CHAPTER 3

EXPERIMENTAL

3.1 Materials and Chemicals

3.1.1The materials and chemicals for hapten synthesis

3.1.1.1 Synthesis of DDT-haptens

1) 2,2′-Bis(4-chlorophenyl)ethanol (DDOH), 188883, Aldrich, Sigma-

Aldrich Co. USA.

2) Dichloro benzhydrol (DCBH), TCI, Japan (kindly given by Prof. Dr.

Takahiko Takatori, School of Medicine, University of Tokyo)

3) Succinic anhydride, 14089, Fluka, Sigma-Aldrich Co. USA.

4) Glutaric anhydride, 49670, Fluka, Sigma-Aldrich Co. USA.

5) 4-Dimethyl amino pyridine (DMAP), 39405, Fluka, Sigma-Aldrich,

Germany.

6) Hydrochloric acid (HCl), 37%, proanalysis, K405566517, Merck,

Germany.

7) Sodium chloride (NaCl), K41042404 021, Merck, Germany.

8) Magnesium sulphate (MgSO4), 63136, Fluka, Sigma-Aldrich,

Germany.

9) Thin-layer chromatography (TLC) silica gel 60F254 20 x 20 cm

aluminium sheet, Merck, Germany.

10) Silica gel 60F254, Merck, Germany.

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3.1.1.2 Synthesis of cypermethrin-haptens

1) Methyl-3-(2,2-dichlorovinyl)-2,2-dimethyl-(1-cyclopropane)

carboxylate, CAS no. 61898-95-1, Acros, Germany.

2) β-alanine methyl ester hydrochloride, 05210, Fluka, Germany.

3) 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride

(EDC), 03450, Fluka, Sigma-Aldrich, Germany.

4) Sodium hydroxide (NaOH), K2433898 735, Merck, Germany.

5) Thin-layer chromatography (TLC) silica gel 60F254 20 x 20 cm

aluminium sheet, Merck, Germany.

6) Silica gel 60F254, Merck, Germany.

3.1.1.3 The materials and chemicals for preparation of immunogens and

coating antigens

1) Bovine serum albumin (BSA), A-7906, Sigma Chemical Co.,

Germany.

2) Albumin from hen egg white (egg albumin; OVA), 05440, Fluka,

Sigma-Aldrich Chemie GmbH, Germany.

3) Dimethylformamide (DMF), Merck, Germany.

4) 4- dimethylaminopyridine, 39405, Fluka, Sigma-Aldrich Co. USA.

5) N, N – dicyclohexylcarbodiimide, 36650, Fluka, Sigma-Aldrich Co.

USA.

6) N- hydroxysuccinimide, 56480, Fluka, Sigma-Aldrich Co. USA.

7) 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride

(EDC), 03450, Fluka, Sigma-Aldrich, Germany.

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8) Spectra/Por

membrane tubing, Spectrum, Spectrum Laboratory, Inc

9) Bio-Rad protein assay, 500-0006, Bio-Rad laboratories,Inc.

3.1.1.4 Organic solvents

1) Toluene, 9460-03, Actual analysis, J.T. Baker, USA.

2) Ethyl acetate, 9280-03, Actual analysis, J.T. Baker, USA.

3) Petroleum ether, 9268-05, Actual analysis, J.T. Baker, USA

4) Acetic acid, K30802863 224, Pro-analysis, Merck, Germany.

5) Pyridine, EC-No. 203809-9, BDH, England.

6) Sulfuric acid (H2SO4), K31004031, Pro-analysis, Merck, Germany.

7) Hexane, 9309-03, Actual analysis, J.T. Baker, USA.

8) Methanol, 9093-68, Actual analysis, J.T. Baker, USA.

9) Dimethyl sulfoxide (DMSO), D/4121/PB17, Analytical grade, Fisher

Scientific, United Kingdom.

10) Tetrahydrofuran (THF), 9731, Merck, Germany.

3.1.1.5 Standard insecticides

1) o,p’-DDE, ch-10311, 99.8%, Laboratory of Dr. Ehrenstorfer, Augburg,

Gemany.

2) o,p’-DDT, c-12081000, 98.0%, Laboratory of Dr. Ehrenstorfer,

Augburg, Gemany.

3) o,p’-DDD, c-120820, 99.3%, Laboratory of Dr. Ehrenstorfer, Augburg,

Gemany.

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4) p,p’-DDE, PS-696, 99.5%, Chem Service Inc., West Chester, PA,

USA.

5) p,p’-DDT, PS-699, 99%, Chem Service Inc., West Chester, PA, USA.

6) p,p’-DDD, 49009, 98.5%, Supelco, Bellelofente, PA, USA.

7) p,p’-DDA, 35484, 99.9%, Riedel-de Haen, Sigma-Aldrich Co. USA.

8) p,p’-DDM, 35488, 99.1%, Riedel-de Haen, Sigma-Aldrich Co. USA.

9) p,p’-DBP, 45421, 99.7%, Riedel-de Haen, Sigma-Aldrich Co. USA.

10) Dicofol, 36677, 96.9%, Riedel-de Hean, Sigma-Aldrich Co. USA.

11) Cypermethrin, c11890000, 91.0%, Laboratory of Dr. Ehrenstorfer,

Augburg, Gemany.

12) Permethrin, c15990000, 94.0%, Laboratory of Dr. Ehrenstorfer,

Augburg, Gemany.

13) Cyfluthrin, c11850000, 99%, Laboratory of Dr. Ehrenstorfer, Augburg,

Gemany.

14) Deltamethrin, c12120000, 99%, Laboratory of Dr. Ehrenstorfer,

Augburg, Gemany.

3.1.1.6 Immunological materials

1) Immuno plate Maxisorp 96F, 442404, NUNC, Denmark.

2) HRP- Conjugate anti - mouse Ig G antibody (H+L), 81-6520, Zymed

Laboratories Invitrogen immuno detection, USA.

3) Ortho-phenyleneline diamine, P-1526, Sigma Chemical Co., USA.

4) Tween

20, 63158, Sigma, United Kingdom.

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5) Complete Freund’s adjuvant (CFA), F-5881, Sigma Chemical Co.,

USA.

6) Incomplete Freund’s adjuvant (IFA), F-5506, Sigma Chemical Co.,

USA

3.1.1.7 Equipments

1) UV-2101 PC UV/vis spectrophotometer, Shimadzu Scientific

Instrument, Inc. Japan.

2) Micro plate spectrophotometer, Sunrise, TECAN, Austria.

3) Gas chromatograph 5890, Hewlett Packard, USA.

4) Gas chromatograph 7890A, Agilent Technologies, USA.

5) Mass-spectro detector (MSD), 5975C, Agilent Technologies, USA.

6) 1H and

13C NMR, Bruker, Bruker analytic GmbH, Germany.

7) VACELUTE, SPS24, Varian, USA.

8) Bond elute-C18, Part no. 12102028, Varian, USA.

3.2 Methods

3.2.1 Synthesis of haptens

3.2.1.1 Synthesis of haptens for anti-DDT antibody production

Haptens with chemical structure resembling DDT were designed and

modified structures were produced by spacer arm attachment. Two kinds of

hapten were synthesized with modified from Beasley et al. (1998)

(Figure 3.1), hapten I and hapten II were synthesized by attaching hydroxyl

(-OH) to DCBH. Haptens were synthesized by linking hydroxyl group of

44

DDOH with glutaric anhydride and DCBH with succinic acid anhydride by

esterification in dry pyridine. These substitutes act as spacer arm joining the

two aromatic rings through the carbon atom of carboxylic acid. Structures of

haptens were confirmed by 1H and

13C Nuclear Magnetic Resonance

Spectroscopy (NMR) and gas chromatography/Mass- spectrometry (GC/MS).

The synthesis of the two haptens is described in details below.

i) Hapten I: 4-(bis(4-chlorophenyl)methoxy)-4-oxobutanoic acid.

Hapten I was synthesized according the previous reported

procedures (Hongsibsong et al., 2010). A mixture of 200 mg (0.78

mmol) of DCBH, 750 mg (7.5 mmol) of succinic anhydride, and 10

mg (0.08 mmol) of dimethylaminopyridine (DMAP) in 10 ml dry

pyridine was stirred overnight at room temperature. Twenty milliliters

of water was then added and the mixture was evaporated dry. Crude

was rinsed twice with 10 ml toluene and dissolved in 10 ml ethyl

acetate. Crude solution was washed once with 10 ml of cool HCl,

twice with 10 ml water, and twice with 10 ml saturated sodium

chloride, respectively. The product was dry over magnesium sulphate

(MgSO4). Purity of the product was confirmed by TLC. Crude product

was purified by column chromatography on silica gel using ethyl

acetate: hexane (40:60 v/v) solvent. Single-compound fraction was

collected and evaporated dry. The structure of the compound was

confirmed by 1H and

13C NMR, and GC/MS.

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ii) Hapten II: 5-(bis(4-chlorophenyl)methoxy)-5-oxopentanoic acid was

synthesized using the same procedure as hapten I but glutaric anhydride

was substituted for succinic anhydride.

iii) Hapten III: Pentanedioic acid mono-[2,2-bis-(4-chloro-phenyl)-

ethyl] ester:

This hapten was synthesized according to diagram shown in

Figure 3.1. 2,2′-Bis(4-chlorophenyl) ethanol (DDOH, 100 mg,

0.37 mmol) was reacted with glutaric anhydride (375 mg, 3.74 mmol)

in 5 mL of dry pyridine with 5 mg of Dimethyl aminopyridine

overnight at room temperature. Twenty milliliters of water was then

added to the mixture and evaporated to remove pyridine. The crude

compound was rinsed with toluene and solvent was removed by

evaporation and then dissolved in ethyl acetate. The miture was

washed with 1 M HCl, water, and brine, before drying over MgSO4.

Crude product was purified by column chromatography on silica gel

using ethyl acetate: petroleum ether (40:60 v/v) with 0.01% acetic acid

as mobile solvent. Single-compound fraction was collected and

evaporated to dryness. The structure was confirmed by 1H-,

13C-NMR

and GC/MS.

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DCBH

CH

OH

ClClO

O

OCH ClCl

O

CO(CH2)nCO2HDMAPdry pyridine

Hapten I, n=2Hapten II, n=3

O OO

DMAPdry pyridine

HC

CH2OH

Cl ClHC

COCO(CH2)3CO2H

Cl Cl

DDOH Hapten III

(CH2)n

Figure 3.1 Synthesis of targeted haptens using DDOH and DCBH as precursor

3.2.1.2 Synthesis of haptens for anti-cypermethrin antibody production

The structure of synthetic pyrethroid insecticides has two major parts:

cyclopropane ring and benzene ring connected to cyano (-CN) group. The target

hapten was synthesized from the chemical that had structure similar to these two

parts.

i ) Hapten 1: 3-((1S,3S)-3-(2,2-dichlorovinyl)-2,2-dimethyl cyclopropane

carboxamido) propanoic acid

The reaction was shown in Figure 3.2. The precursor, 3-(2, 2-

Dichloro-vinyl)-2, 2-dimethyl-cyclopropane carboxylic acid, was prepared by

modified from Shan and Hammock (2001) and Hong et al. (2002). A mixture

47

of 200 mg 3-(2, 2-Dichloro-vinyl)-2, 2-dimethyl-cyclopropanecarboxylic acid

methyl ester and 62.0 mg NaOH in 5 mL THF:MeOH:water (3:2:1, v/v/v) was

stirred for 4 hours at room temperature. The crude compound was purified by

thin layer chromatography on silica gel using ethyl acetate and hexane (40:60,

v/v). Then, a mixture of 50 mg beta alanine methyl ester and 65 mg EDC in

3 ml THF was stirred for 10 minutes. The 59 mg 3-(2, 2-dichlorophenyl) 2, 2-

dimethyl–(1-cyclopropane) carboxylic acid was slowly added to mixture

solution on ice bath (0C). The mixture solution was stirred for 10 hour in

heating box (50C) and evaporated to dryness. The crude was redissolved in

dichloromethane and then washed twice with 10 mL of cool HCl and twice

with 10mL water. The product was dry over magnesium sulphate. The target

hapten was confirmed by thin layer chromatography (TLC) and then purified

by column chromatography on silica gel using ethyl acetate and hexane

(10:1, v/v) solvents. Single compound fraction was collected and evaporated

to dryness. The structure and molecular mass of the obtained compound was

confirmed by 1H and

13C nuclear magnetic resonance (NMR) spectrometry

and GC-MS, respectively. Hapten obtained was a white-yellow powder

compound with Rf value of 0.26 (ethyl acetate:hexane:acetic acid, 1:10:0.01,

v/v).

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Cl

Cl

O

O

Cl

Cl

O

OH

Cl

Cl

O

NHO

O

Cl

Cl

O

NHO

OH

NaOHTHF:MeOH:water

beta

-ala

nine

m

ethy

l es

ter

ED

C

THF:MeOH:water

3-(2,2-Dichloro-vinyl)-2,2-dimethyl-cyclopropanecarboxylic acid methyl ester

3-(2,2-Dichloro-vinyl)-2,2-dimethyl-cyclopropanecarboxylic acid

3-{[3-(2,2-Dichloro-vinyl)-2,2-dimethyl-cyclopropanecarbonyl]-amino}-propionic acid methyl ester

3-{[3-(2,2-Dichloro-vinyl)-2,2-dimethyl-cyclopropanecarbonyl]-amino}-propionic acid

Figure 3.2 Synthesis of targeted haptens (H1) for producing antibody to cypermethrin

ii) Hapten 2: Succinic acid mono-[cyano-(3-phenoxy-phenyl)-methyl]

ester

The hapten synthesized was modified from Beasley et al. (1998)

and Gao et al. (2006). The precursor, 3-phenoxy-benzaldehyde

cyanohydrin was prepared by adding 2.38 g 3-phenoxybenzaldehyde and

1.17 g potassium cyanide into a mixture of 7 ml tetrahydrofuran (THF)

and 0.5 ml water and stirred in ice bath, and 1.3 ml concentrated HCl will

be added all at once. After a short time, the mixture became basic and

mild exoterm accompanied the formation of the cyanohydrin. The mixture

was then allowed to warm to room temperature with stirring over 30 min.

The mixture was diluted with CH2Cl2 and water, acidified with 3 N HCl

(caution, HCN released), washes three times with water, dried briefly over

anhydrous MgSO4, and stripped to colorless oil. Then, 113 mg of

3-Phenoxy-benzaldehyde cyanohydrin was added with 120 mg succinic

acid anhydride and 190 μL triethylene diamine in 500 μL chloroform.

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The mixture was stirred for overnight (16 hour) and washed 3 times with

0.1 M HCl, filter through MgSO4 and evaporated to dryness. Crude

product was purified with TLC plate. Single compound fraction was

collected and evaporated to dryness. The structure and molecular mass of

the obtained compound was confirmed by 1H and

13C nuclear magnetic

resonance (NMR) spectrometry and GC-MS, respectively. The reaction

was show in Figure 3.3.

OO

KCN

OOCO

CO

OH

N

DM

AP

OH

N

O

O

O

O

pyri

di n

e

THF/water

HCl

3-Phenoxy-benzaldehyde 3-Phenoxy-benzaldehyde cyanohydrin

Succinic acid mono-[cyano-(3-phenoxy-phenyl)-methyl] ester

H

Figure 3.3 Synthesis of targeted haptens (H2) for producing antibody to cypermethrin

3.2.1.3 Preparation of immunogens and coating antigens

To prepare immunogens and coating antigens, Hapten I and II were

conjugated to proteins (BSA, or OVA). In all cases, an active

N-hydroxyscuccinimide (NHS) ester method was used to couple the carboxylic

acid moieties of the haptens to proteins (Haas and Guardia, 1968). For the

preparation of immunogens and coating antigens, 25 µM of the hapten were

incubated overnight at RT with stoichiometric amounts of 75 µM NHS and 75 µM

50

dicyclohexylcarbodiimide (DCC) in 0.5 mL of dimethylformamide (DMF).

The mixture was then centrifuged at 10,000 rpm for 10 min. Four hundred

microliters of supernatant containing the active NHS ester of hapten was collected

and then slowly added to 2 mL of 15 mg/mL BSA or OVA in 50 mM carbonate

buffer, pH 9.6. The mixture was allowed to react at RT for 4 h with stirring.

The mixture was dialyzed overnight in PBS, pH 7.2. Concentrations of protein

were determined by Bradford protein assay.

i) Bradford protein assay

Dye reagent was prepared by diluting 1 part of concentrate dye

reagent (BioRad) with 4 parts of water. The working reagent was filtered

through Whatman paper #1 to remove particles. Standard BSA range

from 0.1 mg/mL to 0.5 mg/mL (0.1, 0.2, 0.3, 0.4, 0.5 mg/mL) was

prepared. Protein solutions were analyzed in duplicate. Ten microliters

of various dilutions of standard proteins or test samples were added to

a microtiter plate in the presence of 300 μL diluted dye reagent.

The mixture was mixed and incubated at RT for 5 min. Absorbance at

595 nm was read on ELISA plate reader. Protein concentration was

calculated from a standard curve.

ii) Hapten density assay

Ten micrograms per milliliter of DCBH and 100 μg/ml of BSA,

KLH, OVA, and immunogen were prepared in 1% methanol-PBS,

pH 7.5. The hapten-protein conjugates were examined individually by

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UV/Vis spectrophotometer to confirm coupling. The spectra of

conjugates, free hapten, and native proteins were compared.

The absorbance of DCBH was measured at various

wavelengths to determine the wavelength of maximum absorbance

(λmax). The absorbance of other solutions was read at this wavelength,

and the hapten density was calculated according to Beer’s law.

A = εbC

A = Absorbance at λ max

ε = Molar absorbtivity

b = Path length of radiation

C = analyte concentration

UV/Vis spectral data were used to confirm the structures of

the final conjugates. Assuming that the molar absorptivity of haptens

was the same for the free and conjugated forms, the hapten densities

(the number of hapten molecules per molecule of protein) of the

conjugates were estimated directly by the mole absorbance ε (Moreno

et al., 2001)

Formula for hapten density calculation:

Hapten density =

εconjugation – ε protein

ε hapten

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3.2.1.4 Antibody production

Antibody were produced and characterized by the following procedures.

BALB/c mice (8-10 weeks old) were immunized with immunogen. The first

doses for one mouse consisted of 30 µg protein of immunogen emulsified in

complete Freund’s adjuvant injected subcutaneously. Mice were given the

subsequent injections with immunogen emulsified in incomplete Freund’s

adjuvant at 3 week intervals. Blood was collected from mouse tail vein at 7 days

interval and sera were stored at -20oC

3.2.1.5 Detection of antibody by non-competitive indirect ELISA

Non-competitive indirect ELISA was performed similarly to previously

described protocol (Kramer et al., 2004). One hundred microliters of coating

antigen in 50 mM carbonate-bicarbonate buffer (pH 9.6) were added to Maxisorp

Immunoplate (Nunc). The assay plate was incubated overnight at room

temperature. On the following day, plate was washed with washing buffer (0.05%

Tween-PBS) and blocked by incubation with 200 µL/well of 1% gelatin in PBS at

37oC for 1 h. The blocked buffer was discarded and 100 µL of 1:10,000 serum in

washing buffer were added in duplicate wells of the assay plate. The plate was

incubated at 37oC for 1 h and was then washed 4 times with washing buffer, and

100 μL of 1: 5,000 horseradish peroxidase (HRP) conjugated goat-anti-mouse

immunoglobulin G (H+L) in washing buffer were added to each well. The plate

was incubated at 37oC for 1 h, washed 4 times, and 100 μL of the OPD substrate

solution were then added to each well. The plate was incubated in the dark at RT

53

for 30 min and then 50 μL of 2 N H2SO4 stop solution were added to each well.

Absorbance before and after stopping reaction were measured at 492 nm.

3.2.1.6 Development of indirect competitive ELISA (ic-ELISA) to detect p,p’-

DDE in human milk samples

The pAb was detected by non-competitive indirect ELISA and were

tested for specificity to cypermethrin by competitive indirect ELISA. In brief, 96

well Maxisorp Immunoplates were coated with coating antigen in 50 mM

carbonate-bicarbonate buffer (pH 9.6) at 4oC overnight. The wells were washed 4

times by using 0.05% Tween20/PBS, pH7.2 as washing buffer and then were

blocked by incubation with 200 µL/well of 1% gelatin in PBS. Fifty microlitters

of competitors having similar structure to DDE at different concentration and

50µL/well 1:2,500 diluted antibody 1:5,000 final dilution was dispensed into each

well, and the wells were then incubated at room temperature (25oC) for 1 hour 30

mins. The wells were washed and 100 µL of 1:5,000 HRP-conjugated goat anti-

mouse IgG in washing buffer were added to each well. After incubation at 25oC

for 1 hour, the plates were washed and 100 μL of OPD solution were added to the

wells. The reaction was allowed to continue for 30 min and was stopped by

adding 50 µL of 2 N H2SO4. The absorbance was read at 492 nm. Antibody from

a mouse that gave the best sensitivity and specificity to the target compounds was

selected for further study. The sensitivity of pAb was detected and calculated

inhibition concentrations at 50% (IC50) were fit to a four parameter logistic

equation.

54

The relative cross-reactivity (CR) was calculated by the following equation:

%CR = (IC50 of target compound /IC50 of related compound) x 100

i) The effect of coating antigen and organic solvent on activity of antibody

Effect of coating antigen: Two types of coating antigens, s-DCBH-

OVA and g-DDOH-OVA, were compared by ic-ELISA. p, p’-DDE was used

as a competitor.

Effect of organic solvent: DMSO is often used as an organic modifier

of the buffer formulation in the extraction of less hydrophilic analytes from

samples. To determine optimal concentrations of DMSO in the buffer, non

competitive indirect ELISA was carried out using various solutions of DMSO

(10%, 20%, 30%, 40%, and 50%) with 0.05% Tween-20 (v/v). DMSO at the

concentration that had no effect to antibody reaction was used as diluent

composition. The standard p,p’-DDE was diluted at the optimal concentration

of DMSO in PBS with or without 0.05% Tween-20 (v/v). The assays were

done in duplicate wells.

ii) Recovery and precision assay

Recovery assay was carried out by spiking three concentrations of

p,p’- DDE (10, 50 and 100 ng/mL) and three triplicate. After extracted with

C18 solid phase extraction, the residue was re-dissolved in DMSO.

The developed ELISA was performed to determine p,p’-DDE against standard

curve of p,p’-DDE. Recovery was reported in percent and precision would be

55

reported in %CV. Variations day to day were done by using the same

extracted milk samples from recovery assay for 3 days continue.

iii) Analysis of DDT and its metabolites in human milk samples

Human milk samples

Human milk samples were part of samples collected from health care

clinics of the Shoklo Malaria Research Unit (SMRU) in Maela camp, 50 km

north of Mae Sot district (at the Thai-Myanmar Border), Tak province,

northern Thailand, in 2004 to 2008. The study was approved by the Ethics

Committee of Faculty of Tropical Medicine, Mahidol University, Bangkok,

Thailand and the Oxford Tropical Research Ethics Committee, University of

Oxford, the United Kingdom. In brief, the purpose and methods of the survey

were explained to all participants in their own language, mostly Tai ethnic

group. Breast-milk samples were collected by manual expression into glass

tubes. The samples were mixed and aliquoted into Eppendorf tubes, and then

kept frozen at -20 C in Maela camp and transported to the SMRU office in

Mae Sot district. The collected samples were stored at -80 C in the freezer.

Samples were shipped frozen on dry ice to Toxicology lab of the Research

Institute for Health Sciences, Chiang Mai University for the analysis of DDT

and its metabolites using GC-ECD.

56

GC-ECD analysis

Human milk samples were extracted using the method described by

Prapamontol and Stevenson (Prapamontol and Stevenson, 1991) with slight

modification. In brief, 2 mL human milk was extracted with 10 mL (ethyl

acetate: methanol: acetone =1:2:2) and clean up with C18 solid phase

extraction (Bondelute, Varian, USA). Lipid residue in the eluate was treated

with 300 μL concentrated sulfuric acid. One microlitter of clean eluate was

injected for analysis on GC-ECD-63

Ni (HP 5890 A series II, USA) equipped

with an automatic sampler (HP 7673, USA), and a fused silica capillary

column (Ultra 2: 25 m x 0.32 mm i.d., with 0.52 μm film thickness) for

separation. Spiked skimmed cow milk was used for standard calibration curve

construction. The calibration curve for p,p’-DDE was constructed at 3,

6, 9, 12, 15 and 30 μg L-1

. The high concentration of p,p’-DDE in milk

samples will be diluted and repeated run again. The recovery of p,p’- DDE at

concentrations of 6 and 21 μg L-1

were 98.2 0.2 %and 89.1 ± 0.4 %,

respectively. The limit of detection (LOD) as described by signal to noise

ratio of 10:1 was 0.04 μg L-1

. As a part of quality assurance of analysis,

besides internal quality control, Environmental Research Group, Research

Institute for Health Sciences, Chiang Mai University, Chiang Mai, Thailand

participated in the German External Quality control (G-EQUAS, program 40

in 2007), University of Erlengen-Nuremberg, Erlangen, Germany and

obtained qualified results.

57

3.3 Data analysis and statistics

3.3.1 Calculation of antibody response as sensitivity to target compound were

analyzed by 4-parametric logistic regression and reported in IC50 by using

Graphpad prism version 4.0 software.

3.3.2 Correlation between p,p’-DDE data obtained from ic-ELISA and GC-

ECD were conducted with SPSS for WINDOWS (SPSS Inc., Chicago, IL;

Version 11.5)