HPTLC/AMD-EDA - HPLC-qTOF/MS in water and ... Landeswasserversorgung HPTLC/AMD-EDA - HPLC-qTOF/MS in...

ZweckverbandLandeswasserversorgung

HPTLC/AMD-EDA - HPLC-qTOF/MS in water and environment analysis

Stefan C. Weiss, Wolfgang Schulz and Walter H. Weber

Zweckverband Landeswasserversorgung Laboratory for Operation Control and Research

Langenau, Germany

[email protected]

Club de CCM - 15èmeannée 6 Juin 2013


Content

Introduction

Ground water contamination from synthetic turf

Identification of pesticides in ground water

Effect-Directed Analysis with Vibrio fischeri

Ozonation reaction products

Conclusion


Landeswasserversorgung is situated in South Germany

www.wikipedia.de

Paris Langenau


Distribution area in South West Germany

3 Million customers within distribution area

Long-distance water fraction approx. 50%

Distribution area of LW Pipelines of LW N

Hessen Bayern

Rheinland-Pfalz

Frankreich

Schweiz Österreich

Heidel- berg

Tauber- bischofsheim

Bad Mergentheim

Crailsheim Schwäb. Hall Karlsruhe

Heilbronn

Pforzheim

Stuttgart Tübingen

Baden- Baden

Freuden- stadt

Aalen

Ulm Reutlingen

Offen- burg

Rottweil

Freiburg Tuttlingen

Biberach

Ravensburg

Friedrichshafen Singen

Lörrach

Konstanz

Bayern

SBH Osterbuch SBH Aufhausen

Egau- Wasserwerk

VPW Burgberg VPW Niederstotzingen WW Langenau

RWP Leipheim

SBH Amstetten

WW Blaubeuren

SBH Heuberg

BH Schönbühl

Distribution area of LW in South West Germany

Langenau Waterworks


Danube water treatment

Ground water Blaubeuren Ground water Burgberg Spring water Egau waterworks

Groundwater Donauried

External sources

90

80

70

60

50

40

30

20

10

0

Million m3 / Year

2007 1999 2000 2001 2002 2003 2004 2005 2006

100 92,0

2008

4,7

28,0

18,7

9,9

2,7

28,0

4,0

31,2

18,5

10,0

3,0

26,2

92,9

4,0

28,0

16,1

10,9

3,1

31,3 93,4 94,0

29,2

3,0

10,8

16,7

30,3

4,0 4,2 4,1 3,7

32,5

17,4

11,3 4,0

32,9 102,3

27,0

15,0

11,0

3,2

38,3 98,6

27,9

15,6

9,2

2,6

37,0 96,0

3,7

29,8

15,4

7,4

2,7

36,1 95,1

3,4

28,5

15,5

6,6

2,5

37,3 93,8

3,4

32,0

17,4 3,4

2,1

33,4 91,7

Water treatment from different resources


Contact Slurry

Slurry Treatment

Slurry Treatment

Groundwater decarbonized

Groundwater

Amstetten

Osterbuch

Activated Carbon

Flocculation - agent (Fe +++ ) Lime Milk Flocculation aid

Flocculation - agent (Fe +++ )

Lime Milk

Flocculation aid

Ozone


Hydroanthrazit Quarz sand Donau

Chlorine dioxide

Chlorine dioxide Orthophosphate

Raw Water Pump Station

Filtrate Tank Pure Water Tank Pump Station

Filter Unit Ozonation Unit

Flocculation Unit 1 Raw Water Tank

Pre-mixing Unit Flocculation Unit 2

Q FL2 Q FL1

Danube

Treatment of Danube water at Langenau waterworks


Drin

king

Wat

er, G

roun

dwat

er

Sur

face

Wat

er

Detection Separation Enrichment

Solid Phase Extraction

Light phase perforator

UV-Scan

Toxicity

MS MS/MS

ECD, FID, PND

Liquid/Liquid-Extraction

SEC

HPTLC

CC

HPTLC

GC

HPLC

Clean-up

„Multidimensional Screening“


Content

Introduction





Conclusion


Map of ground water monitoring wells in the water protection area


60 µl TBME-Extrakt

0

100

200

300

400

500

600

700

800

0 0.125 0.25 0.375 0.5 0.625 0.75 0.875 1Rf

AU

190 nm200 nm220 nm240 nm260 nm280 nm300 nm

Schematic diagram of the HPTLC/AMD analysis

Development AMD 2

Detection Scanner 3

Application TLC Sampler 4


HPTLC/AMD fingerprint of a ground water sample


? HPTLC/AMD fingerprint of a ground water sample


nano-Electrosprayion source

Quadrupole

Flight tubewith reflector

Tandem mass spectrometerwith full-scan capabilityMass accuracy: 2 ppm

Eluent from HPLC pump

H. Luftmann (2004) Anal. Bioanal Chem., 378, 964

Transfer of substances via HPTLC-MS interface and MS analysis

Plunger with cutting edge and filter frit

Seal with carrier material


m/z 173.0379 Molecular formular:C8H10N2O4S

Asulam (Herbicide)

Identification via HPLC-qTOF/MS

Confirmed by reference


Content

Introduction





Conclusion


Direct abstraction of Danube water in the downstream of the Cities of Ulm and Neu-Ulm

Neu-Ulm

Ulm

Langenau

Günzburg

Leipheim

Wiblingen

Erbach

Dellmensingen

Abstraction point

To waterworks Langenau

Central sewage treatment plant


Time variation curve of Benzotriazoles in the Danube (Leipheim, 2010)

N = 359

Con

cent

ratio

n in

ng/

L


Contact Slurry

Slurry Treatment

Slurry Treatment


Groundwater

Amstetten

Osterbuch

Activated Carbon



Lime Milk

Flocculation aid

Ozone



Chlorine dioxide







Q FL2 Q FL1

Danube



Schematic illustration of the experimental ozonation reactor

NH

NN

CH3

+ O3 ?


Separation of the main ozonation products via „2D-chromatograpy“

Sample

HPLC (RP)

HPTLC/AMD (NP)

HPTLC/AMD (NP)

Derivatization

HPLC-QTOF/MS

HPLC-QTOF/MS


Separation of HPLC-fractions via HPTLC/AMD (λ = 254 nm)

λ = 254 nm La

ufst

reck

e

HPLC Fractions

1 2 3 4 5 6

Mig

ratio

n


[M+H]+268.3

[M+Na]+290.3

0.0

0.2

0.4

0.6

0.8

1.0

Intens.

1.0

x108

150 200 250 300 350 400 m/z

[M+H]+268.3

[M+Na]+290.3

0.0

0.2

0.4

0.6

0.8

1.0

Intens.

1.0

x108

150 200 250 300 350 400 m/z

H. Luftmann (2004), Anal. Bioanal Chem., 378, 964

Schematic diagram of the HPTLC-MS coupling Development

AMD 2 Transfer and Detection

TLC-MS interface and ESI-MS Application

TLC Sampler 4


Localisation of the postulated oxidation product M147 on the developed HPTLC plate

Proposed structure

Retention time

Inte

nsity

[cou

nts]

Inte

nsity

[cou

nts]

Mass spectrum

Mig

ratio

n


Confirmation of the carbonyl group with 2,4-DNPH*

NN

NH

N

NH

NO2O2N

NN

NH

OH

NH2

NH

NO2O2N

+H2SO4

-H2O

M = 147,0433 Da [M-H]- = 146,0360 Da

M = 327,0716 Da [M-H]- = 326,0643 Da

M = 198,0389 Da [M-H]- = 197,0316 Da

* 2,4-Dinitrophenylhydrazin


Confirmation of the carbonyl group with 2,4-DNPH

Extraction of the derivatization product (m/z 326) for the oxidation product M147

Retention time

Inte

nsity

[cou

nts]

Inte

nsity

[cou

nts]

Mig

ratio

n

Mass spectrum


Content

Introduction





Conclusion


Luminescence bacteria Vibrio Fischeri

• Marine bacterium

• Lives in symbiosis with marine life forms

• Continuous bioluminescence

• Bioluminescence is coupled to

energy metabolism

Inhibition of electron transport Isolation

Narcotic effects

Suspension of Vibrio fischeri in water

Reference: Reinecke, N. (2003), Dissertation, Universität Hamburg


Contact Slurry

Slurry Treatment

Slurry Treatment


Groundwater

Amstetten

Osterbuch

Activated Carbon



Lime Milk

Flocculation aid

Ozone



Chlorine dioxide







Q FL2 Q FL1

Danube



10 min

pH3 pH8 pH3 pH8

Metoprolol Propranolol

200µl 200µl 100µl 100µl

Standards

M P 134 ng/µl 10 ng/µl

O

OH

NH

CH3

CH3

OCH3

Ozonation by-products of ß-blockers


10 min

CH3N

N

O

Cl

NH2

Ozonation by-products of the main metabolite of the pesticide Chloridazon


Potential ground water contamination by synthetic turfs

500 µl500 µl

Leaching test: (analog DIN 38414-S4)

Elution of 40 g sample with 80 g water Duration: 24 h Liquid/liquid extraction of the eluate Extraction solvent: MTBE Blank: Ultra pure water


Fingerprint of waste water samples (Δt = 6 month)

2. Sample series 1. Sample series


Content

Introduction





Conclusion


Ref.: Google Maps

Ground water Catchment

Aerial photo of the region of interest

Sports field


Simulation of leaching synthetic turf with water

Leaching test: (analog DIN 38414-S4)

Elution of 40 g sample with 80 g water Duration: 24 h Liquid/liquid extraction of the eluate Extraction solvent: Tert-butyl-methyl-ether Blank: Ultra pure water

Synthetic turf:

PUR rubber granulate (elastic layer)

Publication in Preparation


Weisemann, C., Kreiss, W., Rast, H-G., Eberz. G.;“Analytical Method for Investigating Mixtures for Toxic Components.” European Patent No: EP 0 588 139 B1.

Development AMD 2

Immersion Vibrio fischeri

Application TLC Sampler 4

Detection Bioluminizer

Schematic diagram for the HPTLC-AMD analysis with bioluminescence detection


HPTLC/AMD analysis

Eluents: MeOH:NH3 (4%) Dichloromethane n-Hexane

Chromatography:

Detection: - UV absorption (multiple wavelength scan) - Fluorescence quenching at 254 nm - Fluorescence, excitation at 366 nm - Luminescence inhibition test



Detection of luminescence inhibition (Vibrio fischeri)

Rf =1

Rf =0

λ = 254 nm

Bla

nk e

xtra

ct

20 µ

l TB

ME

extr

act

40 µ

l TB

ME

extr

act

60 µ

l TB

ME

extr

act

Bla

nk tr

ack

Ref

eren

ce M

ix



InhibitionInhibition−

=Γ100

Gamma-Value:

Gamma value chromatogram (proportional to luminescence inhibition)

40 µl TBME extract


[M+H]+268.3

[M+Na]+290.3

0.0

0.2

0.4

0.6

0.8

1.0

Intens.

1.0

x108

150 200 250 300 350 400 m/z

[M+H]+268.3

[M+Na]+290.3

0.0

0.2

0.4

0.6

0.8

1.0

Intens.

1.0

x108

150 200 250 300 350 400 m/z

H. Luftmann (2004), Anal. Bioanal Chem., 378, 964

Schematic diagram of the HPTLC-MS coupling Development

AMD 2 Transfer and Detection

TLC-MS interface and ESI-MS Application

TLC Sampler 4


0

200

400

600

800

1000

4 5 6 7 8 9 10 11 12 13

Retentionszeit [min]

Mas

se

Non-Target-Screening via LC-QTOF

EIC: 143.0502

Retentionszeit [min] 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Inte

nsitä

t

TIC

EIC: 695.3117

EIC: 430.9735

EIC: 242.0129

0

800

700

600

500

400

300

200

100

m/z

EIC: 143.0502

Retentionszeit [min] 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Inte

nsitä

t

TIC

EIC: 695.3117

EIC: 430.9735

EIC: 242.0129

0

800

700

600

500

400

300

200

100

m/z

522 Compounds

Nano-LC

QTOF

Top view


Mass/Retention time diagram of all compounds in the eluate

522 Komponenten

Einzeichnen von Benzothiazolen


Mas

s

Retention time [min]

522 Compounds


Rf =1

Rf =0

NP

RP

HP

TLC

HPLC

Bioluminescence detection

Detection of luminescence inhibition (Vibrio fischeri) and transfer of the substances via HPTLC-MS interface to MS



0

10

20

30

40

50

60

70

80

90

100

200 250 300 350 400 450 500 550 600

Abs

orpt

ion

0

10

20

30

40

50

60

70

80

90

100

200 250 300 350 400 450 500 550 600

Abso

rptio

n

Identification of the unknown compound

UV/VIS

λ [nm]

λ [nm]

137.0243 (M+H) + 137.0243

(M+H) +

3 x10

0

1

2

3

4

5

6

7

8 136.0213 (M+H) +

Counts vs. Mass - to - Charge ( m/z ) 136 136.5 137 137.5 138

3 x10

0

1

2

3

4

5

6

7

8 136.0213 (M+H) +


137.0243 (M+H) + 138.0276

(M+H) +

3 x10

0

1

2

3

4

5

6

7

8 136.0213 (M+H) +


3 x10

0

1

2

3

4

5

6

7

8 136.0213 (M+H) +


137.0243 (M+H) + 137.0243

(M+H) +

3 x10

0

1

2

3

4

5

6

7

8 136.0213 (M+H) +


3 x10

0

1

2

3

4

5

6

7

8 136.0213 (M+H) +


137.0243 (M+H) + 138.0276

(M+H) +

Empirical formula: C7H5NS

137.0245 ( M+H) + 138.0276

( M+H) +

5 x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1 136.0216 ( M+H) +

Counts vs. Mass - to - Charge (m/z) 136 136.5 137 137.5 138

5 x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1 136.0216 ( M+H) +

Counts vs. Mass - to - Charge (m/z) 136 136.5 137 137.5 138

137.0245 ( M+H) + 138.0276

( M+H) +

MS

Reference

Sample

Database query Database query

Benzothiazole

N

S



Rf =1

Rf =0

NP

RP

HP

TLC

HPLC

Bioluminescence detection




0

10

20

30

40

50

60

70

80

90

100

200 250 300 350 400 450 500 550 600[ ]

Abs

orpt

ion

UV/VIS

λ [nm]

λ [nm]

Empirical formula: C7H5NOS MS

Reference

Sample

Database query Database query

N

SOH

2-Hydroxybenzothiazole

Identification of the unknown compound (II)




522 Komponenten


N

S

N

SNH2

N

SOH

Benzothiazole

2-Aminobenzothiazole


l


Mas

s



Target Analysis for Benzothiazole via Triple Quadrupol MS

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0Time, min

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e65.2e6

2-Aminobenzothiazole


2-(Methylthio)-benzothiazole

2-Mercaptobenzothiazole

Eluate of the elastic layer Q1 q2 Q3

N

SOH

N

SNH2

N

SSH

N

SS CH3


Rf =1

Rf =0

NP

RP

HP

TLC

HPLC




Combination of the Non-Target-Screening Strategies

Rf =1

Rf =0

Sportplatzbelag HPTLC-Extrakt bei 36 mm

0

200

400

600

800

1000

4 5 6 7 8 9 10 11 12 13

Retentionszeit [min]

Mas

se

52 Compounds



Mass spectra from the HPTLC zone with a migration distance of 36 mm



522 Komponenten


N

S

N

SNH2

N

SOH

Benzothiazol

2-Aminobenzothiazol

2-Hydroxybenzothiazol

Unknown Substance

Mas

s



MS/MS from the HPTLC zone with a migration distance of 36 mm 4 x10

0 0,2 0,4 0,6 0,8

1 1,2 1,4 1,6 1,8

2 2,2 2,4 2,6 2,8

3 3,2 3,4 3,6 3,8

4 4,2 4,4 4,6 4,8

…

123.0663 193.1192

Counts vs. Mass-to-Charge (m/z) 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460

177.0880

5 x10

0 0.25 0.5

0.75 1

1.25 1.5

1.75 2

2.25 2.5

2.75 3

3.25 3.5

3.75 4

4.25 4.5

4.75 5

5.25 5.5

5.75 6

6.25 6.5

6.75 7

7.25 7.5

7.75 8

8.25 8.5

8.75 9

9.25 9.5 359.2047

360.2070

Counts vs. Mass-to-Charge (m/z) 358.4 358.6 358.8 359 359.2 359.4 359.6 359.8 360 360.2 360.4 360.6 360.8 361 361.2 361.4 361.6 361.8 362

MS/MS

MS


1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0Time, min

0.0

000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

7.0e4

7.5e4

Water from the ground water catchment Q1 q2 Q3

Target Analysis for Benzothiazole via Triple Quadrupol MS


1 2 3 4 5 6 7 8 9 10 11 12 13 14Time, min

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e6

5.5e6

6.0e6

6.5e6

7.0e6

7.5e6

8.0e6

8.5e6

Mass transition 359.2 / 177

359.2 177

Eluate of the elastic layer


1 2 3 4 5 6 7 8 9 10 11 12 13 14Time, min

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e6

5.5e6

6.0e6

6.5e6

7.0e6

7.5e6

8.0e6

8.5e6

Eluat

1 2 3 4 5 6 7 8 9 10 11 12 13 14Time, min

0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

320

340

360

380

400

420

440

460

0.170.53

359.2 177

Dilution factor > 50000

Mass transition 359.2 / 177

Water from the ground water catchment


Content

Introduction





Conclusion


Conclusion

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0Time, min

0.0

000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

7.0e4

7.5e4

1 2 3 4 5 6 7 8 9 10 11 12 13 14Time, min

0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

320

340

360

380

400

420

440

460

0.170.53

359.2 / 177

Rf = 0

Rf = 1

3.0 2.5 2.0 1.5 1.0 0.5

Γ-Wert

N

S

Unknown Substance

Benzothiazole


Alexander Müller and the member of staff at the Laboratory for Operation Control and Research

Acknowledgments


Thank you for your attention!

HPTLC/AMD-EDA - HPLC-qTOF/MS in water and ... Landeswasserversorgung HPTLC/AMD-EDA - HPLC-qTOF/MS in...

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