Extraction and Fractionationin Effect-Directed Analysis

Martin Krauss & Werner Brack

Department Effect-Directed Analysis,

Helmholtz Centre for Environmental Research - UFZ,

Leipzig, Germany

EDA @ UFZ

Main focus on:

Identification &

assessment

of toxicants in

complex mixtures

Mainly

sediments & water

Our EDA approach

Biologicalanalysis

Biologicalanalysis

Chemicalanalysis

Fractionation

Confirmation

Toxicant

Complex

Sample

Extraction

Extraction

Exhaustive extractions result in heavy matrix load: cleanup required

“Total” concentrations

do not account for real exposition situation in sediments

do not consider bioavailability (bioaccessibility) of compounds

Extraction and preconcentration

are essential for biotesting (& chemical analysis)

Water samples:

Solid-phase extraction / (liquid-liquid extraction)

Sediment samples:

Solvent extraction / Pressurized liquid extraction (ASE)

Effect of cleanup on biotest result

Mutagenicity of sediment extracts spiked with Benzo(a)pyren

Ames fluctuation assay TA98 + S9

Masking of mutagenicity by matrix (cytotoxicity, …)

Loss of BaP (& other mutagens?) by GPC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 --

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 200.0

0.2

0.4

0.6

0.8

1.0Bitterfeld

inh

ibit

ion

ASE

TENAX

pe -

Green algae growth inhibition of sediment extract fractions

non-bioaccessible

PAHs

NH

N-phenyl-2-naphthylamine

Sch

wab e

t al., E

T&

C, 20

09

Considering bioavailability in extraction

TENAX extraction:

Rapidly desorbable fraction

Zhao & Pignatello, 2004

Extraction

EDA on sediments EDA on food

Matrix removal / cleanup

is often necessary

Cleanup methods are

available

Including bioaccessibility is

relevant for realistic exposure

Concepts and methods are

available

Matrix removal / cleanup

is necessary

Cleanup methods are

available or transferable

Is bioavailability an issue?

If yes, different concepts and

methods have to be applied

25.00 30.00 35.00

100 200 300

260

130

268

239134

100 300200

TIME

A

B

Extract

F2.5.8

Fractionation helps to

dramatically reduce

complexity

O

S

Fractionation

Wanted:

� Individual fractions with low overlap and high recovery

� Separation according to well defined properties → helps to identify compounds

� High throughput (many samples/time) and high capacity

(much sample)

Reducing complexity by fractionation

Colum Chromatography/Solid-phase extraction

(Semi-)preparative LC

Preparative capillary GC

NP-LC (NO -Phenyl)2

F2.1

.1 n

icht

h

alo

genie

rt

F2.1

.2

F2.1

.3

F2.1

.4

F2.1

.5 F2.1

.6

F2.1

.7 F2.1

.8

F2.1

.9

F2.1

.10

F

2.1

.11

Elektrondonor-Akzeptor-LC (PYE)

EXTRAKT

F1: unpolareAliphaten

F2: unpolare Aromaten

F3, F4: polareFraktionen

Säulenchromatographie (Al O ) 2 3

F2.1 PCDD/Fs

PCBs, PCNs

F2.2 PAHs

MW 152-166F2.3 PAHs

Mw178

F2.4 PAHs

MW 202

F2.5 PAHs

MW 226/228

F2.6 PAHs

MW 252

PAHs

MW 276>

PCBs: Cl + , ortho Cl - Tetra- bis Octa- CN, CDD/F

F1.8

.2 T

CD

F,

F1.8

.1T

CD

D,

F1.8

.3 P

eC

N

F1.9

.3 H

xC

N

F1.9

.2 P

eC

DF

F1.9

.1 P

eC

DD

F1.1

0.1

H

xCD

D

F1.1

0.2

H

xCD

F

F1.1

0.3

HpC

N

Größenausschluss-LC

Brack et al. J Chrom A (2003)

Reducing complexity by complex fractionation

AhR-receptor mediated toxicity of a sediment extract

NP-LC (NO -Phenyl)2

F2.5

.1

e.g

. TR

PF

2.5.

2 e.

g. B

aA

F2.

5.3

e.g

. C

HR

F2.5

.4 e.g

. 2M

BaA

F2.5

.5

e.g

. 10M

BaA

F2.5

.6 e

.g. 4M

CH

RF

2.5.7

e

.g. 3M

CH

RF

2.5

.8

e.g

. 9M

BaA

F2.

5.9

e

.g. 2M

CH

RF

2.5

.10

e

.g. 121

2D

NF

RP-LC (C18, endcapped, Polymer)

EXTRAKT

F1: unpolareAliphaten

F2: unpolare Aromaten

F3, F4: polareFraktionen

Säulenchromatographie (Al O ) 2 3

RP-LC (PYE)

F2.1 PCDD/Fs PCBs, PCNs

F2.2 PAHsMW 152-166

F2.3 PAHsMw178

F2.4 PAHsMW 202

F2.5 PAHsMW 226/228

F2.6 PAHsMW 252

PAHsMW 276>

F2.5.8.1 9MBaA

F2.5.8.2 22NBT

F2.5.8.3 2123DNF

F2.5.8.4 1MCHR

Reducing complexity by complex fractionation

AhR-receptor mediated toxicity of a sediment extract

Brack et al. J Chrom A (2003)

PyrenylNitrophenyl

PyrenylNitrophenyl

PyrenylNitrophenyl chlorinated diaromatic compounds (PCBs, PCDDs/Fs)

alkanes, alkenes, sulphur, non-planar diaromatic compounds

Sampleinjection keto-, hydroxy-, nitro-PAHs,

N-heterocyclic PACs, quinones

Nitrophenyl stationary phase (NO)

Porous Graphitised Carbon stationary phase (PGC)

Cyanopropyl stationary phase (CN)

N

CH 3

CH3

N+

O-

O

Step 1

Step 2

Step 3-5

PAHs, S-, O-heterocyclic PACs

Functional groups

CN

NO

PGC electron-rich surface

High effort of fractionation procedure

⇒ on-line combination of three different NP-columns

Lübcke-von Varel et al. 2008 J Chrom A 1185:31-42

Reducing complexity by fractionation

Results in 18 fractions

0

20

40

60

80

100

120

P1

P2

P3

P4

P5

P6

P7

P8

P9

P10

P11

P12

P13

P14

P15

P16

P17

P18

fractions

% o

f contr

ol

0

500

1000

1500

2000

2500

3000

3500

4000

P1

P2

P3

P4

P5

P6

P7

P8

P9

P1

0

P1

1

P1

2

P1

3

P1

4

P1

5

P1

6

P1

7

P1

8

pm

ol T

4 e

q/g

dry

sed

imen

t

0

5

10

15

20

25

30

35

40

P 1

P 2

P 3

P 4

P 5

P 6

P 7

P 8

P 9

P 1

0

P 1

1

P 1

2

P 1

3

P 1

4

P 1

5

P 1

6

P 1

7

P 1

8

% m

axim

um

in

du

cti

on

thyroid

hormone

disturbing

potency

estrogenicity

tumor promotion

(inhibition of GJIC)

0

10

20

30

40

50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

nu

mb

er

of

po

sit

ive w

ells

TA98

TA98 with metabolic

activation

mutagenicity

PCBs, PCDD/Fs, halogenated pesticides

PAHs polar compounds

Multi-endpoint EDA in Elbe sediment extracts

Lübcke-von Varel 2011, ES&T 45:2348

Polar compounds play dominant role for adverse effects of sediment contamination.

Page 14

N+

O-

O

N+

O-

O

N+

O-

O

N+O

-

O

F14.6

Two fractionation steps later(RP-C18, followed by NP-Pyrenyl):

1.8-dinitropyrene 1,6-dinitropyrene

Isolation and quantitative confirmation of 1,8- and 1,6-dinitropyrene

as cause of mutagenicity.

Significant contributorsto mutagenicity of other fractions:

N+

O-

O

N+

O-

O

1,3-dinitropyrene

3-nitrobenz-anthrone

Lübcke-von Varel 2012, Environ. Int. 45, 31

Multi-endpoint EDA in Elbe sediment extracts

Fractionation

EDA on sediments EDA on food

Fractionation is successful to

isolate toxicants and allow

identification

Fractionation methods are

available

Fractionation will be necessary

to isolate unknown toxicants

and allow identification

Fractionation methods are

transferable

Page 16

br_extrac t_pos 28.07.2009 10:23:05 PM

RT: 0.00 - 75.01

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75

Tim e (m in)

0

10

20

30

40

50

60

70

80

90

100

Rela

tive A

bundance

28.97

4.36

3.43

4.9242.90

5.86 28.13 35.11 44.58 48.48 49.0842.396.32 24.36 40.42 55.3233.06 58.6321.248.03 59.2814.27 19.83 62.68

3.04 66.16 73.51

NL:

9.29E7

TIC F : FTM S + p

A PCI c orona Full

m s

[100.00-400.00]

M S

br_ex trac t_pos

br_ex trac t_pos #30-6966 RT: 0.34-74.58 AV : 1734 NL: 5.62E5

F: FTM S + p A PCI corona Full m s [100.00-400.00]

120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

m /z

0

5

10

15

20

25

Rela

tive A

bundance

163.04130.16

117.09

344.34

395.37

283.26 376.29134.06 251.05187.04171.15 369.35205.09 341.21

111.04

327.20

150.03

277.09212.12 289.09 298.31379.34237.08

221.10

193.12 313.27

LC-HRMS: about 10 000 masses

detected, mostly unknowns

Mutagenic extract of water

sample in the River Elbe

A full scale EDA example

Bases

Cation

exchange

Acids & neutrals

Anion

exchange

Acids NeutralsBases

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

Acid fraction Basic fraction Neutral fraction

Nu

mb

er

of

Re

ve

rta

nts

pe

r p

late

(m

ax

=4

8) Without S9

With S9

Reducing complexity by fractionation

Mutagenic extract of water sample from River Elbe

Mutageniticy (Ames fluctuation assay, TA98)

0

5

10

15

20

25

30

35

40

45

N1 N2 N3 N4 N5 N6 N7 N8 N9 N10 N11

Num

ber

of

revert

ents

10 g of BR without S9

10 g of BR with S9

N2 mutagenic only with

activation, fraction eluted early

⇨ more hydrophilic compounds

N7 mutagenic only without activation, fraction eluted later

⇨ more lipophilic compounds

LIPOPHILICITY

Reducing complexity by fractionation

Neutral

fraction

Prep LC

C18 column

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

N2-1 N2-2 N2-3 N2-4 N2-5 N2-6 N2-7 N2-8 N2-9 N2-10 N2-11 N2-12 N2-13 N2-14 N2-15 N2-16

Nu

mb

er

of

revert

an

ts p

er

pla

te (

max=

48)

10 g of BR with S9

5 g of BR with S9

Three mutagenic fractions, N2-8 same number of revertants for 10 and 5 g of BR

⇒ Priority for the identification, 20-60 peaks per fraction

Reducing complexity by fractionation

Prep LCPhenyl-Hexyl

column

• Accurate mass

• Isotope patterns

• Accurate mass fragments

LC-HRMS(n)

Molecular formula

e.g. C13H10O2N2S• Spectral libraries

• Compound databases

(e.g., ChemSpider)

• Structure generation

(MOLGEN) Large number of

candidate structures

Step 1: Identification of candidate structures

Compound identification in the EDA context

SEITE 21

Predicted mutagenic potential

Retention timeprediction

MS fragmentation

prediction

Predicted KOW- and

pKa-values of fractions

Eff

ect-

based

2 Peaks with one candidate

each for aromatic aminesFinal Candidates

Confirm Identity & Effect

Ch

em

ical-

an

aly

ticall

y b

ased

20 Peaks,

5-150 Candidate structures

Aromatic amines

Predicted mutagenic potential

Stability of the Nitrenium ion of arom. amine

• Nitrenium ion is ultimate mutagen after metabolic

activation

• Stability of nitrenium ion is correlated with mutagenicity

P450Ar-NH2 ArNH+

Mutagenicity likely if

(∆∆∆∆EArNH+−∆−∆−∆−∆EArNH2) < (∆∆∆∆EPhNH+−∆−∆−∆−∆EPhNH2)

Aromatic Amines likely

Comparison of strains TA98 / YG1024 / YG1041

Step 2: Selection of candidates

Compound identification in the EDA context

Where are we now in EDA

Our EDA approach is working and the combination of fractionation, biotesting and nontarget chemical analysis is suitable to identify toxicants

No routine application, but site- and case-specific

Time- and resource-consuming:

large amount of sample material

large number of fractions to be tested and analyzed

Automatization of sample extraction & fractionation

Miniaturized and automated biotests would be very useful

5.00 10.00 15.00 20.00 25.00 30.00 35.000

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

5500000

6000000

6500000

7000000

7500000

8000000

8500000

9000000

9500000

1e+07

Time-->

Abundance

TIC: WER046.D

mut

agen

TA98m

utage

n TA

98 + S

9

mut

agen

TAM

ixm

utag

en T

AMix

+ S

9

estro

gen

andro

gen

anti-a

ndro

gendi

oxin-

like

thyr

oid

antib

iotic

Pardubice

Přelouč

Jirkow

Most

Jorba

Martorell

St. Joan Despí

Rundvaartbrug

Eenhoorn

Hansweert

Terneuzen

Making EDA easier for many samples

Use multivariate statistics to precede and guide

“classical EDA” approach

multivariate analysis of chemical and biological fingerprints

Current developments

Funding

TxB X