
2D = 2 m
Peak capacity (nc) = 1Dnc x 2Dnc
= 50 x 25 = 1250 peaks
Instrumental Setup
Splitless
ECD
1D
DB1- 40 m
0.25mm ID x 0.25µm
1Tt
R
LN2-Quad-Jet
Modulator
~ 50 min
PM = 4 sec
2D
Rtx-PCB 1.6 m
0.18mm ID x 0.18µm
Injector
Detector
LN2 supplier
1st dimension
2nd dimension
2nd Oven
1st Oven
Trapping
Injector
Detector
LN2 supplier
1st dimension
2nd dimension
2nd Oven
1st Oven
Releasing
Injector
Detector
LN2 supplier
1st dimension
2nd dimension
2nd Oven
1st Oven
Refocusing
2nd Oven
Modulator
Peak Modulation
•
Original peak is “chopped” into
3 peaks
•
Sensitivity enhancement
occurs through focusing
•
Second dimension peaks are
only 400 ms wide
•
Need detector capable of
defining peaks
1544
0.72
1544
0.74
1546
0.70
– Hundreds of spectra/sec
ms = millisecond
59
59
First Dimension Modulation
Signal Intensity
2t X
R
2t X
R
2t
2t X
R
RY
2t Y
R
2t Y
R
2t X
R
2t Y
R
2t X
R
2t Y
R
2t X
R
Retention Time (tR)
2t Y
R
1t
R
PM
60
60
Second Dimension Modulation
Signal
Fi
rst
X
di
m
en
sio
n
Y
re
ten 1t
tio R
n
tim
e
2t
X
1t
Y
RX
1t
RY
2t
61
R
Second dimension retention time
RX
2t
RY
61
Comprehensive GC x GC
Form of 2DGC: Orthogonal Column Setup
1st Dimension (Column)
Standard (10–60 m, 0.25mm, 0.25 um)
Non-polar (DB-1, Rtx-5)
2nd Dimension (Column)
Very short (1–2 m)
Narrow bore, thin film (0.10–0.18mm, 0.10–0.18um)
Polar or shape selective (DB-1701, Rtx-PCB)
62
62
1D vs. 2D analysis for PCB/OC/CBz - Biota
PCBT
analysis
OC_Rtx-CLP 1
PCBc – DB-5
2nd Dimension (s)
3
Toxaphene bands
2
PCBs/OCs
PCBc_DB1701
OC_Rtx-CLPII
1
0
676
1176
1676
2176
1st Dimension (s)
72
PCBs Challenging Separations
DB5-10M/0.18/0.18_DB17-2M/0.1/0.1
73
73
Rtx-PCB >
Dioxin-Like PCBs vs. Other PCBs
m/z 292 326 360 394
126
156
77
81
169
189
157
105
114
167
118
123
Rtx-1 >
74
74
Organochlorine Pestcides
DB5-10M/0.18/0.18_DB17-2M/0.1/0.1
ORGANOCHLORINE PESTICIDES
75
75
PCB Standard by GCxGC-ECD – Orthogonal Elution
PCB STD (BP‐MS)
Hexa169
Penta126
Tetra81
74 70
101
Di-
22
28
15
54*
8
19
18*
33
44
49
87 110*
99
155* 119
95
149
128
167
105
114
118
123
Tri37
77
138
156 157
158
Hepta189
170
194
205*
180 191*
177
171
183*
187
153
168*178*
199*
Octa206*
Nona209
Deca-
208*
201
202
151*
188*
52 104*
*to be confirmed by GCxGC/TOF‐MS
76
76
Between – Class Separation
by GCxGC-ECD: PCB/OC/CB Mix Standard Solution
PCB/OC/CB STD Mix
→ OCs; → CBs; not marked peaks: PCBs
77
7777
SRM1944 Analysis – Within Run (n=10)
78
CNS312 Analysis – Within-run Run
79
SRM1944 Analysis – Between Run
80
Sediments by GCxGC-ECD
PCNs, PCDEs
CBz
2nd Dimension (s)
3
Unknown
compounds
2
1
PCBs/OCs
Dioxins/Furans
0
676
1176
1676
2176
1st Dimension (s)
81
Aroclor 1242 @ 150ng/ml
PCBT = 145 ng/ml
Aroclor 1248 300ng/ml
PCBT = 298 ng/ml
PS1 (1248:1254:1260=1:1:1) 300ng/ml
PCBT = 328 ng/ml
Sludge sample – GCxGC-µECD
CBz
3
2nd Dimension
(s)
PCBs/OCs
Chlorinated
2
Terphenyls
1
0
220
720
1220
1st Dimension (s)
85
2220
Sludge Samples – PCB/OC/CB Analysis
2D Chromatogram – Sludge Sample
Triclosan
86
Halogenated Flame Retardants
Brominated Flame Retardants
• Polybrominated Diphenyl
Ethers (PBDEs)
• Polybrominated Biphenyls
(PBBs)
• Hexabromocyclododecane
(HBCD)
• “replacements”
Chlorinated Flame Retardants
• Mirex (Dechlorane)
• Dechlorane Plus
• Dechloranes
87
Some Current HFRs
PBDEs
Dechlorane Plus
PBB-153
TBBPA
HCDBCO
BEHTBP
,,-HBCD
88
ATE
Background
• >95,000 industrial chemicals used in commerce
• Halogenated organics make up a large
percentage
• >50% of the flame retardants are Halogenated
Flame (HFRs)
• >1 mega tonne of HFRs produced 1992 – 2002
• HFRs are the most effective at charring and
neutralizing free radicals
• HFRs or degradation products can be persistent,
bioaccumulative or toxic
89
Analytical Methods
Current analytical methods
1. Gas Chromatography- Mass Spectrometry (GC-MS) is widely
used to detect halogenated flame retardants (HFRs).
Advantages: High chromatographic resolution
Excellent sensitivity
Limitations: Thermal stability => HFR decomposition.
Isomerization => quantitative issues.
2. Liquid Chromatography – Tandem Mass Spectrometry (LC-MSMS)
Electrospray Ionization (ESI) – Limited analytes
Atmospheric Pressure Chemical Ionization (APCI) - Wider application
Atmospheric Pressure Photo Ionization (APPI) – Limited availability
90
Fragmentation Reactions
(1) Displacement reactions – Most HFRs
M + O2•- → [M - R + O]M + O2•- → [M - R + O2]R = Br, [Br + HBr], or Cl
(2) Elimination reactions – TBBP-A, HBCDs, Octa, Nona, BDE-209
M + O2•- → [M - R]R = H, or part of a molecule
(3) Association reactions – HCDBCO, HBCD, DP
M + O2•- → [M + O2]•-
91
APCI – Chromatogram – HFRs
4500
4000
5.75
a-DP
DP
3500
3000
5.92
s-DP
2500
2000
1500
1000
500
0
0
25000
1
2
3
4
5
6
α-
7
β-
14000
20000
5.06
BDE-100
BDEs
10000
Br
Br
Br
O
HBCD
10000
Br
Br
4.29
-HBCD
12000
5.27
BDE-99
15000
γ-
Br
4.43
-HBCD
8000
O
Br
Br
4.15
-HBCD
Br
6000
4000
Br
2000
5000
0
0
0
0
92
1
2
3
4
5
6
7
1
2
3
4
5
6
7
Comparison with GC-HRMS
Wastewater samples (APCI) and fish tissues (APPI)
1000.0•
100.0
Use existing extracts (optimized for PBDE analysis by
GC-HRMS)
External standard calibration1000.0
BDE-154
BDE-99
BDE-100
10.0
1.0
0.1
0.1
1.0
10.0
100.0
1000.0
Concentrations by LC-APCI-MS/MS (pg/mL)
(High Resolution Mass Spectrometry = HRMS)
93
Concentrations by GC-HRMS (pg/mL)
Concentrations by GC-HRMS (pg/mL)
•
100.0
BDE-209
BDE-183
BDE-153
10.0
1.0
0.1
0.1
1.0
10.0
100.0
1000.0
Concentrations by LC-APCI-MS/MS (pg/mL)
APCI – Chromatograms – Wastewater Sample
I (cps)
1200
(A)
a-DP
BDE-71
800
TBBP-A
BDE-66
400
s-DP
DBDPE
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
I (cps)
100000
(C)
BDE-99
80000
(B)
I (cps)
6000
BDE-183
EHTeBB
60000
BDE-154
BDE-209
40000
4000
BDE-100
20000
BDE-85
BDE-47
BDE-153
2000
0
BEHTBP
1
T (min)
0
1
94
2
3
4
5
6
7
8
9
10
11
12
13
14
2
3
4
5
6
7
8
9
10
BDE 209 & BEHTBP Calibration Curve
BDE-209 calibration curve
BEHTBP calibration curve
1.2E+05
5.0E+05
y = 230.89x - 5195.9
R2 = 0.9995
y = 51.694x - 1270.8
R = 0.9991
4.0E+05
8.0E+04
Peakintensity(cps)
Peak intensity (cps)
1.0E+05
2
6.0E+04
3.0E+05
2.0E+05
4.0E+04
1.0E+05
2.0E+04
0.0E+00
0
0
500
1000
1500
Injected amount (pg)
95
500
1000
1500
Injected amount (pg)
0.0E+00
2000
2500
2000
2500
Sample Data – APPI
Application in real samples
1000.00
Concentration (ng/g)
100.00
BDE-47
BDE-99
BDE-100
BDE-153
BDE-154
BDE-209
4PC-BDE-208
a-DP
s-DP
10.00
1.00
0.10
0.01
mussel-1
96
mussel-2
mussel-3
sludge-1
sludge-2
Instrument Detection Limits (pg/ul)
APPI
APCI
Polybrominated diphenylethers (PBDEs) – 16 congeners
0.5-40 (2)
0.5-40 (3)
1
1
4-10 (8)
4-10 (8)
0.5
0.5
Allyl 2,4,6-tribromophenyl ether (ATE)
4
4
2-Bromoallyl-2,4,6-tribromophenyl ether (BATE)
2
2
2,3-Dibromopropyl-2,4,6-tribromophenyl ether (DPTE)
20
40
Octabromotrimethylphenylindane (OBIND)
1
4
Pentabromoethylbenzene (PBEB)
2
4
Hexabromobenzene (HBB)
0.5
0.5
1,2-Bis (2,4,6-tribromophenoxy) ethane (BTBPE)
0.5
0.5
4
4
4-20
4-20
Hexachlorocyclopentadienyl-dibromocyclooctane (HCDBCO)
20
10
2-Ethylhexyl-2,3,4,5-tetrabromobenzoate (EHTeBB)
1
2
Bis(2-ethly-1-hexyl)tetrabromophthalate (BEHTBP)
0.5
0.5
2
4
2,2',4,4',5,5'-Hexabromobiphenyl (BB-153)
Hexabromocylcododecane (HBCD)
Tetrabromobisphenyl-A (TBBPA)
Decabromodiphenylethane (DBDPE)
Dechlorane Plus (DP), (anti, syn)
2,2',3,3',4,5,5',6,6'-Nonabromo-4'-chlorodiphenyl ether (4PC-BDE208)
97
Dioxin Screening
98
•
“Dioxin” is classically analyzed by gas chromatography-high
resolution mass spectrometry (GC-HRMS)
•
A single analysis costs about $700–$1000 and can take 8 to 10
days to complete a set of 10 samples.
•
Required detection limit for unrestricted fish consumption in Ontario
is 2.3 picograms (10-12g) per gram total dioxin toxic equivalents.
•
>90% of the fish restrictions in Great Lakes samples is from dioxinlike compounds
•
Bioassay, Immunoassay and polymerase chain reaction (PCR) have
been used to screen contaminated soil for site cleanup
PCR Correlation with GC-MS for Soils
5
10
4
pg/g TEQ (AhR-PCR)
10
2
R = 0.94
3
10
2
10
1
10
0
10
-1
10
-1
10
0
10
1
10
2
10
3
10
pg/g TEQ (GC-MS)
99
4
10
5
10
Dioxin Screening by PCR
•
The PCR screen mimics the binding of the aryl hydrocarbon receptor
“AhR”
•
The dioxin modified DNA is fluorescent tagged
•
The tagged DNA is replicated by PCR and the concentration is
determined by a spectrofluorometer
•
Extracts from PCB/organochlorine fish samples will be used potentially
reducing costs to $50–$100 per screening test.
•
PCR screening will be used to increase dioxin analysis capacity of fish
by up to a factor of 10
100
Eichrom Procept Assay
Add activation solution
to sample in glass vial.
1
Cl
AhR
Cl
O
Shake for 30 minutes.
Cl
O
Cl
Cl
O
Cl
Cl
3
Cl
O
Cl
Cl
O
Cl
AhR
ARNT
ARNT
AhR and ARNT form complex
with dioxin and DNA
5
Transfer to capture strip.
AhR
Wash Capture Strip.
Cl
O
Cl
Cl
O
Cl
Cl
O
Cl
Cl
O
Cl
AhR
AhR
ARNT
ARNT
AhR-Dioxin complex bound
to capture strip.
101
Shake for 1 hour at
room temperature.
Cl
ARNT
DNA
4
O
2
Removes unbound DNA, AhR
and ARNT
Plastic capture strip.
6
Run PCR ~ 1.5 hours.
Add
PCR
Reagents
PCR duplicates and measures
DNA with each cycle.
Dioxin Screening by PCR
•
Other assay methods (bioassays and immunoassays)
require solvent exchange to methanol or water.
•
PCR can operate using hydrocarbon solvents which are
better for dissolving dioxins.
•
PCR dioxin screening is expected to reduce detection limits
over other assay methods (bioassays and immunoassays)
because it manufactures the analyte being measured.
•
Detection limits can be achieved, extra care must he taken
with interferences from PAH and rogue DNA
102
Environomics
Environomics – The statistical investigation of
differences and similarities in the metabolome of an
organism in response to environmental stressors.
Metabolome – The complete set of molecules observed
in an organism (ie. amino acids, hormones, proteins,
DNA…)
103
Why use Metabolomics?
Daphnia
Magna
PCB
Rainbow Trout
PCB
Toxicity
Hg2+
Hg
Toxicity
Water Contamination
Chemical exposure within the food web
Multiple analyses
How is this contamination identified?
• Collect water/fish samples
• Extract the samples
• Quantify contaminant concentrations
104
1 analysis
Metabolomics as a screening tool
• Easy/fast extraction/analysis
• Specific or Non-specific?
• May give preliminary Identification of
stressor
Analytical Procedure
FT-ICR mass spectrum of
Daphnia Magna extracts
Create PCA scores plot that
displays differences between
the samples visually
Daphnia
Magna
Effect
(copper toxicity)
No Effect
Control
Copper
exposure
Taylor et al. 2009 Metabolomics 5:44-58
105
Principal Component
Analysis of mass spectra,
which identify similarities and
differences.
Extraction
Polar metabolites
Sulfur metabolites
(antioxidants)
Amino Acids
(protein building blocks)
Nonpolar metabolites
50:50
Methanol:water
Isoprostanes
(oxidized fatty
acids)
Fatty acids
Chloroform
DNA
O
H
N
damage
N
NH2
O
H
N
H
Guanine
106
N
H
[O]
DNA digest
NH
O
[O]
NH
O
H2N
N
NH2
Analysis
Targeted LC-MS/MS Analysis
Polar metabolites
Sulfur metabolites
(antioxidants)
Amino Acids
(protein building blocks)
Inform mode of toxicity
Nonpolar metabolites
Isoprostanes
(oxidized fatty acids)
Fatty acids
Oxidative stress
• Upregulation of sulfur metabolism
• Amino acids consumed to produce
upregulated protective enzymes
• Increased isoprostane concentrations
• Oxidative DNA lesions
Other metal-specific modes of toxicity
Metabolic Fingerprint?
O
DNA damage
H
N
NH
LC-FT-ICR-MS
O
[O]
N
H
[O]
Guanine
107
Identify novel species of interest
• Novel DNA lesions?
O
H
N
H
NH
O
H2N
Analysis
NH2
N
N
NH2
Analysis
108
Analysis
109
Plastimet Fire - 1997
Kendrick Plot
Kendrick plot - vegetation exposed to fallout from the 1997 Plastimet fire
Kendrick mass = mass x (35 / 34.9689)
1
0.9
Kendrick Mass Defect
0.8
0.7
unassigned
0.6
CH
CHCl
0.5
CHClO
0.4
CHO
0.3
0.2
0.1
0
150
200
250
300
350
Nominal Kendrick Mass
400
450
500
550
Kendrick Plot - Plastimet Ash Extract
0.4
0.38
Benzopyrenes
Kendrick Mass Defect
Ter-phenyls
0.36
Tetracenes
0.34
Benzofluoranthenes
0.32
Pyrenes
0.3
0.28
0.26
Anthracenes
Dioxins
Dibenzofurans
0.24
Biphenylenes
0.22
Chloro
Naphthalenes
Bromo/Chloro
0.2
150
200
250
300
350
400
Nominal Kendrick Mass
450
500
550
Summary of Method Enhancements
113
•
Fast GC, Parallel GC and GCxGC can significantly reduce sample
analysis times and costs while increasing analytical capacity.
•
GCxGC can increase selectivity and sensitivity and can also be used
for analytical triage. Extract fractionation may not be required.
•
LC-MSMS can be used for compounds that are difficult to analyze by
standard GC methods.
•
Most POPs sample preparation procedures can be automated or semiautomated and many can be combined to save money and time
•
Screening techniques can be used to increase lab capacity
•
These enhancements can save time, costs and reduce the use of
solvents and reagents.
Thank You !
[email protected]
GCxGC Workshop – MOE
January 10, 2012