Geosciences / Environmental Analytical Chemistry · Sylvain Merel, Christian Zwiener Environmental Analytical Chemistry Tracking the Occurrence and Fate of Contaminants in Water with

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


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


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


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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


Context & Background E CEnvironmental


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?


Sample

Collection

Workflow E CEnvironmental



E CEnvironmental


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


Sample

Collection

Sample

Preparation

Centrifugation

Analysis without preconcentration

Triplicate field samples

Before Ozonation

After Ozonation

After

Biofiltration

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Workflow


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

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Workflow


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

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Workflow

Total Ion Chromatogram (TIC)

Distinguish 20-50 major compounds

Extracted Ion Chromatogram (EIC)

Individual chromatogram for each compound found (1000-3000)

MFE


Sample

Collection

Sample

Preparation

Sample Analysis

Data Processing



Compound Alignment

Initial Filtering


Compound Alignment

Filtering





MassHunter


Mass Profiler

Professional

Low

High

Low High

RT window (min)

Mass

win

dow

(ppm

)

Excessive

Alignment

Insufficient

Alignment

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Workflow


Sample

Collection

Sample

Preparation

Sample Analysis

Data Processing



Compound Alignment

Initial Filtering


Compound Alignment

Filtering





MassHunter


Mass Profiler

Professional

Mass Profiler

Professional

MassHunter


Recurs

ion

E CEnvironmental


Workflow


Sample

Collection

Sample

Preparation

Sample Analysis

Data Processing



Compound Alignment

Initial Filtering


Compound Alignment

Filtering





MassHunter


Mass Profiler

Professional

Mass Profiler

Professional

Mass Profiler

Professional

MassHunter


Recurs

ion

E CEnvironmental


Workflow


Compound Distribution E CEnvironmental


1 3 5 7 9 11 130

200

400

600

800

1000

Compound mass vs. Retention time (min)

Overview of compound distribution


Overall Impact of Ozonation E CEnvironmental


-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


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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


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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


E CEnvironmental


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


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

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Search For N-oxides


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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


Rela

tive

Abundance (

%)

10.0 10.2 10.4 10.6 10.8 11.0

0

50

100

150

50

100

150


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


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Product Ions Shifted by O Formation of New Specific Product Ions

If no standard is available for the N-oxide, proceed by diagnostic evidence


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Identity confirmed for 10 precursors and their N-oxides

These N-oxides are not retained or degraded by further filtration


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


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


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


2 3 4 5 6

0

20

40

60

80

100

120


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


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




E CEnvironmental


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


Clarithromycin

N-oxide

Library spectrum

(20 V)

Over 100 compounds

identified with the All

Ion Fragmentation

Persticides

Personal care products

Pharmaceuticals

Metabolites

E CEnvironmental


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)


Acknowledgment E CEnvironmental


Environmental Analytical Chemistry Group

Thank You for your attention!


Documents

Geosciences / Environmental Analytical Chemistry · Sylvain Merel, Christian Zwiener Environmental Analytical Chemistry Tracking the Occurrence and Fate of Contaminants in Water with