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Sylvain Merel, Christian Zwiener
Environmental Analytical Chemistry
Tracking the Occurrence and Fate of Contaminants in Water
with High Resolution Mass Spectrometry
Geosciences / Environmental Analytical Chemistry
E CEnvironmental
Analytical Chemistry
E CEnvironmental
Analytical Chemistry
Increasing concern about water resources worldwide
Quantity
Quality
Context & Background
Agilent’s Saturday Innovation Symposium ASMS, Indianapolis, Indiana, USA June 3, 2017
CONTEXT & BACKGROUND E CEnvironmental
Analytical Chemistry
Approaches for the detection of trace organic contaminants
Detects contaminants
As many as possible
At trace level
Reliably
Quickly
Target Screening Non-target Screening
Science 291, 5007 (2001) 1221-1224
Detects contaminants
As many as possible
At trace level
Reliably
Quickly
Find a needle in the haystack
Find the “interesting hayes“ in the haystack
Available tools through two examples
Identifying transformation products in wastewater
Screening for these transformation products in river
Agilent’s Saturday Innovation Symposium ASMS, Indianapolis, Indiana, USA June 3, 2017
E CEnvironmental
Analytical Chemistry
Reduction of
contaminants at the
source
Always new drugs and new
industrial compounds
Design wastewater
treatment plants to
remove trace organic
contaminants
Context & Background
Merel and Snyder, 2016 Environment International 96, 98-117
Agilent’s Saturday Innovation Symposium ASMS, Indianapolis, Indiana, USA June 3, 2017
Context & Background E CEnvironmental
Analytical Chemistry
Secondary
Wastewater
Treatment
Receiving Water
Ozonation + Filtration
O3 c
onta
cto
r
Dual F
ilter
TOrCs TOrCs
TOrCs
Ozonation
by-products ?
Research Questions
How do ozone and filtration affect water quality?
Agilent’s Saturday Innovation Symposium ASMS, Indianapolis, Indiana, USA June 3, 2017
Sample
Collection
Workflow E CEnvironmental
Analytical Chemistry
Agilent’s Saturday Innovation Symposium ASMS, Indianapolis, Indiana, USA June 3, 2017
E CEnvironmental
Analytical Chemistry
Secondary
Wastewater
Treatment
Receiving Water
Ozonation + Filtration
O3 c
onta
cto
r
Dual F
ilter
TOrCs TOrCs
TOrCs
Ozonation
by-products ?
One major German wastewater treatment plant
Pilote for ozonation and biofiltration
Sampling
Agilent’s Saturday Innovation Symposium ASMS, Indianapolis, Indiana, USA June 3, 2017
Sample
Collection
Sample
Preparation
Centrifugation
Analysis without preconcentration
Triplicate field samples
Before Ozonation
After Ozonation
After
Biofiltration
E CEnvironmental
Analytical Chemistry
Workflow
Agilent’s Saturday Innovation Symposium ASMS, Indianapolis, Indiana, USA June 3, 2017
Sample
Collection
Sample
Preparation
Sample Analysis
Triplicate field samples
Before Ozonation
After Ozonation
After
Biofiltration
LC-QTOF Analysis
Inject 100 µL of sample
Reversed phase C18 column, gradiant water/acetonitrile with 0.1% formic acid
Electrospray Ionization, positive mode, m/z 50-1,000
Agilent 1260 infinity LC
Agilent 6550 QTOF
E CEnvironmental
Analytical Chemistry
Workflow
Agilent’s Saturday Innovation Symposium ASMS, Indianapolis, Indiana, USA June 3, 2017
Sample
Collection
Sample
Preparation
Sample Analysis
Data Processing
Sample
Collection
Sample
Preparation
Sample Analysis
Data Processing
Data Quality Assessment
Find Compound (By Molecular Feature)
Compound Alignment
Initial Filtering
Find Compound (By Ion)
Compound Alignment
Filtering
Sample Comparison Principal Component Analysis
Hierarchical Clustering
Statistical Analysis (Anova, T-test, Fold Change)
Find Similar Entities
MassHunter
Qualitative Analysis
E CEnvironmental
Analytical Chemistry
Workflow
Total Ion Chromatogram (TIC)
Distinguish 20-50 major compounds
Extracted Ion Chromatogram (EIC)
Individual chromatogram for each compound found (1000-3000)
MFE
Agilent’s Saturday Innovation Symposium ASMS, Indianapolis, Indiana, USA June 3, 2017
Sample
Collection
Sample
Preparation
Sample Analysis
Data Processing
Data Quality Assessment
Find Compound (By Molecular Feature)
Compound Alignment
Initial Filtering
Find Compound (By Ion)
Compound Alignment
Filtering
Sample Comparison Principal Component Analysis
Hierarchical Clustering
Statistical Analysis (Anova, T-test, Fold Change)
Find Similar Entities
MassHunter
Qualitative Analysis
Mass Profiler
Professional
Low
High
Low High
RT window (min)
Mass
win
dow
(ppm
)
Excessive
Alignment
Insufficient
Alignment
E CEnvironmental
Analytical Chemistry
Workflow
Agilent’s Saturday Innovation Symposium ASMS, Indianapolis, Indiana, USA June 3, 2017
Sample
Collection
Sample
Preparation
Sample Analysis
Data Processing
Data Quality Assessment
Find Compound (By Molecular Feature)
Compound Alignment
Initial Filtering
Find Compound (By Ion)
Compound Alignment
Filtering
Sample Comparison Principal Component Analysis
Hierarchical Clustering
Statistical Analysis (Anova, T-test, Fold Change)
Find Similar Entities
MassHunter
Qualitative Analysis
Mass Profiler
Professional
Mass Profiler
Professional
MassHunter
Qualitative Analysis
Recurs
ion
E CEnvironmental
Analytical Chemistry
Workflow
Agilent’s Saturday Innovation Symposium ASMS, Indianapolis, Indiana, USA June 3, 2017
Sample
Collection
Sample
Preparation
Sample Analysis
Data Processing
Data Quality Assessment
Find Compound (By Molecular Feature)
Compound Alignment
Initial Filtering
Find Compound (By Ion)
Compound Alignment
Filtering
Sample Comparison Principal Component Analysis
Hierarchical Clustering
Statistical Analysis (Anova, T-test, Fold Change)
Find Similar Entities
MassHunter
Qualitative Analysis
Mass Profiler
Professional
Mass Profiler
Professional
Mass Profiler
Professional
MassHunter
Qualitative Analysis
Recurs
ion
E CEnvironmental
Analytical Chemistry
Workflow
Agilent’s Saturday Innovation Symposium ASMS, Indianapolis, Indiana, USA June 3, 2017
Compound Distribution E CEnvironmental
Analytical Chemistry
1 3 5 7 9 11 130
200
400
600
800
1000
Compound mass vs. Retention time (min)
Overview of compound distribution
Agilent’s Saturday Innovation Symposium ASMS, Indianapolis, Indiana, USA June 3, 2017
Overall Impact of Ozonation E CEnvironmental
Analytical Chemistry
-60 -40 -20 0 20 40 60-50
-30
-10
10
30
50
Component 1 (37%)
Com
ponent 2 (
25%
)
Principal Component Analysis Venn Diagram
After ozonation
Before treatment
After biofiltration
506 239232
963
95 242
35
After ozonation
Before treatment
Tap water
After biofiltration
PCA shows
difference between sample types
good reproducibility (replicates clustering together)
Consider features in 100% replicates of at least one sample type
2,312 features left
Distribution of feature through Venn Diagram
Agilent’s Saturday Innovation Symposium ASMS, Indianapolis, Indiana, USA June 3, 2017
E CEnvironmental
Analytical Chemistry
Perform ANOVA
(p-value <0.05)
Keep the feature with
statistically significant
difference
Before
treatment
After
ozonation
After
biofiltration
Lower concentration Higher concentration
Key
1692 compounds
Cluster 1
Compound
s formed
during
ozonation
and stable
during
biofiltration
Cluster 2
Compounds
released
during
biofiltration
Cluster 3
Compounds transformed
during ozonation and
potentially further attenuated
during biofiltration
Cluster 4
Compounds
transformed
during
ozonation but
regenerated
during
biofiltration
Cluster 5
Compounds with limited
alteration during ozonation and
biofiltration
Cluster 6
Compounds
attenuated
mostly during
biofiltration
Overall Impact of Ozonation
Agilent’s Saturday Innovation Symposium ASMS, Indianapolis, Indiana, USA June 3, 2017
E CEnvironmental
Analytical Chemistry
Perform ANOVA
(p-value <0.05)
Keep the feature with
statistically significant
difference
Before
treatment
After
ozonation
After
biofiltration
Lower concentration Higher concentration
Key
1692 compounds
Cluster 1 Cluster 2 Cluster 3 Cluster 4 Cluster 5
Clu
ste
r 6
Before
treatment
After
ozonation
After
biofiltration
Overall Impact of Ozonation
Cluster 1 Cluster 2 Cluster 3 Cluster 4 Cluster 5 Cluster 6
Agilent’s Saturday Innovation Symposium ASMS, Indianapolis, Indiana, USA June 3, 2017
E CEnvironmental
Analytical Chemistry
Relevance of N-oxide formation during ozonation?
Tertiary
amine N-oxide
R3N
R2
R1
R2
R3 N+
O
R1O
3
Search For N-oxides
Match precursor with transformation product and find addition of mass shifted by n*16
Mass in the IUPAC system
H 1.007825
C 12.000000
N 14.003074
O 15.994915
Kendrick mass (based on O)
H 1.008145
C 12.003815
N 14.007526
O 16.000000
Agilent’s Saturday Innovation Symposium ASMS, Indianapolis, Indiana, USA June 3, 2017
100 200 300 400 500
0.2
0.3
0.4
Precursors TPs
Kendrick Mass (O)
Kendri
ck M
ass D
efe
ct
100 200 300 400 5000.1
0.2
0.3
0.4
Precursors Oxides
Kendrick Mass (O)
Kendri
ck M
ass D
efe
ct
100 200 300 400 5000.1
0.2
0.3
0.4
Precursors N-oxides
Kendrick Mass (O)
Kendri
ck M
ass D
efe
ct
100 200 300 400 500
0.2
0.3
0.4
Precursors TPs
Kendrick Mass (O)
Kendri
ck M
ass D
efe
ct
100 200 300 400 5000.1
0.2
0.3
0.4
Precursors Oxides
Kendrick Mass (O)
Kendri
ck M
ass D
efe
ct
100 200 300 400 5000.1
0.2
0.3
0.4
Precursors N-oxides
Kendrick Mass (O)
Kendri
ck M
ass D
efe
ct
100 200 300 400 500
0.2
0.3
0.4
Precursors TPs
Kendrick Mass (O)
Kendri
ck M
ass D
efe
ct
100 200 300 400 5000.1
0.2
0.3
0.4
Precursors Oxides
Kendrick Mass (O)
Kendri
ck M
ass D
efe
ct
100 200 300 400 5000.1
0.2
0.3
0.4
Precursors N-oxides
Kendrick Mass (O)
Kendri
ck M
ass D
efe
ct
O-shift Probability Precursors → Oxides Precursors → (N-)Oxides (without restriction) (RTprecursor < RToxide)
1 Very High 42 → 34 27 → 27
2 High 26 → 21 5 → 7
3 Average 19 → 12 1 → 1
4 Low 18 → 11 4 → 3
5 Very Low 3 → 2 -
All - 99 → 60 39 → 37
How do we know O is fixed added to N
and not another Element?
Apply retention time filter
Find precurors and their oxides
KMoxide = n.16 + KMprecursor
KMDoxide = KMDprecursor
E CEnvironmental
Analytical Chemistry
Search For N-oxides
Agilent’s Saturday Innovation Symposium ASMS, Indianapolis, Indiana, USA June 3, 2017
E CEnvironmental
Analytical Chemistry
Identify precursor
Suspect search
(Stoff-Ident)
library match
Standard
Confirm identity
of the N-oxide
(standard, library
match, diagnostic
evidence)
Check abundance
of the precursor
and N-oxide after
filtration
5.6 5.8 6.0 6.2 6.4
150
100
50
0
50
100
150
Retention Time (min)
Rela
tive
Abundance (
%)
7.0 7.2 7.4 7.6 7.8 8.0
150
100
50
0
50
100
150
Retention Time (min)
Rela
tive
Abundance (
%)
10.0 10.2 10.4 10.6 10.8 11.0
0
50
100
150
50
100
150
Retention Time (min)
Rela
tive
Abundance (
%)
100 150 200 250 300
0
50
100
150
50
100
150
m/z
Rela
tive
Abundance (
%)
50 100 150 200 250 300
0
50
100
150
50
100
150
m/z
Rela
tive
Abundance (
%)
50 200 350 500 650 800
0
50
100
150
50
100
150
m/z
Rela
tive
Abundance (
%)
100 150 200 250 300
0
50
100
150
50
100
150
m/z
Rela
tive
Abundance (
%)
50 100 150 200 250 300
0
50
100
150
50
100
150
m/z
Rela
tive
Abundance (
%)
50 200 350 500 650 800
0
50
100
150
50
150
100
m/z
Rela
tive
Abundance (
%)
Tiapride N-oxide Clarithromycin N-oxide
Ch
rom
ato
gra
mM
S/M
S s
pectru
m (2
0eV
)M
S/M
S s
pectru
m (4
0eV
)
Tramadol N-oxide
Ozonated Water
Analytical Standard
Ozonated Water
Analytical Standard
Ozonated Water
Analytical Standard
Ozonated Water
Analytical Standard
Ozonated Water
Analytical Standard
Ozonated Water
Analytical Standard
Ozonated Water
Analytical Standard
Ozonated Water
Analytical Standard
Ozonated Water
Analytical Standard
Identification of N-oxides
Agilent’s Saturday Innovation Symposium ASMS, Indianapolis, Indiana, USA June 3, 2017
E CEnvironmental
Analytical Chemistry
Product Ions Shifted by O Formation of New Specific Product Ions
If no standard is available for the N-oxide, proceed by diagnostic evidence
Identification of N-oxides
E CEnvironmental
Analytical Chemistry
Identity confirmed for 10 precursors and their N-oxides
These N-oxides are not retained or degraded by further filtration
Identification of N-oxides
Name Formula
Sulpiride C15H23N3O4S
Tiapride C15H24N2O4S
Lidocaine C14H22N2O
Amisulpride C17H27N3O4S
Tramadol C16H25NO2
Venlafaxine C17H27NO2
Diphenhydramine C17H23NO
Citalopram C20H21FN2O
Clarithromycin C38H69NO13
Clindamycin sulfoxide C18H33N2O6SCl
N-oxides in Surface Water
15th EuCheMS International Conference on Chemistry and the Environment Leipzig, Germany
E CEnvironmental
Analytical Chemistry
Collision Cell
-
Fragmen
tation
of
selected io
n
MS/MS Spectrum
Ion source
Mobile Phase
Quadrupole
Form
ation
of
precu
rsor io
ns
Sequ
ential
isolatio
n
of n
precu
rsor io
ns
Collision Cell
Fragmen
tation
of
selected io
n
MS/MS Spectrum
Fragmen
tation
of
selected io
n
Collision Cell MS/MS Spectrum
Compound specific MS/MS
spectrum
Direct interpretation and
comparison to library
Sensitivity impaired by the
number of precursor ions
Usual Targeted MS/MS approach
September 20-24, 2015
15th EuCheMS International Conference on Chemistry and the Environment Leipzig, Germany September 20-24, 2015
E CEnvironmental
Analytical Chemistry
Data processing through multiple step algorithm
All-Ions MS/MS approach
Ion source
Mobile Phase
Quadrupole
Form
ation
of
precu
rsor io
ns
No
isolatio
n o
f p
recurso
r ion
s
Collision Cell
Simu
ltaneo
us
fragmen
tation
of
all precu
rsor io
ns
Mass Spectrum
Complex and
non-specific
MS/MS
spectrum
Low MS/MS
specificity
Sensitivity
independent of
the number of
precursor ions
0 eV 10 eV 20 eV 40 eV
N-oxides in Surface Water
2 3 4 5 6
0
20
40
60
80
100
120
Retention Time (min)
Ab
un
da
nc
e (
%)
256 257 258 259 260
0
20
40
60
80
100
120
m/z
Ab
un
da
nc
e (
%)
2.0 2.5 3.0 3.5 4.0
1000
1000
1000
1000
100
Retention Time (min)
Ab
un
da
nc
e (
%)
Chromatogram of [C9H7Cl2N5 + H]+ m/z 256.0151
Peak found at retention time: 3.226
min
Precursor ion
Product ion 1
Product ion 2
Product ion 3
Product ion 4
Acquired spectrum Theoretical spectrum
Step 1 Formula From Library
Lamotrigine: C9H7Cl2N5
Step 2 Check potential occurrence
0 eV [M+H]+ [M+Na]+
Step 3 Examine MS spectrum
Number of characteristic ions
Mass accuracy
Isotope relative abundance
Step 4 Confirm identity with MS/MS information
Obtain production ions from Library
Extract relevant chromatogram
Co-elution precursor vs product ion
(0eV)
(20eV)
(40eV)
(40eV)
(40eV)
Minimum
2 product ions
Provide
identification score
at MS level
S/N threshold
Extended& relevant
15th EuCheMS International Conference on Chemistry and the Environment Leipzig, Germany September 20-24, 2015
E CEnvironmental
Analytical Chemistry
N-oxides in Surface Water
Agilent’s Saturday Innovation Symposium ASMS, Indianapolis, Indiana, USA June 3, 2017
E CEnvironmental
Analytical Chemistry
Fast Screening of Suspect Compounds in Water Samples
0 200 400 600 800 1000 12000
5.010 4
1.010 5
1.510 5
2.010 5
2.510 5
Distance from source (kms)
Peak a
rea
N-oxides in Surface Water
Clarithromycin
N-oxide
Library spectrum
(20 V)
Over 100 compounds
identified with the All
Ion Fragmentation
Persticides
Personal care products
Pharmaceuticals
Metabolites
E CEnvironmental
Analytical Chemistry
Conclusions & Perspectives
Conclusions
High resolution mass spectrometry allows collecting information on unknown chemicals
Software packages allow traight forward data processing and visualization
Quick statistical analysis with Mass Profiler Professional
Large volume of data can be restricted to isolate specific chemicals (TPs, Intermediates...)
N-oxide proved to be significant ozonation transformation products
N-oxide also found in river with fast suspect screening approach (All Ion Fragmentation)
Agilent’s Saturday Innovation Symposium ASMS, Indianapolis, Indiana, USA June 3, 2017
Acknowledgment E CEnvironmental
Analytical Chemistry
Environmental Analytical Chemistry Group
Thank You for your attention!
Agilent’s Saturday Innovation Symposium ASMS, Indianapolis, Indiana, USA June 3, 2017