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ZweckverbandLandeswasserversorgung

HPTLC/AMD with Vibrio fischerias an example for bioactivity-based detection –

A new dimension in analytics

Wolfgang Schulz, Wolfram Seitz, and Walter H. Weber

Zweckverband Landeswasserversorgung, Langenau, Germany

International Symposium for HPTLC11. – 13. June 2008, HelsinkiInternational Symposium for HPTLC11. – 13. June 2008, Helsinki

ZweckverbandLandeswasserversorgungStructureStructure

Advanced data evaluation

Introduction

Vibrio fischeri HPTLC test / Calculation of inhibition profiles

Conclusions

Aspects of the luminescence inhibition test / Application examples


Landeswasserversorgung is situatedin South GermanyLandeswasserversorgung is situatedin South Germany

www.wikipedia.de

Helsinki

Langenau


Distribution area inSouth West Germany

3 Million customerswithin distribution area

Long-distance waterfraction approx. 50%

Distributionarea of LW

Pipelinesof LW N

HessenBayern

Rheinland-Pfalz

Frankreich

SchweizÖsterreich

Heidel-berg

Tauber-bischofsheim

Bad Mergentheim

CrailsheimSchwäb.

HallKarlsruheHeilbronn

Pforzheim

StuttgartTübingen

Baden-Baden

Freuden-stadt

Aalen

UlmReutlingen

Offen-burg

Rottweil

FreiburgTuttlingen

Biberach

Ravensburg

FriedrichshafenSingen

Lörrach

Konstanz

Bayern

SBH OsterbuchSBH Aufhausen

Egau-Wasserwerk

VPW BurgbergVPWNiederstotzingenWW Langenau

RWP Leipheim

SBH Amstetten

WW Blaubeuren

SBH Heuberg

BH Schönbühl

Distribution area of LW in South West GermanyDistribution area of LW in South West Germany

Langenau Waterworks


4,0

Surface water(Danube)

Ground waterBlaubeurenGround waterBurgberg

Spring waterEgau waterworks

Ground waterDanube Reed

Other

90

80

70

60

50

40

30

20

10

020021993 1994 1995 1996 1997 1998 1999 2000 2001

2,0 5,9 7,3 6,5 6,44,7 4,04,0

30,1

32,5 34,028,6

27,123,7

28,031,2

28,0

18,8

20,0 20,5

16,816,2

15,0

18,718,5

16,1

10,4

9,6 8,9

9,8 9,912,1

10,0

10,98,8

3,1 2,8

2,9 3,3 2,73,0

21,3

19,315,9 24,0 26,5

31,6 28,0 26,2 31,3

82,6

90,488,0 88,3 89,6

91,8 92,0 92,9 93,4100

3,0 3,1

4,0

30,3

16,7

10,8

29,394,0

3,0

2003

5,9

11,3

102,3

Use of different resources for drinking water productionUse of different resources for drinking water production

32,5

17,3

4,0

32,9

ZweckverbandLandeswasserversorgungRiver Danube in South Germany (Leipheim)River Danube in South Germany (Leipheim)

Pump Station

Danube


Direct abstraction of river water (near Leipheim)Direct abstraction of river water (near Leipheim)


Concept of „Multidimensional Screening“for water contaminantsConcept of „Multidimensional Screening“for water contaminants

Gro

und

wat

erSu

rfac

e w

ater

DetectionSeparationEnrichment

Solid phaseextraction

Light phasePerforation

UV/VIS

Bioactivity

MS, MS/MS

ECD, FID, PND

Liquid liquidExtraction

SEC

TLC

CC

TLC

GC

HPLC

TLC

GC

HPLC

Clean-up



Introduction


Conclusions


ZweckverbandLandeswasserversorgungSelected bioactivity-based tests for HPTLCSelected bioactivity-based tests for HPTLC

Neurotoxicity testAcetylcholinesterase(enzyme)

Cytotoxicity test for fungicidesRhodoturula rubra(yeast)

Cytotoxicity test for antibiotic or cytostaticsubstances

Bacillus subtilis(bacterium)

Screening test for bioactivityVibrio fischeri(luminescent bacterium)

PurposeTest

Reference: C. Weins (2006), Dissertation, University of Basel, Switzerland

ZweckverbandLandeswasserversorgungLuminescent bacteria Vibrio fischeriLuminescent bacteria Vibrio fischeri

• Marine bacterium

• Lives in symbiosis with marine life forms

• Continuous bioluminescence

• Bioluminescence is coupled to

energy metabolism

Inhibition of electron transportIsolation

Narcoticeffects

Suspension of Vibrio fischeriin water

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

ZweckverbandLandeswasserversorgungVibrio fischeri in water analysisVibrio fischeri in water analysis

• Classic application: Cuvette test (DIN 11348)

Detection of combination effects of toxic compounds(synergistic effects)

(1) LUMIStox 300(2) LUMIStherm


Reference: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.

Test Kit:BioluminexTM, ChromaDex, CA

Development ImmersionApplication(TLC Sampler)

Sample(extract)

Luminescence inhibition test on TLC platesLuminescence inhibition test on TLC plates


TLC plateafter immersioninto bacteria suspension

CCD cameraCamag Bioluminizer(typical detection time 40 sec)

Detection of luminescenceDetection of luminescence


Data evaluation –Calculation of inhibition profilesData evaluation –Calculation of inhibition profiles

ReferenceSample volume [µL] 10 20 30 40 50 60 70 80 90 100


233 159,35 153,31 154,55 156,95 2,32234 153,15 144,92 146,45 149,80 3,26235 144,05 137,28 139,55 141,80 3,19236 137,75 132,97 134,9 136,33 2,46237 136,45 133,89 133,35 134,90 0,75238 139,35 137,83 136,95 138,15 0,23239 146,4 146,19 146,1 146,25 0,04240 154,75 154,36 155,8 155,28 0,59241 162,5 162,92 163,8 163,15 0,14242 169,95 170,97 171,55 170,75 -0,13

Rn

SnP

n ii1I −=

I = Inhibitioni = Light intensityS = SampleR = Referencen = Number of pixel

Calculation of inhibition profilesCalculation of inhibition profiles

Nr. UGl Track6 UGr MW UG Inhibition [%]1 192,87 191,72 187,5 190,19 -0,812 193,1 191,97 188,35 190,73 -0,653 192,05 191,72 188,65 190,35 -0,724 195,05 192,47 190,75 192,90 0,225 196,15 192,69 192,1 194,13 0,746 197,8 189,25 193,7 195,75 3,327 197,05 184,14 193,65 195,35 5,748 198,2 177,42 193,7 195,95 9,469 199,4 171,47 195,35 197,38 13,12

10 199 168,53 195,95 197,48 14,6611 199,25 167,58 196,65 197,95 15,3412 200,25 166,64 197,6 198,93 16,2313 201,75 168,14 199,35 200,55 16,16


Inhibition chromatogramInhibition chromatogram

Track 6

-10,00

0,00

10,00

20,00

30,00

40,00

50,00

60,00

70,00

80,00

90,00

100,00

0 50 100 150 200 250Number of pixel

Inhi

bitio

n [%

]



Introduction


Conclusions



)ln(11

DosisbaeccR ⋅−+

−+=

bba ceEC /1/50 )21( ⋅−⋅=

R: Response (inhibition) c: Controla: Location parameterb: Slope parameter

Comparison of sensitivity of cuvette test and HPTLC testComparison of sensitivity of cuvette test and HPTLC test

0

10

20

30

40

50

60

70

80

90

100

0 1 10 100 1000

Concentration in ng/µl

Inhi

bitio

n in

%

(1) LUMIStox300(2) LUMIStherm

Dose-effect relationshipof Bromoxynil– Cuvette test –

(1)(2)

(1)(2)

EC50 = 15 ng/µl


Br

Br

OH CN0

10

20

30

40

50

60

70

80

90

100

1 10 100 1000

Area concentration in ng/cm²

Inhi

bitio

n in

%

Dose-effect relationshipof Bromoxynil–HPTLC plate test –

1,32 cm²

0,56 cm²

0,16 cm²

1,32 cm²

0,56 cm²

0,16 cm²

EC50 = 77 ng/cm²

∑

∑−=

2

1

2

11 n

n

Rn

n

n

Sn

S

i

iI

∑

∑−=

2

1

2

11 n

n

Rn

n

n

Sn

S

i

iI

ISRin

InhibitionSampleReferenceIntensityPixel number

= ====


010

2030405060

708090

100

1 10 100 1000Area concentration in ng/cm²

Inhi

bitio

n in

%

1,32 cm²

0,56 cm²

0,16 cm²

1,32 cm²

0,56 cm²

0,16 cm²

0

10

20

30

40

50

60

70

80

90

100

0 1 10 100 1000

Concentration in ng/µl

Inhi

bitio

n in

%

(1) LUMIStox 300(2) LUMIStherm

Dose-effect relationshipof Bromoxynil– Cuvette test –

(1)(2)

(1)(2)

EC50 = 15 ng/µl

Calculation of the iso-inhibition value curveCalculation of the iso-inhibition value curve

Dose-effect relationshipof Bromoxynil–HPTLC plate test –


y = 0.346x - 17.459R2 = 0.9976

0

50

100

150

200

250

300

350

0 200 400 600 800 1000

Area concentration in ng/cm² (TLC test)

Con

cent

ratio

n in

ng/

µl (c

uvet

te te

st) A

VmF ⋅=

m: SlopeV: Sample volumeA: TLC application area

F = 1440

V = 500 µLA = 0,12 cm²

Curve of iso-inhibition valuesCurve of iso-inhibition values


ca. 180Ibuprofen

ca. 1650Fenazaquin

ca. 311Ioxynil

ca. 1440Bromoxynil

StructureSensitivity factorSubstance

Sensitivity factor for selected substancesSensitivity factor for selected substances


Chloridazon

Desphenyl-chloridazon

Methyl-desphenyl-chloridazon

1 2 3 4 5 6 7 8 9 10

Effect of incubation time on theinhibition of luminescenceEffect of incubation time on theinhibition of luminescence

Standard incubation time: 10 min

ZweckverbandLandeswasserversorgungChloridazon and metabolitesChloridazon and metabolites

N

N

OCl

H2N

N

NH

OCl

H2N

N

N

OCl

H2N CH3

N

NH

OCl

H2N

N

N

OCl

H2N

N

N

OCl

H2N

Chloridazon

Methyl-desphenyl-chloridazon(Metabolite B1)

Desphenyl-chloridazon(Metabolite B)

Degradation in soil

Transformationin soil

- Herbicide

- Application for sugar beets

- Used since 1964

- In 2005 approved for further 10 years in Germany


001 min

Comparison of different incubation timesComparison of different incubation times


010 min



180 min



300 min


Luminescence inhibition may change to intensification


Same amount of substance(approx. 25 nmol )

-80

-60

-40

-20

0

20

40

60

80

100

0 100 200 300 400 500 600

Incubation time [min]

Inhi

bitio

n [%

]

ChloridazonDesphenyl-ChloridazonMethyl-Desphenyl-Chloridazon


Inhibition

Intensification


Application example: Potential ground water contamination by coatings of sports fieldsApplication example: Potential ground water contamination by coatings of sports fields

500 µl500 µl

Sample preparation:

• 40 g granulate in 80 g H2O 24 h shaking

• Eluate filtered (0,4 µm membrane)

• Liquid-liquid extraction with TBME approx.. 1 ml extract


010 min

pH3 pH8 pH3 pH8

Metoprolol Propranolol

200 µl 200 µl 100 µl 100 µl

References

M P134 ng/µl 10 ng/µl

Application example: Bioactivity test of ozonation by-productsof beta-blocker substances

Application example: Bioactivity test of ozonation by-productsof beta-blocker substances


Application example: Fingerprint of waste water samples (Δt = 6 month)Application example: Fingerprint of waste water samples (Δt = 6 month)

2. Sample series1. Sample series



Introduction


Conclusions


ZweckverbandLandeswasserversorgungAdvanced data evaluationAdvanced data evaluation

• In general, quantitation requires reference standardsWhat a about unknown compounds?

• The semi-quantitative evaluation of the HPTLC chromatograms with bioactivity detection required a new data evaluation procedure:

(1) Linearization of the dose-effect relationship

(2) Calculation of the iso-inhibition volume V(50)

(3) Introduction of the reciprocal iso-inhibition volume 1/V(50)

This enables the comparison of the concentration of the same unknown substance in different samples


(1) Linearization of the dose-effect relationship –Gamma value(1) Linearization of the dose-effect relationship –Gamma value

jijijiji Vba ,,,, lglg ⋅+=Γ

0

10

2030

405060

7080

90100

1 10 100 1000Area concentration in ng/cm²

Inhi

bitio

n in

%

1,32 cm²

0,56 cm²

0,16 cm²

1,32 cm²

0,56 cm²

0,16 cm²

Gamma value

Extract (sample)jSubstance zonei

Gamma valueApplication volume (µl)VInhibition (%)I

ji

=

=

=Γ

=

=

,

II−

=Γ100

Dose-effect relationship

-2,5000

-2,0000

-1,5000

-1,0000

-0,5000

0,0000

0,5000

1,0000

1,5000

-1,2000 -1,0000 -0,8000 -0,6000 -0,4000 -0,2000 0,0000 0,2000 0,4000

lg Area concentration

lgG

amm

a

-2,5000

-2,0000

-1,5000

-1,0000

-0,5000

0,0000

0,5000

1,0000

1,5000

-1,2000 -1,0000 -0,8000 -0,6000 -0,4000 -0,2000 0,0000 0,2000 0,4000

lg Area concentration

lgG

amm

a

Linearised dose-effectrelationship

ZweckverbandLandeswasserversorgung(1) Linearised dose-effect relationship(1) Linearised dose-effect relationship

-2,5000

-2,0000

-1,5000

-1,0000

-0,5000

0,0000

0,5000

1,0000

1,5000

-1,2000 -1,0000 -0,8000 -0,6000 -0,4000 -0,2000 0,0000 0,2000 0,4000

lg Application volume

lgG

amm

a va

lue

Rf = 0,28Rf = 0,42Rf = 0,47Rf = 0,60Rf = 0,72Rf = 0,91


(2) Calculation of the iso-inhibition volume(2) Calculation of the iso-inhibition volume

jijijiji Vba ,,,, lglg ⋅+=Γ

1=Γ

Effect concentration 50

50=I

Gamma value Linearised dose-effectrelationship

Iso-inhibition volume

)50(lg0 ,,, jijiji Vba ⋅+=

iiii VcmI ⋅=≈

jiji bajiV ,, /

, 10)50( −=

Inhibition is proportional to theapplicated sample volume

Extract (sample)jSubstance zonei

Gamma valueApplication volume (µl)VInhibition (%)I

ji

=

=

=Γ

=

=

,

II−

=Γ100


(3) Introduction of the reciprocal iso-inhibition volume(3) Introduction of the reciprocal iso-inhibition volume

)50()50()50()50(

2,2,2,

1,1,1,

iii

iii

VcmVcm⋅=

⋅=)50()50( 2,1, ii mm =

)50()50( 2,2,1,1, iiii VcVc ⋅=⋅Substance zonei

Application volume (µl)VConcentration (ng/µl)c

=

==

)50(1

)50(1

2,

1,

2,

1,

i

i

i

i

V

Vcc

=

Reciprocaliso-inhibition volume

Comparison of substance i in two samples

The same mass for the same substance and the same inhibition


pH 7

K2

pH 2 pH 7

K1

pH 2

Reference

pH 7

Blanc water

pH 2

Methanol/Aceton 90:10

Comparison of different TBME extractsfrom wastewaterComparison of different TBME extractsfrom wastewater


Comparison of two extracts from wastewaterusing reciprocal iso-inhibition volumesComparison of two extracts from wastewaterusing reciprocal iso-inhibition volumes

0.01

0.1

1

10

100

1000

10000

1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96

hRf

1/V(

50) *

100

0 [1

/µL]

Extract K1 (pH 7) Extract K2 (pH 7)

Reciprocal iso-inhibition volume is proportional to thesubstance concentration

F = 500

ZweckverbandLandeswasserversorgungConclusionsConclusions

• Application of luminescent bacteria in HPTLCScreening for bioactivity of single substances even in complex

mixtures possible

• Vibrio fischeri HPTLC test shows high sensitivity compared tothe classic cuvette test

• Incubation time proved to be an essential parameterLuminescence inhibition may change to intensification

• New semi-quantitative evaluation procedure applying the concept of “reciprocal iso-inhibition volume”

Comparison of different samples by means of inhibition patternEvaluation of the concentration ratios of a substancein different samples

ZweckverbandLandeswasserversorgungThank you for your attention!Thank you for your attention!

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