analysis of estrogens and phytoestrogens using different ... · the tests format are 96 well...

Analysis of estrogens and phytoestrogensusing different ELISA kits in comparison

with UPLC-Q-TOF-MS and LC-MS/MS (QqQ)

Marinella Farré1,4, Rikke Brix1, Yasuhiro Goda2, Fernando Rubio3,Damià Barceló1

1 Department of Environmental Chemistry, IIQAB-CSIC, Barcelona, Spain2 Japan EnviroChemicals, Ltd., Osaka, Japan

3 Abraxis, LCC. 54 Steamwhistle Warminster, PA 18974, USA4 Sensors and Biosensors Group, UAB, Bellaterra, Spain

AQUAbase Workshop on ANALYTICAL METHODS18 - 19 January 2006, Aachen

http://www.uab.es/



Outline Presentation:

• 5 commercial ELISA kits from Japan EnviroChemicals, Ltd, (Tokyo,Japan), and from Abraxis (Warmister, USA) have been evaluated

• Natural and spiked samples of wastewater, river water, well waterand standard solutions were analyzed

• 2 different extraction procedures based on SPE involved in the studywill be presented and discussed




Outline Presentation:

• The concentrations measured using the different ELISA kits were compared withresults by LC-MS/MS (QqQ) (1,2,3)

• A newly developed method based on Ultra Performance Liquid Chromatography(UPLC) coupled to a quadrupole time-of flight mass spectrometry (Q-TOF mass spectrometer) will be presented for confirmatory identity of estrogens, andthe investigation of the presence of other structurally related compound, such as isoflavones

(1) M.S. Díaz-Cruz, M.-J. López de Alda, R. López, D. Barceló, J. Mass Spectrom. 38 (2003) 917-923(2) S. Rodriguez-Mozaz, M.-J. López de Alda, D. Barceló, Anal. Chem, 76 (2004) 6998-7006(3) M.-J. López de Alda, M.S. Díaz Cruz, M. Petrovic, Damià Barceló J. Chromatography A, 1000 (2003) 503-526


ELISA KITS

• 17-β-estradiol (E2)• Estrone (E1)• 17-α-ethinyl estradiol (EE2)• Estrogens (E1) + (E2) + estriol (E3)

• 17-β-estradiol (E2)-------------------------Polyclonal

E2 E1 E3 EE2

Monoclonal



CH3H OH

HO

OCH3

HO

CH3OH

HOH

HO

CH3 OHC

HO

CH




CH3H OH

HO E2

OCH3

HO E1

CH3OH

HOH

HO E3

CH3 OHC

HO

CH

EE2

O

OOH

HO

OH

GEN

O

OOH

HODAID

NPHO

C9H




CH3H OH

HO

OCH3

HO

CH3OH

HOH

HO

CH3 OHC

HO

CHO

OOH

HO

OH

GEN

O

OOH

HODAID

NPHO

C9H




CH3H OH

HO

OCH3

HO

CH3 OH

HOH

HO

CH3 OHC

HO

CH

O

OOH

HO

OH

O

OOH

HOHO

C9H




Samples:

• 5 wastewater treatment plants (WWTP)• 3 river samples• well water • spiked river samples• spiked WWTP effluents• spiked well water• standard solutions

Real samples

Fortified real samples

Standard solutions




Sample Magnetic particles ELISA

Clean-upSPE

LC-MS/MS UPLC-Q-TOF-MS

Dilu

tion

Dilu

tion

(1/1

0 or

mor

e) EVA

LUA

TIO

N

ELISA kits




500 mL of water sample Washing: 5 mL water and 5 mL hexane

C18 preconditioned: 7mL ACN7 mL MeOH

5 mL H2O

1000 mL of water sampleWashing: 5 mL water and 5 mL hexane

C18 preconditioned: 5 mL MeOH10 mL H2O

Elution: 5 mL dichloro methane

Evaporation N2

Reconstitution 1 mL MeOH 5mL MeOH

Evaporation

Reconstitution to 1 mL with MeOH

Elution: 5 mL dichloro methane5mL MeOH

Evaporation

Reconstitution to 300 µL with MeOH

Method A Method B

NH2-SPE




4 ELISA kits based on monoclonal antibodies for:• E1• E2, • EE2• estrogens mixture (E1+E2+E3)

The tests format are 96 well microtiter platesTime of assays 90 minutes

Analytes present in the sample and the enzymatic tracer are premixed

The mixture is added into each well, and allowed to compete for the antibodies immobilized on the surface of the wells

Color development is inversely proportional to the analyte concentration


http://www.uab.es/



E10.3041

C=(1.090+0.001647/A+0.001647)-1)1/-0.3041

E1+E2+E30.06014

C=(2.202+0.1177/A+0.1177)-1)1/-0.4621

EE20.09545

C=(1.250-0.2803/A-0.2803)-1)1/-1.37

E20.1249

C=(1.357-0.02119/A-0.02119)-1)1/-1.003

ESTRONE (E1)

-3 -2 -1 0 10.0

0.5

1.0

1.5

E1(µg/L)

Abs

.

17β-ESTRADIOL (E2)

-4 -3 -2 -1 0 10.0

0.5

1.0

1.5

E2(µg/L)

Abs

.

E1+E2+E3

-2.5 0.0 2.50

1

2

3

E1+E2+E3(µg/L)

Abs

.

Ethynyl 17β- Estradiol (EE2)

-4 -3 -2 -1 0 10.0

0.5

1.0

1.5

EE2 (µg/L)

Abs

.




Estrogens quantification by LC/ESI-MS/MS:

Method based on triple-quadrupole (QqQ)(1) operating in negative mode

Compound Instrumental detection limits(ng/mL)

SRM transitions (m/z)(precursor ion product ion)

Cone Coll.

Estrone 1 269 145269 143

50 40

Estradiol 1 271 145271 183

50 45

Ethynyl estradiol 2 295 145295 159

50 40

Estriol 1 287 171287 145

50 45

(1) M.S. Díaz-Cruz, M.-J. López de Alda, R. López, D. Barceló, J. Mass Spectrom. 38 (2003) 917-923




E2

0

50

100

150

200

250

Mue

stra

s

A1=E

ff M

arto

rell

A2=I

gua*

Est

A3=

Eff I

gua

A4=M

eOH

A5=E

ff Ig

uala

da

A6=N

P

A7=I

n M

arto

rell

A8=E

ff Ig

ua*E

ST

A9=G

enis

tein

a

A10=

Est 1

0

A11=

EST

1

Beso

s In

Beso

s O

ut

Dep

urba

ix In

Dep

urba

ix O

ut

Mol

ins

Rei

(B) S

PIKE

D

Mol

ins

Rei

(B)

Cas

tellb

isba

l (A)

SPI

KED

Cas

tellb

isba

l (B)

SPI

KED

Cas

tellb

isba

l (B)

Pont

Dia

ble

(B)

Pont

Dia

ble

(B) S

PIKE

D

Pont

Dia

ble

(A) S

PIKE

D

Pont

Dia

ble

(A)

Mol

ins

Rei

(A)

Mol

ins

Rei

(A) S

PIKE

D

Agua

Pont

Dia

ble

(B) S

PIKE

D2

Pont

Dia

ble

(A) S

PIKE

D2

Dep

urba

ix O

ut (A

) SPI

KE2

Dep

urba

ix O

ut (B

) SPI

KE2

ng/L

LC/MS/MSELISA




0

5

10

15

20

25

A1=

Eff

Mar

tore

ll

A2=

Igua

*Est

A3=

Eff

Igua

A4=

MeO

H

A5=

Eff

Igua

lada

A6=

NP

A7=

In M

arto

rell

A8=

Eff

Igua

*ES

T

A9=

Gen

iste

ina

A10

=Est

10

A11

=ES

T 1

Bes

os In

Bes

os O

ut

Dep

urba

ix In

Dep

urba

ix O

ut

Mol

ins

Rei

(A)

Mol

ins

Rei

(B)

Mol

ins

Rei

(A) +

EE

2

Mol

ins

Rei

(B) +

EE

2

Mol

ins

Rei

(A) +

E2

Mol

ins

Rei

(B) +

E2

Mol

ins

Rei

(A) +

EE

2+ E

2

Mol

ins

Rei

(B) +

EE2

+E2

Cas

tellb

isba

l (A

)

Cas

tellb

isba

l (B

)

Cas

tellb

isba

l (A

) +E

2

Cas

tellb

isba

l (B

) + E

2

Pon

t Dia

ble

(B)

Pon

t Dia

ble

(A)

Pon

t Dia

ble

(B) +

E2

Pon

t Dia

ble

(A) +

E2

Pon

t Dia

ble

(A) +

EE

2

Pon

t Dia

ble

(A) +

EE

2

Wel

l wat

er (A

) + E

E2

Wel

l wat

er (A

) + E

E2

Dep

urba

ix In

(A)+

EE

2

Dep

urba

ix In

(A)+

EE

2

ng/L

ELISA

LC-MS/MS



0

10

20

30

40

50

60

A1=

Eff

Mar

tore

ll

A2=

Igua

*Est

A3=

Eff

Igua

A4=

MeO

H

A5=

Eff

Igua

lada

A6=

NP

A7=

In M

arto

rell

A8=

Eff

Igua

*ES

T

A9=

Gen

iste

ina

A10

=Est

10

A11

=ES

T 1

M-s

pike

d

M-s

pike

d2

Bes

os In

Bes

os O

ut

Dep

urba

ix In

Dep

urba

ix O

ut

Mol

ins

Rei

(B) S

PIK

ED

Mol

ins

Rei

(B)

Cas

tellb

isba

l (A

) SP

IKE

D

Cas

tellb

isba

l (B

) SP

IKE

D

Cas

tellb

isba

l (B

)

Pon

t Dia

ble

(B)

Pon

t Dia

ble

(B) S

PIK

ED

Pon

t Dia

ble

(A) S

PIK

ED

Pon

t Dia

ble

(A)

Mol

ins

Rei

(A)

Mol

ins

Rei

(A) S

PIK

ED

Agu

a

M-S

pike

d3

M-S

pike

d4

(ng/

L)

ELISALC-MS/MS



E1+E2+E3

0

50

100

150

200

250

300

A1=E

ff M

arto

rell

A2=I

gua*

Est

A3=

Eff I

gua

A4=M

eOH

A5=E

ff Ig

uala

da

A6=N

P

A7=I

n M

arto

rell

A8=E

ff Ig

ua*E

ST

A9=G

enis

tein

a

A10=

Est 1

0

A11=

EST

1

Mol

ins

de R

ey (B

)+ E

2

Mol

ins

de R

ey (B

)

Cas

tellb

isba

l (B)

+ E2

Cas

tellb

isba

l (B)

Pont

del

Dia

ble

(B)

Pont

del

Dia

ble

(B)+

E2

Cas

tellb

isba

l (A)

+E2

Pont

del

Dia

ble

(A)+

E2

Pont

del

Dia

ble

(A)

Mol

ins

Rey

(A)

Mol

ins

De

Rey

(A)+

E2

Cas

tellb

isba

l (A)

ng/L

LC-MS/MSELISA




Method A y = 1,2821xR2 = 0,9642

0

50

100

150

200

0 20 40 60 80 100 120 140

LC-MS/MS (ng/L)

ELIS

A E

2 (n

g/L)

Method B y = 1,4138xR2 = 0,9396

0

100

200

300

0 50 100 150LC-MS/MS (ng/L)

ELIS

A E

2 (n

g/L)




Method (A) y = 1,266xR2 = 0,788

0

10

20

30

40

50

60

0 10 20 30 40 50

LC-MS/MS (ng/L)

ELI

SA (n

g/L)

Method (B) y = 1,5738xR2 = 0,9652

0

10

20

30

40

50

60

70

0 10 20 30 40 50

LC-MS/MS (ng/L)EL

ISA

(ng/

L)




Method A Method B ELISA Target

compounds

Relation LC-MS/MS vs. ELISA

R2

Recovery

%

Relation LC-MS/MS vs. ELISA

R2

Recovery(1)

%

E1 y=1.266x 0.788 79 y=1.5738x 0.9652 100.44 E2 y=1.2621x 0.9642 81 y=1.4138x 0.9396 87.48

EE2 *y=1.0347x 0.9928 75 *y=1.2238 0.994 78.88 E1+E2+E3 y = 1,4298x 0.9871 E3=86 y = 1,6994x 0.8718 88.16 *ELISA compared with spike leves (1) M.-J. López de Alda, D. Barceló, J. Chromatogr. A 892 (2000) 391-406

Better recovery using SPE method BMore overestimation using SPE method B


Magnetic particle based ELISA that can be used as a quantitative, semi-quantitative assay for the detection or measurement of 17β-Estradiol

The test format: tubes

Time of assays 160 minutes

Range of assay 2.5-25 ng/L

Limit of Detection: 1.5 ng/L 1- Pre incubation-antibodies attached to magneticparticles and the analyte

2- Addition of enzymatic tracer and incubation

3- Washing and color substrate addition

4- Color development, stop color development andread 450 nm

1 2 43






E2 - Paramagnetic particles

-4 -3 -2 -1 0 1 2 3 4

0.01

0.02

0.03

0.04

0.05

Log [E2 ng/L]

A

Linear range: 2,5-25 ng/LEC50= 5,5 ng/L

E2- ELISA

01

23

45

6

ng/L

LC-MS/MS (ng/L) ELISA (ng/L)Eff Martorell 0,6 0,96Eff. Igualada+E2 1,8 2,1Eff Igualada+NP 0 0,04MeOH 0 0Eff. Igualada 0 0,03NP 0 0,036In. Martorell 1,1 1,4Eff. Igualada+E2+NP 1,8 2,04GEN 0 0,04Besos In 0,48 1,7Besos Out 0 0,13Depurbaix In 0,7 5,52Depurbaix Out 0 2,36 AQUAbase Workshop on ANALYTICAL METHODS

18 - 19 January 2006, Aachen



Magnetic particles Direct ELISA E2 – SPE-LC-MS/MS

E2

0

5

10

15

20

25

Beso

s In

Beso

s O

ut

Dep

urba

ix In

Dep

urba

ixIn

+10E

2

Dep

urba

ix O

ut

Cas

tellb

isba

l

Cas

tellb

isba

l

Pont

del

Dia

ble

Pont

del

Dia

ble

+ E2

Mol

ins

de R

ei

Mol

ins

de R

ei

Wel

l Wat

er

Wel

l Wat

er+

E2

Milli

Q +

E2

Milli

Q

ng/L

ELISA LC-MS/MS



• Column: Acquity UPLCTM (sub 2µm particles)• Elution: Water-Acetonitrile• Separations were achieved in less than 7 minutes

in positive and negative modes for the selected compounds.

• Run time 15 - 16 minutes • ESTROGENS: ES-NI conditions• PHYTOESTROGENS: ES-PI conditions




Further confirmation of identity of detected compounds by TOF MS-MS

Optimized conditions

Source:Capillary voltage 3000V

Sample cone 30 VExtraction cone 1V

Collision Voltages:45 V in ES-38 V in ES+

Temperatures:Source 120 ºCDesolvatation 350 ºC

Gas Flows:Cone 50 L/hrDesolvatation 600 L/hr


Analysis of estrogens and phytoestrogens using different ELISA kits in comparison


Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00

%

0

100

Estrogens mixture1: TOF MS ES-

TIC2.08e5

5.360.342.69

1.804.964.864.472.81 6.26

5.99

E2 1: TOF MS ES-271

4.80e44.47

4.694.96

E3 1: TOF MS ES-287

1.86e42.81

4.47

EE2 1: TOF MS ES-295

5.13e44.86

E1 1: TOF MS ES-269

5.97e44.96

5.36

Pat-1ppm

m/z266 268 270 272 274 276 278

%

0

100patromiren 81 (3.690) 1: TOF MS ES-

9.16e3269.1540

267.1379

270.1580

271.1589 272.1801276.9310274.9038



Mass (exp) Mass(calc)

Error(ppm)

DBE Formula Compound [M]

255.0663 255.0657 2.2 10.5 C15H10O4 Daidzein

271.0602 271.0606 -1.7 10.5 C15H10O5 Genistein

285.0755 285.0763 -2.8 10.5 C16H12O5 Biochanin A

269.1540 269.1542 -0.6 8.5 C18H22O2 Estrone

271.1690 271.1698 -3 7.5 C18H24O2 Estradiol

287.1619 287.1647 2,7 7.5 C18H24O3 Estriol

295.1703 295.1698 1.7 9.5 C20H24O2 EthynylEstradiol



Entrada Besos

Time2.00 4.00 6.00 8.00 10.00 12.00

%

0

100

2.00 4.00 6.00 8.00 10.00 12.00

%

0

100

2.00 4.00 6.00 8.00 10.00 12.00

%

0

100

2.00 4.00 6.00 8.00 10.00 12.00

%

0

100MFU15 N 1: TOF MS ES-

287 0.10Da2.67e4

2.81

2.31 3.42

MFU15 N 1: TOF MS ES-287 0.50Da

2.67e42.81

2.311.62

6.833.42

MFU15 N 1: TOF MS ES-287

1.80e32.59

2.45

0.34 2.271.422.81 3.38

4.078.137.99

4.637.026.51

8.589.039.53 10.10 11.35

MFU15 N 1: TOF MS ES-TIC

3.58e54.554.293.78

3.202.330.36 1.17

5.935.72 6.41 7.068.317.75 8.58

9.39 9.5512.96

m/z100 150 200 250 300 350 400 450 500

%

0

1001: TOF MS ES-

1.83e4287.2466

154.9860 288.2501

Estriol




ng/L DAID GEN BIO-A E1 E2 E3

In WWTP-1 Bdl Presence4.9

Bdl Bdl Bdl Presence1.2

Eff. WWTP-1 Bdl Bdl Bdl Presence3.4

Bdl Bdl

In WWTP-2 Presence10.2

Presence6.8

Bdl Presence13.87

Bdl *0.6

Bdl

Eff.WWTP-2 Presence12.0

Bdl Bdl Presence7.9

Bdl Bdl

In WWTP-3 Bdl Bdl Bdl Bdl Bdl *0.5

Bdl

Eff-WWTP-3 Bdl Bdl Bdl Presence9.4

Bdl Bdl

Pont del Diable Bdl Bdl Bdl Bdl Bdl Bdl

Castellbisbal bdl Bdl Bdl Bdl Bdl Bdl




Technique Advantages Limitations Application

Magnetic particle ELISA for E2

Sensitive, rapid cost effective

Matrix effects andCross reactivity (E1, NP, Genistein)

Semi-quantitativeCan be applied directly for non complex matrix samples

ELISA kits E1,E2,EE2,(E1+E2+E3)

Rapid, robust, cost effective

Matrix effects andCross reactivity

ScreeningCan be applied for very complex samples using a proper SPE

LC-ESI(NI)-MS/MS Very sensitive, robust Ion suppressionTime consuming

Quantitative analysis of target compounds

UPLC-Q-TOF-MS Exact mass measurements, rapid, sensitive, less ion suppression than LC-MS/MS

Less sensitivity than LC-MS/MS

Quantitative, confirmatory analysis and investigation of non-target compounds




Conclusions:

• Good agreement was obtained comparing the different procedures

• In general an overestimation was obtained for immunoassays, but it can be partially minimized using the proper clean-up.

• The presence of close related compound in the same extracts can not beavoid, as was determined by UPLC-Q-TOF-MS, and the sum of differentinterferences produces a final degree of overestimation


• Comparing the 2 extraction procedures: Whereas method A showed better agreement with the chromatographic values, the recovery percentage of method B was higher.

• The sensitivity and high reproducibility of monoclonal based assays for E1, E2, EE2, and (E1+E2+E3) make these assay an excellent tool for the screening level analysis.

• The high sensitivity of the polyclonal based ELISA, allows the applicationof this assay direct without SPE, for the screening of E2 in drinking water,well water, and river water.

• Sometimes the colour suppresion in very complex samples makes extra clean-up steps nessessary.






• Considering all the results of this study, we conclude that the different ELISA kits assessed here are suitable for the screening analysis of environmental samples, including more complex matrices.




Acknowledgements:

This work has been supported by the EU project SWIFT-WFD,contract SSPI-CT-2003-502492.Marinella Farré would thanks the support from the MINISTERIO DE EDUCACIÓN Y CIENCIA, through the Juan de la Ciervaprogram.

This article reflects only the authors views and the EU is not liable for any use that maybe made of the information contained therein.


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