American Journal of Analyt ical Chemistry, 2011, 2, 18-26
doi:10.4236/ajac.2011.21003 Published Online February 2011 (
Copyright © 2011 SciRes. AJAC
Rapid HPLC Method for Monitoring Relevant Residues of
Pharmaceuticals Products in Environmental Samples
Juana Rodríguez Flores, Ana Maria Contento Salcedo, Lorena Muñoz Fernández
Department of Analytical Ch emistry and Foods Technology, University of Castilla-La Mancha, Ciudad Real, Spain
Received September 3, 2010; revised December 20, 2010; accepted January 10, 2011
This work presents a multi-residue analytical method based on solid phase extraction (SPE) followed by
high-performance liquid chromatographic (HPLC) with diode array (DAD) detection for the simultaneous
determination of a group of pharmaceutical products that include ten antidepressants and three anticanceri-
genic in environmental samples (water and soil). Baseline separation of the studied compounds was obtained
on an ultrabase C18 (4.6 mm i.d. × 150 mm, 5 μm particle) column using acetonitrile:phosphate buffer pH 2.5
(35:65, v/v) as mobile phase with a flow rate of 1.5 mL/min. Different aspects including linearity, accuracy,
precision and detection and quantification limits were examined in order to validate the proposed method.
Detection limits between 1 and 50 ng/mL were obtained for all the target compounds. This method was ap-
plied to the analysis of environmental samples as waters and soils of different precedence. Prior, the HPLC
determination the samples were purified and enriched using SPE or liquid-liquid extraction (LLE) of the tar-
get compounds.
Keywords: High-Performance Liquid Chromatographic (HPLC), Breast Cancer, Antidepressant,
Environmental Samples
1. Introduction
The occurrence of residues of pharmaceuticals in the
aquatic environment has attracted considerable interest in
recent years [1]. The disposal of unused medication via
the toilet seems to be of minor importance but many of
the pharmaceuticals applied in human medical care are
not completely eliminated in the human body. Often they
are excreted only slightly transformed or even unchanged
mostly conjugated to polar molecules. These conjugates
can easily be cleaved during sewage treatment and the
original pharmaceutically active compounds (PhACs)
will then be released into the aquatic environment mostly
by effluents from municipal sewage treatment plants
(STPs). Several investigations have shown some evi-
dence that substances of pharmaceutical origin are often
not eliminated during wastewater treatment and also not
biodegraded in the environment [2]. Under recharge
conditions, residues of pharmaceutically active com-
pounds may also leach into groundwater aquifers. Thus,
they have already been reported to occur in ground and
drinking water samples from water works using bank
filtration or artificial groundwater recharge downstream
from municipal STPs.
The presence of PhACs from human medical care in
groundwater may, however, also be caused by other
sources such as landfill leachates [3-5] or manufacturing
residues [6]. Nowadays, and especially in the industrial-
ized countries, strong regulations and advanced manu-
facturing practices shall prevent such spills. In the past,
regulations were not as strong and in several cases the
release of production residues was either tolerated or
even accepted. But the occurrence of pharmaceutical
residues in the environment may also be caused by agri-
culture applying large amounts of PhACs as veterinary
drugs and feed additives in livestock breeding. Figure 1
shows possible sources and pathways for the occurrence
of PhAc residues in the environment.
The elimination efficiency of conventional wastewater
treatment does not provide a significant reduction of the
concentration of pharmaceuticals and their metabolites
before they are to re-enter the environment as literature
data prove. With the knowledge that wastewater treat-
ment plants discharge pharmaceuticals into surface wa-
Copyright © 2011 SciRes. AJAC
Figure 1. Scheme showing possible sources and pathways for the occurrence of pharmaceutical residues in the
aquatic environment.
ters like rivers and lakes [7,8] as well as into the sea
[9,10], strategies for an advanced elimination of these
pollutants became overdue.
However effluents from wastewater treatment plants
(WWTPs) can be considered one of the most important
sources of pharmaceuticals in the environment.
In the recent years, the presence of pharmaceuticals in
WWTPs effluents have been reported [11-16]. Concen-
tration of pharmaceuticals in the environment, their
temporary evolution and their possible synergic and an-
tagonist effects depend not only in the amount dis-
charged from WWTPs but also on the geographical area
and climate conditions. Because of that the study of the
concentration evolution of these pharmaceuticals, related
to climate conditions in the studied area, is necessary to
evaluate the risk of negative environment effects.
Accurate and sensitive methods are necessary for the
determination of pharmaceuticals in environmental sam-
ples, in order to, evaluate the amount of these compounds
that are being discharged into the aquatic environment.
HPLC-MS [12,13,17-22] and GC-MS [14,15,23] have
been used to determine pharmaceutical in water samples.
However, when analyzing highly contaminated samples,
such as wastewater, a suppression of the electrospray
ionization is likely to occur. Besides, these instruments
are still very expensive and consequently not widely dis-
tributed. On the contrary, almost, every laboratory has a
common HPLC system, thus our alternative proposed in
to apply HPLC with diode array detector to determine
pharmaceutical residues in environmental samples.
Antidepressants and anticancerigenic are class of phar-
maceuticals that is extensively used in industrialized
countries and they can be too environmental pollutants
and cause adverse effects on ecological systems.
So, in this paper, a method for routine determination
of ten antidepressants (fluvoxamine, fluoxetine, citalo-
pram, trazodone, paroxetine, sertraline, clomipramine,
imipramine, doxepine, venlafaxine) and three antican-
cerigenic used for breast cancer (letrozole, anastrozole,
exemestane) in environmental samples is proposed.
The method involves sample pre-treatment by solid
phase extraction (SPE) for water samples and analytical
determination by HPLC with diode array (DAD) detec-
The aim of this work was to provide an accurate, sen-
sitive and inexpensive alternative to the use of HPLC-
MS in the routine determination of these pharmaceuticals.
The proposed method has been conveniently validated in
waters of several precedence and soils.
2. Experimental
2.1. Chemicals and Reagents
All chemicals and solvents used were of analytical re-
agent grade. All reagents were from Panreac (Barcelona,
Clomipramine (CLO), citalopram (CIT) and fluvox-
amine (FLV) were supplied by Tocris. Imipramine (IMI)
and letrozole (LE) were kindly supplied by Novartis
Laboratories. Fluoxetine (FLX), paroxetine (PAR), tra-
zodone (TRA) and anastrazole (ANA) were purchased
from Sigma-Aldrich, Glaxosmithkline, Farma-Leporl and
Astrazeneca laboratories respectively. Exemestane (EXE)
and sertraline (SER) were supplied by Pfizer and doxepine
(DOX) was purchased from Farmasierra S.A.
Standard stock solutions were prepared by dissolving
the appropriate amount of the pure substance in 100 mL
to give a final concentration of 100 mg/L. LE and EXE
were dissolved with ethanol-water 50/50 (v/v); TRA,
CIT, FLV, IMI, VEN, ANA and FLX were dissolved
with Milli-Q water and DOX, CLO, PAR and SER with
methanol. All the solutions were stored under refrigera-
tion at 4˚C.
Working standard solutions were prepared daily by
dilution of the stock standard solution with Milli-Q wa-
Mobile phases and buffer solution were prepared from
analytical-grade-reagent Na2HPO4, NaH2PO4 and H3PO4
from Panreac (Barcelona, Spain) and HPLC-grade ace-
tonitrile from Panreac. The buffers and acetonitrile solu-
tions were filtered through 0.45 μm filters (HNWP mem-
brane filters). This type of membrane filter was pur-
chased from Millipore.
2.2. Chromatographic Conditions
A Thermo FinniganTM Surveyor® Plus HPLC system
with diode-array detector was utilized. The system was
monitored by means of a computer equipped with Chrom
Quest 5.0 software, which was used for all measurements
and data treatment. Compounds were separated on a 4.6
mm i.d. × 150 mm, 5 μm particle, ultrabase C18 reversed
phase column (Análisis Vínicos, Ciudad Real, Spain)
with acetonitrile and 70 mM phosphate buffer, pH 2.5
(35:65, v/v), as mobile phase. Isocratic elution was per-
formed at a flow rate of 1.5 mL/min. The volume injected
was 20 μL. Use of diode-array detection enabled extrac-
tion of chromatograms at different wavelengths. The op-
timization process was made monitoring the analytes at
230 nm and the validation procedure at 215 and 230 nm.
All the analyses were made by duplicate and peak areas
were used for the quantification.
2.3. Treatment of the Samples
The environmental samples objects of study were water
samples from different origin (tap, sea and wastewater)
and ground samples.
Water samples were collected using glass bottles pre-
rinsed with ultra-pure water.
Extraction phase-solid (SPE) was used. The SPE car-
tridges (Sep-pack Plus tC18, waters) were conditioned
using 5 mL of methanol and 5 mL of 10 mM phosphate
buffer solution (pH 7.0). The water samples were trans-
ferred to the SPE cartridges through a Teflon tube using
a vacuum manifold system (Supelco VisiprepTM Sep-
pack system, Madrid, Spain) coupled to a vacuum pump
(Millipore XF 54 23050).
The wastewater samples, prior to extraction were fil-
tered through 0.45 μm of size pore filters. Concerning
tap and sea water samples the pH was adjusted to 6.5
with HCl and only tap water samples was necessary to fit
the ionic strength to 50 mM by addition of NaCl.
After the conditioning step, water samples (50 mL)
were percolated through the cartridges at a flow rate of
10 mL/min. Only for wastewater samples, the loaded
cartridges were washed with 8 mL of 10 mM phosphate
buffer (pH 7.0) and 2 mL of methanol:water (30:70, v/v).
Finally, the elution was performed with 2 mL of metha-
The soil samples belong to the province of Ciudad
Real (Spain) and were collected using bottles of polyeth-
ylene. First, it was come to the breakage of aggregates
with a wood mortar. Later, the soil samples were intro-
duced in a furnace to dry them and afterwards were ex-
tended until the humidity balances with the one of the
atmosphere, removing from time to time. Finally these
samples were sifted with a mortar until reduce the size of
soil particles.
Next, 0.5 g of the soil samples was placed in a conical
bottom glass tube and 5 mL of methanol was added. Af-
ter, 10 min of vertical agitation the samples were centri-
fugated (5000 rpm, 10 min) and the supernatant was
transferred into another conical glass tube, this process
was repeated three times. Finally the extract was evapo-
rated under nitrogen stream and reconstituted with 5 mL
of methanol.
3. Results and Discussion
3.1. Optimization of Chromatographic
To optimize the chromatographic separation of the thir-
teen analytes studied, several of preliminary experiments
was performed testing different mobile phases consisting
of methanol, acetonitrile or mixture of both as organic
phase and different buffer solution at various concentra-
The optimal separation of 13 compounds studied was
achieved using an isocratic elution with 70 mM phos-
Copyright © 2011 SciRes. AJAC
Copyright © 2011 SciRes. AJAC
phate buffer (pH 2.5) and acetonitrile (65:35, v/v).
Elution with the same isocratic conditions at different
flow rates were made and optimal performance based on
a compromise between the speed, separation, efficiency,
peak width and column backpressure was obtained using
1.5 mL/min. In the same way, the effect of temperature
of the chromatographic column on the separation was
studied varying this parameter between 18 - 30˚C. A
tem- perature of column of 20˚C was found advantageous
over the others temperatures essayed in terms of better
resolu- tion between peak of the target compounds and
analysis time not too long. An example of a chroma-
tograms corresponding to a standard solution containing
3 mg/L of each studied compound is shown in Figure 2.
As can be seen very good separation was achieved in an
analysis time of 25 min.
3.2. Optimization of Extraction and
Preconcentration Procedure
Generally, the methodologies developed for the drugs
residue analysis in environmental samples includes ex-
traction and enrichment steps followed by chromato-
graphic determination of target analytes. In our cases,
SPE has been employed in order to eliminate possible
matrix interferences and enrich the studied compounds in
water samples. SPE was optimized by spiking 50 mL
aliquots of each water samples (tap, sea or wastewater)
with 3 mg/L of the drugs studied. In the case of waste-
water the samples were filtered before that the SPE was
optimized. So, preliminary experiments demonstrate that
this step not affect to analytes. Extraction of thirteen
drugs studied from water samples was performed in re-
versed-phase C18 cartridges, which were conditioned
prior to use with 5 mL of methanol followed by 5 mL of
10 mM phosphate buffer solution (pH 7.0). Then, 50 mL
of water sample at several pH and ionic strength were
slowly loaded into the conditioned cartridge. Once the
retention had been completed, the cartridge was submit-
ted to several washing steps depending the precedence of
water samples.
The best results were obtained when the pH water was
adjusted to 6.5 and only the loaded cartridges with waste-
water were necessary washed with 8 mL of 10 mM
phosphate buffer (pH 7.0) and 2 mL of methanol:water
(30:70, v/v). Finally, different solvents (acetone, metha-
nol and acetonitrile) were tested in order to elute the
analytes. Methanol was chosen because allows the best
extraction recoveries of most of the analytes studied.
A chromatogram corresponding to an extract of a
wastewater sample spiked with 500 ng/mL of the ana-
lytes studied is show in Figure 3.
Respect to the soil samples, the extraction procedure
of analytes was optimized using a sample obtained by
spiking 2 mg/L of the drugs studied over 0.5 g of soil
Several extraction procedures using different solvents
(acetone, methanol, water, 2-propanol, carbon tetrachlo-
ride) at several percentages were essayed and the recov-
eries them were calculated. The best results were ob-
tained using a volume of 5 mL of methanol for the ex-
traction of the analytes from the soil samples; this ex-
periment was repeated three times.
Figure 2. Chromatogram of a standard solution containing 3 mg/L of each compound, under the optimized
conditions, recorded at 215 nm.
Figure 3. Chromatogram corresponding: A) blank of wastewater B) extracts from wastewater analyzed
spiked with 0.5 mg/L for all analytes.
All the extraction procedures are exhaustively descri-
bed in the Experimental section.
3.3. Validation of the Method
The proposed method was adequately validated in all the
environmental samples object of study.
Validation was performed by measuring peak areas at
the wavelength of maximum absorbance of each of the
analytes, to maximize sensitivity. The wavelength used
was 230 nm for trazodone, citalopram, doxepine, par-
oxetine, fluvoxamine, imipramine, fluoxetine, sertraline
and clomipramine, and 215 nm for venlafaxine, anastro-
zole, letrozole and exemestane.
In order to evaluate the precision of the proposed
method within-laboratory repeatability and reproducibil-
ity were estimated. To ensure correct quantification of
studied analytes, in the environmental samples a spiked
extract at 0.12 mg/L of each sample was injected nine
times in the same day and nine times in different days.
Comparison of the two sets of data was carried out by
applying the Snedecor F-test on relative standard devia-
tion (RSD) values obtained for migration times and peak
In terms of repeatability, it is remarkable that all the
relative standard deviations were lower than 3.95% for
peak areas and 0.89% for retention times. In terms of
reproducibility, the comparison of the averages by means
of the Snedecor F-test did not provide any significant
difference between both series for a signification level of
0.05 (n = 18).
Limits of detection (LOD) and limits of quantification
(LOQ) were calculated using the maximal sensitivity
allowed by the system and calculating the standard de-
viation (SD) of this response. LOD and LOQ were esti-
mated by multiplying the SD of blanks by a factor of 3
and 10, respectively. Under these conditions LODs and
LOQs obtained were subsequently validated separately
by the analysis of six standards prepared at their respect-
tive concentrations of all the compounds.
The linearity in the response was studied using ma-
trix-matched calibration solutions prepared by spiking
environmental samples extracts at six concentration lev-
els for every compound, ranging from 35 to 1500 ng/mL
in the samples. The linear regression equations were
calculated using the least-squares method and coeffi-
cients of correlation values higher than 0.99 were ob-
tained. In Table 1 analytical parameters obtained with
the proposed method for the analysis of studied com-
pounds in wastewater are shown. Similar results were
obtained for the other environmental samples studied.
In order to test the accuracy of the proposed method
the environmental samples object of study were spiked
with studied compounds at several concentrations levels.
These samples were analysed using the extraction and
chromatographic procedure optimized in this work. Sig-
nals obtained from spiked samples were compared with
the peak areas obtained by injecting standard solutions
directly. Recoveries obtained from soil and wastewater
samples spiked at several concentration levels are shown
in Table 2. As can be seen, good recoveries ranged be-
tween 85 and 100% were obtained for wastewater. Simi-
lar results were achieved when tap and sea water were
analysed. However, with the extraction procedure opti-
mized in this work, poor extraction recoveries of citalo-
pram were obtained in soil samples. Whereas recoveries
Copyright © 2011 SciRes. AJAC
Table 1. Analytical parameters obtained with the propose d method for the analysis of studied compounds in wastewater.
%RSD; (n = 9) Reproducibility
%RSD; (n = 18)
Compounds Equation r2 LOD
(ng·mL–1) LOQ
(ng·mL–1) Tr* PA** Tr* PA**
VEN Y = 66.1X + 102.9 0.9992 10.0 30.0 0.47 0.77 1.68 1.11
TRA Y = 930.0X 44.1 0.9983 1.0 3.0 0.43 0.36 1.99 0.57
CIT Y = 214.8X + 80.9 0.9997 10.0 30.0 0.49 1.79 2.72 1.82
DOX Y = 255.9X + 80.8 0.9993 8.0 24.0 0.74 0.93 2.81 1.04
PAR Y = 30.8X 57.1 0.9980 10.0 30.0 0.77 2.02 3.29 2.37
FLV Y = 109.4X 9.9 0.9986 10.0 30.0 0.59 0.60 3.20 2.18
IMI Y = 511.8X + 63.7 0.9984 9.5 28.5 0.85 1.39 3.38 1.41
ANA Y = 242.1 + 191.7 0.9995 7.0 21.0 0.30 0.75 1.88 1.55
LE Y = 171.1X + 46.9 0.9994 5.0 15.0 0.30 0.92 1.85 2.82
FLX Y = 49.6X + 25.9 0.9994 10.0 30.0 0.82 3.95 3.83 3.99
SER Y = 144.7X 20.8 0.9996 10.0 30.0 0.89 1.86 3.86 2.68
CLO Y = 226.1X + 116.9 0.9990 40.0 120.0 0.81 1.15 4.02 2.96
EXE Y = 127.6X + 187.5 0.9961 50.0 150.0 0.48 1.10 2.31 1.25
Table 2. Recoveries obtained for wastewater and soil.
Recovery (%) Recovery (%) Recovery (%) Recovery (%)
Analyte Added
(ng·mL–1) Waste
water Soil Added
(ng·mL–1) Waste
water Soil Added
water Soil Added
(ng·mL–1) Waste
water Soil
VEN 35.0 94.5 59.1 50.0 101.5 60.875.0 91.7 60.2 100.0 93.3 58.3
TRA 35.0 89.5 85.2 50.0 104.1 80.675.0 101.2 82.3 100.0 102.4 84.1
CIT 35.0 98.5 8.1 50.0 100.2 7.1 75.0 101.3 8.2 100.0 98.6 7.7
DOX 35.0 98.0 71.2 50.0 105.9 76.175.0 103.4 76.9 100.0 105.8 77.6
PAR 35.0 93.5 96.0 50.0 99.5 62.975.0 103.1 61.2 100.0 104.4 59.6
FLV 35.0 99.6 60.7 50.0 94.9 61.275.0 97.1 59.8 100.0 103.9 55.9
IMI 35.0 92.2 71.3 50.0 98.2 70.475.0 101.2 69.9 100.0 103.6 65.5
ANA 35.0 99.0 90.4 50.0 101.5 93.575.0 93.7 92.8 100.0 104.2 90.5
LE 35.0 91.1 93.5 50.0 97.6 90.875.0 94.1 91.2 100.0 100.8 94.8
FLX 35.0 96.8 78.8 50.0 102.6 80.175.0 98.7 79.8 100.0 91.0 80.6
SER 35.0 99.6 96.6 50.0 102.8 97.375.0 96.9 85.4 100.0 102.2 88.5
CLO 160.0 103.1 97.5 200.0 102.2 100.2300.0 92.8 99.1 500.0 100.2 97.5
EXE 160.0 97.4 94.9 200.0 98.5 92.6300.0 104.6 96.3 500.0 102.9 95.7
of the others studied drugs were in the ranged from 59%
to 103%.
The selectivity of the method was verified and not in-
terferences were found at the retention times of the 13
analyzed drugs. As can be seen in the Figures 3, 4 and 5
where are shown the chromatograms corresponding to
two different extracts from blank waters and the last one
from soil extract. In these figures is possible to see the
Copyright © 2011 SciRes. AJAC
Figure 4. Chromatogram corresponding: A) blank of seawater B) extracts from seawater analyzed spiked
with 0.5 mg/L for all analytes.
Figure 5. Chromatogram corresponding: A) blank of soil B) extracts from soil analyzed spiked with 2 mg/L
for all compounds.
differences after an spiked of different amounts of our
drugs in waters (0.5 mg/L) and soil (2 mg/L). The selec-
tivity was also determined, by measurement of peaks
homogeneity using the techniques of normalization and
comparison of spectra from different peak sections and
absorbance measures at two wavelengths [24]. Both te-
chniques proved again to have a high level of purity of
the peak corresponding to the studied compounds in all
the samples. In conclusion the proposed method showed
a good selectivity for the environmental samples chosen
for our analysis.
4. Applications
To demonstrate the applicability of the described method
in this work, several environmental samples as water of
different precedence (tap, sea and waste) and soils be-
long to different zones from the province of Ciudad Real
Copyright © 2011 SciRes. AJAC
were analyzed. The water samples analysed were sam-
ples in different weather stations. All the samples were
fortified with ranged between 35 and 1000 ng/mL con-
centrations of studied compounds and submitted to the
analytical procedure described for each case in this work.
The recoveries obtained in all the water samples ana-
lyzed were ranged between 90 and 109% for all com-
pounds studied.
Chromatogram of a sea water sample fortified with
500 ng/mL of drugs studied is shown in Figure 4 as ex-
As can be observed in the figure the high selectivity
provided by the proposed method allows a reliable iden-
tification of the target compounds in wastewater and
obtaining similar results in all the water samples ana-
Respect to soil samples analyzed, good recoveries
were obtained (ranged between 59 and 103%) for all the
target compounds except for citalopram that was around
A chromatogram corresponding to a spiked soil sam-
ple is shown in Figure 5. Good selectivity was observed
too, in this type of samples.
5. Conclusions
In the present work, a simple and fast multi-residue
method based on SPE step followed by an HPLC-DAD
determination has been developed for the simultaneous
extraction and analysis of ten antidepressants (fluvox-
amine, fluoxetine, citalopram, trazodone, venlafaxine,
paroxetine, doxepine, sertaline, imipramine and clomi-
pramine) and three aromatase inhibitors (letrozole, an-
astrazole and exemestane) in environmental samples. It
has been shown that this method represents an easy and
fast analytical approach, viable for routine analysis, us-
ing instrumental very simple, available in almost every
The proposed method was exhaustively validated in
terms of linearity, accuracy, specificity and precision in
environmental samples. Quantitative recoveries were ob-
tained for all the target compounds ranging from 59 and
103% except citalopram determination which is ex-
tracted from soil samples with recoveries around of 8%.
Linearity with R2 > 0.994 and precision of with the RSD
(%) between 0.30% and 3.99% was very satisfactory.
The SPE step optimized allows not only the elimina-
tion of hydrophobic interferences but also an important
sample preconcentration which results in LOD(s) in wa-
ter samples between 1 and 50 ng/mL. HPLC-DAD is an
inexpensive analytical technique compared to HPLC-MS
and, because of this is a useful and affordable alternative
to HPLC-MS for routine analysis of pharmaceuticals. It
can be a useful tool to known the amount of these com-
pounds discharged from wastewater treatment plants
(WWTPs) to the aquatic environment and to evaluate the
effect of WWTPs in the elimination of pharmaceutical
In conclusion, methods are being developed to en-
hance the capabilities for measuring emerging chemical
contaminants and their associated degradation products
in the environment. Therefore, prioritization of com-
pounds investigated as the analyzed drugs requires care-
ful evaluation of the potential for their environmental
occurrence and persistence, potential health effects, and
the appropriate level at which they should be measured.
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