REVIEW

Biological matrices for the evaluation of exposureto environmental tobacco smoke during prenatal lifeand childhood

Heura Llaquet & Simona Pichini & Xavier Joya &

Esther Papaseit & Oriol Vall & Julia Klein &

Oscar Garcia-Algar

Received: 13 March 2009 /Revised: 26 April 2009 /Accepted: 30 April 2009 /Published online: 24 May 2009# Springer-Verlag 2009

Abstract The measurement of nicotine and its majormetabolites cotinine and trans-3´-hydroxicotinine togetherwith other minor metabolites (e.g., cotinine N-oxide,cotinine, and trans-3´-hydroxicotinine glucuronides) inconventional and nonconventional biological matrices hasbeen used as a biomarker to assess the exposure toenvironmental tobacco smoke during childhood. Thedetermination of these substances in matrices such asamniotic fluid, meconium, and fetal hair accounts forprenatal exposure to cigarette smoking at different stagesof pregnancy. Nicotine and its metabolites in cord blood,neonatal urine, and breast milk are useful for determiningacute exposure to drugs of abuse in the period immediatelybefore and after delivery. Cotinine measurement in child-ren’s blood and urine and nicotine and cotinine measure-

ments in children’s hair constitute objective indexes ofacute and chronic exposure during infancy, respectively.However, for monitoring and categorizing cumulativeexposure to environmental tobacco smoke during the entirechildhood, including the prenatal period, the assessment ofnicotine in teeth has been proposed as a promisingnoninvasive tool. This article reviews the usefulness ofmeasurement of nicotine and its metabolites in differentfetal and pediatric biological matrices in light of noninva-sive collection, time window of exposure detection, andfinally clinical application in pediatrics.

Keywords Biological matrices . Environmental tobaccosmoke . Prenatal . Childhood

AbbreviationsAPCI atmospheric pressure chemical

ionizationETS environmental tobacco smokeGC gas chromatographyHPLC high-performance liquid

chromatographyLC liquid chromatographyMS mass spectrometryMS/MS tandem mass spectrometryNPD nitrogen—phosphorus detectionPPHN persistent pulmonary hypertension

of the newbornRIA radioimmunoassaySPE solid-phase extraction

Anal Bioanal Chem (2010) 396:379–399DOI 10.1007/s00216-009-2831-8

H. Llaquet :X. Joya : E. Papaseit :O. Vall :O. Garcia-Algar (*)Unitat de Recerca Infancia i Entorn, IMIM-Hospital del Mar,Pg. Maritim 25–29,08003 Barcelona, Spaine-mail: 90458@imas.imim.es

H. Llaquet :X. Joya : E. Papaseit :O. Vall :O. Garcia-AlgarDepartament de Pediatria, Universitat Autònoma Barcelona,Barcelona, Spain

S. PichiniDepartment of Therapeutic Research and Medicines Evaluation,Istituto Superiore di Sanità,Rome, Italy

J. KleinDrug Testing Consultants,Toronto, Canada

Introduction

Addiction to tobacco is a major global public health issue.The health risks of smoking are well documented in thescientific literature. Smoking increases the risk for cancer,cardiovascular disease, chronic pulmonary obstructivedisease, etc. Despite the much publicized risks associatedwith tobacco use, more than 25% of the world populationsmoke cigarettes, making smoking one of the largestpreventable causes of human morbidity and mortality [1] .The incidence of tobacco-related diseases is growingrapidly around the world, with devastating health andeconomic costs.

Tobacco is also a major burden to people who do notsmoke. Passive exposure to environmental, side-stream, orsecond-hand cigarette smoke is associated with significanthealth risks such as cancer, heart disease, asthma, andrespiratory illnesses.

The pediatric population is highly affected, passiveexposure to cigarette smoke being a major cause of illnessamong children [2]. The exposure may start in utero andcontinue after birth, during the entire childhood.

The negative impact of smoking during pregnancy isamply described in the literature. Maternal smoking isassociated with increased risk of spontaneous abortion,intrauterine growth retardation, preterm birth, perinataldeath, and sudden infant death syndrome [3]. It may alsoincrease the risk of cognitive and neurodevelopmental delay[4]. Later on, during childhood, it has been shown thatexposure to environmental tobacco smoke (ETS) is associ-ated with increased rates of respiratory infections, asthma,ear and sinus infections, etc. [5].

As a first attempt to identify ETS and appreciate itsmagnitude, maternal/paternal self-reports are used widely.However, as it has been shown previously, maternal reportsregarding smoking are often inaccurate [6]. Many parentswill conceal or underreport their smoking habits during andafter pregnancy because of social pressure, guilt, orembarrassment.

Similar to other drugs of abuse, licit or illicit, in the pasttwo decades, biological markers specific to tobacco smokehave been identified to prevent reporting bias. Nicotine, amajor component of cigarette smoke, and its majormetabolites cotinine and trans-3´-hydroxicotinine havebeen used as biomarkers for ETS and measured inconventional and nonconventional matrices. The primarypathway of nicotine metabolism is presented in Fig. 1.

We report on the “state of the art” of nicotine andmetabolites testing in different matrices as biologicalmarkers of ETS exposure of infants and children duringthe prenatal and the postnatal period. The analyticalprocedures are summarized in Table 1 (prenatal exposure)and Table 2 (postnatal exposure).

Biological matrices for the evaluation of prenatalexposure to environmental tobacco smoke

Cord blood

The concentrations in fetal blood can be indicative oftransplacental passage of nicotine and its metabolites duringpregnancy. But cord blood has two major inconveniences:(1) it accounts only for fetal tobacco exposure during theprevious days or hours before collection and not for chronicexposure during the entire gestation and (2) it is easier tocollect (and more useful) infant urine and meconium thancord blood [7]. Concentrations of nicotine metabolites incord blood are found in the order of nanograms permilliliter (the concentrations are 2 or 3 orders of magnitudelower than those detected in the amniotic fluid). Differentcutoffs (14 and 21.5 ng/mL) have been proposed todifferentiate between passive and active smokers. Todifferentiate between exposure and nonexposure to ETS, acutoff value of 1 ng/mL has been proposed [8–10].

Several studies have been performed in the last 10 yearswith the aim of quantifying cotinine in cord serum. The firststudies determined that cord serum cotinine concentrationsare related to daily smoking rate during pregnancy, to infantbirth weight, and to antepartum and perinatal complica-tions. Perkins et al. [11, 12] described the prevalence ofsmoking among 3,220 Canadian pregnant women. Theymeasured maternal and umbilical cord cotinine levels byradioimmunoassay (RIA) and determined that mean infantbirth weight was inversely correlated to maternal serumcotinine level. Bearer et al. [9] determined that tobaccosmoke products place a newborn at risk for persistentpulmonary hypertension of the newborn (PPHN). Cordblood cotinine was measured in infants with PPHN and in acomparison group (healthy infants). Cotinine concentra-tions were higher than in the comparison group and it was

Fig. 1 The primary pathway of nicotine metabolism

380 H. Llaquet et al.

Tab

le1

Detectio

nwindo

wandanalytical

procedures

formon

itoring

prenatal

expo

sure

toenvironm

entaltobaccosm

okein

biolog

ical

matrices

Biological

matrix

Detectio

nwindo

w[126]

Collection

Metabolite

Extraction

metho

dAssay

metho

dLOD

and/or

LOQ

(cutoffin

the

case

ofim

mun

olog

ical

metho

ds)

Intra-assaycoefficientof

variation

andinterassay

coefficiento

fvariation

Biomarkerlevelsforthe

differentexpo

sure

grou

psReferences

Cord

serum

Hou

rsto

days

Easyand

noninv

asive

(atbirth)

COT

Liquid—

liquid

GC-

MS/

MS

LOQ

=0.1

ng/

mL

Intra-assayand

interassay

<15

%COTlevelsin

cord

serum

indicatedfetal

expo

sure

totobacco.

Goo

dcorrelation

amon

gCOTlevelsin

maternalurine,

plasma,

andcord

serum

Cutoffused

(ng

COT/m

Lcord

serum):

[9,14

,15

]No

expo

sure:LOD-1

low,

1–14

;medium/

gigh

,>14

MeanCOT

(ng/mL):no

nexp

osed

7.6[10];expo

sed76

.0[10]

[8]

COT

Liquid—

liquid

GC-

MS

LOD

=1

ng/m

LNA

[9]

COT

Liquid—

liquid

GC-

MS

LOQ

1.5

ng/m

LNA

[10]

COT

RIA

LOD

=10

ng/

mL

Intra-assay<8.7%

,interassay

8.1%

[11,

12]

COT

RIA

NA

NA

[13]

Thiocyanate

Colori-

metric

NA

NA

COT

RIA

LOD

=0.2

ng/

mL

NA

[14,

15]

COT

Liquid—

liquid

HPLC

LOD

=0.07

8ng

/mL

Intra-assay<8.4%

[16]

Liquid—

liquid

GC-

NPD

LOD

=0.20

0ng

/mL

NA

ELISA

LOD

=0.46

4ng

/mL

NA

COT

Liquid—

liquid

HPLC

LOD

=0.07

8ng

/mL

Intra-assay<8.4%

[18,

19]

Neonatal

urine

1–3

days

before

deliv

ery

Easyand

noninv

asive

COT

RIA

LOD

=0.2

ng/

mL

Interassay

6–10

%Close

correlation

betweenNIC

andCOT

maternalandneon

atal

concentrations

and

infant

irritabilityMean

COT(ng/mL):

nonexp

osed

1.9[20],

0[24];expo

sed17

0.5

[20],50

0[24]

[15]

COT

RIA

LOD

=0.2

ng/

mL

Interassay

6–10

%[20]

NIC,COT,

OH-COT

Liquid—

liquid

HPLC

NA

NA

[24]

Neonatal

hair

In newbo

rns,

last

trim

ester

of pregnancy

In child

ren,

mon

ths

Easyand

noninv

asive

Perform

edun

derdirect

observation

Hair-washing

toremov

eexternal

sourcesof

contam

ination

NIC,COT

Liquid—

liquid

GC-

MS

NA

NA

HairNIC

rather

than

hairCOTisauseful

markerMeanNIC

(ng/

mghair):no

nexp

osed

0.41

[32],0.41

[33],

4.75

[34],0.12

[36];

high

lyexpo

sed3.62

[32],2.41

[33],4.32

[34],0.20

[36]

[27–29

]

NIC,COT

RIA

NIC:LOD

=0.25

ng/m

gCOT:LOD

=0.1

ng/m

g

NA

[31,

32]

NIC,COT

RIA

NA

NA

[33]

NIC,COT

RIA

COT:0.05

ng/

mghairNIC:

0.05

ng/m

ghair

NA

[34]

NIC,COT

Liquid—

liquid

LC-

MS/

MS

NIC:LOD

=0.16

ng/m

g;LOQ

=0.28

ng/m

gCOT:

Inter-assayNIC

<13

.4%,

COT>10

.2%

[35,

36]

Biological matrices for the evaluation of exposure to environmental tobacco smoke during prenatal life and childhood 381

Tab

le1

(con

tinued)

Biological

matrix

Detectio

nwindo

w[126]

Collection

Metabolite

Extraction

metho

dAssay

metho

dLOD

and/or

LOQ

(cutoffin

the

case

ofim

mun

olog

ical

metho

ds)

Intra-assaycoefficientof

variation

andinterassay

coefficiento

fvariation

Biomarkerlevelsforthe

differentexpo

sure

grou

psReferences

LOD

=0.07

ng/m

g;LOQ

=0.10

ng/m

gNew

born

nails

Last

trim

ester

of pregnancy

Easyand

noninv

asive.

Requires

organic

washing

toascertain

external

contam

ination.

Pulverizatio

nbefore

analysis

NIC,COT

SPE

GC-

MS

NIC:LOD

=0.02

5ng

/mgCOT:

LOD

=0.02

5ng

/mg

Interassay

andintra-assay<15

%NIC

andCOTwere

present

[38]

Amniotic

fluid

Mon

ths

(1stand

2nd

trim

esters)

Invasive

collection

procedure

NIC,COT

RIA

NA

NA

Hum

anfetusexpo

sedto

high

erNIC

concentrations

than

the

smok

ingmothers

[45]

COT

RIA

LOD

=25

ng/m

LIntra-assay<10

%interassay

<10

%[47]

Mecon

ium

2ndand

3rd

trim

esters

of pregnancy

Easyand

noninv

asive.

May

bedelayedun

til3

days

COT,

OH-

COT

RIA

NA

Intra-assayCOT7.62

%NIC

metabolites(nam

ely

COT)in

mecon

ium

discriminatesm

okers

from

ETS-exp

osed

orETS-non

-exp

osed

grou

psMeanCOT:

nonexp

osed

<LOD

[53],6.0

ng/g

[55];

high

lyexpo

sed63

2pm

ol/g

[53],42

.6ng

/g[55]

[48]

COT,

OH-

COT

SPE

GC-

MS

NA

NA

COT

SPE

EIA

NA

NA

[49]

COT

ELISA

NA

NA

[50]

NIC,COT

Liquid—

liquid

HPLC

COT:10

ng/m

LNIC:

NA

NA

[52]

NIC,COT,

OH-COT

Liquid—

liquid

HPLC

LOD

=0.02

ng/g

Intra-assayCOT16

%;sum

a22

%[53]

NIC,COT,

OH-COT,

norN

IC,

norCOT

SPE

LC/

APCI-

MS/M

S

NIC:LOD

=1.25

ng/

g;LOQ

=5

ng/g

Intra-assayCOT7.4%

,OH-COT

7.4%

,NIC

6.7%

,no

rNIC

5.5%

,no

rCOT3.9%

Interassay

COT

5.8%

,OH-COT5.5%

,NIC

7.4%

,no

rNIC

7.8%

,no

rCOT:5.3%

[54,

55]

COT:LOD

=LOQ=

1.25

ng/g

OH-COT:LOD

=LOQ

=1.25

ng/g

norCOT:LOD

=LOQ

=1.25

ng/g

norN

IC=LOD

=LOQ

=5

ng/g

APCIatmosph

eric

pressure

chem

ical

ionizatio

n,COTcotin

ine,

EIA

enzymeim

mun

oassay,ETSenvironm

entaltobacco

smok

e,GC

gaschromatog

raph

y,HPLC

high

-perform

ance

liquid

chromatog

raph

y,LC

liquidchromatog

raph

y,LOD

limitof

detection,

LOQ

limitof

quantification,

MSmassspectrom

etry,MS/MStand

emmassspectrom

etry,NAno

tavailable,

NIC

nicotin

e,no

rCOTno

rcotinine,

norN

ICno

rnicotine,

NPD

nitrog

en—

phosph

orus

detection,

OH-COTtran

s-3´-hyd

roxy

cotin

ine,

RIA

radioimmun

oassay,

SPEsolid

-phase

extractio

naLevelsof

COT+NIC

+OH-COT

382 H. Llaquet et al.

Tab

le2

Detectio

nwindo

wandanalytical

procedures

formon

itoring

post-natal

expo

sure

toenvironm

entaltobaccosm

okein

biolog

ical

matrices

Biological

matrix

Detectio

nwindo

w[126]

Collection

Metabolite

Extraction

metho

dAssay

metho

dLOD

and/or

LOQ

(cutoffin

thecase

ofim

mun

olog

ical

metho

ds)

Intra-assay

coefficientof

variationand

interassay

coefficientof

variation

Biomarkerlevelsforthedifferent

expo

sure

grou

psReferences

Breast

milk

Hou

rsEasyand

noninv

asive

NIC,COT

Liquid—

liquid

GLC

NIC

LOD

=COT

LOD=1

ng/m

LIntra-assayNIC

7.3%

,COT

8.5%

Amou

ntof

NIC

transferredto

theinfant

increasedwhenmothers

smok

edbefore

breast-feeding

.Bothnu

rsingandpassive

smok

ingcontribu

teto

theexpo

sure

ofETS.

Infantsbreast-fed

show

edconcentrations

10-

fold

high

erthan

thosebo

ttle-fedMeanCOT

(ng/mL):breast-fed

0.2[61];no

tbreast-fed

495.0[61]

[21]

NIC,COT

Liquid—

liquid

GLC

NIC:LOD

=0.2

ng/m

LCOT:

LOD

=5

ng/m

L

NA

[59]

COT

Liquid—

liquid

GLC

NIC:LOD

=0.2

ng/m

LCOT:

LOD

=5

ng/m

L

NA

[60]

COT

RIA

NA

NA

[61]

COT

Liquid—

liquid

GLC

LOD

=0.1

ng/m

LIntra-assay7.7%

[62]

NIC,COT

Liquid—

liquid

HPLC

NIC:LOQ

=10

ng/m

L,

LOD

=8

ng/m

LCOT:LOQ

=1

2ng

/mL,LOD

=10

ng/m

L

Intra-assayNIC

<7%

,COT

<8%

.Interassay

NIC <9%

,COT<9%

[64,

65]

NIC,COT

Liquid—

liquid

GLC

NIC:LOD

=COT

LOD

=1

ng/m

LIntra-assayNIC

7.3%

,COT

8.5%

[66]

NIC

SPE

LC—

UVDAD

LOD

=13

ng/m

L,

LOQ

=34

ng/

mL

Intra-assay4.1%

,interassay

5.8%

[67]

NIC,

COT,

OH-

COT,

COT-N-

OX

Liquid—

liquid

LC-M

S/

MS

LOD

(all

metabolites)

=1.6

ng/m

L,LOQ

(allmetabolites)

=5

ng/m

L

Intra-assayNIC

<5%

,COT

<10

%,

OH-COT

<8.3%

,COT-N-O

X<1.6%

nterassayNIC

<10

.2%,COT

<6.6%

,OH-

COT

<3.3%

,COT-N-

OX <11.6%

[68]

NIC,COT

SPE

LC-M

S/

MS

NIC:LOD=

1.6

ng/m

LIntra-assay

NIC<5%

,[69]

Biological matrices for the evaluation of exposure to environmental tobacco smoke during prenatal life and childhood 383

Tab

le2

(con

tinued)

Biological

matrix

Detectio

nwindo

w[126]

Collection

Metabolite

Extraction

metho

dAssay

metho

dLOD

and/or

LOQ

(cutoffin

thecase

ofim

mun

olog

ical

metho

ds)

Intra-assay

coefficientof

variationand

interassay

coefficientof

variation

Biomarkerlevelsforthedifferent

expo

sure

grou

psReferences

LOQ=5

ng/m

LCOT:LOD

=1.6

ng/m

LLOQ

=5

ng/m

L

COT<10

%Interassay

NIC

<10

.2%,COT

<6.6%

NIC,COT

Liquid—

liquid

GC-N

PD

NIC:0.2

ng/m

LCOT:5

ng/m

LNA

[98]

Urine

1– 3days

Easyandno

ninv

asive

Requiresdirect

observation

todeteradulteratio

n

COT

RIA

LOD

=0.2

ng/m

LNA

BothNIC

andCOTdiscriminatebetween

child

renexpo

sedandno

texp

osed

totobacco

smok

e.A

sing

lepo

intmeasure

ofCCRdo

esno

tadequately

describe

theinfant’spassive

ETSexpo

sure.Cutoffof

passivesm

oking

forthesum

ofCOT+OH-COTof

10ng

/mLMeanCOT(ng/mL):no

nexp

osed

0.33

[91];high

lyexpo

sed15

.47[91]

[14]

COT

Liquid—

liquid

GLC

LOD

=0.1

ng/m

LIntra-assay7.7%

[79]

COT

ELISA

LOD

=5

ng/m

LIntra-assay5.2%

,interassay

7.7%

[80]

COT

RIA

NA

NA

[82]

NIC,COT

SPE

HPLC

NIC:LOD

=1

ng/

mLCOT:LOD

=1

ng/m

L

Intra-assayNIC

<25

%,COT

<13

.9%

Inter-

assay

NIC

<18

%,

COT

<5.4%

[84]

COT

RIA

NA

Intra-assayCOT

2.5%

,NIC

3%,

OH-COT3%

Interassay:COT

4%,NIC

6%;

OH-

COT6%

[85]

NIC,

COT,

OH-COT

SPE

HPLC

NA

Intra-assayCOT

2,5%

,NIC

3%,

OH-COT3%

Interassay

COT

4%,NIC

6%,

OH-

COT6%

NIC,

COT,

OH-COT

Liquid—

liquid

HPLC

NA

NA

[86]

COT

SPE

HPTLC

LOD

=6

ng/m

LIntra-assay2.9%

,interassay

3.2%

[87]

NIC,COT

Liquid —

liquid

GC-M

SNIC

LOD

=COT

LOD=0.16

ng/m

L;

Intra-assayNIC

4.6%

,COT

[88]

384 H. Llaquet et al.

Biological

matrix

Detectio

nwindo

w[126]

Collection

Metabolite

Extraction

metho

dAssay

metho

dLOD

and/or

LOQ

(cutoffin

thecase

ofim

mun

olog

ical

metho

ds)

Intra-assay

coefficientof

variationand

interassay

coefficientof

variation

Biomarkerlevelsforthedifferent

expo

sure

grou

psReferences

NIC

LOQ

NIC

=COTLOQ

=1.25

ng/m

L

10.9%

NIC,COT

GC-N

PD

NA

NA

[89]

NIC,COT

RIA

*NIC

LOD

=COT

LOD=0.37

ng/m

LIntra-assay5%

,interassay

5%[90]

COT

Liquid—

liquid

HPLC/

APCI-

MS

LOD

=0.05

ng/m

LNA

[91]

NIC,COT

SPE

GC-N

PD

NA

NA

[93]

COT,

OH-

COT

Liquid—

liquid

LC-M

S/

MS

COT:LOQ

=0.2

ng/

mLOH-COT:

LOQ

=0.2

ng/m

L

Intra-assayCOT

12%,OH-COT

4%COT

Liquid—

liquid

HPLC

LOD

=0.5

ng/m

LIntra-assay

<2.14

%,

interassay

<4.23

%

[96,

97]

Oralfluid

0.5– 36

hEasyandno

ninv

asive

Perform

edun

derdirect

observation

COT

Liquid—

liquid

Packed

column

GLC

LOD

=0.1

ng/m

LIntra-assay7.7%

SalivaryCOTmoresensitive

than

NIC.

Average

COTin

saliv

adifferentiates

betweenchild

renexpo

sedandno

texpo

sed

toETSMeanCOT(ng/mL):no

nexp

osed

0.44

[102],0.66

[101];bo

thparents3.38

[102],1.76

[101]

[79]

COT

ELISA

LOD

=1

ng/m

LIntra-assay5.2%

,interassay

7.7%

[80]

NIC,COT

RIA

NIC

LOD

=COT

LOD

=0.37

ng/m

LIntra-assay5%

Interassay

5%[90 ]

COT

RIA

0.5

ng/m

LNA

[101]

COT

Liquid—

liquid

GLC

NA

NA

[102]

COT,

NIC

Liquid—

liquid

Capillary

column

GLC

NIC

LOD

=COT

LOD

=0.1

ng/m

LIntraassay

COT

0.92

%,N

ICNA

[103]

COT

RIA

NA

NA

[104]

COT,

OH-

COT

SPE

LC-M

S/

MS

COT:LOD

=0.05

ng/m

LOH-

COT:LOD

=0.1

ng/m

L

Intra-assay

<17

%,

interassay

<21

%

[106]

Hair

Mon

ths

Easyandno

ninv

asive.

Perform

edun

derdirect

observation.

Hair-washing

toremov

eexternal

sources

ofcontam

ination

NIC

SPE

GC-M

SNA

NA

NIC

inhaircanbe

used

todifferentiate

betweeninfantsexpo

sedandno

texpo

sed

toETS.HairNIC

was

amoreprecise

biom

arkerof

expo

sure

toETSthan

urine

cotin

inelevelsMeanNIC

(ng/mg):

nonexp

osed

0.53

[91];high

lyexpo

sed5.95

[91]

[75]

NIC,COT

SPE

HPLC

NIC:LOD

=0.2

ng/

mgCOT:LOD

=0.1

ng/m

g

[30]

NIC,COT

RIA

NIC:LOD

=0.1

ng/

mgCOT:LOD

=0.05

ng/m

g

[109]

NIC

RIA

0.1

ng/m

gInterassay

<10

%[110

]

NIC

Liquid—

liquid

HPLC-

ECD

NIC:LOD

=0.05

ng/m

gIntra-assay

<10

.2%,

[113

]

Biological matrices for the evaluation of exposure to environmental tobacco smoke during prenatal life and childhood 385

Tab

le2

(con

tinued)

Biological

matrix

Detectio

nwindo

w[126]

Collection

Metabolite

Extraction

metho

dAssay

metho

dLOD

and/or

LOQ

(cutoffin

thecase

ofim

mun

olog

ical

metho

ds)

Intra-assay

coefficientof

variationand

interassay

coefficientof

variation

Biomarkerlevelsforthedifferent

expo

sure

grou

psReferences

interassay

<19

.8%

NIC,COT

Liquid—

liquid

GC

NIC:LOD

=0.00

5ng

/mgCOT:

LOD

=0.01

ng/m

g

Intra-assayNIC

<7.9%

,COT

<8.0%

Interassay

NIC

<8.0%

,COT

<9.0%

[127

]

NIC

Liquid—

liquid

HPLC

LOD

=0.05

ng/m

g,LOQ=0.1

ng/m

gIntrassay

<10

.2%,

interassay

<19

.8%

[128

]

NIC,COT

Liquid—

liquid

HPLC/

APCI-

MS

NIC:LOD

=0.05

ng/m

gCOT:

LOD

=0.02

ng/m

g

NA

[91]

NIC,COT

RIA

NIC:LOD

=0.05

ng/m

gCOT:

LOD

=0.01

ng/m

g

NA

[115]

COT

RIA

COT:LOD

=0.1

ng/

mg

NA

[111]

COT

RIA

NA

NA

[118]

COT

RIA

COT:LOD

=0.01

ng/m

g[120

]

COT

RIA

COTLOQ

=0.00

5ng

/mg

NA

[119]

NIC,COT

Liquid—

liquid

GC-M

SNIC:LOQ

=0.05

ng/m

gCOT:

LOQ

=0.02

5ng

/mg

NA

[129

]

NIC

NA

GLC

LOD

=0.07

ng/m

gNA

[130

]

NIC

Liquid—

liquid

GC-M

SLOD

=0.02

ng/m

gNa

[131

]

NIC,COT

Liquid—

liquid

LC-M

S/M

SNA

[132

]

Teeth

Years

Easyandno

ninv

asive.

Requiresorganicwashing

toascertainexternal

contam

ination.

Requires

pulverizationbefore

analysis

NIC,COT

Liquid—

liquid

GC-M

SNIC:LOD

=0.35

ng/

mL,LOQ

=1.2

ng/

mLCOT:LOD

=0.31

ng/m

L,LOQ

=1.1

ng/m

L

Intra-assayNIC

<10

%,COT

<10

%Interassay:NIC

NA,COTNA

NIC

indicatedcumulativeexpo

sure

totobaccosm

oke.

COTdidno

thave

astatistically

sign

ificantrelatio

nshipto

expo

sure

totobaccoMeanNIC

(ng/g

teeth):no

nexp

osed

grou

p:15

.0[124],

14.11[125];bo

thparentssm

okers42

.3

[123

]

NIC,COT

Liquid—

GC-M

SNIC:LOQ

=2

ng/g

NA

[124

]

386 H. Llaquet et al.

concluded that both active and passive smoking duringpregnancy are a risk factor for PPHN. Nafstad et al. [13]measured cord blood thiocyanate to quantify ETS, but the useof thiocyanate as a marker for tobacco smoke exposure ishampered by the fact that it is nonspecific (high baseline levelseven in nonexposed nonsmokers or environmental sourcesother than tobacco smoke) and insensitive (the incrementalchange owing to ETS exposure is small compared with thebaseline levels). Recently, Franchini et al. [8] used the cordserum cotinine to assess the impact and effectiveness of Italiansmoke-free policy. Puig et al. [14, 15] investigated theassociation between cotinine in cord serum and in maternaland newborn urine samples. Cord serum cotinine (measured byRIA) appeared to be the most adequate biomarker of fetalexposure to smoking at the end of pregnancy, distinguishing notonly active smoking from passive smoking, but also exposure toETS from nonexposure. Other studies [16, 17] carried out incohorts of pregnant women and comparing the usefulness ofcord blood and urine have corroborated these observations.Recently, some other studies have used high-performanceliquid chromatography (HPLC) [18, 19] or gas chromatogra-phy (CG)—mass spectrometry (MS) [10] to compare maternalplasma cotinine concentrations during pregnancy and deliverywith cord blood cotinine concentrations. A pattern of elevatedcotinine concentrations in the plasma of pregnant women (fromthe beginning of the pregnancy to the delivery) was observed,and this correlated significantly with the cotinine levels in theumbilical cord blood.

Neonatal urine

Urine formation starts in the fetus at the eighth week ofgestation, but it is not until the 16th week that urineproduction is sufficient to account for most of the amnioticfluid [7]. Traditionally, urine has been considered thespecimen of choice for neonatal drug testing for severalreasons: even though urine collection is difficult, it is superiorto the invasive serum collection and cotinine analysis inneonatal urine is an easy, rapid, and low-cost test [20].

A disadvantage of urine is that the time window ofdetection is short, reflecting drug use only a few daysbefore delivery. Analysis of the neonatal urine may producefalse-negative results depending not only on the time of thelast ingestion of the drug by the mother but also on the lengthof time after birth when the specimen was collected [15].

Urinary cotinine has been used as a biomarker ofexposure to ETS during pregnancy and it has beencompared with other biological matrices such as breastmilk or cord blood. In some studies, neonatal urinarycotinine has been related to the amount of tobacco taken bythe mother during the pregnancy. For example, Dahlströmet al. [21] studied newborn infants and measured nicotineand cotinine concentrations in mothers’ plasma, neonatalB

iological

matrix

Detectio

nwindo

w[126]

Collection

Metabolite

Extraction

metho

dAssay

metho

dLOD

and/or

LOQ

(cutoffin

thecase

ofim

mun

olog

ical

metho

ds)

Intraassay

coefficientof

variationand

interassay

coefficientof

variation

Biomarkerlevelsforthedifferent

expo

sure

grou

psReferences

[124],27

.49[125]

liquid

COT:LOQ

=2

ng/g

NIC,COT

SPE

LC-M

S/

MS

NIC:LOD

=3.3

pg/

mg,

LOQ

=10

pg/

mgCOT:LOD

=1.6

pg/m

g,LOQ

=5

pg/m

g

Intra-assayNIC

<14

%,COT

<11%

Interassay

NIC

<15

%COT

<10

%

[125]

COT-N-O

Xcotin

ineN-oxide,ECD

electrochemical

detection,

GLC:gas—

liquidchromatog

raph

y,UVDAD

diod

e-arrayUV

detection

Biological matrices for the evaluation of exposure to environmental tobacco smoke during prenatal life and childhood 387

urine, and breast milk. Concentrations were measured byGC [22]. Cotinine but not nicotine concentrations inmothers’ plasma, breast milk, and infants’ urine reflectedthe smoking habits during pregnancy. However, in otherstudies this relationship was not corroborated: Pichini et al.[15] studied 429 mothers and their newborns and measuredcotinine concentrations in neonatal urine and cord serum.Urinary cotinine was measured using a double-antibodyRIA according to a method described by Van Vunakis et al.[23]. Cotinine concentration in urine from newborns andfrom their nonsmoking mothers did not show a significantdifference between mothers exposed and not exposed toETS. Cord serum cotinine appeared to be the most adequatebiomarker of fetal exposure to smoking at the end ofpregnancy, distinguishing not only active smoking frompassive smoking, but also exposure to ETS from non-exposure. Recently, Mansi et al. [20] examined the relationbetween neonatal urinary cotinine (measured by RIA) andaltered behavior and concluded that neonatal behavior canbe significantly altered in a dose-dependent manner evenafter modest prenatal exposure to tobacco smoke.

Other nicotine metabolites have also been measured inneonatal urine: Köhler et al. [24] used HPLC to determinenicotine, cotinine, and trans-3´-hydroxicotinine in maternaland neonatal urine. A close correlation (which did notdepend significantly on the time of postpartal urinecollection) was found between maternal and neonatalnicotine and cotinine concentrations. The extent of currentprenatal smoke exposure attributable to active maternalsmoking was best reflected by the sum of nicotinemetabolites (cotinine and trans-3´-hydroxycotinine) with-out adjustment for creatinine.

Neonatal hair

Neonatal hair is a sensitive biological matrix that can definecumulative exposure to drugs during the last trimester ofintrauterine life. In the fetus, hair starts growing atapproximately 6 months of gestational age and reachesthe scalp surface after approximately 3 weeks. Although thedetection window is smaller than for meconium, hair hasthe advantage of being collected at any point during thefirst 3 months of life, after which time infant hair replacesneonatal hair [7]. Specimens are easily collected and can bestored at room temperature However, sometimes mothersare not willing to consent to testing the hair of their infantsfor aesthetic or cultural reasons. In addition, the neonatemay not have sufficient hair; sometimes not more than 5–10 mg of hair is available and the best extraction andsubsequent analysis is achieved when 20–50 mg of hair isused.

Nicotine in hair could originate from deposition fromfetal blood into the growing hair shaft or from contamina-

tion of hair by amniotic fluid. In either case, externalcontamination is not an issue for monitoring drug exposurein neonatal hair because the only source of the drug is frommaternal ingestion.

Both nicotine and its metabolites are incorporated intothe hair fibers by diffusion from the systemic circulationthrough the hair bulb, but as with other xenobiotics, theparent compound is incorporated more easily than the morepolar metabolites and is found in hair at a higherconcentration than cotinine or other metabolites. Nicotineconcentration has been shown to be higher in pigmentedthan in unpigmented hair [25]. Because hair is a solidmatrix, a chemical digestion is required to extract xeno-biotics from the hair shaft. Acid digestion has beenperformed at temperatures ranging from 37 to 100ºC andfor times varying from 1 h to overnight [26].

In 1993 Kintz et al. [27–29] studied the possibility ofmonitoring the transfer of maternal nicotine through theplacenta by measuring nicotine concentration in neonatalhair using GC-MS with selected ion monitoring.

During the 1990 s, immunological methods were utilizedfor quantifying nicotine and its metabolites in neonatal hair.The disadvantage of the application of immunologicalscreening techniques for hair analysis is the high false-positive rate. Immunoassay techniques designed for urinehave been modified for use with hair, many times withoutextensive validation or confirmation. Currently, the analyt-ical method most frequently used for hair analysis is GC-MS owing to its superiority compared with other analyticalmethods in terms of sensitivity, specificity, and selectivity.Usually electron impact mass detection is used, but positiveand negative chemical ionization mass detectors have alsobeen used. In recent years, tandem MS (MS/MS) and LC-MS have been used for hair analysis to increase sensitivityand detect GC-unstable compounds. Generally, drugs inhair are quantified by selected ion monitoring owing to thelow amounts of drug present. Therefore, the deuteratedtarget drugs are often used as the internal standards [30].

Using RIA, Eliopoulos et al. [31] measured maternal andneonatal hair concentrations of nicotine and cotinine in 94mother—infant pairs. A significant correlation was foundbetween maternal and neonatal hair concentrations ofnicotine. Mothers were grouped in active smokers, non-smokers, and passive smokers. Furthermore, it was shownthat the infants of passive-smoking mothers were at risk ofexposure to cigarette smoke. Later, with the aim todetermine the extent of fetal exposure to cigarette smoke,Eliopoulos [32] measured concentrations of nicotine andcotinine in maternal and neonatal hair and associatedintrauterine exposure to tobacco smoke with lower birthweight, smaller head circumference, shorter length, andperinatal complications. Klein and Koren [33] used RIA toquantify nicotine and cotinine in maternal and neonatal hair

388 H. Llaquet et al.

and also in a cohort of older, asthmatic children. Inagreement with the above-mentioned reports, there was asignificant correlation between maternal and neonatal hairconcentration for both metabolites. Similarly, Jacqz-Aigrainet al. [34], using RIA, measured nicotine and cotinineconcentrations in the maternal and neonatal hair at birth in182 mothers and infants. As it had been reported before, inmaternal hair, both nicotine and cotinine concentrationswere associated with cigarette consumption during the thirdtrimester of pregnancy.

Immunoassay results should be carefully interpreted andconfirmed by a more specific method such as GC-MS, LC-MS, or MS/MS. In 2006, a LC-MS/MS method detectednicotine and cotinine with a low limit of detection using asmall amount of hair similar to the hair available inneonates [35]. Recently, Seong et al. [36] using the methodmentioned above investigated fetal exposure to paternalsmoking at home during pregnancy in 63 Korean families.To determine chronic exposure to ETS, nicotine andcotinine concentrations in hair were measured by LC-MS/MS. Surprisingly, there was no significant differencebetween neonatal nicotine concentrations in the smokingand nonsmoking groups. However, in the indoor-smokinggroup, neonatal nicotine concentrations were significantlyhigher than in the outdoor-smoking and nonsmokinggroups. These findings indicate that paternal smokinginside the home leads to significant fetal and maternalexposure to ETS and may subsequently affect fetal health.

Neonatal nails

Nails are formed during the last trimester of pregnancy andare, thus, supposed to reflect the exposure only in thisperiod. Human toenails grow at a rate of 1 cm every 9–12 months and their use could reflect a relatively longercumulative exposure period [37]. However, similar to hair,incorporation of drugs in the nails of the fetus might bemediated by the fetal blood and/or by the amniotic fluid.Compared with hair, which is not always present on thescalp of newborns or is lost in significant amounts duringthe first months of life, and may encounter some resistancein collection for aesthetic or cultural reasons, nails have thenet advantage of always being present and considered“discarded” material.

Recently, Mari et al. [38] investigated for the first timethe usefulness of newborn nails for monitoring the exposureto drugs of abuse during pregnancy. Toenail samples wereobtained from children abandoned immediately after birthand from babies born at the local maternity hospital.Newborn nail samples were extracted by solid-phaseextraction (SPE) and were subjected to GC-MS analysis,but no quantification was performed for biomarkers oftobacco compounds. Their results support the use of nails

for identifying chronic exposure to drugs of abuse duringpregnancy.

Amniotic fluid

Up to 20 weeks of gestation, maternal secretions are themost important mechanism of amniotic fluid formation,with some additional secretion components from the fetus.After this period, fetal urine and lung fluid secretionbecome the two primary sources of amniotic fluid, withfetal swallowing and intramembranous absorption as thetwo primary routes of amniotic water clearance [39]. Morethan any other compartment, the amniotic fluid accumulateswater-soluble drugs; it may also contain to some extentparent compounds and their metabolites that are not water-soluble. At 10 weeks of gestation, the volume of amnioticfluid is 30 mL, which increases up to 800–1,000 mL atweek 37. Because amniotic fluid is already formed in thefirst weeks of pregnancy, the presence of drugs in this fluidcan account for exposure during early fetal life [40–42].Although it is quite dangerous for the fetus, sampling ofamniotic fluid can be done at any time during pregnancy,with parent drugs and metabolites present in the fluidindicating that the fetus is continuously exposed to thesubstances detected in the maternal circulation. Severalstudies have focused on detection of prenatal exposure tonicotine in the amniotic fluid . Both parent drug andmetabolites have been identified, establishing cotinine asthe most important metabolite of nicotine [43, 44].

For the first time, in 1985, Luck et al. [45] analyzed (in acohort of 69 pregnant women) nicotine and cotinine inplacental tissue during the first trimester, in amniotic fluidduring the second trimester, and in placental tissue and fetalserum at birth. Nicotine concentrations in placenta, inamniotic fluid, and in fetal serum were higher than thecorresponding maternal serum values. The ratio did notdepend on the time elapsed between the last cigarettesmoked and the sampling. Significant correlations werefound between nicotine concentrations in amniotic fluidand maternal serum. Their results indicate that the humanfetus is exposed to higher nicotine concentrations than theconcentrations found in the serum of smoking mothers. In1990, Jordanov [46] measured cotinine in amniotic fluidand compared the results with the values for the urine of 31pregnant women and the urine of their offspring. Amnioticfluid cotinine was 8 times higher in active smokers and 2.5times higher in passive smokers than in nonsmokers. Ingeneral, amniotic fluid cotinine was considerably higherthan urinary cotinine both in active and in passive smokers.In 1999, Jauniaux et al. [47] studied 85 pregnant womenwho aborted for psychosocial reasons during the first halfof pregnancy between 7 and 17 weeks of gestation. Theauthors reported on the concentration of cotinine in

Biological matrices for the evaluation of exposure to environmental tobacco smoke during prenatal life and childhood 389

amniotic fluid and compared the fetal and maternal cotininelevels in passive and active smokers. The study indicatedthat cotinine accumulates in the fetal compartment as earlyas 7 weeks of gestation in both active and passive smokers.

Although amniotic fluid analysis has been used toconfirm fetal exposure to nicotine and cotinine, thisbiological fluid has not gained popularity as a practicaltool for identifying prenatal drug exposure. The mostimportant reason is the difficulty encountered in thecollection of this specimen, which requires an invasiveprocedure that can be potentially harmful to the fetus,unless it is collection is performed at birth [7].

Meconium

Recently, meconium became the specimen of choice fordetecting drug exposure in neonates. Meconium is the firstfecal matter passed by the neonate and pertains to a largetime window in prenatal metabolism. Meconium is primar-ily composed of mucopolysaccharides, water, bile salts, bileacids, epithelial cells, and other lipids. Meconium can becollected between 1 and 5 days after birth, and its collectionis easy and noninvasive. Drug concentrations in meconiumare generally higher than in urine because of accumulationover several months of gestation. For these reasons,meconium testing provides more complete information ontobacco exposure during pregnancy than neonatal urine orcord blood analyses [7].

As meconium is not homogenous, specimens should bemixed thoroughly before analysis. Meconium is complexand recovery of drugs is highly dependent on the extractiontechnique. Generally, drug concentrations in meconiumremain stable when it stored at −20ºC.

RIA is the most commonly employed screening assay.The principal disadvantage of screening techniques for theanalysis of meconium is the high false-positive rate. Aswith other matrices, the results should be carefullyinterpreted and confirmed by a more specific method suchas GC-MS, LC-MS, or LC-MS/MS. In this context, Ostreaet al. [48] screened for cotinine and trans-3´-hydroxycoti-nine in meconium by RIA and confirmed their results byGC-MS in newborns of smoking mothers. The cotinineconcentration in meconium was used to categorize thedegree of active smoking by the mother, but the level wassimilar in passive smokers and light active smokers. Nospecimen tested positive for trans-3´-hydroxycotinine andlimits of quantification were not provided.

Dempsey et al. [49] measured cotinine levels in 102meconium samples by immunoassay using a nonhydrolysisscreening procedure. Separated aliquots were hydrolyzed.Of the nonhydrolyzed samples, 33% were positive forcotinine, while in the hydrolyzed samples 79% werecotinine-positive. It was hypothesized that cotinine forms

reversible Schiff base bonds with free amino functions onproteins, therefore, hydrolysis of meconium would benecessary for the detection of “free” cotinine. Commondrugs of abuse did not interfere with the analysis.

Derauf et al. [50] assessed the agreement betweenmaternal self-reported tobacco use and ethanol intakeduring pregnancy. Cotinine and fatty acid ethyl esters weremeasured in 434 meconium samples. Moderate agreementwas found between reported tobacco use during the thirdtrimester and the cotinine level detected; however, therewas no agreement between the reported ethanol intakeduring the third trimester and fatty acid ethyl estersdetected.

Sherif et al. [51] correlated neonate meconium cotininelevels with urinary and salivary maternal cotinine levels.The mean maternal urinary cotinine levels, measured usingRIA, differed significantly between the three groups (activesmokers, passive smokers, no tobacco exposure), as did themean salivary cotinine and mean cotinine levels inmeconium. There was a significant positive correlationbetween cotinine levels in meconium and both maternalurinary and salivary cotinine levels.

Cotinine, but not nicotine, was detected in meconium in30 samples analyzed by Baranowski et al. [52] using HPLCcoupled with diode-array detection. Similarly, using HPLC,Köhler et al. [53] measured levels of nicotine, cotinine, andtrans-3´-hydroxycotinine in meconium of 115 newborns toidentify prenatal smoke exposure. With the time of passageafter birth, the percentage of nicotine in meconiumdecreased significantly, and the percentage of trans-3´-hydroxycotinine increased, whereas the increase in cotininewas only marginal. The sum of the three compoundsenables the estimation of the intensity of prenatal smokeexposure, irrespective of which meconium sample isavailable for analysis.

Recently Gray et al. [54, 55] developed and validated thefirst LC/atmospheric pressure chemical ionization (APCI)—MS/MS method for simultaneous quantification of nicotine,cotinine, trans-3´-hydroxycotinine, nornicotine, and norcoti-nine in meconium. They quantified concentrations ofnicotine and the four metabolites with and without hydroly-sis simultaneously in meconium from tobacco-exposed andnonexposed neonates. Nicotine, cotinine, and trans-3´-hydroxycotinine were the most prevalent and abundantmeconium tobacco biomarkers and were found in higherconcentrations in tobacco-exposed neonates. Unconjugat-ed nicotine, cotinine, or trans-3´-hydroxycotinine meco-nium concentrations above 10 ng/g most accuratelydiscriminated active from passive and nonexposed neo-nates. There was no significant correlation betweennicotine and metabolites in meconium and neonatal out-comes (although the presence of a nicotine biomarkerpredicted decreased head circumference).

390 H. Llaquet et al.

Biological matrices for the evaluation of postnatalexposure to environmental tobacco smoke

Breast milk

Milk is a complex physiological liquid that simultaneouslyprovides nutrients and bioactive components that facilitatethe successful postnatal adaptation of the newborn infant bystimulating cellular growth and digestive maturation, theestablishment of symbiotic microflora, and the developmentof gut-associated lymphoid tissues. Milk is composed ofcertain vitamins, specific proteins, bioactive peptides,oligosaccharides, and organic (including fatty) acids.

The major reason for drug investigation in human milk isto calculate excretion of certain compounds in this fluidand, consequently, the approximate dose ingested by thebreast-feeding infants. Nicotine and its metabolites (coti-nine, trans-3´-hydroxycotinine, and cotinine N-oxide) passthrough the epithelial cells of the mammary glands into themilk. The rate of diffusion across the mammary glands maybe modulated by the physiochemical properties of themolecules and their concentration in milk depends on thedose ingested, the duration of the consumption, the amountof milk excreted daily, the mother’s health, and hergenotype. Nicotine, which is a weak base (pKa1 = 8.0), isconcentrated in the slightly acidic milk (pH 6.8) throughion trapping. The half-life of nicotine in milk slightlyexceeds the half-life in serum (approximately 2 h), whilethe cotinine concentration remains constant during a4-h interval without smoking; therefore, cotinine is a betterbiomarker. After smoking, the nicotine concentration isconsistently higher in the mothers’ milk than in their blood(3:1), but only 10% of the maternal nicotine dose isexcreted into breast milk [56]. The rapid transfer of nicotinefrom serum into milk and the short half-life of nicotine inmilk indicate that mothers who cannot refrain fromsmoking during the nursing period should attempt toprolong the time between the last cigarette smoked andbreast-feeding to minimize the exposure of the nursinginfant to tobacco products [57].

In 1976, Ferguson et al. [58] were the first to measurenicotine and its major metabolite cotinine in milk of nursingsmokers using gas—liquid chromatography (GLC). Luckand Nau [59] also analyzed breast milk samples fromnursing smokers by GLC. There was a linear correlationbetween both cotinine and nicotine concentrations in serumand in milk. The nicotine concentrations in milk wereconsiderably higher than the corresponding serum concen-trations, while the cotinine concentrations were lower.During the early 1990 s several studies [21, 60–62]followed cohorts of nursing and not nursing smokingmothers. These studies measured simultaneously nicotineand cotinine in breast milk and in the infants’ urine to

estimate the amount of nicotine metabolites incorporated bythe milk. Cotinine concentrations in breast milk have beenrelated to the number of cigarettes smoked daily. Urinaryexcretion of cotinine was 5–10 times higher in fully breast-fed infants whose mothers smoked than in those whosemothers smoked but did not breast-feed. The urinarycotinine excretion in the breast-fed infants was in the rangefor adult smokers, while cotinine excretion in the urine ofinfants from smoking but not breast-feeding mothers washigher than that of adult passive smokers. Therefore, ininfants, breast-feeding is the most important source ofexposure to nicotine, passive smoking being the secondmost important. Also during the 1990 s drug of abusedetection in breast milk was used to help the diagnosis ofcocaine or opiate intoxication. Dickson et al. [63] deter-mined cocaine, nicotine, opiates, and other drugs of abusein breast milk in four clinical cases. The concentrations ofmetabolites were measured by immunoassay drug screeningand confirmed by liquid—liquid extraction with GC withflame ionization detection and GC-MS analysis. Yearsafterwards, Page-Sharp et al. [64] developed a method tomeasure nicotine and cotinine in human milk by HPLCusing liquid—liquid extraction combined with back-extraction into acid, and followed by reverse-phase chro-matography with UV detection of analytes. The amount offat in the milk did not affect the recovery. This method wasused by the same group [65] to assess the use of nicotinepatches in breast-feeding mothers. The results indicate thatundertaking maternal smoking cessation with the nicotinepatch is a safer option than continued smoking. Use of thenicotine patch had no significant influence on the milkintake by the breast-fed infant. Dahlström et al. [66]quantified that the mean intake of nicotine via milk is7 μg/kg per day and recommended not breast-feeding rightafter smoking because the milk nicotine concentration willincrease. Aresta et al. [67] determined simultaneouslycaffeine, theobromine, theophylline, paraxanthine, andnicotine in human milk by LC with diode-array UVdetection. Pellegrini et al. [68] developed a LC-MS/MSmethod and determined nicotine and its principal metabo-lites cotinine, trans-3´-hydroxycotinine, and cotinine N-oxide, as well as caffeine and arecoline in breast milk.Cotinine was the most prevalent biomarker of tobaccosmoke in the breast milk of smoking mothers, whilenicotine was present only in the milk samples of smokingmothers who had declared recent active smoking. CotinineN-oxide and trans-3´-hydroxycotinine were detected for thefirst time in breast milk and appeared as minor metabolites.This method was used by Vagnarelli et al. [69] to diagnosea case of withdrawal of nicotine syndrome in a breast-feeding infant.

Nicotine absorbed by the infant through breast milk isshown to produce short-term symptoms of restlessness,

Biological matrices for the evaluation of exposure to environmental tobacco smoke during prenatal life and childhood 391

insomnia, nausea, vomiting, diarrhea, and rapid pulse andpossible long-term symptoms of physical and mental handi-caps. Quantification of cotinine in breast milk has also beenused to relate exposure to ETS to infants’ cardiorespiratoryhealth problems. In this context, Dahlström et al. [70] foundrecently that in breast-fed infants of smoking mothers themean arterial pressure is significantly lower than in infants ofnonsmokers and concluded that postnatal exposure to nicotineinfluences autonomic cardiovascular control in infants.

Urine

Urinary nicotine content reaches its maximum level 2 hafter smoking and decreases subsequently. Urinary cotininereaches its maximum level 4 h after smoking [71]. Nicotineis not an appropriate marker to determine passive smokerstatus as it has a short half-life (about 2 h). The half-life ofcotinine was reported to be about 20 h. A urinary cotininelevel of 100 ng/mL is known as a cutoff for active smokers,while less than 5 ng/mL indicates nonexposure to ETS [72].Urinary cotinine depends on renal function, flow rate, andurinary pH. Although reporting urinary cotinine adjustedfor creatinine may correct, in part, for differences indilution effects, creatinine excretion is also variable. Inaddition, a low level of creatinine in children (relative to thelevel in adults) results in high cotinine to creatinine ratiosthat are falsely indicative of active smoking [73]. Althoughthe elimination half-life for cotinine does not differ betweeninfants and older children, infants, compared with olderchildren, take up a higher relative dose of nicotine becauseof the higher relative ventilation rate and because they staywith their smoking parents for longer periods. Othernicotine metabolites that have been suggested for measur-ing ETS exposure are trans-3´-hydroxycotinine [74] andthe sum of cotinine and trans-3´-hydroxycotinine [75]. Asreported above, in serum, cotinine reaches its maximumconcentration 1.5 h after nicotine application, whereastrans-3´-hydroxycotinine attains its peak level in serumafter about 18 h. Thus, the ratio between cotinine andtrans-3´-hydroxycotinine in saliva or serum is a function oftime; cotinine prevails at the early stage, whereas in thefurther course the trans-3´-hydroxycotinine concentrationbecomes equal to, or even exceeds, the cotinine level. Thistime pattern also turned out to hold true for the urinaryconcentrations of cotinine and trans-3´-hydroxycotinine. Inthe case of smoke exposure about 24 h earlier, the cotinineconcentration may already have been below the detectionlimit. However, trans-3´-hydroxycotinine (reaching itsmaximum later and having a urinary level, on average, 3times higher than that of cotinine) is still detectable at thistime. Sampling problems, interpersonal variations, and agevariations in the half-life of cotinine complicate the use ofcotinine measurements in urine among small children [76].

There are several fast and relatively inexpensive methodsto analyze urinary cotinine, such as colorimetric methodsand different immunoassays, but the most reliable are thechromatographic methods HPLC and GC, especially whenthe identity of the peaks can be verified by MS. Anotheradvantage of chromatography is the simultaneous quantifi-cation of nicotine and cotinine in a single analysis. Thereagent costs are generally low and the limits of detectionare low, which makes these methods very suitable forpassive-smoking studies [77]. However, the chromato-graphic methods require the use of costly equipment andthe resource of skilled personnel for routine analysis

RIA has been much used to compare the concentrationof nicotine and cotinine in saliva and urine of tobaccosmoke exposed and unexposed infants, but immunoassayvalues overestimate cotinine concentrations by about 2.9times compared with GC-MS [78]. Before 1990 GLC[79] and ELISA [80, 81] were used for measuring urinaryand salivary cotinine in children to distinguish exposedchildren from unexposed children. Peterson et al. [82]measured urinary cotinine by RIA in a population-basedcohort of children every other month from birth to 2 yearsof age. All analyses were performed using the cotinine tocreatinine ratio expressed as nanograms of cotinine permilligram of creatinine. Using the cutoff suggested byHenderson et al. [83], values equal to or less than 32 ng/mg indicated no exposure, while values greater than32 ng/mg indicated exposure. The data showed that asingle point measure of the cotinine to creatinine ratiodoes not adequately describe the infant’s passive ETSexposure. During the 1990 s a HPLC method wasdeveloped and used in children: Hariharan et al. [84]described a HPLC method with UV detection for thesimultaneous analysis of nicotine and cotinine in urine andcompared the results for 20 urine samples from childrenunder 14 years old with the results of GC coupled withnitrogen—phosphorus detection (NPD) and did not noticesystematic differences. Zuccaro et al. [85] applied theSPE procedure for the determination of nicotine,cotinine, and trans-3´-hydroxycotinine in urine samplesby HPLC. Köhler et al. [86] considered the advantageousof using the sum of nicotine metabolites (cotinine andtrans-3´-hydroxycotinine) as a marker for passive-smokeexposure and defined 10 nmol cotinine plus trans-3´-hydroxycotinine per milliliter of urine as the cutoff pointbetween exposed and unexposed children. Their studyshowed that in a severe chronic disease such as cysticfibrosis, only as little as 35% of the parents admitted topassive smoke exposure of their children; therefore,objective biomarkers were clearly needed to classifybetween exposure and nonexposure to ETS.

Bazylak et al. [87] described a high-performance thin-layer chromatography screening assay for urinary cotinine.

392 H. Llaquet et al.

With respect to sensitivity, the proposed method was notsuitable for the adequate detection of incidental home ETSexposure.

Also during the 1990 s Hutchinson et al. [88] used GC-MS for the simultaneous measurement of nicotine andcotinine in urine and serum. Stepans [89] followed 15infants of smoking mothers from birth to 6 weeks of age.Exposure to ETS was assessed by using a smoking habitsquestionnaire, cigarette “butt” collection, infant urinarynicotine and cotinine levels (measured by GC-NPD), andambient nicotine levels (personal air monitors). Cigarettebutt collection was strongly correlated to infant urinarynicotine and cotinine levels when the infants were 2 weeksof age. There was no correlation between ambient nicotineconcentrations collected in the passive air monitors andmeasures of maternal smoking behavior or infant urinarycotinine levels. There was an increase in cotinine excretionduring the first 6 weeks of life. Other authors had reportedthat cotinine excretion increases during the first year of life[90].

Matt et al. [91] tried to determine whether contaminateddust, surfaces, and air contribute to ETS exposure in infantsand whether smoking parents can protect their infants bysmoking outside and away from the infant. ETS contami-nation was measured by nicotine in household dust, indoorair, and household surfaces. Infants were aged between 2and 12 months and urinary cotinine was measured byHPLC/APCI-MS [92]. Urinary cotinine levels of infants inthe indirect exposure group were approximately 8 timeshigher than in the no-exposure group but were 6 timeslower than the cotinine levels in the direct exposure group.Smoking outside the home and away from the infantreduced but did not fully protect a smoker’s home fromETS contamination and a smoker’s infant from ETSexposure. Matt et al. [93] examined ETS contamination inthe homes of 150 children of smoking mothers and thedistribution of urinary cotinine and trans-3´-hydroxycotinineby LC-MS/MS. They suggested that cotinine, trans-3´-hydroxycotinine, and the sum of the two metabolites areapproximately equivalent and equally strong biomarkers ofETS exposure in children. Boyaci et al. [94] examined therelationship between parent-reported estimates of children’sexposure to ETS and children’s urinary cotinine andconcluded that parents’ reports are not reliable. Joseph etal. [95] measured urinary cotinine levels in 104 infants of12 weeks of age. On average, babies of smoking parents(and specially of smoking mother) excreted nearly 6 times asmuch cotinine in the urine as did the babies of nonsmokingparents. Recently, Puig et al. [14] reported the results of acord serum and neonates’ urinary cotinine analysis by RIAin a prospective birth cohort (487 infants). At 4 years of age,the median urinary cotinine level in children increased 1.4 or3.5 times when the father or the mother smoked, respectively.

Cotinine levels in children’s urine statistically differentiatedbetween children from smoking mothers and exposed homesand nonexposed homes. Urinary cotinine had been used tomeasure the prevalence of exposure to ETS. In Thailand,Anuntaseree et al. [96] performed a survey to assess theexposure to ETS of 1-year-old infants using HPLC [97]. Ofthe 725 infants, it was reported that 73.3% were exposed tohousehold smoking and 40.7% had detectable urinarycotinine, with 3.4% having urinary cotinine in the range ofadult heavy smokers.

Urine has also been used as a control matrix in differentcomparisons with other alternatives matrices such as salivaor breast milk. Schwartz-Bickenbach et al. [60] found thatthe highest urinary excretion of cotinine was observed ininfants fully breast-fed by smoking mothers. These resultswere confirmed by Schulte-Hobein et al. [62], whomeasured cotinine in children’s urine and in breast milkby GC procedures similar to those of Luck and Nau [59,98] and Feyerabend et al. [77]. Recently, Ilett et al. [65]analyzed cotinine and nicotine in breast milk and infant(6 weeks old) urine samples by GC. The concentrations ofcotinine in infant urine correlated with the dose of nicotineingested by breast-feeding [22].

Oral fluid

Oral fluid is a composite tissue consisting primarily ofsaliva, mixed with gingival fluid, buccal and mucosaltransudates, cellular debris, bacteria, and residues ofingested products. In this sense, the oral fluid is aninteresting ETS marker for acute consumption that occurredin the hours previous to collection, is less invasive and ismore cost-effective than blood. Transfer of tobacco smokeconstituents and their metabolites from blood to salivaoccurs primarily by passive diffusion and is dependentupon numerous factors, including chemical properties ofthe drug, salivary pH, concentration of unionized drug, anddrug—protein and membrane characteristics [99]. Althoughthere are a number of factors that affect drug concentrationin oral fluid, there appears to be a reasonable correlationbetween blood and oral fluid concentrations of drugs.Salivary monitoring has been shown to be useful for thedetermination of a large number of drugs that havetraditionally have been measured in plasma/serum.

For the first time, in 1984, Greenberg et al. [90] measuredthe urinary and salivary concentrations of nicotine andcotinine in infants exposed and not exposed to ETS byRIA. The concentrations were significantly higher in theexposed group than in the nonexposed group, with the bestindicator of chronic exposure being the urinary cotinine tocreatinine ratio. Salivary cotinine was less sensitive thaturinary cotinine. In agreement with this, in 1987, Coultas etal. [100] measured salivary cotinine levels by RIA in 2,029

Biological matrices for the evaluation of exposure to environmental tobacco smoke during prenatal life and childhood 393

children. Their results showed detectable levels of cotinine inchildren from households with smokers. Cotinine could bedetected in the saliva of nonsmokers’ children, even amongthose not living with a smoker.

In 1994, Ronchetti et al. [101] determined the relationshipbetween salivary cotinine levels in 146 schoolchildren aged9–14 years, and individual and entire household smoking asmeasured by the number of cigarettes smoked as well as therelationship with the season (RIA). The frequency ofdetectable cotinine both in "nonsmokers" and in "activesmokers" was significantly correlated with the number ofcigarettes smoked by household members. In "nonsmokers,"the proportion of subjects with detectable cotinine decreasedsignificantly in spring compared with winter, a finding notobserved in "active smokers." In conclusion, they demon-strated that passive exposure renders cotinine more detect-able in winter, mainly related to smoking in the presence ofchildren. Conversely, salivary cotinine in spring appears tobe derived mainly from active smoking.

Jarvis et al. [102], during the 1980 s, developed differentmethods for the quantification of cotinine in saliva. In1985, in a cohort of 569 nonsmoking schoolchildren theymeasured cotinine concentration in saliva by GLC. Thestudy showed that when neither parent smoked the meanconcentration was lower than that for either parent or thesum of the concentration for both parents. One year later[79], the same group described a method for the analysis ofcotinine in plasma, saliva, and urine using GLC. A fewyears later, in 1990, the same group [103] developed acapillary column GLC for the simultaneous measurement ofnicotine and cotinine in the same matrices with theobjective of minimizing the sample volume needed todetect nicotine and its principal metabolite. In conclusion,their studies demonstrated that cotinine concentrations innonsmoking schoolchildren presented a clear and signifi-cant dose relationship to passive exposure due to theirparents’ smoke.

In 1988, Langone et al. [80] used a monoclonal-antibody-based ELISA test to measure cotinine in saliva and urine of70 children (exposed and not exposed to tobacco smoke) andtheir parents. Once again, cotinine levels in saliva and inurine distinguished between exposed and unexposed chil-dren. The concentrations of salivary cotinine in passivelyexposed children were consistent with those reported usingRIA [81] or chromatographic techniques, but the concentra-tion of urinary cotinine in exposed children was much lowerthan those reported previously

In 1999, Suárez et al. [104] evaluated the exposure ofchildren and their parents to cigarette smoke by measuringcotinine levels in saliva by RIA. Cotinine levels werehigher in the children of passive smokers than in thenonexposed group. A significant correlation was foundbetween salivary cotinine and the total number of cigarettes

smoked daily by both parents, the number of cigarettessmoked daily at home, and the number of cigarettes smokedat home in the 24 h prior to saliva sampling.

During the 1990 s, McAdams [105] developed acapillary GC-MS method using selected ion monitoringfor the analysis of cotinine in urine, serum, and oralsamples. Later, in 1999, Bentley et al. [106] validated anassay for the determination of cotinine and trans-3´-hydroxicotinine in human saliva using automated SPE andLC-MS/MS detection. The results from the analysis ofsaliva samples using this assay were consistent with thesubjects’ self-reported ETS exposures, enhancing theapplicability of cotinine as a biomarker for the assessmentof low-level ETS exposure.

Oral fluid accounts only for acute consumption thatoccurred in the hours previous to collection, and repetitivesampling is needed to verify a suspected repetitive maternalabuse of drugs. For this reason, although theoreticallyinteresting, maternal oral fluid testing has not yet beenapplied extensively to assess maternal abuse of illicit drugsduring pregnancy and fetal exposure [7].

Hair

Hair testing has been considered the “gold standard” toassess chronic ETS. Because cotinine accumulates in hairduring hair growth, it is a unique measure of long-term,cumulative exposure to tobacco smoke. The major potentialadvantage of the hair test, when compared with saliva andserum measurements, is its ability to reflect long-termexposure (months) rather than short-term exposure (hoursor days). It is estimated that hair grows at approximately1 cm/month [30]; therefore, hair can be analyzed inmonthly, 1-cm segments, creating a “calendar” of nicotineexposure. The measured concentration of nicotine must berelated to the length of the hair segment and its distancefrom the scalp [26]. Lack of segmental hair analysis resultsin an inability to match hair nicotine levels to a specificexposure period [107].

For the first time, Nafstad et al. [75] measured hairnicotine levels with the aim of evaluating the limitationsof methods currently used for the estimation of ETS(questionnaire and urinary cotinine). Their results indicatea correlation between urinary cotinine and hair nicotine.Comparing the results of the objective biomarkers andself-reports, they observed an underreporting of ETSexposure. Going one step further, Pichini et al. [108]measured nicotine and its principal metabolite usingHPLC in 24 children aged 3–36 months attending anursery school in the suburbs of Rome. For the first time,in the pediatric population, nicotine measurement in hairwas used to categorize different statuses of chronicexposure to ETS.

394 H. Llaquet et al.

Nicotine and related compounds deposit in the hair ofchildren when tobacco smoke is part of their environment.In this context, Nafstad et al. [109] estimated quantitativelythe relationship between hair nicotine concentrations inmothers and children and tobacco smoke exposure assessedby questionnaires. Children’s nicotine levels were linearlyrelated to the daily number of cigarettes smoked at home byboth mothers and fathers. Similarly, Al-Delaimy et al. [110]assessed the relationship between nicotine and cotininelevels in hair and reported exposure to ETS; moreover, aquestionnaire of smoking habits of household adults wasalso completed. This report concluded that hair nicotinelevels rather than hair cotinine levels provide an informa-tive and objective measure of ETS exposure. In this sense,the main analytes generally found in hair are the parentcompounds rather than their more polar metabolites, whichusually predominate in blood or urine [26].

Al-Delaimy et al. [110, 111] measured nicotine hairlevels in 117 children and evaluated the effect of avoidancestrategies such as smoking outside the household. Levels ofnicotine in hair of children reportedly exposed to smokerswere higher than levels of unexposed children. Whetherhousehold members smoked outside or inside the house hadno significant effect on hair nicotine levels of children. It iswell known that the best understood route of exposure toETS is the inhalation of contaminated indoor air. Contam-inated air also contains ETS particles, and subsequentlythese particles become respirable suspended particles thatcannot be easily filtered and removed by the protectivemechanisms of the nose and throat. Infants of smokingparents are at risk of second-hand smoke exposure throughcontaminated house dust and surfaces. During their firstyear of life, infants spend much time indoors, are in closeproximity to contaminated dust and objects (e.g., blankets,carpets, floors), and are in close physical contact with theirsmoking parents. As infants and young children are highlyactive close to the floor, they may also be exposed to higherlevels of resuspended floor dust than adults. Thesesurprising results were expanded by Matt et al. [91] incomparing three types of households: nonsmokers whobelieve they have protected their children from ETS;smokers who believe they have protected their childrenfrom ETS; and smokers who expose their children toETS. For this study, ETS contamination was estimatedby measuring nicotine in household dust, indoor air, andhousehold surfaces. The results show that dust andsurfaces in homes of smokers are contaminated withETS. For this reason, infants of smokers are at risk ofETS exposure in their homes through dust, surfaces, andair. Smoking outside the home and away from the infantreduces but does not completely protect a smoker’s homefrom ETS contamination and a smoker’s infant from ETSexposure.

In 2004,Wood et al. [112], quantified and compared levelsof ETS exposure in mothers and their healthy children lessthan 3 years old using hair cotinine as an objective measureof exposure. Cotinine in hair is derived only from what anindividual has actually inhaled and metabolized, andtherefore it measures actual systemic exposure to nicotine.Hair cotinine levels of children correlated with maternal haircotinine levels and were greater than or equal to the mothershair cotinine levels. These findings resemble a saturationeffect found in chemical reactions. For minimal exposure,the cotinine levels in children’s hair were consistently higherthan those of maternal hair cotinine. At higher levels ofexposure, the children’s hair cotinine levels were no differentfrom maternal hair cotinine levels.

Biomarkers are becoming increasingly popular for ETSexposure measurement, as they avoid many sources of biasand may provide greater sensitivity than questionnaires. Inthis sense, the nicotine assay in hair is relatively recent. Adisadvantage of hair analysis is the need to use analyticalmethods based on chromatography techniques coupled withMS methods that require qualified personnel. To avoid thisproblem, Al-Delaimy et al. [113] aimed to compare hairnicotine with urinary cotinine and studied the relationshipof these two “epidemiologically cost effective” biomarkerswith the questionnaire assessment of ETS exposure.Although questionnaires are not considered very accurate,they have certain validity and have been used for describingmost of the associations between ETS and illnesses. Thehair nicotine biomarker was better correlated to variables ofreported exposure compared with urinary cotinine levels,and these variables, individually and collectively, predictedbetter hair nicotine levels than urinary cotinine. Moreover,nicotine hair testing is more precise than the currently usedurinary cotinine testing and hair is more easily collected.

Exposure to ETS is associated with increased rates ofrespiratory infections, asthma, ear and sinus infections, andsudden infant death syndrome in young children [5].During the last decade several studies have shown thatchildren whose parents smoke have higher rates of asthma.In 1998, Knight et al. [114] investigated whether asthmaticchildren are different from nonasthmatic children exposedto similar degrees of passive smoking in the way theirbodies handle nicotine, a constituent of cigarette smoke.Hair concentrations of cotinine were measured by RIA.Parents of asthmatic children tended to report a lower dailynumber of cigarettes and this fact agreed with the trend ofurinary cotinine. However, children with asthma had onaverage twofold higher concentrations of cotinine in theirhair than control children. These data suggest that of allchildren passively exposed to ETS, those who exhibitasthma have a higher systemic exposure to nicotine,possibly due to lower clearance rate. This is the firstevidence of pharmacokinetic predisposition to ETS as an

Biological matrices for the evaluation of exposure to environmental tobacco smoke during prenatal life and childhood 395

etiological factor in pediatric asthma. Recently, Spanier etal. [115] evaluated the relation of environmental factorssuch as ETS with exhaled nitric oxide concentrationsamong asthmatic children. Contrary to the findings ofKnight et al., there was no correlation between hair cotinineand exhaled nitric oxide concentrations.

It is considered that Latino children are at particularlyhigh risk for asthma, a condition exacerbated by cigarettesmoke. Latinos have lower rates of insurance coverage andare less likely to have access to preventive health care andpublic health programs. In 2003, Conway et al. [116], in acohort of Latino children from San Diego, measured hairlevels of nicotine and the major metabolite cotinine with theaim of evaluating the effectiveness of an interventionapproach based on sessions with health advisers. Therewere no differences in the measurements of hair nicotinebetween the intervention and the control group. Theintervention was concluded as not effective. Recently,Woodruff et al. [117, 118] in the same cohort assessed theusability and impact of a feedback-based ETS reductionapproach for Latino families. Parents’ reports of exposureand children’s hair nicotine levels showed a statisticallysignificant reduction and it was concluded that thisfeedback approach to ETS reduction warranted furtherstudy with a more rigorous design.

African-American children, although having a lowerreported exposure to tobacco compared with whites, sufferdisproportionately from tobacco-related illnesses. Wilson etal. [119] tested whether African-American children havehigher levels of serum and hair cotinine, after accountingfor ETS exposure and various housing characteristics. Theyinvestigated the level of cotinine in both hair and serum in acohort of 222 children with asthma. The results show thatAfrican-American children with asthma had significantlyhigher levels of both serum and hair cotinine than whitechildren. Identifying causes and consequences of increasedcotinine may help explain the striking differences intobacco-related illnesses between African-American andwhite children. Wilson et al. [120] expanded their results bytaking ambient measures of tobacco smoke for each case.Air nicotine levels and housing volume were independentlyassociated with higher levels of cotinine.

ETS-exposed children have an increased incidence ofpneumonia, bronchiolitis, asthma, and otitis media witheffusion, with greater rates of hospitalization and a longertime to recovery from these conditions than unexposedchildren. For this reason, The American Academy ofPediatrics (Elk Grove Village, IL, USA) advised physiciansthat tobacco prevention and control activities should ideallybegin at the first pediatric visit. Accordingly, theysuggested that pediatricians assess tobacco use and ETSexposure in their patients’ extended family and environ-ment, encourage smokers to smoke outside the home and

consider quitting, and record this information in thepatient’s medical record. Despite these recommendations,simple, specific, and validated screening procedures forETS exposure in the pediatric office setting have not beendeveloped. Groner et al. [121] developed a screening forchildren’s exposure to ETS in a pediatric primary caresetting using hair cotinine levels as the gold standard ofexposure.

Deciduous teeth

Teeth demonstrated the potential of this biological matrix asan important deposit of exogenous substances, which canaccumulate both in the pulp and in the calcified tissues[122]. Indeed, with respect to drugs of abuse, some authorscould identify opiates (morphine and codeine) and/orcocaine in teeth of former drug users to assess and verifyself-reported chronic consumption [7]. This evidencesupports the role of teeth from a toxicological point ofview. For the first time, in 2003, Pascual et al. [123]developed and validated a procedure for analysis ofnicotine and cotinine in deciduous teeth by GC-MS. Themethod was applied by Garcia-Algar et al. [124] for theanalysis of nicotine and cotinine in deciduous teeth fromchildren of both nonsmoking and smoking parents. Theresults support nicotine analysis in teeth as a promisingnoninvasive tool for monitoring and categorizing cumula-tive exposure to ETS from fetal life (when tooth formationstarts) through the entire childhood (deciduous teeth aregenerally lost between 6 and 8 years of age).

Recently, Marchei et al. [125] developed a LC electro-spray ionization MS/MS method for simultaneous determi-nation of minute amounts of nicotine and cotinine in teeth.requiring a smaller quantity of teeth and achieving a lowerlimit of detection. The validated assay was applied to thenicotine and cotinine analysis in deciduous teeth of childrenfrom the Asthma Multicenter Infant Cohort Study, whichassociates biomarkers of prenatal and postnatal exposure toETS with the inception of atopy and asthma. Preliminaryresults confirmed the fact that only tooth nicotine differen-tiated statistically between children exposed and nonex-posed to parental smoking.

Conclusions

Using objective biomarkers in a wide variety of matricesalready in the prenatal period and continuing testing postna-tally has been a tremendous step in assessing the deleteriouseffects of ETS in children. Evaluation of prenatal exposure toETS using different unusual matrices is at times difficult sincesampling may be challenging. Although amniotic fluid startsto form at the beginning of pregnancy, therefore providing

396 H. Llaquet et al.

information about exposure throughout the entire pregnancy,it is difficult and invasive to obtain; therefore, it is not widelyused. Meconium is easily obtainable, and gives informationabout exposure in the second and the third trimester of thepregnancy. Neonatal hair, formed in the last trimester, is avalued biomarker for exposure in the later stage of pregnancy;however, in some newborns it is not available in sufficientamounts. Neonatal urine, indicating exposure to ETS in thefew days before birth, may also be difficult to collect. Ideally,as many matrices as possible should be sampled to obtain anaccurate picture of exposure in the entire prenatal period.

Postnatally, breast milk analysis proved to be of greatvalue in advising breast-feeding smoking mothers how totime the smoking of cigarettes to transmit the least amountof toxic substances through breast milk.

Further research is needed in many areas, such as theinvestigation of potential genetic, ethnic, and gendersusceptibility to tobacco-related disease. The existing andfuture biological markers of ETS in different matrices inchildren of all age groups will play a major role inaddressing these issues.

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