Science of the Total Environment 433 (2012) 281–289

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Science of the Total Environment

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QuEChERS: A new sample preparation approach for the determination of ibuprofen

and its metabolites in soils
Idalina Bragança 1, Alexandra Plácido 1, Paula Paíga, Valentina F. Domingues, Cristina Delerue-Matos ⁎
REQUIMTE, Instituto Superior de Engenharia do Porto, Rua Dr António Bernardino de Almeida, no. 431, 4200-072 Porto, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: Ibuprofen is one of the most used active pharmaceutical ingredients worldwide. A new method for the anal-
Received 1 April 2012 ysis of ibuprofen and its metabolites, hydroxyibuprofen and carboxyibuprofen, in soils is presented. The ex-
Received in revised form 8 June 2012 traction of these compounds from the soil matrices was performed by using a modified quick, easy, cheap,
Accepted 11 June 2012
effective, rugged, and safe (QuEChERS) method. The method involves a single extraction of the investigated
Available online 15 July 2012
compounds with purified water (acidified at pH 2.5 with hydrochloric acid), and a slow and continuous ad-
Keywords:
dition of the QuEChERS content, followed by the addition of acidified acetonitrile (1% acetic acid), prior to the
Ibuprofen determination by liquid chromatography coupled with fluorescence detection (LC–FLD).
Metabolites Validation studies were carried out using soil samples with a range of organic carbon contents. Recoveries of
QuEChERS the fortified samples ranged from 79.5% to 101%. Relative standard deviations for all matrix–compound com-
Soil binations did not exceed 3%. The method quantification limits were ≤ 22.4 μg kg − 1 in all cases. The developed
Extraction method was applied to the analysis of sixteen real samples.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction
Pharmaceutical products (PP) play an important role in the treat-
ment and prevention of diseases in both humans and animals (Jelic et
al., 2009). It is well-known that pharmaceuticals, after administration,
are absorbed and undergo metabolic reactions (e.g. hydroxylation,
cleavage or glucuronation) to produce metabolites, which can be
even more harmful than the original compounds or can be trans-
formed back to the original active drugs (Diaz-Cruz and Barcelo,
2005). Large fractions of the administrated dosage are, in fact, not as-
similated or metabolized, and are released into influents of municipal
wastewater treatment plants (WWTPs). Conventional WWTPs are
not specifically designed to remove PPs, so they often do not elimi-
nate them efficiently; as a result, a portion of PPs disseminate into
the environment via effluents from WWTPs in a dissolved form or
sorbed to sewage sludge. Most sewage sludges are treated to produce
biosolid and are subsequently applied to agricultural lands because of
their fertilizing value. The presence of pharmaceuticals in the envi-
ronment is also due to the improper disposal of surplus- or expired
products. Consequently, PPs, their metabolites and degradation prod-
ucts, are present in different environments (Buchberger, 2007).
⁎ Corresponding author. Tel.: +351 228340500; fax: +351 228321159.
E-mail address: cmm@isep.ipp.pt (C. Delerue-Matos).
1
These authors equally contributed to this work.
During the last decades the European Union and the U.S. Food and
Drug Administration have been introducing requirements for an envi-
ronmental risk assessment as a prerequisite to obtain marketing au-
thorization for new medical products (Santos et al., 2010). In fact, in
June 2010, the European Commission elaborated a shortlist of 19 pos-
sible new priority substances, on which ibuprofen (IBP) is included.
Furthermore, it is considered whether to set limits on these drugs,
which are commonly found in aquatic ecosystems (SCHER, 2011).
IBP (2-(4-(2-methylpropyl)phenyl)propanoic acid) is one of the most
widely used non-steroidal anti-inflammatory drugs, which does not re-
quire a medical prescription (Bushra and Aslam, 2010). According to the
Portuguese Medicine Regulatory Agency, IBP has doubled sales between
2000 and 2009 and moved from the 15th to the 8th position on the list
of the most sold active pharmaceutical ingredients (Infarmed, 2009).
Only 1 to 8% of IBP is excreted unchanged and 14% is excreted as glucuro-
nides. HydroxyIBP (2,4(2-hydroxy-2-methylpropyl)phenylpropionic
acid) and carboxyIBP (2,4(2-carboxypropyl)phenylpropionic acid) were
identified as the main metabolites of IBP present in sewage and environ-
mental samples (Winkler et al., 2001). There are several reports indicating
the presence of IBP in ground- and drinking water (Karnjanapiboonwong
et al., 2011), and in both primary and secondary sludge (Jones-Lepp and
Stevens, 2007) as well as in sewage sludge (Nieto et al., 2010), in the latter
case, with a maximum concentration of 548 μg kg− 1 (Nieto et al., 2010).
IBP was also the most frequently found PP in sediments in Spain
(Vazquez-Roig et al., 2011).
Soil is a complex and heterogeneous matrix with a porous structure
that contains both inorganic and natural organic components (Correia-
Sá et al., 2012). The adsorption capacity of soils is strongly governed by

0048-9697/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.scitotenv.2012.06.035
282 I. Bragança et al. / Science of the Total Environment 433 (2012) 281–289
their sand-, clay- and organic matter contents (Pinto et al., 2010). Soils
play an important role in the environment by preventing the contamina-
tion of adjacent ecosystems. Currently soils are subject to intensive pollu-
tion with chemical compounds. When contaminating agents reach the
soil, they become involved in several types of physicochemical interac-
tions with mineral and organic components (Pinto et al., 2011). The im-
pact of such compounds is not limited to direct adverse effects on
human and animal health; they also deteriorate the properties of the
soil to a significant extent and clean-up is complicated, expensive, and
time-consuming (Pinto et al., 2011).
There is often strong interaction between drug residues and
soils or sediments, so the compounds are difficult to extract. In
order to detect, identify and quantify pharmaceutical residues re-
leased into the environment, analytes need to be isolated from
the sample matrix. Several extraction methods have been devel-
oped to determine the concentration of pharmaceutical com-
pounds in soil samples (Diaz-Cruz and Barcelo, 2007; Sanchez-
Prado et al., 2010). Regarding IBP the following extraction methods
have been developed: solvent extraction (Karnjanapiboonwong et
al., 2011), solid–liquid extraction using sonication (Vazquez-Roig
et al., 2011) pressurized liquid extraction (Nieto et al., 2010), and
microwave-assisted extraction (Sanchez-Prado et al., 2010).
In 2003, Anastassiades et al. introduced the quick, easy, cheap,
effective, rugged, and safe (QuEChERS) method for the analysis of
pesticide residues in fruits and vegetables (Anastassiades et al.,
2003). The three primary QuEChERS methods are: i) the original
method in which acetonitrile (ACN) is used as extraction solvent
and sodium chloride (NaCl) is included in the QuEChERS composi-
tion to enhance extraction and to reduce polar interferences
(Anastassiades et al., 2003), ii) the dispersive AOAC 2007.01 meth-
od, in which a 1% acetic acid ACN solution is the extraction solvent
and anhydrous sodium acetate (CH3COONa) buffer, to protect base
sensitive analytes from degradation and provides superior recov-
ery for pH sensitive compounds, is included in the QuEChERS com-
position (Method AOAC, 2007), and iii) the European Norm EN
15662 includes NaCl to limit polar interferences and several citrate
buffering reagents to preserve base sensitive analytes and also ap-
plies ACN as extraction solvent (EN 15662, 2007). The QuEChERS
method has several advantages over most traditional extraction
methods: high recoveries (> 85%) are achieved; very accurate re-
sults are obtained; high sample throughput is possible; solvent
usage and waste generation is very low; no chlorinated solvents
are used; a single person can perform the method without much
training or technical skill; it is quite rugged because extract clean-
up is performed to remove organic acids; very little bench space is
needed, thus the method can be carried out in a small mobile labo-
ratory if needed; ACN is added by a dispenser to an unbreakable
vessel that is immediately sealed, thus solvent exposure to the
worker is minimal; inexpensive reagents are used; and few equip-
ment are needed (Anastassiades et al., 2003; Lehotay, 2011). Al-
though the QuEChERS method has mainly been used in the
determination of pesticides, other compounds, such as several
pharmaceuticals (Plossl et al., 2006), β-lactam antibiotics (in bo-
vine kidney) (Fagerquist et al., 2005; Mastovska and Lightfield,
2008) or veterinary drugs (Aguilera-Luiz et al., 2008; Kinsella et
al., 2009; Mastovska and Lightfield, 2008; Posyniak et al., 2005;
Stubbings and Bigwood, 2009) have also been determined in sever-
al matrices (blood, chicken muscle, milk, animal tissue and liver).
The aim of this study was the development and optimization of a QuE-
ChERS extraction method of IBP, hydroxyIBP, and carboxyIBP from soil
and sediment samples with a range of different physicochemical proper-
ties. This is a very convenient extraction technique that due to its simplic-
ity and rapidity is being applied in the analysis of complex matrices and
replacing traditional extraction methods. The extracts were analyzed by
liquid chromatography coupled with fluorescence detection (LC–FLD).
The matrix effects on the QuEChERS extraction were also assessed.
2. Experimental
2.1. Reagents, solvents and materials
ACN (purity≥ 99.9%), n-hexane (purity≥99.0%), and acetone (puri-
ty ≥99.7%) were obtained from Merck (Darmstadt, Germany), methanol
(MeOH) (purity ≥99.9%) and ethyl acetate (purity ≥99.5%) were
obtained from Sigma-Aldrich (Steinheim, Germany), hydrochloric acid
37% and glacial acetic acid (purity ≥ 99.7%) were obtained from Carlo
Erba (Rodano, Italy) and orthophosphoric acid 85% was obtained from
Panreac (Barcelona, Spain). For organic carbon (OC) determinations, an-
hydrous D(+)-glucose from Scharlab (Sentmenat, Spain) and anhydrous
sodium carbonate from Nacalai Tesque (Kyoto, Japan) were used as total
carbon (TC) and inorganic carbon (IC) standards. IBP, hydroxyIBP, and
carboxyIBP were purchased from Sigma-Aldrich (Steinheim, Germany).
Deionized water (15.0 MΩ cm) was produced using an Elix 3 Ad-
vantage system (Millipore, Molsheim, France) and was further purified
(18.2 MΩ cm) using a Simplicity 185 system (Millipore, Molsheim,
France). All chromatographic solvents, ACN and purified water, were fil-
tered through a 0.20 μm nylon membrane filter (Ø 47 mm, Supelco,
Bellefonte, PA, USA) using a Dinko D-95 pump (Barcelona, Spain) and
degassed for 15 min in an ultrasonic bath (Raypa® Trade; Terrassa,
Spain). All standards and sample extracts were filtered through an Oli-
mPeak syringe filter, PTFE, 0.20 μm, from Teknokroma (Ø 13 mm, Bar-
celona, Spain). For homogenization, a VWR vortex mixer (Radnor,
Delaware, USA) was used.
Stock solutions of the individual standards (IBP, hydroxyIBP, and
carboxyIBP, in the order of mg L − 1) were prepared by exact weighing
of the solid compounds followed by dissolution in ACN and storage at
−20 °C. A multicomponent intermediate working standard solution
(20 mg L − 1 of each compound) was prepared by appropriate dilution
of the stock solutions with ACN. This intermediate solution was stored
at −20 °C and used to prepare the working standard solutions (concen-
tration in the μg L− 1 range). These solutions were prepared daily in
ACN, stored at 4 °C prior to analysis, and used for fortification of the
samples and for the preparation of the analytical calibration curves.
Amber glassware was used to prevent light degradation.
Four different types of QuEChERS' compositions were tested (all
contained in 50-mL Teflon centrifuge tubes): QuEChERS 1 (6 g of mag-
nesium sulfate (MgSO4) and 1.5 g of CH3COONa); QuEChERS 2 (4 g of
MgSO4 and 1 g of NaCl); QuEChERS 3 (6 g of MgSO4, 1.5 g of NaCl,
1.5 g of sodium citrate dehydrate (NaCit) and 0.750 g of sodium citrate
sesquihydrate (Na2Cit)) and QuEChERS 4 (4 g of MgSO4, 1 g of NaCl, 1 g
of NaCit and 0.50 g of Na2Cit). QuEChERS 1 to 3 were obtained from Unit
Chemical Technologies (UCT), Inc. (Bristol, PA, USA) and QuEChERS 4
was obtained from Agilent Technologies (Santa Clara, PA, USA).
2.2. Soil sampling and characterization
Soils samples were chosen based on the OC content, soil type (from
agricultural to sediments) and location. The sampling sites were located
in the north of Portugal. Fifteen different soils (Table 1) were collected
during 2011. At each sampling site only the upper (0–10 cm) was col-
lected with a spade. After air-drying and sieving to a grain size of
2 mm, samples were stored at 4 °C until analysis. Soil V (washed sea
sand, thin grain QP, 0.25–0.30 mm particle size, 0.2% soluble matter in
hydrochloric acid, 0.2% loss on drying) was obtained from Panreac (Bar-
celona, Spain).
Macro parameters, such as water content, organic matter (OM), TC,
OC and IC contents and pH, were evaluated (Hesse, 1972; Nelson, 1996)
and are shown in Table 2. For soils, a conversion factor of 1.724 was used
to convert OC to OM based on the assumption that OM contains 58% OC
(OM amount = OC amount × 1.724) (Nelson, 1996).
For the determination of OC in soils a dry oxidative combustion
method was used. A Shimadzu TOC analyzer (model VCSN, Shimadzu,
Japan) with a solid sample module (SSM-5000A) was used. For TC
 I. Bragança et al. / Science of the Total Environment 433 (2012) 281–289 283

Table 1 peak, were measured by the TOC–Control V software. For IC mea-

Characterization and geographical location of the soil samples. surements, the samples were acidified with a small amount of
Soil sample Location GPS coordinates orthophosphoric acid (85%) and heated to 200 °C, and the evolved
CO2 was detected by NDIR. With the TOC-analyzer the OC content is
I Agricultural Povoa de Varzim 41°25′ 8°46′
40.96″N 1.24″W the difference between TC and IC. Eight anhydrous D(+)-glucose
II Agricultural Lever 41°4′ 8°29′ standards were used for the measurement of TC and ten sodium
4.07″N 4.46″W carbonate standards were used for the measurement of IC. All de-
III Agricultural Vagos 40°33′ 8°42′
terminations were made in triplicate.
0.56″N 9.19″W
IV Agricultural Oliveira de Azeméis 40°56′ 8°25′ Water content was determined by two methods: (i) drying in an oven
23.34″N 43.89″W (JP Selecta; Barcelona, Spain) until constant weight, and (ii) using
V Washed sea sand a moisture analyzer (Kern MLS 50-3IR160, Balingen-Frommern,
supplied by Panreac Germany).
VI Sediment Moscoso Stream (Ria de 40°49′ 8°24′
Soils I to V were used for extraction method development and val-
Aveiro tributary) 24.81″N 8.16″W
VII Sediment Rabaçal River (Douro 41°34′ 7°15′34″ idation with a previously optimized procedure to evaluate the resi-
tributary) 00″N W dues of the target analysts in these soils.
VIII Sediment Canidelo 41°8′ 8°39′
10.75″N 38.50″W
2.3. Liquid chromatography
IX Sediment Foz 41°8′ 8°40′
58.91″N 35.40″W
X Sediment Madalena 41°6′ 8°39′ Working standard solutions and extracts were analyzed (in tripli-
35.68″N 48.84″W cate) using a Shimadzu LC system (Shimadzu Corporation, Kyoto,
XIa Urban Santa Maria da Feira 40°55′ 8°32′ Japan) equipped with an LC 20AB pump (high-pressure gradient sol-
49.26″N 57.18″W
vent delivery module equipped with two dual-plunger tandem-flow
XIIb Urban Santa Maria da Feira 40°55′ 8°32′
54.09″N 49.65″W pumps), a DGU-20A5 degasser, a SIL 20A autosampler, and a RF-10AXL
XIII Urban Estarreja 40°44′ 8°34′ fluorescence detector (FLD). Separations were performed using a Luna
52.65″N 15.68″W column (C18, 5 μm particle size, 4.60 × 150 mm, Phenomenex, Torrance,
c
XIV Sediment Uíma River (Douro 41°3′ 8°29′
California, USA) with a security guard cartridge (C18, 5 μm particle size
tributary) Point 1 37.10″N 14.14″W
XVd Sediment Uíma River (Douro 41°3′ 8°29′
4 × 3.0 mm, Phenomenex, Torrance, California, USA) at room tempera-
tributary) Point 2 35.75″N 16.26″W ture (20 ± 1 °C). The injected volume was 20 μL and LCsolution soft-
XVI Sediment Lima River 41°44′ 8°38′ ware version 2.1 (Shimadzu Corporation, Kyoto, Japan) was used for
17.53″N 54.90″W control and data processing. For the detection and quantification of
WWTP — wastewater treatment plants. IBP and its metabolites, hydroxyIBP and carboxyIBP, the chromato-
a
Near a WWTP1 of a hospital. graphic conditions were optimized in order to separate the analytes in
b
Proximity of a WWTP2 (not near to the effluent discharged).
c the shortest possible analysis time, to avoid peak tailing, and to obtain
Proximity of a WWTP3 effluent discharged into Uíma River (Douro tributary).
d
Collected at 50 m of soil sample XIV. adequate resolution, peak shape and reproducibility. Different mobile
phases, using isocratic and gradient elution, comprising several combi-
nations of ACN and purified water, were tested to provide better sepa-
analysis the sample was heated to 900 °C in the presence of an oxida- ration. In order to obtain the highest IBP peak area, the optimization
tion catalyst and the evolved CO2 was carried by synthetic air to the of fluorescence emission and excitation wavelengths was performed
non-dispersive infrared (NDIR) gas analyzer for detection. The peak by changing the emission wavelength in the range from 230 to
areas of the NDIR outputs, an analog detection signal that forms a 500 nm and keeping the excitation wavelength constant at 220 nm.
The highest peak area was obtained using an emission wavelength of
290 nm. Fixing the emission wavelength at 290 nm and varying the ex-
Table 2
Macro parameters of the various soils. citation wavelength between 215 and 350 nm, confirmed that the
highest IBP peak areawas obtained at 220 nm. The pH of the mobile
Soil pH Water content OC = TC contentb OM content
phase was adjusted between 2 and 4 with different acids (formic,
samplea (n ≥ 3) (n = 3) (n = 3)
hydrochloric and acetic) and the used flow rates were 0.8 mL min− 1
Oven (105 °C) Balance
and 1.0 mL min− 1. All standards and samples were filtered through a
X ± RSD (%) X ± RSD (%) X ± RSD (%) X ± RSD (%) 0.20 μm syringe filter prior to the injection in the chromatographic
I 7.29 0.766 ± 5.4 0.720 ± 8.69 2.00 ± 1.8 3.45 ± 1.8 system.
II 7.52 11.3 ± 3.6 11.759 ± 2.42 3.12 ± 1.6 5.39 ± 1.6
III 4.87 5.04 ± 3.4 5.674 ± 6.67 0.659 ± 3.6 1.14 ± 3.6 2.4. QuEChERS method
IV 6.23 0.23 ± 4.8 0.554 ± 8.73 4.71 ± 1.3 8.11 ± 1.3
V 6.41 n.d. 0.291 ± 7.67 n.d. n.d.
VI 5.62 8.11 ± 0.63 8.076 ± 2.25 0.458 ± 1.1 0.789 ± 1.1 Several procedures and QuEChERS compositions were tested through
VII 5.85 9.22 ± 2.9 9.313 ± 4.40 1.21 ± 2.0 2.08 ± 2.0 recovery studies. Three ratios of mass (g) of soil I per volume (mL) of ex-
VIII 7.61 6.60 ± 4.2 6.273 ± 7.84 0.205 ± 4.8 0.353 ± 4.8 traction solvent were evaluated: 1.00; 0.75 and 0.50, using 10.0, 7.5 and
IX 7.88 2.93 ± 4.7 2.627 ± 5.15 0.104 ± 6.7 0.179 ± 6.7 5.0 g±0.1 g of soil, respectively.
X 8.05 5.68 ± 1.0 6.104 ± 8.99 0.374 ± 2.6 0.645 ± 2.6
XI 5.02 7.97 ± 3.0 9.175 ± 9.72 2.02 ± 1.4 3.49 ± 1.4
In preliminary tests different extraction solvents and three QuEChERS
XII 3.78 25.3 ± 2.0 25.090 ± 2.48 2.71 ± 1.7 4.66 ± 1.7 were tested using soil I namely ACN, ACN–MeOH (60:40%, 50:50% and
XIII 7.05 12.1 ± 1.3 13.823 ± 1.07 0.785 ± 2.0 1.35 ± 2.0 40:60%, v/v), MeOH, n-hexane–acetone (50:50%, v/v), ethyl acetate,
XIV 6.76 36.1 ± 3.4 37.940 ± 1.31 0.545 ± 4.0 0.939 ± 4.0 and acetone; and QuEChERS 1, 2 and 3 (see Section 2.1 “Reagents,
XV 6.34 40.2 ± 4.4 38.808 ± 1.65 0.840 ± 3.5 1.45 ± 3.5
solvents and materials”). For blanks, 1 mL of pure ACN was added to
XVI 5.78 3.06 ± 1.1 3.399 ± 6.82 0.156 ± 6.8 0.269 ± 6.8
the soil, and for fortified soils (200 μg kg− 1 of each analyte) 1 mL of
X — Average, n — number of samples, n.d. — not detected, and RSD — relative standard working standard solution (1000 μg L− 1 of each analyte in ACN) was
deviation.
a
Soils I to V were used for extraction method development and validation.
added to 5.0±0.1 g of soil.
b
In all samples, IC content wasn't detected and therefore, the results of TC and OC All of the ACNs were evaporated with a gentle stream of nitrogen
are equal (OC = TC − IC, IC = 0 → OC = TC). to ensure that only the analytes (in the fortified soil) were in contact
284 I. Bragança et al. / Science of the Total Environment 433 (2012) 281–289
with the soil sample. A total amount of 10.0 mL of extraction solvent
and QuEChERS content was added.
The influence of extraction time was evaluated in soil I testing 1, 2,
3, 4, 5 and 10 min, using a vortex mixer as homogenization device.
The process of the addition of the QuEChERS to the Teflon centri-
fuge tube containing the sample was evaluated. The QuEChERS con-
tent was added: (i) immediately and (ii) slowly with simultaneous
homogenization. These studies were performed on soils III and V.
The performance of the QuEChERS extraction method for soils with
higher OC contents was carried out using soil II. The studied steps were:
(i) increased homogenization by including an ultrasound step, (ii) addi-
tion of water to the sample prior to the QuEChERS extraction and acid-
ification of the extraction solvent, and (iii) the study of QuEChERS 4.
After QuEChERS extraction, the extracts were centrifuged in a Sar-
torius 2.16 centrifuge (Sigma, Goettingen, Germany) during 10 min at
4000 rpm, the pH of the supernatant was measured, and an aliquot
(5 mL) was transferred to a dark vial and evaporated to dryness
with a gentle stream of nitrogen. The dried extract was dissolved in
500 μL of ACN and the resulting solution was shaken vigorously
using a vortex mixer and filtered through a 0.20 μm syringe filter.
The extract was then placed into an insert inside the vial.
For QuEChERS optimization only one extraction for each test was
performed. After the optimization, extractions were performed in
triplicate.
2.5. Method validation
Once the best conditions for the analysis of IBP, hydroxyIBP, and
carboxyIBP were optimized, the validation of the method was carried
out according to the procedure described below.
The calibration curves and linear ranges of the detector response
for IBP and the two metabolites were evaluated by analyzing working
standard solutions (75 to 2000 μg L − 1; n = 8) in triplicate. Another
calibration curve was constructed using fortified extracts with seven
concentration levels: 100, 250, 500, 750, 1000, 1250 and 1500 μg L − 1.
The method detection limits (MDLs) and method quantification
limits (MQLs) were determined using the standard deviation of the
linear regression (Miller and Miller, 2000).
The precision in terms of repeatability was evaluated by carrying
out the extraction and analysis of fortified samples (100, 200 and
300 μg kg − 1) using three replicates, injecting each extract in tripli-
cate. The accuracy of the analytical method was evaluated through re-
covery studies for all tested soils.
3. Results and discussion
3.1. Soil characterization
Each soil sample was characterized by analyzing pH, water con-
tent, TC, OC, IC, and OM. The results are summarized in Table 2. The
pH values ranged from 3.78 (soil XII) to 8.05 (soil X), water content
ranged from n.d. (soil V) to 40.2% (soil XV) using the oven (105 °C)
and from 0.291 (soil V) to 38.808% (soil XV) using the moisture ana-
lyzer. None of the soil samples' IC was detected, so the OC content
was obtained solely from TC. TC contents (humid base) ranging
from n.d. (soil V) to 4.71% (soil IV) and OM contents ranging from
n.d. (soil V) to 8.11 (soil IV) were obtained.
The amount of OM in surface, mineral soils can vary from less than
1% in coarse-textured, sandy soils to more than 5% in fertile, prairie
grasslands (Mitchell and Everest, 1995). According to the German
norm DIN 18196, sandy soils can be considered organic when they
contain over 3% of OM (Myslinska, 2003), as can be observed in soil
I. In the studied soils the OM content ranged from 1.14 to 8.11%
(humid base), obtaining the highest percentage for agricultural soils.
The water content, ranging from 2.63 (soil IX) to 38.8% (soil
XV), increased in samples taken in the proximity of watercourses.
Good correlation between the results using the oven (at 105 °C)
and the moisture analyzer was obtained (water content moisture
analyzer = 0.996 × water content oven + 0.3586 with a correlation
coefficient of 0.998).
3.2. Chromatographic optimization
The best separation was achieved with a linear gradient by varying
the mobile phase composition from 20–80% to 50–50% ACN–water
(pH 3.0, adjusted with acetic acid) in 20 min, after which the final
composition was maintained for 14 min. Initial conditions for the
next injection were reached within 1 min and maintained for 5 min
before the next run. The total time of analysis was 40 min at a flow
rate of 1.0 mL min − 1. Using the optimized experimental conditions,
the retention times of hydroxyIBP, carboxyIBP, and IBP were 10.52,
11.75 and 29.00 min, respectively.
3.3. Extraction procedure
Several parameters were studied to optimize the performance of the
extraction method, such as the ratio of mass of sample per volume of ex-
traction solvent, the extraction solvent, the QuEChERS composition, the
extraction time, the extraction process, and the addition of ceramic
pieces to prevent agglomeration. A previous work using the QuEChERS
method (Ramalhosa et al., 2009) established an optimized extraction
time of 3 min. In preliminary studies of the present work this time
was adopted, but afterwards this parameter was also studied.
3.3.1. Ratio of mass of sample per volume of solvent
A preliminary study was performed testing different ratios (1.0,
0.75 and 0.50) of mass (g) of sample per volume (mL) of extraction
solvent, using 10.0, 7.5 and 5.0 ± 0.1 g of soil, 10 mL of solvent, QuE-
ChERS 1, and an extraction time of 3 min. The best ratio was found
to be 0.50 because the most suitable dispersion and the best homog-
enization between the soil and the extraction solvent were obtained.
3.3.2. Extraction solvent and QuEChERS content
For the extraction of the desired analytes and to guarantee minimal
co-extraction of matrix interferences, the selection of an appropriate sol-
vent is a crucial point in the procedure (Anastassiades et al., 2003;
Lehotay, 2011). Usually the experiments start using the unbuffered origi-
nal approach (Lehotay, 2011). ACN has shown to offer excellent perfor-
mance for the extraction of the broadest range of compounds showing
the least interference (Correia-Sá et al., 2012; Fernandes et al., 2011;
Ramalhosa et al., 2009). If ACN does not provide adequate recoveries,
other solvents, depending on the residues to be extracted, can be
employed, namely: ethyl acetate, acetone and MeOH (Lehotay, 2011).
An n-hexane–acetone (50:50%, v/v) mixture already showed to be an
efficient solvent system for the extraction of different pollutants from
environmental samples (EPA Method 3546, 2000). As far as the authors
know, the QuEChERS method has not been tested using MeOH, ACN–
MeOH or n-hexane–acetone as extraction solvents for soils. For initial
tests, QuEChERS 1, 2, and 3 (see Section 2.1 “Reagents, solvents and
materials”) and different solvents were tested (Table 3): ACN, ACN–
MeOH (60:40%, 50:50% and 40:60%, v/v), MeOH, n-hexane–acetone
(50:50%, v/v), ethyl acetate, and acetone. As can be seen in Table 3,
QuEChERS 1 provided the lowest recoveries for almost all solvents,
however a good recovery using n-hexane–acetone (50:50%, v/v) was
obtained for IBP (98.3%) but with low recoveries (b35%) for the two me-
tabolites. No results were obtained with MeOH and the 50:50% and
40:60% (v/v) ACN–MeOH mixtures using QuEChERS 1 because in the
evaporation step a viscous solution was formed, which made it unsuitable
for injection. The use of QuEChERS 2 generally improved the extraction ef-
ficiency of the three analytes, but the highest recoveries were obtained
with QuEChERS 3. Comparing ACN, MeOH and the three ACN–MeOH ra-
tios, the best results were achieved for ACN–MeOH (50:50%, v/v) in
 I. Bragança et al. / Science of the Total Environment 433 (2012) 281–289 285

Table 3
Influence of extraction solvent and QuEChERS content on the recovery of hydroxyIBP, carboxyIBP, and IBP from fortified soil I at 200 μg kg− 1 (n = 1).

Extraction solvent QuEChERS 1 QuEChERS 2 QuEChERS 3

pHa % Recovery pHa % Recovery pHa % Recovery

HydroxyIBP CarboxyIBP IBP HydroxyIBP CarboxyIBP IBP HydroxyIBP CarboxyIBP IBP

ACN 9.04 23.3 n.d. 52.6 5.14 65.0 44.2 80.1 4.58 47.4 14.5 68.5
ACN–MeOH (60:40%, v/v) 7.83 7.14 19.3 16.4 5.10 50.6 32.7 69.4 3.44 55.7 51.9 66.5
b b b
ACN–MeOH (50:50%, v/v) 7.53 4.88 70.2 44.7 78.8 3.73 67.0 70.2 77.2
b b b
ACN–MeOH (40:60%, v/v) 8.25 5.03 47.0 40.7 67.3 3.68 61.6 37.5 67.0
b b b
MeOH 6.93 5.51 30.8 19.0 57.5 4.13 41.6 36.3 60.3
n-Hexane–acetone (50:50%, v/v) 8.22 34.1 6.05 98.3 5.41 55.8 6.50 75.1 3.63 52.9 15.8 78.9
Ethyl acetate 8.26 19.1 6.85 47.2 6.29 13.7 4.48 51.9 4.65 22.3 5.53 62.1
Acetone 9.34 17.9 3.74 34.5 7.06 35.7 14.9 60.9 4.50 45.2 43.3 64.0

n — number of samples.
a
pH value of the QuEChERS extract.
b
No results were obtained because in the evaporation step, a viscous gum-like solution was formed and the evaporation was not completed. Therefore, no injection was made
into the LC–FLD.

combination with QuEChERS 2 and QuEChERS 3. Concerning n-hexane–
acetone (50:50%, v/v), ethyl acetate and acetone, the best results for the
tested QuEChERS was obtained using n-hexane–acetone (50:50%, v/v).
For QuEChERS 1 increased recoveries were obtained for: acetoneb ethyl
acetateb n-hexane–acetone (50:50%, v/v) and for QuEChERS 2 and 3:
ethyl acetateb acetoneb n-hexane–acetone (50:50%, v/v).
The best overall results were achieved using ACN–MeOH (50:50%,
v/v) and QuEChERS 3, obtaining recoveries of 67.0, 70.2 and 77.2%, for
hydroxyIBP, carboxyIBP and IBP, respectively. In an attempt to evalu-
ate the effect of the soil matrix on the results, a QuEChERS extraction
without soil sample was performed. The obtained recoveries were
99.7, 99.0 and 103% for hydroxyIBP, carboxyIBP, and IBP, respectively.
and to ensure adequate partition in dry samples (cereals, dried fruits,
tobacco, teas, etc.) (UCT, 2008). Another parameter related to the addi-
tion of water is the pKa of the compounds to be extracted (pKa (IBP) =
4.31 and pKa of hydroxyIBP and carboxyIBP were not available
(Matamoros et al., 2008)). Attending to these parameters, several mod-
ifications in the extraction procedure were performed after fortification
of the soil sample: 3 mL of purified water (pH 2.5, adjusted with
hydrochloric acid), QuEChERS 3, and 7 mL of ACN–MeOH (50–50%, v/v)
(pH 2.5, adjusted with hydrochloric acid) were added. The obtained
recoveries were 49.4%, 51.6% and 46.7%, for hydroxyIBP, carboxyIBP,
and IBP, respectively. Thus, the addition of acidified water and acid-
ified ACN–MeOH (50:50%, v/v) improved the recoveries.

3.3.3. Extraction time 3.3.7. Combination of AOAC and EN 15662

The influence of the extraction time was studied for soil I using QuE-
ChERS 3 and ACN–MeOH (50:50%, v/v). The lowest recoveries for the
three compounds were obtained using an extraction time of 1 min
(the time used in the original QuEChERS (Anastassiades et al., 2003)).
Recoveries improved when the extraction time was increased from 1
to 4 min for hydroxyIBP, from 1 to 3 min for carboxyIBP and from 1 to
2 min for IBP, remaining constant thereafter. Because hydroxyIBP, car-
boxyIBP, and IBP are extracted simultaneously, and to ensure maximum
recovery of each analyte, the adopted extraction time was 4 min, apply-
ing the vortex mixer for homogenization.
3.3.4. Organic carbon content
It has been described that OC affects the recoveries of the QuEChERS
method (Correia-Sá et al., 2012). Using the optimized procedure for a
soil with a high OC content (soil II, OC = 3.12%), low recoveries were
obtained: 16.2% for hydroxyIBP (RSD = 4.9%, n = 4), 25.9% for car-
boxyIBP (RSD = 13%, n = 3), and 18.6% for IBP (RSD = 11%, n = 4).
3.3.5. Homogenization technique
Additional tests on soil II were performed to improve the extraction
of the three analytes. The first approach was the improvement of the
homogenization procedure. As mentioned in Section 3.3.3, recoveries
did not increase for extraction times longer than 4 min when the vortex
mixer was used. To improve the obtained results (Section 3.3.4), the ex-
traction mixture was placed, after vortex mixing, in an ultrasonic bath
for an additional 4 min. The recoveries increased to 34.2%, 36.5% and
37.9% for hydroxyIBP, carboxyIBP, and IBP, respectively. Therefore, all
further tests were performed using the ultrasonic bath after vortex
homogenization.
3.3.6. Addition of water to the soil and acidification of the extraction
solvent
The addition of water to the sample prior to the QuEChERS extrac-
tion is used to weaken interactions of the analytes with the matrix
The original QuEChERS method was performed in this work (using
QuEChERS 2) but low recoveries were achieved (Table 3). Some mod-
ifications of the original QuEChERS have already been described to
ensure efficient extraction of pH dependent compounds and to mini-
mize their degradation (EN 15662, 2007; Method AOAC, 2007). Using
the dispersive AOAC 2007.01 method, which includes 1% acetic acid
in ACN and CH3COONa buffer in the QuEChERS composition (QuE-
ChERS 1), the results (not shown), were comparable with the ones al-
ready obtained using QuEChERS 1 (Table 3).
The extraction method was further modified by combining the sol-
vent indicated in the AOAC 2007.01 method (1% acetic acid in ACN)
and the QuEChERS composition (including citrate salts) used in the
EN 15662 method. The procedure consisted of adding, to the soil II sam-
ple, 3 mL of purified water (pH 2.5, adjusted with hydrochloric acid),
7 mL of extraction solvent and QuEChERS 3. Good recoveries were
achieved for this modified method (Table 4). It was also observed that
the pH value of the QuEChERS extract was lower when the modified
method was used. The method was further modified by using QuE-
ChERS 4, obtaining recoveries close to 100% for two types of soils
(soils I and II) with high and low OC content.
3.3.8. pH of the extraction solvent
Considering that the pKa of the compounds is related to solvent
affinity, several tests were performed with pH adjustment of the pu-
rified water or/and ACN. The parameters optimized in the previous
sections were used to evaluate recoveries and the results are pres-
ented as Supplementary material (Table SM A.1).
Using ACN (10 mL), without adjusting the pH, low recoveries were
obtained (b40%), which increased when purified water (3 mL) was
added (b70%). Better results were observed when ACN (without pH
adjustment) and purified water at pH 2.5 (adjusted with hydrochloric
acid) was used (b79%). In the second group of extractions, ACN was
acidified (1% acetic acid) and recoveries increased (b96%) when
3 mL of purified water (acidified or not) was added. Therefore, the
286 I. Bragança et al. / Science of the Total Environment 433 (2012) 281–289
Table 4
Recoveries achieved using the original QuEChERS and original and modified AOAC 2007.01 and EN 15662 methods.
Experiment QuEChERS method QuEChERS Extraction solvent (10 mL)
1 Original QuEChERS QuEChERS ACN
2b
2 Original AOAC 2007.01 QuEChERS 1% acetic acid in ACN
1b
3 Original EN 15662 QuEChERS ACN
3b
4 Modified EN 15662/AOAC QuEChERS 3 mL of water (pH 2.5, adjusted with hydrochloric acid) and 7 mL of ACN
2007.01 3c with 1% acetic acid
5 QuEChERS
4c
6 QuEChERS
4b
a
pH value of the QuEChERS extract.
b
Soil I.
c
Soil II.
pH adjustment of ACN was sufficient and more important than the
acidification of the water. The best approach for QuEChERS extraction
was obtained using 3 mL of purified water (with or without adjusted
pH) and 7 mL of acidified ACN (1% acetic acid).
3.3.9. Application to different soils
The QuEChERS method was applied to five soils with different phys-
icochemical properties (OC = n.d. (soil V); OC= 0.659% (soil III);
OC= 2.00% (soil I); OC= 3.12% (soil II); and OC= 4.71% (soil IV)).
Tests were performed using the optimized procedure and the
highest recoveries, between 95.2 and 98.7%, were obtained for soils
I and II with RSD b 3% for all analytes. The low recoveries for soil IV
(highest OC) were probably due to the difficulty to extract the
analytes because of higher interactions with OM. The low recoveries
for soils III and V were due to another aspect, which is described in
the next sub-section.
3.3.10. Agglomeration
During extraction, vigorous homogenization is required, other-
wise poor consistencies and recoveries are obtained. During several
of the previous tests some agglomerates were formed, especially in
soils III and V, which compromised the extraction. The use of ceramic
pieces to facilitate sample homogenization and to break-up salt ag-
glomerates more efficiently has been described (UCT, 2008). There-
fore, extractions with the addition of ceramic pieces were performed
and the recoveries for each analyte increased slightly, but no significant
differences were observed (Supplementary material, Table SM A.2). To
avoid the formation of agglomerates the QuEChERS content was
added slowly and continuously. The centrifuge tube with soil and
3 mL of acidified purified water (pH 2.5, hydrochloric acid) was placed
in the vortex mixer, set at slow speed, and the QuEChERS content was
added. After the complete addition of the QuEChERS content, the vortex
was set at maximum speed and the mixture was homogenized for
4 min. Then, the tube was placed in the ultrasonic bath, and after
4 min, 7 mL of acidified ACN was added and the rest of the procedure
executed as described previously. The results are indicated in detail in
the Supplementary Material (Table SM A.2). Good recoveries were
obtained for all types of soils using the optimized procedure shown in
Fig. 1.
3.4. Method validation
3.4.1. Calibration curves
In this study calibration curves were constructed using (i) eight work-
ing standard solutions (concentrations between 75 and 2000 μg L− 1)
prepared in ACN, and (ii) six working standard solutions (concentrations
between 100 and 1500 μg L− 1) prepared in the blank QuEChERS extracts.
pHa % Recovery (n = 1)
HydroxyIBP CarboxyIBP IBP
5.14 65.0 44.2 80.1
6.46 8.67 4.37 24.7
4.58 47.4 14.5 68.5
3.62 68.6 77.7 75.1
3.12 94.1 95.2 95.6
3.10 98.3 98.2 97.9
To obtain the matrix-matched calibration curves, three soils (I (interme-
diate OC), III (low OC) and IV (high OC)) were analyzed and no residues
of the selected compounds were detected. Data of the calibration curves
as well the MDLs, MQLs and matrix effects are presented in the Supple-
mentary Material (Table SM A.3). As can be seen, the detector response
was linear for hydroxyIBP, carboxyIBP, and IBP in the studied ranges
with correlation coefficients higher than 0.999. Analyzing the matrix-
matched calibration curves, the obtained MQLs ranged from 6.10 (soil I)
to 22.4 μg kg− 1 (soil IV), from 14.6 (soil I) to 16.9 μg kg− 1 (soil IV), and
from 11.1 (soil IV) to 14.4 μg kg− 1 (soil III), for hydroxyIBP, carboxyIBP,
and IBP, respectively.
Matrix effects (MEs) were originally discussed by Tang and
Kebarle (1993) (Tang and Kebarle, 1993) and can lead to a significant
increase or decrease in the detector response because of the presence
of interferents which co-elute along with the analyte of interest in a
sample compared to a pure standard solution (Tang and Kebarle,
1993). Evaluation of MEs is required as part of quantitative method
development (Annesley, 2003). One way to evaluate the MEs is
through the comparison of the slopes obtained in the calibration
with matrix matched-standards with those obtained in calibration
with standards prepared in the solvent (Romero-Gonzalez et al.,
2011). Comparing the ratios of the slopes for the various calibration
curves (Supplementary material, Table SM A.3) no significant matrix
effects were observed (ratios between 0.971 and 1.06).
3.4.2. Precision and accuracy
To further validate the developed method, the precision, in terms
of repeatability, and the accuracy of the method were evaluated at
three concentration levels (100, 200 and 300 μg kg − 1) for five types
of soils (n ≥ 3) (Supplementary material, Table SM A.4). The results
of the recovery studies for soil IV were lower than for the other four
soils. This might be due to the relatively high OC content of this soil
(Table 2), which can affect the extraction efficiency of the compounds
under study. Based on the obtained results it can be concluded that an
increase of OC content leads to stronger adsorption of the analytes
and consequently results in lower extraction efficiencies. The adsorp-
tion capacity of soils is strongly governed by their sand-, clay- and or-
ganic matter contents (Garcia Pinto et al., 2010). The average
recoveries of all compounds at the three fortification levels ranged
from 97.0 to 101% for soil I, 93.3 to 97.2% for soil II and 79.5 to
87.8% for soil IV, while the recovery for soils with lower OM content
(soils II and V) varied from 89.0 to 97.0%. To evaluate the precision
of the method at least three replicate extractions were performed
for each fortification level of real soil samples (Table SM A.4), and
the relative standard deviations (RSD) were calculated. RSD values
were lower than 4% for all the soils at all spike levels, indicating
that the analytical procedure provides precise results. Analyzing the
 I. Bragança et al. / Science of the Total Environment 433 (2012) 281–289
Fig. 1. Scheme of the QuEChERS procedure for the extraction of hydroxyIBP, carboxyIBP, and IBP from soils. Description of the illustration. In this figure a detailed optimized
QuEChERS procedure for the extraction of IBP and its major metabolites from soils is shown.
results for each soil sample, no considerable percentage differences
were observed for the three fortification levels. Therefore, the results
obtained for the studied soils, which comprised a range of soil types,
could be extrapolated to most soils.
3.4.3. Application to soil samples
The developed method was applied to the analysis of sixteen dif-
ferent soil samples, ranging from agricultural soils to sediments (near
the river or WWTPs). Some samples contained impurities that may in-
teract with the analytes, affecting the detector's response. A fortified
sample (200 μg kg− 1) was always determined along with the sample
to check the accuracy and precision of the methodology. The recovery
for all soils was between 82.2 and 101% with relative standard devia-
tions lower than 4.0%. CarboxyIBP was detected at 46.1 μg kg− 1 in sam-
ple XV, which was collected in the proximity of a WWTP. The effluent of
this WWTP is discharged into the Uíma River. Fig. 2 shows the LC–FLD
chromatogram without and with fortification of this sample. Our results
suggest that this WWTP is not adequate to remove metabolites of IBP.
These results are according to the considerations of Madureira et al.
(2010), who concluded that WWTPs are a source of pharmaceutical re-
lease into the aquatic environments due to the fact that conventional
treatments are not specifically designed to degrade and/or remove
pharmaceuticals (Madureira et al., 2010). Furthermore, Weigel et al.
(2004) detected hydroxylated and carboxylated metabolites of IBP at
concentrations as high as 1.27 μg L− 1 in WWTP effluents and at a con-
centration of 7.0 ng L − 1 in seawater in Norway (Weigel et al., 2004).
These two metabolites of IBP were also detected in the WWTP effluent
and in the receiving river water in Sweden (Bendz et al., 2005).
After the administration of IBP approximately 90% is metabolized to
two major metabolites; hydroxyIBP and carboxyIBP (Ternes et al.,
2004). The major route of excretion is via the kidney, with 95% of IBP
being excreted in the urine, of which 35% is excreted as hydroxyIBP
(15% free, 20% conjugated), 51% as carboxyIBP (42% free, 9% conjugated)
and 9% as IBP (1% free, 8% conjugated) (APO-IBUPROFEN, 2007)
287
Therefore, as carboxyIBP has the highest percentage of excretion, it
is more likely to find carboxyIBP than hydroxyIBP and IBP in the
environment.
4. Conclusions
To our knowledge, this is the first analytical report in which QuE-
ChERS and LC–FLD were applied to the simultaneous determination
of IBP and its two major metabolites (hydroxyIBP and carboxyIBP)
in soil samples with very different physicochemical properties. The
proposed method has been validated and allows a reliable determina-
tion of IBP and its metabolites in soils with a wide range of OC con-
tent. The MQLs values were ≤22.4 μg kg − 1 for all target analytes.
Satisfactory validation parameters such as accuracy, precision, and
linearity proved fitness for purpose of the developed method. The
method was applied to sixteen real samples and only carboxyIBP
was detected at 46.1 μg kg − 1 in a sediment collected at 50 m of a
WWTP gate effluent.
The results of this study show that the QuEChERS methodology is
characterized by a streamlined procedure. Some complicated analyt-
ical steps employed in traditional methods have been replaced by
easier ones, achieving an efficient process which only requires 7 mL
of organic solvent for each extraction without any further clean-up
steps before LC analysis.
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.scitotenv.2012.06.035.
Acknowledgments
This work has been supported by Fundação para a Ciência e a
Tecnologia through grant no. PEst-C/EQB/LA0006/2011 and project
PTDC/ECM/103141/2008.
288 I. Bragança et al. / Science of the Total Environment 433 (2012) 281–289
Fig. 2. LC–FLD chromatograms corresponding to the analysis of soil XV without and with fortifications (200 μg kg− 1 of each analyte). Description of the illustration. In this figure the
chromatograms of sample XV (without and with fortification) using LC–FLD are shown.
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