Journal of Chromatography A, 1124 (2006) 3–10

Optimisation of a solid-phase microextraction method for the determination

of parabens in water samples at the low ng per litre level
P. Canosa, I. Rodrı́guez ∗ , E. Rubı́, M.H. Bollaı́n, R. Cela
Departamento de Quı́mica Analı́tica, Nutrición y Bromatologı́a, Instituto de Investigación y Análisis Alimentario,
Universidad de Santiago de Compostela, Santiago de Compostela 15782, Spain
Available online 4 April 2006

Abstract
A procedure for the determination of five esters of p-hydroxybenzoic acid (parabens) in water samples is presented. Analytes (methyl, ethyl,
propyl, butyl and benzyl paraben) are concentrated on a solid-phase microextraction (SPME) fibre, converted on their tert-butyldimethylsilyl
derivatives and selectively determined using gas chromatography in combination with tandem mass spectrometry (GC–MS/MS). Influence of
different factors on the efficiency of extraction and on-fibre derivatization steps is described in detail. For all species, the highest enrichment
factors were achieved using a polyacrylate (PA) fibre exposed directly to stirred water samples, containing 150 mg/ml of sodium chloride, at
room temperature. Performance of the further on-fibre derivatization reaction was also maximum at room temperature, considering a short
exposition period of the SPME fibre to vapours of the silylation reagent. Under optimised conditions, the proposed method achieved quan-
tification limits from 0.001 to 0.025 ng/ml and it was free of matrix effects; therefore, external calibration can be used as the quantification
technique. From our knowledge, this work describes the first application of SPME and gas chromatography to the determination of parabens
in water. The analysis of a limited number of real samples revealed the presence of parabens in raw sewage water at concentrations up
to 3 ng/ml.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Parabens; SPME; On-fibre silylation; GC–MS/MS; Water analysis

1. Introduction
Alkyl and aryl esters of p-hydroxybenzoic acid are commer-
cially known under the name of parabens. These compounds
are extensively employed as preservatives and bactericides in
personal care products (e.g. deodorants, bath gels, shampoos,
creams and tooth pastes) and also in processed food and bev-
erages [1–3]. In vivo studies have proven that parabens show a
certain estrogenic activity, which, however, is relatively weak in
comparison to that of 17␤-estradiol [4–6]. In spite of the above
assumption, a considerable interest in toxic effects of a continu-
ous exposition to low-levels of these compounds has arisen after
the recent discovery of parabens in tissue samples from human
breast tumours [7].
From the environmental point of view, available information
regarding levels and potential long-term effects of parabens in
wild-life is still scarce [8,9]. Laboratory studies have confirmed
∗ Corresponding author. Tel.: +34 981 563100x14387; fax: +34 981 595012.
E-mail address: qnisaac@usc.es (I. Rodrı́guez).
that the presence in the same sample of parabens and other
species with relatively weak estrogenic potencies (e.g. genis-
tein, benzophenone, bisphenol A, etc.), at concentration below
their non predicted effect level, might result in a considerable
global estrogenic activity for the sample due to concentration
additive effects [10]. Therefore, parabens must be added to the
list of household chemicals whose environmental levels should
be maintained under surveillance. Urban wastewater constitutes
one of the main sources of these species in the biosphere; there-
fore, it must be the first compartment to be investigated in order
to known the levels of parabens and understand their fate in the
aquatic media.
Available methods for the determination of parabens in water
samples rely on the use of solid-phase extraction (SPE) as the
concentration technique. After the enrichment step, analytes are
determined using liquid (LC) or gas chromatography (GC) with
mass spectrometry detection [8,9,11,12]. For GC based tech-
niques, derivatization of parabens allows to reduce the achieved
detection limits. In the only reported application, it was car-
ried out after the SPE step, and required a previous solvent
exchange [12].

0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2006.03.045
4 P. Canosa et al. / J. Chromatogr. A 1124 (2006) 3–10
During the last decade, solid-phase microextraction (SPME)
has been firmly established as a valuable alternative to SPE
methods in the analysis of water samples. The combination
of SPME and GC is particularly suitable for the determination
of volatile and semi-volatile, non-polar compounds as well as
those whose polarities can be decreased previously, or simulta-
neously, to the microextraction process [13,14]. In addition, the
use of on-fibre derivatization reactions has broaden the appli-
cation field of SPME and GC to polar species, which are not
easily derivatized in presence of water [15–20]. Up to now, the
only application of SPME to the determination of parabens has
been developed for personal care products. Analytes are first
concentrated in a SPME fibre and then desorbed directly in
the drift tube of a ion mobility spectrometer, without a pre-
vious chromatographic separation [21]. This method provides
quantification limits in the 15–30 ng/ml range and it proba-
bly constitutes the faster approach for the determination of
parabens in pharmaceutical and personal care products. How-
ever, it lacks of enough sensitivity for the analysis of water
samples.
The aim of this manuscript is the development of a sensi-
tive method for the determination of five parabens in surface
and sewage water samples. SPME was chosen as the sample
preparation method and GC, combined with tandem MS–MS
detection, as the determination technique. The use of MS–MS
detection, instead of single MS, is expected to increase the
selectivity of the determinations for complex matrices, such as
raw sewage water, decreasing the quantification limits of the
proposed method. An on-fibre silylation step, using N-methyl-
N-(tert-butyldimethylsilyl)trifluoroacetamide as derivatization
reagent, was included in the analytical procedure to improve the
performance of GC separations. Influence of different parame-
ters on the efficiency of the microextraction is described in detail
and the performance of the method compared to that achieved
in previous works.
2. Experimental
2.1. Standards and materials
HPLC-grade methanol and ethyl acetate for trace analysis
were supplied by Merck (Darmstadt, Germany). Methyl (MeP),
ethyl (EtP), propyl (PrP), butyl (BuP) and benzyl (BzP) esters
of 4-hydroxybenzoic acid, as well as the derivatization reagent
N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTB-
STFA), were purchased from Aldrich (Milwaukee, WI, USA).
Individual solutions of each analyte were prepared in methanol.
Further dilutions and mixtures of the five compounds were
made in methanol and ethyl acetate. The first were employed
for preparing spiked water samples. Optimisation of chromato-
graphic and MS–MS fragmentation conditions was accom-
plished using silylated standards of the analytes. They were
obtained by addition of 50 ␮l of MTBSTFA to aliquots (500 ␮l
volume) of paraben standard solutions prepared in ethyl acetate
[22].
A manual SPME holder and fibres coated with different
polymers: poly(dimethylsiloxane) (PDMS, 100 ␮m film thick-
ness), polyacrylate (PA, 85 ␮m film thickness), carboxen-PDMS
(CAR-PDMS, 75 ␮m film thickness), PDMS-divinylbenzene
(PDMS-DVB, 65 ␮m film thickness) and Carbowax-DVB (CW-
DVB, 75 ␮m film thickness), were obtained from Supelco
(Bellefonte, PA, USA). Before their first use each fibre was con-
ditioned following the supplier specifications.
Ultrapure (Milli-Q), river and urban wastewater samples
were employed throughout this study. River and sewage
water samples were filtered using cellulose acetate membranes
(0.45 ␮m pore size) and stored in the dark at 4 ◦ C until
analysis.
2.2. Sample preparation
Under final optimised conditions, microextractions were
accomplished in 10 ml volume glass vessels containing a mag-
netic stir bar and filled completely with the water sample. The
pH and sodium chloride content were adjusted to 6 units and
150 mg/ml, respectively. In the case of spiked samples, the
percentage of methanol in the SPME vessel was limited to a
maximum of 1%. Extractions were carried out in the direct sam-
pling mode, at room temperature, using a PA fibre. After an
exposition period of 40 min, the SPME fibre was retracted into
the needle of the holder syringe, water drops attached to the
metallic needle were removed using a soft paper tissue and the
fibre was exposed to the headspace of a 1.5 ml GC autosam-
pler vessel containing 20 ␮l of MTBSTFA. During this step
the hydroxyl moiety, contained in the structure of parabens,
was converted into the corresponding tert-butyldimethylsilyl
derivative. The reaction was completed in 10 min at room
temperature.
2.3. Equipment
Analytes were determined by GC–MS/MS. The employed
system was a Varian CP 3900 Gas Chromatograph (Walnut
Creek, CA, USA) connected to an ion-trap mass spectrometer
(Varian Saturn 2100). Separations were carried out using a HP-5
MS capillary column (30 m × 0.25 mm I.D., df : 0.25 um) sup-
plied by Agilent (Wilmintong, DE, USA). Helium (99.999%)
was used as carrier gas at a constant flow of 1 ml/min. The GC
oven was programmed as follows: 3 min at 70 ◦ C, at 10 ◦ C/min
to 270 ◦ C (held for 10 min). The GC–MS interface and the
ion trap temperatures were set at 270 ◦ C and 220 ◦ C, respec-
tively. SPME fibres were desorbed for 3 min, in the splitless
mode, using the following temperatures: 260 ◦ C for PDMS and
PDMS-DVB, 280 ◦ C for PA and CAR-PDMS, and 220 ◦ C for
CW-DVB. The temperature of the injector was fixed at 280 ◦ C
for direct injection of silylated standard mixtures (1 ␮l volume)
prepared in ethyl acetate. In this case, the splitless time was
reduced to 2 min. The mass spectrometer was operated in the
electron impact mode (70 eV). MS spectra were recorded in the
range from 90 to 550 m/z units. The parent ion for each silylated
specie was isolated with a m/z window of ±3 units and submitted
to collision induced dissociation. Working MS–MS conditions,
as well as m/z ratios for parent and product ions, are given
in Table 1.
 P. Canosa et al. / J. Chromatogr. A 1124 (2006) 3–10 5

Table 1
MS–MS working conditions and instrumental quantification limits of the GC–MS/MS system, considering an injection volume of 1 ␮l, defined for a signal to noise
ratio of 10
Compound Parent ion (m/z) Excitation storage level (m/z) Excitation amplitude (V) Product ions (m/z) Quantification ions (m/z) Q.L. (ng/ml)

MeP 209 92 0.5 195, 177, 149 177 0.1

EtP 223 98 0.5 195, 177, 163, 151 151 + 177 + 195 0.5
PrP 237 104 0.5 195, 163, 151 195 + 151 0.3
BuP 251 110 0.5 195, 151 195 + 151 0.4
BzP 285 90 0.4 241, 225, 163 241 + 163 1.0

3. Results and discussion containing 20 ␮l of MTBSTFA for 15 min at room temperature.

The variability of the process for the five considered analytes
3.1. GC–MS/MS detection ranged from 4 to 11% (n = 5 replicates). These values were con-
sidered as acceptable to proceed with the optimisation of sample
Optimisation of GC–MS/MS conditions was accomplished preparation.
using a mixture of silylated parabens prepared in ethyl acetate
and containing around 0.5 ␮g/ml of each compound. MS spectra 3.2.2. Microextraction conditions
of target species showed a predominant signal at m/z [M-57]+ The effect of different parameters on the efficiency of the
corresponding to the loss of the tert-butyl moiety. Other ions microextraction step was investigated using spiked aliquots of
appeared at lower m/z ratios and showed very weak intensities. ultrapure water (2 ng/ml level) and fixing the on-fibre derivati-
Therefore, in order to improve the selectivity of the determi- zation conditions on values reported in the previous paragraph.
nations, the MS–MS detection mode was chosen. The [M-57]+
ion, for each silylated paraben, was isolated in the ion trap and 3.2.2.1. Fibre coating and sodium chloride addition. Selection
submitted to further fragmentation using a resonant waveform. of an appropriate coating is one of the most important stages in
Optimisation of MS–MS conditions was carried out using the the development of a SPME method. In a first optimisation step,
automated method development tool implemented in the soft- the efficiency of five different coated fibres (PDMS, PDMS-
ware of the Saturn GC–MS Workstation. In the case of methyl,
ethyl, propyl and butyl paraben their MS–MS reaction patterns
consisted of replacement of the alkyl chain, bonded to the ester
group, by a proton, followed by further losses of CO2 , O2 or
water, Fig. 1A. The most intense signals in their MS–MS spec-
tra were thus detected at 195, 151, 163 and 177 m/z units. The
MS–MS fragmentation behaviour of benzyl paraben seems to
be more complex, the intense signal at m/z [M-57-44]+ sug-
gests the removal of CO2 from the parent ion (m/z 285) with
a re-arrangement in the structure of the compound, Fig. 1B.
The ion at m/z [M-57-44-78]+ points to a further loss of an
aromatic ring, Fig. 1B. Working MS–MS conditions and instru-
mental quantification limits of GC–MS/MS are summarized in
Table 1.

3.2. Optimisation of the SPME method

3.2.1. Preliminary experiments

Performance of microextraction methods, particularly when
they involve also a derivatization stage, is potentially affected
by a large number of factors. In order to asses whether an exper-
imental factor plays a significant influence on the yield of sam-
ple preparation step, or not, the repeatability of whole method
(extraction plus derivatization) should be previously evaluated.
This study was accomplished using aliquots of ultrapure water
spiked with the analytes at the 2 ng/ml level. Departure microex-
traction and derivatization conditions were chosen considering
optimal values reported for other phenolic species [17]. In brief,
extraction was carried out for 30 min using a PA fibre exposed
directly to the spiked sample. After that, derivatization was per-
formed by placing the fibre at the headspace (HS) of a vessel Fig. 1. MS–MS spectra for: ethyl (A) and benzyl paraben (B).
6 P. Canosa et al. / J. Chromatogr. A 1124 (2006) 3–10
DVB, PA, CAR-PDMS and CW-DVB) for the concentration
of parabens was compared using direct extraction, at room
temperature, for 20 min. Experiments were accomplished using
samples adjusted at pH 2.5, without addition of salt. The lower
extraction efficiencies corresponded to PDMS and CW-DVB
fibres followed by PDMS-DVB and CAR-PDMS ones. By far,
the highest responses for all compounds were achieved with the
PA fibre, data not shown. The influence of the ionic strength on
the yield of the extraction for this fibre was further investigated
using samples containing increasing concentrations of sodium
chloride, from 0 to 300 mg/ml. The behaviour of parabens fol-
lowed two different patterns. In the case of methyl and ethyl
paraben, which are the most polar species of the group (log Kow
1.9 and 2.4, respectively), the yield of the extraction raised steady
with the concentration of sodium chloride. For the other ana-
lytes, it increased until NaCl levels of 100–150 mg/ml and then
remained stable, case of propyl paraben (log Kow 2.9), or started
to decrease for butyl (log Kow 3.5) and benzyl paraben (log Kow
3.6), Fig. 2. Addition of sodium chloride to the water samples
affects to both the kinetic and the thermodynamic of the SPME
process. The improvement in the efficiency of the extraction can
be explained from the thermodynamic point of view, considering
the salting out effect which is specially relevant for polar and non
ionic species. On the other hand, the presence of salt increments
the viscosity of the sample slowing down the diffusion of the ana-
lytes to the fibre. This last effect is responsible for the observed
diminution in the responses of butyl and benzyl paraben at high
salt concentrations. In view of the above results, the concentra-
tion of NaCl added to water samples was fixed at 150 mg/ml. The
extraction yield for the three best suited fibres (PA, PDMS-DVB
and CAR-PDMS) was re-evaluated using samples containing
this concentration of sodium chloride and a longer exposition
time (40 min) than in the first fibres comparison study. Again,
the PA fibre provided the highest responses for all compounds,
Fig. 3. This behaviour matches with data obtained for polymer
stabilizers, hormones and other bactericides containing phenolic
moieties in their structures [17,23,24]; however, it is in dis-
agreement with the work of Lokhnauth and Snow, who reported
better extraction efficiencies for PDMS-DVB and PDMS coated
fibres than for the PA one, in the extraction of parabens from
Fig. 2. Influence of NaCl concentration on the efficiency of the SPME for ethyl,
propyl and benzyl paraben. Direct sampling at room temperature for 40 min using
a PA fibre. Responses have been normalised to the maximum signal achieved
for each specie.
Fig. 3. Comparison of SPME efficiencies for PA, PDMS-DVB and CAR-PDMS
fibres. Responses normalised to the PA fibre. Direct extraction at room temper-
ature for 40 min using spiked samples containing 150 mg/ml of NaCl, n = 3
replicates.
diluted solutions of shampoos and other personal care products
[21].
Carryover effects on the PA fibre were evaluated by desorbing
it twice. Negligible signals (below 0.1% of peak areas obtained
in the first run) were observed for all compounds in the second
desorption step. Anyhow, to reduce the risk of cross contamina-
tion between samples containing very different concentrations
of the analytes, fibres were additional heated for 5 min at 280 ◦ C
in the injector of a non-operative GC instrument, after finishing
the analytical desorption step and before being exposed to the
next sample.
3.2.2.2. Sample pH. The effect of pH on the SPME process
was investigated at three different levels (2.5, 4.5 and 6.5 units)
below the pKa of the analytes (from 8.2 to 8.3). Extractions
were performed in triplicate for 40 min using samples containing
150 mg/ml of sodium chloride. Within the considered pH inter-
val, significant differences in the peak areas of the target species
were not observed. In further extractions, river and sewage water
were adjusted at pH 6, whereas Milli-Q water was employed
directly without any pH adjustment.
3.2.2.3. Sampling mode, stirring and sample volume. Efficien-
cies of direct SPME at room temperature and HS SPME, room
temperature and 100 ◦ C, were compared considering an extrac-
tion period of 40 min and those conditions optimised in previous
sections. The responses obtained for MeP in the HS mode repre-
sented around 10 and 20% (for extractions at room temperature
and 100 ◦ C, respectively) of those achieved using direct extrac-
tion. For the rest of analytes, independently of the temperature,
the yield of HS extractions was negligible, figure not shown.
Stirring is another parameter which may affect to the kinetic
of the SPME since it fasten the transference of the analytes to
the fibre. Considering an exposition time of 40 min, the effect of
stirring on the yield of the SPME was compound dependent. For
methyl paraben, its influence was negligible, whereas for the rest
of species higher responses were obtained for stirred samples,
Fig. 4. Therefore, the importance of the agitation increases with
 P. Canosa et al. / J. Chromatogr. A 1124 (2006) 3–10
Fig. 4. Influence of stirring on the yield of the SPME.
the molecular weight of the analytes. The effect of the sam-
ple volume was evaluated using vessels with capacities of 10
and 22 ml and the same internal diameter (2 cm). A magnetic
stir bar was placed in each vial and they were filled com-
pletely with aliquots of the same spiked sample. For all species,
obtained responses were independent of the sample volume,
figure not shown. Therefore, according to the thermodynamic
theory of SPME (n = Kfs Vf Vs Co /Kfs Vf + Vs ) [25], it seems
that the product of the distribution constant (Kfs ) and fibre vol-
ume (Vf ) is lower than the volume of the sample (Vs ). In further
experiments the smaller vessels were employed.
3.2.2.4. Extraction time. Fig. 5 depicts the responses obtained
for each compound as function of the extraction time. In the
Fig. 5. Extraction profiles using a PA fibre. Direct SPME at room temperature,
for samples containing 150 mg/ml of sodium chloride.
7
case of methyl paraben equilibrium between concentrations in
the fibre and in the sample was achieved after 60 min of expo-
sition. The rest of species showed slower extraction kinetics. In
order to maintain the duration of the sample preparation stage
in reasonable values, the enrichment step was limited to 40 min.
3.2.3. On-fibre derivatization
Temperature, time and volume of MTBSTFA are factors
which might affect to the performance of the on-fibre derivatiza-
tion step. Their potential effects on the responses of the silylated
parabens were simultaneously evaluated using a mixed mode
3 × 22 factorial experimental design with a central point [26].
The variable temperature was tested at three levels: 20 (room
temperature), 45 and 70 ◦ C, whereas the other two factors were
only considered at two levels: 10–40 min and 20–80 ␮l for time
and volume of MTBSTFA, respectively. This design, consist-
ing of a total of 13 experiments, provides information regarding
numerical values of the main effect for each factor, two-factor
interactions and the quadratic term associated to the variable
temperature, as well as their statistical significance. In gen-
eral, two-factor interactions presented small and non-significant
effects (at the 95% confidence level), meaning that the three
variables play independent effects on the derivatization reac-
tion. Standardized values corresponding to the main effects of
time, temperature and volume of MTBSTFA are given in Table 2.
The absolute value of each effect is proportional to the change
in the response of a given paraben (as silylated derivative) when
the considered factor changes from the low to the high level
in the domain of the design. Its sign indicates if the response
increases (positive sign) or decreases (negative sign). As shown
in Table 2, all compounds followed a similar pattern: temperature
and time played a negative effect on the yield of the derivatiza-
tion, the first factor resulted statistically significant for methyl,
ethyl and propyl paraben, whereas the second overpass the sig-
nificance bound for ethyl and benzyl paraben. The importance
of the quadratic term associated to the variable temperature was
practically null, meaning a linear decrease in the response of
the compounds with the increase of this factor, data not given.
The diminution in the peak areas of the silylated compounds for
high temperatures and long derivatization times might indicate a
partial desorption of the extracted compounds from the PA fibre
during the derivatization step [23]. Although positive, the role of
MTBSTFA volume was negligible, Table 2. Room temperature,
10 min and 20 ␮l of MTBSTFA were selected as the optimal
on-fibre derivatization conditions.
3.3. Performance of the method
The linearity of the method was evaluated using samples of
ultrapure water spiked with increasing concentrations of the ana-
lytes, at seven different levels, from 0.02 to 5 ng/ml. Obtained
correlation coefficients ranged from 0.996 to 0.999 depending
on the compound, Table 3. Precision was studied using sam-
ples spiked at three different concentration levels from 0.02 to
0.5 ng/ml. Relative standard deviations of peak areas between
2 and 13% were achieved. Quantification limits of the method,
defined for a signal to noise (S/N) of 10, ranged from 0.001
8 P. Canosa et al. / J. Chromatogr. A 1124 (2006) 3–10

Table 2
Experimental domain and standardized values for main effects of factors considered in the optimisation of the on-fibre derivatization reaction
Factor Range of values Compound

Low Medium High MeP EtP PrP BuP BzP

Temperature (◦ C) 20 45 70 −2.46a −4.36a −2.79a −2.24 −1.90

Time (min) 10 − 40 −1.81 −2.70a −2.12 −2.20 −2.43a
Volume MTBSTFA (␮L) 20 − 80 0.62 0.24 0.44 0.18 0.21
a Statistically significant factors at the 95% confidence level.

Table 3
Linearity, repeatability and quantification limits (S/N 10) of the method
Compound Regression coefficient (R2 ) Repeatability, RSD (%), n = 4 replicates Q.L. (ng/ml)

Added concentration (ng/ml)

0.02a 0.2 0.5

MeP 0.999 11.9 10.7 8.3 0.025

EtP 0.997 7.3 8.5 6.3 0.005
PrP 0.998 6.6 1.1 2.4 0.002
BuP 0.996 10.4 5.5 4.7 0.001
BzP 0.999 7.6 13.3 9.0 0.005
a 0.05 ng/ml in the case of MeP.

Table 4
Study of potential matrix effects
Compound Normalised responses with their relative standard deviations (RSD%)

Milli-Q River Treated wastewater Raw wastewater

MeP 100 (8) 114 (3) 92 (2) 92 (3)

EtP 100 (10) 99 (2) 96 (2) 92 (3)
PrP 100 (9) 99 (7) 93 (5) 95 (9)
BuP 100 (10) 98 (13) 87 (5) 96 (8)
BzP 100 (12) 102 (4) 88 (10) 104 (7)

Comparison of signals for different water samples spiked with the analytes at the 2 ng/ml level, n = 3 replicates.

to 0.025 ng/ml, Table 3. With the exception of methyl paraben, any epoxy resins, would serve to asses the validity of above
these values are lower than the detection limit of 0.01 ng/ml assumptions.
(defined for a signal to noise ratio of 3) reported by Lee et al. Efficiency of SPME methods can be affected by the com-
using solid-phase extraction, solvent evaporation and derivati- position of the sample matrix. Table 4 compares the responses
zation of the analytes with pentafluoropropionic anhydride [12]. obtained for different water samples spiked with the analytes at
In the case of ethyl, propyl, butyl and benzyl paraben quantifi- the 2 ng/ml level. Each sample was processed in triplicate and
cation limits of the developed method were controlled by the results normalised to those obtained for ultrapure water. Non-
enrichment efficiency of the SPME step and the performance spiked raw wastewater showed signals for the four alkylated
of the GC–MS/MS instrument. The value achieved for methyl parabens. The average response for each compound (n = 3 repli-
paraben was determined by the signal obtained for this com- cates) was substrated from that measured for spiked aliquots
pound in blank samples. Ultrapure water, SPME vessels and of the same sample, and then, the difference compared to the
salt were excluded as responsible for this contamination. In
fact, the direct exposition of a new SPME fibre to the vapours
of MTBSTFA, followed by its desorption in the GC–MS/MS Table 5
Accuracy of the method for spiked river water
system, produced a small peak for this compound prevent-
ing its quantification at levels below 0.025 ng/ml. According Compound Concentrations (ng/ml) with their standard
to data given in the literature, decomposition of epoxy resins deviations, n = 3 replicates
employed to stick the silica fibre to the steel needle of the Added Measured Added Measured
SPME holder and/or the presence of methyl paraben in the atmo- MeP 0.21 0.22 (0.02) 1.01 1.13 (0.03)
sphere of the laboratory might explain this problem [27,28]. EtP 0.21 0.23 (0.01) 1.01 1.06 (0.08)
Anyhow, the exact source of this contamination could not be PrP 0.23 0.22 (0.03) 1.12 1.26 (0.06)
BuP 0.21 0.20 (0.02) 1.02 1.09 (0.04)
determined. In a close future, it is expected that the availabil-
BzP 0.21 0.20 (0.03) 1.02 1.08 (0.09)
ity of titanium coated SPME fibres, which does not contain
 P. Canosa et al. / J. Chromatogr. A 1124 (2006) 3–10 9

Fig. 6. Overlay of GC–MS/MS chromatograms for a blank (A), a non-spiked raw wastewater (B, code 7 in Table 6), and the same sample after addition of the
analytes at the 0.5 ng/ml level (C).

Table 6
Concentrations of alkyl parabens in sewage water samples, n = 4 replicates
Code Sampling point Sampling date Concentrations (ng/ml) with their standard deviations

MeP EtP PrP BuP

1 Hospital sewer 20/12/04 2.4 (0.19) n.d. 0.98 (0.10) n.d.

2 Hospital sewer 16/6/05 0.065 (0.008) 0.056 (0.006) 0.81 (0.07) n.d.
3 Hospital sewer 23/6/05 1.48 (0.17) 0.10 (0.01) 1.22 (0.11) 0.019 (0.001)
4 Hospital sewer 15/9/05 0.073 (0.003) n.d. 0.18 (0.03) n.d.
5 STP influent 17/6/05 2.92 (0.20) 0.21 (0.01) 0.81 (0.14) 0.086 (0.015)
6 STP effluent 17/6/05 n.d. n.d. n.d. n.d.
7 STP influent 24/7/05 0.43 (0.04) 0.052 (0.004) 0.23 (0.02) 0.020 (0.001)
8 STP effluent 24/7/05 n.d. n.d. 0.064 (0.001) n.d.

STP: sewage treatment plant; n.d.: under detection limits.

response obtained for Milli-Q water. Taking into account the
precision of the developed method, matrix effects are negligible
and thus quantification can be performed using external calibra-
tion.
Accuracy of the method was investigated using river water
samples spiked at two different concentration levels. Non-spiked
aliquots of this sample were also analysed and none of the com-
pounds was detected over the quantification limits of the method.
Calculated concentrations, determined using external calibra-
tion, were in acceptable agreement with the theoretical values
added to the samples, considering the precision of the developed
method, Table 5.
3.4. Analysis of real samples
GC–MS/MS chromatogram for a non-treated wastewater sam-
ple. Obtained data are given in Table 6. Benzyl paraben remained
under detection limits of the method in all samples, whereas con-
centrations up to 2.9 ng/ml were found for the rest of species in
raw wastewater, with the highest levels corresponding to methyl
and propyl paraben. Values shown in Table 6 are in the same
order of magnitude than the semi-quantitative data reported for
non-treated domestic grey wastewater samples collected in Den-
mark [9]. Comparison of concentrations measured for the two
pairs of composite samples taken in the inlet and outlet of the
sewage treatment plant, revealed very high removal efficiencies
of parabens. This behaviour is in agreement with the tendency
described by Lee et al. in several sewage plants from Canada
[12].

The proposed method was applied to investigate the levels 4. Conclusions

of parabens in sewage water. Flow proportional 12 h compos-
ite samples were taken in the main sewer of a hospital (codes
1–4) and in the inlet and outlet streams of an urban wastewater
plant equipped with primary and secondary (activated sludge)
treatments (codes 5–8). Influent and effluent samples were taken
with a delay of 12 h, similar to the hydraulic retention time of the
plant. After reception samples were filtered and stored at 4 ◦ C
for a maximum of 5 days before being analysed. Fig. 6 shows the
SPME combined with GC–MS/MS detection is as a valu-
able approach for the determination of five parabens in water
samples. The developed method avoids the use of organic sol-
vents, achieves quantification limits at the low ng per litre level,
presents an acceptable precision and it is free of matrix effects.
Of the two stages involved in the sample preparation scheme:
SPME enrichment and on-fibre silylation, the first is the one,
10 P. Canosa et al. / J. Chromatogr. A 1124 (2006) 3–10
which controls the duration of the procedure. Preliminary results
for a reduced number of real samples confirmed the presence of
four of the five analytes in non-treated wastewater samples; how-
ever, their removal efficiencies in conventional sewage water was
practically quantitative, consequently, the presence of significant
concentrations of these compounds in river water seems rather
improbable, unless they receive direct discharges of non-treated
sewage water.
Acknowledgments
Financial support from projects BQU 2003-02090, DGICT
CTQ2005-00425 and PGIDIT04PXIC23701PN is acknowl-
edged. P.C. acknowledges a FPU grant from the Spanish Min-
istry of Education.
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