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\DOI{10.5802/crchim.286}
\datereceived{2023-11-29}
\daterevised{2023-12-05}
\datererevised{2023-12-08}
\dateaccepted{2024-01-19}
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\dateposted{2024-02-29}
\begin{document}

\begin{noXML}

%\makeatletter
%\def\TITREspecial{\relax}
%\def\cdr@specialtitle@english{French Women in Chemistry in 2024}
%%\def\cdr@specialtitle@french{Les chimistes fran\c{c}aises en 2024}
%\makeatother

\title{Development of an analytical method for the simultaneous
determination of 22 Polycyclic Aromatic Hydrocarbons (PAHs) in maternal
and~umbilical cord blood}

\alttitle{D\'{e}veloppement d'une m\'{e}thode analytique pour la
d\'{e}termination simultan\'{e}e de 22 hydrocarbures aromatiques
polycycliques (HAP) dans le s\'{e}rum et le sang de cordon ombilical de
la m\`{e}re}

\author{\firstname{St\'{e}phanie} \lastname{Swiha}}
\address{Laboratory of Analytical and Bioanalytical Sciences and Miniaturization,
UMR CBI 8231 CNRS -- ESPCI Paris, PSL University, Paris, France}
\address{INSERM U1139, Facult\'{e} de Pharmacie, Paris, France}

\author{\firstname{Val\'{e}rie} \lastname{Pichon}\IsCorresp}
\addressSameAs{1}{Laboratory of Analytical and Bioanalytical Sciences and Miniaturization,
UMR CBI 8231 CNRS -- ESPCI Paris, PSL University, Paris, France}
\address{Sorbonne Universit\'{e}, Paris, France}
\email[V. Pichon]{valerie.pichon@espci.fr}
\curraddr[V. Pichon]{LSABM, UMR CBI 8231 CNRS -- ESPCI Paris, PSL
University, 10 rue Vauquelin, 75231 Paris cedex 05, France}

\author{\firstname{Thierry} \lastname{Fournier}\CDRorcid{0000-0002-7515-339x}}
\addressSameAs{2}{INSERM U1139, Facult\'{e} de Pharmacie, Paris, France}
\address{Universit\'{e} Paris Descartes, UMR-S 1139, Sorbonne Paris Cit\'{e},
Paris, France}
\address{Fondation PremUP, Paris, France}

\author{\firstname{Sophie} \lastname{Gil}}
\addressSameAs{2}{INSERM U1139, Facult\'{e} de Pharmacie, Paris, France}
\addressSameAs{4}{Universit\'{e} Paris Descartes, UMR-S 1139, Sorbonne Paris Cit\'{e},
Paris, France}
\addressSameAs{5}{Fondation PremUP, Paris, France}

\author{\firstname{Nathalie} \lastname{Delaunay}\CDRorcid{0000-0003-3008-0286}}
\addressSameAs{1}{Laboratory of Analytical and Bioanalytical Sciences and Miniaturization,
UMR CBI 8231 CNRS -- ESPCI Paris, PSL University, Paris, France}

\thanks{The authors gratefully acknowledge the R\'{e}gion Ile-de-France
and the DIM Analytics for their financial support.}

\begin{abstract}
Pregnant women and their fetuses, a very fragile population, are
exposed to polycyclic aromatic hydrocarbons (PAHs) and subject to
potentially serious health risks. Consequently, the determination of
PAHs in maternal serum and umbilical cord blood is necessary but
challenging, due to their highly varied physico-chemical properties and
trace presence. The aim of this project was to develop a method
enabling, for the first time, the simultaneous determination of 22
listed PAHs from small volumes (100 ${\upmu}$L) of maternal
serum and umbilical cord blood, including a sample pre-treatment
reducing as far as possible the quantity of solvents used. A
solid-phase extraction step on C18-grafted silica was optimized by
studying the effect of different parameters (nature and proportion of
organic solvent added to the sample, washing and elution conditions) on
extraction yields. The evaporation step was carefully controlled to
limit the loss of the most volatile PAHs. Then, to break any
interactions between PAHs and serum proteins, a precipitation step
prior to solid-phase extraction was optimized using a design of
experiments. The final sample processing procedure led to extraction
yields of between 27 and 57\% and between 34 and 69\% for 22 PAHs in
serum and umbilical cord blood, respectively, with standard deviation
values of less than 11\%. Limits of quantification between 0.2 and 3.1
${\upmu}$g/L were achieved for PAHs fluorescing in spiked real
samples, close to the concentrations expected in biological samples.
\clearpage
\end{abstract}

\begin{altabstract}
Les femmes enceintes et leurs f\oe{}tus, une population tr\`{e}s
fragile, sont expos\'{e}s aux hydrocarbures aromatiques polycycliques
(HAP) et soumis \`{a} d'\'{e}ventuels risques sanitaires graves. Par
cons\'{e}quent, la d\'{e}termination des HAP dans le s\'{e}rum maternel
et le sang du cordon ombilical est n\'{e}cessaire mais repr\'{e}sente
un d\'{e}fi en raison de leurs propri\'{e}t\'{e}s physico-chimiques
tr\`{e}s vari\'{e}es et leur pr\'{e}sence \`{a} l'\'{e}tat de traces.
Ce travail visait \`{a} d\'{e}velopper une m\'{e}thode permettant pour
la premi\`{e}re fois la d\'{e}termination simultan\'{e}e de 22 HAP
r\'{e}pertori\'{e}s \`{a} partir de faibles volumes (100 ${\upmu}$L) de
s\'{e}rum maternel et de sang de cordon ombilical, incluant un
pr\'{e}traitement de l'\'{e}chantillon r\'{e}duisant autant que
possible la quantit\'{e} de solvants utilis\'{e}s. Ainsi, une \'{e}tape
extraction sur phase solide sur silice greff\'{e}e C18 a \'{e}t\'{e}
optimis\'{e}e en \'{e}tudiant l'effet de diff\'{e}rents param\`{e}tres
(nature et proportion du solvant organique ajout\'{e} \`{a}
l'\'{e}chantillon, conditions de lavage et d'\'{e}lution) sur les
rendements d'extraction. L'\'{e}tape d'\'{e}vaporation a \'{e}t\'{e}
soigneusement contr\^{o}l\'{e}e pour limiter la perte des HAP les plus
volatils. Ensuite, pour rompre toutes les interactions entre les HAP et
les prot\'{e}ines s\'{e}riques, une \'{e}tape de pr\'{e}cipitation
avant l'extraction sur phase solide a \'{e}t\'{e} optimis\'{e}e \`{a}
l'aide d'un plan d'exp\'{e}riences. La proc\'{e}dure finale de
traitement des \'{e}chantillons a conduit \`{a} des rendements
d'extraction compris entre 27 et 57\% et entre 34 et 69\% pour 22 HAP
dans le s\'{e}rum et le sang de cordon ombilical, respectivement, avec
des valeurs d'\'{e}cart-type inf\'{e}rieures \`{a} 11\%. Des limites de
quantification comprises entre 0,2 et 3,1 ${\upmu}$g/L ont \'{e}t\'{e}
atteintes pour les HAP qui fluorescent dans des \'{e}chantillons
r\'{e}els dop\'{e}s, ce qui est proche des concentrations attendues
dans les \'{e}chantillons biologiques.
\end{altabstract}

\shortrunauthors

\keywords{\kwd{Polycyclic aromatic hydrocarbons}
\kwd{Maternal serum}
\kwd{Umbilical cord blood}
\kwd{Solid phase extraction}
\kwd{Liquid chromatography}}

\altkeywords{\kwd{Hydrocarbures aromatiques polycycliques}
\kwd{S\'{e}rum maternel}
\kwd{Sang de cordon ombilical}
\kwd{Extraction sur phase solide}
\kwd{Chromatographie liquide}}

\maketitle

\twocolumngrid

\end{noXML}

\section{Introduction}\label{mark-1.}

Polycyclic Aromatic Hydrocarbons (PAHs) are pollutants that are emitted
naturally or produced by human activities, due to incomplete combustion
or pyrolysis of organic materials (industries, road traffic,
heating\,{\ldots})~\cite{1}. They are present in environment, but also in
food, leading to a constant exposure of humans~\cite{2,3,4}. They are
carcinogens and genotoxic substances. After entering the human body,
due to their hydrophobic properties, PAHs are transported to all
lipid-rich tissues. This is why a long-term exposure may lead to their
accumulation in adipose tissue, liver, and kidney~\cite{4,5}. They can
also undergo different metabolic transformations resulting to the
formation of reactive electrophilic intermediates able to bind cellular
macromolecules such as proteins and nucleic acids. It is now well
established that these pollutants are chemical hazard to human health.
Long-term exposure increases the risk of a series of cancers. Moreover,
an exposure during pregnancy can lead to fetal malformations,
prematurity and even disorders in children and adults~\cite{6,7,8}.
This may come from a contamination of the placenta, which ensures
multiple functions directly involved in the initiation and outcome of
gestation, fetal growth and possibly the initiation of parturition,
and/or of the fetal tissues through a transfer of PAHs via the
placenta. This is why it is highly interesting to determine PAHs in
both maternal and umbilical cord bloods and also in placenta.

Today, 24 different PAHs are listed as priority pollutants: 16 by the
United State Environmental Protection Agency (US-EPA) and 16 by the
European Food Safety Authority (EFSA), eight of them belonging to both
lists. The 24 listed PAHs belong to a wide range of Log~P values
reported in Figure S1 (see Supplementary Information)~\cite{9,10,11,12}, 
thus rendering their determination in biological
samples particularly difficult but necessary as human contamination has
multiple sources and because the EFSA PAHs are quite different from the
US-EPA PAHs (higher number of aromatic cycles). Focusing on the
determination of PAHs in blood, in most of the cases, only the US-EPA
PAHs were targeted~\cite{13} except Pleil and coworkers who quantitated
also 6 EFSA PAHs~\cite{14}. Typical blood, plasma, and serum used
volumes range from 1 to 12 mL, which are quite large values,\break limiting
the access to epidemiological cohort samples, with one exception of a
study using pipette-tip solid-phase extraction (SPE) and only 200
${\upmu}$L of blood to determine the 16 US-EPA HAPs~\cite{15}. The most
used technique for sample pretreatment consists in extracting the PAHs
by liquid--liquid extraction
(LLE)~\cite{6,14,16,17,18,19,20,21,22,23,24,25,26}, but with some
drawbacks such as the large volumes of used solvent and being time
consuming as it involves several successive LLE steps, often three, to
improve the extraction yields. Most of the time, the LLE steps were
followed by SPE using C18-bonded silica, silica or silica/alumina
packed in cartridge~\cite{17,18,19,21,24,25,26} to improve the clean-up
of the sample extract by removing matrix interferents that could
disturb the PAH quantitation.

SPE can also be used alone as an alternative approach to LLE to extract
the PAHs from blood, plasma or serum prior to their chromatographic
analysis\break \cite{27,28,29,30,31}. It requires a lower volume of solvent
than LLE and simple less-time consuming manipulations. Indeed, 1
mL~\cite{30} or 2 mL~\cite{28,29,31} of blood, plasma or serum were
percolated on an apolar sorbent, C18-bonded silica when this
information is given~\cite{27,28,29}. The SPE step was followed by an
analysis with either gas chromatography coupled to mass 
spectrometry~\cite{27,28,29,31} or liquid chromatography (LC) coupled to a
fluorescence detection (FD)~\cite{30}. Unfortunately, in some of these
papers, several analytical data are missing, such as the nature of the
SPE sorbent~\cite{30,31}, the enrichment factors~\cite{28,31}, the
extraction yields in real samples~\cite{31}, and the limits of
quantitation (LOQs) in the biological matrices~\cite{28,31}, whereas
these data are necessary to be sure that the presented results are
consistent. Moreover, only between seven and 16 PAHs were targeted and
they only belong to the US-EPA list~\cite{27,28,29,30,31}, even for the
last study dealing with miniaturized pipette-tip SPE format involving
200 ${\upmu}$L of sample~\cite{15}.

Concerning the detected concentrations of PAHs in blood, plasma or
serum, they were most often \mbox{normalized} with lipid content and reported
in ng/g lipid. A study demonstrated that the mean lipid content in 283
maternal sera was 7.33 ${\pm}$ 1.86 g/L~\cite{32}. Using this value for
the lipid content, it is therefore\break possible to convert the data
reported in each study in the same unit and then to compare these\break
estimated values, with the hypothesis that all the\break reported values are
consistent. The highest mean values were observed by Song 
{et~al.}\ with up to 37~${\upmu}$g/L of benzo{[}a{]}anthracene (BaA) in the
blood of heavy-taste food consumers and a minimum mean value of 0.3
${\upmu}$g/L of pyrene (PYR) in the blood of Chinese 
non-smokers~\cite{27}. Similar values, between 0.05 and 2.7~${\upmu}$g/L, were
determined for each of 13 US-EPA PAHs in blood plasma collected from
Hong Kong residents~\cite{29}. These values are in accordance with the
ones determined in sera of autopsied people in Tennessee, where the sum
of the concentrations of 13 US-EPA PAHs varied between 0.1 and 12
${\upmu}$g/L~\cite{31}. Focusing on both maternal and cord sera, there
are two Chinese studies reported values between 0.1 and 
2.5~${\upmu}$g/L for 11 of the 16 US-EPA PAHs (the five other US-EPA PAH
concentrations were below the LOQs)~\cite{28} and between 0.1 and 9.5
${\upmu}$g/L for the 9 targeted US-EPA PAHs~\cite{30}. This gives an
idea of the PAH concentrations that one can expect in these kind of
samples. 

Due to the lack of consistent data of contamination level in
literature, especially for the PAHs listed for food, there is still a
great societal need to determine PAH concentrations in biological
tissues or fluids to increase knowledge of their health effects,
especially on at-risk populations such as pregnant women and their
fetus. Therefore, the objective of this study is to develop a simple
and low solvent consuming sample preparation step able to extract
simultaneously the maximum of the 24 listed PAHs from maternal and
umbilical cord sera. One of the challenges of this work is to obtain
the best possible sensitivity from low volumes of sera in order to
access to precious samples from epidemiological cohorts. For this, a
protocol involving protein precipitation, SPE, and
evaporation-redissolution before PAH analysis was developed. The key
parameters of each sample pretreatment step were optimized. At last, to
highlight the potential of the developed method, the extraction yields
and LOQs were determined in both real spiked maternal and umbilical
cord\break sera.

\section{Materials and methods}

\vspace*{-2pt}

\subsection{Chemicals}

\vspace*{-2pt}

HPLC grade methanol, acetonitrile (CH$_{3}$CN), propan-2-ol, and
tetrahydrofuran (THF) were\break supplied by Carlo Erba (Val de Reuil,
France). High purity water was dispensed by a milli-Q purification\break
system (Millipore, Saint Quentin en Yvelines, France). The PAH
standards were supplied by Cluzeau Info Labo (Sainte-Foy-La-Grande,
France): acenaphthylene (ACY) 99\%, cyclopenta{[}cd{]}pyrene (CPcdP)\break
99\%, benzo{[}c{]}fluorene (BcF) 97\%, benzo{[}j{]}fluorene (BjF)
99.7\%, dibenzo{[}a,l{]}pyrene (DalP) 99.8\%, dibenzo{[}a,e{]}pyrene
(DaeP) 99.8\%, dibenzo{[}a,h{]}\break pyrene (DahP) 99\%,
dibenzo{[}a,i{]}pyrene (DaiP) 99\% at 10~mg/L in CH$_{3}$CN and a
standard mixture of the 16 US-EPA PAHs at 100~mg/L in CH$_{3}$CN.

A stock solution mixture containing 100~${\upmu}$g/L of each PAH was
prepared in CH$_{3}$CN and stored at 4~\textdegree C until further use.
An aqueous solution containing 10\% (w/v) sodium dodecyl sulfate (SDS)
was purchased from Thermofisher Scientific (Villebon-Courtaboeuf,
France). A synthetic serum was prepared from Earle's balanced salt
solution containing albumin at 25~g/L and spiked with a mixture of 16
representative PAHs at 4 ${\upmu}$g/L for each PAH in CH$_{3}$CN. 
\looseness=-1

\vspace*{-2pt}

\subsection{Apparatus and analytical conditions}\label{sec2.2}

\vspace*{-2pt}

\begin{table*}
\caption{\label{tab1}LOQs (S/N~$=$~10) in ${\upmu}$g/L obtained with
fluorescence and UV detections and estimated by injecting 10 ${\upmu}$L
of a solution of PAHs at 1, 5, and 20 ${\upmu}$g/L in CH$_{3}$CN:water
(1:1, v/v)\vspace*{-2pt}}
\fontsize{10}{11.6}\selectfont
\begin{tabular}{lcccc}
\thead
Compounds & ${\lambda}_{\mathrm{UV}}$ (nm) &
$\lambda_{\mathrm{ex}}$, $\lambda_{\mathrm{em}}$ (nm) &
LOQ$_{\mathrm{FD}}$ (${\upmu}$g/L) & LOQ$_{\mathrm{UV}}$ (${\upmu}$g/L)\\ 
\endthead
NAPH & 220 & 269, 327 & 1.2 & \02.2 \\ 
ACY & 230 & - & - & \02.1 \\ 
ACE & 230 & 227, 315 & 0.5 & \02.3 \\ 
FLO & 260 & 227, 315 & 1.1 & 13.2 \\ 
PHE & 250 & 250, 364 & 0.4 & \01.5 \\ 
ANT & 250 & 250, 364 & 0.9 & \00.7 \\ 
FLT & 230 & 234, 454 & 0.9 & \04.8 \\ 
PYR & 240 & 237, 388 & 0.2 & \00.9 \\ 
BcF & 230 & 237, 388 & 0.3 & \08.0 \\ 
CPcdP & 230 & - & - & \06.8 \\ 
BaA & 275 & 265, 376 & 1.5 & \08.7 \\ 
CHR & 270 & 265, 376 & 0.1 & \06.3 \\ 
5MCHR & 270 & 265, 376 & 0.2 & \05.5 \\ 
BjF & 240 & - & - & \04.9 \\ 
BbF & 260 & 290, 440 & 0.3 & \04.6 \\ 
BkF & 240 & 290, 440 & 0.1 & \05.7 \\ 
BaP & 260 & 290, 440 & 0.2 & \04.5 \\ 
DahA & 295 & 292, 428 & 0.3 & \03.6 \\ 
DalP & 315 & 292, 428 & 0.4 & \06.7 \\ 
BghiP & 285 & 292, 428 & 0.3 & 15.9 \\ 
IcdP & 300 & - & - & \09.3 \\ 
DaeP & 310 & 280, 404 & 0.8 & \05.7 \\ 
DaiP & 240 & 390, 439 & 0.4 & \06.2 \\ 
DahP & 310 & 260, 456 & 0.4 & \02.0
\botline
\end{tabular}
\vspace*{-3pt}
\end{table*}

The LC-diode array detector (DAD)/FD analyses were performed using an
Agilent 1200 series system (Agilent Technologies, Massy, France) that
was controlled by the Chemstation software. The separation was
performed on a Pursuit PAH column ($100 \times 2.1$~mm, 3~${\upmu}$m, Agilent
Technologies) maintained at 35~\textdegree C with a column oven
(Croco-cil, Interchim, Montlu\c{c}on, France). Samples were analyzed
using a linear gradient elution with water (A) and a mixture of
CH$_{3}$OH:CH$_{3}$CN (6:4, v/v) (B). The gradient started at 50\% of B
and increased to 90\% of B in 27~min, held for 13~min, and increased to
100\% of B in 1~min, held for 10~min, and finally returned to initial
composition within 1~min and let 10~min to equilibrate the system. The
flow-rate was set at 0.2~mL/min and the injection volume was 5 or
10~${\upmu}$L. ACY, CPcdP, BjF, and indeno{[}1,2,3-cd{]}pyrene (IcdP)
were quantified by UV, at 230 nm for both ACY and CPcdP, 240~nm for
BjF, and 300 nm for IcdP. For the other PAHs, a time program of the
excitation and emission wavelengths was performed and they were
quantified with FD. The wavelengths are reported in Table~\ref{tab1}.
\looseness=-1

\vspace*{-2pt}

\subsection{SPE procedure}\label{sec2.3}

The SPE cartridges were Hypersep (25~mg of trifunctional octadecyl
non-end-capped silica, 40--63~${\upmu}$m, 60~\AA{},
520~m$^{2}$/g, 21--23\% carbon, ThermoFisher Scientific),
Versaplate (25~mg of trifunctional octadecyl end-capped silica,
40~${\upmu}$m, 60~\AA{}, 500~m$^{2}$/g, 17.4\%\break carbon,
Agilent Technologies) and BondElut SPEC (30 mg of trifunctional
octadecyl non-end-capped silica, 70~\AA{}, 220~m$^{2}$/g,
7\% carbon, Agilent Technologies). The first SPE developments were
carried out by percolating ultra-pure water spiked at 5~${\upmu}$g/L
with PAHs on the Hypersep cartridges. Except when it is mentioned, the
protocol was as follows: the cartridge was conditioned with
300~${\upmu}$L each of THF, CH$_{3}$OH, and H$_{2}$O:CH$_{3}$CN (1:1,
v/v). After percolating 100 or 200~${\upmu}$L of a H$_{2}$O:CH$_{3}$CN
(1:1, v/v) mixture spiked with PAHs, a washing step with 100~${\upmu}$L
of H$_{2}$O:CH$_{3}$CN (1:1, v/v) was carried out and the cartridge was
next dried. Finally, the PAHs were eluted with 300~${\upmu}$L of THF.
The elution fraction was diluted with water to obtain a THF:H$_{2}$O
mixture (1:1, v/v) before injection in LC-DAD/FD system or subjected to
evaporation. 

\subsection{Optimization of the evaporation step} 

To enrich the SPE elution fraction, the optimization of an evaporation
step was carried out by evaporating 200~${\upmu}$L of THF spiked with
5~${\upmu}$g/L of 20 PAHs to dryness or partially when 10 or 40
${\upmu}$L of an aromatic solvent (xylene, trimethylbenzene, or
toluene) were added to THF. For both approaches, after the evaporation
step, the extract was recovered in CH$_{3}$CN before its injection in
LC-DAD/FD (5~${\upmu}$L). 

\subsection{Optimization of the protein precipitation procedure with a
design of experiment}

The optimization of the protein precipitation parameters was performed
with a design of experiment (DOE). The DOE was constructed with two
levels for the percentage of CH$_{3}$CN and heating and three levels
for the percentage of SDS. The DOE was realized with an Earle's
balanced salt solution containing albumin at 25~g/L (synthetic serum)
and spiked at 4~${\upmu}$g/L with 16 PAHs (acenaphthene (ACE), fluorene
(FLO), phenanthrene (PHE), anthracene (ANT), fluoranthene (FLT), PYR,
BcF, chrysene (CHR), 5-methylchrysene (5MCHR), benzo{[}b{]}fluoranthene
(BbF), benzo{[}k{]}fluoranthene (BkF), benzo{[}a{]}pyrene (BaP),
dibenzo{[}a,h{]}anthracene (DahA), DalP, DaeP, and DahP) to prevent
from wasting precious sera samples. For these experiments, only 16 PAHs
among the 24 were selected in order to cover the whole range of
hydrophobicity, except the less hydrophobic compounds, naphthalene
(NAPH) and acenaphthylene (ACY) because of their higher LOQs. All
samples were introduced in 2 mL Protein Lobind tubes (Eppendorf,
Montesson, France). The denaturation and the precipitation of the
albumin were carried out by adding an aqueous solution containing 10\%
of SDS (w/v) to achieve a final concentration of 0.1 or 0.2\%. Some
samples were next heated at 60~\textdegree C during 25~min. Then,
different volumes of CH$_{3}$CN were added to obtain a final content of
50 or 70\% (v/v) and samples were vortexed. Finally, a centrifugation
at 4500 rpm at 4~\textdegree C during 30~min was carried out and the
supernatants were collected. Before SPE, as the samples already
contained 700~${\upmu}$L of CH$_{3}$CN, a dilution with water to
achieve a final content of 50\% of CH$_{3}$CN in the sample was
implemented. After conditioning the SPE cartridge, 400~${\upmu}$L of
the diluted supernatant were percolated. A washing step with
100~${\upmu}$L of H$_{2}$O:CH$_{3}$CN (1:1, v/v) was done and the
cartridge was dried. Finally, the PAHs were eluted with 300~${\upmu}$L
of THF and a partial evaporation with the addition of 40~${\upmu}$L of
toluene and a recovered volume of 35~${\upmu}$L of CH$_{3}$CN was
performed before {injection} in LC-DAD/FD system (5~${\upmu}$L). The
measured responses were the PAH extraction recoveries. The data
treatment was performed using JMP 10.0 (S.A.S Institute Inc, Cary, NC,
USA) software.

\vspace*{-5pt}
\subsection{Extraction of PAHs from maternal and umbilical cord sera} 

\subsubsection{Sample collection and storage}

Biological samples were obtained following informed written patient
consent and approval from our local ethics committee (CPP
2015-Mai-13909). Umbilical cord and maternal blood were collected with
harmonized procedures from patients delivered in Obstetric Units of
Paris' Hospitals. Sera were obtained after blood centrifugation at 3000
rpm during 15~min and stored at ${-}$20~\textdegree C in Protein
Lobind tubes.

\subsubsection{Sera pretreatment procedure}

100~${\upmu}$L of maternal or umbilical cord serum were spiked at
10~${\upmu}$g/L with each PAH except at
20~${\upmu}$g${\cdot}$L$^{-1}$ for CPcdP and IcdP. A solution
containing 10\% of SDS was next added to achieve a final concentration
of 0.2\% (w/v). Sample was vortexed and 225~${\upmu}$L of CH$_{3}$CN
were added. A centrifugation at 4500 rpm during 30~min at 
4~\textdegree C was carried out. 300~${\upmu}$L of supernatant were then
collected and 120~${\upmu}$L of water were added to obtain a final
content of 50\% of CH$_{3}$CN (v/v). 400~${\upmu}$L of this sample were
percolated on SPE cartridge. A washing step with 100~${\upmu}$L of
CH$_{3}$CN:H$_{2}$O (1:1, v/v), a drying and an elution with
300~${\upmu}$L of THF were implemented. 40~${\upmu}$L of toluene were
added to the elution fraction that was next vortexed and evaporated.
The final extract was suspended in 100~${\upmu}$L of
CH$_{3}$CN:H$_{2}$O (1:1, v/v) before injection in LC-DAD/FD system
(10~${\upmu}$L).

\vspace*{-5pt}
\section{Results and discussions}

\subsection{Limits of quantitation of the LC-DAD/FD analysis}

The analysis of the 24 PAHs by LC-DAD/FD was carried out with an
Agilent Pursuit PAH ($100 \times 2.1$~mm, 3~${\upmu}$m) column containing a
polymeric C18-bonded silica stationary phase, chosen for its shape
selectivity towards PAHs~\cite{33}. Considering the wide polarity range
of the 24 targeted PAHs with log P values from 3.3 up to 7.7 (see
Figure S1)~\cite{9,10,11,12}, different temperatures (30 and
35~\textdegree C), mobile phases (CH$_{3}$CN and\break water, CH$_{3}$OH and
water, CH$_{3}$OH plus CH$_{3}$CN and water), and gradients were tested
to find optimal conditions implementing a ternary gradient with water,
CH$_{3}$CN, and CH$_{3}$OH (see Figure S2). 

The FD detection schedule was optimized by studying the excitation and
emission fluorescence spectra of each PAH. Nevertheless, some
compromises were necessary as it is only possible to change the
wavelengths when the time gap between two peaks is sufficient. Table~\ref{tab1}
presents the LOQ values (defined as the concentration level that gives
a signal to noise ratio of 10) determined with the optimized
fluorescence schedule. They ranged from 0.1 to 1.5~${\upmu}$g/L for an
injection volume of 10 ${\upmu}$L. It is worthwhile to notice that no
values are given for four PAHs (ACY, CPcdP, BjF, and IcdP) in FD.
Indeed, ACY and CPcdP do not fluoresce while BjF have a poor
fluorescence. Concerning IcdP, its quantification in UV was preferred
because the resolution was insufficient to change the excitation and
emission wavelengths in order to better detect it in FD. The LOQs were
also determined in UV, at the maximal absorption wavelength of each PAH
(see Table~\ref{tab1}), and are comprised between 0.7 and 15.9~${\upmu}$g/L. As
expected, except for ACY, CPcdP, BjF, IcdP and ANT, these LOQ values
are significantly better in FD than in UV, by factors of 1.8 to 63.
This is why FD was used for quantitation of all these PAHs. For ANT,
LOQs in LC-UV and -FD are very close and FD was chosen in order to
exploit its specificity for the analysis of real samples. It can also
be noticed that the obtained LOQs for these standard solutions are
close to the expected concentrations in sera already determined in
literature thus indicating that the sample pretreatment method does not
necessitate to provide a high enrichment factor. This should favor the
possibility of using reduced sample volumes, which is often required by
cohort leader.  

\subsection{Development of the SPE procedure}

In order to determine PAHs at trace level in sera that are complex
biological samples, a purification and extraction by SPE on C18-bonded
silica-based sorbent was optimized in order to remove as much as
possible matrix components. 

\subsubsection{Choice of the percolation solvent}\label{sec3.2.1}

{The solubility of some PAHs is very low in aqueous solutions,
particularly for the high molecular weight PAHs. This can induce their
adsorption on the vial or cartridge walls and consequently their loss
and then the underestimation of their concentration in samples. The
strategy to avoid their adsorption consists in adding some organic
solvent in the sample. Moreover, the treatment of serum samples as
envisaged in this study, generally required a first precipitation step
usually carried out in organic solvent that will also prevent the risk
of loss of PAHs by adsorption to proteins. Therefore, the critical
parameters are the solvent nature and its proportion to add in the
sample without reducing too much the breakthrough volume of the lowest
molecular weight PAHs. Preliminary experiments were performed by
percolating water containing 30\% of CH$_{3}$OH, CH$_{3}$CN or
isopropanol and spiked with CHR, BaP, and DaiP, i.e. three PAHs
selected to be representative of some of the most hydrophobic targeted
ones. For the washing step, an CH$_{3}$CN:H$_{2}$O (1:1, v/v) mixture
was chosen to remove some potential interferents that could be present
latter in real samples since it has a slightly higher eluent strength
than the percolation media. A volume of 100~${\upmu}$L of this solution
was introduced to limit the risk of reaching the breakthrough volume of
the most polar PAHs. For the elution step, one has to select a solvent
able to disrupt the interactions between the analytes and the sorbent
with a volume as low as possible to either lead to some enrichment or
to limit the time of the potential subsequent evaporation step. In
literature, THF~\cite{34} or mixtures of hexane:CH$_{2}$Cl$_{2}$
(1/1)~\cite{28,29} and CH$_{3}$CN:butyl chloride (55/45)~\cite{31} or
ethyl acetate~\cite{30} were used. THF was selected, with an elution
volume of 300 ${\upmu}$L. Figure~\ref{fig1} shows the resulting average
extraction recoveries obtained with this SPE protocol, percolating 
100~${\upmu}$L of hydro-organic mixtures (7:3, v/v) spiked at 
5~${\upmu}$g/L with the three PAHs on 25 mg of a C18-bonded silica
(Hypersep). It appears that the DaiP concentration in the elution
fraction was below the LOQ whereas it was not the case with CH$_{3}$CN
and isopropanol. CH$_{3}$OH appears as too polar to ensure the
solubilization of this highly hydrophobic PAH (log P(DaiP)~${=}$~7.3),
leading to its adsorption on the vial or cartridge walls. Therefore,
CH$_{3}$OH was removed for the end of the study.}\looseness=-1

\begin{figure*}
\vspace*{-4pt}
\includegraphics{fig01}
\vspace*{-4pt}
\caption{\label{fig1}Effect of the nature and the volume of organic
solvent (30\% or 50\%) introduced in spiked water on the extraction
recoveries. SPE procedure (Hypersep cartridge): percolation of
100~${\upmu}$L of water containing 30 or 50\% of the organic studied
solvent and spiked with 5 ${\upmu}$g/L of each PAH, washing with
100~${\upmu}$L of CH$_{3}$CN:H$_{2}$O (1:1, v/v), and elution
with 300 ${\upmu}$L of THF, dilution of the THF extract with water
(THF:H$_{2}$O, v:v, 1/1) ($n=3$). *~not quantifiable PAH.}
\vspace*{-4pt}
\end{figure*}

An ANOVA test ($\alpha = 5$\%) demonstrated that the
recoveries of the three PAHs obtained after percolating them in 30\% of
CH$_{3}$CN or isopropanol were not significantly different.
Nevertheless, CH$_{3}$CN led to lower standard deviation (SD) values
than isopropanol and was thus preferred. Its proportion in the sample
was next increased up to 50\% to favor the PAH solubility, especially
for the most hydrophobic ones. It appears in Figure~\ref{fig1} 
that the obtained
extraction recoveries did not significantly change for CHR and BaP
(ANOVA test, $\alpha = 5$\%), but was increased for DaiP.
Therefore, for the rest of the development of the SPE step, a content
of 50\% of CH$_{3}$CN was added in the percolation media.\looseness=-1

\subsubsection{Choice of the eluting solvent}

Another solvent with a higher elution strength
(${\varepsilon}_{0}$), CH$_{3}$CN, was evaluated for the desorption
of the retained PAHs on the cartridge. The experiments were performed
with a spiked CH$_{3}$CN:H$_{2}$O (1:1, v/v) mixture
solution and the same washing conditions described in 
Section~\ref{sec3.2.1} ($n = 3$). 
It was observed that CH$_{3}$CN did not allow the elution of
DaiP, and led to not significantly different extraction recoveries for
CHR, and lower ones (47\%) for BaP (ANOVA test, $\alpha = 5$\%)
compared to THF (68\%). Therefore, THF was the best choice as eluting
solvent and the results were in good agreements with the
$\varepsilon_{0}$ values of CH$_{3}$CN (${\varepsilon}_{0} =
3.1$) and THF (${\varepsilon}_{0} = 3.7$), which give a measure of
their elution strength on a C18-based sorbent~\cite{35}. 

\subsubsection{Comparison of three different SPE sorbents}

In order to select a C18-based sorbent favoring the PAH extraction and
leading to the best recoveries, three SPE cartridges containing 25 or
30~mg of C18-bonded silica from different suppliers that mainly differ
by their carbon content were evaluated (see Section~\ref{sec2.3}) by applying
the optimized conditions. For these experiments, nine PAHs among the 24
were selected in order to cover the whole range of hydrophobicity,
except the less hydrophobic compounds, NAPH and ACY, because of their
higher LOQ (see Table~\ref{tab1}). After conditioning the SPE sorbents,
200~${\upmu}$L of H$_{2}$O:CH$_{3}$CN (1:1, v/v)
spiked with the nine PAHs were percolated. After washing and drying,
PAHs were eluted with 300~${\upmu}$L of THF. Results are reported in
Figure~\ref{fig2}. High extraction recoveries between 65\% and 98\% were
obtained. An ANOVA test ($\alpha = 5$\%) demonstrated that the
recoveries obtained on the three sorbents for each PAH were not
significantly different. The single exception was for 5MCHR, which has
a significantly higher extraction recovery with the Hypersep cartridge.
Therefore, the Hypersep cartridge was preferred for the end of the
study.\looseness=-1

\begin{figure*}
\vspace*{-4pt}
\includegraphics{fig02}
\vspace*{-3pt}
\caption{\label{fig2}Comparison of the extraction recoveries obtained
using three different SPE sorbents. SPE procedure: percolation of
100~${\upmu}$L of CH$_{3}$CN:H$_{2}$O sample (1:1,
v/v) spiked with 5 ${\upmu}$g/L of each PAH, washing with
100~${\upmu}$L of CH$_{3}$CN:H$_{2}$O (1:1, v/v) and
elution with 300 ${\upmu}$L of THF ($n=3$).}
\vspace*{-3pt}
\end{figure*}

\subsubsection{Study of the evaporation step} 

At this stage of the study, the SPE elution fraction had never been
evaporated before its LC\break analysis. The introduction of an evaporation
step was next evaluated to improve the enrichment factor and the method
sensitivity, but this step can also induce some loss of the most
volatile PAHs. To evaluate this risk, 200~${\upmu}$L of THF was spiked
with 20 PAHs and was evaporated to dryness and further reconstituted in
50~${\upmu}$L of CH$_{3}$CN before analysis. Results are reported in
Figure~\ref{fig3}. This evaporation step led to the partial loss of the five
most volatile PAHs (NAPH, ACE, FLO, PHE, and ANT). Their recoveries
ranged from 15\% (NAPH) to 54\% (ANT) with SD values up to 20\%. For
the other PAHs, the losses were lower and the recoveries were higher
than 70\%. To improve the recoveries of the most volatile PAHs, a small
volume of an aromatic solvent having a low volatility and able to
develop ${\uppi}$--${\uppi}$ interactions with PAHs was added in the
extract before starting the evaporation and the evaporation was stopped
before a complete dryness in order to trap all PAHs in this small
residual volume of the aromatic solvent, this volume being controlled
thanks to the shape of the used vials. For these experiments, three
aromatic solvents were selected: toluene (boiling point (BP):
110.6~\textdegree C, vapor pressure (VP): 0.29~kPa), xylene (BP:
144.4~\textdegree C, VP: 0.8~kPa), and trimethylbenzene (BP:
175~\textdegree C, VP: 0.18~kPa). First, 10~${\upmu}$L of each aromatic
solvent was added in 200~${\upmu}$L of THF spiked at 5~${\upmu}$g/L
with each PAH. The evaporation was stopped before dryness (last drop)
and the extract was diluted with 50~${\upmu}$L of CH$_{3}$CN before
analysis. Figure~\ref{fig3} presents the obtained results. 

\begin{figure*}
\vspace*{-4pt}
\includegraphics{fig03}
\vspace*{-4pt}
\caption{\label{fig3}Effect of the nature and the volume of the
aromatic solvent added in 200 ${\upmu}$l of THF and spiked with 5
${\upmu}$g/L of each PAH on extraction recoveries. The extract was
recovered in 50 ${\upmu}$L of CH$_{3}$CN before analysis ($n=3$).
* not quantifiable PAHs.}
\vspace*{-4pt}
\end{figure*}

With xylene, the quantitation of NAPH, BcF, and BaA was not possible
because of the presence of\break co-eluting peaks in the chromatogram due to
xylene and its impurities. There was a similar problem with
trimethylbenzene, which prevents from the quantitation of ACE and FLO.
Therefore, toluene was selected. Nevertheless, 10~${\upmu}$L of toluene
were not sufficient to ensure high recoveries for NAPH, ACE, FLO, and
ANT. Then, the effect of the addition of 40~${\upmu}$L of toluene was
evaluated. In that case, the recoveries for the five most volatile PAHs
were improved: about 50\% for NAPH, 60\% for DahP, and above 80\% for
all the other PAHs. Only DahP has a lower recovery than without the
addition of an aromatic solvent. The addition of 40~${\upmu}$L of
toluene in the SPE extract before its evaporation was then selected to
limit the loss of most of the PAHs. 

Then, different volumes of CH$_{3}$CN (50, 100, and 150~${\upmu}$L)
were tested for the dilution of the extracts after evaporation and
before injection in LC. According to the ANOVA test ($\alpha =
5$\%), the extraction yields were similar for all the tested volumes
except for DahP (data not shown) for which a volume of 150~${\upmu}$L
of CH$_{3}$CN gave higher extraction recoveries (87\%). Therefore,
150~${\upmu}$L of CH$_{3}$CN was selected for the optimization of the
next step. 

\subsubsection{Optimization of the elution volume} 

As described previously, two elution solvents were tested, CH$_{3}$CN
and THF, and THF was preferred. At this stage, 300~${\upmu}$L of THF
were used for the elution but it is important to ensure that this
volume is sufficient to elute the most hydrophobic PAHs from the SPE
cartridge. This is why an increase in the elution volume was studied.
Three volumes of THF were tested: 300, 400 and 500~${\upmu}$L and
Figure~\ref{fig4} presents the obtained recoveries. An ANOVA test ($\alpha
= 5$\%)\break demonstrated that they are not significantly different except
for NAPH and IcdP. NAPH is the most polar and the most volatile PAH and
its recovery decreases when the elution volume increases. This cannot
be due to a problem of desorption from the SPE cartridge but rather to
the subsequent evaporation step that takes a longer time, leading to an
increasing lost of this volatile compound. For IcdP, there is a
decrease in the recovery with the increase in the elution volume. This
unexpected result for this highly hydrophobic compound may be also due
to the evaporation step. Indeed, with a higher elution volume to
evaporate, the compound may adsorb on the entire walls of the vials and
may become more difficult to recover within a resuspension volume of
150~${\upmu}$L of CH$_{3}$CN even after vortexing. This is why a volume
of 300~${\upmu}$L of THF was finally\break selected.

\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}Optimization of the SPE elution volume. SPE
procedure (Hypersep cartridge): percolation of 200~${\upmu}$L of
CH$_{3}$CN:H$_{2}$O (1:1, v/v) spiked with 5 ${\upmu}$g/L of each PAH
except ACY, CPcdP, BjF, and IcdP at 50~${\upmu}$g/L, washing with
100~${\upmu}$L of CH$_{3}$CN:H$_{2}$O (1:1, v/v), elution with THF, and
partial evaporation (addition of 40 ${\upmu}$L of toluene in the
elution fraction). The extract was recovered with 150 ${\upmu}$L of
CH$_{3}$CN before analysis ($n=3$).}
\vspace*{7pt}
\end{figure*}

\subsubsection{Improvement of the enrichment factor}

At this stage of the study, almost all the parameters of the
pretreatment steps were optimized except the sample volume and the
final volume of solvent to recover compounds after the evaporation just
before analysis. Both are key parameters for the recoveries and
enrichment factors of the SPE procedure, that have to be as high as
possible to detect the PAHs present at trace levels in real samples.\break
The loading volume was first increased from 200 to 400~${\upmu}$L. The
obtained extraction recoveries are presented in Figure~\ref{fig5}. For the first
four less hydrophobic PAHs, a dramatic decrease was observed. Indeed, a
loading volume of 400~${\upmu}$L followed by a washing\break volume of
100~${\upmu}$L may exceed their breakthrough volumes. For the other
PAHs, the obtained extraction recoveries were quite similar and SD
values were between 2 and 17\% for both conditions. As the aim of this
study was to quantify the 24 listed PAHs at trace levels, a compromise
had to be made and a loading volume of 400~${\upmu}$L was chosen to
favor the enrichment factor. \looseness=-1

\begin{figure*}
\includegraphics{fig05}
\caption{\label{fig5}Optimization of the SPE percolation volume. SPE
procedure (Hypersep cartridge): percolation of 200~${\upmu}$L or 400
${\upmu}$L of CH$_{3}$CN:H$_{2}$O sample (1:1, v/v) spiked with 5
${\upmu}$g/L of each PAH except for ACY, CPcdP, BjF, and IcdP at 50
${\upmu}$g/L, washing with 100~${\upmu}$L of CH$_{3}$CN:H$_{2}$O (1:1,
v/v), elution with 300 ${\upmu}$L of THF and partial evaporation
(addition of 40 ${\upmu}$L of toluene in the elution fraction). The
extract was recovered with 150 ${\upmu}$L of CH$_{3}$CN before analysis
($n=3$).}
\end{figure*}

The volume needed for recovering all the PAHs after the evaporation and
before analysis was finally studied. For this, three volumes of
CH$_{3}$CN were tested: 35, 100, and 150~${\upmu}$l. According to the
results presented in Figure~\ref{fig6} and the ANOVA test ($\alpha =
5$\%), the extraction recoveries were not significantly
different. To improve LOQs, the injection volume of 5~${\upmu}$L used
for all the injection achieved for the development of the SPE procedure
was increased to 10~${\upmu}$L, which prevents from the use of
35~${\upmu}$L as volume use to dilute the final extract after the
evaporation step. Therefore, a volume of 100~${\upmu}$L was selected.
However, an injection volume of 10~${\upmu}$L of PAHs in CH$_{3}$CN
induced a peak deformation for the first eluted PAHs. Therefore, the
100~${\upmu}$L of CH$_{3}$CN was replaced by the addition of
50~${\upmu}$L of CH$_{3}$CN for resuspension followed by 50~${\upmu}$L
of H$_{2}$O. Finally, the optimized SPE protocol with spiked pure
media, at this stage, was: percolation of 400~${\upmu}$L of
H$_{2}$O:CH$_{3}$CN (1:1, v/v),\break washing with 100~${\upmu}$L of
H$_{2}$O:CH$_{3}$CN (1:1, v/v),\break drying, elution with 300~${\upmu}$L of
THF, addition of 40~${\upmu}$L of toluene in the elution fraction,
partial evaporation (last drop), and dilution of the extract with
100~${\upmu}$L of H$_{2}$O:CH$_{3}$CN (1:1, v/v) before analysis. 

\begin{figure*}
\includegraphics{fig06}
\caption{\label{fig6}Optimization of the resuspension volume after
evaporation before LC analysis. SPE procedure (Hypersep cartridge):
percolation of 400 ${\upmu}$L of CH$_{3}$CN:H$_{2}$O sample (1:1, v/v)
spiked with 5 ${\upmu}$g/L of each PAH except for ACY, CPcdP, BjF, and
IcdP at 50 ${\upmu}$g/L, washing with 100~${\upmu}$L of
CH$_{3}$CN:H$_{2}$O (1:1, v/v), elution with 300 ${\upmu}$L of THF, and
partial evaporation (addition of 40 ${\upmu}$L of toluene in the
elution fraction). The extract was recovered with 35, 100 or 150
${\upmu}$L of CH$_{3}$CN ($n=3$).}
\end{figure*}

\subsection{Optimization of the protein precipitation procedure by a DOE}

Previous results were obtained for spiked pure media. Nevertheless, the
analysis of real sera is \mbox{targeted.} Therefore, it is necessary to
proceed first to the precipitation of the proteins contained in the
sera minimizing PAHs losses that may occur due to their potential
interactions with the serum proteins. The traditional approach involves
the addition of an organic solvent, but also of a surfactant, such as
SDS~\cite{36,37,38,39}. The CH$_{3}$CN percentage, SDS concentration,
and temperature may affect these interactions. A DOE-based approach was
selected to study the potential effects and interactions of these three
factors and determine the optimum precipitation\break conditions. 

According to literature and the conventional protocols used for protein
precipitation, the low and high levels for the CH$_{3}$CN content was
fixed at 50 and 70\% (v/v), respectively~\cite{36,37}, the three levels
of SDS were fixed at 0, 0.1, and 0.2\% (v/w)~\cite{38}, and the
precipitation was carried out at 25~\textdegree C (rt) for 2~min or at
60~\textdegree C during 25~min~\cite{39}. Table~\ref{tab2} presents the coded
and non-coded values for each factor. The \mbox{experiments} were next carried
out with 16 PAHs representative of the log P range of the 24 targeted
ones (except for NAPH and ACY, the less hydrophobic compounds because
of their higher LOQs) and, it is worthwhile to notice it, with a
synthetic serum (Earle's balanced salt solution containing albumin at
25~g/L). Indeed, to carry out this DOE with sera, 4.5~mL
(300~${\upmu}$L~$\times$~15 experiments) would have been necessary
whereas sera are highly precious samples. 

\begin{table*}
\caption{\label{tab2}$2\times 2\times 3$ factorial design with the
different factors studied for their potential impact on the PAH
recoveries during the protein precipitation (test with a synthetic
serum: Earle's balanced salt solution containing albumin at 
25~g/L spiked with 16 PAHs at 4 ${\upmu}$g/L each)}
\begin{tabular}{cccccccc}
\thead
\xmorerows{1}{Order} & \multicolumn{3}{c}{Coded values} &&
\multicolumn{3}{c}{Experimental values}\\
\cline{2-4}\cline{6-8}
& {[}CH$_{3}$CN{]} & \parbox[t]{2cm}{\centering Precipitation conditions} 
& {[}SDS{]} && \parbox[t]{4.3em}{\centering {[}CH$_{3}$CN{]} (\%,~v/v)} & 
\parbox[t]{2cm}{\centering Precipitation conditions} & 
\parbox[t]{1.3cm}{\centering [SDS] (\%,~w/v)}\vspace*{2pt} \\ 
\endthead
\01 & $+$1 & $+$1 & 3 && 70 & 25 min at 60~\textdegree C & 0.2 \\ 
\02 & $+$1 & $+$1 & 1 && 70 & 25 min at 60~\textdegree C & 0 \\ 
\03 & $+$1 & $+$1 & 1 && 70 & 25 min at 60~\textdegree C & 0 \\ 
\04 & $-$1 & $-$1 & 2 && 50 & 2 min at 25~\textdegree C & 0.1 \\ 
\05 & $+$1 & $-$1 & 2 && 70 & 2 min at 25~\textdegree C & 0.1 \\ 
\06 & $+$1 & $-$1 & 3 && 70 & 2 min at 25~\textdegree C & 0.2 \\ 
\07 & $+$1 & $-$1 & 3 && 70 & 2 min at 25~\textdegree C & 0.2 \\ 
\08 & $-$1 & $-$1 & 3 && 50 & 2 min at 25~\textdegree C & 0.2 \\ 
\09 & $-$1 & $+$1 & 1 && 50 & 25 min at 60~\textdegree C & 0 \\ 
10 & $-$1 & $+$1 & 1 && 50 & 25 min at 60~\textdegree C & 0 \\ 
11 & $+$1 & $-$1 & 1 && 70 & 2 min at 25~\textdegree C & 0 \\ 
12 & $+$1 & $+$1 & 2 && 70 & 25 min at 60~\textdegree C & 0.1 \\ 
13 & $-$1 & $+$1 & 2 && 50 & 25 min at 60~\textdegree C & 0.1 \\ 
14 & $-$1 & $+$1 & 3 && 50 & 25 min at 60~\textdegree C & 0.2 \\ 
15 & $-$1 & $-$1 & 1 && 50 & 2 min at 25~\textdegree C & 0 
\botline
\end{tabular}
\vspace*{2.5pt}
\end{table*}

The considered responses were the recoveries of the 16 PAHs and these
data are reported in Table S1. A polynomial model was used to identify
the factors impacting the PAH recoveries during the protein
precipitation step. Using a classical least-squares multiple linear
regression, the model coefficients were estimated to evaluate factor
effects. Coefficient significance was evaluated using a $t$-test
comparing the coefficient estimate to its standard-deviation obtained
from the repeated experiments and the results are presented Figure S3.
A factor or an interaction was considered as significant with a 5\%
risk of the first kind if the value of $P > t$ was lower
than 0.05. First, the change from 50 to 70\% of CH$_{3}$CN had no
significant effect for all the studied PAHs except for FLO. It seems
then that 50\% of CH$_{3}$CN are sufficient to disrupt the PAH--protein
interactions. Similarly, the SDS content had no significant effect on
the PAH recoveries. Considering temperature, a negative effect on PAH
recoveries was observed for all the PAHs except DahP. Moreover, the
interaction between SDS content and temperature had also a significant
negative effect for 11 of the 16 studied PAHs. Finally, the interaction
between the SDS content and CH$_{3}$CN percentage had a negative
significant effect for\break seven PAHs. 

The Derringer's desirability function was next used to determine the
precipitation conditions\break giving rise to the highest PAH recoveries.
Each response was transformed on a scale between 0 (corresponding to
the most undesirable value) and 1 (representing the value that most
fulfilled the requirements). The value of each transformed response was
called ``desirability value''. The global desirability was calculated
by multiplying all the individual desirability values. Finally, this
global desirability was maximized to determine the predicted optimum
(see Figure S4): the coded values of the predicted optimum were (1, ${-}$1,
3), which correspond to 70\% of CH$_{3}$CN (v/v), 0.2\% of SDS (w/v),
and precipitation done at rt for 2~min. 

This optimum corresponded to one of the experimental points tested
during the DOE, for which it appeared that the obtained PAH recoveries
are very close to the ones obtained with another experimental condition
using the same content of CH$_{3}$CN (70\%) and no heating, but with
only 0.1\% of SDS. Therefore, both experimental conditions were tested
again, but with the 24~PAHs at a spiking level of 10~${\upmu}$g/L. For
the predicted optimum, extraction recoveries were between 28 and 82\%
with SD values lower than 16\%, in agreement with the predicted
recoveries, whereas for the other experimental condition, they were
between 33\% and 81\% with SD values lower than 14\%. An ANOVA test
($\alpha = 5$\%) demonstrated that the extraction recoveries were
not significantly different for both conditions (data not shown).
Therefore, the predicted optimum by the DOE was preferred. 

\subsection{Extraction of the PAHs from maternal and umbilical cord sera}

After the optimization of the different steps of the sample handling
with spiked pure media and a synthetic serum, the extraction efficiency
of this procedure was evaluated in real biological fluids for the 
24~PAHs by spiking pooled maternal and umbilical cord sera at
10~${\upmu}$g/L for each PAH except for CPcdP and IcdP at
20~${\upmu}$g/L. 100~${\upmu}$L of pooled maternal and umbilical cord
sera were used for each experiment carried out in triplicate. The
protein precipitation was made by adding 233~${\upmu}$L of CH$_{3}$CN
plus 0.2\% SDS. 300~${\upmu}$L of the supernatant were taken and water
was added to decrease the CH$_{3}$CN proportion from 70 to 50\%.
400~${\upmu}$L of this mixture were percolated through the SPE
cartridge and were recovered after elution and evaporation in
100~${\upmu}$L of H$_{2}$O:CH$_{3}$CN (1:1, v/v) before analysis.
Figure~\ref{fig7} shows the resulting chromatograms and
Table~\ref{tab3} the calculated extraction recoveries. It is worthwhile
to notice that the pooled sera were also analyzed without spiking to
evaluate the potential presence of endogenous PAHs and\break correct
the resulting extraction recoveries, but no PAHs were detected in this
pooled sample. 

\begin{figure*}
\vspace*{3pt}
\includegraphics{fig07}
%\vspace*{2pt}
\caption{\label{fig7}LC/UV/FD chromatograms of the extracted pooled
maternal (A) and umbilical cord (B) sera spiked with 24 PAHs at 10
${\upmu}$g/L except CPcdP and IcdP at 20 ${\upmu}$g/L. Other
conditions: see Section~\ref{sec2.2}. 1, NAPH; 2, ACE; 3, FLO; 4, PHE; 5, ANT;
6, FLT; 7, PYR; 8, BcF; 9, BaA; 10, CHR; 11, 5MCHR; 12, BbF; 13, BkF;
14, BaP; 15, DahA; 16, DalP; 17, BghiP; 18, DaeP; 19, DaiP; 20, DahP.}
%\vspace*{1.8em}
\end{figure*}

\begin{table*}
\caption{\label{tab3}Extraction recoveries ($R$\%) and SD ($n=3$) of 24
PAHs in spiked CH$_{3}$CN:H$_{2}$O mixture, pooled maternal and
umbilical cord sera}
\begin{tabular}{lcccccccc}
\thead
\xmorerows{1}{PAHs} & 
\multicolumn{2}{c}{Spiked CH$_{3}$CN:H$_{2}$O (1:1, v/v)} && 
\multicolumn{2}{c}{Spiked maternal serum} && 
\multicolumn{2}{c}{Spiked umbilical cord serum} \\
\cline{2-3}\cline{5-6}\cline{8-9}
 & $R$ (\%) & SD (\%) && $R$ (\%) & SD (\%) && $R$ (\%) & SD (\%) \\
\endthead
NAPH  & 27 & 13 && 46 & 11 && 47 & 11 \\ 
ACY  & 24 & \05 && 31 & \02 && 34 & \02 \\ 
ACE  & 35 & 10 && 40 & \07 && 48 & \04 \\ 
FLO & 38 & \07 && 40 & \05 && 48 & \04 \\ 
PHE  & 47 & \05 && 45 & 10 && 51 & \02 \\ 
ANT  & 51 & \04 && 39 & \05 && 53 & \02 \\ 
FLT & 64 & \05 && 52 & \08 && 62 & \05 \\ 
PYR & 66 & \09 && 57 & \06 && 68 & \09 \\ 
BcF & 62 & \07 && 53 & \05 && 64 & \04 \\ 
CPcdP & 64 & \06 && NQ & - && NQ & - \\ 
BaA  & 55 & \05 && 51 & \04 && 68 & \02 \\ 
CHR & 67 & \07 && 55 & \03 && 69 & \03 \\ 
5MCHR & 71 & \08 && 51 & \06 && 64 & \04 \\ 
BjF & 71 & \02 && 41 & \06 && 60 & \02 \\ 
BbF & 68 & \07 && 51 & \03 && 69 & \05 \\ 
BkF & 69 & \08 && 51 & \04 && 67 & \04 \\ 
BaP & 68 & \08 && 48 & \03 && 65 & \04 \\ 
DahA & 74 & \09 && 46 & \02 && 68 & \07 \\ 
DalP & 70 & \06 && 33 & \01 && 66 & 10 \\ 
BghiP & 82 & \08 && 40 & \01 && 59 & \03 \\ 
IcdP & 75 & \06 && NQ & - && NQ & - \\ 
DaeP & 77 & \09 && 37 & \02 && 61 & \04 \\ 
DaiP & 72 & \08 && 28 & \01 && 54 & \05 \\ 
DahP & 67 & \08 && 27 & \02 && 47 & \03 
\botline
\end{tabular}
\tabnote{Precipitation step: 0.2\% of SDS and 70\% of CH$_{3}$CN. SPE
\textit{(Hypersep cartridge)}: percolation of 400~${\upmu}$L of spiked
sample with 10 ${\upmu}$g/L of each PAH except for CPcdP, BjF and IcdP
at 20 ${\upmu}$g/L, washing with 100~${\upmu}$L of CH$_{3}$CN:H$_{2}$O
(1:1, v/v), elution with 300 ${\upmu}$L of THF and partial evaporation
(addition of 40 ${\upmu}$L of toluene in the elution fraction). The
extract was further recovered with 100~${\upmu}$L of
CH$_{3}$CN:H$_{2}$O (1:1, v/v). NQ: Non-quantifiable.\vspace*{-.8pt}}
\end{table*}

Extraction recoveries were between 27 and 57\% for maternal sera and
between 34 and 69\% for umbilical cord sera, with SD values lower than
11\% thus highlighting the reliability of the developed method applied
to complex samples. These recoveries are quite inferior to the ones
obtained with spiked pure media (between 24 and 82\%) (Table~\ref{tab3}), but
for which no precipitation was involved. This may also result from a
matrix effect. Moreover, the extraction recoveries for maternal sera
are lower than those obtained with umbilical cord sera, especially for
BaA to DahP. Therefore, the matrix effect seems to be different for
both kinds of sera. Unfortunately, it was not possible to quantitate
CPcdP and IcdP, because some impurities co-eluted with them. This is
why it is possible to conclude that the developed whole sample
pretreatment led to good extraction recoveries for 22 of the 24 listed
PAHs. These extraction recoveries are inferior to the values given in
literature~\cite{6,19,20,24}, ranging from 77 to 122\%, but it is
worthwhile to notice that here 22 compounds with a wide range of log P
were targeted against only between five and 16 US-EPA PAHs in
literature. 

The whole sample pretreatment led to ratio between the sample volume
and the final extract volume of 0.95. Considering the extraction
recoveries, enrichment factors ranged from 0.3 to 0.5 and from 0.3 to
0.7 for maternal and umbilical cord sera, respectively. This led to
LOQs in sera between 0.2 and 3.1~${\upmu}$g/L for the 20 fluorescent
PAHs and between 7.0 and 14.5~${\upmu}$g/L for the two others PAHs
(calculation for a signal to noise ratio equal to 10), which are close
to the expected concentrations in real 
samples~\cite{6,14,21,27,28,29,30,31}. In literature, LOQ values between 0.01
and 2.5~${\upmu}$g/L were reached, but requiring a serum volume at
least 10 times larger, i.e.\ 1 or 2~mL of serum~\cite{6,20,21,24}.

The optimized sample handling, separation, and detection steps require
a total analysis time of 1 h 40 and the SPE and analytical steps can be
automated. This constitutes also a significant improvement over the
literature where serum sample handling always required at least three
LLE steps followed most often by an additional SPE 
step~\cite{6,19,20,21,24,25}. After validation, it should allow the analysis
of a large number of real samples.

\section{Conclusions}

The analysis of the 24 listed PAHs by LC-DAD/FD was developed. Then,
different parameters governing the SPE procedure were studied to
optimize the\break extraction recoveries on C18 silica of the 24 PAHs
belonging to a large range polarity (lop P values between 3.3 and 7.7).
The use of an aromatic solvent was proposed to minimize the loss of the
most volatile PAHs during the evaporation step. A DOE was used to
optimize the protein precipitation\break conditions. The performances of the
whole sample pretreatment were determined for 22 PAHs and for a reduced
volume of 100~${\upmu}$L of maternal and umbilical cord sera. The LOQs
obtained for both matrices were between 0.2 and 3.1 ${\upmu}$g/L for
the fluorescent PAHs and between 7.0 and 14.5 ${\upmu}$g/L for ACY and
BjF (UV detection). The optimized sample handling,\break separation, and
detection steps require a total analysis time of only 1 h 40 and the
SPE and analytical steps can be automated. The next step will consist
in validating this method, which will allow its use for the analysis of
numerous real samples. The consistency of the resulting data will
enable to carry out epidemiologic studies. 

\section*{Abbreviations}
ACE, acenaphthene; ACY, acenaphthylene; ANT, anthracene; BaA,
benzo{[}a{]}anthracene; BaP, benzo{[}a{]}pyrene; BbF,
benzo{[}b{]}fluoranthene; BcF, benzo{[}c{]}fluorene; BghiP,
benzo{[}g,h,i{]}pyrene;  BjF, benzo{[}j{]}fluorene; BkF,
benzo{[}k{]}fluoranthene; CHR, chrysene; CPcdP,
cyclopenta{[}cd{]}pyrene; DaeP, dibenzo{[}a,e{]}pyrene; DahA,
dibenzo{[}a,h{]}anthracene; DahP, dibenzo{[}a,h{]}pyrene; DaiP,
dibenzo{[}a,i{]}pyrene; DalP, dibenzo{[}a,l{]}pyrene; FLO, fluorene;
FLT, fluoranthene; IcdP, indeno{[}1,2,3-cd{]}pyrene; 5MCHR,
5-methylchrysene; NAPH, naphthalene; PHE, phenanthrene; PYR, pyrene;
SDS, sodium dodecyl sulfate.

\section*{Declaration of interests} 

\vspace*{-2pt}

The authors do not work for advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

\vspace*{-2pt}

\section*{Ethical approval}

\vspace*{-2pt}

All procedures performed in studies involving human participants were
in accordance with the ethical standards of the institutional and/or
national research committee and with the 1964 Helsinki declaration and
its later amendments or comparable ethical standards. This article does
not contain any studies with animals performed by any of the authors.

\vspace*{-2pt}

\section*{Informed consent}

\vspace*{-2pt}

Informed consent was obtained from all individual participants included
in the study.

\back{}

\vspace*{-2pt}

\section*{Supplementary data}

\vspace*{-2pt}

Supporting information for this article is available on the journal's
website under \printDOI\ or from the author.  

\CDRsupplementaryTwotypes{supplementary-material}{\cdrattach{crchim-286-suppl.pdf}}

\vspace*{-2pt}

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