International Journal of Environmental Analytical

Chemistry

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Combined method of solid-phase extraction and

GC-MS for determination of acidic, neutral, and
basic emerging contaminants in wastewater
(Poland)

Katarzyna Nosek, Katarzyna Styszko & Janusz Golas

To cite this article: Katarzyna Nosek, Katarzyna Styszko & Janusz Golas (2014) Combined
method of solid-phase extraction and GC-MS for determination of acidic, neutral, and basic
emerging contaminants in wastewater (Poland), International Journal of Environmental Analytical
Chemistry, 94:10, 961-974, DOI: 10.1080/03067319.2014.900680

To link to this article: https://doi.org/10.1080/03067319.2014.900680

Published online: 04 Apr 2014.

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Intern. J. Environ. Anal. Chem., 2014
Vol. 94, No. 10, 961–974, http://dx.doi.org/10.1080/03067319.2014.900680

Combined method of solid-phase extraction and GC-MS for determination

of acidic, neutral, and basic emerging contaminants in wastewater (Poland)
Katarzyna Nosek, Katarzyna Styszko and Janusz Golas*

Department of Coal Chemistry and Environmental Sciences, AGH University of Science and Technology,
Al. Mickiewicza 30, 30-059 Krakow, Poland
(Received 15 November 2013; final version accepted 27 February 2014)

In this study an analytical procedure of solid-phase extraction (SPE) followed by gas

chromatography mass spectrometry (GC-MS) was elaborated and validated for simultaneous
determination of 11 acidic, neutral and basic emerging contaminants in wastewater. The most
frequently used pharmaceuticals were studied, i.e. five anti-inflammatory drugs – ibuprofen,
diclofenac, ketoprofen, naproxen and salicylic acid, an antiepileptic drug carbamazepine,
clofibric acid, antibacterial triclosan, a plasticiser bisphenol A and two β-blockers – propra-
nolol and metoprolol. Sample enrichment was performed using Oasis HLB sorbent. Sample
pH and sorbent washing step during the solid-phase extraction were optimised on real
wastewater samples. Recoveries of the most polar acidic compounds diminished substantially
when the alkalinity of the sample loaded into the cartridge increased. Thus finally wastewater
was extracted at pH 2.0. Before elution, sorbent was washed subsequently with 5% methanol
in water and n-hexane, which resulted in best recoveries of most of the target compounds and
reduced a co-elution phenomena with respect to β-blockers. The optimised method was
successfully applied to influent and effluent samples from wastewater treatment plant,
Krakow, Poland. All target compounds except propranolol were identified in wastewater at
a concentration up to 12.8 µg L−1.
Keywords: emerging contaminants; gas chromatography-mass spectrometry; GC-MS; HLB
sorbent; solid-phase extraction; SPE; pharmaceuticals; wastewater

1. Introduction
In recent years, more and more attention has been given to the presence of pharmaceutical
residues as emerging contaminants in the aquatic environment and their potential impact on
organisms involved in the water cycle. These contaminants became a topic of particular
concern, owing to their specific chemical properties and their continuous flow into water bodies,
principally by wastewater discharges [1,2]. The most frequently identified pharmaceuticals in
the aquatic environment are steroids, analgesics and anti-inflammatory drugs, antibiotics,
β-blockers, lipid regulators, antiepileptic drugs, contrast media [3–5]. They have been recog-
nised at low concentrations, below μg L−1 level, in sewage effluents, surface waters and even in
drinking water [6–11]. As biologically active compounds, these pharmaceuticals may have the
ability to cause adverse effects on non-target organisms. The toxicology of pharmaceutical
contaminants has not been comprehensively studied yet, but first evidences of their harmfulness
have been revealed. For instance, prolonged exposure to diclofenac at µg L−1 concentration
leads to histopathological organ lesion of rainbow trout and bioaccumulation [12], psychoactive
compounds affect sexual and nervous functions, as well as the hormonal system [13]. Special

*Corresponding author. Email: jgolas@agh.edu.pl

© 2014 Taylor & Francis

962 K. Nosek et al.

attention has been given to hormones and endocrine disruptors which lead to fish feminisation,
and antibiotic agents which develop resistance of pathogens at very low concentration [13–15].
Due to their high water solubility pharmaceuticals are able to circulate in surface water and
leach into groundwater, thus they may cause a potential risk to drinking water consumers [16].
Risk assessment related to pharmaceuticals as environmental contaminants is a problematic
process in terms of chronic life-long exposure, low exposition and possible synergic impact of
pharmaceutical mixtures as well as transformation products. To that end, a detailed multi-
purpose studies are required, starting with development of sensitive and selective methods for
determination of pharmaceuticals in water samples.
So far, many individual analytical methods for pharmaceutical determinations in an aqueous
environment have been developed and described in the literature [17–20]. Recently, attention
has been paid to simultaneous analysis of pharmaceuticals belonging to different therapeutic
groups [21–23]. Obtaining the most suitable method for all diverse chemical compounds
requires a compromise, but it results in a shorter overall analysis time, reduced sampling and
lower cost. These factors make the whole procedure more practicable for routine analysis, on the
understanding that pharmaceutical contaminations might lead to enforcement of future legisla-
tion. The starting point for multi residue analysis is the development of an effective sample
clean-up procedure. As of today the most popular and suitable technique for rapid and selective
sample preparation used in pharmaceuticals determination in water bodies is Solid-Phase
Extraction (SPE) [24,25].
Extraction efficiency, described usually as an ‘analyte recovery’, can be affected by several
variables such as sample pH, type of sorbent and type of solvent used, sample volume loaded to
the SPE cartridge. Recently, polymeric sorbents became widespread in pharmaceuticals extrac-
tion from environmental samples. In comparison to popular silica phases (octadecyl C18, octyl
C8), polymeric materials are stable in the entire range of pH and demonstrate better wetting
characteristics for larger loading capacities. Polymeric sorbents can offer high surface area and
thus higher retention capabilities of analytes [26,27]. Among many commercially available
sorbents, hydrophilic-lipophilic-balanced Oasis HLB is the one of the most commonly used
polymeric sorbents for simultaneous extraction of acidic to basic pharmaceuticals including
the neutral ones [27–29]. Protonation and deprotonation of compounds should be suppressed
before SPE process to ensure hydrophobicity of analytes, thus, it is recommended to pre-
concentrate acidic pharmaceuticals under acidic conditions opposite to basic ones. As expected,
extraction efficiency of neutral compounds should not be influenced by variations in pH [27],
although some authors found extraction of neutral carbamazepine pH dependent [30,31]. For
simultaneous determination of acidic, neutral and basic compounds, the effect of pH was
studied with the range of 1.0–12.0. Most often, best recoveries were obtained at pH 7.0 using
HLB sorbent [29,32]. Neutral pH has several advantages: simple and quick sample
processing, no enhanced risk of acidic hydrolysis of susceptible analytes and significant reduc-
tion of co-extraction of matrix components (humic, fulvic acids) in comparison to pH 2.0 [27].
Nowadays, a liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) is
the most preferable choice for the determination of pharmaceuticals in complex environmental
matrices, however gas chromatography coupled with mass spectrometry (GC-MS) is still widely
used technique, owing to a widespread availability of GC-MS instruments in environmental
laboratories, and also due to the signal suppression phenomena seen during analysis in liquid
chromatography system [23]. Because of the low volatility of polar pharmaceuticals a deriva-
tisation step is required before GC-MS analysis.
Although a lot of analytical methods were proposed so far, there are many inconsistencies
among the results of different authors, thus further studies on analytical procedures for pharma-
ceuticals determination are still necessary and required. The authors of this study aim to develop
 International Journal of Environmental Analytical Chemistry 963

a sensitive and simple analytical method, permitting the simultaneous determination of 11

compounds (mainly pharmaceuticals) with acidic, neutral and basic character in wastewater at
the ng L−1 level. The analytical procedure includes a solid-phase extraction following by
GC-MS detection. Elaboration of solid phase extraction conditions were begun with adapting
existing analytical procedures described in the literature. After the own optimisation studies the
SPE methods were modified and adjusted to the group of target compounds. All experiments
were performed on real wastewater samples in order to include possible matrix effects and make
analysis more reliable. Further, the established analytical procedure was checked thoroughly in
order to evaluate the method performance and consider its future application for the control of
pharmaceuticals introduced to aquatic environment. So far very few papers on the presence of
drugs in Polish water bodies has been published [33–38]. Except several preliminary studies
[39,40], such a research will be a novel for the Polish waters and for Krakow in particular but it
also has several significant more general aspects. Krakow is the second Polish city in terms of
size and population and huge load of diverse contaminants is continuously discharged with
effluents to surface water. Contaminants which are the subject of this work are the most
frequently used drugs and afterwards disposed to the aquatic environment. These include anti-
inflammatory ibuprofen, diclofenac, ketoprofen, naproxen, and salicylic acid, clofibric acid –
metabolite of the lipid regulators (clofibrate, etofibrate, and etofyllinclofibratelipid), β-blockers
– propranolol and metoprolol, antibacterial triclosan, antiepileptic carbamazepine and plasticiser
bisphenol A (Table 1). They represent a wide range of organic chemicals with diverse physi-
cochemical properties – polarity, water solubility, acidity and alkalinity [4,41].

2. Experimental
2.1 Materials and chemicals
Reference standards were of the following origins: ibuprofen, diclofenac sodium, ketoprofen,
naproxen, metoprolol tartrate and carbamazepine from Sigma-Aldrich (USA), salicylic acid and
bisphenol A from Dr. Ehrenstorfer (Germany), clofibric acid and propranolol hydrochloride
from Acros (USA) and triclosan from Merck (Germany). 1-hydroxypiren, used as an internal
standard was obtained from Sigma-Aldrich (USA). Individual stock solutions (1000 µg mL−1)
of tested compounds were prepared by dissolving a 10 mg of each compound in ethyl acetate
(with the exception of β-blockers which were dissolved in methanol). Working solution of
10 µg mL−1 was prepared in ethyl acetate. All standard solutions were stored at 4°C.
All solvents used during the entire analytical procedure were of analytical grade of high
residue purity: methanol, n-hexane and hydrochloric acid were of POCH (Poland), ethyl acetate
was of Merck (Germany), ultrapure water was obtained from an HLP5 system (Hydrolab,
Poland).
Glass-fibre filter papers GF-4 and GF-1 (Ø = 45 mm) used in samples pre-treatment were of
Macherey-Nagel (Germany).
For solid-phase extraction Oasis HLB 60 mg from Waters (USA) was used.
Silylating agent, N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) was of
Sigma-Aldrich (USA).

2.2 Gas chromatography

The chromatographic separation and analysis were performed with HP 5890 series II gas
chromatograph equipped with a split/splitless injector, HP-5MS capillary column
 964 K. Nosek et al.

Table 1. Characteristics of tested compounds.

M, g log Water solubility,

Therapeutic class Compound Structure mol−1 pKa KOW g L−1, 25°C

Non-steroidal anti- salicylic acid 138.12 2.97 2.26 2.2

inflammatory (SAL)
drugs (NSAIDs)

ibuprofen 206.29 4.91 3.97 0.021

(IBF)

diclofenac 296.16 4.15 4.51 0.002

(DCF)

ketoprofen 254.29 4.45 3.12 0.051 (22°C)

(KET)

naproxen 230.27 4.15 3.18 0.016

(NPX)

Lipid regulators clofibric acid 214.65 3.18 2.57 0.58

metabolite (CLA)

β-blockers propranolol 295.81 9.5 0.74 3.0

(PRO)

metoprolol 267.37 9.6 1.69 4.7

(MET)

Antiepileptic drugs carbamazepine 236.28 13.9 2.45 0.017

(CBZ)

Antibacterials triclosan (TRC) 289.55 7.8 4.76 0.01 (20°C)

Endocrine bisphenol A 228.29 9.6 3.32 0.12

compounds (BPA)

Source: http://chem.sis.nlm.nih.gov/chemidplus/chemidheavy.jsp, http://www.syrres.com/what-we-do/databaseforms.aspx?id=386

 International Journal of Environmental Analytical Chemistry 965

(30 m × 0.25 mm i.d., 0.25 µm film thickness, 5% phenyl 95% dimethylpolysiloxane stationary
phase) and HP 5971 quadrupole mass selective detector (Agilent Technologies, USA).
The injection port and transfer line temperature were set to 250ºC and 260ºC respectively.
The GC oven temperature started at 70ºC and was held at 70ºC for 2 min. After this time
temperature was elevated to 280ºC at a heating rate of 15ºC/min and held at 280ºC for 1 min.
Samples in 1 µL aliquots were manually injected in splitless mode and then carried to the
column by helium gas at a constant flow rate of 1 mL/min. 1-hydroxypyrene was applied as an
internal standard to control sample volume during manual injection.

2.3 Mass spectrometry

Identification and quantification of analytes were performed in the selected ion monitoring
(SIM) mode based on time retention and the characteristic ions of target compounds (Table 2).
In order to improve their validation parameters the MS ions were grouped and analysed
separately. The electron impact ionisation potential was set to 70 eV. Mass spectrometer started
to detect in 9 min (solvent delay) of the total run time. All compounds were successfully
separated in 17 min.

2.4 Sample collection

Wastewater samples were collected from Plaszow Wastewater Treatment Plant, Krakow, Poland.
The raw wastewater passes through conventional treatment processes, including pre-treatment
step followed by a secondary activated sludge treatment. It treats approximately 165 000 m3 of
urban wastewater per day, which is over 70% of the total volume of the city’s wastewater.
Effluents discharged to the Drwina river, which is a discharge channel to the Vistula river.
Wastewater samples were drawn before and after all treatment stages, in June 2012. Equal
aliquots of wastewater were grabbed every hour over a 24 hour period, collected in PE-HD
bisphenol A free containers with UV protection and stored at 4ºC until collection process was
finished. Then, the samples were transported to the laboratory and processed instantly. In the
first step, before the solid-phase extraction, wastewater was homogenised and filtered with glass
fibre filters to remove any particulate matter.

Table 2. GC-MS data: retention time, m/z ratio of characteristic ions of target
compounds, instrument detection limit (IDL).

Retention time m/z for characteristic ions (for IDL, pg

Compound (min) quantification) injected

Clofibric acid 9.90 143, 271 119

Ibuprofen 10.15 263, 161 29
Salicylic acid 10.86 309, 195 1
Metoprolol 12.86 72, 324 32
Naproxen 12.94 287, 185 2
Triclosan 13.25 347, 200 56
Propranolol 13.46 72, 316 47
Ketoprofen 13.67 311, 295 32
Carbamazepine 14.01 193, 293 31
Diclofenac 14.23 352, 214 16
Bisphenol A 15.05 207,441 21
1-hydroxypyrene 15.60 275, 332 −
966 K. Nosek et al.

2.5 Solid-phase extraction

The authors initially adapted the SPE procedure for six acidic and neutral pharmaceuticals
presented by Santos et al. [42]. Reproduction of this method did not give satisfactory results.
Thus, the procedure was modified and adjusted to the group of target compounds taking into
account our previous experience and the results of optimisation experiments.
Optimisation of the solid-phase extraction was conducted on effluent samples including all
target analytes of different characteristic. The influence of the pH value on extraction efficiency
was tested (pH 2.0, pH 7.0, pH 9.0) in order to compare to the results of the literature studies
and to find the most suitable extraction method for every presented group of compounds. After
pH optimisation a 5:95 (v/v) solution of methanol in water and n-hexane as a washing solvents
were tested. This experiment was triplicated at a concentration level of 1 µg L−1. Finally five
different SPE combinations were studied using a 60 mg HLB cartridge (Table 3). After elution
with 3 mL ethyl acetate the eluates were evaporated to dryness.

2.6 Derivatisation
To increase volatility and stability of analytes, silylation with the N-(tert-butyldimethylsilyl)-N-
methyltrifluoroacetamide (MTBSTFA) was performed before analysis. Silylation is the prevalent
type of derivatisation used in GC-MS analysis of hydroxyl compounds. Therefore, dry residues of
wastewaters were dissolved in 50 µL of MTBSTFA. Samples were heated at 65ºC for 40 min
using digital dry bath thermo-block Labnet (USA) and subsequently analysed in GC-MS system.

2.7 Blank measurements

Before analysis of target compounds in wastewater, blank measurements were applied for
identification of possible impurities of the eluent and derivatisation reagent and chromato-
graphic processes. To this end 3 mL of eluent reagent was evaporated to dryness and the dry
residue was derivatised under the same conditions as the wastewater samples. Several impurities
were found in the blanks, at salicylic acid and triclosan retention time at a concentration levels
less than MDL values. Nevertheless, blank corrections were taken into consideration in the final
calculations of analytes concentrations in wastewater.

Table 3. Tested SPE conditions.

Method Conditioning Loading Washing

1. 3 mL ethyl acetate, 500 mL effluent, pH 2.0 3 mL methanol:water 5:95 (v/v),

3 mL methanol, 3 mL n-hexane
3 mL deionised water, pH 2.0.
2. 3 mL ethyl acetate, 500 mL effluent, pH 7.0 3 mL methanol:water 5:95 (v/v),
3 mL methanol, 3 mL n-hexane
3 mL deionised water, pH 7.0.
3. 3 mL ethyl acetate, 500 mL effluent, pH 9.0 3 mL methanol:water 5:95 (v/v),
3 mL methanol, 3 mL n-hexane
3 mL deionised water, pH 9.0.
4. 3 mL ethyl acetate, 500 mL effluent, pH 2.0 3 mL methanol:water 5:95 (v/v)
3 mL methanol,
3 mL deionised water, pH 2.0.
5. 3 mL ethyl acetate, 500 mL effluent, pH 2.0 no washing
3 mL methanol,
3 mL deionised water, pH 2.0.
 International Journal of Environmental Analytical Chemistry 967

3. Results and discussion

3.1 Role of sample pH
Results of pH optimisation study are shown in Figure 1.

3.1.1 Acidic compounds

Extraction efficiency of pharmaceuticals with acidic character is strongly pH dependent. It was
expected that acidic compounds would obtain best results at pH 2.0 and it became especially
observable for the most polar ones (low octanol water partition coefficient, Kow): salicylic acid,
clofibric acid. Recoveries of analytes with carboxyl group are satisfactory at pH 2.0 and they
ranged from 57% for diclofenac to 98% for naproxen. These results correspond to those
obtained by Rodriguez et al. [18] under comparable SPE conditions for ibuprofen, naproxen,
clofibric acid and in a measure for ketoprofen. Similar recoveries were also reported by Öllers
et al. [43] for ibuprofen, clofibric acid, ketoprofen, naproxen, diclofenac. In this study increase
of alkalinity of water sample to pH 7.0 resulted in that no relevant recoveries of clofibric acid
and salicylic acid were observed, recovery of diclofenac was reduced more than halved,
recovery of ibuprofen was reduced about 40%, whereas recoveries of naproxen and ketoprofen
did not suffer much when sample pH was adjusted to 7.0. These results do not fully correspond
to Santos work who found recoveries of diclofenac, ibuprofen at acidic pH similar to that
obtained at neutral pH [42]. Significantly different results were presented by Togola et al. [30],
where ketoprofen, ibuprofen, naproxen and diclofenac did not recovered at all from HLB
sorbent at pH 7.0.

3.1.2 Neutral and phenolic compounds

It was shown that the extraction efficiency of neutral carbamazepine did not vary significantly
under three tested pH conditions. The recoveries were very successful as they ranged from 91%
to 100%. Santos et al. also reported no significant pH dependence on carbamazepine, comparing
pH 2.0 to neutral pH [42], whereas Togola et al. reported that SPE at acidic pH was totally
ineffective to carbamazepine [30], similar to Sebok et al. who claimed that carbamazepine could
be recovered only at pH 7.0 (comparing to pH 2.0, 4.0) [31].

Figure 1. Influence of sample pH on extraction recovery.

Abbreviation: CLA – clofibric acid, IBF – ibuprofen, SAL – salicylic acid, MET – metoprolol, NPX –
naproxen, TRC – triclosan, PRO – propranolol, KET – ketoprofen, CBZ – carbamazepine, DCF –
diclofenac, BPA – bisphenol A.
968 K. Nosek et al.

Recoveries of bisphenol A and triclosan were not extremely responsive to pH variations.

Recoveries of triclosan ranged from 66% at pH 2.0 to 90% whereas recoveries of bisphenol A
varied from 58% at pH 7.0 to 74% at pH 2.0.

3.1.3 Basic compounds

β-blockers as a basic compounds are extracted more efficiently under basic condition, although
they recovered a maximum of up to 47% at pH 9.0. Perhaps ethyl acetate did not remove
efficiently basic analytes from the sorbent. Usually methanol is successfully applied for elution
of β-blockers from SPE cartridge than ethyl acetate [27,44]; however, previous studies (not
reported) showed that methanol as an eluent is generally less effective for acidic compounds
(recoveries dropped about 30%) and causes a huge co-elution phenomena due to high content of
humic and fulvic acids in eluates.
As shown, the results on pH study do not fully accord to previously reported results within
similar conditions, which is probably due to different wastewater matrix or other factors difficult to
explain. Considering simultaneous extraction of presented group of compounds, pH 2.0 was chosen
for further experiments, because salicylic and clofibric acids did not trap on the sorbent at higher pH.

3.2 Washing step optimisation

The results of this experiments presented in the Figure 2 showed that purification step can be
crucial for the extraction performance of several target analytes. When no purification step was
applied a relative low recoveries was observed for salicylic acid and bisphenol A. Applying 5%
methanol-water solution (v/v) proved to be effective for these two analytes as their recoveries
increased relevantly. Moreover, this small addition of methanol significantly reduced humic and
fulvic impurities which are easily retained on sorbent during extraction at acidic conditions. It
has been reported previously, that the recoveries of acidic pharmaceuticals and carbamazepine
were not affected by methanol concentration in washing solution up to 20% [43] or even 40%
[30] and what is more, a higher concentration of methanol significantly reduced background
noise making analysis more reliable. Similar studies conducted for acidic and neutral pharma-
ceuticals (not reported) also showed no considerable differences in extraction yield after
washing with 5%, 15% or 30% methanol-water solution. Washing with 5% addition of methanol

Figure 2. Influence of clean-up solvent on extraction recovery.

Abbreviation: CLA – clofibric acid, IBF – ibuprofen, SAL – salicylic acid, MET – metoprolol,
NPX – naproxen, TRC – triclosan, PRO – propranolol, KET – ketoprofen, CBZ – carbamazepine,
DCF – diclofenac, BPA – bisphenol A.
 International Journal of Environmental Analytical Chemistry 969

caused significant diminution of β-blockers recoveries down to 23–30%, whilst neglect of

sorbent washing led to abnormally high recoveries what clearly has to be attributed to the co-
elutions phenomena. It seems obvious that a purification step is necessary but more compre-
hensive clean up studies are needed towards increasing recoveries of basic compounds without
substantial losses of acidic ones. For propranolol and metoprolol, an optimisation of washing
step have to be done carefully, because polar solvents might presumably run the risk of the loss
of polar analytes. N-hexane, a highly non-polar solvent was used previously at sorbent washing
step to remove hydrophobic matrix components from wastewater [27,42]. Since a lot of oil
phase was present in collected influent samples, application of n-hexane was tested in order to
check its effectiveness and neutrality to analytes. In turn, no lipid phase was observed in eluates.
With the exception of β-blockers and triclosan, even slight improvement in recoveries of target
compounds was achieved. Recoveries of metoprolol and propranolol dropped about 4% after
n-hexane application, which seems to have crucial importance here, since recoveries of these
two analytes were relatively low. However, comparing analyte signal responses (IDLs, Table 2)
and considering the fact that clofibric acid is usually determined in effluent at two times lower
concentration than β-blockers [2] it was finally decided to apply n-hexane for sorbent purifica-
tion. Taking into consideration the whole group of analytes and all advantages, the clean up step
was applied in further analysis by using both 5% methanol and n-hexane solvent.

3.3 SPE performance and validation study

Ultimately, the SPE conditions were set as in the method 1 (Table 3). In comparison to the
method presented by Santos et al. and initially adapted here [42], samples were acidified to pH
2.0 before extraction and two times smaller aliquots of wastewater were passed through the
sorbent – 250 mL of influent and 500 mL of effluent, to avoid clogging and overloading of the
HLB sorbent capacities. In this study recovery studies were performed for influent and effluent
independently by spiking wastewater samples at a concentration of 1 µg L−1 prior to the SPE
and just before the derivatisation step. Recovery rates were calculated for each compound by
comparing signal response of spiked samples before and after SPE, taking corrections associated
with blanks. The developed SPE method gives significantly better performance for pharmaceu-
ticals of acidic character – recoveries in the range of 58% for diclofenac to 109% for clofibric
acid, comparing to basic β-blockers, whose recovery was much lower – from 19% for propra-
nolol to 35% for metoprolol. The recovery yield for neutral carbamazepine ranged from 89% to
99%. In general, recovery of all investigated compounds from the effluents was slightly lower
than from the influent samples. This might be associated with the higher volume of effluent
samples loaded into the SPE cartridge. Finally, comparing the extraction performance of target
analytes studied also in the work of Santos et al. [42] (in effluents only), apart from diclofenac
similar or slightly better recoveries were obtained for all compounds.
Due to complicated matrix effects and non-availability of ideal blank samples standard
additions technique was used for quantification. Although the most recommended quantifi-
cation procedures involves the use of isotopically labelled internal standards, standard
addition method still gives satisfactory final results [17]. Six calibration points were
analysed, in the concentration range of 0.1–15.0 µg L−1 for influents and 0.05–10.0 µg L−1
for effluents. Finally, three of the most suitable points (i.e. in the range of approximately
50–150% of target analytes) were chosen for quantification. Correlation coefficients R2 were
in the range of 0.9523–0.9999. Relative standard deviations (RSDs) for three consecutive
injections of wastewater sample ranged from 0.2% for naproxen to 9.0% for diclofenac,
which are satisfactory for a real wastewater samples. Instrumental detection limits (IDLs)
were estimated from the injection of 1 µL of a standard solution (5.0 µg mL−1) as three
970 K. Nosek et al.

Table 4. Recoveries, method detection limits (MDL), method quantification limits (MQLs) , correlation
coefficients (R2) of the analytical method.

Influent Effluent

MDL, MQL, MDL, MQL,

Compound Recovery µg L−1 µg L−1 R2 Recovery µg L−1 µg L−1 R2

Salicylic acid 90% 0.017 0.058 0.9999 83% 0.003 0.010 0.9952
Ibuprofen 103% 0.006 0.020 0.9955 89% 0.014 0.048 0.9972
Diclofenac 69% 0.013 0.043 0.9913 58% 0.020 0.068 0.9796
Ketoprofen 97% 0.011 0.036 0.9906 83% 0.056 0.186 0.9866
Naproxen 98% 0.008 0.025 0.9999 99% 0.013 0.044 0.9963
Clofibric acid 109% 0.142 0.474 0.9893 92% 0.080 0.267 0.9647
Propranolol 32% 0.081 0.269 0.9913 19% 0.181 0.604 0.9866
Metoprolol 35% 0.122 0.406 0.9922 27% 0.166 0.555 0.9765
Carbamazepine 89% 0.022 0.074 0.9974 99% 0.011 0.037 0.9523
Triclosan 91% 0.015 0.051 0.9998 66% 0.051 0.170 0.9996
Bisphenol A 101% 0.012 0.041 0.9974 74% 0.007 0.024 0.9995

values of background noise (measured ± 0.5 min around the analyte retention time)
expressed in the number of pg injected and detected. The IDLs varied between 1 pg
(salicylic acid) and 119 pg (clofibric acid) injected. Method detection limits (MDLs) and
method quantification limits (MQLs) were determined as the minimum detectable concen-
tration of analyte in spiked influent and effluent samples (including all steps of the analysis
procedure) with a signal-to-noise ratio of 3:1 and 10:1, respectively. For influent MDLs
varied from 0.006 µg L−1 (ibuprofen) to 0.142 µg L−1 (clofibric acid) whereas for effluent
MDLs ranged from 0.003 µg L−1 (salicylic acid) to 0.181 µg L−1 (propranolol).All valida-
tion parameters are shown in Table 4.

3.4 Analysis of wastewater

The method developed in this work was applied to wastewater influent and effluent from a
municipal wastewater treatment plant in southern Poland in order to evaluate its suitability for
analysis of real environmental samples. The results are shown in Table 5 and chromatograms of
effluents are presented in Figure 3. Most of studied compounds have been detected in both influent

Table 5. Concentration of investigated compounds in wastewater ± standard error of

predicted concentration in the regression, µg L−1.

Compound Influent June 2012 Effluent June 2012

Salicylic acid 12.765 ± 0.234 0.452 ± 0.013

Ibuprofen 2.496 ± 0.003 < 0.042
Diclofenac 0.249 ± 0.031 0.545 ± 0.001
Ketoprofen 0.508 ± 0.066 0.485 ± 0.029
Naproxen 0.696 ± 0.007 0.313 ± 0.015
Clofibric acid < MDL 0.395 ± 0.007
Propranolol < MDL < MDL
Metoprolol <0.406 1.304 ± 0.012
Carbamazepine 0.525 ± 0.074 2.873 ± 0.031
Triclosan 0.339 ± 0.003 0.430 ± 0.004
Bisphenol A 1.393 ± 0.095 0.813 ± 0.004
 International Journal of Environmental Analytical Chemistry 971

Figure 3. GC-MS chromatograms of effluent at characteristic ions of each detected compound.

Abbreviation: CLA – clofibric acid, IBF – ibuprofen, SAL – salicylic acid, MET – metoprolol,
NPX – naproxen, TRC – triclosan, KET – ketoprofen, CBZ – carbamazepine, DCF – diclofenac,
BPA – bisphenol A.
972 K. Nosek et al.

and effluent samples in the range of 0.25–12.8 µg L−1. In influent the most abundant compounds
were salicylic acid (12.8 µg L−1), ibuprofen (2.5 µg L−1), and bisphenol A (1.4 µg L−1), whereas
the highest concentrations found in effluent correspond to carbamazepine (2.9 µg L−1), metoprolol
(1.3 µg L−1) and bisphenol A (0.81 µg L−1). Propranolol was not detected in influent, neither in
effluent samples possibly due to low recovery and MS signal, and thus relatively high
detection limits.
A comparative study among other reports available in the literature indicates that concen-
trations of target compounds in European wastewater usually vary in the range of low ng L−1 to
high µg L−1, and none of target compounds was detected in Krakow wastewater beyond this
range. Previously reported influent and effluent concentrations of all tested compounds in Spain
[11,32,45], Poland [38], UK [46], Sweden [7,47,48], France, Greece and Italy [7], Switzerland
[43], Norway [49] and Germany [6,49,50] were generally similar or higher (up to three order of
magnitude) than those reported in this study with the exception of clofibric acid, carbamazepine
and metoprolol concentrations in treated wastewater. In Spain [45], UK [46] and Switzerland
[43] clofibric acid was observed in effluent at several times lower concentrations than those
reported in this work. Carbamazepine was also determined at up to ten times lower concentra-
tions in Spain [11], Switzerland [43], Italy and Sweden [7] than in Krakow. Metoprolol was
detected in German treated wastewater at a corresponding magnitude [6], but in most reports did
not exceed the concentration of 0.41 µg L−1 [7,45,46,48,49].
The presence of target analytes in effluents clearly proves that treatment processes in
Krakow wastewater plant are insufficient to remove all of discussed emerging contaminants. It
was previously reported that several pharmaceuticals can exhibit no elimination or poor
elimination at municipal wastewater plants (up to 30–40%) and these are especially carbama-
zepine, metoprolol, diclofenac, clofibrate acid, ketoprofen [2,51,52]. Presumably, it could be
the one reason of high concentration of several pharmaceuticals in effluents (metoprolol,
carbamazepine, diclofenac) comparing to influent samples, having in mind that influent and
effluent samples represent here different portion of wastewater. In the literature, an increased
concentration of some compounds in effluents is also explained by transformation of glucur-
onide conjugates of metabolites to parent pharmaceuticals during treatment processes
[31,51,53,54], although metoprolol for example is excreated with no evidence for excretion
of conjugates [55]. It clearly needs to be confirmed through detailed study and long term
observations, which is within the scope of the plans for our future work.

4. Conclusions
The authors developed the method for simultaneous determination of 11 chemically diverse
emerging contaminants in wastewater, including nine pharmaceutical compounds. The method
combines a solid-phase extraction and GC-MS detection. The proposed method was success-
fully applied to real wastewater allowing the identification of most of the target compounds,
both in influent and effluent in the concentration up to several μg L−1.
The work confirmed that achievement of the best conditions for each of the compounds
individually is extremely hard or even unrealisable in terms of simultaneous extraction of
compounds with acidic, neutral and basic forms. Thus, the selection of experimental conditions
generally requires a compromise, and the final evaluation should not be based solely on analytes
recovery rates as such. The low method detection limits of target analytes as a whole should be
the principal indicator of the method’s performance. In this regard, further works will be
continued and a mixed-mode polymeric sorbents with cation-exchange groups will be studied
under different conditions with the aim to achieve a higher sensitivity for the detection of all
target compounds but especially basic analytes. Although many individual methods as well as
 International Journal of Environmental Analytical Chemistry 973

several multi-class solid-phase extraction procedures were proposed in the literature, it is still
not clear how to effectively deal with a diverse group of pharmaceutical contaminants. It was
shown that the results published so far can vary significantly within the same analytes and
similar extraction conditions. Thus one recommends to conduct an optimisation tests before
determination of the target pharmaceuticals in environmental samples.

Acknowledgements
This work was supported by the National Science Centre under grant no N N305 452340. The authors give
special acknowledgments to the Krakow Municipal Water and Sewage Company and its personnel for the
access to GC-MS system and for supplying wastewater samples.

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