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AFLATOXIN M1 DETERMINATION

IN RAW MILK USING A

FLOW INJECTION IMMUNOASSAY SYSTEM

AFLATOXIN M1 DETERMINATION

IN RAW MILK USING A

FLOW INJECTION IMMUNOASSAY SYSTEM

Mihaela BadeaUniversity of Rome Tor Vergata - Italy

Mihaela BadeaUniversity of Rome Tor Vergata - Italy


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CONTENT

1. Aflatoxin M1(AFM1) problematic

2. Methods for AFM1 determination

3. Principle of Flow Injection Immunoassay (FI-IA)

4. Optimisation of enzymatic label detection

5. Optimisation of AFM1 determination

6. Analytical parameters of FI-IA method

7. Real samples analysis

8. Conclusions


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O

O

OO

O

OMe

Aflatoxin B1

O

O

OO

O

OH

OMe

Aflatoxin M1

Aflatoxins are mycotoxins produced byAspergillus flavus, A.

parasiticus and A. nomius found in corn, peanuts, grains and spices.

Aflatoxin M1 is found in milk of animals that ingested feed

contaminated with Aflatoxin B1.

Aflatoxins are classified as Group 1 cancerogens (WHO, 1987).

Aflatoxin M1 is a potent liver carcinogen.

Aflatoxin M1 is relatively stable during pasteurization, storage

and preparation of various dairy products.


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Aflatoxin M1 maximum admissible level in milk (raw milk,

milk for the manufacture of milk-based products and heat-

treated milk as defined by Council Directive 92/46/EEC, as

last amended by Council Directive 94/71/EC) is 0.05

g/Kg.

COMMISSION REGULATION (EC) No 466/2001 of 8 March 2001

setting maximum levels for certain contaminants in foodstuffs

Amendments to this regulation are also proposed to bemade with respect to aflatoxin M1 (0.025 g/kg) in infantmilk and dietary products.


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8. Conclusions


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Method

Sample

treatment Detection

Detectionlimit (ppt)

Obs.

TLC

1. Extraction

2. Clean-up

- Visual

- Densitometric

- Fluorimetric

5 - 10

Lowrecoveries

HPLC

1. Extraction on C18 cartridge

2. Derivatization- Fluorimetric

5 - 20 - Normalphase

- Goodrecoveries

1. Extraction on C18

cartridge

- Fluorimetric 15 - Reversephase

1. Extraction onimmunoafinitycolumn

- Fluorimetric 2

- Reverse

phase

TLC and HPLC methods for AFM1 determination in milk


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ELISA methods for AFM1 determination in milk

Competition Conjugate Detection Detectionlimit (ppt) Obs

Indirect - onmicrotiter plate

AFM1 - BSA Colorimetric

(405 nm)

50 - Extraction inmethanol

Indirect in aflow - throughdevice

AFB1 - BSA Colorimetric

(450 nm)

50 - clean-up stepintegrated

Direct on

microtiter plate

AFB1 - HRP Colorimetric

(450 nm)

2 - clean-up and

evaporation

Direct onmicrotiter plate

AFM1 - HRP Colorimetric

(450 nm)

10 - centrifugation


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8. Conclusions


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buffer

GC electrode+100 mV vs Ag/AgClTMB

H2O2

Protein G

Ag ( )+ Ag* ( ) + Ab ( )

Ag = AFM1

Ag* = AFM1 HRP

Ab = Rat Monoclonal Anti-Aflatoxin

1) E. Burestedt, C Nistor, U. Schagerlf and J. Emnus,Anal. Chem., 2000, 72, 41714177

2) M. Badea, C. Nistor, Y. Goda, S. Fujimoto, S. Dosho, A. Danet, D. Barcel, F.Ventura, J. Emnus,Analyst, 2003, 128, 849856


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1. Off-line incubation of the sample containing

AFM1 with fixed amounts of AFM1- HRP (Ag*)

and Anti-AFM1 (Ab)

2. Injection of the mixture in the flow injection

system

3. Separation of the free Ag* from the bound

Ag*Ab in the protein G column

4. Free Ag* monitoring by amperometricdetermination of HRP activity using 3,3,5,5-

tetramethylbezidine (TMB) and hydrogen

peroxide


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8. Conclusions


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Sub H2(reduced) + H2O2 Sub (oxidised) + 2 H2OHRP

- two oxidation peaks at + 250 and + 400 mV vs Ag/AgCl

CH3

CH3CH3

CH3

NH2

NH2

TMB

G. Volpe, D. Compagnone, R. Draisci, G. Palleschi,Analyst, 1998, 123, 1303 - 1307

- a working potential of + 100 mV vs Ag/AgCl was

selected for HRP activity determination


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Carrier selection-0.05 M phosphate citrate buffer, pH 5.0

-0.05 M phosphate buffer, pH 7.4


-0.05 M phosphate buffer, pH 6.5 with 0.1M NaCl

-0.05 M phosphate buffer, pH 6.5 with 0.1M NaCl, 10 mM MgCl2

H2O2 solution



-0.05 M phosphate citrate buffer, pH 5.0 with 0.1 M NaCl

TMB solution


-Distilled water


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Optimisation of TMB concentration

TMB concentration (M)

0 5x10-4 10-3 2x10-3 2x10-3

C = 10-2 mol l-1H2O2

HRP = 0.05 IU ml-1

Flow rate = 0.9 ml min-1


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H2O2 concentration influence on HRP activity

determination

0,0 5,0x10-3

1,0x10-2

0

50

100

150

200

250

Current(nA)

H2O

2concentration (M)

cTMB = 510-4 mol l-1

HRP = 0.05 IU ml-1



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Influence of length of mixing and reaction coils

0 50 100 150 200 250

50

100

150

200

250

300

350

400

mixing coil (for a reaction coil = 200cm)

reaction coil (for a mixing coil = 50 cm)

curren

t(nA)

Length (cm)

cTMB = 510-4 mol l-1

HRP = 0.05 IU ml-1


C = 10-2 mol l-1H2O2


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0,00 0,02 0,04 0,06 0,08 0,100

100

200

300

400

500

600

700

0,0000 0,0005 0,00100

5

10

Cu

rrent(nA)

HRP activity (IU mL-1)

Calibration graph for HRP activity determination

cTMB = 510-4

mol l-1

HRP = 0.05 IU ml-1

Flow rate = 0.9 ml min

-1

C = 10-2 mol l-1H2O2

Mixing coil = 50 cm

Reaction coil = 200 cm

I (nA) = 3.23 + 6730.68 EA HRP (IU ml-1) ; R = 0.9992

LOD = 0.25 mU mL-1 HRP (RSD = 2.34 %, n=10)


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Selection of the Ag* (AFM1 HRP) dilution

Compromise between

High dilution

Signal sufficient high (S/N >100)

AFM1 - HRP dilution

Current(nA)

0

5

10

15

20

25

30

1/200 1/100 1/501/66

Dilution 1/200 of Ag*

RSD = 1.65 % (n=10)

Using 0.03 % (v/v) Tween 20

in carrier and H2O2 solutions


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4. Optimisation of enzymatic label detection5. Optimisation of AFM1 determination



8. Conclusions


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0 20 40 60 80 100 120

5

6

7

8

9

10

11

12

current(nA)

incubation time (min)

Influence of incubation time of Ag* with Ab

Ag *(1/200) + Ab (1ppm)

Incubation media:

PBS 0.05 M, pH = 6.5without Tween 20

Temperature:room temperature(22 1 C)

0 00 0 50 1 00 1 50 2 00


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total flow rate (ml min-1)

0,00 0,50 1,00 1,50 2,00

Influence of flow rate on Ag*Ab retaining in theprotein G column

Ag *(1/200) + Ab (1ppm)

Incubation media:PBS 0.05 M, pH = 6.5

without Tween 20

Temperature:room temperature(22 1 C)

Incubation time = 1 h


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8. Conclusions


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Aflatoxin M1(ppt)

10 100 1000 10000

60

Calibration graph for AFM1 determination

1

b

a df d

x

c

= +

+

c = IC50

Analyte concentration (ppt) at50 % binding of Ag*

50% change in signal vs.the response in the absence of the analyte

AFM1 + Ag* (1/200) + Ab (1ppm)


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The main characteristics of the FI-IA for AFM1 determination

Assay 1 Assay 2 Assay 3

Calibration points (N) 12 13 12Replicates 3 3 3

LOD a / g l-1 10.4 0.9 11.1 0.9 10.9 1.0Dinamic range b / g l-1 20 - 500 20 500 20 -500IC50 / g l-1 160.6 16.6 165.8 22.1 181.3 17.9% CV cintra-assay 3.80 4.54 4.21

% CV cinter-assay 7.90

a the analyte concentration that gives a response equal with three times the standard deviation

of the zero doseb the concentration range that led to a relative change in signal between 20% and 80 %, when

compared with the zero dose

c calculated only within the dynamic range of the response


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8. Conclusions


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0 50 100 150 200 250

0

50

100

150

200

250

FI-IAmethod

HPLC method

Comparison with HPLC method (AOAC 2000.08)- Immunoaffinity column AFLA M1 AFLATOXIN TESTING- Reversed phase HPLC analytical column Nova-Pak C18 60 , 4 m, 3.9 x 300 mm- Loop injection system (50 L)- Fluorescence detection with 365 nm excitation and 435 nm emission- Flow rate 0.8 ml min-1

- Mobile phase: H2O CH3CN CH3OH (65:25:10)


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8. Conclusions


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Work in progress

Designing of an automated FI-IA

system for Aflatoxin M1

Validation of the proposed method


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ACKNOWLEDGEMENTS

Financial support:

European Community (Contract No QLK1-CT-2001-01617)

Financial support:

European Community (Contract No QLK1-CT-2001-01617)

Research team:

Prof. Giuseppe Palleschi

Dr. Laura Micheli

Prof. Emanuele Marconi

Dr. Maria Cristina Messia

Dr. Tiziana Candigliota

Special aknowledgements to:

Prof. Jenny Emnus

University of Rome Tor Vergata - Italy

University of Molise - Italy

University of Lund - Sweden

1st t


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International Scientific CommitteInternational Scientific Committe

G. Palleschi, Italy (Symposium Chair)

A. Amine, Morocco

S. Crouch, U. S. A.

G. Evtugyn, Russia

L. Gorton, Sweden

G. Guilbault, Ireland

J. Hart, England

M. Karayannis, Greece

A. A. Karyakin, Russia

V. Magearu, Romania

J. L. Marty, France

M. Mascini, Italy

H. Mottola, U. S. A.

K. Van Staden, South Africa

Local Organizing CommitteLocal Organizing Committe

University of Rome Tor Vergata

D. Moscone

S. Alarcon

M. Badea

J. Calvo Quintana

A. Lupu

L. Micheli

S. Piermarini

F. Ricci

F. Valentini

G. Volpe

LocationLocation

The conference will be held at Villa

Mondragone, one of the most beautiful"Ville Tuscolane". Now owned by the

University it has, through the centuries,

been a theatre of important events as well

as the the residence of popes and famous

families.

Built on the ruins of the Villa of the

Quintili, who were Roman consuls, it

enjoyed its maximum splendour during the

epoch of the Borghese family in the 18th

century.

In 1981 it was sold by the Order of the

Jesuits to the University of Tor Vergata.

Villa Mondragone:

Universit degli Studi di RomaUniversit degli Studi di Roma

Tor VergataTor Vergata

KINETICS INKINETICS INANALYTICAL CHEMISTRYANALYTICAL CHEMISTRY

8th International Symposium o

8-10 July 2004

ROME, ITALY

1st announcement

Villa Mondragone
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Scientific ProgrammeScientific Programme

Deadline Schedule:

15 January: 2 announcement 15 March: Abstract submission

Pre-registration FormPre-registration Form

Last name: .......................................

First name: ......................................

Institute: ..........................................

..........................................................

Department:.....................................

Address: ............................................

Postal Code: ......................................

City: ...................................................

Country: ............................................

Fax number: .......................................

e-mail address: ...................................

88thth KAC - Conference SecretariatKAC - Conference Secretariat

Communications should be directed to:

Francesca Dominici

Nancy Downer

University of Rome Tor Vergata

Via della Ricerca Scientifica, 00133

Rome, Italy

Tel. +390672594422

Fax +390672594328

e-mail: [email protected] .it

Further details, including

registration form, scientific

programme, travel and

accomodation, will be sent with

second announcement and will soon

be available on the KAC-2004 web

site

(www.uniroma2.it/dipartim/BEAT/KAC2004).

The scientific programme will include

plenary lectures, oral presentations and

poster sessions to cover the following

topics:Analytical chemistry applications

based on the kinetics of:

Electrochemical reactions

Spectrometric systems

Antigen-Antibody interaction

Redox reactions

Enzyme systems

This form can be sent either by fax: +390672594328 or by email:

[email protected] as attached file.
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